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 DERMATOLOGY - LABORATORY AND CLINICAL RESEARCH

ENCYCLOPEDIA OF DERMATOLOGY
(6 VOLUME SET)

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DERMATOLOGY -
LABORATORY AND CLINICAL RESEARCH

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 DERMATOLOGY - LABORATORY AND CLINICAL RESEARCH

ENCYCLOPEDIA OF DERMATOLOGY
(6 VOLUME SET)

MEGHAN PRATT
EDITOR

New York
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 CONTENTS

Preface xiii
Chapter 1 Cellular and Histological Changes in Dermis Aging 1
C. M. Bernal-Mañas, C . Ferrer, E. Beltrán-Frutos,
V. Seco-Rovira and L. M. Pastor
Chapter 2 Non-Invasive Methods in the Study of the
Dermal Structure and Composition 43
Jalil Bensaci and Georgios N. Stamatas
Chapter 3 Dermal and Epidermal Interaction:
A Critical Role for Skin Homeostasis 63
Carla Abdo Brohem and Márcio Lorencini
Chapter 4 Melanogenesis and Natural Hypopigmentation Agents 83
H. M. Chiang, H. W. Chen, Y. H. Huang, S. Y. Chan,
C. C. Chen, W. C. Wu and K. C. Wen
Chapter 5 Fungal Melanins: Biosynthesis and Biological Functions 159
Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk
and Rosely Maria Zancope-Oliveira
Chapter 6 The Coat Color Genes Regulate Eumelanin and
Pheomelanin Synthesis in Melanocytes 191
Tomohisa Hirobe
Chapter 7 The Role of Melanin Production in Gaeumannomyces Graminis
Infection of Cereal Plants 221
Hanafy Fouly, Shelby Henning, Osman Radwan, Henry Wilkinson
and Bruce Martin
Chapter 8 Skin Anatomy and Physiology Research
Developments in Melanocytes 249
Naoki Oiso and Akira Kawada
Chapter 9 Optical Spectroscopy and Structural Properties of Synthetic
and Natural Eumelanin 271
Giuseppe Perna and Vito Capozzi
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vi Contents

Chapter 10 Melanic Pigmentation in Ectothermic Vertebrates:

Occurrence and Function 293
Classius de Oliveira and Lilian Franco-Belussi
Chapter 11 Fairness in a Natural Way -- Novel Polyherbal Ingredients
Inhibiting Melanin Synthesis and Transfer 307
S. Gokulshankar, M. S. Ranjith, Babu, M. A. Deepa,
B. K. Mohanty and G. Prabhakaran
Chapter 12 The Melanocortin-1 Receptor: A Key Melanoma Risk Determinant
and a Critical Regulator of the UV DNA Damage Repair Response 323
Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker
and John D’Orazio
Chapter 13 MC1R, EDNRB and Kit Signaling in Pigmentation Regulation
and related Disorders 365
Javier Pino and Lidia Kos
Chapter 14 Multiple Genes and Diverse Hierarchical Pathways Affect
Human Pigmentation 389
C. Ganesh, Anita Damodaran, Martin R. Green,
Sheila Rocha, Nicole Pauloski and Shilpa Vora
Chapter 15 Acquired Skin Pigmentation 413
Hideo Nakayama
Chapter 16 The Pro-Opiomelanocortin (POMC) and Melanocortin System in
Regulation of Human Skin Pigmentation 441
Han-En Tsai, Elsa C Chan, Gregory J. Dusting
and Guei-Sheung Liu
Chapter 17 Overview on the Melanocyte Precursor Migration from the
Neural Crest 455
Toyoko Akiyama and Ai Shinomiya
Chapter 18 Radiation Treatment and Alopecia – Past and Present Concerns 473
Paula Boaventura, Dina Pereira, José Teixeira-Gomes
and Paula Soares
Chapter 19 Psychosocial Aspects in Alopecia Areata: Studies on Stress
Involvement in Adults and Children 489
Liana Manolache
Chapter 20 The Power of the Gene: The Origin and Impact of Genetic
Disorders Alopecia: Causes, Diagnosis And Treatment 505
Naoki Oiso and Akira Kawada
Chapter 21 Alopecia Areata: Treatment Options 521
Emina Kasumagić-Halilovic and Nermina Ovcina-Kurtovic
Chapter 22 The Genetic Basis of Alopecia Areata 535
F. Megiorni, M. Carlesimo, A. Pizzuti and A. Rossi

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 Contents vii

Chapter 23 Ocular Rosacea: Recent Advances in Pathogenesis and Therapy 545

Alejandro Rodriguez-Garcia
Chapter 24 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 571
Mayra Cuéllar Cruz, Guillermo Quindós
and Everardo López Romero
Chapter 25 Candida Parapsilosis Complex 617
D. V. Moris, M. S. C. Melhem, M. A. Martins and R. P. Mendes
Chapter 26 Oral Candidiasis: Conventional and Alternative Treatment Options 655
C. E. Vergani, P. V. Sanitá, E. G. O. Mima, A. C. Pavarina
and A. L. Machado
Chapter 27 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 687
Dorota Olczak-Kowalczyk, Maria Roszkowska-Blaim,
Małgorzata Pańczyk-Tomaszewska, Maria Dąbkowska,
Ewa Swoboda-Kopeć, Beta Pyrżak, Ewa Krasuska-Sławińska
and Renata Górska
Chapter 28 Oxidative Stress and the Development of Antifungal Agents
for the Treatment of Candidiasis 717
Maxwel Adriano Abegg and Mara Silveira Benfato
Chapter 29 Inhalation and Topical Steroid Therapy and Oral Candidiasis:
A Brief Overview 735
Arjuna N. B. Ellepola, H. M. H. N. Bandara
and Hugh D Smyth
Chapter 30 Fluorescent Staining for the Diagnosis of Oral
Erythematous Candidiasis 749
Yoichi Nakagawa
Chapter 31 Cyanosis: Causes, Symptoms and Treatment 761
K. R. Ramanathan
Chapter 32 Perinatal Cyanosis: Neuropsychological Functioning 767
Ashlee R. Loughan, Robert Perna and Hana Perkey
Chapter 33 Laryngomalacia: A Cause of Cyanosis in Pediatric Age 789
Marco Berlucchi, Diego Barbieri, Daniela Tonni,
Silvana Molinaro, Patrizia Bardini and Nader Nassif
Chapter 34 The Visual Recognition of Cyanosis and the Influence of
Lighting and Color Vision 805
Stephen J. Dain
Chapter 35 Keratinocytes in Psoriasis: Key Players in the Disease Process 815
Inas Helwa, Meg Gullotto and Wendy B. Bollag
Chapter 36 Types, Triggers and Treatment Strategies of Psoriasis 871
Spyridoula Doukaki and Maria Rita Bongiorno
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viii Contents

Chapter 37 A New Strategy for the Treatment of Psoriasis —

Keratin 17 (K17)-Targeting Therapy 911
JiXin Gao and Gang Wang
Chapter 38 Narrow-Band Ultraviolet Light B (UVB) and Psoralen Plus UVA
Effect in the Circulating Levels of Biological Markers in Psoriasis 937
Susana Coimbra and Alice Santos-Silva
Chapter 39 Psoriasis Vulgaris Investigated by
Electron Paramagnetic Resonance 959
Kouichi Nakagawa and Daisuke Sawamura
Chapter 40 Psoriasis and Comorbidities 981
Nayra Merino de Paz, Marina Rodríguez-Martín
and Patricia Contreras Ferrer
Chapter 41 Nutrition and the Treatment of Psoriasis 999
Emily de Golian, Maryam Afshar and Nancy Anderson
Chapter 42 Psoriasis and Cardiovascular Disease - Update 1009
Manisha R. Panchal, Helen Coope, Anton B Alexandroff
and John McKenna
Chapter 43 Bullous Pemphigoid: An Overview 1017
Alexandre Carlos Gripp, Aline Bressan,
Cândida Naira Lima e Lima-Santana
and Daniele do Nascimento Pereira
Chapter 44 Bullous Pemphigoid Due to Anti-TNFαlpha 1025
Vincenzo Bettoli, Stefania Zauli, Michela Ricci
and Annarosa Virgili
Chapter 45 Desquamative Gingivitis as an Oral Manifestation of Mucous
Membrane Pemphigoid: Diagnosis and Treatment 1031
Hiroyasu Endo, Terry D. Rees, Hideo Niwa, Kayo Kuyama,
Hirotsugu Yamamoto and Takanori Ito
Chapter 46 Associations between Bullous Pemphigoid and Internal
Malignancies: A Literature Review 1045
Yuta Kurashige, Norihiro Ikoma, Tomotaka Mabuchi,
Akira Ozawa and Kenichi Iwashita
Chapter 47 New Therapeutic Advances in the Management of Acne 1051
Vincenzo Bettoli, Stefania Zauli and Annarosa Virgili
Chapter 48 A Large-Scale European Observational Study to Describe the
Management of Acne in Clinical Practice 1069
S. Seité and B. Dreno
Chapter 49 Skin Aging 1079
Samira Yarak and Carolina A. Pontes da Silva

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 Contents ix

Chapter 50 A Procedure for the Assessment of Skin Aging 1093

Natsuko Kakudo, Satoshi Kushida, Nobuko Saito, Kenji Suzuki
and Kenji Kusumoto
Chapter 51 Aged Skin and Strenuous Exercise: Can the Skin Handle the Heat? 1099
Stuart A. Best and Martin W. Thompson
Chapter 52 New Insights on the Regulation of Extracellular Matrix Proteins
During Skin Aging 1121
Connie B. Lin and Michael D. Southall
Chapter 53 Improved Cell Metabolism and Strengthening of the Extracellular
Matrix by Nicotinamide, and Copper for Anti-Skin Aging 1141
Neena Philips, Philips Samuel, Halyna Siomyk, Harit Parakandi,
Hui Jia, Sesha Gopal and Hossam Shahin
Chapter 54 Skin Morphology of Caucasian Women during Aging 1157
H. Zahouani, R. Vargiolu, C. Guinot, E. Tschachler
and F. Morizot
Chapter 55 Molecular Understanding of the Development of “Age Spots” 1179
Connie B. Lin and Miri Seiberg
Chapter 56 Skin Rejuvenation – Ultrastructural Study 1197
Tokuya Omi and Shigeru Sato
Chapter 57 The Role of Sun Exposure in Skin Aging 1215
Raja Dahmane, Ruza Pandel, Polonca Trebse
and Borut Poljsak
Chapter 58 Photoprotection Practices 1245
Jacqueline Selph, Ritva Vyas and Meg Gerstenblith
Chapter 59 Risk Factors for Sun Exposure During Spring Break among
College Students 1265
Marvin E. Langston, Stephanie G. Lashway
and Leslie K. Dennis
Chapter 60 Sun Exposure and Protection Habits and Vitamin D Levels in
Children and Adolescents With a History of Malignancy 1283
Yael Levy-Shraga and Dalit Modan-Moses
Chapter 61 The Surgeon General’s Call to Action to Prevent Skin Cancer:
Facts for Consumers 1301
Surgeon General of the United States
Chapter 62 The Surgeon General’s Call to Action to Prevent Skin Cancer 1305
Meg Watson, Erin Garnett, Gery P. Guy and Dawn M. Holman
Chapter 63 False and Misleading Health Information Provided to Teens
by the Indoor Tanning Industry: Investigative Report 1415
U.S. House of Representatives Committee on Energy
and Commerce-Minority Staff
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x Contents

Chapter 64 Metabolomic Assessment of Sunscreen Efficacy 1429

Manpreeet Randhawa and Michael D. Southall
Chapter 65 The History and Evolution of Sunscreen 1445
Mary Laschinger and Anna H. Chacon
Chapter 66 Psychology Behind the Use of Sunscreens, Tanning and
Skin Cancer Prevention 1457
Shailee Patel, Tulsie Patel and Katlein França
Chapter 67 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1467
Ana Flo Sierra, Víctor Flo Sierra,
Ana Cristina Calpena Campmany
and Beatriz Clares Naveros
Chapter 68 UV Filters, Their Degradation Reactions and
Eco-Toxicological Effects 1505
Albano Joel M. Santos and Joaquim C. G. Esteves da Silva
Chapter 69 Assessment of Sunscreen Safety by Skin Permeation Studies:
An Update 1523
Lucia Montenegro
Chapter 70 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1541
Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić
and Đurđica Parac-Osterman
Chapter 71 Light Conversion for UV Protection by Textile Finishing and Care 1571
Tihana Dekanić, Anita Tarbuk, Tanja Pušić,
Ana Marija Grancarić and Ivo Soljačić
Chapter 72 The Potential of Mycosporine-Like Amino Acids as UV-Sunscreens 1601
Rajesh P. Rastogi, Ravi R Sonani, Datta Madamwar
and Aran Incharoensakdi
Chapter 73 Guidelines for School Programs to Prevent Skin Cancer 1619
Karen Glanz, Mona Saraiya and Howell Wechsler
Chapter 74 Shade Planning for America’s Schools 1651
Centers for Disease Control and Prevention
Chapter 75 Sun Safety for America’s Youth Toolkit 1703
Centers for Disease Control and Prevention
Chapter 76 Burn Diagnosis, Management, and Research 1739
Amy L. Strong, Kavitha Ranganathan, Eric T. Chang,
Michael Sorkin, Shailesh Agarwal and Benjamin Levi
Chapter 77 Pediatric Burn in Bangladesh: A Tertiary Level Hospital Experience 1775
Kishore Kumar Das, M Quamruzzaman
and Syed Shamsuddin Ahmed

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 Contents xi

Chapter 78 Mulligan's Mobilisations with Movement: A Manual Therapy

Approach to the reatment and Management of Hand Burn Injuries 1789
Natalia Montes Carrasco, Maria Jesús Trancón Bergas,
Carmen Oreja Sánchez, Maria Virginia Vicente Blanco
and Javier Nieto Blasco
Chapter 79 Epidemiological Characteristics of Burn Injuries 1803
Bishara Atiyeh and Michel Costagliola
Chapter 80 Current and Future Directions of Burn Resuscitation and
Wound Management 1813
Jeanne Lee, Leslie Kobayashi and Raul Coimbra
Index 1823
 PREFACE

This encyclopedia presents important research on dermatological advances. This six set
volume includes discussions on the structure and composition of the dermis layer of the skin;
the biosynthesis, functions and health benefits of melanin; the genetics, as well as the
geographic variation and disorders, of skin pigmentation; the causes, diagnosis and treatment
of alopecia, rosacea, candidiasis, cyanosis, psoriasis, and bullous pemphigoid; new research
on skin aging; risk factors, protection practices and health effects of sun exposure; skin cancer
prevention; the use of sunscreen; skin cancer prevention guidance for schools and youth; and
the epidemiology, management and impact on muscle and joint functions of burns.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 1

CELLULAR AND HISTOLOGICAL CHANGES IN

DERMIS AGING

C. M. Bernal-Mañas1,2,, C. Ferrer2, E. Beltrán-Frutos2,

V. Seco-Rovira2 and L. M. Pastor2
1
Department of Pathology, Complejo Hospitalario
Universitario de Cartagena, Murcia, Spain
2
Department of Cellular Biology and Histology, IMIB, Aging Institute,
Medical School, Regional Campus of International Excellence
“Campus Mare Nostrum,” University of Murcia, Murcia, Spain

ABSTRACT
Skin, which is in continuous evolution throughout our lifetime, suffers changes with
age and may develop malignancies. The part of the skin most involved in its
biomechanical functions is the dermis. The knowledge of the histopathological changes
that occur in the dermis with age is essential to develop regenerative or aesthetic
techniques which will minimize or delay the effects of cutaneous aging from a
physiological point of view. During embryogenesis, the dermis undergoes changes as the
amount of collagen and elastic fibers increases and it becomes less cellular. It is in the
dermal-epidermal junction where the major alterations with age occur. Together with that
fact, the dermis changes are crucial in cutaneous aging, resulting in the loss of its
biomechanical properties. The dermis becomes thinner, more acellular and avascular,
while collagen, elastin and ground substance are altered, and cutaneous appendages
decrease. Elastic fibers decrease in number and size in the papillary dermis, which shows
signs of elastolysis. The fibroblasts show an altered metabolism by reducing their
lifespan, number and their capacity to produce collagen and to divide. All this give rise to
elongated collagen fibrils, provoking the loss of skin elasticity. Collagen fibers are
increasingly fragmented and disorganised, diminishing the overall percentage of type I
and III collagen. The bundles of collagen become thicker and stiffer, especially in the
reticular dermis. All these alterations make the skin less stretchable, less resilient and,
therefore, physiologically prone to wrinkling - changes that are known as the Net effect.


E-mail: [email protected].
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2 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

Along with this, several external agents provoke early skin aging, such as smoking or,
particularly, ultraviolet radiation, which damages the collagen and elastic fibers and
hinders the development of elastosis and telangiectasias. Photoaging consists of the
destruction of fibers in the papillary dermis, with a corresponding increase in intercellular
substance and moderate inflammatory infiltrate.
In the chapter, we will analyze in detail the main histological changes that take place
in the different elements of the dermis as a result of aging, and look at the cellular and
histological basis of certain anti-aging skin treatments which target the dermis. For this,
we bring together the information that exists on this topic, providing researchers and
dermatologists with rapid access to current knowledge concerning the mechanisms which
explain, at cellular and tissular level, part of their deterioration with age.

1. INTRODUCTION
The skin is the largest organ of the body, representing approximately 15% of body weight
[1]. Its functions are many [2-4]: for example, it acts as a mechanical barrier against physical,
chemical and biological external agents, and as an immunological organ, it regulates body
temperature and is involved in electrolyte homeostasis. We must not forget that it is the most
important organ with which we relate to others and is the ideal marker of chronological age
[5]. As the rest of the body, the skin changes throughout life, reflecting not only the age
(intrinsic aging) but also the exposure to hazardous agents such as ultraviolet radiation,
among other agents (photoaging). While much attention has been paid to the epidermis in
relation with skin cancer, the dermis has received less attention, although the changes that
occur with age mean that things have changed in recent years because of the possibilities
offered by surgery and aesthetic medicine. Therefore, we consider that it is important to know
the changes that occur in the dermis during aging, differentiating the changes caused by sun
exposure from those produced by the passage of time, because knowledge of the cellular,
histological and molecular basis of the same may help in the development of effective agents
that reduce or delay skin aging. The goal of this chapter is to explain current knowledge on
cellular and histological changes that take place in the various elements of the dermis due to
aging, mentioning briefly the histological bases of certain anti-aging treatments specifically
targeted at the dermis.

2. HISTOLOGY OF THE SKIN

Traditionally, it has been considered that the skin is made up of three zones: the
epidermal layer, the dermal layer and the hypodermal or subcutaneous cellular tissue layer
[6].

2.1. Epidermal Layer

The epidermis is a keratinized stratified squamous epithelium [6, 7], which can be
divided into four regions: basal cell layer, spinous stratum, granular stratum and cornified
stratum [6]. It is composed of four cell types: keratinocytes, melanocytes, Langerhans cells

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 Cellular and Histological Changes in Dermis Aging 3

and Merkel cells. Keratinocytes are found in the most numerous (90%), followed by
melanocytes, Langerhans cells (4-5% each) and a very low proportion of Merkel cells (less
than 1%). Keratinocytes act as a protective barrier against external agents, thanks to the
connection between them provided by desmosomes; the melanocytes transfer melanin
pigment to the keratinocytes, thus exercising a protective action [8]. It is separated from the
dermis by the basement membrane.

2.2. Dermal Layer

The dermis is a dynamic structure, divided into two parts: papillary or superficial dermis
and reticular or deep dermis, which are separated by the superficial vascular plexus. The
dermis consists of an extracellular matrix (ground substance and fibers, mainly collagen and
elastic fibers) and cells (fibroblasts, macrophages, dendritic cells, mast cells and
inflammatory cells) [4, 6]. The dermis is a supportive connective tissue for vessels, nerves
and skin appendages. Most components of the dermis are also observed in extracutaneous
tissues [6].

2.2.1. Extracellular Matrix of the Dermis

The extracellular matrix tissue is involved in the cohesion and in the regulation of
intercellular communication via different signal pathways and allows the binding of growth
factors, enzymes and other molecules [9]. The interactions between cells and the extracellular
matrix are important for normal growth and cellular differentiation [9]. Amorphous ground
substance is made up of glycosaminoglycans and acid mucopolysaccharides, usually non-
sulfated (predominantly hyaluronic acid) and sulfated (mainly chondroitin sulfate) [4, 10].
This can be visualized among collagen bundles by histochemical staining using alcian blue or
toluidine blue [1], although in pathological conditions, such as lupus erythematosus,
granuloma annulare, and dermal mucinosis, it can be observed by Hematoxylin-Eosin
staining as a string of bluish material [1]. Ultrastructurally, the dermis contains cells,
amorphous ground substance and fibers (collagen, elastic and reticular fibers) [11]. The
ground substance has a reticular filamentous framework organized into vacuoles surrounded
by a dense substance [11]. The collagen fibers are formed of tropocollagen filaments, and
vary in diameter, between 200 and 1500 Å. Longitudinally, they show an axial striation of
640 Å (up to 14 sub-bands per striation period). These striations are due to the fact that
tropocollagen molecules have no end-to-end connections, but are partially overlap. Also,
there may be fibers with an antiparallel orientation, which are important for cohesion of the
fibrillar bundles [11]. Ultrastructurally, the reticulin fibers are similar to the collagen fibers
[11]. The elastic fibers in the dermis are composed of an amorphous matrix of elastin and
intertwining bundles, measuring 10 to 14 nm in diameter [12-17]. The filaments are arranged
longitudinally and in the peripheral portion are bounded by a dense area composed of
amorphous material [11]. Elastin is found in the amorphous electronlucent matrix [18, 19].
During elastogenesis, elastin deposition occurs around or between the microfibrillar structure
[20, 21]. Microfibrils are biochemically composed of fibrillin-1 (located at the periphery),
fibrillin-2, fibrillin-3 and other microfibril-associated glycoproteins [20, 22, 23]. Elastic fibers
are mainly composed of elastin, fibrillin-1 and amyloid P component. Elastin is the insoluble
and amorphous component of elastic fibers, is rich in hydrophobic non-polar amino acids, and
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4 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

is constituted by the cross-linkage of repeating of desmosine and isodesmosine [14, 24].

Fibrillin-1, a 350 kDa glycoprotein, is the major structural component of microfibrils in
elastic fiber [20-23]. The amyloid P component is located at the periphery of the elastic fibers
[25]. The elastic system is constituted by three different fiber types: oxytalan, elaunin and
elastic fibers, which represent different stages during the histogenesis of elastic fibers [26,
27]. These distinct fibers of the elastic system have different staining characteristics and
ultrastructural patterns [27]. Oxytalan fibers are rich in microfibrils [12] and do not contain
elastin. The elaunin fibers contain numerous bundles of microfibrils intermingled with scarce
and amorphous material (elastin) [27].

2.2.2. Cells in the Dermis

In the extracellular matrix, bipolar or polydendritic cells [28] correspond to:

 Fibroblasts: derived from the mesoderm, these represent the main structural cell type
of the dermis [29]. Fibroblasts are responsible for providing the necessary elements
for the extracellular matrix composition (Table 1), synthesizing and contributing
glycosaminoglycans, elastin, fibronectin, laminin and, primarily, collagen [29]. They
also secrete several humoral factors, such as prostaglandins, leukotrienes and
cytokines [30, 31]. These cells play an important role in cell proliferation and
migration, as well as in autocrine and paracrine interactions with their neighboring
cells [9, 32].
 Langerhans cells: CD1+ or CD1- cells, (antigen-presenting cells) [28].
 Dermal melanocytes: less frequent than other cells [28].
 Mast cells: usually located around blood vessels, mast cells are characterized by a
granular cytoplasm with metachromasia, as seen by giemsa staining. These granules
contain a variety of vasoactive substances, histamine, heparin and certain cytokines
[33]. Among their functions are anticoagulation, the regulation of angiogenesis, the
modulation of mononuclear cell trafficking, extracellular matrix deposition and
remodeling, and the mediation of cytotoxicity.
 Stem cells: in the skin stem cells are usually adult stem cells. In general they play an
important role in homeostasis of the tissues, and are required for tissue replacement
throughout the lifespan of the organism [29]. They are multipotent cells in a stage of
quiescence, and divide asymmetrically and differentiate in response to extracellular
signals, losing their capacity for self-renewal [29]. The balance between quiescence
and activity is critical, and intrinsic and extrinsic signals regulate the balance of self-
renewal and differentiation [34]. In the skin, stem cells are found in the epidermis
[29, 35], hairs [36-38], sebaceous glands [39, 40], and in the dermis. As regards the
dermal stem cells, the following can be distinguished: multipotent stem cells, skin-
derived progenitor cells, dermis-derived multipotent stem cells and fibrocytes (Table
2). Dermal stem cells can express nestin, vimentin and fibronectin, as well as other
markers that induce their differentiation into various tissues [41, 42]. 6.4% of the
dermal clones derived from single cells are tripotent and represent 0.3% of dermal
fibroblasts [43]. Fibrocytes, the major progenitors of fibroblasts, can migrate from
blood to an area of the damaged dermis, infiltrate it and take part in its restoration
[42, 44], although their relationship between them with stem cells is unclear. Some

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 Cellular and Histological Changes in Dermis Aging 5

authors have suggested that dermal fibroblasts are a heterogeneous cell population
containing progenitors with different levels of differentiation: osteoblastic,
adipogenic and chondrogenic [43, 45-47]. Furthermore, the gene expression profile
of fibroblasts and mesenchymal stem cells is very similar, differing only in 4 genes:
FOXA2, KRT15, NCAM1 and NOTCH1 [48]. Unrepaired gene lesions in stem cells
are transmitted to their self-renewing daughter cells and accumulate with aging [29],
so that these cells acquire a potential for neoplastic transformation.

Other cells observed in the dermis are smooth muscle cells, lymphocytes and Schwann
cells in the nerves.

2.2.3. Structures Adnexal, Nerves and Vasculature

Skin appendages observed in the dermis include pilosebaceous units, sweat glands
(eccrine mostly, but also apocrine in certain regions of the human body such as the: armpits,
genital and periareolar areas). There are also nerves and specializations of the nervous tissue,
such as tactile sensory receptors (Meissner corpuscles) in the superficial dermis of acral skin,
and pressure receptors or Pacinian corpuscles located in the deep dermis and hypodermis [6].
As regards vascular structures, of note is the superficial vascular plexus formed by arterioles,
venules and capillaries, which extend to the dermal papillae, as well as lymph vessels.
Another vascular plexus: the deep vascular plexus separates the reticular dermis from the
subcutaneous tissue. Vascular anastomoses lie between both vascular plexuses [6].

Table 1. Principals dermal components synthesized by fibroblasts:

extracellular matrix and humoral factors

Extracellular matrix
Collagen (I, III, IV, VI)
Glycoprotein (laminin, fibronectin, thrombospondin)
Proteoglycan (glycosaminoglycan, Hyaluronic acid, Heparan sulfate, Chondroitin
sulfate)
Matrix modifying proteins (matrix metalloproteinases and inhibitor)
Secreted factors
Cytokines (IL-1, 6, 10, TNFα)
Growth factors (TGFβ, GM-CSF, PDGF, IGF-1,2, VEGF)
Chemokines (IL-8, etc.)
Inflammatory mediators (Phospholipase-A2, Prostacyclin, NO)

Table 2. Stem cell in dermal tissue

Stem cells in dermal tissue Differentiates Marker expression

Adipocytes, osteoblasts,
Multipotent dermal cells condrocytes, neural cells, Nestin -, Vimentin +
hepatocytes.
Adipocytes, smooth muscle cells,
Skin-derived progenitor cells neural cells (glial cells and Nestin +, Fibronectin +, Vimentin +
Schwann cells).
Adipocytes, osteoblasts,
Dermis-derived multipotent
condrocytes, neural cells, Desmin -, αSMA -, Collagen-Type II -
stem cells
pancreatic cells, hepatocytes.
Fibrocytes Fibroblasts Collagen-Type I, III +, Vimentin +
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6 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

Figure 1. A) Scalp of 13 years old male. The papillary and reticular dermis with abundant collagen
fibers can be recognized. B, C) Male, 23 years old: (B) lower region and (C) upper region of the dorsal
skin. Hardly any histological differences can be observed in the dermis of different regions of the skin.
In the reticular dermis of the lower back area abundant disorganized collagen fibers and little
disorganized elastic fibers are evident, while in the dermis of the upper dorsal area, there is a major
disruption of the collagen fibers, mild chronic inflammatory infiltration perivascular, and disorganized
elastic fibers with initial signs of basophilic degeneration thereof. D) Male, 62 years old, with a
common wart on the back; the dermis shows abundant collagen fibers in thick bundles and fragmented
(arrow) and irregular and abundant elastic fibers with basophilic degeneration of the same (arrowhead).
E) Woman, 68 years old, back of the hand: the patient shows a squamous cell carcinoma in the dermis,
with decreased number of collagen fibers (arrow), which are disorganized and fragmented, and
abundant thickened elastic fibers with basophilic degeneration and irregularly arranged (arrowhead). F)
Woman, 87 years, leg: squamous cell carcinoma and prominent solar elastosis in the dermis. G, H, I)
Woman, 79 years, phototype I- II skin of temporal region: microinvasive squamous cell carcinoma
(circle) in the epidermis and a severe solar elastosis in upper reticular dermis seen as elastotic
amorphous mass (asterisk): in deep reticular dermis disorganized elastic fibers are abundant with sun
damage between skin appendages; Masson Trichrome staining shows an amorphous mass and staining
for elastic fibers points to an amorphous mass that presents abundant and disorganized thicker
amorphous elastic fibers, whereas in the papillary dermis dense collagen fibers are observed.
Abreviations: Ep: epidermis; PD: papillary dermis; RD: reticular dermis; S: sebaceous gland; F: hair
follicle. Magnification: A, B, C, G) H&E 10X. D,E,F) H&E 20X. H) Masson Trichrome 10X. I) Elastic
fibers stain 10X.

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 Cellular and Histological Changes in Dermis Aging 7

2.2.4. Papillary Dermis

The papillary or superficial dermis and the periadnexal dermis present poorly organized
collagen bundles, predominantly composed of type III collagen fibers [49-51], mixed with
type I collagen and few fine elastic fibers. The elastic fibers, which are dispersed throughout
the dermis, have a characteristic structure in this portion of the dermis [18]. These fibers are
arranged vertically but, near the epidermis they form an arcade or band running parallel to the
skin surface, resulting in the so-called elaunin plexus [18], from which fine oxytalan fibers
branch to travel perpendicularly to the basal lamina of the epidermis [12].

2.2.5. Reticular Dermis

The reticular or deep dermis is thicker than the papillary dermis. It is composed of
multiple layers of well-organized thick bundles of collagens [6], predominantly type I
collagen, running parallel to the surface [49-51]. There are also elastic fibers of greater
thickness and more fragmented than in the papillary dermis, which can be detected by elastic
fiber or orcein staining protocols [1]. These elastic fibers are thicker than the oxylatan and
elaunin fibers [18], and are key players in the upper reticular dermis of the sunlight-exposed
skin in the development of senile or solar elastosis [12, 16, 52].

2.3. Hypodermis or Subcutaneous Tissue

Hypodermis or subcutaneous tissue is composed of mature adipocyte lobules separated

by thin bands of dermal connective tissue that constitute the interlobular septa [1].

3. HISTOLOGICAL VARIATIONS ACCORDING

TO ANATOMICAL REGION

It is important to bear in mind the histological characteristics of the skin according to the
anatomical region:

 The skin of the scalp contains numerous hair follicles which extend through the
dermis into the subcutaneous tissue [1].
 Facial skin shows characteristically abundant pilosebaceous units, with large
sebaceous glands on the nose [1].
 The eyelids have a thin epidermis and modified apocrine glands (Moll’s glands) and
hair follicles in the dermis.
 The skin of the back shows a thicker reticular dermis of greater density, such as the
periumbilical skin [1].
 The dermis of the external genitalia and areola of the nipple contain smooth muscle
fibers.
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8 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

4. HISTOLOGICAL METHODS TO STUDY SKIN VARIATIONS

There are various techniques that can be used to study the skin histologically; for
example, histochemical staining, immunofluorescence and immunohistochemistry.

4.1. Histochemical Stains

Depending on the findings of basic staining with hematoxylin-eosin (H&E), specific

histochemical techniques are used to visualize other structures in detail [1]:

 PAS: stains the basement membrane, glycogen (diastase-susceptible) and fungi

(diastase-resistant).
 Gomori's methanamine silver: stains fungal organisms.
 Giemsa: stains mast cells and protozoan organisms.
 Mucicarmine: stains mucin.
 Alcian Blue: stains mucopolysaccharide (pH 2.5) and sulphated mucopolysaccharide
(pH 0.5) acids.
 Congo red: stains amyloid.
 Elastic van Gienson: stains elastic fibers.
 Fontana-Masson: stains melanin.
 Von-Kossa: stains calcium.
 Picrosirius ultrared: stains collagen [5, 53].

4.2. Immunofluorescence

Immunofluorescence techniques are used to assess autoimmune disorders and blistering

diseases. The tissue, which must be fresh, is examined for the presence of immunoglobulins
A, G and M, fibrinogen and complement proteins [1].

4.3. Immunohistochemistry

These techniques are useful for differentiating specific elements that cannot be seen
correctly with H&E. They serve to establish poorly differentiated malignant tumors, among
other phenomena.
Immunohistochemical stains are used as prognostic markers and for specific
differentiation. Mainly:

 Epithelial markers: usually cytokeratin, carcinoembryonic antigen (CEA) and

epithelial membrane antigen (EMA).
 Melanocytic markers: S-100 protein, Melan A (MART-1), HMB-45.
 Mesenchymal markers: Vimentin, Factor XIIIa, CD31, CD34, S-100.

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 Cellular and Histological Changes in Dermis Aging 9

 Lymphoid markers, neuroendocrine cells markers.

 Cell proliferation markers: among them, Ki67 is an excellent marker for determining
cell proliferation [54].

5. EMBRYOLOGICAL DEVELOPMENT
The skin begins to develop at 30 days of gestation, as it is transformed from a monolayer
of undifferentiated ectoderm to an epidermal layer [55]. The elements that constitute the
dermis originate from the mesoderm. It develops later than the epidermis, and at 43 days of
gestation the dermis is characterized by numerous stellate mesenchymal cells dispersed
within a collagen-poor matrix of basophilic hyaluronic acid [6]. By the end of the first
trimester, the dermis has acquired the pattern observed in adults [55, 56]. During
embryogenesis, the dermis becomes less cellular and shows an increased amount of collagen
and elastic fibers. In newborns the dermis is more cellular than in adults, with a higher
concentration of ground substance [1], being composed of small bundles of collagen lying
parallel to the skin surface [57]. The number of eccrine glands is also higher at birth, while
apocrine glands are not well developed until puberty [58]. Sebaceous glands are developed in
childhood, but secretion begins at puberty influenced by androgen stimulation [59]. The
subcutaneous tissue is thinner than in adults, and there is much brown fat, which exerts an
important role in thermogenesis as fat molecules are degraded into fatty acids.

6. CELLULAR AND HISTOLOGICAL CHANGES

IN THE DERMIS WITH AGING

Manifestations of skin aging vary from person to person, depending on skin type,
lifestyle, the presence of disease, and genetic and environmental influences [60]. The clinical
appearance of aging differs in men and women [61-63]. Changes are observed in different
parts of the skin due to atrophy and the decrease in most skin elements [64, 65].
It is very important to remember that skin aging derives from two processes: a)
chronological or intrinsic aging, and b) photoaging [66-68]. In both processes, tissular,
cellular, ultrastructural and molecular changes occur in the different elements of the skin
(summarized in Table 3).

6.1. Causes of the Cellular and Histological Alterations

of the Dermis with Skin Aging

As already mentioned, skin homeostasis is “attacked” by the intrinsic aging process,

ultraviolet-light and other external irritants that provoke a dramatic change in the dermis
structure [29]. A distinction must be made between disorders caused by intrinsic or
chronological aging in skin areas not sun-exposed, and skin disorders caused by extrinsic
causes (such as ultraviolet irradiation) or photoaging, in sun-exposed skin regions [69].
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10 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

Table 3. Principal changes associated with aging in the dermis

of sun-protected and sun-exposed skin

Sun-protected dermis Sun-exposed dermis

Tissular and
ultrastructural changes
Collagen fibers Disruption and fragmentation. Decreased collagen I and III.
Decreased solubility. Reduction and fragmentation.
Decreased collagen I and increased
collagen III
Decreased density.
Decreased contrast between cross
striations and separation of the fiber ends
into filaments
Elastic fibers Papillary dermis: reduction and Increasing density and percentage
fragmentation. of areas with elastic fibers.
Reticular dermis: fragmentation, Accumulated and fragmented.
thickening and disorganization. Papillary dermis: dermal papillae
-Elastin: preserved or slightly decreased. loss. Absence of oxytalan fibers.
-Fibrillin-1: preserved. Reticular dermis: Pinkus elastin
-Amyloid P component: conserved, globes. Massive elastic fibers
increased. proliferation with increased
-α1-antitrypsin: expression in degenerated basophilia (solar elastosis).
fibers. Grouping and thickening.
-Elastin: decrease in papillary
Cystic spaces (lacunae) between fibers. dermis.
Granular degeneration of fibers, blurred. -Fibrillin-1: decrease.
Sun-protected dermis Sun-exposed dermis
Papillary dermis: decrease in oxytalan -Amyloid P component: decrease
fibers. Fibrillin-1 expression, electron- in papillary dermis.
dense strands and degenerative granules. -Lysozyme: expression in
Cylindrical and vertical fibers. reticular dermis.
Reticular dermis: decreased number of -α1-antitrypsin: increase in areas
peripheral microfibrils, decreased elastin, with extensive solar elastosis.
elongation and branching in large sheets. -Vitronectin: increase in areas
with extensive solar elastosis.

Decreased microfibrils and

vacuolization.
Elastin matrix: condensed strands
with granulations.

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Table 3. Principal changes associated with aging in the dermis of sun-protected and sun-
exposed skin

Sun-protected dermis Sun-exposed dermis

Extracellular matrix Dehydration. Increased inflammatory cells.
Decrease in high molecular weight
proteoglycans (Chondroitin-Sulfate).
Increase in low molecular weight
proteoglycans (Dermatan-Sulfate):
increased collagen synthesis.
Hyaluronic acid decreases.
Cellular changes
Fibroblasts Decreased number. Decreased number.
Altered metabolism. Elongation and thinning.
Decreased life expectancy. Decreased metabolism.
Decrease ability to divide. Oxidative phosphorylation
Alteration in collagen synthesis ability. dysfunction.
Numerous vacuoles and increase
in rough endoplasmic reticulum.
Stem cells Loss of replicative capacity. Similar alterations to those seen
Decreased pool. in sun-protected dermis, but
more intense.
Molecular changes
Genes transcription Cell cycle: alteration of genes that regulate the cell cycle, GOS2 increases
in replication of dermal fibroblasts.
Increase in apoptosis regulatory genes (FoxO1).
Alteration of cytokine regulation: Stat3 regulation increases.
Increased Sprr: increase extracellular matrix synthesis.
Cytoskeletal changes: senescent fibroblast replication.
Caveolin Increase: cellular hyporeactivity.
AGEs Accumulation of AGEs, residues: cell elasticity loss.
Elastin Photodamaged skin: increased polar amino acids.

6.1.1. Sun-Protected Skin (Intrinsic Aging)

Intrinsic aging of the skin is a natural process that results in slow and irreversible tissues
degeneration [68, 70], as occurs in other organs. There are more than 300 theories concerning
aging [71]. The etiopathogenetic factors responsible for intrinsic aging are multiple, and it is
possible to differentiate between internal and external agents (Table 4). One of the most
accepted theories is the free radical generation theory or oxidative stress [72-74]. Other
factors that have been implicated in the same include mild chronic inflammation which would
cause long term tissue alterations [75], changes in the nuclear and mitochondrial genome
[74], e.g., telomere attrition, a phenomenon observed in keratinocytes in cell cultures but not
in vivo [76].
The metabolism also plays an important role, as has been observed in rodents, in which
caloric restriction is associated with an increased lifespan [77]. Hormonal changes have also
been associated with accelerated cutaneous aging [63, 78], while molecular and enzymatic
alterations, e.g., abnormal expression of matrix metalloproteinases, have also been observed
in aged skin [79].
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12 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

Table 4. Principal factors associated with cutaneous aging

(intrinsic and photoaging)

INTRINSIC AGING Alterations

Internal factors
Nuclear and mitochondrial genome Shortened telomeres.
Alteration in cell cycle control.
Cytoskeletical changes.
Inflammatory response alterations.
Signaling and metabolism alterations.
Growth factors: Fos, Jun, Myc, Max and Ehf
(expression or repression).
Stem cells Changes in the stem cell niche
Molecular and enzymatic Increase in matrix metalloproteinases
alterations
Metabolism Hormonal changes: estrogen deficits
External factors
Free radicals Cell damage by oxidative stress.
Chronic inflammation Cell damage, generation of free radicals.
Metabolism Dietetic changes: caloric restriction postpones aging
and reduces oxidative damage.
Smoking Irritable and dry skin.
Increased plasma neutrophil elastase activity.
Increase in matrix metalloproteinases.
Vasoconstriction and cutaneous ischemia.
PHOTOAGING
Ultraviolet radiation p53 suppresses tumoral gene mutation (prone to
neoplasms).
DNA repair pathways alteration.
Increased matrix metalloproteinases (collagen
degradation).
Induction of transcription factors: C-Jun (inhibition of
procollagen I and III synthesis).
Free radicals generation: alteration of elastin gene
transcription.
Inflammatory response: free radical generation.
Photon ultraviolet absorption Immunosuppression and skin cancer. Quantitative and
qualitative alterations in structural macromolecules.
Damage in fibroblasts: abnormal elastin.
UVA DNA damage.
Oxygen free-radical generation.
Fibroblast apoptosis.

It has been reported that the expression of 105 genes changes with age (43 showing
down-regulation and 62 up-regulation) [74]. These changes in gene expression illustrate how
several cellular processes become deregulated with aging, leading to alterations in cell cycle
control, cytoskeletal changes, and alterations in the inflammatory response, cellular signaling
and metabolism [74].
Many genes involved in aging are associated with the expression or suppression of
growth factors, among them Fos, Jun, Myc, Max and Ehf [74]. As regards mitochondrial
changes that accompany aging, a deficit in mitochondrial DNA polymerase has been

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 Cellular and Histological Changes in Dermis Aging 13

observed in diseases characterized by accelerated aging, resulting in the accumulation of

mitochondrial DNA mutations [80]. It seems, then, that both DNA repair and genomic
stability have an enormous influence on aging and longevity [81].
Changes in diet have been associated with aging, caloric restriction increasing the
lifespan and improving the health in rodents and in other species, while some authors even
consider the same to slow down the aging process [82]. It has been suggested that caloric
restriction prevents the accumulative oxidative damage generated during aging [83].
Women, it has often been observed, show a greater predisposition to wrinkling compared
with men [61, 62, 84], aspect that has been related with the hormonal changes that occur in
women. Collagen levels decrease as a result of estrogen deficiency in postmenopausal
women, which may aggravate the severity of wrinkling [63, 78]. Treatment by hormone
replacement therapy (HRT) reduces the risk of wrinkling, although, in contrast, it appears to
increase the severity following full term pregnancies [63].
Smoking has been linked to several skin effects [84, 85], and a more pronounced skin
aging effect has been observed in smokers [86, 87, 88]. It is an independent risk factor for
premature wrinkling of the skin after controlling for sun exposure, age, sex and skin
pigmentation [87], and its cutaneous impact is dose-related. So, the more cigarettes a person
smokes per day the more pronounced the skin aging [87]. Moreoever, women are more
susceptible to the harmful cutaneous effects of smoking [61]. Smoking causes elastosis and
telangiectasia in the dermis [61, 84], although the exact mechanism responsible is still unclear
[61, 84]. The mechanism by which smoking causes the skin aging could be due to the irritant
or drying effect of smoke [84]. It has been observed that smoking can damage the collagen
and elastin in the lung parenchyma [85] and so it could cause similar changes in the skin. As
mentioned, the elastic fibers of the reticular dermis change with smoking and these changes
are similar to those associated with solar elastosis, although in this case the papillary dermis is
not affected [85]. Also snuff smoke causes an increase in the plasma neutrophil elastase
activity [89]. At molecular level, an increase in matrix metalloproteinases has been observed
in the skin of smokers [90, 91], just as in sun-exposed skin, which leads to further degradation
of dermal collagen. Another smoking effect is vasoconstriction in the microvasculature of the
skin [92]. Nicotine increases blood levels of vasopressine, which is a potent vasoconstrictor,
causing decreased blood flow and leading to chronic ischemia in the dermis [93-95]. Such
ischemia, in turn, could cause a proliferation of small blood vessels, leading to the appearance
of telangiectasias [84].

6.1.2. Sunlight-Exposed Skin (Photoaging)

As mentioned above, various external agents may be responsible for premature skin
aging, especially ultraviolet irradiation, particularly type A ultraviolet radiation.
Ultraviolet irradiation generates genetic damage that will provoke alterations that will
depend on the dose, the type of irradiation, the duration thereof and the cell type it is acting
on. For tissue changes to occur, the skin must be white or of a low phototype (I-II-III) and be
situated in an anatomical region usually exposed to sunlight [96]. The main mechanisms
involved in photoaging are summarized in Table 4.
Ultraviolet radiation induces photoaging and suppresses the systemic immune function,
resulting in photocarcinogenesis [97]. The deleterious effects caused by the absorption of
ultraviolet photons are immunosuppression and skin cancer, while photo-oxidative damage is
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14 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

responsible for the quantitative and qualitative changes in cells and in the structural
macromolecules of the dermal connective tissue [98].
It has been observed that ultraviolet radiation damages fibroblasts, causing them to
synthesize abnormal elastin, although the presence of altered elastin, histologically
manifested as elastotic material, may also be due to chronic enzymatic digestion of the
extracellular matrix by proteases induced by inflammatory mediators [99].
There are three types of ultraviolet radiation according to wavelength:

 Ultraviolet A (UVA, wavelength 315-400 nm): these wavebands are those with the
least energy and which reach deeper levels of the dermis [9]. They are responsible
for tanning and premature skin aging [100]. The initial events that cause the
histological changes and typical clinical manifestations of photoaging are DNA
damage and the generation of reactive oxygen species [101]. It has been observed
that UVA radiation induces apoptosis in the fibroblasts located in the superficial
region of the dermis [102, 103].
UVA is also involved in the development of skin tumors, but of minor severity, while
its carcinogenic role involves the generation of oxygen free radicals [100, 104],
generating 8-hydroxyguanine residues through the oxidation of guanine [100, 105].
 Ultraviolet B (UVB, wavelength 280-315 nm): these wavebands are of intermediate
energy and penetrate the epidermis. This is the main type of ultraviolet radiation
responsible for cutaneous neoplasms. It acts directly and indirectly in carcinogenesis
through different pathways: DNA damage, cell cycle alteration, oxidative stress,
inflammation and immunosuppression [100, 104, 105]. It directly damages the DNA
and generates 6-4 photoproducts between adjacent pyrimidine residues, and
pyrimidine or cyclobutane dimers. These cause mutations, cell cycle alterations and
local and systemic immunosuppression due to the generation of IL-10 and the
isomerization of trans- to cis-uroconio, which affects to the skin cells and immune
system functioning [100, 106].
Ultraviolet B radiation causes an increase in epithelial keratins, elastin and
metalloproteinases, as well as type I, IV and VII collagen degradation [107]. It also
produces an increase in leukocyte infiltration and elastase secretion by leukocytes or
dermal fibroblasts [108].
 Ultraviolet C (UVC, wavelength of 100-280 nm): these wavebands have the most
energy and are the most dangerous to health, but they are absorbed by the ozone
layer and hardly reach the earth's surface [100].

It has been observed that UV irradiation generates specific mutations (through pyrimidine
dimers) in the tumor suppressor p53 gene [109, 110]. Keratinocytes with damaged DNA
sequences in the epidermis undergo apoptosis, but those with dysfunctional p53 do not,
leading to clonal expansion, which clinically manifests as actinic keratoses [110]. The
uncontrolled proliferation of functioning abnormal cells could lead to the development of
squamous cell carcinoma.
Another important aspect is the alteration in the genetic material repair pathways that
occurs under UVA radiation through base excision repair, or, under UVB radiation through
nucleotide excision repair [100].

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 Cellular and Histological Changes in Dermis Aging 15

It has also been observed that relatively short exposure to UV radiation provokes an
increase in matrix metalloproteinase accompanied by the significant degradation of type I
collagen [70], and an inhibition of type I and III procollagen synthesis through the induction
of c-Jun, which interferes in procollagen transcription [111].
The generation of free radicals has also been associated with photoaging: ultraviolet
radiation induces elastin gene transcription through the generation of free radicals by the
xanthine/xanthine oxidase system [112, 113], which causes an increase in elastin deposition
replacing the degenerated collagen fibers [70]. Ultraviolet radiation is responsible for
generating various reactive oxygen species, such as hydrogen peroxide and singlet oxygen
[101, 114]. Likewise, a decrease in antioxidant catalase has been observed in the hydrogen
peroxide detoxification in the cornified layer of chronically sun-exposed skin [115], mainly
due to UVA irradiation [116, 117], as well as in creatine kinase activity. The loss of this
activity could be prevented by antiglycation actives [115]. Oxidative damage can also arise
from an inflammatory response, characterized by neutrophil and macrophages recruitment
and the release of superoxide radicals and nitric oxide [115], presumably, formed from the
xanthine oxidase and nitric oxide synthase enzymatic pathways, respectively [118].

6.2. Changes in the Extracellular Matrix of the Dermis Aging

6.2.1. Sun-Protected Skin (Intrinsic Aging)

6.2.1.1. Dermoepidermal Junction

The main changes due to intrinsic aging are observed at the dermoepidermal junction [70,
119, 120], where flattening results from the disappearance of the dermal papillae and
epidermal crests [121, 122].
At the dermoepidermal junction, too, a reduplication of the lamina densa and anchoring
fibril complex beneath both keratinocytes and melanocytes has been observed with aging
[123], along with the absence of projections to the basal layer of the epidermis [57, 123].

6.2.1.2. Dermal Layer

Several changes have been observed in the different papillary and reticular dermis
elements. The dermis is atrophied due to a loss of collagen, degeneration of the elastic fiber
network [64, 124], alteration of the ground substance by dehydration [68]. The most
prominent changes are observed in the collagen and elastic fibers [70].

 Collagen fibers: The most significant change observed with aging is the diminished
amount of collagen [5, 125, 126]; these fibers act as markers of the functional
capacity of the dermal fibroblasts [126], and are directly associated with the
appearance of wrinkles and with the laxity of the skin. The amount of bundles of
collagen decreases approximately 1% per year during adulthood [125], such changes
becoming more prominent from the age of fifty [5, 70, 127], while major changes
take place above the age of eighty [128]. However, some authors wrongly describe
an increase in the density of the collagen network [57], probably due to the
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compression of the collagen bundles, which imparts a more compact appearance as a

result of the loss of intercellular amorphous substance [57].
Also, changes can be observed in the type of collagen, the amount of type I collagen
fibers, which are fundamentally concentrated just beneath the epidermis, decreasing
with age, while the amount of type III collagen increases [70]. At first, type I
collagen shows an ordered structure. However, this gradually becomes more
fragmented and, in later stages, the disorganization and fragmentation of these fibers
is accompanied by an abundance of type III collagen fibers [5].
Elongated collagen fibrils cause a decrease in skin elasticity [122], although other
authors consider that the collagen bundles become thicker and stiffer [1]. The
collagen becomes less soluble and shows a lower swelling capacity, making it more
resistant to digestion by collagenase. The hexosamine: collagen ratio is low [129].
Furthermore, in aged skin collagen fibers are spaced further apart than in young skin
[130].
An abnormal expression of matrix metalloproteinases has been observed in aged skin
[79], associated with an increase in the degradation of collagen fibers and a decrease
in the ability of fibroblast growth [131], mechanisms that could explain dermal
thinning and wrinkling [123, 132, 133].
 Elastic fibers: Elastic fibers show structural and biochemical alterations that cause
changes in the elasticity of the skin [1]. No change has been observed with aging in
the percentage of the dermis areas occupied by the elastic fibers, although Suwabe et
al. [18] noted that in the upper reticular dermis the occupied area increased from 8.1
to 18.1%, while in the inferior reticular dermis it increased from 6.9 to 13.1% [18].
To study elastic fibers exhaustively, it necessary to differentiate the changes
observed according the dermal localization (papillary or reticular dermis).
Thus, in the elastic fibers of the papillary dermis gradually show a decrease in
numbers and diameter [134-136] and may even become fragmented [121]. These
elastic fibers shrink, become looser, while the deepest part of the papillary dermis
seems to separate from its main structure and attached itself to the epidermis [137].
This subepidermal elastic network regression [121] is accompanied by a decrease in
the dermal papillae [18]. Along with these changes in the dermal papillae, oxytalan
fibers in the papillary dermis decrease [18, 138], initially becoming shorter, but with
the fibrillar nature of individual elastic fibers remains unchanged. However, after the
6th decade the number of oxytalan fibers progressively decreases [70] until they
almost disappear.
Meanwhile, in the reticular dermis, the elastic fibers increase in number and diameter
[136]. The fibers show thickening, fragmentation, branching and disorganization
[121, 135], constituting an abnormal elastic network with the loss and/or focal
proliferation of some fibers [16].

As regards the components of the elastic fibers components, several changes have been
observed to result from intrinsic skin aging. These are set out below:

 Elastin and fibrillin-1: the expression of these components does not change with age
in sun-protected skin, although some authors consider that the amount of elastin in
the skin not exposed to sunlight decreases [70].

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 Cellular and Histological Changes in Dermis Aging 17

 Amyloid P component: this component is not detected in the sun-protected skin of

young people, e.g., the back, while with age its expression increases in the dermal
elastic fibers [18, 20, 21, 139, 140].
 α1-antitrypsin: occasionally the presence of α1-antitrypsin has been observed in
degenerated elastic fibers. These molecules play a protective role during the
formation of elastic fibers by attenuating extracellular proteolytic enzymes [140].
 Ground substance: The dermis ground substance becomes dehydrated [68] and there
is an overall loss of extracellular matrix [135]. Some authors believe that there is an
early rapid decrease in ground substance, which gradually slows down [141, 142],
although such a reduction is controversial. High-molecular weight proteoglycans
decrease (chondroitin sulfate) and low-molecular weight proteoglycans (dermatan
sulfate) increases, provoking the synthesis of collagen fibers and, at the same time,
their elongation, thus contributing to diminished skin elasticity [122]. The total
amount of hyaluronic acid decreases with age, as does the amount of hexose [143].

This set of skin changes associated with aging is known as the net effect, the result of
which makes the dermis less stretchable, less resilient, more lax and prone to wrinkling [144].
At ultrastructural level, the following changes in collagen and elastic fibers can be
distinguished:

 Collagen fibers: these fibers fragment and become disorganized [145]; the synthesis
of type I procollagen decreases, matrix metalloproteinases activity increases and
there is degradation of the collagen matrix in aging skin [146, 147]. As to the
ultrastructure of the collagen fibers, there is a decrease in collagen fiber density and a
diminution in the contrast between the cross striations, while the fibers at the end of
these striations separate into filaments [148].
 Elastic fibers: morphological changes in the elastic fibers occur throughout life [15,
16, 17, 149], although the most apparent ultrastructural changes are observed from
30 to 50 years of age [52].
In the papillary dermis, oxytalan fibers decrease with age, but maintain their
morphology (predominantly microfibrils) [18] and also their immunoreactivity to
fibrillin-1, which is detected in electron-dense strands and strongly expressed in the
degenerative condensed spots [18]. Scanning electron microscopy has shown that the
elastic fibers are cylindrical in shape and oriented vertically.
In the reticular dermis, elastic fibers form an amorphous matrix around the
microfibril bundles or form into electron-dense strands [15]. There is a gradual
decrease in microfibrils numbers located at the periphery of the elastic fibers [18]
and they lose immunoreactivity for elastin. Scanning electron microscopy shows the
elastic fibers to be more elongated and branched, and even arranged into large bands
in the deeper reticular dermis [57].

In general terms, transmission electron microscopy identifies cystic spaces developing

between the bundles of elastic microfibrils as aging progresses, resulting in lacunas due to the
gradual separation of the elastic fibers, and microfibrils show areas of irregularly arranged
fibers grouped into a short electron-dense amorphous material [16]. The periphery of elastic
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18 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

fibers undergoes finely granular fibrillar degeneration [16]. Finally, the elastic fibers present a
fuzzy appearance due to the deposition of a granular material on the surface of the fibers or to
the effect of elastolytic enzymes [16, 57].

6.2.1.3. Adnexal Structures, Nerves and Vasculature

Other structures associated with the dermis are also altered during aging, such as hair
follicles, eccrine glands and apocrine glands, the vascularization of the dermis and the nerve
structures.

 Cutaneous appendages:
a) Hair follicles: The number and rate of growth of hair follicles decrease. Vellus
hair develops into terminal hair, causing cosmetic problems, particularly in the
ear, nose and nostrils [1].
b) Secreting glands: The eccrine and apocrine glands also decrease in number, as
well does the functionality of the same, reducing their secrection. However, the
sebaceous glands increase in size, causing clinically sebaceous hyperplasia,
although secretion is lower due to reduced functional activity [64, 150].
 Vascularization: With aging, the cutaneous blood supply decreases, reducing the
inflammatory response, and the absorption and the clearance of the skin [151].
Microcirculation blood vessels collapse and become disorganized, even there and
avascular areas may appear [137]. Changes in the capillary bed are mainly observed
in the papillary dermis, while no changes have been observed in the reticular dermis
[152]. The capillary area decreases after 60 years of age (by up to 33%), and between
the 4th and 9th decades by 65% [152] and the distance between the capillaries is
increased [152].
The causes of these changes in the capillary bed include shortening of the capillary
loops surrounding the flattened dermal papillae, as well a decrease in their numbers
[152].
Changes in cutaneous irrigation compromise the inflammatory response during the
healing of tissue damage, increasing the amount of elastin and fibrillin II. This
affects the proximal dermal vessels, restoring the structure of the papillary dermis
and facilitating wound healing [122].
 Neural specializations: Meissner corpuscles and Vater-Pacini corpuscles show lower
functionality [153].

6.2.2. Sunlight-Exposed Skin (Photoaging)

Sun-exposed skin ages early, showing striking changes at tissue level in different regions.

6.2.2.1. Dermoepidermal Junction

Ultraviolet radiation causes changes in the structure of the dermal-epidermal junction.
Flattening of the region has been observed, with a decrease in the type IV collagen fiber
network [122], especially in the depths of the wrinkles [154]. Moreover, in sun-exposed skin
there is a decrease in type VII collagen fibers (responsible for making up the anchoring fibrils
which establish the connection of the basement membrane with the papillary dermis) [122],

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thus contributing to the formation of wrinkles [154]. Another phenomenon observed is the
depletion and reorganization of the fibrillin in the dermal-epidermal junction [154].

6.2.2.2. Dermal Layer

The tissue changes that take place in the dermis due to photoaging are multiple. Several
elements of the dermis are altered, including the ground substance and, especially, the
collagen and elastic fibers of the dermis. Ultraviolet radiation damages the collagen and
elastic fibers, giving rise to the condition known as solar elastosis, and provokes the
appearance of telangiectasia [84]. All these changes due to sun exposure are grouped under
the term dermatoheliosis. In general, there is an increase in elastotic material in the upper
dermis, a destruction of the fibril structure, an increase in the intercellular ground substance
and the presence of a moderate chronic inflammatory infiltrate [122].
The principal changes observed in the different elements of the dermis are listed below
(Table 3):

 Collagen fibers: the amount of type I and III collagen in sun-exposed skin diminishes
to a greater extent than in sun-protected skin [70].
 Elastic fibers: Changes in elastic fibers are typical of morphological findings in the
photoaged dermis. Solar elastosis consists of thickened elastic fibers with a tangled
arrangement, which finally become amorphous granular masses [16, 155, 156].
Furthermore, accumulation of this material is accompanied by elastotic degeneration
of the surrounding collagen fibers [101].

The density of elastic fibers in the dermis of the sunlight-exposed skin is greater than that
observed in the non sun-exposed skin [18], the elastic fibers fragmenting [138] and
accumulating due to their reduced degradation, and the overproduction [122] and a selective
increase of elastin [135], which contrasts with the decrease observed in the elastic fibers of
non-exposed skin [70]. Likewise, a high percentage of the dermis occupied by elastic fibers
has been observed in the more photoexposed skin areas, such as the face, especially in the
upper reticular dermis [18].
As regards changes in the papillary dermis, there is a greater loss of dermal papillae in
the sun-exposed skin [18], and no oxytalan fibers are observed in this region [18]. The elastic
fibers show a loss of fibrillar structure [70] and the same vacuolar degeneration as seen in the
subepidermal (subpapillary) zone. The fibers take on the form of cell basophil degenerative
globules, known as “elastin globes of Pinkus” [18], which correspond to the final stage of the
degradation of the elastic fibers in the sun-exposed skin [157]. Also, condensation of the
elastic fibers of the superficial vascular plexus has been observed, along with an increase in
the thickness of the fibers of photodamaged skin. Furthermore, the oxytalan fibers and the
elaunin matrix can only be observed with difficulty [70].
In the reticular dermis, the elastic fibers proliferate massively, but show alterations and
increased basophilia, in process being referred to as actinic or solar elastosis [122]. In the
upper reticular dermis there is a pronounced degree of aggregation and thickening of the
elastic fibers [18]. However, the degree of solar elastosis is lower in areas with large wrinkles
and higher in areas of deep wrinkling, and even more advanced in areas without wrinkles
[158].
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20 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

Regarding the components of elastic fibers, the following changes were observed with
photoaging:

 Elastin: disappears in the papillary dermis in photodamaged skin, but is not altered in
the reticular dermis [18].
 Fibrillin-1: the expression of immunoreactivity for this elastic fiber disappears in the
papillary dermis and upper reticular dermis of the photoaged dermis suffering severe
solar elastosis [18]. Together with the decrease in microfibrils, such disappearance is
considered one of the fundamental mechanisms in the irregular thickening and
vacuolar disruption of the fibers in the skin with solar elastosis [18, 159].
 Amyloid P component: its expression is lost in the papillary dermis [18], but remains
unchanged in the reticular dermis.
 Lysozyme: no expression is observed in the papillary dermis, while in the reticular
dermis of the photoaged areas it increases its expression [18], especially in areas with
solar elastosis and with pseudoxanthoma elasticum [140, 160]. Lysozyme deposition
is associated with an increased in basophilia in elastic fibers [18, 160].
 α1-antitrypsin: its expression is restricted to sun-exposed areas of skin and skin with
severe damage to the elastic fibers, fundamentally in the upper reticular dermis [18];
however in the inferior reticular dermis it is not expressed.
 Vitronectin: is deposited on the elastic fibers in the dermis of the sun-exposed skin,
but not on the elastic fibers in sun-protected skin. It presents the same expression
pattern as amyloid P component [20, 161, 162].
Summarizing, skin areas suffering extensive solar degeneration, such as elastin
globes of Pinkus, contain amyloid P component, lysozyme and α1-antitrypsin [18].
 Extracellular Matrix: Shows an increase in chronic inflammatory infiltrate,
generally, perivascular and perifollicular, and abundant mast cells. These cells
synthesize and release mediators that directly or indirectly modulate the production
of extracellular matrix and its degradation [163].

In the sun-exposed the skin ultrastructural changes in the dermis are accelerated by aging.

 Collagen fibers: photoaging decreases the amount of collagen fibers and fragment
them, changes that occur in the same areas that contain many elastic fibers [70].
 Elastic fibers: These consist essentially of a matrix of elastin with microfibrils
dispersed through the matrix and at the periphery of the fibers [70]. The matrix is
more homogeneous and electron-dense, with shorter, straight elongated microfibrils
arranged in the same direction as the fibers [70]. With aging, the elastic fibers take
on an irregular thickness, and become elongated and winding. Microfibrils are lost
and degenerative vacuoles of differing sizes appear [18, 52, 70]. Condensed strands
and several sizes of granulations have been observed in the matrix of elastic fibers
[18], the granulations showing greater intensity centrally and intermediate density at
the periphery [18]. Lastly, in severely photoaged skin (post7th decade) the elastin
matrix takes on a granular appearance with short and fragmented microfibrils, some
fibers showing prominent vacuoles [70], its density diminishes with a great loss of
condensed spots and microfibrillar structures [18].

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 Cellular and Histological Changes in Dermis Aging 21

Ultrastructurally, the elastotic material consists of microfibrils masses, 8-11 nm in

diameter [123]. Fibroblasts in these areas have been seen to contain numerous vesicles and
abundant rough endoplasmic reticulum around widely dilated cisternae filled with flocculent
material. These findings refer to active fibroblasts in the process of elastogenesis [164, 165].
The elastic fibers are separated from the collagen bundles and surrounded by an
amorphous material, which is probably composed of a type of glycosaminoglycans [166].
Oxytalan fibers decrease and finally disappear [52, 167].

6.3. Cellular Changes

6.3.1. Sun-Protected Skin (Intrinsic Aging)

The changes observed in dermal cells with aging include a decrease in the number of
fibroblasts, dendritic cells and mast cells [1].

 Fibroblasts: Despite decreasing in number [128, 168], fibroblasts continue to

produce collagen. Their altered metabolism shortens their life expectancy [131], and
diminishes their ability to divide and synthesize collagen (Figure 2) [122], which
results in the decreased synthesis of type I collagen and enhanced synthesis of type
III collagen. However, the decrease in collagen synthesis may also be due to several
other factors, including its degradation as a result of the synthesis of matrix
metalloproteinases, which increases with aging with no corresponding increase in
their inhibitors [169].
 Stem cells: As in other organs, the role of stem cells is important in the aging skin.
The functionality of stem or progenitor cells changes due to intrinsic and extrinsic
factors including the stem cell niche [170]. Several changes have been observed in
which the stem cells lose their replicative capacity with age, which is responsible for
many of the aspects of aging [29]. The existence of exhausted stem cells may be the
result of forced proliferative stress to maintain normal homeostatic mechanisms [29].
This depletion of the stem cell pool with age has been observed in peripheral blood
stem cells [171, 172], in skeletal muscle satellite cells [173] and in putative stem
cells of the skin [174].

6.3.2. Sunlight-Exposed Skin (Photoaging)

The cells of the different layers of the skin suffer specific changes as a result of
ultraviolet radiation, leading to skin photoaging and the development of neoplasms.
The different cells that compose the dermis undergo changes with photoaging,
particularly the fibroblasts and stem cells.

 Fibroblasts: decrease in number, and become elongated and thinner, but retain their
ability to synthesize collagen, although there is a drop in type I and III collagen fibers
[175]. Furthermore, there is growing dysfunction of their capacity for oxidative
phosphorylation [176], as well as a diminution in the energetic metabolism of the
skin in response to mild ultraviolet radiation stress [177].
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22 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

 Stem cells: greater replicative dysfunction and decreased ability to maintain dermal
homeostasis compared with sun-protected skin [178].

Figure 2. A) Dermis of Wistar rat control group, with abundant collagen bundles (arrow) and few
fibroblasts (arrowhead). B) Dermis of Wistar rats subjected to a cycle of bipolar radiofrequency with
heat production treatment. Increased amount of fibroblasts (arrowhead). Radiofrequency treatment
points to an increase in the proliferation of fibroblasts and therefore collagen synthesis. Abreviation: S:
sebaceous glands; arrows: collagen fibers. Magnification: H&E 20X.

6.5. Molecular Changes

During aging, changes occur in the genomic material, especially in the genes that regulate
the expression of cell proliferation transcription factors, apoptosis, cell cycle regulators, genes
of the cell cytoskeleton synthesis, among others. In recent years the significance of changes in
molecules such as the caveolins, and the synthesis of advanced glycation products (AGEs)
has received growing attention. The changes observed are usually due to intrinsic cellular
aging, but are magnified and evolve more quickly in the sun-exposed skin, although some
specific molecular changes are photoaging-related, among them:

 Changes in genes expression: Genes involved in the cell cycle and its control
undergo changes during cutaneous aging, and the protein GOS2 (Golgi SNAP) it has
also been observed to intervene. This protein is involved in cell cycle progression in
the G0/G1 phase, and its expression is necessary for cells to enter in the G1 phase of
the cell cycle [179]. GOS2 has been shown to be up-regulated in dermal senescent
fibroblast replication [74, 180].
As for the genes that regulate apoptosis, changes in their transcription have been
observed; for example, changes in some transcription factors such as FoxO1, which
is an important regulator of apoptosis [181], provoke an increase in apoptosis in aged
sun-exposed skin [74].
Some of the transcription factors regulated by genes altered during aging are
hyperexpressed in tumoral cells with a high proliferation index, such as transcription
factor and Myc proto-oncogene [74]. Genes involved in Stat3 expression, which is a

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 Cellular and Histological Changes in Dermis Aging 23

component that combines elements of transcription and cytokine signaling, and is

involved in tumorigenesis [74], are also overexpressed. In aged skin an increase in
the regulation of Stat3 signaling has been observed, and is considered an important
event during skin aging [74].
Genes involved in the synthesis of extracellular matrix components are modified by
aging, e.g., Sprr genes (small proline-rich), particularly Sprr2 genes, which are
induced during intrinsic skin aging and after ultraviolet radiation [182].
Cytoskeletal modulation through transcription genes seems to have an important role
during skin aging, since changes in keratin filaments [74] are involved in incorrect
cell motility, in actin cytoskeleton reorganization, in wound healing and cutaneous
fragility. With aging, cytoskeleton gene transcription can undergo upward or
downward regulation or not suffer any alteration in expression [145]. In other words,
no common pattern has been identified for the changes that cytoskeletal protein
expression shows with skin aging [145]. Most of the cytoskeletal changes associated
with aging are found in skeletal muscle, in senescent fibroblast replication and in
keratinocytes irradiated by ultraviolet light [180, 183, 184]. Down-regulation in
keratin 2A expression has been observed, while keratins 6 and 16 showed up-
regulation [74]. The expression of keratin 19 gene, a biochemical marker of skin
stem cells in vivo and in vitro [185] has not been identified in aged skin [145].
Adaptive changes in keratins 16 and 19 with aging have been associated with their
functions in the physiology of skin [145]. Besides changes in the expression of
keratin, changes in actin expression, as well as in proteins related with its function,
such as transgelin or SM22, which are up-regulated, have been seen [186, 187].
 Caveolins: High levels of caveolins, which are responsible for the hyporeactivity of
senescent cells through the modulation of receptor-mediated endocytosis, have been
observed during aging [188-190]. The functional and morphological deterioration is
affected by the status of caveolin, its increase or decrease being responsible for
several aging manifestations [191]. These molecules are considered as tentative
molecular gatekeepers [188, 192], in the same way as amphiphysin and some G
proteins [193, 194].
 Advanced glycation end products: Advanced glycation end products (AGEs) have
been identified in both sun-protected and sun-exposed skin [195, 196]. These are
residues created by the crossover of a non-enzymatic glycation reaction in the
extracellular matrix of the dermis. The AGEs are one of the factors responsible for
the loss of elasticity and other changes in the dermis during aging [195].
 Elastin: It has also been observed that photoaged elastin contains small amounts of
sugar and fat, and an abnormally high level of polar amino acids [197].

7. CLINICAL MANIFESTATIONS OF THE CUTANEOUS AGING

Systemic factors (endocrine, metabolic, nutritional) and local conditions (vascular,
neurological) affect the manifestations of skin aging (summarized in Table 5). Different parts
of the skin are affected [96]: actinic keratoses develop in the epidermis, solar elastosis in the
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24 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

dermis, telangiectasia in blood vessels and comedons in the sebaceous glands, while
melanocytes are activated and pigmentations or age spots or solar or senile lentigines appear.
During aging, the skin becomes thinner, and relatively acellular and avascular [1]. Some
authors have observed a decrease in the thickness of the dermis, mainly in the elderly [120].

Table 5. Principal cutaneous manifestations of aging

and histological changes

Skin changes Clinical manifestations Histologic changes

Intrinsic aging Thinning of the skin Dermal atrophy
Wrinkles Decreased collagen
Transparency Decreased elastic fibers
Increased fragility Decreased inflammatory
Loss elasticity response
Healing difficulty Melanocyte loss
Photoaging Increased wrinkles Decrease collagen. Increased
Redness elastic fibers.
Telangiectasia Dermal blood vessel
Rough skin tortuosity.
Irregular pigmentation Solar elastosis.
Benign (keratosis) and malignant Irregular distribution of
(melanoma and non-melanoma melanocytes.
skin cancer) neoplasms Keratinocyte displasia.

In sun-protected skin, cutaneous aging is manifested as a thinner, laxer and finally

wrinkled skin, which is transparent and shows clinical signs of increased fragility and loss of
elasticity [145]. The decrease in collagen synthesis causes atrophy in the dermis and poorer
wound healing [198]. Dermal atrophy manifests itself as a decrease in superficial skin marks
or fingerprints [199]. Furthermore, there is a decrease in fibronectin in cutaneous scars due to
a lower inflammatory response and reepithelialization, so that healing becomes more difficult.
Chronic sun damage manifests itself as skin photoaging and photocarcinogenesis. The
consequences of sun exposure are both cosmetic and medical. On the one hand, the cosmetic
changes include increased wrinkles and redness, the appearance of telangiectasia, severely
damaging the skin [96]. The aging skin shows a variety of clinical manifestations from a
rough, wrinkled, uneven pigmentation, yellow discoloration and telangiectasia, to a variety of
benign or malignant lesions [63, 200]. On the other hand, the medical consequences include
the development of non-melanomas skin tumors, melanoma, keratosis, and general weakness
of the skin, which is easily irritated [96]. In immunocompromised individuals these effects
are much more pronounced and severe, particularly in renal transplant patients [201, 202].

8. HISTOLOGICAL CHANGES OF THE DERMIS

WITH AESTHETIC TREATMENTS

There are many aesthetic treatments that attempt to palliate or disguise the clinical
manifestations of intrinsic aging and photoaging.

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Among the traditional treatments to improve the appearance of the skin are chemical
peeling and agents such as retinoic acid and glycolic acid [203], which aim to improve the
appearance of the skin after dermabrasion and photoaging. Currently new treatments based on
hormesis [204-211], cellular therapy using stem cells and fibroblasts [48], non-ablative
radiofrequency [53], photorejuvenation using 5-aminolevulinic acid [212] and caloric
restriction [213] are being developed:

 Classic aesthetic treatments: It has been suggested that the benefit of retinoids on
aged skin is due to its inhibitory effect on matrix metalloproteinases [169].
 Hormesis: Hormesis is defined as the life-supporting beneficial effects due to the
cellular response to mild stress [204-207]. Low levels of stress from physical,
chemical and biological stressors often lead to the functional improvement of cells,
tissues, organs and organisms (physiological hormesis) [214-216]. This encompasses
adaptive responses of cells and organisms to mild to moderate stress, including heat
(thermal hormesis), irradiation, hypoxia, oxidative stress and caloric restriction
[217]. Stress-mediated activation of different pathways, stimulates repair
mechanisms, thereby reducing the cumulative molecular damage associated with age
[2008].
Hormesis would explain the beneficial effects of food, spices, flavanoids and
polyphenols [209, 210, 211, 218, 219]. There are seven main response pathways to
intrinsic stress: heat shock response, unfolded protein response, autophagy,
antioxidant response, inflammatory response, DNA repair response and sirtuin
response [220, 221]. These responses are specific to a given harmful agent or process
[220, 221. These agents and processes are known as hormetins and have an anti-
aging potential. They include heat shock, irradiation, hypergravity, curcumin,
kinetin, rosmarinic acid, ferulic acid, ginseng and caloric restriction [208, 211, 222 -
227].
The application of hormesis in aging interventions based on knowledge of the
biological mechanisms involved in aging is the key to developing new cosmetic
products for skin care [208].
 Stem cells: The activity of stem cells in skin regeneration is essential and so it is
necessary to know the mechanisms involved when using stem cells in medical and
cosmetic procedures. Currently, stem cells are used to test the effectiveness of anti-
aging and rejuvenating products [48]. The self-renewal and multilineal differentiation
of skin stem cells, both fetal and adult, increase their attractiveness for studying
aging and for use in regenerative medicine, tissue repair, gene therapy and cellular
therapy with adult autologous stem cells, among other areas [228].
As we have mentioned, progenitor cell activity declines with age, although this can
be modulated by systemic factors that change with aging; for example, it has been
suggested that progenitor cells can be rejuvenated by exposure to a “young systemic
environment” [29, 229]. Control of dermal stem cells may permit us to modify the
extracellular matrix, manage humoral factor composition, and so maintain and
regenerate dermis homeostasis [29]. In addition, exchange and co-activity between
stem cells of the dermis and other cell types could cause a general activation and
rejuvenation of the skin, allowing the correction of wrinkles and slacks.
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26 C. M. Bernal-Mañas, C. Ferrer, E. Beltrán-Frutos et al.

Stem cell treatment could be considered useful for reducing wrinkles, lines and scars,
as well as for correcting facial contour problems, acne scars and other defects of the
dermis [48, 230].
 Fibroblasts: Fibroblasts have been used as filler in autologous cosmetic treatments
[48], and the application of allograft fibroblasts is considered safe and effective in the
treatment of diabetic foot ulcers [231], resurfacing of the skin with severe burns
[232] and in cutaneous cosmetic repairs [230].
 Non-ablative radiofrequency techniques: Recent years have seen the development of
non-ablative techniques in the field of aesthetic medicine. Some have been used for
facial rejuvenation by non-ablative laser or by non-ablative radiofrequency [53, 233,
234]. These aesthetic techniques cause an increase in the heat affecting the tissues
and induce a response, which produces collagen denaturation and hyalinization,
followed by a phase of wound healing and remodeling of the treated connective
tissue [235, 236]. Low-power radiofrequency has been found to cause inflammation
and subsequent neocollagenesis [237], or, in less aggressive conditions, edema,
vascular congestion and collagenization [238]. The number, proliferation and
biosynthetic activity of fibroblasts increase, phenomena that increase the cell density
of connective tissue [53]. It has not been observed that the application of low-energy
radiofrequency causes histopathological lesions in the skin [53], although, after long
sessions, occasional vascular congestion and edema in the papillary dermis have been
identified, accompanied by an increase in mucopolysaccharide acids, collagen and
elastic fibers [238]. The application of high-energy systems has been observed to
cause damage in the dermis, including periadnexal and perivascular inflammation,
ultrastructural studies showing that some collagen fibers are damaged immediately
after radiofrequency application but recover within six weeks [239]. The effect of the
low frequency bipolar radiofrequency treatment could be an adaptive phenomenon of
hormesis in the skin in response to a harmful agent [53]. Low power radiofrequency
(with or without producing heat) increases fibroblast proliferation and activates heat
shock protein [53, 240]. When bipolar radiofrequency techniques are applied, an
increase in the number of Hsp47 (heat shock protein 47) positive cells, has been seen,
the expression of which reflects the initiation, synthesis and deposition of collagen
on the extracellular matrix [53]. Hsp47 is a chaperone, which acts as an
antiinflammatory agent and stimulates proteasome degradation pathways, thus
maintaining its anti-aging benefits [241, 242]. This protein is a good marker of
fibroblast activation during collagen synthesis, wound healing and fibroblast aging
[243, 244]. The induction of Hsp70 by hormetins in dermal fibroblasts lengthens
their lifespan, maintains their morphology, enhances cell functionality and maintains
their ability to repair [208, 245].
 Photodynamic Therapy: The use of 5-aminolevulinic acid for photodynamic therapy
of photodamaged skin causes an increase in collagen fibers and the normalization of
elastotic materials [212]; its use also normalizes the ultrastructural morphology of
damaged fibroblasts, thus diminishing the dilation of the endoplasmic reticulum
[212].
 Caloric restriction: Another possible treatment is caloric restriction, an approach that
has mainly been studied in rodents [213]. Changes resulting from caloric restriction

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 Cellular and Histological Changes in Dermis Aging 27

and the effect of exercise lead to changes in the cellular cholesterol content and
promote methylation status, which could influence the cellular status of caveolin
[188]. Furthermore, the senescent phenotype of the response of growth factors may
be altered, thus by reducing the caveolin status [246]. So, if this status in aged cells is
adjusted, cell efficiency is improved by adjusting the transduction signals, and also
the structural pattern is improved by the modulation of focal adhesion complexes
[188-190].

CONCLUSION
Knowledge of the cell and tissue mechanisms of aging and its manifestation in the dermis
is crucial for developing cosmetic treatments that alleviate the effects of intrinsic aging and
photoaging in the skin. In the aged dermis the main changes are structural and quantitative
alterations of the collagen fibers (decreased in both intrinsic aging and photoaging) and
elastic fibers (decreased in intrinsic aging and increased in photoaging). Current anti-aging
treatments attempt to stop these changes.

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[238] Alvarez, N., Ortiz, L., Vicente, V., Alcaraz, M., Sánchez-Pedreño, P. (2008). The
effects of radiofrequency on skin: experimental study. Lasers in Surgery and Medicine
40, 76-82.
[239] Zelickson, B.D., Kist, D., Bernstein, E., Brown, D.B., Ksenzenko, S., Burns, J., Kilmer,
S., Mehregan, D., Pope, K. (2004). Histological and ultrastrutural evaluation of the
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effects of a radiofrequency-based nonablative termal remodeling device. Archives of

Dermatology 140, 204-209.
[240] Perez, F.P., Zhou, X., Morisaki, J., Jurivich, D. (2008). Electromagnetic field therapy
delays celular senescence and death by enhancement of the heat shock response.
Experimental Gerontology 43, 307-316.
[241] Daugaard, M., Rohde, M., Jaattela, M. (2007). The heat shock protein 70 family:
Highly homologous proteins with overlapping and distinct functions. FEBS Letters 581,
3702-3710.
[242] Liberek, K., Lewandowska, A., Zietkiewicz, S. (2008). Chaperones in control of protein
disaggregation. The EMBO Journal 27, 328-335.
[243] Miyaishi, O., Ito, Y., Kozaki, K., Sato, T., Takechi, H., Nagata, K., Saga, S. (1995).
Age-related attenuation of HSP47 heat response in fibroblasts. Mechanisms of Ageing
and Development 77, 213-226.
[244] Kuroda, K., and Tajima, S. (2004). HSP47 is a useful marker for skin fibroblasts in
formalin-fixed, paraffin-embedded tissue specimens. Journal of Cutaneous Pathology
31, 241-246.
[245] Beedholm, R., Clark, B.F.C., Rattan, S.I.S. (2004). Mild heat stress stimulates
proteasome and its 11S activator in human fibroblasts undergoing aging in vitro. Cell
Stress & Chaperones 9, 49-57.
[246] [Cho, K.A., Ryu, S.J., Park, S.J., Jang, I.S., Ahn, J.S., Kim, K.T., Park, S.C. (2003).
Senescent phenotype can be reversed by reduction of caveolin status. The Journal of
Biological Chemistry 278, 27789-27795.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 2

NON-INVASIVE METHODS IN THE STUDY

OF THE DERMAL STRUCTURE
AND COMPOSITION

Jalil Bensaci, PhD and Georgios N. Stamatas, PhD

SkinCare R&D, Johnson & Johnson Santé Beauté France, Issy-les-Moulineaux, France

ABSTRACT
Situated just below the epidermis, the dermis forms the largest layer of skin,
providing its structural and mechanical support. It is composed primarily of connective
tissue rich in tough collagen bundles and elastic fibers (elastin and fibrillin). The dermis
contains an extensive network of blood vessels, neuronal fibers, and lymphatic vessels
and supports the cutaneous follicular and eccrine structures. Early studies on dermal
structure involved invasive biopsies to provide material for histological sections. In
recent decades non-invasive methods have been developed as alternative means of
interrogating the properties of the dermal tissue. Non-invasive methods that have been
used in vivo in dermatological research include those based on principles of
ultrasonography, optical coherence tomography, confocal reflectance microscopy, higher
order optical microscopy (two-photon fluorescence and second harmonic generation),
fluorescence spectroscopy, and Raman confocal microspectroscopy. Analysis of the
resulting data to yield useful information about collagen and elastin fiber structure is an
evolving research area on its own right. In this chapter, we will review the principles of
these in vivo non-invasive methods and data analysis with emphasis on their application
in investigations of dermal structure, function, and composition. Particular applications in
dermatological research will be highlighted, including skin aging, infant skin maturation,
and dermal scars.

1. INTRODUCTION
Skin is the largest organ of the human body. It serves as the principal communication
interface between the internal organs and the environment. It is typically divided into three
major layers. From the outside to inside, they are: the epidermis, the dermis, and the subcutis.
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44 Jalil Bensaci and Georgios N. Stamatas

The epidermis is directly in contact with the external environment and its principal function is
to provide a continuously renewable barrier to the penetration of external aggressors and to
internal water loss. The dermis comes immediately below the epidermis and is thicker. Its role
is to provide structural, mechanical, and nutritional support to skin. The subcutaneous layer is
essentially composed of fat tissue and its main function is to provide cushioning, thermal
insulation, and energy storage.
The dermis is basically composed of a connective tissue, rich in collagen bundles and
elastic fibers associated with a complex network of ground substance (muccopolysaccharides
and glycoproteins). This connective tissue is crisscrossed by nerve fibers, blood and
lymphatic vessels, and skin eccrine glands and follicles. Besides these structures the dermal
connective tissue is sparsely populated by cells. The principle dermal cell type is fibroblasts
producing different types of fibers: collagen (protein providing strength and resistance), and
elastin (protein providing flexibility). In the dermis collagen fibers are primarily type I (80 to
90%) and type III (10 to 20%) [1]. Histologically, the high proportion of collagen and the
cellular dearth in the dermis visually discriminates it from the epidermis [2].
Even though it might be hard to make the histological distinction, two levels can be
defined in the dermis: the papillary and the reticular dermis. The stratum papillare is located
immediately under to the epidermis and provides the substrate (basal lamina) where
epidermal basal cells are attached. It is made of loose connective tissue, contains capillaries
providing nutrients to the epidermis and nerve fiber terminals. The stratum reticulare is a
thicker and dense layer of connective tissue, in which the bundles of collagen are arranged
parallel to the surface. It contains larger blood and lymphatic vessels, tightly interlaced elastin
fibers, and circulating immune cells including mast cells, and macrophages. The ground
substance is surrounding all the components and provides viscosity and hydration. Other
structures are also present in the dermis such as the hair follicles with an arrector pili muscle
attached to each follicle, the sebaceous (producing sebum) glands, and the apocrine and the
eccrine (sweat) glands that play an important role in the thermoregulatory capacity of the
dermis. For a long time, the only possible way to study the dermis involved the collection and
removal of tissue specimen, i.e., invasive biopsies. A main disadvantage of these methods is
that the results might be altered by the inflammation process induced by the biopsy. Another
is that the observation cannot be done in vivo. During the last decades an increasing number
of methods have been developed as alternatives to biopsy, to study skin properties in a non-
invasive way. Depending on the targeted information type (composition, structure or
function), one can orient oneself toward the most appropriate method. Each method has its
benefits and drawbacks that will be discussed.
In this chapter, we will consider methods relating to:

a) Mechanical properties, focusing on the use of the Cutometer (skin elasticity) and the
Ballistometer (stiffness).
b) Composition, with emphasis on in vivo spectroscopic methods including
Fluorescence Spectroscopy, Near Infra-Red (NIR) Spectroscopy, and Confocal
Raman Micro-Spectroscopy.
c) Structure, where we will present the principles of Ultrasonography, Optical
Coherence Tomography (OCT), Confocal Reflectance Microscopy (CRM), and
Higher Order Microscopy including Two-Photon Fluorescence Microscopy and
Second Harmonic Generation (SHG) Microscopy.

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2. MEASURING DERMAL FUNCTION: MECHANICAL PROPERTIES

Several devices have been developed for measuring the skin mechanical characteristics.
These instruments use different principles such as torsion, suction, and indentation.
Nevertheless, only a few are able to give specific information about the dermal mechanical
properties. Among these are the Cutometer and the Ballistometer.

2.1. The Cutometer

The principle of the Cutometer® (Courage and Khazaka, Electronic GmbH, Köln,
Germany) is based on suction. Its main purpose is to provide quantitative information/data on
the elasticity and the firmness of skin. The instrument generates a negative pressure inside a
probe drawing the skin into it and after some time releasing the pressure to let the tissue
return back to a relaxed state (Figure 1). The probe can have several opening sizes (2, 4, 6 or
8 mm), with the larger ones recommended to study the deepest layers of the skin. The vacuum
varies from 50 to 500 mbar. After a defined time the skin is released. The displacement of
skin inside the probe is measured by a light optical system. The system is composed of a
source of light (infrared) and two prisms facing each other, through which the light will pass
to reach a light receptor. The light intensity varies as a function of skin depth. Skin firmness
is defined as the level of resistance of the skin structure to the imposed deformation due to the
suction process, while skin elasticity is defined as its capacity to return to its original state.

Figure 1. Schematic graph representing the deformation of skin due to the Vacuum effect of the
Cutometer and the transition time for skin to return to normal.

The tool itself is connected to a computer for data transfer and processing. The results are
presented as skin displacement over time and several parameters are calculated including:
elastic deformation, retraction, and viscoelasticity. Typically ratios of parameters are
preferred. Stress versus Strain graphs can also be calculated.
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46 Jalil Bensaci and Georgios N. Stamatas

2.2. The Ballistometer

Ballistometry is based on the principle of impacting the targeted area of study with a
constant force. The purpose is to use the indentation and the rebound effect respectively as
measures of firmness and dynamic resilience. Originally, the Ballistometer was presented as a
weight system depending on gravity to provide a constant level of force when hitting the
tissue. A second generation version, the “Torsional Ballistometer” (Dia-Stron limited,
Andover, UK), includes a torsional wire mechanism, making it independent of gravity and
allowing users to select the level of energy to be generated as a function of the targeted layer.
The Ballistometer essentially provides a short tap on the skin, registering the subsequent
oscillations and describing them in terms of frequency and amplitude.
The probe has a length of 25 cm and contains a rigid low mass arm suspended at its
balance point on a torsion wire. The impact force and the dynamic properties of the test site
are considered to be the most influential parameters on the results. The first one is modulated
through a mechanical switch that is suspended into the probe. This allows the delivery of a
constant level of energy, making the data only influenced by the test site type.
The Torsional Ballistometer has been used in testing skin anti-ageing products, cellulite,
and in the evaluation of medical conditions such as scleroderma and edema, and the quality of
wound formation.
One main drawback of these instruments (Cutometer and Ballistometer) comes from the
fact that phenomena like stratum corneum moisturisation may affect the mechanical
properties measures.

3. MEASURING DERMAL COMPOSITION

3.1. Fluorescence Spectroscopy

“Fluorescence” describes the phenomenon that when light of wavelength excitation

interacts with certain substances (fluorophores), light of longer wavelength (emission) may be
emitted. The excitation light source is often in the ultraviolet to visible range (200-700 nm)
oriented toward the sample to stimulate the fluorophores. The photons will be absorbed by the
targeted molecule making an electron of the outer orbit to go briefly from its ground state
(low energy state) to its excited state (high energy state). When the electron returns to its
basic state, it releases a new photon. In each energy state (low and high), there are several
vibrational levels where the electron might find itself. As a function of the final vibrational
level reached in the ground state following the excitation, the emitted photons can occupy
energy levels described by different frequencies (or equivalently by different wavelengths).
The measurement of these spectra (fluorescence emission frequencies and their related
intensities) under a specific excitation light source is called fluorescence spectroscopy.
Using monochromators we can select the wavelength of light at the excitation or the
emission light path. There can therefore be three different types of fluorescence experiments
with regards to experimental setup: a) when the excitation wavelength is held constant and the
measurement is made for various emission wavelengths to collect an “emission spectrum”; b)
when the emission wavelength is kept constant and the measurement is done as a function of

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different excitation wavelengths to collect an “excitation spectrum”; and c) when both

excitation and emission wavelengths are changed at the same time keeping a constant
difference between the two ( = excitation – emission) in which case the experiment is a
“synchronous” scan.

Figure 2. Schematic diagram of a fluorescence spectrometer device.

A fluorescence spectrometer (also known as spectrofluorimeter) used for in vivo studies

of human skin is typically equipped with a bifurcated fiber optic probe that delivers the
excitation light to the tissue and collects the emitted light to guide it back to the spectrometer
(Figure 2). The principal fluorophores, native to the dermis, are chemical crosslinks on the
collagen and elastin molecules [3]. The deposition of fluorescent active ingredients can also
be studied with this method [4].
Fluorescence spectroscopy is fast, non-invasive, with portable instrumentation and can be
used for the chemical identification and quantification of the concentration of the
fluorophores under investigation.
One should be vigilant however, as extensive exposure to excitation (particularly in the
ultraviolet range) may photobleach the fluorophores and lead to erroneous measurements.
Moreover, fluorescence intensity can be sensitive to fluctuations in pH and temperature.

3.2. Near Infrared Spectroscopy

NIR spectroscopy refers to diffuse reflectance spectroscopy using light in the wavelength
range from about 700 to 3000 nm. NIR spectrometry is related to overtones and combination
bands derived from fundamental vibrations that are detected in the mid-infrared region of the
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48 Jalil Bensaci and Georgios N. Stamatas

electromagnetic spectrum. Mostly –CH, –NH, –OH, and –SH functional groups are the
stronger absorbers in the NIR.
There are two dominant vibrating modes in the NIR: “stretching” that is a continuous
variation in the interatomic distance along the axis of the bond between two atoms and
“bending” that is a change in the bond angle. Overtones occur roughly at 2 or 3 times the
frequency (or half and third the wavelength) of the fundamental vibrations. Combination
bands are most likely to be detected near the sum of 2 or 3 fundamental bands.
The NIR spectrometer for in vivo skin measurements is composed of a light source that is
usually a tungsten-halogen lamp, a bifurcated fiber optic probe that comes in contact with the
skin surface and a detector. These detectors might be of silicon, lead sulfide indium gallium
arsenide [5]. The instrument also includes a monochromator to separate the polychromatic
NIR spectral region into monochromatic frequencies [6]. NIR active molecules in the dermis
include water (OH bond), lipids (CH), and proteins (CH and NH). Spectral band positions and
shapes have been used to study changes in molecular structure and conformation of skin
proteins and lipids [7].
The main advantage of NIR in studying the dermis is the penetration depth even in highly
scattering tissue such as skin. The depth of the sampled volume in the skin depends on the
separation distance between the fibers of the probe that are used for illumination and those
that are used for light collection: the larger the distance the deeper the sampled volume.
The main drawback is that although NIR absorption bands are bond-specific, they are
broad and one cannot easily identify the individual molecules that contain those bonds.

3.3. Raman Spectroscopy

When an incident ray of light is scattered, a very small fraction of the scattered photons
(1 out of 107) are scattered at a different energy level (often lower) than the incident ones.
This “inelastic scattering” is known as the “Raman Effect” and it was discovered by
experiments of the Indian physicist Chandrasekhara Venkata Raman in 1928 [8]. Raman
scattering is due to the perturbation of a molecule’s electric field by the incident photon. If the
scattered photon has lower energy than the incident one, then the phenomenon is called
“Stokes scattering,” if it has higher energy, we refer to it as “anti-Stokes scattering.”
The Raman spectroscopy principle is based on the measurement of the energy shift, in
wavenumbers, of the incident light following Raman scattering events and it involves
electronic vibrations or rotations of the molecule’s chemical bonds. Raman active molecules
are the ones that allow for a change in the polarizability of their chemical bonds electronic
vibrations, in the presence of an electromagnetic field (visible or infrared light) [9]. The
Raman spectrum is a graph of the scattered light intensity versus the energy delta
(wavenumber shift).
A confocal microscopy configuration has been used as endpoint probe to collect Raman
spectra from skin in vivo at various depths from the surface. Analysis of such spectra using
chemometric methods can result in depth resolved biochemical information about the
composition of skin. Water is a molecule with a known Raman signal and Confocal Raman
spectroscopy has been recently used to measure in vivo the dermal water content, showing
that elderly dermis had significantly higher water content than young dermis [10].

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Raman spectroscopy presents many advantages of which the fact that the studied sample
needs no preparation and that there is no risk of photodamage. It provides both qualitative and
quantitative information. The spectra can be collected quickly inducing a much reduced
exposure time for the sample. Moreover, water does not interfere with Raman spectra due to
its weak scattering indexes.
The principal drawback of the method is that the Raman effect is very weak; hence its
detection needs a very sensitive and highly optimized instrumentation. Skin fluorescence
(particularly from melanin in the epidermis) can “hide” the Raman signal making it
impossible to acquire.

4. MEASURING DERMAL STRUCTURE

4.1. Ultrasonography

Ultrasound is defined as a sound wave with a frequency higher than 20 KHz, the human
hearing limit. The acoustic energy is produced by a “transducer” that is a crystal made of
piezoelectric materials (plastic polymers, lithium, ceramics or quartz) through which an
electric current is applied [11, 12]. The latest instruments allow reaching wavelengths of
higher frequencies and hence a better level of resolution, although it implies a loss of
penetration depth. Depending on the purpose, one must choose the right balance. Cutaneous
structures are evaluated with high-frequency transducers running in the 20- to 100-MHz range
[13, 14].
The extracted information depends on the acoustic behavior of the studied tissue. For skin
tissues, it is determined by the density, composition, homogeneity, and spatial organization of
skin’s structural elements. The ultrasound signal is emitted with the transducer through a
probe into the skin and as a function of the tissue structure it will undergo different
phenomena: reflection, absorption and scattering.
The ultrasound devices use the pulse echo system principle. The signal is a short burst of
ultrasonic energy (pulse) generated by a transducer. The “vibration” moves as a wave through
the tissue and can be reflected or refracted at the tissue limits. When the wave returns back
(the echo) to the transducer, it gets converted into an electric signal that is processed and
stored. The structure and composition of the different media imply different impedances and
therefore different levels of reflection/refraction [15].
The resolution of such systems is generally defined as a function of two orientations,
axial and lateral. The axial resolution is function of the duration of the pulse (and hence of the
frequency) and is the measure of the smallest thickness possible. The lateral resolution
depends not only on the frequency but also on the bandwidth. It is the measure of the smallest
structure width that can be resolved [16].
The variations of voltage in the transducer are recorded and converted into images. The
conversion can be done following three modes [13] that define the three types of devices
usually available:

1) A-mode (A-scan) for amplitude: proposing amplitude curves of each reflection

(echo) as a function of depth [17].
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50 Jalil Bensaci and Georgios N. Stamatas

2) B-mode (B-scan) for brightness: providing an image of a cross section of the skin by
combining A-scans and displaying each pixel regarding to its brightness or color
using a gray scale or a pseudo-color scale [18, 19].
3) C-mode (C-scan) for constant depth: are two dimensional but in a plane parallel to
the skin surface and are derived from multiple B-scans [20].

4.2. Optical Coherence Tomography

Unlike ultrasonography, OCT uses electromagnetic waves (such as light) instead of

mechanical waves (such as sound). It allows imaging of tissues in vivo, using near infrared
lasers. It is based on the principle of interferometry and more specifically the Michelson
configuration [21]. A ray of coherent light (the laser) is separated by a partially reflecting
mirror (the beam splitter) into two different directions (the arms). The newly formed beams
cross different distances. The first one (the reference arm) travels through air and is reflected
by a moving reference mirror, while the second one (the sample arm) goes to the targeted
tissue (the skin) on which it gets reflected as well. The two beams are then recombined
forming an interference fringe on the light detector (Figure 3). Depending on the optical
distances crossed by each beam, the result will be different. If the distance is the same, they
interfere constructively (same phase) giving bright spots; if not, they interfere destructively
(out of phase) giving darker spots [22].

Figure 3. Schematic diagram of an Optical Coherence Tomography device.

OCT in dermatology typically uses a low coherence light source, so the signal is only
observed on a limited range of distances (depth), and hence the distance at which the laser is
reflected in the tissue can be evaluated. Scanning both the mirror of the reference arm and the
final interference fringe permits the reconstruction of an image of a slice of the studied tissue
equivalent to an A-scan in ultrasound.

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The succession of scans allows obtaining cross sectional images (B-scan) of

morphological features at the micrometer scale [23]. The axial resolution of OCT devices is
nowadays able to reach levels up to 3mm [24] and even 1.2 mm when combined with
intensity-based Doppler variance [25].

4.3. Confocal Reflectance Microscopy

The interest in this instrument is that it allows real-time visualization of the skin
microstructures with details that approach histology. Contrary to the wide-field microscope, a
confocal arrangement permits to focus on one point of the studied sample and eliminate the
out-of-focus light reflected from the sample.
The basic principle of confocal microscopy was first developed by Marvin Minsky [26],
but the first real use of a laser for CRM was first reported by Rajadhyaksha and his team [27].
The usual system consists of a source of light (low intensity laser), sending beams through an
objective lens over a defined part and depth of a sample. The rays reflected back are then
“filtered” by a pinhole aperture (condenser), so that only the light from the targeted sample
site reaches the photoreceptor.
Several factors may influence the penetration depth of light as the reflectivity and the
scattering properties of the sample but also the selected wavelength. A long wavelength will
decrease the light scatter and hence will go deeper but will reduce the resolution at the same
time.
By moving the laser successively through the x and y axis, a whole area of reflected
signal is produced corresponding to a thin optical section of the tissue. By repeating the
operation at different defined depths (z axis), the CRM generates a series of “en-face” planes
(sections parallel to the surface) of the sample with a thin depth and high lateral resolution.
One main advantage of CRM is the possibility to image unlabeled living tissue without
suffering the presence of photobleaching artifacts (when fluorescence is used).
In terms of drawbacks, CRM is not specific. It relies on the natural variations in
refractive indices of tissue microstructures for contrast. In human skin, there are several
endogenous sources of contrast for confocal imaging: melanin being the strongest, but also
keratin, small structures like mitochondria and cytoplasmic organelles, chromatin in the
nuclei, and collagen and elastin fibers in the dermis [28].

4.4. Higher Order Microscopy

Higher order microscopy is based on the interaction of two incident photons at the same
point in space that result in a fluorescence or scattering event that would be equivalent to the
same event induced by a single photon with energy equal to the sum of the energies of the
two original photons. In terms of instrumentation, the difference with the regular confocal
microscopy is mainly on the source of light. In higher order microscopy a high repetition rate
(100 Mhz), ultrafast (femtosecond or picosecond pulse widths) laser like the “Titanium-
Sapphire” laser is used. This type of technology allows maximizing the two-photon excitation
events while minimizing the power deposition (photodamage) on the studied sample.
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52 Jalil Bensaci and Georgios N. Stamatas

4.4.1. Two Photon Fluorescence Microscopy

The excitation source wavelength is selected so that it will induce the excitation of a
fluorophore in the sample, only after absorption of two photons of that wavelength
successively (time window around 10-18 sec). A new photon is then released with energy
equal to the sum of the absorbed ones (high energy state). As the strength of absorption
depends on the square of the light intensity, it is considered as a nonlinear optical process.
Note that the occurrence of such an event (2 photons fluorescence) under natural or arc-lamp
light is quasi impossible, therefore it is mandatory to use light source producing photon flux
of 1020 to 1030 photons/cm²/sec [29]. The use of a mode-locked laser that emits photons
intermittently (pulsed light) in high intensity bursts is typically used, rather than in a
continuous beam, so the excitation takes place only where needed (in the focal spot) and
hence all fluorescence will come from the localized volume [30].
In the study of the dermis, the main fluorophores include collagen and elastin crosslinks
and their detection is wavelength dependent. Typically a wavelength is chosen that will excite
elastin crosslinks only and the collagen signal is captured by the SHG process (see below).
An example of such a combined image is shown in Figure 4.

Figure 4. Typical image of two-photon fluorescence (green) and second harmonic generation (red) in
the dermis.

The use of infrared light allows the imaging of living tissue up to a depth of 1 mm,
minimizing artifacts such as light scattering, photobleaching, and photodamage to the sub-
femtoliter volume. It actually does not need a pinhole aperture (contrary to the CRM).

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As a drawback it has a lower spatial resolution than the CRM because it demands the use
of excitation at twice the one-photon wavelength which leads to, more or less, half the
resolution.

4.4.2. Second Harmonic Generation Microscopy

SHG microscopy is a nonlinear optical process which requires an environment without a
center of symmetry to produce signals. It is associated to the use of pulsed light too, but
unlike fluorescence microscopy, it does not need the “absorption” part and uses the induced
scattering that might provoke a harmonic up-conversion instead (in collagen fibers for
example). SHG is coherent and sensitive to phase, allowing it to provide information on the
studied sample that is not accessible by fluorescence [31]. Another difference with
fluorescence microscopy is that the contrast is not obtained through the variations of optical
density within the sample, but through its capacity to generate second harmonic effect from
the incident light. Like in two-photon fluorescence, it is focal plan selective.
Since in SHG microscopy there is no excitation of molecules, there is a lesser risk of
phototoxicity. It can be used without labeling the targeted sample and given that no energy is
lost in SHG there is almost no risk of photobleaching [32].
However, few biological materials are able to give a clear SHG signal, such as collagen.
That is why this method is typically used in association with two-photon fluorescence to
provide a wider range of information from a single sample.

5. APPLICATIONS
5.1. Skin Aging

Skin senescence can be considered as a complex multifactorial degenerative process that

is both physiological and structural. It results from both “natural” aging (intrinsic) and
external aggressions (extrinsic) like excessive exposure to the sun. Over the years the dermis
undergoes cumulative such deteriorations leading to high disorder in the structure of collagen
and elastin fibers [33].
Crisan and team [34] have studied the aging of skin using high frequency ultrasound. The
point of the study was to measure quantitatively and as a function of different age classes, the
modifications of skin thickness, dermal density, and echogenicity. The study was made on a
160 volunteers divided in four age classes: less than 20, 21-40, 41-60 and 61-80. Three areas
where targeted: the dorsal forearm, the medial arm and the zygomatic area. They succeeded in
identifying markers of skin aging such as the LEPs/LEPi ratio (number of low echogenic
pixels in the upper dermis/number of low echogenic pixels in the lower dermis) that increases
significantly with age for all the studied areas.
In their study on skin senescence, Yang et al. [35] propose a new method to determine the
collagen content and the health of skin through fluorescence and reflectance spectra analysis.
They demonstrate that the effectiveness of collagen absorption by a healthy skin is increased
when collagen is mixed with an adequate concentration of L-ascorbic acid.
In another study, Miyamae and his collaborators [36] have used NIR diffuse reflectance
spectroscopy to monitor quantitative and qualitative variations in collagen within the skin and
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54 Jalil Bensaci and Georgios N. Stamatas

succeeded in quantitatively evaluate the influence of both photoaging and physiological aging
on the skin. The study was performed on 86 female Japanese patients aged from 23 to 69
years. They measured both a sun exposed site (outer forearm) and a sun protected site (inner
upper arm) on each subject, in spectral frequencies ranging from 8000 to 4000 cm-1. Their
results show that photoaging and chronological aging can be differentiated by a score plot of
principal component analysis of NIR diffuse reflectance data. They were able to distinguish
the degree of photoaging and physiological aging due to degenerative changes in protein
elasticity and reduction in protein quantity, respectively.

5.2. Skin Photoaging

Photoaging focuses on damages provoked by excessive exposure to the sun. Many

studies have been conducted in order to better understand the complex processes involved.
Gonzales et al. [37] demonstrated that the use of Raman spectroscopy for hydration and
protein structure measurement was able to provide information of equivalent quality when
compared to 3 mm punch biopsies analysis. The study was performed on 21 healthy patients,
and on both exposed and protected skin sites. The results obtained from Raman spectra were
analyzed by principal component analysis and were correlated with the histological data.
More recently, Zhuo et al. [38] demonstrated the use of the SHG signal to quantify
collagen changes between photodamaged and normal skin. SHG was used to generate three
dimensional images, highlighting significant differences in the collagen content and the fine
structure. Although the study was made on ex vivo samples coming from eight participants
aged from 55 to 66 years, the authors’ conclusion recommend the use of this method to
establish clinical diagnosis of photoaging on in vivo samples.
Another way to investigate the photoaging processes is to combine different methods like
multi-photon fluorescence and SGH on the same target. Lin and his team [39] studied the
superficial dermis on 3 patient faces. They were respectively aged 20, 40, and 70 years.
Working on collagen-specific SHG signal and the elastin-specific autofluorescence (AF)
signal, they obtained AF and SHG images showing collagen gradually replaced by elastin
fibers that is consistent with previous histological results. They were able to quantify their
results and to propose an index, namely the “SHG to AF Aging Index” (SAAID) as an
indicator of photoaging severity.

5.3. Edema

Under physiological conditions, the lymphatic system removes the excess of fluid
seeping from blood vessels in order to avoid liquid accumulation in the interstitial tissue
spaces and the resulting edema [40]. However in certain cases, due to infections, trauma,
surgery, radiotherapy, cancer or even congenital reasons, the lymphatic system can suffer a
dysfunction [41, 42]. Following that dysfunction the interstitial fluid gets accumulated
creating edema (tissue swelling).
NIR spectral imaging has been successfully used for the in vivo monitoring of edema and
its clinical treatments [43]. A histamine-induced edema model was used to demonstrate this

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 Non-Invasive Methods in the Study of the Dermal Structure … 55

application. The method allowed mapping the concentrations of hemoglobin and tissue water
in the area of concern.
Another team [44] using fiber optic near-infrared Fourier transform Raman spectroscopy,
showed that it was possible to quantitatively discriminate several degrees of positive patch
test reactions (inducing edemas) by estimating the relative water content. In this case,
measurements were performed at 63 patch test sites and were visually compared in 19
patients with suspected allergic contact dermatitis. The results revealed the possibility to
monitor the dynamics of the patch test reactions with a continuous grading of reaction
intensity that is appropriate for clinical studies at 48 and 72 hrs.
A new interesting method has been proposed to determine real time quantitative in vivo
depth distribution of serum albumin in case of severe burn injuries [45]. Using ultrasound
photoacoustic molecular imaging method, these authors worked on a rat deep burn model
where they measured albumin and also tissue water content and urine volume for reference
purpose. Three areas where targeted: burn, non-burn, and their boundaries. Photoacoustic
imaging allowed the researchers to follow how albumin diffused in each area type showing
two clear increases in the burn regions and their boundaries directly after burn and from 24 to
72h after burn, while it stayed limited in the non-burn regions.

5.4. Diabetes

Diabetes has been declared a global epidemic by the World Health Organization due to
its rapidly increasing incidence. It is a major cause of mortality in the age group of 20–79
years. Therefore, the regular and frequent monitoring of blood glucose is essential to avoid
diabetic complications such as diabetic retinopathy, kidney damage, heart diseases, stroke,
neuropathy, and birth defects [46].
NIR spectroscopy has been proposed for non-invasive glucose monitoring [47]. The idea
was to obtain NIR diffuse reflectance spectra based on numerical simulation of light diffusion
in skin, instead of using the usual calibration models built on in vivo experimental data. Using
multivariate analysis these authors obtained a regression coefficient vector (the calibration
model) with a very characteristic peak at around 1600 nm. Interestingly, the feature of this
peak happened to be very similar to the one obtained experimentally for the absorption band
of blood glucose. The calibration method was then validated experimentally with a very good
correlation between predicted and measured blood glucose levels (r² = 0.87; standard error of
prediction = 12.3 mg/dL). The results are supporting the idea of creating, through numerical
simulations, accurate NIR Spectroscopy calibration models for each patient, and thus
providing a better non-invasive blood glucose monitoring.
In a recent study [48], the authors developed a new imaging system associating different
optical methods: Raman spectroscopy, confocal reflectance microscopy, and multi-photon
microscopy. The objective was to acquire accurate spectral measurement with confocal
Raman under the guidance of two-photon microscopy and SHG real time imaging that
allowed defining very precise regions of interest. The team succeeded on measuring in vivo
several microstructures as much as deriving blood flow velocity and blood glucose
concentration on very precise areas.
Interestingly, the time taken for glucose measurement with current non-invasive glucose
monitoring techniques is much higher than the time taken by glucose meters. This is a
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56 Jalil Bensaci and Georgios N. Stamatas

limitation concerning most of these techniques and more intensive research efforts are still
required to develop robust non-invasive devices for highly precise glucose measurements
[46].

5.5. Scars

Scaring can be the result of the natural healing process occurring in skin following
trauma. It results from overgrowth of fibrous tissue and represents an exuberant response
[49]. Scars might be affected by several factors: biochemical, genetic and physiological ones,
influencing the type of scar such as keloid extending beyond the border limits, hypertrophic
not extending, atrophic, contracted or fine line [50]. Given that patients can become
psychologically distressed and even functionally restricted by their scars, one might easily
understand the importance of developing non-invasive methods to assess them and monitor
the efficiency of treatments.
On scars resulting from burns, an interesting study was performed to evaluate the
development of pathological scarring and to assess the treatment response progression [51].
The authors used OCT to quantify the vascularity in the region of the burn scars. The
microvessels were delimited through three-dimensional OCT speckle de-correlation imaging
[52] allowing to determine both the diameter and the density of the vasculature. Results
showed a clearly increased density of blood vessels in the hypertrophic scar tissues at 38%
instead of 22% in normal skin. They also noted a rise in vessels larger than 100 µm in the
scars that was nonexistent in the unscarred skin.
Since collagen and elastin fibers are deeply involved in the scarring process, a research
report has focused on the pathophysiology of hypertrophic scarring using higher order
microscopy [53]. Although the method was applied on ex vivo human skin samples, the
authors state that it can be applied for in vivo clinical assessment. The study was performed
on 30 skin samples of 30 µm thicknesses, obtained from 6 patients aged from 10 to 50 years
undergoing plastic surgery. These samples were analyzed using SHG for collagen imaging
and two-photon fluorescence for elastin imaging. The study provided useful quantitative
insights on the amount, the distribution, and the orientation of both fiber types. Results clearly
revealed the microstructure and spectral features of collagen and elastin and showed obvious
differences between normal skin and hypertrophic scars.

5.6. Wounds

Wound monitoring is essential to evaluate the efficacy of therapeutic treatments and to

help establishing a pertinent diagnosis. The attractiveness of optical methods for that matter
has been growing with time. Indeed, as we have previously seen, these methods can provide
fast and accurate measures non-invasively, unlike biopsies that risk perpetuating the existence
of the wound while increasing the risk of infection.
Recently, a study [54] has shown the application of confocal reflectance microscopy to
assess in vivo skin dynamic changes on patients suffering of acute radiation dermatitis. The
investigation was made including 6 women aged from 45 to 80 years, diagnosed with breast
cancer and undergoing radiotherapy. The first histopathological signs of radiation dermatitis,

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 Non-Invasive Methods in the Study of the Dermal Structure … 57

such as dendritic shaped cells, broken papillae, epidermal architectural disarray,

melanophages, and hyperpigmentation of the basal layer, were detected through confocal
microscopy after 15 days only, while the clinical ones appeared after 30 days.
Confocal laser scanning microscopy (CLSM) was used recently [55] to asses and monitor
the wound healing process in skin of different groups of patients. Fifteen patients were
recruited in three different groups: 1) 5 healthy individuals, 2) 5 with skin cancer receiving
split skin grafts, and 3) 5 Patients with chronic leg ulcers. The types of studied wounds were
thus, including acute and chronic, superficial and deep dermal ones. Parameters assessed by
CLSM included cellular, morphological and architectural wound repair characteristics, and
other dynamic processes such as blood flow. The observations were performed on both
wound bed and margin areas allowing visualizing different aspects of the wound healing
process like cutaneous inflammation, neovascularization, dynamics of wound closure, and the
time point of completed tissue repair. However, the authors point out some complications
they encountered including the difficulty of evaluating the wound bed of superficial and deep
dermal wounds due to significant crust and slough formation, and the challenge to have an
adequate positioning and immobilization of the CLSM objective for a better accuracy of the
optical resolution.
Other studies focused on bacterial infections [56] and their negative impact on wound
healing time. OCT was used to monitor the recovery of skin wounds infected by
Staphylococcus Aureus. The study was performed on mice skin samples in vitro and in vivo
using tape stripping to cause the wound, followed by bacterial infection and focusing on
collagen birefringence changes. The observations were made after 2, 4, and 10 days, using
polarization-sensitive OCT imaging that were compared to histological results, and real-time
OCT that allowed in vivo monitoring of the healing celerity of the infected wounds. The OCT
imaging system allowed the assessment of the different wound healing phases: inflammation,
collagen remodeling, and epithelium reconstitution.

CONCLUSION
Although skin biopsy may still be considered as the gold standard, it is invasive, very
demanding to evaluate, not repeatable on the same site, and brings variable results depending
on location. Considering these complications, there has been a substantial drive to develop
non-invasive in vivo methods. Currently, they even tend in many cases to replace the invasive
ones, being faster, repeatable, and more comfortable for both patients and clinicians.
In this review, we have seen numerous methods including the Cutometer and the
Balistometer for mechanical properties; Raman, NIR and Fluorescence Spectroscopy for
composition studies; and finally, Ultrasonography, Confocal Reflectance and High Order
Microscopy for structural parameters. All of them have been developed to answer the need
for non-invasive assessment and we have seen some examples of their applications in the
field of dermal research. Over time they become more accurate and reliable. Taken on their
own, none of these methods are able to provide the complete set of information needed to
address the complexity of the dermis. However, the combination of the appropriate devices
can propose a thorough set of data that might offer more complete and valuable answers.
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58 Jalil Bensaci and Georgios N. Stamatas

Their fields of application are numerous and diverse, allowing to improve both qualitatively
and quantitatively the pathological progression and the evaluation of therapeutic treatments.
It is certain that the continuous improvements of these methods image qualities and
understanding of their content will offer the necessary holistic approach allowing the dermis
comprehension. Nevertheless, a major issue lies in the nonexistence of standard
ways/protocols for the analysis of the tremendous quantity of gathered data.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 3

DERMAL AND EPIDERMAL INTERACTION:

A CRITICAL ROLE FOR SKIN HOMEOSTASIS

Carla Abdo Brohem and Márcio Lorencini†

Grupo Boticário, R&D Department,
São José dos Pinhais, Paraná, Brazil

ABSTRACT
The skin is responsible for human body survival because it performs critical
functions such as forming a protection barrier against pathogens and UV exposure,
controlling thermoregulation and evaporation, and performing sensation and metabolic
functions. The skin is organized into a complex stratified structure composed mainly of
an impermeable barrier called the epidermis and also by the dermis, which confers
physical and functional support to the skin. Other structures, such as the hypodermis
layer, and appendages, for instance hair follicles, sweat and sebaceous glands, nerves and
lymphatic and blood vessels, are also constituents of the skin. Recent studies indicate that
for the correct function and regulation of skin homeostasis, communication between the
dermal and epidermal layers is essential—the so-called epithelial-mesenchymal
interactions. Complex signaling networks established between the two major cellular
components of skin (keratinocytes and fibroblasts) have proven to be critical for
numerous processes, such as skin cell growth and differentiation, tissue repair, wound
healing and aesthetic features such as the presence of wrinkles and firmness.
Disturbances in these communication networks can cause pathological conditions ranging
from malformation during the development of the organism to cancer; these disturbances
can also trigger the loss of biological function, as observed during skin aging. Wound
healing is a well-studied process in the field of skin interactions and consists of multiple
steps, starting with inflammation and passing through the proliferation phase, followed
by maturation and scar remodeling. During this process, keratinocytes induce growth
factor expression by fibroblasts, promoting cyclical keratinocyte responses, characterized


Corresponding author: Carla Abdo Brohem. Address: Rua Alfredo Pinto, 1500. Postal Code: 83065-150. São José
dos Pinhais, Paraná, Brazil. Telephone number: 554133759173. Facsimile number: 554133757987E-mail:
[email protected].
†
E-mail: [email protected].
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64 Carla Abdo Brohem and Márcio Lorencini

by cell proliferation and correct tissue repair. Considering skin complexity as a whole,
other cell types are involved in a multiple co-regulation system. Hair induction, growth
and regeneration are also dependent on epithelial-mesenchymal interactions. Fibroblast-
derived factors are crucial for the correct regulation of melanocyte differentiation, and
stromal fibroblasts can be used as a target for melanoma therapy because they play an
important role in tumor progression. This review describes the epithelial-mesenchymal
interactions, showing the importance of communication between these two main skin
layers for normal and unimpaired biological functions.

1. INTRODUCTION
The skin is a self-renewing tissue that is the largest organ of the human body and is
responsible for numerous physiological functions such as thermoregulation, protection from
pathogens and UV radiation, tactile sensations, secretion and excretion, among others.
Additionally, as the most exposed part of the body, the skin is also an important indicator of
the health and well-being of the individual, with significant aesthetic and psychosocial
impacts (Farage et al., 2010). Skin imperfections have a negative impact on self-esteem and
can significantly affect the quality of life of individuals, causing anxiety, depression or even
social isolation (Jobling & Naldi, 2006; Bilgiç et al., 2011; Farage et al., 2010).
As a highly dynamic organ in the reception and elaboration of responses to external
stimuli, the skin has a complex signaling network between its different layers and cell types.
In this chapter, one of the primary forms of dermal function regulation will be addressed:
communication between dermis and epidermis. The main aspects of this interaction described
in the scientific literature will be considered and contextualized with relation to their
importance to skin homeostasis.

2. EPITHELIAL-MESENCHYMAL INTERACTIONS
The skin is organized into histological layers called epidermis and dermis, which differ in
flexibility, thickness and strength and provide a structured architecture that results in a variety
of skin functions. The epidermis is the outermost skin layer and acts as a selectively
permeable barrier between the body and the environment. The dermis is the underlying layer,
responsible for the physical and nutritional support of the epidermis. It is composed of
conjunctive tissue and has an abundant extracellular matrix rich in fibers such as collagen and
elastin, which confer strength and flexibility to the skin (Balasubramani et al., 2001; Ajani et
al., 2007; Brohem et al., 2010).
Communication between the two primary skin layers is essential for correct functioning.
In receiving signals from the external environment, the epidermis activates specific
mechanisms, such as cytokine production when exposed to UV radiation, which reach the
dermis and stimulate a biological response. The activation of this intra- and intercellular
signaling cascade can generate feedback stimuli to the epidermis, forming a cycle of
continuous interactions and mutual regulation between the layers. Furthermore, the dermis,
with its rich fibrous structure and blood vessels, provides constant support and guarantees the
supply of nutrients for the viable maintenance of the epidermis. Maintaining the hydroionic

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balance is another functional example of interaction between epidermis and dermis. Water
exchanges between the different skin compartments and the external environment depend on
three factors: (1) the moisture content of the external environment, (2) the ability to replace
water lost by evaporation (movement of water from the inside to the outside, or from the
blood vessels) and (3) the intrinsic ability of the stratum corneum to prevent or reduce
transepidermal water loss (Bouwstra et al., 2008). For all of these processes to occur, complex
signaling networks are established between the two primary cell components of the skin:
keratinocytes and fibroblasts. These interactions have been demonstrated to be essential to
numerous processes, such as cell growth and differentiation, tissue repair and wound healing,
in addition to the development and treatment of various diseases.

2.1. Regulation of Cell Growth and Differentiation

Studies of epithelial-mesenchymal interactions or epithelial-stromal interactions indicate

that this strong relationship plays a critical role in skin homeostasis, mediated by soluble
factors that act as autocrine and/or paracrine regulators of the growth, function and
differentiation of fibroblasts and keratinocytes (Maas-Szabowski et al., 1999; Ghahary &
Ghaffari, 2007).
The initial understanding of the majority of these interactions was achieved with in vitro
cell culture models. Keratinocytes are difficult cells to cultivate due to their innate tendency
to differentiate. In 1975, a large step in the biological study of the skin was achieved with the
in vitro isolation and proliferation of keratinocytes, described by James G. Rheinwald and
Howard Green. However, the presence of fibroblasts was necessary for keratinocyte growth
in cell culture. The success of this model consists of controlling fibroblast proliferation with
the use of lethally irradiated 3T3 cells at the correct density, with later plating of the
keratinocytes on this layer. Several years later, while reporting problems encountered with the
culture of keratinocytes, the same authors stated that epithelial cells are not independent cell
types and that their poor culturability may be due to a lack of fibroblasts to provide adequate
support (Green et al., 1977). Keratinocyte cultivation improved over time with the discovery
of essential growth factors such as epidermal growth factor (EGF) and keratinocyte growth
factor (KGF), among others (Rheinwald & Green, 1977; for a current review of in vitro
keratinocyte culture models, see Rasmussen et al., 2013).
Subsequently, numerous studies facilitating the use of these cells for the clinical
treatment of burns and for increasing knowledge regarding the skin’s physiology and
mechanisms were developed. The extensive use of this technique and its improvement
allowed many people with severe burns a chance for recovery (Martínez-Santamaría et al.,
2012; Lootens et al., 2013).
Keratinocytes are known to produce a series of soluble proteins in vivo and in vitro,
which modulate their own growth and that of other cell types. Initially, the primary function
attributed to these cytokines was the mediation of inflammatory diseases, including
interleukin (IL)-1, IL-6, IL-8, granulocyte macrophage colony stimulating factor (GM-CSF),
transforming growth factor (TGF)-α and -β, nerve growth factor (NGF) and platelet-derived
growth factor (PDGF), among other members of the fibroblast growth factor (FGF) family.
These proteins were shown to play a role in skin repair through epithelial-mesenchymal
interactions. Maas-Szabowski et al. (1999) demonstrated that, through the release of IL-1,
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66 Carla Abdo Brohem and Márcio Lorencini

keratinocytes induce an increased expression of growth factors in fibroblasts, especially

keratinocyte growth factor (KGF), in addition to inducing keratinocyte proliferation in a
positive feedback response.
The interaction between the main skin components can be better observed in in vitro
models of reconstituted skin. The model consists of fibroblasts embedded in a collagen
matrix, forming a dermal compartment that receives keratinocytes after polymerization
(Figure 1A). This cell culture is then exposed to the air-liquid interface where keratinocyte
differentiation occurs, forming the epidermal layers: basal, spinous, granulous and stratum
corneum. (Brohem et al., 2010) (Figure 1B). Some companies commercialize the
reconstituted epidermis model. However, there is great difficulty implementing the epidermis-
only model in the majority of research laboratories, which do not have access to a matrix or
culture medium sufficiently enriched for keratinocyte cell proliferation and differentiation,
with the consequent failed formation of an epidermis containing all layers found in the in vivo
epidermis.

Figure 1. Reconstructed skin model. (A) Fibroblasts are embedded in a collagen matrix, forming a
dermal compartment to receive keratinocytes after polymerization. Exposition to the air-liquid interface
promotes keratinocyte differentiation. (B) Epidermis in detail, showing the main layers: basal, spinous,
granulous and stratum corneum.

The epidermis is strongly dependent on the presence of fibroblasts for the long-term
survival of reconstituted skin. Interactions between keratinocytes and fibroblasts are mutually
important to the cell proliferation and organogenesis processes, in addition to being
influenced by the appropriate microenvironment for the correct functioning of the epidermal
tissue in reconstituted skin models (Boehnke et al., 2007).
Melanocytes are another cell type regulated by epithelial-mesenchymal interactions.
Studies performed by Yamaguchi et al. (2004, 2008 and 2009), demonstrated that the protein
dickkopf 1 (DKK1; inhibitor of the canonical Wnt signaling pathway), which is produced by
fibroblasts originating from the palmoplantar region, decreases melanocyte growth and
differentiation via regulation of microphthalmia-associated transcription factor (MITF) and β-

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 Dermal and Epidermal Interaction 67

catenin. Additionally, the authors observed that treatment of keratinocytes with DKK1
increases their proliferation and decreases melanin absorption, whereas the treatment of
reconstructed skin with DKK1 induces a thicker and less pigmented epidermis through
increased expression of keratin 9, increased a-Kelch-like ECT2-interacting protein (aKLEIP)
and decreased expression of β-catenin, glycogen synthase kinase 3b, protein kinase C and
proteinase-activated receptor-2 (PAR-2) in keratinocytes.
The use of certain drugs or agents that generally target a specific cell type can,
surprisingly, yield different effects when the target cell type is either isolated or in the
presence of other cells. One example is a derivative of the F2alpha prostaglandin - latanoprost
(LT), used to treat glaucoma, which can induce pigmentation in the iris of patients and in skin
regions near the application of the eye drop, where the skin may be in contact with the
molecule. Pigment responses to LT were examined in human iridial melanocytes alone or in
co-culture with epithelial cells or mesenchymal cells. The results revealed that only in the
presence of mesenchymal cells (that is, of fibroblasts) is there a significant increase in dopa
oxidase activity in response to the treatment, thereby demonstrating that these cells are
partially responsible for the increased pigmentation of this region (Smith - Thomas et al.,
2004).
Cells of the immune system can also be regulated by interaction with other skin cells. A
recent study demonstrated that E-cadherin is necessary to induce the expression of proteins
CD1a and Langerin, which are present in Langerhans cells (LC) but not in monocytes. LCs
are induced from monocytes after three days in culture with TGF-β1. Co-culture with
epidermal keratinocytes that express E-cadherin or the addition of soluble E-cadherin
increases the expression of Langerin, suggesting that E-cadherin interactions in the epidermis
are essential for the differentiation of LCs (Mayumi et al., 2013).

2.2. Tissue Repair and Healing

The most studied model of dermal-epidermal interaction and its components in the skin is
tissue repair and/or healing. The crosstalk between different dermal and epidermal
components enables cell recruitment and proliferation, as well as the production of matrix
elements necessary for adequate scar formation (Figure 2).
After a lesion in a specific area of the skin, a dynamic sequence of specific and complex
biological processes is initiated (Clark et al., 1996), involving soluble mediators, extracellular
matrix components, local cells (keratinocytes, fibroblasts, endothelial cells and nerve cells)
and infiltration-derived leukocytes, which will act on different phases of the healing process
(Gillitzer et al., 2001). Tissue repair can be divided into three sequential phases:
inflammation, proliferation and maturation (Witte et al., 1997; Chang et al. 2000; Gillitzer, et
al. 2001).
At the beginning of the process, resident fibroblasts proliferate from the wound margin
and migrate through a provisional matrix composed of a fibrin clot. A few days after
wounding, the provisional matrix is replaced with a newly formed conjunctive tissue, known
as the granulation tissue, essentially composed of small vessels, extracellular matrix and
spindle-shaped cells known as myofibroblasts (Hinz et al., 2001). The myofibroblasts have
contractile structures that are represented primarily by smooth muscle actin (α-SMA) (Singer
& Clark, 1999; Hinz et al., 2001; Shephard et al., 2004). This transformation involves TGF-
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68 Carla Abdo Brohem and Márcio Lorencini

β1, which is recognized as a stimulator of myofibroblast differentiation related to the α-SMA

expression level (Desmouliére, 1996; Kurosaka et al., 1998). Smad family proteins, primarily
Smad-3, are involved in TGF-β1-induced α-SMA expression (Hu et al., 2003). After the
wound is completely closed, the population of myofibroblasts disappears, most likely through
selective apoptosis (Shephard et al., 2004).
In addition to its effect on fibroblasts, TGF-β1 promotes a decrease in keratinocyte
proliferation, and therefore, re-epithelialization (Brunner & Blakytny, 2004; Yang et al.,
2012). Epidermis formation in the in vitro reconstituted skin model (that is, epidermogenesis)
suppresses the expression of α-SMA in a fibroblast-rich dermal matrix, except close to the
dermal-epidermal junction.
The α-SMA-positive cells in the dermal-epidermal junction contribute to the
hyperproliferative phenotype of the epidermis. By contrast, this epidermis expresses more
TGF-β1, which is responsible for myofibroblast differentiation (Yang et al., 2012). Therefore,
TGF-β1 appears to have two primary functions in wound repair: 1) in the initial stages, TGF-
β1 recruits inflammatory cells to the wound area, apparently in parallel to a delay in re-
epithelialization and 2) in later stages, TGF-β1 acts on fibroblasts to promote the production
of conjunctive tissue, wound contraction, and scar formation. Thus, overall healing process is
accelerated (Brunner & Blakytny, 2004), and TGF-β1 clearly acts on more than one cell type
(Figure 2).
Another cytokine that has an important role during wound healing and that has been
extensively described in the literature is interleukin-1 (IL-1). This cytokine is produced by
keratinocytes and induces the expression of KGF derivatives and other growth factors
produced by fibroblasts that will stimulate keratinocyte proliferation. In a co-culture model of
fibroblasts and keratinocytes, blocking this interleukin has been shown to increase α-SMA
expression; however, IL-1 completely suppresses the induction of α-SMA expression when
added exogenously (Shephard et al., 2004). Keratinocyte-produced IL-1 is also capable of
activating the expression of peroxisome proliferator – activated receptor β/δ (PPAR β/δ) in
the underlying fibroblasts, which in turn inhibit the mitotic activity of keratinocytes through
inhibition of the IL-1 signaling pathway. PPAR β/δ stimulates production of the IL-1 receptor
antagonist, leading to an autocrine decrease of the IL-1 signaling pathways, and consequently
decreases the production of mitogenic factors secreted by fibroblasts. This signaling is
necessary for the adequate healing of wounds and can regulate the tumor formation, as well as
homeostasis of normal human keratinocyte proliferation (Chong et al., 2009).
As cited in the regulation of cell growth and differentiation topic, KGF is a key protein in
keratinocyte proliferation, being a member of the fibroblast growth factor family (FGF7).
KGF was also shown to be essential during the tissue repair and healing process because its
expression is significantly increased through interactions between fibroblasts and
keratinocytes. This process leads to an increase in keratinocyte proliferation and migration
with triggering of local re-epithelialization and an increase in collagen deposition in the
granulation tissue (Canady et al., 2013). KGF expression is increased in people that develop
keloids when compared to healthy controls. Following KGF stimulation, the keratinocytes
produce and secrete oncostatin M (OSM) that acts on fibroblasts, inducing expression of
collagen type I-α1 and fibroblast activation protein, as well as increasing cell migration. Thus,
there is a two-way paracrine effect in which the fibroblast signaling induces keratinocyte
proliferation, which in turn stimulates an exacerbated increase of collagen production by the
fibroblasts in keloids (Canady et al., 2013).

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Figure 2. Wound healing process. A) After an injury, the healing process begins with the migration of resident fibroblasts and
subsequent proliferation. B) TGF-β1/Smad-3 signaling increases α-SMA expression, leading to the transformation of fibroblasts into
myofibroblasts. Regarding epidermis - dermis interaction, there is a communication between keratinocytes and fibroblasts that
modulates their proliferation ratio. (1) TGF-β1 recruits inflammatory cells to the wound area, apparently in parallel to a delay in re-
epithelialization; (2) Keratinocyte-produced IL-1 is also capable of activating the expression of PPAR β/δ in the underlying fibroblasts
(3), which in turn inhibit the mitotic activity of keratinocytes through IL-1 reduction (4). C) In the late wound healing stages, there is an
increased amount of IL-1 produced by keratinocytes (1) inducing the production of KGF by fibroblasts (2), as well as reduced
expression of α-SMA. Following KGF stimulation, the keratinocytes produce and secrete OSM (3) that acts on fibroblasts, inducing
expression of collagen type I-α1 (4). D) To complete the healing process, myofibroblasts apotosis occurs, as well as extracellular matrix
organization and proliferation of epidermal keratinocytes.
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70 Carla Abdo Brohem and Márcio Lorencini

Other proteins with a paracrine effect that participate in the epithelial-mesenchymal

interaction are found in the literature on keloid formation, including Smad protein, which has
a more marked role than Smad3 and is capable of inducing fibroblast proliferation and
activating collagen production in the presence of keloid-derived keratinocytes. This finding
suggests Smad signaling suppression as a new therapeutic approach in keloids (Phan et al.,
2005). The stratifin protein released by keratinocytes is involved in the overexpression of
matrix metalloproteinase-1 (MMP-1) through the expression of c-Fos and c-Jun activity in
fibroblasts. This effect is mediated at least partially by p38 mitogen-activated protein kinase
(MAPK). Other members of the MMP family are also regulated by stratifin (Medina et al.,
2007).
Finally, paracrine interactions between keratinocytes and fibroblasts direct and balance
matrix proliferation and degradation processes related to collagen metabolism and, finally,
scar formation. One study demonstrated that the activity of certain MMPs involved in tissue
repair is significantly induced when keratinocytes and fibroblasts are co-cultured, and these
increases are associated with decreased collagen levels (Tandara & Mustoe, 2011).
A clinical therapy for treating and preventing cutaneous hypertrophic scarring is
occlusion of the epidermis with a silicone gel, and its most likely mode of action is by
increasing the hydration state of the epidermal keratinocytes (Tandara & Mustoe 2008;
Gallant- Behm et al., 2011). Hydrated keratinocytes can modulate the behavior of fibroblasts,
including collagen synthesis, through the production and release of pro-inflammatory
cytokines (Gallant- Behm et al., 2011).

3. DISTURBANCES OF THE COMMUNICATION NETWORKS:

SKIN AGING, MALFORMATION, SKIN TUMORS, AND OTHER
RELATED DISEASES
As described previously, an extremely efficient communication network between cells
and other signaling components must be in perfect harmony to maintain skin homeostasis.
However, failures in this network can cause disruptions that are related to skin problems such
as the formation of keloids and hypertrophic scars, malformation during embryonic
development, skin tumors and skin aging, among others. Elucidating these processes and their
key elements can facilitate the understanding and treatment of skin disturbances, providing
scientific advancements and thus a better quality of life to the affected patients.

3.1. Skin Aging

Many morphological aspects are used to characterize skin aging, such as the appearance
of wrinkles and expression lines, decreased skin hydration (which leads to a drier
appearance), thinner and less elastic skin (which is often more susceptible to infection), spots
and non-uniform pigmentation, among other characteristics. Aging is caused by intrinsic (i.e.,
genetic) and extrinsic factors (mainly sun exposure).
Internally, the skin displays changes over time that can be observed in its different layers,
including decreased blood flow, reduced thickness of the dermis and epidermis, alterations in

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 Dermal and Epidermal Interaction 71

the organization of collagenous and elastic fibers, decreased activity of enzymes that act on
the post-translational modification process, formation of protein aggregates, modifications in
the deposition of glycosaminoglycans that tend to interact less with water molecules, and
changes in lipid content (Waller & Maibach, 2005 and 2006). Regarding the skin structure,
alterations occur, such as a reduced fat content in the subcutaneous tissue, an increase in the
elastolytic substance in the upper dermis, destruction of the fibrillar structure, an increased
amount of intercellular fluid and moderate inflammatory infiltration. A broad study that
evaluated 45 distinct skin samples from men and women with ages between 17 and 81 years
observed that, with aging, there is a decrease in the thickness and number of layers of viable
cells in the epidermis, an increased number of keratohyalin granules, flattening of the dermal-
epidermal junction, a greater presence of elastolytic material in the dermis, an increased
inflammatory infiltrate (with the presence of thicker fibrous trabeculae), and atrophy of the
hypodermis. Chronological aging also alters the metabolism of fibroblasts (reducing their
longevity), the ability for cell division and the potential for collagen production. Furthermore,
during aging, the increased collagen fibril thickness decreases the skin elasticity (Levakov et
al., 2012).
IL-1α secretion increases as the cells age. This protein is secreted by aged keratinocytes
that can stimulate the paracrine production of hepatocyte growth factor (HGF) in dermal
fibroblasts and the autocrine production of endothelin-1 (ET-1) by keratinocytes, inducing
melanocyte proliferation and increasing tyrosinase activity for melanin production. Thus, the
increase in IL-1α secretion by aged keratinocytes in the aged skin can play a role in the
marked cutaneous pigmentation and other aspects of skin aging (Okazaki et al., 2005).
UV-irradiated epidermal keratinocytes release pro-inflammatory cytokines and indirectly
promote the production of MMPs, specifically MMP-1, by dermal fibroblasts. MMPs break
down dermal collagen and other proteins, thus impairing the functional and structural
integrity of the extracellular matrix. Continuous sun exposure causes the accumulation of
dermal damage that eventually results in the appearance of wrinkles characteristic of
photoaged skin. Studies demonstrate that IL-1α produced by keratinocytes is capable of
inducing MMP-1 activation in fibroblasts through activation of MAPK and the AP-1
transcription factor (Wang & Bi, 2006).
Although there is a direct correlation between aging and an increased rate of skin
carcinomas, some authors are intrigued by why it occurs. Malaquin et al. (2013) found a
greater frequency of post-senescent keratinocytes, which are cells with transformed and
tumorigenic properties, in medium conditioned by autologous senescent dermal fibroblasts. In
addition, the emergent cells showed enhanced migratory properties and a more marked
epithelial-mesenchymal transition. The authors also observed that MMP-1 and MMP-2,
which are involved in the late stages of tumor invasion and metastases, are responsible for
migratory events through the activation of PAR-1. Finally, they found that MMPs and PAR-1
have greater expression in older individuals compared with young individuals.
The relationship between aging of the epidermis and the rate of keratinocyte proliferation
is so significant that it may even be relevant to the onset of some types of cancer. In 2011,
Lewis et al. demonstrated that the skin of older individuals has a greater number of senescent
fibroblasts, leading to a decrease in the activation of the type-1 insulin-like growth factor
receptor (IGF-1R) in keratinocytes through IGF-1 silencing. It also changes the response
potential to genotoxic stress caused by UV radiation, enabling the appearance of mutated cells
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72 Carla Abdo Brohem and Márcio Lorencini

that could initiate tumor development. Therefore, coordinated activation between the dermis
and epidermis is required for the appearance of aging-related carcinomas.

3.2. Malformation

For the development of an embryonic cell into a full human being with its complexities,
signaling and communication networks between the different cells of the human body were
defined over the course of evolution. Communication between cell components can be
obtained by cell-cell interactions, cell contact with extracellular matrix components or by
soluble signaling factors that bind to specific receptors in the plasma membrane. Any
disturbance of these intercellular communication signals can cause pathological disorders,
resulting in impaired organism development and frequently leading to premature death,
physiological disabilities, aging or disease onset.
During development, ectodermal cells depend on instructive signals from the underlying
mesenchyme to first form an epithelial cell line and, subsequently, form a fully differentiated
epidermis with its cutaneous appendages. In the skin of an adult, epithelial-mesenchymal
interactions are involved in the maintenance of the epidermal barrier function through
regulation of the keratinocyte proliferation rate and, consequently, the suprabasal
keratinization steps. The signals exchanged by these interactions are critical for skin repair
and development. (Shepard et al., 2004).

3.3. Skin Tumors

The role of epidermal-mesenchymal interactions in tumor development for numerous

tissues has been described over time. For skin tumors, co-culture of keratinocytes with
fibroblasts isolated from a basal cell carcinoma tissue induces keratinocyte alterations such as
the expression of keratin 19, which is generally expressed in these tumors types (Lacina et al.,
2007). Additionally, the tumor cells can induce an increased expression of certain proteins by
normal cells, for example, the production of cathepsin K by fibroblasts in the presence of
squamous cell carcinoma. This cysteine protease has a strong effect on collagen degradation
and matrix remodeling during the tumor invasion process (Xie et al., 2011). As another
example, fibroblasts that undergo a process of UV-induced senescence can induce the
activation of extracellular signal-related kinase (ERK) and PI3K/AKT signaling and the
modulation of focal adhesion kinase (FAK) and other cytoskeletal proteins in keratinocytes,
thus leading to an increased proliferation of these cells, which may culminate in skin cancer
(Kang et al., 2008).
In squamous cell carcinoma, epithelial cells and fibroblasts are separated by the basal
membrane; this barrier is permeable to oxygen, nutritional substances, metabolites or
signaling molecules. Morphologically, the epithelium is thicker in the region where
concentration of fibroblasts is higher, and communication between the layers occurs through
the production of specific growth factors via Wnt or hedgehog proteins. The fibroblasts
obtained from distinct areas of the body control the specific ectopic expression of keratins in
keratinocytes (Plzák et al., 2010).

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Another important example of cell interaction and regulation is that of melanocyte

proliferation by the keratinocytes surrounding them, through a complex network of paracrine
growth factors, interactions between cells and cell adhesion to the extracellular matrix.
However, when the process of malignant transformation of melanocytes occurs (leading to
the emergence of tumor cells), one of the proteins, called E-cadherin, which plays a key role
in this regulation process, is downregulated and another protein, N-cadherin, is upregulated—
thus allowing greater communication between the malignant and tumor-associated
melanocytes. Melanoma is not only formed by tumor cells but also by fibroblasts, endothelial
cells and inflammatory cells, which contribute to the tumor structure, as well as to tumor
invasion, survival and growth. This niche allows the tumor cells to escape the cytotoxic
effects of radiation, chemotherapy and specific treatments. New therapies for melanoma are
using tumor-stromal cells as a target to fight against the tumor because fibroblasts are
genetically more stable and will not develop resistance to the medications (Smalley et al.,
2005; Paraiso & Smalley, 2013).

3.4. Other Skin Diseases

Regarding disease development, in addition to cancer, numerous examples can be found

in the literature, demonstrating the importance of the interaction between epithelial and
mesenchymal cells as a cause of some anomalies found in humans. The hyperproliferation of
keratinocytes in cholesteatomas is induced by the overexpression of a protein, namely
epiregulin, which is produced by subepithelial fibroblasts (Yoshikaw et al., 2013).
Another autoimmune disease characterized by epidermal thickening due to keratinocyte
hyperproliferation is psoriasis. Studies have revealed that keratinocytes are not solely
responsible for this disease, and understanding the role of dermal and inflammatory cells is
essential to mitigate the effects of the disease and to make more effective treatments
available. Sugai et al. (1998), performed a subcutaneous transplant of four mixtures in
immunodeficient mice, divided into the following groups: normal keratinocytes and normal
fibroblasts (NK/NF), psoriatic keratinocytes and normal fibroblasts (PK/NF), normal
keratinocytes and psoriatic fibroblasts (NK/PF) and psoriatic keratinocytes and fibroblasts
(PK/PF). The researchers observed the formation of cysts, and the histological analysis
revealed a variation in the cyst structure in the mixtures containing psoriatic keratinocytes.
Additionally, mixtures containing psoriatic fibroblasts may be partially responsible for the
epidermal thickening. In vitro studies with reconstituted skin demonstrated the role of
fibroblasts in a phenotype similar to that of in vivo psoriasis. Krueger and Jorgensen (1990)
demonstrated that fibroblasts derived from individuals with psoriasis can alter the phenotype
of keratinocytes from normal to psoriatic. Other variations of reconstituted skin models have
been subsequently published, containing cells derived from individuals with psoriasis or
using cytokines that induce a condition similar to psoriasis (Barker et al. 2004, Jean et al.,
2009; van den Bogaard et al., 2012).
The role of interleukins in psoriasis has been widely described, and these molecules have
been used as therapeutic targets for treating the most severe cases. Studies relate the role of
ILs not only in immune cells and keratinocytes but also in fibroblasts, as well as in the
increased and/or decreased production of key cytokines for disease development by
fibroblasts only or for their potentiation when in the presence of other cell types, such as IL-8
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74 Carla Abdo Brohem and Márcio Lorencini

(Glowacka et al., 2010), IL-17A (Kirkham et al., 2013), IL-23 (Schirmer et al., 2010) and IL-
36 (Towne & Sims, 2012), among others.
Another disease affected by this interaction is epidermolysis bullosa, which is
characterized by skin fragility with the presence of mechanically induced blisters and erosions
of the skin and mucous membranes. This disease is also characterized by mutations in genes
encoding components of the basement membrane. Among the functions of the basement
membrane is the control of epithelial-mesenchymal interactions in normal and pathological
physiological conditions (Bruckner-Tuderman & Has, 2013).

4. MULTIPLE CO-REGULATION SYSTEM: CAPILLARY INDUCTION,

GROWTH AND REGENERATION
Hair follicles can be considered mini-organs associated with the skin. All hair follicles
have the same basic structure: a permanent region, which consists of the infundibulum and
isthmus; and a variable region, containing differentiated epithelial cells, the hair matrix and
the dermal papilla (DP). The hair follicle is also a large reservoir of stem cells that are
maintained in specific niches, such as epithelial cells in the permanent bulge region,
melanocyte precursor cells derived from the neural crest in the sub-bulge region and
mesenchymal stem cells in the DP region (Asakawa et al., 2012). Epithelial progenitor cells
give rise to multiple intermediary cell lineages that make up the hair shaft and its guiding
channel (Sennetta & Rendl, 2012). Thus, among the cells that control this cyclical behavior of
hair are the cells present in the mesenchymal compartment of the follicle, that is, from the DP
(Figure 3).
These cells are essential for the determination of the development pathways of
ectodermal cell lineages during the hair follicle formation and cycle (Tobin et al., 2003;
Enshell-Seijffers et al., 2010; Asakawa et al., 2012; Rompolas et al., 2012). The exchange of
molecular signals between epithelial and mesenchymal cells begins during embryogenesis,
and many of the essential signaling programs necessary for hair morphogenesis are
evolutionarily conserved between distinct species (Sennetta & Rendl, 2012).
Hair follicles undergo cycles, with a growth phase (anagen), a regression phase (catagen),
a quiescent stage (telogen) and, finally, regeneration. During the anagen phase, the mature
hair follicle is composed primarily of keratinocytes arranged in concentric layers of
differentiated cells that make up the hair shaft (HS), inner root sheath (IRS) and outer root
sheath (ORS) (Enshell-Seijffers et al., 2010). With this continuous cycle, the hair follicle can
recapitulate its embryogenesis many times over its lifetime whenever it enters the anagen
phase (Paus et al., 1999; Tobin et al., 2003) (Figure 3).
Much attention has been given to DP cells due to their plasticity and signaling to the
entire follicle structure. The keratinocytes in direct contact with DP cells at the base of the
hair follicle undergo asymmetric divisions, yielding a compartment of stem cells with the
potential to generate descendant cells that migrate far from the DP and undergo some cell
divisions prior to differentiation into the HS and IRS (Enshell-Seijffers et al., 2010)

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Figure 3. Hair Follicle embryogenesis and cycling. (A) During embryogenesis, epithelial and
mesenchymal interactions are essential to hair follicle formation. Epithelial progenitor cells give rise to
multiple intermediary cell lineages that make up the hair shaft and its guiding. Among the cells that
control this cyclical behavior of hair are the cells present in Dermal Papilla (DP). (B) Hair follicles
undergo cycles, with a growth phase (anagen), a regression phase (catagen), a quiescent stage (telogen)
and, finally, regeneration (early anagen).

In an in vitro model study, which had the objective of investigating the role of DP cells in
epidermal morphogenesis, the authors found that isolated DP cells were capable of
differentiating into adipogenic and osteogenic cells, inducing tubule-like structures in a three-
dimensional model in vitro, and reorganizing the collagen matrix. The conditioned medium
collected from actively proliferating cells and from immortalized DP cells was capable of
inducing tubulogenesis after prolonged cultivation of keratinocytes (Chermnykh et al., 2010).
During hair growth, the dermal papilla increases in size due to the duplication of its cells,
much of which occurs before the intrafollicular proliferation of the papilla cells. This
indicates that some papilla cells originate and migrate to the proliferative pool of the
fibroblasts in the connective tissue sheath. The mesenchyme of the hair follicle displays a
large plasticity associated with the capillary cycle. Modulations of these cell locations may be
important for transformations of the hair follicle, such as in androgenic alopecia (Tobin et al.,
2003).
One gene that appears to be essential in this regulation is β-catenin, expressed by dermal
papilla cells. In an animal model, Enshell-Seijffers et al. (2010) demonstrated that the
inactivation of β-catenin in hair follicles results in a decreased proliferation of cells that
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76 Carla Abdo Brohem and Márcio Lorencini

generate the hair shaft and, subsequently, early induction of the destructive phase of the hair
cycle, i.e., the catagen phase. Two signaling pathways for regulating this process were
observed, namely, FGF and IGF, which perform the mediation of signaling from DP cells to
keratinocytes. Therefore, a reciprocal signaling loop uses signaling via Wnt / β-catenin in
both epithelial progenitor cells and in their mesenchymal niche to govern and coordinate
interactions that are essential to the function of these two compartments.
Mesenchymal signals, including FGF7 and FGF10, and BMP inhibitors are regulators for
the beginning of the hair regeneration cycle. Using a non-invasive imaging model, a study
demonstrated hair follicle regeneration in real time in live rats, monitoring the behavior of
epithelial stem cells and their descendants during physiological regeneration of hair, and
analyzing how the mesenchyme influences their behavior. The stem cells are at rest during
the initial stages of hair regeneration while their descendants are actively dividing. In addition
to cell divisions, the coordinated movements of the progenitor cells allow rapid growth of hair
follicle (Rompolas et al., 2012).
In a mouse model, it was possible to verify that PPARβ and Akt1 are highly expressed in
the follicular keratinocytes over the entire morphogenesis of the hair follicle. PPARβ
inhibition significantly delayed the hair follicle development due to an increase in early
apoptosis of follicular keratinocytes. PPARβ anti-apoptotic function is mediated by Akt1
signaling, and HGF secreted by the mesenchyme leads to the temporally coordinated
activation of PPARβ at the beginning of hair follicle maturation by increasing COX-2
expression. Therefore, the epithelial-mesenchymal interactions regulate PPARβ / δ expression
during hair follicle development and provide support to the study of molecular relationships
between the different capillary compartments (Di-Poï et al., 2005).
In 2012, Asakawa et al. demonstrated that bioengineered hair follicle germs, performed
from skin-derived embryonic epithelial and mesenchymal cells, can develop hair follicles that
are histologically equivalent to those normally found in mice when transplanted, going
through the entire hair follicle cycle, in addition to being associated with nerves and the
arrector pili muscle. Therefore, the study revealed that these bioengineered hair follicles may
assist in the surgical treatment of alopecia by restoring the physiological functions of the hair
follicle.

CONCLUSION
The interaction between dermis and epidermis is essential for the perfect functioning of
the largest organ of our body, the skin. The signal regulation network between these different
compartments is necessary from the moment of embryogenesis, allowing a coordinated
regulation of cell proliferation and differentiation, production of matrix elements and
signaling to other cells, among many other yet-to-be-discovered functions.
The volume of scientific production in this area has been growing annually, as there is
increasing evidence that the response of compartments or isolated cells may not correspond to
the in vivo conditions. Therefore, more complex models have emerged that facilitate studying
the fine modulation between the various skin components in an attempt to mimic the skin in
the most accurate manner possible.

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 Dermal and Epidermal Interaction 77

Alterations in these signaling networks can induce changes in the skin that vary from
aging to pathologies such as cancer. At the same time, targets that were not recognized before
as potential molecules for treating various diseases have been demonstrated to be effective,
such as in the case of fibroblast-targeted therapy for combating the most aggressive form of
skin cancer, melanoma.
Concluding, many additional studies are necessary to fully explore the complex
communication process between cell elements and their surrounding environment.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 4

MELANOGENESIS AND NATURAL

HYPOPIGMENTATION AGENTS

H. M. Chiang, H. W. Chen, Y. H. Huang, S. Y. Chan,

C. C. Chen, W. C. Wu and K. C. Wen
Department of Cosmeceutics, China Medical University, Taichung, Taiwan

ABSTRACT
Human melanin is synthesized in melanosomes located in melanocytes of the skin,
hair, eyes, ears, and leptomeninges. Melanin not only determines skin color, but also
protects the skin from UV damage by absorbing UV light. Congenital pigmentary
disorders that result in skin and hair depigmentation, such as Hermenksky Pudluk
Syndrome, Chediak Higashi Syndrome, and Griscelli Syndrome are due to various gene
mutations that cause defects in melanin synthesis. Excessive production of melanin,
which occurs in response to UV-induced DNA damage, inflammation, or other skin
injuries, however, can result in skin hyperpigmentation including freckles, melasma, solar
lentigo, age spots, and post-inflammatory hyperpigmentation. In this article we review
the synthesis of melanin, the signaling pathways related to the regulation of
melanogenesis, the factors influencing melanogenesis and various pigmentation
disorders, as well as the effectiveness of various natural products at reducing
hyperpigmentation.

ABBREVIATIONS
ACTH, adrenocorticotropin melanocyte stimulating hormone;
AHA, α-hydroxy acids;
ASP, agouti signaling protein;
ATP, adenosine 5'-triphosphate;


Correspondence to: Professor Kuo-Ching Wen. Department of Cosmeceutics. China Medical University.
Taichung, Taiwan 404. E-mail: [email protected]. Telephone: 886-4-22053366 ext. 5302. Fax:
886-4-22078083
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84 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

BBI, Bowman Birk inhibitor;

bFGF, basic fibroblast growth factor;
BHAs, β-hydroxy acids;
cAMP, cyclic AMP;
CRE, cAMP response element;
CREB, cAMP-response element binding protein;
CRH, corticotropin-releasing hormone;
DCT, TRP2,DOPAchrome tautomerase;
DHI, 5,6-dihydroxyindole;
DHICA, 5,6-dihydroxyindole-2-carboxylic acid;
DKK 1, dickkopf-related protein 1;
DPPH, 1,1-diphenyl-2-picryl-hydrazyl;
ECE, ET converting enzyme;
ERK2, extracellular signal-regulated kinase 2;
ET-1, endothelin-1;
ETBR, endothelin B receptor;
FOXD3, forkhead-box transcription factor D3;
GM-CSF, granulocyte-macrophage colony-stimulating factor;
GSK3β, glycogen synthase kinase-3β;
HGF, hepatocyte growth factor;
HPS, Hermansky-Pudlak syndrome;
HQ, hydroquinone;
IL, interleukin;
ITF2, immunoglobulin transcription factor-2;
L-DOPA, 3,4-dihydroxyphenylalanine;
LEF-1, lymphoid-enhancing factor-1;
LIF, leukemia inhibitory factor;
LT, leukotrienes;
MAP kinase, mitogen-activated protein kinase;
MC1R, melanocortin 1 receptor;
MITF, microphthalmia-associated transcription factor;
MOPB, methylophiopogonanone B;
NGF, nerve growth factor;
NHKC, normal human keratinocytes;
NHMC, normal human melanocytes;
NO, nitric oxide;
NRG, neuregulin;
PAR-2, protease activated receptor 2;
PAX3, paired box 3;
PGs, Prostaglandins;
PIAS3, protein inhibitor of activated STAT3;
PKA, protein kinase A;
PKC, protein kinase C;
PLA2, phospholipase A2;
POMC, pro-opiomelanocortin;
ROS, reactive oxygen species;

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 Melanogenesis and Natural Hypopigmentation Agents 85

RSK, ribosomal S6 kinase;

SA, salicylic acid;
SCCE, stratum corneum chemotrypic enzyme;
SCF, Stem cell factor;
SEM, skin equivalent model;
SOX, Sry-related HMG box;
STAT3, signal transducer and activator of transcription 3;
STI, soybean trypsin inhibitor;
TGF-β1, transforming growth factor-β1;
TNF-α, tumor necrosis factor α;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
TRP1, tyrosinase-related protein 1;
UV, ultraviolet;
α-MSH, α-melanocyte-stimulating hormone;

INTRODUCTION
Variations in human skin, hair, and eye color are due to the type, amount, stage, and
distribution of melanin [1]. Melanin, one of the most widely distributed pigments, is a
heterogeneous polyphenol-like biopolymer with a complex structure and color varying from
yellow to black [2]. More than 150 genes regulate and contribute to skin pigmentation [3, 4].
In addition to contributing to the color of skin and hair, melanin also protects skin from
physical (such as ultraviolet (UV) irradiation damage), chemical (such as environmental
pollutants, heavy metals, and oxidative stress), and biochemical (such as bacteria) challenges
[5, 6]. Overexposure to solar UV irradiation can result in photoaging, mutagenesis, and
photocarcinogenesis in human skin [7, 8].
The incidence of skin cancer is increasing at a rate of 3% to 4% per year, and the
mortality rate associated with skin cancer (melanoma) is increasing more rapidly than the
mortality rate associated with any other cancer [9]. Melanocytes transfer melanosomes
through their dendrites to surrounding kerotinocytes where they form melanin caps. This
accumulation of melanin plays a protective role against UV irradiation by absorbing and
transforming UV energy into harmless heat. Melanin can also scavenge toxic xenobiotics and
reactive oxygen species (ROS) as well as bind to drugs, thereby protecting human skin
against chemical and biochemical challenges [5, 6, 10-12]. However, excessive production of
melanin and its accumulation in the skin can cause pigmentation disorders, including
melasma, solar lentigo, and post-inflammatory hyperpigmentation [13]. Overproduction of
melanin is not only a dermatological issue but also poses esthetic problems, especially among
patients in Asian cultures.
In this article we review the synthesis of melanin, the signaling pathways related to the
regulation of melanogenesis, the factors influencing melanogenesis and various pigmentation
disorders, as well as the effectiveness of various natural products at reducing
hyperpigmentation.
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86 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

Melanosynthesis

Variations in dermal pigmentation depend on the number, size, composition, and

distribution of melanocytes as well as the activity of melanogenic enzymes.
Melanin synthesis by melanocytes within membrane-bound organelles (melanosomes)
and their transfer to keratinocytes within the epidermal melanin unit determines cutaneous
pigmentation.
Melanin synthesis is characterized by an increased number of melanocytes in the basal
layer of the epidermis, the size, maturation, and number of melanosomes, the production of
melanin, the dendricity of melanocytes, the transfer of melanosomes from melanocytes to
keratinocytes, the proliferation of keratinocytes, and the thickening of the epidermis and
stratum corneum.

Melanocytes and Melanosomes

Melanin is synthesized in melanocytes, which are localized at the basal layer of the
epidermis.
Each melanocyte is functionally related to underlying fibroblasts in the dermis and to
keratinocytes in the epidermis. Each melanocyte transfers pigment-containing melanosomes
via dendritic melanocytes to approximately 36 basal and suprabasal keratinocytes – the so-
called epidermal melanin unit [3, 14, 15] (Figure 1).
This inter-cell cross-talk regulates the function and phenotype of human skin [16].
Protease-activated receptor 2 (PAR-2) plays an important role in melanosomal transfer [17,
18]. PAR-2, a G protein-coupled receptor, mediates the phagocytosis of melanosomes in a
Rho-dependent manner [19]. The amount and type of melanin produced and transferred to the
keratinocytes with subsequent incorporation, aggregation, and degradation influences skin
complexion coloration [20]. Melanoblasts, melanocyte precursor cells, are derived from the
neural crest and migrate to target sites such as dermis and eyes [21]. Melanoblasts
differentiate into melanocytes when they reach their destination and start to produce
melanosomes, the organized elliptic membrane-bound organelles where melanin is
synthesized. Melanin synthesis starts with the exportation of structural proteins from the
endoplasmic reticulum to the cytosol, where they fuse with melanosome-specific regulatory
glycoproteins that have been released in coated vesicles from the Golgi apparatus. Melanin
synthesis ensues subsequent to the sorting and trafficking of these proteins to melanosomes
[22, 23]. Melanosomes are divided into four maturation stages according to their structure and
to the type and amount of melanin produced [24, 25]. ‘Early’ melanosomes (stages I and II)
present with little or no pigment, while ‘late’ melanosomes (stages III and IV) present with
some to complete pigment. Stage I melanosomes are spherical vacuoles lacking tyrosinase
activity and internal structural components. Stage II melanosomes are elongated, fibrillar
organelles containing tyrosinase and little melanin [26, 27]. After stage II, melanin synthesis
starts. Stage III melanosomes have uniformly deposited pigment on the internal fibrils.
Mature melanosomes (stage IV) are either elliptical or ellipsoidal in shape, are electron-
opaque due to complete melanization, and have minimal tyrosinase activity. Highly
pigmented melanocytes are rich in Stage IV melanosomes which are transferred by
melanocyte dentrites to keratinocytes [16].

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 Melanogenesis and Natural Hypopigmentation Agents 87

Figure 1. Melanosome transfer.

The trafficking of sorting vesicles to their target organelles is controlled by two classes of
microtubule-associated motor proteins – kinesins and cytoplasmic dyneins [28]. Kinesins
power plus-end-directed microtubule-based motility, while cytoplasmic dyneins drive minus-
end-directed motility [29, 30]. Dyneins and kinesins also play roles in retrograde and in
anterograde transport of melanosomes [31-34], whereas dyneins and spectrin dominate the
movement of early melanosomes [35].
The methods of melanosome transfer from melanocytes to keratinocytes include
cytophagocytosis of melanocyte dendrite tips [36, 37] and exocytosis of melanosomes into
the extracellular space and their subsequent uptake by phagocytosis into keratinocytes [38,
39], either by filopodia-mediated melanosome transfer [40-42] or the filopodial-phagocytosis
model [43]. Rab, melanophilin, and myosin Va have been shown to be involved in the
movement of melanosomes [37, 44].

Melanin Biosynthesis

Melanins are polymorphous, multifunctional biopolymers. The major types of melanins

include eumelanin, pheomelanin, a combination of eumelanin and pheomelanin (mixed
melanin), and neuromelanin (Figure 2). Eumelanin is a blackish-brown heterogeneors
polymer consisting of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid
(DHICA). Pheomelanin is yellowish-red in color and consists of sulfur-containing
benzothiazine derivatives [2, 45].
Neuromelanin is produced in dopaminergic neurons of the human substantia nigra, the
dorsal motor nucleus of the vagus nerve, and the median raphe nucleus of the pons.
Neuromelanin has the capacity to chelate redox-active metals such as Cu, Mn, and Cr as well
as toxic metals such as Cd, Hg, and Pb to avoid neuron degeneration [46]. If the level of
neuromelanin decreases, dopamine synthesis may be diminished, resulting in diseases
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88 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

associated with neuronal degradation. Massive loss of dopamine-producing pigmented

neurons in the substantia nigra has been found in patients with Parkinson’s disease [47].

Figure 2. Pathway of melanin biosynthesis.

The biosynthetic pathway governing melanin formation is well established [48-52]

(Figure 2). Synthesis of melanin starts with the conversion of the amino acid L-tyrosine to
dopaquinone by tyrosinase, a copper-containing glycosylated type I membrane-bound
glycoprotein that catalyzes the rate-limiting step of melanin biosynthesis [53, 54]. Tyrosinase
is synthesized by melanosomal ribosomes on the rough endoplasmic reticulum [55]. The
enzyme is glycosylated en route to and within the Golgi apparatus, and subsequently
delivered to melanosomes via coated vesicles [55, 56]. Tyrosinase is the most common target
for therapeutic agents intended to alleviate hyperpigmentation [57-59]. Tyrosinase catalyzes
two distinct oxidation reactions. First, tyrosinase catalyzes the oxidation of monophenol (L-
tyrosine) to o-diphenol (3,4-dihydroxyphenylalanine, L-DOPA (monophenolase activity)).
Second, L-DOPA is oxidized to o-quinone (dopaquinone) (diphenolase activity). Tyrosinase
gene transcription has been shown to correlate with the differentiation of lysosomes and/or
peroxisomes into melanosomes [60, 61]. Tyrosinase-related protein 1 (TPR-1) and
DOPAchrome tautomerase (DCT, also known as TRP-2) subsequently metabolize
dopaquinone into eumelanin through a process referred to as eumelanogenesis. Dopaquinone
is transferred to DHI via multiple processes including decarbxylation, oxidation, and
polymerization and DOPAchrome is converted to DHICA. Pheomelanogenesis refers to the
process through which dopaquinones conjugate with thiol-containing cysteines or
glutathiones to form pheomelanin. As mentioned above, dopaquinone plays pivotal roles both
in eumelanogenesis and pheomelanogenesis [16]. Eumelanogenesis involves the activation of
tyrosinase, TRP-1, and TRP-2 whereas the synthesis of pheomelanin only requires the
activation of tyrosinase [16, 62]. Following the synthesis of those pigments, melanin-
containing melanosomes are transferred to neighboring keratinocytes. However, without
successful transfer of melanosomes to keratinocytes, the skin can appear essentially
unpigmented [63].

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 Melanogenesis and Natural Hypopigmentation Agents 89

Factors Regulating Melanin Biosynthesis

UV radiation from the sun stimulates melanin synthesis in skin. After UV exposure,
melanocytes increase their expression of pro-opiomelanocortin (POMC, the precursor of α-
MSH) and its receptor melanocortin 1 receptor (MC1R), tyrosinase, TRP-1, protein kinase C
(PKC), and other signaling factors [64-66] (Figure 3). Upon exposure to UV irradiation,
fibroblasts release the above-mentioned cytokines, growth factors, and inflammatory factors,
which then stimulate melanin production and/or stimulate melanin transfer. UV also
stimulates the production of endothelin-1 (ET-1) and POMC in keratinocytes, factors that
then act in a paracrine manner to stimulate melanocyte function [67, 68]. Other keratinocyte-
derived factors that regulate the proliferation and/or differentiation of melanocytes include α-
MSH, adrenocorticotropin melanocyte stimulating hormone (ACTH), basic fibroblast growth
factor (bFGF), nerve growth factor (NGF), endothelins, granulocyte-macrophage colony-
stimulating factor (GM-CSF), steel factor, leukemia inhibitory factor (LIF), and hepatocyte
growth factor (HGF) [69]. Melanocytes have been shown to increase the production of
intracellular nitric oxide (NO), which in turn triggers signal transduction cascades to initiate
melanogenesis [70, 71] through the enzyme tyrosinase. In addition, human melanocyte
proliferation requires cross-talk between several signaling pathways including the
cAMP/PKA, PKC, and tyrosine kinase pathways; therefore, the mechanisms by which
various factors increase skin pigmentation are closely inter-related [52, 72-75].

Figure 3. Factors regulating melanin biosynthesis.

UV radiation has been shown to influence melanogenesis through a paracrine regulation

process involving keratinocytes [52, 76] (Figure 3). Both autocrine and paracrine cytokine
networks are involved in UV-induced upregulation of melanogenesis [77]. α-MSH is a major
mediator of the response of melanocytes to UV [78]. The POMC gene is activated in the
pituitary gland but POMC-derived peptides are also generated in keratinocytes and
melanocytes [79, 80]. The POMC gene encodes a large precursor protein, which is then
enzymatically cleaved to form several different peptides including α-MSH, ACTH,
melanocortin, and β-endorphin [80]. The binding of α-MSH and ACTH to MC1R on the
melanocyte membrane [81] activates intracellular adenylate cyclase through G proteins,
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90 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

which then catalyze the conversion of adenosine triphosphate to cyclic AMP (cAMP) [82].
cAMP exerts its function through protein kinase A (PKA) [83]. The intracellular elevation of
cAMP increases the protein expression of microphthalmia-associated transcription factor
(MITF), tyrosinase, and TRP-2, but not tyrosinase or TRP-2 mRNAs[84]. PKA promotes the
activation of the cAMP-response element binding protein (CREB) that binds to the cAMP
response element (CRE) that is present in the M promoter of the MITF gene [85, 86]. MITF is
a transcription factor with a basic helix-loop-helix-leucine zipper motif. MITF regulates
melanocyte cellular differentiation and the transcription of melanogenic enzymes such as
tyrosinase, TRP-1, and TRP-2 and the transcription of melanosome structural proteins
including MART-1 and Pmel17 [87-90]. Pmel17 is a structural matrix protein and an amyloid
protein required for the generation of the internal fibril [91]. The promoter sequences of
tyrosinase, TRP-1, and TRP-2 share a highly conserved motif known as the M-box, which
contributes to their melanocyte-specific expression [92, 93] (Figure 4). TRP-1 promoter
activity is up regulated by paired box 3 (PAX3) [94]. The M-box (AGTCATGTGCT) is an
extended E-box (ACATGTGA) and is necessary for promoter up-regulation by MITF [16,
95]. The E-box is more important than the M-box in promoting the transcription factor MITF
[96] (Figure 4). MITF is exclusively expressed in melanocytes. It binds to the M-box
promoter elements of tyrosinase and modulates TRP-1 and TRP-2, resulting in
hyperpigmentation[97-99]. In addition to the process of melanization, MITF also regulates
melanocyte proliferation, differentiation, development, apoptosis, and survival [100-102].

Figure 4. Tyrosinase gene expression.

A transient increase in MITF leads to the up-regulation of tyrosinase, TRP-1, and TRP-2
[103] as well as to increased dendricity [88]. Many transcription factors including Sry-related
HMG box (SOX) 9 and 10, PAX3, signal transducer and activator of transcription 3
(STAT3), protein inhibitor of activated STAT3 (PIAS3), lymphoid-enhancing factor-1 (LEF-
1), immunoglobulin transcription factor-2 (ITF2), and forkhead-box transcription factor D3
(FOXD3) are able to modulate the expression and/or transcriptional activity of MITF in vivo
[104] (Figure 4). The transcription factor SOX9 may play an important role in UVB-induced
melanocyte differentiation and pigmentation through MITF regulation [105]. SOX10
regulates the expression of MITF and TRP-2. SOX10 has been demonstrated to activate the

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 Melanogenesis and Natural Hypopigmentation Agents 91

TRP-2 promoter-reporter construct and to work in synergy with MITF [106, 107]. PAX3
binds to the MITF gene promoter to regulate MITF expression [108]. In addition, PAX3 has
been shown to act in synergy with SOX10 to up regulate the expression of MITF [109]. The
transcriptional activity of MITF is regulated through the interaction between STAT3 and
PIAS3. LEF-1, a transcription factor involved in the Wnt signal transduction pathway,
initiates and facilitates MITF expression, while ITF2 and FOXD3 down regulate MITF
expression [104, 110-112].
The transcriptional activity of MITF is regulated by phosphorylation of tyrosinase
residues on extracellular signal-regulated kinase 2 (ERK2) following signals from c-kit
(tyrosinase-type receptor) and then by phosphorylation of the 73rd serine residue in the N
terminal domain of MITF [113] (Figure 4). The tyrosinase gene and TRP-1 promoter zones
share a CATGTG motif. When MITF is activated, binding to the formed dimmers serves to
regulate the expression of the tyrosinase gene TRP-1. MITF is also regulated at the
transcriptional level by interleukin-6 (IL-6) and the Wnt signaling pathway and it is post-
transcriptionally regulated by phosphorylation via ribosomal S6 kinase (RSK), glycogen
synthase kinase-3β (GSK3β), p38 stress signaling, and the mitogen-activated protein kinase
(MAP kinase) pathways [89, 90, 98, 114-116] (Figure3). α-MSH also stimulates p38 MAP
kinase, which in turn phosphorylates upstream transcription factors that bind to the tyrosine
promoter [52].
Human placental lipid upregulates p38 activation and subsequent tyrosinase expression,
thereby promoting melanogenesis [117]. Down-regulation of p38 expression leads to an
increase in expression of biomarkers associated with differentiation such as tyrosinase and
tyrosinase-related proteins.
The mechanism involved in the p38-mediated regulation of melanogenesis is the
ubiquitin–proteasome pathway, through which melanogenic enzymes are degraded [118]. In
addition, inhibition of ERK and AKT signaling via MITF up-regulation plays a key role in
inducing hyperpigmentation [119]. ERK activation results in phosphorylation of MITF and its
subsequent ubiquitination and degradation [120]. Sphingosine-1-phosphate, C2-ceramide, and
sphingosylphosphorylcholine activate ERK and may play important roles in the inhibition of
melanogenesis [120-122].
Transforming growth factor-β1 (TGF-β1) inhibits melanogenesis by mediating the down-
regulation of MITF promoter activity as well as by reducing the production of tyrosinase,
TRP-1, TRP-2, and MITF protein levels. In addition, TGF-β1 inhibits the expression of PAX
3, which in turn inhibits melanogenesis [123]. It has been reported that TGF-β1 influences the
ERK pathway and down regulates MITF and the production of melanogenic enzymes [115,
124, 125].
The agouti signaling protein (ASP) can down regulate MITF gene expression and
compete with α-MSH in binding to MC1R, causing inhibition of α-MSH signaling on the
MC1R receptor. ASP modulates the frequency, rate, and extent of eumelanin and
pheomelanin generation [4].
Studies have demonstrated that high levels of ASP are associated with yellow-pigmented
bands in mouse hair because ASP inhibits α-MSH binding to MC1R [16]. Thus, MC1R and
its ligands, α-MSH and ASIP, regulate the switch between eumelanin and pheomelanin
synthesis in melanocytes [79, 126].
UVB exposure activates the transcription factor p53, which in turn induces the expression
of POMC. Expression of that α-MSH precursor leads to the secretion of α-MSH and the up-
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92 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

regulation of melanogenesis via MC1R in keratinocytes [127, 128]. In addition, p53 directly
stimulates the expression of the genes encoding tyrosinase and TRP1 in melanocytes [129].
UVB also induces the expression of corticotropin-releasing hormone (CRH) in melanocytes,
which is mediated by the CREB–PKA signaling pathway with consequent stimulation of
POMC expression through the CRH-R1 receptor. The POMC gene has been shown to be p53-
responsive following UV irradiation [130].
Kichina et al. demonstrated that stable transfection of wild-type p53 into pigmented
melanoma cells leads to overexpression of wild-type p53 and a decrease in tyrosinase mRNA
levels and tyrosinase activity [131].
Khlgatian et al. have shown that UV irradiation results in increased p53-dependent
tyrosinase mRNA levels in melanoma cells and that p53 is required for the thymidine
dinucleotide-induced increase in tyrosinase function in mouse epidermis [132]. They also
reported that tanning is part of a p53-mediated adaptive response of mammalian skin to UV-
induced DNA damage [132].
Other hormones, such as steroids and sex hormones, can influence pigmentation [79, 127,
133], and it has been reported that cholesterol is capable of increasing the expression of MITF
and its target genes in melanocytes through the up-regulation of the CREB protein [134]. Two
fibroblast-derived paracrine factors, namely dickkopf-related protein 1 (DKK1) and
neuregulin-1 (NRG1), regulate melanogenesis. DKK1 is a factor secreted by fibroblasts.
DKK1 has been shown to suppress growth of melanocytes, strongly inhibit melanin
production, and inhibit binding of Wnt proteins to their receptors, which results in down-
regulation of melanogenesis [102, 135].
In addition, DKK1 suppresses melanocyte growth and function by inhibiting the Wnt/b-
catenin signaling pathway [136, 137]. DKK1 has also been shown to regulate the expression
of PAR-2 [137].

Pigmentary Disorders

Hyperpigmentation disorders are characterized by the overproduction of melanin and

include melasma, postinflammatory hyperpigmentation, freckles, moles, chloasma, age spots,
and lentigines [138-140]. Hypopigmentation disorders are characterized by the
underproduction of melanin and include disorders such as oculocutaneous albinism,
Hermansky–Pudlak syndrome, Griscelli syndrome, Chediak-Higashi syndrome, and
Waardenburg syndrome.
Oculocutaneous albinism is an inherited autosomal recessive disorder characterized by
deficiency or complete absence of melanin [61]. At least 10 types of oculocutaneous albinism
exist. Patients with the disorder present with hypopigmention of the skin, hair, and eyes as
well as reduced visual acuity with nystagmus and photophobia. Furthermore, in these patients
there is often complete lack of tyrosinase activity [141, 142].
Oculocutaneous albinism type 2, which is characterized by a congenital reduction or
absence of melanin pigment in the skin, hair, and eyes, is the most common type and the
incidence is highest in black Africans [16].
Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous group of related
autosomal recessive conditions. It is divided into eight types according to the HPS genes that
carry mutations [143].

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 Melanogenesis and Natural Hypopigmentation Agents 93

Defects in proteins encoded by these genes can affect the biogenesis or function of
intracellular organelles such as melanocytes and retinal pigment epithelial cells. HPS is also
associated with lung disease, inflammatory bowel disease, renal disease, and bleeding
problems due to platelet dysfunction [16].
Griscelli syndrome is an autosomal recessive disorder characterized by pigmentary
dilution of the skin and the accumulation of large and abnormal end-stage melanosomes in the
center of melanocytes [144].
It may be caused by defects in the formation of the Rab27a–Mlph–MyoVa protein
complex in melanocytes, an important protein that connects melanosomes to the actin
network [144]. Chediak-Higashi syndrome is an autosomal recessive disorder similar to
oculocutaneous albinism [145].
Patients with this syndrome are susceptible to infection because they lack natural killer
cell function and are at risk for developing lymphofollicular malignancy and peripheral
neuropathies [146, 147].
Mutations in the human homolog of the MITF gene are associated with auditory and
pigmentary abnormalities in patients with Waardenburg syndrome type IIA [109, 148, 149].
Mutations in the PAX3 gene are associated with Waardenburg syndrome type I, while SOX
10 mutations are characteristic of Waardenburg syndrome type IV [4, 51].

Mechanisms of Depigmentation

Studies on the processes of cellular melanogenesis and the response of pigment-

producing cells to UV radiation have been instrumental in promoting the development of
depigmenting agents [57, 58, 114, 150, 151].
The mechanisms of action by which biological and chemical agents cause
hypopigmentation include (i) tyrosinase inhibition, maturation, and enhancement of its
degradation; (ii) inhibition of tyrosinase mRNA transcription; (iii) inhibition of MAP kinases,
TRP-1, TRP-2, and MITF; (iv) downregulation of MC1R activity; (v) interference with
melanosome maturation and transfer; and (vi) melanocyte loss and desquamation [57, 114,
150-153].
Tyrosinase inhibition is the most common approach to achieve skin hypopigmentation as
this enzyme catalyses the rate-limiting step of pigmentation [114, 152]. Tyrosinase inhibitors
can be classified as competitive, uncompetitive, mixed type, and non-competitive inhibitors
[57, 154].
Tyrosinase can be inhibited at the transcriptional and post-transcriptional levels by
inhibiting tyrosinase mRNA transcription and disrupting tyrosinase glycosylation by using
competitive or non-competitive inhibitors to attenuate the catalytic activity of tyrosinase, by
accelerating tyrosinase degradation, and by modulating tyrosinase stability [155, 156].

Natural Hypopigmentation Agents

Hydroquinone, ascorbic acid, and retinoic acid have been shown to be effective skin-
whitening agents; however, they are associated with harmful side effects, thereby limiting
their clinical use [155]. Compounds derived from natural products, on the other hand, have
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94 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

been shown to be as effective as chemical-based products at reducing hyperpigmentation.

Some natural skin-depigmenting products have been reported to directly effect
melanogenesis; enhance tyrosinase degradation; interfere with melanosome maturation and its
transfer; inhibit inflammation-induced melanogenesis; and accelerate skin desquamation [51,
154, 157-159].

EFFECT ON MELANOGENESIS
As shown in Table 1, whitening agents derived from natural products can be divided into
three groups: phenols, polyphenols, and others [58]. Table 1 also presents the plants from
which the compounds are derived, the mode of action including tyrosinase inhibition, other
enzyme inhibition (OEI (TRP1 and TRP2), melanin inhibition (MI), and other mechanisms of
action, as well as the IC50 values of said compounds. The mechanisms of tyrosinase inhibition
can be evaluated by measuring enzyme inhibition kinetics using Lineweaver-Burk plots with
varying concentrations of L-DOPA as the substrate. Moraceae, Anacardiaceae,
Chloranthaceae, Ericaceae, Lamiaceae, Sapindaceae, and Fabaceae are rich in phenols and
polyphenols that have anti-melanogenesis activity. Most studies used B16 melanoma cells as
a model to investigate the mechanism of action governing melanin inhibition. Some of the
studies used mouse melan-a or mel-ab melanocyte cultures or normal human melanocytes
(NHMC) as experimental models.
Data from studies that involved the use of NHMC cells are probably more reliable
because those cells mimic the response to stimuli seen in vivo. Human melanocyte
proliferation and enhancement of melanin synthesis require cross-talk between several
cytokines and hormones that are released from keratinocytes. Co-cultures of melanocytes and
keratinocytes from mouse [160, 161] or human skin [162] also more closely mimic the
response seen in vivo.
The brownish guinea pig (GP) model is commonly used to study the effects of skin-
whitening agents on reducing hyperpigmentation induced by UV or exposure to exogenous α-
MSH (Table 1). In human studies, the activities of skin-whitening agents are normally
investigated by evaluating skin color changes using a Chromameter or a Mexameter or by
histochemical investigations of DOPA positive cells [163, 164]. Beginning in September
2009, the Commission of the European Communities established a prohibition to test finished
cosmetic products and cosmetic ingredients on animals (European Commission - Consumer
Affairs). Commercially available skin equivalent models (SEMs), a keratinocyte and
melanocyte co-culture system [165], and MatTek's MelanoDerm™ (MatTek Corporation), a
human three dimensional skin-like tissue structure, are useful in vitro models for evaluating
the ability of cosmetic and pharmaceutical agents to modulate skin pigmentation. A common
vertebrate model organism that is used for whitening studies is the zebrafish, which has been
[165, 166] proved to be a useful model for demonstrating the in vivo toxicity of whitening
agents.

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 Table 1. Whitening ingredients from natural sources effect on melanogenesis

Compounds (phenol, Source Mode of action Refs.

polyphenols, others) TI OEI (TRP- other MI
1, TRP-2)
Phenols
Anacardic acid, 6- Anacardium Yes (c) [249]
[8(Z),11(Z),14- occidentale
pentadecatrienyl]-salicylic cashew fruit
acid, 5-[8(Z),11(Z),14- (Anacardiaceae)
pentadecatrienyl] resorcinol
10’(Z)- Rhus succedanea Yes Yes [250]
heptadecenylhydroquinone (Anacardiaceae) IC50= 37 μM IC50= 40 μM
[HQ17(1)]
2-hydroxy-4- Rhus vulgaris Yes (m) [251]
methoxybenzaldehyde Meikle IC50=0.03 mM
Sclerocarya caffra
Sond
(Anacardiaceae)
Mondia whitei (Hook)
Skeels
(Asclepiadaceae)
3,4- Ilex pubescens Yes Reduction of Yes [252]
Dihydroxyacetophenone (Aquifoliaceae) IC50= 10 μM TYR and
MITF protein
level
p-Coumaric acid Panax ginseng Yes (m) [253]
(Araliaceae) IC50= 3.65 mM
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI (TRP-1, other MI
TRP-2)
p-Coumaric acid Sasa quelpaertensis Yes (c) Reduction of Yes [254]
(Gramineae) TYR protein
level
2’,4’,6’- Greyia flanaganii Yes [255]
trihydroxydihydrochalcone (Greyiaceae) IC50= 69.15 μM

protocatechuic Salvia miltiorrhiza Yes (c) [256]

aldehyde (Lamiaceae) IC50=19.92 µM
protocatechualdehyde Phellinus linteus Yes (c) [257]
(Hymenochaetaceae)
protocatechuic acid methyl Black Rice Bran Yes [258]
ester IC50= 0.28 µM
phloroglucinol (1), Ecklonia stolonifera. Yes [ (1) and (2), -- Yes [259]
eckstolonol (2), brown alga extracts (c) ]
eckol (3), (Laminariaceae) (1) IC50= 92.8 μg/mL
hlorofucofuroeckol A (4), (2) IC50= 126 μg/mL
ieckol (5) Yes [ (3) , (4) and (5)
-- (n) ]
(3) IC50=33.2 μg/mL
(4) IC50=177 μg/mL
(5) IC50=2.16 μg/mL
Phloroglucinol, Ecklonia cava Yes Reduction of Yes [260]
dieckol, eckol, (Lessoniaceae) Dieckol (88.9% of UV-B
TYR at 50 μM) induced
cell damages

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI (TRP- other MI
1, TRP-2)
7-phloroeckol Ecklonia cava Yes (nc) Yes [261]
(Lessoniaceae) IC50 =0.85 μM
cinnamaldehyde(1), Cinnamomum cassia Yes [262]
2-methoxy cinnamaldehyde (Lauraceae) (1) IC50=0.52 ±0.03
(2) mM,
cinnamic acid (3) (2) IC50=0.42 ±0.02
O-coumaric acid (4) , mM,
icariside DC (5), (3) IC50=0.41 ±0.01
dihydromelilotoside (6), mM,
dihydromelilotoside (7) (4) IC50=0.67 ±0.03
mM,
(5) IC50=0.71 ±0.03
mM,
(6) IC50=0.57 ±0.01
mM,
(7) IC50=0.63 ±0.02
mM
Mulberroside F (moracin Morus alba leaves Yes Superoxide Yes [263]
M-6, 39-di-O-β-D- (Moraceae) TYR (mushroom) Scavenging (30.6% of MI
glucopyranoside) IC50=0.29 µg/mL ; Activity at 1 mg/mL)
TYR (mammalian)
IC50=68.3 µg/mL
4-Substituted resorcinols Artocarpus incises Yes (c) [264]
(Moraceae)
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Macelignan Myristica fragrans Yes TRP-1 Reduction Yes [265]
(Myristicaceae) IC50=30 µM TRP-2 of TYR, IC50=13 µM
TRP-1 and
TRP-2
protein
level
Americanin A (1), 3,3’- Morinda citrifolia Yes SOD-like [266]
bisdemethylpinoresinol (2) seeds (1) IC50 =2.7 mM activity
(Rubiaceae) (2) IC50 =0.3 mM
3-caffeoylquinic acid green coffee beans Yes [267]
4-caffeolyquinic acid 5- (Rubiaceae)
caffeoylquinic acid 5-
feruloylquinic acid
3,4-dicaffeoylquinic acid
3,5-dicaffeoylquinic acid
4,5-dicaffoylquinic acid
3,4-dihydroxycinnamic acid Pulsatilla cernua Yes (nc) [268]
(1), 4-Hydroxy-3- (Ranunculaceae) (1) IC50=0.97 mM
methoxycinnamic acid (2) (2) IC50=0.33 mM
4-acetonyl-3,5-dimethoxy- Synsepalum dulcificum Yes [269]
p-quinol (1), cis-p- (Sapotaceae) (1) IC50=208.1 μM,
coumaric acid (2), trans-p- (2) IC50=197.9 μM,
coumaric acid (3), p- (3) IC50=168.7 μM,
hydroxybenzoic acid (4), (4) IC50=358.6 μM,
Vanillic acid (5) (5) IC50=174.4 μM

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Cardamonin Alpinia katsumadai Yes MITF Yes [270]
Hayata
(Zingiberaceae)
Isopanduratin A (1) , 4- Kaempferia pandurata. Yes Reduction Yes [271]
hydroxypanduratin A (2) ( Zingiberaceae) (1) IC50=10.5 µM of TYR (1) IC50=10
(2) IC50>30 µM protein .64 µM
level (2) IC50=23
.25 µM
Curcumin, Chouji Syzygium Yes (c) [272]
yakuchinone A, aromaticum (Myrtaceae) (Curcumin and
yakuchinone B, eugenol and Yakuchi Alpinia yakuchinone B)
ferulic acid oxyphylla
(Zingiberaceae)
polyphenols
1,2,3,4,6-penta-O-galloyl- Galla rhois Yes (nc) [273]
â-D-glucose (Anacardiaceae)
2,3,4,6-tetra-O-galloyl-D- Rhus chinensis Yes (nc) Yes [274]
glucopyranose (1), 1,2,3,6- (Anacardiaceae) (1) IC50 = 54 μM,
tetra-O-galloyl-beta-D- (2) IC50 = 30 μM,
glucopyranose (2), (3) IC50 = 15 μM
1,2,3,4,6-penta-O-galloyl-
beta-D-glucopyranose (3)
Tannic acid Rhus javanica Yes (c) [275]
leaves IC50= 22 µM
(Anacardiaceae)
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Icariside I (1), Epimedium Yes [276]
Icariside II (2), grandiflorum (1) IC50= 49.04
Icaritin (3) (Berberidaceae) µM,
(2) IC50= 10.53
µM,
(3) IC50= 11.13
µM
Xanthohumol Humulus lupulus L. Yes Reducti Reduction Yes [277]
(Cannabaceae) on of of cAMP,
TRP-1 MITF
and protein and
TRP-2 its mRNA
mRNA expression
level and TYR
protein
expression
GB-2 (biflavanones) Garcinia kola Yes [278]
Seed IC50 = 582 µM
(Clusiaceae)
GS contained 2 biflavonoids; Garcinia subelliptica Yes [279]
2R,3S-5,7,4',5'',7'',3''',4'''- (Clusiaceae) (1) IC50 = 2.5 µM
heptahydroxy-flavanone[3-8''] (2) IC50 = 26 µM
flavone (1),
5,7,4',5'',7'',3''',4'''-
heptahydroxy[3-8'']
biflavanone (2)

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
3 flavonols: Heterotheca inuloides 3 favonols-(c) [280]
Quercetin (1), Kaempferol (Asteraceae) (1) ID50 = 0.07 mM
(2), (2) ID50 = 0.23 mM
Morin (3), (3) ID50 = 2.32 mM
2 flavones: 2 flavones-(n)
Luteolin (4), (1) ID50 = 0.19 mM
Luteolin 7-O-glucoside (5) (2) ID50 = 0.50 mM

Luteolin Yes Inhibition Yes [281]

of adenyl
cyclase
activity
N-feruloylserotonin (1), Carthamus tinctorius Yes Yes [282]
N-(p-coumaroyl)serotonin L. (1) IC50 = 0.023 mM (1) IC50 = 0.191
(2), acacetin (3) (Asteraceae) (2) IC50 = 0.074 mM mM
(3) IC50 = 0.779 mM (2) IC50 = 0.245
mM
(3) IC50 > 20 mM

Inulavosin Inula nervosa Mistargetin Yes [283]

(Asteraceae) g of
tyrosinase
to
lysosomes
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Anastatin A (1), Isosilybin A Anastatica Isosilybin Yes [284]
(2), Isosilybin B (3), Luteolin hierochuntica A (2) and (1) IC50 = 16 µM,
(4), Quercetin (5), (+)- (Cruciferae) Isosilybin (2) IC50 = 10 µM,
Dehydrodiconiferyl alcohol B (3) (3) IC50 = 6.1
(6), (+)-Balanophonin (7), inhibit the µM,
3,4-Dihydroxybenzaldehyde mRNA (4) IC50 = 14 µM,
(8) expression (5) IC50 = 15 µM,
of TRP-2. (6) IC50 = 16 µM
(7) IC50 = 15 µM
(8) IC50 = 17 µM

Silymarin Silybum marianum Yes Reduction Yes [285]

(milk thistle) of TYR IC50= 28.2 μg/mL
(Asteraceae) protein
levels
5,2’,4’-trihydroxy-2’’,2’’- Dalea elegans Yes [286]
dimethylchromene- (Fabaceae) [(m) L-tyrosine IC50=
(6,7:5’’,6’’)-flavanone 0.26 μM]
[(nc) L-DOPA IC50=
18.61 μM]
Kuraridin (1), Sophora flavescens Yes [287]
Kurarinone (2), Norkurarinol (Fabaceae) (1) IC50= 1.1 μM
(3) (2) IC50= 1.3 μM
(3) IC50= 2.1 μM

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Sophoraflavanone G (1), Sophora flavescens Yes [288]
Kurarinone (2) (Fabaceae) (1) nc- IC50 = 4.7 µM,
Kurarinol (3) (2) nc- IC50 = 2.2 µM
(3) c- IC50 = 0.1 µM
Sophoraflavanone G (1), Sophora flavescens Yes [289]
Kuraridin (2), (Fabaceae) (1) IC50 = 6.6 µM
Kurarinone (3) (2) IC50 = 0.6 μM
(3) IC50 = 6.2 µM
Kurarinone (1) Sophora flavescens Yes (1)-nc [290]
Kushnol F (2) (Fabaceae) (1) IC50 = 4.6 µg/mL
(2) IC50 = 9.0 µg/mL
Kurarinol (1), Kuraridinol Sophora Yes (1,2)-nc Yes [291]
(2) flavescens (1) IC50 =8.60±0.51 (1) IC50=29 μM,
(Fabaceae) µM (2) IC50=17 μM
(2) IC50 =0.88±0.06 μM
5,2’,4’-trihydroxy-2’’,2’’- Dalea elegans Yes [286]
dimethylchromene- (Fabaceae) [(m) L-tyrosine IC50=
(6,7:5’’,6’’)-flavanone 0.26 μM]
[(nc) L-DOPA IC50=
18.61 μM]
Kuraridin (1), Sophora flavescens Yes [287]
Kurarinone (2), (Fabaceae) (1) IC50= 1.1 μM
Norkurarinol (3) (2) IC50= 1.3 μM
(3) IC50= 2.1 μM
N-Feruloyl-N′-cis-feruloyl- Sophora japonica Yes (m) [292]
putrescine (Fabaceae) IC50 = 85.0 μM
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Dalbergioidin Lespedeza cyrtobotrya Yes (nc) Yes [293]
(Fabaceae) IC50 =20 μM IC50 =27 μM
Haginin A Lespedeza cyrtobotrya Yes (nc) TRP-1 Reduction Yes [294]
(Fabaceae) IC50 =5.0 µM protein of TYR, Melan-a cells
level and MITF IC50 = 3.3 µM ;
protein HEMn cells IC50
level, = 2.7 µM
Induction
of ERK and
Akt/PKB
protein
level
Glycyrrhisoflavone (1), Glycyrrhiza uralensis Yes Yes [295]
Glyasperin C (2) (Fabaceae) (2) IC50 = 0.13 μg/mL (1) 63.73 ±
6.8%
inhibition at
5 μg/mL
(2) 17.65 ±
8.8% at 5
μg/mL
Licuraside (1), Glycyrrhiza uralensis Yes [296]
Isoliquiritin(2), (1-2) 1, 2 and 3 (c)
Licochalcone A (3) Glycyrrhiza inflate (3) (1) IC50 =0.072 mM
(Fabaceae) (2) IC50 =0.038 mM
(3) IC50 =0.0258 mM

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Calycosin Astragalus Yes Yes [297]
membranaceus IC50 = 38.4 µM IC50 = 40 µM
(Fabaceae)
Butin Spatholobus suberectus Yes Reducti Reduction Yes [298]
(Fabaceae) IC50 = 35.9 µM on of of TYR 29.26% at 100
TRP-1 protein and µM
and mRNA
TRP-2 level
protein
and
mRNA
level
Gallocatechin (1), Epi- Distylium racemosum Yes [299]
gallocatechin gallate(2), (Hamamelidaceae) (1) IC50 = 4.8 μg/ mL,
Quercitrin (3) (2) IC50 = 30.2 μg/
mL,
(3) IC50 = 37.7 μg/ mL
Quercetin(1) Marrubium velutinum Yes [300]
Tiliroside (2) and Yes
Marrubium cylleneum 100% inhibition
(Lamiaceae) (1) 49.67 ± 1.16 mM
(2) 30.19 ± 9.60 mM
Kaempferol Crocus sativus L. Yes (c) [301]
(Iridaceae) ID50 =0.23 mM
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Bibenzyl xyloside-1 (1), Chlorophytum Yes [302]
Bibenzyl xyloside-2 (2), arundinaceum (1) IC50 =1.6 µM
Bibenzyl xyloside-3 (3) (Liliaceae) (2) IC50 =0.43 µM
(1) IC50 =0.73 µM
Resveratrol (1), Veratrum patulum Yes [303]
Oxyresveratrol (2) (Liliaceae) (1) IC50 = 43.5 µM
(2) IC50 = 1.2 µM
2''- O-Feruloylaloesin, Aloe extracts Yes (n) [304]
aloesin Aole vera (Liliaceae)
Aloesin Yes in vitro Yes [305]
pigmented skin
equivalent model
Artocarpfuranol(1), Artocarpus Yes [306]
dihydromorin (2), heterophyllus (1) IC50 = 47.93 µM
steppogenin (3), (Moraceae) (2) IC50 = 10.34 µM
norartocarpetin (4), (3) IC50 = 0.57 µM
artocarpanone (5), (4) IC50 = 0.46 µM
artocarpesin (6), (5) IC50 = 1.54 µM
isoartocarpesin (7) (6) IC50 = 0.52 µM
(7) IC50 = 0.66 µM
Norartocarpetin (1), Artocarpus Yes [307)
Resveratrol (2) gomezianus
(Moraceae)

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
3-prenyl luteolin Artocarpus Yes Yes [308]
heterophyllus IC50 = 76.3 µM IC50 = 57.6 µM
(Moraceae)
1,3-diphenylpropanes: Broussonetia kazinoki. Yes (c) [309]
kazinol C (1), (Moraceae) (1) IC50 = 15.5 µM
kazinol F (2), broussonin C (2) IC50 = 0.96 µM
(3), (3) IC50 = 0.43 µM
kazinol S (4) (4) IC50 = 17.9 µM
chlorophorin Chlorophora excelsa Yes (c) [310]
(Moraceae) IC50 = 1.3 µM
4-[(2’’E)-7”-hydroxy-3,”7” Chlorophora excelsa Yes (c) [310]
-dimethyloct-2” -enyl]-2’ (Moraceae) IC50 = 96 µM
,3,4’,5-tetrahydroxy-trans-
stilbene
(±)2,3-cis-dihydromorin Cudrania Yes [311]
(1), cochinchinensis (1) IC50 = 31.1 μM
2,3-trans-dihydromorin (2), (Moraceae) (2) IC50 = 21.1 μM
Oxyresveratrol (3) (3) IC50 = 2.33 μM
2,4,2',4'-Tetrahydroxy-3- MORUS NIGRA Yes (c) Yes [312]
(3-methyl-2-butenyl)- (MORACEAE) IC50 =0.95 µM
chalcone
Oxyresveratrol Morus alba L. Yes (nc) [313]
(Moraceae) IC50 = 1 µM
Polyphenols: Morus lhou Yes (c) [314]
Compound 1,5,9 (Moraceae) (1) IC50 = 1.3 µM
(5) IC50 = 1.2 µM
(9) IC50 = 7.4 µM
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Betulinic acid Morus alba L. and Yes [315]
Morus rotundiloba K.
(Moraceae)
Crude extract (C-AP) Malpighia emarginata. Yes Yes [316]
Anthocyanins: acerola fruit (C-AP) IC50=15 μg/mL, (data no shown)
cyanidin-3-alpha-O- (Malpighiaceae) (1) (2) – (nc)
rhamnoside (1), (1) IC50=40 μM,
pelargonidin-3-alpha-O- (2) IC50=19.1 μM
rhamnoside (2)
2R,3S-5,7,4',5'',7'',3''',4'''- Hibiscus tiliaceus Yes [279]
heptahydroxy- (Malvaceae)
flavanone[3-8''] flavone,
and 5,7,4',5'',7'',3''',4'''-
heptahydroxy[3-8'']
biflavanone
Globulusin A (1), Eucalyptus globules Yes [194]
Eucaglobulin (2) (Myrtaceae)
Kaempferol (1), quercetin Paeonia suffruticosa Yes [317]
(2), (Paeoniaceae) (1) to (5) --(c)
mudanpioside B (3), (1) IC50 = 0.12 µM
benzoyl-oxypaeoniflorin (2) IC50 = 0.11 µM
(4), mudanpioside H (5), (3) IC50 = 0.37 µM
pentagalloyl-β-D-glucose (4) IC50 = 0.45 µM
(6) (5) IC50 = 0.32 µM
(6) -- (nc)
(6) IC50 = 0.06 µM

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
2,3-dihydro-4’,4’’’-di-O- Podocarpus Yes Reductio Yes [318]
methylamentoflavone macrophyllus var. IC50=0.10 mM n of
macrophyllus TRP-2
(Podocarpaceae) mRNA
Anthraquinones Polygonum cuspidatum Yes [319]
(Polygonaceae)
(2R,3R)-(+)-taxifolin Polygonum hydropiper Yes [320]
L. (Benitade) IC50=0.24 mM
(Polygonaceae)
3,4-Dihydroxycinnamic Pulsatilla cernua Yes (nc) [268]
acid (1), (Ranunculaceae) (1) IC50 = 0.97 mM
4-hydroxy-3- (2) IC50 = 0.33 mM
methoxycinnamic acid (2)
Quercetin Rosa canina L. Yes Yes [321]
(Rosaceae) Reducing melanin
content to 64% at
10 µM, 34.5% at
20 µM, 17.5% at
17.7% at 40 µM
3,3'- Morinda citrifolia Yes [266]
Bisdemethylpinoresinol (Rubiaceae) (1) IC50 = 0.3 mM,
(1), (2) IC50 = 0.1 mM
Quercetin (2)
Nobiletin Peel of Citrus fruit Yes [322]
( Rutaceae) IC50=46.2 μM
Betulin, Lupeol, Guioa villosa Yes [323]
Soyacerebroside I (Sapindaceae)
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
(+)-epi-Syringaresinol (1), Synsepalum dulcificum Yes [269]
N-cis-Feruloyltyramine (2) (Sapotaceae) (1) IC50=200 μM,
(2) IC50=215.5 μM
Acetone extract, Sideroxylon inerme Yes yes [324]
epigallocatechin gallate (Sapotaceae) Acetone extract
(1), Procyanidin B1(2) IC50=63 µg/mL,
(1)IC50=30 µg/mL
(2)IC50＞200 µg/mL
Negundin A (1), Vitex negundo Linn. Yes [325]
Negundin B (2), 6- (Verbenaceae) (1) IC50 = 10.06 μM
hydroxy-4-(4-hydroxy-3- (2) IC50 = 6.72 μM
methoxy)-3- (3) IC50 = 7.81 μM
hydroxymethyl-7-methoxy- (4) IC50 = 9.76 μM
3,4-dihydro-2- (5) IC50 = 3.21 μM
naphthaledehyde (3), (6) IC50 = NA
Vitrofolal E (4), (+)- (7) IC50 = 15.13 μM
lyoniresinol (5), (+)- (8) IC50 = 5.61 μM
lyoniresinol-3α-O-β-D-
glucoside (6), (+)-(－)-
pinoresinol (7),
(+)-diasyringaresinol (8)
isopanduratin A (1), Kaempferia pandurata Yes Yes [271]
4-hydroxypanduratin A (2) ( Zingiberaceae) (1) IC50=10.5 μM (1) IC50 = 10.64
(2) IC50 > 30 μM μM,
(2)IC50 = 23.25
μM

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Gentol Gnetum genus Yes Yes [326]
( Zingiberaceae) IC50=4.5 μM
(-)-epigallocatechin gallate Green tea Yes (c) [327]
(EGCG) (1) (1) IC50 = 34.10 μM
(-)-gallocatechin 3-0- (2) IC50 = 17.34 μM
gallate (GCG) (2) (3) IC50 = 34.58 μM
(-)-epicatechin gallate
(ECG) (3)
1,2,3,6-Tetra-Ogalloyl-b- 10 Chinese Galls Yes (nc) Yes [274]
D-glucose (1), (1) IC50=30 μM
1,2,3,4,6-Penta-O-galloyl- (2) IC50=15 μM
b-D-glucose, (2) (3) IC50=54 μM
2,3,4,6-
Tetra-O-galloyl-D-glucose
(3)
N,N′-dicoumaroyl- Corn bran yes Yes [328]
putrescine (DCP), (DCP) IC50=181.73 μM (DCP)
N,N′-diferuloyl-putrescine (DFP) IC50=291.3 μM IC50 =3169.5 μM
(DFP) (DFP)
IC50 =733.64 μM
7,8,4’- Korean fermented Yes Yes [329]
Trihydroxyisoflavone (1), soybean paste (1) IC50 = 11.21 ± 0.8 μM (1) IC50 =
7,3’,4’- (Doenjang) (2) IC50 = 5.23 ±0.6 μM 12.23±0.7 μM
Trihydroxyisoflavone (2), (2) IC50 = 7.83
Genistein (3) ±0.7 μM
(3) IC50= 57.83
±0.5 μM
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
5,7-dihydroxyflavone propolis Blockin Yes [330]
(chrysin) g (51.6% at10 μM,
adenyly 40.90% at
l 100μM)
cyclase
activity
Others
Eextract Salicornia herbacea Yes Yes [331]
(Amaranthaceae)
70% Acetone extract Rhus chinensis Yes Yes [274]
(Anacardiaceae) IC50= 22 μg/mL

Isoimperatorin Angelica dahurica Yes Reducti Yes [332]

Imperatorin ( Apiaceae) on of
TYR
mRNA
levels
Anisic acid Pimpinella anisum Yes (u) [268]
(Apiaceae) IC50=0.68 mM
Anisaldehyde Pimpinella anisum Yes (nc) [268]
(Apiaceae) IC50=0.38 mM
Cumic acid (1), Cuminum cyminum Yes (nc) [333]
Cuminaldehyde (2) (Apiaceae) (1) IC50 = 0.26 mM
(2) IC50 = 0.05 mM

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
ethanolic extract Areca catechu Yes Yes [334]
(Arecaceae) IC50 = 0.48 mg/mL
Lichen species: Yes [335]
Graphina glaucorufa,
Graphina multistriata,
Graphina
salacinilabiata,
Graphis assamensis,
Graphis nakanishiana,
Phaeographopsis indica
(2Z,8Z)-Matricaria acid Erigeron breviscapus Yes Yes [336]
methyl ester (Asteraceae) IC50=25.4 μM
selina-4(14),7(1)-dien-8- Atractylodis Rhizoma Yes TRP-1, yes [337]
one Alba. TRP-2
(Asteraceae)
Extract Lepidium apetalum Yes Reducti yes [338]
(Brassicaceae) on of
TYR
mRNA
and
MITF
protein
level
2 germacrane-type Chloranthus henryi Yes [339]
sesquiterpenes (Chloranthaceae) IC50=325 μM and 269 μM
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Tianmushanol (1), Chloranthus Yes [340]
8-O-methyltianmushanol tianmushanensis (1) IC50=358 ±3 µM
(2) (Chloranthaceae) (2) IC50=312 ±3 µM
3β,21,22,23- Amberboa ramosa Yes [341]
tetrahydroxycycloart- (Asteraceae) IC50=1.32 μM
24(31),25(26)-diene
1β-Hydroxy arbusculin A Saussurea lappa Clarke Yes [342]
(1), costunolide (2), (Asteraceae) (1) IC50 = 11
reynosin (3) µg/mL,
(2) IC50 = 3.0
µg/mL
(3) IC50 = 2.5
µg/mL
methanolic extract fraction Arbutus andrachne L. Yes [343]
(Ericaceae) IC50=1000 mg/mL
Esculetin Euphorbia lathyris L. Yes(c) [344]
(Euphorbiaceae) IC50 = 43 µM
three steroids: stigmast-5- Trifolium balansae Yes [345]
ene-3 beta,26-diol (1), (Fabaceae) (1) stronger than (2) and
stigmast-5-ene-3-ol (2), (3)
campesterol (3) (1) IC50 =2.39 μM

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
Stryphnodendron barbatimao, Yes [346]
Entada africana
Prosopis africana
(Fabaceae)
Cariniana brasiliensis,
(Lecythidaceae)
Portulaca pilosa,
(Portulacaceae)
Trifolirhizin Sophora flavescens Yes Yes [291]
(Fabaceae) IC50 =506.77±4.49 (3) IC50 = 36 µM
µM
methyl gallate Distylium racemosum branches Yes [299]
(Hamamelidaceae) IC50 = 40.5 μg/ mL
5-hydroxymethyl-2- Phellinus linteus Yes (nc) [257]
furaldehyde (Hymenochaetaceae) IC50 = 90.8 μg/mL
crocusatin-K Crocus sativus Yes [347]
(Iridaceae ) IC50= 260 μM
Trans-cinnamaldehyde Cinnamomum cassia Yes (c) [348]
(Lauraceae)
linderanolide B and Cinnamomum subavenium Yes Yes [349]
subamolide A (Lauraceae )

Extract Portulaca pilosa Yes [346]

(Lecythidaceae)
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-1,
TRP-2)
(-)-N-formylanonaine Michelia alba D.C. Yes DPPH, yes [350]
(Magnolianceae) IC50= 74.3 μM reducing
power, and
chelating metal
ions.
1',3'-dilinolenoyl-2'- Flammulina velutipes Yes [351]
linoleoylglycerol (Marasmiaceae) IC 50 =16.1 ±
0.5µg/mL
ethanolic extract of Morus alba Yes [352]
mulberry twigs (EEMT), (Moraceae)
ethanolic extract of
mulberry root bark
(EEMR)
A series of α,β-unsaturated Olea europaea L. (Oleaceae) Yes(n) their ability to [353]
aldehydes form a Schiff
base with a
primary amino
group in the
enzyme
(2E)-alkenal (C 7 ) Oliva olea L. Yes (nc) [353]
(Oleaceae)
acetonic extract Osmanthus fragrans Yes (u) Yes [354]
(Oleaceae) IC50= 2.314
mg/mL

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI (TRP- other MI
1, TRP-2)
methanol extract Lichen species: Yes [355]
Usnea ghattensis IC50= 8.5 μg/mL
(Parmeliaceae )

methanol extract Lichen species: Yes [355]

Arthothelium IC50= 17.8 μg/mL
awasthii (Parmeliaceae)
Sesamol (3,4- Sesamum indicum L. Yes(c) Yes [356]
methylenedioxyphenol) (Pedaliaceae) IC50 = 1.9 µM 63%
decreased
in 100
mg/mL
5-(Hydroxymethyl)-2- Dictyophora indusiata Yes (nc) [357]
furfural (Phallaceae) ID50=0.98 mM
piperlonguminine Piper longum Yes Reduction of Yes [358]
(Piperaceae) TYR mRNA, and
MITF protein
level,
phosphorylates
CREB
geranic acid Cymbopogon citrates Yes [359]
(Poaceae) IC50=0.14 mM (trans)
IC50=2.3 mM (cis)
Extract Coccoloba uvifera Yes [360]
(Polygonaceae) IC50= 68.84 µg/ml
Ethanol extract and Ganoderma lucidum Yes [361]
distilled water extract (Polyporaceace) IC50 =0.32 mg/mL
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI (TRP- other MI
1, TRP-2)
Extract Dimocarpus longan Yes [364]
(Sapindaceae) IC50= 2.9–3.2 mg/mL
1-O-methyl- Schisandra chinensis Yes via activation of Yes [365]
fructofuranose (Turcz.) Baill MEK/ERK and
(Schisandraceae) PI3K/Akt
signaling
pathway and
subsequent MITF
downregulation.
ethanol extract, water Stichopus japonicas Yes (m) [366]
extract, (Stichopodidae) ethanol extract 0.49–
adenosine (1), 0.61 mg/mL,
Ethyl-α-D- water extract 1.80–
glucopyranoside (2) 1.99 mg/mL,
(1) IC50= 0.13 mg
/mL,
(2) IC50=0.19 mg /mL
hirsein A, hirsein B Thymelaea hirsuta Yes TRP1, Decrease PKC Yes [367]
(Thymelaeaceae) TRP2 activity, MITF,
TRP1, TRP2,
Metallothionein Aspergillus niger Yes (m) [368]
(protein) (Trichocomaceae)
9-Hydroxy-4- Angelica dahurica Yes (nc) [369]
methoxypsoraln (Umbelliferae) IC50=2.0 μg/mL

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Compounds (phenol, Source Mode of action Refs. Refs.
polyphenols, others) TI OEI (TRP- other MI
1, TRP-2)
Alpinia galanga Alpinia galanga Yes Yes [370]
extract Rhizome IC30=18.5
(Zingiberaceae) μg/mL

Extract Curcuma aromatica Yes Yes [370]

Rhizome IC30=8.9
(Zingiberaceae) μg/mL

partial purification Curcuma longa Yes TRP Phosphorylates Yes [371]

(Zingiberaceae) MEK, ERK1/2 and
Akt, MITF, and TRP-
2 protein level
triacylglycerols; triolein Sake lees Yes (nc) [372]
(1), TI 2 > 1
trilinolein (2) (1) IC50=30 μM
(2) IC50=8.4
μM

aqueous extracts green asparagus Yes (m) radical scavenging, [373]

IC50= 1.21 chelating activities
mg/mL and protected
liposome
against oxidative
damage.
rsolic acid Yes Tyrosinase mRNA Yes [374]
and protein
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Table 1. (Continued)

Compounds (phenol, Source Mode of action Refs. Refs.

polyphenols, others) TI OEI other MI
(TRP-
1,
TRP-
2)
San-bai-tang San-bai-tang Yes TRP1, MITF Yes [375]
IC50= 215.6 ± 10.3 TRP2 IC50=
µg/mL 254.8
± 14.5
µg/mL
TI: tyrosinase inhibiton, (c) competitive (u) uncompetitive (nc) noncompetitive and (m) mixed mode, OEI: other enzyme inhibition,
MI: melanin inhibition, TRP-1: tyrosinase related protein-1, TRP-2: tyrosinase related protein-2, PKC: protein kinase C, MITF:
microphthalmia-associated transcription factor.

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 Melanogenesis and Natural Hypopigmentation Agents 121

ENHANCING TYROSINASE DEGRADATION

Fatty acids are ubiquitous components of cell membranes and serve as a biological
energy source. They also play important roles in intracellular signaling and as precursors for
ligands that bind to nuclear receptors [152, 167-169]. Fatty acids act as intrinsic factors that
modulate the proteasomal degradation of membrane glycoproteins such as tyrosinase. In
addition, they regulate the selective degradation of melanogenic enzymes through the
ubiquitin-proteasome pathway [170]. Ando et al. found that fatty acids regulate the
ubiquitination of tyrosinase and are responsible for modulating the proteasomal degradation
of the enzyme [170] and that they had remarkable regulatory effects on melanogenesis in
cultured B16F10 murine melanoma cells by modulating proteolytic degradation of tyrosinase
[171]. Physiological doses of oleic acid and linoleic acid have been shown to increase the
proteolytic activity of 20S proteasomes in rat skeletal muscle [172].

INTERFERENCE WITH MELANOSOME MATURATION AND TRANSFER

Table 2 presents the natural products that have been shown to interfere with melanosome
maturation and transfer.

Table 2. Whitening agents from natural sources interference with melanosome

maturation and transfer

Source Compounds Mode of action Refs.

maturation transfer others
Soybean Bowman Birk inhibitor yes [175]
extract (BBI), soybean trypsin
inhibitor (STI)
Achillea Centaureidin Yes Yes Inhibition of [177, 179]
millefolium, melanogenesis
Yarrow and reduction
the amount of
tyrosinase.
Ophiopogon Methylophiopogonano Yes Yes [179]
japonicus ne B
Root of Niacinamide Yes [162, 180-
vegetable and 182]
yeast
Lectins and Yes [162, 376,
Neoglycoproteins 377]

Soybean Extract

Protease-activated receptors (PARs) are a subfamily of related G protein-coupled trans-

membrane receptors that are proteolytically activated by serine proteases (including trypsin or
mast cell tryptase). PAR-2 is expressed in keratinocytes but not in melanocytes.
Stimulation of this receptor enhances the rate of phagocytosis of keratinocytes, which in
turn leads to increased melanin transfer [173]. Soybean contains small serine proteases, such
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122 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

as Bowman Birk inhibitor (BBI) and soybean trypsin inhibitor (STI, Kunitz-type trypsin
inhibitor), that have been shown to inhibit the PAR-2 pathway in keratinocytes.
Interference with the PAR-2 pathway was shown to induce depigmentation by reducing
the phagocytosis of melanosomes by keratinocytes, thereby diminishing melanin transfer [17,
174-176]. Interestingly, only unpasteurised soybean milk exhibits this activity.

Centaureidin

Centaureidin (5,7,3'-trihydroxy-3,6,4'-trimethoxyflavone), a flavone from yarrow, has

been shown to reduce melanosome transfer and melanocyte dentrite outgrowth [177].
Centaureidin either directly or indirectly activates Rho, a small GTP-binding protein that acts
as a master regulator of dendrite formation. Ito et al. reported that activation of Rho in cells
exposed to centaureidin resulted in dendrite retraction and reduced melanocyte trafficking of
melanin to keratinocytes [178]. In addition, Saeki et al. found that centaureidin inhibited
melanogenesis and reduced the total amount of tyrosinase, but not TRP-1 [177].

Methylophiopogonanone B (5,7-Dihydroxy-6,8-Dimethyl-3-(4-
Methoxybenzyl)Chroman-4-One, MOPB)

Studies have shown that MOPB-induced activation of Rho causes reversible dendrite
retraction, microtubule disorganization, and tubule depolymerization, which in turn leads to
reduced melanosome transfer. The effect MOPB has on melanogenesis, however, is not the
same as the effect centaureidin has on melanin synthesis. Ito et al. showed that MOPB did not
influence melanin synthesis or the expression of melanogenic enzymes [179].

Niacinamide

Niacinamide (nicotinamide; 3-pyridinecarboxamide), the amide form of vitamin B3, is a

biologically active form of niacin found in many root vegetables as well as in yeast. Studies
have shown that niacinamide down regulates melanogenesis via inhibiting the transfer of
melanosomes from melanocytes to keratinocytes [162, 180]. Other studies have reported that
niacinamide is a tyrosinase inhibitor [181, 182].

Lectins and Neoglycoproteins

Cellular recognition between melanocytes and keratinocytes is an important process in

melanosome transfer. Lectins and neoglycoproteins are glycosylated residues on melanocyte
and keratinocyte membranes that play inhibitory roles in the process of receptor-mediated
endocytosis, a process that facilitates melanosome transfer [63]. Specifically, plasma
membrane lectins and their glycoconjugates are thought to interrupt melanocyte and
keratinocyte contact and interaction by binding to their specific plasma membrane receptors,

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 Melanogenesis and Natural Hypopigmentation Agents 123

resulting in inhibition of melanosome transfer [183]. This inhibition is reversible and has
been shown to be enhanced in the presence of niacinamide [162].

INHIBITION OF INFLAMMATION-INDUCED MELANOGENESIS

Some mediators produced by keratinocytes after exposure to primary inflammatory
stimuli or UV exposure, such as interleukin-1α (IL-1α), tumor necrosis factor α (TNF-α), ET-
1, and Stem cell factor (SCF) are able to promote melanogenesis. ET-1 shows a unique
behavior in exerting stimulatory effects both on DNA synthesis and melanization in human
melanocytes [65, 184-186]. Activation of epidermal ETs is determined by the enzymatic
cleavage of inactive prepolypeptides by an endopeptidase termed ET converting enzyme
(ECE), which is regulated by the primary inflammatory cytokine IL-1α [187]. The SCF
expressed in keratinocytes is involved in melanocyte growth and the synthesis, migration, and
maintenance of melanin. UV exposure stimulates the overexpression of SCF, which binds to
its receptor, c-kit, resulting in enhanced melanogenesis [188]. Arachidonate-derived chemical
mediators, namely the cysteinyl leukotrienes (LTC) LTC4 and LTD4, and thromboxanes,
such as TXB2, are released from membrane phospholipids by phospholipase A2 (PLA2).
Leukotrienes not only significantly up-regulate tyrosinase, but also enhance the transfer of
melanosomes to keratinocytes. These results suggest that PLA2 itself triggers melanin
synthesis following UV irradiation or inflammation, thereby resulting in hyperpigmentation
[52, 189]. Prostaglandins (PGs) synthesized from arachidonic acid by cyclooxygenase are
responsible for regulating cellular growth, differentiation, and apoptosis. In the skin, PGs
(especially PGE2, PGF2α) are produced and rapidly released by keratinocytes after exposure
to UV irradiation, resulting in hyperpigmentation [190]. Therefore, anti-inflammatory
compounds could be useful for the prevention or treatment of post-inflammatory
hyperpigmentation.
Table 3 lists some natural products that have been shown to be effective treatments for
inflammation-induced hyperpigmentation. Topical application of Matricaria chamomilla
extract has been shown to inhibit UVB-induced pigmentation by supprerssing ET-1-induced
DNA synthesis. The extract, however, did not affect IL-α-induced ET-1 production or
tyrosinase activation [184].
Hachiya et al. reported that a 50% ethanol extract of Sanguisorba officinalis root
inhibited UVB-induced pigmentation of brownish guinea pig skin. The results of their study
suggest that the mechanism governing the inhibition of ET-1 production in human
keratinocytes is via the suppression of endothelin-converting enzyme-1α [191]. Kobayashi et
al. reported that a 45% 1,3-butylene glycol extract of Althaea officinalis roots inhibited both
the secretion of ET-1 from normal human keratinocytes (NHKC) and the action of ET-1 on
NHMC, mainly by suppressing ET-1-induced calcium mobilization. They found that binding
of ET-1 to the endothelin B receptor (ETBR) on the cell surface of NHMC induced the
mobilization of intracellular calcium [192]. Fucoxanthin, a carotenoid derived from edible sea
algae, exhibited anti-pigmentary activity when applied either topically or orally in an animal
model of UVB-induced melanogenesis. This effect of fucoxanthin may be due to suppression
of PGE2 synthesis and melanogenic stimulant receptors (neurotrophin, PGE2 and MC1R)
[193, 194].
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124 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

Table 3. Whitening agents from natural sources inhibiting on inflammation-induced

melanogenesis

Source Compounds Mode of action Refs.

Matricaria Matricaria Antagonist for ET-receptor (in vitro and in [184]

chamomilla chamomilla extract vivo)
Sanguisorba Suppression of endothelin-converting [191]
officinalis L. enzyme-1α (in vitro and in vivo)

Althaea roots extract Inhibits both the secretion and action of ET- [192]
officinalis L. 1 (in vitro)

sea algae fucoxanthin Suppression of prostaglandin (PGE2) synthesis [193]

and melanogenic stimulant receptors
(neurotrophin, PGE2 and α-MSH). (p.o.)
Fenugreek steroidal saponins Inhibition of TNF-α and melanogenesis (in [195]
seed vitro)
(Trigonella
foenum-
graecum L.)
Eucalyptus Globulusin A and Anti-inflammatory and anti-melanogenesis [194]
globulus eucaglobulin activity (in vitro) 改小寫
Azadirachta nimolicinol Inhibition of melanogenesis (in vitro) and [378]
indica seed TPA-induced inflammation (in vivo)
Guava leaves extract Suppression of skin inflammation and [196]
(Psidium melanogenesis (p.o.)
guajava L.)
ANTI-MELANOGENESIS MAY DUE TO ANTIOXIDANT ACTIVITY
glabridin superoxide anion Inhibition of UVB-induced pigmentation [379]
productions and and erythema (in vivo), inhibition of
cyclooxygenase superoxide anion productions and
activities cyclooxygenase activities (in vitro)

luteolin Inhibiting adenyl cyclase induced by MSH, [281]

anti-oxidant activity in DPPH, NBT/XO
and intracellular ROS and xanthine oxidase
(in vitro)
pine bark Pycnogenol Inhibition of tyrosinase and melanin [380]
(catechin, biosynthesis, suppressing ·O2, NO·,
epicatechin and ONOO−, and ·OH in (in vitro)
epicatechin-4-(2-
hydroxyethyl)thio
ether)
Ecklonia cava Phlorotannins Inhibition of tyrosinase activity and [381]
(brown alga) (dieckol) reduction of intracellular ROS induced by
UV-B radiation (in vitro)

Ishige diphlorethohydrox Inhibition of tyrosinase activity and [382]

okamurae ycarmalol reduction of intracellular ROS induced by
(marine algae) UV-B radiation (in vitro)
ginger [6]-Gingerol Decreasing ROS level and suppressing [383]
TYR activity (in vitro)

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 Melanogenesis and Natural Hypopigmentation Agents 125

Globulusin A and eucaglobulin, monoterpene glycosides isolated from Eucalyptus

globules, not only have DPPH free radical scavenging activity, thereby inhibiting phorbol
myristate acetate-induced expression of tumor-necrosis factor-α and interleukin-1β, but also
inhibit melanogenesis in vitro [194]. In addition, a methanolic extract and its steroidal
saponins, 26-O-β-D-glucopyranosyl-(25R)-furost-5(6)-en-3β,22β,26-triol-3-O-α-L-rhamno-
pyranosyl-(1′′→ 2′)-O-[β-D-glucopyranosyl-(1′′′ → 6′)-O]-β-D-glucopyranoside, minutoside
B, and pseudoprotodioscin isolated from Fenugreek seed (Trigonella foenum-graecum L.
Fabaceae) inhibited the production of phorbol-12-myristate-13-acetate-induced inflammatory
cytokines, namely TNF-α and melanogenesis in vitro [195]. Guava leaf extracts have been
shown to suppress UVB-induced skin inflammation. Takashi et al. found that the skin color
of guinea pigs that had been exposed to UVB irradiation followed by treatment with guava
extract (p.o.) became lighter as a result of the tyrosinase inhibitory activity of guava leaf
extract [196]. Nimolicinol, a limonoid isolated from Azadirachta indica seeds, shows
inhibitory effects both on melanogenesis in B16 melanoma cells and on 12-O-
tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice.
Many studies have found that compounds with potent free radical scavenging activities
inhibit tyrosinase expression. Some of the most potent compounds with free radical
scavenging ability and tyrosinase inhibiting activity include glabridin, diarylheptanoids and
phenolic compounds from Acer nikoense; luteolin and pycnogenol from pine bark;
phlorotannins from Ecklonia cava; diphlorethohydroxycarmalol from Ishige okamurae; and
[6]-gingerol from ginger (Table 3).

ACCELERATING SKIN DESQUAMATION

Desmosomes, which are classified as a molecular complex of cell adhesion proteins
consisting of desmoglein and desmocollin, are mainly responsible for the adhesion between
epidermal cells. As the cells move upward from the basal layers to the stratum corneum, the
desmosome attachments become weaker. This weakening action is accelerated by enzymes,
namely the stratum corneum chemotrypic enzyme (SCCE) and Cathepsin D, by breaking the
bonds of the desmosomes, resulting in the sloughing off of cells. Keratinization refers to the
turnover of the stratum corneum and begins at the basal layer and gradually moves upward to
the stratum corneum corneocytes. This desquamation process normally takes about four
weeks and is normally more efficient in younger skin. The process stimulates the growth of
newer cells at a deeper level; however, in skin of advanced age, the intercellular desmosomes
become glue-like in their ability to cement cells together. As a result, cell sloughing becomes
more difficult, which leads to a thicker skin with a dull appearance. The stratum corneum has
a pH of 7 at the bottom layer and a pH ranging from 4.5-5.4 at the surface [197]. The optimal
pH for SCCE and Cathepsin D activity in the final desquamation stage ranges from 4 to 6,
which explains why those enzymes are most active at the surface of the stratum corneum
[198-200].
The capability of a compound to accelerate the turnover of epidermal layers and/or
disperse melanin pigment can result in skin lightening. Depigmenting agents lighten the skin
by stimulating the removal of pigmented keratinocytes [155, 201]. Pigmented spots, such as
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126 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

freckles or actinic lentigines, melasma spots, and post-inflammatory hypermelanosis macules

may be removed by the peeling of corneocytes and epidermal keratinocytes.

Chemical Exfoliants And Their Mode of Action

(1) α-Hydroxyacids
α-Hydroxyacids (AHA, i.e., lactic acid, glycolic acid, and malic acid) are weak organic
acids found in fruits, plants, and milk [202]. Studies on cell cohesion and skin pH changes
indicate that keratin bonds may became weaker at low pH values. AHA solution is activated
under low pH conditions and may dissolve the desmosome protein linkages causing a burst in
skin exfoliation. AHAs have also been used to successfully treat pigmentary lesions such as
solar lentigenes, lesions caused by melasma, and post-inflammatory hyperpigmentation
macules. AHAs promote exfoliation by decreasing corneocyte cohesion and by stimulating
dermal cell growth in the basal layer at low concentrations, while at higher concentrations
AHAs promote epidermolysis and dispersal of basal layer melanin. The accelerated
desquamation of the stratum corneum by AHAs is complemented by a direct inhibition of
tyrosinase, without influencing mRNA or protein expression [201-203]. Lactic acid can be
isolated from sour milk [201]. Glycolic acid can be isolated from natural sources, such as
sugarcane, sugar beets, pineapple, cantaloupe, and unripe grapes. Both glycolic acid and
lactic acid affect the skin layers in the same manner as described above. Furthermore,
additional beneficial effects unique to lactic acid include an increase in dermal
glycosaminoglycans (GAGs-natural moisturizers) and ceramides (epidermal barrier lipids),
and improved water barrier properties. Glycolic acid stimulates collagen synthesis in a
manner similar to that of lactic acid [204]. Yamamoto et al. studied the histological
differences between patients who received a six-week treatment of topical AHA, glycolic
acid, lactic acid, or citric acid as treatment for photo-aged skin and found that patients who
had received AHA showed increased epidermal thickness, decreased melanin deposition, and
up-regulated collegen levels relative to patients who received topical glycolic acid, lactic acid,
or citric acid [205]. In addition, the authors found that AHA treatment not only decreased
melanin deposition, but also resulted in the remodeling of the epidermis and the acceleration
of desquamation [205]. The Cosmetic Ingredient Review, a panel endorsed by the Esthetics
Manufacturers and Distributors Alliance of the American Beauty Association suggests that
consumers should not use glycolic acid or lactic acid products with concentrations exceeding
10% or at a pH of 3.5; for professional use, the limits are extended to 30% and the lowest
advisable pH value is 3.0.

(2) β-hydroxyacids (BHAs)

Salicylic acid (SA) is a β-hydroxyacid (BHA) found in willow bark and sweet birch. It is
also a phytohormone that acts similar to hormones that regulate cell growth and
differentiation. SA functions as a desquamating agent by penetrating and dissolving the
intercellular matrix of the stratum corneum [114, 202]. Unlike lactic acid, salicylic acid does
not hydrate the skin and does not help to normalize epidermal anatomy or physiology.
Salicylic acid, which is primarily a keratolytic agent, dissolves the stratum corneum layer by
layer from the outside in, resulting in a thinning of the stratum corneum. The effect of

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 Melanogenesis and Natural Hypopigmentation Agents 127

salicylic acid on hyperpigmentation inhibition has been demonstrated in a number of studies,

but only at very high concentrations (50%). SA is more lipophilic than AHAs, enabling it to
penetrate sebaceous substances in the hair follicles and exfoliate the pores. The water
solubility of AHAs is lower than that of SA. Since SA has a much stronger comedolytic effect
than AHAs, it can be used in acne therapy.

(3) Retinol
Retinol (Vitamin A) is a potent skin exfoliant and antiaging agent. Retinol has been
shown to improve the visible signs of photoaging as well as normal chronological aging when
used on a daily basis. Studies have shown that retinol slows down collagen degradation in
skin that has been chronically exposed to sunlight. In addition, retinol has been demonstrated
to inhibit enzymes that are responsible for the degradation of collagen, such as collagenase
[206].

(4) Liquiritin
Liquiritin, a flavonoid glycoside derived from liquorice, significantly reduces
hyperpigmentation in patients with bilateral and symmetrical idiopathic epidermal melasma
[207]. Zhu et al. found that a 20% liquiritin cream was effective at inducing skin lightening
by dispersing melanin in a clinical trial involving patients with melasma [208]. The proposed
mechanisms involve melanin dispersion by means of the pyran ring of its flavonoidal nucleus
and acceleration of epidermal renewal.

WHITENING AGENTS VERIFIED BY CLINICAL TRIALS

Whitening agents derived from natural products that have been tested in clinical trials are
listed in Table 4 and described below:

Arbutin and Its Derivatives

The compound 4-hydroxyanisole has been shown to act as an alternative substrate for
tyrosinase both in vivo and in vitro [209]. However, 4-hydroxyanisole and other phenolic
compounds have the potential to generate toxic quinone products and have, therefore, been
used in various studies to evaluate the toxic effects mediated by tyrosinase in melanoma cells
[210, 211].
Hydroquinone (HQ) was widely used as an effective skin-whitening agent before it was
banned by the US Food and Drug Administration in 2006 because animal studies in South
Africa, the United Kingdom, and the USA revealed that HQ was a potential carcinogen and
was associated with an increased incidence of ochronosis. HQ is defined as a drug since its
cancer-causing properties have not yet been proved in humans. Other phenolic compounds
that have been used to evaluate the toxic effects mediated by tyrosinase include arbutin, kojic
acid and ascorbic acid derivatives (Table 4).
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128 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

Table 4. The effect of whitening agents have been verified by clinical trials

Compounds Indication of clinical trials Refs.

3% Arbutin Treating hyperpigmentary disorders, such [212]
as melasma
3% Deoxyarbutin Acceleration of the fading of UV-induced [384]
tan
1% Kojic acid Treating hyperpigmentary disorders, such [216]
as melasma, post-inflammatory
hyperpigmentation, age spots, and freckles
10% Magnesium L- Effective for reducing melasma and age [220]
ascorbic acid 2- spots
phosphate
SLM (skin lightening Reduction of hyperpigmented spots on [385]
moisturizer containing the face
3% magnesium
ascorbyl phosphate
0.3% Rucinol Treating hyperpigmentary disorders, such [222]
as melasma
0.5% Ellagic acid Effective for treating UVB-induced [224]
hyperpigmentation of the skin
Ellagic Acid (200 mg, Inhibitory effect on a slight pigmentation [386]
100 mg/oral in the human skin caused by UV
administration ) irradiation
Ellagic acid Melasma [387]
0.5% Chamomilla Effective for treating UVB-induced [225, 226]
extract hyperpigmentation of the skin
0.5% 5,5’-Dipropyl- Effective for treating UVB-induced [228]
biphenyl-2,2’-diol hyperpigmentation of the skin
0.5% 5,5’-Dipropyl- Effective in treating hyperpigmentary [229]
biphenyl-2,2’-diol disorders, such as melasma and senile
lentigo
2% Rhododendrol Effective for treating UVB-induced [153]
hyperpigmentation of the skin
20% Azelaic acid Melasma [115]
Tranexamic acid Treating melasma [388]
3% adenosine Effective for treating hyperpigmentary [239]
Monophosphate disorders, such as melasma
Disodium Salt
4% N-acetyl-4-S- Melasma [240]
cysteaminylphenol (4-
S-CAP)
0.1% Linoleic acid Effective for treating melasma and to [241-243]
lighten UVB-induced hyperpigmentation
of the skin

5% Glycolic acid Whitening [244]

10% Glycolic acid Melasma [245]
Lactic acid, full Peeling agent in the treatment of melasma [246]
strength (92%; pH
3.5),
8% Glycolic acid and Hypopigmentation [247]
8% lactic acid
30% Salicylic acid Skin whitening [248]
peels

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 Melanogenesis and Natural Hypopigmentation Agents 129

Arbutin is a glycosylated form of HQ that is present in bearberry extracts but it can also
be synthesized from HQ by glucosidation. Its principal modes of action are competitive
inhibition of tyrosinase and TRP-1 activity, inhibition of UV-induced formation and
elongation of melanocyte dendritric processes and inhibition of production of ‧O2- and ·OH. It
has been shown that a 3% arbutin-containing formulation is effective for treating
hyperpigmentary disorders, such as melasma [212]. A combination therapy comprising a
YAG laser and 7% α-arbutin solution has been shown to be an effective and well-tolerated
treatment for refractory melasma [213]. Deoxyarbutin inhibits tyrosine hydroxylase and
DOPA oxidase activities of tyrosinase. In vitro studies have demonstrated that the inhibition
constant (Ki) of mushroom-derived tyrosinase is 350-fold lower than the Ki of arbutin. In a
human clinical trial, topical treatment with Deoxyarbutin for 12 weeks resulted in a
significant reduction in overall skin lightness in a population of light-skinned individuals and
a slight reduction in overall skin lightness and improvement in solar lentigines in a population
of darkskinned individuals [214, 215].

Kojic Acid

Kojic acid is a γ-pyrone compound produced during the fermentation of aspergillus

species, penicillium species and filiform bacteria. Kojic acid exerts a slow-binding inhibition
of tyrosinase activity, mainly by chelating copper, and inhibits the polymerization of DHI and
DHICA. In a clinical trial, Mishima et al. showed that a 1% kojic acid-containing formulation
was effective at treating melasma, post-inflammatory hyperpigmentation, age spots and
freckles [216]. In 2003, however, the Japanese Ministry of Health, Labor and Welfare
notified suppliers of kojic acid to delay manufacture or import of the product because of
concerns about possible carcinogenic effects in animals [217]. However, in 2005, kojic acid
was deemed to be a safe cosmetic ingredient and continues to be used as a skin-lightening
quasi-drug [218].

Ascorbic Acid and Its Derivatives

Ascorbic acid is highly unstable when exposed to heat or highly acidic conditions;
derivatives of ascorbic acid, however, are much more stable. Some of the more commonly
administered ascorbic acid derivatives include magnesium ascorbyl phosphate, ascorbyl
glucoside, sodium ascorbyl phosphate and 3-O-ethyl ascorbic acid. Ascorbic acid is a potent
reducer of DOPA quinone and melanin.
It has been reported that ascorbyl glucoside releases ascorbic acid gradually through
hydrolysis due to the action of α-glucosidase in living organisms [219].
In a clinical trial, Kameyama et al. found that a 10% magnesium ascorbyl phosphate-
containing formulation was shown to be effective at reducing the number of melasma patches
and age spots [220]. In another clinical trial, Miyai et al. found that a 2% ascorbyl glucoside-
containing cream was effective at accelerating the disappearance of UVB-induced
hyperpigmentation [221].
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130 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

Rucinol

Rucinol (4-n-butylresorcinol) has been reported to be an inhibitor of tyrosinase and TRP1

activity. Katagiri et al. found that a 0.3% Rucinol®-containing lotion was effective at
alleviating UV-induced pigmentation and melasma patches [222].

Potassium Methoxysalicylate

Hideya et al. found that potassium methoxysalicylate inhibits melanin synthesis via a
mechanism involving competitive inhibition of tyrosinase activity.
This mechanism is similar to the mechanisms governing the modes of action of arbutin
and Rucinol [153].

Ellagic Acid

Ellagic acid, a polyphenolic compound, is found in strawberries, apples and a variety of

plants.
Shimogaki et al. demonstrated that ellagic acid is a potent antioxidant and that it inhibits
tyrosinase activity through copper chelation [223]. Kamide et al. showed that application of
0.5% ellagic acid-containing cream was effective for treating UVB-induced
hyperpigmentation and melasma patches [224].

Chamomilla Extract

Chamomilla extract is a crude plant extract.

It inhibits melanin synthesis by binding to endothelin receptors and by inducing the
synthesis of inositol triphosphate. Ichihashi et al. demonstrated that a 0.5% chamomilla
extract-containing cream was effective at treating UVB-induced hyperpigmentation in
humans [225, 226].

5,5’-Dipropyl-Biphenyl-2,2’-Diol (Magnolignan®)

5,5’-Dipropyl-biphenyl-2,2’-diol is a biphenyl compound isolated from Magnolia

heptapeta.
It has been shown to inhibit melanin synthesis by interfering with the process of
tyrosinase maturation [227]. Takeda et al. found that a 0.5% Magnolignan®-containing
formulation was effective at treating melasma, senile lentigo and UVB-induced
hyperpigmentation in humans [228, 229].

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 Melanogenesis and Natural Hypopigmentation Agents 131

Rhododendrol (4-(4-Hydroxyphenyl)-2-Butanol)

Rhododendrol is a phenolic compound derived from White Birch and Nikko Maple.
Rhododendrol inhibits melanin synthesis through competitive inhibition of tyrosinase
activity.
In 2010, Kanebo Cosmetics Inc. obtained approval from the Japanese Ministry of Health,
Labor and Welfare to use Rhododendrol as a whitening agent [153].

Azelaic Acid

Azelaic acid is a naturally occurring saturated nine-carbon dicarboxylic acid. Its use
originated from the finding that Pityrosporum species can oxidize unsaturated fatty acids to
dicarboxylic acids, which competitively inhibit tyrosinase. Azelaic acid was initially
developed as a topical drug for the treatment of acne. However, because of its effect on
tyrosinase, it has also been used to treat melasma, lentigo maligna and other hyperpigmention
disorders [230, 231]. In addition, azelaic acid has been shown to be effective at treating
postinflammatory hyperpigmentation due to acne by inhibiting the production of free radicals
[232, 233]. In the USA, 20% azelaic acid is only indicated for treatment of acne, although it
has off-label use for hyperpigmentation. However, studies have found that 20% azelaic acid is
equivalent to or better than 2% hydroquinone for the treatment of melasma [233, 234].

Tranexamic Acid and Tranexamic Acid Cetyl Ester Hydrochloride

Plasmin, a protease found in blood serum, not only enhances the intracellular release of
arachidonic acid, a precursor of prostaglandins(235), but also elevates the levels of α-MSH
[236]. Tranexamic acid has been shown to inhibit UV-induced plasmin activity in
keratinocytes by preventing the binding of plasminogen to keratinocytes, which ultimately
results in less free arachidonic acid and a diminished ability to produce PGs, thereby
decreasing the activity of tyrosinase in melanocytes [189, 237]. Both arachidonic acid and α-
MSH can activate melanin synthesis in melanocytes. Therefore, the anti-plasmin activity of
tranexamic acid is thought to play a role in its topical effectiveness at treating melasma. The
effect of tranexamic acid cetyl ester hydrochloride in treating hyperpigmentary disorders is
due to its ability to inhibit UVB-induced inflammation, leading to the quiescence of active
melanocytes. This mechanism is similar to the mechanisms of action of chamomilla extract
and tranexamic acid.

Adenosine Monophosphate Disodium Salt

Adenosine is the building block of adenosine 5'-triphosphate (ATP), the main

intracellular source of energy. Since energy is essential for cell proliferation and maturation,
supporting ATP levels with topical adenosine safely accelerates epidermal turnover [238].
Adenosine monophosphate has the potency to increase the amount of intracellular glucose
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132 H. M. Chiang, H. W. Chen, Y. H. Huang et al.

uptake, which is necessary for the biosynthesis of adenosine triphosphate. Therefore,

adenosine monophosphate disodium salt accelerates epidermal turnover by elevating
intracellular energy metabolism, which leads to the excretion of melanin from the skin. A
clinical trial found that topical administration of a 3% adenosine monophosphate disodium
salt-containing formulation was effective at treating hyperpigmentary disorders, such as
melasma [239].

N-Acetyl-4-S-Cysteaminylphenol

N-acetyl-4-S-cysteaminylphenol is a tyrosinase substrate, and, on exposure to tyrosinase,

it forms a melanin-like pigment. The depigmentation effect of N-acetyl-4-S-
cysteaminylphenol is associated with a decrease in the number of functioning melanocytes
and in the number of melanosomes transferred to keratinocytes. A 4% N-acetyl-4-S-
cysteaminylphenol emulsion (O/W) was shown to be effective for treating melasma [240].

Linoleic Acid

Linoleic acid accelerates tyrosinase degradation, resulting in the down-regulation of

melanin synthesis. In clinical trials, topical application of a 0.1% linoleic acid-containing
liposomal formulation alleviated melasma symptoms [241] and UVB-induced
hyperpigmentation of the skin [242, 243].

AHAs and BHAs

Many clinical studies on the effectiveness of AHAs such as glycolic acid and lactic acid
as peeling agents for accelerating skin desquamation have been conducted in patients with
pigmentation disorders. For example, a 5% glycolic acid topical cream was shown to improve
skin texture and photoaging-induced discoloration [244]. In addition, a 10% glycolic acid
lotion has been reported to be effective at improving symptoms of melasma [245].
Furthermore, a 92% lactic acid (pH 3.5) formulation has been shown to be effective at
treating melasma [246]. A combination of 8% glycolic acid and 8% lactic acid creams has
been shown to be modestly useful in ameliorating mottled hyperpigmentation, sallowness,
and roughness due to chronic cutaneous photodamage [247].A clinical trial showed that 30%
salicylic acid in absolute ethanol was effective at treating acne and postinflammatory
hyperpigmentation [248].

CONCLUSION
In this article we have reviewed the synthesis of melanin, the signaling pathways related
to the regulation of melanogenesis, the factors influencing melanogenesis and various
pigmentation disorders, as well as the effectiveness of various natural products at reducing

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 Melanogenesis and Natural Hypopigmentation Agents 133

hyperpigmentation. An important issue regarding crude extracts or fractions from natural

products used in cosmetics is the standardization of cultivation, harvesting, collecting, storage
and extraction processes of the plants. Isolation of the active components from natural
products for skin-whitening formulations will clarify the effect and mechanism on
hypopigmentation. In addition, multi-functional formulations may increase the efficacy of
skin-whitening products.

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Antioxidant and antityrosinase activity of aqueous extracts of green asparagus. Food
Chem., 127, 141-146.
[374] Pinon, A., Limami, Y., Micallef, L., Cook-Moreau, J., Liagre, B., Delage, C., Duval, R.
E. & Simon, A. (2011). A novel form of melanoma apoptosis resistance: melanogenesis
up-regulation in apoptotic B16-F0 cells delays ursolic acid-triggered cell death. Exp.
Cell Res., 317, 1669-1676.
[375] Ye, Y., Chu, J. H., Wang, H., Xu, H., Chou, G. X., Leung, A. K., Fong, W. F. & Yu, Z.
L. (2010). Involvement of p38 MAPK signaling pathway in the anti-melanogenic effect
of San-bai-tang, a Chinese herbal formula, in B16 cells. J. Ethnopharmacol., 132, 533-
535.
[376] Minwalla, L., Zhao, Y., Cornelius, J., Babcock, G. F., Wickett, R. R., Le Poole, I. C. &
Boissy, R. E. (2001). Inhibition of melanosome transfer from melanocytes to
keratinocytes by lectins and neoglycoproteins in an in vitro model system., Pigment
Cell Res., 14, 185-194.
[377] Brenner, M. & Hearing, V. J. (2008). Modifying skin pigmentation–approaches through
intrinsic biochemistry and exogenous agents. Drug Discov. Today Dis. Mech., 5, 189-
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[378] Akihisa, T., Noto, T., Takahashi, A., Fujita, Y., Banno, N., Tokuda, H., Koike, K.,
Suzuki, T., Yasukawa, K. & Kimura, Y. (2009). Melanogenesis inhibitory, anti-
inflammatory, and chemopreventive effects of limonoids from the seeds of Azadirachta
indicia A. Juss. (neem). J. Oleo. Sci., 58, 581-594.
[379] Yokota, T., Nishio, H., Kubota, Y. & Mizoguchi, M. (1998). The inhibitory effect of
glabridin from licorice extracts on melanogenesis and inflammation. Pigment Cell Res.,
11, 355-361.
[380] Kim, Y. J., Kang, K. S. & Yokozawa, T. (2008). The anti-melanogenic effect of
pycnogenol by its anti-oxidative actions. Food Chem. Toxicol., 46, 2466-2471.
[381] Heo, S. J., Ko, S. C., Cha, S. H., Kang, D. H., Park, H. S., Choi, Y. U., Kim, D., Jung,
W. K. & Jeon, Y. J. (2009). Effect of phlorotannins isolated from Ecklonia cava on
melanogenesis and their protective effect against photo-oxidative stress induced by UV-
B radiation. Toxicol. Vitro, 23, 1123-1130.
[382] Heo, S. J., Ko, S. C., Kang, S. M., Cha, S. H., Lee, S. H., Kang, D. H., Jung, W. K.,
Affan, A., Oh, C. & Jeon, Y. J. (2010). Inhibitory effect of diphlorethohydroxycarmalol
on melanogenesis and its protective effect against UV-B radiation-induced cell damage.
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[383] Huang, H. C., Chiu, S. H. & Chang, T. M. (2011). Inhibitory Effect of [6]-Gingerol on
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Boissy, R. E. (2006). Comparative efficacy and safety of deoxyarbutin, a new
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 5

FUNGAL MELANINS: BIOSYNTHESIS AND

BIOLOGICAL FUNCTIONS

Rodrigo Almeida-Paes1, Joshua Daniel Nosanchuk2

and Rosely Maria Zancope-Oliveira1
1
Laboratorio de Micologia, Instituto de Pesquisa Clínica Evandro Chagas,
Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
2
Department of Medicine (Division of Infectious Diseases) and
Department of Microbiology and Immunology,
Albert Einstein College of Medicine, Bronx, NY, US

ABSTRACT
Melanins are hydrophobic polymers of high molecular weight, formed by oxidative
polymerization of phenolic and indolic compounds, produced by organisms in all
Kingdoms. They are typically black or dark brown in color and their molecular structures
are diverse. Several fungi can produce melanins and the functions of this pigment
enhance microbial survival under diverse unfavorable environmental and host conditions.
The major melanin type encountered among fungi is the 1,8-dihydroxynaphthalene
(DHN) melanin that is synthesized from acetyl-coenzyme A via the polyketide pathway.
This melanin is generated by several human pathogenic fungi, such as Fonsecaea
pedrosoi, Exophialla dermatitidis, Aspergillus fumigatus, Histoplasma capsulatum and
Sporothrix schenckii. It is also present in phytopathogenic fungi such as Colletotrichum
spp., Magnaporte orizae and Ascochyta rabiei. In addition to DHN melanin, fungi can
also produce melanin via dihydroxyphenylalanine (DOPA), in which tyrosinases or
laccases hydroxylate tyrosine via DOPA to dopaquinone that then auto-oxidizes and
polymerizes, resulting in a polyphenolic heteropolymer of black color known as
eumelanin. Cryptococcus neoformans is the best known fungus to produce this type of
melanin, but other fungi such as Candida albicans, Paracoccidioides brasiliensis and S.
schenckii can also produce eumelanin. A type of soluble fungal melanin is produced from
L-tyrosine through p-hydroxyphenylpyruvate and homogentisic acid. This pigment is
called pyomelanin and it is similar to alkaptomelanin produced by humans. A. fumigatus,
Madurella mycetomatis and Yarrowia lipolytica are examples of fungi that can produce
this type of pigment. Fungal melanins play an important role in the protection of fungi
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160 Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk et al.

from several environmental stresses, such as desiccation, UV irradiation, heavy metals,

temperature fluctuation and digestion by hydrolytic enzymes. Melanins also play a role in
the virulence of a broad range of pathogenic fungi. These pigments protect the fungi from
host defense mechanisms and antifungal agents. Although melanins challenge the
immunological strategies of host defense, they are also targets for alternative
antimicrobial strategies, by the use of antibodies against melanin or inhibitors of melanin
synthesis.

INTRODUCTION
Melanins are ubiquitous pigments produced by a broad range of living organisms from
bacteria to humans [1, 2]. They are typically dark brown or black in color [3] and have a high
molecular weight [4]. Melanins are synthesized by several pathways, all converging on the
oxidative polymerization of phenolic or indolic compounds [5, 6]. Some of physical and
chemical properties of melanins include a negative charge, hydrophobicity and insolubility in
both aqueous and organic solvents [7].
To date, no definitive structure has been found for any type of melanin because of their
insolubility, which makes studies on melanins very difficult [2, 6]. In general, fungal
melanins are studied after digestion of cells with glycolitic and proteolytic enzymes followed
by extraction with guanidinium isothiocyanate and hot concentrated acid (hydrochloric acid
6N). This treatment yields dark particles retaining the original cellular shape, but devoid of
cytoplasm or organelles, and are referred to as melanin ghosts [8].
Structurally, melanins appear to represent a mixture of high molecular weight polymers
and this structure makes them very stable and resistant to several destructive physicochemical
processes such as oxidant agents, desiccation, extreme temperatures, UV light, heavy metals
and other drugs [6, 9]. Electron spin resonance (ESR) characteristics have been used to define
pigments with stable organic free radicals as melanins [2]. This technique generates
distinctive signals (Figure 1) due to the presence of unpaired electrons in the polymer [10].

Figure 1. Electron spin resonance analysis of melanin particles generated from a representative S.
schenckii strain IPEC 26449 on its yeast phase, cultured in minimal medium (15 mM glucose, 10 mM
MgSO4, 29.4 mM K2HPO4, 13 mM glycine, and 3.0 mM thiamine, pH 5.5) with 1 mM L-3,4-
dihydroxyphenylalanine at 37ºC during 10 days.

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 Fungal Melanins: Biosynthesis and Biological Functions 161

Many fungi are able to synthesize melanin through different pathways, and functions of
this pigment are related to survival under diverse environmental and host conditions [1, 5].
This chapter will focus on the pathways used by different fungi to produce these dark
pigments, and also on their biological function on the fungi and implications for their hosts.

TYPES AND BIOSYNTHESIS OF FUNGAL MELANINS

Various types of melanin can be found in nature. The major type of melanin found within
the Kingdom Fungi is the 1,8-dihydroxynapthalene (DHN) melanin synthesized from acetyl-
coenzyme A (CoA) or malonyl-CoA via the polyketide pathway [9, 11]. Fungal polyketides
are synthesized by a process similar to fatty acid biosynthesis.
The biosynthesis of this type of melanin (Figure 2) begins with the conversion of
malonyl-CoA into 1,3,6,8-tetrahydroxynaphtalene (THN) by the enzyme polyketide synthase.

Figure 2. General biosynthetic pathway of fungal DHN melanin. Acetyl- and/or malonyl-CoA are
converted by at least two enzymatic steps to 1,3,6,8-THN that after two reductions and two dehydration
enzymatic reactions is converted to the 1,8-DHN precursor of melanin synthesis. The reduction steps
marked with * can be blocked with tricyclazole.

Then, by successive steps of reduction and dehydration, this compound is converted to

1,8-DHN. Subsequent steps are thought to involve a dimerization of the 1,8-DHN molecules,
which are finally polymerized by a fungal laccase to form the DHN melanin [11-13].
This metabolic pathway can be inhibited by the commercialized inhibitors tricyclazole
(Figure 3) and fenoxanil [13, 14]. It is important to note that, since the biosynthesis of this
type of melanin starts with the product of essential metabolic pathways such as glycolysis and
the pentose phosphate pathway, DHN melanin can be synthesized without the presence of any
precursor.
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162 Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk et al.

Figure 3. Influence of tricyclazole on melanization of a representative S. schenckii strain, IPEC 26449.

Numbers indicate concentration (mg/L) of tricyclazole on cultures. Ethanol (the tricyclazole diluents)
concentration was 0.6% on all media.

Other types of fungal melanins are synthesized only if a specific precursor is present
during fungal growth. The most common precursor for fungal melanin synthesis is L-
tyrosine. In fact, two types of melanin can be formed with this amino acid. Many fungi are
able to synthesize black or dark- brown pigments from L-tyrosine via
dihydroxyphenylalanine (DOPA).
The pathway for this type of melanin (Figure 4), called eumelanin, requires that
tyrosinases or laccases hydroxylate tyrosine via DOPA to dopaquinone.

Figure 4. General biosynthetic pathway of fungal eumelanins. In this pathway, tyrosine is converted to
L-DOPA and this compound to dopaquinone directly or via dopamine. Dopaquinone is converted via
other intermediates to dihydroxyindole that oxidates and polymerizates to generate melanin.

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 Fungal Melanins: Biosynthesis and Biological Functions 163

Then, after a series of cyclyzation, oxidation, tautomerization and polymerization

reactions, eumelanin is produced [11].
Other fungi, however, have the ability to produce brown pigments from tyrosine in a
pathway leading to the accumulation and auto-oxidation of intermediates of tyrosine
catabolism [15, 16]. This fungal pigment, known as pyomelanin, is similar to the human
pigment alkaptomelanin.
Actually, pyomelanin and alkaptomelanin are considered different designations for the
same pigment [16]. In general, homogentisic acid is the accumulated product of tyrosine
catabolism (Figure 5) that, after oxidation in benzoquinoacetate and polymerization, leads to
the production of pyomelanin.

Figure 5. General biosynthetic pathway of fungal pyomelanins. Phenylalanine and tyrosine catabolisms
generate homogentisic acid that can lead to the production of pyomelanin through benzoquinone acetic
acid after oxidation and polymerization. The step marked with * can be blocked with sulcotrione.

It is important to note that all biosynthesis models presented are general in nature, and the
pathways may vary slightly from fungus to fungus. Fungi can also produce other types of
melanin, such as allomelanins, nitrogen-free macromolecular polymers of simple phenols,
which have not been related to fungal virulence. The remainder of this chapter will focus on
the major human fungal pathogens and also on some plant pathogens, emphasizing the
importance of melanins for the fungus-host interactions and also on their implications for
human health.

Melanized Fungi

Cryptococcus Neoformans
Cryptococcus neoformans is a free-living ubiquitous yeast-like organism with a
characteristic polysaccharide capsule that can survive in a variety of environmental niches
such as soils contaminated with avian excreta and certain tree species [17-19].
This fungus and the closely related species, Cryptococcus gattii, cause cryptococcosis, a
disease that is relatively common in individuals with suppression of the cellular immune
system, but can also affect immunocompetent individuals.
The major complication of this disease is a life-threatening meningoencephalitis [20].
C. neoformans and C. gattii differ in biochemical, molecular characteristics, ecology and
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164 Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk et al.

geographic distribution [21], though melanization properties are similar between these two
species [22, 23].
C. neoformans is the most studied fungus in terms of melanization, with more than 210
published references in this field up to June 2011. Melanization was identified in this
organism as early as 1962 [4]. This fungus is unable to synthesize DHN-melanin and,
therefore, is incapable of de novo melanogenesis, relying on the presence of phenolic
compounds, such as L-DOPA (Figure 6), for making eumelanin [4, 24].

Figure 6. Cryptococcus neoformans [strain ATCC 24607 (serotype D)] produces a melanized
phenotype when cultured in minimal medium with L-DOPA (left) and an albino phenotype when
cultured in absence of L-DOPA (right). All cultures were maintained at 30ºC during 14 days in the
dark.

This particular feature can be utilized in the diagnosis of cryptococcosis, because when
C. neoformans is cultured in media such as Staib Agar, rich in phenolic compounds, yeast
colonies grow dark-brown whereas other pathogenic yeast produce white colonies [25, 26].
Over the past decade, researchers have advanced the concept that virulence and other
aspects associated with the relationship between certain fungi and mammalian host originated
from interactions between fungal cells and environmental organisms, such as bacteria,
protozoan and also nematodes [27-30]. For instance, C. neoformans can survive and replicate
within macrophages in a manner similar to that within amoebae [30]. Furthermore, the
interaction between C. neoformans and the gram-negative bacterium Klebsiella aerogenes
results in fungal melanization [22]. Also, C. neoformans is able to produce melanin using the
bacterial melanin precursor homogentisic acid in a laccase dependent way [23]. The
generation of pigment from bacterial products may in part explain the fact that this yeast is
melanized in the environment [31, 32], since this microbe produces melanin only from
exogenous substrates. As C. neoformans is a free-living organism that does not require
mammalian parasitism in its life cycle, melanization would protect this fungus primarily from
disadvantageous environmental conditions. In fact, melanization protects C. neoformans from
enzymatic degradation by antagonist microbes in the environment [33] and by damage from
UV light [34] as well as heat and cold [35].
Melanin production, as well as capsule growth (another important virulence factor of C.
neoformans), is regulated by a G-α protein-cAMP-PKA (cAMP-dependent protein kinase A)

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signaling pathway [36]. Melanin is produced when a laccase of 75-kDa encoded by the
CNLAC1 gene catalyzes the oxidation of L-DOPA or dopamine to quinones, which then
polymerizes to form melanin [37]. This fungus also possesses another laccase-encoding gene,
CNLAC2, whose transcript is a 65-kDa laccase that is also associated with melanogenesis
[38]. C. neoformans L-DOPA melanin is very similar to mammalian DOPA melanin, but the
cryptococcal melanin does not contain any phaeomelanin, a thiol containing melanin type
derived from tyrosine and cysteine, that is also present in mammalian melanin [11]. It is
important to note that some fungi are able to synthesize eumelanin from tyrosine, but the C.
neoformans laccase is considered a diphenol oxidase, since it produces pigment from
phenolic compounds with two hydroxyl groups but not from tyrosine [39]. Some of the
substrates used by C. neoformans laccase include L- and D-DOPA, methyl-DOPA, catechol,
dopamine and norepinephrine [8, 40, 41].
In C. neoformans, melanin is deposited in a layer internal to the fungal cell wall, next to
the plasma membrane [24, 42]. The current model for melanin synthesis is that the pigment is
formed in vesicles that are secreted and retained by the chitin cell wall [43], generating a
structure comprised of several layers of granular particles. Melanin porosity is a property of
the assembly of these particles, with absence of specialized pore structures for nutrient
acquisition. Small nutrient molecules, such as sugars and amino acids, can enter the cell by
passing through the spaces between the melanin particles [42]. During asexual reproduction,
melanin in the parent cell is not carried to the daughter cells, but rather is synthesized de novo
in buds. Hence, melanin remodeling occurs during fungal cell growth in a process requiring
degradation and synthesis at sites of budding [44].
Melanin is synthesized during mammalian C. neoformans infection [45, 46] and this
black pigment is highly associated with C. neoformans virulence. In fact, amelanotic C.
neoformans mutant strains are severely attenuated in animal models of infection [24, 28].
Cryptococcal melanin also impacts diverse host responses. Cryptococcal melanin is
immunogenic. For instance, it activates the alternative complement pathway [47], and also
elicits antibodies, some of which can inhibit fungal growth [48, 49]. It is noteworthy that
monoclonal antibodies (mAbs) have been generated to cryptococcal melanin [45] that are
reactive against a wide spectrum of melanin types [4]. Moreover, melanization is associated
with lowered levels of pro-inflammatory cytokines in animal models of infection [50].
Melanization decreases the rate of phagocytosis and killing of C. neoformans by macrophages
[24], probably because melanized C. neoformans cells are less susceptible than nonmelanized
cells to the fungicidal effects of nitrogen- and oxygen-derived oxidants [51]. Melanin also
down regulates immune responses early in infection [50]. Together, these studies indicate that
melanin in C. neoformans increases virulence by reducing its susceptibility to host defense
mechanisms and interfering with the development of successful immune responses [1, 4, 7,
32].
In addition to increasing resistance of C. neoformans to immune defenses, melanin also
reduces the efficacy of certain antifungal drugs, such as amphotericin B and caspofungin [52-
54].
Thus, melanization has clinical implications for C. neoformans infections in terms of the
alteration in immunologic responses and the interference with the potency of antifungal
drugs.
However, the pigment is also a potential drug target, either by antibody binding to
melanin [49] or by disruption of the melanization pathway [55].
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166 Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk et al.

Members of the Order Chaetothyriales

The Order Chaetothyriales is composed by fungi with dark mycelium [56]. The dark
coloration of the mycelium (Figure 7) is due to the production of melanin.

Figure 7. Fonsecaea pedrosoi, a member of the Order Chaetothyriales. Note the dark pigment
(melanin) on the cell walls of hyphae, conidiophores and conidia. Bar 10µm.

These fungi generally contain DHN melanin [56], although melanin derived from L-
DOPA has also been described in Exophiala dermatitidis [57]. Additional studies are
necessary to access whether or not other members of Chaetothyriales produce other types of
melanin. Melanin is an important factor associated with virulence of some members of this
order, however the presence of melanin alone is not sufficient to explain the pathogenicity of
these fungi, and additional factors, such as thermotolerance, must be involved in the
pathogenesis of disease [58]. This order contains several species of medical importance,
mainly Fonsecaea pedrosoi and E. dermatitidis.

Fonsecaea Pedrosoi
Fonsecaea pedrosoi is the main agent of chromoblastomycosis, an important
subcutaneous mycosis that is endemic worldwide although its prevalence is higher in tropical
countries. The disease begins with the traumatic inoculation of pigmented moulds into the
skin, and the most important species are F. pedrosoi, Phialophora verrucosa and
Cladophialophora carrionii. Even though chromoblastomycosis is typically not fatal, it is
characteristically chronic, extremely difficult to treat, and it can be complicated by lymphatic
damage and neoplastic transformation [59]. This disease is characterized by the presence of
muriform sclerotic bodies in tissue [60]. F. pedrosoi produces olivaceous to black mycelia
colonies that under microscopic analysis present conidia formed from swollen denticles,
giving rise to secondary and tertiary conidia; conidia may be also formed on sympodial
conidiophores and occasionally from discrete phialides [56].
Inhibition of melanin synthesis with tricyclazole [61, 62] together with de novo
melanogenesis [63, 64] confirms that F. pedrosoi synthesizes DHN melanin. The fungus is
able to synthesize melanin on hyphae, conidia and sclerotic bodies [62, 63]. Constituents of
melanin from F. pedrosoi comprise aromatic, aliphatic and glycosidic structures with a

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predominance of the latter [63]. Both extracellular and cell-wall associated melanin [62, 65,
66] are produced inside melanosome-like compartments associated with Ca2+ and Fe2+ [67].
The dispersed melanin over the fungal cell-wall is thought to have a valuable role in cross-
linking distinct cell wall compounds that help maintain the normal shape of the cell [62].
Melanin from F. pedrosoi is immunologically active. It can elicit a humoral immune
response, giving rise to human antifungal antibodies that can impair fungal growth in vitro
and enhance the antifungal functions of phagocytes [64]. Melanin can influence the
complement system activation by the alternative pathway [68]. Moreover, it has the ability to
interact with immune system cells. Some studies have shown that F. pedrosoi melanin
inhibits nitric oxide production by macrophages [69, 70] and that melanized cells are more
resistant to phagocytosis [61, 65]. On the other hand, soluble melanin leads to high levels of
fungal internalization by macrophages associated with an enhanced oxidative burst [64].
Thus, there are different effects of soluble and cell-wall associated melanin in F. pedrosoi.
Another role for the cell-wall associated melanin in F. pedrosoi is the reduction of
specific antibody recognition. In fact, melanin masks cerebroside recognition by antibodies,
conferring resistance of sclerotic cells to the antimicrobial effects of antibodies to
monohexosylceramides [71]. Together, these results show a diverse role for melanin on
chromoblastomycosis due to F. pedrosoi.

Exophiala Dermatitidis
Species within the genus Exophiala are frequently referred to as black yeasts, due to the
ability of several species to form budding yeast-like cells in addition to hyphal forms during
their life cycle. Exophiala (Wangiella) dermatitidis is phenotypically characterized by its
mucoid colonies, an ability to grow at 40ºC, and a lack of nitrate assimilation as well as
forming yeast cells surrounded by capsules [56]. This species causes phaeohyphomycosis and
it is of particular concern as an agent of brain infections in patients from East Asia [58]. This
fungus has previously been known as Wangiella dermatitidis, however according to a recent
revision about dematiaceous fungi, this is an obsolete name [56].
The first DHN-melanin pathway in a pathogenic fungus was described for E. dermatitidis
[72]. A polyketide synthase encoded by the gene WdPKS1 catalyses the reactions on the first
steps of the pathway [14, 73]. Detailed new molecular approaches show that E. dermatitidis
produces DHN melanin in a pathway that requires hexaketide 2-acetyl-1,3,6,8-
tetrahydroxynaphtalene as a precursor of THN [74]. These results show that, despite the high
degree of similarity among fungal polyketide synthases, they have different ways to produce
the necessary precursors for melanin synthesis.
In this fungus, melanin is polymerized exclusively on the fungal cell wall, and an
effective chitin synthase gene is necessary for the correct deposition of the pigment [75].
Also, albino mutant strains of E. dermatitidis present thinner cell-walls compared to the wild
type [57]. Melanin biosynthesis does not affect cell wall permeability. However, melanin
affects the development of E. dermatitidis within the host as it is associated with invasion of
the fungus in vitro and in vivo [76].
Several approaches have been used to successfully correlate melanin and virulence in E.
dermatitidis. Initially, wild type and UV generated melanin-deficient mutant strains were
used in mouse studies. These melanin deficient mutants were significantly less virulent than
the wild type, with a few hyphae observed in brains of mice infected with amelanotic strains
[77-79].
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168 Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk et al.

The molecular cloning and characterization of the WdPKS1 gene revealed the importance
of melanin synthesis during a mouse model of infection [73], probably because of its
protective effects against antifungal agents [57]. Interestingly, melanin did not influence
phagocytosis, but the presence of melanin protected E. dermatitidis from killing within the
phagolysosome of neutrophils [80]. Additionally, melanin deposition on the cell wall protects
this fungus from environmental stresses, such as lysing enzymes, heat and cold [57].
Treatment of phaeohyphomycosis, especially cerebral cases, is difficult, and needs early
diagnosis and aggressive therapy [81]. Studies on antifungal susceptibility using UV
generated melanin-deficient mutants and an agar dilution technique showed that only for
itraconazole the minimum inhibitory concentration (MIC) for mutant strains was lower than
for the wild type, with MICs for fluconazole, amphotericin B, amorolfine, flucytosine,
terbinafine and ketoconazole being similar for melanized and non-melanized strains [82].
On the other hand, another study using a molecular approach to knock-out the WdPKS1
gene determined by time-kill assays that melanin protects E. dermatitidis from amphotericin
B and voriconazole [57].

Dimorphic Fungi

Dimorphic fungi comprise a special group of microbes that can reproduce in either a
mycelial or a yeast-like state. Usually the mycelial saprotrophic form is present at 25°C, and
the yeast-like or spherule pathogenic form is found at 37°C.
Several dimorphic fungi are also pathogenic for humans and other mammals and can
cause diseases like sporotrichosis, histoplasmosis, paracoccidioidomycosis, blastomycosis,
coccidioidomycosis and penicilliosis.

Sporothrix Schenckii
Sporothrix schenckii in its saprophytic stage or when cultured at 25ºC is composed of
hyaline, septate hyphae with conidiogenous cells arising from the undifferentiated hyphae that
form conidia in groups of small, clustered denticles. Often, brown thick-walled conidia arise
alongside the hyphae. Macroscopically filamentous colonies are smooth and wrinkled, white
to creamy at first, but turn into brown to black after a few days, after the dematiaceous
conidia are produced. This fungus is evident in both human and animal tissue as budding
cigar shaped yeasts causing sporotrichosis, a subcutaneous mycosis commonly acquired by
traumatic implantation of the fungus into the skin [83]. Recently, S. schenckii was found to be
a complex of species that have morphological, physiological and molecular differences. The
new described species are Sporothrix brasiliensis, Sporothrix mexicana, Sporothrix globosa
and Sporothrix luriei [84, 85].
Both morphological forms of S. schenckii have the ability to synthesize melanin. Melanin
production on S. schenckii dematiaceous conidia occurs through the DHN pathway [86].
Macroscopicaly, only the mycelial phase of the fungus is melanized, however melanin
production on yeast cells has been demonstrated in vitro and during infection [87]. Recently,
our group has shown that S. schenckii can also produce melanin, both on filamentous and
yeast forms, using phenolic compounds such as L-DOPA as a precursor. Of particular interest
is that on the fungal filamentous form, only conidia form melanin by the DHN pathway.

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Nevertheless, if L-DOPA is accessible during fungal growth, hyphae can be melanized as

well (Figure 8) [88].

Figure 8. Melanin particles of mycelial forms of Sporothrix schenckii strain IPEC 18782A grown on
minimal medium with (left) and without (right) L-DOPA. Bars, 10µm.

Melanization of S. schenckii has been proposed as a virulence characteristic originating in

response to interactions with environmental predators. Yeast cells of S. schenckii when
ingested by Acantamoeba castellanii, a free-living soil amoeba, are able to survive within the
protozoan after ingestion, and are capable of killing the amoeba and using it as a source of
nutrients. This behavior is not shared by pathogenic fungi that do not have the soil as habitat,
such as Candida albicans or by non primary pathogenic fungi such as the yeast
Saccharomyces cerevisiae [29].
In view of the fact that S. schenckii is a soil-habiting fungus that does not require host
parasitism to complete its life cycle, fungal melanization must also play an important role for
survival in response to unfavorable environmental conditions, since the fungus is mycelial in
nature [87].
Environmental sources that enhance melanization might promote this virulence factor,
contributing to the success of possible encounters between the organism and a host [89]. In
vitro studies show that the melanization process in S. schenckii can be enhanced by several
factors such as temperature, pH and nutrient conditions [88]. Moreover, similar culture media
from different suppliers can yield differences in melanization within a single S. schenckii
strain [90].
Conidial melanization enhances their resistance to phagocytosis by macrophages [86].
Melanization also has a role in the pathogenesis of cutaneous sporotrichosis, since pigmented
wild type S. schenckii has a greater invasive ability than an albino mutated strain in a rat
experimental model of sporotrichosis. The albino strain also was restrained in the core of
granulomas, whereas the melanized strain produced multifocal granulomas [91]. Interestingly,
a laboratory worker who handled large numbers of both pigmented and albino strains of S.
schenckii developed cutaneous sporotrichosis with a dematiaceous strain [92].
Historically, S. schenckii melanization in vivo was suspected based on the identification
of a brown halo on the yeast cell wall by tissue staining with Fontana-Masson, a
histopathological technique originally used to demonstrate melanin on C. neoformans [93].
More recently, melanization has been confirmed by the findings that Sporothrix melanin
ghosts can been isolated from tissues of infected animals and antibodies to melanin have been
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170 Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk et al.

detected in sera from human patients with sporotrichosis [87, 88]. These sera have antibodies
reacting preferentially against melanin derived from L-DOPA rather than DHN-melanin [88].

Histoplasma Capsulatum
Histoplasma capsulatum is the anamorphic form of Ajellomyces capsulatum. A.
capsulatum is a heterothallic fungus with two distinct mating types: (+) and (-) [94]. Although
there historically have been three H. capsulatum varieties (H. capsulatum var. capsulatum,
the etiological agent of classic histoplasmosis, a cosmopolitan fungal infection with areas of
high endemicity; H. capsultaum var. duboisii, the etiological agent of African histoplasmosis;
and H. capsulatum var. farciminosum, the etiological agent of epizootic lymphangitis of
horses and mules [95]), recent phylogenetic work has demonstrated significant molecular
interdispersion leading to the suggestion that rather than assigning strains to a variety that we
recognize instead the existence of genetically distinct geographical populations or
phylogenetic species [96]. In immunocompetent individuals, most primary infections result in
mild or asymptomatic respiratory disease, however there is a broad spectrum of clinical
manifestations of histoplasmosis, ranging from a self-limited pulmonary infection that
resolves without treatment to chronic pulmonary infection to widespread disseminated lethal
disease [97].
The filamentous form of H. capsulatum occurs at temperatures below 35ºC or in the
environment. This form is composed by hyaline septate hyphae that produce two different
hyaline asexual reproduction structures: round to pear-shaped microconidia and large, thick-
walled, round macroconidia. These macroconidia are typically tuberculate, knobby or with
short cylindrical projections, though they occasionally may be smooth. In parasitism or when
cultivated at 37ºC in specific enriched media, H. capsulatum forms small hyaline ovoid yeast
cells with a narrow base at the smaller end [98].
In both saprophytic and parasitic stages, H. capsulatum must face assorted challenging
environmental conditions. In response, this dimorphic fungus produces several molecules
with biological activities such as siderophores to survive iron starvation, catalase to survive
oxidative stress conditions and orotidine 5-monophosphate pyrophosphorylase to endure
uracil limitation [99]. Moreover, this fungus produces melanin on both conidia and yeast cells
despite the production of hyaline structures on both morphological phases [100].
Melanization has been associated with the pathogenesis of histoplasmosis since 1962,
when it was observed that brown phenotype filamentous H. capsulatum colonies were more
virulent in a rabbit infection model than the albino phenotypes of H. capsulatum [101]. In its
filamentous form, H. capsulatum can perform de novo melanogenesis, thus probably it
produces DHN melanin on these structures. Since H. capsulatum conidia synthesize melanin
in the absence of exogenous phenolic substrate, it is probable that conidia are melanized in
the environment, a theory supported by the observation that melanin production genes are
induced in the mycelial phase of fungal growth [102]. Thus, melanization may protect the
conidia from environmental insults. Melanization of the yeast form, where a laccase-like
enzymatic activity has been observed, requires compounds such as L-DOPA, (-)-epinephrine
or phenolic compounds present on brain heart infusion medium [100]. Yeast melanization
appears to contribute to virulence by reducing H. capsulatum susceptibility to host defense
mechanisms and the antifungal drugs amphotericin B and capsofungin [53, 103]. Moreover,
yeast L-DOPA melanin can elicit an antibody response in mice infected with H. capsulatum
[100].

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Paracoccidioides Brasiliensis
Paracoccidioides brasiliensis is the dimorphic fungus agent of paracoccidioidomycosis,
the main systemic mycosis on Latin America and a fungal infection that initiates with the
transition of the inhaled infective P. brasiliensis conidia into the lungs. Upon inhalation, the
conidia transform into peculiar yeast-like cells with multiple buds into the lungs. Subsequent
dissemination to other organs may occur, giving rise to secondary lesions on the skin, lymph
nodes and adrenal glands preferentially [104].
Melanin or melanin-like pigments can be found in conidia and yeast of P. brasiliensis.
Treatment of conidia with proteolytic and glycolitic enzymes, denaturant and hot
concentrated acid results in a the isolation of particles retaining the size and shape of the
original conidia [105]. Since the conidia are obtained from a culture medium with only water
and agar, conidia are thought to produce DHN-melanin.
The first analyses of P. brasiliensis yeast using Fontana Mason staining indicated that
they were not melanized, in contrast to the positive staining for melanin in a related fungus,
Lacazzia loboi, the etiologic agent of lobomycosis [106]. However, a more recent study has
shown that, in the presence of L-DOPA, yeast cells in agar darken after 8 days, with a dark-
brown pigment in the cytoplasm and in the cell wall. These cells also yield dark particles after
enzymatic, denaturant and hot-acid treatments, which react with antibodies to melanin and
produced the characteristic free radical signal of melanin by ESR spectroscopy [105]. P.
brasiliensis melanin is located external to the cell wall [103]. Although a laccase-like activity
has been demonstrated on cytoplasmic yeast extracts of P. brasiliensis by two different
methods [105, 107], the enzymatic pathway to synthesize melanin has not yet been
established.
Melanin is synthesized in vivo by P. brasiliensis, as demonstrated by the recognition of
yeast cells by melanin-binding antibody, by the recovery of dark particles in infected tissues
[105] and by the observation that melanin synthesis genes (e.g., tyrosinase gene) are up
regulated during a mouse model of systemic infection [108]. Actually, this tyrosinase over
expression and the aromatic L-amino acid decarboxylase expression when this fungus is in
contact with human plasma [109] are strong evidences of eumelanin production by P.
brasiliensis yeast through L-DOPA or other phenolic compounds during parasitism. The
functions of these pigments have been associated with protection from the fungicidal and
fungistatic effects of phagocytic cells as well as from the antifungal drugs amphotericin B,
ketoconazole, fluconazole and itraconazole and also the sulfonamide antibiotic
sulfamethoxazole [107]. The resistance to phagocytosis is in part due to the protective effect
of melanin against nitric oxide and other reactive oxygen species, such as hypochlorite and
hydrogen peroxide [110]. Finally, nonmelanized P. brasiliensis yeast are less pathogenic than
melanized yeast cells [110].

Other Dimorphic Fungi

Blastomyces dermatitidis is an endemic dimorphic fungal pathogen found in central USA
that is the etiological agent of blastomycosis, a systemic mycosis that ranges in disease
manifestations from asymptomatic cases to fatal pneumonia in immunocompetent individuals
[111]. B. dermatitidis is mycelia in the environment and produces yeast cells of 8-10μm in
diameter that display broad-based budding. Coccidioides posadasii and its relative species C.
immitis are endemic to the USA, Mexico and desert and semiarid areas in Central and South
America. They grow as filamentous form in soils and, after inhalation of the infective
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172 Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk et al.

arthroconidium by a mammal host, they convert to a peculiar spherule phase in the lungs,
causing coccidioidomycosis, an infection that may be asymptomatic or result in an atypical
pneumonia in more than 50% of the immunocompetent infected individuals [112].
Penicillium marneffei is a dimorphic fungus restricted to the Southeast Asia that causes
infections in both immunocompetent and immunodeficient individuals, although penicilliosis
caused by P. marneffei in the non-HIV infected populace is extremely unusual [113].
These three fungi produce hyaline structures when observed by bright field microscopy,
however their conidia, after treatment with enzymes, denaturant and hot concentrated acid
result in melanin ghosts retaining the original size and shape from the intact original conidia.
Also, melanin or melanin-like pigments are observed on their parasitic phases both in vitro
and in vivo with the techniques developed to study melanization described earlier in this
chapter for other fungi [114-116]. P. marneffei possesses a DHN melanin-biosynthesis gene
cluster [117], but the pathways to synthesize melanin in B. dermatitidis and C. posadasii have
not been elucidated, although they do not require exogenous phenolic compounds to
melanize. However, C. immitis has a putative gene with 80% similarity to a laccase from
Botrytis cinerea and to the enzyme Lac2 of C. neoformans [38]. In B. dermatitidis, melanin
reduces susceptibility to amphotericin B, but not to voriconazole or itraconazole [114].
Melanin has been posited to play a role in the virulence of B. dermatitidis, C. posadasii and
P. marneffei, thus affecting their pathogenesis.

Candida Albicans and Other Yeasts

Several members of the genus Candida are commensal microorganisms in humans and
other mammalians, co-existing with the host without any overt damage. This balance can be
broken, however, if the defense mechanisms of the host are compromised [118]. The
polymorphic fungus Candida albicans is the major agent of candidemia and candidiasis
worldwide. This species is characterized by germ tube and chlamidospore production and has
morphological, genetic and carbohydrate assimilation profiles that permits distinguishing
from other species within the genus [119].
C. albicans was long believed to be a non-melanin producer, and was used as a negative
control in several experiments on melanin synthesis [22, 45, 100, 105, 107]. However, it has
now been shown that this yeast produces melanin in vitro and during infection. Melanin
particles extracted from C. albicans yeast cells, unlike the other fungi described in this
chapter up to now, does not retain the shape and size of the original cells, presenting a quarter
of the size of the initial yeasts. These melanin particles were obtained from yeast cells both in
vitro and in vivo, but hyphae do not yield melanin. Other peculiar aspect about C. albicans
melanization is that the small spheres of melanin obtained do not accumulate beneath the cell
wall, being more similar to melanosome structures [120]. More recently, it has been
demonstrated that these melanin bodies are produced when the fungus is incubated in medium
containing L-DOPA as a substrate and this melanin is externalized from the fungal cells in a
chitin-dependent mechanism, where the product of the CHS3 gene, short chitin rodlets, is
required for melanin externalization and the product of the CHS8 gene, long chitin
microfibrils, impairs the process [121]. It is due to these unusual aspects of C. albicans
melanization that led earlier studies to conclude that this species was a non-melanin producer.
Melanin production was also observed in Candida glabrata and Candida famata when
cultured in the presence of L-DOPA [122]. Another yeast species, Yarrowia lipolytica,
produces a black pigment that results from the extracellular accumulation and oxidation of an

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intermediate of tyrosine catabolism [15]. This pigment was shown to be pyomelanin, formed
by the oxidation and polymerization of homogentisic acid accumulated on the culture medium
during fungal growth [123]. Y. lipolytica is also able to convert L-tyrosine to L-DOPA,
producing small amounts of melanin after the process [124].
However, no association between melanization and virulence has been defined for C.
albicans [121] or other hemiascomycete yeasts. C. albicans yeast cells secrete complex
polymers into biofilm structures that alter antifungal susceptibilities [125], and melanin may
play some role in this process, since it is externalized by this yeast [120].

Aspergillus
Members of the genus Aspergillus are among the most abundant and widely distributed
organisms in the world. Several of them produce metabolites with diverse applications. Many
Aspergillus species have the ability of degrade agricultural products and some cause a disease
known as aspergillosis in immunocompromised hosts, especially in patients receiving
chemotherapy or with cystic fibrosis [126]. Melanin production by species of the genus
Aspergillus was first reported in 1969, when melanization of A. nidulans was detected in
batch and chemostat cultures [127, 128]. This fungus is one of the most important species for
studying eukaryotic cell biology [129] and its melanin has been shown to have an antioxidant
activity [130].
A. fumigatus is the major clinically relevant fungal pathogen, being the main etiological
agent of human and animal aspergillosis. Although this fungus lacks some virulence traits
present in other fungal species, A. fumigatus is able to successfully establish infection in
immunosupressed patients due to its virulence factors and modulation of innate and adaptive
immunological responses [131]. The putative virulence factors of A. fumigatus include
secretion of hydrolytic enzymes and toxins, such as gliotoxin, the presence of extra-cellular
matrix adhesion molecules on cell surface, and the production of pigments [132].
The DHN-melanin synthesis pathway in A. fumigatus is very well characterized [13,
133]. Biosynthesis of this type of melanin requires the products of six different genes, located
in a cluster that is expressed during fungal conidiation. For this reason, A. fumigatus produce
DHN melanin only in the conidia as demonstrated by the methods used to generate melanin
ghosts [134]. The first characterized gene of this pathway is named arp1 and its expression
yields a scytalone dehydratase, an enzyme that converts scytalone on 1,3,8-THN [135].
Another important gene in this pathway is alb1 whose transcript is a polyketide synthase
characterized latter as a naphthopyrone synthase [136, 137]. Mutations on this gene leads to
an albino conidial phenotype [136]. The abr2 gene is also characterized and codes for a
laccase that is not essential for virulence, indicating that the intermediates of the DHN
pathway confer some scavenging activity to reactive oxygen species [138].
DHN-melanin plays an important role in the pathogenesis of aspergillosis. In vitro
experiments show that melanin protects the fungus against phagocytosis and decreases its
susceptibility to reactive oxygen species produced by phagocytic cells, such as alveolar and
monocyte-derived macrophages and neutrophil granulocytes [136, 139, 140]. A. fumigatus
melanin also impedes apoptosis pathways, contributing to fungal dissemination within the
host [141]. The melanin interferes with cellular responses to some fungal antigenic ligands
[142]. Additionally, the melanin has an indirect effect on the pathogenesis of aspergillosis, as
it allows for the correct assembly of the cell wall layers of conidia, thus permitting correct
expression of laminin adhesins and other virulence factors at the conidial surface [13].
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174 Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk et al.

Together, these results may explain the findings of a lower virulence of alb1 mutants of A.
fumigatus in a mouse model of disseminated aspergillosis [143]. Attachment of melanin-
binding antibodies to conidia within tissue sections from patients with nasal aspergilloma also
suggests that melanization occurs during infection [134]. Melanin is produced by A.
fumigatus growing in biofilms [144, 145]. Interestingly, DHN-melanin appears to be a
specific virulence factor of A. fumigatus in mammalian disease, since strains with mutations
in the genes of the DHN-melanin synthesis cluster are more virulent than the wild type strain
in an insect model of Aspergillus infection using Galleria mellonella as the host [146].
Pyomelanin has been described in A. fumigatus. In the presence of L-tyrosine or L-
phenylalanine, this species expresses enzymes related to the degradation of these amino acids,
leading to the production of pyomelanin, with homogentisic acid as the major intermediate of
the pathway. This pigment probably protects the germlings and hyphae of A. fumigatus from
the oxidative products of neutrophils [16]. This species can also utilize tyrosine and DOPA
melanins as sole carbon sources, leading to the production of a third type of melanin, a fungal
allomelanin, that turns typically pale mycelia dark [147]. Whether allomelanin production
occurs in vivo is not known and no role for this type of melanin is established in the
pathogenesis of aspergillosis.
A. niger is another species that causes aspergillosis. Melanin ghosts have been extracted
from its conidia and they have the ability to activate the alternative complement pathway
[47]. In an elegant study, the melanin contents of A. niger from two environments at Mount
Carmel, Israel, receiving different levels of solar radiation was measured and showed that
isolates with higher levels of solar radiation have higher melanin concentration and resisted
long wavelength UV radiation better than the lower radiation treatment [148]. A polyketide
synthase gene, albA, from A. niger has been characterized and it is an ortholog of alb1 gene
of A. fumigatus, responsible for the production of melanin and other naphtho--pyrone family
of polyketides [149], confirming the capacity of this fungus to produce DHN melanin.

Other Human Pathogenic Fungi

Pneumocystis jirovecii is a peculiar fungus that is unable to grow in vitro, which grows in
a yeast-like from in vivo and causes a life-threatening pneumonia in immunocompromised
humans. Pneumocystis carinii, a closely related species that is able to cause infection in rats,
is able to produce melanin at its cell wall, as shown by the generation of melanin ghosts from
microorganisms isolated from the lungs of infected rats and by labeling of cells in tissue
sections by melanin-binding antibodies [150]. Subsequent to the demonstration of melanin in
P. carinii in rats, melanin has also be demonstrated in Pneumocystis isolated from mice and
ferrets, as well as for P. jiroveci in patient biopsy specimens [151]. Additionally, melanized
Pneumocystis are less susceptible to UV irradiation or desiccation compared to non-
melanized yeasts, suggesting a role for melanin against a range of stressors [151].
Scytalidium dimidiatum is a pigmented dematiaceous coelomycete that typically causes
chronic superficial skin diseases and onychomycosis, but sometimes also causes deeper
infections, such as subcutaneous abscesses. This fungus produces melanin on hyphae and
artroconidia. In vivo melanization of S. dimidiatum is supported due to the detection of
melanin in the skin of a patient with subcutaneous disease. Interestingly, S. hyalinum, a
species with similar morphology to S. dimidiatum, but does not produce pigmented
mycelium, yielded no dark particles after treatment with denaturant and hot acid, supporting
the theory that S. hyalinum is an albino mutant of S. dimidiatum [152].

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The dermatophytes are a group of fungi classified in three anamorphic genera

(Epidermophyton, Microsporum and Trichophyton) that have the ability to invade keratinized
tissues to cause dermatophytosis (or ringworm). Most recently, melanin production has been
demonstrated in the microconidia and macroconidia of T. rubrum, T. mentagrophytes, E.
floccosum and M. gypseum [153]. Also, these species produce melanin or melanin-like
pigments during infection [153]. Therefore, melanin may be a putative virulence factor for
dermatophytes, as for other pathogenic fungi.
Madurella mycetomatis is the main agent of black grain eumycetoma. Melanization of M.
mycetomatis occurs on both hyphae and grains, presumably by the DHN and pyomelanin
pathways. L-DOPA has been shown to be toxic to this species. Melanization appears to be
involved in fungal morphogenesis as M. mycetomatis cells cultured in the presence of melanin
inhibitors are longer, less branched and slimmer than the melanized phenotype. Other
functions of melanin in this fungal species include protection against oxidant compounds and
to the antifungal agents itraconazole and ketoconazole, the main antifungal drugs used in the
treatment of eumycetomas [154].
Melanization has also been described in other medically important fungi, such as the
yeasts of the genus Malassezia [155], Hortaea werneckii [156], Lacazzia loboi [106] and the
opportunistic fungal pathogens Paecilomyces lilacinus, Scedosporium prolificans, Curvularia
lunata and Alternaria alternata [157-160]. In H. werneckii melanin appears to be responsible
for the reduction in the permeability of the cell wall to glycerol, which might be one of the
features that facilitates the osmotic adaptation of this halophilic fungal species [156].
Biological functions for melanin during infection caused by these species, however, are not
established and more studies are necessary to determine their impact on pathogenesis.

Phytopathogenic Fungi
Given that melanin is able to protect fungi against numerous environmental stresses, it is
not surprising that melanization occurs in phytopathogenic fungi.
Melanin synthesis related enzymes, especially polyketide synthase genes, are abundant in
fungal genomes and are more abundant in phytopathogenic ascomycetes than in saprobic
fungi [161]. DHN-melanin is required for pathogenicity of fungi that produce pigmented
appressoria, such as Colletotrichum lagenarium and Magnaporte oryzae. The cell wall
melanin protects and stabilizes these fungi against the enormous pressures required to build
and release appressoria that enable the pathogen to penetrate plant leaves [162, 163].
Gaeumannomyces graminis var. tritici is hyaline in culture, however, pathogenic strains
invade host roots with melanized macrohyphae, and dark infection cushions that are
composed of clustered hyphopodia, which are appressorium-like structures, except that they
come from the vegetative hyphae [164].
Interestingly, several phytopathogenic fungi do not require melanin as a virulence factor,
as albino mutants can be as pathogenic as wild type strains. Melanization is thought to have
an indirect effect on the virulence of these fungi, protecting them from environmental insults
and thus positively selecting for them in nature [164].
For instance, Ascochyta rabiei, which causes infection of chickpeas, produces DHN-
melanin in pycnidia and sexual fruting bodies, but it does not augment plant infection;
however this pigment is able to protect the reproduction cells within the fruting bodies from
UV light [165]. Additionally, Bipolaris oryzae induces expression of the 1,3,8-THN reductase
gene involved in melanin biosynthesis upon exposure to ultraviolet radiation [166]. Melanin-
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176 Rodrigo Almeida-Paes, Joshua Daniel Nosanchuk et al.

deficient mutants of Monilinia fructicola yield lesions on fruit, but their conidia are
vulnerable to high temperature, desiccation, freezing, UV, mechanical pressure and hydrolytic
enzymes [164].

Implications for Human Health

As described in this chapter, fungal melanins are important virulence factors for leading
human fungal pathogens. Melanins contribute to fungal virulence through diverse
mechanisms, both directly impacting the fungus’ capacity to protect their cellular structures
and functions and by modifying host effector responses. However, our understanding of the
physicochemical properties and biological functions of melanins sets the stage for our being
able to directly therapeutically target these amorphous polymers or interfere with their
synthesis.
Since melanins are important for the pathogenesis of several fungi, disrupting their
synthesis should be an interesting mechanism to combat pathogenic fungi. Glyphosate is a
widely used herbicide that inhibits growth of several microbes [167] and also interferes with
C. neoformans melanization by direct inhibition of the autoxidation of L-DOPA, oxidation of
epinephrine and consequently melanin polymerization [55]. This drug has therapeutic effects
in mice systemically infected with C. neoformans. Administration of glyphosate to infected
mice prolonged animal survival and reduced lung fungal burdens [55]. Melanin affects lung
inflammatory reaction during cryptococcal infection by eliciting high levels of interleukins
and greater numbers of leukocytes [168] and interestingly glyphosate treatment lowered
inflammation in mice lungs, where only a few defective melanin ghosts were observed [55].
Other indirect evidence that treatment with drugs that block melanin synthesis results in
better outcomes for fungal infections is the fact that voriconazole, a broad-spectrum triazole
that inhibits cytochrome P450 dependent 14α lanosterol demethylation and is highly active
against C. neoformans [169, 170], inhibits melanization by a direct interaction with the fungal
laccase that inhibits the enzymatic synthesis of melanin [171]. Therefore, fungal melanin-
synthesis pathways appear to be promising new targets for antifungal design. Recently, a new
compound ptilomycalin A, a spirocyclic guanidine alkaloid extracted from the marine sponge
Monanchora arbuscula that acts synergistically with amphotericin B, has been shown to
suppress melanogenesis in C. neoformans by functioning as a potent laccase inhibitor [172].
Another important observation regarding melanin-inhibiting compounds as treatments is the
fact that passive immunization with melanin-binding monoclonal antibodies prolonged
survival and reduced the C. neoformans fungal burden on infected mice [49]. Hence, targeting
melanin or melanin synthesis appears to be an excellent approach to combat melanotic fungi.
Actually, the inhibitory effects of antifungal drugs on melanin synthesis can be utilized
also in the treatment of non-fungal diseases. Miconazole, an imidazole antifungal drug of
topic use commonly used to treat cutaneous fungal infections, inhibits tyrosinase activity and
tyrosinase expression in B16 melanoma cells, slowing melanin biosynthesis and, therefore,
may have beneficial effects in the treatment of hyperpigmentation disorders such as melasma
and ephelis [173]. On the other hand, amphotericin B induces de novo synthesis of tyrosinase
by neuroretinal cells, allowing these cells to produce melanin [174], suggesting that different
antifungal drugs may have antagonist roles on melanin production by animal cells.

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Melanized fungi inhabit some remarkably extreme environments on the planet, including
Arctic and Antarctic regions and the walls of the damaged reactor at Chernobyl, where they
are exposed to a high and constant radiation [175]. Melanized fungi can display increased
growth relative to non-melanized cells after exposure to ionizing radiation [176], because
chemical composition, free radical quenching and spherical spatial arrangement of melanins
protects the fungi from the radiation and the energy absorbed through these interactions can
fuel fungal growth [177]. These observations led to the creation of rationally designed
melanins as novel radioprotectors, that were able to protect mammalian cells against ionizing
radiation of different energies [178]. In fact, melanin-covered nanoparticles offered protection
of bone marrow from ionizing radiation during external radiation or radioimmunotherapy,
whereas no tumor protection by these unencapsulated melanins was observed [179].
Radiolabeled melanin-binding peptides were also successfully studied in the treatment of
melanoma [180]. Another way to exploit the resistance of melanized fungi to radiation is their
use in bioremediation of radioactively contaminated sites and the cleanup of industrial
effluents [181].
Melanins are able to chemically bind diverse compounds especially those used in several
therapies, such as antifungals, antibiotics, antipsychotic and antineoplasic drugs [53, 154,
182, 183]. In fact, the capacity of melanin to adsorb a vast variety of compounds is similar to
that of medicinal activated charcoal [6].
A. fumigatus has the ability to degrade melanin [147]. Studies on cosmetic development
have used melanin degrading extracts isolated from A. fumigatus and S. cerevisiae. These
extracts can significantly reduce UVB induced pigmentation of human skin, suggesting the
usefulness of these extracts in the development of new whitening cosmetics to modify skin
color and tone [184].

CONCLUSION
Melanins are important virulence factors for several human and plant fungal pathogens.
Virtually all fungi produce melanin under specific growth conditions and several important
pathogens can synthesize melanin in the absence of phenolic or other substrates. Interestingly,
some fungi accumulate melanin on their conidia, others on conidia and hyphae, some only in
the yeast form, and a few secrete melanin to the external medium. In general, DHN melanin is
produced during fungal growth in the environment, whereas melanins derived from L-DOPA
or tyrosine appear to be preferentially (but not exclusively) expressed during pathogenic
stages of fungal growth where they can interact with the immune responses of the host.
Despite increasing fungal pathogenicity, melanins are interesting targets for new drug
development and treatment strategies for fungal infections and their properties allow the use
of melanins as adjuvants in other diseases, especially cancer.

REFERENCES
[1] Nosanchuk JD, Casadevall A. The contribution of melanin to microbial pathogenesis.
Cellular microbiology. 2003 Apr;5(4):203-23.
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 6

THE COAT COLOR GENES REGULATE EUMELANIN

AND PHEOMELANIN SYNTHESIS IN MELANOCYTES

Tomohisa Hirobe
Radiation Risk Reduction Research Program, National Institute of Radiological Sciences,
Chiba, Japan

ABSTRACT
In mice, eumelanin and pheomelanin synthesis is regulated by numerous coat color
genes. Eumelanin and pheomelanin contents were measured in cultured melanocytes and
in the epidermis/dermis and hairs of C57BL/10JHir (B10) and its congenic mice carrying
the coat color genes. Eumelanin contents in agouti and dilute melanocytes are similar to
black melanocytes, whereas the contents in brown, pink-eyed dilution, slaty and ruby-eye
2d melanocytes are reduced to one third~one thirthieth. In contrast, pheomelanin contents
in agouti, dilute, slaty and ruby-eye 2d melanocytes are similar to its content in black
melanocytes, whereas the content in brown melanocytes is increased. Eumelanin and
pheomelanin contents in cultured epidermal melanocytes correlate well with those in skin
and hair of the congenic mice, except that agouti melanocytes do not synthesize
pheomelanin in culture, the pink-eyed dilution allele does not affect pheomelanin content
in hairs, and the ruby-eye 2d allele increases pheomelanin content in hairs. These results
suggest that eumelanin and pheomelanin synthesis in melanocytes is regulated by
numerous coat color genes in a complicated manner.

Keywords: Melanoblast/melanocyte/coat color gene/melanocyte-stimulating hormone

INTRODUCTION
Melanocytes are neural crest-derived cells that synthesize melanin pigments (Rawles,
1947; Mayer, 1973; Hearing, 1993, 2000; Ito, 2003). Undifferentiated precursors of
melanocytes, melanoblasts, are derived from neural crest cells in embryonic skin (Rawles,


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192 Tomohisa Hirobe

1947; Mayer, 1973; Steel et al., 1992; Aoki et al., 2009; Motohashi et al., 2011).
Melanoblasts invade the epidermis (Mayer, 1973) and colonize at the same place. Mouse
epidermal melanocytes are known to differentiate from melanoblasts around the time of birth
(Hirobe, 1984a). Fully differentiated melanocytes are characterized by pigmentation and well-
developed dendrites and can be seen mainly in hair bulbs of the skin where they secrete
mature melanosomes into surrounding keratinocytes giving rise to melanized hairs (Mann,
1962; Slominski and Paus, 1993; Hirobe, 1995; Peters et al., 2002). Hair bulb melanocytes
are derived from epidermal melanoblasts and melanocytes (Hirobe, 1992a). Epidermal
melanocytes are found only during the early weeks after birth in the hairy skin of mice
(Hirobe, 1984a). In glabrous skin, such as the ear, nose, foot sole and tail of mice, epidermal
melanocytes are also found even in adult mice (Quevedo and Smith, 1963).
Melanin synthesis is mainly controlled by tyrosinase (Tyr), Tyr-related protein 1 (TRP1,
Tyrp1) and TRP2 (Tyrp2, dopachrome tautomerase (Dct); Hearing, 1993, 2000; Ito, 2003; Ito
and Wakamatsu, 2011). Tyr initiates melanin synthesis by catalyzing oxidation of L-tyrosine
(tyr) to dopaquinone (Cooksey et al., 1997). Tyrp1 possesses 5,6-dihydroxyindole-2-
carboxylic acid (DHICA) oxidase activity (Jackson et al., 1990). In contrast, TRP2 possesses
dopachrome tautomerase (Dct) activity (Jackson et al., 1992; Tsukamoto et al., 1992;
Kroumpouzos et al., 1994), which converts dopachrome (DC) to DHICA (Korner and
Pawelek, 1980). Melanocytes produce two types of melanin: brownish-black eumelanin and
yellow-reddish pheomelanin (Ito, 1993, 2003; Ito and Wakamatsu 2011). Although
differences exist in molecular size and general properties, these melanins arise from a
common metabolic pathway in which dopaquinone is a key intermediate (Ito and Prota, 1977;
Hearing and Tsukamoto, 1991; Ito and Wakamatsu, 2008, 2011).
Melanin synthesis occurs in specialized organelles called melanosomes (Seiji et al.,
1963). Melanosome maturation is categorized into four stages: stages I and II include
unmelanized immature premelanosomes, while melanized melanosomes are classified into
stages III and IV (Fitzpatrick et al., 1969). In mice, coat colors are regulated by melanosome
transfer from melanocytes to neighboring keratinocytes in hair bulbs (Silvers, 1979; Hirobe,
1995). Melanosomes are produced in varying sizes, numbers and densities in melanocytes.
Melanosomes in hair bulb melanocytes are passed on to the hair shaft where the final
distribution patterns of the pigment are determined. This distribution plays an important role
in determining the coat coloring of mice (Silvers, 1979). Eumelanin-containing melanosomes
(eumelanosomes) are elliptical with longitudinal depositions of pigments in intraluminal
fibrils (Hearing et al., 1973; Sakurai et al., 1975; Hirobe and Abe, 1999). In contrast,
pheomelanin-containing melanosomes (pheomelanosomes) are spherical with granular
depositions of pigments in multivesicular bodies found in yellow phase agouti melanocytes as
well as in yellow (lethal yellow (Ay/-) and recessive yellow (Mc1re/Mc1re)) melanocytes
(Sakurai et al., 1975; Takeuchi, 1985). Thus, the differences in melanin synthesis correspond
to those in melanosome morphology.
The proliferation and differentiation of mouse melanocytes during development is
regulated by numerous epigenetic and genetic factors (Hirobe, 1992a). Epigenetic factors
from the surrounding tissue environment, such as keratinocytes (Imokawa, 2004; Hirobe,
2005, 2011a; Yamaguchi and Hearing, 2009; Kondo and Hearing, 2011) and fibroblasts
(Imokawa, 2004; Hirobe, 2011a, Yamaguchi and Hearing, 2009; Kondo and Hearing, 2011),
the blood supply of hormones and other substances from the pituitary gland and other organs
(Snell and Bishitz, 1960; Hirobe, 1996, 2011a; Hirobe and Abe, 2000; Hirobe et al., 2004a),

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 The Coat Color Genes… 193

minerals, especially iron (Hirobe, 2007, 2009a, b, c, 2011b) and environmental factors such
as ultraviolet (UV) radiation (Quevedo and Smith, 1963; Szabo, 1967; Gilchrest et al., 1996;
Hachiya et al., 2001; Naganuma et al., 2001; Furuya et al., 2002, 2009; Imokawa, 2004;;
Hirobe et al., 2002a, 2004b; Choi et al., 2010) and ionizing radiation (Chase, 1949; Quevedo
and Isherwood, 1958; Hirobe and Zhou, 1990; Hirobe, 1994a; Hirobe et al., 2004c, 2011a, b;
Inomata et al., 2009) are also important for the regulation of melanocyte proliferation and
differentiation. Among the genetic factors, semidominant genes controlling melanocyte
numbers are involved in regulating the melanocyte and melanoblast-melanocyte populations
in the epidermis of newborn mouse skin (Hirobe, 1982, 1988a, b, 1995). The coat color genes
are the most important (Silvers, 1979; Hirobe and Abe, 1999; Lamoreux et al., 2001, 2010;
Bennett and Lamoreux, 2003; Hirobe, 2011a). In mice, more than 300 genes are involved in
melanocyte proliferation and differentiation; about one half of these genes have been cloned
and their functions are clarified (Mouse Genome Informatics). However, many unknown
genes and their functions in melanocyte proliferation and differentiation still remain to be
investigated. In this chapter, studies of the control of melanin synthesis in differentiated
melanocytes by the coat color genes are reviewed and their role in melanin synthsis is
discussed in detail.

REGULATION BY THE COAT COLOR GENES

To clarify the mechanisms of the regulation of melanin synthesis by the coat color genes,
characteristics of melanin synthesis in mouse epidermal melanocytes in serum-free primary
culture of epidermal cell suspension (Hirobe, 1992b, c) were compared between two different
strains of mice that possess the same genetic background except for one allele in the topical
coat color locus, that is, comparison was made between C57BL/10JHir (B10) and its
congenic strains. In the initial stage of this serum-free primary culture of epidermal cell
suspension of B10 mice, keratinocytes proliferate well and epidermal melanoblasts and
melanocytes start to proliferate around the keratinocyte colony, and after 8-9 days
keratinocytes gradually die, then pure cultures of melanoblasts or melanocytes are obtained
after 14 days (Hirobe, 1992b, c). Pure culture of many melanocytes is obtained by
melanocyte-proliferation medium (MDMD), consisting of Ham’s F10 supplemented with 10
g/ml insulin (bovine), 0.5 mg/ml bovine serum albumin (Fraction V), 1 M ethanolamine, 1
M phosphoethanolamine, 10 nM sodium selenite, 0.5 mM dibutyryl adenosine 3’:5’-cyclic
monophosphate (DBcAMP) is used. Pure culture of numerous undifferentiated melanoblasts
is obtained by melanoblast-proliferation medium (MDMDF) consisting of MDMD
supplemented with 2.5 ng/ml bFGF (Hirobe, 1992b, 1994b). The differentiation and
melanogenesis/dendritogenesis of mouse epidermal melanocytes are induced by cAMP-
elevating agent such as -melanocyte-stimulating hormone (-MSH, Hirobe, 1992c),
DBcAMP (0.1~1 mM, Hirobe, 1992c), 3-isobutyl-1-methylxanthine (IBMX, Hirobe, 1992c)
or adrenocorticotrophic hormone (ACTH)/ACTH fragments (Hirobe and Abe, 2000).
Eumelanin and pheomelanin contents in the cultured melanocytes using MDMD as well as in
the epidermis, dermis and hairs derived from skins of congenic mice were measured and
compared with those in B10 mice, and the role of coat color genes in the regulation of
eumelanin and pheomelanin synthesis was studied.
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194 Tomohisa Hirobe

Alleles from 7 important coat color loci, namely agouti (A), brown (b/Tyrp1b), albino
(c/Tyrc), dilute (d/Myo5ad), recessive yellow (e/Mc1re), pink-eyed dilution (p/Oca2p) and
slaty (slt/Dctslt) were introduced to B10 background by repeated backcrosses in author’s
laboratory, and congenic lines of B10 mice, namely, B10-A/A, -Tyrp1b/Tyrp1b, -Tyrc/Tyrc, -
Myo5d/Myo5d, -Mc1re/Mc1re, -Oca2p/Oca2p and -Dctslt/Dctslt were prepared (Hirobe, 1986,
2011a). The ruby-eye 2d (ru2d/Hps5ru2-d) allele is a spontaneous autosomal recessive mutation
that occurred in B10 mice in my laboratory (Hirobe et al., 2011d). Studies using C57BL/6J
(B6) congenic lines (Lamoreux et al., 2010) as well as noncongenic strains are also reviewed
in this chapter.

Agouti, Mahogany, Mahoganoid and Subtle Grey

Although there are a number of loci which affect melanin synthesis in mice, two major
loci is known to control the nature of the pigment formed. Namely, the numerous alleles of
the agouti and extension loci are involved in regulating the relative amount and distribution of
pheomelanin in hairs of the coat. In the coat color of wild type (A/A) mice, individual hairs
possess a subterminal yellow band in otherwise black. This phenotype is called agouti pattern.
The agouti pattern formation is altered by genic substitutions at the agouti locus (Sakurai et
al., 1975). Animals homozygous for the a allele produce black eumelanin only (Silvers,
1979). The switch between eumelanin and pheomelanin synthesis is regulated by -MSH and
agouti protein or agouti signaling protein (Asip), the product of the A allele expressed in the
hair bulb (Barsh, 1996). The Asip is produced and released from dermal papilla cells in the
hair bulb. A recent study showed that loss and gain of function of -catenin in dermal papilla
cells resulted in yellow and black mice, respectively. In addition, -catenin activity in dermal
papilla cells regulates melanocyte activity (eumelanogenesis) via a mechanism that is
independent of the Asip (Enshell-Seijiffers et al., 2010).
These results suggest that -catenin plays an important role in the agouti pattern
formation as well as in eumelanogenesis. Eumelanin content in agouti hairs did not differ
from black mice, but pheomelanin content in agouti hairs increased dramatically (Table 1).
It appears that no influence of the genetic background in the content of eumelanin and
pheomelanin in agouti hairs, since no difference in the content was observed between B10
and B6 mice (Table 1).
These results suggest that -catenin plays an important role in the agouti pattern
formation as well as in eumelanogenesis. Eumelanin content in agouti hairs did not differ
from black mice, but pheomelanin content in agouti hairs increased dramatically (Table 1).
It appears that no influence of the genetic background in the content of eumelanin and
pheomelanin in agouti hairs, since no difference in the content was observed between B10
and B6 mice (Table 1).

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Table 1. Effects of the coat color genes on eumelanin and pheomelanin synthesis in cultured melanocytes and in the
epidermis, dermis and hairs of mice

Gene Strain Eu Eu Eu Eu Eu Pheo Pheo Pheo Pheo (d) Pheo Reference

(in) (out) (e) (d) (h) (in) (out) (e) (h)
A B10 Ozeki et al., 1995; Hirobe
→ → → → → → ↑↑ ↑↑
et al., 2004d
A B6 → ↑↑ Lamoreux et al., 2001
Ozeki et al., 1995; Hirobe
Tyrp1b B10 ↓ ↓ ↑ ↑
et al., 1998
Tyrp1b
B6 ↓ ↑ Lamoreux et al., 2001
Ozeki et al., 1995; Hirobe
Tyrc B10 0 0 0 0 0 0 0 0
et al., 1998
Tyrc-2J
B6 0 0 Lamoreux et al., 2001
Tyrc-ch
B6 ↓ ↓ Lamoreux et al., 2001
d
Myo5a Ozeki et al., 1995; Hirobe
B10 → → → →
et al., 1998
Mc1re B10 ↑ → ↓↓ ↓↓ ↓↓ ↓ → ↑ ↑↑↑ ↑↑↑ Hirobe et al., 2007a, b

Mc1re B6 ↓↓ ↑↑ Ozeki et al., 1995

Oca2p B10 ↓↓ → ↓↓↓ ↓ → → Hirobe et al., 2003, 2011c

Dctslt B10 ↓ ↓ ↓ ↓↓ ↓ → ↑ ↓ ↓ → Hirobe et al., 2006

Dctslt B6 ↓ → Ozeki et al., 1995

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Table 1. (Continued)

Gene Strain Eu Eu Eu Eu Eu Pheo Pheo Pheo Pheo (d) Pheo Reference

(in) (out) (e) (d) (h) (in) (out) (e) (h)
Dctslt-lt B6 ↓ ↑ Lamoreux et al., 2001
Hirobe, 2011a; ; Hirobe et
Hps5ru2-d B10 ↓ ↑ ↓↓ ↓↓↓ ↓ → → ↓ ↓ ↑
al., 2011d
Pmel17si B6 → → Lamoreux et al., 2001

Ay B6 ↓↓↓ ↑↑ Lamoreux et al., 2001

Effects of the coat color genes on eumelanin (Eu) and pheomelanin (Pheo) synthesis in melanocytes (B10 congenic mice) cultured in
MDMD for 14 days as well as in the epidermis (e), dermis (d) and hairs (h; 5-week-old) of B10 or B6 congenic mice. PTCA and
AHP (or 4-AHP) were measured in cultured melanocytes (Eu (in), Pheo (in)) and in culture supernatant (Eu (out), Phe (out)), and,
in addition, in the epidermis (Eu (e), Pheo (e)), dermis (Eu (d), Pheo (d)) and hairs (Eu (h), Pheo (h)) as described in the text. →, no
effects; ↑, slightly increased (~×10); ↑↑, increased (~×100); ↑↑↑, greatly increased (~×1000);↓, slightly decreased (~×1/10); ↓↓,
decreased (~×1/100); ↓↓↓, greatly decreased (~×1/1000). Effects of the coat color genes were compared with control melanocytes
(B10 mice) cultured in MDMD and with control epidermis (B10), dermis (B10) and hairs (B10 or B6) of mice.

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 The Coat Color Genes… 197

The number of melanoblasts and melanocytes in the epidermis after birth does not differ
between black and agouti mice (Hirobe and Abe, 1999). The proliferation of agouti
melanocytes cultured in MDMD is also similar to that of black melanocytes. Agouti
melanocytes exhibit normal morphology (dendritic, polygonal or epithelioid) and a similar
degree of pigmentation to black melanocytes is observed. Moreover, there is no difference in
Tyr, Tyrp1, Dct and Kit activity between black and agouti melanocytes (Hirobe, 2011a).
Melanosomes of black and agouti melanocytes are evenly distributed within the melanocytes,
and they are elliptical in morphology (Hirobe and Abe, 1999; Hirobe, 2011a).
Chemical analysis of melanin produced in cultured epidermal melanocytes revealed that
the pyrrole-2,3,5-tricarboxylic acid (PTCA, a degradation product of eumelanin; Ito and
Fujita, 1985; Ito and Wakamatsu, 1994) content in agouti melanocytes is similar to that in
black melanocytes (Hirobe et al., 2004d). Also, the 4-aminohydroxyphenylalanine (4-AHP, a
degradation product of pheomelanin; Wakamatsu and Ito, 2002; Wakamatsu et al., 2002)
content in agouti melanocytes cultured in MDMD is similar to that in black melanocytes
(Hirobe et al., 1998), as are the PTCA/AHP ratios. However, a 1.5-fold increase in AHP, and
a 37-fold increase in 5-S-cysteinyldopa (5-S-CD, a precursor of pheomelanin), was observed
in culture media derived from agouti melanocytes cultured in MDMD (Hirobe et al., 2004d).
Moreover, a 11-fold increase in AHP content in the epidermis of 3.5-day-old agouti mice and
a 95-fold increase in the epidermis of 5.5-day-old agouti mice were observed compared with
black mice (Hirobe et al., 2004d).
Analysis of the A allele using reverse transcription-polymerase chain reaction (RT-PCR)
revealed that cultured epidermal keratinocytes and melanocytes did not express the A allele.
Moreover, the Asip was expressed in the dermis of 0.5-, 3.5- and 5.5-day-old agouti mice, but
not in the dermis of black mice or in the epidermis of agouti or black mice (Hirobe et al.,
2004d). These results suggest that epidermal melanoblasts of agouti mice can be influenced
by the Asip produced in the dermis, and can continue to synthesize pheomelanin in culture
conditions. Pheomelanin production in the epidermis of 3.5- and 5.5-day-old agouti mice may
be derived from the influence of the Asip produced in the dermis.
The master regulator of pigment-type switching is the receptor for -MSH, melanocortin-
1 receptor (Mc1r). When -MSH binds to Mc1r on melanocyte membrane, adenylate cyclase
is activated through the stimulatory G-protein, raising levels of cAMP, thereby activating the
melanogenic transcription factor, microphthalmia-associated transcription factor (Mitf;
Bertolotto et al., 1998). This leads to the upregulation of many genes required for melanin
synthesis such as Tyr, Tyrp1, Dct and many other genes (Levy et al., 2006). The Asip is a
competitive antagonist that inhibits the eumelanogenic effect of -MSH by competing with
-MSH for binding to the Mc1r. When viable yellow (Avy/-) mice producing a mixed-type
melanin were injected with -MSH, Tyr activity increased 2-fold and more eumelanic hair
was produced with a concomitant increase in total melanin (TM). When these viable yellow
mice were injected with bromocriptine (inhibitor of -MSH production in the pituitary), Tyr
activity was greatly reduced and pheomelanic hair was produced along with a decrease in TM
(Burchill et al., 1986). These results suggest that Tyr activity is important for controlling
mixed melanogenesis; higher tyrosinase activities favor eumelanogenesis, while lower
activities pheomelanogenesis.
The Asip requires two accessory proteins for pigment type switching; products of the
mahogany (mg) and mahoganoid (md) loci (Walker and Gunn, 2010). The mahogany locus
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198 Tomohisa Hirobe

was identified as the mouse orthologue of the human attractin (ATRN) gene, and the
mahoganoid locus encodes a novel RING-domain containing protein. Mice homozygous for
mahogany and heterozygous for lethal yellow produce a mixed type melanin with a low level
of eumelanin (ca. 15% of black) and have a reduced level of pheomelanin (ca. 60% of lethal
yellow). Similarly, Gunn et al. (2001) found that three Atrn mutants, either homozygous or
compound heterozygous, showed a pheomelanin content 5- to 10-fold lower than wild-type
agouti C3H⁄ HeJ mice. Another control point in the regulation of eumelanogenesis and
pheomelanogenesis is cysteine concentration in melanosomes (del Marmol et al., 1996).
Chintala et al. (2005) showed that the murine subtle gray (sut) mutation arose because of a
mutation in the Slc7a11 gene that encodes the plasma membrane cystine⁄glutamate exchanger
xCT. The resulting low rate of extracellular cystine transport into sut melanocytes reduces
pheomelanin synthesis with minimal or no effect on eumelanin synthesis. In fact, the effect of
the sut mutation on pheomelanin synthesis was greatly emphasized by the Ay ⁄a background,
decreasing pheomelanin levels in hairs to one-sixth of the control level.

Brown

B (Tyrp1), the wild type allele at the brown locus, produces black eumelanin, while b
(Tyrp1b), the recessive allele, produces brown eumelanin. The coat color of brown mice is
lighter than that of black mice, whereas tyrosinase activity in brown mice is higher than in
black mice (Foster, 1965; Hirobe, 1984b; Tamate et al., 1989). Eumelanin content in brown
hairs is decreased compared with black hairs, whereas pheomelanin content is increased in
both B10 (Ozeki et al., 1995) and B6 (Lamoreux et al., 2001) background (Table 1). The
proliferation rate of brown (B10-Tyrp1b/Tyrp1b) melanocytes cultured in MDMD is similar to
that of black (B10-Tyrp1/Tyrp1) melanocytes (Hirobe, 2011a). Brown melanocytes in culture
possess normal morphology (dendritic, polygonal or epithelioid), but their pigmentation is
lower than that of black melanocytes (Hirobe et al., 1998). Tyr, Dct and Kit activities in
brown melanocytes in culture do not differ from that in black melanocytes in culture, but
Tyrp1 activity is greatly reduced (Hirobe, 2011a). Although brown melanosomes are evenly
distributed within melanocytes, their morphology is very different from that of black
melanosomes. Elliptical melanosomes and mature stage IV melanosomes are rarely observed
(Hirobe, 2011a). Brown melanosomes are mostly spherical stage III melanosomes with
granular or lamellar depositions of pigments. In addition, eumelanin is decreased 3-fold in
brown melanocytes, whereas pheomelanin is increased 3- to 4-fold (Tamate et al., 1989;
Ozeki et al., 1995; Hirobe et al., 1998). The PTCA/AHP ratio in brown melanocytes is one-
tenth of that in black melanocytes. The formation of elliptical eumelanosomes requires plenty
of eumelanin and higher Tyrp1 activity.
Tyrp1 is believed to act as a DHICA oxidase in mice (Jimenez-Cervantes et al., 1994;
Kobayashi et al., 1994a). The brown mutation encodes Tyrp1 that is not properly translocated
to melanosomes, resulting in no functional Tyrp1 activity and decreased tyrosinase function
(Jackson et al., 1990). Brown melanocytes seem to inhibit eumelanin synthesis (TM and
PTCA values). The brown mutation does not significantly alter the proportion of DHICA in
the eumelanin synthesized, but rather, brown eumelanin seems to possess a smaller molecular
size compared to black eumelanin (Ozeki et al., 1997). Although the exact function of Tyrp1

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 The Coat Color Genes… 199

is not known well, it is thought to stabilize tyrosinase and Dct (Lamoreux et al., 1995;
Kobayashi et al., 1998).

Albino

The albino mice lack pigment in the coat and eyes. The inability of albino mice to
produce pigment is derived not from an absence of melanoblasts, but from a deficiency of Tyr
activity (Tanaka et al., 1990; Hirobe and Abe, 1999). The enzyme Tyr is encoded at the
albino/tyrosinase (C/Tyr) locus in mice. C (Tyr), the wild-type allele of the albino locus,
produces melanin, while c (Tyrc), the recessive allele, produces no pigment in the coat and the
eyes (Silvers, 1979; Yamamoto et al., 1989; Tanaka et al., 1990; Table 1). The Tyrc allele is a
point mutation at nucleotide residue 387 (G to C transversion) causing a Cys to Ser
substitution at position 85 in one of the cysteine-rich domains of the Tyr molecule (Shibahara
et al., 1990). This mutation reduces Tyr activity completely. We studied the effects of the c
mutation on the proliferation of melanoblasts cultured in MDMD and MDMDF, and found
that the proliferation rate of albino melanoblasts was about one-half that of black melanocytes
(Hirobe et al., 1998), suggesting the possibility that cell proliferation is active in epidermal
melanocytes with full melanogenesis such as black melanocytes, but not in epidermal
melanoblasts with no melanogenesis such as albino melanoblasts. In other words,
proliferation and differentiation of epidermal melanocytes in culture may be linked. Albino
melanoblasts exhibit normal morphology (dendritic, polygonal or epithelioid), but no
pigmentation was observed (Hirobe et al., 1998; Hirobe, 2011a). Expression of Tyr in albino
melanoblasts is not observed, whereas expression of Tyrp1, Dct and Kit is similar level to that
in black melanocytes (Hirobe, 2011a). Melanosomes are evenly distributed within albino
melanoblasts, and morphology of stage I and II melanosomes is similar to that of black
melanocytes (Hirobe, 2011a). Moreover, the number of stage I and II melanosomes is greatly
increased compared with black melanocytes (Hirobe, 2011a), probably due to the inhibition
of stage III and IV formation by the Tyrc mutation.
The chinchilla allele (cch/Tyrc-ch) at the albino locus encodes a partially functional Tyr
whose activity is one half of that of wild type, due to a point mutation (Ala464Thr) that
makes it susceptible to proteolytic cleavage (Muller et al., 1988). Therefore, this is a good
model to examine the specific effects of Tyr activity on pigmentogenesis. Tyrc-ch/Tyrc-ch hairs
possessed eumelanin content about one half of that observed in Tyr/Tyr hairs (Lamoreux et
al., 2001). Brown chinchilla (Tyrp1b/Tyrp1b; Tyrc-ch/Tyrc-ch) hairs possessed lower eumelanin
content than in black chinchilla (Tyrp1/Tyrp1; Tyrc-ch/Tyrc-ch) hairs (Lamoreux et al., 2001).
However, Tyrc-ch/Tyrc-ch hairs possessed similar amount of pheomelanin as Tyr/Tyr hairs
(Lamoreux et al., 2001). These results suggest that functional Tyrp1 is also necessary, in
addition to high levels of Tyr, for maximal production of eumelanin. In chinchilla mice, the
degree of eumelanogenesis is proportional to Tyr activity under low cysteine concentration.

Dilute, Leaden and Ashen

The recessive allele of the dilute locus, d/Myo5ad elicits a lighter hair pigmentation in
mice. Despite the fact that the dilute mutation possesses a dilution effect when introduced into
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200 Tomohisa Hirobe

wild-type mice producing intensely pigmented eumelanic and pheomelanic hairs, this effect is
not due to a decrease in eumelanin and pheomelanin content (Table 1). We investigated the
effects of the dilute allele on the proliferation and differentiation of melanoblasts and
melanocytes (B10-Myo5ad/Myo5ad) cultured in MDMD/MDMDF and found that the
proliferation rate of dilute melanoblasts and melanocytes was similar to that of black
melanoblasts and melanocytes (Hirobe et al., 1998). The rate of differentiation and the
reactivity to dopa and dopa-premelanin reactions of dilute melanocytes cultured in MDMD
was also similar to that of black melanocytes (Hirobe et al., 1998). Dilute melanocytes were
dendritic, polygonal or epithelioid in morphology, but their melanosomes were distributed
around the nucleus (Hirobe, 2011a). A few melanosomes were observed in the peripheral
region of the cytoplasm as well as in dendrites. Expression of Tyr, Tyrp1, Dct and Kit in
dilute melanocytes was similar to that in black melanocytes (Hirobe, 2011a). Dilute
melanosomes were distributed around the nucleus, and the number and morphology of stage
I–IV melanosomes was similar to that of black melanosomes (eumelanosome type; Hirobe,
2011a). These results suggest that the dilute allele is involved in regulating the transport of
melanosomes from the perinuclear region to the dendrites, rather than in regulating dendrite
formation. These findings are consistent with results of molecular analyses of the dilute allele
(Mercer et al., 1991; Provance et al., 1996; Wei et al., 1997; Wu et al., 1997). The dilute gene
encodes myosin Va which is a dimer of two 190 kDa heavy chains. The N-terminal head
region consists of actin- and ATP-binding sites and functions as a motor domain for short
range movement along actin filaments of the cytoskeleton (Westbroek et al., 2001; Wu et al.,
1997).
Leaden (ln) is also dilute mutation when homozygous and it transforms the intensely
pigmented nonagouti coat color to bluish-grey (Murray 1931). The effects of the leaden allele
on hairs are essentially the same as in dilute except that some leaden genotypes, such as
chocolate leaden animals, are a little lighter in color than the corresponding dilute type
(Silvers 1979). This appears to be due to a more pronounced pigment lag in ln/ln hairs rather
than any noticeable differences in pigment clumping (Silvers 1979). Ashen (ash), recessive
mutation arose in strain C3H/DiSn (Lane and Womack, 1977). The coat color of these mice
mimics that of dilute and leaden. Recent molecular analysis revealed that leaden (lacking
melanophilin (Mlph) protein) and ashen (lacking Rab27a protein) exhibited similar
melanosome distribution and these proteins appeared to anchor to MyoVa motor on the
melanosome (Wu et al., 2002; Hume et al., 2007). This mechanism allows melanosomes to be
retained in dendrites and to make short myosin-driven movements along actin filaments
towards the plasma membrane prior to transfer to keratinocytes.

Recessive Yellow and Lethal Yellow

The phenotype that produces mostly pheomelanin is regulated by two alleles, namely,
recessive yellow (e/Mc1re) at the extension locus and lethal yellow (Ay) at the agouti locus.
The extension (E/Mc1r) locus increases eumelanin in hair follicular melanocytes when
dominant, but it blocks eumelanin synthesis, extending the range of pheomelanin when
recessive (Silvers 1979). The recessive yellow allele results from a frameshift in Mc1r that
produces a prematurely terminated, nonfunctioning receptor (Robbins et al., 1993). In
addition to the frameshift mutation, the Mc1re allele possesses a conservative point mutation,

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 The Coat Color Genes… 201

Val101Ala (Robbins et al., 1993). Moreover, the Mc1re allele stimulates pheomelanin
synthesis in the epidermis and dermis as well as hair follicles in B10-Mc1re/Mc1re skin
(Hirobe et al., 2007a). In B6-Mc1re/Mc1re mice, epidermal and dermal melanoblasts and
melanocytes are greatly reduced in number (Tamate et al., 1986).
Since we could not obtain a pure culture of yellow melanocytes producing pheomelanin
only from Mc1re/Mc1re mice, we investigated the proliferation and differentiation of cultured
recessive yellow melanocytes producing mainly eumelanin. The addition of DBcAMP to
culture media can elicit upregulation of the PKA pathway and stimulate eumelanogenesis in
melanocytes (Tamate and Takeuchi, 1984). The proliferation rate of Mc1re/Mc1re
melanoblasts or melanocytes cultured in MDMDF or MDMD was decreased (by one-half)
compared with that of Mc1r/Mc1r melanoblasts and melanocytes (Hirobe et al. 2007b).
Differentiation of melanocytes cultured in MDMD was also delayed in Mc1re/Mc1re mice
(Hirobe et al., 2007b). Although the expression of Tyr and Kit in Mc1re/Mc1re melanocytes
was similar to that in black melanocytes, expression of Tyrp1 and Dct was decreased (Hirobe
et al., 2007b). The number of stage III melanosomes did not change, while the number of
stage IV melanosomes was decreased (Hirobe et al., 2007b). Excess L-tyr added to MDMD
rescued the reduced proliferation rate of Mc1re/Mc1re melanocytes. L-tyr also stimulated Tyr
activity and expression of Tyrp1, Dct and Kit as well as maturation of stage IV melanosomes
and eumelanin synthesis (Hirobe et al., 2007b). These results suggest that the Mc1re mutation
affects the proliferation and differentiation of melanocytes and L-tyr rescues the reduced
proliferation and differentiation of Mc1re/Mc1re melanocytes by stimulating Tyr activity and
expression of Tyrp1 and Dct as well as melanosome maturation and eumelanin synthesis.
Even at the higher cAMP levels elicited by DBcAMP-supplemented MDMD and MDMDF,
the proliferation of Mc1re/Mc1re melanoblasts and melanocytes was greatly inhibited,
suggesting that the PKA pathway elicited by excess DBcAMP in Mc1re/Mc1re melanocytes is
different from the PKA pathway elicited by wild-type Mc1r in Mc1r/Mc1r melanocytes. The
altered PKA pathway in Mc1re/Mc1re melanocytes may affect crosstalk with protein kinase C
(PKC) or MAP kinase (MK), and consequently the proliferation and differentiation may be
inhibited. L-tyr is thought to rescue the altered PKA pathway as well as the altered crosstalk
between PKA and PKC/MK.
Eumelanin content in cultured Mc1re/Mc1re melanocytes in MDMD was higher than in
Mc1r/Mc1r melanocytes. However, eumelanin content in culture supernatant did not differ
between Mc1re/Mc1re and Mc1r/Mc1r melanocytes (Hirobe et al., 2007b). In contrast,
pheomelanin content in cultured Mc1re/Mc1re melanocytes was lower than in Mc1r/Mc1r
melanocytes. However, pheomelanin content in culture supernatant did not differ significantly
between Mc1re/Mc1re and Mc1r/Mc1r melanocytes (Hirobe et al., 2007b; Table 1).
Eumelanin contents in the epidermis and dermis of Mc1re/Mc1re mice were much lower than
those of Mc1r/Mc1r mice, whereas pheomelanin contents in the epidermis and dermis of
Mc1re/Mc1re mice was much greater than those of Mc1r/Mc1r mice (Hirobe et al., 2007a).
Eumelanin content in Mc1re/Mc1re hairs (5-week-old) was much lower than in Mc1re/Mc1re
hairs, whereas pheomelanin content in Mc1re/Mc1re hairs was much greater than in
Mc1r/Mc1r hairs (Hirobe et al., 2007a; Table 1).
Eumelanin and pheomelanin content in dorsal hairs of female B10-Mc1re/Mc1re mice is
greater than that seen in male mice, suggesting that the expression of the recessive yellow
allele is regulated in a sex-dependent manner (Hirobe et al., 2007a). We have suggested that
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202 Tomohisa Hirobe

estrogen is a main factor in determining the higher content of eumelanin and pheomelanin in
the hair of female Mc1re/Mc1re mice (Hirobe et al., 2010).
Lethal yellow (Ay) represents the top dominant of the agouti locus. Phenotypically Ay/-
mice produce mostly pheomelanic hairs. Ay/Ay embryos can be formed but display
characteristic abnormalities at the morula or blastocyst stage (Kirkham 1919) and die early on
the sixth day of gestation. In the Ay mutation, there is a chromosomal rearrangement that
results in the production of chimeric RNA expressed in nearly every tissue of the body. The
5’ portion of this chimeric RNA contains highly expressed novel 5’ sequences, but the 3’
portion retains the protein-coding potential of the wild-type allele. Thus, Ay/- mice produce a
plenty of the Asip and suppress the action of -MSH toward Mc1r, consequently produce
pheomelanin only during normal hair growth (Miller et al., 1993). Eumelanin and
pheomelanin contents in hairs from B6-Ay/a and B6- Mc1re/Mc1re are similar level (Ozeki et
al., 1995; Table 1), suggesting that melanin synthesized in hair bulb melanocytes does not
differ from Ay/a and Mc1re/Mc1re.

Pink-Eyed Dilution, Underwhite and Silver

Pink-eyed dilution mutant was discovered in the mouse fancy, and is known to reduce the
pigmentation of both the coat and the eyes. The eyes of pink-eyed dilution mice resemble
those of albinos, possessing a pink tent. However, in contrast to albino eyes, pink-eyed
dilution genes are not entirely free of pigment (Silvers 1979). P (Oca2), the wild-type allele at
the pink-eyed dilution locus, produces an intense pigmentation of both eumelanin and
pheomelanin in the skin and eyes, while p (Oca2p), the recessive allele, greatly reduces
pigmentation in both the coat and the eyes (Silvers, 1979). The pink-eyed dilution locus
controls melanin synthesis, melanosome morphology and Tyr activity (Ozeki et al., 1995;
Hirobe and Abe, 1999; Chen et al., 2002; Toyofuku et al., 2002). The product of the Oca2
allele is an integral membrane protein that localizes in melanosomes (Rosemblat et al., 1994);
its predicted secondary structure is a 12-transmembrane domain protein similar to a channel
or transporter (Gardner et al., 1992; Rinchik et al., 1993). The Oca2 protein seems to control
processing and transport of Tyr (Toyofuku, 2002), but may not be a tyr transporter (Gahl et
al., 1995). Sitaram et al. (2009) reported that the Oca2 protein is active in melanosomes and
its activity might be limited by additional sorting to lysosomes. The pH of melanosomes is
abnormal in Oca2p mutant melanocytes (Puri et al., 2000).
The proliferation and differentiation of mouse melanocytes cultured in MDMD is greatly
inhibited by the Oca2p mutation (Hirobe, 2011a) and L-tyr rescues both proliferation and
differentiation (Hirobe et al., 2002b), though most of melanins and their precursors fail to
accumulate in Oca2p/Oca2p melanosomes (Wakamatsu et al., 2007). Moreover, in
Oca2p/Oca2p melanoblasts, only a few stage I and II melanosomes are observed (Hirobe et
al., 2002b), whereas L-tyr greatly increases the number of stage II, III and IV melanosomes
(Hirobe et al., 2002b). The Oca2p allele greatly inhibits eumelanin synthesis, but not
pheomelanin synthesis (Hirobe et al., 2011c). Production of pheomelanin in Oca2p/Oca2p
melanocytes is not influenced by the agouti, nonagouti black and recessive yellow allele
(Hirobe et al., 2011c).
Pink-eyed dilution melanoblasts possess smaller but more numerous mitochondria than
black melanocytes (Hirobe et al., 2008). Treatment of Oca2p/Oca2p melanoblasts with L-tyr

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decreased the number of mitochondria (Hirobe et al., 2008). Media supplemented with 2,4-
dinitrophenol (DNP), an inhibitor of mitochondrial function, stimulated both proliferation and
differentiation of Oca2p/Oca2p melanoblasts, and simultaneous DNP and L-tyr treatment
dramatically induced the differentiation of Oca2p/Oca2p melanocytes (Hirobe et al., 2008).
These results suggest that the Oca2p allele is involved in regulating the function of
mitochondria.
Since mitochondria are well developed in Oca2p/Oca2p melanoblasts and melanocytes,
the possibility exists that apoptosis occurs in Oca2p/Oca2p melanoblasts and melanocytes.
Inhibitors of apoptosis, such as caspase-9 inhibitor (C9I) and Bax-inhibiting peptide (BIP),
stimulated the proliferation and differentiation of cultured Oca2p/Oca2p melanoblasts, but not
of Oca2/Oca2 melanoblasts and melanocytes. The number of apoptotic melanoblasts and
keratinocytes in culture derived from Oca2p/Oca2p mice was greater than that derived from
Oca2/Oca2 mice (Hirobe 2011a). Apoptotic melanoblasts and keratinocytes in Oca2p/Oca2p
mice could be decreased by treatment with C9I and BIP. Moreover, expression of caspase-9
and Bax in Oca2p/Oca2p melanoblasts and keratinocytes was greater than in Oca2/Oca2
melanoblasts and keratinocytes (Hirobe 2011a). These results suggest that the increased
apoptosis is related to the reduced proliferation and differentiation of Oca2p/Oca2p
melanoblasts.
Eumelanin content in cultured Oca2p/Oca2p melanocytes in MDMD was much lower
than in Oca2/Oca2 melanocytes. However, eumelanin content in culture supernatant did not
differ between Oca2p/Oca2p and Oca2/Oca2 melanocytes (Hirobe et al., 2003). In contrast,
pheomelanin content in cultured Oca2p/Oca2p melanocytes was lower than in Oca2/Oca2
melanocytes. However, pheomelanin content in culture supernatant did not differ significantly
between Oca2p/Oca2p and Oca2/Oca2 melanocytes (Hirobe et al., 2003; Table 1). Eumelanin
content in Oca2p/Oca2p hairs (5-week-old) was much lower than in Oca2p/Oca2p hairs,
whereas pheomelanin content in Oca2p/Oca2p hairs did not differ from that in Oca2/Oca2
hairs (Hirobe et al., 2003; Table 1).
Underwhite (uw) is an autosomal recessive mutation that arose spontaneously in the B6
strain (Dickie 1964). The dorsum of uw/uw mice is a light buff color, whereas the ventrum is
white. The eyes of uw/uw mice are unpigmented at birth, but darken to a dark reddish color at
maturity (Green 1966a). Molecular analysis revealed that underwhite regulates Slc45a2
protein. The Slc45a2 locus in mice encodes a membrane-associated transporter protein (Matp)
that has a 12-transmembrane-spanning structure (Newton et al., 2001). All of the three
mutations (uw, uwd and UWdbr) at the underwhite locus reduce the production of eumelanin
more than 90% compared to wild-type mice (Lehman et al., 2000). The hypopigmentary
effect of the underwhite mutation is independent of Oca2p, because double mutant mice at
Slc45a2 and Oca2p exhibit an albino appearance. However, Costin et al. (2003) reported that
processing and trafficking of Tyr to melanosomes is disrupted and Tyr is abnormally secreted
from uw/uw melanocytes in a similar process to that seen in Oca2p/Oca2p melanocytes.
Mutations at the silver (si/Pmel17) locus affect eumelanin production only slightly (20%
reduction) on a nonagouti background. In contrast to nonagouti silver mice, where the
animals become progressively more silvered, in agouti and yellow silver mice the silvering
decreases markedly as the animals get older (Dum and Thigpen, 1930). The effects become
more pronounced (40–50% reduction) when interacting with the brown locus (Lamoreux et
al., 2001). Thus, the effects of the mutations at the brown and silver loci are additive. The
silver protein is important for the biogenesis of early stage melanosomes (Kobayashi et al.,
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204 Tomohisa Hirobe

1994b; Theos et al., 2005), and being the primary component of the matrix fibrils in
eumelanosomes (Theos et al., 2006).

Slaty

Slaty (slt/Dctslt) is the recessive autosomal mutation occurred in a heterogeneous stock

carrying limb-deformity (ldj) and mahogany (mg). On a nonagouti background, slaty
homozygotes possess a slightly diluted coat pigmentation (Green, 1972). The slaty locus
encodes Tyrp2/Dct and thus wild-type animals produce DHICA-rich eumelanin. The slaty
mutation greatly reduce the PTCA value with a mild reduction in TM. Therefore, the
PTCA/TM ratio was reduced four to six-fold, suggesting that DHICA-pour eumelanin is
produced in Dctslt/Dctslt melanocytes. In addition to the original slaty mutation, slaty light
(Sltlt/DctSlt-lt; more severe effect) and slaty 2J (slt2J/Dctslt-2J; similar phenotype) have been
identified (Budd and Jackson, 1995). The slaty mutation is known to change an arginine to a
glutamine in the first copper-binding domain of Dct, which converts DC to DHICA in the
eumelanin synthesis pathway (Korner and Pawelek, 1980; Jackson et al., 1992; Tsukamoto et
al., 1992); it also yields about 10–30% of the activity of wild-type Dct in eye extracts
(Jackson et al., 1992). Dct is produced by both wild-type and slaty mutant cDNA, but the
protein level of Dct in the slaty mutant is greatly reduced (Kroumpouzos et al., 1994).
The slaty mutation does not affect the proliferation of cultured epidermal melanoblasts
and melanocytes in MDMD (Hirobe et al. 2007c). However, the differentiation and Tyrp2
expression of cultured slaty melanocytes is greatly inhibited (Hirobe et al., 2006). The slaty
mutation affects both eumelanin and pheomelanin synthesis in a developmental stage-specific
and skin site-specific manner (Hirobe et al., 2007c). Eumelanin content in cultured
Dctslt/Dctslt melanocytes in MDMD was lower than in Dct/Dct melanocytes. However,
eumelanin content in culture supernatant did not differ between Dctslt/Dctslt and Dct/Dct
melanocytes (Hirobe et al., 2006, Table 1). In contrast, pheomelanin content in cultured
Dctslt/Dctslt melanocytes did not differ from that in Dct/Dct melanocytes. However,
pheomelanin content in culture supernatant did not differ significantly between Dctslt/Dctslt
and Dct/Dct melanocytes (Hirobe et al., 2006; Table 1). Eumelanin and pheomelanin contents
in the epidermis and dermis of Dctslt/Dctslt mice were lower than those of Dct/Dct mice
(Hirobe et al., 2006; Table 1). Eumelanin content in Dctslt/Dctslt hairs (5-week-old) was
smaller than in Dct/Dct hairs, whereas pheomelanin content in Dctslt/Dctslt hairs did not differ
from Dct/Dct hairs (Hirobe et al., 2006; Table 1).
In slaty melanocytes, numerous spherical melanosomes with granular depositions of
pigments, black type elliptical melanosomes with longitudinal depositions of pigments in
intraluminal fibrils and a mix of the two melanosome types are observed (4:1:1) (Hirobe and
Abe, 2006). Moreover, in slaty melanocytes, mature stage IV melanosomes greatly decrease,
while immature stage III melanosomes are more numerous than in black melanocytes (Hirobe
and Abe, 2007a). In slaty melanocytes, spherical and mixed type melanosomes gradually
decrease after birth, whereas elliptical melanosomes gradually increase. These results suggest
that the slaty mutation blocks melanosome maturation at stage III and affects melanosome
morphology (elliptical or spherical) in a developmental stage-specific manner.
Inhibition of eumelanin synthesis by the slaty mutation can be partly restored by the
addition of excess L-tyr to MDMD (Hirobe et al., 2006). Eumelanin and pheomelanin may be

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accumulated with difficulty in slaty melanocytes and are easily released during skin
development. L-tyr is thought to stimulate this release. Perhaps, L-tyr acts directly on
melanoblasts and melanocytes and activates factors involved in regulating eumelanin
synthesis (Coughlin et al., 1988; Imokawa, 2004; Hirobe, 2005). Another possibility is that L-
tyr acts on the tissue environment, especially keratinocytes, and stimulates synthesis of
melanogenic factors controlling eumelanin synthesis (Imokawa 2004; Hirobe, 2005).
When L-tyr is added to MDMD, it stimulates melanosome maturation and increases
elliptical melanosomes, but decreases spherical melanosomes (Hirobe and Abe, 2007b),
suggesting that L-tyr restores the reduced melanosome maturation and changes the altered
morphology of melanosomes affected by the slaty mutation. L-tyr may act directly on
melanocytes and activate factors involved in regulating pigmentation. Since excess L-tyr
restores maturation of stage IV elliptical melanosomes, slaty melanosomes are thought to
possess a normal pathway related to L-tyr transport (Hirobe and Abe, 2007b).
Thus, the possibility exists that L-tyr transport system from the cytoplasm to
melanosomes is affected by the slaty mutation. If this is true, melanin synthesis would be
increased by excess L-tyr, and maturation of stage IV melanosomes would be stimulated.
Furthermore, L-tyr increases the total number of melanosomes, suggesting that L-tyr
stimulates de novo melanosome formation. It has been reported that -MSH stimulates
differentiation of epidermal melanocytes of black mice in vivo (Hirobe and Takeuchi, 1977).
Differentiation stimulated by -MSH is associated with an increase in the total number of
melanosomes. Similar mechanisms in -MSH and L-tyr seem to be involved in the
stimulation of de novo melanosome formation by -MSH and L-tyr.

Ruby-Eye 2d, Beige and Mottled

In 2006, a spontaneous autosomal recessive mutant of brown coat color with ruby eyes
occurred in B10 mice in my laboratory (Hirobe et al., 2011d). The phenotype of this mutant
was similar to that of ruby-eye (ru/Hps6ru) or ruby-eye 2 (ru2/Hps5ru2). Human Hermansky-
Pudlack syndrome (HPS) is a recessively inherited disease that affects several organs such as
the skin (hypopigmenation), eyes (low visual acuity), blood cells (prolonged bleeding) and
lungs (interstitial pulmonary fibrosis) (Wei, 2006). Many distinct types of human HPS have
been described (Wei, 2006). In mice, many naturally occurring hypopigmentation models of
HPS have been characterized (Wei, 2006). Human HPS5 corresponds to mouse Hps5ru2 (ru2),
and HPS6 to Hps6ru (ru) (Zhang et al., 2003). RT-PCR analysis revealed that this novel
mutation named ru2d/Hps5ru2-d is a frameshift mutation by 997G deletion in Hps5 (Hirobe et
al., 2011d).
Mouse Hps5 gene is on chromosome 7 and possesses a 3381-bp open reading frame
(ORF) with 23 exons, encoding a 1126 amino acid (aa) protein (127.4 kDa), 81% homologies
to the human sequence (126.3 kDa) are observed (Zhang et al., 2003). All tissues (heart,
brain, spleen, lung, liver, skeletal muscle, kidney and testes) examined contained the 4.8 kb
transcript. Nine murine mutations in the Hps5 (Ru2) gene are known until now: the
ru2mr/Hps5ru2-mr allele is a spontaneous recessive mutation with undefined molecular
characterization (Bateman 1957); the ru2hz/Hps5ru2-hz allele leads to a predicted loss of 118 C-
terminal aa (frameshift by insertion of CCGG at E900) (Dickie, 1965; Zhang et al., 2003); the
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206 Tomohisa Hirobe

ru2/Hps5ru2 allele contains a 1.0-kb insertion (K867) of the H2A histone sequence
immediately preceding codon 868 of exon 18 (Lilly, 1966; Zhang et al., 2003); the
ru2J/Hps5ru2-J allele leads to a predicted loss of 311 aa at the C terminus (frameshift by
⊿G757) (Eicher and Fox, 1977; Zhang et al., 2003); the ru28J/Hps5ru2-8J allele is a
spontaneous recessive mutation with undefined molecular characterization (Cook 1995); the
ru211J/Hps5ru2-11J allele is an N-ethyl-N-nitrosourea (ENU)-induced recessive mutation with
undefined molecular characterization (Gwynn et al., 2004, Mouse Genome Informatics); the
ru2Btlr/Hps5ru2-Btlr allele leads to T- to C- transition in the donor splice site of intron 9 (ENU-
induced recessive mutation) (Eidendchenk et al., 2008 in Mouse Genome Informatics); and
the ru22Btlr/Hps5ru2-2Btlr allele leads to A- to T- transversion at nucleotide position 2337 (ENU-
induced recessive mutation) (Blasius et al., 2008 in Mouse Genome Informatics). We first
reported the tenth allele occurred in mice, ru2d/Hps5ru2-d, caused by frameshift by deletion
(⊿G997). The Hps5ru2-d mutation makes large molecule protein to smaller one by a premature
termination codon, and reduces mRNA expression.
Human HPS1, 2, 3, 4, 7 and 8 correspond to mouse pale ear (ep/Hps1ep), pearl
(pl/Ap3b1pl), cocoa (coa/Hps3coa), light ear (le/Hps4le), sandy (sdy/Dtnbp1sdy) and reduced
pigmentation (rp/Bloc1S3rp), respectively (Wei 2006). All the HPS mutations are
characterized by hypopigmentation and several diseases, and in mice, Hps is a disorder of
organelle biogenesis in which hypopigmentation, bleeding and pulmonary fibrosis are
resulted from defects in melanosomes, platelet dense granules and lysosomes (Wei 2006).
The difference in the coat color in the Hps mutant seems to be due to the inhibition of
melanosome formation (inner structure) and maturation. Zhang et al. (2003) reported that in
the retinal pigment epithelium and choroid of B6-Hps5ru2/Hps5ru2 mice, melanosomes were
fewer and immature, and their shape were mostly spherical. Nguyen et al. (2002) reported
that in the hair follicle melanocytes of the dorsal skin of 4-week-old B6-Hps5ru2/Hps5ru2
mice, stage IV melanosomes decreased in number, and their morphology remained spherical.
However, in Hps5ru2-d/Hps5ru2-d melanocytes, melanosomes were elliptical, but they were
fewer and immature, suggesting that the Hps5ru2-d allele controls the maturation of
melanosomes, but not their internal structure. The severity of the lesion in Hps5ru2-d allele
(melanosome formation and maturation) may be less than that of Hps5ru2 allele.
To clarify the mechanism of the hypopigmentation, the characteristics of the proliferation
and differentiation of Hps5ru2-d/Hps5ru2-d epidermal melanoblasts and melanocytes cultured in
MDMD and MDMDF were investigated. The proliferation of Hps5ru2-d/Hps5ru2-d
melanoblasts and melanocytes did not differ from that of Hps5/Hps5 (Hirobe et al., 2011d).
However, the differentiation of Hps5ru2-d/Hps5ru2-d melanocytes was greatly inhibited. Tyr
activity, expression of Tyr, Tyrp1, Dct and eumelanin synthesis were markedly decreased in
Hps5ru2-d/Hps5ru2-d melanocytes (Hirobe et al., 2011d). However, the addition of excess L-tyr
to MDMD rescued the reduced differentiation via increased Tyr activity, expression of Tyr,
Tyrp1, Dct and Kit and eumelanin synthesis (Hirobe et al., 2011d). These results suggest that
the Hps5ru2-d allele inhibits melanocyte differentiation, though the impaired differentiation is
rescued by excess L-tyr.
In Hps5ru2-d/Hps5ru2-d melanocytes, elliptical melanosomes were observed, though many
immature stage III melanosomes and less stage IV melanosomes were observed (Hirobe et al.,
2011d). The number of stage IV melanosomes was much smaller than in Hps5/Hps5
melanocytes. The total number of melanosomes in Hps5ru2-d/Hps5ru2-d melanocytes was also
less than in Hps5/Hps5 melanocytes. However, L-tyr markedly increased the number of stage

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 The Coat Color Genes… 207

IV melanosomes and the total number of melanosomes in Hps5ru2-d/Hps5ru2-d melanocytes

(Hirobe et al., 2011d). These results suggest that the Hps5ru2-d mutation markedly inhibits
melanosome formation and maturation, but its inhibition can be restored by L-tyr.
Eumelanin content in cultured Hps5ru2-d/Hps5ru2-d melanocytes in MDMD was lower than
in Hps5/Hps5 melanocytes. However, eumelanin content in culture supernatant was greater
than that of Hps5/Hps5 melanocytes (Hirobe et al., 2011d, Table 1). In contrast, pheomelanin
content in cultured Hps5ru2-d/Hps5ru2-d melanocytes in MDMD as well as in culture
supernatant did not differ from that in Hps5/Hps5 melanocytes (Hirobe et al., 2011d, Table
1). Eumelanin contents in the epidermis, dermis and hairs (5-week-old) of Hps5ru2-d/Hps5ru2-d
mice were much lower than those of Hps5/Hps5 mice (Hirobe et al., 2011d; Table 1).
Pheomelanin contents in the epidermis and dermis of Hps5ru2-d/Hps5ru2-d mice were lower
than those of Hps5/Hps5 mice. However, 2- to 3-fold increase in pheomelanin content in hairs
of 5-week-old Hps5ru2-d/Hps5ru2-d mice was observed (Hirobe, 2011a; Table 1). These results
suggest that pheomelanin synthesis in Hps5ru2-d/Hps5ru2-d mice is increased in hair bulbs.
These results are consistent with the results that 5-S-CD level in plasma of Hps5ru2-d/Hps5ru2-d
mice was greater than that of Hps5/Hps5 mice (Hirobe et al., unpublished). We first presented
the evidence that the Hps5ru2-d allele stimulates pheomelanin synthesis in mouse hair bulb
melanocytes.
Beige (bg/bg) is a recessive mutation affecting both coat and eye color. The eye color of
bg/bg mice was light at birth and varied from ruby to almost black in adults. bg/bg mice also
display reduced pigmentation in the ear and tail, and the coat color is lighter than wild-type
mice, particularly at the base of the hairs (Kelly, 1957). In retinal pigment epithelia and hair
bulb melanocytes of bg/bg mice, melanosomes decrease in number and this reduction is due
both to the synthesis of fewer lysosomes and to the fusion of lysosomes into progressively
larger lysosomes (Pierro, 1963). The beige gene is homozygous to Chediak-Higashi
syndrome gene, and these genes are encoded by Lyst gene (Barbosa et al., 1996; Nagle et al.,
1996). Lyst encodes a protein with a carboxy-terminal prenylation motif and multiple
potential phosphorylation sites. The Lyst protein is predicted to form extended helical
domains, and possesses a region of sequence similar to stathmin, a coiled-coil
phosophoprotein that is thought to act as a relay integrating cellular signal response coupling
(Barbosa et al., 1996).
The mottled (Mo) mutation occurred in females among the progeny of a cross-
segregating for albinism, piebald (s/Ednrbs), brown and hairlessness (hr) (Fraser et al., 1953).
The female was Tyrp1/-; Ednrb/- and possessed many regions of light-colored hair scattered
in an apparently patternless fashion over the entire body. The depth of color of the hairs in
these regions varied between regions. The mottled (Mo/Atp7a) gene locates in X
chromosome. Females which are heterozygous for the Mo gene possess, to varying degrees, a
mottled coat with patches of white, light-colored and full-colored hairs, as well as
intermingled hairs of different colors (Silvers, 1979). The activity of the copper-dependent
enzyme, cyrochrome c oxidase and superoxidase dismutase are reduced in this mutant mice.
The mottled gene is homologous to the gene related to human Menkes disease that is an X-
linked recessive copper deficiency disorder caused by mutations in the ATP7A (MNK) gene.
Thus, the new symbol for the mottled allele is Atp7a. The MNK gene encodes a copper-
transporting P-type ATPase, MNK, which is localized predominantly in the trans-Golgi
network (TGN). The MNK protein relocates to the plasma membrane in cells exposed to
elevated copper where it functions in copper efflux (Petris et al., 2000).
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208 Tomohisa Hirobe

Sash

Sash forms a dominant spotting pattern (W-locus). This mutation occurred spontaneously
in a pair set up to provide a (C3H × 101) F1 hybrid stock (Silvers, 1979). The original mutant
had a broad sash of white around its body in the lumbar region and produced offspring like
itself when bred with a normal animal. The semi-dominant sash mutation is characterized by
a sequence inversion near the Kit gene that leads to ectopic expression of Kit (Duttlinger et
al., 1993). Homozygous B10-KitW-sh/ KitW-sh mice possess almost all-white body hair except
for the ear, and in heterozygous mice, the center of the body is covered with white hair.
Primary cultures of epidermal cell suspensions of sash mice have not detected any
melanoblasts or melanocytes (Hirobe, 2011a). However, co-culture of black
melanoblasts/melanocytes with sash keratinocytes stimulated proliferation of black
melanoblasts/melanocytes in MDMDF (Hirobe, 2011a). These results suggest that the sash
allele affects early melanoblast development without affecting the production of mitogens for
melanoblasts in keratinocytes. Moreover, human epidermal melanocytes can be grown in hair
follicles of B10-KitW-sh/ KitW-sh mice. After plucking out all the reconstituted hairs, the
secondary hairs were regrown in the same area and their colors were lighter than the first
reconstituted hairs (Ideta et al., 2006). These results also support the assumption that sash
keratinocytes possess a normal function in the melanocyte environment.

CONCLUSION
The coat color genes that were the focus of this chapter mostly act directly on
melanocytes, whereas the agouti and nonagouti black alleles act on the tissue environment,
especially on fibroblasts in dermal papilla.
The sash and slaty alleles affect melanoblast migration and differentiation. The albino
and pink-eyed dilution alleles influence melanoblast proliferation. The brown, pink-eyed
dilution and slaty alleles control formation of stage I and II melanosomes in melanoblasts.
The albino, pink-eyed dilution, recessive yellow, slaty and ruby eye 2d alleles affect
expression and activity of Tyr in melanocytes. The brown, pink-eyed dilution, slaty and ruby
eye 2d alleles affect melanosome maturation, especially stage IV melanosome maturation.
The agouti, lethal yellow, nonagouti black and recessive yellow affect the types of
melanin synthesized (eumelanin or pheomelanin).
Finally, the dilute allele is involved in regulating melanosome transfer from melanocyte
dendrites to keratinocytes. Eumelanin and pheomelanin synthesis are regulated by numerous
coat color genes in mice. Eumelanin contents in agouti and dilute melanocytes are similar to
black melanocytes, whereas the contents in brown, pink-eyed dilution, slaty and ruby-eye 2d
melanocytes are reduced to one-third~one thirthieth.
In contrast, pheomelanin contents in agouti, dilute, slaty and ruby-eye 2d melanocytes are
similar to its content in black melanocytes, whereas the content in brown melanocytes is
increased.
Eumelanin and pheomelanin contents in cultured epidermal melanocytes correlate well
with those in the epidermis/dermis and hairs of the mice, except that agouti melanocytes do
not synthesize pheomelanin in culture, the pink-eyed dilution allele does not affect

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 The Coat Color Genes… 209

pheomelanin content in hairs, and the ruby-eye 2d allele increases pheomelanin content in
hairs. Thus, eumelanin and pheomelanin synthesis in melanocytes is regulated by numerous
coat color genes in a very complicated manner.

ACKNOWLEDGMENTS
The author expresses his thanks to Prof./Drs. Takeuchi (Tohoku University, deceased in
1996), Ito/Wakamatsu (Fujita Health University), Abe (Yamagata University),
Kawa/Mizoguchi/Soma (St. Marianna University), Takeuchi/Hotta/Yoshihara (Okayama
University), Furuya/Akiu/Naganuma/Fukuda/Ideta/Ifuku/Hara/Horii (Shiseido), Nishikawa
/Osawa (RIKEN), Eguchi-Kasai/Sugaya/Murakami (National Institute of Radiological
Sciences), Ogawa/Ishizuka (Joetsu University of Education) and Ootaka/Terunuma/Kiuchi
(Chiba University) for their collaboration in the original papers.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 7

THE ROLE OF MELANIN PRODUCTION IN

GAEUMANNOMYCES GRAMINIS INFECTION OF
CEREAL PLANTS

Hanafy Fouly1, Shelby Henning2, Osman Radwan2,

Henry Wilkinson3 and Bruce Martin1
1
Department of Entomology, Soil and Plant Sciences, Clemson University, SC, US
2
Department of Crop Sciences, University of Illinois, Urbana, IL, US
3
Department of Natural Resources and Environmental Sciences, US

ABSTRACT
Gaeumannomyces graminis var. graminis (Ggg) is an ascomycete that causes black
sheath rot disease of rice (Oryza sativa L.) and take-all root rot of several turfgrass
species. G. g. var. graminis synthesizes melanin and deposits it in hyphal cell walls. Our
research indicates that the nature of the association between Ggg and plant root is
parasitic, but can change to pathogenic and ultimately terminate as saprophytic. Melanin
plays several roles during fungal growth and throughout infection and colonization of
plant roots. First, hyphal morphology (diameter, shape and melanin concentration)
appears to change as the fungus invades and colonizes the tissues of the root. Second,
melanin appears to be a determinant of fungal pathogenicity. Wild-type isolates of Ggg
were pathogenic, and colonized plants showed more severe symptoms of infection while
isolates lacking melanin were able to ectotrophically colonize and penetrate roots as a
parasite, but no macroscopic symptoms of take-all were observed to indicate
pathogenicity.

INTRODUCTION
Gaeumannomyces graminis (Sacc.) Arx & D.L. Olivier var. graminis (Ggg) is an
ascomycete that infects roots of rice (Oryza sativa L.) and several turfgrasses (Hawksworth,
1995; Walker, 1981). It is an aggressive pathogen of rice causing black sheath rot disease.
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222 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

Gaeumannomyces graminis var. graminis is an aggressive colonist but a somewhat weak

pathogen of turfgrasses including centipedegrass (Eremochloa ophiuroides (Munro) Hack.)
(Wilkinson, 1994), bermudagrass (Cynodon dactylon L.) (Elliot 1991), zoysiagrass (Zoysia
japonica Steudel) (Wilkinson 1993), and St. Augustinegrass (Stenotaphrum secundatum
(Walt.) Kuntze) (Elliot, et al. 1993). It is a primary colonist, forming a perennial association
with vegetatively cloned grasses and an annual association with rice. In general, the pathogen
acts as a primary colonist of newly formed roots and crowns. As an aggressive colonist, it
mantles the root surface with highly melanized, ectotrophic hypha. Ectotrophic colonization
is supported by endotrophic root colonization of the epidermal and cortical tissues.
Subsequent invasion and colonization of the endodermis and stele tissues results in vascular
occlusion which compromises the host’s capacity to conduct water and store, transport, or
utilize available photosynthates (Jones & Clifford, 1978). Foliage discoloration and root
rotting are followed by plant death only when drought and/or heat stress occur over time..
Finally, seed formation is severely limited, or inhibited if root colonization is extensive.
Melanins are dark colored pigments produced by various organisms of all biological
kingdoms (Hill, 1992). Chemically, there are three different kinds of melanins that are
produced by living organisms (Bell and Wheeler, 1986). Brown and black pigments
manufactured from dihydroxyphenylalanine (DOPA) are termed eumelanins. Red and yellow
pigments derived from DOPA and cysteine are known as phaeomelanins. Melanins derived
from phenols and catechols which lack nitrogen are known generically as melanin (Bell and
Wheeler, 1986). In addition to cellulolytic and pectinolytic enzymes that aid in the infection
of host cells, it is important to acknowledge the presence of melanin in the hyphae of
Gaeumannomyces. Cellular synthesis of the biopolymer melanin has been linked to the
pathogenicity of fungi (Brush and Money, 1999; Henson et al., 1999; Hill, 1992; and Hornby,
1998). For example, melanin deficient mutants of the rice-blast fungus, Magnaporthe grisea,
have been demonstrated to be avirulent (Henson et al., 1999). Due to the fact that
Gaeumannomyces species are characteristically melanizied, the presence of melanin in
hyphae may play a role in the pathogenicity of Gaeumannomyces graminis (Henson et. al.,
1999).
Gaeumannomyces graminis melanin is formed by the 1, 8 DHN pathway (Henson et al.,
1999). Using wild-type and melanin deficient mutant isolates of Ggg, Frederick, et al. (1999)
showed melanin was deposited on Ggg hyphal cell walls while Bell and Wheeler (1986)
reported melanin was deposited as a layer at the exterior surface of the fungal cell wall and/or
as electron dense granules distributed within the cell wall of the melanized yeast
Phaeococcomyces. The potential benefits that DHN melanins could confer to hypha that
synthesize them are considerable. Melanin protects fungal hypha from the negative effects of
UV irradiation (Bell and Wheeler, 1986), temperature extremes (Hill 1992), over-and under-
abundance of moisture (Hill 1992), toxic concentrations of metal ions (Caesar-Tonthat et. al.,
1998), attack from antagonistic microbes (Henson et al., 1999), and extreme pH conditions
(Frederick et al., 1999).
While melanin has been implicated in the fungal colonization and infection of plants, it
has also been shown in work using melanin deficient mutatnts, that that the presence of
melanin in hyphae may not be required for infection (Frederick et al., 1999; Henson et al.,
1999). However, restoring melanin production restored pathogenicity in some non-melanized,
non-pathogenic mutants that evidently depend on the presence of melanin to penetrate host
tissues. In one study, a melanin-deficient mutant of the human pathogen, Wangiella

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 The Role of Melanin Production ... 223

dermatitidis was non-pathogenic (Brush and Money, 1999). However, when melanin
production was restored, it was able to penetrate and colonize animal tissues.
This research was divided into two phases. The objectives of the first phase were to
determine the role of melanin on linear growth, hyphal width and branch formation, and to
quantify melanin in wild-type isolates and melanin-minus mutants of Ggg. The objectives of
second phase were: to observe and measure changes in the melanin content of
Gaeumannomyces graminis var. graminis (Ggg) during pathogenesis (inoculation through
colonization of the stele) to determine if melanin content had an effect on the ability of Ggg
to infect and colonize host roots and to;. A third objective of the second phase determine the
nature of the host and parasite association as it is affected by the ability of Ggg to produce
melanin.

MATERIALS AND METHODS

Fungal Isolates and Culturing

The fungal isolates of Gaeumannomyces graminis var. graminis used in this research
were designated WT1+, M1-, WT2+, and M2- (Table 1). Isolates WT1+ and M1- were
obtained from Joan M. Henson, Department of Microbiology, Lewis Hall 109 Bozeman, MT
59717. Isolate WT1+ was originally isolated from soybean (Glycine max L.). Isolate WT1+ is
a wild-type fungus that was used to produce the hyaline, melanin deficient mutant M1- using
nitroquinolene oxide (NQO) as the mutagenic agent (Epstein, et al., 1994). Isolate WT2+ is a
wild-type of Ggg obtained from Monica Elliot, University of Florida, Fort Lauderdale REC
3205 College Avenue, Ft. Lauderdale FL 33314. Isolate M2- is a hyaline, melanin deficient
mutant produced from WT2+ by the method of Frederick, et.al. (1999). All isolates were
maintained on potato dextrose agar (PDA) (Sigma-Aldrich, St. Louis, MO, USA) and
transferred every 7 days to fresh media.

Hyphal Morphology and Vegetative Growth Rate

To determine the effect of melanin on hyphal morphology, measurements of hyphal

width (W) and distance between hyphal branches (DBB) were recorded where isolates grew
in culture. Each isolate was cultured and evaluated on three different media: Luria-Bertani
agar (LBA, 5g tryptone, 10g NaCl, 5g yeast extract, 15g agar/1L water), vegetable juice agar
(V8, 200ml V8 juice, 1.8g CaCO3, 15g agar/1L water) and Czapek-Dox agar (CDA, 3g
NaNO3, 0.5g KCl, 0.5g MgSO4, 0.01g FeSO4, 1g K2HPO4, 30g sucrose, 15g agar/1L water)
using 3 repetitions (1 petri-plate = 1 repetition). When the leading edge of a colony had
extended to the perimeter of the petri plate, or 7 days had elapsed, measurements of hyphal
width and distance between branching were recorded. Distance between branching was
defined by two consecutive points of intersection formed between the main hypha and the
hyphal branch. Within these randomly selected areas of a culture, 10 measurements per area
were recorded. Each experiment was replicated 3 times and repeated 3 times. Measurements
were made using an ocular micrometer and an Olympus BH-2 light microscope (40 x).
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224 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

Table 1. Gaeumannomyces graminis var. graminis isolates

Desig- Plant Source Descri-ption Coloration Hypho-podia Reference/Source

nation* of Adult
Thallus
WT1+ Soybean wild type black lobed, Frederick, 1999
melanized
M1- Soybean NQO mutant hyaline lobed, Frederick, 1999
of WT1+ melanized
WT2+ Bermu- wild type black lobed, M. Elliot, FL.
dagrass melanized
M2- Bermu- NQO mutant hyaline simple, S. Henning
dagrass of WT2+ hyaline
* WT= wild-type; reference number; + = pigmented; and - = non-pigmentation,
NQO = 4-Nitroquinoline-1-oxide.

To determine the effect of melanin on the vegetative growth of isolates in culture, studies
comparing radial growth on agar media were conducted. Each isolate was cultured on LBA,
CDA, and V8A media. The diameter (mm) of each colony was recorded using digital
calipers. Measurements were recorded every 24h from the time of seeding and until the
leading edge of a colony had reached the edge of the petri plate or 7 days had elapsed.
Growth experiments were repeated 3 times, with 3 replications per experiment. The data were
used to calculate mean daily growth rate (mm).

MELANIN QUANTIFICATION
Purification of Melanin from Wild-type Hyphae

Melanin concentration was estimated using Azure A as a melanin binding agent. Melanin
was produced by culturing WT1+ in LB broth (LBB, 5g tryptone, 10g NaCl, 5g yeast
extract/1L water). The LBB was seeded with 10 culture plugs (1.0 mm diameter) of WT1+
taken from leading edge of a colony growing on LBA. The LBB cultures were incubated at
room temperature (20-23°C) on an orbital shaker (150 rpm) for 7 days. The LBB medium
was then separated from the hyphae by gentle vacuum filtration and discarded. The fungal
mat was cut into 5 mm pieces, submersed in acetone and heated to 50°C for 30 minutes. The
acetone was then separated from the hyphae by vacuum filtration and discarded. The fungal
mass was then washed three times by pouring 100 ml distilled water (20°C) over the fungal
tissue. The fungal tissue was then immersed in 200 ml absolute ethanol and heated to 85°C
for 3 hours in a hot water-bath. The fungal tissue was separated and washed as described
previously. The fungal tissue was then placed into a 500 ml single neck boiling flask
equipped with a dry reflux condenser. To the fungal mass, 200 ml 38% HCl were added via
the reflux condenser and heated to 85°C for 18 hours in a fume hood. The resulting melanin
granules were collected from the resulting black suspension by ultra-centrifugation (13,200

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 The Role of Melanin Production ... 225

rpm, 60 sec.), washed 3 times with 38 ml distilled water, dried over anhydrous CaCl2 under
vacuum and stored at -80°C. The resulting material was considered concentrated melanin.

Absorption of Azure A by Melanin

The melanin-Azure A binding coefficient was determined from a reaction of concentrated

melanin and a stock solution of Azure A (4.75 mg Azure A/1L 0.1M HCl). The Azure A
solution (4.75 µg Azure A/1ml 0.1M HCl) had an absorbance of 0.6 O.D. at 610 nm. Serial
dilutions of the reaction solution resulted in a proportional decrease in absorbance with a
lower limit of detection estimated at 10 µg/ml. Triplicate samples of melanin (250, 500, 1000
µg) were each placed into 15 ml Corex tubes and 3 mls of Azure A stock solution was added.
The reactions were incubated for 60 minutes at 20°C with slight shaking (50 rpm). The
melanin-Azure complex was then separated from the Azure A solution using an ultra-
centrifuge (13,200 rpm, 60 sec.). The optical density of the Azure A remaining in solution
was measured at 610 nm and recorded. It was calculated that a 1 milligram of concentrated
melanin absorbed 873 µg of Azure A in solution and that 1 milligram of melanin would
decrease the optical density (610 nm) of the Azure A stock solution by 0.13 units.

Quantification of Melanin in Mycelia

Measurement of mycelial melanin was made using a modification of melanin

quantification reported by Butler and LaChance (1986). Fungal tissue used for melanin
quantification was cultured in Erlenmeyer flasks (125 ml) containing 60 ml LBB. Cultures
were started with 3 plugs of an isolate taken from the leading edge of a colony on LBA using
a Pasteur pipette and sterile technique as previously described. Cultures were shaken at 150
rpm and maintained at laboratory temperature (20-22°C) for 7, 14, 21, or 28 days. At the end
of each growth period, fungal material was collected by removing the LBB using vacuum
filtration, washed as previously described and then lyophilized for 24 hours. Hyphal melanin
was assayed by reacting triplicate samples of lyophilized hyphae (2000 µg) and Azure A
stock solution as described above. The reactions were incubated at 20°C for 60 minutes with
orbital shaking (50 rpm). The hyphae were then separated from the Azure A solution using an
ultra-centrifuge (13200 rpm, 60 sec.). The optical density of the Azure A solution was
measured (610 nm) and recorded. The loss in optical density of the Azure A solution was
compared with losses in optical density resulting from concentrated melanin to determine the
melanin concentration (µg melanin/mg hyphae).
Morphological experiments were complete randomized designs with sub-sampling and
three replicates. Isolate and media type were the fixed factors for both experiments with
treatment comparisons performed using contrast statements. Growth rate and melanin content
were analyzed over time for isolate and media combinations using linear regression. All
statistics were performed using general linear model or regression procedures of SAS
statistical software (SAS Institute Inc., Cary, NC, USA). Every experiment was repeated at
least once.
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226 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

PATHOGENICITY TESTS
Inoculation of Rice Using Conetainer Assay

The fungal isolates of Gaeumannomyces graminis var. graminis WT2+ and its melanin
deficient counterpart isolate WT2- were used in this study. Inoculum was produced in
Erlenmeyer flasks containing millet (Panicum miliaceum L.) seed (50 ml) and deionized
water (50 ml), autoclave-sterilized (32 psi, 161°C) for 1 hour. The moist grain was allowed
to cool for 24 hours and then was autoclaved a second time. Upon cooling, the grain was
seeded with culture plugs of WT2= or WT2- (5 plugs ca. 5 mm square) excised from the
leading edge of a young fungal colony. The flasks were vigorously shaken every 24 hours for
the first 2 days to uniformly distribute inoculum with the millet. After the millet appeared
covered with fungal mycelium, it was removed from the container and dried under a laminar
flow hood for 48 hours. The dried inoculum was stored at room temperature in the dark and
periodically evaluated for contamination and viability by plating 1-20 millet kernels on the
surface of PDA.
The procedure used for host inoculation was a modification of a conetainer assay
previously reported by Wilkinson et al. (1985). A cotton ball was placed at the bottom of a
small conetainer (16 x 4 cm) (Ray Leach, Inc. Canby, OR). The conetainer was then filled to
within 4 cm of the top with double-autoclaved vermiculite. Five colonized millet seeds were
placed on top of the vermiculite layer. Upon this layer of inoculum, a 0.5 cm thick layer of
double-autoclaved vermiculite was added. Three surface-sterilized rice (Oryza sativa
‘Cypress’) seeds (1 minute soaking in 2.5% sodium hypochlorite, rinsed with sterile water
until no smell of bleach remained) of a host species are placed on top of the vermiculite and
covered with an additional 0.5 cm thick layer of double-autoclaved vermiculite. The filled
conetainer was then placed into a holding rack. A total of 16 conetainers were prepared for
each treatment. Eight additional conetainers were prepared lacking the pathogen and these
served as control treatments. The conetainers were placed on a mist bench (10 seconds misted
water/15 minutes) until the vegetative growth of each species was approximately 2.54 cm tall.
Then, the conetainers were placed in a growth chamber (15 or 30C, 18 hours of light/6 hours
of darkness cycle). Each conetainer was kept moist by topical applications of distilled water.
Inoculated rice plants were rated for disease severity using a modified version of a
previously reported assay (Wilkinson et al., 1985) using a randomized block design. Each
week, for a total of 4 weeks after being placed in the growth chamber, 4 conetainers per
treatment as well as 2 non-inoculated control treatments were randomly removed from
incubation, the roots washed free of vermiculite, and the roots rated for disease severity as
follows: (no disease present, DS=0); (1-25% of roots with necrotic tissue, DS=1); (26 –50%
necrotic, DS=2); (51-75% necrotic, DS=3); (76-100% necrotic, DS=4). All treatments were
replicated 3 times as 3 independent biological replicates. Statistical analysis of the data was
performed by SAS statistical analysis software (SAS Institute Inc., Cary, NC, USA) using
analysis over time and standard deviations are given.

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 The Role of Melanin Production ... 227

Inoculation of Rice Using a Petri-plate Assay

Rice was also inoculated using a Petri plate assay. Isolates used for the plate assay were
wild-type WT2+ and WT1+, and their respective melanin-deficient counterparts M2- and
M1-. Rice seeds were prepared by removing the outer husk and surface disinfesting them in 3
percent aqueous solution of hydrogen peroxide containing 100 ul of polyoxyethylenesorbitan
(Sigma# P139). The disinfesting solution was decanted and the seeds allowed to dry on sterile
paper towels. Three surface disinfested seeds were then placed at the outer edges of 90 mm
Petri plates containing 10 ml potato dextrose agar. Plates were then placed under constant
fluorescent lighting until germinated roots were approximately 6 cm long. Using sterile
technique, seedling roots were inoculated with one 2-mm2 plug cut from the leading edge of
an actively growing colony cultured on PDA. The inoculum plug was placed in the center of
the Petri pate containing germinated seeds. Inoculated plates were then placed under
fluorescent lighting and monitored to determine when the fungus intersected a root. Plants
were harvested 28 days following the initial contact between the fungus and the root. Samples
were then embedded and sectioned for microscopicobservation.

Wax Embedment and Sectioning of Rice Roots Harvested from Conetainer

Assay

Roots harvested from the conetainer assay were submerged in formalin-acetic acid-
alcohol (FAA) solution for 48 hours to fix both host and fungus tissues. The fixed samples
were dissected. Pieces (2 cm in length) of the main root from the area closest to the inoculum
were excised, initially dehydrated in a graded water/ethanol series, and finally dehydrated a
graded ethanol/xylene series. Dehydrated samples were prepared for sectioning by infiltrating
them with molten Paraplast (Sigma# P3558) at 60°C over a 24-hour time period. The
infiltrated samples were placed into hand folded cube-shaped tin-foil molds, and embedded in
molten paraplast. Thin section (10 µm) were then cut with a hand-operated rotary microtome,
floated on 7% formaldehyde solution on gelatin (Sigma# G6144) coated slides, and incubated
at 30°C for 24 h. Paraplast was removed from sections on slides by immersing them in
several changes of xylene until no paraplast was observed when viewed at 400 X. Sections
were stained by immersing slides in hematoxylin solution (0.2% aqueous hematoxylin
(Sigma# H3136), and 0.2% potassium iodide) for 2 hours, followed by rinsing briefly under
gently flowing tap-water. Sections were further stained by placing them in 1% aqueous Fast
Green (Sigma# F758) for 30 seconds, and rinsing briefly. Hematoxylin-Fast Green stained
slides were quickly dehydrated in an ethanol/water series (70:30, 95:5, 100:0; 20-30 seconds
in each solution), dipped in xylene, and allowed to air dry. Slides were then mounted in 3
drops of permount (Electron Microscopy Sciences, 1560 Industry Rd., Box 550 Hatfield, PA
19440), covered with 80-mm cover slips and allowed to dry overnight previous to
examination with an Olympus BH-2 compound microscope (40-100X).
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228 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

Agarose Embedment and Sectioning of Rice Roots Harvested from Petri-

plate Assay

Roots from plants harvested from the Petri plate assay were excised and embedded in
molten 3% agarose contained in 2.5 ml cryovials (Sigma# V9380). Following agarose
solidification (about 15 minutes), the embedded roots were removed from cryovial containers
and hand-sectioned underwater using a half of a double-edged razor blade under
magnification (dissecting microscope at 40x). Sections were placed on microscope slides,
observed at 100-1000X, and digital images photographically captured. Samples were
observed either stained or not. Samples were stained by placing one drop of Azure A stain
(Sigma# A918, 1g Azure A/L in 95% ethanol) on them prior to application of a cover slip.

RESULTS
The Effect of Melanin Hyphal Width

There was no significant difference in hyphal width between Ggg wild-type isolates when
cultured on LBA or V8A media (Table 2). There were significant differences in hyphal width
between the two wild-type Ggg’s cultured on CDA where WT1+ was wider (27%) than
WT2+. Wild-type WT1+ and its corresponding melanin-deficient mutant (M1-) displayed
significant differences in hyphal width on all tested media. Wild-type WT1+ hyphae were
wider than M1- on CDA (19%) and V8A (28%) media. Melanin-deficient mutant M1- was
wider (26%) compared to WT1+ on LBA media. Wild-type WT2+ and its’ corresponding
melanin-deficient mutant M2- showed differences in hyphal width. When cultured on CDA,
M2- hyphae were wider (24%) compared to wild-type WT2+. When grown on V8A, WT2+
had wider (19%) hyphae than M2-. There were no differences in hyphal width between
WT2+ and M2- cultured on LBA medium.

Table 2. Mean Hyphal width of wild-type and melanin deficient Gaeumannomyces

graminis var. graminis isolates

Isolate Contrasts CDA LBA V8A

WT1+ 3.90 2.89 3.63
WT2+ 2.85 3.12 3.86
** NS NS
WT1+ 3.90 2.89 3.63
M1- 3.16 3.90 2.61
** ** **
WT2+ 2.85 3.12 3.86
M2- 3.78 3.28 3.12
** NS **
Mean hyphal widths (um) were calculated using the datda from three separate experiments. Each
experiment was replicated 3x and repeated 3x (n=90). * and ** represent an alpha level of 0.05 and
<0.001, respectively. NS = not significant. Ten measurements of hyphal diameter were recorded by
randomly selecting 10 different hyphae from within each of those randomly selected areas of a
culture. WT = wild type and M = mutant ;CDA= Czapek-Dox Agar, LBA= Luria-Bertani agar, and
V8A= vegetable juice agar.

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The Effect of Melanin on Hyphal Distance between Branches

In general, the distance between hyphal branches was longer for wild-type isolates than
their respective mutants. Distances between hyphal branches (DBB) were significantly
different between the two wild-type isolates cultured on each of the three media tested (Table
3). On CDA, WT1+ had an 18% longer DBB than WT2+. On LBA and V8A, respectively,
WT2+ exhibited 12% and 27% longer DBB, respectively, compared to WT1+. In comparing
the wild-type with their respective mutants, WT1+ displayed longer DBB on both CDA
(22%) and LBA (26%) media compared to M1-. On V8A medium, WT1+ and M1- showed
no differences in DBB. Isolate WT2+ exhibited a significantly longer DBB on CDA (41%),
LBA (50%), and V8A (67%) compared to M2-.

Fungal Vegetative Growth in Different Cultures

All fungal isolates were grown on LBA medium displayed differences in hyphal growth
rates (Table 4). WT2+ (15.3 mm/day) grew faster than WT1+ (12.2 mm/day) on LBA. Isolate
WT2+ grew faster (15.3 mm/day) than M2- (0.5 mm/day) on LBA medium. M1- grew faster
(14.6 mm/day) than WT1+ (12.2 mm/day).

Table 3. Mean Distance between branches of wild-type and melanin deficient

Gaeumannomyces graminis var. graminis

Isolate Contrasts CDA LBA V8A

WT1+ 64.88 67.72 59.32
WT2+ 53.29 77.11 82.08
** * **
WT1+ 64.88 67.72 59.32
M1_ 50.38 49.96 57.54
** ** NS
WT2+ 53.29 77.11 82.08
M2- 31.45 38.86 27.14
** ** **
Mean hyphal widths (um) were calculated using the data from three separate experiments. Each
experiment was replicated 3x and repeated 3x (n=90). * and ** represent an alpha level of 0.05 and
<0.001, respectively. NS = not significant. Ten measurements of hyphal diameter were recorded by
randomly selecting 10 different hyphae from within each of those randomly selected areas of a
culture. WT = wild type and M = mutant ;CDA= Czapek-Dox Agar, LBA= Luria-Bertani agar, and
V8A= vegetable juice agar.

When fungal isolates were cultured on CDA medium, there were significant differences
in growth rate between the wild-type isolates and between each wild-type isolate and their
corresponding melanin deficient mutant (Table 5). WT1+ grew significantly faster (10.8
mm/day) compared to WT2+ (3.6 mm/day). Wild-type isolates grew significantly slower than
their corresponding melanin deficient mutants. Isolate WT1+ grew at a slower (10.8 mm/day)
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230 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

than M1- (15.2 mm/day) when cultured on CDA. Wild-type WT2+ grew slower (3.6
mm/day) compared to M2- (5.3 mm/day) on CDA.

Table 4. Regression equations and contrasts of analysis of growth rate of

Geaumannomyces graminis var. graminis isolates cultured on Luria-Bertani agar

Isolate Regression equation

WT1+ y = 12.168x -3.968
M1- y = 14.562x -1.696
WT2+ y = 15.326x -11.88
M2- y = 0.502x -8.26
Contrasts of Growth Rates p-value
WT1+ vs. WT2+ < .0001
WT1+ vs. M1- 0.0025
WT2+ vs. M2- < .0001
Regression equations were generated from regression lines fitted to growth rate (mm/day) of wild-type
(WT1+ &WT2+) and melanin deficient mutant (M1- & M2-) Gaeumannomyces graminis var.
graminis. Experiments were repeated 3 times, with 3 repetitions per experiment. Alpha level =
0.05.

Table 5. Regression equations and contrasts of analysis of growth rate of

Geaumannomyces graminis var. graminis isolates cultured on Czapek-Dox agar

Isolate Regression equation

WT1+ y = 10.774x -3.447
M1- y = 15.169x -7.016
WT2+ y = 3.61x + 0.103
M2- y = 5.292x +3.192
Contrasts of growth rate p-value
WT1+ vs. WT2+ < .0001
WT1+ vs. M1- 0.0333
WT2+ vs. M2- < .0001
Regression equations were generated from regression lines fitted to growth rate (mm/day) of wild-type
(WT1+ &WT2+) and melanin deficient mutant (M1- & M2-) Gaeumannomyces graminis var.
graminis. Experiments were repeated 3 times, with 3 repetitions per experiment. Alpha level =
0.05.

Quantification of Melanin in Gaeumannomyces Graminis var. graminis

Hypha

Melanin concentration was calculated using changes in the optical density at 610 nm of
Azure A in solution. Extracted, purified, and concentrated Ggg melanin had a binding
coefficient of 873 µg Azure A/mg melanin. Both wild-type isolates of Ggg showed no
significant difference in melanin concentration throughout the experiment (Figure 1). Wild
type isolates and their corresponding melanin deficient mutants were not significantly

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 The Role of Melanin Production ... 231

different in melanin production at the first sampling(7days of growth). Melanin concentration

appeared to reach a maximum by day 14 for the wild-type isolates (Figures 2 & 3). After 14
days of fungal growth, each wild-type had a mean of 233 µg melanin/mg hyphae. Melanin
deficient mutants M1- and M2- displayed means of 65 and 33 µg melanin/mg hyphae,
respectively (Figures 2 & 3).

500
Melanin concentration (ug melanin/mg hyphae)

400

300

200

100

0
7 14 21 28

Culture age (days)

Figure 1. Melanin concentration (ug melanin/mg hyphae) of wild-type WT1+ (●) and WT2+ (○)
Gaeumannomyces graminis var. graminis cultured in Luria-Bertani broth. Each point represents the
mean of 3 repeated trials with 3 replications per treatment. Where standard error bars cross, there is no
statistically significant difference in the data.

500
Melanin concentration (ug melanin/mg hyphae)

400

300

200

100

0
7 14 21 28

Culture age (days)

Figure 2. Melanin concentration (ug melanin/mg hyphae) of wild-type WT1+ (●) and melanin-deficient
M1- (○) Gaeumannomyces graminis var. graminis cultured in Luria-Bertani broth. Each point
represents the mean of 3 repeated trials with 3 replications per treatment. Where standard error bars
cross, there is no statistically significant difference in the data.
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232 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

500

Melanin concentration (ug melanin/mg hypae)

400

300

200

100

0
7 14 21 28

Culture age (Days)

Figure 3. Melanin concentration (ug melanin/mg hyphae) of wild-type WT1+ (●) and melanin-deficient
M2- (○) Gaeumannomyces graminis var. graminis cultured in Luria-Bertani broth. Each point
represents the mean of 3 repeated trials with 3 replications per treatment. Where standard error bars
cross, there is no statistically significant difference in the data.

Disease Severity and Ectotrophic Colonization of Rice in Conetainer Assay

Rice plants generated by conetainer assay were examined macroscopically after 28 days
of incubation (n=24). Plants inoculated with wild-type WT2+ were all severely diseased, with
all rated a 4 for mean disease severity (MDS), ..., and those inoculated with melanin-deficient
M2- were free of disease, with all rated a 0 (MDS) for each experiment (n=24). Using the
conetainer assay, wild-type WT2+ displayed extensive ectotrophic colonization (Figure 4) of
the root epidermis at the time the roots were prepared for histopathological observation (ca 28
days incubation)(Figure 4). The roots of plants inoculated with the wild-type isolate were a
uniform black color. Ectotrophic large diameter runner hyphae (5um) were darkly pigmented,
and branched extensively forming a mantle of mycelia on the root epidermis. Plants
inoculated with Ggg isolate M2- (melanin-deficient) displayed no evidence of lesions, or
other symptoms commonly observed for root ectotrophic colonization after 28 days of
incubation (Figure 4). Plants inoculated with M2- were indistinguishable from controls at 28
days of age. At the time they were prepared for sectioning, roots of M2- inoculated rice plants
exhibited no observed mycelium on the outer surface of the roots, and the roots displayed no
symptoms. Uninoculated rice plants displayed no symptoms of disease or discoloration
(Figure 4). Roots were uniform in their appearance among treatments.

Histopathological Observations of Rice Inoculated in Conetainer Assay

Unstained roots inoculated with the wild-type WT2+ isolate exhibited darkly pigmented
runner hyphae on their epidermal surfaces. Darkly pigmented hyphopodia were also detected
on roots inoculated with WT2+. Unstained root sections inoculated with WT2+ did not show
infection hyphae in the epidermis, cortex, or stele. Mycelia of the melanin-deficient isolate

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 The Role of Melanin Production ... 233

M2- were not detected in any tissues of inoculated, unstained, sectioned, root material (n=24).
Unstained sections of controls did not show the presence of fungi on or in any root tissues.
Colonizing mycelium in inoculated root samples were elucidated by staining with
Hematoxylin/Fast Green. Stained hypha were readily identified both ecto- and
endotrophically. Dark runner hyphae were easily detected without staining, and their
appearance was enhanced by staining with hematoxylin (Figure 5). Hematoxylin stained
runner hyphae were a rich, dark brown color. Runner hyphae were also observed to develop
infection pegs on the host surface, and these exhibited the same staining reaction (dark
brown) as runner hyphae (Figure 5). Infection hyphae of the wild-type fungus did not appear
melanized, and were visible in the epidermal, cortical, and vascular tissue of infected plants
only after treatment with fast green (Figure 5; n=24). Infection hyphae stained by fast green
were green/blue in color, and colonized root tissues in an intracellular manner (Figure 5).

A. B.

C.

Figure 4. Rice inoculated with Gaeumannomyces graminis var. graminis using a container system and
incubated at 15C. Uninoculated controls (A.); wild-type inoculated WT2+ (B.); melanin-deficient M2-
inoculated rice (C.).
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234 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

Figure 5. Rice roots after inoculation with Gaeumannomyces graminis var. graminis wild-type isolate
WT2+. Longitudinal section stained with hematoxylin and fast green (A.) shows infection pegs (IP);
runner hyphae (RH); and infection hyphae (IH). Longitudinal section stained with hematoxylin and fast
green (B.) shows infection pegs (IP); runner hyphae (RH); infection hyphae (IH), and xylem (X).

The fungus appeared hyaline as it traversed the endodermis and entered the stele. A color
change indicating re-melanization of hyphae at the endodermal tissue layer was not detected
in roots inoculated with the wild-type fungus after 28 days of incubation. Hematoxylin did
not appear to cause a staining reaction in infection hyphae as they traversed the endodermis
and entered the stele. Identification of infection hyphae was easiest in longitudinally
sectioned samples. After treatment with fast green, sectioned samples of rice roots inoculated

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 The Role of Melanin Production ... 235

with M2- did not exhibit stained hyphae inter- or intracellularly colonizing the epidermis,
epidermal, cortical, or vascular tissues. Sections produced from M2- inoculated roots (n=24)
and un-inoculated controls (n=4) were microscopically indistinguishable from each other.

Figure 6. Rice inoculated with Gaeumannomyces graminis var. graminis: un-inoculated (A); wild-type
WT2+ (B); melanin-deficient M2- (C). Red circle denotes border of fungal colony. Germinated seeds
are labeled as “S.”

Disease Severity and Ectotrophic Colonization of Rice in Petri-plate Assay

Rice plants inoculated with Ggg and incubated in the Petri plate assay were extensively
colonized by ectotrophic hyphae after 28 days (Figure 6). As cultures grew across the Petri-
plate, they grew over and obscured the rice roots on the plate with a mycelial mat. Inoculated
plants removed from the plates were entirely mantled by the fungi and their roots were often
embedded in the agar. The hyphae formed a much denser mantle around the roots compared
to the colonization observed on roots cultured in the conetainer assay. Wild-type isolates
WT1+ and WT2+ produced ectotrophic mycelium that was melanized and mantled the roots.
Wild-type isolates (WT1+ & WT2+) caused macroscopic lesions on black roots in inoculated
plants that coalesced (n=18 each). Melanin-deficient mutant isolates M1- and M2- were also
observed colonizing the exterior root surface and forming a mantle. These hyphae were
hyaline. The densities of the mycelial mantle of wild-type and melanin-deficient isolates were
judged to be equivalent. Melanin-deficient isolate M1- inoculated roots showed reddish
colored lesions While M2- displayed no color change or other symptoms (n=18).

Histopathological Observation of Rice inoculated in Petri-plate Assay

Unstained rice roots inoculated with Ggg isolate WT1+ showed uniformly light brown
hyphae in the epidermal, cortical, and vascular tissues by 28 days of incubation. Melanized
runner hyphae were not observed to be produced by WT1+ on the surface of inoculated roots
cultured in the Petri plate assay. Hyphae inside of plant roots appeared the same diameter (6
µm) in each tissue. A change in hyphal coloration (darkening) indicating re-melanization of
WT1+ hypha when entering the stele was not detected in any unstained sections (n=18).
Unstained sections of WT2+ infected plant material inoculated with wild-type isolate WT2+
showed that it had colonized all tissues of the root at the time of sectioning (Figure 7; n=18).
Melanized runner hyphae were not observed to be produced by WT2+ on the surface of
inoculated roots cultured in the Petri plate assay. In unstained sections, WT2+ hypha were a
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236 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

uniform brown color in each tissue (epidermis, cortex and stele) of an infected root (n=18).
These hyphae appeared to be the same diameter (6 µm) in each tissue. Sections of rice roots
indicated that WT2+ hyphae did not change hyphal coloration throughout the process of
pathogenesis. In addition, a change in hyphal coloration (darkening) indicating re-
melanization of WT2+ hypha when entering the stele was not detected in sections of
unstained inoculated roots (n=18). Unstained root sections of melanin-deficient mutant M1-
inoculated plants did not indicate the presence of hyphae in any of the roots tissues. Unstained
root sections of melanin-deficient mutant M1- inoculated plants showed lignitubers being
produced by the host in epidermal and cortical tissues at 28D of inoculation. Unstained root
sections of melanin-deficient mutant M2- inoculated plants did not indicate the presence of
hyphae in any of the roots tissues.
Staining with Azure A allowed the fungus to be readily observed in sections produced
from plants in Petri-plate assay. Azure A stained the anticlinal plant cell walls dark purple
while fungal hyphae were stained with light purple color (Figure 8). Stained roots inoculated
with Ggg isolate WT1+ showed light purple hyphae in the epidermal, cortical, and vascular
tissues by 28 days of incubation (Figure 8). Hyphae inside plant roots appeared the same
diameter (6 µm) in each tissue. A change in hyphal coloration (darkening) indicating re-
melanization of WT1+ hypha when entering the stele was not detected in any stained
sections. Azure A stained sections of WT 1+ inoculated plants showed colonizing mycelia
infecting the root in an intracellular manner. Stained roots inoculated with Ggg isolate WT2+
showed light purple hyphae in the epidermal, cortical, and vascular tissues by 28 days of
incubation. Hyphae inside of plant roots appeared the same diameter (6 µm) in each tissue. A
change in hyphal coloration (darkening) indicating re-melanization of WT2+ hypha when
entering the stele was not detected in any stained sections.

Figure 7. Rice inoculated with wild-type Gaeumannomyces graminis var. graminis isolate WT2+ and
cultured in the Petri-plate assay. The transverse sections of rice roots (A. & B.) were unstained. Root
morphology is labeled as follows: Plant cell walls (PCW), epidermis (E); cortex (C); stele (S); vascular
bundles (V). Fungal hyphae are labeled as “H.”

Azure A stained sections of WT 2+ inoculated plants showed colonizing mycelia

infecting the root in an intracellular manner. Hyphae appeared to be the same diameter (6 µm)
in each tissue. The melanin deficient isolate M1- could infect and colonize the epidermal and
cortical cells of the root by day 28 (Figure 9). The hyphae produced by M1- during infection
were hyaline in color and could not be detected without staining. Azure A stained the
anticlinal plant cell walls dark purple, and fungal hyphae were stained a light purple color.
Infective hyphae of M1- were produced intracellularly in the host root. At 28 days of

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 The Role of Melanin Production ... 237

incubation, the melanin-deficient isolate M1- was able to infect epidermal and cortical cells,
but was stopped prior to entering the stele by l]lignified host tissue (lignitubers) around its
hyphae (Figure 9; n=18). The hyphae produced by M2- during infection were hyaline in color
and could not be detected without staining. Azure A stained the anticlinal plant cell walls dark
purple, and fungal hyphae were stained a light purple color. The melanin-deficient isolate
M2- was rarely able to infect single epidermal cells intercellularly (Figure 10; n=1/18).

Figure 8. Rice inoculated with wild-type Gaeumannomyces graminis var. graminis isolate WT1+ and
cultured in the Petri-plate assay. The transverse sections of rice roots (A. & B.) were stained with Azure
A. Plant cell walls (PCW) are dark purple, fungal hyphae (H) are light purple. Root morphology is
labeled as follows: epidermis (E); cortex (C); stele (S); vascular bundles (V).

DISCUSSION
Nature of Mutations Used (Pigmentation and Morphology)

Both melanin deficient mutants (M1- and M2-) were produced from their corresponding
wild-type parents using 4-nitroquinolene-1-oxide (NQO) as the mutagenic agent.
Nitroquinolene oxide is an electrophile and a powerful carcinogen and mutagen (Sugimura,
1981). It mimics the mutagenic action of ultraviolet light and forms charge-transfer
complexes with 5'-deoxyribonucleotides (Winkle & Tinoco, 1979). Nitroquinolene oxide
(NQO) forms DNA adducts and can cause a wide range of DNA “lesions” including single-
strand breaks, pyrimidine-dimer formation, abasic sites, and oxidized bases. In bacteria and
yeast NQO has been shown to be a base substitution mutagen acting at guanine residues,
inducing mainly guanine to adenine transitions (Fronza et al. 1992). The genetic basis for the
pigmentation (melanin) changes in both M1- and M2- have not been determined. Due to the
nature of NQO chemical mutagenesis, mutations in addition to conferring changes in
pigmentation could have occurred, but have not been identified or characterized. For instance,
the melanin mutant produced by Frederick et al. (1999) also exhibited hyphopodia and other
morphological variation different from those of their parent cultures. Epstein et al. (1994)
reported mutants that differed not only in hyphopodia, but pigmentation, compared to the
parent culture. While the pigmentation mutations for these isolates have been characterized, it
is unknown whether they possess additional DNA mutations. Epstein’s isolates were selected
from either Benomyl (2 of 1000 transformants) or Phleomycin (1 of 42 transformants)
resistant transformants. The hyphopodial mutation was an artifact of the transformation
process, not the main goal. Analysis of the transformants indicated that there was a single
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238 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

insertion in each case, but the exact location of the insertion in the transformants was not
described. Bal et al. (1977) reported that NQO is a “good” mutagen for Aspergillus nidulans
(Eidam) Winters because it induces mutations at a high frequency (0.5% of treated cells) and
generates a broad spectrum of morphological and physiological changes.

Figure 9. Rice inoculated with melanin-deficient Gaeumannomyces graminis var. graminis isolate M1-
and cultured in the Petri-plate assay. The transverse sections of rice roots (A., B., C., & D.) were
stained with Azure A. Plant cell walls (PCW) are dark purple, fungal hyphae (H) are light purple. Root
morphology is labeled as follows: lignituber (L); epidermis (E); cortex (C); stele (S); vascular bundles
(V). Fungal hyphae are labeled as “H.”

Figure 10. Rice inoculated with melanin-deficient Gaeumannomyces graminis var. graminis isolate
M2- and cultured in the Petri-plate assay. The transverse sections of rice roots (A. & B.) were stained
with Azure A. Plant cell walls (PCW) are dark purple. Root morphology is labeled as follows:
epidermis (E); cortex (C); stele (S); vascular bundles (V). Fungal hyphae are labeled as “H.”

Our mutant cultures were observed to be stable for melanin content for 36 months. M1- is
similar to the hyphopodial mutant reported by Epstein et al. (1994). M1- (generated by
Frederick et al. 1999) exhibited an increased frequency in production of lobed, melanized
hyphopodia on the bottom of the polystyrene culture dish when cultivated on solid media as
compared to its WT1+ parent culture. M2- is hyaline, and did not produce hyphopodia when

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 The Role of Melanin Production ... 239

cultivated on solid media, while its corresponding parent culture WT2+ is heavily pigmented
and produces lobed hyphopodia under these conditions. M2- formed only simple hyaline
hypal tips: M1- had hyaline mycelia and lobed, pigmented hyphopodia. Further, it appears
that melanin production could be genetically segregated within a thallus of G. graminis. This
segregation appears to be non-temporal as M2- demonstrated stable hyaline mycelia and
hyphopodia for at least 36 months. Such genetic segregation could be based in the poly-
nucleic nature of ascomycetes or in the N + N status of the thallus if melanin production is a
dominant trait. Finally, it is very interesting that WT2+, a true Ggg, when transformed to M2-
appears morphologically like Gaeumannomyces graminis var. avenae (Gga) or
Gaeumannomyces graminis var. tritici (Ggt). This raises the question about the relationship
between Gga, Ggt and Ggg. Fouly et al. (1997) showed genetic dissimilarities between these
groups, but there was also a great deal of genetic similarity. Are these isolates really all Ggg
sub-species, with Gga and Ggt lacking some of the functional genes of Ggg?
Isolate WT2+ is a wild-type isolate of Ggg from bermudagrass (Table 1) and a pathogen
of this host. WT1+ and WT2+ both produced about the same amount of melanin in their
respective hyphae. Therefore differences in their behavior as reported here (growth, hyphal
width and DDB), would not be expected to be assigned to melanin content.

Effect of Melanin on Fungal Hyphal Morphology and Vegetative Growth

Rate

Measurements of hyphal width were used as a means to evaluate the effect of melanin
content on hyphal morphology. The basis for using hyphal width is that melanin, a wall
component, is suspected of imparting a more rigid wall structure, which could allow for
higher turgor pressure within a hypha. Further, it has been reported (Skou, 1981) that root-
infecting hypha are melanin-less and smaller in diameter than melanized, ectotrophic hypha.
This suggests that Gaeumannomyces species have melanin regulatory mechanisms that are
environmentally sensitive. While infectious hypha appear to be devoid of melanin, it is
unclear if they still are producing low levels of this pigment.
In general, the WT isolates developed hypha with similar widths, except when grown on
CDA. The basis of this difference could reflect the heterogenous nature of the Ggg isolates as
reported by Fouly and Wilkinson (2000). More interestingly, there were significant
differences between the WT isolates and their respective melanin-deficient mutants in terms
of hyphal width. For both isolate couplets, the WT generally displayed larger diameter hypha
than the corresponding mutant. However, there were some inconsistencies in this pattern
when considering behavior in different media. However the general trend toward larger hypha
with melanin suggests that melanin may in fact allow hypha to grow larger while fungal walls
deficient in melanin will support smaller diameter hypha. The smaller diameter of hypha for
melanin-less isolates reported here supports observations that melanin-less infecting hypha
are also smaller diameter. Epstein et al. (1994) reported that wild-type and corresponding
single gene insertion melanin/hyphopodial mutants did not show a differences in hyphal
width when cultured on dilute V8A. A reason for this difference compared to our
measurements could have resulted from different experimental growth conditions. Epstein
cultured isolates under thin layers of diluted V8A and measured hyphal widths after the agar
layer was removed from the hypha. Both the dilution of the V8A and the sub-agar culturing
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240 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

could affect the osmotic and hydration conditions of the fungal environment resulting in
moisture limitations. If melanin functions to control osmotic potential and/or allows for
greater endogenous turgor pressure, then in a dilute osmotic medium, the loss of melanin
would not be expected to correspond to a reduction in hyphal width. The comparison of our
results with those of Epstein et al. (1994) are further limited by the fact that the width of wild-
type and mutants reported were 6.4 +/- 0.05 µm whereas we reported that the wild-types here
were 3.9 and 3.6 µm on V8A and the mutants were 3.6 and 3.1 µm, respectively.
The primary site of nutrient uptake for a fungal colony is at the thin-walled hyphal tip
(Sietsma et al. 1995). This is also the only site where the fungal mycelium is actively
elongating (growing) using a complicated physio-chemical process that involves hydrostatic
pressure generated and controlled by osmotic regulation. The number of hyphal tips for a
given thallus is determined by the frequency that hyphal branches are formed.
Gaeumannomyces is characterized as having both septa, and branches. Septa are generally
intercalary to the branches. In general, fungi form more branches when exposed to optimal
growth conditions. The frequency of branching or distance between branches (DBB) is, in
part, dependent on the physical-chemical nature of the medium, and the genetic-based ability
to exploit it for growth and development (Rayner et al. 1994). As DBB increases, there are
fewer hyphal tips being produced per thallus. Our results showed that WT2+ and WT1+ did
not form branches at the same rate and that each formed branches at variable rates depending
on the growth medium. To determine the range of DBB among Ggg isolates, a large
population of isolates would be required for comparative purposes. However, the DBB data
for WT and their corresponding mutants did show that the loss of melanin resulted in
significantly shorter DBB compare to WT isolates. Further, this behavioral pattern was only
slightly affected by WT1+ in V8A medium. Epstein et al. (1994) reported that wild-type Ggg
and corresponding single insertion mutants also displayed no differences in hyphal DBB
when cultured on dilute V8A. Their work focused on the differentiation and pigmentation of
hyphal tips to form hyphopodia. Their primary objective focused on the frequency, shape,
color and stimuli of hyphopodia. However their mutants all produced melanin, although in
different degrees. For example, isolate JH849 produced melanized hyphopodia, but not in
“sufficient quantity.” Upon further examination of their work, JH849, did produce a reduced,
but unquantified amount of melanin, and was the only mutant reported to branch about half as
often (DBB = 158 µm) compared to wild-type or two other mutants of Ggg (DBB = 97, 96,
and 71 µm, respectively). Mutant isolate JH2982, produced as much or more melanin than the
wild-type (JH2033) and exhibited the shortest DBB (71 µm). While not statistically tested, it
would appear from Epstein’s work, that both hyphal width and DBB were affected similarly
by reductions in melanin content of the hypha compared to the M1- and M2- mutants used in
this study.
The three media (CDA, V8 and LBA) that were used in this research were also used by
other researchers that investigated melanin and its role in Ggg morphology (Epstien et al.
1994; Frederick et al. 1999; Money et al. 1998). These media are among the most commonly
used for fungal cultivation and in particular for culturing Gaeumannomyces. Each isolate was
cultured and evaluated for hyphal width and distance between branching when grown on
LBA and CDA. In general, as medium type became more defined, wild-type isolates
produced narrow hyphae and with a shorter distance between branches. There was no single
factor that could be attributed to the effect of medium type, melanin, and growth rate, though

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 The Role of Melanin Production ... 241

the data indicate that in general the more defined the medium, the slower the growth rate of
isolates.
The reported effect of melanin on the vegetative growth rate of Gaeumannomyces is
variable. High levels of DNH-melanin have been implicated in limiting the uptake of
nutrients by mycelium (Frederick et al. 1999; Henson et al. 1999). A constitutive melanin
producing Ggg mutant exhibited slower radial growth in culture than a melanin minus mutant
and the corresponding wild-type (Frederick et al. 1999). In addition, the melanin deficient
mutant exhibited a faster growth rate in culture as compared to the wild-type isolate of Ggg.
Epstein et al. (1994) showed that wild-type and melanin/hyphopodial mutant Ggg’s, when
cultured on dilute V8A on glass slides did not have different growth rates. However, the
culture conditions (see above) and the incompleteness of melanin disruption preclude this
work from being considered definitive in terms of the impact of melanin on vegetative growth
rate.
In data presented here, melanin deficient M1- was the fastest growing isolate, growing
faster than both wild-type isolates. However, the melanin deficient isolate, M2-, was the
slowest growing isolate under most tested conditions. This phenomenon could be due to the
presence of melanin as evidenced in the pigmented hyphopodia of this isolate. Still, the effect
of melanin on growth rate based on the data collected is not definitive. Wild-type and melanin
deficient isolates displayed variable responses when tested on different media. The pair
consisting of WT1+ and M1- generally showed the melanin deficient M1- growing faster on
different media compared to WT1+. The pair consisting of WT2+ and M2- showed the
melanin deficient strain growing slower on all but the CDA medium. In the case of CDA
medium, the mutant strain grows slightly faster than its melanin producing parent. The
complexity of the media combined with the likely multiple mutations in both M1- and M2-
preclude assigning any role of melanin in the determination of growth rate. Single insertion
mutants with disrupted genes involved in the DHN-melanin synthesis pathway along with
testing isolates for growth rate using defined media could allow for a determination of the
role of melanin in vegetative growth rate of Ggg.

Melanin Quantification

The melanin content of the wild-type and mutants reported here was measured using an
indirect method (Butler & LaChance, 1986). The binding of solubilized Azure A dye to
hyphal melanin was used to estimate the concentration of melanin in hyphae. Wild-type
isolates were not significantly different from each other in hyphal melanin content. The
mutants M1- and M2- both showed very little melanin per unit mass of mycelium and the
melanin content of WT isolates were considerably higher than the mutants. Melanin
concentration reached a constant value after 14 days in culture. Wild-type WT1+ (JH2033)
was also analyzed for melanin by Frederick, et al. (1999) who reported a concentration of 155
µg melanin/mg hyphae. The melanin concentration of the wild-type Ggg isolates reported
here were 250 and 115 µg melanin/mg hyphae, respectively, thereby supporting the use of the
Azure A method for mycelial melanin determination. While the mutants M1- and M2-
reported here produced significantly less melanin compare to their respective parent cultures,
they did produce an average of 65 and 30 µg melanin/mg mycelium respectively at 14-28
days of age according to the Azure A melanin assay. Bell and Wheeler (1986) and Frederick
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242 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

et al. (1999) also reported that melanin-deficient mutants of Ggg showed a similar degree of
Azure A binding when compared to M1- and M2- reported here. This can perhaps be
explained by cell wall components other than melanin absorbing some of the Azure A, but not
an amount comparable to the binding coefficient of melanin. Hyphopodia were not produced
by liquid-grown isolates, and this precludes the affect the melanin status of these appendages
could produce in the assay. Furthermore, when living cultures of M1- and M2- were stained
with Azure A and examined at 400X, both the cell walls and the cytoplasm absorbed some
Azure A. We also immersed Pythium aphanidermatum (Edson) Fitzp. cultures in Azure A
solution, and it absorbed stain as well. The reason for this may be due to the cell walls of
Pythium being composed primarily of beta-glucans and cellulose, not chitin (a polymer of N–
acetylglucosamine), as in filamentous fungi. These compounds may absorb Azure A to a
greater extent than chitin. The melanized yeast, Phaeococcomyces, was reported to bind
Azure A to melanin located in its cell walls (Butler & LaChance, 1986). Melanin-deficient
mutants of this yeast did not take up stain at the cell wall, though dead or impaired cells
showed staining.
The issues dealing with melanin as a determinant of Ggg morphology and vegetative
growth reported here give a strong indication that melanin is important to the basic growth
and development of the fungus. To further test this using a more definitive approach, single
DNA insertion into one or more of the enzymes of the DHN melanin pathway should be used.
Using these defined mutants would also allow non-specific binding of Azure A to be assigned
to mycelial components other than melanin.

The Effect of Melanin on the Histopathology of Gaeumannomyces Graminis

The location and quantification (+/-) of hyphal melanin in sectioned tissues during
pathogenesis was difficult to determine. Melanized runner hyphae were readily seen on rice
plants inoculated with a wild-type isolatein the conetainer assay. In the conetainer assay, the
wild-type exhibited no discernable melanization of hyphae when it had gained entry to the
plant. Re-melanization of hyphae prior to infection of the stele, as seen by Wilkinson
(personal communication) in regard to Ggt was not observed. Yet, it is common knowledge
that Gaeumannomyces form melanized hyphae in necrotic plants at the later stages of
pathogenesis. One aspect not included in these studies was induction ofphysical/physiological
stress applied to the host. It is possible that had heat or drought stress been applied to the
colonized host, the hyphae might have reacted differently.
Staining of fungal elements (macro and micro-hyphae, infection pegs, hyphopodia) was
investigated though several methods in order to determine if stains would enhance
observation of fungal melanin. Some staining procedures were not useful due to their
interference with the visual detection of melanin. Melanin specific stains such as Masson-
Fontana and Schmorl’s used to stain these samples precluded enhanced observations of
melanin deposition on hyphae because they stain all fungal tissues black and therefore
obscure melanin. This was also reported by Masatomo et al. (1998) and Gupta et al. (1985).
Periodic acid-Shiff staining was attempted to contrast melanized and non-melanized hyphae,
but was discarded as it stained all tissues bright crimson and did not enhance the visualization
of hyphal melanin. Azure A, a melanin specific stain, was also found not to be useful in
selectively staining for melanin. Azure A stained plant tissues as well as fungal elements and

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proved valuable for enhancing observation of the fungus in rice roots inoculated in Petri-plate
assay. This is interesting because infection hyphae are hyaline in appearance. It was recorded
(see Chapter 1) that melanin-deficient strains of Ggg can show a degree of Azure A binding.
The staining of hyaline infection hyphae by Azure A appears to support this finding.
Fluorescence microscopy was also investigated. Melanin will fluoresce if oxidized (Kyatz et
al., 2001). It was postulated that by inducing fluorescence, melanin could be pinpointed as to
where it was being deposited by the fungus. Unfortunately, oxidizing melanin with hydrogen
peroxide to enhance visualization was not useful because plant tissues fluoresced brightly as
well, and melanin in hyphae could not be discerned.
It has been stated that there are three phases for fungal nutrition during plant infection
(Solomon et al. 2003). These phases are germination, proliferation, and sporulation. These
phases can be compared to the seven stages of pathogenesis and are useful as a context for
studying the production of melanin by Ggg in situ. The first, or germination, phase can be
compared to melanized Ggg hyphae growing towards a host from the tissues they have
overwintered upon (dissemination, stage 2). During this time, external nutrient sources are
likely to be in short supply, and the fungus is at a disadvantage to compete with host defenses
and competitive microorganisms. The production of melanin at this time may assist the
pathogen in survival during inoculation (stage 3) and pre-penetration (stage 4), penetration
(stage 5), and infection, (stage 6). The second phase, proliferation, takes place after the
fungus has gained entry to the host. At this stage, nutrients are not limiting, and competing
microorganisms will be reduced, if not eliminated, compared to stage one. After this stage,
compatibility (stage 7) is determined, and melanin may not be necessary for the advance of
the fungus throughout the rest of the host. At this time, the production of melanin may either
hinder the progress of disease or be unnecessary for pathogenesis. This may be why Ggg is at
first hyaline when it invades the cortex and stele of infected plants. The production of spores
(sporulation, stage 3) by Ggg is not thought to be a major determinant in the spread of
Gaeumannomyces (Skou 1981). Instead, the re-melanization of hyphae in host tissues post-
mortem is most likely most comparable to this stage of pathogenic nutritional requirements.
At this point, the protective role melanin biosynthesis serves may be necessary for the
pathogen in order for it to complete the disease cycle. In this case, melanin will protect the
fungus as it competes with other organisms in the soil for nutrient sources, and aid in the
overwintering process, thus completing the disease cycle. This is why it remains to be
determined when Ggg resumes melanin production between colonization of the stele and
necrosis.

Effect of Melanin on Fungal Pathogenicity

Pathogenicity of Ggg isolates was affected by their ability to produce melanin. It has
been shown that melanin is necessary for Ggt to cause disease (Kelly 1997).
Gaeumannomyces graminis var. tritici may have to re-melanize upon encounter of the stele in
order to penetrate vascular tissue (disease), while Ggg may not. Frederick et al. (1999)
showed that melanin was not necessary for Ggg to cause disease in rice, but did not examine
infected plants histopathologically to determine melanin status of infective hyphae, or where
they were produced. Infected plants may have been colonized by the fungi they were
inoculated with, and not actually diseased. Here, melanized wild type isolates were
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244 Hanafy Fouly, Shelby. Henning, Osman Radwan et al.

pathogenic, and isolates lacking in melanin were reduced in their ability to infect and colonize
the host. Melanin can bind to and inactivate chemical agents (Hill 1992). The inability of
melanin deficient strains to colonize all tissues of host roots (parasitic vs. pathogenic) may be
a result of failure to resist host-defense chemical mechanisms. This phenomenon has been
described in rice (Datta et al., 2001; Nishizawa et al., 1999). Also, since melanin has an effect
on hyphal turgor pressure (Brush and Money, 1999), it may be that non-melanized isolates
could not produce the necessary mechanical force to penetrate through the lignified host
response (lignitubers) in the epidermal and cortical layers. In contrast to this, melanized
isolates were able to infect all tissues of rice roots by 28 days. Genetic analysis of the
mutations in the mutant isolates would help to characterize the mutation of the mutant Ggg
strains and perhaps their influence on the infection process.
It is important to point out that there were differences in the way the fungus behaved
between the conetainer and plate assay. In conetainer assay, the wild-type produced runner
hyphae, infection pegs, and non-melanized infection hyphae. This differentiation of hyphae is
consistent with those found in “naturally” infected plants in the field. In Petri plate assay,
only one type of hyphae was seen. These hyphae were distinguishable from those that
proliferated on the culture medium only by the fact that they were produced in the roots of
plants. Some researchers (Frederick et al., 1999) have stated that since melanin serves in a
protective role for hyphae, unmelanized strains would be at a disadvantage in a natural
setting. This may be true since M2- did not infect in conetainer assay, where the environment
is not as conducive to the survival of the fungus as it is in culture (Petri-plate assay). While
melanin deficient (M1- & M2-) Ggg strains were able to parasitize roots in plate assay,
designation of these fungi as parasites may be correct only in an environment where the host
is at an extreme disadvantage to repel the fungus. In a more natural setting, it appears that
melanin may be required not only as a determinant of pathogenism, but for survival outside of
culture.
This research indicates that the common rating system of virulence (i.e., darkening of
roots) may not be adequate for assessing whether a particular fungus is a parasite or a
pathogen. Rating roots for infection by Ggg based solely on epidermal darkening may not
illustrate the actual relationship between host and fungus. When acting as parasites, melanin-
deficient strains did not induce characteristic blackening of host roots. This interaction of
host/pathogen would not be properly characterized by using this traditional scale.
It is undisputed that the fungus will at some point re-melanize in host tissues (Skou,
1981.). This has been previously documented in the fact that wild type fungi will often
develop “cessation structures” if they are unable to advance past a certain point in host roots.
This is often seen in another melanized monocot root pathogen, Magnaporthe poae. These
structures are also produced, seemingly at random, in compromised host roots by Ggg (Skou
1981). While their function remains unknown, it is postulated that they may function as
survival structures (overwintering) for the fungus in temperate zones (Skou 1981). It may be
that the time-frame involved in both assays reported here are too short for this phenomenon to
be evidenced and that re-melanization takes a longer amount of time than we used. Frederick
et al. (1999) reported that a non-melanized mutant derived from WT1+ (JH4300) was
pathogenic 28 days post-inoculation. Unfortunately, this isolate is no longer living and thus is
precluded from our studies. It should be noted that their conditions for host/pathogen
interaction was rated by root dry weights, at a time of incubation at 25C for 28D. Melanized
wild-type isolates were capable, under our conditions, of colonizing the stele (i.e., causing

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 The Role of Melanin Production ... 245

disease) by 28 days and melanin-deficient isolates were not. It may be that after six months
the non-melanized isolates might be able to compromise host defenses, but the apparent
advantage that melanin confers to the fungi that synthesize it are clear: melanin assists in the
ability of Ggg to cause disease.
The objective of this research was to determine if melanin plays a role in the
pathogenicity of Ggg. The evidence presented here indicates that the ability of wild-type
isolates to produce melanin has a marked effect on their ability to produce disease symptoms
on rice roots. This effect on disease severity caused by Ggg is uniform over the plants
evaluated. Melanin producing wild-types caused extensive root rotting in most host roots,
while melanin-deficient strains did not. Melanin-deficient strain WT1- caused slight
discoloration of inoculated oats, wheat and rice roots (data not presented). This differed from
the coloration caused by wild-type isolates. Wild-type Ggg caused characteristic blackening
of host roots. In some cases, wild-types were able to completely kill and rot host roots into an
unrecognizable state. In contrast, melanin-deficient strain WT1- induced a light brown, rather
than black color to inoculated roots, a phenomenon especially noticed in rice. These lesions
macroscopically appeared to be limited in their deleterious effect on the roots (Wilkinson,
personal communication).
The effect of melanin on the pathogenicity of Gaeumannomyces graminis var. graminis
based solely on symptoms of host roots as indicated by this research is that melanin is
required by Ggg to induce disease symptoms in host roots. This research shows a dependence
on melanin by Ggg in order to cause characteristic blackened and rotted roots in host plants.
In conetainer assay conducted with melanized wild-type fungi and their melanin-deficient
counterparts, there was a significant difference in the ability of wild-type and melanin-mutant
isolates to colonize rice plants.

REFERENCES
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Bell, A. A., and Wheeler, M.H. 1986. Biosynthesis and Functions of Fungal Melanins. Ann.
Rev. Phytopathology. 24: 411-51.
Brush, L, and N.P. Money. 1999. Invasive hyphal growth in Wangiella dermatitidis is
induced by stab inoculation and shows dependence upon melanin biosynthesis. Fungal
Genetics and Biology 20:190-200.
Butler, M.J., and Lachance, M.A. 1986. Quantitative binding of azure A to melanin of the
black yeast Phaeococcomyces. Exp. Mycol. 10: 166-170.
Caesar-Tonthat, T., Kloeke, F.V.O., Geesey, G.G. and Henson, J.M. 1998. Melanin
production by a filamentous soil fungus in response to copper and localization of copper
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Datta, K., J.M. Tu, N. Oliva, I. Ona, R. Velazhahan, T.W. Mew, S. Muthukrishnan, and S.K.
Datta. 2001. Enhanced resistance to sheath blight by constitutive expression of infection-
related rice chitinase in transgenic elite indica rice cultivars. Plant Science v. 160, p. 405-
414.
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Elliot, M.L. 1991. Determination of an etiological agent of Bermudagrass decline.

Phytopathology. 83: 414-418.
Elliot, M.L., Hagan, A.K., and Mullen, J.M.. 1993. Association of Gaeumannomyces
graminis var. graminis with St. Augustine grass root rot disease. Plant Dis. 77: 206-209.
Epstein, L., Kaur, S., Goins, T., Kwon, Y.H., and Henson, J.M. 1994. Production of
hyphopodia by wild-type and three transformants of Gaeumannomyces graminis var.
graminis. Mycologia. 86: 72-81.
Fouly, H. M., Wilkinson, H. T. & Chen, W. 1997. Restriction analysis of internal transcribed
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Fouly, H.M., & Wilkinson, H.T. 2000. Detection of Gaeumannomyces graminis varieties
using polymerase chain reaction with variety-specific primers. Plant Dis. 84, 947-951.
Frederick, B., T. Caesar-TonThat, M.H. Wheeler, K.B. Sheehan, W.A. Edens, and J.M
Henson. 1999. Isolation and characterization of Gaeumannomyces graminis var. graminis
melanin mutants. Mycological Research 103: 99-110.
Fronza, G., Campomenosi, P., Iannone, R., & Abbondandolo, A. 1992. The 4-nitroquinoline
1-oxide mutational spectrum in single stranded DNA is characterized by guanine to
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Hawksworth, D.L., Kirk, P.M., Sutton, B.C., & Pegler, D.N. (Editors).1995. Ainsworth and
Bisby's Dictionary of the Fungi. CAB International, Wallingford.
Henson, J., Butler, M.J., & Day A.W. 1999. The dark side of the mycelium: melanins. Annu.
Rev. Phytopathology. 37: 47-71.
Hill, H.Z. 1992. The Function of melanin or six blind people examine an elephant. BioEssays.
14: 49-56.
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Kelly, C. 1997. Genetics of pigmentation and pathogenicity in G. graminis. PhD thesis,
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is associated with changes in hyphopodial permeability, wall rigidity, and turgor pressure
in Gaeumannomyces graminis var. graminis. Fungal Genet. Biol. 24: 240-251.
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Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by
constitutive expression of rice chitinase. Theoretical Applied Genetics v. 99 p. 383-390.
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The Growing Fungus. Edited by N.A.R. Gow &.G.M. Gadd Chapman & Hall, London.
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Sugimura, T. 1981. Carcinogenesis: A Comprehensive Survey. Vol. 6. Raven, New York.

Walker, J. 1981. Taxonomy of take-all fungi and related genera and species. in Biology and
Control of Take-all. Edited by M.J.C. Asher & P.J. Shipton. Academic Press, London. pp.
15-74.
Wilkinson, H.T. 1994. Root rot of centipedegrass (Eremochloa ophinroides (Munro) Hack.)
caused by Gaeumannomyces graminis ((Sacc.) Arx & Olivier) var. graminis. Plant Dis.
78: 1220.
Wilkinson, H.T., & Kane, R.T. 1993. Gaeumannomyces graminis infecting zoysiagrass in
Illinois. Plant Dis. 77: 100.
Wilkinson, H.T., J.R. Alldredge, and R.J. Cook. 1985. Estimated distance for infection of
wheat roots by Gaeumannomyces graminis var. tritici in soils suppressive and conducive
to take-all. Phytopathology 75: 557-559.
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deoxyribonucleotides. Biochemistry 18: 3833-3839.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 8

SKIN ANATOMY AND PHYSIOLOGY RESEARCH

DEVELOPMENTS IN MELANOCYTES

Naoki Oiso and Akira Kawada

Department of Dermatology, Kinki University Faculty of Medicine,
Osaka-Sayama, Osaka, Japan

ABSTRACT
In mice models of pigment anomalies, over 800 phenotypic alleles are known. This
indicates that skin color is distinctly regulated by more than 800 genes. This requires
several steps; (i) distribution of melanoblasts into skin in embryo, (ii) construction of
melanosomes in melanocytes, (iii) production of melanin granules in melanosomes, (iv)
translocation of melanosomes from perinuclear to peripheral region in melanocytes, (v)
transfer of melanosomes from melanocytes to keratinocytes and (vi) translocation of
transferred melanin granules from a peripheral to a supranuclear region in keratinocytes.
The damage in each step induces pigment anomalies. We summarize biogenesis and
function of melanin granules with pigment anomalies; piebaldism and Waadenburg
syndrome caused by inadequate distribution of melanoblasts in embryo; Hermansky-
Pudlak syndrome, Chediak-Higashi syndrome, and oculocutaneous albinism type 2 and 4
by improper biogenesis of melanosomes and melanin granules; and Griscelli syndrome
by inappropriate intercellular translocation of melanosomes. Aberrant intercellular
transfer of melanin granules is shown in a case of pediatric erythema dyschromicum
perstans (ashy dermatosis). Aberrant translocation inside keratinocytes is present in
Dowling-Degos disease. Unregulated melanogenesis is present in disorders affected in
KITLG-KIT signaling and RAS-MAPK signaling. The loss or decreased enzymatic
function in melanogenesis induces oculocutaneous albinism types 1 and 3. Pheomelanin-
dominant production is present in red hair color phenotypes showing fair skin, poor
tanning ability and elevated risk of freckles, malignant melanoma, basal cell carcinoma
and squamous cell carcinoma. This section will provide the current findings to recognize
the function and the health effect of melanin granules as well as the pathogenesis of
pigmentation-associated disorders.


Address correspondence to: Naoki Oiso, MD., PhD. Department of Dermatology, Kinki University Faculty of
Medicine, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan. Tel: +81 72 366 0221. Fax: +81 72
368 2120. Email: [email protected]
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250 Naoki Oiso and Akira Kawada

1. INTRODUCTION
More than 800 phenotypic alleles are now identified in mice models of pigment
anomalies [1]. This indicates that skin, hair and eye color is distinctly regulated by multiple
genes. The melanocyte-precursor melanoblasts derived from the neural crest migrate and
localize as melanocytes in the basal layer of the epidermis in embryo. Melanocytes contain
melanosomes where melanin granules are produced. During maturing from melanosome stage
I to stage IV via stage II and III in melanocytes, melanosomes shift from perinuclear region to
peripheral dendritic processes. The matured melanosomes containing melanin granules are
transferred from the tip of the process of melanocytes to the peripheral region of
keratinocytes. The transferred melanin granules are transported from peripheral to a
supranuclear region in the same keratinocytes or are transferred to the other keratinocyts.
Both intrinsic and extrinsic factors influence production of melanin granules, eumelanin and
pheomelanin. Genetic and environmental factors influence melanogenesis. This chapter
summarizes human pigment anomalies in the view of biosynthesis, functions and health
effects of melanin granules.

2. THE DISORDERS IN ABERRANT MIGRATION OF MELANOBLASTS

Hereditary disorders in aberrant migration of melanoblasts include piebaldism and

Waadenburg syndrome (WS). Piebaldism is a rare autosomal dominantly inherited disorder,
characterized by congenital leukoderma, most commonly involving the forehead, abdomen,
and knees. Patients with piebaldism have mutations of the KIT gene, which encodes stem cell
growth factor (KIT ligand (KITLG)) receptor, a type III transmembrane receptor tyrosine
kinase with an extracellular domain that binds KITLG [2-9]. The complete depigmented
patches in piebaldism represent regions lacking in melanocytes, the result of defective
melanoblast differentiation, migration, proliferation, or survival during embryonic
development [10]. KITLG acts as a chemokinetic factor for melanoblast migration, and KIT
promotes melanocyte movement and acts as a chemokinetic or motogenic receptor [11].
Waardenburg syndrome (WS) is syndromatic disorder and is sub-classified into type 1 to
4. WS is characterized by localized pigment abnormalities of the hair, skin, and eyes, and
congenital sensorineural hearing loss with or without other symptoms.
The association of hearing loss and pigment abnormalities results from an abnormal
proliferation, survival, migration, or differentiation of neural crest-derived melanocytes [12].
WS type 1 (WS1) is characterized by the presence of dystopia canthorum: WS type 2
(WS2) is the absence of dystopia canthorum; WS type 3 (WS3) is the presence of dystopia
canthorum and musculoskeletal abnormalities of the upper limbs, and WS type 4 (WS4) have
the complication of Hirschsprung disease [12].
Heterochromia and white forelock may be present in WS1 and WS2. WS1 is caused by
mutations in PAX3 [13, 14]; WS2 by MITF [15-17], SLUG [18], SOX10 [19] and PAX3 [13];
WS3 by PAX3 [20, 21]; and WS4 by EDB3 [22, 23], EDNRB [24, 25], and SOX10 [26, 27].
A mutation in SOX10 may cause severest phenotype of PCWH including peripheral
neuropathy, mental retardation, cerebellar ataxia, and spasticity [28]. SLUG-associated WS2

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shows an autosomal recessive inheritance, and PAX3-associateed WS3 shows both autosomal
dominant and recessive forms. Others are autosomal dominant forms.

3. THE DISORDERS IN BIOGENESIS OF MELANOSOMES IN

MELANOCYTES
Biogenesis of melanosomes in melanocytes is distinctly regulated. Abnormal biogenesis
of melanosomes is present in Hermansky-Pudlak syndrome (HPS), Chediak-Higashi
syndrome (CHS), possibly oculocutaneous albinism (OCA) type 2 (OCA2) and OCA type 4
(OCA4). Melanosomes belong to cell-specific lysosome-related organelles (LROs). LROs
include lytic granules in cytotoxic T lymphocytes and natural killer cells, MHC class II
compartments (MIICs) in antigen presenting cells, dense granules in platelets, lamellar bodies
in lung epithelial type II cells, azurophil granules observed in neutrophils, and others [29, 30].
HPS and CHS have OCA and specific features because of the dysfunction of affected LROs.
In HPS, prolonged bleeding times related to platelet dysfunction is caused by absence of
platelet-dense granules [31], and pulmonary dysfunction related to lung epithelial type II cells
is possibly caused by decreased secretion from lamellar bodies [32, 33]. In CHS, repeated
infections related to immunological deficiency is caused by enlargement of lytic granules,
MIICs and azurophil granules [31].
Human HPS is autosomal recessive disorder which has been sub-classified into 9 types.
The function of HPS-assciated proteins has been studied with mice, rats, Caenorhabditis
elegans, and yeast Saccharomyces cerevisae. HPS-associated proteins assemble heteromeric
complexes except Rab38 [34]. Two of five complexes, adaptor protein (AP)-3 and homotypic
vacuolar protein sorting (HOPS) or the class C VPS complex, are conserved from yeast to
humans, whereas the remaining three complexes, biogenesis of lysosome-related organelles
complex (BLOC)-1, BLOC-2, and BLOC-3, seem not to be conserved in unicellular
eukaryotes [35, 36]. AP-3 complex is composed of (a mutation identified in mouse mocha
, β3A (mouse pearl [38] and human HPS-2 [39]), σ3, and μ3A subunit; HOPS is
composed of Vps33a (mouse buff [40]), Vps11, Vps16, Vps18, Vps39 and Vps41; BLOC-1 is
composed of dysbindin (mouse sandy and human HPS-7 [41]), BLOS3 (mouse reduced
pigmentation [42] and human HPS-8 [43]), pallidin (mouse pallid [44] and human HPS-9
[45]), cappuccino (mouse cappuccino [46]), muted (mouse muted [47]), snapin [42], BLOS1
[42] and BLOS2 [42]; BLOC-2 contains HPS3 (mouse cocoa [48] and human HPS-3 [49]),
HPS5 (mouse ruby-eye-2 [50] and human HPS-5 [50]) and HPS6 (mouse ruby-eye [50] and
human HPS-6 [50]) and BLOC-3 contains HPS1 (mouse pale ear [51] and human HPS-1
[52]) and HPS4 (mouse light ear [53] and human HPS-4 [53]).
CHS is autosomal recessive syndromatic disorder caused by mutations in the lysosomal
trafficking regulator (LYST) gene [54-57]. As LYST proteins act as negative regulators of
fusion by limiting the heterotypic fusion of early endosomes with post-lysosomal
compartments, the loss of function inducts excess fusion and large LROs [58].
Non-syndromatic autosomal recessive OCA is a heterogeneous disease with
hypopigmented skin, hair, and eyes [59]. It is sub-classified into 4 types caused by mutations
of four genes; the tyrosinase gene (TYR) for OCA type 1 (OCA1), the P gene for OCA2, the
TYRP1 (tyrosinase-related protein 1) gene for OCA type 3 (OCA3), and the MATP
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252 Naoki Oiso and Akira Kawada

(membrane-associated transporter protein) or SLC45A2 (solute carrier family 45, member 2)

gene for OCA4 [59]. The function of the products encoded by the P and MATP gene is still
unknown. Recently, genetic interaction was studied between mutant alleles causing
deficiency in OCA2 (pink-eyed dilution unstable), AP-3 (pearl), BLOC-1 (pallid), and
BLOC-2 (cocoa) in C57BL/6J mice [60]. The study suggested that functional links between
OCA2 and these three protein complexes (AP-3, BLOC-1, and BLOC-2) involved in
melanosome biogenesis [59]. SLC45A2 (MATP), a solute carrier family member, and P
proteins may implicate in the control of eye, hair, and skin pigmentation via the regulation of
melanosome pH [61].

4. THE DISORDERS IN THE TRANSLOCATION OF MELANOSOMES IN

MELANOCYTES
The defect of melanosome transport in melanocytes is present in Griscelli syndrome
(GS). It is a rare autosomal recessive disorder caused by mutations in either the myosin 5A
(MYO5A) in GS type 1 (GS1) [62], RAB27A in GS type 2 (GS2) [63] or melanophilin
(MLPH) in GS type 3 (GS3) [64]. It is characterized by pigment dilution of the skin and hair
color because of perinuclear accumulation of melanosomes in melanocytes and presence of
large clumps of pigment in hair shafts. GS1 represents cutaneous albinism with a primary
neurologic deficit [62]; GS2 dose cutaneous albinism with immune impairment [63]; and GS3
dose only cutaneous albinism [64].
In melanocytes, the RAB27A-MLPH-MYO5A tripartite protein complex is involved in
the intramelanocytic melanosome transport [65]. Mature melanosomes connecting activated
Rab27a move to the cell periphery on microtubules via the motor protein kinesin [65]. At the
cell peripherally, Rab27a-Mlph-Myo5a tripartite protein complex is formed that captures the
melanosomes in the actin-rich dendritic tips [65]. The loss of function of Rab27a-Mlph-
Myo5a tripartite induces the accumulation of melanosomes at the perinuclear regions and
induces cutaneous albinism.
The transportation of synaptic vesicles in a neuron is shown to be regulated by Rab3a-
Rabphilin3A-Myo5a tripartite protein complex. The loss of function in myosin 5A is
therefore associated with a primary neurological deficit [66].
A variety of Rab27 effector proteins have been identified:

Exophilin1/Rabphilin-3a,
Exophilin2/Granuphilin-a/Slp4-a,
Exophilin3/Melanophilin/Slac2-a,
Exophilin4/Slp2-a,
Exophilin5/Slac2-b,
Exophilin6/Slp3,
Exophilin7/JFC1/Slp1,
Exophilin8/MyRIP/Slac2-c,
Exophilin9/Slp5, Noc2, and Munc13-4 [67-69]. GS2 is characterized by defects in
melanosome transport in melanocytes, defects in granule secretion by cytotoxic T
lymphocytes [63, 69, 70] and excessive phagocytosis in macrophages [71]. As
phenotypic diversity and different binding ability for myosin 5A and melanophilin is

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present in each mutation in Rab27a [72], further functional investigation would be

needed for elucidating the relationship between immunologic impairment and the binding
ability to various Rab27a effector proteins in each mutation in Rab27a.

5. THE DISORDERS IN THE TRANSFER OF MELANOSOMES VIA

INTERCELLULAR SPACES
The precise mechanism of melanosome transfer from melanocytes to keratinocytes and
between keratinocytes has not been completely elucidated. However, possible mechanisms
have been suggested; (i) pinching off of melanocyte dendrites containing melanosomes by
keratinocytes; (ii) direct inoculation of melanosomes into keratinocytes via keratinocyte-
melanocyte membrane fusions through nanotubular filopodia; and/ or(iii) melanosome release
into the extracellular space followed by their phagocytosis by keratinocytes [73]. Protease-
activated receptor-2 (PAR-2), a seven-transmembrane G-protein coupled receptor expressing
in keratinocytes but not in melanocytes, is a key regulator of melanosome transfer via
keratinocyte phagocytosis [74].
We examined electron microscopic features in erythema dyschromicum perstans (ashy
dermatosis) in a Japanese pediatric patient [75]. It showed melanosomes transferred from a
melanocyte to a keratinocyte following a forced curve resembling a filopodial-phagocytosis
model [76, 77], but did not identify melanosomes transferred between keratinocytes, possibly
because of the intercellular spaces and the retracted melanosomes [75]. Some acquired
pigment disorders may be caused by the improper transfer of melanin granules [78].

6. THE DISORDERS IN THE TRANSLOCATION OF MELANOSOMES

IN KERATINOCYTES

The mechanism of intracellular distribution of melanosome in keratinocytes is poorly

understood. Dowling-Degos disease (DDD) may be caused by aberrant translocation in
keratinocytes. It is a rare autosomal dominant keratinocyte pigmentation disorder, and is
caused by haploinsufficiency by a mutation in the keratin 5 (KRT5) gene [79]. The mutation
in KRT5 affects melanosome distribution in keratinocytes but not the integrity of the keratin
cytoskeleton [79, 80]. Keratin 5 is a component of the intermediate filament (IF) cytoskeleton
in the basal layer of the keratinocytes. Dysfunction of the IF cytoskeleton causes aberrant
distribution of melanin granules in keratinocytes [79, 81]. Further study will shed light on the
accurate mechanism of translocation of melanin granules from peripheral to supranuclear
region in keratinocytes.

7. THE DISORDERS OF UNREGULATED MELANOGENESIS

Skin pigmentation is controlled by a complex melanogenic paracrine network between
mesenchymal and epithelial cells, which regulates melanocyte survival, proliferation, and
melanogenesis [82, 83]. Keratinocyte-derived factors that act as activators of melanocytes
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254 Naoki Oiso and Akira Kawada

include KITLG, basic fibroblast growth factor, hepatocyte growth factor, granulocyte-
macrophage colony-stimulating factor, nerve growth factor, α-melanocyte stimulating
hormone (α-MSH), adrenocorticotropic hormone, endorphin, endothelin-1, prostaglandin
(PG) E2/PGF2α and leukemia inhibitory factor [84, 85]. Among melanogenic growth factors,
KITLG and its receptor KIT signaling that triggers RAS-MAPK (mitogen-activated protein
kinase) signaling pathway plays crucial roles in the control of physiological and pathological
skin pigmentation [82, 83]. The unregulated melanogenesis is present in disorders affected in
KITLG-KIT signaling and RAS-MAPK signaling. The disorders in aberrant KITLG-KIT
signaling include familial progressive hyper- and hypopigmentation (FPHH), familial
progressive hyperpigmentation (FPH), and possibly dyschromatosis universalis hereditaria
type 2 (DUH2) [82, 83]. The disorders in anomalous RAS-MAPK signaling (neuro-cardio-
facial-cutaneous syndrome) comprise (i) pigment anomaly-related Leopard syndrome type 1
to 3, neurofibromatosis type 1, neurofibromatosis type 1-Noonan syndrome, and
neurofibromatosis type 1-like syndrome (Legius syndrome) and (ii) pigment anomaly-
unrelated Noonan syndrome, cardio-facial-cutaneous syndrome and Costello syndrome.
FPHH is autosomal dominantly inherited disorder. It is characterized by diffuse,
progressive hyperpigmentation that begins at an early age [82, 83, 86]. It may show café-au-
lait macules and generalized lentiginosis intermixed with larger hypopigmented ash-leaf
macules [82, 83, 86]. FPHH is caused by a gain-of-function mutation in KITLG [83]. FPH is
an uncommon dominantly inherited disorder characterized by progressive hyperpigmentation
similar to that seen in FPHH, but without hypopigmented lesions [83]. FPH is linked to two
loci on chromosome 19p13-pter and on 12q21.31-q23.1 [87, 88]. A six-generated family with
FPH is caused by a gain-of-function mutation in KITLG [88]. DUH, characterized by
depigmented and hyperpigmented features on the trunk and extremities, is mapped on two
loci on chromosome 6q24.2-q25.2 (initially reported as dyschromatosis symmetrica
hereditaria) and on chromosome 12q21-q23 where KITLG is located [89, 90].
RAS genes are cancer-related genes due to their frequent activation in human cancers and
play a central role in the RAS-MAPK signaling cascade, which has a pivotal role in cell
proliferation, differentiation, survival, and cell death [91, 92].
Neuro-cardio-facial-cutaneous (NCFC) syndrome is proposed for disorders caused by
mutations in the genes involved in the RAS-MAPK signaling pathway. NCFC syndrome
includes several phenotypically overlapping, but clinically distinct disorders [93].
Leopard syndrome (LS) is a rare multiple congenital anomalies condition, mainly
characterized by skin, facial and cardiac anomalies [94]. LEOPARD is an acronym for the
major features including multiple Lentigines, ECG conduction abnormalities, Ocular
hypertelorism, Pulmonic stenosis, Abnormal genitalia, Retardation of growth, and
sensorineural Deafness [94]. LS type 1 (LS1) is caused by a mutation in PTPN11 [95]; LS2 in
RAF1 [96]; and LS3 in BRAF [97]. LS mutants in PTPN11 are catalytically defective and act
as dominant negative mutations [98] and are associated with multiple granular cell tumors
[99].
Noonan syndrome (NS), characterized by short stature, congenital heart defect, and
developmental delay and related disorders such as cardio-facio-cutaneous syndrome (CFCS)
and Costello syndromes, are caused by a mutation in genes involving in the RAS- MAPK
signaling cascade [92]. Mutations in PTPN11 are identified in NS [100]; RAF1 in NS [101];
and BRAF in NS [97] and CFCS [102].

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Neurofibromatosis type 1 (NF1) is autosomal dominant disorder caused by a loss-of-

function mutation in neurofibromin encoded by the NF1 gene [103-107].
It is characterized by café-au-lait spots, axillary freckling, Lisch nodules in the eye, and
multiple neurofibromas on the skin. The affected persons are susceptible to other benign and
malignant tumors. The incidence of NF1 is high, 1 in 2,500 to 1 in 3,000 individuals [108].
Neurofibromin acts as RAS-GTPase, catalyzing RAS-GTP into RAS-GDP. The loss-of-
function of neurofibromin induces excess presence of RAS-GTP accelerating the RAS-
MAPK signaling cascade [109, 110].
Neurofibromatosis type 1-Noonan syndrome (NFNS) is an entity characterized by the
presence of features of NS in individuals in NF1 [111-113]. Most cases are caused by a
mutation in NF1 [114-116] and rare in both PTPN11 and NF1 [117].
Neurofibromatosis type 1-like syndrome or Legius syndrome is an autosomal dominant
disorder caused by inactivating sprouty-related EVH1 domain-containing protein 1 (SPRED1)
mutations, which is initially identified in individuals presenting mainly with café-au-lait
macules, axillary freckling, and macrocephaly [118].
Subsequent studies identify that a high SPRED1 mutation detection rate is present in NF1
mutation-negative families with an autosomal dominant phenotype of café-au-lait macules
with or without freckling, and no other NF1 features including neurofibromas [119-122].
Legius syndrome is not associated with the peripheral and central nervous system tumors
seen in NF1 [121]. As SPRED-1 negatively regulates RAS-MAPK activation [123-125], the
loss-of-function of SPRED-1 inducts the activation of RAS-MAPK signaling cascade.

7. THE DISORDERS OF NO OR DECREASED MELANOGENESIS

Melanin granules are produced in melanosomes with tyrosine as a substrate and
tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1)/ 5,6-dihydroxyindol-2-carboxylic
acids (DHICA) oxidase, and dopachrome tautomerase/ 5,6-dihidroxyindole (DCT) as
enzymes. Tyrosine is converted into dopaquinone via dopa by tyrosinase. Eumelanins are
constructed from dopaquinone without glutathione or cysteine. Pheomelanin is produced from
dopaquinone with glutathione or cysteine. Eumelanin are defined as black or brown
nitrogeneous pigments and are insoluble in all solvents. Pheomelanins are yellow to reddish
brown pigments and are soluble in alkali solvents.
The decreased or non-enzymatic function in TYR and TYRP1 results in OCA1 and
OCA3, respectively. OCA1 is autosomal recessive pigment disorder caused by mutations in
TYR [126-128] and subclassified into tyrosinase-negative OCA1A, tyrosinase-positive
OCA1B. Individuals with OCA1A are the most severe phenotype, because they cannot
produce any melanin granules. The clinical features are (i) white skin without tanning, (ii)
white hair, eyelashes and eyebrows, and (iii) light blue to almost pink and fully translucent
irises with severe photophobia and decreased visual acuity [129]. Persons with OCA1B are a
milder phenotype, because they can produce a few melanin granules. The clinical
characteristics are gradual pigmentation of the skin and hair to some extent during growing,
and the gradual color change of irises from blue to green or brown. Temperature-sensitive
variants manifest as having depigmented body hairs, and pigmented hairs on hands and feet
due to lower temperatures [129]. OCA3 is autosomal recessive pigment disorder caused by
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256 Naoki Oiso and Akira Kawada

mutations in TYRP1 [130, 131]. OCA3 is present mostly in African individuals as rufous or
red OCA [130, 131] and rarely in Caucasian [132] and Asian [133, 134]. African individuals
with rufous or red OCA have red hair and reddish brown skin [129]. The synergic effect in P
and TYRP1 is present in a family, suggesting both are modifier genes of each other [135].

8. THE VARIANTS IN HAIR, EYES AND SKIN COLOR AND

ASSOCIATED DISORDERS
Hair, eyes and skin color are different between African individuals, Asian and Caucasian.
The determinants of hair, eyes and skin color have been investigated. Variants of the MSH
receptor gene (MC1R) are shown to be associated with red hair and fair skin with a poor
tanning response in Caucasian [136]. The loss-of-functional variants are associated with
production of pheomelanin in Caucasian. Subsequently, variants in MC1R are identified as a
susceptibility gene of malignant melanoma [137-139], squamous cell carcinoma [140, 141],
basal cell carcinoma [140, 141], and freckles [142]. So far, red hair color phenotype having
specific variants in MC1R shows the association between red hair and increased risk of
melanoma and skin cancer [143].
Using genomewide association study for hair, eyes and skin color determinants in the
European population, variants located in six loci are identified; MC1R associated with red
hair color, blond hair color, fair skin, skin sensitivity to sun, and freckles; 6p25.3 with
freckles; tyrosinase with eye color and freckles; SLC24A4 with eye and hair color; OCA2 (P)
with eye, hair and skin color; and KITLG with hair color [144]. Another study identified
variants in TPCN2 associated with hair color and a variant at the ASIP locus associated with
skin sensitivity to sun, freckling and red hair [145]. With genetic studies, melanoma
susceptibility genes have been identified; MC1R [137-139, 148], ASIP [146], TYR [146, 148],
MATP/SLC45A2 [147], CDKN2A [148], and common sequence variants on 20q11.22 [149].
The combination of susceptible variants in MC1R and mutations in BRAF [150] or CDKN2A
[151] are associated with the high frequent development of melanoma. Similarly, basal cell
carcinoma susceptibility genes have been identified; MC1R [140, 141], ASIP [146], TYR
[146], KRT5 [152], CDKN2A [152], CDKN2B [152], KLF14 [152], MATP/SLC45A2 [152],
and the TERT-CLPTM1L locus [152].

9. OTHER DISORDERS
(1) Dyschromatosis Symmetrica Hereditaria

Dyschromatosis symmetrica hereditaria (DSH) (or reticulate acropigmentation of Dohi)

is a pigmentary genodermatosis of autosomal dominant inheritance. It is characterized by a
mixture of hyperpigmented and hypopigmented macules on the dorsal aspects of the hands
and feet [153]. It is caused by a heterozygous mutation of the adenosine deaminase acting on
RNA 1 (ADAR1, previously called double-stranded RNA-specific adenosine deaminase
(DSRAD)) gene [154]. The precise pathogenesis is still unknown, even though adenosine-to-
inosine (A-to-I) RNA editing is a widespread modification of the transcriptome [155].

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 Skin Anatomy and Physiology Research Developments 257

Dermoscopy to the hyper- and hypopigmented macules on the dorsal hands showed
round and variously pigmented spots 0.5-1.5 mm in diameter connected to each other [156].
The different and unregulated A-to-I RNA editing may induct different melanocytes function
in each pigmented spots.

(2) Reticulate Acropigmentation of Kitamura

Reticulate acropigmentation of Kitamura (RAK) is autosomal dominant dermatosis. It is

characterized by pigmented and irregular freckle-like lesions with atrophy on the surface,
arranged in a reticular pattern on the dorsa of the hands and feet [157]. As some cases overlap
clinical and histological features of both DDD and RAK, these disorders may be phenotypic
diversity to DDD and RAK [158, 159].

10. FUNCTION AND HEALTH EFFECT

The production of melanin granules by melanosomes in melanocytes is important for
humans to survive in the sun-exposed surroundings. The adequate quality and quantity of
melanin granule is needed for hair, eyes and skin function. The production of melanin
granules in the eyes prevents photophobia and decreased visual acuity, and the synthesis in
the skin protects sun-burn and damage of DNA from ultraviolet exposure.

CONCLUSION
The melanin synthesis, translocation, and transfer are distinctly regulated by various
proteins. The genetic and environmental factors influence not only the quantity but also the
quality of melanin granule. Syndromatic symptoms are present in disorders caused by
mutations in the genes having the function in other cells, tissues or organs. As over 800
phenotypic alleles are known in mice models of pigment anomalies, more players will be
identified. Studies in melanocytes will give a gift to humans to reduce the mobility and the
mortality of malignant melanoma and skin cancers.

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melanocytes from an individual with brown oculocutaneous albinism: a new subtype of

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Steinberg, S., Pálsson, S., Jonasson, F., Sigurgeirsson, B., Thorisdottir, K., Ragnarsson,
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V., Hopper, J. L., Ingvar, C., Kanetsky, P. A., Kefford, R. F., Landi, M. T., Lang, J.,
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van Nieuwpoort, F. A., Novakovic, S., Olsson, H., Puig, S., Weiss, M., van Workum,
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Bastian, B. C., Peris, K., and Landi, M. T. (2008). MC1R variants increase risk of
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 9

OPTICAL SPECTROSCOPY AND

STRUCTURAL PROPERTIES OF SYNTHETIC AND
NATURAL EUMELANIN

Giuseppe Perna and Vito Capozzi

Dipartimento di Scienze Biomediche, Università degli Studi di Foggia, Foggia, Italy

ABSTRACT
Optical properties of synthetic and natural eumelanin are presented and compared, in
order to investigate the structural organization of eumelanin, which is related to the
function of this biopolymer. Synthetic eumelanin is produced by oxidation of tyrosine
with hydrogen peroxide, whereas natural eumelanin is extracted from Sepia Officinalis
and from Rana Esculenta. Vibrational spectroscopy techniques (as Raman scattering and
infrared absorption) show that both types of biopolymer include chemical functional
groups characteristic of the monomeric units of eumelanin, although natural eumelanin
includes also protein-related groups, proportionally to the protein content. X-ray
diffraction spectra are in agreement with the hypothesis that eumelanin monomers
assembly themselves and form protomolecules consisting of stacked layers (distant 3 – 4
Å each other) of indolic sheets. Absorption measurements, characterized by a monotonic
increase of optical density from near-IR to UV range, support the model that eumelanin
consists of a distribution of aggregates of oligomeric structures having different size and
chemical composition. The estimated values of the optical gap indicate that the natural
eumelanins are characterized by a larger structural disorder than the synthetic one.
Fluorescence spectra confirm that the biopolymer consists of ensembles of chemically
distinct oligomer systems, which can be selectively excited. This result is also supported
by Dynamic Light Scattering measurements, which permit to visualize the distribution of
particles size. In fact, the nanoaggregate systems of natural eumelanin have a larger size
than those of synthetic eumelanin. This might be related to the biological functions of
such a biopolymer, particularly as far as photoprotective action is concerned.
 Free ebooks ==> www.Ebook777.com
272 Giuseppe Perna and Vito Capozzi

INTRODUCTION
The melanins are a class of biological pigments that provide coloration to animals and
plants [1]. In particular, eumelanin (a brown-black pigment containing nitrogen) and
pheomelanin (a brown-red pigment containing also sulphur) are the predominant forms of
melanin in humans. Eumelanin, the most diffuse form, is formed inside specialized organelles
called melanosomes. The key enzyme of melanogenic pathway is the tyrosinase [2]. This
enzyme catalyzes the first rate-limiting steps of melanogenesis, the hydroxylation of L-
tyrosine and the subsequent oxidation of the intermediate L-dopa to yield L-dopaquinone.
Several other proteins located in the melanosomes are involved in melanogenesis process.
Therefore, eumelanins are considered to be firmly bound to proteinaceous components,
through covalent or ionic bonds [3]. In fact, purification processes can be used to isolate
eumelanin from the protein component, although a complete separation is hardly achievable.
A method to prepare eumelanin without protein is to synthesize it non-enzymatically, for
example through a process starting from oxidation of tyrosine with hydrogen peroxide. The
sample obtained in this way is called synthetic eumelanin.
In humans, eumelanin pigment is found in skin, hair and eyes, where it acts mainly as an
excellent photoprotectant [4], because it largely absorbs UV and visible light by converting
the light energy into heat. However, eumelanin plays a dual role with respect to the sunlight’s
UV radiation: on one hand, it is beneficial because it absorbs the UV radiation, thereby
reducing the UV damage in skin cells [5]; on the other hand, it is deleterious by acting as a
photosensitizer that generates active oxygen species capable of causing DNA strand breaks,
although such a role occurs mostly in pheomelanin [5]. The balance of these two processes
determines whether a beneficial action or a malignant transformation activated by the oxygen
species occurs.
Eumelanin, besides its important biological role, has very attractive physical properties
for material science applications. In view of such applications, synthetic eumelanin has been
widely investigated [4, 6, 7–11]. It exhibits broad UV and visible absorption spectra [4, 7, 8],
electrical conductivity similar to that of amorphous silicon [9], strong non radiative relaxation
[6] and photoconductivity [10], as it occurs for amorphous semiconductors; therefore, it has
been proposed for photovoltaic and optoelectronic applications. Recently, hysteresis
behaviour of the current–voltage characteristic of synthetic eumelanin-based structures allows
to foresee the possible integration of eumelanin in memory devices [11].
The properties of synthetic eumelanin, although interesting for technological
applications, cannot be directly generalized for the natural pigment, because of the presence
of protein component in the latter. Instead, the investigation of synthetic eumelanin can be
used as starting point to study the physical properties of natural eumelanin and the role of
proteins in the structural organization of the natural biopolymer, which is partly debated yet.
In fact, it is accepted that eumelanin is a biopolymer resulting from aggregation of indolic
monomers, such as 5,6-dihydroxyindole (DHI), 5,6-dihydroxyndole-2-carboxylic acid
(DHICA), 5,6-indolequinone (IQ) and semiquinone (SQ). A schematic representation of these
monomers is shown in Figure 1. Nonetheless, it is still not well known how these monomer
units are connected together to form eumelanin pigment. Until the last decades of the XX
century, it was still unclear whether eumelanin was actually a highly cross-linked extended
heteropolymer [1] or it was composed of much smaller oligomers condensed into

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 Optical Spectroscopy and Structural Properties … 273

nanoaggregates [12]. In the last decade, several theoretical [8, 13] and experimental [14, 15]
studies have been published to support the structural model of eumelanin as consisting of
stacked oligomeric nanoaggregates. In particular, it has been recently confirmed that both
synthetic and natural eumelanin are organized according to planar sheets of varying
dimensions which stack each other with inter-sheet spacing values between 3.7 and 4.0 Å
[16]. Furthermore, it has been established that the lateral extension of the indolic sheets in
synthetic eumelanin is less than 10 nm, whereas it is about one order of magnitude larger in
natural eumelanin (from bovine epithelium and ink sacks of Sepia officinalis) [16].

Figure 1. Schematic representation of the structure of the basic monomeric building blocks of
eumelanin: 5,6-dihydroxyindole (DHI), 5,6-dihydroxyndole-2-carboxylic acid (DHICA) and the redox
forms 5,6-indolequinone (IQ) and semiquinone (SQ).

An accurate investigation of the structural organization of eumelanin can be provided by

optical spectroscopy techniques. In fact, such techniques are non destructive (and,
consequently, the same sample can be analysed by different techniques) and they require a
very small amount of material for such analysis. In particular, vibrational, absorption and
fluorescence spectroscopy are useful analytical tools to investigate the presence of monomer
and oligomer components inside the eumelanin biomolecules. This can be accomplished by
means of a comparison with theoretical models and calculations about the eumelanin
structure.
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274 Giuseppe Perna and Vito Capozzi

In fact, Raman and Fourier Transform Infrared (FTIR) spectra provide information about
the energy of vibrational modes of the chemical bonds involved in the structure of the
investigated sample: therefore, the spectral position of Raman and FTIR features of
eumelanin spectra allows to identify the functional groups present in the structure of
eumelanin components. Several works exist about Raman [17-19] and FTIR [17, 20-24]
investigation of the melanin. In particular, the article from Powell et al. [17] reports a first
principles density-functional calculation of the Raman and FTIR spectrum of the eumelanin
monomers. These calculated spectra consist of several very narrow peaks, because they are
related to gaseous phase monomers. Nonetheless, such calculated spectra can be compared
with the experimental ones in order to attribute the spectral peaks, even if the broadening and
shifting effects should be considered as well when a solid biopolymer is formed.
The absorption properties of all types of melanin is characterized by a very broad
absorption, whose intensity increases from visible to UV [6, 25]. In addition, such absorption
band is structureless, i.e., it includes no resolved absorption peaks related to some specific
component of the biopolymer. Some authors have remarked that melanins are indeed
disordered organic semiconductors [26, 27], because of such similar absorption properties
(and also electrical conductivity and photoconductivity [28]). Therefore, absorption spectra
are not sensitive probes to reveal specific components of the eumelanin structure.
Nevertheless, several attempts have been performed to simulate the monotonic behaviour of
eumelanin absorption spectra. This was achieved by the convolution of a basis set of numeric
atomic functions [8, 13] or several broadened Gaussian functions [4], each with different peak
position and intensity, related to highest occupied molecular orbital–lowest unoccupied
molecular orbital (HOMO-LUMO) gaps of distinct eumelanin monomers and/or oligomers.
The good agreement of calculated absorption spectra with the experimental ones have
suggested that an ensemble of similar but chemically distinct species can explain the observed
monotonic, broad band absorbance. Linh Tran et al. [29] introduced the term “chemical
disorder model” to explain the eumelanin structure as consisting of many chemically distinct
oligomers, each with a different HOMO–LUMO energy gap. According to this model, the
broadband absorption characteristics is due to overlapping of a large number of HOMO-
LUMO transitions associated with each of the eumelanin components.
Fluorescence (FL) spectroscopy technique has been widely used to investigate the
structural organization of eumelanin [6, 30–33], although the radiative quantum yield is
extremely low [6]. The eumelanin FL spectra support the hypothesis that radiative emission in
synthetic eumelanin is related to chemically distinct oligomeric units that are selectively
excited, in agreement with the chemical disorder model. So, the emission bands are due to the
convolution of many different narrower features, each one corresponding to the FL of a
different eumelanin component. However, the results obtained for synthetic eumelanin cannot
be directly generalized to the natural pigment, because of the presence of protein component
in the latter.
In addition to optical techniques, also structural techniques as Dynamic Light Scattering
(DLS), X-ray Diffraction (XRD) and Atomic Force Microscopy (AFM) can be properly
applied to investigate the size of the structural components of the biopolymer. In fact, DLS
technique is usually used to determine the size distribution profile of small particles or
polymers in suspension. X-ray scattering measurements [34-36] of eumelanin samples have
revealed the presence of nanometric particles consisting of 3 or 4 planar layers of few
indolequinone units having lateral size of 15–20 Å and stacked each other of about 3.4 Å.

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Also AFM images of eumelanin samples have revealed the coexistence of nanometric
particles having different sizes, from about tens nanometers to few hundred nanometers, and
different shape, as spherical and filamentous ones [12, 15, 37-39]. Therefore, the structural
techniques suggest a model for eumelanin structure, as consisting of nanoaggregates of
different shape and size comprising several protomolecules each one formed by 3 or 4 planar
layers of few monomer units having lateral size of 15–20 Å and stacked each other of about
3.4 Å.
In this article, we review our recent results concerning the optical and structural
properties of the synthetic and natural eumelanin biopolymer, by focusing on important
aspect such as its capability to absorb, scatter and emit light and including its molecular and
supramolecular structure. We have found that several physical properties are influenced by
the protein content of the biopolymer. In particular, the protein content result to influence the
structural organization of eumelanin, by aiding to construct aggregates having a larger size.

EXPERIMENTAL
Materials

Three types of eumelanin samples have been investigated: i) synthetic eumelanin

samples, obtained from commercial eumelanin powder produced by oxidation of tyrosine
with hydrogen peroxide (Sigma-Aldrich): nominally it doesn’t contain proteins; ii) natural
eumelanin samples obtained from commercial eumelanin powder extracted from ink sacs of
Sepia Officinalis (Sigma-Aldrich): for such samples a protein content of about 8% is reported
[40]; iii) natural eumelanin samples extracted from liver of Rana esculenta according to the
method of Cicero et al. [41]: for such samples a protein content larger than that of Sepia
officinalis has been estimated [42].

Experimental Techniques

The Raman spectra were measured at room temperature by means of a Raman confocal
micro-spectrometer using the 632.8 nm line of He–Ne as laser source and a notch filter (200
cm-1 line-with) to suppress the laser scattered light. The Raman signal was detected by means
of a cooled CCD (at 223 K). The laser beam was focused, by an Olympus optical microscope
with a x100 objective, on the investigated sample, obtaining an illuminated spot of few m
diameter. The laser power at the sample was about 0.1 mW, corresponding to a laser intensity
of about 103 W/cm2 on the surface of samples. The mean spectral resolution was 4 cm-1. The
samples for Raman measurements were prepared starting from eumelanin powders mixed
with HPLC-grade water: a continuous melanin dispersion in water was produced, because it is
well known that melanin is essentially insoluble in any solvent [37]. Such dispersions were
sonicated for 15 min to improve a little solubility. Successively, a drop of each type of
eumelanin dispersion was deposited on glass substrate and air dried before performing Raman
measurements.
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276 Giuseppe Perna and Vito Capozzi

Mid-Infrared absorption spectra at room temperature were performed in a Fourier

Transform Interferometer Vertex70 (Bruker Optics), operating in the wavelength range 900–
4000 cm-1. The samples were prepared as pellets (13 mm diameter) of eumelanin powders (4
mg) in KBr (100 mg).
UV, visible and near-IR absorption spectra in the 240–1100 nm spectral range at room
temperature were measured by means of a JASCO V-530 double beam spectrophotometer, at
a scan rate of 400 nm/min and with a slit band-width of 2 nm. The samples for absorption
measurements consisted of synthetic and natural eumelanin solutions prepared in HPLC-
grade water, obtained by sonication for 15 min and centrifugation (Heraeus sepatech
‘‘Biofuge 13’’) at 13000 rpm for 30 min of the starting dispersions, in order to remove large
suspended aggregates. The supernatant eumelanin resulting from centrifugation was a
homogeneous solution; such samples were used for optical measurements. The optical density
spectrum was obtained by using the well known Lambert–Beer law.
Fluorescence spectra of eumelanin samples were measured by means of a Cary Eclipse
fluorescence spectrophotometer, by using the same eumelanin solutions prepared for
absorption measurements. All spectra were measured by using a 1cm x 0.5cm rectangular
quartz cuvette. The measurements were performed at several excitation energies between
4.960 and 2.296 eV. A band pass width of 10 nm and an integration time of 1s were used.
Background scans were performed under identical instrumental conditions using the HPLC-
grade water. The fluorescence spectra have been also normalized to account for excitation
beam penetration depth and emission re-absorption, according to the procedure described in
the literature [32, 33].
Dynamic Light Scattering (DLS) measurements were performed using a Zetasizer-NanoS
correlator from Malvern operating with a 4 mW He–Ne laser (633 nm wavelength) and a
fixed detector angle of 173o (non invasive backscattering geometry NIBSTM) and with the
cell holder, containing the eumelanin solutions (prepared as for absorption and fluorescence
measurements) at a proper concentration, maintained at 25 oC by means of a Peltier
thermostatic element. Data were collected after having optimized the instrumental parameters
(attenuator, optics position and number of runs). Usually, the time Autocorrelation Function
(ACF) of scattered light intensity was the average of 12–16 consecutive runs of 10 s each.
The ACF of scattered light intensity was converted into the ACF of scattered electric field.
From this last quantity, the fraction of the light intensity scattered by particles of different size
(i.e., the size distribution by intensity) has been recovered by taking the inverse Laplace
transform of the ACF using the software implemented by the manufacturer.
XRD spectra were performed by using the CuK radiation ( =1.5406 Å) of a -2
diffractometer. Samples of synthetic eumelanin for XRD measurements consisted of pellets
obtained by pressing the powders at a pressure of about 500 MPa for about 3 min, whereas
the natural eumelanin samples consisted of an air-dried deposit of eumelanin extracted from
Rana esculenta on a quartz substrate.
An atomic force microscope (Perception, by Assing S.p.A.) was used for AFM imaging.
Its lower unit contains the sample holder mounted on top of a cylindrical piezoelectric
scanner having a maximum x–y scan range of 3 × 3 μm and an z range of 0.6 μm. The upper
unit contains a cantilever holder, the mirror deflection system and a four-sector position-
sensitive photodiode, used as the deflection detector. An electronic feedback loop is used to
integrate the optical signal and maintain a constant cantilever deflection during the image
acquisition. The measurements were performed in air, with the microscope working in the

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non-contact mode. Gold-coated Si3N4 cantilevers with a spring constant of 40 N/m and a
statistical apical radius of 5–20 nm were used. Constant force images were acquired with a
scan rate of 3.0–4.0 s/row. The samples for AFM measurements were obtained by suspending
1 mg of melanin in 1 ml of HPLC-grade water. The mixture was firstly sonicated for 15 min
and then centrifuged at 18000g for 30 min. 10 μl of the supernant, containing only the tiny
aggregates, were dropped on freshly cleaved mica substrate and air-dried in dark at room
temperature before the measurements.

RESULTS AND DISCUSSION

Vibrational Spectroscopy: Raman and FTIR

The Raman spectra of air dried drops of synthetic, Sepia officinalis and Rana esculenta
eumelanin at room temperature are shown in Figure 2a, 2b and 2c, respectively. The spectra
have been normalized by subtracting the contribution of the FL emission. Such spectra
present a strong similarity to the Raman spectrum of amorphous carbon, which is dominated
by two bands, centred at 1350 and 1550 cm-1 [43], both related to vibrational modes of the
carbon atoms arranged in graphitic-like domains. Although carbon is the main constituent of
the eumelanin samples, several atoms other than carbon (such as O, H and N) are bonded to
the carbon atoms in the biopolymer. Therefore, the eumelanin Raman spectra can present
contributions from Raman active vibrational modes involving different atoms. In fact, the
three spectra in Figure 2 are very similar: they are dominated by the two strong and broad
bands centred at about 1400 and 1600 cm-1, with other lower intensity bands appearing at 950
and 1200 cm-1. The Raman intensity of the two main bands are similar, except for the Rana
esculenta spectrum in Figure 2c, where the band at 1600 cm-1 presents an intensity value
larger than that at 1400 cm-1. Moreover, the two main bands at 1400 and 1600 cm-1 have a
larger intensity in the natural eumelanin spectra comparing with the synthetic one.
The features of the Raman spectra can be assigned to vibrational modes related to the
structural units of the biopolymer; in addition, vibrational modes of the protein content should
be considered when analysing the spectra of natural eumelanin samples [44, 45]. In particular,
the overall shape of the Raman spectra are similar to that calculated by Powell et al. [17], so
confirming the presence of the eumelanin monomers DHI, IQ and SQ inside the analysed
samples. By considering the spectral position of vibrational modes inside functional groups
similar to those present in eumelanin monomers, the dominant mode in the high wavenumber
region, centred at about 1600 cm-1, can be attributed to ring vibrations of the indole structure
[46]. Moreover, the C=O stretching bond, present in the quinone structure of IQ and SQ and
in the carboxylic acid group of DHICA, also weakly contributes to the high wavenumber side
of the strong band at 1600 cm-1. Indeed, the weak spectral feature at about 1700 cm-1 in
Figure 2, can be assigned to the C=O stretching vibrational mode, which is reported in the
1655-1690 cm-1 range for a quinone group and in the 1680-1715 cm-1 range for a carboxylic
acid group [46]. Instead, the low wavenumber side of the band at 1600 cm-1 is also broadened
by the C=C and C=N in plane vibrations of the pyrrole structure: indeed, such vibrational
modes are reported in the 1460-1510 cm-1 and 1380-1430 cm-1 spectral ranges [46]. The main
contribution to the low wavenumber region of the Raman spectrum results from the band
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278 Giuseppe Perna and Vito Capozzi

centred at about 1400 cm-1, which is due to the overlapping of several vibrational modes,
mainly i) the C=C and C=N in plane vibrations of the pyrrole structure descrived above, ii)
the C–N stretching mode of the pyrrole structure (reported in the 1325-1367 cm-1 spectral
range [19, 47, 48]) and iii) combination bands due to C–O stretching and O–H deformation of
the carboxylic acid (reported in the region of 1250 cm-1 [49]). Finally, the low intensity bands
at about 950 and 1200 cm-1 are related to O-H out-of-plane deformation mode (reported at
935 cm-1 [49]) and C–H bending modes (reported in the 1145-1200 cm-1 region [50],
respectively.

Figure 2. Micro-Raman spectra at room temperature of a air dried drop of synthetic (a), Sepia officinalis
(b) and Rana esculenta eumelanin (c).

In addition to vibrational modes of the eumelanin structure, also Raman modes

characteristic of functional groups of proteins contribute to spectra in Figure 2, especially for
the natural eumelanins in Figure 2b and 2c. In fact, several vibrational modes due to protein
components are reported in the spectral regions covered by the bands centred at 1400 and
1600 cm-1, as CH2 (1436-1460 cm-1) and CH3 (1335-1343 cm-1) deformation modes, C-C and
C-N breathing modes (1573-1582 cm-1) and C=C mode (1615-1618 cm-1) [51]. The
contribution of such protein modes can be responsible of the intensity increase of the two
main bands with respect to the other bands in the two natural eumelanins, particularly for the
band at 1600 cm-1 in the Rana esculenta spectrum in Figure 2c.

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Figure 3. Absorption FTIR spectra at room temperature of a sample of synthetic (a), Sepia officinalis
(b) and Rana esculenta (c) eumelanin. The samples are pellets (13 mm diameter) of eumelanin powders
(4 mg) in KBr (100 mg).

These differences between synthetic and natural eumelanins are also more evident in the
FTIR spectra, shown in Figure 3. The analysis of the synthetic eumelanin spectrum in Figure
3a provides information about the main functional groups characteristic of eumelanin
structure, according to the spectral position of absorption peak, as reported in the literature
[21, 22]. So, it can be deduced that the synthetic eumelanin sample includes aromatic systems
(C=C stretching at about 1620 cm-1), indole rings (C–N stretching at about 1360 cm-1) and
carboxyl groups (C=O asymmetrical stretching at about 1710 cm-1, C=O symmetrical
stretching at about 1400 cm-1 and –C–OH stretching at about 1280 cm-1). In contrast, the
Sepia officinalis eumelanin spectrum, shown in Figure 3b, differs from the synthetic one for
several features. First of all, the peaks related to carboxyl groups are much less evident, so
indicating the small amount of DHICA monomers in this sample. In addition, the peak at
about 1600 cm-1 is broader with respect to the corresponding peak in the synthetic sample;
such broadening can be due to a contribution of amide II (1550 cm-1) and amide I (1660 cm-1
[22]) vibrations, typical of protein groups. The presence of protein content is more evident in
the FTIR spectrum of Rana esculenta sample, shown in Figure 3c. Indeed, such spectrum is
dominated by peaks related to proteins, as the amide I peak at 1650 cm-1 and the amide II
peak at 1550 cm-1: these two peaks overlaps to the C=C stretching group. The contribution of
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280 Giuseppe Perna and Vito Capozzi

carboxyl groups is also scarcely resolved: C=O asymmetrical stretching is visible as a

shoulder at about 1710 cm-1, C=O symmetrical stretching overlaps to the amide III peak at
about 1380 cm-1 and CH bending groups at about 1450 cm-1, whereas the –C–OH stretching
is resolved at about 1250 cm-1. Moreover, the peak at about 1080 cm-1 is related to C-O-C
groups of protein components.

Absorption Spectroscopy

Figure 4. Optical density spectra at room temperature of synthetic (a), Sepia officinalis (b) and Rana
esculenta (c) eumelanin solutions.

The absorption spectra of natural and synthetic eumelanin solutions at room temperature
in the spectral region from near-IR to near-UV are reported in Figure 4. All solutions present
an increase of the optical density versus the energy, with a very strong and broad UV–visible
absorption. Such a behaviour promotes the photoprotection function of the biopolymer. In
particular, the absorption of synthetic melanin decreases monotonically from UV to IR
energies, without any resolved peak: only a weak absorption shoulder is visible at about 4.6
eV, due to absorption on behalf of residual tyrosine in the synthetic sample. Instead, the
spectra of both natural eumelanins present a broad absorption band at about 4.6 eV. Such
band is due to light absorption of residual proteins, which are not completely removed during
isolation and purification processes. In fact, several aromatic aminoacids, as phenylalanine,

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tyrosine and tryptophan, present absorption features in this spectral region [52]. Moreover,
the absorption spectra of eumelanin from Sepia officinalis and Rana esculenta show weak
features at about 3.9 eV and 3.1 eV, respectively. Such shoulders are also related to the
protein coat, which cannot be fully removed during the preparation of the biopolymer sample.

Fluorescence Spectroscopy

Figure 5. Fluorescence emission spectra at room temperature of synthetic (a), Sepia officinalis (b) and
Rana esculenta (c) eumelanin solutions, measured by the excitation light energy of 4.960 eV.

The presence of protein components in natural eumelanins influences also the

fluorescence properties, as it is evident in Figure 5, which shows the FL spectra of the three
types of eumelanin solutions measured at room temperature and with the same excitation
energy (4.96 eV). The spectra show similar spectral features, except for the presence of a high
energy band, at about 3.50 eV and 3.70 eV, observed in Sepia officinalis and Rana esculenta
eumelanin, respectively. Such feature is lacking in synthetic eumelanin sample. According to
the different chemical composition of the eumelanin samples, we can attribute such high
energy bands to radiative emission of the residual protein components. In fact, some
aminoacids, as tryptophan and tyrosine, when excited by UV wavelengths, have fluorescence
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282 Giuseppe Perna and Vito Capozzi

emission in this spectral range [52]. Such attribution is also in agreement with the larger
intensity of the high energy band in Rana esculenta with respect to Sepia officinalis: indeed,
the protein content of the former is larger than that of the latter. A broad low energy band at
2.45 eV dominates the spectrum of the synthetic eumelanin; it is blue-shifted at 2.68 eV in the
spectrum of the two natural ones. Finally, all spectra in Figure 5 are characterized by a
shoulder at about 2.85 eV. FL spectral features of eumelanin are due to radiative transitions
between energy levels of the molecular groups present in the biopolymer; hence, the energy
of radiative emission depends on the HOMO–LUMO transition, which is also related to the
specific molecular species and the degree of delocalization of HOMO and LUMO
wavefunctions. For example, when monomeric units are linked to form an oligomer,
delocalization of HOMO and LUMO wavefunctions occurs if the oligomer presents a planar
structure. In this case, a redshift of the HOMO–LUMO gap of the oligomer species with
respect to that of the monomer units results. In addition, larger the number of coplanar
monomers forming the oligomer, lower the energy of the corresponding radiative emission.

Figure 6. Fluorescence spectra at room temperature of a synthetic eumelanin solution at different

excitation energies. The excitation energy value is reported on the right hand side of each spectrum and
the sensitivity factor is reported on the left hand side of each spectrum.

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The attribution of the FL features can be obtained by investigating the FL emission of

eumelanin samples as a function of the excitation energy, shown in Figs. 6, 7 and 8 for
synthetic, Sepia officinalis and Rana esculenta eumelanin, respectively [53, 54]. The FL
spectra at different excitation energies have similar behaviours for the three samples. In fact,
at lower (visible) excitation energies, a single broad band characterizes the FL spectra,
whereas a few bands are present in the spectra at higher (near-UV) excitation energies.
Furthermore, the spectral position of the FL bands does not change at higher excitation
energies, whereas it red shifts as the excitation energy decreases; the intensity and full-width
at half-maximum (FWHM) of the FL emission decrease with excitation energy.

Figure 7. Fluorescence spectra at room temperature of a Sepia officinalis eumelanin solution at different
excitation energies. The excitation energy value is reported on the right hand side of each spectrum and
the sensitivity factor is reported on the left hand side of each spectrum.
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284 Giuseppe Perna and Vito Capozzi

Figure 8. Fluorescence spectra at room temperature of a Rana esculenta eumelanin solution at different
excitation energies. The excitation energy value is reported on the right hand side of each spectrum and
the sensitivity factor is reported on the left hand side of each spectrum.

Such behaviour can be explained according to the “chemical disorder model” [29], which
involves the presence of HOMO–LUMO radiative recombinations related to slightly different
chemical species, as monomer units and oligomer groups, each one contributing to the
emission process with a narrow FL peak. The overlapping of different peaks causes the broad
bands observed in the FL spectra. In addition, the contribution of radiative emission due to
monomeric units and small oligomer groups can be discriminated from the contribution of
polymeric groups in the spectra shown in Figs. 6, 7 and 8. In particular, the FL band observed
at about 2.85 eV only in the spectra measured at higher excitation energies for all the

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eumelanin samples is due to FL from monomers and small oligomer groups. In fact, many
monomers (DHI, IQ, DHICA), with different stable tautomeric forms, can coexist in melanin
aqueous solution [55]. In contrast, the FL band observed at higher excitation energies at about
2.45 eV in Figure 6 and at about 2.68 eV in Figures 7 and 8 arises from radiative
recombination of polymeric groups, i.e., entities made of a higher number of coplanar indolic
units. In fact, red-shift of the HOMO–LUMO gap occurs in the polymerization form [4].
The independence of the spectral position of FL bands on the excitation energies
observed at excitation in the UV region occurs because the HOMO–LUMO gap energy of
each polymeric species is lower than the energy of exciting light. Consequently, all the FL
centres can absorb incident light and are involved in the emission process. Therefore, the
band energy and FWHM remain almost constant, independently of the excitation energy. On
the contrary, the excitation energies in the visible region are not large enough to excite all the
polymeric species and their selective excitation with lower HOMO–LUMO gap occurs as the
excitation energy decreases. The decrease of the number of species having larger HOMO–
LUMO gap (i.e., smaller size) causes a red-shift of the peak energy and a bleaching of the
peak intensity. At the same time, by lowering the excitation energy, the number of species or
radiative centres that can absorb the excitation light (i.e., the centres having lower HOMO–
LUMO gaps) decreases and this effect causes a narrowing of the FL band.
The spectral narrowing and shift (about 0.23 eV) of the low energy band in the natural
eumelanin samples (at 2.68 eV) with respect to that of the synthetic ones (at 2.45 eV), in the
spectrum obtained at the largest excitation energy can be related to the fact that natural
eumelanins include oligomer systems (inside which radiative recombinations occur) whose
size distribution is narrower with respect to the corresponding systems in synthetic
eumelanin. In particular, a narrower size centre distribution corresponds to a narrower
HOMO–LUMO gap distribution in natural eumelanin samples with respect to the synthetic
one. So, since the fluorescence band results from the convolution of radiative recombinations
from the distribution of the excited HOMO–LUMO gaps, a narrower fluorescence band
occurs in the natural eumelanins than in the synthetic one. The shift of the low energy band in
the spectra of the two kind of samples can be related to a different density of states connected
to the HOMO–LUMO transitions occurring inside the chemical components of the natural
and synthetic biopolymer.

Macromolecular Structure: Dynamic Light Scattering

The structural organization of the biopolymer and the role of proteins have been
investigated also by means of DLS measurements, which allows to estimate the size
distribution of the macromolecular components of the three eumelanin samples in solution, as
shown in Figure 9 [54]. It is evident that the distributions of eumelanin particles have slightly
different modal values, i.e., 70 nm for synthetic eumelanin and 120 nm for natural
eumelanins. The latter value is quite in agreement with analogous results of the
macromolecule sizes of Sepia officinalis eumelanin as obtained by SEM, TEM and AFM
techniques [12, 16]. It is also confirmed that the size of natural eumelanin nanoaggregates is
larger than that of synthetic ones, as reported in [16] for indolic planar sheets, although the
lateral extent of synthetic sheets was estimated to be less than 10 nm large. Although the
modal value of the particle sizes obtained for synthetic eumelanin is slightly lower than that
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286 Giuseppe Perna and Vito Capozzi

got for the natural ones, the distribution of the particle sizes for synthetic eumelanin results
broader than that for natural eumelanins (full width at half maximum of about 180, 147 and
152 nm for synthetic, Sepia officinalis and Rana esculenta samples, respectively). In addition,
a more intense tail towards higher size values can be observed in Figure 9 for the distribution
of the synthetic eumelanin with respect to the natural ones. Since the main difference between
natural and synthetic eumelanin is the larger protein content of the former with respect to the
latter, DLS results suggest that the presence of proteins modifies the chemical environment in
which the indolic sheets are assembled. Further, they aid in the connection of each monomer
units and build up large oligomers. Such protein control of oligomers assembly is lacking in
synthetic eumelanin. Consequently, the modal value of the size distribution of the synthetic
eumelanin particles is lower than the corresponding value of the natural eumelanins, as well
as the size distribution is broader in the former than in the latter cases.

Figure 9. Distribution of particle size for synthetic (continuous line), Sepia officinalis (dashed line) and
Rana esculenta (dotted line) eumelanin solutions, obtained by Dynamic Light Scattering measurements.

Macromolecular Structure: X-Ray Diffraction

The XRD spectra at room temperature of synthetic and Rana esculenta eumelanin are
shown in Figure 10a and 10b, respectively. Both spectra are characterised by a broad peak (as
it occurs in amorphous and disordered materials), centred at about 25.6o for synthetic
eumelanin and 21.5o for natural one. Such peaks are due to X-ray diffraction from parallel
planar layers [56]. The peak position can give information about the interlayer spacing d,
according to the Bragg equation:

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2d sin   m (1)

where  is the diffraction angle, m is the diffraction order and  is the X-ray wavelength. By
considering the first order diffraction (m=1) we obtain d =3.5 Å for synthetic eumelanin and d
=4.0 Å for natural one. In particular, the value of 3.5 Å is in good agreement with the
literature value of the interlayer spacing in the stacked sheets model of the melanin [15]. In
contrast, the increase of the d distance occurring in natural eumelanin is probably due to the
presence of residual molecules (e.g., proteins from the purification procedure) intercalated
between the layers, which increases the interlayer distance.

Figure 10. X-ray Diffraction spectra of a pellet of synthetic eumelanin (a) and a air dried deposit of
Rana esculenta eumelanin (b).

An estimate of the average melanin grain size D can be obtained from the Debye–
Schrerrer relationship [57]:

0.9
D (2)
( FWHM  cos  )

where FWHM is the full width at half maximum of the diffraction peak. The obtained D
values are 13.5 Å for synthetic eumelanin and 10.1 Å for natural one. These values support
the nanoaggregate model of eumelanin: in fact, they may correspond to the lateral or height
extension of the eumelanin stacked sheets protomolecules. In particular, the value D=13.5 Å
obtained for synthetic eumelanin corresponds to about four stacked sheets of planar
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288 Giuseppe Perna and Vito Capozzi

structures. The average size of the natural eumelanin protomolecules is lower than that of
synthetic eumelanin probably for the presence of proteic residual in the former: it weakens the
bonding forces between the layers and, consequently, counteracts the stacking.

Macromolecular Structure: Atomic Force Microscopy

AFM images of height data for synthetic and Rana esculenta eumelanin samples are
shown in Figs. 11 and 12, respectively. In particular, the comparison between Figure 11 and
Figure 12 reveals that the two samples comprise aggregates having similar morphology and
size, whose values range from few nanometers of height size and few tens nanometers of
lateral size (see cross section 2 in Figure 11 and cross sections 2 and 3 in Figure 12) to about
ten nanometers of height size and few hundred nanometers of lateral size (see cross section 1
in Figure 11 and cross section 1 in Figure 12). The main difference between the two images is
that synthetic eumelanin comprises nanoaggregates more isolated than those present in
natural eumelanin, i.e., the former seems less organized than the latter. Such behaviour could
be related to the role of proteins, which are absent in synthetic eumelanin and largely present
in Rana esculenta eumelanin. So we can assume that the proteins, which act either as
scaffolding matrix for eumelanin deposition or as enzymes involved in melanogenic pathway,
are important in defining the assembly of natural pigment.

Figure 11. AFM picture measured in non-contact mode topography and cross section profiles
(corresponding to the straight lines marked on the AFM images) of a synthetic eumelanin sample,
obtained from a drop of eumelanin solution deposited on mica substrate and air dried. The scale bar of
AFM image is reported on its right hand side.

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 Optical Spectroscopy and Structural Properties … 289

Figure 12. AFM picture measured in non-contact mode topography and cross section profiles
(corresponding to the straight lines marked on the AFM images) of a Rana esculenta eumelanin sample,
obtained from a drop of eumelanin solution deposited on mica substrate and air dried. The scale bar of
AFM image is reported on its right hand side.

CONCLUSION
In this paper review we have described some recent results concerning the optical
properties of eumelanin and the structural organization of this biopolymer. The observed
properties are explained by considering eumelanin as formed by many distinct nanometric
protomolecules, each one consisting of several monomer units arranged in different manner.
Indeed, the vibrational spectra confirm the presence of the functional groups present in the
main monomer units (DHI, DHICA, IQ and SQ) of eumelanin, whereas the optical absorption
spectra evidence the overlapping contribution of many distinct HOMO-LUMO transitions.
Moreover, the fluorescence spectra measured as a function of excitation energy indicate that
the radiative emission is due to both ensembles of large oligomer systems and to monomers
and small oligomer systems. So, a large degree of chemical heterogeneity and structural
disorder characterize the nanoaggregate macromolecules building up the biopolymer. Such
large degree of disorder characterizes the optical spectra of both synthetic and natural
eumelanin. Therefore, it can be deduced that this structural and chemical disorder is a
property of eumelanin organization, independently of the presence of proteins. Instead, the
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290 Giuseppe Perna and Vito Capozzi

structural analysis performed by means of DLS, AFM and XRD techniques indicates that the
size distribution of the natural eumelanin has a larger modal value and it is narrower than the
corresponding size distribution occurring in synthetic eumelanin. Therefore, the role of
protein content in the structural organization of natural eumelanin accounts for the linkage of
the large oligomer systems, in order to achieve a size distribution centered at a typical value
(about 120 nm in Sepia officinalis and Rana esculenta eumelanin), which is larger than the
typical value of the size distribution of synthetic eumelanin (about 70 nm). This might be
related to the biological functions of such a biopolymer, particularly as for its photoprotective
action. Today, the physical and chemical properties of eumelanin are known enough and
technical applications finalized to obtain eumelanin-based devices capable of long range
absorption (from UV to IR) are currently being defined.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 10

MELANIC PIGMENTATION IN ECTOTHERMIC

VERTEBRATES: OCCURRENCE AND FUNCTION

Classius de Oliveira1 and Lilian Franco-Belussi2

1
São Paulo State University – UNESP, IBILCE – São José do Rio Preto, Brazil
Department of Biology. São José do Rio Preto, Brazil
2
Post-Graduate Program in Animal Biology (UNESP/IBILCE-SJRP)

ABSTRACT
Ectotherms have specialized chromatophores whose pigments are responsible for the
different colors of the epidermis. Melanocytes are one type of chromatophore that
produce and store melanin in organelles called melanosomes. In ectotherms, cells
containing melanin pigments occur in several organs and tissues. These cells are found in
the capsule and stroma of the organs, giving it a dark coloration. The function of visceral
pigment cells is poorly known, but melanomacrophages are known to perform
phagocytosis in hematopoietic organs and also act against bacteria, due to melanin. In
addition, the distribution of visceral melanocytes varies with physiological factors, such
as age, nutritional status; and also environmental one, such as temperature and
photoperiod. On the other hand, the pigmentation in some organs seems to be
conservative, and may help in phylogenetic reconstructions.

Keywords: Chromatophores, melanin, melanocytes, melanomacrophages, extracutaneous

pigmentary system

INTRODUCTION
Chromatophores are specialized cells found in invertebrates and ectothermic vertebrates
that store pigments. These cells have many cytoplasmic projections, giving it a dendritic
aspect. Chromatophores originate in the neural tube during embryonary development and
later migrate to the skin. In the adult, chromatophores are found in the epidermis and dermis
[1].
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294 Classius de Oliveira and Lilian Franco-Belussi

Chromatophores have many types of pigments. At least five types are described in
vertebrates. The melanophores are black or brown colored cells with melanin granules. They
are found in fish, amphibians, and reptiles. The erythrophores are reddish cells with pteridine
pigments. The xanthophores also have pteridine, along with carotenoid pigments arranged in
vesicles, which gives it a yellow color. The iridophores have a metallic color due to purine
deposited in reflective crystals. As erythrophores and xanthophores, iridophores are also
found in fish, amphibians, and reptiles. The leucophores are white colored cells with purine
granules, and only occur in fish [1, 2].
Chromatophores are found preferably in the dermis of animals. The xanthophores are the
most superficial cells of this skin layer, located just below the basement membrane. Deep
inside the skin there are iridophores, cells that have iridescent appearance. Even more deep,
there are the melanophores. The arrangement of these pigment cells in the skin layers are
closely related to their pigment type and which wavelengths they reflect or absorb [2].
In this chapter, we will discuss some hypotheses posed to explain the function of these
cells in internal organs of ectothermic vertebrates.

MELANOPHORES: COLOR CHANGES AND HORMONAL CONTROL

Melanophores are found in the deepest layer of the dermis and in visceral organs of
ectotherms. These cells are dendritic in shape (Figure 1A), and in dermis are responsible for
the dark color and the quick color change. The arrangement of these pigment cells in the skin
layers are closely related to their pigment type and which wavelengths they reflect or absorb.
This feature dictates its function [2].
Some ectotherms can quickly change the body color through the regulation of
chromatophores. For example, the stimulus for aggregation or dispersion of pigments in fish
may come directly from innervation or alternatively from hormonal control [3]. Contrarily,
pigment migration in amphibians only occurs by hormonal control [2]. This quick color
change is physiological and involves numerous types of chromatophores. It is also related to
camouflage and social signaling [4]. Physiological color change occurs in animals that can
quickly change their coloration through the bidirectional migration of pigment granules
within pigment cells. Environmental stimuli that evoke these changes are mainly associated
with light intensity, background color or social context [5, 6].
Color change may also happens by means of morphological change in vertebrates. It is
slower but lasting than the physiological one. A change in morphological coloration is
defined as a gradual color change resulting from the increase or decrease in the number of
pigment cells or the amount of pigment within cells. Such a change is usually associated with
ontogenetic, sexual, feeding, or seasonal changes [5, 6]. Melanosomes are dispersed and
transferred to skin cells in mammals and amphibians. In these animals, the dispersion of
pigments also stimulates the production of new pigment cells in the long term. The
morphological change in color is also modulated by hormones, although the regulation of
pigment cell differentiation and the transfer of pigments are dependent on intracellular levels
of cyclic AMP [6].

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 Melanic Pigmentation in Ectothermic Vertebrates 295

Figure 1. Visceral pigmented cells of Eupemphix nattereri. A: Visceral pigmented cells of parietal
peritoneum showing dendritic shape. B and C: Electron micrograph of melanocyte of testicle showing
association with fibroblast and presence of melanossomes in the citoplasm. D-H: Stages of
melanossome development. The granules become electron dense by melanin accumulation. M:
Melanossomes. N: Nuleous. Na: Nuclear area. Nu: Nucleolus.

Three main hormones modulate color change in vertebrates. The α-Melanocyte

stimulating hormone (α-MSH) is among the best known of these. This hormone regulates
both the cutaneous pigmentation of ectotherms and extracutaneous pigmentary system. In the
cutaneous pigmentation, α-MSH regulates the dispersion of pigments in melanophores,
xanthophores, and erythrophores [2]. In the extracutaneous pigmentary system, α-MSH
increases the expression of tyrosinase gene, as demonstrated by Guida et al. (2004) in the
liver of Pelophylax lessonae. The melanin-concentrating hormone (MCH), which has an
antagonistic effect to α-MSH, is a cyclic neuropeptide synthesized as a pre-hormone in the
hypothalamus of vertebrates [7]. As a result, MCH influences the aggregation of
melanosomes [3]. Melatonin, a pineal hormone produced in darkness, is responsible for skin
whitening in amphibians [8]. Melatonin also induced the aggregation of melanosomes in
cultures of melanophores from fish and amphibians [9, 10].

Melanossomes: An Organelle That Synthesizes and Stores Melanin

Melanosomes are organelles that produce and store melanin pigments. These organelle
posses distinct stages of maturation, with different shapes, sizes an electron densities [11, 23]
(Figure 1B-H). The initial, or pre-melanosomes, have no pigments but distinctive features. At
the Stage I, melanosomes have irregular fibrous structures and internal membranous vesicles
that resemble typical late multivesicular endosomes (also known as multivesicular bodies).
Stage II melanosomes are easily defined in transmission electronic microscopy due to their
regular, elongated and parallel fibers. These fibers serve as a mold for the deposition of
eumelanin in mature melanosomes. As a result, melanossomes at Stage III are dark and thick.
The accumulation of melanin causes a masking of the intraluminal structure of melanossomes
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296 Classius de Oliveira and Lilian Franco-Belussi

at Stage IV. Thus, the formation of melanosomes with characteristic fibers precedes the
eumelanin synthesis and is an essential step in eumelanogenesis [2, 11].
Melanosomes are mobile structures and move inside cells by the action of motor proteins
guided by microtubules: the cytoplasmic dynein, kinesin II, and myosin V. Each protein act
differently in the movement of melanosomes, and also the aggregation and dispersion of
pigments. Kinesin is responsible for the centrifugal transport, dispersion of melanosomes and
consequent darkening of the animal. Dynein is the antagonist of kinesin and are responsible
for the centripetal transport of melanosomes, aggregation of pigment cells, and consequent
bleaching of the organism [2, 12]. On the other hand, actin molecules are responsible for the
short or cross transport. This transport is conducted by actin molecules because this route is
deslocated from the axis of microtubules. It also allows a greater dispersion of pigments
throughout the cytoplasm. In this type of transport, myosin V binds to melanosomes allowing
the myosin-melanosome set to slide along the actin filament [2, 12].

MELANIN IN ECTOTHERMIC VERTEBRATES

Melanin is an endogenous complex polymer [13] that occurs in multifunctional forms
[14, 15] in both vertebrates and invertebrates. The biosynthesis of this substance is initiated
by hydroxylation of L-phenylalanine into L-tyrosine or directly from L-tyrosine, which is
hydroxylated to L-dihydroxyphenylalanine (L-DOPA) by the tyrosinase enzyme. Lately, L-
DOPA is oxidized to dopaquinone by the tyrosinase enzyme, which diverges into the
synthesis of eu- or pheomelanin [14, 15]. Melanin may absorb and neutralize free radicals,
cations, and other potentially toxic agents derived from the degradation of phagocytosed
cellular material [16]. Melanin is also a key agent against bacterial components in ectothermic
vertebrates, due to the action of hydrogen-peroxidase and their quinone precursors, which act
as a bactericide [17].
A unique characteristic of ectothermic vertebrates is the presence of an extracutaneous
pigmentary system [18] in various tissues and organs composed of many cells with melanin-
filled cytoplasm. The melanin often present in the liver, spleen, kidneys, peritoneum, lungs,
heart, blood vessels, thymus, gonads, and meninges constitute the visceral pigmentation [17,
19, 20, 21, 22] (Figure 2). Visceral melanocytes are localized closed to vessels and
conjunctive membranes (Figure 3) and these cells are distributed in both surface and
interstitium of the organs’ stroma (Figure 4).

Visceral Pigmentation: Anatomical Patterns in Anurans

In anurans, visceral pigmentation is present in several organs of the abdominal cavity, we

hypothesize that this pigmentation has a similar pattern of occurrence within taxonomic ranks
(species, genera and families). In fishes, the presence of extracutaneous pigment is highly
variable, even among closely related species [16].

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 Melanic Pigmentation in Ectothermic Vertebrates 297

Figure 2. Organs and structures of the abdominal cavity of distinct anurans species showing variation in
visceral pigmentation in categories according to Franco-Belussi et al. 2009. Category 0: absence of
melanic pigmetnation. Category 1: Presence a few pigmented cells. Category 2: Moderated
pigmentation. Category 3: Intense melanic pigmentation. O a: Odontophrynus americanus; E n:
Eupemphix nattereri; H m: Hylodes magalhaesi; H s: Hylodes sazimai, P cv: Physalaemus cuvieri; I j:
Ischnocnema juipoca; P o: Physalaemus olfersii; PS f: Pseudopaludicola falcipes; E b:
Eleutherodactylus binotatus; R o: Rhinella ornata; PR b: Proceratophrys boiei; PR m: Proceratophrys
melanopogon; L fr: Leptodactylus furnarius; L b: Leptodactylus bokermanni; D m: Dendropsophus
minutus; D n: Dendropsophus nanus.

Such studies found that the pigmentation on the surface of testes of several anurans varies
among species and genera. Members of genera Adelphobates, Colostethus, Dendropsophus,
and Leptodactylus no present pigmentation on the testes [24, 25, 26]. On the other hand,
members of the family Leiuperidae and other Dendrobatidae have melanic pigments on the
testes in various degrees of pigmentation [24, 25].
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298 Classius de Oliveira and Lilian Franco-Belussi

Figure 3. Presence of melanic pigmented cells in conjuntive membrane of nerves of the lumbar plexus
(A) and associated with renal blood vessels (B) of Eupemphix nattereri. C: Vertebral column. K:
Kidney. N: Nerve of the lumbar plexus. P: Lumbosacral parietal peritoneum. R:Renal blood vesels.
Arrow: pigmented cells.

Figure 4. Melanic pigmentation in testicles of Eupemphix nattereri showed intense color black on the
surface (A, D) and pigmented cells in interstitium of this organ (B) with associated with blood vessels
(C) and presence of pigmented cells in surface (D). L: Seminiferous locule. T: Testicle. V: Blood
vessel. Arrow: pigmented cells. B-D: Stained with Hematoxilin-eosin.

So, these findings suggest that the visceral pigmentation shows a phylogenetic signal.
Although, studies reported that the pigmentation on testes varies. This variation is related
with breeding season in the bufonids Rhinella schneideri [27] and Atelopus spp. [28]. In
Physalaemus cuvieri testicular pigmentation is present and not varies during breeding season
[27]. In the kidneys, similar to testes, the pigmentation on the dorsal surface increased from
the beginning towards the end of the breeding season in Dendropsophus nanus [27]. On the

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 Melanic Pigmentation in Ectothermic Vertebrates 299

heart, testes and kidneys pigmentation in Eupemphix nattereri increase following

administration with E. colli’s lippopolysacaride [26].
Pigmented cells in the epidermis and various organs are similar to melanocytes [29, 21]
originated from the ectodermal neural crest [30]. These cells are able to produce and store
melanin [31]. However, additional studies about its occurrence and anatomical distribution
are needed in order to determine their biological functions.

THE MELANIN IN HEMATOPOIETIC ORGANS

As mentioned previously, a unique feature of ectothermic vertebrates is the presence of
an extracutaneous pigmentary system in various tissues and organs composed of many cells
with melanin-filled cytoplasm. In hematopoietic organs, a pigmentary cell types with
phagocytic activity are found. Few studies have investigated the functions of the pigmentary
system in animals. The majority of studies have focused on the pigmented cells of the spleen
and liver.
These macrophage-like cells [32] are derived from hematopoietic stem cells [30] and
often aggregate in pigmented nodules called melanomacrophage centers [33]. These centers
belong to the mononuclear phagocyte system and its main function is related to the
phagocytosis of cellular material derived from catabolism [34]. This evidence suggests that
melanomacrophage centers are responsible for the detoxification or recycling of both
endogenous and exogenous products [35]. There are also evidences that melanomacrophage
centers are involved in the resistance against bacterial pathogens, parasites, and spores [36].
The storage of iron after erythrophagocytosis is also reported [31].

Figure 5. Liver sections showing melanomacrophages in the tissue (A) and presence of hemosiderin (B)
and lipofuscin inside of pigmented cell. H: Hepatocytes. S: Sinusoids. Coloration: Acid ferrocyanide
solution (B) and Schmorl’s solution (C).
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300 Classius de Oliveira and Lilian Franco-Belussi

The presence of melanomacrophages is reported in the spleen, liver, and kidneys. These
cells are known as Kupffer cells in the liver and have phagocytic activity. They can be
classified as “small” or “large” Kupffer cells, according to their melanin content [37, 38].
These cells have peroxidase, lipase [18], and melanin granules, along with other substances,
such as hemosiderin and lipofuscin, derived from the degradation of phagocytosed cellular
material (Figure 5) [21, 39, 40].
Haemosiderin is a granular substance composed of iron hydroxide and proteins. It is
generated in tissues saturated with iron ions and have to accumulate in granules to remain
stored inside the cell [41]. The hemosiderin have protein derived from the catabolism of
hemoglobin, and therefore it is an intermediate metabolite in the recycling of components in
the erythropoiesis [42]. The production of granules of denatured hemoglobin occurs during
the catabolism of red cells, which takes place in digestive vacuoles. These vacuoles are
yellow-brownish due to iron hydroxide and bile pigments. The color tends to fade out within
three to four days, although some partially digested granules may remain in the tissue,
producing a yellow color due to the absorption of bilirubin [41].
Lipofuscin, also known as the age pigment, is an intra-lysosomal pigment that are neither
degraded by lysosomal hydrolases nor exocitated [43]. This pigment is produced by the
oxidative polymerization of polyunsaturated fatty acids and accumulates in post-mitotic cells
[44]. During the normal autophagic degradation of mitochondria and iron-containing proteins
in lysosomes, iron is released in lysosomes, in which it may react with hydrogen peroxide to
form hydroxyl radicals. Depending on the rate of formation of these highly reactive radicals,
they can bind to lysosomal material to form lipofuscin or these reactive radicals can
destabilize the lysosomal membrane, inducing apoptosis and necrosis [43, 45].
Some studies have described drastic structural and functional alterations in the Kupffer
cells during the hibernation cycle, a period characterized by low temperatures and reduced
food supply. In an experiment with three amphibian species (Rana esculenta, Ichthyosaura
alpestris, and Triturus carnifex), there were much more pigments in the hepatic parenchyma
in the hibernation than in the active period [46, 47]. In addition, an increase in liver
pigmentation may be related to hemocatereses [48, 49]. For example, the activation of hepatic
melanogenesis in salamanders may be related to hypoxia [50]. Accordingly, the increase of
melanin pigments in melanomacrophage centers in fish has been associated with diseases
[36].

THE FUNCTIONS OF MELANIN IN VISCERAL PIGMENTATION

Moresco and Oliveira (2009) analyzed the extracutaneous pigmentation pattern of three
species of anuran amphibians (Dendropsophus nanus, Physalaemus cuvieri, and Rhinella
schneideri) during the breeding season. In that study, the change in the pigmentation of
structures during the reproductive period could not be associated with or compared among
species, since the occurrence of pigmentation was different for each species. The authors
reported that the pigmentation varied during the reproductive period in the toad R. schneideri.
However, the same study showed that the testicular pigmentation was evenly distributed
throughout the breeding season in P. cuvieri.

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 Melanic Pigmentation in Ectothermic Vertebrates 301

Accordingly, the gonads of D. nanus, D. minutus, D. elianeae, and D. sanborni had no

pigmentation during the reproductive period [19]. These differences between species of
distinct families can be related with similar phenotypic traits, in species that lives in similar
environmental conditions [51]. Ours studies showed that is possible determine a pattern for
each species, and identify a relationships among within of the taxon. These description of
visceral pigmentation represent helpful information to evaluate biological relations in a
phylogenetic and evolutionary perspective.
Pigment cells are not found in the gonads of the majority of anuran species (e.g., Franco-
Belussi et al. 2011). When present, visceral melanocytes are closely related to the vascular
system, as well as blood vessels of other organs and associated conjunctive membranes.
Specifically, there is an intense pigmentation in the interstitium and the tunica albuginea of
the gonads of Eupemphix nattereri, Physalaemus cuvieri, and P. marmoratus, giving the
testicle a full or mixed black color [23, 52, 53, 54].
These cells make up the connective tissue of the organ itself or of tissues associated with
it, such as the tunica adventitia or serous membranes. Pigmented cells of most organs, such as
gonads and rectum have typical dendritic melanocytes, which differentiate it from
melanomacrophages of organs, which have a punctuated appearance. Melanocytes are
distributed in both the surface and interstitium of the organs’ stroma. Its occurrence may vary
from a few to a large concentration of cells, when an intense blackish color is observed on
structures.
The visceral pigmentation on testes, heart, and kidneys of anurans increases after the
administration of lipopolysaccharide (LPS) from Escherichia coli [26]. These cells responded
to LPS intoxication promoting a rapid increase of pigmentation on the surface of the testes
after 2 hours, followed by a decrease in the pigmentation after 24 hours of administration.
These changes are probably related to the bactericide role of melanin, which neutralizes LPS
effects [26].

CONCLUSION
Finally, the pigmentation is an anatomical feature of an organism. However there are
very complex relationship between chromatophores and the organs in which they occur.
Certainly melanocytes have multiple functions still waiting to be determined. Additional
studies about its occurrence and anatomical distribution are needed in order to determine their
biological functions.

ACKNOWLEDGMENTS
The authors are indebted to FAPESP (São Paulo Research Foundation – grant #
02/08016-9, 05/02919-5, 09/13925-7, 06/57990-9, and 08/52389-0) and CNPq (Brazilian
National Council for Scientific and Technological Development – grant # 475248/2007-4 and
473499/2010-0) for financial support and fellowships. LFB received a doctoral fellowship
from FAPESP (#2011/01840-7) during the final preparation of this chapter. We would like to
thank Msc. Diogo Borges Provete for suggestions in the chapter and revision of the English
 Free ebooks ==> www.Ebook777.com
302 Classius de Oliveira and Lilian Franco-Belussi

language, and Dr. Lia Raquel de Souza Santos, Dr. Rodrigo Zieri, Msc. Rafaela Maria
Moresco for contributing for the research project.

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Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 11

FAIRNESS IN A NATURAL WAY -- NOVEL

POLYHERBAL INGREDIENTS INHIBITING
MELANIN SYNTHESIS AND TRANSFER

S. Gokulshankar1, M. S. Ranjith1, Babu2, M. A. Deepa3,

B. K. Mohanty1 and G. Prabhakaran4
1
Faculty of Medicine, AIMST University, Semeling, Malaysia
2
R andD Center, Cholayil Private Limited, Chennai, India
3
Department of Life Sciences, Kristu Jayanti College, Bangalore, India
4
Department of Biotechnology, Faculty of Applied Sciences,
AIMST University, Malaysia

ABSTRACT
Melanocytes produce melanin that determines the skin color. Skin color can be
mildly manipulated by use of fairness creams with skin lightening/ whitening ingredients.
Some of skin lightening ingredients are harmful to skin and health due to their deleterious
effects. Yet the ‘quest for fairness’ is global and that puts the research on safe skin
lightening products as one of the pinnacles in the billion dollar cosmetic industry.
Tyrosine is the precursor in the sequels of biochemical pathways that lead to the
formation of melanin pigment. Tyrosinase is the key enzyme that mediates two steps in
the biochemical conversion of tyrosine to melanin. Hence most skin lightening
ingredients exhibit their mode of action by tyrosinase inhibition. Melanocytes are
dendritic cells and they are involved in the transfer of melanosomes to the keratinocytes.
This process is aided by the dendrites in the melanocytes. Any qualitative and or
quantitative changes to the dendrites in the melanocytes would effect the transfer and
thereby the melanization of the skin. Besides understanding the tyrosinase modulating
activity, it is also necessary to study the effect of the skin lightening agents on the
dendrites in the melanocytes. The extracts of several plants such as bearberry, cranberry,
mulberry or blueberry are used in the skin lightening formulations. We studied the
tyrosinase inhibitory effects of the extracts of Hemidesmus indicus, Decalepis hamiltonii,
Raphanus sativus var. longipinnatus (white), Raphanus sativus var. sativus (Red),
Curcuma zedoaria and Aloe vera individually and in different permutation combinations.
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308 S. Gokulshankar, M. S. Ranjith, Babu et al.

Tyrosinase inhibition assay, melanocyte cell culture assay, measurement of dendrite

length and number of melanocytes were used as methods to evaluate the efficacy of the
extract combinations. Tyrosinase activity was assayed spectrophotometrically by
following the oxidation of DOPA to dopachrome at 475 nm. B16F10 murine melanoma
cells were cultured in Eagles minimal essential medium with supplements. The extracts
treated melanocytes were examined under microscope, the number and relative length of
dendrites in each melanocyte were recorded at random and statistically compared with
untreated control. The polyherbal extract combinations of Curcuma zedoaria, Aloe vera
and Decalepis hamiltonii was found to be effective in inhibiting the melanin synthesis
and may also have a suggestive role in preventing the melanin transfer to the
keratinocytes thereby could bring about the desired skin lightening benefit.

INTRODUCTION
Melanin, a pigment which is ubiquitous in nature determines the skin colour of man. The
most common form of biological melanin is Eumelanin and is produced by man, animals and
certain microorganisms. In man, melanin pigments are derivatives of the amino acid tyrosine.
Skin color in man is of cosmetic significance as fairness and its perception as the symbol
of beauty and the pursuit to achieve it through skin lightening cosmetics are universal. Skin
color results from the transfer of the melanin-containing melanosomes, produced by the
melanocytes, into the keratinocytes in the epidermis and their ensuing degradation.
Interestingly, there is only a little variation in the number of epidermal melanocytes between
light and dark-skinned individuals [1].
Stratum basale (also known as the stratum germinativum) is the basement layer of the
skin that separates the epidermis from the dermis that consists of keratinocytes and
melanocytes. The keratinocytes-melanocytes are sometimes referred as "the epidermal
melanin unit.” Synthesis of melanin by melanocytes, within highly organized elliptic
membrane-bound organelles called melanosome, is a complex pathway [2] (Figure 1).
Melanin-containing melanosomes move from the perinuclear region to the dendrite tips and
are transferred to keratinocytes. It has been estimated that each melanocyte is in contact with
∼40 keratinocytes [3].
Any qualitative and or quantitative changes to the dendrites in the melanocytes would
affect the transfer and thereby the melanization of the skin. Besides understanding
the tyrosinase modulating activity, it is also necessary to study the effect of the skin
lightening agents on the dendrites in the melanocytes.
There is roughly a distribution of 2000 epidermal melanocytes/mm2 on the head and
forearm of the man and 1000 epidermal melanocytes/ mm2 on the other parts of the body. It is
also been found that these differences are usually present at birth and exist till death in the
normal conditions1. Thus, all persons, be it either extremely dark skinned individuals or light
skinned ones, have the same total number of melanocytes in them. Then, what makes the
difference is only the distribution and location of melanosomes [1].
In lighter skin people, keratinocytes of both the thigh and volar skin exhibit complexed
melanosomes while the remaining skin usually processes singly dispersed melanosomes. In
contratry to that keratinocytes from the thighs of dark-skinned individuals display singly
dispersed melanosomes. But invariably keratinocytes from the lighter volar skin have
complexed melanosomes. Interestingly it is amazing to know that the melanosomes in the

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 Fairness in a Natural Way 309

minimally pigmented volar skin of dark-skinned individuals closely resemble the

melanosomes of lighter-skinned individuals. Such findings reiterate the fact that the skin
color has direct correlation with the distribution of melanosomes [1].
Further, the investigators found that the skin complexion was affected both by epidermal
melanin concentration and to a smaller extent by the deoxyhemoglobin residing in the
superficial venous plexus. Skin color can be mildly manipulated by use of fairness creams
with skin lightening/whitening ingredients. Several skin lightening ingredients of
synthetic/herbal origin [4, 5, 6] are widely used in fairness/skin lightening creams, underarm
lightening creams/ facial and body massage creams (Table 1).

Table 1. Popular Skin lightening ingredients currently in the market

Active Ingredient Source Mode of action
Pre-melanin synthesis
Tretinoin (all-trans retinoic acid) Synthetic (acid form of vitamin A) Unknown
During melanin synthesis
Hydroquinone Synthetic Cytotoxic to melanocytes
Glycyrrhizin Glycyrrhiza glabra Tyrosinase inhibitor
Beta-Arbutin Uva ursi (bearberry) extract, Morus Tyrosinase inhibitor
bombycis (mulberry), Morus alba
(white mulberry), and Broussonetia
papyrifera (paper mulberry)
Kojic Acid By product in the manufacturing Makes melanocytes
of ‘Sake’ (Japanese rice wine) nondendritic and decreases
melanin content
Glabridin Licorice (Glycyrrhiza glabra) Tyrosinase inhibitor
Emblica Phyllanthus emblica Tyrosinase inhibitor
Tyrostat Rumex occidentalis extract Tyrosinase inhibitor
Azelaic acid Wheat, Rye, Barley, Malassezia spp. Tyrosinase inhibitor
Alpha-Arbutin Bio-synthetic Tyrosinase inhibitor
Vitamin C (Magnesium ascorbyl Synthetic Acts as reducing agent on
phosphate, L-ascorbic acid, melanin pathway intermediates
ascorbyl glucosamine, and
ascorbic acid)
Melanostat Synthetic (peptide obtained by amino Tyrosinase inhibitor
acid synthesis)
Paper mulberry extract Broussonetia kazinoki Tyrosinase inhibitor
Post melanin synthesis
Niacinamide Synthetic (amide of nicotinic Inhibition of melanosome
acid (vitamin B3 / niacin) transfer
Alpha hydroxyl acids (lactic/ Synthetic Remove hyperpigmented cells
glycolic acid) by exfoliation
Monobenzone Synthetic (Monobenzyl ether of Destruction of melanocytes
hydroquinone)
Mequinol Synthetic (Monomethyl ether of Destruction of melanocytes
hydroquinone)
Soy extract Soya bean Interaction with PAR-2 of
keratinocytes
Pycnogenol Pinus pinaster (French maritime pine Removes existing melanin
Septiwhite MSH® Undecylenoyl phenylalanine Alpha MSH(melanotropin)
antagonist

Sometimes extracts of the plants are also used in these formulations. Some of the skin
lightening ingredients are reported to be harmful to skin and health due to their deleterious
 Free ebooks ==> www.Ebook777.com
310 S. Gokulshankar, M. S. Ranjith, Babu et al.

effects. Yet the ‘quest for fairness’ is global and that puts the research on safe skin lightening
products as one of the pinnacles in the billion dollar cosmetic industry.
Tyrosine is the precursor in the sequels of biochemical pathways that lead to the
formation of melanin pigment. Tyrosinase is the key enzyme that mediates two steps in the
biochemical conversion of tyrosine to melanin (Figure 1).

Figure 1. Pathway of melanin synthesis.

Hence most skin lightening ingredients exhibit their mode of action by tyrosinase
inhibition (Table 1). Extracts of several plants belonging to different geographical locations
have been screened and reported to have anti-tyrosinase activity (Table 2). Further, the anti-
tyrosinase activity depends on the plant parts, the solvents used for the extraction and the
process of extraction.
Hence, anti-tyrosinase compunds that acts as a tyrosinase inhibitor either by inhibiting
the conversion of tyrosine to DOPA or DOPA to Dopaquione and eventually into melanin. So
far, a number of anti-tyrosinase compounds are identified, manufactured/extracted and used
in the cosmetic formulations. Some are synthetically produced while some are isolated from
the plants. Agents such as hydroquinone, salicylhydroxaminc acid, azealic acid, retinoids,
arbutin, glabaridin are synthetically manufactured tyrosinase inhibitors whereas kojic acid,
quercetin, extracts of several plants such as bearberry, cranberry, mulberry or blueberry are
natural tyrosinase inhibitors.

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 Fairness in a Natural Way 311

Table 2. Plants screened and reported to have anti-tyrosinase

activity from different countries

Country Name of the plant

Anacardium occidentale, Etlingera eliator, Etlingera fulgens, Etlingera littoralis, Etlingera
maingayi, Etlingera rubrostriata, Garcinia mangostana, Hibiscus mutabilis, Hibiscus rosa-
Malaysia sinensis, Hibiscus sabdarifa, Hibiscus tliaceus, Macaranga gigantea, Macaranga pruinosa,
Macaranga tanarius, Macaranga triloba, Psidium guaava, Pulchea indica, Quercus
infectoria

Aloe vera, Aspidisra sutepensis, Boesenbergia pandurata, Blumea balsamifera,

Coriandrum sativus,Cucumis sativum, Curcuma aromatica, Cymbopogon citratus, Daucus
carota ssp.sativus, Duguetia uniflora, Eurycoma longifolia, Garcinia mangostana,
Herperethusa cernulata, Hibiscus esculentus, Hibiscus sabdariffa, Lycopersicon
Thailand esculentum, Mabea nitida, Mentha cordifolia, Momordica charantia, Nelumbo nucifera,,
Ocimum basilicum, Piper longum, Piranhea trifolia,Psophocarpus tetragonolobus,
Rapanea parviflor, Raphanus sativus, Ruprechtia sp.,Schefflera leucantha, Sesbania
grandiflora, Trigonostemon reideoides

Cornus walteri, Cudrania tricuspidata,Distylium racemosum, Ficus erecta var.sieboldii,

Limonium tetragonum, Maackia faurier, Morus bombycis, Morus alba,Myrica rubra,
Korea Phormium tenax, Rhus javanica, Rumex crispus, Toxicodendron succedaneum, Veratrum
patulum
Allophylus timorensis, Asparagus cochinchinensis, Bidens pilosa var. radiate, Calophyllum
inophyllum, Carex pumila, Cassytha filiformis, Cerbera manghas, Clerodendrum inerme,
Crinum asiaticum var. japonicum, Crossostephium chinense, Exocoecaria agallocha,
Flagellaria indica, Garcinia subelliptica, Hernandia nymphaefolia, Hibiscus tiliaceus,
Ipomoea pes-caprae subsp. Brasiliensis, Ishchaemum muticum, Ixeris lanceota, Lactuca
formosana, Limonium wrightii var. arbusculum, Liriope spicata, Lysimachia mauritiana,
Japan Maytenus diversifolia, Morus australis var. glabara, Pandanus tectorius var. tectorius,
Pemphis acidula, Peucedanum japonicum, Pongamia pinnata, Scaevola taccada, Sesuvium
portulacastrum, Sophora tomentosa, Spinifex littoreus, Stenotaphorum secundatum,
Terminalia catappa, Thespesia populnea, Tournefotia argentea, Vigna marina, Vitex
trifolia var. Trifolia, Wedelia biflora

Aloe vera, Asparagus racemosus, Curcuma zedoaria, Holarrhena antidysenterica, Lippia

India nodiflora, Pachygone ovata
Gentiana macrophylla, Glycyrrhiza uralensis, Lithospermum erythrorhizon, Morus alba,
China Pharbitis nil, Sophora japonica, Spatholobus suberectus
Camellia sinensis, Citrus grandis, Koelreuteria henryi, Malus doumeri var. formosana,
Taiwan Rhodiola rosea
Cymbopogon citrates, Piper longum, Raphanus sativus, Aloe vera, Sesbania grandiflora ,
Ocimum basilicum, Momordica charantia, Hibiscus esculentus, Abelmoschus esculentus ,
Boesenbergia pandurata, Psophocarpus tetragonolobus, Lycopersicon esculentum,
Thailand Coriandrum sativum, Cucumis sativus, Ocimum sanctum, Mentha cordifolia, Daucus
carota ssp.sativus

Vitex negundo Linn.

Pakistan

The natural tyrosinase inhibitors are believed to be relatively safer and known as mild
agents for treating hyper pigmentation disorders and used extensively as cosmetic agents for
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312 S. Gokulshankar, M. S. Ranjith, Babu et al.

skin whitening effect. Synthetic compounds like hydroxyquinone are completely eliminated
in modern cosmetics as they can cause allergic reactions, cytotoxicity and mutagenicity.

Figure 2. Decalepis hamiltonii – Root.

Figure 3. Hemidesmus indicus – Root.

Figure 4. Aloe vera – Whole plant.

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 Fairness in a Natural Way 313

Figure 5. Curcuma zedoria – Rhizome.

Figure 6. Raphanus sativus (white) – Root.

Figure 7. Raphanus sativus (Red) – Root.

Melanocytes are dendritic cells and they are involved in the transfer of melanosomes to
the keratinocytes. This process is aided by the dendrites in the melanocytes. Any qualitative
and or quantitative changes to the dendrites in the melanocytes would affect the transfer and
thereby the melanization of the skin. Besides understanding the tyrosinase modulating
activity, it is also necessary to study the effect of the skin lightening agent on the dendrites in
the melanocytes. Six plants commonly grown/ cultivated in India (Hemidesmus indicus,
Decalepis hamiltonii, Raphanus sativus var. longipinnatus (white), Raphanus sativus var.
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314 S. Gokulshankar, M. S. Ranjith, Babu et al.

sativus (Red), Curcuma zedoaria and Aloe vera) were identified for the present study(Figures
2-7) and investigated for their effect on melanin synthesis and transfer. The extracts of these
plants/plant parts were tested both individually and in different combinations.

Decalepis hamiltonii Wight and Arn. (Asclepiadaceae)

Vernacular names – Sanskrit – Svetasariva; Tamil – Mavilikizhangu, Mahalikizhangu.

Habit - Climbing shrub with aromatics roots. Leaves elliptic-obovate. Flowers whitish-
brown, in paniculate cymes. Follicles stout, short.
Distribution in India– Peninsular India, Tamilnadu and Andhra Pradesh.
Chemical constituents – Roots contains aldehyde inositol, saponins, tannins, crystalline
resin acid, sterols.

Hemidesmus indicus (L.) Schult. (Periplocaceae)

Vernacular names: Sanskrit – Sariva; English - Indian Sarsaparilla- Tamil – Nannari.

Habit – Twining or prostrate or semi-erect laticiferous herbs. Leaves linear-lanceolate,
often with white streaks above. Flowers yellow to brownish, in cymes. Follicles slender,
divaricate.
Distribution – India (Upper to Gangetic plains eastwards to Bengal and from Madhya
Pradesh to South India), Srilanka.
Chemical constituents – Roots contain hexatriacotanes, lupeol,α-amyrin, β-amyrin,
sitosterol, p-methoxysalicylic aldehyde as the major constituents.

Aloe vera (L.) Burm. F. (Liliaceae)

Vernacular names: Sanskrit – Kumari; English - Aloe ; Tamil – Katrazhai.

Habit – Perennial herbs with fleshy ensiform leaves. Flowers reddish-yellow, in long
scapes.
Distribution – Native to West Indies and is now naturalized in India.
Chemical constituents – Plant contains aloesone and aloesin. Leaves contain barbuloin,
glycoside and isobarbaloin and β –barbaloin, free anthraquinone like aloe emodin, iso-
emodin.

Curcuma zedoria (Berg.) Rosc. (Zingiberaceae)

Vernacular names: Sanskrit – Kumari; English - Zedoray; Tamil – Kachora,

Kicchilikizhangu.
Habit – Herbs with rhizome bearing palmately branched sessile cylindric tubers. Leaves
with long petiole, oblanceolate. Flowers yellow, in spikes; flowering bracts cymbiform, green
tinged with red.

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 Fairness in a Natural Way 315

Distribution – Cultivated throughout India.

Chemical constituents – Rhizome contains sesquiterpenoids – curcumol, zederone,
fyranodiene, cumolone, curcumenone, zedoarol etc.

Raphanus sativus Linn. (Brassicaceae)

Habit – Annual herb with fleshy fusiform tap root. Roots 22-25 cm long, 3-5cm dia.,
cylindrical, skin pure white and smooth; flesh snow white crisp, solid and mild in flavour.
Leaves in rosette, radical, lyrate. Flowers pink, in racemes. Fruit cylindrical, gibbous at base.
Distribution – Uttar Pradesh, Punjab, Maharastra and Baroda (India).
Chemical constituents – Roots contains Vit-A,C and proteins.

Table 3. Details of plants and parts used for extraction

Botanical name Common English name Part used

Hemidesmus indicus Indian Sarsaparilla Root
Decalepis hamiltonii Swallow root Root
Raphanus sativus var. Radish (White) Root
longipinnatus
Raphanus sativus var. sativus Radish (Red) Root
Curcuma zedoaria Zedoary (White Rhizome
turmeric)
Aloe vera Aloe, Burn plant Leaves

Cell Culture

B16F10 murine melanoma cells were cultured in Eagles minimal essential medium
supplemented with 10% heat inactivated fetal bovine serum and 2mM L-glutamine at 37ºC in
a humidified atmosphere containing 5% CO2. Different concentrations of the extracts of the
herbs were used for the study on as is basis and in different permutation combinations.
Extracts ranging from 1-5 µl were added to the culture after the cells were seeded. The cells
were incubated for 24, 48 or 72 hrs. After incubation the cell numbers were determined by
counting using a haemocytometer chamber. Melanin contents and Tyrosinase activities were
also determined7 in triplicate for each treatment as detailed below.

Melanin Measurement

Melanin content was measured as per the method described below. Approximately 10 [7]
cells were pelleted by centrifugation at 1000 g for 5 minutes and then washed twice with
phosphate buffered saline.
After further centrifugation, the supernatant was decanted, the precipitated cells were
suspended in 200 μl of distilled water, and 1 ml of ethanol- ether 1:1 was added to remove
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316 S. Gokulshankar, M. S. Ranjith, Babu et al.

opaque substances other than melanin. The mixture was then stored and suspended at room
temperature for 15 minutes.
After further centrifugation at 3000 g for 5 minutes, the precipitate was solubilized by
treatment with 1 ml 1N NaOH/10% dimethyl sulfoxide at 80°C for 30 minutes in a capped
tube. The absorbance was measured at 400 nm and the melanin content per cell was
calculated and expressed as percentage of control (=100%) using standard procedure7.

Tyrosinase Assay

Tyrosinase activity was assayed as DOPA oxidase activity [7]. Approximately 107 cells
were pelleted and then washed twice with phosphate buffered saline. After centrifugation, the
supernatant was decanted. The cell pellet was dissolved in 1.0 ml of 0.5% sodium
deoxycholate in distilled water and allowed to stand at 0°C for 15 minutes.
Tyrosinase activity was assayed spectrophotometrically by following the oxidation of
DOPA to dopachrome at 475 nm. The reaction mixture consisting of 3 ml of 0.1% L-DOPA
in 0.1 M phosphate buffer, pH6.8 was mixed with cell lysate.
Assay was performed at 37°C in a spectrophotometer. The reaction rate was measured
during the first 10 minutes of the reaction while it was linear. Corrections for auto oxidation
of L DOPA in controls were made. Specific activity was defined as the amount of
DOPAchrome formed per 10 min per cell, and is expressed as percentage control (=100).

Dendrite Length and Number Measurement

The extracts treated melanocytes were examined under microscope and the number and
relative length of dendrites in each melanocytes were recorded at random and compared with
untreated control7.

A New Breakthrough in Skin Lightening Benefit: Synergy of Poly Herbal

Combination in Inhibiting Melanin Synthesis and Transfer

1) Curcuma zedoaria recorded the highest anti-tyrosinase activity followed by

Decalepis hamiltonii (Table 4)
2) Based on the above findings the following combinations were made with three herbal
extracts (in 1:1:1 ratio) keeping Curcuma zedoaria and Decalepis hamiltonii in all
the four combinations.

a) Aloe vera, Curcuma zedoaria and Decalepis hamiltonii

b) Raphanus sativus var. longipinnatus, Curcuma zedoaria and Decalepis hamiltonii
c) Raphanus sativus var. sativus, Curcuma zedoaria and Decalepis hamiltonii
d) Hemidesmus indicus, Curcuma zedoaria and Decalepis hamiltonii

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 Fairness in a Natural Way 317

Table 4. Effect of individual plant extracts on Tyrosinase inhibition,

dendrite length and number

Tyrosinase Dendrite Dendrite

Plants
inhibition % length (µm) number
Hemidesmus indicus 51 70 ±2 12 ±3
Decalepis hamiltonii 64 68 ±4 7 ±2
Raphanus sativus var.
longipinnatus 40 85 ±3 15 ±3
Raphanus sativus var. sativus 41 96 ±3 15 ±2
Curcuma zedoaria 68 65 ±4 8 ±2
Aloe vera 49 87 ±3 11 ±3
Control - 108 ±4 17 ±3

Aloe Vera was found to be synergistic with the combined poly herbal extract of Curcuma
zedoaria and Decalepis hamiltonii in exhibiting anti-tyrosinase activity and inhibiting the
dentrite length and numbers in the melanocytes which indirectly affects the melanin transfer
to the keratinocytes. A similar synergistic activity was also recorded with the poly hebal
combination of Hemidesmus indicus, Curcuma zedoaria and Decalepis hamiltonii (Table 5).

Table 5. Effect of polyherbal extracts on Tyrosinase inhibition,

dendrite length and number

Tyrosinase Dendrite Dendrite

Extract combinations
inhibition % length (µm) number
Control - 108 ±4 17 ±3
Aloe vera, Curcuma zedoaria
and Decalepis hamiltonii 73 61 ±2 6 ±2
Raphanus sativus var.
longipinnatus, Curcuma
zedoaria and Decalepis
hamiltonii 68 75 ±3 10 ±3
Raphanus sativus var.
sativus, Curcuma zedoaria
and Decalepis hamiltonii 65 80 ±3 11 ±2
Hemidesmus indicus,
Curcuma zedoaria and
Decalepis hamiltonii 70 60 ±2 7±2

3) The dendrite length (µm) of the control was 108 ±6 while the best of the polyherbal
combinations showed a reduction in the length to a level of 60-61 ±4. A similar
reduction of the dendrite number 6-7±2 was observed as against the control which
recorded 17±3.
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318 S. Gokulshankar, M. S. Ranjith, Babu et al.

4) Aloe vera, Curcuma zedoaria and Decalepis hamiltonii polyherbal extract

combination recorded a maximum reduction of 71% in the melanin content assay
5) Hakozaki et al8 have reported that niacinamide reduces cutaneous pigmentation by
suppression of melanosome transfer. Such an effect can occur because of reduced
number of dendrites on melanoctyes or reduction in their length. Krishnamoorthy et
al7 are the first to record that poly herbal extracts can bring about reduction in the
dendrite length and number. In our present study we have reestablished the fact that
poly herbal extracts combination can bring about suppression of melanosome transfer
effectively. A schematic representation of the reduction in dendrite length and
number in extract treated melanocyte in comparison to the control is represented in
Figures 8 and 9.

Table 6. Effect of polyyherbal extracts on melanin content

Melanin
Plants
inhibition (%)
Aloe vera, Curcuma zedoaria and Decalepis hamiltonii 71
Raphanus sativus var. longipinnatus, Curcuma zedoaria and Decalepis
hamiltonii 64
Raphanus sativus var. sativus, Curcuma zedoaria and Decalepis hamiltonii 56
Hemidesmus indicus, Curcuma zedoaria and Decalepis hamiltonii 69

Figure 8. Schematic representation of Melanocyte with normal dendrites and melanosmoes – Control.

Figure 9. Schematic representation of Melanocyte with reduced number of dendrites with shortened
length- treated with polyherbal extract.

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 Fairness in a Natural Way 319

CONCLUSION
Beauty is a qualitative element that boosts the self esteem of a person and is a joy for the
eyes of the beholder. Aesthetic appearance is indeed the pleasure to the senses and the
esteemed status that beauty offered to people is commendable since the primordial times [9]
Cosmetology, the science of alteration of appearance has gained popularity with time. In
countries like India, the art of beautifications finds its origin from the traditional fields of
medical science like Siddha and Ayurveda. The secrets of the glorious medicinal herbs were
confidentially written on palm leaves and were carefully handed over for ages from Guru
(teacher) to Shishya (taught) in a closed circuit. Hence the age of herbal cosmetics is much
older than the modern cosmetics. In recent years there is a rediscovery of this traditional
knowledge and the market research shows the growing interest on herbal cosmetics and
natural beauty among consumers world wide [10]. The current study in fact highlighted the
cosmetic potential of poly herbal extracts especially in skin care management. There are
several herbal formulations/ingredients available in the market that confers skin lightening
benefit by inhibiting melanin synthesis at different stages. One of the most obvious cellular
targets for depigmenting agents is the enzyme tyrosinase [11]. Since a huge number of
tyrosinase inhibitors have been developed [12-15], clarifying the validation of these inhibitors
in skin-whitening efficiency has become more relevant and important [16]. The current study
reveals the need to adopt the following approaches for better and effective results on skin
lightening benefit and highlights the possibility of exploring polyherbal extracts as skin
lightening ingredients in cosmetic formulations

a) It is important to screen the time tested herbs of the traditional system of Medicine
like Siddha and Ayurveda through modern research methods and establish their
cosmetic claims through in vitro substantiation studies and thereby rediscover the
secrets of the traditional knowledge in modern cosmetology.
b) It is better to use a combination of poly herbal extracts that act at different stages of
melanin synthesis than using a stand alone herbal extract in a skin lightening
formulation with a single mode of action viz not only in effective tyrosinase
inhibition (during the process of melanin synthesis) but also in preventing the
melanin transfer to the keratinocytes (post melanin synthesis)
c) It is necessary to screen and study the poly herbal extract for a combined synergistic
effect for effective functional benefits in cosmetics.
d) It is ideal to screen the anti-tyrosinase activity/melanin synthesis inhibition activity
of plant products extracted using non- toxic solvents like water and propylene glycol
as used in the present investigation. Such extraction enables easy miscibility of the
poly herbal extract in the same solvents used for extraction during formulation of
cosmetics which makes the cosmetic more skin and eco-friendly.
e) Poly herbal extracts of Aloe vera, Curcuma zedoaria and Decalepis hamiltonii or
Hemidesmus indicus, Curcuma zedoaria and Decalepis hamiltonii were found to be
effective in inhibiting the melanin synthesis and may also have a suggestive role in
preventing the melanin transfer to the keratinocytes thereby could bring about the
desired skin lightening benefit. The reduction in melanin synthesis is evident from
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320 S. Gokulshankar, M. S. Ranjith, Babu et al.

tyrosinase inhibition assay and the melanin measurement done on the treated and
control cells
f) Poly herbal extracts of Aloe vera, Curcuma zedoaria and Decalepis hamiltonii or
Hemidesmus indicus, Curcuma zedoaria and Decalepis hamiltonii can be further
evaluated for skin lightening benefits by formulating these synergistic herbal extracts
in cosmetic formulations and tested for the functional claims through clinical trials.

REFERENCES
[1] Leslie Baumann, Sogol Saghari (2009), Skin Pigmentation and Pigmentation Disorders
Leslie Baumann eds. Cosmetic Dermatology: Principles and Practice, ISBN: 978-0-07-
164128-9 McGraw-Hill.
[2] Hearing, V. J. (1997) The regulation of melanin production. Hori, W. eds. Drug
Discovery Approaches for Developing Cosmeceuticals, Advanced Skin Care and
Cosmetic Products, 3.1.1-3.1.21 IBC Library Series Southborough, Massachusetts.
[3] Fitzpatrick, T. B., Breathnach, A. S. (1963) The epidermal melanin unit system.
Dermatol. Wochenschr., 147:481-489.
[4] Kim, Y.J.; Uyama, H. Tyrosinase inhibitors from natural and synthetic sources:
structure, inhibition mechanism and perspective for the future. Cell. Mol. Life Sci.,
2005, 62, 1707-1723.
[5] Parvez, S.; Kang, M.; Chung, H.S.; Bae, H. Naturally occurring tyrosinase inhibitors:
mechanism and applications in skin health, cosmetics and agriculture industries.
Phytother. Res., 2007, 21, 805-816.
[6] Rendon, M.I.; Gaviria, J.I. Review of skin-lightening agents. Dermatol. Surg., 2005, 31,
886-889.
[7] Krishnamoorthy J.R., Ranjith M.S., Gokulshankar S (2010) Extract combinations of
Curcuma zedoaria and Aloe vera inhibit melanin synthesis and dendrite formation in
murine melanoma cells Journal of Applied Cosmetology:25:95-100.
[8] Hakozaki T, Minwalla L, Zhuang J, et al. (2002). "The effect of niacinamide on
reducing cutaneous pigmentation and suppression of melanosome transfer.” Br. J.
Dermatol., 147 (1): 20–31. doi:10.1046/j.1365-2133.2002.04834.x. PMID 12100180.
[9] Prashant LK, Hemant RJ, Prasad T, et al. (2005) Cosmetics potential of herbal extracts.
Natural Product radiance, 4(4): 315-321.
[10] Ashawat M.S., Madhuri Banchhor, Shailendra Saraf, Swarnlata Saraf, (2009) Herbal
Cosmetics "Trends in Skin Care Formulation" Pharmacognosy Review, 3(5), 82-89.
[11] Nico Smit, Jana Vicanova and Stan Pavel (2009) The Hunt for Natural Skin Whitening
Agents International Journal of Molecular Sciences, 10,5326-5349.
[12] Kuo-Hsien Wang, Rong-Dih Lin , Feng-Lin Hsu et al. (2006) Cosmetic applications of
selected traditional Chinese herbal medicines Journal of Ethnopharmacology.106,353–
359.
[13] Vuthy TY, Natalia Drouart, Mathieu Leti et al. (2011) Screening of anti-tyrosinase
activity of Cambodian plants. Mekong Health Congress, University of Health Sciences
of Cambodia.

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 Fairness in a Natural Way 321

[14] Nithya Narayanaswamy, Arun Duraisamy, Balakrishnan KP (2011) Screening of some

Medicinal Plants for their Antityrosinase and Antioxidant activities International
Journal of PharmTech Research, 3(2) 1107-1112.
[15] Ji-Young Moon, Eun-Young Yim, Gwanpil Song et al. (2010) Screening of elastase and
tyrosinase inhibitory activity from Jeju Island plants. Eur.Asian Journal of Bio.
Sciences.,,4, 41-53.
[16] Te-Sheng Chang (2009) An Updated Review of Tyrosinase Inhibitors International
Journal of Molecular Sciences. Mol. Sci., 10, 2440-2475; doi:10.3390/ijms10062440 .
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 12

THE MELANOCORTIN-1 RECEPTOR: A KEY

MELANOMA RISK DETERMINANT AND A CRITICAL
REGULATOR OF THE UV DNA DAMAGE
REPAIR RESPONSE

Stuart G. Jarrett, PhD, Alexandra Amaro-Ortiz, BS,

Jason Tucker, BS and John D’Orazio, MD, PhD*
University of Kentucky College of Medicine, the Markey Cancer Center, the Graduate
Center for Toxicology and the Departments of Pediatrics and of Molecular and
Biomedical Pharmacology, Lexington, KY

ABSTRACT
Malignant melanoma of the skin represents one of the most aggressive and deadly
malignancies, affecting persons of all ages with increasing frequency. Ultraviolet
radiation (UV) is a major etiological risk factor, but despite public educational campaigns
aimed at limiting UV exposure, melanoma incidence has been steadily increasing for
several decades. The melanocortin-1 receptor (MC1R) has emerged as a major genetic
risk factor of melanoma. It encodes a Gs-protein coupled receptor expressed on the
surface of the melanocytes that relays survival and differentiation signals to melanocytes
through the second messenger cAMP. The MC1R gene is highly polymorphic with
variant alleles linked with red hair, fair skin, poor UV tanning, and elevated melanoma
risk. MC1R activation after UV exposure results in adaptive melanization (tanning) to
provide enhanced protection against cutaneous UV penetration and damage. Defective
MC1R signaling results in inadequate melanization of the skin, which allows UV-induced
DNA changes to accumulate that can lead to mutations and carcinogenesis. However,
MC1R signaling also protects against UV-mediated carcinogenesis independent of
pigmentation, suggesting other roles for MC1R in the UV DNA damage response. This

*
Author to whom correspondence should be addressed: John D'Orazio, M.D., Ph.D., Associate Professor of
Pediatrics, University of Kentucky College of Medicine, Markey Cancer Center, Combs Research Building
204, 800 Rose Street, Lexington, KY 40536-0096, Phone (859) 323-6238, Fax: (859) 257-8940, E-mail:
[email protected].
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324 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

review assesses the consequences of MC1R allelic variants in light of the newly
identified crosstalk between MC1R signaling and melanocytic DNA repair.

DESCRIPTIVE PARAGRAPH
Melanoma remains one of the few malignancies that continue to increase in the
developed world over the last several decades [1]. Due to its propensity to spread from its site
of origin and the high mortality rate once it has metastasized, it has become a cancer with
significant socioeconomic impact. Its presentation is often insidious, heralded only by a
change in a preexisting mole, making early detection difficult [2]. Worst of all, melanoma is
increasingly affecting young adults in the primes of their lives; melanoma of the skin is now
the leading cause of death by cancer of American women in their early twenties. In the United
States, the overall incidence of melanoma has been steadily increasing, with a 1 in 1500
lifetime risk in 1935 increasing to roughly 1 in 50 in 2011 [3]. In 2012, an estimated 76,250
individuals will develop melanoma of the skin, with women under the age of 40 showing an
alarming increase in malignancies, surpassing even breast cancer. The major etiologic risk
factor for melanoma is thought to be exposure to intermittent erythemogenic doses of UV,
especially during childhood and early adolescence years [1]. Interestingly, the extent of
cutaneous protection afforded against UV exposure differs profoundly between individuals
with varying skin pigmentation [4]. A link has been identified between genes that regulate
pigmentation and cellular DNA defense and repair mechanisms [5-6]. Specifically, mutations
in the melanocortin 1 receptor (MC1R) influence complexion, ability to tan and importantly,
the efficiency by which melanocytes can protect themselves as well as recover from
mutagenic UV damage [6-9].

MELANOMA- A GROWING PROBLEM

Skin cancers are by far the most common of all human malignancies with at least three
million diagnosed in the United States each year. Though melanoma accounts for less than
5% of all cases, it is responsible for roughly three quarters of all deaths from skin cancer [10].
Roughly 70,000 Americans will be diagnosed with melanoma in 2012 and approximately
9,000 will die of the disease according to National Institutes of Health estimates. Melanoma
incidence has been on a steady rise over the last several decades (Figure 1) [11], until now it
is estimated that roughly one American in fifty will be diagnosed with melanoma at some
point in his/her life [12-13]. It is mainly a disease of adulthood, although patients of any age
can be affected and its incidence has been increasing even among children, adolescents and
young adults [14-15]. Much can be learned about melanoma by studying its epidemiology.
With the exception of very rare melanoma predisposition syndromes such as inherited p16
deficiency [16-18] or xeroderma pigmentosum [19-20], three main risk factors stand out
above all others in determining melanoma risk in the general population: age, skin
complexion and UV exposure (Table 1). In fact, the global distribution of melanoma cases
can largely be explained by these three factors, with most cases prevalent in UV-rich Western
nations made up of a good proportion of elderly individuals and in which a large portion

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 The Melanocortin-1 Receptor 325

Data are based on NCI Surveillance, Epidemiology and End Results (SEER) reports.

Figure 1. Lifetime Risk of Malignant Melanoma of the Skin: US Incidence since the 1930’s. The
number of cases of cutaneous melanoma has steadily increased over the past several decades, most
likely due to a variety of factors including better surveillance, increased reporting, an aging population
and increased UV exposure.

of the population is descended from fair-skinned Northern European ancestry [21]. Thus,
melanoma burden is predictably largest in Australia, New Zealand, the countries of northern
Europe and the United States, and [22-23]. When one considers the alarming rate at which
melanoma cases seem to be increasing and the fact that a large number of cases might be
prevented by the adoption of reasonable UV-avoiding behaviors, melanoma should be viewed
as an emerging public health concern for Western nations [24].

Table 1. Melanoma Risk Factors

Age  The incidence of melanoma increases dramatically with age. This may
reflect the long latency period typical for environmentally-induced
cancers, declining tumor surveillance with age, or other factors.
UV Radiation  Occupational or recreational UV exposure, living in UV-rich
geographies (e.g., equatorial locations), living at altitude are all risk
factors for melanoma.
 Acute intermittent UV exposure seems particularly relevant for
melanoma risk. One or more blistering sunburns as a child or teenager
increases risk.
 A propensity to burn rather than tan after sun exposure correlates with
increased melanoma risk.
 Tanning bed use: first exposure to indoor tanning before 35 years of age
raises lifetime risk of melanoma by 75%
Fair Skin  Deficiency of the highly UV-protective eumelanin epidermal pigment
Complexion allows more UV to penetrate into the skin and promote mutagenesis.
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326 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

Table 1. (Continued)

Personal or  Once a person has been diagnosed with a melanoma, their risk for others
family history is heightened. Up to 10% of melanoma patients will develop a second
melanoma in their lifetime.
 Inherited CDKN2A defects (the gene that encodes the p16INK4A tumor
suppressor) is associated with familial melanoma.
 Many melanomas appear to arise from pre-existing moles. Benign nevi
and melanoma both frequently exhibit gain-of-function mutation in the
B-Raf gene. Having a large number of moles increases the risk for
melanoma, particularly if they are dysplastic.
Immune  Immunosuppressive conditions (particularly T lymphocyte deficiency) or
suppression immunosuppressive therapies are associated with melanoma. Solid organ
transplant recipients, for example, have much higher rates of melanoma
than the general population.
DNA repair  Xeroderma pigmentosum (XP) patients have a 2,000-fold increased risk
deficiency of skin cancers, including melanoma. XP is caused by homozygous
deficiency of one of at least eight enzymes in a common nucleotide
excision DNA repair (NER) pathway.
Heavy metals  Chronic exposure to chromium, cobalt, arsenic and other metals may
promote oxidative mutagenesis in melanocytes and possibly interfere
with genomic integrity.

Age and Melanoma

As with most other cancers linked to an environmental carcinogen, there is typically a

long latency between exposure (UV radiation) and cancer (melanoma) development [25]. For
this reason, and possibly because of other factors (e.g., declining immune function and tumor
surveillance with aging [26-27]), melanoma is primarily a disease of later adulthood [3, 28-
29] (Figure 2). According to NCI SEER data, the average age at which most melanomas get
diagnosed is 61 years, however rates of increase in incidence in younger adults are among the
highest of any age groups [30]. For women and men between the ages of 20-29, melanoma is
the second and third most commonly diagnosed cancer respectively [31]. One recent study
found startlingly high increases in melanoma incidences (400 and 800 percent for young men
and women respectively) over the past forty years [30]. Many hypothesize that recent
increases in melanoma among young adults may be due to higher tanning bed use and
environmental UV exposure [32-33]. In any case, melanoma now ranks among the
commonest cancers in young adults [10]. Similar to trends in young adults, melanomas are
also being diagnosed more and more in children and adolescents, with some estimating as
much as an 85% increase in melanoma incidence among late adolescents over the last 20-30
years [34-35]. Fortunately, melanomas are highly unusual in prepubertal children except
those with very rare melanoma predisposition syndromes such as xeroderma pigmentosum
(XP). Nonetheless, it is widely accepted that UV exposure in the pediatric years may strongly
influence melanoma risk later in life [36-46].

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Figure 2. US melanoma incidence by age and race. Incidence rates based on the year 2000, NCI SEER
data. Note the marked increase in melanoma incidence with increasing age and the tremendous
discrepancy in melanoma incidence between persons of fair- and dark-skinned complexions.

Ultraviolet Radiation (UV) As a Carcinogen

The International Agency for Research on Cancer, an affiliate of the World Health
Organization classifies UV as a Group 1 carcinogen together with other dangerous and
significant cancer-causing environmental substances such as asbestos, benzene,
formaldehyde, mustard gas, radon and plutonium [47]. UV in the form of ambient sunlight
and artificial tanning salons has been unequivocally linked to melanoma risk in several
scientifically rigorous studies [1, 48-52]. UV causes melanoma and other skin cancers
because of its photochemical characteristics and ability to alter DNA and other biological
macromolecules.
UV photons lie between x-rays and visible light on the electromagnetic energy spectrum
(Figure 3). UV energy can be broken down into three distinct but partially overlapping
species based on the wavelength of its photons. Thus, UVA photons carry the least amount of
energy and are the longest in wavelength (315 - 400 nm). UVB (280 - 315 nm) and UVC
(200 – 280 nm) are the shortest, highest energy photons of UV radiation [53]. Sunlight is
comprised of all three UV species, however because of the UVC-filtering effect of ozone in
the stratosphere, natural ambient sunlight that strikes the surface of the Earth and therefore
most biologically relevant to human disease is roughly 95% UVA and 5% UVB [54-56].
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328 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

Figure 3. Electromagnetic spectrum of UV radiation and biologic effects on the skin. The diagram
shows the subdivision of the solar UV spectrum with the shorter UV wavelengths (i.e., UVC) being
entirely absorbed by stratospheric oxygen, and the majority of UVB (> 90%) being absorbed by ozone.
Most of the UV that reaches the Earth’s surface as sunlight is UVA. UV light penetrates the skin and is
absorbed by different layers in a wavelength- dependent manner. UVA penetrates deeply into the
dermis reaching the dermal stratum papillare whereas UVB is almost completely absorbed by the
epidermis, with less than a quarter reaching the epidermal stratum basale. UVA is able to generate
reactive oxygen species that can damage DNA via indirect photosensitizing reactions. UVB is directly
absorbed by DNA which causes molecular rearrangements forming the specific photoproducts
cyclobutane dimers and (6,4)-photoproducts. Mutations and cancer can result from a variety of UV-
induced modifications to DNA.

UV and Oxidative Damage

UV has both direct and indirect effects on DNA and other macromolecules including
RNA, proteins and lipids [57]. Through the generation of free radicals and oxidative species,
UV exposure results in significant oxidative damage to macromolecules including DNA,
RNA, proteins and lipids in the skin [58-59]. Oxidative species such as the superoxide radical
(O2), hydrogen peroxide (H2O2) and the hydroxyl radical (OH; Figure 4) are all generated
after UV exposure, and each of these highly reactive molecules can interact with lipids,
proteins, RNA and DNA to interfere with their function and promote instability [59]. Not
only can UV-induced oxidative damage directly cause mutations in DNA by modifying
existing nucleotides in the DNA to change their base-pairing specificity (to cause abnormal
transcription and mutagenesis upon DNA replication), but oxidative damage can further
promote genomic instability by inactivating DNA repair proteins through oxidative damage
[60-61].

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Figure 4. Free radical oxidative species generated by UV. UV converts oxygen to several reactive
species which can then interact with macromolecules including lipids, proteins, RNA and DNA. In
general, oxidative changes to macromolecules alters their structure and interferes with their function.

Oxidation-induced mutagenesis is considered indirect DNA damage, since UV must first

create reactive oxidative species which then can interact with the double helix. The
predominant ROS-induced lesions formed are oxidized bases together with DNA single and
double strand breaks. In fact, many different nucleotide changes result from oxidative
damage. Some examples of oxidative changes include thymine glycol, 5-hydroxycytosine, 2-
hydroxyadenine and cytosine glycol. Oxidative alteration of guanine, in particular the
generation of 8-oxo-2'-deoxyguanosine (8-oxo-dG) is among the most numerous and
biologically significant DNA changes caused by oxidative free radicals [62-63]. 8-oxo-dG
promotes G:C→T:A transversion mutations [64-65], which have been documented in several
UV-induced tumors [66-68]. It is thought that UVA is responsible for the bulk of oxidative
damage caused by ambient UV radiation [69-73].

UV and DNAPhotodamage

In addition to indirect damage, UV photons can also interact with the structure of the
double helix and have direct effects on nucleotides within DNA [1, 74-75]. Specifically, the
5-6 double bond of pyrimidines is susceptible to cleavage by higher-energy UV photons,
especially UVB and UVC. If this double bond is disrupted in two adjacent pyrimidines, a
stable covalent ring structure referred to as a cyclobutane pyrimidine dimer can be formed
[76]. Since this occurs primarily in adjacent thymines, this structure is also commonly called
a thymine dimer (Figure 5A). Thymine (cyclobutane) photodimers are the main mutagenic
DNA photoproduct formed by UV, but UV can also form pyrimidine 6-4 pyrimidone lesions
(often termed (6,4)- photoproducts) that also affect base pairing and are mutagenic. (6,4)-
photoproducts result when the 5-6 double bond in a pyrimidine opens and reacts with the
exocyclic moiety of an adjacent 3' pyrimidine to form a covalent 6-4 linkage [77-78]. Both
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330 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

thymine dimers and (6,4)-photoproducts cause distortion of the double helix and abnormal
base-pairing, and if left unrepaired, both cause characteristic transition mutations between
adjacent pyrimidines. These “UV signature mutations” which most often involve T-to-C or C-
to-T changes are a predictable genetic consequence of UV exposure and are commonly found
in UV-induced malignancies [79-82]. Because UVC is not an appreciable component of
ambient sunlight and UVA is much less efficient at forming photodimers [83], it is assumed
that most UV signature mutations result from the UVB spectrum [78]. UV signature
mutations in cancer-relevant genes such as the p53 tumor suppressor gene are found in a large
number of skin cancer isolates, particularly keratinocyte malignancies such as basal cell
carcinoma and squamous cell carcinoma [84-86], clearly establishing a causal link for UV in
these tumors.
With respect to melanoma, an epidemiologic link with UV exposure has long been
appreciated. Two thirds of melanomas arise in UV-rich anatomic areas of the body [87-90],
suggesting a strong link between UV and melanoma. Melanoma risk is particularly linked
with acute intermittent UV exposures. Risk apparently doubles with five or more lifetime
sunburns [91] and a history of pediatric blistering sunburns more than doubles risk melanoma
risk later in life [36, 92]. As with keratinocyte skin malignancies, mutational screening of
melanoma samples confirms the presence of UV-induced mutations on a molecular level [93].
State of the art techniques such as whole-genome and exome sequencing reveal large-scale
UV photoproduct-based mutational signatures in a variety of primary melanoma tumors and
cell lines [87, 94]. Taken together, evidence suggests that UV is causal for a substantial
fraction of human melanomas [95].

Figure 5. Overview of the structure (A) and repair (B) of UV-induced photodimers. UV energy,
particularly shorter wavelengths in the UVB and UVC spectra, interact with DNA in such a way as to
promote the formation of abnormal covalent interactions between adjacent pyrimidines. Cyclobutane
photo-dimers (A) distort the three-dimensional structure of the double helix and can promote mutations
through altered complementary base pairing. Such lesions are corrected mainly by the nucleotide
excision DNA repair pathway (NER). NER is accomplished through the following basic steps: (1)
Lesion recognition by the XPC-hHR23B heterodimer which scans the genome for DNA helix-distorting
lesions. Photolesions in actively transcribed strands can also be identified by stalling of RNA
polymerases and subsequent recruitment of NER enzymes. (2) Lesion unwinding and stabilization by
the TFIIH complex containing XPB and XPD helicases that unravel local DNA, XPA and RPA that
stabilize and orient the unwound strand for excision. (3) Strand excision by ERCC1/XPF and XPG
endonucleases to remove the oligonucleotide containing the photolesion. (4) DNA synthesis by DNA
polymerase to fill in the resulting gap using the undamaged opposing strand as a template and ligation
by DNA ligase to seal the strand.

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UV and Geography

Since UV radiation can be absorbed, reflected back into space or scattered by particles in
the atmosphere, ambient UV doses on the surface of the Earth vary according to the amount
and nature of atmosphere sunlight must pass through. The more atmosphere solar radiation
must traverse, the weaker the corresponding UV content of the sunlight realized on the
surface of the Earth will be in that geographic location. Because of the spherical nature and
varied topography of Earth, the UV content of sunlight differs greatly between geographical
locations.
Equatorial regions receive the highest doses of UV because sunlight hits the Equator
most perpendicularly from the sun, traversing the least amount of atmosphere on its way to
the Earth’s surface. Distance away from the Equator correlates with weaker ambient UV
doses because sunlight strikes Earth increasingly tangentially toward the poles, traveling at
oblique angles through abundant atmosphere (Figure 6). Thus, UV content of sunlight is most
powerful in equatorial locations and weakest in polar extremes [96-100]. Melanoma risk
seems to follow this geographic pattern, highest in UV-rich environments inhabited by people
of fair complexion such as Australia [101-103]. One study examining the low rates of
melanoma in Scandinavia, for example, suggested that ambient UV levels in Norway were
significantly lower than most of the world because of its high latitude [104]. Equatorial
locations are also typically the hottest environments, therefore people living in such places
tend to wear lesser amounts of clothing and expose more skin surface to UV. Although the
reasons for the burgeoning prevalence of melanoma over the last several decades is almost
certainly multifactorial and complex, some hypothesize that increased ambient UV radiation
may be an important factor [105].
Gradual depletion of stratospheric ozone over the last several decades has resulted in
higher levels of solar UV radiation reaching the Earth’s surface [106] and a warming climate
introduces more opportunities for outdoor occupational and recreational activities [56, 106-
110].
Inhabiting temperate climates is associated with a reduced risk for melanoma because the
UV dosage in ambient sunlight is weaker in such locations and because people living in these
cooler climates cover themselves with more clothing during the year [100].

Sun Tanning and Melanoma Risk

For nearly 100 years, having a tan appearance has been associated with youth, fitness and
well-being in Western civilizations. The association between enhanced pigmentation and
attractiveness has, not surprisingly, served as a powerful incentive to seek a suntan,
particularly among adolescents and young adults. However, there currently is no safe way to
induce a natural tan without the mutagenic and carcinogenic risks of UV radiation [111]. The
desire to tan has been matched by a proportionate increase in commercial and recreational
venues for UV exposure. No longer must a person seek the sun to get tan; man-made UV
lamps are as effective as the sun at stimulating tanning of the skin. In fact, UV radiation
emitted by tanning lamps can be even more powerful than sunlight with some estimating that
thirty minutes in a tanning booth being equivalent as 300 minutes of unprotected sun [112].
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332 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

However commercial tanning equipment is largely unregulated and highly variable.

Levels of UVA/UVB emitted by tanning bed lamps are unpredictable, widely unregulated,
and sometimes much higher than environmental exposure. One study of 62 tanning salons in
North Carolina, for example, found that the average UVA and UVB outputs of tanning beds
were 2-4 times as powerful as summer solar output at noon in Washington D.C) [113].

Figure 6. Relationship between latitude and solar UV strength. The intensity of UV radiation varies by
geographic placement on the Earth. Solar UV can be blocked or scattered by ozone and atmospheric
particles. Consequently, the more atmosphere sunlight must travel through on its way to the surface of
the Earth, the weaker its UV component will be. The highest UV doses in sunlight are found at the
equator, where the sun hits the Earth directly.

In the United States alone, the tanning industry is a multi-billion dollar business, made up
of more than 25,000 facilities, more than 150,000 employees and represented by a powerful
lobby intent on maximizing commercial profits by allaying concerns over the dangers of UV
radiation [114]. In fact, the proliferation of indoor tanning facilities may be an important
contributor to the increases in melanoma incidence observed over the last several years [111].
It is estimated that only 1% of Americans ever used a tanning bed in 1988. Now over a
quarter of Americans have used a tanning bed (nearly thirty million people), despite
incontrovertible evidence linking UV exposure and tanning to multiple kinds of skin cancers,
most importantly melanoma [115]. Tanning bed use is clearly associated with skin cancers of
all varieties. People who have ever used a tanning bed have a 50% increased risk of basal cell
carcinoma and more than a 100% increased risk of squamous cell carcinoma [116]. Similarly,
risk association between melanoma development and indoor tanning is well substantiated
[117-119]. Using an indoor tanning salon just once increases a person’s chances of
developing melanoma by 20 percent, and risk rises an additional two percent with each
session during the same year [120].

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Importantly, tanning can clearly be addictive, reinforced not only by the healthy glow and
self-esteem of a robust tan but also by the natural morphine-like endorphins produced
physiologically in the skin during the natural tanning process [121-129]. Overall, users of
tanning beds are 74 percent more likely to develop melanoma than those who have never
practiced indoor tanning [130], and lifetime melanoma risk rises by 87 percent if indoor
tanning is started before the age of 35 years (as most patrons do) [120]. Of the nearly thirty
million users of tanning beds in the US, it is estimated that two to three million are teenagers
[131]. There currently is no “safe” way to tan by UV without the inherent risk of
photodamage and malignancy. Decreasing UV radiation exposure, from both sun exposure
and artificial UV light, may be the single best preventable factor for decreasing the incidence
rate of melanoma [132]. Besides cancer risk, UV is associated with other health problems
including photoaging, wrinkling, skin atrophy and immunosuppression [133].

UV and Vitamin D

The paradox of UV is that in addition to its significant health risks, there are important
health benefits from UV exposure [134-137]. UV directly catalyzes the chemical conversion
of 7-dehydrocholesterol into previtamin D3 in the epidermis, which represents the major non-
dietary source of vitamin D in the body [138-140]. Vitamin D deficiency causes rickets, a
disease in which defective calcium metabolism leads to osteomalacia and osteoporosis that
result in delayed growth, chronic pain, muscle weakness and disfiguring skeletal
abnormalities [141]. In fact, it is likely that risk of rickets was the major evolutionary driver
of fair skin complexion, offering an explanation as to why ancestral populations underwent
lightening of their skin as they migrated away from equatorial regions to more polar locations
with weaker ambient UV [142-143]. Having less epidermal melanin would favor more
penetration of UV into the skin and maximize vitamin D synthesis in such locations [144].

Sunburns

Melanoma risk has been linked to numbers of blistering sunburns, particularly during
childhood [32, 36, 45, 96, 145-149]. Sunburns represent inflammatory reactions to
intermittent, high intensity UV exposure and are mediated by a complex series of pro-
inflammatory mediators such as eicosanoids, prostaglandins, interleukins and nitric oxide
[150-152].
Curiously, long-term cumulative UV exposure seems to correlate more so with
keratinocyte malignancies such as basal or squamous cell carcinoma of the skin [153-156]. In
fact, long-term low-dose UV exposure may even be somewhat protective against melanoma,
perhaps because of enhanced vitamin D levels in chronically sun-exposed skin and
subsequent enhanced efficiency of DNA repair in vitamin D- exposed melanocytes [157-
158]. Because of the relative sun sensitivity of their young skin and their outdoor recreational
habits, children and adolescents are particularly at risk for sunburn, with one study finding
that almost three quarters of adolescents experienced sunburn the previous summer [159].
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334 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

Skin Complexion

Risk of sunburn, like other UV-related health effects, correlates with basal melanin
content of the skin, occurring preferentially in lightly-pigmented people during recreational or
occupational activities with inadequate UV protection [160]. Melanoma risk can similarly be
predicted by skin complexion (Figure 7).
Skin color can be described semi-quantitatively by use of the “Fitzpatrick Scale,” which
divides skin color into six distinct phototypes based on pigmentation (Table 2) [161-162].
Minimal erythematous dose, abbreviated “MED,” is a measure of the skin’s response to UV
using inflammation and redness (i.e., erythema) at 24h after UV exposure as an endpoint.
MED and UV sensitivity correlate fairly well with Fitzpatrick phototype [163-164]. Thus,
more UV radiation is needed to “burn” dark skin than fair-skinned persons [165-167]. Besides
basal pigmentation, melanoma risk correlates with MED with higher Fitzpatrick skin tones
being protected from both acute (i.e., sunburn) and chronic (i.e., melanoma) UV injury [160].
Individuals from any race can be affected by melanoma, but risk is much higher in fair-
skinned persons [168-170]. In the United States, for example, current overall lifetime risk of
melanoma in the United States is roughly 2% (1 in 50) for Caucasians, 0.5% (1 in 200) for
Hispanics and 0.1% (1 in 1,000) for persons of darkest complexion [10].
Skin pigmentation is mostly determined by the amount of melanin pigments present in
the epidermis. Melanins are manufactured by interfollicular melanocytes residing in the deep
layers of the epidermis. These very cells responsible for the production of protective melanin
pigments in the skin are likely the source of melanoma in the skin. Melanocytes make two
major forms of melanin in the skin- eumelanin and pheomelanin (Figure 8). Both eumelanin
and pheomelanin are large biopolymeric pigments derived from the amino acid tyrosine
[171]. Melanogenic biosynthetic reactions are catalyzed by pigment enzymes. Tyrosinase
catalyzes the first two steps in melanogenesis wherein tyrosine is converted into DOPA and
then into DOPAquinone and is thought to be the rate-limiting step for melanin synthesis [172-
173]. The final amount and type of melanin found in the epidermis depends upon both
inherited and environmental factors [174]. Eumelanin is a brown-black chemically inert and
poorly-soluble pigment polymer that is preferentially expressed in persons of darkest
complexion.
In contrast, pheomelanin is a lighter, red-yellow colored compound due to the presence of
a sulfur moiety introduced by incorporation of cysteine into the melanin pigment [175]. Since
it absorbs UV much more efficiently than pheomelanin, eumelanin serves as a better
“sunscreen” and as a result, UV resistance is mainly determined by the absolute amount of
eumelanin in the skin, explaining why dark-skinned individuals are comparatively UV-
resistant [176]. In fact, epidermal pheomelanin levels are fairly similar between light-and
dark-skinned persons.
Fairness of skin, therefore, is mainly a reflection of inadequate eumelanin deposition.
Besides being a poorer blocker of UV energy, pheomelanin may actually contribute to UV-
induced cellular and DNA damage. UV radiation of pheomelanin is associated with
generation of reactive oxidative species [177-178] and pheomelanin photosensitized UV-
induced DNA damage when added to melanocytes in vitro [179]. In a recent study, Fisher and
colleagues found that expression of pheomelanin alone was sufficient to promote melanoma
formation, even without UV radiation, presumably through oxidative DNA damage and
mutagenesis [180].

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The contribution of pheomelanin to UV-induced carcinogenesis is an ongoing area of

investigation, but what is clear is that approaches to enhance eumelanin in the epidermis hold
the promise of reducing UV sensitivity and melanoma risk.

Figure 7. US Melanoma incidence by race. Note that the disease correlates with skin tone and that most
of the increase in melanoma incidence over the last several years is almost exclusively among fair-
skinned persons.
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Table 2. Fitzpatrick Scale of Skin Phototypes

Fitzpatrick Epidermal MED Melanoma

Pigment Phenotype Cutaneous response to UV
Phototype eumelanin (mJ/cm2 )a risk
• Bright white unexposed skin • Always burns
• Blue/green eyes typical • Peels
I +/- 15-30 ++++
• Freckling frequent • Never tans
• Northern European/British
• Unexposed skin is white • Burns easily
• Blue, hazel or brown eyes • Peels
II + 25-40 +++/++++
• Red, blonde or brown hair • Tans minimally
• European/Scandinavian
• Unexposed skin is fair • Burns moderately
• Brown eyes • Average tanning ability
III ++ 30-50 +++
• Dark hair
• Southern, Central European
• Unexposed skin is light brown • Burns minimally
• Dark eyes • Tans easily
IV +++ 40-60 ++
• Dark hair
• Mediterranean, Asian, Latino
• Unexposed skin is brown • Rarely burns
• Dark eyes • Tans easily and
V • Dark hair ++++ substantially 60-90 +
• East Indian, Native American, Latino,
African
• Unexposed skin is black • Almost never burns
• Dark eyes • Tans readily and profusely
VI ++++++ 90-150 +/-
• Dark hair
• African, Aboriginal ancestry
a
Minimal erythematous dose (MED) is defined as the least amount of UVB radiation that will result in reddening and inflammation of the skin 24h after exposure (i.e.,
the lowest UV dose that causes sunburn). High MED’s correlate with large amounts of epidermal eumelanin, darker skin, higher Fitzpatrick phototype and
melanoma resistance.

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Figure 8. Melanin Biosynthesis. Melanin is a large bioaggregate composed of pigmented chemical

species synthesized from the amino acid tyrosine. It is found in two major forms: (1) the brown/black
highly UV-protective “eumelanin” pigment and (2) the red/blonde UV-permeable “pheomelanin.”
Tyrosinase is rate-limiting for melanogenesis and is defective in the most common type of albinism.
Both eumelanin and pheomelanin are derived from the amino acid tyrosine. Incorporation of a cysteine
into pheomelanin results in the retention of a sulfur moiety into the pigment, which may contribute to
UV-mediated oxidative injury. The melanocyte stimulating hormone (MSH) - melanocortin 1 receptor
(MC1R) signaling axis is a major determinant of the type and amount of melanin produced by
melanocytes in the skin, with eumelanin favored in conditions of abundant cytoplasmic cAMP.

Inherited Determinants of Skin Color

Many pigmentation genes were identified through detailed study of coat color mutations
in mice and other model organisms [181]. Most of the genes known to influence skin color
are associated in some way with melanocyte survival, melanin synthesis or melanin
packaging and transfer to keratinocytes (Table 3) [182-185]. Defects in genes responsible for
melanocyte survival or development lead to drastic pigment phenotypes such as piebaldism in
which the embryologic development and/or anatomic placement of melanocytes in the skin is
disrupted in discreet anatomic patterns. In contrast, loss of function of melanogenic enzymes
generally leads to dilutional pigmentary effects rather than complete absence of melanocytes.
The most profound pigmentary defect caused by defective melanogenesis is oculocutaneous
albinism type I (OCA1). OCA1 is caused by homozygous deficiency of tyrosinase [186-187],
therefore melanocytes, though present in normal numbers and distribution in the skin, fail to
make any melanin pigments at all. As a result, individuals with this severe form of albinism
are highly UV-sensitive and must avoid occupational and recreational UV exposure as much
as possible throughout life [188]. SLC45A2, also known as MAPT, encodes a membrane-
associated transporter protein (MATP), which when defective causes a milder form of
albinism known as OCA4 [189-190]. Many pigment genes regulate either the ratio or absolute
levels of eumelanin or pheomelanin expressed in the skin. For example, solute carrier family
24 member 5 (SLC24A5), purported to encode a cation exchange protein in melanosomes,
accounts for up to 40% of skin color differences between races [191]. Skin color is also
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338 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

influenced by loss-of-function polymorphisms in TYR, OCA2, MC1R, ASIP and IRF4 [192-
194], emphasizing that pigmentation is a complex and multigenic phenotype.

Melanocytes

Cutaneous melanocytes are cells derived from the neural crest and are defined by their
ability to produce melanin. Comprising 5-10% of total cells in the epidermis, there are
probably between 1,000 and 2,000 melanocytes in every square millimeter of human skin
[195]. Melanocytes are found both in dermal hair follicles where they impart pigment to hair
and in the interfollicular epidermis where they manufacture pigments that color the skin.
Interestingly, the numbers of epidermal melanocytes are similar across Fitzpatrick
phototypes, therefore skin pigmentation is a reflection of melanin synthesis rather than
melanocyte numbers or location. Via dendritic projections, epidermal melanocytes may be in
intimate contact with as many as 30-50 maturing keratinocytes. The “epidermal melanin unit”
describes the close association between one melanocyte and numerous keratinocytes in the
epidermis [196].

Table 3. Major genetic determinants of human pigmentation

Pigmentation
Gene Proposed Function General Structure
Disorder
Tyrosinase (TYR) Oculocutaneous Rate-limiting enzyme in Type I transmembrane
albinism type 1 melanin biosynthesis protein
(OCA1)
Tyrosinase-related Oculocutaneous Melanin biosynthesis; Type 1 transmembrane
protein-1 (TRP1) albinism type 3 tyrosinase stabilization protein
(OCA3)
Microphthalmia Waardenburg Myc-like master basic-helix-loop-helix-
(MITF) syndrome type 2 transcription factor essential leucine-zipper
for melanocyte transcription factor
differentiation and survival
Dopachrome Unknown Melanin biosynthetic Type 1 transmembrane
tautomerase (TRP2) enzyme protein
Solute carrier family Fair skin Melanosomal cation Membrane transporter
24 member 5 exchange
(SLC24A5)
Stem cell factor/ kit Piebaldism Transmits survival and Membrane tyrosine
ligand (KITLG) differentiation signals to kinase
melanocytes
Pmel17 (gp100; Unknown Striation formation; melanin Type 1 transmembrane
ME20) polymerization protein
P/OCA2 Oculocutaneous Melanosome acidification 12-transmembrane
albinism type 2 domain-containing
(OCA2) protein
OA1 receptor Ocular albinism (OA) Maintenance of melanosome G-protein-coupled
size receptor
Melanocortin 1 Red hair, freckling, Binds to α-MSH and 7 transmembrane Gs-
receptor (MC1R) defective tanning generates cAMP signal coupled receptor

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Numerous studies confirm a complex physiologic relationship between melanocytes and

keratinocytes in an epidermal melanin unit. With respect to pigmentation, as melanocytes
manufacture melanin pigments, they transfer melanin to keratinocytes in little packets known
as melanosomes [197]. Melanosomes originate in the main body of the melanocyte, and move
centripetally away from the nucleus down the dendrites [198]. As they move, melanin
pigments accumulate in the lumen of the melanosomes, manufactured by and processed by
biosynthetic enzymes so that by the time melanosomes are transferred intact to neighboring
keratinocytes, their melanins have matured into eumelanin and/or pheomelanin [199]. With
respect to cancer, epidermal melanocytes are thought to be the precursor cells that, upon
malignant degeneration, develop into melanoma.

Melanocortin 1 Receptor (MC1R)

The MC1R locus has emerged as a critical determinant for pigmentation, tanning
response and melanoma risk[200-201]. Rees and colleagues showed that loss-of-function
polymorphisms of MC1R correlated directly with UV sensitivity and melanoma risk [169,
202]. The MC1R is a seven-transmembrane domain Gs-protein-coupled receptor belonging to
the melanocortin receptor subfamily. Ligand-mediated signaling through MC1R involves G-
protein signaling, adenylyl cyclase activation and resultant increases in levels of intracellular
cAMP as a second messenger. α-melanocyte stimulating hormone (α-MSH) is the high-
affinity physiologic ligand for the MC1R, initiating the enzymatic activity of adenylyl cyclase
and generation of cAMP [203]. In melanocytes, cAMP levels control many aspects of
differentiation, including melanin production. cAMP levels directly correlate with pigment
enzyme levels/activity and eumelanin production. In the setting of low cytoplasmic cAMP,
melanocytes produce pheomelanin through a default pathway of melanin biosynthesis.
However, when cAMP levels rise, either through MC1R engagement by MSH or
pharmacologically in some manner, eumelanin synthesis is potently enhanced [6, 204-210].
The MC1R directly influences pigment phenotype by controlling the ratio of eumelanin
and pheomelanin made by melanocytes [200-201]. This is clearly demonstrated genetically by
the extension (lethal yellow) mouse, in which a premature truncating inactivating mutation in
MC1R leads to almost complete pigment switching from eumelanin to pheomelanin [211-
212]. In humans, fairness of skin, ability to tan after UV exposure and melanoma
susceptibility all correlate with the signaling ability of the MC1R. Loss of function
polymorphisms of the MC1R are commonly found in fair-skinned UV-sensitive, melanoma-
prone individuals [169, 202].
In fact, over 50 non-synonymous polymorphisms have been identified in MC1R, with the
vast majority of allelic variation occurring in European and Asian populations [213]. MC1R
variants have also been shown to act as modifier alleles, increasing the penetrance of other
melanoma-relevant alleles such as CDKN2A (p16) [214-216]. Thus, co-inheritance of MC1R
loss-of-function variants R151C, R160W or D294H with CDKN2A mutations decreased
latency for melanoma by approximately 20 years [217].
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340 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

MC1R and Adaptive Melanization

Since the skin’s ability to respond to UV radiation correlates with melanoma risk, people
with a defective “tanning response” are at heightened risk of melanoma. One model of the
tanning response involves p53-induced POMC and α-MSH production in UV-exposed
keratinocytes and downstream binding of α-MSH with MC1R on melanocytes (Figure 9). α-
MSH, the major physiologic agonist of MC1R, is a product of the proopiomelanocortin
(POMC) precursor protein, which is made by the anterior pituitary as well as in other tissues
[218]. In the skin, POMC and α-MSH are produced upon UV exposure, presumably as part of
a global damage-response pathway [219]. The skin’s adaptive tanning response depends on
MC1R signaling, as MC1R-defective mice were unable to tan in response to UV radiation
[209]. Normally, the binding of α-MSH to MC1R on the surface of melanocytes promotes
increases in cytoplasmic cAMP and enhancement of melanin synthesis, particularly
eumelanin if cAMP signaling is intact. If MC1R signaling is defective, then cAMP signaling
downstream of MSH is blunted and there is no UV-induced enhancement of pigmentation
that would protect the skin against further UV insult [209]. Curiously, however, the POMC-
defective mouse does not exhibit a pheomelanotic phenotype in sharp contrast to the MC1R-
defective extension mouse [220], raising the possibility that some degree of ligand-
independent MC1R signaling may be sufficient for basal pigmentation (in contrast to the
tanning response). In humans, there are three common polymorphisms of the MC1R:
Arg151Cys (R151C), Arg160Trp (R160W) and Asp294His (D294H) [168]. These “red hair
color” (RHC) mutations correlate with red hair, freckling and tendency to burn rather than tan
after UV exposure [221]. Most importantly, these alleles correlate with melanoma risk [202].
People with defective MC1R signaling have up to a four-fold higher risk of melanoma than
their MC1R-intact counterparts [201, 222-225]. Molecularly, these RHC MC1R variants
display a muted ability to activate adenylate cyclase after MSH binding, and thus are
associated with a blunted cAMP signaling response [226].

α-MSH and POMC

An oligopeptide hormone of only thirteen amino acids (Ac-SYSMEHFRWGKPV-NH2),

α-MSH is derived from a much larger protein precursor known as proopiomelanocortin
(POMC) [218, 227-229]. The POMC pro-hormone is composed of 241 amino acids, itself a
cleavage product of the 285 amino acid pre-POMC protein product. Cleavage of POMC into
its constituent peptide subunits (including α-MSH) is accomplished by the enzymatic action
of subtilisin-like enzymes known as prohormone convertases (PCs) that regulate the activity
of target proteins by cleavage and liberation from inhibitory peptide sequences within the
target proteins themselves. In this way, one protein product such as POMC can be cleaved
into several bioactive products. In the case of POMC, major polypeptide products include
adrenocorticotropic hormone (ACTH), β-lipotropin, β-endorphin and α-MSH (Figure 10).
Tissue levels of these protein products therefore are dependent not only on POMC gene
expression but also on PC activity and the efficiency of POMC processing [230]. α-MSH, in
fact, is a cleavage product of ACTH [231].

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Figure 9. The adaptive tanning response. Robustness of adaptive melanization correlates with reduced
melanoma risk, probably because MC1R is relevant to both processes. When UV strikes the skin,
macromolecules in epidermal keratinocytes are damaged, and damage response pathways, including
p53 are activated. There is evidence that p53 mediates transcriptional activation of the pro-
opiomelanocortin (POMC) gene. The POMC gene encodes a propeptide that is cleaved into three
bioactive protein products: β-endorphin, adrenocorticotropic hormone (ACTH) and α-melanocyte
stimulating hormone (α-MSH). MSH is produced and secreted from keratinocytes to bind with high
affinity to melanocortin 1 receptors (MC1Rs) on interdigitating melanocytes in the basal epidermis. α-
MSH binding induces generation of the second messenger cAMP via activation of adenylyl cyclase. In
melanocytes, elevated cAMP levels trigger a number of downstream events including activation of
protein kinase A which in turn activates the cAMP responsive binding element (CREB) and
microphthalmia (MITF) transcription factors. CREB and MITF mediate increased melanin production
by inducing tyrosinase and other melanin biosynthetic enzymes. In this way, MSH-MC1R signaling
leads to enhanced pigment synthesis and transfer of melanin to epidermal keratinocytes to result in
greater UV resistance. MSH-MC1R signaling may also enhance DNA repair and maintenance of
genomic stability in melanocytes to reduce mutagenesis and carcinogenesis.

Two PCs have been specifically linked to POMC processing in mammalian skin: PC1
and PC2 [232-233]. PC1 processes the POMC precursor to yield ACTH, β-LPH, and β-
endorphin, while PC2 further processes ACTH to yield α-MSH [234]. PC1 and PC2 have
been shown to be increased in expression after UV exposure [235-236], which would be
consistent with increased epidermal production of α-MSH after UV exposure. Evidence for
POMC expression in the skin was first appreciated by Thody and coworkers who noted that
concentrations of α-MSH did not significantly decrease in murine skin following removal of
the pituitary gland [237]. Numerous studies have since reported that the epidermal
keratinocytes produce POMC and its peptide products [238-240]. POMC expression increases
in the skin after UV exposure [241], and this has recently been found to be dependent on the
DNA damage response protein and tumor suppressor p53 [219].
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342 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

Figure 10. Proopiomelanocortin (POMC) processing. α-MSH is a 13-amino acid cleavage product of
the proopiomelanocortin precursor polypeptide. The POMC gene product is cleaved at discreet sites
(indicated by the arrows) by two “proconvertase” enzymes, PC1 and PC2 to yield α-MSH and other
bioactive moieties including adrenocorticotropic hormone (ACTH) and β-endorphin (βEND).

MC1R Antagonists

The MC1R is a particularly interesting melanocytic cell surface receptor because its
signaling activity can be modified by a variety of peptide and protein ligands [242-244].
While cAMP signaling is robustly induced upon α-MSH binding, MSH signaling is inhibited
by two other proteins: agouti signaling protein (ASP) and beta-defensin 3 (βD3). The primary
antagonist for the MC1R receptor is ASP [245-246], which is expressed in the skin as well as
several other tissues [247]. ASP is a well-characterized mediator of pheomelanotic
pigmentation across animal species [242, 248-255]. Mechanistically, ASP antagonizes MSH
binding and downstream cAMP signaling at the MC1R locus [256-257], and ASP has
predictable effects on melanocytes, including reduced pigment enzyme levels, reduced
eumelanin synthesis and pheomelanin pigment switching [258]. Mice that over-express ASP,
for example, exhibit yellow fur due to the excessive presence of pheomelanin and low levels
of eumelanin induced by ASP-mediated antagonism to the MC1R [256].
βD3 is a member of the β-defensin family, antimicrobial peptides expressed in the skin
and other locations throughout the body that are important mediators of innate immunity. β-
defensins directly inhibit the growth of certain microorganisms [259] and their production by
keratinocytes in the skin is up-regulated following UV exposure or inflammation [260]. The
contribution of βD3 in melanocyte physiology and pigmentation was noted by the Barsh
group who determined that coat color in dogs was influenced by βD3. Specifically, they
reported that βD3 acted as a neutral antagonist to the MC1R, interfering with α-MSH
signaling through MC1R and encouraging pheomelanin pigment switching [261]. Since that
time, others have confirmed that βD3 interacts with MC1R, blunting the signaling effects of
α-MSH and diminishing cAMP second messenger generation [262-263]. Taken together,
studies suggest that βD3 production in the skin may serve as a negative regulator of MSH-
MC1R signaling and downstream melanocytic UV responses.

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Nucleotide Excision DNA Repair

The rate of mutations caused by UV or any carcinogen is determined by two main

factors: (1) the amount of exposure and (2) physiologic ability to reverse DNA changes
induced by the carcinogen that might cause mutations if left unrepaired. Nucleotide excision
repair (NER) is the evolutionarily-conserved DNA repair pathway responsible for ridding
UV-exposed DNA of photodimers and (6,4)-photoproducts [264-265]. Defective NER is a
clear risk factor for the development of melanoma and other UV-induced malignancies [266].
There are other excellent reviews that detail the intricate molecular mechanisms of NER
[267-270], therefore we will outline the basic overview here. The NER pathway involves the
following basic steps: (1) recognition of damage and recruitment of a multiprotein repair
complex to the damaged site, (2) nicking the damaged strand several nucleotides away on
each side of the damaged base(s) and excision of the damaged region between the two nicks,
3) filling in the resultant gap by a DNA polymerase using the non-damaged strand as a
template and (4) ligating the nick to seal the strand (Figure 5B). The importance of the NER
pathway to genomic stability of melanocytes is best appreciated by considering the natural
history of patients with Xeroderma pigmentosum (XP) who have defective NER caused by
homozygous deficiency of any one of eight or more genes central to NER function [266]. XP
patients exhibit profound UV sensitivity beginning in early childhood and despite a
significant amount of UV avoidance necessitated by their UV sensitivity, patients develop
significant UV-mediated pathologies in childhood, including epidermal thinning,
telangiectasias, lentigenes and pigmentary changes adolescence[271]. Importantly, XP
patients have a markedly elevated risk of melanoma and other skin malignancies, and these
cancers often begin appearing in childhood [20, 272]. Moreover, the contribution of UV to
these malignancies is incontrovertible, with clear evidence of UV-induced mutagenesis at the
DNA level [273]. Fortunately XP is rare, on the order of 1 in 106, however the natural history
of the disease highlights the central relevance of the NER pathway in melanoma resistance.
The contribution of polymorphisms in NER enzyme levels and activity in sporadic melanoma
is an area of active investigation [274-276].

MC1R and NER

Besides influencing pigment synthesis, MC1R also determines the ability of melanocytes
to recover from UV-mediated cellular injury. The last decade has seen compelling evidence
of MCIR-cAMP-mediated signaling in promoting the early repair response to UV-induced
DNA damage. Such a DNA repair function for MC1R was initially suggested by the
observation that RHC variants had a reduced ability to remove UV-induced CPD lesions
independent of the cellular melanin content [277]. Numerous studies have been published
describing an impaired UV-induced DNA damage response in the setting of defective MC1R
function [278-279]. Furthermore, MC1R agonists and cAMP stimulants enhance melanocytic
NER [5, 277, 279-280]. Determining the molecular mechanisms linking MC1R signaling and
NER function remains a critically unanswered question. Many downstream effects of MC1R-
cAMP stimulation in melanocytes are driven by microphthalmia (MITF), a cAMP-responsive
transcription factor that regulates a number of pigmentary, survival and differentiation genes
including genes required for UV-induced DNA damage check points and repair [281].
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344 Stuart G. Jarrett, Alexandra Amaro-Ortiz, Jason Tucker et al.

A recent RNA-seq study showed that siMITF silencing resulted in down-regulation of

numerous genes involved in the UV DNA damage repair response, implicating molecular
links between MC1R, MITF and a variety of NER genes [281]. It is likely that MSH-MC1R
influences on NER are mediated at least in part through MITF.

MC1R and Double Strand Break Repair

Emerging evidence suggests that other DNA repair pathways may also be impacted by
MC1R signaling [7, 258, 279]. Double-strand breaks, formed when both strands of the DNA
duplex are simultaneously broken in the same area, are highly genotoxic lesions that can
result in significant genomic instability. Double-strand breaks are repaired by the double
strand break repair pathway [282-284], of which two participant proteins BRCA1 and Ddit3
were found to be down-regulated by the MC1R antagonist ASP, suggesting a link between
MSH-MC1R signaling and DSB repair [258]. Noteworthy, ASP also down-regulated NR4A3,
a member of the NR4As superfamily of nuclear receptors known to participate in UV DNA
damage repair and DSB repair [285]. Taken together, it seems as though MC1R and cAMP
signaling may be important to DSB repair.

MC1R and Defense against Oxidative Damage

MSH-MC1R signaling and cAMP generation may influence oxidative burden and the
robustness of antioxidant defenses in many ways. One of the best studied molecular links
involves nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that serves as
a global positive regulator of antioxidant genes and phase 2 detoxifying enzymes [286].
cAMP stimulation either by α-MSH or by the adenylyl cyclase activator forskolin prevented
UVB-induced down-regulation of Nrf2 and Nrf2-dependent antioxidant gene expression in
melanocytes [287]. Similarly, cAMP signaling mediated up-regulation of catalase, the
enzyme responsible for detoxifying hydrogen peroxide (H2O2) [288-289].
MC1R signaling may also impact the ability of melanocytes to reverse oxidative DNA
damage and mutagenesis by enhancing base excision repair (BER), a highly conserved
genomic maintenance pathway primarily responsible for the removal of bases altered by free
radicals within the genome [290-291]. There are five basic steps to BER: (1) recognition of
altered bases by glycosylase enzymes that cleave abnormal nucleotides away from the
phosphodiesterase backbone to form an abasic site in the DNA, (2) removal of the abasic site
by an AP-endonuclease, (3) excision of the dexyribose phosphate residue by a
phosphodiesterase, (4) insertion of the correct base by DNA polymerase using the non-
damaged complementary strand as a template, and (5) sealing of the strand by DNA ligase
[292]. Much as is the case with NER, variations in the resistance or repair of UV-induced
oxidative lesions may be relevant in determining mutagenesis in the skin and subsequently
risk of melanoma [8, 289, 293-294].
MITF has recently been shown to regulate a set of genes required for BER, specifically
apurinic apyrimidinic endonuclease (APE1), DNA ligase I and 8-oxoguanine DNA
glycosylase [8, 281, 295]. Recently the p53 tumor suppressor was implicated as a central
mediator of MC1R effects on BER and oxidative damage [7]. Taken together, there is

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mounting evidence that MSH-MC1R signaling results in enhanced anti-oxidant defenses in

melanocytes.

Pharmacologic Manipulation of MC1R Signaling

Pharmacologic manipulation of melanocytic cAMP levels represents a promising and

novel approach to alter UV sensitivity and melanoma risk. Pharmacologic MC1R mimetics
include both small peptides that mimic MSH agonist activity [296] as well as agents that
bypass the MC1R to directly manipulate melanocyte cAMP levels [209]. We reported that
topical application of the adenylate cyclase activating drug forskolin restored melanocytic
pigmentation in an animal model of the fair-skinned human and that this “sunless tanning”
was potently protective against UV damage and carcinogenesis of the skin [209]. More
recently, Khaled and coworkers showed that a similar UV-protected phenotype could be
induced not by induction of cAMP generation, but rather by pharmacologic interference with
clearance of cAMP by topical application of a phosphodiesterase inhibitor [210]. Small
molecule-based approaches of cAMP manipulation offer a critical advantage over MSH
peptide mimetics in that fair-skinned, UV-sensitive persons most at risk of melanoma are
frequently defective in MC1R signaling ability, and thus would not be expected to generate a
brisk cAMP response upon MSH peptide binding. Of course, such agents are broad-acting
and would therefore have effects in cells other than melanocytes, thus the more melanocyte-
targeted approach of the MSH mimetics may offer selective advantages. In any case, rational
development of pharmacologic agents capable of safely manipulating cAMP levels in
epidermal melanocytes might offer UV- and melanoma protection through a variety of ways.
First, by up-regulating melanin in the skin, fair-skinned individuals would be better protected
from UV. Second, sunless tanning by small molecules would represent a way to uncouple
tanning from the mutagenic effects of UV exposure. Fair-skinned persons seeking tans would
no longer need to sunbathe or frequent tanning salons to enjoy the cosmetic and UV-
protective benefits of improved skin pigmentation. Lastly, stimulation of the MC1R pathway
would enhance the ability of melanocytes to repair indirect and direct UV-induced DNA
damage and would therefore be expected to result in fewer mutations and ultimately less
melanoma in UV-exposed skin. We and others are working to develop safe and effective
strategies to reduce melanoma incidence through rational drug design strategies.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 13

MC1R, EDNRB AND KIT SIGNALING

IN PIGMENTATION REGULATION
AND RELATED DISORDERS

Javier Pino and Lidia Kos

Department of Biological Sciences, Florida International University, Miami, FL, US

ABSTRACT
Skin and hair pigmentation results from the presence and distribution of melanin in
keratinocytes. Melanin is produced by melanocytes in well-defined chemical reactions
where tyrosinase is the rate-limiting enzyme, and then transferred to keratinocytes in
small vesicles called melanosomes. Melanocytes are derived from the neural crest, a
transient embryonic population of cells that emanate from the forming central nervous
system. During development the proliferation, survival, migration and differentiation of
melanocyte precursors are regulated by a series of molecules secreted in the local
environment that trigger the activation of intracellular signaling cascades. Once
melanocytes reach their final destination in the skin and hair follicles, a combination of
these and other signaling molecules produced by the neighboring keratinocytes,
fibroblasts and vascular endothelial cells regulate their physiological functions including
melanin production. Genetic variants or mutations in the genes that code for the various
components of these signaling pathways lead to pigmentary disorders and increased risk
for melanoma. Here we will review three of the major signaling pathways involved in the
establishment of mammalian skin and hair coloration via the regulation of tyrosinase
activity. The Melanocortin 1 Receptor pathway acts in differentiated melanocytes
regulating the type of melanin produced. Particular variants in this gene are responsible
for fair skin and red hair traits, which have been associated with high risk for skin cancer.
The Endothelin Receptor B and KIT signaling pathways play essential roles during
melanocyte development and when mutated lead to the hypopigmentation phenotypes in
Waardenburg-Shah syndrome and piebaldism, respectively. We will discuss how these
three pathways may interact at the cellular level to produce the final pigmentation
patterns observed in mice and humans.


Corresponding author: Lidia Kos, [email protected].
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366 Javier Pino and Lidia Kos

INTRODUCTION
Throughout the three domains of life, organisms display a variety of colorations and
patterns because of the pigments they produce. Pigments are found in single celled organisms,
plants, and animals. In some prokaryotes and plants pigments are used in the production of
nutrients using photosynthesis [1, 2]. In animals, pigments play a large role in camouflage,
and most importantly, confer overall fitness and survival. Brighter pigmented male house
finches were shown to have a greater reproductive success than males with less vibrant colors
[3]. Similar observations were made with male mandrills that displayed higher dominance
and hierarchical success in harems [4]. Pigmentation can also be used as warning signals or
camouflage to avoid predation and increase survivorship as shown in poison frogs and reef-
dwelling fish, respectively [5, 6].
Pigment acts as a barrier to provide defense against ultraviolet (UV) radiation that may be
harmful to melanocytes, keratinocytes and other cells found in the mammalian skin [7-9].
Although UV exposure is essential for providing vitamin D for bone health, increased
immunity, and the prevention of cancers and heart disease, there are also various adverse
effects associated with it [10]. Recently studies have shown that UV radiation negatively
affects the ability of the skin to act as a mechanical barrier by changing its cell cohesion
properties and the mechanical integrity of the skin cells [11]. UV exposure increases
oxidative stress and mutations in skin cells that can ultimately lead to cell death and the
formation of various skin cancers [12]. An increase in the activity of signaling molecules
involved in the normal production of pigment follows UV radiation resulting in a rise in
pigment levels found in the skin, also known as tanning [13, 14]. These molecules are mostly
secreted by the keratinocytes and include alpha-Melanocyte Stimulating Hormone (-MSH),
Endothelins (EDNs), KIT ligand (KITL), Hepatocyte Growth Factor, LIF, Granulocyte-
Macrophage Colony-Stimulating Hormone, and basic Fibroblast Growth Factor. Other
neighboring cells such as fibroblasts, endothelial cells, inflammatory cells and neurons also
secrete factors that contribute to the overall levels of pigmentation [15, 16].
The amount of cell damage caused by UV radiation is inversely related to the amount of
pigment found in the skin [17]. Higher levels of pigment give greater protection from DNA
damage by preventing the formation of pyrimidine dimers and other photoproducts that may
cause mutations [18]. People with fairer skin tend to have higher susceptibility to UV-induced
cell damage than those with darker skin. Pigment in the upper epidermis prevents underlying
cells within the epidermis from UV damage and undergo more apoptosis than cells with less
pigment [19]. This allows for a higher turnover of cells preventing those with mutations from
turning carcinogenic. Genetic studies suggest that evolutionarily, humans who lived closer to
the equatorial line had darker skin pigmentation in comparison to those in higher latitudes in
order to protect the skin from the higher levels of UV exposure [20, 21].

Pigment Production

In mammals, melanocytes can be divided into two major groups: the cutaneous
melanocytes that are found in the skin and hair, and non-cutaneous melanocytes associated
with other parts of the body such as the inner ear, eye, and valves of the heart [22-24].

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 MC1R. EDNRB and Kit Signaling in Pigmentation Regulation ... 367

Irrespective of their location, the major differentiation characteristic of these cells is the
production of the pigment melanin. Melanin is produced and stored in melanocyte-specific
organelles, known as melanosomes. Protein analysis confirmed that melanosomes contain
proteins specific to other organelles such as endoplasmic reticulum, lysosomes, and
endosomes [25]. Melanosomes undergo a four-stage maturation process with gradual
accumulation of pigment [26]. The latest stage melanosomes are transported to the cell
membrane via cytoskeletal filaments and are finally transferred to keratinocytes. Although the
transfer is essential for the final distribution of pigment in the skin the exact mechanism and
the factors involved with this process are still not fully understood [27]. Some hypotheses
have been put forth such as the release of melanosomes into the extracellular space and their
uptake through phagocytosis by the keratinocytes and filopodia mediated melanosome
transfer [28, 29]. Recent studies suggest that pigment globules made up of densely packed
melanosomes bud out of various parts of the dendrites and are phagocytized by the
keratinocytes [30].
Melanocytes produce two types of pigment: the black/brown eumelanin and the
yellow/red pheomelanin [31]. The production of melanin involves the oxidation of tyrosine to
DOPA and DOPA into DOPAquinone in the presence of the enzyme Tyrosinase (TYR).
Eumelanin synthesis involves the activity of the Microphtalmia Transcription Factor (MITF),
the tyrosinase related enzymes Tyrosinase Related Protein 1 (TYRP1),
Dopachrometautomerase (DCT) and the melanosome-associated protein PMEL17. The
process of pheomelanin production lacks these melanocytic genes. Melanocytes that produce
higher amounts of pheomelanin present less dendricity, have lower survival rates and exhibit
lower photoprotective properties. The regulation of all the melanogenic genes and resulting
pigment production is dependent on the signaling pathway activated downstream of the
Melanocortin 1 Receptor (MC1R), a transmembrane G-coupled receptor found on the cell
surface of melanocytes [32].

Pigment Cell Development

The melanocytes are derived from a multipotent population of cells, the neural crest
(NC), that arise at the dorsal aspect of the developing neural tube and the border with the
prospective epidermis. In the mouse, NC cells migrate from the neural tube between
embryonic day (E) 9-E9.5 [33]. In the trunk of the mammalian embryo, the first group of cells
that leave the neural tube take a ventral pathway and give rise to neurons and glial cells of the
peripheral nervous system, and some endocrine cells. They are followed by the melanocyte
precursors, the melanoblasts, which take a dorsolateral pathway [34]. In mice, these cells start
expressing melanocyte markers as early as E9.5, as observed by the expression of Mitf [35].
Melanoblasts enter the ectoderm at E16.5 to populate the epidermis of the skin and the hair
follicles as differentiated melanocytes and start producing melanin [36]. A recent study
suggests the existence of a second cell lineage for the population of melanocytes that migrate
to the skin [37]. Instead of taking the dorsolateral pathway, these cells follow the ventral
pathway and are found along the developing nerves. Depending on the presence or absence of
neuregulin signaling they can either become Schwann cells or melanocytes.
During development, various signaling pathways are involved in the specification and
differentiation of melanocytes including those triggered by the ligands WNT, Endothelin 3
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368 Javier Pino and Lidia Kos

(EDN3), and KITL [38-42]. All the pathways contribute to the transcriptional activation of
MITF, which in turn regulates the expression of most of the melanogenic genes including
TYR, TYRP1, DCT and PMEL17. These same pathways continue to play a role in the adult
organism regulating pigment production and maintenance of the pigmentation patterns.
Although the cascade of intracellular events that occur downstream of the activation of
the major signaling pathways that control pigmentation have been well characterized
biochemically and molecularly, how these pathways interact in vivo to produce the ultimate
pigmentation phenotypes has not been fully established. In this review, we will focus on three
of the major pathways that contribute to establish normal pigmentation in mice and humans
and have been shown to interact genetically. We will also describe how alterations in these
pathways lead to pathological conditions that include pigmentation manifestations. MC1R
and its ligand α-MSH are at the center of the establishment of pigmentation patterns and are
critical in the switch between eumelanin and pheomelanin production. Deactivation of this
pathway, through mutation in the receptor and the presence of Agouti Signaling Protein
(ASIP in humans and ASP in mice), leads to variations in pigmentation. The Endothelin
Receptor B (EDNRB) along with its ligand EDN3 cooperate with the tyrosine kinase receptor
KIT and its ligand KITL to properly specify melanocytes, expand the population of
precursors, and coordinate their migration to the skin. Deactivation of the EDNRB pathway is
responsible for humanWaardenburg-Shah syndrome and the piebald phenotype in mice [43]
while human piebaldism results from mutations in KIT [44].

THE MELANOCORTIN 1 RECEPTOR PATHWAY

Melanocortin receptors are involved in various processes in mammalian body systems
including inflammatory response, steroid secretion, nervous system function, exocrine
function and the production of pigments [45]. MC1R plays an essential role in pigment
production on the cell membrane of skin and hair follicle melanocytes [32].
The agonist ligand for MC1R is α-MSH, one of the many melanocortins derived from the
proopiomelanocortin (POMC) precursor [46]. POMC RNA is found in both melanocytes and
keratinocytes at low levels and cultured keratinocytes are able to produce α-MSH in vitro [47,
48]. α-MSH has a high binding affinity to MC1R and, when bound, leads to the synthesis of
eumelanin [49]. The binding of ligand to receptor activates cyclic adenosine monophosphate
(cAMP) which binds to the regulator subunit of protein kinase A (PKA) allowing for the
initiation of catalytic activity [50]. PKA then enters the nucleus activating the cAMP response
element binding protein (CREB) leading to the production of MITF (Figure 1). MITF is
considered a master regulator in melanocytes and is essential for the production of TYR and
most other genes required for pigment production [51-54].
Melanocortin receptors have two antagonist ligands, namely ASP and Agouti-related
protein (AgRP). AgRP is not involved in pigment production and only binds to MC3R and
MC4R [55]. Binding of ASIP to MC1R in melanocytes causes a shift in melanin production
from eumelanin to pheomelanin [56]. ASIP competitively inhibits α-MSH from binding to
MC1R by using the cysteine-rich carboxyl terminus to bind to the receptor, deactivating the
signaling pathway [57, 58]. Binding of ASIP results in a decrease in cAMP and TYR activity,
which are required at lower levels in the production of pheomelanin [59].

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 MC1R. EDNRB and Kit Signaling in Pigmentation Regulation ... 369

In vitro studies in which ASIP was applied to melanocytes did not result in cultures
solely producing pheomelanin suggesting that other molecules that are present in vivo may
also be involved in pigment type switching [56, 60]. This is further corroborated by studies
carried out with mouse mutants. Attractin, a type 1 transmembrane protein encoded by
Mahogany, has low affinity for ASP binding but is essential for ASP to bind to Mc1r [61, 62].
Mice lacking attractin protein are not able to produce pheomelanin and only produce
eumelanin even in the presence of ASP [63, 64]. Another mutant that produces eumelanin in
the presence of ASP has been identified in the Mahoganoid gene [63]. Mahoganoid encodes
for an intracellular E3 ubiquitin ligase known as Mahogunin ring finger-1, but its exact
function is not yet known. Another ligand, β-defensin, was recently discovered and
characterized in dogs and transgenic mice [65]. β-defensin was shown to have high affinity to
Mc1r producing a dark pigmentation phenotype and the production of eumelanin in dogs.

Figure 1. MC1R, EDNRB and KIT signaling pathways in melanocytes. Molecular interactions among
the pathways that converge on activation of the MITF gene and protein that leads to the regulation of
melanogenic genes and pigment production.

MC1R in Pigmentation

Tyrosinase activity is the rate-limiting component in pigment production and its

activation is mostly dependent on the MC1R signaling pathway. Tyrosinase oxidizes tyrosine
into DOPA and later into the melanin precursor, DOPAquinone [66-69]. Once DOPAquinone
is oxidized, the production of pigment is specified: if cysteine is present pheomelanin is
synthesized and in its absence DOPAquinone is transformed into leucodopachome and later
dopachrome. During the oxidation cascade following dopachrome in the production of
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370 Javier Pino and Lidia Kos

eumelanin, the melanogenic genes TYRP1, DCT and PMEL17 are activated [70]. Tyrp1 and
Dct are found in the melanosome and are mapped to the mouse brown and slaty loci,
respectively [71, 72]. Mutations in the brown and slaty loci affect eumelanin production and
cause changes in the pigmentation phenotype resulting in lighter coat colors [72-74]. TYRP1
is necessary for the production of black over brown pigment [75]. DCT oxidizes
DOPAchrome into 5,6-dihydroxyindole-2-carboxylic acid (DHICA), preventing the
conversion of DOPAchrome into 5,6-dihydoxindole (DHI). TYRP1 then oxidizes DHICA
[76]. PMEL17 is found in the matrix of the melanosome and is encoded by the Silver locus
[77]. Mutations in Silver lead to a progressive greying of coat color in mice due to
melanocyte loss of function [78].

Disruption of the MC1R Signaling Pathway in Mice and Humans

A functional MC1R signaling pathway results in the production of eumelanin, while an

interrupted pathway leads to the production of pheomelanin. This change in pigment
production is the result of the agouti (a) and extension (e) loci. Point mutations in either locus
cause variations of yellow coat color in mice [79, 80]. Lethal yellow mice (Ay) carry a
mutation at the agouti locus resulting in the over production of ASP. The mutation consists of
a deletion that removes the entire Raly gene except for its promoter and noncoding first exon.
Raly maps close to the 3’ end of the agouti gene and lies in the same transcriptional
orientation [81, 82]. In Ay mice, the coding region of the agouti gene ends up being under the
control of the ubiquitous Raly promoter. Heterozygous Ay mice (Figure 2A) display a longer
body size, resistance to insulin, obesity and a yellow coat color as a consequence of ASP
overexpression [83]. Homozygous Ay mice are embryonic lethal because of the absence of
Raly product [81, 84].
Mutations in the e locus result in lack of function of Mc1R due to an early termination of
the fourth transmembrane domain that prevents the coupling of the G-proteins to the receptor
[80, 85]. Recessive yellow mice have mutations in the e locus and display a yellow coat color.
Application of α-MSH to hair follicle melanocytes of e/e mice does not rescue eumelanin
production, while it does for melanocytes from Ay heterozygous mutants confirming that e/e
mice have mutations in the Mc1R receptor, while Ay mutants do not. Upon exposure to
cAMP, both types of mutant melanocytes respond by producing eumelanin indicating that
these mutations are at the level of the receptor and/or ligand and not in downstream factors
[86]. a locus mutants have lighter hair color than those with mutations in the e locus [87].
This shows that ASP acts more than as a simple antagonist to Mc1r signaling. It can also be
considered as an agonist by causing the extreme opposite effect of the binding of α-MSH.
Mechanisms regulating pigmentation in humans work very similarly to those found in
mice. Melanocytes found in the hair follicles and epidermis of humans also synthesize both
types of pigments and give the visible pigmentation variations [88].
The gene that encodes for MC1R in humans is highly polymorphic with various
mutations in comparison to that found in the mouse [89, 90]. Many of these mutations result
in a loss of function receptor that can no longer bind α-MSH. These mutations result in the
loss of eumelanin production and the production of red hair [91, 92]. Humans with
homozygous and heterozygous alleles for MC1R display paler skin and have increased risk of
acquiring skin cancers [93, 94]. The null phenotype for MC1R has been found to be red hair

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 MC1R. EDNRB and Kit Signaling in Pigmentation Regulation ... 371

[95]. Specific alleles of ASIP have been associated with pigmentation characteristics such as
dark hair [96], basal cell carcinoma risk [97] and those generally affected by variants in
MC1R including skin sensitivity to sun, freckling and red hair [98].

THE ENDOTHELIN RECEPTOR B PATHWAY

EDN signaling is triggered by the binding of any of the three ligands, EDN1, EDN2 or
EDN3 to the two major receptors, EDNRA or EDNRB. The process of ligand production
starts with the cleavage of prepolypeptide precursors by prohormone processing hormones to
produce big EDNs. Big EDNs display low activity and are cleaved into smaller, active EDNs
by Endothelin Converting Enzyme-1 [99-101]. Final EDN products are made up of 21 amino
acid residues that result from the proteolytic cleavages between Trp-21 and Val/Ile-22. EDN1
was the first described and the best functionally characterized. It was originally shown to be a
potent vasoconstrictor and to be involved in hypertension, congestive heart failure, and
ovarian cancer [102, 103]. EDNRA and EDNRB are seven-transmembrane G-coupled protein
receptors. Both receptors display different binding affinities to the EDNs. EDNRA has higher
affinity to EDN1 than EDN2 and EDN3, with EDN3 being the lowest presenting about 100
times lower affinity than EDN1 [104, 105]. EDNRB shows the same affinity for all three
EDNs, although EDN3 is the one most readily available to the receptor [106].
In melanocytes, upon binding of EDN1 or EDN3 to EDNRB, two possible signaling
transduction pathways may be elicited (Figure 1). Activation of cAMP may occur following
the activation of PKA, as seen in MC1R signaling, leading to the activation of CREB and the
transcription of MITF [107]. The second pathway involves the activation of Protein Kinase C
(PKC), which acts along with RAF activating the Mitogen Activated Protein Kinase (MAPK)
signaling pathway [107-109]. MAPK phosphorylates MITF, and subsequent activation of a
variety of melanocytic and melanogenic genes, including EDNRB itself [107].

EDNRB in Melanocyte Development and Pigmentation

EDNRB signaling plays various roles in the development of melanocytes from NC cells.
It is not responsible for the initial commitment of NC cells to the melanocytic fate but
participates in most cellular processes after the initial commitment step such as survival,
migration, proliferation and final differentiation [43]. EDNRB is expressed in the neural tube
and in most NC derivatives as they migrate to their final destinations, including the
melanoblasts. In vitro, EDN3 markedly increases the proliferation of pluripotent NC cells,
stimulates the production of large numbers of melanocyte precursors and eventually leads to
their differentiation as pigmented cells [110, 111]. In spontaneous Ednrb homozygous null
mouse mutants (piebald lethal, Ednrbsl/sl) as well as in mice in which the LacZ gene was
inserted downstream of the endogenous Ednrb promoter by homologous recombination there
is a drastic reduction in the number of melanocyte precursors by E12.5 [112, 113]. A study in
which Ednrb was expressed at different stages of embryogenesis under the control of the
tetracycline inducible system showed that its expression is critical between E10.5 and E12.5
for the generation of a normal coat color [114]. The over-expression of Edn3 during this same
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372 Javier Pino and Lidia Kos

embryonic period is also required for the skin hyperpigmentation phenotype obtained in an
inducible transgenic mouse [115].
While activation of EDNRB signaling by EDN3 is critical during melanocyte
development, adult skin or UV-induced melanogenesis seems to be mostly maintained or
induced by the release of EDN1 from keratinocytes [108, 109, 116]. This is further supported
by the findings that EDN1 is downregulated in the hypopigmented skin of the palms and soles
and upregulated in the hyperpigmented skin of lentigo senilis [117, 118].

Disruption of the EDNRB Pathway in Mice and Humans

Mice with mutations in the Edn3/EdnrB signaling pathway have hypopigmentation

defects. Ednrbsl mutants do not produce Ednrb and, as homozygous, display a white coat
color with pigmented spots on the head or rear [119] (Figure 2B). Additionally, these mice
lack the enteric ganglia of the distal colon and develop megacolon causing premature death.
The spontaneous lethal spotting (Edn3ls) mice, do not produce Edn3 and present with partial
loss of pigmentation, possibly because of compensation from Edn1 [120]. Humans carrying
mutations in EDNRB or EDN3 present with Waardenburg Syndrome type IV also known as
Waardenburg-Shah syndrome [133, 134, 135]. Patients show patchy hypopigmented areas
generally in the hair, forehead and chest, dystopia canthorum, and light eyes [121]. Patients
may also have hearing defects, due the lack of melanocytes in the stria vascularis of the inner
ear. Most patients also present with Hirschsprung disease as a result of the lack of proper
innervation of the distal portion of the colon [122]. Both heterozygous and homozygous
mutants of either EDNRB or EDN3 alleles have been shown to display Waardenburg-Shah
syndrome [123, 124]. Homozygous mutants have more distinct phenotypes while
heterozygous still display some signs of disease, resulting in “not fully recessive-not fully
dominant” mutations as described by Jabeen et al. [121]. Missense mutations of EDNRB have
also been described in patients with Waardenburg-Shah syndrome, mutations are seen
throughout various encoding regions for different components of the receptor [125, 126].

THE KIT RECEPTOR PATHWAY

KIT signaling is involved in the differentiation, proliferation and survival of a wide
variety of cell types during development. These cell types include mast cells, germ cells,
interstitial cells and melanocytes [41, 127-129]. KIT signaling has also been shown to be
associated with the formation of various cancers. KIT is a type III receptor in the protein-
tyrosine kinase receptor family [130]. It has three components: the extracellular domain made
up of five immunoglobulin-like (Ig-like) motifs, the transmembrane portion, and the
intracellular domain that contains an ATP and a phosphotransferase region [130, 131]. KIT
signaling is initiated by the interaction of KITL, also known as Stem Cell Factor, Mast Cell
Growth Factor, and Steel Factor, with the first Ig-like motif on the extracellular domain of the
receptor [132-135]. KITL is biologically active during development in two isoforms,
membrane-anchored and soluble [136, 137]. The cleavage of the mature, membrane-anchored
isoform of the post-translated sequence at Ala-164 results in the soluble isoform of KITL

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 MC1R. EDNRB and Kit Signaling in Pigmentation Regulation ... 373

[138]. Soluble KITL activates KIT quicker but leads to a faster degradation of the receptor in
comparison to the membrane-bound isoform [139]. Since the membrane-bound isoform
maintains signal transduction for a longer period of time, the production of downstream
signaling molecules, such as MAPK, occurs more commonly [140, 141].
Binding of KITL to the receptor causes dimerization and activation of the receptor’s
tyrosine kinase activity. Autophosphorylation of the receptor allows for signaling proteins
containing a Src homology 2 (SH2) domain to bind to the tyrosine residues located on the
intracellular domain of the receptor [142]. These proteins lead to the recruitment of RAS,
RAF and subsequent activation of the MAPK pathway ultimately leading to the
phosphorylation of MITF at Ser-73 [141] (Figure 1).

KIT Signaling in Melanocyte Development and Pigmentation

KIT signaling is required for the proper migration, proliferation, differentiation and
survival of melanocytes and their precursors [143]. In the mouse Kit is expressed at all
developmental stages and into adulthood. In situ hybridization studies in embryos showed the
expression of Kit in melanoblasts migrating along the dorsolateral pathway [144, 145]. One
study identified a small population of Kit positive cells on the most dorsomedial aspect of the
neural tube that subsequently migrate exclusively into the developing dermis and express
melanocyte differentiation markers. This finding indicates that some NC cells are already
committed to the melanocytic fate prior to emigration from the neural tube [146].
Kit signaling plays a role in the migration of melanocyte precursor cells into the dermis,
epidermis and hair follicle during mid to late development [144, 147, 148]. Transgenic mice
that overexpress Kitl in the keratinocytes had increased numbers of melanocytes in the
epidermis and displayed hyperpigmented footpads and oral epithelium, which do not
normally display pigmentation [149]. The administration of functional Kit antibodies during
development and postnatally resulted in apoptosis of melanocytes in vivo [150].
Not many studies have directly addressed the role of KIT in skin and hair pigmentation
but its direct link to MITF regulation would implicate it as an important contributor. The
analysis of mouse follicular skin showed an increase in membrane-bound Kitl and the
application of Kit functional antibody caused reversible hair depigmentation in mouse hairs
and human hair organ culture supporting a role for Kit signaling in the maintenance of hair
follicle pigmentation [151]. As for EDN1, the expressions of KITL and KIT were found to be
downregulated in areas of hypopigmented skin in humans such as the palms and soles further
suggesting the importance of this pathway in the maintenance of skin pigmentation [117]. In
the pathological condition of dermatofibroma where the overlying skin is hyperpigmented,
KITL along with hepatocyte growth factor were found to be over-expressed in the underlying
dermis further suggesting the involvement of KITL in pigment production the human skin
[152]. UV exposure leads to enhanced KITL secretion resulting in an increase in pigment
production in human melanocytes in vitro and the skin of guinea pigs in vivo. Application of
KIT inhibitory antibodies to guinea pig skin prevented the production of pigment [153].
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374 Javier Pino and Lidia Kos

Disruption of the KIT Signaling Pathway in Mice and Humans

The Dominant White Spotting (W) and Steel (Sl) loci encode for Kit and Kitl in mice,
respectively [133, 154]. There are several alleles that arose spontaneously and are mostly
semidominant. Heterozygous mutants have spotting in the trunk area of the coat, while
homozygous mice are generally lethal, but those that survive are completely white with black
eyes (Figure 2C). These mutants are also anemic and sterile due to accompanying defects in
the red blood cell and germ cell lineages [155]. The Dickie mouse mutant has a 4-kb deletion
of the sequence encoding for Kitl resulting in a translated protein that only has the
extracellular domain of the ligand and blocks the existence of the membrane-bound form
[156]. Heterozygous mice have spotted coats but are fertile while homozygous are completely
white and die perinatally.
In humans KIT mutations cause piebaldism [157]. Piebaldism is a rare, autosomal
dominant disorder that results in the loss of melanocytes in midline areas, resulting in a loss
of pigmentation [158]. Areas affected include the medial portion of the forehead, eyebrows,
chin, chest, abdomen and the extremities. Some patients with piebaldism also present with
deafness due to the lack of melanocytes in the inner ear where they are required for the
maintenance of the endochoclear potential [159]. Most piebald patients carry heterozygous
mutations in KIT [158]. The site of the mutation in the KIT gene is often associated with the
severity of the clinical phenotype. Mild forms of piebaldism have been shown to result from
mutations in the extracellular ligand-binding domain while the most severe ones are caused
by dominant negative missense mutations in the tyrosine-kinase domain.
Recently, studies have shown that gain of function mutations in the gene encoding KITL
result in familial progressive hyperpigmentation and hypopigmentation (FPHH) [160, 161].
FPHH is characterized as hyperpigmentation of melanophages and keratinocytes in the
dermis at an early age that becomes progressively darker and larger as aging occurs [162].
FPHH is also associated with lentiginosis, hypopigmentation and café-au-lait macules. The
hypopigmented macules display limited hyperpigmentation in the basal epidermis with few to
no melanophages in the upper dermis.

INTERACTIONS AMONG SIGNALING PATHWAYS

The various signaling pathways that regulate pigment production in melanocytes act
synergistically in many instances and for the most part, converge on the activation of the
transcription factor MITF which in turn is responsible for activation of all the melanogenic
genes [163] (Figure 1). This does not, however, mean that pigment production relies
exclusively on MITF. Other transcription factors such as PAX3 and SOX10 directly regulate
the expression of TYR and its related genes [164-167]. It is also likely that some of the
signaling pathways controlling pigmentation have effects on the melanogenic genes such as
post-transcriptional and post-translational modifications that are independent of MITF [168].
For example, the enzyme diacylglycerol kinase, which phosphorylates diacylglycerol and
may act downstream of different hormones and growth factors, seem to have a melanogenic
effect by modulating the posttranslational processing of TYR [169].

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 MC1R. EDNRB and Kit Signaling in Pigmentation Regulation ... 375

Figure 2. Mouse Mutants reveal the role of signaling pathways in pigment production. Ay mouse mutant
displays a yellow coat color phenotype due to the inhibition of Mc1r signaling (A) while Ednrbsl/sl (B)
and Kit Wv/Wv mutants (C) have white coat colors because of improper melanocyte precursor
development.

When specific ligands or receptors are mutated and become dysfunctional, overt effects
on skin and/or hair pigmentation are observed underscoring the significant contribution of
each individual pathway. Some of these effects can be compensated by the over-activation of
a different pathway demonstrating the utilization of common downstream intracellular targets
for the generation of a normal pigmentation phenotype. The EDNs and KITL pathways act
synergistically on melanocyte proliferation and skin pigmentation. Treatment of cultured
human melanocytes with EDN1 and KITL caused enhanced activity of RAF-1, MEK and
MAPK [170]. The addition of KITL and EDN1 to human skin xenografts on SCID mice led
to a significant increase in melanin content and TYR gene expression when compared to
treatment with each factor alone [171]. The cross-talk appears to occur at least partially at the
KIT receptor level with its phosphorylation resulting from EDN1 binding to EDNRB [170].
Another possible point of convergence is the activation of RAF by PKC that occurs after the
stimulation of melanocytes with EDN1 [172] and subsequent MITF phosphorylation. This
interaction is further supported by experiments carried out with NC explant cultures [173].
Murine NC cells lacking Ednrb are not capable of producing Tyr positive melanocytes. The
addition of Kitl to these Ednrb deficient cells can, however, rescue their capacity to produce
Tyr. In vivo, the over-expression of Edn3 or G-proteins associated with Ednrb could partially
rescue the complete absence of dermal skin melanocytes and pigmentation observed in mice
with Kit mutations [115, 174, 175]. The coat, however, remained devoid of melanocytes and
pigmentation demonstrating that Edn3 is not capable of promoting the entry of melanocytes
in the epidermis and hair follicles.
The activation of cAMP downstream of MC1R and EDNRB is essential for
transcriptional regulation of MITF mediated by CREB and pigment production [50, 107].
This convergence may explain why the over-activation of Edn3 signaling can compensate, at
least partially, for the lack of Mc1r signaling in Ay mice [115, 176]. Since in Ay mice Mc1r is
not defective and the excessive amounts of ASP block the activation of the downstream
events, over-expression of Edn3 could lead to increased expression of Mc1r and make more
receptors available at the membrane for -Msh binding [177]. The partial rescue could also
be explained by the phosphorylation of Mitf that occurs downstream of Ednrb via the
activation of the MEK/ERK pathway [107]. The involvement of the latter pathway
exclusively does not seem to be sufficient for eumelanin production given that mice deficient
in Mc1r signaling crossed to transgenic mice that over-express Kitl do not present with
darkened skin [178]. However, over-activation of downstream components of the KIT
pathway such as RAS and RAF do cause hyperpigmented skin in mice [179-181] and café-au-
lait spots or macules in patients with Rasopathy syndromes such as Noonan, Legius, Leopard
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376 Javier Pino and Lidia Kos

and Neurofibromatosis type 1 [182, 183]. It is unclear whether hair pigmentation is also
affected in the transgenic mice and human patients with altered RAS and RAF activity. It has
been proposed that particular subgroups of melanocytes respond differently to signaling
molecules [24, 184] and this maybe another example where the production of eumelanin in
hair melanocytes requires higher levels of activation of the cAMP pathway than what is
necessary in skin melanocytes.
MC1R also interacts with downstream targets of the WNT signaling pathway by the
elevation of cAMP levels. In human melanocytes, it induced the phosphorylation of -
catenin, the stabilization of -catenin protein, and the attenuation of GSK3, further
stimulating the activity of -catenin in the nucleus where it binds to the MITF promoter [185].
-catenin has also been shown to be a part of pigment type switching regulation by acting
upstream of Mc1r [186]. In the dermal papilla -catenin suppresses ASP expression and
activates Corin, a negative regulator of ASP. -catenin loss of function in the dermal papilla
leads to a yellow coat color and its gain of function results in a darkened coat phenotype. In
mouse melanocytes, another consequence of the elevation of cAMP levels that occur upon the
binding of -Msh to Mc1r is the activation of the MAPK pathway. In human melanocytes
and melanoma cells, the MAPK pathway is activated downstream of MC1R independently of
changes in cAMP via Src tyrosine kinase-mediate transactivation of KIT [187]. These and
other still to be uncovered functional links among the different signaling pathways
demonstrate that the production of hair and skin pigmentation is a complex process.
Nevertheless, the establishment of a complete picture of the many functional links involved
will facilitate the development of optimal strategies for correcting and ameliorating
conditions of hypo- and hyperpigmentation.

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of Mc1r in UV-induced tanning. Nature 443, 340 (Sep 21, 2006).
[179] Powell M. B. et al., Hyperpigmentation and melanocytic hyperplasia in transgenic mice
expressing the human T24 Ha-ras gene regulated by a mouse tyrosinase promoter.
Molecular carcinogenesis 12, 82 (Feb, 1995).
[180] Dhomen N. et al., Oncogenic Braf induces melanocyte senescence and melanoma in
mice. Cancer cell 15, 294 (Apr 7, 2009).
[181] Milagre C. et al., A mouse model of melanoma driven by oncogenic KRAS. Cancer
research70, 5549 (Jul 1, 2010).
[182] Tartaglia M., Gelb B. D., Zenker M., Noonan syndrome and clinically related
disorders. Best practice & research. Clinical endocrinology & metabolism25, 161
(Feb, 2011).
[183] Martinez-Quintana E., Rodriguez-Gonzalez F., LEOPARD Syndrome: Clinical
Features and Gene Mutations. Molecular syndromology 3, 145 (Oct, 2012).
[184] van Raamsdonk C. D., Barsh G. S., Wakamatsu K., Ito S., Independent regulation of
hair and skin color by two G protein-coupled pathways. Pigment cell & melanoma
research 22, 819 (Dec, 2009).
[185] Bellei B., Pitisci A., Catricala C., Larue L., Picardo M., Wnt/beta-catenin signaling is
stimulated by alpha-melanocyte-stimulating hor- mone in melanoma and melanocyte
cells: implication in cell differenti- ation. Pigment cell & melanoma research 24, 309
(Apr, 2011).
[186] Enshell-Seijffers D., Lindon C., Wu E., Taketo M. M., Morgan B. A., Beta-catenin
activity in the dermal papilla of the hair follicle regulates pigment-type switching.
Proceedings of the National Academy of Sciences of the United States of America 107,
21564 (Dec 14, 2010).
[187] Herraiz C. et al., Signaling from the human melanocortin 1 receptor to ERK1 and
ERK2 mitogen-activated protein kinases involves transactivation of cKIT. Mol Endo-
crinol 25, 138 (Jan, 2011).
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 14

MULTIPLE GENES AND DIVERSE HIERARCHICAL

PATHWAYS AFFECT HUMAN PIGMENTATION

C. Ganesh*1, Anita Damodaran1, Martin R. Green2,

Sheila Rocha3, Nicole Pauloski3 and Shilpa Vora1
1
Unilever R&D Bangalore, India
2
Unilever R&D Colworth, UK
3
Unilever R&D Trumbull, US

ABSTRACT

One of the most easily visible and recognized human physical attributes is skin color
which is largely determined by the amount and type of melanin in skin and the influence
of haemoglobin. Human genetic adaptation, multiple geographic origins and intermixing
during migration of human population has resulted in a naturally wide palette of skin
color. Exposure to sunlight (which varies in intensity across geographical locations),
immune reactions, hormonal changes and aging alter skin appearance and pigmentation
through multiple mechanisms. Many genes are involved in the control of the type and
amount of melanin synthesized in melanocytes and its subsequent transfer to and
distribution within keratinocytes. We report our observations on the differential changes
in melanin content in human melanocytes, on modulating various pigmentation pathways.
Further, IL-1 was shown to be an upstream regulator of many of these pigmentation
pathways. Multiple SNPs have been mapped in the genes linked to human pigmentation
including MC1R, TYR, SLC45A2, KIT, EDNRB and SLC24A5. Our ground breaking
studies demonstrated that variation of the non-synonymous SNP rs1426654 in SLC24A5
encoding the NCKX5 protein amino acid change A111T, accounted for over 30% of the
variance in the constitutive skin color of South Asians. Diverse hyper- and hypo-
pigmentation disorders have been well documented as local spots, vitiligo, melasma and
mosaic pigmentation. Our investigations of such disorders have highlighted the role of
genes involved in pigmentation, cell adhesion and communication, immune processes
and lipid metabolism. Leveraging scientific advances in functional genomics has led to
increased awareness of the intricate regulation of human pigmentation and altered

*
Correspondence: [email protected]; [email protected].
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390 C. Ganesh, Anita Damodaran, Martin R. Green et al.

modulation in pigmentary disorders. As understanding of pigmentary processes

improves, our investigations will also translate discoveries into more effective safe
cosmetic and pharmaceutical interventions for skin pigmentation benefits.

INTRODUCTION
By virtue of its immediate striking visibility, pigmentation has captured the attention of
both scientists and laymen alike. There occurs naturally, wide variations in pigmentation and
many pigmentary diseases and disorders have been well described [Goldsmith et al., 2012].
These have triggered intense discussion, hypotheses, commentaries and rigorous scientific
investigations across centuries. It is evident that such complex phenomena are an outcome of
complex genetics and environmental pressures. Multitudes of pigmentary genes have been
identified, cloned and characterized [Lamoreux et al., Eds. 2010]. Complementary
biochemical and cell biological investigations have uncovered fundamental processes
governing pigmentation and its control. Excellent texts [Levine and Maibach, 2003; Nordlund
et al., Eds., 2006; Quevedo WC and Holstein TJ, 2006; Borovansy and Riley, 2011] and other
references [Yamaguchi and Hearing, 2009; Kondo and Hearing, 2011] abound in such areas
of scientific investigations.
The most striking example of phenotypic change due to underlying mutations in
pigmentation genes, is seen in human albinism. Oculocutaneous albinism type 1 caused by
mutations in the Tyrosinase gene, is one example of albinism. It is amongst the best
understood [Oetting et al., 2003] and malfunction of tyrosinase compromises the quality of
life (e.g., sunburn, vision problems etc.) due to little or no production of melanin. The
fundamental importance of tyrosinase and related proteins to pigmentation have been well
reviewed [Hearing, 2011] and it is understood that tyrosinase is regulated at multiple levels of
transcription, post translation and enzymatic activity [Schallreuter et al., 2007; Ebanks et al.,
2009]. Melanogenic proteins are the culmination points of diverse cellular signaling pathways
in the melanocytes [Imokawa, 2004; Lin and Fisher, 2007; Schiaffino, 2010] which are
regulated by intricate diverse interactions between melanocytes, keratinocytes and fibroblasts
[Hirobe, 2005; Kondo and Hearing, 2011].

MAJOR PATHWAYS IN PIGMENTATION

The origins of signaling events in skin can be traced as responses to triggers such as UV
radiation, hormones, growth factors and inflammation [Slominski et al., 2004; Yamaguchi
and Hearing, 2009]. Classical studies have examined signaling systems such as MSH:MC1R
[Abdel-Malek et al., 1995; Abdel-Malek et al., 1999; Suzuki et al., 1996; Millington, 2006;
Eves and Haycock, 2010; Dessinioti et al., 2011], SCF:cKIT [Giebel and Spritz, 1991; Spritz
et al., 1993; Lennartsson and Rönnstrand, 2012], WNT:FZD [Dorsky et al., 2000; Yamaguchi
et al., 2008; Yamaguchi et al., 2009] and EDN:ENDR [Imokawa et al., 1992; Imokawa et al.,
1995; Imokawa et al., 1996; Imokawa et al., 1997]. The cognate receptors are predominantly
of the GPCR variety. In addition, literature describes the effect of mutations in pigmentation
genes on melanin production [Lamoreux et al., 2010]. Some mutations result in unique
changes in pigmentation, with albinism as the most severe phenotype. For long, these

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 Multiple Genes and Diverse Hierarchical Pathways … 391

signaling pathways and their significance in the control of melanogenesis have been
investigated, usually one pathway at a time. In tune with the paradigm shift in biology,
integrated systems biology approaches now allow investigations on the extensive cross talk
and relationship between pigmentation pathways [Imokawa et al., 2000; Bellei et al., 2011;
Herraiz et al., 2011]. Physiological pathways operate not as isolated individual entities, but as
coordinated interactive units.
We chose the human primary melanocyte as the model system to discern contributions
from multiple signaling pathways to melanin content. Either signaling pathways agonists and
antagonists or siRNA (receptor gene specific transient knockdown) intervention was used,
followed by quantitative photometric assessments of resultant changes in melanin content (the
physiologically relevant end point). (figure 1 and table 1). While MSH increased melanin
content and cKit phosphorylation inhibitor (ISCK03) led to substantial decrease in melanin
content, other treatments resulted in no change (figure 1). Experiments using agonists and
antagonists are challenging to a certain extent, as typical cell culture media are replete
complex mixtures, including critical growth factors. Necessary inclusion of such material in
the media could result in an artificially blunted response, saturation of or even no effects in
regard to an added test material.

Figure 1. Effect of select receptor specific agonist or antagonist on cellular melanin content in human
primary melanocytes. Cells in culture were treated with the indicated amounts of either the agonist or
antagonist, for three days. Total cellular melanin was estimated using the regular A405nm method and
normalized with respect to control (reference 100%), after accounting for changes in cell count (neutral
red assay). Only MSH (MC1R agonist) and ISCK03 (cKIT RTK activity inhibitor) altered melanin
content. Results were from at least triplicate measurements.

A complementary approach is to alter cognate receptor levels by altering their gene

expression (siRNA). Table 1 depicts the observed differential levels of reduction in melanin,
upon reducing of the expression of melanocyte receptors. Interestingly, we observed that the
knockdown of type A endothelin receptor was far more effective in reducing melanin content,
than type B receptor. A complex pattern of results was observed that was dependent on
siRNA duplex and time point.
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392 C. Ganesh, Anita Damodaran, Martin R. Green et al.

Table 1. Effect of siRNA against select major melanogenic receptors on cellular melanin
content in human primary melanocytes. The maximal extent of reduction in melanin
content is shown. It should be noted that the maximal reduction in different cases
occurred at different time points and with different concentrations of siRNA oligos
(purchased from Invitrogen). Mirus reagent was used to transfect the siRNA oligos into
melanocytes and each case was standardized with respect to oligo concentrations and
time. % reduction in gene expression were measured by qRTPCR and melanin content
was estimated by A405nm method

Our observations overall indicate that diverse signaling pathways contribute to melanin
contentand that EDNRA and FZD agonist/antagonists do not exhibit the effect of mRNA
knock down experiments.

CYTOKINE REGULATION OF MELANOGENESIS

Epidermal keratinocytes are known to release many growth factors involved in
inflammation and melanogenesis [Takashima & Bergstresser, 1996; Ansel et al., 1990]. It is
well documented that external stimuli such as UV and allergic contact dermatitis can induce
keratinocytes to secrete mitogens and pro-melanogenic factors including ET-1 [Imokawa,
1992], NGF [Tron et al., 1990], NO [Romero-Graillet et al., 1997], -MSH [Rousseau et al.,
2007] and lipid mediators such as prostaglandins [Tomita et al., 1992; Scott et al., 2005],
which increase proliferation, melanin synthesis or dendricity by melanocytes [reviewed in
Imokawa, 2004; Hirobe, 2005; Yamaguchi and Hearing, 2009]. Melanocytes express specific
receptors for growth factors and the ligand receptor interactions then signal for various
melanocyte functions.
UV initiates several signaling cascades which are also common to various growth factor
and cytokine mediated pathways. However, how UV initiates these signals is still unclear
[Gilchrest et al., 1996: Rosette and Karin 1996; Fischer et al., 2002]. Various intermediates
speculated in UV responses are reactive oxygen species, ROS [Gross et al., 1999], cytokines
like IL-1 [Griswold et al., 1991] or lipid mediators [Tomita et al., 1992; Scott et al., 2005]. In

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 Multiple Genes and Diverse Hierarchical Pathways … 393

skin keratinocytes, IL-1 exists as preformed protein complex, which can be released
immediately in response to a noxious stimulus [Brink et al., 2000; Ashida et al., 2001; Luo et
al., 2004; de Jongh et al., 2007] and plays an important role in the pathophysiology of skin
inflammation and wound healing [Kominine et al., 2000; Murphy et al., 2000; Freedberg et
al., 2001]. Also, it has been reported [Okazaki et al., 2005] that the amount of IL-1 secreted
by keratinocytes from aged volunteers was higher than the young volunteers, which suggests
a role of Il-1 in age related changes in skin. Studies have revealed that both release [Murphy
et al., 1989] and synthesis [Griswold et al., 1991; Lew et al., 1995] of IL-1is maximum 1
hr. post UV exposure in epidermis. Meanwhile, others studies report extended release and
synthesis of IL-1 for 3-72 hrs. [Imokawa, 1995; Luo et al., 2004] in skin epidermis.
Since IL-1 is the only factor which is preformed and stored in keratinocytes, we
hypothesised IL-1 to be one of the intermediate as well as a common initiator, through
which UV effects both inflammatory and melanogenic changes in epidermis. Our studies
demonstrated that keratinocytes do indeed constitutively produce large amounts of IL-
1(Figure 2). UV irradiation further increased the production and secretion of IL-1α in a
short period (4hrs.) without any change in cell viability, suggesting a specific mechanism for
release of preformed IL-1 by keratinocytes in response to UV.
The IL-1 gene family comprises of IL-1α & β and the IL-1Ra receptor antagonist. IL-1α
& β are formed as a 31 kDa precursor protein [Dinarello, 2002]. This leaderless peptide gets
processed into active 17 kDa forms by the action of proteases, specifically Caspase-1 [Faustin
and Reed, 2007; Martinon and Tschopp, 2007]. A set of proteins forming the
‘inflammasomes’, a multi-protein innate immune complex, have been implicated in the
maturation and secretion of IL-1in various immune cells [Martinon and Tschopp, 2007]. It
has been demonstrated [Feldmeyer et al., 2008] that UVB mediated enhancement of
cytoplasmic Ca+2 is required for the activation of caspase 1 by the inflammasomes, for
maturation of IL-1 in keratinocytes and implicated the process in UV induced sunburn.
Recently, new mechanisms involving cAMP and NO activating NLRP3 inflammasome in the
processing of IL-1 has been suggested [Leavy, 2013]. However, it is unclear if the process is
same for IL-1maturationin keratinocytes, though it has been demonstrated that both proIL-
1 and processed IL-1 are functionally active [Werman et al., 2004].
UV induced IL-1 has been demonstrated in various studies in skin [Murphy et al., 1989;
Griswold et al., 1991; Lew et al., 1995] and keratinocytes [Kupper et al., 1987 and Figure 2].
However, unlike an earlier report [Swope et al., 1994], we did not detect IL-1 or  in
melanocytes (Figure 3). The receptors for IL-1 were expressed by both keratinocytes and
melanocytes, suggesting that IL-1 produced by keratinocytes can regulate functions of both
keratinocytes and melanocytes in autocrine and paracrine manners respectively. Thus the
keratinocyte appears to be the major and most important source of active preformed IL-1 in
skin [Murphy et al., 2000].
Earlier studies have demonstrated that IL-1α can induce the production of ET-1
[Imokawa et al., 1995], POMC/MSH/ACTH [Funasaka et al., 1998, Scholzen et al., 2000],
SCF [Da Silva et al., 2003] and COX2 [Kessler-Beckker et al., 2004]. ET-1, SCF and POMC
are melanogenic mediators produced by keratinocytes upon UV exposure. However, the
mechanism or signaling events involved in their synthesis is unclear. In our study, expression
of these molecules was induced by IL-1 (Figure 4) to a higher level than by UV during early
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394 C. Ganesh, Anita Damodaran, Martin R. Green et al.

time points. This suggests that the direct effect of IL-1 is faster than that of UV as the latter
needs a preceding build up of IL-1. Further, induction by UV was inhibited by anti-IL-1
antibody confirming that UV induction of these melanogenic molecules is under the
regulation of IL-1.

Figure 2. Constitutive and UV induced release of IL-1 α by keratinocytes. Confluent HaCaT (immortal
transformed human keratinocyte cell line) and primary human keratinocytes (1o K)were irradiated (100
mJ/cm2 UVA and 20mJ/cm2 UVB). Culture supernatant media was collected 4 hrs. post UV exposure
and analyzed by ELISA for IL-1α. HaCaT cells were a kind gift from Dr. Norbert Fusenig.

Figure 3. (Continued).

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 Multiple Genes and Diverse Hierarchical Pathways … 395

Figure 3. Endogenous levels of IL-1α mRNA and IL-1 mRNA in melanocytes and keratinocytes.
Total RNA was extracted from confluent cultures of HaCaT cells, primary human melanocytes and
keratinocytes. Real time PCR was carried out to determine the endogenous level of IL-1(A) and IL-
1 (B) mRNA.

Figure 4. Induction of COX-2, SCF, POMC and ET1 in keratinocytes on IL-1 treatment. Confluent
primary keratinocytes were treated with 10ng of IL-1 and 2g of IL-1antibody for 1 hr. and UV
(100 mJ/cm2 UVA and 20mJ/cm2 UVB). Total RNA was isolated and semi-quantitative PCR was
performed using specific primers for COX-2, SCF, POMC, ET1 and GAPDH (internal control). PCR
products were then separated through 2% agarose gel and visualized by ethidium bromide staining.
Photocap software was used to digitize the data and converted to fold changes. Lanes 1-4 are: Control,
10ng IL-1treatment, UV followed by 2g of IL-1 Antibody and UV treatment.
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396 C. Ganesh, Anita Damodaran, Martin R. Green et al.

IL-1 regulation of melanogenesis has been reported in organ cultured guinea pig skin
[Maeda et al., 1996] and in vitro in mouse melanoblasts [Hirobe and Ootaka, 2007], while it
has been demonstrated that IL1 treatment reduced tyrosinase activity in human melanocytes
[Swope et al., 1991; Abdel-Malek et al., 1993]. In our study, we observed that UV and IL-1
exposed keratinocytes induced increase in melanin content (Figure 5) and tyrosinase activity
(data not shown), which could be abolished by anti-IL-1 antibody in co-cultures. Primary
human keratinocytes were treated with IL-1 for 6hrs, supernatant collected and primary
melanocytes were treated with those supernatant for 4hrs. Tyrosinase expression in
melanocytes was quantified by qRTPCR (GAPDH reference gene). We observed that
tyrosinase expression increased to ~3, 9 and 7 fold upon treatment of melanocytes with spent
media from control keratinocytes or IL1 treated keratinocytes or IL1 treated keratinocytes
respectively (reference 1-fold in untreated melanocytes). By contrast, direct addition of IL-
1/ to melanocyte culture did not affect either melanin synthesis or tyrosinase activity or
expression.

Figure 5. Melanin content estimation in co-cultures where keratinocytes were treated with IL-1 prior
to melanocyte addition. Primary keratinocytes were treated with IL-1α, UV and UV followed by
addition of anti-IL-1α antibody (ab). After 24hrs., melanocytes were added to the keratinocytes culture
and incubated for further 72hrs, prior to the estimation of melanin content (change by IL1 treatment
was significant over control at p<0.01).

Our study also demonstrated an autocrine effect of IL-1 on keratinocytes resulting in

production of more IL-1 (data not shown) as well as other melanogenic gene products,
suggesting a self sustained keratinocyte initiated IL-1 signal cascade. It is known that IL-1R
levels on the keratinocyte surface increase 9-20 fold on differentiation, although IL-1 protein
level does not change much on cell differentiation [Blanton et al., 1989]. This suggests that
differentiated keratinocytes, on external stress, release IL-1 and can be activated in an
autocrine manner, to produce keratinocyte and melanocyte growth and melanogenic factors.
This serves to propagate the UV signal via IL-1. We have thus identified and confirmed the
role of IL-1α in regulating melanogenesis by activation of keratinocytes to release
melanogenic factors. Further studies are required to identify the molecular mechanism
involved in IL-1α induction of melanogenic factors.

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GENES INFLUENCING NATURAL VARIATION

IN HUMAN SKIN COLOUR

Human, visible skin colour variation is largely due to amount and type of melanin in skin
[Alaluf et al., 2002a]. Darker skin colours are dominated by darker eumelanin while the
lighter yellow/orange pheomelanin contributes to fairer skin types. Blood and the organisation
of blood-vessels can provide a reddish hue while carotenoids contribute to skin yellowness
[Alaluf et al., 2002b], and other structures in paler skin such as ‘blue’ veins are caused by
differential light reflection and absorption. Over the past 30,000y skin colour variation has
been thought to be due to human adaptation to variations in sun light intensity particularly
UVB, as mankind moved north and occupied a wide range of geographical locations [Beleza
et al., 2012]. The ancestral colour of human skin is considered to be black with lightening
suggested to be driven by need for UVB catalysed vitamin D synthesis and/or protection of
folate in skin. However recently the ‘vitamin D’ theory has been challenged by the hypothesis
that pigmentation is essential to prevent dehydration and sun burn, by protecting the skin
epidermal water barrier from the destructive power of sun light. Universally, females within
populations appear to be lighter in skin colour, an observation variously ascribed to sexual
selection or the need by females for higher levels of vitamin D for successful reproduction.
Many gene products are required for and influence the amount and nature of melanin
production in skin. For example the color-genes web site http://www.espcr.org/micemut/ on
Dec 2012 listed 378 loci linked to pigmentation of which 171 were cloned genes having
human homologues. A siRNA based functional genomics study identified at least 92 genes
affecting the degree of melanin production by human melanocytes (MNT-1 cells).
Interestingly despite the number of genes required for pigment production, human albinism is
restricted to inactivating mutations in just 4 genes (TYR, OCA2, TRP1 and SLC45A2) the
protein products of all of which are specifically located in functional form in the melanosome.
Melanosomes are lysosome-related organelles [Raposo et al., 2007] and melanin production
has parallels with autophagy [Ganesan et al., 2008]. One might speculate that other
inactivating mutations that might give rise to albinism in humans are selected against because
of essential, life sustaining function(s) in other cellular pathways.
Several pigmentation gene variants have shown a remarkable degree of selection in
human populations with some gene variants moving to fixation very recently, that is within
the last 11 to19,000y [Beleza et al., 2012]. Such genetic adaptation of skin colour genes is
almost certainly driven by the human survival advantages noted above. Other sets of genes
may also have been subject to recent selection for example the worldwide variation in human
mitochondrial DNA responding to climate [Balloux et al., 2009]. A striking example of
variant selection linked to skin colour is provided by SLC24A5 which codes for the
potassium-dependent sodium-calcium exchanger family member 5 (NCKX5). In a genome
wide association study of skin pigmentation in a South Asian population, SLC24A5 was
found to account for >30% of the variance in dichotomously defined light and dark skin
colour [Stokowski et al., 2007]. Data from the HapMap project [International HapMap
Consortium, 2005] shows that alternate alleles of the non-synonymous (ns) SNP rs1426654 in
the SLC24A5 gene are present almost mutually exclusively in African and European
populations.
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Table 2. Genes linked to normal (constitutive) variation of skin-colour in human populations

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400 C. Ganesh, Anita Damodaran, Martin R. Green et al.

A different inactivating mutation in the zebrafish homologue is responsible for the lighter
golden pigment phenotype [Lamason et al., 2005]. Recently a predicted inactivating 4-
nucleotide insertion into SLC24A5 has been described in a person showing extreme
cutaneous hyperpigmentation (Mondal et al., 2012).
The nsSNP (rs1426654) changes the coding amino acid at position 111 in NCKX5 from
alanine to threonine (pA111T) and results in a greatly reduced exchange function of the
protein expressed in ‘High Five’ insect cells [Ginger et al., 2007]. NCKX5 is primarily
located in the trans-Golgi network and the mechanism by which altered ion exchange activity
regulates pigmentation remains unconfirmed, though Lamason et al., [2005] have proposed a
role in melanosomal calcium uptake and a location for NCKX5 in the melanosomal
membrane. Surprisingly NCKX5 knockdown perturbs sterol and particularly cholesterol
metabolism in melanocytes [Wilson et al., 2012] a lipid which has been shown to regulate
melanogenesis [Hall et al., 2004; Jin et al., 2008; Schallreuter et al., 2009]. Apart from
SLC24A5 other pigment genes showing a high degree of selection in human populations are
ASIP, KITLG, MC1R, OCA2, SLC45A2, TYR, TYRP1 [Norton et al., 2007; Sturm et al.,
2009; Edwards et al., 2010; Donnelly et al., 2012; see also table 2].
Given the complex genetic nature of many continuous human traits, remarkably few
genes to date have a confirmed role in natural, constitutive variation in human skin colour.
Genes are listed in table 2 as confirmed if there is at least two independent pieces of evidence
from human skin colour studies, or candidates if the link is inferred, of borderline significance
and/or supported by only one reference. The table also lists pigmentation informative SNPs,
the function of the SNP if known, and the human populations affective by the alternate allele.
Accordingly there are so far perhaps 8 confirmed (ASIP, IRF4, KITG, MC1R, OCA2,
SLC24A5, SLC45A2, TYR) and 9 candidate genes that have had variants selected through a
process of human adaptation to new environments.
As hundreds of genes are required for pigmentation but so few are linked to natural
human skin colour variation (Table 2 and Figure 6), it is instructive to ask why this may be.
Although further genes influencing constitutive human skin colour variation may remain to be
discovered it is highly likely that all the major skin colour ‘effect’ genes such as SLC24A5,
have been discovered in genome wide association investigations. It is possible that over the
last 30,000y the stochastic gene variant selection process may not have sampled all the
available possibilities leaving safe intervention targets still to be discovered. Alternatively and
more likely is that very few genes essential for pigment production can have their function
altered to reduce pigmentation in a safe and effective manner without adversely affecting
other important cellular processes in the human body. As noted above, melanosomes are
lysosomal-related organelles [Raposo et al., 2007] and pigment synthesis shares processes
with autophagy [Ganesan et al., 2008] and both processes that might therefore be adversely
affected by changes in pigment genes. Brinkman et al., [2006] argued a parallel case by
suggesting that the study of the human phenome [the set of all human phenotypes] and
associated underpinning genetic variation is a good place to start in order to discover safe and
effective drug targets. Hence it is probable that many gene variants have been ‘tested’ by
survival pressures as human adapted to new environments, but very few of those genes
variants have been ‘selected’ having safe function and provided sufficient advantage to
become established in the human population.

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Figure 6. Genes whose variants are associated with natural variations in human skin colour. Both the
well documented genes and candidate genes have been depicted. It is interesting to note that although
most genes are expressed in melanocytes, they participate in diverse stages of melanogenesis.

HYPERPIGMENTATION OF SKIN
Hyperpigmentation is a localised darkening of the skin. It is most common in people with
darker skin tones of Asian, Mediterranean, or African descent. It is a major clinical problem
that can affect the life of a person by altering his or her appearance. The visual impact of
hyperpigmentation often causes considerable psychological distress [Brown et al., 2008]. In
fact it has been ranked the top cosmetic dermatological concern for which people seek a
dermatologist. Hyperpigmentation can result from many factors and can present itself in
different forms throughout a person’s life. Chronic sun damage, inflammation due to acne
vulgaris or other skin injuries and hormonal imbalances can all lead to some form of
hyperpigmentation. It can be diffuse or local and is often associated with underlying medical
conditions. However, ultimately it is strongly linked to an over production of melanin by the
melanocytes. It can occur anywhere on body especially on sun exposed skin, but the areas of
greatest esthetic concern are the face, hands and upper body.
Hyperpigmentation has been associated with numerous diseases or conditions. It has been
linked to adrenal hormonal imbalance in cases of Addison’s disease and Cushing’s disease. It
is a symptom of sex hormone imbalance as in cases of melasma or chloasma and Linea nigra,
associated with insulin resistance in cases of Acanthosis nigricans and linked to many other
syndromes or disorders such as Nelson’s syndrome. Other forms of hyper pigmentation such
as Leopard syndrome, freckles, and nevi have been linked to genomic mutations or SNPs.
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402 C. Ganesh, Anita Damodaran, Martin R. Green et al.

However, the forms of hyperpigmentation that impact even-skin tone in the majority of the
population, are melasma, post-inflammatory hyperpigmentation (PIH) and solar lentigines.
Melasma is a patchy brown, tan, or blue-gray discoloration that occurs mainly on the
face. It is most common in women, however more recently there have been reports of
melasma in men [Vachiramon et al., 2012]. Some reports link a genetic predisposition as a
major factor in male melasma [Sarkar et al., 2010]. Although a high number of men with
melasma have a family history of the condition, interestingly none of them reported melasma
in their fathers [Vazquez et al., 2010]. Therefore, there is a high potential for an X-linked
chromosome aberration responsible for the condition in men. In the female population it is
most common among pregnant women with olive or darker skin tone such as Hispanic,
Asian, and Middle Eastern individuals [Grimes 1995]. Despite a great deal of research in the
area, the exact cause of melasma remains largely unknown. It has been reported to be
triggered by several factors such as pregnancy, birth control pills, hormone [HRT and
progesterone] etc. and predisposition due to family history and race. Sun exposure is also a
key factor, especially in individuals with a genetic predisposition for developing melasma.
Once present it is responsive to sun exposure and therefore darkens during summer months
and fades slightly over the winter months.
Post-inflammatory hyperpigmentation (PIH) can occur as a consequence of exposure to
certain chemicals or inflammatory agents such as in acne or fungal infections. PIH has also
been linked to shaving or plucking in the axilla [Evans 2012]. The sun has been implicated in
exacerbating various forms of PIH. It is less characterized because it is a sensitive area to
biopsy without significant risk of further worsening the condition. It is believed that the
inflammatory cytokines released by the inflammatory cells that infiltrate the area in response
to the skin challenge can trigger the melanocytes to produce more melanin.
Chronic sun exposure is responsible for the development of another form of
hyperpigmentation, solar lentigines. Solar Lentigines or age spots develop on sun-damaged
skin of the face, the back of the hands, lateral forearms, the back and chest. They are
characterized by a hyperpigmented basal layer, elongated rete ridges and increased numbers
of melanocytes. Aside from the apparent activated state, the melanocytes appear otherwise
normal with melanosomes present in all stages of maturation in the cytoplasm and in
dendrites. Increased expressions of melanogenesis-specific genes and proteins such as
POMC, TYR, TYRP-1, DCT, PMEL-17, and OCA2 have been confirmed. It has been
proposed that there is a perturbation in keratinocyte differentiation. Genomic profiling studies
have been completed to aid in characterizing solar lentigines. Insights generated in these
studies confirm the up regulation of melanogenesis specific gene and inflammatory pathways.
In order to expand on this work we have investigated the expression profile of microRNAs of
solar Lentigines.
Only a subset of the genes in the human genome has been confirmed to be active in any
cell type and it is now known that around 98% of the human genome consists of non-protein
coding regions (The ENCODE project consortium, 2012). However approximately 76% of
human DNA is actively transcribed into functional primary RNA transcripts or non-coding
RNAs (ncRNAs). Some of these ncRNAs are involved in post-transcriptional regulation,
mainly small interfering RNAs (siRNAs) and microRNAs (miRNAs). MicroRNAs are small
endogenous RNA molecules that play essential roles in regulation of gene expression and a
wide range of cellular processes. They are believed to be coded for about 1-3% of the
mammalian genome. It is estimated that one-third of the protein-coding mRNAs are regulated

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 Multiple Genes and Diverse Hierarchical Pathways … 403

by miRNAs. Although their functions have not been completely elucidated it has been
confirmed that many regulate mRNAs by targeting them for degradation or inhibiting their
translation.
MicroRNAs have recently been shown to play a pivotal a role in a variety of skin
diseases. Several miRNAs have been implicated in wound healing and inflammatory skin
conditions. In order to gain insight into the genomic profile of hyperpigmented lesions we
explored the expression profile of miRNAs and mRNAs in solar lentigines compared and
photo-exposed and [peri-lesion] and photo protected skin.
Through the analysis of over 160 skin biopsy samples we have identified characteristic
expression profiles in these hyperpigmented lesions that can guide our understanding of its
etiology. The microRNA profile for age spots reveals that there is a gradual increased
expression of specific microRNAs from photo-protected to peri-lesional skin to solar
Lentigines (Figure 7). The profile of these microRNA reveal a significant change in
microRNAs that regulate genes involved in lipid and fatty acid metabolism as well as
inflammation.
Further studies are on going to understand the role of microRNAs in the etiology of solar
Lentigines.

Figure 7. miRNA array data on solar lentigines. Statistical testing was performed on the array data
assuming a linear model of condition as an ordinal variable [photo-protected < peri-lesion < lesion] and
subject to test the effects of each condition relative to photo-protected. Probes were filtered to ensure
FDR < 1% and monotonic increase or decrease across the three conditions. 49 miRNAs were
considered significant. On the heat map, red indicates relatively higher expression and green indicates
relatively lower expression.

Although several topical over the counter and prescription creams are marketed for
ameliorating various forms of hyperpigmentation, to date the most efficacious treatments
require in office visits to dermatologists. In the cases that involve hormonal imbalance,
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404 C. Ganesh, Anita Damodaran, Martin R. Green et al.

medical treatments for these conditions usually lead to some relief of the hyperpigmentation.
For the aesthetic conditions that do not respond to those types of treatments the only options
available today are chemical peels, cryosurgery or laser treatments. Therefore, in order to
provide more efficacious, specific and less invasive treatment options for hyperpigmentation
conditions, further characterization of each type of hyperpigmentation is needed.

MOSAIC HYPOPIGMENTATION OF SKIN

Depigmentation of skin is also widespread in human population. Classical examples of
hypopigmented skin conditions include the earlier discussed albinism as well as vitiligo
[Gawkrodger et al., 2010; Guerra et al., 2010]. However, there exists other distinct
hypopigmented conditions, which are being characterized. We have observed and described
one such [Mollet et al., 2007] mosaic hypo pigmentary pattern, characterized by
hypopigmented patches in the background of normal pigmentation in hands, legs and torso.
This pattern is present from birth and persists through the life of the affected individuals, who
otherwise lead normal healthy lives. The condition is very similar to nevus depigmentosus
[Lee et al., 1999; Khandpur and Sumanth, 2005; Kim et al., 2006] but distinct from vitiligo,
according to the opinion of expert dermatologists. A family tree (Figure 8) analysis suggests
possible genetic association, in a family native to the southern part of India.

Figure 8. Family tree of individuals from southern part of India, who display blotchy skin
hypopigmentation patterns. Numbers indicate individuals whose skin samples were analyzed by
histology as well as microarray.

It was evident from Mason-Fontana stained sections of the normal and hypopigmented
patches (data not shown) that the overall melanin content in the lighter patches was much
reduced but not completely absent compared to adjacent normal skin. The regular basal layer

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in normal skin was seen enriched in melanin, while melanin was grainy in the lighter patches.
RNA was isolated from such skin biopsies and analyzed by microarray. It was observed that
~200 genes were differentially regulated (comparable numbers up and down regulated).
Broadly, those genes were under the categories of cell adhesion, phosphatidyl inositol
signaling, lipids metabolism, immune function related, steroid metabolism etc. In terms of
gene ontology, significant categories include signal transduction (GO:4871) and lipid/fatty
acid metabolism (GO:6629/31).
Genes with links to organelle biology/intracellular transport were also found to be
differentially regulated. In contrast, although several melanogenic genes were represented by
multiple spots in the microarray chip, their expression did not vary uniformly between
samples. It is clear that substantial reduction in pigmentation/skin colour can be observed,
even when the expression of critical core melanogenic genes are unaffected. As noted
elsewhere pigmentary differences can arise from altered cellular signaling enhancing the
possibility of intervening at different levels, to safely modulate skin colour.

CONCLUSION
Our work discussed in this chapter exemplifies the complex nature of signaling pathways
related to pigmentation. Although multiple pathways are involved, there appears to be a
graded contribution towards melanin content.
Being a primary organ for the defence of the body, skin is unique by virtue of its
intrinsically highly visible nature. Human adaptation to new geographic environments has
generated a continuous palette of skin colours biologically enabled by highly regulated
intricate signaling pathways. Deeper and wider understanding of cellular signaling events in
pigmentation can open up new vista to safely modulate skin colour and tackle a wide range of
problems associated with pigmentation.
Both Pharma and Cosmetics domains can benefit by leveraging a wide variety of
scientific approaches and modern genomic tools to address the fundamental biology of
pigmentation.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 15

ACQUIRED SKIN PIGMENTATION

Hideo Nakayama
Nakayama Dermatology Clinic, Shinyo CK, Tokyo, Japan

INTRODUCTION
Pigmentation of the face is not fatal, except for melanoma, however they are disastrous
diseases, as nobody wants to have abnormal pigmented spots or diffuse or bizarre
pigmentation on the face. It is so with other sites of the body, including the trunk and
extremities. There are several causations to produce such pigmentation, as are listed in
Table1.
Most of these causations have been clarified, however, some of them are still kept
unknown. Treatments and prevention are quite different according to the diseases of
pigmentation, as the mechanism of the production of the pigmentary disorder is much
different.

1. MELASMA
Melasma is a flat brown hyperpigmentation of the face, without any sign of inflammation
such as erythema or itching. It occurs mostly on middle-aged women, and average age was, in
Japan, 43. It means that female hormone must have relationship in production of melasma, as
it is rare before 20 years of age and after 70. The most common site of melasma is the area
around the eyes, especially the lower section of the lower eyelids and the upper site of the
cheeks. Melasma often appears also on the forehead, and most severe cases show the
configuration of goagle around the eyes [Figure 1a].


Corresponding author: Hideo Nakayama, M.D., Chief Dermatologist, Nakayama Dermatology Clinic, Shinyo CK,
building 6F, 3-3-5, Kami-Ohsaki, Shinagawa-ku, Tokyo 141-0021, Japan.
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414 Hideo Nakayama

Histopathology

Melanin deposition is clearly noted in epidermal cells showing sometimes just above the
nuclei as if melanin pigments were a cap for nuclei protecting from sunlight. The number of
melanocytes is normal, and hyperplasia of melanocytes is not present [1, 2].
This fact means that hyperpigmentation of melasma is due to hyperfunction of each
melanocyte, and that when hyperfunction is interrupted medically, cure or much improvement
of melasma is expected.
Mild inflammatory infiltrates composed of lymphocytes and histiocytes are sometimes
noted in the upper dermis, however, this is not an original pathological process. It is regarded
as a secondary change due to cosmetics or medication for melasma [Figure 2].

a b

Figure 1. a: Melasma of goagle-like configuration in a 44-year-old woman. b: She applied 1% kojic

acid cream everyday, and almost complete cure was noted 2 years later.

Table 1. Acquired hyperpigmentation with short summaries

1. Melasma
Mainly occurs on middle aged women. Causation is increase in progesterone on luteal phases.
Hypersensitive to UV-B at 20% of the cases. Laser is not effective, however, long term usage
of depigmenting cream is effective.
2. Pigmented contact dermatitis
An unique type of allergic contact dermatitis mainly caused by textile finishes, washing
powder components and metals. Allergic reaction occurs at the basal layer of the epidermis,
and incontinentia pigmenti histologica is the mechanism of pigmentation. Patch test to find
out causative allergens and their elimination is necessary
3. Pigmented cosmetic dermatitis
Old name was Melanosis faciei feminae (Noma, 1947). Acquired dark faces of women of
black, bluish purple, and brown in tint, and diffuse or reticular or spotty in configuration.
When causative cosmetic allergens are found by patch testing cosmetic series allergens,
allergen-free soaps and cosmetics for the patients should be used exclusively for a long time.
This allergen control is effective. Laser is not effective.

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4. Pigmented Purpric lichenoid dermatitis

Occurs mainly on middle aged and old people on the legs. Histology shows accumulation of
hemosiderin in the upper dermis. Causative allergens are dyes, rubber components and other
textile finishes. Finding out socks which are allergen-free for the patients is needed in severe
cases.
5. Dirty neck of atopic dermatitis
Severe atopic dermatitis patients may develop black hyperpigmentation of the neck, when
only antisymptomatic treatment is continued for years. When serum IgE is elevated, and
RAST shows high unit of allergy to house dust mites (Dp, Df), the responsible allergens
reside in the own homes. Mite fauna is better be investigated, followed by reasonable
environmental improvement, and atopic eczema with pigmentation has been reported to
improve slowly.
6. Solar lentigo
Sun exposure for many years produces increase in melanocytes locally to result in macular
brown spots. Regarded as a benign mild tumor, and may develop to seborrheic keratosis some
years later. Laser is the first choice for the treatment with excellent effect.
7. Others
Ochronosis, Berloque demratitis, Posttraumatic hyperpigmentation, Tatoo, Pigment-syphilis,
Lichen planus cum pigmentatione, Amyloidosis, etc.

Figure 2. Histopathology of melasma shows increased melanin pigment at the basal layer and the lower
part pf prickle layer of the epidermis.

Causation

(1) Hormonal Disturbances

The main cause of melasma is considered to be an increase in progesterone (P4) in the
serum at luteal phases. Sato [1] measured various hormones by tritium (3H)
radioimmunoassay in two groups of age-matched middle aged women (average age 43) with
and without melasma on the seventh days of the ovarial and luteal phases.
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416 Hideo Nakayama

Significant differences were only present in the increased levels of progesterone (P4) and
17OH progesterone in the plasma in the luteal phases of melasma patients as compared to the
age-matched female controls without melasma [Figure 3].
Other hormones, such as estradiol, follicle stimulating hormone, luteinizing hormone,
prolactin, androstenedione, and cortisol, showed no differences between groups during the
ovarial and luteal phases [3].
The increase in plasma progesterone may be attributed to the fact that melasma is
exacerbated by pregnancy where plasma progesterone is increased or by contraceptive pills
that occasionally contained progesterone; there is gradual decline of melasma after
climacterium by 70 years of age.

Figure 3. Plasma P4 and E2 levels of melasma patients and matched controls in follicular and luteal
phases.

(2) Photohypersensitibity
Melasma patients often claim that melasma on the face worsened by strong sunlight. It
occurs in summer, however, usually spontaneous improvement is not seen in winter,
therefore, sun care is apparently necessary to prevent worsening, and improvement cannot be
expected by a simple procedure such as long term avoidance of sunlight.
When minimum erythema dosis (MED) was measured in melasma patients, 18 (24.7%)
of the 73 melasma patients showed clear photohypersensitivity by lowered MED and
minimum pigmentation dosis (MPD) to a mixture of UVA and UVB. Further study showed
that reactivity to UVA was normal but hypersensitivity to UVB was remarkable in all 15
patients. With such photohypersensitive melasma patients, MED was lowered to
approximately one-third of normal persons in summer, and a palpable erythema was observed
above 2 MEDs of UVB which produced long-lasting hyperpigmentation for weeks.
Therefore, 2 MEDs were almost equal to 1 minimum quaddel dosis (MQD) and to 1 MPD
(Table 2; Figure 4). All these patients did not have any medication when MED was measured,
uroporphyrin and coproporphyrin levels were normal in urine, and the effect of common

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photoallergens such as musk ambrette or thiazides was denied. Plasma 17OH progesterone
levels were elevated only in one case, out of 10 cases studied [1].

Table 2. MED and MPD with Melasma Patients

Therefore, the mechanism of UVB photohypersensitivity in melasma should be

investigated in the future.
Thus melasma on the faces of middle aged women has been speculated to have been
caused by the increase in serum progesteron (P4) at luteal phases, and about 25% (one out of
four patients) of them are aggravated by UVB.

Treatment
The perorally administered Vitamin C and transamine failed to successfully cure
melasma patients, therefore, topically applied whitening agents have been tried to cure the
disease, and they were in most cases successful [Table 3].
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418 Hideo Nakayama

Figure 4. The results of MED and MPD [Table 2] showed that UVB hyperpigmentation was
demonstrated at 1 week of UV irradiation. Note that no reaction occurred under UVA irradiation, even
though the doses were the same.

The best topically applied whitening agent was ellagic acid, at 1% cream used twice a
day for three to six months. The rate of improvement was 92.6% when 68 melasma patients
entered for the trial. Amazingly, the side effect of ellagic acid to produce contact dermatitis
was 0%, and it was proven to be the most safely and effectively used whitening cream.
Unfortunately, ellagic acid has not been commercially sold, presumably because it was
difficult to be synthesized for its complicated chemical structure.

Table 3. Results of in vitro and animal assay studies for depigmentation agents

*
n-2,4-acetoxyphenyl thioethyl acetamide.

The second most, successful whitening agent was kojic acid used at 1% cream. Kojic acid
was discovered as a whitening agent at a fermentation company in Kyushu Island in Japan in

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mid 20th Century. When workers having darker complexion were newly employed and started
working in fermentation process of Japanese liquor “sake,” their complexion turned to
ordinary normal skin color after 6 months of labor.
The causation of this “occupational” whitening was later analyzed to have been brought
by the contact to a ferment “koji” of the fermentation process.
The main component of koji was kojic acid having pyrone ring, and showed whitening
effect in several experimental methods. Firstly, the activity of isolated mushroom tyrosinase
was inhibited by kojic acid with dose responses.
Secondly, streptomyces fervents produces melanin when it is cultured in liquid medium,
and the melanin synthesis can be inhibited by the presence of depigmentation agents. The
important fact is that streptomyces was alive in all the culture mediums, even though black
eumelanin was not produced or decreased in production after kojic acid was added in various
concentrations: when streptomyces was transferred to another culture medium without kojic
acid, it produced melanin, turning the color of the medium to black again [1].
Thirdly, cultured B-16 melanoma cells are also excellent material for visually confirming
the melanogenesis inhibition in vitro. A recommended method is to culture B-16 cells in
Eagle’s MEM with 10% fetal bovine serum, and depigmentation agents are added in the
culture medium at different concentrations.
After 5 days of the culture, the cells are fixed by formalin and stained by ammonical
silver nitrate, then premelanosome can be visually stained in black. When the cells are alive,
and such premelanosome stain is negative with the presence of depigmentation agents,
melanogenesis is recognized as having been successfully inhibited [4, 5].
More dramatic effects of melanogenesis inhibition can be recognized when a
depigmentation agent is added to the water in which black goldfish are kept. The addition of
kojic acid required a month or two for the black goldfish to turn to yellowish brown; since
they were alive and vivid, this demonstrated that only melanogenesis was inhibited, not
systemic metabolism [Figure 5]. The chemical structures of main depigmentation agents are
shown in Table 4.

Figure 5. Black gold fish (rear as a control) changed color from black to brown, when Kojic acid was
added in the water at 0.25% for a month (front fish).
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420 Hideo Nakayama

Table 4. Chemical structures of main depigmentation agents

Clinical Evaluation of Depigmenting Agents

Depigmentation agents can be screened in vivo by tyrosinase inhibition tests or various
other methods that clearly demonstrate the inhibition of melanogenesis; however, what is
most important is that not only they show definite melanogenesis inhibition in vitro, but also
they improve the hyperpigmentation of melasma in clinical evaluation.

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 Acquired Skin Pigmentation 421

When there is no clinical effect of depigmentation, they are of course useless, even
though they showed excellent results in vitro trials. Laser is not effective to melasma, whereas
laser is very effective to solar lentigo and to nevus of Ota to which depigmentation agents are
less effective or ineffective. Therefore, the best of target for depigmentation agents is
apparently melasma.
First, for that purpose, depigmentation agents should be mixed in vehicles, normally
creams or lotions, without any alteration of color or effectiveness. They should be put into
production without producing impurities.
They should pass acute, subacute, and chronic toxicity tests, skin and eye irritation tests,
skin sensitization tests (maximization and similar tests), oncogenicity tests (Ames test,
micronuclei tests, carcinogenicity tests), teratogenicity tests, and stability tests.
These tests are all required to develop new drugs and likewise with depigmentation
agents. It is because depigmentation agents require several months to exhibit their effects and
consumers may use them for several months or even several years.
Double-blind clinical tests for melasma usually are not appropriate because it takes more
than 3 months for the effect to be recognized. Actually, depigmentation agents like kojic acid,
hydroquinone, and arbutin can improve the brown hyperpigmentation of melasma by
continual usage for 3-12 months. Theoretically it is possible to give active depigmentation
agents to one group while a second group is given a placebo cream for 3 to 12 months [1];
there should be no significant differences between the backgrounds of the melasma patients
as to age, severity, and sun exposure. It is ethically acceptable to use a placebo when another,
effective treatment is given.
However, when melasma patients are involved in the clinical trial, they have the right to
see improvement in a short period of time. Therefore, the long-term use of placebo cream was
abandoned because it apparently deceived patients who anticipated the effect. Double-blind
tests are all right when the test ends in a week or so (as with corticosteroid ointments or
antibiotics), especially when some other reliable basic treatment is given or the placebo is a
competing drug having a definite effect.
The evaluation of the treatment of pigmentary disorders of the face is not easy. With
melasma, the brown pigmentation fades so slowly that patients often do not recognize the
effects of depigmentation agents after 6 months of continual, twice-a-day application.
The best way to evaluate is to take color photographs of the faces of melasma patients
from three angles─front, 45°right, and 45° left. When the same camera, flashlight, and color
film are used, the effect of depigmentation agents can surely be recognized [1].
First the color of the melasma turns from brown to yellowish brown or normal skin color,
and second, the contrast at the border of the melasma becomes obscure. When colorimetry is
used, it is possible to recognize the change of tint, but when the place of measurement differs
at times of measurement, correct change of color is difficult to be obtained. Mapping the
human cheeks and forehead to determine the same spots at each time of measurement is
usually difficult.
On the other hand, pattern recognition using color photographs from the same three
angles of the face is much easier. When past color photographs from the same three angles of
the patient’s face are shown at the time of the revisit of the patient for evaluation, the effect of
whitening is easily recognized. At the very least classification (“cured, almost cured,
remarkably effective, effective, slightly effective, no effect, and exacerbation”) is possible.
Figures 1a and 1b illustrate such evaluations. Table 5 shows the clinical effect of 1% kojic
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422 Hideo Nakayama

acid cream on melasma patients studied in one clinic and one hospital in Tokyo. It showed an
excellent whitening ability as is demonstrated in Figure 1. Historically, betacyclodextrin was
once introduced to incorporate kojic acid to release it constantly. The device enhanced the
whitening effect, however, contact sensitization was seen, which was very rare when
betacyclodextrin was not used. Therefore, today, kojic acid is used at 0.3% concentration
without using a newly discovered adjuvant, betacyclodextrin.
The mixture usage of another whitening agent liquiritin at 0.15% showed again excellent
whitening effect without producing contact allergy to kojic acid, as is shown in Table 6. Such
whitening agent should be used till melasma disappears completely for one or two years [6,
7]. Strong sunlight should be avoided as possible by daily behavior, wearing a cap, holding
sun umbrella, and applying sunscreaning cream with SPF more than 20. These are necessary
when melasma patients enjoy mountaineering, golf, tennis and fishing.

Table 5. The Effect of 1% kojic acid cream II with an improved base cream on Melasma
Patients (1994)

Effect Cases treated Duration of treatment (Months, Mean) %

Complete cure 0 ─ 0.0
Remarkably improved 48 11.5
80.9
Improved 58 11.1
No effect 25 12.1 19.1
Worsened 0 ─ 0.0
Total 131 11.4 100.0
Side effect: Those who were contact sensitized by having previously used kojic acid cream containing
betacyclodextrin also developed erythema and itching by the usage of 1% kojic acid cream Ⅱ. The
rate of the dermatitis was 2 out of 131 patients in the table (1.5%). Those who had not used
betacyclodextrin-containing kojic acid cream had not produced contact dermatitis. 1% kojic acid
cream II did not contain betacyclodextrin.

Table 6. Mixture usage of whitening agents is possible. The effect of 0.45KL cream
showed the rate of effectiveness at 93.0% on melasma patients in the year 2012

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 Acquired Skin Pigmentation 423

2. PIGMENTATED CONTACT DERMATITIS

Asian people are easily pigmented when they suffer from allergic contact dermatitis, as
melanin is much more contained in the epidermis than caucasians. Hyperpigmentation after
skin inflammation is very clearly noted with mongoloids, compared to those who have
originally darker skin as Africans. Therefore, when pigmentation caused by dermatitis is clear
and persistent, it is a problem for Asian people.
Usually, hyperpigmentation after severe allergic contact dermatitis with brown
pigmentation is transient. The histopathology is increase in melamin in the basal layer of the
epidermis, and melanin incontatinence and accumulation in the upper dermis. Such acute
hyperpigmentation after dermatitis is often seen with the contact to primula obconica, textile
finishes such as Biocheck 60, formaldehyde, various dyes of black, blue and red.
On the other hand, when small amount of strong contact sensitizers contacts almost daily,
preceding allergic erythematous and itchy dermatitis is not prominent. Rather, such contact
sensitizers gradually produce brown or dark or blueish-grey hyperpigmentation, and such
pigmentary disorder lasts for a long time and is sometimes incurable by the application of
corticosteroid ointments. Such a disease has been described as pigmented contact dermatitis.
The first case was reported by Osmundsen in Denmark in 1969 [8, 9]. There was
hyperpigmentation of mostly covered areas, and patch test showed the patients were
sensitized by CH3566, an optical whitener in the washing powders they used. The optical
whitener, to make the shirts and underwear whiter than white, remained on the textile at very
small amounts even after washing, causing sensitized people to result in persistent
pigmentation.
The next pigmented contact dermatitis was reported by Ancona-Alayόn in Mexico. In a
textile factory, 12 among 53 workers suffered from this new spotted pigmentary disorder
without pruritus [10]. 8 mild cases were found. This disorder appeared 4 months after the
introduction of a new dyeing process of azo-coupling on textiles, and most of the patients had
contact with azo-dyes on weaving machines. The site of pigmentation was, in contrast to the
previously described first case in Denmark, mostly exposed areas, such as face, neck and
arms.
Patch tests showed that 24 of the 53 workers were positive to Naphthol AS 5% in water,
while the other 29, as well as 10 controls, were negative to Naphthol AS. The dermatoses
disappeared after the dyeing process was changed so that the workers did not directly touch
Naphthol AS, an azo dye coupling agent.
In the early 1980s, pigmented contact dermatitis due to Naphthol AS appeared in central
Japan, but this time it was not occupational. A textile factory manufacturing flannel
nightwear, a traditional Japanese garment called yukata, economized on water for washing the
products after the process of azo-coupling using Naphthol AS. This modification of
production resulted in the appearance of pigmented contact dermatitis of the covered areas of
skin of people living in the districts where the products were distributed and worn. Kawachi
et al. [11] and Hayakawa et al. [12] reported such cases, and the hyperpigmentation was
mainly located on the back and neck. The factory was said to have improved the washing
process and the materials quickly, but the presence of such cases indicates that whenever the
textile industry uses Naphthol AS, and at the same time economizes on water for washing the
products, there must be a risk of producing pigmented contact dermatitis of the covered areas.
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424 Hideo Nakayama

According to Hayakawa et al. [12] the amount of Naphthol AS detected in the patients’
nightwear was 4,900–8,700 ppm, a considerable amount. A case due to Naphthol AS in a
pillow case was later reported [13].
In 1984, the city of Tokyo decided to investigate new textile finishes which seemed to
have produced contact dermatitis of the covered skin areas, including pigmented contact
dermatitis [14, 15]. Based on information about the textile finishes which actually came into
contact with the patients’ skin or were very commonly used, 115 chemicals were finally
chosen and patch tested. The test materials included 50 dyes of all colors, 13 whiteners, 5
fungicides, 32 resin components, 13 softening agents, and 15 other miscellaneous textile
finishes which were widely used at that time by the textile industry in Japan. They were
chosen from approximately 1,200 textile finishes, either imported or produced in Japan. They
were checked as to solubility in water, ethanol, acetone, etc., diluted to 5% (except
bactericides, fungicides, and other pesticides for textiles which were diluted to 1%), and then
applied to dry paper discs 8 mm in diameter, to make dry allergen-containing discs named
“instant patch test allergens.” They were peeled off silicon-treated covering paper before use.
The results obtained from five hospitals in and around Tokyo revealed that several new
contact sensitizers were responsible for producing textile dermatitis and secondary
hyperpigmentation. These textile finishes included Biochek 60, a very toxic fungicide which
seemed also to have acted as a sensitizer, a phosphite polymer of pentaerythritol and
hydrogenated bisphenol A (PPP-HB), impurities in a dye CI Blue 19 (or Brilliant Blue R),
and mercury compounds [Figure 6].
The research on these 115 chemicals was performed in the 5 hospitals on 80–101
persons, among whom 51–62 were patients suffering from textile contact dermatitis, and the
rest, 29–39, were controls with atopic dermatitis and dermatitis due to causes other than
textiles. Among those with textile contact dermatitis, 27–33 had pigmented contact dermatitis.
Such cases had been deliberately chosen for patch testing because the investigators hoped to
find out the causative contact sensitizers producing such hyperpigmentation. Of these
pigmented contact dermatitis patients, 9 showed positive reactions suggestive of an allergy to
Biochek 60, and 1 to several textile finishes. The results were rather disappointing, but they
did show that it is not easy to discover the contact sensitizers producing pigmented contact
dermatitis from contact with textile finishes. The discoveries of CH3566 and Naphthol AS
can be regarded as having been important and valuable. Pigmented contact dermatitis due to
blue dyes, Blue 106 and 124 was reported by Kovacevic et al. in 2001 [16].

a b

Figure 6. (Continued).

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 Acquired Skin Pigmentation 425

Figure 6. a: Pigmented contact dermatitis on the trunk of a 55-year-old man. Itching was not severe,
however, diffuse spotted brown pigmentation was severe. He was later cured by the exclusive usage of
allergen controlled shirts (Allerion®) for 10 months. b: Histopathology of pigmented contact dermatitis
on the back due to B-60 shows mild acanthosis, remarkable incontinentia pigmenti histologica and
moderate infiltration of lymphocytes and histiocytes in the upper dermis. c: Patch test showed that he
was positively sensitized by Biochek60 (B-60), a bactericidal used very commonly on textiles in 1970s
and 1980s in Japan. The photograph shows a clear positive reaction to B-60 at 0.2% still remain on 15th
day of the patch test.

After a joint meeting among Japan Consumer Center, Safety Section of the City of
Tokyo, dermatologists and textile industry members, the usage of Biochek 60 and PPP-HB
was agreed to be abolished. It was said that several tons of Biochek 60 were still kept in a
warehouse at that time, and later it was believed that Biochek 60 has never been used in
Japan, however, the evidence of its destruction or disposal has never been reported.
Various textile finishes are secret even to the textile industry itself, therefore, textile
industry sells the products not knowing all the applied textile finishes on their merchandise.
This is why we still see pigmented contact dermatitis patients of the covered area occasionally
in the 21st Century. Milder cases have been called ashy dermatosis for a long time.

Treatment

Whenever we see acquired pigmentary disorders of the covered area or hands and arms,
we have to find out causative contact sensitizers of the disease. Apparent eczematous
dermatitis may not precede the pigmentation, according to the chemicals which were contact
sensitizers. The substances which come into contact to the pigmentation area should be asked,
and primula obconica, metals, perfumes, and rubbers are important. When pigmentation is
persistent in the covered areas, the species of the textile to contact the areas should be
investigated. In some rare cases, a dye for new swimming suits were the causative allergen.
Patch test is useful to find out the causative allergens, and a battery for textile contact
dermatitis should be prepared, and patch tested. When causative allergens are widely used by
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426 Hideo Nakayama

the textile industry without recognizing the hazard of contact sensitization, it is really a
disaster for patients. Such condition was present when Biochek 60 was widely used in Japan
in 1980s. The solution was that two textile companies produced set underwear using only 4
textile finishes which showed patch test negative results with 47 textile dermatitis patients.
Clinical evaluation test was carried out, and it showed that 36 out of 47 textile dermatitis
patients cleared their relapsing itchy dermatitis during the exclusive usage of this allergen
controlled underwear [17]. These quite safe underwear have been sold in Japan by the
commercial names, Allerion® (Shikibo Company) and E-earth® (Wacoal Company). With the
latter, the metal line of the lower margin of brassiere was eliminated in addition to the
elimination of common and rare sensitizers from the textile itself, and it was apparently a
good device to stop the recurrent dermatitis, when the patients were hypersensitive to nickel
or cobalt. It is ideal that such allergen controlled wearing apparel is produced and be available
in many countries [18].

3. PIGMENTED COSMETIC DERMATITIS

The most commonly seen hyperpigmentation due to contact dermatitis in the history of
dermatology must be the pigmented cosmetic dermatitis which affected the faces of Oriental
women [19, 20, 21].
Innumerable patients with this pigmentary disorder presented in the 1960s and 1970s in
Japan, and similar patients were also seen in Korea, India, Taiwan, China, and the US.
The signs of pigmented cosmetic dermatitis are diffuse or reticular (Figure 7, 8), black or
dark brown hyperpigmentation of the face, which cannot be cured by laser or the use of
corticosteroid ointments or the continuous ingestion of vitamin C or corticosteroids. The
border of pigmented cosmetic dermatitis is not sharp, as in lichen planus or melasma, and it is
not spot-like as in nevus of Ota tardus bilateralis.
Slight dermatitis is occasionally seen with hyperpigmentation, or dermatitis may precede
hyperpigmentation. In contrast to Addison’s disease, pigmented cosmetic dermatitis does not
show any systemic symptoms such as weakness, fatigue, and emaciation. Laboratory findings
such as full blood count, liver function tests, daily urinary excretion of 17-ketosteroid and 17-
hydroxy corticosteroid, and serum immunoglobulins and electrolytes are normal in the
majority of patients with pigmented cosmetic dermatitis [21].
Histopathological examination of pigmented cosmetic dermatitis shows basal liquefaction
degeneration of the epidermis and incontinentia pigmenti histologica.
The epidermis maybe mildly acanthotic, however it is sometimes atrophic, presumably
the effect of frequently applied corticosteroid ointments for the treatment of itchy dermatitis
of the face. Cellular infiltrates of lymphocytes and histiocytes are seen perivascularly, as are
often seen in ordinary allergic contact dermatitis [Figure 9c].
In some cases, the dark brown or black hyperpigmentation is also seen on skin other than
on the face. The neck, chest, and back can be involved and, in a few exceptional cases,
hyperpigmentation may extend to the whole body. In these cases, the allergens cinnamic
alcohol and its derivatives sensitize the patients first to cosmetics and then provoke allergic
reactions to soaps, domestic fabric softeners, and food, all of which sometimes contain
cinnamic derivatives.

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 Acquired Skin Pigmentation 427

The ingestion of 1 g cinnamon sugar from a cup of tea in a supermarket was enough to
provoke a mild focal flare of dermatitis at the sites of diffuse reticular black
hyperpigmentation of the whole body in one reported case [22].

a b

Figure 7. a: Diffuse brown and black hyperpigmentation of pigmented cosmetic dermatitis in a 41-year-
old woman. The causation was unknown, and it was regarded as incurable, when this photograph was
taken. b: A project to discover new allergens to produce this disease started in 1969, demonstrated
contact allergy to patchouli oil (No.7), benzyl salicylate (No.16), jasmin absolute (No.17) etc. with this
case by patch testing cosmetic series allergens. c: She was recommended to use allergen controlled
soaps and cosmetics (Acseine®) exclusively. By this allergen elimination, she recovered normal skin
color, and it was maintained till 8 years later. This case was the worlds’ first case who was cured by
allergen control.
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428 Hideo Nakayama

a c

Figure 8. a: Reticular hyperpigmentation of pigmented cosmetic dermatitis in a 33-year-old woman.

The disease could not have been cured in the past three years. b: Patch test showed that she was
strongly sensitized to cinnamic alcohol, cinnamic aldehyde, cinnamic acetate and cinnamal cinnamate,
and these were all cinnamic derivatives. This photograph shows such strong positive reactions still
observed on 14th day of the patch test. These reactions turned to small dark spots on the 50 th day of the
patch test. c: The exclusive usage of allergen controlled soap and cosmetics led the pigmented skin to
normal skin one year and half later. Normal skin was maintained 3 years later as is shown in this
photograph.

When one of the common potent sensitizers producing pigmented cosmetic dermatitis, D
and C Red 31 (Japanese name R-219), was discovered, a focal flare of dermatitis at the site of
facial hyperpigmentation was occasionally noted by patch testing 5% R-219 in petrolatum.
These findings show that the allergen could provoke the dermatitis not only by contact
with the skin surface but also from within the skin, by allergens transported via blood vessels,
just as allergic contact dermatitis can be provoked by the administration of small amounts of
nickel or drugs.

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 Acquired Skin Pigmentation 429

Causative Allergens

The term “pigmented cosmetic dermatitis” was introduced in 1973 for what had
previously been known as melanosis faciei feminae when the mechanism (type IV allergy),
most of the causative allergens, and successful treatment with allergen control for this
miserable pigmentary disorder were clarified for the first time [20, 21]. The name was
adopted by modifying Osmundsen’s designation, pigmented contact dermatitis, which was
caused by CH3566 on the trunk.
Historically, the first description of the disease goes back to 1948, when Japanese
dermatologists encountered this peculiar pigmentary disorder for the first time, and were
greatly embarrassed as to diagnosis. Bibliographical surveys showed that Riehl’s melanosis,
described in 1917 [23], seemed probable, because World War II had ended just 3 years before
the investigation.
Subsequently, the disease was erroneously called Riehl’s melanosis for almost 30 years
in Asian countries. Riehl’s melanosis, however, was a dark brown hyperpigmentation
observed during World War I in Caucasian men, women and children, when food was
extremely scarce and the patients had to eat decayed corn and weed crops instead of the
normal food of peacetime. Besides hyperpigmentation of the face, ears and scalp, there were
nodules and, histopathologically, dense cellular infiltration was present in the dermis.
Cosmetics could be excluded as a cause, because it was during World War I, and it was
not possible for all these people, especially the men and children, to have used cosmetics
before they had the disease. Riehl could not discover the true cause of this pigmentary
disorder, but suspected the role of the abnormal wartime diet [23]. Riehl’s melanosis
disappeared when World War I ended, when people obtained normal food again, to reappear
for a short period in France during the German occupation in World War II, when food again
became scarce.
Consequently, Riehl’s melanosis, a wartime melanosis, having no relationship to
cosmetic allergy, should not be confused with pigmented cosmetic dermatitis, which involved
many Asian women in peacetime for many years. In 1950, Minami and Noma [24] designated
the disease melanosis faciei feminae, and recognized the disease as a new entity. The
causation was not known for many years.
However, Japanese dermatologists gradually became aware of the role of cosmetics in
this hyperpigmentation. First, it occurred only on those women, and very exceptionally men,
who used cosmetics and, secondly, even though the bizarre brown hyperpigmentation was so
conspicuous, the presence of slight, recurrent, or preceding dermatitis was observed. The
problem for the dermatologists at that time was that the components of cosmetics were
completely secret, and the kinds of cosmetic ingredients were too many (more than 1,000) for
their allergenicity to be evaluated.
Finally, in 1969, a research project was set up to identify the causative allergens from 477
cosmetic ingredients by patch and photopatch testing [19]. It was a new idea, because
melanosis faciei feminae had been regarded as a metabolic disorder rather than a type of
contact dermatitis. This was 7 years before Finn chambers became available; therefore, small
patch test plasters of 10.2 cm with six discs 7 mm in diameter (Miniplaster) were put into
production to enable 48–96 samples to be patch tested at one time on the backs of volunteer
control subjects and patients.
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430 Hideo Nakayama

Many cosmetic ingredients, adjusted to nonirritant concentrations with the cooperation of

30–40 volunteers, were subsequently patch and photopatch tested on the patients. Fragrances
were selected from 418 fragrant materials, judging from the much quantity of the production
and imports of the previous year.
Results for each ingredient were obtained from 172–348 patients, including 79–121 with
melanosis faciei feminae. Statistical evaluation brought to light a number of newly discovered
contact sensitizers amongst the cosmetic ingredients, mainly fragrant materials and pigments,
including jasmine absolute, ylang-ylang oil, cananga oil, benzyl salicylate, hydroxycitronellal,
sandalwood oil, artificial sandalwood, geraniol, geranium oil, D and C Red 31, and Yellow
No. 11 [14, 19, 20, 21].
Other rare causations of pigmented cosmetic dermatitis include fragrances, musk
ambrette [25], musk moskene [26] and diisostearyl maleate in the lipsticks.

Treatment

The above-mentioned research project at the same time included a plan to produce soaps
(acylglutamate) and cosmetics for the patients from which the causative allergens had been
completely eliminated, as even those who suffered from severe and bizarre
hyperpigmentation usually could not accept abandoning their use of cosmetics to remove this
pigmentary disorder. Patch testing with a series of 30 standard cosmetic ingredients to find
the allergens causing the disease [20, 21, 27], followed by the exclusive use of soaps and
cosmetics that were completely allergen-free for such patients, designated the allergen control
system, produced dramatic effects. Around 1970, most textbooks of dermatology in Japan
said that melanosis faciei feminae was very difficult to cure and that the causation was
unknown.
However, after allergen control was introduced, the disease became completely curable.
Table 7 shows the effect of allergen control in 165 cases reported to the American Academy
of Dermatology in 1977, and also the long-term follow-up results of allergen control obtained
by Watanabe after 3–11 years (mean, 5 years) [28]. In 50 cases of pigmented cosmetic
dermatitis cured by allergen control (i.e., patch test with 30 cosmetic series patch test
allergens [20] followed by the exclusive use of allergen-free soaps and cosmetics, Acseine®
in Japan and Hong Kong), there were, on average, 2.5 allergens for each patient. It usually
required 1–2 years for a patient to regain normal nonhyperpigmented facial skin [Figure 7, 8].
Contamination with ordinary soaps and cosmetics was the most influential and decisive factor
inhibiting therapy, because such ordinary daily necessities contained the allergens that were
producing the disease. The patients were therefore requested to visit the dermatologist once a
month to be checked for improvement, and were persuaded every time to avoid such
contamination, including products used in beauty parlors [21, 28].
In 1979, Kozuka [29] discovered a new contact sensitizer, phenyl-azo-2-naphthol (PAN),
as an impurity in commercial supplies of D and C Red 31. Its sensitizing ability and ability to
produce secondary hyperpigmentation were as great as those of Yellow No. 11, and therefore
many industries began to eliminate or considerably decrease the amount of PAN and Yellow
No. 11 in their products.
The legal partial restriction of Red No. 31 and Yellow No. 11 by the Japanese
government and the voluntary restriction by cosmetic companies of the use of allergenic

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fragrances, bactericides, and pigments resulted in a remarkable decrease in pigmented

cosmetic dermatitis after 1980.

Table 7. Effect of allergen-controlled cosmetics on pigmented

cosmetic dermatitis patients

Nakayama 1977 Watanabe 1989 [28]

Total 165 53
Complete cure 52 40
Almost complete cure 21 0
Remarkable improvement 51 13
Improvement 22 0
Not Effective 19 0
3-11 years
Follow-up 3 months to 5 years
(mean 5 years)

One of the reasons for the proposal to change the name from “melanosis faciei feminae”
to “pigmented cosmetic dermatitis” [19] was that the latter name makes it easier for the
patients to understand the causation of the disease and, at the same time, for industry to
recognize the danger of cosmetics in producing such disastrous pigmentary disorders through
contact sensitization.
The discovery of various potent contact sensitizers to produce pigmented cosmetic
dermatitis in 1970s and 1980s was valuable, since owing to the cooperation of industries to
decrease claims on cosmetics to eliminate main responsible allergens, the positive rates of
common cosmetic sensitizers decreased to one fifth then to one tenth in the last 20 years of
the 20th Century [30]. Pigmented cosmetic dermatitis was a common disease in the outpatient
clinics in Japan in 1960s and 1970s, however, it became a rare disease at the end of 20th
Century, and first decade of 21st Century.
From the year 2010, however, pigmented cosmetic dermatitis patients started to reappear
[Figure 9]. Of course these were all new patients, and patch test revealed that some of them
again exhibited positive reaction to phenyl-azo-naphthol, benzyl salicylate, other fragrances
and yellow No.10, which contained notorious yellow No.11 at 20% at the maximum. Again
the allergen controlled cosmetics, Acseine® cosmetics, and acylglutamate soap, which had all
eliminated common strong cosmetic sensitizers described previously, were useful to cure the
disease. It turned out that recent technicians of cosmetic industries do not know what are the
dangerous ingredients to produce pigmented cosmetic dermatitis since all the skillful
technicians who made efforts to eliminate common sensitizers in the 20th Century retired, and
information to avoid such allergens was not inherited to the next generation in many cosmetic
companies.
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432 Hideo Nakayama

a b

c d

Figure 9. a: In 2012, a nightmare revived. Diffuse severe pigmented cosmetic dermatitis had not been
seen since 1985, however, a 72-year-old woman was seen after a long interval. b: Patch test showed she
was positively sensitized to phenyl-azo-naphthol (PAN). c: Biopsy of the face showed liquefaction
degeneration of the basal layer of the epidermis, melanin incontinence at the upper section of the
dermis, and perivascular mononuclear cell infiltrates in the dermis. d: Again she was requested to use
allergen controlled soaps and cosmetics exclusively, and 10 months later, her facial pigmentation was
observed to have much improved.

Thus, history is repeating itself. Therefore, at the beginning of the 21st Century, the
dermatologists should know what is pigmented cosmetic dermatitis, what are its causative
allergens, what should be patch tested to find out the causative allergens [Table 8], and how
the treatment of allergen control should be performed, for the patients to recover the normal
facial skin color without bizarre hyperpigmentation. By law, only Red 31 and Yellow No.11
were banned, and it is not sufficient to exterminate pigmented cosmetic dermatitis. The
education on cosmetic contact allergens to the technicians of cosmetic industries will be
necessary forever, to prevent ordinary cosmetic dermatitis and pigmented cosmetic dermatitis.

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Table 8. Chemical structures of contact allergens to produce pigmentation

4. PIGMENTED PURPURIC LICHENOID DERMATITIS

In 1886 Majocchi described purpura annularis telangiectodes and, 4 years later,
Schamberg described a progressive pigmentary dermatitis which is now well known as
Schamberg’s disease.
The pigmentation in this dermatitis is due to the intradermal accumulation of
hemosiderin, the predominant sites being the legs and thighs. Later, Gougerot and Blum
described a similar dermatosis as pigmented purpuric lichenoid dermatitis.
The disease was rare but most often occurred in middle-aged or elderly men. However,
when a similar disease occurred in many British soldiers during World War II, especially in
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434 Hideo Nakayama

those who sweated freely or experienced friction when wearing khaki shirts or woolen socks,
with severe pruritus, dermatitis and pigmentation due to purpura, dermatologists became
aware that some textile finishes must have been responsible for the disease [31, 32]. Patch
tests and use tests revealed that a blend of vegetable oils and oleic acid seemed to have been
responsible.
In 1968, Batschvarov and Minkov [33] reported that rubber components such as N-
phenyl-N´-isopropyl-p-phenylenediamine (IPPD), N-phenyl-b-naphthylamine (PNA), 2-
mercaptobenzothiazole (MBT) and dibenzothiazole disulfide (DBD), i.e., derivatives of p-
phenylenediamine, naphthylamine, and benzothiazoles, were the allergens responsible for a
purpuric dermatitis around the waist underneath the elastic of underwear [Table 9].

Table 9. Causation of pigmented contact dermatitis, pigmented purpuric lichenoid

dermatitis and dirty neck

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 Acquired Skin Pigmentation 435

A similar pigmented dermatitis was recognized in the shoulders, breasts, groins, and
thighs. The capillary resistance (Rumpel-Leede) test was positive in all 23 cases studied.
Similar test results were obtained in a smaller proportion of patients with the khaki dermatitis
mentioned above.
In Bulgaria, over 600 patients were recorded, and the necessity for dermatologists to
investigate contact allergens in textiles to solve the problem of purpuric dermatitis of covered
areas of skin was stressed [33]. A dye, blue 85, was reported as a causation in 1988 [34]. A
case due to a textile finish of socks is demonstrated [Figure 10]

Causation and Treatment

Allergic contact dermatitis is a very itchy disease, therefore, one causation of marked
microscopical hemorrhage must be scratching. In atopic dermatitis, however, with severe
cases, itching is tremendous, and still such microscopical hemosiderin retention in the
upperdermis is rare.

a b

Figure 10. a: Reticular brown hyperpigmentation of pigmented purpuric lichenoid dermatitis on an 80-
year-old male. Biopsy showed marked hemorrhage around capillaries of the upper dermis, along with
the cellular infiltrates composed of lymphocytes and histiocytes. b: Patch test revealed strong contact
hypersensitivity to paratertiarybutyl phenolformaldehyde resin at 1% petrolatum (No.8). It had been
positive from D2 to D14 and confirmative patch test was again strongly positive. Exposure to the
contact allergen was considered to have been from the textile finishes of his socks. The exclusive usage
of well-washed white cotton socks gradually improved the dermatitis. Complete blood count (CBC) and
liver function test results were normal. This case indicates the importance of patch test of textile
finishes, if available, for the treatment of this pigmentary disorder.

Considering the predominant site of purpuric dermatitis, which is known under several
names historically, the gravity is suspected to produce microscopical hemorrhage when
lymphocytes and histiocytes come out from the capillaries at the sites of contact allergic
reactions.
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436 Hideo Nakayama

As this purpuric pigmented dermatitis is considered to be ordinary allergic contact

dermatitis plus microscopical hemorrhage from capillaries, in treating the disease, patch test
is needed to find out the causative allergens as possible, along with the usage of
antisymptomatic treatment composed of corticosteroid ointments and antihistamins. Socks
should be changed to white cotton socks having no rubber strings, considering the reported
cases were occasionally sensitized by dyes or rubber components. At the upper parts of socks,
elastic components are necessary, therefore, when rubber strings are avoided, usage of
spandex, non rubber elastic filaments, is allowed.

5. DIRTY NECK OF ATOPIC DERMATITIS

Atopic dermatitis is a multifactorial itchy dermatitis, and when severe cases are treated
with antisymptomatic treatment only, reticular or diffuse brown or black hyperpigmentation
often appears around the neck, and it is very difficult to cure [35]. Such pigmentation has
been called “dirty neck,” and histopathology usually shows slight acanthosis, lymphocyte and
histiocyte infiltration around the vessels in the upper dermis with incontinentia pigmenti
histologica [14]. Congo red stain does not show the clear presence of amyloid, however, very
small amount of amyloid was detected by electron microscopy [36].
Increase in serum IgE is a characteristic abnormality found at 70 to 80% of severe cases
of atopic dermatitis. The average serum IgE of severe 83 cases of atopic dermatitis was 2,947
IU/ml, while average serum IgE levels of 18 asthma bronchiale, 53 allergic rhinitis, 54
urticaria patients were 336, 323, 350 IU/ml respectively. Such remarkable increase in serum
IgE was analyzed by RAST to have shown that 75 to 87% of elevated IgE was due to house
dust mite allergy. This fact means that dermatophagoides pteronyssinus (Dp) and
dermatophagoides farinae (Df), in average 89% of all house dust mites, are considered to be
the main causation of the increase in serum IgE with severe atopic dermatitis patients [37,
38]. Today, IgE is known to be present on the surface of epidermal dendrinc cells to provoke
eczematous allergic reactions [39, 40], therefore, Dp and Df are considered to be most
important causative allergens in producing and maintaining atopic eczema and secondary
hyperpigmentation. In addition, when live mites were patch tested on atopic dermatitis
patients, clear eczematous positive reactions appeared, which were negative on controls [41,
42]. The analysis of mite body components revealed the presence of primary contact
sensitizer, α-acaridial, from one of house dust mites, Tyrophagus putrescentiae (Tp) [38].
Today α-acaridial is refrained from patch testing, as it turned out to be a strong primary
sensitizer, however, when it was patch tested at 0.3% in petrolatum, it easily produced prurigo
Besnier which lasted for a few months when positive reactions appeared [38, 43]. These are
all excellent evidences that house dust mite allergy is the most important causation of severe
atopic dermatitis including “dirty neck.”
The treatment of “dirty neck” is not easy. When the mite fauna were investigated by a
new methylene blue agar method in the homes of atopic dermatitis patients, and
environmental improvements were made to decrease the mite numbers to fewer than 20/m2 at
20 second aspiration using a 320-W cleaner, 88% of severe atopic dermatitis patients showed
considerable improvement in their severe dermatitis when they were followed up for 1–2
years [38, 44]. The statistically significant effect of house dust mite elimination in atopic

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 Acquired Skin Pigmentation 437

dermatitis with controls was also reported by Tan et al. [45]. The “dirty neck,” however, was
difficult to cure even with this method, and it can be regarded as the last symptom to improve
for atopic dermatitis. The combination therapy of environmental improvement based on the
the mite fauna investigation, and a long-term application of depigmenting agents has been
most effective to “dirty neck” [14] [Figure 11].

a b

Figure 11. a: So-called “dirty neck” of a 30-year-old atopic dermatitis patient. As she was strongly
hypersensitive to house dust mites, mite fauna was investigated, and environmental improvement was
performed. b: Along with this allergen elimination, a whitening agent, 1% ellagic acid cream was
applied on the dark neck skin twice a day. One year and three month later, the pigmentation was
observed to have much improved.

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[32] Twiston Davies, J. H., Neish Barker, A. (1944) Textile dermatitis. Br. J. Dermatol. 56:
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[33] Batschvarov, B., Minkov, D. M. (1968) Dermatitis and purpura from rubber in clothing.
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[34] Van der Veen, J. P. W., Neering, H., DeHaan, P., et al. (1988) Pigmented purpuric
clothing dermatitis due to Disperse Blue 85. Contact Dermatitis 19: 222–223
[35] Manabe, T., Inagaki, Y., Nakagawa, S., et al. (1987) Ripple pigmentation of the neck in
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[36] Humphreys, F., Spencer, J., McLaren, K., Tidman, M. J. (1996) An histological and
ultrastructural study of the dirty neck appearance in atopic eczema. Clin. Exp.
Dermatol. 21: 17–19
[37] Nakayama, H. (1995) The role of the house dust mite in atopic eczema. Practical
contact dermatitis. McGraw-Hill, New York, pp 623–630
[38] Nakayama, H., Kumei, A. (2003) House dust mite – an important causation of atopic
dermatitis. SP World 31: 13–20
[39] Bruynzeel-Koomen, C., VanWichen, D. F., Toonstra, J., et al. (1986) The presence of
IgE molecules on epidermal Langerhans cells in patients with atopic dermatitis, Arch.
Dermatol. Res. 278: 199-205
[40] Novak, N., Bieber, T., Kraft, S. (2004) Immunoglobulin E-bearing antigen-presenting
cells in atopic dermatitis, Curr. Allergy Asthma. Rep. 4: 263-269
[41] Vincenti, C., Trevisi, P., Guerra, L., Lorenzi, S., Tosti, A. (1994) Patch testing with
whole dust mite bodies in atopic dermatitis. Am. J. Contact Dermatitis 5: 213–215
[42] Sakurai, M. (1996) Results of patch tests with mite components in atopic dermatitis
patients (in Japanese with English abstract). Allergy 45: 398–408
[43] Nakayama, H., Kumei, A. (2008) Importance of Mite Allergy in Atopic Dermatitis,
Skin Research Suppl. 10: 16-23
[44] Kumei, A. (1995) Investigation of mites in the house of atopic dermatitis (AD) patients,
and clinical improvements by mite elimination (in Japanese with English abstract).
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[45] Tan, B. B., Weald, D., Strickland, I., Friedmann, P. S. (1996) Double-blind controlled
trial of effect of housedust-mite allergen avoidance on atopic dermatitis. Lancet 347:
15–18
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 16

THE PRO-OPIOMELANOCORTIN (POMC)

AND MELANOCORTIN SYSTEM IN REGULATION
OF HUMAN SKIN PIGMENTATION

Han-En Tsai1, Elsa C Chan2, Gregory J. Dusting2,3

and Guei-Sheung Liu2,3,
1
Institute of Biomedical Science, National Sun Yat-sen University, Taiwan
2
Centre for Eye Research Australia
3
Department of Ophthalmology, University of Melbourne, Australia

ABSTRACT
Pro-opiomelanocortin (POMC) is a precursor protein which produces many
biologically active peptides through a series of enzymatic steps in a tissue-specific
manner, including melanocyte-stimulating hormones (MSH), corticotrophin (ACTH) and
β-endorphin.
Melanocyte stimulating hormones such as α, β and γ-MSH are encoded by the
POMC gene which is known to be one of the main regulators of skin pigmentation. MSH
exert their effects by activating melanocortin receptors (MCRs), the smallest family
members of the seven transmembrane G-protein-coupled receptors (GPCR), of which
there are five MCR subtypes. Accumulating evidence suggests mutations in the POMC
gene could lead to fair skin and red hair in humans. Further, the melanocortin receptor-1
(MC-1R) gene is highly polymorphic in human populations, and allelic variations have
been associated with hair and skin color phenotypes, freckles, and a risk of melanoma
and non-melanoma skin cancers.
In this chapter, we will briefly describe the roles of POMC and melanocortin system
in pigmentation.


Correspondence to: Guei-Sheung Liu, Ph. D. Centre for Eye Research Australia and Department of
Ophthalmology, University of Melbourne, Level 1, 32 Gisborne Street, East Melbourne, VIC 3002, Australia,
Tel : 61-3-99298488 ; Fax: 61-3-9662 3859, Email: [email protected]
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442 Han-En Tsai, Elsa C Chan, Gregory J. Dusting et al.

ABBREVIATIONS
(ACTH) Adrenocorticotropic hormone;
(AGRP) Agouti-related protein;
(CREB) cAMP response element binding protein;
(CRE) cAMP response element;
(CRH) Corticotropin-releasing hormone;
(CLIP) Corticotrophin-like intermediate lobe peptide;
(GPCR) G-protein couple seven transmembrane receptor;
(DCT) Dopachrome tautomerase;
(β-EP) β-endorphin;
(FGD) Familial glucocorticoid deficiency;
(LH) Lateral hypothalamic area;
(LPH) Lipotrophin;
(MSH) Melanocyte stimulating hormone;
(MCRs) Melanocortin receptors;
(MITF) Microphthalmia-associated transcription factor;
(Met-ENK) Met-enkephalin;
(MT IandII) Melanotan IandII;
(POMC) Proopiomelanocortin;
(PC) Prohormone convertase;
(TYR) Tyrosinase;
(TYRP1) Tyrosinase-related protein 1;
(TEM) Transmission electron microscopy ;

INTRODUCTION
The existence of a precursor molecule for adrenocorticotropin, melanotropins and
analgesic peptides such as β-endorphin, designated ‘pro-opiomelanocortin’ (POMC), was
confirmed in 1979 by the cloning of bovine POMC.
The physiological significance of POMC-derived peptides has been known since 1950s,
when the effects of purified ACTH on adrenal function were first recognized. Apart from the
regulation of adrenal function, POMC-derived peptides have also been shown to possess
other pleiotropic functions, including regulation of energy homeostasis, and modulation of
immunity, particularly in skin pigmentation.
The importance of the POMC-melanocortin systems in skin and hair pigmentation is
evidenced clinically in patients with POMC gene mutations, who exhibit pale skin and red
hair in addition to early-onset obesity and adrenal insufficiency, as well as melanocortin
receptor mutations. In addition, some patients with Addison’s disease and Nelson’s
syndrome, where excessive concentrations of POMC are detectable in the circulation, have
marked hyperpigmentation. In this chapter, we will briefly review the roles of POMC and
melanocortin system in pigmentations.

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PRO-OPIOMELANOCORTIN (POMC)
Melanocortins are ancient peptides that are conserved evolutionarily, and can be traced
back to the appearance of the first vertebrates [1]. They are derived from a larger precursor
molecule known as the POMC protein, first identified in common ancestors of lampreys and
gnathostomes about 700 million year ago [2]. POMC is a multifunctional poly-cistronic gene
located on human chromosome 2p23 and it was found in 1979 by the cloning of bovine
POMC cDNA [3, 4]. POMC is a 31 kDa prohormone of various neuropeptides including
adrenocorticotrophic hormone (ACTH), melanotrophins (α-, β- and γ-melanocyte-stimulating
hormone (MSH)), lipotropins and β-endorphin (Figure 1). POMC has been detected in the
hypothalamus, pituitary and periphery including the immune system, spleen, lungs,
melanocytes and the gastrointestinal tract [5]. Post-translational processing of the POMC
protein ultimately leads to the generation of melanocortins, β, γ-lipotrophin (LPH) and β-
endorphin (β-EP) [1].
The melanocortins include ACTH, α-melanocyte-stimulating hormone (α-MSH), the N-
terminal proteolytic cleavage fragment of ACTH, as well as other melanocortins not directly
derived from ACTH (ie. β-MSH and γ-MSH) [6]. The enzymes that regulate expression of
these neuropeptides are prohormone convertases 1 and 2 (PC1 and PC2), which belong to an
evolutionary conserved family of serine proteinases of the subtilisin/kexin type [7].
Melanocortins are polypeptide hormones and neuropeptides that all share the core sequence
HFRW, which is a key pharmacophore necessary for receptor binding of all melanocortins
[8].
Coll et al. showed how POMC regulate these aspects by generating an independent line
of mice lacking all POMC-derived peptides (Pomc-/-) [9]. Pomc-/- mice are obese and show
enhance food intake, along with altered pigmentation and adrenal insufficiency. These mice
also showed a significantly higher amount of fat and lean tissues when compared to the age-
matched wildtypes. Additionally, the basal oxygen consumption, as an index of metabolic
rate was 23% less in Pomc-/- mice than those of wildtype mice when corrected for body
weight, and total plasma T4 [9].

Figure 1. Processing of the POMC precursor yields various bioactive peptides which regulate biological
function by mediating different melanocortin receptor subtypes MC-1R to MC-5R.
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444 Han-En Tsai, Elsa C Chan, Gregory J. Dusting et al.

MELANOCORTIN RECEPTORS (MC-RS)

The melanocortin pathway has been implicated in the regulation of diverse physiological
functions including obesity, inflammation, sexual function, pigmentation, cardiovascular
function, and steroidogenesis [10]. The melanocortins exert their effects by activating the
seven transmembrane G-protein-coupled receptor (GPCR) melanocortin receptors (MC-Rs).
MC-R is the smallest family of GPCR because it has a short second extracellular loop and
short amino- and carboxy- terminal ends [11]. Five melanocortin receptor subtypes (MC-1R
to MC-5R) have been identified [12, 13] and they are highly conserved in mammals [14]. All
MC-Rs are functionally coupled to adenylyl cyclase and their effects are mediated primarily
by activating a cAMP-dependent signaling pathway. These receptors increase intracellular
cAMP upon stimulation by melanocortins [15]. Different forms of MSH have different
binding affinity for MC-Rs. -MSH has the highest binding affinity for MC-1R and MC-5R
over ACTH, β-MSH and γ-MSH while β-MSH has the highest affinity for MC-4R when
compared to the  and γ forms. MC-Rs are widely distributed in peripheral tissues including
melanocytes, adipocytes and adrenal glands and the brain [12] (Table 1). Such a wide
distribution pattern of MC-Rs reflects the diverse biological actions of the melanocortin
system. We have summarized the phenotypes of mice with a deletion of POMC or any of the
MC-R genes to highlight functional roles of the melanocortin system (Table 2).
MC-1R is the only subtype found in melanocytes and is involved in the regulation of skin
and hair pigmentation [16]. MC-1R is also expressed in other tissues and cells including
macrophages and glial cells, where it mediates inflammatory responses via an endogenous
circuit involving α-MSH [17]. So far it has been shown that a functional MC-1R is not
required for survival as mice with a non-functional MC-1R due to a frameshift mutation
between transmembrane domains IV and V. The mice with frameshift mutation of MC1-R
appear to have a normal phenotype, despite having a yellow coat which highlights a role of
MC-1R in the regulation of coat colour phenotype [18, 19]. However, MC-1R mutant mice
having a frameshift mutation showed a significant aggravation of inflammation [20, 21]. The
specific allelic variants of the MC-1R gene might also be associated with a higher risk of
developing inflammatory bowel disease, especially intriguing as the MC-1R gene is located
on human chromosome 16, the same chromosome which a definitive susceptibility gene for
Crohn’s disease, namely the NOD2/CARD15 gene [20]. However, the role of MC-1R in
melanocortin-mediated functional regulation still needs to be clarified.

Table 1. The affinity and distribution of melanocortin receptors

for POMC-derived peptides

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Table 2. Phenotypes with a deletion in POMC gene or any of MC-Rs gene

MC-2R has a major role in the postnatal development of adrenal glands and is involved
in the stimulatory effect of ACTH on the biosynthesis and secretion of glucocorticoids from
the adrenal gland [22]. Mice lacking MC-2R were born normal but three quarters of newborns
did not survive to the age of weaning. Hypoglycemia appears to contribute to neonatal
mortality because blood glucose levels and expression of liver enzymes for gluconeogenesis
were remarkably low compared to wildtypes, suggesting an impaired utilization of glucose
for nutritional needs by the neonates [22]. On the other hand, MC-2R knockout mice
surviving to adulthood exhibited macroscopic abnormalies in the adrenal glands with
atrophied zona fasciculate but fairly normal zona glomerulosa and medulla. The zona
fasciculate showed microscopic defects including inactive mitochondria and reduced lipid
droplets in their cells. Surviving adult MC-2R knockout mice also had undetectable basal
levels of corticosterone despite high levels of ACTH and had no hormonal responses
following injections of ACTH [22]. Interestingly, microscopic examination of adrenal glands
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446 Han-En Tsai, Elsa C Chan, Gregory J. Dusting et al.

of newborns from MC-2R knockouts and wildtypes were similar, suggesting that MC-2R was
involved in the development and steroidogenesis of adrenal glands during the postnatal stages
rather than the embryonic stages. The phenotypes of MC-2R knockouts resemble some of the
characteristics of patients suffering from familial glucocorticoid deficiency (FGD) with
adrenal insufficiency, of which 25% showed mutations in MC-2R. Therefore MC-2R
knockout mice may provide a useful animal model to study FGD for developing better
treatment strategies [22].
MC-3R is expressed in the brains, placenta, pancreas and the gastrointestinal tract.
Previous studies demonstrated that homozygous-null MC-3R (Mc3r-/-) mice have a unique
phenotype, in that although they are not significantly overweight, they have an increase in fat
mass and a reduction in lean mass compared with WT mice. Two groups also reported a
reduction in the body length of Mc3r-/-mice [23, 24]. Activation of MC-3R on B lymphocytes
has also recently been shown to mediate the inhibitory effect of α-MSH on antigen-induced
lymphocyte proliferation in humans [25], highlighting a role of MC-3R in inflammation and
immunity.
MC-4R is mainly found in the CNS and muscles. Using homologous recombination to
delete the coding sequence of Mc4r, Huszar and colleagues generated a mouse that developed
a maturity-onset obesity syndrome associated with increased food intake, hyperinsulinemia
and hyperglycemia [26]. Mc4r-/- had similar basal corticosterone levels, but showed an
increase in linear growth compared with wildtypes [26]. In addition, mice heterozygous for
the Mc4r-null mutation had an intermediate phenotype between wildtype and Mc4r-/- for body
weight, linear growth, and insulin, strongly suggestive of a gene dosage effect [27]. Clinical
data also indicates that the mutation in MC-4R associated with obesity in human is consistent
with previous finding in MC-4R knockout mice [28-30].
Finally, MC-5R is widely distributed in peripheral tissues including spleen, adrenals,
sexual organs, muscles and the brain [31]. MC-5R knockout mice show gland dysfunctions
with decreased sebaceous lipid production [29, 32].

ROLE OF THE MELANOCORTIN SYSTEM IN SKIN PIGMENTATION

Human skin exists in a wide range of colors and gradations, ranging from white, brown to
black. This is due to the presence of a chemically inert and stable pigment known as melanin.
This is produced deep inside the skin but is displayed as a mosaic at the surface of the body.
The melanin in the skin is produced by specialized dendritic cells of neural crest origin,
known as melanocytes, which are found in the basal layer of the epidermis. Because melanin
is an aggregate of smaller molecules, there are many different types of melanin with differing
proportions and bonding patterns of these component molecules. Both pheomelanin (yellow/
red pigment) and eumelanin (brown/ black pigment) are found in human skin and hair, but
eumelanin is the most abundant melanin in humans, and is likely to be deficient in albinism.
The key regulatory enzyme of melanogenesis is known as tyrosinase. There are also
numerous reports of the stimulation of human melanocyte tyrosinase and melanogenesis by
POMC peptides [33]. In human epidermis, α-MSH [34, 35] and ACTH [16] are produced in
and released by keratinocytes and are involved in the regulation of melanogenesis and/or
melanocyte dendrite formation. α-MSH and ACTH bind to a melanocyte-specific receptor,

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MC-1R(36), which activates adenylate cyclase through G protein, which then elevates cAMP
from adenosine triphosphate. Cyclic AMP exerts its effect in part through protein kinase A
(PKA), which phosphorylates and activates the cAMP response element binding protein
(CREB) that binds to the cAMP response element (CRE) present in the M promoter of the
microphthalmia-associated transcription factor (MITF) gene [37].
Increased expression of MITF and its activation by phosphorylation stimulates the
transcription of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and dopachrome
tautomerase (DCT), which together produce melanin. Melanin synthesis takes place within
specialized intracellular organelles named melanosomes [38, 39]. Melanin-containing
melanosomes then move from the perinuclear region to the dendrite tips and are transferred to
keratinocytes (Figure 2). Critical structural proteins of the melanosome involved in its
biogenesis and transportation that have been characterized include the SILV and MLANA loci
encoding the pMel17/gp100 and MART1 proteins, respectively. Determination of the
spectrum of proteins present during melanosome biogenesis has been attempted [40],
revealing that each stage contains ~600 proteins, with ~100 shared at each stage thus defining
the essential proteome component of the melanosome [41].
The functions of melanins relate to their physical and chemical properties. These include
anti-oxidant, free-radical scavenging behavior and an ability to absorb visible and broad band
UV wavelengths. Other properties are capacity to relax during non-radioactive and photo-
excited electronic states at the molecular, supramolecular and aggregate levels [42]. Indeed α-
MSH analogs, MTI and MTII, can also regulate melanogenesis. Their potencies are related to
their resistance to enzymatic breakdown, which prolongs their durations of action at the MC-
1R compared with the endogenous α-MSH molecule [43]. Stimulation of the MC-1R
promotes melanogenesis both by stimulating melanocyte proliferation and by upregulating
tyrosinase activity [44]. This pharmacological intervention thus harnesses eumelanization at a
point downstream of DNA damage [45].

Figure 2. The signaling pathway of POMC-derived peptide in the regulation of melanogenesis in

melanocytes. POMC-deived peptides such as MSH and ACTH activate adenylate cyclase (AC) by
binding to MC-1R to stimulate adenylate cyclase to induce subsequent increases in cAMP production.
PKA activation then leads to CREB phosphorylation which regulates MITF gene expression. MITF
gene upregulation activates melanin forming enzymes TYR, TYRP1, and DCT to induce
melanogenesis.
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448 Han-En Tsai, Elsa C Chan, Gregory J. Dusting et al.

MUTATION OF POMC/MC-1R ON SKIN PIGMENTATION

AND DISORDER

Loss of function mutations of the MC-1R gene are associated with red hair in a variety of
animals, and recently loss of function mutations of the ligand precursor, POMC, were shown
to lead to a red-hair phenotype in humans. Two patients were described by Krude et al. who
had congenital corticotroph insufficiency, massive obesity and red hair suggested a causal
relation with the lack of POMC-derived peptides [46]. In general, skin pigmentation can be
influenced by an activation of the MC-1R, expressed on melanocytes in the epidermis and
hair follicles, but whether a defect in its receptor binding affinity to ligands such as α-MSH
plays a role has not been examined. In this context, the red hair phenotype in the POMC-
deficient patients seems to substantiate an important role of POMC-derived peptides as potent
agonists at the MC-1R, capable of inducing pigment changes in humans, as has already been
established in mice [47, 48]. Similarly, we have shown an overexpression of POMC led to
melanogenesis in mouse melanocyte and melanoma cells [49] (Figure 3).
A second recent concept of skin physiology seems to be substantiated by the finding of
altered pigmentation in POMC-mutant patients [46]. Based on immunological and mRNA
expression studies, keratinocytes of the skin have been identified as an active site of POMC
gene expression and processing, leading to the hypothesis that ligands of the MC-1R are
generated within the target organ itself and might act in a paracrine manner [50, 51].
Consistent with this local production of POMC peptides is the clinical observation that in
patients with a traumatic or tumor-associated loss of pituitary function, changes in
pigmentation do not occur. In these cases, the endogenous dermal generation of MC-1R
ligands will maintain the individual level of pigmentation. On the other hand, loss-of-function
mutations of the POMC gene lead to an ubiquitous lack of POMC-derived peptides and with
the consequence of red-orange hair and pale skin phenotype.

Figure 3. Overexpression of POMC derived peptides induced melanogenesis by adenovirus-mediated

gene delivery in B16-F10 cells. After adenovirus gene delivery for 48 hours, POMC gene delivery
elevated the protein levels of MITF, TYR and TYRP1 as demonstrated by western blot analysis (A).
POMC gene delivery induced melanin synthesis. The TEM ultrastructure and melanin content of
transduced melanoma cells were evaluated in Ad-POMC-infected melanoma cells. Scale bar represents
2 m (B and C).

Genetic variability of MC-1R is associated with skin-colour variation in humans.

Unsurprisingly, specific MC-1R variants with impaired function are linked with skin cancer

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epidemiology. The human MC-1R is more polymorphic than several other pigment genes,
including tyrosinase, suggesting its importance in determining constitutive pigmentation in
humans [52-54]. More than 30 allelic variants of the human MC-1R have been identified
mainly in northern European populations and in Australia [54-58]. Some of these variants,
namely Arg160Trp, Arg151Cys, and Asp294His, were overexpressed in individuals with red
hair, as well as fair skin and reduced tanning ability in several populations [59]. Of
significance is the association of this phenotype with a high risk for skin cancer. Risk ratios
have typically been in the range of 2-5 fold, with clear evidence of a heterozygote effect [60].
The relation between a change in MC-1R sequence and skin cancer has been the subject of
different interpretations and has not reached to a consensus. What is not in dispute is that the
alleles mentioned above are associated with a variety of human skin cancers including basal
cell carcinoma, squamous cell carcinoma, and melanoma.

CONCLUSION
The mechanism involved in the regulation of skin pigmentation is still under intensive
investigations for clinical significances. The goal is to develop measures and treatment
strategy such as safe hypopigmenting and/or tanning cosmetics to cure and prevent pigment
disorders. The discovery of the contribution of POMC-derived peptides to the regulation of
pigmentation in humans will also help to understand the complex interactions of the
melanocortin system in pigmentation. It is clear that mutations of the melanocortin receptor
such as MC-1R may be linked with increased incidence of the three major skin cancers
namely basal cell carcinoma, squamous cell carcinoma, and melanoma. However, it is not
known whether mutations in either POMC or its processing system have similar pathogenic
impacts. The cutaneous POMC system offers much complexity for further investigation.

ACKNOWLEDGMENTS
This work was supported by grants from the Ophthalmic Research Institute of
Australia (ORIA) and Early Career Researcher grant (from University of Melbourne). Centre
for Eye Research Australia acknowledges the Victorian State Government’s Department of
Innovation, Industry and Regional Development’s Operational Infrastructure Support
Program.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 17

OVERVIEW ON THE MELANOCYTE PRECURSOR

MIGRATION FROM THE NEURAL CREST

Toyoko Akiyama* and Ai Shinomiya

Department of Biology, Keio University, Yokohama, Japan

ABSTRACT
In vertebrates, melanocytes in the trunk region originate from the neural crest as a
member of pluripotent cells during the developmental process. Melanoblasts, a precursor
of melanocytes, have been known to originate from the neural crest, migrate through the
dorsolateral pathway and settle in the body’s integument. But recently, in the lower
vertebrates, many pigment cells are found in pleura, intestine and connective tissues, etc.
in addition to skin, feather or scales. These pigment cells in the internal body are thought
to migrate through the dorsovental pathway and locate in the inner organs and connective
tissues. Even in many lines of chicken or wild quail, we found considerable numbers of
melanocytes in the inner organs. Furthermore, the unique chicken, Silky, is known to
display the ectopic hyperpigmentation in the internal organs (Fibromelanosis; Fm). In the
Silky chicken, huge numbers of melanoblasts migrate through the dorsoventral pathway
and proliferate in the inner organs. To understand the gene responsible for Fm, we
mapped it as 1.64Mb on chromosome 20. Furthermore, we also detected a Silky specific
duplicate region with 130 Kb in the area. Endothelin 3 is included in the region and
shows a two-fold expression compared to the wild type. Endothelin 3 is known as a
strong mitogen for melanoblast and it is supposed to be the responsible gene of Fm. Here
we summarize the involvement of the paracrine or autocrine factors from the
environment and dorsoventral pathway during pigment cells migration and
differentiation. In this review, we will describe and discuss the melanocyte differentiation
process from recent reports and our data using fish and chicken, that is, 1) melanoblast-
migration through both pathways from the neural crest, 2) differentiation to melanocytes,
3) factors involved in these processes of pigment cells.

*
Corresponding author’s e-mail; [email protected]
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456 Toyoko Akiyama and Ai Shinomiya

INTRODUCTION
In vertebrates, neural crest cells migrate extensively and contribute to diverse derivatives,
including the craniofacial skeleton, peripheral neurons and glia, and pigment cells.
Melanocytes in the trunk region originate from the neural crest (NC) as a member of
pluripotent cells during the embryonic developmental process (Mayer, 1973; Le Douarin and
Teillet, 1974, Carlson, 1988; Hearing, 1993, 2000; Erickson, 1993; Raible and Eisen, 1996;
1996; Nordlund et al. 2006, Le Douarin & Kalcheim, 2009) Generally, NC cells are known to
migrate in two different pathways during embryonic development: one part of the neural crest
derivatives pass near the neural tube within the anterior part of the somites in the dorsoventral
route and differentiate into Schwann cells, peripheral ganglia and nerve cells. The other part,
the melanoblasts, precursors of the melanocytes, migrate between the ectoderm and the
dermomyotome and invade the subectodermal mesenchyme through the dorsolateral pathway
(Weston 1963; Le Douarin and Teillet 1974, Le Douarin & Kalcheim, 2009) (mediolateral
route; Dupin and Le Douarin, 2003). The latter cells, the melanoblasts, start to migrate at a
slightly (1 day or more after) later stage than the former cells, differentiate into melanocytes,
and settle in the integumental basal layer of most of the body. (Erickson et al. 1992; Erickson
and Goins 1995; Le Douarin and Kalcheim 2009). This classic concept, that the melanocytes
migrate specifically through the dorsolateral route, seems to be correct in mammals. For a
long time, the melanocytes had been believed to migrate only through the outer route (Le
Douarin and Kalcheim, 2009). But in the lower vertebrates, it is known that many pigment
cells are also found in many internal organs, especially connective tissues or the sheath of
visceral tissues (Collazo et al., 1993, Raible and Eisen 1994, 1996; Kelsh 2004; Akiyama et
al. 2006; Tomlinson et al. 2009; Reyes et al. 2010), although its cell numbers vary from
species to species. These pigment cells, in the internal body, have been recently discovered to
migrate through the dorsoventral pathway and enter the inner organs and connective tissues
(Collazo et al., 1993). We could find considerable numbers of melanocytes in the inner
organs, connective tissues and the sheath of the organs in addition to the skin and the attached
organs such as hair, feathers and scales in birds or fishes. Therefore, we propose that it is
possible for melanoblasts to migrate through both routes from the neural crest but the
mechanism to sort the each melanocyte precursor into the different migration route is
unknown.
Furthermore, the unique chicken, Silky, is known to display ectopic hyperpigmentation in
the internal organs. In the chicken, huge numbers of melanoblasts migrate through the
dorsoventral pathway and proliferate in the inner organs (Reedy et al. 1998; Faraco et al.
2001; Ortolani-Machado et al. 2007; Ortolani-Machado et al. 2009). This phenotype is called
Fibromelanosis (Fm) (Hutt 1949). To know the mechanism of the pigment cell migration and
differentiation system in the chicken, we analyzed the gene responsible for Fm by linkage
mapping. We suggest the involvement of the gene product in pigment cell proliferation and
differentiation. The Silky chicken is certainly a useful model to discover how and when the
fate of the pigment cells are determined and how these cells are differentiated during
migration through one of the pathways.
The melanocyte differentiation process, that is, determination of cell destiny and
migration route, is discussed in association with recently reported factors.

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 Overview on the Melanocyte Precursor Migration from Neural Crest 457

Figure 1. An illustration of the transverse section through the trunk region of the avian embryo showing
the migration routes from the neural crest cells. (Reproduced from B.M. Carlson, 1988)

MELANOBLAST-MIGRATION THROUGH BOTH ROUTES FROM THE NC

NC cells have been thought to migrate through two pathways; cells in the first group that
migrate through the dorsoventral pathway are the precursors of the dorsal root ganglion,
sympathetic ganglion, Schwann cells, sensory neuron, Chromaffin cells in the adrenal
medulla, prevertebral plexus, and parasympathetic plexus in the gut. The second group,
migrating through the dorsolateral path, is the precursor of the pigment cells (Figure 1). These
NC derivatives are detected in their localizations by immunofluorescence using antibodies;
HNK-1 (Bronner-Fraser, 1986; Erickson et al., 1989, 1992; Newgreen et al., 1990), anti-
neurofilament, Mel-EM (Nataf et al., 1993,1995) or MEBL-1 (Kitamura et al., 1992) and
endothelin receptor B2 (Akiyama et al., 2002) against all NC cells, nerve cells, melanoblasts
and melanocytes, respectively. Erickson et al. (1992) reported the dispersion of NC cells
along the dorsolateral pathway and entry into the ectoderm in the chick embryo using HNK-1.
We also investigated the localization of the melanophores of goldfish (Carrassius auratus)
during embryogenesis on their differentiation and migratory behaviors from NC (embryonic
shield) cells by means of immunofluorescence using the HNK-1 and melanoblast specific
antibody, MEBL-1. In a preliminary assay, using cells derived from NC explants, NC cells
under migration unequivocally cross react with HNK-1 and melanoblasts with MEBL-1 until
the onset of melanogenesis, respectively. To disclose melanophore behavior more precisely,
cryosectioned embryos and larvae were assayed and we obtained following results: (1) the
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458 Toyoko Akiyama and Ai Shinomiya

HNK-1 positive cells first appear in the dorsal ridge of the neural tube at stage 20 and are then
distributed over all sites of both dorsolateral and ventral pathways, (2) the MEBL-1 positive
cells first appear among the NC derivatives distributing outside of the neural tube, suggesting
the melanization started in the route of migration, (3) huge number of melanoblasts/phores
migrate through dorsoventral route in goldfish, different from avians and mammals. (4) The
melanoblasts and melanophores are close at starting point of migration at stage 20 and are
later found in a more migrated position, suggesting the presence of a pioneer, which develops
the routes. The result shows that melanoblasts/cytes in goldfish migrate through not only
through the dorsolateral route but also through the dorsoventral route. The functions of the
pigment cells that invade into internal organs remain unknown.
As described above, the pigment cells migrating through both pathways from NC
recently been found in the lower vertebrate and in fishes or amphibians; the major part of the
pigment cells migrate through dorsoventral pathway (Figure 2, Figure 3) (Collazo et al., 1993,
Akiyama et al., 2006). But the cell number of the melanocyte precursor migrating through the
inner route is different in each vertebrate species and shows a wide variation in each species.

Figure 2. An illustration showing the neural crest cell derivatives and their migration paths in the trunk
region of a Xenopus embryo. Pigment cells migrate through both dorsoventral and dorsolateral routes.
(Reproduced from Collazo et al., 1993)

In fish, melanophores are commonly found in connective tissues or sheath of the inner
organs (Figure. 3). In the case of the teleost, melanophores are found in the epidermis
(Figures 3a, b), but a larger numbers of melanocytes are located in connective tissues,
surrounding the veins and pleura (Figures 3c, d). In particular, the pleura looks to be lined by
a black layer of black pigment cells. The layer is often constructed with melanophores and
iridophores in fish. These NC cells are detected by immunofluorescence labeling using HNK-
1. Positive labeled cells are located on the ganglia, row of the somite and epidermis in
Medaka fish (Figure 4) and surrounding the neural tube and ventral region in goldfish (Figure
5). In addition, labeling with the MEBL-1 antibody can identify the melanoblasts, before
heavy melanization. In goldfish, positive labeled melanoblasts were found in both pathways

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 Overview on the Melanocyte Precursor Migration from Neural Crest 459

from the neural crest (Figures 6, 7). Also, larger numbers of the melanophores were found in
the ventral area than in the ectoderm or epidermis (Figures 4-7). It is suggested that the major
part of the melanoblasts migrate through the dorsoventral pathway (Collazo et al., 1992;
Akiyama et al., 2006). The number of the melanophores in the inner organs do not equate to
the outer body color. Generally, red or bright colored fish tend to have larger numbers of
melanocytes in the inner body than black colored fish.

Figure 3. Cross-sectioned pattern of trunk region in medaka adult. Paraffin sectioned and Hematoxylin-
Eosin stained. a) Whole pattern of the cross section. Bar; 200μm. b) Upper region of a). There are
melanophores at the epidermis (arrowheads), c) Gut region of a). Melanophores in the gut (thin arrows).
d) Abdominal lining area in a). Melanophores form a black line (thick arrows). Bar: 100μm.
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460 Toyoko Akiyama and Ai Shinomiya

If the number of precursor melanoblasts was determined in NC, the total number of
melanoblasts migrating through the dorsoventral and dorsolateral routes might be constant. If
the fish or chicken has a dark color in the integument, skin, scale or feather, there aren’t many
melanophores or melanocytes in the inner organs. It has been suggested that melanoblasts
may eventually migrate to both routes in embryonic development and their total numbers are
regulated as constant in the species. If the final destiny of the melanoblast precursors is
determined in the NC at the same time as the peripheral neurons, glia and craniofacial
skeleton, the cell-fate-committed melanocyte precursors migrate through the same
dorsoventral route with the precursor of other derivatives from NC. On the other hand, Dupin
et al., (2000) and Dupin and Le Douarin (2003) reported the reversibility of the melanoblasts
from their results that endothelin 3 induces the reversion of melanocytes to glia through a
NC–derived glial-melanocytic progenitor. Also, Le Douarin et al., (2004) proposes the
concept of NC cell plasticity or reversibility from the fact that endothelin 3 affected the
differentiated cells such as melanocytes or glia to induce a pluripotent precursor state. The
cell fate determination of melanoblasts at the NC may be far from conclusive and the cells
still maintain the flexibility to differentiate. If it is true, there may be some factors or genes to
induce specific phenotypes of the NC derivatives during migration.

Figure 4. Immunofluorescence patterns of medaka embryos by HNK-1 antibody. a) Horizontal and b)

vertical sections parallel to the long axis of trunk region at stage 26. There are positive areas in
ganglions, dorsoventral pathways along somite and ectoderm. c) Phase contrast pattern of the cross-
sectioned trunk region at stage 26. d) Immunofluorescence pattern of c). Positive cells are found in both
pathways from neural crest. e) Phase contrast pattern of the vertical section along long axis from head
to tail at stage 27. Melanophores are located in the epidermis and ventral area. f)g) Immunofluorescence
patterns of e). Positive cells are found at the epidermis and each segment of the somite and surrounding
area of neural tube. No: notochord, NT: neural tube. Bar: 100μm.

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 Overview on the Melanocyte Precursor Migration from Neural Crest 461

Figure 5. Immunofluorescence patterns of cross-section in trunk region of goldfish embryos using

HNK-1 antibody. a) Immunofluorescence pattern at stage 20, b) phase contrast of a), c)
Immunofluorescence pattern at stage 22, d) phase contrast of c). Positive cells (arrows) are found in
both pathways from neural crest. e) Immunofluorescence pattern of vertical section along long axis at
stage 26, f) phase contrast of e). Melanophores located at the ventral area and border to the yolk, g)
illustration of e) and f). NK: neural keel (presumptive neural tube), NT: neural tube, NO: notochord.
Bar: 100μm.

Figure 6 Immunofluorescence patterns of cross-sectioned goldfish embryos in trunk region using

MEBL-1. a) Immunofluorescence pattern of stage 23, b) phase contrast pattern of a), c)
immunofluorescence pattern of stage 26, d) phase contrast pattern of c). Positive labeled melanoblasts
are located on both dorsolateral and dorsoventral pathways. NT: neural tube, NO: notochord. Bar;
200μm.
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462 Toyoko Akiyama and Ai Shinomiya

Figure 7. Immunofluorescence patterns of cross-sectioned goldfish embryos at stage 26 in trunk region

using MEBL-1. Melanoblasts revealing positive labeling are found in the inner pathway and epidermis.
NT: neural tube, NO: notochord. Bar: 200μm.

In the lower vertebrates and Aves, melanocytes migrate through both routes from the
neural crest but the melanocytes are sparsely located in the skin and inner body. Therefore,
we encounter the question; how can the melanocyte precursor migrate both pathways in these
animals? To study the reason, the Silky chicken that reveals hyperpigmentation in its inner
organs and dermal tissues is an extremely useful tool.
Also, many lines of colored chickens have more or less melanocytes in their inner organs
and connective tissues (Figure 8). These chickens demonstrate tendencies similar to bright
colored birds like GSP or YL, which show high numbers of melanocytes in their inner organs.
The Black Minorca, covered with black feathers, has a small number of melanocytes. Of
course, Silky (White Silky: WS and Black Silky; BS in Figure 8) is a discriminating
exception of the melanocyte differentiation since the chicken has heavy melanization in inner
organs regardless of the feather color. Pigmentation in Silky is described in the next section.
In conclusion, it is supposed that the precursor of the melanoblasts was emitted from NC over
a long period and the first group of melanogenic cells starting migration are induced to the
inner route (Figures 1, 2) with other derivatives of the NC although these cells may still have
plasticity to differentiate to glial cells (Le Douarin et al., 2004; Thomas and Erickson, 2009).
The second group definitively determined their fate as a precursor of melanocytes a little later
than the first group.

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 Overview on the Melanocyte Precursor Migration from Neural Crest 463

Figure 8. Inner organs and feather colors in five chicken lines. Upper line: patterns of the high-
magnified pleura. Bar: 100μm. Middle line: patterns of low magnified pleura. Bottom line: Chicks
showing feather color. a, f, k) White Silky [2 week(2W) chick], b, g, l) Black Silky (2W), c, h, m), GSP
(1W), d, i, n) YL (1W), e, j, o) Black Minorca (1W). There are considerable variations of the
melanocytes in inner organs between these chicks but both Silky reveal peculiar heavy melanization in
inner organs.

HYPERPIGMENTATION IN SILKY CHICKEN

In the case of the Silky chicken, it is a unique mutant on the pigment cell migration. The
bird shows heavy melanization in inner organs such as pleura, blood vessel, sheath of organs
and connective tissues in addition to normal pigmentation in skin and feathers (Figure 9).
Cells having melanosomes in internal organs in Silky contain various stages of immature
melanosomes in their cell bodies (Reedy et al. 1998; Faraco et al. 2001; Ortolani-Machado et
al. 2007; Ortolani-Machado et al. 2009), indicating that melanization occurs inside these cells.
Therefore, these cells could be judged as melanocytes. The melanocytes are located in the
same position as fibroblasts (Figure 9). The hyperpigmentation phenotype in the inner body is
called “Fibromelanosis; Fm” (Hutt, 1949). In the embryonic development, lots of
melanoblasts migrate through the dorsoventral route at first with the other derivatives from
the neural crest (Figure 10). Afterwards, melanoblasts in the second group translocate through
the dorsolateral route to the integument of the entire body surface. There are white and black
color variants in Silky lines but melanocytes exist in the skin in both variants. Since White
Silky has c/c locus (tyrosinase incomplete recessive, Chang et al. 2006), there are a small
number of melanocytes in skin but no pigmentation in the feathers. Dorshorst et al. (2010,
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464 Toyoko Akiyama and Ai Shinomiya

2011) analyzed the genomic region associated with the dermal hyperpigmentation and we
also independently mapped the responsible gene for Fm as 130 kb duplicate region in
chromosome 20 (Sinomiya et al., 2012). 5 genes including endothelin 3, HIVEP1, slowmo
homolog2 (SLMO2), H+ transporting F1 ATP synthetase epsilon subunit (F1ATPase-e), and
tubulin beta 3 (TUBB3) are located in the region and 4 out of 5 genes are expressed in stage
18-20, almost twice as much as the wild type. Since the endothelin 3 is known as a strong
mitogen for melanoblasts (Baynash et al., 1994; Lahav et al., 1998; Dupin & Le Douarin,
2003; Nordlund et al., 2006), we proposed it is the first candidate for the Fm phenotype. The
signal of the EDN3 is received by the endothelin receptor (EDNR). Defects in the gene
encoding the endothelin-B receptor have been known to produce aganglionic megacolon and
pigmentary disorders in mice and humans. Baynash et al. (1994) reported that a targeted
disruption of the mouse endothelin-3 ligand (EDN3) gene produces a similar recessive
phenotype of megacolon and coat color spotting. Birds are known to have EDNR-A, -B and
B2. The antagonist for the EDNRB suppressed the hyperpigmentation in Silky chicken. On
the other hand, supplementing with excess EDN3 to the organ culture of the neural tube from
the Road Island Red line (wild type with fm/fm) clearly activated pigmentation in the ventral
area. From these results, we suggested that the duplicate endothelin 3 gene, expressing two-
fold product, leads to excess endothelin 3/endothelin receptor signaling. Then, by triggering
the excess endothelin 3, the related genes in the downstream will be induced to express
sequentially to exhibit the Fm phenotype. For example, the expression of intracellular matrix
or adhesion substances like fibronectin, collagen, cadherin or integrin etc. and genes related to
melanin production such as tyrosinase, 5,6-dihydroxyindole-2-carboxylic acid (DHICA; TRP-
1), DOPA chrome tautomerase (TRP-2), MITF (Nordlund et al., 2006) may be induced.

Figure 9. Cross-sectioned patterns in the inner organs of the Silky chicken. Paraffin sectioned and
Hematoxylin-Eosin stained. a) Feathers, epidermis and dermis, b) dermis, c) notochord, d) cartilage,
e) muscle, f) vein. Arrows: melanocytes.

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 Overview on the Melanocyte Precursor Migration from Neural Crest 465

Figure 10. Immunofluorescence patterns of cross-sectioned trunk region in Silky embryo at stage 36
using MEBL-1 antibody. Melanoblasts retaining positive labeling (green fluorescence) with MEBL-1
are located at side of neural tube and ventral area in addition to the epidermis at the top of the neural
tube. Yellow cells; Blood cells

Figure 11. Expression of EDN3 and EDNRB2 transcripts in Black Silky or Barred Plymouth Rock
derived fibroblasts or melanocytes. Fibroblasts and melanocytes expressed EDN3 and EDNRB2,
respectively.
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466 Toyoko Akiyama and Ai Shinomiya

Figure 12. Immunofluorescence patterns of the trunk region in Black Silky embryo at stage 36 by anti-
EDNRB antibody (Sigma E-2764) (a) Melanocytes are labeled with green fluorescence and blood cells
reveal yellow color. b) Phase contrast image of a). NT; Neural tube, NO; Notochord.

FACTORS INVOLVED IN MELANOCYTE DIFFERENTIATION

From the results of Figures 6, 7, the precursor of the melanoblasts at the top of the neural
tube showed melanoblast specific positive labeling. It indicates that their cell fate was already
determined before migration although the precursor may retain some flexibility to
differentiate glia or other derivatives. But we still have a question on how the NC derived
cells determined their cell fate and migration route and how these cells start to migrate at the
right time. To consider the answer, Thomas et al., (2008, 2009) suggested that FDN3 plays an
important role in determining the cell fate of melanogenic cells at NC. In addition to factors
like Wnt and BMP signaling, Kit, EDN3 is involved in the differentiation (induction of MITF
expression) of the melanogenic cells. Pla et al. (2005) and Kawasaki-Nishihara et al. (2011)
also reported that ET3 (EDN3)/Ednrb2 signaling is critically involved in regulating
melanophores migration in Xenopus.
The starting time for the emission from NC may be one factor to specify the cell type of
the derivatives. Before the emission time, NC cells may be exposed with many paracrine or
autocrine factors in the neural tube and may change their cell destinies gradually during the
incubation time. Recently, Shoval and Kalcheim (2012) indicated the roles of Rho and Rac
GTPases in the transition from NC delamination to migration. The environment of NC at the
slightly late phase may induce the NC cells to melanoblasts or glial cells. Another possibility
may be the environment of the migration route. Special substances of the route may permit
passage of only the specific cell precursor or may lead to a special cell type. Rogers et al.,
(2012) reported that Elk3 is essential for the progression from progenitor to definitive NC

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 Overview on the Melanocyte Precursor Migration from Neural Crest 467

cell. The adhesive substances like cadherin or integrin or extracellular matrix like fibronectin
or vitronectin etc., may positively induce their pathway specifically and work to determine
their final cell fate (McKeown et al., 2013; Hochgreb-Hägele and Bronner, 2013).
But, if the precursor of the melanocyte migrates through both pathways from the NC and
if one part of the melanocyte precursors migrates through the inner route with other
derivatives, the possibility of the incubation effects in NC or of the environment of the route
may become invalid. Therefore, it is suggested that cell type commitment occurs roughly at
the NC and differentiates into their final destination during the migration phase. On the other
hand, Aoki et al. (2009) suggested that the non-cutaneous and dermal melanocytes equipped
different signaling responsiveness from epidermal melanocytes. They described that
melanocytes in the eye, ear and harderian gland were revealed to be less sensitive to Kit
signaling than cutaneous melanocytes and that these non-cutaneous melanocytes were
stimulated more effectively by endothelin 3 (EDN3) or hepatocyte growth factor (HGF)
signals than by Kit signaling. Otherwise, space for each pathway from the neural crest may
produce different timing and each temporal tunnel-like space may make it easy for the
specific migration. If the cell fate commitment occurs early, precursors of melanocytes
migrate with other derivatives through the dorsoventral pathway, which is the only possible
way to pass through. If the cell commitment occurs a little later, these cells invade into the
dorsolateral pathway, which they are able to pass through.
Pigment cells in vertebrates emigrate from NC and differentiate during migration in
embryonic development. To find the secret factors playing important roles in the
differentiation process, NC cells were cultured and their derivatives induced with a variety of
supplements in vitro. These factors are produced from keratinocyte, fibroblasts such as
paracrine factors and from melanocytes such as autocrine. EDNs, leukemia inhibitory factor
(LIF), steel factor (SLF or kit ligand), hepatocyte growth factor (HGF) and granulocyte-
macrophage colony-stimulating factor (GMCSF, Csf2) are produced by keratinocytes and ß-
fibroblast growth factor (ß-FGF) and EDN3 are produced by fibroblasts (Hirobe 2011). Other
effective factors for melanocytes differentiation or proliferation are hormones, α-MSH,
melanocortin, and insulin-like growth factor, etc. (Nordlund et al., 2006)

Figure 13. Illustration showing the migrating routes of the precursors of the melanocytes and related
factors.
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468 Toyoko Akiyama and Ai Shinomiya

Although melanoblasts in vertebrate differentiate under these complex regulation

mechanisms (Figure 13), melanocytes are often found in inner organs in the lower vertebrate.
In some animal species, like lower vertebrate or Aves, the ventral pathway seems to be the
major route for migrating melanoblasts during embryogenesis. The melanogenic cell
migration from NC are not determined clearly and specifically in these animals. One of the
features in these animals may be the existence of EDNRB2. Mammals have EDNB, but not
B2, so EDN3 signals may not be received effectively. On the contrary, if an excess of EDN3
signals are received by EDNRB and B2, the downstream gene for migration, proliferation and
differentiation for melanocyte/phore may be easily elicited.

ACKNOWLEDGMENTS
This work was supported in part by the grants from Keio University. The authors deeply
thank MS. Mizuho Nakamura, Drs. Yasunori Kayashima and Yasuho Taneda for their
valuable help and support for the preparation of specimens. We also appreciate chicken
supply from Avian Bioscience Research Center in Nagoya University.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 18

RADIATION TREATMENT AND ALOPECIA – PAST AND

PRESENT CONCERNS

Paula Boaventura1, Dina Pereira1, José Teixeira-Gomes1

and Paula Soares1,2
1
IPATIMUP (Institute of Molecular Pathology and Immunology),
Porto, Portugal
2
Medical Faculty, University of Porto, Portugal

ABSTRACT
The use of radiation to induce epilation was discovered shortly after Wilhelm
Roengten found out about X-rays in 1895. In 1897 the roentgentherapy began to be used
for tinea capitis treatment, a fungal disease, in order to eliminate the infected hair, and
facilitate the therapeutic ointments application [1]. The method at that time was very
intense, and frequently led to permanent alopecia and radiodermitis. Later on, the
Keinbock-Adamson technique was adopted, a method considered safe to induce scalp
epilation without permanent alopecia or other possible side effects [1, 2]. Several studies
performed years after this epilation treatment have shown that, in fact, there are several
possible side effects that can be related to this treatment, namely head and neck tumors
[3-6] and permanent alopecia [7, 8]. Nevertheless, in the case of tinea capitis epilation
treatment, we observed, for the first time, that the type of tinea diagnosis (favus tinea)
could cause a higher risk of alopecia than the radiation treatment itself [8]. In the cases of
transient alopecia, the real purpose of this intervention, there have been reports of the
regrowth of curly and/or white hair.
The radio-induced epilation, very well described for tinea capitis treatment, was also
used in the beauty shops, for the purpose of treating superfluous hair [9]. This hair that
appeared in the nipple-region or in the arm pits is a common phenomenon, so its epilation
surely involved the X-ray exposition of appreciable areas of the breasts, and this could
have been a preventable cause of breast cancer [9].
Today we deal with a different situation, as radiation to induce epilation is no longer
acceptable, but alopecia can appear as a sequel from radiation therapy and interventional
radiology. There are several studies on this issue. Approaches are being addressed to
established protocols to minimize these side effects [10, 11].
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474 Paula Boaventura, Dina Pereira, José Teixeira-Gomes et al.

THE USE OF X-RAYS TO INDUCE EPILATION

The Tinea Capitis (or Scalp Ringworm) Epilation Model

Three months after Wilhelm Roengten´s publication about his discovery of X-rays in
December 1895, its depilatory effect was described [12]. In 1897 the roentgentherapy began
to be used for tinea capitis treatment, a fungal disease, in order to eliminate the infected hair,
and facilitate the therapeutic ointments application [1].
Tinea capitis was a highly contagious mycosis with long evolution that infected mainly
children’s scalp [13], as adult infection was considered exceptional [14]. It did not produce an
alteration in the general health status of the child, nor even a considerable cutaneous damage
in most of the cases, but it would interfere with the children’s welfare due to its high
contagiousness [13], commonly causing epidemic infections in schools and orphanages.
Unless isolated, a child under long-term topical therapy could infect countless others before
cure was achieved [2].
The disease could be caused by several fungi. The main agents of tinea capitis in Portugal
were, by order of frequency, Trichophyton violaceum, Microsporum canis, Trichophyton
tonsurans and Trichophyton schoenleinii [15, 16]. Trichophyton violaceum was also the most
important agent of scalp ringworm all round the Mediterranean and in Eastern Europe [17].
The clinical expressions and evolution of these infections lead to the classification of the
disease in two main types: tonsure tinea and non tonsure tinea [18]. Tonsure tinea caused hair
breaking by opposition to the non tonsure tinea, where normally the hair did not break [19].
Tonsure was then subdivided in trichophytic tinea and microsporic tinea. Trichophytic tinea
was caused by several Trichophyton species and the microsporic tinea was caused mainly by
M. canis and M. felineum [20-22].The non tonsure tinea (favus tinea) was caused by T.
schoenleinii.
Favus tinea infection could attain the entire scalp, destroying the hair follicles, leading to
the transformation of the scalp into scar tissue of definitive alopecia [14]. It was more prone
to recurrence [1]. This was considered the worst form of tinea capitis disease, followed by
trichophytic tinea, being the more benign form the microsporic tinea [18].
There was no oral antifungal treatment. The topical treatment was performed with
different compounds, namely iodine tincture and an ointment with sulphur salts. Due to the
profound penetration of mycelia in the hair follicle, it was difficult for any fungicidal to enter
to the necessary profundity in order to produce the adequate disinfection [14, 23], if previous
epilation was not applied [14].
The first oral treatment, griseofulvin, appeared only in 1958, and from that time on the X-
ray epilation was slowly abandoned, as it was no longer needed [18]. So, the X-ray irradiation
of the scalp to obtain temporary epilation was the best therapeutic approach for the tinea
capitis disease from 1897 to the end of the 5th decade of the XX century.
The most common technique was the Kienbock-Adamson technique that was of proven
worth in the control of the epidemic [2, 23, 24]. This method allowed the irradiation of the
entire scalp in a short period of time, using only five fields, with a dose of approximately 300-
400 Roentgen per field (R) [1, 2, 25-27]. The five fields were in the frontal, vertex, occipital
and parietal regions and were determined by marking five points, far off between 11 and 13
cm from each other, depending on the dimension of the head [26]. The head was set in

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 Radiation Treatment and Alopecia – Past and Present Concerns 475

successive positions so the vertical rays from one field would be perpendicular to the vertical
rays from the other fields [1]. The irradiation of the fields set in an antero-posterior
orientation was performed in ventral decubitus. The ears were pulled to the front and kept still
against the face with adhesive tape, after what they were protected with an appropriate lead
cast. It was recommended that the face should also be protected [1]. The hair began to fall,
commonly 15-26 days after the X-ray irradiation [1, 24, 28] and regrowth occurred from 5-8
weeks after the falling [1, 13, 24, 28].
At that time is was considered that when this Kienbock-Adamson technique was carried
out meticulously by a qualified operator, permanent alopecia [2] or other side effects, such as
brain damage [1, 2], needed not to be feared. The main difficulties in the application of this
method were the availability of the X-ray equipment and of trained personnel for a mass
treatment [23], the objection of the parents against the irradiation of the head [23, 29], and it’s
almost impossible application to children below three years of age [1, 23]. The method was
not recommended in children approaching puberty, as spontaneous cure was expected to
occur [24]. The mechanism of the cure was not fully understood, but could be probably due to
chemical alterations on the scalp chemical composition occurring through the action of the
sex glands [14].
This treatment for tinea capitis was used extensively between 1910 and 1959, and
approximately 200,000 children worldwide received this form of treatment [30].

The Beauty Shops

The radio-induced epilation, very well described for tinea capitis treatment, was also used
in the beauty shops, for the purpose of treating superfluous hair [9].
Cleveland [31] became aware of the use of X-rays for the removal of superfluous hair
from the face, in the so called Marton Laboratories, during the autumn-winter of 1930-31.
Having drawn the attention of the Vancouver Health Department to this issue, an
investigation took place that allowed finding out that the proprietor of the Marton
Laboratories was herself the operator of the X-ray machine, without having any competence
for it. Nevertheless the license of these Marton Laboratories could not be cancelled and later
on they were included in a well known beauty parlour with the new name of Arnold Dermic
Laboratories.
In the USA, California and Washington states, the same concern had occurred about the
effect of the so called Tricho System, which represented another designation for the same
procedure. Some operators had been already law-suited for damage to elastic tissue, causing
wrinkling and other disfigurements. Few years later, thanks to the testimonial of several
women that were treated in these laboratories, the Arnold Dermic Laboratories closed.
Nevertheless Cleveland, in 1948 [31], said that it was not unreasonable to expect that
“institutions” and laboratories of the same character would continue to appear under new
guises and disguises. In fact the author ends is paper saying that new ones had recently
opened in various cities of the United States and Canada.
Some years later, Lapidus [32], reported having observed five women submitted to the
Tricho System epilations 30 to 40 years before to treat facial hypertrichosis, in Pennsylvania.
The device was manufactured, sold, and proclaimed safe by its physician-inventor. All the
women developed, at varying intervals, radiodermatitis and basal cell and/or squamous cell
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476 Paula Boaventura, Dina Pereira, José Teixeira-Gomes et al.

carcinoma at the sites of epilation. The same pattern of radiodermitis and skin cancer [33, 34],
as well as cancer other than skin – thyroid, parathyroid, oral cavity, facial skeleton and breast
[34, 35] – was observed by other authors that alerted to the danger of this epilation treatment.
Interestingly a report on the indiscriminate use of X-rays in the treatment of hypertrichosis
had already been published as early as 1925, by a committee appointed by the New York
Academy of Medicine, stating that this method was dangerous and constituted an improper
method of treatment, entailing great hazards to the patient [36]. The committee, and the
recognized dermatologists of the world, did not use or recommend X-rays for the treatment of
hypertrichosis. Removal of superfluous hair by Roengten therapy could not be accomplished
without permanent skin injury [31].
Hypertrichosis in the nipple region and in the arm pits is a common phenomenon, so
Gofman [9] refers that the X-rays were also used for its removal, and that appreciable areas of
the breasts were hit by the X-ray beam during the procedure.
It is not possible to know how many such beauty shops were operating and for how many
years, but it was an important source of breast irradiation from 1920 to 1960 [9].

Possible Side Effects Related to the Epilation Treatment

Studies to evaluate possible side effects from the X-ray epilation treatment have started
soon after the treatment discontinuation, at least for the tinea capitis cohorts. One of such
studies was the one from Albert et al. [37] that has shown a substantially larger number of
cases of cancer, mental disease and permanent damage of the scalp hair in the irradiated
group compared to controls. In a second follow-up of tinea capitis irradiated patients, Shore et
al. [38] found an excess incidence in the irradiated individuals of tumors of the head and
neck, namely of the skin, brain, thyroid and parotid, although no difference was found in
mortality due to malignant disease, or any other cause, between the two groups.
These follow-up studies have been performed until today, namely the large cohort from
Israel that includes 10,000 individuals [3, 7, 39-41]. They have shown a 2 to 10-fold
increased risk for head and neck neoplasias, namely for basal cell carcinoma (BCC) [40-43],
meningioma [7, 44-46] and thyroid carcinoma [3, 47]. The thyroid is highly sensitive to
radiation, especially when the exposition occurs at a younger age [3, 5, 48, 49]. An
association between radiation exposure and parathyroid hyperplasia has also been described
[50-53] in small cohorts other than the Israeli cohort. The latency period between radiation
exposure and neoplasia diagnostic can be as long as 20-40 years for non-melanoma skin
cancer [54-56] and for meningioma [4, 46, 57]. We have been clinically observing a cohort
submitted to the X-ray epilation treatment for tinea capitis infection in an health institution in
the North of Portugal [58]. Our findings on thyroid cancer prevalence and thyroid disease
[58], as well as on basal cell carcinoma prevalence [59], are in accordance with the increased
prevalence of these neoplasias presented in the above referred studies.
Besides the most well studied tumor-related effects, non-cancer effects have also been
shown. We have already mentioned mental disease, in the Albert et al. study [37], that found
a significantly higher amount of major mental disorders (psychosis and personality disorders)
in the irradiated individuals when compared to the non-irradiated ones. Later on Omran et al.
[60] investigated the late effects of the X-ray epilation treatment upon subclinical mental
disorders and found was an excess of psychiatric symptoms, paranoid orientation, work

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 Radiation Treatment and Alopecia – Past and Present Concerns 477

problems, and treated psychiatric illnesses in the irradiated group, but only in the white
individuals. In their study, Yaar et al., suggested permanent change in EEG activity,
following average doses of 130 rads of childhood X-irradiation to the brain [61]. Continuing
in the scope of mental disease, in a recent study Sadetzki et al. [62] did not find support for an
association between exposure to ionizing radiation and risk of schizophrenia. The authors say
that more research on possible effects of early exposure to ionizing radiation on schizophrenia
risk as well as in brain tissue injury is needed.
Other sequelae from scalp irradiation are now being unraveled, namely due to the
recently reported association between low dose radiation and cardiovascular disease. Shai et
al. [63] found, in the Israeli tinea capitis cohort, that childhood scalp irradiation was a
significant and thus far underestimated risk factor for adult carotid atherosclerosis disease.
They emphasize the need for physicians to be aware of the existence of such high risk
populations.
Another important side effect from this treatment, due to its emotionally troublesome
effects, is permanent alopecia. At the time the tinea capitis epilation treatment was applied it
was considered that the margin of safety between a dose causing temporary loss of hair and
one causing permanent hair loss was considerable, so the treatment was safe [2]. Strauss and
Kligman [64] stated that the necessary dose to produce permanent alopecia was above 1000 R
for each of the five points. More recent studies refer that lethal doses for hair follicles vary
between 7 and 16 Gy [10, 65, 66]. The dose for which 50% of the patients develop definitive
alopecia was considered to be 43Gy at the follicle level (4.5 mm under the skin) [67, 68]. As
has been previously mentioned, the doses reported to cause permanent hair loss vary widely
[67].
When the radiation treatment had to be repeated, due to disease relapse, it should be
performed only 6 months after the first treatment, to prevent the appearance of radiodermitis
that could result in definitive alopecia [1]. Albert et al. [37], using a phantom head built
around the head of a seven year old child, have calculated that the dose applied to the scalp
ranged from 500 to 800 rad (6-8 Gy). However, the doses could be higher due to technical
problems. The most common error appeared to be overlapping of the irradiation fields
[69,70], but also faulty or poorly calibrated equipment occurred, and both could lead to
permanent alopecia [70].
In the follow-up studies permanent alopecia has been observed [7, 8, 46] and was
positively associated with higher irradiation doses [8, 67, 71], received when the treatment
had to be repeated (2-3 sessions).
In our series of tinea capitis irradiation we have observed 670 women (to avoid the higher
confounding effect of androgenetic alopecia in male gender) and we found overall prevalence
of alopecia of 6.7% (95% CI 4.6–8.3%) [8]. Women who received an irradiation dose ≥ 630
R were more likely to develop alopecia than those who received an irradiation dose < 630 R
(dose ≥ 630 vs. < 630 R, RR 5.50, 95% CI 2.96–10.22) and the relationship was maintained
after adjustment for tinea diagnosis and age at diagnosis (dose ≥ 630 vs. < 630R, RR 3.93,
95% CI 2.61–5.91). Nevertheless, we observed, for the first time, that the type of tinea capitis
agent (favus tinea vs tricophytic/microsporic) could cause a higher risk of alopecia than the
radiation treatment itself.
In the cases of transient alopecia, the real purpose of the X-ray epilation treatment, a
curious aspect mentioned by several patients from our cohort (11 out of 100 – 11%)
(unpublished data), was a change in the hair pattern when it grew back. They stated that
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478 Paula Boaventura, Dina Pereira, José Teixeira-Gomes et al.

straight hair grew back curly and/or white hair appeared. Hair changes related to scalp
radiation therapy have been referred but references are scarce. Zeligman observed the graying
of hair in 3 out of 37 children with tinea capitis [72], Alexander observed the regrowth of
dark hair in a patient that previously had white hair after scalp radiotherapy [73] and
Möhrenschlager et al. observed curly hair appearance after brain radiotherapy [74].
The possible side effects from the epilations performed at the beauty shops are not so
well documented. The main sequelae were mentioned in the previous item and were observed
soon after the superfluous hair removal. These treatments in the beauty shops were scattered
in many “clinics” and there are no registered and extensively studied cohorts as we found in
the tinea capitis model. Although the number of these “beauty shops Roengten departments”
is not known, nor the number of epilations performed, Gofman [9] states that these
procedures have caused a number of totally preventable breast cancers that appeared in
subsequent decades.

ALOPECIA AS A SEQUEL OF RADIOLOGICAL INTERVENTIONS

Diagnostic/Therapeutic Techniques and Accidents Can Cause Alopecia

Interventional radiology is widely used in the treatments of various diseases worldwide.

Cardiac angiography is known to produce one of the largest radiation exposures of any
diagnostic X-ray procedure; a patient may receive up to 6 Gy during a prolonged coronary
angioplasty [66]. So, these interventional procedures in radiology and cardiology often apply
high radiation doses to patients’ skin [75]. Gavagan et al. [76], in a study about hair loss in
neuro-interventional procedures, found out that 7% of the 958 patients were reaching the
threshold for temporary epilation, that is suggested to be 2-3Gy [65, 76-79]. The radiation
skin exposure is not only for patients, as hair loss has been described in cardiologists´ legs
[66].
Radiotherapy is a common modality in cancer treatment and more than 50% of affected
patients will eventually receive some form of radiotherapy as definite, preoperative,
postoperative or palliative treatment [80]. Radiation-induced skin changes and associated hair
loss are severe complications of this treatment, but, unfortunately, to achieve a curative dose
to the tumor, some degree of damage in the surrounding tissues occurs [81]. Hair loss may be
transient after therapy, with new hair continuing to return for up to one year, or may be
permanent, due to follicular fibrosis [82].
In any case it is a feared side effect for cancer patients, so feared that some patients may
even refuse treatment because of the risk of developing hair loss [83]. Hair loss, even if not
permanent, may be identified by the patients as a visible reminder of their cancer that causes
distress both by identifying them as a cancer patient as well as by confronting them with the
seriousness of their disease [84].
Temporary epilation was reported among atomic bomb survivors in Hiroshima and
Nagasaki. The historical data from Hiroshima have shown that epilation appeared with
estimated doses as low as 0.75Gy [85].

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 Radiation Treatment and Alopecia – Past and Present Concerns 479

Radiation-Induced Alopecia – Clinical Features and Pathogenesis

Radiation dermatitis is a skin reaction that can occur upon the radiation
treatment/intervention, being the reddening of the skin and dry desquamation its initial signs
[86, 87]. The skin changes caused by radiation dermatitis are associated with an increase in
transepidermal water loss that precedes the onset of dermatitis [88]. The barrier impairment is
comparable to the changes observed with UV radiation exposure but exhibits an even more
delayed course.
The dryness and hair loss are secondary to injury to sebaceous glands and hair follicles.
Permanent or cicatricial alopecia can be defined as any degree of alopecia persisting for >
12 months after radiotherapy completion [67]. This cutting point of one year may be not
enough, as it has been shown that dermal atrophy, with associated loss of hair follicles,
continues to progress beyond 52 weeks after exposure to ionizing radiation [89].
The permanent alopecia results from irreversible damage to epithelial stem cells locating
in the bulge region of the hair follicle [90]. When it occurs during a more generalized
destructive event within the skin, such as thermal burn, trauma, infection, ionizing radiation,
it is designated as secondary [91, 92]. The clinical features of this disorder include destruction
of the hair follicles, progressive hair loss, and permanent replacement of the follicle with
fibrous tissues [93]. Histologically we can observe that hair follicles and sebaceous glands are
absent throughout large areas [10]. The only remnant of the pilosebaceous apparatus is the
arrector pili muscle, often embedded in a pear-shaped mass of collagen.
Scalp hair grows in cycles with 3 distinct phases: anagen when active hair growth occurs
(lasts for 2 to 8 years), catagen that represents involution (lasts for 4 to 6 weeks), and telogen
that is the resting phase (lasts for 2 to 3 months) [94]. Radiation-induced alopecia is due to
high susceptibility of anagen follicles to radiation [95]. Loss of dystrophic hairs (anagen
effluvium) due to acute damage to actively dividing matrix cells of anagen follicles (active
hair growth) is followed by telogen shedding due to premature catagen entry of follicles in
late anagen [95, 96]. Complete hair regrowth generally occurs 2−4 months after irradiation in
the reversible type of radiation-induced alopecia [95].
Hair follicles from the scalp appear to be more radiosensitive than those in other parts of
the body [97-99].

Prevention and Treatment of Radiation-Induced Alopecia

The most common factor associated with radiation-induced skin injury is the duration of
the exposition at a single site of the skin, especially if the same target is repeatedly irradiated
[100]. In the mouse model, Nakayama et al. [96] also found that the extent of follicle damage
occurred in a radiation dose-dependent manner. Lawenda et al. [67] have analyzed several
variables that could be associated with radiation-induced alopecia and found out that the
follicle dose was the only one that was statistically significant, although a personal history of
alopecia (before the radiation treatment) and chemotherapy treatment were of borderline
statistical significance. In fact, numerous chemotherapeutic agents have been shown to cause
temporary, or even permanent, alopecia [67], many of which are considered radiosensitizers,
as they enhance the effects of the radiation on normal tissues [101]. Increased awareness of
potential interactions between radiotherapy and concomitant chemotherapy has led no new
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480 Paula Boaventura, Dina Pereira, José Teixeira-Gomes et al.

treatment schedules designed to maximize antineoplastic effects while minimizing skin

toxicity [86].
In the fluoroscopically guided interventional procedures, when performing repeated
exams for the same target vessel or organ, the orientation of the X-ray beam should be
adjusted whenever possible to avoid overlapping [109]. Nevertheless, even with the use of
dose-spreading techniques, different areas of irradiation can still overlap on the skin surface
that will receive a higher irradiation dose [100].
Concerning the radiotherapy treatments, it has been reported that the skin reaction is less
severe with dose fractionation [99]. Moreover the use of stereotactic radiotherapy, a modality
that combines the accurate focal dose delivery of stereotactic radiosurgery with the biological
advantages of conventional radiotherapy [102], minimizes the dose to non targeted tissues.
The use of arcs can minimize the follicle dose, because arcs typically do not overlap, and the
surface dose is distributed over the length of the arc length [67]. The newer intensity-
modulated radiation therapy (IMRT) has resulted in small volumes of normal tissue receiving
the full treatment dose, with a consequent decrease in acute dermatitis [87].
Some biological factors that influence the threshold for skin effects have been referred by
radiation biologists such as topographic localization of the exposed site, nutrition status,
concomitant diseases, age, genetic factors including predisposition to DNA repair genes
defects, oxygen status, capillarity density and probably also hormonal status [68, 99].
Several studies have been conducted to assess the outcome of interventions for the
prevention and management of radiation skin reactions [103]. Histamine, an important
inflammation mediator, has been shown to be involved in the development of dermatitis
[104], but in a mouse model of acute radiation dermatitis, the radiation induced hair loss was
not inhibited by histamine antagonists [105]. Substance P, a product of preprotachykinin-A
mRNA, recently considered as an important regulator of the hair growth cycle, and its
receptor NK1R (neurokin-1 receptor) may play important roles in the development of
radiation-induced hair loss [106]. Again in a mouse model, fibroblast growth factor-1 (FGF1)
could prevent radiation-induced hair follicle apoptosis [96]. However, the structural
instability of the wild type form of this molecule may diminish its potential for practical use.
The use of topical corticosteroids to prevent or treat radiation dermatitis is somewhat
controversial [86, 103]. They have been used due to its anti-inflammatory effects and
inhibition of IL-6 upregulation [107]. However, comparisons of different topical steroids,
either used to prevent or treat acute radiodermitis, have shown contradictory results [86]. In
any case, it could be seen that at best steroids could ameliorate dermatitis, but do not prevent
it [108]. Prostaglandins are potent radioprotective agents [109]. In a mouse model of
radiation-induced hair loss, Geng et al. [110] have shown that these agents enhanced hair
regrowth following irradiation, both with systemic and topical applications. They concluded
that these compounds might provide some protection of hair follicles lying within the
radiation therapy field. A subsequent study from these authors confirmed that prostaglandins,
but also thiol compounds, provided a significant protection of hair follicles [109].
Nitroxides (stable free radicals) were tested, again in an animal model (guinea pig), in a
fractionated radiation treatment [111]. The authors showed that the topical application of the
nitroxides reduced the post-irradiation hair loss in their model and suggested that these
compounds could be useful in a clinical setting to reduce radiation-induced alopecia. In
humans, a nitroxide (Tempol) was tested in a Phase I study [112] that demonstrated that its

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 Radiation Treatment and Alopecia – Past and Present Concerns 481

topical application to the scalp before whole brain irradiation was safe, well tolerated, and
was showing up protection against radiation-induced alopecia.
More recently, the protective effect of vitamin D3 was investigated in a rat model of skin
radiation injury [81]. The authors showed that the administration of vitamin D3 may protect
hair follicles from radiation toxicity and stated that further clinical trials are needed to prove
the preventive effect of vitamin D3 as well as dosing and timing of the agent on radiation-
induced alopecia.
Calendula officinalis was found to be highly effective for the prevention of acute
dermatitis of grade 2 or higher in breast cancer patients [113], but its topical application was
considered difficult by the patients and no other studies have been conducted to date [103].
Scalp cooling for hair loss prevention and 2% topical minoxidil for quicker hair regrowth
are currently the best studied and most effective options for treatment for chemotherapy-
induced alopecia [84]. However, these interventions are not commonly used for radiation-
induced alopecia. For scalp cooling, only a pilot study was found, assessing its use for
palliative whole brain therapy [114]. The study involved only seven patients and the authors
concluded that the effect of scalp cooling on radiotherapy associated alopecia remained
uncertain, although they observed some hair preservation over the parietal regions of the
patient’s scalp. Minoxidil use for radiation induced permanent alopecia was referred in one
study, in one patient, but it revealed ineffective [115]. Radiation-induced hair loss has been
much less investigated than the chemotherapy-induced alopecia [81]. Moreover, a lack of
trials in human populations has been making it difficult for the application of the most
promising interventions that are being experimented in animal models [84].

CONCLUSION
Radiation induced epilation is no longer at use but individuals submitted to this
intervention, either for aesthetic or therapeutic reasons, are still alive, and the data strongly
suggest that they should be followed-up for increased risk of head and neck cancer and well
as other diseases.
Prevention of radiation induced alopecia is an actual concern in routine medical measures
such as intervention radiological procedures and in radiation therapy, and more approaches
are under current investigation in order to be able to manage this most disturbing side effect.

ACKNOWLEDGMENTS
This work was supported by a grant from Calouste Gulbenkian Foundation (ref. 76636)
and FCT (project: PIC ⁄IC ⁄83154 ⁄2007) and further funding from the Portuguese Foundation
for Science and Technology (FCT), by a grant to P.B. (SFRH ⁄BPD ⁄34276 ⁄2007).
IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology
and Higher Education and is partially supported by the FCT.
The authors thank Ana Reis for proofreading.
Prize ACS-MERCK SERONO in Cancer Epidemiology, 2010.
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482 Paula Boaventura, Dina Pereira, José Teixeira-Gomes et al.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 19

PSYCHOSOCIAL ASPECTS IN ALOPECIA AREATA:

STUDIES ON STRESS INVOLVEMENT
IN ADULTS AND CHILDREN

Liana Manolache
Cetatea Histria Polyclinic, Bucharest, Romania

1. INTRODUCTION
Hair is very important in our lives, even since childhood, so hair loss could affect both
self-image and social relations.
The psychosocial aspects of alopecia could be described by stress as a potential cause or
effect of the disease, the anxiety or depression of patients, or the impact of alopecia
(especially alopecia areata) on patient’s quality of life.
Stress is an abnormal or extreme physiological adjustment in animals to cope with
adverse effects and management of their environment. The reaction to stress could be
influenced by genetics and also by someone’s perception. The stressors could be
environmental, behavioral or psychological [1].
The aetiopathogenesis of alopecia areata is complex, and includes genetic factors,
autoimmune processes, infectious factors and psychological factors (stress and personality
characteristics of patients).
First observations are dating from early ‘60’s when alopecia areata was related to mental
stress [2, 3]. It took about 15 years to come again to the idea of “alopecia areata and stressful
events” [4] or correlating hair loss in children to underlying emotional disturbance [5].
In 1980 the combination “psyche and skin“ [6] appeared, mentioning case reports of
alopecia areata as psychosomatic dermatoses.
Studies on personality traits of alopecia areata are not concordant. Some of them describe
patients with alopecia areata as having psychopathological morbidity more often than in the
general population. Such morbidity includes depression [7-10], anxiety [7-13], social phobia
[8], paranoid disorder [8] and adjustment disorders [9]. Other studies have not found any
difference in anxiety or depression compared to controls [14].
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490 Liana Manolache

Patients with alopecia areata are considered by some authors to lack symbolic or
language schemes of representation for experiences of separation and loss, which affects
personality and creates a devoid-of-affect impression. There is an inability to link sorrow and
body pain [15]. Alopecia areata patients have high rates of alexithymia and avoidant behavior
that could reduce the ability to cope with stress [16-18].
There are different opinions regarding the involvement of stress in alopecia areata. Some
believe that general events could appear in up to 80% of cases with alopecia areata [19]. On
the other hand, Tan’s study [20] found that stressful events preceded hair loss in only 9.8% of
132 alopecia areata patients. van der Steen et al. [21] did not correlate the pathogenesis of
alopecia areata with emotional stress. As for children and adolescents are even fewer reports
regarding stress, starting from no correlation with stress [22] to involvement of stressful
events in up to 80% of children [23]. No data available of stress involvement in diffuse hair
loss in children.
We performed some studies to add to this knowledge, with the purpose of observing
stress involvement before the onset of childhood and adulthood alopecia areata and also of
childhood diffuse alopecia. Furthermore, we relate the results to the psychosocial aspect of
alopecia areata.

2. STUDIES OF STRESS INVOLVEMENT IN ALOPECIA AREATA IN

CHILDREN, ADULTS AND DIFFUSE ALOPECIA IN CHILDREN
Our study was performed at the Department of Dermatology of Cetatea Histria Polyclinic
in Bucharest, Romania. Patients (children and adults) were referred to the polyclinic by
general practitioners in the city and its surrounding areas (approximately 500,000
inhabitants).
There were two different studies: a) one conducted between March 2001 and December
2005 for the adults (≥ 15 years old) and b) one conducted between March 2001 and December
2006 for children (≤ 14 years old). We have decided to enroll only children up to 14 years old
(included) because usually around 15 years old in Romania there is an important exam to pass
from secondary school to high school that could have influenced a lot the situation of stressful
events involved.
The studies design was case-control, with each patient having an age- and gender-
matched counterpart. Controls had skin diseases with a well-established etiology with a
presumably low psychosomatic component, or had skin diseases unrelated to stress
(e.g.bacterial, viral, and fungal infections, Tables 1, 3).
For the adults, Holmes and Rahe’s social readjustment rating scale [24] was used for both
cases and controls. For children, we performed interviews with patients and parents. We have
taken in consideration the potential stressful situations or life events occurring during the year
before the evaluation were included and those occurring after the onset or exacerbation were
excluded. Susceptibility to illness and mental health problems can be influenced by increase
in stress levels caused by life events. We divided situations described in Holmes and Rahe’s
scale into three groups: family matters, personal problems, and job or financial problems.
After patients had filled in the questionnaire they were also interviewed in order to clarify
situations that were not included in the scale (or slightly different, for example, spouse

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 Psychosocial Aspects in Alopecia Areata 491

beginning to work outside home; meaning attached to going to work abroad), but were
considered important in their life. The situations reported by children and parents were
classified into: events related to school and education, family changes, personal
illnesses/accidents/surgeries, and psychosocial trauma (frightening situations to children).
This classification, made after the collection of data, could be considered arbitrary without
other references, but we determined this categorization to underline the importance of events
related to events of importance in childhood.
Odds ratios were calculated and χ2 and t tests were used in order to study the differences
between the groups, and used the standard significance value of p ≤ 0.05.

2.1. Study on Adults

123 cases of alopecia areata were found in 16910 new dermatology consults in patients (≥
15 years old). The incidence of alopecia areata was 0.72% of all dermatologic conditions. 45
patients with recent onset/recurrence not longer than 3 months before the evaluation were
included in our analysis.

2.1.1. Demographics
There were 27 females (60%) and 18 males (40%) in the alopecia areata group. Mean age
was 30.6 years old (standard deviation, SD=11.96). For the control group the mean age was
31.04 years old (SD=12.32).

Table 1 General data about the groups in adults

Alopecia areata (AA)

women men Total (%)
Socio-professional level
Pupil/student/High 10 11 46.7
Average 13 5 40
Low (housewife, unemployed or retired) 4 2 13.3
Localization
1. multiple patches 6 1 15.6
2. beard 0 4 8.9
3. parietal /vertex 14 8 48.9
4. occipital 5 4 20
5. temporal 1 0 2.2
6. ophiasis 0 1 2.2
7. universalis 1 0 2.2
Age %
1. 15-20 years old 6 7 28.9
2. 21-30 years old 10 4 31.1
3. 31-45 years old 4 5 20
4.>45 years old 7 2 20
women men
Controls
Superficial mycosis 19 12
Benign tumors 8 6
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492 Liana Manolache

There was no significant difference (p=0.3) between the mean age in women (32.07
years, SD= 12.75) and men (28.38 years, SD = 10.76).
Data regarding distribution according to age group, lesion type, socio-professional level
was collected (Table 1). In the alopecia areata group there was a female predominance in
patients between 21 and 30 years of age and in those > 45 years of age. Almost half of
alopecia areata patients had a single patch situated in the parietal or vertex area (48.8%). One
male patient (2.2%) with alopecia areata had a family history of alopecia areata. Eight
patients (17.7%) (seven females and one male) had previous episodes of alopecia areata.

2.1.2. Stress Involvement

In alopecia areata group, 31 o f 45 (68.9%) patients identified stressful events compared
to 10 of the controls (22.2%). The difference was statistically significant (χ2 =17.919,
p<0.0001). The odds ratio was 7.75 [95% CI: 3.0135-19.931]. There was no significant
statistical difference (p=0.36) between men and women (stress involvement in 74.1% of
female cases and 61.11% of male cases). We found a significant difference in the mean
number of stressful events between alopecia areata patients and controls (P=0.005). This
difference was maintained when men (P0.05) and women (P=0.001) were analysed
separately.
The presence of one event (P <0.001) with a greater impact before the onset of alopecia
areata seems to be more important than multiple potential stressful situations (77.4% for
alopecia areata and 50% for controls).
Regarding the types of events mentioned (Table 2), the most important matters in the
alopecia areata group were related to family (45.6% of stressful situations) (P=0.0004),
especially in women (P=0.0002). Among family problems, the most often noted were death
of a family member and family disputes (together comprising two-thirds of issues). Personal
matters were mentioned by 35.7% of the alopecia areata group, and there was a statistically
significant difference between patients and controls (P0.04). Exam periods were noted by
both patients and controls as potential stressful situations. In the control group, the most
important problems were almost equally shared among the three categories of problems.
Patients commented on other potential stressful situations than those listed by Holmes and
Rahe.
Working abroad is a new and increasing potential stressful situation in Romania, with
pressure for both the person working outside the country and for the partner staying at home
(both situations were mentioned in our results). This situation even increases since 2005 in
the EU conditions. Overworking is another potentially stressful situation, particularly for
young men who are developing their own firms and for those who have pressure to succeed.

2.2. Study on Children/Adolescents

53 cases of alopecia areata were found in 6917 new dermatology consults in patients (≤
14 years old). The incidence of alopecia areata was 0.76% of all dermatologic conditions.
From those 53 children with alopecia areata we have chosen 43 with the onset not more than
9 months before the evaluation. We have included the same number of children with diffuse
alopecia.

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 Psychosocial Aspects in Alopecia Areata 493

Table 2. Comparison between Alopecia areata and Control Group regarding

the Types of Events involved

Types of events Alopecia areata Control group p

group
F M T F M T Fem. males total

1. Family problems 18 3 21 5 1 6 0.0002 - 0.0004

Death of a family member 7 0 7 0 0 0
Illness of a family member 3 1 4 3 0 3
New person in the family 0 0 0 0 1 1
Family disputes 4 2 6 1 0 1
Reconciliation 1 0 1 0 0 0
Partner’s unemployment 1 0 1 0 0 0
death of a friend 1 0 1 0 0 0
member of the family leaving 1 0 1 1 0 1
home
2. Personal problems 8 6 14 3 3 6 0.08 0.25 0.04
personal illness/accident 1 1 2 0 0 0
pregnancy/birth 1 0 1 1 0 1
sexual problems 2 0 2 0 0 0
exams/ ending school 0 5 5 0 3 3
change of residence 1 0 1 1 0 1
major changes of sleep 2 0 2 1 0 1
major changes of food habits 1 0 1 0 0 0
3. Job/financial problems 8 3 11 5 2 7 0.34 0.63 0.29
dismissing/ unemployment 0 0 0 1 0 1
resignation 1 0 1 0 0 0
change of economic status 2 2 4 0 0 0
change of office 0 0 0 1 0 1
incapacity to pay the debts 0 0 0 1 0 1
change of responsibility at 1 0 1 0 0 0
work
problems with superiors 1 0 1 0 0 0
change of schedule/job 1 0 1 1 2 3
conditions
debts less than 10 times average 1 0 1 1 0 1
salary
Overworking 0 1 1 0 0 0
Working abroad 1 0 1 0 0 0

2.2.1. Demographics
There were 25 girls (58.14%) and 18 boys (41.86%) with alopecia areata and 40 girls
(93.02%) and 3 boys (6.98%) with diffuse alopecia. The youngest child for both types of
alopecia was 18 months old.
Data regarding mean age, distribution according to the age group, types of lesions,
families, onset of lesions are presented in the Table 3. The mean age for alopecia areata group
was 7.78 years old (SD=3.46). Diffuse hair loss group has the mean age of 9.04 (SD=3.82).
There was an almost significant difference between the mean ages in the two groups of
alopecia (p=0.06).
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494 Liana Manolache

Table 3. General data about the children groups

Alopecia areata (AA) Diffuse alopecia

girls boys Total girls boys Total
Family
Only child 9 12 24 1
Socio-professional level %
High 4 3 16.27 4 9.31%
Average 12 8 46.51 21 2 53.48%
Low (mother housewife, one 4 7 25.58 8 1 20.93%
parent unemployed or
retired)
Separated/divorced parents 3 0 6.97 7 0 16.27%
One parent deceased
2 0 4.65 0 0
Onset of lesions
1. < 3 months 20 13 76.74 4 1
2. 3-6 months 4 4 18.6 29 2
3. 6-9 months 1 1 4.66 7 0
Localization
8. Alopecia areata patients
9. multiple patches 9 6 34.88
10. parietal /vertex 9 5 32.55
11. occipital 3 6 20.93
12. temporal/frontal 3 1 9.12
13. totalis 1 1 2.52

Mean age 7.37 years 8.33 years 7.78 years 9 years 9.66 years 9.04 years
(SD=3.14) (SD=3.7) (SD=3.46) (SD=3.88) (SD=2.86) (SD=3.82)

Age %
1. <5 years 6 4 39.53 6 0 13.95%
2. 5-9 years old 11 6 37.2 14 1 34.88%
3. 10-14 years old 8 8 23.25 20 2 51.16%
Girls boys Girls boys
Controls
Mycosis
1. tinea pedis 1 3 2 1
2. tinea manuum 1 0 0 0
3. tinea corporis/faciei 4 4 6 0
4. pityriasis versicolor 7 6 11 0
Verruca 7 2 8 1
Impetigo 5 3 13 1

Most of the children had multiple patches or parietal hair loss. During the evaluation
period, there was a boy with the extension of hair loss from multiple patches to alopecia
totalis.
There were 2 boys (4.7%) with a family history of alopecia areata. Five cases (2 girls and
3 boys)-11.62% had previous episodes of alopecia areata.

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 Psychosocial Aspects in Alopecia Areata 495

In both groups of alopecia patients, children were coming from families with an average
socio-professional level- Table 3. Important to mention that in diffuse alopecia there was a
high rate of patients with separated/divorced parents.

2.2.2. Stress Involvement

Twenty-five children with alopecia areata from the group of 43 had mentioned a stressful
situation before the onset of hair loss (58.13%), compared to 7 children in controls (16.27%).
The odds ratio was 7.14 [95% CI: 2.53-22.60]. There was no difference between girls
(60.0%) and boys (55.5%); χ 2 =14.36, p=0.000 2.
Twenty-nine children with diffuse alopecia from the group of 43 had mentioned a
stressful situation before the onset (67.4%) compared to 9 children in controls (20.9%). The
odds ratio was 7.82 [95% CI: 2.95-20.70]; χ 2 =17.02, p <0.001.
88% of alopecia areata children, 86% of diffuse alopecia children and almost all controls
had mentioned only one event. The comparison between the two hair loss groups was not
statistically significant (p=0.21). The mean number of stressful events has reached a statistical
significance in both groups of hair loss (p<0.0001).
56% of the events in alopecia areata group were related to school. The other half was
shared among family problems, psycho-traumas or other illnesses or accidents. For control
patients there were only situations regarding school-Table 4.
54% of the situations in diffuse alopecia group were related to school/kindergarten. It is
important to mention the presence of previous illnesses/accidents or operations or parent’s
separation as potential stressful events that could lead to diffuse hair loss. For girls (most of
patients) in both alopecia areata and diffuse alopecia the events related to school were
statistically significant different compared to controls (p=0.02, p=0.03).

Table 4. Comparison between Patients’Group and Control Group regarding the Types
of Events involved

Type of event AA group Control p DA group Control p

group group
g b T g b T g b T g b t g b t g b t
related to school/kindergarten 11 2 13 4 2 6 0.02 1 0.06 17 1 18 8 2 10 0.03 0.5 0.06
- beginning 5 0 5 3 1 4 7 1 8 4 1 5
- exams 1 0 1 0 0 0 5 5 3 1 4
- change school/class 1 0 1 0 0 0 1 1 0 0
- problems/over-solicitation 4 2 6 1 1 2 4 4 1 1

family problems 3 3 6 9 9
- death of a family member 0 1 1 0 o
- disputes 2 2
- financial problems 2 1 3 1 1
- parents left for work abroad 1 0 1 0 0
- separations/divorces 0 1 1 4 4
- illnesses in the family 1 1
- change of residence 1 1

personal 2 1 3 6 6
illness/accident/operation

psycho-trauma 2 2 4
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496 Liana Manolache

3. DISCUSSION
The spectrum of incidence for alopecia areata ranges from 0.7% in India [25] (similar to
our result), 0.94% in China [26], 1.5% in Korea [27] up to 3.8% in Singapore [20].
As for the pediatric population, the results of the literature are much higher than in our
report 2.4% in Thailand [28] and 6.7% in Kuwait [29].
Most studies are reporting alopecia areata more often in males [25-27, 30], but the study
of Tan [20] has mentioned females mostly affected by patchy hair loss.
In childhood alopecia areata appeared mostly in girls in some studies, like in our results
[25, 29]. But there are also reports of alopecia areata more frequent in boys [31-33].
The mean age of our adult cohort (30.6 years old) is similar to the one of Yang [26]
(28.98 years old). In a study [25] on more than 800 patients, 88% of them were below 40
years old. High incidences in the third (41.8%) and forth (20%) decades are also reported by
Ro [27].
Our result of mean age (7.78 years old) for alopecia areata in children is higher than in
other studies (about 5 years old) [29, 34], but lower than Tan’s mean age of 11.2 years old
[32].
A family history of alopecia areata (2.2% in adults and 4.7% in children) is a lower rate
than all other studies (from 4.6% to 24% in adults, from 10% up to 50% in children) [20, 25-
27, 29-34]. The dimensions of our study sample could be considered a limitation and account
for differences in demographics from other studies.

3.1. Stress Involvement

Genetic factors could be strong in alopecia areata, but there are also other factors
involved, such as infection, socioeconomic factors and also psychological stress [26, 35].
In 1991, there is a case-control study on 92 Saudi patients associating atopy and
psychological stress to alopecia areata [36].
Our study on adults suggests the importance of stressful events as precipitating or
aggravating factor in more than 65% of alopecia areata cases. There was no statistically
significant difference between men and women. In a previous study we identified stressful
events before the onset of alopecia areata in 55% of cases (40 patients in study) compared to
67% of cases with diffuse alopecia (56 patients in study) [12]. In other preliminary data on 58
patients with alopecia areata (adults and children) we found stressful events in 75% of
patients compared to controls, with 20% involvement [13].
Our findings are comparable with those of Wygledowska [19] who reported the mention
of an important general event by 80% of alopecia areata patients, with 62% stating this as a
serious event. There are also studies that are correlating stress with alopecia areata in less than
10% of cases [20] or not at all [21].
It seems that stress in alopecia areata is not recent (i.e., during the past year), the
‘aetiology’ being much more insidious. Old stressful situations are reported more often,
revealing a chronic stress [16]. A case-control study on 90 patients reported total lifetime and
early childhood traumatic disease, alopecia areata patients having a higher score of the global
impact to their traumatic experiences than controls [37].

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We aimed to search for potential stressful life events and their involvement in the onset
or recurrence of alopecia areata. Some studies mention the importance of perceived stress,
which is sometimes even more important than the stressful situation itself for both the first
episode and recurrence [11, 14]. Gupta cited a study by Andersen, in which only 23% of
subjects had recent stresses that occurred less than 3 months before disease onset [38].
Our results showed a significant difference in the mean number of stressful events
between patients with alopecia areata and controls. However, Picardi et al. [17] did not find
significant differences between the same two groups when comparing the total number of
stressful events and the number of undesirable or major events (21 cases studied). Moreover,
the control group had a greater number of uncontrollable events. The authors support the idea
of the influence of personality characteristics (alexithymia, avoidance of attachment
relationships) or poor social support on individual susceptibility to stressful situations.
Our high odds ratio of involvement in stressful events for alopecia areata patients is
important, particularly as we carried out age- and sex-matched control comparisons: many
other observations on stress as a precipitating or aggravating factor have not been related to
control groups.
As well as the impact of stressful life events, psychological vulnerability of the patient is
another factor that could influence the onset of psychosomatic diseases. There is a high
degree of perceived distress among patients with alopecia areata (both first onset or
recidivism) than in healthy control group [11]. Gupta et al. [39] described alopecia areata
patients in their study as having high reactivity to stress; these patients also had higher scores
for depression.
Some of the life situations listed by Holmes and Rahe are related to psychological
characteristics of patients (family disputes, exams, different kinds of changes in activities,
etc.). As we have not investigated any psychological vulnerability, we are unable to comment
on the impact of an event in association with psychological traits.
In alopecia areata group the occurrence of one event with a high impact before onset
seems to be more important than multiple potential stressful situations. It may be that this
event had huge consequences (like death of a family member or dismissal), or that patients
with multiple life events had developed coping mechanisms that helped them to overcome
problems and prevent onset of the disease.
There are no comparative studies on stressful events in relation to dermatological
disorders. In other studies we have found 54.4% stressful events in psoriasis (OR=4.92, χ2
=42.71, p<0.0001), 65.6% in vitiligo (OR=6.81, χ2 =10.73, p=0.0011), 67.3% in lichen planus
(OR=7.44, χ2 =17.58, p<0.001). In terms of the types of events mentioned, the most important
matters in the vitiligo group were personal problems (47.2% of stressful situations). Among
personal problems, exams represented a third of problems. As for psoriasis and lichen planus,
comparing the presence of major life events (separation, death or illness of a family member,
pregnancy, personal illness/accident, dismissing/unemployment, debts > 10 times average
salary), there was a significant difference (p<0.0001) between patients and controls.
Regarding the types of events mentioned, the most important matters in the psoriasis and
lichen planus group were related to family (>42% of the stressful situations). Among family
problems, most often cited were death/illness of a family member and also family disputes.
There is a lack of studies in pediatric dermatology to which to compare our data. Our
report, which found that 58% of pediatric patients reported stress involvement in the natural
history of their disease, was statistically significant: χ2 =14.36, p=0.0002 with an odds ratio
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498 Liana Manolache

of 7.14. It seems to be important to mention that 64% of alopecia areata boys were single
child in the family. The use of interviews instead of scale to show the presence of stressful
situations before the onset of hair loss could be considered as a limitation, but the results are
statistically significant comparing patients with controls.
There are reports that alopecia areata pediatric patients experienced more stressful events
[40]. In studies regarding alopecia areata in children and teenagers stress seemed to be a
precipitating factor in 9.5% of cases (up to 3 months prior to onset of disease) [34] up to 80%
of cases [23].
Liakopolou et al. [41] correlate the alopecia areata in children with the lack of positive
events during the time before the onset (33 cases). There are other studies [22] that had found
no significant difference between the mean number of positive or negative life events in
children with alopecia areata (12 cases compared to a normative sample).
The types of events noticed by children with alopecia areata were mostly related to
school (beginning school or kindergarten, exams at the end of gymnasium, change of class or
school, problems with school-mates or teachers, too many classes or homework, children
feeling over-solicited).
The difference between patients and controls had reached a statistical significance in girls
(p=0.02). Other potential stressful situations were related to family (death of a family
member, family disputes, financial restrictions, parents being abroad for work). Other
illnesses, accidents or surgery in children, or situations involving a great feeling of fright were
also mentioned.
The study of Andreoli [23] on 180 children and teenagers has proposed as potential
stressful events: separations (from people, pets, habits, things or familiar environment) in
37% of cases, relational problems (in family, school, with friends) in 32% of cases, but also
the difficulties for the child to fulfill the parents’ expectations (especially in school activity)
in 24% of cases.
Other data [34] had found similar types of events involved before the onset of alopecia
areata in children: family disputes, starting school, parent’s divorce, operation, but also
different kinds (birth of a sibling, commencement of speech therapy).
Our results regarding diffuse alopecia in children are the first, so there is no data to refer
to, yet. Situations related to school/kindergarten were mentioned in more than 50% of patients
in diffuse alopecia group. Previous illnesses/accidents or operation or parent’s separation
were also reported as related to diffuse hair loss.
We also studied, in a similarly designed case-control study, children with vitiligo (41
cases) and psoriasis (41 cases). In the vitiligo group, we found stress involvement in 53% of
cases (17% in controls).
This difference was significant (χ2 =7.79, p=0.005). The odds ratio was 5.25 [95% CI:
1.73-15.92] [42]. The types of events reported by children with vitiligo were mostly related to
school, i.e., beginning school or kindergarten, exams, change of class or school, too many
classes or homework. In children with psoriasis, stress was present in 41% of cases (17% in
controls). The difference was statistically significant (χ2 =4.77, p=0.028). The odds ratio was
3.44 [95% CI: 1.23-9.57].
Girls with psoriasis vulgaris and boys with guttate lesions were more often affected by
stressful situations. Family issues (death, illnesses, disputes, parents working abroad,
financial restrictions) were more often described, but school- related problems (exams or
beginning school) were also prevalent.

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3.2. Psychiatric Symptomatology

As well as the impact of stressful life events, psychological vulnerability of the patient is
another factor that could influence the onset of psychosomatic diseases. Gupta et al. [39]
described alopecia areata patients as high reactors to stress and also having depressive
symptoms.
Alopecia areata patients tend to have high scores for anxiety [7-11, 43, 44], depression
[7-10, 43, 44], obsessive-compulsive disorder [10], and adjustment disorder [9]. Furthermore,
phobic states [43], social phobia [8], paranoid disorder [8] could also appear in alopecia
areata patients.
Patients with alopecia areata also have high rates of alexythimia [17, 45, 46]. They have a
tendency to avoidant relationships and poor social support. Alexithymic patients may suffer
from unnoticed chronic stress with impaired immune response [46].
There are also reports that found no difference between alopecia areata patients and
controls regarding the degree of anxiety and depression [14, 46]. Other notes are mentioning
psychiatric disorders (anxiety, affective disorder, substance use disorder) in first-degree
relatives of alopecia areata patients [7]. Patients with alopecia areata could have an important
risk of a family disfunction [47].
Alopecia areata patients seem to have more often Behaviour pattern A indicating a
certain type of relations between the individual and the environment and raising the risk for
the disease [48].
As for pediatric patients with alopecia areata there are a few studies with a high rate of
major depressive disorder (up to half of cases) and obsessive-compulsive disorder (in a third
of cases) [49]. Children with alopecia areata seem to be more anxious or depressed,
withdrawn, aggressive and delinquent. They could have problems in social relations and in
attention. Children are more worried and have difficulties in concentration [41]. In a previous
study we have described in children with alopecia areata anxiety, depressive symptoms,
inhibition, fear of confrontation, relational problems (conflicting relationships), need for
support and security, adjustment troubles [13].
Other reports found rates of anxiety and depression in normal limits [22].
Overall, the psychiatric comorbidity is requiring psychiatric evaluations of alopecia
areata patients. A close relationship between dermatologist and psychiatrist could offer a
better management of the disease. There are mentions of hypnosis as associated method to
improve and maintain the psychological well-being in refractory alopecia areata [50, 51].

3.3. Quality of Life

Hair loss could have an important impact on the patients’ self-image. There are not so
many studies on the influence on the quality of life.
Patients have certain beliefs regarding their diseases. Referring to alopecia areata ones,
more than 75% of them believed that the role of the stress was the cause of their hair loss.
More than half of the patients believed that their illness had major consequences on their
lives, only 57% of them considering treatments to be effective [52].
It is important to measure the impact of the disease on the patient’s life. Dermatology
Life Quality Index (DLQI) is the first dermatology-specific quality of life questionnaire
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500 Liana Manolache

developed in 1994, composed of 10 simple questions validated in different languages. The

scores range from 0 to 30 (0-1: no effect on patient’s life, 2-5: small effect, 6-10: moderate
effect, 11-20: very large effect, 21-30: extremely large effect). There is a study with
significant differences between the mean DLQI score in severe forms of alopecia areata than
in controls [53]. These forms have to be treated very early, requiring complex approach and
psychological support.
Sometimes, our questionnaires are not correlating with patients’ own evaluation. As in
women with alopecia (alopecia areata, effluvium telogenum or androgenic alopecia) that
scored more severe than the dermatologist both the severity and the impact on the quality of
life, revealing a different perception [54].
Alopecia areata patients who cope well with their condition (especially with extensive
hair loss) have higher self-esteem. There is a study reporting satisfactory adaptation to the
illness with few repercussions in family or social life, work or sexual adjustment. Poor
adjustment was associated with dependent or antisocial personality, generalized anxiety and
depression [9].
A new study [55] on patients’ coping reports that alopecia areata patients do not have
dysfunctional coping strategies in general, but they could benefit from psychological
interventions focusing on training general and alopecia areata- specific coping competences
and regulating negative emotionality and insecurity, especially at the first onset of hair loss.
The clinical presentation of pediatric hair disorders ranges from subtle to disfiguring.
Management of hair disorders requires a holistic approach to the child and family. Young
children usually lack self-awareness and it may be the parent who, projecting their own
concerns onto the child, most acutely feels any associated anxiety. Hair loss for the older
child can lead to low self-esteem, depression and humiliation [56].

CONCLUSION AND LIMITATIONS

The psychological and cosmetic importance of hair in our society is immense.
Stressful situations can be correlated with the onset or recidivism of alopecia areata.
Often, one stressful situation could have an impact on the emotional balance of the patient,
triggering or exacerbating the hair loss. Family problems in adults or periods of adjustments
to new conditions (beginning education) for children could play an important role and require
special attention and the appropriate intervention. The psychological profile and
comorbidities could be also relevant, influencing the impact of potential stressful events in
patients’ lives. Small samples and the use of the questionnaire for adults could be limitations
comparing to larger views offered by in-depth interviews. The recollection of data could also
intervene as a limitation. Case-controls studies, especially in the pediatric area are not so
many, so our results can offer a comparative perspective.

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[50] Willemsen R, Vanderlinden J, Deconinck A, Roseeuw D. Hypnotherapeutic
management of alopecia areata. J. Am. Acad. Dermatol. 2006 Aug;55(2):233-7. Epub
2006 Mar 20.
[51] Willemsen R, Haentjens P, Roseeuw D, Vanderlinden J. Hypnosis and alopecia areata:
Long-term beneficial effects on psychological well-being. Acta Derm. Venereol. 2011
Jan;91(1):35-9.
[52] Firooz A, Firoozabadi MR, Ghazisaidi B, Dowlati Y. Concepts of patients with
alopecia areata about their disease. BMC Dermatol. 2005 Jan 12;5:1.
[53] Al-Mutairi N, Eldin ON. Clinical profile and impact on quality of life: Seven years
experience with patients of alopecia areata. Indian J. Dermatol. Venereol. Leprol. 2011
Jul-Aug;77(4):489-93.
[54] Reid EE, Haley AC, Borovicka JH, Rademaker A, West DP, Colavincenzo M,
Wickless H. Clinical severity does not reliably predict quality of life in women with
alopecia areata, telogen effluvium, or androgenic alopecia. J. Am. Acad. Dermatol.
2011 May 21. [Epub ahead of print].
[55] Matzer F, Egger JW, Kopera D. Psychosocial stress and coping in alopecia areata: a
questionnaire survey and qualitative study among 45 patients. . Acta Derm. Venereol.
2011 May;91(3):318-27.
[56] Harrison S, Sinclair R. Optimal management of hair loss (alopecia) in children. Am. J.
Clin. Dermatol. 2003;4(11):757-70.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 20

THE POWER OF THE GENE: THE ORIGIN AND

IMPACT OF GENETIC DISORDERS
ALOPECIA: CAUSES, DIAGNOSIS AND TREATMENT

Naoki Oiso and Akira Kawada

Department of Dermatology, Kinki University Faculty of Medicine,
Osaka-Sayama, Osaka, Japan

ABSTRACT
Alopecia areata is an acquired disorder involving in the fair follicles and sometimes
the nail apparatus. It is currently believed to be caused mainly by a T-cell mediated
autoimmune mechanism toward hair follicles and sometimes to the nail. It is typically
characterized by asymptomatic well-defined patches showing non-scaring alopecia and
no obvious epidermal changes. Alopecia areata is classified into patchy alopecia areata,
alopecia totalis, alopecia universalis, and other variants of ophiasis type, ophiasis
inversus (sisapho) type, a diffuse thinning type, and acute diffuse and total alopecia. It is
frequently associated with other autoimmune diseases, atopic disease and a psychiatric
morbidity. Autoimmune diseases associated with aropecia areata includes autoimmune
thyroid disease such as Hashimoto’s thyroiditid and Graves’ disease, pernicious anemia,
Addison’s disease, psoriasis, systemic lupus erythematosus, vitiligo, celiac disease,
ulcerative colitis, and multiple sclerosis. This indicates that the presence of genetically
determined susceptibility to not only alopecia areata but also to other autoimmune
disorders. A genome-wide association study recently identified the predisposing genes,
which were classified into hair follicle-associated and immune-associated genes. Some of
the predisposed genes are shared by other autoimmune disorders. Dermatologists are
required to diagnose the affected alopecia precisely and to examine the complicated
disorder. Treatment for aropecia areata is recommended to deicide referring the age of the
patients, the extent of scalp involvement, and the response to the chosen first-line
therapies. Here, we summarize the current understanding of classification, pathogenesis,
and treatment in alopecia areata.


E-mail: [email protected], Tel: +81 72 366 0221, Fax: +81 72 368 2120
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506 Naoki Oiso and Akira Kawada

1. INTRODUCTION
Alopecia areata is an acquired disorder showing patchy to complete hair loss. It is
believed to be caused mainly by a T-cell mediated autoimmune mechanism to hair follicles. It
is frequently associated with other autoimmune diseases. A genome-wide association study
recently identified the predisposing genes which can be classified into hair follicle-associated
and immune-associated genes. Here, we briefly summarize the current understanding in
alopecia areata.

2. EPIDEMIOLOGY
Alopecia areata is one of the common dermatologic disorders associated with genetic and
environmental factors. In the general population, the affected people with alopecia areata
were about 0.1% to 0.2% [1]. The lifetime risk was about 2% [2]. Alopecia areata is
frequently present in individuals having autoimmune disorders, atopic dermatitis and
psychiatric disorders [3, 4]. Alopecia areata can be affected in children and mainly in twenties
to forties.

3. CLASSIFICATION
The classification is based on the extent or pattern of hair loss [5, 6]. The extent of hair
loss can classify alopecia areata into patchy alopecia areata (Figures 1-5), alorcia totalis
(Figures 6a, 6b) or alopecia universalis (Figures 7a, 7b). Patchy alopecia areata is defined as a
partial loss of scalp hair. Alopecia totalis is all loss of scalp hair. Alopecia universalis is all
loss of scalp and body hair. The pattern of hair loss can classify alopecia areata into ophiasis
(Figure 8) [7], sisapho (ophiasis inversus) [8], diffuse [9], and acute diffuse and total alopecia
[10].

Figure 1. Case 1: Clinical appearance of single patchy alopecia on the scalp in a 25-year-old Japanese
female.

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 The Power of the Gene 507

Figure 2. Case 2: Clinical appearance of single patchy alopecia on the scalp in a 43-year-old Japanese
female. The affected lesion was poorly circumscribed.

Figure 3. Case 3: Clinical appearance of multiple patchy alopecia on the scalp in a 51-year-old Japanese
female.

Figure 4. Case 4: Clinical appearance of rapidly progressive multiple patchy alopecia on the scalp in a
25-year-old Japanese female. The affected lesions were poorly circumscribed.

Aloprcia areata ophiasis shows band-like hair loss from the peripheral border of a
primarily hair growing region toward a central region the in parieto-temporo-occipital area.
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508 Naoki Oiso and Akira Kawada

Aloprcia areata sisapho (ophiasis inversus) demonstrates band-like hair loss in the fronto-
parieto-temporal area mimicking male alopecia. Diffuse alopecia areata occurs partially or
totally. Acute diffuse and total alopecia is characterized by a favorable prognosis and rapid
and spontaneous recovery even without treatment [10]

Figure 5. Case 5: Clinical appearance of rapidly progressive multiple patchy alopecia on the scalp in a
40-year-old Japanese female. The affected lesions were poorly circumscribed.

Figures 6a and 6b. Case 6: Clinical appearance of alopecia totalis on the scalp in a 36-year-old Japanese
male. The affected lesions were restricted on the scalp. The eyebrows and eyelashes were intact.

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4. ASSOCIATED DISORDERS
Alopecia areata is associated with nail changes, autoimmune diseases, atopic disease and
psychiatric disorders.

Nail Change

Hair and nails have much similarity of their origin, anatomical structures, and common
involvement [11]. Both are similarly made up of keratinous fibrils embedded in a sulfur-rich
matrix [11]. The nail unit was comparable in several respects to a hair follicle sectioned
longitudinally and laid on its side [11-14]. The nail changes are present in alopecia areata
ranging from 7% to 66% [15]. The most representative disorder is nail pitting and other main
changes are onychorrhexis, Beau lines, longitudinal ridging, onychodystrophy, and
trachyonychia [15, 16]. Individuals having severer alopecia areata tend to have severer nail
alterations [15, 16].

Autoimmune Diseases

Autoimmune diseases associated with aropecia areata includes autoimmune thyroitis such
as Hashimoto’s thyroiditid and Graves’ diseases, pernicious anemia, Addison’s disease,
psoriasis, systemic lupus erythematosus, vitiligo, celiac disease, ulcerative colitis, and
multiple sclerosis [5, 17].

Atopic Dermatitis

An association of alopecia areata with atopic disease (Figure 9) has been described [18-
21]. Betz et al. showed that alopecia areata in patients with atopic dermatitis with loss-of-
function variants in the filaggrin gene tend to become severer types [21].

Psychiatric disorders

The clinical data indicates that stress dose dot strongly trigger the onset of alopecia arata
onset [22]. However, in some cases, aberrant psychosocial disorders including depression,
anxiety, and aggression are associated with the development of alopecia areata [22-24].

5. GENETIC FACTORS
Family and twin studies strongly signified that genetic factors are involved in the
development of alopecia areata as a polygenic fashion [25, 26]. Christiano and colleagues
undertook a genome-wide association study in a sample of 1,054 cases of alopecia areata and
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510 Naoki Oiso and Akira Kawada

3,278 controls and identified 139 single nucleotide polymorphisms that exceeded genome-
wide significance (p < 5 × 10-7), which clustered in eight regions across the genome and
implicated genes of the immune system, as well as genes that are expressed in the hair
follicle: (i) the CTLA4 gene on chromosome 2q33.2; (ii) the IL2/IL21 locus on chromosome
4q27; (iii) the HLA class II region on 6p21.32; (iv) the cytomegalovirus UL16-binding
protein (ULBP) gene cluster on chromosome 6q25.1; (v) the STX17 gene encoding syntaxin
17 on chromosome 9q31.1; (vi) the IL2RA gene on chromosome 10p15.1 7; (vii) the PRDX5
gene encoding peroxiredoxin 5 on chromosome 11q13; and (viii) the IKZF4 gene on
chromosome 12q13 [27, 28]. The study identified two major groups; genes of STX17 and
PRDX5 associated with the expression in the hair follicle, and genes associated with innate
and acquired immunity, most of which were shared with other autoimmune disorders [27, 28].
The proteins encoded by ULBP3 and ULBP6 genes are believed as having the function of
NK-activating ligands in the hair follicles. In non-segmental vitiligo, susceptible genes were
classified into a gene of TYR encoding tyrosinase expressing specifically in the melanocytes,
and genes associated with innate and acquired immunity [29-34]. These findings in both
alopecia areata and non-segmental vitiligo indicate that the susceptible genes include the
targeted proteins expressing in each specific functional tissue and the targeting proteins
expressing in autoimmune related cell lines. The current candidate genes are summarized in
Table 1 [35-71].

Table 1. Genes associated with non-segmental vitiligo

Involved in other
Gene Function autoimmne Reference
disease
(1) autoantigen
A divergent member of the syntaxin family, SNAREs
STX17 (soluble N-ethylmaleimide-sensitive factor-attachment 35, 36
protein receptors)
A member of the peroxiredoxin family of antioxidant
PRDX5 enzymes reducing hydrogen peroxide and alkyl CD, MS 37-39
hydroperoxides
(2) immunity
CD, CeD, GD,
Major histocompatibility complex class II proteins being 30, 32, 40-
MHC MS, PA, PS, RA,
indespensable for antigen presentation 51
SLE, T1D, VIT
CeD, GV, HD,
A member of the immunoglobulin superfamily. The
CTLA4 MS, RA, SLE, 52-58
encoded protein transmits an inhibitory signal to T cells.
T1D
A functional marker for naturally occurring, thymus- GD, RA, PA, PS,
IL2RA (CD25) 48, 58-61
selected CD4+CD25+FOXP3+ regulatory T cells SLE, T1D
A protein reducing self-reactive T cells by binding to CD, CeD, MS,
IL2 62-68
CD25 (IL2R) expressed in the regulatory T cells. PS, RA, T1D
ULBP6/ULBP3 (UL16-binding protein 6/ UL16-binding
ULBP6/ULBP3 protein 3) genes encoding ligands of the activating 69, 70
immunoreceptor NKG2D.
A member of the Ikaros family of transcription factors,
which includes Eos. Eos interacts directly with Foxp3 and
71
IKZF4 induces chromatin modifications that result in gene
silencing in CD4+CD25+FOXP3+ regulatory T cells.
abbreviation; Crohn's disease (CD), celiac disease (CeD), Graves disease (GD), Hashimoto thyroiditis
(HT), interleukin (IL), multiple sclerosis (MS), psoriasis (PS), psoriatic arthritis (PA), rheumatoid
arthritis (RA), systemic lupus erythematosus (SLE), type 1 diabetes (T1D), ulcerative colitis (UC)

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6. PATHOGENESIS
The hair cycle comprises three key phases of anagen (the growth phase), catagen (the
regression phase), and telogen (the resting phase). Three aberrant hair growth cycle patterns
are present in alopecia areata; (i) hair follicles in dystrophic anagen by mild inflammatory
reaction cannot produce significant hair fiber: (ii) hair follicles in truncated anagen by
moderate inflammatory reaction result in rapid cycling and short hair fiber growth; and (iii)
hair follicles in prolonged telogen develop chronic alopecia areata [5]. Hair follicle
inflammation in alopecia areata is believed to be caused by a T cell-mediated autoimmune
mechanism.
Anagen follicles located at the margin of progressing alopecia areata usually illustrate
perifollicular and intrafollicular inflammatory cells infiltrate as “swarm of bees” in the
horizontal section.
The infiltrated lymphocytes are mainly CD8+ and CD4+ lymphocytes which are induced
by up-regulated expression of HLA class I and class II. It is believed that CD8+ T
lymphocytes are responsible for the follicular damage, because CD8+ T cells predominate in
the follicular epithelium in active alopecia areata [72, 73]. Alli et al. constructed a new model
for alopecia areata derived from C57BL/6J mice and showed that clonal CD8+ T
lymphocytes independently mediated follicular destruction from alopecia arata toward
aropecia universalis.
Hair follicle immune privilege in the dermal papilla and the bulbar epithelium and in the
hair bulge areas is the key concept for alopecia areata and cicatrial aropecias. In alopecia
areata, the dermal papilla and the bulbar epithelium in the hair follicles are mainly damaged
by the infiltrated inflammatory cells.
The potential of hair regrowth is found in alopecia areata, because the stem cells in the
hair bulge areas are not involved. In cicatrial alopecias, the hair bulge areas containing stem
cells are chiefly injured.
Therefore, the potential of hair regrowth is lost in cicatrial alopecias. Hair follicle
immune privilege is maintained by several factors including no expression of HLA class I in
the proximal outer root sheath (ORF) and matrix cells [74]. Hair follicle immune privilege
can only be present in the proximal epithelium of anagen hair follicules, but not during
catagen or telogen [74].
The collapse of immune privilege is associated with induction and maintenance of
alopecia areata. Alopecia areata may develop in psoriasis patients treated with TNF-
blockers (infliximab, etanercept or adalimumab) [75]. TNF- blockers may induce adverse
immune-mediated diseases such as leukocytoclastic vasculitis, systemic lupus erythematosus,
and paradoxical psoriasiform eruption [75, 76].
We recently reported a case showing a spontaneous alternation from psoriasis to alopecia
universalis (Figures 7a, 7b), a subsequent treatment-mediated change from alopecia areata to
psoriasis, and a spontaneous swing with the feature of the Renbök phenomenon (normal hair
growth in psoriatic plaques in patients with both aropecia areata and psoriasis) [77] after
stopping the treatment. A swinging from psoriasis to alopecia areata can occur as a result of
change of cytokine balance, even though the mechanism has not been elucidated [78].
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512 Naoki Oiso and Akira Kawada

a b

Figures 7a and 7b. Case 7: Clinical appearance of alopecia universalis in a 45-year-old Japanese female.
The affected lesions were spread beyond the scalp. The eyebrows and eyelashes were affected. The
patient draws eyebrows and uses artificial eyelashes.

8. TREATMENT
A variety of treatments are applied for alopecia areata. First-line therapies for alopecia
areata are usually intralesional or topical corticosteroids and topical immunotherapy [79].
Second-line therapies are topical minoxidil, anthralin, photochemotherapy of psoralen plus
ultraviolet A (PUVA), oral glucocorticoids, sulfasalazine, cyclosporine, and methotrexate
[79]. Alkhalifah and colleagues summarized treatment algorithm for aropecia areata involving
the scalp [80]. This algorithm is currently very useful for treatment for alopecia areata.

Figure 8. Case 8: Clinical appearance of alopecia areata ophiasis on the occipital region of the scalp in
an 11-year-old Japanese female.

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 The Power of the Gene 513

Figure 9, Case 9: Clinical appearance of alopecia universalis in a 36-year-old Japanese male with atopic
dermatitis. The eyebrows and eyelashes were affected, but a beard, a mustache, and body hair were
intact.

CONCLUSION
Extensive progress is being made towards understanding the pathogenesis of alopecia
areata. A well-designed genome-wide association study identified a number of susceptible
genes. However, we have no genotype-phenotype relationship in alopecia areata. Further
study would elucidate the relationship between the destruction of the hair follicle immune
privilege and the development of alopecia areata, and the factors progressing patchy alopecia
areata towards alopecia totalis and alopecia universalis. In the future, we can anticipate
further progression in the association between disease-susceptible genes and gene-
environment interfaces.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 21

ALOPECIA AREATA: TREATMENT OPTIONS

Emina Kasumagić-Halilovic
and Nermina Ovcina-Kurtovic
Department of Dermatovenerology,
Sarajevo University Clinical Center Sarajevo,
Bosnia and Herzegovina

ABSTRACT
Alopecia areata (AA) is common cause of reversible hair loss afflicting
approximately 1-2% of the general population. It commonly present as round patches of
hair loss which can be the first manifestation of a more severe alopecia totalis or
universalis. The etiology of AA is unknown but is characterized by hair cycle dysfunction
and the presence of peribulbar and perifollicular mononuclear cell infiltrates. Much
evidence suggests that AA is tissue restricted autoimmune disease. Current traditional
therapies are predominantly immunomodulating modalities, including corticosteroids,
topical immunotherapy, anthralin, and photochemotherapy (PUVA). These treatments
stimulate hair growth but do not prevent hair loss and probably do not influence the
course of the disease.
A nonspecific modality is topical minoxidil, which prolongs anagen and promotes
growth of longer and wider hair. Improved future treatments may be immunosuppresive
or immunomodulatory or they may otherwise protect hair follicles from the injurious
effects of inflammation. Biologic therapies target specific immunologic responses and
offer new strategies for treating pathogenic T cells and the cytokines they produce. The
choice of therapy depends primarily on the patient's age and the extent of the hair loss.
The aim of this article is to review available data on current and potential agents for the
treatment of alopecia areata.

Keywords: Alopecia areata, therapy


Corresponding author: Emina Kasumagić-Halilovic, University Clinical Center Sarajevo, Department of
Dermatovenerology, Bolnička 25, 71 000 Sarajevo, Bosna i Hercegovina, Tel/Fax: + 387 33 470 872, E-mail:
[email protected]
 Free ebooks ==> www.Ebook777.com
522 Emina Kasumagić-Halilovic and Nermina Ovcina-Kurtovic

INTRODUCTION
Alopecia areata (AA) is a common cause of reversible hair loss afflicting approximately
1-2% of the general population [1] and it is also expressed in several non-human mammals
[2]. It ranges in severity from small, round patches of hair loss that regrow spontaneously to
persistent, extensive patchy involvement to the loss of all scalp hair (alopecia totalis) or all
scalp and body hair (alopecia universalis). Characteristic nail changes may also accompany
hair loss. AA affects both sexes equally and occurs at all ages, although children and young
adults are affected most often.
The etiology of AA is unknown but is characterized by hair cycle dysfunction and the
presence of peribulbar and perifollicular mononuclear cell infiltrates. Scalp biopsies from
patients show a heavy presence of type 1 cytokines, including interleukin-2 (IL-2), interferon-
γ (IFN-γ) and tumor necrosis factor α [3].
The most widely accepted hypothesis is that AA is a T-cell mediated autoimmune
condition that is most likely to occur in genetically predisposed individuals. Although acute
phases of hair loss are followed by spontaneous hair regrowth in most patients, the disorder
may persist for many years or even for life when severe.
But even in these cases hair loss is potentially reversible, because the disease usually
does not result in destruction of hair follicles or scaring [4]. The prognosis of AA is
influenced by several factors in particular by the type and extent of AA [5]. We aimed to
review evidence for the management of alopecia areata and discuss which treatments may
help patients. We also discuss potentially interesting new treatments that require further
investigation.

TREATMENT OF ALOPECIA AREATA

Treatment of AA is still a difficult task for every dermatologist. Current traditional
therapies are predominantly immunomodulating modalities, including corticosteroids,
anthralin, topical sensitizers, and photochemotherapy (PUVA). A non-specific modality is
minoxidil, which prolongs anagen and promotes growth of longer and wider hair. These
treatments stimulate hair growth but do not prevent hair loss and probably do not influence
the course of the disease [6, 7]. Improved future treatments may be immunosuppressive or
immunomodulatory targeting of the autoimmune pathogenesis of AA, or they may otherwise
protect hair follicles from the injurious effects of inflammation (4). Any treatment has to be
suitable for long-term therapy, because AA is a disease that can persist for many years or
even for life. The choice of therapy depends primarily on the patient’s age and the extent of
the hair loss [5, 8].

Corticosteroid Therapy

Corticosteroids are probably most popular form of treatment for patchy AA [4, 6]. The
mechanism of steroid effect in AA is speculated to be immunomodulatory. Corticosteroids
decrease production and/or secretion of interleukin 1, interleukin 2 and monocyte chemotactic

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 Alopecia Areata 523

factor [6]. Topical steroids decrease Langerhans cell members as well as Langerhans cell-
dependent T-lymphocyte activation [7]. Which of these or perhaps other steroid effects are
relevant to hair regrowth in AA is unknown at this time. Corticosteroids can be administered
in four different ways: topically as a cream, foam or lotion, intralesionally as local injection
into the bald patches, and systemically either as injections into a muscle or taken orally.
These different methods of application vary in their potency.

Topical Corticosteroids
Topical corticosteroids are widely used to treat all types of AA and they are the mildest
form of steroid treatment. Response to topical steroid in therapeutic trials has been mixed.
Reports of nearly 100% response for prepubertal children have been reported alongside a
response rate of just 33% in adults [6, 7]. The failure of topical corticosteroids is most likely
duo to the insufficient penetration of topically applied drugs forms ointments, creams or
lotions into the hair bulb [4]. In order to increase the effect of topical corticosteroids, an
occlusive dressing technique can be applied in the treatment Side effects of topical steroids
include local folliculitis, acne outbreaks, local atrophy and very occasionally hypertrichosis.
If doses of topical steroids are too high there is a small risk of systemic absorption and the
potential associated effects [7]. Topical corticosteroids are the treatment of choice in children.

Intralesional Corticosteroids
Intralesional corticosteroids were first described in 1958 with the use of hydrocortisone
[9]. This method is very popular compromise between topical application and systemic use. It
involves the injection of a steroid solution (usually triamcinolone acetonide) intradermally
every four to six weeks. The intention is to get as much of the steroid directly to the root of
the affected hair follicles where the associated inflammatory infiltrate is present.
Corticosteroids suppress the T-cell mediated immune attack on the hair follicle. The
recommended dose per treatment is up to 3 mL of a 5 mg/mL solution injected into the mid-
dermis in multiple sites 1 cm apart. The amount injected into each site is 0.1 mL. For the
eyebrows and face, 2.5 mg/mL can be used. After each injection, a gentle massage of the
treated area is recommended to help prevent treatment-induced atrophy. Initial regrowth is
often seen in four weeks although it can take up to two months before noticeable hair growth
develops [7]. Several studies reported hair regrowth at the site of injection in the majority of
cases [3, 4, 6]. Other dermatologist report less successful response rates [4, 6, 7]. Injections in
frontoparietal areas are not recommended because of the potential risk of thrombosis in the
central retinal artery due to the formation of crystalline deposits. Ferrando and Moreno
propose the use of mesotherapy multi-injectors with 5 or 7 needles, an approach which has
the advantage of optimizing the process while economizing on product use and ensuring
homogeneity, with a shorter application time and a decrease in painful injections [10]. Side
effects can include pain from the injections and atrophy of the skin around the injection site.
However, a topical anesthetic cream such as lidocain –prilocaine can be applied 30 to 60
minutes before treatment to reduce discomfort. This is particularly useful when treating the
pediatric population. The risk of atrophy can be minimized by injecting into the mid-dermis
rather than into the epidermis or the subdermal fat [11]. Use of steroid injections is a popular
form of treatment for eyebrow hair loss [8]. This treatment is not appropriate in rapidly
progressing or very extensive forms. Children under ten years of age not usually treated with
intralesional steroids.
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524 Emina Kasumagić-Halilovic and Nermina Ovcina-Kurtovic

Systemic Corticosteroids
Dillaha and Rothman introduced into the treatment of alopecia areata systemic
corticosteroids since 1952 [12]. Systemic application (oral, intravenous, intramuscular) is the
most powerful form of corticosteroid treatment. They are frequently effective, but their use is
limited because of the high relapse rate after reduction of the dose [8]. In addition, the
presence of side effects with long-term systemic steroid therapy may suggest a dose reduction
or even discontinuation of the treatment. To avoid these complications, pulse-therapy had
been introduced. Several modalities for use of high doses in the form of pulses in different
oral and intravenous regiments have been reported.

Photochemotherapy (PUVA)

Photochemotherapy (PUVA) is term for a combination of using UV-A light with a

photosensitizing drug psoralen (P). The mechanism of action of PUVA on AA is believed to
be a photoimmunologic action. It may affected T-cell function and antigen presentation and
possibly inhibits local immunologic attack against the hair follicle by depleting Langerhans
cells [6].
Psoralen inhibits mitosis by binding covalently to pyrimidine bases in DNA when
photoactivated by UV-A [13]. This drug has been administrated topically (1% 8-MOP
ointment or 0.1% solution) or orally (0.6 mg/kg 8-MOP) and followed in 1 or 2 hours,
respectively, by UV-A radiation. Treatments are given two to three times weekly with a
gradual increase in UV-A dosage and clinical responsiveness is usually seen within 20 to 40
treatments. Success rates with PUVA treatment have varied from 20% to 50%, although the
relapse rate is high [7, 8].
Side effects include nausea with orally administrated drug and burning erythema [7]. The
possibility of skin cancer formation with prolonged treatment should also be considered.
PUVA-turban is a method of administrating a dilute psoralen solution (8-MOP 0.0001%)
selectively to the scalp for 20 minutes using a cotton towel as a turban. The patient's scalp is
then exposed to UV-A radiation [14].
It is a well tolerated therapy with minimal local phototoxic side effects and without the
systemic side effects of PUVA. Technical improvements, such as a comb emitting UV-A
light have been tried, but so far no results have been reported. PUVA is generally limited to
patients over age 12.

Topical Sensitizers

Topical immunotherapy (contact sensitization) is one of the more effective treatments for
patients with chronic AA affecting more then 50% of the scalp [7]. The patients are sensitized
by application to the scalp of a potent contact allergen, and allergic contact dermatitis is
subsequently elicited by weekly applications of the same agent [15]. Initial hair regrowth is
usually visible after 8-12 weeks [4].
Response rates of treatment with contact sensitizers varie from 29% to 78% [5, 15, 16].
The differences may be explained in part by the different extent and duration of AA prior to

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treatment for the patients in each study, and in part by differences in methods of treatment [4].
In 1978, Daman at al. first reported the use of dinitrochlorbenzene (DNCB) to induce hair
growth in two patients with AA [17].
This chemical was first found to be a potent contact allergen in 1912. Today,
diphenylcyclopropenone (DPCP) or squaric acid dibutylester (SADBE) are widely used. The
mechanism by which contact sensitization suppresses AA is uncertain but may involve the
generation of nonspecific suppressor T-cells or the inhibition of proinflammatory cytokines
[15, 16]. Happle proposed the concept of "antigenic competition" where an allergic reaction
generates suppressor T cells that non-specifically inhibit the autoimmune reaction against a
hair follicle constituent [18]. Contact allergens also tend to attract a new population of T cells
in to the treated areas of the scalp, and thus enhancing a clearance of putative follicular
antigen [19].
The other events noted are a decrease in the raised interferon γ levels, increase in mRNA
expression of interleukins 2, 8, 10, and tumor necrosis factor α in the lesional skin [20].
Recent studies demonstrated that treatment with a contact sensitizer induces apoptosis in
perifollicular T cells [21]. The adverse effects of topical immunotherapy include itching,
vesicular or bullous reaction, urticaria, facial and scalp edema, pigmentary disturbances and
cervical lymphadenopathy, which are invariably present [7, 16].

Anthralin

Anthralin is the only irritant substance generally agreed to induce hair regrowth in AA
[22]. The mechanism of anthralin effect in AA can only be speculated on. Anthralin induces
inflammation in a possibly unique manner, primarily by generation of free radicals [23].
Reactive oxidants are potent antiprolifferative and immunosuppressive agents that inhibit
chemotaxis, IL-2 production, cytotoxic activity of natural killer cells, and mitogen induced
transformation of B and T lymphocytes [6, 23, 24]. Anthralin has been shown to be toxic to
Langerhans cells. Concentrations of between 0.25 and 1% applied overnight, can be used.
Alternatively, so called "short-contact therapy" may be used, which involves applications of
30 minutes with progressive increases until reaching an exposure of 1 hour. When therapy is
effective, new hair growth is usually seen within 2 to 3 months after the start of treatment,
and about 25% of patients may have cosmetically acceptable growth in about six months [6,
15]. It takes 24 or more weeks for a cosmetically acceptable response [7]. Application of
topical anthralin may cause pruritus, erythema, scaling, folliculitis and regional
lymphadenopathy [25]. Anthralin is a good choice for children or for those individuals with
extensive AA.

Minoxidil

Minoxidil is a piperidinopyrimidine derivate that acts as a smooth muscle vasodilatator in

the treatment of hypertension. Although minoxidil has been used as a hair regrowing
treatment for more than 20 years, its mechanism of action of on hair growth promotion is still
unclear. It does not have any hormonal effects or immunosuppressant effects; rather, it has
direct effects on the proliferation and differentiation of follicular keratinocytes in vitro, and
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526 Emina Kasumagić-Halilovic and Nermina Ovcina-Kurtovic

regulates hair physiology independently of blood flow influences [7]. Both topical and oral
minoxidil therapy have been tried in AA [26]. Topically, concentrations ranging from 1 to 5%
twice daily have been used. The results reported in the literature have been highly variable,
ranging from little benefit to response rates of over 50% [7, 8].
The average time to response with topical minoxidil is 2 to 3 months [6]. The time to
maximum response is generally about 1 year, although it can be longer [15]. This drug may
be useful in treatment of patchy AA but not alopecia totalis or universalis. It is generally used
at a concentration of 5% in combination with a topical corticosteroid or anthralin, which
enhance its action by increasing absorbtion. Topical minoxidil has also been used in
combination with systemic corticosteroids because this seems to limit hair loss after
suspending corticosteroid therapy [27].
Side effects are limited to mild local irritation and, less frequently allergic contact
dermatitis, and localized facial hypertrichosis. Oral minoxidil (a dose of 10 mg/d) may result
in more extensive and more rapid hair growth. Adverse effects, including fluid retention,
head-ache, depression, palpitations and tachycardia, may make oral minoxidil an
unacceptable mode of therapy for AA [7, 27].

New Immunomodulatory Therapies

Remarkable progress during the last two decades, has brought much progress in the
understanding of the immunopathogenesis of alopecia areata, leading to the development of
more targeted therapies. These therapies have a common therapeutic goal: to reduce or
eliminate the pathogenic effects of T cells in alopecia areata.
Topical immunomodulators are a relatively new class of agent that acts locally on T cells
by suppressing cytokine transcription [28]. They are now emerging as the therapy of choice
for several immune-mediated dermatoses, because of their comparable efficacy, ease of
application and greater safety than their systemic counterparts [29]. The two most studied
topical immunomodulators are tacrolimus and pimecrolimus. A third new member of this
group is topical cyclopsporine A (CsA). All three drugs inchibit calcineurin, thereby
inchibiting interleukin-2 production and limits CD4 lymhocyte cell activity [30].

Topical Tacrolimus (Protopic)

Tacrolimus is macrolide lactone, produced by Streptomyces tsukabaensis, a fungus found

in the soil of Mount Tsukuba, the science city of Japan, where initial isolation and
characterization of this drug was performed in the year 1987. The name of the drug is
neologism, composed of tsukuba, macrolide and immunosuppression.
Tacrolimus is an immunosuppressive agent that can be applied topically to the skin. It
acts directly on T-cells to inhibit IL-2 transcription, which results in decreased growth and
proliferation of T lymphocytes in response to foreign antigens [31]. It also inhibits other
cytokines, including TNF-α and IFN-γ, both important in T-cell activation. Moreover, topical
application of tacrolimus also has a hair growth stimulatory effect, independent of its T-cell
suppressive effect [32]. Tacrolimus ointment does not cause skin atrophy, pigment changes,
or teleangiectasia. Therefore, tacrolimus is promising candidate for the treatment of AA [33].

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 Alopecia Areata 527

Topical Pimecrolimus (Elidel)

Pimecrolimus is a semi-synthetic product of ascomycin, which is fermentation product of

Streptomyces hygroscopicus var. ascomycetes. Similar to tacrolimus, it is a cell-selective
cytokine inhibitor developed for the treatment of inflammatory skin diseases [34]. It binds to
macrophilin-12, inhibits calcineurin, inhibits synthesis of inflammatory cytokines, such as IL-
2 and IFN-γ, and inhibits Tpcell and mast cell activation. Pimecrolimus has high skin-specific
anti-inflammatory activity with low potential for affecting the systemic immune response
[30]. The cream 1% formulation is safe and effective and does not cause skin atrophy or
teleangiectasia.
Unfortunately, the cream is not expected to be effective for hair regrowth because it
permeates no lower than the superficial dermis, which is an insufficient depth for targeting T-
cells involved in AA [30].

Topical Cyclosporine A (Psorban)

Cyclosporine A (CsA), isolated from the fungus Tolypocladium gams, is a lipophilic

cyclic polypeptide and calcineurin inhibitor. CsA is a potent immunomodulatory agent whose
mechanism involves inhibition of T-4 lymphocyte activation [35]. Cyclosporine therapy
reduces the number of T cells infiltrating the hair follicle and the perifollicular area, and CsA
is a potent inhibitor of interleukin 2, a cytokine that stimulates the proliferation of T
lymphocytes [36].
It is known that cyclosporine stimulates T cells and the pilosebaceus unit, thereby
inducing hypertrichosis and sebaceus hyperplasia. Although systemic CsA appears to be
effective in AA, the adverse effect profile, recurrence rate after treatment discontinuation and
inability to produce long-term remissions make CsA unattractive for the treatment of AA. In
the past topical formulations of CsA were ineffective because of poor skin penetration. To
surmount this problem, a heptamer of arginine was conjugated to CsA thought a pH-sensitive
linker designed to release CsA at psysiologic pH within the skin [37].
The oligoarginine transporters enable full-skin-thckness penetration of CsA into cells
throughout the epidermis and dermis of human skin, with functional inhibition of cutaneous
inflammation [38]. A recent publication reports the use of mixture of ethanol and
phospholipids in the formulation of new topical cyclosporine preparations in order to increase
penetration [39].

New Biologic Therapies

Biologics are pharmacologically active proteins extracted from animal tissue or

synthesized through recombinant DNA techniques. They are designed to mimic the action of
normal human proteins or to interact with circulating proteins or cellular receptors. There are
three distinct classes of biologic agents: monoclonal antibodies, fusion proteins and
recombinant cytokines or growth factors [40].
They include Etanercept, Infliximab, Efalizumab and Alefacept. Biologic therapies target
cell surface receptors, and their theoretical advantage is that their greater specificity will
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528 Emina Kasumagić-Halilovic and Nermina Ovcina-Kurtovic

provide better safety profiles [30]. The advent of the new biologic medications raised hopes
for successful control of many immune-mediated diseases, including AA. Based on current
hypothesis regarding AA immunopathogenesis, two main therapeutics approaches have
emerged: modulating either T cells activation or cytokines.
Owing to the suspected involvement of tumor necrosis factor α in the pathogenesis of
alopecia areata, one might expect that biologic therapies with anti TNF-α agens might be
beneficial [41]. On the other hand, there is evidence suggesting that other biologic therapies
that target T cells may represent an effective treatment modality for AA. Some clinical trials
are ongoing to evaluate the efficacy of the newer biologic therapies in the treatment of AA.

Liposomes

Another novel approach in treating AA is to create a vehicle that allows penetration to the
subcutaneous fat where the bulbs of anagen hair follicles are located and where the
pathomechanism takes place [4]. Liposomal drug delivery may increase penetration of skin
and allow slow release of active compound locally with diminished toxicity.
At present, liposomes seem to be the best candidate as a vehicle topical treatment.
Topically applied liposomes have been shown to deliver melanin, proteins, genes and various
small molecules selectively to hair follicles and hair shafts of mice in vivo [42].
Liposome-targeting of moleculas to human hair follicles has been demonstrated in human
scalp in histoculture [43]. However, future experiments have to show whether liposomes are
able to deliver molecules to the hair bulb in human scalp in vivo.

Miscellaneous Agents

Sulfasalazine
Sulfasalazin is an anti-inflammatory agent composed of a sulfonamide and salicylate. It
was developed in 1938 for the treatment of rheumatoid arthritis [44]. Sulfasalazine has both
immunomodulatory and immunosuppressive actions that include suppression of T cell
proliferation and reducing the synthesis of cytokines, including interleukin 1, 2, 6, and 12,
tumor necrosis factor α [45]. It also inhibits the release of prostaglandin E2 and atibody
production. Several reports showed good hair regrowth with sulfasalazine in the treatment of
AA. Sulfasalazine was started at 500 mg twice daily for one month, 1 g twice daily for one
month , and then 1 g three times daily [46]. Treatment with sulfasalazine is generally well
tolerated and characterized by a lower incidence of serious side effects in comparison with
other systemic therapies like corticosteroids and methotrexate. Side effects include
gastrointestinal distress, fiver, dizziness and headache. Sulfasalazine could be considered as a
therapeutic alternative in the treatment of AA, because of its safety profile, cosmetically
acceptable efficacy, and good tolerability.

Inhibition of the Fas-Fasl System

Induction of hair follicle apoptosis by the Fas-FasL system seems to be involved in the
pathogenesis of AA [47]. Therefore, inhibition of the Fas-FasL system might protect hair

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 Alopecia Areata 529

follicles from injury caused by the inflammatory infiltrate. However, such treatment could
only be applied topically and specifically limited to hair follicles, because systemic inhibition
would disturb essential control mechanisms of lymphocyte homeostasis [4].

Imiquimod
Imiquimod is the first member of a new class of immune response modifier, it was first
improved 1997 for the topical treatment of genital warts. It is a synthetic molecule, which
enhances both innate and acquired immune response, in particular, cell mediated pathways,
by stimulating monocytes and macrophages via binding to cell surface receptors to produce
several specific cytokines including IFN-α, IL-1, 6, 8, 10, 12 and tumor necrosis factor [48],
resulting in local immunoregulatory activity. Imiquimod also stimulates natural killer and B
cells and enhances migration of Langerhans cells. The clinical outcomes obtained with
imiquimod have been inconsistent, some authors have reported regrowth whereas others have
found no response [49, 50] In the future, imiquimod and newer generation of
imidazoquinolines (resiquimod) require further investigation for potential clinical utility in
treating AA.

Bexarotene
Bexarotene is a member of a subclass of retinoids that selectively activate retinoid X
receptor. It was noted that topical bexarotene yielded significant hair regrowth when used to
treat patients with follicular mucinosis or folliculotropic mycosis fungoides, and thus it was
theorized that topical bexarotene may also induce hair regrowth in AA [51]. Although the
mechanism for its action in AA is not completely understood, bexarotene induced T-cell
apoptosis. Topical bexarotene 1% gel application is well tolerated and possibly effective [52].
Mild irritation is a common side effect. A randomized placebo-controlled trial should be
conducted.

Nonpharmacologic Methods

Laser Therapy
The 308-nm excimer laser is a system that offers high doses of long-wave
monochromatic UVB radiation. Gundogan et al. are the first to discuss successful treatment
of two patients with AA with the 308-nm xenon chloride eximer laser [53]. The laser induces
T-cell apoptosis in vitro, which is analogous to topical treatment of AA. In another study, the
authors observed hair regrowth in all patients with patchy AA, whereas no hair regrowth was
observed in patients with either AA totalis or universalis [54]. Each lesion was treated twice a
week for a maximum of 24 sessions. The untreated areas did not show any regrowth,
suggesting that the regrowth observed was most probably not a spontaneous phenomenon.
The only side effects described were erythema and mild hyperpigmentation. Treatment was
well tolerated and so the authors suggested that this type of laser could be a good therapeutic
option. Also, the use of excimer laser in children with AA has been reported to have a good
success rate [55]. However, further studies are needed to confirm these findings.
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530 Emina Kasumagić-Halilovic and Nermina Ovcina-Kurtovic

Cosmetic Treatments
Cosmetic treatments for patients with AA include dermatography and hairpieces
transplants. Dermatography has been used to camouflage the eyebrows of patients with AA.
The procedure is relatively easy, provides permanent camouflage, and is generally devoid of
any significant adverse effects [56]. Hairpieces and transplants may be the only options
available for persons with severe disease that remains unresponsive to available medical
treatments [57]. Patients with extensive disease may wear wigs, or other scalp coverings.

CONCLUSION
Alopecia areata is a non scarring inflammatory hair disease, frequently recurrent.
Because of their psychological stigmatization, the medical attendance and therapy of patients
who suffer from distinct form of AA is difficult to challenge. Although spontaneous
remission is possible, it occurs rarely. The success of treatment depends on the age of onset of
the disease and the extent of hair loss. The most important prognostic factors are the extent
and pattern of disease. Alopecia totalis, alopecia universalis, and ophiasis have the worst
outcomes, with lower rates of spontaneous remission and poorer responses to therapy than
other presentations. Onset before puberty, long disease duration, co-existing atopy, nail
dystrophy, associated autoimmune diseases, and positive family history are risk factors for
more severe disease. At present, corticosteroids are the most popular form of treatment and
can be given topically, intralesionally, or, in rare case systemically. Minoxidil has had limited
success in stimulating hair regrowth without altering the course of AA. Topical
immunotherapy with diphenylcipropenone or PUVA therapy may be effective in longstading
and wide speared disease. Unfortunately, none of these agents is curative or preventive. These
treatments stimulate hair growth but do not prevent hair loss and probably do not influence
the course of the disease. The treatment of patchy AA is usually successful. However, the
therapy for extensive AA may be prolonged and difficult. In selected cases observation and
supportive therapy may be indicated. An individualized treatment approach is recommended
for each patient.
As long as no causal treatment is available, future approaches should focus on a more
specific targeting of the underlying pathomechanism with a topical action around the hair
bulbs and without serious side-effects. New immunomodulators and biologic therapies target
specific immunologic responses and offer new strategies for treating pathogenic T cells and
the cytokines they produce. Future success in treating of AA will require continued research
on the regulation of the hair-growth cycle and basic hair biology, the development of new
therapeutic approaches, and the judicious use of existing drugs.

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therapy in alopecia areata. J. Am. Acad. Dermatol. 2004; 51: 837-838.
[55] Al-Mutairi N. 308-nm excimer laser for the treatment of alopecia areata in children.
Pediatr. Dermatol. 2009; 26: 547-550.
[56] Garg G, Thami GP. Micropigmentation tattoing for medical purposes. Dermatol. Surg.
2005; 31: 928-931.
[57] Coskey RJ. Drake LA, Hordinsky MK, Rosenberg EW, Solomon AR, Chancho-Turner
ML. Guidelines of care for alopecia areata. J. Am. Acad. Dermatol. 1992; 26: 247-250.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 22

THE GENETIC BASIS OF ALOPECIA AREATA

F. Megiorni1,, M. Carlesimo2, A. Pizzuti1 and A. Rossi2

1
Department of Experimental Medicine
2
Department of Internal Medicine and Medical
Specialities, - “Sapienza” University of Rome, Italy

ABSTRACT
Alopecia areata (AA) is a common autoimmune disorder, characterized by circle
patches of hair loss, in which genetic and environmental factors influence the disease
development and progression. In this chapter, we will focus on the genetic loci that have
been associated with AA. Some of these loci contain genes involved in innate and
adaptive immunity and are shared with other autoimmune diseases, suggesting an overlap
of the genetic mechanisms involved in the development of such disorders. Linkage and
association studies underline the major region of AA susceptibility coming from the HLA
system (6p21.32), specifically HLA-DQB1*03 alleles coding for DQ7 heterodimers.
Modern technological innovations have advanced our understanding of the genetic basis
of AA. Genome wide association studies have recently identified new chromosomal
regions linked to AA liability in 2q33.2 (CTLA4), 4q27 (IL-2/IL-21), 6q25.1 (ULBP),
10p15.1 (IL-2RA) and 12q13 (IKZF4). A significant association was also evident for
single-nucleotide polymorphisms in 9q31.1 and 11q13, harboring genes expressed in the
hair follicle (STX17 and PRDX5, respectively), and in an intronic region of SPATA5 gene.
These association studies may provide mechanistic insights into the AA pathogenesis and
can improve the predictive models of the genetic risk. Follow-up of individuals with a
high genetic risk of AA could also help to elucidate the role of environmental factors
(such as stressful events, diet, infections etc) with the general aim to develop novel
clinical approaches for AA treatment.


Corresponding author: [email protected].
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536 F. Megiorni, M. Carlesimo, A. Pizzuti et al.

INTRODUCTION
Alopecia Areata (AA) is a common tissue-specific autoimmune disease which is
characterized by the sudden appearance of areas of hair loss on the scalp and other hair-
bearing areas. AA has a prevalence of 1-2% among Caucasians and affects genetically
predisposed individuals [1-3]. Indeed, as for many autoimmune disorders, AA is not inherited
in a Mendelian way but has a multifactorial etiology in which environmental as well as
genetic factors are involved, each acting in an additive fashion to generate the disease clinical
manifestations [4, 5]. Infection, nutritional deficiencies and psychological stress have been
suggested as triggering factors of AA onset and/or exacerbation [6-9]. The importance of the
genetic component is supported by the epidemiological indication that AA shows a familial
aggregation, with 10-47% of patients having a positive family history, and the recurrence risk
is greater among close relatives [10-13]. The incidence of AA in the offspring, siblings and
parents of severely affected probands has been reported as 2%, 3% and 7% respectively, with
an estimated lifetime risk of 6% for children [4, 11]. The observation of a significantly higher
concordance rate among monozygotic than in dizygotic twins (42-55% vs. 0-10%) also points
to a genetic influence on AA occurrence [6, 14]. Moreover, an increased prevalence of
autoimmune conditions, such as psoriasis, vitiligo, type 1 diabetes mellitus, celiac disease,
thyroid disorders, has been reported in AA cases and their relatives, mainly first-degree
individuals, suggesting that common genetic determinants for autoimmunity are likely shared
[15, 16]. Traditional approaches such as family-based linkage studies and population-based
candidate gene association studies have been extensively used to identify multiple loci and
alleles that contribute to AA liability [2]. Recently, genome-wide association studies
(GWASs), based on the identification of single nucleotide polymorphisms (SNPs)
determining susceptibility to common diseases, have been applied to a large cohort of AA
patients and controls allowing to increase our acknowledgement of the genetic markers
involved in the phenotypic expression of this complex disorder [17-19]. This chapter
summarizes the current findings of the Alopecia Areata genetic architecture.

ALOPECIA AREATA AND HLA GENES

Alopecia Areata pathogenesis is likely related to the activation of CD4+ and CD8+ cells
that infiltrate around and inside anagen hair follicles, providing a link between AA
development and the Human Leukocyte Antigen (HLA) class I and class II genes. Moreover,
aberrant expression of HLA heterodimers has been reported in AA affected scalp tissues [20,
21]. HLA genes map on human chromosome 6p21.3 and code for cell surface glycoproteins
important in the antigen presentation and self-recognition by lymphocyte immune cells. HLA
class I molecules, specifically recognized by CD8+ T cells, are encoded by the HLA-A, B and
C loci while HLA class II heterodimers, bound by CD4+ T cells, are specified by genes in the
HLA-D region that comprehends the HLA-DP, DQ and DR genes [22]. The genetic load of
the HLA region in Alopecia Areata was initially noted in the late 1970s and since that time
hundreds of studies have been performed in order to more precisely understand the role of
HLA in the disease. As regarding HLA class I, some studies have indicated an association
with AA that was not confirmed in other reports [23, 24]. The results on the investigated

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 The Genetic Basis of Alopecia Areata 537

antigens were different in different ethnic groups, as HLA-B12 in Finnish patients and B18 in
Jerusalem [25, 26]. HLA-A1, A2, A28, B40, B62, Cw3 and Cw7 associations have also been
described but not replicated [27-30]. Conversely, various HLA-DQB1 and -DRB1 alleles have
been recurrently suggested to confer a high risk of developing disease by both case-control
and family-based studies [4]. The first analyses, performed at serological levels, indicated that
DR4, DR5, DR6, DR7 and DQ3 were at-risk heterodimers for AA development [28, 31-36].
More recent studies, performed at molecular level, have shown a significant increased
frequency of DRB1*11:04 allele in patients with AA [32, 33, 35], mainly linked to the early-
onset form and to a higher familial recurrence risk [35, 37]. The genetic association analyses
between extension of the disease and particular DRB1 alleles have generated different results
with DRB1*11:04 variant being strongly correlated to AA totalis (AT) or universalis (AU)
phenotypes in some population [38] but not in others [37], and DRB1*04:01 variant (DR4
molecules) generally associated with the more aggressive clinical manifestations [35]. Among
the HLA-DQ genes, the DQB1*03 variants, serologically related to DQ7 heterodimers, have
been extensively analyzed and confirmed as the major risk allele for AA onset in different
population studies. DQ7-positive status is known to confer the greatest genetic effect in
Caucasians with a prevalence of up to 85% in patients compared with 46% in the general
population [36]; moreover, the strongest associations have been found between
DQB1*03(DQ7) allele and severe AA phenotypes [32, 35, 39, 40]. Interestingly, only the
DQB1*03(DQ7) variants encode a beta-chain carrying a glutamic acid at position 45 in one of
the two extracellular domains which might display an increased binding affinity with hair
follicle antigens and, therefore, explain the molecular mechanisms underlying the DQ7-
mediated AA genetic susceptibility [40, 41]. In addition, an increased frequency of the
DQB1*02:02 allele, frequently co-inherited with DRB1*07 variant, has been observed in
DQ7-negative AA patients [38, 40]. Furthermore, certain HLA class II variants, such for
HLA-DRB1*03:01, DRB1*13 and DQB1*06 alleles, have been described as protective
against the development of AA [38-40]. Differences highlighted in the various studies are
likely attributable to the different ethnic characteristics of the analyzed populations and the
study design. Overall, it is difficult to determine with certainty if the numerous HLA
associations are due to the disease heterogeneity or are casual effects owing to the strong
linkage disequilibrium (LD) across the HLA region. Literature data give evidence that
different populations have characteristic frequencies not only for individual alleles but also
for preferential allelic combinations. Among Caucasians, DQB1*03:01 variant (DQ7 in
serology) is almost always found in DR11/12 haplotypes and less frequently with DR4 so that
the DRB1*11:04 association with the risk of AT/AU might be due to the tight LD with
DQB1*03:01 allele [33, 38]. Even if all these variants are common in the healthy population,
the DQB1*03(DQ7) allele involved in the risk haplotype is over-represented in cases
compared to controls suggesting that HLA-DQ status is primarily associated with AA.
The strong correlation between variants in the HLA region and AA has also been
confirmed by recent genome-wide genetic analyses [17, 19, 42]. In particular, the AA
associated SNPs are physically close to the DRB1 and DQB1 loci and are highly correlated
with the HLA-DQB1*03 allele, serologically corresponding to the HLA-DQ7 glycoprotein.
Hence, GWAS findings are consistent with the very early AA associations with classical
HLA alleles.
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538 F. Megiorni, M. Carlesimo, A. Pizzuti et al.

ALOPECIA AREATA AND NON-HLA GENES

Particular HLA alleles have been widely established as AA at-risk factors even if they are
not sufficient to explain the entire genetic susceptibility of the disease. Classical linkage and
case-control analyses have led to the identification of several non-HLA linked chromosomal
regions and candidate genes that might have a role in the AA pathogenesis [2]. Starting from
the observation that Alopecia Areata is more common in Down syndrome and in autoimmune
polyglandular syndrome type I than in general population, many studies have been focused on
chromosome 21q22.3 as the more likely susceptibility region for AA. Indeed, a significant
association between AA and the intronic polymorphism +9959 in the MX1 gene has been
reported and the impact of MX1 in the disease onset has been also supported by the
observation of a strong expression of the MX1 protein only in patients’ lesional hair follicles
but not in normal scalp [43]. The investigation of different SNPs in the AIRE gene at
chromosome 21q22.3 has identified specific alleles and haplotypes that strongly predispose to
AA [44, 45]. Since AIRE transcription factor controls expression and presentation of self-
antigens in the thymus, its deregulation may impair the central tolerance and contribute to the
autoimmune processes in AA.
However, many classical genetic studies are limited by relatively small sample size,
candidate-driven selection bias, low statistical power and resolution for variants of modest
effect. GWASs, in which a large number (104-106) of SNPs across the entire genome are
examined in thousands of individuals, have rapidly increased our understanding of the AA
genetic background by the identification of novel at-risk loci outside the HLA region showing
a consistent association with the disease [17-19]. Most of these candidate non-HLA genes,
such as IL-2/IL-21, IL-2RA, IKZF4, ERBB3 and ULBP [17], are localized in genomic blocks
that are in moderate linkage disequilibrium and are involved in distinct signaling networks
comprising cytokine production and activation/proliferation of regulatory T cells which play
an essential role in the control of the immune response and in the maintenance of self-
tolerance [46]. ULBP proteins are also able to bind the natural killer (NK) cell receptor
NKG2D and to favor the development of NK-acquired dysfunction. Indeed, NK cell depletion
in C3H/HeJ mice, a well-established animal model of AA, significantly accelerated the onset
of the disease highlighting the potential involvement of NK cells in autoimmune skin-
diseases [47]. Altogether these genetic factors overlap with those for other autoimmune
diseases and support that both innate and adaptive immune responses are involved in the AA
etiology. In this regard, a high-resolution association analysis of the cytotoxic T lymphocyte-
associated antigen 4 (CTLA4) locus has recently shown that specific polymorphisms
(rs12990970, rs231775, rs3087243 and rs1427678) significantly influence the risk of AA in a
large set of patients from the Central Europe [18], being mainly correlated with the more
aggressive forms of the disease. CTLA4 molecules play a key role in the fine tuning of T-cell
immunity by negatively interfering with intracellular signal transduction events, so that
genetic variants able to modulate CTLA4 activity are likely to be a link between dysregulated
T-cell response and AA disease onset. Interestingly, particular SNPs located in DNA
sequences encompassing PRDX5 and STX17, two genes that are expressed in the hair follicles
[17], and in the SPATA5 gene, coding for a protein with an acid ATPase domain likely
involved in the regulatory subunit of the 26S protease, also achieved the genome-wide
statistical significance for AA association [19]. Several papers have revealed oxidative status

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 The Genetic Basis of Alopecia Areata 539

to be affected in AA patients, rendering the antioxidant enzyme PRDX5 a relevant molecule

in the pathogenesis and progression of AA [48].
Replication studies of the identified SNP markers in different populations and
expression/functional analysis of candidate genes are needed to establish their weight in the
genetic background of AA and the precise role in the disease pathogenesis.

CONCLUSION
Alopecia Areata is a complex genetic condition caused by genetic and environmental
interactions (Figure 1). Several genes have been correlated so far with AA liability, however,
when examined individually each of these loci only confer modest disease risk. Efforts in
future studies should be addressed to a clear definition of the AA genetic heritability in order
to better understand the pathogenesis and to develop novel laboratory tests and therapeutic
treatments for the AA clinical management [49]. Currently, the only available molecular test
for AA susceptibility may be the HLA-DQB1 typing since most AA patients are positive for
DQB1*03 alleles coding for DQ7 heterodimers. However, considering that many healthy
subjects in the general population also carry these alleles, HLA test may be an important
marker only in the identification of individuals who belong to at-risk groups such as first-
degree relatives of AA patients.
Moreover, the strong relationship between DQB1*03(DQ7) and the disease severity
seems to suggest the prognostic value of this genetic test in patients with Alopecia areata. One
goal is, therefore, to design a DNA analysis that combines the individual effects of different
and well-validated loci of susceptibility into a global genetic risk able to accurately
discriminate individuals with a very high likelihood of developing AA as well as to give
important information on the onset and aggressiveness of the disease. This will also help the
clinicians to decide on patient-specific monitoring and appropriate treatments.

Figure 1. Multifactorial etiology of Alopecia Areata. Gene-gene and gene-environmental interactions

among known factors are depicted.
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540 F. Megiorni, M. Carlesimo, A. Pizzuti et al.

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enzymes and lipid peroxidation in the scalp of patients with alopecia areata. J.
Dermatol. Sci. 29:85-90.
[49] Petukhova, L., Cabral, R. M., Mackay-Wiggan, J., Clynes, R., Christiano, A. M. (2011)
The genetics of alopecia areata: What's new and how will it help our patients?
Dermatol. Ther. 24:326-36.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 23

OCULAR ROSACEA: RECENT ADVANCES IN

PATHOGENESIS AND THERAPY

Alejandro Rodriguez-Garcia, MD

Director of the Immunology & Uveitis Service
Senior Ophthalmic Research Coordinator
Instituto de Oftalmología y Ciencias Visuales
Escuela de Medicina y Ciencias de la Salud
TEC Salud. Tecnológico de Monterrey, México

ABSTRACT
Ocular rosacea forms part of the clinical spectrum of rosacea. It is characterized by a
chronic and recurrent inflammation of the eyelids, conjunctiva and cornea.
Approximately 50% of rosacea patients present ocular manifestations, and the condition
is most frequently diagnosed when cutaneous signs and symptoms are present. However
in 20% of patients, ocular manifestations may precede the cutaneous disease. Most
frequent ocular symptoms are: red eyes, burning, foreign body sensation, photophobia
and blurred vision.
Chronic blepharitis with meibomian gland dysfunction is the most frequent ocular
manifestation of the disease, and produces evaporative dry eye with consequent ocular
surface damage. Corneal inflammation and scarring may be a cause of severe visual loss.
In addition to therapeutic strategies for the cutaneous disease, ocular rosacea treatment
involves, lid hygiene, topical macrolides and tetracyclines as eyelid gels or ointments,
lubricant eye drops, and short-term topical steroids, depending on the severity of
blepharitis, conjunctivitis and keratitis. Prognosis and visual outcome depend on the
severity of the disease, early diagnosis and appropriate treatment.


Correspondance: Instituto de Oftalmología y Ciencias Visuales. Centro Médico Zambrano Hellion (1er Piso
Oriente). Av. Batallón de San Patricio No. 112. Col. Real de San Agustín. San Pedro Garza García, Nuevo
León. C.P. 66278. MEXICO. Tel. 52(81) 8888-0551 y 0552. Fax 52(81) 8356-1799. E-mail:
[email protected]
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546 Alejandro Rodriguez-Garcia

DEFINITION
Ocular rosacea is defined as the clinical spectrum of ocular manifestations considered
subtype-4 rosacea by the National Rosacea Society [1]. The syndrome is characterized by
chronic inflammation involving the eyelids and to a variable extent, the conjunctiva and
cornea [2, 3]. Its clinical course is one of exacerbations and remissions over a long period of
time with poor correlation between the occurrence of ocular manifestations and the facial
flushing [4, 5].

HISTORY
The ocular manifestations of rosacea were recognized much later than the dermatologic
ones. Rosacea was probably first evoked by the famous British writer Geoffrey Chaucer
(1387) in his description of the Summoner’s face in “The Canterbury Tales”[6] and painted
one century later by the Florentine renaissance painter Domenico Ghirlandaio (1490), in “The
Old Man and his Grandson”(Musée du Louvre, Paris), where he portraits a grandfather with a
prominent rhinophyma [7]. The first person known to describe rosacea as a medical condition
was a prominent medieval French surgeon, Dr. Guy de Chauliac (1300-1368) who called it
“goutterose” which means, “pink droplet” [8]. In the 18th century, the dermatologist J.J.
Plenck (1738-1807) called it “gutta rosacea” [9, 10]. Later on, both terms were replaced in the
English literature by Batemann (1778-1821) who first called it “acne rosacea” [9, 10], a term
that was later discarded due to the lack of evidence of a relationship between acne and
rosacea [11].
The first scientific description known of the ocular manifestations of rosacea came from
Arlt (1864), who first noted conjunctivitis and keratitis in patients with facial disease [12, 13].
Sir W. Stewart Duke-Elder (1898-1978) first stated that “ocular rosacea is more common than
reported, and is frequently undiagnosed,” [14, 15] and affirmation that unfortunately is still
valid today [16-18]. Only few case reports or literature reviews were written before 1980 in
ophthalmology journals [3, 13, 15, 19-21]. But on the last two decades of the last century, the
number of publications in both ophthalmologic and dermatologic journals started to raise,
showing an increase scientific interest for the ocular manifestations of rosacea [2, 5, 22-27].
Finally, from the last decade until present time, many reports reviewing particular aspects of
ocular rosacea like, quality-of-life, pathophysiologic mechanisms and treatment modalities
have increased the awareness, and improved our knowledge of the disease [28-37].

EPIDEMIOLOGY
Approximately 50% (range, 3 – 58%) of rosacea patients present ocular manifestations
[19, 38, 39]. Ocular rosacea is most frequently diagnosed when cutaneous signs and
symptoms of the condition are present [40, 41]. However, it may persist undiagnosed for a
long period of time when cutaneous manifestations presented earlier in the course of the
disease and are not prominent at the time of ophthalmic examination [14, 18, 42]. It is
important to note that in 20% of patients, the ocular symptoms may be the initial

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manifestation of the disease, and in 27% of them, the cutaneous and ocular manifestations
may occur simultaneously [2, 19, 39].
Even though rosacea has been more commonly described in populations from the north
and west of Europe, and in countries with a large European descent, particularly the United
States [11], there are no precise data on the prevalence variation among races [5]. It appears
like rosacea may occur less frequently in other ethnic groups, particularly in dark-skin
individuals [25]. Previous reports have found that approximately 4% of rosacea patients are of
African, Latin, or Asian descent [43].
In general, women without ocular involvement are more frequently affected than men
[13, 44], and although ocular rosacea appears to affect both sexes equally, the ocular
manifestations tend to be more severe in man [2, 35, 45]. Ocular rosacea can appear at any
age, including childhood [31, 46, 47]. Pediatric rosacea is a poorly defined condition, and it is
probably underestimated as flushing and facial erythema are frequently confused with a
“healthy glow” in children [11]. Ocular manifestations of rosacea in children are similar to
adults, but sight-threatening complications are more frequently found at this age group [47,
48]. Children are likely to have a family history of rosacea, and the condition may persist
through adulthood [49, 50]. In general, rosacea flushing usually initiates during the second
decade of life, becomes troublesome at the third decade, and may continue to progress
thereafter [51]. The peak incidence of the disease occurs later, between the fifth to seventh
decades of life, when most cutaneous and ocular morbidity occurs [52, 53]. There appears to
be a strong correlation between the degree of ocular involvement and the tendency to flush
[54].
Finally, ocular rosacea significantly affects the quality of life of affected patients [4].

CLINICAL MANIFESTATIONS
Ocular manifestations are mainly present in the eyelids and the external ocular surface,
comprising the conjunctiva and cornea [2], and they can precede skin changes in a minority of
patients; develop a the same time; develop later than skin manifestations; or occur
independently from skin flushing [55, 56]. Most frequently, ocular manifestations occur after
skin flushing, or coexist with an advanced cutaneous stage (subtype-3 or phymatous) of
rosacea [35] (Figure 1).
Problems range from minor irritation, dryness, and blurry vision to potentially severe
ocular surface disruption and inflammatory keratitis [35, 46, 57].
Ocular rosacea symptoms usually present bilaterally, but asymmetry or alternate
inflammation is not uncommon [58, 59]. As would be expected, reports from ophthalmology
clinics indicate a higher prevalence of ocular manifestations than dermatologic ones [2, 5, 29,
45, 60]. The most prominent symptoms of ocular rosacea are redness, tearing, foreign body
sensation and irritation [5, 35, 56].
Other symptoms equally important in these patients are: eyelid margin redness and
pruritus, swollen eyelids, visibly dilated conjunctival vessels, mucus and/or watery discharge,
grittiness, pain and burning, dryness, photophobia, blurred vision, recurrent hordeolum or
chalazion, among others [2, 5, 42, 56].
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548 Alejandro Rodriguez-Garcia

Figure 1. Patient with subtype-3 (Phymatous) rosacea showing a prominent rhinophyma, and severe
ocular involvement OS>OD.

Symptoms most typically worsen early in the morning after awakening, but also by
hostile environmental factors such as, air pollution, air conditioning and fans air flow, dry and
hot climate, among others [58, 59, 61, 62]. These aggravates are related to meibomian gland
inflammation and dysfunction (MGD), which typically worsen overnight to became more
symptomatic early in the mornings, and to tear film instability causing evaporative dry eye
[53, 55, 60, 62-64].

Figure 2. Inferior eyelid margin of a patient with ocular rosacea, showing grade-3 meibomian gland
dysfunction with meiboum stagnation in most gland orifices.

Chronic blepharitis and conjunctivitis are most common signs of patients with ocular
rosacea [31, 57, 59]. Signs may be divided according to the adnexa and ocular structure
affected by the disease. (Table 1) Eyelid findings include, chronic anterior and posterior
blepharitis, MGD, hordeolum and chalazion, eyelid margin erythema, crusting, scales,

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telangiectasias, meibomian duct obstruction with keratinization, squamous metaplasia, and lid
border irregularity [2, 5, 58] (Figure 2). Chronic blepharitis with associated MGD has been
recognized as the most important feature of ocular rosacea [28, 53, 65, 66]. Meibomian gland
dysfunction is seen in up to 78 to 83% of ocular rosacea patients [5, 35], while approximately
60% of patients with chalazion have rosacea [67].
The reported prevalence of most relevant signs of ocular rosacea is shown in Table 1.

Table 1. Ocular Rosacea Manifestations and Prevalence

Eyelids Manifestations Prevalence Range References

Blepharitis 47 – 93% [5,13,35,42,55]
Meibomitis or meibomian gland dysfunction (MGD) 4 – 83.7% [5,42,55]
Secretions (collarette, sleeves, crusts, scales) 56.3% [35]
Telangiectasias 63 – 81% [5,35,42]
Meibomian duct obstruction with keratinization NA ---
Margin erythema NA ---
Lid border irregularity NA ---
Margin squamous metaplasia NA ---
Hordeolum and chalazion 7.3 – 71.4% [5,31,35,55]
Conjunctival Manifestations
Hyperemia 45 – 86% [5,13,35,42,55]
Papillary / follicular reaction 10.9% [35]
Phyctenula 0.7% [5]
Nodule NA ---
Granuloma 0.7% [5]
Bacterial conjunctivitis 16.3% [35]
Cicatrizing conjunctivitis 9 – 20% [5,57,69]
Corneal Manifestations
Sicca or tear film instability 26 – 62.5% [5,28,32,35,55]
Superficial punctate keratitis (SPK) 15 – 50.9% [5,35,42,55]
Interstitial keratitis 28.5% [31]
Pannus formation 20 – 65.2% [31,35]
Vascularization and subepithelial infiltrates 6.5 – 67% [5,13,35,42,55,63]
Vascularization and stromal thinning 7.2 – 10% [35,42]
Recurrent epithelial erosion 5 – 12% [5,42]
Ulceration 5 – 14.2% [5,31,35]
Peripheral ulcerative keratitis (PUK) 9 – 19.4% [35,42,63]
Scarring and leukoma formation 21 – 56.5% [5,35,63]
Perforation Case reports [5,35]
Other Manifestations
Episcleritis 8 – 8.1% [5,63]
Scleritis 0.7% [5]
Iritis 2 – 10% [5,13,55]
NA = not available.

The conjunctiva frequently shows bulbar and tarsal hyperemia, a mixed follicular and
papillary reaction, and to a lesser extent, phlyctenula, nodule and granuloma formation [13,
35, 42, 68]. Some patients may also experience from acute bacterial to chronic cicatrizing
conjunctivitis [57, 69].
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Figure 3. Large central corneal leukoma and stromal vascularization, obstructing the visual axis of a
patient with chronic ocular rosacea.

Although site-threatening corneal findings are not common in patients with rosacea, the
cornea has been involved in up to 50% of patients, and may vary from superficial punctate
keratitis to severe corneal ulceration, scarring with dense leukoma formation, and even globe
perforation [5, 35] (Figure 3).
Other important corneal findings are: pannus formation, recurrent epithelial erosions,
sub-epithelial infiltrates, interstitial keratitis, stromal thinning, vascularization, and peripheral
ulcerative keratitis [35, 53] (Figure 4).

Figure 4. Inferior peripheral ulcerative keratitis with multiple corneal stromal infiltrates and
vascularization in a patient with severe ocular rosacea.

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As mentioned before, some of these corneal changes may produce serious visual
impairment. Keratoconjunctivitis sicca associated with rosacea has been reported in as many
as 40% to 56% of patients [55, 70]. This finding is due to a mixed form of lacrimal
dysfunction (aqueo-mucinous deficiency and increased tear evaporation) [64, 70]. The
evaporative part is directly related to chronic blepharitis and MGD seen commonly in these
patients, and is responsible for symptoms exacerbations and aggravation of the ocular surface
disease [5, 28, 35]. Other less frequent but not least important ocular manifestations of
rosacea are episcleritis, scleritis and iritis [5, 40, 58].

CLINICAL COURSE, STAGES AND GRADING

Ocular rosacea is considered subtype-4 of the disease by the National Rosacea Society
Classification Committee, and the different subtypes do not necessarily follow one another in
a consecutive manner [1]. Like the cutaneous disease, ocular rosacea is characterized by a
chronic inflammation of the eyelids and ocular surface that waxes and weans throughout the
clinical course of the disease [5, 45]. The role of known cutaneous triggers on ocular rosacea
exacerbations is uncertain. In general, triggering factors are taken into account by
ophthalmologists when following and managing the ocular inflammation [2, 5]. Ocular
inflammation may last from weeks to months, and the longer the uncontrolled inflammation,
the eye becomes more prone to sight-threatening complications due to corneal involvement
[2, 58]. Skin manifestations may or may not be present at the time of ocular disease, and
severity of skin flushing does not correlate with ocular manifestations [31, 59]. The National
Rosacea Society has divided the clinical course of rosacea into one pre-rosacea stage, and 3
progressive stages of the disease [71]. Ocular changes are part of the second stage, along with
cutaneous features like, persistent, spreading erythema; edema, papules, pustules; and
enlarged pores [71]. (Table 2)

Table 2. Stages of Rosacea*

Stage Symptoms and Signs

Pre-rosacea Frequent flushing
Irritation caused by topical preparations
Stage 1 Transient facial erythema that becomes more persistent

Slight telangiectasias
Increased skin sensitivity
Stage 2 Persistent, spreading erythema
Edema, papules, pustules
Enlarged pores
Ocular changes
Stage 3 Large inflammatory nodules and furuncles
Tissue hyperplasia, fibroplasias
Rhinophyma
* Standard classification of rosacea: Report of the National Rosacea Society Expert Committee on the
Classification and Staging of Rosacea [1].
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552 Alejandro Rodriguez-Garcia

The severity of ocular manifestation of rosacea has also been analyzed by the National
Rosacea Society Expert Committee on the Grading of Rosacea [71, 72] (Table 3). The
purpose of grading the severity of the ocular manifestations is for specific recommendations
on adequate therapeutic intervention.

Table 3. Grades of Ocular Rosacea*

Grade 1: Mild itch, dryness, or grittiness of eyes; fine scaling of lid margins;
telangiectasia and erythema of lid margins; mild conjunctival injection
(mild congestion of conjunctival vessels).

Grade 2: Burning or stinging of eyes; crusting or irregularity of lid margins, with

erythema and edema; definite conjunctival hyperemia or injection; formation of chalazion
or hordeolum.

Grade 3: Pain, photosensitivity, or blurred vision; se- vere lid changes, with loss of lashes;
severe conjunctival inflammation; corneal changes, with potential loss of vision;
episcleritis or scleritis; iritis.

* Standard grading system for rosacea: report of the National Rosacea Society Expert Committee on the
classification and staging of rosacea [71].

PATHOPHYSIOLOGY
The pathophysiology of rosacea and its ocular manifestations is complex, and involves a
closed interaction among innate immune defense mechanisms, innervation, adaptive
immunity, and vascular biology [73]. Despite recent advances in our understanding of the
complexities of neuro-immune communication, acute and chronic inflammation, immunity,
and tissue repair mechanisms, the pathogenesis of rosacea remains inconclusive [74-80].
Both, vascular dysregulation and altered immune system responses and consequent
inflammatory changes have been majorly involved in the skin and the ocular surface [81].
Recent research has shown an upregulation of pro-inflammatory and vasoregulatory genes in
rosacea patients [80, 82, 83]. The concept of an altered vascular function or a vascular
hyperreactivity, is supported by the characteristic facial flushing, persistent erythema, and
telangiectasias seen in these patients [83]. More recent studies point toward a vascular
endothelial growth factor (VGEF)-mediated angiogenesis and lymphangiogenesis [83, 84].
Since IL-17 mediates the induction of VEGF in fibroblasts in vitro, there is a potential role of
this cytokine in rosacea-associated angiogenesis [85, 86]. Interestingly, increased vascular
densities were correlated with the papulo-pustular and ocular rosacea subtypes [87]. Different
chemokines have been shown to exert bimodal functions on the vasculature. Whereas
interferon-inducible and CXCR3-binding chemokines such as CXCL9, CXCL10, and
CXCL11 have been classified as angiostatic, CCL2, CCL11, CXCL1, CXCL8, CXCL12, and
CXCL1 promote angiogenesis [88].

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Nevertheless, the role of these chemokines in the vascular dysregulation, angiogenesis or

lymphagiogenesis in rosacea remains to be elucidated.
On the other hand, alterations in the innate immune system responses include an
overabundance of cathelicidin (an antimicrobial peptide), along with kallikrein-5 (KLK5), an
enzyme involved in processing cathelicidin [78]. There is strong evidence for the role of
proteases in rosacea, in particular, KLK5 which is able to activate signaling pathways, leading
to the production of inflammatory mediators, cytokines, and LL-37 [78]. Kallikrein-5 also
activates several matrix metallo-proteases, which cause shedding of several epithelial growth
factor receptor (EGFR) ligands. These EGFR ligands may also induce more LL-37
production. However, the mechanism that enhances KLK5 in rosacea is not fully understood
[78]. In addition, it has been shown that toll-like receptor-2 (TLR2) activity of the innate
immune system is upregulated in patients with rosacea [89, 90]. TLR2 activation leads to the
expression of abnormally high levels of cathelicidin-LL37, which promotes leukocyte
trafficking through the induction of CXCL8, and induce angiogenesis [77, 91]. In this respect,
immunohistochemical staining for a variety of vascular markers and for toll-like receptor-4
(TLR4), performed to eyelid biopsies of ocular rosacea patients, and normal controls has
shown a statistically significant staining for ICAM-1 and CD105 among arterioles but not
venules on ocular rosacea patients compared to controls [34]. And the correlation between the
number of TLR4 positive cells and each vascular marker was also statistically significant
[34]. These findings suggest that ICAM-1 and CD105 mediate the vascular abnormalities
seen in the eyelids of ocular rosacea patients, and that the innate immune system may govern
the cutaneous effects of ocular rosacea [34].
Ocular surface inflammation in patients with rosacea has also been related to increased
levels of interleukin-1 (IL-1), matrix metalloproteinase-8 (MMP-8), and gelatinase-B (pro-
MMP-9) activity in the tears of these patients [24, 27, 92]. Gelatinase-B activity correlates
with a delayed tear clearance and tear fluid concentration of interleukin-1, a pro-
inflammatory cytokine that has been reported to increase the production and activity of
certain enzymes of the matrix metalloproteinase (MMP) family, including collagenases and
gelatinases, like MMP-8 and MMP-9 [27]. These enzymes degrade extracellular matrix and
may contribute to the development of eyelid and ocular surface irritation, recurrent corneal
epithelial erosions, corneal vascularization and persistent epithelial defects, as well as corneal
ulceration seen in some of these patients [27, 92, 93]. In addition, delayed tear clearance, a
common feature of MGD present in almost all ocular rosacea patients, has shown a strong
correlation with decreased corneal and conjunctival sensitivity scores [64, 94]. Although the
cause of decreased ocular surface sensation has not been established, the inflammation of the
ocular surface that develops in these patients is a possible cause [95]. Inflammatory factors,
including IL-1 have been reported to alter sensory neural threshold [96]. The importance of a
reduced ocular sensation is a decreased stimulation reflex to the lacrimal glands for tear
production, which further decreases tear clearance rate, creating a vicious cycle [97, 98].
More recently, impression cytology combined with flow cytometry performed to samples
from eyes with active ocular rosacea, showed a significant increase of HLA-DR and ICAM-1
expression by epithelial cells compared to normal eyes [28]. Both markers were well
correlated with each other and inversely correlated with the tear break-up time (TBUT) and
Schirmer test [28]. Also, the percentage of goblet cells in the conjunctiva was significantly
decreased in rosacea patients compared with the normal group, with a significant negative
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554 Alejandro Rodriguez-Garcia

correlation with both HLA-DR and ICAM-1 markers [28]. The goblet cell deficiency has
been further corroborated by significantly lower levels of the peptide core of the conjunctival
mucins (M1/MUC5AC), as compared to normal controls [28].
A variety of rosacea triggers have been described including eyelid colonization with
Demodex mites [99, 100] and Staphyloccocus epidermidis [101, 102]. A recent study looking
at the concentration of cytokines and chemokines in tears of patients with Demodex
blepharitis demonstrated increased levels of IL-2, IL-7, and IL-17 as compared with
Demodex-free blepharitis, suggesting that specifically IL-17, may play a role in the
inflammation of the lid margin and ocular surface [99]. Helicobacter pylori is well recognized
as a causative factor for gastritis and duodenal ulcers, and rosacea has long been associated
with gastritis [103]. The prominent English ophthalmologist Sir Stewart Duke-Elder stated,
"digestive troubles have frequently been cited as a causal factor of rosacea" [14]. However,
the proposed link of H. pylori to rosacea is intriguing but unproven so far [103, 104]. Few
studies have been carried out to clarify this controversy. In an uncontrolled, non-comparative
study of a short cohort of patients, eradication of Helicobacter pylori improved rosacea in
some patients. Based on these results, it was then suggested that the organism may play a role
in the pathogenesis of the disease [105]. On the contrary, a further well-controlled,
comparative clinical assay was not able to establish a direct relationship between the
organism and rosacea [106, 107]. Finally, the eradication of Helycobacter pylori from seven
patients with ocular rosacea, whom at the same time had clinical and serological evidence of
systemic infection with H. pylori, resulted in improvement of ocular symptoms far better than
the cutaneous response, raising again the possibility of the pathogenic role of this
microorganism in the disease [108].
The role of well-established clinical cutaneous flushing triggers such as, microbes, cold,
heat, alcohol, coffee and caffeine-containing beverages, tobacco, spicy foods containing
Capsicum, vasodilating medications and emotional stress, has not been well studied for ocular
rosacea [79, 109]. However, it is generally assumed that they all can also contribute to the
exacerbation of ocular signs and symptoms of rosacea.
The same is true for UV-light exposure, which directly decreases the competence of
already dilated vasculature, increasing persistent erythema and telangiectasias. UV-irradiation
also induces the expression of IL-1, IL-6, IL-10, TNF, and CXCL8 in a time and dose-
dependent manner [110]. In particular, IL-1 and TNF are known to induce the expression
of a subset of pro-inflammatory chemokines (CXCL1, CXCL8, CCL20, CCL27) in
keratinocytes [110]. CXCL8 may induce neutrophil recruitment [111]. However, the specific
role of these cytokines and chemokines in the pathogenesis of rosacea and its ocular
manifestations is unclear at the present time.

DIAGNOSIS
Ocular rosacea is frequently undiagnosed because of its large diversity of non-specific
clinical manifestations; the lack of specific diagnostic tests; and the difference in timing
between the appearance of cutaneous and ocular signs and symptoms [16, 17, 32]. Indeed,
ocular rosacea is most frequently diagnosed when cutaneous findings typical of rosacea are
also present at the time of diagnosis [2, 45, 59].

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Table 4. Differential Diagnosis of Ocular Rosacea

Ocular Disease Similarities Differences

Chronic blepharitis Identical eyelid changes Facial flushing and skin changes
Staphyloccocal Collarette secretion, eyelid erythema; Facial flushing and skin changes.
blepharitis conjunctival hyperemia. Phlyctenula formation
Seborrheic blepharitis Crusts and scaling, eyelid margin and Foamy conjunctival discharge.
conjunctival hyperemia, MGD, Scaling, eczematous changes of
telangiectasias. paranasal, nasolabial, and
extrafacial distribution.
Sebaceous gland Recurrent chalazia, MGD, eyelid margin Tendency to invade periocular
carcinoma erythema and irregularity, yellowish region; pagetoid infiltration of the
secretion. conjunctival epithelium or skin
epidermis.
Meibomian gland Obstructed meibomian ducts, turbid or Indistinguishable, apart from
dysfunction paste meiboum secretions; increased tear facial skin changes.
film break-up time, SPK.
Recurrent chalazia Identical hordeolum or chalazion Indistinguishable, apart from
characteristics. facial skin changes.
Allergic conjunctivitis Conjunctival hyperemia, papillary Ocular pruritus, Horner-Trantas
reaction, watery discharge dots; tarsal giant papillae.
Bacterial conjunctivitis Conjuntival hyperemia, mucopurulent Indistinguishable, apart from
discharge, sticky eyelids. facial skin changes.
Viral conjunctivitis Conjunctival hyperemia, follicular Conjunctival pseudo-membrane or
reaction, watery discharge membrane formation
Chlamydia Chronic conjunctival hyperemia, Follicular reaction, tarsal fibrosis,
conjunctivitis mucopurulent discharge. Herbert pits, trichiasis.
Ocular mucous Chronic red eye, foreign body sensation, Subepithelial fibrosis, fornix
membrane pemphigoid graining and stinging. foreshortening, symblepharon
formation, trichiasis/dystrichiasis.
Atopic Chronic red eye, foreign body sensation, Pruritus, photophobia, tarsal
keratoconjunctivitis graining and stinging. papillae, subepithelial fibrosis,
corneal conjunctivalization.
Keratoconjunctivitis Chronic red eye, foreign body sensation, Filamentary keratitis, low tear
sicca graining and stinging. meniscus, reduced
Schirmer test
Recurrent corneal Red eye, watery discharge, photophobia, Facial flushing and skin changes
erosion foreign body sensation
Bacterial keratitis Conjunctival injection, mucopurulent Facial flushing and skin changes
discharge, corneal stromal infiltrate,
photophobia and blurred vision.
Peripheral ulcerative Crescentric peripheral corneal ulceration, Indistinguishable, apart from
keratitis ciliary injection. Foreign body sensation, facial skin changes
photophobia.
Episcleritis Episcleral edema and vasodilation; ocular Indistinguishable, apart from
hypersensitivity. facial skin changes
Scleritis, autoimmune Scleral edema, vascular ingurgitation, Indistinguishable, apart from
related ocular pain. facial skin changes
Acute iritis, idiopathic Ciliary injection, photophobia, pain and Indistinguishable, apart from
or autoimmue blurred vision. facial skin changes
SPK = superficial punctate keratitis.
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The problem arises when the patient experiences skin flushing much earlier in the course
of the disease and by the time ocular manifestations occur, facial flushing may not be
prominent or even absent. In such cases, a definitive diagnosis of ocular rosacea becomes a
difficult task to the ophthalmologist due to the lack of specific ocular manifestations of the
disease [16, 17]. Moreover, in many cases of ocular rosacea, the severity of ocular symptoms
does not correlate to cutaneous findings, and skin manifestations are not a prerequisite for the
ocular diagnosis [1, 3, 45, 56].
To complicate even more the diagnosis, many other ocular surface disorders may mimic
ocular rosacea. (Table 4). According to the Report of the National Rosacea Society Expert
Committee on the Classification and Staging of Rosacea [1], the diagnosis of ocular rosacea
should be considered whenever the following signs and symptoms are present in both eyes of
any patient regardless of cutaneous manifestations: ocular redness, watery discharge, foreign
body sensation, burning or stinging, dryness, itching at the palpebral margin; photophobia,
blurred vision, swollen and erythematous eyelids with scaling; telangiectasias, eyelid margin
irregularity, and chronic blepharitis with MGD; recurrent chalazion or hordeolum; keratitis,
episcleritis, scleritis, and iritis [3, 11, 29, 72, 112].
In summary the diagnosis of ocular rosacea is established clinically and is often aided by
dermatologic findings [58, 59]. There are no laboratory tests directly related to the diagnosis,
but in some cases, eyelid margin scraping and culture for Staphylococcus aureus and
coagulase-negative, Demodex folliculorum, and other potentially involved microorganisms
can be performed [2, 3, 99].

Figure 5. Positive inferior lisamine green staining pattern typical of chronic blepharitis with meibomian
gland dysfunction associated to ocular rosacea.

From the ophthalmological perspective, the diagnosis of ocular rosacea can also be aided
by careful examination of the eyelid margins looking for signs of chronic blepharitis, like
secretions (collarette, sleeves, crusts and scales), telangiectasia formation, and posterior
displacement, keratinization and/or inspissation of the meibomian gland excretory orifices

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[66]. Also, manual expression of meibomian gland ducts need to be done, looking at the
amount of functional glands, and the appearance of meiboum secretions for grading severity
of MGD [66, 113, 114]. Meibography can also be performed with halogen trans-illumination,
infrared light or confocal microscopy in order to measure meibomian gland atrophy and
dropout [115-118].
Patients with ocular rosacea frequently experience an aqueous deficient type of dry eye
[55, 70]. The Schirmer test, although unreliable because of its variability [97, 119], and lack
of repeatability [120], has been used to demonstrate an aqueous tear deficiency in patients
with ocular rosacea [55, 60]. Using fluorescein dye, the tear break-up time must be assessed,
as well as corneal staining in order to measure tear film stability and corneal damage [121].
Other vital dyes, like lisamine green may be used to detect the amount of conjunctival cell
damage due to dryness [122]. The typical staining pattern of the inferior interpalpebral
quadrants seen in chronic blepharitis may also be considered for the diagnosis in patients with
ocular rosacea [114] (Figure 5).

HISTOPATHOLOGY
The first histopathologic analysis from the conjunctiva of patients with ocular rosacea
was performed by Brown and Shahinian in 1978 [3]. They found that 6 out of 12 conjunctival
biopsies stained positive for IgG, IgA, or IgM by direct or indirect immunofluorescence, and
5 out of 6 biopsies also stained positive for C3. At that time, they concluded that the
significance of their findings was unclear postulating that possibly, local autoimmune
mechanisms were responsible for at least part of the initial inflammatory signs or were the
result of prolonged inflammation, and that these eyes might respond more violently to further
episodes of the disease [3].
Although there are not specific immunohistopathologic features for ocular rosacea, the
conjunctiva is frequently infiltrated by inflammatory cells. The conjunctival epithelium is
attenuated and significantly infiltrated, mainly by lymphocytes-T (CD4+), phagocytic cells
and antigen presenting cells (CD14, Mac-1) [23]. In the stroma, there is increased vascular
dilation and large sub-epithelial infiltrates of chronic inflammatory cells with granulomatous
changes, resembling a type IV hypersensitivity reaction. All cell types are present in larger
proportions than normal controls, but especially T-helper (CD4+) lymphocytes. There is a
3.5-fold increase in the lymphocyte-CD4+ to CD8+ ratio in the conjunctiva of patients with
rosacea [23].

DIFFERENTIAL DIAGNOSIS
The differential diagnosis of ocular rosacea is related to most common forms of chronic
blepharitis, and/or ocular surface inflammatory diseases. (Table 4) Most frequently, ocular
rosacea is misdiagnosed in patients with chronic staphylococcal and seborrheic blepharitis
with meibomian gland dysfunction. Meibomitis-related keratoconjunctivitis (MRKC),
characterized by sub-epithelial cellular infiltrates and superficial vascularization of the cornea
associated to Propionibacterium acnes needs also be considered in the differential [123, 124].
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558 Alejandro Rodriguez-Garcia

Another very important diagnosis is sebaceous gland carcinoma, a disease frequently

misdiagnosed as recurrent chalazia which needs to be biopsied for histopathology
confirmation [58, 125, 126].
Other causes of infectious conjunctivitis with chronic or recurrent conjunctival
hyperemia, as well as cicatrizing conjunctivitis may resemble ocular rosacea, like mucous
membrane pemphigoid, pemphigus vulgaris, lichen planus, and atopic keratoconjunctivitis
[127-129]. On the other hand, many disorders resembling cutaneous rosacea may also exhibit
ocular manifestations in a diverse clinical presentation. Diseases like acne vulgaris,
seborrehic dermatitis, steroid rosacea, contact dermatitis, photodermatitis, systemic lupus
erythematosus, dermatomyositis, mucous membrane pemphigoid, and pemphigus vulgaris
[11, 130].

TREATMENT
Ocular rosacea remains incurable and disease stabilization remains elusive despite its
relatively common nature and its severe potential consequences [131]. Our limited
understanding of the pahogenic mechanisms responsible for the onset and development of
rosacea has resulted in an array of non-specific and less than optimal therapeutic strategies to
minimize ocular damage [89, 131, 132]. Therefore, no precise treatment algorithm has
become the standard of care, and ocular management remains empirical and limited to
symptomatic control and resolution of complications only [132].
The treatment of ocular rosacea may be divided into three main categories: 1) avoidance
of triggers to reduce exposure of the eyelids and ocular surface to the disease; 2) conservative
measures to minimize the damage caused by rosacea and alleviate active symptoms; and 3)
therapies to revert the damage that has already occurred [131]. Avoidance of known triggers
of rosacea exacerbation such as, prolonged exposure to UV-sunlight; intake of alcohol and
caffeine-containig beaverages; and physical or emotional stress among others, should be
recommended to all patients with ocular rosacea [133]. Because most ocular rosacea patients
suffer from chronic blepharitis and MGD, conservative measures intended to unclog the
meibomian gland ducts, improve the outflow of meiboum, and stabilize the tear film through
the application of warm compresses to the eyelids and lid hygenie (scrubs and massage) are
mainstay therapy for these patients [66]. It is very important to instruct the patient to warm
the eyelids with a hot-humid compress in order to liquefy the solidified meiboum in the gland
ducts and to dilate the ducts. Then, the patient must massage the lids to mechanically force
the meiboum and debris from the plugged or stagnant ducts, and to clean the eyelid margins
[5]. Massage can be done by rubbing with the fingers or with special eyelid pads design for
that purpose [66, 134]. Topical antibiotics such as bacitracin and erythromycin ointments, as
well as fusidic acid viscous eyedrops, may be effective to control bacterial overgrowth and
reduce staphylococci colonies from the eyelid margins [5, 135, 136]. Conventional
management of chalazion and hordeolum with warm compresses, and incision with curettage
when necessary should take place if these lesions occur [5, 67].
According to the National Rosacea Society Comittee, topical medication should be
reserved for grade 1 (mild) disease; systemic medication for grade 2 (moderate) rosacea; and
all patients with persistent grade 1 or 2 disease, or suspected to have grade 3 (severe) disease

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should be referred to the ophthalmologist [71]. From the ophthalmological standpoint, this
therapeutic strategy is difficult to follow if we consider that the evaluation of the ocular
manifestations of rosacea needs the expertise of an specialized physician, the use of a
biomicrosocope for detailed assessment of the eyelid margins, the conjunctiva and the cornea,
and the clinical tests like fluorescein and lisamine green staining, as well as the Schirmer test,
among others [35, 72]. Since the ocurrence of ocular rosacea has been documented prior to
cutaneous manifestations, and the severity of the ocular disease does not correlate well with
the cutaneous findings, we recomend that all patients with rosacea who experience ocular
signs or symptoms, regardless of the grade of disease progression, be referred to an
ophthalmologist for a thorough evaluation and adequate management [35].
For patients with moderate to severe MGD and ocular surface disease characterized by
lacrimal dysfunction with conjunctival and corneal epithelial irregularity or defects, appart
from lid hygiene and topical management to the eyelids, patients also need artificial tears,
gels, and lubricant ointments [28, 63, 70]. Since ocular rosacea patients suffer from decreased
numbers of goblet cells, decreased tear production, and increased tear breakup time, which
are all clinical features of chronic dry eye, lubricants are applied frequently and for prolonged
periods of time therefore, preservative-free eyedrop formulations are preferred [70, 131].
Also, medications containing hyaluronic acid, chondroitin sulfate, aprotinine, and
dexpanthenol, which are intended to reestablish and heal epithelial defects are frequently
needed [137, 138]. Additionally, nutritional supplementation with fish oil and flax seeds
containing omega-3 fatty acids has been reported to improve the symptoms of blepharitis,
MGD, and dry eye [135, 139, 140-142].
If the ocular surface inflammation is significant, particularly in patients with rosacea
keratitis, a short course of topical corticosteroids should be considered [133, 143].
Formulations like, preservative-free dexamethasone phosphate 0.1%, loteprednol etabonate
0.2% and 0.5%, and flurometholone phosphate 0.1%, are adequate for these patients when
used for short periods of time and at the lowest possible concentration, under close
monitoring of intraocular pressure [135]. It is important to note that patients with rosacea are
prone to rapid corneal melting at high corticosteroid concentrations [135, 144]. Topical
corticosteroids are also useful in managing episcleritis and iritis associated to rosacea [2, 3,
131]
Other topical anti-inflammatory medications can also be used as adjunctive therapy for
severe ocular rosacea. In a double-masked, randomized, comparative clinical trial,
cyclosporine 0.05% showed a significant improvement in the ocular surface disease index
(OSDI) scores, corneal staining patterns, tear break up time, and tear-production levels in
patients with ocular rosacea and secondary chronic inflammation of the ocular surface and
dry eye [145]. Moreover, a retrospective clinical study of chronic active ocular rosacea
patients who failed to respond to various combinations of traditional therapies, showed a
significant improvement in the ocular signs and symptoms in 31% of patients, and resolved
the inflammation in 18% of them, after treatment with topical cyclosporine 0.05% and low-
dose oral tetracycline for 6 months [146].
The indirect ocular effects of facially applied metronidazole, was prospectively studied in
20 patients with ocular rosacea. After 8 weeks of facial metronidazole 0.75% gel application,
there was improvement in ocular signs and symptoms and the tear break-up time in ocular
rosacea patients as compared to normal controls [147]. In another prospective study,
metronidazole 0.75% gel was applied topically to one eyelid margin, while lid hygiene alone
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560 Alejandro Rodriguez-Garcia

was perfomed to the fellow eye of 10 patients with ocular rosacea [26]. There was a
significant improvement in the eyelid score, but not in the ocular surface inflammation of
both the treated and control groups. When the pre-treatment and post-treatment eyelid and
ocular surface scores were combined, there was a significant improvement in the treated eyes
but not in the control eyes. No adverse effects on the metronidazole treatment group were
encountered in this study [26].
Another alternative of topical therapy for ocular rosacea is azythromycin 1.5% eyedrops.
In a propective and comparative study, a significant improvement in tear break up time,
meibomian gland plugging, and Oxford score associated with symptom reduction was
reported by all patients after one month of topical azythromycin therapy [148]. Except for
mild burning after instilation, azythromycin 1.5% eyedrops were well tolerated by all patients
[36]. In another retrospective trial, 16 children with ocular rosacea and phlyctenular
blepharokeratoconjunctivitis were treated with lid hygiene plus azithromycin 1.5% eye drops:
3-day treatments (1 drop twice a day) every 10 days, reduced based on efficacy to one
treatment every 15 days and then to one treatment per month [149]. Ocular inflammation was
controlled by azithromycin alone in 15 patients; bulbar conjunctival hyperemia resolved
completely within one month in all eyes, whereas phyctenules and keratitis took longer to
improve, with complete resolution within 3 to 10 months post-therapy. Therapy was stopped
after a median of 6 months (range, 4 to 10 months) without recurrence of inflammation [149].
Despite these promising results with different therapeutic modalities, a Cochrane
Database Review of interventions in chronic blepharitis found no strong evidence for any of
the treatments in terms of curing [150]. According to this analysis, commercial products are
marketed to consumers and prescribed to patients without substantial evidence of
effectiveness. The review suggests that further research is needed to evaluate the
effectiveness of such treatments. Medical interventions and commercial products should be
compared with conventional lid hygiene measures, such as warm compresses and eyelid
margin washing, to determine effectiveness, as well as head-to-head to show comparative
effectiveness between treatments [150].
More advanced and severe cases of ocular rosacea generally require oral antibiotics
which remain the mainstay of therapy, even though the mechanisms of action of these
medications are unclear and often non-specific [131]. Considerable controversy still exists of
whether the improvement in ocular surface inflammation is due to the antimicrobial effect of
these agents or their anti-inflammatory and anti-angiogenic properties [151, 152]. In either
case, oral oxy-tetracycline, minocycline or doxycycline, initiated at full dose for several
weeks, and then gradually decreased (titrated to clinical response); a combination of a 30mg
dose of standard doxycycline and 10mg of sustained-release doxycycline, or a 40mg slow-
release dose of doxycycline alone, have all shown to be effective in the treatment of ocular
rosacea and chronic blepharitis with MGD [22, 37, 153-161]. Nonetheless, these medications
require prolonged use and may be associated with side effects including, infections, allergy,
multi-drug resistance, gastrointestinal distress, photosensitivity, among others [151, 155].
These inconveniences, rise the necessity for targeted therapeutic strategies with safer profiles,
that can be well tolerated for prolonged periods of time. In this respect, a comparative study
of the effects of tetracycline and doxycycline on ocular rosacea patients showed that
tetracycline alleviated the symptoms faster, but doxycycline caused less gastrointestinal side
effects and hence showed better compliance [162]. Another study showed relatively similar
side effects of both tetracycline and doxycycline [5].

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One small cohort clinical trial showed some clinical benefit of oral metronidazole (20-
30mg/kg/day for 3 to 6 months), as adjunctive therapy in ocular rosacea children with MGD,
keratitis, and corneal ulceration [163].
A recent Cochrane Database Review found that the majority of assessed rosacea
treatment trials were at high or unclear risk of bias however, there was some evidence to
support the effectiveness of topical metronidazole, azelaic acid, and oral doxycycline (100mg
or 40 mg) in the treatment of moderate to severe rosacea, and cyclosporine 0.05% ophthalmic
emulsion for ocular rosacea [164]. The review concluded that further well-designed,
adequately-powered randomized controlled trials are required to elucidate the efficacy of
different topical and systemic medications in the management of rosacea [164].
As we expand our knowledge on the pathogenic mechanisms that govern the ocular
manifestations of rosacea, new and more specific therapeutic strategies with fewer side
effects will emerge. Ideally, new therapies would focus on tackling the biochemical and
immunological alterations that ultimately lead to chronic inflammation and damage to the
eyelids and the ocular surface.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 24

INVASIVE CANDIDIASIS
EPIDEMIOLOGY, DIAGNOSIS AND TREATMENT

Mayra Cuéllar Cruz1*, Guillermo Quindós2

and Everardo López Romero1
1
Departamento de Biología, División de Ciencias Naturales y Exactas,
Universidad de Guanajuato, Guanajuato, México
2
Laboratorio de Micología Médica (UFI 11/25, Microbios & Salud), Departamento de
Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología,
Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), Bilbao, España

ABSTRACT
Invasive candidiasis (IC) is associated with high morbidity and mortality in
immunocompromised and hospitalized patients, being Candida albicans the most
frequent species in humans. However, other species such as Candida glabrata, Candida
krusei, Candida parapsilosis and Candida tropicalis have recently emerged as common
causes of IC. In critically ill neonates, C. parapsilosis predominates as the etiologic agent
of candidemia. Factors favoring an increased incidence of IC are most frequently found
in the hospital environments. Multiple risk factors have been identified that predispose
patients to IC including indiscriminate use of broad spectrum antibiotics, antitumor
chemotherapy, organ transplantation, and the use of catheters and other medical devices.
Moreover, since IC does not present with defined clinical symptoms that allow a timely
diagnosis, treatment is often difficult. Thus, it is imperative to find a specific, early
etiological diagnosis to establish an appropriate treatment, improve the prognosis, and
reduce the morbidity and mortality associated with this invasive disease. A prompt
diagnosis is even more critical in neonates and elderly patients with suspected IC. Since
culture-based methods for the diagnosis of IC have a rather limited benefit, non-culture
methods like antigen, antibody, (1,3)-β-D-glucan, and nucleic acid detections seem

*
Corresponding author: Mayra Cuéllar-Cruz. Mailing address: Departamento de Biología, División de Ciencias
Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato. Noria Alta S/N, C.P. 36050,
Guanajuato, Guanajuato, México. Phone: (+52) 473 73 20006 Ext. 8159. E-mail: [email protected] or
[email protected].
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572 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

promising for rapid diagnosis. In this chapter, we review the epidemiology of IC, its
predisposing factors, the mechanisms for Candida dissemination, its main clinical
presentations, and the advantages and disadvantages of current diagnostic methods, as
well as care and therapeutic approaches.

1. INTRODUCTION
The term invasive candidiasis (IC) defines those invasive mycoses caused by the genus
Candida. This genus includes opportunistic yeasts that can cause acute or chronic infections,
mainly in immunocompromised and critical ill patients. IC may be confined to a unique organ
(endophthalmitis, endocarditis, meningitis osteomyelitis, peritonitis, etc.) or become
disseminated through the bloodstream to multiple organs and viscera. Disseminated
candidiasis can be acute, frequently presented as candidemia, or chronic, also called
hepatosplenic candidiasis [1-3]. Candida albicans remains the predominant species causing
IC, and nearly half of all cases are due to this species. However, other species of Candida,
such as Candida parapsilosis and Candida glabrata, and less frequently Candida tropicalis
and Candida krusei, have emerged as common causes of IC. Over 90-95% of candidemias are
caused by these five species. Moreover, C. glabrata, C. krusei and several less common
species of Candida can exhibit lower susceptibility or even resistance to fluconazole and
other current antifungal drugs [4, 5].
Patients at highest risk of suffering from Candida bloodstream infections are usually
patients hospitalized in neonatal, surgical or hematological wards or burn units. The increased
rate of IC is likely multifactorial and it is closely associated to recent changes in clinical
practice, such as the incremented use of long-term central venous catheters, use of broad-
spectrum antibacterial agents, corticoids, monoclonal antibodies and other immunosuppressor
therapies, increases in patients suffering from human immunodeficiency virus (HIV) infection
or other immunodeficiencies, cancer, neutropenia, low weight newborns, and older adults.
The incidence of IC has also grown due to the increasing use of various diagnostic and
therapeutic tools (prosthetic heart valves, stents and other cardiac devices, urinary, peritoneal
and vascular catheters, parenteral nutrition, prosthetic joints, endotracheal intubation, and
major surgery) [6, 7].
Moreover, there has been an improvement in the laboratory procedures for culture and
identification of pathogens that has facilitated the isolation of unusual Candida species. An
extremely important issue is the mortality due to IC that remains unacceptably high, being a
leading cause of nosocomial mortality worldwide. Two important contributors for this poor
outcome are the delay in diagnosis and in the beginning of an appropriate antifungal therapy
[8, 9].
Important risk factors for IC have been characterized and can be useful for guiding
prophylaxis and early empirical therapy. However, improved diagnosis is needed to expand
the potential for early therapy [10, 11].

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 573

2. EPIDEMIOLOGY OF IC. DIFFERENCES IN THE DISTRIBUTION AND

PATHOGENICITY BETWEEN THE SPECIES IN NEONATES, CHILDREN
AND ADULTS: CANDIDA ALBICANS, CANDIDA GLABRATA, CANDIDA
KRUSEI, CANDIDA PARAPSILOSIS, AND CANDIDA TROPICALIS

Wenzel and Edmond have estimated that 5% (2.5-10%) of all patients admitted to
hospitals will be affected by a nosocomial infection. One out of ten of these patients will
suffer from bloodstream infections: 8-10% caused by Candida [12]. Since the 1980s,
Candida has ranked the fourth most common cause of bloodstream infections in USA and
Europe, accounting for 85-95% of all fungemias [13-17]. Over the period 1980-1990, there
was a steady increase in the rate of nosocomial mycoses, from 1.4 to 3.8 episodes per 1,000
discharges. Studies from 1996 to 2003 show remarkably consistent incidence rates from 8-10
cases per 100,000 inhabitants. Then, the incidence of IC has remained similar or even has
decreased slightly in Australia, Canada, many European countries and USA. Meanwhile, the
incidence of IC shows a continuous growing in the rest of the world. Most published data
derive from individual hospitals, as this is not a notifiable disease in most countries.
However, there are sentinel and population-based studies that can help to determine the
current importance of IC and the etiological changing trends (Tables 1 and 2). In population-
based studies, all patients resident in a defined surveillance area are included and as a result,
selection bias is minimized. These surveillances also provide the opportunity to accurately
define incidence in specific risk groups [18].
The incidence of candidemia (CA) in Australia, Europe and Canada was significantly
lower than in the USA. Two exceptions are Denmark and, more recently, Spain, where IC
incidences are similar to those reported in USA. Incidences of 6-10 per 100,000 have been
reported in most population-based studies in the USA [19-21]. One major exception is a study
that reported an incidence of 7.1 per 100,000 in Connecticut and 24 per 100,000 in Baltimore,
despite the fact that the incidences calculated in terms of number of discharges were
comparable [22]. In most European surveys, incidences of 1.4-5.7 per 100,000 have been
reported [23-27]. However, a notable incidence of fungemia, 10 episodes per 100,000
inhabitants, has been reported in different chronological studies from Denmark and in the
more recent Spanish survey [28-30]. Conversely, in other Nordic countries, the incidence of
CA has always been in the range of 1.4-5.7 per 100,000, with 70% of cases being caused by
C. albicans [31-34]. Something similar have been reported from Australia (1.8 IC per
100,000 inhabitants) and Canada (2.9 IC per 100,000 inhabitants) [35, 36]. Arendrup et al.
hypothesized that the use of centrally drawn blood cultures from patients in ICUs may
contribute to a high incidence of fungemia [28]. However, they observed that the proportion
of blood cultures positive for coagulase-negative staphylococci, which are microorganisms
that are frequently associated with catheter infection, was 25-30%, which suggests that this is
not the unique explanation for the high incidence of IC in Denmark. In this country, the
highest incidences of candidemia were observed among elderly patients, reaching an
incidence of 36.9 episodes per 100,000 among the population aged > 65 years. A high
incidence was also observed in neonates (16.3 IC per 100,000 inhabitants) [28]. It is not clear
why rates of IC are higher in USA but may partially be related to the different rates of
sampling, distribution of risk factors in the populations studied, the age distribution, or in the
study methodologies [35]. However, in every reported survey an increase in incidence was
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574 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

documented over the course of the study with a stabilization of candidemia rates in the last
years. Similarly, an increase in incidence has been observed in Argentina, Brazil, Colombia,
Mexico and other American countries or in reports from China and Japan (Table 2).
The highest incidence of candidemia and IC occurred at the extremes of the age spectrum
(infants < 1 year and adults > 65 years old). Kao and colleagues examined the incidence of
candidemia among neonates (<30 days of age) and found a remarkably high incidence of 466
ICs per 100,000 neonates, higher among black (960 per 100,000) than white neonates (238
per 100,000) [19]. This discrepancy could be due in part to the elevated incidence of
prematurity and low birth weight among black infants [19, 22]. There has also been
documented a higher incidence of candidemia among cancer patients (71 per 100,000) and
diabetic adults (28 per 100,000), as well as the presence of central venous catheters in most
patients diagnosed with candidemia. Neutropenic cancer patients and hematopoietic stem cell
transplant recipients are no longer the most vulnerable due to the widespread use of antifungal
prophylaxis during neutropenia in these settings [19, 22, 25].

Table 1. Selected population-based epidemiological studies on candidemia and invasive

candidiasis

Incidence (no. of cases / 100,000 /year)

Location Year Children < 1 Patients ≥ 65 References
Total
year old years old
America
Canada 1999-2004 2.9 20 21.3 [35]
70 (Black: 165 vs. 26 (Black: 40 vs.
USA 1992-1993 8 [19]
white 41) white 10)
USA 1998-2001 6 [21]
Black: 157 vs. Black (92) vs.
USA 1998-2000 10 (7-24) [22]
white 33 white (30)
Europe
Denmark 2004-2006 10.4 16.3 36,9 [28]
Denmark 2004-2009 8.6 11.3 27.7 [56]
Finland 1995-1999 1.9 9.4 5.2 [32]
Finland 2004-2007 2.86 6.9 12.2 [31]
Iceland 1980-1989 1.4 12.7 [33]
Iceland 1990-1999 4,9 11,3 19.3 [33]
Iceland 2000-2011 5,7 20,7 18.1 [27]
Norway 1991-2003 2.4 10.3 7 [34]
Spain 2002-2003 4.3 38.8 12 [25]
Spain 2010-2011 10.1 90 25 [30]
Scotland
2005-2006 4.8 55.9 [26]
(UK)
Oceania
Australia 2001-2004 1.8 24.8 13.7 [36]

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Table 2. Selected epidemiological studies on candidemia and invasive candidiasis

Incidence (no. of cases / 1,000

Location Year References
admissions /year)
America
Argentina 2005-2008 1.15 (0.35-2.65) [54]
Brazil 1997-2007 0.74 (0.41-1.21) [199]
Brazil 2006-2010 0.54 (0.41-0.71) [47]
USA 1998-2000 0.15 [22]
Asia
China 1998-2007 0.026 [59]
Taiwan 2000-2010 0.351 [60]
Europe
Austria 2001-2006 0.27-0.77 [55]
Denmark 2004-2006 0.51 [28]
Denmark 2004-2009 0.41 [56]
England (UK) 2005-2008 0.11 [200]
Germany 1998-2008 0.47 [201]
Iceland 1980-1989 0.15 [33]
Iceland 1990-1999 0.55 [33]
International 1997-1999 0.20-0.38 [23]
Scotland (UK) 2005-2008 0.59 [26]
Spain 2002-2003 0.53 [25]
Spain 2008-2009 1.09 [202]
Spain 2010 0.92 [203]
Spain 2010-2011 0.90 [30]
Oceania
Australia 2001-2004 0.21 [36]
Australia 1999-2008 0.45 [51]

Trick et al. studying the incidence of nosocomial CA in intensive care units (ICU) in
USA from 1989 to 1999 demonstrated a decline in frequency almost entirely due to an
absolute decrease in C. albicans CA [37]. IC is mainly a hospital-acquired infection and
approximately two-thirds of candidemias and IC have its origin in different hospital wards but
communitary IC is increasingly reported. This growth of the number of episodes of
communitary IC could be related to the increasing at home healthcare [10, 38]. Hajjeh et al.
observed in a population-based study that 36% of candidemia occurred in the ICU, and a third
of them were of community onset [22]. Candidemia occurs more frequently in males (60.6%).
Patients who have been hospitalized in an ICU, particularly in surgical, trauma and burn
units, and neonatal ICUs have the highest rates of candidemia. The incidence of candidemia
in ICU patients was found to be 6.9 episodes per 1,000 patients [39]. A recent SENTRY study
including 79 medical centers between 2008 and 2009 reported a total of 1,752 Candida
isolates distributed nearly equal from ICU and non-ICU settings. The frequency of ICU-
associated candidemia was higher in Latin America (56.5%) compared with Europe (44.4%)
and USA (39.6%) [10, 38, 40].
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576 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

Table 3. Distribution of the five most frequent species of Candida on selected

epidemiological studies on candidemia and invasive candidiasis

No. of Species (%)

Location Year References
isolates CA CP CT CG CK
America
Argentina 2005-2008 683 41.3 24.3 19.9 6.3 0.6 [54]
Argentina 2007-2008 461 38.4 26 15.4 4.3 0.4 [204]
Brazil 1997-2007 151 44 22 15 9 6 [199]
Brazil 2006-2007 300 34 26 24 7 3 [47]
Brazil 2006-2010 313 44 14.4 21.7 11.2 3.5 [61]
Canada 1999-2004 209 51.1 6.2 5.7 21.5 4.8 [35]
Colombia 2001-2007 921 44.7 13.6 13.6 1.7 2.1 [205]
Mexico 2004-2007 398 31.9 37.9 14.8 8 2.7 [5]
USA 1998-2000 1143 45 13 12 24 2 [22]
USA 2004-2007 108 47 12 6 29 0 [20]
USA 2001-2009 453 50 13 11 22 1 [9]
Asia
China 1998-2007 102 57.8 10.8 12.8 10.8 0 [59]
Israel 2006-2007 444 44.4 16.6 17.1 15.3 3.1 [126]
Taiwan 2000-2010 2856 50 13.2 19.3 16.1 1.4 [60]
Taiwan 2001-2010 154 32 12 46 7 4 [36]
Turkey 2010-2011 39 11.5 22.2 5.9 1.78 2.1 [53]
Europe
Austria 2001-2006 283 70 8.1 4.9 13.8 0 [55]
Denmark 2004-2006 1133 59.8 4 4.6 20.5 4.1 [28]
Denmark 2004-2009 2820 57.1 3.7 4.8 21.1 4.1 [56]
England (UK) 2005-2008 106 43 20 3 31 2.5 [200]
Finland 1995-1999 479 70 5 3 9 7 [32]
Germany 1998-2008 35 45.7 17.1 5.7 14.3 0 [201]
Iceland 1980-1999 172 64.4 9.6 5.6 12.4 1 [33]
Iceland 2000-2011 222 56 9 13 16 1 [27]
Italy 1992-1997 208 53.8 48.1 5.3 6.7 0 [206]
Italy 1998-2001 162 48.1 17.9 4.7 6.6 0.6 [206]
Scotland (UK) 2005-2006 300 52 11.7 2 22.7 1 [26]
Spain 2002-2003 345 51 23 10 8 4 [25]
Spain 1990-2003 555 42.3 36.3 4.4 9.7 4 [42]
Spain 2008-2009 984 49.1 20.7 10.7 13.6 2.1 [202]
Spain 2009 752 45 24 8 13 2 [30]
Sweden 2005-2006 403 60.8 8.9 2 20.1 1.2 [207]
Oceania
Australia 2001-2004 1068 47.3 19.9 5.1 15.4 4.3 [36]
Australia 1999-2008 1137 45.4 26.9 5.2 13.4 2.8 [51]
CA = Candida albicans, CP = Candida parapsilosis, CT = Candida tropicalis, CG = Candida glabrata,
CK = Candida krusei.

There have been described more than 200 species inside the genus Candida. Twenty of
them are considered human pathogens. C. albicans remains the most isolated species from
blood cultures, with frequencies varying from 32% in Mexico and Taiwan to more than 60%
in Austria and Sweden (Table 3). However, the candidemias caused by other species of

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 577

Candida are rising and there is a wide variation in the etiology of IC in different group of
patients and distinct hospital settings. C. parapsilosis, C. tropicalis, C. glabrata and C. krusei
have become significant causes of human infection. Other species, such as Candida
dubliniensis, Candida orthopsilosis, Candida guilliermondii, Candida metapsilosis, Candida
inconspicua, Candida lusitaniae, Candida norvegensis, Candida nivariensis or Candida
bracarensis, have been isolated from some patients [41-43].
In the 2008-2009 SENTRY study including Candida isolates from 79 medical centers,
approximately 90-95% of isolates belonged to five species: C. albicans, C. glabrata, C.
parapsilosis, C. tropicalis and C. krusei [20]. C. albicans caused 56% (43-67%) of the
episodes of candidemia in a recent European study covering France, Germany, Austria, Spain,
Sweden and the UK, [23]. A recent Australian population-based surveillance found that C.
albicans was the predominant species (47.3%), followed by C. parapsilosis (19.9%) and C.
glabrata (15.4%) [36]. However, the specific incidence and the distribution of C. albicans
and non-C. albicans Candida (NCAC) causing candidemia vary enormously between
hospitals and patients (Table 3). There has been a significant shift in the etiology with an
increase in those infections caused by NCAC: although C. albicans was previously associated
with 70-90% of blood isolates, nowadays C. albicans and NCAC each account for
approximately half of all episodes of candidemia and IC [2, 4, 6, 7, 40, 44-49].
An interesting feature of most NCAC species is that they exhibit patient-specificity and a
specific geographical distribution of their frequencies (Figure 1 and Table 3). C. parapsilosis
is primarily isolated from central venous catheters in neonates and patients receiving
parenteral nutrition and predominates in Australia, Latin America and the Mediterranean
countries of Africa, Asia and Europe. C. parapsilosis is more important in neonates in
neonatal ICUs [30, 36, 42, 47, 50-54]. C. glabrata and C. krusei are associated with recent
major abdominal surgery, cancer, older patients, neutropenic neonates, transplant recipients,
and patients treated with corticosteroids. C. glabrata predominates as second cause of
candidemia in USA and the countries of the North and Center of Europe [27, 28, 55, 56]. The
proportion of C. glabrata has remained constant worldwide at 9-12% but C. glabrata is more
common in USA (21.1%) than in the rest of the world (7.6-12.6%) [57, 58]. A significant
variation in distribution of C. glabrata in blood isolates between hospitals using BACTEC
and those using BacT ⁄ ALERT has been reported from Denmark [28]. In these Northern
European countries, C. dubliniensis, a species close-related to C. albicans can exceed 2-3% of
blood isolates [41, 56]. Finally, C. tropicalis has been isolated from patients with solid tumors
or hematologic diseases and it has been reported as the second etiological agent of IC in Asia
and some parts of Latin America (Colombia and Brazil). The overrepresentation of C.
tropicalis candidemia in patients aged >70 can be related to the increased frequency of
tumors and hematologic diseases in the elderly population [59-61]. Mixed infections
involving two or more fungal species have been described. Usually these polyfungal
infections do not exceed 5% of candidemias. C. albicans is the species most frequently
isolated in combination with other yeasts, with C. glabrata accounting for the majority of
episodes [28]. Moreover, another important feature of NCAC is that some of these species,
such as C. glabrata and C. krusei, are more resistant than C. albicans to antifungal agents and
its potential therapeutic implications [50, 56, 62-68].
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578 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

Figure 1. Geographical distribution of the most frequently isolated species of Candida, excluding
Candida albicans. The dots represent those sites where Candida dubliniensis represents more than 2%
of blood isolates.

Two of these emerging species, C. parapsilosis and C. glabrata are in fact complexes of
species with special clinical and demographic characteristics [42, 69-72]. C. parapsilosis
forms a complex composed of three separate species; C. parapsilosis sensu stricto, C.
metapsilosis and C. orthopsilosis. The exact importance of C. orthopsilosis and C.
metapsilosis as human pathogens remains unknown. Recent data from a nationwide study
during 2009 in 44 Spanish hospitals reported that the incidence of C. parapsilosis and C.
orthopsilosis were 0.22 and 0.02 per 1000 admissions, respectively. Interestingly, C.
orthopsilosis was the fifth most frequently isolated from blood, preceding C. krusei (0.018
cases of candidemia per 1000 admissions) [72]. Moreover, the incidence of C. orthopsilosis
and C. metapsilosis infections have increased since 2004, with prevalence rates ranging from
2.3 to 9% and 0.9 to 6.9% of isolates of C. parapsilosis sensu lato, respectively [72]. The
prevalence of C. orthopsilosis is apparently higher in warmer Mediterranean countries than in
the cooler countries of the Atlantic, Central and North Europe. However, other factors could
be responsible for local specificities, such as differences in hospital services (presence or
absence of ICU or surgical wards) and the patient population (transplant recipients and other
immunodeficient patients). This variability in prevalence has been reported in other parts of
the world, with a higher prevalence of C. orthopsilosis among isolates of C. parapsilosis
sensu lato, in those countries with hot and humid climates, such as Taiwan (8.5%), Brazil
(9.1%) and Malaysia (24.4%) [42]. C. parapsilosis is usually susceptible to most antifungal
agents, but there are reports of clinical isolates with decreased susceptibility to azoles and
echinocandins [72, 73].

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 579

C. glabrata is a complex species that includes C. glabrata sensu stricto and two newly
described species, C. bracarensis and C. nivariensis [42, 70, 71, 74]. Lockhart et al. in their
analysis of 1,598 C. glabrata isolates from 29 countries observed that C. bracarensis and C.
nivariensis isolates constituted a very small percentage (0.2%) of the C. glabrata clinical
isolates [75]. However, these cryptic species could be more prevalent in specific regions.
Most reports have underlined the lower susceptibility of C. glabrata, C. bracarensis and C.
nivariensis to the most commonly used azoles. Some authors have linked institutional or
individual fluconazole use in the selection of C. glabrata, especially in cancer centers [48,
62]. In a recent SENTRY study including 79 medical centers and a total of 1,752 Candida
isolates, C. glabrata was the only species in which resistance to azoles and echinocandins was
reported [57].
The attributable mortality rate of CA is estimated to be >30% (range 24–60%, median
38%), with a crude mortality rate of >50% (range 13–90%, median 55%). These figures
exceed widely the reported for most bacterial infections. Since 1989, a 50% reduction in
mortality rates for IC has been reported, following a steady increase in mortality in the
previous decades reaching 0.62 deaths per 100,000 persons. A similar decline in rates of
death from systemic candidiasis associated with HIV infection occurred (0.04 per 100,000).
The explanation for decreased mortality in both HIV infected and non-infected patients could
be related to the increased awareness, earlier diagnosis, and the enhanced therapy. CA in
neutropenic patients is a life-threatening infection that is associated with acute disseminated
candidiasis, a sepsis-like syndrome, multiorgan failure, and death. Infections due to C.
parapsilosis tend to be associated with reduced lethality (23%). Patient outcomes appear to be
worst for C. glabrata and C. tropicalis infections, and to a lesser extent C. krusei IC. CA not
only increases patient mortality, but also extends the length of stay and increases the total cost
of medical care [10, 30, 76, 77].

3. RISK FACTORS FOR INVASIVE CANDIDIASIS

IC is an opportunistic infectious disease facilitated by the concurrence in the pediatric
and adult patients of several and very different underlying diseases and risk factors. Among
the most relevant are included prolonged neutropenia, the widespread use of broad-spectrum
antibiotics and the increased use of central venous catheters. However, age, severity of the
underlying disease, length of stay in the hospital (above all stay in the ICU), neoplasia and
antitumor chemotherapy, corticosteroids, total parenteral nutrition, acute pancreatitis, and
prior fungal colonization, are also relevant. Some risk factors facilitate Candida colonization
(v.g., corticoids), others contribute to tissue or bloodstream invasion (v.g., intravascular
catheters), while others impair host defenses [10, 76].
Another group of patients with a high incidence of IC are those who have suffered major
abdominal surgery such as those of colon or pancreas, severe peritonitis, or suture failure.
In these patients, the factors most frequently associated with IC are the presence of
intravenous catheters, treatment with corticosteroids and chemotherapy agents, malnutrition,
admission to ICU, neutropenia and hemodialysis [4, 10, 78-80].
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580 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

Table 4. Risk factors involved in the development of invasive fungal infection

Factor Mycosis
Ambient exposure Histoplasmosis
Coccidioidomycosis
Paracoccidioidomycosis
Blastomycosis
Aspergillosis
Hialohyphomycosis
Feohyphomycosis
Anatomic barriers –skin and mucosa- disruption (surgery, Candidiasis
catheters, antitumor chemotherapy, mechanical Aspergillosis
ventilation, hemodialysis, continuous ambulatory
peritoneal dialysis)
Microbiota unbalance (antimicrobial chemotherapy) Candidiasis
Aspergillosis
Neutrophil dysfunction (quantitative –neutropenia- or Candidiasis
qualitative) Aspergillosis
Trichosporonosis
Geotrichosis
Hialohyphomycosis
Feohyphomycosis
Cellular immunodeficiency (HIV infection and AIDS) Cryptococcosis
Pneumocystosis
Histoplasmosis
Coccidioidomycosis
Immune immaturity (low weight neonates) or immune Candidiasis
senescence (old persons)
Diabetes mellitus and other metabolic diseases Mucormycosis
Candidiasis

In Table 4 the association between the major invasive mycoses and risk factors is
presented. Basically, IC is associated to the presence of risk factors related to the alteration
and disruption of the anatomic barriers, an unbalance of the host microbiota, quantitative
and/or qualitative dysfunctions of neutrophils, and immune alterations (immaturity,
immunosenescence and/or cellular immunodeficiency). Tables 5 and 6 reflect the main
factors associated to a major risk for suffering IC in hospitalized patients. There is a great
variability in the patient population with high susceptibility to fungal infections, and this
complicates an adequate stratification of the patients at risk. For instance, in a prospective
study conducted in six surgical ICUs, multivariate analysis revealed increased risk with prior
abdominal surgery, relative, acute renal failure, total parenteral nutrition, and use of a triple
lumen catheter as the major risk factors. However, prior fungal colonization, considered an
important predisposing condition, could not be demonstrated in this study as a risk factor,
whereas receipt of any systemic antifungal drug was associated with reduced risk [64, 65, 81].
Candida colonization and infection are facilitated by host barrier disruptions, as in major
burn. In most cases, colonizing and infecting strains are identical, with the time from
colonization to infection typically brief. NCAC occurs more commonly in patients who have

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 581

received azoles, and in those with prolonged neutropenia. Cutaneous and mucosal
colonization by Candida in at least two different anatomical regions is considered a risk
factor for IC in hematology and in ICU patients [22, 82-84]. The colonization index quantifies
the degree of colonization as the number of distinct body sites with cultures positive for
Candida, and is helpful in the setting of ICU and general surgical patients. Another index, the
‘Candida score’, has been proposed for guiding antifungal prophylaxis in critically-ill non-
neutropenic patients with Candida colonization. This index utilizes is based on risk factors
with variable weightings in the score. A value of >2.5 may could distinguish between patients
with IC and those only colonized [76, 85-87].
Moreover, there are specific predisposing factor for some clinical presentations of IC. For
instance, the predisposing factors of Candida endocarditis include underlying valvular
disease, prosthetic cardiac valves, prolonged presence of intravascular catheters, heroin
intravenous user, cancer chemotherapy, pre-existing bacterial endocarditis,
immunocompromise, abdominal surgery and low birth weight. Risk factors involved in the
development of peritonitis include recent or concomitant antimicrobial therapy, inoculum
size, and surgery for acute pancreatitis. Pancreatic transplantation, especially with enteric
drainage, is associated with intraabdominal Candida abscesses, intrapancreatic abscesses and
infection of pseudocysts [10, 76, 77].

Table 5. Invasive candidiasis and candidemia risk factors for hospitalized patients

Clinical setting Risk factors

Immunodeficiency Neutropenia (intensity, duration and dynamics)
Lymphopenia (intensity, duration and dynamics)
Cellular and humoral immunodeficiencies
Malnutrition
Senescence
High doses of chemotherapy and/or corticosteroids
Immune suppressors (alemtuzumab, infliximab, antithymocytic
globulin)
Immunomoduladory virus infection (CMV, EBV, HHV6, HHV7,
HHV8)
Organic Skin and mucosal disruption and mucositis by surgery, radiotherapy,
dysfunctions chemotherapy, GVHD, HHV1 infection, intravascular catheters…
Renal and/or hepatic dysfunction
Digestive obstruction
Hypoesplenia or asplenia
Respiratory distress associated to viruses
Microbial Antimicrobial agents
colonization and Gastric acid suppressors
reactivation of Prolonged hospitalization or ICU stay
latent infections Reactivation of latent infections caused by mycobacterium,
Toxoplasma, CMV, EBV, HHV1, VZV, HBV or HCV
Previous invasive mycoses
CMV: Cytomegalovirus, EBV: Epstein-Barr virus, GVHD: Graft versus Host disease, HBV: Hepatitis
B virus, HCV: Hepatitis C virus, HHV1: Human Herpesvirus 1, HHV6: Human Herpesvirus 6,
HHV7: Human Herpesvirus 7, HHV8: Human Herpesvirus 1, VZV: Varicella-Zoster virus.
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582 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

Table 6. Risk factors and patient populations at high risk for suffering from invasive
candidiasis

General factors Severity of acute disease

Age (< 1 year and > 65 years old)
Major surgery (mainly gastrointestinal)
ICU stay
Indwelling catheters
Underlying diseases: diabetes mellitus, cirrhosis, malnutrition
Multiple blood transfusion
Parenteral nutrition
Mechanical ventilation
Patients at higher risk Low weight neonate (<1,500 g)
Prolonged neutropenia (< 500 cells per mm3)
Central venous catheter
Chemotherapy
Treatment with corticosteroids and/or inmunosupressors
Pancreatitis, visceral perforation
Renal failure, hemodialysis
Polytraumatisms
Extended burns
Treatment with broad spectrum antibacterial agents
Multiple Candida colonization

Alterations to host cellular defenses and host microbiota typically precede Candida
colonization. Polymorphonuclear cells are the first line defense against intravascular spread of
Candida by damaging and killing fungal cells. Prolonged neutropenia or neutrophil
dysfunctions are high-risk factors for developing IC. Patients suffering from cellular
immunodeficiencies are predisposed to mucocutaneous candidiasis, but seldom develop IC.
Protective immunity against fungal pathogens is achieved by the integration of two distinct
arms of the immune system, the innate and adaptive responses. T-helper lymphocytes (Th) 1
secrete cytokines like interleukin2 (IL2) and interferon-gamma, whereas Th2 produce IL4,
IL5, IL6, IL10, and IL13. Th1 responses are significant in protection against C. albicans and
recovery from infection. Th2 responses apparently are related to the blockade of
dissemination of candidiasis. Moreover, IL-17-producing T cells (Th17) have pivotal roles in
both innate and acquired immune response to fungal infections. The Th17 pathway may play
an inflammatory role previously ascribed to unrestrained Th1 responses. Regulatory T cells
have become recognized as providing the host with immune defense mechanisms adequate
for protection, without necessarily eliminating fungal pathogens or causing an unacceptable
level of tissue damage [88,89]. Host immune responses depend upon specific ligand–receptor
systems, which activate intracellular signaling pathways with distinct differences in the
immune consequences. Complement and immunoglobulins are necessary for efficient
opsonization and intracellular killing of Candida, and deficiency of either, can be associated
with complicated candidiasis. Cells of the innate immune system express a variety of pattern-
recognition receptors (PRRs) which play a crucial role in host recognition of Candida via
pathogen-associated molecular patterns (PAMPs) located in the cell wall. Recognition of

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 583

PAMPs by PRRs leads to a variety of signaling pathways generally mediated by differential

cytokine production [76, 90].
The most important is an ever-expanding population with immunocompromise due to
mucosal or cutaneous barrier disruption, defects in the number and function of neutrophils or
in cell-mediated immunity, metabolic dysfunction, and extremes of age. Increasing use of
broad-spectrum antibiotics, cytotoxic chemotherapies, and transplantation further increases
the risk for IC. In patients with hematologic diseases, risk factors for mortality include
allogenic bone marrow transplant, septic shock, and the absence of antifungal prophylaxis
[91,92]. The high mortality in these critically ill patients is due to many factors that favor the
development of IC, such as the underlying severity of their illness, the presence of central
venous catheters, parenteral nutrition, broad-spectrum antibiotics, transfusions, acute
pancreatitis, intestinal fistulas, and prior colonization [66, 85, 87, 93-96].
The patients admitted to the ICU who have a high risk of contracting IC are preterm
infants, not only because of the ICU environment but also because their still immature
immune system with chemotaxis defects, cytokine production, antibodies and defects in
phagocytosis. This condition compromises the integrity of the skin, and causes
gastrointestinal tract diseases leading to chronic malnutrition. This may require long term use
of central venous catheters and endotracheal intubation, as well as other neonatal care
practices, particularly related to septic processes [97-100]. However, some authors believe
that the origin of candidemia in these patients may be endogenous, and that other factors are
involved in catheter colonization such as local trauma, the use of parenteral nutrition and
broad-spectrum antibiotics [101]. High mortality rates have been reported for IC patients due
to infection related to the use of catheters. One third of these patients with catheter infection
develop serious complications, including septic shock, chronic infection, septic
thrombophlebitis, metastatic abscesses, endocarditis, and arteritis. IC primarily affects
immunocompromised patients, especially those with some form of cancer and recipients of
liver, bone marrow and lung transplants. The incidence for recipients of kidney and heart
transplants has decreased in recent years [102]. Renal transplanted patients rarely present IC
symptoms, whereas urinary tract infections from bacteremia are more common [103].
Hematological transplant recipients have an increased incidence of IC. Since hematopoietic
cell transplantation is being used more frequently in the treatment of hematological diseases,
these patients require intensive immunosuppressive therapy, have high rates of severe graft-
versus-host disease and therefore have increased risk of infection [104]. The main risk factor
for the development of IC in these patients is neutropenia, the alteration of mucosal barriers,
and the use of broad spectrum antibiotics [105, 106]. Liver transplant recipients are at risk for
IC during the immediate postoperative period [107, 108]. It has been shown that 80% of the
invasive fungal infections in these patients are IC [109]. For liver recipients, the risk factors
for developing IC include the complexity and duration of the transplant surgery, prolonged
use of broad-spectrum antibiotics and hospitalization, post transplantation dialysis,
retransplantation, colonization of medical devices, cytomegalovirus damage, blood
transfusions, and the refusal of treatment [109, 110]. In patients with lung transplants, graft
loss is a potential problem for the development of IC. When the graft loses function,
retransplantation may be a therapeutic option. However, both single-lung and double-lung
retransplantation are associated with high morbidity and mortality and development of IC. In
general, in recent years the incidence of IC in patients with solid organ transplantation has
been reduced to approximately 2%, a value significantly lower than the 4 and 6% rate of
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584 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

previous decades. For these patients, the overall survival rate at 12 months after suffering IC
is only 66% [108, 111].
Other immunosuppressed patients who have high incidence of IC are those with some
type of cancer. Several studies have shown that in patients with hematolymphoid diseases
such as leukemia and lymphoma, neutropenia, therapy with corticosteroids, and the use of
anaerobicides are predisposed to develop candidemia. In patients with solid tumors, use of
anaerobicides was independently associated with candidemia [112]. Currently, candidemia
and IC is considered to be a rare complication of AIDS, typically associated with advanced
stages of disease and the use of central venous catheters [113]. However, in recent years an
increase in IC has been observed due to lymphoproliferative damages in these patients. IC
also occurs in people with metabolic abnormalities like diabetes mellitus. Thus, it has been
reported that this disease affects the proper functioning of iron metabolism as well as cellular
and humoral immunities, which together with diabetic angiopathies favor the development of
IC.

4. MECHANISMS OF CANDIDA DISSEMINATION

Candida species are opportunistic fungal pathogens that normally live as commensals in
the human digestive tract. However, when there is an alteration of the host immune system,
they become pathogenic and cause infection in a variety of tissues. C. albicans has been
isolated from the mouth and the gastrointestinal tract of around 30-50% of healthy persons
and it is also a member of the vagina microbiota. The isolation increases in patients receiving
medical attention and during their hospitalization. Individual patients tend to harbor the same
genotype of Candida over long periods of time. For instance, more than 60% of patients with
candidemia have positive cultures for the same Candida genotype as the genotype isolated
from various anatomic sites prior to developing candidemia [114]. C. albicans is less
commonly isolated from the ambient than other species of Candida but it survives for up to 4
months in the hospital environment. C. parapsilosis can be a member of the human
microbiota of the skin and mouth, and many infections caused by this species are associated
with its carriage on the hands of health care workers. This species is commonly related to
catheter- and intravenous hyper alimentation-associated candidemia due to its capability to
adhere to and develop biofilms on the surfaces of intravascular devices and to contaminate
and grow in parenteral nutrition solution [42, 115, 116]. Candidiasis typically originates from
the endogenous microbiota of the patients. The major steps in the pathogenesis of invasive
candidiasis include increasing colonization, characteristically secondary to broad-spectrum
antimicrobials; failure of skin and mucosal barriers, often a result of the use of intravascular
devices, severe burns or recent surgery and; immune dysfunction (v.g., neutropenia) that
enables fungal access and ultimately allows dissemination. In neutropenic persons and
patients following abdominal surgery without intravascular central catheters, digestive tract
colonization is likely to be the source of most cases of candidemia. Conversely, the skin is
thought to be the source in patients with skin colonization and contaminated intravascular
catheters. Other sources could be intraabdominal abscesses, peritonitis, and uncommonly, the
urinary tract and contaminated intravenous solutions. Candida can be recovered from the

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 585

hospital environment, including food, furniture, air-conditioning vents, floors, respirators, and
from hands and gowns of medical staff [77].
The presence of other microorganisms appears to inhibit virulence factors such as
adhesion and colonization, which are essential for tissue invasion and subsequent
dissemination of Candida. This is the one of the main reasons that explains why that the
indiscriminate use of broad spectrum antibiotics predisposes to IC. There are several
mechanisms of dissemination of Candida which, like other organisms, can colonize internal
organs and blood. Among endogenous mechanisms, the invasion of the peritoneal cavity
through a discontinuity is one of the most commons. Candida accesses to the peritoneal
cavity through a perforation of the gastrointestinal tract that may occur during abdominal
surgery or by infection or perforation of an organ connected to the intestine or by contiguity,
for instance renal or perinephric abscess, ovarian tubes, liver, spleen, or pancreas. Another via
to reach the internal organs is translocation. This mechanism is probably the most accepted as
it has been observed in various microorganisms. Translocation is the passage of the
microorganism or their metabolic through intact intestinal wall to the lamina propria reaching
the mesenteric lymph nodes, blood and other tissues [117]. In surgical patients with damaged
mucosal barriers, and suffering from mucositis, diarrhea, or intestinal graft-versus-host
disease, the translocation of Candida through the digestive tract to the bloodstream and
internal organs is favored. Moreover, translocation has been described in persons after
ingesting a large inoculum of C. albicans [95, 117-119]. Translocation could occur when
Candida directly penetrates through the enterocytes by a unique process that differs from
classical phagocytosis [117]. First, it appears that Candida adheres to epithelial microvilli,
which are distorted during adhesion. Subsequently, in an almost noticeable process, the
pathogen passes through the enterocyte membrane into the cytoplasm, locating within a
vacuole. The overlying microvilli are distorted suggesting a cytoskeleton dislocation. Then
the yeast migrates through the enterocyte cytoplasm, reaches the basal membrane and enters
the lamina propria, and finally into the blood stream. As dissemination continues remote
metastatic foci are produced involving kidneys, urinary tract, eyes, lungs, bladder, liver, bone,
lymph nodes and blood [120]. After 24 hours, some yeast cells reach the serosal surface,
already in the peritoneal cavity. The lymphatic system of the lamina propria and submucosa
would destroy those cells that cross the intact mucosa by a similar mechanism. When the
physiological system that destroys microorganisms and their metabolic degradation products
is saturated, IC appears. Additionally, it has been shown that translocation is favored when
the patient has hemorrhagic shock, intestinal obstruction, hyperpyrexia, burns, administration
of antibiotic therapy, endotoxins or cytotoxic drugs [117 ,121, 122].
Transmission of Candida from person to person has also been reported [99]. The main
mechanisms of cross infection include direct patient to patient transmission, transmission
from a colonized or infected person to a susceptible one via a third person, often a healthcare
professional or a medical tool; and simultaneous transmission to two or more patients from a
common source, such as a contaminated intravenous infusion. In many nosocomial outbreaks
due to C. albicans in ICU, the source has been the hands of healthcare staff. Conversely, in
most C. parapsilopsis infection outbreaks the cross infection was related to medical devices,
such as vascular catheter used for parenteral nutrition. Although, there are reports of C.
parapsilopsis cross infection related to hand carriage by medical or nursery staff. DNA typing
has also confirmed acquisition of nosocomial Candida from environmental and human
sources. Candida has been recovered from 25% to 50% of inanimate surfaces sampled and
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586 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

cultured from patients’ rooms prior to patient acquisition of the same strain. Surfaces in
contact with hands of personnel or patients commonly harbored Candida. Identical strains of
Candida have also been recovered from patient food prior to patient acquisition. However,
except in outbreak settings, the source of Candida is usually from the host’s endogenous
microbiota [77].
Candida species that persist within the biofilm matrix (e.g., on intravascular catheters)
show reduced apoptosis. Biofilms could play an important role in the development and
establishment of IC. Biofilm cells develop several other unique phenotypic and genotypic
characteristics, including resistance to most antifungals. Tissue invasion involves enzymatic
activation of lipases, proteinases and adhesins. Invasion of epithelial and sub-epithelial tissues
involves cell filamentation, which likely enhances the pathogen ability to invade the organism
via burrowing-like processes. Following yeast angioinvasion, budding via unicellular buds
facilitates hematogenous dissemination. This process typically evolves by a miliary pattern
with fungal abscesses and granulomata. Finally, it has been shown that most infection-related
changes in C. albicans gene expression reflect environmental adaptation; initial yeast contact
with the host, and disease progression are associated with fungal metabolic and stress (e.g.,
heat shock proteins) adaptation responses [76, 123, 124].

5. CLINICAL PRESENTATIONS OF INVASIVE CANDIDIASIS

Acute disseminated candidiasis is the most common form of IC and usually presents as a
serious, rapidly progressing infection. However, this is not the unique clinical presentation of
IC that can evolve in a chronic condition (chronic disseminated), or present as deep organ
candidiasis. The difficulty of reaching an early diagnosis and treatment refractoriness can lead
to high morbidity, prolonged hospital stay and elevated mortality. Candidemia is defined as
the isolation of Candida species from one or more blood cultures. The presence of Candida in
the bloodstream can lead to the development of deep-organ candidiasis or to disseminated
candidiasis with infection of two or more different organs [125, 126]. Hematogenous spread
occurs at some stage in the evolution of IC but only catheter-related candidiasis and acute
disseminated candidiasis are associated with documented candidemia. This fact causes an
underestimation of the true incidence of IC. In hematological patients, candidemia usually
occurs after few days of fever and chemotherapy-induced neutropenia. In these patients, IC is
often the result of mucositis and Candida digestive translocation. In the rest of cases, IC
occurs in patients with advanced diseases or after major abdominal surgery, often in the
setting of an ICU stay, generally after weeks of hospitalization. This IC is more frequently the
result of an intravenous catheter-related candidemia or from Candida draining during or after
surgery [125, 127].
Patients suffering from acute IC can present with fever alone without specific
manifestations, or a wide spectrum of symptoms. Acute disseminated candidiasis is
indistinguishable from bacterial septicemia. Moreover, clinical manifestations are frequently
superimposed on those symptoms and signs of the underlying illnesses and concomitant
infections. Patients with neutropenia have a significantly higher rate of visceral and cutaneous
dissemination. Candidemia can occur from any source, but frequently it follows intravascular
catheter infection with a better resolution following catheter removal [66]. Prolonged

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candidemia, especially when blood cultures remain persistently positive on appropriate

antifungal therapy, suggests a persistent focus, such as an intravascular catheter, an abscess,
or suppurative thrombophlebitis, endocarditis, severe neutropenia, and rarely, antifungal
resistance, especially with some CNCA [10].
IC in neonates presents with subtle symptoms. Two distinct syndromes have been
described, neonatal systemic candidiasis and congenital cutaneous candidiasis. Neonatal
systemic candidiasis develops either by uterine infection prior to birth or from colonization
during the birth, with hematogenous dissemination of Candida in the first days of life.
Previous antibiotics, presence of a central catheter or endotracheal tube, and center were
strongly associated with IC. Candida meningitis has been reported in > 60% of low birth-
weight neonates with IC. This meningitis has a high mortality and neurologic defects are
common in survivors. Conversely, the congenital cutaneous candidiasis presents within a few
hours of birth with a diffuse maculopapular, erythematous rash that can evolve to pustular or
vesicular lesions with desquamation. In low birth-weight neonates, systemic involvement is
frequent and neonatal systemic candidiasis should be excluded [18, 77, 128-130].
Chronic disseminated candidiasis, often referred to as hepatosplenic candidiasis, develops
as a complication of IC after profound and prolonged neutropenia, which is more often seen
in patients with acute hematological malignancies, especially acute leukemia, that have been
treated with cytotoxic chemotherapy, and received antibacterial therapy during an episode of
febrile neutropenia. Many patients have no documented candidemia. The lesions established
during neutropenia do not resolve, and become prominent, especially in the liver, spleen, and
kidneys, when patients recover from neutropenia. This syndrome is now less frequent because
of the widespread antifungal prophylaxis and early empirical antifungal therapy in patients
with febrile neutropenia [131].
IC can present as an infection of a unique organ or multiple ones. The organs more
frequently infected are kidney, heart, brain and meninges, eye and the peritoneum. Candida
can be also an infrequent cause of pulmonary infection, osteomyelitis and arthritis,
cholecystitis and cholangitis, and pyomyositis [111, 132, 133].
Renal candidiasis most commonly follows hematogenous dissemination of Candida to
the kidneys. Around 80% of patients with IC develop renal infection and this condition
should be suspected in septic patients with persistent candiduria. Moreover, indwelling
urinary catheters serve as a portal of entry for Candida as most catheters become colonized if
left in place long enough. Catheterization allows the migration of Candida into the bladder
along the external surface of the catheter from the periurethral areas. Ascending infection can
also lead to acute pyelonephritis and, rarely, candidemia. In adults, the symptoms of renal
candidiasis include fever, rigors, and lumbar and abdominal pain. The presence of a fungus
ball in the renal pelvis or the urether can complicate infections.
Candida endocarditis is the commonest form (30-50%) of fungal endocarditis, with high
mortality, increased frequency of aortic and mitral valves involvement and of large vessel
embolization. Three groups of patients are prone to develop this condition, those patients with
underlying native valve disease, patients with prosthetic heart valves and intravenous drug
users. In the latter, the tricuspid valve id often involved. C. parapsilosis has a tropism for
prosthetic endovascular surfaces. Candida myocarditis is the result of hematogenous
dissemination with development of abscesses or microabscesses. Candida can reach the
pericardium from adjacent endocarditis or myocarditis but most frequently pericardial
involvement is the result of hematogenous seeding or direct inoculation at the time of cardiac
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588 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

surgery. Phlebitis is common and often is associated with tunneled subcutaneous catheters
[77].
Candida meningitis can be a complication of neurosurgery or a head trauma but it is
more frequently the result of hematogenous dissemination. Single or multiple abscesses cab
be found scattered throughout the brain. Other presentations are thrombosis, vasculitis,
hemorrhage, fungus balls, and mycotic aneurysms, following the use of catheters, intravenous
drugs, and dialysis. Brain abscess presents with fever, altered mental status, and/or focal
manifestations depending on size and site of the lesions. Candida meningitis can be acute
with meningismus or chronic resembling tuberculous or cryptococcal meningitis with an
indolent course. Patients present with chronic headache, fever, and nuchal rigidity [134].
Endogenous endophthalmitis and chorioretinitis occurs in 30-45% of patients with
nosocomial candidemia, including neonates, and intravenous drug users. Several of the
patients with an initially negative eye examination developed signs of ocular candidiasis 1-2
weeks later. Symptoms include visual blurring, floaters, scotomata and blindness.
Fundoscopic examination usually reveals yellow-white, cotton-like lesions. Untreated
candidiasis can progress to retinal necrosis and visual loss [18, 77, 135-137].
Candida abdominal sepsis may occur as monomicrobial or polymicrobial peritonitis and
result in single or multiple abscesses. Peritoneal contamination with Candida species usually
follows spontaneous gastrointestinal perforation or surgical opening of the gut. Peritonitis is
more likely to follow proliferation of accompanying bacterial pathogens but can occur with
Candida alone. With Candida peritonitis, Candida usually remains localized to the peritoneal
cavity; dissemination occurs in approximately 25% of patients. CA complicating
intraabdominal infection is associated with a high mortality.

6. DIAGNOSTIC METHODS: FROM THE IDENTIFICATION

OF CANDIDA TO THE NUCLEIC ACID DETECTION

Diagnosis of IC is a difficult challenge for the clinician because of the very varied and
non specific clinical presentations. Traditional microbiological methods are slow and present
a low diagnostic sensitivity. Moreover, there are important difficulties for the interpretation of
results and to differentiate colonization from invasion by Candida. In most cases, the
diagnosis is based on a combination of clinical, radiological, microbiological and
histopathological findings. These findings may include a positive blood culture result, or a
positive culture for a specimen obtained from a normally sterile site, or microscopic
examination showing polymorphic cells, including budding yeasts, pseudohyphae or hyphae
consistent with Candida in a biopsy or aspirate from deep lesions. Laboratory diagnosis is an
essential step in establishing the etiology of IC, because it allows the identification of the
organism by direct observation and traditional culture procedures based on their
morphological and biochemical characteristics [76, 116, 138-142]. Conventional
microbiological methods could be complemented with molecular methods in the rapid and
definitive identification of fungal isolates. Biomarkers (1,3)-β-D-glucan –BG-, mannan,
antigerm tube antibodies –CAGTA-) are very useful in patients suffering from an IC (Tables
7 and 8). Disadvantages are the rapid elimination of antigens by the patient and the low
production of antibody titers by some severely immunosupressed persons, so in many

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 589

occasions it is necessary to analyze serial serum samples for facilitate a reliable diagnosis
[141-147].
Suggested criteria for diagnosis of IC include; culture of Candida species from normally
sterile sites, clinical signs of infection at sites of Candida isolation and absence of other
potential pathogens. Currently the gold standard for diagnosis of IC is culture of Candida
species from a normally sterile body site. The performance of conventional diagnostic
procedures depends on the possibility of obtaining samples from deep tissues. Isolation of
Candida from sterile sites, such as blood, cerebral spinal, pleural or peritoneal fluids, points
to IC. Isolation of Candida species from a single blood culture is considered diagnostic of
CA, results are usually available in 24-72 h, but blood cultures are only positive in 50-75% of
autopsy-proven IC. The sensitivity of blood cultures decreases in neutropenic patients and
when patients are undergoing antifungal prophylaxis. Then, negative blood cultures do not
rule out IC. Lack of sensitivity of blood culture can be related to intermittent presence of
Candida in the blood-stream, uptake of Candida in circulating leukocytes and monocytes or
low counts of Candida cells per milliliter of blood [77, 148]. The likelihood of systemic
invasion has been correlated with increasing numbers of positive blood cultures. The number
of blood cultures recommended in a single session is three [141, 149]. A blood cultures set
comprises of 40-60 ml blood for adults obtained in a single session within a 30-min period
and inoculating 10 ml of blood in each of 3 aerobic and 3 anaerobic bottles. The volume of
blood should be lower for children (2-4 ml for children under 2 Kg, 6 ml between 2-12 Kg,
and 20 ml between 12-36 Kg). The frequency recommended is daily when CA is suspected,
and the incubation period must be at least five days. The sensitivity of blood cultures may be
worse in children, since much less blood can be collected. There is a strong recommendation
in most diagnostic guides to use an automated validated blood culture system, such as
BACTEC or BacT/Alert [141, 148]. When both automated blood culture systems have been
compared, the highest performance was obtained using the mycological medium. However,
routine usage of this medium is not usual in the microbiological laboratories [150].
The presence of fungal structures in biopsies of infected tissues and organs can provide a
direct evidence of IC and consequently a proper antifungal therapy can be promptly initiated.
However, these specimens may contain small numbers of Candida cells with resultant
negative cultures. Tissue sections can be stained with calcofluor white, Gomori-Grocott
methenamine silver, or periodic acid-Schiff stains to facilitate the observation of yeasts and
hyphae of Candida. This histopathological approach has several limitations as organic lesions
and fungal structures are difficult to interpret for those unfamiliar with fungal infections and
the diagnosis is made many times when the fungal burden is too high and the tissue damage
nearly irreversible. The use of specific antibodies marked with fluorescein, DNA probes or
PCR using primers specific for fungal DNA can help in some situations [141, 142].
The emergence of species, such as C. glabrata and C. krusei with reduced susceptibilities
or resistance to current antifungal agents makes species-specific identification very important,
as the antifungal susceptibility of clinical isolates can be inferred and predicted from their
identity. A schematic procedure for laboratory identification of clinical isolates is shown in
Figure 2. The isolation of Candida in culture media gives invaluable information on main
characteristics of the clinical isolates, such as virulence factors, specific genotypes and the
antifungal susceptibility. This information permits to understand the pathogenesis and
epidemiology of these infections, and to build local and global epidemiological patterns. Most
species of Candida grow in standard cultures, but Sabouraud dextrose agar can facilitate their
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590 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

isolation and facilitate the next steps for identification. CHROMagar Candida (CHROMagar,
France), ChromID Candida (BioMérieux, France), and the Chromogenic agar for Candida
(Laboratorios Conda, Spain) and other chromogenic agars can be used for primary culture and
to rapidly identify the most common species of Candida, such as C. albicans, C. dubliniensis,
C. glabrata, C. krusei, and C. tropicalis, based on colony colors (Figure 3) [151-153].

Microscopy (Germ tube test)

B

Carbohydrate assimilation

Culture and isolation

Latex agglutination (antigens)

PCR (nucleic acids)

Antifungal susceptibility testing PNA-FISH

MALDI-TOF

Figure 2. Scheme for laboratory diagnosis of invasive candidiasis. A) Specimen processing. B) Culture
and identification of clinical isolates.

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 591

CHROMagar Candida A
Candida albicans

Candida dubliniensis
Candida tropicalis

Candida lusitaniae

Candida glabrata

Candida krusei

Candida guilliermondii
Candida kefyr

ChromID Candida B
Candida albicans

Candida dubliniensis
Candida tropicalis

Candida lusitaniae

Candida glabrata

Candida krusei

Candida guilliermondii Candida kefyr

Figure 3. Colonial morphology of Candida albicans and other relevant species of Candida on
chromogenic agars. A) CHROMagar Candida. B) ChromID Candida.

A rapid, but nonspecific identification of C. albicans can be made by germ tube test,
growing the yeast in serum at 37°C during 2-3 h and observing for the formation of germ
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592 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

tubes and young hyphae. However, C. dubliniensis and some C. tropicalis isolates generate
germ tubes eliciting false-positive results. A rapid trehalose test allows for the presumptive
identification of C. glabrata within a few hours. There are rapid immunological agglutination
tests for C. albicans, C. dubliniensis and C. krusei (Bichro-latex albicans, Bichro-dubli or
Krusei color (Fumouze, France) that allow a reliable identification of isolates within 5-10 min
[154, 155]. Carbohydrate fermentation and assimilation assays, such as API 20C, ATB
ID32C or Vitek 2 allow the identification of the different species of Candida with more
precision. The use of specific primers, DNA probes or mass-spectrometry (matrix-assisted
laser desorption/ionization time of flight or MALDI-TOF) allows a rapid identification of
clinical isolates from cultures [142, 156-165]. Susceptibility testing has an important role in
the management of IC. There are two standardized susceptibility testing, CLSI method M27-
A3 and the EUCAST method, and several commercialized tests, such as Sensititre Yeast One,
ATB Fungus, VITEK 2 system, etc. [141, 164, 166, 167].
Candida identification using peptide nucleic acid fluorescence in situ hybridization (PNA
FISH) techniques from blood culture bottles is rapid (within 1.5 to 2.5 h) and is highly
specific and sensitive. The assay uses PNA probes targeting Candida-specific rRNA. The C.
albicans / C. glabrata PNA FISH (AdvanDx, USA) distinguishes between C. albicans and C.
glabrata from blood culture bottles that have signaled positive and demonstrate yeast on
Gram staining.
Polymerase chain reaction (PCR) and DNA probes have the advantage of being able to
detect small amounts of Candida DNA in either blood or tissues. Despite the fact that PCR
assays have been shown to be highly sensitive, they continue to have problems of
standardization. Sequencing of fungal DNA from positive blood culture bottles, by rapid PCR
techniques can be useful, thereby potentially facilitating early diagnosis of fungemia.
However, contamination difficulties, sample volume and sample imprecision, optimum
sampling frequency, and difficulty distinguishing colonization and infection for validation
purposes, mostly limits utility of PCR to the research setting. Moreover, little agreement
exists regarding technical aspects of Candida PCR testing [141, 142]. The SeptiFast (Roche
Molecular Diagnostics, USA) multiplex PCR assay is now available commercially; although
there is limited but promising data on performance of this system [150, 168, 169].
There are several methods for detecting Candida antigens and antibodies against these
fungal antigens. At least three of them are available, Platelia Candida Antigen Plus and
Antibody Plus (Bio-Rad Laboratories, France) for detecting mannan and antimannan
antibodies and Candida albicans IFA IgG (Vircell, Spain) for detecting CAGTA, that have
been evaluated with promising results but important differences on their diagnostic usefulness
according to the patients group studied (Tables 7 and 8) [170-175]. Detection of antimannan
antibodies has a low sensitivity in some groups of patients with IC. Mannan is a major
component of the cell wall that can be detected by latex agglutination or enzyme
immunoassay tests in patients with IC. The circulation of detectable concentrations of
mannan is of short duration in blood, this fact diminished its diagnostic value, and serial
determinations may be necessary. However, the combined detection of mannan and
antimannan antibodies is considered to be useful for the diagnosis of IC using serum samples,
with sensitivity, specificity, and negative predictive value rates around 80-85%. Mannan and
anti-mannan antibodies can be positive 6 days on average prior blood cultures [174]. This
combination has been recommended for the diagnosis of candidemia in adults and neonates as
it could be used to establish the absence of the disease to reduce the unwarranted use of

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 593

antifungal agents in prophylactic and empirical regimens in ICU [148, 175]. In a group
including pediatric and adult patients, sensitivity of mannan detection and antimannan
antibody alone was very low (less than 50%). However, in combination, sensitivity reached
75% [140]. In a study including 70 critical-ill neonates, sensitivity and specificity values were
94.4% and 94.2, respectively, for patients with proven and probable IC. Positive results for
mannan were obtained a median of 8 days before blood culture becomes positive in near 50%
of patients [176].
BG is a major cell wall component of most fungal species, with exception of
Cryptococcus and the mucorales that is released in blood and tissues in the course of invasive
mycoses. This test is a panfungal biomarker not specific for IC but it cannot identify the
infecting species. Moreover, occurrence of BG is widespread in the environment and small
amounts can be found in human serum of healthy adults [150]. There are several techniques
on the market for the detection of BG in serum. In Europe and America, the most used is
Fungitell (Associated of Cape Cod, USA) (Table 8). Meta-analyses on the diagnosis of CA
have been undertaken using data from cross-sectional, case-control and cohort studies. With a
cut-off value of 80 pg/ml of BG, sensitivity was 50-90% and specificity 70-100% for proven
or probable IC [127, 177]. The test seemed of greater utility in patients who did not have
hematological diseases such as surgical or medical ICU patients suffering from Candida
infections [171, 178, 179]. BG detection, twice a week, is very useful for ruling out infection
in adults, but the test has not been validated in children [148, 175, 180, 181]. In a study,
evaluating the BG concentrations in pediatric and adult patients with and without invasive
mycoses, mean BG concentrations were higher in immunocompetent uninfected children (68
pg/ml) than adults (48 pg/ml) [182]. Mokkadas et al. have detected higher BG values due to
Candida colonization. However, other authors have reported that BG may be higher in
children with proven invasive mycoses than in those uninfected. Furthermore, positive results
for BG were obtained before CT scan findings or culture of fungi in more than 70% of
patients [150, 183, 184].

Table 7. Grading quality of evidence and strength of recommendations for diagnostic

tests and therapy recommendations

Strength of recommendation
A Good evidence to support a recommendation for or against use.
B Moderate evidence to support a recommendation for or against use.
C Poor evidence to support a recommendation.
Quality of evidence
I Evidence from ≥ 1 properly randomized, controlled trial.
II Evidence from ≥ 1 well-designed clinical trial, without randomization; from cohort or
case-controlled analytic studies (preferably from > 1 center); from multiple time-series; or
from dramatic results from uncontrolled experiments.
III Evidence from opinions of respected authorities, based on clinical experience, descriptive
studies, or reports of expert committees
Adapted from the Infectious Diseases Society of America (IDSA) / US Public Health Service Grading
System for ranking recommendations in clinical guidelines [125].
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594 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

Table 8. Indications and diagnostic limits of selected serological biomarkers for invasive
candidiasis

Indications & (1-3)-β-D-glucan detection (BG) Mannan and anti- Anti-germ tube
diagnostic limits mannan antibodies antibodies (CAGTA)
Quality of Early diagnosis of invasive mycoses Early diagnosis of IC Early diagnosis of IC
evidence and (Panfungal biomarker): IC and (AII-BII). (AII-BII).
strength of invasive aspergillosis (AI-BII), and
recommendations pneumocystosis (BII).
Methods and Fungitell (Associates of Cape Cod Enzyme immune assay Indirect immune-
suggested use Inc., USA), Wako WB003 (Wako (Platelia Candida fluorescence (Candida
Pure Chemical Ind., Japan). Antigen Plus and albicans IFA IgG,
Fungitec G (Seikagaku Kogyo Co., Antibody Plus, Bio-Rad Vircell, Spain).
Japan). B-G Star (Maruha Co., Lab., France). Detection each 4-7
Japan). Detection each 3-4 days. days.
Detection each 3-4 days.
Diagnostic value Mycological criterion of probable Mycological criterion of Detection of CAGTA
invasive mycoses according to probable IC according to previous to positivity
ESCMID and EORTC/MSG ESCMID and in other tests.
definitions. EORTC/MSG The combination of
Detection of BG previous to definitions. BG and CAGTA
positivity in other tests. Detection of mannan + increases the diagnosis
Positive: > 60-80 pg/ml (Fungitell) o antimannan antibodies of IC.
7 pg/ml (Wako). previous to positivity in Positive: > 1/160
other tests. Detection of CAGTA
and later decrease of
titers are considered
sign of good prognosis.
Prognostic value BG decrease is considered as good Decrease of titers is Decrease of titers is
prognosis. considered as good considered as good
prognosis. prognosis.
False positive Contamination of laboratory ware. Allergic aspergillosis. Allergic aspergillosis.
Gram-positive (Streptococcus) and
Gram-negative (Alcaligenes and
Pseudomonas aeruginosa)
bacteremia.
Contact with fat and sponge during
surgery. Hemodialysis (glucose
acetate filters and membranes).
Intravenous albumin,
immunoglobulins, coagulation
factors plasmatic proteins.
Antineoplastic (lentinan and K
polysaccharide) and antimicrobial
agents (amoxicillin-clavulanic acid
or piperacillin-tazobactam).
False negatives Hiperpigmented serum (↑ bilirrubin Some immunodeficient Some immunodeficient
and/or triglycerides). patients patients
Antifungal prophylaxis or empiric
treatment.
Intravenous azithromycin or
pentamidine.
References: [127,141,142,148,150,170-175,177,178,183,184]
EORTC/MSG: European Organization for Research and Treatment of Cancer / Mycoses Study Group
of the National Institute of Allergy and Infectious Diseases. ESCMID: European Society of
Clinical Microbiology & Infectious Diseases.

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 595

7. CARE AND TREATMENT OF PATIENTS. CONCERNS ON ANTIFUNGAL

RESISTANCE AND NEW THERAPEUTIC APPROACHES
Among the most important measures for care and treat patients suffering from IC are to
minimize those risk factors that can be avoided or modified. Few of these factors are
preventable but a wise use should be done of all invasive procedures that disrupt the integrity
of skin and the gastrointestinal tract. Moreover, the administration of broad spectrum
antibiotics that can select and help Candida proliferation at mucosal surfaces by eliminating
part of the bacterial microbiota should be limited to clearly indicate treatment approaches.
Hyperalimentation should be changed to early enteral nutrition as soon as possible. Careful
hand hygiene should be encouraged between hospital staff and patients’ visitors and after
contact with infectious materials and specimens, and other adequate infection control
practices are important prevention strategies [18, 76]. When the IC has been established,
antifungal therapy is a key tool for recovery of the patient by eliminating Candida from blood
and tissues. The general patterns of in vitro antifungal susceptibility for the most relevant
species of Candida are shown in Table 9. There are some antifungal drugs that have clearly
proved their efficacy in the treatment of IC, such as the polyene amphotericin B, the triazoles
fluconazole, itraconazole, posaconazole and voriconazole, and the candins, anidulafungin,
caspofungin and micafungin (Figures 4 & 5).

Table 9. General pattern of antifungal susceptibility of relevant Candida species

Antifungal agent
Species
AMB FLU VOR POS AND CAS MIC
Candida albicans S S S S S S S
Candida parapsilosis S S S S S-R S-R S-R
Candida glabrata S-I SDD-R S-R S-R S S S
Candida tropicalis S S S S S S S
Candida krusei S-I R S S S S S
Candida guilliermondii S-I S S S S-R S-R S-R
Candida dubliniensis S S-R S S S S S
Candida lusitaniae S-R S S S S S S
S: susceptible, SDD: susceptible dose-dependent, I: intermediate susceptible, R: resistant. AMB:
amphotericin B, AND: anidulafungin, CAS: caspofungin, FLU: fluconazole, MIC: micafungin,
POS: posaconazole, VOR: voriconazole.
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596 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

Figure 4. Antifungal targets in Candida.

Figure 5. In vitro activity of systemic antifungal drugs against relevant species of Candida. A) Azole
drugs. B) Candins. Resistance is represented by white areas, and decreased susceptibility by grey areas.
AMB: amphotericin B, ANI: anidulafungin, CAS: caspofungin, FCZ: fluconazole. ITZ: itraconazole,
MIC: micafungin, POS: posaconazole, VCZ: voriconazole.

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 597

Amphotericin B deoxycholate is an amphiphilic organic molecule belonging to the

polyene group. This property enables amphotericin B to bind to ergosterol at the fungal cell
membrane and its fungicidal activity results from creating pores and altering the permeability,
causing leakage of cellular components and ultimately cell death. Analysis by nuclear
magnetic resonance has suggested that eight molecules of amphotericin B bind to eight
molecules of ergosterol forming hydrophilic sides with a central channel of 70-100 nm in
diameter. The formation of this pore destabilizes the plasma membrane and results in leakage
of intracellular components such as K+ ions, responsible for cell lyses. Amphotericin B has
been considered as the gold standard because of its broad antifungal activity at doses of 0.5-1
mg/Kg/day. Some clinical isolates from C. lusitaniae, C. glabrata and C. krusei may have
reduced susceptibility or resistance to amphotericn B. The major disadvantages of
amphotericin B deoxycholate include nephrotoxicity, electrolyte disturbances and acute
infusion-related side-effects that can be alleviated by using continuous infusions of
amphotericin B. A recent retrospective study has evaluated the nephrotoxicity of
amphotericin B deoxycholate and its association to characteristics of patients [185]. Lipid
preparations offer better tolerance and particularly less nephrotoxicity which allow using
higher doses in less time. There are two lipid formulations broadly used in therapy, liposomal
amphotericin B and amphotericin B lipid complex. Nephrotoxicity with lipid formulations of
amphotericin B is minimized due to the preferential accumulation of these drugs within
organs of reticuloendothelial system, rather than the kidneys.
Amphotericin B lipid complex is given in a dose of 5 mg/Kg/day for the treatment of IC.
Liposomal amphotericin B is usually prescribed in a dose of 3–5 mg/Kg/day to treat IC.
Higher doses of lipid formulations of amphotericin B are generally recommended to treat C.
krusei and C. glabrata infections. The use of amphotericin B is not recommended for
recipients of solid organ transplants because of its nephrotoxicity, particularly in kidney
transplants. Interestingly, in cases of neonatal IC amphotericin B is the most widely used
antifungal because of its efficacy and good tolerance [125, 186, 187].
Triazoles, such as fluconazole, itraconazole, voriconazole or posaconazole, inhibit the
cytochrome p450 dependent enzyme lanosterol 14-α-demethylase, impairing the biosynthesis
of ergosterol, resulting in a fungistatic action by increased membrane permeability and
inhibition of cell growth and reproduction. Fluconazole may be used in clinically stable
patients who are not or have not recently been on azole prophylaxis and with proven
fluconazole-susceptible species of Candida. The usefulness of fluconazole for empiric
therapy is limited by its reduced activity against the emerging species C. krusei and C.
glabrata. Moreover, some initial isolates of Candida species that are susceptible to
fluconazole can develop secondary resistance. Adverse events usually occur in doses >400
mg/day (headache, nausea and abdominal pain, mild elevation of transaminases, and rarely
sudden and fatal hepatitis), with neurotoxicity reported mostly in doses >1200 mg/day.
Itraconazole has improved activity against C. glabrata, although it lacks reliable activity
against C. krusei. The use of itraconazole is limited by its poor oral bioavailability and the
reduced clinical information about the parenteral formulation. Voriconazole and posaconazole
are well tolerated and show a broad spectrum, including the fluconazole-resistant isolates.
Overall, more than 95% of all Candida isolates are susceptible to voriconazole and less than
3% are resistant, usually related to the azole cross-resistance observed in some Candida
glabrata isolates. Voriconazole is as effective as and safer than amphotericin B in the
treatment of IC in non-neutropenic patients and it has an excellent activity against C. krusei.
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598 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

Conversely, its efficacy against fluconazole-resistant C. glabrata is controversial and cross-

resistance can occur. Voriconazole is metabolized by cytochrome P-450 with significant drug
interactions. Side effects include hepatotoxicity, visual disturbances or rash, generally
transient and reversible. Posaconazole, the newest triazole, is available only as an oral
suspension. This fact and the erratic oral absorption limits its utility to the treatment of those
IC caused by isolates resistant to other antifungal drugs [188-191].
Candins or echinocandins are lipopeptides that act against the fungal cell-wall BG
synthase, leading to osmotic instability and fungal death. These drugs are considered first-line
agents for treatment of IC among patients who are critically ill, clinically unstable, or have a
history of recent azole exposure or colonization or infection with a Candida with reduced
susceptibility to azoles [67, 125]. Candins are effective due to their potential to treat those
species, such as C. krusei and C. glabrata, with intrinsic resistance or reduced susceptibility
to fluconazole and other triazole antifungal drugs [67, 125]. Moreover, candins are the
preferred alternative antifungal agents, in patients taking other drugs that utilize P-450
pathways. Side effects of candins are generally mild, and include fever, thrombophlebitis,
headache, and elevated liver enzymes. In current guidelines (Table 10), all three
echinocandins are labeled as equally effective for the treatment of most cases of candidemia
and IC [111, 125]. However, the recent European guidelines introduce some differences on
their therapeutic indications according to the accumulated information from clinical studies
[125, 186, 187]. There are some species, such as C. parapsilosis and C. guilliermondii, with
higher minimum inhibitory concentrations but apparently this fact has no clinical importance
[192]. Caspofungin is as effective as amphotericin B in IC. Anidulafungin was found to be
superior to fluconazole in the treatment of IC in a group of predominantly non-neutropenic
patients. It was also found to be at least equivalent to liposomal amphotericin B for the
treatment of IC. In a large prospective trial, micafungin was compared with liposomal
amphotericin B as first-line therapy for IC and CA and was shown to be as effective but less
toxic than liposomal amphotericin B [190-194]. In Table 10, the main recommendations made
in recent guidelines for treatment are shown. There is a clear differentiation of treatments in
relationship to age and clinical status of patients and the acute or chronic clinical presentation
of IC [125, 186, 187].
Current expert consensus recommends the removal of IV catheters in candidemic
patients, whenever feasible, due to the high affinity of Candida species to prosthetic material.
Catheter preservation has been associated with prolonged candidemia and worst outcomes
especially so in critically ill unstable patients [66]. Inadequacy of antifungal therapy and lack
of removal of central lines have been associated with poorer outcomes in non-neutropenic
patients with candidemia. Daily blood cultures after initiating therapy are recommended. If
blood cultures remain positive, then a search for metastatic foci, such as deep abscesses or
endocarditis should be undertaken. Antifungal therapy should be continued for at least 2
weeks after the last positive blood culture and after resolution of all clinical signs and
symptoms of infection [76, 125].
Due to the limited range of the available antifungal and increasing reports of resistance,
the option of combination therapy has been explored to improve cure rates, allow for dose
reduction, and thus, toxicities. However, the combination fluconazole plus amphotericin B
versus fluconazole alone has shown a trend towards improved clinical success with
combination therapy. Early data on echinocandin and triazole combination therapy are also
encouraging. However, combination therapy should not generally be used outside the context

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 Invasive Candidiasis Epidemiology, Diagnosis and Treatment 599

of a clinical trial, until better data on efficacy and safety from prospective, randomized
clinical trials demonstrate superiority over monotherapy [76, 195, 196].

Table 10. Current recommendations for the treatment of candidemia and

invasive candidiasis

Clinical presentation Treatment

First-line Alternative
Candidemia in non- AND / CAS /MIC (AI)1 LAMB (BI) or FLC (CI) or
neutropenic patients FLC or AND / CAS /MIC (AI)2 ABLC (CII) or VOR (BI)1
LAMB or ABLC or VOR (AI)2
Suspected IC in non- FLC / AND / CAS /MIC (CII)1
neutropenic patients FLC / AND / CAS /MIC (BIII)2
Candidemia in CAS / MIC (AII)1 AND / LAMB /MIC (BII) or
neutropenic patients AND / CAS /MIC / LAMB (AII)2 VOR (CII)1
FLU or VOR (BIII)2
Suspected IC in LAMB / CAS (AI)1 ABLC / VOR (BI) or MIC /
neutropenic patients LAMB / CAS / VOR (BI)2 AMB (BII)1
FLU or ITR (BI)2
Chronic LAMB (AIII)1 FLC / VOR (BIII)1
disseminated FLC / LAMB / AMB (AIII)2 AND / CAS /MIC (BIII)2
candidiasis
Candida biofilm and Early catheter removal (AII)1 Catheter retention + AND / CAS
catheter-associated /MIC / LAMB (CII)1
candidemia
Neonatal candidiasis LAMB / MIC / CAS (AI)1 VOR / FLC (BI) or AND /
AMB (AII) or FLC (BII)2 ABLC (BII) or AMB (CI)1
LAMB (BIII)2
Candidiasis in AMB / LAMB / FLC / MIC (BII)1 CAS / ABLC (CII)1
children
1
European Society of Clinical Microbiology and Infectious Diseases [186,187,208].
2
Infectious Diseases Society of America guidelines [125].
ABLC: amphotericin B lipid complex, AMB: amphotericin B deoxycholate, AND: anidulafungin, CA:
candidemia, CAS: caspofungin, FLC: fluconazole, IC: invasive candidiasis, ITR: itraconazole,
LAMB: liposomal amphotericin B, MIC: micafungin, POS: posaconazole, VOR: voriconazole.

Resistance to antifungal agents is a matter of concern. Reports of IC caused by clinical

isolates with in vitro resistance or decreased susceptibility are common. However, the real
importance of this decrease antifungal susceptibility apparently is low [67]. For instance,
amphotericin B is a broad-spectrum compound that has been used for many years without any
noticeable selection of resistance. Moreover, a lower susceptibility of C. parapsilosis sensu
stricto to the echinocandins has been widely described, but the number of isolates with MICs
>2 μg/ml is low [68, 75, 192, 197]. The increase in azole use that occurred during the last
decades has been associated with a shift in species distribution from a preponderance of C.
albicans to more frequent isolation of less azole-susceptible species of Candida, such as C.
glabrata [28]. The intrinsic resistance of some of these species, such as C. krusei, or the
acquisition of resistance to fluconazole, such as C. glabrata, may have contributed to the
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600 Mayra Cuéllar Cruz, Guillermo Quindós and Everardo López Romero

emergence of these species and of new ones. However, in recent surveys, overall, fluconazole
resistance was detected in 5% of ICU isolates and 4.4% of non-ICU isolates. Apart from C.
krusei which is intrinsically resistant to fluconazole, C. glabrata was the only species in
which resistance to azoles and echinocandins was reported. Concern regarding C. glabrata
must now include resistance to echinocandins as well as azole antifungal agents. However,
the increase in the incidence of infections caused by azole-resistant species has not been
reported in many hospitals, despite the widespread use of fluconazole, and significant
regional differences have been observed in the distribution and pattern of susceptibility to
antifungal agents among the different species. Furthermore, C. glabrata and C. krusei are
very infrequent in the pediatric setting, and fluconazole is still a reasonable option for
fungemia treatment before species identification, except in children with prior azole exposure.
Unfortunately, data about epidemiology or antifungal susceptibility patterns in pediatric
patients are scarce, and empirical treatment in children with suspicion of IC frequently is
instituted by extrapolating information from adult patients [198]. The use of fluconazole
would be safe if restricted to cases in which susceptibility testing has already been
undertaken, or susceptibility is reliably indicated by the species identification or a close
surveillance of the patient suggests that fluconazole resistant yeasts are a rare cause of
fungemia [28, 67].

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 25

CANDIDA PARAPSILOSIS COMPLEX

D. V. Moris1,3, M. S. C. Melhem2, M. A. Martins2

and R. P. Mendes 1
1
Departamento de Doenças Tropicais e Diagnóstico por Imagem –
Faculdade de Medicina de Botucatu – UNESP, Botucatu,
São Paulo State, Brazil
2
Instituto Adolfo Lutz, São Paulo, Brazil
3
Faculdade de Biomedicina, Universidade do Oeste Paulista –
Presidente Prudente, São Paulo State, Brazil

ABSTRACT
History. C. parapsilosis complex was first isolated by Ashford (1928) from the feces
of a Puerto Rican patient with diarrhea, as a species of Monilia parapsilosis that was
incapable of fermenting maltose and therefore reclassified as Candida parapsilosis by
Langeron and Talice (1932).
Ecology and Epidemiology. C. parapsilosis complex has an extensive distribution in
nature, having been isolated from domestic animals, insects, soil, and marine
environments. Known as a normal human commensal, it is also one of the most
frequently isolated fungi from the subungual space of human hands. Its transient
colonization of human integument is the basis of much debate as to whether or not C.
parapsilosis is a pathogen or bystander in certain infections.
C. parapsilosis complex has been reported as a contaminant of high concentration in
glucose solutions and prosthetic material. Several nosocomial infections have been
associated with its presence in contaminated hospital equipment via external sources such
as medical devices or fluids, hands of health care workers, prosthetic devices, and can
occur without prior colonization. C. parapsilosis complex is highly associated with the
biofilms formation on various surfaces. Biofilms display a very complex three-
dimensional architecture, affecting the nutrient fluxes and waste products as well as the
sensitivity of cells to antifungal drugs. Biofilms are already formed when C. parapsilosis


Corresponding author: Daniela Vanessa Moris, Departamento de Doenças Tropicais e Diagnóstico por Imagem.
Faculdade de Medina de Botucatu-UNESP, Distrito de Rubião Junior s/n, Botucatu-SP Brazil, CEP: 18600-
000, Telephone: [5514] 38116212/ [5514] 38116437/Fax: [5514] 38159898, Email: [email protected]
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618 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

cells grow in means containing high glucose and lipid concentrations, which is associated
with the increased prevalence of bloodstream infections in patients receiving parenteral
nutrition.
C. parapsilosis complex is the second most common Candida species isolated from
normally sterile body sites of hospitalized patients. It accounts for 15.5% of Candida
isolates in North America, 16.3% in Europe, and 23.4% in Latin America, outranked only
by C. albicans (51.5%, 47.8%, and 36.5%, respectively) and by C. glabrata (21.3%) in
North America.
Virulence factors. The pathogenesis of invasive candidiasis is facilitated by a number
of virulence factors, mainly by adherence to host cells, biofilm formation, and secretion
of hydrolytic enzymes - proteases, phospholipases, and lipases.
Mycology. C. parapsilosis complex is formed by white and creamy colonies with
variable morphology. It grows as oval cells or pseudohyphal filaments, but in contrast to
C. albicans, it does not form true hyphae.
Prior to 2005, C. parapsilosis complex was separated into groups I to III. Further
genetic studies revealed sufficient differences that have led to the separation of the
groups into closely related, distinct species: C. parapsilosis, C. orthopsilosis, and C.
metapsilosis. The identification of C. parapsilosis complex is performed by conventional
morphological and physiological methods and by molecular typing for identification of
C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis species.
Antifungal susceptibility. C. parapsilosis complex is usually susceptible to most
antifungal compounds but decreased responsiveness to fluconazole and caspofungin was
reported. It has also been suggested that C. orthopsilosis and C. metapsilosis could be
more susceptible to amphotericin B and echinocandins than C. parapsilosis sensu stricto,
which could affect therapeutic choices.
Clinical manifestation. C. parapsilosis complex can cause several infections such as
bloodstream infections, endocarditis, meningitis, onychomycosis , endophthalmitis,
peritonitis, arthritis, otomycosis, vulvovaginitis and urinary tract infections.

Keywords: C. parapsilosis complex, ecology, epidemiology, virulence factors, antifungal

susceptibility and clinical manifestation

I. HISTORY
The first isolate of the Candida parapsilosis species complex was described in Puerto
Rico by Ashford (1928) from diarrheic human feces (Ashford, 1928). This species, previously
designated Monilia parapsilosis, is characterized by its incapacity to ferment maltose, among
other features, and was reclassified as Candida parapsilosis by Langeron and Talice in 1932
(Langeron and Talice, 1932). Although it was initially considered nonpathogenic, C.
parapsilosis was identified as the causative agent of a fatal case of endocarditis in an
intravenous drug user in 1940 (Joachim and Polayes, 1940). Even at this early point,
investigators associated these infections with exogenous introduction of C. parapsilosis,
which astutely foreshadowed the link between C. parapsilosis and invasive medical
instrumentation and hyperalimentation solutions (Trofa et al., 2008). However, C.
parapsilosis is also the yeast species that is isolated most frequently from the skin, especially
from the subungual space in healthy individuals (McGinley et al., 1988), and it is considered
to be part of the normal microflora (Moris et al., 2008, 2012). Isolates of this yeast are
commonly found in skin and nails (including on the hands of health-care workers) and on the

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surface of medical plastics, prosthetic devices and blood. C. parapsilosis is considered to be

an emerging fungal pathogen, as it is increasingly associated with a wide range of infections,
such as fungemia, vaginitis, endocarditis, endophthalmitis, septic arthritis, and peritonitis
(Weems 1992; Trofa et al., 2008). Modern molecular tools have allowed for the classification
of C. parapsilosis isolates into groups I through III (Tavanti et al., 2005), which are currently
considered to be closely related but distinct species: C. parapsilosis, C. orthopsilosis, and C.
metapsilosis.

II. ECOLOGY AND EPIDEMIOLOGY

The C. parapsilosis complex exhibits a wide distribution in nature, having been isolated
from domestic animals, insects, soil, and marine environments. This fungus is known as a
normal human commensal organism, and it is one of the fungi that is most frequently isolated
from the subungual space on human hands (Nosek et al., 2009). C. parapsilosis is usually
associated with invasive procedures or prosthetic devices (Canto´n et al., 2011) and with
neonatal infections in the northern hemisphere, and it is isolated from patients of all ages in
Latin America (Almirante et al., 2005; Nucci et al., 2010). The transient colonization of the
human integument by C. parapsilosis has given rise to much debate regarding whether this
species is a pathogen or bystander in certain infections. The C. parapsilosis complex
represents the second most common Candida species group isolated from normally sterile
body sites in hospitalized patients, as it accounts for 15.5% of the Candida isolates recorded
in North America, 16.3% in Europe, and 23.4% in Latin America, being outranked only by C.
albicans (51.5%, 47.8%, and 36.5%, respectively) and by C. glabrata (21.3%) in North
America (Messer at al., 2003).
In the period from 2001 - 2006, 1,929 isolates of C. parapsilosis were analyzed by the
ARTEMIS Global Surveillance Study. These isolates came from 89 study centers in 29
countries on six continents. C. parapsilosis accounted for 1,762 (91.3%) of the isolates, while
117 (6.1%) were C. orthopsilosis, 34 (1.8%) C. metapsilosis, and 16 (0.8%) were L.
elongisporus (Lockhart 2008), showing that the percentage of C. parapsilosis complex
isolates that were classified as C. orthopsilosis increased longitudinally during this
surveillance study. Whereas during the first 4 years of the study, the average percentage of
the isolates identified as C. orthopsilosis was 4.5%, the average percentage over the last 2
years was 8.3% (p = 0.01). Among the isolates for which the site of isolation was provided,
77% of the C. orthopsilosis isolates and 60% of the C. metapsilosis isolates came from
bloodstream infections, compared to 79% of the C. parapsilosis isolates. C. orthopsilosis was
also isolated from ascites fluid, abscesses, catheters, cerebrospinal fluid, and pulmonary
sources (e.g., pleural and bronchoalveolar lavage fluids) and C. metapsilosis from abscesses,
ascites fluid, bronchial alveolar lavage fluid, and joint fluid (Lockhart et al., 2008). For Asia,
6.1% of isolates were identified as C. orthopsilosis, while 10.9% of the isolates from South
America were classified as C. orthopsilosis. The distribution on the latter continent varied
from 16.5% and 16.0% C. orthopsilosis isolates found for Venezuela and Brazil, respectively,
to 4.3% and 3.1% C. orthopsilosis isolates for Ecuador and Argentina, respectively. In North
America, 5.0% of the C. parapsilosis complex isolates were identified as C. orthopsilosis.
More specifically, 4.9% of the isolates from the United States and 10.7% from Mexico were
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620 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

C. orthopsilosis, whereas none of the 52 isolates from Canada were found to be this species.
A total of 17 (3.5%) C. orthopsilosis isolates were recorded from Europe and the Middle East,
almost half of which came from Italy, where 8.0% of the isolates were C. orthopsilosis. No C.
orthopsilosis isolates were recovered from Finland, the United Kingdom, Poland, Portugal,
Russia, Hungary, or Israel, despite a combined total of 177 isolates of C. parapsilosis being
reported for these countries. Although 146 C. parapsilosis complex isolates were obtained
from multiple centers in South Africa, only a single isolate of C. orthopsilosis was identified
from that country.
C. metapsilosis isolates could also be found on all six continents. Only 4 of 646 (0.6%)
North American isolates and 1 of 146 (0.7%) South African isolates were identified as C.
metapsilosis. Although no C. orthopsilosis isolates were found for Canada, 2 C. metapsilosis
isolates were attributed to this country. Europe presented almost as many C. metapsilosis
isolates, 14 (2.9%) as C. orthopsilosis isolates. Poland exhibited the highest percentage, with
13.8% of all of the C. parapsilosis complex isolates being identified as C. metapsilosis. C.
metapsilosis isolates were also recorded from Spain, Portugal, Turkey, and Italy. Australia
displayed more C. metapsilosis than C. orthopsilosis isolates, with 6.8% of its C. parapsilosis
complex isolates being found to be C. metapsilosis. Both China and Taiwan presented C.
metapsilosis isolates, and 2.6% of all of the Asian isolates corresponded to C. metapsilosis.
Despite the large number of C. orthopsilosis isolates recorded from South America, only
1.7% of the C. parapsilosis complex isolates from this country were identified as C.
metapsilosis: four isolates from Brazil and one from Argentina. Pires et al. (2011) isolated
100 strains of C. parapsilosis from a hemodialysis unit, and using molecular analysis, 53% of
these isolates were found to be C. parapsilosis sensu lato, while 47% corresponded to C.
orthopsilosis.
In the FUNGEMYCA study carried out by Canto´n et al. (2011), 400 out of 1,356
isolates were identified as C. parapsilosis sensu lato (29.5%), and this species was the second
most frequently isolated species obtained from blood in Spain, after C. albicans. Of these 400
isolates, 364 were identified by molecular methods: C. parapsilosis sensu strict represented
90.7%, C. orthopsilosis 8.2% and C. metapsilosis 1.1% of the isolates.
Recently, Moris and co-workers demonstrated that 53.3% of 15 oral cavity isolates from
HIV-infected individuals in Brazil initially identified as belonging to the C. parapsilosis
complex, were, in fact, C. metapsilosis, whereas no C. orthopsilosis isolates were observed
(Moris et al., 2012). This finding suggests that C. metapsilosis could be a human commensal
organism, though its importance as a pathogen has yet to be confirmed.

III. VIRULENCE FACTORS

The pathogenesis of invasive candidiasis is facilitated by a number of virulence factors,
the most important of which are adherence to host cells, biofilm formation, pseudo-hyphae
formation, and secretion of hydrolytic enzymes, such as proteases, phospholipases, and
lipases. Despite intensive research to identify pathogenic factors in these species, little is
known about the virulence determinants of the C. parapsilosis complex.

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 Candida Parapsilosis Complex 621

1. Adherence

The primary factor involved in the fungal colonization of human tissues is adherence to
host surfaces. This process is controlled and induced by several cell-signaling cascades in
both the fungus and the environment. In addition, Candida species, particularly those in the
C. parapsilosis complex, can adhere to the surfaces of medical devices and form biofilms.
The initial attachment of Candida cells is mediated by non-specific factors (hydrophobicity
and electrostatic forces) and promoted by specific adhesins present on the surface of fungal
cells that recognize ligands such as proteins, fibrinogen and fibronectin (Li et al., 2003). The
phenomenon of adhesion is exhibited by specialized surface proteins (adhesins) that
specifically bind to amino acids and sugars on the surface of other cells or promote adherence
to abiotic surfaces (Verstrepen and Klis, 2006).

2. Biofilm Formation

Biofilms are surface-associated communities of microorganisms within an extracellular

matrix and represent the most prevalent type of microbial growth (Kuhn et al., 2002a). This
type of structured microbial community attached to surfaces has increasingly been found to
be a source of infection by Candida, especially in view of the vast number of biomaterials
that are used in the medical industry. Biomaterials such as stents, catheters, and orthopedic
joints serve as excellent substrates for microbial adhesion and subsequent biofilm formation
(Cardial et al., 1996; Ells, 1996; Leonhardt et al., 1999).
Biofilms are specific and organized communities of cells under the control of signaling
molecules, as opposed to random accumulations of cells resulting from cell division (Davies,
1998). Cell-cell signaling, particularly quorum sensing, is essential for biofilm formation, and
homoserine lactones act in a concentration-dependent manner, which a threshold
concentration triggering the formation of a biofilm (Miller et al., 2001; Riedel et al., 2001). A
quorum-sensing molecule is produced by planktonic cultures of C. albicans (Hornby et al.,
2001). This molecule, farnesol, has been shown to prevent the germination of yeast cells into
mycelia, a phenomenon that may be pertinent to C. albicans biofilm formation. Farnesol is
associated with mycelial development and is an important regulatory (quorum-sensing)
molecule involved in C. albicans biofilm formation (Ramage et al., 2002).
The generation of C. albicans biofilms is associated with the dimorphic shift from
growing as yeast to hyphal growth, and the structure of the biofilm formed involves two
distinct layers: a thin, basal yeast layer and a thicker, less compact hyphal layer (Baillie and
Douglas, 1999).
In contrast, C. parapsilosis strains produce quantitatively fewer and structurally less
complex biofilms than C. albicans (Hawser and Douglas, 1999; Kuhn et al., 2002a).
However, certain filamentous (pseudohyphal) C. parapsilosis phenotypes generate biofilms
more frequently and are more invasive in agar than strains remaining predominantly in the
yeast form (Laffey and Butler, 2005; Sardi et al., 2013).
Biofilms are readily formed when C. parapsilosis cells are grown in media containing
high glucose and lipid concentrations, which is associated with an increased prevalence of
bloodstream infections in patients receiving parenteral nutrition. The selective preference of
this species for medical plastics is of particular interest, as biofilm formation enhances the
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622 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

microbe’s capacity to infect catheters and intravascular lines (Weems 1992; Trofa et al.,
2008). In contrast to C. albicans, C. parapsilosis biofilms are thinner, less structured, lack
true hyphae, and consist exclusively of aggregated blastospores (Kuhn et al. 2002a). The
extracellular matrices of these biofilms contain large amount of carbohydrates and exhibit a
lower protein content. The formation of biofilms is highly strain dependent, which is a
phenomenon that has not been observed in related yeasts (Silva et al., 2009). Transcriptional
profiling indicated that the expression pattern of C. parapsilosis genes during biofilm
formation resembles their expression in hypoxic conditions. In both cases, cells exhibit
increased expression of genes involved in the ergosterol synthesis, fatty acid metabolism, and
glycolysis as well as in cell wall biogenesis (Rossignol et al., 2009). The formation of
biofilms is affected by farnesol, a quorum-sensing molecule found in C. albicans. Although
C. parapsilosis cells do not secrete any significant amount of farnesol (Weber et al., 2008),
treatment with externally applied farnesol causes growth arrest, but without any apparent
effect on C. parapsilosis morphology. A transcriptomic analysis of farnesol-treated cells
revealed upregulation of the oxidoreductases GRP2 and ADH7 and altered expression of
genes involved in the metabolism of lipids, amino acids, and sugars as well as ribosome
biogenesis. However, farnesol does not affect the orthologs of genes implicated in hyphal
growth in C. albicans, pointing to substantial differences in the control of morphogenetic
programs between these yeasts (Nosek et al., 2009).
Biofilms cause clinical problems that are of concern because they increase resistance to
antifungal therapy, and the mechanism underlying biofilm resistance to antimicrobial agents
is not fully understood. One hypothesis is that the presence of the matrix restricts the
penetration of drugs through the formation of a diffusion barrier (Kojic and Darouiche, 2004;
Sardi et al., 2013), and only the most superficial layers are therefore in contact with lethal
doses of antifungal compounds. Despite their less complex structure, C. parapsilosis biofilms
show similar resistance to C. albicans biofilms regarding conventional antifungals, such as
amphotericin B and azole compounds (Katragkou et al., 2007; Ruzicka et al., 2007).
However, therapeutic levels of echinocandins can inhibit the metabolic activity of C.
parapsilosis biofilms (Cocuaud et al., 2005; Katragkou et al., 2007), and lipid formulations of
amphotericin B have shown activity against C. parapsilosis biofilms (Kuhn et al., 2002b).

3. Secreted Enzymes

Extracellular enzymes secreted by microbial pathogens have received significant

attention due to their potential role in pathogenesis and as possible targets for the design of
synthetic inhibitors to treat infection. These enzymes include aspartic proteinases (Saps),
phospholipases, and lipases.

A. Aspartic Proteinases
Ten aspartic proteinase (Sap) isoenzymes are responsible for the proteinase activity of C.
albicans. These proteins exhibit molecular masses between 35 and 50 kDa and are encoded
by the SAP1–10 genes.
The roles of the SAP1–6 genes are related to adherence, tissue damage and changes in the
immune response. The function of SAP7 is not yet fully understood, but it has been reported
that SAP9 and SAP10 are not secreted proteinases; instead, they are involved in preserving the

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 Candida Parapsilosis Complex 623

regulatory surface integrity of yeast cells (Naglik et al., 2003). Saps facilitate the invasion and
colonization of host tissue by disrupting host mucosal membranes (Ruchel et al., 1992) and
degrading important immunological and structural defense proteins, such as immunoglobulin
G heavy chains, α2-macroglobulin, C3 protein, β-lactoglobulin, lactoperoxidase, collagen,
and fibronectin (Pichova et al., 2001). Compared to C. albicans, C. parapsilosis presents less
Sap activity (Odds et al., 1987; Ruchel et al., 1986).
Three Saps have been identified in C. parapsilosis, two of which remain largely
uncharacterized (Merkerova et al., 2006), whereas the Sapp1p isoenzyme has been
biochemically characterized (Pichova et al., 2001; Trofa et al., 2008). Although SAPP2P was
originally classified as a pseudogene, it produces a functional proteinase, Sapp2p, which was
found to account for approximately 20% of the Saps isolated from a culture supernatant
(Fusek et al., 1993). Furthermore, the proteolytic activity of the SAPP2P gene product
exhibits a different activation mechanism than that of the SAPP1P product (Merkerova et al.,
2006). No study has yet analyzed or characterized SAPP3 or Sapp3p. Sap production varies
among the isolated strains of C. parapsilosis, and the involvement of Saps in pathogenesis
remains unclear.
However, there is a trend relating Sap production and the site of isolation, as both
vulvovaginal and skin isolates of C. parapsilosis exhibit higher in vitro Sap activity than
blood isolates (De Bernardis et al., 1989; Dagdevirem et al., 2005; Trofa et al., 2008). This
has significant implications for infection models, as in vaginal infections in rats, blood C.
parapsilosis isolates are cleared during the first or second week post-challenge, while skin
isolates generate sustained infection (De Bernardis et al., 1989). No significant differences in
vaginopathic potential were found between vaginal C. parapsilosis isolates showing high Sap
production and a vaginopathic C. albicans isolate (De Bernardis et al., 1999). Saps appear to
be less important for pathogenesis in bloodstream infections than in localized invasive
disease, particularly in vaginal infections. Interestingly, all four C. parapsilosis strains
isolated from patients with candiduria in Sao Paulo, Brazil described in one study exhibited
proteolytic activity (Silva et al., 2007).
Inhibitors of Saps have been tested as antimycotic drugs. Of the four HIV aspartic
protease inhibitors ritonavir, nelfinavir, indinavir, and saquinavir, only ritonavir and
saquinavir were found to affect Sapp1p activity (Pichova et al., 2001) . Another group found
that ritonavir reduced Sap activity but that saquinavir did not (Asencio et al., 2005). Pepstatin
A, a specific aspartic proteinase inhibitor, blocks the initial penetration of C. albicans and C.
parapsilosis through mucosal surfaces and reduces histopathological alterations during
experimental cutaneous candidiasis (Gacser et al., 2007).

B. Phospholipases
Phospholipases are enzymes that are capable of hydrolyzing one or more ester linkages in
glycerophospholipids. The function of phospholipases during infection is not well
understood, although it is believed that they are involved in the disruption of host membranes
(Ghannoum et al., 2000). Seven phospholipase genes have been identified (PLA, PLB1,
PLB2, PLC1, PLC2, PLC3 and PLD1).
However, the role of the enzymes encoded by these genes remains unclear
(Samaranayake et al., 2006). PLB1 has been described as playing a role in virulence in animal
models of candidiasis. Plb1p is an 84 kDa glycoprotein present at hyphal tips during tissue
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624 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

invasion that displays hydrolase and lysophospholipase-transacylase activity (Ghannoum,

2000).
Phospholipases have been shown to affect C. albicans virulence in a murine infection
model, in addition to adhesion to epithelial cells (Dagdevirem et al., 2005; Trofa et al., 2008),
the invasion of a reconstituted human oral epithelium (Jayatilake et al., 2005), host signal
transduction (Schaller et al., 2005), and host cell penetration (Pugh and cawson, 1977).
The role of phospholipases in C. parapsilosis pathogenesis is less clear. There have been
contradictory findings reported on this topic, with some investigators detecting phospholipase
activity in as many as 51% of C. parapsilosis strains (Ghannoum et al., 2000; Trofa et al.,
2008) and others finding no activity (Kantarcioglu et al., 2002; Samaranayake et al., 1988).
Additionally, only one of four isolated C. parapsilosis strains reported to cause candiduria in
Sao Paulo, Brazil, was found to exhibit phospholipase activity (Silva et al., 2007).
Such inconsistencies in the available data could be the result of relatively small sample
sizes as well as the biological differences between the tested strains. Furthermore, variations
in the production of phospholipases have also been found when comparing systemic versus
superficial isolates, with some investigators identifying phospholipase activity only in
bloodstream isolates (Dagdevirem et al., 1993) and others describing significantly higher
activities in superficial C. parapsilosis isolates than in systemic isolates (Fernanado et al.,
1999).

C. Lipases
The roles of extracellular microbial lipases include the digestion of lipids for nutrient
acquisition, adhesion to host cells and tissues, nonspecific initiation of inflammatory
processes through affecting immune cells and self-defense via lysing competing microflora
(Stehr et al., 2004; Ga´cser et al., 2007).
Lipases catalyze both the hydrolysis and synthesis of triacylglycerols and are
characterized by their stability at high temperatures and in organic solvents, high
enantioselectivity, and resistance to proteolysis (Brockerhoff et al., 1974). In C. parapsilosis,
two lipase genes, CpLIP1 and CpLIP2, have been identified, although only CpLIP2 encodes
an active protein (Brunel et al., 2004).
In addition, lipase inhibitors significantly reduce tissue damage during the infection of
reconstituted human tissues (Ga´cser et al., 2007). Furthermore, homozygous mutants for the
CpLIP1-CpLIP2 genes form thinner and less complex biofilms, exhibit reduced growth in
lipid-rich media, are more efficiently ingested and killed by macrophage-like cells, and are
less virulent compared to wild-type C. parapsilosis organisms, both in infections of
reconstituted human oral epithelia and in a murine intraperitoneal challenge model (Gacser et
al., 2007b).

D. Hemolysin
The production of hemolysin plays an important role in virulence. This protein is
essential for survival and is related to the acquisition of iron (Vaughn and Weinberg, 1978).
Hemolysins are proteins produced by microorganisms to destroy red blood cells. Iron, an
inorganic element, is essential for the development of microorganisms, including yeasts, and
the ability to obtain this element is essential for the establishment of the infectious process
(Manns et al., 1994).

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 Candida Parapsilosis Complex 625

IV. MYCOLOGY
1. Colony Morphology and Dimorphism

C. parapsilosis forms white and creamy colonies with variable morphology. It grows as
oval cells or pseudohyphal filaments, but in contrast to C. albicans, it does not form true
hyphae. C. parapsilosis undergoes dramatic changes in its cellular and colony morphology in
response to a specific subset of amino acids (Kim et al., 2006). Interestingly, several studies
have suggested that in addition to their role as building blocks in protein synthesis, amino
acids might also exhibit morphogenetic activity. Characterization of the morphology of cells
obtained from C. parapsilosis colonies revealed that out of the 19 amino acids tested, only
arginine, aspartic acid, glutamine, histidine, leucine, lysine, phenylalanine and proline led to
the formation of elongated cells. (Kim et al., 2006). Transport analyses have shown that
amino acid-mediated morphogenesis does not require transport of the ligand across the
plasma membrane, which indicates that amino acids could be recognized by membrane
receptors that activate signal transduction pathways controlling cell differentiation (Nosek et
al., 2009). The switch between different colony morphologies is most likely associated with
alterations in the cell wall architecture and changes in cell-to-cell, cell-to-surface, and cell-to-
host tissue adhesion, which is mediated by cell wall glycoproteins (Chaffin 2008; Nather and
Munro 2008). Bioinformatic searches for pathogen-specific gene families in Candida species
revealed a number of genes encoding cell wall proteins (Nosek et al., 2009). In C.
parapsilosis, these genes encode 5 Als (agglutinin-like sequence)-, 17 Hyr (hyphally
regulated)-, and 6 Pga30 (predicted GPI anchored protein 30)-like proteins (Butler et al.,
2009). Importantly, the observed changes in colony morphology are associated with the
dimorphic transition of cell morphology, which varies between single-cell forms and
pseudohyphal filaments. Molecular mechanisms implicated in this morphological switching
and the dimorphic transition are closely associated with the formation of biofilms (or slimes)
on various surfaces (Nosek et al., 2009).

2. Genetics

C. parapsilosis forms a complex (the Candida parapsilosis complex) composed of three

genetically distinct groups that have been recognized as separate species: C. parapsilosis
sensu stricto or Candida parapsilosis (C. parapsilosis sensu stricto), Candida metapsilosis and
Candida orthopsilosis.
Since the 1980s, several studies have demonstrated genetic heterogeneity within the
Candida parapsilosis species via different molecular techniques. Using whole-cell DNA from
Candida spp. digested with the restriction endonuclease EcoRI, followed by electrophoresis
of DNA fragments, Scherer and Stevens (1987) observed that C. parapsilosis isolates
presented differences and divided them into three groups. The data obtained in this study
suggested that the isolates identified as C. parapsilosis are genotypically more heterogeneous
than C. albicans or C. tropicalis isolates. Various studies have shown that genetic
discrimination between isolates of C. parapsilosis can be accomplished through
electrophoretic karyotype (EK) analysis, DNA fingerprinting by digesting chromosomes with
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626 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

restriction endonucleases followed by pulsed field gel electrophoresis (PFGE) and randomly
amplified polymorphic DNA (RAPD) methods (Carruba et al., 1991, Lott et al., 1993;,
Branchini et al.,1994; Shin et al., 2001.
To investigate the possibility of a single-source outbreak that presented a high incidence
of C. parapsilosis as the etiologic agent of nosocomial infections at the Veterans
Administration Medical Center in San Antonio, Texas, for several years, Lin and co-workers
(1995) conducted multilocus enzyme electrophoresis (MLEE) and DNA sequencing of
internal transcribed spacer (ITS) sequences flanking the 5.8S RNA gene. These authors
revealed the existence of three genetically distinct isoenzyme-defined groups of C.
parapsilosis.
Some of these isolates had been analyzed previously via RAPD, and due to the genetic
heterogeneity within C. parapsilosis, they were divided into three distinct groups based on
their RAPD profiles (group I, II, III) (Lehmann et al.,1992). Subsequently, nuclear DNA base
composition, DNA reassociation, and DNA RFLP analyses of isolates of C. parapsilosis from
diverse sources, including representative isolates from groups I through III reported by Lin et
al. (1992), confirmed the unrelatedness of the three groups at the species level (Roy et al.,
1998).
Another study using a complex DNA fingerprinting probe, Cp3-13, for Southern blot
analysis also found differences among C. parapsilosis isolates and divided them into three
groups (Enger et al., 2001). To distinguish these three species from each other, Tavanti et al.
(2005) proposed analysis of the restriction polymorphisms in the SADH gene, which encodes
a secondary alcohol desidrogenase that is common to all three species. Briefly, a 716 bp
fragment of SADH was amplified via polymerase chain reaction (PCR). The restriction
fragment length polymorphisms (RFLPs) among the SADH PCR products observed following
BanI restriction enzyme digestion generated three unique RFLP patterns upon
electrophoresis: two bands (521 and 196 bp) for C. parapsilosis group I; one band (716 bp)
for C. parapsilosis group II; and four bands (370, 188, 93, and 60 bp) for C. parapsilosis
group III . Based on the extensive differences between the three subgroups of C. parapsilosis,
these researchers proposed that the name C. parapsilosis be used for only group I isolates,
and two new species names, C. orthopsilosis and C. metapsilosis, be used in place of the
former groups II and III, respectively. Since this report, surveillance data on the prevalence
and distribution of the three genomic species in different countries have begun to emerge
(Asadzadeh et al., 2009, Bonfietti et al., 2012a, 2012b; Chen et al., 2010; Gonçalves et al.
2010; Gomez-Lopez et al., 2008; Hensgens et al., 2009; Kocsubé et al., 2007; Lockhart et al.,
2008; Miranda-Zapico et al., 2011; Moris et al., 2012; Pires et al., 2011; Purisco et al., 2012;
Silva et al., 2009; Tavanti et al., 2007; Toro et al., 2010; Yong et al., 2008). In the same year,
based on sequence data for the ITS1 region, Iida et al. (2005) found that some isolates
belonged to a different phylogenetic group, which was designated group IV. The sequences of
the Dl and D2 domains of the large ribosomal subunit DNA region (Kurtzman et al., 1998)
confirmed this finding and suggested that further molecular and taxonomic studies addressing
these isolates might allow the proposal of a third new species.
Lodderomyces elongisporus has been recognized as a four-species complex, and it is the
only species in that complex that forms ascospores (Lockhart et al., 2008). Lodderomyces
elongisporus was previously classified as a teleomorph of C. parapsilosis (Lin et al., 1995),
but data obtained from small rRNA subunit gene sequencing showed that it is a distinct
species that is closely related to C. parapsilosis (James et al., 1994). Despite the many studies

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 Candida Parapsilosis Complex 627

addressing the genetic heterogeneity within the Candida parapsilosis species complex, only
two studies have mentioned this species. Using MLLE, Lin et al. (1995) excluded a close
relationship of any of the three groups to Lodderomyces elongisporus, and Tay et al. (2009)
demonstrated the existence of four RAPD profile groups and identified this species was
through sequencing of the ITS region.
Another molecular method for rapid identification of these three species was recently
developed based on a simple PCR method using species-specific primers derived from unique
sequences within the ITS1–5.8S rRNA–ITS2 region (Asadzadeh et al., 2009). PCR-RFLP
(Tavanti et al., 2005) and sequencing of the ITS region (White et al., 1990) are currently the
molecular methods that are most commonly used to identify species of the Candida
paraspilosis complex. Other molecular techniques, such as EK with PFGE, PFGE in
conjunction with restriction endonucleases such as SfiI or BssHII, RAPD, DNA fingerprinting
with Cp3-13 probes and amplification fragment length polymorphisms (AFLPs), can also
distinguish the three (or four) species of the Candida parapsilosis complex, but they are more
useful for identifying the same strain in independent isolates, discriminating unrelated
isolates, and identifying microevolution in infecting populations. These tools are important in
the investigation Candida parapsilosis outbreaks for determining whether the same or
different strains are responsible for persistent or recurrent fungemia and for determining
whether the central venous catheter or the hands of healthcare professionals as the source of
nosocomial candidemia, as identical profiles are presented by isolates from blood and CVC as
well as blood and human hands (Almirante et al., 2006; Chen et al., 2010; Shin et al., 2001;
van Asbeck et al., 2007).
Tavanti et al. (2007) analyzed sequential isolates of C. orthopsilosis obtained from the
same patients via AFLP and suggested the existence of clonal recombination as well as strain
maintenance at different body sites in a single individual. Another study concluded that
recombination occurs in C. metapsilosis genetic populations, giving rise to variability
detected through AFLP genotyping (Hensgens et al., 2009). Several authors have observed
low genetic variability among isolates (Kocsubé et al., 2007; Lehmann et al., 1992; Tavanti et
al., 2005, 2007; Tay et al., 2009) and genetic heterogeneity in C. orthopsilosis and C.
metapsilosis (Iida et al., 2005; Lasker et al., 2006; Rycovska et al., 2004; Tavanti et al., 2005,
2007; van Asbeck et al., 2008).

V. ANTIFUNGAL SUSCEPTIBILITY
Antifungal resistance of infective strains is becoming recognized as a key factor involved
in clinical failure, and the occurrence of clinical resistance and the appearance of in vitro-
resistant strains have been described systematically around the world (Lin et al., 1995; Pfaller
et al., 2001; Tortorano et al., 2006; Tay et al., 2009). However, few studies have addressed
the global epidemiology and antifungal susceptibility profile of C. parapsilosis (Silva et al.,
2009; Almirante et al., 2002, Pfaller et al., 2008). Overall, C. parapsilosis isolates have
remained reliably susceptible to flucytosine, azoles (except for itraconazole), and
echinocandin antifungal agents in recent years (Diekema et al. 2009a; Diekema et al. 2009b;
Pfaller et al. 2002b; Pfaller et al. 2002d; Pfaller et al. 2004e; Pfaller et al. 2005a; Pfaller et al.
2005c; Pfaller et al. 2007b; Pfaller et al. 2008a; Pfaller et al. 2008e). Some differences in drug
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628 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

susceptibility between the three species in this complex may influence the therapeutic choices
that are made, and careful species identification and the use of a reference antifungal
susceptibility method are therefore recommended to support clinical treatment. The European
Committee on Antibiotic Susceptibility Testing (EUCAST) and the Clinical Laboratory
Standards Institute (CLSI) have developed standard methods based on broth dilution for
susceptibility testing of yeasts (CLSI, 2008; Rodriguez-Tudela et al., 2008). The
susceptibility results obtained by EUCAST are in close agreement with those generated
following the procedures of the CLSI (Cuenca-Estrella et al., 2002). The results are expressed
in terms of the minimal inhibitory concentration (MIC) of each antifungal agent.

Amphotericin B

The MIC interval for amphotericin B against C. parapsilosis isolates is typically between
0.03 and 2 mg/L. The interpretation of breakpoints for amphotericin B represents a
problematic issue, as they have not been established for individual fungal species. However,
Candida isolates for which the MICs are >1 mg/mL are unusual and possibly “resistant,” and
they may require high doses of amphotericin B for optimal treatment (Pappas et al. 2009; Rex
et al. 2002; Spellberg et al. 2006). Resistance to amphotericin B among Candida members
may be determined through agar-based methods such as Etest®, which have proven to be
more sensitive and reliable methods in comparison with reference broth microdilution
methods (Clancy et al. 1999; Krogh-Madsen et al. 2006; McClenny et al. 2002; Park et al.
2006; Pfaller et al. 1998a; Pfaller et al. 2004c; Wanger et al. 1995). The available reference
methods (M27-A and E.Def 7.1) fail to detect resistance to amphotericin B, likely due to the
narrow MIC range involved. In contrast to the decreased susceptibility to amphotericin B
observed for C. glabrata, C. krusei, and C. rugosa or the acquired amphotericin-B resistance
reported for C. guilliermondii and C. lusitaniae bloodstream isolates, the situation is much
more favorable for strains of C. parapsilosis (Atkinson et al. 2008; Dick et al. 1985; Diekema
et al. 2009b; McClenny et al. 2002; Pfaller 2005; Pfaller et al. 2004c; Pfaller et al. 2007a,;
Pfaller et al. 2006d). Rare high amphotericin B-MIC concentrations (MIC, 2 mg/L) are
occasionally reported for C. parapsilosis (Silva et al., 2009; Córdoba et al., 2011).
Some data suggest that for C. parapsilosis sensu stricto, the amphotericin B MIC is
higher than for C. orthopsilosis and C. metapsilosis (Gomez-Lopez et al., 2008; Lockhart et
al., 2008; van Asbeck et al., 2008; Tay et al., 2009; Silva et al., 2009, Chen et al., 2010,,
Pfaller et al., 2011). Little information has been published for C. metapsilosis, most likely due
its low prevalence as an invasive agent. However, low MICs for amphotericin B are
commonly found for C. metapsilosis isolates (Gonçalves et al., 2010; Lin et al., 1995).
Further investigation with a greater number of C. metapsilosis isolates will be necessary to
achieve a better understanding of the pattern of susceptibility among invasive C. metapsilosis
strains.

Fluconazole

The use of polyene therapy for the treatment of invasive candidemia has markedly
diminished with the availability of newer azoles and a new class of antifungal agents, the

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echinocandins (Chandrasekar, 2011). Fluconazole and echinocandins are the preferred

options for first-line therapy according to the guidelines of the Infectious Diseases Society of
America (IDSA) for the management of candidiasis in non-neutropenic patients (Pappas et
al., 2009).
Large global studies have revealed that all C. orthopsilosis and C. metapsilosis isolates
are susceptible to fluconazole (Lockhart et al., 2008; Pfaller ad Diekema, 2010; Moris et al.,
2012). The published range of fluconazole MICs is 0.06 to 16 mg/L for the C. parapsilosis
complex, and azole resistance has been detected only among C. parapsilosis sensu stricto
isolates (Silva et al., 2009; Dimopoulos et al., 2009; Bonfietti et al., 2012a). The emergence
of C. parapsilosis strains with decreased susceptibility to fluconazole may be related to the
extensive use of this drug (Arendrup et al., 2002). No resistance has been reported among C.
orthopsilosis or C. metapsilosis strains.
The differences in fluconazole susceptibility patterns reflect the distinct affinity of azoles
for the enzymes involved in the ergostherol metabolic pathway (van Asbeck et al., 2008).

Voriconazole

The MICs of voriconazole generally fall between 0.02 and 1 mg/L, and it was recently
suggested that voriconazole use affects neither the distribution nor antifungal drug
susceptibility of Candida spp. (Fournier et al., 2011). The lower susceptibility to voriconazole
described in some studies for C. orthopsilosis (Toro et al., 2011) in comparison to C.
parapsilosis sensu stricto has not been reported in other publications (Bonfietti et al., 2012a).

Echinocandins

Three echinocandin-class drugs, anidulafungin, caspofungin, and micafungin, are

licensed for the treatment of invasive candidiasis. Echinocandins have been suggested for use
as an initial therapy in neutropenic patients until the species of the Candida isolate involved is
identified (Pappas et al., 2009). For infections caused by C. parapsilosis, fluconazole therapy
is advised, and polyenes are recommended as an alternative therapy in cases of intolerance or
limited availability of other antifungal agents. Although resistance to echinocandins is rare
(Gomez-Lopez et al., 2008; Lockhart et al., 2008; van Asbeck et al., 2008; Tay et al., 2009;
Chen et al., 2010), a few case reports describing breakthrough C. parapsilosis bloodstream
infections after prolonged caspofungin therapy have suggest that C. parapsilosis could be
clinically resistant to this class of drugs (Cheung et al. 2006; Kabbara et al., 2008; Moudgal et
al., 2009). Despite the limited existing data linking growing caspofungin usage and increases
in bloodstream infection by C. parapsilosis (Forrest et al., 2008; Paugan et al., 2011), it is
accepted that there is selective pressure on this species (Pfaller et al. 2008a; Pfaller et al.
2008e). Moreover, in a recent study, the increase in caspofungin use was found to be
significantly correlated with increases in the caspofungin MIC for C. parapsilosis (Fournier et
al., 2011).
The wild-type distribution of C. parapsilosis with respect to all three echinocandins,
defined as the MIC distribution for isolates that exhibit no acquired/mutational resistance to
the drug, is clearly separated from those of the other Candida species (Arendrup et
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630 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

al.,2010;Pfaller et al.,2010b). The MIC distributions demonstrate that high MICs are typical
for C. parapsilosis for all three echinocandins, with that for anidulafungin ranging between
0.003-4 mg/L, for caspofungin from 0.015-4 mg/L, and for micafungin from 0.015-2 mg/L. In
fact, the echinocandins exhibit intrinsically low activity against C. parapsilosis isolates in
vitro, which is generally attributed to the natural polymorphism in the fks1 gene sequence of
this organism (Pfaller et al., 2006a; Walker et al., 2010). The MIC is in the same range as for
mutant isolates of the other Candida members (Garcia-Effron et al., 2008a).
Although C. parapsilosis sensu stricto is not considered to be a resistant species, the
caspofungin MICs found for C. orthopsilosis and C. metapsilosis are relatively lower
(Lockhart et al., 2008; Silva et al., 2009; Pfaller et al., 2011; Bonfietti et al., 2012b). The
interspecies differences in the susceptibility to caspofungin could result from distinct
components in the structure of the cell wall or variations in the affinity for the key enzyme 1-
3 β glucana (van Asbeck et al., 2008). For anidulafungin, high MIC values have been found
for C. parapsilosis (MIC, 4 mg/L) (Pfaller and Diekema, 2007; Córdoba et al., 2011).
Additionally, the description of multiechinocandin-resistant and multiazole-resistant
phenotypes of C. parapsilosis sensu stricto is of clinical relevance (Silva et al., 2009;
Moudgal et al., 2005, Ghannoum et al., 2009, Vazquez et al., Bonfietti et al., 2012).
Given the growing incidence of critically ill patients showing a high risk of invasive
fungal infection, leading to increased antifungal drug usage, particularly in intensive care
units, the impact of this practice on the distribution or drug susceptibility of Candida species
should be closely and systematically monitored around the world.

VI. CLINICAL MANIFESTATIONS AND TREATMENT

The clinical manifestations of candidiasis caused by C. parapsilosis will be presented
regarding the site of infection. Thus, candidemia, endocarditis, meningitis, onychomycosis,
endophthalmitis, peritonitis, arthritis, otomycosis, vulvovaginitis, and urinary tract infection
will be addressed.
General measures, antifungal susceptibility and the site of the infection should be
considered in the treatment of infections caused by C. parapsilosis.
The general measures applied vary according to the site of infection and will be presented
for each organ. Sensitivity to antifungal compounds has to be carefully considered. First,
epidemiological data indicating the predominant Candida species in the region the patient
comes from and its susceptibility can be used as a guide for initiating treatment. Second,
appropriate clinical specimens should be collected prior to the introduction of the antifungal
compound to allow etiological diagnosis and susceptibility analysis to be performed. These
results will play a decisive role in the maintenance or replacement of the previously
introduced antifungal drug. Patients with mild cases can generally wait for identification of
the causative agent to begin treatment, while moderate and severe patients should be
immediately treated. Subsequent etiological identification and susceptibility tests, in addition
to the initial clinical response to the treatment, will aid in the choice of the most appropriate
antifungal compound. Polyene antibiotics (nystatin, amphotericin B and its lipid
formulations), flucytosine, systemic azoles (ketoconazole, fluconazole, itraconazole,
voriconazole, posaconazole) and topic azoles (butaconazole, clotrimazole, miconazole,

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tioconazole, terconazole and econazole), and echinocandins (caspofungin, anidulafungin,

micafungin) can be used for the treatment of infections caused by C. parapsilosis.

1. Candidemia

Candidemia is defined as isolation of Candida species from the bloodstream. It has

mainly been diagnosed in patients hospitalized for long periods, receiving antibiotics,
immunosuppressive therapy and total parenteral nutrition, and subjected to invasive medical
procedures, mainly central venous catheter (Colombo et al., 2006; Chow et al., 2008).
The incidence and the etiology of candidemia vary according to the region where a
hospital is located, period evaluated, and patients’ age distribution (Colombo et al., 2006).
The C. parapsilosis complex (16.9%), together with C. glabrata (16.6%), represents the
second most prevalent Candida species group, preceded only by C. albicans (C Trofa et al.,
2008). However, neonatal candidemia is caused mainly by C. albicans (57.5% of cases),
followed by C. parapsilosis (34.3%), while the other Candida species present a much lower
prevalence (Trofa et al., 2008). A study performed in Brazil showed C. albicans (41%), C.
tropicalis (24%) and C. parapsilosis (21%) to be the most common Candida species among
adult patients with cadidemia, while C. albicans (40%) and C. parapsilosis (21%) were most
common among pediatric cases (Colombo et al., 2006). Compared with C. albicans, C.
parapsilosis is less likely to be found in patients subjected to mechanical ventilation (31%
versus 41%; p=0.03) or corticotherapy (25% versus 34%; p=0.04) (Colombo et al., 2006).
However, some studies carried out in patients with candidemia (Mujica et al., 2004; Medrano
et al., 2006) have detected a similar prevalences of C. parapsilosis and C. albicans, while in
others, C. parapsilosis has outranked the latter species (Levy et al., 1998, Ng et al., 2001). In
addition, cases of C. parapsilosis outbreaks have been reported (Solomon et al., 1984, 1986;
Weems et al., 1987; Welbel et al., 1996; Plouffe et al., 2002), which have been increasing in
pediatric intensive care units over the years (san Miguel et al., 2005).
Fever (100%), cardiovascular arrest (22%) and renal failure (10%) are the main clinical
findings in patients with candidemia caused by C. parapsilosis (Trofa et al., 2008).
Hematogenous candidiasis is characterized by a wide spectrum of clinical findings,
varying from isolated episodes of detecting Candida species in the bloodstream, to the
recurring presence of the fungus in the bloodstream, spreading to one or more organs. As
most of the publications on this topic refer to candidemia, this term will be used.
Non-neutropenic patients. The echinocandins are the drugs of choice for the treatment of
candidemia in non-neutropenic patients (Nguyen and Yu,1995; Mora-Duarte ET AL., 2002;
Kuse et al., 2007; Rebolli et al., 2007). In spite of the high MICs found for echinocandins
when used against C. parapsilosis, the rate of therapeutic success is satisfactory (Gonçalves et
al., 2010; Colombo et al., 2010). However, in the case of persistent positive hemocultures,
echinocandins should be replaced by another class of antifungal compounds (Colombo at al.,
2012). Caspofungin should be administered at a dose of 70 mg once a day on the first day
(initial treatment), followed by 50 mg once a day thereafter (maintenance therapy);
anidulafungin at a dose of 200 mg on the first day, followed by 100 mg once a day; and
micafungin at a dose of 100 mg for both initial and maintenance therapy. The length of the
treatment should be at least 14 days after achieving a clinical cure, characterized by the
disappearance of the previously symptomatology presented by the patient and negative
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632 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

hemocultures. Thus, serial hemocultures should be performed periodically, including on the

3rd and the 5th days after the initiation of the treatment. Lipossomal amphotericin B,
administered in daily doses of 3 mg/kg for adults, is an alternative for the treatment of the
candidemia (Kuse at al., 2007).
Neutropenic patients. These patients should be treated very early, preferentially with
fungicidal compounds (Colombo et al., 2012). Lipossomal amphotericin B, amphotericin B
lipid complexes and echinocandins are the drugs of choice in such cases (Bates et al., 2001;
Hughes 2002; Colombo et al., 2012).
The treatment of patients with candidemia and endophthalmitis, meningitis, endocarditis
or arthritis will be analyzed in the next sections.

2. Endocarditis

The reported incidence of fungal endocarditis has been increasing in recent decades both
because the associated predisposing factors are increasing and because better culture systems
are now available (Garzoni et al., 2007). Fungi account for 1.3 to 6.0% of infective
endocarditis cases, 94.1% of which are attributed to Candida species. C. parapsilosis has
been found to be responsible for 17% of etiologically identified cases, making it the second
most common species after C. albicans (Pierrotti et al., 2002).
A comparative review of 124 cases of fungal endocarditis caused by C. parapsilosis (72
cases) and C. albicans (52 cases) revealed no difference in the prevalence of the predisposing
factors (Garzoni et al., 2007). However, C. parapsilosis showed a tendency toward a higher
prevalence associated with intravenous parenteral nutrition usage (6.9% versus 0.0%; p=0.06)
and a lower prevalence related to previous valvular disease (4.8% versus 13.5%; p=0.06). The
prevalence of the valves involved showed no difference according to the Candida species, but
combined lesions were less prevalent in cases of C. parapsilosis infection (0.0% versus
11.5%; p=0.05). Mortality rates were also not found to differ in terms of the Candida species
involved (41.7% versus 33.0%; p>0.05), which is a finding that conflicts with other reports
(Pierrotti et al., 2002). Embolic or hemorrhagic complications were reported to be present in
43.8% of the patients for whom this information was available, mostly in the lower limbs
(37.8% of the cases with this clinical picture) and the brain (21.4%), followed by the lungs
(10.7%) and the upper limbs (7.1%). Intracranial hemorrhage was observed in 25% of the
patients, possibly associated with mycotic aneurysm (Garzoni et al., 2007). This complication
is a classical indication for a surgical procedure, as are large vegetations.
Amphotericin B deoxycholate (daily doses of 0.5 -1.0 mg/kg), flucytosine (25.0 – 37.5
mg/kg q.6 hrs) and fluconazole (400 mg q.12 hrs in the first day, followed by 100-200 mg
q.12 hrs or 6 – 12 mg/kg daily) were the antifungal compounds that were most frequently
used. Newer antifungal drugs, such as lipid formulations of amphotericin B, voriconazole and
caspofungin, were also employed in a few instances. Combined surgical-clinical treatment
was carried out in 58.3% of the patients. Surgical procedures predominated in native over
prosthetic valves (72.4% versus 45.7%; OR=3.1; 1.0 – 8.9; p=0.05). A multivariate analysis
of the variables associated with mortality in patients with C. parapsilosis infection
demonstrated the importance of the adjuvant surgery (OR=0.33; 0.01 – 1.02; p=0.05)
(Garzoni et al., 2007), in agreement with other authors and recommendations (Rex et al.,
2000; Steinbach et al., 2005). The resistance of C. parapsilosis biofilms, mainly where

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prosthetic valves are involved, can explain the best results when a surgical procedure is
carried out (Trofa et al., 2008).

3. Meningitis

Autopsy studies of patients with invasive candidiasis showed central nervous system
(CNS) involvement in less than 15% of adults (Lipton et al., 1984) but in 64% of neonates
(78). However, C. parapsilosis is rarely the cause of meningitis. Individual casereports
(Bosch et al., 1990; Faix, 1983; Jimenez-Mejias et al., 1993) and publications describing
small series of patients (Dorko et al., 2002) have suggested an increasing incidence of this
Candida species.
Recently, C. parapsilosis meningitis was reported to be the AIDS-defining disease in one
patient. This patient was hospitalized with a 15-day history of intense holocranial headache
associated with multiple episodes of vomiting and recurrent high fever (Sidrim et al., 2011).
One day prior to admission, he presented paresthesia in his right fingers. Physical
examination revealed left facial palsy and neck stiffness with signs of meningeal irritation,
including Kernig and Brudzinsky signs and several whitish plaques in the oral cavity,
consistent with severe oropharyngeal candidiasis. ELISA and Western blot serological testing
confirmed HIV infection. Lumbar puncture showed increased intracranial pressure,
cerebrospinal fluid (CSF) that was clear in appearance, pleocytosis of 10 leukocytes/mm3,
consisting of 43% lymphocytes, 2% monocytes and 55% neutrophils, hypoglycorrhachia (52
mg/dL), hyperproteinorrhachia (72 mg/dL) and structures suggestive of Candida species in
Gram-stained smears. These fungi were then isolated in culture and identified using
phenotypic and molecular methods as C. parapsilosis. Another case of C. parapsilosis
meningitis in an AIDSpatient was previously reported (Baradkar et al., 2008), but not as an
AIDS-defining disease.
Due to the increasing incidence of C. parapsilosis meningitis and the associated potential
for morbidity and mortality, mainly in case of a late diagnosis, this condition should be
investigated when suggestive epidemiological data are obtained.
The combination of amphotericin B deoxycholate (0.7 – 1.0 mg/kg once a day) or
liposomal amphotericin B (3.0 – 5.0 mg/kg once a day) with flucytosine - 5.FC (25.0 mg/kg
q.6 hrs) is the regimen of choice for treating Candida infection of the CNS (Pappas et al.,
2004; Smego et al., 1984). Serum levels of flucytosine should reach 40 - 60 µg/mL (Francis et
al., 1992). Administration of amphotericin B deoxycholate via the intraventricular route is
recommended only for exceptionally severe cases. Fluconazole has been indicated as a
maintenance therapy (Marr et al., 1994). Removal and replacement of infected ventricular
devices through a two-step process is indicated (Nguyen et al., 1995; Sanchez-Portocarrero et
al., 1994). The treatment should be administered for at least 4 weeks after complete clinical
recovery due to a tendency to relapse.

4. Onychomycosis

Onychomycosis is defined as infection of the nail by fungus (Zaias, 1972).

Epidermophyton, Microsporum and Trichophytos species are universally recognized as
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634 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

etiologic agents of this condition, but nondermatophytic fungi, such as Candida species, have
been rarely identified as a cause of onychomycosis. One of the four clinical types of
onychomycosis is characterized by the involvement of all the nail plate, is caused by C.
albicans and is seen in patients with chronic cutaneous candidiasis, a syndrome associated
with dysfunction of cell-mediated and humoral immunity, neutrophils and serum factors. This
condition involves a variable number of dystrophic nails, including fingernails and toenails
(Zaias, 1972).
In addition to this clinical presentation, some patients show a distal subungual lesion that
is not associated with the chronic cutaneous candidiasis syndrome, from which yeasts have
been isolated. Hyphae have been observed in keratinous subungual debris yielding both C.
parapsilosis (predominately) and C. albicans (Zaias, 1972).
As C. parapsilosis is one of the main species in the microflora inhabiting the subungual
space (McGinley et al., 1988), its isolates can be interpreted only as commensal and not as
pathogenic yeast. In spite of this consideration, several reports have described C. parapsilosis
as the most common Candida species causing onychomycosis (Trofa et al., 2008; Mujica et
al., 2004; Mugge et al., 2006; Figueiredo et al., 2007 ), with a prevalence comparable to that
presented by some dematophytes (Mugge et al., 2006).
Although data related to the treatment of onychomycosis caused by C. parapsilosis are
scarce, pulse therapy involving 1 week of treatment with 200 mg itraconazole twice a day for
3 or 4 months is efficacious (De Doncker et al., 1995; Roseeuw et al., 1993; Pappas et al.,
2004).

5. Endophthalmitis

Hematogenous spreading of Candida species to the eye, first reported in 1943, was
observed in 9 (28.1%) of 32 patients with confirmed candidemia and was caused by C.
albicans in 6 cases, by C. parapsilosis in 1 and by Candida non-albicans in 2 (Brooks, 1989).
The candidemic patients, both with and without endophthalmitis, were similar regarding their
age, gender, risk factors, the use of a central venous catheter, broad spectrum antibiotics and
hyperalimentation, and prior surgery. However, patients with endophthalmitis showed a
higher fungal load, characterized by an increased frequency of cases with ≥4 positive blood
cultures. Initial ophthalmologic examination revealed unilateral eye involvement in 7 and
bilateral involvement in 1 of the 9 cases. One patient with normal eyes upon initial evaluation
showed unilateral lesions on the second examination. Fluffy infiltrates, retinal involvement
and Roth spots were observed. These findings suggest that periodic eye examination should
be performed in patients with confirmed candidemia.
Endophthalmitis was also observed as a complication following intraocular lens
implantation (IOLI). An outbreak of C. parapsilosis endophthalmitis was detected in 13
(3.9%) out of 333 patients following cataract extraction and IOLI performed using a balanced
salt solution (GBR-BSS) contaminated with this yeast as an intraoperative ophthalmic
irrigation solution (McCray et al., 1986; O’Day et al., 1987). The incubation period ranged
from 2 to 97 days (median of 9 days), and the main complaints were decreased vision (62%),
eye pain (62%), red eye (54%) and visual floaters (15%). Most patients presented a gradual
onset, with mild and nonspecific signs of inflammation, making an early diagnosis difficult.
Ophthalmologic examination showed anterior chamber cells and flare (100%), keratic

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precipitates (85%), and ciliary injection (54%). Clinical signs of vitreous cells and vitreous
“snowballs” were reported in 38% and 31% of the cases, respectively. C. parapsilosis was
identified in both the aqueous and vitreous humor and in 6.7% of the lot of bottles containing
the GBR-BSS. The applied treatment varied among the 13 patients. Topical steroids were
used by all of these individuals, subconjunctival or subtenons steroids by 6, and systemic
steroids only by 2. Antifungal compounds were administered to 11 of the 13 patients, which
consisted of intraocular amphotericin B in 11 cases, topical amphotericin B in 5, and oral
ketoconazole or flucytosine in 10. Partial or extended vitrectomy was carried out in 10 of the
13 patients. Decreased visual acuity (6 cases), retinal detachment (1) and glaucoma (1) were
the observed sequelae.
The treatment of ocular candidiasis depends on a clear distinction between chorioretinitis,
which responds to systemic therapy alone, and endophthalmitis, which may require
intravitreal in addition to systemic administration of antifungal compounds, mainly when a
vitreous abscess is present (Christmas and Smiddy, 1996; Darling et al., 2000). Fluconazole
and voriconazole are the antifungal compounds that show the highest levels in ocular fluids
(Akler et al., 1995; Riddell et al., 2011). Amphotericin B deoxycholate should be
administered at a daily dose of 0.7 – 1.0 mg/kg intravenously and fluconazole at a daily dose
of 6 – 12 mg/kg either intravenously or orally. Treatment regimens with a duration of 4 – 6
weeks (Colombo et al., 2012), or 6 – 12 weeks after surgery (Pappas et al., 2004) have been
indicated. Follow-up should be performed by an ophthalmologist to better characterize the
duration of the treatment and the therapeutic response (Colombo et al., 2012).

6. Peritonitis

Candida peritonitis is an important cause of morbidity, with a mortality rate that can
reach 44% (Wang et al., 2000). This infection has been observed as a complication of
peritoneal dialysis, gastrointestinal surgery or perforation of an abdominal viscus (Bayer et
al., 1976). Continuous ambulatory peritoneal dialysis (CAPD) has been found to be an
important predisposing factor for this condition, as is previous antibiotic therapy, which
presumably promotes Candida overgrowth (Amici et al., 1994; Wong et al., 2000;
Kaitwatcharachai, 2002).
Clinically significant infections should be differentiated from peritoneal contamination
with Candida species. The presence of persistent fever, peritoneal signs, altered abdominal
radiography, cloudy ascitic fluid, and leukocytosis contribute to this differentiation (Bayer et
al., 1976).
The Candida species responsible for peritonitis appear to have changed over the years,
with an increasing prevalence of C. parapsilosis being observed. Patients on CAPD show a
higher prevalence of C. parapsilosis than C. albicans, with C. parapsilosis being detected in
29% to 50% of cases (Greaves et al., 1992; Yinnon et al., 1999; Wang et al., 2000; Manzano-
Gayosso et al., 2003; Chen et al., 2004; Chen et al., 2006, Trofa et al., 2008).
The clinical manifestations of C. parapsilosis peritonitis include a cloudy dialysate
effluent, abdominal pain, fever, and bowel obstruction, which are symptoms similar to those
presented by patients infected by other Candida species or bacteria (Trofa et al., 2008). In the
case of a misdiagnosis with bacterial peritonitis, systemic antibacterial drugs may be
introduced, resulting in further progression of the Candida disease (Trofa et al., 2008).
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636 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

Treatment of C. parapsilosis peritonitis related to peritoneal dialysis should be based on

a) catheter removal, considering the propensity of C. parapsilosis to form biofilms, mainly in
high-glucose environments, such as the peritoneal cavity (Kaitwatcharachai, 2002; Colombo
et al., 2012); b) intensive antifungal treatment due to the higher risk of abscess formation and
greater frequency of patients responding slowly (Trofa et al., 2008); c) the fact that treatment
complications are substantially higher associated with C. parapsilosis (100%) than other
Candida species (29%) [C]; d) the administration of amphotericin B at 0.1 – 1.0 mg/kg daily
and fluconazole 400 – 800 mg daily, as the main choices for treatment; caspofungin at 50 or
100 mg daily should also be used; e) treatment regimens of 4 to 6 weeks are indicated; and f)
replacement of the peritoneal catheter can only be performed 4 to 6 weeks after starting the
antifungal therapy (Colombo et al., 2012; Wong et al., 2008).
Peritonitis after surgical procedures is usually associated with gastrointestinal
intervention or perforation of an abdominal viscus. Candidal peritonitis is more commonly
associated with perforation of the upper digestive tract than the ileum and appendix (Sandven
et al., 2002; Colombo et al., 2012). The recommended antifungal compounds and regimens
are the same as presented above (Sandven et al., 2012; Colombo et al., 2012).

7. Arthritis

Arthritis is a rare clinical presentation of a fungal disease and is usually associated with
Candida species. The knowledge of this condition is based on reports of individual cases or
small series of patients. The direct intra-articular inoculation of Candida species into a joint,
mainly in elderly patients, and the spread of the disseminated disease, mostly in
immunosuppressive individuals, constitute the physiopathology of articular candidal
involvement.
Implantation of a prosthesis, joint injection, arthrocentesis and arthroplasty are associated
with C. parapsilosis infection (Cushing and Fulgenzi, 1997; Brooks and Pupparo, 1998;
Wada et al., 1998; Yang et al., 2001). In addition, immunosuppressed patients with healthy
joints usually present articular involvement as a consequence of the dissemination of Candida
species, as was observed in a kidney transplant recipient (Vasquez et al., 2002) and an AIDS
patient (Legout et al., 2006). Osteoarticular candidiasis has also been reported in heroin
addicts but was caused by C. albicans (Dupont et al., 1985).
Swelling, tenderness and decreased joint motion are the complaints reported by a patient
with arthritis caused by C. parapsilosis in a previously healthy structure (Chow et al., 2008;
Legout et al., 2006). The response to treatment depends on early diagnosis and initiation of
general care and antifungal compounds. Arthroscopic irrigation and debridement, together
with local and systemic administration of antifungal compounds are indicated (Chow et al.,
2008; Legout et al., 2006).
Arthritis caused by C. parapsilosis in a previously affected joint can be initially
misdiagnosed as being associated with the underlying disease and only subsequently as a
bacterial infection. After general care and antifungal compounds are administered, resection
arthroplasty should be considered. In some cases, subsequent joint instability can require an
amputation (Trofa et al., 2008).

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 Candida Parapsilosis Complex 637

The best results of the treatment of fungal arthritis are observed when medical and
surgical approaches are combined. Open or arthroscopic debridement and drainage, including
open drainage of the hips, are indicated (Pappas et al., 2004).
Administration of amphotericin B deoxycholate (0.5 – 1.0 mg/kg/day) or lipid
formulations of amphotericin B (3.0 – 5.0 mg/kg/day), either combined with flucytosine or
not, for 2 or 3 weeks, followed by fluconazole (6 – 12 mg/kg/day) for a total of 6 – 12 months
is indicated for the treatment of native joint arthritis (Pappas et al., 2004). Fluconazole was
previously used successfully as the only antifungal compound (Weigl, 2000). Appropriate
synovial concentrations of these antifungal compounds are achieved following parenteral
administration, discouraging their intra-articular injection.
The treatment of Candida infections of prosthetic articulations should follow the
antifungal regimens proposed for infected native joint arthritis. In addition, resection of the
arthroplasty is necessary (Tunkel et al., 1993), and a new prosthesis can later be inserted.
Voriconazole and posaconazole (Denes et al., 2002, Sili et al., 2007) as well as
caspofungin (Cornely et al., 2007; Dumaine et al., 2008) have been used in the treatment of
Candida species that are resistant to fluconazole; however, the experience with these new
compounds is still limited.

8. Otomycosis

Otomycosis is a disease of the middle and/or outer ear characterized by lesions produced
by fungal infection. The isolation of a fungus from these sites opens the discussion regarding
whether its role is pathogenic or commensal. However, patients with chronic inflammation
showing erythema, edema and desquamation of meatal epithelial tissues exhibit resolution of
these signs after treatment with topical antifungal compounds (Vennewald et al., 2003).
Patients with chronic hyperplastic (polipoid) inflammation are especially susceptible to
infection with pathogenic fungi, as the increased production and buildup of mucus favors the
fungal colonization (Vennewald et al., 2003).
C. parapsilosis has been associated with cases of otomycosis in some studies. Two of
these studies showed prevalences of 42.5% and >50.0%, which was superior to those
presented for C. albicans (Garcia-Martos et al., 1993; Vennewald et al., 2003). In 2 other
studies, the prevalences were 27.5% and 23.5%, which was lower than for infection by C.
albicans (Dorko et al., 2004; Martin et al., 2005).
Treatment of otomycosis includes general measures, such as intense debridement and
cleansing, in combination with the application of topical antifungal compounds for 7 to 14
days, which is extended to 4 weeks for tympanic membrane infections (Vennewald et al.,
2003).

9. Vulvovaginitis

Vulvovaginitis is an infection of the vagina, which also typically involves the vulva
and/or vulvovaginal glands. Candida species are the second most common agents of vaginal
infection, after bacteria (Sobel, 2007). C. albicans remains the most common species
responsible for vulvovaginitis, showing prevalences ranging from 66.2% to 89.3% (median of
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638 D. V. Moris, M. S. C. Melhem, M. A. Martins et al.

68.3%), while C. parapsilosis continues to be infrequent, presenting prevalences ranging

from 1.2% to 8.9% (median of 5.1%) (Otero et al., 1998; Namkinga et al., 2005; Bauters et
al., 2002; Abu-Eteen et al., 1997; Nyirjesy et al., 2005; Richter et al., 2005).
The role of C. parapsilosis as a vulvovaginal pathogen has been discussed because it is
also a commensal yeast. However, the increased secretion of aspartyl proteinases by this
species can compromise vaginal integrity by hydrolyzing mucosal immunoglobulin A, which
is one of the most effective barriers against infection, contributing to the pathogenic capacity
of the fungus (Agatensi et al., 2001). In addition, women infected by C. parapsilosis show
symptomatic relief after clearing the yeast using azole derivatives (Nyirjesy et al., 2005).
Itching (53%), burning (43%), dyspareunia (31.4%) and abnormal discharge (21.6%) are
the main complaints presented by patients with this condition (Nyirjesy et al., 2005).
Uncomplicated vulvovaginitis usually responds to a short course of topical or oral
treatment. Butaconazole, clotrimazole, miconazole, tioconazole, terconazole and econazole
can be applied topically in regimens ranging from 1 to 7 days (Watson et al., 2002; Colombo
et al., 2012). Oral azoles are a safe and efficacious alternative to topical therapy. Fluconazole
at 150 mg once, itraconazole at 200 mg twice daily for 1 day or itraconazole at 200 mg daily
for 3 days is indicated.
Treatment of moderate or severe clinical forms of vulvovaginitis or immunosuppressed
patients (complicated forms) should be carried out for 7 to 14 days using either topical
regimens or oral azoles [either fluconazole at 150 mg once a day every 3 days (2 or 3 doses)
or itraconazole once daily for 3 days] (Colombo et al., 2012). Recurrent vulvovaginitis
requires the eradication of underlying factors as far as possible, followed by the use of
antifungal compounds. The initial treatment can be conducted as for the complicated forms,
and maintenance therapy should be performed with 150 mg fluconazole weekly for 6 months
(Watson et al., 2002; Sobel et al., 2004).

10. Urinary Tract Infection

Urinary tract infection (UTI) is an expression that is applied to a variety of clinical

conditions, ranging from asymptomatic bacteriuria to acute pielonephritis with sepsis.
The prevalence of UTI caused by Candida species varies but is generally very low, for
example, corresponding to 0.9% of 6,281 strains isolated from in-patients at an Italian
hospital (De Francesco et al., 2007). C. parapsilosis was found to be the causative agent for 4
(8.9%) out of the 45 Candida species identified, being outranked by C. albicans, C. tropicalis
and C. glabrata. In a pediatric hospital in São Paulo, Brazil, C. parapsilosis was identified in
only 4% of 100 evaluated cases of candiduria (Silva et al., 2007). These isolates presented
strong proteinase and phospholipase activity. C. parapsilosis was also shown to be the
causative agent of a renal fungal ball that was not eliminated by amphotericin B but was
cured with fluconazole (Weintrub et al., 1994).
Therapeutic recommendations are related to four scenarios in patients (Sandven et al.,
2002; Colombo et al., 2012). First, in patients with no previous risk factor for candiduria,
such as individuals with no underlying diseases, no previous use of antibiotics or
corticosteroids, and who have not been subjected to catheterization, a new specimen of urine
should be evaluated. If the Candida infection persists, the possibility of a mucosite should be
investigated. Second, in patients with a predisposition to candiduria, but with improbable

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 Candida Parapsilosis Complex 639

disseminated candidiasis, such as individuals who were subjected to catheterization or other

risk factors, the risk factors should be removed and the individuals monitored, and patients
with symptomatology compatible with cystitis should receive antifungal compounds. Third,
in patients showing a predisposition to candiduria with probable systemic dissemination, such
as critical patients with risk factors for disseminated infection, invasive candidiasis should be
investigated in serial hemocultures; vesical catheters should be removed; and systemic
therapy must be introduced. Four, in patients showing asymptomatic candiduria and risk
factors (neutropenia, low weight neonates, urologic procedures) treatment should be initiated
and image evaluations should be performed to monitor possible complications. Fluconazole at
200 mg daily, either orally or intravenously, for 7 to 14 days is the regimen of choice. Patients
who show intolerance to fluconazole, are refractory to the treatment or exhibit resistant C.
parapsilosis should be treated with intravenous amphotericin B at 0.3 to 1.0 mg/kg daily for 7
to 14 days, or through vesical irrigation of amphotericin B at 50 mg daily, with continuous
irrigation for 48 to 72 hours. Current experience with voriconazole and echinocandins is very
limited (Pappas et al., 2009).

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Weber K, Sohr R, Schulz B, Fleischhacker M, Ruhnke M. Secretion of E, E-farnesol and
bioWlm formation in eight diferent Candida species. Antimicrob. Agents Chemother
2008; 52:1859–1861.
Weems Jr JJ. Candida parapsilosis: epidemiology, pathogenicity, clinical manifestations, and
antimicrobial susceptibility. Clin. Infect. Dis. 1992; 14:756–766.
Weems Jr JJ, Chamberland ME, Ward J, Willy M, Padhye AA, Solomaon SL. Candida
parapsilosis fungemia associated with parenteral nutrition and contaminated blood
pressure transducers. J. Clin. Microbiol. 1987; 25: 1029 – 32.
Weigl JA. Candida arthritis in a premature infant treated successfully with oral fluconazole
for six months. Ann Acad Med Singapure 2000; 29: 253 – 5.
Weintrub PS, Chapman A, Piecuch R. Renal fungus ball in a premature infant successfully
treated with fluconazole. Pediatr Infect. Dis. J. 1994; 13: 1152 – 4.
Welbel SF, McNeil MM, Kuykendall RJ, Lott TJ, Pramanik A, Silberman R, Oberle AD,
Bland LA, Aguero S, Arduino M, Crow S, Jarvis VR. Candida parapsilosis bloodstream
infections in neonatal intensive care unit patients: epidemiologic and laboratory
confirmation of a common source outbreak. Pediatr Infect Dis. J. 1996; 15: 998 – 1002.
White, T. J., Bruns, T. D., Lee, S. B. and Taylor, J. W. (1990). Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a Guide
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orthopsilosis isolated from blood culture. Mycopathologia 2008; 165:81-7.
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Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 26

ORAL CANDIDIASIS:
CONVENTIONAL AND ALTERNATIVE
TREATMENT OPTIONS

C. E. Vergani, P. V. Sanitá, E. G. O. Mima,

A. C. Pavarina and A. L. Machado
Araraquara Dental School, UNESP – Univ Estadual Paulista,
Araraquara, SP, Brazil

ABSTRACT
Candidiasis is a common opportunistic infection that affects mainly oropharyngeal
and vaginal mucosa, but can also be an invasive systemic and life-threatening disease
(candidemia). Nowadays, disseminated candidiasis is highly associated with mortality,
especially in immunocompromised and hospitalized patients. Among all candidal
infections, oral candidiasis is the most common form and affects especially denture
wearers and severe ill patients, such as those infected by HIV virus, under antibiotic or
chemotherapy, and submitted to organ transplantation. Additionally, candidiasis is of
great clinical importance in patients with the systemic disease Diabetes Mellitus.
Clinically, this superficial infection may be characterized as erythematous lesions or
white patches and, despite the fact that oral candidiasis is frequently asymptomatic,
patients may complain of slight bleeding and swelling in the involved area, mucosal
burning or other painful sensations.
Usually, candidiasis has been treated with topical or systemic antifungal agents, such
as those belonging to polyenes (nystatin, anphotericin B) or azoles, which are divided
into imidazoles (clotrimazole, miconazole, and ketoconazole) and triazoles (fluconazole
and itraconazole). A new class of antifungals, the echinocandins (caspofungin,
micafungin, and anidulafungin), is also clinically available. However, besides the side
effects and high cost, the indiscriminate use of these agents, especially the azoles, has led
to the development of fungal resistance. Although these antifungal drugs are aimed at
treating the infection in the oral mucosa, it is widely known that strict oral hygiene and
denture disinfection measures are crucial to the treatment of oral candidiasis. Thus, to


Corresponding author: Carlos Eduardo Vergani, Email: [email protected]
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overcome the limitations of these standard medications, the search for alternative
therapies has directed the interest to physical methods of denture disinfection. In this
context, the effectiveness of denture microwave disinfection has been demonstrated in
several in vitro and in vivo studies. Another promising modality is the photodynamic
therapy, which combines a photosensitizing agent with light of appropriate wavelength in
the presence of oxygen, resulting in reactive species that are toxic to microbial cells.
Moreover, immunotherapy, natural bioactive molecules, and vaccines have also been
investigated.
Based on the information given above, this book chapter will provide to the readers
relevant and current scientific information about candidiasis. The topics that will be
addressed are: oral candidal infection among healthy subjects, denture wearers, and
immunocompromised patients; the frequent symptoms reported by infected individuals;
and the available conventional and alternative treatment options for this common and
clinically important disease.

ORAL CANDIDAL INFECTION: PREVALENCE,

SYMPTOMS, AND ETIOLOGICAL FACTORS
Candida species, particularly Candida albicans, are the most common etiologic agent of
a large percentage of fungi mediated oral, esophageal, and systemic diseases in human.
Usually, these microorganisms live in a symbiotic relationship with healthy individuals. It
was demonstrated that patients can harbor an abundance of yeasts in the oral cavity, even in
the absence of clinical signs of infection [1]. However, if there are predisposing conditions
related to the host, such as local conditions [2-12] or systemic diseases that lead to
immunossupression [13-18], they can become opportunistic pathogens, invading tissues and
causing infections. Among all candidal infections, oral candidiasis is the most common form
and affects especially elderly individuals [6-9, 11]. The high prevalence of oral candidiasis
does reflect the impact of the epidemic. Epidemiologic data show that this infection has been
reported in about 65% of denture wearer patients [19] and in a high percentage of
immunocompromised individuals [16, 17]. The manifestation of oral candidiasis can occur in
many different forms, including acute pseudomembranous, acute atrophic, chronic
hyperplastic, chronic atrophic (known as denture stomatitis), median rhomboid glossitis, and
angular cheilitis [20]. Among them, denture stomatitis is the most common. According to the
criteria proposed by Newton [21], denture stomatitis is clinically classified in type I, petechiae
dispersed throughout all or any part of palatal mucosa in contact with the denture (localized
simple inflammation); type II, macular erythema without hyperplasia (generalized simple
inflammation); and type III, diffuse or generalized erythema with papillary hyperplasia
(inflammatory papillary hyperplasia). Often, this infection is symptomless, however, patients
may complain of halitosis, slight bleeding, and swelling in the involved area, mucosal
burning or other painful sensations, dryness in the mouth, and taste alterations (dysgeusia)
[22].
There are a number of local and host factors that are known to predispose oral
candidiasis. Among the local factors, the use of removable total or partial prosthesis and
subsequently biofilm formation on epithelial surfaces and prosthetic devices is critical in the
development of oral candidiasis [2-4, 10]. Candidal adherence to surfaces is a crucial first
step in the initiation and propagation of oral candidiasis [23, 24] and, since Candida spp. have

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the ability to adhere to the denture tissue surface, dentures may act as reservoirs that harbor
microorganisms, enhancing their infective potential [6-9, 11, 25, 26]. When Candida species
are accumulated in its pathogenic form (pseudohyphae and hyphae) in the denture surface, an
intense immunological and inflammatory response is observed [27]. The presence of a
removable denture in the oral cavity also decreases the salivary pH [28] and saliva flow rate
[29], and impedes the mechanical cleaning of the soft tissue surfaces by the tongue [27]. In
addition, denture induced trauma may reduce tissue resistance against infection because of
increasing the permeability of the epithelium to soluble candidal antigens and toxins [28].
There are other important risk factors that may predispose the onset and progression of
oral candidiasis. Among them, age, female gender, and the age of the dentures can be cited.
Clinical studies that evaluated different groups of patients with oral candidiasis showed that
the mean age of these patients was higher than 60 years [7-9, 11, 30, 31]. In fact, it has been
proposed that older patients had an increased risk of yeast infection [3] due to the greater
number of denture wearers from the sixth decade of life [4, 13, 32]. In addition, it has been
found that elderly women presented more oral lesions than men [3, 5, 9, 11, 12, 31, 32]. The
hormonal factor and the great incidence of iron deficiency in women could be responsible for
this increased risk in women [5, 25]. It has also been suggested that the higher prevalence in
women may be due to the fact that female patients wear their dentures more often and perhaps
for longer periods of time for esthetic purposes [4]. The age of dentures has also been related
to the occurrence of oral candidiasis [4, 5, 8, 9, 11, 12]. Tissue trauma, frequently detected in
patients with poorly fitting dentures and non-balanced occlusion, can affect the occurrence of
this infection [5]. Old dentures are also more difficult to keep clean because of the greater
tendency to porosities in the denture base [33], favoring Candida colonization. It was
observed that only 25% of individuals using dentures for less than one year were diagnosed
with denture stomatitis, while more than 84% of those using dentures for more than 5 years
had the disease [12].
Further than these local factors, tobacco smoking, dry mouth complaint or xerostomia,
nocturnal wear of the dentures, and poor hygiene habits have also been found to be important
etiological factors in oral candidiasis [3, 8, 9, 11, 29, 34]. Tobacco smoking associated with
denture friction on the oral mucosa alters the mucosal surface, leading to contamination by
Candida spp. [34]. Clinical studies found that tobacco smoking is associated not only to an
increased frequency of oral candidiasis [10], but also to the severity of the infections [35].
Salivary secretion plays a significant role in oral mucosa immunity due to its physiological
functions [29]. Moreover, studies have shown that saliva reduces the adhesion of C. albicans
to acrylic [36]. Thus, an inadequate salivary production, which is frequently observed in oral
candidiasis patients [9], may favor the colonization by Candida spp. [37] and the
development of numerous oral and pharyngeal disorders, such as oral candidiasis [3]. It is
also common that oral candidiasis patients have the habit of using their denture during sleep
[9,38], thus facilitating the disease process [10]. In fact, continuous denture wearing might
cause this infection by increasing the local injury [39], but it might also cause an increase in
the time of mucosal exposure to denture biofilm [40]. It has been shown that edentulous
patients who wear the dentures during sleep showed an increase in the density of C. albicans
on the fitting surface of maxillary dentures [22] and in the severity of the infection [35]. Poor
denture hygiene is another undesirable habit observed among denture wearers [5, 9, 41] and it
is frequently cited as a relevant local etiological factor for oral candidiasis [40]. Some clinical
studies demonstrated a higher frequency of oral candidiasis in patients who did not clean their
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658 C. E. Vergani, P. V. Sanitá, E. G. O. Mima et al.

dentures properly [2, 38]. In addition, when an improved oral hygiene protocol was adopted
by denture wearers, an overall decrease in Candida spp. colonization and a significant
reduction in the number of patients with candidiasis were observed [42].
When considering the host systemic factors, oral candidiasis frequently affects
immunocompromised patients, such as those infected by HIV virus, under antibiotic or
chemotherapy, and submitted to organ transplantation [14-18]. In AIDS patients, the body
defenses are depressed, particularly the cellular immune response. Under these conditions,
yeasts can quickly change from friendly commensals to harmful pathogens [18].
Oropharyngeal candidiasis has been considered a predictor of HIV infection and
immunossupression and it is associated with CD4+ T-lymphocytes count less than 200
cells/μL (< 15%) [43, 44]. However, some studies have demonstrated that HIV viral load is a
more important factor for developing oropharyngeal candidiasis than the CD4+ cell count
[44, 45]. Fortunately, the incidence of candidiasis in HIV-infected subjects has declined with
antiretroviral therapy, but it remains high in patients with limited resources or with poor
immunologic response and resistance to HIV drugs [43, 44]. Additionally, patients who
undergo organ transplantation are exposed to an intensified immunosuppressive regimen and,
consequently, to a lifetime of chronic immunosupression [14]. This occurs because these
patients are treated with immunosuppressive drugs, which also hindrance the immunologic
defense mechanisms, including defense against mycosists [15]. In fact, investigators who
compared the prevalence of Candida infections between denture wearer patients submitted or
not to organ transplantation showed higher infection rates among the immunocompromised
individuals [14]. This local fungal infection also has a high prevalence among patients with
Diabetes Mellitus, which has been considered a global public health problem [46]. It has been
estimated that the number of adults with diabetes worldwide is expected to increase to 300
million in the next 15 years [46]. Besides damaging many organs and systems in the body
[47], the consequences of diabetes are strongly associated with several local alterations in the
oral mucosa, and, in this context, oral candidiasis is of great clinical importance [48]. Diabetic
patients are more susceptible to fungal infections [13] and show a higher prevalence of
Candida colonization in the oral cavity compared with non-diabetic individuals [13, 30-32,
49]. There are several mechanisms that predispose the diabetics to fungal infections. Salivary
glucose levels in diabetic patients favors yeast growth due to increased number of available
receptors for Candida [50, 51]. Consequently, buccal cells from diabetic patients have shown
an increased adherence of C. albicans compared to buccal cells from non-diabetics [52, 53].
The micro-vascular degeneration found in histological examination of diabetic patients may
also predispose to Candida colonization [51], making them more susceptible to infections.
Another host factor which may promote the oral carriage of Candida in diabetics is the
possible defects in candidacidal activity of neutrophils [51, 54], particularly in the presence of
glucose [54]. The reduced salivary flow, associated with diabetes, may also play a role in
Candida colonization and, consequently, in the pathogenesis of oral candidiasis in these
patients [29, 36, 37, 49]. The presence of a denture in the oral cavity, associated with the local
alterations of the oral mucosa and the systemic complications, may render the denture wearer
patients with diabetes even more prone to candidal infection [31, 32, 53]. A significantly
higher incidence of Candida infection and increased levels of Candida spp. were found in
diabetic patients wearing removable denture [31, 32, 53]. It is also important to mention that,
besides the local and systemic host factors that predispose oral candidiasis, several virulence
factors may influence the infective ability of the Candida spp. yeasts. Among them,

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phenotypic switching, filamentation, adhesion to host surfaces (epithelial cells and denture
surfaces), and biofilm formation are considered primordial functions of Candida spp. yeasts
that can be determinant to the onset of oral candidiasis [23, 52, 53, 55]. Phenotypic switching
is an epigenetic conversion from white cells to opaque cells in need for sexual mating [56].
White and opaque cells show differences in cell and colony morphology, metabolism, and
interaction with the host [56, 57]. Another important virulent factor observed in C. albicans is
the ability of filamentation. C. albicans is a polymorphic microorganism, altering between
different morphological types: the yeast and the filamentous (hyphae and pseudohyphae)
forms. The filamentous form is characterized by elongated cells, but hyphae are narrow cells
with parallel walls showing absence of constriction at the septation, while pseudohyphae are
wider cells with constriction [56, 58]. The yeast form is associated with the early steps of
infection whilst filamentous forms are responsible for tissue invasion and deep infection [56,
59]. The ability of Candida to produce extracellular hydrolytic enzymes, such as
phospholipases and secreted aspartyl proteinases, is another important virulence factor [55].
These exoenzymes have an active role in the infection process because they have the potential
to cause the rupture of the epithelial cell membrane and permit the penetration of the fungi
cell into the cytoplasm [55, 60-62]. Another important virulence factor that can be related to
the onset, development, or recurrence of oral candidiasis is antifungal resistance. Clinically,
antifungal resistance can be defined as persistence or progression of an infection despite
appropriate antimicrobial therapy [63, 64]. In terms of laboratory setting, antifungal resistance
is defined as the highest concentration of drug required to inhibit the pathogen growth, which
is measured by standard protocols known as minimal inhibitory concentration (MIC) [56, 63,
65]. According to the literature, antifungal resistance is commonly related to the uncontrolled
prescription of medications, especially azoles. Another aspect related to antifungal resistance
and infection recurrence is the ability of Candida spp. to form biofilms on surfaces [64, 66,
67]. A biofilm has been defined as a community of microorganisms organized at interfaces,
enclosed in a self-produced polymeric matrix and adhered to an inert or living tissue [23]. The
presence of an exopolymeric matrix couple with the organization of layers of cells may confer
protection to organisms in the inner layers contributing to antifungal resistance [66].
Together, all these virulence factors determine the pathogenicity of the Candida yeasts
and their ability to cause infections. C. albicans is considered the most virulent and pervasive
of all the Candida spp. [55], which is the reason for its pre-eminent position in the hierarchy
of prevalence [6, 8, 9, 11, 13, 52, 68]. It has been shown that this Candida species was
isolated from more than 90% of patients with oral candidiasis [8]. C. albicans expresses
several virulence factors that contribute to its pathogenesis and very high prevalence. These
factors include host recognition biomolecules (adhesins), morphogenesis (the reversible
transition between unicellular yeast cells and filamentous, growth forms), and aspartyl
proteinases and phospholipases production [55, 69]. Phenotypic switching is accompanied by
changes in antigen expression, colony morphology, and tissue affinities in C. albicans, which
might provide cells with a flexibility that results in the adaptation of the organism to the
hostile conditions imposed by the host and treatment modality [55]. In addition, C. albicans
has the ability to adhere to mucosal and denture surfaces [6-9, 11, 25, 26, 68], which is
considered as the first step in the pathogenesis of oral candidiasis.
Although C. albicans is the major pathogen, infections with species other than C.
albicans, notably C. glabrata and C. tropicalis, have been increasingly described, both in
compromised and non-compromised hosts [6, 8, 9, 11, 26, 68]. Moreover, studies of
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epidemiologic surveillance have showed that the proportion of non-albicans species is

increasing among the HIV-infected patients [70] and diabetics [8, 9]. In fact, recent studies
showed that both, C. glabrata and C. tropicalis, were frequently isolated from diabetics and
non-diabetics with oral candidiasis [6, 8, 9, 11, 68]. In addition, both C. tropicalis [8] and C.
glabrata [68] were associated with more severe oral infections and has demonstrated to
display higher potential for dissemination and mortality rates than C. albicans and other
species [71-73]. The reason for this epidemiological change is not clear, although the reduced
susceptibility of this species to commonly used antifungal agents, such as fluconazole, may
have led to their selection [71]. Some virulence factors associated with these non-albicans
Candida species could also be responsible for this shift. Different non-albicans Candida
species strains have demonstrated a high cell-surface hydrophobicity, which is involved in the
adherence of microorganisms to different surfaces and considered an important pathogenic
attribute of yeasts [74, 75]. It has been demonstrated that C. tropicalis and C. glabrata
obtained from the oral cavity of denture wearers with denture stomatitis were more adherent
to buccal ephitelial cells than those obtained from patients without signs of disease [76].
Other important virulence factor of these species is its capability to produce degradative
enzymes, such as phospholipase [77] and proteinase [77, 78], which are directly correlated to
the invasion and destruction of host tissue [55, 60-62]. The ability of these non-albicans
Candida species to form biofilm on different surfaces [6-9, 11, 23, 26, 67, 79, 80] is another
potential virulence trait which is related to both, the onset of infection and the increased
resistance to antifungal treatment [67, 81, 82]. It has also been demonstrated that, in general,
non-albicans Candida species are less susceptible to antifungals than C. albicans [83, 84]. In
addition, clinical isolates of Candida from HIV positive [85, 86] and diabetic patients [87, 88]
showed a higher resistance to antifungals than those from individuals without systemic
complications. Understanding the mechanisms involved in the etiology of oral candidiasis is
crucial to determine strategies for prevention, control, and treatment of this type of fungal
infection. Despite the fact that oral candidiasis is a superficial infection, if left untreated, it
may has the potential to contribute to the dissemination of infection through the bloodstream
or upper gastrointestinal tract. Candida spp. within biofilms on the dentures can be released
into the oral fluids and aspirated into the lower respiratory tract, thus causing systemic
infections such as fungemias, severe infections with significant morbidity and mortality rates
[72, 73]. In fact, nowadays, disseminated candidiasis is highly associated with mortality,
especially in immunocompromised and hospitalized patients [71-73].

CONVENTIONAL AND ALTERNATIVE TREATMENT OPTIONS

FOR ORAL CANDIDIASIS

Conventional Treatments for Oral Candidiasis

Antifungal Agents
The conventional treatment of candidal infection involves the treatment of the oral
mucosa by means of the administration of topic or systemic antifungal agents. Topic agents
are selected for superficial infections, while systemic drugs are usually administrated for
invasive and recurrent infections. In general, topical application of antifungal solutions is

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indicated before systemic drugs are used. These antifungal agents are divided in three
different classes: polyenes (nystatin and amphotericin B), azoles that include imidazoles
(clotrimazole, miconazole, and ketoconazole) and triazoles (fluconazole and itraconazole),
and echinocandins (caspofungin, micafungin, and anidulafungin).
Polyenes were the first antifungal agents available for clinical use since 1950, and are
still the first choice for treating oral candidiasis [89]. They bind strongly to ergosterol, the
main sterol compound of fungal membrane [90], promoting membrane channel [91-93] and
leakage of cellular ions [93]. These changes would not only reduce the ability of candidal
adhesion to buccal epithelial cells [92] and denture acrylic surfaces [91], but also supress
active budding and multiplication [91, 92]. Further, polyenes can perturb germ tube formation
[94], modulate the cell surface hydrophobicity [95], and supress the proteolitic activity of
Candida [96]. It is important to highlight that the polyenes has been widely used for the
treatment of oral and disseminated candidiasis [20, 90, 97] and, despite this, Candida
resistance is rare. Nonetheless, polyenes showed high renal toxicity, especially amphotericin
B, that was the only drug available for managing serious fungal infection for years [90,98].
The similarity between ergosterol and cholesterol in the human cell membrane has been
attributed to the nephrotoxicity of polyenes [56, 99]. The lack of a topical preparation for
dental use and the limited tolerance of the oral-rinse product [97] may also limit its clinical
application. In an attempt to overcome these shortcomings, new formulations of these drugs
have been developed, such as those based on lipid-complexes polyenes [56, 99]. There are
several studies that evaluated the effectiveness of polyenes to treat oral candidiasis and the
most commonly used is nystatin [9, 11, 25, 97, 100, 101]. Some of these investigations found
that treatment with nystatin in a daily basis reduced the numbers of Candida on cultures from
the palates and dentures of patients with oral candidiasis and the clinical signs of the disease
[9, 11, 100].
The azoles are the largest class and the most popular antifungal agents. With the
development of triazoles in 1980s it became possible to treat persistent infections to polyenes
[98]. Concerning the treatment of oral candidiasis, studies that used systemic azoles indicated
fluconazole as the first choice drug [102, 103]. It was verified that a systemic approach with
fluconazole (50 mg once a day for 14 days) in conjunction with topical treatments (hexetidine
mouthrinses [102] or denture hygiene with chlorhexidine solution [103]) improved the palatal
inflammation and decreases candidal colonization from saliva, dentures, and palates. There
are also reports of the use of topical azoles, such as clotrimazole and miconazole, in the
treatment of this local infection [7, 104-106]. The azoles inhibit the biosynthesis of ergosterol
and change the fungal membrane permeability [56, 99]. The azoles also have the ability to
decrease candidal adhesion to buccal epithelial cells [76, 95] and denture acrylic surfaces
[91], and the production of phospholipase, an enzyme that plays an important role in the
tissue invasion process and, consequently, in the pathogenicity of Candida spp. [55] Unlike
other azoles drugs, fluconazole has distinctive features, since it is very well absorbed in
gastrointestinal tract, reach many sites in the body, and is excreted by kidneys, which avoid
the possibility of hepatotoxic effect [89]. However, the efficacy and safety of azoles,
especially fluconazole, have expanded their clinical use for both the treatment of fungal
infections, mainly in HIV-infected subjects, and as prophylactic agent for high-risk patients.
This widespread use and the fungistatic effect of azoles have favored the development of
resistance in Candida spp. [56, 90, 99]. Differences in the in vitro susceptibility to azoles
have been reported in the literature, ranging from 70% to 100% of oral isolates, depending on
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662 C. E. Vergani, P. V. Sanitá, E. G. O. Mima et al.

the groups of patients studied [70, 77, 78, 84, 87, 88, 107-109]. In fact, the higher levels of
resistance were verified in Candida isolates from immunocompromised patients, such as
those HIV positive [70] or with advanced cancer [107].
Echinocandins are the newest class of antifungal agents and the first drugs that act
specifically on fungal cells, since they inhibit the biosynthesis of (1,3)β-D-glucan
polysaccharides [110], a component present in fungal cell wall, but not in mammalian cells
[56, 98, 99]. Thus, due to this high specificity, a high safety has been reported. It is also
important to mention that these drugs, despite expensive, are well tolerated by the patients
and have few drug-drug interactions [111]. Echinocandins also showed ability to reduce the
adhesion of Candida spp. to human cells by about 40-90%, depending on time of exposure
and drug concentration [112]. Considering the importance of Candida adhesion to buccal
cells during the infectious process, this is another relevant mechanism of action of these
medications. Anidulafungin has been recommended for severe ill patients with invasive
candidiasis, such as candidemia, due to its higher efficacy compared with fluconazole [113].
Nonetheless, due to their short time in clinical use, no longitudinal study is available and
resistance of Candida spp. to echinocandins has been reported [56, 63]. Caspofungin has
demonstrated effectiveness for the treatment of esophageal and invasive candidiasis [111,
114], including those in HIV positive patients [115, 116]. There are also some reports
concerning its efficacy against fluconazole resistant Candida [117] and to several other
species, including C. albicans, C. tropicalis, C. glabrata, and C. krusei [77]. Nonetheless,
echinocandins are only available as intravenous formulations [44] and thus they are not the
first choice for oral candidiasis.
Although the variety and the effectiveness of topical and systemic antifungals in
alleviating the symptoms and signs of oral candidiasis, there are several disadvantages
associated with the use of these agents. The major disadvantage is the development of fungal
resistance. Microorganisms may exhibit primary (intrinsic) resistance or secondary
(developed) resistance. Microorganisms show primary resistance when they are resistant to a
drug before being exposed to the drug, which is observed in C. glabrata and C. krusei in
relation to fluconazole [56, 64, 118]. Secondary resistance is developed in response to
exposure to an antimicrobial agent over long periods [64], as observed for C. albicans [56,
64, 118]. While resistance to polyenes is rare, several mechanisms of resistance to azoles
have been proposed, such as alterations of drug target, overexpression of drug transporters
(efflux pumps), cellular stress response pathways, and also mechanisms associated with
biofilms [56, 63, 64]. Additionally, other mechanisms of secondary resistance have been
described in the literature. Goldman et al. [119] identified four mutations previously
described and fourteen novel mutations in fluconazole-resistant isolates of C. albicans
obtained from AIDS patients. Replacement of C. albicans by C. dubliniensis has been
documented in a considerable number (27%) of patients treated with fluconazole who failed
to develop fluconazole-resistent C. albicans [120]. Hunter et al. [86] found that exposure to
fluconazole provides a positive selection pressure for non-albicans yeasts, described as a
replacement of fluconazole-susceptible C. albicans strains with other species that are
intrinsically less fluconazole sensitive – for example C. glabrata and C. krusei.
Besides antifungal resistance, there are other problems related to the use of these
medications. The recurrence of infection shortly after treatment has been frequently observed
[7, 9, 11, 25, 100, 101, 103] and is attributed to re-emergence of the original infecting strain
[121, 122]. The topical agents may reach transient response due to the action of the

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surrounding musculature or due to the presence of local secretions, such as saliva, which may
reduce the concentrations of the drug to subtherapeutic levels [89]. In addition, topical agents
frequently require multiples doses and their taste may cause nausea, which can lower the
patient’s compliance [101]. The systemic agents may be toxicity to liver and kidney [98,
123]. Thus, these medications must be administered with caution. Moreover, while antifungal
drugs are aimed at treating the oral mucosa, they do not erradicate the Candida that colonizes
the denture [7, 9, 11, 25]. Considering all the above and the fact that removable prostheses
may be the principal Candida source of oral candidal infection, there are conventional
treatments of this fungal infection based on the concept of denture disinfection.

Denture Hygiene
For a long time, the maintenance of health includes a proper denture hygiene by means of
brushing it with a soft toothbrush and soap [123-125] or a disinfectant solution [127-129].
Soaking the dentures in chemical agents have also shown to be a effective procedure in
decreasing the number of contaminating organisms [129-133] and to treat oral candidiasis
[25, 26, 103, 134].
The literature shows that mechanical plaque removal by means of a toothbrush is
considered the most common method of controlling plaque development [135] and
maintaining denture hygiene [128, 129, 136]. It has been suggested that the mechanical action
provided by the direct contact between the bristle tips and the accumulated biofilm and the
hydrodynamic shear forces of the fluid flow during brushing [137] are the main factors in the
biofilm-removing process. Under these conditions, biofilm may be mechanically disrupted
from the acrylic resin surface. Corroborating this observation, an in vitro study showed at
least 96% reduction on the viability of a 48h C. albicans biofilm on acrylic disks after
brushing it with a toothbrush and water or dentifrice [129]. Besides the mechanical action of
the toothbrush, this could also be attributed to the additional antimicrobial effect of the
dentifrice, which commonly contains sodium monofluorophosphate (1450 ppm) and sodium
lauryl sulphate. Fluorides have demonstrated some antimicrobial effects, such as metabolic
interference and reduction of biofilm acidogenicity [138-140]. Dentifrices detergents like
sodium lauryl sulphate have a variety of functions, including the removal of organic material
on the tooth surface, antimicrobial effects, and a moderate biofilm inhibitory action [141-
143]. However, in the study of Paraskevas et al. [128], brushing with water or dentifrice
showed a lower reduction (50%) on the viability of a more complex in vivo mature biofilm
[23]. Thus, it can be suggested that, when a more complex biofilm is present, which is very
common in the dentures of oral candidiasis patients [6, 8, 9, 11], the use of an antimicrobial
cleansing agent in association to the brushing method must be necessary. In fact, evidences
from clinical studies that evaluated the efficacy of denture hygiene alone in the treatment of
oral candidiasis indicated that scrubbing the dentures with coconut soap for 15 [124] or 30 [7]
days had no significant effect on the inflammation severity of the palatal mucosa of the
patients. In addition, this treatment did not reduce the proportion of mycelial forms and
density of Candida from the palates and dentures of these patients [7]. Thus, denture hygiene,
by itself, may not be enough to treat oral candidiasis.
There is also another problem related to the use of brushing with dentifrice for denture
hygiene. The literature contains substantial data about the adverse effects of this method on
acrylic resins and artificial teeth [144-147]. The friction between the inorganic phosphate and
sulphate contents and the denture surface during brushing may result in severe damage on
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664 C. E. Vergani, P. V. Sanitá, E. G. O. Mima et al.

acrylic materials, such as wear (weight loss) of denture base resins and artificial teeth, with
increase in roughness [144-148]. Surface roughness is known to be a factor in the entrapment
of microorganisms on surfaces and their protection from shear forces, which can result in a
higher propensity for Candida colonization and biofilm accumulation [149-150]. Thus, the
use of less or non-abrasive cleansing agents are recommended to overcome this disadvantage
of the dentifrices and to improve the effectiveness against Candida spp. In this context,
several chemical agents have been proposed. Among them, sodium hypochlorite and
chlorhexidine gluconate are the most widely used [129-131, 151-153]. For denture
disinfection, they can be used either for denture soaking or in association to a brushing
method.
Sodium hypochlorite solution has been intensely used in dentistry as a denture cleanser
[129-132, 151-154]. There are also clinical investigations that evaluated its efficacy in the
treatment of oral candidiasis [26, 124]. The antimicrobial mechanism of action of sodium
hypochlorite has been related to its physicochemical characteristics and its reaction with
organic tissues and microorganisms. Sodium hypochlorite is a strong base (pH>11) and its
high pH alters the integrity of the cytoplasmic membrane by means of either chemical injuries
to organic components and transport of nutrient, or degradation of phospholipids or
unsaturated fatty acids of the cytoplasmic membrane. This causes an irreversible enzymatic
inhibition and biosynthetic alterations in cellular metabolism, resulting in cell death [154].
Besides being bactericidal and fungicidal [129, 130, 151-154], it dissolves mucin and other
organic substances [156]. Due to this broad mode of action, sodium hypochlorite has been
considered useful as denture cleanser solution because it inactivates bacterial plaque and other
microorganisms, including the methicillin-resistant Staphylococcus aureus (MRSA) [129-
131], removes stains, and inhibits calculus formation on dentures [152, 156]. There are
several clinical protocols recommended in the literature concerning the use of sodium
hypochlorite for denture disinfection. Pavarina et al. [131] and Pelizzaro et al. [129]
established a protocol of disinfection in which 10 minutes of immersion at 1% concentration
solution was effective in removing in vivo biofilm from complete dentures [131] and C.
albicans mature biofilm from acrylic resin disks [129]. Other studies found that 4 minutes of
immersion at 0.5% concentration solution achieved complete disinfection of acrylic disks
inoculated with C. albicans and other bacteria [151, 152]. The studies in which this chemical
agent is proposed for the treatment of oral candidiasis used different regimens. In the study of
Weeb et al. [26], patients had their complete dentures immersed in 0.02% sodium
hypochlorite during night for 1 week. By contrast, Barnabé et al. [124] used a protocol in
which the patients’ dentures were soaked in 0.05% sodium hypochlorite for 10 minutes for 15
days. In the latter study, this disinfection procedure was made in association with brushing the
dentures with coconut soap [124]. It is important to mention that both clinical studies
demonstrated that the use of sodium hypochlorite was effective to reduce the clinical signs of
oral candidiasis in the patients’ palatal mucosa [26, 124]. Brushing with a 1% solution also
proved to be very effective against C. albicans mature biofilms, since a complete reduction
(100%) on its viability was observed with this method [129].
Despite the antimicrobial effect of sodium hypochlorite, some problems have been
related to the use of this chemical solution. It has been frequently related to corrosion of the
metal parts of dentures [156, 157], bleaching of denture acrylic resin [156, 157], and some
other detrimental effects on acrylic materials. A decrease in hardness and increase in
roughness were observed after immersion of acrylic materials in 2% sodium hypochlorite for

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5 minutes [158-159]. A lower concentration of 1% sodium hypochlorite solutions for a longer

period of time (8 hours) also influenced the color stability and flexural strength of acrylic
resin [160]. By contrast, no significant changes in hardness and roughness of acrylic resins
were observed when the 1% concentration was used for 10 minutes [161]. Furthermore,
unpleasant taste and odor of sodium hypochlorite are frequent complains reported by some
patients. It is possible that certain components of the disinfectant solutions may penetrate the
acrylic resin material and not be completely eliminated by rinsing [131]. Consequently, these
components may be unintentionally introduced to the oral cavity, resulting in those
complaints.
Chlorhexidine gluconate is another agent commonly recommended for denture
disinfection. This chemical agent posses a broad spectrum of antimicrobial activity, being
able to eliminate species of Candida and a wide range of bacteria from the genus
Streptococcus, Pseudomonas, Bacillus, Acinetobacter, Escherichia, and Staphylococcus,
including MRSA [130, 152, 153, 162]. In fact, investigators found that immersion of
complete dentures in 4% chlorhexidine gluconate for 10 minutes [131], 0.12% for 20 minutes
[163], and 2% for 5 minutes [163] was satisfactory in controlling oral and C. albicans
biofilms. In an in vitro study conducted by Mima et al. [164], chlorhexidine solutions at 2%,
1%, and 0.2% were effective in disinfecting complete dentures inoculated with fluconazole-
resistant C. albicans after 10 minutes of immersion. The concentration of 2% was the most
effective, since it resulted in the highest number of dentures without fungal growth after 7
days [164]. Evidences from clinical studies also demonstrated the effectiveness of
chlorhexidine gluconate to treat patients with oral candidiasis [25, 103, 134, 165]. Uludamar
et al. [134] instructed their oral candidiasis patients to mouthrinse with 0.2% chlorhexidine
gluconate twice daily for 1 minute and soak their dentures overnight in the solution for 15
days. The authors observed that this protocol was effective for the management of the
infection. In other studies, rinsing 0.2% chlorhexidine gluconate 4 times a day [165] or
soaking denture overnight in the solution [25] were effective as a co-adjunct to nystatin to
treat patients with oral candidiasis. This solution was also effective to improve the palatal
inflammation and decrease the candidal colonization from dentures of oral candidiasis
patients when used at 2% concentration in the inner surface of the dentures twice a day in
association with fluconazole for 2 weeks [103]. As a co-adjunct treatment for oral candidiasis,
brushing the patients’ dentures with a 2% chlorhexidine gluconate should also be an effective
alternative, since it demonstrated to completely inactivate mature C. albicans biofilms [129].
The effect of this solution can be attributed mainly to its chemical mechanism of action
against the fungal cell. MacNeill et al. [166] observed that, after the contact with
chlorhexidine gluconate, Candida cells exhibited a severe cytoplasmic degeneration
(fragmentation and clumping of the contents, vacuolization, lipid accumulation, and
condensation) and fragmentation and desquamation of the cell wall, resulting in cell death.
As for sodium hypochlorite, there are some problems related to the use of chlorhexidine
gluconate solution. The protocol of disinfection that used 10 minutes of immersion at 4%
concentration solution [131] has shown to negatively affect the hardness and roughness of
acrylic resins [158-159]. Considering that the deleterious effects on acrylic resins are affected
by concentration and exposure time, the protocols that showed the antimicrobial efficacy with
lower concentrations and periods should be used [25, 103, 129, 130, 134, 163, 165]. The use
of chlorhexidine gluconate has been also limited by some side effects that could affect the
patients’ compliance. Discolorations of the tongue [167] and natural teeth [168] have been
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666 C. E. Vergani, P. V. Sanitá, E. G. O. Mima et al.

reported after prolonged use (1 to 6 months). Moreover, its bitter taste can reduce the
patients’ compliance [168]. Such disadvantages are clearly a concern, since a disinfection
method should be effective without having any detrimental effect on denture materials or
discomfort and side-effects to the patients.
Given all of the above, it can be stated that, when selecting a disinfection procedure,
besides the antimicrobial effect, its effect on the denture must be carefully considered. In
addition, the fact that fungal resistance is emerging alarmingly and surpassing the
development of new specific-drugs [56, 169], the search for new therapeutic modalities for
control and management of candidal infections is an important challenge.

Alternative Treatment Options for Oral Candidiasis

Efforts have been devoted to investigate alternative therapies for oral candidiasis.
Considering the fact that removable prostheses are potential sources of oral infection [6-9, 11,
25, 26, 68], physical methods of denture disinfection, such as microwave irradiation [1, 7, 9,
11, 25, 26, 130, 170-172] and photodynamic therapy [6, 173, 174], have proven to be very
effective alternatives. More recently, adjunctive modalities, such as immunotherapy [98,
175], natural bioactive molecules [176], and vaccines [98, 169, 177] have also been reported.

Microwave Irradiation for Denture Disinfection and Oral Candidiasis Treatment

Microwave irradiation is as an effective, simple, fast, safe, and inexpensive method for
prosthesis disinfection, since requires only a domestic microwave oven and water. In
addition, it was also demonstrated that microwave energy can be a more effective method of
inactivating microorganisms on dentures than soaking it in sodium hypochlorite [19].
However, as for the chemical disinfectants, when a denture is microwaved, an important
concern to be taken into account is the mechanical properties of denture base materials, such
as dimensional stability of acrylic resin. Thus, in the course of time, several regimens have
been advocated.
In the context of denture microwave disinfection, the first studies were performed in
order to demonstrate the effectiveness of microwave irradiation in inactivating
microorganisms adhered to complete dentures [19, 178, 179]. Rohrer and Bulard [178], in
1985, verified that 15, 10, and 8 minutes of microwave irradiation at 720W sterilized acrylic
dentures contaminated with C. albicans suspension. Nevertheless, Thomas and Webb [179]
observed that microwave energy for 10 minutes can produce unacceptable dimensional
changes in complete dentures. Silva et al. [172] then observed that complete dentures
contaminated with individual suspensions of C. albicans showed sterilization after 6 minutes
of microwave irradiation at 650W. Furthermore, this protocol of microwave irradiation has
been evaluated as a method for eradicating C. albicans from the surfaces of hard and soft
chairside reline resin [180-181]. Despite the effectiveness, it has been observed that this
procedure decreased the flexural strength of a hard chairside reline resin [182] and the surface
hardness of 5 brands of acrylic resin denture teeth [183]. The results from another study
showed that the linear dimensional changes of a denture base material significantly increased
after microwave disinfection for 6 minutes, with values ranging from 0.98% to 1.43% [184].
Shrinkage of such a degree could probably cause pressure on the supporting tissues and thus
discomfort to the patient [185].

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It is likely that the deleterious effects on acrylic resins at medium setting are slighter as
the exposure time decreases. Therefore, reduced times of microwave irradiation for denture
disinfection have been investigated. Sterilization of wet acrylic resin specimens inoculated
with individual suspensions of four microorganisms, including C. albicans and other bacteria,
was achieved after 3 minutes at 650W of microwave irradiation [180]. Similarly, three
minutes of irradiation at 650W was also effective in the disinfection of dentures contaminated
by different species of Candida, including the intrinsically resistant C. glabrata and C. krusei,
and other bacteria [170-172]. An in vivo study also showed that this microwave protocol
inactivated the denture biofilm of 30 individuals [1]. Considering the positive results, several
investigations evaluated the effect of this protocol of microwave disinfection on the
mechanical properties of acrylic resins. Ribeiro et al. [186] demonstrated that the flexural
strength and hardness of different acrylic resin specimens were not detrimentally affected by
irradiation with microwaves for 3 minutes in a wet condition. It had also no effect on the
hardness of acrylic resin denture teeth [183] and on the dimensional stability [187] and
porosity [188] of the denture base materials. Therefore, from these studies, an effective,
drugless, and safe protocol for the treatment and prevention of oral candidiasis was
established.
Banting and Hill [25] conducted the first study that evaluated the effectiveness of
microwave energy for denture disinfection as a co-adjuvant to topical nystatin in the
treatment of oral candidiasis. They observed that disinfection of dentures for one minute
irradiation at 850W, three times, for 14 days, reduced the clinical signs of infection when
compared to disinfection with chlorhexidine. These findings are in agreement with those
found by Webb et al. a few years later [26], who observed no differences between microwave
irradiation (10 minutes at 350W) and 0.02% sodium hypochlorite for denture disinfection in a
daily basis during 1 week. The authors stated that these procedures reduced Candida spp.
counts on dentures and palates and also improved palatal inflammation. A more recent study
conducted by Neppelenbroek et al. [7] also evaluated the effectiveness of dentures microwave
disinfection in the treatment of patients with oral candidiasis. The authors immersed the
dentures in water during microwave irradiation for 6 minutes at 650W in order to improve the
disinfection. In agreement to Banting and Hill [25] and Webb et al. [26], they observed that
denture microwave disinfection was effective for the treatment of oral candidiasis [7]. These
authors found that this disinfection procedure resulted in a significant reduction of Candida
spp. density from the dentures and palates of the patients when compared to the treatment
with topical miconazole and improved the signs of palatal inflammation. Another interesting
finding observed is that the risks of re-infestation of the denture tissue surface by the invasive
form of Candida (mycelial) and re-infection of the adjacent soft tissue were dramatically
reduced for patients whose dentures were microwaved [7, 25]. These studies then provided
the baseline for controlling of microorganisms and candidal infection.
Considering the positive in vitro results of the 3 minutes at 650W protocol for denture
disinfection and that it did not affect the mechanical properties of the denture materials, two
recent investigations also tested this regime to treat oral candidiasis patients. In addition,
since there is a relevant etiological relationship between systemic status and fungal infection,
this treatment was evaluated in healthy individuals [11] and in those with well-controlled type
2 Diabetes Mellitus [9]. The investigations compared the effect of denture microwave
disinfection, one [11] or three times a week [9, 11], for 14 days, to the effect of topical
application of nystatin. When dentures were microwaved 3 times a week, a significant
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668 C. E. Vergani, P. V. Sanitá, E. G. O. Mima et al.

reduction in Candida spp. from the dentures and palates and an improvement of the palatal
inflammation were verified for diabetics and non-diabetics [9, 11]. Also, there were no
significant differences in the number of cured patients at the end of the treatments with
microwave energy and nystatin [9, 11]. Moreover, denture disinfection in a reduced
frequency (once a week) also showed similar results when compared to the 3 times a week
frequency and to treatment with topical nystatin [11]. Hence, both investigations concluded
that microwaving dentures was as effective as nystatin for treating denture stomatitis of
diabetic and non-diabetic individuals.
Unlike drugs, microwave irradiation is a physical method for prosthesis disinfection and
its lethal action is well established in the literature [1, 6-9, 11, 170-172, 180, 181]. Its
mechanism of destruction is not completely understood, however, the lethal effects of such
radiation have been attributed to a combination of effects. Some investigators stated that the
extremely elevated internal temperatures produced by the vibration of water molecules of the
microbial cells, when they are exposed to microwaves, is responsible for the changes in cell
morphology and cell disintegration [189]. In addition, depending on the composition and
volume of their surrounding medium, the cells may be selectively heated by microwave
irradiation [190]. Others believe that non-thermal mechanisms are also involved [190, 191]
and that microwaves may cause a mechanical disruption of the cell wall, due to the
oscillations of the cells in electromagnetic field [191]. A recent study also verified that
microwave irradiation of Candida suspensions produced changes in structural integrity and
permeability of cell membrane and cell metabolism, resulting in cell death [192]. Although
studies demonstrated similar results among denture microwave disinfection and topical
antifungals in the treatment of oral candidiasis, microwave disinfection may provide further
advantages. Antifungals act directly on the oral mucosa, which is often less colonized than the
dentures [8, 9, 11]. There is also the problem related to patient compliance [101]. Further,
investigations have demonstrated that microwave irradiation produces a broad, non-selective
activity against several microorganisms, including several Candida spp. [1, 170, 171],
Staphylococcus aureus [1, 170], including MRSA [130], Pseudomonas aeruginosa [1, 170],
Bacillus subtilis [170], and Escherichia coli [190]. In spite of infection by Candida being
considered the main etiologic factor of oral candidiasis, the presence of other microorganisms
may also be secondarily involved in the pathogenesis of this infection [103, 193-195]. In oral
candidiasis, the bacteria possibly favor the adhesion of blastopores (commensal form of
Candida) to the tissue surfaces of dentures by co-aggregation [195]. With fungal adhesion,
there is an increase in microflora virulence by synergetic interaction, and the blastopores alter
their morphology to mycelial, which results in damage to the epithelial cells and,
consequently, invasion of the buccal tissues [195]. This suggests that the treatment of oral
candidiasis by means of microwave irradiation should simultaneously eliminate the mycelia
Candida and inhibit bacterial growth in the tissue surfaces of dentures [26]. Finally, one of
the most important advantages of microwave irradiation is, given that it is a physical method
of disinfection, the emergence of resistant microorganisms would be avoided.

Photodynamic Therapy for Denture Disinfection and Oral Candidiasis Treatment

Another promising modality for microbial infections is Photodynamic Therapy (also
known as PDT). PDT employs a chemical agent that absorbs light (photosensitizer or PS) and
light of appropriate wavelength (the same of the PS absorption) in the presence of oxygen.
Microbial cells are first treated by a determined concentration of the PS for a specific period

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 Oral Candidiasis 669

of time (pre-irradiation time), in a process called photosensitization, in order to the PS be

bound or taken up by the cell. Following, cells are illuminated by a determined light fluence.
The interaction between the PS and light generates reactive oxygen species, such as singlet
oxygen and other free radicals, which are toxic to the cells [196, 197]. Due to this mechanism
of action, development of resistance to PDT seems to be improbable. PDT was first described
more than 100 years ago when Oscar Raab observed inactivation of Paramecium caudatum
by acridine and sunlight [198], and nowadays it has been successfully employed for treating
cancer. Nevertheless, antimicrobial PDT was forgotten during the last century due to the
discovery of penicillin by Alexander Fleming in 1928 and the following Golden Age of
antibiotics [199]. It was only with the advent of AIDS and opportunistic infections and also
with the development of resistant strains that alternative antimicrobial modalities, including
PDT, began to be extensively investigated. Nowadays antimicrobial PDT is still more an
academic issue than a clinical method, and the first topical application of PDT that was
approved by US FDA was the 5-aminolevulinic acid (ALA), a precursor of protoporphyrin
IX, for the treatment of actinic keratoses in 1999 [199]. In recent decades, several PS were
investigated against bacteria, both Gram-positive and Gram-negative, fungi, and viruses.
It has been demonstrated that fungi are more difficult to photoinactivate than bacteria due
to nuclear membrane that acts as an additional barrier for photosensitization, the greater cell
size, and the reduced number of targets for reactive oxygen species in yeasts [200-202].
Phenothiazinium dyes (toluidine blue O, methylene blue) are often used as PS associated with
laser or light-emitting diodes (LED) for antimicrobial PDT against Candida spp. [203-206].
Munin et al. [207] observed that methylene blue-mediated PDT inhibited the germ-tube
formation of C. albicans and Jackson et al. [66] verified that hyphaes are more susceptible to
PDT than yeasts, since the filamentous form of C. albicans required lower concentrations of
methylene blue than yeasts for photoinactivation. Nonetheless, dyes have the undesirable
effect of staining teeth, lips, tongue, buccal mucosa, and prosthetic devices; thus a nondye PS
would be more suitable for the oral cavity. Porphyrins are the first generation PS widely used
in anticancer PDT. In vitro studies have demonstrated their efficacy in photoinactivating
Candida spp. [208-210] and fluorescent microscopy analyses showed that the cytoplasm
membrane is the target of PDT [210]. Using Photogem and LED light, Dovigo et al. [211]
demonstrated that planktonic cultures of C. albicans, C. tropicalis, and C. dubliniensis were
completely killed by PDT, while for C. krusei only a significant reduction was achieved.
Another investigation verified that fluconazole-resistant C. albicans and C. glabrata strains
were less susceptible to Photogem-mediated PDT than reference strains and that biofilms of
these strains were more resistant than their planktonic counterparts [212]. The association of
Photogem with LED light also resulted in disinfection of dentures inoculated with different
species of Candida in vitro [173].
Although several in vitro investigations have demonstrated the candicidal effect of PDT,
only few in vivo studies are available. Using methylene blue and laser light, complete
photoinactivation of azole-resistant C. albicans in an immunodeficient murine model of oral
candidiasis has been reported [213]. In addition, fewer epithelial alterations and lower
inflammatory response in rats with buccal candidiasis submitted to PDT [214] has been
found. When using Photogem and LED light, PDT promoted significant reduction of C.
albicans in a murine model of oral candidiasis without harming the tongue tissue [215] and
with no toxic effect to rat palatal mucosa [216]. PDT mediated by erytrosine and LED light
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670 C. E. Vergani, P. V. Sanitá, E. G. O. Mima et al.

also resulted in significant reduction of C. albicans in immunossupressed mice and reduced

the adherence of C. albicans to buccal epithelial cells [217].
Despite all these studies with animal models, only recent few clinical investigations are
available. Ribeiro et al. verified effective clinical disinfection of dentures by PDT when
Photogem in suspension or in gel formulation was associated with LED light [218]. Another
study reported successful treatment of five patients with denture stomatitis after six sessions
of PDT [174]. In a randomized clinical trial [6], the effectiveness of PDT was compared with
a more conventional antifungal therapy (topical nystatin) for treating denture stomatitis. In the
PDT group, dentures and palates were subjected to six sessions of PDT, three times per week,
using Photogem as PS and LED light. Both treatments reduced Candida spp. from dentures
and palates, with PDT showing 45% of clinical success in improving palatal inflammation
when compared with 53% of clinical success using nystatin. In terms of recurrence, palatal
inflammation was observed in 75% and 78% of the patients in the nystatin and PDT groups,
respectively, during the follow up [6]. Scwingel et al. [219] reported that one session of PDT
mediated by methylene blue and laser light eradicated colonies of Candida spp. from HIV-
infected patients, decreased the clinical signs of candidiasis, and no recurrence was observed
until 30 days after treatment, while fluconazole for 15 days improved the signs and symptons
of infection but did not prevent recurrence, and no efficacy was achieved with one session of
laser therapy.
Another PS that has recently shown promising outcomes is Curcumin, which is a natural
compound isolated from rhizomes of the Curcuma longa plant and used worldwide as a
cooking spice, flavoring agent, and colorant. It has been demonstrated that Curcumin exhibits
potential therapeutic applications such as anti-inflammatory, antioxidant, antimicrobial,
antifungal [220], and anticancer properties [221]. Curcumin was effective when used as PS
for photoinactivation of Candida spp. biofilms [222-224] and also against C. albicans in a
murine model of oral candidiasis in association with LED light [225]. Other PS that has
exhibit candicidal effects are phtalocyanines [226-228] and nanoparticles [229-230].
Although several PS has been successfully employed for photoinactivation of Candida,
further clinical trials are still required to corroborate outcomes obtained in vitro and from
animal studies.

Adjunctive Modalities for Oral Candidiasis Treatment

Another possibility for treating candidal infection is immunotherapy. In high-risk
patients, antifungal drugs are often ineffective due to impairment of immune system. The
improved understanding of host defense mechanisms against fungal pathogen has encouraged
the development of immunotherapies. Hence, improving host protection by using antibodies
and cytokines has been studied as treatment modality for candidiasis [98, 175]. Nonetheless,
most investigations are restricted to animal models and the few clinical available studies have
evaluated this modality as adjunctive therapy in association with antifungal drugs [98, 175].
Vaccines are also under investigation against Candida using live attenuated strains and
also cell wall antigens and promising outcomes have been mostly reported in preclinical
studies using murine models [98, 169, 177]. However, unlike mice, humans are colonized by
Candida spp., which live in human body as commensals, i.e., without being pathogens.
Additionally, immune system from mice and humans are considerably different. Therefore,
other animal models are required in order to develop effective vaccines for human infections
caused by Candida. Furthermore, Phase I clinical trials are beginning to be tested and

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evaluation of vaccines in humans encounters some obstacles, such as high costs for
developing antigens for clinical trials according to suitable standards of manufacturing and
lack of commercial interest [169, 177].

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[2] Abaci O, Haliki-Uztan A, Ozturk B, Toksavul S, Ulusoy M, Boyacioglu H.
Determining Candida spp. incidence in denture wearers. Mycopathologia. 2010; 169:
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[3] Campisi G, Panzarella V, Matranga D, Calvino F, Pizzo G, Lo Muzio L, Porter S. Risk
factors of oral candidosis: a twofold approach of study by fuzzy logic and traditional
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[4] Coelho CM, Sousa YT, Daré AM. Denture-related oral mucosal lesions in a Brazilian
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[5] Figueiral MH, Azul A, Pinto E, Fonseca PA, Branco FM, Scully C. Denture-related
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[6] Mima EG, Vergani CE, Machado AL, Massucato EM, Colombo AL, Bagnato VS,
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[7] Neppelenbroek KH, Pavarina AC, Palomari Spolidorio DM, Sgavioli Massucato EM,
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[8] Sanitá PV, Pavarina AC, Giampaolo ET, Silva MM, Mima EG, Ribeiro DG, Vergani
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 27

CANDIDA SPP. IN ORAL CAVITY OF

CHILDREN WITH IMMUNODEFICIENCIES

Dorota Olczak-Kowalczyk,1 Maria Roszkowska-Blaim,2

Małgorzata Pańczyk-Tomaszewska,2 Maria Dąbkowska,3
Ewa Swoboda-Kopeć,3 Beta Pyrżak,4
Ewa Krasuska-Sławińska5 and Renata Górska6
1
Department of Pediatric Dentistry,
Warsaw Medical University, Poland
2
Department of Pediatric Nephrology,
Warsaw Medical University, Poland
3
Department of Medical Microbiology,
Warsaw Medical University, Poland
4
Department of Pediatric Endocrinology,
Warsaw Medical University, Poland
5
Dental Surgery Clinic for Children, Warsaw
Children’s Memorial Health Institute, Poland
6
Department of Periodontology and Oral
Medicine, Warsaw Medical University, Poland

ABSTRACT
Oral yeast-like fungi do not produce lesions in immunocompromised subjects.
Colonization progresses to infection when the host-fungus balance has been impaired.
The chapter presents yeast pathogenic determinants and main elements of host
antifungal defense, emphasizing the role of innate and specific immunity. Local and
general factors of systemic diseases, increasing the susceptibility to Candida species
infections, oral candidiasis clinical presentation (according to Samaranayake), and basic
diagnostic methods, including direct mycological and serological tests, are discussed. The
use of serological methods is limited to patients with immunodeficiencies, because of
antigenic similitude of fungi with bacteria, and of a decreased production of detectable
antibodies.
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688 D. Olczak-Kowalczyk, M. Roszkowska-Blaim, M. Pańczyk-Tomaszewska et al.

A short characteristic of respective immunodeficiencies is presented, as well as

factors increasing the susceptibility to yeast infections, candidiasis frequency and clinical
presentation of candidiasis, and Candida species isolated from the mouth. Primary
immunodeficiencies include: phagocytic disorders: congenital neutropenia, chronic
granulomatous disease, and a decreased count of an impaired function of T lymphocytes:
Severe Combined Immunodeficiency, Job’s Syndrome, Chronic Mucocutaneous
Candidiasis, Nijmegen Syndrome); secondary immunodeficiencies include: anticancer
chemotherapy, pharmacological immunosuppression, liver vs. kidney recipients, Graft-
Versus-Host-Disease, chronic liver disorders, nephrotic syndrome and diabetes.
According to the present study, among primary immunodeficiencies, oral candidiasis was
most often present in APS type 1 (100%) and in congenital neutropenia (50%), and,
among secondary immunodeficiencies, in kidney recipients (11.42%). In
immunodeficiencies, yeast infections are often associated with oral mucous membrane
lesions. Cyclosporine A, kidney transplants, and mucositis, occurring during
antineoplastic treatment, all increase the susceptibility to yeast infections.
Yeasts are isolated in the mouth of 45.4% of children with nephrotic syndrome and
14.8 with Type 1 diabetes. Oral candidiasis occurred respectively in 13.3% and 11.1% of
them.

INTRODUCTION
Candida fungi are often considered as non-pathogenic - in a healthy mouth they are
commensal and can be considered as part of physiological flora; at other times, they influence
the patients’ general condition, despite the absence of symptoms, being classified as
pathogens causing systemic mycoses [1-2].
The gut contains a balanced mix of yeasts and good bacteria. Disrupting that balance, for
example with antibioticotherapy, might lead to fungal overgrowth and its translocation - the
most common tissue invasion mechanism. A healthy fungal flora constantly varies in quality
and quantity, depending on age, eating habits, and the presence of oral biological factors,
interacting synergistically or antagonistically [2].
Oral yeast colonization does not automatically imply infections. When host defense
mechanisms are efficient, yeasts do not cause any lesions. Clinical symptoms of an infection
appear only when host-fungus balance is disrupted.

1. FACTORS INFLUENCING HOST-FUNGUS INTERACTIONS

Three groups of genes are responsible for fungal virulence:

I enabling fungal existence and growth in living and healthy organisms,

II initiating its capability to infect deep layers of tissue,
III determining host immune responses.

There most probably exist groups of genes responsible for immunosuppressive metabolite
production, and, depending on host immune responses, they can influence fungal virulence.
Candida krusei, a low-virulence yeast-like fungus, requires deep immunosuppression to
proliferate and infect [4].

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 689

Pathogenic yeasts have a more complex virulence “gene,” determining their ability to
overcome host natural defense mechanisms. Virulence is determined by pathogenic
characteristics such as a specific fungal cell membrane construction, adhesion and adherence
properties, ability to produce two different antigenic forms: yeast and hyphae, biofilm
formation properties, and also yeast proteolytic and lipolytic activities [2-12].
Yeast pathogenicity is determined by:

1. Adhesion and adherence properties, among others, to epithelium, bacteria, neutrophil

granulocyte and macrophage cells, to extracellular proteins, such as in saliva and
extracellular matrix (collagen, fibronectin, and elastin), to serum proteins
(fibrinogen, albumin, and transferrin), and to synthetic polymers. Fungal cell wall
mannoproteins participate in adhesion and adherence processes, and are considered
to be the main adhesins. Mannan, present in yeast cell walls, disrupts neutrophil
granulocyte functions and destroys host tissues. Those processes are also
accompanied by proteolytic enzyme and lipase (phospholipases and
lysophospholipases) secretions. Candida albicans has the strongest adhesive
properties; those of C.tropicalis and C.krusei are slightly weaker. Strains with
stronger adhesive properties are more pathogenic. Yeast adhesion is facilitated by the
secreted lipases and phospholipases, facilitating penetration into host tissues [4, 6-9,
11].
2. Fibroblast and epithelial cells endocytosis – might be responsible for recurrent
mycoses and take part in cancerogenesis.
3. Virulence, i.e., yeast existence as blastopore, pseudohyphae (chain of elongated
cells) and hyphae/mycelial form, are considered to be more pathogenic as it
penetrates more easily through tissues and has the binding complement component 3
on its surface. A higher virulence of the mycelial form most probably results from its
ability to digest and penetrate host tissue. Both blastopore and mycelial forms are
important in candidiasis pathogenesis - yeasts are resistant to phagocytes, and the
mycelial form penetrates deep levels of tissue [4, 6-11].
4. Proliferation, extracellular polysaccharide production, and biofilm formation
(Candida albicans) abilities. Candida species form a biofilm on the surface of
prostheses, such as dentures. Biofilm cells display diverse biochemical activity. Cells
located in superficial layers are more active. Those located in deeper layers display a
lower biochemical activity and a higher drug resistance. Cells containing genetic
information are found in the inner layer. Dead cells, used as nutrients, may be present
in all biofilm layers [13].
5. Hydrolytic enzyme secretion, facilitating tissues (proteases, phospholipases,
lipophospholipases) and toxin invasion. Aspartic proteases (key hydrolytic enzyme),
are most active in C. albicans, then in C. tropicalis, C. lusitaniae and C. krusei [8-10,
14-20]. Phospholipases are mainly produced by C. albicans. Other species secrete
that enzyme in smaller quantities. Jayatilake JA and Samaranayake YH presented
ultrastructural characteristics of oral candidiasis, using tissue cultures [20]. They
worked on the reconstitution of the human oral epithelium (RHOE) that they
implanted with Candida albicans S.C. 5314. After a forty-eight hour incubation,
Candida albicans had penetrated into the RHOE. Candida albicans phospholipase,
localized during tissue invasion and tested cytochemically, were active at the hyphae
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extremity and was budding. This discovery confirms that complex cell interactions,
such as tchigmotropism and extracellular phospholipase cooperate and co-decide
about invasive candidiasis. Other hydrolytic enzymes, secreted by Candida, N-
acetyl-β-glucosaminidase and α-glycosidase, inhibiting neutrophil granulocyte
migration to infected areas, also influence the course of infection [21].
6. Weakening of host defense mechanisms. Candida proteases may impair multinuclear
leukocyte activity and cause macrophage lysis. They also participate in inflammation
processes. Mannoproteins disrupt neutrophil granulocyte activity by inhibiting
myeloperoxidase secretion and lymphocyte T proliferation.
7. Carbohydrate fermentation to acid metabolites, which by lowering saliva pH favor
fungal existence [22]. Pathogenic Candida albicans use a source of energy - the N–
acetyl-glucosaminidase amino sugar. During Candida albicans infections, mucous
membranes are rich in amino sugars. This specific fungal adaptation reflects their
high pathogenicity [23]. Fungi are of little threat to organisms with efficient immune
systems.

Local environment, immune mechanisms related to phagocyte activity, and specific cell
response play an important part in host antifungal defense (Table 1).
Neutrophils directly destroy yeasts through phagocytosis, neutrophil extracellular trap
(NET), and induction and regulation of the specific immune response [25, 26].
The intracellular killing of yeast cells phagocytized by neutrophils is performed by
producing reactive oxygen metabolites (respiratory burst) and by liberating enzymatic
proteins from lysosomal granules. Neutrophil granulocyte’s ability to kill yeasts increases the
tumor necrosis factor (TNF-α). NET is created by extracellularly liberating nuclear
chromatin, which ‘immobilizes’ pathogens and exposes them to killer proteins, coming from
neutrophil lysosomal granules. Macrophages possess receptors binding certain yeast cell wall
sugars, such as Candida albicans β–glucan. Yeast cells are destroyed with the participation of
Fc IgG and C3b receptors. Natural killer cells are also directly toxic towards some fungi.
Cellular response (CD8+ and CD4+ lymphocytes) is crucial in preventing superficial
candidiasis. Helper cells Th1 prevent invasive Candida inflammations (IFN-γ activates
macrophages, IL-2 increases lymphocyte cytotoxic activity), whereas T helper 17 cells
secrete IL-17 and IL-22, which participate in preventing superficial mycoses by improving
non-specific antifungal defense of the mucous membrane [26, 27].

Table 1. Host anti-fungal defense factors

Antifungal defense
continuity of oral mucous membrane,
epithelium exfoliation,
local oral saliva (mechanical fungus removal, IgA aggregating fungal
environment cells hindering adhesion, antifungals; lactoferrin,
1 line
lactoperoxidase, liposome, histatins, β-defensins, iron) [24],
rivalry for an ecological niche with oral flora
immunocompetent cells (macrophages, neutrophil granulocytes,
innate immunity
natural killers, and complement component)
2 line specific immunity cellular immune response (lymphocytes T CD4+ and CD8+)

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 691

These results were confirmed by tests in mice and observations in patients possessing
neutralizing antibodies to Th17 cytokines due to thymoma or AIRE deficiency, and with
predisposition to oral candidiasis [28-31]. Humoral response is insignificant in yeast fighting.
Local factors increasing susceptibility to yeast infections, related to systemic diseases
might include: epithelial damage, diminished salivary secretion and lowered pH, high
carbohydrate diet, inhaled steroid treatment and changes in oral flora composition (such as
during antibioticotherapy), improper oral hygiene, and dental caries [32, 33].
Systemic factors include: nutritional deficiency, iron, folic acid or vitamin C deficiencies,
hormonal perturbations, immune deficiencies and pharmacologic immunosuppression
(especially medication impairing cellular response), and neoplasms [32-36].
Depending on the level of host immune system impairment and fungal pathogenicity,
infections may take the form of light superficial candidiasis and affect just one organ (such as
mouth or throat) or of multifocal infections that might be associated with candidemia [32, 37].
Potential Candida pathogenicity manifests itself most probably when a couple of factors
concur in a patient with immune deficiency. Candida mycoses, both superficial (chronic or
recurrent) and invasive, suggest primary (genetically conditioned) or acquired (such as AIDS)
immune deficiency.
In case of severe immunodeficiencies, Candida species might even cause life-threatening
candidemia (about 25%). Invasive candidiasis is the most common invasive fungal infection
(about 70-90% of all invasive mycoses) [37, 38].

2. CLINICAL SYMPTOMS OF ORAL CANDIDIASIS

Candida infection symptoms might include pseudomembranous, white lesions, tightly
attached to their support, vivid red erythematous plaques on the mucous membrane, white
keratose-like growths, angular cheilitis, and median romboid glossitis [32-35, 39].
There are various classifications of oral candidiasis. Samaranayake divided them in
primary and secondary (Table 2) [33, 36].
Pseudomembranous candidiasis manifests as creamy white plaques, which once removed,
leave a vivid red, painful and bleeding surface. It occurs in infants, HIV- positive patients,
those taking glucocorticosteroids, immunosupressants or cytotoxins. It might be also caused
by broad-spectrum antibiotics.
Erythematous candidiasis (EC), acute or chronic, manifests as reddish areas, sometimes
giving a burning sensation, often localized on the dorsum of the tongue (with concurrent loss
of papillae), palate, or buccal mucosa. It could be a “progression” of the pseudomembranous
form. It occurs in patients with AIDS, taking cytotoxins and glucocorticosteroids, and after
chronic broad-spectrum antibioticotherapy.
Chronic hyperplastic candidiasis (CHC), formerly known as ”Candida-leukoplakia,” is
quite particular.
Primary CHC appears in adults, and secondary in children with primary
immunodeficiencies. CHC is characterized by translucent, homogeneous or nodular white
plaques, which cannot be rubbed off, localized mainly in the commissural regions of the oral
mucosa, buccal mucosa, or less often on the dorsum of the tongue. Hyphal invasion of the
epithelial surface is characteristic in CHC [33, 36, 40].
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Table 2. Classification of oral candidiasis modified according to Samaranayake (1991)

Primary Oral Candidosis Secondary Oral Candidosis

(Group I) (Group II)
The ‘Primary Triad’: Condition Subgroup
Familial chronic mucocutaneous
Pseudomembranous (mainly acute) 1
candidosis
Diffuse chronic mucocutaneous
Erythematous (acute/chronic) 2
candidosis
Hyperplastic (mainly chronic) Candidosis endocrinopathy syndrome 3
- Plaque-like Familial mucocutaneous candidosis 4
- Nodular/speckled Severe combined immunodeficiency 5a
Candida-associated lesions Di George syndrome 5b
Denture stomatitis Chronic granulomatous disease 5c
Angular cheilitis Acquired immunodeficiency syndrome 6
Median rhomboid glossitis
Linear gingival erythema

It is accompanied by an elevated risk of neoplastic transformations and developing oral

squamous cell carcinoma. Candida albicans is said to be mainly implied in cancerogenesis.
However, its pathogenetic role in neoplastic transformations has not been sufficiently
investigated. Candida albicans ability to synthesize carcinogens, such as nitrosamines, could
be responsible for that [40].
Median rhomboid glossitis manifests as atrophic filiform papillae lesions on the dorsum
of the middle of the tongue, creating a diamond or elliptic shaped area, anterior to the
circumvallate papillae. Angular cheilitis manifests as redness, cracks, and, in chronic cases,
also as hyperplastic lesions. They are caused either by Candida, or by bacterial infections,
most often Staphylococcus aureus. Linear gingival erythema (LGE) manifests as a red line,
about 2 mm wide, adjacent to the gingival margin, first described in patients with AIDS [41].

3. ORAL CANDIDIASIS DIAGNOSIS

Oral candidiasis is diagnosed based on lesions and additional testing, mainly
mycological, and sometimes also cytologic and histopathologic [32, 33, 35, 36, 42].
Mycological testing includes:

● microscope slide preparation directly from clinical material.

● culture of the collected clinical material on universal Sabouraud agar with
gentamycin and chloramphenicol. Fungal identification, based on morphological and
biochemical characteristics, are performed with commercial micro tests, such as ID
32C, API Candida (bioMeriéux), or using commercial differentiation medium, such

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 693

as CHROMagar Candida (Becton Dickinson), or Candi Select (BIO-RAD), API C

AUX (bioMeriéux),
● the assessment of antimycotic drug resistance with yeast-like fungi diagnostic kits
ATB Fungus (bioMeriéux), Fungitest (BIO-RAD) and E-tests (AB BIODISK) for
Candida fungi and yeasts, determining MIC values (Minimal Inhibitory
Concentration).

In patients with immunodeficiencies, it is also important to diagnose invasive mycoses.

Diagnostic tests, including mycological and serological, fungal metabolite (D-arabinitol and
D-mannitol) detection, and molecular biology, are all helpful [21].
Serological testing is performed at the same time as microbiological testing, and results
are interpreted while confronted with the patient’s clinical condition. Fungal or bacterial
antigen relationships might result in false positive results.
There are many factors that might give erroneous serologic results such as:
galactomannan – Aspergillus antigen, discovered in some intravenous preparations of
piperacillin and amoxicillin with clavulanic acid, in some cereal products, and in powdered
milk, therefore test results are to be interpreted taking into account patient’s eating habits.
Many factors account for false positive Cryptococcus antigen tests, such as rheumatoid factor,
concurrence of some cancers, Trichosporon beigelii infections, Gram-negative OX+
fermenting rods, Capnocytophaga canimorsus (DF-2), and OX- Klebsiella pneumoniae [43].
False positive results for anti-Candida antibodies result from yeast proximity, omnipresent in
the environment and colonizing mucous membranes.
Patients with immunodeficiencies do not produce antibodies detectable with the methods
in use; therefore, a negative result does not automatically exclude fungal infections. The use
of serological testing in diagnosing remains controversial. Diagnosis of fungal infections uses
latex, ELISA immunoenzymatic, and double diffusion in agar gel tests, and secondary
immunofluorescence.
Should conventional diagnosis methods fail, molecular biology methods are used. Their
main advantage is their high sensitivity, minimal amount of clinical material necessary, and
the result-waiting period.
This might be both an advantage and a disadvantage, depending on circumstances. The
smallest contamination of clinical material, destined to be tested with exogenous DNA, might
result in fake positive results [44]. Molecular biology methods are used for epidemiological
testing. Their high costs and harmonization difficulties, do not allow routine use.

Use of D-Arabinitol Fungal Metabolite in Diagnosing Fungal Infections

Very few centers in the world use chemical marker detection of microorganisms,
including metabolic products. D-arabinitol is a typical metabolite of several Candida
pathogens (Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida
pseudotropicalis). Determination of urinary D-/L–arabinitol ratio, measured with gas
chromatography or mass spectrometry, is used as a biomarker for systemic and disseminated
infections in clinical diagnosis. The method is useful with a 93.3% sensitivity, and a 97%
specificity.
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The material to be tested is obtained in a non-invasive way, which is important in infants

and premature babies. 10 – 20 µL of urine is enough, and the test takes 2 hours. Samples can
be prepared frequently, the infection can be diagnosed early, and antimycotic treatment can
be monitored [21, 45].

4. YEAST INFECTIONS IN IMMUNODEFICIENCIES

Immunodeficiencies with high risk of yeast infection include:

● primary: severe neutropenia (ANC<500/μl), chronic granulomatous disease,

decreased count/-impaired function of T lymphocytes,
● secondary: AIDS, diabetes, secondary neutropenia, pharmacological
immunosuppression (especially chronic CsA treatment with glucocorticosteroids),
intensive or chronic broad-spectrum antibioticotherapy, graft-versus-host-disease
[46, 47].

4.1. Study Synopsis

Candida species are to be found in about 1/3 of healthy children and teenagers. Oral
colonization is higher in immunodeficiencies. Yeasts were isolated in all patients with
autoimmune polyendocrine syndrome type 1 (APS type 1). In healthy patients, Candida
albicans was the only isolated species; in those with immunodeficiencies several strains were
isolated. In patients with primary immunodeficiencies, yeast infections were most often seen
in APS type 1 and congenital neutropenia; in those with secondary immunodeficiencies – in
kidney recipients.
There is also a high Candida species and yeast infection incidence in patients with oral
inflammatory lesions, especially in those with mucositis and undergoing antineoplastic
treatment (Table 3).
Lesions in patients with immunodeficiencies are varied and hard to define. It is difficult to
determine just one cause of infection, especially since that cause is modified by numerous
systemic factors, disease and/or treatment-related. Apart from Candida infections, erythematous
and white pseudomembranous lesions on oral mucosa might be caused by contact allergies,
anemia, autoimmunization, and even pre-cancer and cancer processes [55-57].
The present study confirmed Candida infections in 48.6% of yeast lesions
(pseudomembranous plaques, erythema, angular cheilitis) in children and teenagers with
secondary immunodeficiencies (kidney or liver diseases/recipients). Candida was more often
isolated from erythematous lesions than from white pseudo-membrane.
In 25.7% of the cases, candidiasis was associated with other changes in mucous
membrane (RAS minor, ulcers, black or geographic tongue). CsA and kidney transplants
increase the susceptibility to concomitant lesions [53].

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 695

Table 3. Study results presenting frequency of Candida oral colonization and

candidiasis in healthy and immunodeficient children

% of patients with
Reference Average age N of Candida
Candida species **
number in years patients in oral oral
cavity candidiasis
48 8.9±3.7 39 33.3. 2.6 C albicans
Healthy
49 10.8 ± 4.2 70 27.14 1.4 C. albicans
Primary immunodeficiencies
C albicans,
Neutropenia* 10.8±6.6 6 50. 50
C. dubliniensis
48
C albicans,
CGD* 11.5±3.8 16 43.7 18.7
C. incospienta
CGD (retrospective
50 4-17 27 - 22 -
test)
51 APS 1 12.95±4.0 8 100 100 C. albicans
52 NBS 11.3 21 38 28.6 -
Secondary immunodeficiencies
C. albicans,
Renal failure 10.0±5.,2 13 38.4 0
C. parapsilosis
Liver failure 11.8±3.7 23 60.8 8.7 C. albicans
48 Kidney recipients 10.9±3.9 22 18.2 9.1 C albicans
C. albicans
Liver recipients 10,7±4.2 22 40.9 4.5 C. parapsilosis, C.
glabrata
C. albicans,
Kidney recipients 14.5±3.7** 105 32.38 11.42 C. parapsilosis,
C. glabrata
C. albicans
49
C. dubliniensis
Liver recipients 11.2±5.5** 80 36.25 10 C.tropicalis, C.
Parapsilosis,
C. crusei
Purposive sampling (criteria: lesions in oral mucosa
Inflammations:
in chronic liver disease 10.94±4.94 20 50 50. C albicans
in nephrotic syndrome 10.94±3.52 12 58.3. 41.6. C albicans
53 in kidney recipients 14.82±4.69 38 42.1. 26.3. C. parapsilosis,
C. dubliniensis, C.
in liver recipients 10.99±6.08 32 34.4 31.2 crusei,
C. tropicalis,
Stomatitis in kidney or C. albicans,
11.9 ±6.1** 20 50 20
liver recipients C. inconspicua
54
Mucositis (anticancer C. albicans
9.3±4.25** 14 78.5 78.5
chemotherapy) C. tropicalis
*
undergoing antibacterial antibioticotherapy.
**
data base second analysis.
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4.2. Primary Immunodeficiencies

4.2.1. Phagocyte Impairment

Congenital neutropenia manifests as a permanent or temporary (at least 6 months)
decrease in neutrophil granulocytes, below 1500/μl (absolute neutrophil count, ANC). It
might manifest as an isolated symptom, or one of the symptoms of genetic disorders (e.g.,
glycogenosis type Ib, hyper IgM syndrome, Shwachman-Diamond syndrome) [58]. There is
an increased risk of fungal infection in severe neutropenia (ANC<500/μl). Factors increasing
the susceptibility in children with congenital neutropenia include: concomitant cellular
immunodeficiencies, chronic and prolonged broad-spectrum antibioticotherapy of bacterial
infections, glycogenosis type 1b, and also a diet rich in carbohydrates [47]. Glycogenosis type
1b is a glucose-6-phosphate translocase insufficiency, causing glycogen degradation
perturbations in the liver, and in progenitor cells, hematopoietic stem cells, neutrophil
granulocytes and monocytes. It results in hypoglycemia, lactic acidosis, neutropenia and
granulocyte and monocyte function impairment [59].
The frequency of oral candidiasis in neutropenia, and during antibacterial
antibioticotherapy was established at 50% [48]. In further observations oral candidiasis was
diagnosed in 2 out of 8 patients with neutropenia and in 2 out of 3 patients with glycogenosis
type 1b (erythematous candidiasis - 3, pseudomembranous candidiasis with concomitant
angular cheilitis - 1) (Figure 1, 2). Candida albicans was isolated in 2 patients, and Candida
dubliniensis in another two. In chronic granulomatous disease (CGD) phagocytes lose their
ability to kill intracellularly, which normally depends on oxygen influence, following damage
to nicotinamide adenine dinucleotide phosphate-oxidase (NADPH). The disease is caused by
a mutation of NADPH oxidase protein coding genes, and is recessive (NCF1, NCF2, CYBA
gene mutations) or more often X-linked recessive (CYBB gene mutation) [60-62]. A
decreased NADPH oxidase activity in neutrophil granulocytes increases the susceptibility to
bacterial and fungal infections, more often Aspergillus than Candida [46, 63].

Figure 1. Acute erythematous candidiasis in a patient with agranulocytosis, undergoing antibacterial

antibioticotherapy (photo by D. Olczak-Kowalczyk).

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 697

Figure 2. Angular cheilitis in glycogenosis type 1b (photo by D. Olczak-Kowalczyk).

Figure 3. Gingivitis and Candida albicans infection in CGD (photo by E. Krasuska-Sławińska).

Since inflammations have a heavy course and are sometimes even life-threatening,
patients with CGD undergo preventive antibacterial and antimycotic antibioticotherapy [64].
Candida is considered to be the most common cause of meningitis (20%) and the fourth most
frequent cause of death in patients with CGD. Invasive candidiasis benefits from intravascular
catheters use and prolonged antibiotic and steroid antibacterial treatment [46, 61]. Candidiasis
in oral cavity occurs in 12.3% of the cases - thrush in 22%, angular cheilitis in 7.4% (Figure
3) [65, 66].

4.2.2. Decreased Count of Impaired Function T Lymphocytes

Severe combined immunodeficiency (SCID) concerns cellular and humoral immunity
and is recessive or sex-linked recessive. It might be caused by one of many mutations in 10
different genes. One of its typical symptoms is the occurrence, between ages 3 to 6 months, of
pseudomembranous candidiasis (thrush) [46, 47]. Candida yeasts and viruses often cause
severe recurring infections in patients with SCID. They might cause thrush, pneumonia, or
meningitis. Antibiotics are used preventively, similarly as in CGD (Figure 4).
Hyper IgE syndrome (HIES, Job’s syndrome) occurs sporadically or as an autosomally
inherited trait, either recessive (AR) or dominant (AD). It might result from gene mutation
(STAT3, Tyk2, DOCK8) and manifests as an elevated IgE level (>2000 IU/ml), recurring
pneumonia, and viral and bacterial infections. The AD pattern is additionally accompanied by
connective tissue and skeletal disorders, and teeth abnormalities [28, 67]. Mainly, Candida
albicans mycoses were detected in 83% of patients with an AD pattern and in 70% of patients
with an AR pattern [28, 67, 68]. Yeast infections might manifest only in the mouth or as
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698 D. Olczak-Kowalczyk, M. Roszkowska-Blaim, M. Pańczyk-Tomaszewska et al.

mucosal candidiasis (Figure 5). In HIES, Th 17 cell and antimicrobial protein (AMPs - β-
defensin 2 and histatins) deficiencies predispose patients to oral yeast infections [68].
Candida organisms, mainly Candida albicans, might lead to candidemia and
endocardidtis, endophthamitis, visceral candidiasis and disseminated diseases with pulmonary
nodules or changes in the liver [46].
Chronic mucocutaneous candidiasis (CMC) refers to a group of heterogeneous diseases
characterized by recurrent or persistent infections of the skin, nails and mucous membranes
with Candida, usually Candida albicans. It results from inadequate production of Th1 or
Th17 cytokines, as a response to yeast presence [69, 70].

Figure 4. Pseudomembranous candidiasis in SCID (photo by E. Krasuska-Sławińska).

Figure 5. Lateral tongue ulceration and pseudomembranous candidiasis in HIES (photo by D. Olczak-
Kowalczyk).

There is little tendency in CMC for the infection to spread, but there is a considerable risk
of cancerogenesis [72]. CMC is connected to hormonal and autoimmune disorders, and

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 699

immunodeficiencies (e.g., Addison’s disease, HIES, DiGeorge syndrome, SCID, AIDS) [33,
46].
Kirkpatrick divided them in:

● Familial CMC, inherited in a recessive or dominant pattern

● Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome
(autoimmune polyendocrine syndrome type 1, APS type 1)
● Chronic localized candidiasis
● CMC with thymoma (in adults)
● CMC with keratitis [72].

APS type 1 is inherited in a recessive pattern. The disease is caused by one of the 40
described mutations in the autoimmune regulator gene (AIRE0 21q22.3 [73]. At least two
diseases, among Addison’s disease, mucosal candidiasis, and hypoparathyroidism, manifest
themselves in APS type 1. They can be associated to other autoimmune diseases, such as
hypothyroidism, diabetes mellitus, anemia, vitiligo, and baldness.
Mycotic skin lesions most often appear as rings, and are often covered in scabs. They
might also manifest as infiltrations, tumors, and excessive keratosis. In patients with CMC,
oral candidiasis manifests as: white hypertrophic and tumorous lesions with deep cracks on
the tongue and on cheek mucous, thrush, erythematous and pseudomembranous candidiasis,
chronic hyperplastic candidiasis, and angular cheilitis (Figure 6) [51, 74, 75]. In the present
study, Candida albicans was responsible for oral candidiasis in patients with APS type 1.

Figure 6. Median rhomboid glossitis in APS type 1 (photo by D. Olczak-Kowalczyk).

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Figure 7. The linear gingival erythema in NBS (photo by D. Olczak-Kowalczyk).

Nijmegen breakage syndrome (NBS) is a rare, inherited in an autosomal pattern, disease

characterized by spontaneous chromosomal aberrations and cancerogenesis. NBS is caused
by biallelic mutations in the NBN (formerly NBS1) gene located on the chromosome 8q21,
which encodes the NBN protein (previously called nibrin) involved in processing and
repairing DNA double strand breaks (DNA DSBs). In patients with NBS, cellular and
humoral arms (B- and T cells) display various anomalies, with the tendency to progress over
time. Dysgammaglobulinemia, with or without decrease in IgG, IgA, IgE, and rarely IgM
occurs. A decrease in CD4+ and CD8+ T cell count is another type of immunodeficiency [52,
76, 77].
Oral candidiasis (pseudomembranous candidiasis, erythematous candidiasis, angular
cheilitis and linear gingival erythema) was diagnosed in 38% patients (Figure 7). In all of
those cases, Candida albicans was isolated.

4.3. Secondary Immunodeficiencies

Secondary immunodeficiencies are caused by numerous systemic diseases, viral

infections, pharmacological immunosuppression, glucocorticoid or cytostatic treatments,
protein-energy malnutrition, vitamin insufficiencies and stress. Those factors might impair
both the innate immune system, related to phagocytes, and the specific immune system.

4.3.1. Pharmacological Immunosuppression Cytostatics Used in Neoplasms

Antineoplastic therapies present a high risk of invasive, even life-threatening, fungal
infections. The most common infection is candidiasis (58%-69%) [78, 79]. C. albicans, C.
glabrata and C tropicalis are most often isolated. There is a risk of fungemia in patients
medicated with cytostatics such as: mucositis, neutropenia, broad-spectrum
antibioticotherapy, glucocorticoid therapy, and invasive medical procedures [79, 80].
Mucositis occurs in 40% of patients treated with conventional chemotherapy and in more
than 70% of patients undergoing conditioning regimens for bone marrow transplants. That is
directly caused by the influence of cytostatics on the rapid cell division in oral epithelium and
apoptosis induction, and indirectly by: inflammatory mediator release, decrease in salivary

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protective function and neutropenia. Chemotherapy also lowers pH and salivary buffering
capacities, and immunoglobulin (sIgA, IgG), lactoperoxidase, lysosome and lactoferrin levels
[81]. Cytostatics causing inflammation in oral mucous membrane and strongly mucotoxic
include: fluorouracil with/without folic acid, methotrexate, doxorubicin, etoposide,
melphalan, cytarabine, and cyclophosphamide. Methotrexate and etoposide might also be
secreted in saliva, which intensifies mucotoxicity.
All cytostatics cause myelosuppression with varying degrees of severity. Neutropenia
increases the risk of Candida infections. They damage rapidly dividing cells, such as late
blood cell progenitors, which leads to granulocytopenia 7-14 days after starting treatment.
Cytostatics such as busulfan, melphalan, thiotepa, carmustine, and lomustine, acting on early
progenitor or stem cells lead to myelosuppression about 25–40 days after chemotherapy.
Cytostatics, not depending on division cycles are even more myelosuppressive than those
depending on cell cycle phases. Bleomycin, vincristine, asparaginase, and cisplatin have the
lowest myelosuppressive effect.
Radicals are also said to play an important destructive role, apart from directly damaging
the cytostatics on DNA in rapidly dividing cells of gastrointestinal tract epithelium.
Fragmentation of fibronectin might cause the release of pro-inflammatory cytokines,
including TNF-alpha and metalloproteins. The process starts 3–10 days after chemotherapy.
Children with neoplasms are in a state of immune insufficiency, because of their primary
disease. Cytostatics increase immunosuppression, cause lymphopenia, monocytopenia, and
above all, a decrease in granulocyte count and their function impairment. The risk of
infectious complications, including fungal, increases with the decrease in granulocyte count
in peripheral blood, in neutrophil phagocytic efficiency, granulocytopenia intensity and
duration, patient immunity within humoral and cellular response, and perturbations in
mechanical defense barriers [82, 83].
Lesions in oral mucous membrane appear about 7-10 days after the beginning of
chemotherapy. There are four phases in mucosistis development: inflammatory (vascular),
epithelial, erosive and healing. The erosive phase, also called microbiological, presents the
highest risk of fungal infection because of simultaneous neutropenia. The neutropenia-yeast
infection relationship was confirmed by Alberth et al.; oral samples from children and
adolescents with acute lymphoblastic leukemia or solid tumors before and after neutropenia
episodes, did not test positive for Candida spp. Yeasts were isolated in 84.4% of oral cultures
in neutropenic patients [84].
Children are said to be more prone to mucositis and superficial fungal infections than
adults. It most probably results from their fast epithelial mitosis and a higher number of
epidermal growth factor receptors. And hematopoietic neoplasms, often requiring prolonged
and intensive myelosuppression, occur more often in children than in adults.
Yeast occurrence in mucositis in patients treated with cytostatics for leukemia is about
26-51.5%, and in those treated with bone marrow transplants – 36.7% [85, 86]. According to
Chen, fungal infections occur in 21.8% of patients with mucositis, viral and fungal infections
in 9.9% of patients [87].
In the present study, Candida species were present in 78.5% of children with mucositis
undergoing antineoplastic chemotherapy in medulloblastoma, B-cell non-Hodgkin
lymphomas and hepatoblastoma treatment. Angular cheilitis and pseudomembranous
candidiasis are the most frequent clinical manifestations of oral candidiasis in children
undergoing cytostatic treatments (Figure 8).
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702 D. Olczak-Kowalczyk, M. Roszkowska-Blaim, M. Pańczyk-Tomaszewska et al.

Figure 8. Angular cheilitis and pseudomembranous candidiasis in a boy undergoing chemotherapy for
B-cell non-Hodgkin lymphomas (photo by D. Olczak-Kowalczyk)..

There seem to be no direct relation between mucositis intensification, patient age, type of
neoplastic disease and cytostatic agent received [54, 88].
According to Olczak-Kowalczyk et al. C. tropicalis was isolated from the mouth of
7.14% of patients, and C. albicans in all the other cases [54]. González Gravin et al. isolated
C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, C. krusei, and C. lusitaniae. In 9.3%
of children - more than one yeast species was isolated [88]. Alberth et al. established Candida
albicans was replaced by non-albicans species (C. kefyr, C. lusitaniae, C. sake, and C.
tropicalis) in extended severe neutropenic conditions [84].

Immunosupressants in Glomerulonephritis and Transplantology

The immunosuppressive treatment is to suppress immune responses in the
pharmacotherapy of renal disorders such as glomerulonephritis, tubulointerstitial nephritis,
systemic diseases affecting the kidneys, and in prevention and treatment of kidney transplant
rejection. Immunosupressants, because of their mechanism of action (MOA), might cause
yeast infections, especially those affecting cellular response (Table 4).
Immunosupressants might be classified according to their MOA:

● inhibit the transcription of genes responsible for cytokine production:

glucocorticoids, immunophilin binding: calcineurin inhibitors (cyclosporine,
tacrolimus), mTOR inhibitors (sirolimus, everolimus),
● cell division inhibitors: azathioprine, cyclophosphamide, sodium mycophenolate
● biological factors - antibodies: polyclonal (ATG) and monoclonal: anti-CD3 (OKT3),
anti IL-2R (daklizumab, basiliximab),
● misc. drugs: anti CD-20 (rituximab), anti- CD52 (alemtuzumab).

Studies on the relationship between immunosuppressant type and Candida species oral
colonization, and the occurrence of candidiasis, are heterogeneous. Children undergoing long-
term immunosuppressive treatments, especially CsA, glucocorticoid and rapamycin, are prone

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 703

to develop oral candidiasis [49, 53]. However, according to Dongari-Bagtzogolu et al., such a
correlation does not exist [93].
Immunodeficiencies in children undergoing immunosuppressive treatments are often not
just consequences of drug actions.
Vascularized organ transplantation has to always be accompanied by pharmacological
immunosuppression. In organ recipients, immunity might be impaired by additional factors
connected to the main disease that led to organ failure (such as cholestasis in liver recipients,
chronic kidney disease in kidney recipients) or other intercurrent systemic disease (such as
diabetes), nutritional and vitamin deficiencies, frequent hospitalizations, antibioticotherapies,
and viral infections (CMV and HHV-1). Around the time of the operation, stress, intervention
length and extent and general anesthesia also affect the immune system.

Table 4. Mechanism of immunosuppressive drugs used [89-92]

Drug Immunosuppressive activity

inhibit lymphocyte T proliferation
 IL-1, IL-3, IL-4. IL-6, IL-8, IGF, TNF, IFN-gamma, CD40 ligand, GM-
Glucocorticoids CSF, cell adhesion particles (selectins E, VCAM-1, ICAM-1, and MHC
 phagocytosis, monocyte migration, macrophage activity; prostaglandin
production
inhibits IL-2 IL-3, IL-4, IFN-γ, TNF-α production,
increases TGF-β synthesis
Cyclosporine A
 cellular and humoral response by mainly inhibiting T helper cells, cytotoxic
T cells and lymphocytes B
inhibits interleukin (IL-2 IL-3, IL-4, IL-5), GM-CSF, TNF-α and IFN-γ
Tacrolimus synthesis and secretion
inhibits cellular and humoral response
Sirolimus inhibits cell cycle of stimulated lymphocytes in G1phase
inhibits nucleic acid biosynthesis
 lymphocyte T and B proliferation
Azathioprine  IL-2 production
 immunoglobulin production
pro-apoptotic action
inhibits lymphocyte T and B proliferation
Cyclophospha increases Th2 response depending on IL-4, IL-5, IL-10 increase and cytokine
-mide Th3 levels, indispensable for regulatory T cell (Treg) response.
 IFN-γ and IL -12 secretion by monocytes
selectively and reversibly inhibits ionosine monophosphate dehydrogenase,
which takes part in guanine nucleoside synthesis, indispensable for DNA
construction
decreases lymphocyte and monocyte count at inflammatory site
inhibits lymphocyte T and B proliferation
Mycophenolate mofetil
inhibits dendritic cell maturation
antibody production stimulated by mitogens and antigens
induces activated lymphocyte T apoptosis
inhibits the glycation of adhesion molecules, mediating in binding active
lymphocytes T to endothelial cells.
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Organ recipients are at high-risk of developing invasive fungal infections (5%-42%),

especially in the post-transplant period [94-96]. Those infections are mainly caused by
Candida species (C. albicans, C. glabrata, and C. tropicalis) and Aspergillus. The highest
incidence of Candida infections is reported in liver and small bowel transplant recipients, the
lowest in renal recipients [97, 98]. Endogenous sources of colonization, mainly the
gastrointestinal tract, including the mouth, are the principal sources of infection.
Oral candidiasis and oral mucous membrane breakdown, coupled with oral yeast
occurrence, might lead to fungemia. In organ recipients, the frequency of oral yeast
colonization and oral candidiasis are higher than in non-patients.
Candida albicans is most often isolated in oral candidiasis; C. dubliniensis, C. parapsilosis,
C. crusei, C. tropicalis, C. glabrata, C. famata, and C. guillermondi are less frequently isolated
[73, 94, 99, 100]. Candida albicans is also the most common cause of oral yeast infections.
Al.-Mohaya et al. isolated yeasts in the mouth of 74.1% of kidney recipients (aged from
16 to 62), and erythematous candidiasis and angular cheilitis in 15.5% of them [99]. In
children with renal insufficiency and after a kidney transplant, oral candidiasis occurred in
31.82% of cases [101]. In the present study, in children after a kidney transplant, oral
colonization occurred in 27%-32% of them, and oral candidiasis in 11%-22%. In pediatric
liver recipients, Candida species was present in 30%-36% of the patients, and oral candidiasis
in 10%-23% [49, 98]. Oral candidiasis might occur at any time after transplantation, but the
risk is the highest in the first month following the operation. Candidiasis in pediatric organ
recipients most often manifested as erythematous candidiasis (atrophic candidiasis) and
angular cheilitis; pseudomembranous candidiasis and median rhomboidal glossitis occurred
less often (Figure 9). The occurrence of Candida species is also associated to black hairy
tongue, coated tongue, atypical ulcerations, RAS minor, and RAS major. It also often
accompanies Herpesviridae viral infections, which additionally impair immune mechanisms
[102]. Graft-versus-host-disease (GVHD) is a complication that can occur after an allogeneic
hematopoietic stem cell transplant. The causes of the disease are not fully known. There are
two types of GVHD: acute and chronic. Cytokines, which increase the expression of the
antigens of the HLA system and activate the cells presenting the antigen of the donor, take
part in the acute form development.

Figure 9. Pseudomembranous candidiasis in liver recipient (photo by E. Krasuska-Sławińska).

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 705

Discrepancies between the presented antigens of the HLA system activate donor T cells
and the gene transcription for IL-2, INF-, and their receptors. IL-2 and INF- trigger the
release of pro-inflammatory cytokines by monocytes and macrophages, and induce cytotoxic
T cells (CTL), and NK cells.
As a consequence, CTL and NK cells directly influence recipient cells, such as the
renewing ones of the oral mucosal epithelium, hematopoietic, lymph and endothelial cells,
which results in their injury and apoptosis.
In most recipients, chronic GVHD develops as a consequence of acute GVHD. Thymic
autoreactive Th2 cells, favoring the production of antibodies by lymphocytes B, most
probably play the main role in that process. GVHD symptoms remind those of autoimmune
and immune diseases and autoimmune and primary immunodeficiencies. Immunosuppressive
medication to treat mild and severe diseases additionally impairs host defense mechanisms
[103, 104].
GVHD patients are at risk of fungal infections, both superficial and invasive [105]. The
main cause of oral candidiasis in GVHD patients are immunosuppressive treatments,
localized glucocorticoid use, xerostomia, and also in case of viral infections – their additional
immunosuppressive action. Candida infections might cause both pseudomembranous and
erythematous candidiasis (Figure 11). GVHD is hard to diagnose because it manifests as
white (impetigo-like and hyperkeratoic) and red (self-limited or diffuse erythema) lesions.
Cytology exams are useful to diagnose yeast infections, especially the erythematous form
[106].
Chronic liver diseases (CLD) in children might be caused by congenital or metabolic
disorders, infections, neoplasms, intoxications, adverse drug reactions, nutritional
insufficiencies, or traumas. CLD causes liver failure, whose manifestations depend on the
degree of liver damage.
Compensated cirrhosis often does not present any clinical symptoms, however the
decompensated one is associated with malnutrition, ascites, edemas, esophageal varices,
coagulation disorders, and sometimes jaundice.

Figure 10. Candidal leukoplakia in GVHD (photo by D. Olczak-Kowalczyk).

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706 D. Olczak-Kowalczyk, M. Roszkowska-Blaim, M. Pańczyk-Tomaszewska et al.

Figure 11. Pseudomembranous candidiasis and median rhomboid glossitis in CLD and mucoviscidosis
(photo by E. Krasuska-Sławińska).

Immunodeficiencies in CLD might result from primary disease, cholestasis, malnutrition,

or immunosuppressive treatments in autoimmunological diseases [107].
In cirrhosis, oral candidiasis occurs in 8.7%-20% of pediatric patients. C. albicans was
the only species to be isolated (Figure 12) [108]. The nephrotic syndrome is a disorder
manifesting with proteinuria of an intensity exceeding the body’s ability to compensate
(protein leak over 50 mg/kg/day). In children between ages 1-12, the idiopathic nephrotic
syndrome (INS) occurs in most cases. It is characteristic of the INS to recur.
Immunodeficiencies in children with INS are a direct consequence of proteinuria, associated
with hypoproteinaemia or dyspoproteinaemia, hyperlipidemia and shifts in immunoglobulin
composition (including decreasing IgG level), and of immunosupressants, such as
glucocorticoids, cyclosporine A, cyclophosphamide, and mycophenolate mofetil [109].
Immune system impairment increases the susceptibility to fungal infections. A reduced salivary
secretion might also additionally impair the oral immune system in patients with INS.

CANDIDA SPECIES AND INS

Material and Method

An oral candidiasis and Candida species (direct mycological test) occurrence was
evaluated in 44 patients with INS (average age 9.36 ± 4.5 years). The administered
medication is presented in table 5.

Results

Candida species were isolated in 45.5% children - much more often in children treated
with a single-drug than with combination therapy (Chi2 p<0,01) (Table 5). The isolated
species was Candida albicans.

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 707

In one child, treated exclusively with CsA, C. albicans was accompanied by C.

parapsilosis. Oral candidiasis was diagnosed in 6 patients (13.3%), most often medicated
with three and two immunosupressants than with just one (Table 5, Figure 13). Diabetes is a
metabolic disease, characterized by hyperglycemia caused by defective insulin secretion or
activity, or by the combination of both. Diabetes Type 1, one of the most common chronic
diseases of childhood and adolescence, is an autoimmune disease in people with a genetic
predisposition, in which the beta cells of pancreatic islets are randomly destroyed. Factors
increasing the susceptibility to oral yeast infections are: reduced salivary secretion and
changes in its composition, and impaired neutrophil granulocyte activity and cellular
immunity [109, 110].
Diabetes manifests as a reduced salivary secretion and changes in its composition, i.e.,
increased glucose level, lowered pH, increased salivary amylase activity, and also oral
mucous membrane breakdown (capillary epithelial tissue thickening, which restricts oxygen
circulation, and insufficient waste removal from the body).
Infections are also said to be caused by dental plaque microorganisms, which might
increase the susceptibility to yeast infections. Patients with diabetes have an altered oral flora.
By producing leucotoxins, bacteria might impair neutrophil granulocyte activity, which
results in inflammations [110]. Impairment of neutrophil granulocyte activity, such as
phagocytosis, intracellular killing of microorganisms, adhesion, or chemotaxis [112-113].
That function impairment is closely related to the degree of diabetes compensation; it occurs
in patients with metabolic decompensation, increased HbA1c level, and prolonged diabetes.
In hyperglycemic conditions many proteins undergo nonenzymatic glycation.
AGE, the advanced glycation end product, plays an important role in cell migration and
phagocytic activity of macrophages/ monocytes, resulting in an increase in oral bacterial and
fungal flora. Inflammations increase oxidative stress levels and insulin resistance, resulting in
impaired regulation of pro-inflammatory TNF-α and IL-1 cytokine secretion.

Table 5. Candida species and oral candidiasis occurrence in patients with INS

Candida
Immunosuppr Patients Oral candidiasis
Drug species
ession
N=100% N/% N% Clinical appearance (N)
Median rhomboid glossitis
Glucocorticoids 19 5/26.3 1/5.2
(1)
1 drug
CsA 2 1/50 0 -
total 21 6/28.6 1/4.76 -
Erythematous candidiasis
Glucocorticoids+ with angular cheilitis (1)
19 11/73.3 3/20
CsA Pseudomembranous
2 drugs candidiasis (2)
Erythematous candidiasis
CSA+ MMF 1 1 1
(1)
total 20 12/60 20 -
Glucocorticoids Erythematous candidiasis
3 drugs 3 2/66.6 1/33.3
+CsA+MMF (1)
Total 44 20/45.5 6/13.3 -
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708 D. Olczak-Kowalczyk, M. Roszkowska-Blaim, M. Pańczyk-Tomaszewska et al.

Figure 12. Erythematous candidiasis in INS medicated with CsA and glucocorticoids (photo by D.
Olczak-Kowalczyk).

Oral infections might be caused by hyperglycemia, insulin resistance, AGE protein

development and accumulation resulting in degradation, destruction and proliferation of
connective tissue proteins.
Nonenzymatic glycation leads to imbalanced synthesis, development and homeostasis of
collagen, resulting in its decreased resistance to vascular integrity and modifications [114-
116]. Diabetes displays a higher glycation level of immunoglobulin, benefiting IgM
immunoglobulin, and disadvantaging the IgG one, and impaired agglutination of IgM. The
high level of glycated IgM is said to have a negative impact on immune response in the early
stage of acute infection [117].
In response to Candida antigens, a Th1 immune response, characterized by an increase in
interleukin 2 and interferon-gamma production, and a concurrent interleukin 4 and 10
shortage or deficiency, is activated. Additionally, CD4+ T regulatory cells decrease in count.
In decompensated diabetes, catabolic processes overcome the anabolic ones, which might
also cause inflammations.

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 Candida Spp. in Oral Cavity of Children with Immunodeficiencies 709

Table 6. Candida species and oral candidiasis in patients with diabetes type 1

Patient
Candida spp. Oral candidiasis
HbA1c number
N=100% N /% N/% clinical presentation
<7% 4 0 0 -
7-8% 9 0 0 -
>8% 13 4/30.7 3/ 23.1
angular cheilitis
Total 27 4/14.8 3/11.1

CANDIDA SPECIES AND DIABETES

Material and Method

The level of glycated hemoglobin (HbA1c), oral yeast (direct mycological test) and oral
candidiasis occurrence was evaluated in 27 patients with diabetes type 1 (mean age 13.3 ± 2.8
years). The criteria for compensated diabetes were HbA1c < 7% - good, 7-8% average, >8%
bad (Table 6).

Results

Candida species was present in the mouth of 4 patients (14.8%), and 3 patients had
angular cheilitis (23.1%). Candida albicans was the only species to be isolated. Yeasts and
oral candidiasis symptoms occurred only in patients with HbA1c >8% (Table 6).

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 28

OXIDATIVE STRESS AND THE DEVELOPMENT OF

ANTIFUNGAL AGENTS FOR THE
TREATMENT OF CANDIDIASIS

Maxwel Adriano Abegg*1 and Mara Silveira Benfato2

1
Institute of Science and Technology, Federal University of Amazonas,
Itacoatiara, AM, Brazil
2
Department of Biophysics, Federal University of Rio Grande do Sul,
Porto Alegre, RS, Brazil

ABSTRACT
In comparison with antibacterial antibiotics, only a small number of antifungal
agents are available, mainly due to the fact that eukaryotic fungal cells present fewer
targets that may enable the generation of drugs with selective toxicity. The drugs
currently used to treat localized or systemic fungal diseases mainly comprise groups of
polyene, allylamines, azoles, flucytosine and griseofulvin, and candin and mycin drugs.
Most of these drugs, such as azoles, inhibit the biosynthesis of ergosterol. Despite an
established arsenal of potent antifungal drugs, the popularity of the most employed group,
azoles, has been compromised by the development of resistance, particularly among
species of non-albicans Candida, limiting treatment options. Therefore, it is fitting to
consider new options to obtain antifungal agents, and the development of the next
generations of antifungals will require a greater understanding of the biology of fungal
pathogens. In this context, some authors claim that the virulence factors of Candida
species are interesting as alternatives to traditional targets. In living yeast cells, reactive
oxygen species (ROS), including singlet oxygen, hydrogen peroxide, and hydroxyl
radicals, are generated as metabolic byproducts from endogenous or exogenous sources.
ROS derived from the intracellular metabolism of oxygen act as signal transducing
molecules, contributing in the activation of transcription factors leading to gene
expression. ROS normally exist in the cell in balance with antioxidants, but when this
critical balance is disrupted due to excessive ROS generation, it generally incurs
significant damage known as oxidative stress. Recent studies have reported that major

* Corresponding author: [email protected]

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718 Maxwel Adriano Abegg and Mara Silveira Benfato

classes of antifungals contribute to induce programmed cell death via the production of
ROS. In fact, it is suggested that the effectiveness of azoles is due in part to the
sensitization of Candida albicans to oxidants produced by phagocytes. The production of
ROS has, for example, proven to be important in the antifungal activity of miconazole
and fluconazole; several peptides with the anti-Candida effect also seem to act via
generation of oxidative stress. Antioxidant defense mechanisms also appear to be
associated with resistance to antifungal agents. There are several indications that there is
a contribution of certain products of genes involved in oxidative stress response to the
development of azole antifungal resistance. The polyene antifungal resistance can
involve, among other mechanisms, increased intracellular catalase activity in the fungus,
preventing the formation of free radicals responsible for pore formation. The
understanding of the association of oxidative stress and antifungals is important in future
approaches to the development of antifungal drugs. In this chapter, we aim to present
updated information about the exploitation of oxidative stress in the development of new
anti-Candida antifungals.

INTRODUCTION
Depending on the methods of sampling and the sites of the organism, Candida spp. are
commensals in up to 71% of the healthy population (Calderone 2002). On average, 25% to
30% of individuals are carriers of Candida albicans, the main pathogenic species of the genus
in the oral cavity, with the highest incidence in young children, infants and people with AIDS.
It is estimated that healthy humans have a colonization index ≥ 50% in the gastrointestinal
tract, and a colonization index between 10 and 20% in the rectal tract and vagina (Odds
1988). Specifically, between 25 and 30% of adolescent and adult women have vaginal
colonization at some point, the latter being particularly prevalent during pregnancy.
Adolescent and adult women have the same distribution of Candida species in the vagina
(Nantel et al., 2002; Fidel, Jr., 2004).
C. albicans is an extraordinarily versatile opportunistic pathogen that does not appear to
possess habitats outside of warm-blooded animals (Cheng et al., 2007). This species causes
most of the fungal infections in humans, and this yeast is the most frequently isolated from
biological samples (Calderone 2002). In general, the studies indicate that around 60% of
cases of infection involve this species, although this is variable according to the site of
infection. However, in addition to C. albicans, a much less virulent non-albicans species of
Candida, such as Candida krusei, were reported as causing systemic candidiasis (Nantel et
al., 2002; Sardi et al., 2013).
In fact, although C. albicans remains the most common infecting species, the
epidemiology of Candida infections has changed considerably in recent years (Sardi et al.,
2013). Candida species rarely associated with human infections in the past have emerged as
serious pathogens, and some of these microorganisms were more difficult to remove than C.
albicans with current therapies. Candidiasis caused by these species is a clinical problem
(Kremery & Barnes, 2002; Redding, 2003). This change can be observed, for example, in
studies with oral swabs of cancer patients, where the non-albicans species hit a percentage of
approximately 25% of cases (Davies et al., 2006).
The pathogenicity of the Candida species is attributed to certain virulence factors, such as
the ability to evade host defenses, adherence, biofilm formation (on host tissue and on

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 Oxidative Stress and the Development of Antifungal Agents for the Treatment … 719

medical devices) and the production of tissue-damaging hydrolytic enzymes such as

proteases, phospholipases and haemolysin (Ribeiro et al., 2012) and antioxidant enzymes
(Abegg et al., 2010).

CANDIDA SPP. AND OXIDATIVE STRESS

As stated previously, C. albicans has a commensal relationship with homeothermic
organisms and thus is expected to live in a relatively stable environment in terms of
temperature and osmotic conditions. In contrast, the oxidative stress can often be challenging
to cells of C. albicans when these become targets of action of phagocytes, for example, which
must occur in the case of other potentially pathogenic and saprophytic Candida species under
normal conditions (Enjalbert et al., 2003). The response of the host towards potentially
dangerous C. albicans and other Candida species includes the recruitment of phagocytes from
blood and tissues, considered as the first line of defense (Miramón et al., 2012).
Currently, it is understood that death by oxidation of fungal cells represents a major line
of elimination of pathogenic microorganisms. Not surprisingly, correlations have been made
between the functioning of the oxidative stress response in certain pathogenic fungi and their
ability to proliferate in the host. Moreover, the study of oxidative stress in yeast, especially
the role of mitochondria, clearly assists in understanding the mechanisms involved in aging,
apoptosis and disease in higher organisms (Moye-Rowley 2003).
The C. albicans phagocytosis is mediated by several receptors, opsonic and non-opsonic.
Complement fixation and activation is mediated by the alternative pathway of complement
activation and is especially important for the chemotaxis and opsonization of C. albicans, but
not to lyse. Furthermore, although C. albicans is recognized by the mannose binding lectin,
the lectin pathway of complement activation probably exerts only a minor effect on the
engulfment of this species by phagocytes. Many membrane-bound receptors contribute to C.
albicans phagocytosis. Among them, dectin-1, mannose receptor (MR), and C-type lectin
receptor (DC-SIGN) demonstrated to directly mediate the engulfment of fungal particles
(Frohner et al., 2009).
According to Naoum (1996), after intake, the death of C. albicans occurs through both
oxidative and non-oxidative mechanisms. The respiratory burst mechanism is an essential
antifungal effector that results in the production of toxic oxidants and proteases in the
activation of granules that can kill C. albicans. The death of this fungus also occurs in the
extracellular environment through undefined actions of fungi pattern recognition receptors
(FPRRs) and galectin-3.
Regarding the importance of oxidative stress in the killing mechanisms of Candida,
Thompson & Wilton (1992) demonstrated that the ability to eliminate C. albicans by
polymorphonuclear cells (PMNs) and macrophages was higher under aerobic conditions than
under anaerobic conditions. Furthermore, they revealed that death by macrophages was
reduced by inhibitors of O2•- and H2O2, and death by PMNs was decreased by inhibitors of
O2•-, H2O2, HOCl and HO•, suggesting that these cells use ROS to eliminate C. albicans and
that the chemical arsenal used differs between these cell types.
Kusch et al. (2007) established the proteomic profile of the oxidative stress response of
C. albicans induced by non-lethal concentrations of H2O2 and diamide. The expressions of
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720 Maxwel Adriano Abegg and Mara Silveira Benfato

proteins 57 and 45 were modified by exposure to these stressors, respectively. The signatures
of induction were almost identical, whereas the repression showed little overlap. Among the
induced proteins were enzymes with antioxidant functions, such as catalase and thioredoxin
reductase and a set of oxidoreductases.
Enjalbert et al. (2007) examined the extent to which the individual cells of C. albicans
activate a response to oxidative stress in specific microenvironments during a systemic
candidiasis process, using reporter genes fused to green fluorescent protein (GFP). The results
indicated that the species is significantly exposed to oxidative stress after phagocytosis by
neutrophils, but few fungal cells are exposed to oxidative stress after exposure to
macrophages or when a kidney infection is established. For these authors, it seemed clear that
oxidative stress is not a constant threat during systemic infection, and fungal cells are most
likely exposed to oxidative stress in the early establishment of a systemic infection when they
come into contact with circulating neutrophils. At this stage, the ability to adapt appropriately
to oxidative stress can promote pathogen survival and the subsequent development of deep
infection. They conclude that the oxidative stress response in C. albicans is niche-specific
during the establishment and progression of systemic infection.
According to Brown et al. (2009), it is now known that the interaction with phagocytes
occupies a large portion of the cycle of disease in candidiasis, so that to survive the infection
sites requires, among other things, inducing signaling pathways that detect oxidants (inlet)
and the downstream production of antioxidants (output) that detoxify these oxidants. Also,
with respect to how relevant the in vitro studies are, there are several genes for which the
roles in in vitro adaptation (generally determined by spot-plate studies) are known, but the
roles in the survival in front of phagocytes were only determined for some genes. Correlations
between in vitro and ex vivo (pool of human phagocytes) functions were made by building
mutants and testing each for susceptibility to oxidants in vitro and survival against phagocytes
ex vivo. Each mutant tested avirulent in murine models of invasive candidiasis. Also,
stationary phase cells of C. glabrata and C. albicans are more resistant to ROS than cells in
the exponential phase. This suggests that there may need to be a vital balance of growth with
stress resistance to proliferate in the host. Chauhan et al. (2006) interprets that there is a
relationship between the data with mutants and data from the in vitro studies.
Interestingly, the inactivation of the enzyme iNOS-2 does not make mice more
susceptible to infection with C. albicans, suggesting that the production of reactive nitrogen
species (RNS) may not be the primary defense against candidiasis and apparently ROS would
be more important. However, RNS are protective in oral candidiasis (Brown et al., 2009).
Miramón et al. (2012) stated that upon phagocytosis, the phagosome fuses with
preformed granules containing several enzymes (e.g., cathepsin G, neutrophil elastase) and
antimicrobial cationic peptides (a-defensins). Then, the neutrophils produce copious amounts
of oxidants in the respiratory burst process. This production involves the assembly of the
NADPH oxidase enzyme complex on the plasma and phagosomal membranes of the
phagocyte. The NADPH complex produces the highly reactive superoxide anion (O2•-), which
is further metabolized to form hydrogen peroxide (H2O2). Other reactive species are produced
inside the neutrophil, such as peroxynitrite (ONOO-), which is formed upon production of
nitric oxide (NO•) by the inducible nitric oxide synthase (iNOS). Additional compounds with
oxidative properties (for example, hypochlorous acid, HClO), are produced by
myeloperoxidase, an enzyme that is highly abundant in neutrophil granules. Neutrophil
activities involve the degranulation and secretion of peptides and enzymes stored in the

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 Oxidative Stress and the Development of Antifungal Agents for the Treatment … 721

neutrophil granules. Among the granule components, myeloperoxidase, lactoferrin and

azurocidin are known to have candidacidal properties. The production of neutrophil
extracellular traps (NETs) provides another mechanism by which neutrophils contain
infection, which involves the extrusion of chromatin-scaffolding, net-like structures with
antimicrobial proteins.
However, according to Miramón et al. (2012), the relative contributions of neutrophil
activities to fungal clearance and the relative importance of the fungal responses that
counteract these activities remain unclear. This group studied the contributions of the intra-
and extracellular antifungal activities of human neutrophils using diagnostic GFP-marked C.
albicans strains. They found that a carbohydrate starvation response, as indicated by the
upregulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus.
Similarly, the nitrosative stress response was only observed in internalized fungal cells. In
contrast, the response to oxidative stress was observed in both phagocytosed and non-
phagocytosed fungal cells, indicating that oxidative stress is imposed both intra- and
extracellularly. This may demonstrate the high importance of oxidative mechanisms in the
clearance of Candida.
In one study, on the issue involving the greater effectiveness of neutrophils compared to
monocytes/macrophages in their ability to kill C. albicans, Fradin et al. (2005) compared the
gene transcription profiles of C. albicans incubated with different human blood cells and
whole blood. Among the many findings, the fraction of PMNs closely resembled the events
that occurred in whole blood with respect to the growth of the organism and gene
transcription. Yeast cells in whole blood were predominant, while in erythrocytes, monocytes
or plasma, the shape of hyphae was predominant. Approximately 97% of yeast cells remained
in PMNs. Also in these cells, 16 of 18 antioxidant genes were induced, while only 2 of 18
were induced in mononuclear cells (MNs). Thus, the transcriptional events that occur in
human neutrophils and monocyte populations are significantly different, and these differences
appear to correlate with the increased growth inhibitory activity of C. albicans by PMNs
compared to MNs.
Accordingly, Koh et al. (2008) observed that the selective depletion of neutrophils
combined with a disruption of the gastrointestinal mucosa resulted in infection by C. albicans
and 100% mortality in a model for gastrointestinal colonization in rats. The selective
depletion of neutrophils, macrophages, lymphopenia or gastrointestinal disruption alone
resulted in no mortality.
Xiong et al. (2000) reported that systemic candidiasis also occurs in hosts with normal
neutrophil function, suggesting that cells other than these also play an important role in the
host defense system. When hosts are neutropenic, mononuclear cells, especially
monocytes/macrophages, contribute to the defense against infections. These authors
hypothesized that the Candida species that differed in pathogenicity could differentially
induce immune regulatory cytokine production of human monocytes and demonstrated that C.
krusei, but not C. albicans, clearly induces IL-12 production of monocytes, indicating that
species such as C. albicans have the ability to create an environment rich in IL-10 and low in
IL-12 and IFN-γ, which could generate a state in the host more susceptible to candidiasis.
Candida species also demonstrate differential responses to oxidative agents in vitro, which
may correlate with their invasion and infection capacities (Abegg et al., 2010; 2011; 2012).
Fungal pathogens have different routes of infection, and this influences their response to
the chemical arsenal of phagocytes. This may also have contributed to the evolutionary
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722 Maxwel Adriano Abegg and Mara Silveira Benfato

divergence of the stress response in different species. In fact, the data suggests that different
organisms evoke different responses in the host cell or different organisms respond differently
to the same neutrophil oxidative environment, for example, and use different mechanisms to
detoxify ROS and ensure their survival (Moye-Rowley, 2003; Rubin-Bejerano et al., 2003).
Also, Brown et al. (2009) suggest that responses to ROS and RNS make contributions to the
differential pathogenicity, depending on the type of pathogen, the gateway to the host and the
type and stage of infection.
It is possible to note that Candida species require ROS detoxification systems to succeed
as potential pathogens. These systems include enzymatic antioxidants (superoxide
dismutases, catalases, peroxidases, glutathione peroxidase, etc.) and non-enzymatic
antioxidants (glutathione, peroxiredoxin, etc.), which assure the rapid turnover of ROS to
maintain the redox homeostasis (Aguirre et al., 2005; Scott & Eaton, 2008). Mutants
defective in pathways required for detecting oxidative stress often have reduced virulence, for
example, as in the case of mutants associated with the stress-activated MAP kinase of C.
albicans (Alonso-Monge et al., 2003).

THE OXIDATIVE STRESS AND THE DEVELOPMENT OF

ANTI-CANDIDA DRUGS
The invasive fungal infections are historically associated with high morbidity and
mortality, due in part to the limitations of antifungal therapy available and the difficulties in
making a rapid and accurate diagnosis. Compared with antibacterial antibiotics, only a small
number of antifungal agents are available, mainly due to the fact that eukaryotic fungal cells
present a smaller number of targets at first sight that may enable the generation of drugs with
selective toxicity (Sable et al., 2008).
According to Gauwerky et al. (2009), drugs commonly used for the treatment of systemic
or localized fungal diseases mainly comprise groups of polyene, allylamines and azoles,
flucytosine and griseofulvin. Most of these drugs, such as azoles, inhibit the biosynthesis of
ergosterol.
Vandeputte et al. (2012) states that, despite extensive research dedicated to the
development of new therapeutic strategies, there are only a limited number of available drugs
to fight against invasive fungal infections. Indeed, only four molecular classes that target
three distinct fungal metabolic pathways are currently used in clinical practice to treat
essentially systemic fungal infections: fluoropyrimidine analogs, polyenes, azoles, and
echinocandins. Several other classes, such as morpholines and allylamines are only used as
topical agents due to either poor efficacy, or severe adverse effects when administered
systemically.
Flucytosine inhibits fungal protein synthesis at the level of DNA/RNA, with well
characterized resistance problems and has a very limited spectrum of action. Griseofulvin
interferes with the production of intracellular microtubules, inhibiting fungal mitosis. This
drug only has an effect against dermatophytes (Gauwerky et al., 2009). Table 1 presents the
drugs most commonly used in candidiasis.

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 Oxidative Stress and the Development of Antifungal Agents for the Treatment … 723

Table 1. Antifungal agents

anti-Candida site of action example structure

drugs
allylamines* biosynthesis of terbinafine
ergosterol

azoles biosynthesis of fluconazole

ergosterol
and ROS

candin cell wall echinocandin

hydroxypyridone Chelation of metal ciclopirox

cations(?) and
ROS(?)

micin cell wall nikkomycin

polyene cell wall and amphotericin

ROS(?) B

flucytosine DNA/RNA flucytosine

synthesis

griseofulvin microtubules griseofulvin

production

*only used as topical agents, all the others are also used for systemic infections.
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724 Maxwel Adriano Abegg and Mara Silveira Benfato

Certain new antifungals work in the production/integrity of the fungal cell wall as
candins (echinocandin, pneumocandin and papulacandin) and micin drugs (nikkomycin,
pramidicin and benanomicin). According to Gauwerky et al. (2009), it can be concluded that
a full arsenal of powerful antifungal drugs was established during the last 50 years. However,
the popularity of the most widely used group of antifungals, the azoles, has been
compromised by the development of resistance, particularly among species of non-albicans
Candida, limiting treatment options in many cases. Since treatment options are limited, the
development of the next generation of antifungals will require a greater understanding of the
biology of fungal pathogens (Aratani et al., 2002; Cowen et al., 2002).
Thus, fungal diseases remain a challenge, and it is interesting to proceed with the
development of antifungal agents that interfere with the production of the main elements of
the cell membrane or cell wall. In light of these increasing problems with resistance to
antifungal agents, however, some authors argue that it is also mandatory to consider new
options. In this context, the virulence factors offer interest as alternatives to traditional
targets; according to Gauwerky et al. (2009), these include antioxidant enzymes such as
catalases, aspartic proteinases (from C. albicans, particularly), melanin and phospholipases.
According to Saijo et al. (2010), it is assumed that the oxidative stress response is directly
related to pathogenicity, and thus the inhibition of this response can lead to attenuation of
virulence. Still, these authors point out that clarifying the mechanisms of oxidative stress
responses in pathogenic fungi could assist in meeting targets for new antifungal agents.
As presented in this chapter, some classes have or might have a mechanism of action
involving damage to Candida cells throughout the generation of ROS (Table 1). In fact, the
mechanisms of action of antifungal drugs are still not fully understood (Gray et al., 2012), and
the participation of ROS in the effectiveness of anti-Candida drugs still needs to be
investigated.
There is data that maintains that cell damage caused by antifungals increases sensitivity
to oxidative damage and is part of the mechanism of action of azoles (Rogers & Barker,
2002) and a number of other substances. Until the present, there have been diverse
descriptions associated with the antifungal actions of substances throughout prooxidant or
inhibition of the antioxidant activity of Candida spp. Some of these substances are currently
used as antifungals, and others have been recently described as having anti-Candida
effects.Among currently used antifungals, according to Roilides et al. (1990), azoles do not
suppress the function of PMN and may even select functions of these cells in vitro. It has also
been suggested that the effectiveness of azoles is due in part to sensitizing C. albicans to
oxidants produced by macrophages. Also, the different sensitivities to oxidants and
neutrophils was reported for opaque and white cell types associated with one strain of C.
albicans (WO-1) (Jamieson et al., 1996).
The production of ROS is important in the antifungal activity of miconazoles, and it is
likewise important that fluconazole is able to induce the production and accumulation of ROS
in C. albicans (Kobayashi et al., 2002). Azoles change the activity of antioxidant enzymes
such as cytochrome c oxidase, peroxidase and catalase (Thomas, 1986; Ghannoum & Rice,
1999).
Ciclopirox inhibited the growth of C. albicans yeast and hyphal cells in a dose-dependent
manner. This effect was reduced: (i) by the addition of iron ions or the metabolic inhibitor 2-
deoxy-D-glucose to growth media, (ii) in media that lacked glucose, and (iii) for cells that
were pre-incubated with hydrogen peroxide or menadione (which caused induction of

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 Oxidative Stress and the Development of Antifungal Agents for the Treatment … 725

proteins involved in the detoxification of ROS). In contrast, cells pre-cultured under poor
oxygen conditions (which had decreased activity of proteins involved in ROS detoxification)
were more susceptible to ciclopirox (Sigle et al., 2005).
The hydroxypyridone antimycotics, ciclopirox, rilopirox and piroctone, belong to the
antimicrobials that show remarkable activity against fungi, including Candida species. Sigle
et al. (2006) also reported that the mode of action of hydroxypyridone antimycotics,
particularly rilopirox and piroctone, is not fully understood. Data from this group indicates
that an active metabolism in the target cell and a presence of oxygen are important factors.
Especially ROS seem to play an important role in the fungicidal action of rilopirox and
piroctone.
Fluconazole is often used as the first-line therapy for candidiasis. Among non-albicans
species, C. krusei is the only species that is predictably fluconazole resistant. C. krusei
appears to be resistant to ROS and to possess a potent antioxidant system (Abegg et al.,
2010). Recently, Costa-de-Oliveira et al. (2012) showed that fluconazole induced a low
percentage of ROS formation by C. krusei cells. These results may suggest that the fungistatic
mechanism of this azole is not based upon ROS formation. However, when the alternative
oxidase enzyme (AOX) of C. krusei was inhibited, an increase in the intracellular ROS levels
was noted. The authors conclude that AOX activity allows the yeast cells to reduce ROS
accumulation when challenged by antifungals like fluconazole, leading to drug tolerance and
suggests that the alternative respiratory pathway (ARP) is a potential target that should be
taken into account when considering the development of new therapeutic strategies in the
case of C. krusei infections. Future approaches may lead to species-specific, anti-Candida
drugs.
Some research has suggested that polyene drugs are able to induce an oxidative stress
(particularly in C. albicans), and their activity seems to be reduced in hypoxic conditions
(Vandeputte et al., 2012).
In some cases, for even widely used antifungals, the mechanisms of action still need to be
fully described and the participation of oxidative stress needs to be evaluated. For example,
amphotericin B (AmB) is a prototypical small molecule, natural product that can form ion
channels in living eukaryotic cells and has remained refractory to microbial resistance,
despite extensive clinical utilization for more than half a century in the treatment of life-
threatening fungal infections. It is accepted that AmB kills yeast primarily via channel-
mediated membrane permeabilization. Gray et al. (2012), by the synthesis of a functional
deficient derivative group of this natural product, recently discovered that channel formation
is not required for potent fungicidal activity. Alternatively, AmB primarily kills yeast by
simply binding ergosterol, and membrane permeabilization via channel formation represents a
second complementary mechanism that further increases drug potency.
In addition to the fungal cellular membrane, the cellular antioxidant system can also be a
viable target in the antifungal action of amphotericin B (AmB). Co-applications of certain
redox-potent natural compounds with AmB actually increases efficacy of the drug through
chemosensitization. Some redox-potent chemosensitizers and AmB perturb common cellular
targets, resulting in the synergistic inhibition of fungal growth. Kim et al. (2012) tested
chemosensitizing activities of four redox-potent benzaldehydes against clinical and reference
strains of C. albicans, C. krusei, C. tropicalis, and Cryptococcus neoformans in combination
with AMB. Two dihydroxybenzaldehydes (DHBAs), i.e., 2,3-DHBA and 2,5-DHBA,
significantly enhanced the activity of AMB against most strains.
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Some substances show potential as new antifungal drugs, and have actions related to
oxidative stress. Strong evidence demonstrates that for the fungicidal action of histatin-5, the
formation of ROS is the final step and essential for the effect (Helmerhorst et al., 2001).
Calera et al. (2000) suggested that the identification of novel targets for drug discovery is
also important, and one possibility is Ssk1p protein, which plays an important role in
virulence control and protection against ROS in neutrophils.
Peng et al. (2012) demonstrated that results of antifungal susceptibility showed that the
inhibition of alternative respiration with salicylhydroxamic acid enhanced azole’s
susceptibility to C. glabrata. All azole-resistant isolates studied were respiratory-deficient,
with a reduced generation of ATP and ROS and decreased oxygen consumption. The authors
concluded that the alternative respiratory pathway plays an important role in the decreased
susceptibility to azole antifungals.
Shirai et al. (2012) describe the action of gemini-pyridinium salts by ROS in C. albicans.
According to Tebbets et al. (2012), the identification of novel antifungals is hindered by
the limited number of drug targets that are unique to fungi due to the close evolutionary
relationship between fungi and mammals. Oxidative damage appears to be involved in the
mode of action of a compound, compound 13, obtained by this group. This compound
showed robust activity against numerous fungal genera including Candida spp., Cryptococcus
spp. and molds such as Aspergillus fumigatus and Rhizopus oryzae. Drug-resistant C.
albicans from patients were also highly susceptible to compound 13. The compound was also
highly active against C. albicans biofilm, in vitro and in vivo, and exerted synergy with
fluconazole, which was inactive alone.
Kim et al. (2013) recently reported the production of a biotransformation product of
rhapontin, called rhapontigenin, which was active against C. albicans isolates in vitro
(minimal inhibitory concentration - MIC values of 128-256 μg/ml). The authors detected
increased ROS levels in yeast cultures treated with rhapontigenin at the MIC. As expected for
a drug mechanism involving ROS generation, the substance inhibited DNA, RNA, and
protein synthesis, especially RNA synthesis, and induced morphological changes and
apoptosis.
Also against non-albicans Candida, there are examples of substances that work
throughout the generation of ROS. Kang et al. (2011) described the action of metergoline, a
serotonin receptor antagonist, which has a good anti-Candida krusei effect.
Antimicrobial peptides (AMPs) are ubiquitous and multipotent components of the first
line of defense against an invasion by pathogens for most living organisms. Park & Lee
(2010) described the action of melittin, a naturally occurring antimicrobial peptide, which is
the principal toxic component of the European honeybee venom, Apis mellifera. Melittin also
seems to act in C. albicans cells throughout increases in the generation of ROS.
The peptide psacotheasin has been reported to exhibit broad antimicrobial activity against
human pathogenic microbial strains, and to have membrane-active activity by the perturbation
and disruption the plasma membranes in C. albicans. Hwang et al. (2011a) presented that one
mode of action of this peptide occurs via apoptosis through induction of ROS.
Mello et al. (2011) have reported the induction of ROS and nitric oxide in fungal cells
treated with the peptide PvD1.
Ribeiro et al. (2012), described a 6,000 Da peptide, named CaTI, isolated from Capsicum
annuum L. seeds. This peptide showed potent inhibitory activity against trypsin and
chymotrypsin. The group determined the effect of CaTI on Saccharomyces cerevisiae, C.

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 Oxidative Stress and the Development of Antifungal Agents for the Treatment … 727

albicans, C. tropicalis and Kluyveromyces marxiannus cells. CaTI inhibited the growth of S.
cerevisiae, K. marxiannus as well as C. albicans. They used dyes that indicated the presence
of ROS to evaluate whether the action mechanism of CaTI involved the induction of
oxidative stress. CaTI induced higher levels of nitrosatives, such as NO, and oxidative
species, which may be involved in different metabolic mechanisms of yeast growth arrest or
death. Aerts et al. (2009) also utilized the dye DAF-2 DA to demonstrate the generation of
ROS by C. albicans treated with the peptide Rs-AFP2. This group has demonstrated that the
growth inhibition of C. albicans by Rs-AFP2 is a consequence of apoptosis and showed that
the process is independent of the metacaspase-1 pathway. These findings provided a direct
link between ROS generation and the antifungal effects of certain antifungal peptides.
Among natural products, Sharma et al. (2010) described the antifungal effects of a natural
polyphenol, CUR (curcumin), against albicans and non-albicans species of Candida and have
shown its ability to inhibit the growth of all the tested strains. This study provides the first
evidence that CUR acts as an antifungal agent, via generation of oxidative stress.
Several in vivo and in vitro studies have shown the great effectiveness of garlic against a
broad spectrum of yeasts. At least in part, this effect may be related with the generation of
ROS (Cardelle-Cobas et al., 2010).
Hwang et al. (2011a; 2012) have described different natural products that work as anti-
Candida substances throughout the generation of ROS. One of those substances is the
phytochemical (+)-Medioresinol, a furfuran-type lignan identified and isolated on the stem
bark of Sambucus williamsii (Hwang et al., 2012). Indole-3-carbinol (I3C), found in all
members of the cruciferous vegetable family, which includes cabbage, broccoli, Brussel
sprouts, cauliflower and kale, demonstrated a significant capacity to increase the ROS and
hydroxyl radical accumulation in C. albicans treated with this substance hand also has anti-
cancer effects (Hwang et al., 2011b).
In cells exposed to pleurocidin, derived from the skin mucous secretion of the winter
flounder Pleuronectes americanus, intracellular ROS, which is a major cause of apoptosis,
were increased, and hydroxyl radicals were especially a large part of ROS. The increase of
ROS induced oxidative stress and mitochondrial membrane depolarization which causes a
release of pro-apoptotic factors (Cho et al., 2011).
Although Candida species exhibit a planktonic unicellular form, they commonly show
filamentous growth or complex, multicellular structures in the infected tissues. These
structured microbial communities, known as biofilms, can attach to surfaces and encase
within a matrix of exopolymeric materials. The latter can also form on various implanted
medical devices such as vascular and urinary catheters, joint prostheses, cardiac valves,
artificial vascular bypass devices, and those being topically used including contact lenses and
dentures. The complex structure of biofilms makes them resistant to both host defense and
commonly used antifungal drugs. According to Sun et al. (2012), to date, several papers have
reported the fungicidal effect of non-thermal plasma against C. albicans. This group also
found that non-thermal plasmas can effectively inactivate strains of Candida species
including fluconazole-resistant C. albicans, C. glabrata and C. krusei in their planktonic
form. A direct current atmospheric pressure He/O2 (2%) plasma microjet (PMJ) was used to
treat Candida biofilms in a 96-well plate. ROS, such as hydroxyl radicals, superoxide anion
radicals and singlet molecular oxygen, were detected by electron spin resonance (ESR). The
authors concluded that He/O2 (2%) plasma alone, as well as in combination with common
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728 Maxwel Adriano Abegg and Mara Silveira Benfato

antifungal drugs, is able to rapidly inactivate Candida biofilms. The generation of ROS is
believed to be one of the underlying mechanisms for the fungicidal activity of plasma.
In another situation, Zhang et al. (2009) show that, considering the proximity between
ROS and various diseases, there is a continuing interest in finding antioxidants as preventive
or therapeutic drugs. According to the MDL Drug Data Report (MDDR), seven antioxidant
drugs were introduced by 2009, most of these (lipoic acid, policosanol, acetylcysteine,
probucol and idebenone) directly or indirectly derived from natural antioxidants. Exploring
how organisms use antioxidants to combat ROS is therefore of great importance to drug
discovery.
On the question whether knowledge about the mechanisms of adaptation to oxidative
stress can be applied in immune potentiation, Chauhan et al. (2006) point out that the
pretreatment of C. albicans with a monoclonal antibody directed to oligomannan acid-labile
cell walls increases the organism's death by neutrophils (Caesar-Tonthat & Cutler, 1997).
Thus, the passive transfer of antibodies may be protective in high-risk patients.
Pfaller & Diekema (2012) revealed that the antifungal susceptibility testing of Candida
has been standardized and refined and may now play a useful role in managing Candida
infections. Important new developments include validation of 24-h reading times for all
antifungal agents, and the establishment of species-specific epidemiological cutoff values
(ECVs) for the systemically active antifungal agents and both common and uncommon
species of Candida. According to Pfaller & Diekema (2012), in association with the
development of new drugs with alternative mechanisms of action, including through
oxidative stress, there have been improvements in the ability of antifungal susceptibility
testing methods to detect emerging resistance patterns, coupled with molecular
characterization of resistance mechanisms. These advances would provide adjuncts to
optimize the effectiveness of antifungal therapy in candidiasis.
Antioxidant defense mechanisms also appear to be associated with resistance to
antifungal agents. The resistance to polyene antifungals (AmB, nystatin) may involve, among
other mechanisms, an increase in the intracellular catalase activity of the fungus, preventing
the elevation of levels of free radicals responsible for pore formation (Kerridge & Nicholas,
1986; Sokol-Anderson et al., 1988). The antioxidant enzyme superoxide dismutase (Cu/Zn
SOD) is required for the occurrence of oxytetracycline resistance in S. cerevisiae (Avery et
al., 2000).
A comprehensive profile analysis of gene expression in C. albicans revealed the
coordinated regulation of genes associated with the gradual acquisition of resistance to azoles,
especially fluconazole, in clinical isolates of the fungus. Several of these genes are involved
in the oxidative stress response, such as GPX1 encoding the enzyme glutathione-peroxidase
(GPx), suggesting that reduced susceptibility to oxidative damage can contribute to the
acquisition of resistance (Rogers & Barker, 2002). The CAP1 gene of C. albicans is involved
in multidrug resistance and also in the oxidative stress response in this yeast. Still, the data
indicates that it may be important to understand the antioxidant defense system. Furthermore,
the adaptive response ability in non-albicans Candida species is of medical importance, since
these mechanisms may be species specific and probably contribute to antifungal resistance
mechanisms (Alarco & Raymond, 1999).
Linares et al. (2006) demonstrated that antineoplastic methotrexate increased the activity
of catalase and the antifungals fluconazole and amphotericin B increased the activity of SOD

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 Oxidative Stress and the Development of Antifungal Agents for the Treatment … 729

and catalase in strains of C. dubliniensis and C. albicans resistant to these antifungals,

indicating an oxidizing effect of these drugs.
In fact, there are several indications that there is a contribution of certain products of
genes involved in oxidative stress response with the development of resistance to azole
antifungals, and even the production of ROS, in the mechanism of action of antifungals. The
understanding of this possible association is important to fully elucidate the mechanisms that
generate resistance to these drugs and future approaches to a rational development of
antifungal drugs (Kovacic & Becvar, 2000; Chauhan et al., 2006).

CONCLUSION
Despite that antifungals developed to inhibit antioxidant molecules of Candida cells or to
exploit the generation of free oxygen radicals against this fungi are still not in clinical use, the
search for novel drugs with alternative mechanisms of action seems to be an interesting field
of exploration that could lead to species-specific anti-Candida antifungals for the treatment of
candidiasis caused by Candida strains naturally resistant to currently used antifungals.

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Linares, CEB; Griebeler, D; Cargnelutti, D; Alves, SH; Morsch, VM; Schetinger, MRC.
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 29

INHALATION AND TOPICAL STEROID THERAPY AND

ORAL CANDIDIASIS: A BRIEF OVERVIEW

Arjuna N. B. Ellepola1,, H. M. H. N. Bandara2

and Hugh D Smyth2
1
Faculty of Dentistry, Health Sciences Center, Kuwait University, Kuwait
2
College of Pharmacy, The University of Texas at Austin, Austin, TX, US

ABSTRACT
Candida-induced infection in the oral cavity is by far the commonest human fungal
infection, which manifests in a variety of clinical guises. The foremost reason for its high
occurrence appears to be the wide array of predisposing factors, which facilitate the
transformation of oral commensal Candida to a parasitic existence. One such
predisposing factor is the prolonged usage and off-targeting of inhaled corticosteroids,
where oral candidiasis is a common side effect of such therapy. Due to the anti-
inflammatory and immunosuppressive effects steroids are used in the management of
bronchial asthma and oral mucosal diseases. In this chapter we briefly discuss the clinical
and laboratory findings on the relationship between steroids inhalers, other topical
steroids and oral candidiasis, possible mechanisms of pathogenicity following such
exposure to steroids as well as the precautions that could be taken to minimize this oral
side effect of steroid therapy.

INTRODUCTION
Candidiasis is by far the commonest oral fungal infection in man and manifests in a
variety of clinical guises. These range from pseudomembranous (thrush), erythematous, linear
gingival erythema associated with HIV infection to Candida-associated denture stomatitis and
median rhomboid glossitis and angular stomatitis, possibly of multifactorial origin. The main
*
Address of corresponding author: Dr. Arjuna Ellepola (BDS, PhD, FIBiol), Department of Bioclinical Sciences,
Faculty of Dentistry, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait. Email: [email protected];
Telephone: 00965 24636714; Fax: 00965 25326049.
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736 Arjuna N. B. Ellepola, H. M. H. N. Bandara and Hugh D Smyth

reason for the high incidence of oral candidiasis appears to be the multiplicity of predisposing
factors, which facilitates the conversion of commensal Candida to a parasitic existence. Thus,
all forms of oral candidiases are considered opportunistic. For instance, the advent of the
human immunodeficiency virus (HIV) infection has resulted in a resurgence of oral candidal
infections. It has been reported that more than 90% of HIV-infected individuals develop oral
candidiasis, as one of the commonest and earliest manifestations [1]. Further, the increasing
prevalence of other compromised patient groups in the community, common endocrine
disorders such as diabetes mellitus and severe nutritional deficiencies have resulted in
resurgence of oral candidiasis as a relatively common affliction [2]. Apart from this, oral
candidiasis can manifest as an adverse effect of drug therapy such as broad-spectrum
antibiotics, cytotoxics and corticosteroids [2].
In this chapter we briefly discuss the clinical and laboratory findings on the relationship
between steroid inhalers, other topical steroids and oral candidiasis as well as the possible
mechanisms of pathogenicity following steroid exposure. The precautions that could be taken
to minimize this adverse side effect of topical steroids and alternative treatment options are
also outlined.

THE EFFECT OF CORTICOSTEROIDS IN THE INFLAMMATORY AND

IMMUNE RESPONSE
Corticosteroids inhibit many of the processes associated with inflammation and the
immune response. The anti-inflammatory and immunosuppressive properties of
corticosteroids reside mainly with their inhibitory action on eicosanoid synthesis and cytokine
production from lymphocytes. The inhibition of these compounds also has further effects for
other mediators of inflammation and the immune responses [3]. Briefly, in its early stages
they reduce the capillary permeability caused by histamine and bradykinin, which in turn
reduce edema. They also inhibit both bradykinin formation and the migration of white blood
cells into the site of inflammation. In its latter stages, steroids reduce granulation tissue
formation by inhibiting the proliferation of fibroblasts and blood vessels. As for the immune
system, steroids decrease the production of interleukin (IL) -2, which subsequently decrease
the action of T-helper cells and the clonal proliferation of T cells. They also decrease the
synthesis of other cytokines and tumor necrosis factor-α (TNF-α) and decrease the
complement proteins in plasma. Further, they reduce the efficacy of cytokine-activated
macrophages [3].

STEROID INHALATION THERAPY

Corticosteroid inhalers are increasingly used in the management of bronchial asthma in
patients requiring continuous treatment [4, 5]. It is now recognized that suppression of the
inflammatory cascade should be the cornerstone of management of bronchial asthma and
therefore inhaled corticosteriods are the most effective and widely used form of anti-
inflammatory therapy in such patients [4]. Many preparations of steroid inhalers are
commercially available for the management of bronchial asthma. These include

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 Inhalation and Topical Steroid Therapy and Oral Candidiasis 737

beclomethasonedipropionate, fluticasone propionate, budesonide, triamcinolone acetonide,

betamethasone valerateand mometasonefuroate.
Aerosol steroid preparations have reduced or eliminated the need for systemic steroids
and their resultant complications in many patients [6, 7]. It also improved the compliance of
the patients. Interestingly, the new formulations are making the usage of inhaled
corticosteroids more convenient to asthmatic patients. For example, fluticasone furoate once
daily administration (400µg/day) was as effective as twice a day administration of fluticasone
propionate (200µg/day) and showed an increase in FEV1 (Forced Expiratory Volume in 1
second) by 200ml after 8 week of treatment [8]. However, usage of inhaled corticosteroids in
chronic obstructive pulmonary disease (COPD) is still controversial. According to an
extensive analysis done by Yang 2012, inhaled corticosteroids did not consistently reduce the
rate of decline in FEV1 in COPD patients. It also failed to improve mortality though long
term usage reduced exacerbations and lowered the rate of decline of quality of life [9].
Conversely, when fluticasone furoate combined with vilanterol, a long acting β2 agonist to
develop an inhalational therapy to both COPD and asthma, it showed statistically significant
improvement of FEV1 after 4 weeks suggesting that combination drug therapy would be
more effective in treating COPD [10]. However the age restrictions must be applied for drug
combination therapy as it would increase the risk of oral steroid treated exacerbations and
hospital admissions in children under the age of 12 years [11]. Nonetheless, long term
inhalational therapy of mometasonefuroate and beclomethasonedipropionate in children
below 12 years old was well tolerated with minimum side effects [12]. One of the most recent
developments in treating asthma is to have small-particle inhaled corticosteroids. Over
conventional inhaled corticosteroids, small-particle steroids are able to reach small airways
thus, having improved total lung deposition and low quantities administered. It also provides
high safety margin and low incidence of oropharyngeal candidiasis though some studies
question the improved safety of small particle inhaled steroids [13].
However, there is considerable clinical and experimental evidence to suggest that inhaled
corticosteroids enhance the development and severity of oral candidal infections [12]. For
instance inhaled corticosteroids are accountable for oral candidiasis in about 10–30% of
patients on these drugs [7]. In the fluticasone furoate/vilanterol treatment, 8% of patients
showed oral candidiasis after 4 weeks of treatment [10]. Although oral candidiasis responds
well to treatment, it can be bothersome, sometimes painful and disconcerting for the afflicted
patients [6, 14].
Although inhaled corticosteroids are intended to be administered to the airways, and
more specifically, the deep lung, considerable fractions of the dose delivered to the patient
does not reach that target site. Typically, for currently marketed and approved technologies,
an inhaler device will only deliver approximately 20-40% of the emitted aerosol to the lungs.
The remaining 60-80% is deposited in the oropharyngeal region and is directly related to the
development of oral candidiasis side effects. The physics of aerosol generation are complex
and depend on the type of inhaler technology being used. There are three main modes of
aerosol generation for inhaled corticosteroids: (1) aqueous droplet formation from airjet,
ultrasonic, or vibrating plate nebulizers, (2) hydrofluoroalknane (HFA) propellant driven
metered dose inhalers (pMDIs), and (3) dry powder inhalers (DPIs) [15-17].
Most inhaled corticosteroid therapy is currently administered using pMDIs and DPIs due
to their convenience, improved portability, rapid dose administration, and relatively low cost.
pMDIs are typically formulated as suspension systems whereby the steroid active is
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738 Arjuna N. B. Ellepola, H. M. H. N. Bandara and Hugh D Smyth

micronized to approximately 1-5 microns and suspended in the propellant. Generation of the
aerosol from pMDIs is achieved by the patient by depressing the pressurized canister into the
actuator which opens a precise metering valve that empties the rapidly expanding formulation
through a spray nozzle. This action results in propellant droplet formation due to propellant
cavitation and flashing [18]. The droplets contain either microcrystalline drug particles or the
drug in solution. The solution based aerosols generate smaller particle sizes and allow great
deposition in the deep lung, and less off targeting to the oropharynx. As a result of this
improved aerosol performance and as a consequence of reduced oropharyngeal deposition,
beclomethasonedipropionate HFA solution systems appear to have lower rates of
oropharyngeal candidiasis and dysphonia [19]. However, propellant based metered dose
inhalers require the patient to coordinate actuation of the device with their inhalation
maneuver to prevent loss of the aerosol to the mouth. Use of spaces can minimize off-
targeting due to coordination issues but there is many studies suggesting that patient
compliance and acceptance of these devices is poor.
Dry powder inhalers were originally developed in the late 1960’s and early 1970’s and
have the advantage of being patient breath activated – thus obviating the need for excellent
coordination on the part of the patient. DPIs dramatically increased in their importance as the
pharmaceutical industry searched for replacement technologies as the chloroflourocarbon
propellants were phased-out under the Montreal protocol due to their adverse effects on the
stratospheric ozone layer [17].
Dry powder inhalers now are among the leading device technologies used in asthma and
for inhaled corticosteroids. For example, Advair™ (fluticasone and salmeterol) and
Symbicort™ (budesonide and formoterol) have been leading dry powder inhaler products in
the USA and European markets respectively. Advair™ has been in the top 10 of all
prescription drugs for US sales since 2003. However, currently marketed dry powder inhalers
also suffer from poor lung delivery efficiencies and typically deliver 70-80% of the drug to
the oropharynx. Because particles must have aerodynamic diameters between 1-5 microns to
be delivered to the deep lung, inter-particle forces causes significant aggregation of these
powders. This aggregation is difficult to disrupt and re-disperse the particles so that they are
not impacted upon the mouth and throat upon inhalation. Recent developments in dry powder
inhaler design may solve these issues of poor aerosolization whilst maintaining the advantage
of being patient breath activated [20].

TOPICAL CORTICOSTEROID THERAPY

Due to the anti-inflammatory and immunosuppressive effects steroids are used topically
in the management of oral mucosal diseases such as recurrent oral ulceration, erosive lichen
planus, erythema multiforme and pemphigus [3], which could in turn lead to clinical oral
candidiasis in some patients [21]. These topical preparations include hydrocortisone sodium
succinate 2.5 mg, triamcinolone acetonide 0.1%, betamethasone sodium phosphate 0.1%,
fluocinoloneacetonide 0.1% (topical), hydrocortisone acetate and, triamcinolone hexacetonide
(intralesional).

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 Inhalation and Topical Steroid Therapy and Oral Candidiasis 739

STEROID INDUCED ORAL CANDIDIASIS WITH INHALED

CORTICOSTEROIDS
Dennis and Itkin [22] first drew the attention of the clinical community to the relationship
between steroid inhalers and oral candidiasis when 20% of the patients whom they treated
with dexamethasone inhalers for asthma, developed oral candidiasis during therapy. Since
then a substantial body of information has appeared either confirming or refuting these
observations. Nevertheless a review of literature including all trials with more than 100
patients inhaling steroids for more than 6 months indicates 4–16% incidence or prevalence of
oral candidiasis in these cohorts. Though it is difficult to directly compare these studies due to
differences in population groups, absence of control populations, concurrent antibiotic usage,
previous exposure to steroid therapy and varied sampling techniques, the consensus would
appear to be that steroid inhalers in general promote the oral carriage of Candida and/or
clinical infection, which may usually present as the erythematous (previously atrophic) or the
pseudomembranous variant. Other general points of interest are that some investigators have
revealed that the propensity to develop clinical candidiasis is greater in patients
harbouringCandida intra-orally prior to usage of steroid inhalers [6], that the lesion is
generally localized in areas where the aerosol is deposited [23], and the degree of candidiasis
is probably related to dosage and frequency of therapy [24]. Recent research also proved the
dose dependence of the incidence of isolating Candida and the severity of candidiasis in
asthmatic patient under treatment of inhaled steroids (Fluticasone propionate FP) [25, 26].
The patients who received 500µg/day of FP showed significantly higher Candida in their
pharyngeal swabs compared to untreated asthmatic and healthy individuals. Patients who
were receiving 200µg/day did not show any significant finding [25]. In addition, there is a
possibility that the Candida colonization after a corticosteroid inhalation therapy depends on
the type of the drug used. According to Ohbayashi and Adachi asthmatic patients treated with
fluticasone carried significantly higher Candida colonies in their retopharyngeal walls
compared to patients treated with beclomethasone [26]. In contrast, Mullaoglu et al.
investigated the risk of esophageal candidiasis in asthma patients who are on inhaled steroids
without any other risk factors [27]. They identified that the prevalence oropharyngeal and/or
esophageal candidiasis in the cohort was low and there were no statistical significance in
isolating Candida in oropharyngeal or esophageal environments in asthmatic patients under
inhalational steroid therapy for at least a month [27]. In a similar study performed in a cohort
of children who received inhaled corticosteroids, there were no significant increase in the
prevalence and the number of Candida spp. Isolated in their oral cavity compared to healthy
children. Hence, the usage of inhaled steroids appears safe in terms of Candida infections.
Nevertheless commonly isolated Candida species were C. albicans followed by C.
guilliermondii,C. parapsilosis and C. stellatoidea in both steroid inhaled and control groups
while; Rhodotorularubra and C. lusitaniae in the treated group [28]. These findings create an
important question whether steroid therapy predisposes the patients for opportunistic
infections especially Candida colonization in oropharyngeal and esophageal environments.
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740 Arjuna N. B. Ellepola, H. M. H. N. Bandara and Hugh D Smyth

STEROID INDUCED ORAL CANDIDIASIS WITH TOPICAL STEROIDS

Whilst the foregoing pertains to the effect of aerosol steroids on oral candidiasis there are
few reports on the impact of topical steroids on this infection. In one study, patients requiring
topical steroid treatment (ie, dexamethasone, triamcinolone) for various oral conditions were
monitored for the development of clinical oral candidiasis [29]. A significant percentage of
patients (P= 0.01) developed oral candidiasis, and of the patients identified as carriers prior to
treatment only 20% developed candidiasis whilst others remained asymptomatic. These
workers concluded that the incidence of oral candidiasis secondary to topical oral use of
steroids might be expected to be greater than that associated with inhaled aerosols, due to
greater contact time in the oral cavity. In another study 20 patients with oral lichen planus
were treated topically with fluocinoloneacetonide 0.1% and were compared with 20 others
treated with triamcinolone acetonide 0.1% [21]. They found that acute pseudomembranous
candidiasis during treatment was common with both drugs and could be cured with
antifungals in every case. On the other hand, in patients with mucosal inflammatory
conditions, the presence of secondary oral candidosis may increase the local symptoms and
may prevent successful management of the mucosal lesion. Hence, appropriate early
management of secondary candidiasis is recommended [29]. Moreover, a study conducted in
patients with systemic lupus erythematosus (SLE) with steroids and immunosuppressive
treatments indicated that there is no difference between opportunistic pathogen counts of C.
albicans, Staphylococci, Enterobacteria and Pseudomonas spp. between SLE patients and
healthy controls or between corticosteroid treated patients and untreated SLE patients [30].
The use of corticosteroids to reduce the morbidity associated with laryngotracheo-
bronchitis (croup) is rather controversial. Yet, recent literature does support a decreased
morbidity and favorable clinical response when short-term steroids are used for this purpose
[31]. As a prophylactic measure against bacterial superinfection, antibiotics are commonly
utilized in the treatment of croup. However, there is a reported case of an otherwise healthy
infant with severe croup treated with steroids as well as antibiotics developing Candida
laryngotracheitis [31]. This emphasizes the necessity for close monitoring of patients
aggressively treated with both steroids and antibiotics.

MANAGEMENT OF STEROID INDUCED ORAL CANDIDIASIS

Generally these lesions are superficial and are amenable to topical antifungal therapy
[32]. For instance it has been shown that nystatin pastilles and suspensions were equally
efficacious both clinically and microbiologically and that the potential for enhanced drug
delivery to the oro-pharynx may be useful in patients in whom poor compliance seems likely
[33]. Further, a patient who had precipitins to C. albicans prior to aerosol treatment with
triamcinolone acetonide who developed oropharyngeal candidiasis was successfully treated
with a gargle containing nystatin [34]. This finding is significant, as some researchers have
speculated that patients with serum precipitins prior to administration of aerosolized steroids
may be at a higher risk of developing oral candidiasis during such therapy [6]. In contrast
there is a report where two cases of oral candidiasis failed to respond to nystatin when used in
combination with triamcinolone acetonide [35]. The C. albicans isolates obtained from these

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 Inhalation and Topical Steroid Therapy and Oral Candidiasis 741

patients showed high in vitro resistance to nystatin when tested in combination with
triamcinolone acetonide. However, when treated with miconazole nitrate a mycological and
clinical cure was obtained in one of the cases [35]. Development of nystatin resistance in
Candida is extremely rare, whereas such resistance is relatively common in the azole group of
drugs (eg, miconazole). It is difficult to speculate as to why Candida developed resistance to
nystatin although the possibility of inactivation of the antifungal due to combination with
triamcinolone acetonide cannot be ruled out; a mechanism akin to deactivation of nystatin
when combined with chlorhexidine [36]. Gargling with amphotericin B (1:50) solutions was
also appeared to be effective in eliminating Candida in asthmatic patients treated with either
fluticasone or beclomethasone [26]. However gargling with water or low concentrations of
amphotericin B is not effective in preventing Candida colonization in the throats. Rather, it
would be necessary to use concentrations higher than 1/100 of dilutions of the regular dose of
amphotericin B [37]. Conversely, the unpleasant taste of the drug would adversely affect the
compliance of the patient.

ANIMAL MODELS: EXPERIMENTALLY INDUCED ORAL CANDIDIASIS

WITH STEROID TREATMENT

The relationship between steroids and oral candidiasis in animal models has been studied
by a few [38]In an early study Budtz-Jorgensen [39], investigated the effect of triamcinolone
acetonide given intramuscularly on the development of oral candidiasis in a group of adult
Macaque monkeys wearing acrylic palatal plates inoculated with C. albicans. Whilst the
erythematous candidiasis which developed in the control group of monkeys resolved
spontaneously within 2–3 weeks the test group wearing appliances developed
pseudomembranous candidiasis which persisted for a prolonged period. Histological findings
of the lesional tissue revealed only a minor inflammatory infiltrate. This study indicated that
triamcinolone acetonide may potentiate oral candidiasis and its mechanism of action may
entail suppression of both the non-specific inflammatory response and cellular immunity. In
another animal experiment mucosal candidiasis was induced in mice by intraoral inoculation
following treatment with beclomethasonedipropionate aerosol [40]. Histologically, in
hormone treated mice the adherence of the pathogen to the mucosal surface was found during
the first hours after inoculation. This was followed by the formation of the germ tubes and
invasion of the epithelial layer. Psedomycelial invasion of the superficial epithelium was
accompanied by a leukocyte response, which limited further spread of the fungal cells [40].
However, in the control group of mice, the inoculation was not followed by the effective
attachment of the fungal cells to the mucosal surface or induction of infection. This study
illustrates that the enhanced adherence of fungal blastospores to epithelial cells of hormone
treated mice, may be a pathogenic mechanism contributing to oral candidiasis seen in steroid
inhalation. However independent validation of these observations is warranted. In a more
recent study a topical application of a corticosteroid (Topsyn gel) to the oral mucosa of 75
mice twice daily for 21 days resulted in a 400-fold increase in the residual population of
Candida by day 21, virtual disappearance of resident population of intraepithelial CD4+
T cells in the oral mucosa and massive depletion of T cells in the lymph nodes [41]. Further,
the cessation of treatment resulted in restoration of normal levels of both Candida and
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742 Arjuna N. B. Ellepola, H. M. H. N. Bandara and Hugh D Smyth

intraepithelial CD4+ T cells. Hence it was concluded that resistance to superficial invasion by
Candida is linked to the presence of a primary defense barrier in the oral mucosa and that the
topical application of corticosteroids may dramatically shift the host-parasite relationship in
favour of Candida [41]. These animal studies reinforce the clinical observations in humans
and re-emphasize the critical importance of mucosal defense barriers against oral Candida
infections.

MECHANISMS OF PATHOGENESIS OF STEROID

INDUCED ORAL CANDIDIASIS
The putative mechanisms by which either inhaled or topically applied steroids predispose
to oral candidiasis have not been fully clarified, as yet. There is little doubt that generalized
immunosuppressive and anti-inflammatory effects of steroids play a major role in the
pathogenesis [3, 4, 42]. For instance in a recent study when free cortisol concentrations in
unstimulated whole saliva samples from unmedicated HIV-positive and control subjects were
measured, it was found that the mean cortisol level was significantly higher in the HIV-
infected individuals than in the control subjects and that two HIV-infected individuals with
pesudomembranous candidiasis had the highest saliva cortisol concentrations [43]. Cortisol
induced immunosuppression, provides selective growth advantage for Candida and could be
one possible reason for the presence of oral candidiasis during steroid inhalation and topical
usage [43]. However, oral candidiasis may be related to the deficiencies in topical immunity
such as salivary IgA. Fukushima et al. studied the effect of inhaled corticosteroids used in
bronchial asthma in salivary IgA levels and candidiasis [44]. There was no difference of total
salivary IgA and Candida-specific IgA between culture negative steroid-treated asthmatic
patients. Salivary total IgA was significantly lower in Candida positive asthmatic patients
than Candida-negative asthmatic patients though Candida-specific IgA was not affected [44].
Thus, local immunosuppressive effect due to inhaled corticosteroids may affect candida
colonization in oropharyngeal regions but other host factors may play important role in
candidal pathogenesis. However, these may not be the only mechanisms by which
corticosteroids aggravate the disease process. For instance, whilst triamcinolone acetonide
does not significantly affect the viability of C. albicans, it increases its calcium uptake and
protects the yeast against the action of econazoles [45, 46]. Others have shown that
dexamethasone promotes growth of C. albicans [47] while some have contradicted this
finding [48]. Interestingly, dexamethasone-grown C. albicans was found to be more virulent
than the control yeasts initiating keratitis in a mouse model [47], a finding others were unable
to reproduce [49]. Early reports have indicated that patients treated with corticosteroids have
a higher level of salivary glucose than controls [50] and this may also promote growth,
proliferation and adhesion of Candida to oral mucosal cells [51]. It has also been
demonstrated that adherence of Candida species to buccal epithelial cells in vitro is enhanced
when the yeasts are grown in media supplemented with dexamethasone and triamcinolone
acetonide [52]. Others have shown that dexamethasone is incorporated into the outer surface
of the yeast [53] thus promoting adherence probably via a surface receptor interaction. The
previously quoted in vivo studies in mice [40], also supports these observations. In addition,
low pH levels consequential to high salivary glucose concentrations are conducive for the

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 Inhalation and Topical Steroid Therapy and Oral Candidiasis 743

secretion of aspartyl proteinases and phospholoipase of Candida [54], which are potent
enzymes that contribute to candidal pathogenicity. It has also been found that direct oral
muosalabsorption of an inhaled steroid, fluticasone propionate, is not an important
contribution to systemic bioavailability [55], implying a poor mucosal absorption of the drug
and a short, transient effect on the oral mucosa. This observation is substantiated by a number
of studies where no clinical oral candidiasis was observed following inhalation therapy. Such
observations may also explain, at least partly, the candidalsuperinfection, as the lesions when
present are particularly restricted on the oral mucosa directly exposed to the aerosol spray
[23], signifying a localized effect.

PREVENTIVE MEASURES
A number of preventive measures may be adopted to minimize oral candidosis during
steroid inhalation. Using a spacer device that can be attached to the inhaler could reduce the
local effect of steroids in causing oral candidiasis [56]. This controls the total amount of drug
reaching the patient as well as the area of the lung deposition. Further a spacer device when
attached to a metered dose inhaler or a reduces oropharyngeal deposition of the drug and
increase lung deposition, thus reducing the oral side effects such as candidiasis. However,
colonization of Candida in spacers is not affected by inhaled corticosteroids and topical
corticosteroids promote candidal growth in spacers made of polycarbonate or polyethylene
than metal [57]. Hence metallic spacers would be a better choice for patients receiving topical
corticosteroid therapy. As mentioned previously, however, ample evidence suggests that
spacer devices are not well accepted or routinely used by patients prescribed them.
A reduction in the corticosteroid dosage may also be possible [4]. For example, the turbuhaler
achieves lung deposition approximately twice that of a metered-dose inhaler for patients who
can achieve sufficient inhalation forces to correctly use this device [42]. Corticosteroids with
improved deposition profiles in comparison to conventional formulations would also reduce
the side effects by reducing the inhaled dose [58]. Drug combination therapy would also help
reducing the dosage of inhaled corticosteroid.
The latter combined with long acting β-2 agonists showed a reduction in the incidence of
oral candidiasis compared to higher dose of corticosteroid inhalational therapy [11]. Washing
out the mouth with a prescribed mouth wash following each use of steroid inhalers is a simple
measure to reduce local and systemic adverse effects when using dry powder devices
especially at high doses [4]. A study conducted in denture wearing asthmatic population
demonstrated that there is a significant amount of fluticasone propionate (FP) and
hydrofluoroalkane-beclomethasonedipropionate (HFA-BDP) retention after the inhaled
corticosteroid therapy. Full dentures caused more retention than partial dentures. Hence, three
times gargling after inhaled corticosteroids was recommended to remove all residual
corticosteroid captured in dentures specially when treated with HFA-BDP to reduce the
occurrence of candidiasis [26]. It was also suggested that immediate rinsing of the mouth
would be more effective in removing residual drugs in the oral cavity as amount of drug
washed away with gargling was significantly associated with the time lag between inhalation
and the mouth washing [59]. In contrast others have shown that there is no rationale to
employ mouth rinsing to reduce its systemic effects though it may be of value for reducing
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744 Arjuna N. B. Ellepola, H. M. H. N. Bandara and Hugh D Smyth

oral candidiasis [55] and of some prophylactic benefit for alleviating candidiasis induced by
(metered-dose) steroid inhalers [60]. In contrast, as mentioned in elsewhere, Kurt et al. [25],
suggested that the most important determinant of the occurrence of Candida in patients
treated with inhaled steroids were not rinsing the throat after the treatment or the duration of
inhalational therapy used but the dosage of the corticosteroid used in treatment.
It has been documented that prolonged steroid therapy could damage mucosal barriers
predisposing to oropharyngeal candidiasis, which can be prevented by controlled
administration of topical antimycotics such as nystatin [29, 61]. The initial use of this
medication is sufficient to control the signs and symptoms. However, if the antifungal is
discontinued recurrences could occur. Hence it is necessary to continue the use of nystatin
throughout the course of a steroid regimen [29]. The systemic administration of antifungals is
an alternative for those who do not respond. Both oral ketoconazole and intravenous
amphotericin B have proven to be effective [61]. For more extensive management regimens
in this regard the reader is refereed to reviews on oral candidiasis and antifungal agents [62-
64]. Further, the development of laryngeal and pharyngeal candidiasis following steroid
therapy is a cause for concern because it is difficult to ascertain the clinical effect of the
respiratory tract infection. For this reason caution with steroid aerosols would seem advisable,
particularly the long term high dosage regimens. There have been calls for prudent use of
these extensive regimens in patients with lung damage such as bronchiectasis, in which fungal
infections could be difficult to eliminate [65]. Candida oesophagitis is also an important
problem and oral nystatin suspension can be helpful in mild cases. In others oral ketoconazole
and intravenous amphotericin B can be used [61]. A whole range of other preventive
measures such as combined use of H2 receptor antagonists and antifungal prophylaxis is also
available [61]. However, the effects of H2 receptor antagonists on salivary secretion and oral
health are needed to be studied. The details of these are beyond the remit of this chapter.

CONCLUSION AND FUTURE DIRECTIONS

The exact relationship between oral candidiasis and locally acting steroids is still unclear.
However, the incidence of oral candidiasis in patients receiving such therapy necessitates
probing into the effects of steroids on local oral immune mechanisms, and the mechanisms by
which Candida proliferate intraorally consequential to steroid therapy. For instance, there is
no substantial information on the effect of steroids on the virulence attributes of oral Candida,
such as germ tube formation and enzyme production. Work in these areas as well as related
genomic studies are warranted to further understand the mechanisms of steroid induced oral
candidiasis.

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[21] Thongprasom, K., et al., Relative efficacy of fluocinolone acetonide compared with
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lupus erythematous: correlation with treatment and disease activity. Lupus, 2012. 21(9):
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[31] Burton, D.M., et al., Candida laryngotracheitis: a complication of combined steroid and
antibiotic usage in croup. Int. J. Pediatr. Otorhinolaryngol., 1992. 23(2): p. 171-5.
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298(6671): p. 403-4.
[33] Thompson, P.J., et al., Assessment of oral candidiasis in patients with respiratory
disease and efficacy of a new nystatin formulation. Br. Med. J. (Clin. Res. Ed), 1986.
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dependent asthma. Chest, 1977. 72(1): p. 36-44.
[35] Martin, M.V. and R.C. Dinsdale, Nystatin-resistance of candida albicans isolates from
two cases of oral candidiasis. Br. J. Oral Surg., 1982. 20(4): p. 294-8.
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Candida albicans. Oral Surg. Oral Med. Oral Pathol., 1989. 67(3): p. 279-81.
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albicans in asthmatic patients on steroid inhalation therapy. Respiration, 2001. 68(5): p.
465-70.
[38] Samaranayake, Y.H. and L.P. Samaranayake, Experimental oral candidiasis in animal
models. Clin. Microbiol. Rev., 2001. 14(2): p. 398-429.
[39] Budtz-Jorgensen, E., Effects of triamcinolone acetonide on experimental oral
candidiasis in monkeys. Scand. J. Dent. Res., 1975. 83(3): p.
171-8.

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[40] Bykov, V.L. and E.V. Velichko, [Cytological mechanisms of the development of
mucosal candidiasis after exposure to a steroid aerosol preparation]. Biull. Eksp. Biol.
Med., 1987. 103(3): p. 369-71.
[41] Deslauriers, N., et al., Topical application of a corticosteroid destabilizes the host-
parasite relationship in an experimental model of the oral carrier state of Candida
albicans. FEMS Immunol. Med. Microbiol., 1995. 11(1): p. 45-55.
[42] Davis, K.C. and R.E. Small, Budesonide inhalation powder: a review of its
pharmacologic properties and role in the treatment of asthma. Pharmacotherapy, 1998.
18(4): p. 720-8.
[43] Enwonwu, C.O., V.I. Meeks, and P.G. Sawiris, Elevated cortisol levels in whole saliva
in HIV infected individuals. Eur. J. Oral Sci., 1996. 104(3): p. 322-4.
[44] Fukushima, C., et al., Salivary IgA and oral candidiasis in asthmatic patients treated
with inhaled corticosteroid. J. Asthma, 2005. 42(7): p. 601-4.
[45] Raab, W. and B. Gmeiner, Evaluation of econazole by Warburg assay; comparison with
other antimicrobials. Mykosen, 1976. 19(7): p. 238-40.
[46] Berdicevsky, I. and M. Silbermann, Effect of glucocorticoid hormones on calcium
uptake and the morphology of Candida albicans. Cell Biol. Int. Rep., 1982. 6(8): p. 783-
90.
[47] Khosla, P.K., et al., The effect of dexamethason and oxytetracyclin on the growth of
Candida albicans. Mykosen, 1978. 21(10): p. 342-8.
[48] Ghannoum, M., G. Burns, and K. Abu Elteen, Growth of Candida albicans in
dexamethasone-supplemented media. Sabouraudia, 1985. 23(4): p. 313-5.
[49] Gupta, P.N., et al., Effect of pretreatment with dexamethasone on corneal pathogenicity
of Candida albicans. J. Commun. Dis., 1983. 15(3): p. 200-4.
[50] Knight, L. and J. Fletcher, Growth of Candida albicans in saliva: stimulation by glucose
associated with antibiotics, corticosteroids, and diabetes mellitus. J. Infect. Dis., 1971.
123(4): p. 371-7.
[51] Samaranayake, L.P. and T.W. MacFarlane, Hypothesis: on the role of dietary
carbohydrates in the pathogenesis of oral candidosis. FEMS Microbiol. Lett. 1985. 27:
p. 1-5.
[52] Ghannoum, M.A. and K.A. Elteen, Effect of growth of Candida spp. in the presence of
various glucocorticoids on the adherence to human buccal epithelial cells.
Mycopathologia, 1987. 98(3): p. 171-8.
[53] Ghannoum, M.A., S. Mudher, and G. Burns, Incorporation of dexamethasone by
Candida albicans. Microbios., 1985. 42 (168): p. 103-9.
[54] Wu, T. and L.P. Samaranayake, The expression of secreted aspartyl proteinases of
Candida species in human whole saliva. J. Med. Microbiol., 1999. 48(8): p. 711-20.
[55] Dempsey, O.J., et al., Evaluation of the buccal component of systemic absorption with
inhaled fluticasone propionate. Thorax., 1999. 54(7): p. 614-7.
[56] Toogood, J.H., et al., Comparison of the antiasthmatic, oropharyngeal, and systemic
glucocorticoid effects of budesonide administered through a pressurized aerosol plus
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[57] de Vries, T.W., et al., The influence of inhaled corticosteroids and spacer devices on the
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[58] Prenner, B.M. and J.A. Bernstein, Clinical benefits of switching to an inhaled
corticosteroid extrafine aerosol; a case series. Eur. Rev. Med. Pharmacol. Sci., 2002.
6(4): p. 61-5.
[59] Yokoyama, H., et al., Influence of mouth washing procedures on the removal of drug
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1923-5.
[60] Yamada, Y., et al., [Effects of mouth wash on the removing beclomethasone
dipropionate delivered by pressurized aerosol metered-dose inhaler in the mouth].
Yakugaku Zasshi, 1999. 119(6): p. 436-43.
[61] Prentice, A.G., Oral and gastrointestinal candidosis: prophylaxis during
immunosuppressive therapy. Mycoses, 1989. 32 Suppl 2: p. 42-6.
[62] Ellepola, A.N. and L.P. Samaranayake, Oral candidal infections and antimycotics. Crit.
Rev. Oral Biol. Med., 2000. 11(2): p. 172-98.
[63] Ellepola, A.N. and L.P. Samaranayake, Antimycotic agents in oral candidosis: an
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[64] Ellepola, A.N. and L.P. Samaranayake, Antimycotic agents in oral candidosis: an
overview: 2. Treatment of oral candidosis. Dent. Update, 2000. 27(4): p. 165-70, 172-4.
[65] McAllen, M.K., S.J. Kochanowski, and K.M. Shaw, Steroid aerosols in asthma: an
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 30

FLUORESCENT STAINING FOR THE DIAGNOSIS

OF ORAL ERYTHEMATOUS CANDIDIASIS

Yoichi Nakagawa
Department of Clinical Pathophysiology,
Tsurumi University School of Dental Medicine, Yokohama, Japan

ABSTRACT
The diagnosis of candidiasis is based on clinical findings, and the diagnosis is
confirmed by the identification of pseudohyphae in stained smears sampled from a lesion,
by the identification of colonies cultured on Sabouraud’s medium or by histological
examination. There are several staining techniques for the microscopic examination of
smears, including the Giemsa, Gram and periodic acid-Schiff (PAS) stains. Fungiflora Y
is a fluorescent stain. The solution binds to ß-linked polysaccharides, such as chitin and
cellulose, which are components of the fungal cell wall. The usefulness of Fungiflora Y
staining for the diagnosis of erythematous candidiasis is described in this chapter. The
sensitivity, specificity and positive and negative predictive values were calculated using
fungal culture as the gold standard, and the results were compared with the staining
results using the modified Giemsa staining system.

Keywords: Candida, candidiasis, culture, fluorescence staining, exfoliative cytology,

Fungiflora Y, quantitative analysis, dry mouth

1. INTRODUCTION
Oral candidiasis is classified into three major variants: pseudomembranous (Figure 1),
erythematous (Figure 2) and hyperplastic candidiasis [1]. The erythematous forms of the


Corresponding author: Yoichi Nakagawa, Department of Clinical Pathophysiology, Tsurumi University School of
Dental Medicine 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan E-mail address: nakagawa-
[email protected]
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750 Yoichi Nakagawa

disease occur more commonly than the pseudomembranous type [2]. Denture stomatitis,
angular cheilitis and median rhomboid glossitis are regarded to be candida-associated lesions.
The diagnosis of candidiasis is based on the clinical findings, and the diagnosis is
confirmed by the identification of blastospores and pseudohyphae in stained smears sampled
from a lesion, by the identification of colonies by culture on Sabouraud’s medium or by
histological examination [3, 4]. Smears are valuable for differentiating between the yeast and
hyphae forms, but are less sensitive than culture methods [5]. Because the number of Candida
isolates is smaller in erythematous candidiasis than in pseudomembranous candidiasis,
negative results are occasionally obtained by direct examination of erythematous candidiasis
[6]. In the diagnosis of erythematous candidiasis, examinations are reported to yield false-
negative results in 25% of culture tests and 42.5% of microscopic examinations [6]. Thus, a
more sensitive staining method for the microscopic examination has been needed. The
usefulness of fluorescent staining using Fungiflora Y for the detection of Candida in the
smear samples from erythematous candidiasis is described in this chapter.

Figure 1. Clinical manifestations of pseudomembranous candidiasis.

Figure 2. Clinical manifestations of erythematous candidiasis.

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 Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 751

2. EXAMINATION OF CANDIDA IN THE DIAGNOSIS

OF ORAL CANDIDIASIS

2.1. Culture Examinations

The interpretation of the results of microbial examinations is often complicated, because

Candida is present commensally in the oral cavities of up to 40% of individuals [1, 4]. There
is an overlap in the candidal counts from carriers and individuals showing infection, and there
are no established criteria to differentiate candidal commensalism from disease [1, 2]. Hence,
the isolation of Candida from the mouth is not confirmatory evidence of infection, and the
diagnosis of candidiasis is based on the combination of mycological examinations and
assessments of the clinical signs and symptoms [1, 2, 7].
The techniques available for the isolation of Candida within the oral cavity include the
use of a plain swab, an imprint culture, the collection of whole saliva, concentrated oral rinse
and oral mucosal biopsy [5]. The collection of whole saliva and concentrated oral rinse are
appropriate methods for evaluating the quantitative level of Candida in the whole mouth,
while swab and imprint culture can detect Candida from infected lesions.
For fungal cultures, the swab is directly inoculated onto an agar, such as Sabouraud’s
dextrose agar. The Candida species can be identified by the colony colors on the
CHROMagar Candida plates (Chromagar Microbiology, Paris, France; Figure 3). In our
department, the number of Candida colonies on the CHROMagar Candida plates is counted
after incubation at 37°C for 48 h and the results are expressed as the CFU/plate.

Figure 3. Candida colonies on the CHROMagar Candida plates. The swab was directly inoculated onto
the agar, the number and color of the colonies was then observed after incubation at 37°C for 48 h. A:
Candida albicans, B: Several species are seen in the agar.

2.2. Microscopic Examinations

Samples for exfoliative cytology are obtained by scraping the organisms, along with the
superficial epithelial cells. The samples are then smeared onto microscope slides. Several
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752 Yoichi Nakagawa

staining methods are used for visualization of the organisms. The presence of pseudohyphae
is considered to be a positive finding for candidiasis.

2.2.1. Conventional staining

For microscopic examinations, a smear of a sample that was taken from a lesion site is
fixed onto a microscope slide and then stained either by Gram staining or by the periodic acid
Schiff (PAS) technique [5]. A rapid staining test, the CytoQuick staining system (Muto Pure
Chemicals Co., Ltd., Tokyo, Japan), which is a modification of the Giemsa stain, is also
useful for the diagnosis of candidiasis [6, 8].
The CytoQuick staining system (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) is
composed of solution A (eosin) and solution B (methylene blue). The slides are placed into
solution A for 5 s, rinsed with tap water, placed into solution B for 15 s, and then examined.
Representative examples of the microscopic findings are shown in Figure 4, where
CytoQuick staining dyed the fungus body dark blue, while the cell walls remained colorless.
The exfoliated epithelial cells and bacteria were stained dark blue.

Figure 4. Modified Giemsa staining in exfoliative cytology. CytoQuick staining dyed the fungus body
dark blue, while the cell walls remained colorless. The exfoliated epithelial cells and bacteria were
stained dark blue.

2.2.2. Fluorescent Staining

Several fluorescent dyes that are specific for fungal cell wall polysaccharides have been
reported to be effective in the screening of clinical specimens for fungi [7, 9]. These include
Calcofluor white, Blankophor and Fungiqual [7, 9-11]. It has been recognized that fluorescent
techniques have significant advantages over traditional staining methods with respect to
rapidity, cost and the absence of interference with permanent fungal stains [12]. The
usefulness of fluorescent dyes has therefore been recognized as an effective tool not only in
histopathology, but also in cytopathology [13].
Fungiflora Y (Biomate Co., Ltd., Tokyo, Japan) is a nonspecific fluorescent stain for
fungi in cytological specimens. The solution binds to ß-linked polysaccharides, such as chitin

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 Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 753

and cellulose, which are components of the fungal cell wall [14, 15]. Fungiflora Y staining
offers a method for the rapid screening of cytological specimens for fungi and Acanthamoeba
because of its speed and technical simplicity [16-19]. However, Fungiflora Y has not been
used clinically for the diagnosis of oral candidiasis, and the application of the fluorescent dye
has so far only been performed in animal studies [14, 20]. We applied this staining method to
detect oral Candida and examined its usefulness.

2.2.3. The Staining Method Using Fungiflora Y

To stain samples using Fungiflora Y, a smear taken from the lesional site is fixed onto
microscopic slides, and Fungiflora Y is placed onto the slide for one minute. A cotton swab
cannot be used for sampling, because Fungiflora Y stains cotton fibers. The slide is then
observed under a fluorescent microscope at a wavelength of 365 nm using a CyScope®
(Partec GmbH, Münster, Germany). A CyScope® is a portable fluorescence microscope,
which enables it to be used at the bedside or at the side of a dental chair. A darkroom is not
necessary to observe stained slides, because the fluorescent intensity of the Fungiflora Y stain
is stable, and its fluorescent attenuation is smaller than that of the other fluorescence staining
methods [16]. A BZ−9000 fluorescent microscope (Keyence Corporation, Tokyo, Japan) was
used to obtain the images for this manuscript.
Fungiflora Y staining clearly shows fungal walls, because the compound specifically
attaches to polysaccharides, such as cellulose or chitin, which are present in the fungal cell
walls. Under fluorescent microscopy, typical hyphae and yeast of the Candida species display
brilliant green fluorescence, which readily differentiates them from exfoliated cells (Figure
5). Although Fungiflora Y nonspecifically binds to epithelial cells and to bacteria, the binding
is very weak.

Figure 5. Fluorescent staining in exfoliative cytology. Fungiflora Y staining clearly showed the fungal
walls, because the fluorescent molecules specifically attached to polysaccharides, such as cellulose or
chitin, which are present in fungal walls. Under fluorescent microscopy, typical hyphae and yeast of the
Candida species display brilliant green fluorescence, which readily differentiates them from exfoliated
cells. This specimen is from a case of erythematous candidiasis.
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754 Yoichi Nakagawa

Therefore, the fungi appear to stand out against the background. This allows for detection
even at low magnifications. Furthermore, the margin of the fungal body emits fluorescence
that is brighter than the inner portion because the dye binds to the fungal wall, thereby
showing the characteristic fungal form [14]. This makes it easy to discriminate fungi from
nonspecific foreign bodies.
For samples of suspected erythematous candidiasis, a 60–70 µL drop of sterile water was
dropped onto the surface of the lesion, usually the dorsum of the tongue, and was swabbed
using a dental mirror.

3. DIFFERENCES IN THE CYTOLOGICAL FINDINGS BETWEEN

PSUEDOMEMBRANOUS AND ERYTHEMATOUS CANDIDIASIS
One helpful clinical feature of the pseudomembranous type of candidiasis (Figure 1) is
the ability to scrape off the superficial white plaques. In these white plaques, there are
numerous superficial Candida hyphae, and these are more likely to result in a positive smear
test (Figure 6). On the other hand, the cytological findings of erythematous candidiasis are
completely different from the findings of pseudomembranous candidiasis (Figure 5).
Negative results are occasionally obtained by fungal examinations for this type of infection,
because the number of Candida isolates is smaller in atrophic candidiasis than in
pseudomembranous candidiasis [6]. Based on this background, we have developed
fluorescent staining methods for microscopic examinations.
The studies performed in our department were as follows; comparison of Fungiflora Y
and modified Giemsa staining of erythematous candidiasis, and quantitative evaluation of
Candida by microscopy in erythematous candidiasis. The outline of these studies will be
described in the following sections.

Figure 6. Fungiflora Y staining in a case of pseudomembranous candidiasis.

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 Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 755

4. COMPARISON OF FUNGIFLORA Y WITH MODIFIED GIEMSA

STAINING IN ERYTHEMATOUS CANDIASIS
The usefulness of Fungiflora Y staining for the diagnosis of erythematous candidiasis
was investigated. The study subjects were recruited from consecutive patients who had
subjective dry mouth and visited the Dry Mouth Clinic at Tsurumi University Dental
Hospital. Microscopic and cultural examinations were performed in the cases that were
clinically diagnosed with erythematous candidiasis, which was based on findings such as the
redness of the oral mucosa, an atrophic or smooth tongue, as well as soreness or pain of the
tongue [21]. A total of 48 patients [nine males and 39 females; age ranging from 26 to 96
years; mean ± SD = 68.0 ± 12.0] who were clinically diagnosed with oral erythematous
candidiasis were enrolled in this study. Of the 48 patients, 37 (77.1%) complained of pain in
their mouths. All of the patients had soreness of their tongue.
The sensitivity, specificity and positive and negative predictive values were calculated
using fungal culture as the gold standard, and the results were compared with the staining
results obtained using the CytoQuick system. The accuracy was calculated, and the
differences between CytoQuick and Fungiflora Y groups were examined using contingency
tables and the chi square test.

4.1. Comparison of the Reliability of Staining with Fungiflora Y and

Modified Giemsa Staining

The inter-observer agreement was assessed to compare the reliability of each staining
method. The microscopic findings of the presence (positive) or absence (negative) of
pseudohyphae on the smear specimens were evaluated by an oral surgeon (Dr. S. Yamachika)
and a general dentist (Dr. M.R. Okamoto). Dr. Yamachika had 30 years of experience in the
oral surgery field and Dr. Okamoto had one year of experience in general dental practice.
Kappa values were calculated in order to determine the inter-observer agreement.
The inter-observer agreement was poor for the CytoQuick staining and fair for the
Fungiflora Y staining; the kappa values for the assessment of the presence of pseudohyphae
or yeast were 0.47 and 0.61, respectively. A kappa value less than 0.40 indicates poor
agreement, 0.40–0.59 indicates fair agreement, 0.60–0.74 indicates good agreement and 0.75–
1.00 indicates excellent agreement. Thus, the results suggested that the Fungiflora Y staining
was a superior method for the microscopic examination of erythematous candidiasis
compared to the CytoQuick staining method.

4.2. Comparison of the Accuracy of Fungiflora Y with Modified Giemsa

Staining

Positive microscopic or cultural examinations confirmed the diagnosis of candidiasis in

38 (80.9%) of the 48 patients. The rest of the patients were thought to be false negatives or to
have other oral diseases, such as burning mouth syndrome. The CytoQuick staining
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756 Yoichi Nakagawa

examination detected pseudohyphae with occasional yeast in 20 cases (41.7%). In contrast, 31

cases (64.6%) were positive on the microscopic examination using Fungiflora Y staining.
The sensitivity and specificity of the CytoQuick staining was 0.51 and 0.91, respectively;
the positive predictive value was 0.95, and the negative predictive value was 0.36 (Table 1).
The positive likelihood ratio was 5.65, and the negative likelihood ratio was 1.87. The
accuracy was (19 + 10)/48 = 0.60.
The sensitivity and specificity of the Fungiflora Y staining were 0.84 and 1.0,
respectively; the positive predictive value was 1.00, and the negative predictive value was
0.65 (Table 2). The positive likelihood ratio was more than 10 (infinity). The accuracy was
(31 + 11)/48 = 0.88, which was superior to that of CytoQuick (p = 0.0052). These results
suggest that the detection of fungal elements from erythematous candidiasis was more
accurate using Fungiflora Y staining compared to traditional staining.
Although it is not clear which method is the most accurate among the various fluorescent
staining methods, Fungiflora Y seems to have an advantage, because Fungiflora Y only
involves a one-step process, whereas the Calcofluor method involves two steps [11].
Moreover, one minute is adequate for the Fungiflora Y staining. Therefore, the microscopic
examination of a smear specimen using Fungiflora Y staining was useful for the diagnosis of
oral erythematous candidiasis.

Table 1. Contingency table created from the results of the CytoQuick

staining and culture

Culture
Positive Negative Total PPV NPV
CytoQuick Positive 19 1 20 19/20 = 0.95
Negative 18 10 28 10/28 = 0.36
Total 37 11 48
Sensitivity 19/37 = 0.51
Specificity 10/11 = 0.91
Positive likelihood ratio 5.65
PPV, positive predictive value; NPV, negative predictive value.

Table 2. A contingency table created from the results of the Fungiflora Y

staining and culture

Culture
Positive Negative Total PPV NPV
Fungiflora Y Positive 31 0 31 31/31 = 1.0
Negative 6 11 17 11/17 = 0.65
Total 37 11 48
Sensitivity 31/37 = 0.84
Specificity 11/11 = 1.0
Positive likelihood ratio >10
PPV, positive predictive value; NPV, negative predictive value.

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 Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 757

5. QUANTITATIVE EVALUATION OF CANDIDA

BY MICROSCOPY IN ERYTHEMATOUS CANDIDIASIS

Light microscopy has been used for a differential diagnosis of fungal infections,
especially in the dermatological field, because it is quick.. However, the quantitative
evaluation of fungus in terms of cytology has not been studied. The Gaffky’s scale is widely
used for quantitative evaluation of Mycobacterium tuberculosis in sputum smears. The
presumption of the number of bacteria with quantitative relevance by Gram staining and
bacterial culture has also been studied. In a textbook on urinary tract infections, it is stated
that a number of investigators have reported that staining methods correlate with quantitative
culture in about 80 to 90% of cases [22]. Based on this background, the quantitative
relationship between Candida detected in microscopic and cultural examinations was
investigated to evaluate the usefulness of exfoliative cytology. This study may also elucidate
why negative results are occasionally obtained by direct examination of erythematous
candidiasis [6].
The subjects in this study were recruited from consecutive patients who had subjective
dry mouth and visited the Dry Mouth Clinic at Tsurumi University Dental Hospital.

5.1. Subjects and Methods

5.1.1. Subjects
Samples were obtained from a total of 54 patients (three males and 51 females; mean age
± SD = 68.4 ± 12.5) at the Dry Mouth Clinic, Tsurumi University Dental Hospital. The
patients were clinically diagnosed to have erythematous candidiasis and underwent both
microscopy of a smear specimen stained with a fluorescent dye, Fungiflora Y, and fungal
cultural examination. Of the 54 patients, 34 (77.1%) had redness of the tongue, 36 atrophy of
the tongue papillae, 15 angular cheillitis, six redness of the lips, 14 redness of the palate and
11 had redness of the buccal mucosa. All complained of pain in their mouths, and all had
soreness of their tongue. Of the 54 patients, Candida were detected in 51 (94.4%) of the
cultures, so that these cases were diagnosed to have oral candidiasis. In the cytological
examination, candidal hyphae were detected in 46/51 of these cases (90.2%).

5.1.2. Methods for Microscopic Examination

The specimens were obtained from surface of the dorsum of the tongue by swabbing. The
number of Candida was expressed as the number of fungi/field of vision (FOV) by
microscopy, and colony-forming units (CFU) in the culture. For microscopic examination of
the Fungiflora Y staining of the exfoliative cytology, a specimen was observed under
magnification of 200-power and the number of the fungi was counted. At first, the
examination with the fluorescent microscope was performed for more than four FOV in
succession in the top and bottom directions. Then, the observation was continued for more
than four FOV in the right and left direction. When no cell bodies were found, the number of
the FOV was further increased. As a result, more than 18 FOV were observed, for an average
of 51 FOV, in each specimen.
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758 Yoichi Nakagawa

5.2. Relationship between the Microscopic and Cultural Examinations of

Candida

The results of the microscopic and cultural examinations demonstrated that there were 0 to
3.7 fungi/FVO and 0 to 1992 CFU/plate, respectively. The correlation coefficient of the
examinations was 0.569 (p<0.001); the regression equation was [CFU/plate] = 330.4x
[number of fungi/FOV] + 124.6 (Figure 7). please remove ¶Because this result demonstrated
that there was a quantitative correlation between the microscopic and cultural examination,
the possibility of quantitative evaluation of Candida by cytology was suggested.
Since numerous factors are involved in the establishment of candidiasis, such as the
number of organisms, the enzyme produced from Candida and the impairment of the host
defense, a diagnosis of oral candidiasis cannot be made based only on the amount of detected
Candida [23-25]. However, a decrease in the number of Candida is important for the
management of oral candidiasis, as well as elimination of the risk factors for further infection.
Quantitative culture of saliva has been thought to be helpful in the diagnosis of oral
candidiasis. This is because patients with candidiasis have more than 400 CFU/ ml of saliva,
whereas carriers of Candida albicans have less than 400 CFU/ ml [26]. The establishment of
a cut-off point will be helpful for the daily oral care of dry mouth patients in order to prevent
the risk of erythematous candidiasis [27]. Since it is recognized that there is a relationship
between the amount of Candida and the clinical signs, the quantitative analyses are useful for
managing oral candidiasis. Rapid microscopic examinations will give helpful information for
the treatment and prevention of oral candidiasis.
Because the number of Candida isolates is smaller in cases of erythematous candidiasis
than pseudomembranous candidiasis, negative results are occasionally obtained by direct
examination of erythematous candidiasis [6]. In our present study, the intercept of the
regression equation was 124.6 for erythematous candidiasis (Figure 8).
Culture (CFU / plate)

Microscope (Number of fungi / FOV)

Figure 7. The correlation between the numbers of Candida detected in microscopic and cultural
examinations.

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 Fluorescent Staining for the Diagnosis of Oral Erythematous Candidiasis 759

This result suggested that the sensitivity of exfoliative cytology was relatively low.
However, even when only one mycelium is recognized from the lesion by microscopy, the
lesion is regarded to be due to oral candidiasis. This means that the microscopic examination
has high specificity.

CONCLUSION
Exfoliative cytology has been recognized to be of value in differentiating between the
yeast and hyphal forms of infection, and is advantageous due to the rapid examination.
Additionally, our study suggested that the direct examination of a smear was helpful, even for
quantitative evaluations, because a positive correlation was recognized between the number
of Candida detected by microscopic and cultural examinations. Although the number of
Candida isolates is smaller in erythematous candidiasis than in pseudomembranous
candidiasis, and negative results are sometimes obtained, an examination of smears from the
lesion is still useful, because a sensitive fluorescence staining option is available. Fungiflora
Y staining clearly shows the presence of fungal walls, because the fluorescent molecule
specifically binds polysaccharides, such as cellulose or chitin, which are present in fungal
walls. Under fluorescent microscopy, typical hyphae and yeast of the Candida species display
brilliant green fluorescence, which readily differentiates them from exfoliated cells.

REFERENCES
[1] Ellepola AN, Samaranayake LP (2000) Oral candidal infections and antimycotics. Crit.
Rev. Oral. Biol. Med. 11:172-98.
[2] Giannini PJ, Shetty KV (2011) Diagnosis and management of oral candidiasis.
Otolaryngol. Clin. North Am. 44:231-40.
[3] Worthington HV, Clarkson JE, Eden OB (2007) Interventions for treating oral
candidiasis for patients with cancer receiving treatment. Cochrane Database Syst.
Rev.:CD001972.
[4] Scully C. (2004) Oral and maxillofacial medicine. London: Elsevier: 252-68.
[5] Williams DW, Lewis MA (2000) Isolation and identification of Candida from the oral
cavity. Oral. Dis. 6:3-11.
[6] Terai H, Shimahara M (2009) Usefulness of culture test and direct examination for the
diagnosis of oral atrophic candidiasis. Int. J. Dermatol. 48:371-3.
[7] Olsen I, Stenderup A (1990) Clinical-mycologic diagnosis of oral yeast infections. Acta
Odontol. Scand. 48:11-8.
[8] Terai H, Shimahara M (2010) Chronic oral ulcer associated with Candida. Mycoses
53:168-72.
[9] Gulec AT, Demirbilek M, Seckin D, Can F, Saray Y, Sarifakioglu E, Haberal M (2003)
Superficial fungal infections in 102 renal transplant recipients: a case-control study. J.
Am. Acad. Dermatol. 49:187-92.
[10] Coleman T, Madassery JV, Kobayashi GS, Nahm MH, Little JR (1989) New
fluorescence assay for the quantitation of fungi. J. Clin. Microbiol. 27:2003-7.
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760 Yoichi Nakagawa

[11] Hamer EC, Moore CB, Denning DW (2006) Comparison of two fluorescent whiteners,
Calcofluor and Blankophor, for the detection of fungal elements in clinical specimens
in the diagnostic laboratory. Clin. Microbiol. Infect. 12:181-4.
[12] Lynch DP, Gibson DK (1987) The use of Calcofluor white in the histopathologic
diagnosis of oral candidiasis. Oral. Surg. Oral. Med. Oral. Pathol. 63:698-703.
[13] Kumar RS, Ganvir S, Hazarey V (2009) Candida and calcofluor white: Study in
precancer and cancer. J. Oral. Maxillofac. Pathol. 13:2-8.
[14] Nishiyama Y, Aoki Y, Yamaguchi H (1995) Morphological aspects of cell wall
formation during protoplast regeneration in Candida albicans. J. Electron Microsc.
(Tokyo) 44:72-8.
[15] Kimura H, Furuta I, Furuta T, Teramura K, Maekura S, Satou T, Hashimoto S (1996 )
New fluorescent stain for fungi in tissue sections. JARMAM 8:21-5.
[16] Inoue T, Asari S, Tahara K, Kiritoshi A, Inoue Y, Shimomura Y (1999) Utility of
Fungiflora Y stain in rapid diagnosis of Acanthamoeba keratitis. Br. J. Ophthalmol.
83:632-3.
[17] Kimura M, Sano A, Maenishi O, Ito H (2007) Usefulness of Fungiflora Y to detect
fungus in a frozen section of allergic mucin. Pathol. Int. 57:613-7.
[18] Kimura M, Takeda T, Maekura S, Hashimoto S (1996) Detection of hyphae in pus with
Fungiflora Y. Acta Cytol. 40:1327-8.
[19] Shiraishi A, Kobayashi T, Hara Y, Yamaguchi M, Uno T, Ohashi Y (2009) Rapid
detection of Acanthamoeba cysts in frozen sections of corneal scrapings with
Fungiflora Y. Br. J. Ophthalmol. 93:1563-5.
[20] Hisajima T, Ishibashi H, Yamada T, Nishiyama Y, Yamaguchi H, Funakoshi K, Abe S
(2008) Invasion process of Candida albicans to tongue surface in early stages of
experimental murine oral candidiasis. Med. Mycol. 46:697-704.
[21] Okamoto MR, Kamoi M, Yamachika S, Tsurumoto A, Imamura T, Yamamoto K,
Kadomatsu S, Saito I, Maeda N, Nakagawa Y (2012) Efficacy of Fungiflora Y staining
for the diagnosis of oral erythematous candidiasis. Gerodontology.
[22] Lewis JF, Alexander J (1976) Microscopy of stained urine smears to determine the need
for quantitative culture. J. Clin. Microbiol. 4:372-4.
[23] Holmes AR, Bandara BM, Cannon RD (2002) Saliva promotes Candida albicans
adherence to human epithelial cells. J. Dent Res. 81:28-32.
[24] Jayatilake JA, Samaranayake YH, Samaranayake LP (2005) An ultrastructural and a
cytochemical study of candidal invasion of reconstituted human oral epithelium. J.
Oral. Pathol. Med. 34:240-6.
[25] Hibino K, Samaranayake LP, Hagg U, Wong RW, Lee W (2009) The role of salivary
factors in persistent oral carriage of Candida in humans. Arch. Oral. Biol. 54:678-83.
[26] Epstein JB, Pearsall NN, Truelove EL (1980) Quantitative relationships between
Candida albicans in saliva and the clinical status of human subjects. J. Clin. Microbiol.
12:475-6.
[27] Kimori H, Nakagawa Y, Yamamoto K, Oshima T (2009) Establishing the cut-off point
for the Candida swab test for daily oral care in dry mouth patients. Oral. Therap.
Pharmacol. 28:17-25.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 31

CYANOSIS: CAUSES, SYMPTOMS AND TREATMENT

K. R. Ramanathan
National University of Singapore, Department of Cardiac
Thoracic and Vascular Surgery, National University Heart
Centre, National University Hospital, Singapore

ABSTRACT
Cyanosis refers to the bluish discoloration of the skin, nails or mucus membrane due
to an increased amount of reduced hemoglobin [> 5 g%] in capillary blood. Cyanosis is
broadly classified as being central, peripheral or differential but can also be caused by
abnormal pigments circulating in the blood. Central causes of cyanosis are mainly cardiac
or pulmonary while peripheral causes arise from local vasoconstriction or lack of
peripheral blood supply. Differential cyanosis happens in specific cardiac conditions.
Cyanosis is usually a sign of an underlying condition rather than being a disease in itself.
Patients with cyanosis may have other features like breathlessness, shortness of
breath, bluish or purple discoloration of the oral mucous membranes, rapid and shallow
breathing etc.
There is general tiredness or weakness in patients who suffer from long term
cyanosis. There may be episodes of headaches as well.
Treatment of cyanosis focuses on the underlying disease rather than the symptom
alone. Initial stabilization requires oxygenation. Treatment of central cyanosis due to
congenital heart defects may often involve surgery. Peripheral cyanosis brought about by
exposure to cold may be treated symptomatically using gentle warming of the fingers and
toes.
Antibiotics are prescribed for treatment of pneumonia and other infections. The
chapter analyses the various causes of cyanosis and the possible treatment options for
patients when they are cyanosed.


Dr. Ramanathan K.R.: Consultant CTICU, A/Prof – National University of Singapore, Department of Cardiac
Thoracic and Vascular Surgery, National University Heart Centre, National University Hospital, 1E Kent
Ridge Road, NUHS Tower Block, Level 9, Singapore 119228.
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762 K. R. Ramanathan

INTRODUCTION
Cyanosis refers to the bluish discoloration of the skin or mucus membrane due to an
increased concentration of deoxygenated hemoglobin in the capillary bed [1]. Cyanosis can
be caused either by reduced oxygenation of the arterial blood or by sluggish peripheral
circulation with increased oxygen extraction. The normal adult has a hemoglobin
concentration of 15 gm% of which 95% is saturated with oxygen in the arterial blood.
The capillaries contain 2-3 gm% of reduced or deoxygenated hemoglobin. When the
level of reduced hemoglobin exceeds 5 gm% in the capillaries the blood appears dark giving
the tissues a bluish hue. This discoloration is seen earliest in warm areas with increased
capillary circulation e.g., palate, tongue, inner side of lips or conjunctiva and is referred to as
central cyanosis. Peripheral cyanosis occurs due to the slowing of blood which allows more
time for removal of oxygen, so cyanosis is more visible on the tip of the nose, ear lobule, tip
of the finger and nail bed.

CAUSES OF CYANOSIS
Cyanosis is broadly classified as central, peripheral or differential, but also can be caused
by abnormal pigments in the blood.

Central Cyanosis

a. Cardiac: Circulatory conditions may cause cyanosis by mixing oxygenated and

deoxygenated blood
1 Congenital cyanotic heart disease
2 Acute pulmonary edema
3 Congestive heart failure

b. Pulmonary
1 Hypoxia due to reduced fraction of inspired oxygen
2 Obstruction of airways as in Chronic Obstructive Pulmonary Disease
3 Intrinsic Lung disease as in fibrosis of lung or bronchogenic carcinoma
4 Impaired lung expansion as in tension pneumothorax.
5 Pulmonary vascular abnormalities as in primary pulmonary hypertension.
6 Hypercoagulable states like pulmonary embolism

c. High Altitude

Peripheral Cyanosis

a. Cold
b. Shock

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 Cyanosis: Causes, Symptoms and Treatment 763

c. Raynaud’s disease
d. Polycythemia

Mixed Cyanosis

Mixed Cyanosis: Refers to a mix of central and peripheral cyanosis.

1 Acute Left Ventricular Failure

2 Mitral Stenosis [right heart failure with peripheral vasoconstriction]

Differential Cyanosis

Differential Cyanosis: Refers to cyanosis of upper or lower limbs.

a. Of lower limbs: Patent Ductus Arteriosus [PDA] with reversal of shunt

b. Of upper limbs: PDA with reversal of shunt in transposition of great arteries.

Cyanosis Due to Abnormal Pigmentation

1 Methemoglobinemia
2 Sulhemoglobinemia

APPROACH TO A PATIENT WITH CYANOSIS

History — Several historical features help determine the cause of cyanosis.

 Age – Cyanotic congenital heart disease and polycythemia are much more common
etiologies for life-threatening central cyanosis in neonates. Acrocyanosis and cold
exposure are common causes of peripheral cyanosis.
 Trauma – Chest wall or upper airway trauma may cause central cyanosis due to lung
injury or upper airway obstruction.
 Exposures – Smoke inhalation or exposure to other low oxygen environments
suggests central cyanosis from decreased inspired oxygen.
 Medications – Blue skin color may occur in patients undergoing treatment with
amiodarone.
 Prior lung disease – Exacerbation of pre-existing lung disease (e.g., asthma,
bronchopulmonary dysplasia) is a common cause of respiratory distress and central
cyanosis.
 Congenital heart disease – Cyanotic congenital heart disease may explain profound
central cyanosis in some patients.
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764 K. R. Ramanathan

Physical examination — Cyanosis is always a concerning finding and is often less

apparent in the skin of patients with darker pigmentation. For this reason, examination should
include the nail beds, tongue, and mucous membranes, which are less affected by
pigmentation.
Patients with peripheral cyanosis typically have cyanotic nail beds, decreased peripheral
perfusion, and cold extremities in conjunction with a pink tongue and oral mucous
membranes.

 Fever – Fever is often present in patients with intrinsic pulmonary conditions (e.g.,
pneumonia, bronchiolitis) and septic shock.
 Lung examination – Tachypnea is seen in patients with either respiratory or
circulatory causes of cyanosis. Similarly, flaring, grunting, and retractions are
nonspecific indicators of respiratory distress. Rales and/or wheezing suggest lower
airway disease or pulmonary edema.
 Cardiac examination - A cardiac murmur and a second heart sound that is loud or
single is heard in many patients with cyanotic structural heart disease and/or
pulmonary hypertension.

Myocardial dysfunction with pulmonary edema is suggested by the presence of a gallop

rhythm, palpable cardiac thrill, and/or laterally displaced point of maximal impulse.

 Skin examination – Central cyanosis with a slate gray appearance to the skin is
characteristic of methemoglobinemia [2].

Figure 1. Approach to a patient with cyanosis.

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 Cyanosis: Causes, Symptoms and Treatment 765

Figure 2. Differential Diagnosis for central cyanosis.

MANAGEMENT OF PATIENT WITH CYANOSIS

An approach to diagnosis of the etiology in patients with cyanosis is outlined in Figures 1
and 2.
Treatment of cyanosis focuses on the underlying disease rather than the symptom alone.
Initial stabilization requires oxygenation.
Treatment of central cyanosis due to congenital heart defects may often involve surgery.
Peripheral cyanosis brought about by exposure to cold may be treated symptomatically using
gentle warming of the fingers and toes.
Antibiotics are prescribed for treatment of pneumonia and other infections.
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766 K. R. Ramanathan

REFERENCES
[1] Driscoll, D. J. Evaluation of the cyanotic newborn. Pediatr. Clin. North Am.
1990;37(1):1.
[2] Dahshan, A., Donovan, G. K. Severe methemoglobinemia complicating topical
benzocaine use during endoscopy in a toddler: a case report and review of the literature.
Pediatrics, 2006;117(4):e806.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 32

PERINATAL CYANOSIS:
NEUROPSYCHOLOGICAL FUNCTIONING

Ashlee R. Loughan1, MEd., PhD,

Robert Perna2, RN, PhD, and Hana Perkey1, M S
1
NeuroBehavioral Associates, LLC., Augusta, Georgia, US
2
TIRR Memorial Herman, Houston, TX, US

ABSTRACT
Perinatal cyanosis is the expression of pathological low arterial blood oxygen
saturation in children surrounding the time of birth. The birth process is very complicated
and through many different mechanisms can lead to low oxygen levels in the fetus or
newborn. The passage through the birth canal and potential placental or umbilical cord
issues can interrupt CNS blood supply. Moreover, after birth the newborn must
oxygenate its own blood for the first time and many newborns have lungs which are not
sufficiently mature for this process. Thus, any respiratory immaturity or cardiac
anomalies may quickly diminish efficient oxygenation. While acute medical issues can
often be corrected with the newborn appearing fully intact, a brief period of insufficient
oxygen to the brain can result in issues which are not readily apparent. Research suggests
that instability of circulation or oxygenation is the leading cause of perinatal brain
damage (1 – 6 out of every 1000 newborns) and can result in long-term neurological
consequences, significant cognitive dysfunction, and subsequent academic challenges.
Although medical practitioners often investigate the cause of a cyanotic event, parents
and educators are frequently ill-informed as to the future developmental and long-term
learning and social implications. This chapter will investigate perinatal cyanotic
populations (i.e., cardiac, respiratory, prematurity, and hypoxic-ischemic) and their
distinct neurocognitive and behavioral profiles. Specific focus will be placed on empirical
literature which identifies cognitive, academic, and emotional deficits. The impact of
perinatal cyanosis on all domains of cognitive functioning (i.e., intellect, language,
motor, memory, attention and executive functioning) will be explored. Finally, general
developmental and academic recommendations will be reviewed.


Address all correspondence to: Ashlee R. Loughan, M.Ed., Ph.D. Neurobehavioral Associates, 639 13 th Street,
Augusta, GA, Email: [email protected]
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768 Ashlee R. Loughan, Robert Perna and Hana Perkey

Perinatal or neonatal cyanosis is an expression of pathological low arterial blood oxygen

saturation in children surrounding the time of birth and may provide the earliest sign of major
medical pathology. The recognized underlying causes of cyanosis can vary and include, for
example, intrauterine hypoxia; asphyxia, often combined with decreased cerebral blood flow
(CBF); or respiratory distress (The World Health Organization, 1993). These conditions often
result from a number of medical pathologies involving the heart and lungs or other issues
related to adverse events during childbirth (e.g., uterine rapture), prenatal infections (e.g.,
rubella), umbilical cord issues, immature lungs, congenital heart disease, and poorly
controlled gestational diabetes. The nature and duration of a cyanotic episode, as well as
oxygen saturation levels determine the severity of its consequences (Perna & Cooper, 2012).
Though quick correction of the cyanotic episode will cause a newborn to turn a healthy shade
of pink, the temporary depletion of oxygen can result in negative long-term consequences.
Research suggests that instability of circulation or oxygenation is the leading cause of
perinatal brain damage (1 – 6 out of every 1000 newborns) and can result in long-term
neurological consequences, significant cognitive dysfunction, and academic challenges. The
outcomes of cyanosis have been shown to range from mild, transient changes in behavior
(e.g., irritability, inattention, poor feeding, or excessive crying) to severe acute effects,
profound long-term deficits (e.g., seizures & intellectual disabilities), or even death (Triulzi,
Parazzini, & Righini, 2006, 2013; van Handel, Swaab, de Vries, & Jongmans, 2007; Zhang et
al., 2013). Although medical practitioners often investigate the cause of a cyanotic event,
parents and educators are frequently ill-informed as to the future developmental and long-
term learning and social implications.

COMMON CYANOTIC POPULATIONS

Cardiac Etiologies

Cardiac pathology is among the most common and researched causes of perinatal
cyanosis. Typical brain growth and development is dependent upon adequate volume and
content of oxygenated blood supply (Limperopoulos, 2009). In order for the brain to receive
ample oxygenated blood, it requires sufficient circulation from the heart, which can be
negatively impacted by an abnormal cardiovascular system or event (Limperopoulos, 2009).
Poor oxygen supply or cardiac procedures, such as those resulting from a congenital defect,
can produce a significant impact on a child’s developing cognitive abilities. Specific
neurologic abnormalities have been found by multiple congenital heart defects (CHD) group
and longitudinal studies (Bellinger et al., 1995, 1999, 2003; Green, 2004; Hovels-Gurich et
al., 1997, 2002; Limperopoulos et al., 1999, 2000).
Approximately eight out of every 1,000 newborns have congenital heart defects, which
can range from mild to severe (Green, 2004; Hoffman & Kaplan, 2002; Karsdorp, Everaerd,
Kindt, & Mulder, 2006; Limperopoulos et al., 1999; Miatton, de Wolf, Francois, Thiery, &
Vingerhoets, 2006). A multitude of heart defects have been known to cause some level of
cyanosis (e.g., coarctation of the aorta, hypoplastic left heart syndrome, Tetralogy of Fallot,
transposition of the great arteries, etc.). As a result, researchers have begun investigating the
neuroanatomical structures of children with CHD and found irregularities both pre- and post-

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surgical intervention (Donofrio et al., 2003; Donofrio & Massaro, 2010; Johnston, 2007;
Limperopoulos et al., 2010; McQuillen & Miller, 2010). This suggests that children with
CHD may be exposed to numerous cyanotic risk factors including poor circulation in utero,
oxygen deprivation post-delivery, surgical procedures during correction, and for some, all of
the above (Donofrio et al., 2003; Donofrio & Massaro, 2010; Johnston, 2007; McQuillen &
Miller, 2010). Though the cardiac issues may be fully corrected, there is the risk of persistent,
yet often subtle effects on brain functioning. Compared to peers in the general population,
children with CHD have an increased probability of abnormalities in the central nervous
system resulting in some form of brain injury (Green, 2004; Majnemer et al., 2008). Identified
reasons include “chronic hypoxemia, congestive heart failure, poor nutrition, polycythemia,
right-to-left shunting with the risk of embolic events or brain abscess, episodes of arrhythmia
or cardiac arrest, and other organ system abnormalities” (Bellinger et al., 1991, p.702). A
common factor investigated is the insufficient circulation prior to and after surgical repair as
even brief periods of hypoxia can cause adverse effects and neurologic injury (McElhinney &
Wenovsky, 2001; Perna & Cooper, 2012).

Respiratory Etiologies

Respiratory distress in neonates interferes with effective intake of oxygen which often
leads to cyanosis. Neonatal respiratory distress is quite frequent and, in its more severe
manifestations, contributes significantly to infant mortality rates (Hermansen & Lorah, 2007;
Kumar & Bhat, 1996). The most common cause of respiratory distress in newborns is from
incomplete removal of fluid from the lungs following the dilation of lymphatic vessels post-
delivery. The resulting transient tachypnea typically resolves in a matter of a few hours to two
days. A more serious issue leading to neonatal respiration difficulties is respiratory distress
syndrome (RDS). Associated with structural and functional lung immaturity, RDS is most
prevalent in infants born before the 28th week of gestation. In more severe cases, RDS can
develop into bronchopulmonary dysplasia and chronic oxygen dependence (Campbell,
McAllister, Volpe, 1988; Hermansen & Lorah, 2007).
Meconium Aspiration Syndrome (MAS) is another common cause of infant respiratory
distress (Hermansen & Lorah, 2007). Related to the release of meconium into amniotic fluid,
MAS can lead to hypoxia if aspiration occurs in utero (Hermansen & Lorah, 2007; Karatekin,
Kesim, & Nuhoḡlu, 1999). Current research links more severe cases of MAS to other factors
(e.g., prolonged fetal compromise or intrauterine infections) rather than the aspiration of
meconium alone (Ghidini & Spong, 2001). Other, less frequent, conditions, such as
infections, pulmonary hypertension, or pneumothorax, can also compromise respiration
following delivery and increase the risk for hypoxia and cyanosis. Current literature has
established a relationship between more severe cases of respiratory distress and deficits of
neurological functioning (Campbell, McAllister, Volpe, 1988; Meisels, Plunkett, Roloff,
Pasick, & Stiefel, 1986). Specifically, RDS and bronchopulmonary dysplasia are both linked
to subsequent motor and cognitive developmental delays, and, in some cases, progressive
neurological disease (Campbell, McAllister, Volpe, 1988; Meisels, Plunkett, Roloff, Pasick,
& Stiefel, 1986).
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770 Ashlee R. Loughan, Robert Perna and Hana Perkey

Prenatal Etiologies Related to Low Birth Weight and Nenonatal Prematurity

As a group, infants who are born preterm and/or have low birth weight overlap
significantly with the two previously mentioned populations—children with CHD and
pulmonary disease; as both low birth weight and neonatal prematurity are associated with
cardiac and pulmonary pathology. According to Cole, Hagadorn, and Kim (2002) and
Fanaroff et al. (2007), the majority of neonatal complications (specifically surrounding
preterm birth) include collapsed lungs or chronic lung disease, cardiac abnormalities, shallow
or arrested breathing, infection or nutritional disturbances, and brain abnormalities evident in
neonatal cranial ultrasounds (i.e., intraventricular hemorrhage, periventricular leukomalacia,
& ventricular dilation). This cerebral pathology, especially in neonates with low birth weight,
is attributed primarily to hypoxic-ischemic events which can result from many of the cardiac
and respiratory issues described above (Inder & Volpe, 2000). Brain damage most often
occurs in the subcortical structures and circuits connecting these structures to the frontal and
parietal regions. Dysfunction in these areas has the potential to negatively impact the
development of executive functions, attention, and self-regulation.
Both preterm birth and low birth weight are complex variables that may carry different
risks for different individuals. Highly correlated with each other, many additional factors
(e.g., previous miscarriages and preterm pregnancies, maternal age, maternal stress, other
psychosocial factors, and exposure to toxins; Perna, Loughan, Perkey, & Tyson, 2014; Pitzer
et al., 2001; Wadhwa, Sandman, Porto, Dunkel-Schetter, & Garite, 1993) can play a role in
preterm and low birth weight delivery and thus increase the risk for cyanosis. In recent
decades, advances in neonatal intensive care, including resuscitation, assisted ventilation,
drug treatments, intravenous nutrition, and phototherapy for jaundice (Hack & Fanaroff,
1999) have led to increasing survival rates of children with low birth weight (2,500g) or
preterm birth (<37 weeks). According to the National Center for Health Statistics, infants
with low birth weight comprise 1-8% of US live births, and 2-12% of those, depending on
exact classification requirements met the criteria for preterm births. Along with the
decreasing mortality and morbidity, these statistics suggest that the preterm and low birth
weight group constitutes a large and continually growing high risk population for cyanotic
attacks and associated outcomes. For example, children with very low birth weight have been
shown to be at much higher risk for brain insults and abnormalities in cognitive development,
behavior, and learning compared to their normal weight peers (Aarnoudse-Moens, Weisglas-
Kuperus, van Goudoever, & Oosterlaan, 2009). Additionally, although prematurity can result
in a generalized cognitive impairment, many children display more subtle deficits.

Hypoxic-Ischemic Brain Injury

Many of the conditions discussed above result in cerebral insults that fall under the
umbrella of hypoxic-ischemic brain injuries. This term signifies the importance of the role
that both reduced levels of oxygen (i.e., hypoxia) and/or diminished cerebral blood perfusion
(i.e., ischemia) play in the type and severity of the outcomes (Arciniegas, 2012; Stevens, Raz,
& Sander, 1999) from these injuries. The same two factors---hypoxia and ischemia---are also
prerequisite in the manifestation of cyanosis. The incidence rates for hypoxic-ischemic
encephalopathy (HIE) are reported at two – five per 1000 live births in the current literature

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(Graham, Ruis, Hartman, Northington, & Fox, 2008; Huang et al., 2013; Zhang et. al., 2013).
Research identifies neonatal HIE as one of the most prominent causes of moderate to severe
neurological deficits in children (Huang et al., 2013; de Vries & Jongmans, 2010).
Furthermore, even mild cases of HIE have been associated with later cognitive impairments
(Huang et al., 2013, de Vries & Jongmans, 2010). Because the long brain developmental
period, these impairments may not occur for several years.
Hypoxic-ischemic brain injuries are described and investigated in both animal (Huang et
al., 2013; Zhang et al., 2013) and clinical research (Kurinczuk, White-Koning, & Badawi,
2010; Triulzi et al., 2006; de Vries & Groenendaal, 2010). The current animal models identify
subcortical brain, hippocampus and areas surrounding lateral ventricle as vulnerable to
hypoxia-ischemia, manifesting decreased brain-derived neurotrophic factor levels,
leukoaraiosis (white matter abnormalities), abnormal neural cell arrangement, neuron
degeneration, and cell death (Huang et al., 2013; Zhang et al., 2013). These ischemic injuries
are associated with motoric and memory impairments in later development (Huang et al.,
2013; Zhang et al., 2013). Animal model researchers have also highlighted the fact that the
extent and location of primary damage varies depending on the gestational phase and fetal
maturity at the time of hypoxic-ischemic event (Towfighi, Mauger, Vannucci, & Vannucci,
1997).
Similarly, clinical research has identified developmental stages as an important factor in
HIE outcomes (Triulzi et al., 2006). Specifically, Triulzi and colleagues (2006) found that the
premature brain was more vulnerable to white matter damage, whereas full-term infants
showed more grey matter involvement. Additionally, the location of insult could be further
specified by the severity of the hypoxic-ischemic event (Triulzi et al., 2006). Investigators
have also noted a general trend of progression from more focal and circumscribed injuries
resulting from mild to moderate hypoxia-ischemia to diffuse brain damage symptom pattern
in more severe cases (Triulzi et al., 2006). From a developmental perspective there is also a
potential for a more diffuse presentation with increasing age. Recent research using more
advanced methods of imaging (e.g., diffusion-weighted imaging, DWI, and magnetic
resonance imaging, MRI) have also described distinct patterns of damage, which were related
to specific deficits in later development (de Vries & Groenendaal, 2010; de Vries &
Jongmans, 2010). Specifically, two main patterns emerged in the MR imaging: a basal-
ganglia-thalamus pattern (BGT), which was most common in cases that included an acute
sentinel event (e.g., placental abruption, uterine rapture), and the watershed predominant
pattern of injury (WS), typically seen in cases of prolonged partial asphyxia (de Vries &
Groenendaal, 2010; de Vries & Jongmans, 2010). The former was associated with more
severe disabilities, such as dyskinetic cerebral palsy and epilepsy, as well as learning
disabilities, while the latter, involving the vascular WS zones, evidenced suboptimal head
growth, as well as language, and behavioral problems (de Vries & Groenendaal, 2010;
deVries & Jongmans, 2010). Although the focus of investigations often seems to be on the
more serious HIE and the associated severe impairments, research does suggest that even
mild-to-moderate incidents of perinatal and neonatal hypoxia-ischemia of either short or
prolonged duration can have adverse effects on development and later cognitive functioning
(Bass et al., 2004; Perna & Cooper, 2012).
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772 Ashlee R. Loughan, Robert Perna and Hana Perkey

Neurocognitive Implications

A review of the literature shows that severity of neurocognitive outcomes largely

corresponds with the severity of the diagnosed neonatal HIE (Pin, Eldridge, & Galea, 2009;
de Vries & Jongmans, 2010). Utilizing primarily the Sarnat classification of mild, moderate,
and severe (Sarnat & Sarnat, 1976), researchers have shown that the short-term outcomes
(i.e., under 3 years) for infants with mild HIE do not include adverse cognitive effects. Infants
with moderate HIE classification tend to experience more variable outcomes, and all infants
diagnosed with severe HIE incur some adverse cognitive consequences (i.e., at least two
standard deviations below their age group on a measure of cognitive functioning; Dixon et al.,
2002; Pin, Eldridge, & Galea, 2009; de Vries & Jongmans, 2010). The longer-term effects of
perinatal and neonatal HIE are less empirically investigated, however similar relationships
between the classification of the neonatal HIE and the outcomes are evident. While school-
age children that were classified with mild HIE continue to perform cognitively on par with
their peers, non-disabled survivors with moderate HIE and disabled children with severe HIE
show a pattern of weakness in specific cognitive domains (de Vries & Jongmans, 2010,
Marlow, Rose, Rands, & Draper, 2005; Mañeru, Junqué, Botet, & Guardia, 2001).

Intelligence

With regard to overall intellectual ability, researchers have found that elementary school-
age children who had a medical history of moderate HIE performed similarly to healthy
peers (controls) in all domains of intellectual functioning (e.g., spatial and verbal reasoning;
Marlow et al., 2005). Conversely, those with a history of severe HIE performed significantly
poorer than the normal developing peers (controls) as well as the moderate HIE children in all
measured domains. The only exception was a non-significant difference between moderate
and severe HIE children on a subscale measuring verbal ability. Similarly to Marlow’s et al.
(2005) findings, Mañeru and colleagues (2001) reported significant differences from control
group in perceptual reasoning for their moderate HIE group, but not for their participants with
history of mild HIE.
In the low birth weight population, children showed a greater downward displacement of
the IQ distribution with decreasing birth weight when Taylor et al. (2004) examined 3 groups
of children born at different gestational weights (<750g; 750-1499g, & full-term). In addition,
a bimodal distribution of scores was most evident in the group with < 750g birth weight. The
elevated subset of more severe IQ deficits suggests that those children possibly sustained
more severe brain insult; however, as previously mentioned, children with low birth weight
who suffered a cyanotic episode, may also have had other issues which affected brain
development.
Children with congenital heart disease (CHD) have also been investigated in terms of
their overall cognitive abilities. Researchers have revealed, in general, age-expected
intellectual functioning (Forbess et al., 2002; Oates, Simpson, Cartmill, & Turnbull, 1995).
Interestingly, even with mean IQ scores falling in the average range, children with CHD fall
below the average full scale IQ of 100, more typically floating around a full scale IQ of 90;
especially in specific groups of more severe cardiac pathology (Bellinger et al., 1999, 2003;

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Hovels-Gurich et al., 1997; Hovels-Gurich et al., 2002; Majnemer et al., 2008; Silbert et al.,
1969; Wray & Sensky, 1999).

Language

Language appears to be an area of significant weakness for the neonatal HIE population
with deficits in both expressive and receptive functioning. Marlow and colleagues (2005)
found that both children with moderate and severe HIE scored significantly lower on a scale
of language functioning than the controls, while they did not significantly differ from each
other.
With regard specifically to the preterm population, researchers have revealed that preterm
children have more limited social and communicative interactions than term-birth controls
(Landry, Smith, Miller-Loncar, & Swank, 1997). However, speech and language impairments
appear to be less consistently evident during the school years than was demonstrated
originally in early childhood (Luoma, Herrgard, Martikainen, & Ahonen, 1998). Some of
these children may have sufficient cognitive reserve to eventually fully compensate for the
early life hypoxic induced brain injury.
In the CHD population, Bellinger et al. (1999, 2003), Hovels-Gurich et al. (1997, 2002),
and Majnemer et al. (2008) revealed decreased language ability in comparison to healthy
peers with results suggesting both expressive and receptive delays. Additionally, speech
dysfunction was found to be a noted concern in this population.

Motor

Many children with history of severe HIE and some in the moderate classification
develop physical disabilities that dramatically impact their motor functioning (Marlow et al.,
2005; de Vries & Jongmans, 2010). For example, in Marlow’s et al. study (2005) more than
20% of the participants suffered with cerebral palsy with major disabilities (i.e., quadriplegia,
hemiplegia, and diplegia). Mañeru et al. (2001) investigated differences between children
with mild and moderate neonatal HIE and a control group on visuo-motor task (i.e., the
Purdue Pegboard test; Tiffin & Asher, 1948) and a pre-motor function assessment (Luria’s
hand sequencing task). Their findings supported a significant difference in performance for
the moderate HIE group, but not the participants with mild HIE (Mañeru et al., 2001).
Similarly, low birth weight children have demonstrated deficits in motor skills, particularly at
young ages (Landry et al., 1997).
Conversely, the research literature suggests that motor functioning of children with CHD
is variable Although some research has found that motor skills fall within the normal range
(Forbess,Visconti, Bellinger, Howe, & Jonas, 2002a; Forbess et al., 2002b; Silbert et al.,
1969), most find both fine and gross motor function delays. During school age, Majnemer et
al. (2006) reported that difficulty with balance and/or coordination may limit the recreational
activities that CHD children can successfully participate in, affecting social interaction with
their peers. Thirty to 60 percent of classroom activities involve fine motor skills, thus a
reduction in motor ability can have a large impact on learning and academic success. At a 5
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774 Ashlee R. Loughan, Robert Perna and Hana Perkey

year follow-up, 49.4% of their population presented with gross motor deficits and 39% had
fine motor challenges (Majnemer et al., 2006).

Memory

The hippocampus is one of the brain structures identified as vulnerable to HIE damage
(Huang et al., 2013; Mañeru et al., 2003; Vargha-Khadem et al., 1997; Zhang et al., 2013).
For example, researchers discovered hippocampal atrophy in subjects with antecedent
moderate HIE that corresponded with poorer long-term memory; however in the absence of
other cognitive deficits this impairment did not affect development and academic functioning
(Mañeru et al., 2003; Vargha-Khadem et al., 1997). Conversely, other investigations with
participants who displayed a number of cognitive impairments discovered more substantial
differences in memory functioning that likely affected academic achievement. For example,
Marlow et al. (2005) noted impairment in short-term auditory memory as well as poor
orientation. Similarly, Mañeru et al. (2001) found that participants with history of moderate
HIE performed more poorly than controls on measures of delayed recall of a word-learning
task as well as the Visual Reproduction Test (Wechsler Memory Scale-Revised, WMS-R;
Wechsler, 1987). Additionally, the moderate HIE subjects achieved lower scores on a
measure of working memory (Digit Span, Wechsler Intelligence Scale for Children-Revised,
WISC-R; Wechsler, 1974), even though they performed significantly differently only on the
forward condition and not on the backward portion of the test (Mañeru et al., 2001). Yet other
investigators have reported case studies of subjects with moderate antecedent HIE who
experienced a significant impairment of episodic memory (Gadian et al., 2000).
Memory, assessed over both the short and long term on psychometric measures, is a
cognitive domain that appears to be relatively intact within the CHD population when
compared against their peers. Forbess et al. (2002) found memory and learning to be within
normal limits in a sample of 243 children who underwent repair or palliation of CHD.
Bellinger et al. (2003) also examined memory at the eight year follow up and found no
significant difference between the control and CHD groups.

Attention and Executive Function

Research suggests that this domain of cognitive functioning is affected most in children
who had been classified as having severe HIE, while children with history of moderate HIE
tend to score similarly to controls on measures of attention and executive functioning
(Marlow et al., 2005). Correspondingly, Mañeru and colleagues (2001) found no differences
in performance of participants with mild and moderate HIE and the control group.
Similarly, the most frequent deficits found in premature children and/or low birthweight
children were impairments in working memory, attention, and executive functioning. In fact,
during the “School-Age Follow-up Project,” out of the four composite scores investigated
(Language, Memory, Visual-Motor, and Executive Functioning), there was a significantly
larger group difference in the Executive Functioning deficits than any other category (Taylor
et al., 2004). Children in the <750g birthweight group were more prone to specific deficits
including slower rate of learning, impulsive responses, slowed processing times, and poorly

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organized approaches to multistep tasks (Taylor et al., 2004). Elevated rates of inattention and
hyperactivity have also been demonstrated across multiple studies. For example, Botting,
Powls, and Cooke (1997) found that 23% of low birthweight children were diagnosed with
ADHD compared to 6% of their matched full-term peers. These executive deficits remain
evident even when IQ is controlled for (Edgin et al., 2008; Espy, Stalets, McDiarmid, Senn,
Cwik, & Hamby, 2002; Vicari, Caracale, Carlesimo, Casadei, & Allemand, 2004).
Minimal research has been completed on the investigation of attention and executive
functioning skills in children with CHD. Overall, few researchers have suggested attention,
impulse control, and organization deficits (Bellinger et al., 2003; Bernstein & Waber, 1996;
Hovels-Gurich et al., 2007; O’Dougherty, Berntson, Boysen, Wright, & Teske, 1988).

Academic

Differences in academic achievement between children with neonatal HIE and

comparison peers have been supported by research findings. Marlow et al. (2005) discovered
lower levels of achievement in both moderate and severe HIE groups; however, while the
severe HIE group evidenced poorer performance on all indicators of attainment, the moderate
HIE participants underperformed on measures of spelling and reading only (Marlow et al.,
2005).
Follow-up studies indicated that children with low birthweight continued to demonstrate
pervasive cognitive problems both at age of school entry (Msall, Buck, Rogers, & Catanzaro,
1992; Saigal, Szatmari, & Rosenbaum, 1992; Wolke & Meyer, 1999) and throughout their
academic years (Saigal, 2000; Taylor, Minich, Klein, & Hack, 2004). Testing of reading,
spelling, math, written expression, and handwriting were all demonstrated as areas of lower
scores when compared to the full-term controls (Anderson et al., 2006; Feder et al., 2005;
Grunau, Whitfield, & Davis, 2002; Hack et al., 1992; & Taylor et al., 2006). In fact, Saigal et
al. (2000) found that 58% of their sample of extremely low birthweight children (ages 12-16)
had either repeated a grade or were receiving special education services. This is compared to
only 13% of their peers.
For the CHD population, although overall functioning often falls within the normal range,
van der Rijken et al. (2007) proposed that problems for children with CHD most likely appear
at school age when they have to “rise up” and meet specific cognitive and social demands
being placed upon them. Multiple researchers have suggested that children with CHD are
performing below average in academic functioning and require school services. Wright and
Nolan (1994) found that surgically corrected CHD children performed significantly worse on
measures of academic functioning compared to their peers who had cardiac murmurs not
requiring surgery. More specifically, Hovels-Gurich et al. (2002) revealed that 23.3% of their
investigated population had below average academic knowledge, with 18.3% below one
standard deviation from the mean and 5% below two standard deviations. During the Boston
Circulatory Arrest Trail, 37% of parents reported that their child received remediation in
school and 10% had repeated a grade by age eight (Bellinger et al., 2003). These numbers are
similar to the study conducted with Dutch children showing 26% of CHD children were
receiving special education along with an increase in the number of children repeating a grade
(van der Rijken et al., 2007).
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776 Ashlee R. Loughan, Robert Perna and Hana Perkey

Psychosocial Functioning

Existing literature also supports an association between neonatal moderate to severe HIE
and deficits in psychosocial functioning. Marlow et al. (2005) reported a significantly higher
incidence in behavioral problems, mostly manifested as increased hyperactivity, more
emotional difficulties and less pro-social behavior, with higher impact on daily functioning in
the severe group. Conversely, children with antecedent moderate HIE faired comparably to
the control group (Marlow et al., 2005).
Similarly, behavioral consequences are also evident for children with low birthweight as
early as 3 and 6 months post delivery (Wolf et al., 2002). Later behavioral sequelae have also
been seen in adaptive behavior skills, social competence, hyperactivity, both internalizing and
externalizing problems, and atypical behavior patterns when compared to term children
(Bhutta, Cleves, Casey, Cradock, & Anand, 2002; Klebanov, Brooks-Gunn, & McCormick,
1994; Taylor, Hack, & Klein, 1998). For example, parent reports showed elevated rating of
social, thought, and attention problems up to 0.5 to 1.2 standard deviations above controls
(Hille et al., 2001). On personality questionnaires, Allin et al. (2006) revealed lower scores on
extraversion (i.e., sociability), higher scores on neuroticism (i.e., anxiety, low mood, and low
self-esteem), as well as higher scores on the “lie” scale when compared to full-term controls.
Additionally, more symptoms of anxiety and depression have also been reported in low
birthweight children compared to their full-term peers (Elgen, Sommerfelt, & Markestad,
2002; Indredavik et al., 2004).
Psychosocial difficulties also appear to be a common finding in those with CHD
(DeMaso et al., 1990; Green, 2004). Behavioral difficulties were found in 33% of children
with CHD and included increased activity level, irritability, and oppositional/defiant behavior
(Limperopoulos et al., 2000), as well as internalizing behaviors, including anxiety,
withdrawal, sadness, low self-esteem, and somatic complaints (Kramer, Awiszus, Sterzel, van
Halteren, & Calarben, 1989; Majnemer et al., 2008; van der Rijken, Maassen, Walk, Daniels,
& Hulstijn-Dirkmatt, 2007). In addition, Utens et al. (1998) examined the relationship of
cardiac variables to psychosocial outcomes and found that the number of operations appeared
to be a significant predictor of the intensity of internalizing, and externalizing behavior
patterns; with those who had more operations showing increased externalizing patterns.

INTERVENTIONS
Interventions should always be individualized, but there are many general rules which
warrant mention. Children who have experienced a cyanotic episode should be screened for
early developmental delays. Some research suggests that those children who have
experienced cyanosis may have a higher incidence of developmental disorders. Those that
have emerging developmental disorders should receive early assessment and interventions.
These children may be at risk for a variety of comorbidities, and this risk likely warrants a
neuropsychological assessment in their early education. Some of these children will have
specific learning disorders, and many will likely be responsive to intervention. Still, others
may need early speech or occupational therapy. A common long-term symptom of an early
life brain injury is disruption of the attention systems of the brain and the potential for

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difficulty with self-regulation. While there is very strong empirical research supporting the
benefits of stimulant medications for these children, many behavioral strategies can be very
effective also in helping support their learning. Some of the research suggests that the
behavioral strategies may actually help these children develop long-term self-regulation
skills, self-esteem, and a level of self-efficacy that would not be appreciated if only
medication was used.
Often appropriate academic accommodations can help the child make the necessary
academic progress. A structured schedule and perhaps structured tasks or cognitive and
academic exercises will serve to help the child to make age appropriate educational gains.
There is often a need for an Individualized Educational Plan (IEP) for many of these children.
Again, earlier implementation of treatment and accommodations, if needed, may help create
the setting for academic success. The following are some academic and behavioral
recommendations/accommodations which may be beneficial to those with a history of
cyanotic insults:

Language

 Dysfunction in the language areas of the left hemisphere of the brain can result in a
diverse range of impairments at different ages. From ages 1 to 3 language issues
involve delayed speech or poor speech articulation, then potentially delayed speech
fluency, problems listening, and then difficulty reading.
 Speech and language problems warrant early intervention for best outcomes.
 Acquiring well developed speech requires good role modeling and feedback.
 Difficulty with reading requires many hours of structured practice.
 Seek the services of a Speech and Language Pathologist to identify specific language
deficits and practice individualized strategies. Often children attend private sessions
weekly to assist with either expressive or receptive language deficits.
 Regularly practice Rapid Auditory Processing tasks and various speaking and
reading drills.

Motor

 Motor delays can take very subtle forms and may start as low much tone in infants
and poor head control, but later during the first year may involve a delay in crawling
or walking. Later it may involve gross motor issues like clumsiness and then fine
motor issues like difficulty effectively using eating utensils and then difficulty with
writing skills.
 Working with an Occupational Therapist can be quite beneficial to assist with fine
and gross motor skills; particularly in daily living activities.
 Assistance with motor coordination tasks (i.e., cutting, tying shoes, buttoning/zipping
coat)
 Having access to a Letter Strips to refer to and graph paper to write on when needing
help on letter formation. Make sure the parents have the same strip at home during
homework time.
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778 Ashlee R. Loughan, Robert Perna and Hana Perkey

 Handwriting requirements can be limited by allowing the child to give answers orally
and/or having them write every other problem.
 Taking breaks during lengthy or demanding gross motor activities to reduce motor
fatigue (i.e., gym class or recess)
 Practice cutting both at school and at home. Make sure you are using child scissors
(small handles and shorter blades). You can start by playing puppet games to build
muscle strength and memory. When practicing cutting, begin with short straight
lines, then longer lines, then finally to curvy lines before proceeding to actual shapes
or pictures.
 Play Simon Says, Hokey Pokey, or Animal Walk often and incorporate gross motor
skills.
 Introducing assistive technology early may assist with fine motor deficits and
difficulty with handwriting. This could include a word processor or verbal recording
devices when required to write lengthy assignments; especially as they advance in
the grade levels.

Memory

 Make the use of Acronyms. When using Acronyms to help students remember
information, it is frequently found that the more unusual the phrase, the better. If a
student is non-verbal, then helping them create a visual representation of the
information (i.e., cartoon) may also be a useful tool.
 Use external memory compensatory strategies including recording devices, smart
phones, tablets, daily planners, and To-Do lists. This can help a student who cannot
retain large amounts of information or hold information in their mind long enough to
take sufficient notes. Recording the lesson and listening to it later will assist
retention.
 It is important to make learning meaningful experience. Children who can relate a
topic to an area of interest or to an event or a topic which is already solidified in their
brain is going to be beneficial when required to store new information while learning.
 Particular placement of objects in specific locations to help the child find things and
more effectively complete routines like getting ready for school. For example,
placing a child’s coat in front of their backpack will help ensure they see their coat,
and remember to bring their coat, before grabbing their bag on the way out to the
dismissal bus. Getting school supplies ready and in place the night before school and
establishing a specific morning (before school) routine may also greatly help children
compensate for forgetfulness.

Attention / Executive Functioning

 To-Do lists or Visual lists are very helpful for students with short working memories.
Often time’s children will forget their routines or which step is next in a multi-step
directional activity. Teachers can post to-do lists in the classroom for students to
refer to. Individual children can also have personal post-it notes on their desks with
reminders or tasks to complete. Checklist can be effectively used for nearly any

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academic task including how to get out of the house on time each morning. Some
parents say posting a checklist of the morning routine can be a sanity saver: make
your bed, brush your teeth, get dressed, have breakfast, grab your lunch, get your
backpack.
 A time sheet is often helpful for students to help plan their homework time and
schedule. Specific time-cards can be used where each student learns to fill in
timeslots with specific activities needing to be performed each night before bedtime
and/or each week. Models and specific instruction are even more important for use
when children enter middle and high school where independent planning is expected
and required in order to juggle multiple deadlines at a time.
 Planning as a class which step should be completed and when it should, will help
facilitate proper planning.
 Using scoring rubrics can also help children learn to plan what needs to be included
in their assignment or projects.
 When organizing class work materials, specific systems can be taught including color
coding, using separate binders to organize subjects and projects, and using
assignment books.
 Breaking down the writing process explicitly with organizers (i.e., essay webs),
templates, and assignment sheets would be helpful to all students, not only those with
organization deficits.
 Teaching organizational note-taking strategies can also assist students during lectures
and when reading textbooks independently. These strategies ensure that the students
are interacting with their text instead of just skimming the material passively.

Emotional / Behavioral

 Seek out the services of a Licensed Clinical Psychologist to provide individualized

coping and motivational strategies.
 Develop a positive management program for appropriate emotions and behaviors.
The use of “marbles” or “tokens” can help reinforce a student’s appropriate
behaviors.
 Parents and teachers should utilize a ratio of five praise statements to each correction
or command to assist with motivation. This 5:1 ratio has been shown in research to
be highly effective at treating behavioral difficulties and reducing future emotional/
mood difficulties.
 Help the child with concrete visual strategies to understand their emotions. For
example, the child might work with a professional to develop a “thermometer” or
“speedometer” metaphor for measuring emotions. They should label each
temperature or speed to reflect degrees of specific emotions (i.e., anger, anxiety,
etc.). Each level should then be tied to a specific concrete behavior, such as counting
to delay responses, terminating the conversation, seeking adult intervention, or
immediately leaving the situation.
 It is important to identify triggers and observe when children are becoming
overloaded and frustrated. Triggers often exacerbate the emotions and behaviors,
leading to increased irritability, inattention, impulsivity, inflexibility, or aggression.
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780 Ashlee R. Loughan, Robert Perna and Hana Perkey

When they become overloaded, provide them with a break or down time away from
other children in which they can engage in quiet pleasurable activities.

CONCLUSION
Frequently, perinatal and postnatal cyanosis is the manifestation of low arterial blood
oxygen saturation in neonates. Passage through the birth canal can induce trauma. Numerous
problems can negatively affect the placenta and the umbilical cord, leading to an interruption
of CNS blood supply. Moreover, upon emerging, the newborn must oxygenate its own blood
for the first time. Thus, any respiratory immaturity or cardiac anomalies may quickly
diminish efficient oxygenation. While acute medical issues can often be corrected with the
newborn appearing fully intact, a brief period of insufficient oxygen to the brain can result in
issues which are not readily apparent. As outlined in this text many medical issues can cause
cyanosis and some may be sufficiently severe enough to cause a hypoxic-ischemic episode
and result in brain dysfunction. The effects of these episodes can be very apparent in some
cases such as when a child develops cerebral palsy. Yet, often the consequences of hypoxia-
ischemia may not be fully apparent in newborns and infants. In fact, in some cases, the full
impact of the injury may not be evident for many years. Documentation of the event may be
important and may subsequently allow for the correct diagnosis and intervention. Physicians
should be made aware of children who have experienced an episode of hypoxia-ischemia and
monitor their development with follow-up assessments. Such ongoing evaluation and care
may afford the timely implementation of any necessary interventions. There is certainly at
least some risk that children with antecedent hypoxic-ischemic injuries may be misdiagnosed
at some point during their childhood if the examining clinician is not alerted to their medical
history and the hypoxic-ischemic event. Possible misdiagnoses may include ADHD or other
disorders involving executive dysfunction, impulse control, or even psychiatric issues. Thus,
inquiry during the clinical interview should always explore possible early life HIE, especially
when there are unexplained cognitive impairments or delays.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 33

LARYNGOMALACIA:
A CAUSE OF CYANOSIS IN PEDIATRIC AGE

Marco Berlucchi1,*, Diego Barbieri2, Daniela Tonni1,

Silvana Molinaro3, Patrizia Bardini3 and Nader Nassif1
1
Department of Pediatric Otorhinolaryngology, Spedali Civili, Brescia, Italy
2
Department of Otorhinolaryngology, University of Brescia, Brescia, Italy
3
Department of Pediatric Anesthesiology, Spedali Civili, Brescia, Italy

ABSTRACT
Laryngomalacia is the most common congenital laryngeal anomaly and the most
frequent cause of stridor in pediatric age. The disorder involves 45−75% of all infants
with congenital stridor. Signs and symptoms appear usually within the first 2 weeks of
life and the spectrum of disease presentation, progression, and outcomes is variable. Most
cases are mild and resolve spontaneously without surgical procedures. Such patients
undergo only medical treatment for the gastroesophageal reflux that is generally
associated with this disease. Only severe cases with intolerable symptoms such as
dysphagia, failure to thrive, dyspnea, cyanosis, intermittent complete obstruction and/or
cardiac failure must undergo surgery. Surgical therapy is in relation to the type and
severity of laryngomalacia. The authors describe the different clinical pictures that every
clinician can observe and summarize the different treatments available.

INTRODUCTION
Characterized by weak laryngeal tone, laryngomalacia is the most common congenital
laryngeal anomaly. The pathological condition favors collapse of supraglottic structures into
the airway causing inspiratory stridor and airway obstruction 1, 2 and represents the most

*
Corresponding author: Marco Berlucchi, MD; Department of Pediatric Otorhinolaryngology, Spedali Civili,
Piazza Spedali Civili 1, 25123 Brescia – Italy. Tel. +390303996226; Fax +390303996009; E-mail:
[email protected]
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frequent cause of stridor in pediatric age. Its symptomatology ranges from simple laryngeal
stridor to severe dyspnea associated with cyanosis and difficultly in feeding. Thus, treatment
of the disease is in relation to its clinical picture.

TERMINOLOGY AND INCIDENCE

The term laryngomalacia, or soft larynx in Latin, replaced the more antiquated term
congenital laryngeal stridor, which had previously been used to describe the condition. First
coined by Jackson and Jackson in 1942 3, this term differentiated the disorder from other
causes of stridor and more clearly depicted the flaccidity of the larynx 4.
Its incidence is unknown because it has been estimated in only cohorts of infants affected
by stridor referred for specialist consultation. Holinger reported that 59.8% of congenital
laryngeal anomalies in children presenting laryngeal stridor were due to laryngomalacia 5.
Furthermore, in a large series of congenital laryngeal anomalies, the incidence of
laryngomalacia ranged from 50% to 75% 6, 7, 8. Laryngomalacia affects males more than
females 5, 8, 9, 10, 11, 12, 13, 14, but the importance of this is not known. Considering
racial demographics, in 2007 Thompson showed that laryngomalacia is more common and
more severe in Caucasian infants 2.

PATHOLOGICAL MECHANISM
To date, the precise pathophysiologic anomaly causing laryngomalacia is still unknown.
Several theories have been proposed to explain its pathogenesis. Anatomic, histological, and
neurologic factors can all contribute to the development of laryngomalacia. Regarding an
anatomic theory, this has been challenged. Since an anatomic theory is based on abnormal
placement of flaccid tissue in the larynx 15, it does not explain why some children with
typical anatomic laryngeal findings of laryngomalacia do not have the characteristic
symptoms of laryngomalacia. Several classifications of laryngomalacia have been proposed
12, 16, 17, 18, 19, 20, 21, 22, 23. Among these, Holinger’s one is the most widely used and
categorizes laryngomalacia into the following 5 types 16:

Type 1: anterior prolapse of the arytenoid folds and corniculate cartilages (Figure 1).
Obstruction occurs as the cuneiforms are drawn inward during inspiration;
Type 2: tubular epiglottis which curls upon itself, often associated with type 1 (Figure 2);
Type 3: anteromedial collapse of the arytenoids (Figure 3);
Type 4: posterior inspiratory displacement of the epiglottis against the posterior
pharyngeal wall or vocal folds (Figure 4-5);
Type 5: short aryepiglottic folds leading an overly acute angle of the epiglottis at the
laryngeal inlet (Figure 6).

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Figure 1. Anterior collapse of the arytenoids and corniculate cartilages.

Figure 2. Tubular epiglottis curling upon itself associated with type 1.

Figure 3. Anteromedial collapse of the arytenoids associated with edema retrocricoid area due to
gastroesophageal reflux.
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792 Marco Berlucchi, Diego Barbieri, Daniela Tonni et al.

Figure 4. Posterior inspiratory displacement of the epiglottis into the laryngeal vestibule.

Figure 5. Close view of the posterior epiglottis displacement.

Figure 6. Short aryepiglottic folds.

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Even if one usually dominates, two or more of these anomalies can be present
simultaneously.
The histologic theory suggests that the immaturity of laryngeal cartilage may be the cause
of collapse upon inspiration, but conclusive evidence of histologic abnormalities has not been
found 24. Moreover, only a few studies have documented altered histologic structure in
laryngomalacia specimens without significant conclusions 24,25. In addition, recent studies
have shown histologically normal fibroelastic arytenoid cartilage in infants with symptomatic
laryngomalacia 26.
At present, the neurologic theory is the best supported by the available evidence and
proposes that neurosensory dysfunction leads to a lack of neuromuscular coordination of the
supraglottic airway 2, 27. Increased laryngopharyngeal sensory thresholds have been
observed in patients with laryngomalacia indicating that peripheral afferent function of
laryngeal sensation is altered. Such an alteration leads to changes in laryngeal motor function
that appears as weak laryngeal tone in patients affected by laryngomalacia.

SYMPTOMATOLOGY
Laryngomalacia presents with inspiratory stridor, which is the audible symptom produced
by the rapid, turbulent flow of air through a narrowed segment of the respiratory tract. The
symptoms usually begin at birth or within the first few weeks of life, worsen at 4−8 months,
improve between 8−12 months, and generally resolves by 12−24 months 4, 28. However,
the clinical picture can sometimes persist for several years 29. Stridor presents as a harsh,
high-pitched, musical, vibrating, multiphase, inspiratory noise that typically worsens with
feeding, crying, supine positioning, agitation, and flexion of the cervical spine, and improves
by extension of the cervical spine, prone position, and quiet breathing. Cyanosis,
supraclavicular, intercostal, and substernal retraction are signs of increasingly severe
obstruction, and pectus excavatum is an example of the extreme degree of obstruction that
can occur 24. Chronic hypoxia from airway obstruction can lead to pulmonary artery
hypertension if not recognized and managed 30.
However, stridor is not a specific symptom of this illness 31. In fact, there are different
causes of airway obstruction that can lead to stridor during different phases of respiration.
Thus, one of the most useful ways to differentiate the causes of noisy breathing is to identify
in which phase of the respiratory cycle (i.e., inspiration, expiration or both) the sound is
heard. If stridor is present during inspiration, it is usually caused by partial obstruction at the
level of supraglottic tissues such as in laryngomalacia. In these cases, negative pressure is
created in the airway lumen causing the collapse of supraglottic structures. On the other hand,
if stridor is present during expiration, it is caused by obstruction in the lower tracheal airway
such as in the tracheomalacia. In this pathological situation, intrathoracic positive pressure
during expiration leads a collapse of the affected portions of the trachea. Finally, if stridor is
biphasic, the reason is due to fixed lesions at the level of glottic or subglottic plane that do not
change dynamically with respiration. The most frequent is a viral croup, but even subglottic
stenosis, subglottic cyst, subglottic hemangioma, vocal cord paralysis, laryngeal web, or
respiratory papillomatosis may be present 32, 33, 34. Moreover, stridor should be
differentiated by two other noisy breathing conditions: wheezing and stertor. Wheezing is
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794 Marco Berlucchi, Diego Barbieri, Daniela Tonni et al.

characterized by a coarse whistling sound heard on the phase of expiration and is usually due
to lung disease. Stertor is a grunting or a snoring sound and is present during expiration. This
sound is typically caused by adenotonsillar disease.
Other symptoms, which are related to feeding, include regurgitation, emesis, cough,
choking, and slow feeding. In infants with laryngomalacia the delicate balance between the
suck-swallow sequence and respiration is often disrupted 35. The increased metabolic
demand of coordinating eating and breathing against the obstruction can result in a weight
loss and failure to thrive. The severity and progression of laryngomalacia are especially
influenced by feeding and obstructive symptoms, APGAR score, presence of other airway
lesions, or other comorbidities 2, 36.
Thompson 28 divided laryngomalacia into mild, moderate, and severe categories. Mild
laryngomalacia occurs in 40% of patients and presents as inspiratory stridor with occasional
feeding-related symptoms of cough and choking or regurgitation. The average resting oxygen
saturation is 98−100%, and 70% of patients with mild laryngomalacia will have an uneventful
course with resolution by 12 months of age. The management of these patients is based on
clinical observation. The remaining 30% who present with reflux symptoms and a baseline
resting SAO2 ≤96% can progress to the moderate disease category.
Moderate laryngomalacia occurs in 40% of patients. These patients complain of
inspiratory stridor associated with feeding–related symptoms like cough, choking,
postprandial regurgitation, and cyanosis during feeding. Their resting average SAO2 is 96%,
and 72% of these patients will have resolution of symptoms by 12 months with feeding
modification and acid suppression therapy. About 28% of these patients will develop severe
disease requiring surgical intervention despite feeding modifications and acid suppression
therapy 35.
Laryngomalacia is severe in 20% of affected infants. These patients show inspiratory
stridor associated with recurrent cyanosis, apnea, feeding difficulty, aspiration, and failure to
thrive. Suprasternal and subcostal retraction can lead to pectum excavatum. Their resting
SAO2 is about 85%. Chronic hypoxia leads to pulmonary hypertension and cor pulmonary.
These patients require surgical intervention in addition to acid suppression therapy 33.
Patient factors that do not influence severity, progression, or outcomes of the disease are:
prematurity or gestational age at birth, birth weight, gender, or race 2. On the other hand,
APGAR scores, number of medical comorbidities, present of secondary air lesion 36, and
baseline resting SAO2 affect evolution of the disease 28.

SYNCHRONOUS AIRWAY LESIONS

Laryngomalacia may be associated with other anomalies of the airway such as laryngeal
dyskinesia, vocal cord paralysis, subglottic stenosis, tracheomalacia, microretrognathism,
glossoptosis, vallecular cyst, palatal anomaly, and choanal atresia 37.
The incidence of synchronous airway lesions (SALs) in children with laryngomalacia is
estimated to be 19−27% 38, 39, 40, but is higher in infants with severe laryngomalacia
requiring surgery. Toynton et al. 41 reported that the incidence of SALs in patients
undergoing supraglottoplasty was 47%. Dickson et al. [36] reported associated lesions in 79%
of cases of severe laryngomalacia and in 28.8% of cases of laryngomalacia with few signs of

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severity. Shroeder et al. found similar results in a cohort of 60 patients 37: 58% of these
children had SALs of whom 77% had subglottic stenosis, while 47% had tracheomalacia,
bronchomalacia, or both.
SALs have an accumulative effect on airway block, leading to greater airway obstruction.
Infants with mild or moderate disease that have a SAL is 4.8 times more likely to require
surgical intervention [36]. Diagnosis of SAL may lead to earlier intervention and ultimately
affect progression.

COMORBIDITIES
The most common associated medical comorbidity is gastroesophageal reflux disease
(GERD), which is present in 23−100% of patients with laryngomalacia 12, 16, 31, 42, 43,
44, 45. GERD is common in neonates and infants and is characterized by emesis, dysphagia,
chocking, gagging, and failure to thrive. The pathophysiology of GERD in laryngomalacia is
caused by negative intrathoracic pressure that potentiates gastroesophageal and
laryngopharyngeal reflux (LPR). Frequent LPR events result in inflammation and edema of
laryngeal tissues, potentially leading to airway compromise 44, 46.
Neurologic diseases including hypotonia, developmental delay, cerebral palsy, mental
retardation, microcephaly, and quadriparesis are the second most commonly reported medical
comorbidities, with an incidence of 8−50% 9, 11, 12, 13, 18, 31, 42, 47, 48. Moreover,
congenital anomalies and genetic disorders occur in infants with laryngomalacia with an
estimated incidence of 8−20% 12, 49. Down syndrome appears to be the most commonly
reported associated genetic disorder. Other syndromes associated with laryngomalacia include
CHARGE association, Pierre Robin Sequence characterized by micrognathia 9, 12, 49, 50,
51, 52, George syndrome 51, 53, Larsen syndrome 54, 55, 56, 57, and arthrogryposis 49,
58.

DIAGNOSIS
Diagnosis of laryngomalacia is suggested by its typical history, but it is confirmed by
direct examination of the larynx. Awake flexible laryngoscopy is the gold standard
examination (Figure 7). During execution of laryngeal endoscopy using a flexible fiberoptic
nasopharyngolaryngoscope, a topical anesthetic such as lidocaine can worsen symptoms. For
this reason, the use of a topical anesthetic is not indicated during this procedure 22.
Endoscopic evaluation of the upper airway in the office setting includes assessment of nasal
fossae, choanae, nasopharynx, tongue base and posterior pharyngeal wall, hypopharynx, and
larynx. Laryngeal dynamics is studied for supraglottic collapse and vocal fold motion. The
checklist includes assessment of respiratory epithelium and the presence of edema. Routine
use of rigid bronchoscopy is not usually necessary. However, in patients with severe
laryngomalacia, who require surgical intervention, complete airway evaluation, including
direct laryngoscopy and rigid broncoscopy, is required in order to assess the possible presence
of SALs. In the operating room, endoscopic examination starts with flexible laryngoscopy
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without anesthesia for evaluation of vocal fold function. Following this, a careful examination
of the pharynx, larynx, trachea, and bronchi is carried out under general anesthesia.
Other diagnostic procedures may be considered. Whenever aspiration events are present,
modified barium swallow or functional endoscopic evaluation of swallowing can be carried
out.
Furthermore, impedance pH for laryngopharyngeal reflux is reserved for patients who
have failed medical treatment or who have refractory symptoms after supraglottoplasty and/or
supraglottoplasty failure. Finally, the diagnostic work-up of laryngomalacia may be
completed by cardiological, ophthalmological, and genetic assessment.

Figure 7. Execution of a flexible laryngoscopy in an infant held in the arms of a nurse in the office.

TREATMENT
The treatment of laryngomalacia depends on its severity, and comprises non-
pharmacological, pharmacological and/or surgical procedures. Non-pharmacological
treatment is reserved for children affected by mild laryngomalacia. These patients are
managed with lifestyle, dietary measures, and regular monitoring of the disease with flexible
laryngeal endoscopy. Lifestyle changes includes maintenance of posture after eating, no
bottles of water before lying down, raising the head of the bed or mattress and weekly weight
checks, whereas feeding procedures consist in the use of high-calorie formulas with smaller
volumes.
Since the association between laryngomalacia and GERD has been well documented
59, pharmacological therapy of laryngomalacia involves the use of anti-reflux drugs. The
treatment should be administered in patients with a confirmed diagnosis of GERD and/or in
those with feeding difficulties and symptoms related to GERD. No studies have determined
the optimal dose and duration H2 histamine antagonist or proton pump inhibitor (PPI) therapy
or the preferred agent. Ranitidine can be used at a dose of 3 mg/kg TID and PPI can be used

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at the dose of 1 to 2 mg/kg/day 2. A proton pump inhibitor is used for both refractory and
breakthrough symptoms. At present, a combination of daytime PPI therapy and nighttime
histamine type-2 receptor antagonist therapy is used 28. Most infants are kept on acid
suppression therapy for an average of 9 months 28.
Although laryngomalacia is a benign and self-limiting disorder, 10−20% of patients
affected by severe laryngomalacia require surgical intervention 18, 41. The presence of
dyspnea with permanent and severe intercostal and/or xyphoid retraction, difficulty in
feeding, failure to thrive, sleep apnea or obstructive hypoventilation, bradycardia, cyanosis,
cor pulmonale, and uncontrollable reflux disease are considered indications for surgical
treatment 13, 18, 60.
In the past, tracheotomy was considered the gold standard for severe laryngomalacia,
although in recent years more conservative managements have been proposed. In 1898,
Variot performed first the resection of excess mucosal tissue from the aryepiglotic folds based
on the post-mortem findings in a neonate. In 1922, Iglauer first proposed partial
epiglottectomy. After 6 years, Hasslinger performed successful endoscopic division of the
aryepiglottic folds by forceps in three patients 61. In the 1970s, Fearon and Ellis 62
described the suture of the epiglottis to the base of the tongue. In France, in the same years,
hyomandibulopexy was reported with satisfactory initial results 8, but was subsequently
abandoned. In 1981, Templer et al. carried out a resection of the epiglottis, ventricular folds,
and aryepiglottic folds via lateral pharyngotomy in an 18-year-old patient with satisfactory
results 63. In 1984, Lane et al. reported the endoscopic resection of the excess supra-
arytenoid and epiglottic mucosa using micro instruments 24. In 1985, Seid et al. divided the
short aryepiglottic folds by CO2 laser in 3 patients 64. Following this, several authors used
endoscopic treatment to resolve severe laryngomalacia. At present, this technique, called
supraglottoplasty, is considered the standard surgical treatment 16, 48, 65. The mean timing
of supraglottoplasty is approximately 3 months 2, but can range from 1−12 months of age
66.
Different methods can be used for anesthesia: 1) mechanically controlled ventilation via a
small caliber endotracheal tube is rarely used, as it may interfere with the surgical procedure;
2) spontaneous breathing anesthesia is generally the technique of choice; 3) the intermittent
apnea technique provides only a limited time to the surgeon to perform the surgical procedure
before re-intubation; and 4) jet ventilation 67, 68.
Although jet ventilation, spontaneous breathing anesthesia, and apnea techniques have
the advantage of providing optimal view, maximum space, and operating time, the airway is
not protected by tracheal intubation. Thus, particulate debris, smoke, gastric contents and/or
blood may contaminate the patient’s lungs.
After inhalation induction of anesthesia and intravenous access are established, the
mobility of the true vocal folds should be assessed using a flexible fiberoptic
nasopharyngoscope with the patient spontaneously breathing. This procedure also allows
observation of the dynamic collapse of structures of the supraglottis into the airway. After
this, anesthesia is deepened and the patient is placed supine, with a shoulder roll, head drape,
and eye protection in place. The larynx is exposed with a Parson’s laryngoscope, and the
laryngeal inlet and supraglottic structures are sprayed with lidocaine (maximum 4 mg/kg) to
blunt sympathetic stimulation 67. Next, the larynx is exposed by inserting a Parson’s or
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Benjamin-Lindholm laryngoscope. At this point, endoscopic procedures in the supraglottic

area may be carried out using different surgical techniques.
Surgical techniques such as division of short aryepiglottic folds, resection of excess
supra-aryetenoid mucosal tissue, sectioning of the median glossoepiglottic ligament with
suspension of the epiglottis to the base of the tongue, partial epiglottectomy 69 and/or a
combination of these procedures are in relation to the laryngeal structures involved 70.
Supraglottoplasty is usually bilateral, but some authors have also proposed a unilateral
technique highlighting the lower risk of supraglottic stenosis after unilateral treatment 13. In
1995, Kelly and Gray first analyzed the results of unilateral supraglottoplasty, reporting a
success rate of 94% in a cohort of 18 patients. Only 3 children required a bilateral approach
71.
Several instrumented can be used for resection of excess tissue, and the use of cold
microinstruments, microdebriders 70, or CO2, thulium 72, and diode laser all provide
similar results 73. However, some authors believe that management of laryngomalacia by
CO2 laser and a microdebrider can reduce the risk of intraoperative bleeding and
postoperative edema. Laser and microdebriders are more expensive than traditional laryngeal
microsurgery instruments. Furthermore, laser techniques require special precautions to avoid
the risk of fire during surgery. For this reason, a special orotracheal tube must be utilized.
Complications of supraglottoplasty are rare and are divided into major and minor groups
67. Major complications include intraoperative and postoperative complications. The former
comprise airway fire, whereas the latter include supraglottic and glottic stenosis. Damage to
teeth or gums from pressure caused by the laryngoscope, airway edema, hoarseness,
aspiration, bleeding, infection, granuloma, and granulation tissue represent the minor
complications. Moreover, pneumonia can be present in 7−10% of cases 47, 73, 74.
Finally, revision supraglottoplasty and/or tracheotomy is required in 19−45% of infants
and is influenced by the number and type of medical comorbidities 75. In particular,
tracheotomy is performed in patients who continue to have life-threatening airway obstruction
and who fail to improve after supraglottoplasty.

NON-INVASIVE VENTILATION
Although there are no guidelines, several authors have proposed the use of non-invasive
ventilation (NIV) for children affected by severe laryngomalacia [68, 76]. The aim of NIV for
these patients is to decrease the exertion of breathing, which is increased due to collapse of
sopraglottic structures during inspiration. In particular, positive-end-expiratory-pressure
(PEEP) keeps the airway patent and increases the residual functional capacity of the patient
[77, 78].
This therapeutic procedure can be utilized when a patient is waiting for surgical
treatment, there are severe comorbid diseases, and/or whenever surgery has been unsuccessful
[68].
PEEP can be performed by the following NIV-techniques: 1) continuous positive airway
pressure (CPAP) and 2) bilevel positive airway pressure (BiPAP). The use of CPAP provides
positive pressure throughout the respiratory cycle, while inspiration is not supported [79]. On
the other hand, BiPAP involves the use of a ventilator that provides two different levels of

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pressure in view of the diverse patient efforts during the respiratory cycle. During inspiration,
ventilatory support is greater, whereas it is less during the expiratory phase [80].
The choice of the devices with which to perform NIV is fundamental. Facial devices
must ensure minimal air leak and at the same time be well tolerated. Nasal devices are often
preferred in younger patients and are better tolerated than total-face masks and/or oro-nasal
methods. Moreover, it is important to keep in mind that children are growing, and thus facial
deformities that could be favored by pressure exerted by the mask on the facial structures
must be avoided [81, 82].
NIV can be used in case of sudden progression and/or long-lasting disease. In this latter
situation, NIV is generally carried out during sleep [80]. Finally, it is sometimes necessary to
use NIV at the home. In this case, training of caregivers and compliance of parents is
mandatory [83].

CONCLUSION
Laryngomalacia is the most frequent laryngeal congenital disease. Clinical picture ranges
from simple laryngeal stridor to severe dyspnea associated with cyanosis. Its treatment, which
includes non-pharmacological, pharmacological and/or surgical managements, is in relation
to symptomatology. Every children affected by this disorder must be carefully assessed to
decide his/her more adequate therapy.

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[49] Master, IB; Chang, AB; Patterson, L; Wainwright, C; Buntain, H; Dean, BW et al.
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[67] Rawlings, BA; Derkay, CS; Chu, MW; John, J. Surgical treatment of laryngomalacia.
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[70] Zalzal, GH; Collins, WO. Microdebrider-assisted supraglottoplasty. Int. J. Pediatr.
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[71] Kelly, SM; Gray, SD. Unilateral endoscopic supraglottoplasty for severe
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[73] O’Donnell, S; Murphy, J; Bew, S; Knight, LC. Aryepiglottoplasty for laryngomalacia:
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[74] Whymark, AD; Clement, WA; Kubba, H; Geddes, NK. Laser epiglottopexy for
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 34

THE VISUAL RECOGNITION OF CYANOSIS

AND THE INFLUENCE OF LIGHTING
AND COLOR VISION

Stephen J. Dain
School of Optometry and Vision Science,
University of New South Wales, Sydney, NSW, Australia

ABSTRACT
The use of pulse oximeters has minimized the need for the visual recognition of
cyanosis. However, there are times when this is still valuable. Lighting is important for
this visual task and it is also a difficult task for some people with color vision
deficiencies. There were fluorescent tubes that permitted the accurate recognition of
cyanosis but they used less efficient halophosphate technology. In the 1990s the change
in fluorescent tube technology to give greater energy efficiency introduced tri-phosphor
technology. This created some problems most notably for anesthetists. A standard for
lighting to permit the accurate identification of cyanosis was written (Australian/New
Zealand Standard 1680.2.5:1997) based on measurements of isolated blood that were
later confirmed by measurements of lips, nail beds and palm creases. Using these data,
the basis of the problem for people with color vision deficiencies was also illustrated. The
standard introduced the concept of the Cyanosis Observation Index (COI), its calculation
and compliance values. Since then, there have been many attempts to produce complying
sources for hospital lighting using fluorescent tube technology. The solutions tended to
be energy inefficient (using halophosphate technology) or expensive. As a consequence,
there was no successful solution to the problem using fluorescent lamps (straight tube or
compact fluorescent). The other, less demanding, visual tasks in clinical observation (like
observation of rashes etc.) were satisfied using modified tri-phosphor technology. The
future of lighting is now firmly with light emitting diode (LED) technology. LEDs are
more efficient than fluorescent sources and have advantages of compactness and no
warm-up time. There are a number of white LED products available that have a COI that


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806 Stephen J. Dain

easily complies with AS/NZS 1680.2.5. As a consequence, there has been a renewed
interest in the COI as an appropriate and achievable measure in hospital lighting.
In this chapter the basis of the COI will be reviewed. The issues with color vision
deficiencies will be discussed. The reasons why tri-phosphor technology fails to deliver a
good solution will be outlined and the results of suitable LED sources will be provided.

INTRODUCTION
Technology has largely reduced the need for visual observation of patients and the
unfortunate events like the demise of Mrs. Arnott-Smith [1] are, hopefully, now largely a
thing of the past. However, visual observation of the color of lips, skin, nail beds and palm
creases is still needed [2, 3].
In the measurement of color, there are a number of influences;

1. spectral reflection characteristics of the object viewed,

2. spectral characteristics of the lighting and
3. spectral characteristics of the observer.

For the perception of color, we need to add;

4. size of the object,

5. state of visual adaptation of the observer and
6. color of the background.

1. THE SPECTRAL REFLECTION CHARACTERISTICS

OF THE OBJECT VIEWED

This is the primary feature and the direct relation of blood oxygen concentration. The
measurements of spectral reflectance characteristics of such small and heterogeneous objects
as lips, nail beds and palm creases on a subject that is not totally motionless is a challenging
exercise for a color metrologist. In the past such an exercise took a few minutes per subject
and the need for that subject to hold still and for the blood oxygen concentration to be
maintained constant made the task very difficult, if not impossible. As a proxy for in vivo
color, the author’s early work on lighting for cyanosis observation used the spectral
reflectance of blood [4, 5]. It seemed safe to assume that the overlying dermis and epidermis
(or nail) constituted a constant so that any color change would be directly related and
proportional to the color change of the blood. It was actually suggested, albeit a long time
ago, that visual examination of drawn blood is a better measure than visual examination of the
lips etc. [6] The ability to measure spectral reflectances in the visible range for oxygen
saturations from 0.7 to 99.4% provided the basis to an averaged set of data in 10% oxygen
saturation. The upper and lower limits of this scale are shown in Figure 1 along with the
difference between the two, to highlight that the vast majority of the change in reflectance is
in the red part of the spectrum, 600-700 nm.

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 The Visual Recognition of Cyanosis and the Influence of Lighting … 807

From this, the change in color of the blood with oxygenation can be calculated. This is
shown in Figures 2 and 3 (redrawn from [4]). In Figure 2, CIE L* is the measure of lightness
and is related to the total amount of light reflected. It is a perceptually linear scale. This
shows that L* reduces, almost linearly, with decreasing oxygen saturation. This is a
quantification of the visual observation that blood (and lips etc.) gets darker with decreasing
oxygen saturation. In Figure 3, a* is redness (+ve) - greenness (-ve) and b* is yellowness
(+ve)-blueness (-ve). Again the scale is perceptually approximately linear and quantitatively
the same as Figure 2. That is, a change of a given magnitude looks the same whether it is in
L*, a* or b* or a combination of them. The origin has been included in Figure 2. This
represents greys. It may be seen that the overall change in color is a move towards the origin,
so the change in blood color is that it becomes less colorful, loses redness and yellowness,
rather than is changed in hue, becoming bluer. Color metrologists use the term “saturation”
[7] for this metric, but in the present context, that would be confusing. That is, it does not turn
blue despite the popular term. The total change in lightness (Figure 2) is about 10 units and
the total change in chromaticity (Figure 3) is about 37 units, so the change in chromaticity is a
much more visible clue than the change in lightness. This difference may also be seen
ophthalmoscopically. In the eyes, veins look a darker red rather than blue. The apparent
visual blueness in cyanosis arises, therefore, because of the structures overlying the blood
supply and also the colors that surround the structure observed.
The measurements on isolated blood were made and used because the problems in
measuring the color of lips, nail beds and palm creases were considerable. As the technology
for making spectral reflectance measurements improved, the viability of making in vivo
measurements has improved. The measurement now takes about 5 seconds [8] rather than the
3-4 minutes previously [4, 5]. Spectral reflectance measurements in vivo have been reported
for chronically cyanosed patients before and after exercise [8]. In general, they confirm the
direction of color shifts, but the measurements still show a high degree of variability.

Figure 1. Spectral reflectance of blood in the visible region for two oxygen levels and showing the
difference in reflectance (which is primarily in the region around 650 nm.
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808 Stephen J. Dain

Figure 2. Change of CIE Lightness L* as a function of oxygen saturation. The reduction is about 10
units and in a monotonic fashion.

Figure 3. Change of CIE a* and b* as a function of oxygen saturation. The reduction is about 37 units
and in a monotonic fashion. Extrapolated it passes close to the origin which is the location of grey.

2. THE SPECTRAL CHARACTERISTICS OF THE LIGHTING

This is normally characterized by the correlated color temperature (CCT, Tc) expressed in
Kelvin and the CIE color rendering index (Ra). The CCT is the temperature of the ideal source
or of a reference daylight most closely resembling the source under test. It is a convenient
way of characterizing the color of a nominally white source in one number. Typical examples
are set out in Table 1. A CCT of about 4000 K has become the benchmark for clinical
observation (see later).

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 The Visual Recognition of Cyanosis and the Influence of Lighting … 809

Sources with identical CCTs are not necessarily spectrally the same. In an extreme case, a
mixture of monochromatic blue and yellow-green could form a white source and this will
perform differently from a white source that has approximately equal energy at all visible
wavelengths. This phenomenon is known as “metamerism.” The measure of the fidelity with
which a source renders colors is the CIE Color Rendering Index, Ra. This is calculated from
the change in color of 8 reference samples under the test source and reference source [9].
There are a further 6 reference samples that are not included in the general color rendering
index but may be used for specific situations. The reference sample 9 is a red and R9 is
traditionally accepted as a good indication of the quality of a source for assessing skin tones.
Color rendering is a scale running from 100 for a perfectly color rendering source to 0 or even
negative. For many applications Ra ≥ 80 is considered adequate and for the more critical
applications Ra ≥ 90 is often specified. The model numbers of modern fluorescent sources
now often incorporate these numbers so that the first digit is the more significant digit in the
color rendering and the second and third digits are the two most significant digits in the
correlated color temperature. Thus a fluorescent tube designated 840 has a color rendering
index in the range 80 to 89 and a correlated color temperature of 3500 K to 4500 K.
In 1965, a Medical Research Council study recommended an optimal light source for
clinical purposes. The study was based on the observations of dermatology and pathology
specimens and a few cyanosed patients. The recommended light source had a CCT of around
4000 K. The Crawford method of specifying color rendering was used at first to indicate
acceptable color rendering limits of the light source [10-12]. The Crawford method has since
been superseded by the Internationally accepted Commission Internationale de l’Éclairage
(CIE) Color Rendering Index (CRI) [9] and the recommended sources were defined in the
new system as a function of CCT and CRI. The form of this specification, in this case in an
Australian Standard) may be seen in Figure 4 [13]. Consistent with the advice of the time,
hospitals were often uniformly lit using the same fluorescent tube type [14].

Table 1. Examples of the correlated color temperature of common light sources

Correlated color Typical description Example sources

temperature
About 2850 K Tungsten filament lamps
2900-3300 K Warm white Quartz halogen tungsten filament lamps
Fluorescent tubes
Compact fluorescent lamps
Light emitting diodes
Around 4000 K White Fluorescent tubes
Around 5000 K Cool white Compact fluorescent lamps
5500 to 7500 K Daylight Light emitting diodes
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810 Stephen J. Dain

Figure 4. The limits of Correlated Color Temperature and CIE Color Rendering Index for clinical
observation [13].

The technology in fluorescent tubes at the time was halophosphates. The spectral
emittance was a combination of the mercury discharge lines and the broad-band emission of
the phosphors coating the tube. See Figure 5. It was relatively easy to design fluorescent
lighting with high values of color rendering and a wide range of CCTs. Their broadband
emission was not dissimilar to the reference 4000 K source (see Figure 5). However, these
sources were relatively inefficient. They have been superseded by the triphosphor technology
that is cheaper to produce and more energy efficient. This technology includes a red phosphor
that has a “spikey” spectral emittance that is also relatively deficient in the 650-700 nm
region of the spectrum, Figure 5, which is where the reflectance change of blood occurs.
There are some modified triphosphor lamps that have higher general color rendering but low
energy levels in this wavelength region are still relatively low and R9 is low. Dissatisfaction
with these tubes led to the studies reported above [4, 5]. From the measurements on blood and
the conventional wisdom on which tubes were successful, a measure called the “Cyanosis
Observation Index” (COI) was developed and incorporated into a new standard [15].
Elsewhere, studies in hospital lighting reported difficulty making measurements relating to
cyanosis (probably because the light sources in use had little energy in the significant spectral
region) and the recommendations were made on the basis of the needs of dermatological
observation rather than cyanosis [16-18]. For surgical luminaires, the limits are set as 3000 K
≤ Tc ≤ 6700 K and 85 ≤ Ra ≤ 100 [19]. Cyanosis observation is more likely to be carried out
under general lighting.
In the author’s experience, there have been no triphosphor tubes that comply with the
COI requirements, so the measure did not achieve much recognition. What has changed the
prospect is the coming of light emitting diode (LED) sources. Where the fluorescent tubes use
a mercury arc to “pump” the phosphor, white LEDs contain a blue (around 470 nm) or, more
recently, a violet (around 445 nm) LED as the pumping source and a yellow phosphor or
mixture of phosphors. These have a broadband emission more in the style of the

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 The Visual Recognition of Cyanosis and the Influence of Lighting … 811

halophosphate. There are a number of different ways of obtaining the required CCT, CRI >
90 and a COI < 3.3 to comply with AS/NZS 1680.2.5. They also embody all the other
advantages of LEDs including higher efficiency, no warm up time, long life, stability and
robustness. Figure 5 includes such a white LED source. Table 2 contains the CCT and CRI
values for these sources and also a modified triphosphor with a good CRI. Measurements
were made with a Topcon SR-3 telespectroradiometer having a wavelength accuracy ± 0.9
nm (mercury and neon spectral lines), spectral half band width of 1.1 (mercury line at 546.1
nm) and calibrated using sources calibrated at the National Measurement Laboratory of
Australia. The COI superiority of the halophosphate and LED sources is evident in Table 2.

Table 2. Characteristics of some white sources. See also Figure 5

Measure Halophosphate Triphosphor LED

840 940
Correlated color temperature K 3986 3691 3970 3982
General color rendering index Ra 87.7 83.0 90.5 94.5
R9 88.4 10.6 55.8 78.7
Cyanosis observation index 0.9 6.4 3.9 1.6
AS/NZS 1680.2.5 Pass Fail Fail Pass

Figure 5. Spectral emittance of some representative sources for clinical observation. The halophosphate
is an old technology inefficient source that is compliance with AS/NZS 1680.2.5 [15]. Color 840 is a
source with moderate colour rendering and considered suitable for dermatological observation but
which is deficient in the region of 650 nm and not compliant. The LED source is a source that is
compliant. See also Table 2.

This illustrates that, in addition to their superior characteristics in efficiency and

longevity, LEDs can also meet the correct color for hospital lighting.
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812 Stephen J. Dain

3. THE SPECTRAL CHARACTERISTICS OF THE OBSERVER

Just as sources vary and may be described as being metameric, observers are also
described as having metamerism because they also vary. For a visual task such as
identification of cyanosis, the inter-observer variation in normal color vision does not result in
significant differences. However, the greater variations exhibited in congenital color vision
deficiencies have the potential to affect detection and recognition of cyanosis significantly.
The common congenital color vision deficiencies are in two distinct types termed protan
and deutan. They are both “red-green deficiencies.” In protans, the “red” receptor that is
modified (anomalous trichromats) or missing (dichromats) and in deutans it is the “green”
receptor that is modified or missing. They are called protanomalous, deuteranomalous,
protanopic and deuteranopic, respectively. Both types of anomalous trichromat come in a
range of severities from those in which the alteration of the cone is minor, with a mild color
vision deficiency, through to those who lack the red or green cone entirely and for whom the
world is actually in shades of blueness, yellowness and neutrals (although they may use other
clues, e.g., a yellow always reflects or emits more light in total and is seen as brighter than a
red or green). The problems for color vision deficient practitioners in the detection and
recognition of cyanosis have been identified by Spalding, himself a deuteranope [20-24].
A theoretical analysis was undertaken to assess the propensity for error by comparing the
direction of color change of blood with oxygenation using the same blood data [25]. The
change of lightness (L*) is little changed for deuteranopes but is about 40% reduced for
protanopes. The color difference for protanopes is only about 9% that for normal observers
and deuteranopes are reduced to about 23% of that for the normal observer. From this it might
be concluded that deutans are the more disadvantaged than protans. However, the anecdotal
reports are always about deutans [22]. The study went on to assess the possibility of
enhancing the color differences to aid the color vision deficient by changing the light source.
The rate of change of L* is increased only when a warm incandescent source is used. For
color vision normals, such a source is too red, tending to make even the cyanosed look
healthy. In addition, the warmer sources tend to reduce the chromaticity changes for color
vision normals. The reduction in color difference is less dramatic for deuteranopes and is
more affected by changing the source. The bluer the source, the greater the chromaticity
difference for deutans. However, the bluer the source, the less the rate of change of L*. It is
not clear which is the more powerful clue for the deutan. However, extremes of yellowness
and blueness in the source disqualify its use for color vision normals.
A practical experiment using digitally altered photographs [26] confirmed that some
color vision deficient subjects are less accurate at identifying cyanosis. It also showed that
new ambulance officer recruits perform less well than experienced ambulance officers, so the
identification of cyanosis is a learned skill unlike, for instance, signal color naming [27].

Influences on Perception

The effects of size, adaptation and surround have received little attention. While the
colorimetric system provides for stimuli with 2° or 10° subtense [28], the calculated effects of
increasing size are seen, almost entirely, in blue-yellow discrimination not red-green.

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 The Visual Recognition of Cyanosis and the Influence of Lighting … 813

At low lighting levels, color vision is impeded, however, the levels that are typically
recommended for hospital lighting [15] are well within the region of optimal color vision
performance.
The effects of the color of the surround, such as operating theatre drapes, have been
commented upon but not systematically investigated [3].

CONCLUSION
The color changes that provide visible clues to the possibility of cyanosis in a patient are
relatively subtle and subject to many influences. The variable of illumination color may be
taken into account by the use of limitations on the CCT and CRI of the source or by applying
the requirement of the COI. While COI has effectively excluded fluorescent tube sources as a
validated lighting option, the availability of LED sources (as fluorescent tube replacements
and spotlight replacements and the like) means that simple, effective, energy efficient and
colorimetrically accurate options are now available. Many (if not most) practitioners with a
color vision deficiency are able to recognize cyanosis as accurately as those with normal color
vision. However, those with the more extensive color vision deficiencies need, at least, to be
aware of their potential limitations to be able to be more guarded in their dependence on
visual color.

REFERENCES
[1] Transcript of Inquest before coroner and jury upon request of relatives. Deceased:
Margaret Arnott-Smith. 1979: City Coroner’s Court, Glebe. Coroner: Margaret Mary
Sleeman.
[2] O'Donnell, C. P. F., et al., Clinical assessment of infant colour at delivery. Arch Dis
Child Fetal Neonatal Ed., 2007. 92: p. F465-467.
[3] Changizi, M. and Rio K., Harnessing color vision for visual oximetry in central
cyanosis. Med Hypotheses, 2009. 74: p. 87-91.
[4] Dain, S. J., et al., A Method for Evaluating the Acceptability of Light Sources for
Clinical Visual Evaluation of Cyanosis. Color Res Appl, 1998. 23: p. 4-17.
[5] Dain, S. J. and Hood J. W., Lighting for cyanosis identification Lighting, 1998. 65: p.
18-24.
[6] Morgan-Hughes, J. O. and Bartlett M. C., The colour of blood in syringes as a guide to
hypoxaemia. Brit J Anaesth, 1968. 40(5): p. 310-314.
[7] Commission Internationale de l’Éclairage., ILV: International Lighting Vocabulary.
2011, Commission Internationale de l’Éclairage.: Vienna.
[8] McNamara, R., et al., Colour change in cyanosis and the confusions of congenital
colour vision deficient observers. Ophthal Physiol Opt, 2010. 30(5): p. 699-704.
[9] Commission Internationale de l’Éclairage., Method of measuring and specifying colour
rendering properties of light sources. 1995, Commission Internationale de l’Éclairage.
Vienna.
 Free ebooks ==> www.Ebook777.com
814 Stephen J. Dain

[10] Crawford, B. H., Measurement of Color Rendering Tolerances. J Opt Soc Am, 1959.
49(12): p. 1147-1156.
[11] Crawford, B. H. and Palmer D. A., Further Investigations of Colour Rendering, and the
Classification of Light Sources. Stud Conserv, 1961. 6(2-3): p. 71-82.
[12] Crawford, B. H., The colour rendering properties of illuminants: the application of
psychophysical measurements to their evaluation. Brit J Appl Phys, 1963. 14: p. 319-
328.
[13] AS, 1765, Artificial lighting for clinical observation. 1965, Standards Australia:
Sydney.
[14] Kelman, G. R. and Nunn J. F., Clinical recognition of hypoxaemia under fluorescent
lamps. Lancet, 1966. 287(7452): p. 1400-1403.
[15] AS/NZS, Interior lighting Part 2.5. Hospital and medical tasks. 1997, Standards
Australia/Standards New Zealand: Sydney.
[16] Lovett, P. A., et al., The effect on clinical judgements of new types of fluorescent
lamps: I. Experimental arrangements and clinical results. Lighting Res Tech, 1991. 25:
p. 35-51.
[17] Lovett, P. A., Halstead M. B., and Hill A. R., The effect on clinical judgements of new
types of fluorescent lamps: II. Colour measurements and statistical analysis. Lighting
Res Tech, 1991. 23: p. 53-67.
[18] Lovett, P. A., Hill A. R., and Halstead M. B., The effect on clinical judgements of new
types of fluorescent lamps: III. Further statistical measurements leading to a new
specification for lamps. Lighting Res Tech, 1991. 23: p. 69-80.
[19] IEC, 60601-2-41, Medical electrical equipment Part 2-41. Particular requirements for
the basic safety and essential performance of surgical luminaires and luminaires for
diagnosis. 2013, International Electrotechnical Commission: Geneva.
[20] Spalding, J. A., The doctor with an inherited defect of colour vision: effect on clinical
skills. Brit J Gen Pract, 1993. 43(366): p. 32-33.
[21] Spalding, J. A., Colour vision deficiency in the medical profession. Brit J Gen Pract,
1999. 49: p. 469-475.
[22] Spalding, J. A., Confessions of a colour blind physician. Clin Exp Optom, 2004. 87(4-
5): p. 344-349.
[23] Spalding, J. A., Medical students and congenital colour vision deficiency: Unnoticed
problems and the case for screening. Occup Med (Lond), 1999. 49(4): p. 247-252.
[24] Spalding, J. A., Cole B. L., and Mir F. A., Advice for medical students and
practitioners with colour vision deficiency: a website resource. Clin Exp Optom, 2010.
93(1): p. 39-41.
[25] Dain, S. J., Color changes in cyanosis and the significance of congenital dichromasy
and lighting. Color Research and Application, 2007. 32(6): p. 428-432.
[26] Dain, S. J., Recognition of simulated cyanosis by color-vision-normal and color-vision-
deficient subjects. J Opt Soc Am A, 2014. 31(4): p. A303-306.
[27] Dain, S. J., et al., Color Vision and the Railways, 1. The Railway LED Lantern Test.
Optometry Vision Sci, 2015. in press.
[28] ISO, Colorimetry Part 1, CIE standard colorimetric observers. 2008, International
Standardization Organisation: Geneva.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 35

KERATINOCYTES IN PSORIASIS:
KEY PLAYERS IN THE DISEASE PROCESS

Inas Helwa2, Meg Gullotto3 and Wendy B. Bollag1,2,4,5,

1
Charlie Norwood VA Medical Center, Augusta, US
2
Department of Oral Biology, Georgia Regents University, Augusta, US
3
Department of Medical Illustration, Georgia Regents University, Augusta, US
4
Department of Physiology, Medical College of Georgia,
Georgia Regents University, Augusta, US
5
Departments of Cell Biology and Anatomy, Medicine (Dermatology) and Orthopaedic
Surgery, Medical College of Georgia, Georgia Regents University, Augusta, US

ABSTRACT
Psoriasis is a common chronic hyperproliferative inflammatory disease that affects
skin, nails and joints. This disorder affects about 1%-3% of the general population and
prevalence varies among countries and races. Although psoriasis can occur at any age,
two peaks of disease incidence are observed: one between 15 and 30 years and the second
between 50 and 60 years. Psoriasis has been described for many years as an autoimmune
disorder; however, due to lack of convincing evidence regarding autoantibodies in the
disease, it has recently been more precisely described as an “immune-mediated disorder.”
The pathogenesis of psoriasis involves a strong crosstalk between immune cells (mainly
T cells and dendritic cells) and lesional keratinocytes. However, the exact etiology
regarding which cell initiates the disease is still an unresolved issue. The lack of a widely
accepted animal model is one obstacle towards an exact understanding of the disease
mechanism, although it is known that genetic and environmental factors are involved.
Since skin is the primary barrier against environmental insults and keratinocytes are
major contributors to the innate immune response, there is an evolving hypothesis that the
keratinocyte is a key player and may be, in some cases, the initiator of the disease process
rather than simply a bystander in an active T cell-mediated immune response. This
evolving hypothesis may provide a new avenue for establishing promising treatment


To whom correspondence should be addressed: Wendy B. Bollag, Georgia Regents University, Department of
Physiology, 1120 15th Street, Augusta, GA 30912, TEL: (706) 721-0698, FAX: (706) 721-7299,
Email: [email protected]
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816 Inas Helwa, Meg Gullotto and Wendy B. Bollag

options that have negligible adverse effects. Such treatments are critical since long-term
management of psoriasis is required in most cases and a complete cure of the disease is
rare. Thus, safer treatment options with minimal side effects are in increasing demand.
Options that directly target keratinocytes may have fewer adverse effects than the newer
biologic agents that reduce immunity, thereby putting patients at risk of fatal infections as
well as lymphomas. In this chapter, we will discuss types of psoriasis as well as the
evidence supporting the developing theory of the keratinocyte being a major player. We
will also be summarizing possible treatment options including those that may be directly
targeting keratinocytes. Understanding what is known and unknown concerning the exact
etiology of the disease may lead to new perspectives and novel insights regarding
potential research directions and treatment options for this chronic and debilitating
disorder.

I. SKIN HOMEOSTASIS AND REGULATION OF FUNCTION

The skin is a vital organ that spans a surface area of approximately 2 m2 in the average
adult [1]. It is the body’s primary defensive barrier that protects the internal organs from
physical, chemical and microbial insults [2]. However, the skin does not act solely as an inert
barrier but also serves as an immunologically active sensory and excretory organ. It aids in
regulating body temperature and transepidermal movement of water and electrolytes
preventing dehydration [1, 3]. The multilayered structure of the skin is adapted to serve these
functions, which are essential for sustaining life.
The skin consists of 3 major layers that include the epidermis, dermis, and underlying
subcutaneous tissue (or hypodermis), with barrier functions of the skin largely confined to the
epidermis. The dermis is the connective tissue layer that supports the epidermis and provides
nervous and vascular supply to the avascular epidermis. It is also the site of epidermal
derivatives such as hair follicles and sweat and sebaceous glands [4]. The primary constituent
cells of the epidermis are the keratinocytes which are arranged in layers of the following
order: stratum basale (basal cell layer, the deepest layer adjacent to the dermis), stratum
spinosum, stratum granulosum, and stratum corneum (the outermost protective layer) [1].
The thickness of the epidermis is maintained as a result of the balance between
proliferation in the stratum basale (SB) and differentiation in the upper sub-layers of the
epidermis; namely, the stratum spinosum (SS) and stratum granulosum (SG) with sloughing
in the upper most stratum corneum (SC) layer [2, 5]. It is a fine balance between keratinocyte
proliferation, differentiation and desquamation that maintains skin thickness and an efficient
epidermal barrier [6].
In normal skin the journey from the stratum basale to the stratum corneum, at which site
the keratinocytes undergo terminal differentiation, takes about 28-30 days, whereas in
pathological conditions this time span may be massively reduced as in the case of psoriasis
where the entire process takes just 3-5 days [2, 7]. This dramatic reduction in maturation time
does not allow sufficient time for keratinocyte maturation as reflected by the absence of the
granular cell layer and a thick cornified layer in psoriatic lesions. Therefore, the precise
regulation and differentiation of the epidermis is crucial for proper stratification and barrier
formation.

 Calcium in Epidermal Homeostasis

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 Keratinocytes in Psoriasis 817

The migration of keratinocytes from the basal layer until they become cornified and reach
the skin surface is triggered by poorly understood signals. However, calcium is one of a few
known signals and a key regulator of this balance. A calcium gradient has been documented
in human and murine epidermis [6, 8-13]. In vitro, it has been shown that the epidermal
differentiation process is regulated by the concentration of extracellular calcium ions where
keratinocytes cultured in low calcium concentrations (<90 µM) show an undifferentiated,
basal cell-like phenotype, and raising medium calcium concentrations to between 90 and 120
µM results in a terminal differentiation process that is almost identical to the in vivo process
[14].
The concentration required for differentiation in vitro differs somewhat in human versus
murine keratinocytes [15]. A relationship between elevated calcium ion concentration and
keratinocyte differentiation is observed both in vivo and in vitro [8, 10]. Higher calcium levels
occur in the differentiated granular and spinous layers as compared to the undifferentiated
basal layer. The epidermal calcium gradient, however, not only regulates the epidermal
differentiation process, but also regulates the homeostasis of the epidermal permeability
barrier through maintaining epidermal cohesion. Calcium appears to act in keratinocytes by
binding to and activating a G-protein coupled receptor, the Ca2+-sensing receptor. The
importance of the calcium receptor and its splice variant for normal differentiation of
keratinocytes has been reported [13, 16]. Indeed, ablation of the Ca2+-sensing receptor gene in
knock-out mice results in an epidermal phenotype characterized by impaired keratinocyte
differentiation and barrier function and increased proliferation [12].
Since a recent report by Celli et al. [17] challenges the existence of a calcium gradient in
the skin, clearly additional work is needed to establish whether or not such an epidermal
calcium gradient exists as well as the role of calcium in differentiation in vivo. In addition,
impaired calcium homeostasis is an important factor in the pathogenesis of many skin
diseases including Darier disease, Hailey-Hailey disease, atopic dermatitis and psoriasis [2, 3,
6, 18-22].

II. PSORIASIS
Psoriasis is a common lifelong multifactorial inflammatory hyperproliferative immune-
mediated disorder thet affects skin, nails and joints. It is characterized by periods of
remission, relapse and exacerbation [23-30].

1. Epidemiology

Psoriasis has a total prevalence of 1-3% of the population worldwide. It affects 25 million
individuals in North America and Europe [31, 32] with lowest prevalence among Asians and
greatest among Scandinavians [33]. It is considered the most prevalent immune-mediated skin
disease in adults, with men and women equally affected [32, 34]. Psoriasis can typically
affect any age group although it is most common before age 40 with the highest incidence in
the second decade [25]. It has two peak incidences: one between 15 and 30 years and another
between 50 and 60 years [27, 35]. Psoriasis can also occur in childhood with about one third
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818 Inas Helwa, Meg Gullotto and Wendy B. Bollag

of all patients exhibiting onset of the disease in the first or second decade of life [36, 37]. The
median age of occurrence among children is 7 to 10 years with a prevalence of 0.7% of
affected patients, and family history may predict early onset of the disease [38, 39].
Although the rate of incidence in children is low, this age group is the most
psychologically affected [36]. The need for safe treatment options with minimal short- and
long-term adverse effects, as well as encouraging maximum compliance, is more critical in
children and adolescents. Furthermore, psychological rehabilitation is a major factor in the
therapeutic strategy.
Psoriatic arthritis (PsA) is a disabling joint disorder commonly occurring in psoriatic
patients, but is not common in children. The prevalence of psoriatic arthritis (PsA) among
psoriatic patients is about 25% and it can progress to polyarticular disease and loss of
function [40]. Psoriatic arthritis is usually seronegative for rheumatoid factor. Cutaneous
lesions precede arthritis in 60-70%, and arthritis may precede skin lesions in about 20%, of
patients [41].

2. Clinical Features

The psoriatic lesion typically appears as a sharply demarcated chronic erythematous

plaque covered by silvery white scales. It has common predilection sites including elbows,
knees and scalp [7, 32, 41, 42]. However, patients can still exhibit a myriad of clinical
phenotypes reflecting the dynamic spectrum of the disease [27].

3. Psoriasis and the Quality of Life

Psoriasis is rarely life threatening but it is life destroying in a large number of cases [43].
Nevertheless, the psychological impact of psoriasis is not sufficiently appreciated by health
care practitioners, although these psychological effects might be of great impact not only on
the patient’s quality of life but also on disease management [44, 45]. Thus, psychological
sequelae of psoriasis may affect the response to treatment, with patients exhibiting high levels
of anxiety having significantly worse treatment outcomes in response to therapy [46].
Psoriasis patients report an impaired quality of life equal to or worse than patients with
chronic conditions including cancer and heart disease [47]. Patients have a tendency for social
withdrawal as they are embarrassed by their appearance. Patients also suffer from multiple
side effects of the long-term therapy of the disease, which is often required lifelong. Together
with the increased levels of unemployment relative to non-psoriatic individuals and the
functional disability secondary to psoriatic arthritis, the disease increases the risk of
depression, anxiety, and suicidal thoughts, especially with the young age of disease incidence.
If we also consider the combined costs of long-term therapy and the social costs of the
disease, we can then appreciate the disease’s serious impact on patients, their families, health
care systems and society as a whole.
In 2004, the annual direct medical costs of psoriasis in the United States exceeded one
billion dollars and the indirect costs resulting from missed work days and loss of productivity
at work has largely exceeded this annual amount [48]. This makes psoriasis not just a

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cosmetic problem but a life-destroying disorder with a high degree of morbidity and a serious
social and economic impact [42, 49-51].

4. Psoriasis-Associated Co-Morbidities

In addition to the already known skin and joint symptoms and the psychological burden
of psoriasis, there is increasing evidence that psoriasis is more than “skin deep” [52, 53].
There is accumulating epidemiologic evidence of an association between psoriasis and other
systemic disorders, including cardiovascular diseases like atherosclerosis, coronary artery
calcification and stroke [54, 55]. There is also an association with depression and metabolic
syndrome and obesity [56-63]. However, this association does not per se establish a causal
relationship. A causal relationship between these disorders and psoriasis must be strengthened
with additional epidemiologic analyses.
Some studies suggest that psoriasis patients not only have significant inflammation in
their skin but also have subclinical inflammation in the liver, joints, tendons, and vascular
tree, even after adjusting for traditional cardiovascular risk factors [61]. Others have
suggested the hypothesis that psoriasis-initiated skin and systemic inflammation directly
promotes endothelial cell dysfunction. Endothelin-1 levels are increased in psoriasis, and this
vasoconstrictor is thought to be produced by keratinocytes, thereby predisposing to insulin
resistance and endothelial dysfunction. Thus, anti-inflammatory therapies of psoriasis may
also provide potential cardio-protective effects [64].
Taking many recent studies into consideration, psoriasis is strongly suggested to be an
independent risk factor for mortality as a result of the predisposing systemic complications,
and the issue now is to explore the possible pathways that may be mediating this link [52, 65,
66]. It is not yet well understood whether the associated co-morbidities are directly related to
the disease pathophysiology or related to other associated factors like poor nutritional habits,
increased alcohol consumption and tobacco smoking. All these behaviors may be aggravated
by the psychological factors associated with the disease and may account for the disease-
associated co-morbidities [67-69]. More studies are needed to elucidate the exact etiology of
the association between psoriasis and the previously mentioned co-morbidities. This will help
in establishing preventive measures for these risks.
Crohn’s disease is another co-morbidity that seems to be associated with psoriasis. Both
diseases share common genetic susceptibility factors, and 10% of patients with Crohn’s
disease have a first degree relative with psoriasis, whereas there is only a 2.9% incidence in
healthy control individuals [70]. Several studies have also suggested lymphoma as a common
co-morbidity in psoriasis patients. However, it is not yet well understood whether the risk of
lymphoma is due to the disease itself or is an adverse effect of some of the systemic therapies
used for treatment [71, 72].
The clinician developing a plan for treatment of a psoriatic patient must consider the
possibility of all these comorbidities. Psoriasis is thus considered a systemic disease, and the
patient must be closely monitored. Patient management cannot focus only on the apparent
dermatological manifestations but instead must be multidisciplinary and account for all issues
related to the disease [55].
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5. Histologic Features

Psoriasis presents characteristic histological hallmarks including:

1. Thickening of the epidermis (acanthosis) and parakeratosis (abnormal retention of

nuclei in the squames of the stratum corneum), with an abnormal increase in basal
keratinocyte turnover and elongation of rete ridges. These features are clinically
reflected by the adherent silvery scales covering the skin lesions.
2. An exaggerated vascularity of the lesion due to neo-angiogenesis, as clinically
manifested by pin-point bleeding upon scratching or accidental dislodging of scales
(Auspitz sign) [37]. The redness in some areas of the lesions is due to an increased
number of tortuous capillaries that lend the skin their color through the markedly
thinned areas of the epidermis.
3. Immune cell infiltration, mainly of dendritic cells, macrophages and T helper cells
within the upper papillary dermis, with some neutrophils and T-cells in the dermis
[27, 73].

It is obvious from the histological hallmarks as well as the clinical manifestations that the
disease process is maintained by the interplay between three major cell types: keratinocytes,
endothelial cells and immune cells, with genetic and environmental factors playing a major
role in predisposition to the disease and the initiation of the inflammatory cascade [42, 74-
76].

6. Psoriasis Phenotypes

Psoriasis has been considered a single disease entity that is characterized by a spectrum
of well-defined clinical symptoms. However, since major issues about the disease process
remain unresolved, this assumption might not be accurate. Indeed the clinical variants of
psoriasis represent dynamic distinctive features of the disease and some patients may present
with more than one variant.
Unresolved issues include the primary etiology of the disease, particularly as to whether
epithelial or immunologic in origin and whether the disease is actually autoimmune, the role
of genetic versus environmental factors, and the impact of all these factors on the response to
therapy [27, 73]. The alternative hypothesis is that psoriasis represents a common clinical
expression of different inflammatory skin diseases each with a different pathophysiology and
genetic determinants and thus responds differently to the different treatment options [27, 77].
This hypothesis is supported by the finding that manipulation of different distinct molecular
pathways in mouse models can result in inflammatory skin conditions similar to psoriasis.
Many genetic loci have also been identified as susceptibility factors in developing psoriasis
lesions [23, 78]. Identifying the different psoriatic phenotypes is a critical step for
determining the appropriate therapy. Psoriasis has been classified according to various
assessment criteria. Classification according to the clinical phenotype is the main method of
assessment and helps to classify the disease into various categories, which are discussed
below [79, 80].

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Classification according to the age of onset into early- and late-onset psoriasis is another
way of distinguishing the disease type. Early onset psoriasis occurs before the age of 40 years
and represents 75% of patients [81-83]. An alternate classification is according to human
leukocyte antigen HLA-Cw6, which is commonly known as PSORS1. According to this
susceptibility locus, psoriasis can be classified into type I and type II [84]. Type I is HLA-
Cw6 positive and represents approximately 35-50% of the heritability of the disease and
almost 65% of the psoriasis population [27, 42]. It is usually associated with a younger age
group of patients (below 30 years), family history, onset following streptococcal sore throat
and association with guttate lesions. Type II, which is HLA-Cw6 negative, is typically
associated with an older age group (above 40 years) with no family history or history of
recent streptococcal sore throat and is clinically characterized by chronic plaques (plaque
psoriasis) with nail and joint involvement [27].
There are also some indices used for classifying psoriasis according to various factors
that reflect the patient’s improvement in response to treatment. These indices include
Psoriasis Area Severity Index (PASI), which describes the area covered by psoriatic lesions.
However, the area of the body covered by psoriatic plaques is as important as the severity of
the lesions. Total Severity Score (TSS) is another index that includes all the signs and
symptoms associated with the disease. The third index is Investigator Assessment of Global
Improvement (IAGI), and this is the least commonly used index.
Another index that has recently gained attention for the assessment of psoriasis patients
as well as for other patients with immune-mediated disorders is the Health Related Quality of
Life index (HRQOL). The importance of the psychological impact of psoriasis has been lately
appreciated, especially with the observation that its impact does not always correlate with the
severity of the disease or the surface area of lesions. Also, psychological factors affect the
outcome of treatment and may also add co-morbidity risks to the disease, such as depression
and suicidal thoughts [43]. The HRQOL and each of the previously mentioned indices have
their own scoring criteria.

CLASSIFICATION OF PSORIASIS ACCORDING

TO THE CLINICAL PHENOTYPE

I. Chronic Plaque Psoriasis

Chronic plaque psoriasis (Figure 1) is the most common and typical psoriatic lesion in all
age groups including children, accounting for 80% of all cases. It is characterized by well-
demarcated adherent silvery scales of various sizes. It commonly has a symmetric distribution
involving the extensor surfaces such as the elbows and knees and can affect the scalp, trunk
and intergluteal cleft. The lesions may persist for months to years in the same location and
only about 5% of patients report complete remission for up to 5 years [35, 85]. Plaque
psoriasis can be further classified into mild, moderate and severe plaque psoriasis. This
distinction is based on one or more clinical metrics such as the Psoriasis Area and Severity
Index (PASI), which accounts for the percentage of the body surface area affected, or the
Dermatological Life Quality Index [86]. In children, lesions are often smaller, thinner and less
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822 Inas Helwa, Meg Gullotto and Wendy B. Bollag

scaly, making diagnosis more challenging. Most affected children have pruritus [37], whereas
otherwise pruritus is a symptom in about 70% to 90% of psoriatic patients [87].

II. Inverse Psoriasis

Inverse psoriasis (Figure 2) is characterized by suddenly appearing erythematous plaques

involving skin folds, including axillary, genital, perianal, intergluteal and inframammary
regions. Its main clinical feature is pink to red plaques with minimum scaling, and it is more
common in children [35, 37]. Topical corticosteroids remain the gold standard for the
treatment of inverse psoriasis [88].

III. Guttate Psoriasis

Guttate psoriasis (Figure 3) usually arises suddenly following streptococcal pharyngitis.

It is known to have better prognosis than other types of psoriasis in that it resolves
spontaneously after 3-4 months, but most patients experience recurrence with subsequent
streptococcal infections [37, 41]. This type is characterized by scattered small tear-drop-
shaped lesions that are well-demarcated, red and scaly. Some cases may progress to plaque
psoriasis [89, 90]. This variant occurs in fewer than 2% of patients younger than 30 years
[35].

IV. Erythrodermic Psoriasis

Erythrodermic psoriasis (Figure 4) is one of the rarest and most severe forms of psoriasis,
with an estimated prevalence of 1% to 2.25% of patients with psoriasis [91]. The most
prominent feature of this type is generalized erythema affecting more than 75% of the body
and associated with superficial desquamation, hair loss, nail dystrophy, and systemic
symptoms like fever, chills, malaise and even high-output cardiac failure [35]. This type can
be triggered by medical illness, withdrawal of systemic and topical corticosteroids, PUVA
phototoxic reactions, emotional stress and discontinuation of methotrexate along with other
causes [92]. It has an increased risk of mortality since patients tend to lose the protective
barrier functions of the skin and can succumb to infection or severe loss of fluids and
nutrients [91]. Sepsis is a common complication of erythrodermic psoriasis [93].

V. Childhood Psoriasis

Childhood psoriasis is a frequent condition; however, limited epidemiologic data are

available [37, 94]. Most of the subtypes of the disease occur similarly in adults and children,
but the relative frequency of particular types and patterns of presentation differ [37]. Frequent
and longer durations of remission are more common in the pediatric group (disease onset
before 16 years) as compared to adult-onset psoriasis (disease onset after 16 years) [95].

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Plaque psoriasis, guttate psoriasis and diaper psoriatic rash are the most common types of
pediatric psoriasis [96]. Childhood psoriasis is more frequently pruritic and is more common
in girls than boys. Lesions are usually thinner, softer and less scaly [94]. Face and flexural
involvement is common [36] with a common site of facial involvement being under the eye
[96]. Erythrodermic psoriasis and psoriatic arthritis are less frequent in children [37]. A
special clinical variant in young children is psoriatic diaper rash, which usually occurs until
the age of 2. It responds poorly to conventional treatment, and the erythematous lesions
commonly disseminate to the whole body.

VI. Pustular Psoriasis

Pustular psoriasis (Figure 5) is an uncommon form of psoriasis characterized by a large

collection of neutrophils in the stratum corneum, which clinically present as sterile pustules.
The pustules may be localized within or at the edges of existing plaques or may be
generalized (von Zumbasch variant) [37, 41]. It is considered a form of the disease that is
difficult to treat due to its strong inflammatory process, and conventional therapy usually fails
[97]. Severe cases of pustular psoriasis may be as serious as erythrodermic psoriasis, where
the patient is susceptible to dehydration and infections. Some localized forms involve the
palms and soles and can be quite debilitating due to the difficulty of walking or manipulating
objects with the hands [41].

VII. Nail disease (Psoriatic Onychodystrophy)

Nail disease can occur in all psoriasis subtypes. Fingernails are involved in about 50% of
all patients and toenails are involved in about 35% of all patients [98]. Approximately 90% of
patients with psoriatic arthritis have nail changes [35].

VIII. Psoriatic Arthritis (PsA)

PsA is an inflammatory seronegative spondyloarthropathy associated with psoriasis [99].

It can result in erosion of joints in about 60% of cases and functional disability in
approximately 20%, so it represents a serious comorbidity in a considerable population of
psoriasis patients. Although PsA can develop at any time including childhood, it is most
common between the ages of 30 and 50 years and affects men and women equally [99].
Epidemiological studies of PsA are very limited due to population heterogeneity and varying
methods of disease classification as well as a lack of accepted criteria for diagnosing the
disease [50]. There is a wide variation in incidence among countries and different regions of
the world [100], as well as wide prevalence ranging from 6% to 42% among psoriatic patients
or an overall prevalance of about 1% of the general population [65].
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7. Therapeutic Armamentarium

Due to the dynamic spectrum of psoriasis and the diversity of its phenotypes there is
more than one approach to treatment of the disease (also because there is no ideal treatment
for psoriasis). Patients often do not respond identically to treatment options and there is no
commonly approved regimen of therapy among dermatologists. Treatment options differ
according to the age of the patient, the stage and severity of disease, the area covered by
lesions, the body site, the availability, adverse effects and cost of treatment and the patient’s
compliance. Therapies for psoriasis include topical, systemic (traditional and biologics),
phototherapy, combination treatments and herbal remedies [101]. In summary, treatment must
be tailored to meet individual needs. We will be discussing the most common and generally
accepted treatment options available and promising treatment options that are still under
investigation.

i. Topical Therapy
Typically, topical therapies are considered the first-line therapy for patients with mild
psoriasis (<5% body surface area involvement), but as a monotherapy topicals tend to be
ineffective in cases of moderate to severe or even limited recalcitrant disease [102]. However,
they can be used adjunctively with other treatment options like phototherapy or systemic
medications in severe cases [103]. They are usually sufficient to control pediatric psoriasis
with minimal side effects. Topical treatments have the advantage of being targeted to the
affected area, generally effective, safe and well-tolerated [104]. It should also be noted that
80% of the patients affected with psoriasis have mild to moderate disease, and the majority
can be treated with topical agents [103].
Topical therapies [29, 83] include:

a. Topical corticosteroids
b. Tars
c. Anthralin (Dithranol)
d. Topical vitamin D analogues (Calcitriol, Tacalcitol, Calcipotriol)
e. Emollients
f. Topical retinoids
g. Calcineurin inhibitors (Pimecrolimus and Tacrolimus)
h. Keratolytics (Salicylic acid, Urea)

a) Topical corticosteroids

Topical corticosteroids are considered the cornerstone of treatment for the majority of
patients, especially those with limited disease, and are the most commonly prescribed
psoriasis drug in the USA [105]. They are used both as a monotherapy and as a complement
to systemic therapy. They have the advantage of being available in diverse strengths and
formulations, and their potency in treating mild lesions improves patients’ adherence to
treatment regimens [103]. According to Stoughton-Cornell classification, the potency ranking
of topical steroids (from weak to super potent) is based on their ability to produce
vasoconstriction [106].

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 Keratinocytes in Psoriasis 825

 Safety:

Topical corticosteroids are considered the first choice treatment for children especially
for highly pruritic lesions that are mostly localized in flexures. Topical corticosteroids are
also safe during pregnancy as well as breastfeeding for the control of localized psoriasis [107,
108]. Although commonly used in children, adolescents and adults with mild psoriasis,
prolonged use of highly potent corticosteroid formulations is not recommended to avoid
possible adverse effects. These complications include skin atrophy and striae rurae distensae
at the site of application and/or suppression of the hypothalamic-pituitary-adrenal axis.
Withdrawal from corticosteroids should be done slowly, with gradual reduction of the dosage
to avoid psoriasis disease rebound [37].

 Mechanism of action:

The mechanism of action of corticosteroids includes anti-inflammatory, anti-proliferative,

immune-suppressive, and vasoconstrictive effects. Corticosteroids act at the cellular level on
genomic and non-genomic pathways. The genomic pathway refers to the activation of the
glucocorticoid receptor (GR) by cortisol and related glucocorticoids. This binding initiates a
cascade of transcriptional events that eventually suppresses the expression of pro-
inflammatory genes responsible for the production of cytokines, growth factors, adhesion
molecules, nitric oxide and prostanoids. This pathway is responsible for the long-term effects
of corticosteroids. The non-genomic pathway is responsible for the rapid effects of
glucocorticoids, which occur within a few minutes of application. This pathway functions
through membrane-bound receptors and second messengers and requires no de novo protein
synthesis. Stimulation of this pathway directly modulates the activation and responsiveness of
target cells such as T-cells, monocytes and platelets. These two pathways ensure both
immediate relief and long-term potency of topical corticosteroid treatments [83].

b) Vitamin D analogues:

The role of synthetic vitamin D analogues in treating psoriasis was originally based on
the observation that treating a patient with oral vitamin D significantly improved his lesions
[109]. Since then, the idea of using topical vitamin D (1, 25 dihydroxyvitamin D3; Calcitriol
is the biologically active form) was introduced, and subsequently these agents have been used
as a standard topical therapy for psoriasis. The exact mechanism of action is not yet fully
understood, although it is thought that vitamin D analogues act by binding to the vitamin D
receptor (VDR). VDR is a ligand-dependent transcription factor that forms a heterodimer
with the retinoid X receptor (RXR) and translocates to the nucleus. This complex binds to the
promoter regions of responsive genes, ultimately resulting in the initiation or suppression of
transcription, cell differentiation and proliferation, immunomodulation and mineral
homeostasis [110]. This signaling pathway is believed to mediate both an inhibition of
keratinocyte proliferation and an enhancement of keratinocyte differentiation [103]. These
agents are less efficacious than topical corticosteroids; however, they are often used
adjunctively with them to enhance efficacy and reduce the risk of atrophy with long-term use
[103].
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826 Inas Helwa, Meg Gullotto and Wendy B. Bollag

c) Anthralin (Dithranol):

Dithranol is a herbal-derived therapy extracted from Andira Araroba and is known as

Cignolin in Germany and Anthralin in the US [111]. Its use has been declining over recent
years due to its staining effect, making its utilization difficult in an outpatient setting,
especially with the availability of more cosmetically acceptable alternatives [103]. Its exact
mechanism is not yet fully understood, but recent studies suggest its ability to prevent T-
lymphocyte activation, induce keratinocyte apoptosis and enhance differentiation through a
direct effect on mitochondria [103, 112].

d) Keratolytic agents (e.g., salicylic acid):

Salicylic acid is thought to work through two mechanisms both of which lead to
desquamation of corneocytes [103]. The first mechanism is by reducing intercellular
cohesiveness of corneocytes and the second is by reducing pH and thus increasing the
hydration and softening of plaques [105]. These two effects lead to reduced scaling and
softening of psoriatic plaques; thus, salicylic acid represents a purely symptomatic treatment.

e) Emollients:

The use of non-medicated moisturizers such as aloe vera gel is a standard adjunctive
approach to manage mild to moderate psoriasis. Such emollients have the advantage of being
the safest option during pregnancy and lactation as well as for children, yet their efficacy and
their active ingredient are unknown [113]. These agents help to retain moisture in the stratum
corneum to cause softening of the psoriatic plaques. The use of emollients is considered a
symptomatic approach rather than a therapeutic approach. However, there are conflicting
reports regarding the efficacy of aloe vera itself in the treatment of psoriasis, and this topic is
still under investigation by many groups [111, 114, 115].

f) Topical retinoids:

Topical retinoids have been used for treating chronic skin conditions including psoriasis
for almost 3 decades. The first topical retinoid approved by the US Food and Drug
Administration (FDA) in 1971 was Tretinoin. Tazarotene is another commonly prescribed
topical retinoid that was approved in 1997 [103, 116].

 Mechanism of action:

The biologic effects of retinoids are mediated by nuclear hormone receptors (retinoic acid
receptors or RARs and RXR). Binding of retinoids to these receptors elicits the transcription
of retinoic-acid responsive genes. Retinoids influence proliferation and differentiation of cells
and help to reverse the abnormal desquamation of keratinocytes in psoriasis [116].

 Safety:

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Skin irritation is considered the main adverse effect of topical retinoids and may be
reduced by using lower doses or combining them with moisturizers [103]. The safety of using
topical retinoids during pregnancy and lactation and in children below 12 years of age has not
been validated by clinical reports [116].

g) Calcineurin inhibitors:

Calcineurin inhibitors are non-steroidal immune-modulating agents that act by blocking

the enzyme calcineurin, thus inhibiting the production of IL-12 and subsequent T-cell
activation and proliferation. They are considered safe for use in children [36, 117].

ii. Phototherapy
The idea of using ultraviolet (UV) light for the treatment of psoriasis arose in antiquity as
patients and physicians observed lesions improving following sun exposure. Historically,
phototherapy was the first line for treating psoriasis; however, long-term therapy was limited
by dose-dependent toxicity [118]. Modifications now exist, including Psoralen plus UVA
(PUVA), broadband UVB (BB-UVB), and narrowband UVB (NB-UVB). Phototherapy can
be administered in the hospital, outpatient clinic, or in the patient’s home [119].

 The Goekermann regimen:

In 1925, the application of topical crude coal tar and subsequent UV irradiation was
introduced by Goeckerman and became a standard therapy for psoriasis for almost half a
century, especially in the USA. However, this treatment modality has been abandoned due to
the carcinogenic potential of coal tar and the associated time constraints and mess [119].
The use of coal tar alone has also been reported to achieve moderate relief, but the
mechanism of action is unclear [105].

 Psoralen plus ultraviolet A irradiation (PUVA):

The concept of using a photosensitizer was introduced by ancient Egyptians and Indians
as they used psoralen in combination with sunlight for treating vitiligo [120]. Psoralen’s
modern form was introduced for the treatment of psoriasis by Pinkus in 1951 and for vitiligo
in 1974 [119, 121]. It is most often used in patients who are unresponsive to UVB or have
thick plaques with involvement of some areas like the hands, soles or nails [119].

ULTRAVIOLET B RADIATION (UVB)

 Safety:

Broad-band UVB (BB-UVB) is considered the first and safest line of treatment for
pregnant women who suffer plaque or guttate psoriasis requiring systemic intervention. This
is considered an advantage of UVB over PUVA treatment, which is contraindicated during
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828 Inas Helwa, Meg Gullotto and Wendy B. Bollag

pregnancy and lactation [120]. Neither BB-UVB nor narrow-band UVB (NB-UVB) is known
to have teratogenic effects [107, 108, 122].

 Adverse effects:

Acute

Acute side effects of both UVB and PUVA are quite similar and include erythema,
itching, burning and dry skin. The use of protective eyeglasses is necessary to decrease the
risk of UVB-related cataracts [122].

Chronic

Long-term side effects include photo-aging and carcinogenesis. Patients treated with
PUVA have an increased risk of skin cancer and require lifetime monitoring [120].

 Mechanism of action:

The mechanism of action of phototherapy includes anti-inflammatory and anti-

proliferative effects. UVB interferes with the synthesis of proteins and nucleic acids leading
to decreased proliferation of keratinocytes. This happens through formation of pyrimidine
dimers, membrane lipid peroxidation, activation of signaling cascades and induction of
transcription factors. These are considered early changes which are followed by delayed anti-
inflammatory changes, including alteration of antigen-presenting cells and signaling
pathways. UVB causes a reduction in the number of Langerhans cells, thus inhibiting the
antigen-presenting capacity of skin dendritic cells. Likewise, it down-regulates Th17 cells,
which play a central role in the pathogenesis of psoriasis, and alters the secretion of cytokines
in macrophages [122].
The concept of sensitizing the skin with psoralen relies on the idea that psoralen itself
reacts with DNA in three steps. First, psoralen incorporates into DNA in the absence of UV
radiation. Second, after irradiation cyclobutane monoadducts (MA) with pyrimidine bases are
formed and some of these MA can form psoralen DNA cross-links after absorbing a second
photon. Third, excited psoralen can then react with reactive oxygen, and the reactive oxygen
species formed by this reaction tend to damage cell membranes. These DNA-psoralen cross
links inhibit DNA replication causing cell cycle arrest. It was found that PUVA can also
induce apoptosis in lymphocytes and its pro-apoptotic action is even more potent in
lymphocytes than in keratinocytes [123]. Thus, the use of a photosensitizer such as psoralen
(8-methoxypsoralen) increases the efficiency of UVA and allows the use of lower doses of
radiation.

iii. Systemic Therapy

Systemic therapies are considered in cases of more extensive or refractory psoriasis when
topical therapy and phototherapy show lack of efficacy. A systemic approach is considered

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not only when lesions involve a large surface area (more than 10%) but also in a subset of
patients with limited disease affecting critical sites like the palms and soles or with severe
scalp psoriasis. In these cases the lesions may be limited but result in debilitating symptoms
that affect the patient’s quality of life and increase the appropriateness of a systemic approach
[30].
Systemic therapies can be classified into:

1. Traditional systemic therapies (Non-biologic therapies)

2. Biologics

a) Traditional systemic therapies:

Traditional systemic therapies are considered non-biologic agents that are used as a first
line of systemic therapy for moderate to severe psoriasis. They represent important players in
a psoriasis treatment plan due to their oral route of administration and low cost as compared
to biologics [30] and include:

1. Methotrexate
2. Cyclosporine (CsA)
3. Acitretin

a) Methotrexate

Methotrexate is considered a cornerstone in the treatment of rheumatoid arthritis as well

as other rheumatic diseases; yet it has also been considered the primary agent for systemic
treatment of moderate to severe psoriasis for decades [124, 125].

 Mechanism of action:

Methotrexate is a folate antagonist that was initially used as an anti-carcinogenic drug. It

acts by inhibiting the enzyme dihydrofolate reductase thus decreasing the synthesis of folate
cofactors that are essential for nucleic acid synthesis [126-128]. Thus, methotrexate has an
anti-proliferative effect on epidermal cells as well as inflammatory cells like lymphocytes and
macrophages. It is also believed to have anti-inflammatory actions mediated via the inhibition
of polyamine synthesis. This pathway is separate from its action as a folate antagonist.

 Safety:

The most common side effects of methotrexate, which usually start on initiation of
therapy, include nausea, anorexia, fatigue and malaise. These side effects are dose dependent
and can be minimized by taking the medication several hours before bedtime or by
concomitant folic acid administration. Liver toxicity is common especially in psoriatic
patients, and this enhanced toxicity is probably attributed to higher rates of diabetes and
alcoholism in these patients [129]. Methotrexate is an independent risk factor for cutaneous
squamous cell carcinoma, and this risk is increased in patients treated with PUVA. The use of
methotrexate as a second option for treatment of patients with a previous PUVA exposure
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830 Inas Helwa, Meg Gullotto and Wendy B. Bollag

must be considered with caution [130]. Methotrexate is absolutely contraindicated during

pregnancy and breast-feeding due to its teratogenic potential and increased risk of
spontaneous abortion during the first trimester. Women of child-bearing age should consider
contraception during methotrexate treatment. Even for men, methotrexate has an anti-
spermatogenic effect and may cause mutagenesis, and it is recommended that both sexes
postpone conception at least 3 months after the cessation of therapy [131]. Susceptibility to
infections like tuberculosis or recurrence of infections such as hepatitis B virus are common
in patients treated with immunosuppressive drugs like methotrexate [132]. Mucocutaneous
ulceration can occur without adequate folic acid supplementation or in cases of over dosage
[133, 134]. Neurological side effects including headache and dizziness are also common
either at the initiation of the medication or as a result of its long-term use [135]. Whether
methotrexate is an independent risk factor for cardiovascular disease is still controversial and
requires further extensive population studies and deeper investigation into the existing
correlation between psoriasis and its therapy and methotrexate in particular [136-139]. Some
studies suggest possible beneficial effects of methotrexate on cardiovascular health and
attribute this benefit to its anti-inflammatory effects. In addition, methotrexate improves the
patient’s quality of life, which contributes to a better psychological status and exercise
compliance, both of which improves the patient’s overall health [138, 139]. Methotrexate is
not the most recommended treatment for children due to its multiple long-term side effects
that include hepatotoxicity [36].

b) Cyclosporine (CsA):

Cyclosporine is an immune-suppressive drug that was initially used to avoid rejection

following organ transplant. Its effectiveness in the management of psoriasis has gained
attention since 1979 [30, 140]. It has been approved by the FDA as an anti-psoriatic
medication since 1997, and it is also used off-label for other inflammatory skin diseases
including but not limited to atopic dermatitis [141]. Cyclosporine is recommended because of
its high efficacy and rapid onset, making it suitable for short-term treatment of inflammatory
skin diseases, particularly psoriasis.

 Mechanism of action:

Cyclosporine acts as a calcineurin inhibitor to act selectively on T-cells, particularly T-

helper cells, although T-suppressor cells may also be a target. Cyclosporine forms a complex
with cyclophilin to inhibit the phosphatase activity of calcineurin. As a result calcineurin is
unable to dephosphorylate nuclear factor of activated T-cells (NFAT), a transcription factor
upstream of interferon gamma (IFN-γ) and granulocyte-macrophage-colony stimulating factor
(GM-CSF).
Accordingly, cyclosporine depletes lymphocytes and macrophages in the epidermis and
dermis to result in an anti-inflammatory effect. It also inhibits the release of histamine from
mast cells, and down-regulates the expression of cellular adhesion molecules on dermal
capillary endothelium to inhibit neo-angiogenesis. The anti-inflammatory and anti-angiogenic
effects are also associated with anti-proliferative effects on keratinocytes. Thus, cyclosporine
affects the three key events in the pathogenesis of psoriasis [30, 142-145].

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 Safety:

The most significant side effects of cyclosporine are hypertension and nephrotoxicity,
which are due to its vasoconstrictive effects on renal arterioles. Thus, patient selection is very
critical before the initiation of therapy [146-149]. Patients taking CsA that have had a history
of PUVA treatment are at increased risk of cutaneous squamous cell carcinoma. However, the
risk of malignancy with adherence to the published guidelines and patient selection criteria
remains controversial [150].
The majority of information about cyclosporine safety during pregnancy is derived from
studies in which CsA is used to prevent organ transplant rejection. These patients already
have multiple complications and are taking an array of drugs, which makes it difficult to
assume that any birth defects or prenatal complications are due solely to CsA [30]. The
nephrotoxic effect of CsA on the newborn is still questionable, and CsA is suggested for use
during pregnancy only if the potential benefits over-ride the possible risks to the fetus [151].
The majority of studies regarding the use of cyclosporine in children have investigated its
efficacy, although its exact efficacy is still ambiguous and seems to differ from one type of
psoriasis to another and from one patient to another. Safety issues are not adequately
investigated and data are sparse [152, 153]. Some studies suggest that adverse effects in
children are the same as in adults. Taking into consideration that children may need to be
treated for a longer term, which will tend to enhance adverse effects, CsA might not be the
drug of choice for pediatric psoriasis.

c) Acitretin:

Acitretin is an oral vitamin A derivative used to treat psoriasis since 1980 [30]. Its exact
mechanism of action is not yet well understood; however, recent studies have suggested an
influence on T-helper cells (Th17 and Th2).

 Mechanism of action:

According to Niu et al. [154], Acitretin reduces Th-1 and Th-17 infiltration and
attenuates their cytokine secretion in the skin and peripheral blood of psoriasis vulgaris
patients. Th17 is a major player in the psoriatic cytokine network [42]; thus, reducing its
secretion will have a significant effect in reducing inflammation and improving treatment
outcome. Acitretin as a monotherapy is not as effective as other traditional systemic
medications; however, it is a safe and efficient option for patients with human immune-
deficiency syndrome as it appears to have no immunosuppressive properties [155].

 Safety:

Acitretin is teratogenic and totally unsafe during pregnancy and breast-feeding, with risks
of possible malformations and growth retardation outweighing any possible benefits. It is
even contraindicated to conceive within 3 years after the cessation of Acitretin therapy.
Accordingly, Acitretin should not be used under any circumstances for women of child-
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bearing age. Mucocutaneous lesions and hyperlipidemia are also common side effects of this
drug.
Acitertin can be used in children with severe pustular psoriasis. It is recommended as a
combination therapy with topicals. Long-term treatment in children can lead to growth
retardation with premature epiphyseal closure and impaired bone growth. Follow-up has to be
maintained carefully to avoid undesirable side effects in children treated with Acitretin [36,
37].

d) Biologics:

Although the exact etiology and primary cell type responsible for initiating psoriasis are
unknown, lesions appear to evolve as a result of interplay between various inflammatory cells
and immune mediators (inflammatory cytokines), known as the hypothesis of the cytokine
network. A cross-talk between innate and adaptive immunity shapes the inflammatory
infiltrate. Among the key players in this cascade, in addition to epidermal keratinocytes, are
immune cells such as dendritic cells, T-cells and macrophages and key cytokines such as
interferons (IFN-α and-γ), tumor necrosis factor-α (TNF-α) and interleukins (IL-1β, IL-20,
IL-6, IL-23, IL-12, IL-17A, IL-17F and IL-22). Topical and traditional systemic therapies
have not met the needs of all patients but seem empirically to improve symptoms, sometimes
without a clear understanding of targets and mechanisms and/or possibly multiple
targets/mechanisms. As a result, biologics have recently been added to the therapeutic
armamentarium for treatment of psoriasis [42, 84, 156-159].
Biologics are proteins that possess pharmacological potential and are extracted from
animal tissue or produced by recombinant DNA technology. Biologic therapies target the
immune system, so they are contraindicated in patients with active or serious infections
(infections that require antibiotics). It is important to use all approaches to reduce the risk of
infection, including providing vaccinations throughout the treatment course. In case of
infection, cessation of treatment is mandatory. Live vaccinations should be avoided under all
circumstances, and proper screening must be performed in all patients before commencing
biologics. These tests include liver function tests, complete blood count including platelet
count and tests for hepatitis and tuberculosis (TB) [98].
Since inflammatory events involving T-cells and cytokines play a central role in the
pathogenesis of psoriasis, biologics target the inflammatory pathway and may have one or
both of the following targets:

1. T-cells; attempting to correct the immune deviation towards a more balanced

response.
2. Cytokines; attempting to block or neutralize inflammatory cytokines like TNF-α
[156].

According to the National Psoriasis Foundation (webpage updated in 2012), there are
currently 5 biologic drugs approved by the FDA for the treatment of psoriasis and psoriatic
arthritis (PsA), and these drugs are:

1. Enbrel (etanercept)
2. Humira (adalimumab)

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 Keratinocytes in Psoriasis 833

3. Remicade (infliximab)
4. Simponi (golimumab)
5. Stelara (ustekinumab)

Efalizumab (Raptiva) is a monoclonal antibody to the CD11a subunit of LFA-1, which

has been withdrawn from both the European and American markets because of long-term
safety issues [160].

 TNF blockers:

The importance of TNF in the progression of psoriasis is obvious based on the elevated
levels of TNF-α observed in both the affected skin and the serum of psoriatic patients and the
reduction of these levels following successful treatment [98].
The group of biologics targeting TNF-α includes:

1. Enbrel (Etanercept)
2. Humira (adalimumab)
3. Remicade (infliximab)
4. Simponi (golimumab)

Etanercept is Discussed Below as A Representative of this Class of Biologic Therapies

Etanercept (brand name Enbrel) is a fully human soluble tumor necrosis factor (TNF)
receptor fusion protein with the constant Fc region of immunoglobulin G1, which neutralizes
TNF-α thus blocking its inflammatory outcomes. This protein exerts different effects on TNF-
α expressing cells including cytotoxicity, complement-dependent cytotoxicity (CDC) and
antibody-dependent cell-mediated cytotoxicity (ACDC) [161]. According to the National
Psoriasis Foundation, Enbrel is FDA-approved for the treatment of psoriatic arthritis,
rheumatoid arthritis, juvenile rheumatoid arthritis and ankylosing spondylitis. It is
administered as an injection once or twice a week according to each individual case.

 Safety:

Etanercept has shown good tolerability in individual short- and long-term clinical trials.
This drug has not exhibited any significantly increased rate of serious infections or
malignancies as compared to placebo [162].

 IL12/IL23 inhibitor:

 Ustekinumab:

Ustekinumb (brand name Stelara) is the latest biologic approved by the FDA for the
treatment of moderate to severe psoriasis. It targets the p40 subunit that is shared by IL12 and
IL23. It is a monoclonal antibody that blocks the interaction of these cytokines with their
receptors, thus blocking signaling, differentiation and cytokine production in both Th-1 and
Th-17 cells that contribute to the pathogenesis of psoriasis [156].
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834 Inas Helwa, Meg Gullotto and Wendy B. Bollag

 Limitations of biologic therapy:

Increased risk of serious fungal and bacterial infections, such as tuberculosis, is a major
concern for patients receiving biologics. There is also risk of other opportunistic infections
such as histoplasmosis, candidiasis, listeriosis and non-TB mycobacterial infections. Safety
warnings include possible reactivation of hepatitis B infection [163, 164]. The risk of
malignancy associated with biologic agents is complex and controversial. Chronic
inflammatory diseases may confer an increased risk regardless of the treatment, such that
inflammatory conditions including autoimmune disorders seem to have an increased risk of
certain malignancies including non-hemolytic lymphoma [165]. Psoriasis in itself is
considered a possible risk factor for malignancies like lymphoma, so it is not obvious whether
lymphoma is a risk factor of the disease or of the treatment options. However, the
immunosuppressive potential of drugs may contribute to the possible risk, and patient
monitoring is essential, especially with long-term treatment over the course of the disease.
Thus, definitive conclusions regarding the risk of lymphoma in psoriasis patients and the role
of biologics as a risk factor are still controversial [72, 166]. Other safety concerns regarding
the risk of cardiovascular and neurological disorders are also still controversial [163]. As for
childhood psoriasis, the use of biologics is not generally approved. However, recent studies
have reported some successful cases treated with biologics.
In summary, biologics are considered the third treatment option for moderate to severe
psoriasis. They have achieved promising success rates in controlling the disease and
improving the patient’s quality of life. Safety concerns are still an issue due to lack of
sufficient long-term safety data, as the biologics are an only recently introduced therapy
option [160]. Biologics directly target the immune system (cytokines or cells), yet the role of
the immune system as a primary cause of psoriasis has not yet been definitely proven.
Psoriasis is a dynamic disease and more investigation is required to determine the primary
etiology of the disease, which should help in developing more selective treatment options that
can be individualized to each patient’s needs.

8. The Mystery of the Disease Origin

The primary etiology of psoriasis is still an unresolved issue. Three major factors are
believed to contribute to the disease origin:

1. Environmental factors
2. Genetic predisposing factors
3. Immune system-keratinocyte cross talk

 Predisposing environmental factors:

Several known environmental triggers predispose to the development of psoriasis lesions

or worsen existing disease. These triggers include streptococcal infections in that guttate
psoriasis is common following streptococcal sore throat [167, 168]. Physical trauma gives
rise to the Koebner phenomenon, which is the development of psoriatic lesions in formerly

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uninvolved skin of psoriatic patients after cutaneous trauma [169]. This reaction is commonly
seen in areas of tattoos or surgical incisions. Some medications are known to induce psoriasis
such as anti-depressants, β-blockers and anti-cytokine therapy like IFN-α used for the
treatment of hepatitis C virus [170]. Smoking and alcohol are also known to trigger or worsen
psoriasis [171, 172].

 Predisposing genetic factors:

In a genetically predisposed individual various elements in the epidermis and the dermis
may be dysregulated [25, 173, 174]. This dysregulation may include defects of skin barrier
integrity that lead to an abnormal response to environmental or antigenic insults. Some genes
associated with a susceptibility to psoriasis are discussed below.

 Immune system-keratinocyte cross talk:

As mentioned earlier, the psoriatic disease process seems to involve interplay between
keratinocytes and immune cells. The initiation of the disease and maintenance of the psoriatic
cytokine network depend on close communication between these two cell types. It is
extremely challenging to track the primary cell type responsible. There is evidence supporting
the role of each cell type in disease initiation. In contrast to the well-known T-cell mediated
origin of the disease, a suggested theory of a keratinocyte-mediated origin has evolved with
numerous supporting data. This theory may open new therapeutic avenues for targeting
keratinocytes as key players in the disease process.

 The T-cell mediated origin:

As mentioned earlier, although psoriasis has been studied for decades, a major unresolved
issue is whether the defect resides primarily in epidermal keratinocytes or in T-cells [23-25,
173, 175-177]. Many investigators believe that psoriasis is primarily a T-cell disease or even
an auto-immune disease [23, 75, 177]. However, due to lack of convincing evidence
regarding autoantibodies, psoriasis has been more precisely described as an “immune-
mediated disorder” triggered by genetic and environmental factors [31]. Some of the psoriasis
genetic susceptibility loci that have been identified so far are HLA-C, IL12B, IL23A, IL23R,
IL2, TNFAIP3, TNIP1, and ZNF313. In addition, recently identified loci include NOS2,
FBXL19 and PSMA6 [25, 174, 178]. HLA class 1 allele is associated with human leukocyte
antigen. Psoriasis is strongly associated with the HLA-C allele, Cw6. Early studies performed
in Northern European populations showed that the frequency of HLA-Cw6 was 46% in
patients with psoriasis vulgaris as compared to 7.4% in controls [179]. So genetic variation in
the MHC (major histocompatibility locus antigen cluster) increases the risk of developing
psoriasis and psoriatic arthritis [180]. IL12B, IL23A and IL23R encode interleukin 12,
interleukin 23 and alpha subunit p19 interleukin 23 receptor, respectively. The two subunits
(IL12B and IL23A) heterodimerize to form IL-23. IL-23 binds the IL-23 receptor (a
heterodimer of IL23R and IL12RB1) on naïve CD4+ T-cells to induce the development of
Th17 cells. Th17 cells play a major role in driving the disease process in psoriatic skin [181].
Accordingly, all these genetic predisposing factors may be playing a major role in the
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836 Inas Helwa, Meg Gullotto and Wendy B. Bollag

dysregulation of the inflammatory cascade of the disease. TNFAIP3 encodes TNF-α-induced

protein 3 and TNIP1 encodes TNFAIP3-interacting protein-1. These two genes are associated
with psoriasis and are likely to enhance NFкB activation [182]. ZNF313 is a paralog of
TRAC1, a ubiquitin ligase that regulates T-cell activation, and it is thought that ZNF313 has a
similar role [181]. NOS2 and FXL19 encode regulators of innate immunity [181, 183, 184]
All these loci are related to immune cell activation, making the immunological origin of
psoriasis more likely. However, there are other susceptibility genes that are expressed by
keratinocytes. Among these genes is the β-defensin cluster. β-defensins are secreted by
keratinocytes in response to cytokines and/or an inflammatory environment [181]. In
addition, genes of the epidermal differentiation complex are up-regulated in psoriatic lesions,
suggesting underlying alterations in the coordinate regulation of the epidermal cornified
envelope structure [185]. SLC12A8 (solute carrier family 12 member A8) is another
candidate gene for psoriasis susceptibility and is suggested to be related to keratinocyte
function. SLC12A8 encodes a protein sharing homology with a family of cation-chloride-
coupled co-transporters that are responsible for electro-neutral transport [186]. The exact
function and tissue distribution of SLC12A still requires investigation in order to address its
possible role in the pathogenesis of psoriasis [187].
In summary, many identified susceptibility genes make the immunologic origin of
psoriasis likely. On the other hand, other sets of identified genes are closely related to
keratinocytes, making the keratinocyte-mediated origin of psoriasis an idea that requires
further investigation. More data regarding the keratinocyte-mediated origin of psoriasis will
be discussed in the following section.

 The keratinocyte-mediated origin:

However and despite the evidence that supports the T-cell-mediated concept, an
epidermal keratinocyte-mediated origin cannot be excluded for many reasons [188].

 Keratinocytes: pro-inflammatory potential:

Psoriasis is an inflammatory disease and keratinocytes are a major source of a wide

spectrum of pro-inflammatory mediators [189-191]. They produce interleukin (IL)-1, 6, 7, 8,
10, 12, 15, 18 and 20 as well as TNF-α. Keratinocyte cytokine production has many
consequences including induction of inflammatory cell migration and promotion of
keratinocyte proliferation and differentiation. These cytokines have their effect on other
keratinocytes (feedback loop) as well as on immune cells to trigger the production of
additional cytokines and chemokines (inflammatory cascade) [192]. IL-6 secretion by
keratinocytes occurs under various conditions including UVB exposure as well as with
diseases like lichen planus or upon treatment with transforming growth factor-α (TGF-α)
[193]. IL-6 stimulates keratinocyte proliferation, is involved in epidermal hyperplasia and is
an important factor for wound healing. Local over-expression of IL-7 in basal keratinocytes
results in infiltration of the epidermis with skin-derived T-cells. IL-8 attracts neutrophils and
is produced by keratinocytes after exposure to various irritants like sodium lauryl sulfate
[194].

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 Keratinocytes in Psoriasis 837

In summary, cytokines and chemotactic factors produced by keratinocytes in response to

local irritation or injury can initiate an inflammatory process. The ability of keratinocytes to
release cytokines suggests these cells can stimulate resident inflammatory cells such as
dendritic cells and attract T-cells to initiate an inflammatory reaction. A feed-back loop and
cross-talk between keratinocytes and immune cells maintains this inflammatory cascade and
can establish what is known as the psoriatic-cytokine network [42]. There are studies
reporting the up-regulated production of pro-inflammatory cytokines and their receptors in
keratinocytes in various skin diseases including psoriasis, and these released cytokines may
have effects on other inflammatory cells.
Keratinocytes can modulate the function of splenic antigen-presenting cells through the
production of IL-10 [195]. IL-12 production is also up-regulated in eczematous skin. IL-12 is
a pro-inflammatory cytokine that can be released by macrophages, dendritic cells and
keratinocytes in response to various allergens [196]. In addition, the levels of the IL-20
receptor are elevated in psoriatic keratinocytes [197]. Keratinocyte-derived cytokines and
over-expression of their receptors may therefore play an important role in various skin
diseases that are thought to be primarily initiated by immune cell defects. The role of
keratinocytes has been recently appreciated in terms of the mediation of some disorders that
have long been regarded as immune- mediated or strictly related to the dermis. There is
increasing evidence that the epidermis plays a crucial role in modulating collagen synthesis,
with inflammation in the overlying epidermis predisposing to cutaneous scarring [198].
Keratinocytes can be activated by tissue injury, and activated keratinocytes are
hyperproliferative. Activated keratinocytes then produce paracrine signals to alert fibroblasts,
endothelial cells, melanocytes, and lymphocytes, as well as autocrine signals targeting
neighboring keratinocytes. The affected cell types, in turn, produce their own autocrine and
paracrine signals, which modify the actions of activated keratinocytes. Keratins K6 and K16
are markers of the keratinocyte active state [199]. Eventually, having responded to the injury,
keratinocytes receive a ``de-activation'' signal and revert to their normal differentiation
pathway. Any dysregulation in this process will cause hyperproliferation of keratinocytes and
may initiate a disease process.
It is also reported that HaCaT cells (an immortal keratinocyte cell line) respond to acute
thermal injury by producing platelet-activating factor (PAF) [189]. Activation of the PAF-
receptor can further stimulate the production of other proinflammatory mediators including
IL-6, IL-8, prostaglandin E2 and TNF-α [200, 201]. PAF feeds back and augments the
inflammatory effect to induce more PAF production [202]. Regarding these functions,
keratinocytes have been described using the term “cytokinocyte,” which describes the
abundance of cytokines produced by keratinocytes [203].
In conclusion, a possible dysregulation of the normal keratinocyte pro-inflammatory
potential might be the primary cause of psoriasis. This dysregulation leads to an exaggerated
cytokine response of keratinocytes upon exposure to certain irritants or insults such as
physical trauma to result in enhanced immune system activation. These abnormalities may be
due to inherited genetic defects.

 Keratinocytes, genetic defects and barrier integrity:

Genetic background is now considered a definite predisposing factor for psoriasis, and
studies are continuing to identify additional genetic determinants as well as the cells affected
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838 Inas Helwa, Meg Gullotto and Wendy B. Bollag

by these variations. At least six different risk loci have been identified (PSORS1-PSORS6)
[204]. The primary genetic susceptibility locus reported is related to the major
histocompatibility complex (MHC) and includes the HLA-Cw6 allele [179]. HLA-Cw6 was
identified in 46% of psoriasis vulgaris cases and 73% of patients with guttate psoriasis.
However, having HLA-Cw6 is not sufficient to develop psoriasis [25]. This confirms that
psoriasis is a multifactorial disease that involves interplay of diverse factors.
Genome–wide association studies have recently identified numerous risk loci outside the
MHC with no direct relation to immune cells. Some of these loci are closely related to the
regulation of normal keratinocyte development [25, 173]. The epidermal differentiation
complex (EDC), which is located on chromosome 1q21 within PSORS4, was proven to be
associated with the late cornified envelope (LCE) complex. LCE3B and LCE3C deletion is
associated with psoriasis in European and Chinese populations [205, 206]. This suggests that
a compromised skin barrier may be a susceptibility factor for psoriasis. Moreover, there is an
association between LCE3C_LCE3B-del and rheumatoid arthritis, which may suggest that a
disruption in the epidermal barrier may further be a predisposing factor for autoimmune
diseases [207, 208]. In addition, psoriatic skin shows altered expression of occludin, an
integral membrane protein that is expressed at the maculae occludentes in the granular cell
layer. This protein is a component of tight junctions and plays a major role in keratinocyte
differentiation. Other defects in tight junction components are also associated with various
skin diseases that involve an inflammatory aspect, such as psoriasis and atopic dermatitis
[209].
The atopic march is a phenomenon in which asthma occurs in association with
preexisting eczema. Individuals carrying one (or more) mutated allele(s) in one of the
epidermal differentiation markers (filaggrin) display atopic march and develop asthma. Since
filaggrin is not expressed in bronchial epithelium, it is believed that asthma is a secondary
consequence of filaggrin deficiency in the skin [210], with the idea that the impaired barrier
function of the skin in these patients allows for sensitization to environmental allergens,
resulting in asthma.
Therefore, in summary, a disruption of the skin barrier may enhance the keratinocyte
reaction to environmental stimuli, which in turn activates dermal inflammatory cells leading
to immune-mediated skin disorders with increased circulating inflammatory mediators
predisposing to other immune-mediated systemic diseases [25, 173, 206-208, 211]. Thus, the
integrity of the epidermal barrier not only plays a passive protective role but is also involved
in other immunological processes. Dealing with psoriasis as a “barrier organ disease” may
open new insights into therapeutic strategies to regulate the response of the patient to
infectious diseases or microbial flora [212].
Another set of genes that is found to be associated with psoriasis and at the same time is
closely related to keratinocyte inflammatory cytokines is human beta-defensin (hBD) [213,
214]. hBDs are antimicrobial peptides that have cytokine-like properties and are encoded by
DEFB genes. DEFB1 (encoding the protein hBD-1), DEFB4 (encoding the protein hBD-2)
and DEFB103 (encoding the protein hBD-3) are expressed constitutively in skin. The beta-
defensins are induced in cultured keratinocytes by cytokines or bacterial lipopolysaccharides,
and minor skin injury or infection may trigger the release of beta-defensins in skin in vivo.
hBD-2, hBD-3 and hBD-4 stimulate keratinocytes to release the pro-inflammatory mediators
IL-8, IL-18 and IL-20, and these cytokines are known to have an important role in the
development of psoriasis [214-216]. It was observed that psoriasis patients tend to have

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higher genomic copy numbers for the beta-defensin genes [213]. These data empower the
hypothesis that an inherited disruption in the skin barrier as a result of a genetic defect may be
a major contributor to the initiation of the disease process. A disrupted skin barrier may even
predispose to systemic inflammatory disorders as in the case of the ‘’atopic march’’. Since
the role of genetics in mediating psoriasis is established, genetically manipulated animal
models are currently being developed in an attempt to study disease progression and to test
treatment options.

 Keratinocytes and calcium homeostasis:

As mentioned earlier calcium is one of the major signaling molecules regulating

keratinocyte proliferation and differentiation. An epidermal calcium gradient is necessary for
regulating homeostasis and impairment of this gradient leads to the development of
dermatological disorders such as psoriasis. In fact, psoriatic lesions show a disturbed calcium
gradient as compared to normal skin from healthy individuals as well as uninvolved skin from
psoriasis patients [8]. This defect presumably favors enhanced proliferation and abnormal
terminal differentiation of keratinocytes. Furthermore, recent studies have shown psoriatic
keratinocytes have an inborn error of calcium metabolism such that the functioning of store-
operated calcium channels and capacitative calcium entry is disturbed [217]. This suggests
that psoriatic keratinocytes are defective and that this defect may be a major reason behind
psoriasis pathogenesis.

 Animal models and keratinocyte-mediated origin of the disease:

Animal models are powerful tools for studying human diseases. However, psoriasis is a
strictly human disease with two reported exceptions in monkeys [23, 218-220]. Accordingly,
all the available animal models of psoriasis are either spontaneous mutations, genetically
engineered, immunologically reconstituted or xenograft models [221]. The unresolved
complex nature of the disease, together with the lack of a recognized naturally occurring
animal model, hampers research into the pathogenesis of and therapeutic approaches to the
disease [222].
However, to date animal models reproducing psoriasis have so far highlighted the role of
keratinocyte defects as a disease-initiating factor independent of T-cells. For example,
STAT3 is involved in cell proliferation and survival and plays an essential role in wound
healing [223]. In addition, STAT3 has been found to be activated in psoriasis. In 2005, Sano
et al. [224] developed a transgenic mouse with constitutively active STAT3 expressed in
basal keratinocytes under the control of the keratin 5 promoter. These mice exhibited
psoriasis-like skin lesions, characterized by keratinocyte hyperplasia, dilated blood vessels
and infiltration of leukocytes, neutrophils and T-cells into the dermis, which represent the
major hallmarks of psoriasis [224].
Another transgenic animal model that supports the role of keratinocytes in the
development of a psoriasis-like skin condition is the conditional JunB/c-Jun double knock-out
mouse [204]. JunB is a component of the AP-1 transcription factor localized in the PSORS6
locus, and c-Jun is the proposed antagonist of JunB [225] Activator protein 1 (AP-1) proteins
play key roles in the regulation of cell proliferation and differentiation [226, 227]. As reported
in healthy human skin [226], JunB was found ubiquitously expressed in all layers of the
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840 Inas Helwa, Meg Gullotto and Wendy B. Bollag

epidermis of unaffected skin of psoriatic patients with highest levels in the basal and spinous
layers. However, in lesional skin of severe psoriasis JunB is highly reduced, whereas c-Jun
was weakly expressed in normal human epidermis, but quite prominent in psoriatic skin. This
shows a strong reduction of the JunB/c-Jun ratios in psoriatic skin, most prominently in the
basal layer [204]. (Nevertheless, this result is somewhat controversial as other studies have
reported opposite findings [228], and more subjects should be examined in order to confirm
the exact role of JunB and/or c-Jun in mediating epidermal proliferation as well as its role in
psoriasis.) Epidermal-specific ablation of these two genes was targeted to the basal layer of
keratinocytes using keratin 14 promoter-driven expression of Cre recombinase. All of the
double mutant mice had scaly plaques and showed the hallmarks of psoriasis involving the
epidermis and dermis, as well as intra-epidermal T-cells with epidermal micro-abscesses.
Moreover, psoriatic arthritis was also observed in these mice. In addition, these epidermal
specific JunB/c-Jun double knock-outs in a T-and B-cell deficient background (Rag-2 knock-
out) [229] or tumor necrosis factor receptor (TNFR)-deficient background exhibited a milder
phenotype, especially regarding arthritis, but the features of the skin lesions were maintained
[230, 231]. Therefore, although there may be a role of immune cells (mainly T-cells and
dendritic cells) in the systemic progression of the disease and the maintenance of the
inflammatory milieu, keratinocytes may be contributing to the disease initiation in the skin in
at least some cases of psoriasis.
An inducible mouse model generated using tetracycline-regulated transcriptional
transactivators driven by the keratin 5 (K5) promoter (K5-tTA and K5-rTA) allows inducible
and conditional expression of genes of interest specifically in the epidermis. Bicistronic
mouse models are then generated by breeding a transgenic K5-tTA or K5-rTA mouse (with
the tTA activating expression in the absence, and rTA in the presence, of doxycycline) with a
transgenic mouse possessing a tetracycline-responsive promoter element-controlled gene
[232]. Using this technology, bicistronic transgenic mice have been generated to allow
inducible tissue-specific overexpression of Tie2, a tyrosine kinase receptor that has been
shown to be up-regulated in psoriasis [233]. This receptor binds angiopoietins and plays a
role in controlling angiogenic remodeling [234]. Two mouse models overexpressing Tie2
have been engineered; in one inducible expression is confined to keratinocytes and in the
other, to endothelial cells. Both of these models exhibited a significant increase in dermal
vasculature consistent with the role of Tie2 in angiogenesis. However, only the epidermal-
specific Tie2-overexpressing mice developed a cutaneous psoriasiform phenotype [235].
These epidermal-specific Tie2 transgenic mice spontaneously develop a psoriasiform skin
phenotype, and skin sections of these mice show histological features of psoriasis with an
inflammatory T-cell infiltrate. Thus, confining Tie2 expression solely to keratinocytes is
sufficient to initiate psoriasis lesions with all the major hallmarks of the disease including the
inflammatory infiltrate. The epidermal-specific Tie2 transgenic model has been used to study
the association between skin inflammation, vascular inflammation and thrombosis. This same
group has also demonstrated the development of aortic root inflammatory lesions in the
epidermal-specific Tie2 mice, with decreased collagen content, and increased elastin
fragmentation and pro-inflammatory cytokines and chemokines in the skin as well as in the
peripheral blood [236]. This article, together with other reports of the high prevalence of
cardiovascular diseases in psoriasis patients [52, 54, 64], highlights the possible association
between psoriasis and systemic diseases, with an emphasis on the possible role of

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keratinocytes not only in initiating the disorder but also in predisposing to systemic
complications.
The xenograft model is another mouse model that provides an alternative approach to
transgenic manipulation. In this model uninvolved non-lesional psoriatic skin or plaque
psoriatic skin is transplanted onto severely immune-deficient mice. Currently, this model is
considered the closest to incorporating the genetic, phenotypic and immune processes of
psoriasis [23]. Investigators initially used athymic nude mice. These mice have vestigial
thymi so they possess no mature T-cells. The mice maintained the psoriatic features of the
transplanted skin for more than 2 months [237]. Later on, severe combined immune-deficient
(SCID) mice were used. These mice have a mutation in the DNA-dependent protein kinase
essential for T-and B-cell development. Therefore, these mice lack both humoral and cellular
immunity. Injecting activated autologous immunocytes under transplanted skin causes
development of psoriasis plaques in skin biopsied from psoriasis patients (PN) but not from
normal donors (NN). Uninvolved skin grafts from psoriatic patients developed features of
psoriasis when grafted into nude mice and injected with pre-activated T-cells [237]. Pre-
activation was performed using IL-2 and bacterial derived antigens (SEB and SEC2) [177].
The data collected from this animal model have shown that T-cells are not able to induce
psoriasis without previous activation by inflammatory mediators [177]. In addition, pre-
activated T-cells from the majority of psoriasis patients (although a small sample size) were
incapable of inducing psoriasis in normal skin from healthy control individuals but only in
uninvolved skin from psoriasis patients, which suggests that keratinocytes from psoriasis
patients behave differently from normal patients. Indeed, calcium metabolism has been
reported to be dysregulated in psoriatic keratinocytes [22].
Thus, it can be concluded that T-cells alone may not be able to initiate psoriasis but are
rather important for disease maintenance. Keratinocytes may be the hidden player behind
disease development.

 Psoriasis and the abnormal epidermal calcium gradient:

Psoriatic lesions exhibit an abnormal localization and profile of calcium distribution [8].
The calcium gradient is known to play a major role in the regulation of epidermal
differentiation and maintenance of skin homeostasis. Normal human and uninvolved psoriatic
epidermis showed increased calcium-containing precipitates in the uppermost stratum
granulosum; in contrast, all psoriatic suprabasal layers displayed heavier than normal
concentrations of calcium, which may provide an explanation of the parakeratosis observed in
psoriatic skin. On the other hand, the basal layer of psoriatic lesions contained less
extracellular calcium than normal; this may also explain the hyperproliferation of the basal
cells in psoriasis [238]. It was reported that cultured psoriatic keratinocytes and psoriatic
plaques have reduced expression of the calcium-sensing receptor and of calcium channels as
compared to normal controls [22]. This finding may underlie the abnormal desquamation and
permeability barrier defect in psoriasis. The difference in the calcium profile between
involved psoriatic skin and uninvolved skin suggest that this defect is an inherited variation
specific for psoriatic skin, and that the skin defect itself initiates the disease process [238].
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842 Inas Helwa, Meg Gullotto and Wendy B. Bollag

9. Emerging Drugs

Based on the evolving concept of the “keratinocyte-mediated disease origin,” emerging

drugs that directly target keratinocytes can be effective while displaying fewer adverse
effects. Moreover, the fact that psoriasis involves an interplay between three different cell
type makes targeting the 3 cell types by a single agent a challenging yet promising therapeutic
goal. A better understanding of disease etiology and pathogenesis may give hope for
developing new drugs or even insight into the function of some conventional drugs, the
mechanism of action of which is not well understood. Fumaric acid esters (FAE) are in the
category of agents that have been used for decades, but their mechanism of action is as yet
unresolved.
FAE have been used to treat psoriasis since 1959, as this drug was first successfully used
by the German chemist Schweckendiek to treat his own psoriatic lesions [239]. A mixture of
dimethylfumarate (DMF) and three salts of monoethylfumarate (MEF) has been licensed in
Germany since 1994 under the product name Fumaderm® and marketed by the
pharmaceutical company Biogen Idec. Fumaderm® is an oral drug available in two strengths
and used for the treatment of moderate to severe psoriasis resistant to topical therapy [240].
Fumaderm® is not licensed in other European countries like the United Kingdom or in
many other countries worldwide including the US. A lack of knowledge about the
pharmacokinetic and pharmacodynamic properties of FAE, as well as the exact mechanism of
action may be the main reason behind the drug’s limited use worldwide [241]. Recently, FAE
have gained interest in the US, especially with increased multicenter studies revealing the
drug’s efficacy and limited safety concerns in most patients [241-251].
Although DMF is the main ingredient of the drug, DMF does not seem to be the active
ingredient. Fumaric acid esters are almost completely absorbed in the small intestine where
DMF is rapidly hydrolyzed by esterases to monomethylfumarate (MMF). DMF has a short
half-life, and it cannot be detected in blood long-term, whereas MMF can be detected for up
to 36 hours with a peak concentration between 5 and 6 hours. In addition, DMF and free
fumaric acid cannot bind to serum proteins while up to 50% of MMF can be found bound to
serum proteins [249]. Taken together, it appears more likely that MMF, a DMF byproduct, is
the active ingredient of Fumaderm®.

THE CONTROVERSIAL ROLE OF DMF

In vitro, DMF seems to be more effective than MMF, especially on immune and
endothelial cells. However, as mentioned above DMF has a very short half-life in vivo as it is
completely hydrolyzed in the intestine. Therefore, in vivo it seems unlikely that DMF is the
active agent.
This controversy has recently been explored by analyzing the levels of the DMF
metabolite [N-acetyl-S-(1,2-dimethoxycarbonylethyl)cysteine or NAC-DMS] in the urine of
human subjects 210-240 minutes after drug intake [252]. As NAC-DMS is detectable in
urine, a possible explanation for DMF’s potential activity in vivo is that a portion of the drug
is not hydrolyzed after oral intake but instead enters the portal circulation and binds to blood
cells such as T-cells and monocytes.

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 Keratinocytes in Psoriasis 843

DMF is known to have the ability to deplete cells of their glutathione (GSH) content
which correlates with an induction of apoptosis [253, 254]. This may explain the role of DMF
in reducing T-cell counts and their potential to produce pro-inflammatory cytokines.
However, more studies are required to validate this hypothesis.

 Mechanism of action:

As mentioned earlier, the exact pharmacokinetic and pharmacodynamic properties of

FAE are not well understood nor is the mechanism of action clear. Nevertheless, studies have
shown interesting effects of these compounds on keratinocytes, immune cells and endothelial
cells [249, 255].

 Fumaric acid esters and keratinocytes:

FAE (DMF, MMF and MEF) induce a transient rise in intracellular free calcium in
cultured human keratinocytes. The increase seems to be the result of a release from
intracellular calcium stores as it was not blocked by chelation of extracellular calcium by
EGTA. This effect may account for the pro-differentiative and anti-proliferative effects of
MMF on keratinocytes and may help to explain, in part, its anti-psoriatic action [256].

 Fumaric acid esters and immune cells:

Fumaric acid esters also have anti-inflammatory effects that may be of benefit in
hindering the psoriatic cytokine network. DMF immuno-modulates cytokine secretion away
from the Th1 cytokine IFN-γ towards the Th2 cytokine IL-10 [255]. Th2 cytokines have
antagonistic effects to inhibit the secretion of Th1 cytokines (IL-2, IL-12, and IFN-γ) that
play a major role in the psoriasis process. Surprisingly, this immune-modulatory effect
occurred only in co-cultures of HUT 78 T cells and psoriatic keratinocytes and was not
observed with control keratinocytes or in unstimulated monocultures. This shows that
psoriatic keratinocytes may have an inherent and distinct inflammatory role in the psoriatic
cytokine network. Moreover, fumaric acid esters may be directly targeting keratinocytes to
modulate their inflammatory potential [26]. Similarly, Litjens et al. [257] have shown that
MMF down-regulates Th1 lymphocyte responses by affecting the polarization of monocyte-
derived dendritic cells. As compared to control dendritic cells, MMF-treated cells favor Th2-
related production of IL-4 and IL-10 at the expense of IFN-γ. This effect was shown in co-
cultures of MMF-treated dendritic cells and naïve Th lymphocytes. The mechanism
underlying this effect is not fully elucidated. Another group has previously shown that DMF
is an inhibitor of cytokine-induced nuclear translocation of nuclear factor-kappa B1 (NFKB1)
[258]. NFKB1 is a major regulator of immune responses, inflammation, cell proliferation, and
apoptosis. Thus, NFKB1 inhibition could also account for the anti-inflammatory effect of
DMF’s hydrolysis byproduct MMF. Nibbering et al. [259, 260] have shown that MMF has
antagonistic effects on granulocytes. The authors have speculated that this action is initiated
by binding of MMF to a pertussis toxin (PTX)-sensitive receptor on the plasma membrane of
granulocytes. This receptor may be present on keratinocytes. However, they could not explain
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844 Inas Helwa, Meg Gullotto and Wendy B. Bollag

the mechanism by which MMF mediates intracellular calcium release or modulates the
inflammatory potential of keratinocytes.
In 2008 Tang et al. [261] provided evidence that MMF is an agonist of the G-protein
coupled receptor GPR109A.They also showed that the expression of this receptor is increased
in psoriatic lesions (although the sample size was small). This receptor is expressed on
immune cells such as Langerhans cells of the skin and granulocytes. It is also expressed on
keratinocytes, retinal pigment epithelial cells, hepatocytes, colon epithelial cells and
adipocytes. GPR109A is thought to play an important role in these cells and hence may be
mediating the pharmaceutical actions of MMF, especially in the skin. However, the exact
function of this receptor is not yet well understood. This receptor has recently been renamed
the hydroxy-carboxylic acid (HCA) receptor 2 (HCA2) [262]. One of the major challenges
facing a complete understanding of MMF is to understand the relationship between HCA2 in
the skin and the anti-psoriatic effects of MMF [263].

 Fumaric acid esters and endothelial cells:

The third important player in the psoriatic process is the endothelial cells, which serve as
a pro-angiogenic cell type. DMF was found to have an inhibitory effect on the proliferation,
differentiation and migration of endothelial cells and hence inhibits angiogenesis. Treating
endothelial cells with DMF leads to impaired lymphocyte rolling and a lack of firm adhesion
to endothelial cells due to down-regulated adhesion molecule expression.
Thus, the interaction between human lymphocytes and endothelial cells is reduced. This
effect interferes with a central pathogenic event in psoriasis. From this perspective, DMF may
be regarded as an indirect antagonist of TNF-α, which is a key cytokine of the psoriatic
cytokine network [264]. DMF also has an anti-angiogenic effect by inhibiting tubal formation
as well as migration of endothelial cells [265]. These functions are essential for the formation
of new blood vessels (neo-angiogenesis), a key step in psoriasis pathogenesis.
These results taken together suggest that fumaric acid esters (Fumaderm®) may be a
promising drug targeting the three key steps in developing a psoriatic lesion; namely,
hyperproliferation of keratinocytes, inflammation and activation of the immune system and
neo-angiogenesis by the vasculature. More studies are required to complete the complex and
incompletely understood puzzle of the mode of action of fumaric acid esters. Exploring more
into the mechanism of this drug may expand its registration to other countries and may even
extend its use beyond the treatment of psoriasis to include other similar inflammatory
conditions. Preliminary results certainly offer evidence of its potential efficacy in a multitude
of dermatological as well as non-dermatological diseases such as atherosclerosis [255, 266,
267].

CONCLUSION
The skin is a vital organ that plays a major role in the health and well-being of humans.
Any dysregulation in normal epidermal homeostasis will lead to skin diseases that will
severely affect the patient’s quality of life and may predispose to other systemic conditions.
Psoriasis is a common skin disorder that affects 1-3% of the general population worldwide. It

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 Keratinocytes in Psoriasis 845

is a lifelong multifactorial inflammatory hyperproliferative immune-mediated disorder with

onsets of remissions, relapses and exacerbations and affecting skin, nails and joints. Although
it is simple for dermatologists to diagnose psoriasis, its treatment is considered a real
challenge. The major obstacle facing the treatment is the disease’s unresolved primary
etiology.
The disease process involves interplay among three major cell types: keratinocytes,
immune cells and endothelial cells. This makes it difficult to identify the primary cell type
responsible. Identification of the initiating cell type in the disease process may allow the
development of a selective treatment option that targets the primary cause of the disease
rather than being symptomatic.

Figure 1. Chronic Plaque Psoriasis (Psoriasis Vulgaris). Illustrated is the appearance of chronic plaque
psoriasis (right panel) as well as the common distribution pattern of the lesions (left panel).

Figure 2. Inverse Psoriasis. Illustrated is the appearance of inverse psoriasis (right panel) as well as the
common distribution pattern of the lesions (left panel).
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846 Inas Helwa, Meg Gullotto and Wendy B. Bollag

Figure 3. Guttate Psoriasis. Illustrated is the appearance of guttate psoriasis (right panel) as well as the
common distribution pattern of the lesions (left panel). Note the tear-drop shape of the lesions.

Figure 4. Erythrodermic Psoriasis. Illustrated is the appearance of erythrodermic psoriasis (right panel)
as well as the common distribution pattern of the lesions (left panel). This type of psoriasis can
represent a dermatologic emergency.

Figure 5. Pustular Psoriasis. Illustrated is the appearance of pustular psoriasis (right panel) as well as
the common distribution pattern of the lesions (left panel). Note that the pustules are sterile.

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 Keratinocytes in Psoriasis 847

Figure 6. Cross-Talk between Key Cells and Mediators of the Psoriatic Cytokine Network. This figure
illustrates the interaction between keratinocytes, immune cells and endothelial cells, which mediate the
initiation, progression and maintenance of the inflammatory process. A stimulus such as trauma or an
infection can initiate an inflammatory response in a genetically susceptible person, for instance, those
individuals with absent or mutant epidermal differentiation complex (EDC) genes such as late cornified
envelope 3 (LCE3). Keratinocytes are active contributors capable of initiating an immune response
through the production of various cytokines, chemokines and anti-microbial peptides. These pro-
inflammatory mediators trigger the resident immune cells, which in turn, produce their own cytokines
including iterleukin-12 (IL-12), IL-23 and vascular endothelial growth factor (VEGF). IL-12 and IL-23
stimulate the differentiation of T-cells into type 1 and type 17 helper T-cells, respectively. VEGF
stimulates a tissue-angiogenic response as well as recruitment of neutrophils. Th1 and Th17 produce
additional cytokines, which feed back on the keratinocytes, inducing further activation and production
of cytokines. Moreover, activated keratinocytes produce VEGF and exhibit increased expression of
intercellular adhesion molecule-1 (ICAM-1), which plays a role in angiogenesis and promotes
neutrophil migration into the epidermis. All of these events amplify the immune response and stimulate
epidermal remodeling with altered differentiation and increased proliferation. Together with neo-
angiogenesis, these changes (keratinocyte hyperproliferation and aberrant differentiation, neutrophil
infiltration and angiogenesis) represent the three major hallmarks of psoriasis. This cross-talk together
with the dynamic flow of cytokines converts the pre-psoriatic skin into a psoriatic plaque. In addition,
the interplay between the key cells and inflammatory mediators creates a vicious cycle, with
keratinocytes playing a key role in the initiation, progression and maintenance of the disease process
[27, 32, 42, 84, 159, 269].
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848 Inas Helwa, Meg Gullotto and Wendy B. Bollag

There is increasing evidence that keratinocytes play a pivotal role in the induction of
psoriasis. As a matter of fact, keratinocytes can release an array of pro-inflammatory
mediators in response to multiple stimuli. Accordingly, there is an established inflammatory
crosstalk between keratinocytes and T-cells such that accurate detection of the origin of the
disease may be extremely challenging. In the meantime, there is no drug that can cure
psoriasis, and this is attributed to lack of knowledge about the complex etiology of the disease
as well as lack of a naturally occurring or widely accepted animal model that recapitulates the
complexity of the disease process. Most of the drugs used currently target T-cells and include
methotrexate, cyclosporine and alefacept. Other drugs target different pro-inflammatory
mediators and cytokines such as ustekinumab, which targets IL-23, and infliximab, which
targets TNF-α [268].
A major problem with these immuno-suppressive drugs in the treatment of psoriasis is
the fear of fatal infections possibly associated with their prolonged use. Thorough knowledge
of the exact nature of psoriasis and the major cell type initiating the disease may result in the
development of more selective and hence more efficacious drugs with fewer side effects. In
addition, identifying the exact role keratinocytes in the disease process may even lead to the
development of safer treatment options. Drugs targeting both the immune pathway and
keratinocytes, like Fumaderm® (fumaric acid esters), show promise. Fumaderm® has shown
good long-term efficacy and tolerability during its use in Germany since 1994 and this drug in
fact, has shown minimal adverse effects. Its exact mechanism is still unclear although it is
suggested to have effects on all of the cell types contributing to the disease, namely,
keratinocytes, immune cells and endothelial cells. Animal models, wider genetic analyses and
development of novel efficacious drugs have revealed much about the disease pathogenesis in
psoriasis. More studies are required to solve the controversies and bring together all the clues
to uncover the mystery behind this multifactorial complex disease and to enrich the
therapeutic armamentarium with more selective, safer and effective treatment options.

Table 1. Treatment Options for Psoriasis

Treatment Target/Mechanism Benefits Drawbacks

of Action
Topical Induction of anti- Availability in diverse Skin atrophy, suppression of
Corticosteroids inflammatory and forms, potency and the HPA, possible rebound
other effects through efficacy, safety upon cessation of treatment,
GR possible compliance issues
Topical Coal Tars Unknown Efficacy may be Aesthetics (odor and
similar to staining), irritation,
corticosteroids and increased sun sensitivity,
Vitamin D analogs possible carcinogenicity,
possible compliance issues
Topical Vitamin D Induction of anti- Efficacy, safety, Irritation, possible (although
Analogs proliferative, pro- targeting of both unlikely) calcemic effects
differentiative effects keratinocytes and with application to large
in keratinocytes and immune cells surface area, possible
immunomodulation compliance issues
through VDR

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Treatment Target/Mechanism Benefits Drawbacks

of Action
Topical Anthralin Largely unknown, Possibility of long- Aesthetics (staining),
(Dithranol) prevention of T-cell lasting improvement irritation, unknown
activation, induction of in symptoms mechanism, possible
keratinocyte apoptosis compliance issues
Topical Keratolytics Improvement of Improvement of Purely symptomatic
(e.g., salicylic acid) desquamation by symptoms and approach, toxicity when
reducing cohesion and appearance of applied over a large surface
pH to increase psoriasis, enhance- area, possible compliance
hydration of plaques ment of penetration of issues
other medications
Topical Emollients Increase in moisture Safety (safest Purely symptomatic
retention to soften treatment option) approach, possible
plaques compliance issue
Topical Retinoids Regulation of Efficacy comparable Irritation, possible safety
proliferation, to corticosteroids and concerns in children or
differentiation and Vitamin D analogs, during breast-feeding,
desquamation of lack of rebound upon possible teratogenicity,
keratinocytes through cessation of treatment possible compliance issues
RAR and RXR
Topical Calcineurin Inhibition of Safety (use in children Possible increased risk of
Inhibitors (e.g., calcineurin (a protein and in facial and lymphoma and skin cancer,
Tacrolimus and phosphatase) to intertriginous areas), possible compliance issues
Pimecrolimus) prevent T-cell tolerability
activation
Phototherapy Induction of anti- Efficacy, safety (use Erythema, pruritus, burning,
(e.g., PUVA, BB- proliferative effects in of BB- and NB-UVB dry skin, photoaging,
UVB, NB-UVB) keratinocytes, anti- in pregnancy) possible increased risk of
inflammatory effects in skin cancer, time required
T cells, reduction in for treatment
Langerhans cells
Systemic Antagonism of folate Efficacy, targeting of Nausea, anorexia, fatigue,
Methotrexate synthesis, inhibition of both keratinocytes and malaise, liver toxicity,
proliferation of immune cells, ease of muco-cutanous ulceration,
keratinocytes and treatment increased risk of skin cancer
immune cells, anti- and infection, contra-
inflammatory effects indication during pregnancy
Systemic Inhibition of High efficacy and Hypertension,
Cyclosporine calcineurin to prevent rapid onset of action, nephrotoxicity, increased
T-cell activation ease of treatment, risk of infection and possibly
targeting of skin cancer
keratinocytes and
immune and
endothelial cells
Systemic Acitretin Largely unknown, Ease of treatment, lack Hyperlipidemia,
(oral Vitamin A inhibition of Th1 and of immunosuppression mucocutaneous lesions,
derivative) Th17 immune (use in immune- teratogenicity in pregnancy,
pathways compromised patients) contra-indication during
breast-feeding
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850 Inas Helwa, Meg Gullotto and Wendy B. Bollag

Table 1. (Continued)

Treatment Target/Mechanism Benefits Drawbacks

of Action
Systemic Largely unknown, Ease of use, Unknown mechanism, lack
Fumaderm® inhibition of tolerability, targeting of knowledge concerning
(fumaric acid esters) keratinocyte of keratinocytes and pharmacokinetics and
proliferation and immune and pharmacodynamics
angiogenesis, endothelial cells
immunomodulation
Systemic Biologics Inhibition of TNF- or Efficacy, generally Increased risk of infection
(e.g., Etanercept, IL-12/IL-23 signaling good compliance and possibly lymphoma
Ustekinumab)
Abbreviations: GR, glucocorticoid receptor; HPA, hypothalamic-pituitary axis; VDR, vitamin D
receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; PUVA, psoralen (a
photosensitizer) with ultraviolet A therapy; BB-UVB, broad-band ultraviolet B; NB-UVB, narrow-
band ultraviolet B; Th1, type 1 T helper cells; Th17, T helper 17 cells; TNF-, tumor necrosis
factor-; IL-12, interleukin-12; IL-23, interleukin-23.

Table 2. Evidence for a Keratinocyte- or Immune-Mediated Etiology of Psoriasis

Keratinocyte Etiology Immune Cell Etiology

of Psoriasis of Psoriasis
Some susceptibility loci expressed by Some susceptibility loci expressed by immune cells
keratinocytes (e.g., corneodesmosin) (e.g., HLA-Cw0602)

Delayed barrier recovery with stress (and Disease initiated and/or exacerbated by bacterial
psoriasis amelioration by occlusion) suggests a infection and HIV (but no autoantibodies detected)
possible barrier involvement

Epidermal expression of certain growth factors Disease reproduced in mouse psoriatic uninvolved
(e.g., amphiregulin, VEGF) or other genes (e.g., skin xenograft models by injection of pre-activated
constitutively active Stat3 ) in transgenic animals T cells
mimics disease

Epidermal-specific knockout of some genes in Transgenic expression of certain immune

transgenic animals mimics disease (e.g., c-Jun constituents (e.g., B27/hb2m) mimics disease
and JunB)

Disruption of calcium gradient detected in Association of certain other immune-mediated

psoriatic lesions diseases with psoriasis (e.g., Crohn’s)

Intrinsic changes in calcium metabolism Biologic therapies targeted at the immune system
observed in psoriatic keratinocytes in vitro used successfully to treat psoriasis

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 Keratinocytes in Psoriasis 851

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 36

TYPES, TRIGGERS AND TREATMENT STRATEGIES

OF PSORIASIS

Spyridoula Doukaki and Maria Rita Bongiorno*

Department of Dermatology, University of Palermo, Palermo, Italy

ABSTRACT
Psoriasis is a common chronic, immune mediated, inflammatory disease,
characterized by periods of exacerbation and remission. Patients with psoriasis have a
significantly impaired quality of life; the disease is associated with substantial burden in
terms of disability or psychosocial stigmatization. Moreover, in a percentage of patients
that varies between 5% and 42%, depending on the population studied, psoriatic arthritis
may occur.
Psoriasis is found worldwide but the prevalence varies among different ethnic
groups. It has a strong genetic component but environmental factors such as β-haemolytic
streptococcal infection, HIV, stress, and drugs, including β blockers, antimalarials and
lithium can play an important role in the presentation of disease.
Onset may occur at any age, although two peaks in incidence have been described,
one in early adulthood, and the other between the ages of 55–60 years.
Skin disease can be highly variable in morphology, distribution, and severity. Plaque
type psoriasis, characterized by papulosquamous plaques well-delineated from
surrounding normal skin, is the most common form. It accounts for 80% of cases.
However, the morphology can range from small tear shaped papules (guttate psoriasis) to
pustules (pustular psoriasis) and generalised erythema and scale (erythrodermic
psoriasis). In addition, these different forms of psoriasis may be localised or widespread.
Approximately 80% of patients with psoriasis have mild to moderate disease, whereas
20% have moderate to severe disease. About 25-50% of patients with psoriasis have
distinctive nail changes related to the disease. Psoriatic nail disease occurs most
commonly in patients with psoriatic arthritis.
Patients with mild disease can be treated with topical agents while systemic agents,
including cyclosporine, methotrexate, and acitretin, or phototherapy are usually required

*
Corresponding author: Bongiorno Maria Rita, Department of Dermatology, University of Palermo, Via del Vespro
131, 90123, Palermo, Italy. Tel +39 091 6554 001; Fax +39 091 6554 022; E-mail: [email protected]
 Free ebooks ==> www.Ebook777.com
872 Spyridoula Doukaki and Maria Rita Bongiorno

in patients affected by moderate to severe psoriasis. With our increased understanding of

the immunopathogenesis of psoriasis multiple biologic agents, which target specific
molecules necessary for the development of psoriatic lesions, have been introduced
during the past years.

INTRODUCTION
Psoriasis is a chronic, disabling, relapsing and remitting, immune-mediated, multisystem,
inflammatory disease characterized by an extremely increased rate of epidermal turnover,
vascular changes involving elongation and dilatation of capillaries in the papillary dermis,
and migration of activated neutrophils and T lymphocytes into the dermis and the epidermis.
In 5%−42% of the patients, the skin findings are associated with inflammatory arthritis
(psoriatic arthritis, PsA) which is characterized by synovitis, enthesitis, dactylitis and
spondylitis and is usually negative for rheumatoid factor [1-2-3].
Pathogenesis is not fully elucidated but multiple genetic, immunologic, and
environmental factors are thought to be involved and act in an integrated way with
overproduction of proinflammatory cytokines, including interleukin (IL)-2, IL-12, IL-17, IL-
21, IL-22, IL-23, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-α, adhesion
molecules, growth factors like nerve growth factor (NGF) and neuropeptides [ 3].
Psoriasis affects millions of people across the world with prevalence rates varying
between countries and races. The highest psoriasis prevalence was observed in the Arctic
Kasach'ye population (11.8%), whereas the lowest rates of psoriasis were seen in certain
African (<0.1%) and Samoan populations (0%). In the USA, it is estimated that
approximately 2% of the population is affected by psoriasis. Similar prevalence values have
been obtained in Europe, with the exception of slightly higher values seen in Norway and
the Faeroe Islands. Caucasians are more frequently affected than any other racial or ethnic
group. In a recent US population-based survey, the prevalence of psoriasis in African
Americans was 52% lower compared to Caucasian Americans. Studies investigating Asian
populations also report lower prevalence rates than those observed in Caucasians (0.1% –
0.3%) [4-5-6]. Most studies have found that males and females are affected equally [5].
Psoriasis can clinically present at any age; it has been reported at birth and in people of
advanced age. Approximately 75% of patients experience their reported onset before the age
of 40 years [6].
In recent years, much emphasis has been placed on age at onset in discriminating
between different types of psoriasis. It has been suggested that age at onset has a bimodal
distribution: type I psoriasis, occurring before the age of 40 years, and type II, presenting later
with a peak at 55-60 years. This has been taken as evidence for etiologic heterogeneity. Early
onset of disease (type I psoriasis) is associated with female gender and affected first-degree
relatives. Patients more likely experience severe and recurrent disease, a higher incidence of
guttate psoriasis, nail involvement, and greater psychosocial impact. In comparison, patients
who develop late onset (type II) psoriasis, usually have a milder and more stable course, lack
affected relatives, but more frequently experience palmoplantar pustulosis. In addition, strong
associations have been reported with human leucocyte antigen (HLA)-Cw6 in patients with
early onset, compared with later onset of psoriasis [4-5-7].

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 Types, Triggers and Treatment Strategies of Psoriasis 873

TRIGGERING FACTORS
The severity of psoriasis show wide variations, from minor localized patches to
generalized skin involvement; triggering factors can lead to different clinical manifestations
in predisposed individuals [8]. Epidemiological studies have identified a wide range of factors
incriminated in precipitating and worsening psoriasis, including infections, drugs, cutaneous
trauma, alcohol, cigarette smoking, stressful life events [9].

Infections

A number of epidemiological studies suggest that various microorganisms can trigger or

exacerbate psoriasis. The strongest association is that of Streptococcus pyogenes. The
resulting psoriasis is commonly of the guttate pattern, but existing plaque psoriasis may also
be exacerbated. There is similar evidence to suggest that guttate flares in chronic plaque
psoriasis are also triggered by streptococcal throat infections.
Two thirds of patients with guttate psoriasis, have had an acute sore throat 1 to 2 weeks
before the eruption, positive throat swabs and serological evidence of a recent streptococcal
infection. Furthermore, a small number of case reports, have shown induction of guttate
psoriasis as a result of an infection of perianal skin by Streptococcus pyogenes.
It has been shown that guttate psoriasis is induced not only by Lancefield group A β-
hemolytic streptococci but also by groups C and G. Group A, C and G streptococci express
one of several antigenically distinct M proteins on their surface; however, no association has
yet been found between particular M serotypes and the triggering of guttate psoriasis by
streptococci. Streptococcal M protein is a virulence factor characterized by repeats of α-
helical coiled-coil structures and several tandem repeats of nearly identical amino acid
sequences. Epithelial keratins share this structure with M protein and an extensive amino acid
homology has been reported between M protein and keratins 16 and 17. Neither is present in
normal epidermis, but both are markedly up-regulated within psoriatic lesions. It has been
postulated that cross-reaction between M protein and human epidermal keratin leading to skin
autoimmunity may play a role in the pathogenesis of psoriasis. A possible mechanism was
suggested when it was discovered that M protein is a T cell–activating superantigen that bind
to the T cell receptor via the variable region of the β chain (Vβ) and also induce the
expression of a skin homing receptor, cutaneous lymphocyte-associated antigen (CLA) on T
cells. Indeed, an increased frequency of interferon-γ–producing T cells specific for keratin-
derived peptides that share sequences with streptococcal M proteins has been reported in the
peripheral blood mononuclear cells of psoriatic patients [10-11-12].
Viral infections may also play a role in the etiology of psoriasis, as severe exacerbation of
psoriasis can be a manifestation of human immunodeficiency virus (HIV) infection.
Interestingly, the prevalence of psoriasis in HIV infection is similar of that of the general
population, indicating that the infection is not a trigger for psoriasis but rather a modifying
factor. The course of the disease is different in immunosuppressed patients. Psoriasis is
increasingly more severe, worsening with the progression of immunodeficiency and can remit
in the terminal phase. This paradoxical exacerbation may be due to loss of regulatory T cells
and increased activity of CD8 T-cell subset. Psoriasis can be treated with combination
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874 Spyridoula Doukaki and Maria Rita Bongiorno

antiretroviral therapy, implicating a possible direct role for the HIV virus in disease
exacerbation. HIV infected immune cells could release the neuropeptide substance P which
can stimulate keratinocyte proliferation and modulate various immune and inflammatory
functions mediating psoriasis exacerbation [12].
Finally, psoriasis may appear at the sites of chicken pox or herpes zoster, but this is likely
to represent a Koebner reaction, rather than a specific effect of the virus [12].

Drugs

There is a growing list of drugs that may result in exacerbation of pre-existing psoriasis,
in induction of psoriatic lesions on clinically uninvolved skin in patients with psoriasis, or in
precipitation of the disease in persons without family history of psoriasis or in predisposed
individuals [13]. A distinction has been proposed between drugs influencing the onset of
psoriasis and those causing relapse of the disease. However, this distinction is to some extent
artifactual since the real onset of a condition like psoriasis, which may have a long subclinical
phase, may be difficult to define [14].
In view of their relationship to drug-provoked psoriasis, therapeutic agents may be
classified as drugs strongly related to psoriasis, drugs about which there are considerable but
insufficient data to support the induction or aggravation of the disease, and drugs that are
occasionally reported to be associated with aggravation or induction [13].
The most common drugs encountered to induce psoriasis are β-blockers. They have been
reported to aggravate psoriasis and to induce a psoriasiform dermatitis, which occurs more
frequently in patients with no past or family history of the disease, questioning whether this is
true psoriasis. Moreover, β-blockers have been reported to transform plaque psoriasis into
pustular psoriasis. Finally, topical application of timolol, a β-blocker used in the treatment of
chronic open angle glaucoma was reported to induce psoriasis and to transform psoriasis
vulgaris into psoriatic erythroderma, probably through the passage of timolol into the
systemic circulation via the conjunctiva, nasal mucosa, or uveal circulation.
Latency periods vary from 1 to 18 months after starting to take the drug. In most patients,
the psoriasiform eruption clears after several weeks of discontinuing the medication.
Reexposure with oral challenge resulted in recurrence within a few days. The drug-
aggravated psoriasis improves on discontinuing medication, but it did not clear completely.
The mechanism by which β-blockers might induce or exacerbate psoriasis is largely
unknown. It has been postulated that β-adrenergic receptors are present in the skin and are
blocked by β-blockers resulting in a decrease of cellular cyclic adenosine monophosphate
(cAMP) levels that leads to a decrease in intracellular calcium and consequently increased
cellular proliferation and lack of differentiation as seen in psoriasis [12-13-15].
Psoriasis is the commonest skin side effect of litium. It may induce a new onset of
psoriasis; exacerbate pre-existing psoriasis; or cause nail changes, psoriasis pustulosa, and
erythroderma. The incidence of exacerbating or inducing psoriasis due to lithium treatment
has been reported to range from 3.4 to 45%. The true relationship between lithium and onset
of new disease has been questioned. It has been reported that the induction of psoriasis
without preexisting disease is less common than exacerbation of existing disease. Not all
patients with preexisting psoriasis have a flare when starting lithium, and psoriasis is not

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 Types, Triggers and Treatment Strategies of Psoriasis 875

considered to be a contraindication to lithium treatment. These observations imply that host

factors may influence the induction or aggravation of psoriasis with lithium.
The latency period between starting lithium and the exacerbation of psoriasis is relatively
long, on average 20 weeks. In lithium-induced psoriasis, the latency period is longer, on
average 48 weeks. Lithium provoked pustular psoriasis on the palms and soles has a relatively
short latency period. Psoriasis that has flared with lithium appears to be more resistant to
standard treatment modalities [12]. The mechanism by which lithium induces or exacerbates
psoriasis is not exactly known but its role in modulating secondary messenger systems such
as adenyl cyclase and inositol monophospate mediated pathways resulting in the alteration in
the calcium homeostatis have been reported. Lithium, also has mitogen properties and acts by
blocking cell differentiation. Recent studies have shown that is involved in the dysregulation
of proinflammatory cytokines by triggering the secretion of transforming growth factor
(TGF)-a, IL-2, IL-6, and IFN- γ. The increased production of these cytokines could contribute
to the deterioration of psoriasis [12-13].
In contrast to lithium and β-blockers, antimalarials do not induce psoriasis de novo, but
only trigger already existing psoriasis It is estimated that up to 42% of patients with psoriasis
would develop an exacerbation of their disease following antimalarial therapy. Psoriasis is
now considered a contraindication for the use of antimalarials. It has been suggested that the
chemical structure of the antimalarial drugs is very similar to that of dansylputrescine, a
potent transglutaminase inhibitor in the skin. Transglutaminase is thought to influence cellular
proliferation. Therefore, inhibition of this enzyme probably triggers psoriasis [12-13].
The evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) exacerbate or induce
psoriasis is not as strong as with the drugs discussed above. Also, induction of generalized
pustular psoriasis have been associated with the use of NSAIDs. Considering the widespread
use of NSAIDs, both topical and systemic, the experience of many dermatologists does not
suggest that NSAIDs have any obvious adverse effect on psoriasis. When psoriasis is
adversely affected by NSAIDs, effects are experienced within 2 weeks. Arachidonic acid can
be metabolized to form either prostaglandins via the cycloxygenase pathway or leukotrienes
via the 5-lipoxygenase pathway. NSAIDs inhibit the metabolism of arachidonic acid by the
cycloxygenase pathway, leading to an accumulation of leukotrienes, which have been
postulated to aggravate psoriasis [12-13].
There are many other drugs that have been incriminated in the exacerbation or induction
of psoriasis. In many instances, the associations are simply case reports. The influence of
antibiotics on the course of psoriasis is controversial. Tetracyclines (doxycycline, penicillin,
amoxicillin, and ampicillin) been implicated in psoriasis. It has to be remembered that
infections, particularly those caused by streptococci, which may have prompted the use of
antibiotics, can themselves precipitate or aggravate psoriasis. In addition to lithium, other
psychotropic drugs including fluoxetine, carbamazepine, and olanzapine, were found to be
associated with psoriasis in a case-control study.
Angiotensin-converting enzyme (ACE) inhibitors, also have been implicated in a case-
control and case-crossover study. A recent study suggested that patients with an ACE gene
genotype of low ACE activity, which seems often to occur in patients with familial psoriasis,
were more susceptible to the onset of psoriasis [15]. Other drugs with a weak association
include digoxin, clonidine, and amiodarone.
Recently, the induction or exacerbation of psoriasis by cytokine therapy has also been
reported. Interferons (IFNs) have been increasingly used to treat a wide array of diseases.
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Recombinant DNA-derived IFN-α treatment and exacerbation/onset of psoriasis in patients

with metastatic renal carcinoma has been reported. Such observations and the report of
induction or exacerbation of psoriasis by recombinant IFN-γ stresses their role in this disease.
However, the impact of IFN therapy in psoriasis induction/exacerbation is still a subject of
research and the exact mechanism of action remains obscure.
Acute withdrawal of systemic or potent topical corticosteroids has been reported to
induce psoriatic erythroderma or generalized pustular psoriasis [13].
Tumor necrosis factor (TNF)-α antagonists are successfully utilized in the treatment of a
wide variety of chronic autoimmune diseases and inflammatory conditions, including
psoriasis. Paradoxically, TNF-α inhibitors may induce or aggravate psoriasisform eruption
and palmoplantar pustular psoriasis. The incidence of TNF-α inhibitor- induced psoriasis is
2.3 to 5%. The underlying pathophysiologic mechanisms responsible for this phenomenon
remain elusive. A treatment-induced cytokine imbalance may be involved: inhibition of TNF-
α can induce overexpression of cutaneous INF-α, which in turn predisposes to psoriasis [16].
Finally, imiquimod cream, an immune response modifying drug which demonstrates
potent antiviral and antitumorous activity, has been reported to exacerbate psoriasis, affecting
both treated sites, and distant previously unaffected skin [17-18].
Current evidence suggests that imiquimod, as a selective toll-receptor (TLC) 7 agonist,
exert its immunomodulatory properties principally through the induction of a cascade that
leads to the production of cytokines such as IFN-α, IFN-γ and IL-12, which promote a Th-1
immune response. These cytokines are capable of inducing psoriasis [17]. Systemic
absorption of topical imiquimod can occur and may account for psoriasis occurring at sites
distant from the treated areas [19].

Cutaneous Traumas

In about 30% of patients, lesions are reported to have appeared at a site of skin injury.
This phenomenon was first noted by a physician named Koebner in 1872, and it is now
known as the Koebner phenomenon. It is also called the isomorphic response because it has
the same configuration as the injury. Provoking factors include not only physical trauma, but
also burns, friction, insect bites, surgical incision, tattoos, allergic and irritant reactions, and
radiation exposure. The period from injury to psoriatic lesion development is generally
between 10 and 20 days, but may range from 3 days to 2 years [20].
There are no anatomical site of preference for Koebnerization. It may involve either the
classic areas of psoriatic involvement (scalp, elbows, knees) or involve regions usually spared
such as the face. Finally, patients who reacted to one experimental stimulus, would react to
all. Conversely, lack of a positive response to a known Koebner inducing stimulus predict
negative response to other stimuli. This positive and negative reactivity was termed “the all or
none phenomenon.” However, patients may transfer from a state of Koebner positivity to
negativity, and vice versa. The Koebner phenomenon is an indicator of disease activity and
may have a prognostic value. Despite its common occurrence, the specific mechanisms
underlying it have not been elucidated; cytokines, stress proteins, adhesion molecules or
autoantigens may be involved. For the phenomenon to occur, both the epidermis and the
dermis need to be involved in the injury [7-20-21].

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Stress and Psychosomatic Factors

Among risk factors, psychological stress or an abnormal response to stressors are also
believed to play a role in psoriasis. Although many patients believe that stress might cause or
exacerbate their psoriasis, this idea has received only preliminary support from a small
prospective and some controlled retrospective studies [22]. Proportion of patients with
psoriasis in whom stress plays an important role as a trigger vary from 40 to 80% and is
difficult to estimate, because of the problems in defining stress and because diverse stressful
situations affect individuals differently, depending on their personality. No direct correlation
has been observed between the severity of stress and time to onset or exacerbation of
psoriasis, possibly because of considerable individual variation in coping skills and the
importance of the emotional meaning rather than the intensity of the life events.
It is still unknown how psychic stress affects the first occurrence or the exacerbation of
psoriasis. The stress reaction in patients is probably mediated by the hypothalamic–pituitary–
adrenal relationship with immunologic effects. Psychological stress causes phenotypic
changes in circulating lymphocytes and is regarded as an important trigger of the T-helper 1
cell-polarized inflammatory skin diseases as psoriasis [13].

Alcohol

While alcohol has been suspected to act as a promoting factor in various medical
conditions, the actual data related to its precise role in psoriasis are somewhat contradictory;
it is still unknown whether alcohol misuse represents a true risk factor or merely is an
epiphenomenon or consequence of psoriasis. Nevertheless, many authors found both a higher
alcohol intake and an increased prevalence of alcoholism among psoriatic patients. Regarding
prognosis, mortality from alcohol-related causes were significantly higher in patients with
psoriasis than in normal controls. In particular, the relative risk of alcohol-related mortality
was 4.46 for men and 5.60 for women. It is well known that psoriasis has a relevant impact on
patients’ quality of life. The psychological and social difficulties, which include
stigmatization, embarrassment, social inhibition and vulnerability, might increase the risk of
psychiatric pathologies and substance abuse. Thus, alcohol assumption might represent a
stress response, elicited by the patient in order to cope with the disease. A number of studies
show the possible influence of alcohol on the severity and phenotype as well as the course and
prognosis of psoriasis, concluding that alcohol can not only trigger psoriasis but also a
drinking habit appears to exacerbate a preexisting disease. Heavy drinkers have a tendency to
develop more severe, more extensive, and more inflamated manifestations [23-24].
The exact molecular mechanisms by which alcohol triggers or exacerbates psoriasis are
not fully elucidated. Recent evidence suggests that alcohol misuse may induce immune
dysfunction with resultant relative immunosuppression, but acute and chronic alcohol
consumption has opposite effects on inflammatory cell activation. It seems that acute alcohol
exposure is inhibitory, whereas chronic alcohol exposure is stimulatory in inflammatory cell
response. Alcohol may also enhance the production of inflammatory cytokines and cell cycle
activators, such as cyclin D1 and keratinocyte growth factor, which could lead to epidermal
hyperproliferation. Additionally, the increased susceptibility to superficial infections
commonly observed in alcoholics, such as those caused by Streptococcus and trauma, may be
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implicated in the development of psoriasis. Moreover, the misuse of alcohol in patients with
psoriasis has been shown to be associated with decreased response to treatment [25-26].

Smoking

Significant epidemiological evidence suggests a link between cigarette smoking and

psoriasis. Furthermore, a significant association between the intensity and duration of
smoking and clinical severity of psoriasis also has been reported; smoking more than 20
cigarettes per day confers twice the risk of more severe involvement. There are several
speculated mechanisms by which cigarette smoke may affect the immunopathogenesis of
psoriasis: it increases oxidative damage, promotes inflammatory changes, and enhances
expression of genes associated with psoriasis. Smoking initiates formation of free radicals
that stimulate cell signalling pathways active in psoriasis including mitogen-activated protein
kinase (MAPK), nuclear factor-κB (NF-κB) and Janus kinase/signal transducers and
activators of transcription (JAK-STAT). Smoking damages the skin by increasing formation
of reactive oxygen species and decreasing the gene expression of antioxidants. Nicotine also
stimulates innate immune cells integral to the pathogenesis of psoriasis including dendritic
cells, macrophages and keratinocytes. These cells release cytokines that activate T
lymphocytes and perpetuate a cycle of chronic inflammation. Smoking also enhances
expression of genes known to confer an increased risk of psoriasis, including HLA-Cw6,
HLA-DQA1*0201 and CYP1A1 [27-28].

CLINICAL VARIANTS
The clinical phenotype of psoriasis may manifest in several forms depending on disease
morphology, activity, location, and severity. The characterization of psoriasis restricted to
lesional morphology distinguishes 2 main morphologies: pustular e non-pustular psoriasis.
Multiple subtypes of psoriasis have been described, based on a combination of morphology,
distribution, and pattern. These subtypes often occur alone. However, there may be an overlap
or transition from one subtype to another due to various triggers or evolution of the disease
[5].
In its most classic morphologic presentation, non-pustular psoriasis is characterised by
red papules, patches and plaques with a grey or silvery-white, dry scale. These correlate to the
inflammation, vascular dilatation, and altered epidermal proliferation and differentiation seen
histologically. Different combinations of both erythematous and squamous components
determinate the typical polymorphism of psoriatic lesions.
The second morphologic presentation is that of discrete and/or confluent, superficial,
yellow-white pustules, either on a smooth erythematous and edematous base or overlying
normal-appearing skin. Pustules are characterized by intra-epidermal neutrophil accumulation
with only mild epidermal hyperplasia on histology [4].
Common symptoms associated with psoriasis include pruritus, irritation, burning, pain,
and bleeding. The discomfort caused by the skin lesions may result in impaired sleep,
concentration, and overall reduced quality of life. Pruritus is the most commonly reported

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symptom in psoriasis. Studies report that as high as 80% of psoriasis patients experience
pruritus and two thirds of patients experience itching often or constantly [5].
Activity of the psoriatic disease may vary with time. Increased activity in “acute” —
unstable—disease is associated both with enlargement of existing lesions and appearance of
new lesions within a short time. In “chronic”—stable—disease, lesions may persist
unchanged for months or years and new ones do not appear although the psoriasis may still be
very extensive, i.e., widespread large-plaque disease. Patients have a great variability in
regard to relapses. A small percentage of patients have lifelong, inherently unstable disease
whereas others have more stable disease with only occasional recurrence [29].

Psoriasis Vulgaris-Chronic Plaque Psoriasis

Psoriasis vulgaris, also named chronic plaque psoriasis, is the most common clinical
variant of psoriasis. It occurs in more than 80% of affected patients. It is characterized by
discoid, round or irregularly oval, well demarcated erythematous papules and plaques covered
by loosely adherent silvery-white scales. Early lesions frequently start as small pinpoint
erythematous macules or papules, which, soon in their evolution, show scaling. The initial
lesions extend peripherally, and coalesce to form plaques of one to several centimetres in
diameter [1-7].
With gradual peripheral extension, plaques may develop different configurations
including: psoriasis gyrate, in which curved linear patterns predominate; annular psoriasis, in
which ring-like lesions develop secondary to central clearing; psoriasis follicularis, in which
minute scaly papules are present at the openings of pilosebaceous follicles.
There can be great variation in the intensity of erythema (ranging from light pink to
bright red or deep purplish red), elevation of the lesion (flat to very thick and elevated), and
amount of scale (scattered light diffuse white scale overlying the lesion to thick micaceous,
hyperkeratotic scales) [1]. Indeed, the terms rupioid and ostraceous relate to distinct
morphological subtypes of plaque psoriasis. Rupioid plaques are small (2–5 cm in diameter)
and highly hyperkeratotic, resembling limpet shells. Ostraceous psoriasis refers to
hyperkeratotic plaques with relatively concave centres, similar in shape to oyster shells [4].
The distribution is typically symmetric, and sites of predilection include the extensor
surfaces of the extremities, particularly elbows and knees, the lumbosacral area, the scalp, the
nape of the neck, and to a lesser extent the remainder of trunk, genitalia, face, and ears [1-6].
Plaque psoriasis may occur as single lesions at predisposed sites (e.g., extensor aspects of
knees and elbows) or disseminated (generalized) over the body.
Additional features of psoriatic plaques include the Auspitz sign and the Woronoff’s ring.
Auspitz sign is the presence of pinpoint bleeding at the base of a plaque after scale is forcibly
removed. Woronoff’s ring refers to the presence of a white blanching ring occasionally seen
around lesions and usually associated with treatment, most commonly topical corticosteroids
or UV radiation. The Koebner phenomenon is present in 30% of patients with psoriasis [6].
On clearing, a temporary hypopigmentation, called psoriatic leukoderma, is frequent in the
healed cutaneous areas.
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Guttate Psoriasis

Guttate psoriasis, also named eruptive psoriasis, is characterized by an acute eruption of

small (< 1cm) round to oval-shaped well-defined erythematous scaly papules. Lesions are
usually distributed in a centripetal fashion on the upper trunk, but the limbs and face may also
be involved and vary in number from few scattered papules to hundreds. This form of
psoriasis has the strongest association to HLA-Cw6 and classically, occurs 2 to 4 weeks after
an acute group β-haemolytic streptococcal infection of the pharynx or tonsils. It can be the
presenting episode of psoriasis in children or, occasionally, adults. Guttate psoriasis accounts
for 2% of cases of psoriasis. In children, an acute episode of guttate psoriasis is usually self
limiting; in adults, guttate flares may complicate chronic plaque disease. Although few
studies have assessed the long term prognosis of children with acute guttate psoriasis, one
small study revealed that 33% of patients with acute guttate psoriasis eventually developed
chronic plaque disease [1-4-6].

Small Plaque Psoriasis

This form of psoriasis, similar in morphology to guttate psoriasis, is characterized by

discrete papules and plaques, as large as 1 to 3 cm in size. However, small plaque psoriasis
represents a chronic form of psoriasis rather than an acute eruptive process. It can be
distinguished by its onset in older patients and by its chronicity. This variant may not have
the pattern of accentuation on the extensor extremities, scalp, elbows, and knees as in classic
psoriasis, and may have a more randomly distributed, scattered, and diffuse pattern [6].

Inverse Psoriasis

Inverse (flexural, intertriginous) psoriasis is characterized by glossy, sharply demarcated

erythema localized predominantly to intertriginous regions including the axillae,
inframammary regions, gluteal cleft, genitals, abdominal and inguinal folds. Scaling is
usually minimal or absent. Painful fissures may appear at the apex of the fold. Maceration
occasionally occurs. The presentation may initially be confused with candidal, intertrigo, and
dermatophyte infections [1-4-6].

Seborrhoeic Psoriasis

Seborrhoeic psoriasis (‘sebopsoriasis’), so called because of its similarity in morphology

and anatomical distribution to seborrhoeic dermatitis, may occur either in isolation or
associated with plaque psoriasis. It presents with erythematous plaques with greasy scales
localized to seborrheic areas, including scalp, along the hair margin, eyebrows, glabella,
nasolabial folds, nose, ears, presternal and interscapular regions [1-29-30].

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Scalp Psoriasis

Scalp psoriasis is the most common manifestation of plaque psoriasis and may be present
in up to 79% of psoriatic patients. Similarly to other clinical variants, scalp psoriasis is
characterized by sharply demarcated erythematous lesions with silvery-gray scaling. The
disease can cover the entire scalp or multiple lesions of varying size may be seen, especially
in the fronto-parietal and occipital regions. Scalp psoriasis results in shedding of scale. It is
frequently pruritic and is associated with significant impact on the quality of life [5]. A
particular variety, called pseudotinea amiantacea, presents with thick, asbestos-like scales
adherent to tufts of hair; scales are moist at times and lack the characteristic silvery aspect.
This form is more frequent in children and requires evaluation to distinguish severe
seborrheic dermatitis or tinea capitis [1].

Palmoplantar (Non Pustular) Psoriasis

Involvement of palms and soles by psoriasis occurs with or without lesions elsewhere and
may appear as confluent redness and scaling, discrete plaques, ill-defined scaly/fissured areas,
or confluent plaques extending to the wrists and to the margins of the soles and heels [1].

Nail Psoriasis

Nail involvement in psoriasis is common. The incidence varies from 25 to 50%. It is

thought to correlate with age, duration and extent of disease and psoriatic arthritis; it may
occasionally occur without skin lesions. Psoriatic nail disease occurs more commonly in the
fingernails than the toenails. The lesions may be seen in the nail bed or in the nail plate, as
a result of psoriasis affecting the nail matrix [5].
Nail pits are the most common features of psoriasis, involving the fingers more often than
the toes. Pits are round depressions within the surface of the nail plate, approximately of the
size of a pinhead. They may be single or multiple and are thought to occur as a result of
psoriasis affecting the nail matrix. Pitting can be found in other diseases such as eczema,
alopecia areata, and lichen planus.
Onycholysis is characterized by distal separation of the nail plate from the nail bed; it
may affect single or multiple nails and involve a small area under the nail or extend to 90% of
the nail plate. If extensive, the nail may be lost, but another will regrow, and is also likely to
show onycholysis. Occasionally in onycholysis, bacteria grow under the nail plate and give
rise to green or black nail discoloration.
Oil drops are yellowish-brown translucent areas seen under the nail plate due to small
areas of psoriasis, giving rise to parakeratosis of the nail bed.
Subungual hyperkeratosis, characterized by thickening of the nail plate with
hyperkeratosis of the nail bed, is commonly seen in the toe nails. It often leads to gross
deformity of the nail, which may interfere with normal function.
Other signs of nail psoriasis include red spots in the lunula representing dilated tortuous
vessels associated with psoriasis, vertical or transverse ridging, and, finally, ‘splinter’ nail bed
hemorrhages [1-5-6-30].
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Mucous Membranes

Involvement of mucous membranes in psoriasis is very rare. The forms most commonly
encountered are fissured tongue, characterized by deep longitudinal fissures, and geographic
tongue, also named benign migratory glossitis, characterized by geographic patterns of
sharply circumscribed gyrate red patches with a white-yellow border and loss of filiform
papillae on the dorsum of the tongue. The patches may evolve and spread and change on a
daily basis [6].
Scaling of the lips is sometimes seen in erythrodermic psoriasis. In generalized pustular
psoriasis, geographical tongue and discrete denuded areas with white slightly elevated
edges on the dorsal and ventral tongue, buccal mucosae, and gingivae may occur.
Ocular lesions are rare in psoriasis, but blepharitis and keratitis have been reported;
whether these are of a primary nature, or complications of psoriasis treatment, is debatable.
Psoriasis may rarely involve genital mucosae (glans or labia) as small well-demarcated,
erythematous patches varying from 0.5 to 2cm in diameter. The patches have a shiny
appearance due to the absence of scales, as is the case of flexural psoriasis [1-30].

Pustular Psoriasis

Traditionally, two major clinical variants of pustular psoriasis are recognized: generalized
and localized pustular psoriasis.
Generalized pustular psoriasis is an heterogeneous group of severe pustular psoriasis
which include eritrodermic generalized pustular psoriasis (von Zumbush), anular pustular
psoriasis, impetigo herpetiformis and exanthematic pustular psoriasis.
Eritrodermic generalized pustular psoriasis (von Zumbush) is a rare, serious, acute
variant of pustular psoriasis. It may either be preceded by plaque psoriasis or arise de novo.
Episodes can be triggered by local irritants (i.e., ultraviolet light from sunlight or
phototherapy), abrupt withdrawal of systemic steroids, infections of the upper respiratory
tract and drugs. Attacks are characterized by fever and generalized eruption of numerous
pustules over the trunk and extremities. Pustules are sterile, flat, non-follicular, 2 to 3 mm in
diameter and arise on highly erithematous skin. Pustules may become confluent, producing
lakes of pus. They are easily ruptured leaving a glazed, smooth erythematous surface on
which new crops of pustules may appear. Characteristically, the disease occurs in waves of
fever and pustules. The erythema often spreads and becomes confluent, leading to
erythroderma. Oral lesions and subungual pustules may also appear.
Leukocytosis and high erythrocyte sedimentation rate (ESR) are commonly encountered.
This form of psoriasis carries an increased morbidity and mortality [7].
Annular pustular psoriasis is a rare variant characterized by pustules raised at the
periphery of erythematous annular lesions. It can, also present within the context of
generalized pustular psoriasis. Interestingly, a greater proportion of cases of pustular psoriasis
in children are of this subtype compared to adults. The course may be characterized by
recurrences, and the severity is generally less intense than that of eritrodermic generalized
pustular psoriasis (von Zumbusch) [6].
Impetigo herpetiformis is a rare form of pustular psoriasis occurring in pregnancy. Onset
is usually on third trimester and resolves at delivery. Morphologically, the lesions are

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erythematous patches or plaques with peripheral pustules that initially begin in flexural areas
and ultimately generalize. Patients are often affected earlier and more severely with
subsequent pregnancies. It is associate with hypocalcaemia and hypoparathyroidism.
Increased foetal mortality is an important complication. There have also been reports of
recurrences associated with monthly menses and oral contraceptives [6].
Exanthematic pustular psoriasis is characterized by a sudden onset of widespread
pustules with generalized plaque psoriasis in patients without history of psoriasis. It generally
occurs after a viral infection and is considered a benign form of psoriasis with no
constitutional upset. Unlike the von Zumbusch pattern, the disorder does not tend to recur.
There is an overlap between this form of pustular psoriasis and acute generalized
exanthematous pustulosis, a type drug of eruption [4-6].
A localized area of pustulation may occur in plaque psoriasis on the trunk and limbs
(distinct from the chronic form on the palms and soles). The localized area usually occurs
anywhere preexisting or new plaques are developing, generallyafter the application of an
irritant, e.g., dithranol, or following the withdrawal of potent topical steroids.
Two main clinical varieties are reported as localized pustular psoriasis: acrodermatitis
continua of Hallopeau and palmoplantar pustulosis. Localised forms of pustular are chronic,
not life- threatening, debilitating and resistant to therapy.
Acrodermatitis continua of Hallopeau, is a rare, pustular eruption of the fingers and toes.
Often, it begins after a localized trauma starting at the tips of one or two fingers, less often on
the toes. Pustules on bursting leave an erythematous, shiny area in which new pustules
develop. Pustules may coalesce to form lakes of pus, and, over time, they may spread
proximally. Pustulation of the nail bed and nail matrix often is associated with
onychodystrophy and even anonychia of the involved digits. Over time, patients may develop
osteolysis of the distal phalanx that underlies the eruption. Successive eruptions may become
generalized. Acrodermatitis continua may be associated with generalized pustular psoriasis of
the von Zumbusch subtype.
Palmoplantar pustulosis is characterized by sterile, yellow pustules on a background of
erythema and scaling affecting the palms and/or soles. The pustules are tender and fade to
form dark brown coloration with adherent scale/crust. Approximately 25% of cases are
associated with classic psoriasis vulgaris, but classification of palmoplantar pustulosis within
the spectrum of psoriasis is controversial. Today, palmoplantar pustulosis is regarded as its
own entity; genetic studies shown no association with HLA-Cw6 or other markers on
chromosome 6p linked to chronic plaque and guttate psoriasis; palmoplantar pustulosis more
commonly affects women (9:1) and presents between the ages of 40 and 60 years; has a very
striking association with smoking, either current or past, in up to 95% of subjects.
Palmoplantar pustulosis is associated with SAPHO syndrome (synovitis, acne, pustulosis,
hyperostosis, and osteitis) [4-6-7].

Erythrodermic Psoriasis

Total or subtotal (more than 90%) involvement of the skin by active psoriasis is known
as erythroderma. The most common precipitating factors of erythrodermic psoriasis are
discontinuation of systemic medications (such as corticosteroids, cyclosporine, or
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methotrexate), phototherapy-related toxicity, irritants (such as tar), and systemic illness or

infection.
The erythrodermic phase is dominated by loss of peculiar clinical features of psoriasis
and by generalized erythema and skin inability to maintain its homeostatic functions. The skin
is bright red and is covered by superficial, fine, flaky scales, rather than the thick, silver-
white, coarse scales of classical, chronic plaque psoriasis. Associated clinical findings may
include lymphadenopathy, fever or hypothermia, tachycardia, and peripheral edema.
Laboratory abnormalities include elevated erythrocyte sedimentation rate, hypoalbuminemia,
leukocytosis or leukopenia, anemia, electrolytes imbalance and elevations of lactate
dehydrogenase, liver transaminases, and uric acid. Significant morbidity may occur due to
dehydration from extensive fluid and electrolyte disturbances, protein losses, high-output
cardiac failure, and infection [6-31].

TREATMENT
General Considerations

There is no cure for psoriasis; therefore, the aim of treatment is to minimize the extent
and severity of the disease to the point at which it no longer substantially disrupts the
patient’s quality of life. The determination of severity is important in classifying patients for
study purposes and directing guidelines and decision-making in determining the course of
therapy. Yet, there is no uniformly accepted definition or guidelines for the severity of
psoriasis. Disease severity has been measured using various tools over the years. Of these, the
2 most common measurements in practice are the percentage of body surface area (BSA)
involved, either measured by the full hand print (in which the palm and digits equal
approximately 1% BSA) or the ‘rule of 9’s’ (in which different regions of the body are equal
to 9% or a multiple of 9% of the BSA), and the Psoriasis Area and Severity Index (PASI).
PASI measures erythema, infiltration, scaling, and extent of involvement of the four body
areas (head, trunk, arms, and legs). The PASI scale ranges from 0 to 72. The appreciation that
psoriasis impacts the quality of life of the individual, and therefore its impact on the overall
severity of the disease, has led to the use of multiple questionnaire-based measures. The most
common quality of life measure used in clinical trials is the Dermatology Life Quality Index
(DQLI).
When evaluating disease severity, along with scoring schemes for quantifying skin
and/or joint symptoms, or impairment of quality of life, should also be considered a number
of other parameters, i.e., affected site (visible area, genital region), symptoms (pruritus),
treatment response, burden of disease, prior need for inpatient and rehabilitative measures,
as well as the necessity of continued care and therapy [32-33]. Approximately 80% of
patients have mild to moderate disease, with 20% having moderate to severe psoriasis
affecting more than 5% of the body surface area (BSA) or affecting crucial body areas such as
the hands, feet, face, or genitals.
Recently, patients with psoriasis reported to have cardiometabolic disturbances including
hypertension, obesity, insulin resistance, and dyslipidemia. This constellation of risk factors,
referred to as the metabolic syndrome, increases the risk for atherosclerotic cardiovascular

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disease and type 2 diabetes mellitus, together the leading causes of mortality in the Western
world. In addition, evidence has suggested that psoriasis is an independent risk factor for
myocardial infarction when controlling for the aforementioned risk factors. Interestingly, the
risk remained elevated despite excluding patients treated with medications that elevate lipids
and blood pressure, specifically retinoids and cyclosporine [6].
Because psoriasis is a chronic disease requiring lifelong treatment, the selection of a
suitable therapy should be made according to various parameters such as age, sex, course and
activity of the disease, previous therapies, comorbidities, concomitant medications, burden of
the disease, presence of psoriatic arthritis and treatment-related toxicities [32].
At present, most clinical studies have used PASI 75. a 75% reduction from baseline
PASI, as the goal of therapy. The majority of patients with PASI 75 also have a relevant
improvement in quality of life, measured as improved DLQI. The successful establishment of
treatment goals requires that a minimum target be defined which must be achieved by
therapy. Most guidelines, take PASI 50 as the minimum target. If it is not reached within a
given amount of time, the therapy must be modified. Various forms of adjustment include
increasing the dosage, initiation of combination therapy, or transitioning to another drug or
procedure [32].
Strategies such as rotation of systemic therapies, combination therapy, sequential therapy,
and treatment regimens incorporating periods off therapy may be also used in an attempt to
reduce patients’ cumulative exposure to each agent [34].
A board spectrum of anti-psoriatic treatments, both topical and systemic is available for
the management of psoriasis.
Topical therapy is the mainstay of treatment for mild to moderate psoriasis and often the
initial treatment for severe psoriasis. In cases of severe, extensive psoriasis, where topical
therapy is either impractical or not sufficiently effective, phototherapy or systemic treatment
may be warranted. Patients treated with phototherapy or systemic agents, including biological
agents, can also be managed with topical agents as adjunctive therapy [32-33]

Topical Treatments

Corticosteroids
Topical corticosteroids are the cornerstone of treatment for the majority of patients with
psoriasis, particularly those with limited disease. They are available in many strengths and
formulations (lotions, solutions, creams, ointments, gels, sprays, and foams), which provide
clinicians with substantial flexibility in their approach to treatment. The mechanisms of action
of corticosteroids include anti-inflammatory, antiproliferative, immunosuppressive, and
vasoconstrictive effects. These effects are mediated through their binding to intracellular
corticosteroid receptors and regulation of gene transcription of numerous genes, particularly
those that code for proinflammatory cytokines [35]. The potency of topical steroids is based
on their ability to produce vasoconstriction, when applied to the skin of healthy volunteers,
according to the Stoughton-Cornell classification system. Topical steroids potency ranges
from weak (class 7), over-the-counter preparations, such as 1% hydrocortisone, to superpotent
(class 1) preparations, such as clobetasol propionate.
The choice of the appropriate potency corticosteroid and its vehicle should take into
consideration the disease severity, the location being treated, patient preference, as well as the
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age of the patient. Lower potency corticosteroids should generally be used for limited periods
of time on the face, intertriginous areas, areas with thin skin, and in infants. In adults and in
other areas, mid- or high-potency agents are generally recommended as initial therapy.
Patients with thick, chronic plaques often require treatment with the highest potency
corticosteroids. Use of class I steroids should be limited to an initial treatment course of
twice-daily application for 2 to 4 weeks and no more than 50 g/wk; if used for longer periods
of time there is an increased risk of both cutaneous side effects and systemic absorption.
However, longer durations of therapy are frequently utilized in clinical practice with
appropriate supervision and attention to potential side effects.
A wide range formulations are available; the chosen vehicle is of crucial importance both
for therapeutic efficacy and patients’ compliance. Corticosteroids in ointment formulation are
more commonly used and particularly indicated for thick lesions, since their hydrating power
enhance their absorption. Cream formulations are versatile and more cosmetically acceptable,
particularly for skin folds or friction areas. Finally, topical corticosteroids formulations in
solutions and foams are generally used in the treatment of scalp psoriasis because of their
high effectiveness.
Topical corticosteroids are associated with potential side effects that can limit their use.
Local cutaneous side effects are more commonly seen at steroid-sensitive sites, (face and
intertriginous areas), as well as in any areas that are treated over the long term. These include
skin atrophy, telangiectasia, striae distensae, acne, folliculitis, and purpura. Topical
corticosteroids may exacerbate preexisting or coexistent dermatoses, such as rosacea, perioral
dermatitis, and tinea infections and may on occasion cause contact dermatitis.
Systemic side effects occur less frequently than cutaneous side effects; they occur when
locally applied corticosteroids become absorbed through the skin and enter the circulatory
system. The greatest risk of systemic side effects occurs when ultra-high-potency or high-
potency corticosteroids are used over a large surface for a prolonged period or are used under
occlusion. Systemic effects have also been observed with widespread and extended use of
mid-potency corticosteroids. Well-known but relatively uncommon systemic side effects of
topical corticosteroid usage include Cushing’s syndrome, osteonecrosis of the femoral head,
cataracts, and glaucoma. Because of their increased skin surface–to–body mass ratio, infants
and young children are at increased risk of local and systemic side effects. Several approaches
have been utilized to minimize the side effects of topical corticosteroids, including
transitioning to weaker potency agents after clinical improvement, intermittent usage
(weekend only), and combination with other non steroidal agents. Another possible concern
with the use of topical corticosteroids in the treatment of psoriasis is rebound, wherein disease
recurs worse than the pretreatment baseline after the topical corticosteroid is discontinued.
Although rebound is known to occur most typically when topical corticosteroids are abruptly
discontinued, its frequency and severity are poorly characterized. All topical corticosteroids
are pregnancy category C and are of unknown safety in nursing women [32-33-36-37].

Vitamin D Analogues
The vitamin D analogues are also considered first-line topical agents and include
calcipotriol, calcitriol, and tacalcitol. Calcipotriol is available as cream, ointment, or solution.
Tacalcitol comes as ointment or lotion (emulsion), and calcitriol as ointment [32].
The vitamin D analogues act through vitamin D receptors present in keratinocytes and
lymphocytes to correct epidermal hyperproliferation, abnormal keratinization and

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 Types, Triggers and Treatment Strategies of Psoriasis 887

angiogenesis; in addition, apoptosis is induced in inflammatory cells. Vitamin D analogues

can also modulate the inflammatory process in psoriasis by a number of mechanisms,
including decreasing IL-1 and IL-6 levels, changing the balance between pro-inflammatory
and anti-inflammatory T-helper cell cytokines, and reducing CD45RO and C8+ T cells.
Moreover, vitamin D analogues increase transforming growth factor-b1 and -b2 levels,
thereby inhibiting epithelial cell growth [38].
Calcipotriol is given twice a day;. the preparation should be applied in a thin layer to the
affected areas of the skin. Several short-term studies have shown that calcipotriol is more
effective than coal tar, short-contact ditranol, and tacalcitol. Calcipotriol given twice a day
takes up to 12 weeks to reach the maximum treatment success; significant improvement is
seen after 1-2 weeks of treatment. To prevent hypercalcemia, the maximum recommended
weekly dosage of calcipotriol is 100 g; it means that can be treated adequately those patients
who have a total body involvement of approximately 30% or less.
The most common advesre effects include lesional and perilesional irritation, itching,
burning, pruritus, edema, dryness, and erythema and disappear after discontinuation of
treatment.
Calcipotriol has become a widely prescribed treatment in association with phototherapy.
Its combination with PUVA therapy demonstrated a dramatic benefit; it results in a lower
total ultraviolet (UV) dose and a faster onset of response. The sequence in which treatments
are administered is important because UV inactivates calcipotriol. Therefore, it should be
pointed out to apply calcipottiol after UV exposures, not before.
Tacalcitol is applied once daily. The maximum recommended quantities per day for adult
is 10 g (15 – 20% of the body surface area to treat). Long-term studies have demonstrated that
tacalcitol is suitable for topical long-term management of mild-moderate psoriasis for up to
18 months. If tacalcitol is administered for more than eight weeks a maximum of 15% of the
body surface area should be treated with up to 3.5 g/daily. Local side effects of tacalcitol
seem to be comparable to calcipotriol, although tacalcitol seems to cause less skin irritation if
applied on sensitive skin areas.
Calcitriol is applied twice daily. The maximum recommended quantity is 30g daily on up
to 35% of the body surface area. Some clinical trials have demonstrated the safety,
tolerability and efficacy of calcitriol ointment for the treatment of plaque psoriasis that
involve sensitive areas such as face, hairline, retroauricular, axillary, inguinal submammary
and popliteal areas [32-36].
An important advantage of the vitamin D analogues is their potential to function in a
corticosteroid-sparing fashion. This observation has led to the development of a two-
compound ointment (calcipotriol/betamethasone dipropionate) for the treatment of patients
with psoriasis. This new association is able to reduce the irritation effect of the vitamin D
derivate with a clinical improvement after a week from baseline and results continue to
improve throughout the therapy. It is administrated once daily; after 4 of treatment, PASI75 is
achieved in 72% of patients, which maintented this result, after 12 weeks, in 68% of cases.
Clinical trials shown that betametasone monotherapy has slightly lower effect than the
combination therapy after 4 weeks. Finally, the application of the combined formula for 4
weeks followed by the application of calcipotriol results in a more efficient maintenance of
clinical results than tacalcitol for 8 weeks.
In recent years, the cosmetic acceptability and ease of use of topical therapies have
improved greatly compared to earlier; however, complete adherence to the prescribed
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888 Spyridoula Doukaki and Maria Rita Bongiorno

regimen is still a problem and new formulations may help improve this aspect of clinical care
[32-36].
To address the potentially negative effect on adherence that may be associated with
psoriasis treatments formulated in an ointment, a two-compound gel vehicle was developed. It
has the same active ingredients as the two-compound ointment (calcipotriol ⁄ betamethasone
dipropionate); like the two-compound ointment, the two-compound gel is administered once
daily. Whether an ointment or gel is ‘better’ depends on the individual patient. Some patients
will adhere to treatment with the gel, some patients may feel that the ointment works better
for them and some will prefer occlusion [38].

Tazarotene
Tazarotene, a potent third generation topical retinoid became available for the treatment
of psoriasis in 1997. Tazarotene is thought to function by normalizing abnormal keratinocyte
differentiation, diminishing hyperproliferation, and by decreasing expression of inflammatory
markers. The systemic absorption of tazarotene following non-occlusive topical application
is less than 1%. It is not stored in the fatty tissue. Compared to etretinate, tazarotene is 1.000
times less lipophilic. The half life is 18 hours.
Tazarotene is available in 0.05% and 0.1% gels and creams formulation. Effects are seen
soon after beginning therapy and last for 12 weeks after the end of therapy. Clinical trials
compared the efficacy of tazarotene cream 0.05% and 0.1% to placebo. They shown that
58.8% of the patients who were treated with 0.1% cream and 47.6% of the patients who
were treated with 0.05% cream demonstrated a >50% improvement of the lesions after 12
weeks. In the placebo group 26.2% of the patients improved [32-36].
Tazarotene should be applied as a thin film to the affected sites once daily in the evening
(not more than 10% of the body surface). If used as monotherapy, irritation (itching, burning
and erythema) at the application sites develops in a significant proportion of patients. This
retinoid dermatitis is dose-dependent. Therefore, treatment should be started with the weaker
preparation (0.05%). If this is well tolerated or the clinical efficacy is not sufficient, after
about 1–2 weeks, the stronger (0.1%) form can be employed. The combination of tazarotene
with topical corticosteroids has been studied for the purpose of avoiding retinoid
dermatitis [32-37]. Use with potent corticosteroids has proven to be helpful. The response rate
is higher when tazarotene is applied in the evening and the corticosteroid in the morning; the
adverse drug reactions are lower and the remission phase longer. The best results are
produced by combinations with betamethasone dipropionate cream (78% of the patients
display a 50% improvement) and mometasone (66% of the patients display a 50%
improvement).
Typical adverse drug reactions of oral retinoids do not occur with topical application.
There are no reports of phototoxic/photoallergic reactions. The safety of use during
pregnancy and breastfeeding has not been ascertained [32].

Salicylic Acid
Salicylic acid is the most widely keratolytic agent used in the topical treatment of
psoriasis. Keratolytic agents help to remove accumulated scales or hyperkeratosis. Their
precise mechanism of action is not fully understood; it is thought that they act by decreasing
the cell-to-cell cohesion in the stratum corneum, favoring the physiologic keratolysis. There
are no placebo-controlled studies verifying the efficacy and safety of salicylic acid used

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 Types, Triggers and Treatment Strategies of Psoriasis 889

alone; salicylic acid is often combined with other topical therapies, including corticosteroids.
The improvement in efficacy of combination therapy compared with salicylic acid alone is
likely due to the increased skin penetration that occurs because of the keratolytic effects of
salicylic acid.
Salicylic acid can be applied to palms, soles and scalp in concentrations of 2–10%. It can
be used at a concentration up to 20% when psoriatic hyperkeratosis is massive and cohesive,
as in palmo-plantar areas.
Side effects include local irritation and burning sensation. Systemic toxicity (salicylism)
is rare; it can occur, when salicylic acid is applied to more than 20% of the body surface or in
patients with abnormal hepatic or renal function. Salicylic acid should not be used in
combination with other oral salicylate drugs and should be avoided in children because the
risk of systemic absorption and toxicity is greater. Salicylic acid decreases the efficacy of
UVB phototherapy because of a filtering effect; therefore it should not be used before UVB
phototherapy. Salicylic acid appears to be a safe choice for the control of localized psoriasis
in pregnancy [32-36].

Topical Calcineurin Inhibitors

The topical calcineurin inhibitors, tacrolimus and pimecrolimus, have been approved to
treat atopic dermatitis. Their use in the treatment of psoriasis vulgaris is based on the
results of clinical studies which demonstrated efficacy under occlusion. They do not
appear effective for treating plaque-type psoriasis when simply applied as commercially
available. Subsequent investigations demonstrated the efficacy of topical calcineurin
inhibitors in the treatment of psoriasis lesions on face, intertriginous areas and anogenital
region.
Tacrolimus and pimecrolimus are topical immunomodulators; function by blocking the
synthesis of numerous inflammatory cytokines that play an important role in the pathogenesis
of psoriasis. However, neither medication has yet to be approved by the FDA for this
indication.
Tacrolimus is available as a ointment in concentrations of 0.03% and 0.1%; pimecrolimus
as a cream with a concentration of 1%. They are generally applied once-twice daily. In a
double-blind, randomized, vehicle-controlled study 65% of patients with facial and
intertriginous psoriasis treated with tacrolimus 0.1% ointment were clear or almost clear after
8 weeks of therapy as compared with 31% of patients treated with placebo. In another double-
blind, randomized, vehicle-controlled study of patients with intertriginous psoriasis, 71% of
the patients treated with pimecrolimus 0.1% cream were clear or almost clear after 8 weeks of
twice-daily treatment as compared with 21% of patients treated with placebo. There are no
routine clinical studies of calcineurin inhibitors under occlusion available. Therefore, their
use under occlusion can not be recommended for routine practice.
The most common side effect for both medications is burning and itching that generally
reduces with ongoing usage. Only in few patients persistent burning make necessary to stop
therapy. This side effect appears to be more significant in patients treated with tacrolimus
ointment as compared with patients treated with pimecrolimus cream. Calcineurin inhibitors
disrupt the local immune system by inhibiting T- cell activation and bacterial (folliculitis) and
viral infections (HPV-induced diseases and herpes simplex) are more common. Some animal
studies suggest that the concomitant use of calcineurin inhibitors and ultraviolet light may
lead to an increased risk of epithelial tumors; there are no similar observations in humans.
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890 Spyridoula Doukaki and Maria Rita Bongiorno

Nevertheless, calcineurin inhibitors should not be used in combination with UVB or PUVA.
Tthere is no evidence of a teratogenetic effect of calcineurin inhibitors. Because of a lack of
experience, it is recommended not to administer pimecrolimus and tacrolimus topically
during pregnancy or lactation [32-36].

Anthralin
While anthralin has been a mainstay for the topical treatment of psoriasis, typically in the
inpatient setting, its use has declined in recent years because of the availability of more
cosmetically acceptable alternatives (corticosteroids, vitamin D3 analogs). The exact
mechanism of action of anthralin is not fully understood; recent studies suggest that they act
by preventing T-lymphocyte activation and normalizing keratinocyte differentiation. Several
doses and preparations of anthralin are available; however, owing largely to issues of
cosmesis and convenience, anthralin is most commonly used as short contact (20-30 minutes)
therapy in the outpatient setting, starting at 1% concentration with increasing concentration
over time as tolerated [32-36].
The most common side effects of anthralin are skin irritation and staining of lesional and
perilesional skin, nails, clothing, and other objects with which patients come into contact. If
the psoriatic plaques are well defined, the surrounding normal skin can be protected by the
use of an agent such as zinc oxide paste. Anthralin should be applied with caution to face and
intertriginous areas because of the risk for severe skin irritation. There is no evidence of any
long-term toxicities related either to skin exposure or to systemic issues. Anthralin is
pregnancy category C [32-36-37].

Coal Tar
Coal tar, a distillation product from coal, is a mixture of thousands of compounds which
may differ in composition from one preparation to the next. Coal tar has been used for
approximately 100 years in the treatment of psoriasis. Although the mechanism of action of
coal tar is not well understood, it is known to suppress DNA synthesis by lessening the
mitotic labeling index of keratinocytes. Elements of coal tar are absorbed percutaneously and
are also effective after the preparation has been removed from the skin. They are fat-soluble,
metabolized and excreted through the kidney. Psoriasis vulgaris is treated with tar ointments
of varying dosages. Coal tar increases the effectiveness of subsequent UVB radiation
(Goeckerman regimen)
Coal tar products are often poorly tolerated by patients because of cosmetic issues,
including staining of clothes and the tar odor that is present in almost all products. Other
potential adverse effects include irritant contact dermatitis, and folliculitis. Coal tar is
carcinogenic in animals; however, in humans it has only been described in the squamous
carcinoma of the scrotum following occupational exposure [32-36].

Phototherapy

Various spectra of UVB and UVA wavelengths have been used to treat psoriasis.
Originally, broadband ultraviolet B (BB-UVB) light with wavelengths of 280 – 315 nm was
used for treatment of psoriasis. Goeckerman first described the use of broadband (BB)-UVB

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 Types, Triggers and Treatment Strategies of Psoriasis 891

in combination with day and night applications of crude coal tar. During the years after the
development of the Goeckerman regimen, several modifications and simplifications were
made. In the 1950s, the Ingram regimen was developed, which replaced crude coal tar with
anthralin. In the 1980s, a new type of UVB bulb with a narrow emission between 311 and 313
nm was developed; this new UVB treatment is now commonly referred to as narrowband
(NB)-UVB therapy[32-39].
Phototherapy is locally immunosuppressive by its direct effects on Langerhans cells and
indirect effects on numerous cytokines and adhesion molecules, which can lead to a switch
from a Th 1 to a Th 2 phenotype. Other effects of UV light include inhibition of both
epidermal hyperproliferation and angiogenesis. Furthermore, UV light causes a selective
reduction in T lymphocytes within psoriatic skin via apoptosis. BB-UVB, NB-UVB, and
psoralen plus UVA (PUVA) can all induce apoptosis of T lymphocytes, which may play an
important role in the mechanism involved in remissions of psoriasis [39].

UVB Phototherapy
BB-UVB phototherapy has been used for the treatment of psoriasis for more than 75
years. This therapy remains one of the safest treatments for cutaneous psoriasis, but requires
treatments at least three times per week for several months to be effective. The most effective
wavelengths of UVB light used for the treatment of psoriasis fall in a very narrow range,
311– 313 nm (NB-UVB phototherapy). Many studies evaluating the efficacy of NB-UVB
phototherapy shown that it is superior to conventional BB-UVB phototherapy. Most studies
were right left half body comparisons of NB-UVB with BB-UVB. One such study
demonstrated that 40% of patients treated with NB-UVB had superior results. Other similar
studies have demonstrated more rapid clearing in patients treated with NB-UVB compared
with those treated with BB-UVB and treatment with NB-UVB was more likely than BB-UVB
to lead to histopathological resolution of psoriasis lesions (88% compared with 59%) [39].
Analysis of recent comparative clinical trials suggested that the efficacy of NB-UVB was
slightly lower than but approached that of PUVA. Concomitant use of systemic retinoids or
topical therapy does not increase the efficacy of NBUVB; however, combination therapy may
reduce patients’ cumulative UVB dose, leading to improved long-term safety. There is some
evidence that concomitant treatment with topical corticosteroids may be associated with a
higher relapse rate; therefore, this combination should be avoided [34].
The dosage of UVB may be administered according to the Fitzpatrick skin type and the
minimal erythema dose (MED), with subsequent dosages adjusted accordingly. Basic
phototherapy education should be given to all patients. This must include education about the
use of goggles in all patients and the use of genital shields in male patients [39].
Erythema, burning, pruritus, dry skin, occasional blistering and increased recurrence of
herpes simplex eruption have been reported as short-term side effects of NB-UVB
phototherapy. Topical corticosteroids may be used to treat painful erythema, and non steroidal
anti-inflammatory drugs and systemic corticosteroids have been used in severe case. The use
of eye protection with goggles is required to decrease the risk of UVB-related cataract
formation. Long-term side effects include photodamage and increased incidence of skin
cancer. It is estimated that the excess annual risk of nonmelanoma skin cancer associated with
UVB radiation is likely to be less than 2%. NB-UVB phototherapy is contraindicated in
patients with xeroderma pigmentosum or systemic lupus erythematosus, and should be
avoided in patients with a history of skin cancer. Current guidelines recommend careful
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892 Spyridoula Doukaki and Maria Rita Bongiorno

monitoring of patients considered to be at risk of skin cancer, and particular caution for
patients with skin types I–II, and blonde or red hair [34].
Pregnancy is not a contraindication to the use of UVB therapy. NB-UVB therapy has
been used successfully in the treatment of psoriasis in pregnancy and should be considered
first-line therapy in pregnant patients with plaque and guttate psoriasis who need a systemic
approach to treatment.
In recent years, excimer lasers which emit monochromatic UVB light (308 nm) have
been tested. The chromophore for the excimer laser is cellular DNA. Breakage of strands of
DNA in T lymphocytes and expression of mitochondrial proteins related to cell death has
been noted after exposure to the 308-nm laser. After psoriatic lesions are exposed to 308-nm
excimer light, there is T-cell depletion accompanied by decreased epidermal proliferation The
dose of energy delivered is guided by the patients’ skin type and thickness of the plaque. The
frequency of treatment is 2 to 3 times a week, with a minimum of 48 hours between
treatments. In several small studies and in one larger non-randomized study, after several
weeks of therapy there was a good response rate at treated sites, ranging from partial
remission to complete clearance of skin lesions.
Due to technical reasons, the excimer laser has the advantage of treating only involved
skin, therefore minimizing potential risks of exposing normal-appearing skin to UV radiation.
Adverse effects are limited to the area irradiated and include erythema, burning, and
hyperpigmentation. Blisters are noted more often with the use of higher fluences. The long-
term safety of excimer laser therapy has not yet been fully established [32-39].

Photochemotherapy
Photochemotherapy (PUVA) involves oral or topical administration of a photosensitizing
psoralen followed by exposure to long-wavelength (320–400 nm) UVA irradiation. Psoralens
are tricyclic furocoumarins that occur naturally in some plants and are also synthetically
produced. Currently, are available two systemic psoralen for the treatment of dermatological
disease, methoxsalen (8-methoxypsoralen) and 5-methoxypsoralen which is characterized by
a lower potential for phototoxicity. Trimethylpsoralen is used for bath water-delivered PUVA
[32-39]. The mechanism of action of PUVA therapy in patients with psoriasis has not been
fully elucidated; however, PUVA is known to have antiproliferative, anti-inflammatory and
immunosuppressive effects [34] .
8-methoxypsoralen should be administered 1.5 hours before exposure to UVA radiation.
During the clearance phase, treatments are usually given 2 to 3 times weekly with at least 48
hours between treatments allowing sufficient time to assess for the degree of erythema
induced by the previous dose.
Clinical trials suggest that PUVA therapy is effective for most forms of psoriasis and
induces complete or partial remission in 70–90% of patients with psoriasis. Concurrent
retinoid plus PUVA therapy appeared to be more effective in clearing psoriasis than either
regimen alone, and the efficacy of PUVA therapy was increased by concomitant use of
topical treatments such as corticosteroids and vitamin D3 analogues. Research has shown that
combination PUVA–methotrexate therapy may also be highly effective; however, this
combination may be limited by the risk of excessive immunosuppression and additive
carcinogenesis [34]. Topical PUVA therapy (direct application of psoralen to the skin
combined with subsequent exposure to UVA) is another form of PUVA. Bath PUVA with

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 Types, Triggers and Treatment Strategies of Psoriasis 893

trimethylpsoralen is commonly used in Scandinavian countries for generalized psoriasis to

reduce systemic psoralen exposure and thereby minimize toxicities.
PUVA therapy may be associated with several immediate and delayed adverse effects.
Common acute side effects include erythema, pruritus, xerosis, irregular pigmentation, and
gastrointestinal symptoms such as nausea and vomiting and can be managed modifying the
dosage of the psoralen or the UV light, using emollients and antipruritic agents. Patients with
gastrointestinal symptoms may experience improvement by dividing their 8-methoxypsoralen
dosage over 15 minutes or taking it with food, particularly milk. Other acute toxicities may
include blisters, photo-onycholysis, and melanonychia. Hepatic toxicity from psoralens is
uncommon.
Long-term effects include skin cancer, photodamage and premature ageing of the skin,
characterized by elastosis, poikiloderma and dark brown to black macules known as PUVA
lentigines. There is a clear relationship between cumulative PUVA exposure and an increased
risk of skin cancer. Several PUVA trials revealed a 14-fold increased incidence of
spinocellular carcinoma in patients who received high-dose PUVA compared with those who
received low-dose PUVA. The risk of spinocellular carcinoma of the male genitalia is
particularly elevated without shielding of this area during PUVA treatments. Whether
exposure to oral PUVA increases the risk of developing melanoma is an area of controversy.
PUVA treatment is contraindicated in patients with known lupus erythematosus,
porphyria, xeroderma pigmentosum and idiosyncratic reactions to psoralen compounds.
Caution should be exercised in patients with skin types I and II who tend to burn easily and
patients with a history of arsenic intake or previous treatment with ionizing radiation therapy.
In addition, patients with a history of melanoma or multiple nonmelanoma skin cancers,
severe liver disease that could lead to toxic levels of psoralens and patients previously treated
with cyclosporine or methotrexate should be approached with caution. As topical PUVA can
be associated with significant toxicity if not correctly administered by fully trained personnel,
patients need to be appropriately educated about the potential risks. The potential effects of
systemic methoxsalen on the fetus and ⁄or female reproductive capacity are not known;
therefore, oral methoxsalen is contraindicated during pregnancy and lactation.
Drug interactions with PUVA therapy may occur when patients are concurrently being
treated with other photosensitizing agents such as nonsteroidal anti-inflammatory drugs,
diuretics, antifungals, neuroleptics, and certain antibiotics such as the tetracyclines and the
fluoroquinolones
All patients who are considered for treatment with phototherapy or photochemotherapy
must have a complete history and physical examination. Monitoring associated with oral
PUVA requires ophthalmological and routine laboratory evaluation prior to treatment and at
regular intervals during therapy. Patients should be monitored regularly for early detection of
skin cancer [32-34-39].

Systemic Oral Agents

Methotrexate
Methotrexate was introduced several decades ago for the treatment of psoriasis. It is a
structural analogue of folic acid and thereby competitively inhibits dihydrofolate reductase.
Such inhibition ultimately influences the conversion of homocysteine to methionine and the
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894 Spyridoula Doukaki and Maria Rita Bongiorno

synthesis of polyamines. In addition, methotrexate directly inhibits thymidylate synthase,

which converts deoxyuridylate to deoxythymidylate and glycinamide ribonucleotide
transformylase, which are important in the de novo synthesis of purines. Methotrexate also
influences the activity of the enzyme methylentetrahydrofolate reductase.
Methylentetrahydrofolate reductase is necessary for the generation of 5 methyl-
tetrahydrofolate, which is the methyl donor for the conversion of homocysteine to methionine
[40].
Its mechanism of action in psoriasis is not fully understood. Because the effects of
methotrexate are most dramatic on rapidly dividing cells, it was originally thought that its
beneficial effects in psoriasis were a result of the inhibition of epidermal proliferation.
However, it is now believed to act primarily as an immunosuppressant [34]. It is known that
there is little effect on epidermal cells, but there is significant inhibition of the proliferation of
lymphoid tissue at concentrations of methotrexate that are typically achieved with low dose
weekly methotrexate. These findings support the concept that the therapeutic effect of low-
dose methotrexate in psoriasis is a result of its effects on the immune system [41]. In addition
to inhibition of T- and B-lymphocyte replication and function, methotrexate has potent anti-
inflammatory effects, most of which involve an intracellular elevation of adenosine as a key
step. Adenosine may then influence leukotriene production and adhesion molecule expression
by vascular endothelial cells which are crucial in the development of psoriasis.
Although the literature is sparse, there are a number of recent studies demonstrating
methotrexate efficacy for psoriasis [42]. Methotrexate may be administered as either a single,
oral, subcutaneous, or intramuscular dose once a week, or as 3 divided oral doses over a 24-
hour period once a week. There is little or no evidence to substantiate which regimen, if any,
is superior. Intramuscular administration is helpful when there is gastrointestinal intolerance
to oral dosing; subcutaneous injection is equally effective and can be self-administered at
home. Nowadays, most dermatologists use a once weekly single oral dosage schedule.
Methotrexate therapy may be started as a single weekly dose of 2.5 or 5 mg to evaluate for
significant bone marrow suppression. The dose may be gradually increased to achieve
optimal control of psoriasis symptoms. There are no established maximum or minimum
dosages of methotrexate; however, weekly dosages usually range from 7.5 to 25 mg. In
general, a total weekly dose of 30 mg should not be exceeded. After an increase in
methotrexate dose, it may take up to 4 weeks for a clinical response to occur [34-41].
Methotrexate therapy may be associated with a number of significant side effects that
demand close supervision of all patients treated with this therapy. The risk and severity of
toxicity may be related to the dose. Methotrexate is renally excreted and should therefore not
be administered to patients with impairment of kidney function. Common and manageable
side effects include nausea, stable leucopenia, and mild elevation of liver transaminases.
Addition of folic acid (5 mg daily) can help to alleviate gastrointestinal side effects, and close
monitoring of liver function and full blood count allow continue therapy in most. Rarely,
hematopoietic suppression can be significant, particularly as a result of overdosing. In this
situation, folinic acid administered intramuscularly is the treatment of choice. Nausea can
lead to discontinuation of methotrexate therapy; antiemetics may help to alleviate this
problem.
Although bone marrow toxicity is the most serious short-term side effect of methotrexate
therapy, hepatotoxicity is the most common long-term adverse effect. Consequently,
methotrexate should either be avoided or given with extreme caution in patients with liver

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 Types, Triggers and Treatment Strategies of Psoriasis 895

disease. Active alcoholism is a contraindication to the administration of methotrexate.

Patients who are obese or diabetic can have an increased risk of cirrhosis, and pretreatment
liver biopsy should be considered in patients with a history of hepatitis. In the most recent
guidelines, patients with normal liver function tests and without a history of liver disease or
alcoholism should not undergo liver biopsy until they have been treated with a cumulative
methotrexate dose of 1 to 1.5 g. For patients at increased risk of hepatotoxicity, liver biopsy
may be justified earlier during therapy. Repeat biopsies should be performed approximately
every 1 to 1.5 g thereafter if liver function tests and biopsy findings are normal; with usual
dosages this means a liver biopsy every 2 years. The presence of pathological changes
detected by the liver biopsy will direct the decision of whether or not to continue
methotrexate therapy. Although, liver biopsy is the standard for detecting fibrosis and
cirrhosis, it is associated with significant morbidity and rarely mortality. One possible
alternative to the liver biopsy consists in monitoring of serum levels of type III procollagen
amino terminal propeptide (PIIINP) in serum. However, the diagnostic value of this method
of determining an onset of liver damage is controversial, as the interpretation of the individual
values is not always easy.
Pulmonary fibrosis is another risk during methotrexate treatment. Other rare
complications, when a weekly dosing schedule is used, include oral ulcerations or stomatitis,
anagen alopecia, cutaneous ulceration, folliculitis, infection and reactivation of tuberculosis,
ataxia, depression, and other psychotic symptoms, osteopathy, and mutagenicity.
Methotrexate osteopathy presents as a triad of severe pain localized to the distal tibia,
osteoporosis, and compression fractures of the distal tibia. Withdrawal of methotrexate
appears to be the only treatment. There are several reports of malignant lymphomas
developing in patients treated with methotrexate. Considering the immunosuppressive effects
of methotrexate, it should not be surprising that lymphoproliferative disorders are
occasionally reported.
Methotrexate is both a teratogen and an abortifacient; therefore, it is contraindicated in
pregnancy. Pregnancy should be avoided during treatment and for one ovulation cycle after
discontinuation in female patients; partners of male patients taking methotrexate should not
become pregnant for at least 3 months after discontinuation of treatment. Methotrexate is also
contraindicated during breastfeeding due to the potential for serious adverse effects in the
infant.
Several drugs may influence methotrexate metabolism and ⁄or potentiate methotrexate-
induced toxicity, including some nonsteroidal anti-inflammatory drugs and salicylic acid.
Therefore, physicians prescribing methotrexate and patients using the drug should be aware
of a number of potential drug-drug interactions, including interactions with trimethoprim/
sulfamethoxazole, cyclosporine (resulting in additive immunosuppression), oral retinoids
(increased risk of hepatotoxicity), and penicillins. Particular caution is warranted for patients
who may be at increased risk of methotrexate-induced toxicity due to impaired renal function
or folate deficiency [34-37-40-42].
Before initiating therapy with methotrexate laboratory tests, including a complete blood
cell (CBC) count with differential, creatinine, liver function tests including albumin and
bilirubin should be obtained. Screening for hepatitis B and C should be considered when there
is evidence of viral hepatitis such as elevated liver function test results. As methotrexate is an
immunosuppressive drug some experts recommend screening for latent tubercolosis. A chest
radiograph is important for patients with underlying pulmonary disease. Pretreatment liver
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896 Spyridoula Doukaki and Maria Rita Bongiorno

biopsy should only be performed in patients who have abnormal liver function test results,
chronic hepatitis, and a history of greater than moderate alcohol intake.
Ongoing laboratory studies should include a CBC count every 2 to 4 weeks for the first
few months, then every 1 to 3 months, depending on leukocyte count and patient’s stability.
Some suggest that laboratory studies be performed on the fifth to sixth day of the weekly
methotrexate cycle, to detect the leukopenia nadir, and because liver chemistry values may be
elevated 1 to 2 days after a dose of methotrexate. The frequency of blood count monitoring
may be slowly decreased over time as long as there is no toxicity or changes in the medical
history. Patients with risk factors for hematologic toxicity need closer monitoring, particularly
at the onset of therapy and after increasing the dosage of methotrexate. Liver chemistries
including alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and
serum albumin levels should be performed every 4 weeks. Patients with significant renal
impairment require careful monitoring by obtaining blood counts before the second dose.
Serum urea nitrogen (BUN) and creatinine should be obtained at 2- to 3-month intervals. For
those patients with normal values, who may be at risk for decreased renal function, such as
the elderly or those with a decreased muscle mass, a glomerular filtration rate should be
calculated [41].

Acitretin
Acitretin is an oral retinoid approved for the treatment of psoriasis. Acitretin is the
principle active metabolite of the prodrug, etretinate, the first retinoid introduced for the
treatment of severe psoriasis and replaced in 1988 by acitretin [43]. The exact mechanism of
action in the treatment of psoriasis is unclear. Retinoids are known to modulate epidermal
proliferation and differentiation and to have immunomodulatory and anti-inflammatory
activity. It is likely that the biological effects of retinoids are to a large extent promulgated by
binding to nuclear retinoic acid receptors [41]. Acitretin directly affects epidermal
keratinocytes in psoriasis, effectively normalizing their hyperproliferation and loss of
differentiation. Retinoids may also influence angiogenesis by inhibiting the action of vascular
endothelial growth factor via activator protein 1 transcription factors. Vascular endothelial
growth factor promotes angiogenesis, and elevated levels are found in plaques of psoriasis.
Finally, it has been shown that retinoids can also modulate T-cell responses, inhibit
chemotactic responses, and activation of polymorphonuclear leukocytes [40].
The efficacy of acitretin is dose dependent. Retinoids can be dramatically effective as
monotherapy for generalized pustular psoriasis and for erythrodermic psoriasis. They are
much more slowly effective for plaque and guttate psoriasis but can dramatically improve the
response to PUVA and UVB.
In patients with generalized pustular psoriasis the initial dose required is 25 to 50 mg per
day. A rapid resolution of generalized pustular psoriasis is achieved usually within 10 days at
which point tapering of the dose to around 10 to 25 mg/d is often adequate as maintenance
therapy. Similar dosing regimens are effective in the treatment of exfoliative erythrodermic
psoriasis. Acitretin is also effective for the treatment of palmoplantar pustulosis, where
decreases the level of pustulation and controls coexistent hyperkeratosis. In resistant cases of
palmoplantar pustulosis, the addition of PUVA therapy is useful.
Acitretin, in combination with other forms of treatment, is more effective than
monotherapy for plaque psoriasis. Acitretin, at a dose of 25 mg/d for 2 weeks followed by up
to 10 weeks of 25 mg every other day or 10 mg daily, can be combined successfully with

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 Types, Triggers and Treatment Strategies of Psoriasis 897

PUVA. This regimen has been shown to be superior to PUVA alone and to reduce the
cumulative dosage of UVA required for clearance and. The increases in ultraviolet radiation
should be more gradual and cautious than in patients not taking oral retinoids because of an
increased risk of generalized erythema. This is not a true photosensitivity, but probably
represents increased epidermal transmission of ultraviolet radiation because of altered optical
properties of the stratum corneum caused by the retinoid. Concomitant treatment with
acitretin and topical calcipotriene may help reduce psoriatic plaques better than with either
alone [40-43].
Because of a lack of significant immunosuppression, acitretin is generally considered
effective and the treatment of choice in HIV-positive patients with severe psoriasis [41].
Although there is a high incidence of nuisance side effects, acitretin therapy continues to
have an important role as a therapy for psoriasis. Mucocutaneous side effects of acitretin
occur in almost all patients to varying degrees and may include cheilitis and dryness of other
mucous membranes such as nose, eyes, mouth, throat, and vagina. These mucocutaneous side
effects are dose related. Higher doses may induce exfoliative cheilitis, balanitis, urethritis,
gingivitis, and corneal ulceration. A paronychia can occur, and in some instances, this is
severe enough to necessitate cessation of therapy. Periungual pyogenic granulomas may occur
during long-term use of acitretin. Thinning of palmar and plantar skin combined with nail
fragility is a common complaint. Patients may also have stickiness and fragility of the skin.
Alopecia is a relatively common side effect necessitating withdrawal of retinoids.
The most common laboratory abnormality seen in patients treated with acitretin is
hyperlipidemia, with as many as 25% to 50% of patients experiencing increases in serum
triglycerides. The risk is increased in the setting of diabetes mellitus, obesity, and increased
alcohol intake. Lifestyle change to prevent/ reduce hyperlipidemia, i.e., low-fat diets, reduced
alcohol intake, and exercise, should be encouraged in patients with psoriasis who are being
treated with oral retinoids. Lipid-lowering drugs may be used in cases of more significant
hypertriglyceridaemia and hypercholesterolaemia. Liver function should also be monitored
during retinoid therapy. Minor elevations of hepatic transaminases occur in 13% to 16% of
patients. They are of little importance and return to normal on cessation of treatment. It has
been reported that 1.5% of patients on acitretin therapy may develop a toxic hepatitis,
although examination of liver biopsies from such patients revealed no histological evidence of
hepatotoxicity.
Patients treated with high doses for long periods may develop skeletal abnormalities such
as anterior spinal ligament calcification and osteophyte formation, similar to those seen in
diffuse idiopathic skeletal hyperostosis. These skeletal changes are usually, but not always,
asymptomatic. Periodic spinal radiographical assessment should be considered if long-term
therapy is planned. Arthralgia and myalgia are common musculoskeletal complaints,
occurring in up to 25% of patients treated with retinoids. Acitretin induced osteoporosis has
been subject of controversy; the risk is possibly highest in those receiving high-dose retinoids
for long time periods.
Pseudotumor cerebri has been reported in patients treated with acitretin. It seems that the
risk is increased if retinoids are taken alongside tetracyclines. Symptoms include severe
headache, nausea, vomiting, and visual disturbance. Papilloedema may be evident on
ophthalmologic examination.
Systemic retinoids are highly teratogenic. Fetal abnormalities consequent on retinoid
therapy during pregnancy include cardiovascular, ocular, and auditory malformations, facial
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898 Spyridoula Doukaki and Maria Rita Bongiorno

dysmorphia, meningomyelocele, meningoencephalocele, and bony malformations, with the

greatest risk occurring between the third and sixth weeks of gestation [37-40]. Although the
half-life of acitretin is 49 hours, acitretin may transform either spontaneously, or as a result of
alcohol ingestion, into etretinate, which has a half-life of 168 days. Based on this long half-
life, it can take up to 3 years after discontinuing treatment for etretinate to be eliminated from
the body. The minimum amount of alcohol consumption for this conversion to take place is
not known and inadvertent exposure to alcohol-containing products is difficult to avoid. For
these reasons, acitretin is contraindicated in women who intend to become pregnant during
therapy or at any time for at least three years following the discontinuation of therapy. In
addition, acitretin must not be used by females who fail to use adequate contraception for 3
years after discontinuing acitretin [37-40-43].
Drug interactions may be relevant, particularly drugs that interfere with cytochrome P450
metabolism, such as ciclosporin and drugs that compete for plasma protein binding such as
phenytoin. Acitretin has been reported to potentiate the glucose-lowering effect of
glibenclamide. Because of the risk of hypervitaminosis A, concomitant administration of
vitamin A and/or other oral retinoids with acitretin must be avoided [41].
Pretreatment laboratory studies should include pregnancy testing, liver and renal function
tests and lipid studies. If woman of childbearing potential are treated baseline and monthly
pregnancy testing is appropriate. After starting treatment, patients should be monitored with
every other- week for lipid profiles and liver enzymes. After 8 weeks of every-other-week
monitoring, monitoring of lipid profiles and liver enzymes every 6 to 12 weeks can be
instituted. CBC count and renal function test results should be obtained every 3 months [41].

Cyclosporine
Cyclosporine (CSA), an undecapeptide derived from the soil fungus Tolypocladium
inflatum Gams, has been discovered in 1970 and originally used as an immunosuppressive
agent in organ transplantation. It belongs to the family of immunosuppressant drugs known as
calcineurin inhibitors. It was first shown to be effective for psoriasis in 1979. CSA induces
immunosuppression by inhibiting the first phase of T-cell activation. It binds to the
intracellular receptor cyclophillin. The CSA/cyclophillin complex binds to and inhibits the
activity of a key cytoplasmic enzyme, calcineurin phosphatase, responsible for the
dephosphorylation of nuclear factor (NFAT) of activated T-cells. Dephosphorylation permits
translocation of NFAT from cytoplasm to nucleus, thereby activating the T-cell leading to
production of cytokines such as IL-2 and INF-γ. Blocking interleuckin production, therefore,
is a key part of cyclosporine’s ability to disrupt the pathogenic process of psoriasis [40-41-
42].
Despite the recent development of multiple new therapeutic modalities, CSA remains an
important option in treating psoriasis. CSA therapy appears to be effective for all types of
psoriasis, including erythrodermic and pustular psoriasis as well as psoriatic arthritis. CSA is
very useful in severe flare of psoriasis, and in the rapid treatment of psoriasis unresponsive to
other modalities, i.e., as interventional therapy. CSA also has been used in patients who
suddenly need to terminate another systemic therapy or as rotational therapy to reduce the
side effects that may accumulate with the use of anyone systemic therapy [32-41-42].
Current clinical practice dictates that cyclosporine is best used as short-term (induction)
therapy, with long-term (maintenance) therapy being the exception. A microemulsion
formulation is better absorbed from the gastrointestinal tract and shows a superior

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 Types, Triggers and Treatment Strategies of Psoriasis 899

pharmacokinetic profile than older formulations. The recommended initial ciclosporin dosage
is 2.5 to 3 mg/kg in two divided doses. Another approach to dosing of CSA used in patients
with more severe disease is to initiate treatment at the highest dosage, typically 5 mg/kg/day,
with stepwise decreases after adequate disease control is achieved. The higher dosage results
in a better and faster clinical response; it is, however, associated with a higher rate of adverse
effects [41]. A clear improvement of the psoriasis findings is to be expected after about four
weeks and the maximum therapeutic success is seen after about 8–16 weeks. If the patient
does not respond satisfactorily to the initial therapy phase over 4–6 weeks with the lower
dosage (2.5–3 mg/kg), the dosage can be increased by 0.5–1.0 mg/kg/daily every 2–4 weeks
if the laboratory parameters are satisfactory. If the skin response is still unsatisfactory after an
additional four weeks, the treatment with cyclosporine should be discontinued [32-34].
With the short-term therapy (induction therapy) the patient is treated until an adequate
therapeutic success is achieved, generally 10–16 weeks and then cyclosporine is discontinued.
Some studies have indicated that the relapse rate, defined as loss of 50% of the improvement
initially achieved with therapy, is higher and the period until relapse is shorter if cyclosporine
is abruptly discontinued rather than with a slow reduction of the dosage. “Fade-out regimens”
include a reduction of 1 mg/kg every week over 4 weeks or a reduction by 0.5–1 mg/kg every
2 weeks.
Long-term therapy of psoriasis with cyclosporine should only be performed as an
exception and after consideration of other therapeutic options, because of possible adverse
effects [32].
CSA is a drug that requires careful patient selection and subsequent monitoring to be
used safely. Therefore, a careful assessment of psoriasis disease severity is critical when
assessing the risk-benefit ratio of treatment with CSA. The most common cutaneous side
effect of CSA is hypertrichosis, occurring in about 6% of patients; headache occurs in 15% of
patients; paresthesia in 7%, and musculoskeletal pain in 5%.8 Rare incidences of
pseudotumor cerebri in young patients taking concomitant tetracyclines for acne have been
noted. Other neurologic side effects include tremor, asthenia, and fatigue. Gingival
hyperplasia occurs most commonly in patients who have poor oral hygiene; Pulmonary and
respiratory symptoms (cough, rhinitis, and dyspnea) occur in about 5% of patients.
Gastrointestinal side effects include abdominal pain, nausea, vomiting, and diarrhea.
Elevation of serum triglycerides may occur in 15% of patients, whereas
hypercholesterolemia occurs in less than 3% of patients. Importantly, these changes in lipid
levels are reversible after CSA is discontinued. Hypomagnesemia and hyperuricemia may
occur.
CSA’s most serious side effects are nephrotoxicity and hypertension. These two toxicities
are thought to be mediated by CSA’s vasoconstrictive effects on renal arterioles. It is
accepted that risk of renal toxicity is directly related to the dose of cyclosporine, particularly
greater than 5 mg/ kg/day, and the length of treatment. A reduction (25%-50%) in dose of
cyclosporine is recommended if serum creatinine increases by greater than 30% above
baseline value, even if the increased level of creatinine remains within the normal laboratory
range.
Hypertension is another common side effect of CSA therapy that often resolves in
patients treated with short courses of CSA. CSA-induced hypertension occurs more
commonly in older patients. Patients who develop hypertension (measured on two separate
occasions) and who have no history of hypertension should have their CSA dose reduced by
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900 Spyridoula Doukaki and Maria Rita Bongiorno

25% to 50%. If the blood pressure does not normalize after lowering the dose on several
occasions, the package insert recommends stopping CSA. Another approach could be
continuation of CSA as long as the hypertension is appropriately treated and monitored.
Calcium channel blockers are the preferred treatment for CSA-induced hypertension because
of their effect on vasodilation. Other options for treating hypertension include beta blockers
and angiotensin-converting enzyme inhibitors. The use of thiazide diuretics should be avoided
as they can lead to increased nephrotoxicity when combined with CSA. Potassium-sparing
diuretics should also not be used as they act synergistically with CSA to cause hyperkalemia.
Patients with psoriasis treated with cyclosporine may be at increased risk of developing
nonmelanoma skin cancer as compared with the non psoriatic population. This risk is
observed exclusively in patients who have had significant prior PUVA therapy.
Because of these concerns, CSA is a drug that requires careful patient selection and
subsequent monitoring to be used safely. CSA is contraindicated in patients with abnormal
renal function, uncontrolled hypertension or malignancies, and should be avoided in patients
with active infection and immunodeficiency. CSA is not absolutely contraindicated in
pregnancy; the FDA has ranked CSA as pregnancy category C. Increased prenatal and
postnatal mortality and reduced fetal weight have been found in animal studies and there has
been an increased risk of premature birth in babies exposed to CSA in utero. As ciclosporin is
excreted in breast milk, treatment is contraindicated during breastfeeding [32-41].
Numerous drugs interact with CSA. Drug interactions may be of particular concern for
elderly patients with psoriasis, many of whom have a large number of concomitant
medications. CSA is metabolized by hepatic cytochrome P450 enzymes. Drugs that modulate
the activity of this enzyme can shift CSA concentrations outside the narrow therapeutic range.
Thus, drugs (e.g., erythromycin, methylprednisone and amiodarone) may increase serum
levels of CSA by inhibition of cytochrome P450 and conversely some drugs (e.g.,
phenobarbital, orlistat and St John’s wort) may lower cyclosporine levels by induction of
cytochrome P450. Finally, several medications (e.g., nonsteroidal anti-inflammatory drugs
administered long-term) may potentiate renal dysfunction in patients receiving concomitant
CSA therapy [34].
Current consensus guidelines recommend that a range of laboratory values, in particular
blood pressure and serum creatinine, BUN, urinalysis, CBC count, magnesium, potassium,
uric acid, lipids, liver enzymes, and bilirubin including possible exposure to tuberculosis and
hepatitis B or C, should be monitored prior to treatment. After starting CSA, patients should
be monitored with every-other-week blood pressure, BUN, and creatinine measurements,
along with monthly levels of CBC count, uric acid, potassium, lipids, liver enzymes, serum
bilirubin, and magnesium. After 3 months of every-other-week monitoring of blood pressure,
BUN, and creatinine, monthly monitoring of these parameters can be instituted. Particular
care should be taken with elderly patients due to the possibility of renal impairment and with
patients receiving concomitant nephrotoxic drugs [34].
Vaccinations given concomitantly with CSA may be less effective. However, because of
the immunosuppression in patients treated with CSA, killed vaccines may prevent severe
infection and their administration appears to be safe [41].

Other Systemic Drugs

Fumaric acid esters have been approved for the treatment of psoriasis in Europe for
several years. PASI scores improve by up to 80%, but many patients discontinue treatment

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 Types, Triggers and Treatment Strategies of Psoriasis 901

because of gastrointestinal side effects, including abdominal pain and diarrhea as well as
flushing. Lymphocytopenia and eosinophilia are common [44].
Other systemic therapies are used less commonly in the treatment of psoriasis. These
include hydroxyurea, mycophenolate mofetil, 6-thioguanine, sulfasalazine, and azathioprine,
as monotherapy or in combination, on rotational or sequential regimens [44-46].

Biologics
Elucidation of the immunopathogenesis of psoriasis has led to the emergence of new
therapies targeting the immune cells and molecules that induce and maintain the clinical
changes seen in psoriatic plaques. Biologic agents are proteins that can be extracted from
animal tissue or produced by recombinant DNA technology and possess pharmacologic
activity [45]. In psoriasis, these agents are designed to block specific molecular steps
important in the pathogenesis of psoriasis. Many of these drugs are also effective in treating
PsA.
Biological therapies for the treatment of psoriasis and/or PsA are defined by their mode
of action and are classified into three categories, the T-cell modulating agents (alefacept and
efalizumab), the inhibitors of TNF-ablockers, (adalimumab, certolizumab, etanercept,
golimumab, and infliximab), and the inhibitors of IL-12 and IL-23 (ustekinumab and
briakinumab) [47].

Alefacept
Alefacept is a recombinant dimeric fusion protein that consists of the extracellular CD2-
binding portion of lymphocyte function-associated antigen (LFA)-3 linked to the Fc portion
of human IgG1 [33]. It binds to extracellular human CD2 and the Fc portion of human
immunoglobulin IgG1. Alefacept blocks signalling between LFA-3 on antigen presenting
cells and the CD2 molecule on T cells (primarily CD45RO+). Subsequently, the activation
and proliferation of CD45RO+ T cells, which account for approximately 75% of T
lymphocytes in psoriatic lesions, are inhibited. Furthermore, alefacept decreases the number
of pathogenic T cells by binding CD2 on CD45RO+ T-lymphocytes to the FcgRIII (CD16)
receptor on natural killer cells, resulting in granzyme-mediated apoptosis of T cells [47].
Because CD2 expression is higher in memory-effector T lymphocytes than in naive T
lymphocytes (CD45RA1), it selectively causes apoptosis of memory-effector T lymphocytes
[41]. Thus, alefacept reduces circulating CD4+ and CD8+ memory effector T cells and
specifically CD45RO+ T cells, with no changes in naive CD45RA+ T cells or B cells [31].
Alefacept was the first biologic agent approved by the FDA for the treatment of psoriasis
[48]. It is intended for intermittent use. A 12-week course of alefacept given intramuscularly
and dosed at 15 mg/wk allows for a 50% to 75% reduction in PASI in approximately one-
quarter of patients; this improvement may be maintained in some patients for a median
duration of 10 months. Treatment courses may be repeated as often as twice a year. Patients
who respond to alefacept can achieve additional benefit from successive 12-week treatment
course. However, the number of such responders is difficult to estimate. Alefacept leads to
full clearance of symptoms and signs of psoriasis in only a small minority of patients. Several
studies demonstrated that alefacept has a very low onset of action, with maximum response at
18 weeks after starting therapy, i.e., 6 weeks after the last intramuscular shot of the 12- week
course.
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902 Spyridoula Doukaki and Maria Rita Bongiorno

Alefacept has an excellent safety profile when CD4 counts are monitored. A baseline
CD4 lymphocyte count should be performed before treatment and repeated every other week.
Dosing of alefacept should be withheld whenever the CD4 count decreases below 250
cells/mL and dosing should be discontinued if the CD4 count remains below 250 cells/mL for
4 consecutive weeks. Alefacept therapy is not indicated for patients with a CD4 T-
lymphocyte count below normal or in those who are infected with HIV because of the
potential for acceleration of disease progression as a result of CD4 T-lymphocyte count
reduction induced by alefacept. Caution should be exercised in patients who are at risk for or
have a history of malignancy or infection, especially clinically significant infections.
The most common adverse effects are injection site reactions, chills, headache, pturitus,
pharyngitis, flu like syndrome and non specific infections. However, alefacept has been
associated to malignancies (lymphoma and basal cell or squamous cell cancers), and
hypersensitivity reactions (urticaria and angioedema). There also have been some reports of
liver injury and hypersensitivity reactions (urticaria and angioedema). Alefacept should not be
used in conjunction with other immunosuppressive agents because of the risk of excessive
immunosuppression. Alefacept is pregnancy category B [42-45-49].

Efalizumab
Efalizumab is a recombinant humanized IgG1 monoclonal antibody binding to the human
CD11a subunit in leukocyte function antigen-1 (LFA-1) and therefore blocks the binding of
LFA-1 to intracellular adhesion molecule-1 (ICAM-1). Blockade of LFA-1 interferes with T-
lymphocyte activation, trafficking to sites of inflammation and T-lymphocyte recirculation.
Efalizumab also decreases epidermal hyperplasia, ICAM-1 and keratin-16 expression.
Efalizumab is administered subcutaneously by the patient. The recommended dose is 0.7
mg/kg for the initiation dose followed by weekly 1-mg/kg doses thereafter. PASI 75 scores
observed in phase III studies ranged from 22 to 39% at week 12. Efalizumab is not effective
in treating psoriatic arthritis and psoriatic arthritis may develop or recur in a small percentage
of patients during efalizumab treatment of psoriasis.
Efalizumab was well tolerated by most patients. The most common adverse events
comprised flu-like reactions, upper respiratory infections and arthralgias. In 2009, three cases
of confirmed progressive multifocal leukoencephalopathy have been reported in patients on
long-term efalizumab treatment (3 years of treatment) with consequent withdrawal of the
market in Europe and the United States [33-47-50].

Adalimumab
Adalimumab is a fully human anti-TNF-α-monoclonal antibody that specifically binds to
soluble and membrane-bound TNF-α and blocks TNF-α interactions with the p55 and p75
cell surface TNF receptors. The dosing schedule for psoriasis is 40 mg every other week,
beginning 1 week after a loading dose of 80 mg. The dosage is not adjusted for obese
patients (>100 kg) [32].
Adalimumab is a highly effective treatment for chronic plaque psoriasis in adults. There
are no controlled studies available on the use of adalimumab in children with psoriasis [32].
Onset of action is rapid, with significant improvements in disease severity evident within 2
weeks of treatment initiation and maximal disease response seen between weeks 12 and 16.
Response is dose related with 69% of patients achieving PASI 75 at week 12 with
adalimumab 40 mg every other week, and 80% achieving PASI 75 with adalimumab 40 mg

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 Types, Triggers and Treatment Strategies of Psoriasis 903

weekly. Clinically relevant improvements in health-related quality of life indicators are also
reported [50]. Adalimumab is used continuously. Rebound does not typically occur when
adalimumab is discontinued, however, clinical benefits are better maintained with continuous
use and there is loss of efficacy after restart of adalimumab. Continued therapy beyond 16
weeks should be carefully considered in non responding patients within this time period [50].
Compared with methotrexate and placebo, adalimumab proved to have higher rates of 75%,
90%, and 100% PASI improvement and a lower rate of adverse events [47].
Reactions at the injection site are reported in up to 15% of patients. These reactions
usually resolve spontaneously within the first 2 months of therapy. Adalimumab therapy is
associated with an increased rate of infections, including infections of the urinary tract, the
upper respiratory tract, and bronchitis. Severe infections (pneumonia, septic arthritis, post-
operative infections, erysipelas, phlegmonous infections, diverticulitis, and pyelonephritis)
can also occur. Rarely reported hematological effects include thrombocytopenia and
leukopenia. Severe allergic reactions are rare and include exanthema, urticaria, pruritus,
respiratory distress, tightness in the chest, as well as swelling of the mouth, face, lips, or
tongue.
Adalimumab therapy can induce auto-antibodies (ANA, anti-dsDNA antibodies) and
in rare cases lupus-like-syndrome [32]. Anti-adalimumab antibodies develop in 8.4% of
patients and are associated with increased clearance and reduced efficacy of adalimumab (but
not specific adverse events). The addition of methotrexate to adalimumab results in reduced
immunogenicity (i.e., a lower rate of anti- adalimumab antibody formation) and increased
effectiveness (in part due to reduced clearance of adalimumab) with no increase in adverse
events in rheumatoid arthritis patients [50].
Very rare side effects include malignancy, especially lymphoma. Adalimumab is
contraindicated in pregnant women given lacking information on its effects. After stopping
treatment, women should continue to use contraception for up to five months. If pregnancy
occurs during therapy, treatment must be stopped. There are no toxic effects on the embryo
or fetus, and thus normal development of the fetus may be expected (FDA classification:
B). Adalimumab can enter breast milk and should not be used by breastfeeding women.
Women should avoid breastfeeding for at least five months after discontinuing adalimumab
therapy [32].

Etanercept

Etanercept is a a dimeric fusion protein consisting of a portion of TNF-α receptor linked

to the Fc portion of IgG1; It has anti-inflammatory and immunosuppressant properties.
Etanercept has been used successfully in the treatment of several inflammatory diseases and
is currently approved for treatment of moderate to severe plaque psoriasis, psoriatic arthritis,
rheumatoid arthritis, juvenile rheumatoid arthritis, and ankylosing spondylitis. Recentrly, it
has been approved in treatment of chronic severe plaque psoriasis in children and adolescents
from age eight onward who do not tolerate or have not responded adequately to other
systemic therapies or phototherapy.
The recommended adult dosage for the treatment of plaque psoriasis is etanercept 25 mg
given subcutaneously biweekly or 50 mg once weekly. If there is high disease activity, or the
patient is overweight, an initial dose of 50 mg biweekly may be given for up to 12 weeks,
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904 Spyridoula Doukaki and Maria Rita Bongiorno

followed by 25 mg biweekly or 50 mg once weekly thereafter. Onset of action is slow with

clinically significant improvement in disease severity scores evident between 4 and 8 weeks
after initiation of treatment. The maximum efficacy of the drug appears to be reached after 18
– 24 weeks. The efficacy of etanercept has been demonstrated in many clinical trials. At week
12, in 34% of the etanercept group receiving 25 mg twice weekly and 49% of the etanercept
group receiving 50 mg twice weekly achieved PASI75. The clinical responses continued to
improve with longer treatment. At week 24, 44% of those in the 25 mg twice weekly group,
and 59% in the 50 mg twice weekly group achieved PASI75 or more. Continuous therapy
beyond 24 weeks may be appropriate for some adult patients. Overall, continuous therapy
provides better disease control and higher levels of patient satisfaction compared with
interrupted therapy. Some patients will show a loss of clinical response after 12 weeks when
the weekly dose is reduced from 50 mg twice weekly to 50 mg once weekly. Rebound does
not typically occur when etanercept is discontinued. When treatment is stopped, disease
relapses slowly: median time to disease relapse as defined by loss of PASI 50 in those who
achieved PASI 75 after 24 weeks of continuous etanercept 25 or 50 mg biweekly, was 85 and
91 days, respectively. In clinical trials, on retreatment, mean PASI scores were similar, with
the majority of patients achieving equivalent efficacy after 12 further weeks. The 25 mg twice
weekly and 50 mg once weekly dosing regimens are probably interchangeable given that their
pharmacokinetic profiles are comparable [50].
In a study of etanercept treatment for children and adolescents (ages 4-17 years) with
plaque psoriasis who were dosed once weekly with 0.8 mg/kg of etanercept (up to a
maximum of 50 mg), 57% of patients receiving etanercept achieved PASI75 as compared
with 11% of those receiving placebo [32-33].
Given the role of TNF in adipocyte homeostasis and the elevated levels of TNF in obese
patients, the fixed (non weight adjusted) dosing regimen used for etanercept may attain
decreased response rates in heavier patients, particularly with low-dose etanercept [50].
An important issue to consider with etanercept, as with other TNF inhibitors, is the
potential loss of efficacy over time, possibly related to the development of antibodies.
Antibodies to the drug are detected in up to 6% of patients. The antibodies are not
neutralizing and in most patients are only transitory. There appears to be no association
between antibody formation and clinical response or side effects [32-33-50].
Injection site skin reactions occur in up to 37% of patients treated with etanercept and are
mild to moderate, generally not requiring drug discontinuation. Mean duration of reactions is
3 to 5 days; these reactions generally occur in the first month of drug administration and
subsequently decrease [33].
As with other anti-TNF drugs, the potential increased risk of infection is a concern.
Autoimmunity (eg, formation of antinuclear antibodies and antibodies to double-stranded
DNA) has also been observed to occur after treatment with etanercept, although the impact of
treatment with etanercept on the development of autoimmune diseases is currently unknown.
Special care should be taken when administering etanercept to patients with a history of
congestive heart failure (CHF), as treatment with this agent has been associated with
exacerbation of existing CHF and rare cases of new- onset CHF. Vaccination with live
vaccines is also contraindicated [2].
Due to the pharmacokinetics of etanercept, there is no need for dosage modification in
patients with impaired renal and liver function. There have been no reports of increased
etanercept concentrations in patients who experienced acute kidney or liver failure during

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 Types, Triggers and Treatment Strategies of Psoriasis 905

treatment. It is not certain whether etanercept use can increase the incidence of lymphoma.
Undesirable hematological effects and demyelinating diseases are effects of all TNF-blockers
and hence can occur with etanercept [32].

Infliximab
Infliximab is a chimeric (mouse/human) monoclonal antibody against TNF-α. It is an
IgG1 immunoglobulin with human sequences in the constant regions and murine sequences in
the complementarity determining regions of the light and heavy chains. Infliximab binds to
both the soluble and the transmembrane TNF-α molecules, thereby neutralizing the effects of
TNF-α
There are indications that infliximab is not only effective in chronic plaque psoriasis, but
also in erythrodermic and pustular psoriasis. Clinical studies have shown a good level of
efficacy in patients with nail involvement. The dosage used is weight-dependent. Infliximab
is administered intravenously at a dose of 5 mg/kg over 2 to 3 hours at weeks 0, 2, and 6 and
then every 8 weeks [32]. Onset of action is rapid, with evidence of significant improvement
within the first 2 weeks of treatment and maximum benefit by week 10 when 79% of patients
achieve PASI 75. Infliximab is one of the most effective treatments available for induction
therapy in psoriasis vulgaris. The response is largely maintained over time with 74% and 53%
achieving PASI 75 at 6 and 12 months, respectively. Thus, it is also suitable for long-term
therapy [50].
Loss of efficacy over time may occur and correlates with development of antibodies to
infliximab. Administration of low-dose methotrexate concurrently decreases the formation of
antibodies against infliximab which occurs in 19% of patients treated and helps to
maintaining clinical efficacy over time [33].
The commonest side-effects of ifliximab are headache, upper respiratory tract infections,
and increased hepatic enzymes. Acute infusion-related reactions occur in 3–22% of patients
with psoriasis; usually these reactions are mild with chills, headache, flush, nausea, dyspnea,
or infiltration at the infusion site. Anaphylactic shock and delayed hypersensitivity have been
reported rarely. The probability of an infusion reaction is increased in patients with infliximab
specific antibodies. During the infusion, and for one hour after, the patient should be
monitored with the potential for emergency intervention if an infusion reaction occurs. Serum
sickness may occur three to 12 days following infusion. After a long treatment-free interval,
renewed treatment initiation may lead to arthralgia, myalgia, Quincke edema, or other acute
reactions. Administration of an antihistamine or low-dose methotrexate (5 – 10 mg/weekly)
can reduce or prevent a moderate infusion reaction.
Infliximab therapy has been associated with soft tissue infections, candidiasis, fungal
infections, and serious infections including pneumonia, bronchitis, peritonitis, septicaemia,
pyelonephritis, cellulitis, systemic fungal infection and herpes zoster. There are rare reports of
opportunistic infections such as listeriosis, histoplasmosis, cryptococcosis, and pneumocystis
carinii pneumonia. The use of infliximab carries a risk of re-activation and generalization of
pre-existing latent tuberculosis.
A transient and asymptomatic elevation in liver transaminases is well recognized to occur
with infliximab therapy. Rare cases of severe hepatitis and acute liver failure resulting in
transplantation or death have been reported. Infliximab has been associated with exacerbation
of existing cardiac insufficiency. The use of infliximab is not advised in patients with pre-
existing cardiac insufficiency NYHA III-VI. There are also occasional reports of an
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906 Spyridoula Doukaki and Maria Rita Bongiorno

association between infliximab and demyelinating diseases; multiple sclerosis may be

exacerbated by infliximab. There is no indication from registry data of increased risk of
lymphoma with infliximab. An increased risk of skin cancer has been reported. As with other
anti-TNF-α, there are reports of lupus-like reactions. The use of infliximab is not advised in
pregnant or breastfeeding women. Women of childbearing age should use adequate
contraception [32-33-50].

Ustekinumab
Ustekinumab is a recombinant human IgG1 κ antibody. It binds with high specificity and
affinity to the common p40 subunit of the cytokines IL-12 and IL-23 impairing the IL-12 and
IL-23 signaling-dependent maturation and expansion of Th1- and Th17-cells.
Ustekinumab is available as a 45 mg/0.5 ml or 90 mg/1.0 ml injection solution in a pre-
filled syringe. It is given as a subcutaneous injection in the abdomen or thigh. An initial dose
of 45 mg is recommended in week 0, followed by a 45 mg dose in week four and then every
12 weeks. In patients who weigh more than 100 kg the dosage is 90 mg per injection [32].
Both doses of ustekinumab (i.e., 45 mg and 90 mg) are highly effective; onset of action is
evident within 2 weeks, with 67% and 72% of patients achieving PASI 75 by week 12 for the
45 mg and 90 mg doses, respectively, and maximal efficacy evident between week 20 and
week 24. Disease responses are maintained with continued therapy. On cessation of therapy,
median time to relapse (i.e., loss of PASI75) is 15 weeks, with no reports of rebound
psoriasis. Similar response rates are achieved on re-treatment. Dicontinuation should be
considered for those who have not responded by week 28. A large randomized clinical trial
directly comparing ustekinumab to etanercept indicates that ustekinumab is more effective
than etanercept in the short-term and is probably of comparable efficacy to adalimumab and
infliximab, but safety data are very limited. Ustekinumab should therefore be reserved for
patients who have failed or cannot use TNF antagonists.
Safety of ustekinumab in psoriasis has been evaluated in two phase III trials, the
PHOENIX-1 and PHOENIX- 2 trials. Common adverse events reported include upper
respiratory tract infection, nasopharyngitis, arthralgia, cough and headache. Injection site
reactions were uncommon (1,5%), perhaps because of the infrequency of drug administration.
Neutralizing antibodies developed in approximately 5% of patients and were associated with
poorer responses to therapy, but do not correlate with injection site reactions. Occasional
serious side effects reported in the PHOENIX-1 trial included two infections (bilateral
erysipelas of the legs and herpes zoster), both of which were successfully bought under
control. In the PHOENIX-2 trial, one patient given ustekinumab developed a serious
infection, in this case also erysipelas. In both studies together, there were 15 malignancies,
including 11 cases of skin cancer, during the total observation period. No cases of
tuberculosis, demyelination or lymphoma were identified. There was no association with
lymphocytopenia nor there were any cumulative toxic effects reported. There are insufficient
data on the use of ustekinumab in pregnant women. It is also unclear whether the drug can
enter breast milk. The package insert recommends that women of childbearing age use
contraception during treatment and for up to 15 weeks after stopping ustekinumab [32-33].

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 Types, Triggers and Treatment Strategies of Psoriasis 907

CONCLUSION
Several other anti–TNF-α agents are in various phases of development, including
golimumab, a human monoclonal antibody similar to infliximab for subcutaneous
administration, certolizumab, a pegylated Fab fragment of a humanised anti-TNF monoclonal
antibody that neutralises the activity of TNF, and briakinumab, a recombinant fully human,
IgG1 monoclonal antibody targeting the shared p40 subunit of IL-12 and IL-23 [47-51-52].
When planning to initiate treatment of a patient with psoriasis with a biologic it is
important to obtain an age appropriate history and physical examination along with an
updated medication list. In addition, it is also important to obtain a reliable set of baseline
laboratory studies that will allow the clinician to detect and be aware of any underlying
conditions or risk factors [33].
Tests that should be obtain before commencing treatment with biologics include:
chemistry screen with liver function tests, complete blood cell count including platelet count,
a hepatitis panel, and tuberculosis testing. These should be repeated with variable frequencies
thereafter.
Because biologic therapies target the immune system, any steps that can be taken to
prevent infection, such as vaccinations, should be considered. In patients with who need
vaccination, it is preferable to perform these before initiating biologic therapy. Once patients
have begun biologic therapies, physicians should consider the advantages and disadvantages
of administering killed virus vaccines such as influenza. Administration of live vaccines must
be avoided in patients being treated with biologics under all circumstances. While being
treated with biologics, patients need to be periodically re-evaluated for the development of
new symptoms including infection and malignancy [33-53].

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Griffiths CE. Systemic therapies for psoriasis: methotrexate, retinoids, and

cyclosporine. Clin Dermatol. 2008;26(5):438-47.
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[47] Weger W. Current status and new developments in the treatment of psoriasis and
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[48] Villaseñor-Park J, Wheeler D, Grandinetti L. Psoriasis: evolving treatment for a
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[49] Hsu S, Papp KA, Lebwohl MG, Bagel J, Blauvelt A, Duffin KC, Crowley J,
Eichenfield LF, Feldman SR, Fiorentino DF, Gelfand JM, Gottlieb AB, Jacobsen C,
Kalb RE, Kavanaugh A, Korman NJ, Krueger GG, Michelon MA, Morison W, Ritchlin
CT, Stein Gold L, Stone SP, Strober BE, Van Voorhees AS, Weiss SC, Wanat K, Bebo
BF Jr; National Psoriasis Foundation Medical Board. Consensus guidelines for the
management of plaque psoriasis. Arch Dermatol. 2012 ;148(1):95-102.
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AY, Griffiths CE, Jackson K, McHugh NJ, McKenna KE, Reynolds NJ, Ormerod AD;
(Chair of Guideline Group). British Association of Dermatologists' guidelines for
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[51] Myers WA, Gottlieb AB, Mease P. Psoriasis and psoriatic arthritis: clinical features
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[53] Lebwohl M, Bagel J, Gelfand JM, Gladman D, Gordon KB, Hsu S, Kalb RE, Kimball
AB, Korman NJ, Krueger GG, Mease P, Morison WL, Paller A, Pariser DM, Ritchlin
C, Strober B, Van Voorhees A, Weinstein GD, Young M, Horn L. From the Medical
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 37

A NEW STRATEGY FOR THE

TREATMENT OF PSORIASIS—
KERATIN 17 (K17)-TARGETING THERAPY

JiXin Gao and Gang Wang

Department of Dermatology, Xijing Hospital,
Fourth Military Medical University, Xi’an, China

ABSTRACT
Keratin 17 (K17) is an intermediate filament protein present in epithelial cells, which
is mainly expressed in the basal cells of complex epithelia such as nail beds, hair
follicles, sebaceous glands, and eccrine sweat glands, in normal skin. In contrast, in
psoriatic lesions, K17 is aberrantly expressed in the suprabasal keratinocytes.
Furthermore, Keratin 17 is also closely associated with the immune system and plays an
important role in the pathogenesis of psoriasis. Th17 and Th22 cells derived IL-17A, IL-
22 and IFN-γ, could up-regulate K17 mRNA and protein levels in keratinocytes in a
dose-dependent manner. Moreover, these effects are partially blocked with STAT1- and
STAT3-specific inhibitors, as well as small interfering RNA (siRNA) targeting STAT1
and STAT3. On the other hand, the HLA DRB1*04 and/or *07 positive patients show
significant T cell responses to two peptides from the K17 protein selected on the basis of
predicted HLA DRB1*04 and/or *07 bindings. These indicate a K17/T cells/cytokine
autoimmune loop, in which ectopically expressed K17 impacts on the maintenance of
psoriasis by activating autoreactive T cells. Furthermore, it has been found that altered
peptide ligands, which are produced through single alanine residue substitutions at a
critical TCR contact position, abolish the T cell proliferation and IFN-γ production
induced by K17 pathogenic peptides. K17-specific antisense ODNs and RNAi suppress
K17 mRNA and protein expression in psoriatic skin in vivo, which coincides with
marked clinical and histological improvement. In summary, blocking this K17/T
cells/cytokine autoimmune loop could serve as a potential novel strategy in treating
psoriasis.


Corresponding author: Gang Wang, Department of Dermatology, Xijing Hospital, Fourth Military Medical
University, 127 Changlexi Road, Xi’an 710032, China. Tel: 86 29 84775401; Fax: 86 29 84775401; E-mail:
[email protected].
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912 JiXin Gao and Gang Wang

Keywords: Keratin 17, psoriasis, immunoregulation, keratinocytes, positive feedback loop

INTRODUCTION
Psoriasis is a chronic, inflammatory skin disorder characterized by hyperproliferation and
aberrant differentiation of keratinocytes (KCs) (Guttman-Yassky et al., 2011). The overall
prevalence of this condition is 2–3% worldwide, with American and Canadian populations
having a higher rate (4.6–4.7%) than African and Asian populations (0.4–0.7%)
(Christophers, 2001; Perera et al., 2012). In China, the prevalence of psoriasis was 0.47%,
and the prevalence of psoriasis in males and females was 0.54% and 0.44% respectively
(Ding et al., 2012).
The physical manifestation of psoriasis includes the presence of raised, well-demarcated,
erythematous oval plaques with adherent scales, which causes itching and suffering (Bolognia
et al., 2012). Histological manifestations including parakeratosis, acanthosis, and
telangiectasis show that it is a non-infectious disease characterized by abnormal behavior of
keratinocytes (McKee et al., 2005).
Psoriasis is a complicated skin disease, with a high recurrence rate, causing great pain to
patients both physically and mentally. Since traditional therapy usually fails to control
recurrence, new therapies must be explored. Nowadays successful and effective new
treatment explorations offer a better understanding of the mechanisms of the occurrence and
reoccurrence and process of the disease.
However, the extremely complicated mechanism of psoriasis keeps bringing heavier and
heavier difficulties to researchers. The cause of psoriasis is still far from clear, but it has
already been accepted that skin lesions are due to the deregulated interplay between
immunocytes and keratinocytes (Lowes et al., 2007).
In respect to immunocytes, psoriasis has long been thought to be mediated by T cell
related immune imbalance. Later advances confirmed that Th1, Th17, and Th22 cells play a
major role in the development of psoriasis (Guttman-Yassky et al., 2011). The classically
accepted pathogenic mechanism of psoriasis involves stressful stimuli to keratinocytes in
susceptible individuals, which may lead to the production of DNA-antimicrobial peptide
complexes that activate the plasmacytoid dendritic cells (pDCs) (Gilliet et al., 2008).
Activated myeloid dendritic cells (mDCs) induce the differentiation of Th1, Th17, and Th22
cells in the draining lymph nodes (Nestle et al., 2009), and these T cells home into the skin
where they secret cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ,
interleukin (IL)-17, and IL-22. These cytokines activate the keratinocytes and promote their
proliferation and the production of chemokines resulting in the recruitment of more immune
cells into the lesion (Nestle et al., 2009).
Regarding keratinocytes, the changes in the keratin expression profile raises concern.
One of the important features of activated keratinocytes is the change in the keratin
expression profile from keratins 1 and 10 to keratins 6, 16, and 17 (Perera et al., 2012).
Among these, keratin 17 is most closely associated with the pathogenesis of psoriasis.
As a cytoskeletal protein, K17 is overexpressed in psoriatic lesional epidermis, but is not
found in healthy epidermis. So it can be taken as a hallmark of psoriasis (de Jong et al., 1991).

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 A New Strategy for the Treatment of Psoriasis — Keratin 17 (K17) … 913

More importantly, Th1 hyperactivity is also positively related with K17 expression in
psoriatic plaques, as the resolution of plaque commonly accompanied by the polarization to a
Th2 response and the loss of K17 expression at the same time (Nickoloff, 2007). Thus,
overexpressed K17 may take part in the deregulated interplay between KCs and T cells.
While any new insight into this area might offer direct or indirect help to the exploration
of new therapies, it may not happen that fast. The elucidation of the role of Keratin 17 (K17)
in the process of psoriasis might be this kind of contribution.

1. KERATIN 17
1.1. Expression and Location of Keratin 17

Keratin is a family of fibrous structural proteins, which are present in epithelial cells in
general. Keratin is the key structural material making up the outer layer of human skin, and is
widely distribute in various tissues including hair, nails, feathers, and scales, etc. Keratin
monomers assemble into bundles to form intermediate filaments, which are tough and
insoluble and form strong unmineralized tissues found in reptiles, birds, amphibians, and
mammals. In skin, keratins provide mechanical support to keratinocytes to maintain the
integrity of the skin (Schweizer et al., 2006; Windoffer et al., 2011). It is an important marker
of the proliferation of human epithelial cells (Karantza, 2011) (see details at the Human
Intermediate Filament Database, (Szeverenyi et al., 2008) http://www.interfil.org/index.php).
According to the differences in structure, human keratins can be divided into over 50
types. In 1982, Moll et al. for the first time, mapped the keratin profiles using 2D isoelectric
focusing and SDS-PAGE. They grouped the basic-to-neutral type II keratins as K1–K8 and
the acidic type I keratins as K9–K19 (Schweizer et al., 2006). Within which, Keratin 17 (K17;
Mr 48,000) was classified into the group of human type I (acidic) epithelial keratins. Human
K17 contains 432 amino acids and can be divided into 3 domains including head, rod, and tail
(Figure 1) (Gu et al., 2007).
Compared with a mouse, human K17 amino acid sequences are 88%, 96%, 97% alike in
the head, rod, and tail domains, respectively (McGowan et al., 1998). In human and bovine
orthologous K17, the amino acid sequences are also 95% alike in the rod domain and 93%
alike in the tail domain, respectively.
In total, K17 is highly evolutionarily conserved. This high homology usually indicates
vital biological functions of K17.
Although KCs are the main producer and container of keratins, K17 is not found in
normal epidermis. Instead, it is mainly expressed in skin appendages, such as the basal cells
of complex epithelia such as nail beds, hair follicles, sebaceous glands, and eccrine sweat
glands (Troyanovsky et al., 1989; Kurokawa et al., 2011) and is located in the apex of the
matrix and nail bed (De Berker et al., 2000).
Locations of K17 inn the hair follicles and sebaceous glands are suprabasal part of the
infra-infundibulum, sebaceous duct, the outer root sheath below the opening of the sebaceous
ducts, and the companion layer (Kurokawa et al., 2011).
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Figure 1. The homology analysis and immune function on IFN-γ of K17. K17 contains the head, rod,
and tail domains and is of high homology among Human, Murine and Bovine. S1 and S4: Peptide
epitope S1 and S4 induce T-cell and keratinocyte proliferation and IFN-γ production. S2: Peptide
epitope S2 induces T-cell proliferation and IFN-γ production.

In the eccrine sweat gland, K17 is found situated in the luminal cells of the acrosyringium
and intradermal ducts, and myoepithelial cells of the secretory portion (Bragulla et al., 2009).

1.2. Functions of Keratin 17

Keratins are proteins of multiple functions, and their malfunctions are related to various
tissue-specific diseases (Omary et al., 2004). Besides serving as cell framework, more and
more evidence indicates that keratins are also very important to cell behaviors like apoptosis,
wound healing, tissue polarity, and remodeling, not only to cell proliferation and
differentiation (Porter et al., 2003; Chamcheu et al., 2011).
Unsurprisingly, K17 is also a multifunctional protein. According to its locations, K17 is
apparently involved in the differentiation and development of epithelial appendages. Clinical
research disclosed that mutations in K17 gene could result in skin appendage diseases like
steatocystoma multiplex and pachyonychiacongenita type II, autosomal dominant inherited
appendage-related disorders (McLean et al., 1995; Omary et al., 2004; Zang et al., 2011).
Experimental conditions also showed that artificial knock-out of K17 in mice would lead to
severe alopecia during the first week after birth (McGowan et al., 2002). K17 also takes part
in the process of wound healing. K17 could increase cytoplasmic levels of 14-3-3 proteins by
binding with these proteins and consequently activate PI3K/Akt/mTOR signaling and the
upregulation of the protein synthesis rate (Tong et al., 2006). K17 expression is also highly

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upregulated under stress conditions such as viral infection, injury, tumor growth, and
psoriasis, suggesting that it is closely associated with response to stress (Fan et al., 2009;
Mcnairn et al., 2011; Pan et al., 2011; Ide et al., 2012). Finally, K17 could promote basaloid
skin tumor growth, including basal cell carcinoma (Depianto et al., 2010).
K17, together with concomitant changes in cell surface receptors expressions, represents
a characteristic phenotype of activated keratinocytes (Tomic-Canic et al., 1998). This K17-
associated activation process also participates in wound healing, and is essential in re-
epithelialization of the wound area (Perera et al., 2012). Activated keratinocytes are
hyperproliferative and migratory, with cytoskeleton change, amplified surface receptors, and
higher production of constituents of the basement membrane. After successful repairment,
keratinocytes must be deactivated and returned to a state of differentiation. This process is
known as KC activation cycle. In wound healing, the KC activation cycle is completed and
the activation of KCs is a rapid, self-limiting process. By contrast, the failure to resolve the
deregulated inflammatory response in psoriasis leads to the persistent activation of
keratinocytes, which is characterized by prolonged K17 expression (Fu et al., 2012).

1.3. Keratin 17 As an Immune Regulator

Besides other functions, K17’s role as an immune regulator is raising more and more
concern. In the mechanism of causing alopecia, K17 also display immune regulating
behavior. K17 in the cytoplasm could sequest the TNF receptor-associated-death-domain
(McGowan et al., 2002). This sequestering would hamper TNF-α function and rescue
apoptosis in the hair bulb (Tong et al., 2006). This is the reason of the K17 null mice’s
alopecia.
Related evidence also came from studies in basaloid skin tumors. As a very common kind
of skin tumors, basaloid skin tumors can be divided into well-known basal cell carcinoma
(BCC) and basaloid follicular hamartoma, and had long been known to be associated with
aberrant Hedgehog (Hh) signaling. K17 were found in BCC and co-polymerizes with K5 in
vivo more genetic variants in BCC. In K17-KO mice, Hh signaling in epidermis is still
working, while the basaloid follicular hamartoma tumor initiation and growth were delayed.
Profile of inflammatory cytokines was also polarized to a Th2-dominated from Th1- and
Th17-dominated, while the total inflammation was reduced. Hyperplasia and inflammation in
acute dermatitis models constructed on K17 KO mice are also lighter. And reconstitution of
K17 expression in KC induces Th1 chemokines (Depianto et al., 2010).
Our lab has long been concerned with the role of K17 in the pathogenesis of psoriasis.
According to our research, we disclosed the existence of a “K17/T cells/cytokines”
autoimmune loop, which plays an important role in the pathogenesis of psoriasis, and offers a
better interpretation of the persistent and recurrent psoriasis lesions (Shen et al., 2006). In
later sections, the current understanding of K17 with regard to the pathogenesis of psoriasis
and a review of related therapeutic strategies are summarized. Areas of potential
breakthrough in this field, such as upstream regulatory factors and interactive molecules are
also part of the discussion.
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916 JiXin Gao and Gang Wang

2. KERATIN 17 AND PSORIASIS

2.1. Expression of K17 in Psoriasis

Psoriasis is a disease with keratinocytes proliferation and dysdifferentiation, or in another

word, failure to accomplish terminal differentiation.
This defect in maturation is accompanied by a series of cell marker change. Within these,
keratins, as the major product and the most important cell marker of keratinocytes, display a
serial change in their profile.
In normal skin, K5 and K14 are the main keratins expressed in basal cells, and K1 and
K10 are the major ones expressed by suprabasal cells, which are known as differentiation-
related keratins (Figure 2A). However, in psoriasis lesions, there is a great change in this
profile, within which, the reduction of K1 and K10, and the evaluation in K6, K16 and K17
expressions are outstanding (Figure 2B) (Stoler et al., 1988; Thewes et al., 1991). These three
keratins are all known as hyperproliferation-associated keratins, and the exact location of
them in the upper layer of the suprabasal compartment, just beneath the cornified layer
(Figure 2B).

Figure 2. Keratin expression profiles in normal and psoriasis epidermis. (A) In normal skin, K5 and
K14 are expressed in basal layer, and differentiation-related keratins K1and K10 are expressed in the
suprabasal parts. (B) In psoriatic lesions, K5 and K14 expression are elevated and distributed all over
the epidermis including stratum corneum, and K1 and K10 expression generally decreased, while in the
cells in upper spinous and granular layers, K6, K16 and K17 expression are induced, which are abscent
in normal skin.

Among these, K17 is of special importance because it is highly expressed in psoriatic

lesions, but not normally expressed in healthy epidermis. Early in the 1990s, K17 was for the
first time found to be expressed in the psoriatic epidermis (Wilson CL, 1990), which indicate
the special relationship between K17 and psoriasis. K17 expression was later found positively

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associated with the severity of psoriasis, by comparing the expression of K17 in lesional
psoriatic epidermis before and after the treatment of anthralin or Vitamin D3- analogue
MC903, which is markedly reduced during the treatment (de Jong et al., 1991). K17’s
expression in suprabasal epidermis of psoriasis lesions was further verified both in vivo and
in vitro in the following study (Leigh et al., 1995).
These associations support K17 taking part in the pathogenesis of psoriasis, and make it
one of the markers of keratinocyte hyperproliferation in psoriasis (Leigh et al., 1995). But the
significance of K17 expression in psoriasis and the way it affect the process of psoriasis still
call for deeper exploration.

2.2. K17 and Psoriasis-Associated Cytokines

Though K17 has long been known to be highly expressed in psoriasis, in atopic
dermatitis, which is another non-infectious skin disease with similar clinical and pathological
manifestation, K17 expression was not found elevated (Tomic-Canic et al., 1998). This
selectivity indicates the high expression of K17 is not a non-specific result of common
inflammatory response, but under a subtle regulation of local cytokine milieu. The local
cytokine milieu contains cytokines secreted from local extruding immunocytes and
keratinocytes themselves. As mentioned above, Th1, Th17, and Th22 cells are the key
contributors to the pathogenesis of psoriasis. These cells can release a group of inflammatory
cytokines such as IFN-γ, IL-17A, and IL-22 to promote keratinocyte proliferation,
recruitment of inflammatory cells (Bowcock et al., 2005). Overwhelming these cytokines
contribute to psoriatic lesion formation; this supports their importance in the pathogenesis of
the disease (Johnson-Huang et al., 2012; Leonardi et al., 2012; Mitra et al., 2012; Papp et al.,
2012) and call for investigations on what they act on K17 expression.

2.2.1. Regulation of K17 Expression by IFN-

As a well known cytokine, IFN-γ is at first known as a critical cytokine for innate and
adaptive immunity against viral and intracellular bacterial infections and for tumor control. Its
immunostimulatory and immunomodulatory effects are also gaining more and more concern.
Aberrant IFN-γ expression is associated with a number of autoinflammatory and autoimmune
diseases (Schoenborn et al., 2007). In psoriasis, IFN-γ is also a traditional well-known central
cytokine. Early in 1990s, Fierlbeck et al. found that after a month of repeated IFN-γ injections
as a treatment to psoriatic arthritis at that time, visible development of a psoriasis-like lesion
was formulated at the site of injection (Fierlbeck et al., 1990).
Even a single intradermal injection of IFN-γ to an area of clinically normal, non-lesional
(NL) skin of psoriasis patients could induce an psoriatic inflammatory state with increasing
expression of a number of genes in the skin, and many chemokines concomitant with an
influx of T cells and inflammatory DCs, though without visible lesions (Johnson-Huang et al.,
2012).
This revealed that IFN-γ is a key pathogenic cytokine that can induce many features of
the inflammatory cascade of psoriasis (Johnson-Huang et al., 2012). IFN-γ’s central role
hasn’t even been shaded by the discovery of the IL-17A/Th17 axis which has been a great
breakthrough in the pathogenesis of psoriasis (Perera et al., 2012). As a Th1-derived cytokine,
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918 JiXin Gao and Gang Wang

IFN-γ used to be thought to inhibit Th17 cell development in vitro. This point conflicts with
their simultaneously increasing and collaborative contributions in psoriasis. Later research
disclosed that IFN-γ could program myeloid APCs to induce human IL-17+ T cells by
enhancing production of IL-1 and IL-23 and other pathways. IFN-γ also stimulates APC
production of CCL20 to support the migration of IL-17+ T cells (Kryczek et al., 2008).
Direct evidence of the regulation effects of IFN-γ on K17 expression was discovered
sooner. In 1995, researchers discovered that IFN-γ could upregulate the expression of a novel
keratin class I gene that encodes a 432 amino acid protein in the HeLa cell line (Flohr et al.,
1992), which is coincidental with the K17 gene profile found in the same year (Troyanovsky
et al., 1992). Later research on in vitro HaCaT cells (Bonnekoh et al., 1995; Vogel et al.,
1995) further confirmed IFN-γ overexpression in psoriatic lesions consequently induced the
aberrant K17 expression. Compared to atopic dermatitis, a higher expression of K17 in
psoriatic lesions is shown to be associated with higher levels of IFN-γ than atopic dermatitis
(Komine et al., 1996). These clues would be further supported by other studies that showed
that IFN-γ strongly induced the promoter of K17 genes, which will be discussed in a later
section. Notably, K17 is the only keratin reported to be induced by IFN-γ, while no genes of
other keratin were induced. This also further supports the role of K17 as a kind of psoriasis-
associated keratin (Komine et al., 1996).

2.2.2. Regulation of K17 Expression by IL-17 and IL-22

Besides IFN-γ, recently another group of cytokines are getting more and more concern,
IL-17 and IL-22. These two cytokines and their producing cells play great roles in the process
of psoriasis and affect various aspects of keratinocytes’ behavior (Teunissen et al., 1998;
Homey et al., 2000; Nograles et al., 2008).
The most important producing cell of IL-17 is Th17 cells, which are activated by IL-23
and derived its name from producing lineage-defining cytokine IL-17A (Burgler et al., 2009).
Th17 cells also produce cytokines shared with other Th cell subsets, such as IL-22 and IFN-γ
(Volpe et al., 2008; Boniface et al., 2010). However, the direct relationship between IL-17
produced by Th17 cells and K17 expression hasn’t been entirely elucidated. Our lab has long
been concerned with this subject. We found that IL-17A up-regulates K17 on both mRNA
and protein levels in the keratinocytes cell line (HaCat) in a dose-dependent manner. Similar
results were also obtained in both primary human keratinocytes and in a mouse model (Shi et
al., 2011).
IL-22, a cytokine mainly produced by Th22 cells and Th17 cells, is another cytokine that
extensively affects keratinocytes. Recently, our lab disclosed for the first time that IL-22
could also upregulate the expression of K17 (Zhang et al., 2012).
To sum up, Th1, Th17, and Th22 cell cytokines including IFN-γ, IL-17A, and IL-22 are
inclined to promote K17 expression, while Th2 cytokines like IL-4 and IL-10 do not. This
comforts the local infiltration profile of immunocytes in psoriatic epidermis. Therefore, K17
expression arises as a result of a complex interplay of different cytokine networks between
and within cell players (Fu et al., 2012) and serve as a vital link connecting these psoriasis-
associated cytokines and skin lesions.

2.2.3. Signal-Regulated Kinase Pathway of K17 Expression

What is worth more concern is that K17 is the only keratin specifically induced by these
psoriasis-associated cytokines, while other kinds of keratins with higher expression in

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 A New Strategy for the Treatment of Psoriasis — Keratin 17 (K17) … 919

psoriasis lesions like K6 and K16 do not show a clear reaction to these cytokines. This further
supports the unique role of K17 in psoriasis, and calls for further investigation on the
mechanism of how these cytokines work.
The earliest mechanism research focused on IFN-γ. In the K17 promoter sequence, the
IFN-γ activation site (GAS) was identified. This binding site is responsible for IFN-γ
regulation and binds the transcription factor STAT-1 (Jiang et al., 1994). The role of STAT1
on regulation of K17 by other cytokines was later disclosed; cytokines able to induce the
phosphorylation of STAT1 such as IL-6 and leukemia inhibitory factor (LIF) could also
promote K17 expression. Cytokines which do not induce phosphorylation of STAT1, such as
IL-3, IL-4, IL-10, IFN-α, IFN-β, and granulocyte macrophage colony stimulating factor (GM-
CSF), had no effect on K17 transcription (Komine et al., 1996). These data highlighted the
role of STAT1 pathway in the regulation of K17 expression by IFN-γ.
At the mention of IL-17A, our lab found that the IL-17A binds to and upregulates the
activity of K17 promoter DK17p2 (Shi et al., 2011). There are at least three pathways that
have been found in the IL-17A signaling; JAK/STAT, mitogen-activated protein kinase, and
NF-kB pathways (Shen et al., 2008; Ivanov et al., 2009; Shi et al., 2011). Research on U937
monocytic leukemia cells disclosed that among these pathways, IL-17A, in particular, induces
tyrosine phosphorylation of STAT1, 2, 3, and 4 (Subramaniam et al., 1999; Miyoshi et al.,
2011). As far as psoriasis was concerned, data of our lab showed that HaCaT cells pretreated
with IL-17A express phosphorylated proteins of STAT1 and STAT3, whereas untreated ones
do not. This indicates that K17 regulation by IL-17A is also mediated by the STAT1 and
STAT3 signal pathway (Shi et al., 2011).
Applications of STAT1/3 specific inhibitors (Fludarabine to STAT1 and piceatannol to
STAT3) or siRNA targeting to STAT1 and STAT 3 could both potently suppress the effect of
IL-17A on K17 expression. Though p65 and p38 are found to be increasingly phosphorylated
in IL-17A-pretreated KCs, application of their inhibitors (PDTC to p65 and SB203580 to
p38) do not block the increased K17 expression induced by IL-17A at both mRNA and
protein levels (Shi et al., 2011). Thus, STAT1 and STAT3 signaling is an important
transduction that pathway mediates IL-17A’s up-regulation effect on K17 expression.
What’s more, IL-22’s effect on up-regulation of K17 expression is also shown to be
mediated by the STAT 3 signaling pathway (Zhang et al., 2012).
Taken together, regulations of K17 expression by IFN-γ, IL-17A and IL-22 are mediated
by different members of the STAT family (IFN-γ, STAT-1; IL-17A, STAT1/STAT3; IL-22,
STAT3) (Figure 3).
IL-22’s effect on upregulation of K17 expression is also shown to be mediated by the
STAT 3 signaling pathway. What’s more, the ERK1/2 signaling pathway is also found
participating in this process (Zhang et al., 2012). This, for the first time, disclosed ERK1/2
signals as another signaling regulation pathway involved in the K17 expression modulation,
and is being further investigated.
Other research also worth mentioning is an in vitro research on the role of IL-1 in the
IFN-γ’s inducing K17 expression. By using a skin organ culture model, Wei L et al. (Wei et
al., 1999) found that the K17 expression induced by IFN-γ completely rely on endogenous IL-
1β production.
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920 JiXin Gao and Gang Wang

Figure 3. The intracellular STATs signaling pathways mediating K17 transcription regulated by IFN-γ,
IL-17A and IL-22. Binding of IFN-γ, IL-17A and IL-22 to their receptors activate associated JAK
kinase, and consequently phosphorylate STAT molecules. Of which, IFN-γ activates STAT1, and IL-22
activates STAT3, while IL-17A activates both. The phosphorylated STATs form dimers, which
translocate to the nucleus, then bind to the K17 promoter to modulate its transcription.

However, IL-1 was not able to induce the K17 expression in the HaCaT cell line in vitro.
The conflict between these two results may be attributed to different keratinocyte responses
or participation of other immunocytes or cytokines. It remains to be investigated whether IL-
17A and IL-22’s regulation also share the IL-1-dependent pathway.
To sum up, local cytokine milieu in psoriatic lesions plays an important role in regulating
K17 expression at the molecular level. IFN-γ, IL-17 and IL-22 are major players in the
milieu. The regulation of these cytokines is mediated mainly by STATs pathways.
However, there may be other pathways involved in the regulation of K17 expression,
because our attempts failed to totally inhibit the elevated K17 expression induced by IL-17A
and IL-22 by blocking either the STAT 1/3 or STAT3 pathway (Shi et al., 2011).
Now, our group is concerned with other possible pathways, such as the c-Jun N-terminal
kinase and the extracellular signal-regulated kinase pathways. What’s also worth concern
might be the origin of these cytokines and their feedback on the cells producing them.

2.3. Impact of K17 on Psoriatic Autoreactive T Cells

Abnormal activation of psoriatic autoreactive T cells is taken as the central mechanism of

abnormal keratincyte behavior. Dominant T cell clones infiltrate and persist within psoriatic
skin lesions and reappear in psoriasis relapses, but are absent from uninvolved skin in

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psoriasis patients (Chang et al., 1994; Menssen et al., 1995; Vollmer et al., 2001). These T
cell clones are usually found with a conserved amino acid motif in the third complementarity-
determining region (CDR3) of clonally expanded lesional TCR β-chain rearrangements (Prinz
et al., 1999), which indicate that the psoriatic T cell response is directed against dominant
psoriatic autoantigens.

2.3.1. Classic Antigens of Psoriatic T Cells in Different Types of Psoriasis

As a complicated non-infectious skin disease with systemic clinical manifestation and
classification, psoriasis cases of different types or development stages might have different
mechanisms and influencing factors.
Guttate psoriasis is characterized by the formation of small psoriatic lesions roughly 2
weeks after a throat infection. 97% of patients suffering from this type of psoriasis could have
isolated streptococci from their throats (Tervaert et al., 1970). The superantigens from these
streptococci were found activating V beta 2+ T cells in acute guttate skin lesions (Leung et al.,
1995) and further deteriorated this type of psoriasis (Davison et al., 2001).
Psoriasis vulgaris, which consist in over 90% of cases of total psoriasis (Griffiths et al.,
2007), is also often found accompanied by hemolytic streptococci infection at a rate of about
29.3%, while this rate in the common population is only 2.6% (Gudjonsson et al., 2003).
Suffering a ten-times higher infection rate potentially supports group A beta-hemolytic
streptococci’s role of worsening chronic psoriasis vulgaris.
Psoriasis as a non infectious skin disease. The way is influenced by group A beta-
hemolytic streptococci, is a process not executed by those facilitating the infection, but
through inducing a cross-reaction to autoantigens by the M-protein, which is a major
superantigen in psoriasis (Roberson et al., 2010).

2.3.2. Sequences in K17 Serving As Psoriatic T Cell-Epitopes

The vital reason why streptococci M protein induces an autoimmune response in psoriasis
is that some keratins share homologue sequences with the M protein (Valdimarsson et al.,
1995). T cells from the peripheral blood of psoriasis patients displayed a much higher
frequency of activation and production of IFN-γ under stimulation of short K17 peptides,
compared with health controls. What’s more, when epidermal T cells were cleared or at least
diminished by treatment with UVB therapy, the responses to the K17 peptides were also
diminished (Valdimarsson et al., 1995; Gudmundsdottir et al., 1999). This highlighted K17 as
an autoantigen in initiating autoimmune T cell response in psoriasis. Compared with other
peptides, the M and K peptides sharing the “ALEEAN” and “GLRRxLD” amino acid
sequence homolog to streptococci M-proteins could induce IFN-γ production by T cells and
keep T cell responses in a state of higher strength and longer duration (Gudmundsdottir et al.,
1999; Johnston et al., 2004). This explains the unique role of K17 in the pathogenesis of
psoriasis because it serves as a major target for autoreactive psoriatic T lymphocytes. After a
local infection of group A beta-hemolytic streptococci, T cells within local lymphoid tissues,
like the pharyngeal tonsils, are primed to the M-protein, followed by the induction of skin-
homing characteristics. These cells migrate into the skin where they cross-recognize the
ALEEAN sequence in K17 and secrete IFN-γ, then promote the production of psoriasis
related cytokines like IL-17A and IL-22, causing abnormal keratinocytes behavior (Figure 1)
(Johnston et al., 2004).
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922 JiXin Gao and Gang Wang

In 2004, we panned a semi-synthetic phage antibody library, which is against human

epidermal keratin, extracted from psoriatic scales, then cloned and expressed as anti-K17
human monoclonal antibodies (Wang et al., 2004). Later we further broadened the T cells
epitope profile in K17 sequence. As psoriasis is shown to be strongly associated with the
HLA DRB1*04 and HLA DRB1*07 alleles, we searched for HLA DRB1*04, *07-restricted
T cell epitopes by scanning the entire K17 molecule. This routine lead us to the discovery of 3
new immunodominant T cell epitopes on psoriatic K17, which induced a much stronger
proliferation and IFN-γ production by T cells from psoriasis patients, but nearly negligible
responses of T cells from non-psoriatic controls. One of these peptides contained the
ALEEAN sequence mentioned above, while others did not, but had an amino acid sequence
that has not been reported to be recognized by psoriatic T cells (Shen et al., 2005). Maybe
K17 has common immunogenic characteristics with other antigens, but not only with
streptococci. To further verify the effect of these peptides, we used K17-specific analogues of
peptides with T-cell receptor contact residue substitutions (altered peptide ligands). We found
these modified peptides could inhibit psoriatic T-cell proliferation and production of Th1-type
cytokines (Shen et al., 2006). This finding further confirmed T cell epitopes exist on K17
(Figure 2A).
Our findings support that K17 containing T cell epitopes may act as an autoantigen
targeting psoriatic autoreactive T cell, and therefore play a vital role in the
immunopathogenesis of psoriasis. Although antigen present cells like various DCs have long
been found to extensively exist in normal skin and with higher infiltration in psoriatic lesions,
and maybe the primary roles in the pathogenesis of psoriasis (Robert et al., 1999). The
mechanism of how K17 antigens are presented to psoriatic T cells still needs further
investigation.

2.3. K17 and Autoimmune Positive Feedback Loops in Psoriasis

To sum up the interaction between K17 and other psoriasis related cytokines and
immunocytes, and to better understand K17’s role in psoriasis, we constructed K17/T
cells/cytokine autoimmune loops which might be a vital mechanism in the pathogenesis of
psoriasis (Figure 4), based on former findings. Within this loop, T cells activated and
extravasated into the skin producing Th1-, Th17-derived cytokines, to induce K17 expression
in KC, which is in part mediated by activation of the STAT signal pathway. As feedback, up-
regulated K17 containing psoriasis-related T-cell epitopes serving as superantigens then
further enhance and maintain the activated state of pathologic T cells, and further production
of cytokines mentioned above.
This loop offers help on understanding the part of this complicated mechanism more
comprehensively, and also helps disclose knowledge gaps demanding further concern and
exploration. The relationship between this vicious loop and other pathological mechanism of
psoriasis also calls for further investigation.
For example, the way T cells are presented in K17 antigens still lacks direct evidence.
Related clues pointed to skin dendritic cells (DCs).
As the most important innate immune first-line defense barrier, the skin is full of ample
antigen present cells, including dendritic cells in different types, like mDCs and pDCs. DCs

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have long been thought to serve as primary contributors to the pathogenesis of psoriasis
(Robert et al., 1999).

Figure 4. “K17/T cells/cytokines” autoimmune positive feedback loops. The basic scheme of these
loops is the “K17/T cells/cytokines” loop, in which K17 expression is elevated in keratinocyes activated
by IFN-γ. IL-17A, and IL-22 from autoreactive T cells. And up-regulated K17 expression further
activates T cells and maintain their cytokine production. To supplement the details how K17 exposed,
dentritic cells (DCs) could be taken into account. They were activated under stress conditions, and
recognized K17 released from damaged keratinocytes. Then activated DCs containing K17 epitopes
induce T cell proliferation and cytokines production. These further form a “K17/DCs/T cells/cytokines”
autoimmune positive feedback loop. Activated keratinocytes also produce chemokine to further recruit
T cells from blood circulation, which further supplement the origin loop into a “K17/chemokines/T
cells/cytokines” loop.

In psoriasis, DCs were found being activated by antimicrobial peptides secreted by

psoriatic keratinocytes, like cathelicidin (LL-37) (Morizane et al., 2012), accompanied by
production of IFN, TNF-α and IL-6 and consequently amplified Th cells and high Th
cytokines production like IL-1, IL-17 and IL-22 (Lande et al., 2007, Ganguly et al., 2009;
Zaba et al., 2009). These DCs might also recognize and present K17 antigens containing T
cell epitopes, serving as the bridge between T cells and K17.
By inducing pDC activation and mDC maturation, K17 might also serve as the
pathogenic cross talk between stressed keratinocytes and recruited DCs, linking the vicious
with other complicated mechanisms of psoriasis.
The main stream explanation of psoriasis pathogenesis is the immune disorder
dominantly mediated by T cells (Eyerich et al., 2011; Quaglino et al., 2011; Raychaudhuri,
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924 JiXin Gao and Gang Wang

2012), including those of CD8+ T cells, CD4+ T cells, and NKT cells (Balato et al., 2009; Res
et al., 2010; Gunther et al., 2012).
As mentioned above, Th1, Th17, and Th22 and cytokines produced like IFN-γ/TNF-α
(Th1), IL-23/IL-17A (Th17), and IL-22 (Th22) are especially highlighted (Kagami et al.,
2010; Perera et al., 2012). These cytokines are often shown pleiotropic fuctions (Zheng et al.,
2007; Ouyang, 2010; Johnson-Huang et al., 2012; Krueger et al., 2012). They not only
promote keratinocytes expressing K17, but also upregulate a variety of chemokines and
adherence factors production, to recruit pathologic immune cells. They also act on immune
cells themselves directly to produce specific other cytokines that enhance the Th1, Th17, or
Th22 cell reaction. As another important link in this feedback loop, keratinocytes are also
immunocompetent cells that secrete many chemokines under the stimulation of cytokines, and
these chemokines then continue to recruit more immune cells.
By supplementing proper details mentioned above, this loop could be further enriched.
Taking DCs into account, under the stimulation of T cells’ IFN-γ induced by K17 (Shen et al.,
2005), DCs could produce IL-1 and IL-23, which further promote terminal differentiation,
maintenance, and pathogenicity of Th17 and Th22 cells (Perera et al., 2012; Zielinski et al.,
2012). Then a K17/T cells/IFN-γ autoimmune loop could be constructed. The STAT1/3 or
ERK1/2 signaling pathways’ role in up-regulating K17 expression (Shi et al., 2011; Zhang et
al., 2012) disclosed K17/T cells/IFN-γ/DCs/IL-17A or the K17/T cells/IFN-γ/DCs/IL-22
autoimmune positive feedback loops (Figure 4). Once new related knowledge could be
supplemented, we would likely get a more and more comprehensive understanding of the
development of psoriasis.

3. K17 AND TREATMENT OF PSORIASIS

Taking together the unique important role of K17 in psoriasis as mentioned above, and
the expression change after medical treatment, going deeper into what happen to K17 in the
treatment and related mechanisms is helpful to better understand the mechanism of medical
treatment and offer a new break point in exploration of new strategies.

3.1. Treatments in Psoriasis

Topical ointments (such as corticosteroids, calcipotriene, and tacrolimus), systemic

medicines (such as acitretin, methotrexate, and cyclosporine), phototherapies, and biologic
therapies are the four common choices for psoriasis treatment. Among which, biologic
treatments are the latest ones and have been developing fast under the double push from
increasing demand in better treatment effects together with less side effects, and the fast
advance in the knowledge of psoriasis pathogenesis. Currently five biologic agents are
approved by the Food and Drug Administration: alefacept (an anti-CD2 human fusion
protein), efalizumab (a recombinant humanized monoclonal antibody directed against
CD11a), adalimumab (a fully human anti-TNF-α monoclonal antibody), etanercept (a
recombinant human TNF-α receptor protein fused with the Fc portion of antibodies that binds
to TNF-α), and infliximab (a chimeric antibody that binds to TNF-α).

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 A New Strategy for the Treatment of Psoriasis — Keratin 17 (K17) … 925

Detailed information regarding the treatment of psoriasis is available from the

“Guidelines of care for the management of psoriasis and psoriatic arthritis” published in The
Journal of the American Academy of Dermatology (Menter et al., 2008; Menter et al., 2009;
Menter et al., 2009; Menter et al., 2010; Menter et al., 2011).

3.2. K17 in the Treatments of Psoriasis

Dithranol and dimethylfumarate are commonly used gradients in topical ointments

treating psoriasis, and they are shown to suppress upregulated K17 expression induced by
IFN-γ in an in vitro study (Bonnekoh et al., 2001), but without deeper exploration.
Tacrolimus, another effective topical drug treating psoriasis, was also found inhibiting
theSTAT1 phosphorylation induced by IFN-γ in keratinocytes (Tu et al., 2011). The STAT1
pathway is a vital pathway mediating IFN-γ and IL-17’s induction of K17 expression,
tacrolimus might also downregulate K17 expression in keratinocytes. It worth considering
whether other topical drugs share similar mechanism.
There is even less concern on the relation between traditional systemic drugs and K17.
As a classic potent immune inhibitor, cyclosporine A’s effects on K17 also raised concern. A
study on normal human epidermal keratinocytes (NHEK) disclosed that cyclosporine A could
inhibit the production of CXCL1 and CXCL8 induced by IL-17A in keratinocytes, while
vitamin D3 and glucocorticoids also displayed similar effects (Takei-Taniguchi et al., 2011).
This suggests that treating effects might depend on interference of the IL-17A signaling
pathway, and further the downstream protein, K17.
Thus, the change in K17 expression in the treatment of psoriasis still lacks proper
attention while its interaction with drugs also calls for further investigation. Our group is
currently concerned with corticosteroids or calcipotriene (topical ointments)’s effects on of
K17, which might help understand their pharmacological action better.

3.3. K17 As a Therapeutic Target for Psoriasis

The role for K17 as a psoriatic T cell autoantigen and its vital role in the K17/T
cells/cytokine autoimmune loops make it an attractive target for novel therapies treating
psoriasis. If expression of K17 or its relevant events were blocked, the feedback loop might
be interference and consequently improve the lesions.
Our group tried two different strategies targeting K17 or its downstream events (Figure
5). In the first strategy, we used altered analogues peptide ligands of immunodominant
autoantigenic epitopes, which bind to pathogenic autoreactive T cells in competition with
native peptide epitopes.
Altered peptide ligands were shown to abolish the T cell proliferation and IFN-γ
production in autoimmune disease, although the biochemical and cellular mechanisms
underlying the T-cell responses to altered peptide ligands are not clear.
Inspired by this, we adopted single alanine residue substitutions to synthesize several
psoriatic altered peptide ligands.Among which, altered peptide ligands 119R and 355L
effectively down-regulate psoriatic T cells proliferation (Shen et al., 2006).
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926 JiXin Gao and Gang Wang

What’s more, these peptide ligands also significantly down regulate IFN-γ and IL-2 level,
as well as up-regulate IL-4, IL-10, and TGF-β. Even supernatants from PBMCs stimulated
with these altered peptides suppressed keratinocyte proliferation.

Figure 5. K17-targeting strategies for the treatment of psoriasis. (A) Altered peptide ligands compete
with wild-type K17 epitope binding and modulate K17-specific T cell response. (B) K17-specific
antisense ODN (ASODN) and siRNA inhibit K17 gene translation in keratinocytes. K17-ASODN and
siRNA specifically binds to the coding region of K17 mRNA, then hinder the translation process or
causes mRNA cleavage, respectively.

These result indicate that the altered peptide ligands could be adopted as a effective
immunomodulatory drug treating psoriasis.
The second attempt targeted the expression stage of K17. We constructed K17-specific
antisense ODN (ASODN) and siRNA and found that both ASODN and siRNA reduced K17
expression both at mRNA and protein levels, and consequently inhibited growth and induced
apoptosis in keratinocytes, (Chang et al., 2011). Feasibility examination is further played on
the SCID-hu xenogeneic transplantation model, and this in vivo verification succeeded again.
The mouse model showed marked clinical improvement, with an almost complete clearance
of erythema and scales, while the control group still had typical psoriatic lesions. The
pathological performances were also greatly improved after treatment, as proven by relevant
disease parameters, such as epidermal thickness, number of inflammatory cells and
parakeratosis in the transplantation model.
The disclosure of the theoretical feasibility of the two strategies mentioned above present
an optimistic aspect of K17 targeting therapy. More strategies following different routines
should be further explored, as well as careful and positive further clinical trials. These
strategies might offer potential efforts to the future treatment of psoriasis.

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4. FUTURE PERSPECTIVES
Until now, the mechanism of K17 up-regulation and its biological role in psoriasis have
already been highlighted. Analysis of this data led us to propose the existence of a K17/T
cells/ cytokine autoimmune loop.
However, a comprehensive understanding of this relationship depends on the further
investigation of the regulation mechanism and immunomodulation of K17 in psoriasis. Future
studies are needed to investigate K17 related cytokines, transcription factors and signaling
pathways, immunomodulation, and exploitation of new therapeutic strategies targeting K17.
Although IFN-γ, IL-17, and IL-22’s dominant role of affecting K17 is hard to shake, the
actions of other cytokines on K17 regulation in both healthy and psoriatic keratinocytes, and
how these cytokines affect immunocytes themselves, are still worth study. What’s more, as a
famous multifunctional cell type, keratinocytes are also worth further exploration as to
whether they themselves could produce such cytokines, to provide positive feedback on
immunocytes or act upon themselves in the way of an autocrine. Therefore, further studies on
its regulation on T cell response will allow better understanding of the pathomechanisms
involved in psoriasis, and reinforce the role of K17 as a therapeutic target for psoriasis.
As transcription factors and signaling pathways involved in the pathogenesis of psoriasis
are complicated, the transcription modulation of K17 expression still calls for further
investigation. Besides previously disclosed STAT1/3 and ERK1/2 signaling pathways and
IFN-γ activation sites (GAS) on K17 promoters (Jiang et al., 1993), there are more promoter
binding sites and related signaling pathways and transcription factors worth further
investigation. A systematical investigation of the promoter region of the human K17 gene
disclosed 8 protein binding sites in total, of which 5 were binding sites for known
transcription factors NF1, AP2, and Sp1, while the left 3 were binding sites for as yet
unidentified proteins (Milisavljevic et al., 1996). Other transcription factors including Gli and
BRCA1 were also shown to directly bind the K17 promoter and consequently activate the
K17 transcription in a study on basal cell carcinoma and breast cancer (Callahan et al., 2004;
Gorski et al., 2010). Further investigations on these mechanism would help gain more
comprehensive understanding of K17 functions, and their role in psoriasis or other non
infectious skin diseases.
Post-translational level regulation of K17 expression is also worth deeper concern. K17-
Ser44 is shown to be a phosphorylation target of ribosomal protein S6 kinase (RSK1) through
the ERK1/2 signal pathway under stress conditions, and its phosphorylation is associated with
stress related protein synthesis and cell growth of keratinocytes. The stress factors activating
these pathways might come from serum, epidermal growth factor, 12-O-tetra-
decanoylphorbol-13- acetate, UV irradiation, and hydrogen peroxide (Pan et al., 2011). The
development of psoriasis is also highly related with stress conditions, so further investigation
of K17 phosphorylation’s role in the pathogenesis of psoriasis is worthy.
K17’s role as immunomodulator is steadily raising more advances and new gaps. Besides
the modulation function mentioned above (Depianto et al., 2010), K17 is also found
modulating the PI3K/AKT/mTOR pathway via direct interaction with 14-3-3δ protein in
keratinocytes (Kim et al., 2006), and interacting with a novel partner, AnxA2, to contributes
to functions downstream from the epidermal growth factor receptor activation in tumor cells
(Chung et al., 2012). These results further support K17’s role as an immunomodulator, which
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928 JiXin Gao and Gang Wang

might act through proper signal transduction pathways via direct interactions with other
proteins. These immune-modulation functions of K17 may also take part in the pathogenesis
of psoriasis and might depending on its interaction with specific proteins and by regulating
the cell signaling pathways in psoriatic keratinocytes, such as the PI3K/AKT/mTOR pathway
and 14-3-3 protein. Thus the positive feedback loop could be further supplemented into a new
autoimmune positive feedback loop, “K17/chemokines/T cells/cytokines” (Fig 4), on which
our group is still working.
Common treatments to psoriasis may inhibit K17 expression in keratinocytes, leading to
therapy exploitation targeting K17 and related mechanisms.
Our research disclosed two routines against K17’s malfunction, while there should be
other rountines to explore. For that it is unclear how the K17 antigen is exposed to APCs and
T cells, the strategy of blocking K17 antigen exposure calls for related fundamental research
which had not yet begun.

CONCLUSION
As keratins have a unique relationship with psoriasis, K17’s multiple functions and
complicated relations with other pathological cytokines and immunocytes in psoriasis make it
an object of both fundamental and clinical research. To date, IFN-γ, IL-17A, and IL-22 are
known as major cytokines inducing K17 expression in psoriatic keratinocytes, mainly through
STAT1/3 signaling pathways.
Several psoriasi-specific T cell epitopes existing in K17 amino-acid sequence make it an
indispensable activator to autoreactive T cells. Consequently the elevated cytokines,
mentioned above, produced by these activated T cells, majorly Th1, Th17 and Th22 cells
further induce higher K17 expression. Thus a K17-related positive K17/T cells/cytokine
autoimmune feedback loop is constructed, which straightened the complicated mechanism of
psoriasis to a large extent. The concept of this loop also offered theoretical feasibility for
strategies targeting K17 to treat psoriasis, which our group is studying, and positive advances
have been obtained. Future studies to better understand the detail of the feedback loop and
interference ways would facilitate new strategies of exploitation targeting K17 and related
regulation pathways to improve psoriasis symptoms.

ACKNOWLEDGMENTS
The authors are grateful to Dr. Xiaowei Shi, Dr. Liang Jin, Dr. Meng Fu, Dr. Zhu Shen,
Dr. Ting Chang, Dr. Wei Zhang and M. S. Erle Dang for their contributions to the research.
The study was supported by the National Natural Science Foundation of China (No.
30972805, 30376334 and 81174114, Gang Wang).

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 38

NARROW-BAND ULTRAVIOLET LIGHT B (UVB) AND

PSORALEN PLUS UVA EFFECT IN THE CIRCULATING
LEVELS OF BIOLOGICAL MARKERS IN PSORIASIS

Susana Coimbra1,2* and Alice Santos-Silva1,3

1
Instituto de Biologia Molecular e Celular (IBMC),
Universidade do Porto, Porto, Portugal
2
Instituto Politécnico da Saúde Norte (IPSN), Cooperativa de Ensino Superior Politécnico
e Universitário (CESPU), Gandra-Paredes, Portugal
3
Departamento de Ciências Biológicas, Laboratório de Bioquímica, Faculdade de
Farmácia, Universidade do Porto, Porto, Portugal

ABSTRACT
The main goal of psoriatic therapies is to try to control the disease and its clinical
manifestations, contributing to improve the quality of life of the patients. The choice of
psoriasis therapy depends on many factors, including the severity of the disease, the skin
type, the effect on patient’s quality of life, the response to previous psoriatic treatments,
patient’s age and clinical history. A chronic, unpredictable course of the disease and the
need for periodical alternation of drugs or classes of drugs, make psoriasis difficult to
treat.
A variety of approaches are available for its treatment, ranging from topical agents,
for milder and limited forms of psoriasis, to phototherapy, photochemotherapy, systemic
and biological agents, for moderate and severe psoriasis. The ultraviolet light B (UVB)
irradiation, phototherapy, which includes broad-band UVB irradiation and narrow-band
UVB irradiation (NB-UVB), and the photochemotherapy, with psoralen plus UVA
(PUVA) irradiation are usually limited to patients with moderate and severe psoriasis.
The UVB irradiation leads to a reduction in the synthesis of DNA, RNA, and
proteins, decreasing the rate of mitosis of the epidermal cells. Furthermore, UVB has the
ability to reverse the disturbed vessel architecture of the skin in psoriasis plaques,
bringing the elongated capillary loops back to a normal state, and exerts

*
Corresponding author: Email: ([email protected]).
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938 Susana Coimbra and Alice Santos-Silva

immunomodulatory effects, suppressing the pro-inflammatory axis. NB-UVB offers

significant therapeutic advantages over broad-band UVB, with lower erythemogenecity
and with faster clearing of the lesions, and a more complete disease resolution. Moreover,
the immunomodulatory effect appears to be more pronounced with NB-UVB than with
broad-band UVB. PUVA, combining the UVA irradiation with the photosensitising agent
psoralen, is known to have immunomodulatory effects and to promote inhibition of DNA
synthesis, preventing replication of keratinocytes and inducing death of activated T cells
in the skin. Moreover, it seems that PUVA inhibits angiogenesis.
The majority of PUVA versus NB-UVB studies evaluated the clinical improvement
of psoriasis, and their results are sometimes controversial. Studies evaluating and
comparing the impact of NB-UVB and PUVA in the levels of important biological
markers are less common. In studies previously performed by our team, promising
biological markers of psoriasis severity were found. In this review, we will focus on the
effects of NB-UVB and PUVA in the levels of some potential biomarkers, as any attempt
to identify predictive biologic markers of severity and monitoring of psoriasis should be
encouraged.

INTRODUCTION
Psoriasis, a chronic erythematosquamous dermatitis that affects about 2-3% of the world
population, is histologically characterized by epidermal hyperplasia (acanthosis), dilated and
prominent blood vessels in the dermis, and an inflammatory infiltrate of leukocytes,
predominantly in the dermis. Nowadays, it is believed that the onset of psoriasis is similar to
an immune reaction, which is composed of a sensitizing phase, a silent phase and an effector
phase [1]. The latter presents also three stages - skin infiltration of immune cells, immune cell
activation and keratinocyte response. It has been proposed that different cells play a dominant
role in psoriasis at different stages [1], and that the interleukin (IL)-23/T-helper (Th)17 axis is
crucial in psoriasis pathogenesis. Its inhibition appears to be critical for therapeutic
achievement [2, 3].
The main goal of psoriatic therapies is to try to control the disease and its clinical
manifestations, contributing to improve the quality of life of the patient. The choice of
treatment for psoriasis depends on many factors, including the severity of the disease, the skin
type, the effect on patient’s quality of life, the response to previous psoriatic treatments,
patient’s age and clinical history. A chronic, unpredictable course of the disease and the need
for periodical alternation of drugs or classes of drugs, make psoriasis difficult to treat.
A variety of approaches are available for its treatment, ranging from topical agents, for
milder and limited forms of psoriasis, to phototherapy, photochemotherapy, systemic and
biological agents, for moderate and severe psoriasis. These therapies can help to minimize the
lesions and to prolong the remission periods of the disease. The associations of different
therapies, as well as, the alternation of the type of therapies are common clinical practices, to
maximize beneficial effects and minimize adverse reactions.
There are three types of ultraviolet (UV) radiation, the UVA (400-320 nm), the UVB
(320-290 nm) and the UVC radiation (290-200 nm), but the latter does not reach the land
surface. UVA radiation can be classified in UVA I (400-340 nm) and UVA II (340-320 nm).
Concerning UVB radiation, it can be used as broad-band (BB)-UVB (290-320 nm) and
narrow-band (NB)-UVB irradiation (311-313 nm), which was more recently introduced.
Phototherapy includes the irradiation of patients with UVA, UVB or those submitted to

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 Narrow-Band Ultraviolet Light B (UVB) and Psoralen Plus UVA Effect … 939

sunlight exposure. In photochemotherapy, photosensitising agents are associated to non-

ionizing radiation; the combination of psoralens with UVA (PUVA) is the combination more
frequently used. The photochemotherapy PUVA and the NB-UVB are among the more used
therapies in moderate and severe psoriasis.
The UV radiation is known to present immunosuppressive/ immunomodulatory
properties. There are immediate and delayed effects of UV irradiation in psoriasis patients.
Membrane lesions induced by lipid peroxidation, DNA lesion and transcription of induction
factors are some of the immediate effects. The delayed effects reflect the action induced in the
cells, due to immediate effects of UV, with modulation of the psoriatic lesion
microarchitecture.

PHOTOTHERAPY – A BRIEF HISTORIC APPROACH

The benefits of sunlight for psoriasis were known long before phototherapy units were
introduced for the treatment of psoriasis. The first reports about medicinal use of sunlight are
dated of 1400 B.C. BC. During centuries, from the Han to the Tang Dynasty, the Chinese
civilization used sun radiation therapeutically, believing in the mythical power of the sun.
There are also, reports about sunlight use from Old Egyptian, the Greek and the Roman
civilizations. In the XVII, XVIII and XIX centuries, several findings related to sunlight
components and characteristics gave an all-new perspective for its clinical applications [4, 5].
The history of modern phototherapy begun at XIX century, when was described that the
sunlight beneficial effects resulted from gamma radiations from the ultraviolet zone. Niels
Finsen used for the first time artificial ultraviolet light, carbon arc lamps, in the treatment of
Lupus vulgaris. The first book about phototherapy, from Willibald Gebhardt, referred several
applications for its use, namely in psoriasis therapy, but also in syphilis, acne, leprosis and
pellagra. Sunlight radiation was progressively substituted for artificial radiation, first carbon
arc lamps and later for more efficient ones, such as mercury vapour lamps. During the first
and second world war, phototherapy was used to treat cutaneous ulcers. With the finding of
the first antibiotic, the use of phoptotherapy declined, but it raised again with its application
to treat venous leg ulcers [4, 5].
In 1925, Goeckerman used the combination of UVB with coal tar in psoriasis therapy,
which was used over fifty years, until PUVA photochemotherapy was described. Parrish and
co-workers, in 1974, used, successfully, for the first time, the combination of UVA radiation
with 8-methoxipsoralen in the treatment of psoriasis; the use of 5-methoxipsoralen was
posterior. Van Weelden and Green, in 1988, implemented the use of NB-UVB [5]. At end of
the eighties, BB-UVB, NB-UVB, PUVA and UVA-1 were available as options for psoriasis
treatment; even nowadays, although some modifications in the protocols were made to
achieve more efficiency, security and less toxicity, its use is widely spread. Indeed, according
to Carvalho et al. [6], these forms of intervention remain essential methods of treatment for
psoriasis vulgaris in the 21st century.
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940 Susana Coimbra and Alice Santos-Silva

UVB THERAPY
The UVB therapy is a good choice for patients with moderate and severe forms of
psoriasis, and for those that do not respond to topical treatment. The irradiation with UVB
leads to a reduction in the synthesis of DNA, RNA, and proteins, decreasing the rate of
mitosis of the epidermal cells. Furthermore, UVB has the ability to reverse skin disturbed
vessel architecture in psoriasis plaques and to bring the elongated capillary loops back to a
normal state [7]. It is generally accepted that UVB suppresses both contact hypersensitivity
and delayed-type hypersensitivity responses in humans and animal models [8]. UVB
irradiation is known to induce the production of the anti-inflammatory cytokine IL-10 [9],
which inhibits the production of IL-1, IL-1, IL-6, IL-8 and tumor necrosis factor (TNF)-
[10]. The general immunomodulatory effect of UVB seems to be a shift in the cytokine
profile, from Th1 to Th2 type cytokines [11]. UVB interferes with cytokine expression,
suppressing type-1 cytokine responses in normal skin and in psoriatic lesional skin, leading to
a decrease in the number of T cells invading the skin [12-14]. In summary, UVB irradiation is
beneficial in psoriasis, by decreasing the production of pro-inflammatory cytokines, by
inducing the production of anti-inflammatory cytokines, and by suppressing and interfering
with the activity of several cells, like T cells.
The initial dose of UVB irradiation and the increment of UVB irradiation in each session
of treatment in phototherapy regimens, depends on Fitzpatrick skin types or on minimal
erythema dose [15], and requires, to be effective, at least three treatments per week, for a
significant period of time.
NB-UVB offers significant therapeutic advantages over BB-UVB, with faster clearing,
more complete disease resolution, and with lower erythemogenecity [16]. The exposure to
NB-UVB also reduces natural killer cell activity [17], and seems to switch the activity of
neutrophils, thus, acting also on the innate defences [7].
Dermal T cells obtained after exposure to NB-UVB irradiation, displayed a reduced
capacity to express interferon (IFN)-, as compared to dermal T cells isolated before pre NB-
UVB irradiation; thus, a change occurred in the phenotype of the T cells remaining in the skin
after NB-UVB exposure [14].
Broad-band UVB has been proposed to have immunosuppressive effects in psoriasis
through induction of apoptosis of T cells, whereas NB-UVB has been reported to be directly
cytotoxic to T cells in vivo, leading to a higher depletion of dermal and epidermal T cells in
psoriatic skin. NB-UVB has also an immunomodulatory effect, by suppressing the pro-
inflammatory axis, and by reducing the pro-inflammatory cytokine production by individual
T cells [13]. This immunomodulatory effect appears to be more pronounced with NB-UVB
than with BB-UVB [16].
The NB-UVB initial dose is also dependent on the minimal erythema dose testing or on
Fitzpatrick skin types, and an increasing dose schedule is used, normally, with three sessions
per week, until reaching a maximum dose of 2.5 J/cm2.
In NB-UVB phototherapy, the clearing of psoriasis lesions seems to be achieved with a
median of 28 exposures [18]. The median time to relapse is about 4 months [18], and very
few patients are still clear 6 months after the treatment [19]. Usually, it is well tolerated, but
side effects can occur, namely, erythema and drying of the skin, which can cause pruritus.

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Some concern has been raised about the potential rise for skin cancer of psoriasis patients
treated with NB-UVB. As it is a recent type of therapy, there are few epidemiological studies
addressing this issue. Ferahbas et al. [20], by performing the micronucleus test, showed that
NB-UVB treatment ´may cause a detectable chromosome damaging effect. Multicentre
studies are required, involving several thousands of new patients per year, followed for 10
years or more. Other studies, such as cytogenotoxic studies are also required to clarify the
mutagenic potential of phototherapy [19]. Nonetheless, NB-UVB has been used in almost any
patients, regardless of comorbidity, and it has been used also in pregnant women and children
[7].
Broad-band and NB-UVB can be used alone or combined with a variety of topical and
systemic agents, to achieve faster and more effective results. However, caution must be used
when combining phototherapy with other agents, to avoid adverse effects, including increased
photosensitivity and burning, or even a shortened remission. When retinoids are combined
with UVB phototherapy, the doses of retinoids can be dramatically reduced, sparing patients
to the side effects of high-dose oral retinoids, and a more rapid and more effective clearing of
psoriasis may be achieved. As referred, phototherapy can also be combined with coal tars,
known as the Goeckerman regimen, which can improve the clinical response and provide
long-lasting results. However, as it requires a significant effort of the patient, because coal
tars are unpleasant to use and can compromise patient’s compliance, it is best suited for those
who are very motivated, with diffuse or severe psoriasis. Another therapeutic combination
that may improve efficacy, is the use of anthralin in combination with UVB - the Ingram
method [21].

PUVA THERAPY
PUVA photochemotherapy, is usually used in patients with moderate and severe
psoriasis, with a long history of the disease, with more than 30% of body surface
involvement, with thick plaques, with involvement of hand, soles, and/or nails, and in patients
that were previously unresponsive to UVB [7].
As referred, PUVA therapy combines the UVA irradiation (320-400 nm) with psoralen,
as photosensitising agent. The psoralens, also known as furocouramins, are naturally
occurring or synthetic tricyclic aromatic compounds with photosensitization activity. The 8-
methoxipsoralen, 5-methoxipsoralen and trimethylpsoralen are the most frequently used.
These agents can be administered orally or topically, by applying a lotion directly in the
lesion or by immerging the lesion in a bath. After administration, the psoralen reaches a peak
level in the skin between 1 to 3 hours, depending on the formulation used. After a medium
period of time of 1 hour and half, patients are exposed to UVA, which activates psoralen.
The exact mechanism of action of PUVA is not completely clarified, but it is known to
have anti-proliferative, anti-inflammatory and immunosuppressive properties. Psoralen, once
activated, induces the formation of pyrimidine dimmers and the crosslinking with DNA
strands, preventing replication of keratinocytes and inducing death and impairment of
activated T cells in the skin [22]. PUVA leads also to a depletion of Langerhans cells from
epidermis. In vitro studies showed that it induces apoptosis of human microvascular
endothelial cells [23], suggesting that it inhibits angiogenesis. PUVA also normalizes the
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942 Susana Coimbra and Alice Santos-Silva

enhanced chemotaxis of neutrophils and monocytes in psoriasis [24, 25]. Moreover, PUVA
seems to regulate the expression of adhesion molecules [26]. PUVA is considered a remissive
therapy, as it is able to induce long-lasting improvements in the symptoms of psoriasis, after
treatment discontinuation [27].
The dose of psoralen, by oral administration, is defined by body weight (0.6 mg/kg) or by
body surface area (25 mg/m2) of the patient. The 5-methoxipsoralen absorption rate is 25% of
the absorption rate of 8-methoxipsoralen, if both were administered in the same dose;
therefore, 5-methoxipsoralen would be less efficient that 8-methoxipsoralen when used in the
same dose [28]. The ingestion of food one hour before and one hour after 8-methoxipsoralen
ingestion must be avoided, since it delays and diminishes its absorption [29]. The initial dose
and the increment in UVA irradiation during the treatment depends on the minimal
phototoxic dose or on the Fitzpatrick skin type. The patients should be treated three to four
times per week, usually, for a significant period of time [30], until a maximum dose of 12
J/cm2. Eyes and genitals must be shielded during the irradiation procedures.
The side effects more referred for PUVA treatment are erythema, itch, irregular
pigmentation, xerosis, and gastrointestinal alterations, such as nausea and vomiting. The
gastrointestinal effects of psoralen can be controlled by dividing the dosage or by ingesting
foods, like milk, at the time of psoralen administration. Another potential side effect is related
with its phototoxicity. To avoid it, patients should not be exposed to sunlight on the days of
psoralen administration, to prevent burns. With long-term therapy, many patients develop
PUVA lentigines, which are small black macules in PUVA-exposed sites [21]. If protection
of the eyes is not used during UVA exposure, PUVA therapy can cause cataracts. Male
genitalia and lower limbs are sensitive to the development of squamous cell carcinomas, and
they should also be shielded. Indeed, PUVA therapy has been associated with a high risk for
squamous cell carcinomas of the skin, and the risk of other non-melanoma cutaneous
malignancies appears to increase also [31]. An increased risk of malignant melanomas has
been correlated with the number of treatments and the time of exposure of the patients [32].
Hamurcu et al. [33] concluded that PUVA treatment causes a detectable chromosome
damaging effect on the relatively profound cells/tissues of patients. No doubt, the side effects
that raise more concern for the clinicians are carcinogenesis and photoaging.
Several conditions are a contraindication for PUVA therapy, like photodermatoses,
history of skin cancer, trichothiodystrophy, pregnancy, child age, concomitant
immunosuppressive therapy, history of oftalmologic disease and disturbance of hepatic
function [7]. Some drug interactions can occur if the patient is under treatment with other
photosensitising agents, as some neuroleptics, diuretics, non-steroidal anti-inflammatory,
antifungal and antibiotics drugs.
A topical photosensitising method with psoralen can also be used, offering advantages
over the oral PUVA - psoralen blood levels are lower and the UVA dose needed is also lower,
reducing the cumulative doses. The topical application of psoralen is performed by immersion
of either localised areas or the whole body in water containing 8-methoxypsoralen capsules,
prior to UVA exposure. In case of immersion of localised areas, an unnecessary exposition to
UVA radiation of the areas without lesions and the adverse symptoms, such as nausea are
avoided. The immersion in the bath should be performed for 15 minutes and the exposure to
UVA light should occur between 20 minutes to 3 hours after the bath. The topical application
of psoralen presents a higher photosensibility and, therefore, lower doses of UVA radiation
should be used, as compared to oral PUVA. Despite its advantages, the provision of bathing

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facilities has economic, logistic and sanitary implications, reducing its application in clinical
practice.
The use of a cream psoralen-UVA combined therapy has been developed as a new variant
for topical PUVA therapy, which is easy to perform and less expensive [34].
The advantages of topical PUVA therapies are the lack of gastrointestinal and hepatic
side effects, no need for eye protection and, since it requires smaller cumulative UVA doses,
it appears to have a lower risk of skin cancer [35, 36]. The importance of topical PUVA lies
in the fact that it can be used to treat local disease spots, like soles and palms, without
exposing healthy body skin to UVA irradiation.
PUVA can be associated with other drugs, such as oral retinoids. This combination is
synergetic, reduces the side effects for both PUVA and retinoids [21].

NB-UVB VERSUS PUVA

As referred, UV radiation presents with immunosuppressive and immunomodulatory
effects. UV light is thought to exert an immunomodulatory effect, via acute and sub-acute
changes according to UV exposures. Long exposures or high doses of UV radiation induce
acute changes, such as membrane damage, production of cytoplasmic transcription factors,
and isomerisation of urocanic acid; the sub-acute changes include alteration of the antigen
presenting cell populations, modification of cell-cell signalling, and epidermal depletion of
Langerhans cells and T cells [9].
UVB radiation is known to promote alterations in the epidermis, while UVA radiation is
known to induce also dermic alterations. UVA radiation affects cells present in the dermis,
like fibroblasts, dendritic and endothelial cells, and some inflammatory cells, such as T cells,
mast cells and granulocytes. UVB radiation affects the function of epidermal keratinocytes
and Langerhans cells, the principal antigen-presenting cells of the epidermis [37]. UVB target
is nuclear DNA, inhibiting DNA synthesis and, consequently, diminishing the proliferation of
keratinocytes from the epidermis. Thus, UVB radiation leads to a reduction in the synthesis of
DNA, RNA and proteins, and therefore, to a diminished cell division rate. Erkin et al. [38]
compared the effect of PUVA and NB-UVB on dendritic cells and activated lymphocytes in
psoriatic lesions, and both types of treatment showed equal capacity to reduce lymphocytes,
macrophages and dendritic cells; PUVA also decreased epidermal Langerhans cells. Both UV
radiations were referred to be capable of interfering with Langerhans cells, affecting their
antigen-presenting function. Both UVB and PUVA therapies are capable of inducing
alterations in T cells, and after these treatments a cutaneous reduction of T cells seems to
occur. Bukulmez et al. [39] found a significant decrease in the levels of adenosine deaminase,
which is a non-specific marker of T cell activation, after PUVA therapy, but they did not
study the effect of NB-UVB radiation. A significant reduction on natural killer cell activity
was found in psoriasis patients receiving NB-UVB treatment, but not for those receiving
PUVA [40]. UVB radiation exerted enhancing effects on the production of a complement
component, C3, by IFN- -stimulated cultured human epidermal keratinocytes, in contrast to
PUVA therapy that showed suppressive effects, which in part may explain the efficacy of
PUVA in the treatment of inflammatory dermatoses such as psoriasis [41].
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944 Susana Coimbra and Alice Santos-Silva

UV radiation also seems to interfere with the cytokine profile present at the epidermis in
psoriasis, inducing an immunosuppressive effect. UVB increases the production of pro-
inflammatory substances, like TNF-; in opposition, UVA irradiation seems to reduce the
production of TNF-.
The mutagenic and carcinogenic properties of UVA light are established. UVB exposure
apparently is less harmful, but the mechanisms behind these effects remain a matter of debate.
The impact of UVB radiation, in experimental induction of squamous cell carcinomas,
was inferred from the characteristic point mutations in p53 tumor suppressor gene [42]. In
contrast to UVB radiation, much of the mutagenic and carcinogenic action of UVA radiation
appears to be mediated through reactive oxygen species [43]. Agar et al. [44] reported that the
basal location of UVA-rather than the suprabasal UVB-induced DNA damage, suggests a role
for UVA in human skin carcinogenesis. No proeminent differences were found between UVA
and UVB for anti-mutagenic cellular responses, such as DNA repair and apoptosis; data
suggested that the less effective anti-mutagenic cellular responses, in particular different and
shorter-live cell cycle arrests, renders pyrimidine dimers induced by UVA
more mutagenic than pyrimidine dimers induced by UVB [45].
There are few studies about the mutagenic and carcinogenic properties of NB-UVB, as its
use is more recent. Long-term exposure to NB-UVB showed to induce a higher frequency of
skin cancer in mice than BB-UVB, and it was suggested that this is mediated through the
formation of cyclobutane pyrimidine dimers (CPDs); indeed, NB-UVB induced highly
malignant tumors caused by p53 dipyrimidine mutations through the formation of CPDs [46].
It was observed that the serum levels of 5-S-cysteinyldopa, which have been used as a
biological marker of melanoma progression, were significantly increased by NB-UVB
exposure, and these sustained high levels appear to reflect the degree of skin injury during
NB-UVB therapy [47]. However, Weischer et al. [48] did not found evidences for an
increased skin cancer risk for patients treated with either broadband or NB-
UVB phototherapy. Moreover, no evidences were formed for increased skin cancer risk in
Korean with skin phototypes III-V treated with NB-UVB phototherapy [49]. Emerit et al. [50]
reported that plasma clastogenic activity, which evaluates chromosomal breakage, persisted
after PUVA therapy in a follow-up study, while after NB-UVB, plasma-adjusted clastogenic
scores for psoriasis patients returned to values even lower than baseline. In summary, data in
the literature shows that there is an increased risk of skin cancer following PUVA, and this
risk may be, at least in part, explained by the high UVA dose exposure and by the phototype
of the treated patients [51]. The lack of prospective studies in psoriasis patients treated with
NB-UVB constitutes a barrier to the assessment of carcinogenic risk of this phototherapy
technique [51].
Several studies compared the clinical improvement of psoriasis in patients treated with
PUVA versus those treated with NB-UVB. However, data from these studies is not always
consensual. Some of the studies showed PUVA as therapeutically more effective than NB-
UVB [18, 52-54] and others showed that NB-UVB was as effective as oral or topical PUVA
therapy [55-59]. According to others [60, 61], NB-UVB is more effective than bath-PUVA
and is better tolerated. NB-UVB was proved to be a suitable alternative to treat patients who
cannot access bath PUVA therapy [62]. Some of the controversial results are probably due to
different treatment protocols used, for example dose of irradiation, time of exposure, number
of treatments per week and localisation of the lesions. For instance, in the van Weelden et al.

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study [55] was demonstrated that psoriasis lesions on the extremities respond better to PUVA,
and that lesions on the trunk respond better to NB-UVB. PUVA seems to have up-to 20%
higher remission rate, as compared with UVB phototherapy [63]. With PUVA treatment,
clearance of the lesions is achieved in more psoriasis patients with fewer treatment sessions,
and results in longer remission periods, as compared to NB-UVB [18]. Indeed, in a study
performed by our team, we found that oral PUVA prolonged the remission period, as
compared with NB-UVB and topical therapy [64], which is in accordance with the results of
Brazzelli et al. [65]. Moreover, when PUVA was given twice or thrice weekly, was more
effective than NB-UVB phototherapy in psoriasis treatment [52, 53]. Bath PUVA and NB-
UVB showed to have a systemic effect, decreasing peripheral CD4+ T cells, but this effect
was more pronounced with bath PUVA [54]. PUVA is the remaining supportive therapy for
patients with severe psoriasis, with high Psoriasis Area and Severity Index (PASI) scores,
who do not respond or are difficult to control adequately by NB-UVB therapy [56].
Psoriasis vulgaris deeply affects the health-related quality of life of the patients; thus,
improving patient’s quality of life is the primary goal of psoriasis therapy. We found that both
PUVA and NB-UVB treatments were effective, mainly NB-UVB, contributing to a
significant improvement of psoriatic patient’s quality of life [66]. Mckenna and Stern found
that a moderate to high relative impact on total quality of life was more often reported by
patients who had recently used UVB phototherapy than by those using PUVA or
methotrexate [67]. Other authors also reported an improvement in quality of life after NB-
UVB therapy [68, 69].

PUVA AND NB-UVB EFFECTS ON BIOMARKERS

Studies evaluating the impact of NB-UVB and PUVA, as well as studies comparing their
effects in the levels of important biological markers are less common. As the identification of
predictive biologic markers of severity and of monitoring of the treatment are important for
clinical evaluation of psoriasis, the studies evaluating the effects of NB-UVB and PUVA, as
well as of other therapies, in some potential biomarkers should be conducted and encouraged.
The effect of these 2 therapies on several inflammatory markers has been addressed.
However, the number of studies that simultaneously evaluated NB-UVB and PUVA effects in
their levels is small; several studies evaluated only one of these two therapies.
One of the studied biomarkers is TNF-, which is known to influence the proliferation,
activation and differentiation of several cell types, to stimulate apoptosis, to enhance the
synthesis of some cytokines and the expression of some adhesion molecules [70]. The
proliferation of local T cells is dependent on the local production of TNF- [71]. This
cytokine increases keratinocyte proliferation, the production of pro-inflammatory cytokines
by T cells and macrophages, and the production of adhesion molecules by vascular
endothelial cells [72]. Serwin et al. [73] found that after NB-UVB therapy there was a
decrease in the soluble TNF-α receptor type 1 (sTNF-R1) blood levels and in the
concentration of TNF-α converting enzyme. According to these authors, PUVA and NB-UVB
were associated with a decrease in sTNF-R1 levels, but in both treatment groups the decline
in sTNF-R1 was significant only in patients in whom the duration of skin lesions was less
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946 Susana Coimbra and Alice Santos-Silva

than 3 months, suggesting that the value of serum sTNF-R1 as a marker of response to
phototherapy may depend on the duration of skin lesions [74].
The studies about TNF- plasma levels in active psoriasis are not consistent [75-79].
Some authors suggested that this cytokine is mainly produced and act locally [70], and,
therefore, plasma levels might be lower than the levels achieved at the inflammatory area.
Studies performed by our team [3, 80] found that both NB-UVB and PUVA therapy were
associated with a significant decrease in TNF-α levels. Rotsztejn et al. [81] found a decreased
production of TNF-α and a decreased number of CD4+CD25+ T cells in the blood of
psoriasis patients after PUVA therapy.
The typical erythema of psoriatic lesions is due to the increased, dilated, and tortuous
capillaries that extend between the epidermal columns into the dermis. The superficial dermal
microvascular plexus in psoriasis is due to an active vasoproliferative process, known as
angiogenesis [82]. The vascular endothelial growth factor (VEGF), a major epidermis-derived
vessel-specific growth factor, released from keratinocytes, appears to contribute to the
vascularization of the lesions [83]. This growth factor can stimulate epidermal hyperplasia,
vascular growth and leukocyte infiltration in the skin [84], and, through its receptors, it
appears to play an important role in regulating psoriatic keratinocyte activity [85]. In
psoriasis, VEGF levels are significantly high in skin lesions, and its concentration in plasma
is raised in the active stage of the disease [3, 86-88]. Furthermore, in chronic plaque psoriasis
patients, a significant positive correlation was found between PASI and VEGF levels in
psoriasis plaques and in peripheral blood [83]. As angiogenesis is particularly dependent on
VEGF [89], its role in psoriasis is crucial, as a key factor linking inflammation and
angiogenesis [90]. VEGF levels appear to be significantly decreased in psoriatic patients
submitted to PUVA therapy [91], while the effect of UVB irradiation in VEGF levels is more
controversial [91-93]. According to our data [3], a significant decrease for VEGF levels at the
end of PUVA and NB-UVB treatments was observed, and in both cases, VEGF values were,
still, significantly higher than those of the control, suggesting that angiogenic alterations may
persist after a successful treatment. Rácz et al. [94] reported a decrease in the epidermal
expression of VEGF receptor (VEGFR)2, VEGFR3 and E-selectin after NB-UVB therapy.
The effect of PUVA and NB-UVB in the concentrations of some interleukins, known to
be raised in the active forms of psoriasis, has also been studied. Peripheral blood mononuclear
cells from psoriasis patients treated with NB-UVB secreted larger amounts of the anti-
inflammatory cytokine IL-10, and showed a markedly decreased production of IL-1β, IL-2,
IL-5 and IL-6, compared to the pre-treatment values, and a trend towards a decrease in the
production of IFN-, IL-8 and IL-12p70 [9]. Piskin et al. [14] found a significant decrease in
the expression of IFN-, and, concomitantly, a significant reduction of the IFN- inducers, IL-
12, IL-18 and IL-23, after NB-UVB therapy. The improvement in psoriatic skin lesions
following NB-UVB therapy, seemed to be also due to a reduced capacity of the surviving
dermal T cells to express the pro-inflammatory cytokine IFN- [12]. UVB exposure of
psoriatic skin appears to induce IL-4 expression by neutrophils [95]. The UVB-induced
growth inhibition of keratinocytes in hyperproliferative skin disorders may, in part, be related
to downregulation of CXCR-2, a specific cell surface receptor of IL-8, which seems to be
increased in psoriasis [96].
Concerning PUVA, Olaniran et al. [97] showed an association between PUVA-induced
resolution of psoriasis and a decrease in the levels of various cytokines (IL-2, IL-6, IL-8,

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TNF-α and IFN-) highly expressed in psoriatic lesions. PUVA, both in vitro and in vivo,
suppress the production of the proinflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α by
peripheral blood mononuclear cells [98]. A decrease in the epidermal staining for IL-6 was
observed in skin lesions, during and after PUVA treatment [99, 100]. The mRNA gene
expression of IL-10, an anti-inflammatory cytokine, in peripheral blood mononuclear cells of
psoriasis patients increased markedly after PUVA and NB-UVB successful therapies [101].
As referred, the IL-23/Th17 axis is believed to be crucial in psoriasis pathogenesis, and
its inhibition appears to be central to therapeutic achievement. IL-23 is an important regulator
of Th17 lymphocytes, which influence the cutaneous immune system by production of IL-
17 and several other proinflammatory cytokines. The biologic drug ustekinumab, which
targets the p40 subunit of IL-12 and IL-23, has been used successfully for the treatment of
moderate to severe psoriasis [102]. Moreover, other IL-23 pathway inhibitors are being
studied, such as apilimod [103], briakinumab [104] and secukinumab [105]. Ravić-Nikolić et
al. [106] showed that the immunosuppressive effect of PUVA therapy was associated with a
significant decrease in the expression of IL-12p40, IL-23p19 and IFN-γ in epidermis and
dermis of psoriatic lesions, revealing an impact of PUVA therapy in Th17 and Th1 pathways.
Moreover, a study performed in K5.hTGF-beta1 transgenic mice, which exhibit a skin
phenotype and cytokine abnormalities, with strong similarities to human psoriasis, indicated
that inhibition of the IL-23/Th17 axis, as well as, induced regulatory T cells involving
CTLA4 (cytotoxic T-lymphocyte-associated antigen 4) signaling, are central for the
therapeutic action of PUVA [107].
Concerning NB-UVB, it decreases the numbers of CD11c(+) dendritic cells and their
products, IL-20, inducible nitric oxide synthase, IL-12/23p40, and IL-23p19, and suppressed
IL-17 and IL-22 mRNAs, which are strongly correlated with lesion resolution; thus, NB-UVB
suppressed several parameters of the IL-23/IL-17 pathway [108]. Moreover, clinical
improvement of psoriasis by NB-UVB was linked to the suppression of Th17 and type I and
type II IFN signaling pathways, since i) downregulation of Th17 signaling pathway in
psoriatic epidermis during NB-UVB therapy, ii) strong inhibition of the Th17 pathway by
UVB confirmed in an ex vivo organ culture system, and iii) inhibition of the Th17-dependent
psoriasis-like dermatitis in mice, were observed [109]. It was reported that NB-UVB caused a
marked, although non-significant, decrease of IL-17 and IL-1β in psoriasis lesions [110]. IL-
17, as well as, IL-22, TNF-α, IFN-, IL-12, IL-18 and VEGF correlated with psoriasis
severity [77, 111-113].
The suppression of the Th17 pathway appears to be a promising treatment option;
therefore, there are several compounds under study, namely brodalumab, a human anti-
interleukin-17-receptor monoclonal antibody and ixekizumab, a humanized anti-interleukin-
17 monoclonal antibody [114, 115]. We found that during the treatment with both NB-UVB
and PUVA, a decrease in IL-23 and TNF- was observed after 3 weeks of treatment,
followed by a decrease in IL-22 and IL-17, 6 weeks after initiating therapy, and, finally, a
decrease in VEGF and IL-8 levels at the 12th week of treatment [3]. According to our data, the
reduction in IL-23 (the first change observed with NB-UVB and PUVA treatment) seems to
be crucial for the following changes observed for the other cytokines, strengthening the vital
role of the IL-23/Th17 axis. Despite the similar final changes observed for both therapies, the
improvements were higher in patients treated with PUVA, especially for IL-23, what may
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948 Susana Coimbra and Alice Santos-Silva

explain its higher remission rate, as compared with NBUVB. We also found a statistical
significant decrease in IL-6 levels after PUVA, but not after NB-UVB therapy [80].
Psoriasis is clearly an inflammatory condition, as showed by the enhanced levels of C-
reactive protein (CRP), total leukocyte and neutrophil count, as well as, of neutrophil
activation products, such as elastase and lactoferrin, and of anti-proteases systems, like α1-
antitrypsin [116, 117]. In accordance with Chodorowska et al. [118], we found a statistical
significant reduction in CRP and α2-macroglobulin levels after PUVA therapy; moreover, our
data revealed that PUVA treatment was associated with a significant decrease in the values of
elastase, lactoferrin, 1-antitrypsin and leukocyte and neutrophil counts. For patients under
NB-UVB treatment, we found similar results, except for total leukocytes and neutrophil
count, which did not reduced significantly [116]. However, according to Gasior-Chrzan et al.
[119], UVB and PUVA treatment had no significant influence on the serum levels of α2-
macroglobulin in psoriatic patients. Also, some studies have suggested that PUVA has no
significant effect on neutrophils, while others suggest that PUVA enhances neutrophil activity
[120-122]. The trauma-induced and leukotriene B4 (LTB4)-induced intra-epidermal
accumulation of polymorphonuclear leukocytes was quantified after UVB and PUVA
treatments, using elastase as a marker enzyme; both caused a profound inhibition of trauma-
and LTB4-induced polymorphonuclear leukocytes accumulation [123]. Romani et al. [124]
also found a significant decrease in CRP levels after NB-UVB therapy. It should be
highlighted that increasing CRP levels were associated with increasing PASI scores,
suggesting that CRP is a good marker of psoriasis severity; moreover, CRP value, after
treatment, seems to be an important determinant of the length of remission of psoriasis for
patients treated with phototherapy or topical therapy [64, 118, 125]. Additionally, it seems
that CRP is a good marker for monitoring psoriasis treatment.
Pentraxin 3 (PTX3) is a long-chain pentraxin, produced by macrophages, dendritic cells
and endothelial cells, in response to inflammatory signals. PTX3 seems to reflect aspects of
the inflammatory process that are different for CRP [126]. Data in the literature reported that
PTX3, as occurs with CRP, is increased at the psoriasis exacerbation stage and that there is a
positive significant correlation between PTX3 levels and PASI [125, 127, 128]. After PUVA
and NB-UVB treatments, we observed similar modifications, a decrease in CRP and PTX3
levels, inducing a significant reduction in the inflammatory state. Our data also suggested that
for the severer forms of psoriasis, even after a successful treatment, a residual inflammation
still persists, with PTX3 and CRP levels remaining higher. In accordance, Ctirad et al. [127]
found that PTX3 and CRP levels decreased significantly after Goeckerman's therapy and that,
after therapy, PTX3 and CRP remained significantly increased for patients with PASI scores
of 20.9  8.4 (mean ± standard deviation), confirming the importance of these two
inflammatory markers for the psoriasis clinical evaluation process.
The effect of psoriasis therapies in the levels of some inflammation-related markers, such
as markers of redox status and adipokines, has also been addressed. We reported that PUVA
exposure induced a significant decrease in the values of TBA (thiobarbituric acid reactivity)
and TBA/TAS (total antioxidant status) and a trend towards a decrease in oxidized low-
density lipoprotein (oxLDL) and oxLDL/LDL ratio, as well as, a significant increase in
adiponectin levels, which seem to be a consequence of a reduction in inflammation, as
suggested by the significant decrease observed in CRP levels. For NB-UVB, we found a
significant decrease in the values of TBA and TBA/TAS [80, 129]. Kawashima et al. [130]

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reported that phototherapy decreased significantly resistin levels. UVB radiation proved to
significantly suppress IFN-- or TNF-α-induced nitric oxide production, and to downregulate
IFN-- or TNF-α-induced inducible nitric oxide synthase expression at both the mRNA level
and the protein level [131].
Membrane-bound hemoglobin (MBH) and the erythrocyte membrane band 3 profile were
proposed as good cumulative markers of erythrocyte aging and/or damage in several
inflammatory and oxidative stress conditions [132-135]. Differences in the band 3 profile
(high-molecular-weight aggregates, band 3 monomer and proteolytic fragments) have been
associated with the age and condition of red blood cells (RBCs). Older and damaged RBCs
have higher band 3 aggregation and lower fragmentation, whereas younger RBCs showed
reduced aggregation and higher fragmentation. We reported that after treatment of psoriasis
patients with NB-UVB and PUVA therapy, a significantly different band 3 profile was
observed, as compared with the band 3 profile observed before initiating the therapy. In both
cases, after therapy, the band 3 profile was associated with a younger/less damaged RBC
population. Moreover, both PUVA and NB-UVB patients presented with a significant
decrease in MBH. Considering the erythrocyte parameters, after PUVA therapy, but not after
NB-UVB, a significant increase was observed for reticulocyte count and reticulocyte
production index [136]. This rise in erythropoiesis and the younger/less damaged RBC
population observed after PUVA exposure appear to be due to the more pronounced clearing
of the lesions, as suggested by PASI scores, probably a result of a significant improvement of
the inflammatory process.

Table 1. The impact of psoralen plus ultraviolet light A (PUVA) and narrow-band UVB
(NB-UVB) therapies in some biological markers

PUVA NB-UVB
Angiogenesis  VEGF levels  VEGF levels
(controversial results)
TNF and TNF-  sTNF-R1 levels  sTNF-R1 levels
R1  TNF-α levels  TNF-α levels
IFN-  INF- levels  INF- levels
IL(s)  IL-1β, IL-2, IL-6, IL-8 levels  IL-1β, IL-2, IL-6 produced
by PBMC
IL-8 levels
IL-23/Th17 axis  IL-23, IL-22, IL-17 levels   IL-23, IL-22, IL-17 levels 
 IL-12p40 and IL-23p19 epidermal and  of the CD11c(+) dendritic
dermal expression cells products:
IL-12/23p40 and IL-23 p19

Inflammation  CRP levels  CRP levels

 PTX3 levels  PTX3 levels
 elastase, lactoferrin, α1-antitrypsin  elastase, lactoferrin, α1-
levels antitrypsin levels
 leukocyte and neutrophil counts no significant effect
Redox status  TBA and TBA/TAS levels  TBA and TBA/TAS levels
oxLDL and oxLDL levels no significant effect
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950 Susana Coimbra and Alice Santos-Silva

Table 1. (Continued)

PUVA NB-UVB
Adiponectin  adiponectin levels no significant effect
Erythrocyte improved band 3 profile improved band 3 profile
aging and/or  MBH levels  MBH levels
damage
Erythrocyte  reticulocyte count and RPI no significant effect
production
the effect was higher in patients treated with PUVA;
, decrease; , trend towards a decrease.
(CRP; C-reactive protein; IL, interleukin; INF, interferon; LDL, low-density lipoprotein; MBH,
membrane-bound hemoglobin; oxLDL, oxidized LDL; PBMC, peripheral blood mononuclear
cells; PTX3, pentraxin 3; RPI, reticulocyte production index; TAS, total antioxidant status; TBA,
thiobarbituric acid reactivity; Th, T helper; TNF, tumor necrosis factor; sTNF-R1, soluble TNF-α
receptor type 1; VEGF, vascular endothelial growth factor)

A brief summary of the effects of PUVA and NB-UVB therapies in some biological
markers is presented at Table 1.

CONCLUDING REMARKS
In summary, inflammatory indicators appear to be good markers for assessing the
severity and monitoring psoriasis therapy. According to data in the literature, CRP levels, the
PASI score, eventually complemented by the values of other inflammatory markers, such as
those related with the IL-23/Th17 axis, seem to be good markers for evaluating severity,
monitoring treatment and to predict the length of remission in psoriasis patients, particularly
for those treated with PUVA and NB-UVB. Further studies are warranted to test this
hypothesis and it would be interesting to study psoriasis patients treated with other
therapeutic agents.
PUVA seem to be associated with a higher therapeutic efficacy and long-lasting periods
of remission, which seem to be mainly due to a more marked reduction of the inflammatory
process. Indeed, PUVA has a more pronounced effect than NB-UVB in several markers of
inflammation. The significant improvement in inflammatory process may, at least in part,
explain why PUVA is a remissive therapy, capable of inducing long-lasting improvements in
the symptoms of psoriasis after treatment discontinuation.

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gamma, IL-12p40 and IL-23p19 in psoriatic plaques. Eur J Dermatol, 2011, 21(1), 53-
7.
[107] Singh, T.P. et al. 8-methoxypsoralen plus ultraviolet A therapy acts via inhibition of the
IL-23/Th17 axis and induction of Foxp3+ regulatory T cells involving CTLA4
signaling in a psoriasis-like skin disorder. J Immunol, 2010, 184(12), 7257-67.
[108] Johnson-Huang, L.M. et al. Effective narrow-band UVB radiation therapy suppresses
the IL-23/IL-17 axis in normalized psoriasis plaques. J Invest Dermatol, 2010, 130(11),
2654-63.
[109] Racz, E. et al. Effective treatment of psoriasis with narrow-band UVB phototherapy is
linked to suppression of the IFN and Th17 pathways. J Invest Dermatol, 2011, 131(7),
1547-58.
[110] Vahavihu, K. et al. Narrow-band UVB treatment improves vitamin D balance and alters
antimicrobial peptide expression in skin lesions of psoriasis and atopic dermatitis. Br J
Dermatol.
[111] Takahashi, H. et al. Serum cytokines and growth factor levels in Japanese patients with
psoriasis. Clin Exp Dermatol, 2009,
[112] Wolk, K. et al. IL-22 regulates the expression of genes responsible for antimicrobial
defense, cellular differentiation, and mobility in keratinocytes: a potential role in
psoriasis. Eur J Immunol, 2006, 36(5), 1309-23.
[113] Caproni, M. et al. Serum Levels of IL-17 and IL-22 Are Reduced by Etanercept, but not
by Acitretin, in Patients with Psoriasis: a Randomized-Controlled Trial. J Clin
Immunol, 2009, 29(2), 210-4.
[114] Leonardi, C. et al. Anti-interleukin-17 monoclonal antibody ixekizumab in chronic
plaque psoriasis. N Engl J Med, 2012, 366(13), 1190-9.
[115] Papp, K.A. et al. Brodalumab, an anti-interleukin-17-receptor antibody for psoriasis. N
Engl J Med, 2012, 366(13), 1181-9.
[116] Coimbra, S. et al. C-reactive protein and leucocyte activation in psoriasis vulgaris
according to severity and therapy. J Eur Acad Dermatol Venereol, 2010, 24(7), 789-96.
[117] Rocha-Pereira, P. et al. The inflammatory response in mild and in severe psoriasis. Br J
Dermatol, 2004, 150(5), 917-28.
[118] Chodorowska, G., D. Wojnowska, and M. Juszkiewicz-Borowiec, C-reactive protein
and alpha2-macroglobulin plasma activity in medium-severe and severe psoriasis. J Eur
Acad Dermatol Venereol, 2004, 18(2), 180-3.
[119] Gasior-Chrzan, B., L.K. Dotterud, and E.S. Falk, Serum alpha 2-macroglobulin levels
in psoriatic patients treated with UVB and PUVA. Riv Eur Sci Med Farmacol, 1996,
18(3), 125-8.
[120] Silny, W. et al. Effect of PUVA treatment on the locomotion of polymorphonuclear
leukocytes and mononuclear cells in psoriasis. J Invest Dermatol, 1980. 75(2), 187-8.
[121] Kapuscinska, R. et al. [Cytofluorimetric assay for evaluation of CD16 receptor
expression and myeloperoxidase (MPO) activity of neutrophils in patients with
psoriasis vulgaris treated with PUVA]. Wiad Lek, 2004, 57(11-12), 599-602.
[122] Bredberg, A. and A. Forsgren, Effects of in vitro PUVA on human leukocyte function.
Br J Dermatol, 1984. 111(2), 159-68.
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[123] Chang, A., J.A. Alkemade, and P.C. van de Kerkhof, PUVA and UVB inhibit the intra-
epidermal accumulation of polymorphonuclear leukocytes. Br J Dermatol, 1988,
119(3), 281-7.
[124] Romani, J. et al. Effect of narrowband ultraviolet B therapy on inflammatory markers
and body fat composition in moderate to severe psoriasis. Br J Dermatol, 2012, 166(6),
1237-44.
[125] Coimbra, S. et al. Inflammatory markers of cardiovascular disease risk in Portuguese
psoriatic patients – relation with NB-UVB and PUVA therapy Int J Dermatol In press.
[126] Jenny, N.S. et al. Associations of pentraxin 3 with cardiovascular disease and all-cause
death: the Cardiovascular Health Study. Arterioscler Thromb Vasc Biol, 2009, 29(4),
594-9.
[127] Ctirad, A. et al. Goeckerman's therapy for psoriasis with special reference to serum
pentraxin 3 level. Int J Dermatol, 2008, 47(10), 1011-4.
[128] Bevelacqua, V. et al. Long pentraxin 3: a marker of inflammation in untreated psoriatic
patients. Int J Mol Med, 2006, 18(3), p. 415-23.
[129] Coimbra, S. et al. Psoriasis therapy and cardiovascular risk factors: a 12-week follow-
up study. Am J Clin Dermatol, 2010, 11(6), 423-32.
[130] Kawashima, K. et al. Phototherapy reduces serum resistin levels in psoriasis patients.
Photodermatol Photoimmunol Photomed, 2011, 27(3), 152-5.
[131] Yamaoka, J., M. Sasaki, and Y. Miyachi, Ultraviolet B radiation downregulates
inducible nitric oxide synthase expression induced by interferon-gamma or tumor
necrosis factor-alpha in murine keratinocyte Pam 212 cells. Arch Dermatol Res, 2000,
292(6), 312-9.
[132] Santos-Silva, A. et al. Altered erythrocyte membrane band 3 profile as a marker in
patients at risk for cardiovascular disease. Atherosclerosis, 1995, 116(2), 199-209.
[133] Santos-Silva, A. et al. Erythrocyte damage and leukocyte activation in ischemic stroke.
Clin Chim Acta, 2002, 320(1-2), 29-35.
[134] Belo, L. et al. Band 3 as a marker of erythrocyte changes in pregnancy. Eur J
Haematol, 2002, 69(3), 145-51.
[135] Rocha-Pereira, P. et al. Erythrocyte damage in mild and severe psoriasis. Br J
Dermatol, 2004, 150(2), 232-44.
[136] Coimbra, S. et al. Erythroid disturbances before and after treatment of Portuguese
psoriasis vulgaris patients: a cross-sectional and longitudinal study. Am J Clin
Dermatol, 2012, 13(1), 37-47.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 39

PSORIASIS VULGARIS INVESTIGATED

BY ELECTRON PARAMAGNETIC RESONANCE

Kouichi Nakagawa1 and Daisuke Sawamura2

1
Department of Radiological Life Sciences,
Graduate School of Health Sciences, Hirosaki University, Hirosaki, Japan
2
Department of Dermatology, Graduate School of Medicine,
Hirosaki University, Hirosaki, Japan

ABSTRACT
EPR (electron paramagnetic resonance) is useful for elucidating structural aspects of
skin. Non-invasive spectroscopic characterization of the outermost layer of the stratum
corneum (SC) as well as nail is an important subject in dermatology and cosmetology.
However, there is no feasible spectroscopic method to evaluate initial changes of SC and
severity of nail with psoriasis. EPR (electron paramagnetic resonance) might be feasible
for evaluating the conditions in the patients with psoriasis.
A little, broad three-line pattern of the psoriasis vulgaris SC (pv-SC) was observed.
The spectral pattern is quite different from those of other SC reported. The spectral
pattern suggests that the 5-DSA is mobile or less rigid in the SC. The reasonable
agreement between the experimental and simulated spectra was obtained. The S0 value
obtained for 5-DSA in the SC was approximately 0.20. It is noted that the lower value of
the S0 indicates the less rigid (abnormal) structure of the pv-SC. We found that the pv-SC
is less rigid of the structure than that of the control SC, indicating abnormal architecture
of psoriasis vulgaris stratum corneum. The statistical analysis using Student’s t-test
suggests that the value of pv-SC is significantly smaller than that of the control (p <
0.01). In the case of the finger nail, EPR spectra were analyzed using the intensity ratio of
the two motions (fast and slow) at the peaks of the lower magnetic field.
We observed two distinguishable sites in the nails. In addition, EPR simulation was
performed to analyze the spectra obtained. The present EPR results and the detailed
analyses show that there are rigid and fragile sites in the nail. In the case of nail psoriasis,
the fragile components are 2 ~ 3 times more than those of the control. Therefore, we
suggest that the EPR assay is of great use for evaluating SC and nail function.


Phone and Fax: 81+172-39-5921, E-mail: [email protected]
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960 Kouichi Nakagawa and Daisuke Sawamura

INTRODUCTION
Stratum corneum (SC) is the outermost layer of skin and the skin barrier against
chemicals, surfactants, UV irradiation, and environmental stresses. The SC has a
heterogeneous structure composed of corneocytes embedded in the intercellar lipid lamellae
as illustrated in Figure 1. The morphology of the SC lipids is closely associated with the main
epidermal barrier. Knowledge of the lipid structure is important in understanding the
mechanism of irritant dermatitis and other SC diseases. The structural information of the SC
lipid is obtained by the analysis of aliphatic spin probes incorporated into intercellar lamella
lipids using EPR (Electron Paramagnetic Resonance) [1-6]. EPR in conjunction with spin
probe method non-destructively measures the mobility of the lipid bilayer of SC.
EPR (or ESR: Electron Spin Resonance) utilizes spectroscopy, which measures the
freedom of an unpaired electron in an atom or molecule. The principles behind magnetic
resonance are common to both EPR and nuclear magnetic resonance (NMR), but there are
differences in the magnitudes and signs of the magnetic interactions involved.
EPR probes an unpaired electron spin, while NMR probes a nuclear spin. EPR can
measure 10-9 M (moles per liter) concentration of the probe and one of the most sensitive
spectroscopic tools. Therefore, EPR is able to elucidate skin lipid structures as well as
dynamics.
It is important to know the composition of SC lipid as well as its structure in relation to
depth. The various components, such as ceramides, cholesterol, and free fatty acids of SC
lipids have been investigated by TLC (thin-layer chromatography) [7, 8].
Structural information organized by the components is essential for knowing the detailed
functions of SC. The role of the intercellular SC lipid bilayer in relation to barrier function
has been investigated by IR (infrared) spectroscopy [9, 10] and X-ray diffraction [11]. IR
examination showed that the outer layers were less cohesive and the intercellular lipids are
more disordered compared with the deeper membrane, based on the C-H stretching
absorbance of the methylene groups of the lipid acyl chains. The X-ray approach is somewhat
limited to model lipid membranes containing water or in vitro SC specimens, and it is
difficult to obtain information about depth-related changes of the SC.

Intercellular Sebaceous secretion

route

SC
Corneocytes Fatty acid

Intercellular
space Lipid Bilayer

Figure 1. Schematic representation of the modified “Brick and Mortar” model of the stratum corneum
(SC) is shown. Also, there is shown the most likely probe location in the lipid bilayer and pathways of
drug (or spin probe) permeation through intact stratum corneum.

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 Psoriasis Vulgaris Investigated by Electron Paramagnetic Resonance 961

On the other hand, the EPR probe method can provide insight into the SC lipid
organization as well as its dynamics. The physicochemical properties of intercellar lipids of
SC as a function of various surfactants [1, 2], water contents [3], various kinds of spin probes
[4], and fluidity change of the SC lipid [1, 4-6] were investigated. EPR is a reliable, sensitive,
and non-destructive technique to measure the probe in the lipids at ambient temperature.
An introduction on EPR spectroscopy and its application in conjunction with slow-
tumbling simulation to elucidate the organization of SC lipids are discussed next. This
technique provides confirmatory and complementary information about structure and
physicochemical properties on a molecular level. The advantage of using the spin probes is
that not only the structure but also the acyl chain motion in the stratum corneum (SC) lipid
can be determined. These studies provided the fluidity related behaviors of SC at the different
conditions by measuring EPR signals. EPR measurements and the simulation analysis can
potentially provide further quantitative insight into the skin-lipid structures.

APPARATUS
EPR apparatus consists of a klystron to generate microwaves, electromagnet, resonant
cavity, microwave detector, amplifier, A/D converter, and PC as shown in Figure 2. The
microwaves from the klystron have a constant frequency, and those microwaves reflected
from the resonant cavity are detected, changed to an electronic signal, amplified and then
recorded.
In contrast to NMR, substances which contain unpaired spin can be observed by EPR.
Paramagnetic substances including transition metal complexes, free radicals,
macromolecules, and photochemical intermediates are observed. Approximately 10-13 mole of
a substance gives an observable signal, thus EPR has great sensitivity.
Momentum of electron spin in a magnetic field orients only two quantum states: ms = ½
and - ½.

Phase
Shifter
Circulator
Microwave
Detector
Source
Amplifier
Signal
Sample
A/D

Magnet S N

Cavity

Figure 2. Block diagram of EPR spectrometer.

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962 Kouichi Nakagawa and Daisuke Sawamura

Application of an oscillating field perpendicular to a steady magnetic field (H) induces

transitions between the two states provided the frequency () of the oscillating field satisfies
the resonance condition:

E  E 1  E 1  gH ,

2 2 (1)

Thus,

h  E  gH , (2)

where E is the energy-level separation, h is Planck’s constant, g is a dimensionless constant

called the g-value, and  is the electron Bohr magneton, and H is the applied magnetic field.
The interaction of an electron spin in resonance with a neighboring nuclear spin in a
molecule is called hyperfine coupling. In the case of nitroxide spin probe, 14N of the probe
has three quantum states: mI = +1, 0, and -1. Each quantum state interacts with an electron
spin and further splits into two sets of energy states as shown in Figure 3. The selection rules
for transitions in hyperfine coupling are ms = 1 and mI = 0.

mI = 1
0
E1/2
Energy Levels

-1
ms = 1/2

ms = -1/2 mI = -1
E-1/2 0
1
A

mI = 1 0 -1

0 Magnetic Field
Figure 3. Hyperfine levels and transitions for a nitroxide nitrogen nucleus (14N) of I = 1 with positive
coupling constant. An observable EPR observable spectrum is shown.

Thus, one can observe three transition (resonance) lines for fast tumbling nitroxide spin
probe in a spectrum.
The interval of the resonance lines is called the hyperfine coupling constant (A). The
EPR spectra are usually recorded as the first derivative of the absorption spectrum as shown
in lower part of Figure 3.

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 Psoriasis Vulgaris Investigated by Electron Paramagnetic Resonance 963

Stratum Corneum Cyanoacrylate Glue Stripping

The sampling method was first utilized by Marks and Dawber [12] to obtain SC sheets.
Recently, Yagi, Nakagawa, and Sakamoto developed a process to study SC properties [6].
The SC specimens were successively removed from the mid-volar forearm and shank of the
volunteers, who had given informed consent to the procedure [6]. All subjects had normal
skin, as judged by visual assessment. A glass plate (7 mm x 37 mm; Matsunami Glass Ind.,
Ldt., Tokyo, Japan) on which a single drop (~1.2 mg) of a commercially available
cyanoacrylate resin had been uniformly spread was used to strip the SC sheet as depicted in
Figure 4. Only approximately 1 mg of SC sample is required for the studies. Once the glue
has solidified, no significant signal arise from the cured resin or from the spin probe dissolved
in the resin; the only signal observed arise from the spin probe in the attached SC sheet. This
method has the advantage of avoiding prior exposure of the SC to enzymes. EPR intensity
slightly depends on how thick a sample is removed by each stripping, but it can be adjusted
by the amount and areas of glue on the glass plate.

Mid-volar forearm EPR

A glass plate

Cyanoacrylate Incubation with probe

Figure 4. Schematic representation of SC sample procedures and the EPR spectrum.

Preparation of SC Sheets for EPR Measurements

One piece of stripped SC (~ 5 x 22 mm2) was incubated in ~50 M of a spin probe

(Figure 5) aqueous solution for about 60 minutes at 37 C (Figure 4). The probe solution was
dropped on the SC sheet. The SC sheet repels the aqueous solution but the probe goes into the
lipid phase during the incubation. After rinsing with distilled water to remove excess spin
probe, the SC sample was mounted on an EPR cell.

Spin Probes

Organic free radicals containing the nitroxide group are called spin probes or spin labels.
The fluidity of the lipid bilayer is obtained with doxylstearic acid (DSA) which most
commonly used. Commercially available spin probes, 5-doxylstearic acid (5-DSA) and 3β-
doxyl-5α-cholestane (CHL), were used to obtain the mobility of the SC lipid. The chemical
structures of 5-DSA and CHL are depicted in Figure 5. Changes of the lipid chain mobility
are able to monitor using various probes. The orientation of spin probe reflects the local
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964 Kouichi Nakagawa and Daisuke Sawamura

molecular environment and should serve as indicator of conformational changes in lipid

bilayers.

O 1 5 5-DSA
O N O
O

O CHL
N

O H

Figure 5. Chemical structures of 5-doxylstearic acid (5-DSA) and 3β-doxyl-5α-cholestane (CHL) spin
probes.

Description Approx. tumbling Approx. mobility

of spectra time (ns) parameter
2A

Immobilized 0.5 0.7

2A

Moderately 2.5 0.3

Immobilized

Weakly 5.0 0.1

Immobilized

Figure 6. Nitroxide EPR line-shape as a function of tumbling time and mobility parameter. The parallel
and perpendicular hyperfine couplings, 2A and 2A, are also indicated for an anisotropic (immobilized)
EPR spectrum.

EPR Line-Shapes due to Spin Probe Motion

The line-shapes and line-widths can vary under certain spin probe environments. When
line broadening arises from incomplete averaging of the g-value and the hyperfine coupling
interactions within the limit of rapid tumbling in a medium, EPR line-shape starts changing
from the triplet pattern. EPR spectra of nitroxide radicals for different tumbling times as well
as different mobility parameters are presented in Figure 6. Schematic illustration of lipid

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 Psoriasis Vulgaris Investigated by Electron Paramagnetic Resonance 965

bilayer structures and corresponding EPR spectra is also shown in Figure 7. If a spin probe is
oriented (immobilized) in a lipid membrane, EPR spectrum is an anisotropic pattern which
clearly shows parallel (2A) and perpendicular (2A) hyperfine coupling structures (the top
spectrum in Figure 6). The mobility parameter is approximately 0.7 or higher. If a spin probe
tumbles relatively fast (weakly immobilized) in a lipid membrane, EPR spectrum is a triplet
pattern with unequal intensities. The mobility parameter is usually very small (~0.1).

Qualitative Mobility Parameter (S)

The inclination of the principal axis of the nitroxide radical to the rotational axis of the
long-chain probe molecule represents a measure of the order-disorder of the molecular
assemblies of a membrane. The mobility parameter indicates the membrane chain dynamics
and microenvironment of the medium in which the spin probe is incorporated.

Disordered structure Ordered structure

Order Parameter: S ≈ 0 : Lipid Order Parameter: S ≈ 1

: Spin probe

mobile Immobilized

Figure 7. Schematic representation of lipid bilayer structures as a function of lipid mobility. The
corresponding EPR spectral patterns were also indicated.

The conventional mobility parameter (S) is determined from the hyperfine coupling of the
EPR signals according to the following relations [13]:

AII  A a
S  ', (3)
AZZ   AXX  AYY  a
1
2
A  2 A
a '  II , (4)
3

where a is the isotropic hyperfine value, (AXX + AYY + AZZ)/3; AXX, AYY, and AZZ are the
principal values of the spin probe. The following principal components were used for 5-DSA
[14].

AXX, AYY, AZZ = (0.66, 0.55, 3.45) mT (5)

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966 Kouichi Nakagawa and Daisuke Sawamura

The experimental hyperfine couplings of 2A and 2A are obtained from the experimental
spectrum (as shown in Figure 6). The mobility parameter indicates that the S value increases
with increasing anisotropy of the probe site in the membrane. On the other hand, the S value
becomes zero for completely isotropic motion of the nitroxide radical. Since the spin probe is
incorporated into the highly oriented intercellular lipid structure in normal skin, in which the
probe cannot move freely due to the rigidity of lipid structure, its EPR spectrum represents
the microscopically oriented profile as depicted in Figure 7. When the normal structure is
completely destroyed by chemical and/or physical stress, the EPR spectral profile changes to
three sharp lines because the probe mobility is unrestricted. Thus, the EPR spectral profile
reflects the rigidity of the environment of the probe moiety. However, conventional analysis
measuring 2A and 2A from the observed spectrum gives limited information concerning the
probe moiety in the membrane, and may not reveal subtle differences in the overall
experimental spectra related to the membrane chain mobility [6].

Quantitative Mobility Parameter (S0) by Slow-Tumbling Spectral Simulation

The slow-tumbling motions on the order of 10-7 s of the aliphatic spin probes in
membranes were evaluated by using the nonlinear least-squares fitting program NLLS to
calculate the EPR spectra based on the stochastic Liouville equation [15, 16]. The EPR
spectra for spin probes incorporated into the multilamellar lipid bilayer were calculated
according to various distribution of the probe in the membrane. The spectrum of a sample can
be regarded as the superposition of the spectra of all of the fragments. The lipid and 5-DSA
molecules in the lipid bilayer experience ordering (or fluidity) potentials, which restrict the
amplitude of the rotational motion. The ordering potential in a lipid bilayer determines the
orientational distribution of molecules with respect to the local ordering axis of the bilayer
[17]. The overall orientation of the probe can be expressed by the order parameter (S0), which
is defined as follows [16, 18]

1
S0   (3 cos 2   1)
2

 d exp(U / kT ) D
2
00
 ,
 d exp(U / kT ) (6)

which measures the angular extent of the rotational diffusion of the nitroxide probe moiety.
Gamma () is the angle between the rotational diffusion symmetry axis and the z-axis of the
nitroxide axis system as shown in Figure 8. The  = (, , ) are the Euler angles between the
molecular frame of the rotational diffusion tensor, U is the ordering potential, and D is a
Winger rotation matrix element. In addition to S0, the simulation calculates slow-tumbling
motions of the probe in the bilayer, providing rotational diffusion coefficients, as described in
detail elsewhere [19]. The values of the rotational diffusion coefficients (dynamic values) are
in relation to the S0 values. The A and g of the principal components were used for the
simulation of 5-DSA [14].

AXX, AYY, AZZ = (0.66, 0.55, 3.45) mT (7)

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 Psoriasis Vulgaris Investigated by Electron Paramagnetic Resonance 967

gXX, gYY, gZZ = (2.0086, 2.0063, 2.0025). (8)

Bilayer Surface

z 
O
N y
O
x

Figure 8. A schematic representation of a conformation of DSA spin probe in the SC membrane, where
Z-axis of the acyl chain is parallel to z-axis of the nitrogen 2Pz orbital.

The local or microscopic mobility of the nitroxide probe in the multilamellar lipid bilayer
is characterized by the S0 value. A larger S0 value indicates highly rigid structure and a
smaller S0 shows less ordered structure (less rigid or mobile). Changes of the lipid structural
mobility of SC are able to be monitored using the aliphatic probes. The orientation of spin
probe reflects the local molecular environment and should serve as indicator of
conformational changes in lipid bilayers of the SC. The modern simulation takes into account
overall experimental intensities, line-widths, and hyperfine coupling values and provides the
quantitative information regarding the probe environment. Therefore, S0-value reflects the
local mobility of the lipid structure in the membrane. The error of the spectral simulation is a
few percent in the case of the dipalmitoylphosphatidylcholine membrane [19]. In the presence
of fast motion of the probe in the SC, the simulation may result in the deviation from the
experimental spectra.

RESULTS AND ANALYSES

Qualitative Mobility Parameter (S) and Quantitative Mobility Parameter (S0)
of SC Lipids

The modified “Brick and Mortar [20] model of the SC is illustrated in Figure 1. SC
intercellular lipids arrange themselves into bilayer and pack into lamellae. The single-chain 5-
DSA normally dissolves into lipids and fat phases. The most likely location of the single-
chain probe in the SC. The aliphatic probe will be located in the lipid phase and fat like
sebaceous secretion of the SC.
Figure 9 shows the experimental and simulated EPR spectra of 5-DSA in the SC. The
reasonable agreement of the experimental and simulated spectra suggests that simulation
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968 Kouichi Nakagawa and Daisuke Sawamura

analysis can provide detailed information regarding the SC lipids. The S0 value changes from
0.61 to 0.96, while the S value is in the range of 0.56 to 0.59. The conventional S value was
obtained by the Eq. 3 measuring the hyperfine values from the observed spectrum.
There are significant differences between the conventional and simulated mobility
parameters. Because the slow-tumbling simulation calculates the total line-shape of the
spectrum, it is able to extract more detailed information about the SC structure than the
conventional analysis, which is normally ambiguous in distinguishing the two hyperfine
components (parallel and perpendicular) from the experimental spectrum due to the presence
of weak and broad signals [5]. Thus, the S0 values (0.2 ~ 0.9) obtained by the simulation
suggest that the outermost SC layers are less rigid (or more mobile, S0 ~ 0.2), while the
deeper lipid layers (S0 ~ 0.9) have more rigid and oriented structures.
The arrow in the spectrum indicates the characteristic peak, which is prominent only for
the first strip (Figure 9). This peak diminishes in intensity with increasing depth in the SC.
The marked peak appears near the center of the spectrum because the probe embedded in the
first sample stripped has greater freedom of motion. The other two lines of the nitroxide probe
overlaid the central region of the spectrum. Further investigation of the characteristic peak
was performed. Figure 10 (a) shows the EPR spectrum of the first strip from SC. The strong
and broad peak observed for the SC sheet from the human forehead is shown in Figure 10 (b).
The peak intensity decreases after washing the SC with soap (Figure 10 (c)). Thus, the
characteristic signal can be attributed to sebaceous secretion [6]. The strength of the signal is
considered to reflect the abundant sebaceous secretion at the forehead compared with that of
the forearm.

Simulated
Stripping Order Parameter
number S0
1 0.61

3 0.96

5 0.96

1 mT

Figure 9. Experimental (solid line) and simulated (dashed line) EPR spectra of 5-DSA probe. Stripping
numbers show consecutively stripped SC from the surface downwards. The arrow of stripping number
1 indicates the characteristic peak. The EPR spectra were obtained with the single scan.

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 Psoriasis Vulgaris Investigated by Electron Paramagnetic Resonance 969

Figure 10. Experimental EPR spectra of 5-DSA in the first stripped SC from human mid-volar forearm
(a), the first stripped SC from human forehead pre-washing (b), and the first stripped SC from human
forehead after-washing (c). The short dashed line corresponds to the characteristic signal. The long
dashed line corresponds to the probe incorporated into the SC lipids.

Quantitative Mobility Parameter (S0) Related to SC Lipid Structure

One can calculate the angle (γ in Figure 8) between the rotational diffusion symmetry
axis (the lipid in SC) and the z-axis of the nitroxide axis system. Figure 11 represents the
schematic illustration of the bilayer distance in relation to the angle. The simulated S0 value
of 0.61 can be the angle of 30°. The value of 0.96 is the angle of 9.4°. The angle suggests that
the SC lipids align nearly perpendicularly to the bilayer surface. The larger S0 value yields
larger distance between the lipid bilayer.
The analysis implies that the longer distance of the lipid bilayer can be related to the
well-oriented SC structure.


Lipid Bilayer
Simulated value
Distance
S0 = 0.61 ( = 30 º)

Figure 11. Schematic illustration of relative lipid bilayer distances and the values of simulated mobility
parameter (S0) related to the angles (γ) between the bilayer surface and the single-chain probe.

Figure 12 shows that human SC stripped from lower-leg presents typical EPR spectra of
5-DSA incorporated in the SC lipids. The EPR spectrum about stripping number 1 is slightly
different from that of number 3. The characteristic peak indicated by the arrow in the
spectrum is prominent for the first strip.
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Stripping
number

S0 = 0.28

S0 = 0.60

2 mT

Figure 12. Experimental (solid line) and simulated (dashed line) EPR spectra of 5-DSA in the first and
the third stripped human SC from lower-leg. The EPR spectrum was obtained with the single scan.

The reasonable agreement of the simulated and experimental spectra suggests that
simulation analysis can provide comprehensive information regarding the SC lipids. The S0
value changes from 0.28 to 0.60, while the S value is in the range of 0.63 to 0.64. The S0
values of 0.28 and 0.60 are the angle of 44° and 31°, respectively. The higher S0 value implies
that the lower SC lipids have less rigid structure than those of the upper SC lipids.
Satisfactory agreement between the experimental and calculated spectra can provide a
quantitative S0, which reveals the microscopic mobility in association with the structure of the
SC lipids.
The EPR simulation can potentially provide further insight into skin-lipid structures. The
mobility parameter (S0) of spin probe will provide the useful index about structural
dependence as a function of the SC depth. It is notable that the value is not the absolute index
for living animals. The value may differ from sample to sample. However, the relative value
of the particular SC sample as a function of the depth could provide a useful index of the SC.
Next, interaction between keratin solution from human epidermis and 5-DSA was
examined. Figure 13 shows EPR spectra of the keratin/5-DSA and 5-DSA stock solutions.
EPR spectrum of 5-DSA stock solution shows typical nitrogen triplet pattern of the probe in
H2O solution as presented in Figure 13 (A).
The EPR spectrum of keratin/5-DSA solution also shows the triplet pattern (Figure 13
(B)) and stays the same after one hour. The similar spectra for both experiments provide that
5-DSA probe does not strongly interact with human keratin in the solution. The results
suggest that 5-DSA probe most likely do not permeate keratin in the period [21].

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 Psoriasis Vulgaris Investigated by Electron Paramagnetic Resonance 971

(A)
5-DSA/H2O

(B)
Keratin/5-DSA/H2O

2 mT

Figure 13. EPR spectra of (A) 5-DSA stock solution and (B) keratin/5-DSA solution are shown. EPR
spectra were taken at ambient temperature.

Other Applications of the EPR Method

Effects of Mild Surfactants on SC Lipids

EPR in conjunction with a slow-tumbling simulation was utilized for examining the
effect of diluted detergent on stratum corneum (SC) lipid structure. SC from the back of
hairless mouse (HOS:HR-1) was stripped consecutively from one to three times. EPR
spectrum of 5-DSA incorporated in the control SC demonstrated a characteristic peak for the
first strip. A slow-tumbling simulation for 5-DSA showed slight differences in mobility
values (S0) of the SC for the control and detergent treated SC. The S0 values were 0.15 and
0.32, respectively. EPR spectra of the detergent treated SC showed that the characteristic
component was eliminated. Thus, the EPR method along with the simulation analysis
revealed the differences in mobility of the detergent treated SC.
Different types as well as mixtures of surfactants change the SC structure of the lipid
bilayer differently. Kawasaki et al. examined the influence of anionic surfactants, sodium
lauryl sulfate (SLS) and sodium lauroyl glutamate (SLG), on human SC by the EPR spin
label method [1]. The qualitative mobility parameter obtained by 1.0% wt SLS-treated
cadaver SC was 0.52. On the other hand, the high S value of 0.73 for 1.0% wt SLG was
obtained. The results suggest clear surfactant effects on the mobility of the lipid bilayer. In
addition, a reasonable correlation between the qualitative mobility parameters and human
clinical data (visual scores and transepidermal water loss values) was demonstrated.

Effects of Skin Penetration Enhancers on SC Lipids

Interaction of skin penetration enhancer correlates with the fluidity of the intercellular
lipid bilayers. Nakagawa and Anzai investigated the effects of terpenes, -terpineol and (+)-
limonene, on SC lipids utilizing the EPR spin probe method [23]. The EPR spectra of -
terpineol treated SC were totally different from those of untreated SC. The results suggest that
-terpineol increases in the penetration of local bilayers surrounding 5-DSA.
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972 Kouichi Nakagawa and Daisuke Sawamura

The -terpineol enhanced permeation of the single chain 5-DSA about three times than
that of the control as shown in Figure 14. However, EPR spectra of CHL in the SC did not
show a clear difference for each strip, except for the signal intensity. The results imply that
CHL permeates SC lipid differently from 5-DSA. The enhancement of the 5-DSA is more
significant than that of CHL [23]. Therefore, the present results can be useful for various drug
administrations via the skin.

Figure 14. A comparison of 5-DSA EPR spectra for control, limonene treated, and α-terpineol treated
SC is presented. The EPR spectra were obtained for the first stripping of the SC.

SC with Psoriasis Vulgaris

Psoriasis vulgaris is classified as a disorder of keratinization although its pathogenesis
has not been fully elucidated. We usually recognize tick scale in psoriasis lesions and found
hyperkeratosis and parakeratosis are found histologically. Turnover time of psoriatic
keratinocytes decreased approximately 7 times and the increase of proliferating cell
components in the epidermis may cause differentiation abnormalities of keratinocytes. In fact,
many results concerning differentiation abnormalities including increase of K6 and K16
expression, and decrease of profilaggrin expression were found in psoriatic epidermis [24-
27].
Figure 15 (A) shows 5-DSA in aqueous solution. A sharp three-line signal of the 5-DSA
aqueous solution was observed. Figure 15 (B) shows the typical EPR spectrum of 5-DSA in
psoriasis vulgaris SC (pv-SC). A little, broad three-line pattern was observed. The spectral
pattern is quite different from those of other SC reported [4-6].
The spectral pattern suggests that the 5-DSA is mobile or less rigid in the SC. The red
dashed-line is the simulated spectrum. The reasonable agreement between the experimental
and simulated spectra was obtained as shown in Figure 15 (B). The S0 value obtained for 5-
DSA in the SC was approximately 0.20. It is important note that the lower value of the S0
indicates the less rigid structure of 5-DSA probe moieties.

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 Psoriasis Vulgaris Investigated by Electron Paramagnetic Resonance 973

Figure 15. (A) EPR spectrum of aqueous 5-DSA stock solution is presented. (B) EPR spectrum of (B)
psoriasis vulgaris SC is presented. Experimental (solid line) and simulated (dash line) EPR spectra of
5-DSA probe are shown. (C) EPR signal due to 5-DSA of the cyanoacrylate on the glass plate. (D) EPR
spectrum of 5-DSA of the typical mid-volar forearm SC (control) is presented. All EPR spectra were
obtained with the single scan.

One can recognize additional small peaks at lower and higher magnetic fields as
indicated by the arrows in Figure 15 (B). These peaks can be due to 5-DSA located in the
rigid site in the sample. These spectral differences can be related to the structural differences
in the pv-SC. Thus, a part of 5-DSA is immobile site in the case of pv-SC.
Figure 15 (C) is EPR spectrum of the mid-volar forearm (control). The EPR pattern is
very similar to those for the forearm SC previously reported [4-6]. The red dash-line is the
simulated spectrum. Good agreement between the experimental and simulated spectra was
obtained. The S0-value obtained was 0.42. The quantitative structural rigidity (0.42) of the SC
lipids also implies that the probe moiety is relatively rigid. In addition, the signal intensity of
the control is weaker based on the S/N than that of the pv-SC and does not show the strong
three-line pattern. The weak signal demonstrates the low amount permeation of 5-DSA in
control SC.
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974 Kouichi Nakagawa and Daisuke Sawamura

Figure 16. Plot of simulated or mobility parameter (S0) of the control and psoriasis vulgaris SC. The
statistical results obtained for the control SC and the psoriasis vulgaris SC are 4.9 ± 0.90 and 2.0 ±
0.25, respectively. Each value represents mean ± SD three measurements. The S0 values of the control
SC show significantly higher values than those of pv-SC (p < 0.01).

Figure 17. (a) EPR spectrum of psoriasis vulgaris SC is presented. (b) EPR spectrum of control SC is
presented. (c) Add EPR spectrum of (0.7×(a) + 0.3×(b)) is presented. The dash lines indicate
immobilized components of the spectrum. The spectrum is re-presentation of the spectrum of Figure 14
(B).

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 Psoriasis Vulgaris Investigated by Electron Paramagnetic Resonance 975

Figure 16 shows the bar chart of the S0 values corresponding to the control and the pv-
SC. The low S0 value of 0.19 for the pv-SC is associated with irregular structure of the lipids
in the SC. Student’s t-test analysis suggests that the 0.19 value of pv-SC is significantly
smaller than the 0.48 value of the control (p < 0.01). The statistical analysis is consistent with
those of the experimental spectra obtained.
Figure 17 shows that the detailed comparison of pv-SC (a) and control (b). In each case,
peak areas (double intregral) of the spectrum are normalized to 1. In the case of pv-SC, 5-
DSA is in the mobile site of the pv-SC. Figure 17 (c) shows the added spectrum of 0.7 times
(a) and 0.3 times (b). The spectrum (c) is very similar to the Figure 15 (B), except for the
broad line-width. The line-width of Figure 17 (a) is sharp because 5-DSA is originally mobile
in the sample. Contrary in the case of control, 5-DSA is immobile site in the case of control
and EPR spectra show the immobile pattern. These spectral differences can be reflected by
the structural differences in the SC samples. Thus, the added spectrum in Figure 17 (c)
suggests that approximately 30% of 5-DSA in the sample can be in rigid site.
In this study, we found that the pv-SC is less rigid of the structure than that of the control
SC, indicating abnormal architecture of psoriasis vulgaris stratum corneum. This result is
consistent with previous observations [21]. Therefore, we suggest that this EPR assay is of
great use for evaluating SC function and can be extended to other skin diseases with abnormal
keratinization.

Psoriatic Nails
Nail lesions are common features of psoriasis and found in almost half of nail psoriatic
patients. Clinical manifestations include pitting, onycholysis, hyperkeratosis, splinter
hemorrhages and so on. Nail psoriasis is associated with discomfort and causes significant
functional impairments and psychological stress. However, nail involvement is often
overlooked and treatment is focused on cleaning the cutaneous lesions. Furthermore, there is
no feasible spectroscopic method to evaluate changes and severity of nail psoriasis. EPR
(electron paramagnetic resonance) is also useful for elucidating structural aspects of stratum
corneum (SC) [21, 23, 29, 30]. Therefore, we thought that EPR might be feasible for
evaluating nail conditions in the patients with nail psoriasis.
EPR spectral changes are due to the molecular motion of the 5-DSA probe as we
discussed in the section of EPR line-shape due to spin probe motion. In the fast motional
region, EPR spectrum is a clear three line pattern. In the slow motional region, EPR spectrum
shows an asymmetric pattern [28]. EPR spectra obtained from the nails are shown in Figure
18. Both spectra are composed of two components: one is the fast 5-DSA probe motion which
indicates smaller hyperfine coupling, and the other is the slow motion in the nail. In order to
analyze the spectra obtained, we can take the intensity ratio of the two motions at the peaks of
the lower magnetic field:

F ( peak )
(9)
S ( peak )

F and S are peak intensities for fast and slow probe motions, respectively. The peaks are
indicated in Figure 18. The fast motion (F) refers to the relatively fast probe molecules in a
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976 Kouichi Nakagawa and Daisuke Sawamura

nail, and shows three EPR lines. The nitrogen hyperfine coupling of the 5-DSA is smaller due
to the fast probe motion.

Figure 18. (A) EPR spectrum of finger nail with psoriasis obtained after incubation with 5-DSA
aqueous solution is presented. (B) EPR spectrum of control nail. Two sites (fast motion (F) and slow
motion (S)) three-line pattern in the nails for both (A) and (B) spectra are presented. The lowest peak
intensity of each spectrum was taken for the calculation. The nitrogen hyperfine coupling for the fast
motion is smaller than that of the slow motion.

The slow motion (S) refers to the relatively slow probe molecules in a nail, and shows an
anisotropic EPR pattern due to restricted motion in the nail. The spectrum obtained,
composed of parallel and perpendicular hyperfine components, shows an asymmetric pattern.
This asymmetric EPR pattern is always observed for controlled SC [21, 23, 29, 30]. We
obtained a stronger asymmetric pattern for the control nails for those with the nail psoriasis.
Thus, two distinct motions suggest there are two distinguishable sites in the nails.

Figure 19. The bar plot of the relative (F/S) values of the control and nails with psoriasis. The statistical
results obtained for the control nails and the psoriasis nails are 2.02 ± 0.606 and 5.12 ± 1.06,
respectively. Each value represents mean ± SD for five individual measurements. The (F/S) values of
the control nail show significantly smaller values than those of the psoriasis nails (p < 0.01).

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 Psoriasis Vulgaris Investigated by Electron Paramagnetic Resonance 977

The plot of the relative (F/S) values of the finger nails for control and nail psoriasis is
shown in Figure 19. The analyses suggest that the relative intensity of the nails with psoriasis
is ~3 times higher than those of the control. The smaller (F/S) values for the control indicate
that the rigid site is dominant. In the case of the nail psoriasis, the fast component is more
intense than that of the slow component. Student’s t-test suggests that the (F/S) value of nail
psoriasis is higher statistical values than those of the control (p < 0.01).
Figure 20 shows that the calculated EPR spectra for the fast motion (a) and the slow
motion (b). Calculation of EPR spectra were performed using EasySpin 4.0.0 version [31]. In
the case of fast 5-DSA motion, EPR spectrum shows a three-line pattern. Contrarily, the EPR
spectrum shows asymmetric pattern for slow 5-DSA motion. In each case, peak areas (double
intregral) of the spectrum are normalized to 1. The calculation of the spectra shows that
rotational correlation time of the fast motion is approximately 200 times shorter than that of
the slow motion.
Figure 20 (c) shows the added spectrum of 0.6 times (a) and 0.4 times (b). The spectrum
(c) is very similar to the Figure 15 (B), except for the central region. The line-width of Figure
20 (a) is sharp because 5-DSA was originally mobile in the sample. The mobility due to the
probe location also reflects to g-value difference. The difference can be related to the main
spectral discrepancy between the observed and calculated spectra. Contrarily, the 5-DSA
motion is slow and EPR spectrum shows a broad asymmetric pattern. These spectral
differences reflect the structural differences in the clipped nail samples. Thus, the added
spectrum in Figure 20 (c) suggests that approximately ~40% of 5-DSA in the sample can be
in rigid site. Figure 20 (d) shows the sum of EPR spectra of 0.3 times (a) and 0.7 times (b).
The added spectrum in Figure 20 (d) suggests that approximately ~70% of 5-DSA in the
sample can be in the rigid site. Most of the 5-DSA is in the rigid site. Thus, these spectral
differences reflect the structural differences in the nail samples.

Figure 20. The calculated EPR spectra for the fast (a) and the slow motion (b) are presented. In each
case, peak areas are normalized to 1. EPR spectrum (c) is the result of an addition of 0.6×(a) and
0.4×(b). EPR spectrum (d) is the result of an addition of 0.3×(a) and 0.7×(b).
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978 Kouichi Nakagawa and Daisuke Sawamura

Therefore, the present results show that the structure of the psoriasis nail is less rigid
(more fragile) than that of the control. In addition, we scored the NPAS of the examined nails.
Although the sample number was small, the NPAS score tended to be correlated with the
relative EPR (F/S)) values as well as the calculated spectral values. In the case of nail
psoriasis, the fragile components are 2 ~ 3 times more than those of the control. This EPR
method is thought to be a novel and reliable method of evaluating the severity of nail
psoriasis.

CONCLUSION
EPR along with a modern computational analysis provides quantitative insight into the
various SC and nail structures. The EPR spectral pattern contains important information
regarding the probe mobility as well as the SC lipid structure. Satisfactory agreement between
the experimental and calculated spectrum can provide the microscopic lipid structure of the
SC. The SC lipid structures can be related to the SC barrier functions. In addition, the EPR
method recognizes sebaceous exudates [6], detergents [22], penetration enhancers [23], and
pv-SC [29]. Therefore, the EPR technique could in turn provide more comprehensive
information, which would further the understanding of various SC and nails.

REFERENCES
[1] Kawasaki, Y., Quan, D., Sakamoto, K., Cooke, R., and Maibach, H.I. (1999) Influence
of surfactant mixtures on intercellular lipid fluidity and skin barrier function. Skin Res.
Technol., 5, 96-101.
[2] Mizushima, J., Kawasaki, Y., Tabohashi, T., and Maibach, H.I. (2000) Effect of
surfactants on human stratum corneum: electron paramagnetic resonance. Int. J. Pharm,
197, 193-202.
[3] Alonso, A., Meirelles, N.C., Yushmanov, V.E., et al. (1996) Water increases the
fluidity of intercellar membranes of stratum corneum: correlation with water
permeability, elastic and electrical resistance properties. J. Invest. Dermatol., 106,
1058-1063.
[4] Nakagawa, K. (2010) Electron Paramagnetic Resonance Investigation of Stratum
Corneum Lipid Structure, Lipids, 45, 91-96.
[5] Nakagawa, K., Mizushima, J., Takino, Y., Kawashima, T., and Maibach, H.I. (2006)
Chain ordering of stratum corneum lipids investigated by EPR slow-tumbling
simulation. Spectrochimi Acta Part A Mol. and Biomol. Spectroscopy, 63, 816-820.
[6] Yagi, E., Sakamoto, K., and Nakagawa, K. (2007) Depth dependence of stratum
corneum lipid ordering: A slow-tumbling simulation for electron paramagnetic
resonance. J. Invest. Dermatol., 127, 895-899.
[7] Bontė, F., Saunois, A., Pinguet, P., and Meybeck, A. (1997) Existence of a lipid
gradient in the upper stratum corneum and its possible biological significance. Arch.
Dermatol. Res., 289, 78-82.

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[8] Weerheim, A., and Ponec, M. (2001) Determination of stratum corneum lipid profile by
tape stripping in combination with high-performance thin-layer chromatography. Arch.
Dermatol. Res., 293, 191-199.
[9] Bommannan, D., Potts, R.O., and Guy, R.H. (1990) Examination of stratum corneum
barrier function in vivo by infrared spectroscopy. J. Invest. Dermatol., 95, 403-408.
[10] Zhang, G., Moore, D.J., Mendelsohn, R., and Flach, C.R. (2006) Vibrational
microspectroscopy and imaging of molecular composition and structure during human
corneocytes maturation. J. Invest. Dermatol., 126, 1088-1094.
[11] Pilgram, G.S.K., Engelsma-Van Pelt A.M., Bouwstra, J.A., and Koerten, H.K. (1999)
Electron diffraction provides new information on human stratum corneum lipid
organization studied in relation to depth and temperature. J. Invest. Dermatol., 113,
403-409.
[12] Marks, R., and Dawber, R.P. (1971) Skin surface biopsy: an improved technique for the
examination of the horny layer. Br. J. Dermatol., 84, 117-123.
[13] Hubbell, W.L., and McConnell, H.M. (1971) Molecular motion in spin-labeled
phospholipids and membrane. J. Am. Chem. Soc., 93, 314-326.
[14] Ge, M., Rananavare, S.B., and Freed, J.H. (1990) ESR studies of stearic acid binding to
bovine serum albumin. Biochim. Biophys. Acta., 1036, 228-326.
[15] Schneider, D.J., and Freed, J.H., Calculating slow motional magnetic resonance spectra.
In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance Vol. 8, New York:
Plenum Press, 1-76, 1989.
[16] Budil, D.E., Lee, S., Saxena, S., and Freed, J.H. (1996) Nonlinear-least-squares analysis
of slow-motion EPR spectra in one and two dimensions using a modified Levenberg-
Marquardt algorithm. J. Magn. Reson. Ser. A, 120, 155-189.
[17] Meirovitch, E., Igner, D., Igner, E., Moro, G., and Freed, J.H. (1982) Electron-spin
relaxation and ordering in smectic and supercooled nematic liquid crystals. J. Chem.
Phys., 77, 3915-3938.
[18] Crepeau, R.H., Saxena, S., Lee, S., Patyal, B.R., and Freed, J.H. (1994) Studies on lipid
membranes by two-dimensional Fourier transform ESR: enhancement of resolution to
ordering and dynamics. Biophys. J., 66, 1489-1504.
[19] Ge, M., and Freed, J.H. (1998) Polarity profiles in oriented and dispersed
phosphatidylcholine bilayers are different. An ESR study. Biophys. J., 74, 910-917.
[20] Elias, P.M. (1983) Epidermal lipids, barrier function and desquamation. J. Invest.
Dermatol., 80(suppl), 44-49.
[21] Nakagawa, K. (2011) Elucidated Lipid Structures of Various Human Stratum Corneum
Investigated by EPR Spectroscopy, Skin Res. Technol., 17, 245-250.
[22] Nakagawa, K., and Anzai, K. (2011) Stratum Corneum Lipid of Hairless Mouse
Investigated by Electron Paramagnetic Resonance, Appl. Magn. Reson., in press.
[23] Nakagawa, K., and Anzai, K. (2010) Stratum Corneum Lipid Structure Investigated by
EPR Spin-Probe Method: Application of Terpenes, Lipids, 45, 1081-1087.
[24] Iizuka H., Takahashi H., and Ishida-Yamamoto A. Psoriatic architecture constructed by
epidermal remodeling. J. Dermatol. Sci. 2004; 35: 93-9.
[25] Takemoto H., Tamai K., Akasaka E., Rokunohe D., Takiyoshi N., Umegaki N.,
Nakajima K., Aizu T., Kaneko T., Nakano H., and Sawamura D. Relation between the
expression levels of the POU transcription factors Skn-1a and Skn-1n and keratinocyte
differentiation. J. Dermatol. Sci. 2010; 60: 203-5.
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[26] Kim B.E., Howell M.D., Guttman-Yassky E., Gilleaudeau P.M., Cardinale I.R.,
Boguniewicz M., Krueger J.G., and Leung D.Y. TNF-α downregulates filaggrin and
loricrin through c-Jun N-terminal kinase: role for TNF-α antagonists to improve skin
barrier. J. Invest. Dermatol. 2011; 131: 1272-9.
[27] Oyama R., Jinnin M., Kakimoto A., Kanemaru H., Ichihara A., Fujisawa A., Honda N.,
Masuguchi S., Fukushima S., Maruo K., and Ihn H. Circulating microRNA associated
with TNF-α signaling pathway in patients with plaque psoriasis. J. Dermatol. Sci. 2011;
61: 209-11.
[28] Poole Jr C.P., and Farach H.A., Theory of magnetic resonance 2nd ed., John Wiley and
Sons, Inc., New York, 1987; 310-317.
[29] Nakagawa K., Minakawa S., and Sawamura D. Spectroscopic evidence of abnormal
structure of psoriasis vulgaris stratum corneum, J. Dermatol. Sci. 2012; 65; 222-4.
[30] Nakagawa K. Electron paramagnetic resonance studies of skin lipid structure, Chapter
19, Handbook of Cosmetic Science and Technology 3rd Ed, Barel A.O., Paye M., and
Maibach H.I., Eds., Informa Heathcare, New York, 2009; 207-15.
[31] EasySpin 4.0.0 version (an internet available academic program) was used to calculate
the fast and slow motional EPR spectra.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 40

PSORIASIS AND COMORBIDITIES

Nayra Merino de Paz1,, Marina Rodríguez-Martín2

and Patricia Contreras Ferrer1
1
Hospital Universitario de Canarias, La Laguna, Tenerife, Spain
2
Hospital USP Costa Adeje, Adeje, Tenerife, Spain

ABSTRACT
Psoriasis is a chronic cutaneous inflammatory disease that is characterized by
erythematous plaques with grayish scales on their surface.
Nowadays, it is considered a systemic inflammatory disease with metabolic
comorbidities (obesity, diabetes, hypertension and hyperlipidemia) and a high
cardiovascular risk in contrast with non-psoriatic subjects. The activation of Th1 and
Th17 cells with the secretion of inflammatory cytokines has been involved in the
physiopathological basis of psoriasis. The angyogenesis and epidermic hyperproliferation
are secondary to the action of these cytokines. Tumor necrosis factor alpha (TNF-alpha),
interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-23 (IL-23)
and interferon-gamma (INF-gamma) are several of these inflammatory cytokines with
atherogenic action.
Other associated comorbidities are psoriatic arthritis, inflammatory bowel disease,
non-alcoholic fatty liver and psychological disorders. Some malignancies, such as skin
cancer and lymphomas, have shown a high prevalence in patients with psoriasis.. Several
studies have associated unhealthy lifestyles (smoking and alcohol consumption) with
psoriasis patients. An integrated therapy may consider psoriasis systemic comorbidities to
provide appropriate management. So an early diagnosis of these concomitant diseases is
necessary and psoriasis patients should be encouraged to change unhealthy habits to
prevent them.


Corresponding author: Nayra Merino de Paz. E-mail: [email protected].
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982 Nayra Merino de Paz, Marina Rodríguez-Martín and Patricia Contreras Ferrer

INTRODUCTION
Psoriasis is a systemic, immune-mediated disorder, characterized by inflammatory skin
(Figure 1) and joint manifestations. Although the systemic nature of psoriasis often remains
unrecognized, the inflammatory processes involved may be associated with the development
of comorbidities, which, themselves, have a significant impact on the patient's health and
quality of life. Psoriasis has been related to multiple conditions in recent years.
Nowadays, when we evaluate a psoriatic patient, we have to focus not only on cutaneous
manifestations but also on joints, the cardiovascular system, and psychiatric status for optimal
management.
It implies identification and treatment of psychological disorders, addictions and
associated cardiovascular and metabolic diseases. Psoriasis is frequently associated with a
range of comorbidities, including inflammatory bowel disease, heart disease, and obesity,
thus complicating management and negatively impacting mental and emotional health [1].
Recent studies have found that severe psoriasis is significantly associated with a higher
prevalence of comorbidity and nail alterations [2]. Psychiatric comorbidity was found to be
the strongest predictor of poor quality of life, regardless of disease severity [2]. Since
psoriasis is recognized as a systemic disease, patient management must be multidisciplinary.
During the past decade multiple data have shown psoriasis as a systemic disease with an
inflammatory immune mediated-pathogenic basis. This pathogenic origin could contribute to
the inflammatory, cardiovascular, metabolic and neuropsychiatric involvement described,
especially in young patients with severe psoriasis [3].
Other comorbidities significantly associated with psoriasis have been arthritis,
depression, sleep disorder/insomnia, chronic obstructive pulmonary disease, gastroesophageal
reflux disease [4] and malignancy [5].
The hypothesis of an etiologic role of psoriasis in its cardiovascular and metabolic
comorbidities is powered by pathophysiologic concepts establishing a link between chronic
inflammation in psoriasis, endothelial dysfunction, formation of atherosclerotic plaques and
the different compounds of metabolic syndrome [1, 3].

Figure 1. Typical psoriatic plaques.

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 Psoriasis and Comorbidities 983

Psoriasis and Cardiovascular Risk

The relative risks of myocardial infarction (MI) and stroke are increased in patients with
psoriasis compared with the general population. These are especially seen in younger patients
with more severe disease, and are believed to contribute to the 3- to 4-year reduction in life
expectancy among patients with severe psoriasis [5].
The recent results of large studies indicate that the increased cardiovascular (CV) risk is
at least partially attributable to psoriasis and independent of the presence of metabolic
comorbidities [3, 5]. The last systematic review [6] showed that patients with psoriasis
demonstrate a higher prevalence of cardiovascular risk factors and appear to be at increased
risk for ischemic heart disease, cerebrovascular disease, and peripheral arterial disease. This
increase in vascular disease may be independent of shared risk factors and may contribute to
the increase in all-cause mortality [6, 8].
The possible interplay between psoriasis and CV disease is complex. The molecular
mechanisms involved in psoriasis-associated dysregulation of metabolic function are believed
to be due, in large part, to the action of increased levels of proinflammatory factors, such as
tumor necrosis factor-alpha, that are central to the pathogenesis of psoriasis [9]. Large studies
have shown that psoriasis patients were significantly more likely to have cardiovascular
comorbidities, including hypertension, hypercholesterolemia, and diabetes, compared with
non-psoriasis patients [9, 10]. Patients receiving TNF-alpha inhibitors had a 48% reduction in
the risk of myocardial infarction (P = 0.0062) [7]. The presence of joint involvement
increased the risk of myocardial infarction by 42% [7, 8].

Psoriasis and Metabolic Disease

Psoriasis has been related to multiple metabolic disorders, like diabetes, dyslipidemia and
obesity [11-13]. Interestingly, many reports demonstrate that adipose tissue is metabolically
active, representing a source of inflammatory mediators, known as adipokines [11]. Metabolic
diseases such as obesity and diabetes have overlapping genetic predispositions with psoriasis
[10]. Both conditions are likely to also interact at a functional level because obesity and the
up-regulation of pro-inflammatory mediators in psoriasis appear to influence adipocyte
homoeostasis [8]. This may perpetuate psoriatic inflammation, displaying similarities to the
immunopathogenesis of atherosclerosis [1].
Finally, the disturbed adipokine profile and inflammation associated with psoriasis
enhances insulin resistance, causing subsequent endothelial dysfunction, atherosclerosis and
eventual coronary events [8]. Successful treatment with methotrexate appears to lower the
rates of MI in patients with psoriasis [8]. TNF-alpha inhibitors are known to counteract
insulin resistance and emerging studies demonstrate an even higher protective effect of TNF-
alpha antagonist therapy against the development of diabetes or CV co-morbidities in patients
[6, 9]. The latter include TNF-alpha, macrophage chemo-attractant protein-1, plasminogen
activator inhibitor-1 (PAI-1) [12], IL-6, leptin and adiponectin, leading to a pro-inflammatory
status in obese subjects. This evidence supports the idea of obesity as a low-grade
inflammatory disease [11]. In particular, it seems to affect several features of psoriasis, such
as its development, cardiovascular risk and clinical outcome. Recent data suggest that
increased BMI in early adulthood increases the risk of psoriatic arthritis development in
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psoriatic patients, supporting a link between fat-mediated inflammation and joint involvement
[11-14].
Multiple theories could explain the relationship between psoriasis and metabolic
abnormalities. In most obese patients, obesity is associated with a low-grade inflammation of
white adipose tissue (WAT) resulting from chronic activation of the innate immune system,
which can subsequently lead to insulin resistance, impaired glucose tolerance and even
diabetes [11-15]. WAT is characterized by an increased production and secretion of a wide
range of inflammatory molecules including TNF-alpha and IL-6, which may have local
effects on WAT physiology but also systemic effects on other organs [15]. Several factors
derived not only from adipocytes but also from infiltrated macrophages probably contribute to
the pathogenesis of insulin resistance. Conversely, expression and plasma levels of
adiponectin, an insulin-sensitising effector, are down-regulated during obesity. Leptin could
modulate TNF-alpha production and macrophage activation [15]. TNF-alpha is overproduced
in adipose tissue of several rodent models of obesity and has an important role in the
pathogenesis of insulin resistance in these species [14, 15].
Both TNF-alpha and IL-6 can alter insulin sensitivity by triggering different key steps in
the insulin signaling pathway [15]. Adiponectin is highly expressed in WAT, and circulating
adiponectin levels are decreased in subjects with obesity-related insulin resistance, type 2
diabetes and coronary heart disease [15].
In obesity, the pro-inflammatory effects of cytokines through intracellular signaling
pathways involve the NF-kappaB (nuclear factor kappa-light-chain-enhancer of activated B
cells) and JNK (C-Jun N-terminal kinases) systems. Genetic or pharmacological
manipulations of these effectors of the inflammatory response have been shown to modulate
insulin sensitivity in different animal models [15].
Many studies in the last decade have shown a relation between psoriasis and dyslipemia.
The last systematic review about psoriasis and dyslipemia found that psoriasis was
significantly associated with greater odds and incidence of dyslipidemia [12]. Greater
psoriasis severity appeared to be associated with a higher prevalence of dyslipidemia [12].
Diabetes mellitus has also been related to psoriasis. A recent systematic review found that
psoriasis is associated with an increased prevalence and incidence of diabetes. The
association of psoriasis with diabetes may be strongest among patients with severe psoriasis
[13]. The same results have been found with obesity. Obesity may represent an additive
cardio-metabolic risk factor in psoriatic subjects. Abdominal obesity (Figure 2) may also
determine an increased risk of not achieving minimal disease activity, highlighting the role of
abdominal fat accumulation as a negative predictor of good clinical response to biologic
agents [14].
Overall, compared with the general population, psoriasis patients have a higher
prevalence and incidence of obesity. Patients with severe psoriasis have greater odds of
obesity than those with mild psoriasis [14].

Psoriatic Arthritis (PsA)

PsA could be defined as an inflammatory joint disease associated with psoriasis. It

presents a chronic evolution with flares and periods of remission. It is a seronegative
spondyloathropathy related with HLA-B27.

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Figure 2. A patient with abdominal obesity and psoriasis.

It could be a severe disease and it is a criterion for systemical treatment [1-3]. An erosive
evolution has been reported in 66% of patients with PsA developing an important dysfunction
and a high quality of life affectation [16].
The prevalence of PsA is around 0.1-1% of the general population and it is lower than
rheumatoid arthritis prevalence [17].
Around 30% of psoriasis patients will develop PsA and 70% present before the cutaneous
lesions, so a dermatologic evaluation is very important to get an early diagnosis [18-19].
There are not differences by gender, but a light male predominance has been reported. It
could appear at any age but the highest prevalence is in adults between 30 and 50 years old
[3].
Several factors are associated to its pathogenesis. A genetic factor is suggestive by the
high rate of psoriasis and PsA in first-grade family members. The PsA is associated with gene
polymorphisms in HLA area of chromosome. A high association has been described with
HLA Cw6.
Other related factors are traumas and infections [20].
In 1973, Moll and Wright suggested different clinical presentations for the PsA: [7, 21]

1 Asymmetric: This type affects less than five joints, especially knees, ankles and
wrists. It usually presents a mild evolution.
2 Symmetric: It affects more than five joints symmetrically. Clinical manifestation is
similar to rheumatoid arthritis. Differences are: 1) a negative test result of
rheumatoid factor, 2) the asymmetrical affectation of the distal interphalangeal joints
of the hands and 3) the lack of cutaneous nodules. This type usually affects hands,
wrists, feet and hip. It may develop a severe form of arthritis.
3 Distal interphalangeal predominant: It is associated with a severe nail affectation. It
is more prevalent in men.
4 Arthritis mutilans: A short-time severe joint destruction is observed. A typical
radiological image (pencil and cup) is present. It usually affects hands and feet. The
prevalence is higher in men.
5 Spondylitis: It is characterized by the affectation of axial skeleton and sacroiliac
joints. Radiology could show syndesmophytes and paravertebral ossifications. More
prevalent in men.
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Other anatomical structures could be affected by PsA including:

1 Typical peri-articular symptoms: [22-24]

 Enthesitis: Is the inflammation of the sites where tendons or ligaments insert into
the bone. It affects around 40% of patients. The Achilles tendon and plantar fascia
are the preferred sites.
 Dactylitis: Is characterized by the sausage-swelling aspect of fingers or toes. It
affects around 30-40% of patients.
2 Extra-articular symptoms: [25-28]
 Cutaneous. Psoriasis is the major symptom. The most common type of psoriasis
is psoriasis vulgaris or common chronic stable plaque psoriasis.
 Changes on the nails (Figure 3 and 4): It has been observed in the 80% of patients
with PsA. Pitting, hyperkeratosis and “oil drops” are usually present. It could be
measured by Nail Psoriasis Severity Index (NAPSI) scale (Table. I).
 Bowel inflammation: similar to Crohn’s disease. The improvement of the bowel
disease is associated with the articular inflammation.
 Ocular symptoms: Conjunctivitis and acute anterior uveitis.
 Heart symptoms: Aortic valve inflammation may develop a heart failure.

Several clinical factors are associated with a worse evolution: 1) female gender, 2)
polyarticular onset, 3) high levels of acute phase proteins, 4) early joint damage and
dysfunction, 5) a positive test for HLA-B27, B9, DQW3 or C08, 6) radiological erosions and
7) lack of response to treatments [29-33].

Figure 3. Nail matrix disorders: Oil drop (black arrows) and pitting (blue arrows).

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 Psoriasis and Comorbidities 987

Figure 4. Nail bed abnormalities: subungual hyperkeratosis and onycholisis.

A higher rate of mortality has been described in these patients secondary to the great
inflammatory activity associated with other complications such as diabetes, arteriosclerosis
and cardiovascular risk [3].

Table 1. NAPSI score

Each nail is divided into 4 quadrants.

1. Nail matrix examination for the presence of these signs: pitting, leukonychia, red lunulae,
trachyonychia.
Score for each nail: 0 none, 1 in a single quadrant, 2 in 2 quadrants, 3 in 3 quadrants and 4 in all
quadrants.
Nail matrix score: from 0 to 40 for the fingernails and to 80 if toenails are included
2. Nail bed examination for the presence of: onycholysis, subungual hyperkeratosis, oil spots and
splinter hemorrhages.
Score for each nail: 0 none, 1 in one quadrant, 2 in 2 quadrants, 3 in 3 quadrants and 4 in all
quadrants.
Nail bed score: from 0 to 40 for the fingernails and to 80 if toenails are included
Total NAPSI score: from 0 to 80 for the fingernails and to 160 if toenails are included.

The diagnosis is mainly clinical. There is no definitive test to diagnose PsA. Several tests
are used to try to get an early diagnosis: the Psoriasis and Arthritis questionnaire (PAQ), the
Psoriasis Arthritis screening and evaluation (PASE), the Psoriasis Epidemiology screening
tool (PEST) and the Psoriasis Arthritis screening questionnaire (ToPAS).
Finally, in 2006 the CASPAR criteria (Classification criteria for Psoriatic Arthritis) were
proposed by the GRAPPA (Group for Research and Assessment of Psoriasis and Psoriatic
Arthritis). A high sensitivity of 91.4% and a specificity of 98.7% was shown with these
criteria (Table II and Figure 5) [32, 34, 35].
Blood tests such as rheumatoid factor, C-reactive protein (CRP) and erythrocyte
sedimentation rate (ESR) are useful in this entity. Rheumatoid factor is usually negative in
PsA and is useful to differentiate PsA from rheumatoid arthritis.
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However, around 13% of PsA patients may test positive. CRP and ESR are useful to
control the treatment response. Moreover, ESR is associated with high inflammatory activity
and high mortality.

Table 2. CASPAR criteria

Inflammatory articular disease with 3 or more points for the following items:
1. Evidence of current psoriasis (defined by rheumatologist or dermatologist), a personal history of
psoriasis or a family history of psoriasis in a first- or second-degree relative. (2 points)
2. Typical psoriatic nail dystrophy, including onycholysis, pitting and hyperkeratosis, observed on
current physical examination. (1 point)
3. A negative test result for the presence of rheumatoid factor. (1 point)
4. Current dactylitis or a history of dactylitis recorded by rheumatologist (1 point)
5. Radiographic evidence of yuxta-articular new bone formation, appearing as ill-defined ossification
near joint margins (but excluding osteophyte formation) on plain radiographs of the hands or foot. (1
point)

Figure 5. Patient with psoriatic plaques, nails changes and inflammatory articular disease.

Figure 6. The radiologic appearance of PsA (feet and sacroiliac joints involvement).

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Radiologic tests such as X-ray (Figure 6), US, and MRI could be helpful. X-ray evaluates
the joint affectation, the evolution and the treatment response. US and MRI are useful to
assess enthesitis and sinovitis. Sacroiliac joints may be evaluated with MRI [32, 34, 35].
The treatment depends on the clinical features, the type of arthritis, the severity and the
impact on quality of life [34]. Peripheral arthritis is treated with disease-modifying
antirheumatic drugs (DMAD), NSAIDS or oral corticosteroids. Milder cases of the disease
may be treated with NSAIDS alone. Moderate and severe cases (more than three joints
affected, refractory enthesitis or dactylitis) need treatment with a DMAD alone or with oral
corticosteroids at a low dose. Axial arthritis may be treated with NSAIDs, because DMADs
are not effective [35].
Recently, other drugs called biological response modifiers (TNF-alpha inhibitors) have
demonstrated their efficacy and security to treat refractory cases of PsA. Infliximab,
etanercept, golimumab, certolizumab and adalimumab are included in this group of drugs
[36].

Non-Alcoholic Fatty Liver Disease (NAFLD)

NAFLD occurred when more than 5% of the hepatocytes present a deposit of

triglycerides not due to excessive alcohol use [38]. Nowadays it is a sign of metabolic
syndrome. NAFLD is the most frequent liver disease in industrialized countries [39]. A third
of the general population is affected [40-41]. Diabetes mellitus, obesity, insulin resistance and
dyslipidemia have been described as risk factors to present a NAFLD. In fact, NADFLD have
been associated with a high risk of cardiovascular events independent from metabolic
syndrome [42]. The prevalence of NAFLD increases with age and is higher in men [43].
Three clinical patterns have been described: a) Hepatic steatosis: lipid deposits without
inflammation. B) Steatohepatitis: characterized by the inflammation of the hepatic lobules. c)
Cirrhosis: consist on a liver fibrosis and may develop a hepatocellular carcinoma [44]. Over
time, around 30% of patients with hepatic steatosis may develop a steatohepatitis in 7-10
years and around 20% of patients with steatohepatitis may progress to cirrhosis in 7-8 years
[38].
Alcohol abuse, hepatitis B and C, hepatotoxic drugs and auto-immune diseases must be
excluded to make a correct diagnosis. Most patients are asymptomatic. But often an
abdominal pain could be present. Blood test with liver enzymes could be useful to the
diagnosis. Liver enzymes are in normal ranges in around 80% of patients with simple hepatic
steatosis [43]. However patients with steatohepatitis show an increase of liver enzymes, but
not three times higher than the normal range [38]. GGT and AF elevation usually have been
associated with the NAFLD, however bilirrubin and albumin usually stay at normal ranges.
The liver biopsy is the gold standard, but it presents a high risk of bleeding and mortality. So,
other non-invasive tests are preferred, such as US with a high security and sensitivity (60-
94%), TC or MRI. US sensitivity presents a reduction when the fatty infiltration is lower than
30% [45].
NAFLD is associated with diabetes, insulin resistance, hypertension, dyslipidemia,
obesity and metabolic syndrome. These entities are characterized by an increase of
proinflammatory cytokines (TNF alpha and IL6) and a reduction of adiponectin levels [38].
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990 Nayra Merino de Paz, Marina Rodríguez-Martín and Patricia Contreras Ferrer

Several common treatments for psoriasis, such as methotrexate or acitretin, may be

reconsidered in these patients because of their risk of liver damage [46]. NAFLD patients
must follow healthy lifestyle recommendations with a good diet and stop smoking and
drinking alcohol [47].

Crohn’s Disease (CD)

CD is a digestive tract inflammatory disease that may affect any part of the tract, from
mouth to anus. It is included in the inflammatory bowel diseases. It affects young people and
causes a high impact on quality of life [48, 49]. Currently the incidence is 9 cases per 100,000
people per year [50]. CD can present gastrointestinal, systemic or extra-intestinal symptoms.
Diarrhea, fever, fistulization and bowel obstruction are the main symptoms. It is characterized
by flares and remissions. Toxic megacolon, hemorrhages, colon cancer or perforation, are
some of its complications. Extraintestinal symptoms include uveitis, episcleritis, seronegative
spondiloarthropathy, colelytiasis, secondary amyloidosis, nefrolytiasis, deep venous
thrombosis, autoimmune hemolytic anemia, erythema nodosum and pyoderma gangrenosum
[51].
An association between psoriasis and CD has been described [52]. A patient with CD
presents a 7 times higher risk of developing psoriasis and a patient with psoriasis presents a 3
times higher risk of developing CD [53].
Recently, a gene (CARD-15) located in 16q21 chromosome, the same area of PSORS8,
has been reported in these patients. This relation could explain the susceptibility, but it has
not been demonstrated yet.
Several molecules and cytokines as TNF-alpha, INF-gamma, IL-12, T lymphocytes are
implicated in both pathologies [52, 53]. Moreover both entities are associated with other
inflammatory diseases such as ankilosant spondilitis [54].

Erectile Dysfunction

An association has been made between previous psoriasis and erectile dysfunction (ED)
[56]. Both the metabolic and cardiovascular comorbidities described share risk factors with
this condition, so clinicians dealing with psoriatic patients need to be alert to the development
of ED.

Uveitis

Uveitis is characterized by a process of intraocular inflammation resulting from various

causes. Both psoriasis and uveitis are immune-mediated diseases. It seems that psoriasis
without arthropathy is not a risk factor for the development of uveitis [57].
Uveitis tends to develop more frequently in patients with arthropathy or pustular psoriasis
than in patients with other forms of psoriasis [22]. An ophthalmic examination should be
performed periodically in patients with psoriasis and uveitis.

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If ophthalmopathy is diagnosed, the patient should receive adequate treatment with anti-
inflammatory drugs or immunomodulators to prevent vision loss.

Cancer

Lymphoma
Thirty-five types of lymphoma have been described and they are included in two groups:
a) Non-Hodgkin lymphoma and b) Hodgkin lymphoma. Early stages are usually
asymptomatic [38]. Lymphadenophaties are the main sign. Other symptoms such as fever,
loss of weight, malaise, fatigue, dyspnea, anorexia and night sweats may also be present. The
histological study is the gold standard for diagnosis.
Several studies have associated lymphomas and psoriasis. The hypotheses to explain this
relation are: 1) the immunological alteration in psoriasis with a high activity of T-
lymphocytes, dendritic cells, Th1 cytokines and B-lymphocytes. 2) Use of immunosuppressor
drugs (methotrexate, cyclosporine and biological) [55].

Cutaneous Non-Melanoma Cancer (CNMC)

CNMC represents 97% of cutaneous tumors. Basal cell carcinoma (70%) is the most
prevalent tumor. The associated mortality is very low.
The increase of CNMC in psoriasis patients is associated with cyclosporine and
phototherapy (especially UV-A) treatments [38].

Psoriasis and Psychological and Psychiatric Comorbidities

The skin and the central nervous system are embryologically related, and they share
several hormones, neurotransmitters, and receptors. The skin plays a key role as a sensory
organ in the socialization processes throughout the life cycle. Numerous skin changes are
seen in response to emotional stimuli, and skin appearance greatly influences body image and
self-esteem.
It has been reported that psychological stress perturbs the epidermal permeability barrier
homeostasis, thus acting as a precipitant for psoriasis. Psychiatric and psychosocial factors
play an important role in several skin diseases and the prevalence of psychiatric morbidity in
these patients is also very high [15, 58, 59]. The immune and autonomic system may present
changes with stress. Catecholamines can influence cytokine production and increase
lymphocytic Th-1 activity secondary to acute stress.
However, an endocrine response is activated by chronic stress, depending on the
hypothalamic-pituitary-adrenal axis. This pathway produces ACTH, corticosteroids,
decreases the cellular immune response and the induction of the humoral Th-2 one. Recent
studies have described a neural-immuno-cutaneous-endocrine network. In susceptible patients
stress induces the release of neuroimmune substances that may activate the skin inflammation
[60]. About 40-80% of psoriasis patients identify the presence of one or more stressful
factors before the onset of the disease.
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Moreover, stress factors can exacerbate preexisting psoriasis. So, there is a bidirectional
relationship among psychological factors and psoriasis [61].
Psoriasis is a visible cutaneous disease with a high physical, psychological, social and
occupational impact. Psoriasis has been associated with several psychological and psychiatric
comorbidities such as loss of quality of life, disability, trauma, stigmatization, poor self-
esteem, suicidal ideation, social isolation, stress, anxiety, depression, substances abuse and
sexual dysfunction [62].
One of the most important features to evaluate the psychological status of patients with
psoriasis is the quality of life (QOL). Several studies have indicated an association between
psoriasis and the affectation of QOL [2, 63]. The dermatology life quality index (DLQI) scale
is used to quantify this entity (Table III).
It is a simple 10-question validated questionnaire designed for use in adults. The DLQI
can be analysed under six dimensions of life: 1) symptoms and feelings, 2) daily activities, 3)
leisure, 4) work and school, 5) personal relationships and 6) treatment [64].
The impact of psoriasis on QOL is significant even when it involves a relatively limited
body surface area [62]. Several studies have shown that those patients with psoriasis and
psychiatric comorbidities present a high affectation of their QOL.
In fact, psychiatric comorbidity is the major predictor of bad QOL and it is independent
of the psoriasis severity. Psoriasis localization has been described as a relevant feature too.
So, patients with psoriasis located on their scalp present a worse QOL. A reduction of QOL is
associated with a loss in work productivity, higher levels of stress and problems with
treatments [63].

Table 3. Dermatology Life Quality Index (DLQI)

The scoring of each question is as follows: “Very much” (score 3), “A lot” (score 2), “A little” (score
1), “Not at all” (score 0). The question 7 has the choices “Yes” (score 3), “No” or “Not relevant”
(score 0).
1. Over the last week, how itchy, sore, painful or stinging has your skin been?
2. Over the last week, how embarrassed or self conscious have you been because of your skin?
3. Over the last week, how much has your skin interfered with you going shopping or looking after
your home or garden?
4. Over the last week, how much has your skin influenced the clothes you wear?
5. Over the last week, how much has your skin affected any social or leisure activities?
6. Over the last week, how much has your skin made it difficult for you to do any sport?
7. Over the last week, has your skin prevented you from working or studying?
1. If “no” over the last week, how much has your skin been a problem at work or studying?
8. Over the last week, how much has your skin created problems with your partner or any of your
close friends or relatives?
9. Over the last week, how much has your skin caused any sexual difficulties?
10. Over the last week, how much of a problem has the treatment for your skin been, for example by
making your home messy, or by taking up time?
The maximum score (the highest possible impairment of quality of life) is 30 and the minimum 0.
0-1: no impairment on patient’s quality of life
2-5: small effect on patient’s quality of life
6-10: moderate effect on patient’s quality of life
11-20: very large effect on patient’s quality of life
21-30: extremely large effect on patient’s quality of life

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In different large epidemiological studies up to 79% of psoriasis patients had a negative

impact on their lives. Psoriasis was reported to be associated with a stressful life event in 10-
90%, depression in 24-51%, shamefulness and embarrassment over their appearance in 89%,
lack of confidence in 42%, family friction in 26%, wishing to be dead to active suicidal
ideation in 9.7-5.5%, addiction and alcoholism in 18%. There was also a significant impact
upon sexual function, bipolar disorder, delirium and eating disorders [58, 59, 65, 66.]
The high rates of depression may be associated with inflammation substances as TNF-
alpha or INF-gamma [62]. Several studies have confirmed smoking as an independent risk
factor for psoriasis. Toxic cigarette chemicals and nicotine may affect immune response,
activating T-cells and releasing inflammatory cytokines. Moreover, keratinocytes present
nicotinic receptors, which control adhesive properties and epidermal migration. However, the
relation between psoriasis and alcohol is not completely elucidated, but it has an impact on
the psychological comorbidity of patients with psoriasis [60]. Children with psoriasis had a
25-47% higher risk of developing any psychiatric disorder, a 23-62% higher risk of
developing depression, and a 25-32% higher risk of anxiety. Pediatric patients with psoriasis
had an increased risk of developing psychiatric disorders, including depression and anxiety,
compared with psoriasis-free control subjects [63, 66].
Alexithymia consists of a special difficulty to express one’s own feelings. Alexithymic
patients are at special risk for other medical and psychiatric disorders. It is considered a
triggering factor associated with other diseases. The failure of these patients to control their
emotions results in an exacerbated autonomic and neuroendocrine response, producing
several somatic diseases (hypertension, asthma, myocardial infarction, functional
gastrointestinal disorders, fibromyalgia…).
These patients tend to discharge tension with impulsive acts. It can be measured with a
great variety of questionnaires such as the Bermond-Vorst Alexithymia Questionnaire
(BVAQ), the Observer Alexithymia Scale (OAS), the Schalling-Sifneos Personality Scale
(SSPS), the MMPI-Alexithymia Scale (MMPI-AS) or the Toronto Alexithymia Scale (TAS-
26 or TAS-20) (Table IV). The prevalence of Alexithymia is around 10-13% in the general
population. It is associated with poor education and a low-income level and it is more
prevalent in men.
Several studies have demonstrated a higher prevalence of alexithymia between patients
with psoriasis in comparison with control subjects. However, results are not uniform and they
have used different scales to measure the presence of alexithymia [60].
Stigmatization is another of the main problems among psoriatic patients. It is defined as
having a discrediting mark that leads to social discrimination and alienation. The
Stigmatization scale and Feeling of Stigmatization questionnaire help to measure this entity
[67]. Studies have observed higher levels of stigmatization between patients with psoriasis in
comparison with other skin conditions [67-68].
In fact, experiences of stigmatization mediate the association among psoriasis severity
and quality of life score, explaining these cases that present mild psoriasis and a low quality
of life [68].
A high number of psychological interventions have shown promise in recent trials.
Moreover, some preliminary data suggest that several drugs may help psoriatic patients
with depression and stress. Moclobemide, bupropion, selective serotonin reuptake inhibitors,
immunomodulators (such as methotrexate and cyclosporine) and biologic drugs (adalimumab,
etanercept, infliximab and ustekinumab) may improve the psychiatric comorbidities [60].
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Table 4. TAS-20 questionnaire

I am often confused about the way I am feeling inside.

I find it difficult to say how I feel inside.
I feel things in my body that even doctors don’t understand.
I can easily say how I feel inside.
When I have a problem, I want to know where it comes from and not just talk about it.
When I am upset, I don’t know If I am sad, scared or angry.
I am often puzzled by things that I feel in my body.
I’d rather wait and see what happens, instead of thinking about why things happen.
Sometimes I can’t find the words to say how I feel inside.
It is important to understand how you feel inside.
I find it hard to say how I feel about other people.
Other people tell me that I should talk more about how I feel inside.
I don’t know what’s going on inside me.
I often don’t know why I am angry.
I prefer talking to people about everyday things, rather than about how they feel.
I prefer watching funny television programs, rather than films that tell a story about other people’s
problems.
It is difficult to me to say how I really feel inside, even to my best friend.
I can feel close to someone, even when we are sitting still and not saying anything.
Thinking about how I feel, helps me when I want to do something about my problems.
When I have to concentrate on a film to understand the story, I enjoy the film much less.
Each item is rated by this way: 1 point (strongly disagree), 2 points (disagree), 3 points (unsure), 4
points (agree) and 5 points (strongly agree).
Cases of alexithymia are defined by the presence of 61 points or more and possible alexithymia is
considered if the score is between 52 and 60.
Three subscales could be differentiated: F1 or difficulty identifying feelings and distinguishing
between feelings and the bodily sensations of emotional arousal (red items), F2 or difficulty
describing feelings to others (blue items) and F3 or externally-oriented thinking (black items).

It is important that clinicians consider the psychosocial aspects of this illness. The results
shown in multiple studies imply the need for careful examination of the mental state of
patients with psoriasis in order to offer and provide treatment of any concomitant psychiatric
conditions [58-63]. Finally, social interventions to raise awareness that psoriasis is a non-
contagious chronic condition may reduce the feelings of stigmatization [67, 68].

REFERENCES
[1] Krueger, G. G., Koo, J., Lebwohl, M., Menter, A., Sterns, R. S., Rolstad, T. The impact
of psoriasis on quality of life: Results of a 1998 National Foundation patient-
Membership survey. Arch. Dermatol. 2001;137: 280-4.
[2] Hernanz, J. M., Sanchez regaña, M., Izu, R., Mendiola, V., García Calvo, C. Clinical
and Therapeutic evaluation of patients with moderate to severe psoriasis in Spain: The
sequence study. Actas Dermosifiliogr. 2012; 103:897-904.

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[3] Bens, G., Maccari, F., Esteve, E. Psoriasis: a systemic disease. Presse Med. 2012; 41:
338-48.
[4] Wu, Y., Mills, D., Bala, M. Psoriasis: cardiovascular risk factors and other disease
comorbidities. J. Drugs Dermatol. 2008 Apr.;7(4):373-7.
[5] Gottlieb, A. B., Dann, F. Comorbidities in patients with psoriasis. Am. J. Med. 2009
Dec.;122(12):1150.e1-9.
[6] Patel, R. V., Shelling, M. L., Prodanovich, S., Federman, D. G., Kirsner, R. S. Psoriasis
and vascular disease-risk factors and outcomes: a systematic review of the literature. J.
Gen. Intern. Med. 2011 Sep.;26 (9):1036-49. Epub. 2011 Apr. 7.
[7] Lloyd, P., Ryan, C., Menter, A. Psoriatic Arthritis: An Update. Arthritis. 2012; 2012:
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serum aminotransferase activity in the United States en 1999-2002. Am. J.
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Arcaro, G. Prevalence of nonalcoholic fatty liver disease and its association with
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[47] Gisondi, P., Targher, G., Zoppini, G., Girolomoni, G. Non-alcoholic fatty liver disease
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Politi, P., Tsianos, E., Clofent, J., Vermeire, S., Monteiro, E., Mouzas, I., Fornaciari,
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CD8+ clonal expansions indicate a role of antigens in ankylosing spondylitis; a study in
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of the art. t J. Dermatol. 2008 Sep.; 47(9):903-10.
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Cossidente, A. The importance of stressful family events in psoriatic patients: a
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[62] Rieder, E., Tausk, F. Psoriasis, a model of dermatologic psychosomatic disease:
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affects patient's quality of life more seriously in female than in male in Japan. Tokai J.
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psoriasis feel stigmatized. Acta Derm. Venereol. 2012 Jan.; 92(1):67-72.
[68] Vardy, D., Besser, A., Amir, M., Gesthalter, B., Biton, A., Buskila, D. Experiences of
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in psoriasis patients. Br. K Dermatol. 2002 Oct.;147(4):736-42.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 41

NUTRITION AND THE TREATMENT OF PSORIASIS

Emily de Golian*,1, Maryam Afshar2 and Nancy Anderson3

1
School of Medicine, Medical College of Georgia at Georgia Health Sciences University,
Augusta, Georgia, US
2
Department of Medicine, Loma Linda University, Loma Linda, California, US
3
Department of Dermatology, Loma Linda University, Loma Linda, California, US

ABSTRACT
Though many options exist for the treatment of psoriasis, nutrition is a therapeutic
component that should not be overlooked. Dietary modifications, including gluten free
diet and low calorie diet, induce a statistically significant improvement of clinical
psoriasis. Weight loss through a low calorie diet improves psoriasis not only through
control of psoriasis-related metabolic syndrome but also through increasing the efficacy
of cyclosporine in obese psoriatic patients. In addition to various diets, multiple oral and
topical nutritional supplements have been studied in the treatment of psoriasis. Selenium
when combined with coenzyme Q10 and vitamin E has been shown to be beneficial.
Some studies also support the use of omega-3 fatty acids in psoriasis. Most significantly,
oral supplementation with vitamins A and D have long been recognized as affecting this
disease process, although vitamin A in particular is associated with significant side
effects like teratogenicity. These two vitamins are also useful when applied topically, and
vitamin D is regularly used therapeutically in psoriasis with good evidence for its
efficacy. Finally, removing substances from the diet that may exacerbate psoriasis should
be considered for all patients. Counseling on alcohol use, which is associated with
increased morbidity and mortality in psoriasis, is an important component of any
therapeutic regimen. Here, the nutritional aspects of the treatment of psoriasis are
discussed in detail.

* Contact information for corresponding author: Emily de Golian, 827B Edgewood Avenue, Atlanta, GA 30307,
Phone: (404) 391-9687, Email: [email protected]
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1000 Emily de Golian, Maryam Afshar and Nancy Anderson

DISCUSSION
While many nutritional therapies have been proposed and investigated for psoriasis,
literature review yields several in particular with the best evidence for efficacy. Two of the
best studied of these are vitamins A and D, and both are known to be effective in the
treatment of psoriasis. Retinoids act via retinoid receptors to inhibit hyperproliferative
keratinocyte growth, inducing their terminal differentiation and ameliorating the disease
process in psoriasis [1]. The primary barrier to this effective treatment is the side effect
profile, which is significant. Vitamin D has been well established in the treatment of psoriasis
as well, particularly as a topical therapy, and has fewer adverse effects [1].
In addition to vitamins, changes in dietary habits, including modified diet, elimination of
alcohol, and use of specific supplements, may enhance psoriasis therapy [1, 2, 3]. Though the
mechanism for dietary therapy via calorie restriction is not completely understood,
arachidonic acid is thought to play a role. Reducing the intake of arachidonic acid results in
decreased production of inflammatory eicosanoids and an increase in anti-inflammatory
cytokines like interleukin(IL)-4 [2, 3]. Decreased calorie intake also reduces oxidative stress,
thus further ameliorating psoriatic disease [2, 3]. A reduction in alcohol intake as well may
further benefit psoriasis patients. Increased alcohol consumption has been associated with the
presence of psoriasis and increased mortality, and alcohol intake may also detrimentally
enhance the release of inflammatory histamines [1, 3]. Finally, a potentially useful dietary
addition is the combination of supplemental selenium, coenzyme Q-10, and vitamin E, which
has been shown to improve severe forms of psoriasis [4].
Related to this concept of diet, intake, and psoriasis, although causality has not been
determined, is an association between psoriasis and metabolic syndrome. This syndrome is
closely linked to obesity and includes such additional criteria as diabetes mellitus,
hypertension, and hyperlipidemia. Systemic inflammation is increased, therefore, it has been
hypothesized that management of metabolic syndrome may also play a role in treating
psoriasis [5]. The following discussion will review the above concepts as they relate to
improving psoriasis in greater detail.

Vitamin A

Vitamin A therapy is best utilized when complemented by additional treatments, and both
oral and topical preparations have proven effective. Acitretin, a second generation retinoid, is
an oral formulation most commonly used with topical corticosteroids or calcipotriene [6].
However, of the 62% of visits where acitretin was found to be co-prescribed with another
drug, 6% of those drugs were biologics, which include TNF-alpha blockers and monoclonal
antibodies against IL-12 and IL-23 [6]. The advantage to this particular combination is that
acitretin spares the immune system, thus preventing further immunosuppression in patients
already using biologic drugs [6]. Because acitretin may be metabolized to etretinate, which
has a long half-life, the risk of teratogenicity is a contraindication to its use in women who
may become pregnant [7]. When using this therapy, women of childbearing age are
recommended to use oral contraceptives during treatment and for at least two years following
cessation of the drug [7].

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 Nutrition and the Treatment of Psoriasis 1001

Oral retinoids are also known to be effective together with narrow band ultraviolet B
(nbUVB) therapy, which reduces recovery time and allows lower doses of both acitretin and
nbUVB in achieving good results [8]. This combination is particularly useful for guttate and
plaque-type psoriasis, and while the risk of teratogenicity remains, as an anticarcinogenic
agent, the retinoid component may also reduce the risk of skin cancer from regular UVB
exposure [8].
In a literature review examining oral retinoids as related to subtypes of psoriasis, use was
effective for generalized and local pustular psoriasis as well as plaque-type psoriasis [9]. As a
single agent, retinoids may be effective in pustular psoriasis, although as the disease may
spontaneously remit, better studies are needed to confirm their efficacy [9]. When combined
with psoralen plus UVA (PUVA) therapy, retinoids are known to enhance the benefits of
PUVA alone in palmoplantar pustular psoriasis [9]. Similar findings applied to studies
examining plaque-type psoriasis, which was most effectively treated by combination therapy,
as retinoids enhanced PUVA therapy alone and UVB therapy alone [9]. Additionally, dosing
studies in plaque psoriasis illustrated a dose dependent decrease in the psoriasis area and
severity index (PASI) and a dose-dependent increase in significant adverse effects, which
may contribute to treatment withdrawal [9]. Recommended treatment regimens thus suggest
that patients using acitretin start no higher than 25 mg/day, progressing upward with small
dosing changes to reduce the incidence and severity of adverse effects [9]. Of note, retinoids
have not been found to increase the risk of skeletal abnormalities in psoriasis patients [9].
With combination therapy utilizing PUVA, however, an increased risk of skin cancer is a
significant consideration [10]. A meta-analysis of 45 studies considering skin cancer in the
setting of PUVA therapy noted that all studies found an increased risk of non-melanoma skin
cancers (NMSC), most commonly squamous cell carcinoma, therefore patients must be
monitored for abnormal skin changes [10].
In addition to oral therapy, topical retinoids may be useful in the treatment of psoriasis;
however, they may irritate the skin and are not recommended for use in pregnant women [11,
12]. Although there are no systemic side effects, retinoid erythema affects adherence to
therapy and remains a barrier to long term use [11, 12]. A review of the topical retinoid
tazarotene, however, found that in both gel and cream formulations used as monotherapy and
adjuvant therapy, daily application of tazarotene was effective with sustained benefits and
limited local side effects [13]. Its combination with steroids, calcipotriene and phototherapy
are known to be useful in psoriasis [13]. Tazarotene has been shown to upregulate the tumor
suppressor tazarotene induced gene 3, which is overexpressed in psoriasis and skin cancer
[13]. Although some studies note irritation as a preventative factor in adherence to therapy, a
2009 review suggested that adverse effects are limited, non-severe, and that daily application
may yield positive results [13].

Vitamin D

As with vitamin A, both oral and topical formulations of vitamin D are known to be
beneficial in treating psoriasis. Low serum levels correlate with more severe psoriasis, and
oral vitamin D improves psoriasis as well as psoriatic arthritis, in addition to other health
benefits beyond these disease processes [14]. Hypercalcemia remains a potential side effect,
but it is avoidable with appropriate dosing and monitoring, and the benefits of oral vitamin D
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1002 Emily de Golian, Maryam Afshar and Nancy Anderson

are significant [14]. While this treatment is effective, it is not often utilized. Given its utility,
the option of oral vitamin D should not be forgotten.
Most commonly, vitamin D is used topically, and particularly in combination with topical
corticosteroids, topical vitamin D analogues are a mainstay of therapy for psoriasis.
Calcipotriol, a synthetic derivative of 1,25-dihydroxyvitamin D3, is second only to
corticosteroids as the most commonly used drug in psoriasis [11, 12]. A 2012 literature
review of 51 articles found that vitamin D analogues plus steroids are twice as effective as
vitamin D analogues alone, as well as more cost effective [15]. Furthermore, a 2010
symposium in Sweden noted that this combination had the greatest proven efficacy in
randomized clinical trials, reflecting current treatment guidelines in the United States and
Germany for mild to moderate psoriasis [16].
Regarding the mode of efficacy for topical vitamin D treatments, one study examined
human skin biopsies that were untreated or treated with vitamin D analogues [17]. Induction
of thymic stromal lymphopoietin and cathelicidin occurred in psoriatic lesions of those treated
with topical vitamin D3 analogues, thus resulting in suppression of IL-12/23 p40, IL-1α, IL-
1β, and TNF-α [17]. This suppression lead to improvement of psoriatic plaques [17].
Additionally, a 2011 Japanese study found that switching among topical vitamin D3
analogues when treatment was not satisfactory with a particular agent yielded improvement
for plaque-type psoriasis [18]. Although the pharmacological efficacies of the three reagents
do not differ significantly, the authors postulated that rotation therapy may provide a means
for encouraging patient adherence via reexplanation of therapy and anticipation of a new drug
[18]. Overall, evidence for the efficacy of topical vitamin D is abundant, and this therapy
continues to be a mainstay in psoriasis treatment.

Low Calorie Diet

Based on a study of in-patients with low energy versus normal diet, a low energy diet
could be an important adjuvant factor in the treatment and prevention of moderate non-
pustular psoriasis [19]. While all patients were maintained on their normal topical therapies,
half were additionally treated with a low calorie diet and compared to control patients on a
normal diet. Although body weight did not significantly change in either group, a statistically
significant decrease in serum lipids and substantial decrease in clinical skin findings were
noted in low energy diet patients with psoriasis vulgaris [19]. Despite findings such as this,
however, a simple trend towards improvement with low calorie diet alone rather than a
statistically significant difference between low calorie versus free diet was noted in another
study of obese psoriasis patients [20]. This small clinical study of 42 patients, with the goal of
decreasing body mass index (BMI) in the experimental group, indicated that for obese
patients, weight loss alone may not be sufficient for maintaining remission of moderate-to-
severe psoriasis [20].
Low-calorie diet may be used as an adjunctive therapeutic element. In a randomized,
controlled, investigator-blinded clinical trial of 61 patients, obese patients with moderate to
severe psoriasis had a greater response to low-dose cyclosporine when a low calorie diet was
added to their treatment plan [21]. Although findings differ among some studies, low calorie
diet has health benefits beyond the scope of psoriasis treatment, and lifestyle modifications

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 Nutrition and the Treatment of Psoriasis 1003

such as this may actually enhance the benefits of other treatments in patients with psoriasis,
particularly in the obese.

Gluten-Free Diet

Although the precise etiology is unknown, celiac disease (CD) is characterized by

damage to small intestinal absorptive villi secondary to a reaction to gluten, which is found in
wheat, barley, rye, and various other foods and products. In an examination of 130 psoriasis
patients for serum IgG and IgA antigliadin antibodies, IgA antitransglutaminase antibodies,
and IgA antiendomysial antibodies, as well as endoscopic biopsy and clinical exams, psoriasis
patients with celiac disease associated antibodies were positively correlated with a greater
burden of disease [22]. Furthermore, in a large scale study of 12,502 psoriatic patients and
24,285 age and sex matched controls, psoriasis was associated with CD (odds ratio 2.73, 95%
confidence interval 1.65-4.53) [23]. Based on this association, a reasonable hypothesis is that
a gluten free diet (GFD), which should improve CD, will lead to improvement in psoriasis.
Clinical and histologic support for a GFD in treatment of psoriasis has been noted in
clinical studies. Results from 31 psoriasis patients illustrated clinical improvement in patients
with antibodies to gliadin accompanied by significant histological changes for both involved
and noninvolved skin, particularly in the dermis [24]. Moreover, these changes were
significant in patients with no treatment other than GFD, some of whom had not responded to
previous regimens, thus indicating that the noted improvements were diet-induced [24].
Psoriasis then clinically worsened with resumption of normal diet in these patients, giving
further credence to the value of GFD [24]. In an older study by the same author, GFD
improved psoriasis in patients with elevated antigliadin antibodies even without anti-
endomysial antibodies or only slight or absent duodenal biopsy changes [25]. There was a
highly significant decrease in mean PASI after a 3 month trial of GFD, and approximately
half of patients deteriorated with resumption of normal diet, again indicating the positive
effect of GFD in psoriasis [25].
As a springboard for examining this relationship, a 2003 case report describing a
dramatic improvement in psoriasis with GFD also uses a literature review to demonstrate
some conflicting data regarding the efficacy of GFD in psoriasis [26]. Although the case in
question was an example of GFD positively impacting one patient’s clinical psoriasis, the
author does note that some reports question the validity of this association as coincidental
[26].

Metabolic Syndrome

Metabolic syndrome represents a collection of disorders that is strongly associated with

obesity and thus important to discuss in the context of diet and psoriasis. Many, if not all, of
the criteria comprising this syndrome may be affected by weight. The International Diabetes
Federation consensus in 2006 defined metabolic syndrome as central obesity plus any two of
the following: triglycerides >150 or treatment for elevated triglycerides, HDL <40 in men and
<50 in women or treatment for this lipid abnormality, systolic blood pressure >130 or
diastolic >85 or treatment for hypertension, and fasting plasma glucose >100 or diagnosed
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1004 Emily de Golian, Maryam Afshar and Nancy Anderson

type II diabetes [27]. In a retrospective study including over 16,000 psoriasis patients and
over 48,000 controls, multivariate models adjusting for age, gender and smoking status of the
patients demonstrated that psoriasis was associated with metabolic syndrome, ischemic heart
disease, diabetes mellitus, hypertension, and obesity [5]. Each of these components may be
improved by diet and or pharmacologic therapy, and this constellation of findings should not
be overlooked as part of a comprehensive treatment plan for psoriasis patients.

Supplements

A 2009 study of 58 hospitalized patients was the first to find that a combination of
conventional therapy plus supplementation with vitamin E, coenzyme Q10, and selenium
yielded clinical improvement in patients with severe psoriasis, as well as a reduction in
oxidative stress in circulating granulocytes, blood plasma, and the epidermis of psoriasis
lesions [4]. This reduction is significant, as granulocytes release pro-inflammatory reactive
oxygen and nitrogen species, proteases, cytokines, and chemokines, while skin keratinocytes
then react to these markers by releasing inflammatory IL-8, monocyte chemoattractant protein
1 (MCP-1), and chemotactic cytokine ligand 5 (CCL5) [4]. By reducing such an
inflammatory burden with selenium, coenzyme Q10, and vitamin E, uncontrolled
keratinocyte proliferation is reduced and clinical improvement attained [4].
A 2003 study confirmed low selenium status in patients with psoriasis, particularly in
men with disease course greater than three years [28], which suggests the possibility of
selenium supplementation as a therapeutic option. However, adding selenium alone has not
been shown to be effective as an adjuvant therapy [29, 30].
In addition to selenium, research examining ω-3 fatty acids has been pursued, but
evidence in favor of its benefit is equivocal. Regardless, there is some support for ω-3 fatty
acids in the improvement of psoriasis, and it may be considered as an additional therapy. In a
small study of 30 patients, half treated with topical tacalcitol and half with topical tacalcitol
plus two Oravex capsules, a supplement containing omega-3 fatty acids, significant
improvement was noted in all efficacy endpoints for both groups [31]. Favoring the addition
of Oravex, however, improvement was significantly greater in those treated with Oravex
versus the control group [31]. Another series of studies found that intravenous n-3-fatty acid
administration improves psoriasis without severe adverse side effects and with a beneficial
reduction in triglyceride levels, which may be related to changes in inflammatory eicosanoid
generation [32]. Although a meta-analysis of small studies evaluating this type of therapy is
not possible due to differences in dosing, administration, and treatment duration, because
omega-3 fatty acids have been shown to improve psoriasis and are beneficial in the
prevention and treatment of coronary artery disease, hypertension, arthritis, cancer, and other
inflammatory and autoimmune disorders, their use is reasonable [33].

Alcohol

While the previous sections have looked at therapeutic options known to positively
impact psoriasis lesions, the discussion of nutrition and psoriasis would not be complete
without noting dietary factors that negatively impact psoriasis. The most significant of these

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 Nutrition and the Treatment of Psoriasis 1005

is alcohol intake. A meta-analysis of 15 case-control studies showed a statistically significant

association between psoriasis and alcohol consumption across a number of stratified analyses,
including sensitivity analyses assessing the potential effect of varying psoriasis outcome
definitions [34]. Alcohol consumption is indeed associated with an increased risk of psoriasis
[34].
Ethanol and its metabolites are triggers for psoriasis, and alcohol use may also worsen
preexisting psoriasis [35, 36]. Consumption may have a dose-dependent effect on the
incidence and severity of psoriatic disease [35, 36]. The mechanisms of alcohol in this role
are multiple. First, it may affect the immune response, thus predisposing consumers to
infection, which are known triggers of psoriasis flares [35, 36]. Alcohol also stimulates pro-
inflammatory cytokines, including TGF- α, IFN-γ, and IL-6, alters skin barrier function, and
modulates disease via ethanol metabolites [35, 36]. The oxidation of ethanol produces
acetaldehyde and reactive oxygen species, which modulate signal transduction pathways to
upregulate inflammatory cytokines [35, 36]. The ethanol metabolite acetone directly enhances
keratinocyte proliferation and increases the mRNA levels of genes associated with
proliferation, such as alpha-5-integrin, cyclin D1, and keratinocyte growth factor receptor [35,
36].

Pharmacologic Drugs

A final thought to consider regarding oral intake and psoriasis is the fact that many
common and uncommon drugs may affect the disease process. Lithium, gold salts, beta
blockers, and antimalarials are some of the most common drugs known to relate to psoriasis,
and while over 120 drugs have been described as exacerbating preexisting disease, a shorter
list of 21 drugs may even initiate the disease [37]. Some of these are very commonly used and
may be obtained over the counter, such as aspirin and ibuprofen, so patient medication lists
must be considered as possibilities for the etiology of psoriasis in each patient [37].

CONCLUSION
As discussed above, numerous nutritional and lifestyle related factors are worth
consideration in the treatment of psoriasis. While some are very commonly used, like topical
vitamin D, certain dietary changes and supplements are not necessarily considered in the
development of treatment plans. By understanding the effects and significance of vitamin
therapy options, low calorie and gluten free diets, and specific supplement combinations, as
well as factors like alcohol and pharmacologic drugs that may worsen psoriasis, a more
comprehensive and ultimately perhaps more successful approach may be taken to treating
patients with psoriasis.
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1006 Emily de Golian, Maryam Afshar and Nancy Anderson

REFERENCES
[1] Ricketts JR, Rothe MJ, Grant-Kels JM. Nutrition and Psoriasis. Clinics in Dermatology.
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[2] Araujo ML, Burgos MG, Moura IS. Nutritional influences in psoriasis. An Bras
Dermatol. 2009 Jan-Feb;84(1):90-2.
[3] Wolters M. Diet and psoriasis: experimental clinical and evidence. Br J Dermatol.
2005;153:706-14
[4] Kharaeva Z, Gostova E, De Luca C, Raskovic D, Korkina L. Clinical and biochemical
effects of coenzyme Q10, Vitamin E, and Selenium supplementation to psoriasis
patients. Nutrition 2009;25:295-302.
[5] Cohen AD, Sherf M, Vidavsky L, Vardy DA, Shapiro J. Meyerovitch. Association
between psoriasis and the metabolic syndrome. Dermatol 2008;216:152-5.
[6] Ghasri P, Yentzer BA, Dabade TS, Feldman SR. Acitretin for the treatment of psoriasis:
an assessment of national trends. J Drugs Dermatol. 2011 Aug;10(8):873-7.
[7] Berbis P. Acitretin. Ann Dermatol Venereol. 200;128(6-7):737-45.
[8] Monfrecola G, Baldo A. Retinoids and phototherapy for psoriasis. J Rheumatol Suppl.
2009 Aug;83:71-2.
[9] Sbidian E et al. Efficacy and safety of oral retinoids in different psoriasis subtypes: a
systematic literature review. J Eur Acad Dermatol Venereol. 2011 May;25 Suppl 2:28-
33.
[10] Archier E et al. Carcinogenic risks of Psoralen UV-A therapy and Narrowband UV-B
therapy in chronic plaque psoriasis: a systematic literature review. J Eur Acad Dermatol
Venereol. 2012 May;26 Suppl 3:22-31.
[11] Bos JD, Spuls PI. Topical treatments in psoriasis: today and tomorrow. Clin Dermatol.
2008 Sep-Oct;26(5):432-7.
[12] Albrecht L, Bourcier M, Ashkenas J, Papp K, Shear N, Toole J, Vender R, Wasel N;
Canadian Psoriasis Guidelines Committee. Topical psoriasis therapy in the age of
biologics: evidence-based treatment recommendations. J Cutan Med Surg. 2011 Nov-
Dec;15(6):309-21.
[13] Talpur R, Cox K, Duvic M. Efficacy and safety of topical tazarotene: a review. Expert
Opin Drug Metab Toxicol. 2009 Feb;5(2):195-210.
[14] Kamangar F, Koo J, Heller M, Lee E, Bhutani T. Oral vitamin D, still a viable
treatment option for psoriasis. J Dermatolog Treat. 2012 Jan 21.
[15] Devaux S, Castela A, Archier E, Gallini A, Joly P, Misery L, Aractingi S, Aubin F,
Bachelez H, Cribier B, Jullien D, Le Maître M, Richard MA, Ortonne JP, Paul C.
Topical vitamin D analogues alone or in association with topical steroids for psoriasis: a
systematic review. J Eur Acad Dermatol Venereol. 2012 May;26 Suppl 3:52-60.
[16] Murphy G, Reich K. In touch with psoriasis: topical treatments and current guidelines.
J Eur Acad Dermatol Venereol. 2011 Jun;25 Suppl 4:3-8.
[17] Sato-Deguchi E, Imafuku S, Chou B, Ishii K, Hiromatsu K, Nakayama J. Topical
vitamin D₃ analogues induce thymic stromal lymphopoietin and cathelicidin in psoriatic
skin lesions. Br J Dermatol. 2012 Jul;167(1):77-84.
[18] Imafuku S, Kubota Y, Ito K, Koga M, Takahashi A, Nakayama J. Effects of rotation of
topical vitamin D3 in chronic plaque-type psoriasis. J Dermatol. 2012 Mar;39(3):275-7.

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 Nutrition and the Treatment of Psoriasis 1007

[19] Rucevic I, Perl A, Barisic-Drusko V, Adam-Perl M. The role of the low energy diet in
psoriasis vulgaris treatment. Coll Antropol 2003;27 (suppl 1):41-8.
[20] Del Giglio M, Gisondi P, Tessari G, Girolomoni G. Weight reduction alone may not be
sufficient to maintain disease remission in obese patients with psoriasis: a randomized,
investigator-blinded study. Dermatology. 2012;224(1):31-7. Epub 2012 Mar 27.
[21] Gisondi P, Del Giglio M, Di Francesco V, Zamboni M, Girolomoni G. Weight loss
improves the response of obese patients with moderate-tosevere chronic plaque
psoriasis to low-dose cyclosporine therapy: a randomized, controlled, investigator-
blinded clinical trial. Am J Clin Nutr 2008;88:1242-7.
[22] Woo WK, McMillan SA, Watson RG, McCluggage WG, Sloan JN, McMillan JC.
Coeliac disease - associated antibodies correlate with psoriasis activity. Br J Dermatol.
2004;151:891-4.
[23] Birkenfeld S, Dreiher J, Weitzman D, Cohen AD. Coeliac disease associated with
psoriasis. Br J Dermatol 2009:1-4.
[24] Michaëlsson G, Ahs S, Hammarström I, Lundin IP, Hagforsen E. Gluten-free diet in
psoriasis patients with antibodies to gliadin results in decreased expression of tissue
transglutaminase and fewer Ki67+ cells in the dermis. Acta Derm Venereol.
2003;83(6):425-9.
[25] Michaëlsson G, Gerdén B, Hagforsen E, Nilsson B, Pihl-Lundin I, Kraaz W,
Hjelmquist G, Lööf L. Psoriasis patients with antibodies to gliadin can be improved by
a gluten-free diet. Br J Dermatol. 2000 Jan;142(1):44-51.
[26] Addolorato G, Parente A, de Lorenzi G, D'angelo Di Paola ME, Abenavoli L, Leggio L,
Capristo E, De Simone C, Rotoli M, Rapaccini GL, Gasbarrini G. Rapid regression of
psoriasis in a coeliac patient after gluten-free diet. A case report and review of the
literature. Digestion. 2003;68(1):9-12.
[27] Alberti G, Zimmet P, Shaw J, Grundy SM. The IDF consensus worldwide definition of
the metabolic syndrome. Brussels: International Diabetes Foundation, 2006.
<http://www.idf.org/webdata/docs/ IDF_Meta_def_final.pdf>
[28] Serwin AB, Wasowicz W, Gromadzinska J, Chodynicka B. Selenium status in psoriasis
and its relation to the duration and severity of the disease. Nutrition. 2003;19:301-4
[29] Serwin AB, Mysliwiec H, Hukalowicz K, Porebski P, Borawska M, Chodynicka B.
Soluble tumor necrosis factor-alpha receptor type 1 during selenium supplementation in
psoriasis patients. Nutrition. 2003 Oct;19(10):847-50.
[30] Serwin AB, Wasowicz W, Chodynicka B. Selenium supplementation, soluble tumor
necrosis factor-alpha receptor type 1, and C-reactive protein during psoriasis therapy
with narrowband ultraviolet B. Nutrition. 2006 Sep;22(9):860-4.
[31] Balbás GM, Regaña MS, Millet PU. Study on the use of omega-3 fatty acids as a
therapeutic supplement in treatment of psoriasis. Clin Cosmet Investig Dermatol.
2011;4:73-7.
[32] Mayser P, Grimm H, Grimminger F. n-3 fatty acids in psoriasis. Br J Nutr. 2002 Jan;87
Suppl 1:S77-82.
[33] Rackal J, Barankin B. The role of fish oils in psoriasis. Skinmed. 2004 Sep-
Oct;3(5):290-1.
[34] Zhu KJ, Zhu CY, Fan YM. Alcohol consumption and psoriatic risk: A meta-analysis of
case-control studies. J Dermatol. 2012 Sep;39(9):770-3.
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[35] Cassano N, Vestita M, Apruzzi D, Vena GA. Alcohol, psoriasis, liver disease, and anti-
psoriasis drugs. Int J Dermatol. 2011 Nov;50(11):1323-31.
[36] Farkas A, Kemény L. The alcohol metabolite acetaldehyde and psoriasis: another
trigger factor? Clin Exp Dermatol. 2010 Dec;35(8):923-5.
[37] Milavec-Puretić V, Mance M, Ceović R, Lipozenčić J. Drug induced psoriasis. Acta
Dermatovenerol Croat. 2011;19(1):39-42.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 42

PSORIASIS AND CARDIOVASCULAR

DISEASE - UPDATE

Manisha R. Panchal1, Helen Coope2,

Anton B Alexandroff3,* and John McKenna3
1
Department of Dermatology, Sherwood Forest Hospitals,
Kingsmill Hospital, UK
2
Novartis Pharmaceuticals UK Ltd
3
Department of Dermatology, University Hospitals of Leicester,
Leicester Royal Infirmary, Leicester, UK

ABSTRACT
Psoriasis is a common chronic inflammatory disease of skin which affects
approximately three percent of the population in Europe and the United States. Although
traditionally seen as a predominantly skin disorder, it is known to be associated with
seronegative arthritis and inflammatory bowel disease. More recently epidemiological
studies strongly linked psoriasis to cardiovascular disease including ischaemic heart
disease, cerebrovascular and peripheral vascular disease, with the risks of premature heart
disease approaching those in diabetes mellitus. Here we summarise recent data on
cardiovascular morbidity and mortality of patients with psoriasis including attenuation of
atherosclerosis by systemic anti-inflammatory treatments. We also outline novel
biological anti-psoriatic agents which are currently being developed.

LINK BETWEEN PSORIASIS AND ATHEROSCLEROSIS

Psoriasis is a common inflammatory skin disease which affects between 2 and 5 percent
of population in the Europe and the United States of America. It is easily recognised due to
the typical appearance of well-defined salmon pink patches and plaques with silvery scale. It
often affects scalp and nails but may affect any part of the body, including face, skin flexures
*
Corresponding author’s email: [email protected].
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1010 Manisha R. Panchal, Helen Coope, Anton B Alexandroff et al.

and genital area. Curiously psoriasis frequently exhibits a predilection for extensor aspects of
elbows and knees, umbilicus and natal cleft.
The majority of patients are affected by mild and limited psoriasis. However in a small
proportion of patients psoriasis can be extensive, severely affecting the quality of life to the
extent similar to that in patients with diabetes, heart attacks and cancer [1].
Traditionally psoriasis was believed to affect only the skin, scalp, nails and joints,
although associations with some other autoimmune diseases such as ulcerative colitis was
also noted. However 40 years ago McDonald and Calabresi noted that psoriasis may also be
linked to the manifestations of atherosclerosis including ischaemic heart disease,
thromboembolism and cerebrovascular disease [2-4]. Nevertheless it took another 33 years
before Gelfand and co-workers in their seminal population-based study unequivocally showed
a link between psoriasis in general, specifically a severe psoriasis, and myocardial infarction
[5]. A number of confirmatory publications followed linking severe psoriasis with
cardiovascular morbidity and mortality (including cerebrovascular disease, pulmonary
embolism, and peripheral vascular disease) [6, 7]. It is now generally believed that psoriasis,
alongside with other inflammatory diseases including systemic lupus, rheumatoid arthritis and
ankylosing spondylitis, is in fact a systemic inflammatory disease and as such may manifest
itself by atherosclerosis, which is in itself also an inflammatory disease [8-11].
The exact nature of the link between psoriasis and atherosclerosis remains inadequately
understood. It has been suggested however that the common underlying pathogenic
mechanisms of psoriasis and atherosclerosis are common or even almost identical. In
particular, the similarities between these two entities extend to a preferential activation of Th1
and Th17 pathways with a corresponding down-regulation/dysregulation of Th2 and Treg
pathways [10]. In addition, there is remarkable similarity in the activation of a plethora of
cytokines, chemokines, adipokines, adhesion and co-stimulatory molecules, leucocyte subsets
and other proinflammatory molecules [10]. In fact, there is an astonishing resemblance of
common inflammatory pathways between psoriasis, systemic lupus, rheumatoid arthritis and
atherosclerosis (Figure 1) [8]. Interestingly patient with psoriasis appear to have increased
biomarkers of atherosclerosis e.g., carotid artery intima-media thickness and impaired
endothelial function but at the same time myocardial perfusion and left ventricular function
appear to be preserved [12-14].

PSORIASIS IS STRONGLY ASSOCIATED WITH CLINICAL

MANIFESTATIONS OF ATHEROSCLEROSIS
In their seminal publication Gelfand and co-workers demonstrated that an adjusted risk of
myocardial infarction in a 30 year old man with severe psoriasis was 3.1 fold higher than in a
control group (after statistical adjustments for hypertension, diabetes, history of myocardial
infarction, hyperlipidemia, age, sex, smoking, and body mass index) [5]. This is similar to the
risks observed in patients with diabetes. In addition, patients with severe psoriasis also had an
increased adjusted risk of cardiovascular and overall mortality, stroke, and peripheral vascular
disease [adjusted risk are 2.69, 1.5, 1.43, 1.98 respectively] [6, 7, 15, 16].

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 Psoriasis and Cardiovascular Disease - Update 1011

Figure 1. Key pathogenic mechanisms are overlapping in atherosclerosis, psoriasis, rheumatoid arthritis and systemic
lupus. C1q and C3 - components of complement pathway, CCP - cyclic citrullinated peptide, Neu - neutrophils, Rh F -
rheumatoid factor, S100A7 - Psoriasin. [reprinted from 8 with permissions of Novapublisher].

Another confirmation of a link between psoriasis and atherosclerosis comes from the
observation that anti-inflammatory treatment may ameliorate cardiovascular morbidity and
mortality. It has been shown that methotrexate reduces cardiovascular morbidity in patients
with psoriasis, and also cardiovascular morbidity and mortality in patients with rheumatoid
arthritis [6, 17-19]. It is noteworthy that patients with rheumatoid arthritis who responded to
TNF antagonists also had a reduced rate of myocardial infarctions [20].
More recently there was a controversial observation that psoriasis patients treated with
IL-12/23 antagonists might have developed major adverse cardiovascular events (MACE).
However, meta-analyses of randomised controlled studies with biologics did not appear to
show in increased rate of MACE (we discuss this in a recent review of long term safety of
biologics in psoriasis [21]).

BIOLOGIC THERAPIES IN LATE STAGE CLINICAL

DEVELOPMENT FOR PSORIASIS
Three Interleukin 17 (IL17) antagonist therapies are in late stage development for
psoriasis; brodalumab (Amgen), ixekizumab (Eli Lilly) and secukinumab (Novartis) [22-26].
These are all injectable monoclonal antibodies (biologic therapies), their key properties are
shown in the Table 1.
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1012 Manisha R. Panchal, Helen Coope, Anton B Alexandroff et al.

Table 1.

Cytokine
Therapy Molecular Phase 3 clinical
signals
type target study status
impacted
Brodalumab Fully human IgG2 IL17 receptor IL17A Phase 3 Recruitment
[23] monoclonal A IL17F AMAGINE1 open
antibody IL17AF AMAGINE2
IL17E AMAGINE3
Ixekizumab Humanized IgG4 IL17A IL17A Phase 3 Recruitment
[24] monoclonal IL17AF UNCOVER1 open
antibody UNCOVER2
UNCOVER3

UNCOVERA Planned
Secukinuma Fully human IL17A IL17A Phase 3 Recruitment
b IL17AF ERASURE closed
[25;26] antibody FIXTURE extension
SCULPTURE ongoing
STATURE

FEATURE ongoing
JUNCTURE

Genetics studies and analysis of gene expression in disease tissues have implicated the
IL17 cytokine system in the pathogenesis of multiple autoimmune and auto-inflammatory
conditions (reviewed in [27]), including psoriasis where levels of IL17A positive cells are
raised in psoriatic lesions [28]. This is supported by direct and indirect pharmacological
evidence for the role of IL17 in psoriasis: in a mouse model, development of imiquimod-
induced skin lesions (which resemble plaque type psoriasis) is almost completely blocked in
mice genetically lacking IL17 receptors [29]. This is supported by the clinical efficacy of
ustekinumab in psoriasis, ustekinumab blocks the p40 subunit of IL23, which is required for
the generation of Th17 cells which produce IL17. However, since ustekinumab also inhibits
IL12 it is not clear what the relative importance of IL12 and IL23 inhibition is for therapeutic
efficacy in psoriasis.
More recently, data on the efficacy of IL17 antagonists in human psoriasis has been
published [23-26]. The Interleukin 17 family of cytokines encompasses five receptors
(IL17RA to IL17RE) and six cytokines (IL17A to IL17F). Efficacy data from brodalumab,
ixekizumab and secukinumab phase 2 clinical trials each supports the importance of IL17
cytokines in psoriasis, with each IL17 antagonist reporting highest PASI75 response rates in
excess of 80% in these short term studies. The risk-benefit profile in these phase 2 studies
was sufficient for progression to phase 3 clinical trials which will be key to determining the
long term safety and efficacy of these agents. The first regulatory filing for an IL17 antagonist
in psoriasis is likely to be secukinumab, expected late in 2013.

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 Psoriasis and Cardiovascular Disease - Update 1013

BIOLOGIC THERAPIES IN EARLY CLINICAL

DEVELOPMENT FOR PSORIASIS
Further biologic agents are in phase 1 and 2 clinical development for psoriasis [27, 30].
Where the molecular target is specified this is shown in Table 2 below. These include
molecules which specifically target IL23 (via the p19 subunit), IL12, Interferon , B7-related
protein and three therapies targeting the IL17 cytokine system.

CONCLUSION
A significant progress has been made since 1973 when McDonald and Calabresi first
suggested a tentative link between psoriasis and atherosclerosis. Today the link between the
systemic inflammation of psoriasis and atherosclerosis appears to be well established and
accepted both on the molecular and clinical levels. Furthermore it has been postulated and in
some instances confirmed that reducing systemic inflammation may at least in some instances
ameliorate clinical manifestations of atherosclerosis. This opens an attractive possibility of
using anti-inflammatory agents to reduce cardiovascular morbidity and mortality.

Table 2.

Therapy Molecular Stage of

type target development:
CNTO Human monoclonal p19 subunit of IL23 Phase 2
1959 antibody
MK-322 Humanized monoclonal P19 subunit of IL23 Phase 2
antibody
AMG139 Human monoclonal IL23 Phase 1
antibody
RG4934 Humanized monoclonal IL17A Phase 1
antibody
NI-1401 Human monoclonal IL17A and IL17F Phase 1
antibody
SCH Humanized monoclonal IL17A Phase 1
900117 antibody
AMG811 Human monoclonal Phase 1
antibody
AMG557 Human monoclonal B7-related protein 1 Phase 1
antibody (B7RP-1)

Conflict of Interests

A.B.A. is the Lead of the LNR Comprehensive Local Research Networks Dermatology
Specialty Group, a member of the Biologics for Psoriasis Industry Subgroup, and the Steering
Group of the U.K. DCTN; he has received fellowships, plus educational grants, consultancy
 Free ebooks ==> www.Ebook777.com
1014 Manisha R. Panchal, Helen Coope, Anton B Alexandroff et al.

fees, acted as an investigator or a member of advisory board or a director for Abbott

Laboratories, Novartis, Pfizer, Procter & Gamble, Merck Serono, LEO Pharma, Basilea
Pharmaceutica, Apodi, Genus Pharmaceuticals, GlaxoSmithKline, Medefield, EMS Research,
CSD Health Research, XBioCell, Bryter Research, Genactis, Medicus, The Research House,
All Global, Keyquest, Almirall and Galderma.
J.M. is the Deputy Lead of the LNR Comprehensive Local Research Networks
Dermatology Specialty Group; he has received educational grants, consultancy fees, or acted
as an investigator for Novaritis, Galderma, Abbott, Leo Pharma, Genus Pharmaceuticals, and
Almirall.
H.C. is an employee of Novartis.

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 Psoriasis and Cardiovascular Disease - Update 1015

[15] Kaye, JA; Li, L; Jick, SS. Incidence of risk factors for myocardial infarction and other
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[18] Choi, HK; Hernan, MA; Seeger, JD; et al. Methotrexate and mortality in patients with
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[26] Papp, KA; Langley, RG; Sigurgeirsson, B; et al. Efficacy and safety of secukinumab in
the treatment of moderate-to-severe plaque psoriasis: a randomized, double-blind,
placebo-controlled phase II dose-ranging study. The British journal of dermatology,
2013, 168, 412-21.
[27] Patel, DD; Lee, DM; Kolbinger, F; et al. Effect of IL-17A blockade with secukinumab
in autoimmune diseases. Ann Rheum Dis, 2012.
[28] Johansen, C; Usher, PA; Kjellerup, RB; et al. Characterization of the interleukin-17
isoforms and receptors in lesional psoriatic skin. Br J Dermatol, 2009, 160, 319-24.
[29] van der Fits, L; Mourits, S; Voerman, JS; et al. Imiquimod-induced psoriasis-like skin
inflammation in mice is mediated via the IL-23/IL-17 axis. J Immunol, 2009, 182,
5836-45.
[30] conducted February 2012. Citeline trialtrove search. 2013. Ref Type: Online Source
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 43

BULLOUS PEMPHIGOID: AN OVERVIEW

Alexandre Carlos Gripp,1, Aline Bressan2,

Cândida Naira Lima e Lima-Santana3
and Daniele do Nascimento Pereira4
1
MD, Master's Degree in Dermatology. Assistant Professor of Dermatology and Chief of
the Dermatology Ward at Pedro Ernesto University Hospital - State University of Rio de
Janeiro, Brazil. Chief of the Dermatology Service at Pedro Ernesto University Hospital
2
MD, Specialist in Dermatology by the Brazilian Society of Dermatology
Medical assistant of the Imunobiologic Ambulatory and the Imunobiologic
Ward at Pedro Ernesto University Hospital – State University of Rio de Janeiro, Brazil
3,4
MD, Postgraduate student of Dermatology at Pedro Ernesto University Hospital –
State University of Rio de Janeiro, Brazil

ABSTRACT
Bullous Pemphigoid (BP) is an autoimmune blistering disease, preferentially
affecting elderly subjects and rare in childhood. BP is characterized by widespread tense
blister formation and rarely involves mucosa. In the skin biopsy, BP shows subepidermal
clefting as a result of injury caused by autoantibodies against structural components of
the hemidesmosome. The diagnosis is confirmed by immunofluorescence studies.
Corticosteroids (CS) are the mainstay of treatment. Mycophenolate mofetil, azathioprine,
methotrexate, cyclophosphamide are some of the steroid-sparing agents that can also be
used. Despite multiple treatment options, there are few studies supporting their use.

INTRODUCTION
Bullous pemphigoid (BP) is an autoimmune disease which affects mainly older
individuals. It is more common in women and rarely occurs in childhood.


E-mail: [email protected]
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1018 A. Carlos Gripp, A. Bressan, C. Naira Lima e Lima-Santana et al.

EPIDEMIOLOGY
Data from the preliminary studies of BP estimated an annual incidence of 6 to 7 new
cases per million people in Europe [1, 2]. In a 2-year study involving the Swiss population, it
was found an incidence of 12 cases per million people per year [3]. Another prospective study
made in Germany found a similar result, with an incidence of 13.4 [4]. Retrospective studies
in England and France showed a trend of increased incidence of BP in the last decade [5, 6].
It is a disease more common over 60 year with an increment of risk about 297 times higher
over 90 years [2].

CLINICAL ASPECTS
The most characteristic feature of BP is a tense bullae with widespread pruritus even in
healthy or erythematous skin [7-9.] The bullae can persist for days, leaving eroded or crusted
areas [8, 10] and has negative Asboe Hansen and Nikolsky signs [10]. The lesions are
generally symmetrical and can occur in any location. However, it occurs predominantly in the
abdomen, extremities and trunk (figure 1) [7, 8, 10].

Table 1. Clinical variants of bullous pemphigoid

Limited to pretibial area, reminding eczema, bullosis

Pretibial pemphigoid
diabeticorum, contact dermatitis or drug rash
Limited to palmoplantar region, reminding eczema, contact
Dysidrosiform pemphigoid
dermatitis, drug rash or dysidrosiform dermatitis
Excoriated nodules and papules in the limbs and trunk,
Pemphigoid nodularis
which can develop scar, resembling nodular prurigo
Rare, with intertriginous purulent, erythematous, erosive,
Pemphigoid vegetans
well circumscribed vegetating plaques
Erythrodermic bullous pemphigoid Erythroderma with or without blistering
Small tense blisters with a symmetric distribution that
Vesicular pemphigoid
mimicking dermatitis herpetiformis
Vesicles and blisters, resembling dermatitis herpetiformis
Polymorphic pemphigoid
in association with bullous pemphigoid
Large eroded areas on the trunk, buttocks and flexures,
Erosive bullous pemphigoid without pruritus, blisters or urticarial inflammatory lesions,
unwieldy
Typical features of both lichen planus and bullous
pemphigoid, it is speculated that the damage caused during
Lichen planus pemphigoid the liquefaction of the basal layer in lichen planus induces
the formation of antibodies against constituents of the basal
membrane and has relatively benign course
In general, the drug presents thiol group, acting as hapten
Drug induced bullous pemphigoid
in the lamina lucida
It presents greater involvement of mucosae and
Bullous pemphigoid of childhood
palmoplantar region
Adapted from Khandpur S, 2011, Di Zenzo G, 2012; Walsh SR, 2005.

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 Bullous Pemphigoid 1019

Figure 1. Bullous pemphigoid. Note the erythematous skin with tense bullae and eroded areas.

The blisters usually have clear content, but may be hemorrhagic. It is unusual scarring or
milia and usually heals with post-inflammatory dyschromia [7, 8]. The mucous involvement
rarely occurs [7].
The oral mucous membrane involvement occurs in 10 to 30% of patients, usually in
newly diagnosed patients.
The ocular, nasal, pharyngeal, esophageal and anogenital mucosa may be more rarely
affected [11] and has been reported a case of BP with the tracheobronchial mucosa
involvement [12].
In some patients, an initial assessment can occur on a stage without bullae, but with
intense itching, and can express a variety of injuries like excoriated, eczematous, papular or
urticarial lesions [8, 13]. This observation implies the importance of considering the
possibility of BP in patients with chronic itching without frankly blistering.
BP can remain localized, around stomas or in paralyzed limbs or irradiated areas, and
may never reach the generalized form [8, 10]. There are case reports associated with
malignancy, such as renal cell carcinoma, gallbladder, colon, breast, and parotid malignancy
and leukemia [7]. The clinical variants of BP can be evidenced in table 1.
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1020 A. Carlos Gripp, A. Bressan, C. Naira Lima e Lima-Santana et al.

TRIGGER FACTORS
Some factors have been associated to BP, as certain types of local aggression such as
traumas, burns, radiation therapy and ultraviolet radiation, drugs, and certain autoimmune
diseases, such as rheumatoid arthritis, Hashimoto thyroiditis, dermatomyositis, lupus
erythematosus and thrombocytopenia. BP is also closely associated with neurological
diseases such as dementia, Parkinson’s disease, multiple sclerosis, psychiatric disorders and
cerebrovascular injuries. This relationship can be explained by the fact that the Bullous
Pemphigoid Antigen 180 (BP180) and Bullous Pemphigoid Antigen 230 (BP230), antigens
related to BP, are expressed in central nervous system and, with the development of
neurological diseases, there is the phenomenon of epitope spreading by the humoral immune
system which affects the skin [8].

DIAGNOSIS
In the skin biopsy from a fresh blister, stained with haematoxylin and eosin, BP shows
subepidermal clefting and inflammatory infiltrate mainly consisting of eosinophils [14].
These are results of injury caused by autoantibodies against structural components of the
hemidesmosome. The known antigens in BP are BP180, also known as type 2 (BPAg2);
BP230, also known as type 1 (BPAg1) and collagen type XVII [10]. The direct
immunofluorescence of perilesional skin is essential for diagnosis and shows linear
deposition of IgG, IgA and/or C3 along the basement membrane zone. In most cases of BP,
the antigens are detected at the roof of the salt-split [14].
The serum levels of autoantibodies to BP180 and BP230 can be detected by enzyme-
linked immunosorbent assay (ELISA). The titles correlate with disease activity and can be
used to monitor response to treatment in addition to the clinical status of the patient [14].

DIFFERENCIAL DIAGNOSIS
Includes bullous diseases such as linear IgA bullous dermatosis, bullous systemic lupus
erythematosus, dermatitis herpetiformis, epidermolysis bullosa acquisita, gestational
pemphigoid and cicatricial pemphigoid (also called pemphigoid mucous membrane) [7, 8].

MANAGEMENT OF BULLOUS PEMPHIGOID

The choice of treatment depends on age, severity of disease and presence of
comorbidities, since there is increased risk of drug interactions and side effects. The major
goal is controlling symptoms with minimum adverse effects. Multiple options are available,
including immunosuppressive and immunomodulating drugs and agents that reduce
pathogenic autoantibodies.
Corticosteroids (CS) are the mainstay of management; however, they are related to high
incidence of side effects. Prednisone and prednisolone are the most used drugs. Daily dose

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 Bullous Pemphigoid 1021

higher than 0.75 mg/kg have no additional benefit and is related to larger frequency of side
effects [15]. Patients with high levels of antibodies to BP180 and severe disease may require
larger CS doses [16]. On the other hand, prednisone doses higher than 40 mg/kg at discharge
are associated with increased risk of death in the first year after hospitalization [17]. Topical
steroids are also safe and successful in treating BP and some studies have shown similar
efficacy when compare to oral treatment [18]. Nevertheless, there are practical limitations and
higher costs.
In refractory cases or in order to reduce CS doses due to associated side effects, other
drugs can be added or used as monotherapy, such as azathiprine (1-3 mg/kg/day),
mycophanolate mofetil (35-45 mg/kg/day) and methotrexate (7.5-25 mg/week) [14, 19-22].
Low doses of cyclophosphamide (50-100 mg/day) is also an option, as evidenced in a serie of
twenty patients with beneficial results [23].
Immunomodulatory antimicrobials are effective in controlling symptoms and preventing
relapse. They are also safe and have more favorable side effect profile. Nicotinamide (500-
2500 mg/day), tetracycline (500-2000 mg/day), doxycycline (200-300 mg/day) and dapsone
(100 mg/day) can be used with or without CS. However, more controlled trials are necessary
to improve knowledge about their use in the BP treatment [14, 15].
The intravenous immunoglobulin (2 g/kg/day during three consecutive days) should be
considered in cases of treatment failure, significant adverse effects with conventional therapy,
progressive disease and contraindications to the use of CS. The main mechanism of action is
reduction of pathogenic antibodies [14, 21]. In a case report about the use of
immunoadsorption in the management of BP, that was evidenced an excellent clinical
response, although more studies are necessary to prove effectiveness and to define treatment
protocols [24].
The use of monoclonal antibodies in the treatment of BP has also been demonstrated.
Rituximab is a humanized antibody against CD20-expressing B lymphocytes which are
involve in producing pathogenic autoantibodies. There is no specific protocol to patients with
BP, but it has been used in refractory cases [25]. Case series of patients treated with
omalizumab reported positive results [26]. The rational to the use is based on data that
evidenced high levels of IgE in patients with BP [27].
Despite multiple treatment options, there are few controlled trials supporting their use.
The majority of related studies are uncontrolled trials and case reports with multiple
definitions and outcome measures. In this context, an international BP definitions committee
was organized to provide definitions for the stages of disease activity, define therapeutic end
points and an objective disease extent measure [28]. Further studies are needed to compare
the efficacy of BP treatment options and provide more consistent evidence.

REFERENCES
[1] Bernard P, Vaillant L, Labeille B, Bedane C, Arbeille B, Denoeux JP, Lorette G,
Bonnetblanc JM, Prost C. Incidence and distribution of subepidermal autoimmune
bullous skin diseases in three French regions. Bullous Diseases French Study Group.
Arch. Dermatol. 1995 Jan;131(1):48-52.
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[2] Jung M, Kippes W, Messer G, Zillikens D, Rzany B. Increased risk of bullous

pemphigoid in male and very old patients: A population-based study on incidence. J.
Am. Acad. Dermatol. 1999 Aug;41(2 Pt 1):266-8.
[3] Marazza G, Pham HC, Schärer L, Pedrazzetti PP, Hunziker T, Trüeb RM, Hohl D, Itin
P, Lautenschlager S, Naldi L, Borradori L; Autoimmune bullous disease Swiss study
group. Incidence of bullous pemphigoid and pemphigus in Switzerland: a 2-year
prospective study. Br. J. Dermatol. 2009 Oct;161(4):861-8. doi: 10.1111/j.1365-
2133.2009.09300.x.
[4] Bertram F, Bröcker EB, Zillikens D, Schmidt E. Prospective analysis of the incidence
of autoimmune bullous disorders in Lower Franconia, Germany. J. Dtsch. Dermatol.
Ges. 2009 May;7(5):434-40. doi: 10.1111/j.1610-0387.2008.06976.x.
[5] Langan SM, Smeeth L, Hubbard R, Fleming KM, Smith CJ, West J. Bullous
pemphigoid and pemphigus vulgaris--incidence and mortality in the UK: population
based cohort study. BMJ. 2008 Jul 9;337:a180. doi: 10.1136/bmj.a180.
[6] Joly P, Baricault S, Sparsa A, Bernard P, Bédane C, Duvert-Lehembre S, Courville P,
Bravard P, Rémond B, Doffoel-Hantz V, Bénichou J. Incidence and mortality of
bullous pemphigoid in France. J. Invest. Dermatol. 2012 Aug;132(8):1998-2004. doi:
10.1038/jid.2012.35.
[7] Khandpur S, Verma P. Bullous pemphigoid. Indian J. Dermatol. Venereol. Leprol.
2011 Jul-Aug;77(4):450-5. doi: 10.4103/0378-6323.82398.
[8] Di Zenzo G, Della Torre R, Zambruno G, Borradori L. Bullous pemphigoid: from the
clinic to the bench. Clin. Dermatol. 2012 Jan-Feb;30(1):3-16. doi:
10.1016/j.clindermatol.2011.03.005.
[9] Ladizinski B, Lee KC. Bullous pemphigoid. J. Gen. Intern. Med. 2013 May;28(5):733.
doi: 10.1007/s11606-012-2250-y.
[10] Walsh SR, Hogg D, Mydlarski PR. Bullous pemphigoid: from bench to bedside. Drugs.
2005;65(7):905-26.
[11] Di Zenzo G, Thoma-Uszynski S, Fontao L, Calabresi V, Hofmann SC, Hellmark T,
Sebbag N, Pedicelli C, Sera F, Lacour JP, Wieslander J, Bruckner-Tuderman L,
Borradori L, Zambruno G, Hertl M. Multicenter prospective study of the humoral
autoimmune response in bullous pemphigoid. Clin. Immunol. 2008 Sep;128(3):415-26.
doi: 10.1016/j.clim.2008.04.012.
[12] Bonifazi M, Zuccatosta L, Poidomani G, Ranaldi R, Gasparini S. Bullous pemphigoid
with the unusual complication of tracheobronchial involvement. Chest. 2013
Jan;143(1):236-8. doi: 10.1378/chest.12-0226.
[13] della Torre R, Combescure C, Cortés B, Marazza G, Beltraminelli H, Naldi L,
Borradori L. Clinical presentation and diagnostic delay in bullous pemphigoid: a
prospective nationwide cohort. Br. J. Dermatol. 2012 Nov;167(5):1111-7. doi:
10.1111/j.1365-2133.2012.11108.x.
[14] Venning VA, Taghipour K, Mustapa MFM, Highet AS, Kirtschig G. British
Association of Dermatologists’ guidelines for the management of bullous pemphigoid
2012. Br. J. Dermatol. 2012;167:1200-14.
[15] García-Romero MT, Werth VP. Randomized controlled trials needed for bullous
pemphigoid interventions.Arch.Dermatol. 2012 Feb;148(2):243-6.
[16] Miida H, Fujiwara H, Ito M. Association between effective dose of prednisolone, alone
or in conjunction with other immunosuppressants, and titre of anti-bullous pemphigoid

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180 antibody: a retrospective study of 42 cases. Clin. Exp. Dermatol. 2011

Jul;36(5):485-8.
[17] Rzany B, Partscht K, Jung M, Kippes W, Mecking D, Baima B, Prudlo C, Pawelczyk
B, Messmer EM, Schuhmann M,Sinkgraven R, Büchner L, Büdinger L, Pfeiffer C,
Sticherling M, Hertl M, Kaiser HW, Meurer M, Zillikens D, Messer G. Risk factors for
lethal outcome in patients with bullous pemphigoid: low serum albumin level, high
dosage of glucocorticosteroids, and old age. Arch. Dermatol. 2002 Jul;138(7):903-8.
[18] Joly P, Roujeau JC, Benichou J, Picard C, Dreno B, Delaporte E, Vaillant L, D'Incan
M, Plantin P, Bedane C, Young P,Bernard P; Bullous Diseases French Study Group. A
comparison of oral and topical corticosteroids in patients with bullous pemphigoid. N.
Engl. J. Med. 2002 Jan 31;346(5):321-7.
[19] Daniel BS, Borradori L, Hall RP 3rd, Murrell DF. Evidence-based management of
bullous pemphigoid. Dermatol. Clin. 2011 Oct;29(4):613-20.
[20] Bressan AL, Silva RS, Fontenelle E, Gripp AC. Immunosuppressive agents in
Dermatology. An. Bras. Dermatol. 2010 Jan-Feb;85(1):9-22.
[21] Ruocco E, Wolf R, Caccavale S, Brancaccio G, Ruocco V, Lo Schiavo A. Bullous
pemphigoid: associations and management guidelines: facts and controversies. Clin.
Dermatol. 2013 Jul-Aug;31(4):400-12.
[22] Tirado-Sánchez A, Díaz-Molina V, Ponce-Olivera RM. Efficacy and safety of
azathioprine and dapsone as an adjuvant in the treatment of bullous pemphigoid.
Allergol Immunopathol (Madr). 2012 May-Jun;40(3):152-5.
[23] Gual A, Iranzo P, Mascaró JM Jr. Treatment of bullous pemphigoid with low-dose oral
cyclophosphamide: a case series of 20 patients. J. Eur. Acad. Dermatol. Venereol. 2014
Jun;28(6):814-8.
[24] Müller PA, Bröcker EB, Klinker E, Stoevesandt J, Benoit S. Adjuvant treatment of
recalcitrant bullous pemphigoid with immunoadsorption. Dermatology.
2012;224(3):224-7.
[25] Shetty S, Ahmed AR. Treatment of bullous pemphigoid with rituximab: critical analysis
of the current literature. J. Drugs Dermatol. 2013 Jun 1;12(6):672-7.
[26] Yu KK, Crew AB, Messingham KA, Fairley JA, Woodley DT. Omalizumab therapy for
bullous pemphigoid. J. Am. Acad. Dermatol. 2014 Sep;71(3):468-74.
[27] Dimson OG, Giudice GJ, Fu CL, Van den Bergh F, Warren SJ, Janson MM, Fairley JA.
Identification of a potential effector function for IgE autoantibodies in the organ-
specific autoimmune disease bullous pemphigoid. J. Invest. Dermatol. 2003
May;120(5):784-8.
[28] Murrell DF, Daniel BS, Joly P, Borradori L, Amagai M, Hashimoto T, Caux F,
Marinovic B, Sinha AA, Hertl M,Bernard P, Sirois D, Cianchini G, Fairley JA,
Jonkman MF, Pandya AG, Rubenstein D, Zillikens D, Payne AS,Woodley D,
Zambruno G, Aoki V, Pincelli C, Diaz L, Hall RP, Meurer M, Mascaro JM Jr, Schmidt
E, Shimizu H,Zone J, Swerlick R, Mimouni D, Culton D, Lipozencic J, Bince B,
Grando SA, Bystryn JC, Werth VP. J. Am. Acad. Dermatol. 2012 Mar;66(3):479-85.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 44

BULLOUS PEMPHIGOID DUE TO ANTI-TNFΑLPHA

Vincenzo Bettoli, Stefania Zauli, Michela Ricci

and Annarosa Virgili
Department of Medical Sciences, Section of Dermatology,
University of Ferrara, Arcispedale S. Anna, Ferrara, Italy

ABSTRACT
Anti-tumor necrosis factor-α (TNFα) agents are increasingly being used for rapidly
expanding number of autoimmune diseases, principally cutaneous, rheumatic and
gastroenterological. With this use and longer follow-up periods of treatment, there are a
growing number of reports of the development of autoimmune processes related to the
use of anti-TNFα (cutaneous vasculitis, lupus-like syndrome, systemic lupus
erythematosus and interstitial lung disease). Despite anti-TNFα can be used to treat
severe forms of autoimmune bullous skin diseases, few cases of pemphigus vulgaris and
bullous pemphigoid occurring under anti-TNFα therapy have been described. The
triggering role of anti-TNFα blockers remains unclear but it can not be excluded that they
could be an immunologic trigger for autoimmune conditions in predisposed individuals.
These drugs may act as triggers by either modifying the immune response or altering the
antigenic properties of the cutaneous antigens. Based on authors’ experience it seems that
different type of anti-TNFα blockers influences the immune response in different way.
The authors propose our personal experience and a review of the cases of
autoimmune bullous skin diseases induced by anti-TNFα agents reported in literature.

INTRODUCTION
Tumour necrosis factor-alpha (TNFα) is a cytokine that plays a crucial role in causing
inflammation by means of predominantly T-cell-mediated tissue damage. TNFα have been


Correspondence to: Dr. Stefania Zauli MD, Department of Medical Sciences, Section of Dermatology, Azienda
Ospedaliera Universitaria di Ferrara, Arcispedale Sant’Anna, Ferrara, Via Aldo Moro 8, 44124 Cona [FE],
Italy, Tel: +39 0532 688129; Fax: +39 0532 206791, E-mail: [email protected]
 Free ebooks ==> www.Ebook777.com
1026 Vincenzo Bettoli, Stefania Zauli, Michela Ricci et al.

clearly identified as having a pivotal role in different kinds of autoimmune/inflammatory

diseases. For this reason anti-TNFα agents have increasingly being used for rapidly
expanding number of diseases principally cutaneous, rheumatic and gastroenterological. The
autoimmune bullous skin diseases, pemphigus vulgaris and bullous pemphigoid, are included
in the list of diseases that benefiting from the treatment with TNFα blockers.
In particular, in bullous pemphigoid the mast cell are responsible for the secretion of
TNFα. The serum level of TNFα are reported to be correlated with both the severity and
number of lesions, and also blister fluid contains high level of this cytokine [1].
Etanercept, Infliximab, and Adalimumab are the anti-TNFα agents developed to date.
Etanercept is an artificially engineered dimeric fusion protein that mimics the inhibitory
effects of naturally occurring soluble TNFα receptors.
Infliximab and Adalimumab are monoclonal antibodies that work by binding to TNF-α
thus preventing it from activating TNF receptors. Infliximab is a genetically engineered
antibody consisting of 25% murine sequences in the variable region of the antibody, while
Adalimumab is a human antibody.
Probably these drugs with different mechanism of action and biosimilarity, influence the
immune system in different ways [2].
Despite the anti-TNFα drugs have been used successfully in the treatment of different
types of autoimmune/inflammatory diseases, with these use and longer follow-up periods of
treatment, unexpectedly there are a growing number of reports of the development of
autoimmune processes related to the use of anti-TNFα agents. These autoimmune adverse
processes mainly include cutaneous vasculitis, lupus-like syndrome, systemic lupus
erythematosus and interstitial lung disease [3] but only a few cases of pemphigus vulgaris and
bullous pemphigoid occurring under therapy have also been described (Table 1) [4-8].
In 2008, Ramos-Casals et al. reviewed 379 cases of autoimmune diseases secondary to
anti-TNFα agents through a baseline Medline search of articles published between January
1990 and May 2008. Among those cases, there were no reports of autoimmune bullous skin
disease [3].
In 2008, Daulat et al. first described a case of pemphigus vulgaris occurring during anti-
TNFα therapy (Etanercept) [4]. In 2009, Stausbol-Gron et al. first described a case of bullous
pemphigoid occurring during Adalimumab administration [5]. A further case of bullous
pemphigoid due to Etanercept has been described in 2009 by Bordignon et al. in a patient
with rheumatoid arthritis [6].
In 2010, Boussemart et al. described two cases of autoimmune bullous skin diseases
occurring during anti-TNFα therapy: one case of bullous pemphigoid under Adalimumab
treatment and one of pemphigus fogliaceus under Infliximab treatment [7]. A further case of
bullous pemphigoid related to Etanercept in a psoriatic patient has been reported by Kluk et
al. in 2011 [8]. All patients improved after discontinuation of the suspect drug and
administration of systemic corticosteroid treatment [4-8]. A case of pemphigus in a patient
with pustular psoriasis treated with Infliximab has been also reported, but this patient had also
a high malignant non Hodgkin lymphoma. Therefore in this case the pemphigus could be
defined as paraneoplastic [9]. The latency period between drug beginning and the onset of
skin manifestations seems variable, from 2 months [short-time] to 3 years [long-time] [4-8].
Recently, the authors have been described a further case of bullous pemphigoid occurring
in a patient undergoing Infliximab (5 mg/kg) for Ulcerative Colitis. Twenty days after the

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 Bullous Pemphigoid due to Anti-TNFα 1027

second infusion [during the induction phase] she developed an itching urticarial eruption
located on her trunk and limbs.

Table 1. Revision of the cases of autoimmune bullous disease reported in literature [4-8]

Authors, years Disease Anti-TNFα drug Latency Bullous disease

of pubblication [dosage]
Daulat et al. Psoriasis Etanercept 2 years Pemphigus vulgaris
2008 [50 mg sc 1 or 2
weekly]
Stausbol-Gron Psoriatic Adalimumab 12 weeks Bullous pemphigoid
et al. 2009 arthritis [40 mg sc every 2
weeks]
Bordignon Rheumatoid Etanercept 2 years Bullous pemphigoid
et al. 2009 arthritis [25 mg sc twice
weekly]
Boussemart Rheumatoid Adalimumab 3 years Bullous pemphigoid
et al. 2010 arthritis [40 mg sc every 2
weeks]
Boussemart Rheumatoid Infliximab 7 months Pemphigus vulgaris
et al. 2010 arthritis [3 mg/Kg]
Kluk et al. Psoriasis Etanercept 2 months Bullous pemphigoid
2011 [25 mg sc twice
weekly]

Over several days this eruption developed into blisters on erythematous skin. Nikolsky’s
sign was negative. The mucosae were not involved. Histological examination of skin biopsy
revealed a superficial dermal inflammation consisting of lymphocytes. Deposition of IgG and
C3 along the basement membrane was detected by direct immunofluorescence, confirming
the clinical suspect of bullous pemphigoid. In the absence of any other known cause, the
authors considered the chimeric anti-TNFα drug as a possible inducing factor.
For this reason Infliximab was stopped while systemic corticosteroids were started to
keep both dermatologic and gastrointestinal disease under control.
After the resolution of the skin eruption, the patient started another anti-TNFα
[Adalimumab, 40 mg s.c. every other week after the induction phase] obtaining clinical
improvement of Ulcerative Colitis in one month [10]. After five months the patient still
maintains complete clinical remission. Surprisingly vesicular-bullous lesions did not appear
during the treatment with this human anti-TNFα, confirming as probably the two drugs
influence the immune system in different ways [2].
Pemphigus and the pemphigoid group are autoimmune conditions in which
autoantibodies cause skin blistering secondary to loss of connections between skin cells.
Pemphigus is characterized by intraepidermal blister due to antibodies that recognize cell
adhesion molecules, the desmogleins. A small group of patients can develop pemphigus after
certain medications, principally penicillamine and captopril [6]. In the bullous pemphigoid
autoantibodies are specific for the hemidesmosomal bullous pemphigoid antigens BP230 and
collagen type XVII, so the blister detachment is subepidermal. Bullous pemphigoid can be
 Free ebooks ==> www.Ebook777.com
1028 Vincenzo Bettoli, Stefania Zauli, Michela Ricci et al.

induced by systemic ingestion or local use of certain drugs. Two main types of drug-induced
bullous pemphigoid may be distinguished: an acute, self-limited variety showing definitive
resolution after the withdrawal of the culprit drug [drug-induced bullous pemphigoid] and a
chronic type that seems merely to precipitate by drug administration. In the long run it
assumes the characteristics of the classic disease [drug-triggered bullous
pemphigoid] [11].
In predisposed individuals, drugs may act as triggers by either modifying the immune
response or altering the antigenic properties of the cutaneous antigens [12].
The triggering role of anti-TNFα blockers remains unclear. It seems that the ability of an
anti-TNFα agent to induce or to cure an autoimmune disease is associated with the
immunological profile of each patient, and, more specifically, it depends on the level of INFγ
and IL-4 [1].
To conclude, the possibility of an autoimmune cutaneous bullous disease should be
considered when managing patients who are under treatment with biological agents.
Conversely, TNFα antagonist can be an effective alternative therapy for these diseases.
Further studies need to establish the efficacy of anti-TNFα in the treatment of
autoimmune bullous disorders and to solve this controversy.

REFERENCES
[1] Stavropoulos PG, Soura E, Antoniou C. Drug-induced pemphigoid: a review of the
literature. J. Eur. Acad. Dermatol. Venereol 2014;doi:10.1111/jdv.12366.
[2] Sands BE, Blank MA, Patel K, van Deventer SJ. Long-term treatment of rectovaginal
fistulas in Crohn's disease: response to infliximab in the ACCENT II Study. Clin.
Gastroenterol. Hepatol. 2004;2:912-920.
[3] Ramos-Casals M, Brito-Zerón P, Soto MJ, Cuadrado MJ, Khamashta MA.
Autoimmune diseases induced by TNF-targeted therapies. Best Pract. Res. Clin.
Rheumatol. 2008;22:847-861.
[4] Daulat S, Detweiler JG, Pandya AG. Development of pemphigus vulgaris in a patient
with psoriasis treated with etanercept. J. Eur. Acad. Dermatol. Venereol 2009;23:483-
484.
[5] Stausbøl-Grøn B, Deleuran M, Sommer Hansen E, Kragballe K. Development of
bullous pemphigoid during treatment of psoriasis with adalimumab. Clin. Exp.
Dermatol. 2009;34:285-286.
[6] Bordignon M, Belloni-Fortina A, Pigozzi B, Tarantello M, Alaibac M. Bullous
pemphigoid during long-term TNF-alpha blocker therapy. Dermatology 2009;219:357-
358.
[7] Boussemart L, Jacobelli S, Batteux F, et al. Autoimmune bullous skin diseases
occurring under anti-tumor necrosis factor therapy: two case reports. Dermatology
2010;221:201-205.
[8] Kluk J, Goulding JM, Bhat J, Finch TM. Drug-induced bullous pemphigoid: cases
triggered by intravenous iodine and etanercept. Clin. Exp. Dermatol. 2011;36:871-873.

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 Bullous Pemphigoid due to Anti-TNFα 1029

[9] Scholberl A, Allmacher C, Flegl N, Krahl B, Krahl D, Amon U. Paraneoplastic

pemphigus due to high malignant non Hodgkin lymphoma in a patient with pustular
psoriasis treated with infliximab. Aktuelle Dermatologie 2010;36:129-132.
[10] Ricci M, Zauli S, Zelante A, Trevisani L, Virgili A, Bettoli V. Bullous pemphigoid
occurring under anti-tumor necrosis factor-α therapy. Int. J. Colorectal. Dis.
2014;29:1573-1574.
[11] Ruocco V, Sacerdoti G. Pemphigus and bullous pemphigoid due to drugs. Int. J.
Dermatol. 1991;30:307-312.
[12] Lo Schiavo A, Ruocco E, Brancaccio G, Caccavale S, Ruocco V, Wolf R. Bullous
pemphigoid: etiology, pathogenesis, and inducing factors: facts and controversies. Clin.
Dermatol. 2013;31:391-399.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 45

DESQUAMATIVE GINGIVITIS AS AN ORAL

MANIFESTATION OF MUCOUS MEMBRANE
PEMPHIGOID: DIAGNOSIS AND TREATMENT

Hiroyasu Endo1,*, Terry D. Rees2, Hideo Niwa3,

Kayo Kuyama4, Hirotsugu Yamamoto4 and Takanori Ito1
1
Department of Oral Diagnosis, Nihon University,
School of Dentistry at Matsudo, Japan
2
Department of Periodontics, Texas A&M University Baylor College of Dentistry,
Dallas, TX, USA
3
Department of Head and Neck Surgery, Nihon University,
School of Dentistry at Matsudo, Japan
4
Department of Oral Pathology, Nihon University,
School of Dentistry at Matsudo, Japan

ABSTRACT
Mucous membrane pemphigoid (MMP) is one of a group of autoimmune,
subepithelial blistering diseases that predominantly affect mucous membranes.
Desquamative gingivitis (DG) is a common manifestation of MMP. Both
histopathological examination and direct immunofluorescence testing are essential to
establish a final diagnosis. Early recognition and treatment of MMP can improve the
prognosis, but diagnostic delays are common in DG because obtaining a diagnostic
biopsy is technically challenging. The stab-and-roll biopsy technique is designed to
prevent the epithelium from being removed from the biopsy specimen. The complications
caused by scarring and associated loss of function often require surgical intervention in
MMP patients. Early diagnosis of MMP is critical, and immunosuppressive treatment
may prevent serious complications in mucous membranes.

*
Corresponding author: Dr. Hiroyasu Endo, Department of Oral Diagnosis, Nihon University School of Dentistry at
Matsudo, 2-870-1 Sakaecho Nishi, Matsudo Chiba, Japan 271-8587, TEL: 81-47-360-9423, FAX: 81-47-360-
9426, e-mail address: [email protected].
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1032 Hiroyasu Endo, Terry D. Rees, Hideo Niwa et al.

INTRODUCTION
Mucous membrane pemphigoid (MMP) is a group of putative autoimmune, chronic
inflammatory, subepithelial blistering diseases that predominantly affect mucous membranes
[1]. MMP is characterized by linear deposition of IgG, IgA, IgM or C3 along the epithelial
basement membrane zone in direct immunofluorescence testing [1, 2]. Most patients with
MMP are between 60 and 80 years of age. However, on relatively rare occasions, blistering
disorders such as MMP have been reported in children, adolescents or young adults [3]. It
affects women at a greater ratio of at least 2:1 compared to men [1, 4, 5]. Oral lesions are
observed in 85-90% cases, and the primary lesions often appear in the oral cavity [1, 4, 5].
MMP can involve any oral mucosal site: gingival, buccal or labial mucosa, hard or soft palate,
alveolar ridge, or tongue, although the gingiva is affected far more often than other oral
tissues. In more than half of early cases, the gingiva is the only site of lesions [4, 6, 7].
Patients with MMP often initially report only oral symptoms of pain and discomfort (Table 1)
and therefore often visit the dentist before other health care workers. Desquamative gingivitis
(DG) is a common manifestation of MMP, probably because the usual mouth functions such
as chewing, exposure to hot foods and liquids, and oral hygiene measures traumatize the
gingiva, resulting in tissue sloughing [4, 7, 8]. This chapter presents the clinical and
diagnostic features of DG, as a common oral manifestation of MMP. The current literature on
the diagnostic and therapeutic modalities of DG associated with MMP is reviewed.

Table 1. Reported oral symptoms in patients with MMP

gingival pain
burning sensation, particularly after eating salty or spicy foods
easy bleeding
blister formation
redness of gum
gingival desquamation
Modified from Endo et al. [4], Endo and Rees [9], Nisengard and Levine [10]

DESQUAMATIVE GINGIVITIS
DG is a clinical manifestation that is common to several diseases or disorders [9-11]. It is
characterized by localized or generalized epithelial desquamation, erythema, erosion of the
gingival epithelium, and/or blister formation on the gingiva (Figure 1). Nikolsky's sign often
shows a positive reaction in patients with DG (Figure 2). This sign involves the application of
a shearing force on normal-appearing gingiva, producing epithelial desquamation [12]. Most
cases of DG are caused by mucocutaneous diseases [9, 11, 13]. The differential diagnoses
include MMP, oral lichen planus, and pemphigus vulgaris [9, 11, 13]. Contact allergic
reactions to various oral hygiene products have also been reported in the differential diagnosis
of DG [14, 15]. It is impossible to diagnose MMP from the clinical presence of DG lesions
alone. The appropriate use of biopsies to perform histopathologic and direct
immunofluorescence examination of lesional and peri-lesional tissues is required to establish
the final diagnosis [9, 11]. Obtaining diagnostic gingival biopsies from MMP patients is

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 Desquamative Gingivitis as an Oral Manifestation of Mucous Membrane … 1033

technically challenging. The excised gingival tissue tends to be fragile because disruption of
the epithelial cell-to-basement membrane adhesion components is likely to occur. This
situation may often result in detachment of the gingival epithelium from the underlying
connective tissue, causing a failure in biopsy diagnosis. This tissue friability, coupled with an
inadequate surgical technique, surgical site selection or improper tissue handling, may easily
lead to the loss of the gingival epithelium, causing a failure in histopathologic and direct
immunofluorescence diagnosis. Because of this, some authors have stated that if lesions are
present at several mucosal sites, including the gingiva, it is usually best not to use the gingiva
for biopsies [16-18]. However, in approximately 60% of the MMP patients, the gingiva was
the only site of involvement [6] and in these cases, the gingiva should be selected as the
biopsy site. Recently the authors developed and validated a biopsy technique (the stab-and-
roll technique) to maintain the gingival epithelium/connective tissue union in DG patients
[19] (Figure 3).

Figure 1. Clinical presentation of DG associated with MMP. (A) Erythema. (B) Pseudomembrane-
covered erosion. (C) Erosion. (D) Ulceration. (E) Blister formation. (F) Localized blood-filled blister
formation.

In this technique, the operator applies gentle pressure on the gingiva with the tip of a #15
blade until the bone surface is reached, and then the blade is rolled from the tip along the
entire cutting edge. If a larger specimen is needed the tip of the blade can be repositioned and
the rolling stroke extended. This stab-and-roll biopsy technique prevents the occurrence of
lateral shear forces. In contrast in the conventional gingival biopsy technique, the scalpel
blade is pulled across the biopsy site while the tip of the blade is against the bone surface.
This potentially creates a lateral shear force potentially causing the epithelium to be displaced
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1034 Hiroyasu Endo, Terry D. Rees, Hideo Niwa et al.

from the biopsy specimen. A total lack of epithelium has been reported in 40% [20] - 41.2%
[21] of gingival biopsy samples using the conventional biopsy technique whereas in a series
of 52 gingival biopsies only 1.9% of the samples obtained using the stab-and-roll biopsy
technique resulted in epithelium-connective tissue separation [19]. Some authors are
concerned that gingival biopsies may result in permanent periodontal defects [20, 21]. Indeed,
since many DG lesions develop in the anterior facial area, resultant periodontal defects could
be an esthetic problem. To prevent this difficulty, stab-and-roll biopsies are often taken from
perilesional tissues apical to the free gingival margin. This site selection also prevents the
biopsy tissue from being obscured by gingival inflammation (Figure 4).

Figure 2. Positive Nikolsky's sign in a patient with MMP. Gentle palpation with the periodontal probe
elicited some desquamation of the gingival surface.

Figure 3. Histopathologic and direct immunofluorescence features of MMP. Gingival biopsies were
performed using the stab-and-roll technique. (A) Hematoxylin- and eosin-stained section (Original
magnification x400). Subepithelial bulla formation. (B) Direct immunofluorescence section (Original
magnification x200). A linear deposition of IgG along the basement membrane zone.

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 Desquamative Gingivitis as an Oral Manifestation of Mucous Membrane … 1035

SPECIFIC SITE CONSIDERATION

Extraoral MMP lesions have been reported on skin including the external genitalia and
perianal skin as well as on the mucous membranes of the eye, nose, pharynx, larynx,
esophagus, and anus [1, 22]. Although scarring is rarely a feature of oral MMP, in extraoral
sites scar formation may lead to an irreversible loss of function of the affected areas. Sight-
threatening ocular scarring [23-25] and life-threatening upper airway obstruction [26-28] have
been reported. In contrast, only one case report has described oral scarring. Sato et al. [29]
reported microstomia associated with MMP exhibiting anti-laminin 332 autoantibodies. Scar
contracture was ring-shaped and localized on the oral mucosa. A commissuroplasty was
performed in treatment using 5-flap Z-plasty on the upper lip and 2-flap Z-plasty on the lower
lip. The patient was reported to be satisfied with the postoperative esthetics and the size of the
oral aperture.

Figure 4. Periodontal conditions after gingival biopsy. The periodontal defects or recessions did not
occur after the gingival biopsy using the stab-and-roll technique. (A) Before biopsy. The gingival
sample was removed from the dotted-line area. (B) 1 week after biopsy. (C) 6 months after biopsy.
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1036 Hiroyasu Endo, Terry D. Rees, Hideo Niwa et al.

In a previous study, an incidence of only 10.4% (10/96) ocular lesions was reported in
MMP associated DG patients [30]. Consequently, patients with exclusively intraoral MMP
were thought to have a less severe disease that might not be associated with ocular
involvement [7, 31]. However, a recent study indicated 30% (9/30) of patients with oral MMP
had ocular involvement at presentation to an opthalmologist [24]. Another study of 25
patients who initially had only oral involvement reported that 4 (16%) developed ocular
lesions within 5 years [25]. These studies indicate that MMP patients with initial oral lesions
have a risk of developing ocular involvement with a calculated incidence rate from 0.03 [24]
to 0.05 [25] person per year. Some specific oral symptoms are common in individuals likely
to have oral MMP (Table 1). The presence of these symptoms should increase the healthcare
provider’s level of suspicion of a mucocutaneous disease such as MMP. Symptoms
suggestive of possible ocular involvement in patients with MMP are shown in Table 2. MMP
patients with oral involvement, however, frequently have asymptomatic ocular lesions,
especially in the early stages of ocular disease [24, 25]. These observations indicate that all
patients diagnosed with intraoral or extraoral MMP should undergo ophthalmic examination
by an ophthalmologist (Figure 5). Patients with oral MMP should have regular opthalmologic
monitoring every 6 to 12 months, even if no ocular involvement is identified at initial
diagnosis [24].

Table 2. Symptoms possibly related to ocular

involvement in patients with MMP

burning sensation
dryness
foreign body sensation
irritation
excess tearing
mucus production
photophobia
blurry vision
decreased visual acuity
Modified from Fleming and Korman [5],
Kourosh and Yancey [22]

Patients with MMP restricted to the upper airway tract are rarely observed [26, 27]. More
than 84% of MMP patients with upper airway involvement had oral lesions [26, 27]. Despite
this, reports of the upper airway involvement in DG patients are scarce. One report described
a case of MMP in a young patient presenting with DG and laryngeal manifestations that
resulted in severe life-threatening sequels of events [32]. Symptoms that should raise
suspicion regarding the presence of upper airway involvement in patients with MMP are
shown in Table 3. When DG patients complain of these symptoms, they should be seen by an
otolaryngologist for evaluation and possible endoscopic examination of the upper airway tract
(Figure 6).

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 Desquamative Gingivitis as an Oral Manifestation of Mucous Membrane … 1037

Figure 5. Ocular involvement in a DG patient with MMP. (A) Desquamative lesions featuring gingival
erythema. (B) Ophthalmic examination by an opthalmologist revealed lower conjunctiva symblepharon
in the same patient.

Table 3. Symptoms suggestive of possible upper airway

involvement in patients with MMP

nasal stuffiness
nasal bleeding or blood-tinged mucous discharge
cough
hoarseness
difficult or labored breath
continuous inspiratory musical sound of variable pitch
sore throat
pain on swallowing
dysphonia
Modified from Alexandre et al. [26], Higgins et al. [27]
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1038 Hiroyasu Endo, Terry D. Rees, Hideo Niwa et al.

Figure 6. Laryngeal involvement in a DG patient with MMP. (A) Desquamative lesions of soft palate in
addition to gingiva. The patient complained of chronic cough and sore throat. (B) Conventional
endoscopic examination revealed white coat of the epiglottis and the aryepiglottic fold. The
involvements are consistent with MMP early lesions. (C) Narrow band image (NBI) enhanced and
defined white coat MMP.

Figure 7. Topical corticosteroid therapy in a DG patient with MMP. (A) The initial examination
revealed localized erythematous gingiva. (B) The gingival lesions went into remission with the topical
corticosteroid therapy.

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 Desquamative Gingivitis as an Oral Manifestation of Mucous Membrane … 1039

MANAGING PATIENTS WITH DESQUAMATIVE GINGIVITIS

The therapeutic goal for DG lesions is the remission or suppression of the clinical signs
and symptoms. The severity of the DG lesions, the presence or absence of extraoral lesions,
and the medical history of the patient are key factors in determining the selection of a topical
or systemic treatment. In most cases, topical therapy alone is sufficient to achieve resolution
of lesions when MMP is diagnosed and treated in its early stages. When MMP affects the oral
cavity as the sole involvement, moderate to very high-potency topical corticosteroids are
effective and widely used for treatment [6, 9] (Figure 7). The absorption of topical
corticosteroids may increase in the presence of DG, since the continuity of intact epithelium
may be disrupted. In 1990, Plemons et al. [33] studied the systemic uptake of high-potency
topical corticosteroid gel (0.05% fluocinonide gel) applied to oral desquamative diseases
three times daily for 3 weeks. They found no evidence of adrenal suppression in the study
population. Occlusive steroid therapy using a plastic stent may be used to enhance the effect
of topical corticosteroid therapy by maximizing the contact between the corticosteroids
applied on the gingiva [34, 35] (Figure 8). In this therapy, the topical corticosteroid is in
contact with the gingiva for a longer time period and the systemic absorption is probably
increased thus enhancing the effect of the topical agent. To date there are no studies
documenting medical complications related to the intraoral use of very high potency topical
corticosteroids, but caution should be used when providing occlusive steroid therapy for
patients afflicted with hypertension, gastrointestinal ulcers or diabetes mellitus, pending
further study. Secondary candidosis is the most common side effect from topical
corticosteroid therapy (Figure 9). Oral candidosis should be suspected in patients that
continue to complain of oral symptoms despite several days or weeks of topical therapy.

Figure 8. Occlusive steroid therapy using a plastic stent in a DG patient with MMP. (A) Plastic stent for
mandibular arch. (B) Stent in place.
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1040 Hiroyasu Endo, Terry D. Rees, Hideo Niwa et al.

Figure 9. Secondary candidosis in a DG patient with MMP. During the topical steroid therapy period,
gingival candidosis occurred. The symptoms disappeared after an antifungal medication was
administered.

Patients with severe and/or multiple oral lesions, or recalcitrant lesions, may need
aggressive systemic treatment [36]. The presence of extraoral lesions also may require
systemic corticosteroids and/or immunosuppressive drugs for effective MMP management.
Careful medical management is necessary to monitor the patient for adverse effects of
systemic drugs and to manage concomitant systemic diseases. Although extraoral MMP
lesions involving erythema, erosion, ulceration, or blister formation will respond well to
medical immunosuppressive therapy, the treatment will be highly resistant if fibrosis and
scarring has occurred [22]. The complications caused by scarring and associated loss of
function often require surgical and/or medical intervention. Airway obstruction is the most
serious complication and may necessitate an emergency tracheotomy [26-28]. Ocular lesions
occur most often in association with DG and may induce inflammation, loss of tear film,
progressive scarring and adherence of the eyelid to the eyeball (symblepharon), inward
turning of the eyelashes (triachiasis), and inward turning of the eyelids (entropion) [5, 22].
Unless treated aggressively, these lesions can lead to loss of vision in one or both eyes [37].
Early diagnosis of MMP is critical and immunosuppressive treatment may prevent scar
formation in mucous membrane.

Figure 10. Poor oral hygiene status in a DG patient with MMP. Dental plaque and calculus deposits
were recognized around teeth.

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 Desquamative Gingivitis as an Oral Manifestation of Mucous Membrane … 1041

PERIODONTAL CONSIDERATIONS
It is often very difficult for DG patients to clean their teeth due to pain and bleeding, and
patients often fear to create new lesions on the gingiva. Therefore, their oral hygiene is likely
to be ineffective, making it difficult to treat this condition (Figure 10). For this reason, some
authorities have stated that desquamative lesion of the gingiva could lead to periodontal
destruction and bone loss, necessitating tooth extractions [5, 37]. However, little information
is available regarding the periodontal conditions of patients with DG associated with MMP.
The relationship between the existence of MMP lesions and progression of periodontal
diseases is inconclusive. Arduino et al. [38] demonstrated that periodontal status is worse in
MMP patients compared with healthy controls because of substantial differences in oral
hygiene. Conversely, Tricamo et al. [39] and Schellinck et al. [40] suggested that MMP
patients demonstrate higher levels of gingival inflammation, but not chronic periodontitis
compared to healthy age and sex matched controls even after at least a 5 year history of
MMP. Plaque accumulation may be an aggravation factor to make DG worse. Plaque-related
gingivitis is almost universal in patients with painful gingival lesions and an effective
therapeutic protocol should include non-surgical periodontal therapy consisting of oral
hygiene instruction, scaling, and root planing [41]. Orrico et al. [42] affirmed that plaque
control performed by a professional and the application of 0.12% chlorhexidine digluconate
resulted in 90% improvement of gingival lesions in MMP patients. Professional oral hygiene
treatment and detailed oral hygiene instructions are connected with improvement of gingival
status and a decrease in gingival-related pain in patients affected by MMP with DG lesions
[43]. Combined treatment and long-term maintenance of MMP and periodontal disease are
effective at improving and stabilizing the gingival conditions in MMP patients [44-46].

CONCLUSION
Early signs and symptoms of MMP develop in the oral cavity in almost all cases, and DG
is a common manifestation. After MMP is diagnosed from DG or concomitant lesions,
patients should undergo examination by medical specialists including an opthalmologist and
an otolaryngologist, and the presence or absence of extraoral mucosal lesions should be
determined. Scarring may lead to an irreversible loss of function of the affected extraoral
mucous membranes in some MMP cases. Early recognition and treatment of the diseases is
very important and can significantly improve the prognosis.

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[28] Miziara, ID; Sperandio, F; Bohadana, SC; Braga, N; Romano, FR; Miniti, A. Cicatricial
pemphigoid: report of five cases. Ear Nose Throat J, 2002, 81(7), 442-448.
[29] Sato, H; Toriyama, K; Yagi, S; et al. Surgical correction of microstomia in a patient
with antilaminin 332 mucous membrane pemphigoid. Ann Plast Surg, 2014, 72(5), 553-
555.
[30] Nisengard, RJ; Rogers, RS; 3rd. Desquamative gingivitis. In: Beutner EH, Chorzelski
TP, Kumar V, eds. Immunopathology of the skin 3rd ed. New York : A Wiley medical
publication; 1987, 361-369.
[31] Dayan, S; Simmons, RK; Ahmed, AR. Contemporary issues in the diagnosis of oral
pemphigoid: a selective review of the literature. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod, 1999, 88(4), 424-430.
[32] Ojha, J; Bhattacharyya, I; Stewart, C; Katz, J. Cicatricial pemphigoid with severe
gingival and laryngeal involvement in an 18-year-old female. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod, 2007, 104(3), 363-367.
[33] Plemons, JM; Rees, TD; Zachariah, NY. Absorption of a topical steroid and evaluation
of adrenal suppression in patients with erosive lichen planus. Oral Surg Oral Med Oral
Pathol, 1990, 69(6), 688-693.
[34] Endo, H; Rees, TD; Kuyama, K; Matsue, M; Yamamoto, H. Successful treatment using
occlusive steroid therapy in patients with erosive lichen planus: a report on 2 cases.
Quintessence Int, 2008, 39(4), e162-172.
[35] Lamey, PJ; Jones, CM. Desquamative gingivitis treated with occlusive steroid therapy:
a pilot study. Gerodontics, 1988, 4(4),188-190.
[36] Scully, C; Lo Muzio, L. Oral mucosal diseases: mucous membrane pemphigoid. Br J
Oral Maxillofac Surg, 2008, 46(5), 358-366.
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1044 Hiroyasu Endo, Terry D. Rees, Hideo Niwa et al.

[37] Yancey, KB. Cicatricial pemphigoid. In: Wolff K, Goldsmith LA, Katz SI, Gilchrest
BA, Paller AS, Leffell DJ. eds. Fizpatrick's Dermatology in General Medicine, 7th ed.
New York : McGraw Hill Inc; 2008, 481-485.
[38] Arduino, PG; Farci, V; D'Aiuto, F; et al. Periodontal status in oral mucous membrane
pemphigoid: initial results of a case-control study. Oral Dis, 2011, 17(1), 90-94.
[39] Tricamo, MB; Rees, TD; Hallmon, WW; Wright, JM; Cueva, MA; Plemons, JM.
Periodontal status in patients with gingival mucous membrane pemphigoid. J
Periodontol, 2006, 77(3), 398-405.
[40] Schellinck, AE; Rees, TD; Plemons, JM; Kessler, HP; Rivera-Hidalgo, F; Solomon, ES.
A comparison of the periodontal status in patients with mucous membrane pemphigoid:
a 5-year follow-up. J Periodontol, 2009 80(11), 1765-1773.
[41] Rees, TD. Vesiculo-ulcerative diseases and periodontal practice. J Periodontol, 1995,
66(8), 747-748.
[42] Orrico, SR; Navarro, CM; Rosa, FP; Reis, FA; Salgado, DS; Onofre, MA. Periodontal
treatment of benign mucous membrane pemphigoid. Dent Today, 2010, 29(7), 100-102;
quiz 102-103.
[43] Arduino, PG; Lopetuso, E; Carcieri, P; et al. Professional oral hygiene treatment and
detailed oral hygiene instructions in patients affected by mucous membrane pemphigoid
with specific gingival localization: a pilot study in 12 patients. Int J Dent Hyg, 2012,
10(2),138-141.
[44] Damoulis, PD; Gagari, E. Combined treatment of periodontal disease and benign
mucous membrane pemphigoid. Case report with 8 years maintenance. J Periodontol,
2000, 71(10), 1620-1629.
[45] Lilly, JP; Spivey, JD; Fotos, PG. Benign mucous membrane pemphigoid with advanced
periodontal involvement: diagnosis and therapy. J Periodontol, 1995, 66(8), 737-741.
[46] Lorenzana, ER; Rees, TD; Hallmon, WW. Esthetic management of multiple recession
defects in a patient with cicatricial pemphigoid. J Periodontol, 2001, 72(2), 230-237.

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Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 46

ASSOCIATIONS BETWEEN
BULLOUS PEMPHIGOID AND INTERNAL
MALIGNANCIES: A LITERATURE REVIEW

Yuta Kurashige1,, Norihiro Ikoma1, Tomotaka

Mabuchi1, Akira Ozawa1 and Kenichi Iwashita2
1
Department of Dermatology, Tokai University
School of Medicine, Kanagawa, Japan
2
Yochomachi dermatology clinic, Tokyo, Japan

ABSTRACT
The correlation between bullous pemphigoid (BP) and internal malignancies has
been argued over for more than half a century, and remains controversial. In this chapter,
we review the literature in three categories. First, we examine the historical perspective
of the association between BP and malignancies; the first case report was probably
mentioned by Forman in 1960, and the first case series (seven cases) was described by
Parsons and Savin in 1968. Second, we look at the incidence of malignancy in BP
patients; among 17 previous studies, six concluded that BP carries an increased risk of
internal malignancy, while nine denied such an association. The most recent cohort study
found no correlation. Third, we examine the incidence of BP among cancer patients; one
cohort study reported no evidence of overall correlation, but a sub-analysis in the study
suggested that kidney cancer alone was linked to an elevated risk of BP. Considering the
results of these studies, an overall association between BP and malignancies is not
shown, while restricted associations depending on the type of cancer or ethnic
background of the BP patient might be possible.


Corresponding author: Dr. Yuta Kurashige, Department of Dermatology, Tokai University School of Medicine,
143 Shimokasuya, Isehara-shi, Kanagawa 259-1193, Japan. E-mail: [email protected], phone: +81.
463.93.1121, fax: +81.463.93.9387.
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1046 Yuta Kurashige, Norihiro Ikoma, Tomotaka Mabuchi et al.

INTRODUCTION
Bullous pemphigoid (BP), the most common form of acquired autoimmune bullous
dermatosis, occurs mostly in elderly people and is characterized by autoimmune reactions to
hemidesmosomal proteins BP180 and BP230, located at the dermo-epidermal junction [1].
For more than fifty years, some autoimmune bullous dermatoses such as pemphigus and
pemphigoid have been considered to be associated with internal malignancies. Regarding BP,
the literature has described some clinical findings as strongly suggestive of the presence of
internal malignancy, including bullae on gyrate erythema [2], negative findings of indirect
immunofluorescence [3], and existence of autoantibodies against BP180 [4]. A large number
of case reports and clinical studies have examined associations between BP and internal
malignancies, but the association remains controversial. This chapter reviews the publications
that represent the historical perspective and that refer to either the incidence of malignancy in
BP patients or the incidence of BP in cancer patients.

HISTORICAL PERSPECTIVE
In 1953, Lever [5] first established distinct criteria for the diagnosis of BP, distinguishing
this entity from other bullous diseases. To the best of our knowledge, the 1960 report by
Forman [6] represents the first description of BP in association with internal malignancy; a
70-year-old man was diagnosed with rectal adenocarcinoma 1 year after the diagnosis of BP.
In that patient, BP had initially been under successful control. Nevertheless, severe relapse
was seen after surgery. At that time, Wilson [6] mentioned BP with carcinoma of the breast
and stomach, and Hellier [6] also referred to two cases of BP with carcinoma of the breast and
uterus. Hellier suggested that a search for neoplasm should be made in all BP patients. In
1968, as first case series, Parsons and Savin [7] reported seven cases in which BP and
malignancies occurred at the same time: the malignancies involved the skin (two cases),
cervix, bronchus, uterus, gallbladder, and anus. However, the authors denied a close
relationship between BP and malignancies. In the same year, Lim et al. [8] investigated the
incidence of internal malignancy among BP patients using patient data collected from
dermatologists and pathologists in the eastern counties of England. As a result, malignancy
was found in 12 of 103 BP patients and no correlation was apparent. Since then, as shown in
Table 1, various clinical studies have aimed at revealing associations or confirming their
absence.

INCIDENCE OF INTERNAL MALIGNACIES AMONG BP PATIENTS

Table 1 shows 17 representative English-language investigations concerning the
incidence of internal malignancy in BP patients [3, 8-23]. Among these, six simply described
the incidence of internal malignancies, while 10 conducted comparisons of the incidences of
malignancy between BP patient and control groups. In addition, only one study was designed
as a cohort study.

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 Associations between Bullous Pemphigoid and Internal Malignancies … 1047

Table 1. Studies concerning the incidence of internal malignancies in bullous

pemphigoid

Data Incidence of malignancies

Authors Country Institution collection (Total/ With malignancy) Conclusion
period BP Controls
Lim CC et al. Multiple 103/ 12
UK 1950-1965 No controls No association
(1968) [8] hospitals (11.7%)
Stone SP and 146/ 11 (7.5%)
73/ 8
Schroeter AL US Single hospital 1960-1972 Other skin No association
(11.0%)
(1975) [9] diseases
Ahmed AR et al. 33/ 1
US Single hospital ND No controls No association
(1977) [10] (3.0%)
Moss AA and
29/ 3 120/ 11 (9.2%)
Hanelin LG (1977) US Single hospital ND Increased risk
(10.3%) Diabetes mellitus
[11]
Person J and Rogers 84/ 11
US Single hospital 1968-1975 No controls No association
RS 3rd (1977) [12] (13.1%)
Chorzelski TP et al. 110/ 12 Nationwide health Increased risk
Poland ND 1968-1977
(1978) [13] (10.9%) information P< 0.01
Increased risk
Hodge et al. (1981) 124/ 12
UK Single hospital ND No controls OR 3.63, P<
[14] (9.7%)
0.05
Increased risk in
Venencie et al. 93/ 9
US Single hospital 1970-1980 No controls negative IIF
(1984) [3] (9.7%)
patients
Lindelöf B et al. Single national 497/ 61 Nationwide health
Sweden 1975-1985 No association
(1990) [15] laboratory (12.3%) information
Venning VA and 168/ 9 (5.4%)
84/ 15
Wojnarowska F UK Single hospital 1975-1989 Other skin Increased risk
(17.9%)
(1990) [16] diseases
Ogawa H et al. 1113/ 64 Nationwide health
Japan 393 hospitals 1981-1986 Increased risk
(1995) [17] (5.8%) information
172/ 27 (15.7%)
Chang YT et al. 86/ 13 No association
Taiwan Single hospital 1977-1994 Other skin
(1996) [18] (15.1%) P> 0.5
diseases
Cozzani E et al. 32/ 6
Italy 11 hospitals 1996-1997 No controls No association
(2001) [19] (18.8%)
Iwashita K et al. 115/ 12 Nationwide health
Japan Single hospital 1975-2006 Increased risk
(2007) [20] (10.4%) information
No association
89/ 14 (15.7%) OR 1.74, 95%CI
Jedlickova H et al. Czech 89/ 19
Single hospital 1991-2006 Other skin 0.43-6.96,
[21] Republic (21.3%)
diseases p=0.33
(Age<80)
Increased risk
190/ 4 (2.1%)
Li J et al. (2013) 190/ 13 AOR 4.226,
China Single hospital 1992-2012 Other skin
[22] (6.8%) 95%CI 1.176-
diseases
15.180
Nationwide
Nationwide No association
Ong E et al. (2014) hospital 4,720/ 502
UK 1999-2011 hospital admission RR 1.00, 95%CI
[23] admission (10.6%)
dataset 0.92-1.09
dataset
RR; Relative risk, OR; Odds ratio, CI; Confidential interval, AOR; Adjusted prevalence odds ratio.
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1048 Yuta Kurashige, Norihiro Ikoma, Tomotaka Mabuchi et al.

Among these, five studies were from the United States, four from the United Kingdom,
two from Japan, and one each from Poland, Sweden, Taiwan, the Czech Republic, and China.
The incidence of internal malignancies in BP patients varied from 3.0% to 21.3%. It should
be noted that, among these studies, six concluded that BP was associated with an increased
risk of internal malignancy, while nine found no such correlation. Although the majority of
studies used relatively small samples, some made use of large samples or nationwide patient
data. For example, the 1990 investigation by Lindelof et al. [15] reported that 61 of 497 BP
patients had internal malignancies, whereas the expected number of malignancies based on
nationwide information on cancer incidence in Sweden was 82.6 (relative risk [RR], 0.84;
95% confidence interval [CI], 0.65-1.06). Accordingly, the authors concluded that
pemphigoid might not be associated with malignancy. On the other hand, in 1995, Ogawa et
al. [17] reviewed 1113 BP patients recruited from a nationwide survey of patients with
autoimmune blistering disease in Japan. The survey revealed 64 patients with internal
malignancies, and this incidence rate of 5.8% was significantly higher than that of Japanese
controls. These increased risks were corroborated in another Japanese study from our
department [20]. Concerning the cohort study, Ong et al. [23] used nationwide hospital
admission data from the United Kingdom and evaluated the risk of concurrent and subsequent
malignancies. As a result, the “BP cohort” of 4,720 BP patients showed no increased risk
compared with the “reference cohort” of individuals without a diagnosis of BP (RR, 1.00;
95%CI, 0.92-1.09).

INCIDENCE OF BP IN PATIENTS WITH INTERNAL MALIGNANCY

Dermatologists tend to have an interest in whether BP can provide a good predictor of
latent malignancies. Accordingly, a large proportion of studies on BP from dermatologists
have stressed investigation of the incidence of malignancies among BP patients. However,
confirmation of an association between these entities naturally requires investigation of the
incidence of BP among cancer patients. To the best of our knowledge, only one study has
examined this theme; Ong et al. [23] performed another cohort study using the above-
mentioned nationwide dataset. The result showed that the “cancer cohort” of 2.87 million
people with a diagnosis of various types of cancer did not show any increased risk of
concurrent or subsequent BP compared to the “reference cohort” of individuals without
internal malignancy (RR, 0.96; 95%CI, 0.88-1.04). The authors then undertook additional
analyses to investigate whether “sub-cohorts” involving specific cancers showed any
increased risk of BP compared to the reference cohort. Kidney cancer alone showed an
elevated risk of BP (RR, 2.23; 95%CI, 1.48-3.24; p<0.001).

DISCUSSION AND SUMMARY

The argument about associations between BP and internal malignancies has been ongoing
for a long time, but a definitive conclusion remains elusive. As mentioned above, previous
studies have shown inconsistent outcomes between no correlation and increased risk. Factors
that might contribute to this state are as follows. First, the different study designs including

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 Associations between Bullous Pemphigoid and Internal Malignancies … 1049

patient numbers, settings, and statistical methods might lead to different results. Second, the
reliability of the diagnoses of BP and malignancies in early studies might be dissimilar to
those of recent studies, given the advances in medical technologies. Third, because these
studies were undertaken in various countries, differences in patient backgrounds in relation to
lifestyle, environment or genes might have affected the results. The 17 studies are thus not
directly comparable. Nonetheless, from an epidemiological perspective, the results from the
recent, well-designed cohort study by Ong et al. [24] can be plausibly taken into account.
Considering the results from that study and others [8-10, 12, 15, 18, 19, 21], we suspect
that the balance of evidence favors a lack of overall correlation between BP and
malignancies. On the other hand, restricted associations depending on two factors are
plausible: type of cancer, e.g., kidney cancer [23]; and the ethnic background of BP patients,
e.g., Japanese [17, 20].

REFERENCES
[1] Kasperkiewicz, M., Zillikens, D. The pathophysiology of bullous pemphigoid. Clin.
Rev. Allergy Immunol. 2007 Oct; 33(1-2): 67-77.
[2] Gilmour, E., Bhushan, M., Griffiths, C. E. Figurate erythema with bullous pemphigoid:
a true paraneoplastic phenomenon? Clin. Exp. Dermatol. 1999 Nov; 24: 446-448.
[3] Venencie, P. Y., Rogers, R. S. 3rd, Schroeter, A. L. Bullous pemphigoid and
malignancy: relationship to indirect immunofluorescent findings. Acta Derm. Venereol.
1984; 64: 316-319.
[4] Muramatsu, T., Iida, T., Tada, H., Hatoko, M., Kobayashi, N., Ko, T., Shirai, T.
Bullous pemphigoid associated with internal malignancies: identification of 180-kDa
antigen by western immunoblotting. Br. J. Dermatol. 1996 Nov; 135: 782-784.
[5] Lever, W. F. Pemphigus. Medicine (Baltimore). 1953 Feb; 32: 1-123.
[6] Forman, L. Pemphigoid occurring with carcinoma of the rectum. Proc. R Soc. Med.
1960 Jul; 53: 563.
[7] Parsons, R. L., Savin, J. A. Pemphigoid and malignancy. Br. J. Cancer. 1968 Dec; 22:
669-672.
[8] Lim, C. C., Macdonald, R. H., Rook, A. J. Pemphigoid eruptions in the elderly. Trans.
St Johns Hosp. Dermatol. Soc. 1968; 54: 148-151.
[9] Stone, S. P., Schroeter, A. L. Bullous pemphigoid and associated malignant neoplasms.
Arch. Dermatol. 1975 Aug; 111: 991-994.
[10] Ahmed, A. R., Chu, T. M., Provost, T. T. Bullous pemphigoid: clinical and serologic
evaluation for associated malignant neoplasms. Arch. Dermatol. 1977 Jul; 113: 969.
[11] Moss, A. A., Hanelin, L. G. Occult malignant tumors in dermatologic disease. The
futility of radiological search. Radiology. 1977 Apr; 123: 69-71.
[12] Person, J. R., Rogers, R. S. 3rd. Bullous and cicatricial pemphigoid. Clinical,
histopathologic, and immunopathologic correlations. Mayo Clin. Proc. 1977 Jan; 52:
54-66.
[13] Chorzelski, T. P., Jablonska, S., Maciejowska, E., Beutner, E. H., Wronkowski, L.
Coexistence of malignancies with bullous pemphigoid. Arch. Dermatol. 1978 Jun; 114:
964.
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1050 Yuta Kurashige, Norihiro Ikoma, Tomotaka Mabuchi et al.

[14] Hodge, L., Marsden, R. A., Black, M. M., Bhogal, B., Corbett, M. F. Bullous
pemphigoid: the frequency of mucosal involvement and concurrent malignancy related
to indirect immunofluorescence findings. Br. J. Dermatol. 1981 Jul; 105: 65-69.
[15] Lindelöf, B., Islam, N., Eklund, G., Arfors, L. Pemphigoid and cancer. Arch. Dermatol.
1990 Jan; 126: 66-68.
[16] Venning, V. A., Wojnarowska, F. The association of bullous pemphigoid and malignant
disease: a case control study. Br. J. Dermatol. 1990 Oct; 123: 439-445.
[17] Ogawa, H., Sakuma, M., Morioka, S., Kitamura, K., Sasai, Y., Imamura, S., Inaba, Y.
The incidence of internal malignancies in pemphigus and bullous pemphigoid in Japan.
J. Dermatol. Sci. 1995 Mar; 9: 136-141.
[18] Chang, Y. T., Liu, H. N., Wong, C. K. Bullous pemphigoid--a report of 86 cases from
Taiwan. Clin. Exp. Dermatol. 1996 Jan; 21: 20-22.
[19] Cozzani, E., Parodi, A., Rebora, A., Delmonte, S., Barile, M., Nigro, A., Priano, L.,
Troiano, G., Patri, P. L.; Gruppo Ligure di Studi in Dermatologia (GLISID). Bullous
pemphigoid in Liguria: a 2-year survey. J. Eur. Acad. Dermatol. Venereol. 2001 Jul;
15:317-319.
[20] Iwashita, K., Matsuyama, T., Akasaka, E., Mizutani, K., Yamamoto, K., Kondoh, A.,
Nozawa, M., Yagi, Y., Ikoma, N., Mabuchi, T., Shinagawa, H., Tamiya, S., Nuruki, H.,
Ohta, Y., Umezawa, Y., Ozawa, A. The incidence of internal malignancies in
autoimmune bullous diseases. Tokai J. Exp. Clin. Med. 2007 Mar. 20; 32: 42-47.
[21] Li, J., Zuo, Y. G., Zheng, H. Y., Qiu-Ning, S. Association between bullous pemphigoid
and internal diseases. J. Dtsch. Dermatol. Ges. 2013 Mar; 11: 263-264.
[22] Jedlickova, H., Hlubinka, M., Pavlik, T., Semradova, V., Budinska, E., Vlasin, Z.
Bullous pemphigoid and internal diseases - A case-control study. Eur. J. Dermatol.
2010 Jan-Feb; 20: 96-101.
[23] Ong, E., Goldacre, R., Hoang, U., Sinclair, R., Goldacre, M. Associations between
bullous pemphigoid and primary malignant cancers: an English national record linkage
study, 1999-2011. Arch. Dermatol. Res. 2014 Jan; 306: 75-80.

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Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 47

NEW THERAPEUTIC ADVANCES IN THE

MANAGEMENT OF ACNE

Vincenzo Bettoli1, Stefania Zauli1, and Annarosa Virgili1

1
Department of Medical Sciences, Section of Dermatology,
University of Ferrara, Arcispedale S. Anna, Ferrara, Italy

ABSTRACT
The treatment of acne continues to be a challenge to practicing clinicians and
dermatologists.
Among the available treatment oral isotretinoin remains the more effective acne
medication and oral antibiotics the more prescribed treatments. Given the restrictions
placed on the use of isotretinoin and the increase in antibiotic resistant strains of P. acnes,
there is a high clinical need for new treatment.
Acne pathogenesis is a complex mechanism in which different factors play a role. In
the recent years the increased knowledge of the acne pathogenesis lead to the
development of new and targeted drugs such as drugs blocking the activation of Toll-like
receptor, PPAR antagonist, inhibitors of IL-1α and leukocyte chemotaxis, the antagonist
of pro-inflammatory cytokines, the inhibitors of the production of reactive oxygen
species and so on. A number of molecules named with abbreviations are studied and
registered in the official sites.
Another problem is the tolerability of the currently available topical. In order to
increase tolerability of these topical and of consequence to improve the patients’
compliance, new vehicles have been tested.
Finally, also a vaccination killed P. acnes may lead to an innovative approach of
acne management.
This indicates that the research in the field of acne is very active and so it is probably
that in the next years several new drugs will hit the market.
The authors propose a review of the emerging treatments in acne field.


Tel.: +39 0532 688129; Fax.: +39 0532 206791; E-mail: [email protected].
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1052 Vincenzo Bettoli, Stefania Zauli and Annarosa Virgili

INTRODUCTION
Acne is a common chronic inflammatory dermatosis that affects more than 85% of
adolescents and approximately 95% of the population [1].
The increased knowledge about the mechanisms of action regulating the development of
acne lesions have stimulated further research in the hope of finding new active drugs [2].
A number of inducing factors, acting on a genetic predisposition, pave the way to the
development of the following pathogenic events which lead to formation of acne lesions:

1) hypercornification of the infrainfundibulum and sebaceous duct,

2) hyperactivity of sebaceous glands and hyperseborrea,
3) hyperactivity of P. acnes,
4) inflammation and immunological host reaction.

Anatomically, the pilosebaceous unit is the cutaneous entity where the above mentioned
events occur.
Hypercornification of the infrainfundibulum has been considered for a long time as the
first of the sequence of events ending in the formation of inflammatory acne. More recent
data have demonstrated the presence of inflammatory cells around the follicle before the
appearance of the microcomedone [3]. As factors inducing hypercornification like androgens,
growth factors, P. acnes and IL-1α may also directly induce inflammation, probably
hypercornification and inflammation concur simultaneously at the start of the process leading
to acne.
Clinically acne lesions are mainly localized on face, back and chest.
During active phase acne vulgaris is characterized by a mixture of different types of non
inflammatory and inflammatory lesions which are concurrently present giving the dermatosis
a polymorphic aspect. Non inflammatory lesions, also called comedones, include open
comedones (blackheads) and closed comedones (whiteheads) depending on the presence or
not of an evident opening to the surface of the skin. Inflammatory acne lesions may be
superficial and relatively small like papules and pustules or deep and larger like nodules.
After the resolution of the acute inflammatory phase erythematous macules, post-
inflammatory hyperpigmentations and post-acne scars can persist. Post-acne scars occur in up
to 90% of the acne patients and they are socially and estetically relevant in 22% of the
sufferers [4].
On the basis of acne severity, current guidelines for acne treatment suggested to use a
combination of topicals (retinoid plus antimicrobial agents) in patients affected with mild-to-
moderate papulopustolar acne and a systemic antibiotic, usually tetracyclines, associated with
topicals in patients with severe papulopustolar/moderate-to-severe nodular acne. Oral
isotretinoin can be used in patients not responsive or relapsed after the use of previous
systemic treatments [5].
Given that many current treatments have potential side effects as teratogenicity of oral
isotretinoin and experience-reduced responsiveness as the increase of antibiotic resistance,
there is a need to develop safer and effective options to treat this common disorder. Moreover
some of the treatments commonly used are contraindicated in certain groups as pregnant
women.

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 New Therapeutic Advances in the Management of Acne 1053

The Centers for Disease Control defined a chronic disease as a disease “that in general
terms, has a prolonged course, that does not resolve spontaneously, and for which a complete
cure is rarely achieved” [6]. As acne vulgaris is characterized by prolonged course, pattern of
recurrences or relapse, manifestation as acute outbreaks or slow onset, psychological and
social impact, according to the aforementioned definition, it can be considered a chronic
disease.
Based on that, it is reasonable to use all the available “tools” to prevent the recurrences of
acne. Maintenance therapy represents an adequate strategy to maintain the achieved
therapeutical results and to minimize the risk of relapse [6].
The management of acne is a problem also in term of costs. In fact there is a tremendous
number of visits made to both generalists and dermatologists for the treatment of acne. The
direct cost of acne treatment is estimated to be more than $1 billion per year and
approximately $100 millions are spent on over-the-counter acne medication [1]. To follow the
authors propose a review of some emerging treatments is acne field.

OXIDATIVE STRESS
The results of a recent study suggest that also oxidative damage may play a role in the
pathogenesis of acne. In fact the serum levels of malondialdehyde and xanthine oxidase
activity in patients with acne vulgaris resulted significantly higher than those of the healthy
controls. A significantly lower superoxide dismutase and catalase activity has been found in
the acne group than in the control group. It shows that in acne patients is present an
alterations in the antioxidant defence system [7].

Fullerene

Fullerene is a spherical carbon molecule with strong radical sponge activity that
penetrates deep into the epidermis. Oxidative stress plays a role in acne formation, suggesting
that oxygen radical scavengers are potential therapeutic agents. In a vitro study, fullerene
significantly decreases sebum production by 27.4%, suggesting another possible pathway of
fullerene’s effect on acne [8]. On the other hand, fullerene seems not to have antibacterial
activity against P. acnes.
In an open trial including 11 patients with mild to moderate acne, fullerene 1% gel was
given twice a day for 8 weeks [8]. At the end of the treatment comedos’ number decreased
only in 3 patients and this reductions was not statistically significant. Inflammatory lesions
decreased in a statistically significant way in 9 patients. Also pustules decreased to 87.6%
indicating that fullerene strongly suppresses neutrophil infiltration, possibly through its
potential antioxidant effect on skin cells.
No apparent side effects were experienced by all patients.
The authors concluded that fullerene has a mild effect on acne and it could be used as a
skin care product for acne patients.
Recently, fullerenol, a novel polyhydroxylated fullerene, with many hydroxyl groups
capable of potent radical-scavenging activity has been developed. Vitro results suggest that
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1054 Vincenzo Bettoli, Stefania Zauli and Annarosa Virgili

fullerenol could be a beneficial skin care reagent for controlling acne vulgaris by suppressing
sebum in the inflammatory response and by reducing P. acnes lipase activity [9].

Taurine Bromamine

Taurine bromamine (TauBr) is a haloamine generated by eosinophil and neutrophils at

the site of inflammation, that has anti-inflammatory and anti-oxidant properties [10]. In fact
TauBr reacts and inactivates H2O2, induces the synthesis of heme oxygenase-1 (a stress
inducible enzyme with anti-inflammatory and anti-oxidant capacity), suppresses the
productions of cytokines and chemokines, and inhibits the activation of NF-kB. Moreover
TauBr has strong microbicidal activity and, at non-cytotoxic concentrations, is able to kill P.
acnes. In a double blind trials 3.5 mM TauBr cream was evaluated versus 1% clindamycin gel
[11]. Both were applied twice-a-day for 6 weeks. Forty patients affected by mild to moderate
inflammatory facial acne were enrolled. After 6 weeks of treatment, comparable reductions of
acne lesions, 65% and 68%, were observed respectively in the TauBr and clindamycin
groups. No adverse events were observed. The authors support the concept that TauBr can be
used in the treatment of acne, especially in patients who are already developed antibiotic
resistance.

ANTI-MICROBIAL MOLECULES
P. acnes, a gram-positive anaerobic bacterium, attaches to pilosebaceous gland wall and
secretes an extracellular polysaccharide substance, the biofilm. This biofilm acts as a barrier
between the cell populations underlying and the exterior environment. P. acnes is the major
contributor of the inflammation as it releases chemotactic factors, pro-inflammatory
cytochines and corticotrophin releasing hormone (CRH), increases the expression and
activation of Toll-like receptors, produces lipases and Reactive Oxygen Substances (ROS),
stimulates lipogenesis, lipid peroxidation and comedogenesis, drives to production of
enzymes such as metalloproteases.
Moreover membrane fractions of P. acnes could act as superantigens, amplifying the
inflammatory reaction [12-16].
Due to the important role of P. acnes in the pathogenesis of acne, antibiotics have
frequently used in the treatment of acne.
Among topicals clindamycin and erythromycin are the most popular. Other types of
topical antibiotics are available in different countries according to the local national licences,
such as nadifloxacin, tetracyclines, clarithromycin and azithromycin. Among systemic
antibiotics cyclines and macrolides are the most used. Recently a decrease in term of efficacy
of these antibiotics, erythromycin in particular, have been detected due to the development of
antibiotic-resistant propionibacteria [17].
The management of antibiotic-resistant, and of consequence how to used antibiotics in
acne treatment, is a delight question. Anyway the presence of propionibacteria resistant to the
common used antibiotics has led to the development of new molecules with antimicrobial
properties.

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 New Therapeutic Advances in the Management of Acne 1055

Picolinic Acid

Picolinic acid is an intermediate metabolite of the amino acid tryptophan that plays a role
in zinc transport [18]. It chelates transition metal ions and is involved in the absorption and
transport of transition metal ions. This molecule seems to work by perturbing zinc binding in
zinc finger proteins and therefore leads to an alteration in chemokine expression. It has
antibacterial properties and also modifies the immune response. In a open-label study
picolinic acid 10% gel was applied twice daily on the face of 15 acne patients over a 12 week
time [18]. A reduction of 58.2% (P < 0.001) in mean total lesion count, 55.5% (P < 0.001) in
mean inflammatory lesion count and 59.7% (P < 0.005) in non-inflammatory lesion count
was seen in this population. No serious adverse events or clinically significant changes in
laboratory values were noted. The anti-inflammatory and antibacterial properties of lauric
acid are already known, whereas recently also the anti-inflammatory and antibacterial
properties of capric acid were investigated. Although to a lesser extent than lauric acid, also
capric acid has bactericidal and anti-inflammatory activities against P. acnes probably
through the inhibition of NF-κB activation and the phosphorylation of MAP kinases [19].

Calcipotriene

Calcipotriene is a vitamin D topical cream that seems to have antimicrobial properties

and comedolytic activity. A study evaluating the effects of calcipotriene on the face and on
the bacteria that cause acne is currently in phase of recruiting participants. In this study
calcipotriene is compared with a placebo cream in a double blind way [20, 21].

Vaccines

Acne vaccine, obtained by inactivated P. acnes, targets a cell wall-anchored sialidase of

P. acnes. Sialidases are thought to be used by P. acnes in order to catabolise
sialoglycoconjugates to obtain sialic acids that ultimately act as substrates for energy
production. It is also probable that sialidase may facilitate the adhesion of P. acnes to
sebocytes [22].
Nakatsuji et al. demonstrated that intranasal immunization of mice with this vaccine
generated in vivo protective immunity against P. acnes. The antibodies elicited attenuated IL-
8 production in human sebocytes but without effect on P. acnes growth. So, the authors
suggested that these antibodies exhibit anti-inflammatory properties sufficient for clinical
improvement but without an antimicrobial effect [23].
The question is whether the results obtained in mice can be translated to the human
model and if these antibodies have the potential to cross-react with other human cells.
Beside these killed pathogen-based vaccines various immunization-based approaches
have been developed over the last decades, including monoclonal antibodies to the Christie,
Atkins, Munch-Peterson factor of P. acnes and anti-Toll-like receptors vaccines.
The role of P. acnes in acne confers legitimacy on the possible benefits of immunization-
based approaches, which may represent a solution for limiting the development of antibiotic-
resistant P. acnes [24].
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Thiazolidinedione Derivatives

Propionibacterium acnes infections are difficult to treat due to the presence of biofilms at
the infection site and the associated resistance towards conventional antimicrobials.
A recent study has clearly demonstrated the effect of two thiazolidinedione derivatives on
Propionibacterium acnes biofilm formation in vitro. The compounds were shown to have a
moderate to strong anti-biofilm activity when used in sub-inhibitory concentrations. These
compounds do not affect P. acnes attachment but lead to increased dispersal of biofilm cells.
This dispersal results in an increased killing of the P. acnes biofilm cells by conventional
antimicrobials [25].

Antimicrobial Peptides

Natural antimicrobial peptides could be considered as a new type of antimicrobial

reagents for several reasons including their relative selectivity towards targets (microbial
membranes), their rapid mechanism of action and, above all, the low frequency in selecting
resistant strains.
Cathelicidines are a family of antimicrobial peptides acting as multifunctional effector
molecules in innate immunity [26]. Cathelicidin-BF has been purified from snake venoms of
Bungarus fasciatus. It is synthesized by GL Biochem (Shanghai) Ltd. In vitro cathelicidin-BF
exerts a rapid antimicrobial activity against P. acnes. Its MIC against two P. acnes strains is
4.7 μg/ml, which is comparable to the anti-P. acnes potential antibiotics of clindamycin (2.3
μg/ml).
Cathelicidin-BF was found to inhibit O2- production induced by P. acnes. As a
consequence it has anti-inflammatory properties because reduce the production of cytokines
(such as IL-8, TNF-alpha, IL-1β and MCP-1) induced by O2-. In vivo anti-inflammatory
effect of cathelicidin-BF was confirmed by relieving P. acnes-induced ear swelling and
granulomatous inflammation in mice [26].
A designed peptide named LZ1, with 15 amino acid residues, contains strong
antimicrobial activity against Propionibacterium acnes. The minimal inhibitory concentration
against three strains of P. acnes was only 0.6 µg/ml, which is 4 times lower than that of
clindamycin. In experimental mice skin colonization model, LZ1 significantly reduced the
number of P. acnes colonized on the ear, P. acnes-induced ear swelling, and inflammatory
cell infiltration. It ameliorated inflammation induced by P. acnes by inhibiting the secretion
of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β.
Combined with its potential bactericidal and anti-inflammatory properties, simple structure
and high stability, LZ1 might be an ideal candidate for the treatment of acne [27].

ProOxy

ProOxy facial spray is a topical 15% Oxygen solution. A phase I open label pilot clinical
trial evaluating the efficacy and safety of ProOxy spray in the treatment of moderate facial
acne has been recently completed. In this study the spray were used twice daily for 3 months
[28].

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 New Therapeutic Advances in the Management of Acne 1057

Povidone-Iodine

A mainstay in the pathogenesis of acne involves the overgrowth and proliferation of skin
micro-organisms, primarily Proprionibacterium Acnes. Long term antibiotic therapy is
usually prescribed for a period of 3 to 6 months or more. Povidone Iodine with its antiseptic
property represents a promising avenue for the elimination of Proprionibacterium Acnes
without the associated problems of long term antibiotic use, and the development of
antibiotic-resistance. It is cosmetically acceptable, affordable, and easy to use. A phase II
study with the aim to evaluate efficacy and safety of 3% Povidone-Iodine cream (Repigel-
Mundipharma Pte Ltd.) versus placebo applied twice a day during 8 weeks has been
registered in official site. Actually this study is not yet open for participant recruitment [28].

ANTI-INFLAMMATORY
Zileuton

Zileuton (Zyflo™) directly inhibits lipogenesis in human sebocytes and blocks the
activity of 5-lipoxygenase, an enzyme involved in the biosynthesis of the pro-inflammatory
lipids leukotriene B4 (LTB4) and prostaglandin-E2 by arachidonic acid. LTB4 is considered to
be a major player in the development of tissue inflammation, and it is also a natural ligand for
PPAR-alpha [29].
In the first pilot clinical study with 10 patients with papulo-pustular acne Zileuton 4 x
600 mg/day per os for 3 months decreased the acne severity index in a time-dependent
manner being 41% of the initial score at week 12 (P < 0.05). This was mostly due to a
decrease of the number of inflammatory lesions, corresponding to 29% (P < 0.01). In addition
total sebum lipids significantly decreased (35%, P < 0.05) [30].
These data were in agreement with a phase II multicentric clinical study including 101
patients with moderate-to-severe inflammatory facial acne, which showed an average
reduction on the total number of lesions of 25.3% in the Zileuton group and 16.4% in the
placebo group [28]. In all studies Zileuton was found to be safe and well tolerate.

Afamelanotide

Afamelanotide is a super potent α-melanocyte-stimulating hormone (α-MSH) analogue

[31]. α-MSH is a melanocortin peptide that increases skin pigmentation during ultraviolet
light-mediated tanning. However, many experimental in vitro and in vivo studies have
demonstrated that α-MSH have also anti-inflammatory and antioxidative properties [32].
It is further reported as α-MSH have anti-bacterial effects against gram-positive bacteria,
but it is unknown if it has antimicrobial effect also against P. acnes [33]. On the other hand,
α-MSH increased lipid synthesis by human sebocytes in vitro [34]. In a phase II open-label
pilot study, afamelanotide 16 mg was given subcutaneously to 3 patients with mild to
moderate facial acne [31]. Two patients received 3 injections at 3-week intervals, one patient
2 injections at 4-week interval. The total lesion number, as well as the number of
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1058 Vincenzo Bettoli, Stefania Zauli and Annarosa Virgili

inflammatory acne lesions, declined in all patients 56 days after the first injection. Total
lesions decreased from 68 to 30 and inflammatory lesions from 46 to 23.7. The number of
non-inflammatory lesions declined dramatically in 2 patients, while in the third patient only a
transient improvement was seen. No adverse events, except mild and short-term fatigue in
one patient, were observed. Important abnormalities in laboratory data were not detected. A
placebo-controlled trial on a much larger number of acne patients will be needed to confirm
the preliminary data and to determinate the optimal dose of afamelanotide capable to
suppressing skin inflammation.

Apremilast

Apremilast is an oral PDE4-inhibitor agent that has been shown to inhibit the production
of TNF-α, IL-8 and neutrophil infiltration. It is usually use to treat different kind of arthritis.
A phase II, open-label, single-arm pilot study evaluating the efficacy and safety of apremilast
in the treatment of moderate to severe acne has been recently completed [28]. In this study 20
mg of apremilast was assumed twice a day for 12 weeks.

Gevokizumab

XOMA 052 (gevokizumab) is a recombinant humanised anti-interleukin 1β antibody that

seem efficacy to treat different kind of disease such as diabetes, arthritis and Behcet’s disease.
It is a sterile solution administered subcutaneously on day 0, 28 and 56.
A phase II, randomized, double-blind, placebo controlled study to evaluate the efficacy
and safety of gevokizumab in subjects with moderate to severe acne is registered and
currently in phase of recruiting participants [28].

Ectopeptidase Inhibitors

Inhibitiors of dipeptidylpeptidase IV and APN stimulate the expression of IL-1 receptor

antagonist, thus they could be expected to reduce primarily comedogenesis and, secondarily,
inflammation [35]. In vitro these inhibitors suppressed proliferation, enhanced terminal
differentiation and slightly decreased total neutral lipid production, and suppressed
proliferation and IL-2 production of P. acnes-stimulated T cells. Moreover the level of IL-1
receptor antagonist results significantly up-regulated.

OTHER
Talarozole

Talarozole (R115866-Rambazole™) is an inhibitor of cytochrome P450-mediated

catabolism of endogenous all-trans retinoic acid in the skin. So it enhances intracellularly the

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 New Therapeutic Advances in the Management of Acne 1059

endogenous levels of all-trans-retinoic acid. By virtue of this property, and the proven
positive effects of retinoids in the treatment of acne, talarozole could potentially be an useful
drug for acne [28].
It was developed by Barrier Therapeutics Inc initially under license from Johnson and
Johnson and after acquired by Stiefel Laboratories Inc (GlaxoSmithKline) [36].
A phase II clinical trial of an oral formulation of talarozole in patients with psoriasis and
acne, and a phase I clinical trial of a topical formulation have been completed [37].
Oral talarozole 1 mg once daily for 12 weeks was assumed by 17 males affected by
moderate to severe acne in a phase II trial. At the end of the treatment a mean reduction in
inflammatory lesion count of 77.4% (P < 0.001), in non-inflammatory lesion count of 58.3%
(P < 0.001) and in total lesion count of 76.0% (P < 0.001) was observed. Nine patients
complained of side effects, mainly eczema and stomach discomfort, but they were mild in
most of the cases. The authors concluded that talarozole is efficacious in acne treatment and
well tolerate [38].
As far as topical formulation is concerned, gels containing talarozole (0.35% and 0.07%)
were applied once daily for 9 days on the buttock of 16 healthy patients in a phase I, double-
blind vehicle-controlled study [39].
Talarozole treatment increased the mRNA expression of cellular retinoic acid binding
protein 2 (CRABP29), cytokeratins (KRT4), CYP26A1 and CYP26B1 dose dependently, and
decreased the expression of KRT2 and IL-1alpha compared with vehicle-treated skin. Both
the two examined dosages showed a low irritancy [39].
An European placebo-controlled Phase II trial evaluating a topical containing talarozole
0.35% administered once-daily for 12 weeks in 80 acne patients has been conducted.
Twenty-five% of patients in talarozole group and 8% in placebo group resulted to be
clear or almost clear [28].

Cortexolone 17α-Propionate

Cortexolone 17α-propionate (cortodoxone) is a new and the first topical anti-androgen

that competes at the human androgen-receptor level. It seems to have also mild anti-
inflammatory properties.
A randomized, double-blind, parallel-group, comparative trial, with placebo and tretinoin
0.05% cream, has been performed to evaluate the safety and efficacy of 8 weeks treatment
with cortexolone 17α-propionate 1% cream applied once a day in acne patients [40].
Seventy-seven patients with mild to moderate facial acne were enrolled in the study.
Cortexolone 17α-propionate 1% cream decreased the total lesion count and inflammatory
lesion count; the improvement was significantly better than placebo. In comparison with
tretinoin, cortexolone 17α-propionate 1% cream was always clinically more effective without
reaching a statistically significant level.
The time to reach 50% improvement showed significant differences among the groups
with a median time of 42.5 days for cortexolone 17α-propionate 1%, 44.0 days for tretinoin
and 57.0 days for placebo.
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A total of 8 subjects (11%) experienced 14 adverse events which were distributed as

follows: 5 in placebo group, 6 in tretinoin group and 3 in cortexolone 17α-propionate group.
None was judged serious to required treatment discontinuation.
A double-blind, placebo-controlled, dose-escalating Phase II trial in 360 patients with
mild-to-moderate facial acne is ongoing to evaluating the safety and efficacy of cortexolone
17α-propionate for 12 weeks [28].

Epigallocatechin-3-Gallate

Epigallocatechin-3-Gallate (EGCG) is the major polyphenolic constituent of green tea

know as a potent anti-carcinogenic, anti-inflammatory, anti-proliferative and anti-microbial
activities. Also anti-androgenic properties have been reported.
In SEB-1 sebocytes EGCG reduces sebum production by modulating AMPK-SREBP-1
signalling pathway and reduces inflammation by suppressing the NK-kB and AP-1 pathway.
Moreover EGCG decreases the viability of P. acnes, thus targeting almost all the pathogenic
feature of acne.
In a 8-week randomized, split-face, clinical trial EGCG improved acne and it is well
tolerated [41]. Tea tree oil gel is also under study in the treatment of mild to moderate facial
acne. In a phase II open label study the gel is applied twice daily for 12 weeks.

Oligonucleotides

Inhibition of the expression of androgen receptor by antisense oligonucleotides reduces in

vitro the enhanced proliferation of sebocytes challenged by testosterone and DHT [42]. They
could be consider a novel strategy for the blockage of the androgen receptor and could be
represent a specific therapeutic approach in androgen-associated acne.

NATURAL PRODUCTS - HOMOEOPATIC MEDICINE

A long list of natural product have been tested in the treatment of acne and in general in
the treatment of cutaneous disease, including green tea, essential oil, various plant extracts,
etc.
Studies on cell lines revealed that flavonoid, alkaloid, essential oil, phenol and phenolic
compound, tannin, xanthone and xanthone derivative, and the bisnaphthquione derivative are
effective in treatment of acne. Animal studies showed that diterpene acid, phenylpropanoid
glycosides, acteoside and flavonoids have anti-inflammatory activity. Eleven human studies
revealed that Camellia sinensis has 5α-reductase inhibitory and anti-inflammatory activities.
Also anti-bacterial effect has been shown by oleoresin of Commiphora mukul [43].
Below we have listed only some of these studies just to underline how the research of
new anti-acne molecules derived from nature is active.

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Resveratrol

Resveratrol is a natural phytoalexin produced by some spermatophytes, such as grapes

and other plants. It exhibits activity against P. acnes as well as anti-inflammatory properties
[44]. In a pilot single-blind study including 20 patients with facial acne resveratrol
incorporated in a carboxymethylcellulose-based gel was applied daily on the right side of the
face for 60 days.
As control the hydrogel vehicle was applied to the left side [44]. Clinical evaluation
showed a reduction in the acne score of 53.47% on the resveratrol-treated sides and of 6.10%
on the vehicle-treated sides. No adverse events were registered. The authors concluded that
resveratrol could be a valid alternative in acne treatment. This effectiveness should be tested
at different concentration and formulation, and in a larger group of patients.

Curcumin

Curcumin in the vehicles significantly inhibited the growth of P. acnes in the skin when
evaluated by the bioluminescence assay [45].

Ethanolic Rosemary Extract

Ethanolic rosemary extract (ERE) has been showed to significantly suppress the secretion
and mRNA expression of pro-inflammatory cytokines, including interleukin (IL)-8, IL-1β,
and tumor necrosis factor-α in P. acnes-stimulated monocyte THP-1 cells.
In vivo mouse model intradermal injection of ERE attenuated the P. acnes-induced ear
swelling and granulomatous inflammation. Further studies are needed to explore the role of
bioactive compounds of rosemary in mitigation of P. acnes-induced inflammation [46].

Essential Oil and Aromatherapy

A randomized controlled trial involving 192 participants divided in 3 groups (treatment

with essential oil-aromatherapy-wait list control) has been provided high-quality evidence of
the effectiveness of essential oil and aromatherapy in the treatment of acne [47].

Marine-Derived Ingredients

Potential benefits may be offered also by natural, marine-derived ingredients such as

those derived from brown seaweed (Laminaria digitata) and a novel seaweed oligosaccharide-
zinc complex (SOZC) [48].
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Homoeopatic Medicine

The efficacy of some lesser known homoeopatic medicines in acne treatment has been
recent assessed in open label phase I studies. Zingiber officinalis are pills to assumed 4 times
a day for 7 days. The same is for azadurachta indica and lappa arctium [28].

INSTRUMENTAL TREATMENT
Headheld Heat Device

A phase 4 open label study comparing a popular handheld heat devised and a topical
treatment with BPO 4% in the treatment of individual acne lesions is currently registered
under the sponsoritation of the University of British Columbia [28].

Acleara Needle Insert

A open-label study assessing the efficacy of Acleare Needle Insert in acne vulgaris has
been completed. Fifteen subjects were enrolled. Each subject could have up to 5 lesions
treated with this system and received up to 3 follow up visits [28].

Clear Device

Clear device is a light based devise. A clinical research, involving 50 patients, assessed
the efficacy of this device to treat mild to moderate inflammatory acne and determined if the
patients are able to use the device properly.
Two sessions a week for 4 weeks, for a total of 8 sessions, and 2 follow-up visits were
performed. The patients treated themselves with the clear device.
The average improvement of acne lesions was 56.7% after one month and 57.7% after
three months [28].

KLOX Biophotonic System

An opel label split face phase III study evaluating KLOX Biophotonic System versus no
treatment in moderate to severe acne is ongoing. This system provides the application of
KLOX KLGA0105-01 photo-convert gel and after the use of KLOX THERA lamp.
This procedure is performed twice a week for 6 weeks followed by a 6 week follow up
period [28].

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PhotoPneumatic System

The PhotoPneumatic System includes the use of intense pulsed light in combination with
vacuum technology. This system has been used to treat mild to moderate acne in 20 adult
patients in a prospective observational spilt-face study.
A significant reduction in the number of acne lesions were observed on the treated side of
the faces. Most patients experienced global clinical improvement.
No severe side effects occurred during the study, with only a few patients experiencing
transient erythema, purpura and/or exacerbation of pre-existing acne [49].

Photodimanic Therapy

Recently PDT with a photosensitizer, 5-aminolevulanic acid (ALA) or methyl

aminolevulinate (MAL), has been proven useful in the management of inflammatory acne
[50].
Twenty-four subjects with acne on both sides of the face were included in a study with
the aim to evaluate the clinical efficacy and safety of chlorophyll-a photodynamic therapy.
Eight treatment sessions were performed over a 4-week duration. Half of the face was
irradiated using a blue and red light-emitting diode after topical application of chlorophyll-
lipoid complex. The other half underwent only light-emitting diode phototherapy. On the
chlorophyll-a photodynamic therapy-treated side a significant reduction in acne lesion counts,
acne severity grades, and sebum levels were observed in comparison with the side treated
with light-emitting diode phototherapy alone. The side effects were tolerable in all the cases
[51].
Two studies using as photosensitizier respectively lemuteporfin topical solution 15 and
topical visonac have been registered.
More over a study comparing PDT with a conventional acne treatment with topical
adapalene gel 0.1% for 12 weeks plus doxycycline 100 mg/day for three months is ongoing
[28].

Botulin

A phase II and a phase III double-blind randomized studies evaluating the efficacy and
safety of the botulinum neurotoxin type A injections in subjects with acne has been
terminated.
This approach already known in the treatment of post-acne scars is so currently proposed
also in the treatment of active acne lesions [28].

Ultrasound Device

Currently two pilot open label studies assessing, in a small group of subjects, the efficacy
of an ultrasound device in acne treatment have been registered. One has been already
completed whereas the other one is in phase of recruiting of participants.
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1064 Vincenzo Bettoli, Stefania Zauli and Annarosa Virgili

The experimental ultrasound device uses sound waves to heat the acute acne lesion and
the surrounding sebaceous gland without affecting the surface of the skin. The authors hope
that heating the sebaceous glands will reduce their size and the symptoms of inflamed acne
[28].

Plasma Treatment System

A single center pilot study evaluating the efficacy and safety of the antimicrobial plasma
treatment system to treat back acne is ongoing. Plasma is applied to back for up to 20 minutes
twice/week for 4 weeks [28].

Silk'n Blue Device

The Silk'n Blue device has an array of 24 LEDs emitting a spectrum of light in the blue-
violet range of light (405-460 nm). This light based technology seems safe and efficacious as
home device for the treatment of mild to moderate inflammatory acne vulgaris.
The data were obtained by a study involving 70 subjects that have been received eight
treatments with the Silk'n Blue device over a 4-week period. There was a statistically
significant decrease in mean acne counts from baseline through the follow-up visits (p =
0.002).
The 36.4% of the patients have also obtained a complete clearance with the studied
device [52].

Low-Level Laser Therapy (Erchonia EML)

Recent evidence indicates that low-level laser therapy (LLLT) is able to significantly
diminish the expression of COX-2, resulting in the reduction of inflammation.
The ability to modulate the COX-2 pathway via LLLT is believed to inhibit the
production of pro-inflammatory cytokines (i.e., TNF-α and IL- α) present in acne-prone skin.
The Erchonia® EML Laser is a dual-diode 7 mW laser of 635 nm and 405 nm
wavelength. The light emitting diode is manufactured by Coherent and classified by the
Center for Devices and Radiological Health (CDRH) as a Class IIIb laser diode.
A pilot study assessing the efficacy of this system to reduce the number of inflammatory
and non-inflammatory acne lesions has been completed [28].

CONCLUSION
The treatment of acne continues to be a challenge to practicing clinicians and
dermatologists. Among the available treatments oral isotretinoin remains the more effective
acne medication and oral antibiotics the more prescribed treatments. Given the restrictions
placed on the use of isotretinoin and the increase in antibiotic resistant strains of P. acnes,

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there is a high clinical need for new treatments. Acne pathogenesis is a complex mechanism
in which different factors play a role. In the recent years the increased knowledge of the acne
pathogenesis have led to the development of new and targeted drugs overall with anti-
microbial and anti-inflammatory properties. In order to reduce the development of antibiotic
resistant P. acnes also acne vaccine, obtained by inactivated P. acnes, may represent an
innovative approach of acne management in the future. A number of molecules named with
abbreviations, therefore unidentifiable, are studied and registered in the official sites. So, it is
probably, that in the next years several new drugs will hit the market. Finally, various
instrumental approaches have been tested in the treatment of acne range from the use of
different kind of lights to mechanical insults such as ultrasound or needle insert.
All this indicates that the research in the field of acne is very active.
In the future it is likely that other new treatments will continue to appear as our
knowledge of the pathogenic mechanism of acne are in continuous improvement providing an
always wider array of therapeutic targets. Prospective new acne treatment may be represent
by drugs blocking the activation of Toll-like receptor, PPAR antagonist, inhibitors of IL-1α
and leukocyte chemotaxis, antagonist of pro-inflammatory cytokines, inhibitors of the
production of reactive oxygen species, etc. Nanotechnology may facilitate follicular targeting
of such treatments.
Some studies investigating dietary interventions are also registered. So, it is also probably
that in the next years beside to a pharmacological prescription, a modification of patients’
lifestyle could be required.

REFERENCES

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[8] Inui S., Aoshima H., Nishiyama A., et al. Improvement of acne vulgaris by topical
fullerene application: unique impact on skin care. Nanomedicine, 2011; 7: 238-41.
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[9] Inui S., Aoshima H., Ito M., et al. Inhibition of sebum production and
Propionibacterium acnes lipase activity by fullerenol, a novel polyhydroxylated
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[10] Marcinkiewicz J. Taurine bromamine (TauBr)-its role in immunity and new
perspectives for clinical use. J. Biomed. Sci., 2010; 17 Suppl. 1: S3.
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new candidate in the treatment of moderate inflammatory acne vulgaris: a pilot study.
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[12] Pivarcsi A., Bodai L., Rethi B., et al. Expression and function of Toll-like receptors 2
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[15] Isard O., Knol A., Castex-Rizzi N., et al. Cutaneous induction of corticotrophin
releasing hormone by propionibacterium acnes extracts. Dermato-Endocrinology, 2009;
1: 96-99.
[16] Kim J., Ochoa M. T., Krutzik S. R., et al. Activation of toll-like receptor 2 in acne
triggers inflammatory cytokine response. J. Immuno., 2002; 169: 1535-41.
[17] Simonart T., Dramaix M. Treatment of acne with topical antibiotics: lessons from
clinical studies. Br. J. Dermatol., 2005; 153: 395-403.
[18] Heffernan M. P., Nelson M. M., Anadkat M. J. A pilot study of the safety and efficacy
of picolinic acid gel in the treatment of acne vulgaris. Br. J. Dermatol., 2007; 156:
548-52.
[19] Huang W. C., Tsai T. H., Chuang L. T., et al. Anti-bacterial and anti-inflammatory
properties of capric acid against Propionibacterium acnes: a comparative study with
lauric acid. J. Dermatol. Sci., 2014; 73: 232-40.
[20] Youssef D. A., Miller C. W., E-Abbassi A. M., et al. Antimicrobial implications of
vitamin D. Dermatoendocrinol., 2011; 3: 220-9.
[21] Nieves N. J., Ahrens J. M., Plum L. A., et al. Identification of a unique subset of 2-
methylene-19-nor analogs of vitamin D with comedolytic activity in the rhino mouse. J.
Invest. Dermatol., 2010; 130: 2359-67.
[22] Nakatsuji T., Liu Y. T., Huang C. P., et al. Vaccination targeting a surface sialidase of
P. acnes: implication for new treatment of acne vulgaris. PLoS One, 2008; 3: 1551.
[23] Nakatsuji T., Liu Y. T., Huang C. P., et al. Antibodies elicited by inactivated
propionibacterium acnes-based vaccines exert protective immunity and attenuate the
IL-8 production in human sebocytes: relevance to therapy for acne vulgaris. J. Invest.
Dermatol., 2008; 128: 2451-7.
[24] Simonart T. Immunotherapy for acne vulgaris: current status and future directions. Am.
J. Clin. Dermatol., 2013; 14: 429-35.
[25] Brackman G., Forier K., Al Quntar A. A., et al. Thiazolidinedione derivatives as novel
agents against Propionibacterium acnes biofilms. J. Appl. Microbiol., 2013; doi:
10.1111/jam.12378.
[26] Wang Y., Zhang Z., Chen L., et al. Cathelicidin-BF, a snake cathelicidin-derived
antimicrobial peptide, could be an excellent therapeutic agent for acne vulgaris. PLoS
One, 2011; 6: 22120.

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 New Therapeutic Advances in the Management of Acne 1067

[27] Zhang Z., Mu L., Tang J., et al. A small peptide with therapeutic potential for
inflammatory acne vulgaris. PLoS One, 2013; 8: e72923.
[28] clinicaltrials.gov.
[29] Zouboulis C. C. Zileuton, a new efficient and safe systemic anti-acne drug.
Dermatoendocrinol., 2009; 1: 188-92.
[30] Zouboulis C. C., Nestoris S., Adler Y. D., et al. A new concept for acne therapy: a pilot
study with zileuton, an oral 5-lipoxygenase inhibitor. Arch. Dermatol., 2003; 139:
668-70.
[31] Böhm M., Ehrchen J., Luger T. A. Beneficial effects of the melanocortin analogue
Nle4-D-Phe7-α-MSH in acne vulgaris. J. Eur. Acad. Dermatol. Venereol., 2014; 28:
108-11.
[32] Brzoska T., Luger T. A., Maaser C., et al. Alpha-melanocyte-stimulating hormone and
related tripeptides: biochemistry, antiinflammatory and protective effects in vitro and in
vivo, and future perspectives for the treatment of immune-mediated inflammatory
diseases. Endocr. Rev., 2008; 29: 581-602.
[33] Cutuli M., Cristiani S., Lipton J. M., et al. Antimicrobial effects of alpha-MSH
peptides. J. Leukoc. Biol., 2000; 67: 233-9.
[34] Zhang L., Anthonavage M., Huang Q., et al. Proopiomelanocortin peptides and
sebogenesis. Ann. N Y Acad. Sci., 2003; 994: 154-61.
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aminopeptidase N target major pathogenetic steps in acne initiation. J. Invest.
Dermatol., 2007; 127: 1042-51.
[36] Geria A. N., Scheinfeld N. S. Talarozole, a selective inhibitor of P450-mediated all-
trans retinoic acid for the treatment of psoriasis and acne. Curr. Opin. Investig. Drugs,
2008; 9: 1228-37.
[37] Geria A. N., Scheinfeld N. S. Talarozole, a selective inhibitor of P450-mediated all-
trans retinoic acid for the treatment of psoriasis and acne. Curr. Opin. Investig. Drugs,
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[38] Verfaille C. J., Thissen C. A., Bovenschen H. J., et al. Oral R115866 in the treatment of
moderate to severe plaque-type psoriasis. J. Eur. Acad. Dermatol. Venereol., 2007; 21:
1038-46.
[39] Pavez Loriè E., Cools M., Borgers M. et al. Topical treatment with CYP26 inhibitor
talarozole (R115866) dose dependently alters the expression of retinoid-regulated genes
in normal human epidermis. Br. J. Dermatol., 2009; 160: 26-36.
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new potent antiandrogen for topical treatment of acne vulgaris. A pilot randomized,
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Dermatol., 2011; 165: 177-83.
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antisense oligonucleotides regulates the biological activity of androgens in SZ95
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[43] Azimi H., Fallah-Tafti M., Khakshur A. A., et al. A review of phytotherapy of acne
vulgaris: perspective of new pharmacological treatments. Fitoterapia, 2012; 83:
1306-17.
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Dermatol., 2011; 12: 133-41.
[45] Liu C. H., Huang H. Y. In vitro anti-propionibacterium activity by curcumin containing
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Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 48

A LARGE-SCALE EUROPEAN OBSERVATIONAL

STUDY TO DESCRIBE THE MANAGEMENT OF ACNE
IN CLINICAL PRACTICE

S. Seité1* and B. Dreno2

1
La Roche-Posay Pharmaceutical Laboratories, Asnières, France
2
Hôtel Dieu, Nantes, France

ABSTRACT
Background: Acne is one of the major reasons that patients consult a dermatologist.
Current recommendations for the treatment of juvenile facial acne suggest treating mild
acne with topical treatments and moderate acne with a combination of topical treatments
with systemic antibiotics. The aim of this investigational survey was to evaluate how
European dermatologists in private practice currently manage acne.
Method: Dermatologists practicing in 12 European countries were asked how they
manage patients with acne (except those undergoing isotretinoin treatment). Each
dermatologist completed a written questionnaire, about patient characteristics, acne
severity and the therapy they prescribed at baseline and after 2 months of treatment.
Results: In total, 5809 acneic patients were questioned. In 40% of cases
(independent of severity), dermatologists prescribed up to 3 local treatments combined
with up to 2 systemic therapies, and a cosmetic product. In 44% of cases, dermatologists
prescribed only a dermocosmetic product for very mild acne; in 44% of cases of mild
acne they prescribed one treatment, mostly topical one and in 48 and 58% of cases two
treatments (mainly a combination of local and systemic therapy) to patients with
moderate or severe acne respectively.
Conclusion: This observational study illustrates that dermatologists employ complex
treatment regimens to manage acne. Seeing as complex regimens are harder for patients
to comply with, this notably raises the question of adherence, which is a key factor in
successful treatment.

*
Correspondence: S. Seite, Ph.D., La Roche-Posay Pharmaceutical Laboratories, 110 Avenue Henri Barbusse,
92602 Asnières Cedex, France. Phone: + (33) 1.46.88.65.44; Fax: + (33) 1.46.88.29.22; E-mail:
[email protected].
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1070 S. Seité and B. Dreno

Keywords: Acne, management, severity of acne, tolerance, adherence

BACKGROUND
Acne is one of the most frequent reasons patients consult in dermatology (20% of
consultations) and affects approximately 80% of adolescents. Acne interferes with quality of
life, and requires both therapeutic and psychological support [1, 2]. Therapeutic management
must balance efficacy and tolerance, otherwise defined as benefit/risk. The treatment period
can be long as acne is a chronic inflammatory disease and the majority of treatments have a
latent effect. Patient compliance/adherence is imperative to obtain a therapeutic success [3,
4]. Published guidelines and recommendations, review scientific and medical advances and
guide practitioners on the appropriate use of different pharmaceutical products. The
recommendations of the Global Alliance in 2003, updated in 2009 [5, 6] led to an algorithm
for the management of acne [7] and more recently the French group published
recommendations for juvenile acne [8]. These algorithms were based upon the published
literature, National Health Authority recommendations, expert experience in the field, and
also regulatory changes concerning the different treatments. They provide therapeutic
markers for the daily use of anti-acne medication. These guidelines therefore evolve regularly
over time [9, 10] to keep in line with current research. These recommendations have led to
changes in prescribing habits that reflect scientific research in areas such as bacterial
resistance or side effects [11]. additionally, although not included in the treatment algorithms,
dermocosmetic products are often prescribed as part of acne treatment regimens [12].

QUESTIONS ADDRESSED
In this context, it seemed interesting to perform a survey to assess the implementation of
these algorithms in daily practice of European dermatologists for patients with acne. For each
level of acne severity, we set out to evaluate the number and type of medical treatments
prescribed in addition to a dermocosmetic product.

EXPERIMENTAL DESIGN
This observational study was conducted with dermatologists in private practice in 12
European countries including France, Spain and Portugal (Western countries), Poland,
Slovakia, Hungary, Romania and Croatia (Eastern countries) and Germany, Switzerland, Italy
and Slovenia. Patients, with acne, of both sexes, aged seven years or older were invited to
participate. Patients undergoing isotretinoin treatment were excluded. At baseline,
dermatologists were asked to complete a questionnaire to obtain information concerning the
patient profile (age, sex, skin type, age of acne), and clinical severity, using the Global
Evaluation Acne scale (GEA) [13]. Lastly, the prescribed treatment regimen was noted, and
dermatologists were asked to prescribe the dermocosmetic (Effaclar Duo® (La Roche-
Posay)). During the second visit, planned 2 months later, dermatologists re-evaluated the acne

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 A Large-Scale European Observational Study ... 1071

severity, and the overall tolerance and efficacy of treatment prescribed. During these 2 visits,
seborrhea was evaluated with a 10 cm visual analogic scale (VAS).
Data analysis was performed on all patients included for whom both inclusion and final
questionnaires were completed by dermatologists. Some analysis was also done for GEA
grade sub-groups. Variables were expressed by number of subjects (N), percentage (%),
quantitative data by mean ± SD (min - max) and median.

RESULTS
The baseline characteristics of the 5809 patients, with grade 1 to grade 4 acne, questioned
during this survey are shown in Table 1. Most of the patients were female (66%) and 34%
were male, with a mean age of 21 ± 7 years (7-97 years), the average length of time since
their acne was diagnosed was 25 ± 31 months (0.5 to 384 months) and 82% had skin type II
or III. The presence of lesions on the trunk was noted in 30% of the total population.

Table 1. Patient characteristics and degree of

acne severity (GEA scale) [14]

GEA grade Gender Age lesions on the

(n=5763) trunk
F M <20 ≥20 Yes No
Grade 1 (n=663/11.5%) 71% 29% 56% 44% 6% 94%
Grade 2 (n=2494/43.3%) 71% 29% 56% 44% 19% 81%
Grade 3 (n=2332/40.5%) 61% 39% 59% 41% 43% 57%
Grade 4 (n=274/4.8%) 50% 50% 65% 35% 66% 34%

PRESCRIPTION THERAPY
Dermatologists prescribed the dermocosmetic product alone, without supplementary
medical treatments for 15% of the total population. Otherwise, prescribing habits were
complex, particularly for young patients (sometimes up to 6 different treatments prescribed).
No correlation was noticed between the number of treatments prescribed and patient age.
In 35% of cases, dermatologists prescribed up to 3 topical treatments. In 9% of cases, up
to 2 systemic treatments and in 40% of cases, they prescribed up to 3 local treatments
associated with one or 2 systemic therapies.
Prescriptions made in western countries differed from those in Eastern countries (Table
2).
Eastern dermatologists prescribed twice the amount of local therapies alone than western
dermatologists. Conversely, western dermatologists preferred combining topical and systemic
treatments.
Therapeutic regimens prescribed in accordance with the GEA grade are presented in
figures 1 to 4.
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1072 S. Seité and B. Dreno

For patients with virtually no lesions or grade 1 acne, according to the GEA scale (Figure
1), 44% of prescriptions contained only the dermocosmetic product, with no additional
medical treatment. If they prescribed a drug treatment, a topical one was preferred (42%). The
top 3 topical treatments prescribed were: a topical antibiotic, a topical retinoid or benzoyl
peroxide (BPO).

Table 2. Percentage of patients according to

the different treatment regimens

Prescription (n=5809) % n Western Eastern

countries countries
(n=3609) (n=1779)
Systemic only 9% 513 9% 7%
Local only 35% 2036 27% 52%
Local + systemic 41% 2364 52% 23%
No treatment (dermocosmetic only) 15% 896 12% 18%

For patients with mild or grade 2 acne (Figure 2), the dermocosmetic product alone was
prescribed for 18% of prescriptions. However, in most cases (44%), dermatologists prescribed
one drug treatment, either a local antibiotic, a topical retinoid, or BPO. In 31% of cases, they
prescribed two treatments; mostly a combination of a topical with a systemic treatment, most
frequently a retinoid with a cycline. However combination therapy was mix of various
products (a cycline associated with BPO or with BPO plus a retinoid). And for 6% an
association of three treatments was prescribed, essentially the association of two topical with
one systemic treatment.

Figure 1. Regimen prescribed for Grade 1 patients (n=658).

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 A Large-Scale European Observational Study ... 1073

Figure 2. Regimen prescribed for Grade 2 patients (n=2486).

For patients with moderate or grade 3 acne (Figure 3), 6% of dermatologists prescribed
the dermocosmetic product alone. Two-thirds (68%) of the patients were treated with
combined therapy (2 to 6 associated treatments). Cyclines with retinoids or BPO were the
preferred two associated prescriptions for these patients. A quarter of grade 3 patients
received only one treatment, which was often a topical antibiotic (17%) or a cycline (27%).
For patients with severe or grade 4 acne (Figure 4), only 2% of prescriptions contained a
dermocosmetic prescription alone. More than half of the patients were treated with
combination therapy, most often consisting of a topical and a systemic drug treatment. As for
grade 3, cyclines were associated with either retinoid or BPO alone or associated with
Adapalene.

Figure 3. Regimen prescribed for Grade 3 patients (n=2324).

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1074 S. Seité and B. Dreno

Figure 4. Regimen prescribed for Grade 4 patients (n=273).

SECOND VISIT (58 ± 17 DAYS AFTER)

All therapeutic regimens prescribed were efficacious as acne improved for 77% of
patients at the second visit (reduction of one grade or more) (p<0.0001) (Table 4). We also
noticed a decrease in the level of seborrhoea in 89% of cases (p<0.0001) (Table 3). For 94%
of patients, dermatologists found it worthwhile to combine a drug treatment with a cosmetic
product to improve patient comfort and tolerance while maintaining and/or enhancing its
effectiveness. Moreover, 86% of patients were satisfied with the efficacy of the cosmetic
product prescribed. Dermatologists noted the tolerance of treatments as “good” or “excellent”
for 93% of patients and for 90% of patients by auto-evaluation.

Table 3. Clinical progress (GEA Grade) and seborrhoea score between baseline and
Visit 2

No improvement (same grade) 22%

Improvement of 1 grade (reduction of 1 grade) 56%
Improvement of 2 grades 20%
Improvement of 3 grades 1%
Deterioration of 1 grade 1%

Baseline 2nd visit

Mean ± SD Mean ± SD
Seborrhea score (VAS 0-10) 4.6 ± 1.8 2.6 ± 1.5

DISCUSSION AND CONCLUSION

This survey evaluated the current management of 5809 European patients with acne by
dermatologists in private practice. The results provide a better understanding of patient
characteristics related to their acne severity and confirms the value, and ease of use of the

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 A Large-Scale European Observational Study ... 1075

GEA scale ("Global Evaluation Acne Scale") [13]. For example, the presence of acne lesions
on the trunk was more frequent for severer grades (66% versus 6%)(Table 1). For severer
grades, males consulted more often than females and in contrary, for milder grades, females
consulted more often than males (Table 1). This was perhaps either due to consultations for
aesthetic reasons, or because the adult female population included some patients with
premenstrual acne syndrome. This is supported by the fact that grades 2 and 3 acne are
diagnosed for more than twice as many women, aged 30 years or more, than men.
This study indicated that European dermatologists do not usually prescribe medical
treatments to treat grade 1 acne (in 44% of cases, they prescribe only application of the
dermocosmetic product to patients with very mild acne); one drug treatment, usually a topical
one to treat grade 2 acne (in 44% of cases, one treatment was prescribed to patients with mild
acne) and two treatments (a combination of topical and systemic therapy) to those with grades
3 or 4 acne (in 48 and 58% of cases two treatments were prescribed to patients with moderate
or severe acne respectively) (Figures 1-4). We also noticed that in 40% of cases,
dermatologists prescribed several topical treatments (up to 3) associated with one or several
systemic therapies (up to 2), as well as at least one cosmetic product (Table 2). The
complexity of prescribing habits, particularly for young patients raises the question of
adherence, which is a key factor for a successful treatment. Interestingly, data of the evolution
of the clinical condition between the 2 visits (Table 4) seems to indicate that increasing the
number of treatments in mild to moderate acne doesn’t add any extra benefit.

Table 4. Clinical Progress for Grade 2 and 3 patients (n= 2441 / 2266)

Therapy / Grade 2 % of patients GEA (Tf-Ti)*

No treatment 18% -0.96 ± 0.64
1 treatment 44% -0.85 ± 0.62
2 treatments 31% -0.79± 0.64
3 treatments 6% -0.84 ± 0.68

Therapy / Grade 3 % of patients GEA (Tf-Ti)*

No treatment 6% -1.36 ± 0.78
1 treatment 25% -1.32 ± 0.71
2 treatments 48% -1.23 ± 0.66
3 treatments 15% -1.08 ± 0.68
4 treatments 5% -1.07 ± 0.61

This study also demonstrated that the therapeutic regimens prescribed were efficacious
(acne improved for 77% of patients (reduction of one grade or more at the second visit)(Table
3)) and generally well tolerated, although these regimens are somewhat complex and differ
from country to country (Table 2),. Differences seen between eastern and western
dermatologist’s prescriptions were perhaps due to educational reasons, GDP level or problem
of reimbursement.
These data highlight certain points that contradict the published recommendations [5,6],
indicating that dermatologists should not use antibiotics alone and that it is necessary to
favour topical treatments with a single application a day to ensure patients use the treatment
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1076 S. Seité and B. Dreno

regularly. The development of bacteriological resistance and ensuing recommendations from

health authorities limited the use of local and systematic antibiotics, favouring their use
within the framework of combined therapy regimens [7,11], Nevertheless, we noted in 12%
of cases (687 prescriptions) only one local or systematic antibiotic was prescribed.
Finally, this study confirms that dermatologists prescribe dermocosmetic skincare as an
integral part of acne management. A dermocosmetic product was prescribed alone for 44% of
the patients with very mild acne, 18% of the patients with mild acne and 6% of patients with
moderate acne. In 94% of cases, the dermatologists preferred to associate a dermocosmetic
with their drug treatment to improve the tolerance of treatments, for patient comfort whilst
maintaining and/or strengthening the overall efficacy of the regimen.
In conclusion, this study shows that European dermatologists frequently prescribe several
treatments for acne, which is unfavourable for observance particularly for young patients with
acne. Furthermore, in spite of published recommendations, local or systematic antibiotics are
still often prescribed alone. Finally, a dermocosmetic product can be associated with
prescription treatment, and may even provide good results in managing mild acne.

ACKNOWLEDGMENTS
We would like to thank all the European dermatologists who participated in this survey,
Martine Fortuné and Guénaëlle Le Dantec for their administrative support and Catherine
Delva (SYLIA-STAT France) for the statistical analysis. We acknowledge editing assistance
provided by Amy Whereat (Speak the Speech Consulting, Paris, France).

Conflict of interest: This observational study was sponsored by La Roche-Posay. Pr B.

Dreno is consultant for La Roche-Posay and Sophie Seité employee of La Roche-Posay
Pharmaceutical Laboratories.
Funding: This article was funded by La Roche-Posay Pharmaceutical Laboratories, France.

REFERENCES
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Alliance to Improve Outcomes in Acne. Large-scale worldwide observational study of
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[4] Dreno B, Daniel F, Allaert FA, Aube I. Acne: evolution of the clinical practice and
therapeutic management of acne between 1996 and 2000. Eur J Dermatol 2003;13:166-
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[5] Gollnick H, Cunliffe W, Berson D, Dreno B, Finlay A, Leyden JJ, et al. Global Alliance
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therapeutic management of acne between 1996 and 2000. Eur J Dermatol 2003;13:166-
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[11] Dréno B, Bettoli V, Ochsendorf F, Perez-Lopez M, Mobacken H, Degreef H, et al. An
expert view on the treatment of acne with systemic antibiotics and/or oral isotretinoin in
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[12] Poli F. Acné : les soins d'hygiène. Ann Dermatol Venereol 2003; 130:148-50.
[13] Dréno B, Poli F, Pawin H, Beylot C, Faure M, Chivot M, et al. Development and
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 49

SKIN AGING

Samira Yarak1 and Carolina A. Pontes da Silva2

1
Federal University of São Francisco Valley and
Federal University of São Paulo, Brazil
2
Federal University of São Paulo, Brazil

ABSTRACT
Skin aging is a complex process that involves intrinsic and extrinsic factors. The age
at which it begins varies and is influenced by genetics, hormones and the environment.
Intrinsic or chronological aging is an ongoing process resulting from decreased
collagen and elastin production in the dermis. Hormonal changes, such as alterations in
estrogen levels, may be related to intrinsic aging.
Extrinsic aging is mainly due to the cumulative effect of ultraviolet radiation on the
skin, which causes elastosis in the skin, appendages and blood vessels. In addition to the
sun, other factors, such as smoking and gravity, contribute to the extrinsic aging process.
Skin aging is characterized by mild skin atrophy or deep rhytids, facial areas with
muscle hypertrophy and changes in the distribution of subcutaneous tissue, telangiectasia,
solar melanosis, poikiloderma and the development of premalignant and/or malignant
skin lesions.
The literature describes several therapeutic approaches to treating intrinsic and
extrinsic skin aging, including the use of sunscreens, antioxidants, chemical peels, lasers
and intense pulsed light.
Despite advances in research, nothing has succeeded in preventing the aging process
in skin. However, it has become clear that avoiding smoking and ultraviolet radiation can
reduce skin aging.

INTRODUCTION
Medical advances have resulted in an increased life expectancy in both developed and
developing countries. Consequently, the prevalence of diseases associated with aging has
increased, and the clinical and biological understanding of the aging process has improved.
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1080 Samira Yarak and Carolina A. Pontes da Silva

Aging is an irreversible process that affects both the skin and the internal organs. The
aging process has not yet been fully elucidated.
An understanding of skin aging is needed because it is immediately perceptible and thus
has a considerable social impact. Furthermore, the skin is the ideal model organ for
investigating the aging process [1].
Like any other organ, the skin is subjected to progressive morphological alterations, and
its physiological functions reduce with the passage of time [1-2]. However, the natural aging
process might be accelerated in photoexposed skin areas due to the accumulation of
environmental, chemical, physical and mechanical injuries [1-2].
Thus, skin aging is a complex and continuous biological process with a variable age of
onset. Aging is characterized by cellular and molecular alterations that lead to cell senescence
and apoptosis [3, 4]. An organism’s homeostatic ability is progressively reduced due to the
decrease in the skin’s functions and in its ability to tolerate injuries [2]. The factors that play
an important role in this process involve genetic damage or are extrinsic (i.e., environmental)
[4-6].

SKIN AGING: MORPHOLOGICAL AND

PHYSIOLOGICAL ALTERATIONS OF THE SKIN
The skin is the organ responsible for protecting an organism against environmental
injuries and maintaining the body temperature and water and electrolyte balance [7]. It is also
responsible for processing and synthesizing several proteins, glycans and lipids, in addition to
producing vitamin D, growth factors and steroid hormones [8-9].
With the passage of time, the skin loses its structural and morphological characteristics,
and its homeostatic function becomes impaired. The morphological alterations that
accompany such physiological changes might include the following:

1 Alteration of the skin’s permeability due to the reduction of lipids in the corneal layer
and abnormalities in cholesterol synthesis, which impair the skin’s ability to function
as a physiological barrier. Consequently, the skin becomes more susceptible to
mechanical trauma and infectious diseases [10]. It is believed that the imbalance of
sex hormones associated with menopause might also contribute to this change in
permeability [11].
2 Loss of the morphological and functional characteristics of the sebaceous glands with
a consequent decrease in the skin’s lipid content, resulting in xerosis. Xerosis
predisposes elderly individuals to skin diseases, such as chronic itch or eczema [8]. It
is assumed that xerosis in the elderly might also be attributed to hormonal deficiency
because it improves with topical hormone use [8].
3 Decreased ability to produce vitamin D3 and a consequent reduction in the protective
effects of that vitamin. The main source of vitamin D is its endogenous production in
the skin after exposure to ultraviolet B (UVB) radiation. Although the diet
contributes only 20% of an individual’s daily vitamin D needs, it represents an
important alternative source of vitamin D for the elderly. The cutaneous precursor of
vitamin D (7-dehydrocholesterol) produces pre-vitamin D3 upon exposure to UVB

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 Skin Aging 1081

radiation. Pre-vitamin D3 undergoes molecular rearrangement to become vitamin D3

(cholecalciferol). Vitamin D3 is essential for calcium homeostasis and bone integrity,
the immune response, the release of inflammatory cytokines and the regulation of
cellular growth and differentiation. It also inhibits the activation of protein kinases
due to stress [8].
4 Decreased thermoregulation abilities. In the elderly, this reduction seems to be
related to the sudoriferous glands’ reduced response to central or peripheral stimuli
and to structural alterations of these glands and/or the skin cells around them [12].
5 Fewer Langerhans cells and alterations in their structure and function. This is most
likely why the elderly have compromised immune functions and an increased
susceptibility to infection [13].
6 Reduced size and density of blood vessels in the skin. Such alterations are
exacerbated in photoexposed skin, in which the architecture of the vascular plexus is
lost. The architecture is unaltered by the intrinsic aging process [14].
7 Dysregulation of matrix metalloproteinase (MMP) expression and increased
proteinase activity [15].
8 Accumulation of advanced glycated end-products (AGEs). The formation of AGEs is
a predominantly endogenous process. It occurs slowly under physiological conditions
and affects collagen; thus, it plays an important role in the aging process [16-18].
AGEs might also have exogenous sources, such as UV radiation, smoking and diet
(carbohydrates and lipids). AGE formation is initiated by nonenzymatic reactions
involving glucose and intra- and extracellular proteins [18]. AGEs can damage cells
via three basic mechanisms: a) alterations of intracellular structures, which result in
gene transcription modifications; b) interactions with extracellular matrix proteins
that alter the signaling between matrix molecules and the cell, resulting in
dysfunction; and c) alterations of blood proteins and lipids which, upon binding to
specific receptors, result in the production of inflammatory cytokines and growth
factors. Some of the reactions that result in AGE formation can also produce reactive
oxygen species (ROS), thus contributing to oxidative stress and other types of
structural and functional damage to molecules [18]. The body’s defense mechanism
against accumulated damage from AGEs involves enzymes (oxaldehyde reductase
and aldose reductase), which efficiently detoxify the body. It is important to stress,
however, that some reactions that lead to AGE production can generate ROS and
occur in parallel with oxidative stress and other types of structural and functional
molecular damage [16-18].

INTRINSIC AND EXTRINSIC SKIN AGING

Skin aging involves different biological processes that can be classified as intrinsic and
extrinsic [4-6].
Intrinsic skin aging is a genetically determined, continuous, unavoidable and time-
dependent process that results in slow tissue degeneration and reflects the overall aging of the
body. This process occurs naturally and is associated with changes in the endocrine
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1082 Samira Yarak and Carolina A. Pontes da Silva

environment. Intrinsic skin aging is exacerbated by extrinsic skin aging, which is induced by
environmental factors [1-6]
The mechanisms involved in intrinsic skin aging are related to cumulative endogenous
damage caused by the continuous formation of ROS, telomere shortening and hormone level
alterations [1-6, 19, 20]
In the human dermis, intrinsic aging has three main characteristics: dermal atrophy
caused by collagen loss; degeneration of the elastic fibers network; and loss of hydration [19,
2] (Table 1).
Several factors are involved in extrinsic aging, including ionizing radiation, severe
physical and psychological stress, alcohol use, poor nutrition, excessive eating, environmental
pollution and exposure to UV radiation. UV radiation is the most important environmental
factor, and it contributes to up to 80% of extrinsic skin aging (also known as photoaging) as a
function of the characteristics of photoexposed skin [1-6, 19, 20]. The histological
characteristics of extrinsic aging are summarized in Table 2 [2].

Table 1. Morphologic and functional changes in intrinsic aging skin (adapted from
Zouboulis et al., 2011) [2]

Thinning of epidermis Increased vulnerability, fragility

Slow replacement of lipids Disturbed barrier function
Flattening of the dermoepidermal junction Decrease in surface contact area
Decrease and heterogeneity of melanocytes Graying of hair, amelanosis, lentigines
Diminished cutaneous immune
Decrease of Langerhans cells
function
Atrophy of the extracellular
Matrix, reduction of dermis Reduced strength and resiliency
thickness, decrease of fibroblasts
Reduction and disintegration Sensitization to deformational
of collagen and elastic fibers forces, fine wrinkle formation
Reduction of cutaneous microvasculature and Reduction of cutaneous
decrease of skin appendages and their function (e. g., vascular responsiveness and disturbed
sebaceous glands, sweat glands, apocrine glands) thermoregulation and supply with nutrients
Reduction of nerve endings Disturbed sensory function

Table 2. Morphologic changes in extrinsic aging skin (from Zouboulis et al., 2011) [2]

Accumulation of abnormal elastic tissue in the dermis

Sparse distribution of collagen fibers, increased collagen degradation
Stellate phenotype of fibroblasts and increased biosynthetic activity
Increased levels of dysfunctional glycosaminoglycans and proteoglycans
Increased numbers of mast cells and neutrophils
Flattening of the dermoepidermal junction, reduction of anchoring fibrils
Thickening of the vascular walls of postcapillary venules and
of arterial and venous capillaries, increased number of veil
cells, and marked regression and disorganization of small
blood vessels
Impaired proliferation, differentiation, desquamation and
apoptosis of keratinocytes
Thickening of epidermis

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MECHANISMS INVOLVED IN INTRINSIC

AND/OR EXTRINSIC SKIN AGING

1. Telomere Shortening

Telomeres are base pairs at the end of eukaryotic chromosomes. They do not replicate
during mitosis and thus suffer progressive erosion during successive cycles of cellular
replication, which results in progressive shortening and ultimately rupture [4] (Figure 1).
Before senescence, cells are able to multiply countless times. During senescence,
however, although the cells are still viable, they are unable to proliferate. This facilitates end-
to-end chromosomal fusion, which results in karyotype disorders and subsequent apoptosis.
Thus, the telomeres seem to function as a biological clock that determines the duration of
a cell’s proliferative life [5, 6].
Even the quiescent skin fibroblasts lose more than 30% of their telomere length in
adulthood. This process is natural (intrinsic aging) and is accelerated by ultraviolet radiation
and other causes of DNA damage (extrinsic aging) [1, 3, 5, 6].

2. Production of Free Radicals

The normal cellular respiration process for energy production occurs in organelles known
as mitochondria. Mitochondria use molecular oxygen, and a small fraction of it produces
ROS due to incomplete reduction during aerobic metabolism. ROS include the superoxide
anion (O2•-), hydrogen peroxide (H2O2), the hydroxyl anion radical (HO•) and singlet oxygen
(O) [19]
ROS production by mitochondria can cause oxidative damage to proteins, membranes
and mitochondrial DNA [15, 19], thus impairing the mitochondria’s ability to synthetize
adenosine triphosphate (ATP) and perform countless metabolic functions that are important
for the normal operation of most cells.

http://2.bp.blogspot.com/-TBIdKU1IFJk/T7G4H6NOBiI/AAAAAAAAADg/Jc-
8iTTDprc/s1600/chromosome.jpg.

Figure 1. Telomere shortening.

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1084 Samira Yarak and Carolina A. Pontes da Silva

Oxidative damage to mitochondria can also increase the release of intermembrane

proteins, such as cytochrome C (cyt C), into the cytosol, thus activating the cell’s apoptotic
machinery. In addition, ROS might act as second messengers for specific cell signaling
pathways [1, 19].
Increased ROS production or removal might increase the odds of oxidative modification
of intracellular components or cause mutations of the cell genetic material. An imbalance
between ROS production and their detoxification by the antioxidant defense system might
result in a state of exacerbated oxidative stress and consequently increase oxidative damage to
all cells in the body [1, 15, 19].
To summarize, oxidative stress or damage represents an imbalance between oxidants and
antioxidants that favors the former and results in disordered signaling and redox control
and/or molecular damage. Therefore, it is not surprising that mitochondrial oxidative damage
contributes to countless diseases.
The skin is the organ most exposed to the environment; thus, it is important that it have
protective mechanisms against oxidative injury [1, 15, 19].
Several antioxidant enzymes are located in the skin, including glutathione reductase,
catalases and superoxide dismutases. These enzymes are constantly active in maintaining
homeostasis and in minimizing the damaging effects of ROS [1-6, 19, 20]
Polymorphisms of the genes that code for antioxidant enzymes can result in insufficient
ROS detoxification, thus reducing the body’s antioxidant potential [21].
Therefore, alterations that result in intrinsic skin aging are partly caused by cumulative
endogenous damage resulting from the continuous generation of ROS caused by oxidative
cellular metabolism.
Solar radiation, infections and other external stimuli accelerate the production of these
free radicals [15, 19, 20].

3. Solar Radiation

Ultraviolet radiation (UV) is a powerful stimulus for the production of free radicals in the
skin [19] (Figure 2). UVB (280-320 nm) and UVA-2 (320-340 nm) radiation penetrate only
the epidermis and upper dermis, whereas UVA-1 (340-400 nm) reaches the deep dermis. The
DNA and cellular proteins absorb both UVA and UVB radiation and suffer its direct effects
on cell membranes [19]. UVB radiation induces damage in the DNA of melanocytes and
keratinocytes by forming thymidine dimers. With aging, the covalent bonds between
thymidine dimers cannot be dissolved quickly; thus, mutations can accumulate. In turn, UVA
radiation penetrates deeper and damages both the epidermis and the dermis [1-6, 19, 20] The
cumulative effect of UVA and/or UVB rays on cellular metabolism in the skin via
keratinocytes, melanocytes, and fibroblasts results in increases in ROS and the consequent
activation of collagen degrading enzymes, MMPs and inflammatory cytokines (Figure 2).
This effect causes degradation and reduced synthesis of extracellular matrix collagen and
results in the appearance of wrinkles [1-6, 15, 19]. Thus, the senescence process is
characterized by alterations in the structure and amount of extracellular matrix, which is
composed of collagen Types I and III, elastin, proteoglycans, fibronectin and collagen fibrils.
Photoaging causes countless histological alterations. In the epidermis, the spinous layer
becomes thinner, and the dermal-epidermal junction is flattened [22].

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 Skin Aging 1085

Adapt of Chen, Hu and Wang 2012 [31].

Figure 2. Role of reactive oxygen species (ROS) in photoaging.

In turn, the aged keratinocytes become resistant to apoptosis and thus susceptible to DNA
mutations. This process has been implicated in carcinogenesis [1-6, 15, 19, 20]. The number
of melanocytes decreases, and the melanocytic density is altered. The Langerhans cells
exhibit an altered structure and decrease in number with aging, with a consequent impairment
of the immune function [2, 22, 23] (Table 2). At the dermal-epidermal interface, the
epidermal crests are flattened, thereby reducing the area available for nutrient exchange. The
dermis exhibits a loss of volume and collagen, fibroblasts appear, and the number of blood
vessels and sensory and autonomic nerves is reduced [2, 15, 19, 22, 23] (Table 2).

4. Hormonal Factors

The skin responds to steroidal and nonsteroidal hormones, and hormonal activity
decreases with age. The decline of sex hormones, particularly estrogens, is the best-known
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1086 Samira Yarak and Carolina A. Pontes da Silva

age-related phenomenon. Nevertheless, the understanding of estrogens’ effects on the skin is

still poor [8, 24]
It is believed that estrogens affect the skin through the same molecular pathway involved
in other nonreproductive tissues; that is, the skin responds to estrogens via alpha or beta
estrogen receptors [8, 24]. The skin’s lipid layer and hydration ability decrease during
menopause, and the dermis exhibits degeneration of the elastic fibers, reduced thickness and
water retention. The decrease in estrogen levels during menopause seems to contribute to
wrinkle formation, xerosis, atrophy, flaccidity, pigmentation alterations, delayed wound
healing, flushing and vulvar atrophy [8, 24, 25].

5. DNA Errors or Mutations

Some genodermatoses are associated with errors and/or mutations of the DNA repair
genes, thus resulting in premature aging, as in Cockayne syndrome, Werner syndrome, ataxia
telangiectasia and progeria, for example [6].

6. Smoking

Smoking accelerates skin aging, especially in women, and there is a direct correlation
between the number of cigarettes smoked and the degree of aging [6]. Nicotine causes
vasoconstriction; this results in tissue hypoxia, which in turn contributes to the production of
free radicals. In addition, nicotine increases blood viscosity, elastase activity and
hypoestrogenism [26, 27].

CLINICAL FEATURES
Skin aging is characterized by skin atrophy, discrete to deep wrinkles, bone and muscular
tissue alterations, changes in subcutaneous tissue distribution, telangiectasia, melasma,
poikiloderma and the eventual development of premalignant and/or malignant skin lesions
[28].
Glogau (1996) [29] developed a photoaging scale, which is used to clinically classify the
extent of photodamage (Table 3).
Extrinsic aging occurs primarily as a function of the damage caused by the cumulative
effect of ultraviolet radiation on the skin and the consequent elastosis of the dermis,
appendages and blood vessels [30]. In addition to the sun, other factors are associated with
extrinsic aging, including smoking and gravity [1]. Extrinsic aging is characterized by deep
wrinkles, dry and yellowish skin, telangiectasia, poikiloderma, depigmentation, actinic
keratosis and a greater predisposition toward skin cancer (Table 4) [30].
Intrinsic or chronological aging is caused by reduced collagen and elastin production in
the dermis. It is characterized by skin atrophy, fine or discrete wrinkles, xerosis, altered
distribution of subcutaneous tissue, bone reabsorption, flaccidity, depigmentation, graying
and alopecia (Table 4) [30].

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Table 3. Glogau’s photoaging classification (from Glogau, R. G., 1996) [29]

Age: 20s to 30s

Early photoaging
Mild pigmentary changes
I – No wrinkles
No keratosis
Minimal wrinkles
Minimal or no make up
Age: 30s to 40s
Early to moderate photoaging
Early senile lentigines
II – Wringles in motion Palpable but not visible keratoses
Parallel smile lines beginning to
appear laterally to mouth
Usually wears some foundation
Age: 50 or older
Advanced photoaging
Obvious dyschromias,
III- Wringles at rest Wringles even when not moving
Telangiectasias
Visible keratoses
Always wears heavy foundation
Age: 60 or older
Severe photoaging
Yellow-gray skin
IV – Only wrinkles
Precancerous lesions
No normal skin
Can’t wear makeup

THERAPEUTIC APPROACH
Currently, photoprotection is the main approach for preventing skin aging.
Photoprotection involves the use of broad-spectrum sunscreens against UVA and UVB
(sun protection factor ≥ 30) [2, 15, 28] and photoprotective clothing, such as hats, long-
sleeved blouses and sunglasses. Patients must be advised to avoid environmental factors
significantly associated with skin aging, such as smoking and alcohol use, in addition to sun
exposure [2, 15, 28]. Finally, patients should attempt to neutralize the formation of free
radicals via topical or systemic antioxidants; however, the efficacy of these substances has not
been fully established.

ANTIOXIDANTS
ROS appear to represent an important primary factor in aging [2]. Antioxidants can
potentially decrease ROS production induced by UV radiation [31-36]. The antioxidants
naturally present in the skin are superoxide dismutase, catalase, alpha-tocopherol, ascorbic
acid, ubiquinone and glutathione; however, many of these antioxidants may be inhibited by
visible and UV light.
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1088 Samira Yarak and Carolina A. Pontes da Silva

Table 4. Cutaneous affections caused by intrinsic and extrinsic aging (Adapt of

Montagner et al. 2009) [30]

Intrinsic aging Extrinsic aging

Wrinkles Fine Deep
Corneum layer Unchanged Thinning
Dysplastic cells Few Many
Small change in size and Large changes in the size and
Collagen fibers
organization organization
Decreased production and
Elastic fibers Reorganized
increased degeneration
Decreased number and Decrease the number and
Hair follicle
thinning structure and hair loss
Decrease the number and
Melanocytes Normal
melanin
Decreased number and dry
Sebaceous and sweat glands Decrease in the number
skin
Dermo-epidermal junction Mild flattening Significant flattening
Telangiectasia, bruising,
Microvasculature Reduced area perivascular inflammatory
infiltrate
Benign affections Seborrheic keratosis Seborrheic keratosis
Premalignant affections Actinic keratosis
Basal cell carcinoma
Malignat affections
Squamous cell carcinoma

Vitamins and many plants have antioxidant properties and have been widely used to care
for the skin and protect it against ROS [2, 3]. We will only discuss topical antioxidants’ role
in minimizing the effects of oxidative stress on the skin and thus preventing skin aging. The
current trend is to incorporate antioxidants into sunscreens and cosmetics.

Vitamin C

Vitamin C is a water-soluble antioxidant agent that neutralizes free radicals in the

hydrophilic compartments of the skin and contributes to the regeneration of vitamin E. It is a
cofactor in the formation of enzymes involved in collagen synthesis, and it also reduces
elastin accumulation. It further reduces skin hyperpigmentation by inhibiting tyrosine kinase,
and it maintains the hydration provided by the skin’s epidermal barrier [32]. At the molecular
level, 1% topical vitamin C increases collagen synthesis and reduces collagenase expression
[33, 34]. It also participates in cholesterol synthesis and iron absorption, in addition to
increasing the availability of selenium [2].
Topical vitamin C is useful as levogyre ascorbic acid at concentrations of 5 to 15% [1].
However, vitamin C’s penetration into the skin poses a challenge because ascorbic acid must
lose its ionic load and its pH must be lower than 3.5 to penetrate the corneal layer [32].

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Vitamin E

Vitamin E (α-tocopherol) reacts with singlet oxygen and is a fat-soluble antioxidant. For
this reason, it can penetrate the corneal layer and the sebaceous glands. Ultraviolet radiation
can deplete vitamin E, even after a single exposure at a suberythemal dose [2, 34, 35].
Tocopherol acetate is used in topical products at concentrations from 0.2% to 1.5% [2].
The combination of vitamins C and E reduces oxidative stress [18] and doubles the
protection against ultraviolet radiation by reducing erythema, sunburn cell formation and
thymidine dimers [36].

Vitamin A

The two topical varieties of vitamin A are retinoids and carotenoids. Topical retinoids are
found in sunscreens and cosmetics. Retinol and its derivatives (tretinoin, isotretinoin and
tazarotene) exhibit anti-aging properties [31]. Retinoids act by binding to specific nuclear
receptors. They influence several cell processes, including DNA repair; gene expression;
stimulation of the growth and differentiation of keratinocytes, melanocytes and fibroblasts;
and the production of extracellular matrix by fibroblasts [1, 34, 37, 38].
In addition to reducing keratinocyte cohesion, retinoids stimulate cell proliferation, alter
the production of mucin and the composition of keratin, induce vascular alterations, reduce
melanocyte activity and stimulate collagen synthesis [39].

Alpha Hydroxy Acids

Alpha hydroxy acids belong to a group of hydrophilic organic acids. They are used for
their hydrating, exfoliating and keratolytic properties. This group includes the following
acids: glycolic acid, which is extracted from sugar cane; lactic acid, extracted from fermented
milk; mandelic acid, derived from bitter almond extract; citric acid; pyruvic acid; and tartaric
acid [3].
The beneficial effects of alpha hydroxy acids include improving the state of
photodamaged skin, which includes the reduction of wrinkles, skin discoloration and actinic
keratosis; improving pigmentation; increasing collagen density; and improving the quality of
the elastic fibers [40].
Five or 12% lactic acid and 12% ammonium lactate reduce the thickness of the corneal
layer by decreasing the cohesion of keratinocytes and thus increasing the thickness of the
epidermis, improving the softness of the skin, and reducing wrinkles [41].
Glycolic acid is the most widely used agent in cosmetic formulas. Because of its small
molecule size, it penetrates better than the other alpha hydroxy acids [42]. Glycolic acid is
used in cosmetic products at low concentrations (up to 10%), whereas higher concentrations
(above 20%) are used in chemical peels.
There is some evidence that glycolic acid increases extracellular matrix, improves the
quality of elastic fibers and increases the dermal collagen density, resulting in the clinical
improvement of fine wrinkles and hyperpigmentation [3].
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Tea Polyphenols

The polyphenols found in tea hinder the penetration of UVB radiation and thus inhibit its
effects on cells, including immunosuppression. In addition, tea polyphenols exhibit
antioxidant, anti-inflammatory and anticarcinogenic properties, which have been primarily
shown in in vitro or animal studies [43-44]. Like the other antioxidants, tea polyphenols are
unstable when they are used topically and quickly lose their biological activity [31].

Soy Isoflavones

According to some reports, soybean-rich diets protect against some types of cancer and
cardiovascular disease. Soybeans contain isoflavones, such as genistein and daidzein. Topical
application of genistein has been associated with a reduction in UV-related oxidative damage,
such as immunosuppression and inflammation [31].

CONCLUSION
Despite advances in research, it is not yet possible to block the aging process in the skin.
Nevertheless, avoiding smoking and UV radiation might reduce the effects of skin aging.

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[24] Verdier-Sevrain, S., Bonté, F., Gilchrest, B. Biology of estrogens in skin: implications
for skin aging. Exp. Dermatol. 2006:15(2):83–94
[25] Hall, G., Phillips, T. J. Estrogen and skin: the effects of estrogen, menopause, and
hormone replacement therapy on the skin. J. Am. Acad. Dermatol. 2005; 53: 555-68
[26] Francès, C. Smoker’s wrinkles: epidemiological and pathogenic considerations. Clin.
Dermatol. 1998, 16:565-70.
[27] Suehara, L. Y., Simone, K., Maia, M. Evaluation of facial aging related to cigarrete
smoking An. Bras. Dermatol. 2006, 81:34-9.
[28] Yaar, M., Gilcherest, B. A. Photoaging: mechanism, prevention and therapy. British J.
Dermatol. 2007 (157) 874-887
[29] Glogau, R. G. Aesthetic and anatomic analysis of the aging skin. Semin. Cutan. Med.
Surg. 1996 Sep.;15(3):134-830
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1092 Samira Yarak and Carolina A. Pontes da Silva

[30] Montagner, S., Costa, A. Molecular basis of photoaging. An. Bras. Dermatol.
2009;84(3):263-9
[31] Chen, L., Hu, J. Y., Wang, S. Q. The role of antioxidants in photorotection: a critical
review. J. Am. Acad. Dermatol. 2012 10.1016 in press
[32] Campos, P. M., Goncalves, G. M., Gaspar, L. R. In vitro antioxidant activity and in
vivo efficacy of topical formulations containing vitamin C and its derivatives studied by
non-invasive methods. Skin Res. Technol. 2008;14:376-80.
[33] Varani, J., Spearman, D., Perone, P., Fligiel, S. E., Datta, S. C., Wang, Z. Q., Shao, Y.,
Kang, S., Fisher, G. J., Voorhees, J. J. Inhibition of type I procollagen synthesis by
damaged collagen in photoaged skin and by collagenase-degraded collagen in vitro.
Am. J. Pathol. 2001;158:931-42
[35] Manela-Azulay, M., Bagatin, E. Cosmeceuticals vitamins Clinics in Dermatology,
27(5):469-74
[36] Thiele, J. J., Traber, M. G., Packer, L. Depletion of human stratum corneum vitamin E:
an early and sensitive in vivo marker of UV induced photo-oxidation. J. Invest.
Dermatol. 1998;110: 756-61
[35] Lin, J. Y., Selim, M. A., Shea, C. R., Grichnik, J. M., Omar, M. M., Monteiro-Riviere,
N. A., Pinnell, S. R.UV photoprotection by combination topical antioxidants vitamin C
and vitamin E. J. Am. Acad. Dermatol. 2003;48:866-74
[37] Griffiths, C. E., Russman, A. N., Majmudar, G., Singer, R. S., Hamilton, T. A.,
Voorhees, J. J. Restoration of collagen formation in photodamaged human skin by
tretinoin (retinoic acid). N Engl. J. Med. 1993 329:530-5.
[38] Stratigos, A. J., Katsambas, A. D. The role of topical retinoids in the treatment of
photoaging. Drugs 2005 65:1061-72.0
[39] Cucé, L. C., Bertino, M. C., Scattone, L., Birkenhauer, M. C. Tretinoin peeling.
Dermatol. Surg., 2001 Jan.; 27:13-15.
[40] Van Scott, E. J., Yu, R. J. Control of keratinization with alpha-hydroxy acids and
related compounds I. Topical treatment of ichthyotic disorders. Arch. Dermatol.
1974;110:586–590
[41] Smith, W. P. Epidermal and dermal effects of topical lactic acid. J. Am. Acad.
Dermatol. 1996 35:388-91
[42] Ditre, C. M. Glycolic acid peels. Dermatol. Therapy, 2000 13:165-172
[43] Katiyar, S. K., Bergamo, B. M., Vyalil, P. K., Elmets, C. A. Green tea polyphenols:
DNA photodamage and photoimmunology. Photochem. Photobiol. 2001 65:109-14.
[44] Vayalil, P. K., Mittal, A., Hara, Y., Elmets, C. A., Katiyar, S. K. Green tea polyphenols
prevent ultraviolet light-induced oxidative damage and matriz metalloproteinases
expression in mouse skin. J. Invest. Dermatol. 2004 (122):1480-7.

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Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 50

A PROCEDURE
FOR THE ASSESSMENT OF SKIN AGING

Natsuko Kakudo, Satoshi Kushida, Nobuko Saito,

Kenji Suzuki and Kenji Kusumoto
Department of Plastic and Reconstructive Surgery, Kansai Medical
University, Japan 10-15 Fumizono, Moriguchi, Osaka, Japan

ABSTRACT
Skin aging is caused by sunburn damage and by the slowing down of skin cell
metabolism. The symptoms of skin aging include wrinkles; , pigmented spots; , dullness;,
an increased number of open facial pores; , and the debasement of texture, resilience, and
water conservation.
Thus, It is important the development of that objective, reliable, and easy-to-use
methods for to analyzing analyze aging skin be developed is important.; however, it has
been difficult However, problems were encountered during the development to develop
of such methods because skin aging is often associated with minor changes that are
difficult to assess quantitatively. The facial skin aging score is can be evaluated using the
Robo Skin Analyzer®, a system that analyzes digital camera-captured photographic
images. This The device has uses flat lighting, which is achieved using through use of a
diffusion reflector.
The system ensures consistent positioning by using anchor point positioning and
stable contrast by using a gray scale color chart; it clarifies ears and sharpens images of
individuals persons with even the most complicated skin coloration. In addition, the
device recognizes and calculates the various factors associated with skin aging and
records quantified data. In this chapter, we report the clinical application of the Robo
Skin Analyzer® for the assessment of skin aging.


Correspondence to: Natsuko Kakudo, M.D., Ph.D. Department of Plastic and Reconstructive Surgery, Kansai
Medical University, 10-15 Fumizono, Moriguchi, Osaka 570-8506, Japan. Tel: +81-6-6992-1001; Fax: +81-6-
6997-0628; E-mail: [email protected].
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1094 Natsuko Kakudo, Satoshi Kushida, Nobuko Saito et al.

INTRODUCTION
Senescence is the process by which human bodies undergo change as they age. For
example, human skin forms wrinkles, becomes thinner, and loses texture and elasticity as it
ages. Prolonged exposure to sunlight also increases wrinkles and freckles and dries skin,
which loses its healthy shine. In the past, these changes have proven difficult to assess
objectively, which was problematic; however, devices have recently been developed to
analyze physiological and morphological changes in the skin. One of these, the Robo Skin
Analyzer®, can photograph whole faces and analyze the skin with relative ease and under
consistent conditions. In this chapter, we report the clinical application of the device Robo
Skin Analyzer® for the assessment of skin aging.

COMPUTER ANALYSIS OF DIGITAL CAMERA-CAPTURED

PHOTOGRAPHIC IMAGES
The Robo Skin Analyzer® is a system that analyzes digital camera-captured photographic
images. In the field of aesthetic dermatology, one advantage of using the Robo Skin
Analyzer® is that it is possible to photograph a face at a fixed setup and distance. You put in
your The face to be photographed is placed inside the device, that which has a 4 megapixel
CCD camera installed, or and, by when the stand holding up your supporting the face rotating
rotates on its axis, pictures can be automatically taken from three 3 fixed directions can be
automatically taken. The fixing of the face, similar to an that of an optometer, is performed by
sticking yourpressing one’s forehead and chin to against the device. Furthermore, the center
line of the face is lined upaligned with the digital camera’s video mode. This device was is
connected to a Windows-based personal computer via a USB cable. The recorded photos are
instantly saved by the enclosed software (Clinical Suite 2.1 (NIIC, Tokyo, Japan) (Figure 1).
Of the 3 color elements in color images (RBG), the blue signal component most clearly
showed the pore distribution.

Figure 1. Robo Skin Analyzer® system.

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 A Procedure for the Assessment of Skin Aging 1095

Figure 2. Video microscope.

The captured images were analyzed in the color elements by imaging software. Briefly,
in color images, 8-bit (256 color) blue-plane data were are obtained from the 5 megapixel
captured images for analysis. Furthermore, the Robo Skin Analyzer® has its own video
microscope which that can analyze skin texture using the included software (Figure 2).

Analysis of Facial Pores

Using the color information acquired from the digital imaging, it the Robo Skin
Analyzer® software performs a quantitative evaluation by automatically extracting the regions
with visible pores regions from the difference of the brightness of the whole face.

Figure 3. Analysis of facial pores using the Robo Skin Analyzer ® system.
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1096 Natsuko Kakudo, Satoshi Kushida, Nobuko Saito et al.

Pores were were selected according to their density and morphological characteristics as
seen in the monochrome images prepared from these signals. Then, through the embossment
embossing process, round-shaped outlines of the pores were obtained; and the regions with an
area of 0.1–0.6 mm2 and a specific shade (0–100 range within 256 gray levels) were
determined considered as to be conspicuous facial pores. Pores with an area of 0.3–0.6 mm2
were determined classified as “open pores” (Figure 3). According to size and brightness
information of size and brightness, the regions with visible pores regions are classified and
defined into three 3 types; pores, pores with conspicuous opening, and pores with
conspicuous darkening [1].

Analysis of Facial Pigmentation

Using the color information obtained from the digital imaging, it the Robo Skin
Analyzer® automatically extracts hyperthe pigmentationed regions from the differences of
thein data on brightness and shape data, and then performs a quantitative evaluation of the
hyperpigmentation pigmented regions. According to the size of the pigmentationse regions,
they are classified and defined classified as two sizes according to size as either; large and or
small (Figure 4). The number of pigmented regions and their dimensions are displayed As as
analyzed data, the number of pigmented regions and their dimensions are displayed [2].

Analysis of Facial Skin Texture

Facial skin textures can be analyzed through photographs captured by the microscope
attached to the Robo Skin Analyzer®. With this device, facial skin structure is scored on a
scale from 0 to 100. The dark portions of the monochrome image are designated as the sulci
cutis, while the bright portions are designated as the cristae cutis. Each portion is enhanced
separately and an overlay image is prepared for analysis (Figure 5).

Figure 4. Analysis of facial pigmentation using the Robo Skin Analyzer ® system.

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 A Procedure for the Assessment of Skin Aging 1097

Figure 5. Analysis of facial skin texture.

The more closely the composite image resembles the ideal texture model of an equilateral
triangle with sides of 0.4 mm, the higher the score (closer to 100) that is assigned to the
image.

ASSESSMENT OF SKIN AGING USING THE ROBO SKIN ANALYZER®

In addition to measuring facial pores, pigmentation, and skin texture, the Robo Skin
Analyzer® can also assess wrinkles, moisture content, sebum content, and skin tone. The
accompanying Clinical Suite 2.1 imaging software contains a database constructed from
healthy volunteers and facilitates a comparison of patient scores against the average scores of
healthy controls of the same age. Furthermore, the software can calculate a “skin age,” using
proprietary formulas. To support evidence-based medicine, detailed examination of skin
shapes and skin physiology is essential; therefore, objective external assessment of skin
conditions through the use of noninvasive devices is desirable. Analyzing digital images
taken under the same conditions, and thereby quantifying a large amount of information,
enables an objective assessment of skin age. In that sense, the Robo Skin Analyzer® is very
beneficial.

REFERENCES
[1] Kakudo, N., Kushida, S., Tanaka, N., Minakata, T., Suzuki, K., and Kusumoto, K.
(2011) A novel method to measure conspicuous facial pores using computer analysis of
 Free ebooks ==> www.Ebook777.com
1098 Natsuko Kakudo, Satoshi Kushida, Nobuko Saito et al.

digital-camera-captured images: the effect of glycolic acid chemical peeling, Skin Res.
Technol. 17, 427-433.
[2] Kawada, A., Kameyama, H., Asai, M., Shiraishi, H., Aragane, Y., Tezuka, T., and
Iwakiri, K. (2002) A new approach to the evaluation of whitening effect of a cosmetic
using computer analysis of video-captured image, Journal of dermatological science
29, 10-18.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 51

AGED SKIN AND STRENUOUS EXERCISE:

CAN THE SKIN HANDLE THE HEAT?

Stuart A. Best and Martin W. Thompson

Exercise Health and Performance Research Group,
Faculty of Health Sciences, University of Sydney,
Lidcombe, Australia

ABSTRACT
As the number of older adults participating in strenuous exercise and sports
increases, there exists a need to understand the effect of ageing on thermoregulation
during strenuous exercise. The question of whether age affects the capacity to disperse
heat to the external environment has been studied with equivocal results. Most
researchers agree that skin blood flow is diminished with age due to central and
peripheral vasodilatory mechanisms. In the skin this can include vascular stiffness and
decreased activity and sensitivity to vasodilators. Additionally, other studies have also
proposed that due to a reduction in the output of individual sweat glands there is a
diminished sweating response with increasing age. However, where thermoregulatory
differences in older adults have been observed, the response of the older subjects can be
likened to the response seen in untrained younger adults. It is difficult to differentiate
between thermoregulatory differences due to age-related cardiovascular changes, age-
related lifestyle changes, and potential age-related alterations to skin blood flow and
sweat gland function.
The testing of trained adults in different age groups would alleviate lifestyle
differences as trained subjects approach their genetic potential for undertaking physical
work with less apparent physiological strain. Future research should be aimed at
comparing the effect of habitual physical activity and ageing on the response to exercise-
heat stress, passive heat stress or chemically induced skin blood flow and sweating
responses. This will allow future researchers to accurately describe the effect of aging on
thermoregulation and if there are any additional risks for older adults participating in
strenuous exercise and competition.
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1100 Stuart A. Best and Martin W. Thompson

INTRODUCTION
As the human body ages there is a general decline in function and capacity across all
physiological systems. With increased awareness of the health benefits of exercise throughout
an individual’s life including their elderly years, more adults are undertaking or continuing
strenuous endurance training programs as they age. Exercise training has been shown to
reduce the risk or incidence of obesity and many associated lifestyle diseases such as
cardiovascular disease, hypertension and diabetes by limiting or restoring the diminished
function of many of these physiological systems [1]. The thermoregulatory system is one of
these systems that may also decline with ageing. A reduction in thermoregulatory capacity
with age would increase the risk of heat stress leading to serious injury or even death. This
risk may be further increased when the challenge of thermoregulation is combined with
exercise.
The question of how age affects the capacity to disperse metabolic heat to the external
environment can include thermoregulation during normal daily activities and passive heat
stress, thermoregulation during exercise-heat stress and thermoregulatory challenges during
cold stress. It has been shown that the number of reported illnesses and deaths during heat
waves is significantly increased in older adults [2]. Likewise it has been shown that there is
an increase in reported cases of heat stress amongst elderly participants during competitive
exercise events [3]. So the effect of ageing on responses to heat stress at rest, during normal
activities and during strenuous exercise are all valid questions. Thermoregulation can also
include the capacity to regulate body temperatures in response to cold stress and the effect of
ageing on the body’s response to cold environments can and has been investigated [4-9].
While there are many important questions that have been and still remain to be investigated,
for the purpose of this review we will focus solely on the effect of ageing on thermoregulation
during exercise–heat stress. In particular this chapter will review the effect of ageing on heat
dissipation at the skin during exercise-heat stress. In addition this chapter will also consider
how previous exercise training and/or heat acclimatisation may preserve thermoregulatory
capacity in older adults during exercise–heat stress.

HEAT LOSS MECHANISMS IN THE SKIN

Heat is released within the human body as energy is produced through metabolism. There
is a direct relationship between the energy demands of the body and the subsequent heat
generation [10, 11]. Heat storage within the body leads to a subsequent rise in body
temperature [12]. Central regulation of body temperature occurs in the preoptic–anterior
hypothalamus [13] which receives afferent nerve signals from temperature sensitive receptors
in the body ‘core’ and skin and controls the activation of the heat loss effectors [14, 15].
Sweating and increased skin blood flow are the heat loss effectors by which heat loss from the
body occurs but temperature regulation is integrated with many other physiological systems
including the cardiovascular system [16].
Heat loss via an increase in skin blood flow during exercise-heat stress occurs via
temperature gradients between the external environment, the skin and the body core
temperature. Heat is transferred from the warm working muscles and body ‘core’ to the

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 Aged Skin and Strenuous Exercise 1101

cooler blood. The warm blood is then directed to the superficial vessels in the skin where heat
exchange can occur from the blood vessels to the external environment. A “counter-current”
heat exchange process occurs between hot venous blood that is in close proximity with warm
arterial blood which further facilitates a reduction in the rate of heat storage. Thus heat loss
via skin blood flow is determined by the rate of blood flow directed to the skin and the
direction and size of the temperature gradients. The temperature gradient and subsequent
convective heat loss are thereby directly dependent on ambient temperature and if ambient
temperature is greater than core temperature then heat gain will occur.
The rise in skin blood flow during exercise is almost entirely due to increased
vasodilation [17]. The regulation of vasodilation within the cutaneous blood vessels is
controlled via sympathetic cholinergic nerves and it is thought this occurs through a co-
transmitter with acetylcholine [18]. The exact co-transmitter/s remains unclear and while
there is significant evidence that nitric oxide (NO) contributes up to 40% of cutaneous active
vasodilation [19, 20] there is evidence that other co-transmitters also contribute to
vasodilation. Research has shown that vasoactive intestinal peptide [21, 22], histamine [23]
and substance P [24] may all contribute to vasodilation through NO production as well as
other mechanisms. Indeed a recent study showed that NO does not solely activate the soluble
guanylylcyclise (sGC) pathway during active cutaneous vasodilation as previously thought
[25]. In addition to NO, vasodilator prostanoids which are produced by the COX-pathway
have been shown to contribute to vasodilation independently of NO [26]. So although
sympathetic cholinergic nerves are known to regulate skin blood flow the exact mechanism/s
remains unclear.
As body temperatures begin to rise during exercise there is a corresponding rise in skin
blood flow. The internal temperature at which a significant rise in skin blood flow occurs is
referred to as the skin blood flow or vasodilatory threshold [17]. The threshold can change
due to a number of influences including exercise [17, 27] and heat acclimatization [27, 28].
As skin blood flow continues to increase there is a linear relationship between skin blood
flow and core temperature from the skin blood flow threshold to a point at which the rise in
skin blood flow is attenuated [29]. At this point skin blood flow is known to plateau despite
further increases in core temperature [29]. It is agreed within the literature that this plateau is
a preventative mechanism to preserve mean arterial pressure rather than signalling the upper
limit of vasodilatory capacity [29, 30]. The partitioning of blood flow to the skin during
exercise is coupled with a relative vasoconstriction of other vascular beds including the
splanchnic and renal regions [31]. Rowell et al. [31] showed a linear relationship between
decreasing splanchnic blood flow (SBF) and renal blood flow (RBF), and increasing skin
blood flow. However, during exercise the increase in skin blood flow challenges the need for
blood to provide oxygen to the working muscles and the need to thermoregulate whilst still
maintaining central blood pressure. Evidence suggests that a further increase in muscle or
skin blood flow is superseded by the maintenance of central blood pressure [32].
Skin blood flow has typically been measured using venous occlusion plethysmography or
more recently a variety of Laser-Doppler methods including single point, integrated and
topographical perfusion mapping [33]. Venous occlusion plethysmography [34] has typically
been measured on a forearm which is resting at or above the level of the heart during lower
body exercise. It is assumed that during this measurement the changes in forearm blood flow
would be confined to the skin [35]. One advantage of the Laser-Doppler techniques is the
capacity to measure specific sites on the skin, independent of muscle blood flow. Laser-
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1102 Stuart A. Best and Martin W. Thompson

Doppler can also make continuous measurements whereas venous occlusion plethysmography
can only be measured between regular intervals in order to restore normal blood flow before
the next measurement. A disadvantage of Laser-Doppler is the inability to measure absolute
blood flow so measurements are typically compared with relative values as a percentage of
maximal blood flow [33, 36]. The use of Laser-Doppler has been verified as an accurate
alternative to venous occlusion plethysmography [33, 37, 38]. Skin blood flow responses
have been reported as absolute flow rates, relative flow rates (% of maximum skin blood flow
- %SkBFmax) or relative to arterial pressure such as cutaneous vascular conductance (CVC) or
forearm vascular conductance (FVC) [36]. When any of these measures are increased it is an
indication of an increase in skin blood flow.
While the rise in skin blood flow has significant effects on the cardiovascular system it
only accounts for a small proportion of heat loss from the body particularly in high ambient
temperature environments. The primary source of heat loss is the evaporation of sweat which
is secreted onto the skin surface. Fluid (i.e., sweat) is secreted onto the skin via sweat glands,
and this fluid is then heated to such a degree it evaporates [39]. The evaporation of one litre
of sweat releases approximately 2400Kj of energy as heat [40] and is the human body’s most
effective heat loss mechanism.
The secreted sweat will not evaporate but rather drip to the ground or onto clothing if it is
not effectively heated and/or the environmental conditions are not conducive to evaporation
(e.g., high humidity or clothing restrictions). Significant heat dissipation through sweating
only occurs when the secreted sweat is evaporated and thus the effectiveness of sweating is
determined more by vapour pressure (humidity) than ambient air temperature at the skin
surface [41].
Central control of the eccrine sweat glands occurs predominantly through the preoptic–
anterior hypothalamus [13] via sympathetic cholinergic nerves [39, 42-44]. There is also
growing evidence of non-thermal controls of eccrine sweat glands [45, 46] including muscle
mechanoreceptors and metaboreceptors during exercise, baroreceptors, fluid status and
osmolality [45]. It is thought that altering the non-thermal control of eccrine sweat glands
causes no significant contribution to evaporative heat loss during exercise-heat stress [47].
The threshold for the onset of sweating has traditionally been reported as the core
temperature at which a significant rise in sweating occurs [48, 49]. More recently there has
been discussion of whether mean body temperature or some other calculated measure of
temperature or heat storage may be a more accurate measure that reflects the onset of
sweating [50, 51]. The uncertainty stems from the knowledge that core temperature, mean
skin temperature and local skin temperature are all known to affect sweat rate although the
primary input is core temperature [52, 53]. The initial response irrespective of afferent input
is an increase in the number of active sweat glands followed by an increase in sweat output
per gland [54, 55]. Only in times of extreme thermal stress are all sweat glands in a particular
area recruited [54].
Sweat loss has been measured as local or whole body sweat loss. Whole body sweat loss
is commonly measured by changes in body weight with calculated corrections for fluid
consumption, urine loss and respiratory moisture loss [11, 52, 56]. This technique is relatively
easy and effective but dripped sweat makes evaporative sweat loss difficult to accurately
measure. In order to distinguish between evaporated and dripped sweat, the sweat that drips
onto the ground or clothing must be collected and the volume measured. Whilst some have
used varying methods in an attempt to measure dripped sweat, the change in body weight

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 Aged Skin and Strenuous Exercise 1103

without collecting dripped sweat is considered an acceptable measurement. To measure local

sweat rate some researchers have used Dew Point Hygrometry which calculates sweat rate
after measuring water vapour going to and from a sealed capsule on the skin [52, 55, 57-59].
Others have instead used an iodine-starch method to measure local sweat rate as well as the
number of active sweat glands [15, 54, 55]. Another method that has been used is the
bromphenol blue dye method [60]. While the iodine-starch and bromphenol blue dye methods
can be used to measure the number of active sweat glands, dew point hygrometry provides a
continuous measurement of local sweat rate throughout exercise-heat stress.
The combination of increased skin blood flow and sweating as thermoregulatory
mechanisms, stimulated by local, peripheral and central inputs provides a highly adaptable
and efficient system designed to maintain body temperatures within a small range of
comfortable/acceptable temperatures. Because of the complex design of the thermoregulatory
system our knowledge is always expanding and there are many responses and mechanisms
that are not yet completely understood or possibly even discovered. It also means there are
many different ways in which ageing might affect the capacity to dissipate heat from the skin.

AGE AND SKIN BLOOD FLOW DURING EXERCISE-HEAT STRESS

The skin blood flow threshold, rate of increase of skin blood flow or peak skin blood
flow can each affect convective heat loss. Each of these and the mechanisms responsible for
any potential differences with age must be examined. As this review will focus on alterations
at the skin, corresponding cardiovascular adjustments will only be discussed where they are
mechanisms that may/may not contribute to differences in blood flow at the skin.
Ageing has not been shown to effect resting skin blood flow or the skin blood flow
threshold during exercise-heat stress. A number of studies by Larry Kenney and colleagues
found no difference in resting skin blood flow when comparing young and older adults [61-
64]. Likewise, in studies measuring skin blood flow threshold to exercise-heat stress in young
and older adults age has been found to have no effect on skin blood flow threshold [49, 64,
65], including pre and post a period of exercise training in which training significantly
lowered the threshold for both age groups [36]. In contrast, during passive heat stress ageing
has been shown to alter the skin blood flow threshold [66, 67]. The present data do not
suggest that age per se affects resting skin blood flow, the threshold for skin blood flow
during exercise-heat stress or the lowering of the threshold by exercise training. Ageing has
been shown to affect the rate of increase in skin blood flow and peak skin blood flow
response during exercise-heat stress.
The rate of increase in skin blood flow with increasing core temperature can be a viewed
as a measure of the thermal sensitivity of skin blood flow to rising core temperature. First
Kenney [64], and later Thomas et al. [36] showed older adults had an attenuated response to
increasing core temperature when compared to young adults. Thomas et al. [36] further
reported peak skin blood flow and found that this also was significantly lower in older adults.
Although Tankersley et al. [49] found an average reduction in skin blood flow sensitivity of
33% amongst a group of mixed sedentary and active older subjects when compared with
normal young adults the difference was not significant. The attenuated rise in skin blood flow
as shown by Kenney [64] and Thomas et al. [36] decreases convective heat loss during the
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1104 Stuart A. Best and Martin W. Thompson

early stages of exercise-heat stress and may also result in a greater time to peak skin blood
flow. An attenuated rise in skin blood flow has been shown in previous studies conducted
using passive-heat stress [66, 67].
Throughout the research literature the most frequent finding when comparing skin blood
flow responses of young and older adults during exercise-heat stress is a reduction in peak
absolute skin blood flow with increased age. Larry Kenney and colleagues have produced
most of the data in this area and numerous studies from their laboratory have shown a
reduction in peak skin blood flow response amongst older adults (49-78 years) when
compared with young (18-32 years) adults [36, 61-63, 68]. These studies include subjects of
different ages who were matched for maximal aerobic capacity [61-63] and subjects who
weren’t [36, 68]. Exercise intensity (35 - 60%𝑉̇ O2max) and duration (30 – 60+ min) varied
across the studies and one study showed that the difference in skin blood flow between young
and older subjects increased as the exercise intensity increased [63].
In contrast to the reported reduction in absolute peak skin blood flow, relative peak skin
blood flow does not appear to change with ageing. Data from our laboratory has shown that
when normalized to resting skin blood flow there was no difference between young and older
subjects in peak skin blood flow following 60 min cycling at 70%𝑉̇ O2max [69]. Skin blood
flow was measured using a single laser-doppler probe so absolute skin blood flow could not
be measured. Similarly Kenny et al. [65] found no difference in relative peak CVC between
young and middle-aged men when performing exercise at the same metabolic heat production
in 30oC and 35oC. At 40oC there was a trend of higher CVC in the middle-aged subjects. Data
from a study by Kenney et al. [68] showed although peak relative CVC was similar between
young (22 ±1 yrs) and older (66 ±1 yrs) subjects at a control site and bretylium-treated site
(which abolishes local vasoconstrictor activity) they showed an attenuated rise in skin blood
flow that was not observed by Kenny et al. [65]. These differences could be attributed to the
young age of the middle-aged subjects in the Kenny et al. [65] study (45 ±4 yrs) when
compared with the older subjects of the Kenney et al. [68] study (66 ±1 yrs) or the matching
of exercise for metabolic heat production [65] versus relative exercise intensity [68].
In summary, age per se has not been shown to alter resting skin blood flow, the skin
blood flow threshold, or the peak relative skin blood flow. There is strong evidence
suggesting ageing is associated with an attenuated rate of increase in skin blood flow with
increasing core temperature and a reduction in peak absolute skin blood flow. Together these
results suggest ageing affects skin blood flow by reducing the compliance and maximal
vasodilation of the blood vessels in the skin. Possible mechanisms that have been identified
include increased vascular stiffness, an attenuated or decreased response to vasodilators, or a
reduction in the redistribution of blood flow from the renal and splanchnic vascular beds.
In response to passive heating Pierzga et al. [70] found younger subjects (24 ±1yrs)
responded with a greater diameter of vasodilated blood vessels during the early stages of
heating (increase in Tes of 0.6oC) compared with older subject (70 ±1yrs) who were not
matched for 𝑉̇ O2max. The area of the blood vessels was not significantly different at later
stages of heating (increase Tes of 0.8oC) but CVC was lower throughout heating. Pierzga and
colleagues (2003) proposed that this was due to a decreased sensitivity during vasodilation.
The attenuated rise in skin blood flow observed by Pierzga et al. [70] and others [36, 64,
68] could be the result of less compliant blood vessels in the elderly cardiovasculature and
could include microvascular vessels in the skin. Increased vascular stiffness, particularly

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arterial stiffness has been shown to correlate with age [71, 72] and heating has been shown to
increase vascular compliance, particularly in individuals with vascular stiffness in
thermoneutral conditions [73]. Under these conditions it could be theorized that initial
differences in arterial stiffness between age groups are reduced with additional heating. The
vascular stiffness of young and older adults before and after exercise-heat stress and
corresponding skin blood flow responses has not been investigated.
It has been shown that the decreased sensitivity of increasing skin blood flow to the
increase in core temperature is the result of a reduction in the activity and/or sensitivity to
vasodilators and not increased vasoconstriction [36, 68, 70]. While studies have shown
vasoconstriction is attenuated with ageing in response to cold environments [74] it has been
shown that older adults do not respond to exercise-heat stress with a higher level of
vasoconstriction [68]. Therefore it has been concluded that any attenuation in skin blood flow
response due to ageing is not due to vasoconstriction but rather a consequence of diminished
vasodilatory response.
The diminished vasodilatory response has been shown to be the result of decreased
sensitivity and/or output of vasodilatory regulators. These have included nitric oxide [75, 76]
and acetylcholine [77]. The exact mechanism/s by which nitric-oxide regulated vasodilation is
decreased with age remains unknown. While some studies have shown NO has a lower
contribution to vasodilation in older adults [75, 76] others have shown that NO has a greater
contribution with ageing [67]. Black et al. [76] found that the reduction in NO activity was
only present in sedentary men and a cohort of trained older men had the same response as
younger trained men. Furthermore, when the sedentary men completed a period of endurance
training the NO activity was significantly enhanced. Holowatz et al. [67] suggested the
reduction in activity of an unknown vasodilator was the reason for the reduction in skin blood
flow.
Acetylcholine (Ach) sensitivity has also been shown to be decreased in older men. De
Souza et al. [77] found that older men (58 ±2 years) responded to ACh stimulation with a
lower rise in forearm blood flow than their younger counterparts (27 ±1 years). Similar to the
findings of Black et al. [76] there was no difference between elderly-endurance trained men
and young sedentary adults. It should be also be noted that a 13 week training regime in the
older sedentary adults did not improve FBF response. This suggests that if exercise does
restore or improve cutaneous vasodilation it may require a longer-term approach. In contrast
Boegli et al. [78] found that although training improved the response to a NO donor, FBF was
not restored to the level of the younger men.
In a recent series of experiments by Tew and colleagues it has been shown that
microvascular reactivity to vasodilatory stimuli can be maintained with high levels of fitness
in older adults [79-81]. Vasodilation was induced by local heating, occlusion and drug
iontophoresis (ACh and sodium nitroprusside – SNP) and in each condition the older trained
adults displayed a similar response to the young sedentary adults [79]. An additional group of
sedentary older adults showed a diminished response in each condition. The results of a later
study showed that in sedentary older men the attenuated rise in skin blood flow could be due
to impaired sensory nerve regulated vasodilation [80]. A further study did find that in
response to local heating older adults (including trained older adults) displayed an attenuated
initial increase in skin blood flow due to a reduced contribution of noradrenergic sympathetic
nerves which have been shown to play a role in the vasodilatory response to local skin heating
[82]. The responses of the neurotransmitters involved varied with age and aerobic fitness and
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1106 Stuart A. Best and Martin W. Thompson

included norepinephrine (NE) and the cotransmitter neuropeptide Y (NPY). The response to
exercise-heat stress was not measured in these studies [79-82] or those previously discussed
but it is proposed that a combination of these mechanisms could occur during the onset of
exercise-heat stress.
What is apparent from the present data is that although many studies have shown
differences in vasodilatory activity with increasing age [67, 75-82] it is still unclear as to what
the exact mechanism/s are. There is evidence to suggest that whatever the mechanism, regular
endurance training can improve or even restore vasodilatory sensitivity.
While the vascular stiffness and response to vasodilators may occur at the level of the
skin, there is evidence that the redistribution of blood from the renal and splanchnic vascular
beds is also compromised with age in response to exercise-heat stress [62, 63] and passive
heat stress [83]. This would lead to a reduced peak skin blood flow in older adults. The
decreased skin blood flow observed by Ho et al. [63] coincided with a smaller reduction in
renal blood flow and splanchnic blood flow in the older subjects. As with forearm blood flow,
the differences between the young and older subjects were increased further as the exercise
intensity increased.
Although the focus of this review is the effect of aged skin on thermoregulation during
exercise, it should also be noted that the redistribution of the blood flow to the skin results in
significant cardiovascular strain. The competing demands of the thermoregulatory system and
the working muscles must be regulated in order to maintain central blood volume and blood
pressure. The increase in cardiovascular strain is evidenced by decreased stroke volume, and
thus an increase in heart rate in order to maintain cardiac output [84]. There are few data
published demonstrating cardiac output during exercise-heat stress in young and older adults
and the results to date have showed either no change [62] or a reduction [63, 64, 85] in
cardiac output with age, even when exercising at the same relative and absolute exercise
intensity. The reduction (if any) in cardiac output with age is most likely due to a reduction in
stroke volume as relative heart rate response, including the rate of increasing heart rate (heart
rate drift) and final heart rate as a percentage of maximal heart rate [60, 62, 69, 86, 87] has
not been shown to be affected by age during exercise-heat stress. Ageing has also been shown
to have no effect on mean arterial pressure during exercise-heat stress [49, 62, 63, 68, 69].
Finally, it is important to note that in each of the studies in which a difference in skin
blood flow was observed, there was no corresponding increase in core temperature or a
failure to complete the exercise [49, 61, 63, 64, 68]. Given the low amount of heat that is lost
via convection in environments with high ambient heat, it is probable that these small
differences, whilst statistically significant, are not physiologically significant during exercise-
heat stress. Alternatively, older adults could have a lower skin temperature (due to a reduction
in skin blood flow) which would therefore increase the temperature gradient between the
blood, the skin and the environment and thus heat loss could be maintained. This is unlikely
as no significant differences in skin temperature were observed in any of the studies cited [49,
61, 63, 64, 68]. Lastly, it could be that older adults are required to sweat more. If so this
would result in increased risk of heat stress in environments with high humidity.
In summary previous research indicates maximal skin blood flow is compromised with
age. In the skin this can be attributed to a possible decline in vascular compliance and
decreased vasodilator activity and sensitivity in the cutaneous blood vessels. It is difficult to
isolate the effects at the skin however, as it has also been shown that these mechanisms are
not isolated to superficial vessels and there is also a reduction in the redistribution from the

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 Aged Skin and Strenuous Exercise 1107

splanchnic and renal vascular beds. Reduced blood redistribution may be a mechanism by
which central cardiovascular function (central blood volume and blood pressure) is
maintained with age at the expense of peripheral blood flow. Although the reduction in skin
blood flow has been shown, the measurement of how much this reduces convective heat loss
has not been examined. At present it appears unlikely that the reduction in skin blood flow
has a physiologically significant effect on heat loss during exercise-heat stress as impaired
skin blood flow has not been shown to correlate with increased core temperature,
cardiovascular strain or decreased performance.

AGE AND SWEATING RESPONSE DURING EXERCISE-HEAT STRESS

The greatest and most efficient heat loss from the body occurs through the evaporation of
sweat on the skin. Any age-related effects on the sweating mechanism at the level of the skin
would seriously impede the capacity to thermoregulate with increasing age. When
considering the potential effect of ageing on the capacity to evaporate sweat at the level of the
skin one must consider the sweating threshold, number of sweat glands activated, and the
sweat rate of each gland.
Age has not been found to affect the threshold for the onset of sweating during exercise-
heat stress. Numerous studies have shown that the sweating threshold is similar for young and
older subjects during exercise-heat stress [48, 49] and passive heat stress [88-91]. It has been
concluded that ageing does not affect the regulation of the onset of sweating and any age-
related differences in sweating are the result of peripheral adaptations such as the number of
active sweat glands and/or the output of each sweat gland.
Current published research does not suggest that ageing affects the number of heat
activated sweat glands (HASG) in the skin. Using the iodine-starch and bromphenol blue dye
methods a number of researchers have shown that the number of HASG’s are not different in
older adults when compared to young adults [60, 92-94]. Skin locations in which they were
measured included the chest, back, upper abdomen, neck, forehead, forearm and thigh. This is
an important consideration as the number of HASG’s is known to vary throughout the body
with the hands, forearm, upper arm, chest and forehead containing the greatest density of
HASG’s [54]. While there is strong evidence that the number of HASG’s does not alter with
age, the findings on the output of the sweat glands is more ambiguous.
When comparing the effect of age on sweat gland output, and ultimately sweat loss,
during exercise one must be mindful of the exercise intensity in which the exercise is
performed. It has been shown that there is a linear relationship between metabolic heat
generation and sweating [10, 95]. Because submaximal metabolic heat production at a given
relative intensity is determined by an individual’s 𝑉̇ O2max it is no surprise that sweating
during exercise at a given relative intensity (%𝑉̇ O2max) has been found to be proportional to
𝑉̇ O2max during exercise-heat stress [69, 96] and following chemical stimulation of sweating
[97]. This creates a dilemma with ageing as submaximal power outputs and metabolic heat
generation decrease due to the age related decline in maximal aerobic power [98, 99]. If older
and younger subjects are matched for training status (i.e., both sedentary or both trained) and
exercise at the same relative intensity (reduced absolute heat production in the older group), it
is expected they will have similar core and skin temperatures but a reduction in sweating.
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1108 Stuart A. Best and Martin W. Thompson

Alternatively if subjects are matched for maximal aerobic power (𝑉̇ O2max) and exercise at the
same relative intensity (similar heat production in both groups) it is expected that core and
skin temperatures as well as sweat loss would be similar between age groups. It has recently
been suggested that matching subjects for metabolic heat production provides a more accurate
comparison of sweating responses in a compensable environment [100, 101] but the
responses to an uncompensable environment require further investigation. As a result the
metabolic heat production and subject selection when performing exercise in hot conditions
are important considerations when comparing sweat gland output and sweat loss results of
previous research.
The output of individual sweat glands is a difficult measurement during passive heat
stress which drastically increases in complexity during exercise-heat stress. Some studies
have used the iodine-starch and bromphenol blue dye methods immediately following a bout
of exercise [94] or in response to passive heat stress in a study that also involved exercise-
heat stress [60, 93]. The results were contrasting however as one study found no difference in
sweat gland output [94], whilst others observed a decreased sweat gland output with age [60,
93]. The writers of this review are unaware of any studies measuring sweat gland output
during exercise-heat stress. The contrasting results of Inbar et al. [94] and Inoue et al. [60, 93]
reflect the results of other studies comparing sweat loss in young and older subjects during
passive heat stress [88, 91, 102]. Interestingly, Inoue [102] reported significantly reduced
sweat gland density in the thigh but not the back, however sweat gland output was
significantly reduced in the back and not the thigh. Furthermore, although sweat gland density
was similar but sweat gland output on the back was reduced, local sweat rate on the back was
found to be similar between young and older subjects. These results highlight the caution that
must be applied when comparing local sweat rate at a single area of the skin to the entire
body. The findings of Inoue [102] also support findings from their laboratory and others
showing inconsistent regional differences in sweating between young and older adults. There
is a trend that if sweat output with increased age is decreased in may occur in the limbs first
[60, 90, 91, 93, 103]. Given the difficulty in measuring sweat gland output during exercise-
heat stress and assuming HASG density is not altered with age, it could be concluded that any
decrease in sweat rate must be due to a reduced output per gland. Therefore both local or
whole body sweat loss can be considered surrogate measures for sweat gland output, as well
as evaporative heat loss.
Caution must be taken when interpreting local sweat loss as a part of whole body sweat
loss but the advantage of measuring local sweat rate over whole body sweat loss is the ability
to measure sweat rate continuously throughout exercise. Others have used other methods of
measuring whole body sweat loss regularly throughout exercise [88]. The advantage of these
methods is the ability to compare the increase in sweating to the increase in internal
temperature. Of the studies that have measured the rate of increase in sweating during
exercise-heat stress the results show that when subjects are matched for maximal aerobic
fitness there is no difference in the sweating sensitivity with age [49, 65]. During constant
load exercise designed to generate the same metabolic heat load in young (22 ±2 yrs) and
middle aged (45 ±4 yrs) men who were matched for 𝑉̇ O2max, Kenny et al. [65] observed no
difference in the rate of increased sweating (thermal sensitivity) or peak local sweat rate.
These results were observed in a range of temperatures (30 – 40oC) however the rise in core
temperature was low (<0.5oC) in spite of the duration of the exercise (90 min) due to the low

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 Aged Skin and Strenuous Exercise 1109

intensity of the exercise. When young and older subjects are not matched for maximal aerobic
capacity, an attenuated or ‘sluggish’ rise in sweating has been observed [49].
Davies [104] expressed sweat rate as a percentage of maximal sweat rate (measured as a
percentage of the highest recorded sweat rate above 39.3oC). Davies [104] observed a clear
linear relationship between the relative sweat loss and core temperature and the response of
the older athletes mirrored those of the younger athletes. Although the exercise was
performed in thermoneutral conditions (21oC, <50%RH) the high running intensity (76-
78%𝑉̇ O2max) resulted in core temperatures that were higher than most other exercise-heat
stress studies (>39.0oC).
The findings that sweating sensitivity during exercise-heat stress does not appear to be
affected by age support the findings of studies using passive heat stress. Researchers have
reported an attenuated sweating response in older subjects who were not matched to younger
counterparts for 𝑉̇ O2max [90, 91, 93, 103]. Therefore the sweating sensitivity appears to be
more closely associated with an individual’s maximal aerobic capacity than age.
Given that sweating sensitivity and peak sweat rates during exercise-heat stress have not
been shown to be affected by age but are more closely associated with maximal aerobic
capacity, it is not surprising that ageing does not affect total sweat loss during exercise-heat
stress when subjects are matched for 𝑉̇ O2max and exercise at the same relative intensity (i.e.,
similar metabolic heat production). This has been shown repeatedly in the laboratory [61, 62,
64, 69, 105] and outside in closely monitored exercise [106] in a wide variety of ages (17 – 71
yrs), exercise intensities (30 – 70% 𝑉̇ O2max) and prevailing climatic conditions. From these
data there is significant evidence that the sweating response to exercise-heat stress is not
affected by age.
There are a number of studies that have reported contrasting results to those discussed
above but these seem to be explained by differences in experimental methods employed by
the researchers. Data from Kenney and Anderson [86, 87] showed older subjects (56 ±4 and
56 ±1 yrs respectively) responded to prolonged exercise-heat stress (2 hrs) with a decreased
sweat response in a hot-dry environment (48oC, 15% RH) but not a warm-humid environment
(37oC 60%RH). However in a later study [61] conducted in the same hot-dry environmental
conditions but with a reduced exercise duration (30 min) they found no difference in sweat
loss. None of the studies included fluid intake as part of the protocol so it was thought that
older adults may have been more susceptible to the effects of dehydration in the extended
duration. Dehydration is known to cause a reduction in sweat loss during exercise-heat stress
[107, 108].
A study by Pandolf et al. (1988) observed a greater sweating response in older adults (46
±5 yrs) who were matched for 𝑉̇ O2max to a cohort of younger adults (21 ±2 yrs) and performed
exercise at the same relative (45% 𝑉̇ O2max) and absolute intensity. The researchers attributed
these findings to the greater training status and therefore partial acclimation status of the older
group as it coincided with a decreased core temperature, skin temperature and heart rate
response. After a week of heat acclimation, there were no differences between the young and
older groups.
During simulated occupational tasks in young and older subjects who were not matched
for 𝑉̇ O2max, Hellon et al. [109] and Lind et al. [110] found similar maximal sweat rates when
performing the same amount of work. However, both studies observed that during exercise
and recovery periods sweat loss in the older group was lower and higher respectively. A more
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1110 Stuart A. Best and Martin W. Thompson

recent study that also included exercise intervals in the heat found total sweat loss was
reduced in the older subjects (not matched for 𝑉̇ O2max) but sweat loss for each interval and
rest period was not reported [94].
In summary age per se does not appear to affect the sweating response to exercise-heat
stress when subjects are matched for 𝑉̇ O2max and undertake exercise of a similar metabolic
heat production. This includes the threshold for the onset of sweating, the density of heat
activated sweat glands in the skin and sweat gland output as measured by local or whole body
sweat loss. When decreased sweat loss has been observed in older adults the sweat loss is
associated with an age-related decrease in maximal aerobic capacity, a by-product of reduced
maximal stroke volume and heart rate.
The question that then must be asked is whether the reduction in maximal aerobic fitness
directly or indirectly affects the sweating response or is the correlation between maximal
aerobic capacity and sweating the result of two independent physiological systems that appear
to decline at the same rate [69]. Current research suggests this could be due to the reduction in
the training stimulus to the sweat glands with a reduction in maximal aerobic power.
In younger adults it has been shown that sweat loss increases following an exercise
training regime when exercising at the same relative intensity pre and post training [111-113].
Recent research suggests that sweat loss at a given level of metabolic heat production does
not change but rather sweat loss is increased due to the greater metabolic heat produced
following training [100, 101]. Similarly, following a period of detraining, whole body sweat
loss has been shown to decrease [114]. Therefore it would seem logical that with a reduction
in maximal aerobic capacity and an increase in sedentary living with age, there would be a
reduction in metabolic heat production and thus a reduction in the ‘training’ stimulus to the
sweat glands. This would explain that when older adults are matched for 𝑉̇ O2max with younger
adults the young groups do not respond with a greater sweat loss than the older adults [61, 62,
64, 69, 105]. Thus the age-related decline in maximal aerobic capacity would have an indirect
effect on sweating with age. Some researchers have proposed mechanisms by which the
decline in sweating may be directly related to age and these include atrophy of the sweat
gland and/or a reduction in the pharmacological sensitivity of the sweat gland with age [102,
115]. While these mechanisms have been proposed, they have not been measured directly in
young and older adults. Others have also suggested reduced blood flow to the sweat glands
and in particular a reduction in vasodilatation due to a reduction in vasointestinal polypeptide
(VIP) which has been shown to be associated with sweat gland function [116].
In summary, the research suggests that any decline in sweating response with ageing is
closely associated with an age-related decline in maximal aerobic capacity. While there is
strong evidence linking these changes, the question of whether they are causally related or
coincidentally associated is not clear. Furthermore, the precise mechanisms by which
sweating decreases with age remain unknown and may include atrophy of the heat activated
sweat glands, inadequate blood flow to the sweat glands or a yet to be discovered mechanism.

AGED SKIN AND HEAT ACCLIMATISATION

Heat acclimatisation and heat acclimation are processes by which physiological
adaptations occur following repeated exposure to bouts of heat stress. Although heat

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 Aged Skin and Strenuous Exercise 1111

acclimation and heat acclimatization are defined by the environment in which an individual is
exposed to in order to stimulate adaptation [117], for the purposes of this review we will use
the terms interchangeably.
The adaptations that occur via heat acclimatisation include lowering the threshold and
increasing the thermosensitivity of skin blood flow and sweating. As a result the onset of
increased skin blood flow and sweating occurs at a lower core temperature [27, 28] and the
rate and magnitude of the response is greater [84, 113, 118, 119]. Thus thermoregulatory
function is improved, cardiovascular strain is decreased [27, 28, 113, 118] and time to fatigue
in hot and/or humid environments is increased [113].
Heat acclimation can occur through passive heat stress [120, 121] and exercise-heat stress
[27, 28, 113, 118, 119] and is a continual process in response to the environmental and
physiological stresses. Regular strenuous endurance training has been shown to create an
adaptation akin to heat acclimation [27, 28, 122] but the magnitude of the response is
increased significantly when an environmental heat load is added [111-113]. Heat acclimation
is characterised by a plateau in peak core temperature, heart rate and sweating responses
during consecutive bouts of exposure to the same thermal load. An individual’s acclimation
status prior to a heat acclimation period will determine the number of sessions required to
reach an acclimatised state. The rate at which these adaptations occur and the magnitude of
the response are both mechanisms by which age could attenuate the heat acclimation
response.
There are few data published on the effect of ageing on exercise-heat stress before and
after a period of active heat acclimation. Of the studies published some reported post
acclimation results only [86, 87] or the heat acclimation [123] and testing protocols [56] were
not accurately matched. The remaining studies suggest that ageing does not affect the rate or
magnitude of the response to heat acclimation providing subjects have a similar
training/acclimation status, body fatness and hydration at the commencement of a heat
acclimation protocol.
Similar studies by Pandolf et al. [48] and Inoue et al. [60] found that at the end of their
respective heat acclimation protocols the core temperature and sweating responses were
similar between young and older adults. In the study by Pandolf et al. [48] the magnitude of
the change throughout heat acclimation was lower in the older adults (46 ±5 yrs) than the
young adults (21 ±2 yrs), as the older adults displayed decreased thermal stress (signified by
greater sweating and lower core temperature) at the beginning of the heat acclimation period.
The researchers attributed the initial differences between the age groups to the higher training
status (and thus possible acclimation status) of the older adults. Although Inoue et al. [60]
investigated similar groups they only observed a difference in completion time at the
beginning of their acclimation protocol, with all the older, trained subjects (63 ±3 yrs)
completing the full 90 min on day 2 of acclimation as opposed to day 6 for the sedentary
older (67 ±3 yrs) and younger (23 ±1 yrs) adults.
Inoue et al. [60] did observe a difference with ageing in the cholinergic sensitivity of
sweating following heat acclimation. The young group was found to have a significantly
greater increase in sweat output in the thigh following heat acclimation than the fit older (HO)
or more sedentary older (NO) groups. There were no significant differences at any of the
other sites and the number of activated sweat glands was not significantly different pre or
post-acclimation. This suggests there may be age-related regional differences in the
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1112 Stuart A. Best and Martin W. Thompson

adaptation to sweating via heat acclimation although further studies are required to validate
this data.
Studies by Armstrong and Kenney [89] found age did not affect the reduction in skin
blood flow threshold pre and post a 9 day active heat acclimation regime (cycling or treadmill
walking at 40%𝑉̇ O2max) in 2 different age groups (young = 26 ±2 yrs, older 61 ±1 yrs).
However, pre and post-acclimation tests were conducted during passive heat exposure (28-
46oC temperature transient) which induced only a small thermal strain (peak rectal
temperature <0.5oC above resting). No data were reported during the heat acclimation regime.
There remains a clear gap in the literature investigating the question of whether age
affects the physiological adaptations to heat acclimation.

SUMMARY AND FUTURE RESEARCH

The findings of the published research suggest age results in an attenuation of the
increase in skin blood flow but not sweating during exercise-heat stress. Ageing has not been
shown to affect the threshold for the onset of skin blood flow or sweating. The thermal
sensitivity and peak skin blood flow response to exercise-heat stress has been shown to be
diminished and is probably due to a combination of increased vascular stiffness, decreased
sensitivity to vasodilators and a reduction in the partitioning of blood away from the renal and
splanchnic vascular beds. There is evidence to suggest that the maintenance of a regular
exercise regime can improve or even restore skin blood flow response to exercise-heat stress
in older adults. The thermal sensitivity of sweating and sweat gland output appear to be more
closely associated with maximal aerobic capacity than age. Ageing has not been shown to
cause an attenuated or reduced response of skin blood flow or sweating adaptations induced
by heat acclimation.
Whilst there is data to suggest that skin blood flow and sweating are compromised with
age, the matching of subjects for factors such as maximal aerobic power or training status,
acclimation status, as well as body size and composition makes it difficult to conclude that
age per se leads to a direct attenuation of the thermoregulatory systems during exercise-heat
stress.
Further investigation of the mechanisms by which ageing does/does not affect skin blood
flow and sweating during exercise-heat stress and following heat acclimation is warranted.
For skin blood flow the remaining questions regarding the effect of ageing include measuring
the reduction in convective heat loss when skin blood flow is compromised, the exact
mechanisms by which skin blood flow is compromised with age, and whether a regular
exercise training regime can improve or restore the reduction in skin blood flow. When
considering the effect of age on sweating the exact mechanism/s by which sweat gland
function is compromised with age and how this relates to maximal aerobic fitness also
remains unknown. Furthermore there exists a need to further understand any age-related
regional differences in sweat gland density and output.
Irrespective of potential mechanistic differences the data also suggests that age does not
affect core temperature response or exercise performance during exercise-heat stress. In a
majority of the studies the older adults completed the exercise just as well as the young
subjects and there were no significant differences in core temperature [49, 61, 63-65, 68, 69,

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104]. If the ultimate goal of the body during exercise-heat stress is to maintain an internal
temperature within strict boundaries whilst completing the exercise, then ageing does not
affect thermoregulatory capacity during exercise-heat stress. However, very few of these
studies have incorporated exercise intensities and durations akin to exercise training regimes
or athletic competition.
The American College of Sports Medicine currently recommends more than 30 minutes
per day of moderate intensity exercise (46 – 63%𝑉̇ O2max) or more than 20 minutes per day of
vigorous intensity exercise (64 – 90%𝑉̇ O2max) to improve and maintain physical fitness [1].
According to this criteria only 5 studies investigating the effect of age on thermoregulatory
responses in a hot-dry or warm-humid environment include an exercise intensity and duration
that fit the criteria for moderate intensity exercise [36, 62, 63, 68, 94]. Only 2 studies have
been conducted at a vigorous intensity together with environmental heat stress [49, 69] and no
study has investigated the effect of age on thermoregulation during self-paced exercise.
A further consideration that must be made when interpreting the data is that the research
conducted has predominantly compared young and older men with the older men commonly
between the 5th – 7th decades of life. Only a small number of the studies discussed have
investigated the effect of ageing on thermoregulation during exercise-heat stress in a subject
cohort including women [64, 86, 87, 104, 106]. Contrasting results were found in the two
studies in which the effect of ageing and gender were both investigated with one study
reporting lower thermoregulatory capacity in women [106] and another finding no difference
[104]. There remains a need to investigate if sex alters the thermoregulatory changes
associated with ageing, particularly during exercise-heat stress. Furthermore there is only one
study with an average age of the older group beyond 70 years of age [94]. Therefore the
conclusions made from the current research can only be made up to and including the age
groups and exercise intensities that have been investigated and the effect of gender can only
be hypothesized.

CONCLUSION
In summary, an attenuated skin blood flow response but no consistent reduction in
sweating or differences in core temperature and exercise response suggest that despite
possible mechanistic differences aged skin is just as capable of dispersing heat during
exercise-heat stress as younger skin. The difficulty in forming definitive conclusions is the
complexity of the thermoregulatory system, including many different regulators and inputs.
Furthermore there are very few studies involving female subjects, subjects greater than 70
years of age, or exercise intensities and durations performed during training and/or sporting
competition.

REFERENCES
[1] Garber CE, Blissmer B, Deschenes MR, Franklin BA, Lamonte MJ, Lee I-M, et al.
American College of Sports Medicine position stand. Quantity and quality of exercise
for developing and maintaining cardiorespiratory, musculoskeletal, and neuromotor
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1114 Stuart A. Best and Martin W. Thompson

fitness in apparently healthy adults: guidance for prescribing exercise. Med. Sci. Sports
Exerc. 2011;43(7):1334-59.
[2] Conti S, Meli P, Minelli G, Solimini R, Toccaceli V, Vichi M, et al. Epidemiologic
study of mortality during the Summer 2003 heat wave in Italy. Environmental
Research. 2005;98:390-9.
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[74] Thompson CS, Holowatz LA, Kenney WL. Cutaneous vasoconstrictor response to
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[92] Foster KG, Ellis FP, Dore C, Exton-Smith AN, Weiner JS. Sweat responses in the aged.
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[96] Greenleaf JE, Castle BL, Ruff WK. Maximal Oxygen Uptake, Sweating and Tolerance
to Exercise in the Heat. Int. J. Biometerol. 1972;16(4):375-87.
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peripheral sweat production of the human eccrine sweat gland. Age Ageing.
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 52

NEW INSIGHTS ON THE REGULATION OF

EXTRACELLULAR MATRIX PROTEINS
DURING SKIN AGING

Connie B. Lin and Michael D. Southall

The Johnson and Johnson Skin Research Center, Consumer Product Worldwide,
A Unit of Johnson and Johnson Consumer Companies, Inc., Skillman, NJ, US

ABSTRACT
The aging process, especially in the skin, is governed by changes in the epidermal,
dermal-epidermal junction and dermal compartments. Extracellular matrix (ECM)
proteins, which are the major component of dermis, constitute an important target for
intrinsic and extrinsic aging related alterations. Our skin is under continuous assault from
a variety of damaging environmental factors including ultraviolet irradiation and
atmospheric pollutants. Extrinsic factors, particularly sunlight, have been demonstrated to
accelerate the intrinsic aging process, resulting in elastosis, inflammation, and increased
matrix protein degradation. The NF-B (nuclear factor kappa-light-chain-enhancer of
activated B cells) pathway is one of the key signaling pathways that have been implicated
in the regulation of skin ECM production. The NF-B pathway can be activated by
interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-α) and reactive oxygen species,
and has been reported to be the final common pathway for the conversion of
environmental insults into inflammation in the skin. The activity of NF-B in the skin
increases as a function of age, suggesting that sub-clinical levels of inflammation in our
skin slowly go up as we age. Recent studies have demonstrated a direct link between NF-
B activation and suppression of matrix proteins such as elastin and collagen in dermal
fibroblasts, which can lead to an acceleration of the skin aging process. UV-enhanced
matrix degradation is mediated not only by NF-B and activation of activator protein 1
(AP-1), but also by inhibition of transforming growth factor beta (TGF-β) signaling.
TGF-β stimulates the synthesis of ECM including elastin, collagen, proteoglycans, and
glycosaminoglycans. Studies demonstrated that decreased collagen and elastin gene
expression are closely associated with reduced level of TGF- β and its receptor, and
activation of TGF-β signaling leads to increased ECM biosynthesis in human dermal
fibroblasts. New findings on the molecular mechanisms involved in the regulation of
 Free ebooks ==> www.Ebook777.com
1122 Connie B. Lin and Michael D. Southall

ECM production in the skin can help identify new targets to modulate ECM protein
expression and thereby have a significant effect on the skin aging process.

1. INTRODUCTION
Skin is the largest organ of the body. It is organized into three main layers, epidermis,
dermis and subcutaneous layer. The epidermis, an outermost avascular layer, is continually
formed by keratinocyte proliferation at the epidermal basal layer and differentiation into
corneocytes at the outer layer of the epidermis. Besides keratinocytes, the main cells in the
epidermis, the epidermis also contains melanocytes, pigment-producing cells residing at its
basal layer, and immune cells, e.g., langerhans cells. The dermis lies below the epidermis
separated by a basement membrane. It is composed mainly of fibroblasts, and sebocytes,
sebum producing cells, which reside in dermal fibroblasts-derived extracellular matrix (ECM)
proteins, primarily collagens, elastin, and glycosaminoglycans (GAGs)-rich proteoglycans.
The dermis is subdivided into the upper papillary dermis with loose connective tissues and an
underlying reticular dermis with dense connective tissues. Rete ridges, finger-like projections
at the basal layer of the epidermis, extend into dermis to further strengthen dermal-epidermal
junction (DEJ) connection with the ECM of the dermis and provide strength, extensibility and
elasticity to the skin (see review [1]). The subcutaneous layer under the dermis consists of fat
cells in connective tissue.
Skin as a protective layer is subject to both intrinsic (or chronological) aging and
extrinsic aging. Solar radiation is viewed as one of the major environmental factors promoting
skin aging [2]. Skin aging is a cumulative alterations of skin structure, appearance and even
functions such as uneven pigmentation, wrinkles, sagging, skin roughness, laxity and
impaired wound healing. Skin aging is multifactorial, which can be affected by genetics,
hormonal changes, chronological metabolic processes, and by external insults such as solar
irradiation (see reviews [2-6]). The manifestation of skin aging and newly identified
molecular biological processes involved in the regulation of ECM production in healthy and
aged skin are discussed in this chapter.

2. CLINICAL FEATURES OF AGED SKIN

Clinically, intrinsically aged skins appear thin, translucent, dry and manifests a loss of
firmness, which is a slow process, not usually evident until old age, and can be exacerbated
by sun exposure [7]. The characteristic structural change of intrinsically aged skin is a
decrease of skin thickness due to a marked epidermal and dermal atrophy [8-12], slower
epidermal turn over and flattened DEJ [13-15]. The dermis is relatively acellular (reduced in
fibroblast numbers) and avascular [16]. Chronic UV exposure over many years causes
accumulative skin damages and leads to premature skin aging (photoaging). Photoaged skins
overlap and superimpose the changes induced by intrinsic aging, typically with leathery,
coarse appearance, severe wrinkles, reduced recoil capacity and with uneven pigmentation.
Photoexposure-induced aging may occur much earlier than chronological skin aging and the

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severity of photoaging is dependent on the skin color and sun-exposure of the individuals, and
mostly on sun-exposed areas of the body such as face, neck, forearms, and dorsal hands [5-7].

3. DERMAL STRUCTURAL AND FUNCTIONAL CHANGES OF AGED SKIN

Most of aging induced skin structure alterations are related to dermal changes although
UV light has been shown to affect both epidermis and dermis. The prominent epidermal
alterations in photoaged skins are pigmentary changes such as lentigines, actinic keratosis and
seborrheic keratosis [17]. Chronologically aged skin has reduced collagen synthesis due to
both cellular aging and defective stimulating signaling, as shown by isolated dermal
fibroblasts from young and old individuals [18], while photoaging associated reduction of
ECM is mediated by the reduction, aging and apoptosis of fibroblasts [3].

3.1. Composition of Dermal Extracellular Matrix

The ECM proteins are dynamic, large complexes that collectively dictate a tissue’s
mechanical property and regulate cell signaling and adhesion. Collagens are the primary
structural protein of the dermis and provides strength and resiliency to the skin [19]. In young
skin, type I collagen, with lesser amounts of type III collagen, comprises almost 95% of total
skin collagen [19], while collagen IV, an intergral component of DEJ, and collagen VII,
located beneath the DEJ and anchoring fibrils to the underlying papillary dermis, play
important roles in maintaining skin mechanical property [20, 21]. Elastic fibres are composed
of an elastin core and a microfibrillar scaffold (mainly glycoprotein, fibrillin-1) [22]. The
upper papillary dermis contains fine elastin fibers connecting to oxytalan fibres that are
perpendicular to the DEJ, whereas the reticular dermis comprises thick elastin-rich fibres
which run in parallel to the DEJ [23]. The elastin fiber network is responsible for the skin
elasticity, allowing it to resume its shape after stretching or contracting. Various enzymes
including elastase are able to cleave elastic fiber molecules [24]. Along with collagen and
elastin, glycosaminoglycans (GAGs) including hyaluronic acid (HA), dermatan sulphate, and
chondroitin sulphate are found widely distributed throughout the skin. GAGs are produced
mainly by fibroblasts and keratinocytes, and have capacity to bind water up to 100 times their
volume, which are responsible for skin’s cushion property and the outward appearance of the
skin such as skin hydration and softness [25, 26].

3.2. Structural and Functional Changes of Extracellular Matrix in Aged Skin

Dermal photoaging is manifested primarily as the loss and disorganization of collagen

fibrils including loss of fibrillar collagens (I and III in the dermis, and VII anchoring fibrils at
the DEJ) [14, 27, 28], and the accumulation of abundant abnormal amorphous elastin fibers
containing material, namely elastosis, at the junction of papillary and reticular dermis [17, 29-
33]. Elastosis is usually not observed in chronologically aged skin. Sun-exposure-increased
elastin fibers are abnormally located in the areas previously held by collagen [34]. The
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1124 Connie B. Lin and Michael D. Southall

oxytalan fibers at the DEJ are markedly reduced and discrete microfibrillar bundles are rarely
observed in photoaged skin [35]. The loss of elastic fiber integrity leads to a progressive
reduction of skin elasticity and manifests as skin wrinkles.
Comparing to the extensive knowledge on collagen and elastin alterations during aging,
the changes of GAGs and proteoglycans in aged skin are less clear. Bernstein et al. reported
that photoaged skin has reduced levels of hyaluronic acid and elevated levels of chondroitin
sulphate proteoglycans [36].
The decreased hyaluronic acid in dermis in aging leads to its disconnection with collagen
and elastin as well as reduced water binding capacity and contributes to wrinkling and altered
elasticity. The DEJ interface under wrinkles is weakened by a decrease of collagen IV and
VII and a loss of oxytalan fibers.

3.3. Roles of Matrix Metalloproteinases in Extracellular Matrix Degradation

in Aged Skin

Matrix metalloproteinases (MMP), a large family of zinc-dependent endoproteases with

the capacity to degrade all ECM proteins, is expressed by both epidermal keratinocytes and
dermal fibroblasts [37]. The induction of MMPs are responsible for chronological and UV-
induced damage to the connective tissue in skin and an excess of MMP expression induced by
UV irradiation can result in the premature degradation of collagen and elastic fibers,
ultimately resulting in the visible signs of aging [38]. MMP-1 cleaves collagen type I, II, III.
MMP-9, also called gelatinase, further degrades collagen fragments generated by MMP-1, as
well as collagen type IV, V and gelatin. MMP-3, also named as stromelysin 1, degrades type
IV collagen and activates proMMP-1. MMP-2, -9, -12 and neutrophil elastase degrade
elastin. The activity of MMPs is tightly regulated by transcription regulation, activation of the
zymogen, and inhibition of the proteolytic activity [39-41]. The activities of MMPs can be
inhibited by specific endogenous tissue-specific inhibitor of metalloproteinases (TIMPs)
including TIMP-1, TIMP-2, TIMP-3 and TIMP-4 [39-41]. Sun exposure over years results in
repeated induction of collagen-degrading MMPs that are responsible for increased collagen
fragment in sun damaged skin [3, 4, 37, 42-45].

4. MOLECULAR MECHANISM OF SKIN AGING:

IMPLICATION OF NF-B AND TGF- IN DERMAL AGING
4.1. Overview

Although intrinsic and extrinsic aging exhibit both similar and distinct clinical and
histological features, many of the molecular alterations that occur in photodamaged skin
closely resemble those observed in chronologically aged skin [4] and the signal transduction
pathways that stimulate ECM degrading MMPs and decrease ECM protein syntheses are
shared. UV exposure of human skin leads to the induction of transcription factors such as
activator protein 1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells
(NF-B), which are known stimulatory factors for gene transcription and protein expression

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of MMPs within hours post UV exposure [37; 46; 47 48], and amplify the UV response by
stimulating the transcription of inflammatory cytokines [49]. UV exposure also blocks
transforming growth factor beta (TGF-)/Smad signaling pathways, leading to reduce ECM
production.

4.2. Implication of NF-B in the Regulation of Extracellular Matrix in Aged

Skin

The NF-B pathway is a key signaling pathway that has been implicated in the regulation
of skin ECM production and degradation. The activity of NF-B in the skin increases as a
function of age, suggesting that sub-clinical levels of inflammation in human slowly go up
upon aging. Recent studies have demonstrated a direct link between NF-B activation and
suppression of ECM proteins including elastin and collagen in dermal fibroblasts, which can
lead to an acceleration of the skin aging process.

4.2.1. Signal Transduction of NF- B

NF-B was first described in 1986 as a nuclear factor essential for immunoglobulin 
light chain transcription in B cells [50]. Since that initial discovery, NF-B has been found to
be a primary mediator involved in regulating immune responses, apoptosis and cellular
growth, as well as being present in inflammatory diseases such as arthritis and asthma [51].
The NF-B family of transcription factors shares a high-conserved sequence of amino acids
within their amino terminus, which contains a nuclear localization sequence that is involved
in the dimerization with sequence-specific DNA binding and with the inhibitory IB proteins.
In unstimulated cells, NF-κB-family proteins exist as heterodimers or homodimers that
are sequestered in the cytoplasm in an inactive form by virtue of their association with a
member of the IB family of inhibitory proteins , most notably IB, IBand IB52;
53]. About 200 extracellular signals can lead to activation through the dissociation of NF-B
from the IB proteins.
These activating signals include viral and bacterial products, oxidative stress, pro-
inflammatory cytokines including IL-1 and TNF-α, and phorbol esters [54-58], which then
induce the activation of the IB kinase (IKK). UV radiation from sunlight induces IL-1 and
TNF-α, and creates reactive oxygen species, which then lead to NF-κB-mediated
inflammation [59]. The kinase activity of IKK phosphorylates two serine residues (Ser32 and
Ser36) on IB proteins, which results in the ubiquitination and degradation of IB by the
proteasome.
The degradation of IB reveals the nuclear localization sequence of NF-B [52]. Free
NF-B (activated) can then translocate to the nucleus and bind to a NF-B consensus
sequence present within the promoter region of target genes, thereby upregulating the
expression of hundreds of genes, including cytokines (IL-1, -2, -6, etc.), immunoreceptors
(immunoglobin kappa light chain, MHC class I, etc.), cellular adhesion molecules (ICAM-1,
VCAM-1, ELAM-1), and many others [58]. As shown in Figure 1, the NF-B expression is
increased in dermal fibroblasts and thereby could be an important mediator involved in the
skin aging process.
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1126 Connie B. Lin and Michael D. Southall

Figure 1. Increased NF-B P65 protein in dermal fibroblasts as a factor of age. EMSA analysis of NF-
B P65 protein in dermal fibroblasts derived from skin of young and aged donors. NS= non-specific
binding.

4.2.2. Role of NF-B in the Regulation of Extracellular Matrix Protein Degradation

The NF-B pathway is a key regulator of inflammatory mediators in skin cells and has
been reported to be the final common pathway for the conversion of environmental insults
into inflammation in the skin. Activation of NF-B pathway leads to excessive activity of
MMPs, such as collagenase and elastase. In addition, the NF-B pathway also further induces
IL-1 and TNF-α, thus up-regulating its own pathway [60]. Study has demonstrated the
correlated role of NF-B induced MMP expression and the reduced amounts of collagen
produced by the fibroblasts because of its degradation by these enzymes [61]. The NF-B
pathway is therefore a primary factor in regulating the expression of matrix degrading
proteins and mediating intrinsic skin aging.

4.2.3. Role of NF-B in Regulation of Extracellular Matrix Protein Expression

The structure and amounts of extracellular matrix proteins, such as type I collagen, are
known to be disregulated during aging process, due at least in part to a transcriptional control.
A putative role for NF-B on the regulation of human COL1A1 gene was observed when
TNF-α was reported to increase NF-B activity and also produce a down-regulation in the
expression of COL1A1 [62]. Initially the mechanism whereby NF-B downregulated
COL1A1 expression was not well understood since the COL1A1 gene lacked a NF-B
consensus binding sequence in the promoter region; however, a number of other transcription
factors such as c-Krox, CBF, Sp1, and Sp3 can bind to the -112/-61 bp sequence of the
COL1A1 promoter in fibroblasts [63]. In studies of the COL1A1 promoter region, TNF-α was
found to inhibit promoter transcription activity through two elements located between -101 to
-97 bp and -46 to -38 bp of the COL1A1 promoter yet the suppression involved non-
identified protein interactions [64]. Studies in murine NIH-3T3 fibroblasts found that the
TNF-α induced inhibition of collagen promoter transcription activity was mediated by
interaction between the NF-B and Sp1 proteins [65].

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Figure 2. Activation of NF-B Results in Inhibition of ECM Protein expression. QPCR analysis of
collagen1 and elastin in primary normal human dermal fibroblasts treated with TNF to activate NF-
B.

In addition to the interaction with SP1, NF-B can decrease type I collagen expression
with the supplementary participation of CBF, Sp3, hc-Krox on the COL1A1 gene in primary
human skin derived fibroblasts [66]. A similar NF-B mediated inhibition of the COL1A2
gene expression has also been demonstrated [67]. While most published studies have focused
on the regulation of collagen expression, NF-B may be more widely involved in the
regulation of the family of ECM proteins. As shown in Figure 2, TNF-α can reduce
transcription of both COL1A1 and elastin gene transcription in human dermal fibroblasts.
Thus, inhibition of TNF-α and NF-B pathway could block UVB-mediated skin changes.
Indeed, Tanaka K. et al. reported that parthenolide and magnolol (known as NF-B
inhibitors) effectively inhibit the gene expression of MMP-1 mediated by NF-B in the cells
overexpressing p65 [68].

4.3. Implication of TGF- in the Regulation of Extracellular Matrix in Aged

Skin

In addition to NF-B, the pathogenesis of skin photoaging is also closely associated with
activated AP-1 and impaired TGF-/Smad [69-71], which are involved in the reduction, aging
and apoptosis of fibroblasts induced by UVB/UVA radiation and in the UV mediated down-
regulation of ECM production [3].
AP-1 is a transcription factor comprising of Jun and Fos family proteins, in which c-Fos
is constitutively expressed and c-Jun is UV inducible [46, 72-74]. In UV irradiated skin,
elevated c-Jun together with constitutively expressed c-Fos increases activation of AP-1
mediated by UV-activated growth factors and cytokines via MAP kinase pathways [42]. AP-1
expression is increased in aged human skin in vivo and in aged dermal fibroblasts [75].
Activation of AP-1 results in decreases in TGF- and the TGF-receptor, leading to
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1128 Connie B. Lin and Michael D. Southall

upregulation of MMP family members, down-regulation of type I collagen [76, 77], and thus
a decrease in collagen production [37, 46, 78].

4.3.1. TGF- Signaling Pathway and Regulation

The TGF- signaling pathway is involved in multiple cellular processes including
proliferation, and differentiation of fibroblasts [79, 80]. TGF- together with TGF-
receptor and Smad protein jointly constitute TGF-/Smad signaling pathway, through which
TGF- can activate dermal fibroblasts, increase fibroblast proliferation, stimulate collagen
I/III and elastin synthesis in fibroblasts, repress MMP activity, enhance fibronectin
expression, decrease collagen fibers degradation and ultimately upregulate the secretion of
ECM production [71, 80-83]. The TGF- family includes TGF1, TGF2 and TGF3. TGF-
 signaling is mediated by the type-I and type-II receptors, TR1 and 2 [84, 85], while TR3
is indirectly involved in signaling by enhancing the response to TGF- [86]. TR1 and 2 have
intrinsic serine/threonine kinase activity. TGF- binds to TR2, which recruits and
phosphorylates TR1, which can then phosphorylate Smad proteins that propagate the signal
[87, 88]. TGF- effects are mediated through activation of intracellular signal transducers
such as Smad2/3 and are antagonized by Smad7 [89-96]. The expression of Smad7 is
upregulated by TGF- itself, suggesting that the induction of Smad7 may serve as a negative
feedback mechanism in regulating TGF- signaling [97]. Besides activation of Smad
pathways, TGF- induces many other signaling molecules, including p38 mitogen activated
protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt, and Rho-like GTPas [98, 99].
The regulation of TGF- pathways occur at different levels. TGF- is synthesized and
secreted as a latent precursor protein containing the mature domain and a latency-associated
peptide (LAP) region, which needs to be cleaved by the convertase family of endoproteases
before it becomes biologically active. Alkalization of the trans golgi network/endosome
system, e.g., by proton pump inhibitors, could suppress TGF- processing and decrease
mature bioactive TGF- secretion [100, 101]. TGF- binds intracellularly to the latent TGF-
binding protein (LTBP) family proteins to form a large latent complex which associates
extracellularly with fibrillin-rich microfibrils, by which fibrillin microfibrils can mediate
tissue homeostasis via sequestration of TGF- [102, 103]. Activation of TGF- and its
binding to TGF- receptor require the removal of LAP and LTBP from the latent complex.
Therefore, the bioavailability of active TGF- depends on proteolytic processing that releases
active TGF-. Most of the activations of TGF- are mediated by enzymes such as plasmin,
MMP2 and 9 [104]. Upon activation, a number of proteins can bind and modify TGF-
activity, e.g., fibronectin promotes TGF- activity [105], whereas decorin inhibits TGF-
activity [106, 107].
Therefore, the results of damage in elastic fibers may impact not only on the mechanical
but also the biochemical function of the tissue including TGF- signaling. Meran et al.
reported that the TGF--dependent proliferation is also mediated through the HA and HA
receptor CD44 [108]. In addition, TGF- receptors and Smads are subject to post-
translational modifications, including phosphorylation, sumoylation and ubiquitylation [109].
Furthermore, TGF- signal is also associated with a cross-talk with non-Smad pathways such
as MAPK and NF-B [98, 99].

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4.3.2. Physiological Significance of TGF- in the Regulation of ECM

TGF- is the most prominent growth factor that regulates the synthesis of ECM
molecules [110-113]. As shown in Figures 3, TGF- stimulates elastin and collagen gene
expressions in dermal fibroblasts and reverses UVA-induced reduction of ECM gene
expression. TGF- has been shown to stimulate elastin production by up-regulating the
elastin promoter [110, 111], leading to increased levels of tropoelastin (green) and fibrilin-1
(elastin accessory protein, brown) in cultured human skins as shown in Figure 4.

Figure 3. TGF- increases ECM (procollagen I and elastin mRNA) levels and reverses UVA-induced
reduction of ECM gene expression. QPCR analysis of Collagen1 and Elastin in primary normal human
dermal fibroblasts treated with UVA in the absence and presence of TGF-.

Figure 4. TGF- increases tropoelastin and fibrillin-1 protein levels in human skins.
Immunohistochemical staining of tropoelastin (green) and fibrillin-1 (brown) in human skin explants
treated with and without TGF- in the cell culture media.
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1130 Connie B. Lin and Michael D. Southall

TGF- reverses deficient expression of type I collagen in cultured fibroblasts of a patient

with metageria, a cutaneous condition characterized by premature aging [114] and overcomes
UVA induced reduction of ECM gene expression (Figure 3). Furthermore, TGF- can also
stimulate fibroblasts proliferation and restrain fibroblast apoptosis [115].
Among the downstream mediators of TGF-1, connective tissue growth factor (CTGF,
also known as CCN2) is the major growth factor that is responsible for collagen synthesis
[116, 117]. In fibroblasts, TGF- induces CTGF expression, which can bind directly to TGF-
 and enhances its activity. Therefore, CTGF is considered as an important cofactor with
TGF-1 for collagen synthesis. CTGF can also bind to specific intergrins, proteoglycans, and
GAGs in the ECM, resulting in increased binding to TR1 and TRII, and enhanced collagen
synthesis [118]. Quan et al. demonstrated that endogenous production of TGF- and CTGF in
human dermal fibroblasts regulates type I procollagen expression in human skin and the
diminished expression of CTGF and TGF-/Smad signaling is responsible for the progressive
loss of dermal collagen, suggesting the physiological significance of TGF- and CTGF in
maintaining dermal homeostasis [119]. It has been shown that in vitro, TGF- mediates
fibroblast-myofibroblast differentiation, leading to numerous TGF-1-dependent responses,
including reduction in the expression of HA synthase 2 (HAS2) and HA synthesis [120]. On
the other hand, TGF- downregulates the expression of ECM degrading enzymes such as
MMP-1 (collagenase) and MMP-3 (stromelysin) [121]. By enhancing ECM production and
reducing MMPs expression, TGF- plays a pivotal role in the production of ECM in dermis.

4.3.3. TGF- Signaling Pathway in Aged Skin

It has been shown that TGF-1, TRI and TRII are downregulated in aged fibroblasts in
vitro [122, 123] and in aged skin in vivo [4]. Aging related impairment of the TGF-/Smad
pathway, therefore, may play a role in reduced ECM production. UV irradiation has been
shown to impair the TGF- signaling pathway by reducing TR1 expression [44, 69, 124-
126], and its targeted genes such as reduction of CTGF and induction of cysteine-rich protein
61 (also known as CYR61; CCN1) [119, 127-129]. UV increases Smad 7 via AP-1 that
interferes with TGF-/Smad 2/3 signaling, leading to decreased TGF-/smad pathway and
collagen production, and to epidermal hyperplasia in human skin [44, 69, 124; 126].
Activation of AP-1 also decreases TGF- receptors [124]. Decorin is a small leucine-rich
proteoglycan and is distributed along collagen fibrils where it regulates the collagen synthesis
and fibril assembly. Adult human skin contains a truncated form of decorin, which is not
detected in fetal skin [130]. Age-related structure changes in decorin may be involved in
changes in collagen matrix assembly during the aging process [131]. There is evidence
showing that TGF- may be sequestered and become inactive in aged skin by its binding to
decorin [106, 132].
In vivo studies demonstrated that UVA1/UV remarkably reduces TGF- 1 and its
receptor expression, and blocks Smad3/4/7 signaling pathway [133]. It has been reported that
17- estrodiol stimulates TGF- production and signaling, leading to inhibition of bone
resorption in human osteoblast-like cells [134] and topical estrogen improves cutaneous ECM
alterations by stimulating TGF- and TRII expressions in aged human skin in vivo [82].
TGF- 1 neutralizing antibody blocked the increased type I procollagen production induced
by 17 -estradiol in cultured fibroblasts, suggesting that the increased level of TGF- 1 by

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topical estrogen in aged human skin may mediate estrogen-induced collagen synthesis [82],
and reinforces the important role of TGF- in the regulation of dermal ECM in skin.

CONCLUSION
Skin aging has been described as the culmination of 2 independent processes that
combine to produce the phenotypic characteristics of skin aging, namely the development of
wrinkles, fine lines, and loss of elasticity. These 2 processes include the decreased
expressions of ECM proteins [130], and the stimulation and increased expression of MMPs
which degrade ECM proteins [47, 48]. Matrix degradation in aged skin is mediated by NF-
B, activation of AP-1, and by inhibition of TGF-β signaling; while reduction of ECM
synthesis is regulated by disturbed signaling pathways of many growth factors, among which
TGF- is the most prominent. Down-regulation of TGF-, in addition to NF-B and AP-1-
mediated transcriptional expression of MMPs, contributes to reduced ECM production
observed in aged skin.
The “gold standard” clinical treatment of both intrinsically aged and photaged skin is the
topical application of a class of molecules, retinoids [135, 136]. Retinoid compounds have
been shown to negatively regulate AP-1 and MMPs via a post-transcriptional mechanism, in
which retinoid antagonized AP-1 activation by inhibiting c-Jun induction [74, 137, 138]. The
positive effects of retinoids on photodamaged skin are mediated by upregulating both
collagen and elastin (see reviews in [139-142]). New findings on the molecular mechanisms
involved in the regulation of ECM production in the skin can help identify new targets to
modulate ECM protein expression and thereby have a significant effect on the skin aging
process.
It is speculated that NF-B inhibitors could effectively block UV- and NF-B-mediated
gene expression and skin changes, and could be useful in preventing skin aging.
Furthermore, activation of TGF-/CTGF signaling cascade could reverse aging associated
reduction and disorganization of dermal ECM.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 53

IMPROVED CELL METABOLISM AND

STRENGTHENING OF THE EXTRACELLULAR
MATRIX BY NICOTINAMIDE, AND COPPER
FOR ANTI-SKIN AGING

Neena Philips, Philips Samuel, Halyna Siomyk, Harit Parakandi,

Hui Jia, Sesha Gopal and Hossam Shahin
School of Natural Sciences, Fairleigh Dickinson University, Teaneck, NJ, US

ABSTRACT
The skin aging mechanisms include cellular senescence and diminished extracellular
matrix (ECM) integrity from intrinsic and extrinsic factors, such as solar ultraviolet (UV)
radiation that cause oxidative stress, inflammation and damage to DNA. The clinical
manifestations of skin aging are skin thinning or coarseness, wrinkles, impaired wound
healing, and propensity for cancer. The structural ECM proteins are primarily fibrillar
collagens, in order of predominance types I, III, and V; and elastin fibers, which are
formed of elastin and fibrillin. The formation of collagen is closely associated with the
expression of heat shock protein-47 (HSP-47). The matrixmetalloproteinases (MMPs)
degrade the ECM. The predominant classes of MMPs include collagenases (MMP-1, 3)
and gelatinases (MMP-2, 9), which degrade the interstitial collagen and basement
membrane. They are inhibited by tissue inhibitor of metalloproteinases (TIMPs),
especially TIMP-1 and TIMP-2. With skin aging, there is reduced expression of the ECM
proteins by the dermal fibroblasts, and increased ratio of MMPs to TIMPs.
Our laboratory has recently investigated the anti-skin aging effects of copper, and
nicotinamide. Nicotinamide, an amide derivative of niacin or vitamin B 3, is essential for
energy metabolism, and improves skin appearance by reducing wrinkles and increasing
elasticity. Copper is a cofactor to lysyl oxidase (cross links collagen and elastin),
respiratory chain enzymes, and stimulates the remodeling of the ECM in wounds.
Nicotinamide, and copper stimulate structural ECM proteins in dermal fibroblasts, and


Corresponding author: Correspondence Address: Neena Philips, Ph.D.,Professor of Biology, School of Natural
Sciences, Fairleigh Dickinson University, Teaneck, NJ 07666. Phone: 201 692 6494. Fax: 201 692 7349.
Email: [email protected]. [email protected].
 Free ebooks ==> www.Ebook777.com
1142 Neena Philips, Philips Samuel, Halyna Siomyk et al.

would be effective as supplements or topical applications in the alleviation of skin aging

through the improvement of cellular metabolism as well as the structure of the ECM.

Keywords: Skin aging, collagen, elastin, matrixmetalloproteinases, copper, nicotinamide

INTRODUCTION
The basis of skin aging is the cellular response to inflammation and oxidative stress,
which activates inflammatory signal transduction pathways to activate the expression of
proteases that remodel the extracellular matrix (ECM). The atrophy of the ECM results in the
aged appearance of the skin, and compromises skin function. Our current research suggests
that nicotinamide, and low/physiological doses of copper are beneficial to the ECM of the
skin, and thereby to anti-skin aging.
We review our laboratory’s current research on skin aging as (i) oxidative stress and
inflammation; (ii) ECM proteins; (iii) the mediating signal transduction pathways; and (iv)
nicotinamide, and copper for skin health.

I. SKIN AGING: OXIDATIVE STRESS AND INFLAMMATION

One of the major causes of oxidative stress and inflammation in the skin is the
accumulation reactive oxygen species (ROS) due to mitochondrial metabolism, aging, and
exposure to environmental pollutants or ultraviolet (UV) radiation. Skin aging is from the
resultant loss of cell viability, membrane integrity, and ECM structure. The alteration to the
ECM structure is reflected in the loss of structural collagen/elastic fibers and increased ECM
degrading proteases in intrinsic aging; and in addition elastosis, skin thickening, and
coarseness in photoaging (Carbonare et al., 1992; Darr et al., 1997; Fitzpatrick, 1988;
González et al., 1999; Kligman, 1986; Kligman, 1988; Mc Bride et al., 1991; Philips et al.,
2004a; Philips et al., 2004b; Philips et al., 2007; Philips et al., 2009a; Philips et al., 2010a;
Preston et al., 1992). The solar UV spectrum consists of UVC radiation (200-290nm) that is
absorbed by the atmospheric ozone but could penetrate the upper skin surface; UVB radiation
(290-320 nm) that penetrates upto the dermis; and UVA radiation (320-400nm) that
penetrates deeper into the dermis (Nichols et al. 2010). UV radiation also damages
mitochondrial DNA, which leads to senescence and increased accumulation of ROS
(Krutmann, 2001).
The cellular ROS include superoxide, hydroxyl radicals, hydrogen peroxide, and singlet
oxygen, produced by the mitochondria and the tissue infiltrated leukocytes. The ROS cause
direct harm to DNA, proteins and lipids; detected as 8-oxo-7, 8-dihydro-2’-deoxyguanosine
(8-oxo-dG), pyrimidine dimmers, carbonyl amino acid derivatives, lipid peroxidation,
malonaldehyde, and lipid inflammatory mediators (Briganti et al., 2003; Callaghan et al.,
2008; Melnikova et al., 2005; Nichols et al., 2010; Surjana et al., 2010). The oxidative
damage to ECM proteins includes protein oxidation, pathological cross-links, advanced
glycation end products (AGE), increased tissue stiffness, calcification, L-aspartate to D-
aspartate racemization, and mechanical fatigue (Sims et al. 1996; Bailey et al. 2001; Yaar et

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al., 2007; O’Rourke et al., 2007; Robert et al., 2008). The oxidative damage causes cell-cycle
arrest, apoptosis, erythema and premature aged appearance of the skin. UVA and UVB
radiation cause ROS mediated DNA damage and cellular damage. The mechanism to UVA
mediated damage is solely through the generation of ROS, whereas UVB radiation causes
both direct damage as well as damage through ROS (Nichols et al., 2010; Walscheck, 2001)).
ROS is an inducer of pro-inflammatory genes. An amplified cascade results from the
production of inflammatory mediators by ROS signaling, and the formation of more ROS by
inflammation. The inflammatory mediators are released from leukocytes, damaged tissue, and
endothelial lining of blood vessels. They include the plasma mediators (bradykinin, plasmin,
fibrin), lipid mediators (prostaglandins, leukotrienes and platelet activating factor); and the
inflammatory cytokines [interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-
 (TNF-)]. The mediators collectively increase vascular permeability, leukocyte
chemotaxis, release of ROS and proteases by leukocytes, and the release of histamines from
mast cells (Hruza et al., 1993; Kindt et al., 2007).
The counteracting cellular antioxidants, which are outbalanced by ROS in skin aging,
include enzymes such as catalase and glutathione peroxidase, and small molecules such as
glutathione, ascorbate, -tocopherol, and carotene (Callaghan et al., 2008). The prevention of
skin aging has been focused on compounds with anti-oxidant and anti-inflammatory
properties (Clark et al., 1996; Damiani et al., 2006; Dwyer et al., 2001; Ingram, 1994; Jain et
al., 1994; Krinsky, 1994; Svobodova et al., 2006; Toyoda et al., 1995)

II. SKIN AGING: EXTRACELLULAR MATRIX (ECM)

The skin is the outer protective layer of the body, and is composed of the epidermis,
dermis and the subcutaneous tissue. The epidermis consists of differentiating keratinocytes
overlaying the basement membrane, melanocytes and langerhans cells. The dermis, below the
basement membrane, is composed of the ECM, the fibroblasts that synthesize the ECM, and
the vasculature. The subcutaneous layer is primarily adipose tissue (Callaghan et al., 2008).
The predominant structural ECM proteins, which provide skin integrity and function, are
collagen and elastin fibers. Skin aging is primarily from the disintegration of the
collagen/elastin fibers by the matrixmetalloprotienases (MMP).

II.a. Matrixmetalloproteinases/Elastases

The ECM proteolytic enzymes are produced by epidermal keratinocytes, dermal

fibroblasts, and neutrophils in the mediation of skin aging (Brennan et al., 2003; Cho et al.,
2007; Doyle et al., 1997; Jimenez et al., 2006; Khorramizadeh et al., 1999; Labat-Robert et
al., 2000; Lee et al., 2008; Philips et al., 2004a; Philips et al., 2007; Philips et al., 2009a). The
MMPs or matrixins are central to the remodeling of the ECM.
The MMPs are expressed as prepro-enzymes and secreted as inactive pro-MMPs (Nagase
et al., 1999). Most of the MMPs are composed of a propeptide domain, a catalytic domain
with a zinc binding motif, and a C-terminal hemopexin like domain that is essential for its
catalytic activity (Bode et al., 1993; Gomis-Rüth et al., 1996; Herouy et al., 2001; Van Wart
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1144 Neena Philips, Philips Samuel, Halyna Siomyk et al.

et al., 1990). The propeptide domain has a unique cysteine that links it to the catalytic zinc in
its inactive pro-MMP form (Van Wart et al., 1990; Bode et al., 1993; Gomis-Rüth et al.,
1996; Herouy et al., 2001). Proteases, such as plasmin, proteolyze the pro-peptide domain of
MMPs and thereby activate them (Herouy et al., 2001).
The MMPs have been classified on the basis of their susbtrate specificity: the interstitial
collagenases (MMP-1) cleave the fibrillar collagens, the gelatinases (MMP-2, 9) cleave the
basement membrane collagens and elastin, the stromelysines (MMP-3, 10) degrade the
basement membrane proteins, the membrane-type MMPs act on pro-MMPs and fibrillar
collagens, and the other MMPs (metalloelastase: MMP-12) degrade elastin (Bode et al., 1993;
Gomis-Rüth et al., 1996; Herouy et al., 2001; Nagase et al., 1999; Van Wart et al., 1990). The
activator protein -1 (AP-1) transcription factor is an important regulator of the transcription of
MMPs, and thereby to skin aging. MMPs have recently been classified on the basis of their
promoters: group I MMPs (MMP-1, 3, 7, 9, 10, 12, 13, 19 and 26) containing TATA box and
AP-1 site, group II MMPs (MMP-8, 11, 21) without the AP-1 site; and group III MMP-2, 14,
28) without both the TATA box and AP-1 site (Yan et al., 2007).
The pro- and active forms of MMPs are inhibited by the tissue inhibitors of MMPs or
TIMPs (Herouy et al., 2001; Verstappen et al., 2006). Four types of TIMPs (TIMP-1 to 4)
have been identified (Verstappen et al., 2006). Each of the TIMPs binds to most of the
MMPs, though TIMP-1 has preference for MMP-1 and TIMP-2 for MMP2 (Herouy et al.,
2001). Conversely, MMP-2 is activated by TIMP-2 in collaboration with MT1-MMP (Herouy
et al., 2001). The structure of TIMPs consists of an N-terminal conserved region essential to
binding to the active site of MMPs, 6-loops, and a junction between the N- and C- terminal
domains (Verstappen et al., 2006).
The constitutive expression of the MMPs results in the fragmentation of collagen and
elastin fiber proteins with aging. The basal levels of MMPs are higher in aged skin relative to
young skin (Millis et al., 1992). The fragmentation of the collagen and elastin fibers is
accelerated by the increased expression of elastases and MMPs in response to inflammation
and oxidative stress in photoaged skin (Fisher et al., 1996). In addition, exposure to UV
radiation reduces the expression of TIMPs (Philips et al., 2009c).

II.b. Collagen

The predominant skin ECM protein is collagen. It is central to the interstitial ECM as
well as to the basement membrane. There are about 28 types of collagen, the predominant
skin collagen being type I collagen. Collagens are homo or hetero trimeric triple helical
proteins, composed of repeating Gly-X-Y motifs where X or Y being proline or
hydroxyproline (Lodish et al., 2008). The unique properties of the different collagens are
based on the lengths of the triple helical segments, interruptions to the triple helix, and amino
acid modifications.
Collagens are classified as fibrillar collagens (types I, II, III, V), fibril associated
collagens (types VI, IX), sheet forming anchoring collagens (types IV, VII, XV),
transmembrane collagens (types XIII, XVII), and host defense collagens (Lodish et al., 2008).
The basement membrane is composed largely of type IV collagen, and the dermal collagen
fibers are formed of the type I (90%), III, V, and VII collagens (Callaghan et al., 2008).

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 Improved Cell Metabolism and Strengthening of the Extracellular Matrix … 1145

Collagen is synthesized primarily by the fibroblasts (Philips et al., 2009b; Philips et al.,
2010b).
Skin aging is associated with fibroblast senescence, reduced synthesis of collagen, and
increased degradation of collagen fibers by MMPs (Philips et al., 2009b; Philips et al., 2010b;
Philips et al., 2012; Wlaschek et al., 2001). The different types of MMPs collectively degrade
the ECM: MMP-1 degrades interstitial collagenase, MMP-2 and 9 further degrade broken
interstitial collagen and the basement membrane, and MMP-3 degrades type IV collagen
(Fisher et al., 1996).

II.c. Elastin

The elastin fibers provide stretch-recoil properties and resilience to the skin. They are
composed predominantly of an elastin core (90%) and surrounded by fibrillin microfibrils
(Mecham et al., 1994).
Elastin is expressed as soluble hydrophobic tropoelastin, rich in proline, valine, lysine,
alanine, glycine, leucine and isoleucine. It is targeted to the cell surface, transferred to the
microfibril scaffold, and cross linked by lysyl oxidase and transglutaminase (Keeley et al.,
2002; Kielty et al., 2002; Rock et al., 2004).
Lysyl oxidase catalyzes the deamination of the lysine residues in elastin to form
desmosines, and transglutaminase crosslinks elastin to the microfibrils (Keeley et al., 2002;
Kielty et al., 2002; Rock et al., 2004).
The microfibils are composed of fibrillin (FBN). There are three known FBNs (FBN 1-
3), though FBN-1 is the most abundant in mature tissue and FBN2 is most associated with
elastin (Kielty et al., 2002). The FBNs are cysteine rich highly disulphide bonded
glycoproteins, and contain calcium binding epidermal growth factor like domains (Kielty et
al., 2002). They are secreted in pro-forms, processed extracellularly by furine/PACE like
activities, assembled in parallel bundles of head to tail monomers, bound by calcium and
organized into microfibrils (Kielty et al., 2002; Kinsey et al., 2008; Robert et al., 2002). The
assembly of the microfibrils is with the aid of heparin, heparan sulphate and fibronectin, and
it associates with other glycoproteins such as microfibril associated glycoproteins and
fibullins (Kielty et al., 2002; Kinsey et al., 2008; Zhang et al., 1995). The microfibrils
sequester transforming growth factor- (TGF-β) in its latent form, and intereact with the cell
surface integrins through their RGD (Arg-Gly-Asp) domains (Bax et al., 2003; Hubmacher et
al., 2006).
TGF-β is the primarily stimulator of the collagen and elastin fiber formation and
deposition (Philips et al., 2009a; Philips et al., 2009b). The activation of integrins, with ECM
remodeling, results in activation of intracellular signal transduction pathways and cellular
activities (Lodish et al., 2008).
The remodeling of the elastin fibers is by the serine protease neutrophil elastase, and the
matrixmetalloproteinases (MMP) (Kielty et al., 1994; Chakraborti et al., 2003; Tsuji, 2001).
Elastin is reduced with intrinsic aging, and results in the loss of skin firmness and resiliency
(Philips et al., 2007a; Philips et al., 2009a; Philips et al., 2010a; Philips et al., 2010b). UV
radiation depletes the microfibrillar network in the epidermal-dermal layer and the dermis,
which contributes to the aberrant elastic fibers and wrinkles in photoaged skin (Philips et al.,
2009; Philips et al., 2010; Watson et al., 1999; Watson et al., 2001; Yaar et al., 2007). The
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1146 Neena Philips, Philips Samuel, Halyna Siomyk et al.

fragmentation of the elastic fibers results in disorganized elastotic material, with altered
architecture, and the resultant loss of tissue elasticity.

III. SKIN AGING: SIGNAL TRANSDUCTION PATHWAYS

The pathways activated in skin aging are predominantly the mitogen activated protein
kinase (MAPK) pathways, and the NF-kB/p65 pathway (Callaghan et al., 2008; Nichols et al.,
2010). The MAPK pathway is comprised of the extracellular signal-regulated kinase 1/2
(ERK1/2), c-Jun-N-terminal-kinase, and p38 proteins. The activation of MAP kinase
pathway, through the receptor tyrosine kinase, results in the activation of transcription factor
AP-1 (Callaghan et al., 2008). The transcription factor AP-1 stimulates the transcription of
several MMPs that collectively degrade the ECM, such as MMP-1, MMP-2/9 and MMP-3
(Fisher et. al, 1996). Further, AP-1 inhibits the transcription of type I collagen gene
(Callaghan et. al, 2008). Hence, the damage to the ECM and tissue integrity is from the
reduced expression of the structural ECM proteins as well as its enhanced degradation by
MMPs. The JNK and p38 pathways play a major role in the UVA radiation mediated increase
in AP-1 and COX-2 expression, and are targets for chemoprevention of skin aging and cancer
(Bachelor et al., 2004)
The NF-kB pathway is activated by the active cytoplasmic I-kB kinase, which is activated
by oxidative stress and inflammation. Active I-kB kinase phosphorylates and degrades I-kB,
inhibitor of NF-kB (p65/p50 heterodimeric protein) transcription factor (Lodish et al., 2008).
The release of NF-kB, from its inhibitor (I-kB), results in its translocation to the nucleus to
activate the inflammatory cytokines, and cyclooxygenase (COX-2, for the synthesis of
prostaglandins (Lodish et al., 2008). The NF-kB activation is redox sensitive, and its
activation is associated with UVA and UVB radiation mediated oxidative modification of
cellular membrane components (Fisher et al., 1996; Vile et al., 2008).

IV. ANTI-SKIN AGING: NICOTINAMIDE AND COPPER

The manifestation of skin aging is from degeneration of the ECM, which is primarily due
to oxidative stress and inflammation as well as the associated activation of MAPK and NF-kB
pathways. Hence, a beneficial preventive or supplemental regimen for skin health is vitamins
or minerals, which provide antioxidant and anti-inflammatory benefits and/or inhibit the
MAPK and NF-kB pathways. Two such micronutrients are nicotinamide and copper, which
in our recent research indicates to strengthen the ECM and thereby prevent skin aging. In
addition, both nicotinamide and copper are essential to energy metabolism as precursors of
metabolic coenzymes or electron transport components; and provide antioxidant potential by
reducing oxidized cellular antioxidants or as cofactors to antioxidant enzymes. Nicotinamide,
and copper may provide alternatives to retinol, which though prescribed by dermatologists for
anti-skin aging is not well tolerated.

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 Improved Cell Metabolism and Strengthening of the Extracellular Matrix … 1147

IV.a. Nicotinamide

Nicotinamide is essential for energy metabolism, cellular stress response, inhibition of

MAPK and NF-kB pathways, antioxidant activity, and the stimulation of collagen/elastin
fiber components.
Nicotinamide (pyridine-3-carboxamide) is the amide derivative of niacin or Vitamin B3,
which is obtained from diet as niacin or nicotinic acid or through synthesis from tryptophan
(Maiese et al., 2009; Surjana et al., 2010; Benavente et al., 2009). Its predominant
physiological role is as precursor to the coenzymes nicotinamide adenine dinucleotide
(NAD+) and its phosphorylated form NADP+, which are essential to catabolism (glycolysis,
citric acid cycle, electron transport for oxidative phosphorylation or ATP synthesis) and
anabolism (synthesis of biomolecules as NADPH).
NAD+ is required for cellular stress response through the regulation of the activity of
sirtulin 1 (SIRT 1), a histone deacetylase that facilitates cellular stress response, and as a
substrate for poly (ADP-ribose) polymerase (PARP ) that plays a role in DNA repair and
genomic stability (Maiese et al., 2009; Surjana et al., 2010; Liu et al., 2009).
Nicotinamide has antioxidant and anti-inflammatory properties through the inactivation
of the MAPK and NF-kB pathways (Ahn et al., 2003; Grange et al., 2009). It inhibits the
phosphorylation of ERK and JNK MAP kinases, and the degradation of IkB kinase (Grange
et al., 2009). The inhibition of NF-kB in turn inhibits the inflammatory cytokines such as IL-
1, IL-6 and TNF, as well as the lipid inflammatory mediators such as prostaglandins and
cyclooxygenase (Ahn et al., 2003; Lappas et al., 2011). Further, ROS are generated by
NADPH oxidase in the absence of NAD+, in the use of glutamate as an alternate energy
source (Benavente et al., 2008). Nicotinamide can quench singlet oxygen, as well as other
ROS, and inhibit lipid peroxidation and photosensitization induced cell toxicity (Benavente et
al., 2008). NADP and NADPH are essential to the reduction of the oxidized cellular
antioxidant, glutathione (GSSG to GSH), and thereby to the continual scavenging of ROS
(Kamat et al., 1996).
Nicotinamide is an ingredient in skin care creams, and there are several reports on the in-
vivo anti-skin aging effect of nicotinamide. The application of a cosmetic with 4%
nicotinamide reduces wrinkles around the eyes (Kawada et al., 2008). It also reduces facial
wrinkles, hyperpigmented spots, sallowness (yellowing), and aged appearance of skin (Bisset
et al., 2005).
Nicotinamide in combination with retinol and 7-dehydrocholesterol has anti-acne effect
through the reduction of inflammatory cytokines and MMPs, and the induction of TIMPs
(Emanuele et al., 2012). Nicotinamide has a role in retinoic acid biosynthesis, and is more
tolerant than retinoic acid in cosmetics (Fu et al., 2010; Pinkas-Sarafova et al., 2005).
We have investigated the mechanism to the anti-skin aging effects of nicotinamide in
dermal fibroblasts (unpublished data). Nicotinamide stimulates the expression of collagen
(types I, III, V), elastin fiber components (elastin, fibrillin), and inhibits MMPs expression in
non-irradiated and UV radiated fibroblasts (unpublished data). There is potential for
nicotinamide’s presence in cosmetics from its metabolic role and its beneficial regulation of
the ECM, which are essential for anti-skin aging.
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1148 Neena Philips, Philips Samuel, Halyna Siomyk et al.

IV.B. Copper

Copper has anti-skin aging properties, is a cofactor to metabolic enzymes, and is essential
to wound healing. Conversely, it is also considered an environmental pollutant and may cause
oxidative stress and skin aging. Our research indicates that the differential effects of copper
are dose dependent.
Copper powder penetrates the stratum corneum (Hostynek et al., 2006). Copper is
absorbed from topical applications of copper containing ointments, and the use of copper
oxide containing pillow cases improves skin appearance through the release of copper ions
from pillow cases in the presence of moisture (Borkow et al., 2009).
Copper has anti-skin aging effects through the stimulation of collagen and elastin fiber
components, and antioxidant properties. In our laboratory, copper ions at lower
concentrations (0.05nM to 0.5nM) stimulated the expression of collagen (types I, II, and V),
and the elastin fiber components (elastin, fibrillins); and inhibited cellular oxidative effects
such as membrane damage and lipid peroxidation (Philips et al., 2012). Copper also
stimulated heat shock protein-47 (HSP-47), which is essential to collagen fibril formation
(Philips et al., 2012). Copper nanoparticles promote crosslinked elastin matrices (Kothapalli
et al., 2009). The mechanism to copper’s stimulation of collagen and elastin may be through
the induction of transforming growth factor- (TGF-), known to stimulate the deposition of
ECM (Philips et al., 2012).
Copper has also been investigated in complex with a matrix-derived tripeptide (glycyl-
histidyl-lysine or GHK) (Cu-GHK) for its antiaging effects. Cu-GHK (1 pM to 1 nM )
stimulates expression of collagen and elastin in-vivo and in-vitro, stimulates TIMP-1, and
inhibits clinical characteristics of skin aging (Maquart et al., 1993; Maquart et al., 1988;
Pickart et al., 2008; Siméon et al., 2000).
Copper is a cofactor to metabolic and antioxidant enzymes, including lysyl oxidase,
superoxide dismutase, and cytochrome C oxidase (Abreu et al., 2010; Atsawasuwan et al.,
2008; Rucker et al., 1998; Smith-Mungo et al., 1998; Szauter et al., 2005; Zuo et al., 2010).
Lysyl oxidase is essential to the oxidation of lysyl and hydroxyllysine residues in collagen
and elastin for crosslinking (Atsawasuwan et al., 2008; Rucker et al., 1998; Szauter et al.,
2005). Aging and senescence are associated with alterations in lysyl oxidase (Atsawasuwan et
al., 2008; Rucker et al., 1998; Szauter et al., 2005).
Copper is essential to wound healing (Borkow et al., 2008; Borkow et al., 2010;
Voruganti et al., 2005). Wound dressings with copper oxide particles aid wound healing in
diabetic mice, by inducing the expression of pro-angiogenic factors (Borkow et al., 2010;
Gorter et al., 2004). Copper at high concentrations, upto 200M, stabilize fibronection cables
in tissue engineering for wound care (Ahmeda et al., 2004).
However, copper is considered an environmental pollutant, which is released from
industry, fireworks, and agricultural biocidal agents (Davis, 2002; Schiff et al., 2007; Smith,
2000). It can induce oxidative stress through the formation of ROS through Fenton and
Haber-Weiss reactions, reduce glutathione levels, and the induce MAPK pathway (reviewed
in Jomova and Valko, 2011). Myelinopathy has been reported with the accumulation of
copper and lipid peroxidation, followed by inflammation (Viquez et al., 2008). Copper’s role
in oxidative stress and disease is connected to its ratio to zinc in degenerative diseases (Guo et
al., 2011; Mezzeti et al., 1998).

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The negative effects of copper may be at its higher concentrations. Copper stimulated the
expression of MMP-1 at higher concentrations (1-100 M) at the protein, mRNA and
transcriptional level, suggesting regulation at the gene level, and its potential for skin aging
(Philips et al., 2010). Copper did not alter the expression of TIMP-1 to counteract its
induction of MMPs (Philips et al., 2010). Copper at the higher concentrations (30-100 M)
also stimulated the expression of interleukin-8 (IL-8), at the protein, mRNA and promoter
levels, suggesting alterations at the gene levels (Philips et al., 2010). The stimulation of IL-8
may counteract some of the effects of copper’s induction of MMP through the stimulation of
collagen or conversely support inflammation through the chemotaxis of leukocytes. Copper at
low doses would be beneficial to skin aging through its role in the stimulation of collagen and
elastin fiber components, metabolism, and antioxidant activity.

CONCLUSION
Skin aging results from cellular oxidative stress and inflammation, which activates the
MAPK and NF-kB signaling pathways to activate AP-1 and NF-kB transcription factors that
in turn stimulate the expression of MMPs. MMPs are the predominant cause of the atrophy of
the ECM, which is superimposed on the reduced expression of these structural ECM proteins.
The deterioration of the ECM structure manifests as aged skin appearance.
A preventive or supplemental regimen to skin or systemic health is the removal of
inflammation and oxidative stress. Vitamins with their antioxidant and anti-inflammatory
properties would support a preventive or treatment role. Micronutrients that have been found
to be beneficial to the appearance of the skin, in our laboratory current research, are copper
and nicotinamide. Nicotimamide through its role in cellular energetics, cellular stress
response, inactivation of the oxidative and inflammatory MAPK and NF-kB pathways, and
stimulation of the collagen/elastin fiber components as well as the inhibition of the MMPs
would be beneficial to the appearance of skin.
Copper at low doses would be beneficial to skin aging through its role as a cofactor for
metabolic and antioxidant enzymes; as well as its stimulation of collagen and elastin fiber
components.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 54

SKIN MORPHOLOGY OF CAUCASIAN WOMEN

DURING AGING

H. Zahouani1, R. Vargiolu1, C. Guinot2,3, E. Tschachler2

and F. Morizot,4
1
University of Lyon, Laboratory of Tribology and Dynamic of Systems
UMR-CNRS 5513. ENISE – ECL, Ecully, France
2
CE.R.I.E.S, 20 rue Victor Noir, Neuilly Sur Seine cedex, France
3
Computer Science Department, Ecole Polytechnique,
Université de Tours, 64 rue Jean Portalis, Tours, France
4
Department of Dermatology, University of Vienna Medical School,
Akh – Währinger Gürtel Vienna, Austria

ABSTRACT
The structuring of the dermis with a network of collagen and elastic fibres gives a
three-dimensional structure to the skin network with directions perpendicular and parallel
to the skin surface. This three-dimensional morphology prints on the surface of the
stratum corneum a three dimensional network of lines which express the mechanical
tension of the skin at rest. To evaluate the changes of skin morphology, we used a three-
dimensional confocal microscopy and characterization of skin imaging of volar forearm
microrelief. We have accurately characterize the role of skin line network during
chronological aging with the identification of depth scales on the network of lines (z ≤
60μm) and the network of lines covering Langer’s lines (z > 60 microns). During aging
has been highlighted lower rows for elastic fibres, the decrease weakened the tension and
results in enlargement of the plates of the microrelief, which gives us a geometric
pertinent indicator to quantify the loss of skin tension and assess the stage of aging.
The study of 120 Caucasian women shows that ageing in the volar forearm zone
results in changes in the morphology of the line network organisation. The decrease in
secondary lines (z ≤ 60 µm) is counterbalanced by an increase in the depth of the
primary lines (z > 60 µm) and an accentuation of the anisotropy index. The disappearance
of the secondary lines, the change in the line orientation and the expansion of the plateau
area during ageing, probably reflect the alteration in the underlying dermal matrix.. The
changes in the geometry of the line network and the two dimensional size of plateaux
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1158 H. Zahouani, R. Vargiolu, C. Guinot et al.

area, as shown in our study, led us to define a new criterion of ageing: the speed of
ageing, which corresponds to the rate of growth of the plateau area with age. A new index
of skin ageing is proposed that may be used to compare aging of ethnic populations in
future.

1. INTRODUCTION
The longevity of life is a recent unsurpassed demographic phenomenon in the history of
the humanity and impact of which will be considerable. The analysis of ageing of populations
and its social consequences requests an interdisciplinary approach. By gathering capacities of
research in these different domains, it becomes possible to treat in a transverse way the big
questions linked to ageing and to longevity, by understanding better all the factors that drive
to the fragility of the old tissues (origin of diseases, of dysfunctions and handicaps).
Old age is inevitable and a natural period of life characterized by a decrease in physical
function, loss of social role as an adult, changes in physical appearance and routing to a
gradual decrease in capacity. Aging is part of a continuing evolution in the course of human
development, rigorously following embryogenesis, puberty and maturation. Throughout this
process, the organs develop into effect after a specific time. The "program" of aging is
probably mediated by the endocrine hormones, neurotransmitters acting on certain target
organs. Finally the cell is genetically programmed and the programming could be compared
to a turtle horloge. A turtle is programmed for 100 years, a monkey for 20 to 25 years and 120
years for humans. For against this life expectancy is compromised by damage to genetic or
acquired by a malfunctioning biological or enzymatic cell. The first signs that we perceive
aging are changes in the body that they depend on internal changes. It is important to note that
the rate of aging varies with individuals. Thus the change of hair color, which gradually
become gray and then white, and the appearance of wrinkles are a reflection of the inevitable
passing of time. Skin problems, which are more common in the elderly than in younger
individuals, are not only related to the fact that the skin is exposed to a long remained more or
less cumulative dose of ultraviolet rays, but also to change in structures the skin itself
(connective tissue, collagen, elastic fibres, etc..).
The skin is a set of grouped cells in the form of a flexible and resistant fabric, composed
of several layers, and covering the whole body. The skin consists of three distinct parts: the
stratum corneum, the epidermis (epi = above) whose main role is to protect the body, and the
dermis, only one with vessels allowing nutrients (nutrients carried by the blood) to diffuse to
the epidermis. In an adult, the skin weighs between 3.5 kilograms and 4.5 kilograms. Its total
area up to two square meters. It is highly vascularized and also has a large number of glands
producing sweat (sweat glands), sebaceous glands (sebum secreting = fatty substance that
protects the skin) and nerve receptors for tactile sensations and pressure. This cover
(integument) very flexible but strong helps protect the body against external aggression
(infections, temperature variations, etc. ...). The thickness of the skin varies between 1.5 and 4
mm depending on the region of the body in question. The epidermis covering the soles and
palms is thicker than the rest of the body. Below the dermis lies the hypodermis, also called
superficial fascia. It consists of adipose tissue (fat) tissue and looser than the dermis. The
dermis has a role to adapt to the movements of structures located below it (muscles, tendons,
and fascia) but also protect the body shots, thanks to its constitution fat. It is at this level that

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 Skin Morphology of Caucasian Women during Aging 1159

is fat stores of the body, which accumulate in humans in the abdomen, and women on the
thighs and breasts. 1) The epidermis contains different varieties of cells, including
keratinocytes (which are the most numerous), and melanocytes.

1.1. Skin Lines Network

The skin surface shows a specific topography depending on the anatomical site, age and
sex. In general, the skin morphology presents a 3D network of lines, who by his organization
expresses all the multidirectional tensions of elastic fibres and the collagen beams. Micro-
lines, primary lines, fine wrinkle and wrinkle represent, in fact, the special organization of
collagen bundles and elastic fibres in the superficial dermis, and there is a relationship
between the morphology of skin lines and elastic network. Different functions can be
attributed to the lines network. The first function is the retention and drainage canals of the
sebum and sweat. They collect preferentially and retain for long time the substances applied
to the skin: they are thus preferential sites of percutaneous absorption. This reservoir function,
allow the applied topical products to be stored on the skin surface and then eventually to
diffuse in its different layers. The second function is mechanic, during ageing the depth,
width, density and orientation of skin lines change. Some lines become more marked; they
evolve progressively in marked anisotropy connected to the decrease of the elasticity of the
collagen fibres.

1.2. Skin Tension and Anisotropy of Skin Lines Network

When analysing the mechanics of skin in vivo, a significant property is its natural
tension. Discovered by Dupuytren [1], and mapped by Langer [2], the non-uniform skin
tension lines exist. Langer has identified these lines by puncturing the skin with a circular
device, figure (1). The wounds then assume an elliptical shape and by joining the major axes
of the ellipses a system of lines can be drawn, Some authors propose other methods to obtain
these lines, such as wrinkling of the skin by Borges[3]. Skin resistance to traction
predominates in the Langer’s lines direction and varies with body site. On all body sites, the
skin tension is greater in the direction of Langer’s lines, figure (2). This phenomenon is the
source of the Young’s modulus anisotropy[4], whose distribution angle shows a maximum in
the Langer’s lines axis [5],. This result favour a similar orientation of the elastic fibres
involved in the skin. Assuming that the fibres are independent, it has been calculated that, on
the calf, 76% were in the direction of Langer lines and 5.1% perpendicular [6]. Of course the
distribution concerns only the elastic fibres, which are parallel to the skin surface.
Observation of the dermis with scanning electron microscopy confirms this data[7]. In
retracted skin the collagen bundles look tortuous, with no special direction, and sinuous
elastic fibres are fixed to them in several places, especially in their concave portion. In non-
retracted skin, the thinnest collagen bundles as well as the elastic fibers are straightened in the
direction of the Langer’s lines and almost parallel; the thickest bundles remain tortuous and
oriented in all directions, but their shape seems to be modified by the traction from the
oriented bundles and fibres. Contrary to common belief in the past, Langer’s lines do not
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1160 H. Zahouani, R. Vargiolu, C. Guinot et al.

reflect anisotropy of the collagen density but anisotropy of the reticular dermis collagen
bundles’ direction and elastic fibres’ tension.

Figure 1. Detection of Langer’s lines: evolution of circular incisions on the face by Waldorf [4].

Figure 2. Schematic representation of Langer's lines on different areas of the body.

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 Skin Morphology of Caucasian Women during Aging 1161

1.3. Identification of Langer’s Lines

The morphology of lines network reflects the intrinsic tension of the skin, which is
distinct from the additional tension induced by increases in volume of the underlying tissues,
such as muscle contraction, edema, or particular posture which stretches the skin. The
identification of Langer’s lines must therefore, be made on relaxed skin.
Several methods of identification of Langer's lines were developed, as evidenced by the
following works of literature:

 Stark’s Method [8]

According to the definition, the direction of the maximum tension can be found by
stretching the skin in several directions with an equal force, the direction of minimum
elongation is that of Langer’s lines. Stark developed a simple device comparable to a
compass: two branches with claws at their extremities part spontaneously by 30 mm under the
action of a 14.2g/mm spring. He could measure the elongation of the skin into eight directions
quickly, each measurement requiring only 1.5 seconds.

 Borges’ Method[3]

This similar method is less accurate, but even quicker, and consists of creasing the skin
between the thumb and the index in all directions until the furrows are regular and parallel.
They follow Langer’s lines. In the other directions, they are impeded by the skin tension that
makes them irregular.

 Barbenel’s Method [9]

The measurement of the extensibility of the skin using the suction method is valid only if
slip page of the skin into the suction chamber is presented. If, inversely, this movement is
facilitated, the most extensible direction of the skin will appear easily, that is to say
perpendicularly to Langer’s lines. If the contour of the chamber is drawn in during suction,
when the chamber is removed, an oval outline is observed instead of a circle and its main axis
corresponds to Langer’s lines.

 Skin micro- topography : Zahouani’s method [10,11,12]

Apart from the palms and soles, the skin mirorelief is made of plateaus separated by
valleys. The latter are roughly parallel and oriented in different directions, and this layout is
characteristic of each body area. The direction of the deepest valleys matches Langer’s lines.
There may be one or two other preferential directions, indicating an ordered no orthogonal
mechanical anisotropy. This method has an advantage over the others as it is insensitive to
extrinsic skin tensions. Its physiological interpretation is simple. The cutis is normally
retracted (skin tension) and extensible, whereas the epidermis has none of these properties.
Therefore, the epidermal creasing responsible for the micro relief appears to be a
transformation of tension allowing the creases to be flattened by stretching them. The
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1162 H. Zahouani, R. Vargiolu, C. Guinot et al.

superficial dermis, an intermediary zone between epidermis and cutis, is precisely the place
where the skin relief begins. This mechanical transduction is one of its functions.
It is this approach that we developed to study the chronological aging of human skin. The
method uses the wealth of three-dimensional imaging of the skin and the possibility of linking
a signature printed on the surface of the stratum corneum with the organization in volume of
the different skin layers.

2. MORPHOLOGICAL EVOLUTION OF SKIN LINES NETWORK OF

CAUCASIAN FRENCH WOMEN DURING AGING
Several studies have demonstrated changes in the network of lines with age, leading to
deepening of certain lines and the disappearance of others [13-22]. However, quantitative and
detailed descriptions of the modifications of skin lines with age are rarely reported in the
literature since most of the published results are based on standard parameters which give an
overall description of the topography of any surface, without specificity for the skin
morphology. For this reason, we have adapted a 3D confocal microscope working with a high
vertical and lateral resolution, which enables precise characterisation of the skin lines
network. Negative skin replicas were taken with silicone rubber (Silfo®, Flexico Ltd,
England) from 120 Caucasian French women equally divided into six age groups (20-29
years, 30-39 years, 40-49 years, 50-59 years, 60-69 years and 70-80 years). Replicas were
taken from the women’s left volar forearm at the same pre-determined area, after a 30-minute
rest period in an environmentally controlled room (temperature: 21 ± 2ºC and relative
humidity: 50 ± 5%).

Figure 3. Morphology of skin relief of Caucasian women during aging (volar forearm site).

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 Skin Morphology of Caucasian Women during Aging 1163

Resolution refers to the smallest distance the confocal microscope can accurately
measure. It can be considered in terms of lateral or and vertical resolution. The vertical
resolution value of the confocal microscope is about 0.01 µm with a vertical range of 1000
µm. .The lateral resolution depends on the quality of displacement in the plane x, y. In the
case of skin aging, the lateral resolution which was used is 1 µm in the directions x, y.
Figure (3) shows the evolution of skin volar forearm morphology of Caucasian women
aged 20 to 80 years.
The images reflect the three-dimensional character of skin lines network. The scale
height is expressed here by a colour scale. The deepest lines are expressed by the Colour
Blue, heights that are at the top of plates expressed through into colour red – black. The full
scale (pick to valley) can reach 500 µm.

2.1. Multi-Scale Analysis of Skin Lines Network Morphology

Microscopic observations have shown that skin morphology contains a network of lines
whose organisation reflects the multidirectional tensions of elastic and collagen fibres in the
superficial dermis (11). Hashimoto (12) gave a precise four-level classification of the line
network scales: (I) The primary lines are clearly marked and are between 20 and 100 µm
deep, (II) The secondary lines are more discrete and correspond to a depth of 5 - 40 µm, and
are perpendicular to the primary lines, (III) The tertiary lines correspond to the corneocyte
border (about 0.5 µm), (IV) The quaternary lines correspond to the morphology of each
corneocyte (about 0.05 µm). Tertiary and quaternary lines cannot be seen without
magnification.
To study the transformation of the 3D skin line network during aging, it is necessary to
identify all its local motifs. A motif of skin line is defined by the association of two peaks
separated by the hollow of a valley, the height is determined by the difference between the
highest peak and the hollow of the valley [23-24], figure (4) :

Figure 4. Definition of tension lines patterns.

The width of the motif (λ) is given by the distance between both peaks. The direction of
the motif which coincides with the main direction of the line is defined in the orthogonal
direction at the maximal variation of the local gradient, figure (3).
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1164 H. Zahouani, R. Vargiolu, C. Guinot et al.

This quantitative approach enables us to follow the evolution of the different families of
lines in relation to age. The depth Z depends on the skin site and two classes of depth Z1≤ 60
µm and Z2 > 60 µm were adopted for the volar forearm aging. This choice was fixed after
sampling every 10 µm as class depth. The results showed a marked decrease in density of the
family lines of depth less than 60 µm and a net increase in density of the family of lines at
depths exceeding 60 µm:

 Z1 ≤60 µm, related the tension effect of elastic fibers network

 Z2 > 60 µm, related to Langer’s line

Analysis of the morphology of skin tension lines of Caucasian women aged between 20
and 80 years, shows significantly decrease of the density of lines of depth <60 microns and an
augmentation of deep lines beyond 60μm. This important result presented in figure (5) shows
the mechanical role of elastic fibres in maintaining skin tension and firmness of young skin.
This elastic relaxation mechanism of the reduction of elastic fibers according to age is the
basis of the phenomenon of the appearance of wrinkles.

Fig .5. Evolution of the scale lines of tension during aging.

2.2. Changing the Orientation of Lines and Anisotropy during Aging

For better identification of the shift of skin lines network, we privileged to work on the
rest state of the skin of the forearm by characterizing the image of the skin relief at different
stages of aging. The methodology is to identify the local orientation of a pattern of lines and
quantify the overall direction of the network of lines over the 0 ° direction taken as the axis of
the body from head to foot [26, 27]. Two conditions are adopted: i) the direction must
coincide with the local normal of the plan formed by the three features of the motif, figure
(6), ii) if the first peak is point A, the hollow point B and the second peak point C, the normal
vector N is approximately collinear to the vector operation:
  
N  AB  BC (1)

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 Skin Morphology of Caucasian Women during Aging 1165

B
   A 
N  CB  CA C(i,j) N


Figure 6. Detection of the skin line direction.

The direction of the motif must coincide with the direction of the valley which is
composed of a succession of motifs, it is necessary to know if the hollow C(i,j) belongs to the
move of the neighbouring points, figure (7). To achieve this, the intersection points between
half a straight line stemming from C(i,j) and the nearest points are noted. If there is an even
number of these intersection points, then the point is outside the direction of the skin line,
otherwise the hollow is in the direction of the skin line. The number of iterations of this
procedure is equal to the number of valley points of the detected motifs, and the three
elements of the motif Z,  and  are memorised for each motif; these three parameters will
be the fundamental components to build the 3D morphological tree of skin lines network.

Figure 7. Orientation of local motif and skin line tension.

Figure 8. Anisotropy of kin lines network of the forearm of a subject 40 years.

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1166 H. Zahouani, R. Vargiolu, C. Guinot et al.

This approach quantifies the morphology of skin lines according to their orientation and
their depth. The orientation distribution of the skin lines was quantified as a compass rose.
This graphic representation plots in 20° intervals the density of lines between 0° and 180°,
with the body axis used as the principal axis of orientation, figure (8). Thus, the density of
lines in a triangular part corresponds to the percentage of line patterns which have this
orientation.

2.3. Dynamic Rotation of Skin Line Network during Aging

To monitor the rotational dynamics of skin lines during aging, the overall results for the
densities of line orientation from 20 to 80 years were collected for a comprehensive
representation of the dynamic change of direction in function of age. We have chosen to
represent this change for those aged under 60 and over 60 years. Figure (9) shows the
rotational dynamics and the significant decrease in tension lines in the directions between 90
and 180° and the establishment of a marked anisotropy between the directions 20 and 60
degrees. This result demonstrates the relationship between the voltage loss of elastin fibres
and lower voltage lines printed on the plates of the relief and the depth is less than 60
microns.

Figure 9. Aging effect on the dynamic rotation of skin line network of 120 Caucasian women.

2.4. Anisotropy Index of Skin Lines Network during Aging

The parameter resulting from the information on the density of lines according to their
depth and orientation is the anisotropy. To assess the degree of the skin lines anisotropy
during ageing, we introduced an anisotropy index from direction roses. A completely
anisotropic surface gives a rose oriented into only one angular sector. Conversely, a perfectly
isotropic surface leads to a circular direction rose. In consequence, if N is the number of
angular sectors between 0 and , the anisotropy index (A.I) can be defined as [26, 27]

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 Skin Morphology of Caucasian Women during Aging 1167

(2)

where Ri is the rose value corresponding to angular sector i and [26]

(3)

S/N should be the Ri value for all i in the case of a perfectly isotropic surface and the
factor 1/2 derives from the fact that an Ri value greater than S/N must be exactly compensated
by lower values. As a result of the increase of the density of lines deeper than 60 µm and the
diminution of the density of lines < 60 µm, results in the increase of the anisotropy index
significantly during aging, figure (10).

Figure 10. Aging effect on the skin line anisotropy index of 120 Caucasian women.

2.5. 3D Reconstruction of Skin Line Network: Volumetric Anisotropy and

Tree of Skin Tension Network [27, 28, 29, 30]

The dermis is the layer of living skin. It is a supporting connective tissue rich in fiber
which gives the skin elasticity and strength. The dermis contains the appendices of the skin.
Histologically, the dermis can be divided into two layers: the papillary and reticular layer.
Dermis and epidermis are closely meshed into each other through many outgrowths of the
surface ripples of the dermis called papillae. The papillary dermis contains many nerve
endings (thermo-receptors, tactile receptors). The reticular dermis consists of a network of
collagen bundles (thick, wavy, perpendicular to the basal membrane) more visible because
more dense within the reticular dermis. The network of elastic fibres which underlies the
undulations of collagen fibres bundles and around the latter is anchored to their concavity.
The reticular dermis is the strongest part of the dermis. His mobility results from unfolding
the undulating collagen fibres bundles of allowing their extension and their return to their
original position by the action of elastic fibres.
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1168 H. Zahouani, R. Vargiolu, C. Guinot et al.

This structuring of the dermis with a network of collagen and elastic fibers gives a three-
dimensional structure to the skin network with directions perpendicular and parallel to the
skin surface. This three-dimensional morphology prints on the surface of the stratum corneum
a three dimensional network of lines which express the mechanical tension of the skin at rest.
The approach developed specifically to the skin morphology allows the identification of
the lines network anisotropy at different scales of depth and orientation. For each plane at a
certain depth of the skin surface, are determined three parameters of the point belonging to
the line of tension: the density of depth z, the width of the line and the rose of directions
between 0 and 180° [27, 28, 29]. The figure (11A) represents the identification of the network
of skin tension lines in different directions about a 25 year old. The depth of the skin lines is
illustrated by the range of colours from blue to red. One can distinguish the family of skin
lines printed on the plates: secondary lines (colours of green, yellow, and red correspond to a
variation of depths between -17 microns and 50 microns). The family of skin tension lines in
the main colour blue are in a scale between -17 and -84 microns. The identification of the
orientation of skin lines in different directions is illustrated by the figures: 11B, 11C, 11D,
11E. The figure (12), illustrates the anisotropy distribution versus the depth of skin network
families.

Figure 11. Network of skin tension lines.

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 Skin Morphology of Caucasian Women during Aging 1169

Figure 12. Reconstruction volumetric skin tension lines anisotropy.

2.6. Morphological Tree of Skin Line Network [28, 29, 30, 11]

Appropriate and quantitative representation of skin line network has been developed. It
allows reconstructing all network lines as a morphological tree. Each trunk of tree represents
the density of lines in a given direction and for a given depth, figure (13).

Figure 13. 3D Reconstruction: Tree of skin tension lines network.

One family of lines is described as a branch of the tree, in relation to its depth and
direction. With this original method, it is possible for the first time to quantify the different
scales of skin line accurately and to follow the morphological changes of the surface in
relation to age. This multi-morphological decomposition of line network can be used to assess
mechanical tension of elastic fibres and collagen bundle during ageing. In the other hand, this
approach can be used as a preventive test for certain diseases of the elasticity of the skin. The
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1170 H. Zahouani, R. Vargiolu, C. Guinot et al.

examples of figure.(14) shows the use of this approach in the analysis of the transformation of
the 3D line network during ageing [11,26].

Figure 14. Aging effect on the Tree of skin tension lines network [11, 26, 27, 28].

3. PLATES AREA: INDICATOR OF THE LOSS OF ELASTICITY

The works of literature usually describe the skin as a multi-layer tissue by specifying the
role of each layer to facilitate understanding of their functionality. Recently a new vision has
been proposed by Guimberteau [31]. This author proposes a concept based on the vacuole to
describe three-dimensional organization of the tissue and its ability to slip on the underlying
structures, figure (15).To view this three-dimensional fiber structure, Guimberteau has
produced videos in vivo [31] using an endoscope which clearly shows the capacity for
reorganization of the fiber network to best adapt to mechanical stress. The work of this author
shows clearly the role of three-dimensional structure of the fiber network that is responsible
for the development of skin tissue over time but are also following environment.

Figure 15. Structure of the micro-vacuole and fibrilar part.

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 Skin Morphology of Caucasian Women during Aging 1171

According to Guimberteau [31], fibres that constitute the under each vacuole are
continuous with each with the other and consisting essentially of collagen type 1 (70%) and
type 3 and 4 but also elastin around 20%. There is also a high percentage of lipids (4%). They
are continuous and oriented in all directions without any pre-established pattern or in
connection with a logic expected. They are interconnected, vibrate with each other. the
diameters can range from a few microns to tens and lengths are exceedingly variables giving
a messy and chaotic. No reference may be geometric observed. They intersect either very net
or with intermediate areas in sailing said bead tray. Higher magnification reveals changes side
of collagens suggesting that chains of proteoglycans are adhesive and related collagen
The new lighting is quite in line with our work on the relationship between the network
of skin tension lines and the network organization of elastin fibres and collagen that we will
exploit to better describe the natural aging of the human skin.

 The Micro-Vacuoles

The electron microscopy analysis of the skin tissue dehydration after, Guimbrteau found
that this tissue is made up of billions of microvacuoles, ranging in size from a few microns to
several tens of microns, organized available on a chaotic, fractal appearance, apparently
similar but all unique. The volume consists of the vacuolar crossing fibers is inconceivable
that in the three dimensions of space. The vacuole is a volume with walls, a shape, the sides
and a content, figure (15).

3.1. Model of Skin Line and Plates Network as Indicators of Aging

Apart from the palms and soles, the skin microrelief is made of plates (image of vacuole)
separated by valleys. We have accurately characterize the role of the line network in skin
aging with the identification of depth scales on network of lines (z ≤ 60μm) and the network
of lines covering Langer’s lines (z > 60 microns). During aging has been highlighted lower
rows for elastic fibres, the decrease weakened the tension and results in enlargement of the
plates of the microrelief, which gives us a geometric pertinent indicator to quantify the loss of
skin tension and assess the stage of aging.
To describe this morphology one can be inspired by the definition of vacuole used by
Guimberteau, indicating that the skin tissue is composed of fibrillar filaments ranging in all
directions, very chaotic distribution and defining inter-fibrillar spaces which called vacuoles.
By analogy, one can describe the morphology of skin relief as an image of the vacuole
network printed on the stratum corneum. In accordance with the description of Gamberteau,
mapping plates (meaning of the Vacuoles of Gamberteau) have a three-dimensional structure
as shown in figure (16) representing thethree dimensional relief of the forearm.
The morphological approach that has been developed to identify the morphology of
plates (print of vacuole) is based on the algorithm for the detection of the watershed lines
(WL) and catchment basins. This algorithm, which was initially used for the segmentation of
images [32], has been adapted to the identification of skin network. Each watershed line is
identified as a furrow and quantified by its depth and its orientation. The catchment basins are
detected as a 3D motifs surrounded by watershed lines [30, 32], figure (17).
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1172 H. Zahouani, R. Vargiolu, C. Guinot et al.

The area of plates (print of vacuoles) represented by the catchment basins is a new
parameter that is highly relevant, not only as an indicator of ageing through the increase in its
area, but above all as an index of the loss of skin tension during aging, Figure (18) shows the
robustness of the method to identify the network of lines and skin plates of the relief through
the quantification of their areas, will allow us to quantify the evolution of this criterion for the
120 Caucasian women. The three-dimensional character of watershed line is indicated in this
approach by the representation color line points surrounding watersheds (plates or print of
vacuoles).

Figure 16. Three-dimensional character of the plate network.

Figure 17. Skin plates network.

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 Skin Morphology of Caucasian Women during Aging 1173

Figure 18. Network of watershed lines (lines of skin tension) and plates (catchment basins or print of
vacuoles).

To simulate the morphological evolution of the plates area, the figure (19) shows an
illustration of the result of the decrease in the density of elastin fibers (depth lines <60
microns) that manifests a widening of the plates of the microrelief. The parameter area of the
plates, provides for the first time the possibility of measuring the increase in cutaneous aging
such as the variation of the area of the plates per unit time.

Figure 19. Area expansion of plate network (print of vacuoles) during aging [33].

3.2. Dynamic Change of Plate Area: Aging Speed of Caucasian Women [33]

To analyze the growth areas of the plates during aging microrelief we collected data of all
areas of the plates of Caucasian women aged 20 to 80 years, figure (20). On the y axis is
plotted the values of the areas of plates, the x-axis represents time in years, which gives us the
possibility to quantify the growth areas of the plates over time. The results show an evolution
in areas with a certain slope up to age 37 years, followed by a transition accompanied with a
change in slope from that age. This transition marks the acceleration of Caucasian women
aging. From this very relevant result, we are now able to determine a rate of aging as a unit
with the increase of surface area versus time. This is set in solid mechanics by the term: areal
speed with a unit of measurement as mm ² / time.
Plateau areas, did not significantly change until the age of 40 years but increased in a
linear manner after that with a growth rate of ~0.0018 mm2 per year.
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1174 H. Zahouani, R. Vargiolu, C. Guinot et al.

The figure (21) represents the three-dimensional evolution of skin morphology during
aging. This reflects the scenario consequence of the decrease of the elastic fibres density (z
<60 microns) and the increase in the anisotropy of the orientation of the lines. The deep lines
(z> 60 microns) clearly reflect the Langer's lines that are oriented in the main direction of the
tension of the forearm. The decrease in tension of the elastic fibres initiates the increase in
plate area and identifies the parameter of aging rate in mm ² per year.

Figure 20. Evolution of the size of the plates during the aging of Caucasian women.

Figure 21. Summary of the aging dynamics of Caucasian women.

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 Skin Morphology of Caucasian Women during Aging 1175

CONCLUSION
Changes of dermal collagen and elastin content are characteristic for skin aging as well as
for pathological skin conditions. The formation of lines and wrinkles in light-exposed areas
throughout the body, such as the face, throat and hands is a well-known sign of skin aging.
Lines and wrinkles are influenced by both intrinsic and extrinsic factors. The numerous
intrinsic factors are age, gender, genetic disposition and race. In exposed and unprotected
zones of the body such as on the skin of the hands and the face, extrinsic factors such as UV
light, weather and climatic influences, nutrition, tobacco and alcohol abuse, effect the
formation of lines and wrinkles.
With increasing age, the physiology and appearance of the human skin will change.
Alterations in structure, loss in tightness, smoothness and a decrease in the skin's functional
capacity are phenomena which may be attributed to the aging mechanism. An increase in
dryness and thus roughness as well as a loss in elasticity and even pigmentation are also a
sign of increasing skin aging. Wrinkles on flaccid skin develop with growing age. There is a
decline in the subcutaneous fatty tissue. Today, relatively little is known about the exact
biomechanical processes of skin aging. Primarily, changes in the skin's appearance are a
result of a general aging process of the connective tissue of the subcutis. This leads to an
atrophy of the epidermis which adjoins the papillary layer and to an irregular decrease in the
elasticity of the elastic nets which are structures accompanying the collagen fibres in the
connective tissue. As a result of the changed amount and chemical composition of the basic
substance of the connective tissue, a loss of liquids is the result, which consequently leads to a
decrease in glycosaminoglycans, the basic structures of the connective and supporting tissues.
As a consequence, the youthful turgor, i.e., the skin’s tension, is lost. Melanocytes
disintegrate or lose close contact to epidermal cells and finally lead to a spotted pigmentation
of the skin. To evaluate these changes, we used the characterization of skin imaging of
microrelief. The study of 120 Caucasian women shows that ageing in the volar forearm zone
results in changes in the morphology of the line network organisation. The decrease in
secondary lines (z < 60 µm) is counterbalanced by an increase in the depth of the primary
lines (z > 60 µm) and an accentuation of the anisotropy index. The disappearance of the
secondary lines, the change in the line orientation and the expansion of the plateau area
during ageing, probably reflect the alteration in the underlying dermal matrix. This
phenomenon is known to be accelerated by actinic radiation (extrinsic photoageing) which
increases the degradation of the elastic fibres. The changes in the geometry of the line
network and the two dimensional size of plateaux area, as shown in our study, led us to define
a new criterion of ageing: the speed of ageing, which corresponds to the rate of growth of the
plateau area with age. A new index of skin ageing is proposed that may be used to compare
ethnic populations in the same age groups in future.

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epidermique. Dermatologica; 149:266-273.
[24] L. Hashimoto (1974). New methods for surface ultrastructure. Int J Dermatol; 13:357-
381
[25] H. Zahouani (1998), Spectral and 3D motifs identification of anisotropic topographical
components. Analysis and filtering of anisotropic patterns by mophological rose
approach. Int J of Machine Tools & Manufacture; 38:615-623.
[26] H. Zahouani, R, Vargiolu, (2008), “Skin morphology and volume: methods of
evaluation,” Injection Treatments in Cosmetic Surgery, Ed. B. Ascher, M. Landau, B.
Rossi, Informa Health Care, Series in Cosmetic and Laser Therapy, New York, ISBN
9780415386517, (480 pages), pp 13-33.
[27] H. Zahouani, R. Vargiolu, (1998). 3D Morphological tree representation of the skin
relief. A new approach of skin imaging characterization. XXth Congress. International
Federation of the Societies of Cosmetic Chemists, CANNES, France september 14 -18,
1998.Paper N° 30, pp 69 - 80.
[28] H. Zahouani, R. Vargiolu, (2000). Mesures du relief cutané et des rides `` Collection
Explorations Fonctionnelles Humaines. Physiologie de la Peau et Explorations
Fonctionnelles Cutanées. Sous la direction du Professeur Pierre AGACHE. ISBN : 2-
7430-0360X., pp 41-57
[29] H. Zahouani, (2007), Tension des fibres élastiques et lignes cutanées : application au
vieillissement et à la cicatrisation,” Les secrets de l’anti-âge, Ed. J.L Lévy, Groupe
Liaisons SA ISBN 978-2-7184-1161-3, pp 67-79.
[30] J.C. Guimberteau *, J. Sentucq-Rigall, B. Panconi, R. Boileau,P. Mouton, J. Bakhach,
(2005), Introduction à la connaissance du glissement des structures sous-cutanées
humaines. Annales de chirurgie plastique esthétique 50 .19–34
[31] H. Zahouani, S. Mezghani (2004), A new approach to characterize skin surface,
watershed lines and catchment basin. International Society for Bioengineering and the
Skin (ISBS. Orlando, Florida (USA). October. 28-30,. Skin Research and Technology.
Vol 10, Issue 4, pp.6
[32] H. Zahouani, S. Gardinier, I. Le Fur, R. Vargiolu, F. Morizot, L. Lambroisine, C.
Guinot2, E. Tschachler (2005). Determination of Ageing Speed and the Index of
Elasticity Loss of Caucasian and Japanese Women. World Congress on Noninvasive
Studies of the skin 2nd Joint meeting of the ISBS, ISSI and ISDISInternational Society
for Bioengineering and the Skin (ISBS), Philadelphia (USA). September. 28-30,. Skin
Research and Technology. Vol 10, Issue 4, pp.6.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 55

MOLECULAR UNDERSTANDING OF
THE DEVELOPMENT OF “AGE SPOTS”

Connie B. Lin1 and Miri Seiberg2

1
The Johnson & Johnson Skin Research Center, Consumer Product Worldwide, a Unit of
Johnson & Johnson Consumer Companies, Inc., Skillman, NJ, US
2
Seiberg Consulting LLC, Princeton, NJ, US

ABSTRACT
Solar lentigines (SLs, “age spots”) are hyperpigmented lesions which are induced by
sun exposure and are associated with aging. Histologically, SLs exhibit melanocyte
hyperplasia, epidermal finger-like protrusion (rete ridges), and pigment accumulation in
the epidermal basal layer. While SLs are benign in nature, reducing the visibility of such
lesions is strongly desired. Yet no current remedy meets the needs, due to incomplete
knowledge of the molecular mechanism involved in the induction and the development of
SLs. To better understand the pathology of SLs, immunohistochemical staining of
archived human skin biopsies with SLs was performed for the keratinocytes proteins
keratinocyte growth factor and its receptor (KGF and KGFR), the proliferation marker
Ki67, the stem cell marker keratin-15 (K15), stem cell factor (SCF), and protease-
activated receptor-2 (PAR-2), and for the melanocyte protein tyrosinase (TYR). The
expression patterns of these proteins were documented throughout SL development and
progression, and they were compared to those of healthy skins. These expression patterns
suggested a major role for the KGF pathway in SL development, which led to additional
studies. The stimulating effect of KGF on pigment production and epidermal proliferation
was documented in vitro and in vivo using numerous experimental systems. UVB
exposure was shown to increase KGF expression, and KGF treatment was shown to
induce TYR expression in primary melanocytes. Taken together, these results suggest
that the upregulation of the KGF pathway might induce the formation of the two
hallmarks of SLs, the rete ridges and the hyperpigmentation. The reduced levels of all
examined keratinocyte proteins (except K15) within the mature SLs suggest a possible
inactive status of the advanced SL lesion, which could provide novel targets for
intervention.
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1180 Connie Lin and Miri Seiberg

I. INTRODUCTION
Solar lentigines (SLs, also named “age spots,” “liver spots,” “cemetery flowers” or senile
lentigines) are one of the most common age-induced and visible skin phenotypes. SLs are
macular hyperpigmented lesions induced by chronic sun exposure, which evolve slowly over
years. SLs are commonly found on sun-exposed areas (e.g., face and the dorsa of the hands)
of middle-aged and older persons [1-3], therefore, the etiology of SLs is believed to be
associated with sun exposure and aging, as well as with genetic predisposition.
Histopathologically, SLs are defined by a hyperpigmented basal layer and elongated rete
ridges above solar elastosis [4-7]. SLs can be distinguished clinically from seborrheic
keratoses (SKs), which are also age-induced hyperpigmented lesions, by the absence of
epidermal hyperkeratosis.
The mechanisms of SL induction and progression, the triggers for melanocyte
proliferation, accumulation and hyperactivity, and the causes for rete ridges formation are
only partially understood. It has been proposed that local proliferation of both keratinocytes
and melanocytes, and a subsequent increase in hyperactivity of melanocytes, lead to the
formation of SLs [7-9]. Skin pigmentation is regulated via receptor-dependent and
independent mechanisms, in a hormonal, auto-, para-, or intracrine fashion (reviewed in [10,
11]), which could all be altered to induce SL formation. Paracrine interactions among the
skin’s keratinocytes, melanocytes, langerhans cells and fibroblasts play an important role in
regulating epidermal melanogenesis [12, 13], and could be involved in the formation of the
hyperpigmentary basal layer of SLs.
The different histological manifestation of SLs and the dynamic expression patterns of
several candidate genes are discussed in this chapter. These genes are involved in
melanogenesis, melanocyte-keratinocyte interactions and keratinocyte proliferation, and their
expression is altered within the SL lesions, therefore they could play key roles in SL
development. In particular, the role of the KGF/KGFR pathway in inducing
hyperpigmentation and in the development of rete ridges is discussed.

II. Clinical Observations and Treatments of Solar Lentigines

Solar lentigines appear as well-circumscribed macules with flat or slightly depressed

surface. They are most commonly found on UV-exposed body parts, and in particular on the
face, forearms, and dorsa of hands. They gradually increase in size and number with time, and
have variable sizes and shapes (0.2-3 cm in diameter). Their colors range from light yellow to
dark brown, depending on the underlying skin color. Figure 1 shows visual picture and
images of SLs with different sizes and shapes, using different imaging techniques: superficial
pigment is shown in a parallel polarized image, deep melanin is shown in a cross polarized
image and detailed structures are shown using a hi-scope.

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Figure 1. Solar Lentigo: multiple hyperpigmented macules on the dorsal side of the hand and hi-scope,
parallel or cross polarized images of the pigmented lesion.

While SLs represent a benign melanocyte hyperplasia, and therefore they are risk-free,
their appearance suggests an aging phenotype that is cosmetically undesired. Therapy for SLs
is highly demanded, yet no current remedy meets the needs, due to the incomplete knowledge
of the molecular mechanism involved in the induction and the development of SLs. SL
treatments include cryotherapy, laser and pulsed light exposure, chemical peels, and topical
depigmenting agents [14]. However, cryotherapy and laser treatments are associated with
side effects such as erythema and post-inflammatory hyperpigmentation, in particular in
individuals with darker skin types [15-20], and topical depigmenting treatments provide only
short-term improvements, resulting in a high lesion recurrence [21-24].

III. Histological Manifestation of Solar Lentigines

Histologically, the most prominent features of SLs are massive accumulation of pigment
in the basal layer of the epidermis and a characteristic elongation of the rete ridges that form
buds and strands [25]. The SL lesions are complex and continuously evolving with additional
sun exposure and chronological aging, resulting in multiple SLs with different sizes and
progressive “stages” even within a single body location. Figure 2 shows examples of the
epidermal architecture and the pigment deposition levels of normal skin and of progressing
SLs, based on the degree of hyperpigmentation and on the depth and intricacy of the rete
ridges. The presented skin sections are stained with Fontana-Mason (F&M) staining, to
document melanin deposition, which is shown in black. Early-stage areas of the lesion have
lower levels of melanin accumulation, and their rete ridges are shorter and less complex. As
the lesions develop, the later-stage, more developed areas have an increased accumulation of
melanin, many long and complex rete ridges that are protruding into the dermis, and a general
thinning of the epidermis. The progressive stages of SL development are documented in
several studies [5-7] and in different specimens derived from a single individual [7]. It is
important to note that a single SL lesion might be fully developed at the center, while still
evolving at the edges and the perilesional regions [7], explaining the slow increase in lesion
surface area with time.
The histopathology of SLs might be dissimilar at different body locations. Elongation of
the rete ridges is often used for the histological diagnosis of SLs. However, some studies
observed that about 50% of the facial SLs lack the characteristic elongated rete ridges [26,
27]. Interestingly, the flattened epidermis of the facial SLs is significantly thinner, with more
severe solar elastosis and fewer epidermal Langerhans cells, as compared with SLs which
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1182 Connie Lin and Miri Seiberg

have deep rete ridges [27]. It was also reported that the basement membrane of SL lesions is
disorganized [6], and their dermis sometimes contains more melanophages, as compared with
normal skins [28].

Figure 2. Different stages of solar lentigo (SL) progression, cataloged based on pigment deposition
levels and the complexity of rete ridges, and shown using Fontana-Mason (F&M) staining. SL
development is a continuous process, and the presented stages are only points of reference within the
continuum.

IV. MOLECULAR CLUES TO THE PATHOLOGY

OF SOLAR LENTIGINES

A. Overview

The molecular mechanisms involved in the formation of the hyperpigmented and

elongated rete ridges are not completely understood. In addition to the upregulation of known
melanogenic genes such as tyrosinase (TYR), melan-a, endothelin, microphathalmia, stem
cell factor (SCF) and its receptor c-Kit, changes in inflammatory mediators, fatty acid
metabolic genes and growth factors were also reported [4, 29-33], as well as the
downregulation of keratinocyte differentiation markers [4]. The fibroblast derived growth
factor, hepatocyte growth factor, keratinocyte growth factor (KGF) and SCF showed various
degree of increased expression in SL samples [34], suggesting that the cytokine network may
contribute to SLs formation or maintenance. Additionally, heparin sulfate proteoglycans were
implicated in the regulation of paracrine cytokines during SL formation [35]. Mutations in
the fibroblast growth factor receptor-3 (FGFR3) and phosphatidylinositol 3-kinase (PIK3CA)
were reported to be involved in the pathogenesis of SL [36] and SKs [37-39], although the
mutation rates of FGFR3 and PIK3CA in SLs (15% and 7%, respectively) were lower than in
SK (25-90% and 16% respectively). Polymorphism in the promoter region of the
melanocortin receptor-1 gene was also reported to be associated with the development of SLs
[40].

B. Hyperplasia and Hyperactivity of Melanocytes in Solar Lentigines

In skin, tyrosinase (TYR) is expressed in melanocytes only, and is a marker of active,

melanin-producing melanocytes. Immunohistochemical (IHC) staining of skins for TYR
shows similar numbers of TYR-expressing cells and similar levels of TYR protein per cell, in
normal, healthy skins regardless of their pigmentary levels (e.g., see dark and light skins in

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Figure 3, left panels). This confirms that melanocyte numbers and tyrosinase protein
expression in melanocytes have little or no correlation with race and skin color [41]. In
contrast, a marked increase in the number of TYR-positive cells and in the levels of TYR
staining per cell was documented in SLs (see examples in the right panels of Figure 3).
Similar IHC staining of TYR over-expression was also documented within a single and entire
SL lesion, which contains a spectrum of progressing stages of the developing SL. Figure 4
shows several representing sections taken from end to end of one SL lesion. There is a
substantial increase in the numbers of TYR-expressing cells and TYR protein levels per cell
from the margins to the center of the SL lesion, where the more advanced lesions reside. Even
the perilesional skin (the visually healthy edge of the lesion) shows higher TYR staining than
in normal, healthy skin, which could suggest a very early step in the expansion of the lesion
[7]. These observations of increased melanocyte number and activity are consistent with
many studies, which used different melanogenic markers [4, 5, 7, 26, 27]. Other pigmentary
proteins expressed in melanocytes, including tyrosinase-related protein-1, dopachrome
tautomerase, Pmel-17, proopiomelanocortin, and the endothelin receptor B, were all shown to
be expressed at higher levels within SL lesions [33, 42, 43]. While most studies demonstrate
melanocytic hyperplasia (increased melanocyte numbers) in SLs relative to normal sun-
damaged skins [4, 5, 25, 27, 44], few reports document unchanged melanocytes numbers in
SLs [27, 28, 45], possibly due to sampling from different stages of the lesions.

Figure 3. Tyrosinase immunohistochemistry (lower panel, TYR, red) of healthy and solar lentigo (SL)
skins (documented by F&M staining, upper panel), shows increased melanocyte number and TYR
protein levels in SLs.

C. Molecular Mechanism of Hyperpigmentation: The Expression of

Pigmentary Proteins during Solar Lentigines Development

Keratinocyte-melanocyte interactions have a major role in the regulation of pigment

production and distribution. Examples for such keratinocyte-melanocyte interactions include
e.g., the keratinocyte-expressed SCF, and its melanocyte-expressed c-Kit receptor, which are
important in melanocyte biology and melanogenesis [10; 46-48]. Another example is the
keratinocyte protease-activated receptor-2 (PAR-2), which affects skin color by the
phagocytosis and transfer of melanosomes, and which is upregulated and activated by UVB
irradiation [49, 50]
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1184 Connie Lin and Miri Seiberg

Figure 4. Skin sections taken from both edges (images #1 and #6 representing perilesional regions) and
center (images #2-5, representing lesional regions) of one solar lentigo (SL) lesion sample were stained
for TYR expression (red staining), documenting increased melanocyte numbers and TYR protein levels
in the lesional regions.

Figure 5. The expression of pigmentary proteins during solar lentigo (SL) development, as detected by
immunohistchemistry.

Figure 5 shows examples of IHC staining of TYR, SCF, and PAR-2 in SLs with different
development stages and in healthy, lightly-pigmented skins. The TYR staining confirms that
as the SL lesion is progressing, pigment production is gradually increased, until going down
at the very late stage. However, TYR expression and TYR-stained cells in the latest SL stage
are still higher than normal healthy skin (Figure 5). The staining for PAR-2 and SCF revealed
patterns of keratinocyte-melanocyte interactions that correlate with the high pigment
production of SLs. PAR-2 is expressed at the earlier stages of SL development at similar
levels to normal skin, but is reduced in the later SL stages. The reduced PAR-2 levels at the
later SL stages could clue on a regulatory feedback mechanism that is induced in heavily-
melanized keratinocytes. In mature SLs, the melanin-overloaded keratinocytes reduce
additional melanosome transfer [49, 51], which is consistent with the metabolic effects of
melanin [52].
A similar pattern of expression is documented for the keratinocyte SCF protein (Figure
5), which is a positive regulator of pigment production. In response to UV irradiation, both
human keratinocytes and dermal fibroblasts secrete SCF, which may activate melanocytes in

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the overlying epidermis [53]. The role of UV in SL induction could be explained, at least in
part, by increasing SCF secretion. SCF protein levels are increased in SLs. They are
upregulated in early SLs, peak at mid stage, and are strongly reduced at the later stages of SL
development [7, 54] (see Figure 5). IHC staining of c-Kit, the SCF receptor (see Figure 6),
demonstrates increased c-Kit levels in the SL lesion compared to healthy controls, which is in
full agreement with the SCF staining pattern. The increased SCF levels at early and mid
stages suggest its association with the initiation of the hyperpigmentary lesions and that it
might be involved in the induction of melanocyte proliferation and hyperactivity at these
stages. Reports on SCF levels in SLs provide somewhat inconsistent results, including
increased levels [7, 54], no change, or slightly decreased levels [42]. It is likely that the
inconsistent results are related to the sampling of different stages of the SLs in the different
studies.

Figure 6. Immunohistochemical staining of c-Kit in solar lentigo (SL) and normal skin.

D. Molecular Mechanism of Deep Rete Ridges Formation: Dynamic Changes

in Genes Involved in Keratinocyte Proliferation and Differentiation within
the Solar Lentigines

1. The Expression Pattern of Ki76, a Keratinocyte Proliferation Marker, during Solar

Lentigines Development
The molecular mechanism involved in the formation of the elongated rete ridges is not
yet completely understood. It is likely that epidermal hyperplasia might be involved in the
deep rete ridge formation, at least in the initiation of SLs. Therefore, studies were taken to
document the proliferation status of the different SL stages. Ki67 is a nuclear protein
associated with cellular proliferation. As shown in Figure 7, high levels of Ki67 were detected
in the early SL stages, as compared to late-stage or to normal skins. Ki67 is expressed in the
basal layer of the healthy epidermis, but Ki67-expressing cells were also detected in
suprabasal keratinocytes of the early-mid SL stages. This abnormal localization could suggest
a defect in the regulation of keratinocyte proliferation as a possible initiator of SL formation.
Such a defect could lead to an increase in the number of proliferating keratinocytes, and
therefore to the formation of the protruding rete ridges of the SL lesion. Interestingly, Ki67-
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1186 Connie Lin and Miri Seiberg

positive cells are markedly reduced at the late SL stage, suggesting the possible dormancy or
quiescent status of keratinocytes in advanced SLs. The reduced Ki67 expression in SLs was
also documented in other studies [28, 42], and we speculate that these studies used advanced
stages of SLs. Dark and light healthy skins differ in their melanin deposition levels, but not in
their proliferative capacity, while melanin accumulation in SLs correlates with reduced
keratinocytes proliferation [42]. These observations support the hypothesis that melanosomes
and melanin itself might regulate the functional status of the SL epidermis [10, 52].

Figure 7. Immunohistochemical staining of proliferation-related proteins during solar lentigo

development.

2. Increased Expression of Keratin 15, a Hair Follicle Stem Cell Marker, in Solar
Lentigines
Keratin 15 (K15), a hair follicle stem cell marker, is expressed in basal keratinocytes
[55-60]. K15 expression is tightly coupled to the maturity of basal keratinocytes, and is
associated with lateral differentiation within basal cells of stratified epithelia [61]. K15
expression is downregulated at times of hyperproliferation or during wound healing [60, 61].
As shown in Figure 7, in healthy skin, K15-expressing cells are observed mainly in the outer
root sheath of the hair follicles, with a minimal, weak expression within the basal layer of
epidermis. In contrast, the K15 protein is highly expressed in the majority of the SL basal
epidermal cells, with highest levels in the deep rete ridges. K15 expression peaks at SL mid-
stage, suggesting that the elongation of rete ridges could be associated with a clonal
expansion of K15-expressing stem cells in the SL epidermis. The K15 expression pattern is in
concert with the increase in Ki67 positive cells at early SL stages. The increased number of
K15-positive cells could also be indicative of a defect in the SL basal keratinocytes, resulting
in their inactivity and their retaining in the basal layer, and contributing to the permanency of
SLs. It is the melanin-loaded keratinocytes of the advanced SLs that show reduced Ki67 and
increased K15 expression, suggesting a lower proliferative capacity and altered differentiation
ability [4, 7, 28].

3. Increased KGF/KGFR Expression in the Early-Mid Stages of SLs

The keratinocyte growth factor (KGF, also called fibroblast growth factor-7, FGF7) is an
epithelium-specific paracrine growth factor, produced mainly by mesenchimal cells. KGF
binds to the KGF-receptor (KGFR, also called FGF-7 receptor, FGFR2/IIIb) on epithelial

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cells, and mediates epithelial-mesenchymal interactions (reviewed in [62]). KGF is a potent

mitogen for keratinocytes, which induces keratinocytes proliferation [62-64], and is involved
in wound healing processes (reviewed in [65]). KGF promotes melanosome transfer from
melanocytes to keratinocytes [66, 67], and promotes the paracrine activation of the SCF/c-
KIT pathway in melanoma [68]. Therefore, we hypothesized that KGF might be involved in
the initiation of SL formation by inducing keratinocytes hyperproliferation and by enhancing
pigment production and transfer. We therefore evaluated the expression patterns of KGF and
KGFR throughout human SL development and progression.
In normal skin, KGF protein is detected in dermal fibroblasts and in the epidermis. KGF
expression in keratinocytes is controversial, with reports of its presence [7;69] or absence in
keratinocytes [70]. As shown in Figure 7, epidermal KGF levels increase in early stage SLs,
and are maintained at high level with a more focal expression around the nuclei at the mid-
late SL stages. KGF levels decrease at the latest SL stage to a level noticeably lower than that
of normal skins. This kinetics suggests that KGF is involved in the initiation of SL formation,
and may not be required for the maintenance of the mature lesion. The increased KGFR
expression, however, is somewhat delayed relative to KGF (see Figure 7). While KGF is
elevated at the very early stage, KGFR peaks during mid and late stage SLs, suggesting a
possible positive feedback mechanism or an autocrine regulation. KGFR levels are reduced,
but are still higher than in unaffected skin, during the most advanced SL stage. In contrast to
its suprabasal localization in normal skin, KGFR expression is increased across the entire SL
epidermis, with higher levels in the basal layer and rete ridges of SLs. This abnormal
localization further suggests a role for the KGF pathway in the SL pathology. Interestingly,
activating mutations of FGFR-3 and PIK3CA were detected in a subset of SL samples [38,
71, 72]. Our findings of the involvement of other FGFR family members, KGF and KGFR, in
the pathology of SLs suggest the importance of a search for KGF and KGFR mutations in
SLs.

V. THE ROLE OF KGF/KGFR IN THE INITIATION

OF SOLAR LENTIGINES

A. KGF Increases Hyperpigmentation In Vitro

As primary melanocytes express KGF [9, 73], and KGF can activate the SCF/c-KIT
pathway [68], we studied the possible effect of KGF on melanogenesis in vitro. First we
showed that UVB exposure induces KGF expression in cultured melanocytes [9] and that
KGF exposure increases TYR expression in melanocytes (see Figure 8a), suggesting a direct
effect of KGF on melanocytes and melanogenesis. This data suggests that in SL development,
UVB-induced KGF secretion from both melanocytes and fibroblasts, which activates
KGF/KGFR signaling on both keratinocytes and melanocytes, could lead to
hyperproliferation of both keratinocytes and melanocytes, and therefore to increased TYR
levels and hyperpigmentation. Indeed, topical KGF treatments of both pigmented epidermal
equivalents and human skin explants resulted in an increase in melanin deposition [9] (see
Figure 8 b-f).
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1188 Connie Lin and Miri Seiberg

Figure 8. Keratinocyte growth factor (KGF) increases tyrosinase (TYR) expression and melanogenesis
in vitro. (a). QPCR analysis of TYR in primary normal human melanocytes. (b-d). F&M staining of
pigmented skin equivalents exposed to increasing doses of KGF. (e-g). F&M staining of human skin
explants exposed to increasing doses of KGF.

B. KGF Induces Both Hyperpigmentation and Elongated Rete Ridges In Vivo

The hypothesis that KGF could induce hyperpigmentary lesions with complex rete ridges
was evaluated in vivo. Topical KGF treatment of human skins transplanted onto immuno-
compromised mice resulted in histological features that most resemble human SLs [9]. Both
basal layer hyperpigmentation and elongated rete ridges were induced by KGF in this
experimental system (see Figure 9), as well as in swine skin [9]. The increased pigment
deposition was concentrated mainly within the basal layer of the epidermis, which resembles
the pigment distribution observed in human SLs. Similarly, the development of elongated rete
ridges resembles the histological characteristics of human SLs.

Figure 9. Topical treatment of KGF increased melanin deposition in the basal layer and induced rete
ridges formation in human skins transplanted onto immuno-compromised mice.

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UVB, a key player in SL induction (reviewed in [74]), increases the production of IL-1
in keratinocytes [75] and of KGF in melanocytes [20]. IL-1, in turn, increases KGF
secretion from fibroblasts [76]. The increased KGF secretion promotes keratinocyte
proliferation [77] and pigment production (Figure 8). Therefore, it is likely that a positive
feedback loop of UV-induced IL-1 and KGF secretion could contribute to the initiation of
SL formation. Indeed, the in vivo topical treatment of both IL-1 and KGF led to an earlier
onset of lesion formation, and to more significant darkening and rete ridges complexity, when
compared with KGF alone [9].
More evidence on the functional role of KGF in skin pigmentation has emerged lately. It
was found that KGF, but not epidermal growth factor, enabled the development of an active
pigmentary system, suggesting an essential role for KGF in the development of constitutive
pigmentation [78]. KGF also acts on keratinocytes to promote melanosome transfer [66,
79]. This effect is more pronounced in light skin-derived keratinocytes, which express more
KGFR than dark skin-derived keratinocytes [81]. KGF induces PAR-2 expression in
pigmented epidermal equivalents (Lin et al., unpublished), and KGF-induced melanosome
transfer is inhibited by soybean trypsin inhibitor [66, 80], which also inhibits PAR-2
activation [51]. We documented higher levels of the PAR-2 protein expressed in the early SL
stages, which is in agreement with the suggested role of KGF in the initiation and
development of the SL lesion, but not in the maintenance of the mature, melanin-loaded SL.
The enhanced KGF activity at the early stage of SL formation not only enhances pigment
production and rete ridges development. It could also lead to acceleration in melanosome
transfer via PAR-2 activation, which would result in excessive melanin accumulation in the
keratinocytes. Once the melanin load exceeds the keratinocytes capacity, it could affect the
normal balance of proliferation and differentiation in the deep rete ridges, and disable the
keratinocytes from terminal differentiation. The melanin-overloaded keratinocytes would then
remain in the basal layer, resulting in the permanency of the SL lesion.

CONCLUSION
Human SLs are benign Hyperpigmentary lesions. They have an increased numbers of
melanocytes, increased TYR protein levels per cells, and increased SCF protein levels in
early stages; all in agreement with a hyperpigmentary phenotype [4, 44, 45, 81-83]. SLs
could be classified histologically into different, progressive stages, based on their melanin
deposition levels and on the intricacy of their rete ridges. The progression of the SL lesion
development is continuous, with the terms “earlier stages” to “late stages” used only as
representative points within the continuum [7]. This notion is in agreement with, and an
extension of a similar and earlier classification concept [5]. SL lesions are complex, with
progressive, multiple “stages” within one lesion, having a more advanced "stage" by their
centers.
The complexity and intricacy of the SL rete ridges suggests a proliferation imbalance.
Proliferation is increased in the basal and suprabasal cells of the earlier SLs stages, which
could explain the development of the rete ridges. Proliferation is markedly reduced at the
latest SL stages, suggests an additional and later defect in the SL keratinocyte homeostasis
and in terminal differentiation. K15 is a marker of laterally-differentiated keratinocytes. In
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1190 Connie Lin and Miri Seiberg

correlation with the increase in proliferation at early stage, the number of K15-expressing
cells in the basal layer and deep rete ridges is also increased, explaining the lateral expansion
of the basal layer. This expression pattern suggests that the elongation of rete ridges might
result from a clonal expansion of K15-expressing stem cells in the SL epidermis. The
increased number of K15-expressing cells in the late SL stage might explain the retaining of
the SL keratinocytes in the basal layer, which could elucidate on the permanency of SLs. It is
important to note that the levels of all keratinocyte proteins examined, except for K15, were
strongly reduced in late stage SLs, suggesting that the mature SL keratinocytes are relatively
inactive. For example, PAR-2, a keratinocyte receptor involved in melanosome transfer [84],
was markedly reduced in the late SLs, suggesting the inactivation of the keratinocyte-induced
melanosome transfer in mature SLs. The combination of the increased TYR expression and
the reduced keratinocyte proliferation and activity in mature SLs points to an environment of
active melanocytes and very inactive keratinocytes in the deep rete ridges of mature SLs.
KGF is a regulatory protein that could affect pigment production, melanosome transfer,
and keratinocyte proliferation. Therefore we speculate that KGF is a key player in the
induction of the SL lesion. KGF is increased in the epidermis of earlier SLs stages, and is
decreased in the latest stage. The increase in KGF expression could promote keratinocyte
hyperproliferation and the initiation of the complex rete ridges formation, and could enhance
pigment production and melanosome transfer, resulting in melanin accumulations within the
SL proliferating keratinocytes. The reduced KGF/KGFR levels in late SLs might reduce both
proliferation and phagocytosis, reducing both rete ridges elongation and melanosome transfer.
Indeed, the inductive effect of KGF on keratinocyte proliferation and on pigment production
was documented experimentally. Topical applications of KGF in vivo resulted in the creation
of hyperpigmentary lesions with histological similarities to SLs. We hypothesize that KGF is
a key player in the initiation and the early development of the SL lesion, but that is it not
required for maintaining the mature SL lesion. The resulting melanin overload of the late
stage SL keratinocytes could possibly lead to keratinocyte dormancy, which might explain the
lifelong persistency of the SL lesions.
In summary, SLs are characterized by progressive histological features, which correlate
with expression profiles of pigmentary proteins. Expression patterns of KGF/KGFR and other
genes during SLs development reflect on the molecular and cellular mechanisms involved in
SL formation and maintenance. These expression patterns highlight the importance of
epidermal-dermal interactions and demonstrate the unique autocrine and paracrine networks
of the skin that respond to UVB with epidermal proliferation, differentiation and pigment
production. We suggest that the inhibition of the KGF pathway might prevent the formation
of new SLs and possibly slow the development of newly forming SLs. Overcoming the
keratinocytes dormancy and inhibiting hyperactivity of melanocytes in the mature SLs could
lead to an effective treatment of existing SLs.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 56

SKIN REJUVENATION – ULTRASTRUCTURAL STUDY

Tokuya Omi1,2 and Shigeru Sato3

1
Department of Dermatology, Queen's Square Medical Center, Yokohama, Japan
2
Department of Dermatology, Nippon Medical School, Tokyo, Japan
3
Central Institute for Electron Microscopic Researches,
Nippon Medical School, Tokyo,Japan

ABSTRACT
Ablative laser therapy is very effective for skin rejuvenation. But it has some risk for
scar, hyperpigmentation or hypopigmention. From 10 years before, non-ablative lasers
and fractional devices become very popular instruments. Non-ablative light source
therapy for photorejuvenation is widely used because of its rare adverse effects. And
these devices occupy a large market in the world.
There have been few comparisons of these instruments, in this chapter we compare
these devises not only clinical study but histological study with electron microscope.

INTRODUCTION
Skin rejuvenation therapies performed with the aim of improving total skin conditions,
such as reducing wrinkles and freckles, include treatment with topical preparations of vitamin
A (retinol) and kinetin ointments [1], chemical peeling with glycolic acid and lactic acid [2],
non-ablative photo-rejuvenation with near-infrared devices, such as light emitting diodes
(LEDs), Er:glass lasers and diode lasers, in addition to dye lasers, pulsed dye lasers,
potassium titanyl phosphate (KTP) lasers and intense pulsed light (IPL) [3, 4, 5, 6], and
ablative laser therapy [7] with CO2 lasers and Er:YAG lasers, Each of those techniques has
received favorable reports. Furthermore, devices using radio frequencies (RF) [8] have also


Correspond to: Tokuya Omi, Department of Dermatology, Queen’s Square Medical Center, 2-3-5 Minatomirai,
Yokohama, Japan. Zip 220-6208. TEL +81-45-682-4112, E-mail [email protected]
 Free ebooks ==> www.Ebook777.com
1198 Tokuya Omi and Shigeru Sato

been developed, as have devices combining the effects of IPL and RF and of near infrared
light and IPL or RF.
However, the effects of the topical preparations mentioned above are limited, and for
very superficial chemical peeling treatments, such as those used in Japan, multiple (5-6)
treatment sessions are necessary. In addition, non-ablative photo-rejuvenation does not
produce very drastic clinical changes, and patients are often not fully satisfied with the
results. IPL, which is very popular in Asian countries, such as Japan, is widely used due to the
short down time and its significant effect on pigmented lesions, but it is often used
concomitantly with other treatments due to its relatively minor effects on the dermal layer.
Ablative laser therapy is considered to be the gold standard for the so-called rejuvenation,
because of its strong activation of collagen neogenesis and its high efficacy. However,
removal of all epidermal layers causes great heat damage to the dermis, and the epidermal
regeneration takes time, thereby necessitating sufficient postoperative care. In addition,
wound healing takes at least approximately 2 weeks, and pigmentation and scar formation
occur at a high frequency, and also keloids develop at a low frequency, in Asian people such
as Japanese. Therefore, at present, this therapy is not often used in Japan.
The so-called fractional laser devices, which irradiate a laser beam in a dot form over a
grid pattern, have been developed that use several wavelengths [9, 10, 11]. With these
devices, not all the skin layers are targeted, and attempts are made to obtain the effect of
fractional photo-thermolysis (FP) by irradiating the target skin in a grid pattern. Thus, this
method protects intact skin from irradiation, thereby allowing more rapid wound healing and
also reducing the incidence of adverse effects. Among these fractional devices, the 1,550- and
1,440-nm near-infrared devices were developed first, and devices that generate ablative
changes only in the irradiated areas, such as Er:YAG laser and CO2 laser devices, were
developed somewhat more recently.
The above-mentioned devices have been reviewed, and many studies have reported the
clinical efficacies of those devices. In the sections below, the histological and ultrastructural
changes that occur during and after treatment by chemical peeling, pulsed dye laser, LED,
IPL, a combination of IPL plus vacuum incubation, and a fractional CO2 laser are described.
This study was conducted in an attempt to comprehensively review the morphological
changes associated with skin rejuvenation.

MORPHOLOGICAL ASSESSMENT
Considering the previously reported efficacy of these devices, 3-mm punch biopsies were
obtained from 5 to 6 patients each at the following time points: immediately after the first
treatment session, after the second or third treatment session, and 3 weeks after the fifth or
sixth treatment session.
Each specimen was fixed in glutaraldehyde (2.5%) and then in osmium tetroxide (1%).
After dehydration through a graded ethanol series, the specimens were embedded in Epon 812
(Oken Shoji Co., Ltd, Tokyo, Japan), stained with toluidine blue, and examined by light
microscopy. Ultrathin sections were prepared with an Ultracut N ultramicrotome (Reihert-
Nissei, Tokyo, Japan) and a diamond knife. Sections were stained with oolong tea extract

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 Skin Rejuvenation – Ultrastructural Study 1199

(OTE) for connective tissue [12], and with uranyl acetate and lead citrate prior to electron-
microscopic examination (75 kV, Hitachi H-7500, Hitachi, Tokyo, Japan).

Chemical peeling

Chemical peeling [13,14,15] is used frequently not only as a means to treat skin damage
caused by photoaging (acne, wrinkles, pigmentation, etc.), but also for treating solar keratoses
and Bowen’s disease. As the term “peeling” suggests, chemical peeling is considered to exert
its effect by stimulating skin turnover through exfoliation of the horny layer.
The peeling agent used for this study was TH Peeling Gel MD (pH 2.5) (I.C.I.
Cosmetics, Tokyo, Japan) which contains 20% glycolic acid and lactic acid. The peeling
agent was applied for 10 minutes, followed by application of a neutralizing agent, TH Neutral
Gel MD (I.C.I. Cosmetics, Tokyo, Japan). Three minutes after application of the
neutralizing agent, the agents were wiped off the skin. For skin care after peeling, we used
Cleansing Oil and Moisture Essence of the Devancier Series (International Cosmeceuticals,
Cardiff, UK) [1].
The chemical peeling was performed once a week and biopsies were taken 1 week after
the second treatment session and 3 weeks after the fifth treatment session. The horny layer
and upper epidermal layer remained essentially unchanged after the chemical peeling.
Morphologically, all specimens showed vacuolation between the basal cells (Figure 1). At the
ultrastructural level, dissociations between basal cells and the atrophy of basal cells were
noted. Furthermore, lymphocyte infiltrations of the spaces between the basal cells were
observed (Figure 2). On the other hand, the basal epidermal layer showed changes such as the
formation of fissures between basal cells, the atrophy of basal cells, or lymphocyte infiltration
of the intercellular spaces with no horny layer changes.
Increased numbers of vimentin filaments were observed within the fibroblasts and in the
vascular endothelial cells (Figure 3). No increase in the number of collagen fibers was seen,
and there were no morphological changes of the elastic fibers or collagen fibers. In the upper
layer of the dermis, the deposition of melanin granules and phagocytosis by macrophages or
fibroblasts was noted (Figure 4).
In a study of hairless mice conducted by Isoda et al. [16], inflammatory cell infiltration of
the epidermis was noted on day 3 of topical application of 20% glycolic acid, however, it was
no longer seen on day 14. In the same study, necrosis of all epidermal layers was noted on
day 3 of topical application of 35% TCA.
The inflammatory cell infiltration observed on day 3 of topical glycolic acid application
in that study was consistent with the finding of lymphocyte infiltration between basal cells
observed in this study. Moetaz et al. [17] investigated the morphological changes in human
skin: they compared skin conditions before and 3 months after peeling with 30% TCA
(applied at intervals of one week).
They found a slight thickening of the epidermis, which assumed the form of spongiosis,
which is consistent with our finding of the structure of the upper epidermal layer remaining
essentially unchanged. This result suggests that chemical peeling with an α-hydroxy acid
(AHA) is unlikely to cause exfoliation of the horny layer and that the major epidermal change
following chemical peeling with an AHA occurs in the basal layer.
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1200 Tokuya Omi and Shigeru Sato

Figure 1. Vacuolation is visible between basal cells. No inflammatory cell infiltration is seen within the
basal cell layer. (after 2 chemical peeling treatments, toluidine blue staining).

Figure 2. Vacuolation and lymphocyte infiltration are visible within the epidermis. Loss of bonds
between the basal cells and also atrophy of the cells are apparent. (after 2 chemical peeling treatments,
3000).

Changes in the basal epidermal layer included the finding of melanin granule deposits in
the upper layer of the dermis. Melanin granule deposits in the upper layer of the dermis and
phagocytosis by lymphocytes and fibroblasts are apparently associated with diminished
epidermal freckling (pigmentation). It seems likely that this works in combination with other
mechanisms and factors (e.g., reduced melanin production via the suppression of tyrosinase
activity by glycolic acid) to diminish freckling.
The normalization of microfibrils within elastic fibers, detected at the ultrastructural level
by Moetaz et al. [17], was not seen in our study. However, increased numbers of vimentin
filaments within fibroblasts and vascular endothelial cells were noted. The increase in
vimentin filaments apparently reflects elevated cell activity, and this change could induce
subsequent remodeling of the dermis.

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 Skin Rejuvenation – Ultrastructural Study 1201

Figure 3. Vimentin filaments are visible within the fibroblasts and endothelial cells. (after 5 chemical
peeling treatments, 20000).

Figure 4. Melanin granule deposits in the dermis and their phagocytosis by fibroblasts are apparent.
Infiltration of the basal cell layer by monocyte-derived lymphocytes can also be seen. (after 5 chemical
peeling treatments, 8000).

Pulsed Dye Laser (PDL)

Pulsed dye lasers (PDL) have wavelengths of 580-595 nm, which are highly absorbed by
hemoglobin, and therefore the PDL is mainly used to treat vascular lesions, such as vascular
malformations and hemangiomas18). It has also been reported that PDL treatment is effective
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1202 Tokuya Omi and Shigeru Sato

against warts and molluscum contagiosum [19]. The efficacy of low-power irradiation with a
PDL has also been reported, and PDL devices are used for non-ablative laser therapy [20, 21].
In this study, investigations were conducted using Regenlite (Chromogenex, UK).
Irradiation was performed at 3.0 J/cm2 for 350 msec, and 3-mm skin biopsies were taken 3
hours, 2 weeks and 5 weeks after the irradiation. At 3 hours after the PDL laser therapy
(Figure 5), the capillaries showed endothelial cell edema with hemostasis, and the endothelial
cells had become swollen and round in shape. The basement membranes of the capillaries
were thickened and had several lamellar structures. Marked edema was observed around the
capillaries. Neutrophils, monocytes and mast cells were observed in the extravascular dermis.
Two weeks after the PDL laser therapy (Figure 6), the capillaries showed an almost normal
structure. Dermal edema was no longer observed around the capillaries, and new elastic fibers
and collagen fibers appeared around them. At 5 weeks after the PDL laser therapy (Figure 7),
interstitial fibrosis was observed around the capillaries. Numerous lymphocytes and
fibroblasts were also observed.
The above findings at the level of the dermal layer are summarized as follows: at early
stages (a few hours) after treatment, mainly acute inflammatory responses such as edema and
neutrophil recruitment were observed; at 2 weeks after the treatment, the edema was no
longer seen and infiltration by inflammatory cells, mainly mast cells, became apparent; at 5
weeks after the treatment, the aggregation of lymphocytes with fibrosis was found.
Macrophage infiltration was also seen around the lymphocyte aggregates, which could be
suggested to represent granulomas at the micro level, although they were not defined
clinically. Young elastic fibers observed 2 weeks after the treatment were considered to
morphologically represent elaunin fibers.

Figure 5. Three hours after the PDL laser therapy, the capillaries (Cap) showed endothelial cell edema
with hemostasis, and the endothelial cells became swollen and round in shape. The basement
membranes of the capillaries were thickened and had several lamellar structures. Marked edema was
observed around the capillaries. Neutrophils (Ne), and mast cells (M) were observed in the
extravascular dermis.

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 Skin Rejuvenation – Ultrastructural Study 1203

Figure 6. Two weeks after the PDL laser therapy, new elastic fibers () and collagen fibers appeared
around the capillaries (Cap). The number of mast cells (M) was increased.

Figure 7. At 5 weeks after the PDL laser therapy, interstitial fibrosis was observed around the
capillaries (Ca). Many lymphocytes (L) and fibroblasts were also observed.

According to a study of the immunological characteristics of the infiltrating inflammatory

cells, although cultured skin-homing T cells from normal skin were all negative for IL-2 and
IL-4 mRNAs, an increase in IL-2 mRNA and a marked increase in IL-4 mRNA were
observed 1 week after the laser irradiation, suggesting that this might be the mechanism by
which PDL irradiation contributes to local skin immunity [22].
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1204 Tokuya Omi and Shigeru Sato

LED

Thermal low reactive- level laser therapy (LLLT) [23, 24] has been successfully used to
treat a variety of complaints and conditions, based on its effects on the neural network and
lymphatic/blood circulatory systems, and it has been shown to have wavelength-specific
effects on fibroblasts, mast cells and the vascular system. LLLT systems could therefore be
used to deliver the required non-invasive energy to photoaged skin, but they are still
comparatively expensive, and only a small area can be treated per irradiation session. This
makes treatment of large areas, such as in the photorejuvenation of the entire face, very time-
as well as labor-intensive. Development of a new generation of narrow-waveband LED-based
devices now offers promise for the treatment of large areas during a single automated
irradiation therapy session.
In this study, phototherapy was delivered with a red LED-based free-standing unit
(Omnilux Revive, Photo Therapeutics Ltd., Altrincham, UK). The treatment head is
comprised of an array of visible red LEDs (= 633  3 nm, irradiance of 105 mW/cm2, 15
min/session, radiant flux approximately 94 J/cm2). The head is attached to an articulated arm
and is set up with the LED panels approximately 1.5 cm from the target tissue. Each area was
treated with the Omnilux Revive using the above parameters, once per week for 8 weeks, 15
min per treatment session. Skin punch biopsies were obtained from each subject for
ultrastructural study after the second and eighth treatment sessions.
After the 2nd treatment session, the morphology of the skin was basically normal, with
few fibroblasts visualized. Slight perivascular and interstitial edema was noted, with some
mast cells in a mild degranulated state. After the 8th LED treatment session (Figure 8), more
lymphocytes, mast cells and fibroblasts were noted around the capillaries as compared to the
findings after 2 treatments, with some partially degranulated mast cells. Enlargement of the
perivascular interstitium was also observed. As seen after 2 treatments, the morphology was
basically normal with no signs of damage-related morphological changes, but a mild
inflammatory infiltration was noted.

Figure 8. After the 8th LED treatment session, numerous fibroblasts (Fb) and lymphocytes (Ly) are
seen surrounding the capillaries (Ca) in the dermis, as compared to the findings after the 2nd
irradiation. Enlargement of the perivascular interstitium can also be seen, and also some mast cells
(Ma). No damage-related morphological changes were noted, either in the capillaries or in the
fibroblasts. Magnification 2000.

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 Skin Rejuvenation – Ultrastructural Study 1205

TEM photomicrography of non-irradiated skin showed typical fibroblasts with collagen

fibers, both in longitudinal- and in cross-sections, in the extracellular area. Golgi complexes,
rough endoplasmic reticulum and mitochondria were seen in the cytoplasm, but few vimentin
granules or filaments were noted. After the 2nd LED treatment session (Figure 9), more
mitochondria were present in the cytosol of the fibroblasts, with a significant increase in the
number of vimentin filaments as compared with the non-irradiated skin [25].

Figure 9. A fibroblast in a specimen after the second LED therapy session at high magnification
(30000) shows increased numbers of vimentin filaments (arrows), and a higher number of
mitochondria as compared to non-irradiated fibroblasts.

Figure 10. A fibroblast from a specimen after the 8th LED therapy session. More mitochondria are seen
in the cytoplasm. Vimentin filaments (arrows) are significantly increased in number. (Magnification
30000).
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After the 8th LED session (Figure 10), a dramatic increase in the number of vimentin
filaments was noted, with an increase in the number of somewhat more electron-dense
mitochondria. Endothelial cells, after the 2nd treatment session, also showed increased
numbers of vimentin filaments and granules, similar to the findings in the fibroblasts.
However, unlike fibroblasts, the number of endothelial cells did not increase further in the
specimens after the 8th session of treatment, and remained more or less constant thereafter.
At the level of the fibroblasts, our results demonstrated an increase in their metabolic
activity in a treatment-dependent manner, as evidenced by the more abundant mitochondria
and vimentin in the cytoplasm of irradiated compared to non-irradiated fibroblasts. The
increase in the number of mitochondria, the powerhouses of the cells, is reflective of the
greater energy demands that go hand-in-hand with increased metabolism. Vimentin, a
developmentally regulated member of the intermediate filament protein family, is believed to
play a part in the communication and transport between the cell surface and the nucleus by
interconnecting the two organelles. In addition, vimentin copolymerizes with appropriate
desmins to form the constituents of connective tissue, i.e., collagen. The presence of increased
quantities of vimentin in this in vivo trial could therefore be translated into increased collagen
synthesis following low incident doses of 633-nm red light, as has been demonstrated in vitro
in the trial by Trelles and colleagues [26]. The appearance of lymphocytes after the 8th
irradiation session suggests a mild but extended, athermally mediated inflammatory response,
most probably induced by photoaccelerated mast cell degranulation, which has been shown to
occur following irradiation with 633-nm light both in vitro and in vivo [27, 28].

IPL Type

Conventional laser therapy targets water, melanin and hemoglobin, which absorb laser
radiation to variable extents, depending on the wavelength. The laser beam is characterized
by a single wavelength (monochromatic), the absence of diffusion (collimated), and a
constant waveform (coherent). On the other hand, IPL devices are multi-light devices using a
combination of multiple wavelengths, unlike monochromatic laser devices, and in these
devices only certain wavelengths are removed by cut-off filters to create a light source.
Therefore, IPL devices are characterized by a broadband wavelength range of 500 to 1,200
nm, which is absorbed by both melanin and hemoglobin, and they are used in the treatment of
so-called freckles and a ruddy complexion. Also, these devices have a wrinkle smoothing
effect, which is a popular goal in the treatment called photofacial therapy or
photorejuvenation [29, 30].
Freckles encountered in the outpatient setting mainly include melasma, sunlight-induced
(senile) pigmentation, and ephelides. Among them, melasma is often found in women and is
exacerbated by pregnancy, and patients with melasma often visit hospitals wishing to receive
treatments such as laser therapy. However, laser therapy is not only ineffective for treating
melasma, but may also sometimes exacerbate melasma, and generally laser therapy is not
indicated for melasma. Laser therapy is a highly suitable treatment for sunlight-induced
(senile) pigmentation, which is exacerbated by exposure to sunlight, but such treatment has
the disadvantage that the black coloration becomes rather prominent due to the incrustation
caused by the thermal denaturation of melanin.

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 Skin Rejuvenation – Ultrastructural Study 1207

Treatment using IPL devices is characterized by the almost complete absence of

postoperative side reactions, such as incrustation, and the possibility of treatment of a large
area in a short period of time. On the other hand, this treatment has the disadvantage that 5-6
treatment sessions are required owing to the somewhat weak effect obtained with a single
treatment. It has been reported that this treatment is effective not only for sunlight-induced
(senile) pigmentation, but also for nevus spilus and ephelides. In addition, it is particularly
suitable for cases of black pigmented lesions and telangiectasia [30].
In this study, a NatuLight (Lumenis, Israel) was used as the IPL device, and a dose of 22
J/cm2 was delivered 3 times a month. Three-mm skin punch biopsies were obtained from each
subject immediately after the first session and two weeks after the 3rd treatment session for
ultrastructural study.
Electron-microscopic findings showed no marked changes in the epidermis, but showed
stagnant capillaries and a generalized edema of the upper layer of the dermis 3 hours after the
IPL irradiation (Figure 11). Two weeks after the 3rd IPL treatment session, no marked
changes were seen in either the epidermis or the dermis, but melanin granule deposits were
observed immediately under the epidermis, along with melanin-laden macrophages in the
upper layer of the dermis (Figure 12).
As described above, IPL therapy is popular among patients, because almost no
postoperative clinical changes are observed in the skin, except mild erythema, and the so-
called down time is not required.
Electron-microscopic findings showed almost no changes in the epidermis immediately
after the IPL treatment, supporting the clinical findings.

Figure 11. Electron-microscopic findings revealed no marked changes in the epidermis, but showed
stagnant capillaries and generalized edema of the upper layer of the dermis 3 hours after the IPL
irradiation.
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1208 Tokuya Omi and Shigeru Sato

Figure 12. Two weeks after the 3rd IPL treatment session, no marked changes were noted in either the
epidermis or the dermis, however, melanin granule deposits were observed immediately under the
epidermis, along with melanin-laden macrophages in the upper layer of the dermis.

However, after the third treatment session, almost no changes were found in the
epidermis or dermis, but melanin granule deposits and melanin-laden macrophages were seen,
suggesting the effectiveness of IPL devices for treating darkly pigmented lesions. On the
other hand, few changes in fibroblasts or collagen fibers were observed in the dermis, which
could be the reason why patients evaluate IPL therapy poorly as a treatment for wrinkles and
sags. Actually, histological observation revealed no major changes at the microscopic level
after IPL therapy, and IPL therapy is considered to have a weak clinical effect on wrinkles
and sagging of the skin31). This can also be theoretically speculated since IPL therapy has a
weak direct effect on tissues due to the broadband light source, however, IPL covers almost
all wavelengths that are highly absorbed by melanin.
Therefore, it is considered that IPL therapy has a strong therapeutic effect on lesions with
pigmentary changes, but combined use with other techniques and devices is required for skin
rejuvenation aimed at improving wrinkles and sags.

Fractional CO2 Laser

The so-called fractional laser devices, which irradiate a laser beam in a dot form over a
grid pattern, have been developed with several wavelengths [32, 33, 34]. In particular,
ablative laser therapy with a CO2 laser has been considered to be highly effective, however, it
removes all skin layers, and scarring and hyperpigmentation have been observed as
problematic adverse effects. Fractional laser therapy using a CO2 laser has the advantages of
rapid epidermal regeneration and a short down time, because normal skin is left around the

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 Skin Rejuvenation – Ultrastructural Study 1209

laser-irradiated areas in a dot pattern. Many studies have reported the actual clinical effects of
such therapy.
In this study, a fractional CO2 laser device, SmartXide Dot (DEKA, Florence, Italy), was
used with the following irradiation parameters: output power 10 W, pulse width 600 s, dot
spacing 800 m and stack 2 (irradiation output power 0.91 J/cm2). A 1.5-cm square area was
irradiated by a single pass in the scanning mode. A clinical examination and punch biopsy of
each subject was performed before and just after the irradiation, and also at week 3 after 3
irradiation sessions.
Immediately after each fractional CO2 laser irradiation session, degeneration and
desquamation of the epithelium and bleeding were observed in the irradiated area (arrow)
(Figure 13).
The epithelium was desquamated, and the dermis was exposed in the area. In addition,
bleeding was seen between the stratum corneum and the stratum granulosum in the
desquamated epithelium. At 3 weeks, after 3 irradiation sessions, complete regeneration of
the epithelium was observed in the area of degeneration and desquamation. Epithelial cells
undergoing mitosis were also often observed.
At 3 weeks, after 3 irradiation sessions, almost complete regeneration of the epithelium
was noted. Ultrastructural examination of specimens stained with OTE revealed the presence
of elastin in the elastic fibers as electron-dense spherical or rod-shaped masses. Immediately
after each irradiation, the localized disappearance of elastin in the elastic fibers (arrows) was
often observed (Figure 14). At week 3, after 3 fractional CO2 irradiation sessions, elastin was
observed as electron-dense deposits, however, the elastic fibers (arrows) as a whole were
fragmented, showing an elaunin-like appearance (Figure 15).
Desquamation of the epithelium and the exposure and degeneration of the upper layer of
the dermis were confirmed immediately after each irradiation, in accordance with the interval
of the fractional laser irradiation. This was consistent with the ablation mechanism of the
device used in this study being similar to that of the CO2 laser reported previously.

Figure 13. Light-microscopic observations after toluidine blue staining Immediately after each
fractional CO2 laser irradiation session, degeneration and desquamation of the epithelium and bleeding
were observed in the irradiated area (arrow). At week 3, after 3 irradiation sessions, almost complete
regeneration of the epithelium was noted.
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1210 Tokuya Omi and Shigeru Sato

Figure 14. Ultrastructural findings in the superficial dermis. Examination of specimens stained with
OTE revealed the presence of elastin in the elastic fibers, as electron-dense spherical or rod-shaped
masses. Immediately after each fractional CO2 laser irradiation, localized disappearance of the elastin in
the elastic fibers (arrows) was often observed.

Figure 15. At 3 weeks, after 3 fractional CO2 laser irradiation sessions, elastin was observed as electron-
dense deposits, but the elastic fibers (arrows) as a whole were fragmented, showing an elaunin-like
appearance.

Ultrastructurally, degeneration of the epithelium leaving the basal lamina intact in the
irradiated area appeared to cause epithelial regeneration to occur more rapidly after irradiation
with this laser than after treatment with an ablation-type laser, This is similar to the case after
thermal injury, in which the epithelium with the basal lamina regenerates more rapidly.
Evidence of regeneration of the epithelium at 3 weeks, after 3 irradiation sessions, was
observed. Clinically, the formation of crust-like dots was observed after each irradiation
session. The disappearance of elastin was observed in the dermis immediately after
irradiation, and elaunin-like elastin fibers were subsequently observed at 3 weeks, after the 3
irradiation sessions.

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 Skin Rejuvenation – Ultrastructural Study 1211

CONCLUSION
The morphological changes in the skin after the various skin rejuvenation treatments
discussed above can be summarized as follows: 1) inflammatory cell infiltration of the
dermis, and 2) changes in elastic fibers in the dermis were observed, with or without damage
to the epidermis, with almost all methods.
With regard to inflammatory cell infiltration, edema of the upper layer of the dermis
associated with lymphocytic infiltration was observed immediately after treatment by
chemical peeling, and similar edema and infiltration of mainly neutrophils was observed after
the dye laser treatment. Thereafter, infiltration by lymphocytes and histiocytes, as well as
degranulated mast cells, became gradually apparent. Inflammatory cell infiltrations after skin
rejuvenation treatments can be summarized as follows: interstitial edema is observed in the
early stages after treatment, followed by infiltration by neutrophils and mast cells, depending
on the degree of invasion, and later infiltration by lymphocytes with histiocytes is observed.
Mast cells with granulation are also seen during the process of burn wound healing [36],
which is considered to be related to the wound healing process in rejuvenation.
Inflammation is recognized as necessary in the wound healing process, leading to
proliferation and remodeling, both of which have been associated with successful skin
rejuvenation.
During the normal rejuvenation process, immature elastic fibers, i.e., elaunin fibers, are
observed in the dermis and remodeling of the dermis is considered to occur at the same time.
Based on this knowledge, the elaunin-like elastic fibers are considered to reflect dermal
remodeling. The proliferation of degenerated elastic fibers poses a significant clinical
problem. The disappearance of existing elastic fibers and outgrowths of elaunin fibers were
seen immediately after the irradiation treatment. Therefore, elaunin fibers observed after
treatment are considered to represent a sign of rejuvenation.
In IPL therapy, the above-mentioned inflammatory cell infiltrations and elaunin fibers
were not evident, however, on the other hand, many melanin-laden macrophages were seen.
This is considered to serve as clinical evidence supporting the greater usefulness of IPL
therapy for the treatment of hyperpigmented lesions than for rejuvenation as described above.
The comparative ultrastructural study findings also lend support to the clinical treatment
efficacy and are very significant for examining the differences between devices and between
conditions.

REFERENCES
[1] Okuyama M, Omi T, Kawana S et al: Effects on cutaneous condition and wrinkle
reduction of a cream product containing Kinetin and Marixyl. Aesthet Dermatol 13:
161-165, 2003.
[2] Omi T, Sato S, Numano K, Kawana S. Ultrastructural observations of chemical peeling
for skin rejuvenation (ultrastructural changes of the skin due to chemical peeling). J.
Cosmet. Laser Ther, 12(1), 21-24, 2010.
[3] Omi T, Kawana S, Sato S, Honda M: Ultrastructual changes elicited by a non-ablative
wrinkle reduction laser. Lasers Surg. Med. 32, 46-49, 2003
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[4] Negishi K, Kushikata N, Takeuchi K, Tezuka Y, Wakamatsu S.:: Photorejuvenation by

intense pulsed light with objective measurement of skin color in Japanese patients.
Dermatol. Surg. 32, (11):1380-7. 2006.
[5] Takezaki S, Omi T, Sato S, Kawana S: Ultrastructural observations of human skin
following irradiation with visible red light-emitting diodes(LEDs): Apreliminary in
vivo report. Laser Therapy, 14(4), 153-160, 2006.
[6] Papadavid E, Katsambas A: Lasers for facial rejuvenation: a review. Int J. Dermatol.
2003; 42: 480-487.
[7] Kaufmann R, Beier C: Laser skin ablasion: An update on Aesthetics and Medical
Indications. Med. Laser Appl. 19: 212-222, 2004.
[8] Alster TS, Lupton JR: Nonablative cutaneous remodeling using radiofrequency devices.
[9] Gold MH: Fractional technology: a review and clinical approaches. J. Drugs Dermatol.
2007; 6: 849-852.
[10] Clementoni MT, Gilardino P, Muti GF, et al.: Non-sequential fractional ultrapulsed
CO2 resurfacing of photoaged facial skin: preliminary clinical report. J. Cosmet Laser
Ther. 2007; 9: 218-225.
[11] Sasaki GH, Travis HM, TuckerB: Fractional CO2 laser resurfacing of photoagedfacial
and non-facial skin: Histologic and clinical results and side effects. J. Cosmet Laser
Ther 2009; 11: 190-201.
[12] Sato S, Sasaki Y, Adachi A, Dai W, Liu XL, Namimatsu S. Use of oolong tea extract
(OTE) for elastin staining and enhancement in ultrathin specimens. Med. Electron.
Microsc, 2003; 36: 179-182.
[13] Landau M: Chemical peels. Clin. Dermatol. 26: 200-208, 2008.
[14] Furukawa F, Yamamoto Y: Recent advances in chemical peeling in Japan. J. Dermatol.
33: 655-661, 2006.
[15] Kempiak SJ, Uebelhoer N: Superficial chemical peels and microdermabrasion for acne
vulgaris. Semin Cutan Med. Surg. 27: 212-220, 2008.
[16] Isoda M, Ueda S, Imayama S, et al.: New formulation of chemical peeling agent:
histological evalution in sun-damaged skin model in hairless mice. J. Dermatol. Sci.,
27: 60-67, 2001.
[17] Moetaz B, Sameh K, Fatma Y, et al.:Trichloroacetic acid peeling versus dermabrasion:
A histometric, immunohistochemical, and ultrastructural comparison. Dermatol. Surg.,
30: 179-188, 2004.
[18] Athavale SM, Ries WR, Carniol PJ: Laser treatment of cutaneous vascular tumors and
malformations. Facial Plast Surg. Clin. N Am., 19: 303-312, 2011.
[19] Omi T, Kawana S, Honda M et al. Therapy for molluscum contagiosum by using pulsed
dye laser. Japanese Journal of Pediatric Dermatology, 24: 213-216, 2003.
[20] Bjerring P, Clement M, Heickendorff L: Selective non-ablative wrinkle reduction by
laser. J. Cutan. Laser Ther, 2: 9-15, 2000.
[21] Zelickson BD, Kilmer S, Bernstein E: Pulsed dye laser therapy for sun damaged skin.
Lasers Surg. Med., 28: 229-236, 1999.
[22] Omi T, Kawana S, Sato S, Takezaki S et. al. Cutaneous immunological activation
elicited by a low-fluence pulsed dye laser. Br. J. Dermatol, 153, 57-62, 2005.
[23] Ohshiro T, Calderhead RG: Low Level Laser Therapy: Practical Introduction. 1988,
John Wiley and Sons, Chichester, UK.

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[24] Baxter GD: Therapeutic Lasers: Theory and Practice. 1994, Churchill Livingstone,
Edinburgh, UK.
[25] Feroze NG: Ultrastructural pathology of the cell and matrix. Library of Congress
Cataloging-in- Publication Data, 1996, 946-950, Butterworth-Heinemann, UK.
[26] Rigau J, Trelles MA, Calderhead RG, Mayayo E: Changes in fibroblast proliferation
and metabolism following in vitro helium-neon laser irradiation. Laser Therapy, 1991;
3: 25-34.
[27] el Sayed SO, Dyson M. Effect of laser pulse repetition rate and pulse duration on mast
cell number and degranulation. Lasers Surg. Med, 1996; 19: 433-437.
[28] Trelles MA, Mayayo E, Miro L, Rigau J, Calderhead RG. The action of LLLT on mast
cells: a possible pain mechanism examined. Laser Therapy, 1989; 1: 27– 30.
[29] Bitter PH. Noninvasive rejuvenation of photodamaged skin using serial, full-face
intense pulsed light treatments. Dermatol. Surg. 2000; 26: 835-843.
[30] Bierring P, Christiansen K, Troilius A. Intense pulsed light source for treatment of
facial telangiectasias. J. Cosmetic Laser Ther. 2001; 3: 169-173.
[31] El-Domyati M, El-Ammawi TS, Moawad O, Medhat W, Mahoney MG, Uitto J. Intense
pulsed light photorejuvenation: a histological and immunohistochemical evaluation. J.
drigs Dermatol., 10, 1246-1252, 2011.
[32] Gold MH: Fractional technology: a review and clinical approaches. J. Drugs Dermatol.
2007; 6: 849-852.
[33] Clementoni MT, Gilardino P, Muti GF, et al.: Non-sequential fractional ultrapulsed
CO2 resurfacing of photoaged facial skin: preliminary clinical report. J. Cosmet. Laser
Ther. 2007; 9: 218-225.
[34] Sasaki GH, Travis HM, Tucker B: Fractional CO2 laser resurfacing of photoaged facial
and non-facial skin: Histologic and clinical results and side effects. J. Cosmet. Laser
Ther. 2009; 11: 190-201.
[35] Omi T, Kawanami O, Matsuda K, Tsujii A, Kawai A, Henmi H, VJ Ferrans：
Histological characteristics of the healing process of frozen skin allograft used in the
treatment of burns. Burns 22, 206‐211,1996.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 57

THE ROLE OF SUN

EXPOSURE IN SKIN AGING

Raja Dahmane, MD, PhD1,2,, Ruza Pandel, PhD1,2,

Polonca Trebse, PhD1,2 and Borut Poljsak, PhD1
1
Laboratory for Oxidative Stress Research, Faculty of Health
Sciences, University of Ljubljana, Ljubljana, Slovenia
2
Chair of Biomedicine in Health Care Division, Faculty of
Health Sciences, University of Ljubljana, Ljubljana, Slovenia

ABSTRACT
The ultraviolet (UV) spectrum of the solar light is the most damaging exogenous
source for our skin. Photoaging affects the sun-exposed areas and is characterized
clinically by fine and coarse wrinkling, roughness, dryness, laxity, telangiectasia, loss of
tensile strength and pigmentary changes. There is also an increase in development of
benign and malignant neoplasms on photoaged skin.
UV radiation (UVR) penetrates our skin, reaches the cells and is absorbed by DNA,
leading to the formation of photoproducts that inactivate the functions of DNA. UVA
radiation acts mostly indirectly through the generation of Reactive Oxygen Species
(ROS), producing high amounts of singled oxygen, which can further initiate lipid
peroxidation, oxidation of proteins or generation of DNA strand breaks. UVB action is
mostly by direct interaction with DNA via the induction of DNA damage. The epidermis
and dermis are both affected by UVB, but the dermis is also affected to a significant
extent by UVA. It has long been thought that the majority of human photo-lesions are due
to UVB rays; now it is believed that UVA plays a substantial role in photoaging. But
DNA is not the only biomolecule damaged by UVR. Free radicals and oxidants produced
by UVR oxidize also lipids and proteins in the cell. The skin then springs into action with
an inflammatory response, characterized by erythema (sunburn), the release of proteases
and cytokines. Infectious agents that may try to take advantage of this compromised
situation are sought out and destroyed. Then, a temporary period of immune suppression


Corresponding author: Raja Dahmane, MD, PhD, Faculty of Health Sciences, University of Ljubljana,
Zdravstvena pot 5, 1000 Ljubljana, Slovenia. Phone: + 386 40900878, fax: +386 1 3001119, e-mail:
[email protected].
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1216 Raja Dahmane, Ruza Pandel, Polonca Trebse et al.

follows. During that time, repair systems are activated that excise DNA lesions and
replace the damaged DNA, and non-functional proteins are turned-over by proteases.
Hormesis effect activates the synthesis of melanin and antioxidant protection, and
damaged lipids are cleaved and replaced. Irreparable cells are removed by apoptosis.
However, these repair mechanisms are not 100% effective. The problem arises in the
cases of intensive acute sun exposure or in the cases of chronic sun exposure over longer
decades, which manifests as skin photoaging.
To what extent each mechanism—mitochondrialDNA mutagenesis, protein
oxidation, downregulation of collagen synthesis and increased expression of matrix
metalloproteinases—contributes to premature skin aging is still not answered.
UV protection includes not only reduction of sun exposure but also the use of sun
protective filters, UV protective clothes, DNA repair enzymes, and antioxidant
supplementation.

INTRODUCTION
Human skin, like all other organs, undergoes chronological and biological aging. Aging
of the skin is a composite of actinic damage, chronological aging, and internal influences. In
addition, unlike other organs, skin is in direct contact with the environment and, therefore,
undergoes aging as a consequence of environmental damage (Fisher et al., 2002). Factors
contributing to premature aging are dependent on age, sex, pigmentation, smoking, sun
exposure history, alcohol consumption and other environmental, and lifestyle factors (Ernster
et al., 1995). Age-related physiological changes in elderly skin include clinical, histological,
and biochemical changes, as well as changes in neurosensory perception, barrier function,
wound healing and higher incidence of benign and cancerous diseases (Rasche and Elsner,
2010). Skin aging appears to be the result of two types of aging, intrinsic and extrinsic aging.
Extrinsic aging is the skin’s response to external damage and is controllable to a very large
degree by the lifestyle choices we make every day. The rate of aging is significantly different
among different populations, as well as among different anatomical sites, even within a single
individual. The intrinsic rate of skin aging in any individual can also be dramatically
influenced by personal and environmental factors, particularly the amount of exposure to
ultraviolet (UV) light. Photodamage, which considerably accelerates the visible aging of skin,
also greatly increases the risk of cutaneous neoplasm (Farage et al., 2008). Damage to human
skin due to UV light from the sun (photoaging) and damage occurring as a consequence of the
passage of time (chronologic or natural aging) were considered to be distinct entities. The
findings of the study performed by Varani et al. (2000) indicate that naturally aged sun-
protected skin and photoaged skin share important molecular features, including connective
tissue damage, elevated matrix metalloproteinase levels, and reduced collagen production.
The intrinsic (genetically determined) and the extrinsic (UV and toxic exposure mediated)
skin-aging processes are thus overlapped and are strongly related to the increased generation
of free radicals in the skin. Oxidative stress is believed to underlie changes associated with
both photoaging and natural aging and is considered of primary importance in driving the
skin-aging process. Extrinsic skin aging develops due to several factors: ionizing radiation,
severe physical and psychological stress, alcohol intake, poor nutrition, overeating,
environmental pollution, and exposure to UV radiation. It is estimated that among all these
environmental factors, UV radiation contributes up to 80%. UV radiation is the most

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 The Role of Sun Exposure in Skin Aging 1217

important environmental factor in the development of skin cancer and skin aging. After age
45, a thinning of the skin begins, due in part to hormonal changes. This thinning makes the
skin more fragile and vulnerable to damage by abrasion and more sensitive to irritating
environmental factors and allergens. Thin, wrinkled skin is very often attributed to a lack of
collagen. The dermis and overlying epidermis of aging skin are profoundly altered (Schmid et
al., 2002). Slower protein synthesis is one of the most common events observed during aging.
The synthesis of both structural proteins, such as collagen, and enzymes that repair and
maintain the normal metabolic functions of the cell, is slowed down. This leads to the
inefficient removal of damaged molecules and decreased intra- and intercellular signaling
pathways (Rocquet and Bonté, 2002). Synthesis of types I and III procollagen is reduced in
aged skin (Varani et al., 2000).
There is a progressive disappearance of elastic tissue in the dermis due to reduction in
elastin gene expression after the age of 40 to 50. Additionally, decreased proliferative
capacity of skin cells and decreased matrix synthesis contribute to intrinsic skin-aging
process. The moisture-holding proteoglycans and glycosaminoglycans decrease in abundance,
making the skin become dryer and looser. Proteoglycans make up a major part of the
extracellular matrix, the material between cells that provides structural support. Proteoglycans
are heavily glycosylated glycoproteins. This means that they are proteins with chains of
polysaccharides, a kind of carbohydrate, attached. The skin loses fat, so it looks less plump
and smooth. The number of blood vessels in aged skin decreases and the skin loses its
youthful color and glow. Since blood circulation in the dermic layer slows down, the delivery
of nutrients and oxygen to skin cells is decreased.
Skin atrophy is marked only after the fifth decade of human life and shows a plethora of
histomorphologic changes including epidermal thinning, flattening of the dermal-epidermal
junction, loss of melanocytes, and immune-competent Langerhans cells (Bhattacharyya,
2010). There are also dermal changes such as reduced fibroblast population and sebaceous
glands. There are typical ultrastructural changes in microvasculature of elderly people. Two
of the most noticeable changes as skin ages are alterations to pigment production (e.g., age
spots) and the formation of wrinkles.
Altered melanocyte function and reorganized, cross-linked highly structured collagen
matrices directly drive these visible aging changes respectively, but the primary cause of both
is excessive lifetime exposure to the sunlight (Jenkins et al., 2009). The major histological
features of sun-protected, intrinsically aged skin include a thin epidermis, significant
flattening of the dermal-epidermal junction (this results in a reduced exchange of nutrients
and metabolites between these two parts), thinning of the dermis and subcutaneous adipose
layer and reduced numbers of keratinocytes, fibroblasts, Langerhans cells, mast cells and
melanocytes. The dermis appears hypocellular with fewer fibroblasts and mast cells and loss
of dermal volume.
There is a decrease in the number of dermal blood vessels and a decrease in the density of
Pacinian and Meissner’s corpuscles, responsible for pressure and light touch perception (Yaar
and Gilchrest, 2003).
Besides chronological aging, actinic aging, also called photodamage, causes premature
skin aging: thinning of the dermis, a loss of collagen content and protein organization
(Bolognia, 1993). There is also a depressed sensory and autonomic innervation of epidermis
and dermis.
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1218 Raja Dahmane, Ruza Pandel, Polonca Trebse et al.

MECHANISMS OF SKIN PHOTOAGING

Because UV and photoaging play very important roles in skin aging, this chapter
describes their damaging effects on skin in a more detailed way. Unlike chronological aging,
which depends on the passage of time per se, photoaging depends primarily on the degree of
sun exposure and skin pigment. Individuals who have outdoor lifestyles, live in sunny
climates, and are lightly pigmented will experience the greatest degree of UVR skin
penetration and thus suffer from the effects of the photoaging (Fisher et al., 2002).
The term “photoaging” (also known as “Dermatoheliosis”) was first coined in 1986, and
describes the effects of chronic UV light exposure on skin (Kligman and Kligman, 1986).
Photoaging refers to the physiologic and pathological changes that occur specifically in aged
tissue that has experienced chronic sun exposure over time. Human skin aging resulting from
UV irradiation is a cumulative process that occurs based on the degree of sun exposure and
the level of skin pigment.
Clinical signs of photoaging include wrinkles; mottled pigmentation; rough skin, and loss
of skin tone; dryness; irregular, dark/light pigmentation; sallowness; either deep furrows or
severe atrophy; telangiectasia; premalignant lesions; laxity; and a leathery appearance (Yaar
et al., 2002). Other signs include elastosis (a coarse, yellow, cobblestoned effect of the skin)
and actinic purpura (easy bruising related to vascular wall fragility in the dermis) (Gilchrest,
1990). Sun-exposed areas of the skin, such as the face, neck, upper chest, hands, and
forearms, are the sites where these changes occur most often (Helfrich et al., 2008). While
intrinsically aged skin does not show vascular damage, photodamaged skin does. Studies in
humans and in the albino and hairless mouse model for skin aging have shown that acute and
chronic UVB irradiation greatly increases skin vascularization and angiogenesis (Yano et al.,
2002).

Skin Exposure to UVR

The sun is the main source of UVR and the main contributor to the photoaging. In order
to understand sun’s effects on the skin, a brief introduction on basic characteristics of UV
light and its environmental exposure will be presented. Solar radiation reaching the earth’s
surface includes wavelengths in the range 290 to 4000 nm and is divided into three bands: UV
radiation (290 to 400 nm), visible light (400 to 760 nm) and IR (760 to 4000 nm). UVR (100
to 400 nm) comprises only 5% of the terrestrial solar radiation. Sun’s UVR is divided into
categories based on the wavelength.

 UVC Radiation - 100 to 290 nm. UVC radiation is almost completely absorbed by
the ozone layer and does not affect the skin. UVC radiation can be found in artificial
sources such as mercury arc lamps and germicidal lamps.
 UVB Radiation - 290 to 320 nm. UVB affects the outer layer of skin, the epidermis,
and is the primary agent responsible for sunburns. It is the most intense between the
hours of 10:00 am and 2:00 pm, when the sunlight is brightest. It is also more intense
in the summer months, accounting for 70% of a person's yearly UVB dose. UVB
does not penetrate glass.

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 The Role of Sun Exposure in Skin Aging 1219

 UVA Radiation - 320 to 400 nm. UVA was once thought to have a minor effect on
skin damage, but now studies are showing that UVA is a major contributor to skin
damage. UVA penetrates deeper into the skin and works more efficiently. The
intensity of UVA radiation is more constant than UVB without the variations during
the day and throughout the year. UVA is not filtered by glass.

According to Laurent-Applegate and Schwarzkopf (2001), “the UVA has greater

penetration (e.g., about 20% at 365 nm). Whereas UVB is much more damaging to the skin
than UVA if equal exposures are carried out; the deeper penetration of UVA and its greater
abundance in sunlight (about 95% UVA, 5% UVB) suggest that it can also be a significant
contributor to damage. As the photons in the UVA waveband are less energetic, significantly
more photons are needed to cause the same damage as that induced by the shorter
wavelengths in the UVB region. It is important to remember that UVA photons are present in
sunlight in much higher quantities than those of UVB and that these longer wavelengths have
the potential to penetrate into the dermis to a far greater extent than UVB because of its less
energetic potential” (Laurent-Applegate and Schwarzkopf, 2001).
In the middle age, it was known the damaging effect of sun, which caused “farmer’s
skin,” and pale skin was appreciated. In the last decades, this trend changed. Dark skin
complexion was propagated as a sign of “healthy skin.” The exposure of human skin to
environmental and artificial ultraviolet irradiation has increased significantly in the last 50
years. This is not only due to an increased solar UV irradiation as a consequence of the
stratospheric ozone depletion, but also the result of an inappropriate social behavior with the
use of tanning parlors being very popular. Besides this, leisure activities and living style with
travelling to equatorial regions also add to the individual annual UV load. Total UVR load
depends on time of exposure, duration and intensity of exposure.
Since the population in industrialized countries shows an increasing total lifespan, in
parallel the cumulative lifetime dose of solar and artificial UV irradiation is dramatically
augmented (Grether-Beck et al., 2005).
While there is no standard measure, sun exposure can be generally classified as
intermittent or chronic, and the effects may be considered acute or cumulative. Intermittent
sun exposure is obtained sporadically, usually during recreational activities, and particularly
by indoor workers who have only weekends or vacations to be outdoors and whose skin has
not adapted to the sun. Chronic sun exposure is incurred by consistent, repetitive sun
exposure during outdoor work or recreation. Acute sun exposure is obtained over a short time
period on skin that has not adapted to the sun (National Cancer Institute). UV-induced
extrinsic aging is visible on chronically UV-exposed skin areas in persons frequently engaged
in outdoor activities. Exposed skin surface is irradiated differently depending on cultural and
social behavior, clothing, the position of the sun in the sky and the relative position of the
body. Exposure to UVB of the most exposed skin surfaces, such as nose, tops of the ears and
forehead, relative to that of the lesser exposed areas, such as underneath the chin, normally
ranges over an order of magnitude. Ground reflectance plays a major role in exposure to UVB
of the eyes and shaded skin surfaces, particularly with highly reflective surfaces such as snow
(IARC, 2010). The cumulative annual exposure dose of solar UVR varies widely among
individuals in a given population, depending to a large extent on occupation and extent of
outdoor activities (IARC, 2010). For example, it has been estimated that indoor workers in
mid-latitudes receive an annual exposure dose of solar UVR to the face of about 40 to 160
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1220 Raja Dahmane, Ruza Pandel, Polonca Trebse et al.

times “the minimal erythema dose” (MED), depending upon propensity for outdoor activities,
whereas the annual solar exposure dose for outdoor workers is typically around 250 times the
MED. Because few actual measurements have been reported of personal exposures, these
estimates should be considered to be very approximate and subject to differences in cultural
and social behavior, clothing, occupation and outdoor activities. Cumulative annual outdoor
exposures may be augmented by exposures to artificial sources of UVR. For example, the use
of cosmetic tanning appliances increased in popularity in the 1980s. The majority of users are
young women, and the median annual exposure dose is probably 20 to 30 times the MED.
Currently, used appliances emit primarily UVA radiation; prior to the 1980s, tanning
lamps emitted higher proportions of UVB and UVC (IARC, 2010).

Ozone Depletion and UVR

The quality and quantity of UVR at the earth's surface depend on the energy output of the
sun and the transmission properties of the atmosphere. From a biological viewpoint, UVB
radiation is by far the most significant part of the terrestrial UV spectrum, and the levels of
radiation in this waveband reaching the surface of the earth are largely controlled by ozone, a
gas which comprises approximately one molecule out of every two million in the atmosphere
(Diffey, 1991).
Ozone layer in the stratosphere is also the major barrier to UVC (and largely to UVB).
Depletion of this layer may result in more UVB reaching the earth’s surface, with the
corresponding increase in photochemical damage to living organisms. It has been estimated
that each 5% depletion of stratospheric ozone will raise UVB flux at ground level by 10%. Up
to 10% of UVB light falling on the skin can penetrate the epidermis to reach the dermis. In
1974, Molina and Rowland (Molina and Rowland, 1974) first warned that
chlorofluorocarbons (CFCs) and other gases released by human activities could alter the
natural balance of creative and destructive processes and lead to depletion of the stratospheric
ozone layer. Substantial reductions of up to 50% in the ozone column observed in the austral
spring over Antarctica and first reported in 1985 (Farman et al., 1985) are continuing (SORG,
1990).
Coupled with this, there has been a statistically significant downward trend in wintertime
total ozone over the northern hemisphere of about 2% to 3% per decade for the past 30 years,
although summertime ozone levels have remained approximately constant (Frederick, 1990).
In its report in June 1990, the UK Stratospheric Ozone Review Group concluded that
there are serious limitations in our understanding and ability to quantify ozone depletion at
the present levels of contaminant release and in our ability to predict the effects on
stratospheric ozone of any further increases (SORG, 1990). The atmosphere has a profound
effect on the irradiance that reaches the surface of the earth.
In January (in the northern hemisphere) or July (in the southern hemisphere), when the
solar elevation is low, direct UV travels a longer path through the atmosphere, and a large
amount of scattering occurs.
In addition, much of the resultant scattered UV propagates downwards to the earth's
surface at angles to the horizontal that are larger than the solar elevation, hence travelling a
shorter and less absorptive path. This results in large ratios of scattered to direct UV. During

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 The Role of Sun Exposure in Skin Aging 1221

the summer, the ratio of diffuse to direct UV is smaller (International program on chemical
safety, Environmental Health Criteria, 160).

FACTORS AFFECTING TERRESTRIAL UVR

As already mentioned, the spectral irradiance of UVR at the earth's surface is modified by
temporal, geographical and meteorological factors (Frederick et al., 1989).
According to Diffey (1991) the following factors contribute significantly to terrestrial
UVR intensity:

 Time of day. About 20% to 30% of total daily UVR is received one hour either side
of midday in summer, with 75% between 9 am and 3 pm.
 Season. In temperate regions, the biologically damaging UVR reaching the earth's
surface shows strong seasonal dependence. However, seasonal variation is much less
nearer the equator.
 Geographical latitude. Annual UVR flux decreases with increasing distance from the
equator.
 Clouds. Clouds reduce solar irradiance at the Earth's surface, although changes in the
ultraviolet region are not as great as those of total intensity, since water in clouds
attenuates solar infrared much more than UVR. The risk of overexposure may be
increased under these conditions because the warning sensation of heat is diminished.
Light clouds scattered over a blue sky make little difference to UVR intensity unless
directly covering the sun, whilst complete light cloud cover reduces terrestrial UVR
to about one-half of that from a clear sky. Even with heavy cloud cover, the scattered
ultraviolet component of sunlight (often called skylight) is seldom less than 10% of
that under clear sky (Paltridge and Barton, 1978). However, very heavy storm clouds
can virtually eliminate terrestrial UVR, even in summertime (Diffey, 1988).
 Surface reflection. Reflection of UVR from ground surfaces, including the sea, is
normally low (<7%). However, gypsum sand reflects about 25% of incident UVB
and fresh snow about 30% (Doda and Green, 1980, 1981) although other authors
(Blumthaler and Ambach, 1985) have reported that the UVB reflectance of fresh
snow exceeds 80%.
 Altitude. In general, each 1 km increase in altitude increases the ultraviolet flux by
about 6% (Cutchis, 1980). Conversely, places on the earth's surface below sea level
are relatively poorer in UVB content than nearby sites at sea level. This is strikingly
apparent around the Dead Sea, 400 m below sea level (Kushelevsky and Slifkin,
1975).

UVR AND ITS PENETRATION TO THE SKIN

UV incident on human skin can follow one of three courses: it can undergo absorption,
reflection, or scattering. The first step in a photochemical reaction is the absorption of a single
photon by a molecule and the production of an excited state in which one electron of the
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1222 Raja Dahmane, Ruza Pandel, Polonca Trebse et al.

absorbing molecule is raised to a higher energy level. Such radiative transition can only occur
efficiently when the photon energy of the radiation is close to the energy difference of the
atom in the initial and final state (energy level).
The photochemistry that may then occur will, therefore, depend upon the molecular
structure and the wavelength of UV as well as the specific reaction conditions.
The primary products generated by UV absorption are generally ROS. Reflection not
only occurs at the surface of the stratum corneum but at all interfaces changing in refractive
index. Scattering occurs because of the different structural elements, such as hair follicles and
sebaceous glands, and also by cellular components, such as mitochondria and ribosomes.
The remaining UV can penetrate into deeper skin layers (International program on
chemical safety, Environmental Health Criteria, 160).
UV penetrates into the dermis exposing a variety of cells and structures, depending in
part on the thickness of the human stratum corneum and epidermis. The depth of penetration
is wavelength dependent—the longer the wavelength the deeper the penetration (Bruls et al.,
1984).

DAMAGING EFFECT OF UVR

Studies in hairless mice demonstrated the carcinogenicity of exposures to UVR in the
wavelength ranges 315 to 400 nm (UVA), 280 to 315 nm (UVB) and ¾ 280 nm (UVC), UVB
radiation being the most effective, followed by UVC and UVA. UVB radiation is three to
four orders of magnitude more effective than UVA. Nevertheless, both short-wavelength
UVA (315 to 340 nm) and long-wavelength UVA (340 to 400 nm) induced skin cancer in
hairless mice. The carcinogenic effectiveness of the latter waveband is known only as an
average value over the entire range; the uncertainty of this average is about one order of
magnitude. In none of the experiments involving UVC was it possible to exclude completely
a contribution of UVB, but the size of the effects observed indicate that they cannot be due to
UVB alone (IARC, 2010). UVB is three to four times more effective than UVA in producing
erythema. In humans, pigmentation protects against erythema and histo-pathological changes.
People with a poor ability to tan, who burn easily and have light eye and hair color are at a
higher risk of developing melanoma, basal-cell and squamous-cell carcinomas (IARC, 2010).
UVB most commonly causes damage in the form of cyclobutane pyrimidine dimmers. UVA,
on the other hand, primarily causes DNA damage indirectly by the production of short-lived
Reactive Oxygen Species such as singlet oxygen, superoxide and H2O2 via endogenous
photosensitizers. UVA radiation generates more phosphodiester bond breaks in DNA than
would be expected by the total amount of energy directly absorbed by the DNA. Therefore,
most likely there is indirect damage to DNA accomplished by endogenous photosensitizers
such as riboflavin, nicotinamide coenzymes, and rare RNA bases (Laurent-Applegate and
Schwarzkopf 2001).

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 The Role of Sun Exposure in Skin Aging 1223

Skin Response to Acute and Chronic UV Radiation

Skin pigmentation is induced both by UVB and UVA rays. According to Diffey (1991),
the normal responses of the skin to UVR can be classed under two headings: acute effects and
chronic effects. An acute effect is one of rapid onset and generally of short duration, as
opposed to a chronic effect, which is often of gradual onset and long duration. These effects
should be distinguished from acute and chronic exposure conditions, which refer to the length
of the UVR exposure. The acute reactions considered will be sunburn, tanning and vitamin D
production.
Photoaging and skin cancer will be discussed as those chronic reactions produced by
prolonged or repeated UVR exposure (Diffey 1991). Exposure to ultraviolet (UV) radiation
increases skin pigmentation and usually results in an even darkening of the skin.
However, it may also occasionally lead to the development of hyperpigmented lesions
due to a local overproduction of pigment.

Skin Antioxidant Defense

Although the skin possesses an elaborate antioxidant defense system to deal with UV-
induced oxidative stress and immunotoxicity, excessive and chronic exposure to UV light can
overwhelm the cutaneous antioxidant and immune response capacity, leading to oxidative
damage and immunotoxicity, premature skin aging, and skin cancer. Photoaged skin has
significantly reduced concentrations of antioxidant enzymes in the stratum corneum and the
epidermis, while the concentration of oxidized proteins in the upper dermis is increased.
Acute exposure to UV irradiation depletes the catalase activity in the skin and increases
protein oxidation (Sander et al., 2002).

DNA Damage

DNA is constantly exposed to DNA-damaging agents. Environmental DNA-damaging

agents include UV light and ionizing radiation, as well as a variety of chemicals encountered
in foodstuffs, or as air- and water-borne agents. Endogenous damaging agents include
metabolites that can act as alkylating agents and the ROS that arise during respiration.
Since there are always less antioxidants available to neutralize all oxidizing agents
involved in free radical formation and damage, a third line of defense evolved—damage
repair mechanisms. Defense mechanisms are available to cope with constant free radical-
induced damage in our cells. Among them are DNA repair systems to replace damaged DNA
bases and antioxidants to neutralize free radicals. Damaged cells are eliminated by apoptosis
and non-functional proteins are turned-over by proteases. But DNA repair capacity has been
found to decrease with age.
For example, decrease in the level of proteins that participate in nucleotide excision
repair was reported to occur for aged dermal fibroblasts (Goukassian et al., 2000).
Oxidation of DNA can produce different types of DNA damage: strand breaks, sister
chromatid exchange, DNA-protein cross-links, sugar damage, abasic sites, and base
modifications. Cell death, chromosome changes, mutation and morphological transformations
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1224 Raja Dahmane, Ruza Pandel, Polonca Trebse et al.

are observed after UV exposure of prokaryotic and eukaryotic cells. Numerous types of UV-
induced DNA damage have now been recognized that include stand breaks (single and
double), cyclobutane-type pyrimidine dimers, 6-4 pyo photoproducts and the corresponding
Dewar isomer, thymine glycols, 8-hydroxy guanine, and many more. Additionally, the
specific lesions in DNA that can be induced by UVA radiation include pyrimidine dimmers,
single-strand breaks (both not thought to be the critical lesions in UVA radiation-induced
cellular lethality), and, perhaps more importantly, DNA protein cross-links (Peak et al., 1987;
Peak et al., 1988). Solar UV causes formation of ROS, which can oxidize guanine in DNA to
form 8-hydroxy-7,8-dihydroguanine (8-OHdG).
The frequency of this characteristic mutation in human skin increases with cumulative
sun exposure and could be used as internal dosimeter of cumulative sun exposure (Yarosh,
2010). OH˙ can add on to guanine at positions 4, 5 and 8 in the purine ring. Addition to C-8
produces a C-8 OH adduct radical that can be reduced to 8-hydroxy-7,8-dihydroguanine,
oxidized to 8-hydroxyguanine, or undergo opening of the imidazole ring, followed by one-
electron reduction and protonation, to give 2,6-diamino-4-hydroxy-5-formamidopyrimidine,
abbreviated as FAPyG (Halliwell and Gutterigde, 1999). Photoexcitation of cytosine and
guanine may lead to the formation in relatively minor yields of 6-hydroxy-5,6-
dihydrocytosine and 8-oxo-7,8-dihydroguanine, respectively.
A second mechanism that requires the participation of endogenous photosensitizers
together with oxygen is at the origin of most of the DNA damage generated by the UVA (320
to 400 nm) and visible light.
However, it may be expected that the latter oxidized purine lesion together with DNA
strand breaks and pyrimidine base oxidation products are also generated with a lower
efficiency through Fenton type reactions (Cadet et al., 1997). The number of different DNA
modifications that OH˙ is capable of producing appears to be over 100 (Hutchinson, 1985). In
addition, DNA-protein cross-links are produced during UV exposure. Larger scale genetic
alterations include chromosome breakage, sister chromatid exchanges and chromatid
aberrations. Although partial UV action spectra are now available for many of these lesions,
the most studied have been the different types of pyrimidine dimers (International program on
chemical safety, Environmental Health Criteria, 160).

Damage to Elastin and Collagen and Wrinkle Formation

Wrinkling in the skin is caused by habitual photoaging, facial expressions, aging,

smoking, poor hydration, and various other factors (Anderson 2006). The effects of aging on
the dermal layer are significant. Not only does the dermal layer thin but also less collagen is
produced, and the elastin fibers that provide elasticity wear out. These changes in the
scaffolding of the skin cause the skin to wrinkle and sag. Photoaged skin can be associated
with either increased epidermal thickness or pronounced epidermal atrophy. The most
pronounced histological change is the accumulation of elastin-containing material just below
the dermal-epidermal junction, known as solar elastosis (Lavker 1995). Collagen, which
composes over 90% of the skin’s total proteins, becomes disorganized (Bernstein et al.,
1996). While elastin levels are increased in photoaged skin, levels of types I and III collagen
precursors and cross-links are reduced (Talwar et al., 1995). It is likely that such changes in
collagen precursors lead to reduced levels and/or altered organization of fibrillar collagen and

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 The Role of Sun Exposure in Skin Aging 1225

thus may contribute to the wrinkled appearance of photodamaged human skin. The Glogau
classification system was developed to objectively measure the severity of photoaging and
especially wrinkles. Four types of skin depressions can be defined according to their depth:
folds, permanent wrinkles, reducible wrinkles and skin micro-relief. When UV rays from the
sun penetrate the skin, they damage elastic fibers. As the elastin weakens, skin becomes less
elastic and loses its ability to snap back after being stretched. Photoaged skin contains elastic
material in the reticular dermis, and the fibrillin deposits in the reticular dermis are enlarged.
Elastic fibers have a central core of hydrophobic cross-linked elastin surrounded by fibrillin-
rich microfibrils. The papillary dermal microfibrillin-rich microfibril network is truncated and
depleted in photoaged skin.
There are fewer fibrillin-rich microfibrils in wrinkled photoaged skin, probably due to
inflammatory cell proteinases (neutrophil elastase) or activation of matrix metalloproteinase
(Tsuji et al., 2001). Cross-linking causing decreased elasticity could also be involved in
wrinkle formation (Watson et al., 1999). Wrinkles form especially in parts of skin that get
stretched and move a lot (like around eyes, mouth and nose). Anti-aging skin care products
should focus on wrinkles prevention also as anti-sun damage products in order to prevent
elastin and collagen damage. A decrease in the overall ROS load by efficient sunscreens or
other protective agents may represent promising strategies to prevent or at least minimize
ROS-induced photoaging.
For example, the study of Seo et al. (2001) on photoaged skin has shown that UV
irradiation increases the tropoelastin mRNA in keratinocytes and fibroblasts (Seo et al.,
2001). Selective inhibition of skin fibroblast elastase could be one way to fight wrinkle
formation following cumulative ultraviolet B irradiation (Tsukahara et al., 2001). Lysozyme
may alter the elastic fibers in the surface, preventing further degradation and the accumulation
of altered elastic fibers.

Why UV Radiation Accelerates the Aging Process?

As already mentioned, UV radiation can cause significant DNA damage and especially to
mitochondrial DNA (mtDNA). In a study by Berneburg et al. (2004), it was confirmed that
repetitive exposure to UVA light leads to an approximately 40% increase in the level of the
common deletion in skin tissue. Even 16 months after cessation of UV exposure, accumulated
damage was increased in some individuals by 32-fold (Berneburg et al., 2004). The causative
role of oxidative stress for the increased frequency of mtDNA aberrations was demonstrated
also in a variety of other experimental models and in human studies (Blatt et al., 2010). Also,
a causative link between mtDNA mutations and aging phenotypes in mammals was evidenced
in knock-out mice. Age-related phenotypes such as reduced subcutaneous fat, weight loss,
alopecia, reduced fertility and heart enlargement were observed besides increased amount of
point mutations and increased amount of deleted mtDNA (Trifunovic et al., 2004). Increased
formation of oxidative stress due to endogenous or exogenous sources (e.g., UVR) causes
mtDNA lesions in human cells and detrimental changes in mitochondrial respiration. Blatt et
al. (2010) explain that due to a negative feedback loop, damaged mtDNA is a cause and a
consequence of aging as well. Any lack of mitochondrial function impairs cellular ATP
synthesis, thus reducing the “fuel supply” for repair mechanisms. It does further stimulate the
formation of ROS as byproducts of an impaired mitochondrial respiration. More ROS are
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1226 Raja Dahmane, Ruza Pandel, Polonca Trebse et al.

produced, more damage accumulates in neighboring mitochondrial complexes, membranes

and mtDNA, progressive decline in the energy-generating capacity of the cell is observed,
what further accelerates the aging process. Due to impaired ATP formation, the switch to
anaerobic pathway such as glycolysis occurs, but also glycolysis additionally increases the
formation of oxidative stress by the formation of reactive glycolytic intermediates, which
favor the formation of advanced glycation end products (AGEs).
Thus even a single sunburn period could, in theory, start the above-described chain
reaction leading to accelerated aging process in the skin cell.

UVR AND ROS FORMATION

UVR can increase the ROS formation in the skin cells. Oxidative stress is believed to
underlie changes associated with both photoaging and natural aging. According to Pattison
and Davies (2006), UV radiation can mediate damage via two different mechanisms: (a)
direct absorption of the incident light by the cellular components, resulting in excited state
formation and subsequent chemical reaction, and (b) photosensitization mechanisms, where
the light is absorbed by endogenous (or exogenous) sensitizers that are excited to their triplet
states. The excited photosensitizers can induce cellular damage by two mechanisms: (a)
electron transfer and hydrogen abstraction processes to yield free radicals (Type I); or (b)
energy transfer with O2 to yield the reactive excited state, singlet oxygen (Type II) (Pattison
and Davies, 2006).
The primary mechanism by which UV radiation initiates molecular responses in human
skin is via photochemical generation of ROS mainly formation of superoxide anion (O2-˙),
hydrogen peroxide (H2O2), hydroxyl radical (OH˙), and singlet oxygen (1O2) (Hanson and
Clegg, 2002). The main DNA product generated by (1)O2 is 8-oxo-Gua; this is a common
lesion in DNA and is formed by a range of other oxidants in addition to UV. UV light does
not deposit sufficient energy in water molecules to fragment them, in contrast to X-rays and
γ- rays. However, in the presence of H2O2 (formed during normal metabolism), UVB forms
hydroxyl radical OH˙.

H2O2 + UV → OH˙+ OH˙

In vitro, ex vivo and solution-phase studies have found that ROS, such as singlet oxygen
(1O2), H2O2, superoxide (O2-˙) and nitric oxide, are generated after absorption of UV radiation
by chromophores that are also found in keratinocytes (urocanic acid, riboflavin, reduced form
of nicotinamide adenine dinucleotide–reduced nicotinamide adenine dinucleotide phosphate
[NADH/ NADPH], tryptophan) (Hanson et al., 1997; Hanson et al., 1998).
UVA radiation produces cellular modifications that considerably overlap those induced
by oxidative damage (Tyrrell 1991). UVA radiation constitutes an oxidant stress that involves
the generation of active species including singlet oxygen and hydroxyl radicals.
Hydrogen peroxide can be generated by UVA irradiation of tryptophan (McCormick et
al., 1976), and superoxide can be produced by UVA irradiation of NADH and NADPH
(Cunningham et al., 1985). The skin-damaging effects of UVA appear to result from type II,
oxygen-mediated photodynamic reactions in which UVA or near-UV radiation in the

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presence of certain photosensitizing chromophores (e.g., riboflavin, porphyrins, nicotinamide

adenine dinucleotide phosphate (NADPH), etc.) leads to the formation of reactive oxygen
species (1O2, O2-, .OH) (Dalle Carbonare and Pathak, 1992).
Iron-free porphyrins also generate singlet oxygen upon exposure to UVA radiation. Also,
other small molecules have the potential to generate active oxygen intermediates upon UVA
exposure (Tyrell, 1992). For example, the photochemical degradation of tryptophan by
wavelengths that include the more energetic portion of the UVA spectrum is able to generate
hydrogen peroxide and N-formyl kynurenin (McCormick et al., 1976).
Although the level of hydrogen peroxide generated in vivo by such a pathway would
appear to be in the low micromolar range, it could nevertheless be crucial to biological
processes since iron complexes (such as citrate) that are present in the cytoplasm will react
with hydrogen peroxide to generate the highly reactive hydroxyl radical in a superoxide
driven Fenton reaction (Imlay et al., 1988). Since the reaction is driven by the continual
reduction of ferric iron to the ferrous state by superoxide anions, a cellular source of
superoxide anions is also required.
In this context, it should be noted that both hydrogen peroxide and hydroxyl radical are
generated by UVA irradiation of NADH and NADPH (Czochralska et al., 1984; Cunningham
et al., 1985). However, it is not at all clear whether this is really the key source of superoxide
anions or whether the main source is as a consequence of normal cellular metabolism. Iron
liberation and photodamaged skin contribute to oxidative damage and activation of
transcription factors (Halliwell and Gutteridge, 2007).
Indeed, chronic exposure of hairless mice to low levels of UVB increases non-hem iron
content of the skin; increases in iron with age and sun exposure are also observed in human
skin. Topical application of certain iron ion chelators to skin of hairless mice appeared to
delay the onset of UVB-induced damage (Halliwell and Gutteridge, 2007).
Naturally occurring iron complexes can react in vivo with hydrogen peroxide in the
presence of superoxide anion in the Haber–Weiss reaction, which produces the potentially
lethal hydroxyl radical.

MOLECULAR MECHANISMS BY
WHICH UVR CAUSES PHOTOAGING
In previous paragraph, evidence was given that UV radiation generates reactive oxygen
species and ROS can further oxidize cellular components. UV irradiation also directly or
indirectly initiates and activates a complex cascade of biochemical reactions in human skin.
Besides, the UV light-induced ROS interfere with signaling pathways. On a molecular level,
UV radiation from the sun attacks keratinocytes and fibroblasts, resulting in the activation of
cell surface receptors, which initiate signal transduction cascades. This, in turn, leads to a
variety of molecular changes, which causes a breakdown of collagen in the extracellular
matrix and a shutdown of new collagen synthesis (Fisher 2005). UV-induced liberation of
ROS in human skin is responsible for stimulation of numerous signal transduction pathways
via activation of cell surface cytokine and growth factor receptors. UVA or UVB induce
activation (sometimes via peroxides or singlet O2 as signaling molecules) of a wide range of
transcription factors in skin cells (Halliwell and Gutteridge, 2007). This can increase
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production of matrix metalloproteinases that can degrade collagen and other connective tissue
components. For example, the UV light-induced ROS induce the transcription factor activator
protein-1 (AP-1). AP-1 induces upregulation of matrix metalloproteinases (MMPs) like
collagenase-1 (MMP-1), stromelysin-1 (MMP-3), and gelatinase A (MMP-2), which
specifically degrade connective tissue such as collagen and elastin and indirectly inhibit the
collagen synthesis in the skin (Rasche and Elstner, 2010). As indicated by their name, these
zinc-dependent endopeptidases show proteoplytic activity in their ability to degrade matrix
proteins such as collagen and elastin (Krutmann and Gilchrest, 2006). Destruction of collagen
is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion is matrix
metalloproteinase-1 (MMP-1) (Dong et al., 2008). Skin fibroblasts produce MMP-1 in
response to UVB irradiation, and keratinocytes play a major role through an indirect paracrine
mechanism involving the release of epidermal cytokine after UVB-irradiation (Fagot et al.,
2002). MMPs are produced in response to UVB irradiation in vivo and are considered to be
involved in the changes in connective tissue that occur in photoaging (Brinckmann et al.,
1995). They are associated with a variety of normal and pathological conditions that involve
degradation and remodeling of the matrix (Smutzer 2002). UV rays and aging lead to excess
proteolytic activity that disturbs the skin's three-dimensional integrity (Rocquet and Bonte,
2002).
These proteinases are important for breaking down the extracellular matrix during
chronic wound repair, in which there is re-epithelialization by keratinocyte migration. Thus,
MMPs are continuously involved in the remodeling of the skin after chronic aggression.
Photodamage also results in the accumulation of abnormal elastin in the superficial dermis,
and several MMPs have been implicated in this process (Rocquet and Bonte, 2002). ROS
activate cytoplasmic signal transduction pathways in resident fibroblasts that are related to
growth, differentiation, senescence, and connective tissue degradation (Scharffetter-Kochanek
et al., 2000).
As well as causing permanent genetic changes involving protooncogenes and tumor
suppressor genes, ROS activate cytoplasmic signal transduction pathways that are related to
growth differentiation, senescence, transformation and tissue degradation (Scharffetter-
Kochanek et al., 1997). The study of Kang et al. (2003) revealed that UVA/UVB irradiation
of skin causes generation of H2O2 within 15 minutes. AP-1, which leads to increased collagen
breakdown, becomes elevated and remains elevated within 24hours following UV irradiation
(Fisher et al., 1996). Decreased procollagen synthesis within eight hours of UV irradiation
was demonstrated (Quan et al., 2002). Consequently, increased collagen breakdown was
demonstrated (Fisher et al., 1997). It is hypothesized that dermal breakdown is followed by
repair that, like all wound repair, is imperfect. Imperfect repair yields a deficit in the
structural integrity of the dermis, a solar scar. Dermal degradation followed by imperfect
repair is repeated with each intermittent exposure to ultraviolet irradiation, leading to
accumulation of solar scarring and ultimately visible photoaging (Fisher and Voorhees,
1998). While it may seem that the signs of photoaging appear overnight, they actually lie
invisible beneath the surface of the skin for years. UV exposure of the skin causes oxidative
stress, leading to inflammatory reactions, such as erythema, sunburn, and chronic reactions.
Most problematic chronic reactions include premature skin aging and skin cancer
(Oresajo et al., 2010). The amount of photoaging that develops depends on: 1) a person’s skin
color and 2) their history of long-term or intense sun exposure. People with fair skin who
have a history of sun exposure develop more signs of photoaging than those with dark skin.

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 The Role of Sun Exposure in Skin Aging 1229

It should be remembered that solar radiation includes not only UVR but also visible and
infrared radiation. Visible light is thought to be unimportant in photoaging (Kligman, 1986),
but studies have confirmed that infrared radiation can certainly damage the dermal matrix
(Kligman and Kligman, 1984). It could be concluded that photoaging plays a significant role
in skin-aging process.

INTERMITTENT EXPOSURE HYPOTHESIS

Although the evidence from epidemiological studies indicates an association between
melanoma and sunlight exposure, it does not appear that cumulative sun exposure explains
the relationship, as it does for non-melanoma skin cancer (NMSC). Instead, an intermittent
exposure hypothesis proposes that infrequent intense exposure of unacclimatized skin to
sunlight is related to increasing melanoma incidence and is more important than chronic sun
exposure. This hypothesis is supported by the observation that most studies have shown that
an increased risk of melanoma is associated with a past history of severe sunburn in
childhood and adolescence (Armstrong, 1988). This could be attributed also to the effect of
hormesis, which explains action resulting from a response of an organism to low intensity of
a stress (e.g., UVR exposure). Stress response then activates increased protection mechanisms
and DNA repair systems.
According to World Health Organization - International Agency for Research on Cancer,
in the Monographs on the evaluation of carcinogenic risks to humans’ solar and ultraviolet,
radiation is classified:
“Solar radiation is carcinogenic to humans (Group 1). There is sufficient evidence in
humans for the carcinogenicity of solar radiation. Solar radiation causes cutaneous malignant
melanoma and nonmelanocytic skin cancer. There is sufficient evidence for the
carcinogenicity of solar radiation in experimental animals. There is sufficient evidence for the
carcinogenicity of broad-spectrum ultraviolet radiation in experimental animals. There is
sufficient evidence for the carcinogenicity of ultraviolet A radiation in experimental animals.
There is sufficient evidence for the carcinogenicity of ultraviolet B radiation in experimental
animals. There is sufficient evidence for the carcinogenicity of ultraviolet C radiation in
experimental animals. Ultraviolet A radiation is probably carcinogenic to humans (Group
2A). Ultraviolet B radiation is probably carcinogenic to humans (Group 2A). Ultraviolet C
radiation is probably carcinogenic to humans (Group 2A).”
Although DNA damage due to ROS is not a rare event since it is estimated that human
cell sustains an average of 105 oxidative hits per day due to cellular oxidative metabolism
(Fraga et al., 1991), DNA is functionally very stable, so that the incidence of cancer is much
lower than one would expect, taking into account the high frequency of oxidative hits.
Nevertheless, avoidance of excessive cumulative and sporadic sun exposure is important in
reducing the risk of skin cancer and skin aging. Additionally, antioxidants might act by
enhancing the DNA enzyme repair systems through a post-transcriptional gene regulation of
transcription factors (Hirota et al., 1997). The repair capacity of human skin cells, therefore,
directly relates to the probability of initiation of the carcinogenesis process and eventually
tumor formation. Cellular antioxidant defense mechanisms are, therefore, crucial for the
prevention or removal of the damage caused by the oxidizing component of UV radiation.
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1230 Raja Dahmane, Ruza Pandel, Polonca Trebse et al.

The most important strategy to reduce the risk of sun UV radiation damage is to avoid the
sun exposure and to engage in the use of sunscreens. The next step is the use of exogenous
antioxidants orally or by topical application and interventions in preventing oxidative stress
and in enhanced DNA repair. Yarosh (2010) claims that properly conceived efforts to
alleviate skin aging have the benefit of reducing rates of skin cancer since people are more
motivated by improving their physical appearance than lowering their perceived risk of
disease. Thus, the most successful anticancer efforts will arrive as treatments for skin aging.

Sunburn

Sunburn, or erythema, is an acute injury following excessive exposure to solar UVR.

Sunburns are an acute inflammation reaction of the skin and tissue just beneath it that follows
excessive exposure of the skin to UVR. The affected area becomes red, hot, tender, and
swollen, and in severe cases, blisters may form. Low-dose or short exposure to UV irradiation
is tolerated by the skin without noticeable or clinically relevant changes. Only after a certain
threshold is reached does delayed and prolonged vasodilatation develop, allowing passage of
lymphocytes and macrophages into the tissue and induction of an inflammatory response that
is clinically visible as erythema. In epidemiology studies, sunburn is usually defined as burn
with pain and/or blistering that lasts for two or more days. Cumulative sun exposure is the
additive amount of sun exposure that one receives over a lifetime. A frequently used measure
of UV irradiation–induced erythema is determination of the minimal erythema dose (MED).
One MED is the minimal amount of energy required to induce a uniform, clearly demarcated
redness 16 to 24 h after exposure to UV irradiation. Even a single minimum erythema dose (1
MED) can damage the dermal matrix. Cumulative sun exposure may reflect the additive
effects of intermittent sun exposure, or chronic sun exposure, or both. The redness of the skin
that results is due to an increased blood content of the skin by dilatation of the superficial
blood vessels in the dermis, mainly the subpapillary venules (Diffey 1991).
Skin color is an important factor in determining the ease with which the skin will
sunburn. Whereas fair-skinned people require only about 15 to 30 min of midday summer
sunshine to induce an erythemal reaction, people with moderately pigmented skin may
require one to two hours exposure, and those with darkly pigmented skin will not normally
sunburn. Other phenotype characteristics that may influence the susceptibility to sunburn are
hair color, eye color and freckles (Azizi et al., 1988). Based on a personal history of response
to 45 to 60 min of exposure to midday summer sun in early June (Fitzpatrick, 1988),
individuals can be grouped into six sun-reactive skin types (Table 1). Types 1 and 2 are at
high risk of skin cancer, particularly when exposed to intense sunlight.
There are anatomical differences in erythemal sensitivity (Diffey, 1991). The face, neck
and trunk are two to four times more sensitive than the limbs. Vertical surfaces of an upright
person receive about one-half of the ambient UVR, whereas horizontal surfaces, such as the
epaulet region of the shoulder, receive up to 75%.
In addition to erythema and tanning, thickening (hyperplasia) of the epidermis is a
significant component of a mild sunburn reaction (Diffey 1991). A single moderate exposure
to UVB can result in up to a three-fold thickening of the stratum corneum within one to three
weeks, and multiple exposures every one to two days for up to seven weeks will thicken the

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 The Role of Sun Exposure in Skin Aging 1231

stratum corneum by about three- to five-fold (Miescher 1930). Skin thickness returns to
normal about one to two months after ceasing irradiation.
Thickening of the skin (especially of the stratum corneum) after sun exposure can lead to
a significant increase in protection against UVR by a factor of five or even higher. In
Caucasians, skin thickening is probably more important than tanning in providing
endogenous photoprotection, although in darkly pigmented races, skin pigmentation is the
most important means of protection against solar UVR.

Table 1. Fitzpatrick’s skin types

1. Type I: Extremely fair skin, always burns, never tans.

2. Type II: Fair skin, always burns, sometimes tans.
3. Type III: Medium skin, sometimes burns, always tans.
4. Type IV: Olive skin, rarely burns, always tans.
5. Type V: Moderately pigmented brown skin, never burns, always tans.
6. Type VI: Markedly pigmented black skin, never burns, always tans.
Fitzpatrick, 1988.

Tanning

Less intense or shorter duration exposure to UVR results in an increase in skin

pigmentation that provides some protection against further UVR-induced damage. The
increased skin pigmentation occurs in two phases, immediate pigment darkening and delayed
tanning. Intermediate pigment darkening occurs during exposure to UVR and results from
oxidation and redistribution of existing melanin. This reaction may fade rapidly or persist for
several days. Delayed tanning results from increased synthesis of epidermal melanin and
requires 24 to 72 hours to become visible.
Melanocytes are specialized dendritic cells interspersed among basal keratinocytes and
serve the primary function of producing melanin in intracellular organelles melanosomes that
are then distributed to surrounding keratinocytes (Hakozaki et al., 2010).
Following solar UVR exposure, there is an increase in the number of functioning
melanocytes, and activity of the enzyme tyrosinase is enhanced (Fitzpatrick et al., 1983). This
leads to the formation of new melanin and hence an increase in the number of melanin
granules throughout the epidermis. Melanins are the major UV-absorbing chromophores in
skin, exhibiting an extremely broad spectrum of absorption over the UVB, UVA and visible
ranges. Melanins are complex polymeric proteins that are produced by melanocytes and
transferred to keratinocytes. It should be stressed that melanin cannot offer 100% protection
to our skin against harmful effects of solar radiation.
Gilchrest et al. (1999) explained how UVR stimulates melanogenesis in the skin. A direct
effect of UV photons on DNA results in upregulation of the gene for tyrosinase, the rate-
limiting enzyme in melanin synthesis, as well as an increase in cell-surface expression of
receptors for at least one of the several known keratinocyte-derived melanogenic factors,
MSH. Direct effects of UV on melanocyte membranes, releasing arachidonic acid, may also
play a role in the tanning response. The tanning response also relies heavily on UV-stimulated
increased production and release of numerous keratinocyte-derived factors including bFGF,
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NGF, endothelin-1 and the POMC-derived peptides MSH, ACTH, beta-LPH and beta-
endorphin (Gilchrest et al., 1999). Ultraviolet-induced melanogenesis may be one part of a
eukaryotic SOS response to damaging UVR that has evolved over time to provide a protective
tan in skin at risk of further injury from sun exposure (Gilchrest et al., 2006).
The distribution and size of melanin particles also plays an important role in protecting
epidermal cells.
Melanin particles have a distribution within the stratum corneum and epidermal cells
depending upon skin type. In dark skin types (5 and 6), these particles are positioned within
cells to provide optimum optical protection for the cell nuclei and in adequate size in the
stratum corneum (Kollias et al., 1991).
According to Euro melanoma (http://www.euromelanoma.org/uk/home) “fake tan (auto
bronzing) is by far safer than a suntan. A fake tan produces a natural looking tan through a
chemical reaction in the skin. It, therefore, gives the desired cosmetic result in someone who
is very keen to have a tan, without him or her having to sunbathe. Application of fake tan
needs to be carried out every one to two weeks in order to maintain the tan since the top
layers of the skin are constantly being renewed. However, the actual tanning produces by
‘fake tan’ is not at all protective against UV-radiation and consumers should be aware of this
important factor.”

BENEFICIAL EFFECTS OF UV
Until now, just harmful effects of UVR on different cellular molecules of the skin were
discussed. However, small amounts of UV are beneficial for people and essential in the
production of vitamin D. UVR is also used to treat several diseases, including rickets,
psoriasis, atopic dermatitis and jaundice. This takes place under medical supervision, and the
benefits of treatment versus the risks of UVR exposure are a matter of clinical judgment.
According to Diffey (1991), the only thoroughly established beneficial effect of solar
UVR on the skin is the synthesis of vitamin D3. Solar radiation in the UVB waveband
photochemically converts 7-dehydrocholesterol in the epidermis to previtamin D3. This
previtamin immediately isomerizes to vitamin D3 in a reaction controlled by skin temperature
and which takes two to three days to reach completion. Previtamin D3 is photolabile, and
excessive exposure to sunlight causes its photolysis to biologically inert photoproducts,
lumisterol and tachysterol. In fact, production of previtamin D3 is limited to no more than 5%
to 15% of the total 7-dehydrocholesterol content in the skin, no matter how long a person is
exposed to sunlight. Once vitamin D3 is made in the skin, it enters the blood for transport to
the liver to be metabolized to 25-hydroxyvitamin D (Webb and Holick, 1988). If vitamin D3
does not enter the circulation before sun exposure the following day, it can be rapidly
degraded in the skin by sunlight to suprasterol 1, suprasterol 2 and 5,6-transvitamin D3—
products which are believed to be biologically inert (Webb et al., 1989). Thus sunlight,
through its photochemical activity, is able to regulate the production of both previtamin D3
and vitamin D3 in the skin.
Only short exposures to sunlight are required to synthesize vitamin D3 in the skin; from
spring until autumn, 15 min exposure to the hands, arms and face between 9 am and 4 pm is
adequate to provide our vitamin D3 requirement (Diffey 1991). There is still the ongoing

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debate between the adequate sun exposure to create sufficient vitamin D and the risk arising
from skin cancer from moderate increased sun exposure (Grant and Holick, 2005). It has been
roughly estimated that a sufficiency of vitamin D synthesis in skin can be provided by
exposure of 40% of the body to 25% of the UVB minimal erythemal dose (Grant and Holick,
2005). An untanned person with fair skin may receive mild sunburn in as little as 25 minutes
at noon (depending on the time of year and the latitude) but would have to lie in the sun for at
least two hours to receive the same dose after 3 pm (International program on chemical
safety, Environmental Health Criteria 160). Alternative to sun exposure vitamin D production
is the intake of synthetic vitamin D with supplements. But on the other hand, supplements of
vitamin D can cause side effects from excess vitamin D, while sun-exposure cannot lead to
excessive vitamin D amounts. However, it can lead to excessive skin damage formation.

HARMFUL EFFECTS OF SUNSCREENS

Sunscreens contain chemical organic filters, which mainly absorb UVB or UVA
radiation, and physical filters (TiO2 and ZnO), which block the UVB/ UVA part of the solar
radiation by reflection and scattering (Diaz-Cruz et al., 2008, Food and Drug Association
(FDA) documents http://www.cfsan.fda. gov/~lrd/fr990521.html; Salinaro et al., 1999;
Serpone et al. 1999). Such products must be photostable in order to spend energy effectively
mainly through photophysical processes and minimize photochemical ones which involve
singlet oxygen or other ROS or intermediates. They should not penetrate deeper into the skin,
and also may not be transported into the cells. Alternatively, they should prevent UVB and
UVA rays from penetrating into the cell nucleus and damage DNA. Products for skin
protection containing UV filters are in the most countries declared as “cosmetic products”
(Klein, 1992; Lowe, 1990). Sunscreens should always contain these compounds in
combination, because there is no single UV filter, which at present would provide a
sufficiently high SPF according to the legislation (Serpone, 2007). In the formulation of these
compounds the FDA recommends a minimum of 2 mg lotion/cm2 skin present at the ratio of
mg/g to be applied to the skin or hair in large quantities.
Organic UV filters, also regarded as chemical UV filters which are responsible for
absorption of the solar UV radiation, comprise different classes of compounds such as UVA
(benzophenones, anthranilates and dibenzoylmethane) and UVB filters (PABA derivatives,
salicylates, cinnamates and camphor derivatives) that block radiation in UVA or UVB (Díaz-
Cruz et al., 2008). Dibenzoylmethane derivatives are widely used as organic UVA filters,
among them the most common is butyl methoxydibenzoylmethane (avobenzone). With the
increase of industrial production of nano and microparticles, these products have found
applications in sunscreens as well. But still, questions arise about its effects on humans and
animals (Yeats and Mauderly, 2001). The European Commission as early as 2006 estimated
that at the market level there was about 5% of cosmetic products with nanoparticles.
Previous research has shown that some organic UV filters decompose when exposed to
light. In the presence of chlorine and chlorinated medium, like water pools or seawater, direct
photolytic reactions or chlorination of the aromatic ring or the side chain, may also occur.
Chlorination is the most commonly used chemical process for disinfecting swimming pools
and drinking water. The formation of halogenated by-products in chlorinated waters is
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1234 Raja Dahmane, Ruza Pandel, Polonca Trebse et al.

inevitable, especially when they are substituted with phenolic compounds and/or amino
groups (Bedner and MacCrehan, 2006; Lebedev, 2007; Duirk et al., 2013; Zhuang et al.,
2013; Grbović et al., 2013). Direct release of applied sunscreens, as consequence of
recreational activities in swimming pools and sunbathing areas, and indirect discharge
through domestic wastewater (e.g., during showering, clothes washing) represent the main
pathways for organic UV filters to enter the aquatic environment (Díaz-Cruz et al., 2008).
Information on detected environmental concentration of organic UV filters in freshwater (i.e.,
rivers, lakes) and marine ecosystems are summarized in numerous recent publications and are
mainly in ng L-1 range, reaching often levels up to 4-10 and even 24 μg L-1 for specific UV
filtering compound (Fent et al., 2010; Kaiser et al., 2012; Santos et al., 2012; Duirk et al.,
2013; Xiao et al., 2013). On contrary, data regarding the presence of organic UV filters in
swimming pool water are rather limited. Vidal et al. (2010), Cuderman and Heath (2007)
reported values for isoamyl p-methoxycinnamate, benzophenone 3 and 4-methylbenzylidene
camphor of 700 ng L-1, 400 ng L-1and 330 ng L-1, respectively. Higher content of
benzophenone 3, Eusolex 6300 (4-methylbenzylidene camphor) and Eusolex 2292 (octyl-
methoxycinnamate) in swimming pool waters was determined by Lambropoulou et al. (2002)
and Giokas et al. (2004) with concentrations from 2 to 10 μg L-1. Zhuang et al. (2013)
reported about presence of benzophenone 3 in swimming pool waters in concentration of 0.3
and 1.7 μg L-1 and about the presence of 3,5-dichloro benzophenone 3 in the concentration of
6.6 μg/L.
In the last decade there has been extensive discussion regarding the quality of swimming
pool waters. Opinions from different experts representing different sectors are quite opposite.
On one side some of them claim, that the quality of swimming waters is constantly
improving, but on the other side some others warn about the presence of various compounds
pool waters contain, from cosmetics to pharmaceuticals (especially residues and metabolites)
and other contaminants, and about the possible effects of water consumption (also as
inhalation of aerosols), which means increased health risk including an increased incidence of
cancer. A special problem represents chlorinated products formed under conditions of
disinfectant (e.g., swimming pool water) since these compounds are new ones and no
literature on effects of chlorinated compounds are available.
Endocrine disrupting activity of small sized organic and nano sized inorganic UV filters
have been reported. In the literature we found very little data pertaining to estrogen, anti-
estrogen, androgen and antiandrogen effects of parent organic UV filters (Fent et al., 2010;
Zucchi et al., 2011). Due to the high lipophilicity these compounds tend to accumulate or
bioaccumulate in the sediments as well as in the food chain. Some UV filters have been
detected in human breast milk (Schlumpf et al., 2010) and urine (Leon et al. 2010). Just a few
research studies are available on the transformation characteristics and potential health risks
of benzophenone-type UV filters during chlorination disinfection process (Liu et al., 2014).
Data on the effects of these compounds on the skin structure in the scientific literature is not
available. Certain organic UV filters (PABA derivatives, cinnamates, benzophenones, and
octocrylene) have been described to cause photoallergy.
Recent studies have shown on allergic and photoallergic reactions of skin exposed to UV
filters. One of epidemiological studies has shown that avobenzone and some other UV filters
cause photoallergic dermatitis (Gaspar et al., 2013). Many sunscreens also contain
antioxidants (vitamin A, C), which act as photoprotection and maintain or restore a healthy
skin barrier, but at the same time can also form toxic photoproducts and allergens.

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The cosmetic industry is increasingly using new organic compounds and nanoparticles in
products for protection against UV irradiation. For the safety of cosmetic products the
responsibility is on the companies themselves.
The testings’ are carried out by them or by authorized laboratories. Currently, existing
legislation does not provide clear guidance particularly with regard to the use of nanoparticles
in cosmetics. Cosmetic companies, therefore, use tests that apply to other substances, or avoid
the use of nanoparticles or do not advertise their addition.
A potential risk of vitamin D deficiency by sunscreen use has become a major subject of
public health debate (Bens 2013) since 1,25 (OH) 2D stimulates the differentiation and
inhibits the proliferation of cells, although conclusive evidence of vitamin D in cancer
prevention it is not yet definite (Shui and Giovannucci, 2013). Already SPF of 8 inhibits or
even significantly prevents vitamin D production in the skin (Holick, 2004; Sayre and
Dowdy, 2007). Additionally, reducing the exposure of the skin UVB radiation could suppress
the skin’s production of melanin (Autier et al., 1995; Meredith and Riesz, 2004).

TREATMENT OF PHOTOAGING
The primary treatment of photoaging is photoprotection, however the additional
treatment could be achieved with the use of antioxidants as well with some novel compounds
such as polyphenols. Exogenous antioxidants like vitamin C, E and many others cannot be
synthesized by the human body and must be taken up by the diet. Natural antioxidants are
generally considered to be beneficial fruit and vegetable components. It seems that skin’s
antioxidative defense is also influenced by nutritive factors.
Besides vitamins A, C, and E, 𝜂-3 fatty acids certain non-vitamin plant derived
ingredients might have beneficial effect on skin aging, skin sun protection, or skin cancer.
The laboratory studies conducted in animal models suggest that many plant compounds have
the ability to protect the skin from the adverse effects of UVR (Pandel et al., 2013; Bruce,
2008).
Many studies have found that vitamin C can increase collagen production, protect against
damage from UVA and UVB rays, correct pigmentation problems, and improve inflammatory
skin conditions (Poljšak, 2011). Ascorbic acid was a photoprotectant when applied to mice
and pig skin before exposure to ultraviolet (UV) radiation (Elmor 2005).
The application of retinoids might not only clinically and biochemically repair photoaged
skin, but their use might also prevent photoaging (Serri and Iorizzo, 2008). Supplements or a
carotenoid-rich diet decreased sensitivity against UV-induced erythema.
Supplementation with carotenoids contributes to basal protection of the skin but is not
sufficient to obtain complete protection against severe UV irradiation. Beta-carotene acts not
only as an antioxidant but also has unexpected prooxidant properties (Biesalski and
Obermueller-Jevic, 2001). A number of experimental studies indicate protective effects of
beta-carotene against acute and chronic manifestations of skin photodamage. For this reason,
further studies with focus on in vivo 𝛽-carotene-induced prooxidative properties and its
relevance on human health are needed (Pandel et al., 2013).
It was reported that CoQ10 strongly inhibits oxidative stress in the skin induced by UVB
via increasing SOD2 and GPx (Kim et al., 2007). A topical application of CoQ10 has the
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1236 Raja Dahmane, Ruza Pandel, Polonca Trebse et al.

beneficial effect of preventing photoaging (Hoppe et al., 1999) as well as protecting skin
against oxidative stress-induced cell death and enhances the synthesis of basement membrane
components in dermal and epidermal cells (Muta-Takada et al., 2009). Furthermore, CoQ10
appears to have also a cutaneous healing effect in vivo (Choi et al., 2009).
Green tea polyphenols have received attention as protective agents against UV-induced
skin damage. Analysis of published studies demonstrates that green tea polyphenols have
anti-inflammatory and anticarcinogenic as well as antiaging properties (Katiyar et al., 2000).
Topical treatment or oral consumption of green tea polyphenols (GTP) inhibits chemical
carcinogen or UV radiation-induced skin carcinogenesis in different laboratory animal models
(Katiyar, 2003). Green tea itself or caffeine in amounts equivalent to three of five cups of
coffee per day to UVB-exposed mice increased levels of p53, slowed cell cycling, and
increased apoptotic sun burn cells in the epidermis (Lu et al., 2008).
Dietary flavanols from cocoa contribute to endogenous photoprotection, improve dermal
blood circulation, and affect cosmetically relevant skin surface and hydration variables
(Heinrich et al., 2006), but it was reported too that the Cocoa photoprotection against UV-
induced erythema (Heinrich et al., 2006).

CONCLUSION
After skin damage due to sun exposure, repair mechanisms are not 100% effective. The
damaged components are not always completely repaired.
The problem arises in the cases of intensive acute sun exposure or in the cases of chronic
sun exposure over longer decades, which manifests as skin photoaging.
To what extent each mechanism—mtDNA mutagenesis, protein oxidation, down
regulation of collagen synthesis and increased expression of matrix metalloproteinases—
contributes to premature skin aging is still not answered.
As photoaging via sunlight or artificial UV-exposure is the major impacting factor for
skin appearance, new defense strategies have been suggested, including the appropriate
UVA+UVB sunscreen choice in addition to an antioxidant-rich diet, the induction of
photoprotective melanogenesis by means of thymidine-dinucleotide formulations, the use of
phytoestrogens and metal chelating agents to inhibit the collagenase activation, the avoidance
of refined hyperglycemic carbohydrates so as to slow down the glycation/ oxidation of
proteins, the use of aminoguanidine and carnosine formulations to inhibit the collagen cross-
linking as well as retinoic acid to stimulate the DNA repair mechanisms and collagen
synthesis (Ionescu 2005).
UV protection includes not only reduction of sun exposure but also use of sun protective
filters, UV protective clothes, DNA repair enzymes, and antioxidant supplementation.

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Varani, J., Warner, R. L., Gharaee-Kermani, M., Phan, S. H., Kang, S., Chung, J. H., Wang,
Z. Q., Datta, S. C., Fisher, G. J., Voorhees, J. J. (2000). Vitamin A antagonizes decreased
cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates
collagen accumulation in naturally aged human skin. J. Invest. Dermatol., 114(3), 480-6.
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Watson, R., Griffiths, E. M., Craven, N. M., Shuttleworth, A., Kielty, C. M. (1999). Fibrillin-
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 58

PHOTOPROTECTION PRACTICES

Jacqueline Selph, MD,

Ritva Vyas, MBChB and Meg Gerstenblith, MD
University Hospitals/Case Medical Center, Cleveland, OH, US

ABSTRACT
Sun protection practices play a prominent role in dermatology in terms of
photoaging, photocarcinogensis, and photodermatoses. In this chapter, we outline the
various methods of sun protection. Naturally occurring protective agents, such as
geographic and environmental variations create a substantial difference in exposure to
ultraviolet (UV) radiation. Physical protective barriers including glass, clothing, and
sunglasses can provide UV protection, but there is great variability in their defensive
properties. Sunscreens play a major role in sun protection practices and vary substantially
in chemical composition and effectiveness regarding protection against UVA versus
UVB radiation. We will discuss inorganic products versus organic products in sunscreens
as well as their associated advantages and disadvantages. Finally, systemic agents, such
as plant extracts, carotenoids, polyphenols, and other antioxidants may prove to be
promising adjuncts for sun protection in the future.

INTRODUCTION
The electromagnetic spectrum emitted from the sun includes x-rays, ultraviolet radiation
(UVR), visible light, infrared, and microwaves/ radiowaves. UVR, which includes ultraviolet
A (UVA 320-400nm), ultraviolet B (UVB 290nm-320nm), and ultraviolet C (UVC 200-
290nm) has a variety of negative effects on the skin. In addition to being a major
environmental risk factor for melanoma and non-melanoma skin cancers, it also contributes to
skin aging, photosensitivity in certain dermatologic conditions, and immunosuppression.
UVB is the major type of UVR known to cause direct photochemical damage through the
formation of pyrimidine dimers and subsequent gene mutations. UVA can be further
subdivided into UVA2 (320-340 nm) and UVA1 (340-400nm). UVA has indirect effects on
photocarcinogenesis via the formation of reactive oxygen species (ROS).
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1246 Jacqueline Selph, Ritva Vyas and Meg Gerstenblith

Furthermore, evidence suggests that UVA may have a more damaging role compared to
UVB in long-term sun damage. In this chapter, we will outline the various forms of
photoprotection including environmental protections, intrinsic properties of the skin, and
physical, topical and systemic agents.

ENVIRONMENTAL PROTECTIONS
Atmosphere and Geography

The ozone (03) layer, present in the stratosphere at 10km to 50km above the surface of
the earth, absorbs high quantities of short UVB and UVC radiation but very little UVA.
However, the ozone layer is not uniform in thickness, with the concentration increasing
toward polar regions. Chlorofluorocarbons used as aerosol propellants have led to ozone
depletion, especially towards the South Pole. Sunburns and photosensitivity disorders have
been shown to increase after acute, sudden episodes of UVB radiation second to ozone
depletion (Norval et al., 2011). Ozone depleting substances have been phased out, and
therefore a slow recovery of ozone is anticipated over the coming decades (Bais et al., 2014).
Scattering by nitrogen and oxygen molecules also helps to deflect UVC resulting in near
complete attenuation of the UVC reaching the earth’s surface. The atmosphere allows about
10% of UVB light and almost all UVA and visible light to reach the earth’s surface;
therefore, on a summer day >95% of UVR reaching the earth’s surface is UVA, while only
4% is UVB. UVR exposure is greatest at the equator and high altitudes. For every degree
increase in latitude away from the equator, there is a 3% decrease in the transmission of UVB,
and for every 300 meter increase in elevation, there is an approximately 4% increase in the
intensity of UVR, the majority of which is UVB (Rigel, Rigel, and Rigel, 1999).

Time of Day and Season

At the solar zenith, the path of UVR through the ozone is the shortest; thus, substantially
less UVR is absorbed. UVA light penetrates without absorption through the atmosphere;
therefore, its level is constant through daylight hours. However, UVB light varies with
atmospheric absorption; it is strongest from 10 AM to 2 PM, when the path of transmission is
shortest. An observational study in Denmark showed that 50% of the total daily UVR dose
reaches the earth’s surface between 12 PM and 3 PM (Thieden, Philipsen, Heydenreich, and
Wulf, 2004). Likewise, UVR is strongest in the summer because of the elliptical orbit of the
sun (Diffey, 2002).

Clouds, Particulates, and Reflections

Clouds, fog, and haze are estimated to reduce ultraviolet levels between 10%-90%. The
US national weather service calculates that overcast skies allow only 31% of UV
transmission, broken clouds 73%, and scattered clouds 89%; however, very heavy cloud

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cover (i.e., storm clouds), can virtually eliminate ultraviolet exposure (US Environmental
Protection Agency, 2012). While clouds reduce the UVR intensity, the infrared (heat)
intensity is reduced to a much greater extent. Without the warning sensation of warmth from
infrared light, the risk of overexposure to UVR greatly increases on cloudy days.
Pollutants such as soot, nitrogen dioxide, and sulfur dioxide act similarly to clouds,
reducing UVR through scattering. Shorter wavelengths are scattered to a greater extent than
longer wavelengths. Therefore, in large urban areas, UV irradiance is reduced compared to
more underdeveloped areas (Mckenzie et al., 2008).
The reflection of UVR off various surfaces also greatly contributes to overall UVR
exposure. Snow, sand, and metal reflect up to 90% of UVR; almost doubling the UVR
exposure of skiers and beachgoers. However, most surfaces reflect less than 10% of
ultraviolet light.
While little reflection occurs on still water, UVR can penetrate to a depth of one meter,
exposing swimmers to substantial radiation Seawater, due to its motion and relatively high
particulate count, can reflect up to 15% of UVR (Kromann, Wulf, Eriksen, and Brodthagen,
1986; Lautenschlager, Wulf, and Pittelkow, 2007).

Shade

Although data have shown “shade seekers” to be relatively protected from the harms of
UVR, there is still substantial exposure to UVA light in the shade (Turnbull and Parisi, 2003).
Approximately 50% of all exposure to UVA light occurs in the shade. While the sun
protection factor (SPF) of single trees can be as low as 4, dense foliage can increase this
beyond SPF 50, reducing UVR by as much as 95% (Moise and Aynsley, 1999).
An average beach umbrella (not made specifically for UV protection) offers almost no
UVR protection. Newer sun protection umbrellas can provide increased SPF from overhead;
however, with typical beach use, much UVR is reflected from sand and water (Thieden et al.,
2004).

Solar Ultraviolet Index

The global solar ultraviolet index (UVI), developed by the World Health Organization
(WHO) in consortium with various international agencies, takes into account the above
mentioned environmental elements and provides a numerical guide for the level of solar UVR
at any given location and date as well as how much protection is recommended.
The UVI ranges from 1 to 11+ with levels 1-2 requiring no additional protection, levels
3-6 requiring protection, and levels 8 and above requiring extra protection (World Health
Organization, 2014).
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1248 Jacqueline Selph, Ritva Vyas and Meg Gerstenblith

INTRINSIC PHOTOPROTECTIVE PROPERTIES OF THE SKIN

The main human protective barrier to UVR is the skin. However, while the skin serves as
a protective barrier for the internal organs, it absorbs most UVR, sometimes leading to its
own damage. UVB light is primarily absorbed in the epidermis, but UVA light can penetrate
deep into the dermis. As early as half an hour following UVR exposure, apoptotic
keratinocytes, known as sunburn cells, can be observed histologically. This apoptotic process
is a protective mechanism to rid the body of cells with the potential for malignancy second to
DNA damage (Baron and Suggs, 2014).
Chromophores are defined as molecules that absorb light-energy and in turn give off a
color of their own. In the body, the DNA bases purine and pyrimidine act as chromophores
and absorb most of the effects of UVB, which can lead to the development of cyclobutane
and pyrimidine dimers. Such damage, if missed by repair mechanisms, can lead to mutations
or cytotoxicity.
The aromatic amino acids, tryptophan and tyrosine, have significant absorption in the
UVB range, but little UVA absorption. Proteins can photobind to DNA, and there is some
evidence that photoactivation of a protein may be an important step in transcription factor up
regulation (Young 1997).
Melanins are another important chromophore in the skin. Melanin accumulates within
keratinocytes and melanocytes in the perinuclear area and functions as a “cap” to shield DNA
from UVR, absorbing 50%-75% of UV rays; additionally, melanin acts as a free radical
scavenger, antioxidant, and superoxide dismutase that reduces ROS (Brenner and Hearing,
2008).
Eumelanins are brown to black nitrogenous pigments formed from the oxidative
polymerization of 5,6 dihydroxyindoles (DHIs), whereas pheomelanins are alkali-soluble
yellow to reddish-brown pigments formed from the oxidative polymerization of
cysteinyldopas. Pheomelanins, seen in red haired/fair skinned individuals, are formed from
loss of function polymorphisms in the melanocortin 1 receptor gene (MC1R), of which greater
than 100 have been identified (Dessinioti, Antoniou, Katsambas, and Stratigos, 2011;
Gerstenblith, Goldstein, Fargnoli, Peris, and Landi, 2007). Pheomelanin has weak UV
shielding ability compared to eumelanin and also perpetuates damaging ROS.
There is evidence that loss of function polymorphisms in MC1R are associated with an
increased risk of melanoma, indicating a superior protective role of eumelanin over
pheomelanin (Pasquali et al., 2015; Rees, 2000).
Recently, data in mice have suggested that even in the absence of UVR, pheomelanin
contributes to melanoma carcinogenesis through reactive oxidative damage (Mitra et al.,
2012). More darkly pigmented skin, containing mostly eumelanin, is less susceptible to the
damaging effects of UVR than lightly pigmented skin, which in large part explains the
increase in melanoma, squamous cell carcinoma, and basal cell carcinoma observed in fair
skinned individuals.
Other photoprotective agents in the skin include heme and porphyrin. Oxyhemoglobin
and reduced hemoglobin can absorb bands in the UVA/UVB range, as well as blue, green,
and yellow visible light. Porphyrins absorb light generally between 400nm-410 nm, and
generate ROS upon exposure. Unfortunately, these molecules accumulate to high levels in
cutaneous porphyrias causing photosensitivity (Young 1997).

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PHYSICAL PHOTOPROTECTIVE AGENTS

Glass

Although eighty percent of the average day of Americans is spent indoors, contemporary
design increasingly incorporates many large window areas. It has been estimated that
individuals who work indoors receive on average 8.5 standard erythema doses (SEDs) per day
of UVR in spring months and at least 2 per day in winter months (Parisi et al., 2000).
Additionally, a large number of Americans commute, with the average American spending
between 80-90 minutes per day in automobiles. In studies of UVR exposure in cars, an
increased prevalence of facial photodamage, actinic keratosis, melanoma, and non-melanoma
skin cancers were seen on the driver’s exposed side (Butlers ST, 2010; Singer, Hamilton,
Voorhees, and Griffiths, 1994).
The majority of commercially used glass is soda lime glass, made up of a mixture of
silica, salt cake, limestone, dolomite, feld-spar, soda ash, and typically recycled broken glass.
Through melting and slow cooling, the glass develops a random, disorganized non-crystalline
structure, which on its own provides little UVR protection (Tuchinda, Srivannaboon, and
Lim, 2006).
The main types of glass are clear glass, tinted (heat-absorbing) glass, reflective glass,
low-emissivity glass, laminated glass, UVR-blocking coated glass, and insulating glass.
Tinted glass may absorb 40%-50% of incoming solar energy and has less UVR and visible
light transmission compared to clear glass.
Reflective glass uses a metal oxide coating to give glass a mirror-like appearance, which
helps to minimize unwanted solar heat gain and reduces UVR and visible light transmission
(Almutawa, Vandal, Wang, and Lim, 2013). Low Emissivity (Low-E) glass has a surface
coating of microscopically thin transparent layers of silver sandwiched between antireflective
metal oxide coatings. It significantly reduces the loss of generated heat, and may decrease
UVR transmission from 60% down to 20%; however, UVA is largely unimpeded by this
coating (National Glass Association, 2014). Laminated glass is a combination of two pieces
of glass bonded with a tough plastic interlayer. The main benefit is to prevent injury because
if broken, large fragments do not fall free; however, it also filters more than 99% of UVR
without sacrificing visible light transmission. UVR blocking coated glass blocks more than
98% of UVR. Finally, insulated glass combines Low-E glass with UVR-blocking coated glass
to block more than 99% of UVR transmission while eliminating up to 70% of unwanted solar
heat gain (Almutawa et al., 2013; Tuchinda et al., 2006).

Automobile and Airplane Glass

Because of the hazards of broken glass, all automobile windshields are made of laminated
glass, which filters UVB and most UVA radiation below 380 nm. However, side, rear, and
overhead windows are usually made from tempered glass, which block all UVB radiation but
only 21% of UVA radiation. Window tinting can help to further decrease this exposure;
however, it is generally only permitted on rear windows. A subject sitting near the driver’s
side window of non-laminated glass could be exposed to a 5-J/cm2 dose of UVA in 30
minutes; enough to produce an eruption in patients with polymorphous light eruption
(Hampton, Farr, Diffey, and Lloyd, 2004). In 2011, the National Highway Traffic Safety
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1250 Jacqueline Selph, Ritva Vyas and Meg Gerstenblith

Administration mandated stricter requirements to mitigate side and rear window passenger
ejections. As a result, more side and rear windows are being made with laminated glass.
Window films can be applied to side and rear windows, reducing transmission of visible
light and infrared radiation while decreasing interior heat gain and minimize fading. Most
films are made of multiple layers of polyethylene terepththalate (PET), a polyester resin,
which gives only a small reduction in visible light (Almutawa et al., 2013).
However, infrared radiation exposure can contribute to skin aging by decreasing pro-
collagen and increasing cytokines. The primary objective of UVR blocking is accomplished
by adding UVR absorbers or adhesives to the films.
These films must comply with federal and state standards, which mandate no higher than
a 30% blockade of visible light. However, as more automobiles manufacturers use laminated
glass for side and rear windows, window films may become unnecessary for UVR protection
purposes.
Airplane windshields are commonly made of polycarbonate plastic or laminated glass,
which block >99% of UVB. Plastic offers better UVA protection compared to glass; however
UVA transmission as high as 53% has been reported (Nakagawara, Montgomery, and
Marshall, 2007). A recent study demonstrated that pilots flying at 30,000 feet for 56 minutes
were exposed to the same UVA radiation dose as a 20 minute tanning bed session, which may
contribute to carcinogenesis and the increase in melanoma observed in pilots and cabin crew
(Sanlorenzo et al., 2014). Because of the increase in UVR at high altitudes, future
recommendations may mandate UVR absorbing films on airplane windshields.

Architectural Glass for Buildings

All types of glass, as previously mentioned, can be used as architectural glass. The
highest transmission of UVA is through smooth annealed glass (74.3% of UVA) followed by
tempered glass (71.6%), textured annealed glass (44.6%), and finally laminated glass, which
allows <1% UVR through (Tuchinda et al., 2006). These values can vary based on the
thickness and color of the glass. Just as with the tinting of automobile glass, many
architectural buildings apply energy efficient glaze, which reduces heat gain and loss as well
as some UVR protection (Almutawa et al., 2013).

Sunglasses

Several eye disorders are related to sun exposure, such as cataracts, macular
degeneration, pterygium, and keratitis. UVR protection through sunglasses may help to
reduce the incidence of these conditions. Australia leads the way in sunglass guidelines,
establishing the first national standard in 1971. Currently, there are three available national
standards for sunglasses, the American Standard (ANSI Z80.3), the European standard (EN
1836:2005), and the Australian/New Zealand standard (AS/NZS 1067:2003).
The European and Australia/New Zealand standard are very similar, utilizing five lens
categories (from very light tint to very dark tint), with the main differences being the
definition of UVA (315-380 nm per EN and 315-400 nm per AS) and that European standards
allow up to 10% of UVB (defined as 280-315 nm), while Australian standards allow a
maximum of 5% UVB (Dain et al., 2010). The American standards have only four categories
of lens tint but are further differentiated based on use for cosmetic or special purposes.

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Table 1. Common types of glass used in residential and commercial buildings

Types of Glass Comment

Protection from outside elements while allowing visible light
Clear glass
transmission
Absorbs solar energy to  heat gain
Tinted (heat-absorbing) glass
Some  in visible light and UVR
Reflects heat and visible light
Reflective glass
Moderate  in visible light and UVR
Significant in heat loss and  heat gain
Low-emissivity glass
Allows full visible light,  UVB
>99% UVB blocked
Laminated glass no  in visible light
Does not fragment when broken
Allows all visible light
UV blocking coated glass
blocks >98% UVR
UV blocking coated insulated Combines Low-E glass with UV coated glass to both UVR
glass and solar heat gain

Table 2. Summary of Australian, European and United States Sunglass Standards

0 1 2 3 4
(very light tint) (light tint) (medium tint) (dark tint) (very dark tint)
EN
10% LT 10% LT 10% LT 10% LT 10% LT
(280-315 nm)
AS
5% LT 5% LT 5% LT 5% LT 5% LT
(280-315 nm)
US
UVB
(280-315 nm) n/a 12.5% LT 12.5% LT 1% LT 1% LT
Normal Use
US
(280-315 nm) n/a 1% LT 1% LT 1% LT 1% LT
Prolonged Use
EN
LT LT LT 50% LT 50% LT
(315-380 nm)
AS
LT LT LT 50% LT 50% LT
(315-400 nm)
US
UVA
(215-380 nm) n/a LT LT 50% LT 50% LT
Normal Use
US
(215-380 nm) n/a 50% LT 50% LT 50% LT 50% LT
Prolonged Use
*
LT = Luminous Transmittance (the fraction of incident light that passes through the sample).
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1252 Jacqueline Selph, Ritva Vyas and Meg Gerstenblith

Furthermore, the American standards differ depending on whether the lens is to be used
for normal (i.e., commuting from home to work) or prolonged use. A darkly colored special
purpose lens should transmit 1% or less of UVB and 50% or less of UVA; however, a
commonly sold cosmetic lens for normal use can allow up to 12.5% UVB and all UVA light
through (Almutawa and Buabbas, 2014).
Compliance with these standards in the United States is voluntary, whereas compliance
with the European and Australian/New Zealand standards is mandatory. In Europe, quality
assurance to standards can be done by the manufacturer themselves, but AS/NZ mandate
inspection by an independent party (Almutawa et al., 2013). In a recent study testing
European sunglasses that comply with standards, 17% failed to meet these standards when
assessed by an independent party (Dain et al., 2010).
In addition to lens standards, the size and geometry of sunglasses make a significant
difference in UVR exposure.
Maximum UVR to the eye occurs when solar radiation is parallel to the eye; however,
radiation from above or below the eye also makes a sizeable contribution. The best protection
is achieved with wraparound sunglasses or side shields (Almutawa and Buabbas, 2014).
Unfortunately, UVR from other angles can reflect off the inner surface of the lens increasing
exposure to the eye. Australia is the only country with a standard for lens size, which
mandates 28 mm for adults and 24 mm for children (Almutawa et al., 2013).
UVR eye protection is extremely important in young children because their ocular lenses
do not filter UVR as well as developed adult lenses. Visible light and UVR are able to reach
the retina in young children because of their large pupillary size, increasing the risk for
macular degeneration (Rosenthal, Bakalian, Lou, and Taylor, 1988).
While dark lenses often provide more UVR protection, they also result in pupil dilation,
which could increase retinal UVR exposure.

Other Eye Protection

While sunglasses can be an effective method of UVR eye protection, they also reduce the
squint mechanism, which naturally allows less radiation to reach the eye. Newer contact
lenses incorporate some UVR-blocking properties, which helps to mitigate this effect. The
Federal Drug Administration (FDA) mandates that Class I contact lenses must block more
than 90% of UVA (316-380 nm wavelength) and 99% of UVB (280-315 nm), whereas Class
II contact lenses, which are intended for general purposes, must block more than 70% of
UVA and 95% of UVB (Walsh and Bergmanson, 2011).
Polycarbonate is a new thermoplastic material first used in the aerospace industry that is
now being transitioned to use in glasses. Studies of airplane windshields found that
polycarbonate transmitted almost no UVR below 380 nm, therefore blocking all UVB and
almost all UVA. This material is lightweight, strong, and resistant to breakage. Newer
variations of traditional polycarbonate claim to block almost 100% of UVR, and offer an
impact resistant, UVR protective alternative to traditional lenses (Dain 2012).

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Photoprotective Clothing

Over the past twenty years, photoprotective fabric has been developed as a reliable means
of decreasing exposure to UVR. First coined in Australia in 1996, the term “UV protection
factor” (UPF) is used to describe the protection afforded by textiles, which utilizes
spectrophotometric methods to measure transmission of both UVA and UVB in vitro. The
UPF is calculated by combining these transmission data with erythema effectiveness.
Measuring protection of fabrics in vivo is done through exposure to incremental UVB doses
on protected and unprotected skin. The minimal erythema dose (MED) for protected vs.
unprotected skin is measured, and a protection factor is calculated. There are some reports of
good correlation between in vitro and in vivo measurements; however, in vitro measurements
are currently the standard. Unfortunately, not all clothing is equal in photoprotection. One-
third of summer clothing has a UPF of less than fifteen (Gambichler, Altmeyer, and
Hoffmann, 2002) and the average protection afforded by a light-colored cotton shirt was only
UPF 10 (Wright, Hart, and Peirce, 1998). Tightly woven fabrics, dark colors, wool, and
polyester all provide increased UVR protection; however, they are commonly worn in winter
months when solar radiation is diminished (Gambichler et al., 2002). Cotton, linen, acetate,
and rayon generally have a UPF less than 15, whereas thick denim provides a UPF of 1700.
Additives such as Tinosorb FD (BASF, Basel, Switzerland), contained in the product “Sun
Guard” by Rit, absorb UVR significantly decreasing transmission and offer a UPF up to
30. Products like this claim to last up to 20 washes, but the type of detergent used, the weave
of the fabric, and bleaching can significantly decrease longevity.
Hydration can also have a significant effect on the UPF of clothing. When saturated,
linen, viscose, and polyester significantly increase their UPF, making them ideal for beach or
swim wear. However, cotton and polyester fabrics show a significant decrease in UPF when
saturated. It is estimated that a wet cotton shirt provides a UPF of only 3-4 (Gambichler et al.,
2002; Gambichler, Hatch, Avermaete, Altmeyer, and Hoffmann, 2002).

Hats

Hats can be problematic for sun protection because they shade the face, decreasing the
infrared exposure and the feeling of heat, while still allowing significant UVR exposure. A
wide-brimmed hat (>7.5 cm) may only provide an SPF of 7 for the nose, 3 for cheeks, 5 for
the neck, and 2 for the chin; this is due to reflection of UVR off surfaces below the brim of
the hat. A narrow brim hat provides no more than SPF 1.5 for the nose and minimal
protection for other areas (Diffey and Cheeseman, 1992). Additionally, the weave of the hat
can make a significant difference, with a loosely woven straw hat offering little UVR
protection but still significant shade to the face (Jansen, Wang, Burnett, Osterwalder, and
Lim, 2013).
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TOPICAL PHOTOPROTECTIVE AGENTS

Makeup

Most commercial brands of foundation now contain sunscreen, which offers significant
photoprotection and will be discussed subsequently.
Foundation makeup without sunscreen still provides an SPF of 3 to 4 as a result of
pigment in the foundation. However, makeup can give a false sense of protection, as it is only
applied once per day and loses its protective properties within a few hours. Makeup also can
migrate into dermatoglyphs and accumulate in follicles, decreasing its photoprotective
properties. This process happens even more rapidly in situations with increased perspiration,
sebum production, or tearing. Dermatologists recommend using longwearing sunscreen
underneath foundation, even if makeup contains no SPF to increase overall photoprotection.

Sunscreens

The first commercial sunscreen was introduced in 1928; since that time, sunscreen has
become an essential part of sun protection practices. The first commercial sunscreen, an oil
preparation containing benzyl salicylate, was marketed in 1936 by the future founder of
L’Oreal, although it was in the 1970s that the concept of sun protection factor was introduced
broadly, creating a comparable market for sunscreens (Jansen, Osterwalder, Wang, Burnett,
and Lim, 2013).
Currently, there are only 18 approved agents in the FDA monograph, in contrast to 28
different sunscreens in the Europe Union and at least 34 approved in Australia. Since 1978,
the FDA has only approved the addition of three compounds – avobenzone, zinc oxide, and
the more recently approved ecamsule (Food and Drug Administration, 1999). The lag
between the United States and other countries lies in the fact that sunscreens in the US are
treated as over the counter medications, which necessitate a more rigorous investigation than
handling it as a cosmetic product as it is treated in Europe and Australia.
The Sun Protection Factor (SPF) measure is used to evaluate the efficacy of topical
photoprotectants. Unlike the measurement done for clothing (UPF), SPF is measured in vivo
by testing the MED of volunteers with Fitzpatrick type I, II, or III skin. MED, also known as
the sunburn threshold, is the minimal UVR dose, specifically UVB, since it is the primary
erythema inducer, required to produce a faint pink response in the skin (Baron and Suggs,
2014). The UVR dose is calculated:

Dose (mJ/cm2) = Irradiance (mJ/scm2) x exposure time (s)

To measure the MED, adjacent areas of skin are exposed to increasing amounts of UVR
and then 24 hours later, the MED is defined as the amount of UVR that produced visually
apparent erythema. The testing of all sunscreens uses the same density of 2 mg/cm and
measures the MED on treated versus untreated skin (Schalka and Silva dos Reis, 2011). SPF
is then calculated:

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SPF = MED (protected skin) / MED (unprotected skin)

It is important to note that this testing takes place under high intensity solar simulators,
which can cause an erythema dose in as little as two minutes, compared to much less intense
natural sunlight. Therefore, the SPF is not a measure of duration of UV exposure (Baron and
Suggs, 2014; Schalka and Silva dos Reis, 2011). Additionally, the erythema dose is a
combination of UVB and UVA2, but because UVA1 does not cause erythema, it is not
regularly represented in an SPF value (Jansen et al., 2013).
Different countries have imposed differing regulations for UVA testing. Japan currently
uses persistent pigment darkening (PPD) as a clinical endpoint of UVA protection, and rates
sunscreens as PA+ to PA++++ (Japan Cosmetic Industry Association, 1995). The EU also
uses PPD as testing, but simply mandates that all marketed sunscreens contain UVA
protection at least one-third of the labeled SPF (European Cosmetic Toiletry and Perfumery
Association, 2011). In 2011, the US FDA mandated the use of testing for UVA protection
through an in vitro critical wavelength (CW) testing. This test uses a solar simulator to deliver
four-times the MED dose (on a Fitzpatrick type II skin) to the test product, and then measures
transmittances from 290-400 nm (the UVA range). CW is defined as the wavelength at which
90% of the area under the absorbance curve occurs. Sunscreens are then simply classified as
broad spectrum if their critical wavelength is  370 nm (Jansen et al., 2013).

Organic Sunscreen Agents

Organic sunscreen agents primarily absorb UVR through chromophores with a
conjugated -electron system. Generally, the larger the molecule, the more conjugated double
bonds are present, which shifts the absorption toward longer wavelengths. Thus, smaller
molecules are more suitable as UVB filters and larger molecules as UVA filters (Sambandan
and Ratner, 2011). Currently, all organic UVR absorbers are aromatic compounds with
multiple conjugated -electron systems. Photostability is also an important property of
organic agents. If the energy absorbed is not dissipated quickly into heat, it can lead to
degradation of the UVR absorbers.
The formation of a reversible isomer (tautomerization) is used in the menthyl anthranilate
molecule through use of the orthoamino group and in bemotrizinol and bisctrizole through an
orthohydroxy group (Gaspar and Maia Campos, 2006).
Organic agents can be further subdivided into UVA and UVB absorbers. One of the first
widely available and most potent UVB protectors is para-aminobenzoic acid (PABA);
however, it has multiple disadvantages. In addition to staining clothing, it is a common
contact and photoallergen and was found to be a potent carcinogen in vitro, although the in
vivo significance is unknown. It has been largely replaced by the less potent but more
tolerable Padimate O (Jansen et al., 2013). Cinnamates, such as octinoxate and the less
commonly used cinoxate, are less potent UVB absorbers than PABA derivatives but are well-
tolerated and rarely cause irritation. While they do not stain, they also have reduced water
resistance, making frequent reapplication necessary. Salicyclates, such as octisalate,
homosalate, and trolamine salicylate, are the weakest UVB agents but are often used in
combination with each other or other UVR filters to augment UVB protection. In particular,
octisalate and homosalate are highly photostable and when used in combination with other
UVR filters, help reduce photodegradation (Sambandan and Ratner, 2011). The newest UVB
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1256 Jacqueline Selph, Ritva Vyas and Meg Gerstenblith

absorber, ensulizole, provides a lighter and less oily consistency than other organic agents,
and therefore is becoming popular for facial wear (Sambandan and Ratner, 2011).
The first FDA-approved organic UVA1 filter was avobenzone (butyl
methoxydibenzoylmethane), which filters most UVA radiation. Its effectiveness is limited
because it is extremely photolabile, and its protective properties decrease >50% within an
hour of use. Unfortunately, it can also affect the stability of other sunscreen agents; therefore,
much effort has surrounded stabilizing formulas (Palm and O'Donoghue, 2007). One of the
most prominent is the Helioplex stabilizing technology from Neutrogena, which combines
avobenzone, oxybenzone, and diethylhexyl 2,6 napthalate (Jansen et al., 2013).
Ecamsule is the most recently FDA approved agent, which broadly filters UVA but not
UVB. It is therefore commonly combined with avobenzone and octocrylene. Unlike
avobenzone, it is very stable and not subject to photodegradation. Initially, it was only
available as Anthelios in La Roche-Posay products, but it has now been incorporated into
other L’Oreal products as well (Fourtanier, Moyal, and Seite, 2008). Lastly, meradimate, a
weaker UVA2 filter, is in combination with other agents to provide increased UVA protection
(Sambandan and Ratner, 2011).
The most common broad-spectrum UVA and UVB organic agents are in the class of
benzophenones. Currently, oxybenzone, sulisobenzone, and dioxybenzone are approved in
the US, with oxybenzone being the most widely used. Recently, this class has received
attention because of an increase in contact allergies. In addition to being used as sunscreens,
they are also added to many colored personal use products to prevent color degradation;
therefore, it is estimated that 96% of the US population has been exposed to a benzophenone
(Heurung, Raju, and Warshaw, 2014).
Additionally, oxybenzone has been shown in vitro to produce estrogenic and
antiandrogenic effects. In an in vivo study of female rats exposed to oxybenzone, the uterus
size was 23% greater than controls; however, the dosage used far exceeded the normal level
of human exposure (Schlumpf et al., 2001). While overall the evidence is inconclusive, there
is still concern regarding benzophenone’s use.

Inorganic Agents
In contrast to organic agents, inorganic agents (previously known as physical filters)
utilize a film of inert metal particles to form an opaque barrier, which reflects and scatters UV
light. The two widely used inorganic agents are zinc oxide, which offers predominantly UVA
protection, and titanium dioxide, which provides predominantly UVB protection (Sambandan
and Ratner, 2011). Inorganic agents were initially unpopular because they required a thick,
opaque application, which was aesthetically displeasing.
Newer formulations have been able to microsize both titanium dioxide and zinc oxide,
reducing the particle size from 200nm-500 nm to 10nm-50 nm, improving aesthetics (Pinnell,
Fairhurst, Gillies, Mitchnick, and Kollias, 2000). Inorganic agents are not susceptible to
photodegradation, so they provide less variability in photoprotection.
Inorganic sunscreens are recommended for children and sensitive individuals because
they have no known skin irritating or sensitizing potential. Additionally, children, especially
infants, have a higher surface area to volume ratio compared to adults, which causes concern
for absorption of topically applied medications. While the absorption potential of organic

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 Photoprotection Practices 1257

agents such as benzophenone is controversial, inorganic agents have been shown repeatedly
to have no percutaneous penetration (Jansen et al., 2013).
Therefore, in children under two years of age, only inorganic sunscreens should be
applied, and in children less than six months, other photoprotective methods are
recommended (Council on Environmental Health, Section on, and Balk, 2011).

Table 3. FDA approved sunscreen agents

Peak
Range of
FDA approved Absorption
Class Protection Note
ingredient Wavelength
(nm)
(nm)
Inorganic Agents
Titanium
varies 290 – 350 Better UVB protection
Dioxide
Zinc Oxide varies 290 – 400 Better UVA protection
Organic UVB
Stains clothing, contact
PABA 283 260 – 313
PABA allergen
derivatives Largely replaced
Padimate O 311 290 – 315
PABA
Oxtinoxate 311 280 – 310 Decreased water
Cinnamates
Cinoxate 311 270 – 328 resistance
Octisalate 307 260 – 310 Photostable, reduce
photodegredation of
Homosalte 306 270 – 328
Salicylates other agents
Trolamine Water soluble, found in
260-355 260 – 355
Salicylate hair products
Costly, difficult to
Octycrylene 303 287 – 323
incorporate
Ensulizole 310 290 – 340 Lightweight, less oily
Organic UVA
Highly photolabile, in
Avobenzone 360 310 – 400
Helioplex
Meradimate 336 200 – 380 Weaker UVA2 filter
Broad spectrum,
Ecamsule 345 295 – 390
Anthelios
Oxybenzone 290, 325 270 – 350
Broad Spectrum, UVA
Benzophenones Sulisobenzone 366 250 – 380
and UVB protection
Dioxybenzone 352 206 – 380

SYSTEMIC PHOTOPROTECTIVE AGENTS

While currently the most popular form of photoprotection, topical agents have many
limitations. The efficacy of topical agents is drastically reduced with improper application
and reapplication, making a systemic alternative a logical route to pursue. Unfortunately, no
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1258 Jacqueline Selph, Ritva Vyas and Meg Gerstenblith

current systemic therapies provide enough protection to be used as a sole agent; however,
further research in this area is being pursued.

Polypodium Leucotomos

Polypodium leucotomos is perhaps the most well characterized photoprotective agent. As

an extract from the fern leaf, it was used for centuries by Native Americans for its anti-
inflammatory properties. Research on the compound has shown that it acts a scavenger to
absorb free radicals and ROS, protects DNA by inhibiting the formation of pyrimidine
dimers, increases the number of Langerhans cells in the skin, helps to inhibit mast cells from
infiltrating into the skin, and overall reduces the effects of UVR (measured by an increase in
the MED). In a study of 35 patients with long-standing polymorphous light eruption, daily
administration of 480 mg to 1200 mg P leucotomos resulted in a significant proportion of
patients becoming unresponsive to repeated UVA and UVB exposure (Tanew, Radakovic,
Gonzalez, Venturini, and Calzavara-Pinton, 2012). In a larger study of high risk malignant
melanoma patients, 1080 mg of P leucotomos increased the MED in all patients (decreasing
UVR sensitivity) (Aguilera et al., 2013). The SPF of orally administered P leucotomos is
estimated to be between 3-7, making it unacceptable as a sole photoprotective strategy, but an
effective adjuvant therapy.

Carotenoids

Carotenoids are a class of micronutrients that act as antioxidants to provide skin

protection by decreasing the free-radical induced damage to DNA. A class of vitamin A
derivatives, they include lycopene, lutein, zeaxanthin, and betacarotene. Both betacarotene
and lycopene have been show to decrease in skin concentration following UVR. In a large
longitudinal study for 4.5 years, betacarotene supplementation was not shown to decrease
non-melanoma skin cancer development (Green et al., 1999). However, other studies
demonstrated that long term supplementation with betacarotene does provide protection from
UVR-induced erythema (Heinrich et al., 2003).
Overall, the oral administration of carotenoids can provide some photoprotection,
especially in comparison to topical application.

Afamelanotide

Afamelanotide, an -melanocyte stimulating hormone analogue, induces epidermal

melanin formation by binding to receptors on melanocytes, leading to increased melanocyte
proliferation. A relatively new substance, it was granted investigational new drug status by
the FDA in 2009.
Since that time, subcutaneous administration of the 13-amino acid analogue has been
shown to increase tolerance to UVR exposure in patients with erythropoietic protoporphyria
and solar urticaria (Harms, Lautenschlager, Minder, and Minder, 2009). Other -melanocyte

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 Photoprotection Practices 1259

stimulating hormone analogues have been used outside of clinical or research settings by
individuals seeking tan skin and weight loss.
These other analogues are less specific and interact with a wide range of receptor. There
have been several case reports of -melanocyte stimulating hormone analogues causing
eruptive nevi or rapid changes to current nevi, therefore the FDA has issued warnings against
the cyclic peptide (J. Harms, Lautenschlager, Minder, and Minder, 2009; Reid, Fitzgerald,
Fabre, and Kirby, 2013).

Polyphenols

Polyphenols, most commonly phenolic acid, flavonoids, catechins, stilbenes, and

proanthorcyanidins, have anti-oxidant, anti-inflammatory, and anticarcinogenic properties.
Consumption of green tea polyphenols (epicatechin, epicathechin-3-gallate, epigallocetechin,
and epigallocatechin-3-gallate) decreases UVR-induced erythema.
Furthermore, topical tea polyphenols are more potent than both vitamins C and E in
scavenging ROS; however, they have low stability and a short duration of biologic activity.
Interestingly, studies have also shown that human consumption of chocolate rich in flavanols
can provide protection from UVR (Afaq and Katiyar, 2011).

Other Antioxidants

Free radicals damage DNA, lipid membranes, protein structures, and contribute to
photoaging, therefore many cosmeceutical companies have tried incorporating high
concentrations of antioxidants into products to improve skin care. Vitamin C is a water
soluble antioxidant (AO) that neutralizes free radicals, increases collagen synthesis, and
reduces collagenase expression. Topical application of vitamin C has photoprotective effects
inducing reducing erythema and sunburn cell formation. Vitamin E is a lipid-soluble AO,
with the most abundant form being -tocopherol. In contrast to vitamin C, vitamin E readily
reaches the stratum corneum, helping to slow the process of collagen breakdown. Together,
vitamins C and E work synergistically, with vitamin C regenerating oxidized vitamin E
(Chen, Hu, and Wang, 2012).
Other important antioxidants include selenium, silymarin, and soy isoflavones. Selenium
helps optimize glutathione peroxidase and thioredoxin reductase and serves as a cofactor of
vitamin E regeneration. The form L-selenomethionine has superior transepidermal delivery,
and when combined with vitamin E, can reduce UVR induced blistering, pigmentation, and
skin tumors (Chen et al., 2012).
Silymarin, from milk thistle, contains three flavonoids that have potent ROS scavenging
ability and prevent lipoprotein oxidation. Finally, soybeans contain isoflavones, which have
been shown to be anticarcinogenic by scavenging peroxyl and lipid radicals (Jansen et al.,
2013). Other antioxidants are listed in Table 4.
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1260 Jacqueline Selph, Ritva Vyas and Meg Gerstenblith

CONCLUSION
As a skin cancer prevention strategy, there are many methods of photoprotection to
consider. While much progress has been made since the first commercial sunscreen was
introduced in 1928, there are many other agents, including systemic agents, which warrant
further investigation.
Currently, topical protective agents remain the mainstay of photoprotection in the United
States, but ineffective application and the need for reapplication often limit their
effectiveness. Avoidance of sunlight during peak hours, the use of improved architectural and
automobile structures, and the incorporation of photoprotective clothing, hats, and sunglasses
are all important to reduce UVR exposure and enhance protection of the skin.

Table 4. Antioxidants and their functions

Antioxidant
Sources Functions
Compound
Increased collagen production to
Vitamin A reduce photoaging, systemically
Colored fruits and
(retinols, can be used as a preventative
vegetables
carotenoids) measure for skin cancers in
susceptible populations
Cofactor in collagen synthesis,
Vitamin C Fruits, Vegetables reduces erythema and
immunosuppression
Oils, seeds, nuts, Reduce photoaging, reduce cell
Vitamin E
meats membrane lipid peroxidation
Corn, wheat Increases function of endogenous
Selenium
soybean antioxidants, regenerates vitamin E
UV filtering properties to decrease
Silymarin Milk thistle photocarcinogenesis, reduces
immunosuppresion
Reduces erythema, can result in
Tea Polyphenols Isolated from tea
contact and allergic dermatitis
Soy Isoflavones
Soy, red clover, Preserve epidermal proliferation
(genistein,
gingko biloba and repair mechanisms
daidzein, equol)
Coffee beans,
Shown to decrease erythema and
Caffeic Acid propolis plant
immunosuppresion
seeds
Fruits and leafy
Decreases photoaging and
Apigenin vegetables, tea,
photocarcinogenesis
wine
Skin and seeds of Decreases erythema and
Resveratrol
grapes, nuts, fruits photocarcinogenesis

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Sambandan, D. R. and Ratner, D. (2011). Sunscreens: An overview and update. Journal of
the American Academy of Dermatology, 64(4), 748-758. doi:10.1016/j.jaad.2010.01.005
[doi].
Sanlorenzo, M., Vujic, I., Posch, C., Cleaver, J. E., Quaglino, P., and Ortiz-Urda, S. (2014).
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 59

RISK FACTORS FOR SUN EXPOSURE DURING SPRING

BREAK AMONG COLLEGE STUDENTS

Marvin E. Langston, MPH1, Stephanie G. Lashway, MPH1

and Leslie K. Dennis, MS, PhD1,2
1
Division of Epidemiology and Biostatistics,
Mel and Enid Zuckerman College of Public Health,
University of Arizona, Tucson, AZ, US
2
Department of Epidemiology, College of Public Health,
University of Iowa, Iowa City, IA, US

ABSTRACT
In order to look at college students’ behavioral practices prior to a sunny vacation
(during spring break) along with their beliefs and attitudes, we recruited sororities and
fraternities in the Midwestern USA to complete a self-administered questionnaire.
Sorority and fraternity students were expected to have high UVR exposure due to a
strong desire to tan. The questionnaire included information on sun exposure during
spring break, sun-sensitivity, and tanning attitudes and behaviors.
Analyses examined associations between potential risk factors for spending 16 or
more hours in the sun during spring break using logistic regression while controlling for
the clustering effects of sororities and fraternities. Students who tanned mildly were 1.6
times more likely than those with moderate or deep tans to spend 16+ hours in the sun
during spring break, suggesting a strong desire to tan. Students who spent 16+ hours in
the sun during spring break were more likely to have frequented tanning beds (odds ratio
of 2.4 for 11+ times vs. ≤5 times) and to have used self-tanning creams (odds ratio of 2.9)
between New Years and spring break. These data provide evidence that use of artificial
tanning devices and self-tanning creams or sprays among college students are related to
increased intermittent sun exposure (during a spring break vacation) rather than reduced
exposure.
Mistaken beliefs regarding a base tan as potentially beneficial need to be addressed
by excellent science examining the base tan theory and translated to the public.
Replacement of tanning bed use with safer sunless tanning creams may reduce some of
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1266 Marvin E. Langston, Stephanie G. Lashway and Leslie K. Dennis

the harmful UVR exposures. Education alone will not be sufficient to change sun seeking
behavior as was seen here and in other studies.

Keywords: Abbreviations: CI = confidence interval; OR=odds ratio

INTRODUCTION
Sun exposure appears to be the most important risk factor in the development of skin
cancer, with other forms of ultraviolet radiation (UVR) also supporting such associations.
Thus, UVR is the major etiologic risk factor implicated in the development of skin cancer
(Gandini et al., 2005; Boniol et al., 2012; Veierod et al., 2014; Karagas et al., 2014). The
mechanism of sun exposure in relation to skin cancer may work through specific ultraviolet
(UV) wavelengths (van Weelden et al., 1990). UVR is composed of electromagnetic radiation
at various wavelengths with differing possible implications for both melanoma and non-
melanoma skin cancers. UVR from the sun is comprised of UVA (λ=320-400nm), UVB
(λ=280-320nm), and UVC (λ=200-280nm). The energy that each UV type carries is inversely
related to its wavelength. Evidence suggests that both UVA and UVB exposure may
contribute to the development of melanoma, the most aggressive form of skin cancer, albeit
through differing pathways (Zhang & Rosdahl, 2003; Young et al., 1998). UVB seems to
contribute to non-melanoma skin cancers such as squamous cell carcinoma and basal cell
carcinoma (Woodhead et al., 1999).
There are distinct types of sun exposure that have varying degrees of association with
skin cancer types. Sun exposure as a risk factor can be classified as chronic or total sun
exposure and intermittent sun exposure. Most consistently melanoma seems to be caused by
intermittent periods of high sun exposure on unaccustomed skin (Armstrong & Kricker,
2001). This represents the pattern of sun exposure and not just the amount. With intermittent
exposure to the sun, the skin is more vulnerable to the effects of UV radiation as exposure
may result in sunburn, solar keratoses or other sun-induced skin damage (Elwood et al., 1984;
Green, 1984; Holman & Armstrong, 1984; Dubin et al., 1986; Armstrong, 1988). Research on
chronic sun exposure over the life course of individuals has proven less convincing for
melanoma (Armstrong, 1988). The role of sun exposure for basal cell carcinoma is unclear
although some have postulated intermittent sun exposure in addition to childhood sun
exposure as important factors (Madan et al., 2010). However, chronic sun exposure is a major
etiologic factor for squamous-cell carcinoma (Madan et al., 2010).
Other sources of UVR include artificial tanning devices. Artificial UVR tanning or
indoor tanning includes the use of tanning beds, sunlamps, and UV tanning booths at home,
in a salon, or commercial location. From here on we will refer to such use as tanning bed use.
Adolescents and college students have been reported to have the highest rates of UVR from
sun exposure and artificial UVR sources (Cokkinides et al., 2002; Magee, 2007; Dennis et al.,
2009a). Intentional tanning appears to be highest in adolescents and young adults, then
dropping as adults age (Dennis et al., 2009a). Several behavioral studies in adolescents and
college students found strong attitudes that tanning is important (Yoo & Hur, 2014;
Benmarhnia et al., 2013; Dennis et al., 2009b). Most adolescents and young adults prefer to
tan via sunbathing or use of tanning beds over using sunless tanning products (Banks et al.,
1992; Boldeman et al., 2003; Dennis et al., 2009b; Geller et al., 2002; Mawn & Fleischer,

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 Risk Factors for Sun Exposure during Spring Break … 1267

1993). In the United States, nearly 30% of non-Hispanic white female high-school students
used indoor tanning in 2011 and 24.9% of non-Hispanic white women (18-34) used indoor
tanning in 2010 (Guy et al., 2013). In France, nearly 14% of 20-25 year old women used
tanning beds (Benmarhnia et al., 2013). Such UVR exposures have led to studies attempting
to understand why tanning is so important among these age groups.
The objective of this study was to examine factors related to increased amounts of sun
exposure during spring break. Considerations of these factors will help inform the focus of
future interventions. We specifically examined prior tanning bed use and use of sunless
tanning products to see if they were related to an increase or decrease of intermittent sun
exposure during vacation (spring break). We also investigated how knowledge and attitudes
regarding skin cancer prevention efforts related to spring break sun exposure. The analysis
was conducted among sorority and fraternity-affiliated students in the Midwest via a cross-
sectional survey.

METHODS
Our interest in intermittent sun exposure predictors is due to the well described risks of
this factor in melanoma and skin cancers. A University in the Midwestern United States was
chosen for recruitment due to the homogenous population, later life skin cancer risks, and
ability to analyze recent intermittent sun exposures. The student population of the University
under study was 92% non-Hispanic white, 2.2% African Americans, 0.4% American Indians,
3.7% Asians, and 2.5% identified as Latino during the period of data collection.
Students from sororities and fraternities (Greek houses) were recruited to participate in a
survey and educational session on skin care. Participants completed an informed consent
document with the self-administered questionnaire. During the data collection, participants
were provided pizza and were given $10 re-imbursement. This project was approved by the
Institutional Review Board for Human Subjects. Only Greek houses with 10 or more
members were eligible for recruitment. An estimated 80% of those attending their monthly
house meeting met the initial inclusion requirements with 163 students recruited.
The self-administered questionnaire included information on sun exposure during spring
break, sun-sensitivity, sunburns, artificial UVR tanning, sunless tanning cream use and
tanning attitudes. For these analyses the primary outcome was high sun exposure over spring
break. Sun exposure was defined as the self-reported total hours spent outdoors from sunrise
to sunset. High sun exposure during spring break was a reported 16 hours or more during the
week compared to 15 or fewer hours in the sun. The reliability of the self-administered
questionnaire on artificial UVR tanning for specific time-periods ranged in Kappa values
from 0.7 to 0.9 (Dennis et al., 2008), suggesting that these students reported their artificial
UVR tanning practices consistently (Gordis 2013). The self-administered questionnaire also
included several host factors important in studies of sun exposure. Ultimately we are
interested in the amount of UVR that is absorbed by the skin. UV absorption is related to skin
sensitivity to the sun. The amount of UVR an individual needs to obtain abnormal redness of
the skin (the beginning of a sunburn) is called their minimal erythemal dose (MED). Less
UVR is needed to produce abnormal skin redness among fair-skinned individuals than is
required for dark-skinned individuals (Armstrong 1988). However, time of day, season and
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1268 Marvin E. Langston, Stephanie G. Lashway and Leslie K. Dennis

latitude all influence the amount of UV radiation available for absorption (Holick, 2004).
Tendency to sunburn was defined as the reaction of the skin when exposed to strong sunlight
for 30 minutes for the first time each summer with no protection. Tanning ability was
characterized as the ability to tan after repeated and prolonged sun exposure.
All analyses were conducted within SAS version 9.3 (SAS Institute Inc., 2010).
Characteristics of the sample were described using descriptive statistics. Survey sampling
statistical techniques were used because we first recruited from Greek houses, and then
recruited subjects from those members who attended a monthly meeting. Survey sampling
methods including a finite population correction (Tryfos, 1996; SAS Institute Inc., 2010)
were used to describe the means and ranges for analyzed risk factors. Sampling weights were
computed from the selection probabilities at each stage, and were based on both the
recruitment of Greek houses and for the participation rate within each Greek house. These
weights were applied even though we did not attempt to recruit all Greek houses and only
recruited from students attending the monthly house meeting (Tryfos 1996). PROC
SURVEYFREQ was used to analyze the distribution of various risk factors while accounting
for potential clustering by Greek house.
The Kappa statistic was used to show agreement between self-reported tanning ability
and the objectively measured value using the colorimeter. PROC SURVEYLOGISTIC was
used to examine the odds ratios (ORs) for 16+ hours of sun exposure during spring break
(compared to <16 hours). Ninety-five percent confidence intervals (CI) are also reported
around the ORs. Based on the sampling weights used, SAS estimated the total membership of
the Greek Houses.
This estimate was similar to the number reported in student enrollment records. The
Taylor expansion method was used to estimate the standard errors of the estimators used
(Woodruff, 1971; SAS Institute Inc., 2010). The variance estimates also accounted for the
clustering of the students associated with the same Greek houses.
Models run for behaviors, beliefs, and attitudes towards tanning were adjusted for
tendency to sunburn as a measure of sun sensitivity. The amount of sun exposure an
individual experiences and their sun sensitivity are extremely intertwined (Armstrong 1988).
Thus, lack of adjustment or adjustment for measures of sun sensitivity that are not directly
related to the skin’s response to the sun, such as hair and eye color, can cause bias when
trying to understand the health effects of sun exposure.

RESULTS
The majority of study participants were female (72%) and white (99%). They ranged in
age from 18-23 years. Nearly half of all study participants were sophomores in college (49%),
followed by freshmen (29%). About 87% had self-reported deep or moderate tanning ability.
About one third (34%) of participants reported no tendency to sunburn, while 28% burned
before tanning. None of the study participants had a personal history of skin cancer. A family
history of skin cancer was marginally associated with sun exposure over spring break.
Over spring break many students reported engagement in outdoor activities generally
indicative of prolonged periods of sun exposure. The following represents activities students
participated in at least once over spring break, and many students participated in multiple

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 Risk Factors for Sun Exposure during Spring Break … 1269

activities. Some students went swimming, or lay by a pool, lake or ocean (59%). Others
engaged in water or snow skiing (27%) or worked outdoors (45%). About half (51%) toured
other cities or went sightseeing. Finally about a third (34%) went hiking or biking, while
another third (35%) went boating, fishing, or engaged in other water sports. An overwhelming
majority (79%) of students reported participation in at least one of these outdoor activities
over spring break. Participation in these activities was strongly associated with 16+ hours of
sun exposure over spring break. This highlights the problem surrounding outdoor recreational
activities and the need for practicing good sun safety and skin protection.

Artificial Tanning

Increased tanning bed use between New Years and spring break was related to an
increased risk of spending 16+ hours in the sun during spring break (Table 1). This
association did not hold with tanning bed use in the prior 12 months or lifetime use. Sunless
tanning spray use prior to spring break (between New Years and spring break) was shown to
be related to spending 16+ hours in the sun during spring break. This association was also
seen for lifetime sunless tanning spray use (use in the prior 12 months was not assessed). Skin
color and spring break sun exposure were not associated (Table 2). Students who tended not
to burn or burned then tanned upon first exposure to the sun in the summer (Table 2) were
more likely to spend 16+ hours in the sun during spring break, as would be expected.
However, we saw an unexpected increased risk of 16+ hours in the sun among those who
self-reported their tanning ability to be only mild rather than among those who reported to be
moderate or deeply tanned after repeated and prolonged exposure to the sun. Melanin levels
also showed that students who spent more time in the sun during spring break had darker
unexposed skin (upper inner arm or wrist) and exposed skin of the forearm than those who
spent less than 16 hours in the sun.

Reasons for Tanning

Several indicators of tanning attitudes and beliefs seemed to impact sun exposure over
spring break. Twelve percent of participants reported use of artificial UV tanning devices so
that they could spend more time in the sun, and 47% used these devices before they knew
they were going to be in the sun. Those using tanning lamps or sunbeds so that they could
spend more time in the sun (Table 3), were 5.5 times more likely to report 16+ hours in the
sun over spring break. Several aspects of tanning attractiveness were explored here. Personal
beliefs of tanning attractiveness and media perceptions of tanning attractiveness were not
associated with sun exposure over spring break. However, participants that indicated parents
and friends believed tanning was attractive were more likely to spend 16+ hours in the sun
over spring break. Here perceptions on tanning attractiveness from parents and friends
increased the risk of 16+ hours in the sun 1.80 times (95% CI of 1.13-2.86) and 1.54 times
(95% CI of 1.05-2.27) respectively.
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Table 1. Tanning bed and sunless tanning cream use as they are related to sun exposure during spring break among
163 sorority and fraternity students in the Midwest

Sun Exposure Hours 1

16+ <16 OR 95% CI
Between New Years to Spring Break
Tanning bed (# of uses)
<5 times 36 40 Ref -
6 to 10 times 18 13 1.54 (0.67-3.52)
11+ times 37 17 2.42 (1.38-4.23)

Sunless Tanning Cream (# of uses)

None 72 63 Ref -
1+ times 20 6 2.92 (1.51-5.63)

In the Last Year2…

Tanning bed (# of uses)
<5 times 28 25 Ref -
6 to 10 times 10 14 0.64 (0.33-1.23)
11+ times 54 21 1.56 (0.63-3.86)
1
Outdoors in the sun during spring break.
2
Use in the last year was not posed for sunless tanning cream users.

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 Table 2. Host factors as they are related to sun exposure during spring break among 163 sorority and fraternity
students in the Midwest

Host Factors Sun Exposure Hours

16+ <16 OR 95% CI
1
Tendency to sunburn
Severe, moderate, or a mild sunburn 31 32 Ref -
Burn then tan, or no sunburn 62 38 1.68 (1.44-1.97)

Ability to tan2
Deeply, or moderately tanned 79 63 Ref -
No or Mildly tanned 14 7 1.60 (1.03-2.48)
Skin Color
Medium or Dark 45 33 Ref -
Fair 48 37 0.95 (0.83-1.09)

Family history of skin cancer3

No 60 48 Ref -
Yes 30 19 1.26 (1.00-1.59)
1
When skin is first exposed to strong sunlight.
2
After repeated and prolonged sun exposure.
3
Five respondents who were not sure are excluded.
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Table 3. Attitudes, behaviors, beliefs and knowledge as they are related to sun exposure during spring break among
163 sorority and fraternity students in the Midwest1

Sun Exposure Hours

16+ <16 OR 95% CI
Tanning Attitudes and Beliefs
I don’t mind burning if it helps me get a good tan
Disagree or neutral 58 55 Ref -
Agree 35 30 2.44 (1.75-3.39)
I try to sit in the shade, rather than in direct sunlight on a sunny summer day
Agree or neutral 16 30 Ref -
Disagree 76 39 3.49 (1.33-9.18)
I use sunlamps, tanning lamps or sunbeds so that I can spend more time in the sun
No 76 67 Ref -
Yes 17 3 5.50 (2.18-13.87)
Knowledge about Sun Safety 2

Ultraviolet lamps can cause some skin damage

Disagree or neutral 10 2 Ref -
Agree 3 83 68 4.04 (1.57-10.37)

Sunlamps are safer than the sun

True 10 13 Ref -
False 3 82 57 2.09 (0.76-5.75)
Sunburning to get a tan is not harmful
True 89 64 Ref -
False 3 4 6 2.02 (1.12-3.65)

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 Sun Exposure Hours
The strength of the sun’s rays is increased on cloudy days 16+ <16 OR 95% CI
True 71 46 Ref -
False 3 20 23 0.70 (0.49-1.01)
Sunlamps can be dangerous
Disagree or neutral 9 5 Ref -
Agree 3 84 65 1.38 (0.32-5.92)
Sunless tanning creams are safer than the sun
False 14 10 Ref -
True 3 79 60 0.93 (0.34-2.55)
The strength of the sun’s rays is increased at the top of mountains
False 20 23 Ref -
True 3 71 46 1.80 (0.82-3.92)
Knowledge about tanning (overall score) 4
Less than 70% 10 8 Ref -
70% or more 83 62 1.03 (0.62-1.73)
1
All models adjusted for tendency to burn.
2
Modeling the correct response.
3
Correct response to the knowledge question.
4
Correct responses to the knowledge questions presented in the table were summed and a percentage of the total assigned to each
participant.
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1274 Marvin E. Langston, Stephanie G. Lashway and Leslie K. Dennis

Sun Safety

Participants were asked about several sun protective behaviors and risky behaviors to
understand if such behaviors impact their sun exposure. One of the protective behaviors
involved asking them if they were likely to be “sitting in the shade, rather than in direct
sunlight on a sunny summer day.” Participants who disagreed with that statement were more
likely to report 16+ hours of sun exposure over spring break than those that agreed or gave a
neutral response on the 5 point Likert scale (OR=3.49, 95% CI of 1.33-9.18). These
participants would therefore spend 16+ hours outdoors in the sun while also directly in the
sun’s harmful UV rays. Additionally those that didn’t mind burning if it helped to achieve a
good tan were at risk for 16 or more hours in the sun during spring break (OR=2.44, 95% CI
of 1.75-3.39). Participant’s knowledge of sun safety and harmful tanning practices was
assessed as part of the questionnaire. Those who believed ultraviolet lamps could cause some
skin damage were at an increased risk of 16+ hours in the sun over spring break (OR=4.04,
95%CI of 1.57-10.37). Participants who correctly indicated that sunburning to get a tan is
harmful were also at an increased risk of 16+ hours in the sun over spring break (OR=2.02,
95% CI of 1.12-3.65). A summary score of correct knowledge questions was designed based
on responses to 7 component items. Moderate knowledge about sun safety (70% or more
correct answers to component questions) was not associated with sun exposure over spring
break.

DISCUSSION
Artificial Tanning

We found that increased tanning bed use and sunless tanning product use between New
Years and spring break were related to an increased risk of spending 16+ hours in the sun
during spring break. This is important regarding UVR behavior. It would appear that these
students used tanning beds and/or sunless tanning products in an attempt to darken their skin
prior to spring break. Consistent use of sunless tanning products (lifetime use) may indicate
lower tanning ability among a subset with a strong desire to tan. Melanin levels, based on
colorimeter data, showed that students who used both sunless tanning creams and tanning
beds between New Years and spring break had the lightest skin color (both at sun exposed
and unexposed body sites) regardless of the amount of time they spent outdoors in the sun
during spring break. Whereas, those who frequented tanning beds most often, 11+ times
between New Years and spring break, had more melanin (darker skin color) than others (both
on sun exposed and unexposed body sites) regardless of their spring break sun exposure. The
increased sun exposure among students who reported a lower tanning ability (mildly tan)
appears to reflect a desire to tan among those with perceived lighter skin. However, the
melanin data showed darker skin among those students who spent more time in the sun,
suggesting the lighter skin is more perceived than real. The darker skin is likely due to
tanning bed use and sunless tanning spray use prior to spring break along with the increased
sun exposure during spring break. If it was solely or mostly due to sun exposure during spring
break, then we would expect higher melanin rates for exposed skin but not unexposed skin.

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 Risk Factors for Sun Exposure during Spring Break … 1275

Our finding that tanning bed use between New Years and spring break was associated
with an increased risk of spending 16+ hours in the sun is consistent with other research.
Tanning beds users report using tanning beds to prepare their skin for sunny holidays or for
“vacation preparation” (Knight et al., 2002; Mawn et al., 1993; Dissel et al., 2009; Rhainds et
al., 1999; Young et al., 1998; Schneider et al., 2013). A study in France found that 24% of the
population (15-75 years) believed artificial tanning protected them from sunburn on vacation
(Benmarhnia et al., 2013). This proportion drastically increased among tanning bed users
(43%) and slightly decreased (21%) in people who had never used a tanning bed (Benmarhnia
et al., 2013). Another study among all ages of tanning bed users found that about 29% use
them for vacation preparation (Knight et al., 2002). This percentage of utilization increased to
61% among 17-30 year olds (Knight et al., 2002).
Sunless tanning products include fake tans, spray-on tans, and sunless tanning creams and
lotions. They are available over the counter and in tanning salons via spray-on booths as an
alternate to UVR tanning through sun exposure or tanning bed use. Such products can provide
the desired tanned look without UVR exposure to the sun or tanning beds. Dihydroxyaceton
(DHA) is the chemical component of sunless tanning products that darkens the skin with no
known carcinogenic effects (Fu et al., 2004). DHA was show decades ago to increase sunlight
tolerance among UVA sensitive patients and more recently for UV-B, suggesting regular use
along with sunscreen for improved sun safety (Howe et al., 2008; Faurschou & Wulf, 2004).
Sunless tanning products may prove as a healthier alternative to artificial UVR tanning, but in
our study they negatively impacted sun seeking behavior similarly to artificial UVR tanning.
It is unclear if this is only due to picking a population that puts importance on tanning or if
other student populations who use sunless tanning products also spend more time in the sun.
Additionally, since this is an observational study, it is unclear if they would have spent more
time in the sun or using tanning beds if they did not use sunless tanning products. Public
health messages should aim at replacement with sunless tanning, but also other methods to
decrease high sun exposure following application.
The relationship between use of sunless tanning products and sun protection habits is
unclear. While one study found that sunless tanning product users were more likely to use
sunscreens and other sun protection measures, another found a higher rate of sunburn among
users (Stryker et al., 2007; Brooks et al., 2006). It is unclear if the sunburns reflected lighter
skin, which is why these individuals were using the sunless tanning products to tan. So, while
sunless tanning may be a beneficial alternative to indoor or outdoor UVR tanning, more
research is needed to understand the physiologic effects of the products and attitudes and
behaviors of people who use sunless tanning products.

Reasons for Tanning

Reasons for tanning vary with most tanners reporting multiple reasons for tanning. Some
reasons for use of tanning beds mimic those for tanning in general, while others diverge a bit
from general reasons for tanning. Much of the literature on reasons for tanning surrounds
indoor tanning. Reasons reported for tanning bed use have included to feel healthy (Poorsattar
& Hornung, 2007; Rhainds et al., 1999; Dissel et al., 2009), peer pressure or influence of
friends (Knight et al., 2002; Dissel et al., 2009; Mawn et al., 1993; Poorsattar & Hornung,
2007), a desire for a feeling of warmth (Schneider et al., 2013) or as treatment of skin
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1276 Marvin E. Langston, Stephanie G. Lashway and Leslie K. Dennis

diseases (psoriasis, acne, or dermatitis) (Knight et al., 2002). The use of tanning beds to spend
more time in the sun or before exposure to the sun, which we observed, may indicate a belief
in the benefits of “base tanning” prior to periods of high sun exposure. Students who spent
16+ hours in the sun during their spring break vacation had more than 7 times the chance (as
those with <16 hours in the sun) of receiving a sunburn. This would suggest that any
perceived “base tan” they had was not protective of sunburning during their sunny vacation.
If these risky tanning behaviors are influenced by the “base tanning,” educational
interventions may need to address the harmful risks associated with adoption of this theory,
although some recent research shows this may be difficult. The Pool Cool diffusion trial
among lifeguards, across 32 geographic regions in the US, combined educational and
environmental strategies for their multicomponent skin cancer prevention program (Hiemstra
et al., 2012). No association was seen between Pool Cool participation and “It helps to have a
good base tan” (Hiemstra et al., 2012). However, lifeguards who rated “It helps to have a
good base tan” higher at baseline (1 for “not at all” up to 4 for “a great deal”) also rated it
higher at follow-up (Hiemstra et al., 2012). They also found that “It helps to have a good base
tan” at baseline was related to ethnicity (non-Caucasians disagreed with the statement) and
moderate skin cancer risk (Hiemstra et al., 2012). Other studies have found similar behavioral
beliefs where use of tanning beds to gain a perceived “protective” base tan or to spend more
time in the sun is seen (Poorsattar & Hornung, 2007; Dennis et al., 2009b; Knight et al.,
2002). Specifically, a Swedish survey of university students found that individuals who
reported more indoor tanning also reported more outdoor tanning (Jerkegren et al., 1999).
Alternately other studies showed tanning bed use to reduce the frequency of sunbathing
(Dennis et al., 2009b; Knight et al., 2002). More work needs to be done in this area to better
understand the harms of the base tanning theory and to identify the susceptible populations
that have adopted these beliefs.
We found that positive parental and peer tanning behavior were related to increased time
in the sun during student’s spring break vacation. These findings are supported by several
studies that have found that adolescents who had a parent who tanned or permitted tanning
were more likely to intentionally tan (Cokkinides et al., 2002; Cokkinides et al., 2009;
Hoerster et al., 2007; Lazovich et al., 2004; Mayer et al., 2011; Stryker et al., 2004).
Additionally, adolescents with friends who thought a tan was attractive or tanned
intentionally were also more likely to intentionally tan (Geller et al., 2002; Hoerster et al.,
2007; Lazovich et al., 2004; Mayer et al., 2011; O’Riordan et al., 2006; Stryker et al., 2004;
Yoo, 2009). These findings suggest that personal beliefs of attractiveness of tanning do not
influence sun exposure behaviors in this population, but peer pressure and familial pressures
may have an effect. The effect of peer pressure may be exaggerated due to the sample
population chosen in our study. Fraternity and sorority students self-select into these Greek
houses based on social and/or academic similarities, therefore they may be more prone to
adhere to behaviors or beliefs of their peers more than others in this age group.
In our study, personal beliefs regarding attractiveness of a tan were not related to more
time in the sun during their spring break vacation, but other studies have found important
associations with appearance based motivation in adolescent and young adult populations. A
study of female students at a college in Texas found that one of the main motivations for
tanning in that population was appearance-based (felt a tan increased attractiveness) (Yoo &
Hur 2014). Similarly, high percentages of tanning bed users of all ages report using them to
“look better” or for “improved appearance” (Poorsattar & Hornung, 2007; Dissel et al., 2009;

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 Risk Factors for Sun Exposure during Spring Break … 1277

Rhainds et al., 1999; Knight et al., 2002; Mawn et al., 1993; Lazovich et al., 2004;
Monfrecola et al., 2000; Dennis et al., 2009b; Stapleton et al., 2010). A study among tanning
bed users of all ages found that almost 59% of them felt tanning improved their appearance
(Dissel et al., 2009). Poorsattar & Hornung, (2007) found 82% of tanning bed users aged 17-
30 years used them to “look good.” The proportion of tanning bed users who tan because they
“enjoy a tan appearance” increased among college aged users (17-23) to 92% (Knight et al.,
2002). Again, across these studies, an improved appearance was reported as a reason for
tanning bed use more frequently among adolescents and young adults (77% based on 5
studies) than among adults (53% based on 3 studies).
Several studies among adolescents and young adults also suggest use of tanning beds for
relaxation (Poorsattar & Hornung, 2007; Lazovich et al., 2004; Knight et al., 2002; Dennis et
al., 2009b; Dissel et al., 2009; Schneider et al., 2013). This has also been reported in studies
that included people aged 16 to 90 years (Rhainds et al., 1999; Mawn et al., 1993). Pooling
the percentages that report using tanning beds to relax across these seven studies, about 56%
of adolescents and young adults stated they used tanning beds to relax compared to 32%
among studies including all ages. More specifically, the highest percentage of individuals
who felt tanning indoors was relaxing (~75%) was found in an adolescent population aged
14-17 years (Lazovich et al., 2004). The study of female college students in Texas reported
the other main motivation for tanning in that population to be that they found it relaxing
(emotion-based) (Yoo & Hur, 2014). Knight et al.’s (2002) study of college aged (17-22)
tanning bed users found that about 42% used tanning beds for relaxation. Similarly, 44% of
tanning bed users aged 17-30 used them to relax (Poorsattar & Hornung, 2007).

Sun Safety

Our findings suggest that knowledge of harmful tanning practices either increases or has
no effect on the risk of spending more time outdoors in the sun during spring break, which
was unexpected. A study of female university students (18-26) in Australia found that those
who used sunless tanning were more knowledgeable about skin cancer than those who tanned
outdoors (Day et al., 2013). Additionally, students who avoided tanning or applied sunless
tanning products used more sun protection than those who tanned outdoors (Day et al., 2013).
In France, 89.2% of people (15-75) were aware that tanning bed use could possibly cause
cancer, and that tanning bed users were slightly less likely to believe the risk of cancer
(Benmarhnia et al., 2013). However, our findings were consistent with another study among
American female college students who tanned indoors, which showed that tanning bed users
who used the devices for appearance-enhancement were more knowledgeable about both the
health and appearance-damage risks of tanning behaviors (Stapleton et al., 2010). The
students in the study who were not knowledgeable about health or appearance-damage risks
did not tan specifically for appearance or relaxation (Stapleton et al., 2010). In an American
population, risk reduction strategies aiming solely for increased knowledge of sun safety may
not be the most effective as knowledge of correct behavior here does not translate into future
sun protection.
Public health efforts need to be engaged in understanding and designing successful
interventions for risk reduction. Interventions with proven efficacy should be conducted in the
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1278 Marvin E. Langston, Stephanie G. Lashway and Leslie K. Dennis

most high risk populations. This process will take further work at understanding basic
attitudes and behavioral influences on sun exposure and tanning.

Strengths and Limitations

Survey sampling analyses allowed for control of clustering by sex and fraternity/sorority
as students within Greek houses tend to cluster on unknown factors. Our study population
was chosen due to their high interest in tanning and higher disposable income which could
allow them more options than other student populations. Survey data were collected soon
after spring break to reduce problems in recalling spring break activities. Survey items were
shown to be reliable in this population (Dennis et al., 2008). Studying students in the Midwest
directly after spring vacation, allows us to analyze intermittent sun seeking behaviors in a
population without sun exposure prior to their vacation break. However, due to recruitment of
students within sororities and fraternities, the sample is not representative of all students and
may not be generalizable to all student populations.

CONCLUSION
College fraternity and sorority students at a Midwestern University appeared to tan using
tanning beds and/or sunless tanning products before spring break in order to spend more time
in the sun. Mistaken beliefs regarding a base tan as potentially beneficial need to be addressed
by excellent science examining the base tan theory and translated to the public. Replacement
of tanning bed use with safer sunless tanning creams may reduce some of the harmful UVR
exposures. Education alone will not be sufficient to change sun seeking behavior as was seen
here and in other studies. As the rates of UVR related skin cancers rise, the efficacy of
multipronged intervention efforts that include education, adolescent tanning bed bans, and
communication of personal UVR risks should be investigated for future public health
dissemination.

ACKNOWLEDGMENTS
This research was supported in part by the National Cancer Institute, grant number
R03CA099520 and K07CA104556.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 60

SUN EXPOSURE AND PROTECTION HABITS AND

VITAMIN D LEVELS IN CHILDREN
AND ADOLESCENTS WITH A HISTORY
OF MALIGNANCY

Yael Levy-Shraga, MD and Dalit Modan-Moses, MD

Pediatric Endocrinology Unit,
The Edmond and Lily Safra Children's Hospital,
Chaim Sheba Medical Center, Tel-Hashomer, Ramat-Gan, Israel

ABSTRACT
Sun exposure, the main source of ultraviolet radiation exposure, is the major
environmental risk factor for skin cancers, both melanoma and non-melanoma. The
impact of exposure can be dramatically reduced by practicing sun protection behaviors.
Still, recommendations for sunlight exposure are an area of controversy, as sun avoidance
and sun protection practices may lead to inadequate vitamin D levels. This duality is
particularly relevant to childhood cancer survivors, who are particularly vulnerable both
to the hazardous effects of ultraviolet radiation and to deleterious skeletal and extra-
skeletal effects of vitamin D deficiency. These patients are at high risk for developing
non-melanoma skin cancer and therefore are firmly advised to avoid or minimize sun
exposure and adopt skin protection measures. On the other hand, several studies showed
an inverse association between regional UV-B radiation exposure and cancer mortality,
with activation of vitamin D-related pathways by sun exposure proposed as the likely
mechanism. A protective role for vitamin D in the prevention of and recovery from
malignancies has been suggested by observational studies, by clinical trials showing
reduced cancer incidence with vitamin D supplementation, and by laboratory studies
demonstrating that the physiologically active form of vitamin D, 1;25(OH)2D3 has anti-
cancerous effects. In this respect, it is worrisome that a number of studies demonstrated
sub-optimal 25OHD levels in children and adolescents with a history of malignancy.
Regarding sun habits of this population, there is very limited information in the literature


Correspondence to: [email protected]
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1284 Yael Levy-Shraga and Dalit Modan-Moses

comparing sun protection behaviors of childhood cancer survivors to individuals without

a history of cancer. Three studies in adult survivors of childhood cancer showed
inconsistent results. Only three previous studies investigated this issue in children and
adolescents, showing that patients spent less time outside compared to controls, however
adherence to sun protection recommendations was incomplete. Similar findings were
recently observed by our group. We conclude that more attention should be paid to
improve sun protection habits of survivors of childhood cancer throughout their lives. At
the same time, since sunlight avoidance may result in vitamin D insufficiency and might
have deleterious implications for bone health and possibly on cancer survival, dietary
interventions to optimize intake of vitamin D may be required.

ABBREVIATIONS
25OHD - 25hydroxyvitamin D,
1;25(OH)2D3 - 1,25-dihydroxyvitamin D ,
UVR - ultraviolet radiation,
SCC - squamous cell carcinoma,
BCC- basal cell carcinoma,
MM - malignant melanoma,
MED - minimal erythema dose,
CSSS - Childhood Cancer Survivor Study,
BMI - body mass index.

SUN EXPOSURE AND SKIN CANCER

Although skin cancer is the most preventable type of cancer, it is the most common and
rapidly increasing form of cancer diagnosed in the US today - about 3.5 million basal and
squamous cell skin cancers are diagnosed in the US each year [1]. Sun exposure, the main
source of ultraviolet radiation (UVR) exposure, is the major environmental risk factor for skin
cancers, both melanoma [2] and non-melanoma [3]. Epidemiological and laboratory data have
convincingly shown that sunburns are implicated in the pathogenesis of squamous cell
carcinoma (SCC) [4], basal cell carcinoma (BCC) [5, 6], and malignant melanoma (MM) [7,
8]. Chronic sun exposure is the most important cause for the formation of SCC [9], but may
be less important for the development of BCC [6, 10]. Concerning MM, numerous
epidemiologic investigations analyzing solar UV exposure parameters have consistently
reported an association between the development of MM and short-term intense UV
exposure, particularly a history of severe sunburn during childhood [8, 11]. However chronic,
less intense exposure has not been found to be a risk factor for the development of MM and in
fact has been found in several studies to be protective [7, 12]. A population-based study of
survival from melanoma comprising 528 patients suggested that some factors associated with
high levels of sun exposure, such as solar elastosis and, to a lesser extent, sunburns and
intermittent sun exposure, are inversely associated with death from melanoma [12]. The
apparently beneficial relationship between sun exposure and survival from melanoma could
be mediated by the antiproliferative and proapoptotic effects of vitamin D, the skin synthesis

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 Sun Exposure and Protection Habits and Vitamin D Levels … 1285

of which depends on sun exposure. An alternative hypothesis is that sun exposure induces
less aggressive melanoma [12].

RECOMMENDATIONS OF SUN PROTECTION BEHAVIORS

The impact of UV exposure can be dramatically reduced by practicing sun protection
behaviors. Therefore, the World Health Organization [13] , the American Academy of
Dermatology [14] and the American Academy of Pediatrics [15] have issued guidelines
advocating sun-protection measures, such as wearing sunscreen and protective clothing, hats
and sunglasses, in addition to avoiding tanning and sunbathing.
In order to implement these recommendations, public health campaigns were developed
to improve the knowledge of the general population regarding the role of UV radiation in the
development of skin cancer. The first campaigns were established in Australia in the early
1980s [16]. The World Health Organization started a global UV project called INTERSUN
[17], which aimed to encourage countries to take action to reduce UV-induced health risks.
Sun protection policies were implemented in schools, such as 'SunSmart' [18] and 'living with
sun' [19]. A recent study found that initiation of the Danish Sun Safety campaign in 2007
succeeded in reducing high-risk sun-tanning behavior [20]. Still, recommendations for
sunlight exposure are an area of controversy [21]. It has been noted that vitamin D mediated
positive effects of UV light were not adequately considered in most of these campaigns, that
often propose strict 'no sun policy' without giving recommendations on how to prevent
vitamin D deficiency [22]. Indeed, sun protective behaviors were found associated with lower
vitamin D levels [23].
Thus, some investigators recently suggested modification of current sun guidelines,
recommending "sensible" sun exposure enabling adequate vitamin D synthesis [24, 25]. Still,
it is difficult to establish the balance between excessive and insufficient exposure, and
liberalizing the sun safe message may offer people an excuse to over-expose. Therefore,
current guidelines do not recommend intentional sun exposure to induce vitamin D
production, especially in high risk groups [14, 15].

SOURCES OF VITAMIN D
Sun Exposure

The body's requirements of vitamin D are primarily obtained by skin exposure to UVR of
specific wavelength, 290-315 (UVB) [26]. Synthesis of vitamin D depends on latitude, skin
pigmentation, sunscreen use, and time of day of exposure. Synthesis of vitamin D is minimal
during winter months north of 33° latitude in the northern hemisphere and south of 33°
latitude in the southern hemisphere [27]. Maximal synthesis occurs between the hours of
10:00 AM and 4:00 PM in the spring, summer, and fall [28]. UVR skin exposure is measured
as the minimal erythema dose (MED) or the amount of UVR that will cause minimal
erythema of the skin [26]. The amount of UVR exposure that is equivalent to 1 MED depends
on skin pigmentation, duration of exposure and geographical region. In the southern United
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1286 Yael Levy-Shraga and Dalit Modan-Moses

States, 4 to 10 minutes of exposure are needed for pale skin to achieve 1 MED at noon during
summer [24]. Exposure of 40% of the body to one-fourth MED results in generation of
approximately 1000 IU of vitamin D, the minimum amount of daily vitamin D synthesis
necessary to maintain concentration in the reference range [24]. Vitamin D synthesis is
decreased in individuals who spend many hours indoors or wear sun protection when they go
outside. Special attention should be given to individuals that avoid sun exposure due to
cultural reasons and clothing. For example, serum 25hydroxyvitamin D (25OHD) levels have
been shown to be very low in Israeli Ultra-Orthodox young adults [29]. Sun exposure and sun
protection habits can be evaluated using validated questionnaires [30-34]. Sun exposure
habits can be assessed by asking the respondents to indicate the average number of hours they
spend outside in the summer between 10:00 AM and 4:00 PM, and how many times they had
red or painful sunburns during a year. Sun protection habits can be assessed by measuring on
an ordinal scale protective behaviors, such as using sunscreen, wearing a shirt with sleeves,
wearing sunglasses, staying in the shade and wearing a hat. Skin color may be assessed using
a question based on a 4-point Likert scale ranging from “pale or milky white” to “brown, dark
brown or black.” [30-32]. In addition, some studies estimated ambient UV exposure during
the study period using recorded local UV indices [34].

Food

Dietary vitamin D may be beneficial to overcome lack of sunlight exposure. However,

dietary sources of vitamin D are scarce and include mainly fatty fish (e.g., salmon, sardines,
tuna and mackerel) [26]. Under most living conditions, only 10% of body's requirements can
be obtained by diet [22]. Therefore, the intake of vitamin D depends on food fortification and
supplements. All infant formulas are fortified with vitamin D. In the United States some
brands of cow milk, fruit juice, yogurts, cheeses and breakfast cereals are also fortified. For
those who are unable to achieve adequate amounts of vitamin D in their diet or who have
vitamin D deficiency, vitamin D supplements are available in 2 forms: vitamin D2
(ergocalciferol), derived from plants, and vitamin D3 (cholecalciferol), synthesized by
mammals.

Daily Vitamin D Requirements

In 2011, the Institute of Medicine revised the recommended dietary allowances (RDA)
for vitamin D intake to be higher than previous recommendations and the American Academy
of Pediatrics endorsed these recommendations [27]. Adequate vitamin D intake for infants
younger than 1 year is 400 IU/day, for persons between ages 1 year to 70 the requirement is
600 IU/day and for those aged 70 and above 800 IU/day.

Recommendations for 25OHD Levels

Vitamin D status is most reliably determined by measuring serum 25OHD [35], however
there is a debate regarding optimal cutoff values. The Lawson Wilkins Pediatric Endocrine

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 Sun Exposure and Protection Habits and Vitamin D Levels … 1287

Society [26] and the Institute of Medicine [36] set the cut-point for vitamin D deficiency at
circulating concentrations of 25OHD at 20 ng/ml, based on the stipulation that this minimum
level is necessary to support and maintain all the classical actions of vitamin D on bone and
mineral health. However, a level of 30 ng/ml has been suggested as the lower limit of normal
for 25OHD levels by the Endocrine Society guidelines [28], and many experts advocate such
levels to confer the beneficial nonskeletal health effects of vitamin D [37].

Prevalence of Vitamin D Deficiency

National US data revealed increasing prevalence of vitamin D deficiency from the 1988–
1994 survey to 2001–2004 [38]. Cross sectional studies of vitamin D status in adolescents
have found deficiency in 17% to 47% of adolescents [39-42], with lower vitamin D
concentrations in the winter [41-43]. Children and adolescents who are obese are at increased
risk [40, 44], possibly because of sequestration of vitamin D in body fat. Certain medications,
such as anticonvulsants, glucocorticoids, antifungals, and antiretroviral medications, increase
requirements and predispose subjects to deficiency. In studies in adults, at the end of the
winter, 42% of 15- to 49-year-old black girls and women throughout the United States had
25OHD levels below 20 ng/ml [45], and 32% of healthy students, physicians and residents at
a Boston hospital were found to be vitamin D deficient [46].

VITAMIN D ROLES
Skeletal

The most widely established roles of vitamin D are related to serum calcium and
phosphorus homeostasis and bone mineral accretion/mobilization. Without vitamin D, only
10 to 15% of dietary calcium and about 60% of phosphorus is absorbed [47].
The interaction of 1,25-dihydroxyvitamin D [1;25(OH)2D3] with the vitamin D receptor
increases the efficiency of intestinal calcium absorption to 30 to 40% and phosphorus
absorption to approximately 80% [47]. During childhood and adolescence, adequate vitamin
D levels are required due to its important role in cell growth and skeletal structure and
development [48]. Severe vitamin D deficiency is associated with reduced bone mass in
adolescents [49, 50]. On the basis of results of a longitudinal prospective study of 6712
physically active girls aged 9 through 15 years, vitamin D intake during childhood is
associated with reduced risk of stress fractures [51].

Extra-Skeletal

The discovery that most tissues and cells in the body have a vitamin D receptor, and
many possess the enzymatic machinery to convert the primary circulating form of vitamin D,
25OHD, to the active form, 1;25(OH)2D3 , has provided new insights into the function of this
vitamin [47]. A growing body of evidence suggests that vitamin D deficiency is related to
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1288 Yael Levy-Shraga and Dalit Modan-Moses

disease onset or severity of many chronic illnesses [47, 52] as well as to all-cause mortality
[53, 54]. Maintenance of adequate vitamin D levels is required for regulation of several
physiological functions, including regulation of immune function and regulation of cellular
proliferation and differentiation [55].
Low levels of 25OHD are associated with increased risk of type 1 diabetes, multiple
sclerosis and Crohn's disease [56-58]. Better intake of vitamin D was found to be associated
with reduced risk of developing rheumatoid arthritis [59], osteoarthritis [60], type 2 diabetes
[61], and cardiovascular disease [62]. In a recent study, avoidance of sun exposure was
assessed in a cohort of 29,518 Swedish women [63]. The mortality rate amongst avoiders of
sun exposure was approximately twofold higher compared with the highest sun exposure
group. The authors suggested that following sun exposure advice that is very restrictive in
countries with low solar intensity might be in fact harmful. In keeping with these findings, a
recent British study prospectively evaluating 14,641 men and women aged 42-82 for 12-15
years demonstrated an association between baseline 25OHD levels and all-cause mortality,
with a hazard ratio of 0.66 for baseline 25OHD ≥ 36ng/ml (90 nmol/L) [64].

Sun Exposure and Non-Skin Cancer

Numerous studies linked decreased sunlight exposure to non-skin cancer incidence or

survival. This association has been reported as early as 1916, and repeatedly since (reviewed
by Wacker & Holick) [25]. Existing evidence is derived from ecologic studies showing lower
cancer rates in geographical areas with lower daily solar radiation penetrating the atmosphere
(such as high latitude) [65], studies using estimated ground-level UVR exposure from the
Total Ozone Mapping Spectrometer (TOMS) dataset of the National Aeronautics and Space
Administration (NASA) [66], and studies assessing individual history of sun exposure using
questionnaires [67]. Specifically, the protective effect of sun exposure has been shown for
colorectal [65] and breast cancers [67, 68], several sub-types of non-Hodgkin’s lymphoma
(NHL) [69-72], and squamous cell lung, pleural, prostate, kidney, and bladder cancers [66].
The beneficial effects of sunlight against cancer are mainly attributed to its contribution
to vitamin D synthesis, although other mechanisms have been also suggested [73].

VITAMIN D AND CANCER RISK, MORBIDITY

AND MORTALITY

Several lines of evidence suggest that vitamin D has a role in decreasing the risk for
cancer. Many studies demonstrated an inverse association between vitamin D and its
metabolites and cancer morbidity and mortality, particularly colorectal, breast, and prostate
cancer [37, 74, 75]. Furthermore, high 25OHD and high vitamin D intake at the time of
diagnosis and initiation of anti-cancer treatment were associated with improved overall and
recurrence-free survival [76-80] , and a recent meta-analysis demonstrated improved overall
survival as well as cancer-specific survival for breast cancer, colorectal cancer and lymphoma
patients with higher vs. lower (highest quartile vs. lowest quartile) 25OHD levels. For lung
cancer patients results were inconclusive [81]. Only a single study was undertaken in

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pediatric patients, assessing 123 children undergoing hematopoietic stem cell transplantation
(HSCT), and showing a more rapid increase in neutrophil granulocytes and significantly
better overall survival in vitamin D sufficient patients. Furthermore, rejection and relapse
rates were lower in patients with sufficient vitamin D levels [82]. These epidemiological and
observational findings are supported by clinical trials showing reduced cancer incidence [83]
and improved survival [84] with vitamin D supplementation.
The theory that vitamin D can help prevent cancer is biologically plausible, as the
vitamin D receptor is expressed in most tissues [47]. Studies in cell culture and experimental
models suggest that 1;25(OH)2D3 (calcitriol), the active metabolite of vitamin D, promotes
cell differentiation, inhibits cancer-cell proliferation and exhibits anti-inflammatory,
proapoptotic, and antiangiogenic properties [37, 74, 85] – processes that may regulate cancer
development and progression.

VITAMIN D LEVELS IN PEDIATRIC PATIENTS

WITH MALIGNANCY

A number of studies assessed vitamin D status in pediatric patients with malignancy.

Older studies included relatively small numbers of patients, and measured vitamin D levels
mostly in the context of evaluating skeletal health, while other aspects of vitamin D
insufficiency were not considered. Mean 25OHD levels in all studies were in the
insufficiency range [86-93].
With the emerging wealth of data regarding the association between vitamin D and
cancer incidence, morbidity, and mortality, several recent studies investigated this issue more
comprehensively [94-97]. In a cohort of 78 pediatric ALL survivors, 53% of participants were
25OHD insufficient (15-29 ng/dl), and 12% were deficient (<15 ng/dl). Only 27% of
conventional chemotherapy-treated ALL survivors and 8% of HSCT-treated ALL survivors
met RDA for dietary vitamin D intake [94]. Choudhary et al. measured 25OHD levels in 481
survivors of childhood cancer and found that 29% of the participants were 25OHD
insufficient (<20 ng/ml) [96]. Other studies found suboptimal 25OHD levels in 35-95% of
patients [34, 95, 97, 98]. The differences in rates of 25OHD deficiency may be explained by
the chosen definition of optimal levels (20 ng/ml vs. 30 ng/ml), differences in patients
characteristics (patients' ages, recently diagnosed vs. long-term survivors, specific diagnosis
vs. mixed population, etc.), and differences in the reported rates of vitamin D supplements
usage.
Risk factors for vitamin D insufficiency in these studies included older age, non-
Caucasian race, and decreased ambient ultraviolet light exposure [34, 94, 96]. Higher reported
dietary vitamin D intake and use of vitamin D supplementation were associated with higher
serum 25OHD in one study [94], but not in another [34]. Treatment with HSCT was
associated with decreased 25OHD levels in one study [34]. No association was found
between 25OHD levels and gender, body mass index (BMI) [34, 96], cancer diagnoses,
presence of an endocrinopathy, exposure to radiation, or glucocorticoid medications [96].
Our group assessed vitamin D status in a cohort of 211 pediatric patients with a history of
malignancy (aged 12.1±5.8y, Male=69, mean time from diagnosis 4.4±3.8y), and related
25OHD levels to lifestyle habits and to the medical history [107]. Mean 25OHD levels in this
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1290 Yael Levy-Shraga and Dalit Modan-Moses

cohort were 20.6±7.9ng/ml. Vitamin D deficiency (<15ng/ml) was found in 24.6% of the
patients, and insufficiency (15-20ng/ml) in 23.2%. Younger age and amount of sun exposure
were associated with higher serum 25OHD. No association was found with calcium intake,
disease type, gender, BMI-standard deviation score, years since diagnosis, or undergoing
HSCT. 25OHD levels during winter were significantly lower than summer levels. We
considered these findings concerning, given the above discussed possible contribution of
25OHD levels to improved overall and recurrence-free survival. Furthermore, survivors of
childhood cancer are at risk of secondary neoplasms [99], reduced bone mineral density [88,
99], cardiovascular disease and the metabolic syndrome [100] all of which have been
associated with vitamin D deficiency/insufficiency.

CHILDHOOD CANCER SURVIVORS AND SUN EXPOSURE

Survivors of childhood and adolescent cancer are at high risk for developing non-
melanoma skin cancer. In a report from the Childhood Cancer Survivor Study (CCSS), skin
cancer was the most frequently occurring subsequent cancer in the cohort, with non-
melanoma skin cancer accounting for 41% of all confirmed subsequent cancers and
melanoma for 3%. The incidence rate of BCC increased with age, reaching 3785.9/100,000
person-years for survivors who were 45-54 years of age. Radiation therapy, either alone or in
combination with chemotherapy, was associated with an increased risk of BCC compared
with no chemotherapy or radiation, with an odds ratio of 39.8 for subjects who received 35
Gy or more to the skin site vs no radiation. Results were consistent with a linear dose-
response relationship, with an excess odds ratio of 1.09 per Gy. Moreover, 91% of the non-
melanoma skin cancer occurred within the previous radiation fields [101, 102]. These
alarming rates indicate that childhood cancer survivors with a history of radiation treatment
are especially susceptible to skin cancer and may subsequently increase their risk through sun
exposure.
Despite the importance of sun protection in survivors of childhood cancer, there is very
limited information in the literature comparing sun protection behaviors of survivors to
individuals without a history of cancer. It is also unclear if childhood cancer survivors with a
history of radiation treatment utilize sun protection behaviors at equivalent or different rates
than survivors without a radiation history.
Several studies assessed sun-behaviors in adult survivors of childhood cancer. In a study
comprising 835 survivors from the Swiss Childhood Cancer Survivor Study (SCCSS) fewer
survivors than controls reported protecting themselves from sun exposure (78% vs
87%)[103]. In contrast, in a study comprising adult survivors from the USA CCSS (9298
survivors and 2950 sibling controls), survivors and siblings showed similar patterns of
sunscreen use (67% vs 66%), and survivors were significantly less likely to report having
sunbathed in the previous year or use artificial tanning. Survivors with radiation exposure
showed increased use of sunscreen and less sunbathing or artificial tanning compared to those
who were not treated with radiation, suggesting increased awareness of the need of sun
protection in this population. Significant factors for regular sunscreen use in the survivor
population were being female, having lighter skin complexions, having previously been
examined for skin cancer, and having skin that burned when in the sun unprotected [32].

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Finally, a British study assessed sun behavior as a component of a Health Behavior Index in
178 young adult survivors of childhood cancer, finding that survivors lead a healthier lifestyle
compared to controls. However, sun behavior was assessed using a single question - how
many times they were sunburnt in the 12 months preceding the study, and details regarding
sun behavior were not presented [104].
We identified only three studies investigating sun habits in children and adolescents with
a history of malignancy, all suggesting low compliance with sun behavior recommendations.
In a study comprising 75 adolescent survivors of childhood cancer, 37% were non-adherent to
sun protection recommendations. No control group was included [105]. In a study comprising
95 children and adults (44 children/adolescents, 12 young adults and 39 adults) who had
undergone HSCT, mean self-reported hours of sun exposure was 1.1 hours/day. The majority
of participants reported "rarely" or "never" using sunscreen, although a greater percentage of
young adults (50%) reported "always" using sunscreen compared to the percentages of
children/adolescents (16%) or adults (21%). There were no significant differences in average
daily sun exposure by sunscreen use category. Again, no control group was included [98].
Simmons et. al. assessed sun habits in 22 pediatric patients undergoing allogeneic HSCT.
Frequency of sunscreen use was low for both patients and controls, with only 14% of the
patients reporting "always" using sunscreen, and 50% of the patients reporting "never" using
sunscreen. Time spent outside and skin exposure sores were significantly lower for patients
compared to controls [34].
In a recent study we assessed sun habits in 143 children with a history of malignancy and
150 healthy controls (aged 2-21 years) using validated questionnaires. Patients and controls
reported similar sun exposure time during weekdays (94±82minutes/day vs.
81±65minutes/day; p=0.83), however during weekends patients spent significantly less time
outside compared to controls (103±85minutes/day vs. 124±87minutes/day; p=0.015). Time
elapsed from diagnosis positively correlated with time spent outside both during weekdays
(r=0.194, p=0.02) and weekends (r=0.217, p=0.009). Regarding sun protection habits,
patients were more likely than controls to wear a hat when in the sun (34.5% vs. 20.7%
reporting "always" or "frequently"; p=0.009). However, there was no difference between the
two groups regarding the frequency of using sunscreen, wearing a shirt covering the
shoulders, staying in the shade or wearing sunglasses. Age and time elapsed from diagnosis
were identified as risk factors for poor adherence with sun recommendations [106].
Although this study contributes to the understanding of sun protection behaviors in
survivors of childhood cancer, further research is needed to determine factors impacting the
decision to engage in sun protection behaviors, such as perception of benefits and barriers to
utilization, and influence of the past medical illness. These factors have not been adequately
explored in the survivors population and understanding them is imperative and may assist
future interventions in this area.

CONCLUSION -
BALANCING RISKS AND BENEFITS OF SUN EXPOSURE
Survivors of childhood cancer are at a high risk for vitamin D deficiency and
insufficiency. This finding is worrisome, as we consider these patients to be particularly
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1292 Yael Levy-Shraga and Dalit Modan-Moses

vulnerable to the adverse effects of vitamin D deficiency in relation to bone health as well as
possible effects of in the context of malignancy. Monitoring vitamin D levels in this
population is therefore of high importance, and since it is difficult to obtain enough vitamin D
from dietary sources, vitamin D deficiency should be treated by a supplement to achieve
adequate levels.
At the same time, it is important to recognize that beneficial effects of UVR exposure
may not occur solely through UVR-induced vitamin D synthesis. Thus, maintaining current
sun avoidance policies while supplementing with vitamin D may not be sufficient to avoid the
risks of insufficient exposure to UVR [25].
Sun recommendations may need to be individualized according to season, geographical
location, skin type and the medical history. In a healthy population, "sensible" sun exposure,
sufficient for synthesizing the minimum necessary daily amount of vitamin D, has been
suggested by some investigators [24]. Still, sun exposure around midday should be avoided
during the summer in most latitudes. The face should always be protected with a hat or
sunscreen since it is most sun exposed and more prone to skin damage and skin cancer from
sun exposure, while providing very little vitamin D synthesis [25]. Children and adolescents
with history of irradiation are a particular risk group for skin cancer and may need more strict
recommendations. Since our own study, as well as other studies in pediatric cancer survivors
suggest incomplete adherence with sun recommendations, more attention should be paid to
improve sun protection habits of these patients throughout their lives.
More research is warranted to define optimal sun exposure and vitamin D levels as well
as strategies to increase compliance with sun recommendations.

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Relation of dietary intake and serum levels of vitamin D to progression of osteoarthritis
of the knee among participants in the Framingham Study. Annals of internal medicine.
1996;125(5):353-9.
[61] Pittas AG ,Dawson-Hughes B, Li T, Van Dam RM, Willett WC, Manson JE, et al.
Vitamin D and calcium intake in relation to type 2 diabetes in women. Diabetes care.
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[62] Zittermann A. Vitamin D and disease prevention with special reference to
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[64] Khaw KT, Luben R, Wareham N. Serum 25-hydroxyvitamin D, mortality, and incident
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[65] Garland CF, Garland FC. Do sunlight and vitamin D reduce the likelihood of colon
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[67] Knight JA, Lesosky M, Barnett H, Raboud JM, Vieth R. Vitamin D and reduced risk of
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[70] Slager SL, Benavente Y, Blair A, Vermeulen R, Cerhan JR, Costantini AS, et al.
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[74] Deeb KK, Trump DL, Johnson CS. Vitamin D signalling pathways in cancer: potential
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[75] Boscoe FP, Schymura MJ. Solar ultraviolet-B exposure and cancer incidence and
mortality in the United States, 1993-2002. BMC cancer. 2006;6:264.
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[77] Drake MT, Maurer MJ, Link BK, Habermann TM, Ansell SM, Micallef IN, et al.
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[79] Zgaga L, Theodoratou E, Farrington SM, Din FV, Ooi LY, Glodzik D, et al. Plasma
vitamin D concentration influences survival outcome after a diagnosis of colorectal
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Clinical Oncology. 2014;32(23):2430-9.
[80] Shanafelt TD, Drake MT, Maurer MJ, Allmer C, Rabe KG, Slager SL, et al. Vitamin D
insufficiency and prognosis in chronic lymphocytic leukemia. Blood.
2011;117(5):1492-8.
[81] Li M, Chen P, Li J, Chu R, Xie D, Wang H. Review: The Impacts of Circulating 25-
Hydroxyvitamin D Levels on Cancer Patient Outcomes: A Systematic Review and
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[82] Hansson ME, Norlin AC, Omazic B, Wikstrom AC, Bergman P, Winiarski J,
et al. Vitamin D levels affect outcome in pediatric hematopoietic stem cell
transplantation. Biology of blood and marrow transplantation: journal of the American
Society for Blood and Marrow Transplantation. 2014;20.43-1537:)10(
[83] Lappe JM, Travers-Gustafson D, Davies KM, Recker RR, Heaney RP. Vitamin D and
calcium supplementation reduces cancer risk: results of a randomized trial. The
American journal of clinical nutrition. 2007;85(6):1586-91.
[84] Zeichner SB, Koru-Sengul T, Shah N, Liu Q, Markward NJ, Montero AJ, et al.
Improved Clinical Outcomes Associated With Vitamin D Supplementation During
Adjuvant Chemotherapy in Patients With HER2 Nonmetastatic Breast Cancer. Clinical
breast cancer. 2014.
[85] van Ginkel PR ,Yang W, Marcet MM, Chow CC, Kulkarni AD, Darjatmoko S, et al. 1
alpha-Hydroxyvitamin D2 inhibits growth of human neuroblastoma. Journal of neuro-
oncology. 2007;85(3):255-62.
[86] Halton JM, Atkinson SA, Fraher L, Webber CE, Cockshott WP, Tam C, et al. Mineral
homeostasis and bone mass at diagnosis in children with acute lymphoblastic leukemia.
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[87] Halton JM, Atkinson SA, Fraher L, Webber C, Gill GJ, Dawson S, et al. Altered
mineral metabolism and bone mass in children during treatment for acute lymphoblastic
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Society for Bone and Mineral Research. 1996;11(11):1774-83.
[88] Arikoski P, Kroger H, Riikonen P, Parviainen M, Voutilainen R, Komulainen J.
Disturbance in bone turnover in children with a malignancy at completion of
chemotherapy. Medical and pediatric oncology. 1999;33(5):455-61.
[89] Alikasifoglu A, Yetgin S, Cetin M, Tuncer M, Gumruk F, Gurgey A, et al. Bone
mineral density and serum bone turnover markers in survivors of childhood acute
lymphoblastic leukemia: comparison of megadose methylprednisolone and
conventional-dose prednisolone treatments. American journal of hematology.
2005;80(2):113-8.
[90] Othman F, Guo CY ,Webber C, Atkinson SA, Barr RD. Osteopenia in survivors of
Wilms tumor. International journal of oncology. 2002;20(4):827-33.
[91] Marinovic D, Dorgeret S, Lescoeur B, Alberti C, Noel M, Czernichow P, et al.
Improvement in bone mineral density and body composition in survivors of childhood
acute lymphoblastic leukemia: a 1-year prospective study. Pediatrics.
2005;116(1):e102-8.
[92] El-Ziny MA, Al-Tonbary YA, Salama OS, Bakr A, Al-Marsafawy H, Elsharkawy AA.
Low bone mass in children with malignant lymphoma. Pediatric hematology and
oncology. 2007;24(8):577-85.
[93] Gunes AM, Can E, Saglam H, Ilcol YO, Baytan B. Assessment of bone mineral density
and risk factors in children completing treatment for acute lymphoblastic leukemia.
Journal of pediatric hematology/oncology. 2010;32(3):e102-7.
[94] Simmons JH, Chow EJ, Koehler E, Esbenshade A, Smith LA, Sanders J, et al.
Significant 25-hydroxyvitamin D deficiency in child and adolescent survivors of acute
lymphoblastic leukemia: treatment with chemotherapy compared with allogeneic stem
cell transplant. Pediatric blood & cancer. 2011;56(7):1114-9.

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[95] Sinha A, Avery P, Turner S, Bailey S, Cheetham T. Vitamin D status in paediatric

patients with cancer. Pediatric blood & cancer. 2011;57(4):594-8.
[96] Choudhary A, Chou J, Heller G, Sklar C. Prevalence of vitamin D insufficiency in
survivors of childhood cancer. Pediatric blood & cancer. 2013;60(7):1237-9.
[97] Esbenshade AJ, Sopfe J, Zhao Z, Li Z, Campbell K, Simmons JH, et al. Screening for
vitamin D insufficiency in pediatric cancer survivors. Pediatric blood & cancer.
2014;61(4):723-8.
[98] Robien K, Strayer LG, Majhail N, Lazovich D, Baker KS, Smith AR, et al. Vitamin D
status among long-term survivors of hematopoietic cell transplantation. Bone marrow
transplantation. 2011;46(11):1472-9.
[99] Tylavsky FA, Smith K, Surprise H, Garland S, Yan X, McCammon E, et al. Nutritional
intake of long-term survivors of childhood acute lymphoblastic leukemia: evidence for
bone health interventional opportunities. Pediatric blood & cancer. 2010;55(7):1362-9.
[100] van Waas M, Neggers SJ, Pieters R, van den Heuvel-Eibrink MM. Components of the
metabolic syndrome in 500 adult long-term survivors of childhood cancer. Annals of
oncology : official journal of the European Society for Medical Oncology / ESMO.
2010;21(5):1121-6.
[101] Perkins JL, Liu Y, Mitby PA, Neglia JP, Hammond S, Stovall M, et al. Nonmelanoma
skin cancer in survivors of childhood and adolescent cancer: a report from the
childhood cancer survivor study. Journal of clinical oncology : official journal of the
American Society of Clinical Oncology. 2005;23(16):3733-41.
[102] Watt TC, Inskip PD, Stratton K, Smith SA, Kry SF, Sigurdson AJ, et al. Radiation-
related risk of basal cell carcinoma: a report from the Childhood Cancer Survivor
Study. Journal of the National Cancer Institute. 2012;104(16):1240-50.
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Clustering of health behaviours in adult survivors of childhood cancer and the general
population. British journal of cancer. 2012;107(2):234-42.
[104] Larcombe I, Mott M, Hunt L. Lifestyle behaviours of young adult survivors of
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[105] Tercyak KP, Donze JR, Prahlad S, Mosher RB ,Shad AT. Multiple behavioral risk
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30.
[106] Levy-Shraga Y, Pinhas-Hamiel O, Ben-Ami M, Yeshayahu Y, Temam V, Cohen R,
Modan-Moses D. Sun Protection Habits and Calcium Intake in Children with
Malignancy. Horm. Res. Paediatr 2014;82(Suppl. 1):81.
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Vitamin D status in pediatric patients with a history of malignancy. Pediatr Res. 2012
Dec;72(6):620-4.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 61

THE SURGEON GENERAL’S CALL TO ACTION TO

PREVENT SKIN CANCER: FACTS FOR CONSUMERS *

Surgeon General of the United States

Skin cancer is the most common type of cancer the United States. This disease can
greatly reduce quality of life, and it can be disfiguring and even deadly. Medical treatment for
skin cancer is costly for individuals, families, and the nation. The good news is that most
cases of skin cancer can be prevented.

The Surgeon General’s Call to Action to Prevent Skin Cancer calls on partners in
prevention from various sectors across the nation to address skin cancer as a major public
health problem. Government, business, health, education, community, nonprofit, and faith-
based sectors are all essential partners in this effort.
In this Call to Action, the Surgeon General sets forth five main goals that will serve as a
road map for all Americans in their efforts to reverse the rising tide of skin cancer:

 Increase opportunities for sun protection in outdoor settings.

 Provide individuals with the information they need to make informed, healthy
choices about their exposure to ultraviolet (UV) rays.
 Promote policies that advance the national goal of preventing skin cancer.
 Reduce harms from indoor tanning.
 Strengthen research, surveillance, monitoring, and evaluation related to skin cancer
prevention.

With sustained support and a unified approach, we can achieve major reductions in skin
cancer-related illness, deaths, and healthcare costs.
The Surgeon General uses the best scientific information available to promote health,
reduce risk for illness and injury, and to make the nation healthier.

*
This is an edited, reformatted and augmented version of material originally entitled “The Surgeon General’s Call
to Action to Prevent Skin Cancer: Consumer Bulletin” viewed on the following website August 2014:
http://www.surgeongeneral.gov/library/calls/prevent-skin-cancer/consumer-booklet.html.
 Free ebooks ==> www.Ebook777.com
1302 Surgeon General of the United States

More than 1/3 of U.S. adults have been sunburned in the past year alone. Sunburn is a
clear sign of overexposure to UV rays, a major cause of skin cancer.

SKIN CANCER IS COMMON AND COSTLY

Each year in the United States, nearly 5 million people are treated for skin cancer at a
cost that exceeds $8.1 billion.
Melanoma, the deadliest form of skin cancer, is responsible for nearly 9,000 deaths each
year. It is also one of the most common types of cancer among U.S. adolescents and young
adults.

UV (ULTRAVIOLET) EXPOSURE IS A MAJOR CAUSE OF SKIN

CANCER—AND THE MOST PREVENTABLE
The Surgeon General’s Call to Action to Prevent Skin Cancer focuses on UV radiation
because it is the most preventable cause of skin cancer. Genetic factors, such as being fair-
skinned or having a family history of skin cancer, increase a person’s risk. But the most
common types of skin cancer are also strongly associated with exposure to UV radiation. As
many as 90% of melanomas are caused by UV exposure.
UV exposure is the most preventable cause of skin cancer because—unlike genetic
factors—skin cancer risk from UV exposure can be reduced. Take steps to avoid excessive or
unnecessary UV exposures, such as long sun exposure without enough sun protection.
Tanned skin is the body’s response to injury from UV rays. Avoid tanning on purpose, either
with indoor tanning devices or in the sun.
Skin cancer rates are on the rise. Although rates of many of the most common types of
cancers are decreasing in the U.S., rates of skin cancer are increasing.
Skin cancer is more common than all other types of cancer. The number of Americans
who have had skin cancer in the past three decades is estimated to be higher than the number
for all other cancers combined.
For most people in the United States, the sun is the most common source of exposure to
UV rays. UV radiation from indoor tanning devices is a less common but easier-to-avoid
source of exposure than from the sun.

INDOOR TANNING INCREASES THE RISK IF SKIN CANCER,

INCLUDING MELANOMA
Indoor tanning devices, such as tanning beds, tanning booths, and sun lamps, expose
users to intense UV radiation as a way to tan the skin for cosmetic reasons.
Indoor tanning has been linked with skin cancers, including melanoma (the deadliest type
of skin cancer), basal cell carcinoma, and squamous cell carcinoma.
Starting indoor tanning at younger ages appears to be more strongly related to lifetime
skin cancer risk, possibly because of the accumulation of UV exposure over time from more

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1303

years of tanning. Indoor tanning can also can cause other health problems. An estimated
3,000 Americans each year go to emergency rooms with injuries caused by indoor tanning,
including burns to the skin and eye damage.
Every time you tan you increase your risk of getting melanoma. Indoor tanning can also:

 Cause premature skin aging, like wrinkles and age spots.

 Damage your skin texture.
 Increase the risk of potentially blinding eye diseases.

Myths about Tanning

 MYTH: A “base tan” will protect me from a sunburn.

FACT: A “base tan” is not a safe tan. A tan means you have damaged your skin. The
best way to protect your skin from UV rays is by using effective sun protection, such
as staying in the shade, wearing hats and other protective clothing, using broad-
spectrum sunscreen with SPF 15+, and avoiding indoor tanning.
 MYTH: Tanning indoors is safer than tanning in the sun.
FACT: Tanning indoors is not safer than tanning in the sun. Indoor tanning and
tanning outside are both dangerous. You can get a burn from tanning indoors.
Tanned skin is damaged skin.
 MYTH: Tanning is a safe way to get vitamin D, which prevents many health
problems.
FACT: Tanning is not a safe way to get vitamin D. Although it is important to get
enough vitamin D, the safest way is through what you eat. Tanning harms your skin.

Keep your skin healthy. Avoid sunbathing and indoor tanning.

Everyone Can Play a Part in Preventing Skin Cancer

What can policymakers do?

 Incorporate sun-safety education and sun protection into school policies at the district
or state level.
 Enforce existing indoor tanning laws.
 Support shade planning in land use development.

What can businesses and employers do?

 Increase availability of sun protection for outdoor workers.

 Modify work environments and schedules, when feasible, to protect workers from
overexposure to UV radiation.
 Incorporate sun safety into workplace policies, safety trainings, and wellness
programs.
 Free ebooks ==> www.Ebook777.com
1304 Surgeon General of the United States

What can health care systems, insurers, and clinicians do?

 Counsel patients on using sun protection and avoiding intentional tanning in

accordance with U.S. Preventive Services Task Force recommendations.
 Increase awareness of and adherence to melanoma reporting requirements among
providers, especially those in private practice.
 Remain alert to suspicious skin lesions when examining patients.

What can early learning centers, schools, colleges, and universities do?

 Identify opportunities to increase shade through relocating activities or providing

shade structures in key locations.
 Eliminate barriers to individual sun protection (such as policies that prohibit the use
of hats or sunscreen).
 Support sun protection in outdoor athletic settings.
 Discourage indoor tanning by students and reconsider campus practices that may
encourage indoor tanning.

What can community, non-profit, and faith-based organizations do?

 Support effective shade planning in the community.

 Encourage vendors in outdoor recreation areas to sell sun protection products.
 Work collaboratively to support skin cancer prevention in the community.

What can individuals and families do?

While leading healthy, active lives and enjoying the outdoors, choose sun protection
strategies that work:

 Wear a hat, sunglasses, and other protective clothing, and seek shade, especially
during midday hours.
 Use broad-spectrum sunscreen with SPF 15+ to protect any exposed skin; remember
that sunscreen is most effective when used in combination with other methods.

For more information: www.cdc.gov/cancer/skin

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 62

THE SURGEON GENERAL’S CALL TO ACTION TO

PREVENT SKIN CANCER *

Meg Watson, Erin Garnett, Gery P. Guy

and Dawn M. Holman

SKIN CANCER AS A MAJOR PUBLIC HEALTH PROBLEM

Why We Must Act Now

Skin cancer is the most commonly diagnosed cancer in the United States, and most cases
are preventable [1-3].
Skin cancer greatly affects quality of life, and it can be disfiguring or even deadly [1,4-6].
Medical treatment for skin cancer creates substantial health care costs for individuals,
families, and the nation. The number of Americans who have had skin cancer at some point in
the last three decades is estimated to be higher than the number for all other cancers combined
[1, 7, 8], and skin cancer incidence rates have continued to increase in recent years [1, 9].
Each year in the United States, nearly 5 million people are treated for all skin cancers
combined, with an annual cost estimated at $8.1 billion [10]. Melanoma is responsible for the
most deaths of all skin cancers, with nearly 9,000 people dying from it each year [11]. It is
also one of the most common types of cancer among U.S. adolescents and young adults [12].
Annually, about $3.3 billion of skin cancer treatment costs are attributable to melanoma [10].
Despite efforts to address skin cancer risk factors, such as inadequate sun protection and
intentional tanning behaviors, skin cancer rates, including rates of melanoma, have continued
to increase in the United States and worldwide [1, 13-17]. With adequate support and a
unified approach, comprehensive, communitywide efforts to prevent skin cancer can work.
Although such success will require a sustained commitment and coordination across diverse
partners and sectors, significant reductions in illness, deaths, and health care costs related to
skin cancer can be achieved.

*
This is an edited, reformatted and augmented version of a document issued by the U.S. Department of Health and
Human Services, Office of the Surgeon General, July 2014.
 Free ebooks ==> www.Ebook777.com
1306 Meg Watson, Erin Garnett, Gery P. Guy et al.

This document is a Call to Action to partners in prevention from various sectors across
the nation to address skin cancer as a major public health problem. Many partners are
essential to this effort, including federal, state, tribal, local, and territorial governments;
members of the business, health care, and education sectors; community, nonprofit, and faith-
based organizations; and individuals and families. The goal of this document is to increase
awareness of skin cancer and to call for actions to reduce its risk.
The first section describes the problem of skin cancer and its major risk factors. It also
discusses the relationship between exposure to ultraviolet (UV) radiation and health. The
second section describes the current evidence on preventing skin cancer, including current
initiatives in the United States and in other countries. The third section describes the gaps in
research related to skin cancer prevention, highlighting areas of research where more work is
needed. The fourth section identifies specific opportunities to prevent skin cancer by reducing
UV exposure in the U.S. population and calls for nationwide action.
This document also includes six appendices, which provide further detail about specific
topics. For more information about the scope of this document and definitions of commonly
used terms, see Appendix 1. Appendix 2 describes symptoms of skin cancer. Appendix 3
provides a brief discussion of skin cancer screening. Success stories in skin cancer prevention
are discussed in Appendix 4, and current federal efforts on skin cancer prevention are
summarized in Appendix 5. Abbreviations and acronyms are listed in Appendix 6.

Why a Focus on UV Radiation?

Although genetic factors, such as being fair-skinned or having a family history of skin
cancer, contribute to a person’s risk [18-24], the most common types of skin cancer (see
Appendix 1) are also strongly associated with exposure to UV radiation [3, 25-30]. UV
exposure is also the most preventable cause of skin cancer. This Call to Action focuses on
reducing UV exposure, with an emphasis on addressing excessive, avoidable, or unnecessary
UV exposures (such as prolonged sun exposure without adequate sun protection) and
intentional exposure for the purpose of skin tanning (whether indoors using an artificial UV
device or outdoors while sunbathing).
This document focuses on primary prevention of skin cancer through reducing
overexposure to UV, not on early detection or screening. The evidence on skin cancer
screening is growing, and ongoing examinations of the evidence are important. Melanomas
diagnosed at earlier stages are much more treatable than those diagnosed at later stages [6,
31]. It is important for the public to understand that anyone can get skin cancer and to know
the signs, which can be found in Appendix 2 and at http://www.cdc.gov/cancer/ skin/basic_
info/symptoms.htm. Information on screening is available in Appendix 3.
Factors other than UV exposure can increase the risk of skin cancer in certain
populations. Certain uncommon genetic mutations, such as those linked to familial melanoma
and xeroderma pigmentosum, can strongly increase a person’s risk of melanoma [32].
Occupational exposures to ionizing radiation, high doses of UV radiation, or exposure to
certain chemicals during manufacturing processes may increase skin cancer risk beyond that
of the general public [32]. However, this document focuses on reducing the risk of skin
cancer in the general U.S. population.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1307

Sources of UV Radiation Addressed in This Document

UV radiation is a type of electromagnetic radiation emitted by the sun and from some
man-made lights, with wavelengths longer than X-rays but shorter than visible light [33, 34].
For most people in the United States, the sun is the most common source of exposure to UV
radiation. UV radiation from indoor tanning devices is a less common but more easily
avoidable source of UV radiation exposure than from the sun. More information about UV
radiation, including the different types, is provided in the “Exposure to UV Radiation” section
(see page 11). This Call to Action discusses important steps that can be taken to reduce
exposure to the most common sources of UV radiation at the population level.

UV Exposure and Overexposure

UV exposure stimulates melanocytes to produce melanin, often resulting in a tan or
sunburn, both of which indicate overexposure1and damage to the skin, skin cells, and DNA
within those skin cells [35, 36]. The underlying biology of skin cancer risk is directly related
to damage to the skin and its genetic material [37]. Although all UV exposures can affect skin
cancer risk, entirely avoiding UV rays from the sun is neither realistic nor advisable for most
Americans. Spending time outdoors is associated with positive health benefits, such as
increased levels of physical activity and improved mental health [38-40].

Skin Cancer Incidence and Mortality

This document focuses on the three most common types of skin cancers: basal cell
carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma, which together account
for more than 99% of skin cancers (Figure 1) [41, 42]. These three types of cancer are
described in greater detail in Appendix 1. BCC and SCC are the most common types of
nonmelanoma skin cancers (NMSCs).

Nonmelanoma Skin Cancers

In the United States, information on BCC and SCC of the skin is not routinely collected
in population- based central cancer registries, so information on these cancers comes from
medical claims data, survey data, and special studies [2, 10, 43]. Of the 5 million U.S. adults
treated for skin cancer on average each year, an estimated 4.3 million (1.9% of the adult
population) are treated for NMSCs (BCC, SCC, and other rare skin cancers), according to an
analysis of the Agency for Healthcare Research and Quality’s Medical Expenditure Panel
Survey [10]. Among those aged 65 years or older, an estimated 6.9% (9.3% of men and 5.0%
of women) are treated for NMSCs on average each year [10]. Medicare data for 2002–2006
showed that the number of procedures used to treat NMSCs in the Medicare population
increased by 16.0% during that period.2 A special study that examined deaths from SCC and
BCC in Rhode Island during 1988–2000 showed death rates of 0.29 and 0.08 per 100,000 per
year for nongenital SCC and BCC; the rate for melanoma during the same period was 2.6 per
100,000 [5, 44].
 Free ebooks ==> www.Ebook777.com
1308 Meg Watson, Erin Garnett, Gery P. Guy et al.

Figure 1. Types of Skin Cancer.

Basal Cell Carcinomas

BCCs are thought to be more common than any other type of cancer and are generally
treatable [1, 7]. Although national rates are not available, studies have estimated BCC
incidence rates in some states. For example, incidence rates for BCC during 1993–1994 in
New Hampshire were 309.9 per 100,000 among men and 165.5 per 100,000 among women
[45]. Arizona had an estimated rate of more than 900 per 100,000 among men and nearly 500
per 100,000 among women in 1996 [46]. The incidence of BCC appears to be increasing at a
rate of about 2% per year in the United States [1, 13, 47]. About 70%–80% of NMSCs among
males and 80%–90% of NMSCs among females are BCCs [43].

Squamous Cell Carcinomas

SCCs account for about 20% of NMSCs and are the second most common form of skin
cancer. Although both SCCs and BCCs are generally treatable, SCCs are deadly more often
than BCCs [43]. A recent study estimated that at least 4,000 Americans died from SCC of the
skin in 2012 [48].

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1309

Melanoma
Melanoma is the third most common type of skin cancer and is responsible for most skin
cancer deaths [9, 14, 32]. In the United States, according to federal data for 2007–2011, more
than 63,000 people are diagnosed with melanoma, and nearly 9,000 people die from this
disease each year (Tables 1 and 2) [11, 49]. Although melanoma rates overall are highest
among older adults, it is the third most common cancer in adolescents and young adults (aged
15–39 years) [12]. Recent analyses have found increases in incidence across all tumor
thicknesses and stages [9]. If current trends in cancer death rates continue, melanoma will be
the only cancer objective included in Healthy People 2020 that will not meet the targets for
reductions in cancer deaths (http://www. healthypeople.gov/2020/topicsobjectives2020/
objectiveslist. aspx?topicId=5) [50, 51].

Variation by Sex
In 2011, melanoma of the skin was the fifth most common cancer for men, with an
incidence rate of 25.4 cases per 100,000 (31.0 for white men, the group with the highest rates)
and the seventh most common cancer for women, with an incidence rate of 15.7 per 100,000
(20.1 for white women) [52]. During 2002–2011, melanoma incidence increased at an
average annual rate of 1.6% for men and 1.5% for women [52]. This increase is the largest
increase of the 10 most common cancers among men, and it is surpassed only by increases in
thyroid cancer among women (Figure 2) [51, 52]. Although incidence rates for melanoma are
increasing among both males and females (Figure 3), melanoma death rates are only
increasing among males (Figure 4) [9, 14, 51].
Melanoma incidence and death rates are highest among males, especially non-Hispanic
white males (Tables 1 and 2 and Figures 3, 4, and 5) [50]. Increased risk of melanoma in
white males may be related to a variety of factors, including skin type and historical
differences in sun exposure and sun protection behaviors. White men aged 65 years or older
have the highest incidence (130.1 cases per 100,000) and death rates (23.7 per 100,000) for
melanoma [9, 12].

Table 1. Invasive Melanoma Incidence, by Sex and Race/Ethnicity, United States,

2007–2011a,b

Male and Female Male Female

Race/Ethnicity Average Average Average
Rate Annual Rate Annual Rate Annual
Count Count Count
All Races 19.7 63,429 25.1 36,679 15.9 26,750
White 22.2 59,882 27.9 34,842 18.2 25,041
White, 4.4 1,215 4.8 553 4.2 662
Hispanicc
White, Non- 24.7 58,667 30.6 34,289 20.4 24,378
Hispanicc
Black 1.0 336 1.1 145 1.0 191
American 4.7 131 5.8 69 3.9 62
Indian/Alaska
Native
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1310 Meg Watson, Erin Garnett, Gery P. Guy et al.

Table 1. (Continued)

Male and Female Male Female

Race/Ethnicity Average Average Average
Rate Annual Rate Annual Rate Annual
Count Count Count
Asian/Pacific 1.3 177 1.4 84 1.2 93
Islander
Hispanicc 4.3 1,301 4.7 588 4.1 713
a
Rates are per 100,000 people and are age-adjusted to the 2000 U.S. Standard Population (Source: Day
JC. Population Projections of the United States by Age, Sex, Race, and Hispanic Origin: 1995 to
2050. U.S. Bureau of the Census, Current Population Reports, P25-1130. Washington, DC: U.S.
Government Printing Office; 1996).
b
Source: Data are from population areas that meet United States Cancer Statistics publication criteria
(http://www.cdc.gov/cancer/npcr/uscs/ technical_notes/ criteria.htm) for 2007–2011 and were
reported to the National Program of Cancer Registries (Centers for Disease Control and
Prevention) and the Surveillance, Epidemiology, and End Results (SEER) Program (National
Cancer Institute). They cover about 99.1% of the U.S. population.
c
Race and ethnicity are not mutually exclusive. Counts may not always sum to the total due to
rounding and because cases with “other” and “unknown” race are included in the totals.

Table 2. Melanoma Death Rates, by Sex and Race/Ethnicity, United States, 2006–2010a,b

Male and Female Male Female

Race/Ethnicity Average Average Average
Rate Annual Rate Annual Rate Annual
Count Count Count
All Races 2.7 8,776 4.1 5,730 1.7 3,046
White 3.1 8,580 4.6 5,633 2.0 2,947
c
White, Hispanic 0.8 198 1.1 115 0.6 83
White, Non- 3.4 8,375 5.0 5,513 2.1 2,862
c
Hispanic
Black 0.4 131 0.5 63 0.4 68
American 0.9 19 1.2 11 0.7 8
Indian/Alaska
Native
Asian/Pacific 0.4 45 0.4 23 0.3 22
Islander
c
Hispanic 0.8 201 1.1 117 0.6 85
a
Rates are per 100,000 people and are age-adjusted to the 2000 U.S. Standard Population (Source: Day
JC. Population Projections of the United States by Age, Sex, Race, and Hispanic Origin: 1995 to
2050. U.S. Bureau of the Census, Current Population Reports, P25-1130. Washington, DC: U.S.
Government Printing Office; 1996).
b
Source: Surveillance, Epidemiology, and End Results (SEER) Program, National Cancer Institute
(http://www.seer.cancer.gov). SEER*Stat Database: Mortality. Source: Released April 2013.
Underlying mortality data provided by the National Center for Health Statistics, Centers for
Disease Control and Preventionv/nchs).
c
Race and ethnicity are not mutually exclusive. Counts may not always sum to the total due to
rounding and because Hispanic ethnicity for some cases was unknown.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1311

Variation by Anatomic Site

Melanoma is found more often on parts of the skin that get more intermittent, intense UV
exposure, such as the torso and legs, although patterns vary by age [32, 53]. In addition, the
anatomic distribution of melanoma varies by sex, most often occurring on the legs for females
and on the torso for males [32]. Recent research suggests that melanomas among young
women may be particularly increasing on the torso [54]. However, these patterns are complex
and vary among populations [55]. Because acral melanoma (which occurs on palms of hands
and soles of feet) arises in typically unexposed areas of the body, the role of UV exposure in
this cancer is thought to be limited, and acral melanoma may have different risk factors than
other types of cutaneous melanoma [56, 57].

Abbreviation: NOS, Not Otherwise Specified.

a
Calculated by using 1 year for each end point and the weighted least squares method.
b
Source: Data are from population areas that meet United States Cancer Statistics publication criteria
(http://www.cdc.gov/cancer/npcr/uscs/ technical_notes/ criteria.htm) for 2002–2011 and were
reported to the National Program of Cancer Registries (Centers for Disease Control and
Prevention) and the Surveillance, Epidemiology, and End Results (SEER) Program (National
Cancer Institute). They cover about 92.4% of the U.S. population.
c
The average annual percent change is significantly different from zero (2-sided Z test; P  <  0.05).

Figure 2. Average Annual Percent Changea in the 10 Most Common Cancers, 2002–2011b.
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1312 Meg Watson, Erin Garnett, Gery P. Guy et al.

Note: Data after vertical dotted line are projected rates.

Source: Surveillance, Epidemiology, and End Results (SEER) Program, National Cancer Institute
(http://www.seer.cancer.gov). SEER 9 Incidence
Database (1973–2010). November 2011 submission. Nordpred software used to create age-period-
cohort regression models to calculate projections.

Figure 3. Age-Adjusted Melanoma Incidence Rates, Actual and Projected, by Sex, 1975–2020.

Note: Data after vertical dotted line are projected rates.

Source: Surveillance, Epidemiology, and End Results (SEER) Program, National Cancer Institute
(http://www.seer.cancer.gov). SEER*Stat Database: Mortality. Released April 2013. Underlying
mortality data provided by the National Center for Health Statistics, Centers for Disease Control
and Preventionv/nchs). Nordpred software used to create age-period-cohort regression models to
calculate projections.

Figure 4. Age-Adjusted Melanoma Death Rates, Actual and Projected, by Sex, 1975–2020.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1313

Source: Data are from population areas that meet United States Cancer Statistics publication criteria for
2007–2011 (http://www.cdc.gov/cancer/npcr/uscs/ technical_notes/criteria.htm) and were reported
to the National Program of Cancer Registries (Centers for Disease Control and Prevention) and the
Surveillance, Epidemiology, and End Results (SEER) Program (National Cancer Institute). They
cover about 99.1% of the U.S. population.

Figure 5. Melanoma Incidence Rates, by Age and Sex, 2007–2011.

Variation by State
State incidence rates for melanoma among all races vary widely, from 11.9 per 100,000
in Alaska to 31.9 per 100,000 in Utah during 2007–2011 [49]. Reasons for state variations
include differences in populations by race, age, and genetic background; by socioeconomic
status (SES) and health care access; and by patterns of UV radiation and exposure, as well as
differences in collection of data on melanomas by state central cancer registries [53]. Much of
the variation in state rates is because of differences in state populations. Among non-Hispanic
whites, the population at highest risk, higher UV levels are associated with higher melanoma
incidence rates; for this population group, Alaska has the lowest melanoma rate of all states
(14.8 per 100,000), and Hawaii has the highest melanoma rate of all states (66.7 per 100,000)
[49, 58]. States in southern latitudes have the highest death rates for melanoma among non-
Hispanic white populations [52, 59].

Survival
The prognosis for patients with metastatic melanoma remains poor, but has been
improving because of recent advances in treatment [60-62]. Survival is poorest among black
populations, possibly because of later diagnoses and lower perceived risk, and because these
populations are disproportionately diagnosed with certain types of melanoma with poorer
survival rates (acral lentiginous melanoma) (Figure 6) [6, 63, 64].

Economic Burden of Skin Cancer

In addition to causing illness and death, skin cancer is costly to the nation. Skin cancer
treatment is estimated to cost about $8.1 billion in the United States each year, $4.8 billion of
which is for NMSC and $3.3 billion of which is for melanoma [10]. Several new medications
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1314 Meg Watson, Erin Garnett, Gery P. Guy et al.

are available for skin cancer, which increases treatment options but could also lead to higher
costs [65-67].
Skin cancer also results in significant costs beyond those related to treatment. Annual
costs associated with lost workdays and restricted-activity days are estimated at $76.8 million
for NMSC and $29.4 million for melanoma [68, 69]. An individual in the United States dying
from melanoma loses an average of 20.4 years of potential life, compared with an average of
16.6 years for all malignant cancers [70]. Annual productivity losses associated with these
lost years is estimated to cost an additional $4.5 billion ($3.5 billion attributed to melanoma
deaths and $1.0 billion attributed to NMSC deaths) [69, 70].

Risk Factors for Skin Cancer

Genetic Factors
People with certain genetic risk factors are more likely than others to develop skin
cancer. Genetic risk factors for skin cancer include having a lighter natural skin color; blue or
green eyes; blond or red hair; dysplastic nevi (a type of unusual mole) or a large number of
common moles; and skin that burns, freckles, or reddens easily or becomes painful after
excessive time spent in the sun [18, 24]. People with red hair may be at particularly increased
risk of melanoma [24]. In addition, those with a family history or personal history of skin
cancer, especially melanoma, are at increased risk [18-23].

a
Five-year relative survival calculated by actuarial method. Data could not be calculated for 2007–
2010.
Source: Surveillance, Epidemiology, and End Results (SEER) Program, National Cancer Institute
(http://www.seer.cancer.gov). SEER*Stat Database: Incidence – SEER 18 Regs Research Data +
Hurricane Katrina Impacted Louisiana Cases, Nov 2012 Sub (1973–2010 varying).

Figure 6. Trends in 5-Year Melanoma Survival, by Race, 1973–2006a.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1315

Table 3. Fitzpatrick Skin Type

Skin Type Description

I Always burns, never tans, sensitive to ultraviolet (UV ) exposure.
II Burns easily, tans minimally.
III Burns moderately, tans gradually to light brown.
IV Burns minimally, always tans well to moderately brown.
V Rarely burns, tans profusely to dark.
VI Never burns, deeply pigmented, least sensitive.

Skin Type
Skin cancer risk varies by skin type, which is classified by how likely a person is to tan or
burn. The six skin types of the Fitzpatrick skin type classification system are shown in Table
3 [71]. Sunburn often is used as a proxy outcome measure in skin cancer prevention studies
because it takes into account the person’s skin type, as well as the intensity and duration of
UV exposure. Although anyone’s skin can be damaged by UV exposure, people with skin
types I and II are at the highest risk of burns, damage from UV radiation, and skin cancer.
Originally, the Fitzpatrick system was constructed for white populations and had only
four categories (Skin Types I–IV). Types V and VI were added to the system later in
recognition of the wide variety of races and skin types [71]. Because the Fitzpatrick system
was developed to measure the skin types of whites, the terminology used may make it
difficult for blacks or other races to classify their skin type [72]. Although the Fitzpatrick
system is often considered the gold standard for categorizing skin type, it may not always
accurately reflect an indvidual’s risk of skin cancer, and other systems have been proposed
[73, 74].

Race and Ethnicity

Race and ethnicity play an important role in skin cancer risk because characteristics
associated with race and ethnicity (such as skin, hair, and eye color) are indicators of
melanoma risk. Blacks and Asians/Pacific Islanders have the lowest melanoma incidence and
death rates, followed by American Indians/Alaska Natives and Hispanics (Tables 1 and 2).
People of European descent and non-Hispanic whites have the highest melanoma incidence
and death rates because they generally have lighter natural skin color [32, 53].
However, race and skin type do not always align neatly, and wide genetic variation exists
within races [75, 76]. People who identify as being other than non-Hispanic white may still be
at higher risk of skin cancer because of their skin type and may underestimate their risk [63,
64, 77-79]. Some black Americans report being sensitive to the sun [80]. Recent data showed
low reported use of sun protection behaviors among Hispanics, and melanoma may be
increasing among some Hispanic groups [77, 81].

Exposure to UV Radiation
Although genetic risk factors contribute to a person’s skin cancer risk, most skin cancers
are believed to be caused by a combination of genetic factors and exposure to UV radiation,
from the sun and from artificial sources such as indoor tanning. By reducing intentional UV
exposure and increasing sun protection, many skin cancer cases can be prevented.
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1316 Meg Watson, Erin Garnett, Gery P. Guy et al.

Types of UV Radiation
Sunlight is made up of different types of electromagnetic radiation, mostly infrared,
visible, and UV. Exposure to sunlight has both positive and negative effects. Although sun
exposure can have positive effects on mood and stimulates production of vitamin D, exposure
to UV radiation also damages DNA and cell functions, and that damage can lead to cancer.
UV radiation is categorized into three types: UVA (UV radiation with a wavelength of 315
nm to 400 nm), UVB (280 nm to 315 nm), and UVC2 (100 nm to 280 nm) [33, 34].
UVB radiation has intermediate levels of energy and can cause sunburn and direct DNA
damage (Figure 7). Ozone and other components of the atmosphere absorb more than 90% of
UVB from the sun, but the amount absorbed varies widely depending on time, location,
season, and weather. Certain chemical and carbon emissions also have caused depletions in
stratospheric ozone since the 1970s, and evidence suggests that this decrease has led to an
increase in ground-level UVB levels [82, 83]. Further study is needed to determine whether
ozone depletion is contributing to the increasing incidence of skin cancers worldwide [82-85].
UVA radiation has less energy than UVB radiation, but it can also cause skin cancer and
other skin damage. Unlike UVB radiation, nearly all UVA radiation passes through the
atmosphere, and it penetrates to deeper layers of the skin than UVB radiation.

Note: UVC radiation (not shown) is almost completely absorbed by the earth’s atmosphere and does
not generally affect human skin.

Figure 7. Types of Ultraviolet (UV) Radiation and Skin Penetration.

U.S. Environmental Protection Agency’s UV Index

The UV Index developed by the U.S. Environmental Protection Agency (EPA) provides
daily and hourly forecasts of the expected risk of overexposure to UV radiation from the sun.
The UV Index scale describes how to use the UV Index to help avoid harmful exposure to
UV radiation, with a lower UV Index indicating a lower risk on a scale of 0–11 (Table 4). In
winter, the average UV Index is 2 or below, although it can be higher on some days. During
November–January, the daily average for the UV Index is usually 2 or below nearly
everywhere in the United States except Florida and Hawaii (http://www2.epa.gov/sunwise/

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1317

monthly-average- uv-index). However, reflective surfaces, such as snow, water, and sand, and
high altitudes can increase risk of overexposure to UV radiation and sunburn. EPA
recommends that people use more sun protection strategies as UV levels get higher. For
specific recommendations for sun protection at different UV levels, visit http://
www2.epa.gov/sunwise/uv-index-scale.

Table 4. Ultraviolet (UV) Index Levels

UV
COLOR RISK
INDEX
GREEN 0–2 Low
Low danger from the sun’s UV rays for the average
person.
YELLOW 3–5 Moderate
Moderate risk of harm from unprotected sun exposure.
ORANGE 6–7 High
High risk of harm from unprotected sun exposure.
Protection against skin and eye damage is needed.
RED 8–10 Very High
Very high risk of harm from unprotected sun exposure.
Take extra precautions because unprotected skin and eyes
will be damaged and can burn quickly.
PURPLE 11 or more Extreme
Extreme risk of harm from unprotected sun exposure.
Take all precautions because unprotected skin
and eyes can burn in minutes.
Adapted from the U.S. Environmental Protection Agency’s UV Index scale, available at
http://www2.epa.gov/sunwise/uv-index-scale.

UV Exposure and Skin Cancer

Many skin cancers can be avoided by reducing exposure to UV radiation [3, 25-30]. As
many as 90% of melanomas are estimated to be caused by UV exposure [25, 86]. Some
evidence suggests that certain rare skin cancers, such as Merkel cell carcinoma, a rare but
frequently fatal cancer arising from neuroendocrine cells, may also be related to UV exposure
[41, 42]. The degree to which UV exposure increases a person’s risk of skin cancer depends
on many factors, such as individual skin type, the amount and types of sun protection used,
whether exposure is chronic or intermittent, and the age at which the exposure occurs [15, 86-
92].
Ecologic studies have shown that light-skinned people who live in areas with higher UV
exposure, particularly when they are younger, have higher rates of skin cancer, especially
SCC [85, 88, 93]. Similarly, melanoma is thought to be caused by sun exposure throughout
life, possibly with stronger effects in early life, although adult exposures clearly increase risk
as well [87]. Some studies suggest that UV exposures in childhood that do not result in a burn
may be associated with lower rates of future melanomas [94, 95]. Melanoma incidence is also
associated with higher SES, which is a combination of education, income, and wealth. This
association is likely due to the relationship between SES and other risk factors, such as skin
type and patterns of UV exposure [32, 78]. When people with lower SES are diagnosed with
melanoma, they tend to have poorer outcomes, probably because of later detection and poor
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1318 Meg Watson, Erin Garnett, Gery P. Guy et al.

access to treatment [6, 32, 96, 97]. See Table 5 for a comparison of avoidable risk factors for
skin cancer.

Chronic versus Intermittent UV Exposure

The effects of some risk factors are different for different types of skin cancers. Chronic
exposure is defined in different ways in the literature, but usually refers to frequent, extended
outdoor exposures to UV radiation from the sun above a certain number of times a week or a
certain number of days a year [43, 87, 90, 95, 98-103]. Studies of chronic exposure usually do
not include short frequent exposures, such as those experienced by the average person in his
or her commute to work or school. Extended or intense exposures experienced only a few
times a year, such as the sun exposure received on a trip to the beach, are typically classified
as intermittent exposures. Continuous, chronic UV exposure, such as that observed among
outdoor workers, is more strongly associated with SCC, while intermittent or recreational
exposure is more strongly associated with melanoma and BCC [43, 87, 95, 99-103].

Table 5. Excess Health Risks Associated with Ultraviolet (UV) Exposure, by Type of
Skin Cancer and Type of UV Exposure

Excess Risk
Exposure (No. of Studies) Comparison Groups
(95% CI)
MELANOMA
Sun exposurea
Total sun exposure (N = 28) 34% (2, 77) N/A
Intermittent sun exposure (N = 34) 61% (31, 99) N/A
Chronic sun exposure (N = 40) -5% (-13, 4) N/A
Sunburnb
Sunburn in childhood (N = 27) 91% (59, 130) Ever vs never
Sunburn in adolescence (N = 13) 63% (42, 86) Ever vs never
Sunburn in adulthood (N = 13) 44% (27, 63) Ever vs never
Sunburn in past 5–10 years (N = 5) 62% (-1, 165) Ever vs never
Ever sunburned in lifetime (N = 28) 59% (37, 83) Ever vs never
Indoor tanning
Ever indoor tanned (N = 27)c 20% (8, 34) Ever vs never
Ever indoor tanned (N = 8; U.S. studies only)d 23% (3, 47) Ever vs never
Ever indoor tanned (N = 10; studies from year 22% (3, 45) Ever vs never
2000 onward)d
Indoor tanned before age 35 years (N = 13)c 59% (36, 85) Ever before age 35 vs never
before age 35
Frequent indoor tanning (N = 15)c 42% (15, 74) Frequent vs
infrequent/never
Relative risk for each indoor tanning per year (N 2% (0, 4) N/A
= 4)c
>10 lifetime tanning sessions (N = 10)d 34% (5, 71) >10 lifetime tanning
sessions vs never
Indoor tanned >1 year (N = 3)d 61% (-2, 167) Indoor tanned >1 year vs
never
BASAL CELL CARCINOMAe
Ever indoor tanned (N = 10) 29% (8, 53) Ever vs never

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Excess Risk
Exposure (No. of Studies) Comparison Groups
(95% CI)
e
BASAL CELL CARCINOMA
Frequent indoor tanning (N = 4) 50% (-19, 177) Frequent vs
infrequent/never
Indoor tanned before age 25 years (N = 3) 40% (29, 52) Ever before age 25 vs never
before age 25
SQUAMOUS CELL CARCINOMAe
Ever indoor tanned (N = 10) 67% (29, 117) Ever vs never
Indoor tanned before age 25 years (N = 2) 102% (-30, 486) Ever before age 25 vs never
before age 25
Abbreviation: CI, confidence interval. N/A: not applicable; measured as a continuous variable.
Note: We did not differentiate between measures of relative risk (e.g., odds ratio, rate ratio, risk ratio)
for melanoma because it meets the rare disease assumption by which these measures can be
interpreted to be the same. Measures of relative risk for basal cell carcinoma and squamous cell
carcinoma are based on odds ratios.
a
Source: Meta-analysis of risk factors for cutaneous melanoma: II [89].
b
Source: Sunburns and risk of cutaneous melanoma: does age matter? A comprehensive meta-analysis
[87].
c
Source: Cutaneous melanoma attributable to sunbed use: systematic review and meta-analysis [118,
119].
d
Source: The association of indoor tanning and melanoma in adults: systematic review and meta-
analysis [121].
e
Source: Indoor tanning and non-melanoma skin cancer: systematic review and meta-
analysis [120].
Sunburn is a clear sign of overexposure to UV, and it typically occurs after intermittent
exposure; sunburn at any age increases a person’s risk of skin cancer [9, 43, 87, 99, 100].
Indoor workers may receive a substantial proportion of their total UV radiation from
intermittent exposure [104-106]. Cumulative exposure to UVA through glass windows, which
block most UVB, can also cause skin damage over time [107, 108].

Outdoor Workers
Although research clearly indicates that outdoor workers are at increased risk of BCC and
SCC, some studies suggest that outdoor workers might not have an increased risk of
melanoma, or that they may even have a lower risk than indoor workers [43, 109-111]. When
stratified by UV level, outdoor workers in UV-intense areas do appear to be at increased risk
of melanoma [55, 103]. Studies of melanoma risk and outdoor work may be limited by lack of
information on other related factors, which then limit the ability to attribute effects to the
relationship between outdoor work and melanoma [15, 91]. Regardless of these potential
study limitations, outdoor workers often experience excessive UV exposure on the job, and
efforts are needed to ensure that outdoor workers are protected from the sun.

Indoor Tanning
Indoor tanning devices, such as tanning beds, tanning booths, and sun lamps, expose
users to intense UV radiation as a way to tan the skin for cosmetic reasons. Although
reducing UV overexposure from the sun can be challenging for some people, UV exposure
from indoor tanning is completely avoidable. In 2009, the World Health Organization (WHO)
classified indoor tanning devices as Class I human carcinogens on the basis of strong
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1320 Meg Watson, Erin Garnett, Gery P. Guy et al.

evidence linking indoor tanning to increased risk of skin cancer [27, 112]. Meta-analyses
have consistently shown that indoor tanning increases the risk of developing SCC, BCC, and
melanoma (Table 5) [27, 113-121]. The risk increases the more an individual uses indoor
tanning, with younger and more frequent users having more steeply increased risk [113-121].
Findings consistently document a strong association between increased risk of melanoma
and indoor tanning use, although the magnitude of the association varies from study to study,
reflecting different populations and settings. A recent international meta-analysis that
included 31 studies collectively reviewing 14,956 melanoma cases and 233,106 controls
(individuals without melanoma) reported that individuals who reported ever indoor tanning
had a 16% increased risk of melanoma over those who never indoor tanned [121].
The association between indoor tanning and melanoma increased when analysis was
restricted to more recent studies conducted in 2000 or later (22%) or when restricted to
individuals who had used indoor tanning devices 10 or more times in their lives (34%) [121].
When analysis was restricted to the 11 studies from North America, including 4,395
melanoma cases and 79,358 controls, the increased risk of melanoma with ever using indoor
tanning was 23% [121]. In one U.S. study included in the meta-analysis, researchers reported
a 74% increased risk of melanoma among individuals who reported ever using indoor tanning
compared with those who did not tan [116]. Findings from this study also reported a strong
dose-response relationship, with greater risk for more sessions, hours, or years spent tanning
[116].
Indoor tanning also increases the risk of BCC and SCC [122, 123]. For NMSCs, indoor
tanning was found to increase risk of BCC by 29% and of SCC by 67% [120]. A 2014 meta-
analysis estimated that more than 400,000 cases of skin cancer may be related to indoor
tanning in the United States each year: 245,000 BCCs, 168,000 SCCs, and 6,000 melanomas
[124].
Initiating indoor tanning at younger ages appears to be more strongly related to lifetime
skin cancer risk, possibly because of the accumulation of exposure over time from more years
of tanning [114, 116, 118, 119]. The magnitude of increased risk with younger age at
initiation varies because of differences in collection and reporting of data, but studies
consistently show an increase in risk. A frequently cited meta-analysis estimated that tanning
before age 35 increased risk by 59% [118, 119]. This risk estimate is based on a compilation
of data from U.S. and international studies from different settings [118, 119]. One 2010 U.S.
study found that ever using indoor tanning before age 18 increased risk of melanoma by 85%
compared with never indoor tanning; risk for those aged 18–24 years increased by 91% [116].
Years of use of tanning devices appeared to be the strongest predictor of increased risk in this
study, with increased risk of 47% with 1 year of indoor tanning, 64% with 2–5 years of
indoor tanning, 85% with 6–9 years of indoor tanning, and 145% with 10 or more years of
indoor tanning [116]. Harms of indoor tanning may be accelerated for adolescents and young
adults, leading to early-onset skin cancers [115, 125, 126].
Although earlier studies describing the association between indoor tanning and skin
cancer had been criticized for not accounting for skin type and outdoor UV exposure or
sunburns [127], more recent studies have controlled for these factors, and these studies have
also found that indoor tanning increases the risk of melanoma [116, 125, 128-131]. For
example, a 2014 study showed that individuals who tanned indoors without burning had an
increased risk of skin cancer, regardless of lifetime sunburns experienced [128].

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1321

According to 2013 Youth Risk Behavior Survey (YRBS) data from the Centers for
Disease Control and Prevention (CDC), about 13% of high school students, 20% of high
school girls, and 27% of girls in the 12th grade had used an indoor tanning device, such as a
sunlamp, sunbed, or tanning booth (not including a spray-on tan), one or more times during
the previous 12 months [132]. Results from CDC’s 2010 National Health Interview Survey
(NHIS) show that some groups of young adults had high rates of indoor tanning, specifically
non-Hispanic, white women aged 18–21 years (32%) and 22–25 years (30%). Among non-
Hispanic, white indoor tanners, 58% of women and 40% of men did so 10 or more times
during the 12 months before the survey [133]. A study that combined data from the YRBS
and NHIS reported that about one-third of non-Hispanic white women aged 16–25 tanned
indoors each year [134].
No evidence exists to suggest that indoor tanning is safer than tanning outdoors or
confers any substantial protection from future sun exposure. Studies have found that indoor
tanning exposes users to excessive levels of UV radiation, especially UVA [135-138]. The
average intensity of artificial UV radiation was found to correspond to a UV Index of 13 or
14 (extreme), with some devices measuring even higher [135, 136]. Some studies have found
that tanning devices may expose users to 4–13 times the amount of UVA as exposure from
summer noontime sun in the District of Columbia, depending on the type of device used [136,
138]. In studies examining the relationship between UV exposure and skin cancer risk, indoor
tanning is typically classified as intermittent UV exposure (similar to outdoor recreational
exposure) rather than chronic exposure because of the acute intensity of the exposure [90, 98].
An estimated 3,200 people a year in the United States seek care in emergency rooms with
injuries attributed to indoor tanning [139]. In addition to increasing skin cancer risk, indoor
tanning can cause burns to the skin, acute and chronic eye diseases if eye protection is not
used, and, if tanning devices are not properly sanitized, skin infections [139-141].

Other Harms Caused by Excessive UV Exposure

In addition to increasing the risk of skin cancer, UV exposure can have adverse effects on
the skin, eyes, and immune system. Excessive UV exposure can damage the immune system;
cause premature skin aging, including wrinkling, mottled pigmentation, and loss of elasticity;
and increase the risk of actinic keratoses, which can progress to SCC [86, 113, 142, 143].
Excessive UV exposure may reduce the effectiveness of folic acid supplements, which has
potential health consequences for pregnant women and women of childbearing age [144].
Excessive UV exposure can also damage the eye, affecting surface tissues and internal
structures, such as the cornea and lens. Unprotected exposure to excessive UV radiation can
cause photokeratitis (sunburn of the eye) [145]. Chronic exposure to UV radiation can lead to
skin cancer around the eyelids (BCC, SCC, and melanoma), as well as cataracts, conjunctival
cancers, pterygium (abnormal, noncancerous growth in the corner of the eye that can extend
to the cornea and partially block vision), age-related macular degeneration, and possibly
ocular melanoma (melanoma of the eye) [145]. Wearing sunglasses that fit properly and have
100% UVA and UVB protection is the best way to protect eyes from UV damage [146, 147].
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1322 Meg Watson, Erin Garnett, Gery P. Guy et al.

Complex Relationship between Outdoor UV Exposure, Vitamin D, and Human

Health
As well as being a carcinogen, UV radiation can affect other aspects of human health
[148, 149]. UV exposure can stimulate production of vitamin D in the skin, a vitamin
important for bone health and associated with other health outcomes [150-152]. Complete
avoidance of sun exposure may put bone health at risk, although too much exposure increases
risk of skin cancers and eye disease (Figure 8) [82, 86, 142]. UV radiation is sometimes used
as a medical treatment for certain skin or bone ailments [153]. Many people engage in regular
physical activity outdoors, which can lead to UV radiation overexposure if appropriate sun
protection is not used (see the “Reducing the Risk of Skin Cancer” section on page 23 for a
discussion of sun protection methods). Some have also suggested that UV exposure may have
benefits for heart health by reducing blood pressure, but the evidence is still evolving [154,
155]. The following sections summarize the current evidence on UV exposure, vitamin D,
and other health benefits.

Vitamin D
The health benefits of sun exposure are often framed within the context of vitamin D
production. Vitamin D is essential for human health and is synthesized by the skin after
exposure to sunlight [150, 151, 156]. Although the scientific literature has established vitamin
D as an important component of bone health [151, 152, 157], substantial research has also
been devoted to the role of vitamin D in the prevention of numerous chronic diseases,
including autoimmune conditions, obesity, diabetes, high blood pressure, heart disease,
preterm birth, certain types of cancer, and all-cause mortality [155, 158-165]. The results of
this research are primarily based on ecologic studies and are conflicting [151, 160, 166-170].
Some have speculated that low vitamin D concentrations may be a result of ill health, rather
than a cause [166, 171, 172].
In 2010, the Institute of Medicine (IOM) published a report examining dietary reference
intakes and optimal serum 25-hydroxyvitamin D (25OHD) concentrations 3[151]. During
2001–2006, roughly one-quarter of the U.S. population had serum 25OHD values that put
them at risk of inadequacy, 8% were at risk of deficiency, and 1% had a high serum 25OHD
value that may possibly be harmful [172]. Optimal concentrations of serum 25OHD may vary
among individuals [173]. Blacks have the lowest 25OHD concentrations compared with other
racial and ethnic groups [156, 172]. Lower concentrations of vitamin D-binding proteins
among blacks may provide more bioavailable vitamin D, potentially explaining the paradox
of frequently diagnosed deficiency among U.S. black populations, who also tend to have
better bone health than whites [156, 174, 175].
Although maternal concentrations of vitamin D and incidental sunlight exposure are
sufficient for most breastfed infants, some can be at risk of vitamin D deficiency if adequate
vitamin D is not obtained from another source, such as a supplement. The American
Academy of Pediatrics recommends a supplement of 400 IU/day for infants and children not
consuming enough vitamin D-fortified formula or milk to provide the recommended daily
amount of vitamin D [176, 177].

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1323

Source: Recreated from Lucas RM, Ponsonby AL. Ultraviolet radiation and health: friend and foe. Med
J Aust. 2002;177(11):594-598.
© Copyright 2002. The Medical Journal of Australia. Adapted with permission. The Medical Journal of
Australia does not accept responsibility for any errors in adaptation.

Figure 8. Relationship Between Ultraviolet (UV) Radiation Exposure and Disease Burden.

The amount of outdoor sun exposure needed for meaningful vitamin D production
depends on many factors, including time of day, time of year, latitude, altitude, weather
conditions, a person’s skin type, amount of skin exposed to the sun, other individual
circumstances, and reflective surfaces, such as snow, water, and sand. According to WHO, 5
to 15 minutes of casual sun exposure on face, arms, and hands 2 to 3 days a week in the
summer can sustain adequate concentrations of vitamin D in most people [142]. However,
those with dark skin may require 3 to 6 times the amount of sun exposure as those with light
or fair skin [142, 178, 179]. Because the skin of a person with fair complexion is less able to
produce a tanning response [180], the amount of sun exposure needed for a fair-skinned
person to get a tan either indoors or outdoors, even before sunburn, exceeds levels of
exposure needed to synthesize vitamin D [135-137, 181]. In the winter months in northern
latitudes, exposure to sunlight does not result in meaningful cutaneous vitamin D synthesis
[151].
Because the U.S. population contains wide variations in skin tone, and because our nation
covers a wide array of latitudes and geographic conditions, populationwide recommendations
for obtaining vitamin D from sunlight would likely result in too little vitamin D in some
groups and too much sun exposure in other groups because no known threshold of UV
exposure exists that does not also increase skin cancer risk [182]. Exceeding limited levels of
exposure is not advisable because the skin can only produce a finite level of vitamin D, and
increases in UV exposure are not proportional to increases in serum vitamin D concentrations
[183-185].
For individuals and populations who avoid all sun exposure, a dietary source of vitamin
D is necessary to maintain vitamin D status [186]. Although complete sun avoidance can
result in vitamin D deficiency, evidence to date does not suggest that sunscreen use causes
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1324 Meg Watson, Erin Garnett, Gery P. Guy et al.

vitamin D deficiencies. In 2001, the U.S. Food and Drug Administration (FDA) reviewed
seven clinical studies that examined the effect of sunscreen use on vitamin D concentrations
and determined that the studies failed to show that sunscreen use caused vitamin D
deficiencies [187]. Adequate vitamin D can be obtained safely through food and dietary
supplements without the risks associated with overexposure to UV radiation [150, 151].
Research suggests that most people get the majority of the total vitamin D they need from
food rather than from the sun [188].

DIETARY SOURCES OF VITAMIN D

The best natural sources of vitamin D in the diet include fatty fish (such as salmon,
tuna, mackerel, sardines, and catfish) and fish liver oils [173]. Small amounts of vitamin D
are also found in egg yolks, beef liver, some mushrooms, ricotta cheese, and some cuts of
pork. Vitamin D-fortified foods and beverages provide most of the vitamin D in the U.S.
diet. Almost all of the milk in the United States is fortified with vitamin D, and many of
the ready-to-eat breakfast cereals provide a small amount of added vitamin D. In addition,
specific brands of soy beverages, orange juice, yogurt, margarine, and other foods are also
fortified with vitamin D [189].

Medical Uses of UV Exposure

Dermatologists and other doctors sometimes use UV light to treat health conditions, such
as psoriasis, rickets, and eczema. These providers are advised to carefully weigh the risks and
benefits of UV treatment for individual patients and carefully monitor doses [153, 190-196].

Benefits of Being Outdoors

Beyond the benefits directly attributable to UV exposure, spending time in outdoor
environments may also have positive effects on physical and mental health, including higher
levels of physical activity and positive effects on overall health and sense of well-being [38-
40]. These benefits can be achieved while using adequate sun protection, including shade,
protective clothing, and broad spectrum sunscreen with a sun protection factor (SPF) of 15 or
higher to reduce skin cancer risk [38-40, 197-199]. Features like shade trees can make spaces
more attractive and provide protection from the sun and heat. In turn, perceived availability
and “greenness” of spaces are associated with increased physical activity and better mental
and physical health [200-202]. The presence of shade in play spaces for children increases the
use of the play space and children’s activity levels [203, 204]. Thus, changes to outdoor
environments can increase both physical activity and sun protection, aligning important
public health goals.

Risks of Indoor Tanning Outweigh Any Potential Benefits

The benefits to limited UV exposure when outdoors do not extend to indoor tanning
[205]. UV exposure from indoor tanning is particularly intense, the type and intensity of UV
emitted varies between devices, and exposures often exceed limits recommended by FDA or
by states [135-138]. Some tanning lamps emit primarily UVA, which tans the skin but does
not induce vitamin D production or provide even the minimal photoprotection that a UVB-
induced tan provides [150, 206, 207]. Some tanning lamps do emit UVB, but studies suggest

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1325

that vitamin D production is limited and plateaus after brief exposures, so that the amount of
UV radiation needed to tan generally exceeds levels needed for adequate vitamin D
production [183, 208]. Indoor tanning does not appear to be protective against cancer or all-
cause mortality. A recent study found that indoor tanning was not associated with reduced
risks of internal cancer [209]. In addition, a large Swedish cohort study found that, although
outdoor UV exposure was associated with reductions in all-cause mortality, exposure to
artificial UV radiation from indoor tanning was associated with increased mortality [210].
Some people associate tanned skin with attractiveness and health. Some also believe that
a tan provides protection from future UV exposure and sunburn (often referred to as a “base
tan”) [211, 212]. Tanning is the skin’s acute response to damage from UV rays [213-215]. A
UVB-induced tan provides minimal sun protection, equivalent to an SPF of about 3, and thus
does not provide adequate protection against future UV exposure [216, 217]. Belief in the
protection of a “base tan” may lead to a false sense of security and inadequate use of sun
protection while outdoors in the sun [212]. Some studies have found that indoor tanning does
not protect against sunburn [212, 218]. People who engage in indoor tanning before going on
vacation may be more likely to stay out longer in the sun, putting themselves at greater risk of
sunburn [212].
Low levels of sunlight in the winter months may contribute to seasonal affective disorder
(SAD) [219], and as a result, some indoor tanners may attempt to self-treat SAD with UV
exposure through indoor tanning [220, 221]. Medical treatment of SAD frequently
incorporates light treatment, but UV wavelengths are not generally recommended (although
some lights used in treatment of SAD may contain small amounts of UVA and UVB) [153,
219, 222]. In addition, light is thought to affect SAD through the retina, not the skin[219].

Current Trends in Sun Protection, Sunburn, and Indoor Tanning

Data on behaviors related to skin cancer risk among the U.S. population are collected by
CDC through the national YRBS and NHIS. The national YRBS is a cross-sectional, school-
based, biennial survey that monitors the prevalence of health risk behaviors among high
school students. It is a nationally representative survey of students in grades 9–12 attending
public and private schools [223]. This survey includes questions about using sunscreen with
an SPF of 15 or higher and indoor tanning. The NHIS is an annual, cross-sectional, nationally
representative survey of the civilian, noninstitutionalized U.S. population [224]. Interviews
are conducted, mainly in person, with adults aged 18 years or older in each household, with
follow-up interviews by telephone when necessary.
A periodic cancer control supplement to the NHIS includes questions about outdoor sun-
protective behaviors (staying in the shade, wearing a wide-brimmed hat, wearing a long-
sleeved shirt, wearing long clothing to the ankles, and using sunscreen with an SPF of 15 or
higher), indoor tanning, sunburn, and sun sensitivity. This supplement is sponsored by CDC’s
Division of Cancer Prevention and Control and the National Cancer Institute (NCI) in the
National Institutes of Health (NIH).
According to YRBS data, sunscreen use is low among U.S. high school students, with
only 10.1% using sunscreen with an SPF of 15 or higher always or most of the time when
outside for more than 1 hour on a sunny day. Sunscreen use is higher among high school girls
(13.2%) than boys (6.9%) and higher among non- Hispanic whites (11.5%) compared with
non-Hispanic blacks (4.7%) and Hispanics (7.9%). During 1999–2011, a significant linear
decrease occurred in the prevalence of routine sunscreen use (from 13.3% to 10.8%).
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1326 Meg Watson, Erin Garnett, Gery P. Guy et al.

However, prevalence of sunscreen use remained stable from 2011 (10.8%) to 2013 (10.1%)
[132, 225].
NHIS data from 2010 indicate that use of sun protection was also low among U.S. adults
and that about 37% of adults had been sunburned in the past year [226]. Sunburn4 rates were
even higher among adults aged 18–29 years and sun-sensitive groups (defined as those who
burn repeatedly and freckle). Half of all Americans in this age group (about 65% of non-
Hispanic whites, 10% of non-Hispanic blacks, and 35% of Hispanics) reported having had a
sunburn in the past year [227]. Although NHIS data indicate that some sun protection
behaviors have increased among young adults over the past decade (including use of shade,
use of sunscreen with an SPF of 15 or higher, and wearing of long clothing to the ankles), a
corresponding decrease in sunburn has not been reported [227].

Indoor Tanning
According to 2013 YRBS data, 13% of high school students had used an indoor tanning
device, such as a sunlamp, sunbed, or tanning booth (not including a spray-on tan) one or
more times during the previous 12 months [132]. The prevalence of indoor tanning was
higher among female, older, and non-Hispanic white students, with the highest prevalence
among 12th-grade females (27.2%), and among non-Hispanic white females (30.7%). During
2009–2013, a significant linear decrease occurred overall in the prevalence of indoor tanning
device use (from 15.6% to 12.8%). The prevalence of indoor tanning device use did not
change significantly from 2011 (13.3%) to 2013 (12.8%). Data from the 2013 YRBS indicate
that among students who engaged in indoor tanning, frequent sessions were common, with
more than half reporting frequent use (10 or more times during the previous 12 months)
[132].
Results from the 2010 NHIS show that 6% of adults aged 18 years or older had engaged
in indoor tanning in the past year [133]. The prevalence of indoor tanning was higher among
females (9%) than males (2%) and among younger adults than older adults, with the highest
use among adults aged 18–21 years (12%), 22–25 years (12%), and 26–29 years (9%).
Similar to the data for U.S. high school students, rates were higher among non-Hispanic
whites (8%) than among non-Hispanic blacks (<1%) and Hispanics (2%). Certain
demographic groups had high rates of indoor tanning, including non-Hispanic white women
aged 18–21 years (32%) and 22–25 years (30%). Among non-Hispanic white indoor tanners,
58% of women and 40% of men did so 10 or more times during the 12 months before the
survey [133].

REDUCING THE RISK OF SKIN CANCER

Most skin cancers are at least partially caused by UV exposure, so reducing exposure
reduces skin cancer risk. However, one out of every three U.S. adults has been sunburned in
the past year, and most do not take recommended actions to protect themselves from the sun
[227, 228]. In addition, indoor tanning rates are high among some groups, such as young,
non-Hispanic white females, and skin cancer incidence rates are increasing. These facts show
a need to take action to improve sun protection behaviors and address the harms of indoor
tanning.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1327

For Individuals

Sun protection helps prevent the harmful effects of sun exposure, including sunburn, skin
cancer, premature skin aging, and eye damage. When used as part of a comprehensive
approach, well-tailored, individual- focused strategies may be effective for reaching specific
subpopulations [229, 230]. According to WHO’s International Agency for Research on
Cancer (IARC), ideal sun protection involves several behaviors, including

 Wearing tightly woven protective clothing that adequately covers the arms, torso,
and legs.
 Wearing a hat that provides adequate shade to the whole of the head.
 Seeking shade whenever possible.
 Avoiding outdoor activities during periods of peak sunlight (such as midday).
 Using sunscreen (in conjunction with other sun protection behaviors) [231].

Federal agencies and other health organizations in the United States all provide
recommendations for sun protection (see Table A in Appendix 5). These recommendations
vary across agencies and organizations, often reflecting the specific area of focus for each
institution (such as cancer or dermatologic conditions). Strategies for protection are often
listed in varying order and do not always follow guidance from the IARC that sunscreen
should be used in combination with other methods [231, 232].
Recommendations also do not often describe how to use sunscreen appropriately in terms
of the amount to apply, the need to pre-apply some sunscreens before going out into the sun,
and the need to reapply. These differences in sun protection messaging indicate missed
opportunities for coordination across health organizations and highlight the need for more
messaging about sun safety that is consistent and clear [232].

Sun Protection Strategies

Wear Protective Clothing

When possible, wear long-sleeved shirts and long pants and skirts, which can provide
protection from UV rays. Clothes made from tightly woven fabric offer the best protection. A
wet T-shirt offers much less UV protection than a dry one, and darker colors may offer more
protection than lighter colors. Some clothing certified under international standards comes
with information on its UV protection factor.

Wear a Hat and Sunglasses

Wide-brimmed hats that shade the face, ears, and back of the neck provide the most
protection. Tightly woven fabrics, such as canvas, provide the best protection; straw hats with
holes that let sunlight through do not provide adequate protection. A darker hat may offer
more UV protection. In addition to a hat, sunglasses that block as close to 100% of both UVA
and UVB rays as possible can provide extra eye protection. Most sunglasses sold in the
United States, regardless of cost, meet this standard. Wrap-around sunglasses work best
because they also block UV rays from the side.
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1328 Meg Watson, Erin Garnett, Gery P. Guy et al.

Seek Shade
An umbrella, tree, or other shelter can provide protection from the sun and relief during
hot weather. Shade does not block all UV radiation if it does not block all of the sky, nor does
it protect against scattered UV rays. For this reason, shade should be combined with other
methods, such as protective clothing, especially in areas with highly reflective surfaces, such
as snow, water, and sand.

Avoid Times of Peak Sunlight

UV radiation from the sun is most intense during the midday hours of 10 am to 4 pm
(daylight savings) or 9 am to 3 pm (standard time), so scheduling outdoor activities earlier or
later in the day can reduce UV exposure. UV radiation is also more intense during the late
spring and early summer, at higher altitudes, closer to the equator, and when reflected off
surfaces such as snow, water, and sand [233]. Different surfaces have different reflectivity
and can increase exposure. Snow reflects 80%–90% of UV radiation, sand 20%–30%, and
water 5%–7% [233]. Man-made surfaces can also have increased reflectivity. Concrete has
been measured to reflect 14%–15% of UV rays, whereas grass only reflects about 1%–2% of
UV rays [234]. When near highly reflective surfaces, extra care should be taken to protect
from UV exposure.

Use Sunscreen
Sunscreen should be used with other sun protection behaviors and applied to any exposed
skin before going outside. For adequate protection, sunscreen should have an SPF of 15 or
higher. SPF is a measure of how much UV radiation is required to produce a sunburn with
sunscreen applied to the skin in relation to the amount required to produce a sunburn on
unprotected skin. As the SPF increases, the amount of protection increases [235]. Sunscreen
should also have broad spectrum protection, which means that it protects against both UVA
and UVB radiation.
Sunscreen is one of the most common methods of sun protection used by Americans
[227]. When used as directed with other sun protection measures, broad spectrum sunscreen
with an SPF of 15 or higher helps prevent sunburn and reduces the risk of early skin aging
and skin cancer (melanoma and SCCs) associated with UV radiation [99, 231, 236-238].
Sunscreens with lower SPFs, or without broad spectrum protection, also help prevent sunburn
but do not offer sufficient protection against early skin aging and skin cancer.
Sunscreen is most effective when used with other methods of sun protection. Current
recommendations also state that sunscreen should be reapplied every 2 hours and after
swimming, sweating, and toweling off [239]. Some have suggested that a one-time
reapplication 15–30 minutes after the original application may increase sunscreen’s
protectiveness against total UV exposure [240]. When used incorrectly, sunscreen may
provide a false sense of protection, which can ultimately lead to increased duration of sun
exposure [231].
Although concerns have been raised about real-world efficacy (because many people do
not follow label instructions, use enough sunscreen, or reapply it often enough), broad
spectrum sunscreens with an SPF of 15 or higher are effective at reducing the risk of skin
cancer [241]. Future scientific assessments are expected to provide more information about
the long-term safety of frequent sunscreen use in people of all ages.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1329

Avoid Indoor Tanning and Sunbathing

In addition to using sun protection when outdoors, avoiding intentionally tanning can
help prevent skin cancer. Similar to excessive sun exposure, indoor tanning is associated with
an increased risk of melanoma, SCC, and BCC [118-120]. Indoor tanning also causes
premature skin aging, such as wrinkles and age spots [143, 242]. Intentionally tanning the
skin in the sun is an additional source of unnecessary and easily avoidable UV exposure.

Barriers to Using Sun Protection

Many Americans lack a general knowledge or awareness about the risks associated with
sun exposure, or they think they are at low risk of developing skin cancer or sunburn [63, 243,
244]. Some groups of Americans, especially blacks, the elderly, and people with less
education, may perceive themselves to be at low risk of skin cancer [63]. Because of the
perception of low risk and a lack of awareness, these groups tend to be diagnosed with skin
cancer at later stages [78, 96, 245].
A substantial segment of U.S. adults also do not perceive cancer as preventable and thus
may be less likely to engage in skin cancer prevention practices, such as using broad spectrum
sunscreen with an SPF of 15 or higher or covering up [246]. Lack of understanding of the UV
Index is also a barrier to making informed decisions about adequate sun protection while
outdoors [247, 248].
Many Americans either do not use sun protection when outdoors or do not use adequate
protection, and as a result, they experience sunburn [227, 228]. The costs of protective
clothing (e.g., wide-brimmed hats and sunglasses) and sunscreen may pose financial problems
for some [243, 249]. Personal clothing style preferences can also create barriers to people
using certain protective clothing items if they are seen as unfashionable, uncomfortable, or
interfering with sports or other outdoor activities [243]. For some people, protective clothing
may interfere with the body’s ability to cool itself, increasing the risk of heat illness [250,
251].

Reported barriers to sunscreen use include a perception that it is too messy, inconvenient,
or feminine [243, 252]. Some sunscreen users may view sunscreen use as a way to stay out in
the sun longer without getting burned [231, 243]. Others may use sunscreen for protection,
but use it improperly by not applying enough or forgetting to reapply [243, 253]. Sunscreens
may be somewhat less effective than physical barriers, and some people may have skin
sensitivities or concerns about certain chemicals in sunscreens [241, 254].
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1330 Meg Watson, Erin Garnett, Gery P. Guy et al.

High melanoma incidence and death rates among older non-Hispanic white men
demonstrate the need to increase sun protection among males, especially adults. 9]. Higher
rates observed among older men may be due to less use of sun protection and more time spent
outdoors throughout life compared with women [117, 227, 231]. Men are less likely to use
personal care products that contain sunscreen, and they may be less influenced by social
pressures to avoid premature skin aging [231]. For this reason, clothing and wide-brimmed
hats may be particularly important strategies for males. Baseball caps do not provide adequate
sun protection on their own, because they leave the ears and the back of the neck exposed
[228, 255].
If not addressed in a coordinated way, physical activity and sun protection messages can
conflict. Staying out of the sun at peak hours may not be feasible, depending on recreational
and occupational activities and schedules. Reapplication of sunscreen can be difficult during
activities such as sports events or practice [250, 256]. Engaging in physical activity outdoors
is associated with overexposure to UV radiation and sunburn [257]. However, findings from
one study suggest that the promotion of sun safety is not likely to reduce physical activity
among children. [258].

Barriers to Reducing Intentional Tanning

Intentional tanning, which includes both indoor tanning and seeking a tan outdoors, is
strongly associated with a preference for tanned skin and other appearance-focused behaviors
[259-262]. Studies indicate that messages that focus on the effects of indoor tanning that are
related to appearance, such as premature skin aging, may be more effective for tanners than
health-focused messages and may even promote long-term behavior change [263-266].
Patterns of indoor tanning vary, with some people tanning only before special events,
such as proms, and others tanning sporadically or regularly. Strategies that tailor prevention
messages to specific types of tanners are likely to enhance the effectiveness of interventions
[230, 267]. Additional strategies may be needed to prevent the initiation of intentional
tanning. Indoor tanners may incorrectly believe that tanning indoors has health benefits or
that it is safer than tanning outdoors because it is regulated [211, 268, 269].
Researchers are currently examining the psychological effects of indoor tanning and
possible links between indoor tanning behavior and dependence and addiction [270]. Indoor
tanning appears to have reinforcing properties similar to those ascribed to addictive
substances, such as the release of endorphins when the skin is exposed to UV radiation [270-
272]. Endorphins are a type of natural opioid involved in the brain’s reward pathway. Their
production during indoor tanning could create a future incentive to tan [271].

Social Norms Regarding Tanned Skin

Social norms regarding tanned skin as attractive and healthy create barriers to reducing
intentional exposure to UV radiation, whether indoors or outdoors. In many communities and
social groups, tanned skin is considered attractive [273], and social pressures to conform to
this beauty standard can be powerful motivators [274]. Women in particular may experience
greater social pressure to tan and have tanned skin, which likely explains the higher rates of
indoor tanning observed among women than men [133, 134, 259, 273-275].
Social norms regarding tanned skin have changed over time. Before the 1920s, pale skin
was considered beautiful and an indication of upper class lifestyles, while tanned skin was a
sign of working class people who labored outdoors [276]. As the industrial revolution moved

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1331

the working class indoors and into crowded inner cities, pale skin was no longer viewed as a
sign of wealth, but rather an indicator of poverty and poor health [276]. Tanned skin began to
signify a life of leisure and disposable income that allowed time for outdoor sports and beach
vacations [211, 276].
Although messages about the risks associated with excessive sun exposure and indoor
tanning have become more common in recent years, many still consider tanned skin to be a
sign of health, fitness, youth, and attractiveness [211, 276], and this viewpoint is often
perpetuated in popular media [260]. To be successful, future efforts to improve sun protection
behaviors, reduce indoor tanning, and prevent skin cancer will likely need to address the
underlying motives that drive behaviors associated with skin cancer risk, such as the desire to
look attractive and healthy and to conform to societal beauty standards. For example, future
messages could focus on the appearance-related harms of excessive UV exposure and how
most people do not use indoor tanning devices [265, 277-279].
To reduce harms from indoor tanning, some organizations have promoted the use of
topical sunless tanning products as a way to get a tanned appearance without UV exposure
[260]. One concern about this method of tanning is that dihydroxyacetone (DHA), a
commonly used ingredient in sunless tanning products, is approved by FDA for use in
cosmetics and drugs for external application only (21 CFR Part 73) [280]. When this product
is used in spray tanning booths (spray-on tans), inhalation is usually unavoidable [260]. In
addition, the promotion of sunless tanning products does not address the underlying social
norms that drive tanning behaviors. Sunless tanning products are often used in conjunction
with, rather than in place of, UV tanning [281-285]. Furthermore, their use does not appear to
lead to safer outdoor sun exposure and could potentially increase the likelihood of sunburn
[282, 286, 287]. Other methods used to achieve tanned skin, such as pills and injections, have
additional health risks [288]. However, over-the-counter sunless tanning creams and lotions
may be an option for those who want to have tanned skin while avoiding the health risks of
UV exposure and inhaled and absorbed DHA.

For Clinicians

Evidence demonstrates that clinicians can play a role in reducing UV exposure through
individually directed counseling, particularly among adolescent and young adult patients with
fair skin [265, 289]. Federal agencies and the independent U.S. Preventive Services Task
Force (USPSTF) recently conducted a systematic review of the evidence on the effectiveness
of behavioral counseling to prevent skin cancer. Findings from the review indicated that
counseling in primary care settings can increase sun-protective behaviors and decrease
intentional tanning, including indoor tanning [277, 278]. On the basis of these findings, the
USPSTF now recommends that clinicians counsel patients with fair skin aged 10–24 years to
minimize their UV exposure to reduce their risk of skin cancer [265, 277, 278].
Effective interventions are generally of low intensity, are completed almost entirely
during the primary care interaction or visit, and use cancer prevention or appearance-focused
messages to reach specific audiences [265, 277, 278]. Appearance-focused messages are
successful at reducing intent to pursue indoor tanning among late-adolescent women (the
population most likely to engage in indoor tanning) [265, 277, 278]. Efforts are needed to
identify ways to disseminate this type of information to clinicians and provide them with
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1332 Meg Watson, Erin Garnett, Gery P. Guy et al.

effective, user-friendly tools to use with patients. Evidence of the benefits of counseling for
patients older than age 24 is sparse and insufficient to serve as a basis for a recommendation.
Some groups recommend periodic skin cancer screening5either by a health care provider
or by self-examination [290, 291]. Consistent and regular screening identifies melanomas that
are, on average, thinner than those found during usual care. Whether detection of these
lesions leads to fewer cases of disease or death is unknown [292]. For this reason, the
USPSTF has stated that current evidence is insufficient6 to recommend skin cancer screening
by primary care providers among the general U.S. adult population. On May 15, 2014, the
USPSTF released a draft research plan, which will be used to guide a systematic review of the
evidence by researchers [293]. Although screening is not currently recommended, providers
should remain alert to suspicious lesions. For more information on skin cancer screening, see
Appendix 3.

For Communities and Schools

Community-level intervention strategies vary greatly by audience, setting, duration, and

the number and types of included components. For some strategies, sufficient evidence is
available to recommend dissemination. For other strategies, more research is needed to
determine basic effectiveness before the intervention can be disseminated to other
communities. For specific examples of community-level interventions, see Appendix 4.

SKIN CANCER PREVENTION IN ACTION: RECREATIONAL

SETTINGS
Pool Cool: Sun Safety for Outdoor Swimming Pools

Pool Cool is a sun-safety education program for children aged 5–10

years and their parents, as well as for pool staff and other pool users. It is
being used at public pools across the United States. The program is centered
on eight brief sun- safety lessons that are taught at the beginning of regular
swim classes. The program also promotes the creation of sun-safe pool
environments that include shaded areas, signs to promote sun safety, and
sunscreen dispensers.
First piloted in Hawaii and Massachusetts, Pool Cool has been used and
evaluated at more than 400 pools across the country. These evaluations
found that pools that use the program have more protected pool
environments, better sun protection habits among children and parents, and
fewer sunburns among children and lifeguards. For more information about
the Pool Cool program, visit http://www.med.upenn.edu/poolcool/.

Current Evidence on Effective Community-Level Interventions

Federal agencies and the independent Community Preventive Services Task Force have
worked together to conduct systematic reviews of the evidence on the effectiveness of
community-based interventions to prevent skin cancer. Findings from an initial review were

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1333

published in 2003 and 2004 and used as the basis for recommendations for interventions
designed to prevent skin cancer made by The Guide to Community Preventive Services (The
Community Guide7) [28, 294, 295].
The Community Guide states that sufficient evidence exists to recommend
multicomponent, communitywide interventions,8 as well as interventions designed for certain
settings (specifically, child care centers, primary and middle schools, outdoor recreational and
tourism settings, and outdoor occupational settings) [296]. The Community Guide states that
insufficient evidence exists to recommend mass media campaigns alone or to recommend
skin cancer prevention interventions in other settings (high schools, colleges, and health care
settings9) [296]. Although some skin cancer prevention interventions have been shown to be
effective in these settings, more research is needed before these findings can be translated into
evidence-based recommendations for skin cancer prevention interventions [296]. Efforts to
update these recommendations to reflect the latest evidence are ongoing. The current
recommendations for skin cancer prevention, which are based on updated reviews, are
available online at http://www.thecommunityguide.org/ cancer and are summarized in Table
B in Appendix 5. The recommendations provided in this Call to Action are consistent with
The Community Guide.

Prevention Policies in Schools

Sun protection programs for children can have important benefits [297]. Sunburns in
childhood are a clear risk factor for skin cancers later in life, and building healthy habits early
when children are more receptive can lead to increased sun protection into adulthood [297,
298]. Given the amount of time children spend in school settings, much of the skin cancer
prevention efforts for children have focused on sun-safety education in schools and changes
to the school environment to promote sun-safe behaviors. This section provides examples of
the resources available to schools and an overview of policies used in some schools to
promote sun safety.
Sun protection policies can be implemented at the school, community, school district, or
state level. CDC’s School Health Policies and Practices Study (SHPPS) collects data from a
nationally representative sample of public school districts to assess school health policies and
practices in the United States. According to 2012 SHPPS data [299], some U.S. school
districts have policies to promote sun safety among their students. Although very few districts
had policies that required specific sun-safety strategies, many districts had policies that
recommended the following:

 Allowing students to apply sunscreen while at school (44.4%).

 Encouraging students to wear hats or visors (36.1%), protective clothing such as
long-sleeved shirts or long pants (39.6%), and sunglasses (25.0%) when in the sun
during the school day.
 Scheduling outdoor activities to avoid times when the sun is at peak intensity during
the school day (38.3%) [299].

A baseline assessment of school policies collected during 2005–2007 from 112 public
school districts in Colorado and California found that 52% of school districts in California
and 8% in Colorado had at least one policy on sun protection before a randomized
intervention was implemented [300]. After the randomized intervention, districts appeared to
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1334 Meg Watson, Erin Garnett, Gery P. Guy et al.

adopt stronger policies than districts that did not participate in the intervention [301]. Some
states require public schools to provide information on sun safety and skin cancer prevention
[302, 303]. For example, since 2004, Arizona has mandated that all public schools teach
EPA’s SunWise program (http://www.epa.gov/sunwise; SunWise box, page 32) from
kindergarten through eighth grade [303, 304]. Across the United States, teachers taught sun
safety or skin cancer prevention in at least one class as part of required health instruction in
68% of elementary schools, 76% of middle schools, and 78% of high schools in 2006 [305].

Barriers to Interventions in Schools and Communities

Effective strategies can improve sun protection behavior in children and adults,
particularly in child care, school, and outdoor recreational and tourism settings (Table B,
Appendix 5) [306]. But without widespread, comprehensive implementation, these strategies
may have little effect on sun protection behaviors and sunburn prevention at the community
level. Single-component interventions may only have a small effect on behavior change,
which may not be sufficient to reduce skin cancer risk [307]. In addition, school policies can
either support or pose barriers to sun protection. Currently, some schools and school districts
do not allow certain kinds of protection to be easily used, because of rules such as bans on
hats and sunglasses or provision of sunscreen only by prescription or by a school nurse [300].
Policies allowing the use of sun protection in schools can help support broader efforts.
Social and contextual factors within communities can also create barriers to reducing UV
exposure. For example, outdoor environments, such as community parks and school
playgrounds, often lack adequate shaded areas. Providing shade, either in the form of man-
made shade structures or natural shade from trees and shrubs, can help people enjoy the
outdoors at any time of day without the risk of excessive sun exposure [308].

For Outdoor Work Settings

Similar to schools, outdoor work settings are an important setting for efforts to prevent
overexposure to the sun and reduce skin cancer risk. Research has shown that skin cancer
prevention interventions designed to reach outdoor workers can be highly effective at
increasing sun protection behaviors and decreasing sunburns [309].
According to The Community Guide [309], effective interventions include one or more
of the following:

 Educational approaches, such as messages about sun protection delivered to workers

through instruction, small media (e.g., posters, brochures), or both.
 Activities designed to influence the knowledge, attitudes, or behavior of workers,
such as modeling or demonstrating behaviors.
 Environmental approaches to encourage sun protection.
 Workplace policies that support sun protection practices.

A study in Australia found that workers who were more aware of sun safety or who
worked in smaller workplaces were more likely to use protection when they received
instructions on its use [310]. Other studies have found that being employed in a workplace

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1335

that is perceived to be supportive of sun protection is associated with better sun protection
behaviors among workers [311]. In addition to employers, local governments and labor
organizations have played a role in increasing programs for sun protection among outdoor
workers [312, 313].

State and Local Policies, Legislation, and Regulation

Intervention strategies that address social and contextual factors have the potential for
broad public health impact by making the healthy choice the easy or default choice [314].
Policies, legislation, and regulation are examples of such interventions, reaching wide
segments of communities while requiring minimal individual effort compared with
interventions directed at individuals [314].

Sun Protection Policies and Legislation

Sun Protection
Many schools have policies that limit students’ ability to use sun protection, such as dress
codes that prohibit the use of hats or sunglasses or policies about over-the-counter drugs that
prohibit the use of sunscreen [300]. Only a few states, such as California and New York, have
passed legislation requiring that schools allow students to use sun-protective clothing
(California) or sunscreen (California and New York) on campus [315, 316]. The California
School Boards Association recommends that individual school districts adopt specific sun
protection policies for students [317, 318]. In addition, lifeguards in California who get skin
cancer are eligible for workers’ compensation benefits under certain conditions [319].
California law also urges employers to identify and correct workplace hazards connected to
UV radiation [320].
Local policies that address skin cancer prevention vary across the country, and their
effects on the incidence of skin cancer or on intermediate outcomes, such as sun protection
behaviors and sunburn, have not been formally evaluated or documented. However, such
policies could be considered as one component of a larger, more comprehensive skin cancer
prevention initiative within a community.

Education and Awareness

A few states have passed legislation to support sun-safety education programs and skin
cancer prevention awareness. Laws in Arizona and New York mandate instruction on skin
cancer prevention as part of the health education curriculum in public schools [303, 321]. In
2004, Arizona adopted a law requiring implementation of the state’s SunWise school program
(adapted from EPA’s SunWise program; see box, page 32) in grades K–8 in all public schools
[303, 304]. In 2006, Kentucky passed a law encouraging skin cancer education in schools
[322].
Some states have policies that reach beyond children as the audience for education and
awareness. New York mandates sun-safety education for all state employees who spend more
than 5 hours a week outdoors [323]. In 2009, Arkansas began providing grants to
organizations that provide skin cancer education to state citizens [324]. Florida has included
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1336 Meg Watson, Erin Garnett, Gery P. Guy et al.

skin cancer prevention in its health awareness campaign program since 2004, reaching a wide
range of the state’s population. [325].

Indoor Tanning Policies and Legislation

Some states and municipalities in the United States have regulations relating to the use of
indoor tanning devices. As with many public health issues, regulation of indoor tanning is
likely to be most effective if combined with a multifaceted approach. For example,
monitoring use of indoor tanning devices and changes in use over time, restricting use of
tanning devices to protect certain populations (e.g., minors, people with fair skin, people at
increased risk because of a family or personal history of skin cancer), offering safe
alternatives to indoor tanning, warning users about the health risks associated with indoor
tanning, and enforcing existing regulations could help reduce harms [328].

SUNWISE: SUN SAFETY FOR KIDS AND EDUCATORS

SunWise is the most widely used health and environmental education program for sun
safety in the United States. It is designed to teach children aged 5–15 years and their
caregivers how to protect themselves from overexposure to the sun. It uses classroom,
school, and community components to teach sun-safe behaviors. Program participants
receive free materials that promote cross-curricular learning about sun safety, UV
radiation, and ozone science.
SunWise was launched in 2000 by the U.S. Environmental Protection Agency (EPA).
Today, more than 32,000 schools and 6,000 other educational organizations (e.g., camps,
science and children’s museums, scout troops) in all 50 states, the District of Columbia,
and several U.S. territories have received educational materials. Cities, counties, and states
across the country have worked to promote the program’s safety message throughout their
communities. To reinforce its sun-safety message, SunWise partners with community
organizations and nonprofit skin cancer prevention foundations.
The SunWise program has shown success in raising awareness and changing
behaviors related to sun safety [326]. It has also been shown to be cost-effective, with the
potential to prevent as many as 11,000 skin cancer cases among participants and save up
to $4 for every $1 invested [327].
For more information about SunWise, visit the EPA website at
http://www.epa.gov/sunwise.

Considerable variation exists throughout the country in the strength and enforcement of
indoor tanning restrictions, as well as compliance with these restrictions. In October 2011,
California passed the most stringent youth access law in the country, which took effect on
January 1, 2012, and prohibits indoor tanning for anybody younger than age 18 years (Figure
9) [329]. Since then, Vermont, Nevada, Oregon,10 Texas, Illinois, Washington,10 Minnesota,
Louisiana, and Hawaii have also adopted prohibitions on indoor tanning for minors younger
than age 18 years [329-331]. Several additional states proposed legislation to enact bans on
indoor tanning for this age group during the 2013–2014 legislative session [329, 330].
Currently, at least 44 states and the District of Columbia have some kind of law or
regulation related to indoor tanning [329-334], including the following:

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1337

 Bans on indoor tanning for minors under a certain age, ranging from 14 to 18 years.
 Laws for minors requiring parental accompaniment or parental permission.
 Harm-reduction regulations (for all ages) that require use of eye protection or limit
exposure time.

Indoor tanning laws, particularly those that include age restrictions, appear to be effective
in reducing indoor tanning among female high school students, who have the highest rates
[335].
Many states require that tanning salons be licensed or registered and that they provide
information on the risks of tanning to customers; some require that tanners sign a warning
statement before tanning [336]. Other legislative approaches include time limits, UV
irradiance or exposure limits, requirements that warning statements be signed or posted,
mandatory eyewear, mandatory reporting of incidents, penalties for violations of existing
regulations, and training requirements [336]. The strength of state laws varies, and some
states have no laws relating to indoor tanning [336]. Restrictions on indoor tanning also exist
at local levels. For example, indoor tanning is prohibited among minors younger than age 18
in Chicago and Springfield, Illinois, and in Howard County, Maryland [329].

Note: State laws in Oregon and Washington allow minors younger than age 18 years to use indoor
tanning facilities with a doctor’s prescription.
a
Map represents legislation passed before July 10, 2014.
b
Defined as a restriction for any other age group, including for minors younger than age 17, 16, 15, or
14 years.
Source: National Conference of State Legislatures, Indoor Tanning Restrictions for Minors: A State-by-
State Comparison website (http://www.ncsl.org/ research/health/indoor-tanning-restrictions.aspx)
and AIM at Melanoma, 2014 Indoor Tanning Legislation website (http://www.aimatmelanoma.org
/ en/aim-for-a-cure/legislative-accomplishments-in-melanoma/2014-indoor-tanning.html).

Figure 9. Legislative Restrictions on Access to Indoor Tanning by Minors in the United States a.

Evidence suggests that bans on underage tanning are effective in reducing access to and
use of indoor tanning among minors [335, 337]. According to a 2003 telephone survey of
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1338 Meg Watson, Erin Garnett, Gery P. Guy et al.

randomly selected indoor tanning salons in three states—Texas, Illinois, and Wisconsin—that
banned indoor tanning by youth younger than age 13, 14, or 16 years, respectively, 62% of
facilities contacted stated that they would not allow a 12-year-old to tan, whereas only 18% of
facilities in a state without age restrictions (Colorado) would prohibit such use [337]. A study
of the recently enacted under-18 ban in California found that 77% of salons would not allow a
17-year- old to tan [338]. Another recent study found that indoor tanning laws, particularly
those with age restrictions, are associated with lower rates of indoor tanning among female
adolescents [335].
Laws that require parental consent for tanning by youth under a particular age have the
potential to be effective at reducing youth indoor tanning, but more evaluation is needed. In
2009, researchers published results of a study of more than 3,600 indoor tanning facilities
nationwide [339]. Data collectors called the facilities, posing as prospective fair-skinned, 15-
year-old customers who had never tanned before. Of the 20 states with parental consent laws
at the time of the study, facilities sampled in only four states (Louisiana, Maine, New
Hampshire, and South Carolina) uniformly stated that they would require 15-year-old
customers to obtain parental consent to tan [339]. Facilities in Georgia had the lowest level of
compliance (72.5%) [339].
Other smaller studies confirmed low compliance. In a 2005 study, 15-year-old girls
visited 200 indoor tanning facilities in Minnesota and Massachusetts, posing as potential
customers. In 2005, both states had laws requiring parental permission for indoor tanning by
youth (younger than age 16 years in Minnesota or 18 years in Massachusetts). However, 81%
of the facilities sold the girls tanning sessions without parental consent [340]. A 2001 study of
54 salons conducted in San Diego, California, found that 43% of facilities visited would have
enforced the existing parental accompaniment consent law [341] .These data indicate the need
for, and importance of, enforcement of regulations or laws that may be effective at reducing
youth indoor tanning.
Training requirements for tanning facility employees also vary by state. Some require
that a salon must have at least one trained operator on site while tanning beds are in operation
[342]. Others require training for all tanning salon employees [334, 343-345] Likewise, the
extent and rigor of training required varies by state. For example, in Iowa, tanning bed
operators are required to read a document on the risks of tanning provided by the state health
department and complete an assessment [346]. Florida requires that all tanning salon
employees and tanning bed operators complete a training course provided by a preapproved
outside vendor. Many of the vendors are industry groups [345].

Federal Policies, Legislation, and Regulation

Many federal departments and agencies work on efforts related to skin cancer prevention
and control, individually and together. Federal agencies also disseminate information about
what works to prevent skin cancer. The U.S. Department of Health and Human Services
(HHS) and its agencies play important roles in skin cancer prevention at the federal level.
These agencies include the National Cancer Institute (NCI) in the National Institutes of
Health (NIH), CDC, FDA, and the Agency for Healthcare Research and Quality. CDC
supports Comprehensive Cancer Control Programs in states, tribes, and territories, many of
which conduct activities related to skin cancer prevention. Federal entities outside HHS also

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1339

address skin cancer prevention, including the Federal Trade Commission (FTC), EPA, the
National Park Service, and the Occupational Safety and Health Administration (OSHA).
Federal legislation can help support skin cancer prevention and control efforts. For
example, the Affordable Care Act includes a 10% excise tax on indoor tanning services and a
requirement that nearly all health insurance plans cover USPSTF-recommended preventive
services. Recommended services include behavioral counseling for children, adolescents, and
young adults aged 10–24 years with fair skin on how to minimize their exposure to UV
radiation to reduce the risk of skin cancer.
For more information on federal activities related to skin cancer prevention, see
Appendix 5.

Sun Protection Policies and Legislation

Sunscreens sold in the United States are governed by FDA as over-the-counter drugs.
Regulations identify acceptable active ingredients and dosage strengths, provide language and
format for product labels, and establish standardized test methods for determining a product’s
SPF, among other requirements. Products that satisfy regulatory conditions are considered to
be safe, effective, and truthfully labeled and may be marketed without premarket review and
approval by FDA. Products that vary from regulatory conditions may be sold only after FDA
review and approval [187].
Under FDA regulations, all sunscreen products are labeled for use to help prevent
sunburn, and they must state the product’s SPF. Sunscreens that pass a separate test for broad
spectrum (UVA and UVB) protection may also be labeled as “broad spectrum.” In addition,
broad spectrum sunscreens with SPF levels of 15 or higher may be labeled as reducing the
risk of skin cancer and premature skin aging when used together with other sun protection
measures, including limiting time in the sun and wearing long-sleeved shirts, pants, hats, and
sunglasses.
Broad spectrum sunscreens with SPF levels above 2 but below 15 must be labeled with a
“Skin Cancer/Skin Aging” alert in the warning section of the label. This alert states the
following: “Spending time in the sun increases your risk of skin cancer and early aging. This
product has been shown only to help prevent sunburn, not skin cancer or early skin
aging.”[187,347]
FDA regulations do not allow for the terms “waterproof” or “sweat proof” because no
product has been shown to completely retain its effectiveness regardless of the time a person
is immersed in water. Only the term “water resistant,” followed by the length of time of
demonstrated water resistance (40 or 80 minutes), is allowed to appear on sunscreen labeling
[187, 347].

Indoor Tanning Regulations

At the federal level, FDA regulates indoor UV tanning devices under separate authorities,
both as medical devices and as radiation-emitting electronic products. Manufacturers of
indoor tanning devices (also known as sunlamp products) are required to certify that their
products comply with the FDA Performance Standard for Sunlamp Products (21 CFR
1040.20) [348]. FDA originally classified indoor tanning devices as Class I (low risk) medical
devices, suggesting that they posed minimal dangers to consumers. FDA is working to reflect
current science on the risks of indoor tanning, improve the visibility and readability of the
warning label, update and promote compliance with the performance standard, and help
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reduce harms from these devices through regulatory mechanisms. On May 29, 2014, FDA
reclassified indoor tanning devices as Class II medical devices (moderate to high risk) (see
Appendix 5 for more information) [349-352].
Once the reclassification order is effective [350], manufacturers will have to do the
following:

 Include a visible black box warning on the tanning device that people younger than
age 18 years should not use these devices.
 Receive premarket notification 510(k) clearance from FDA for newly marketed
devices (which were previously exempt from any premarket review).
 Show that their products have met certain performance testing requirements.
 Address certain product design characteristics.
 Provide comprehensive labeling that presents consumers with clear information on
the risks of use.

Although the effect of strengthening FDA regulation is currently unknown, estimates

from Australia suggest that strengthening and enforcing regulations restricting indoor tanning
among minors and people with Fitzpatrick Skin Type 1 could result in 18–31 fewer diagnoses
of melanomas per 100,000 and 200–251 fewer diagnoses of SCC per 100,000 each year in
that country [353].

Barriers to Addressing Indoor Tanning Through Policies, Legislation, and

Regulation

Ubiquity of Indoor Tanning Devices

The ubiquity of indoor tanning salons and the low cost of indoor tanning may be
important barriers to reducing harms from indoor tanning. A study found an average of 42
indoor tanning salons in major U.S. cities in 2006 [354]. The study also found that cities with
higher percentages of whites had significantly higher facility densities than those with lower
percentages of whites and that living within 2 miles of an indoor tanning facility was a
significant predictor of indoor tanning among adolescents [354, 355]. In addition, indoor
tanning devices are available for use in unsupervised settings, such as fitness centers and
apartment complexes, which can promote frequent use and raises questions about the ability
to enforce current and future regulations [356].

Enforcement
Lack of enforcement creates a potential barrier to successful implementation of controls
and can limit the effect these efforts could have on reducing indoor tanning. Studies
examining state enforcement of indoor tanning laws and regulations raise concerns about the
sufficiency of enforcement efforts. For example, a 2008 study in 28 cities found that routine
annual inspections of indoor tanning facilities were conducted in only 36% of cities. Thirty-
two percent conducted inspections less than annually, and about 32% did not inspect indoor
tanning facilities for compliance with state laws. Officials in only 50% of cities stated that
they would give citations to tanning facilities that violated laws [357].

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Compliance
FDA recommends limits on maximum exposure times, and FDA regulations require that
the recommended exposure schedule appear on the label and in the instructions for sunlamp
products [358]. However, compliance with existing regulations and recommendations varies
[136, 341, 359, 360]. A study of tanning salons in North Carolina found that 95% of patrons
exceeded FDA exposure recommendations, and 33% of patrons began tanning at maximum
doses recommended for maintenance tanning [136]. Indoor tanning salons often use
promotional pricing packages that promote frequent indoor tanning [260, 359]. A study of 54
tanning salons in San Diego found that 75% of advertisements and 100% of facilities offered
“unlimited” tanning packages [341], which may encourage users to indoor tan in ways that
are inconsistent with the intent of FDA exposure recommendations.
State regulation of indoor tanning devices, including restrictions on youth access, also
varies considerably across the country, and studies examining state indoor tanning laws and
regulations in the United States demonstrate that compliance with these laws is low and not
adequately enforced [262, 360]. The study of tanning salons in San Diego found low
compliance with some state and federal regulations, including posting of warning signs [341].

Marketing
Marketing tactics used by the indoor tanning industry can also be a concern. In 2010,
FTC sued the Indoor Tanning Association (ITA), a trade association representing the tanning
industry, alleging false and misleading advertising about the health risks of indoor tanning
(see Appendix 5) [361]. The settlement reached in this case prohibits the ITA from making
the misrepresentations challenged in the complaint, misrepresenting any tests or studies, or
providing deceptive advertisements to members. These prohibitions are applicable only to the
ITA and related individuals and entities.
Evidence suggests that tanning industry members, including salon chains and individual
salons, continue to make statements about indoor tanning that may be inconsistent with the
available scientific evidence [260, 360]. For example, according to a 2013 report of the
results of a telephone survey of 338 indoor tanning salons in California, 61% denied harms of
UV exposure, and many made claims of health benefits from indoor tanning exposure [338].
A 2012 report of the U.S. House of Representatives Committee on Energy and Commerce,
Minority Committee, described the response of randomly selected tanning salons to calls
from individuals posing as teenaged girls. According to the report, many of the salons stated
that indoor tanning does not increase cancer risk, despite substantial evidence to the contrary
[362].

Lack of a Comprehensive Approach

Lack of a comprehensive, coordinated approach may also be a barrier to successful policy
and legislative efforts. Without enforcement, certain restrictions may be easily circumvented.
Stronger laws to regulate tanning salons and restrict youth access to them will not be as
effective in the absence of increased controls on unsupervised tanning beds and direct sales to
the public. Instead, they may drive people to indoor tan in unsupervised locations, such as
gyms, beauty salons, or common areas of apartment complexes, or to buy tanning beds for
home use. Unsupervised use of a tanning bed or use without a trained operator may lead to
longer, more intense exposure to UV radiation. A qualitative study found that ownership of a
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1342 Meg Watson, Erin Garnett, Gery P. Guy et al.

tanning bed could lead to very high exposures. One participant shared that he would often fall
asleep in his tanning bed, tanning for as long as 40 minutes at a time11[211].
A survey of British youth in 2010, before the United Kingdom enacted restrictions
banning indoor tanning for all minors, found that 23% of youth aged 11–17 years had used an
indoor tanning device at home, and 21% had used unsupervised devices in other setting [363].
To prevent minors from accessing unsupervised tanning facilities where access is not
controlled, WHO has recommended banning unsupervised tanning facilities as a complement
to restricting the use of tanning beds by minors [356].

International Efforts to Prevent Skin Cancer

Other countries have taken a variety of approaches to prevent skin cancer, including
community-based, multicomponent interventions, which are recommended by The
Community Guide [295]. If these types of interventions include some level of continued
support, they have demonstrated an ability to influence sun- protective behaviors [364]. A
study of an Australian skin cancer prevention program called SunSmart estimated that a
national, ongoing program funded at historic levels ($0.12–$0.41 Australian dollars per year
per capita) would save $2.30 in Australian dollars for every $1 invested. The program was
also estimated to save 22,000 life-years in the state of Victoria, Australia, during 1988–2003
[365]. Data from the evaluation of the SunSmart program provide evidence that sustained
funding for a community-level skin cancer prevention initiative can improve health outcomes
and result in long-term savings in health care costs.
Some countries have also used mass media campaigns with varied success, but most of
these efforts have not been formally evaluated. One particularly successful sun-safety
campaign called Reduce Your Sun was implemented in Denmark [366]. Since the campaign
started in 2007, surveys have shown decreases in the percentage of Danes who sunbathe and
who indoor tan [367]. The Danish campaign made extensive use of social marketing and
social media, including provocative videos designed to appeal to adolescents and young
adults [366, 368].
Many countries have laws specifically addressing indoor tanning. In November 2009,
based on WHO’s designation of indoor tanning devices as Class 1 human carcinogens (the
highest risk level), Brazil became the first country to ban indoor tanning for cosmetic
purposes [369].
In February 2012, New South Wales, Australia— home to more than 5 million people—
passed a complete ban on indoor tanning, which will become effective on December 31, 2014
[328]. In addition, as of January 2014, France, Spain, Portugal, Germany, Austria, Belgium,
the United Kingdom, Australia, Iceland, Italy, Finland, and Norway prohibit indoor tanning
for youth younger than age 18 years; most of these laws have been in place since 2003 [328,
369].

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1343

SKIN CANCER PREVENTION IN ACTION: MULTICOMPONENT

EXAMPLE FROM AN INTERNATIONAL SETTING
SunSmart Australia: Lessons from International Success

Australia has the highest incidence of skin cancer of any country, and the disease
costs the country’s health care system more than $294 million in Australian dollars
annually. In 1988, the state of Victoria launched the SunSmart program to encourage
sun-protective behaviors and minimize the human cost of skin cancer.
This multicomponent, communitywide intervention is designed to raise awareness,
change personal behaviors, and influence institutional policy and practices. Activities
include mass media campaigns, programs in schools and work sites, a sports program,
health care provider education, resource development and dissemination, and capacity
building at the community level.
Since SunSmart began, rates of BCC and SCC skin cancers among people younger
than age 45 years have begun to taper off, and increases in melanoma rates have
stabilized [365, 370]. SunSmart is estimated to save $2.30 in health care costs for every
$1 spent [365]. For more information about the SunSmart Program, visit
http://www.sunsmart.com.au.

According to WHO [356], other approaches can include the following:

 Banning unsupervised indoor tanning devices (e.g., devices located in gyms or

apartment common areas, coin-operated devices).
 Requiring eye protection.
 Restricting the use of indoor tanning by people at higher risk of skin cancer (e.g.,
those with Fitzpatrick Skin Type 1).
 Limiting the UV intensity emitted from devices.
 Requiring informational and warning notices.
 Conducting health education.
 Requiring informed consent to ensure that all users are aware of risks.
 Requiring training of tanning salon staff.

GAPS IN RESEARCH AND SURVEILLANCE

Internationally, research has provided strong evidence about what works to reduce the
risk of skin cancer, and there is a growing body of evidence in the United States. However,
additional research and surveillance are needed to maximize the success of future skin cancer
prevention efforts. Important strides have been made in skin cancer prevention in the United
States, but they have not been sufficient to curb the rising rates of skin cancer incidence.
Social and behavioral research can help us better understand some issues, such as ongoing
high rates of sunburn despite improvements in sun protection and ongoing high rates of
indoor tanning despite evidence that it is a human carcinogen. More information is needed
regarding effective message framing and effective policies to promote behavior change.
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Finally, reliable data are needed to measure the effect of prevention efforts. Many skin
cancer cases are not being captured by current surveillance systems, and current behavioral
surveillance systems may not be adequate to track the effect of state and local initiatives, such
as indoor tanning restrictions for minors. Identifying areas where information is lacking is
important in order to improve efforts to prevent skin cancer. This section details the gaps in
what is currently known. The “Calls to Action” section (page 46) will propose strategies for
filling these gaps.

Individuals

Although surveillance data indicate that some sun protection practices have increased,
these behaviors have not been associated with a reduction in the incidence of sunburn [226].
These data indicate inadequate use of sun protection among Americans and are cause for
concern. Research is needed to increase understanding of the factors that underlie inadequate
sun protection behaviors and to identify strategies to increase adoption of sun protection
practices beyond sunscreen use. Current recommended intervals for reapplication may not
maximize the protectiveness of modern sunscreens, and more research that accounts for how
sunscreens are actually applied in real-world situations would help guide future
recommendations [240].
Specific information is needed about effective messaging to influence positive behavior
change related to skin cancer prevention for specific groups, such as frequent tanners, event
tanners (those who tan before a special occasion like a prom or wedding), athletes, people
concerned about vitamin D deficiency, males versus females, and different racial and ethnic
groups. More research on effective communication would allow for specific messaging in
education and communication interventions. Similarly, testing prevention messages would
ensure that only the most effective interventions are disseminated and that they are suitably
tailored for specific groups.
New technologies, such as personalized mobile applications (or “apps”) that measure UV
exposure, may provide opportunities to address messages to people concerned about sun
protection [371, 372]. Behavioral counseling has been shown to be effective, but these studies
were only conducted among female undergraduate tanners. More research is needed to
determine effectiveness among broader populations, especially frequent tanners [306].
Better understanding is needed of the potentially reinforcing properties of indoor tanning,
such as the release of endorphins when the skin is exposed to UV radiation [270-272]. By
adapting questionnaires used for substance- related addiction disorders, researchers found that
study participants who report regular, more frequent indoor tanning tended to report reactions
to tanning such as relaxation, pain relief, stress relief, and a sense of well-being or euphoria
[373]. More research is needed to understand whether these reactions to indoor tanning can
cause users to feel physically or psychologically dependent on indoor tanning. In turn,
alternative approaches may be needed to intervene for these indoor tanners, who may differ
from the broader population [374].

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Parents

For children, adequate sun protection depends largely on their parents’ attitudes and
behaviors [375, 376]. Research has also shown that parental acceptance of tanning has a
strong influence on adolescent tanning behavior [275]. More information is needed to assess
parents’ attitudes and behaviors regarding sun protection and indoor tanning (for themselves
and for their children), as well as parents’ awareness of their children’s use of sun protection
and tanning behaviors. Although parental modeling of tanning behavior is a strong predictor
of their children’s tanning behavior, the prevalence of parents’ indoor tanning with their
children is unknown [275]. Strategies for involving parents in prevention efforts for children
should be formulated and evaluated.

Clinicians

Currently, little information exists on whether clinicians follow USPSTF

recommendations for behavioral counseling for skin cancer prevention. Surveys or other
systems are needed to monitor clinicians’ counseling practices. Given the large time demands
on clinicians, resources and tools are needed to provide guidance on best practices that
clinicians can fit into their short time with patients. These resources should include
information on how to identify patients at high risk and give them appropriate behavioral
counseling on how to prevent skin cancer. Providing computer prompts through electronic
health record (EHR) systems may increase clinicians’ delivery of preventive care, including
behavioral counseling on skin cancer prevention [377].
Skin cancers detected at earlier stages are easier to treat than those diagnosed later [6,
31]. More research is needed to determine who is most likely to benefit from screening (see
Appendix 3), as well as effective ways to increase awareness and early detection.

Schools

Skin cancer prevention interventions that use education and policy approaches in
elementary and middle schools and child care centers have been found to be effective [306].
More information is needed on the long-term effect of these interventions on sun-protective
behaviors and sunburn. More evidence is needed to determine the effectiveness of similar
interventions in high school, college, and university settings. Furthermore, evidence is needed
to identify similar school-based strategies that could effectively address indoor tanning and
complement other efforts to reduce indoor tanning, such as legislation and mass media
campaigns.

Outdoor Workers

Better understanding of how to reduce the risk of skin cancer among outdoor workers is
needed. Although employers are legally responsible for ensuring the protection of their
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1346 Meg Watson, Erin Garnett, Gery P. Guy et al.

workers, they are not specifically required to provide sun protection, such as sunscreen, hats,
or long-sleeved shirts. Understanding the costs and benefits of employer-provided protection
would help determine its effectiveness and cost-effectiveness. More information on the types
and prevalence of sun protection methods among outdoor workers is needed, as well as
information on potential differences in language and culture [378].

Communities and Social Networks

Additional research can provide information to communities on how to maximize the

protective effects of environmental changes in the community, such as how adding shade
structures will affect sun protection, which improvements are most cost-effective, and where
and how shade structures could be best used. Best practices on community shade structures,
similar to guidelines for schools, may be needed. Surveys that ask about common sources of
sun exposure would help identify sites where shade would be most effective.
Researchers need to conduct further studies to identify ways to shift social norms around
tanned skin and sun protection and increase sun protection methods beyond sunscreen use.
Social network research in general and social media research in particular could help capture
real-time responses to media stories and campaigns. This research could help monitor
attitudes on tanned (or untanned) appearance. In turn, social marketing may provide
opportunities for wide dissemination of individualized messages about skin cancer
prevention. Interventions that use text messages have also shown promise in improving
prevention behaviors and may provide opportunities for skin cancer prevention [379]. New
wearable UV sensor technology may provide additional opportunities for research on the
effectiveness of prevention strategies, but their effect on sun protection practices is unknown
[104, 380-382].

Indoor Tanning Legislation and Multilevel Influence

Evidence on the effectiveness of indoor tanning legislation suggests that different laws
may have different effects on tanning behaviors [335].The effects of specific laws, such as
prohibitions on tanning for youth younger than age 18 years or enhanced warning labels, are
unclear. Recent studies have found that age restrictions appear to be associated with
decreased tanning among youth [335, 337]. Evidence regarding the effects of other policies
and legislation on indoor tanning is limited, and some studies that have attempted to examine
the relationship between the presence of local laws and indoor tanning have failed to find an
association [355]. The lack of evidence may be due, in part, to challenges in surveillance and
monitoring of indoor tanning at the state level; wide variation in the stringency, compliance,
and enforcement of laws and regulations; and the relatively recent adoption of restrictive laws
and regulations [335, 355].
Monitoring changes in tanning behaviors over time (both indoor tanning and sunbathing)
as new indoor tanning legislation is adopted can help document the effects of such legislation.
It can also guide future policies and identify unintended consequences, such as replacement of
tanning in salons with indoor tanning at home or sunbathing. Adequate compliance,

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monitoring, and enforcement will be needed to have the greatest effect on behavior and
policies.
The lack of a comprehensive and multilevel approach to reducing indoor tanning may
also be a barrier to successful policy efforts. Without reinforcement by other legislative or
regulatory organizations, certain restrictions on indoor tanning may be difficult to enforce.
For example, without adequate controls to ensure that minors do not tan indoors on their own,
age bans on tanning are easily circumvented [356]. WHO recommends banning the sale of
unsupervised indoor tanning devices as a complement to restricting minors’ use of tanning
beds [356]. The prevalence of unsupervised tanning and home use of tanning beds is also not
well- documented, and surveys or other systems are needed to monitor these types of tanning
behaviors.
Social norms regarding tanning, the desire to have tanned skin, and misconceptions about
the health effects of indoor tanning are more likely to be influenced if policy approaches are
coordinated with comprehensive approaches at local, state, and national levels [260, 262].
Research is needed to identify the effective combination of intervention components at
different levels of influence that can be tailored to specific groups and widely disseminated
[260, 262].

Surveillance

Cancer Surveillance
Counts and prevalence of people treated for NMSCs have been estimated from
individual-level data from the Medical Expenditure Panel Survey [7, 10], but surveillance
data on these cancers are not generally available. Melanoma data are collected by population-
based cancer registries, and these data are needed for long- term evaluation of prevention
efforts. Doctors diagnosing or treating in situ and invasive melanomas are required by law to
report cases to central cancer registries [383]. Hospitals generally have systems set up to
report inpatient cases. However, melanomas diagnosed and treated in outpatient settings are
frequently underreported, highlighting the need for improved awareness of proper reporting
requirements among doctors, especially dermatologists [384, 385].
In 2009, the Centers for Medicare & Medicaid Services (CMS) established incentive
programs to encourage health care providers to adopt, implement, and upgrade the use of
certified EHRs in different stages. (For more information about EHRs, visit
http://www.cms.gov/Regulations-and-Guidance/Legislation/EHRIncentivePrograms/Stage_
2.html.) Reporting cancer cases to a central cancer registry is included in Stage 2 of the
incentive program, so providers have an additional incentive to report melanoma cases
beyond compliance with state law [386, 387]. The most recent estimates of melanoma and
NMSC incidence were conducted in 2011 [7, 9, 53, 388]. New analyses are needed to show
melanoma trends from more recent years, and new methods are needed to better estimate
NMSCs (which are not included in cancer registry collection).

Behavioral Surveillance
Some information on sun-safety behaviors and indoor tanning is included in surveillance
system surveys, but more could be done to use these systems to advance surveillance of
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1348 Meg Watson, Erin Garnett, Gery P. Guy et al.

behaviors related to skin cancer risk. For example, the Behavioral Risk Factor Surveillance
System (BRFSS) is a state-based, random- digit–dialed telephone survey of the
noninstitutionalized, U.S. civilian adult population aged 18 years or older. It is administered
annually to households with landline and cellular telephones by state health departments in
collaboration with CDC [389]. This nationally representative survey is designed to enable
prevalence estimates at the state level and, in some instances, at the local or metropolitan
level.
Inclusion of questions about sun protection and indoor tanning on the BRFSS would
allow better state-specific analysis of indoor tanning prevalence and sun protection behavior,
which would provide more information on the effect of interventions. Likewise, the national
YRBS is designed to be a nationally representative sample of high school students. It
measures indoor tanning frequency and sunscreen use, but does not currently measure other
sun protection behaviors, such as use of protective clothing, hats, and shade. Furthermore, the
survey’s sample design does not yield state-specific measures.
Inclusion of indoor tanning questions on state YRBS questionnaires would allow for
evaluation of state policies. However, only a few states have added such questions to their
surveys, making it difficult to measure prevalence of indoor tanning and monitor the effect of
indoor tanning restrictions for minors at state levels. This lack of data at the state level also
poses barriers for state-level planning and evaluation.
In addition, little information is available on intentional outdoor UV exposure behaviors
(sunbathing or seeking a tan). Measuring this behavior would help researchers quantify any
unintended consequences of changes to indoor tanning legislation or attitudes. Surveillance
systems that monitor indoor tanning attitudes and beliefs among the U.S. population and
among indoor tanners are also needed. Questions designed to collect this information could be
added to existing surveys, such as the Health Information National Trends Survey (HINTS),
the National Survey of Family Growth, or national panel surveys such as HealthStyles.

Surveillance of Environmental Exposure

Little information is available on general outdoor exposure to UV radiation that is
experienced in the course of routine activities, such as playing sports, engaging in physical
activity, gardening, or walking a dog. Research on personalized UV sensor technology and
Global Positioning System devices could be used to better capture UV exposure among
individuals and allow for more accurate measurement of individual UV exposure in the near
future [104, 371, 372, 381]. Research is needed to determine the best way to use existing
technology or expand the way current systems work together. Doing so may improve
surveillance estimates of UV exposure, guide intervention efforts, and possibly enhance
prevention behavior.

Vitamin D and Sun Protection

More research on the relationship between vitamin D and sun protection behaviors is
needed. Although guidelines exist to identify levels of insufficiency and deficiency, optimal
serum concentrations of vitamin D are a matter of scientific debate and most likely vary
among individuals [173]. As previously discussed, FDA concluded in 2011 that clinical
studies on the effect of sunscreen use on vitamin D concentrations were inconclusive [187].

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1349

Although not enough evidence exists currently to determine whether sunscreen use alone can
lead to vitamin D deficiency [187], improving sun protection across the population could
potentially lead to reduced vitamin D concentrations for some individuals if not compensated
for by vitamin D intake from diet or supplements [82, 86, 142, 187]. If populationwide skin
cancer prevention programs were implemented in the future, surveillance data from the
National Health and Nutrition Examination Survey (NHANES) could be used to monitor
vitamin D serum concentrations in the population and document any unintended
consequences of skin cancer prevention interventions, such as increases in vitamin D
deficiency.

Economic Analysis

Estimates of the health and economic benefits of reducing risk factors for skin cancer and
the subsequent reductions in skin cancer incidence and mortality are needed to justify and
guide current and future prevention efforts. For example, economic modeling could be
performed to examine the number of skin cancer cases averted and the costs saved by
implementing various indoor tanning policies and other communitywide efforts.
In addition, the effect of economic interventions, such as the 10% excise tax levied on
indoor tanning services through the Affordable Care Act, is largely unknown [390, 391]. The
only evidence of its effect was limited to tanning salons in one state [391]. A national study is
needed to examine the effect of the excise tax and how increases in the price of indoor
tanning could affect its use, similar to studies conducted on tobacco taxes [392].

Potential Unintended Consequences of Interventions

As with any intervention, increasing sun protection in the population could have
unintended consequences. Some people may become sensitized to the chemicals in
sunscreens, and increased sunscreen use could lead to increased sensitization, resulting in
urticaria (hives) or allergic contact dermatitis [231]. Restrictions on indoor tanning without
changes to social norms about the desirability of tanned skin might encourage tanners to seek
the sun outdoors, to tan in less-regulated settings (such as in homes), or to use sunless tanning
[260]. Ongoing efforts are needed to monitor the effects of skin cancer prevention efforts,
including unintentional and potentially harmful effects, such as reductions in vitamin D
concentrations in the population or reductions in physical activity.

CALLS TO ACTION
This section presents five strategic goals to support skin cancer prevention in the United
States. Federal, state, tribal, local, and territorial governments; members of the business,
health care, and education sectors; community, nonprofit, and faith-based organizations; and
individuals and families are all essential partners in this effort. Strategies that change the
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1350 Meg Watson, Erin Garnett, Gery P. Guy et al.

context or environment to support healthy choices generally have greater reach and are more
effective at the population level than strategies focused on individual behavior [314].
This section also provides education and communication strategies, which will likely be
most effective if used in conjunction with changes to the social context and environment.
Aligning and coordinating efforts for skin cancer prevention across a wide range of partners is
central to achieving success. Involving partners across disciplines, sectors, and institutions
will be essential to addressing the rising incidence of skin cancers in the United States.

Goal 1. Increase Opportunities for Sun Protection in Outdoor Settings

Increasing opportunities for sun protection in outdoor settings can make healthy sun-safe
behaviors the default choice and help Americans enjoy their time outdoors safely with
minimal effort. Changing the context for sun protection may also contribute to changing
social norms regarding the necessity of sun protection while outdoors.

Strategy 1A. Increase Shade in Outdoor Recreational Settings

When spending time outdoors, Americans can be exposed to high levels of UV radiation.
Communities can increase the availability of shade in recreational settings, such as parks and
sports fields, to provide passive protection and increase comfort levels. Strategically planting
trees or building structures to shade frequently used areas can protect people from heat as
well as UV radiation and increase their comfort while outdoors. A shade audit is a systematic
process to determine how much shade is currently available on a site, where more is needed,
and where to place trees and shade structures to be most effective [308, 393, 394]. Existing
shade audit, planning, and policy tools, such as CDC’s Shade Planning for America’s
Schools, could potentially be adapted for broader community use [308, 394]. New
technologies might offer the possibility of strategic shade planning with minimal resources
and investment [393]. Changing the environment by increasing shade can help parents and
caregivers adequately protect their children from excessive sun exposure. Even one bad
sunburn in childhood increases risk of melanoma later in life [87]. Protecting children can be
difficult, as they may be resistant to wearing additional clothing, hats, and sunglasses.
Sunscreen can be difficult to apply and reapply, especially for younger children.

PARTNERS IN PREVENTION
 Federal, state, tribal, local, and territorial governments.
 Businesses, employers, and labor representatives.
 Health care systems, insurers, and clinicians.
 Early learning centers, schools, colleges, and universities.
 Community, nonprofit, and faith-based organizations.
 Individuals and families.

Key partners in prevention include federal, state, tribal, local, and territorial governments;
businesses and employers, especially urban planners and architects; early learning centers,

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schools, colleges, and universities; community, nonprofit, and faith-based organizations; and
individuals and families. These partners can support efforts to do the following:

 Provide shade from trees, nearby buildings, or structures specifically designed to

block the sun, such as canopies and umbrellas.
 Use a shade audit process or tool[308,393,394] to help ensure effective shade
planning.
 Adapt existing shade planning tools[308,393,394] for broader community use and
disseminate these tools widely.
 Ensure ample availability of shade in recreational and play areas to help protect
children from overexposure to UV radiation.

Strategy 1B. Support Sun-Protective Behaviors in Outdoor Settings

Encouraging Americans to enjoy physical activity in outdoor areas, such as parks, fields,
pools, and beaches, is important for physical fitness and health. Just as many recreational
areas take steps to reduce the risk of immediate injury from accidents, other outdoor areas can
take steps to reduce the short- term risks of sunburn and long-term risks of skin cancer by
promoting sun-protective behaviors. Coaches and other organizers of outdoor sports and
recreational activities can change the social context by scheduling routine breaks to reapply
sunscreen and drink water, which also reduces risk of heat illness [239, 250, 251, 256].
Simple modifications to the outdoor environment can help to make sun safety the easy or
default choice.
Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; businesses and employers; early learning centers, schools, colleges, and
universities; and community, nonprofit, and faith-based organizations, can support the
following efforts:

 Establish agreements with vendors in outdoor recreation areas to sell sun protection
equipment, such as protective hats, clothing, and umbrellas, which will support
healthy behaviors and provide additional revenue for communities, businesses, and
sports teams.
 Provide broad spectrum sunscreen with an SPF of 15 or higher in dispensers with
prompts and signs that tell people how to apply sunscreen in high-UV areas, such as
beaches and pools. Sunscreen should not be the only protection method offered.
 Provide prompts and signs about sunscreen to remind people to reapply and to
encourage users to pair sunscreen with other methods of protection, such as
protective clothing and shade.
 Provide routine breaks during outdoor recreational or occupational activities to
reapply sunscreen.

Strategy 1C. Increase Availability of Sun Protection in Educational Settings

Because many children spend substantial amounts of time in schools and child care and
early learning centers, addressing overexposure to UV radiation in these settings is important.
Increasing shade in appropriate locations would provide passive protection from
overexposure to UV and help protect children from heat illness. Shade audits can help
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1352 Meg Watson, Erin Garnett, Gery P. Guy et al.

identify where to place trees and shade structures to be most effective. These audits can also
be used to identify areas that may already be shaded at key times of the day, so that activities
can be located in less UV-intense areas [308, 393]. CDC’s Shade Planning for America’s
Schools provides guidance and tools for using a shade audit to increase availability and use of
shade on school grounds [308].
Schools, colleges, and universities can also support sun protection in outdoor recreational
settings on campus, such as those used for athletics. Support for sun protection at athletic
events has the potential to benefit coaches, athletes, and students, as well as fans and
spectators of all ages. Athletes face many of the same risks as outdoor workers [250].
Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; businesses; early learning centers, schools and school districts, colleges, and
universities; community, nonprofit, and faith- based organizations; and individuals and
families, can support sun protection in educational settings in the following ways:

 Conduct shade audits to find solutions that fit a school or child care system’s budget,
maximize investment, and identify locations where trees and shade structures will be
most effective [308].
 Locate activities in shaded areas or schedule activities during low-UV times of day,
when feasible.
 Provide shade trees or structures in key locations identified through audits. Consider
public-private partnerships or grants to leverage resources.
 Include shade planning in the planning of new school facilities.
 Support sun protection in outdoor athletic settings, especially in high schools,
colleges, and universities.

Strategy 1D. Increase Availability of Sun Protection for Outdoor Workers

Sun protection is of particular importance for outdoor workers, who are at increased risk
of skin cancer. Appropriate protection strategies will depend on the occupation and the work
site. For example, shade for lifeguards can be relatively inexpensive and practical because
they are frequently stationary. For farmworkers, hats and protective clothing are more
appropriate. Sunscreen can make a person’s hands slippery and may interfere with the work
and safety of certain outdoor workers. A readily available hand-washing station to wash
hands after reapplying sunscreen can be considered, as well as other methods of protection.
Heavy, long-sleeved clothing may also increase risk of heat illness by trapping in heat. Sun
protection that takes into account the needs and preferences of workers will likely be most
successful.
Corporate risk managers are accustomed to reducing risk for workers, and they may be
especially attuned to risks of UV exposure. Providing shade for stationary workers or
providing shaded areas for breaks can also reduce the risk of heat illness, but shade alone may
not be sufficient, especially in high-UV areas with reflective surfaces, like snow, water, and
sand. Employers should consider multiple methods of sun protection for workers.
Key partners in prevention, such as businesses and employers, can protect outdoor
workers in the following ways:

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 Provide readily accessible sun protection to all employees, clients, contractors, and
other visitors to outdoor work sites, including uniforms or other protective clothing,
wide-brimmed hats to protect ears and backs of necks, and sunscreen.
 Encourage workers to reapply sunscreen throughout their shifts.
 Modify work environments, when feasible, by increasing the availability of shade
and modifying or covering reflective surfaces to reduce workers’ UV exposure.
 Adapt schedules to protect workers from overexposure to UV radiation, when
feasible. Employers may be able to schedule outdoor work during times of the day
and year when the UV Index is lowest. They may be able to rotate employees
through jobs that require significant sun exposure to avoid excessive exposure to
individual workers.

Goal 2: Provide Individuals with the Information They Need to Make

Informed, Healthy Choices about UV Exposure

Individuals need clear information that is based on the best and most current evidence to
make healthy choices about UV exposure and sun protection. Current skin cancer prevention
messages are broad-based and may not resonate with some groups. Most Americans equate
sun protection with sunscreen alone, so the importance of other strategies, such as shade and
protective clothing, should be emphasized. Messages should also clarify that sunscreen
should be used in combination with other protection, and they can emphasize the importance
of applying and reapplying ample amounts of sunscreen. Current messages also do not
address the need for vitamin D, potentially missing opportunities to highlight the importance
of skin cancer prevention in media coverage of vitamin D issues [395].
Many Americans lack a general knowledge or awareness about the risks associated with
sun exposure [63, 243, 244]. Some groups, especially blacks, the elderly, and people with less
education, may perceive themselves to be at low risk of skin cancer [63]. However, these
groups are at increased risk of being diagnosed with skin cancer at later stages [78, 96, 245].
More comprehensive collection and dissemination of information about skin cancer in the
United States would help underscore how frequently these cancers occur.
People need clear information about how to minimize their risk of skin cancer while
leading healthy, active lives and enjoying the outdoors. A substantial segment of U.S. adults
do not perceive cancer as preventable, and as a result, they may be less likely to engage in
skin cancer prevention practices, such as using sunscreen or covering up [246]. Lack of
understanding of the UV Index is also a barrier to making informed decisions about adequate
sun protection while outdoors [247, 248]. New technologies, such as evidence-based mobile
apps that provide information about sun protection directly to individuals, may provide
opportunities for direct messaging that is based on an individual’s behavior and skin type
[371, 372]. Evidence-based information that is accurate and consistent and provided in
various settings is an important part of reducing excessive UV exposure in the U.S.
population.
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Strategy 2A. Develop Effective Messages and Interventions for Specific Audiences
Mass media campaigns can be very effective at increasing skin cancer prevention
behaviors when they are part of multicomponent, communitywide interventions [396]. Before
conducting any large messaging campaigns, enhanced understanding of which messages will
resonate with specific groups is needed. Because mass media public health campaigns can be
expensive, evidence of the cost-effectiveness of different approaches will be valuable.
Ensuring that messages and the channels used to disseminate them have been proven to be
effective at changing attitudes and behaviors regarding skin cancer prevention in a specific
audience is important before making large investments [365, 397, 398]. Effective messaging
strategies designed to increase sun protection in various populations, including outdoor
workers, specific racial and ethnic groups, younger women, and men across all age groups,
are needed.
Some research has shown that messages that focus on appearance (such as increased risk
of wrinkles and skin aging) can be effective in reducing indoor tanning among college-aged
women [278]. However, more research is needed to determine the most effective messaging
strategies for other demographic groups. Messages should provide specific information on the
most effective methods for sun protection applicable for a specific audience, as well as
information about the limitations of some types of sun protection. Consistent, clear, and
tailored messages with prompts to make specific decisions can encourage people to take the
necessary steps to avoid excessive sun exposure when outdoors [230].
The UV Index was developed to provide such information, but it is not widely
disseminated or understood by the general public [247]. Resources like the UV Index need to
be promoted more widely through simple messages that include action steps for sun
protection, so people understand when and how to take precautions [247, 248]. Information
about the UV Index and the corresponding need for sun protection could be disseminated in
the same way as weather forecasts or air quality reports [247]. Then, people could routinely
consider the UV level just as they consider the weather when they are getting dressed and
preparing to go outdoors. Accurate, up-to-date information about UV levels and appropriate
protection measures could be provided through a variety of media, such as weather reports,
websites, personalized apps for smart phones, and possibly social media [248, 372].
Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; businesses and employers; health care systems, insurers, and clinicians; early
learning centers, schools, colleges, and universities; and community, nonprofit, and faith-
based organizations, can work together to develop and disseminate messages that accomplish
the following:

 Address the misperception that sunscreen alone is the most effective way to protect
skin from the sun.
 Improve sun protection, especially among adult men. Communications designed to
reach men can emphasize the importance of wide-brimmed hats, protective clothing,
and broad spectrum sunscreen with an SPF of 15 or higher on exposed areas when
outdoors for extended periods.
 Improve communication in recreational settings. Prompts in outdoor recreational
areas and in areas such as locker rooms may improve and normalize the use of sun
protection.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1355

 Improve communication about when and how to use sun protection. Simple
messages that include action steps for sun protection could be widely disseminated
across a variety of outlets, including weather reports and online tools.
 Increase understanding about effective use of sunscreens, including what type of
sunscreen to use, how much to use, and how often to reapply.
 Address appearance-related motives and the desire to be tan, especially among young
women.
 Help parents teach their children healthy sun protection behaviors from an early age.

Strategy 2B. Support Skin Cancer Prevention Education in Schools

Education about skin cancer prevention can be incorporated into school curricula and
linked with outdoor activities from an early age, when children are more receptive to such
messages. The education sector can be a key partner in supporting healthy choices for sun
protection through adolescence and young adulthood. It can also influence the community
through connections to families, alumni, and fans.
Educational and behavioral interventions to promote sun protection in child care centers
and in elementary and middle schools have been shown to be effective [295]. These
interventions vary greatly in intensity, duration, and the number of components included. In
addition to influencing children’s behaviors, these interventions may also influence the
practices of adults and caregivers both inside and outside the school.
Key partners in prevention, such as federal, state, tribal, local, and territorial governments
and early learning centers and schools, can support sun protection education in the following
ways:

 Implement skin cancer prevention interventions that are designed for and proven to
be effective in schools.
 Adopt lessons from interventions in daily routines, including during recess, physical
education, and outdoor extracurricular activities.
 Adapt proven skin cancer prevention interventions, such as the SunWise program,
and disseminate them in child care settings.

Strategy 2C. Integrate Sun Safety into Workplace Health Education and Promotion
Programs
Skin cancers cause a significant loss of productivity in the U.S. workforce [69, 70].
Incorporating sun-safety messages into comprehensive workplace health promotion and
protection programs can increase health, safety, and productivity and save money. For
outdoor workers, interventions designed to increase knowledge about sun protection;
activities designed to influence attitudes, behavior, and knowledge of workers; environmental
approaches designed to encourage sun protection (such as provision of shade); and policies
designed to support sun protection practices have been shown to be effective [306].
As an additional benefit, outdoor workers, such as lifeguards, coaches, and ski
instructors, may be able to teach and model skin cancer prevention to customers and clients in
their workplace [399]. Many of these workers have extended interaction with children and are
exposed to high levels of UV radiation.
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Employers of outdoor workers are required by OSHA to train employees on heat illness
prevention. Given the overlap between risk factors for heat illness and risk factors for sunburn
and skin cancer, current policies designed to prevent heat illness in the workplace could be
adapted to incorporate sun safety. Employees who work in direct sunlight through windows
or who drive regularly as part of their job are at risk of overexposure to UVA rays through
glass. Sun-safety messages and training are also appropriate for indoor workers.
Although indoor workers are not usually exposed to UV radiation during the work day,
they may be more likely to have intense, intermittent sun exposure during recreational time
on weekends or during vacations, putting them at increased risk of melanoma.
Key partners in prevention from the business sector, including employers, labor
representatives, risk managers, and employee wellness program managers, can support sun
protection education for workers in the following ways:

 Incorporate sun-safety information into existing workplace wellness programs. Heat

illness prevention programs could be adapted to incorporate sun safety.
 Provide training to outdoor workers about risks of exposure to UV radiation and the
signs and symptoms of overexposure.
 Encourage outdoor workers, such as lifeguards, coaches, and ski instructors, to be
role models and discuss the importance of using appropriate sun protection measures
with others.

Strategy 2D. Partner with Health Care Systems and Providers to Implement and
Monitor Use of Recommended Preventive Services for Provider Counseling on Skin
Cancer Prevention
Currently, the USPSTF recommends provider counseling on skin cancer prevention for
fair-skinned youth aged 10–24 years, and these services are generally covered by health
insurance [265, 277, 278]. However, skin cancer prevention is one of many competing
priorities that health care providers may need to discuss during a medical visit. Providers
could be supported to follow USPSTF guidelines in several ways. For example, tools
developed and disseminated to providers for behavioral counseling on skin cancer prevention
should include appearance-focused messages that have been shown to be effective in reducing
the intent to indoor tan. Some of the materials suggested by the USPSTF have already been
developed and tested [265].
Provider prompts that are part of checklists or EHR systems have been shown to increase
provider adherence to guidelines for other topics and may be applicable to skin cancer
prevention counseling recommended by the USPSTF [400-402].
Key partners in prevention from the health care sector, including health care systems,
insurers, and clinicians (such as dermatologists, primary care physicians, physicians’
assistants, and nurses), can do the following:

 Disseminate counseling messages in accordance with USPSTF guidelines.

 Include specific messages about avoiding indoor tanning when counseling young
fair-skinned adolescents and young women, among whom this behavior is common.

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Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; colleges and universities; health care systems, insurers, and clinicians; and
community, nonprofit, and faith-based organizations, can work together on the following:

 Include provider prompts for counseling to minimize UV exposure for fair-skinned

youth aged 10–24 in checklists or EHR systems.
 Develop and disseminate tools for providers to support behavioral counseling on skin
cancer prevention.
 Collect information on providers’ skin cancer prevention counseling practices to help
identify further opportunities for intervention and conduct behavioral research to
guide and refine tools available for counseling.

Strategy 2E. Establish Partnerships between Public and Private Sectors to Disseminate
Effective Messages About Skin Cancer Prevention
When paired with other interventions at the community level, communication campaigns
can be effective at increasing skin cancer prevention behaviors [295]. Partners in prevention
can work together across all sectors to provide consistent messages to a wide audience.
Dermatologists and dermatologic societies have helped raise public awareness of the issue of
skin cancer, and they and the health care sector will continue to be important contributors to
these efforts. Media and entertainment industries can also be vital strategic partners in efforts
to change social norms related to tanning behaviors. Community, nonprofit, and faith- based
organizations can help tailor communications to select populations or geographic areas,
directly or through innovative partnerships, such as working with various organizations in the
private sector.
To have a broad influence, communication campaigns need to be implemented and
sustained over an extended period. A comprehensive approach is needed to ensure that
opportunities for sun protection are increased along with communications. Leveraging public-
private partnerships may be a cost-effective strategy for such a campaign. Tailored messages
about sun protection may be more effective than broad- based messages to the general
population [230]. Thus, tools that allow for focused messages, such as social and electronic
media, can be important for strategic dissemination, especially to some audiences, such as
adolescents and young adults [372, 379].
Evaluations in Australia have shown that efforts to implement a multicomponent,
communitywide intervention (SunSmart Australia) were successful in reducing skin cancer
rates and were cost-effective [365, 397, 398]. Similar work to implement and evaluate a
large-scale, multicomponent, communitywide intervention is needed in the United States.
Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; businesses and employers; health care systems, insurers, and clinicians;
colleges and universities; community, nonprofit, and faith-based organizations; and
individuals and families, can work together in the following ways:

 Build coalitions to coordinate the efforts of partners across sectors to maximize

communication efforts and use consistent messages.
 Work with media and entertainment industries and the public at large to promote an
understanding that tanned skin is not healthy.
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 Leverage public-private partnerships to support and sustain communications

campaigns.
 Explore new technologies for sharing sun protection messages to specific audiences.

Strategy 2F. Enhance Ongoing Engagement of Federal Partners to Advance Our

Nation’s Skin Cancer Prevention Efforts
This Call to Action on skin cancer prevention is the result of a collaborative effort across
HHS in partnership with other federal departments representing diverse sectors, such as the
environment, recreation, occupational health and safety, and trade (see Appendix 5). Using
this collective leadership and taking specific actions that align with this Call to Action and
with international consensus guidelines will help to advance our nation’s skin cancer
prevention efforts. Consistent skin cancer prevention messages across federal agencies will
also help build consensus behind messaging and streamline the implementation efforts of
partners in the skin cancer community.
Many public health and community initiatives overlap with skin cancer prevention,
providing opportunities for collaboration. For example, public health messages about physical
activity can incorporate sun protection, and sun protection messages can emphasize the
importance of regular physical activity and healthy ways to obtain vitamin D. Heat illness
campaigns can incorporate sun protection messages as well. Continued interagency
coordination is needed to ensure that skin cancer prevention is aligned with other important
public health priorities, such as nutrition, physical activity, and obesity prevention.
Partners in prevention in the federal government, such as HHS and its agencies (e.g.,
CDC, FDA, NIH/NCI), EPA, and OSHA, can work together on the following:

 Reexamine and update current sun protection messages, ensuring consistency across
and within agencies.
 Identify opportunities to promote and enhance cross-agency and departmental
collaborations to plan, implement, and disseminate skin prevention messages and
activities.

Goal 3: Promote Policies That Advance the National Goal of Preventing Skin
Cancer

Efforts to change social norms and increase knowledge about UV exposure are effective
if they are supported by policies that promote healthy behaviors. Policies can establish
support for sun protection from officials, managers, and employees, and they can be used to
set priorities for resource allocation and to promote institutional changes that lead to
increased sun protection. For this reason, policies and procedures in schools, health care
facilities, communities, and workplaces and at state and national levels can have a significant
effect on the success or failure of other skin cancer prevention efforts. Policies should also be
routinely evaluated to assess their effectiveness, compliance, enforcement, and feasibility,
especially if resources change. Skin cancer prevention is one of many other important
concerns, and it should be considered in tandem with policies designed to address other health
priorities, such as nutrition, physical activity, and obesity prevention.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1359

Strategy 3A. Support Inclusion of Sun Protection in School Policies, Construction of

School Facilities, and School Curricula
Rather than relying on individual schools to incorporate sun protection policies and
education, states and school districts can support incorporation of sun-safety education in the
required curriculum, as in Arizona and Florida [303, 325]. Such policies will be most
effective if they are accompanied by input from, and professional development for, teachers
or other school staff who are delivering the sun-safety curriculum [403]. Some school policies
can create barriers to sun protection by prohibiting or limiting the use of sunscreen or hats and
sunglasses [300]. These barriers can be addressed through policies that specifically allow or
encourage students to wear hats, protective clothing, and sunscreen during outdoor activities.
School, school district, and state policies can also encourage the use and planning of shade
structures and could include requirements that school activities be scheduled in areas with
more shade and at less UV-intense times of day. In addition, schools and school districts can
take UV exposure into consideration when new play structures, school buildings, or child care
and early learning centers are being planned as a way to ensure that shaded areas are provided
for outdoor activities.
Parents are influential members of school communities, and they can work with local
schools, school boards, and school administrations to make sure that appropriate, well-
designed shade is considered when new schools are planned and that older schools are
retrofitted. Parents and parent organizations may also be able to influence school policies on
children’s personal use of sunscreen, hats, and clothing while at school; on use of the UV
Index to make decisions about outdoor activities; and on the inclusion of skin cancer
prevention in health or science curricula.
Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; early learning centers, schools and school districts, colleges, and universities;
community, nonprofit, and faith-based organizations; and individuals and families, can do the
following:

 Incorporate sun-safety education into required school curriculum at the district or

state level.
 Address barriers to sun protection in schools.
 Encourage the use of shade through shade-planning policies.
 Involve parents and parent groups in the development and promotion of skin cancer
prevention policies in schools.

Strategy 3B. Promote Electronic Reporting of Reportable Skin Cancers and Encourage
Health Care Systems and Providers to Use Such Systems
Health care providers who diagnose or treat melanomas (both in situ and invasive) are
required to report cases to a central cancer registry in all 50 states and the District of
Columbia. Although hospitals generally have reporting systems established, many providers
in private practice who diagnose skin cancers, including dermatologists and primary care
doctors, are unaware of requirements to report melanomas or are unsure of how to report to
their state or local registry [384, 385]. Additional steps to increase awareness among
providers and to make it easier for providers to report this information are likely to increase
reporting. Implementation of EHR systems for data collection can help increase reporting
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through incentives provided by CMS. In addition, electronic reporting directly from

pathology laboratories to a central cancer registry has been increasing, and this practice
significantly improves capture of melanoma cases [404]. Automation of reporting and other
improvements in health information systems may increase ease and convenience of reporting.
Large national organizations, such as the American Academy of Dermatology, can help
to educate health care providers about mandatory reporting requirements in their states. First
steps can include compiling state- level reporting procedures in an easy-to-reference format
and then disseminating these to providers through affiliated state health care provider
organizations and continuing medical education programs.
Key partners in prevention, such as federal, state, tribal, and territorial governments and
health care systems, provider organizations, insurers, and clinicians, can do the following:

 Increase awareness of reporting requirements, especially among primary care doctors

and dermatologists in private practice.
 Provide clear guidance and specific action steps for reporting melanomas.
 Adhere to policies that require reporting of melanomas (both in situ and invasive).

Strategy 3C. Incorporate Sun Safety into Workplace Policies and Safety Trainings
Employer policies can substantially reduce harms from overexposure to UV in the
workplace for employees, contractors, clients, and other visitors to the work site by ensuring
that strategies for reducing UV exposure among workers are consistently and fairly applied.
Workers or their representatives should be involved in any decisions about policies that
incorporate sun protection to ensure that these policies are functional and effective. Sun
protection policies that take into account the job tasks and other safety and health risks will be
the most useful. Employers who encourage behavioral changes and communicate well with
their workers will increase the success and adoption of new policies.
Employers should also consider policies to reduce UV exposure from other
environmental factors, such as reflective surfaces or the use or presence of substances that
increase sensitivity to UV radiation, such as certain tars, dyes, or pesticides [405]. Policies
that call for work to be scheduled at times of day or year that are less UV-intense can reduce
exposure. In addition, outdoor workers are not the only ones at risk of excessive UV exposure
on the job. Workers in certain occupations, such as drivers, may encounter substantial UV
exposure through windows and may also be at increased risk. Sun protection policies for
these workers can include provision of equipment or modifications to glass in windows [406].
Key partners in prevention in federal, state, tribal, local, and territorial governments and
in the business sector, such as employers, labor representatives, and risk managers, can enact
policies that accomplish the following:

 Support provision of sun protection clothing and equipment for workers, such as
long-sleeved shirts and long pants; hats that shade the face, ears, and back of the
neck; and broad spectrum sunscreen with an SPF of 15 or higher.
 Modify the work environment where feasible to minimize UV exposure.
 Take into account UV levels when scheduling work hours when feasible.
 Encourage the rotation of workers in UV-intense positions to reduce UV exposure to
individual employees.

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Strategy 3D. Support Shade Planning in Land Use Development

Strategic shade planning can maximize the amount of shade provided to key areas of
activity during the times of the day and months of the year with the highest UV levels. If
placement and materials of shade structures are not carefully considered, shade may not
provide sufficient protection, or the structures may not provide shade to the intended space
during times when UV radiation is most intense because the angle of the sun varies
throughout the day and year. In addition, shade needs to complement the way an outdoor
space is used. For example, a shade structure with a low roof built on an area used to play
volleyball or a tree planted in the middle of a space used to play soccer or kickball is clearly
incompatible with the intended use of the space.
State or federal decision makers could support efforts to plan and build effective shade
structures in communities, taking into account local needs for shade and recreational areas.
One model is the Safe Routes to Schools program (http://www.saferoutesinfo.org/), which
provides grants to schools and school districts from federal transportation funding. Any
program used should be evaluated to ensure that the funding is effective in reducing key
adverse outcomes.
Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; businesses and employers; early learning centers, schools, colleges, and
universities; community, nonprofit, and faith-based organizations; and individuals and
families, can do the following:

 Support shade planning in the overall process of designing and building new outdoor
public spaces, such as parks, playgrounds, and schools.
 Implement policies to support increased provision of shade structures in local
communities.

Goal 4: Reduce Harms from Indoor Tanning

Indoor tanning devices, classified by WHO in 2009 as a known human carcinogen,

expose the skin to intense levels of UV radiation. As discussed in the “Reducing the Risks of
Skin Cancer” section, indoor tanning is of strong concern because it has been estimated to be
related to more than 400,000 cases of skin cancer in the United States each year: 245,000
BCCs, 168,000 SCCs, and 6,000 melanomas [124]. In addition to increasing skin cancer risk,
indoor tanning can cause burns to the skin, acute and chronic eye diseases if eye protection is
not used, and, if tanning devices are not properly sanitized, skin infections [139-141]. About
one out of every three non-Hispanic white women aged 16–25 years stated that they had
tanned in the past 12 months, and many said they do so frequently [134].
Unlike sun exposure, indoor tanning provides concentrated UV exposure regardless of
geographical location, time of year, or time of day. Indoor tanning also exposes areas of the
body not normally exposed to intense UV radiation, further increasing risk [115]. However,
indoor tanning can be completely avoided, which allows for points of intervention beyond
those used to reduce sun exposure.
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Strategy 4A. Monitor Indoor Tanning Attitudes, Beliefs, and Behaviors in the U.S.
Population, Especially among Indoor Tanners, Youth, and Parents
Increased data on attitudes and beliefs about indoor tanning would help guide the
development of policies, tailored messages, and programs for indoor tanning prevention and
cessation. Improved understanding of indoor tanners’ motivations, especially subgroups that
have been studied less frequently, such as males or nonwhite tanners, can help guide the
development of messages and policies to address tanning. Monitoring the attitudes and beliefs
of parents and their children is particularly important as part of efforts to reduce indoor
tanning among minors, given parental influence on youth tanning behaviors. Monitoring
attitudes and beliefs can also provide information on the effect of indoor tanning policies on
attitudes and social norms.
Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; health care systems, insurers, and clinicians; colleges and universities; and
nonprofit organizations that conduct science, can support efforts to do the following:

 Examine motivations for indoor tanning among frequent and event tanners, and
examine motivations for not tanning among never-tanners.
 Examine children’s and parents’ attitudes and beliefs about indoor tanning.
 Conduct research to better quantify the effect of indoor tanning legislation on
attitudes, behavior, and social norms.

Strategy 4B. Continue to Develop, Disseminate, and Evaluate Tailored Messages to

Reduce Indoor Tanning among Populations at High Risk
Conflicting messages to the public from industry and health agencies cause confusion
about the risks of indoor tanning. Research shows that appearance-based messaging and
behavioral counseling appear to be effective in reducing tanning behaviors among college-
aged women [263-266]. This strategy has not been tested in other populations of indoor
tanners. Messages that focus on the prevalence of an unhealthy behavior may unintentionally
normalize or reinforce the behavior [407]. For this reason, messages to reduce indoor tanning
may be most effective if they emphasize that most young women do not indoor tan but
instead choose to embrace their natural skin color [279]. This strategy also needs to be tested.
Although teens and young adult women are most likely to engage in indoor tanning, other
populations also indoor tan. Having a mother who tans is a strong predictor of adolescent
girls’ initiation of tanning, so understanding how to present messages to mothers is also
important [408].
Outcomes of effective messaging interventions to reduce indoor tanning should be
evaluated in order to provide information on unintended consequences, such as increased
tanning outdoors. Messaging strategies may vary for different populations of indoor tanners.
People who have accumulated UV exposure from indoor tanning or sunbathing are at
increased risk of skin cancer, and messages focusing on the importance of awareness and
early detection of potentially malignant lesions may be appropriate.
Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; health care systems, insurers, and clinicians; colleges and universities; and
nonprofit organizations that conduct science, can do the following:

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 Develop and test messages for populations at high risk, including current tanners and
those most likely to initiate indoor tanning.
 Develop and test messages for parents.
 Evaluate the long-term effects that messages have on intentional tanning behaviors,
both indoor and outdoor.

Strategy 4C. Support Organizational Policies That Discourage Indoor Tanning by

Adolescents and Young Adults
Colleges and universities sometimes have agreements with tanning salons that allow
students to use university-sponsored debit cards to pay for tanning services [409]. Many
colleges and universities have adopted campus polices that discourage alcohol and tobacco
use, and similar types of policies could address indoor tanning. Colleges and universities
could examine and address the financial incentives, campus policies, systems, and social
norms that directly or indirectly encourage indoor tanning in order to create an educational
environment that is supportive of student health and well-being. Reducing availability of
tanning services on campus could lead to reduced use of indoor tanning by students and to
changes in social norms related to tanning.
Key partners in prevention in the education sector, especially high schools, colleges, and
universities, can address indoor tanning on campus in the following ways:

 Adopt campus policies that discourage indoor tanning by their students on campus.
 Reconsider campus practices that may encourage indoor tanning, such as the use of
school-sponsored debit cards; financial arrangements between student organizations
and members of the indoor tanning industry; and on-campus advertising, incentives,
and promotional materials for indoor tanning.
 Develop an action plan to promote campuswide UV protection strategies.

Strategy 4D. Enforce Existing Indoor Tanning Laws and Consider Adopting
Additional Restrictions
The younger the age of indoor tanning initiation, the more the risk increases [114, 116,
118, 119]. WHO classifies tanning beds as Class I human carcinogens and recommends that
they never be used by anyone younger than age 18 years [27]. Australia and most western
European countries currently prohibit indoor tanning among minors [328, 369]. Currently, at
least 44 states and the District of Columbia have some kind of law or regulation related to
indoor tanning [329-334], including bans on indoor tanning for minors under a certain age,
ranging from 14 to 18; laws requiring parental accompaniment or parental permission; or
regulations that otherwise reduce harms (such as requiring eye protection). FDA now requires
that indoor tanning devices carry a visible black box warning on the device that explicitly
states that the sunlamp product should not be used on people younger than age 18 years [349].
In many locations, such as gyms and apartment complexes, indoor tanning devices are
available for use without the supervision of a trained operator. Although FDA regulations do
not distinguish between tanning devices used in supervised and unsupervised settings,
unsupervised tanning devices are often not held to the same industry standards as indoor
tanning salons, which may be licensed by the state or which may commit to certain standards
as members of indoor tanning associations.
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The inherent risks of indoor tanning are high, but unsupervised tanning locations provide
none of the consumer protection mechanisms created by states and the indoor tanning
industry to reduce risks of acute harm to consumers. The availability of unsupervised use may
also limit the effect of efforts to reduce indoor tanning among minors because of difficulty
enforcing current or future regulations.
Key partners in prevention in the government sector, such as federal, state, tribal, local,
and territorial governments, can do the following:

 Ensure that facilities operate indoor tanning devices in compliance with established
health and safety regulations.
 Adopt evidence-based policies such as age restrictions for minors.
 Investigate and address specific allegations of deceptive advertising by indoor
tanning salons.
 Support efforts to implement and disseminate effective training programs for
operators of indoor tanning devices.
 Educate consumers on FDA warnings and contraindications for indoor tanning
device use.

Industry groups could also help to reduce the harms of indoor tanning in the following
ways:

 Ensure compliance with laws that discourage deceptive and misleading

advertisement.
 Increase communication to consumers on the risks of indoor tanning.
 Ensure compliance with federal and state regulations (such as UV exposure
guidelines, training documentation, and warning labels).

Strategy 4E. Address the Risks of Indoor Tanning with Improved Warning Labels and
Updated Performance Standards
Current warning labels are often not easily visible to customers, or they may be
disregarded [341, 410]. Public health messages should directly address competing health
claims from advertising and should be clear that risks of indoor tanning are substantially
higher than limited sun exposure. Strengthening efforts to communicate the risks of indoor
tanning to consumers at the point of use may be an effective strategy. Stronger warning
labels, visible to consumers and with information describing the risks of indoor tanning,
combined with other outlets of communication about the risks of indoor tanning, could help
make consumers aware of the danger and change perceptions about intense exposure to UV
radiation [352, 411].
Manufacturers of indoor tanning devices (also known as sunlamp products) currently are
required to certify that their products comply with the FDA Performance Standard for
Sunlamp Products (21 CFR 1040.20) [348]. FDA is working to reflect current science on the
risks of indoor tanning, improve the visibility and readability of the warning label, and update
and promote compliance with the performance standard.
As part of FDA’s recent reclassification of indoor tanning devices to Class II medical
devices (moderate to high risk), manufacturers will be required to do the following:

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 Include a visible black box warning on the device that people younger than age 18
years should not use these devices.
 Receive premarket notification 510(k) clearance from FDA for newly marketed
devices (which were previously exempt from any premarket review).
 Show that their products have met certain performance testing requirements.
 Address certain product design characteristics.
 Provide comprehensive labeling that presents consumers with clear information on
the risks of use [349, 350].

Key partners in prevention in the government sector, such as federal, state, tribal, local,
and territorial governments, can do the following:

 Require indoor tanning locations to prominently display the health warnings about
indoor tanning in their facilities and provide verbal and written explanations of health
risks to customers.
 Test different messages for warnings and signs to determine how to most accurately
convey risk to consumers.
 Update performance standards for indoor tanning devices to reflect current science.

Goal 5: Strengthen Research, Surveillance, Monitoring, and Evaluation

Related to Skin Cancer Prevention

This Call to Action proposes both the expansion of existing strategies and the creation of
new strategies for skin cancer prevention in the United States. As with all public health
initiatives, continuing to build on existing research and surveillance activities is critical to
future success in reducing the incidence of skin cancer. As these strategies are implemented,
skin cancer incidence and death rates, as well as trends in risk behaviors related to skin
cancer, will need to be monitored.
Monitoring and evaluation of interventions will allow researchers to learn from the
process of implementation, address weaknesses quickly, track progress over time, and
ultimately determine the success of interventions in changing behavior and preventing
disease, as well as their cost-effectiveness. Federal, state, and local public health agencies,
nonprofit organizations, and academic researchers can work together to conduct this
important research and guide future interventions.

Strategy 5A. Enhance Understanding of the Burden of Skin Cancer and Its
Relationship with UV Radiation
More information is needed on the epidemiology and risk factors for skin cancer and the
risks of different levels of UV exposure. Epidemiologic studies of skin cancers are limited by
underreporting of early-stage melanomas. Increased reporting of melanomas will improve
surveillance of melanoma incidence rates. Overdiagnosis of early-stage lesions has also been
raised as an issue affecting epidemiologic studies, and this problem is suspected because
incidence rates have increased disproportionately to death rates. Better understanding of the
natural history of melanoma would help improve accuracy of diagnoses and therefore
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surveillance. More information on patient history of UV exposure and behavior can help
identify those at risk and provide data on risk factors and prevention.
Lack of national surveillance of BCC and SCC presents another barrier to measuring the
burden of skin cancer, which also inhibits the ability to measure outcomes of interventions.
Data on these cancers can potentially be collected through EHRs as part of sentinel systems in
limited areas. Also, more information is needed to determine the effectiveness of screening
among the general population, as well as the relative benefits and cost benefits of various
screening strategies, particularly in a diverse population like that of the United States, where
wide variations exist in environmental UV exposure and the geographic latitude (and thus the
UV intensity) of where people live.
Although national surveillance of these cancers is not feasible, large health care systems
or other entities could use EHRs to establish systems for collecting their own data. Modeling
studies could be used to better estimate trends in the incidence of BCC and SCC. Economic
analyses could be used to quantify the costs of melanoma, BCC, and SCC. These types of
analyses can incorporate both direct and indirect economic costs, as well as human costs.
Key partners in prevention, such as federal, state, tribal, local, and territorial
governments; health care systems, insurers, and clinicians; colleges and universities; and
nonprofit organizations that conduct scientific research, can do the following:

 Enhance understanding of the burden of skin cancer, including incidence and death
rates from melanoma, BCC, and SCC.
 Continue to monitor the effects of UV exposure on human health.
 Conduct economic analyses to quantify the effects on disease and death rates,
productivity, and health care costs.
 Increase research efforts to determine population groups most likely to benefit from
skin cancer screening and early detection and potential effects on mortality,
especially for populations at high risk.

Strategy 5B. Evaluate the Effect of Interventions and Policies on Behavioral and
Health Outcomes
Although sufficient evidence exists to take action to prevent skin cancer, evaluating the
immediate, intermediate, and long-term effects of skin cancer prevention policies and
interventions remains critical to their success. Evaluations should examine changes in sun
protection behavior and whether they are sustained over time, as well as the effect of
interventions on incidence of sunburn over time and on more distant outcomes, such as skin
cancer incidence. Ongoing monitoring and periodic evaluation provide the opportunity to
learn from and improve on existing interventions and policies and adjust if necessary.
Future interventions and policies should strive to include an impact evaluation
component, whenever feasible. Evaluations can help employers set priorities and allocate
resources toward sun protection strategies that work. Employee education interventions can
be evaluated for efficacy across cultures and languages to address the diversity of the
workforce.
Key partners in prevention, such as public health agencies, colleges and universities, and
nonprofit organizations that conduct scientific research, can evaluate interventions in the
following ways:

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 Examine the duration of the effects of interventions in child care and early learning
centers and elementary and middle schools on sun protection behaviors and sunburn
later in life.
 Continue to evaluate the effectiveness of sun protection interventions in high school,
college, and university settings as evidence evolves.
 Evaluate whether clinicians follow current USPSTF recommendations on counseling
for sun protection among fair-skinned youth aged 10–24 years.
 Determine the health effects and cost-effectiveness of efforts to promote sun
protection among outdoor workers.
 Evaluate the effects of communitywide shade policies.
 Measure the effect of communication interventions on attitudes and behaviors.

Strategy 5C. Build on Behavioral Research and Surveillance Related to UV Exposure

Ongoing surveillance of sun protection behaviors and general outdoor UV exposure in
national surveys would help to measure progress over time and provide direction for future
interventions. To fully understand how best to support the prevention of excessive outdoor
sun exposure, more information is needed on how much (or how little) outdoor UV exposure
the U.S. population in general and subpopulations specifically are receiving.
More in-depth behavioral research is needed in addition to surveillance. Many
interventions to date have used a multicomponent approach that combines strategies directed
at individuals, mass media campaigns, and environmental and policy changes. Research is
needed to determine the contribution of individual components to the observed behavior
change. Determining which components within multicomponent interventions are critical to
eliciting behavior change would help prioritize and maximize use of limited resources.
Current evidence suggests that age restrictions may be more effective than parental
permission and accompaniment laws at reducing indoor tanning among minors [335], but
more evidence is needed.
States could consider including a question on indoor tanning frequency on their YRBS
questionnaire, which would allow for state-level estimates of the prevalence of indoor tanning
among high school students. Similarly, a question on indoor tanning frequency could be
added to surveys such as the BRFSS to monitor indoor tanning in adults. If states begin
collecting data on indoor tanning, they can track changes in indoor tanning behaviors over
time as new policies are implemented. Survey findings can provide evidence on the
effectiveness of indoor tanning policies and can guide future policy decisions at state and
national levels.
Key partners in prevention, such as public health agencies, colleges and universities, and
nonprofit organizations that conduct scientific research, can expand upon current research in
the following ways:

 Increase understanding of indoor tanning behaviors, including when, where, and how
people tan.
 Increase understanding of motivations to tan or not to tan.
 Strengthen collection of information on indoor tanning on national surveys, such as
the state YRBS and the national and state BRFSS.
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 Monitor changes in indoor tanning behaviors and social norms over time to evaluate
the effectiveness of legislation or regulations to reduce intentional tanning, as well as
to guide future efforts.
 Collect and examine information on outdoor tanning to monitor unintended
consequences of indoor tanning restrictions.
 Collect and examine information on average outdoor UV exposure in the U.S.
population.

Strategy 5D. Quantify the Prevalence of Tanning in Unsupervised Locations

Currently, little is known about the prevalence of, access to, and attitudes toward
unsupervised tanning devices. The availability of these devices creates a barrier to successful
implementation of indoor tanning controls and limits the effect of efforts designed to protect
minors from the harms of indoor tanning. Efforts are needed to quantify how many tanning
devices are available for use outside of tanning salons (such as in gyms, apartment
complexes, or beauty salons), where they are located, who has access to them, and whether
they are being used in accordance with FDA performance standards. Similarly, more
information is needed about the prevalence of home ownership of an indoor tanning device
and the standards of devices found in homes.
Key partners in prevention, such as public health agencies, colleges and universities, and
nonprofit organizations that conduct scientific research, can do the following:

 Quantify the prevalence of indoor tanning device use in unsupervised locations.

 Describe patterns of use of unsupervised tanning devices.
 Provide information about indoor tanning device use in private settings, such as
homes.
 Quantify the health effects of unsupervised indoor tanning device use.

CONCLUSION
With this Call to Action, the U.S. Surgeon General emphasizes the need to act now to
solve the major public health problem of skin cancer. Despite efforts to address skin cancer
risk factors, skin cancer rates, including rates of melanoma, have continued to increase in the
United States [1, 13-17]. More than one-third of Americans report being sunburned in the past
year [226], and indoor tanning is common among some groups [132, 133]. We need to work
together to address skin cancer as a public health problem. We know that comprehensive,
communitywide efforts to prevent skin cancer can work, with adequate support and a unified
approach.
To reduce skin cancers in the population, people must get the information they need to
make informed choices about sun protection, policies must support these efforts, youth must
be protected from harms of indoor tanning, and adequate investments need to be made in skin
cancer research and surveillance. Achieving these goals will not be a small task. It will
require dedication, ingenuity, skill, and the concerted efforts of many partners in prevention
across many different sectors. Many of these partners are already enthusiastically involved,
but greater coordination and support are needed to increase the reach of their efforts. The

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strategies outlined in this document are the next steps. We must act with urgency to stop the
ever-increasing incidence of skin cancers in the United States.

APPENDIX 1: SCOPE AND DEFINITIONS

This document focuses on the three most common types of skin cancers: basal cell
carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma, which together account
for more than 99% of skin cancers [41, 42]. This document also focuses only on cutaneous
skin cancers and not other types, such as SCCs that occur on the genitals (and which
generally have different risk factors, notably human papillomavirus) or noncutaneous types of
melanoma, such as ocular.

Types of Skin Cancer

Skin cancer arises from the uncontrolled growth of different types of cells found
normally in skin. The most common types are BCC and SCC. Although these cancers are
rarely deadly, they are very common and potentially disfiguring, and they often recur. People
diagnosed with SCCs and BCCs, especially at younger ages, are at increased risk of
subsequent primary cancers, possibly for genetic reasons [412, 413].

Basal Cell Carcinomas

BCCs arise from the cells in the bottom, or basal, layer of the epidermis. BCCs tend to
occur on skin that is chronically exposed to the sun, such as the face, head, and neck, but they
also frequently occur on the trunk of the body [43]. Because it frequently occurs on the face
and head, BCC and its treatment can result in noticeable disfigurement. This disease can be
classified into five subtypes: nodular, ulcerating, pigmented, sclerosing, and superficial [414].

Squamous Cell Carcinomas

SCCs arise from squamous cells in the outer layers of the epidermis. Similar to BCCs,
SCCs usually occur in prominent, sun-exposed areas, like the face, head, and neck [43]. SCCs
often arise from actinic keratoses, which are rough, scaly patches that occur on sun-exposed
areas [414].

Melanomas
Melanomas develop from melanocytes, the melanin-producing cells that give skin and
eyes their color [32]. These cancers can arise in the skin (cutaneous melanoma) and less
frequently in the eye (ocular melanoma) or mucous membranes. Melanoma can be classified
into several subtypes: nodular and superficial spreading melanomas, which can occur in any
location on the body; lentigo maligna melanoma, which is usually found on the head, neck,
and face; and acral lentiginous melanoma, which arises on the palms of the hands and soles of
the feet, and under nails [32, 57].
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Types of Ultraviolet Exposure

Overexposure
Overexposure or excessive exposure, as used in this document, means any ultraviolet
(UV) exposure that is likely to increase a person’s risk of skin cancer, without conferring
benefits beyond those that can be achieved through more limited outdoor exposures.
Overexposure frequently results in tanned or sunburned skin. Seeking a tan (whether indoor
tanning or outdoor sunbathing) is considered overexposure for the purpose of this document.
Sunbathing and indoor tanning are common methods of skin tanning.
Unnecessary or avoidable exposures, as used in this document, are those UV exposures
that can be easily avoided. Seeking a tan for cosmetic purposes, whether indoors or outdoors,
is considered an unnecessary exposure. Some outdoor exposures are necessary as part of daily
routines, such as walking for transportation, being outdoors for physical activity, or working
outdoors. Sun protection can be used to reduce risks of unavoidable exposure.
Erythema, or sunburn, is defined as an acute cutaneous inflammatory reaction to UV
exposure, with classic signs of inflammation, such as redness, warmth, tenderness, and edema
[415]. Sunburn is a clear indication of overexposure. However, a person can have suffered
overexposure even in the absence of signs of sunburn [128, 183, 184].

Limited Exposure
Limited exposure to UV radiation, as used in this document, refers to very brief (5–15
minute) outdoor exposures received during the course of daily activities or exposures at very
low UV Index levels (2 or less). For most people, the risk of skin cancer from such limited,
incidental UV exposure is likely low. However, damage from UV radiation is cumulative, so
even limited exposures can result in harm over time.
The risks and benefits of UV exposure vary by individual, as well as by environmental
conditions, such as weather, altitude, latitude, and the presence of reflective surfaces, such as
snow, sand, or water. For a very fair-skinned person in the summer sun at low latitudes or
high altitudes, even a very short time outdoors could lead to overexposure, especially in
highly reflective environments, such as areas that are snowy, sandy, or near bodies of water
[135-137, 181].
Conversely, for a very dark-skinned person, short exposures may not be enough to
achieve sufficient vitamin D levels, especially in the winter in northern climates [178]. Skin
pigmentation can also vary within individuals. For example, some people have vitiligo, a
condition in which the melanocytes in some areas of the skin do not function, resulting in
patches of depigmented skin that are sensitive to UV radiation [416]. Current evidence
indicates that UV exposures below the amount needed to induce sunburn, including exposures
that result in a tan, can also increase skin cancer risk [128, 183, 184, 292, 417].

APPENDIX 2: SIGNS AND SYMPTOMS

OF SKIN CANCER

Melanomas diagnosed at earlier stages are much more treatable than those diagnosed at
later stages [6, 31]. Anyone can get skin cancer, and everyone should know the symptoms of

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this disease. Changes in the skin, such as a new growth, a sore that does not heal, or a change
in an existing mole, are the most common signs of skin cancer.
The characteristics of malignant melanoma are often described as the A-B-C-D-Es of
melanoma:

A = asymmetrical. Does the mole or spot have an irregular shape with two halves that
look very different?
B = border. Is the border irregular or jagged?
C = color. Is the color uneven?
D = diameter. Is the mole or spot larger than the size of a pea?
E = evolving. Has the mole or spot changed during the past few weeks or months?

Not all skin cancers look the same. If a person notices a change in the skin, such as a new
growth, a sore that does not heal, a change in an old growth, or any of the A-B-C-D-Es of
melanoma, he or she should consult a doctor.

APPENDIX 3: SKIN CANCER SCREENING

Some groups recommend periodic skin cancer screening,12 either by a health care
provider or by self- examination [290, 291]. Consistent and regular screening identifies
melanomas that are, on average, thinner than those found during usual care. Whether
detection of these lesions leads to fewer cases of disease or death is unknown [292]. For this
reason, the independent U.S. Preventive Services Task Force (USPSTF) has stated that
current evidence is insufficient13 to recommend skin cancer screening by primary care
providers among the general U.S. adult population. On May 15, 2014, the USPSTF released a
draft research plan that will be used to guide a systematic review of the evidence by
researchers [293]. Despite the insufficient evidence supporting screening in the general
population, an estimated 87% of Americans believe that skin cancer screening is
recommended [244].
Although screening is not currently recommended, providers should remain alert to
suspicious lesions. The USPSTF states the following: “Clinicians should remain alert for skin
lesions with malignant features noted in the context of physical examinations performed for
other purposes. Asymmetry, border irregularity, color variability, diameter greater than 6 mm
(ABCD criteria), or rapidly changing lesions are features associated with an increased risk for
cancer. Biopsy of suspicious lesions is warranted.”
A recent study conducted in one state in Germany found that population-based screening
was associated with reduced melanoma death rates [418]. Although the results are promising,
some of these reductions occurred before implementation of the screening portion of the
study, which suggests that some of the reduction could be due to increased awareness among
the population as a result of the communications campaign related to the screening program.
Screening did not reduce melanoma deaths in a recent study in Switzerland, a country with a
particularly high incidence of cutaneous melanoma, implying that other primary and
secondary strategies may be more effective [419].
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Further studies are needed to determine the effectiveness of screening among the general
population. Research is also needed to determine the relative benefits and cost benefits of
various screening strategies, particularly in a diverse population like that of the United States,
where wide variations exist in environmental UV exposure and the geographic latitude (and
thus the UV intensity) of where people live.

APPENDIX 4: SUCCESS STORIES IN SKIN

CANCER PREVENTION
Federal Resources for Skin Cancer Prevention in Schools

A 2002 CDC report, Guidelines for School Programs to Prevent Skin Cancer [420],
reviewed both the scientific evidence on skin cancer prevention and current school practices.
It suggested guidelines for a comprehensive approach to skin cancer prevention in schools
through policies, environmental change, education, family involvement, professional
development, health services, and evaluation.
To support schools’ implementation of these guidelines and to help schools create and
maintain a physical environment that would support sun safety by ensuring that school
grounds have adequate shade, CDC subsequently created a manual for schools called Shade
Planning for America’s Schools [308]. The manual outlines steps that school communities
can take to develop a comprehensive approach to reducing the risk of skin cancer, including
strategies for providing shade at schools, an overview of how to plan a shade project, success
stories from schools and school districts that have completed shade planning projects, and
information on how to conduct a shade audit. CDC also created the Sun Safety for America’s
Youth Toolkit, a resource for local comprehensive cancer control programs [421]. It outlines a
step-by-step process for program planning, suggests strategies for implementing sun
protection education in schools, and provides sample evaluation questions [421].

RAYS Skin Cancer Prevention Program Shines Bright for New Mexico
Schoolchildren

The RAYS (Raising Awareness in Youth About Sun Safety) Project provides funding
and technical support to elementary schools and community organizations across New
Mexico to implement sun-safety education. It is supported by the New Mexico Department of
Health Comprehensive Cancer Program. The RAYS Project uses various evidence-based and
preapproved curricula, including Sunny Days, Healthy Ways, the SunSmart Project, and the
U.S. Environmental Protection Agency’s (EPA’s) SunWise Program.
Many RAYS Project schools have changed policies on their campuses to support sun
protection, including changing recess times to avoid peak UV exposure, allowing students to
wear hats and sunglasses, and providing shade on playgrounds. English- and Spanish-
language materials have been developed for parents and have been distributed to various
audiences statewide. RAYS Project contractors also include sun-safety education from
evidence-based programs in other health-related school and community events [422].

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For more information about the RAYS Project and other CDC-supported Comprehensive
Cancer Control Programs working on skin cancer prevention, visit http://www.cdc.
gov/cancer/ncccp/pdf/success/ SuccessStories.pdf.

City of Toronto Shade Policy

The City of Toronto in Ontario, Canada, enacted a citywide shade policy in 2007. The
shade policy states that providing shade can be an effective way to reduce exposure to UV
radiation and its associated health risks, such as skin cancer. Furthermore, the presence of
shade can encourage physical activity, reduce greenhouse gas and air pollutant emissions,
lessen the urban heat island effect, and reduce energy costs. Under the policy, providing
shade, either natural or constructed, should be an essential element when planning for and
developing new city facilities, such as parks or public spaces, and in refurbishing existing
city-owned and city-operated facilities and sites. Increasing shade in Toronto contributes to a
healthier and more sustainable city.
In 2010, the City of Toronto developed shade guidelines to help implement the shade
policy. For more information, visit http://www1.toronto.ca/health/ shadeguidelines. The shade
policy and guidelines were created by the Ultraviolet Radiation and Shade Working Group of
the Toronto Cancer Prevention Coalition with the support of Toronto Public Health, the
Toronto Board of Health, and the Toronto City Council.

APPENDIX5: FEDERAL DEPARTMENTS, AGENCIES,

AND POLICIES

Appendix 5 presents information about current federal efforts on skin cancer prevention.
Recommendations from federal agencies and from national organizations that are leaders in
skin cancer prevention are summarized in Table A on page 79.

U.S. Department of Health and Human Services: Healthy People

The U.S. Department of Health and Human Services (HHS) and federal agencies within
HHS—including the Centers for Disease Control and Prevention (CDC), National Cancer
Institute (NCI) within the National Institutes of Health (NIH), U.S. Food and Drug
Administration (FDA), and others—work together to develop Healthy People goals and
objectives, which provide science-based, 10-year national objectives for improving the health
of all Americans. Measureable objectives related to skin cancer include reducing melanoma
mortality, reducing the proportion of adults who report sunburn, reducing the proportion of
adults and high school students in grades 9–12 who report using artificial sources of UV light
for tanning, and increasing the proportion of adults and high school students in grades 9–12
who follow protective measures that may lessen the risk of skin cancer.
CDC and NCI work together in ongoing efforts to monitor skin cancer and behaviors
related to known risks and to track progress toward meeting the Healthy People objectives for
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skin cancer. For example, in November 2011, several articles from CDC were published in a
supplemental issue of the Journal of the American Academy of Dermatology
(http://www.jaad.org/ issues?issue_key=S0190-9622%2811%29X0013-0). Some articles
described patterns of melanoma, and others focused on melanoma prevention. Contributors
included partners from the state-based central cancer registries, the American Cancer Society,
NCI, and academic centers.
In addition, CDC and NCI routinely publish analyses of data from the National Health
Interview Survey and its Cancer Control Supplement (NHIS CCS) [133, 227], which they
cosponsor. CDC also publishes analyses of Youth Risk Behavior Survey (YRBS) data related
to both sun protection and indoor tanning [134, 423].

National Cancer Institute

NCI produces the Cancer Trends Progress Report Updates (http://progressreport.

cancer.gov), which summarize the nation’s progress against skin cancer and other cancers in
relation to Healthy People objectives and other targets set by HHS. The first report was
published in 2001, and the data have been updated about every 2 years since then, with annual
updates beginning in 2014. The updates include key measures of progress along the cancer
control continuum, including risk factor monitoring for skin cancer, especially related to sun
protection behavior, indoor tanning, and sunburn. They use national trend data to illustrate
where advances have been made and gaps still remain.
NCI also fields the Health Information National Trends Survey (HINTS), which collects
nationally representative data about the American public’s use of cancer-related information
[246]. For example, HINTS publications show that, despite the large control over behavioral
factors that people may exert—such as avoiding UV exposure, wearing protective clothing,
and applying broad spectrum sunscreen with a sun protection factor (SPF) of 15 or higher—
many adults do not perceive cancer as preventable and are less likely to engage in skin cancer
prevention practices [244].
NCI’s Cancer Control P.L.A.N.E.T. (Plan, Link, Act, Network with Evidence-based
Tools) (http://cancercontrol planet.cancer.gov) is a web-based portal that provides access to
data and resources for cancer control planning efforts. It is a joint effort between NCI, CDC,
the Substance Abuse and Mental Health Services Administration (SAMHSA), and the
Agency for Healthcare Research and Quality (AHRQ). The portal can help planners, program
staff, and researchers to design, implement, and evaluate evidence-based cancer control
programs. State-level melanoma incidence and mortality data and data on skin cancer risk
perceptions are also available on the site through State Cancer Profiles
(http://statecancerprofiles.cancer.gov).
NCI and SAMHSA cosponsor the Research-tested Intervention Programs (RTIPs)
website (http://rtips.cancer. gov/rtips), which is a searchable database of cancer control
interventions and related program materials. Interventions included in the RTIPs database
must meet specific inclusion criteria to ensure research integrity, intervention impact, and
amenability to dissemination in the United States. When possible, the RTIPs page for a given
intervention links directly to corresponding recommendations from The Guide to Community
Preventive Services (The Community Guide) (see Table B on page 81 and
http://www.thecommunityguide. org/cancer/index.html). The website is designed to provide

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1375

program planners and public health practitioners easy and immediate access to research-tested
materials for use in a community or clinical setting.
NCI scientists also have developed an online Melanoma Risk Assessment Tool, which is
designed to help clinicians to evaluate an individual’s risk of melanoma
(http://www.cancer.gov/melanomarisktool/). The tool is limited to estimating risk for non-
Hispanic whites, and it is not appropriate for people with a family history of melanoma.
NCI’s Physician Data Query (PDQ) is a comprehensive cancer database that contains
summaries on a wide range of cancer topics. The PDQ cancer information summary on skin
cancer provides health professionals with comprehensive, peer-reviewed, evidence-based
information about skin cancer prevention. It is intended as a resource to inform and assist
clinicians; it does not provide practice guidelines. See http://www.cancer.
gov/cancertopics/pdq/ prevention/skin/HealthProfessional.
In addition, the NCI website has health education information on skin cancer causes,
prevention, and treatment (http://www.cancer.gov/cancertopics/ types/skin). It includes a
unique resource for people with darker skin, called Anyone Can Get Skin Cancer
(http://www.cancer.gov/cancertopics/ prevention/skin/ anyone-can-get-skin-cancer). NCI also
sponsors extramural research related to health behaviors, including skin cancer prevention
[424].

Centers for Disease Control and Prevention

CDC provides administrative, research, and technical support for the Community
Preventive Services Task Force, an independent, nonfederal panel of public health and
prevention experts. The panel provides evidence-based findings and recommendations about
community preventive services, programs, and policies to improve health. Its members
represent a broad range of research, practice, and policy expertise in community preventive
services, public health, health promotion, and disease prevention.
CDC programs contribute subject matter experts to participate, and sometimes take the
lead, in conducting systematic reviews for The Community Guide
(http://www.thecommunityguide.org/index.html). The Community Guide is a resource that
provides evidence-based recommendations and findings developed by the panel about what
programs and policies work to improve public health and prevent disease in the community
[306]. The Community Guide includes recommendations on effective community-based skin
cancer prevention interventions (see Table B on page 81 and
http://www.thecommunityguide.org/cancer/index. html). Recommendations are periodically
updated to reflect the latest scientific evidence.
CDC has also led communications campaigns designed to reduce UV exposure among
individuals and in communities. HHS and CDC sponsored the Choose Your Cover Campaign,
a 5-year skin cancer prevention and education campaign that ended in May 2003 (some
campaign materials are available at http://www.cdc.gov/ cancer/dcpc/publications/skin.htm)
[425]. In 2014, CDC sponsored The Burning Truth communication initiative, which
encourages individuals to keep their skin healthy and beautiful for life by protecting
themselves from too much exposure to UV rays from the sun and tanning beds. For more
information, see http://www.cdc. gov/cancer/skin/ burningtruth.
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In addition, CDC provides technical assistance to grantees that receive funding under the
National Comprehensive Cancer Control Program (NCCCP). CDC suggests that funded
programs address skin cancer prevention through the use of evidence-based interventions and
promising environmental strategies to reduce UV exposure.

Comprehensive Cancer Control Programs and Coalitions

CDC funds 65 programs in states, territories, Pacific Island jurisdictions, and tribes/tribal
organizations through the NCCCP to establish broad-based comprehensive cancer control
(CCC) coalitions, assess the burden of cancer, and develop and implement CCC plans to
reduce cancer incidence and mortality [426]. Each program uses cancer incidence data to
identify high-priority cancers. Some CCC plans identify melanoma as a problem in their
communities and have objectives that address prevention. A review of published cancer plans
identified 25 CCC plans that addressed sun safety in their goals or objectives, and 18 had
chapters or sections devoted to discussion of UV exposure, sun protection, or skin cancer
prevention [427].
During 2007–2012, nine CCC programs received additional funding to implement skin
cancer programs in various settings. Common intervention settings and strategies include
school-based education and policies, educational outreach in recreational settings (e.g., pools,
beaches, camps, and golf courses), environmental approaches in child care settings to reduce
UV exposure, and education about the risks of indoor tanning [304]. Analysis of NCCCP
programmatic data in 2013 revealed that 16 CCC coalitions have a skin cancer workgroup,
and 13 CCC programs have annual objectives that specifically address preventing skin cancer
in their action plans submitted to CDC [428]. Many objectives use strategies designed to
improve education or knowledge in various settings to educate children, adolescents, and
adults about how to reduce UV exposure from the sun and from indoor tanning.
In addition, many CCC programs are working with partners to address skin cancer
prevention through environmental approaches that reach more people in the community, such
as increased shade in recreational settings and schools. Two CCC programs are working with
their partners on long-term objectives to reduce indoor tanning among adolescent females.
Three NCCCP awardees received funding and technical assistance to use policy and
environmental approaches for cancer control to address skin cancer prevention. Activities
included educating stakeholders about the link between artificial UV exposure and melanoma
and developing shade structure policies.

Agency for Healthcare Research and Quality

AHRQ provides scientific and administrative support for the independent U.S. Preventive
Services Task Force (USPSTF). The USPSTF, in partnership with an AHRQ Evidence-Based
Practice Center, completed a systematic evidence review on behavioral counseling in primary
care to prevent skin cancer. The evidence review, the final USPSTF recommendations, and a
consumer guide are available at http://www. uspreventiveservicestask force.org/
uspstf/uspsskco.htm. Information about skin cancer screening is also available on the
USPSTF website.

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As part of its mission to improve health care, from primary prevention to chronic care
management, AHRQ is exploring how to develop, strengthen, and sustain relationships
among primary care practices, the community, and public health organizations in order to
meet the needs of patients and families. These efforts are valuable to groups working to
improve skin cancer prevention. To learn more, visit Building Relationships Between Clinical
Practices and the Community to Improve Care on the AHRQ website (http://www.
innovations.ahrq.gov/linkingClinicalPractices.aspx).
The AHRQ Innovations Exchange website (http://www.innovations. ahrq.gov) provides
information about specific skin cancer prevention tools, such as how to use interactive kiosks,
provide skin cancer screening and education at beaches, and work with members of the deaf
community to improve cancer awareness (http:// www.innovations.ahrq.gov/innovations_
qualitytools.aspx?search=skin cancer).

U.S. Food and Drug Administration

At the federal level, FDA regulates indoor UV tanning devices under separate authorities,
both as medical devices and as radiation-emitting electronic products. FDA published a
performance standard (21 CFR 1040.20) to set requirements for indoor tanning devices in
1979 and amended this standard in 1985 to accommodate devices that emitted primarily UVA
radiation [348]. FDA’s current performance standard requires that a sunlamp product’s label
include a recommended exposure schedule (see example at http://tanresponsibly.com/uv-
light) [358]. FDA has advised manufacturers that this schedule should provide for exposures
of no more than three sessions in the first week [358]. The performance standard contains
requirements for the warning label on the product, the proportion of shortwave UV radiation,
timer settings, and transmittance requirements for protective eyewear supplied with the
device.
FDA inspectors inspect the manufacturing sites of sunlamp products (as resources allow)
to verify compliance with FDA’s performance standard. FDA also provides “model state
regulations” for sunlamp products as guidance for states to use in their radiation protection
programs. State inspectors routinely inspect tanning salons to ensure that the equipment in the
salons carries appropriate labeling and a timer control as required by the FDA performance
standard, in addition to any state requirements for operator training or procedures to ensure
proper hygiene.
Under its medical device authority, FDA originally classified indoor tanning devices as
low risk (Class I) medical devices [351, 429]. However, based on advice from its advisors and
consultants at a March 2010 Advisory Committee meeting and review by agency experts, the
agency issued a final order in May 2014 that reclassified sunlamp products as moderate to
high risk (Class II) devices [349, 350]. Once effective, the order requires manufacturers of
sunlamps to include a visible black box warning on the device that people younger than age
18 years should not use these devices. Manufacturers must receive 510(k) premarket
clearance from FDA for newly marketed devices, which were previously exempt from any
premarket review. Manufacturers must demonstrate that their products have met certain
performance testing requirements, address certain product design characteristics, and provide
comprehensive labeling that presents consumers with clear information on the risks of use.
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1378 Meg Watson, Erin Garnett, Gery P. Guy et al.

FDA also regulates over-the-counter drugs, including sunscreens. Under labeling changes
that became effective in 2012, sunscreens labeled as both broad spectrum and SPF 15 (or
higher) are labeled as reducing the risk of skin cancer and reducing the risk of early skin
aging caused by the sun if they are used as directed and in combination with other sun
protection measures. All sunscreen products are labeled as helping to prevent sunburn. Any
product that is not broad spectrum, or that is broad spectrum but has an SPF of at least 2 but
less than 14, will have a warning stating that the product has been shown only to help prevent
sunburn, not skin cancer or early skin aging. In 2011, FDA proposed limiting the maximum
SPF on sunscreen labels to “50+,” because FDA did not have adequate data to show that
products with an SPF higher than 50 provide any additional benefit compared with products
with an SPF of 50 or lower (http://www.fda.gov/forconsumers/ consumerupdates/
ucm258416.htm).

Federal Trade Commission

The Federal Trade Commission (FTC) is responsible for investigating false, misleading,
and deceptive advertising claims about products and services, including tanning devices. In
May 2010, the agency issued a complaint and final order against the Indoor Tanning
Association (ITA) [361]. Among other things, the complaint alleged that ITA’s advertising
materials had represented that (1) tanning, including indoor tanning, does not increase the risk
of skin cancer; (2) tanning, including indoor tanning, poses no danger; (3) indoor tanning is
approved by the government; and (4) indoor tanning is safer than tanning outdoors because,
in indoor tanning facilities, the amount of UV light is monitored and controlled. The
complaint charged that these claims were false.
The settlement reached in this case bars ITA from making deceptive claims in the future
and requires certain ITA advertisements to include health disclosures. In response to public
comments opposing FTC’s action, the agency noted that its investigation was informed by a
thorough analysis of the available scientific evidence, including review of relevant scientific
studies and consultation with experts from government, academia, and the industry. In
connection with the ITA case, FTC released a consumer alert, stating that UV radiation from
tanning devices damages the skin and poses serious health risks, including cancer, and that
tanning is not necessary to get the health benefits of vitamin D [430].

U.S. Environmental Protection Agency

To help people plan outdoor activities so they can reduce UV exposure, the National
Weather Service and EPA publish the UV Index, a forecast of the risk of overexposure to UV
radiation from the sun at a given location, date, and time. For more information, visit the
following websites: http://www.epa.gov/sunwise/uvindex. html, http://www.epa.gov/sunwise/
uviresources. html, or http://www2.epa.gov/sunwise/uv-index-scale [431, 432].
The index ranges from 0 to 11+, with higher values indicating greater risk of
overexposure. The forecast is calculated every day on the basis of the angle of the sun, ozone
levels, expected cloud cover, and other local conditions.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1379

In 2000, EPA launched SunWise, a health and environmental education program that
teaches children and their caregivers how to protect themselves from overexposure to the sun.
For more information about SunWise, visit the EPA website at http://www.epa.gov/sunwise.

National Park Service

The National Park Service (NPS), a bureau within the U.S. Department of the Interior,
offers recreational opportunities for about 275 million visitors every year and manages 401
parks throughout the United States. For nearly 100 years, NPS has contributed to the health
and well-being of Americans by providing places that inspire physical activity, promote
physical and mental health, and foster community through the preservation of ecosystems and
interpretation of a shared heritage. Hand in hand with its efforts to provide opportunities for
fun in the most beautiful outdoor environments in the country, NPS is also dedicated to
educating the public about the importance of sun protection.
NPS has collaborated with partners such as the EPA SunWise program and the National
Council on Skin Cancer Prevention to educate the public about ways to enjoy our nation’s
treasures while keeping safe in the sun. In 2012, NPS and its partners created a public service
announcement on sun safety to be used for the annual “Don’t Fry Day” campaign
(http://www2.epa.gov/sunwise/ dont-fry-day). In addition, NPS teamed up with other federal
partners to create a “Healthy Parks, Healthy People” sun-safety window display to educate
the public on how to safely engage in outdoor activities in our national parks while preventing
overexposure to harmful sun rays. NPS’s Safe Adventures program bases its prevention
strategies on science to promote well- being and outdoor recreation while providing tools,
information, and guidance that empower the public to have a safe adventure in national parks.

Occupational Safety and Health Administration

The general duty clause of the Occupational Safety and Health Act states the following in
Section 5(a)(1): “Each employer shall furnish to each of his employees employment and a
place of employment which are free from recognized hazards that are causing or are likely to
cause death or serious physical harm to his employees.”[433] The Occupational Safety and
Health Administration (OSHA) does not mandate employee exposure limits specific to UV
radiation [434]. OSHA’s Campaign to Prevent Heat Illness in Workers is designed to raise
awareness and educate workers and employers about the dangers of working in the heat.
OSHA collaborates with the National Oceanic Atmospheric Administration and other federal
agencies, such as EPA and CDC, for a joint public information notice for sun-safety
awareness and participates in the National Council on Skin Cancer Prevention’s “Don’t Fry
Day” [435, 436].

Affordable Care Act

The Affordable Care Act (Section 10907) created a 10% excise tax on indoor tanning
services, which became effective on July 1, 2010 [390]. The tax is only applicable to UV
 Free ebooks ==> www.Ebook777.com
1380 Meg Watson, Erin Garnett, Gery P. Guy et al.

tanning services, excluding phototherapy sessions performed by a licensed medical

professional. Tanning devices sold directly to consumers, facilities that offer tanning as an
additional service to members without a separate fee, and sunless tanning products are not
subject to the tax. The Affordable Care Act also requires that nongrandfathered health plans
offered in the individual or group market provide benefits for and prohibit the imposition of
cost-sharing requirements for USPSTF-recommended preventive services with a rating of
“A” or “B.” This requirement includes the USPSTF recommendation to counsel children,
adolescents, and young adults aged 10–24 years with fair skin about minimizing their
exposure to UV radiation to reduce risk of skin cancer (B rating). For this type of
recommendation, which only applies to a specific population, decisions about whether an
individual is part of this population and should receive the given preventive service should be
made by the attending provider [437, 438].

Table A. Skin Cancer Prevention Recommendations by Federal Agencies and

National Organizations

Agency Recommendations
FEDERAL AGENCIES
Centers for Disease Control and Stay in the shade, especially during midday hours.
Prevention Wear clothing that covers your arms and legs.
http://www.cdc.gov/cancer/skin/basic Wear a hat with a wide brim to shade your face,
_info/prevention.htm head, ears, and neck.
Wear sunglasses that wrap around and block both
UVA and UVB rays.
Use sunscreen with sun protection factor (SPF) 15
or higher and both UVA and UVB protection.
Avoid indoor tanning.
U.S. Environmental Do not burn.
Protection Agency Avoid sun tanning and tanning beds.
http://www.epa.gov/sunwise/actionst Generously apply sunscreen.
eps. Wear protective clothing.
html Seek shade.
Use extra caution near water, snow, and sand.
Check the UV Index.
Get vitamin D safely.
U.S. Food and Drug Reduce time in the sun.
Administration Dress with care.
http://www.fda.gov/ForConsumers/ Be serious about sunscreen.
ConsumerUpdates/ucm049090.htm Tips for applying sunscreen.
Protect the eyes.
Slip! Slop! Slap! Wrap!
Occupational Safety and Cover up.
Health Administration Use sunscreen.
https://www.osha.gov/Publications/O Wear a hat.
SHA3166/osha3166.html Wear UV-absorbent shades.
Limit exposure.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1381

Agency Recommendations
FEDERAL AGENCIES
American Academy of Seek shade when appropriate.
Dermatology Wear protective clothing.
http://www.aad.org/spot-skin-cancer/ Generously apply a broad spectrum, water-
understanding-skin-cancer/how-do-i- resistant sunscreen.
prevent- skin-cancer Use extra caution near water, snow, and sand.
Avoid tanning beds.
American Academy of Pediatrics Keep babies younger than 6 months out of direct
http://www.healthychildren.org/english sunlight. Find shade under a tree, umbrella, or the
/safety- prevention/at-play/pages/Sun- stroller canopy.
Safety.aspx When possible, dress yourself and your kids in cool,
comfortable clothing that covers the body, like
lightweight cotton pants, long- sleeved shirts, and hats.
Select clothes made with a tight weave—they protect
better than clothes with a looser weave.
Wear a hat or cap with a brim that faces forward to
shield the face.
Limit your sun exposure between 10 am and 4 pm,
when UV rays are strongest.
Wear sunglasses with at least 99% UV protection
(look for child-sized sunglasses with UV protection for
your child).
Use sunscreen.
Set a good example.
American Cancer Society Slip! Slop! Slap! Wrap!
http://www.cancer.org/cancer/ Slip on a shirt.
skincancer- Slop on sunscreen.
melanoma/moreinformation/ Slap on a hat.
skincancerpreventionandearlydetection/ Wrap on sunglasses to protect the eyes and skin
skin- cancer-prevention-and-early- around them.
detection-u-v- protection Seek shade.
Protect your skin with clothing.
Use sunscreen.
Read the labels.
Be sure to apply sunscreen properly.
Wear a hat.
Wear sunglasses that block UV rays.
Avoid tanning beds and sunlamps.
Protect children from the sun.
National Council on Skin Do not burn or tan.
Cancer Prevention Seek shade.
http://www.skincancerprevention.org/ Wear protective clothing.
skin- cancer/prevention-tips Generously apply sunscreen.
Use extra caution near water, snow, and sand.
Get vitamin D safely.
 Free ebooks ==> www.Ebook777.com

Table B. Community-Level Approaches to Preventing Skin Cancer: Recommendations from The Guide to Community Preventive
Services

Target
Setting Intervention Components Task Force Findings
Audience
EDUCATION AND POLICY INTERVENTION STRATEGIES
Child care centers Children, caregivers Include one or more of the following: Recommended based on evidence
(staff, teachers, or parents),  Educational activities through classroom of effectiveness in increasing
or both instruction. children’s protection from
 Small media, such as brochures and flyers. excessive UV exposure
 Activities to influence children’s or (May 2013).
students’ behaviors, such as modeling,
demonstration, or role-playing. Recommended based on evidence
Primary and middle  Activities to change the knowledge, of effectiveness in increasing
schools (kindergarten Children, caregivers attitudes, or behaviors of parents, children’s sun protection behaviors
through 8th grade) (staff, teachers, or caregivers, or teachers. and decreasing UV exposure,
parents),or both  Environmental changes, such as making sunburn incidence, and formation
shaded areas available for outdoor activities. of new moles (August
 Policy changes, such as scheduling outdoor 2012).
activities to avoid hours of peak sunlight or
High schools and Adolescents and young allowing students to wear protective hats Insufficient evidence
colleges adults, teachers, or parents when outdoors. (May 2013).
or a combination of the
three

Health care settings Providers, patients, or Include one or more of the following: Insufficient evidence
clients  Provider education sessions. (July 2002).
 Internet-based education.
 Videos.
 Role-modeling.

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 Target
Setting Intervention Components Task Force Findings
Audience
Outdoor recreational Recreation staff, Include one or more of the following: Recommended based on
and tourism settings adults, and children  Educational brochures. strong evidence of
 Sun-safety training for recreation staff. effectiveness in improving
 Role-modeling by recreation staff. sunscreen use and improving
 Sun-safety lessons. participants’ sun protective
 Interactive activities. behaviors. (February 2014).
 Signs or other prompts encouraging use of sun
protection.
Outdoor occupational Workers Include one or more of the following: Recommended based on
settings  Provision of information to workers through evidence of effectiveness in
instruction, small media, or both. increasing outdoor workers’
 Additional activities intended to change the sun protective behaviors and
knowledge, attitudes, beliefs, intentions, or behaviors in
of workers, such as modeling or demonstrations. reducing sunburn
 Environmental or policy approaches, such as (August 2013).
providing shade and sunscreen.
COMMUNITYWIDE INTERVENTION STRATEGIES
Multicomponent, Communitywide in a A combination of the following across multiple Recommended based on
communitywide defined geographic settings: evidence of effectiveness in
interventions area  Individual-directed strategies. increasing sunscreen use
 Mass media campaigns. (April 2012).
 Environmental and policy changes.
Mass media Communitywide, Dissemination of information and behavioral Insufficient evidence
campaigns (when but may be aimed at guidance to wide audiences through media channels (June 2011).
implemented alone specific audiences; such as the following:
rather than as part of a typically uses broad  Print media (e.g., newspapers, magazines).
multicomponent distribution channels  Broadcast media (e.g., radio, television).
intervention)  Billboards.
 Internet.
 Free ebooks ==> www.Ebook777.com

Table B. (Continued)

Target
Setting Intervention Components Task Force Findings
Audience
INTERVENTION STRATEGIES TARGETING CHILDREN’S PARENTS AND CAREGIVERS
Strategies targeting Children’s parents and Single or multicomponent interventions, including Insufficient evidence
children’s parents and caregivers (e.g., one or more of the following: (July 2002).
caregivers nannies, other family  Educational component using small media (e.g.,
members, lifeguards, educational brochures, newsletters, tip cards, postcard
teachers, coaches) reminders), sun-safety lessons, interactive activities,
and incentives for parents and children.
 Environmental component (e.g., an increase in
available shaded areas, free sunscreen, point-of-
purchase prompts and discount coupons for hats, sun-
safety logo T-shirts, sunscreen).
Source: The Guide to Community Preventive Services (http://www. thecommunityguide.org/cancer/skin/education-policy/index.html).

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1385

APPENDIX 6: ABBREVIATIONS AND ACRONYMS

25OHD 25-hydroxyvitamin D
AHRQ Agency for Healthcare Research and Quality
BCC basal cell carcinoma
BRFSS Behavioral Risk Factor Surveillance System
CCC Comprehensive Cancer Control
CDC Centers for Disease Control and Prevention
CMS Centers for Medicare & Medicaid Services
DHA dihydroxyacetone
EHR electronic health record
EPA U.S. Environmental Protection Agency
FDA U.S. Food and Drug Administration
FTC Federal Trade Commission
HHS U.S. Department of Health and Human Services
HINTS Health Information National Trends Survey
IARC International Agency for Research on Cancer
IOM Institute of Medicine
ITA Indoor Tanning Association
NCCCP National Comprehensive Cancer Control Program
NCI National Cancer Institute
NHANES National Health and Nutrition Examination Survey
NHIS National Health Interview Survey
NIH National Institutes of Health
NMSC nonmelanoma skin cancer
NPS National Park Service
OSHA Occupational Safety and Health Administration
P.L.A.N.E.T. Plan, Link, Act, Network with Evidence-based Tools
RAYS Raising Awareness in Youth About Sun Safety
RTIPs Research-tested Intervention Programs
SAD seasonal affective disorder
SAMHSA Substance Abuse and Mental Health Services Administration
SCC squamous cell carcinoma
SES socioeconomic status
SHPPS School Health Policies and Practices Study
SPF sun protection factor
USPSTF U.S. Preventive Services Task Force
UV ultraviolet
UVA ultraviolet A
UVB ultraviolet B
UVC ultraviolet C
WHO World Health Organization
YRBS Youth Risk Behavior Survey
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1386 Meg Watson, Erin Garnett, Gery P. Guy et al.

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meters does cause less safe tanning habits: a randomized- controlled trial. Photochem
Photobiol., 2008, 84(3), 758-763.
[383] American Academy of Dermatology. Melanoma reporting to state cancer registries.
http://www.aad.org/File%20Library/Global%20navigation/Education%20and%20
quality%20care/State%20cancer%20registries/state-cancer-registries-laws-and-
requirements.pdf. Accessed February 2, 2014.
[384] Cartee, TV; Kini, SP; Chen, SC. Melanoma reporting to central cancer registries by U.S.
dermatologists: an analysis of the persistent knowledge and practice gap. J Am Acad
Dermatol., 2011, 65(5 suppl 1), S124.e001-S124.e009.
[385] Cockburn, M; Swetter, SM; Peng, D; Keegan, THM, Deapen, D; Clarke, CA. Melanoma
underreporting: why does it happen, how big is the problem, and how do we fix it? J Am
Acad Dermatol., 2008, 59(6), 1081-1085.
[386] Centers for Disease Control and Prevention. Meaningful use of electronic health
records. National Program of Cancer Registries, Centers for Disease Control and
 Free ebooks ==> www.Ebook777.com
1410 Meg Watson, Erin Garnett, Gery P. Guy et al.

Prevention website. http://www.cdc.gov/ cancer/npcr/meaningful_use.htm. Accessed

December 12, 2013.
[387] Centers for Disease Control and Prevention. Stage 2 meaningful use fact sheet. Public
health reporting objectives. Centers for Disease Control and Prevention website.
http://www.cdc.gov/phin/library/PHIN_ Fact_ Sheets/Stage%202%20Fact%20Sheet-
09_03_2013.pdf. Accessed February 25, 2014.
[388] Mariotto, AB; Yabroff, KR; Shao, Y; Feuer, EJ; Brown, ML. Projections of the cost of
cancer care in the United States: 2010-2020. J Natl Cancer Inst., 2011, 103(2), 117-128.
[389] Centers for Disease Control and Prevention. Behavioral Risk Factor Surveillance
System. Centers for Disease Control and Prevention website. http://www.cdc.gov/brfss/.
Accessed February 24, 2014.
[390] Internal Revenue Service. Affordable Care Act tax provisions. Internal Revenue Service
website. http://www.irs.gov/uac/Affordable-Care-Act-Tax- Provisions. Accessed June 4,
2013.
[391] Jain, N; Rademaker, A; Robinson, JK. Implementation of the federal excise tax on
indoor tanning services in Illinois. Arch Dermatol., 2012, 148(1), 122-124.
[392] U.S. Department of Health and Human Services. Preventing Tobacco Use Among Youth
and Young Adults: A Report of the Surgeon General. Atlanta, GA: Centers for Disease
Control and Prevention, U.S. Dept of Health and Human Services; 2012. http://www.
surgeongeneral. gov/library/reports/preventing-youth-tobacco-use/full-report.pdf.
Accessed February 24, 2014.
[393] WebShade website. Changing the way we think about shade.
http://www.webshade.com.au/index.html. Accessed Nov 20, 2013.
[394] Toronto Cancer Prevention Coalition. Shade Guidelines. Toronto, Canada: Toronto
Cancer Prevention Coalition; 2010. http://www1.toronto.ca/city_of_ toronto/
toronto_public_health/healthy_public_policy/tcpc/files/pdf/shade_guidelines.pdf.
Accessed March 7, 2014.
[395] Scully, M; Makin, J; Maloney, S; Wakefield, M. Changes in coverage of sun protection
in the news: threats and opportunities from emerging issues. Health Educ Res., 2014,
29(3), 378-387.
[396] Community Preventive Services Task Force. Preventing skin cancer: multicomponent
community-wide interventions (abbreviated). The Guide to Community Preventive
Services website. http://www. thecommunityguide.org/cancer/skin/community-
wide/multicomponent.html. Accessed January 17, 2014.
[397] Cancer Council Victoria. Skin Cancer Prevention: A Blue Chip Investment in Victoria.
Victoria, Australia: Cancer Council Victoria, SunSmart, Centre for Behavioural
Research in Cancer; 2008.
[398] Sinclair, C; Foley, P. Skin cancer prevention in Australia. Br J Dermatol., 2009,
161(suppl 3), 116-123.
[399] Geller, AC; Glanz, K; Shigaki, D; Isnec, MR; Sun, T; Maddock, J. Impact of skin cancer
prevention on outdoor aquatics staff: the Pool Cool program in Hawaii and
Massachusetts. Prev Med. 2001, 33(3), 155-161.
[400] Rand, CM; Blumkin, A; Szilagyi PG. Electronic health record use and preventive
counseling for U.S. children and adolescents. J Am Med Inform Assoc., 2014, 21(e1),
e152-e156.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1411

[401] Pageler, NM; Longhurst, CA; Wood, M; et al. Use of electronic medical record-
enhanced checklist and electronic dashboard to decrease CLABSIs. Pediatrics., 2014,
133(3), e738-e746.
[402] Iglar, K; Katyal, S; Matthew, R; Dubey, V. Complete health checkup for adults: update
on the preventive care checklist form. Can Fam Physician., 2008, 54(1), 84-88.
[403] Community Preventive Services Task Force. Preventing skin cancer: primary and
middle school interventions. Task Force finding and rationale statement. The Guide to
Community Preventive Services website. http://www.thecommunityguide.org/
cancer/skin/education-policy/ RRprimaryandmiddleschools.html. Accessed January 30,
2014.
[404] Merlino, LA; Sullivan, KJ; Whitaker, DC; Lynch, CF. The independent pathology
laboratory as a reporting source for cutaneous melanoma incidence in Iowa, 1977-1994.
J Am Acad Dermatol., 1997, 37(4), 578-585.
[405] Gallagher, RP; Bajdik, CD; Fincham, S; et al. Chemical exposures, medical history, and
risk of squamous and basal cell carcinoma of the skin. Cancer Epidemiol Biomarkers
Prev., 1996, 5(6), 419-424.
[406] Cancer Council Australia. SunSmart position statement on window tinting. Cancer
Council Australia wiki website. http://wiki.cancer.org.au/ prevention/Position_statement
_-_Tinted_windows. Accessed April 11, 2014.
[407] Goldstein, NJ; Cialdini, RB. Using social norms as a lever of social influence. In:
Pratkanis, AR; ed. The Science of Social Influence: Advances and Future Progress.,
New York, NY: Psychology Press; 2007, 167-192.
[408] Stryker, JE; Lazovich, D; Forster, JL; Emmons, KM; Sorensen, G; Demierre, MF.
Maternal/female caregiver influences on adolescent indoor tanning. J Adolesc Health.,
2004, 35(6), 528.e001-528.e009.
[409] Boyers, L; Karimkhani, C; Crane, LA; Asdigian, N; Hollonds, A; Dellavalle, RP.
Buying indoor tanning with university debit cards. J Am Acad Dermatol., 2014, 71(1),
199-201.
[410] Brouse, CH; Basch, CE; Neugut, AI. Warning signs observed in tanning salons in New
York city: Implications for skin cancer prevention. Prev Chronic Dis., 2011, 8(4), A88.
[411] U.S. Food and Drug Administration, Center for Devices and Radiological Health,
Medical Devices Advisory Committee. General and plastic surgery devices panel:
transcript. http://www.fda.gov/downloads/ AdvisoryCommittees/CommitteesMeeting
Materials/MedicalDevices/MedicalDevicesAdvisoryCommittee/GeneralandPlastic
SurgeryDevicesPanel/UCM210232.pdf. Accessed June 4, 2013.
[412] Song, F; Qureshi, AA; Giovannucci, EL; et al. Risk of a second primary cancer after
non-melanoma skin cancer in white men and women: a prospective cohort study. PLoS
Med., 2013, 10(4), e1001433.
[413] Ong, EL; Goldacre, R; Hoang, U; Sinclair, R; Goldacre M. Subsequent primary
malignancies in patients with nonmelanoma skin cancer in England: a national record-
linkage study. Cancer Epidemiol Biomarkers Prev. 2014, 23(3), 490-498.
[414] Wolff, K; Johnson, BE; Saavedra, AP. Precancerous lesions and cutaneous carcinomas.
Fitzpatrick’s Color Atlas and Synopsis of Clinical Dermatology. 7th ed. New York, NY:
McGraw-Hill; 2013.
[415] Honigsmann, H. Erythema and pigmentation. Photodermatol Photoimmunol Photomed.,
2002, 18(2), 75-81.
 Free ebooks ==> www.Ebook777.com
1412 Meg Watson, Erin Garnett, Gery P. Guy et al.

[416] Goldsmith, LA; Fitzpatrick, TB. Fitzpatrick’s Dermatology in General Medicine. 8th
ed. New York, NY: McGraw-Hill Professional; 2012.
[417] U.S. Preventive Services Task Force. Screening for skin cancer: U.S. Preventive
Services Task Force recommendation statement. Ann Intern Med. 2009, 150(3), 188-
193.
[418] Katalinic, A; Waldmann, A; Weinstock, MA; et al. Does skin cancer screening save
lives?: an observational study comparing trends in melanoma mortality in regions with
and without screening. Cancer., 2012, 118(21), 5395-5402.
[419] Bordoni, A; Leoni-Parvex, S; Peverelli, S; Mazzola, P; Mazzucchelli, L; Spitale A.
Opportunistic screening strategy for cutaneous melanoma does not change the incidence
of nodular and thick lesions nor reduce mortality: a population-based descriptive study
in the European region with the highest incidence. Melanoma Res., 2013, 23, 402-407.
[420] Glanz, K; Saraiya, M; Wechsler, H; Centers for Disease Control and Prevention.
Guidelines for school programs to prevent skin cancer. MMWR Recomm Rep., 2002,
51(RR-4), 1-18.
[421] Centers for Disease Control and Prevention. Sun Safety for America’s Youth Toolkit.
Atlanta, GA: Centers for Disease Control and Prevention, U.S. Dept of Health and
Human Services; 2009. http://www.cdc.gov/ cancer/skin/pdf/toolkit/SunSafety
Toolkit_MainText.pdf. Accessed August 9, 2013.
[422] Centers for Disease Control and Prevention. Stories of Success: National
Comprehensive Cancer Control Program: Comprehensive Cancer Control in Action.
Atlanta, GA: Centers for Disease Control and Prevention, U.S. Dept of Health and
Human Services; 2010. http://www.cdc.gov/cancer/ ncccp/pdf/success/SuccessStories.
pdf. Accessed April 13, 2013.
[423] Jones, SE; Saraiya, M; Miyamoto, J; Berkowitz, Z. Trends in sunscreen use among U.S.
high school students: 1999-2009. J Adolesc Health., 2012, 50(3), 304-307.
[424] National Cancer Institute. Behavioral Research, Cancer Control and Population
Sciences: about Health Behaviors Research Branch (HBRB). National Cancer Institute
website. http://cancercontrol.cancer.gov/brp/ hbrb/about.html. Accessed February 3,
2014.
[425] Jorgensen, CM; Wayman, J; Green, C; Gelb, CA. Using health communications for
primary prevention of skin cancer: CDC’s Choose Your Cover campaign. J Womens
Health Gend Based Med., 2000, 9(5), 471-475.
[426] Given, LS; Black, B; Lowry, G; Huang, P; Kerner, JF. Collaborating to conquer cancer:
a comprehensive approach to cancer control. Cancer Causes Control., 2005, 16 suppl 1,
3-14.
[427] Centers for Disease Control and Prevention. Comprehensive Cancer Control Plans: A
Content Review. Atlanta, GA: Centers for Disease Control and Prevention, U.S. Dept of
Health and Human Services; 2005. http://www.cdc.gov/cancer/ncccp/pdf/
CCC_Plans_Content_Review.pdf. Accessed May 16, 2013.
[428] Centers for Disease Control and Prevention. Chronic Disease Management Information
System, DP12-1205 National Comprehensive Cancer Control Program programmatic
data, 2012–2013 Atlanta, GA: Centers for Disease Control and Prevention.
[429] Lim, HW; James, WD; Rigel, DS; Maloney, ME; Spencer, JM; Bhushan, R. Adverse
effects of ultraviolet radiation from the use of indoor tanning equipment: time to ban the
tan. J Am Acad Dermatol., 2011, 64(5), 893-902.

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 The Surgeon General’s Call to Action to Prevent Skin Cancer 1413

[430] Federal Trade Commission. Indoor tanning. Federal Trade Commission website.
http://www.consumer.ftc.gov/articles/0129-indoor-tanning. Accessed June 4, 2013.
[431] U.S. Environmental Protection Agency, SunWise Program. UV index. U.S.
Environmental Protection Agency website. http://www.epa.gov/ sunwise/uvindex.html.
Accessed April 19, 2013.
[432] National Weather Service: Climate Prediction Center. Stratosphere: UV index. Climate
Prediction Center website. http://www. cpc.ncep.noaa.gov/ products/stratosphere/uv_
index/index.html. Accessed April 19, 2013.
[433] Occupational Safety and Health Act of 1970, Public Law 91-596. 84 Statute 1590
(1970).
[434] U.S. Department of Labor, Occupational Safety and Health Administration. Standard
interpretations: 1910.97, 1910.1096. https://www.osha.gov/pls/oshaweb/owadisp.show_
document?p_table=INTERPRETATIONS&p_id=24755. Accessed December 12, 2013.
[435] U.S. Department of Labor, Occupational Safety and Health Administration. Water. Rest.
Shade. The work can’t get done without them. Protective measures to take at each risk
level. Occupational Safety and Health website. http://www.osha.gov/SLTC/
heatillness/heat_ index/protective_high.html. Accessed December 12, 2013.
[436] National Weather Service. Public information notice: excessive heat and sun safety
guidance for 2013 season. National Weather Service website.
http://www.nws.noaa.gov/os/notification/pns13don-t_fry_day.txt. Accessed February 2,
2014.
[437] U.S. Preventive Services Task Force. USPSTF A and B recommendations. U.S.
Preventive Services Task Force website. http://www.uspreventiveservicestaskforce.org/
uspstf/uspsabrecs.htm. Accessed February 3, 2014.
[438] U.S. Department of Labor. FAQs about Affordable Care Act implementation part XII.
U.S. Department of Labor website. http://www.dol.gov/ ebsa/faqs/faq-aca12.html.
Accessed February 3, 2014.

End Notes
1
See Appendix 1 for a definition and discussion of overexposure and for definitions of different types of UV
exposure.
2
Although UVC radiation has the highest energy of the three, it is almost completely absorbed by the earth’s
atmosphere and is not responsible for cancer in the general population.
3
The IOM report states that people with serum 25OHD levels of below 30 nmol/L (12 ng/mL) are at risk of
deficiency relative to bone health and that serum 25OHD levels of 50 nmol/L (20ng/mL) or higher are
sufficient. It expresses concern for values above 125 nmol/L (50 ng/mL).
4
The NHIS defines sunburn as even a small part of the skin turning red or hurting for 12 hours or longer. This
definition is only given to respondents who request more information about what is meant by sunburn.
5
Skin cancer screening is defined as an evaluation of the skin by a medical provider, in the absence of changes to
the skin.
6
The USPSTF concludes that the current evidence is insufficient to assess the balance of benefits and harms of this
service. Evidence is lacking, of poor quality, or conflicting, and the balance of benefits and harms cannot be
determined.
7
The Community Guide is a website that houses the official collection of all Community Preventive Services Task
Force findings and the systematic reviews on which they are based.
8
Multicomponent, communitywide interventions are defined as interventions that include at least two distinct
components that are implemented in at least two different types of settings (e.g., schools, recreation areas) or
that reach the entire community (e.g., mass media campaigns).
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1414 Meg Watson, Erin Garnett, Gery P. Guy et al.

9
Community-based interventions in health care settings were last reviewed and recommendations were updated in
2002. These interventions are different from the provider counseling for fair-skinned youth aged 10–24 years,
which the USPSTF has found to be effective.
10
State laws in Oregon and Washington allow minors younger than age 18 years to use indoor tanning facilities with
a doctor’s prescription.
11
Although indoor tanning devices sold in the United States are required to have timers that would automatically
shut off the device after a certain period of time, these timers may be inoperative or possibly overridden by
users. Data from the National Electronic Injury Surveillance System on visits to emergency rooms related to
indoor tanning contain anecdotal reports of users falling asleep and being burned.
12
Skin cancer screening is defined as an evaluation of the skin by a medical provider, in the absence of changes to
the skin.
13
The USPSTF concludes that the current evidence is insufficient to assess the balance of benefits and harms of this
service. Evidence is lacking, of poor quality, or conflicting, and the balance of benefits and harms cannot be
determined.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 63

FALSE AND MISLEADING HEALTH INFORMATION

PROVIDED TO TEENS BY THE INDOOR TANNING
INDUSTRY: INVESTIGATIVE REPORT *

U.S. House of Representatives Committee on Energy

and Commerce-Minority Staff

I. EXECUTIVE SUMMARY
The World Health Organization and the National Toxicology Program classify indoor
tanning beds as a “known” human carcinogen. The American Academy of Pediatrics calls
indoor tanning beds “generally unsafe for children” and, along with the American Academy
of Dermatology Association, recommends a ban on their use by anyone under 18. Yet despite
the mounting evidence of the dangers of indoor tanning, millions of young people use tanning
salons each year – and this use is on the rise. The most frequent indoor tanners are young
white females.
Rep. Henry A. Waxman, Ranking Member of the House Committee on Energy and
Commerce, Rep. Diana DeGette, Ranking Member of the House Committee on Energy and
Commerce Subcommittee on Oversight and Investigations, and Rep. Frank Pallone, Jr.,
Ranking Member of the House Committee on Energy and Commerce Subcommittee on
Health, along with Reps. Rosa L. DeLauro and Carolyn Maloney, requested this investigation
to determine if tanning salons are providing accurate information about cancer and other risks
to teenage girls who purchase indoor tanning sessions. Committee investigators representing
themselves as fair-skinned teenage girls contacted 300 tanning salons nationwide, including at
least three in each state and the District of Columbia. The investigators asked each salon a
series of questions about its policies and the risks and benefits of tanning. Committee
investigators also reviewed the print and online advertising of tanning salons.

*
This is an edited, reformatted and augmented version of a report prepared for Representatives Henry A. Waxman,
Diana DeGette, Frank Pallone, Jr., Rosa L. DeLauro, and Carolyn Maloney. The report was issued February 1,
2012.
 Free ebooks ==> www.Ebook777.com
1416 U.S. House of Representatives Committee on Energy …

The vast majority of tanning salons contacted by Committee investigators provided false
information about the serious risks of indoor tanning and made specious claims about the
health benefits that indoor tanning provides. Specifically, Committee investigators found:

 Nearly all salons denied the known risks of indoor tanning. When asked whether
tanning posed any health risks for fair-skinned teenage girls, 90% of the salons stated
that indoor tanning did not pose a health risk. When asked about the specific risk of
skin cancer, over half (51%) of the salons denied that indoor tanning would increase
a fair-skinned teenager’s risk of developing skin cancer. Salons described the
suggestion of a link between indoor tanning and skin cancer as “a big myth,”
“rumor,” and “hype.”
 Four out of five salons falsely claimed that indoor tanning is beneficial to a
young person’s health. Four out of five (78%) of the tanning salons claimed that
indoor tanning would be beneficial to the health of a fair-skinned teenage girl.
Several salons even said that tanning would prevent cancer. Other health benefits
claimed by tanning salons included Vitamin D production, treatment of depression
and low self-esteem, prevention of and treatment for arthritis, weight loss, prevention
of osteoporosis, reduction of cellulite, “boost[ing] the immune system,” sleeping
better, treating lupus, and improving symptoms of fibromyalgia.
 Salons used many approaches to downplay the health risks of indoor tanning.
During their calls, Committee investigators representing themselves as fair-skinned
teenage girls were told that young people are not at risk for developing skin cancer;
that rising rates of skin cancer are linked to increased use of sunscreen; that
government regulators had certified the safety of indoor tanning; and that “it’s got to
be safe, or else they wouldn’t let us do it.” Salons also frequently referred the
investigators to industry websites that downplay indoor tanning’s health risks and
tout the practice’s alleged health benefits.
 Tanning salons fail to follow FDA recommendations on tanning frequency. The
Food and Drug Administration recommends that indoor tanning be limited to no
more than three visits in the first week. Despite this recommendation, three quarters
of tanning salons reported that they would permit first-time customers to tan daily;
several salon employees volunteered that their salons did not even require 24-hour
intervals between tanning sessions.
 Tanning salons target teenage girls in their advertisements. The print and online
advertising for tanning salons frequently target teenage and college-aged girls with
student discounts and “prom,” “homecoming,” and “back-to-school” specials. These
youth-oriented specials often feature “unlimited” tanning packages, allowing
frequent — even daily — tanning, despite research showing that frequent indoor
tanning significantly increases the likelihood that a woman will develop melanoma,
the deadliest form of skin cancer, before she reaches 30 years of age.

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 False and Misleading Health Information Provided to Teens … 1417

II. BACKGROUND
A. The Growing Popularity of Indoor Tanning

Tanning salons first appeared in the U.S. in the 1970s. Their popularity grew slowly at
first. By 1988, only 1% of American adults reported using indoor tanning facilities. But by
2007, that number had reached 27%.1
Millions of young people use tanning salons each year — often without full knowledge of
the risks of indoor tanning — and this use is on the rise. The most frequent indoor tanners are
young white females. Researchers consistently find high rates of indoor tanning among white
16- to 18-year-old girls, with some studies reporting that as many as 40% of youth in this
demographic have used indoor tanning facilities.2 Having a parent or guardian who has used
indoor tanning in the last year is associated with a 70% increase in the likelihood that a young
person will visit a tanning salon.3
Tanning salons tend to be concentrated in areas with more teenagers and young women
aged 15 to 24.4 This proximity is itself associated with a 40% increase in likelihood of indoor
tanning among teens.5

B. Cancer and Other Health Risks

Ultraviolet (UV) light is electromagnetic radiation with a wavelength longer than visible
light but shorter than X-rays. Sunlight contains UV radiation and emits three bands of the UV
spectrum: UVA, UVB, and UVC. Exposure to either UVA or UVB light can cause DNA
damage that leads to carcinogenesis.6 The primary culprit in sunburn is UVB, and scientists
once believed it to be the only carcinogenic part of the solar spectrum. Recent research,
however, has confirmed that UVA exposure also contributes to development of skin cancer.7
Indoor tanning is a potent source of ultraviolet radiation, especially UVA. While many
assume that the lamps in tanning beds contain less or similar amounts of light to that emitted
by the sun, the UVA radiation emitted by these devices can be as much as 10 to 15 times
more powerful than midday sunlight. Tanning lights also emit UVB radiation, although
depending on the type of tanning device, the UVB emitted may be similar to or less powerful
than the UVB emitted by the sun.
This radiation makes tanning beds dangerous. Medical research has identified indoor
tanning as a cause of skin cancer, including melanoma, the deadliest form of the disease. The
World Health Organization’s International Agency for Research on Cancer (IARC) classifies
tanning beds as a “Group 1” carcinogen, a category that also includes asbestos, arsenic, and
tobacco smoke.8 Similarly, the National Toxicology Program classifies tanning beds as
“known to be human carcinogens.”9
The risk of melanoma is especially high for youth and young adults who engage in indoor
tanning. According to the IARC, the melanoma risk is “increased by 75% when use of
tanning devices starts before 30 years of age.”10 For those who report having undergone ten or
more indoor tanning sessions in the first three decades of life, the risk of being diagnosed
with melanoma before the age of 30 is six times higher than the risk for those who have never
tanned indoors.11 Scientists have found this risk to persist after controlling for sunburns and
 Free ebooks ==> www.Ebook777.com
1418 U.S. House of Representatives Committee on Energy …

outdoor sunbathing habits of melanoma victims.12 One recent study determined that for young
people diagnosed with melanoma between the ages of 18 and 29 years old, “76% of
melanomas were attributable to sunbed use.”13
Indoor tanning can cause “sunburn,” just like too much sun exposure. Nearly 60% of
indoor tanners report experiencing burns after indoor tanning sessions, a major risk factor for
melanoma.14 The risk of melanoma is highest for women reporting sunburns during
adolescence.
Scientists have also documented a link between indoor tanning and other forms of skin
cancer. Researchers have found that a single use of a tanning bed can increase one’s chance
of acquiring basal cell carcinoma, even after controlling for a history of sunburns, sun
exposure, and sunbathing.15 Recently published peer-reviewed research by scientists at the
Yale Cancer Center showed that young people who have ever tanned indoors see a 69%
increase in risk for developing basal cell carcinoma before the age of 40. Approximately one
in four of these cancers, and 43% of the basal cell carcinomas in young women, could be
prevented if people never used indoor tanning beds.16 The IARC found a similar link between
indoor tanning and squamous cell carcinomas.17 The risk associated with indoor tanning is
especially high for people with fair skin.18
The increased popularity of indoor tanning has coincided with a sharp rise in skin
cancer.19 Melanoma is now the most common form of cancer for white women between the
ages of 15 and 29 years old. Since 1980, the rate of melanoma in this group has increased by
50%.20 Non-melanoma skin cancers have also seen a dramatic rise; by 2007, about 13 million
Americans had had at least one such cancer. According to peer-reviewed research published
in the Archives of Dermatology, the rate of non-melanoma skin cancer in the U.S. is
“reaching epidemic proportions.”21
In addition to increasing cancer risks, tanning can cause ocular damage, premature aging
of the skin, and exacerbate other medical conditions.22
There are no health benefits to indoor tanning that outweigh the risks associated with the
practice. There is no “safe or moderate tan.” Even short exposure to tanning can cause DNA
damage. While many indoor tanners report using tanning beds to develop a “base tan” to
protect against sunburns, researchers have concluded that indoor tanning offers no effective
sunburn protection.
The tanning industry frequently promotes the benefits of Vitamin D and its association
with UV light as an advantage of indoor tanning. Peer-reviewed medical research, however,
shows that indoor tanning is an ineffective source of Vitamin D promotion. Although
exposure to UVB light can produce Vitamin D, those most at risk of Vitamin D deficiency —
people with darker skin — photosynthesize less Vitamin D. Moreover, the amount of UVB
emitted from tanning devices varies, with some popular devices emitting relatively low levels.
For most individuals, five to thirty minutes of midday sun twice each week accompanied by a
healthy diet provides sufficient Vitamin D. For those with Vitamin D deficiency, physicians
recommend oral supplements rather than increased exposure to UV radiation.23

C. Federal and State Regulation

Under the Federal Food, Drug, and Cosmetic Act (FDCA), the Food and Drug
Administration currently regulates tanning beds as Class I medical devices, the most lightly

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 False and Misleading Health Information Provided to Teens … 1419

regulated device category. Other medical products regulated as Class I devices include band-
aids, rubber gloves, and tongue depressors. Class I devices are subject to limited federal
oversight; they are supposed to be those devices that “present minimal potential harm” to the
user.
Tanning beds are subject to FDA’s general controls for medical devices (including rules
about good manufacturing practices, recordkeeping, reporting, adulteration, and misbranding)
and performance standards specific to tanning beds.24 These standards: (1) establish limits on
a tanning bed’s irradiance emissions; (2) require a mechanism by which a user of the device
may terminate the tanning session at any time; (3) mandate that tanning bed manufacturers
include protective eyewear with their products when distributed; (4) mandate the presence of
a timer on each tanning bed (though the regulations state explicitly that “[t]he timer
requirements do not preclude a product from allowing a user to reset the timer”); and (5)
require that all tanning beds include the following warning label:

DANGER--Ultraviolet radiation. Follow instructions. Avoid overexposure. As with

natural sunlight, overexposure can cause eye and skin injury and allergic reactions.
Repeated exposure may cause premature aging of the skin and skin cancer. WEAR
PROTECTIVE EYEWEAR; FAILURE TO MAY RESULT IN SEVERE BURNS OR
LONG-TERM INJURY TO THE EYES. Medications or cosmetics may increase your
sensitivity to the ultraviolet radiation. Consult physician before using sunlamp if you are
using medications or have a history of skin problems or believe yourself especially
sensitive to sunlight. If you do not tan in the sun, you are unlikely to tan from the use of
this product.25

While FDA does not prescribe any particular limits on the frequency or duration of
indoor tanning sessions, it has issued guidance to manufacturers on recommended exposure
frequency during the first week of indoor tanning. FDA requires that manufacturers of
tanning devices provide directions for a tanning device’s use to purchasers. These directions
must include a recommended exposure schedule, and FDA guidance suggests that this
schedule recommend no more than three tanning sessions in the first week of indoor tanning
exposure.26
FDA is presently considering a reclassification of tanning beds, potentially triggering
more stringent protections. On March 25, 2010, the General and Plastic Surgery Devices
Panel of FDA’s Center for Devices and Radiological Health Advisory Committee met to
review recent scientific literature on risks posed by indoor tanning and to recommend whether
changes to the devices’ classification or regulatory controls are needed. The panel considered
a presentation by FDA staff and testimony from the medical community and tanning salon
industry. Testifying on behalf of the American Academy of Pediatrics, Johns Hopkins
University Professor of Pediatrics and Dermatology Bernard Cohen stated that “the Academy
believes that tanning lamps are generally unsafe for children and calls on the Food and Drug
Administration to regulate them as such.” He said the American Academy of Pediatrics
supports a ban on tanning by children and teenagers, testifying: “In order to safeguard
children and adolescents from the dangers of unsafe ultraviolet radiation exposure, the
American Academy of Pediatrics recommends a ban on the use of tanning devices by
individuals under the age of 18, unless under the guidance of their physician.”27
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The FDA advisory panel concluded unanimously that tanning beds should not be Class I
medical devices, with panelists split as to whether they should be Class II devices or Class III
devices, which are subject to the strictest FDA controls. A majority of the panel favored age
restrictions for tanning bed use. The panel also recommended enhanced education, training,
and testing of tanning bed operators and improved labeling of tanning beds. In the words of
one physician on the panel, dermatologist Dr. Erin Walker, such revisions to current
regulations must make clear the medical consensus that “there is no such thing as a safe
tan.”28 The FDA is currently considering these recommendations.
Some states have responded to the growth in the tanning industry and the mounting
medical evidence of a link between tanning and skin cancer with regulations limiting access
to tanning beds by children and adolescents. Over 30 states have enacted legislation
regulating indoor tanning by teens — most commonly, by requiring parental consent for use
of a tanning bed.29 Even in states with these restrictions, the effectiveness of the regulations
remains a concern. Studies of compliance with parental consent laws in Texas, North
Carolina, and Minnesota and Massachusetts have found tanning salon compliance rates of
11%, 13%, and 19%, respectively.30 Despite an increase over the last decade in states
requiring some form of parental permission for indoor tanning, researchers have found no
measurable decrease in indoor tanning among older adolescent girls.
California recently enacted legislation banning indoor tanning by children altogether.31
The law took effect on January 1, 2012. California is the first and only state to protect
children via a ban on indoor tanning. The indoor tanning industry opposed California’s ban,
while the American Academy of Dermatology praised it, commending the state for
“protecting youth from the dangers of indoor tanning.” 32

III. PURPOSE AND METHODOLOGY

Ranking Members Waxman, DeGette, and Pallone, along with Reps. DeLauro and
Maloney, requested that the Democratic Committee staff investigate how tanning salons
communicate risks to teens who seek information about indoor tanning sessions. In response
to this request, Committee staff investigators, including college students interning with the
Committee, telephoned indoor tanning salons across the country representing themselves as
fair-skinned 16-year-old girls considering purchasing indoor tanning sessions for the first
time. Committee investigators spoke with employees at 300 indoor tanning salons
nationwide, including at least three salons in all 50 states and the District of Columbia.
On calls with salons, investigators asked: (1) whether the salon offered discounts to
students or teens; (2) how frequently a new customer would be permitted to use the salon’s
tanning beds; (3) whether indoor tanning posed any risks for people with fair skin; (4)
whether indoor tanning increased one’s risk of acquiring skin cancer; and (5) whether indoor
tanning provided any health benefits. When salons referred callers to information provided on
a website, investigative staff reviewed these materials.
Committee staff also collected and reviewed advertising and promotional material created
by indoor tanning salons. In particular, staff reviewed tanning salon websites, Facebook pages
and posts for and by tanning salons, and print advertising.

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IV. FINDINGS
A. Tanning Salons Provided False Information about the Health Risks of
Indoor Tanning

The vast majority of the 300 tanning salons contacted by Committee staff provided
inaccurate and misleading information about the health risks of indoor tanning. When
Committee staff representing themselves as fair-skinned 16-year-old girls asked tanning
salons whether indoor tanning would present any health risks, 90% of the salons reported that
it presented no risk and only 7% reported that risks were present. The remaining 3% of salons
did not provide clear answers about health risks.
When Committee investigators pressed salons about the specific threat of skin cancer, the
majority of tanning salons provided information that was inaccurate and misleading. More
than half (51%) of the 300 salons claimed that indoor tanning would not increase a young,
fair-skinned person’s risk of developing skin cancer. “No, no, no — that’s not true
whatsoever,” insisted one salon employee. “Tanning beds do not cause melanoma,” another
assured Committee staff. Others described cancer risks as “a big myth,” “rumor,” and “hype”
that had not been “proven.” “People who are meant to get skin cancer are just going to get
skin cancer,” one employee explained. “We wouldn’t offer it if we thought it caused cancer,”
stated another.
Even salons that accurately reported skin cancer risks misleadingly described those risks.
One equated the skin cancer risk associated with indoor tanning as similar to that posed by the
sunlight absorbed while “walking to your car.” Another compared the risk of cancer from
indoor tanning to that presented by “standing in front of the microwave” oven.
Several salons provided misleading advice about who is at risk for skin cancer.
Employees at two salons told investigators representing themselves as 16-year-olds that skin
cancer from indoor tanning is only a concern for “for an old person” or “older people.”
Another suggested that use of sunscreen could actually increase one’s risk for skin cancer,
explaining that “skin cancer rates increased when sunscreen started being promoted.”

Figure 1. 90% of Salons Provided Inaccurate Information about.

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Figure 2. 51% of Salons Denied a Link between Indoor Tanning and Skin Cancer.

In discussing cancer risks, some salons pointed to the regulatory environment for indoor
tanning as evidence of a lack of risk. These salons suggested that the current state of
regulation amounted to confirmation of the practice’s safety, telling Committee investigators:
“If it was incredibly bad for you, you wouldn’t be allowed to do it”; “It’s got to be safe, or
else they wouldn’t let us do it”; “you can get skin cancer from being outside . . . but our
[tanning] beds are certified and regulated”; and “the FDA wouldn’t approve tanning salons if
it weren’t safe.”
Salons also provided false information about skin damage and the risk of burns that might
occur in a fair-skinned, first-time indoor tanner. Several suggested that indoor tanning is
significantly less risky than casual exposure to natural sunlight. Others were unconcerned
about skin damage from any source. One suggested that “aggressive tanning” is necessary
when trying to build a tan in a fair person. Another told the caller that fair-skinned clients
“just have to get that burning out of the way.”

B. Tanning Salons Provided Inaccurate or Misleading Information about

Health Benefits of Indoor Tanning

Tanning salons frequently claimed that indoor tanning would be beneficial to the health
of teenagers, despite medical consensus to the contrary. Overall, 78% of the salons reached
by Committee staff claimed that indoor tanning would provide health benefits. “Tanning is
very good for you,” one salon employee volunteered.
The most common benefit claimed by salons was promotion of Vitamin D production,
with 60% of salons asserting that indoor tanning would be a good source of Vitamin D.
Physicians do not recommend indoor tanning as a source of Vitamin D, however. Those most
at risk of Vitamin D deficiency are least likely to increase Vitamin D levels through tanning
because they typically have darker skin. Moreover, the level of UVB radiation from tanning
devices, which is what can produce Vitamin D, can vary considerably, with several popular

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devices emitting relatively low levels that would not contribute significantly to Vitamin D
production.

Figure 3. 78% of Salons Claimed Indoor Tanning Is Beneficial to Health.

Employees at eleven salons claimed that indoor tanning would prevent cancer. One
named skin cancer, breast cancer, colon cancer, and prostate cancer as diseases that could be
prevented though use of tanning beds.
Other health benefits mentioned by salons contacted by Committee staff include
treatment of depression and low self-esteem, treatment for acne, prevention of and treatment
for arthritis, weight loss, prevention of osteoporosis, “skin tightening,” reduction of cellulite,
“boost[ing] the immune system,” improved sleeping, treating lupus, and improving symptoms
of fibromyalgia.

C. Tanning Salons Regularly Disregarded FDA Safety Recommendations

Three quarters of tanning salons did not follow FDA recommendations on tanning
frequency. The FDA recommends that indoor tanning be limited to no more than three visits
in the first week. Despite this recommendation, 74% of the salons that Committee staff
contacted stated that they would permit first-time, fair-skinned teenage girls to tan daily, and
four salon employees volunteered that their salons did not require 24-hour intervals between
tanning sessions.

D. Tanning Salons Targeted the Teen Market in Advertisements

The tanning salons contacted by Committee investigators frequently targeted youth in

their marketing promotions. Among the tanning salons contacted by Committee investigators,
over half (52%) offered discounts to students or teens.
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Committee investigators reviewed over one hundred tanning salon websites and
newspaper advertisements and found that “prom,” “homecoming,” and “back-to-school”
specials are common. “It’s time to start on that Homecoming tan!!!” states a typical
advertisement. Committee investigators also found that tanning salons are active users of
social media, with many maintaining Facebook pages and Twitter accounts. Salons post
notices about discounts on their own social media sites and also on Facebook pages for
student groups, such as cheerleading squads.

The most common discounts offered to young people in the advertising materials
reviewed by Committee staff were reduced rates on “unlimited” tanning packages, which
allow customers to visit a salon as often as they wish in a particular period of time (typically,
one month). This type of discounting raises concern because, while any use of indoor tanning
increases skin cancer risks, frequent tanning sessions significantly increase the chance of
acquiring melanoma.

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E. Tanning Industry Websites Provide Misleading Information

When presented with requests for health information about indoor tanning, tanning salons
frequently directed investigators to tanning industry websites that create a misleading picture
of the risks and benefits of indoor tanning. Most commonly, they suggested that teens curious
about the health impact of indoor tanning visit www.tanningtruth.com or www.smarttan.com.
Both sites are associated with the “International Smart Tan Network,” a tanning industry trade
association. The sites downplay the cancer risk associated with indoor tanning and tout the
practice’s alleged health benefits.
Visitors to www.tanningtruth.com see a series of large-print pro-tanning statements
running across the top of the screen while navigating the website. The statements begin with
an assertion that “[s]aying sunlight is harmful and therefore we should avoid it is as
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1426 U.S. House of Representatives Committee on Energy …

misleading as saying that water causes drowning, and therefore we should avoid it.”
Statements that follow suggest that medical advice about the use of sunscreen and avoidance
of indoor tanning is driven by the profit motives of pharmaceutical companies and
dermatologists.
The website’s discussion of the health impacts of tanning present a different picture than
that provided by peer-reviewed medical research. Under a tab labeled “What are the real risks
of indoor tanning?” the industry website questions the link between indoor tanning and
melanoma, saying that “the relationship between melanoma and ultraviolet light remains
unclear.” Under a tab labeled “Are there any benefits to indoor tanning?” the trade association
claims that tanning is “nature’s sunscreen,” treats cosmetic skin conditions, and promotes
Vitamin D production. The site then suggests that indoor tanners produce a “sufficient” level
of Vitamin D, “non-tanners” produce a “deficient” level, and dermatologists experience a
“severe deficiency” of Vitamin D.
The other industry website, www.smarttan.com, also provides misleading information
about Vitamin D and tanning. On this website, salon operators may purchase “D-Angel”
training, which “teaches [salon] employees why Smart Tanning is vindicated and why they
should spread the truth about UV and Vitamin D to their friends and family.” It provides a
link to a website for the “Vitamin D Council,” which suggests that Vitamin D promotion
yields a host of health benefits, including prevention of cancer, heart disease, diabetes,
autism, multiple sclerosis, chronic digestive diseases, food allergies, and tuberculosis, as well
as treatment for lupus.

CONCLUSION
Indoor tanning significantly increases skin cancer risks and presents a number of other
significant health concerns. These risks are particularly acute for teenagers and young adults.
Indoor tanning salons, however, regularly deny these risks. When Committee investigators
contacted 300 tanning salons to ask about the risks indoor tanning posed to fair-skinned
teenage girls, the vast majority of salons denied that indoor tanning increases health risks.
The dangers to teenage girls are exacerbated by tanning industry practices. Committee
investigators found that the marketing practices of tanning salons target teenagers and young
adults, often offering back-to-school, homecoming, and prom promotions.

End Notes
1
Denis K. Woo and Melody J. Eide, Tanning Beds, Skin Cancer, and Vitamin D: An Examination of the Scientific
Evidence and Public Health Implications, Dermatologic Therapy (2010) (hereinafter, “Tanning Beds, Skin
Cancer, and Vitamin D”).
2
Id.; Joni A. Mayer et al., Adolescents’ Use of Indoor Tanning: A Large-Scale Evaluation of Psychosocial,
Environmental, and Policy-Level Correlates, American Journal of Public Health (May 2011) (hereinafter,
“Adolescents’ Use of Indoor Tanning”).
3
See Adolescents’ Use of Indoor Tanning.
4
Vilma Cokkinides et al., Indoor Tanning Use among Adolescents in the US, 1998 to 2004, Cancer (Jan. 2009)
(hereinafter, “Indoor Tanning Use among Adolescents”).
5
Indoor Tanning Use among Adolescents; Tanning Beds, Skin Cancer, and Vitamin D; Adolescents’ Use of Indoor
Tanning.

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 False and Misleading Health Information Provided to Teens … 1427

6
Exposure to UVC is also carcinogenic, but UVC rays from the sun do not reach the earth’s surface, so they do not
present the same human health risks as UVA and UVB.
7
See Tanning Beds, Skin Cancer, and Vitamin D.
8
See International Agency for Research on Cancer, Agents Classified by the IARC Monographs, Volumes 1-102
(available online at http://monographs.iarc.fr/ENG/Classification/ ClassificationsGroupOrder.pdf) (visited Jan.
26, 2012).
9
U.S. Department of Health and Human Services, Public Health Service, National Toxicology Program, Report on
Carcinogens, 12th ed.: Exposure to Sunlamps or Sunbeds (2011).
10
Special Report: Policy, A Review of Human Carcinogens — Part D: Radiation, The Lancet (Aug. 2009); see also
Tanning Beds, Skin Cancer, and Vitamin D.
11
Anne E. Cust et al., Sunbed Use During Adolescence and Early Adulthood Is Associated with Increased Risk of
Early-Onset Melanoma, International Journal of Cancer (May 2011) (hereinafter, “Sunbed Use During
Adolescence and Early Adulthood”).
12
See Marit Bragelien Veirød et al., A Prospective Study of Pigmentation, Sun Exposure, and Risk of Cutaneous
Malignant Melanoma in Women, Journal of the National Cancer Institute (Oct. 2003); J Westerdahl, Risk of
Cutaneous Malignant Melanoma in Relation to Use of Sunbeds: Further Evidence for UV-A Carcinogenicity,
British Journal of Cancer (2000).
13
See Sunbed Use During Adolescence and Early Adulthood.
14
See Indoor Tanning Use among Adolescents.
15
See Tanning Beds, Skin Cancer, and Vitamin D.
16
See Leah M. Ferrucci et al., Indoor Tanning and Risk of Early-Onset Basal Cell Carcinoma, Journal of the
American Academy of Dermatology (Dec. 2011).
17
See Tanning Beds, Skin Cancer, and Vitamin D.
18
Rutao Cui et al., Central Role of p53 in the Suntan Response and Pathologic Hyperpigmentation, Cell (Mar.
2007) (hereinafter, “Central Role of p53”); Tanning Beds, Skin Cancer, and Vitamin D.
19
Tanning Beds, Skin Cancer, and Vitamin D.
20
National Cancer Institute, NCI Cancer Bulletin (July 2008).
21
Study Finds “Epidemic” of Skin Cancer, ABC News (Mar. 2010).
22
See James M. Spencer and Rex A. Amonette, Indoor Tanning: Risks, Benefits, and Future Trends, Journal of the
American Academy of Dermatology (1995).
23
See Tanning Beds, Skin Cancer, and Vitamin D.
24
21 U.S.C. § 360c(a)(1)(B).
25
21 C.F.R. § 1040.20(c)-(d).
26
FDA, Consumer Health Information, Indoor Tanning: The Risks of Ultraviolet Rays (Nov. 2009).
27
FDA, Transcript of General and Plastic Surgery Devices Panel Meeting (Mar. 25, 2010) (available online at
http://www.fda.gov/downloads/AdvisoryCommittees/Committees
MeetingMaterials/MedicalDevices/MedicalDevicesAdvisoryCommittee/GeneralandPlasticSurgeryDevicesPan
el/UCM210232.pdf) (visited Jan. 26, 2012).
28
Id.
29
See Indoor Tanning Use among Adolescents; Tanning Beds, Skin Cancer, and Vitamin D. Over twenty states have
enacted laws requiring parental permission for children who wish to purchase indoor tanning sessions, with the
age at which this requirement expires varying from 15 to 18. See, e.g., Ariz. Admin. Code R 12-1-1414 A2;
Ark. Stat. Ann. § 20-27-2202; Conn. Gen. Stat. § 19a-232; Fla. Stat. Ann. tit. § 381.89; Ga. Code Ann. § 31-
38-8; Ind. Code Ann. § 25-8.4-15, 16; Ky. Rev. Stat. § 217.922; La. Rev. Stat. Ann. § 40:2701-18; Md. Health
Code Ann. § 20-106; Mass. Gen. Laws Ann. ch. 111 Pub. Health § 211; Mich. Comp. Laws Ann. §
333.13405; Minn. Stat. Ann. § 325H.08; Miss. Dept. of Health Regs. tit. 15 part III subpart 78 ch. 2; Ohio
Admin. Code 4713-19-09(B); OAR 333-119-0090(2); R.I. Dept. of Health Rules and Regs. for the
Registration of Tanning Facilities, Part III § 9.5; S.C. Code Ann. ch. 61 § 106-4.5; Tenn. Code Ann. § 68-117-
104; Utah Code Ann. § 26-15-13; Va. Code § 59.1-310.3; Wyo. Enrolled Act 26. Several other states require
parental permission for older adolescents and prohibit indoor tanning for very young children, typically under
the age of 14. See, e.g., Del. Code Ann. tit. 16 § 30D; Ill. Admin. Code tit. 77 § 795.190(c); 10-144 Maine
Dept. of Human Servs. Ch. 223 12A(3)(f); N.H. Rev. Stat. Ann. § tit. XXX 313-A:31; N.J. Rev. Stat. §
C.26:2D-82.1; N.Y. Pub. Health Law § 3555; N.C. Gen. Stat. § 104E-9.1; N.D. Cent. Code § 23-39; Tex.
Health and Safety Code Ann. § 145.008. Wisconsin has banned indoor tanning for those under 16, but has no
parental consent requirements for older children. Wis. Code Ann. § 255.08(9)(a).
30
See Indoor Tanning Use among Adolescents in the US; Tanning Beds, Skin Cancer, and Vitamin D.
31
Cal. Bus. and Prof. Code §§ 22706, 2241.3.
32
See California Bans Indoor Tanning for Minors, N.Y. Times (Oct. 10, 2011).
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 64

METABOLOMIC ASSESSMENT OF
SUNSCREEN EFFICACY

Manpreeet Randhawa*, PhD and Michael D. Southall, PhD

1
Johnson and Johnson Consumer Companies, Inc., Skillman, NJ, US

ABSTRACT
UV radiation (UVR) exposure remains the most preventable environmental risk
factor for cosmetic reasons as well for skin cancers. Aside from sun avoidance,
sunscreens remain our best protection. UVR directly damages DNA and cause indirect
cellular damage through the creation of reactive oxygen species, the sum of which leads
to cutaneous immunosuppression as well as phenotypic changes that is referred as
photoaging. The current methodology for looking at sunscreens efficacy against UVR
protection is limited to very few markers at molecular level, whereas erythema is the
main marker validated to current date for clinical purposes. DNA damages at the
molecular level are very well understood (de Gruijl FR et al., 2001). The markers may
provide information about acute exposures, but lack the ability to determine the effect
with chronic exposures. Moreover, it is not necessary that the amount of photon required
for DNA damage, might not be same for some of the other sensitive markers. The correct
determination of the protective ability of a sunscreen product should be a prerequisite to
reliably avoid the appearance of UV-induced injuries to human health, e.g., sunburn,
immunosuppression, skin aging, skin cancer and radical formation.
The chapter summarizes metabolomics: a relatively new branch of omics that studies
the global metabolite profiles in a given biological system (cell, tissue, or organism)
under a given set of conditions. Studies have shown that metabolomic analysis of skin
samples from sun-exposed and sun-protected sites; demonstrated clearly that sun
exposure altered the metabolic profile in the sun-exposed skin biopsies. However the
usage of sunscreen can attenuate the effect of UV exposure and prevents change in
metabolomic profile of explants treated with sunscreen followed by UV exposure.

*
Corresponding author: Manpreeet Randhawa, Johnson & Johnson Consumer Companies, Inc. 199 Grandview
Road, Skillman, NJ 08558; Phone: 908-904-3062 ; Fax: 908-874-1209; Email: [email protected].
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1430 Manpreeet Randhawa and Michael D. Southall

Conflict of interest disclosure: MK Randhawa and M Southall are employees of Johnson &
Johnson Consumer Companies, Inc., the manufacturer of the Ultrasheer Helioplex®
broad spectrum sunscreen SPF 70.

Funding Sources: The study was funded in full by Johnson & Johnson Consumer
Companies, Inc; the preparation of this manuscript was sponsored in full by Johnson &
Johnson Consumer Companies, Inc.

INTRODUCTION
Skin is the largest organ that serves as an important environmental interface providing a
protective envelope crucial for homeostasis. Among environmental stressors UV radiation
which spans 100-400nm of solar spectrum induces a number of phenotypic responses as a
result of biological alterations. Acute exposure to UV leads to clinically perceptible signs like
pigmentation (tanning) and erythema (sunburn), whereas chronic exposure to UV causes
premature skin aging or photoaging and even increases the risk of skin cancer (Zastrow L et
al., 2009a, 2009b). Photoaging is associated with marked cutaneous alterations clinically
characterized by wrinkles, roughness, sallowness, mottled pigmentation, telangiectasia, and a
variety of benign and malignant neoplasms. Histologic and ultrastructural studies have shown
these changes in terms of molecular endpoints in photoaged skin, which were mainly found in
dermal connective tissue (Seité S. & Fourtanier A. 2008). Skin photoaging could be explained
as the consequence of solar UV exposure, where DNA damage has been shown to play an
important role. As a result most of the research is focused on selective DNA related markers
like p53, CPD, TTdimers (7. de Gruijl FR., 2001; Yamaguchi Y et al., 2008) that can provide
some information, but lacks the holistic view about the photodamaged skin.
Sunscreens, the UVR absorbing chemicals attenuate the amount and nature of UVR
reaching viable cells in the skin. They are selected and tested for their ability to prevent
erythema. The correct determination of the protective ability of a sunscreen product should be
a prerequisite to reliably avoid the appearance of UV-induced injuries to human health, e.g.,
sunburn, immunosuppression (Bennett MF et al., 2008), skin aging, skin cancer and radical
formation (Zastro L et al., 2009). But the standard method to characterize sunscreen
protection properties is through determination of the sun protection factor (SPF), which is
significantly influenced by MED and is based on determination of erythema as a clinical
endpoint. On the other hand at molecular level, damages linked to DNA like p53, CPD and
ttdimers (Mouret S et al., 2008) are explored at the biological level to verify the efficacy
of sunscreen for photodamage purposes (Wassberg C. et al., 2003). This, however, could be
disadvantages; as it is taking into account of other biological processes that doesn’t even need
that amount of energy as required for DNA damage to get initiated. On the other hand most of
the DNA related damages are able to repair itself that means it could be a marker for acute
damage, but does not account for low level chronic damages.

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 Metabolomic Assessment of Sunscreen Efficacy 1431

METABOLOMICS A TOOL TO MEASURE UV INDUCED ENDPOINTS

Skin exposure to physical, chemical and environmental stressors induces alterations at
genetic, protein and metabolic level. Photoaging is considered of great cosmetic concern and
has been studied very thoroughly at genetic and proteomic level. Often these UV induced
changes studied through these tools are limited to biological markers including DNA damage
(de Gruijl FR et al., 2001), inflammation, and immune suppression (Bennett MF et al., 2008).
However not much attention has been paid to the change in metabolic activities with UV
exposure.
Metabolomics is a relatively new branch of omics that studies the global metabolite
profiles in a given biological system (cell, tissue, or organism) under a given set of
conditions. Metabolites, are the end product of a complex interplay between the changes and
interactions at genomic and protein levels under a given condition are the result of the
interaction of the system's genome with its environment. These biochemicals are not merely
the end product of gene expression but also form part of the regulatory system in an
integrated manner. These metabolites could be generated or broken down by the cells,
residing in the cells, secreted by the cells or taken up from ECM (Extracelluar Matrix). A
metabolomics investigation provides the ability to assess changes in the abundance of large
numbers of metabolites representing multiple classes of compounds and these changes
capture global shifts such as catabolic or anabolic metabolism and can present an overall
physiological status such as stress or hyperactivity of the biological system. Since a
metabolomic profile is the downstream product of numerous genome-wide or proteome-wide
interactions, so it can be a very proximal snapshot of an organism’s phenotype. Studies have
shown these changes in the context of biochemical networks and pathways, can serve as a
means to identify biomarkers of disease (Wang-Sattler R et al., 2012).
Studies have shown that the contribution of sun exposure to biochemical changes that
result in alterations in skin metabolome have essentially been limited to a few biomolecules
such as glutathione (Zhu M et al., 2006). These changes have often discussed for single
classes of metabolites in relevance to their biological pathway and are described as an
outcome of a particular genetic pathway, yet a holistic approach to understand the effect of a
biochemically related group of metabolites is missing. However these changes can account
for both negative effects like oxidative stress or hyper proliferative phases as well as
modulation of positive effects like acceleration of repair mechanisms in skin.

METABOLOME ANALYSIS OF SUN EXPOSED SKIN

Metabolome analysis of 50 paired skin samples obtained from sun-exposed and sun-
protected sites through principal component anlaysis; demonstrated clearly that sun exposure
altered the metabolic profile in the sun-exposed skin biopsies (Figure 1) (Randhawa M et al.,
2014).
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1432 Manpreeet Randhawa and Michael D. Southall

Figure 1. Principal component analysis (PCA): PCA plot showing separation of the sun exposed and
sun protected skin samples. 1- unexposed inner arm. 2- exposed outer arm.

The identified metabolites belonged to a total of 52 biological pathways and a subset of

42 pathways had one or more metabolite(s) significantly different when biopsies obtained
from sun-exposed regions were compared to the one obtained from sun-protected regions.
These pathways spanned amino acids, nucleotides, sugars, peptides, cofactors, lipid
metabolism and others (Figure 2) (Randhawa et al., 2014).
The study showed significant alterations in multiple classes of metabolites observed
pointing to comprehensive alteration in the metabolic profile of sun-exposed skin. The
metabolomics signature classified the sun-exposed and sun-protected skin samples with very
high accuracy. As expected, in this signature, cis-urocanate; a validated biomarker for UV
damage was significantly higher in sun exposed as compared to sun protected skin.
Additionally, the prioritized metabolite list presented a theme of metabolomic catabolism and
oxidative stress as a result of sun exposure between the two classes, which was further
corroborated when the study analyzed the three major networks. Most of these pathways
suggested increased production of reactive oxygen species (ROS), which resulted in increased
oxidative stress that can be held responsible for changes in the phenotypic appearance of sun-
exposed skin.

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 Metabolomic Assessment of Sunscreen Efficacy 1433

Figure 2. Distribution of the metabolomics data with colors depicting the levels of metabolites.

UROCANIC ACID AS A MARKER OF UV EXPOSURE

The study showed decreased level of histidine and increased level of histamine that
certainly pointed toward increased inflammation as well as increased catabolism for histidine.
The second metabolite, urocanic acid comes from histidine metabolism represented increased
isomerization from trans form to cis form (Figure 3). Trans from of urocanic acid is a major
epidermal chromophore for UVR (Tabachnick, 1957), whereas cis form of urocanic acid act a
mediator of the immunosuppressive effects of UVR in the skin (de Fabo EC, Noonan FP,
1983). The study validated not only one metabolite, but the whole pathway related to the
natural sunscreen present in the epidermis. The various metabolites analyzed in this pathway
not only played role in generating inflammation, but also in building up immunosuppression
at the same time. The whole pathway suggested a very fine line between generating
inflammation and building immunosuppression against generated inflammation (Randhawa et
al., 2014).
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1434 Manpreeet Randhawa and Michael D. Southall

Figure 3. Histidine metabolism: This schematic presents the steps in the Histidine metabolism pathway
to production histamine and urocanic acid.

INCREASED OXIDATIVE STRESS AS A RESULT OF

ADENOSINE DEGRADATION
The study represented significantly higher levels of metabolites related to adenosine
pathway like inosine, inosine monophosphate, xanthine, hypo-xanthine, uric acid but without
any change in adenosine. In this pathway adenosine is deaminated to form inosine that is
converted into hypo-xanthine and subsequently to xanthine which gets further converted into
uric acid. Another purine, guanosine’s degradation can also produce xanthine however; there
was no significant change in the levels of intermediate guanosine degradation metabolite:
guanine when the two sets of metabolomes were compared (Figure 4). The data present in the
study suggested that purine degradation is limited to adenosine and there is no contribution to
purine catabolic products through guanine degradation branch of the pathway (Randhawa et
al., 2014).

Figure 4. Adenine metabolism: This schematic presents the steps in the adenosine and guanosine
pathway leading to production of uric acid. ATP – Adenosine triphosphate, GMP – guanosine
monophosphate, IMP – inosine monophosphate. Red arrows – higher accumulation, green arrow – low
accumulation.

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 Metabolomic Assessment of Sunscreen Efficacy 1435

Purine degradation pathway has been suggested a major biochemical source for ROS
(reactive oxygen species) production (Barnes VM et al., 2009) and can be considered as one
of the main pathways fueling high oxidative stress in sun exposed skin. Xanthine oxidase, the
main enzyme required for degradation of xanthine to hypoxanthine and further to hydrogen
peroxide has been previously shown to induce ROS (Landmesser U et al., 2007). Inhibition of
this particular enzyme has also been shown to decrease ROS in the respective biological
system. Increased ratio of hypoxanthine and uric acid from the data definitely suggest
increased activity of xanthine oxidase, which probably contributes toward phenotypic
appearance of sun exposed skin.
Interestingly, significantly higher levels of fructose were also measured in the exposed
skin samples. Brosh et al. reported that higher consumption of fructose could lead to higher
degradation of adenosine in the liver. Whether fructose plays role in adenosine catabolism in
skin is ascertain, but cannot be ignored and needs to be evaluated further, especially the role
of enzymes and fructose in the catabolic pathway. The source of a simple sugar such as
fructose in this study could not be ascertained. A higher accumulation of fructose could be the
result of carbohydrate degradation through UV exposure or could be contributed by diet
(Randhawa et al., 2014).
Taken together, these measurements suggested that UV exposure is leading to
degradation of adenosine and potentially contributing towards a more oxidized state in the
cell. However finding the exact mechanism and the role of different enzymes and
interestingly the role of diet needs further investigation.

ALTERED HOMOCYSTEINE PATHWAY LEADING

TO ALTERED RATIO OF GLUTATIONE

Methionine and glutathione pathways are connected by the transsulfuration pathway in

which methionine cycle provides sulfur for cystathione formation through homocysteine
(Figure 5) (Ratnam S et al., 2012). The study represented no significant change in the levels
of methionine, S-adenosylmethionine and homocysteine in methionine pathway. Only S-
adenosylhomocysteine had a significantly higher accumulation in the sun exposed skin
samples as compared to sun protected skin samples. However, metabolites further than the
transsulfuration pathway such as cysteine, GSH, GSSG were measured significantly different
between the sun exposed and sun protected skin samples. The ratio of glutathione (GSH) to
oxidized glutathione (GSSG) was lower in the sunprotected samples, suggesting increased
oxidative stress. High levels of Cysteine-glutathione disulfide and the low ratio of GSH to
GSSG reflect prevalence of oxidizing conditions in the samples that were photo exposed.
Additionally, high levels of cysteine, glycine (not statistically significant) and glutamate,
gamma-methyl ester were also detected. These metabolites are part of the glutathione
biosynthesis pathway, which further indicated the pervasiveness of oxidative stress in the
exposed samples (Randhawa M et al., 2014).
A biological system can also utilize an alternate pathway to generate glutathione. In this
pathway, glutathione biosynthesis is achieved through γ-glutamylaminoacids, 5-oxoproline
and glutamate. Levels of various γ-glutamylaminoacids (γ-glutamylalanine, γ-
glutamylleucine, γ-glutamylisoleucine, γ-glutamylphenylalanine and others) were detected at
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1436 Manpreeet Randhawa and Michael D. Southall

low levels in the sun exposed skin samples as compared to the sun protected skin samples.
These findings indicate that the alternate pathway involving γ-glutamylaminoacids is working
at a lower level as compared to the pathway involving cysteine and glycine (Randhawa M et
al., 2014).

Figure 5. Methionine-Glutathione metabolism: This figures details the levels of various metabolites that
were detected in the pathaway. Red arrow indicates higher levels, green arrow indicates lower levels
and black arrow indicates no change in the levels of the metabolite.

Collectively, ratio of glutathione (GSH) to oxidized glutathione (GSSG) definitely points

towards increased ROS as well as oxidative stress and has been documented before in context
of photoaging (Randhawa M et al., 2013). According to the free radical theory of aging, ROS
increases with aging due to the reduced activity of the antioxidant defense enzymes (Harman
D 1956; 1968; 1981), similarly in this case it could be speculated that enzymatic machinery
might be modulated resulting in high oxidative stress.

NICOTINAMIDE PATHWAY SUGGESTS PATHWAY SUGGESTS SKIN

IS USING SALVAGE PATHWAY AS COMPARED TO DE NOVO
PRODUCTION TO CONSUME THE DAMAGED NICOTINAMIDES
In NAD+ metabolism pathway six metabolites were quantified out of which three
metabolites; nicotinamide adenine dinucleotide (NAD), nicotinamide ribonucleotide (NMN)
and nicotine riboside (NR) were observed at significantly higher levels. NR had highest fold
change (2.69) in the exposed samples as compared to the unexposed. All of these metabolites

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 Metabolomic Assessment of Sunscreen Efficacy 1437

belong to the salvage pathway and their higher accumulation indicates hyperactivity of NAD
salvage pathway in the cells of the sun exposed skin samples. The biosynthesis of NAD+
occurs through salvage and/or de novo pathways. In the de novo pathway, NAD is
synthesized from tryptophan through six steps and in the salvage pathway NAD+ is
synthesized by reclaiming degradation products of metabolites having nicotinamide ring
NAD (Lin SJ 2003). Quinolinic acid, nicotinic acid mononucleotide which are critical
intermediates in the de novo synthesis of NAD were not detected in the dataset. Their absence
might indicate hypoactivity or no activity of NAD production through de novo synthesis.

Figure 6. Nicotine metabolism pathway: This schematic presents the de novo and salvage pathways
through which the NAD can be generated. Red arrow indicates higher accumulation and black arrow
indicates no change.

Nicotinamide pathway has been studied very well and its respective metabolites has been
shown the potential to influence cellular processes including DNA repair, genomic stability,
the immune system, stress responses, signaling, transcription, apoptosis, metabolism,
differentiation, chromatin structure and life span (Khan JA et al., 2007). In addition to its
well-known redox functions in energy metabolism, NAD and NADP are also required for the
synthesis of cyclic ADP-ribose and NADP, which are two major mediators of intracellular
calcium signaling pathways (Khan JA et al., 2007). These measurements indicate a theme of
degradation in the cell because the salvage pathway recycles the degraded products of
nicotinamide containing metabolites. Despite higher biosynthesis of NAD+, no significant
change in the NAD+ levels between the two sets of samples. These data indicate that all the
NAD+ produced through salvage pathway is siphoned to produce raw materials for DNA,
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1438 Manpreeet Randhawa and Michael D. Southall

proteins and other cellular processes. Besides UV induced cutaneous proliferation does justify
the need for increased amount of genetic material for new multiplying cells (El-Abaseri TB et
al., 2005). However the increased demand of energy by the respective biological system
cannot be ignored at the same time, hence salvage pathway being more energy efficient and
meets the current needs of the biological system. Moreover this information completely
agrees with previously published studies that reported an increased glycolytic pathway for
energy production instead of TCA cycle in sun exposed skin.

SYSTEM BIOLOGY A NEW APPROACH TO INVESTIGATE

THE EFFICACY OF SUNSCREEN

A second study performed on explants obtained from abdominal plasticity from females
of Caucasian background was analyzed for global metabolomics profile. Metabolome
analysis of explants treated with a photostable UVA/UVB broad spectrum Ultrasheer
Helioplex® sunscreen (Neutrogena Corporation, Los Angeles , CA) followed by UV
treatment revealed that the metabolomics profile of the sunscreen treated skin was most
similar to the metabolomics profile of untreated skin (non-irradiated) and very different from
the metabolomics profile of UV treated skin that did not receive topical sunscreen treatment.
The data analyzed in the study provides the information, which is not limited to few markers,
but also provides a holistic approach about how the skin acts in terms of energy consumption,
oxidative stress and inflammation and role that sunscreen plays to help maintain the system
near to its basal level.

Figure 7. Principal component analysis (PCA): PCA plot showing separation of the (U) untreated, (I)
UV irradiated and (S) Ultrasheer Helioplex® broad spectrum sunscreen protected explants.

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 Metabolomic Assessment of Sunscreen Efficacy 1439

In the topical sunscreen treatment study a total of 190 metabolites were measured under
three conditions. When treatments were compared to the control, the radiation-exposed and
Ultrasheer Helioplex® broad spectrum sunscreen-protected samples presented very different
metabolomics profiles. For the radiation-exposed samples, the metabolic signature matched
the metabolic signature of sun exposed skin discussed earlier. The data analysis from the
respective presented convincing evidence that sunscreen protected the skin explants from the
radiation. In the Principal Component Analysis (Figure 7), sunscreen-protected and
unexposed clustered together and the radiation-exposed explant metabolomes were far away
from this cluster. Interestingly, the photoprotective effect of Ultrasheer Helioplex® broad
spectrum sunscreen was so substantial that it essentially ablated the effect of UV with the
result that the Principal Component Analysis of sunscreen treated skin clustered most closely
with untreated skin (non-irradiated) and was very different from UV exposed skin. These
results demonstrate that even at the Principal Component Analysis level, metabolomics
assessments can strongly differentiate the effects of sunscreens.

SUNSCREEN APPLICATION PREVENTS IRRADIATION INDUCED

ISOMERIZATION OF UROCANIC ACID
The study performed on explants showed significant increase in isomerization of
urocanic acid to cis form almost by 4 folds. However explants exposed to UV after sunscreen
application seems to halts the isomerization of trans to cis form of urocanic acid, hence
representing no change in urocanic acid. Apart from urocanic acid, histamine a marker for
inflammation has also been shown to be induced by UV exposure. Increased levels of
histamine were observed in explants when exposed to UV exposure, whereas no change in
histamine levels was observed in explants when they were exposed to UV irradiation after
sunscreen application (Figure 8). As a matter of fact UV irradiated samples in exvivo model
was shown to mimic the UV irradiated skin biopsies as discussed in earlier sections.

Figure 8. Histidine metabolism from UV irradiated and sunscreen protected explants: This schematic
presents the steps in the Histidine metabolism pathway to production histamine and urocanic acid.
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1440 Manpreeet Randhawa and Michael D. Southall

SUNSCREEN APPLICATION PREVENTS INFLAMMATION PRODUCED AS

A RESULT OF ADENOSINE CATABOLISM INDUCED BY UV RADIATION

Metabolites belonging to Adenosine pathway followed the same metabolomics profile as

discussed before for the invivo study with increased levels of Inosine, Hypoxanthine, and uric
acid. Besides the adenosine pathway, guanosine catabolism was observed in exvivo model
with significant increased levels of Guanosine and Guanine, which also fuels the formation of
uric acid (Figure 9). The lack of imparity between the invivo and exvivo model from
Guanosine pathway could be explained in terms of invivo skin is replenished with internal
source of metabolites, whereas in exvivo model the skin is revived by using media with added
nutrients. However the explants exposed to UV after sunscreen application did not show any
increase in any of the metabolites related to adenine and guanosine pathway.

Figure 9. Adenine metabolism from UV irradiated (upper panel) and sunscreen protected (lower panel)
explants: This schematic presents the steps in the adenosine and guanosine pathway leading to
production of uric acid. ATP – Adenosine triphosphate, GMP – guanosine monophosphate, IMP –
inosine monophosphate. Red arrows – higher accumulation, green arrow – low accumulation.

CONCLUSION
It is well established that exposure of the skin to ultraviolet (UV) light is a major risk
factor for photoaging and well as developing skin carcinomas and sunfilters can provide
effective photoprotection protecting skin from UV damage. Sunfilter efficacy has most
commonly been demonstrated and assessed using a variety of clinical or biomarker endpoints.

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 Metabolomic Assessment of Sunscreen Efficacy 1441

Tool Method Endpoint Advantages Limitations

Metabolomics Invivo: Biopsy, Urocanic acid, Number of targets can Large amount of dataset.
tape stripping, Adenosine be studied at the same
plasma, Exvivo, metabolism, time and can be
invitro histamine, categorized for acute
Nicotinamide and chronic exposure.
metabolism, Does not only serve
Glutathione the purpose for target
metabolism identification, but also
for understanding
sunscreen efficacy.
Molecular targets Invitro, exvivo, p53, CPD, A valuable to tool 1) Number of targets validated to date is
invivo biopsies TTdimers understand the very limited.
physiology of DNA 2) The markers usually repair itself and
changes. the information is lost about the earlier
timepoints.
3) Is it a valuable tool to understand the
suberythemal doses?
Clincal Invivo Erythema, Provides information 1) Expensive and can be done only
pigmentation, under invivo invivo. Usually the markers are looked at
sunburn conditions, which is 24hrs and loses the earlier time windows
very relevant to the to look at other markers.
phenotype. 2) The sunscreens test are limited to
. Caucasian, hence there is a gap to
understand the effect and need for
sunscreen in other ethnicities.
3) Not sufficient Information in generated
for suberythemal doses.

It has been well documented that sunscreens can provide good protection against
sunburn, but other aspects of photoprotection are not simple to assess. In clinical settings the
protection effectiveness of sunfilters can be assessed by the prevention of erythema or
pigmentation produced as result of UVB and UVA respectively in 24hrs and expressed as sun
protection factor (SPF) or pFA respectively. At the molecular level effectiveness of
sunscreens has been assessed using single biomarkers such as DNA damage markers, which
are also measured at 24hrs. Collectively these biomarkers provide only a small part of the
information of sun damage and sunfilter efficacy and often lack a holistic view about the
photoprotective effects of sunfilters on sun exposed skin. Metabolomics can assess changes in
the abundance of large numbers of endogenous metabolites representing multiple biochemical
pathways simultaneously. Metabolomics studies showed that markers for inflammation and
oxidative stress are not limited to single biomarkers, as there are many inflammatory
pathways induced in UV exposed. Moreover the effect of sun exposure and sunfilter efficacy
should not be studied by only focusing on oxidative stress or inflammation since this
approach may miss significant UV effects on other metabolic and biochemical process in
skin. Metabolomics can assess changes in the abundance of large numbers of endogenous
metabolites representing multiple biochemical pathways simultaneously. Metabolomic
profiles of sun-exposed skin have demonstrated significant biochemical changes in
glycolysis, oxidative phosphorylation, and lipid metabolism pathways compared to sun
protected skin. Metabolomics has also identified biochemical changes in skin associated with
sun exposure that were previously unknown or unclear. Thus, global metabolomics profile
could serve a more useful tool to understand the physiology of skin in response to sun
exposure. Sunscreens can prevent or reduce the UV-induced changes in skin metabolomics
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1442 Manpreeet Randhawa and Michael D. Southall

and as such may provide new approaches to investigate the efficacy of sunscreen
formulations. Through providing a holistic approach to simultaneously measure biochemical
pathways in skin, metabolomics may provide a more comprehensive assessment of sunscreen
photoprotection than other single biomarkers. The tool can offer insight into the global
metabolic profile of skin exposed to different wavelengths of UV and also offer great
potential for research to assess the efficacy of sunscreens holistically through different
markers.

ACKNOWLEDGMENTS
The authors would like to thank Dr. Vineet Sangar, Institute for Systems Biology, Seattle,
Washington, for insightful discussions on data analysis and interpretation of metabolomics.
And to Dr. Curt Cole from Johnson and Johnson for extensive discussions on sunfilter
efficacy studies.

REFERENCES
[1] Tabachnick J (1957) Urocanic acid, the major acid soluble, ultraviolet-absorbing
compound in guinea pig epidermis. Arch Biochem Biophys 70:295.
[2] de Fabo EC, Noonan FP (1983) Mechanism of immune suppression by ultraviolet
irradiation in vivo. I. Evidence for the existence of a unique photoreceptor in skin and
its role in photoimmunology. J Exp Med 158:84–98.
[3] Sophie Seité and Anny M.A. Fourtanier (2008) The benefit of daily photoprotection.
Journal of the American Academy of Dermatology. Volume 58, Issue 5, Supplement 2,
Pages S160–S166.
[4] Yamaguchi Y, Coelho SG, Zmudzka BZ, Takahashi K, Beer JZ, Hearing VJ, Miller SA
Cyclobutane pyrimidine dimer formation and p53 production in human skin after
repeated UV irradiation. (2008) Exp Dermatol. 17(11):916-24.
[5] Zastrow L, Groth N, Klein F, Kockott D, Lademann J, et al. (2009) [UV, visible and
infrared light. Which wavelengths produce oxidative stress in human skin?]. Hautarzt
60: 310-317.
[6] Zastrow L, Groth N, Klein F, Kockott D, Lademann J, et al. (2009) The missing link--
light-induced (280-1,600 nm) free radical formation in human skin. Skin Pharmacol
Physiol 22: 31-44.
[7] de Gruijl FR, van Kranen HJ, Mullenders LH (2001) UV-induced DNA damage,
repair, mutations and oncogenic pathways in skin cancer. J Photochem Photobiol B 63:
19-27.
[8] Bennett MF, Robinson MK, Baron ED, Cooper KD (2008) Skin immune systems and
inflammation: protector of the skin or promoter of aging? J Investig Dermatol Symp
Proc 13: 15-19.
[9] Mouret S, Baudouin C, Charveron M et al. (2006) Cyclobutane pyrimidine dimers are
predominant DNA lesions in whole human skin exposed to UVA radiation. Proc Natl
Acad Sci USA 103:13765–13770.

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[10] Wassberg C, Ckvall H, Diffey B, Ponte F, Berne B (2003). Enhanced Epidermal

Ultraviolet Responses in Chronically Sun-exposed Skin are Dependent on Previous
Sun Exposure. Acta Derm Venereol 83: 254–261.
[11] Wang-Sattler R, Yu Z, Herder C, Messias AC, Floegel A, et al. (2012) Novel
biomarkers for pre-diabetes identified by metabolomics. Mol Syst Biol 8: 615.
[12] Zhu M, Zhang Y, Bowden GT (2006) Involvement of mitogen-activated protein
kinases and protein kinase C in regulation of antioxidant response element activity in
human keratinocytes. Cancer Lett 244: 220-228.
[13] Barnes VM, Teles R, Trivedi HM, Devizio W, Xu T, et al. (2009) Acceleration of
purine degradation by periodontal diseases. J Dent Res 88: 851-855.
[14] Landmesser U, Spiekermann S, Preuss C, Sorrentino S, Fischer D, et al. (2007)
Angiotensin II induces endothelial xanthine oxidase activation: role for endothelial
dysfunction in patients with coronary disease. Arterioscler Thromb Vasc Biol 27: 943-
948.
[15] Brosh S, Boer P, Sperling O (1984) Effects of fructose on purine nucleotide
metabolism in isolated rat hepatocytes. Adv Exp Med Biol 165 Pt A: 481-485.
[16] Ratnam S, Maclean KN, Jacobs RL, Brosnan ME, Kraus JP, et al. (2002) Hormonal
regulation of cystathionine beta-synthase expression in liver. J Biol Chem 277: 42912-
42918.
[17] Randhawa M, Southall M, Samaras ST (2013) Metabolomic analysis of sun exposed
skin. Mol Biosyst 9: 2045-2050.
[18] Harman D (1956) Aging: a theory based on free radical and radiation chemistry. J
Gerontol 11: 298-300.
[19] Harman D (1968) Free radical theory of aging: effect of free radical reaction inhibitors
on the mortality rate of male LAF mice. J Gerontol 23: 476-482.
[20] Harman D (1981) The aging process. Proc Natl Acad Sci U S A 78: 7124-7128.
[21] Lin SJ, Guarente L (2003) Nicotinamide adenine dinucleotide, a metabolic regulator of
transcription, longevity and disease. Curr Opin Cell Biol 15: 241-246.
[22] Khan JA, Forouhar F, Tao X, Tong L (2007) Nicotinamide adenine dinucleotide
metabolism as an attractive target for drug discovery. Expert Opin Ther Targets 11:
695-705.
[23] El-Abaseri TB, Fuhrman J, Trempus C, Shendrik I, Tennant RW, et al. (2005)
Chemoprevention of UV light-induced skin tumorigenesis by inhibition of the
epidermal growth factor receptor. Cancer Res 65: 3958-3965.
[24] Randhawa M, Sangar V, Tucker-Samaras S, Southall M. (2014). Metabolic Signature
of Sun Exposed Skin Suggests Catabolic Pathway Overweighs Anabolic Pathway.
PONE DOI 10.1371.0090367.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 65

THE HISTORY AND EVOLUTION OF SUNSCREEN

Mary Laschinger, BS, and Anna H. Chacon, MD

University of Maryland Medical Center, Dept. of Dermatology, Baltimore, Maryland

ABSTRACT.
SUNSCREENS: PROPERTIES, ROLE IN SKIN CANCER PREVENTION
AND HEALTH EFFECTS

The popularization of tanning by Coco Chanel in the 1950s heralded a new era,
where the once fashionable milky complexion was quickly replaced by a darker one,
achieved through baby oil and iodine. Although sunburns were undoubtedly just as
painful as they are today, sunscreen was certainly not seen as a necessity, often barred to
the bottom of the beach bag. As public knowledge of the scientifically proven links
between excessive ultraviolet exposure from the sun and the development of skin cancer
and photoaging expanded, so did the marketing and use of sunscreens. Today topical
sunscreen products remain one of the most widely used forms of photoprotection for the
majority of the public, and within an estimated $1 billion dollar industry, past issues of
variety and availability have been erased, as current and future focus puts safety and
efficacy at the forefront.1
Sunscreen may not have been a common household item decades ago, but the
product has a surprisingly long history. The first commercial sunscreen was introduced in
the United States in 1928, a greasy emulsion containing benzyl salicylate and benzyl
cinnamate. In 1946, Franz Greiter introduced the concept of SPF, and by the 1970s broad
introduction of SPF on sunscreen packaging revolutionized the market, as products
became comparable on a quantitative basis.2 In the past two decades alone, sunscreen
products have undergone fundamental improvements, the most significant of which is the
breadth of protection against UVA I rays. Sunscreens have come a long way since the
days of greasy emulsions, as today’s protection products are not only super effective at
protecting against UV damage, but invisible and aesthetically pleasing to the consumer.
Sun protection is no longer looked at as a product brought out for a beach-day, but
considered a daily-use item, used not only for sun protection but healthy prophylaxis
against aging and skin damage.
Today sunscreen can be found everywhere from daily moisturizers to lip balms, but
although sales have reached an all-time high, skin cancer rates continue to climb. Wild
claims of safety and efficacy together with a history of limited regulation have led to an
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1446 Mary Laschinger and Anna H. Chacon

atmosphere of consumer misconception. In 2012, more than 30 years after its first stab at
sunscreen regulation, the FDA has finally set down legally binding rules on the marketing
on sunscreen products. New mandates touch on areas of broad-spectrum testing, SPF
capping, “waterproof” labeling, and sun safety claims, in hopes that these new rules will
eliminate confusion and establish consumer power.3

REFERENCES
1
Wang S, Tanner P, Lin H. The evolution of sunscreen products in the United States-A 12-
year cross sectional study. Photochem Photobiol Sci. Jan 2013; 12 (1): 197-202.
2
Jacob. Skin care then and now. Skin Inc. May 2013.
3
Stanfield. New trends in sunscreen testing and labeling. Am Acad of Dermatol. Jan 2014.

INTRODUCTION
The popularization of tanning by Coco Chanel in the 1920s heralded a new era, where the
once fashionable milky complexion was quickly replaced by a darker one, achieved through
baby oil and iodine. Although sunburns were undoubtedly just as painful as they are today,
sunscreen was certainly not seen as a necessity, often barred to the bottom of the beach bag.
As public knowledge of scientifically proven links between excessive ultraviolet exposure
from the sun and the development of skin cancer and photo-aging expanded, so did the
marketing and use of sunscreens. Today topical sunscreen products remain one of the most
widely used forms of photo-protection for the majority of the public, and within an estimated
seven hundred million dollar industry, past issues of variety and availability have been erased,
as current and future focus puts safety and efficacy at the forefront.

THE MILLENNIA OF PALLOR

To understand the history of sunscreen, one must first gain a good understanding of the
shifting cultural beliefs and practices when it comes to tanning throughout the past twentieth
century. Numerous cultural and historical factors over the past one hundred years, including
social perceptions, medical knowledge, travel patterns, and fashion trends, have contributed
to the popularization of tanning, dating back to as late as the early 1920s.
In the millennia preceding the twentieth century, the “porcelain beauty” was the standard,
as pallor was deemed a sign of higher social status and a life of leisure spent indoors [1]. Pre-
1900s through 1910, stigma was associated with tanned skin, as it was common among more
working-class individuals who performed manual labor outdoors in farms and fields. This,
and a general negative attitude towards dark-skinned individuals during this time, increased
the appeal of a pale complexion. Fair skin was associated with both social and physical
wellness, and as a result people went to great measures to protect themselves from the sun.
The use of thick clothing which covered a majority of the skin’s surface and parasols were
considered a fashion must, while sun avoidance all together by staying indoors also played a

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 The History and Evolution of Sunscreen 1447

significant role. Even swim and active wear for the outdoors was conservative, covering most
of one’s skin surface. [2]
Cosmetic means of achieving pale skin has a long history, dating back to the ancient
Greek, Roman, and Elizabethan eras, when poisonous lead based whiteners where used to
bleach the skin [3]. In addition to clothing barriers in the pre-1900s, topical sun protectants
were also popular, which usually included heavy powders made up of magnesium, zinc oxide,
or bismuth, designed to prevent any signs of sunburn or freckling. Up until the late 1920s,
evidence of the fashionable nature of fair skin can be found in magazines and newspapers,
which still widely advertised things such as bleaching creams for women. An advertisement
for Elizabeth Arden’s, Bleachine Cream, can be found featured in the July 1, 1920 issue of
Vogue,marketed as “a mild but effective preparation for removing tan. Nourishing as well as
whitening. Excellent for the hands” [4].

BEGINNING OF THE “HEALTHY TAN”

At the turn of the 20th century, new medical paradigms placed sunlight at the forefront as
a both a new treatment modality for many diseases, as well as a preventative health measure,
initiating a shift in social perceptions of sun exposure. The roots of this change can perhaps
be traced to Downes and Blunt, who discovered that direct sunlight could inhibit the growth
of microorganisms, and Palm who was the first to identify the role of sunlight in vitamin D
synthesis, which later became known as the chief cause of rickets [5]. In 1903, Niels Finsen
won the Nobel Prize for the use of ultraviolet radiation (UVR) in the treatment of lupus
vulgaris, igniting the use of UVR phototherapy by physicians for numerous other diseases [6].
UV therapy became widespread, with physicians in both the United States and Europe
advocating for its use in treating an array of cardiovascular, endocrinology, atopic,
gastrointestinal, and rheumatologic diseases.
One of the most noteworthy books published in 1928 was Ultra-Violet Rays in the
Treatment and Cure of Disease [7], on which the editorial staff at the New England Journal
of Medicine deemed a book which they hoped did “not reach laity” [8]. However, that same
year Vogue’s “Burning Question of the Summer” column featured this quote:

“As a substitute for sun there are the ultra-violet ray lamps that have been cleverly
decided to muffle heat rays and give us only the rays that tan. In addition to pleasantly
modish toasting properties, actinic rays are said to stir up sluggish skin, and do all sorts of
desirable things to one’s internal function- reducing colds, stimulating glands, even
improving the condition of such totally unexpected things such as teeth.” [9]
And so was born the phase, “healthy tan.”

THE INDUSTRIAL REVOLUTION AND THE GROWTH

OF OUTDOOR LEISURE

During the same time period that social perceptions towards sunlight and its potential
health benefits were changing, so was the socioeconomics of the United States. With the
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1448 Mary Laschinger and Anna H. Chacon

introduction of the industrial revolution, lower and middle class workers were pushed indoors
into factories and mines [1]. Significant changes in social class activities allowed for tanned
skin to emerge as a symbol of travel, leisure, wealth, and health at this time. Leisure time
shifted from the parlor room to the beaches and parks, and the allure of outdoor leisure was
promoted by the new construction of thousands of athletic fields, tennis courts, baseball
diamonds, and swimming pools [10]. By the 1930s, outdoor athletics to become an integral
part of the American social experience, leading to a quick shift from underexposure to the sun
to overexposure within the matter of decades.

MAKING TAN CHIC

Through promotion in fashion magazines, which continued through the middle of the
twentieth century, a good tan became everybody’s must have accessory. In a 1929 issue of
Vogue, CoCo Chanel endorsed tanning after coming home from a vacation in the French
Rivera with a sunburn, affirming the “the 1929 girl must be tanned” and “the golden tan is the
index of chic” [11]. Some mark this as the beginning of the popularization of tanning from a
fashion and cultural perspective. As a result, clothing became more revealing, allowing
maximum sun exposure to both obtained and show off one’s tanned complexion. Some of the
most significant changes to American clothing around this time was the introduction of the
first T-shirt allowing maximum arm exposure in 1942, and the bikini in the 1946 [12].
Throughout the late twentieth century the fad for tanning showed no signs of burning out.
Celebrities and magazines maintained the allure throughout these decades continuing into
present day. In 1978 tanning took on a whole new perspective when the first indoor tanning
facility opened in Arkansas, allowing people to achieve a tan year round [13]. Indoor tanning
today remains a major public health issue, having been linked to growing numbers of basal
cell carcinoma, squamous cell carcinoma, and melanomas in young populations.

GROWING KNOWLEDGE OF THE DANGERS OF UV RADIATION

Despite the endorsement of UV radiation for the promotion of good health in the early
twentieth century, initial warnings from within the dermatologic community did surface at
this time. Chronic sun exposure as a causative agent of skin cancer was suggested as early as
1864, however, such early claims were largely ignored [3]. As a result of insufficient
scientific evidence for such claims, physicians and scientists clung to clinical observation.
Norman Paul was the first to make such clinical associations in his book published in 1918,
The influence of Sunlight in the Production of Cancer in the Skin [14]. Following in his
footsteps, American dermatologist James McCoy published his observations in 1920 that a
disproportionately higher percentage of skin cancers occurred on sun-exposed areas including
the face, neck, and hands [15]. This and other clinical observations linking UV exposure to
skin cancer formation received little attention in the press, medical community at large, and
public in the early 1900s. This may have occurred given the lack of knowledge when it came
to the mechanisms underlying UV-induced carcinogenesis at the time, as well as widely held

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beliefs that skin cancer only occurred in susceptible individuals, such as those with xeroderma
pigmentosum [3].
By the 1940s, however, things changed as the pathological mechanisms of UV-induced
carcinogenesis was revealed, first by George Findlay, and later Angel Roffo, who were able
to produce skin tumors in animal models by subjecting them to UV radiation [16]. Segments
on television, articles in lay press, and reports in non-dermatologic journals became more
prevalent over the latter half of the twentieth century with focus primarily on the association
between sunburns and skin cancer [16].

THE HISTORY OF SUNSCREEN

With the increased public knowledge of the dangers of UV radiation in the 1940s came a
simultaneous increased interest among consumer product industries to create novel sunscreen
preparations. Although not previously a common household product by this time, topical sun
protectants had been around for thousands of years. Early civilizations have been known to
use plant products to protect their skin, including the use of olive oil by ancient Greeks, and
rice, jasmine, and lupine extracts by ancient Egyptians [17]. Zinc oxide paste and other
physical sun protectants, like titanium dioxide, had also been around for thousands of years
[18].
Early chemical sunscreens, however, were not introduced until 1928, when a blend of
UVB absorbers, benzyl salicylate and benzyl cinnamate was created [17]. These newer
chemical sunscreen were uniquely different in that they worked by absorbing UVB rays
rather than reflecting the rays, which was the primary function of physical blockers, such a
zinc oxide, with the advantage being improved aesthetics. As these new synthetic sunscreens
developed, so did the market, leading to the first major, commercially sold sunscreen in 1936,
created by L’Oreal founder Eugene Shueller [17]. The product was introduced under the
name of Ambre Solaire and was an oil preparation made with benzyl salicylate. Other key
players in the market at this same time include, chemist H.A. Milton Blake who introduced
Hamilton Sunscreen in 1932, and Benjamin Green, who developed a product called Red Vet
Pet for U.S. soldiers in the Pacific tropics at the height of WWII [17, 19]. The most effective
modern sunscreen came out in 1946, developed by Swiss chemist Franz Greiter, called
Gletscher Crème, which later became the basis of his company Piz Buin, which is still today a
marketer of sunscreen products [20].
By 1943, the FDA approved the UVB absorber p-aminobenzoic acid (PABA) for use in
sunscreens [21]. PABA was believed to remain in the stratum corneum while providing UVB
protection for several hours. Although PABA is still approved by the FDA for use in
sunscreens, its popularity sharply declined in the mid-1980s due to its potential for causing
allergies, and its yellowing staining of clothing [20].

EVOLUTION OF SUNSCREEN: AESTHETICS

The first commercially available sunscreens boasted the advantages of being less
conspicuous and more aesthetically appealing to the consumer as compared to the otherwise
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1450 Mary Laschinger and Anna H. Chacon

incredibly effective zinc oxide pastes. The first formulations, however, where still leaps and
bounds from the formulations available today, as sunscreens have come a long way from
these early greasy emulsions in terms of look, feel, and effectiveness.
One of the first marketed sunscreens, Red Vet Pet, short for red veterinary petroleum,
was exactly that, a red, sticky substance similar to petroleum jelly [22]. However, as demand
for sunscreens grew in the latter half of the twentieth century, manufacturers worked hard to
overcome these aesthetic challenges, making more appealing products by the 1950s. Green’s
patented Red Vet Pet was eventually sold to Coppertone who improved and commercialized
the substance under the Coppertone Girl and Bain de Soleil brand in 1953 [22]. This
improved formula introduced cocoa butter and coconut oil into the mix, creating much more
absorbable and better smelling products for consumers. The cosmetic quality of sunscreens
has continued to improve over the years, with today’s products being invisible and
comfortable on the skin. Chemists have continued to work on sunscreens’ improved look and
feel, with some of the most important and recent achievements being the “micronized”
versions of zinc oxide and titanium dioxide, which has eliminated the thick, streaky
appearance formally associated with the highly effective sun protectants, allowing consumers
to now apply them all over the body [20].

EVOLUTION OF SUNSCREEN: EFFICACY

Many of the early sunscreen preparations introduced into the market were not only
aesthetically displeasing, but vastly ineffective. The first sun protection measurement system
was developed in 1934 by Friedrich Ellinger and Rudolf Schulze, which was later adopted by
Greiter in 1974 and re-introduced as the “sun protection factor (SPF)” [20]. The SPF quickly
revolutionized the sunscreen market, as there was now a reliable means of measuring and
comparing a sunscreen’s effectiveness in a quantitative manner. The SPF provided the
consumer with the ability to tell two things: how long they could stay out in the sun and how
well the formula filtered the sun’s rays.
Around this same time, the FDA reclassified sunscreens from cosmetics to over-the-
counter drugs, requiring stricter and more regulated package guidelines [22]. Although their
steps for regulation were limited and many were not enforced, the introduction of mandatory
SPF package labeling was carried out, beginning in 1978 [22]. Coppertone lead the way as
one of the first manufacturers to label their products with SPF numbering, theirs being the
first sunscreen with an SPF of 15, called Coppertone’s Super Shade Lotion [20]. This
represented quantum leaps in efficacy from earlier sunscreens, such as Franz Greiter’s
Gletscher Cream which only sported an SPF of two, and shifted the tone in the market
towards one of safety and effectiveness. Within the last couple decades alone large increases
in SPF value levels have been seen within the sunscreen market, with a twelve year cross
sectional study of U.S. sunscreen products, conducted by Wang et al. noting a general
increase in SPF values of products from 1997 to 2009 alone. It was also found that the
percentage of low SPF products (SPF 4-14) decreased from 27% in 1997 to 6% in 2009 [23].
These trends shed a positive light on the future focus of sunscreen tending towards continuing
increased efficacy and safety.

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In addition to the adoption of SPF as a quantitative means of standardizing sunscreen

efficacy across the market, the Skin Cancer Foundation also introduced its Seal of
Recommendation program in 1981 [20]. This program independently assessed the safety and
efficacy of sunscreen products using guidelines that were not yet monitored and regulated by
the FDA. This provided consumers with an additional way to compare products on the market
at the time.
Another development in sunscreen came in the late 1970s when Johnson and Johnson
introduce the first “water-resistant” sunscreen [14]. The product was marketed as Sundown
Sunscreen. The goal of water-resistant formulations was to allow consumers a longer-lasting,
more reliable protection without the need for reapplication when enjoying outdoor sports,
including swimming. Today many sunscreens claim water resistance or even water proof,
however, regulations on such marketing and packaging claims have been historically lacking.

EVOLUTION OF SUNSCREEN: BREADTH OF COVERAGE

Of the tremendous fundamental improvements sunscreens have undergone in the past
several decades, the most significant may be considered the increased breadth of protection to
include UVA I rays. It was once thought that only UVB radiation, which is the main cause of
sunburn, could cause significant damage to the skin and raise the risk of skin cancer.
Research over the past twenty years, however, has unrevealed a different reality, showing that
cumulative UVA exposure is the key cause of skin aging and wrinkling, while also playing a
role in the development of cutaneous cancers [20]. These new findings have brought upon an
urgent need for the improvement of UVA protection of sunscreens. In fact, cross sectional
studies from over the past two decades show an increase in the number of products containing
a UVA filter, from 5% in 1997 to 70% in 2009 [23].
UVA is considered the long-wavelength ultraviolet radiation, while UVB the short. UVA
can be divided into two groups: UVA I rays with wavelengths ranging from 340-400nm and
UVA II rays ranging from 320-340 nm. Different sunscreens, depending upon their
formulation, either absorb or reflect parts of this ultra-violet radiation spectrum, the broad-
spectrum of which cover more of the UVA wavelengths, thus providing more adequate
coverage.
In the 1980s the only available UVA blockers were zinc oxide and titanium dioxide. Due
to the aesthetically unappealing nature of these products however, manufacturers strived to
create new formulations with different ingredients. In 1980 Coppertone sold the first
UVB/UVA sunscreen, called For Faces Only [20]. This novel formulation contained
oxybenzone, which offered significant although very limited UVA coverage. Following the
introduction of oxybenzone, three other ingredients were created to protect against the UVA
spectrum: sulisobenzone, meradimate, and dioxybenzone [20]. Sunscreens containing any of
these four ingredients began to be marketed as “broad-spectrum.”
Although tremendous headway in terms of increased breadth of coverage against UVR
was being made with the introduction of oxybenzone, the market continued to lack a full
UVA spectrum protectant. In 1988 the FDA approved the chemical avobenzone, which
provided the largest UVA spectrum coverage to date [22]. Eventually concerns about
avobenzone’s photostability sparked interest into finding an even better UVA spectrum
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1452 Mary Laschinger and Anna H. Chacon

protectant, and within the past several years alone chemists have found ways to add
stabilizing ingredients to such sunscreens, such as octocrylene. Today these photostabilized
avobenzone formulations are marketed under names such as Helioplex, Active Photobarrier
Complex, AvoTriplex, SunSure, and Dermaplex [20].

CHANGING ATTITUDES OF SUN PROTECTION

Although the 1940s and 1950s heralded in an era of booming demand and sales for
sunscreen products, these first lotions were definitely not viewed a necessity at the time, but
rather a lotion to have handy in the beach bag to use only after the skin was burned. The first
sunscreen, marketed as “suntan lotions,” were promoted to be used as a means to allow
longer exposure times in the sun without burning, while also allowing for the “healthy tan” so
many still desired during this time [20]. Over time, due to growing evidence of harmful
effects of the sun’s UVR, not only in terms of increased cancer risks, but also skin aging and
wrinkling, suntan lotions were eventually replaced by what we now call sunscreens, metal
reflectors gave way to shade umbrellas, and visors gave way to UV-blocking sunglasses. In
fact, society’s perception of sun protection today is no longer thought of as something only
considered on a beach-day, but rather part of one’s daily routine, providing the benefits of
sun protection against harmful UVR, but also the benefits of prophylaxis against skin aging
and damage.

THE MULTI-MILLION DOLLAR INDUSTRY

Going off societal perceptions of sunscreens as part of a healthy and beautiful lifestyle,
manufacturers have been able to create an estimated 700 million dollar industry [24]. The
sunscreen industry has established itself as an independent industry with little direct
competition, although some indirect competition does come from self-tanning lotions, sun
protectant clothing, and sun protectant shelters. Topical sunscreens currently account for
about 60% of the sun care market, with a predicted annual growth rate of 2.3% [24]. The
average consumer is a fair-skinned woman, ages 35-44, which branding executives have
capitalized on in terms of catering to this demographics health and beauty concerns [24]. The
top sunscreen manufacturers today remain Coppertone and Banana Boat. Branding changes
over the years have led to such manufacturers positioning themselves in the market as either a
health or beauty product, while in some cases both. Issues of variety and availability are no
longer present in the market as they once were. Countless different formulations can be found
on the shelves of the local grocery store, drug store, or even super store. The list of products
containing sunscreen has grown just as much of the number of manufacturers. Today
sunscreen is advertised in moisturizers, lip balms, foundations, and make-up powders to name
a few.

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HISTORY OF REGULATION
Although sales of sunscreen have reached an all-time high, skin cancer rates continue to
climb. Wild claims of safety and efficacy together with a history of limited regulation have
led to an atmosphere of consumer misconception. At the root of these issues is perhaps the
lack of unified FDA regulation over the past thirty years. The FDA’s first stab at regulation
came in 1978 when sunscreens were switched from cosmetic products to over-the-counter
drugs. At this time the FDA tried to establish safety and efficacy guidelines, however, many
fell to the way side, with only the mandate for SPF labeling on packaging withstanding the
process. At this time the FDA did order an advanced notice of proposed rulemaking for the
future stating in the document, “In the long run, suntanning is not good for the skin” [20].
Over the next 30 years FDA regulation was sparse, with the only notable legislation
being the approval of avobenzone as a UVA filter in 1988, and the approval of the marketing
of this ingredient by manufacturers in 1997. In 2006, the FDA missed a deadline set by
Congress to approve proposed guidelines for sunscreens, and it was not until 2013 that the
FDA approved legally binding rules on the marketing of sunscreen products [22]. New
mandates of the regulations touch on areas of broad-spectrum testing, SPF capping,
“waterproof” labeling, and sun safety claims, in hopes that these new rules will help eliminate
confusion and establish consumer power.

2013 FDA SUNSCREEN REGULATION MANDATE [25]

Broad-spectrum testing: All sunscreens must now meet standardized broad-spectrum
testing requirements to be labeled as such. Such testing measures the UVA protection in
relation to the UVB protection, setting a minimum standard for such ratio. In addition, only
sunscreens labeled “broad-spectrum” and with an SPF of 15 or greater can claim to reduce the
risk of cancer and prevent premature aging of the skin.
SPF: At this point the FDA is considering the prohibition of sunscreens being labeled
higher than SPF 50, given consumer misconceptions as to increased sun protection and failure
to realize that protection levels are not proportional to the SPF number directly. Pushback
from manufacturers has forced the FDA to negotiate this point. The FDA argues that a SPF
greater than 50 has been shown to provide minimal additional protection as compared to
sunscreens with SPF 50.
Waterproof terminology: The term “waterproof” will be a thing of the past as the only
acceptable new terms that can be used will be “water-resistant” and “very water-resistant.”
The FDA claims there is no such things a waterproof sunscreen and require products to be
labeled as either resistant for 40 minutes or 80 minutes. Other terms including “sweatproof”
and “sunblock” will also be discarded.
Sun safety claims: Sunscreens can no longer claim immediate efficacy, nor can they
claim to provide more than 2 hours of protection unless otherwise approved by the FDA. This
is in hopes of reminding the consumer of proper application techniques including applying
sunscreens thirty minutes before sun exposure, and the need for frequent reapplication
throughout the day.
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1454 Mary Laschinger and Anna H. Chacon

FUTURE OF SUNSCREEN
Recent industry trends involve the addition of antioxidants to sunscreens. Numerous
manufacturers have begun to embrace this trend, with new marketing strategies aimed at the
inclusion of vitamin C, vitamin E, and other antioxidants in products [26]. The rationale
behind the additions is that such antioxidants serve as another line of protection against
radical oxygen species produced by the UV, helping to diminish the harmful effects of UVR
that gets past the incomplete protection of current filters.
Although the addition of antioxidants seems appealing, a recent study from Wang et al.
demonstrates that the protection against ROS in sunscreens containing antioxidants still
mainly is derived from the UVA filters, concluding that the action of antioxidants in
sunscreens containing such filters is essential non-existent [27]. This is proposed to be due to
either inadequate concentrations of antioxidants, unstable formulations, and use of the wrong
active forms [26]. Studies such as these shed light on the fact that substantial progress must
be made before any significant benefits will likely be seen from adding antioxidants to
sunscreens.

TIMELINE
The following is a timeline outlining the key social, scientific, and regulatory events in
the history and evolution of sunscreen:

1918: Influence of Sunlight in the Production of Cancer of the Skin is published.

1920: Elizabeth Arden’s Bleachine Cream is advertised in July issue of Vogue magazine.
1928: First commercial sunscreen containing benzyl salicylate and cinnamate is
manufactured.
1928: Ultra-Violet Radiation in the Treatment and Cure of Disease is published.
1929: Coco Chanel shows off tanned skin after trip to French Rivera.
1934: Ellinger and Schulze create ultraviolet protection quotient system.
1936: L’Oreal releases Ambre Solaire, the first major commercialized sunscreen on the
market.
1940: Findlay and Roffo undercover pathologic mechanisms underlying UVR
carcinogenesis.
1943: PABA is approved by the FDA.
1944: Green introduces Red Vet Pet for use by U.S. soliders in the Pacific.
1946: Helm and Reard introduce the bikini, ushering in an era of more revealing clothing.
1946: Greiter created Gletscher Crème, the most effective commercial sunscreen to date.
1953: Coppertone debuts Little Miss Coppertone and revolutionizes sunscreen by
creating aesthetically pleasing formulations with cocoa butter and coconut oil.
1972: FDA reclassifies sunscreen from cosmetic products to over-the-counter drugs,
making them subject to regulation.
1974: Greiter adapts Schulze system and coins term SPF.
1977: Johnson and Johnson release the first “waterproof” sunscreen.
1978: First indoor tanning bed facility opens in Arkansas.

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1978: FDA mandates SPF labeling on sunscreen products.

1978: Coppertone releases the first sunscreen with SPF 15 labeling.
1980: Coppertone releases the first “broad-spectrum” sunscreen containing oxybenzone.
1988: FDA approves avobenzone for use as a UVA filter in sunscreen products.
2006: FDA misses deadline for approval of regulator guidelines.
2013: FDA sets legally binding regulations pertaining the marketing of sunscreen
products.

REFERENCES
[1] Chang C MD, Muraku E, Penn L Md, et al. More Skin, More Sun, More Tan, More
Melanoma. Am Journ of Public Health. Nov 2014; 104 (11):92-99.
[2] Martin J, Ghaferi J MD, Cummins D MD, et al. Changes in skinning tanning attitude:
Fashion Articles and Advertisements in the Early 20th Century. Am Journ of Public
Health. Dec 2009; 99 (12): 2140-2146.
[3] Albert MR, Ostheimer KG. The evolution of current medical and popular attitudes
towards ultraviolet light exposure: part 1. J Am Acad Dermatol. 2002; 47(6):930-937.
[4] Elizabeth Arden advertisement. Vogue. July 1, 1920:112.
[5] Hockberger, PE. A history of ultraviolet photobiology for humans, animals, and
microrganisms. Photochem Photobiol. 2002; 76(6):561-579.
[6] Howell J. Niels Ryberg Finsen. In: Fox DM, Rezak I, eds. Nobel Laureates in Medicine
of Physiology: A Biographic Dictionary. New York, NY: Garland Publishing;
1990:181-183.
[7] Hall P. Ultra-violet rays in the treatment and cure of disease. 3rd ed. St, Louis, MO:
C.V. Mosby Co; 1928
[8] Ultra-violet rays in the treatment and cure of disease [book review]. Bos Med Surg J.
1928; 199:591.
[9] The burning question of the summer: Vogue. July 1, 1928: 100.
[10] Durant J, Bettman O. Pictorial History of American Sports: From Colonial Times to
the Present. New York, NY: AS Barnes &Co Inc; 1952.
[11] Segrave K. Suntanning in 20th Century America: Jefferson, NC: McFarland &Co Inc;
2005.
[12] Whiteman, Honor. “Are changes in fashion, tanning perceptions to blame for rising
melanoma rate?.” Medical News Today. MediLexicon, Intl., 6 Oct. 2014. Web.
Available at: http://www.medicalnewstoday.com/articles/283472.php. Accessed 12
Oct. 2014.
[13] Kaminester LH. Suntanning centers. JAMA. 1980;244(11):1258-1259.
[14] Thomas L. Lim HW. Sunscreens. Journal of Drugs in Dermatol.2003; 2,174.
[15] McCoy J. The solar keratosis and cutaneous cancer. Arch Derm
Syphilol.1920;1(2):175-181.
[16] Albert M, Ostheimer KG. The evolution of current medical and popular attitudes
toward ultraviolet light exposure: part 3. J Am Acad Dermatol.2003;49(6):1096-1106.
[17] Shaath, Nadim A., editor (2005). Sunscreens: Regulations and Commercial
Development, Third Edition. Taylor & Francis Group.
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1456 Mary Laschinger and Anna H. Chacon

[18] Craddock, P.T. (1998). 2000 Years of Zinc and Brass. British Museum. p. 27.
[19] MacEachern, W.N.; Jillson, O.F. (January 1964). "A Practical Sunscreen — "Red Vet
Pet".” Arch Dermatol 89 (1): 147–150.
[20] Lim, Henry W. "Quantum Leaps: New, Improved Sunscreens Have Arrived.” The Skin
Cancer Foundation. Accessed Oct, 10, 2014.
[21] Shaath, N. Evolution of modern sunscreen chemicals, 3.
[22] Jacob U. “Skin Care-Then and Now: Sunscreen.” Skin Inc. Apr 2013.
[23] Wang SQ, Tanner PR, Lim HW, Nash JF. The evolution of sunscreen products in the
United State- a 12-year cross sectional study. Photochem Photobiol. Jan 2013;
12(1):197-202.
[24] Mosambuka B. “Sunscreen Market Presentation.” Available at: https://prezi.com/
ebnpw3tew10k/copy-of-sunscreen-market-presentation/. Accessed 13 Oct 2014.
[25] New trends in sunscreen testing and labeling. Am Acad of Dermatol. Jan 2014.
[26] Wang SQ, Hu JY. “Challenges in Making an Effective Sunscreen.” The Skin Cancer
Foundation. Accessed 28 Sept 2014.
[27] Wang SQ, Osterwalder U, Jung K. Ex vivo evaluation of radical sun protection factor
in popular sunscreens with antioxidants. Epub 2011 May 31. J Am Acad Dermatol.
2011; 65(3):525-30.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 66

PSYCHOLOGY BEHIND THE USE OF SUNSCREENS,

TANNING AND SKIN CANCER PREVENTION

Shailee Patel, Tulsie Patel and Katlein França*

ABSTRACT
The benefits of the use of sunscreen have been widely presented in scientific
literature. UV exposure has been related to the development of skin cancers. The use of
sunscreen decreases this risk. Tanning is a social desire in many cultures. Media, fashion
and social behaviors motivate people to get tan. Despite warnings about skin cancer,
many individuals seek for tanning salons and natural sun exposure without adequate sun
protection. Frequent tanners are psychologically and physically dependent on tanning.
When an individual develops a skin cancer after using inadequate sun protection
measures, feelings such as guilt, depression and anxiety may arise. This chapter will
discuss the social psychological aspects related to sunscreen use, tanning and skin cancer
prevention.

SUNSCREEN AND SKIN CANCER PREVENTION

Ultraviolet light is a major factor in the development of skin cancer and sunscreens can
reduce the extent of photocarcinogenesis when sun avoidance is not possible [1]. The
damaging effects from ultraviolet B (UVB) radiation (290-320 nm) are directed to DNA,
which acts as an epidermal chromophore by undergoing conformational changes when
exposed to UVB. This exposure results in the formation of pyrimidine dimers in the double
helix and subsequent mutations [2, 3]. In contrast, ultraviolet A (UVA) radiation (320-400
nm) is less directly absorbed by DNA and causes most of its genotoxic effects by induction of
oxidative stress that indirectly damages DNA and other critical intracellular structures [4, 5].
Without accurate and rapid repair of this damage, mutations in the genome and machinery of

*
Corresponding Author: Katlein França, MD, MSc; Volunteer Faculty- Assistant Professor; Department of
Dermatology & Cutaneous Surgery; University of Miami Miller School of Medicine; Miami, FL, USA
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1458 Shailee Patel, Tulsie Patel and Katlein França

the epidermal cells amass and lead to the development of both nonmelanoma and melanoma
cancers.
Consistent and appropriate use of sunscreens has demonstrated to reduce the risk of
actinic keratosis, nonmelanoma, and melanoma skin cancers [6-11]. Most of the data are
based on randomized trials conducted with a pool of 1,621 patients that were followed for
various amounts of time in Australia [8-11]. The researchers found a statistically significant
reduction in incidence of squamous cell carcinoma at the sites of the sunscreen application
compared to the no daily sunscreen group (rate ratio [RR] = 0.61; 95% confidence interval
[CI], 0.46 to 0.81) [9]. Further analysis eight years after the completion of the initial trial
showed a prolonged protective effect with a 35% lower incidence of squamous cell carcinoma
in the daily sunscreen groups (RR = 0.65; 95% CI, 0.43 to 0.98) [10]. In terms of the
incidence of basal cell carcinoma, the daily sunscreen group compared to the no daily
sunscreen group was not statistically significant in either the four or eight year follow up
study [9, 12]. Interestingly, a 50% reduction in the risk of melanoma was seen in the daily
sunscreen group compared to the control (hazard ratio [HR], 0.50; 95% CI, 0.24 to 1.02; P =
.051) [11].
These studies findings are the first to provide evidence for a reduction in the incidence of
squamous cell carcinoma and melanoma after regular application of broad- spectrum
sunscreen in adults. Of note, Australia has very high UV exposure thus making it difficult to
generalize the data to other regions. In addition, the sunscreen used in the study was SPF 16
with inadequate UVA protection. Another factor to consider is that the control group was
allowed to use sunscreen, so the defensive benefit from sunscreen may be much greater than
the results suggested [9-12]. Nevertheless, there are not enough studies overall to know if
sunscreen recommendations are clearly helpful but considering it has few harms, it would be
reasonable to provide sunscreen counseling to patients at risk of developing skin cancer who
will be exposed to excessive sunlight [13].
Initially sunscreens provided insufficient UVB protection and virtually no UVA
protection, but modern formulations include novel UV filters, UV boosters, and
photostabilizers that deliver substantial coverage against both UVB and UVA radiation [1]. In
regards to UV protection, the main area of progress has been the expansion of coverage of the
long UVA-I (340-400 nm) range with filters such as avobenzone and zinc oxide. Apart from
new filters, the vehicle compounds have increased UV protective properties by using
additives and film formers to ensure even coverage with every application. In addition to the
advancements in superior UV protection, sunscreen technology has also enhanced the sensory
profile with addition of silicones, silicas, and other slipping agents to minimize the unpleasant
sticking feeling. To prevent the opaque white appearance due to the macro-sized titanium
oxide and zinc oxide molecules, sunscreens now use nano-sized molecules for a better
aesthetic finish [1].
The goal of photoprotection through sunscreen use is contingent on the combination of
technological improvements in sunscreen preparation, the regulation of sunscreens, and
proper behavior patterns by the public in regards to the use of sunscreens.

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 Psychology Behind the Use of Sunscreens, Tanning and Skin Cancer Prevention 1459

BEHAVIORAL AND SOCIAL PSYCHOLOGY OF SUNSCREEN USE

Despite improvements in sunscreen technology and regulatory changes, knowledge and
behavior of the individual consumer play the most crucial role in obtaining sufficient
photoprotection. Most of the research on the topic of skin cancer involves studies that focus
on epidemiology, diagnosis and treatment while there is paucity in regards to behavioral
research of the psychological aspects of sun exposure and protection. A better understanding
of the behavioral psychology of sunscreen use would entail investigating the decision-making
processes and communication strategies within and between individuals in a social system.
For example, this could include assessing if fear-based versus appearance-based marketing
improve sunscreen compliance. In addition, determining whether parental versus peer
motivation would encourage better sun safety practices in adolescents. While social
psychology of sunscreen use would evaluate the processes of a social system through impact
of social organization on structural adjustment of the individual and of groups. This may
include public health organizations such as the World Health Organization warning of the
dangers of sun exposure and endorsing photoprotection on a global platform. Another
potential social psychological intervention could address the impact of motivation by
consistent health care provider recommendations of sunscreen use.
One of first studies in 1987 investigating the psychosocial factors in sunscreen use
amongst beachgoers in California found that being a woman, having better skin cancer
knowledge, and knowing someone who had cancer were associated with more sunscreen use.
The higher usage among women may be due to anti-aging goals, association of sunscreen use
with cosmetics, or that sunscreen advertisements are targeted to women. Interestingly,
individuals with anxious mood were also associated with more sunscreen use. This might be
explained by cancer fearing individuals may choose to use sunscreen or the use of sunscreen
reminds individuals of skin cancer which results in anxiety [14]. Similarly, a study of
teenagers in Virginia from 1992 found that female participants used more sunscreen than their
male counterparts. In addition, having a friend who routinely used sunscreen, having parents
who insisted on sunscreen use, and knowing that the maximum time for safe sun exposure is
short were associated with more frequent sunscreen use. However, family history of skin
cancer in this study was unrelated to sunscreen use and overall compliance was poor [15].
Unfortunately over a decade later even after improved efforts from the medical
community and government agencies, less than 40% of teenagers used sunscreen and 83%
reported having experience at least one sunburn in their lifetime [16]. Studies have also
shown that approximately half of children do not protect themselves properly from solar UV
exposure [17-20]. Continuing to empower the public with proper knowledge regarding the
safety and benefits of sunscreen require a lot of effort similar to educating the public about
cigarette smoke causing cancer. Thus targeting elementary school aged children and
implementing basic knowledge during physical education or science class could provide
better long term behavioral outcomes and save millions of lives. In term of adolescents, who
are often motivated by beauty standards set by society such as having a tan can be encouraged
use sunless tanning booths which are a safer alternative to artificial UV induced tanning and
are increasing in popularity [21, 22].
In addition to educating specific populations, debunking myths to help reinforce healthy
attitudes and motivate photoprotective behaviors are necessary. Often times sensationalized
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1460 Shailee Patel, Tulsie Patel and Katlein França

media coverage have been generated from studies that do not represent actual human usage
leading to controversy about the hormonal effects of oxybenzone or phototoxicity of zinc
oxide. For example, it would take 277 years of daily topical application of a sunscreen
containing oxybenzone to reproduce the findings amongst the rats used in the study [23].
Another source of confusion comes from the tanning industry and its supporters trying to
manipulate the public on the benefits of tanning including improved Vitamin D levels [24]. In
reality, the amount of UV exposure needed to produce sufficient Vitamin D levels is small for
example within 5 minutes at noon in June in Boston, Massachusetts and does not justify the
use of artificial UV exposure or overlook the risk of cutaneous malignancy associated with its
use [25]. Moreover, vitamin D supplements can easily avoid any risk of insufficiency even in
those who practice proper photoprotection [26].
Public health interventions including the Surgeon General stance that the rise in skin
cancers is associated with excessive exposure to indoor and outdoor UV is a step in the right
direction but more is needed to ensure sustained momentum for prevention of skin cancer.
Consumer behavior will guide the amount of photoprotection from sunscreens and other sun
protective methods. Effective behavioral changes may take time to permeate through society
by empowerment with knowledge and motivation.

TANNING AND SKIN CANCER

The ability for one to tan is directly related to the amount of melanin (pigment) that is
present in the epidermis. The pigment itself is dependent on the number and size of the
melanosomes that are released by the melanocytes in the basal layer of the skin as opposed to
the actual number of melanocytes themselves [27]. This varied ability to tan is outlined most
clearly by Fitzpatrick. Fitzpatrick created the gold standard classification system for detecting
the degree of tanning or burning of one’s skin following UV exposure [28].
Tanning is due to exposure to UVA and UVB light [29]. This exposure leads to a change
in the skin such that there is a combination of immediate pigment darkening which is
associated with activation of pre-existing melanin followed by tanning, which involves
melanogenesis. Tanning becomes concerning for the development of cancer when the
formation of DNA damage is considered [27]. For example, exposure to UVB is incredibly
dangerous due to the formation of cyclobutane pyrimidine dimers that affect transcription and
replication [29]. Exposure to UVA, on the other hand, may lead to the formation of oxygen
free radicals that lead to breaks in DNA [30]. One innate mechanism for fighting this DNA
damage is dependent on the effects of tumor suppressor, p53. P53 is essential in ensuring that
replication of damaged DNA is halted until repair is complete [27]. Studies have shown that
sunlight can often lead to p53 mutations that lead to unregulated cell growth due to the down-
regulation of the inhibitory function of p53 [31]. It was also recently discovered that these
DNA sequences that are halted by p53 play a direct role in the tanning process as they lead to
tanning regardless of exposure to UV light. Further, a new study indicated that p53 is further
involved in tanning as it leads to the ultimate activation of melanocortin-1 receptor, which
activates melanocytes to produce melanin. In addition, mice models have shown that mice
without p53 are increasingly susceptible to the effects of the damage of UV light on DNA
[27].

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 Psychology Behind the Use of Sunscreens, Tanning and Skin Cancer Prevention 1461

There have been numerous studies indicating the relationship between UV light exposure
and skin cancer. Solar radiation has already been deemed as “carcinogenic to humans” by the
International Agency for Research on Cancer [31]. Over 50% of malignancies that are
diagnosed each year in the United States are from cutaneous malignancies. Although sun
safety measures are easily available, there is also an underlying misunderstanding among
individuals that stems from the belief that tanning is protective against skin cancer. In
actuality, not only is the original tanning behavior dangerous, but ongoing tanning is even
more precarious as relatively unhealthy skin is further exposed to dangerous rays. Further,
patients may be less likely to alter their behaviors given that the effects of UV exposure often
take many years to develop. Childhood exposure often takes 30+ years to appear as a
cutaneous malignancy, therefore making direct relationships between cutaneous injury (as a
child) and the presence of skin cancer (as an adult) difficult [27]. No matter the source of
exposure, whether natural sunlight or artificial sources such as tanning beds, exposure to UV
light is dangerous [31].

PSYCHOLOGICAL ASPECTS OF TANNING

Tanning, like any other behavior, can be attributed to numerous influences [32]. In
popular culture, the term tanorexia has been coined to describe a person’s constant desire to
be excessively tan [33].
Although the term tanorexia seems to indicate a correlation between tanning and the
psychology behind an eating disorder, numerous studies have shown a greater association
between substance abuse disorders and addiction with a disproportionate use of tanning [32,
34]. A recent study actually found that individuals who had abused substances were more
likely to participate in indoor tanning [35]. Further, those with substance abuse disorders and
those who participate in excessive tanning also share other characteristics such as they both
tend to be younger individuals who are negligent of the long-term consequences of their
actions and who are more focused on enhancing their appearance [35-40]. Other common
characteristics include the desire for pleasure in both addiction and tanning [41-43]. For
example, many describe tanning as relaxing and mood boosting [41-43]. These latter effects
have been associated with the concept that tanning dependence stems from the release of
endogenous opioids upon UVR exposure [41, 42, 44, 45]. This idea has been further proven
through the presence of withdrawal symptoms in frequent indoor tanners who were exposed
to opioid blockers [42]. Overall, it has been found that indoor tanners often behave similar to
those facing substance abuse and addiction as they are also more likely to be responding to
social pressure, concerned about their appearance (ex. weight), and are also more likely to be
smokers, drinkers, and drug users [46-48].

SKIN CANCER PSYCHOLOGY

Many studies are being done to determine the effects of a diagnosis with melanoma or
other non-melanoma conditions on the behavior of an individual and those around them.
Although diagnoses with conditions as serious as melanoma are incredibly life altering, it has
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1462 Shailee Patel, Tulsie Patel and Katlein França

been found that it is actually quite difficult to change a person’s behavior [49]. For example, a
recent study showed that patients with dysplastic nevus syndrome, although aware of their
increased risk for skin cancer, were not always willing to change their behavior [50]. Further,
less than 33% of first-degree relatives of melanoma patients were willing to practice better
sun safety precautions [51]. Alarmingly, even some patients with melanoma themselves, were
not always willing to increase their sun safety measures despite an increased chance for
recurrence [49].
However, behavior can be changed. For example, it has been shown that patients who
have been diagnosed with melanoma are often more likely to change their behavior than
others as they are more likely to appreciate the severity of their condition and the possible
complications and results [52-54]. Patients with melanoma are more likely to experience
anxiety and even fear following their diagnosis, thus leading to changes in their behavior [55].
Thus, preventative measures must be undertaken by matching the response that patients have
following diagnosis to the currently limited changes that occur following simple sun safety
education [49, 52-54].

CONCLUSION
There are numerous factors that influence a person’s exposure to UVR. From a quest for
a certain appearance to the availability of indoor and outdoor tanning, exposure to dangerous
UVR is widespread in our culture. Social and psychological influences play a role in these
behaviors. For example, many in our society are now classified as tanorexic due to their
excessive need to tan. Although there are benefits to limited and protected sun exposure, it is
greatly important that proper education be provided to all who are exposed to UVR. It is
imperative that early intervention be implemented in the hopes of establishing safe behaviors
that will help to prevent serious complications including melanoma.

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[20] Weinstock MA, Rossi JS, Redding CA, Maddock JE, Cottrill SD. Sun protection
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[23] Wang SQ, Burnett ME, Lim HW. Safety of oxybenzone: putting numbers into
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[24] Tangpricha V, Turner A, Spina C, Decastro S, Chen TC, Holick MF. Tanning is
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[28] Roberts WE. Skin type classification systems old and new. Dermatologic clinics. 2009
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[31] Woo DK, Eide MJ. Tanning beds, skin cancer, and vitamin D: An examination of the
scientific evidence and public health implications. Dermatologic therapy. 2010 Jan-
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[32] Heckman CJ, Egleston BL, Wilson DB, Ingersoll KS. A preliminary investigation of
the predictors of tanning dependence. American journal of health behavior. 2008 Sep-
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[33] News B. Young 'tanorexics' risking cancer. 2004.
[34] Warthan MM, Uchida T, Wagner RF, Jr. UV light tanning as a type of substance-
related disorder. Archives of dermatology. 2005 Aug;141(8):963-6.
[35] O'Riordan DL, Field AE, Geller AC, Brooks DR, Aweh G, Colditz GA, et al. Frequent
tanning bed use, weight concerns, and other health risk behaviors in adolescent females
(United States). Cancer causes & control: CCC. 2006 Jun;17(5):679-86.
[36] Boldeman C, Jansson B, Dal H, Ullen H. Sunbed use among Swedish adolescents in the
1990s: a decline with an unchanged relationship to health risk behaviors. Scandinavian
journal of public health. 2003;31(3):233-7.
[37] Hillhouse J, Stapleton J, Turrisi R. Association of frequent indoor UV tanning with
seasonal affective disorder. Archives of dermatology. 2005 Nov;141(11):1465.

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[38] Demko CA, Borawski EA, Debanne SM, Cooper KD, Stange KC. Use of indoor
tanning facilities by white adolescents in the United States. Archives of pediatrics &
adolescent medicine. 2003 Sep;157(9):854-60.
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related to behavioral characteristics. Preventive medicine. 1997 Jan-Feb;26(1):114-9.
[40] Zeller S, Lazovich D, Forster J, Widome R. Do adolescent indoor tanners exhibit
dependency? Journal of the American Academy of Dermatology. 2006 Apr;54(4):589-
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[41] Feldman SR, Liguori A, Kucenic M, Rapp SR, Fleischer AB, Jr., Lang W, et al.
Ultraviolet exposure is a reinforcing stimulus in frequent indoor tanners. Journal of the
American Academy of Dermatology. 2004 Jul;51(1):45-51.
[42] Kaur M, Liguori A, Lang W, Rapp SR, Fleischer AB, Jr., Feldman SR. Induction of
withdrawal-like symptoms in a small randomized, controlled trial of opioid blockade in
frequent tanners. Journal of the American Academy of Dermatology. 2006
Apr;54(4):709-11.
[43] Poorsattar SP, Hornung RL. UV light abuse and high-risk tanning behavior among
undergraduate college students. Journal of the American Academy of Dermatology.
2007 Mar;56(3):375-9.
[44] Levins PC, Carr DB, Fisher JE, Momtaz K, Parrish JA. Plasma beta-endorphin and
beta-lipoprotein response to ultraviolet radiation. Lancet. 1983 Jul 16;2(8342):166.
[45] Belon PE. UVA exposure and pituitary secretion. Variations of human lipotropin
concentrations (beta LPH) after UVA exposure. Photochemistry and photobiology.
1985 Sep;42(3):327-9.
[46] Diffey BL, Farr PM. Tanning with UVB or UVA: an appraisal of risks. Journal of
photochemistry and photobiology B, Biology. 1991 Jan;8(2):219-23.
[47] Lazovich D, Forster J. Indoor tanning by adolescents: prevalence, practices and
policies. European journal of cancer. 2005 Jan;41(1):20-7.
[48] Guay AT, Perez JB, Heatley GJ. Cessation of smoking rapidly decreases erectile
dysfunction. Endocrine practice: official journal of the American College of
Endocrinology and the American Association of Clinical Endocrinologists. 1998 Jan-
Feb;4(1):23-6.
[49] Hornquist JO, Wikby A, Andersson PO, Dufva AM. Insulin-pen treatment, quality of
life and metabolic control: retrospective intra-group evaluations. Diabetes research and
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[51] Azzarello LM, Dessureault S, Jacobsen PB. Sun-protective behavior among individuals
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[52] Boggild AK, From L. Barriers to sun safety in a Canadian outpatient population.
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[53] Meyer N, Pruvost-Balland C, Bourdon-Lanoy E, Maubec E, Avri MF. Awareness,
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France. Journal of the European Academy of Dermatology and Venereology: JEADV.

2007 Apr;21(4):520-5.
[54] Smith BJ, Ferguson C, McKenzie J, Bauman A, Vita P. Impacts from repeated mass
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 67

THE ROLE OF ANTIOXIDANTS IN SUNSCREENS:

THE CASE OF MELATONIN

Ana Flo Sierra1, Víctor Flo Sierra1,

Ana Cristina Calpena Campmany1 and Beatriz Clares Naveros2,
1
Pharmacy and Pharmaceutical Technology Department.
Faculty of Pharmacy, University of Barcelona, Spain
2
Pharmacy and Pharmaceutical Technology Department.
Faculty of Pharmacy, University of Granada, Spain

ABSTRACT
The main objective of this chapter is to perform a detailed review on antioxidants in
sunscreens, and more specifically on Melatonin. Melatonin is a neurohormone with
natural antioxidant properties. In order to understand its importance, an initial review of
the structure of the skin and the ultraviolet radiation effects is carried out. Throughout the
chapter the nature of products, mechanisms of action and advantages and disadvantages
of the conventional and novel groups of sunscreens that protect from the ultraviolet
radiation are discussed, (i) organic sunscreens, (ii) inorganic sunscreens (iii) DNA repair
agents (iv) cyclooxigenase-2 inhibitors, (v) iron chelators (vi) osmolytes and (vii) natural
antioxidant substances. Several studies have reported the benefits of Melatonin used
either for ultraviolet radiation protection or in combination with traditional radiotherapy
for the treatment of human cancers. For this reason, in this chapter a thorough review of
the characteristics and properties of this substance is performed in order to underscore the
importance of this promising active for use in sunscreens.

1. HUMAN SKIN
The skin is composed of 3 layers of differentiated tissues. The most external layer is the
epidermis, followed by the dermis and the hypodermis, which is the most internal layer of the


Corresponding Author Address: Campus of Cartuja St, Granada 18071, Spain. Email: [email protected]
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1468 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

skin. Overall, it is a thick, resistant and flexible membrane that in an adult has an approximate
area of 1.5 – 2 m2 [1].

1.1. Epidermis

It is the most external layer of the skin. Its thickness varies from 0.04 mm to 1.6 mm
depending on the zone of the body. The primary cell in the epidermis is the keratinocyte, but
there are additional cells such as melanocytes, Langerhans cells and Merkel cells. In the
epidermis there are three different appendages: the sweat glands, the pilosebaceous follicles
and the nails [1].
Epidermis is composed by 4 distinct layers; Stratum basal is a single layer of columnar
basal cells, which are attached to the basal membrane via hemidesmosomes. It is composed of
epidermal stem cells that by mithosis produce new keratinocytes. These cells evolve as they
move toward the surface layer. When keratinocytes mature, they become in the Stratum
spinosum, the Stratum granulosum and finally the Stratum corneum [2]. The normal turnover
rate of a healthy epidermis is about 28 days [1].
The latter layer, Stratum corneum, formed mainly by corneocytes (terminally
differentiated keratinocytes), is the responsible for the skin barrier that acts protecting the
skin, preventing water loss, maintaining the hydration of the skin and so many other
protective functions [2].

1.2. Dermis

It is located between the subcutaneous and dermal-epidermal junction. It measures from

0.3 mm to 3.0 mm depending on the zone of the body. The dermis is composed in a 90% of
collagen, elastic fibers, blood vessels, lymph vessels, some muscle fibers and pilosebaceous
and sweat glands and a 10% of cellular components, including fibrocyte, monocyte,
histiocyte, Langerhans cells, lymphocytes, and eosinophils, along with the vascular and
lymphaticassociated cells [2].

1.3. Hypodermis

It is the innermost layer of the skin. It invaginates into the dermis, with which is attached
by collagen and elastin fibers. It is composed by fibroblasts, adipose cells and macrophages.
Its principal function is fat storage [2].
The ability of the skin to bear and react to solar radiation depends primarily on the skin
phototype. In Table 1 are classified the 6 different phototypes depending on the ease of
sunburning and skin pigmentation.
The skin, due to its location in the body, is the most important protective barrier against
external aggressions. One of the main aggressions comes from solar radiation, causing
damages such as sunburn, photoaging, skin cancer among others that we be reviewed in the
next section.

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1469

Table 1. Classification of skin phototypes (adaptation from the classification created

by Fitzpatrick and Bolognia [3]

Skin type classification/phototype Sunburn Tans after sun exposure

I Always Seldom
Melano-compromised
II Usually Sometimes
III Sometimes Usually
Melano-competent
IV Seldom Always
V Naturally brown skin
Melano-protected Never
VI Naturally black skin

2. SOLAR RADIATION
The electromagnetic radiation spectrum is classified based on its wavelength in radio
waves, microwaves, infrared (IR), visible light (Vis), ultraviolet light (UV), X-rays and γ
radiation. However, the solar spectrum that reaches the surface of the earth only comprises
wavelengths of electromagnetic energy between 300 and 3000 nm, which include UVB,
UVA, Vis and NIR. The photobiological effects are higher at shorter wavelengths because
they have higher energy.
The main human photobiology studies have been focused on UV but Vis light and NIR
also reach the earth and have photobiological effects [4]. In Figure 1, are summarized the
photobiological effects distributed depending on the wavelength. The main characteristics of
each one of the solar radiations that arrive to the ground are described below.

Figure 1. Solar spectrum that arrives to the Earth and photobiological effects that produces.
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1470 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

2.1. UVB

Wavelength: 280 – 315 nm. Wavelengths between 280 and 295 nm are filtered by the
atmosphere.
Penetration: It penetrates up to the basal layer of the epidermis (Figure 2)
Effects: It generates reactive oxygen species (ROS) and reactive nitrogen species (RNS).
These species create inflammation, sunburn and stimulate skin ageing. The high energy
photons of UVB can be absorbed by DNA bases of the cell, causing mutagenic lesions. These
lesions are normally repaired by nucleotide excision repair. However, a high accumulation of
mutations in skin cells due to UVB exposition could potentially develop a UV-associated skin
cancer [5]. It is responsible of the delayed tanning [6].
Endogen chromophores: Melanin [6].

Figure 2. Penetration of solar radiation in the skin.

2.2. UVA

Wavelength: 315 – 400 nm. The biological effects of the shorter wavelengths of UVA are
very similar to those of UVB, for that reason, some authors differentiates between UVA2
(315-340 nm) and UVA1 (340-400 nm) [4].
Penetration: It penetrates up to the dermis (Figure 2).
Effects: It generates ROS and RNS that alters DNA, proteins and lipids. It also generates
immunosuppression. The oxidative damage that causes in the skin can increase indirectly the

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1471

risk of skin cancer due to the formation of oxidized DNA bases. It also contributes to skin
photoaging [5, 7, 8]. It is responsible of the immediate pigmentation [6].
Endogen chromophores: Melanin, Riboflavin-containing FAD and FMN, trans-urocanic
acid [9].

2.3. Vis Light

Wavelength: 400 – 700 nm.

Penetration: It is approximately the 50% of the solar spectrum [10]. It penetrates deeper
than UV radiation, and a 20% reaches the hypodermis (Figure 2).
Effects: It generated ROS and it also induces inflammatory cytokines such as IL-1, IL-6,
IL-8 and GM-CSF, increases the expression of matrix degrading enzymes (MMP-1 and
MMP-9) and may form oxidized DNA bases [11]. It is also responsible of the pigment
darkening in subjects with phototypes IV and V [12].
Endogen chromophores: Hemoglobin, Melanin, Bilirubin Riboflavin, Porphyrins [13].

2.4. NIR

Wavelength: 700 – 3000 nm.

Penetration: It is approximately the 30% of the solar spectrum, 65% penetrates up to the
dermis and a 10% up to the hypodermis [10] (Figure 2).
Effects: It generates ROS and induces unbalanced gene expression of MMP. It also
decreases collagen gene expression, favors angiogenesis, affects mitochondrial integrity, is
involved in photoaging and it can potentially promote carcinogenesis [14‒16].
Endogen chromophores: Cytochrome C oxydase of the mitochondria [15, 17].

3. SUN EXPOSURE EFFECTS

It is globally accepted that solar radiation has beneficial health effects. For example, it
stimulates the production of cholecalciferol (vitamin D3) [18], necessary for the proper
functioning of bone metabolism and immune system. It is also recommended the controlled
exposure to the sun to treat or reduce the severity of skin diseases such as psoriasis and
vitiligo [19]. But it is also accepted that uncontrolled exposure to sun has harmful
photobiological effects. These effects can be divided into acute or short-term effects and long-
term or chronic effects.

3.1. Short Time Effects

Erythema
This is the best recognized effect of UV radiation in the skin. Commonly known as
sunburn, is primarily caused by exposure to UVB and in lesser extent to UVA2. The signs of
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1472 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

erythema are the classic of an inflammation, such as redness, tenderness, edema and warmth
[20].

Free radical formation

Under sun exposure, the endogenous chromophores absorb the photons produced by
radiation. After photon absorption these excited molecules react with oxygen, resulting in the
generation of ROS. When ROS are generated in an amount that overcome the endogenous
antioxidant defense, it is produced an oxidative stress. ROS include hydroxyl (OH.), singlet
oxygen (1O2) and superoxide anion (O.− 2 ). The 2 last species are also produced by neutrophils
that are increased in photodamage skin. The enzyme superoxide dismutase converts O.− 2 into
hydrogen peroxide (H2 O2). H2 O2 can cross cell membranes and in conjunction with
transitional Fe2+ produces the hydroxyl radical (OH.) Fe3+. Both 1O2 and OH . can interact with
cell membrane lipids producing lipid peroxidation that leads to inflammation [21]. ROS
inactivate TIMPs, which are tissue inhibitors of matrix-metalloproteases and induce the
synthesis and activation of the enzyme MMP responsible of its degradation. Both OH. and
1
O2 are able to directly damage DNA. ROS also damage proteins [22].

3.2. Long Time Effects

Photoaging
ROS promote cytokine cascade producing inflammatory reactions that are the responsible
of photoaging. They cause the activation of matrix metalloproteinase (MMP) and the release
of proinflammatory cytokines and growth factors that modify both collagen and elastin in the
extracellular matrix, ending in the degradation of skin structural integrity and causing
dysfunction of the melanocytes, which leads to skin hyperpigmentation [23].

Immunosuppression
It is known that UV radiation has both effects anti- and proinflammatory on the immune
system of the skin. The irradiation of the skin suppress the immunity mediated by cells
because it alters the Langerhans cell migration, producing suppressor T lymphocytes and
altering the skin cytokine profile [24].

Photocarcinogenesis
Solar radiation induces mutations in DNA and its immunosuppressive properties also
affect host immune system which causes problems recognizing damaged malignant cells. The
association between exposure to UV radiation and the development of skin cancer, including
malignant melanoma, basal cell carcinoma and squamous cell carcinoma is well documented
[25]. Superficial spreading and nodular melanoma are associated with intermittent and intense
solar radiation exposure, while lentigo maligna melanoma and squamous cell carcinoma are
linked to cumulative sun exposure [26, 27].
Ciclooxigenase-2 (COX-2) is an enzyme that is induced in the epidermis by various
cytokines such as IF-γ, TNF-α, IL-1 and growth factors, in various cells and tissues. Some of
these inflammatory mediators are produced in response to UV irradiation. COX-2 is the first
enzyme in the enzymatic cascade that converts arachidonic acid into prostaglandins (PG) and

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1473

thromboxane. A study performed by Tripp et al. [28] demonstrated that keratinocyte

proliferation and apoptosis are regulated by COX-2 after acute UV exposure in vivo, probably
by the production of PG. It suggested that COX-2 induced PG had a significant role in the
pathogenesis of UV-induced epidermal neoplasia.

Photodermatoses
The exposure to UV radiation and Vis light UVR can reduce the quality of life for people
with immunologically mediated photodermatoses, such as solar urticaria, photoallergic drug
reactions and chronic actinic dermatitis among others [29].

4. SUN PROTECTION
Protection against this harmful radiation can be carried out by physical barriers, such as
hats, sunglasses and long sleeved clothing or using photoprotective substances. In the next
section is reviewed the current situation of sunscreens, what types there are, what are their
advantages and disadvantages and how the use of antioxidants improve the current situation
of the photoprotection. The use of sunscreens is the most popular measure of protection to
prevent the undesirable effects of solar radiation. Many studies have shown that regular use of
sunscreens applied correctly reduces the number of cases of actinic keratosis, squamous cell
carcinoma and attenuates the development of new nevi in children. In addition, regular use of
sunscreen prevents premature skin aging [30].
The development of the first sunscreens dates back to the early twentieth century, when
Norman Paul first linked sun exposure with skin cancer. Shortly thereafter, it was proposed
that the responsible for sunburn and skin cancer was only UVB radiation. Following this
discovery, pharmacists began to develop formulations containing substances that are able to
block the UVB radiation. The first sunscreen was commercialized in 1928. Later in the 60s,
the Austrian scientist Franz Greiter introduced the concept of Sun Protection Factor (SPF) to
determine the effectiveness of a sunscreen to suppress UV-induced sunburn. Furthermore,
people began to understand that sun exposure not only caused sunburn and cancer, it also
caused structural damage to the skin which favored the skin ageing. In the 70s, it was
demonstrated that UVA rays also cause premature skin ageing. Therefore, cosmetic
companies started offering sunscreens that protected against UVA and UVB.
Since then, the use of sunscreens has spread worldwide and people are becoming more
aware of the importance of protecting the skin to prevent undesirable effects of solar
radiation.
Nowadays, there are many different sunscreens, with single active substances or
combined with complex combinations.
It is important to take into account that in order to have a complete action preventing all
undesirable effects associated with sun exposure in short and long term, it is necessary to
have a sunscreen that complies with the requirements of:

(i) Providing uniform protection against all the radiation that arrives to the ground.
The major part of the sunscreens that are actually in the market were studied for
its utility absorbing or scattering UVA and/or UVB radiations but they have not
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1474 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

been studied for their usefulness avoiding photobiological effects of IR and Vis
light [10, 16, 31, 32].
(ii) Free radicals scavenging. The photoprotective agents included in the sunscreens
should exert as scavengers of the free radicals formed after exposure to solar
radiation.
(iii) Containing active ingredients that stimulate DNA repair systems. The ideal
sunscreen should include a component capable of directly or indirectly repair
DNA damage as a result of solar exposure.
(iv) Photostability. The substances that act absorbing UV photons, reduce the solar
radiation acting as a chromophore, which absorbs the energy in form of photons
when arriving to the skin during sun exposure. This absorption results in the
excitation of the molecule that has absorbed the energy. The excited filter
dissipates the absorbed energy generally very fast, in the form of heat or light,
returning to its native state. This process, could theoretically continue
repetitively, and this will lead in a photoestable filter [33].
(v) Safety. Sunscreens may be formulated to be retained in the skin, therefore and
will not arrive to plasma. Moreover, they cannot produce photoallergenity.
(vi) In addition, to facilitate the user’s compliance, it is important that it has pleasing
sensory and tactile profiles [31].

The active substances that compose sunscreens are classified depending on how they
exert its effect in: chemical or organic filters, physical or inorganic filters, antioxidants, DNA
repair agents, cyclooxygenase-2 inhibitors, iron chelators and osmolytes.

4.1. Chemical Filters

Usually, this type of filters acts absorbing UV radiation through chemical reactions.
Nevertheless, new developed filters such as Tinosorb M and Tinosorb S can also act
reflecting UV radiation. As an advantage, organic filters are more popular than inorganic
filters because they do not leave white appearance onto the skin. But they have several
disadvantages:

(i) Photoinstability: Some organic filters, when absorbing UV photons can dissipate
the energy by other pathways that lead to the destruction of them or to a partial
or complete reduction of their capacity to absorb energy, through processes such
as fragmentation, isomerization, reactivity with other molecules or free radicals
production [34]. Examples of photounstable organic filters are Octyl
methoxycinnamate, 2-phenylbenzimidazole-5-sulfonic acid, Benzophenone-3,
and avobenzone [35].
(ii) Photodegradation: The photoinstability of some organic filters can lead to
decomposition of the molecule under UV light, potentially leading to the
generation of ROS and toxic derivative compounds [36]. Photodegradation,
which is one of the consequences of photoinstability, can cause among other
phototoxicity, photoirritation and photoallergic reactions that may occur by
contact with the degradation of products formed. The use of sunscreens with

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1475

photodegradable substances may increase the risk of sunburn, but it can also
increase the risk of cancer. For that reason, the photostability of the filters
included in a sunscreen should be always determined in vitro, alone and in
combination, by using absorbance measures [37, 38].
(iii) Absorption through the skin: there are studies that have demonstrated that some
of the most used organic filters are able to penetrate the skin and have been
found in plasma, urine and even in breast women milk [39, 40].
(iv) Estrogenic effects: it has been demonstrated that some of the filters, such as,
Benzophenone-3, homomethyl salicylate, Octyl methoxycinnamate, 3-(4-
methylbenzylidene) camphor have estrogenic effects [35, 41].
(v) Spectrum of action: most of the organic filters absorb only UVB photons and
only a few are able to absorb both UVB and UVA. For that reason, complex
combinations are formulated. Nevertheless, studies have demonstrated that per
se, organic filters do not have protective effect in the NIR range [33].

Usually, a combination of different organic UV filters is used in order to reach sufficient

SPF. After several studies that demonstrated the incompatibility of some filters when are
combined in a sunscreen due to reactions between the molecules that made them
photounstable [33], FDA regulate the combinations of organic filters that could be used for
the preparation of sunscreen and the maximum concentration that can be used for each of
them [42].

4.2. Physical Filters

Inorganic filters are inert materials formed by particles that act as a barrier to UV
radiation because they reflect and scatter the light and also can absorb photons. The photon
absorption results in electron mobility and transitions between electronic states forming
excited species. The two best known inorganic particles used in sunscreens are micronized
zinc oxide (ZnO) and titanium dioxide (TiO2). The problems arisen with ZnO and TiO2 due to
photocalatytic activity and the poor acceptance of these filters, due to the high refractive
index, have led to the development of new inorganic molecules with better stability and low
refractive index, one of them is CePO4. This substance has shown chemical and physical
stability and low interaction with organic filters [43]. Inorganic filters have some advantages:

(i) They do not degrade when exposed to UV radiation

(ii) If they are not used in a nanoparticulate size they are not able to penetrate the
skin
(iii) Broad spectrum. They exert their action in UV, Vis and IR ranges

But these filters can also have some disadvantages:

(i) High refractive index: The inorganic materials with high refractive index tend to
leave the skin with a peculiar white appearance that leads to a low cosmetic
acceptability. It has been tried to reduce the size of the inorganic filter into
nanoscale in order to avoid the undesirable white marks that they leave in the
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1476 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

skin. But these TiO2 and ZnO have been deeply studied because these nanoscale
particles can generate highly oxidizing ROS when exposed to ultraviolet
radiation [44]. Nanoparticles with a surface coating by silica and alumina and/or
doping with vanadium or manganese reduce the generation of ROS.
(ii) Photocatalityc activity: Dunford et al. [45] demonstrated that both ZnO and TiO2
can catalyze oxidative damage to DNA in vitro and in cultured human
fibroblasts.
(iii) ROS generation: in a study performed by Lewicka et al. [44] eight commercial
formulation containing nanoparticles of ZnO and TiO2 were analyzed to
determine the ability to generate ROS, and it was concluded that formulations
containing nanoparticles of ZnO when exposed to UV radiation generate as
much ROS as sunlight.
(iv) Interaction with organic unstable organic filters: The high photocatalytic activity
that facilitates the generation of ROS, can oxidize and degrade other organic
filters included in the sunscreen that would result in instability and unsafe
reactions.

4.3. Antioxidants

In the lasts sections, it has been explained how, most organic filters do not adequately
protect not only against free radicals but also generating free radicals per se due to lack of
photostability. Also, it has been explained that conventional inorganic filters are not popular
because they leave white appearance on the skin and that these filters, when incorporated as
nanoparticles have some toxicological problems, one of them is the production of free
radicals after sun exposure.
Consequently, in the previous years it has increased the necessity of finding new
substances whose main mechanism of action was free radicals scavenging and the oxidative
stress elimination. Molecules that can perform these actions are the antioxidants.
Despite the large amount of antioxidants known, are not yet widely used in commercial
sunscreens. However, there are many articles that demonstrate the goodness of these
substances and describe how they act at the molecular level to scavenge free radicals and
prevent oxidative stress.
Table 2 shows an extensively list of antioxidants which have shown good antioxidant
action against oxidative stress, as well as free radical scavenger in the presence of UV
radiation. Studies also have shown that most of them also work against Vis light and IR
radiation as the main damages of these radiations are oxidative stress and free radical
formation, as shown in Figure 1.
One of the antioxidants that have been more studied is melatonin. Several studies give as
powerful properties as free radical scavenger, oxidative stress reducer and DNA damage
reduction, among others. For this reason, many authors have focused on the study of
melatonin as possible photoprotective, and due to the large number of studies showing his
goodness for this use, in Section 5 will be reviewed this substance in detail.

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1477

Table 2. Antioxidants which good antioxidant action against oxidative stress, and free
radical scavenger in the presence of UV radiation

Aloe poly and oligosaccharide prevent immune suppression induced

Aloe poly/ by exposure to UV by reducing the production of IL-10 in mice. Aloe
[46,47]
oligosaccharides protects against delayed-type hypersensitivity (DTH) in a stable
manner over time, however it is a process that is not yet well studied.
The use combined of apigenin and luteolin inhibits ROS production in
HaCaT cells irradiated with UVA. Keratinocytes pretreated with these
Apigenin flavonoids also inhibit MMP-1 production induced by UVA and
[48]
and luteolin suppresses the expression of c-jun and c-fos, as well as the MAPK
phosphorylation. Flavonoids also reduce the influx of calcium and the
Ca2+/CaMKs phosphorylation.
Astaxanthin is a lipid-soluble carotenoid mainly obtained from
Haematococcus pluvialis. In humans it is administered orally and
topically. It provides a significant inhibition of melanogenesis in age
Astaxanthin spots by suppressing melanocyte oxidative polymerization and [49]
inflammation of the epidermis. A treatment with astaxanthin also acts
protecting keratinocyte differentiation and cornification induced by
oxidative stress.
β-carotene inhibits UVA-induced genetic modulation in the lineages
of human keratinocyte HaCaT. In unirradiated cells, gene regulation
suggests that β-carotene significantly reduce stress signs and
β-carotene [50,51]
degradation of the extracellular matrix, also promotes the
differentiation of keratinocytes. These effects occur through
sequestration 1O2.
It is a synthetic antioxidant used as a preservative in products which
Butylated
contain lipids. It has been shown to inhibit erythema, ornithine [52]
hydroxitoluene
decarboxylase activity, carcinogenesis and photoaging.
Cadmium It is an antioxidant. In mice deficient in MT-1 and MT-2 genes occurs
chloride-induced more sunburn and apoptosis UVB-induced. [53]
MT
This substance applied topically protects from UV-induced erythema
Caffeic acid in vivo and in vitro. Its protective effect comes from its ability to [54]
decrease ROS.
Topical application of caffeine reduces the formation of malignant
and non-malignant tumors, as well as partial reduction of damage by
Caffeine irradiation. Some studies suggest that caffeine facilitates apoptosis in [55]
tumor tissue by inhibiting gene expression of ATR (ataxia
telangiectasia and Rad3 related).
Active form of vitamin D. Topical application of calcitrol in mice
Calcitriol inhibits the sunburn cells formation due to the induction of [56]
melatotionein expression.
Topical application of C. vulgaris extract (4 mg of polyphenols /cm2)
in mice during the 30 minutes before exposure to UVB radiation for
Calluna vulgaris 10 days provides protection to the skin by reducing the levels of TNF-
[57]
L. extract α and IL-6 and the formation of sunburn cells induced by UVB.
Therefore, C. vulgaris extract protects skin from DNA damage caused
by the sun.
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1478 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

Table 2. (Continued)

This genus of Mediterranean shrubs contains flavonoids that are

considered oxidative chain disruptors because they react as
Cistus extracts intermediators and form stable products. The leaf extracts of Cistus [58]
have shown in rat liver microsomes the ability of free radical
scavenging and inhibition of lipid peroxidation.
Volunteers who took this flavonoid for 12 weeks showed a reduction
in UV-induced erythema, improved skin appearance and hydration,
Cocoa powder [59]
increased the thickness of the skin and a reduction in transepidermal
water loss.
It contains arabinose, manose and galactose. It scavenges superoxide
Coptis
and hydroxyl radical in a dose-dependent way, and DPPH radicals. It
chinensis [60]
increases significantly IL-10 and TNF-α. The glycans that compose C.
glycans
chinensis absorb in IR range.
Cynaropicrin prevents photoaging by suppression of light induction of
Cynaropicrin [61]
NF-kB transactivation.
Leaf extract or fruit has a high antioxidant activity against DPPH
radicals and hydrogen peroxide radical. This activity is produced
mainly by its composition in flavonoids, phenols and anthocyanins.
Dacryodes sp. [62]
Ellagic acid present in these species can inhibit the growth of
chemically induced tumors. It has not yet been tested for use in effects
induced by UVR.
It is the topical form of zinc and provides antioxidant photoprotection.
It can replace the redox active, such as iron and copper; on the other
Divalent zinc hand can also induce the synthesis of MT's, sulfhydryl-rich proteins
[63]
ion that protect against free radical molecules. It has also been shown to
efficiently protect mouse skin against the appearance of sunburn cells
induced by UVB and UVA.
It is a derivative of daizein isoflavonoid, metabolized by the intestinal
flora in mammals. Topical application in mice protects against
Equol (4’,7-
inflammation, immunosuppression, and the formation of cyclobutane [64]
isoflavandiol)
pyrimidine dimers. However, its photoprotective effect is lower than
genistein or its precursor, daidzein.
It is used as photoprotective agent in many solar creams and lotions. It
Ferulic acid is a powerful antioxidant that inhibits lipid peroxidation and oxidative [54]
deterioration of cosmetic products and food.
Rich in omega-3 has been shown to have photoprotective properties.
Decreases the formation of sunburn cells and UVB-induced
inflammation after 3 months of ingestion. Reduces response to UVA
Fish oil [65]
in patients with polymorphous light eruptions. However, the effects
are achieved only with large doses of fish oil; therefore, their use is
not widely extended.
The antioxidant activity of flucoxanthin inhibits blood vessel
formation induced by UVB exposure in hairless mice model. The
Fucoxanthin expression of vascular endothelial growth factor decreases with [66]
wrinkle reduction, reducing the hypertrophy of the epidermis caused
by exposure to UV.
Raman spectroscopy has shown that as a defense mechanism against
harmful radiation and environmental factors, topical application of
General
carotenoids increases the potential defense of human epidermis. [67]
carotenoids
However, carotenoids are renowned nutricosmetics, improving the
resistance and hydration of the skin.

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It is a powerful antioxidant found in soybeans. After sun exposure, it

inhibits tyrosine kinase, reduces inflammation and the subsequent
Genistein [68]
immunosuppression. Applied topically, it protects against radiation
damage between 1 and 4 hours after application.
Green tea extract contains variety of active molecules including
catechins, polysaccharides, caffeine, apigenin and luteolin. Dietary
intake of green tea extracts or topical application provides anti-
inflammatory effects, antioxidant, and UVB-induced DNA damage
Green tea
reparation mechanisms. Protection against photoaging related to [69,70]
extract
matrix metalloproteinase (MMP-2, MMP-9) and photoinmunology.
Inhibit foto-increased lipid peroxidation. At 2.5mg/cm2 concentration
on human skin before UV irradiation reduced UV-induced oxidative
damage.
The extract of grape Jacquez wine efficiently prevents oxidative
injury suffered by the skin induced by exposure to UVB radiation.
Jacquez grapes This photoprotective effect is attributed to its high content of
[71]
wine extract polyphenols. Its application, tested in in vitro reconstituted skin helps
to maintain the redox state of the epidermis even after radiation
exposure.
It prevents the appearance of wrinkles and loss of collagen and
Kaempferia
increases the expression of catalase. The treatment with this extract
parviflora [72]
significantly reduces inflammatory mediators NF-KB, IL-1β and
extract
COX-2.
L-carnosine The combination of these extracts modulates the levels of β-
and Rhodiola endorphin, enkephalin, CGRP, substance P, IL-1α, TNF-α and IL-10
[73]
rosea extract in normal human keratinocytes in normal conditions and after
association punctual or chronic exposure to UV radiation.
It has free radical scavenging effects. ECG inhibits keratinocytes
apoptosis induced by UVA and UVB in a dose-dependent manner.
L-epicatechin- For UVA, this mechanism works by inhibiting the production of
3-gallate hydrogen peroxide. For UVB, ECG inhibits peroxidation of [74]
(ECG) membrane lipids, and also blocking the activation of ERK1 / 2, p38
and JNK in keratinocytes. Therefore, ECG has demonstrated an
important antioxidant potential to prevent photodamage.
It is the most active polyphenol of Green tea. It induces the reduction
of H2O2, iNOS, NO, LPO and MPO. It also inhibits the decrease of
cell antioxidant enzymes (catalase, glutathione peroxidase, superoxide
dismutase and glutathione). It has DNA repair properties through
NER mechanism. EGCG promotes survival of keratinocytes and
inhibits UV-induced apoptosis with the help of a dual mechanism: (1)
bad phosphorylation increased via the ERK-AKT dependent pathways
L- (2) ratio Bcl-2 / Bax increased.
epigallocatechi EGCG treatment of human HaCaT keratinocyte cultures reduces
[69,70,75]
n-3-gallate UVB-induced cytotoxicity and also inhibits the expression of p53
(EGCG) mRNA and p21 regulators of apoptosis, and the genes c-fos gene and
blocks the secretion of IL and TNF-α. These data suggest that EGCG
can be used for its anti-aging effects and as a tumor suppressor in
human skin. In addition, EGCG may inhibit /regulate the action of
NF-kB, iNOS gene expression and NO generation in keratinocytes
after UVB exposure. This suggests that EGCG can have an inhibitory
effect of the UVB photodamage caused in the epidermis.
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1480 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

Table 2. (Continued)

In an in-vivo evaluation in humans, the addition of EGCG into a

broad spectrum sunscreen decreased UV-induced damage compared
to the cream alone.

Employed in various formulations for topical use, lycopene has a high

therapeutic potential for recover of epidermal antioxidants lost during
Lycopene [76]
UV exposure. It also acts as a skin protectant against UV damage. It
has been found that lycopene also acts as a preventive agent by
inhibiting the activity of ornithine decarboxylase in the epidermis,
reducing inflammation and maintaining normal levels of cell
proliferation, presumably by preventing DNA damage by apoptotic
lock, after exposure to UVB.
Mice orally treated with mango extract showed a significant ability to
Mangifera indica
modulate the effects of UV radiation by inhibiting epidermal [77]
L. extract
hypertrophy.
The mangiferine is a scavenger of ROS (superoxide radicals and
hydroxyl radicals). In cultures of human HaCaT keratinocytes,
mangiferin inhibits induction of MMP-1 produced by the hydrogen
Mangiferin [78]
peroxide, blocking the binding of AP-1 to DNA. Furthermore,
mangiferin inhibits keratinocyte death by reducing the MEK-ERK and
JNK-SEK pathways.
Myricetin inhibits keratinocyte death UVB-induced in a dose-
Myricetin dependent manner, due to the inhibition of the increase of hydrogen [79]
peroxide and the inhibition of of c-jun activation induced by UVB.
Human skin pretreatment with N-acetylcysteine in combination with
N-acetil cysteine
genistein, blocks UV induction of collagen, indicating the [80]
and genistein
photoprotective potential of these substances.
The treatment of human HaCaT keratinocytes with naringenin extends
the long-term survival of the cells after irradiation with UVB.
Excision of PARP-1, caspase activation and the ratio Bax/Bcl2
induced by UVB are modulated after treatment with naringenin,
Naringenin [81]
indicating an antiapoptotic effect of this molecule. Also, when HaCaT
cells are irradiated with UVB, naringenin increases CPD removal,
indicating that the active ingredient has a protective effect against
DNA damage.
It contains arabinose, xylose, glucose, galactose. It scavenges
Phellodendron
superoxide and hydroxyl radical in a dose-dependent way, and DPPH
amullenses [60]
radicals. It increases significantly IL-10 and TNF-α. The glycans that
glycans
compose C. chinensis absorb in IR range.
The phenylpropanoid glycosides (Verbascoside, forsitoside B,
Phenylpropanoid echinacoside and campneoside I) induce Nrf2 and cytoprotective
[82]
glycosides enzymatic activity and exhibits an antioxidant activity in cultured
human keratinocytes.
Phlebodium Also known as Polypodium leucotomos. Oral administration of P.
aureum L. extract aureum extract in mice for 5 days before exposure to UV and for 2
(synonym - days post-irradiation reduces by 13% the number of proliferating
Polypodium epidermal cells, and promotes an increase in p53 positive cells and an [83, 84]
leucatomos Poir./ increase of 30% in the plasmatic antioxidant capacity. The beneficial
in articles - P. effect of the extract of P. aureum is probably due to its anti-ROS
leucotomos) properties.

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The pretreatments of human HaCaT keratinocytes with this extract

modulate the effects of UVB radiation, in relation to the reduction of
cell viability, intracellular glutathione content and lipid peroxidation.
Pomegranate fruit
It has also been demonstrated the ability of this extract to inhibit the [85]
extract
increase of MMP-1, MMP-2, MMP-9 and MMP-7, the reduction of
TIMP-1, and phosphorylation of MAPK and c-jun induced by UV
radiation.
Coming mainly from grape seeds (GSP's). Human keratinocytes
irradiated with UVB and treated with GSP's inhibit the formation of
hydrogen peroxide, lipid peroxidation, protein oxidation and DNA
damage. It also inhibits the reduction in endogenous antioxidant
Proanthocyanidins compounds such as glutathione peroxidase, catalase, superoxide [86]
dismutase and glutathione. GSP's also inhibit the phosphorylation of
ERK1/2, JNK, p38 and MAPK family proteins, and the activation of
NF-kB/p65 induced by UVB. These results suggest that GSP can
attenuate oxidative stress caused by UV in human skin.
Psidium P. catleianum extract has a high antioxidant capacity against ROS and
catleianum fruit RNS. Its effect may be mainly due to its phenolic content and [87]
extract especially epicatechins and ellagic acid.
It is a flavonoid with powerful antioxidant properties; however it has
not been extensively studied. Some formulations for topical
Quercetin [88]
application with quercetin inhibit UVB radiation-induced damage in
animals.
The red clover extract is a rich source of isoflavones, such as
Red clover genistein, equol, isoequol and dehidroequol, which are able to reduce [89]
edema and immunosuppression caused by UV radiation.
Red orange extract is able to neutralize the UVB-induced response in
human HaCaT keratinocytes. In particular, the events related to
Red orange apoptosis and inflammation, such as translocation of NF-kB and AP-1
[90]
extract and cleavage of procaspase - 3. This activity is probably due to
blocking events related to oxidative stress, showing that red orange
extract can be useful for the photoprotection of the skin.
Resveratrol is a phytoalexin polyphenolic, which is a substance able
to delay or even stop the normal course of skin aging, by blocking
apoptotic mitochondrial events and malfunctions in keratinocytes.
Human skin has specific unions for resveratrol. Studies with human
Resveratrol HaCaT keratinocyte lines have shown that trans-resveratrol is capable [88, 91]
of inhibiting the production of hydrogen peroxide. In humans, while
providing a protective effect against UVA radiation, trans-resveratrol
is even able to improve clinical signs of aging when used associated
with β-cyclodextrin excipient.
The rhubarb extract has anti-radical characteristics and antioxidants
Rheum properties against lipid peroxidation in in vitro studies. The extract
rhaponticum L. also reduces the activity of tyrosinase and inhibits the production of [92]
rizome extract IL-1α, TNF-α, and α-MSH, and tyrosine kinase activity in human
melanocytes exposed to UV radiation.
It is a polyphenol with anti-tumor, anti-inflammatory, antioxidant, and
anti-mutagenic properties. Topically applied it decreases epidermal
Rutin hyperplasia and levels of 4-hydroxynonenal modified protein, which [88, 93]
is characteristic of lipid peroxidation. It also reduces the expression of
iNOS and COX-2 and inhibits the activation of AP-1.
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Table 2. (Continued)

UV irradiated mice were treated orally with a mixture of extract of

fruits of sea buckthorn (SBF), blueberry extract and collagen. Oral
ingestion of SBF reduces wrinkles formation and helps to maintain
Sea buckthorn
skin thickness. SBF treated mice exhibited inhibition of TEWL and an [94]
fruit extract
increase of the skin moisture content. The application of SBF also
reduces expression of MMP-1 and MMP-9, and reduces the levels of
SOD activity.
Silk lutein protects cells exposed to UVB radiation, reducing the
Silk lutein [95]
levels of cytotoxicity and apoptosis.
It is a flavonoid coming from Silybum marianum seed and is
composed of three molecules: silibinin, silidianin and silicristin. In
Silymarins animal models it protects against erythema, DNA damage and [96]
radiation-induced immune suppression. It is an excellent antioxidant,
anti-inflammatory and immunomodulatory.
Soy extract is rich in isoflavones. It inhibits human HaCaT
keratinocyte cell death UVB-induced, as well as phosphorylation of
p38, JNK and ERK1/2. In mice, topical application prior to UV
Soybean extract [97]
irradiation was found to reduce the thickness of the epidermis, the
expression of COX-2 and PCNA, and increases the concentration of
catalase.
T4 endonuclease V is a bacterial DNA repairing enzyme, which
repairs cyclobutane pyrimidine dimers in DNA. When it is
encapsulated in liposomes and applied topically, it can eliminate the
T4 endonuclease DNA dimers formed both in animals and humans. It also prevents the
[98]
V liposomes upregulation of IL-10. The topical application of T4N5 for a year
reduces the proportion of actinic keratosis and basal cell carcinoma.
Also partially protects against formation of sunburn cells, contact
hypersensitivity and DTH. However, it has no effect against edema.
Tannase is an enzyme produced by fungi, yeasts and bacteria that
hydrolyze gallates catechins (EGCG and ECG) of green tea and thus
increase its potential application for the removal of free radicals such
as superoxide and hydrogen peroxide. A formulation containing
Tannase- extract of green tea tannase-converted was used to inhibit oxidative
converted green damage in mouse epidermis induced by UV. The formulation acts to [99]
tea extract prevent the reduction of glutathione levels and controlling hydrogen
peroxide. Treated mice showed significantly reduced levels of
reactive substances of thiobarbituric acid by lipid peroxidation,
compared to UVB-irradiated controls, suggesting that this formulation
is effective in protecting the skin against photoaging.
This substance inhibits the production of proinflammatory cytokines
(IL-6 and IL-8) induced by UVB in cultured human HaCaT
keratinocytes in a dose dependent way. Also inhibits the expression of
Tectroside [100]
COX-2 and JNK phosphorylation. These results suggest that this
component has the potential to protect the skin against UVB induced
inflammation.
CoQ10 is an endogenous electron carrier in cellular respiration.
CoQ10 is used in many creams due to its antioxidant and skin
Coenzyme Q10 [100‒103
protective photoaging properties and. It is able to scavenge ROS and
(CoQ10) ]
to protect cells from oxidative stress, both in absence and in presence
of UV radiation.

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1483

Vitamin C (ascorbic acid) reduces the effects of aging, such as

superficial and deep wrinkles and increases skin elasticity, firmness
and hydration. The evaluation of ascorbic acid and its derivatives, AA
Vitamin C [104]
2-phosphate and AA 2-glucoside, in human HaCaT keratinocytes
subjected to UVB-induced cytotoxicity, showed that ascorbic acid is
not able to inhibit the cytotoxicity, however, their derivatives do.
The antioxidant potential of α-tocopherol, one of the forms of vitamin
E, is widely known. It has been tested the inhibitory role of α-
tocopherol in the regulation of the production of IL-8 and AP-1in
human keratinocytes exposed to UVA, and the results show
Vitamin E [105]
significant inhibition of NADPH oxidase activity, which is
responsible for activating IL-8 and AP-1. α-tocopherol also inhibits
the formation of thiobarbituric acid-malondialdehyde in cells exposed
to UVA radiation.
V. vinifera sprouts extract has an in vitro antioxidant capacity higher
than vitamin C or E. An aqueous extract of V. vinifera tendril, applied
Vitis vinifera L. in human keratinocytes (NCTC 2544) was capable of increasing the
[106]
shoot extract glutathione concentration and the activity of trans-plasma membrane
oxido-reductase, in a time-dose dependent manner, demonstrating that
is has a significant antioxidant activity.
They prevent immune suppression induced by UV exposure by
Xyloglucans [107]
reducing IL-10 production in mice.
An increased lutein intake improves the health of the skin when
administered orally or topically (zeaxanthin and lutein), as evaluated
in the following five physiological parameters: surface lipids of the
skin, skin moisturizing, photoprotective activity, elasticity of the skin
and lipid peroxidation. The oral or topical administration significantly
Zeaxanthin and improves these measures, oral administration results in better
[108]
lutein protection against changes in lipid peroxidation and photoprotective
activity after UV irradiation. However, a combined oral and topical
administration provides a greater degree of protection. Other studies
have shown the protective effect of this combination against
hyperproliferation and inflammation of the epidermis after UVB
exposure in mice.
It is an iron chelator that applied topically prevents erythema, sunburn
2-furildioxime cell, thickness reduction of the skin, inflammation and induction of [109]
ornithine decarboxylase.

4.4. DNA Repair Agents

Some of the antioxidants described in Table 2, such as L-epigallocatechin-3-gallate

(EGCG), T4 endonuclease V liposomes and Green tea extract, besides their antioxidant
properties; they are also able to promote the reparation of damaged DNA.
There are other substances whose main mechanism of action the repair of DNA damage.
Are examples of these substances Photolyase, which is a DNA repair enzyme that applied
immediately after UVB exposure decreases the number of dimers of DNA between 40 and
45% in human skin [110] and Thymidine dinucleotides. Thymidine dinucleotides produce a
mimic effect on the cells as which occurs after exposure to UVR. However, thymidine
dinucleotides pretreatments cause that cells develop a protective response against UV by
activating p53 protein, which is involved in DNA regeneration [111].
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1484 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

4.5. Cyclooxygenase-2 Inhibitors

The results of the study performed Tripp et al. [28] previously commented, suggest that
inhibitors of COX-2 may play a role in the prevention of epidermal skin cancers
development, such as squamous cell carcinoma, and for that reason, it would be interesting to
include these substances in sunscreens.
Kaempferia parviflora extract, Rutin, Soybean extract and Tectroside, included in Table
2, besides their antioxidant properties, they also act inhibiting COX-2, so the use of these
substances in sunscreens would be useful for their different mechanisms of action against the
harmful effects of radiation.
A study performed by Orengo et al. [112] demonstrated that Colecoxib, an inhibitor of
COX-2, orally administrated, inhibits the induced photocarcinogenesis in hairless mouse
model of squamous cell carcinoma and basal cell carcinoma, where COX-2 is overexpressed.

4.6. Iron Chelators

Endogenous iron (Fe2+), under normal conditions, is sequestered by iron-binding

proteins, such as transferrin and ferritin and does not participate in ROS generation.
Nevertheless, when skin is exposed to sun exposure and consequently to oxidative stress, Fe2+
is released from these proteins. This free Fe2+ catalyses hydroxyl radical generation through
Fenton reaction [113]:

H2O2 + Fe2+ → OH + OH− + Fe3+

For that reason, it has been proposed by some authors the use of iron chelators in
sunscreens, which will reduce the free Fe2+ and thus will prevent the Fenton reaction and
consequently hydroxyl radical formation through this reaction. Examples of iron chelators are
2,2′-dipyridyl, 1,10-phenanthroline and 2,2′-dipyridylamine [114].

4.7. Osmolytes

Osmolytes are compounds that protect cells from desiccation by maintaining a high
intracellular osmolality. Compatible organic osmolytes, such as taurine, betaine and
myoinositol are involved in maintaining the cell homeostasis and protecting the cell against
oxidative stress, which could be produced by UV radiation.
An augmented osmolyte uptake has been shown in UV-irradiated keratinocytes and for
that reason it has been suggested that these substances play a role in protecting keratinocytes
against some of the harmful effects induced by UV irradiation [115]. Nowadays, there are not
so many studies of the use of osmolytes in sunscreens.

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1485

5. MELATONIN
Melatonin (N-acetyl-5-methoxytryptamine) actually known as a neurohormone is
secreted primarily at night-time by the pineal gland. It is in fact, a very well conserved
molecule. Its presence has been discovered in very ancient living beings such as a
photosynthetic prokaryote, Rhodospirillum rubrum [116], unicellular organisms, marine
algae, dinoflagellate Gonuaulaz poliedra [117] and yeast (Saccharomyces cerevisae) [118]. In
all these organisms, the main function of melatonin was to protect against the oxidative stress.
With the years, this molecule took new roles such as a chemical signal of light and dark,
immunoestimulation, mediator of the seasonal physiological functions and other functions
that exerts in numerous and extremely diverse biological systems.

5.1. Endogenous Synthesis

Endogenously, melatonin is synthesized from the amino acid tryptophan, not only in the
pineal gland, but also in tissues such as retina [119] and skin [120], among others. Melatonin
synthesis in the skin begins with hydroxylation of tryptophan by the action of the tryptophan-
5-hydroxylase enzyme (TPH) in the skin cells; this process is catalyzed mainly by TPH1,
which is a compound of about 50kD that is degraded to lower molecular weight species. The
TPH is located in the epidermis, the hair follicle and eccrine gland, although the place of
expression is melanocytes. 5-hydroxytryptophan (5-HTP) is rapidly decarboxylated by the
aromatic amino acid decarboxylase (AAD). Serotonin is the product, which is acetylated by
N-acetyl transferase (NAT), and it is expressed in cells of the epidermis, dermis and other
associated compartments. The last step of the synthesis is carried out by the action of the
HIOMT, converting the N-Acetylserotonin in melatonin, this reaction can be reversed by the
action of the CYP450 (Figure 3).

5.2. Skin Metabolism

In the skin, the metabolism of melatonin involves indolic and kynuric pathways and can
be catalyzed by enzymatic or non-enzymatic reactions. The multiple pathways of the
metabolism of melatonin are summarized in Figure 4.
The most important metabolites are 6-hydroxymelatonin, 2-hydroxymelatonin, Cyclic-3
hydroxymelatonin, AFMK and AMK.

6-hydroxymelatonin
In the skin, melatonin can be catabolized to 6-hydroxymelatonin by enzymatic process
involving CYP1B1 or by the interaction with peroxynitrite (ONOO-) [121] and •OH [122].

2-hydroxymelatonin
In the skin, this molecule is produced by the interaction of melatonin with HClO [123]
and •OH [122]. The reaction of cytochrome C with H2O2, uses melatonin and the metabolite
obtained is also 2-hydroxymelatonin [124].
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1486 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

Figure 3. Biochemical synthesis of melatonin in the skin.

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1487

Figure 4. Melatonin metabolism. (a) 2-hydroxymelatonin (b) melatonin 2-indolinone (c) 3-

hydroxymelatonin 2-indolinone (d) melatonin dioxetane (e) cyclic 3-hydroxymelatonin (f) AFMK (g)
6-hydroxymelatonin (h) 5-methoxytryptamine (i) 5-methoxyindoleacetaldehyde (j) 5-methoxyindole
acetic acid (k) 5-methoxytryptophol (l) AMK. (1) CYP1A1, CYP1A2 or CYP1B1 (2) melatonin
deacetylase (3) monoamine oxidase (4) arylamine formamidase, hemoperoxidase and ROS/RNS (5)
aldehyde dehydrogenase (6) alcohol dehydrogenase.

Cyclic 3-hydroxymelatonin
Melatonin scavenges 2 •OH to form this metabolite [125], but can also be produced by
the interaction of melatonin with ONOO‒ [126, 127]. This molecule is readily converted to
AFMK by interaction of ROS and RNS.

AFMK
The metabolite AFMK can be generated by the interaction of melatonin with multiple
agents, such as H2O2 [128], horseradish peroxidase, myeloperoxidase [129, 130], cytochrome
C [122], 1O2 [131, 132], carbonate radical and ONOO‒ [130]. Humans cells also generates
AFMK when are exposed to oxidative stress, such as UVB radiation [133].

AMK
This metabolite is generated by the interaction of AFMK with arylamine formamidase,
hemoperoxidases or interaction with ROS/RNS.
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5.3. Melatonin Effects

Melatonin plays a main role in the control of circadian rhythms and also participates in
the regulation of reproduction, however, in this section is described only the pleiotropic
effects that converts melatonin in a major skin protectant and how it counteracts the solar
radiation effects. The main effects of melatonin in the skin are:

Immunomodulation
The immunoregulatory effects of melatonin represent a line of defense [134, 135]. This
compound has shown to be an efficacious photoprotective agent via modulation of
proinflammatory mediators [136].
Melatonin exerts a stimulating action on the immune response through the activation of
various cell types as B and T lymphocytes, NK, monocytes and reticulum endothelial cells
[135] and the increase of IL-4 production on T helper cells (TCD4) of bone marrow and stem
cells of granulocytic-macrophage line [137].
Other authors have reported that melatonin stimulates the secretion of IL-1, IL-6 and
INFα [138].
AFMK and AMK also have antiinflamatory and immunoregulatory properties. AFMK
inhibits TNF-α and IL-8, AMK inhibit the synthesis of prostaglandins [139] and both
molecules inhibit the gene expression of COX-2 [140].

Antioxidant
The antioxidant effects of melatonin occur by at least two mechanisms. The first
mechanism is its direct antioxidant effects via free radical scavenging by inhibiting their
generation [141]. The second is by enhancing the activity of antioxidants enzymes, which
improve the endogenous antioxidant defense capacity of the organisms and induce up-
regulation of gene expression, thereby increasing the first line of defense against oxidative
damage of the cells [142, 143].

Free Radical Scavenger

The reaction of melatonin with free radicals occurs in all the compartments of the body
due to its lipophilicity and consequently it has wide distribution capabilities. The first
observation that showed that melatonin has antioxidant effects was made in 1991 by Lanas et
al. [144] and was confirmed by Tan et al. in 1993 [145] that showed that melatonin
neutralizes •OH. Since then, numerous papers have been published demonstrating the ability
of melatonin to interact with reactive species, and thus act as an antioxidant molecule.
Melatonin directly reacts with •OH, H2O2, ONOO‒, HClO, 1O2 and O2–, obtaining the
metabolites described before. Free radicals are the main responsible of lipid peroxidation,
protein oxidation, caspase activation and release, apoptosis, mitochondrial instability and
DNA damage. Therefore, melatonin, when scavenge free radicals, acts indirectly reducing the
above processes.

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1489

Endogenous Antioxidant Defense Capacity Improvement

In addition to the intrinsic ability of melatonin to scavenge free radicals, it is also capable
of stimulating the activity and expression of other antioxidant systems, thus establishing a
form to reduce oxidative stress using an indirect action [146].
First, it stimulates glutathione cycle, melatonin up-regulate the activity of Gluthation
peroxidase (GPx), which reduces H202 to water both in rat brain [142] and in chicken tissues
[147] equilibrating the balance of GSSG/GSH [148] and Glutathion reductase (GRd).
It also increases the production of glutathione by stimulating γ-glutamylcysteine
synthase, which is a limiting enzyme in glutathione synthesis pathway [149], and stimulates
glucose-6-phosphate dehydrogenase, which is responsible for generating the NADPH
required by GRd [150]. Melatonin also stimulates other antioxidant enzymes such as SOD
and catalase [151].
The nuclear factor NFkB, induced by ROS is a central and early event in the induction of
inflammatory reactions [152]. NFkB is an oxidative stress sensitive factor that activates
multiple target genes involved in the expression of several proinflammatory mediators.
Melatonin has been reported to inhibit the NFkB activation as action of the cell protection
signaling of melatonin by transcriptional response control [153].
Not only melatonin has antioxidant effects, some of its metabolites also have them. For
example, 6-hydroxymelatonin reduces lipid peroxidation and protects against DNA damage
[154, 155] due to its direct free radical scavenging and antioxidant properties [156‒158]. It
has been found that cyclic 3-hydroxymelatonin is able to prevent the oxidative DNA damage
produced by Fenton regents [154].
AFMK is a powerful antioxidant that can donate 4 or more electrons to interact with free
radicals; it is as potent as melatonin scavenging some free radicals such as O2–·. AFMK also
reduces lipid peroxidation and oxidative DNA [156, 159]. AMK is more efficient as an
antioxidant than AFMK.

Melatonin As an Adjuvant in the Treatment of Cancer

It has been shown in cell studies that both in animals and humans melatonin directly
suppresses and inhibits the tumor cell growth in carcinoma cell lines and inhibits the growth
of colon carcinoma and breast, so it leads to a reduction in the cancer development [138, 160,
161]. Melatonin is also capable of suppressing the proliferation in melanoma cells of different
dignity and growth characteristics; MT1 and RORα receptor-dependent enhance the
suppressive effects [162‒164]. In humans, it has been demonstrated that it has a potential
efficacy against malignant melanoma in patients in stage IV [165].

5.4. Melatonin As a Photoprotective Agent

Several studies have shown the beneficial effects of melatonin counteracting the harmful
effects of sun radiation. In this section are reviewed the different actions of melatonin that
demonstrate its potential to be used as a photoprotective agent.
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1490 A. Flo Sierra, V. Flo Sierra, A. Cristina Calpena Campmany et al.

Erythema
It has been shown that the application of melatonin reduces the erythema induced by UV
radiation [166] in a dose-dependent manner [167]. But this reduction is only produced when
melatonin is applied before UV exposure [168]. The minimum concentration of Melatonin
necessary for a statistically significant reduction in erythema is 0.5%.

Apoptosis
UV radiation increases the apoptosis by damaging cellular structures that undergo
apoptosis. Cellular studies have shown that melatonin applied before UV exposure prevents
apoptosis [169‒177].

Generation of ROS
As it has been previously explained, sun radiation generates ROS and it has been widely
demonstrated that melatonin act as a potent free radical scavenger, not only by itself but also
by its metabolites [170, 172, 174, 175].

Cell Viability
It has been demonstrated in cellular cultures that UV radiation reduces the cell viability
and melatonin prevents this reduction [169, 170, 172, 176, 177].

Gene Expression
When the cells are exposed to UV radiation it is up-regulated the apoptosis of controller
gens, cancer related genes, and oxidative stress response genes. In the presence of melatonin
these up-regulations were prevented [173, 174, 176, 178]. For example, it down-regulates the
expression of genes, that play an important role in the photodamage induced by UV, such as
intersticial collagen (MMP-1), stromelysin (MMP-3), stromelysin (MMP-10) and aldehyde
dehydrogenase 3 type A1 [179].

Mitochondrial Membrane Potential

The exposure to UV radiation leads to a reduction of the mitochondrial membrane
potential that leads to its damage and thus to the activation of the intrinsic pathway of
apoptosis. Melatonin acts preventing the dissipation of the mitochondrial membrane potential
[171, 172, 177, 180].

DNA Synthesis
In cellular studies, it has been shown that the exposure to UV decreases the rate of DNA
synthesis and it has been demonstrated that melatonin prevents this reduction [133, 177].

DNA Damage
UV radiation causes oxidative stress, which subsequently leads to oxidative DNA
damage (represented by 8-OHdG formation). Cellular studies have shown that the oxidative
DNA damage induced by UV decreases when the cells are pretreated with melatonin. For
example, melatonin activates the poly(DP-ribose) polymerase, which is a key DNA-repair-
mediating enzyme [178, 181].

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 The Role of Antioxidants in Sunscreens: The Case of Melatonin 1491

5.5. Melatonin in Sunscreens

As previously mentioned, melatonin is a lipophilic molecule with strong ability to cross

cell membranes, because of this property and the multiple actions as an antioxidant, there are
groups trying to formulate sunscreens containing melatonin as an antioxidant. Although there
isn’t currently marketed formulation, there are a few references to sunscreens containing
melatonin.
Dreher et al. demonstrated in 1998 [182] that melatonin incorporated in a topical
formulation combined with vitamins E and C exerts a synergistic antioxidant action.
In another study, Flo et al. [183] reported an approach on the use of melatonin in a
sunscreen emulsion combined with the common used UV filters octyl salicylate (5%), octyl
methoxycinnamate (5%) and benzophenone-3 (5%). The antioxidant activity assay
demonstrated that the formulation possesses higher antioxidant properties that the formulation
that does not contain melatonin. The in vivo assay demonstrated that irradiated skin areas of
animals treated with this formulation were statistically equivalent to the unirradiated control
areas, thus the photoprotective effect of the formulation was clearly shown.
Another study has demonstrated that melatonin formulated in creams creates a depot
structure in the stratum corneum, in which a small part goes releasing to blood during 24 h
[184]. Therefore, the topical administration is a good candidate for treatment with melatonin
for local action but also as transdermal delivery to reach constant plasma levels.
Due to these direct and indirect antioxidant effects of melatonin and its metabolites, it is a
promising photoprotective agent, acting directly as a free radical scavenger and indirectly by
stimulating antioxidant enzymes [180], inducing down regulation of the gene expression of
enzymes that are involved in the generation of oxidative stress and enhancing the activation
of antioxidant enzyme gene expression [178].
Summarizing, melatonin is a promising photoprotective substance that complies with all
the requirements described for an ideal sunscreen: it provides uniform protection against UV,
Vis and IR, it is a potent free radical scavenging, it stimulates the DNA repair systems,
although not being photostable its metabolites are safe and have per se a potent antioxidant
activity, and it is safe.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 68

UV FILTERS, THEIR DEGRADATION REACTIONS

AND ECO-TOXICOLOGICAL EFFECTS

Albano Joel M. Santos and Joaquim C. G. Esteves da Silva

Centro de Investigação em Química da Universidade do Porto (CIQ-UP),
Departamento de Química e Bioquímica, Faculdade de Ciências,
Universidade do Porto, Porto, Portugal

ABSTRACT
Sunscreens or sunscreen agents are more notoriously known as ultraviolet (UV)
filters, and they are the prime components of many personal care products and
pharmaceuticals. Most UV filters are organic compounds that absorb UV radiation,
therefore protecting us from solar radiation and its nefarious effects on human skin and
health. The protective character of UV filters regarding UV radiation, would presuppose
a stable nature towards alterations in general. However, the compounds are well known
to undergo degradation, and in many cases quite substantially, either by influence of UV
radiation itself (by photolysis or photo-isomerization) or through contact with water
disinfecting agents, such as chlorine. These degradation reactions might be quite
troublesome, since they generate degradation by-products that either do not present the
appropriate UV-protective capabilities, as is the case with photo-isomers, or possess
toxicological profiles potentially damaging for both the human health and the
environment, as is the case with free-radicals or even disinfection by-products (DBP’s).

INTRODUCTION
Ultraviolet (UV) radiation constitutes about 6.2% of the total solar radiation that is able
to reach the Earth’s surface, given the filtration and mitigation capabilities of the protective
ozone layer. Out of this specific portion of the solar radiation, mostly is attributed to UVA
radiation (320-400 nm) while a very small portion is attributed to UVB radiation (290-320
nm).

Corresponding author: Joaquim C.G. Esteves da Silva; [email protected]
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1506 A. J. M. Santos and Joaquim C. G. Esteves da Silva

The highly energetic UVC radiation (100-290 nm) is completely blocked by the ozone
layer and therefore does not reach the surface of the planet 1. Despite the beneficial
character of UV radiation (it enhances the production of vitamin D, improving the human
resistance towards different pathologies; increases the calcium absorption by the organism;
etc.), it is also known to enhance the occurrence of skin cancer, as well as other serious but
less prominent issues like inflammations, sunburns, and allergic reactions 1. It is in this
context that sunscreens or sunscreen agents play a fundamental role, by preventing or
attenuating the damaging effects of UV radiation on the human skin and health 1.
Sunscreen products include complex formulations of different compounds, of which UV
filters are of the utmost importance, since these are the compounds that indeed protect us
from UV radiation. As Salvador and Chisvert [2] refer, a sunscreen is defined as any product
containing UV filters in its formulation, in order to protect the skin from the negative effects
of UV radiation, significantly decreasing its impact on human health 2. The mechanism of
protection, however, is based on two processes that are intimately linked to the two existing
types of filters in question: essentially absorption of UV radiation in the case of the vastly
more numerous organic UV filters; and reflection or scattering in the case of the few existing
inorganic UV filters 3.
UV filters generally display either simple or multiple aromatic structures, often
conjugated with carbon-carbon double bonds or carbonyl groups, which attributes them the
ability to absorb or scatter UV radiation. These compounds will absorb UV radiation,
therefore evolving towards a superior energetic state but returning thereafter to the original
state by emitting energy through vibrational transitions or photochemical reactions 4. As it
was already mentioned, UV filters are classified as either organic (UV-absorbent) or
inorganic (UV-scattering) compounds. The most prominent classes of UV filters are the
benzophenones, salicylates, cinnamates, triazines, p-aminobenzoic acid derivatives, dibenzoyl
methane derivatives and camphor derivatives, and there are globally about 55 filters
approved, regulated and controlled worldwide, out of which merely two are inorganic (zinc
oxide and titanium dioxide) 1, 4, 5.
Table 1 includes all the UV filters currently approved in the EU, as well as all their
relevant physical-chemical properties 3, 6.
As for the nature of UV filters, these compounds present the features common to most
priority organic pollutants (POP’s), such as the presence of aromatic rings in association with
long and unsaturated aliphatic chains. Most of the filters consist of geometrical isomers
(E and Z forms), although the commercial formulations include solely the E isomer. As is
visible in Table 1, these compounds exhibit commonly increased lipophilicity, enabling their
association with particles rich in organic matter content, such as soils and sediments, as well
as high resistance towards biotic degradation, which enhances their accumulation,
concentration and persistence in the environment and the food chain 3, 6, 7.

UV Filter Degradation Reactions

Regarding their purpose of application, protection of human skin from the effects of UV
radiation, there is the assumption that UV filters are quite stable to general degradation.

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 Table 1. Physical-chemical properties of the UV filters currently approved and regulated in and by the EU (adapted from 3 and 6)

Molecular Log Log BCF Log KOC Solubility

Structure INCI name Acronym λmax /nm
weight KOW§ **;§§ ††;§§ /g/L‡‡
Benzophenone-3 BZ3 228.24 3.79 1.38 3.10 0.21 290
Benzophenones
Benzophenone-4 BZ4 308.31 0.88 - - 0.65 240†; 288
p-Aminobenzoic acid PAB 137.14 0.83 - - 915 282
PABA and derivatives PEG-25 PABA P25 277.41 - - - - 310*
Ethylhexyl dimethyl PABA EDP 277.40 6.15 3.74 3.38 2.1×10-3 310
Homosalate HS 262.35 6.16 - - 0.02 -
Salicylates
Ethylhexyl salicylate ES 250.34 5.77 - - 0.028 240*
Ethylhexyl methoxycinnamate EMC 290.40 5.80 5.80 4.10 0.15 306†
Cinnamates
Isopentyl p-methoxycinnamate IMC 248.32 4.06 - - 0.06 -
Camphor benzalkonium methosulfate CBM 409.55 0.28 - - - 288*
Terephtalydene dicamphor sulfonic acid TDS 562.69 1.35 - - 0.014 340*
Benzylidene camphor sulfonic acid BCS 320.40 2.74 - - 0.038 297*
Camphor derivatives
Polyacrylamidomethyl benzylidene camphord PBC - - - - -
4-Methylbenzylidene camphor MBC 254.37 4.95 3.51 3.89 5.1×10-3 300†
3-Benzylidene camphor 3BC 240.34 4.49 9.9×10-3 292*
Ethylhexyltriazone ET 826.10 15.53 - - - 310†
Diethylhexyl butamido triazone DBT 765.98 11.90 - - 4.6×10-7 -
Triazines
Bis-Ethylhexyloxyphenol methoxyphenyl
EMT 627.81 13.89 - - 4.9×10-8 340†
triazine
-5
Drometrizole trisiloxane DRT 225.25 9.79 - - 1.3×10 344; 303
Benzotriazoles Methylene bis-benzotriazolyl
MBT 658.87 14.35 - - 3.0×10 -8
340†
tetramethylbutylphenol
Phenyl benzimidazole sulfonic acid PBS 274.30 0.01 0.50 2.46 0.26 300†
Benzimidazole derivatives
Disodium phenyl dibenzimidazole tetrasulfonate DPD 674.60 - - - - 250
Dibenzoylmethane Butyl methoxydibenzoyl methane BDM 310.39 2.41 4.51 3.23 0.037 358*
derivatives Diethylamino hydroxybenzoyl hexyl benzoate DHH 397.51 6.93 - - 9.5×10-4 360†
Octocrylene OCR 361.49 7.35 - - 2.0×10-4 300†
Others
Polysilicone 15 P15 - - - - 313‡
Data is originated from SciFinder, American Chemical Society, 2008.
UV filters shadowed in green colour, represent the most popular and frequently used compounds in commercial formulations of sunscreen products.
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* Rastogi, S.C., Jensen, G.H. (1998), Identification of UV filters in sunscreen products by high-performance liquid chromatography–diode-array detection, J.
Chromatogr. A 828 (1-2), 311-316.
† De Orsi, D., Giannini, G., Gagliardi, L., Porrà, R., Berri, S., Bolasco, A., Carpani, I., Tonelli, D. (2006), Simple extraction and HPLC determination of UV-A
and UV-B filters in sunscreen products, Chromatographia 64 (9-10), 509-515.
‡ Philippe Maillan Formulation, R&D Cosmetics, DSM Nutritional Products; Measurement of UV Protection in Hair.
§ Octanol-water partition coefficient (KOW); it regards the ratio between the concentration of a substance in octanol and in water, in equilibrium and at a
determined temperature.
** Bio-concentration factor (BCF); it regards the concentration of a substance in an organism and in the water body around it.
†† Organic carbon distribution coefficient (KOC); it regards the ratio between the mass of a substance adsorbed into the soil (by unity of mass of organic carbon
in the soil) and the concentration of the same substance in equilibrium in solution.
‡‡ In water and at 25ºC.
§§ Giokas, D.L., Salvador, A., Chisvert, A. (2007), UV filters: from sunscreens to the human body and the environment, Trends in Analytical Chemistry 26 (5),
360-374.

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 UV Filters, Their Degradation Reactions and Eco-Toxicological Effects 1509

However, such assumptions are not exactly accurate, in fact, it is well reported and
established that UV filters experience degradation from two essential sources: photo-
degradation, upon exposure to UV radiation; and degradation induced by disinfecting agents
such as chlorine, when in contact with these in aqueous solution 3, 4. Figure 1 presents the
paths of degradation and their consequential by-products.

Figure 1. Degradation processes experienced by UV filters.

Photo-Degradation of UV Filters

Photolysis
The direct dissociation of a molecule upon the absorption of a determined amount of
energy from a given type of radiation is ever more likely when that amount is equivalent or
higher than the bonding energy of that same molecule. When this molecule reaches a higher
or excited energy state, it dissociates, and the process is regarded as photolysis 14.
Photolysis is usually a rather complex set of reactions that lead to the formation of
reactive species or fragments, and it can be experienced either by direct or indirect paths.
Direct photolysis occurs upon the absorption of radiation by specific portions of the UV
filters’ structure itself, denominated chromophores. Indirect photolysis occurs upon the
absorption of radiation by other structures or compounds rather than the UV filter, named
photosensitizers, therefore initiating a series of reactions that will induce the transformation
or degradation of the filters 3, 8, 9, 14. Organic compounds will also experience degradation
when in contact with reactive species, such as singlet oxygen, hydroxyl radicals, photo-
excited organic matter and others 8, 9.
This type of photochemical reactions is one of the most important abiotic processes that
control the fate and behaviour of UV filters when in the environment, in particular the aquatic
compartments, and in general their prominence is far more significant than the biotic
processes of degradation 3, 8, which will be approached in more detail further ahead.
There are numerous studies that have dealt with the subject of photo-degradation, with
emphasis on photolysis. The general notion that must be underlined is that as the filters are
exposed to UV radiation, they gradually lose their UV-protective features or capabilities 10,
11, which is also accompanied by the formation of several toxic and harmful by-products, as
it has been demonstrated with EMC 12. Sayre 13 stressed the complexity of the photo-
degradation issue, since UV filters are used as part of a formulation of several different filters
in commercial sunscreen products, and not in singular. In other words, the photochemical
profile and behaviour of a matrix of multiple filters is fundamentally different than that
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1510 A. J. M. Santos and Joaquim C. G. Esteves da Silva

displayed by a single UV filter, since the photo-degradation reactions of many filters are
known to enhance or even induce the degradation reactions of others, even those supposedly
more stable 13. Serpone has also carried out some interesting studies on the photo-
degradation or photo-stability of certain UV filters 10, 11. His approach [11], delved on the
photo-degradation of different UV filters and its extension in aerobic aqueous medium. The
study was carried on the basis of the record of any and every alteration to the UV radiation
absorbance as a function of irradiation time, with any loss of absorbance being subsequently
directly correlated with loss of UV-protective capabilities. Results have shown that, in the
case of the filter PAB, for instance, UV radiation absorbance capacity decreased about 35%
just within the first hour of irradiation, whereas in the case of a very similar filter, EDP, the
UV absorbance decrease was almost complete just after 20 minutes of irradiation. As for
TDS, it was defined as the most photo-unstable UV filter of the study, with 90% UV radiation
absorbance decrease after just 10 minutes of irradiation, while BZ3 was considered the most
photo-stable, with a UV absorbance decrease of 20% and throughout two hours of irradiation.
Photo-degradation studies have always focused on its relation towards photo-protection
alterations [10], ability of the filter mixture to enhance photolysis [10, 13], or the
toxicological potential of by-products [12], but seldom has it focused on degradation in the
environmental context [15]. Sakkas [15] approached simultaneously the disinfection by-
product (DBP) formation as well as the photo-degradation by-products. Results have shown
that photochemical reaction rates depend, not only on the environmental conditions, but also
on the presence of other relevant compounds in solution, in particular dissolved organic
matter (DOM). In the case of the filter EDP, degradation decreased significantly with the
increase of DOM levels in solution, which is easily explained by the fact that DOM actively
competes with any other present organic compound for the incident photons, in regards to
photo-degradation. The authors were also able to identify several photo-degradation by-
products, namely from dealkylation and hydroxylation, and in all the different sources of
water samples studied (distilled water; swimming-pool water; and sea water).

Photo-Isomerization
Contrary to photolysis, photo-isomerization reactions yield new species closely related to
the parental structures, but potentially more toxic and harmful than the original compounds.
Regarding UV filters in particular, this translates essentially into the production of photo-
isomers that may be related but no longer possess the required UV-protective features of the
parental molecules, which is prominently evident in several classes of filters: cinnamates;
salicylates; camphor derivatives; and dibenzoylmethane derivatives [10, 16-18].
The photo-isomerization of UV filters is both a fast and reversible process, when in
aqueous solution, giving origin to a mixture of E and Z isomers in equilibrium. In the
environment, UV filters will always be found in either of these two isomeric forms, given the
existence of carbon-carbon exocyclic double bonds in their structure. However, commercial
formulations of these compounds contain solely the E form of the compounds, despite their
immediate photo-isomerization into the Z form upon exposure to UV radiation [19].
Another notable disadvantage of these reactions is, as approached earlier, apart from the
loss of UV-protective capabilities, the production of potentially more troublesome by-
products. For instance, the isomeric forms of UV filters might be chiral and therefore
enantiomers, with similar physical-chemical properties, but the compounds will display very

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 UV Filters, Their Degradation Reactions and Eco-Toxicological Effects 1511

distinct environmental fate, behaviour and eco-toxicological profile. Contrary to the

biological processes of degradation, that might be stereo-selective or enantio-selective [20],
these abiotic processes are apparently not enantio-selective [19]. In light of this, the stereo-
isomer composition of UV filters in natural waters seems indeed paramount in order to
understand the compounds’ fate and behaviour in the environment, but seldom has the theme
been the subject of serious and focused investigation [3].
Díaz-Cruz [3] reviews the only existing study focused specifically on the subject: Buser
[21] studied the chirality of MBC, showing that the stereo-isomer composition of the filter
depended in fact on biological degradation occurring in waste water treatment plants, other
water bodies like rivers or lakes, as well as plant or animal life.
There are numerous other studies on the photo-degradation or photo-stability of UV
filters in general. One of the most important and popular UV filters, EMC, has been the
subject of several interesting studies [10, 22-25]. In a study already mentioned, Sakkas [15]
investigated the photochemical behaviour of the filter EDP in different water samples (sea
water; swimming pool water; and distilled water) and under natural or artificial solar
radiation. Results demonstrate that the filter degrades photo-chemically, originating several
by-products; influence of dissolved organic matter (DOM) was also evaluated, showing that
its presence decreases the photo-degradation reaction rates, since it competes with the filters
for the incident photons; several by-products were also successfully identified. Huong [24]
studied the photo-isomerization of EMC under artificial solar radiation and in several
different solvents. Results showed significant loss in UV-absorbance capacity after
irradiation, occurrence of chemical environmentally-dependent photo-isomerization E  Z as
well as irreversible degradation of the filter structure; Z isomer displays considerable lower
UV-absorbance capacity; and photo-degradation by-products were also detected and
successfully identified. Pattanaargson [22] also approached the photo-isomerization of EMC
in different solvents and under natural solar radiation. The relevant results were as follows:
photo-isomerization E  Z resulted in significant loss of UV-absorbance capacity; E-Z
equilibrium in solution does occur but it depends on solvent polarity. Pattanaargson and
Limphong [23] approached the photo-stability of EMC on a chromatographic basis, in order
to determine the obtained photo-degradation by-products. The authors have successfully
determined one photo-degradation by-product, identified as the Z form of the filter, referring
that after one day of irradiation, approximately half of the amount of the original E form of
the filter had been transformed into the by-product. No irreversible compound structure
degradation of the filter was detected. Maier [26] carried out a spectroscopically-focused
study on the spectral alterations undergone by a set of sunscreen products upon exposure to
artificial solar radiation, and its reflection on the UV-absorbance capacity. Results have
shown the following: loss in UVB-absorbance capacity never exceeded 5% and considering
all the irradiation times; UVA-absorbance capacity loss was generally much more significant
and frequent; all products displayed increased spectroscopic photo-instability at increasing
wavelengths. Gaspar and Maia Campos [27] evaluated the in vitro photo-stability of different
combinations of UV filters in sunscreen products, under artificial solar radiation. Results have
demonstrated that the interaction between filters within a formulation influences their photo-
stability; and formulations containing the filter OCR increased their UVA-absorbance
capacity. Huong [28] carried out another study similar to the one mentioned before for EMC
[24], but focused now on BDM [28]. As far as the results are concerned, BDM demonstrated
photo-instability in non-polar solvents, with significant alterations in its absorption spectra;
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1512 A. J. M. Santos and Joaquim C. G. Esteves da Silva

these alterations, despite significant, were found to be reversible after protection and storage
of the irradiated solutions in the dark, but also found to be inhibited depending on the solvent
conditions; the general behaviour of BDM was considered analogous to that of EMC, and it
displayed quite significant and irreversible degradation of the filter structure in aqueous
solution; several photo-degradation by-products were detected and successfully identified;
photo-degradation in general was found to be significantly influenced and dependent on the
medium and experimental conditions.
Many other similar studies exist, like Mturi’s and Martincigh’s [29] that dealt with the
photo-stability of BDM in different solvents; or Hojerová’s study [30], that ascertained the
protective efficiency of several sunscreen products containing different UV filter
formulations, and concluded that the sunscreen products’ UV-protective efficiency is quite
distinct from one another, even between commercial products with the same labeled sun
protection factor (SPF); Rodil [25] also evaluated the photo-stability of several UV filters, as
well as the eco-toxicological profile of their photo-degradation by-products in aquatic
microorganisms; Perugini [31] carried out a very interesting study on the effect of
nanoparticle encapsulation of the filter EMC on its photo-stability; and Scalia [32] evaluated
the effect of the natural antioxidant quercetin, on the photo-stability of a combination of two
of the most popular UV filters used worldwide, EMC and BDM.

Degradation Induced by Disinfecting Agents

The water disinfection process has the fundamental purpose of destroying aquatic
microbiological organisms, which represent the ultimate contagion source of disease. This is
contrary to the concept of sterilization, which involves complete destruction of every
microorganism, something that may not always be achievable or even necessary or beneficial
[33].
For more than a century, chlorine has been the most popular disinfecting agent used
worldwide, successfully controlling and even eliminating water-borne infectious diseases
altogether [8]. Despite this, there are several other types of less popular disinfecting agents,
like ozone or even UV radiation, both used in high-scale swimming-pool water disinfection,
but also bromide-based water disinfecting agents, used in lower-scale swimming pools [33].
Although the removal of relevant pathogens and microorganisms is rather effective, the
removal of DOM is not. Removal of organic pollutants is rather complex and varies
significantly [3].
The disinfection process transforms the organic compounds in the water, giving origin to
the so-called disinfection by-products (DBP’s), or in this particular case, chlorinated DBP’s.
Over the years, chlorinated DBP’s have been directly associated with several and serious
potential toxicological effects, which forced authorities to consider the problem of production
of these compounds in the context of drinking water disinfection process [3, 8]. Reports
indicate that exposure to chlorinated DBP’s might be directly associated with the occurrence
of several cancers in human vital organs, so Gopal [33] approached predictive models for
production and kinetics of DBP formation, their health effects, removal techniques, and
guideline implementation.
Given the use of other aforementioned water disinfecting agents, apart from the vastly
more popular chlorine, based on either bromide or iodine, important focus is now being given

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 UV Filters, Their Degradation Reactions and Eco-Toxicological Effects 1513

to brominated and iodinated DBP’s. These compounds are reported to be significantly toxic,
not only in the range of carcinogenicity, but also genotoxicity and cytotoxicity. Such
compounds include iodo-acids, like iodo-acetic acid, bromonitromethanes, iodinated-
trihalomethanes, 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (best known by its
historical name, Mutagen X or MX), halogenated-aldehydes, halogenated-amides, bromate,
and many others [35]. Also a particular reason for concern is yet another by-product of
chlorine disinfection, the production of chloramines, which are originated from the reaction of
chlorine with ammonia. Research has indicated that chloramines potentiate the iodo-acids and
iodinated-trihalomethane production and accumulation within the water compartments [8].
Researchers have previously approached the general pathways of introduction and
movement of synthetic organic pollutants, into and throughout the environment, with
emphasis on the aquatic compartments [6, 8]. Generally speaking, the mode of introduction of
these compounds into the environmental compartments will very much depend on their
pattern of use or application [8]. When in the environment, however, these are transformed,
chemically, photo-chemically or biologically. Usually, these processes will lead to the
compounds’ structure breakdown and subsequent elimination, but at the same time
degradation by-products may also be produced, often more persistent and toxic than the
original structures [6, 8]. Figure 2 represents the pathways involved in the generic fate and
behaviour of UV filters and corresponding by-products in the environment [6].

Figure 2. Relevant pathways of introduction of UV filters and their degradation by-products, into the
environment (inspired by and adapted from [6]; boxes filled in light grey correspond to potential
processes of release into the environment).

An evaluation on the occurrence of DBP’s was carried out in Turkish superficial waters
with low levels of dissolved organic carbon [36]. Results have shown that, given the
susceptibility of the DBP precursors to associate with soils and sediments, events like ground-
level run-off or leaching from soils as well as re-suspension into the watercourse from
sediments, increased their levels in the superficial aquatic compartments. Upon treatment
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1514 A. J. M. Santos and Joaquim C. G. Esteves da Silva

with chlorine disinfecting agents, it led to the production and increase of DBP’s in drinking
water.
There isn’t any data available on the determination of UV filters DBP’s in water
treatment facilities [3]. Nonetheless, given the combined action of solar radiation and the
presence of disinfecting species in solution, UV filters will be readily halogenated, giving
origin to halogenated species of the parental compounds as well as many other DBP’s as a
result of the degradation of the original structure. Up to this moment, there are only four
studies focused on the UV filter degradation reactions in aqueous solution on the presence of
chlorine disinfecting agents [1, 37-39], along with a comprehensive review on both the UV
filter photo-degradation by-products in aqueous solution, and the UV filters’ DBP formation
studies until 2012 [40].
Regarding the study of these compounds, very little is still known about the degradation
reactions induced by disinfecting agents, and what is indeed known is focused solely on a
very limited number of the most popular UV filters [1, 37-39]. More focus should be given
towards the disinfection process and its implications on the degradation of UV filters.

Eco-Toxicological Effects of UV Filters and Their Degradation by-Products

The physical-chemical properties of UV filters, presented in Table 1, such as water

solubility, vapour pressure and polarity, are crucial in order to determine their behaviour in
the environment. As already mentioned, data points to their substantial tendency to
concentration and accumulation within the environment and food chain, which, associated
with their also reported significant potential for eco-toxicity, is quite problematic [3, 6, 8, 15,
41, 42].
Díaz-Cruz and Barceló [43] have reviewed some of the most important existing eco-
toxicological studies, performed both in vitro and in vivo. What follows, is a brief summary
on these and some additional relevant studies and subsequent results and conclusions.

In Vitro Studies
Several UV filters have been reported to display estrogenic activity in vitro [7, 44-48].
The in vitro models applied or used on these and other studies, often revolve around the
highly efficient, sensitive, fast and inexpensive recombinant yeast assay [44, 47, 48], MCF-7
breast cancer cells [45, 48], the human embryonic kidney 293 reporter gene assay (HEK293)
[7], the human endometrial Ishikawa cell line [46], or rat and human primary hepatocytes
[46].
Regarding the UV filters commonly approached in in vitro studies, benzophenones seem
to be quite popular in that regard, with Schultz [44] investigating their estrogenic activity in
specific, although the compounds have been addressed in many other studies, in particular
BZ3 [7, 45, 47].
MBC has also been the subject of some particular studies, regarding its interaction
towards estrogenic receptors [46, 48], amongst other more generic in vitro eco-toxicological
studies [7, 45, 47]. Amidst the most popular UV filters, one must also underline the UV filters
EMC, BDM and EDP, also investigated as to their estrogenic activity [7, 45, 47].
As for the relevant eco-toxicological conclusions arisen from these studies, all have
emphasized the issues subsequent to this context of investigation. These problems include

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 UV Filters, Their Degradation Reactions and Eco-Toxicological Effects 1515

noncommittal results or the clear inability of the general in vitro models to account for the
toxico-kinetics and toxico-dynamics of complex whole organisms, which highlights their
prominent limitations as to relevant predictive value for a mammalian in vivo context.
Overall, nearly all filters have demonstrated dose-dependent estrogenic activity, with
benzophenones being amongst the most active, as was the case of BZ3 [7, 45], with EMC [7]
and BDM [45] being the least active and inactive, respectively, in the same type of studies.
The estrogenic activity seems to be directly linked to the presence of benzene rings
substituted with polar functional groups, particularly in para- position, as well as with the
presence of symmetry in the molecule [44]. MBC, one of the filters studied in specific, has
demonstrated either estrogenic activity analogous to that of other know weak estrogens [46]
or ambiguous results occurring at extremely high levels [48].
Possible effects on thyroid hormonal regulation have also been mentioned [43, 48], but
the data on the subject are rather scarce [49]. This study used a new human recombinant
thyroid peroxidase stably transfected into a human follicular thyroid carcinoma cell line
(FTC-238), in order to assess the possible effects of the filter BZ2.
Very significant disturbance of thyroid hormone homeostasis by inhibition of thyroid
peroxidase was reported, making BZ2 the most potent thyroid peroxidase inhibitor found to
date [49]. A similar study using human FTC-133 thyroid carcinoma cells [50] showed the
opposite results for EMC and MBC.
Evaluation of mere estrogenic activity of UV filters has always been the main focus of
hormonal activity studies of these compounds, but multiple combined hormonal effects
(estrogenic, anti-estrogenic, androgenic and anti-androgenic effects) have seldom been
investigated. Several UV filters have been recently shown to display multiple endocrine-
disrupting behaviour like MBC, which displayed estrogenic and anti-estrogenic activities, or
BZ3 and HS, which demonstrated estrogenic, anti-estrogenic and anti-androgenic activities
[7, 51-53].
In another study [54], out of 19 UV filters and two benzophenone metabolites, all
displayed some kind of hormonal effect, merely two (P25 and PAB) did not demonstrate
multiple hormonal effects, while the vast majority demonstrated multiple effects. However, as
Díaz-Cruz and Barceló argue [43], these effects might be subjective, since individual
activities are directly and significantly dependent on the type of tests conducted.
Regarding the in vitro studies on UV filters’ transformation or degradation by-products,
seldom has it been the subject of any studies. The exceptions are a study by Butt and
Christensen [12], which dealt with the toxicity of photo-degradation by-products of EMC and
BDM in a mouse lymphoma cell line (L5178Y-R), and a more recent study from Nakajima
[38], which focused on the mutagenic activity of EMC and EDP using a mutagenic assay on a
Salmonella typhimurium strain (TA100).
Butt and Christensen [12] demonstrated that exposure to irradiated solutions of UV filter
resulted in increased cell mortality, independently of the irradiation time. Regarding
Nakajima’s study [38], both EMC and EDP were not mutagenic in the referred assay, with the
opposite being exhibited after chlorination. EMC’s mutagenic by-products of chlorination
however, proved to be unstable after 6 hours of completion of the chlorination reactions, since
the mutagenicity of the solutions decreased subsequently.
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1516 A. J. M. Santos and Joaquim C. G. Esteves da Silva

In Vivo Studies
Díaz-Cruz and Barceló [43] mention that benzophenones are the essential focus of study,
regarding in vivo investigations, and these are mostly centered on evaluation of hormonal
effects, effects on reproduction or fertility [54-64].
The usual in vivo models of investigation include mostly fish, particularly juvenile or
mature fathead minnows (Pimephales promelas) [54-58], Japanese rice fish (Oryzias latipes)
[58, 62] or rainbow trout (Oncorhynchus mykiss) [58], with some existing studies also in
ovariectomized rats [59, 60, 61], tadpoles [57, 63] or even fetal rats [64]. Benzophenones are
indeed a special focus of these studies, particularly BZ2 [56, 59, 60] and BZ3 [58, 60], given
the fact that the molecular structure of this class of compounds is quite similar to other known
estrogenic chemicals. But camphor derivatives have also been approached often, with
particular emphasis on MBC [57, 62-64] and 3BC [55, 57, 63, 64]. There are two existing
studies on the extremely popular EMC [61, 62].
Regarding the conclusions of these studies, all have emphasized concerning
considerations, and in general, dose-dependent: significant bioaccumulation factors [55, 56],
which has also been approached in previous chapters (see Table 1 and corresponding
references [3, 6]); quite significant decrease in fecundity or complete cessation of
reproductive ability [55-58]; demasculinization of secondary sexual characteristics [55, 57];
significant induction of Vitellogenin [54, 55, 57, 58]; prominent effects in the masculine and
feminine gonad histology [55, 56, 57]; and development of both oocytes and spermatocytes,
as well as egg production, inhibited [55, 56, 57, 58].
On the other hand, MBC and 3BC displayed no accountable effects on tadpole’s
hormonal and thyroidal systems during metamorphosis [57], which was described as a critical
stage quite susceptible to endocrine disruptions. The filter Ethyl-p-aminobenzoate (yet
another p-aminobenzoic acid derivative) also did not exhibit negative effects on fathead
minnows’ weight and length development, and mortality was not verified either, upon
exposure to the compound [54].
As a counterweight to all the concerning findings, some authors argue that many of the
toxicological effects are found and reported at extremely high levels, sometimes as high as 75
fold the levels previously reported for wastewater effluents [58]. At environmental levels,
however, MBC and 3BC were both studied as to their effects on the hormonal, thyroidal and
sexual systems of tadpoles, during metamorphosis, and after 35 days of exposure, no relevant
negative effects were found [63]. Contrary to this fact, studies have also reported that
significant toxicological effects may indeed be found at low levels, as was the case with 3BC,
which induced prominent histological and reproductive effects in fish and at low
concentrations [57].

FUTURE PERSPECTIVES
In light of what was approached in this review, it is important to emphasize that very
little is still known about the aqueous degradation reactions of UV filters induced by
disinfecting agents. Very few filters have been studied in this context (essentially EMC, BDM
and EDP), and considering the findings of these studies, it is imperative to focus even more
on this context and extend the investigation towards other filters amongst the most popular.

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 UV Filters, Their Degradation Reactions and Eco-Toxicological Effects 1517

Special attention should also be given to the determination of their DBP’s, which is a field of
special concern given their potential hazardous toxicological effects.
Considering the eco-toxicological reviews, it seems clear that additional studies are also
required, given the ambiguous results obtained so far. Both the in vitro and in vivo contexts of
toxicological investigation are of paramount importance, and should therefore be used in
succession in order to achieve a reliable assessment of the eco-toxicity of UV filters and by-
products. However, there are still many issues to consider and solve, particularly regarding
the in vitro models: the typical models used carry significant unclearness regarding
procedures and protocols; many display limited predictive value towards the in vivo results;
and none reflect or replicate the metabolic processes of complex whole organisms, which is
clear from the usually conflicting in vitro and in vivo results.
Naturally, the in vitro models lack the capacity to account for the toxico-kinetics and
dynamics of complete organisms, which represents a comprehensive disadvantage. This fact
stresses the need to carry out studies in vivo, following the investigations made in vitro, for
these often generate inconclusive results and that do not necessarily reflect what may indeed
occur in whole organisms.

ACKNOWLEDGMENTS
A. J. M. Santos wishes to acknowledge Fundação para a Ciência e Tecnologia (FCT) for
the Ph.D. Program in Sustainable Chemistry grant PD/BD/52530/2014.

REFERENCES
[1] Chisvert, A., Salvador, A. (2007), Analysis of Cosmetic Products, Chapter 3, Elsevier,
Amsterdam, The Netherlands.
[2] Salvador, A., Chisvert, A. (2005), Sunscreen analysis – a critical survey on UV filter
determination, Anal. Chim. Acta 537, 1-14.
[3] Díaz-Cruz, M. S., Llorca, M., Barceló, D. (2008), Organic UV filters and their
photodegradates, metabolites and disinfection by-products in the aquatic environment,
Trends in Analytical Chemistry 27 (10), 873-887.
[4] Shaath, N. A. (2010), Ultraviolet filters, Photochem. Photobiol. 9, 464-469.
[5] Shaath, N. A. (2005), The chemistry of UV filters, in: Sunscreens: Regulations and
Commercial Development, 3rd Edition, Taylor and Francis, New York, USA, pp. 217-
238.
[6] Giokas, D. L., Salvador, A., Chisvert, A. (2007), UV filters: from sunscreens to the
human body and the environment, Trends in Analytical Chemistry 26 (5), 360-374.
[7] Schreurs, R., Lanser, P., Seinen, W., Van der Burg, B. (2002), Estrogenic activity of
UV filters determined by an in vitro reporter gene assay and an in vivo transgenic
zebrafish assay, Arch. Toxicol. 76 (5-6), 257-261.
[8] La Farré, M., Pérez, S., Kantiani, L., Barceló, D. (2008), Fate and toxicity of emerging
pollutants, their metabolites and transformation products in the aquatic environment,
Trends in Analytical Chemistry 27 (11), 991-1007.
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1518 A. J. M. Santos and Joaquim C. G. Esteves da Silva

[9] Lam, M. W., Tantuco, K., Mabury, S. A. (2003), PhotoFate:  a new approach in
accounting for the contribution of indirect photolysis of pesticides and pharmaceuticals
in surface waters, Environ. Sci. Technol. 37 (5), 899-907.
[10] Serpone, N., Salinaro, A., Emeline, A. V., Horikoshi, S., Hidaka, H., Zhao, J. (2002),
An in vitro systematic spectroscopic examination of the photostabilities of a random set
of commercial sunscreen lotions and their chemical UVB/UVA active agents,
Photochem. Photobiol. Sci. 1 (12), 970-981.
[11] Serpone, N., Dondi, D., Albini, A. (2007), Inorganic and organic UV filters: their role
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[12] Butt, S. T., Christensen, T. (2000), Toxicity and phototoxicity of chemical sun filters,
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[13] Sayre, R. M., Dowdy, J.C., Gerwig, A.J., Shields, W.J., Lloyd, R.V. (2005),
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[14] Rohatgi-Mukherjee, K.K. (1978), Fundamentals of Photochemistry (Revised Edition),
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[15] Sakkas, V.A., Giokas, D.L., Lambropoulou, D.A., Albanis, T.A. (2003), Aqueous
photolysis of the sunscreen agent octyl-dimethyl-p-aminobenzoic acid. Formation of
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[16] Broadbent, J.K., Martincigh, B.S., Raynor, M.W., Salter, L.F., Moulder, R., Sjoberg, P.
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[17] Deflandre, A. and Lang, G. (1988), Photostability assessment of sunscreens.
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[18] Plaguellat, C., Kupper, T., Furrer, R., Alencastro, L., Grandjean, D. and Tarradellas, J.
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[19] Poiger, T., Buser, H.R., Balmer, M.E., Bergqvist, P.A. and Müller, M.D. (2004),
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[20] Ariens, E. J. (1989), Stereoselectivity of bioactive agents, general aspects, in:
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[21] Buser, H.-R., Müller, M.D., Balmer, M.E., Poiger, T., Buerge, I.J. (2005), Stereoisomer
Composition of the chiral UV filter 4-Methylbenzylidene camphor in environmental
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[22] Pattanaargson, S.T., Munhapol, T., Hirunsupachot, P. and Luangthongaram, P. (2004),
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[23] Pattanaargson, S.T., Limphong, P. (2001), Stability of octyl methoxycinnamate and
identification of its photo-degradation product, Int. J. Cosmet. Sci. 23 (3), 153-160.

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 UV Filters, Their Degradation Reactions and Eco-Toxicological Effects 1519

[24] Huong, S.P., Andrieu, V., Reynier, J.-P., Rocher, E., Fourneron, J.-D. (2007), The
photoisomerization of the sunscreen ethylhexyl p-methoxy cinnamate and its influence
on the sun protection factor, J. Photochem. Photobiol. A 186 (1), 65-70.
[25] Rodil, R., Moeder, M., Altenburger, R., Schmitt-Jansen, M. (2009), Photostability and
phytotoxicity of selected sunscreen agents and their degradation mixtures in water,
Anal. Bioanal. Chem. 395 (5), 1513-1524.
[26] Maier, H., Schauberger, G., Brunnhofer, K. and Honigsmann, H. (2001), Change of
ultraviolet absorbance of sunscreens by exposure to solar-simulated radiation, J. Invest.
Dermatol. 117 (2), 256-262.
[27] Gaspar, L.R., Maia Campos, P.M.B.G. (2006), Evaluation of the photostability of
different UV filter combinations in a sunscreen, Int. J. Pharm. 307 (2), 123-128.
[28] Huong, S.P., Rocher, E., Fourneron, J.-D., Charles, L., Monnier, V., Bun, H., Andrieu,
V. (2008), Photoreactivity of the sunscreen butylmethoxydibenzoylmethane (DBM)
under various experimental conditions, J. Photochem. Photobiol. A 196 (1), 106-112.
[29] Mturi, G.J. and Martincigh, B.S. (2008), Photostability of the sunscreening agent 4-tert-
butyl-4′-methoxydibenzoylmethane (avobenzone) in solvents of different polarity and
proticity, J. Photochem. Photobiol. A 200 (2-3), 410-420.
[30] Hojerová, J., Medovcíková, A., Mikula, M. (2011), Photoprotective efficacy and
photostability of fifteen sunscreen products having the same label SPF subjected to
natural sunlight, Int. J. Pharm. 408 (1-2), 27-39.
[31] Perugini, P., Simeoni, S., Scalia, S., Genta, I., Modena, T., Conti, B., Pavanetto, F.
(2002), Effect of nanoparticle encapsulation on the photostability of the sunscreen
agent, 2-ethylhexyl-p-methoxycinnamate, Int. J. Pharm. 246 (1-2), 37-45.
[32] Scalia, S., Mezzena, M. (2010), Photostabilization effect of quercetin on the UV filter
combination, Butyl methoxydibenzoylmethane-Octyl Methoxycinnamate, Photochem.
Photobiol. 86 (2), 273-278.
[33] Gopal, K., Tripathy, S.S., Bersillon, J.L., Dubey, S.P. (2007), Chlorination by-products,
their toxico-dynamics and removal from drinking water, J. Hazard. Mater. 140 (1-2),
1-6.
[34] Scholz, M. (2006), Wetland Systems To Control Urban Runoff, Chapter 22, Elsevier,
Amsterdam, The Netherlands, pp. 155-162.
[35] Richardson, S.D. (2005), New disinfection by-product issues: emerging DBPs and
alternative routes of exposure, Global NEST J. 7 (1), 43-60.
[36] Ates, N., Kaplan, S.S., Sahinkaya, E., Kitis, M., Dilek, F.B., Yetis, U. (2007),
Occurrence of disinfection by-products in low DOC surface waters in Turkey, J.
Hazard. Mater. 142 (1-2), 526-534.
[37] Negreira, N., Canosa, P., Rodríguez, I., Ramil, M., Rubí, E., Cela, R. (2008), Study of
some UV filters stability in chlorinated water and identification of halogenated by-
products by gas chromatography–mass spectrometry, J. Chromatogr. A 1178 (1-2),
206-214.
[38] Nakajima, M., Kawakami, T., Niino, T., Takahashi, Y., Onodera, S. (2009), Aquatic
fate of sunscreen agents Octyl-4-methoxycinnamate and Octyl-4-
dimethylaminobenzoate in model swimming pools and the mutagenic assays of their
chlorination by-products, J. Health Sci. 55 (3), 363-372.
[39] Santos, A.J.M., Crista, D.M.A., Miranda, M.S., Almeida, I.F., Sousa e Silva, J.P.,
Costa, P.C., Amaral, M.H., Lobão, P.A.L., Sousa Lobo, J.M., Esteves da Silva, J.C.G.
 Free ebooks ==> www.Ebook777.com
1520 A. J. M. Santos and Joaquim C. G. Esteves da Silva

(2013), Degradation of UV filters 2-Ethylhexyl-2-methoxycinnamate and 4-tert-Butyl-

4’-methoxydibenzoylmethane in chlorinated water, Environ. Chem. 10 (2), 127-134.
[40] Santos, A.J.M., Miranda, M.S., Esteves da Silva, J.C.G. (2012), The degradation
products of UV filters in aqueous and chlorinated aqueous solutions, Water Res. 46
(10), 3167-3176.
[41] Balmer, M.E., Buser, H.R., Müller, M.D., Poiger, T. (2004), Occurrence of organic UV
filter compounds BP-3, 4-MBC, EHMC and OC, in wastewater, surface waters and in
fish from Swiss lakes, Agroscope, Swiss Federal Research Station for Horticulture,
Plant Protection Chemistry, CH-8820 Wädenswill, Switzerland.
[42] Giokas, D.L., Sakkas, V.A., Albanis, T.A. (2004), Determination of residues of UV
filters in natural waters by solid-phase extraction coupled to liquid chromatography–
photodiode array detection and gas chromatography–mass spectrometry, J.
Chromatogr. A 1026 (1-2), 289-293.
[43] Díaz-Cruz, M.S., Barceló, D. (2009), Chemical analysis and ecotoxicological effects of
organic UV-absorbing compounds in aquatic ecosystems, Trends in Analytical
Chemistry 28 (6), 708-717.
[44] Schultz, T.W., Seward, J.R., Links, G.D. (2000), Estrogenicity of benzophenones
evaluated with a recombinant yeast assay: Comparison of experimental and rules-based
predicted activity, Environ. Toxicol. Chem. 19 (2), 301-304.
[45] Schlumpf, M., Cotton, B., Consciente, M., Haller, V., Steinmann, B., Linchtesteiger,
W. (2001), In vitro and in vivo estrogenicity of UV screens, Environ. Health Perspect.
109 (3), 239-244.
[46] Müller, S.O., Kling, M., Firzani, P.A., Mecky, A., Durante, E., Shields-Botella, J.,
Delansorne, R., Broschard, T., Kramer, P.J. (2003), Activation of estrogen receptor α
and β by 4-methylbenzylidene-camphor in human and rat cells: comparison with phyto-
and xenoestrogens, Toxicol. Lett. 142 (1-2), 89-101.
[47] Kunz, P.Y., Galicia, H.F., Fent, K. (2006), Comparison of In Vitro and In Vivo
Estrogenic Activity of UV Filters in Fish, Toxicol. Sci. 90 (2), 349-361.
[48] Tinwell, H., Lefevre, P.A., Moffat, G.J., Burns, A., Odum, J., Spurway, T.D.,
Orphanides, G., Sabih, J. (2002), Confirmation of uterotrophic activity of 3-(4-
methylbenzylidine)camphor in the immature rat, Environ. Health Perspect. 110 (5),
533-536.
[49] Schmutzler, C., Bacinski, A., Gotthardt, I., Huhne, K., Ambrugger, P., Klammer, H.,
Schlecht, C., Hoang-Vu, C., Grüters, A., Wuttke, W., Jarry, H., Köhrle, J. (2007), The
ultraviolet filter benzophenone-2 interferes with the thyroid hormone axis in rats and is
a potent in vitro inhibitor of human recombinant thyroid peroxidase, Endocrinology 148
(6), 2835-2844.
[50] Schmutzler, C., Hamann, I., Hofmann, P.J., Kovacs, G., Stemmler, L., Memtrup, B.,
Schomburg, L., Ambrugger, P., Grüters, A., Seidlova-Wuttke, D., Jarry, H., Wuttke,
W., Köhrle, J. (2004), Endocrine active compounds affect thyrotropin and thyroid
hormone levels in serum as well as endpoints of thyroid hormone action in liver, heart
and kidney, Toxicology 205 (1-2), 95-102.
[51] Schreurs, R., Sonneveld, E., Hansen, J.H.J., Seeinen, W., Van der Burg, B. (2005),
Interaction of Polycyclic Musks and UV Filters with the Estrogen Receptor (ER),
Androgen receptor (AR), and progesterone receptor (PR) in reporter gene bioassays,
Toxicol. Sci. 83 (2), 264-272.

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 UV Filters, Their Degradation Reactions and Eco-Toxicological Effects 1521

[52] Ma, R., Cotton, B., Lichtensteiger, W., Schlumpf, M. (2003), UV Filters with
antagonistic action at androgen receptors in the MDA-kb2 cell transcriptional-activation
assay, Toxicol. Sci. 74 (1), 43-50.
[53] Schlumpf, M., Schmid, P., Durres, S., Consciente, M., Maerkel, K., Henseler, E.,
Gruetter, M., Herzog, I., Reolon, S., Ceccatelli, R., Faass, O., Stutz, O., Jarry, H.,
Wuttke, W., Lichtensteiger, W. (2004), Endocrine activity and developmental toxicity
of cosmetic UV filters—an update, Toxicol. 205 (1-2), 113-122.
[54] Kunz, P.Y., Fent, K. (2006), Multiple hormonal activities of UV filters and comparison
of in vivo and in vitro estrogenic activity of ethyl-4-aminobenzoate in fish, Aquatic
Toxicol. 79 (4), 305-324.
[55] Kunz, P.Y., Gries, T., Fent, K. (2006), The ultraviolet filter 3-Benzylidene Camphor
adversely affects reproduction in fathead minnow (Pimephales promelas), Toxicol. Sci.
93 (2), 311-321.
[56] Weisbrod, C.J., Kunz, P.Y., Zenker, A.K., Fent, K. (2007), Effects of the UV filter
benzophenone-2 on reproduction in fish, Toxicol. Appl. Pharmacol. 225, 255-266.
[57] Fent, K., Kunz, P.Y., Gomez, E. (2008), UV filters in the aquatic environment induce
hormonal effects and affect fertility and reproduction in fish, Chimia 62, 368-375.
[58] Coronado, M., De Haro, H., Deng, X., Rempel, M.A., Lavado, R., Schlenk, D. (2008),
UV filters in the aquatic environment induce hormonal effects and affect fertility and
reproduction in fish, Aquatic Toxicol. 90, 182-187.
[59] Jarry, H., Christofell, J., Rimoldi, G., Koch, L., Wuttke, W. (2004), Multi-organic
endocrine disrupting activity of the UV screen benzophenone 2 (BP2) in
ovariectomized adult rats after 5 days treatment, Toxicol. 205, 87-93.
[60] Schlecht, C., Klammer, H., Jarry, H., Wuttke, W. (2004), Effects of estradiol,
benzophenone-2 and benzophenone-3 on the expression pattern of the estrogen
receptors (ER) alpha and beta, the estrogen receptor-related receptor 1 (ERR1) and the
aryl hydrocarbon receptor (AhR) in adult ovariectomized rats, Toxicol. 205, 123-130.
[61] Klammer, H., Schlecht, C., Wuttke, W., Schmutzler, C., Gotthardt, I., Köhrle, J., Jarry,
H. (2007), Effects of a 5-day treatment with the UV-filter octyl-methoxycinnamate
(OMC) on the function of the hypothalamo-pituitary–thyroid function in rats, Toxicol.
238, 192-199.
[62] Inui, M., Adachi, T., Takenaka, S., Inui, H., Nakazawa, M., Ueda, M., Watanabe, H.,
Mori, C., Iguchi, T., Miyatake, K. (2003), Effect of UV screens and preservatives on
vitellogenin and choriogenin production in male medaka (Oryzias latipes), Toxicol. 194,
43-50.
[63] Kunz, P.Y., Galicia, H.F., Fent, K. (2004), Assessment of hormonal activity of UV
filters in tadpoles of frog Xenopus laevis at environmental concentrations, Mar.
Environ. Res. 58, 431-435.
[64] Hofkamp, L., Bradley, S., Tresguerres, J., Lichtensteiger, W., Schlumpf, M., Timms, B.
(2008), Region-specific growth effects in the developing rat prostate following fetal
exposure to estrogenic ultraviolet filters, Environ. Health. Perspect. 116 (7), 867-872.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 69

ASSESSMENT OF SUNSCREEN SAFETY BY SKIN

PERMEATION STUDIES: AN UPDATE

Lucia Montenegro
Department of Drug Sciences, University of Catania, Catania, Italy

ABSTRACT
One of the major concern about the use of sunscreens is their safety. Many
investigations performed with different techniques have addressed this issue providing
conflicting results. To be safe and effective, sunscreens should remain on the skin surface
without penetrating into the underlying living tissue. As these products are normally
applied on large skin areas, even small amounts of UV-filters permeating the skin could
lead to their systemic absorption, making controversial their safety after topical
application. To overcome real or perceived human health concerns arising from the use of
sunscreen products, their margin of safety (MoS) can be easily estimated by assessing
UV-filters skin permeation using in vitro techniques. At present, several in vitro and in
vivo test systems that provide reliable and reproducible results are used by cosmetic and
pharmaceutical industries.
In this chapter, physical and chemical parameters affecting UV-filters ability to
permeate the skin will be discussed, and in vitro and in vivo skin permeation studies
performed on the most commonly used UV-filters will be reviewed. Estimations of UV-
filter MoS from skin permeation studies will highlight the safety of currently used
sunscreen formulations.

INTRODUCTION
The increasing incidence of skin cancer has led the international health authorities to
recommend protection measures to prevent the harmful effects of skin exposure to UV-
radiation. Such measures include avoidance of sun exposure, especially at times when


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disease-inducing wavelengths are more intense, wearing protective clothing and use of topical
sunscreens (González et al., 2008; Lautenschlager et al., 2007).
As sunscreen products have gained the public favor, in the last decades there has been a
notable increase of their consumption and of the number and content of UV-filters present in
cosmetics and toiletries, thus enhancing the potential human exposure to sun-protecting active
ingredients.
To be effective, ideally sunscreens should remain on the skin surface, without penetrating
into the deep skin layers. As these products are often applied to large skin areas, even small
amounts of UV-filters permeating the skin could lead to their systemic absorption and to
adverse reactions.
Reports on the ability of some UV-filters to permeate the skin (Hayden et al., 1997;
Janjua et al., 2004) and on their estrogenic effects (Klann et al., 2005; Koda et al., 2005; Kunz
and Fent, 2006; Schlumpf and Cotton, 2001; Schlumpf et al., 2004) have given rise to a great
concern about the safety of sunscreen products. Therefore, the need to guarantee the safety of
sunscreen products has boosted toxicity studies on UV-filters. A recent review (Gilbert et al.,
2013), illustrating the available toxicity data on the most commonly used UV-filters,
evidenced that their potential estrogenic effects as well as their entrapment into vital organs
are still very controversial.
To perform a risk assessment of sunscreen products, reliable information on the
toxicological profile of individual sunscreen actives are required as well as data from
percutaneous absorption studies designed to mimic actual product use. Therefore,
understanding the basic concept of skin penetration/permeation is an essential requisite to
conduct a rigorous evaluation of the actual safety of sunscreen agents.

THE PROCESS OF PERCUTANEOUS ABSORPTION

In recent years, a growing attention has been focused on the evaluation of permeation
through human skin of exogenous compounds. This evaluation is of significance both in the
pharmaceutical and cosmetic fields to assess the efficacy and the safety of products intended
for application onto the skin. Predicting skin delivery of active ingredients is still a challenge
as many factors are involved in the process of percutaneous absorption. This process relates
to the entering of a molecule from the external environment within the skin (penetration) and
it can be described as permeation if the molecule reaches the systemic circulation. An
exhaustive knowledge of the skin’s barrier function is fundamental both in evaluating the
(trans)dermal delivery of drugs and in making a risk assessment following dermal exposure to
chemicals.
It is well known that the outermost layer of the epidermis, the stratum corneum (SC),
limits skin permeation of xenobiotics, acting as the rate-controlling barrier to skin delivery of
most drugs (Förster et al., 2009). This thin (15 − 30μm) and highly hydrophobic layer consists
of dead keratin cells embedded in a lipid domain and arranged in the so-called bricks and
mortar model (Bouwstra et al., 2000; Wertz, 2000). Lipid content and organization within the
SC and skin thickness are among the factors affecting drug skin permeation and vary for
different anatomical regions in the same individual (intra-subject variability), the same region
of different individuals (inter-subject variability) and among species (Akomeah et al., 2007).

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 Assessment of Sunscreen Safety by Skin Permeation Studies 1525

The underneath viable epidermis is mostly hydrophilic as its water content is > 50% while in
the dermis the water content reaches 70%, thus favoring the uptake of hydrophilic molecules
in these skin layers. Variables such as SC water content, skin extensibility, recovery and
elasticity in individuals of different sex and race could play a significant role, as well
(Berardesca et al., 1991).
Key factors affecting the process of percutaneous absorption involve physicochemical
properties of the applied molecule and type and composition of the vehicle.
As reported in the literature (Guy and Hadgraft 1988; Schaefer and Riedelmayer, 1996),
the physicochemical properties that determine the skin permeation of an active ingredient
include hydrophilic-lipophilic balance, molar mass, molecular size, dipole moment, vapor
pressure and extent of ionization. Molecules showing partition coefficient (log P
octanol/water) values in the range 1-3 have been reported to permeate the skin better than
very lipophilic compounds as they have a sufficient solubility in the lipid domains of the SC
while still having a sufficient hydrophilicity to allow their partitioning into the viable tissue of
the epidermis (Beetge et al., 2000; Bunge and Cleek, 1995). Therefore, depending on its
lipophilic or hydrophilic properties, an active ingredient will accumulate in the SC (very
lipophilic molecules), or remain on the surface (very hydrophilic molecules) or penetrate into
the skin (amphiphilic molecules).
The molar mass and the molecular size of a molecule affect mainly its ability to diffuse
within the SC. Experiments on drugs with different molecular weights evidenced that the
optimal permeability is achieved with molar mass lower than 500 Da (Bos and Meinardi,
2000) while the upper limit for drug ability to penetrate the skin is regarded to be 5000 Da
(Schaefer and Riedelmayer, 1996).
In Table 1, calculated partition coefficients (Log P) (www.chemspider.com) and
molecular weights of some of the most commonly used UV-filters are reported. All these
sunscreen agents show molecular weights lower that 500 Da, suggesting that they would be
able to penetrate the skin. However, penetration of the most lipophilic and the most
hydrophilic UV-filters is likely to be minor as they should accumulate into the SC or remain
on the skin surface, respectively.
The importance of partition coefficients and molecular weights in the structure-skin
permeability relationships has been widely reported (Hadgraft and Lane, 2005; Patel et al.,
2002). In 1992, Potts and Guy made the first attempt at predicting skin permeability from Log
P and molecular weights, calculating the best linear fit for a data set of 93 compounds. The
regression coefficient (0.67) obtained from this data set points at a non-linear relationship,
with the involvement of other physicochemical features of the permeating molecule.
In the same year, Watkinson et al. (1992) developed a mathematical model to predict the
extent of percutaneous absorption of sunscreen agents based on their physicochemical
properties. In this model, some of the most common UV-filters such as benzophenone-3 (BP-
3), octyl methoxycinnamate (OMC), butyl methoxydibenzoylmethane (BMBM), octyl
salicylate (OS), octocrylene (OC), octyl dimethylPABA (OPABA), were analyzed. A rate
constant was assigned to each process involved in skin absorption (partitioning of UV-filter
from the vehicle to the SC; diffusion across the SC; partitioning from the SC to the viable
epidermis; uptake into local circulation and elimination; back partitioning from the viable
epidermis to the SC and from the SC to the vehicle). Rate constants were calculated
considering molecular weight, melting point, and calculated partition coefficients of the UV-
filters. Based on this model, sunscreen absorption ranging from 0.0033 to 83 mg/1.4m2 after a
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1526 Lucia Montenegro

12-hour application was predicted, indicating a potential significant uptake in the systemic
circulation after application to a large surface area for prolonged periods. However, this
model did not account for variables such as effect of the vehicle, repeated application, skin
metabolism of UV-filters or binding to skin sites, evidencing the need for well-designed in
vivo and in vitro studies to perform an accurate evaluation of UV-filter skin absorption, and of
the influence of physiological and formulation factors.

Table 1. Physicochemical properties, molecular weight (MW), partition coefficient (Log

P), water solubility (Sw), UV absorption, maximum concentration allowed (Max conc.),
of commonly used UV-filters

UV-filter Key CAS Max Physical Absorptiona MW Log Sw

conc. form Pb (mg/L)
Octyl OMC 5466- 10 Oily liquid UVB 290.41 5.66 0.15
methoxycinnamate 77-3
Buthyl methoxydi- BMBM 70356- 5 Crystalline UVA 310.39 4.81 1.52
benzoylmethane 09-1 solid
Octyl saliclylate OS 118- 5 Oily liquid UVB 250.34 5.95 <0.1
60-5
Benzophenone-3 BP-3 131- 6 Crystalline UVB- 228.25 3.64 68.56
57-7 solid UVAII
Benzophenone-4 BP-4 4065- 5 Crystalline UVB- 308.31 0.89 25x104
45-6 solid UVAII
4- BC 36861- 4 Crystalline UVB 254.37 4.95 0.57
Methylbenzilydene 47-9 solid
camphor
Octyl dimethyl OPABA 58817- 8 Oily liquid UVB 277.41 6.15 0.20
PABA 05-3
Octocrylene OC 6197- 10 Oily liquid UVB 361.48 7.53 <0.1
30-4
a
UVB (290– 320 nm); UVAII (320– 340 nm); UVA (320– 400 nm).
b
Calculated from www.chemspider.com.

As the main barrier to skin penetration is the non-viable SC, a drug applied in a vehicle
on the skin surface penetrates into the skin by a passive mechanism depending on its
physicochemical properties and according to Fick’s First Law (equation 1).

Equation 1

Equation 1 shows that the flux (rate of transfer per unit area of a chemical at a given time
and position) is proportional to the differential concentration change δC over the differential
distance δx. Therefore, the driving force of the percutaneous absorption process is the
concentration gradient of the active compound between the vehicle and the SC. In Equation 1,
D represents the diffusion coefficient and the negative sign indicates that the flow is in the
direction of decreasing concentration.
When the flux is constant (steady state), it can be calculated from equation 2:

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 Assessment of Sunscreen Safety by Skin Permeation Studies 1527

Equation 2

Jss represents flux at steady state; C1 and C2 represent the concentration of the permeating
molecule in the vehicle and in the SC, respectively; L is the length of the diffusion pathway in
the SC.
C1 depends on the partition coefficient of the active ingredient between the SC and
vehicle (considered equal to Log P for topical formulations) (Roberts et al., 1996) and on the
concentration of the ingredient in the formulation.
Therefore, the extent of skin penetration and/or permeation depends not only on the
physicochemical properties of the active ingredient but also on the vehicle in which it is
formulated and on the interactions between the vehicle and the skin. After topical application
of a pharmaceutical or cosmetic product onto the skin, the first critical step that determines
the skin absorption level of an active ingredient is its release from the formulation and
partitioning between the vehicle and the SC. The vehicle could alter the partitioning of the
compound into the SC lipids and the solubility of the active ingredient in the vehicle will
determine the thermodynamic driving force governing release of the chemical from the
formulation to the skin. In addition, the vehicle itself could modify skin permeability, thus
affecting skin penetration of the active compound.
Additional factors that may affect percutaneous absorption include the way of application
of the formulation onto the skin, the presence of penetration modifiers in the vehicle
(Trommer and Neubert, 2006), which could alter the barrier properties of the SC, the
processes occurring in viable tissues such as metabolism, and biological factors.

SKIN PERMEATION OF UV-FILTERS

Skin permeation can be determined using different methodologies performing a) in vivo
studies in animals or human volunteers, b) in vitro experiments employing excised skin from
human or animal sources, or synthetic model membranes (Godin and Touitou, 2007; Rai et
al., 2010; Varvaresou, 2006).
Because percutaneous absorption depends on the passive diffusion of the permeating
molecule and not on an active transport, the viability of the skin is not a prerequisite for
penetration testing, making the results of in vitro studies predictive of in vivo permeation
(Bronaugh et al., 1982a; Franz, 1975).
Although in vitro tests are preferable for ethical reasons and feasibility and are well
accepted in the European Union, regulatory authorities in the United States and Japan suggest
in vivo penetration studies in a rodent model (EPA 1992).
Skin from a great variety of animals (e.g., pigs and guinea pigs, rats, rabbits, snakes) has
been investigated as a model for human skin (Bartek et al., 1972; Bronaugh et al., 1982b;
Chow et al., 1978; Harada et al., 1993; Itoh et al., 1990; Lin et al., 1992). Pigs and rats show
the most similar skin barrier to that of human skin and therefore are most commonly used for
skin penetration/permeation studies. However, due to the complex nature of the human SC,
animal skin provides only an indication of the diffusion characteristics of chemicals, requiring
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a careful extrapolation to humans. In the following sections, in vitro and in vivo evaluations
of percutaneous absorption through human or animal skin of the most commonly used UV-
filters will be illustrated.

Human Studies

The most widely used sunscreen active ingredients such as octylmethoxycinnamate

(OMC), benzophenone-3 (BP-3), benzophenone-4 (BP-4), octylsalicylate (OS), octyl
dimethylPABA (OPABA), butyl methoxydibenzoylmethane (BMBM), octocrylene (OC), 3-
(4-methylbenzylidene) camphor (BC), have been extensively investigated in vitro and in vivo
in humans to assess their retention into the different skin layers and their permeation through
the skin into the systemic circulation, applying representative vehicles of cosmetic
formulations.
In vivo skin permeation of OMC, BP-3, and BC was evaluated on 32 healthy volunteers
(15 men and 17 postmenopausal women) following daily whole-body topical application of a
cream (2 mg/cm2) without (week 1) or with (week 2) the three sunscreens, each at 10% w/w
(Janjua et al., 2004). At the end of the study, all the sunscreens were detected in urine.
Maximum plasma concentrations were similar for OMC (10 ng/mL in females and 20 ng/mL
in males) and BC (20 ng/mL both in females and males) while they were significantly higher
for BP-3 (200 ng/mL in females and 300 ng/mL in males). The authors observed minor
differences in serum estradiol and inhibin B levels in men only but they concluded that these
differences in hormone levels were not related to sunscreen exposure.
An interesting study on the relationship between in vitro skin penetration and cytotoxicity
of sunscreens was performed to ensure that in vitro cytotoxicity studies examined relevant
doses of these agents to which viable epidermal cells were realistically exposed. With this
aim, in vitro percutaneous absorption through human skin of five commonly used sunscreen
agents (BMBM, OMC, OC, BP-3 and OPABA) was assessed from mineral oil. After 24
hours, 95-98% of the sunscreen agents was recovered on the surface of the epidermis and
detectable amounts of all UV-filters were found in the SC and viable epidermis. BP-3 showed
the most evident epidermal penetration. After determining sunscreen concentration-human
keratinocyte culture response curves, the authors concluded that the concentrations of each
sunscreen found in human viable epidermis after topical application were at least 5-fold lower
than those appearing to cause toxicity in cultured human keratinocytes (Hayden et al., 2005).
Due to its high photo-stability, BP-3 is widely used in commercial sunscreen products.
However, several studies evidenced a higher skin permeation ability of this UV-filter
compared to other commonly used sunscreens. BP-3 percutaneous absorption was evaluated
in nine healthy volunteers after topical application (12.4 mg/cm2) onto the entire surface of
their forearms of a commercial available sunscreen lotion containing BP-3 6% (w/v), OMC
7.5% (w/v), OS 5% (w/v) and OC 7% (w/v) (Hayden et al., 1997). BP-3 was recovered in
urine for 48 h as unchanged BP-3 and as metabolites and the authors concluded that the actual
amount absorbed through the skin over a 10-h period was 1-2% of the applied amount
contained in the product. However, in this study, the amount of formulation applied on the
skin was about six-fold higher than that recommended to evaluate the sun protection factor of
sunscreen products under realistic condition of use (2 mg/cm2).

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 Assessment of Sunscreen Safety by Skin Permeation Studies 1529

An in vitro study through excised human skin was performed to assess skin
penetration/permeation from six commercial milk/lotion sunscreen products for adults and
children of BP-3, along with other UVB-filters (OMC, OS, OC) (Jiang et al., 1999). All these
sunscreens were recovered into the skin (14% of the applied dose) but only BP-3 was able to
permeate through the skin (10% of the applied dose), thus confirming the results obtained
from previous in vivo studies.
A further in vivo investigation on BP-3 skin permeation was carried out on eleven healthy
volunteers who were instructed to apply a commercially available sun-protecting lotion
containing 4% BP-3 over the whole body (2 mg/cm2). Under these experimental conditions,
the average total amount excreted in urine was approximately 0.4% of the applied amount of
BP-3 (Gustavsson Gonzalez et al., 2002).
In vitro studies through human skin proved that BP-3 skin permeation was enhanced by
the concomitant application of the insect repellent N,N-diethyl-m-toluamide (DEET) (Wang
and Gu, 2007). DEET permeation was greater than that of BP-3 and a synergistic
percutaneous enhancement of both compounds was observed when used simultaneously. The
authors reported that the extent of the enhancement effect was dependent on the type of
vehicle (lotion or spay) containing the active ingredients.
Synergy effects on in vitro absorption through human skin between UV filters and other
compounds such as herbicides (Pont et al., 2004) have also been reported.
Vehicle effects on skin permeation of UV-filters have been investigated in many papers.
Chatelain et al. (2003) evaluated the skin penetration of five UV- filters (BP-3, OMC,
BMBM, OS, homosalate) from an O/W emulsion gel and a petrolatum jelly both in vitro and
in vivo. The results of in vitro experiments showed that after applying the formulations under
investigation for 30 min and 6 h, only BP-3 and to a lesser extent OMC were detectable in the
dermis while no skin permeation of these UV filters was observed after 6 h application. In
addition, the authors reported a greater BP-3 penetration from petrolatum. In vivo
investigations, performed by tape stripping of the SC, evidenced a clear vehicle effect on
penetration of the UV-filters into the SC, being the amount of each UV-filter penetrated
greater from the emulsion gel formulation compared to the petrolatum jelly. Treffel and
Gabard (1996) reported similar results studying in vitro and in vivo skin penetration of BP-3
(5%), OMC (7.5%), and OS (3%) from two vehicles (an O/W emulsion-gel and petroleum
jelly) applied (2 mg/cm2) for 2 min to 6 h. The emulsion-gel provided higher epidermal
concentrations of all UV-filters than the petroleum jelly both in vivo and in vitro. However,
the authors noticed only for BP-3 a greater skin permeation from the petroleum jelly than
from the emulsion gel.
The effects of emulsion composition on in vitro skin permeation of different UV-filters
(OMC, BMBM) was evaluated evidencing differences in UV filter penetration/permeation
among various emulsion-type formulations (Marginean-Lazar et al., 1996; Montenegro et al.,
2004).
In vitro percutaneous absorption through human skin of OS was assessed from two
representative sunscreen vehicles (an oil-in-water emulsion and a hydro-alcoholic
formulation) using both the technique of finite and infinite dosing (Walters et al., 1997). The
results of this study evidenced a low skin permeation of OS that was dependent on the
amount of formulation applied (finite or infinite dosing) when a hydro-alcoholic vehicle was
used.
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Animal Studies

Several studies have validated pig skin as a suitable model for human skin to investigate
percutaneous absorption of UV-filters.
Benech-Kieffer et al. (2000) performed in vitro experiments to compare skin permeation
of two UV-filters (OMC and BP-4) through human abdominal skin and pig flank skin. The
authors found a good agreement between data obtained from human and pig skin, as after 16
hours the percentage of the applied dose of OMC and BP-4 recovered in the skin was similar
for both species. However, due to its lipophilicity, OMC showed a higher affinity for the SC
than the hydrophilic BP-4.
Pigskin was used as model to evaluate BP-3 and OMC in vitro percutaneous absorption
from two vehicles, a hydroacloholic and a diisopropyl adipate formulation (Gupta et al.,
1999). BP-3 permeation was greater than that of OMC while OMC was retained to a greater
extent in the SC. It is interesting to note that the authors found that permeation and SC
retention were formulation dependent and the ratio of retained to permeated amount of
sunscreen from a hydroalcoholic formulation after 10 hours was greater when these UV-
filters were present together rather than alone.
Fernandez et al. (2000) evaluated the skin penetration of BP-3 in vitro and in vivo from
six different vehicles, three solvents and three different types of emulsions. In vitro
experiments were performed using pig skin while in vivo data were collected determining BP-
3 concentration in the SC by the stripping method after 30-min application on the forearm of
volunteers. Pig and human skin provided similar results, highlighting a good correlation
between in vitro and in vivo data and between species. In both experiments, significant
differences among the vehicles tested were observed as the highest concentration of BP-3 in
the skin was obtained from the hydrophilic solvent (propylene glycol) and O/W submicron
emulsion while the two oily solvents, W/O emulsion and O/W coarse emulsion provided
lower concentrations of this UV-filter in the skin.
Another in vitro study on OMC percutaneous absorption through pig skin evidenced that
microencapsulating this UV-filter reduced its absorption in comparison with an emulsion
containing free OMC (Jimenez et al., 2004).
The effect two vehicles, an oil-in-water (O/W) emulsion and an alcoholic gel, on in vitro
permeation of BC was assessed through pig ear skin. The results of this study showed that BC
skin penetration was dependent on the vehicle, being more remarkable for alcoholic gel
(Sasson et al., 2009).
In the last decade, several studies aimed at entrapping UV-filters in carriers such as
cyclodextrins (Shokri et al., 2013), lipid microparticles (Scalia et al., 2007) lipospheres (Mew
et al., 2007), lipid microspheres (Mestres et al., 2010; Yener et al., 2003), polymeric
nanocapsules (Siqueira et al., 2011; Weiss-Angeli et al., 2010), microemulsions (Montenegro
et al., 2011), solid lipid nanoparticles (Gulbake et al., 2010; Wissing and Muller, 2002),
polymeric nanoparticles (Vettor et al., 2010) to avoid or at least to reduce skin permeation of
sunscreens. Some of these investigations were performed in vitro or in vivo on rat skin,
pointing out a good correlation between this animal model and human skin. In all these
studies, incorporating UV-filters in different carriers resulted in a decrease of sunscreen
release and penetration into the skin compared to conventional cosmetic formulations.

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 Assessment of Sunscreen Safety by Skin Permeation Studies 1531

NOAEL AND RISK ASSESSMENT OF UV-FILTERS

The risk assessment of sunscreen products requires hazard identification and
characterization, exposure assessment, and risk characterization, the last being a basic step
that brings together hazard characterization and exposure assessment. For sunscreen products,
such evaluations have to be performed taking into account the topical nature of human
exposure.
While hazard identification and characterization lead to a qualitative assessment based on
the available data of a specific chemical, safety evaluation provides a quantitative estimate of
the intake that would not give rise to a significant risk of adverse effects in humans.
Therefore, the margin of safety (MoS) is determined comparing the so-called no adverse
effect level (NOAEL) to the potential human exposure, expressed as systemic exposure dose
(SED).
NOAEL values are obtained from subchronic (90-180 days) or chronic (> 180 days)
toxicity studies that are generally performed in animals, using different oral dosing. NOAEL
is usually defined as the daily dose that does not elicit any toxic effect and is expressed in
mg/kg body weight. However, the definition of NOAEL has been largely debated (Dorato and
Engelhardt, 2005). According to the SCCS's notes of guidance for the testing of cosmetic
substances and their safety evaluation (2012), the no observed (adverse) effect level is defined
as the highest dose or exposure level where no (adverse) treatment-related findings are
observed.
After establishing the NOAEL of UV-filters, their percutaneous absorption has to be
quantified to determine SED, expressed in mg/kg body weight per single application. As
sunscreen products are often applied to large skin areas, SED evaluations are performed
considering the worst scenario, i.e., a total body application.
If the steady state flux through the skin (Jss) is used to estimate SED, the weight and the
skin surface area of a standard human are needed. According to SCCS's notes of guidance for
the testing of cosmetic substances and their safety evaluation (2012), a weight of 60 kg and a
skin area of 18.000 cm2 (Timbrell, 2005) are used to calculate SED.
Therefore, the SED can be determined as follows:

SED = Jss × areaexp × 24 h /body weight Equation 3

where areaexp is the exposed area of the skin. In addition, the SED can be estimated from the
percentage of a topically applied dose that permeates the skin, according to equation 4:

SED = Cproduct ×Aapplied × napplications × areaexp× Perm(%)/body weight Equation 4

where Cproduct is the concentration of UV-filter in the product (%w/w), Aapplied is the amount of
product applied on the skin (mg/cm2), napplications is the number of application per day and
Perm(%) is the permeation percentage of UV-filter.
As many toxicological studies are carried out on animals, uncertainty factors (UF) have
to be applied to convert animal data into an exposure level considered of no toxicological
concern for humans; additional UF have to be considered to account for toxico-kinetic
variability among healthy adults and in children (SCCS, 2012).
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When NOAEL and SED are known, MoS can be calculated as follows:

MoS = NOAEL/SED Equation 5

A MoS < 1 indicates a potential risk associated with a given scenario as the dose
considered safe is lower than the SED. In the safety evaluation of cosmetic products in EU, to
declare a product safe for use, MoS values greater than 100 are required (SCCS, 2012).
Several authors (Gonzalez 2010; Montenegro et al., 2013; Nohynek and Schaefer, 2001;
Walters et al., 1997) have calculated MoS of UV-filters using SED values obtained from
percutaneous absorption studies.
As BP-3 shows a greater skin permeation compared to other UV-filters, an extensive
assessment of its safety has been carried out (El Dareer et al., 1986; Okereke et al., 1994).
Okereke et al., (1995) performed a safety evaluation of BP-3 after topical application in rats
and found it nontoxic when applied at a dose of 100 mg ⁄ kg body weight for 4 weeks. After
evaluating in vitro and in vivo data on BP-3 skin permeation in animals and humans and the
available toxicokinetic data, the Scientific Committee on Consumer Products (SCCP, 2008)
concluded that the use of BP-3 up to 6% in cosmetic sunscreen products does not pose a risk
to the health of the consumer, apart from its contact allergenic and photoallergenic potential.
This conclusion was drawn considering that the mean percentage of the applied dose of BP- 3
permeated was 3.1% plus 2 standard deviations (3.4%). As shown in Table 2, in this
condition a MoS value of 112 was obtained.
OMC and BMBM are the most commonly used UV-filters, being contained not only in
sunscreen products but also in a great variety of cosmetics and toiletries. Although OMC
demonstrated weak estrogenic effects in vivo and in vitro (Gomez et al., 2005; Klammer et
al., 2005), it had no adverse effect on estrus cycle, sperm number, morphology and motility,
differential follicle counts, mating, fertility, gestation and parturition (Schneider et al., 2005).
The NOAEL for fertility and reproductive performance in rats and for systemic parental and
developmental toxicity was determined to be 450 mg/kg/day. The same NOAEL has been
reported for BMBM (Montenegro et al., 2013)
In vitro experiments showed that the percentage of the applied dose of OMC that
permeated through the skin was 0.2–4.5%, using both human and porcine skin (Benech-
Kieffer et al., 2000; Gupta et al., 1999). From in vivo studies, lower OMC skin permeation
was observed (Chatelain et al., 2003; Janjua et al., 2004; Janjua et al., 2008).
As regards BMBM, in vitro studies evidenced that less than 1% of this UV-filter
penetrated into the SC and epidermis while no skin permeation occurred (Montenegro et al.,
2008; Simeoni et al., 2004; Weigmann et al., 2001).
Recently, Nohynek et al. (2010) have pointed out that in vitro experiments tend to
overestimate human systemic exposure. According to the authors, this overestimation was
supported by the results of a study performed in humans, both in vivo and in vitro, on the UV-
filter Mexoryl SX®. In volunteers, 0.014% of the applied filter was systemically available,
while parallel in vitro experiments, performed under identical exposure conditions, provided a
skin penetration rate of 0.37%, suggesting that in vitro results produced a 25-fold
overestimation of the human systemic exposure and of the potential human health risk
(Benech-Kieffer et al., 2003).

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 Assessment of Sunscreen Safety by Skin Permeation Studies 1533

Table 2. Systemic exposure dose (SED) and margin of safety (MoS)

of UV-filters in adults

Parameter UV-filter
BP-3a OMCb BC-3c BC-4d OSe BMBMb Mexoryl
XL (®)f
Typical adult body weight (Kg) 60 60 60 60 60 60 60
Body surface area (cm2) 18000 18000 18000 18000 18000 18000 18000
Average amount (g) of sunscreen 18 36 18 36 16 36 18
applied per day
Maximum allowable 6 10 2 4 5 5 15
concentration in sunscreen
products (%)
Maximum amount applied 1.1 3.6 0.36 1.4 0.8 1.8 1.8
ultraviolet filter (g)
Maximum (in vitro) percutaneous 9.9 4.5 3.29 2.45 0.65 1 0.8
absorption of UV filter (%)
Systemic Exposure Dose (SED) 1.78 0.27 0.21 0.588 0.087 0.03 0.24
(mg/kg/day)
NOAEL of toxicity studies 200 450 7.5 25 250 450 1000
(mg/kg/day)
Margin of Safety 112 1666 36 42.5 2900 15000 4170
(MoS=NOAEL/SED)
a
data obtained from Gonzalez (2010).
b
data calculated from in vitro and in vivo experiments reported in the literature (see text for details).
c
data obtained from SCCS/1513/13 (2013).
d
data obtained from SCCP (2008).
e
data obtained from Walters et al. (1997).
f
data obtained from Nohynek and Schaefer (2001).

However, as according to SCCS (2012) the worst scenario has to be considered,

calculating MoS of OMC and BMBM, the maximum percentage of the applied dose
permeated (4.5% and 1%, respectively) was used (see Table 2). Based on a total body
application of a formulation containing the maximum percentage of UV-filter allowed, the
resulting MoS for OMC and BMBM were 1666 and 15000. A recent investigation on the
effects of commercial O/W emulsions on in vitro skin permeation of OMC and BMBM
evidenced that OMC permeation depended on both its concentration in the formulation and
vehicle composition, while BMBM release from the vehicle was the key parameter that
determined the permeation rate of this UV-filter (Montenegro et al., 2013). All the
commercial products investigated proved safe under normal in use conditions as their MoS
values were greater than 100.
Being one of the oldest UV-filters, the human safety of OS has been extensively
reviewed, highlighting that this UV-filter is well tolerated (Nash, 2006). Investigating OS
skin permeation, Walters et al., (1997) reported that less than 1% of the applied dose of OS
penetrates through the human skin. Using these data, the authors demonstrated the safety of
this sunscreen agent in cosmetic formulations.
Nohynek and Schaefer (2001) reported a thorough risk assessment of a new broad UVA
filter, drometrizole trisiloxane (Mexoryl XL®), listing all the data needed to calculate its
MoS, according to SCCS guidelines.
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As illustrated In Table 2, apart from 3-benzilydene camphor (BC-3) and 4-

methylbenzilydene camphor (BC-4), all the UV-filters mentioned above show MoS values
greater than 100, evidencing their safety of use in sunscreen products at the maximum
concentration allowed in EU.
As regards BC-3, Søeborg et al. (2006), studying in vivo skin permeation of BC-3 in rats,
observed an accumulation of approximately 10% of the applied topical dose in various tissues
after 65 days of treatment. Further studies (Søeborg et al., 2007) pointed out that skin
permeation of BC-3 was higher than that of BC-4. As both UV-filters affected the endocrine
activity, the risk associated with use of BC-3 and BC-4 in sunscreen products was found high.
To evaluate the risk of human exposure to BC-3, the SCCP (2013) used a percentage of
absorption of 3.29% and a NOAEL value of 7.5 mg/Kg/day obtained from maternal toxicity
and embryo-toxicity studies in rats (15 mg/Kg/day), taking into account that BC-3
bioavailability was 50% of the administered dose. Based on the resulting MoS lower than
100, the SCCP declared that the use of BC-3 in cosmetic products in a concentration up 2.0%
is not safe. Therefore, BC-3 has been withdrawn from the list of UV-filters approved in EU.
Using a percutaneous absorption rate of 1.9% along with a NOAEL of 25 mg/Kg/day
(determined in rats) for human risk assessment of BC-4 (SCCP, 2008), a MoS below 100 was
obtained, indicating that BC-4 cannot be considered safe as a UV filter in cosmetic sunscreen
products at 4%. However, as detailed toxicokinetic studies in healthy volunteers demonstrated
that the actual NOAEL in humans was 100 mg/Kg/day, a MoS value of 25 was considered
safe for BC-4. Therefore, as the calculation of the MoS resulted in a value of 42.5, which was
higher than the requested threshold of 25, the use of this UV-filter was regarded safe (SCCP,
2008).

CONCLUSION
In recent years, an increasing attention has been paid to the evaluation of UV-filter
percutaneous absorption to assess the potential risk of human exposure to these sunscreen
agents.
In vitro permeation experiments and animal models, with all their limitations, provide
important tools for screening sunscreen products, making possible to estimate the rate and
extent of percutaneous absorption of their active ingredients.
The safety evaluation of sunscreen products is based on the two factors that contribute to
risk characterization, hazard characterization and potential human exposure and only UV-
filters whose MoS are at least 100 times the NOAEL are accepted as safe.
As evidenced in this chapter, when comparing the toxicological properties of the most
commonly used UV-A and UV-B filters to the potential human exposure, the human risk
caused by exposure to sunscreen products is negligible. Furthermore, modern UV filters are
formulated to be retained on or within the upper layers of the horny layer to achieve high
protection factors, thus reducing their potential of skin permeation into the underlying living
tissue.
Therefore, the benefits of sunscreen products to public health largely outweigh the risk of
human topical exposure to such formulations, supporting the recommendation of a proper use
of sunscreen products to prevent the deleterious effects of UV-radiation.

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 70

UV PROTECTION BY WOOLEN FABRIC DYED

WITH NATURAL DYESTUFF

Ana Sutlović, Anita Tarbuk*, Ana Marija Grancarić

and Đurđica Parac-Osterman
University of Zagreb, Faculty of Textile Technology,
Department of Textile Chemistry and Ecology, Zagreb, Croatia

ABSTRACT
The UV protection by textiles highly depends on large number of factors such are
type of fiber, fabric surface, construction, porosity, density, moisture content, type and
concentration of dyestuff, fluorescent whitening agents (FWA), UV-B protective agents
(UV absorbers), as well as nanoparticles, if applied. The dyes are selective absorbers.
They all absorb visible light, but some absorb light in the near ultraviolet region, as well.
Even though synthetic dyes are cheaper, their usage led to such consequences as
carcinogenicity and some of them are toxic to the environment. Due to increased
awareness of the environmental and health hazards associated with the synthesis,
processing and use of synthetic dyes, most of the commercial dyers have started to re-
looking to the maximum possibilities of using natural dyes for dyeing and printing of
different textiles for targeting niche market. Natural dyes are usually derived from the
plants, animal and mineral sources. The shades produced by natural dyes are usually soft,
lustrous and soothing to the human eye, can be produced a wide range of colors by mix
and match system and are usually renewable and biodegradable. However, it needs longer
dyeing time and excess cost for mordants and mordanting. Applied on textiles, provide
some UV blocking which depends on the structure of dye molecules, type of dye or
pigment, present absorptive groups, depth of dyeing and the uniformity. According to
colour physic principles, darker colors (e.g., black, navy blue and dark red) absorb UV-R
much more strongly than light pastel colors. For that reason, in this chapter the UV
protection by woolen fabric dyed with natural dyestuff extracted from European Ash bark
(Fraxinus excelsior) and European black elderberry berries (Sambucus nigra) was
researched. Since these natural dyes, as most of the natural dyes, are non-substantive and
must be applied on textiles in the combination with mordants i.e., metallic salts, 4

*
Corresponding address: Prilaz baruna Filipovića 28a, HR-10000 Zagreb, Croatia; E-mail: [email protected].
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1542 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

different mordants were applied. The color parameters were measured on remission
spectrophotometer. The fabric UV protection was determined according to AS/NZS
4399:1996 Sun Protective Clothing: evaluation and classification, by UV-A and UV-B
transmission measurement on transmission spectrophotometer and calculation of
Ultraviolet protection factor (UPF).

Keywords: Wool, UV protection, natural dyestuff, Sambucus nigra, Fraxinus excelsior

INTRODUCTION
The application of a number of synthetic dyes is associated with allergies, toxic and
carcinogenic effect as well as impact on the environment. Due to increased awareness of the
environmental and health hazards associated with the synthesis, processing and use of
synthetic dyes, most of the commercial dyers have started to re-looking to the maximum
possibilities of using natural dyes for dyeing and printing of different textiles for targeting
niche market [1-6]. Natural dyes are usually derived from the plants, animal/insects and
mineral sources, and can be classified according to dyeing properties, chemical structure,
origin, hue or application area 1-6. In regards to dyeing properties, most natural dyes can be
sorted into group of mordant dyes; some can be classified as vat, while a small number of
natural dyes belong to groups of direct and basic dyes 6-13.

Table 1. Review of main groups of natural dyes according hues [6, 12, 13, 26, 27]

Name Colour Index Source Mordant Main colouring

matters
Red colour hues
Alkanet root Natural Red 20 roots
Al alkannin
(Anchusa tinctoria L.)
Henna Natural Orange leaves
Al lawsone
(Lawsonia inrmis L.) 6
Kermes Natrual Red 3 insect Al, Sn, Cu, kermesic and
(Coccus ilicis L.) Fe flavokermesic acid
Cochineal Natural Red 4 insect Al, Sn, Cu,
carminic acid
(Coccus cacti L.) Fe
Madder Natural Red 8 root alizarin,
(Rubia tinctorum L.) pseudopurpurin,
Al, Fe, Sn
purpurin,
xanthopurpurin
Brazilwood Natural Red 24 wood
Al, Fe, Sn brazilin, brazilein
(Caesalpinia sappan L.)
Yellow colour hues
Tree of Sorrow Natural Yellow
flower Al crocetin
(Nyctanthes arbor-tristis L.) 19
flowers
Chamomile Natural Yellow luteolin, palulitrin,
and Sn, Al
(Chamaemelum recutica L.) 1 rutin
leaves
Jasmine Natural Yellow
fruit Sn, Al crocetin
(Gardenia jasminoides L.) 6

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1543

Main colouring
Name Colour Index Source Mordant
matters
Saffron Natural Yellow
stigmas Al crocetin
(Crocus sativus) 6
gallotannins,
Pomegranate Natural Yellow
fruit Al anthocyanins,
(Punica granatum) 3
betalains
Curcuma Natural Yellow
root Al, Cu curcumin
(Curcuma longa) 3
Marygold Natural Yellow
flower Al rubixanthin
(Calendula officinalis L.) 27
Ashtree Natural Yellow quercitrin,
bark Sn, Al
(Fraxinus excelsior) 10 isoquercitrin
Spanish broom Natural Yellow
flower Al, Cu isoflavon, genistin
(Spartium junceum L.) 2
Blue colour hues
Logwood (Haematoxylum heartwoo Al, Sn, Cu, haematoxylin,
Natural Black 1
campenhianum L.) d Fe haematein
Indigo indigotin,
Natural Blue 1 leaves -
(Indigofera tinctoria L.) indirubin
Purple colour hues
6,6-
Tyrian purple dibromoindigotin,
(Murex brandraris L. and Natural Violet 1 shellfish - 6,6-
Murex trunculus L.) dibromoindirubin,
6-bromoindigotin
Brown colour hues
green
Walnut-tree
Natural Brown 7 scale or Fe juglone
(Junglans regia)
leaves
Sicilian Sumac chebulinic acid -
Natural Brown 6 leaves Fe
(Ruhus coriaria L.) eutannin

Complexing with metal salts, mordant dyes give different colorations. Aluminum,
copper, iron and tin salts are most usually used mordants. Mordants are usually applied in
protein fiber dyeing as mordant pre-treatment of fibres (prior to dyeing); during the process of
dyeing, or mordant after-treatment. The most common source of these dyestuffs are madder
(Rubia tinctorum), cochenil bug (Dactylopius coccus), as well as herbs from which most
widely used mordant dyes are obtained – flavonoid dyes 14-19. Second important group of
natural textile dyes are in water insoluble vat dyes, which have to be transformed into soluble
form with the addition of reduction agent and alkali. Most commonly known representatives
of this group are indigo and purple 6,6'-dibromindigo dye obtained from murex sea snail
(Murex brandaris and Murex trunculus) 7, 20-23. Among most important natural direct
dyes are turmeric or yellow root (Curcuma longa) and powderd bark or roots of common
barberry (Berberis vulgaris) 7, 24, 25. Review of main groups of natural dyes
[6,12,13,26,27] is shown in Table 1. Some other plants commonly used as natural dyes are:
Dyer’s woad (Isatis tinctoria L.), Lipsticktree (Bixa orellana L.), Brasilwood (Caesalpina
brasiliensis L.), Weld (Reseda luteola L.), Juniper (Juniperus Communis); Elder (Sambucus
Nigra), Oak (Quercus Aegilops), Bramble (Rubus fruticosus), Nettle (Urtica dioica), St. John
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1544 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

Wart (Hypericum perforatum), Hibiscus (Hibiscus L.), Oak (Quercus Aegilops), Bottlebrush
(Callistemon citrinus), Eucalyptus (E. Camaldulensis), etc. [1-37].
When classifying natural dyes according to color hue, the type and process of mordant
should be regarded. Only a few natural dyes are substantive (direct and vat dyes), whilst all
other require inorganic oxides or salts – metal salts (mordant dyes). Division of natural dyes
according to chemical constitution is in accordance to botanical nomenclature. Most
important chemical groups of natural dyes are: carationoide, diaril-methane, benzoquinone,
anthraquinone, indigoide, flavonoide, anthociane, betalaine, neoflavonoide, basic, alcaloide,
benzofenon, galotannine, tannins, chlorophyll, natural pigments, etc. 7,8. Flavonoids and
flavonoid derivatives are most represented compounds in watery herbal extracts [38-42]. In
regards to dyeing properties flavonoids belong to a group of mordant dyes. Most commonly
used dyeing method uses a pretreatment process in which wool is mordanted in watery
solution of metal salts, followed by a process of dyeing in the solution of watery extracted
natural dyes 7, 10-13, 38-41. The combination dye-metal salt has a strong infuence on the
color hue and color fastness properties 10. Depending on the structure of flavonoid
derivative metal complexes 1:1 or 1:2 may be formed 38 (Figure 1).

Figure 1. Tentative structures of the complexes: (A) quercetin complexes (1:1); (B) rutin complexes
(1:1 and 1:2). x =2 for M = Cu (II); x = 4 for M = Fe(II) [42].

Natural dyes represent renewable and sustainable bioresource products with minimum
environmental impact and as such are a potential ‘Green chemistry’ option as an
alternative/co-partner to some extent to synthetic dyes [43-45]. The shades produced by
natural dyes are usually soft, lustrous and soothing to the human eye, can be produced a wide
range of colors by mix and match system and are usually renewable and biodegradable.
However, it needs longer dyeing time and excess cost for mordants and mordanting. There are

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1545

some issues yet to be solved regard optimization of the amount and contents of natural
extracts, increase of dye exhaustion to material, increase in colour fastness, selection and
optimization of mordants used. Although, water is the most usual solvent used to prepare
natural extract it is being substituted with another solvent (ethanol, methanol) in the aim of
increasing the overall amount of the extract, isolation of certain extract components or simply
to remove unwanted impurities such as waxes and lipids from watery extracts 46-49.
During the last few decades, increasing attention has been paid by researchers to various
aspects of natural dye applications. A large number of plant and animal/insect sources have
been identified for extraction of color [2,3] and their diversified use in textile dyeing [9, 29]
and functional finishing [30, 31, 50-56], food coloration, cosmetics, and other application
disciplines [5]. Natural dyes in functional finishing of textiles has become as a result of a
careful balance between compatibility of different finishing products and treatments and the
application processes used to provide eco-friendly textiles with desirable properties, such as
antimicrobial [30, 31, 43, 49-56], insect repellent, deodorizing and UV-protective [54-64]
properties. Several new techniques of modern innovative finishing have been added to dyeing
technologies: CO2, chitosan treatment [65], enzymatic treatment [66, 67], plasma treatment
[68, 69], cationization [70-72], microencapsulation and cross-linking [73], etc. for
enhancement of protective properties of naturally dyed textile materials, e.g., antimicrobial
and UV protection.
The primary cause of skin cancer is believed to be a long exposure to solar ultraviolet
(UV) radiation crossed with the amount of skin pigmentation in the population [74-76].
Intermittent sun exposure in childhood and adolescence is considered to be a stronger risk
factor for melanoma than continuous exposure. In addition to some beneficial effects of UV
radiation it may cause skin and eye damage, especially during the summer time. The proper
and early photoprotection may reduce the risk of subsequent occurrence of skin cancer [76].
Therefore, photoprotection is based on protection from UV-B (from 280 nm to 320 nm) and
UV-A (from 320 nm to 400 nm) radiation, which are reaching the Earth due to diminishing of
the ozone layer.
Textile and clothing show some UV protection, but in the most cases it does not provide
full sun screening properties. A good fabric UV protection depends on a large number of
factors, such as, the type of fiber, fabric surface and construction, porosity, density, moisture
content, type and concentration of dyestuff, fluorescent whitening agent (FWA), UV-B
protective agents, as well as nanoparticles, if applied [57, 77-91].
The dyestuffs are selective absorbers. They all absorb visible light, but some absorb light
in the near ultraviolet region, as well. Applied on textiles, provide some UV blocking which
depends on the structure of dye molecules, type of dye or pigment, present absorptive groups,
depth of dyeing and the uniformity. According to colour physic principles, darker colors (e.g.,
black, navy blue and dark red) absorb UV-R much more strongly than light pastel colors.
Therefore, dyed fabrics protect more than undyed ones and their protection levels rise with
the increase in dye concentration [57-60, 64, 77]. However, most of these results concern
synthetic dyes.
There are only few studies that focused on the UV protection properties of natural dyes.
Sarkar [57] characterized UV protection of plain, twill or sateen weave cotton fabrics dyed
with colorants of plant (madder and indigo) and insect (cochineal) origins. Feng et al. [58] in
an experiment conducted to evaluate UV protection properties of two natural dyes (Rheum
and Lithospermum erythrorhizon) applied on cotton and silk found that these natural dyes
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1546 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

exhibit a comparable UV-absorption performance to benzophenone. Results demonstrated

that UV-protective effect was strongly dependent on absorption characteristics of natural dyes
for UVR. Grifoni et al. [59, 60] studied the effect of color on UVR transmission of cotton,
flax, hemp and ramie fabrics with different construction parameters dyed with some common
natural dyes: dyer’s woad, madder, logwood, lipsticktree, brasilwood, weld and cochineal by
in vitro and outdoor assessments. Metallic salt mordants have been reported to enhance UV-
protective properties of naturally dyed cotton [55], wool and silk [61-63] fabrics to a
substantial account depending on nature of fibre, mordant and natural dye used. Recently,
Hou et al. [75] used natural dyes extracted from orange peel, an abundant, cheap and readily
available agricultural byproduct, for producing highly durable UV protective wool fabrics.
The authors could not found any research considering UV protection of fabric dyed with
European Ash bark (Fraxinus excelsior). On the other hand, there has been ne report
regarding European black elderberry berries (Sambucus nigra) in photoprotective UVA and
UVB; photostability in cosmetic emulsions [92].
For that reason, in this chapter the UV protection by woolen fabric dyed with natural
dyestuff extracted from European Ash bark (Fraxinus excelsior) and European black
elderberry berries (Sambucus nigra) was researched. Since these natural dyes are non-
substantive and must be applied on textiles in the combination with mordants i.e., metallic
salts, 4 different mordants were applied. Additionally, color parameters were measured.

EXPERIMENTAL
Materials

Fabric
Twill woven fabric of 100% wool fibers, having mass per unit area of 162 g/m2 was used
for this research. Warp in the fabric was 30 tex, of density 33 yarns/cm; and weft 25 tex, of
density 28 yarns/cm.

Plant Selection
Natural dyestuff was extracted from European Ash bark (Fraxinus excelsior) and
European black elderberry berries (Sambucus nigra) from Croatia.

Mordants
The chemicals (from Kemika, Zagreb) used as mordants in this research were: KAl(SO4)2
· 12H2O, CuSO4 · 5H2O, FeSO4 · 7H2O and SnCl2 · 2H2O.

Procedure

The dyeing of the wool fabric was done in 3 stages: extraction of dyes from the plant,
mordanting in pre-treatment or after-treatment, and dyeing.

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1547

Extraction of Dyestuff
Air-dryed and powered plants were dried 6 hours at 105°C and cooled. Water extract of
European Ash bark (Fraxinus excelsior) and European black elderberry berries (Sambucus
nigra) was performed in deionized water for 1 hour at the 100°C, in bath ratio (BR) 1:20. The
solution was cooled down and filtered.

Mordanting
The woolen fabrics were treated with metal salts – mordants:

KAl(SO4)2 · 12H2O, CuSO4 · 5H2O, FeSO4 · 7H2O and SnCl2 · 2H2O as pre-treatment or
after-treatment for wool fabric dyed with water extract of European Ash bark (Fraxinus
excelsior); and as pre-treatment for wool fabric dyes with water extract of European black
elderberry berries (Sambucus nigra) due to it is more effective. In pre-treatment, woolen
fabrics were treated with metal salts, followed by dyeing in a water extract of natural dyes. In
after-treatment mordants were applied after the dyeing process.

Procedure of mordating
Mordant was dissolved in deionized water to make the liquor. pH 4.5 was set by adding
2% oxalic acid and 2 % tartaric acid. Wool fabrics were mordanted for 60 min at 100°C using
laboratory dyeing machine (Polycolor, Mathis) at a material to BR 1:20. Mordanting was
carried out with 4 different mordants: Pottasium alum dodecahydrate (KAl(SO4)3 · 12 H2O),
Copper (II) sulfate pentahydrate (CuSO4 · 5H2O), Iron (II) sulfate heptahydrate (FeSO4 · 7
H2O) and Tin(II) chloride dihydrate (SnCl2 · 2H2O)in wide concentration range (0.1% - 5%
owf – over weigth of fabric). The mordant material was than rinsed and dried.

Dyeing
Untereated and woolen fabric treated with mordants were dyed with plant extract solution
of pH value 6.5. Dyeing process was done in laboratory dyeing machine (Polycolor, Mathis)
at a material to liquor ratio of 1:20. The dye bath temperature was raised at a rate of 3°C/min
to 100°C, maintained at this temperature for 60 min and cooled down to room temperature.
Dyed fabrics were rinsed in cold and hot water and allowed to air-dry.

Measurements

The electrokinetic (zeta) potential is part of the total potential drop occurring in the
intermediate surface layer at the boundary of the solid/liquid phases as a consequence of the
ions distribution from the solid surface to the liquid mass. It was measured by streaming
potential/current method using Brookhaven-Paar Electrokinetic Analyzer with a stamp cell
and calculated according to Helmholtz-Smoluchowsky equation [93]. Isoelectric Point (IEP)
is a numeric value of pH where electrokinetic surface potential equals zero and indicate the
nature of solid surface. Therefore, the zeta potential (ZP) was investigated versus pH, and the
Isoelectric Point (IEP) of woolen fabric was determined.
HPLC of plant water extract using Shimadzu HPLC system of LC-10 series was
performed to determine composition of primary dyeing agent in natural dyestuffs. Based on
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1548 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

the best selectivity of flavonoides that should contain these plant extracts, measurement was
performed at 300 nm, bandwidths of 10 nm, in time of 15 min. Column C18 Shodex RSpak
DS-613 were used. Analysis was performed by binary system where the mobiles phase
included water (pH 2 adjusted with phosphoric acid) and acetonitrile in a ratio of 77,5:22,5; at
a flow rate 1 ml/min.
Color parameters of woolen fabrics after dyeing were measured using remission
spectrophotometer SF 600 PLUS CT (Datacolor) under illuminant D65, 8° standard observer
with the specular component excluded and the UV component included. The coordinates used
to determine color values are “L*” for lightness, “a*” for redness (positive value) and
greenness (negative value), “b*” for yellowness (positive value) and blueness (negative
value), “C*” for chroma and “h°” for hue angle in the range of 0° to 360°.
Colour strength (K/S) was calculated by the Kubelka-Munk equation:

K/S = (1-R)2/2R (1)

where R is the remission value (reflectance of the dyed fabric) at max, K is the absorption
coefficient.
The fabric UV protection was determined according to AS/NZS 4399:1996 Sun
Protective Clothing: evaluation and classification. UVA and UVB transmission through
fabric were measured on Varian Cary 50 Spectrophotometer. The ultraviolet protection factor
(UPF) was calculated automatically. Fabrics UV protection was rated accordingly (Table 2).

Table 2. UV protection rating according to AS/NZS 4399:1996

UV-R protection UV-R blocking

UPF range UPF rating
category [%]
< 14 0, 5, 10 non-rateable <93,3
15-24 15, 20 good 93,3-95,8
25-39 25, 30, 35 very good 95,9-97,4
> 40 40, 45, 50, 50+ excellent > 97,5

RESULTS AND DISCUSSION

In this chapter the UV protection by woolen fabric dyed with natural dyestuff extracted
from European Ash bark (Fraxinus excelsior) and European black elderberry berries
(Sambucus nigra) was researched. Prior to dyeing with these natural dyes, the zeta potential
and Isoelectric Point (IEP), a numeric value of pH where electrokinetic surface potential
equals zero, were determined by streaming potential/current method using Brookhaven-Paar
Electrokinetic Analyzer with a stamp cell. The results are shown in Figure 2.
The wool fibre has an anionic character at pH 10 due to the presence of numerous
carboxylate groups. The is also a large number of other chargeable groups, including nitrogen
containing groups, that will protonate and give rise to positive charge at lower pH values. All
these groups result in a high ZP of wool fabric (-48 mV) and IEP = 4.3. The net charge is due
to the balance of these different groups and is the reason for positive zeta potential of wool at
low pH. Isoelectric point, IEP, depends on molecular and supramolecular structure of fibres in

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1549

textile fabric. It is important parameter for fabric dyeability and finishing process. Therefore,
mordating was performed near the isoelectric point, at pH 4.5, what resulted in yellow shade;
whilst in alkali medium it would be brown.

20

ZP [mV] 10

pH
0
0 1 2 3 4 5 6 7 8 9 10 11 12

-10

-20

-30

-40

wool
-50

-60

Figure 2. Zeta potential (ZP, ζ) of the wool fabrics vs. pH of 0.001 M KCl (IEP=4.3).

Based on the retention times of occurrence of peaks of standard solutions of flavonoids

proved to be in alcocholic extract of European Ash bark obtained the strongest signal in the
retention time of 2.61 min, which corresponds to the rutine and less intensity at retention
times 3.44 and 4.40 min corresponding isoquercitrin and quercetin. In the alcoholic extract of
European black elderberry berries resulted in two spades and at retention time 2.61 min
(characteristic of rutin) and 1.85 min which corresponds to anthocyanines. Based on literature
data and HPLC measurement in European Ash bark (Fraxinus excelsior) extract primary
dyeing agents are rutin, quercetin and isoquercitrin [6, 30, 94], and in European black
elderberry berries (Sambucus nigra) rutine and anthocyanin, especially Cyanidin 3-glucoside
and Cyanidin 3-sambubioside [6, 94-96]. The chemical structures of primary dyeing agents
are listed in the Table 3. These compounds were taken account when applied to textiles in the
combination with 4 different mordants.
Natural dyes derived from plants are usually acid mordant dyes which create metal-
flavonoid complexes with metal ions. Depending on the chosen metal the different tones of
the coloration can be obtained. Since all these flavonoid pigments belong to the group of
mordant dyes, 4 different mordants were used in pre-treatment or after-treatment. The woolen
fabrics were pre-treated with metal salts, followed by dyeing in an aqueous extract of natural
dyes. In after-treatment mordants (metal salts) were applied after the dyeing process. In both
cases, it was essential that the flavonoid component from the extract creates colored metal
complex where, depending on the structure of flavonoid derivatives, metal complexes 1:1 or
1:2 may arise.
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Table 3. The primary dyeing agents in European Ash bark (Fraxinus excelsior) and European black elderberry berries
(Sambucus nigra)

OH OH
OH OH

HO O HO O CH 3
O
OH
OH O OH
OH O OH O OH
quercetin dihydrate isoquercitrin
3,3',4',5,7-pentahydroxyflavon quercetin 3-glucoside

OH
OH Cl - OH
O OH +
O HO O
HO O H 2C CH 3 OH
O
OH OH OH
O OH
OH
OH O OH
OH
rutin trihydrate Anthocyanin
quercetin -3-rutinoside Cyanidin

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 OH
OH OH
OH
OH OH
+ O
HO O
O
OH O OH
O O OH
OH OH
O + OH
HO O
OH
OH
OH OH
Cyanidin 3-glucoside Cyanidin 3-sambubioside
(796 mg/100 g) (463 mg/100 g)
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1552 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

Possible copper complexes with quercetin and rutin are shown in Figures 3 and 4. In
neutral aglycone quercetin dihydrate reactivity is [94]: Sn2+ > Al3+ > Fe2+ > Cu2+. On the other
hand, glycoside rutin trihydrate and isoquercetin showed oposite reactivity: Cu2+ > Fe2+ >
Al3+ > Sn2+. The resulting inverse selectivity of aglycone and glycosides in favor of "natural
series" H. Irving and R.J.P. Williams who proved that the stability of metal complexes is
growing in a number of divalent central ions: Mn2+Fe2+Co2+Ni2+Cu2+.

Figure 3. Possible quercetin:copper complex (1:1).

In order to confirm the formation of the colored complex on the dyed woolen fabric,
samples were analyzed with a remission spectrophotometer SF 600 PLUS CT (Datacolor).
The coloristic parameters are given in Tables 4-6. Color depth (K/S) of all dyed woolen
fabrics is presented in Figures 5-9.
From the Tables 4 and 5 can be seen that woolen fabrics dyed in water extract of
European Ash bark (Fraxinus excelsior) resulted with tones in yellow-orange region, a range
of values tons h* = 77 – 87 regardless of mordant or procedure of its application. The effect
of mordant concentration and its application procedure to saturation (C*) can be observed.
The maximum differences are observed for the metal salts of iron and copper which is in
correlation with their ionic potential.

Figure 4. Possible rutin:copper complex (1:1 and 1:2) [97].

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1553

Table 4. Color parameters of woolen fabrics dyed in water extract of European Ash
bark (Fraxinus excelsior) with KAl(SO4)212H2O and CuSO45H2O as mordants

Mordant a* b* L* C* h* X Y Z x y
No mordant 4.42 24.38 61.29 24.78 79.72 30.44 29.58 13.42 0.41 0.40
Pre-treatment KAl(SO4)212H2O
0.1% 5.61 25.77 53.41 26.37 77.71 22.40 21.42 8.61 0.43 0.41
0.5% 5.87 26.36 52.61 27.01 77.44 21.71 20.69 8.08 0.43 0.41
1.0% 5.77 26.41 53.48 27.04 77.68 22.51 21.49 8.47 0.43 0.41
1.5% 5.49 26.23 53.27 26.80 78.17 22.25 21.29 8.42 0.43 0.41
2.0% 5.62 26.28 53.20 26.88 77.94 22.21 21.23 8.38 0.43 0.41
3.0% 5.75 26.25 52.42 26.87 77.65 21.51 20.52 8.02 0.43 0.41
4.0% 5.63 25.99 51.86 26.59 77.79 20.96 20.02 7.84 0.43 0.41
5.0% 5.90 26.27 50.89 26.92 77.35 20.16 19.18 7.35 0.43 0.41
After-treatment KAl(SO4)212H2O
0.1% 4.66 24.07 67.84 24.52 79.05 38.81 37.76 18.19 0.41 0.40
0.5% 4.88 23.74 67.92 24.24 78.39 38.99 37.86 18.40 0.41 0.40
1.0% 5.05 24.02 67.24 24.55 78.13 38.11 36.95 17.75 0.41 0.40
1.5% 4.77 25.03 63.98 25.48 79.21 33.79 32.78 14.96 0.41 0.40
2.0% 5.13 25.03 63.55 25.55 78.41 33.35 32.25 14.66 0.42 0.40
3.0% 4.61 25.43 61.51 25.84 79.72 30.75 29.83 13.18 0.42 0.40
4.0% 4.84 25.68 62.03 26.13 79.32 31.42 30.43 13.42 0.42 0.40
5.0% 4.85 24.31 63.45 24.79 78.73 33.15 32.12 14.87 0.41 0.40
Pre-treatment CuSO45H2O
0.1% 4.27 23.99 54.54 24.37 79.90 23.20 22.49 9.68 0.42 0.41
0.5% 4.60 24.38 54.30 24.81 79.31 23.04 22.26 9.45 0.42 0.41
1.0% 3.85 24.10 54.45 24.40 80.92 23.01 22.40 9.60 0.42 0.41
1.5% 4.10 24.85 53.30 25.18 80.62 21.97 21.32 8.82 0.42 0.41
2.0% 3.84 24.61 53.97 24.91 81.13 22.55 21.95 9.21 0.42 0.41
3.0% 4.23 24.62 54.48 24.99 80.25 23.13 22.43 9.46 0.42 0.41
4.0% 4.58 24.95 55.20 25.37 79.61 23.91 23.12 9.73 0.42 0.41
5.0% 4.54 25.93 54.45 26.32 80.07 23.17 22.40 9.07 0.42 0.41
After-treatment CuSO45H2O
0.1% 3.77 24.20 62.06 24.49 81.16 31.17 30.48 13.99 0.41 0.40
0.5% 3.16 22.63 65.05 22.85 82.06 34.66 34.10 16.70 0.41 0.40
1.0% 2.08 23.00 61.45 23.09 84.82 30.00 29.77 14.05 0.41 0.40
1.5% 1.20 21.37 61.35 21.40 86.79 29.64 29.64 14.61 0.40 0.40
2.0% 1.78 22.56 58.60 22.63 85.50 26.75 26.60 12.41 0.41 0.40
3.0% 2.47 23.24 59.30 23.38 83.94 27.68 27.35 12.59 0.41 0.40
4.0% 1.56 22.48 63.53 22.53 86.04 32.31 32.22 15.67 0.40 0.40
5.0% 2.37 23.36 61.64 23.48 84.22 30.29 29.98 14.03 0.41 0.40

Table 5. Color parametres of woolen fabrics dyed in water extract of European Ash
bark (Fraxinus excelsior) with FeSO47H2O and SnCl22H2O as mordants

MORDANT a* b* L* C* h* X Y Z x y
Pre-treatment FeSO47H2O
0.1% 2.33 15.71 45.23 15.89 81.58 14.93 14.70 7.54 0.40 0.40
0.5% 1.04 12.75 41.77 12.80 85.32 12.37 12.35 6.81 0.39 0.39
1.0% 0.58 10.66 37.55 10.67 86.91 9.80 9.84 5.67 0.39 0.39
1.5% 0.59 9.99 36.98 10.00 86.63 9.50 9.53 5.60 0.39 0.39
2.0% 0.42 10.31 37.54 10.31 87.65 9.78 9.83 5.74 0.39 0.39
3.0% 0.80 10.97 37.04 11.00 85.81 9.55 9.56 5.42 0.39 0.39
4.0% 0.89 12.03 38.52 12.07 85.78 10.39 10.38 5.73 0.39 0.39
5.0% 0.99 12.53 38.20 12.56 85.46 10.22 10.20 5.51 0.39 0.39
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1554 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

Table 5. (Continued)

MORDANT a* b* L* C* h* X Y Z x y
After-treatment FeSO47H2O
0.1% 2.02 16.47 54.91 16.59 83.01 23.05 22.84 12.31 0.40 0.39
0.5% 1.29 13.90 53.72 13.96 84.70 21.76 21.71 12.50 0.39 0.39
1.0% 1.39 14.92 51.12 14.98 84.66 19.44 19.38 10.66 0.39 0.39
1.5% 1.22 16.43 46.74 16.47 85.76 15.86 15.82 8.03 0.40 0.40
2.0% 1.07 16.21 49.21 16.25 86.23 17.77 17.76 9.26 0.40 0.40
3.0% 1.50 17.02 46.50 17.09 84.96 15.73 15.64 7.77 0.40 0.40
4.0% 1.47 18.29 47.34 18.35 85.39 16.37 16.28 7.82 0.40 0.40
5.0% 1.96 18.53 45.32 18.63 83.96 14.94 14.77 6.90 0.41 0.40
Pre-treatment SnCl22H2O
0.1% 6.13 30.06 59.18 30.67 78.48 28.49 27.23 10.27 0.43 0.41
0.5% 5.94 30.46 60.05 31.03 78.97 29.42 28.18 10.61 0.43 0.41
1.0% 5.99 30.86 61.56 31.44 79.01 31.18 29.89 11.32 0.43 0.41
1.5% 6.18 31.49 62.20 32.09 78.90 32.01 30.64 11.48 0.43 0.41
2.0% 6.44 32.62 62.29 33.25 78.83 32.18 30.74 11.15 0.43 0.42
3.0% 6.45 32.42 62.43 33.05 78.74 32.36 30.90 11.30 0.43 0.41
4.0% 6.53 31.59 60.98 32.25 78.33 30.65 29.22 10.76 0.43 0.41
5.0% 7.00 31.75 59.60 32.51 77.57 29.18 27.68 9.97 0.44 0.41
After-treatment SnCl22H2O
0.1% 5.10 24.81 66.92 25.33 78.38 37.70 36.53 17.16 0.41 0.40
0.5% 4.99 26.23 65.64 26.70 79.22 35.98 34.86 15.63 0.42 0.40
1.0% 4.88 26.41 66.08 26.86 79.54 36.52 35.43 15.87 0.42 0.40
1.5% 4.27 25.70 71.05 26.05 80.58 43.25 42.27 20.02 0.41 0.40
2.0% 5.53 26.84 66.99 27.41 78.36 37.93 36.62 16.34 0.42 0.40
3.0% 5.39 26.64 62.12 27.18 78.56 31.69 30.54 13.12 0.42 0.41
4.0% 4.93 26.12 62.40 26.58 79.32 31.89 30.87 13.49 0.42 0.40
5.0% 5.26 26.71 68.00 27.22 78.86 39.22 37.97 17.14 0.42 0.40

14

12

10 9,56
8,26 8,32 8,50 8,55
8,08 8,06
8 7,42
K/S

3,96
4

0
0.0% 0.1% 0.5% 1.0% 1.5% 2.0% 3.0% 4.0% 5.0%

a
KAl(SO4)2  12H2O
Figure 5. (Continued).

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1555

14

12

10

K/S
6
4,34 4,25
3,96
4 3,48 3,59 3,54
2,62 2,55 2,67
2

0
0.0% 0.1% 0.5% 1.0% 1.5% 2.0% 3.0% 4.0% 5.0%

b
KAl(SO4)2  12H2O

Figure 5. K/S values of woolen fabrics dyed in water extract of European Ash bark (Fraxinus excelsior)
mordanted with KAl(SO4)212H2O (0.1% - 5% owf) in a. pre-treatment and b. after-treatment.

14

12

10

8 7,31 7,28
6,91 6,67
K/S

6,51 6,57 6,43 6,55

6

3,96
4

0
0.0% 0.1% 0.5% 1.0% 1.5% 2.0% 3.0% 4.0% 5.0%

a
CuSO4  5H2O
14

12

10

8
K/S

6
4,52 4,40
3,96 3,88 3,89 3,83
4 3,60 3,29
2,94

0
0.0% 0.1% 0.5% 1.0% 1.5% 2.0% 3.0% 4.0% 5.0%

b CuSO4  5H2O

Figure 6. K/S values of woolen fabrics dyed in water extract of European Ash bark (Fraxinus excelsior)
mordanted with CuSO45H2O (0.1% - 5% owf) in a. pre-treatment and b. after-treatment.
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1556 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

14 13,37 13,29
12,54 12,38 12,38
12 11,29

9,85
10
8,76

K/S
6

3,96
4

0
0.0% 0.1% 0.5% 1.0% 1.5% 2.0% 3.0% 4.0% 5.0%

a
FeSO4  7H2O

14
12,56
12 11,52
10,32
10
8,27
8
7,01
K/S

6,13
6
4,60 4,71
3,96
4

0
0.0% 0.1% 0.5% 1.0% 1.5% 2.0% 3.0% 4.0% 5.0%

b
FeSO4  7H2O

Figure 7. K/S values of woolen fabrics dyed in water extract of European Ash bark (Fraxinus excelsior)
mordanted with FeSO47H2O (0.1% - 5% owf) in a. pre-treatment and b. after-treatment.

14

12
10,17 9,95 9,88
9,61 9,47 9,72 9,66
10 9,21

8
K/S

3,96
4

0
0.0% 0.1% 0.5% 1.0% 1.5% 2.0% 3.0% 4.0% 5.0%

a 2  2H2O
SnCl

Figure 8. (Continued).

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1557

14

12

10

8
K/S
6
4,96 4,71
3,96 3,89 3,88
4 3,60 3,51
3,16
2,67
2

0
0.0% 0.1% 0.5% 1.0% 1.5% 2.0% 3.0% 4.0% 5.0%

b 2  2H2O
SnCl

Figure 8. K/S values of woolen fabrics dyed in water extract of European Ash bark (Fraxinus excelsior)
mordanted with SnCl22H2O (0.1% - 5% owf) in a. pre-treatment and b. after-treatment.

However, two-dimensional relationship between hue and saturation, can not make
conclusive experience of color without the third dimension - the ligthness (L*). In general, on
the basis of the coloristic parameters for all samples the L*  60, except for samples
processed with salt FeSO47H2O, L* < 40. It is recognized, based on the x and y, these color
samples are closer to achromatic point, resulting in olive shade. This pheomenon is more
evident if the mordant was applied in pre-treatment. Comparing the procedures of applying
mordants it is evident that pre-treatment results in better saturation than if applied in after-
treatment. This can be considered from two aspects: 1st supstantivity of plant extract to the
wool substrate and 2nd stability of resulting metal complexes. In the complexing was done in
solution of quercetin dihydrate, and isoquercitrin, rutin trihydrate, the highest reactivity was
obtained with the copper salt [94]. However, according to the color measurement, these
results are not the best ones. This confirms that all components in plant extract, as well as the
textile substrate, participate in creating of colored complex on the fiber/textile material.
Mechanism of dyeing with flavonoid dyes is similar to the mechanism of antiradical
behaviour of flavonoids in biological system and chelat bonding of metal ions. In biological
systems the mechanism has not yet been completely explained on molecular level since there
are great differences in chemical properties and significant structural heterogenity. However,
the relationship between the structure and activity of the flavonoid and certain structural
components and properties of bonding free radicals, formation of chelate complexes and anti
oxidation activities have been proofed. Depending on chemical structure and chemico-
morphological characteristics of the fibres being dyed by flavonoid dyes, following chemical
bond are formed: hydrogen bonds are formed among poliphenol hydroxyl groups with free
amino and amid groups of the protein fibre; ionic bonds are formed among free and available
anionic groups in poliphenols and cationic groups of protein fibre 41, 97-100.
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1558 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

Table 6. Color parameters of woolen fabrics dyed in water extract of European black
elderberry berries (Sambucus nigra)

MORDANT a* b* L* C* h* X Y Z x y
No mordant 7.29 21.07 52.10 22.30 70.91 20.65 20.23 11.99 0.39 0.38
Pre-treatment KAl(SO4)212H2O
0.1% 6.67 18.57 45.65 19.74 70.24 12.60 12.28 7.08 0.39 0.38
0.5% 7.58 19.83 44.17 21.23 69.09 14.43 13.96 7.92 0.40 0.38
2.0% 6.45 18.97 42.91 20.04 71.23 13.39 13.10 7.56 0.39 0.38
Pre-treatment CuSO45H2O
0.1% 7.51 18.60 43.17 20.06 68.00 13.73 13.27 7.78 0.39 0.38
0.5% 7.25 18.45 42.73 19.83 68.56 13.39 12.98 7.62 0.39 0.38
2.0% 7.46 19.11 40.58 20.51 68.68 12.05 11.61 6.48 0.40 0.39
Pre-treatment FeSO47H2O
0.1% 6.67 18.57 41.65 19.74 70.24 12.60 12.28 7.08 0.39 0.38
0.5% 5.46 15.62 38.37 16.54 70.72 10.46 10.30 6.40 0.39 0.38
2.0% 4.00 12.88 33.96 13.48 72.75 8.01 7.99 5.28 0.38 0.38
Pre-treatment SnCl22H2O
0.1% 6.67 18.57 41.65 19.74 70.24 12.60 12.28 7.08 0.39 0.38
0.5% 5.61 17.64 42.20 18.51 72.36 12.80 12.63 7.59 0.39 0.38
2.0% 4.82 17.38 40.56 18.04 74.49 11.66 11.59 6.90 0.39 0.38

Considering the mordant concentration, it is evident that optimal color was obtained for
samples pre-treated with 2% of metal salts. By increasing the concentration of mordant, no
significant change in color depth (K/S) occured. If mordant applied in wide concentration
range in after-treatment, the significant change in color parameters occurred if salt
FeSO47H2O was applied. The obtained K/S values can be used as an indicator of water plant
extract supstantivity. Approximately the same color depth (K/S) of woolen fabric achieved by
pre-treated with 2%, and after-treated with 5% of FeSO47H2O, can be attributed to the
reactivity of the whole system. If pre-treated with 2% FeSO47H2O whole system is more
reactive, because in the chelating participate both, the textile substrate and the plant extract.
Considering these observations, mordating was performed only in pre-treatment prior to
dyeing with European black elderberry berries (Sambucus nigra) water extract. Color
parameters are collected in Table 6, and color depth (K/S) is shown in Figure 9.
Considering the color parameters of woolen fabrics dyed with European black elderberry
berries (Sambucus nigra) it can be seen that fabrics have yellow-orange to yellow coloration
(h* = 68 – 75). Samples with the highest lightness values were obtained using aluminum salts
as mordant (L = 45) and the darkest samples were obtained using copper and iron salts, e.g., L
= 34, respectively.
Considering color depth (K/S) of woolen fabrics dyed in water extract of European black
elderberry berries (Sambucus nigra) mordanted in pre-treatment with KAl(SO4)212H2O,
CuSO45H2O; FeSO47H2O and SnCl22H2O it can be seen that the highest K/S value has
fabric mordaned with iron salt.
However, these samples showed lower chromaticity and the subjective visual assessment
noted that the samples have more pronounced grey shade. It is to point out mordating with
SnCl22H2O as well, which resulted in high K/S value of 13.33. Other properties and
observations are similar to the ones dyes with water extract of European Ash bark (Fraxinus
excelsior).

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1559

It is to notice lower reactivity of water extract of European black elderberry berries

(Sambucus nigra) than European Ash bark (Fraxinus excelsior).

16

14

12 11,73

10,02 9,69 9,72

K/S 10 9,59

8
6,69
6,16
6

0
0,0% 0,1% 0,5% 2,0% 0,1% 0,5% 2,0%

KAl(SO4)212H2O CuSO45H2O

a
16
14,74
14 13,33
12,01 12,00 12,01
12 11,58

10
K/S

8
6,16
6

0
0,0% 0,1% 0,5% 2,0% 0,1% 0,5% 2,0%

FeSO47H2O SnCl22H2O

Figure 9. K/S values of woolen fabrics dyed in water extract of European black elderberry berries
(Sambucus nigra) mordanted in pre-treatment with: a. KAl(SO4)212H2O and CuSO45H2O; b.
FeSO47H2O and SnCl22H2O (0.1%, 0.5%, 2% owf).

On the basis of the literature, this lower reactivity of isoquercitrin and rutin trihydrate is a
result of flavonoid glycosidation. Additionally, the berries in a greater proportion contain
anthocyanosides and betalaines, which are considerably more reactive towards metal ions [6,
94-96].
The most of results of the UV protective fabrics concern application of synthetic dyes,
whilst only few studies reports of natural dyes. Since it was found that European black
elderberry berries (Sambucus nigra) helps in cosmetic emulsions giving of photoprotection to
UV-A and UV-B irradiation, its application and European Ash (Fraxinus excelsior) bark were
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1560 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

choosen for the application on light woollen fabric which could protect from harmful UV
radiation.
The fabric UV protection was determined according to AS/NZS 4399:1996 Sun
Protective Clothing: evaluation and classification, by UV-A and UV-B transmission
measurement on transmission spectrophotometer and calculation of Ultraviolet protection
factor (UPF). The results are presented in Tables 7-9.

Table 7. UV protection of woolen fabrics dyed in water extract of European Ash bark
(Fraxinus excelsior) with KAl(SO4)212H2O and CuSO45H2O as mordants

MORDANT Mean UPF  UVA  UVB Stand. Dev. Stand. Err. Calc. UPF UV protection
Wool 32.070 7.634 2.194 1.069 1.326 30.744 30
No mordant 54.800 5.043 1.103 1.481 2.444 52.356 50
Pre-treatment KAl(SO4)212H2O
0.1% 210.273 0.574 0.455 64.604 80.109 130.163 50+
0.5% 188.651 0.651 0.509 47.362 58.728 129.922 50+
1.0% 188.513 0.615 0.481 15.142 18.776 169.737 50+
1.5% 96.801 1.031 0.981 3.529 4.376 92.425 50+
2.0% 134.488 0.812 0.700 12.303 15.256 119.232 50+
3.0% 77.371 1.300 1.237 6.584 8.164 69.207 50+
4.0% 147.360 0.124 0.634 7.927 9.829 137.531 50+
5.0% 70.494 1.449 1.367 7.667 9.507 60.987 50+
After-treatment KAl(SO4)212H2O
0.1% 344.562 0.630 0.206 1.160 54.465 290.097 50+
0.5% 427.916 0.527 0.193 43.923 189.108 238.808 50+
1.0% 265.127 0.771 0.336 152.506 130.034 135.093 50+
1.5% 197.291 0.910 0.418 104.866 64.764 132.528 50+
2.0% 197.056 0.855 0.391 52.229 15.342 181.714 50+
3.0% 364.845 0.567 0.209 12.373 124.600 240.245 50+
4.0% 308.848 0.698 0.299 144.756 179.498 129.350 50+
5.0% 158.789 1.027 0.496 12.907 16.005 142.785 50+
Pre-treatment CuSO45H2O
0.1% 204.600 0.600 0.440 34.831 43.191 161.409 50+
0.5% 147.816 0.759 0.616 10.199 12.647 135.168 50+
1.0% 105.849 1.008 0.890 13.102 16.246 89.648 50+
1.5% 119.909 0.924 0.787 19.677 24.400 95.509 50+
2.0% 150.996 0.752 0.601 12.028 14.915 136.081 50+
3.0% 147.508 0.787 0.642 31.394 38.928 108.579 50+
4.0% 103.013 1.057 0.909 7.773 9.639 93.374 50+
5.0% 140.480 0.786 0.656 10.790 13.379 127.101 50+
After-treatment CuSO45H2O
0.1% 456.846 0.446 0.239 250.687 310.852 145.993 50+
0.5% 408.100 0.529 0.206 152.717 189.369 218.731 50+
1.0% 335.774 0.600 0.303 166.050 205.902 129.872 50+
1.5% 546.108 0.416 0.138 111.925 138.787 407.321 50+
2.0% 488.220 0.398 0.154 58.708 72.798 415.422 50+
3.0% 772.257 0.230 0.116 184.463 228.734 543.523 50+
4.0% 347.056 0.573 0.229 93.859 116.385 230.671 50+
5.0% 939.780 0.163 0.100 15.416 19.115 920.665 50+

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1561

Table 8. UV protection of woolen fabrics dyed in water extract of European Ash bark
(Fraxinus excelsior) with FeSO47H2O and SnCl22H2O as mordants

MORDANT Mean UPF  UVA  UVB Stand. Dev. Stand. Err. Calc. UPF UV protection
Pre-treatment FeSO47H2O
0.1% 156.575 0.699 0.604 28.632 35.504 121.071 50+
0.5% 288.053 0.395 0.306 30.565 37.901 250.153 50+
1.0% 102.195 0.972 0.935 8.843 10.966 91.230 50+
1.5% 125.637 0.786 0.760 2.921 3.622 122.051 50+
2.0% 167.955 0.599 0.570 20.070 24.887 143.068 50+
3.0% 146.659 0.681 0.645 3.414 4.234 142.426 50+
4.0% 168.330 0.593 0.576 18.537 22.986 145.344 50+
5.0% 99.988 0.945 0.970 5.214 6.465 93.523 50+
After-treatment FeSO47H2O
0.1% 665.082 0.275 0.125 83.611 103.677 561.404 50+
0.5% 982.979 0.116 0.100 19.794 24.544 958.435 50+
1.0% 734.727 0.249 0.116 86.981 107.857 626.870 50+
1.5% 967.769 0.119 0.101 41.580 51.559 916.210 50+
2.0% 953.918 0.127 0.101 71.234 60.330 893.588 50+
3.0% 999.824 0.100 0.100 0.497 0.616 999.209 50+
4.0% 949.955 0.119 0.104 66.391 82.325 867.630 50+
5.0% 987.772 0.105 0.101 16.573 20.551 967.222 50+
Pre-treatment SnCl22H2O
0.1% 221.733 0.468 0.419 46.565 57.741 163.991 50+
0.5% 167.333 0.581 0.560 13.917 17.257 150.076 50+
1.0% 136.940 0.700 0.689 12.894 15.989 120.951 50+
1.5% 696.052 0.194 0.128 61.790 76.619 619.433 50+
2.0% 791.720 0.160 0.117 124.593 154.495 637.225 50+
3.0% 305.541 0.345 0.309 66.323 82.241 223.300 50+
4.0% 660.171 0.204 0.138 107.995 133.913 526.258 50+
5.0% 254.974 0.432 0.397 62.367 77.335 168.639 50+
After-treatment SnCl22H2O
0.1% 594.845 0.344 0.128 41.231 514.127 543.719 50+
0.5% 218.308 0.657 0.384 33.397 41.413 176.896 50+
1.0% 317.715 0.512 0.235 27.706 34.355 283.360 50+
1.5% 681.892 0.412 0.106 50.341 62.423 619.469 50+
2.0% 612.764 0.357 0.125 90.445 112.152 500.612 50+
3.0% 443.513 0.384 0.168 15.942 19.769 423.745 50+
4.0% 257.243 0.549 0.325 54.372 67.422 189.821 50+
5.0% 756.154 0.279 0.107 40.552 50.284 821.869 50+

Since no UV-C radiation reaches the earth’s surface due to absorption by oxygen and
ozone in the upper atmosphere, the transmittance of ultraviolet including UV-A and UV-B
through the fabrics was measured on transmission spectrometer Cary 50 (Varian). The
ultraviolet transmittance spectra of the woolen fabric without dyeing and after mordating and
dyeing with European black elderberry berries (Sambucus nigra) and European Ash bark
(Fraxinus excelsior) was compared. As can be seen from Tables 7-9, there was a significant
difference between the dyed fabrics and un-dyed one. Even thoug woolen fabric is the only
one that absorbs radiation throughout the entire UV spectrum even when completely
untreated due to its chemocal composition, it transmit 7.6% of UV-A nad 2.2% UV-B
radiation, resulting in very good UV protection (UPF=32.070).
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1562 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

Table 9. UV protection of woolen fabrics dyed in water extract of European black

elderberry berries (Sambucus nigra)

MORDANT Mean UPF  UVA  UVB Stand. Dev. Stand. Err. Calc. UPF UV protection
Wool 32.070 7.634 2.194 1.069 1.326 30.744 30
No mordant 59.489 4.949 0.826 2.371 1.460 58.029 50+
Pre-treatment KAl(SO4)212H2O
0.1% 856.997 0.167 0.107 76.302 94.689 762.307 50+
0.5% 743.963 0.193 0.124 185.664 230.223 513.739 50+
2.0% 482.273 0.275 0.189 96.842 120.084 362.181 50+
Pre-treatment CuSO45H2O
0.1% 399.652 0.366 0.314 245.563 304.498 95.154 50+
0.5% 164.585 0.616 0.567 10.381 12.872 151.677 50+
2.0% 439.309 0.349 0.309 289.529 359.016 80.293 50+
Pre-treatment FeSO47H2O
0.1% 847.995 0.143 0.112 161.190 199.876 648.119 50+
0.5% 279.710 0.394 0.347 60.479 74.994 204.717 50+
2.0% 952.089 0.119 0.102 49.075 60.853 891.236 50+
Pre-treatment SnCl22H2O
0.1% 740.017 0.179 0.139 247.636 307.068 432.949 50+
0.5% 735.046 0.181 0.140 245.511 304.433 430.613 50+
2.0% 996.544 0.102 0.100 3.978 4.933 991.612 50+

Dyeing with water extract of European Ash bark (Fraxinus excelsior) without mordant,
results already in excellent UV protection (UPF=54.8). It is to point out that UV-B
transmission is lower 50%, whilst UV-A transmission only 30%. As proved in cosmetics,
dyeing with European black elderberry berries (Sambucus nigra), improve UV-B absorption
for 63%, and UV-A for 35%, resulting in excellent UV protection (Highest class 50+).
From the Tables 7-9 it can be clearly seen that the values of spectral transmittance
decrease with all mordants applied, resulting in excellent UV protection. However, it is
possible to evaluate the influence of mordants to UV protection considering the mean UPF
values. Considering mordant concentration, similar behaviour was noticed as for the color
parameters. The concentration of 2% of mordant was selected as optimal one. Therefore,
these results are presented in Figure 10.
The best UV protection has been achieved applying FeSO47H2O as mordant resulting in
UPF almost 1000. The reason for that is the lowest ligthness, suggesting darkest shade with
the highest absorption of UV radiation what results in the lowest UV transmittion. The results
of color parameters confirm that.
For the difference of color depth which was achieved if mordant was applied in pre-
treatment; the significantly better UV protection was achieved if mordant was applied in
after-treatment. Ibrahim et al. [101] research of UV-protective finishing of cotton knits by
addition of the metal-oxide into the finishing bath. It resulted in better UV protection
probably because of ligth scattering [89, 90, 102]. Considering the metal-oxides applied, UV
protection was the next: Cu > Zr > Zn ≫ Al ≈ none. For that reason, if the metal salts were
applied as mordants in after-treatment, it is to assume that ligth scattering form the fabric
surface was higher, what led to better UV protection. In the case of mordating in after
treatment dyed fabrics with water extract of Fraxinus excelsior, UV protection was next: Fe >
Sn > Cu > Al, and significantly higher than if applied as pre-treatment. Again, the difference

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 UV Protection by Woolen Fabric Dyed with Natural Dyestuff 1563

in chelating played an important role. It is to point out mordating with SnCl22H2O as well,
which resulted in higher K/S values, and the highest UV protection as well.
Comparing the influence of European Ash bark (Fraxinus excelsior) and European black
elderberry berries (Sambucus nigra), it can be seen that Sambuctus nigra gave off better UV
protection.

1.000
Wool
900
Fraxinus excelsior (pre-treated)
800 Fraxinus excelsior (after-treated)
Sambucus nigra (pre-treated)
700

600
UPF

500

400

300

200

100

0
No mordant KAl(SO4)2×12H2O CuSO4×5H2O FeSO4×7H2O SnCl2×2H2O

Figure 10. Mean UPF of woollen fabrics dyed with 2% water extracts of Fraxinus excelsior and
Sambuctus nigra pre-treated or after-treated with 4 different mordants.

CONCLUSION
In this chapter the UV protection by woolen fabric dyed with natural dyestuff extracted
from European Ash bark (Fraxinus excelsior) and European black elderberry berries
(Sambucus nigra) was researched. As most of natural dyes water extracts were applied on
textiles in the combination with mordants - KAl(SO4)2·12H2O, FeSO4·7H2O, CuSO4·5H2O
and SnCl2·2H2O. The woolen fabric was characetrized by its zeta potential and isoelectric
point; therefore mordants were applied at pH 4.5. The active components, which are the most
responsible for achieved colour hue, respectively for the forming of coloured chelates, were
determined for the Fraxinus excelsior and Sambucus nigra by HPLC. Analyzed extracts
contained flavonoids substances: quercetin dihydrate, isoquercitrin and rutin trihydrate. It was
confirmed that the water based herbal extracts has got certain substantivity towards woolen
substrates. The influence of a sort of the metal on a hue of a coloured complexes was
confirmed by the color parameters on remission spectrophotometer determination. For
KAl(SO4)2·12H2O and SnCl2·2H2O as mordants, the yellow – orange hues were obtained, for
CuSO4·5H2O orange – brown, and for the FeSO4·7H2O achromatic – chromatic olive green
hues. In dependence to reactivity and property of forming the coloured chelates, the largest
colour depth (K/S) was achieved using Fe2+ ions.
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1564 Ana Sutlović, Anita Tarbuk, Ana Marija Grancarić et al.

Since flavonoids and anthocyanosides have an important role in protecting against

harmful effects of UV radiation the fabric UV protection was determined according to
AS/NZS 4399:1996 Sun Protective Clothing: evaluation and classification, by UV-A and
UV-B transmission measurement on transmission spectrophotometer and calculation of
Ultraviolet protection factor (UPF). It was confirmed that woolen fabrics dyed with natural
dyes, extracted from Fraxinus excelsior and Sambucus nigra, ensure excellent UV protection
(UPF > 50). It is to point out that it offers protection against UVB radiation as well, and
therefore it may reduce the risk of subsequent occurrence of skin cancer. In the case of
application, it is to suggest European black elderberry berries (Sambucus nigra), which gave
maximum UV protection (UPF=1000).

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 71

LIGHT CONVERSION FOR UV PROTECTION

BY TEXTILE FINISHING AND CARE

Tihana Dekanić, Anita Tarbuk, Tanja Pušić,

Ana Marija Grancarić and Ivo Soljačić
University of Zagreb, Faculty of Textile Technology,
Department of Textile Chemistry and Ecology, Zagreb, Croatia

ABSTRACT
The incidence of skin cancer is increasing by epidemic proportions. Its primary cause
is a long exposure to solar ultraviolet (UV) radiation crossed with the amount of skin
pigmentation in the population. It is believed that in childhood and adolescence 80% of
UV-R gets absorbed, whilst in the remaining 20% gets absorbed later in the lifetime. This
suggests that proper and early photoprotection may reduce the risk of subsequent
occurrence of skin cancer. Textile and clothing can show UV protection, but in the most
cases it does not provide full sun screening properties. UV protection highly depends on
large number of factors such are type of fiber, fabric surface and construction, type and
concentration of dyestuff, fluorescent whitening agent (FWA), UV-B protective agents,
as well as nanoparticles, if applied. Based on electronically-excited state by energy of
UV-R (usually 340-370 nm) the molecules of FWAs show the phenomenon of
fluorescence giving to white textiles high whiteness of outstanding brightness by
reemitting the energy at the blue region (typically 420-470 nm) of the spectrum, what
leads to better UV protection. Molecules of UV absorbers are able to absorb the
damaging UV-R range of 290 nm to 360 nm, and convert it into harmless heat energy.
Latest research declares that FWA’s and UV absorbers can be applied in textile care –
washing process as well. Therefore, the UV protective properties of cotton and
cotton/polyester blend fabrics achieved by light conversion in textile finishing and care
was researched in this chapter. For that purpose, three stilbene derivative fluorescent
compounds were selected – fluorescent whitening agent commonly used in textile
finishing, the other one used in detergent formulations, and UV absorber. In textile
finishing process fluorescent compounds were applied by exhaustion procedure in wide


Corresponding address: Prilaz baruna Filipovića 28a, HR-10000 Zagreb, Croatia. E-mail: [email protected],
[email protected]
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1572 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

concentration range. In textile care fluorescent compounds were applied through 9

washing cycles at 60°C with standard ECE reference detergent and commercial detergent.
UV protection was determined in vitro through Ultraviolet protection factor, UPF.
Additionally the influence to fabric whiteness was researched. Since the fabric properties
change in wet state, the discrepancy in whiteness and UV protection was research in
distilled water as well as Adriatic Sea water.

Keywords: UV protection, cotton, polyester/cotton blend, FWA, UV absorber, wet state

INTRODUCTION
The incidence of skin cancer is increasing by epidemic proportions. Basal cell cancer
remains the most common skin neoplasm, and simple excision is generally curative. On the
other hand, aggressive local growth and metastasis are common features of malignant
melanoma, which accounts for 75 percent of all deaths associated with skin cancer [1]. The
reason for that is most likely that in the most cases melanoma was diagnosed in an advanced
stage. The back sides in men and women, as well as the lower limbs in women, are the most
common site for melanomas [2, 3]. The primary cause of skin cancer is believed to be a long
exposure to solar ultraviolet (UV) radiation crossed with the amount of skin pigmentation in
the population [1-6]. Melanoma incidence rates in white populations increase with proximity
to the Equator, and vary across Europe, with the highest rates for both sexes in Switzerland,
Denmark, Norway, Sweden and the Netherlands and the lowest rates in Central and
Southeastern Europe [1].
UV as a whole does not exceed 5% of the total energy emitted by the sun, but their
impact on the organic molecules is very important and it induces significant physiological
responses in all areas of life. In addition to some beneficial effects of UV radiation (UV-R;
wavelengths from 100 nm to 400 nm) on skin it may cause skin and eye damage, especially
during the summer time (UV-C). The UV-C radiation (from 100 nm to 280 nm) is absorbed
by atmosphere. However, diminishing of the Earth´s atmospheric ozone layer raised the UV
exposure health risk, since both, UV-B (from 280 nm to 320 nm) and UV-A (from 320 nm to
400 nm) radiation, are reaching the Earth. Dangerous UV-B rays can cause acute and chronic
reactions and damages such as erythema (sunburn), sun tanning, “photoaging,” DNA and eye
damage, photokeratitis and cataract, and photocarcinogenesis; increase risk factor for
melanoma, or cause various skin cancers [3, 6-33]. Experts estimate about 90% of melanomas
are associated with severe UV exposure and sunburns over a lifetime. Intermittent sun
exposure, especially in childhood and adolescence is considered to be a stronger risk factor
for melanoma than continuous exposure [1]. It is believed that in that period of life 80% of
UV-R gets absorbed, whilst in the remaining 20% gets absorbed later in the lifetime. This
suggests that proper and early photoprotection may reduce the risk of subsequent occurrence
of skin cancer [2].
Textile and clothing are the most suitable interface between environment and human
body. It can show UV protection, but in the most cases it does not provide full sun screening
properties. Literature sources claim that only 1/3 of the spring and summer collections tested
give off proper UV protection [11]. In contact with textile fabric, UV radiation can be
reflected and/or scattered from fabric surface, or get absorbed or transmitted (Figure 1).

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 Light Conversion for UV Protection by Textile Finishing and Care 1573

Figure 1. UV radiation in contact with textile fabric [28].

A good fabric UV protection depends on a large number of factors, such as, the type of
fiber, fabric surface and construction, porosity, density, moisture content, type and
concentration of dyestuff, fluorescent whitening agent (FWA), UV-B protective agents, as
well as nanoparticles, if applied [13-37]. For example, polyester fabric gives off better UV
protection than cellulose one, due to the polyester benzene rings [17, 30].
Fluorescent whitening agents (FWAs), commonly used for reaching higher whiteness
degrees, are chemical compounds that absorb UV-R (usually 340-370 nm), show the
phenomenon of fluorescence, and re-emit in the blue region (typically 420-470 nm) of the
spectrum.
When P. Krais in 1929 discovered fluorescent compound Aesculin by water extraction
from wild chestnut, he wrote “About the new white ....” It was the new white indeed, never
seen before such high whiteness degree. However, he could never assume that this UV-A
absorption of FWA’s would result in better UV protection as well [6, 15-17, 26, 30, 32]. The
phenomenon of fluorescence can be explained by modified diagram according Jablonski
(Figure 2) [38].
The molecules of FWAs go to electronically-excited state by absorbing energy of UV-R.
An electronically-excited molecule can lose its energy by emission of radiation which is
known as “luminescence.” In this case, the emission is fluorescence, which is, according to
Figure 2, an emission process occurring from lowest excited state (S1) to the ground state
(S0). The frequency of fluorescence radiation is lower than that of excitation light (which is
known Stokes Law). For the same compound an ideal emission should be the mirror image of
the absorption band system [39].
Textile finishing agents for UV protection can be incorporated into the fiber matrix, or it
can be applied to the surface of the fabric [3]. Usually sun protection effect is achieved
through the use of UV absorbers [6, 16-18].
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1574 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

Figure 2. Modified Jablonski diagram [38].

UV absorbers are, as a matter of fact, a special type of fluorescent whitening agents and
have the same or similar effect [6, 40]. Molecules of UV absorbers, such as benzotriazole and
phenyl benzotriazole, are able to absorb the damaging UV-R range of 290 nm to 360 nm, and
convert it into harmless heat energy. Therefore, they are even more effective than fluorescent
whitening agents [3]. The impact of fluorescent whitening agents on the whiteness degree of
cotton fabrics, as well as PES/cotton blends, in multiple washing cycles, has been
comprehensively investigated together with the UV protection abilities [26, 29-33]. Recently,
there has been an systematic investigation of the UV absorber resistance to light since the
final effect of fluorescent whitening agents is affected by the exposal to sunlight and by
drying after washing. Exposing the materials treated with fluorescent compounds to sunlight
can cause various photochemical reactions as they are prone to absorb UV radiation [32, 41].
Since the latest research declare that FWAs and UV absorbers can be applied in washing
process as well, the UV protective properties of cotton and cotton/polyester blend fabrics
achieved by light conversion in textile finishing and care was researched in this chapter. UV
protection was determined in vitro through Ultraviolet protection factor, UPF. Additionally
the influence to fabric whiteness was researched. Since the fabric properties change in wet
state, the discrepancy in whiteness and UV protection was research in distilled water as well
as Adriatic Sea water.

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 Light Conversion for UV Protection by Textile Finishing and Care 1575

EXPERIMENTAL
Materials

Two fabrics, cotton (C) and polyester/cotton (65/35) blend (P/C), such are frequently
used during summer time, were used in the investigation. Both fabrics were pre-bleached, in
plain weave, with the following properties: Cotton fabric surface mass of 175.6 g/m2 and yarn
density of (warp/weft 25/25 yarns/cm), and P/C blend fabric surface mass 155.1 g/m2 and
yarn density of (warp/weft 26/25 yarns/cm).
Three fluorescent compounds of stilbene type were used (Table 1): one fluorescent
whitening agent (FWA) commonly used in textile finishing (F1), one FWA commonly used
in textile care in detergent formulation (F2), and UV absorber (F3).

Table 1. Characteristics and structural formula of applied fluorescent compounds

Compound
Structural formula
characteristic

F1 H
HO N N
Fluorescent whitening SO3H HN
agent (FWA) - stilbene N N
N N
type
NH SO3H
CI Fluorescent N N OH
H
Brightener 336 Uvitex
BAM
(Ciba-Geigy AG)
bis(4,4'-triazinylamino)-stilbene-2,2'-disulfonic acid derivative
For textile finishing

O
H
F2 N N N
Fluorescent whitening SO3Na HN

agent (FWA) - stilbene N N

N N
type NH SO3Na
Optiblanc 2MG/LT N N N
H
Extra O
(Sigma 3V)
In detergent disodium 4.4’-bis[(4-anylino-6-morpholino-1.3.5-triazine-2-yl) amino]-
formulation stilbene-2.2’-disulphonate

H
R' N N
SO3H R'
F3 N N
UV absorber N N
- stilbene type R'' SO3H
N N R''
Tinosorb FD H
(Ciba-Geigy AG) R', R'' - differently substituted amines

stilbene disulphonic acid triazine derivative

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1576 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

Procedure

In this chapter, fluorescent compounds – two FWAs and UV absorber, all stilbene
derivatives, were applied to cotton and cotton/polyester blend fabrics in textile finishing and
care.

Textile Finishing
Fluorescent compounds were applied in wide concentration range (c1 = 0.004% owf (over
weight of fiber); c2 = 0.006% owf; c3 = 0.0125% owf; c4 = 0.1% owf; c5 = 0.5% owf; c6 = 1%
owf, c7 = 10% owf, c8 = 50% owf) by exhaustion procedure at 60°C for 30 minutes to achieve
the best whiteness and UV protection. Afterwards, the discrepancy in whiteness and UV
protection was research in distilled and Adriatic Sea water.

Textile Care - Laundering

In textile care fluorescent compounds were applied through 9 washing cycles with ECE
reference detergent 77 for color fastness, without optical brightener, phosphate based, for
application in ISO 105-C06:2010, ISO 6330:1984; and commercial detergent. Formulations
of detergents are given in Table 2.

Table 2. Formulations of detergents

ECE reference detergent Commercial detergent

- 8% Linear sodium alkyl benzene sulphonate - 5% Anionic sufractant

(mean length of alkane chain C11.5) - 1% Nonionic sufractant
- 2,9% Ethoxylated tallow alcohol (14 EO) - 2% Sodium soap
- 3,5% Sodium soap, (chain length - 20% Sodium tripolyphosphate
C12-16 13% - 26% : C18-22 74% - 87%) - 5% Sodium silicate
- 43,8% Sodium tripolyphosphate - 12% Sodium carbonate
- 7,5% Sodium silicate (SiO2:Na2O = 3.3 : 1) - 11% Sodium perborate
- 1,9% Magnesium silicate - 1,2% Carboxy methyl cellulose (CMC)
- 1,2% Carboxy methyl cellulose (CMC) - 2% Etylene diamine tetra acetic acid,
- 0,2% Etylene diamine tetra acetic acid, tetra sodium salt (TAED)
tetra sodium salt (TAED) - 0,6% Enzymes
- 21,2% Sodium sulphate - up to 100%:
- 9,8% Water Sodium sulphate and
*Added 1 g/l of sodium perborate Water

The samples were laundered in the Linitest apparatus, Original Hanau, in the bath of a
5 g/l of detergent. The laundering bath was prepared in the ratio of 1:15, heated from the
initial temperature of 25°C for 15 minutes to 60°C. The fabrics were laundered at 60°C for 15
minutes up to 9 laundering cycles. The samples were rinsed and dried in a Scholl drier for 45
minutes at 40°C. For the purpose of better visibility and review of the results, only the results
after the 1st, 3rd, 6th and 9th laundering cycle are presented. Laundering was done with the
addition of fluorescent whitening agent and UV absorber in different concentration over
weight of detergent. FWA was applied in concentrations of 0.08%, 0.16% and 0.25%; UV
absorber in concentration of 0.20%, and in combination 0.10% of each.

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 Light Conversion for UV Protection by Textile Finishing and Care 1577

Methods

The fabric UV protection was determined according to AS/NZS 4399:1996 Sun

Protective Clothing: evaluation and classification. UV-A and UV-B transmission through
fabric were measured on Spectrophotometer Cary 50 (Varian). This instrument measures
sunlight transmission in the range from 280 to 400 nm. The irradiation applied is a simulation
of a part of sunlight spectrum, as measured at noon on January 17th 1990 in Melbourne,
Australia, while the results obtained indicate the degree of protection offered by the fabric
when worn directly to the skin.
The ultraviolet protection factor (UPF) was calculated automatically according to:

400

 E(  )   (  )  
  280
UPF  400
(1)
 E(  )  T (  )   (  )  
  280

where:

E() = Solar radiation [W m-2 nm-1]

() = relative erythemal spectral effectiveness
T() = spectrum permeability at wavelength 
 = measured wavelength interval [nm]

UPF indicate the ability of fabrics to protect the skin against sun burning saying how
much longer a person can stay in the sun with the fabric covering the skin as compared with
the uncovered skin to obtain same erythemal response.3-20 According to the standards
excellent protection is if UPF is higher 40 (Table 3). However, for the countries with UV
index 7-10 as Mediterranean countries, Australia and USA, the UPF should be 15 times
higher than UV index [17]. Therefore, it is recommended for people who spend eight hours in
the open to use UV clothing with UPF between 105 and 150 if they want excellent UV
protection.

Table 3. UV protection rating according to AS/NZS 4399:1996

UPF range UPF rating UV-R protection category UV-R blocking [%]
< 14 0, 5, 10 non-rateable <93,3
15-24 15, 20 good 93,3-95,8
25-39 25, 30, 35 very good 95,9-97,4
> 40 40, 45, 50, 50+ excellent > 97,5

Remission spectrophotometer SF 600 PLUS CT (Datacolor) was used for measuring

spectral characteristics of cotton and PES/cotton blend fabrics. CIE whiteness degree (WCIE)
was calculated automatically according to ISO 105-J02:1997 Textiles - Tests for colour
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1578 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

fastness - Part J02: Instrumental assessment of relative whiteness. The discrepancy in wet
state was determined through color differences of color coordinates according to:

 
1
E *ab  H *  L *  C *
2 2 2 2
(2)

whereL* is change in lightness, C* change in chroma and H* change in hue.
The relative intensity of fluorescence (Φrel) was calculated from measured fluorescence
on adapted spectrophotometer Specol SV (Carl Zeiss). Illuminant is high voltage Hg bulb
( max = 366 nm). Fluorescent Reference Standard (Datacolor) was used for Φrel. standard = 40,
with amplifying of 200x.

RESULTS AND DISCUSSION

The UV protective properties of cotton and cotton/polyester blend fabrics achieved by
light conversion of fluorescent compounds applied in textile finishing and care was
researched in this chapter. For that purpose, three stilbene derivatives fluorescent compounds
were selected: FWA for cellulosic materials – one commonly used in textile finishing and the
other in detergent formulations; and UV absorber. UV protection was determined in vitro
through Ultraviolet protection factor, UPF. Additionally the influence to fabric whiteness was
researched. Main characteristics of fabrics are collected in Table 4.

Table 4. Main characteristics of cotton (C) and polyester/cotton blend (P/C) fabrics:
Mean UPF, UV-A and UV-B transmission, UV protection rating according to AS/NZS
4399:1996, CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of
remission (Rmax) and wavelength (max)

Fabric Mean UPF UVA UVB UPF rating

C 7.276 16.714 11.969 5: Non-rateable
P/C 18.426 17.148 3.289 15: Good
Fabric WCIE rel Rmax [%] max [nm]
C 74.3 0 86.51 700
P/C 70.7 0 85.34 700

In textile finishing process fluorescent compounds were applied by exhaustion procedure

in wide concentration range. Since the fabric properties change in wet state, the discrepancy
in whiteness and UV protection was research in distilled water as well as Adriatic Sea water.
The UV protective properties of cotton and PES/cotton blend fabric achieved by light
conversion of fluorescent compounds are presented in Tables 5-7. The discrepancy of UV
protection in wet state by distilled (DW) and sea (SW) water is shown in Figures 3-8.
Remission curves of cotton fabric treated with FWA - disodium 4.4’-bis[(4-anylino-6-
morpholino-1.3.5-triazine-2-yl)amino]-stilbene-2.2’-disulphonate (F2) in wide concentration
range as example of Stokes law are presented in Figure 9. CIE whiteness (WCIE), relative
intensity of fluorescence (rel), maximum of remission (Rmax) and wavelength ( max), and the

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 Light Conversion for UV Protection by Textile Finishing and Care 1579

discrepancy of whiteness in wet state of cotton fabrics treated with all fluorescent compounds
are collected in Tables 8-13.

Table 5. Mean UPF, UV-A and UV-B transmission, and UV protection rating according
to AS/NZS 4399:1996 of cotton and PES/cotton fabrics treated with fluorescent
whitening agent - bis(4,4'-triazinylamino)-stilbene-2,2'-disulfonic acid derivative (F1)

Sample Mean UPF UVA UVB UPF rating

C-F1-0,004 9,502 9,472 12,219 5 Non-rateable
C-F1-0,006 9,605 9,407 12,738 5 Non-rateable
C-F1-0,0125 9,379 9,501 11,251 5 Non-rateable
C-F1-0,1 14,005 6,490 5,513 10 Non-rateable
C-F1-0,5 16,219 5,635 4,005 15 Good
C-F1-1 30,061 3,230 2,032 30 Very good
C-F1-10 125,094 0,776 0,439 50+ Excellent
C-F1-50 203,793 0,503 0,333 50+ Excellent
P/C -F1-0,004 35,797 1,366 11,160 30 Very good
P/C -F1-0,006 35,493 1,517 10,231 30 Very good
P/C -F1-0,0125 36,606 1,504 10,185 30 Very good
P/C -F1-0,1 38,589 1,221 9,901 35 Very good
P/C -F1-0,5 45,006 1,383 5,862 45 Excellent
P/C -F1-1 45,122 1,526 4,899 45 Excellent
P/C -F1-10 62,291 1,258 2,883 50+ Excellent
P/C -F1-50 88,355 0,842 1,926 50+ Excellent

Table 6. Mean UPF, UV-A and UV-B transmission, and UV protection rating according
to AS/NZS 4399:1996 of cotton and PES/cotton fabrics treated with fluorescent
whitening agent - disodium 4.4’-bis[(4-anylino-6-morpholino-1.3.5-triazine-2-yl) amino]-
stilbene-2.2’-disulphonate (F2)

Sample Mean UPF UVA UVB UPF rating

C-F2-0,004 9,245 9,776 10,985 5 Non-rateable
C-F2-0,006 9,459 9,600 9,901 5 Non-rateable
C-F2-0,0125 9,690 9,539 8,468 5 Non-rateable
C-F2-0,1 18,578 5,179 2,673 15 Good
C-F2-0,5 62,023 1,554 0,656 50+ Excellent
C-F2-1 63,339 1,589 0,585 50+ Excellent
C-F2-10 497,005 0,268 0,214 50+ Excellent
C-F2-50 548,558 0,208 0,158 50+ Excellent
P/C -F2-0,004 28,589 2,221 9,901 25 Very good
P/C -F2-0,006 31,578 1,833 10,012 30 Very good
P/C -F2-0,0125 32,950 1,552 11,193 30 Very good
P/C -F2-0,1 34,161 2,185 6,341 30 Very good
P/C -F2-0,5 66,276 1,607 0,651 40 Excellent
P/C -F2-1 180,311 0,660 0,291 50+ Excellent
P/C -F2-10 606,725 0,225 0,426 50+ Excellent
P/C -F2-50 98,540 0,836 1,408 50+ Excellent
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1580 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

Table 7. Mean UPF, UV-A and UV-B transmission, and UV protection rating according
to AS/NZS 4399:1996 of cotton and PES/cotton fabrics treated with fluorescent UV
absorber -stilbene disulphonic acid triazine derivative (F3)

Sample Mean UPF UVA UVB UPF rating

C-F3-0,004 12,078 7,389 8,518 10 Non-rateable
C-F3-0,006 11,338 8,118 7,936 10 Non-rateable
C-F3-0,0125 11,022 8,151 9,663 10 Non-rateable
C-F3-0,1 37,246 2,649 2,667 20 Good
C-F3-0,5 90,434 1,057 0,984 50+ Excellent
C-F3-1 122,067 0,867 0,803 50+ Excellent
C-F3-10 424,074 0,260 0,285 50+ Excellent
C-F3-50 1000,000 0,100 0,100 50+ Excellent
P/C –F3-0,004 31,038 1,906 10,376 30 Very good
P/C –F3-0,006 33,758 1,714 9,321 30 Very good
P/C –F3-0,0125 34,136 1,679 9,896 30 Very good
P/C –F3-0,1 41,600 1,470 6,699 40 Excellent
P/C –F3-0,5 81,291 0,773 2,960 50+ Excellent
P/C –F3-1 70,671 1,040 2,671 50+ Excellent
P/C –F3-10 161,614 0,486 1,170 50+ Excellent
P/C –F3-50 160,207 0,536 1,105 50+ Excellent

1000

900

800

700

600
UPF

500

400

300

200

100

0
C F1-0,004 F1-0,006 F1-0,0125 F1-0,1 F1-0,5 F1-1 F1-10 F1-50
Dry 7,28 9,50 9,61 9,38 14,01 16,22 30,06 125,09 203,79
DW 3,79 4,31 4,03 4,37 5,68 7,95 11,02 75,11 713,58
SW 4,34 4,40 4,21 4,32 5,92 7,69 10,94 48,25 633,69

Figure 3. The discrepancy of UV protection in wet state by distilled (DW) and sea (SW) water of cotton
fabric treated with FWA - bis(4,4'-triazinylamino)-stilbene-2,2'-disulfonic acid derivative (F1).

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 Light Conversion for UV Protection by Textile Finishing and Care 1581

1000

900

800

700

600
UPF

500

400

300

200

100

0
C F2-0,004 F2-0,006 F2-0,0125 F2-0,1 F2-0,5 F2-1 F2-10 F2-50
Dry 7,28 9,25 9,46 9,69 18,58 62,02 61,34 497,01 548,56
DW 3,79 4,35 4,20 4,32 7,74 18,87 41,65 967,38 1000,00
SW 4,34 4,24 4,50 4,48 7,75 20,19 40,20 993,92 996,99

Figure 4. The discrepancy of UV protection in wet state by distilled (DW) and sea (SW) water of cotton
fabric treated with FWA - disodium 4.4’-bis[(4-anylino-6-morpholino-1.3.5-triazine-2-yl) amino]-
stilbene-2.2’-disulphonate (F2).

1000

900

800

700

600
UPF

500

400

300

200

100

0
C F3-0,004 F3-0,006 F3-0,0125 F3-0,1 F3-0,5 F3-1 F3-10 F3-50
Dry 7,28 12,08 11,34 11,02 37,25 90,43 122,07 424,07 1000,00
DW 3,79 5,35 5,30 4,56 10,34 58,49 255,49 1000,00 1000,00
SW 4,34 5,32 4,78 5,05 14,49 64,24 282,84 999,70 1000,00

Figure 5. The discrepancy of UV protection in wet state by distilled (DW) and sea (SW) water of cotton
fabric treated with UV absorber - stilbene disulphonic acid triazine derivative (F3).
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1582 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

1000

900

800

700

600
UPF

500

400

300

200

100

0
P/C F1-0,004 F1-0,006 F1-0,0125 F1-0,1 F1-0,5 F1-1 F1-10 F1-50
Dry 18,43 35,80 35,49 36,61 38,59 45,01 45,12 62,29 88,36
DW 20,58 22,53 27,54 24,44 27,27 32,98 39,93 62,49 100,96
SW 20,48 25,02 27,44 23,43 25,68 36,12 45,82 106,86 136,90

Figure 6. Discrepancy of UV protection in wet state – distilled (DW) and sea (SW) water of
polyester/cotton blend fabric treated with fluorescent whitening agent - bis(4,4'-triazinylamino)-
stilbene-2,2'-disulfonic acid derivative (F1).

1000

900

800

700

600
UPF

500

400

300

200

100

0
P/C F2-0,004 F2-0,006 F2-0,0125 F2-0,1 F2-0,5 F2-1 F2-10 F2-50
Dry 18,43 28,589 31,578 32,95 34,161 66,276 180,311 606,725 98,54
DW 20,58 24,66 24,63 24,31 31,98 22,20 43,92 1000,00 395,65
SW 20,48 29,14 24,49 26,46 30,23 21,74 40,21 364,71 85,73

Figure 7. The discrepancy of UV protection in wet state by distilled (DW) and sea (SW) water of
polyester/cotton fabric treated with FWA - disodium 4.4’-bis[(4-anylino-6-morpholino-1.3.5-triazine-2-
yl) amino]-stilbene-2.2’-disulphonate (F2).

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 Light Conversion for UV Protection by Textile Finishing and Care 1583

1000

900

800

700

600
UPF

500

400

300

200

100

0
P/C F3-0,004 F3-0,006 F3-0,0125 F3-0,1 F3-0,5 F3-1 F3-10 F3-50
Dry 18,43 34,14 33,76 31,04 41,60 81,29 70,67 161,61 160,21
DW 20,58 30,70 23,55 29,42 44,02 72,35 63,51 394,42 308,80
SW 20,48 22,88 28,00 25,95 30,29 65,13 74,02 369,08 346,25

Figure 8. The discrepancy of UV protection in wet state by distilled (DW) and sea (SW) water of
polyester/cotton fabric treated with UV absorber - stilbene disulphonic acid triazine derivative (F3).

140
R [%]

120

100

80

60

C C-F2-0,004 C-F2-0,006
40
C-F2-0,0125 C-F2-0,1 C-F2-0,5

20
C-F2-1 C-F2-10 C-F2-50

 [nm]
0
400 420 440 460 480 500 520 540 560 580 600

Figure 9. Remission curves of cotton fabric treated with FWA - disodium 4.4’-bis[(4-anylino-6-
morpholino-1.3.5-triazine-2-yl) amino]-stilbene-2.2’-disulphonate (F2) in wide concentration range.
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1584 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

Table 8. CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of

remission (Rmax) and wavelength (max), and the discrepancy of whiteness in wet state of
cotton fabrics treated with FWA - bis(4,4'-triazinylamino)-stilbene-2,2'-disulfonic acid
derivative (F1)

Fabric WCIE rel Rmax max dE* Discrepancy

[%] [nm]
C 74.3 0 86.51 700 - -
C-DW 61.7 0 83.25 700 2.633 Darker yellow
C-SW 62.9 0 83.47 700 2.413 Darker yellow
C-F1-0.004 77.4 0 85.83 700 - -
C-F1-0.004-DW 67.4 0 83.28 700 2.147 Darker yellow
C-F1-0.004-SW 66.7 0 82.95 700 2.428 Darker yellow
C-F1-0.006 79.0 4.56 85.95 700 - -
C-F1-0.006-DW 69.8 4.23 83.32 700 2.072 Darker yellow
C-F1-0.006-SW 69.7 4.25 83.31 700 2.107 Darker yellow
C-F1-0.0125 83.4 8.85 85.88 700 - -
C-F1-0.0125-DW 76.3 7.28 83.42 700 1.787 Darker less blue
C-F1-0.0125-SW 73.6 7.02 82.78 700 2.336 Darker redder less blue
C-F1-0.1 109.1 27.72 96.84 440 - -
C-F1-0.1-DW 109.4 27.99 95.29 440 2.021 Darker redder bluer
C-F1-0.1-SW 109.4 27.90 95.45 440 1.855 Darker redder bluer
C-F1-0.5 127.1 38.18 106.37 440 - -
C-F1-0.5-DW 127.6 37.80 105.35 440 1.895 Darker redder bluer
C-F1-0.5-SW 130.2 39.32 107.18 440 1.999 Darker redder bluer
C-F1-1 135.9 40.98 112.60 440 - -
C-F1-1-DW 139.1 45.99 113.26 440 2.306 Darker redder bluer
C-F1-1-SW 138.8 45.31 113.43 440 2.146 Darker redder bluer
C-F1-10 143.4 51.53 118.55 440 - -
C-F1-10-DW 146.0 55.15 120.55 440 1.598 Darker redder bluer
C-F1-10-SW 143.0 50.53 118.73 440 1.470 Darker bluer
C-F1-50 132.2 48.73 114.07 440 - -
C-F1-50-DW 134.3 49.29 115.02 440 1.855 Darker redder bluer
C-F1-50-SW 126.7 43.37 110.89 440 1.689 Darker less red less blue

Table 9. CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of

remission (Rmax) and wavelength (max), and the discrepancy of whiteness in wet state of
cotton fabrics treated with FWA - disodium 4.4’-bis[(4-anylino-6-morpholino-1.3.5-
triazine-2-yl) amino]-stilbene-2.2’-disulphonate (F2)

Fabric WCIE rel Rmax max dE* Discrepancy

[%] [nm]
C-F2-0.004 84.7 4.64 85.95 700
C-F2-0.004-DW 77.5 4.22 83.47 700 1.798 Darker less blue
C-F2-0.004-SW 76.1 4.20 83.36 700 2.304 Darker redder less blue
C-F2-0.006 93.0 16.67 88.16 440 - -
C-F2-0.006-DW 86.2 14.44 83.59 440 1.787 Darker redder bluer
C-F2-0.006-SW 84.4 12.98 83.55 700 1.576 Darker redder bluer

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Fabric WCIE rel Rmax max dE* Discrepancy

[%] [nm]
C-F2-0.0125 100.5 27.22 91.95 440 - -
C-F2-0.0125-DW 97.1 22.24 88.97 440 1.833 Darker redder bluer
C-F2-0.0125-SW 94.5 17.76 87.88 440 2.152 Darker redder bluer
C-F2-0.1 134.3 47.07 111.15 440 - -
C-F2-0.1-DW 136.0 49.93 111.15 440 1.857 Darker less green bluer
C-F2-0.1-SW 134.6 46.74 110.27 440 1.450 Darker bluer
C-F2-0.5 147.4 60.66 120.68 440 - -
C-F2-0.5-DW 148.7 62.75 121.00 440 1.756 Darker bluer
C-F2-0.5-SW 147.7 61.53 120.75 440 1.526 Darker less red bluer
C-F2-1 148.1 62.09 122.24 440 - -
C-F2-1-DW 148.2 62.60 122.25 440 1.623 Darker bluer
C-F2-1-SW 147.9 58.87 122.35 440 2.500 Darker less red bluer
C-F2-10 98.4 39.68 105.29 460 - -
C-F2-10-DW 104.6 42.32 105.75 460 2.924 Darker less green bluer
C-F2-10-SW 101.3 40.30 105.04 460 2.419 Darker less green bluer
C-F2-50 82.3 20.01 95.51 460 - -
C-F2-50-DW 72.8 19.93 91.63 460 2.148 Darker less green yellow
C-F2-50-SW 60.0 14.78 87.41 460 4.283 Darker yellow

Table 10. CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of
remission (Rmax) and wavelength (max), and the discrepancy of whiteness in wet state of
cotton fabrics treated with UV absorber - stilbene disulphonic acid triazine derivative
(F3)

Fabric WCIE rel Rmax max dE* Discrepancy

[%] [nm]
C-F3-0.004 93.1 12.20 88.73 440 - -
C-F3-0.004-DW 91.7 11.61 86.49 440 1.916 Darker redder bluer
C-F3-0.004-SW 85.4 9.65 83.76 440 1.945 Darker less blue
C-F3-0.006 96.4 13.72 92.96 440 - -
C-F3-0.006-DW 89.0 11.42 89.69 440 1.721 Darker redder
C-F3-0.006-SW 90.5 12.79 88.24 440 1.922 Darker redder less blue
C-F3-0.0125 102.0 17.27 90.17 440 - -
C-F3-0.0125-DW 98.4 13.75 85.48 440 1.887 Darker redder bluer
C-F3-0.0125-SW 95.6 13.23 85.66 440 1.809 Darker redder bluer
C-F3-0.1 130.0 39.39 107.87 440 - -
C-F3-0.1-DW 131.6 40.61 107.83 440 1.595 Darker redder bluer
C-F3-0.1-SW 131.5 40.60 107.87 440 1.697 Darker redder bluer
C-F3-0.5 146.9 56.95 120.13 440 - -
C-F3-0.5-DW 147.0 58.68 120.21 440 1.411 Darker bluer
C-F3-0.5-SW 144.2 55.55 118.37 440 1.559 Darker less red
C-F3-1 149.2 62.49 122.80 440 - -
C-F3-1-DW 147.1 59.66 121.67 440 1.385 Darker less red
C-F3-1-SW 144.9 56.55 120.59 440 1.579 Darker less red less blue
C-F3-10 99.9 24.20 105.67 460 - -
C-F3-10-DW 66.3 17.44 96.41 460 6.897 Darker greener less blue
C-F3-10-SW 68.1 19.50 97.00 460 6.509 Darker greener less blue
C-F3-50 71.8 13.66 100.18 460 - -
C-F3-50-DW 16.3 4.48 85.38 460 11.017 Darker greener yellow
C-F3-50-SW 15.3 3.83 84.91 460 11.173 Darker greener yellow
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Table 11. CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of
remission (Rmax) and wavelength (max), and the discrepancy of whiteness in wet state of
polyester/cotton blend fabrics treated with FWA - bis(4,4'-triazinylamino)-stilbene-2,2'-
disulfonic acid derivative (F1)

Fabric WCIE rel Rmax max dE* Discrepancy

[%] [nm]
P/C 70.7 0 85.34 700 - -
P/C-DW 62.1 0 83.38 700 1.766 Darker yellow
P/C-SW 61.5 0 83.33 700 1.880 Darker greener yellow
P/C-F1-0.004 75.1 5.23 85.36 700 - -
P/C-F1-0.004-DW 68.5 5.00 83.52 700 1.627 Darker less red yellow
P/C-F1-0.004-SW 67.9 4.93 83.30 700 1.743 Darker less red yellow
P/C-F1-0.006 76.4 6.86 85.28 700 - -
P/C-F1-0.006-DW 67.4 5.55 83.29 700 1.928 Darker less red yellow
P/C-F1-0.006-SW 69.1 5.85 83.45 700 1.661 Darker yellow
P/C-F1-0.0125 77.8 7.65 85.47 700 - -
P/C-F1-0.0125-DW 71.1 7.23 83.45 700 1.617 Darker yellow
P/C-F1-0.0125-SW 70.6 6.92 83.62 700 1.593 Darker yellow
P/C-F1-0.1 91.6 17.76 86.76 440 - -
P/C-F1-0.1-DW 90.3 15.43 85.09 440 1.152 Darker redder bluer
P/C-F1-0.1-SW 89.3 15.09 84.92 440 0.935 Darker redder
P/C-F1-0.5 106.3 18.98 94.12 440 - -
P/C-F1-0.5-DW 106.2 18.82 93.28 440 1.046 Darker redder bluer
P/C-F1-0.5-SW 107.5 19.54 93.45 440 1.524 Darker redder bluer
P/C-F1-1 112.6 20.48 98.07 440 - -
P/C-F1-1-DW 115.1 25.74 98.51 440 1.438 Darker redder bluer
P/C-F1-1-SW 114.7 23.91 98.02 440 1.629 Darker redder bluer
P/C-F1-10 126.9 51.51 107.92 440 - -
P/C-F1-10-DW 132.4 50.14 109.86 440 1.984 Darker redder bluer
P/C-F1-10-SW 132.5 49.53 110.28 440 1.937 Darker redder bluer
P/C-F1-50 124.7 48.73 107.95 440 - -
P/C-F1-50-DW 130.7 49.29 111.29 440 2.196 Darker redder bluer
P/C-F1-50-SW 128.9 38.67 110.00 440 2.103 Darker redder bluer

Table 12. CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of
remission (Rmax) and wavelength (max), and the discrepancy of whiteness in wet state of
polyester/cotton blend fabrics treated with FWA - disodium 4.4’-bis[(4-anylino-6-
morpholino-1.3.5-triazine-2-yl) amino]-stilbene-2.2’-disulphonate (F2)

Fabric WCIE rel Rmax max dE* Discrepancy

[%] [nm]
P/C-F2-0.004 78.3 4.44 85.30 700 - -
P/C-F2-0.004-DW 72.4 4.01 83.57 700 1.401 Darker yellow
P/C-F2-0.004-SW 72.3 3.86 83.70 700 1.421 Darker yellow
P/C-F2-0.006 92.8 14.63 87.55 440 - -
P/C-F2-0.006-DW 83.6 11.98 83.36 700 2.035 Darker less red less blue
P/C-F2-0.006-SW 86.2 12.78 83.32 440 1.743 Darker less red less blue

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Fabric WCIE rel Rmax max dE* Discrepancy

[%] [nm]
P/C-F2-0.0125 98.2 17.65 85.40 440 - -
P/C-F2-0.0125-DW 93.8 15.24 83.77 440 1.315 Darker less blue
P/C-F2-0.0125-SW 94.0 15.66 83.69 440 1.374 Darker less blue
P/C-F2-0.1 112.2 43.07 97.78 440 - -
P/C-F2-0.1-DW 114.1 44.43 97.88 440 1.266 Darker redder bluer
P/C-F2-0.1-SW 113.1 45.01 97.31 440 1.290 Darker redder bluer
P/C-F2-0.5 148.7 60.52 121.66 440 - -
P/C-F2-0.5-DW 148.5 59.74 121.14 440 1.626 Darker less red bluer
P/C-F2-0.5-SW 148.1 58.53 120.84 440 1.882 Darker less red bluer
P/C-F2-1 148.6 57.22 122.66 440 - -
P/C-F2-1-DW 148.6 57.30 122.67 440 1.710 Darker bluer
P/C-F2-1-SW 147.2 53.77 121.92 440 1.623 Darker less red bluer
P/C-F2-10 97.1 49.68 101.34 460 - -
P/C-F2-10-DW 100.3 42.02 104.18 460 1.915 Darker less green bluer
P/C-F2-10-SW 100.4 41.33 99.60 460 2.309 Darker less green bluer
P/C-F2-50 80.7 23.01 91.92 460 - -
P/C-F2-50-DW 85.6 29.18 92.98 460 1.933 Darker less green less yellow
P/C-F2-50-SW 80.2 22.73 91.91 460 1.140 Darker less green less yellow

Table 13. CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of
remission (Rmax) and wavelength (max), and the discrepancy of whiteness in wet state of
polyester/cotton blend fabrics treated with UV absorber - stilbene disulphonic acid
triazine derivative (F3)

Fabric WCIE rel Rmax max dE* Discrepancy

[%] [nm]
P/C-F3-0.004 82.2 11.00 85.19 700 - -
P/C-F3-0.004-DW 75.5 9.63 83.29 700 1.552 Darker yellow
P/C-F3-0.004-SW 75.6 9.90 83.18 700 1.547 Darker yellow
P/C-F3-0.006 89.5 13.31 86.02 440 - -
P/C-F3-0.006-DW 85.5 12.27 83.59 700 1.432 Darker redder less blue
P/C-F3-0.006-SW 86.3 12.75 83.66 700 1.244 Darker redder less blue
P/C-F3-0.0125 96.3 14.28 84.41 440 - -
P/C-F3-0.0125-DW 90.3 13.35 83.85 700 1.321 Darker less blue
P/C-F3-0.0125-SW 91.7 13.86 83.58 700 1.392 Darker redder less blue
P/C-F3-0.1 107.6 22.38 95.51 440 - -
P/C-F3-0.1-DW 106.9 20.64 94.39 440 1.170 Darker redder bluer
P/C-F3-0.1-SW 106.5 18.59 94.28 440 1.016 Darker redder bluer
P/C-F3-0.5 123.7 36.93 105.18 440 - -
P/C-F3-0.5-DW 127.2 45.60 106.43 440 1.565 Darker redder bluer
P/C-F3-0.5-SW 125.7 43.59 106.21 440 1.297 Darker redder bluer
P/C-F3-1 126.5 47.42 107.63 440 - -
P/C-F3-1-DW 129.4 46.44 109.47 440 1.317 Darker bluer
P/C-F3-1-SW 131.2 46.53 110.52 440 1.430 Darker bluer
P/C-F3-10 95.2 23.42 99.07 460 - -
P/C-F3-10-DW 76.7 17.44 93.96 460 3.807 Darker greener less blue
P/C-F3-10-SW 67.0 15.43 92.22 460 5.810 Darker greener less blue
P/C-F3-50 74.6 16.66 95.52 460 - -
P/C-F3-50-DW 39.9 7.83 87.61 460 7.130 Darker greener yellow
P/C-F3-50-SW 32.3 5.82 84.86 460 8.490 Darker greener yellow
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Table 14. Mean UPF, UV-A and UV-B transmission of cotton fabrics treated with
fluorescent compounds in laundering - 1st, 3rd, 6th and 9th washing cycle with ECE
referent detergent

Sample Mean UPF UVA UVB Mean UPF UVA UVB

1st 3rd
C-ECE 7.134 17.056 12.270 7.441 15.515 11.754
C-F2-0.08 8.326 11.694 10.601 11.038 5.972 8.038
C-F2-0.12 8.929 9.575 10.054 14.078 3.978 6.369
C-F2-0.25 9.943 7.762 9.292 19.314 2.468 4.568
C-F3-0.2 14.674 8.196 5.955 41.424 2.380 2.057
C-F2-0.1+F3-0.1 11.498 8.356 7.539 30.264 2.382 2.845
6th 9th
C-ECE 7.135 15.660 12.253 6.970 15.750 12.765
C-F2-0.08 17.575 3.404 5.284 20.845 2.601 4.406
C-F2-0.12 22.768 2.178 3.772 29.965 1.437 2.959
C-F2-0.25 48.240 1.229 1.958 61.834 0.859 1.484
C-F3-0.2 89.895 1.450 0.982 124.911 0.823 0.652
C-F2-0.1+F3-0.1 57.638 1.484 1.474 85.133 0.935 1.093

Table 15. Mean UPF, UV-A and UV-B transmission of PES/cotton fabrics treated with
fluorescent compounds in laundering - 1st, 3rd, 6th and 9th washing cycle with ECE
reference detergent

Sample Mean UPF UVA UVB Mean UPF UVA UVB

st
1 3rd
P/C-ECE 21.034 15.818 2.641 23.013 23.66 13.709
P/C-F2-0.08 27.411 10.643 2.336 39.416 5.812 1.717
P/C-F2-0.12 23.645 10.781 2.938 29.857 6.367 2.451
P/C-F2-0.25 27.319 8.069 2.515 48.332 3.522 1.558
P/C-F3-0.2 34.295 8.223 1.848 37.759 4.895 1.898
P/C-F2-0.1+F3-0.1 24.039 9.200 2.662 42.915 4.271 1.689
6th 9th
P/C-ECE 25.368 13.096 2.207 23.729 13.600 2.423
P/C-F2-0.08 45.893 4.579 1.558 43.958 4.100 1.704
P/C-F2-0.12 30.475 5.503 2.474 30.483 5.108 2.560
P/C-F2-0.25 50.035 3.401 1.542 51.041 2.961 1.601
P/C-F3-0.2 51.245 4.092 1.449 48.923 3.657 1.610
P/C-F2-0.1+F3-0.1 47.058 3.926 1.582 44.036 3.736 1.786

In textile care fluorescent compounds were added to ECE referent detergent or

commercial detergent. It was applied separately or in combination through 9 washing cycles
at 60°C. The achieved UV protections of cotton and PES/cotton blend fabric are presented
through Mean UPF, UV-A and UV-B transmission in Tables 14-17. CIE whiteness (WCIE),
relative intensity of fluorescence (rel), maximum of remission (Rmax) and wavelength ( max)
achieved by repeated laundering are shown in Tables 18-21.

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Table 16. Mean UPF, UV-A and UV-B transmission of cotton fabrics treated with
fluorescent compounds in laundering - 1st, 3rd, 6th and 9th washing cycle with commercial
detergent

Sample Mean UPF UVA UVB Mean UPF UVA UVB

1st 3rd
C-commerc. 9.742 12.351 8.893 10.914 8.708 8.236
C-F2-0.08 11.315 8.921 7.369 16.066 3.990 5.589
C-F2-0.12 10.342 8.923 8.249 16.677 3.415 5.416
C-F2-0.25 13.350 6.175 6.166 29.390 1.564 2.932
C-F3-0.2 18.551 7.473 4.627 49.628 2.042 1.610
C-F2-0.1+F3-0.1 13.189 7.980 6.527 57.378 1.488 1.576
6th 9th
C-commerc. 11.797 7.255 7.584 15.753 6.233 5.739
C-F2-0.08 25.343 2.038 3.704 35.565 2.348 2.562
C-F2-0.12 28.376 1.608 3.197 43.904 2.431 2.038
C-F2-0.25 55.566 0.768 1.601 95.917 1.630 0.926
C-F3-0.2 172.094 0.599 0.509 233.725 1.486 0.329
C-F2-0.1+F3-0.1 101.630 0.711 0.879 175.466 1.579 0.473

Table 17. Mean UPF, UV-A and UV-B transmission of PES/cotton fabrics treated with
fluorescent compounds in laundering - 1st, 3rd, 6th and 9th washing cycle with commercial
detergent

Sample Mean UPF UVA UVB Mean UPF UVA UVB

st
1 3rd
P/C-commerc. 27.308 13.774 2.036 27.408 11.821 2.288
P/C-F2-0.08 26.200 11.901 2.156 32.072 7.495 1.995
P/C-F2-0.12 31.386 10.127 1.801 34.703 6.213 1.744
P/C-F2-0.25 35.318 7.956 1.844 34.593 5.138 2.033
P/C-F3-0.2 28.711 10.934 2.009 41.130. 5.320 1.629
P/C-F2-0.1+F3-0.1 22.038 11.623 2.802 30.837 6.508 2.492
6th 9th
P/C-commerc. 32.068 9.239 1.832 31.309 9.872 2.010
P/C-F2-0.08 40.072 4.893 1.743 40.819 6.003 1.827
P/C-F2-0.12 50.499 3.736 1.343 47.836 5.054 1.528
P/C-F2-0.25 42.789 3.462 1.746 46.379 4.433 1.660
P/C-F3-0.2 60.181 2.974 1.209 51.863 4.583 1.483
P/C-F2-0.1+F3-0.1 38.520 4.092 1.964 43.131 4.655 1.896

The impact of fluorescent compounds – FWAs and UV absorber on the UV protection of

cotton and polyester/cotton blend fabrics was monitored by the UV-A and UV-B transmission
and UPF. From Table 7 it is evident that high effects in textile cleaning of genetic and added
impurities such are waxes, protein substances, pectin and other during scouring and bleaching
in peroxide baths, where pigments are removed [42-44], leads to white cotton. On the other
hand, it leads to low UV protection as well (Table 4). Therefore, chemically bleached cotton
fabric (C) is non-rateable for UV protection since UPF is 7.28. Polyester/cotton blend (P/C)
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1590 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

fabric gives off good UV protection (UPF=18.43 due to the present benzene ring in PES
fibres that absorbs UV radiation.

Table 18. CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of
remission (Rmax) and wavelength (max) of cotton fabrics treated with fluorescent
compounds in laundering - 1st, 3rd, 6th and 9th washing cycle with ECE detergent

WCIE rel Rmax max WCIE rel Rmax max

Sample
[%] [nm] [%] [nm]
1st 3rd
C-ECE 80,00 - 82,71 700 81,39 - 82,49 700
C-F2-0,08 103,23 - 92,39 440 127,49 - 101,85 440
C-F2-0,12 115,32 - 95,03 440 135,94 - 105,17 440
C-F2-0,25 128,11 - 102,88 440 147,45 - 112,33 440
C-F3-0,2 116,75 - 98,65 440 142,34 - 110,13 440
C-F2-0,1+F3-0.1 124,05 - 99,55 440 145,22 - 111,54 440
6th 9th
C-ECE. 80,04 - 85,58 700 79,79 - 85,58 700
C-F2-0,08 138,28 - 115,59 440 146,86 - 122,07 440
C-F2-0,12 147,59 - 121,95 440 154,64 - 127,95 440
C-F2-0,25 156,35 - 129,59 440 160,66 - 134,63 440
C-F3-0,2 149,03 - 123,42 440 155,42 - 129,31 440
C-F2-0,1+F3-0.1 153,87 - 127,45 440 157,67 - 131,52 440

Table 19. CIE whiteness (WCIE), relative intensity of fluorescence (rel),

maximum of remission (Rmax) and wavelength (max) of polyester/cotton fabrics treated
with fluorescent compounds in laundering - 1st, 3rd, 6th and 9th washing
cycle with ECE detergent

WCIE rel Rmax max WCIE rel Rmax max

Sample
[%] [nm] [%] [nm]
1st 3rd
P/C-ECE 76,20 - 82,71 700 76,83 - 82,49 700
P/C-F2-0,08 101,41 - 92,39 440 117,12 - 101,85 440
P/C-F2-0,12 106,36 - 95,03 440 122,28 - 105,17 440
P/C-F2-0,25 119,11 - 102,88 440 131,97 - 112,33 440
P/C-F3-0,2 112,22 - 98,65 440 129,40 - 110,13 440
P/C-F2-0,1+F3-0.1 114,09 - 99,55 440 131,61 - 111,54 440
6th 9th
P/C-ECE 80,50 - 83,44 700 83,73 - 83,19 700
P/C-F2-0,08 125,37 - 106,77 440 129,06 - 109,45 440
P/C-F2-0,12 130,51 - 110,62 440 134,05 - 113,44 440
P/C-F2-0,25 136,24 - 115,58 440 136,88 - 116,52 440
P/C-F3-0,2 133,57 - 112,64 440 135,36 - 112,75 440
P/C-F2-0,1+F3-0.1 134,90 - 113,70 440 135,80 - 115,18 440

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Table 20. CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of
remission (Rmax) and wavelength (max) of cotton fabrics treated with fluorescent
compounds in laundering - 1st, 3rd, 6th and 9th washing cycle with commercial detergent

WCIE rel Rmax max WCIE rel Rmax max

Sample
[%] [nm] [%] [nm]
1st 3rd
C-commerc. 95.36 0 86,32 700 103.80 0 86,04 700
C-F2-0,08 105.33 3,58 96,92 440 124.14 18,83 108,06 440
C-F2-0,12 110.91 8,60 99,93 440 129.88 24,60 111,66 440
C-F2-0,25 123.44 17,25 107,43 440 139.14 39,30 118,46 440
C-F3-0,2 112.33 13,99 100,36 440 136.64 28,16 115,86 440
C-F2-0,1+F3-0.1 110.83 17,99 99,84 440 138.50 32,59 117,95 440
6th 9th
C-commerc. 112.45 32,91 86,56 700 115.24 27,38 86,72 700
C-F2-0,08 140.66 65,63 116,47 440 143.88 45,22 120,53 440
C-F2-0,12 147.56 78,30 121,27 440 150.06 46,50 125,55 440
C-F2-0,25 153.36 89,43 126,91 440 154.34 51,64 130,55 440
C-F3-0,2 150.25 76,55 125,49 440 153.24 44,81 129,06 440
C-F2-0,1+F3-0.1 151.67 82,79 126,78 440 154.83 54,03 130,34 440

Table 21. CIE whiteness (WCIE), relative intensity of fluorescence (rel), maximum of
remission (Rmax) and wavelength (max) of polyester/cotton fabrics treated with
fluorescent compounds in laundering - 1st, 3rd, 6th and 9th washing cycle
with commercial detergent

WCIE rel Rmax max WCIE rel Rmax max

Sample
[%] [nm] [%] [nm]
1st 3rd
P/C-commerc. 85.85 0 85,51 700 96.83 0 85,19 700
P/C-F2-0,08 100.20 2.06 93,26 440 115.28 9,61 102,34 440
P/C-F2-0,12 103.79 2,14 95,21 440 118.81 12,56 104,37 440
P/C-F2-0,25 112.37 12,76 100,39 440 125.70 22,03 109,75 440
P/C-F3-0,2 102.39 4,15 97,39 440 121.77 16,82 105,85 440
P/C-F2-0,1+F3-0.1 105.22 4,62 94,57 440 122.91 18,83 106,53 440
6th 9th
P/C-commerc. 109.52 18,43 85,84 700 104.23 21,17 86,13 700
P/C-F2-0,08 136.60 49,08 108,71 440 129.32 34,23 111,80 440
P/C-F2-0,12 143.16 51,90 111,89 440 133.67 38,54 115,46 440
P/C-F2-0,25 149.62 63,29 116,25 440 135.73 40,06 118,91 440
P/C-F3-0,2 146.44 64,06 116,16 440 135.71 44,91 118,35 440
P/C-F2-0,1+F3-0.1 147.27 53,86 115,28 440 136.31 45,12 118,57 440

From the results in Tables 5 and 6 can be seen that fluorescent whitening agent applied
even in small concentration leads to higher whiteness and higher UPF. By absorbing UV-A
radiation optical brightened fabrics transform this radiation to blue fluorescence what leads to
excellent UV protection in higher concentrations. Optical brightening due to its fluorescence
contributes to the fabric high whiteness and beauty in optimal range of concentration. That is
the concentration of fluorescent compound at which the maximum of Φrel or WCIE are
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observed [16,39]. From the results of the whiteness and fluorescence of FWA treated cotton
fabrics it can be seen that FWA concentration of 1% (up to 10% owf) in relation to mass of
material is the optimum concentration for bis(4,4'-triazinylamino)-stilbene-2,2'-disulfonic
acid derivative (F1), whilst for polyester/cotton blend fabric is 10% owf.
In the case of disodium 4.4’-bis[(4-anylino-6-morpholino-1.3.5-triazine-2-yl)amino]-
stilbene-2.2’-disulphonate (F2) optimal concentration for cotton and polyester/cotton fabric is
0.5-1% owf. Since this UV absorber is fluorescent compound as well, having similar
chemical composition (stilbene disulphonic acid derivative) as FWA, optimum concentration
for UV absorber was determined as well, and it is 0.5-1% owf (Table 7). At the low
concentrations of all fluorescent compounds – FWAs and UV absorber, blue fluorescence
neutralizes the yellowness of bleached fabric giving the high luminosity and “most beautiful”
white. Applied in the higher concentration than optimal one, from results of remission and
wavelength maximums can be seen that the change in emission spectrum occurred. It is a
consequence of well-known bathochromic shift of the remission spectrum (see Figure 9). It
comes to a reduction of remission intensity with FWA and/or UV absorber’s concentrations
causing the extinction of fluorescence by quenching phenomenon, with a consequence of
yellowness.
Cotton fabrics of the highest FWAs concentration have the highest UPF in dry state.
Similar to the results of the cotton fabrics whiteness and fluorescence, at the optimal
concentration of FWA excellent UV protection has been achieved. For bis(4,4'-
triazinylamino)-stilbene-2,2'-disulfonic acid derivative (F1), UPF > 50 was achieved at
concentration 10% owf (UPFF1-0.5=125.094); whilst in the case of disodium 4.4’-bis[(4-
anylino-6-morpholino-1.3.5-triazine-2-yl)amino]-stilbene-2.2’-disulphonate (F2) at 0.5% owf
(UPFF2-0.5=62.023). More benzene rings in the structure of the stilbene-type contribute to
better UV protection. That confirms that FWA insures high protection of UV radiation.
By treating cotton fabric with an UV absorber in the wide concentration range protective
effect is more enhanced. UV absorber offers excellent UV protection if applied in optimal
concentration of 0.1% owf or higher. That is because UV absorbers absorb damaging UV-R
range of 290 nm to 360 nm, and convert it into harmless heat energy. For difference of
FWA’s UV absorbers offer UV-B protection as well. However, the fabrics with the highest
intensity of fluorescence do not show the highest UPF values. In dry state, UV protection
increase with fluorescent compound concentration, regardless of quenching phenomenon.
Treatment of the polyester/cotton blend fabric with fluorescent compounds shows similar
behavior as cotton ones. The presence of benzene ring in PES fibres results in good UV
protection of polyester/cotton blend (P/C) fabric. For that reason, treatment of the
polyester/cotton blend fabric results in very good UV protection for even smallest
concentrations of fluorescent compounds applied. Since these applied fluorescent compounds
are primary for the cellulosic fibers, only the cotton in the blend absorbed it. Therefore, in the
higher concentrations of applied compounds, excellent UV protection has been achieved, but
UPF value is lower than for cotton fabrics.
Washing fabrics with only the detergent, UPF stays mainly the same, and fabrics are non-
rateable UV protection (Tables 14-17). Since commercial detergent contained the soap that
fluorescence as well (can be seen after 6th cycle – Tables 20-21), UPF is little bit higher, but
fabrics are still without UV protection.
UPF significantly grows when washed in a detergent containing fluorescent compounds.
The influence of fluorescent whitening agent in initial launderings on the UPF shows that

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cotton fabrics are non-rateable for UV protection after the first laundering cycle regardless of
applied detergent with fluorescent whitening agents added, and even when an UV absorber is
added to the detergent as well. However, even small addition of disodium 4.4’-bis[(4-anylino-
6-morpholino-1.3.5-triazine-2-yl)amino]-stilbene-2.2’-disulphonate (F2) increase UPF from
7.28 to UPF = 8.32 for ECE detergent and to UPF = 11.31 for commercial detergent.
Application of UV absorber (F3) results in UPF of 14.67 for ECE detergent and to UPF =
18.55 for commercial one. To obtain better UV protection the treatment should be performed
in the course of finishing. UV absorber offers excellent UV protection (UPF>40) after three
laundering cycles regardless of applied detergent, whilst for achieving excellent UV
protection with the fluorescent whitening agent six cycles are necessary.
Polyester/cotton blend fabric shows similar behaviour as cotton ones. The growth of UPF
is not so much prominent as with cotton fabrics, as the fluorescent compounds used act on the
cotton part of the PES/cotton blend only. However, very good UV protection can be achieved
after 1 laundering cycle regardless of applied detergent. The excellent UV protection can be
achieved applying stilbene type FWA (F2) after 6 laundering cycles and/or UV absorber (F3).
The obtained results confirm the well-known fact that fluorescent whitening agents
applied in textile finishing or added into detergents in laundering manage to keep and even
improve basic whiteness degree. This phenomenon is present even when lower concentration
of fluorescent whitening agent is used than recommended. Obviously, whiteness degree
grows with higher concentration. In the case of laundered samples with no fluorescent
whitening agents, it can be seen that basic whiteness degree is somewhat higher, which is due
to the perborate degradation of residual pigments on the cotton fabric. This occurs after the
first laundering cycle already and is more prominent with every succeeding laundering cycle.
It can be seen that higher remission is obtained after the 9th laundering cycle when higher
concentration of fluorescent whitening agent is employed (0.25%), as compared to lower
concentration (0.08%). High whiteness degrees are achieved in the combination of UV
absorbers and fluorescent whitening agents. From the Tables 18-21 it can be seen that the
wavelength of Rmax did not changed after multiple laundering. Stilbene fluorescent whitening
agent, as well as stilbene UV absorber, showed remission maximum at 440 nm. After
repeated laundering and accumulation did not occur bathochromic shift, which confirmed that
the whiteness and UV protective effects stay retain.
Somewhat lower degrees of whiteness are achieved on PES/cotton fabrics than on cotton
fabric. The reason for that is in the fact that PES fibres cannot be optically brightened with
applied FWAs. Since in this research stilbene derivatives were applied in laundering and
finishing, PES fibres were not brightened, and the positive effects are achieved on the cotton
component only. Therefore, the achieved effects on PES/cotton blend cannot compare to the
ones achieved on 100% cotton fabric.
Considering the results of discrepancy of whiteness shown in Tables 9-11 it can be
observed that all fabrics get darker when wet and in general bluer and redder. The reason for
that is lower reflection of light from the fabric. In dry fabric, some of the photons of light are
absorbed, but some are reflected and land on the eye's retina what gives the sensation of
seeing a certain level of brightness. But when the fabric gets wet, the water fills in the
interyarn spacing. When the light falls on the wet fabric, some of it enters the water at one
angle and refracts at other because the light waves travel at a slower speed in water than it
does in air. Fewer photons of light get back to the eyeball, and therefore the wet fabric
"appears" darker than the dry one. When the water gradually evaporates, more and more light
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1594 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

is reflected back to the eyeball, and can be seen brighter again. The amount of refraction,
referred to as the refractive index, is affected by both the salinity and temperature of the
water, and therefore there is a difference between fabrics treated with sea and distilled water.
It is to point out that the salts in Sea water act as quenchers of fluorescence as well.
Therefore, for the highest concentration of applied FWAs - disodium 4.4’-bis[(4-anylino-6-
morpholino-1.3.5-triazine-2-yl)amino]-stilbene-2.2’-disulphonate (F2) 50% owf, degree of
whiteness falls from 82.3 to 60.0. This phenomenon is even more evident for UV absorber
applied in concentrations higher than optimal one. The whiteness for applied 10% owf
decreases from 99.9 to 77.9, and for 50% owf from 71.8 to low 15.3.
Considering the different refractive index in water and in the air it is differently reflected
from the surface as explained before. Because of higher and scattered reflection, transmission
is lower for both water applied, distilled and sea water, resulting in higher UPF values
(Figures 3-8). It can be said that in wet state cotton knit fabrics treated with fluorescent
compounds give off better UV protection than in dry state regardless of the concentration and
type of fluorescent compound applied. This phenomenon is more enhanced for Sea water,
since the refractive index increases with salinity increment and decrease of temperature. That
can be explained by that some of the Sun’s radiant energy is reflected from the water surface;
it is not absorbed, but additionally scattered by molecules suspended in the water, whilst the
other part penetrates the water’s surface, absorb and converse to other forms of energy, such
as heat that warms or evaporates water, or is used by plants to fuel photosynthesis.
Considering the applied concentrations, in general it can be said that higher concentration of
fluorescent compound applied to cotton and polyester/cotton blend fabrics, better UV
protection was achieved in wet state.
It was observed that for the concentrations lower than optimal one, UV protection in wet
state is lower or similar, whilst for the higher concentrations it gets significantly higher. The
achieved UV protection is excellent regardless of the drop and can even obey that request
regarding UV index during the summer time in Mediterranean countries, as well as Australia
and USA, which acquire UPF>UV index*15.
The concentration of 0.08% addition to detergent is the same amount as 0.004% owf
applied in textile finishing, etc. The concentration of fluorescent compound accumulated in 9
laundering cycles is similar to the concentration of 0.1% owf in textile finishing. Therefore,
the comparison of two procedures was done and it is shown in Figures 10 and 11.
As it can be seen in Figure 10, UV protection of cotton fabrics treated with fluorescent
whitening agent - disodium 4.4’-bis[(4-anylino-6-morpholino-1.3.5-triazine-2-yl)amino]-
stilbene-2.2’-disulphonate (F2) is similar regardless of procedure. However, if UV absorber
was applied as addition to laundry detergent it is more efficient, than if applied in the same
concentration in textile finishing. Considering 9x higher concentration and/or accumulation
through 9 washing cycles it is evident that in accumulation UV protection gets significantly
higher. It is to point out that application to commercial detergent resulted in higher UPF than
if applied ECE reference detergent, as a result of fluorescence of the soap in commercial
detergent composition.
On the other hand, the differences in UV protection of polyester/cotton blend fabrics
(P/C), shown in Figure 11, are not so enhanced. The reason for that is in chemical
composition of applied fluorescent compounds. All of them are stilbene derivatives, which
are for application to cellulosic fibers, and therefore, only cotton absorbed it.

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 Light Conversion for UV Protection by Textile Finishing and Care 1595

250
Textile Finishing
Textile Care-ECE detergent
Textile Care - Commercial Detergent
200

150
UPF

100

50

0
C C-F2-0,004 C-F2-0,006 C-F2-0,0125 C-F2-0,1 C-F3-0,0125 C-F3-0,1

Figure 10. The differences in UV protection of cotton fabrics (C) as a result of different application of
fluorescent compounds – FWA (F2) and UV absorber (F3) in textile finishing or textile care.

250
Textile Finishing
Textile Care - ECE detergent
Textile Care - Commercial Detergent
200

150
UPF

100

50

0
P/C P/C-F2-0,004 P/C-F2-0,006 P/C-F2-0,0125 P/C-F2-0,1 P/C-F3-0,0125 P/C-F3-0,1

Figure 11. The differences in UV protection of polyester/cotton blend fabrics (P/C) as a result of
different application of fluorescent compounds – FWA (F2) and UV absorber (F3) in textile finishing
or textile care.
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1596 Tihana Dekanić, Anita Tarbuk, Tanja Pušić et al.

CONCLUSION

Primary prevention and early detection are essential regarding deduction of melanoma
incidence. Considering prevention, especially in childhood and adolescence, it is necessary to
apply sunscreening lotions and wear adequate clothing. Chemically bleached cotton and
polyester/cotton blend fabrics are non-rateable for UV protection. Treatment with fluorescent
compounds, FWA and UV absorber leads to its multifunctionality - high whiteness,
neutralization of yellowness, giving to the fabric the high luminosity and protection against
UV radiation. The fluorescence contributes to the high whiteness and beauty in optimal
concentration. In the range of higher concentration quenching of fluorescence occurs,
resulting in fabric yellowness. In wet state, regardless of applied water – sea or distilled,
fabrics get darker, lowering its whiteness, but because of reflection from water, better UV
protection is achieved. This phenomenon is more evident for Sea water, because of additional
light scattering since it contains about 40% of inorganic salts. UV protection and optical
effects of fabrics laundered in ECE and commercial detergent containing fluorescent
compounds of stilbene type has been achieved by light conversion and cumulative addition
through 9 washing cycles. For that reason, it is to suggest application of these compounds for
prevention of skin cancer incidence, especially in commercial detergent formulation for
protection of wider population.

ACKNOWLEDGMENTS
The authors would like to acknowledge the University of Zagreb for financial support to
research “Optical and Protective Potential of Fluorescent Compounds in Cotton Material
Finishing and Care” (KFPI 1, TP1.89).

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Derived Inorganic-organic Hybrid Polymers Filled with ZnO Nanoparticles as
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[36] Sundaresan, K., Sivakumar A., Vigneswaran, C., Ramachandran, T. (2012). Influence
of nano titanium dioxide finish, prepared by sol-gel technique, on the ultraviolet
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treatment for cotton fabrics. Text Res J 74:97–10.
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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 72

THE POTENTIAL OF MYCOSPORINE-LIKE

AMINO ACIDS AS UV-SUNSCREENS

Rajesh P. Rastogi1, Ravi R Sonani1, Datta Madamwar1

and Aran Incharoensakdi2,*
1
BRD School of Biosciences, Vadtal Road, Satellite Campus,
Sardar Patel University, Anand, Gujarat, India
2
Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry,
Faculty of Science, Chulalongkorn University, Bangkok, Thailand

ABSTRACT
Strong ultraviolet (UV) radiation is one of the most lethal and carcinogenic
exogenous agents that can interact with and alter the normal life processes by means of
its direct or indirect damaging effects. Mycosporine-like amino acids (MAAs) are
important ‘multipurpose’ small biomolecules that provide protection from intense UV
radiation without producing the reactive oxygen species (ROS). A number of MAAs have
been reported from different taxonomic groups. MAAs have great potential in
photoprotection and genome maintenance by minimizing the cellular damage from UV-
induced ROS and thymine dimer formation. Moreover, due to strong UV-
absorbing/screening function, photo-induction, strong antioxidant properties and
resistance to abiotic stressors, MAAs are considered as natural photoprotectants that may
be biotechnologically exploited in cosmetics and other pharmaceutical industries. In the
present article, an attempt has been made to critically review and highlight the recent
updates on various MAAs with respect to their function as potential UV-sunscreens.

Keywords: Mycosporine-like amino acids, UV radiation, oxidative stress, photoprotectants,

sunscreens

*
Corresponding author: Tel.: +66 2 218 5422; fax: +66 2 218 5418 (A. Incharoensakdi), E-mail: [email protected]
(A. Incharoensakdi).
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1602 Rajesh P. Rastogi, Ravi R Sonani, Datta Madamwar et al.

1. INTRODUCTION
The increase in short wavelength solar ultraviolet (UV) radiation on the Earth’s
atmosphere due to anthropogenically increased ozone depleting substances has aroused
tremendous concern about its negative impacts on living organisms in both aquatic as well as
terrestrial ecosystems. UV radiation (280-400 nm) may affect several biochemical and
physiological processes leading to loss of normal life functionality of photosynthetic and non-
photosynthetic organisms including human beings (Rastogi and Sinha, 2011a). It has been
established that solar UV-A radiation (315-400 nm) has less direct effects on living systems
since native DNA of living organisms cannot absorb UV-A. However, UV-A can indirectly
affect the cellular function by the generation of reactive oxygen species (ROS) via
photosensitizing reactions. In contrast, UV-B (280-315 nm) radiation has direct effects on key
cellular machinery such as proteins and nucleic acids (Rastogi et al., 2010a). The short
wavelength solar UV radiation may cause protein modification, membrane disruption,
enzyme inactivation, generation of DNA lesions, alteration of transcription and translation
process, mutagenesis and several other external effects such as sunburn and skin cancer
(Bruce and Brodland, 2000; Rastogi et al., 2010a).
Moreover, several defense mechanisms have been reported in diverse organisms to
counteract the harmful effects of UV radiation (Rastogi and Sinha, 2011b). Biosynthesis of
certain UV-absorbing/screening compounds in different organisms is considered as an
effective mode of defense mechanisms to reduce the damaging effects of solar UV radiation
(Karentz et al., 1991; Rastogi et al., 2010b; Carreto and Carignan, 2011). Mycosporine-like
amino acids (MAAs) are considered as prominent photoprotectants that provide
photoprotection against harmful UV radiation (Cockell and Knowland, 1999; Oren and
Gunde-Cimerman, 2007). In the present chapter, we summarize the eco-biological importance
of the biologically relevant molecules MAAs, with special emphasis on their UV-screening or
photoprotective functions.

2. SOLAR ULTRAVIOLET RADIATION AND BIOLOGICAL EFFECTS

In the past few decades, loss in stratospheric ozone layer due to anthropogenically
released ozone depleting substances such as chlorofluorocarbons (CFCs) and reactive
nitrogen species such as nitrous oxide (N2O) has generated tremendous concern about the
increasing level of short wavelength UV radiations reaching the Earth’s atmosphere
(Ravishankara et al., 2009; Manney el., 2011; Cabrol et al., 2014; Williamson et al., 2014). In
all the groups of UV radiation (280–400 nm), UV-B radiation produces more adverse effects
on diverse habitats, despite the fact that most of the extraterrestrial UV-B is absorbed by the
stratospheric ozone layer (McKenzie et al., 2003). It has been established that UV-C (100-280
nm) radiation is quantitatively absorbed by oxygen and ozone in the Earth’s atmosphere, and
does not show any harmful effects on our ecosystems. Furthermore, UV-A (315-400 nm)
radiation has low efficacy in inducing the DNA damage, as it is not absorbed by native DNA
molecules; however, it can damage DNA via indirect photosensitizing reactions by the
formation of reactive oxygen species (Rastogi et al., 2010a). The data obtained from
European Light Dosimeter Network (Eldonet) dosimeters (Häder et al., 1999) have revealed

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 The Potential of Mycosporine-Like Amino Acids as UV-Sunscreens 1603

the extreme UV-B irradiance in different parts of the Earth (Jacovides et al., 2009; Cabrol et
al., 2014). Several other environmental factors such as aerosols and various tropospheric
pollutants, cloud cover, sun-angle and surface reflectants also affect the intensity of UV-B
radiation reaching the Earth’s surface to a certain extent (Madronich et al., 1998). UV
radiation also induces single- and/or double-strand DNA breaks in various organisms
(Rastogi et al., 2010a; Rastogi and Sinha, 2011a).
Moreover, the global climate change and increase in harmful short wavelength UV
radiation can affect the normal life processes of all organisms inhabiting the aquatic or
terrestrial habitats (Häder et al., 2014, 2015). In addition to UV effects on photosynthetic life
(Rastogi et al., 2013; Rastogi et al., 2014a), a number of UV-induced effects such as
occurrence of melanoma and non-melanoma skin cancer, sunburn, photo-allergy, eye
disorders and immune suppression have also been reported in humans (McAteer et al., 1998;
Lima-Bessa et al., 2008) (Figure 1). Solar UV-B radiation damages cellular DNA inducing
mainly cyaclobutane purine/pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone
photoproducts (6–4 PPs) and their Dewar isomers (Rastogi et al., 2010a). DNA double strand
breaks (DSBs) may lead to loss of genetic information. Increased production of UV-induced
ROS may alter the configuration of cell structure, lipids, proteins and DNA molecules, all of
which are the cause of a number of human diseases (Valko et al., 2007). It has been shown
that CPDs inhibit the progress of microbial and mammalian DNA polymerases (Britt, 1999).
Moreover, UV-A-induced production of CPDs has also been observed in bacteria as well as
in eukaryotic cells (Douki et al., 2003; Courdavault et al., 2004; Rastogi et al., 2014b). In
response to detrimental effects of UV radiation, some organisms have developed certain
defense mechanisms such as DNA repair and synthesis of UV-protective compounds to
mitigate the harmful effects of short wavelength solar radiation (Rastogi et al., 2014c).

3. UV-ABSORBING COMPOUNDS
A number of UV-absorbing/screening biomolecules such as mycosporines/mycosporine-
like amino acids (MAAs), scytonemin, melanins, carotenoids, flavonoids, parietin and usnic
acid, have been reported to be synthesized by diverse organisms (Figure 2) to counteract the
detrimental effects of solar UV-B radiation (Rastogi et al., 2010b). There have been a number
of reviews about diverse classes of photoprotective compounds from natural sources (Karsten
et al., 2000; Bjerke et al., 2002; Gauslaa and McEvoy, 2005; Rastogi et al., 2010b; Rastogi
and Incharoensakdi, 2013; 2014a, 2014b, 2014c); however, their application as
photoprotectants and development of cosmeceutical products has only partially been
elucidated. In the present review, we focus on the occurrence, biosynthesis and commercial
application as potential sunscreens of the important UV-absorbing/screening compounds,
MAAs from various sources.
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1604 Rajesh P. Rastogi, Ravi R Sonani, Datta Madamwar et al.

Figure 1. Effects of solar UV radiation on human health (details in text).

A B Me
O H
H H
N N O
O H
R1 R2 N
N
O
OH H O
OR3 H
R1
C D 1 2
OH

HO O R2
OH

OH
R3 3
OH R4 O
HO
E F
OH O OH
OH O 4

MeO Me
O
Me 5 OH O
G H Me
Me COCH3

HO O OH
N O
H
H COCH3
6 7

Figure 2. UV-absorbing/screening compounds (1 to 7) from different plant sources (A, B-The

cyanobacterium Anabaena sp. and Lynnbya sp., respectively; C-Brown algae; D-Green algae; E-Fungi;
F-Lichens; G-Bryophytes; H-tea leaves) [1 to 7: general structure of MAA, scytonemin, higher plant
flavonoid, luteolin, parietin, lycopodine, and usnic acid, respectively].

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 The Potential of Mycosporine-Like Amino Acids as UV-Sunscreens 1605

3.1. Mycosporine-like Amino Acids

Mycosporine-like amino acids are small, colorless and highly hydrophilic compounds.
They consist of cyclohexenone or cyclohexenimine chromophores, conjugated with the
nitrogen substituent of an amino acid or its imino alcohol. The ring system of MAA includes
a glycine subunit at the 3C atom, though some MAAs also contain sulfate esters or glycosidic
linkages through the imine substituents (Wu Won et al., 1997). The UV absorption spectra of
different MAAs differ due to variations in the attached side groups and nitrogen substituents
(Böhm et al., 1995; Wu Won et al., 1997). Moreover, the precise stereostructure of MAAs
(except palythine and palythene), including amino acids substituents is not completely
elucidated. MAAs are extremely hydrophilic due to their zwitter ionic form derived from the
amino acid substitution. Further hydrophilicity can also be increased by modification with
sulfonic acids or sugar molecules (Böhm et al., 1995; Wu Won et al., 1997). Presently, more
than 25 MAAs (Figure 3) have been reported from diverse organisms; however, a number of
other UV-absorbing/screening compounds with potential application as UV photoprotectants
remain to be explored from nature.

3.2. Occurrence and Distribution of MAAs

Among various UV-photoprotectant biomolecules, the biosynthesis of different MAAs

has been reported from diverse taxonomic groups (Sinha et al., 2007; Rastogi et al., 2010b)
(Table 1). Cyanobacteria are the most dominant photoautotrophs that can synthesize a range
of different MAAs (Shibata, 1969). The MAAs shinorine and porphyra-334 have been found
to be the most dominant MAAs in several species of terrestrial or fresh/marine water
cyanobacteria (Karsten and Garcia-Pichel, 1996; Sinha et al., 2003; Volkmann et al., 2006;
Rastogi et al., 2010b; Chuang et al., 2014; Rastogi et al., 2012). Rastogi and Incharoensakdi
(2014a, b, c) have isolated and characterized some primary MAAs from different
cyanobacteria inhabiting diverse habitats (Figure 4). Several MAAs were isolated from
different species/strains of cyanobacteria inhabiting the hot springs (Rastogi et al., 2012).
Two novel glycosylated MAAs such as a pentose-bound porphyra-334 derivative (MW 478
Da; UVλmax: 335 nm) and other MAAs (MW1050 Da; UVλmax: 312 and 340 nm) consisting
of two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen and
two pentose and hexose sugars were identified in Nostoc commune (Matsui et al., 2011).
Recently, the glycosylated MAAs such as glycosylated porphyra-334 (MW 508 Da; UVλmax:
334 nm) and palythine-threoninehave (MW 612 Da; UVλmax: 322 nm) have also been
reported in the cyanobacterium Nostoc commune (Nazifi et al., 2013).
Besides cyanobacteria, a range of MAAs have also been reported in cyanobacterial
lichens (Coba et al., 2009), micro/macro algae and animals (Sinha et al., 2007) Table 1). A
number of microalgae/phytoplankton such as diatoms (Riegger and Robinson, 1997;
Hernando et al., 2002; Ingalls et al., 2010), dinoflagellates (Klisch and Häder, 2000;
Banaszack and Trench, 2001; Laurion et al., 2004; Banaszak et al., 2006; Laurion and Roy,
2009), chlorophytes (Gröniger and Häder, 2002; Llewellyn and Airs, 2010; Rastogi and
Incharoensakdi, 2013), and prymnesiophytes (Marchant et al., 1991; Riegger and Robinson,
1997; Hannach and Sigleo, 1998) have been found to produce different MAAs. Several
species of macroalgae (Karsten et al., 1998a; Hoyer et al., 2002) belonging to chlorophyceae
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1606 Rajesh P. Rastogi, Ravi R Sonani, Datta Madamwar et al.

(Han and Han, 2005), rhodophyceae (Kräbs et al., 2004; Coba et al., 2009; Yuan et al., 2009;
Cardozo et al., 2011) and phaeophyceae have been reported to synthesize an array of MAAs
and other UV-absorbing compounds.

O O NH NH H3C
NH
OCH 3 OH OCH 3 OCH3 OH OCH 3
OH OCH3

HO HO
HO NH NH HO NH
NH HO HO NH
HO
SO3H HOH 2C COOH
COOH HOH2C COOH
COOH

❶ ❷ ❸ ❹ ❺
H3C
H3C
CO 2H HOOC
NH N
OCH3 N N N
OH OCH3
OH OCH3 OCH 3 OH OCH3
HO HO OH
HO NH NH
HO
HO HO
H3C HO NH HO NH
COOH HO NH
COOH
OH COOH
COOH
COOH
❻ ❼ ❽ ❾ ❿
HOOC HOOC HOOC H3C
N N
H3C H3C H3C
N N NH CH3 OCH3 OCH3
OH OCH 3 CH3 OCH 3 OCH 3
HO HO
HO HO NH NH
HO HO HO
NH NH HO NH
HO HO
COOH COOH
COOH COOH COOH
⓫ ⓬ ⓭ ⓮ ⓯
OH
C H 3 O C H
CH3 OCH3
H 3 H
N N COOH
h e x o se N N H HO
OH N CH3 O
COOH
O CH3
C O O H
HO
HO
O H O O
HO NH O hexose
OH h e x o se
⓰ ⓱ ⓲

Figure 3. Chemical structure of some important MAAs found in different taxonomic groups. [1 to 18:
Mycosporine-taurine, Mycosporine-glycine, Palythine, Palythine-serine, Mycosporine-methylamine-
serine, Mycosporine-methylamine-threonine, Asterina-330, Palythinol, Mycosporine-2-glycine,
Shinorine, Porphyra-334, Mycosporine-glycine-valine, Palythenic acid, Usujirene, Palythene,
Euhalothece-362, Glycosylated palythine-threonine and Glycosylated porphyra-334, respectively].

A B 1.6 a C 3.0 a

2.0
Absorbance

b
1.2 c
Absorbance

1.0
0.8
0.0
250 275 300 325 350 375 400
0.4 Wavelength [nm]
b
c
0.0
0 2 4 6 8 10 12 14
Retention time [min]

Figure 4. The cyanobacterium Lyngbya sp. (A) and high performance liquid chromatograph (B)
showing the UV-absorption maxima (B-inset) of some MAAs such as palythine (a, UV λ max: 320 nm),
asterina-330 (b, UV λmax: 330 nm) and an unknown MAA, M-312 (c, UV λmax: 312) (Adapted from
Rastogi and Incharoensakdi, 2014c).

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 The Potential of Mycosporine-Like Amino Acids as UV-Sunscreens 1607

Table 1. Occurrence and distribution of some common MAAs

in different taxonomic groups

Mycosporine-like amino acids

Taxonomic

MGV
M2G
MG

MT

DL
SH
PR
PE

PL

PT
AS

US

PS
groups

Cyanobacteria + + + + + + + + + +
Green algae + + + + + +
Brown algae + + + + + +
Red algae + + + + + + +
Microalgae/phytoplankton + + + + + + + + + + + +
Copepods + + + + + + + +
Corals + + + + + + + +
Krill + + + + + + + +
Amphipod + + + + + + + +
Molluscs + + + + + + + +
Fish + + + + + + + +
Sea Anemones + + + + + + + + +
Sponge + + + + + + +
(For details please see the references, Sinha et al., 2007; Rastogi et al., 2014c; Rastogi et al., 2014 a,
Rastogi et al., 2010b for details and references therein) [AS, asterina-330; MG,mycosporine-
glycine; M2G, mycosporine-2-glycine; MT, mycosporine-taurine; MGV, mycosporineglycine-
valine; PE, palythene; PR, porphyra-334; PL, palythinol; PT, palythine; SH, shinorine; US,
usurijene; DL, dehydroxylusujirene; PS, palythine-serine]

The accumulation of several MAAs have also been reported in different animals such as
arthropods, rotifers, molluscs, fishes, cnidarians, tunicates, eubacteriobionts, poriferans,
nemertineans, echinodermates, platythelminthes, polychaetes, bryozoans and protozoans
(Sinha et al., 2007). However, it has been thought that MAAs are not directly synthesized by
animals, but rather accumulated in these animals as a result of dietary food acquisition
(Carroll and Shick, 1996; Newman et al., 2000).

3.3. Regulation of MAAs Biosynthesis

The biosynthesis of MAAs in fungi and cyanobacteria are supposed to occur via the first
part of the shikimate pathway, 3-dehydroquinate (DHQ) formed during the early part of the
shikimate pathway. The DHQ molecule is assumed to act as a precursor for the synthesis of
primary MAA, via gadusol or deoxygadusol (Portwich and Garcia-Pichel, 2003). Moreover,
the main steps of the MAAs biosynthetic pathway and their genetic basis have recently been
elucidated in Anabaena variabilis ATCC 29413 (Balskus and Walsh, 2010). A cluster of four
genes (Ava_3858-3855) was found in the cyanobacterium Anabaena variabilis, responsible
for the biosynthesis of a MAA shinorine using the common pentose-phosphate-pathway
intermediate sedoheptulose-7-phosphate via 4-deoxygadusol (Balskus and Walsh, 2010;
Spence et al., 2012). The dehydroquinate synthase homologue Ava_3858 and the O-
methyltransferase Ava_3857 was found to convert the precursor into 4-deoxygadusol (4-DG),
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1608 Rajesh P. Rastogi, Ravi R Sonani, Datta Madamwar et al.

and the ATP-grasp homologue Ava_3856 converts the 4-DG as well as glycine into
mycosporine-glycine (M-Gly) (Figure 5). The MAA M-Gly is converted into shinorine with
addition of serine molecule catalyzed by a nonribosomal peptide synthetase (NRPS) encoded
by Ava_3855 (Figure 5). Recently, the MAA biosynthetic gene cluster has also been reported
in some other cyanobacteria such as Nostoc punctiforme (Gao and Garcia-Pichel, 2011) and
Aphanothece halophytica (Waditee-Sirisattha et al., 2014). In Nostoc punctiforme ATCC
29133, the four gene cluster such as NpR5600, NpR5599, NpR5598, and NpF5597 was
shown to catalyze the biosynthesis of M-Gly in a similar manner as was found in A.
variabilis. However, the NpF5597 was found to encode D-Ala-D-Ala ligase and the direction
of transcription of NpF5597 was opposite to those of NpR5600 and NpR5598 (Gao and
Garcia-Pichel, 2011). In the halotolerant cyanobacterium Aphanothece halophytica, the MAA
gene cluster was capable of synthesizing mycosporine-2-glycine. A unique MAA core 4-DG-
synthesizing gene was separated from three other genes. The identified genes, Ap3857,
Ap3856, and Ap3855, were homologous to Ava_ 3857/NpR5599, Ava_3856/NpR5598, and
NpF5597, respectively. It was found that A. halophytica does not contain a gene homologous
to Ava_3855 encoding NRPS, but contains a gene homologous to NpF5597 encoding D-Ala-
D-Ala ligase (Waditee-Sirisattha et al., 2013).

3.4. MAA Biosynthesis under Different Abiotic Factors

A number of abiotic factors, such as short wavelength UV radiations, temperature,

desiccation, light/dark periods, salt concentrations and various nutrients affect the
biosynthesis of MAAs in cyanobacteria and other organisms (Rastogi et al., 2010b). Several
studies have established the increased production of MAAs under different wavebands of UV
radiation in cyanobacteria (Sinha et al., 2003; Singh et al., 2008a; Rastogi et al., 2010c;
Rastogi and Incharoensakdi, 2014a, b, c; Rastogi and Incharoensakdi, 2013, 2015) and
micro/macroalgae or phytoplanktons (Riegger and Robinson, 1997; Klisch and Häder, 2002).
The production of MAAs in various organisms are highly responsive to UV-B radiation
(Sinha et al., 2001; Singh et al., 2008a; Rastogi and Incharoensakdi, 2015; 2014a, c) (Figure
6); howevr, UV-A induced production of MAAs has also been reported in different organisms
(Kräbs et al., 2004; Rastogi et al., 2010c). Korbee et al. (2005a) studied the accumulation of
MAAs in Porphyra leucosticte and found the favorable role of blue light in the accumulation
of porphyra-334, palythine and asterina-330 in contrast to the accumulation of shinorine
which was observed under white, green, yellow or red light. Contrary to light period, the
biosynthesis of MAAs was found to decrease under dark period (Rastogi et al., 2010c;
Rastogi and Incharoensakdi, 2015), suggesting an energy-dependent process of MAA
synthesis. The biosynthesis of MAAs is also affected under different nutrient conditions
(Rastogi et al., 2010b). A remarkable decrease in MAA content was found under nitrogen
limitations in the marine dinoflagellates (Litchman et al., 2002). Induction of some MAAs in
Anabaena variabilis PCC 7937 was caused by salt and ammonium in a concentration-
dependent manner (Singh et al., 2008b). Increased biosynthesis of certain photoprotective
compounds under enriched ammonium concentrations was also found in the red alga
Porphyra sp. (Korbee et al., 2005b, Peinado et al., 2004). Bio-conversion of a primary MAA
into a secondary MAA was found under sulfur deficiency in A. variabilis PCC 7937 (Singh et
al., 2010). High concentration of certain UV-absorbing compounds has also been reported

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 The Potential of Mycosporine-Like Amino Acids as UV-Sunscreens 1609

under desiccation (Jiang et al., 2008) and warm temperature (Karsten et al., 1998) in the
Rhodophytes Chondrus crispus and Porphyra haitanensis, respectively. Moreover, MAAs
biosynthetic pathway may be regulated by multiple environmental signals.

Figure 5. A proposed pathway of the biosynthesis of some primary MAAs (Adapted from Balskus and
Walsh, Rastogi et al., 2010b, 2010; Spence et al., 2012) (details in text).

Figure 6. Induction of a 324 nm-MAA (A) and shinorine (B) after different durations of UV-B
irradiation in the green alga Tetraspora sp. and Gloeocapsa sp. (Adapted from Rastogi and
Incharoensakdi, 2013, 2014a).
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1610 Rajesh P. Rastogi, Ravi R Sonani, Datta Madamwar et al.

4. MAAS AS SUNSCREENS: DOMINANT ROLE IN PHOTOPROTECTION

Mycosporine-like amino acids (MAAs) are well-known UV-absorbing/screening
compounds for their crucial role in photoprotection. Several properties such as strong UV-
absorption maxima, high molar extinction coefficients, competence to dissipate absorbed
radiation as heat without producing reactive oxygen species (ROS), UV-induction and
stability under different physicochemical factors such as UV radiation, heat, pH, strong
oxidizing agent and different organic solvents strongly support the MAAs as photoprotective
compounds (Gröniger and Häder, 2000; Whitehead and Hedges, 2005; Conde et al., 2007;
Rastogi and Incharoensakdi, 2014a, 2014c). It has been shown that MAAs provide protection
from UV radiation not only to their producers, but also to primary and secondary consumers
through the food chain (Helbling et al., 2002), and may be considered as a broad-spectrum
UV absorbers/protectors. As discussed above, UV radiation can promote the occurrence of
sunburn on skin cells; the application of UV-absorbing compound may provide protection
against intense solar radiation or sunburns (Drolet and Connor, 1992). MAAs may protect the
skin cells from UV-induced cell death. Recently, Rastogi and Incharoensakdi (2013)
investigated for the first time the photoprotective activities of 324 nm-MAA and 322 nm-
MAA isolated from a green microalga, Tetraspora sp., against UV radiation. It was found
that the MAA porphyra-334 along with shinorine can suppress UV-induced aging in human
skin (Daniel et al., 2004). The MAAs were found to protect eggs of the sea hare Aplysia
dactylomela from UV radiation (Carefoot et al., 1998). The MAAs such as shinorine (SH),
porphyra-334 (P-334) and mycosporine-glycine were found to protect the human fibroblast
cells from UVR-induced cell death (Oyamada et al., 2008).
Some MAAs have been reported to have strong antioxidant or free radical scavenging
capacity (Coba et al., 2007a, 2007b; Oren and Gunde-Cimerman, 2007) (Figure 7A). The
MAA mycosporine-glycine (MG) was found to protect biological systems against
photodynamic damage by quenching singlet oxygen with a high efficiency (Suh et al., 2003).
Some MAAs such as porphyra-334 and shinorine showed high antioxidant activity against
free radicals (Coba et al., 2007a, 2007b) generated from UV radiation (Oren and Gunde-
Cimerman, 2007). The antioxidant activities of the MAAs M-gly and usujilene have also been
reported to inhibit lipid peroxidation (Nakayama et al., 1999; Suh et al., 2003). The presence
of the glycosylated MAAs with radical scavenging activity has been reported in the
cyanobacterium Nostoc commune (Matsui et al., 2011). Recently, Rastogi and Incharoensakdi
(2014a) have found the efficacy of MAAs (shinorine + M-307) from Gloeocapsa sp. as a
potential sunscreen. The MAAs (palythine + asterina + M-312) isolated from Lyngbya sp.
were found to act as strong radical scavenger (Rastogi and Incharoensakdi, 2014c).
Recently, some MAAs have been investigated for their potential role in genome
maintainance (Rastogi, 2010). The UV-absorbing compound isolated from a red alga
Porphyra yezoensis was found to block the production of thymine dimer (T<>T) (Misonou et
al., 2003). Recently, Rastogi (2010) has also found the great efficacy of MAAs in reducing
the most genotoxic and cytotoxic DNA lesions, CPD T<>T (Figure 7B). Moreover, some
synthetic analogues of MAAs, such as tetrahydropyridine derivatives, have been developed
for commercial application as suncare products (Bird et al., 1987; Chalmers et al., 1990; Coba
et al., 2007b). Moreover, due to potent UV protecting capacity, MAAs can be a potential
candidate for the commercial development of suncare products.

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 The Potential of Mycosporine-Like Amino Acids as UV-Sunscreens 1611

Figure 7. Free radical scavenging capacity of MAAs shinorine + M-307 (panel A) (adapted from
Rastogi and Incharoensakdi, 2014a) and inhibition of thymine dimer formation (panel B: data not
published). [in panel A; the samples contain 0.2(A), 0.4(B), 0.83(C) and 1.6 (D) mg/ml MAAs; in panel
B; Control cells exposed to UV without (A) and with 0.05 (B), 0.1 (C), 0.25 (D) and 0.5 (E) mg/ml M-
gly].

CONCLUSION
MAAs are multipurpose secondary compounds reported in a number of taxonomic
groups. These compounds are highly stable against a number of physicochemical factors, act
as strong antioxidants, and are able to prevent cellular as well as genomic damage resulting
from UV-induced ROS. Several studies have established the anti-aging role of MAAs,
minimizing the risk of skin cancer. Overall, MAAs can be considered as high value
compounds that have great potential applications as natural photoprotectants and antioxidants
that can be exploited in cosmetics and pharmaceutical industries for the development of novel
cosmeceuticals.

ACKNOWLEDGMENTS
Rajesh P. Rastogi is thankful to the University Grant Commission (UGC), New Delhi,
India, for financial support in the form of Dr. D. S. Kothari Postdoctoral grant. Aran
Incharoensakdi thanks Chulalongkorn University Ratchadaphiseksomphot Endowment Fund
for financial support on the project “Value-added products and bioenergy from microalgae.”

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In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 73

GUIDELINES FOR SCHOOL PROGRAMS

TO PREVENT SKIN CANCER *

Karen Glanz, Mona Saraiya and Howell Wechsler

SUMMARY
Skin cancer is the most common type of cancer in the United States. Since 1973, new
cases of the most serious form of skin cancer, melanoma, have increased approximately
150%. During the same period, deaths from melanoma have increased approximately 44%.
Approximately 65%–90% of melanomas are caused by ultraviolet (UV) radiation. More than
one half of a person’s lifetime UV exposure occurs during childhood and adolescence
because of more opportunities and time for exposure. Exposure to UV radiation during
childhood plays a role in the future development of skin cancer. Persons with a history of 1
blistering sunburns during childhood or adolescence are two times as likely to develop
melanoma than those who did not have such exposures. Studies indicate that protection from
UV exposure during childhood and adolescence reduces the risk for skin cancer. These
studies support the need to protect young persons from the sun beginning at an early age.
School staff can play a major role in protecting children and adolescents from UV exposure
and the future development of skin cancer by instituting policies, environmental changes, and
educational programs that can reduce skin cancer risks among young persons.
This report reviews scientific literature regarding the rates, trends, causes, and prevention
of skin cancer and presents guidelines for schools to implement a comprehensive approach to
preventing skin cancer. Based on a review of research, theory, and current practice, these
guidelines were developed by CDC in collaboration with specialists in dermatology,
pediatrics, public health, and education; national, federal, state, and voluntary agencies;
schools; and other organizations. Recommendations are included for schools to reduce skin
cancer risks through policies; creation of physical, social, and organizational environments

*
This is an edited, reformatted and augmented version of Morbidity and Mortality Weekly Report, April 26, 2002,
Vol. 51, No. RR-4, issued by the Centers for Disease Control and Prevention.
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1620 Karen Glanz, Mona Saraiya and Howell Wechsler

that facilitate protection from UV rays; education of young persons; professional

development of staff; involvement of families; health services; and program evaluation.

INTRODUCTION
Skin cancer is the most common type of cancer in the United States [1]. Since 1973, the
number of new cases of melanoma, the skin cancer with the highest risk for mortality and one
of the most common cancers among young adults, has increased. The incidence of melanoma
has increased 150%, and melanoma mortality rates have increased by 44% [1]. Because a
substantial percentage of lifetime sun exposure occurs before age 20 years [2, 3] and because
ultraviolet (UV) radiation exposure during childhood and adolescence plays an important role
in the development of skin cancer [2, 4], preventive behaviors can yield the most positive
effects, if they are initiated early and established as healthy and consistent patterns throughout
life. Children spend several hours at school on most weekdays, and some of that time is spent
in outdoor activities. Schools, therefore, are in a position to teach and model healthy
behaviors, and they can use health education activities involving families to encourage sun-
safe behaviors at home. Thus, schools can play a vital role in preventing skin cancer.
This report is one of a series of guidelines produced by CDC to help schools improve the
health of young persons by promoting behaviors to prevent the leading causes of illness and
death [5–8]. The primary audience for this report includes state and local health and
educational agencies and nongovernmental organizations concerned with improving the
health of U.S. students. These agencies and organizations can translate the information in this
report into materials and training programs for their constituents. In addition, CDC will
develop and disseminate materials to help schools and school districts implement the
guidelines. At the local level, teachers and other school personnel, community recreation
program personnel, health service providers, community leaders, policymakers, and parents
may use these guidelines and complementary materials to plan and implement skin cancer
prevention policies and programs. In addition, faculty at institutions of higher education may
use these guidelines to train professionals in education, public health, sports and recreation,
school psychology, nursing, medicine, and other appropriate disciplines.
Although these skin cancer prevention guidelines are intended for schools, they can also
guide child care facilities and other organizations that provide opportunities for children and
adolescents to spend time in outdoor settings (e.g., camps; sports fields; playgrounds;
swimming, tennis, and boating clubs; farms; and recreation and park facilities). These
guidelines address children and adolescents of primary-and secondary-school age
(approximately 5–18 years). The recommendations are based on scientific evidence, medical
and behavioral knowledge, and consensus among specialists in education and skin cancer
prevention. In 2003, CDC will publish a chapter on cancer in its Community Guide to
Preventive Services [9], which will summarize information regarding the effectiveness of
community-based interventions geared to-ward preventing skin cancer.
School-based programs can play an important role in achieving the following national
Health Objectives for the Year 2010 related to skin cancer prevention: 1) increase the
proportion of persons who use at least one of the following protective measures that might
reduce the risk for skin cancer: avoid the sun between 10 a.m. and 4 p.m., wear sun-protective

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 Guidelines for School Programs to Prevent Skin Cancer 1621

clothing when exposed to the sun, use sunscreen with a sun-protection factor (SPF) 15, and
avoid artificial sources of UV light; and 2) reduce deaths from melanoma to <2.5 per 100,000
persons [10].

Burden of Skin Cancer

Skin cancer is the most common type of cancer in the United States [11]. The two most
common kinds of skin cancer — basal cell carcinoma and squamous cell carcinoma — are
highly curable. However, melanoma, the third most common type of skin cancer and one of
the most common cancers among young adults, is more dangerous. In 2001, approximately
1.3 million new cases of basal cell or squamous cell carcinoma were diagnosed with
approximately 2,000 deaths from basal cell and squamous cell carcinoma combined.
Melanoma, by contrast, will be diagnosed in 53,600 persons and will account for 7,400
deaths, more than three fourths of all skin cancer deaths [12].
Basal cell carcinoma, which accounts for 75% of all skin cancers [11], rarely
metastasizes to other organs. Squamous cell carcinoma, which accounts for 20% of all skin
cancers, has a higher likelihood of spreading to the lymph nodes and internal organs and
causing death [13], but these outcomes are also rare. Melanoma is nearly always curable in its
early stages, but it is most likely to spread to other parts of the body if detected late.
Melanoma most often appears on the trunk of men and the lower legs of women, although it
also might be found on the head, neck, or elsewhere [14, 15].
In the United States, diagnoses of new melanomas are increasing, whereas diagnoses of
the majority of other cancers are decreasing [16]. Since 1973, the annual incidence rate for
melanoma (new cases diagnosed per 100,000 persons) has more than doubled, from 5.7 cases
per 100,000 in that year to 14.3 per 100,000 in 1998 [1] (Figure). The rapid increase in annual
incidence rates is likely a result of several factors, including increased exposure to UV
radiation and possibly earlier detection of melanoma [17]. Since 1973, annual deaths per
100,000 persons from melanoma have increased by approximately 44%, from 1.6 to 2.3
(Figure). However, over the course of the 1990s, mortality rates have remained stable,
particularly among women (16,18–19]. Although doctors must report other types of cancer
(including melanomas) to cancer registries, they are not required to report squamous or basal
cell cancer, which makes tracking trends in the incidence of these two cancers difficult.
However, death rates for basal cell and squamous cell carcinoma have remained stable [12].

Risk Factors for Skin Cancer

Excessive Exposure to UV Radiation

Skin cancer is largely preventable by limiting exposure to the primary source of UV
radiation, sunlight. Sunlamps and tanning beds are other sources. Persons with high levels of
exposure to UV radiation are at an increased risk for all three major forms of skin cancer.
Approximately 65%–90% of melanomas are caused by UV exposure [20]. The epidemiology
implicating UV exposure as a cause of melanoma is further supported by biologic
evidence that damage caused by UV radiation, particularly damage to DNA, plays a central
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1622 Karen Glanz, Mona Saraiya and Howell Wechsler

role in the development of melanoma [4]. Total UV exposure depends on the intensity of the
light, duration of skin exposure, and whether the skin was protected by sun-protective
clothing and sunscreen. Severe, blistering sunburns are associated with an increased risk for
both melanoma and basal cell carcinoma. For these cancers, intermittent intense exposures
seem to carry higher risk than do lower level, chronic, or cumulative exposures, even if the
total UV dose is the same. In contrast, the risk for squamous cell carcinoma is strongly
associated with chronic UV exposure but not with intermittent exposure.
The two most important types of UV radiation, UV-A and UV-B radiation, have both
been linked to the development of skin cancer. UV-A rays are not absorbed by the ozone
layer, penetrate deeply into the skin, and cause premature aging and possibly suppression of
the immune system [4, 21, 22]. Up to 90% of the visible changes commonly attributable to
aging are caused by sun exposure. UV-B rays, which are partially absorbed by the ozone
layer, tan and sometimes burn the skin. UV-B radiation has been linked to the development of
cataracts [23–25] and skin cancer. Recommended skin cancer prevention measures protect
against both UV-A and UV-B radiation.

*
Rate is age-adjusted to 1970 U.S. population.
†
1973 Incidence rate: 5.7 per 100,000 persons; 1998 incidence rate: 14.3 per 100,000.
§
1973 Mortality rate: 1.6 per 100,000; 1998 mortality rate: 2.3 per 100,000.
Source: Cancer Statistics Review, 1973–1998.

Figure. Melanoma of the skin (invasive): SEER incidence and U.S. mortality rates*, 1973–1998.

Childhood and Adolescent UV Exposure

Exposure to UV radiation during childhood and adolescence plays a role in the future
development of both melanoma and basal cell cancer [26–32]. For example, the risk for
developing melanoma is related strongly to a history of >1 sunburns (an indicator of intense
UV exposure) in childhood or adolescence [27, 28, 33, 34). Similarly, sunburns during these
periods have been demonstrated to increase the risk for basal cell carcinoma [30, 31].
Childhood is the most important time for developing moles, which is an important risk
factor for skin cancer. Sun exposure in childhood might increase the risk for melanoma by
increasing the number of moles [33]. A study supports the use of sun protection during
childhood to reduce the risk for melanoma in adulthood [35].
Children and adolescents have more opportunities and time than adults to be exposed to
sunlight [36–38] and thus more opportunities for development of skin cancer [4, 39, 40].

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 Guidelines for School Programs to Prevent Skin Cancer 1623

More than one half of a person’s lifetime UV exposure occurs during childhood and
adolescence [3, 41].

Skin Color and Ethnicity

Although anyone can get skin cancer, persons with certain characteristics are particularly
at risk. For example, the incidence of melanoma among whites is approximately 20 times
higher than among blacks [1]. Hispanics appear to be at less risk for melanoma than whites; a
study conducted in Los Angeles, California, indicated that the incidence rates for Hispanics
were 2–3 per 100,000, whereas the rate for non-Hispanic whites was 11 per 100,000 [42]. For
basal cell and squamous cell carcinoma, rates among blacks are 1/80 of the rates among
whites [43].
The ethnic differences in observed rates are attributable mostly to skin color. The color of
the skin is determined by the amount of melanin produced by melanocytes, which also protect
the skin from the damage produced by UV radiation. Although darkly pigmented persons
develop skin cancer on sun-exposed sites at lower rates than lightly pigmented persons, UV
exposure increases their risk for developing skin cancer [44]. The risk for skin cancer is
higher among persons who sunburn readily and tan poorly [45], namely those with red or
blond hair, and fair skin that freckles or burns easily [14, 46, 47].

Moles
The most measurable predictors of melanoma are having large numbers and unusual
types of moles (nevi) [48,49]. Usually not present at birth, moles begin appearing during
childhood and adolescence and are associated with sun exposure. Most moles are harmless
but some undergo abnormal changes and become melanomas. A changing mole, particularly
in an adult, is often indicative of the development of melanoma [45].

Family History
The risk for melanoma increases if a person has 1 first-degree relatives (i.e., mother,
father, brother, and sister) with the disease. Depending on the number of affected relatives,
the risk can be up to eight times that of persons without a family history of melanoma.
Nonetheless, only approximately 10% of all persons with melanoma have a family history of
melanoma [45, 50].

Age
The incidence of skin cancer increases exponentially with age because older persons have
had more opportunities to be exposed to UV radiation and they have diminished capacity to
repair the damage from UV radiation [4, 14, 43]. Approximately one half of all melanomas
occur in persons aged <50 years. Melanoma is one of the most common cancers found in
persons aged <30 years [14]; it is the most common cancer occurring among persons in the
25–29 age group and the third most common in the 20–24 age group [51].

Environmental Factors Affecting UV Radiation

Environmental factors that increase the amount of UV radiation exposure received by
humans include a latitude closer to the equator; higher altitude; light cloud coverage (allows
80% of UV rays to go through the clouds); the presence of materials that reflect the sun (e.g.,
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1624 Karen Glanz, Mona Saraiya and Howell Wechsler

pavement, water, snow, and sand); being outside near noontime (UV-B radiation is highest in
the middle of the day and varies more by time of day than does UV-A); and being outside
during the spring or summer [21, 52]. Ozone depletion could potentially increase levels of
solar radiation at the earth’s surface [53, 54].

Artificial UV Radiation
In 2000, the National Institute of Environmental Health Sciences concluded that
sunlamps and tanning beds are carcinogenic [55]. Although limited, epidemiologic evidence
suggests that a causal relation exists between artificial UV radiation and melanoma [55, 56].
The type and amount of UV radiation emitted from some sunbeds appear to be similar to that
of noontime summer sun, and in some cases, the amount is even higher than the sun would
emit [57]. Artificial UV radiation can substantially damage the skin (i.e., cause sunburn) and
has been linked to ocular melanoma [52, 58]. Sunlamps and tanning beds should be avoided.

Protective Behaviors

Options for skin cancer prevention (Box 1) include limiting or minimizing exposure to
the sun during peak hours (10 a.m.–4 p.m.), especially the 1-hour period closest to the noon
hour (11 a.m.–1:00 p.m. when the UV rays are the strongest), wearing sun-protective
clothing, using sunscreens that have UV-A and UV-B protection, and avoiding sunlamps and
tanning beds. Most medical and cancer organizations advocate the use of similar skin cancer
prevention measures [59]. The American Cancer Society [60], the American Academy of
Dermatology [61, 62], the American Academy of Pediatrics [63], the American Medical
Association [64], and the National Cancer Institute [65] all recommend patient education on
UV radiation avoidance and sunscreen use. The third U.S. Preventive Services Task Force is
revising their guidelines on provider counseling for skin cancer prevention and sunscreen use.

BOX 1. SKIN CANCER PROTECTIVE BEHAVIORS

 Minimize exposure to the sun during peak hours (10 a.m.–4 p.m.).
 Seek shade from the midday sun (10 a.m.– 4 p.m.).
 Wear clothing, hats, and sunglasses that protect the skin.
 Use a broad-spectrum sunscreen (UV-A and UV-B protection) with a sun-
protection factor of 15.
 Avoid sunlamps and tanning beds.

Avoiding the Sun and Wearing Proper Clothing and Sunglasses

Some forms of protection (e.g., avoiding the sun, seeking shade, and wearing sun-
protective clothing) are the first approach toward preventing skin cancer. One study has
demonstrated that wearing sun-protective clothing can decrease the number of moles [66];
another study demonstrated that the protective effect of clothing depends primarily on the
construction of the fabric (a tighter weave permits less UV radiation to reach the skin) [67].
Other important factors include fiber type (natural cotton or Lycra™ transmits less UV
radiation than bleached cotton) and color (darker colors transmit less UV radiation);

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 Guidelines for School Programs to Prevent Skin Cancer 1625

additional factors include whether the fabric is wet or stretched (transmission of UV radiation
increases as the fabric becomes more wet and stretched) [68]. Wide-brimmed hats (>3-inch
brim) and Legionnaire hats (baseball type of hat with attached ear and neck flaps) provide the
best protection for the head, ears, nose, and cheeks [69]. In 2001, the Federal Trade
Commission and the Consumer Safety Product Commission assisted in the development of
voluntary industry standards in the United States for rating the UV protective value of
different types of clothing and of shade structures [70]. These standards should help the
public make informed decisions concerning protection against UV radiation [68, 71].
Sunglasses protect the eyes and surrounding areas from UV damage and skin cancer.
Although no federal regulations exist for sunglasses, the American Academy of
Ophthalmology recommends that sunglasses block 99% of UV-A and UV-B radiation. A
chemical coating applied to the surface of the lens is the protective mechanism; protection
does not correlate with the color or darkness of the lens [72]. Sunglasses can reduce UV
radiation exposure to the eye by 80%, and when combined with a wide-brimmed hat or
Legionnaire hat, UV exposure to the face is reduced by 65% [73].
Shade structures and trees can reduce direct UV radiation, but the protection offered is
dependent on the direct and indirect UV radiation from the surrounding surface (e.g., sand
and concrete) [74,75]. For example, umbrellas with more overhang provide more UV
protection than those with less overhang.

Sunscreens
Sunscreens are an important adjunct to other types of protection against UV exposure.
Using sunscreen is one of the most commonly practiced behaviors for preventing skin cancer.
During the previous decade, new studies have contributed to an increased understanding
of the role of sunscreen in possibly preventing skin cancer. The U.S. Preventive Services
Task Force is revising their recommendations on sunscreen use, but the International Agency
for Research on Cancer has concluded that topical use of sunscreens probably prevents
squamous cell carcinoma of the skin. The group drew no conclusions regarding whether the
use of sunscreens reduces the incidence of basal cell carcinoma or melanoma [76] (Appendix
A).
Clinical trials have demonstrated that sunscreens are effective in reducing the incidence
of actinic keratoses, the precursors to squamous cell carcinoma [77, 78]. One randomized
clinical trial demonstrated that sunscreens are effective in reducing squamous cell carcinoma
itself [79]. Another randomized trial demonstrated that, among children who are at high risk
for developing melanoma, sunscreens are effective in reducing moles, the precursors and
strongest risk factor for melanoma [80]. Unfortunately, many persons use sunscreens if they
intend to stay out in the sun longer, and they reduce the use of other forms of sun protection
(e.g., clothing or hats), thereby, acquiring the same or even a higher amount of UV radiation
exposure than they would have obtained with a shorter stay and no sunscreen [22, 76, 81].
The guidelines in this report recommend 1) using various methods (e.g., avoiding the sun,
seeking shade, or wearing protective clothing) that reduce exposure to the full spectrum of
UV radiation as the first line of protection against skin cancer and 2) using sunscreen as a
complementary measure. In some instances, sunscreens might be the only responsible option.
However, to be effective, sunscreens must be applied correctly (Appendix B). For example,
users should apply sunscreen and allow it to dry before going outdoors and getting any UV
exposure [82, 83]. Similarly, users should reapply sunscreen after leaving the water, sweating,
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1626 Karen Glanz, Mona Saraiya and Howell Wechsler

or drying off with a towel. Use of insufficient quantities of sunscreen [84, 85] or use of a
sunscreen with insufficient protection are other concerns. Manufacturers determine the SPF (a
measure of protection from only UV-B radiation) by applying an adequate amount of
sunscreen (1–2 ounces) on humans and testing under artificial light, which is usually not as
strong as natural light [86]. No government standards measure how much protection
sunscreens provide against UV-A rays.
Few studies have been conducted on sunscreens, despite their widespread use, which
make it difficult to estimate the prevalence of allergies to sunscreens. Skin irritation, rather
than an actual allergic reaction, is one of the more commonly reported adverse events [87].
Because the majority of the commercially available sunscreens are a combination of agents
from various chemical groups, persons who might experience adverse effects should be aware
of the active ingredients and try sunscreens with different ingredients. In previous years, the
most commonly reported allergen was para-aminobenzoic acid (PABA) (rarely used today),
whereas the current two most frequently cited allergens are benzophenone-3 and dibenzoyl
methanes [22].

Prevalence of Behavioral Risk Factors, Sun-Safe Behaviors, and Attitudes Related to

Sun Safety
In the United States, sunbathing and tanning habits were established during the early to
mid-1900s [88, 89], most likely reflecting the increased availability of leisure time and
fashion trends promoting tanned skin [89, 90]. In the late 1970s, the majority of the
population had little knowledge concerning their personal susceptibility to skin cancer and
believed that tanning enhanced appearance and was associated with better health [91]. More
recent reports indicate that many Americans feel healthier with a tan and believe that
suntanned skin is more attractive [36, 92, 93].
In 1992, 53% of U.S. adults were “very likely” to protect themselves from the sun by
practicing at least one protective behavior (using sunscreen, seeking shade, or wearing
sunprotective clothing) [94]. Among white adults, approximately one third used sunscreen
(32%), sought shade (30%), and wore protective clothing (28%). Among black adults, 45%
sought shade, 28% wore sun-protective clothing, and 9% used sunscreen [95]. Sun-protective
behaviors were more common among the more sun sensitive, females, and older age groups
among both whites and blacks.
Sun-safety behaviors might be most difficult to change among preadolescents and
adolescents [96]. Teenagers spend a substantial amount of time outdoors, especially on
weekends and during the summer [97, 98]. Many teenagers believe that a tan is desirable
[92]; only teenagers who know persons with skin cancer or who perceive an increased
personal susceptibility to skin cancer are more likely to use sunscreen [98]. However,
teenagers who practice skin cancer prevention tend to only use sunscreen and to use it
infrequently, inconsistently, and incorrectly [97, 98]. Girls tend to use sunscreen more than
boys, but they also use tanning beds more frequently [97–101].
Sunscreen use by children is correlated positively with use by their parents [87, 102].
Some parents know the risks of skin cancer but do not realize that children are at risk [103,
104]. Some parents believe that a suntan is a sign of good health; others use sunscreen on
their children as their only or preferred skin cancer prevention measure [36, 99, 105–107],
even though other measures (e.g., using shade structures and wearing sunprotective clothing)

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 Guidelines for School Programs to Prevent Skin Cancer 1627

are available. Sometimes parents apply sunscreen on their children incorrectly and
inconsistently [22] (e.g., only after a child has experienced a painful sunburn) [97, 108].

Concerns Regarding Promoting Protection from UV Radiation

Sun-safety measures should not reduce student participation in physical activity. Regular
physical activity reduces morbidity and mortality for multiple chronic diseases. Promoting
lifelong physical activity in schools is a critically important public health and educational
priority [8]. Schools might find it difficult to avoid scheduling outdoor physical activity
programs around the midday hours. These schools can focus their efforts on other sun-safety
measures (e.g., seeking shade; and wearing a hat, protective clothing, or sunscreen), which
can be implemented without compromising physical activity while gradually making feasible
scheduling changes.
In addition, because UV radiation plays a role in the synthesis of vitamin D, the
limitation of UV exposure might be of some concern. This limitation might lead to a decrease
in levels of vitamin D and increase the likelihood that rickets, a disorder involving a
weakening of the bones, will develop in susceptible infants and children. However, the
average age for presentation of rickets is 18 months, and the age groups of concern are
typically infants and toddlers, not school-aged children between 5 and 18 years. Although the
major source of vitamin D is through skin exposure to sunlight, supplementing the diet with
foods (e.g., flesh of fatty fish, eggs from hens fed vitamin D, and vitamin D-fortified milk and
breakfast cereal) can provide enough vitamin D to meet adequate intake requirements [109,
110]. The American Academy of Pediatrics [111] recommends vitamin D supplementation
for breast-fed infants whose mothers are vitamin D deficient or for infants who are not
exposed to adequate sunlight. Infants consuming at least 500ml of vitamin D-fortified
formula per day and older children consuming at least 16 ounces of vitamin D-fortified milk
per day will meet the adequate intake of vitamin D.

GUIDELINES FOR SCHOOL PROGRAMS TO PREVENT SKIN CANCER

Schools as Settings for Skin Cancer Prevention Efforts

Epidemiologic data suggest that several skin cancers can be prevented if children and
adolescents are protected from UV radiation [26–32]. Schools can participate in reducing
exposure of young persons to UV radiation from the sun during school-related activities by
offering education and skill-building activities to reinforce the development of healthful
behaviors. School-based efforts to prevent skin cancer can be more effective in the framework
of a coordinated school health program [112, 113] that includes family and community
participation [114] and builds on the context and current practices in the school and
community. Coordinated school health programs aim to create and support environments
where young persons can gain the knowledge, attitudes, and skills required to make and
maintain healthy choices and habits. These programs integrate health education, a healthy
school environment, physical education, nutrition services, health services, mental health and
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1628 Karen Glanz, Mona Saraiya and Howell Wechsler

counseling services, health promotion programs for faculty and staff, and efforts to integrate
school activities with family and community life [113].
Being aware of existing practices for sun exposure and sun protection among teachers,
staff, and students might help define gaps in optimal sun-safety practices. Careful
observations for a few days might also provide important information concerning students’
use of shade areas and sunscreen at recess or lunch time, and staff’s use of hats, shirts, and
sunglasses. Discussions with students and staff who practice sun-safe behaviors might prove
useful in planning and improving implementation of sun-safety practices.
Skin cancer prevention measures vary in both their ease of adoption and relevance.
Schools should not allow an “all or nothing” approach to undermine the effectiveness of their
skin cancer prevention efforts. For sun-safety protection, a shortsleeve shirt and cap might be
better than no hat and a sleeveless top. Being flexible is important while moving in the
direction of optimal skin cancer prevention environments, policies, and programs.

SKIN CANCER PREVENTION GUIDELINES

These guidelines provide recommendations for skin cancer prevention activities within a
coordinated school health program. In addition, these guidelines are based on scientific
literature, national policy documents, current practice, and theories and principles of health
behavioral change [115]. Schools and community organizations can work together to develop
plans that are relevant and achievable. Sustained support from school staff, students,
communities, state and local education and health agencies, families, institutions of higher
education, and national organizations are necessary to ensure the effectiveness of school skin
cancer prevention activities [116].
In this report, seven broad guidelines are included that school programs can use to reduce
the risk for skin cancer among students: 1) policy, 2) environmental change, 3) education, 4)
families, 5) professional development, 6) health services, and 7) evaluation (Box 2). Each
guideline includes suggestions regarding key elements, steps for implementation, and realistic
expectations for change.

BOX 2. RECOMMENDATIONS FOR SKIN CANCER PREVENTION IN

SCHOOLS
1. Establish policies that reduce exposure to ultraviolet radiation.
2. Provide an environment that supports sun-safety practices.
3. Provide health education to teach students the knowledge, attitudes, and behavioral
skills they need to prevent skin cancer.
4. Involve family members in skin cancer prevention efforts.
5. Include skin cancer prevention with professional development of staff (e.g.,
preservice and in-service education).
6. Complement and support skin cancer prevention with school health services.
7. Periodically evaluate whether schools are implementing the guidelines on policies,
environmental change, education, families, professional development, and health
services.

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 Guidelines for School Programs to Prevent Skin Cancer 1629

 Guideline 1: Policy — Establish policies that reduce exposure to UV radiation.

 Guideline 2: Environmental change — Provide and maintain physical and social
environments that support sun safety and that are consistent with the development of
other healthful habits.
 Guideline 3: Education — Provide health education to teach students the
knowledge, attitudes, and behavioral skills they need to prevent skin cancer. The
education should be age-appropriate and linked to opportunities for practicing sun-
safety behaviors.
 Guideline 4: Family Involvement — Involve family members in skin cancer
prevention efforts.
 Guideline 5: Professional development — Include skin cancer prevention
knowledge and skills in preservice and in-service education for school
administrators, teachers, physical education teachers and coaches, school nurses, and
others who work with students.
 Guideline 6: Health services — Complement and support skin cancer prevention
education and sun-safety environments and policies with school health services.
 Guideline 7: Evaluation — Periodically evaluate whether schools are implementing
the guidelines on policies, environmental change, education, families, professional
development, and health services.

The recommendations represent the state-of-the-science in school-based skin cancer

prevention. However, every recommendation is not appropriate or feasible for every school to
implement nor should any school be expected to implement all recommendations. Schools
should determine which recommendations have the highest priority based on the needs of the
school and available resources. As more resources become available, schools could
implement additional recommendations to support a coordinated approach to preventing skin
cancer.

Guideline 1: Policy — Establish Policies that Reduce Exposure to UV

Radiation

Policies can provide sun protection for all persons in a defined population (e.g., a school),
not just those who are most motivated [117]. In addition, policies can involve formal
organizational rules and standards or legal requirements and restrictions related to skin cancer
prevention measures. Policies may be developed by a school, school board, or by other legal
entities (e.g., municipal, state, and federal governments). To be effective, policies need to be
communicated to school personnel, announced to affected constituents (e.g., students and
their parents), managed and implemented, enforced and monitored, and reviewed periodically
[118, 119].
Before establishing healthy skin cancer prevention policies, identify any existing policies
that might deter skin cancer prevention. These existing policies might include outdoor activity
schedules, prohibitions on wearing sunglasses or caps and hats at school, and rules that limit
the use or provision of sunscreen at school (e.g., requiring parental permission, defining
sunscreen as “medicine,” and restricting teachers from applying sunscreen on children).
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1630 Karen Glanz, Mona Saraiya and Howell Wechsler

California enacted a law (effective January 2002) that requires their schools to allow students,
when outdoors, to w ear school-site approved sun-protective hats and clothing. This
legislation was deemed necessary because several school districts had banned hats because
some styles or colors are connected with gang affiliation.
An effectively crafted skin cancer prevention policy provides a framework for
implementing the other six guidelines. The policy demonstrates institutional commitment and
guides school and community groups in planning, implementing, and evaluating skin cancer
prevention activities. Such a policy creates a supportive environment for students to learn
about and adopt sun-protection practices. Although a comprehensive policy is preferable,
more limited policies addressing certain aspects of skin cancer prevention also can be useful.

Developing the Policy or Policies

Skin cancer prevention can be part of a larger school health policy. Although policies
might be initiated by a person or small group, the most effective policies are developed with
input from all relevant constituents. In schools, the constituents include students, teachers,
parents, administrators, coaches, school nurses, health educators and other relevant personnel
as well as community leaders and residents. Schools can also work with community partners
(e.g., recreation and parks departments, health departments, after-school programs, camps,
families, and youth advocacy groups) and others who organize outdoor activities for youth.
Policies require time for development and implementation and might not be as visible as
educational programs [120]. Increased effort in the early stages of policy development might
result in increased adoption [121]. In Australia, health and cancer prevention specialists
developed a sun-protection policy kit for schools and a related staff development module
[120]. Elementary schools were twice as likely to formally adopt a comprehensive sun-
protection policy if they also received the staff development module (44% [kit and module]
versus 21% [kit only]). However, few high schools adopted policies whether they received
just the kit or the kit and the module (11% and 6%, respectively) [120]. Policy development
requires a longterm commitment and sustained efforts and cooperation among all concerned
parties.

Policy Options
Components of skin cancer prevention policies for a school or community to consider
include 1) statement of purpose and goals; 2) schedule and physical environment policies; 3)
policies related to personal protective clothing and sunglasses; 4) sunscreen policies; 5)
education policies; 6) policies on outreach to families; and 7) policies on resource allocation
and evaluation. When implementing a comprehensive policy (which would include all of
these components) is not feasible, schools can start with some of these components and add
others over time.
Policy 1: Statement of Purpose and Goals. Policies usually begin with a statement of
purpose and goals that establish sun safety as a priority and highlight the importance of skin
cancer prevention. In addition, the statement can 1) describe the influence of childhood sun
exposure on the risk for developing skin cancer later in life; 2) identify actions that persons
and institutions can take to reduce the risk for skin cancer; 3) highlight the importance of
establishing a physical, social, and organizational environment that supports skin cancer
prevention; and 4) specify dedicated financial and human resources for skin cancer prevention
and for the other policy options described here.

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 Guidelines for School Programs to Prevent Skin Cancer 1631

Policy 2: Schedule and Structure Policies. Policies can provide the basis for across-the-
board reduction of UV radiation exposure for children and adults in schools and communities
by establishing 1) rules that encourage the scheduling of outdoor activities (including athletic
and sporting events) during times when the sun is not at its peak intensity and 2) building and
grounds codes to increase the availability of shade in frequently used outdoor spaces.
Eliminating the scheduling of outdoor activities during peak sun hours will be difficult, if
not impossible, for many schools to do. For these schools, the best strategy might be to work
toward a gradual shift in scheduling. School board policies could require architects to design
new school buildings with adequate shade coverage adjacent to play and sports fields. Play
and sports fields can be reviewed for existing and potential shade. School and community
organization staff could evaluate frequently used spaces in the community for their UV
protection status and add signs, reminders, or prompts to encourage sun safety. Finally,
volunteer, business, health department, and political support can be secured by school and
community organization staff to generate resources for improving the sun-safety
environment, especially for providing sunscreen and shade.
Policy 3: Policies for Personal Protective Clothing and Sunglasses. Schools can
develop policies that encourage or require students to wear protective clothing, hats, and
sunglasses to prevent excessive sun exposure. These measures could be employed during
physical education classes, recess, field trips, outdoor sports or band events, and camping or
field trips. Some schools, especially in Australia, have a “no hat/no play” policy stating that
students cannot play outdoors if they are not wearing hats [119]. Related policy initiatives
could require the use of athletic, band, and physical education uniforms that reduce or
minimize excessive sun exposure (e.g., long sleeves and broad-brimmed hats). Strategies that
can be implemented to promote the adoption of these policies include gradually phasing-in
new policies that involve students and sports teams designing new uniforms, securing
business sponsorship for sun-safe uniforms, and conducting discussions that promote the use
of hats and sunglasses.
Some schools might have policies that prohibit or discourage students and staff from
wearing hats and sunglasses on school grounds (e.g., because they are associated with
contraband or gang-related items). Possible transmission of head lice among younger children
who share hats might also be a concern; however, policies can be implemented that address
these concerns (e.g., prohibiting both sharing hats and wearing gang-related symbols).
Policy 4: Sunscreen Policies. Policies on sunscreen use at school or for after-school
activities can range from encouraging parents to include sunscreen in required school-supply
kits, using permission slips for students to be able to apply sunscreen at school [122], and
establishing a sunscreen use routine before going outside. Policies also might require teachers
and coaches to use sunscreen for outside activities and require that sunscreen be provided at
official school-sponsored events that occur during midday. Necessary steps that might be
implemented include modifying existing policies that restrict school-based sunscreen
application [123], seeking support for purchasing sunscreen supplies, and supervising
sunscreen use.
Policy 5: Education Policies. The ideal education policy should support planned and
sequential health education to provide students with the knowledge, attitudes, and behavioral
skills needed for skin cancer prevention (Guideline 3). Policies that require teaching skin
cancer prevention within health education courses will need to be balanced with the overall
educational mission of the school.
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1632 Karen Glanz, Mona Saraiya and Howell Wechsler

Policy 6: Policies for Outreach to Families. Schools and other organizations that serve
youth have established methods of communicating with parents and other caregivers. Policies
can ensure that these organizations routinely provide to their youth advice and information
concerning skin cancer prevention. For example, information concerning skin cancer
prevention might be distributed along with other health forms to parents at the beginning of
the year or at parent and teacher visits.
Policy 7: Resource Allocation and Evaluation. Skin cancer prevention efforts will most
likely be sustained if policies exist to guide the allocation of resources for skin cancer
prevention. A funding policy usually includes accountability and ongoing evaluation, thus
providing for periodic review and reconsideration of how effective the resources dedicated to
skin cancer prevention are being used.

Guideline 2: Environmental Change — Provide and Maintain Physical and

Social Environments that Support Sun Safety and that are Consistent with
the Development of Other Healthful Habits

Policies can promote the provision of supportive resources for skin cancer prevention
(e.g., shade, protective clothing and hats, sunscreen at a reduced price or free, and highly
visible information and prompts for sun protection) in the physical and social environment.
These policies help establish routine personal behaviors and social norms that promote skin
cancer prevention in the context of organized group activities.

Physical Environments
The majority of schools in the United States were not designed with sun safety in mind.
Sun protection should be considered in the design of new schools. The design of school
buildings and adjacent grounds, and the availability of natural shade (e.g., trees and
mountains) or constructed shade (e.g., awnings, pavilions, and tall buildings that cast a
shadow) influence potential sun exposure. Students, teachers, and families can identify
opportunities to extend or create new shaded areas. These areas can be temporary or
permanent, natural or constructed. Students might participate in planting trees as part of their
science instruction, in which they learn which trees provide good shade cover, how and where
to plant them, and how long they will need to yield valuable protection. Existing structures
can be modified by constructing roofs on dugouts, installing covers for bleachers, and using
awnings and tarps. An increasing selection of portable or add-on shade structures are
available that school groups can purchase and install. Major construction projects to build
permanent pavilions and play areas can require substantial funding, but they might be the best
option in some settings. School and community partnerships can support these endeavors.
School and community partnerships can facilitate provision of sunscreen that is at a
reduced price or free for staff and students (through sunscreen manufacturers, pharmaceutical
companies, local dermatologist offices, or hospitals) and can make sun safety more accessible
during the school day or recreation period. An alternative school policy could encourage
parents to apply sunscreen to their children in the morning and include it in their children’s
supply kits. In addition, schools and community organizations can provide hats and protective
clothing (e.g., jackets) for persons who forget to bring their own on days with midday outdoor

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 Guidelines for School Programs to Prevent Skin Cancer 1633

activity or field trips. Both hygiene, size, and acceptability are important considerations.
However, if the school has a laundry facility for band and sports uniforms, a laundering
system for emergency sun-safe protective clothing could be instituted.
Information and prompts or reminders can reinforce sun-safety awareness and serve as
reminders to engage in skin cancer preventive practices. Both visual and audio messages
(e.g., sun-safe posters or public address system announcements) can serve as cues to action
for students as well as for families, teachers, and other professionals. After students have
learned about the UV index (an indicator of the intensity of the sun’s rays on a given day)
[124], schools can post and announce the daily UV index to encourage students to practice
sun-protection measures. Some schools and recreation settings also use signs that indicate the
number of minutes a person can be in the sun before sustaining a sunburn.

Social Environments
A supportive social environment involves establishing social norms favoring skin cancer
prevention and including personal preventive behaviors as a part of organized group
activities. Program planners and advocates for skin cancer prevention should serve as role
models, and adults should be invited to lead by example. Schools can also create a social
environment that encourages sun-safety practices through existing peer education groups by
having peer educators teach other students about sun safety and by using periodic recognition
or a special designation to reward teachers, staff, or students who practice sun safety.

Guideline 3: Education — Provide Health Education to Teach Students the

Knowledge, Attitudes, and Behavioral Skills They Need To Prevent Skin
Cancer. The Education Should be Age-Appropriate and Linked to
Opportunities for Practicing Sun-Safety Behavior

Health education that is designed effectively and implemented for youth can increase
their health-related knowledge and contribute to the development of healthy changes in
attitudes and behaviors [125]. Skin cancer prevention is likely to be most effective when it is
taught as part of a comprehensive health education curriculum that focuses on understanding
the relations between personal behavior and health [126] and that provides students with the
knowledge and skills outlined by the National Health Education Standards [112].
The yearly timing of skin cancer prevention education can be tailored to the climate and
linked with opportunities for sun exposure and sun protection. Therefore, in an area with high
altitude where outdoor winter sports are common (e.g., Colorado), skin cancer prevention
could be introduced before winter vacation. In northeastern coastal areas, skin cancer
prevention might be most relevant before summer break. And during the school day, sun-
safety lessons could directly precede recess or outdoor physical education, allowing the class
session to be followed by an opportunity to practice positive sun-safety habits.
Skin cancer prevention can be included as part of a comprehensive health education
curriculum because of the following characteristics:

 Behaviors that lead to UV radiation exposure might be related to other health risk
factors;
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1634 Karen Glanz, Mona Saraiya and Howell Wechsler

 Skin cancer prevention shares many of the key goals of other health education
content areas (e.g., increasing the value placed on health, taking responsibility for
one’s health, and increasing confidence in one’s ability to make healthy behavioral
changes); and
 Skin cancer prevention efforts can incorporate several of the social learning
behavioral change techniques used in other health education domains [126].

In addition to health education classes, skin cancer prevention can be integrated into other
subject areas. For example, a math exercise for students could be to calculate the length of
safe-sun exposure when sunscreen is used at a certain SPF. In history or social studies classes,
students could discuss the social value placed on tanning and fair skin and media portrayal of
tanning. Science classes could explore the light spectrum and discuss how it relates to the risk
for skin cancer, or discuss depletion of the ozone and its effect on UV exposure. This type of
integrated approach requires collaborative planning and curriculum development among
teachers to optimize skin cancer prevention education and to ensure consistency of messages
and practices.

Scope and Sequence

Health education is most effective in promoting positive behavioral changes when it is
repeated and reinforced over time [114]. Short-duration or single-presentation efforts can
increase students’ knowledge regarding sun safety and, in some cases, improve attitudes and
sun-protection behavior immediately after the program. However, these changes are likely to
be short-lived and cannot be expected to translate into sustained positive health behaviors
[125]. Multiunit presentations have been more effective in achieving higher increases in
knowledge and skill acquisition [125].
School-based health education to promote skin cancer prevention is most effective when
it is provided consistently and sequentially and included periodically in every grade, from
prekindergarten through 12th grade. Sequential instruction can build on information and skills
learned previously. Resources for skin cancer prevention programs targeting youth are
included in this report (Appendix C).

Active Learning and Behavioral Focus

In the previous decade, educational programs to encourage children to adopt sun-safety
habits have been implemented and evaluated. Among the school-based studies reported,
interventions have included one-time didactic formats and special events [97, 127, 128]; skin
cancer prevention that is integrated into classroom curricula over time [126, 129, 130]; and
peer-education programs [131, 132]. A majority of these studies have demonstrated that these
interventions increased knowledge and favorable attitudes toward preventive behaviors. In
addition, some of the programs that have multiple lessons and that occur over a longer period
(e.g., 1 year) have yielded improvements in sun-protection behaviors [125]
Actively engaging children and adolescents in the learning process increases the
likelihood for a positive effect. Youth are more likely to consider and adopt new or improved
behaviors when they learn about them through fun, participatory activities rather than through
lectures. For example, a recent study demonstrated increased improvement in knowledge of
the effects of UV radiation among elementary school students who used an interactive

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 Guidelines for School Programs to Prevent Skin Cancer 1635

computer-based program than among those who received the same information in a didactic
format led by a teacher [133]. The students who completed the interactive CD-ROM program
also exhibited significant positive changes in attitudes and a trend toward improvements in
sun-safety behavioral scores [133]. The U.S. Environmental Protection Agency offers an
Internet learning site where students can report and interpret daily measurements of UV
radiation, relate the UV index information to their own community, and correspond with
other participating schools [124, 134].
Health education activities should be tailored to the cognitive and behavioral level of the
students [135]. For example, students in kindergarten through third grades might learn
effectively through repetitious rhyming and learning the ABCs of skin cancer prevention.
Games, puzzles, and contests make learning fun for students of most ages. More intellectually
challenging activities might appeal to high school students, ranging from understanding the
scientific basis of solar radiation and global climates, to making their own video to
communicate sun-protection messages to their peers and communities. Teenagers can learn
about media literacy and different cultures by analyzing images of models in popular
magazines and discussing what sun exposure and a tan means to both white and non-white
racial groups in the United States and worldwide.

School Programs in a Broader Context

The most important long-term objective of skin cancer prevention education in schools is
the adoption and maintenance of sun-protection practices. Therefore, the transmission of
detailed, factual information to students is the foundation of sun-safety practices. In addition,
educational programs and curricula in schools are part of the broader mix of skin cancer
prevention efforts and should not be expected to solely prevent skin cancer. Skin cancer
prevention interventions in recreation, sports, and community settings can complement and
reinforce efforts in the schools [120, 136–140]. Supportive policies, environments, teachers,
and families are essential adjuncts to effectively planned and consistently implemented health
education to prevent skin cancer.

Guideline 4. Family Involvement — Involve Family Members in Skin Cancer

Prevention Efforts

The sun-safety practices of parents are the single most important determinant of the sun-
protection behaviors of children [121, 141]. For younger children, adult family members can
assist and provide sun-protection resources. For adolescents, the direct influence of parents
might decrease and be subordinated by peer influence. Nonetheless, family support plays a
key role in extending the desirable effects of school skin cancer prevention efforts.
Involving family members in skin cancer prevention efforts increases the likelihood that
they will adopt and thus model healthful sun-protection behaviors, and also appears to
favorably influence the sun-protection behaviors of students [122]. At a minimum, parents or
guardians can be informed concerning school initiatives and policies and knowledgeable
regarding how their cooperation is needed to ensure child health. Parents and guardians also
can be encouraged to provide children with sun-protective clothing and sunglasses for
outdoor activities. In addition, parents and guardians can serve as advocates for sun-protective
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1636 Karen Glanz, Mona Saraiya and Howell Wechsler

policies and practices in schools and can also provide volunteer labor for health and
recreation events. Their input and direct assistance can provide support for funding needed for
environmental improvements and educational materials.

Guideline 5: Professional Development — Include Skin Cancer Prevention

Knowledge and Skills in Preservice and In-service Education for School
Administrators, Teachers, Physical Education Teachers and Coaches, School
Nurses, and Others Who Work with Students

Even effectively designed skin cancer prevention programs cannot succeed if they are not
implemented as designed. Therefore, appropriate and effective professional development
efforts should be conducted for decision makers and caregivers at all levels. Professional
development activities, including certification programs and in-service education, are
provided routinely for teachers and other school staff (e.g., coaches and school nurses). Skin
cancer prevention can be integrated into these activities.
All school staff should receive basic information concerning the importance of sun safety
and key strategies for skin cancer prevention. The type of additional professional
development needed will vary, depending on the responsibilities of the various caregivers. In-
service education for principals might address policy implementation and monitoring,
whereas school nurses might highlight proper sunscreen use. Classroom teachers who
implement curricula should receive training that addresses both content areas and teaching
strategies.
As principals, teachers, and other school staff adopt sun-protection behaviors, they can
serve as role models for students. A brief training program, along with participation in
conducting skin cancer prevention activities for children, can result in improved sun-
protection practices among recreation leaders [142].

Guideline 6: Health Services — Complement and Support Skin Cancer

Prevention Education and Sun-Safety Environments and Policies with School
Health Services.

School health services provide an opportunity for nurses, health educators, and school
health resource specialists to promote and reinforce skin cancer prevention practices. A
child’s school health record can include parental permission for the child to use sunscreen
provided by the school as well as a list of possible allergies to sunscreens or their ingredients.
School health services staff also may conduct physical examinations for sports team
eligibility, assist in managing and notifying parents concerning the long-term dangers of a
severe sunburn, and prepare students for field trips. Each of these situations provides an
opportunity to educate and remind students about skin cancer prevention.
Health professionals in the community, including pediatricians, primary care providers,
nurses, pharmacists, and dermatologists are credible sources of information and guidance for
skin cancer prevention. They can be advocates for skin cancer prevention policies,
environmental changes, and programs, and support school programs through presentations,

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professional training, demonstrations, and classroom visits. During their consultation with
children and parents, these health-care professionals can also assess sun-exposure patterns,
reinforce sun-protective behaviors, and provide counseling to persons with sunburns [138,
143].

Guideline 7: Evaluation — Periodically Evaluate Whether Schools are

Implementing the Guidelines on Policies, Environmental Change, Education,
Families, Professional Development, and Health Services

Local school boards and administrators can use evaluation questions to determine
whether their programs are consistent with CDC’s Guidelines for School Programs To
Prevent Skin Cancer. Personnel in federal, state, and local education and health agencies also
can use these questions to 1) assess whether schools in their jurisdiction are providing
effective education to prevent skin cancer and 2) identify schools that would benefit from
additional training, resources, or technical assistance. The following questions can serve as a
guide for assessing program effectiveness:

1. Do schools have a comprehensive policy on skin cancer prevention and is it

implemented and enforced as written?
2. Does the skin cancer prevention program support physical and social environmental
changes that promote sun safety and that are consistent with the development of
other healthful habits?
3. Does the skin cancer prevention education program foster the necessary knowledge,
attitudes, and skills to reduce UV exposure and prevent skin cancer?
4. Is education to reduce UV exposure provided, as planned, in prekindergarten through
12th grade?
5. Is in-service training provided, as planned, for education staff responsible for
implementing skin cancer prevention programs?
6. Do school health services support skin cancer prevention?
7. Are parents or families, teachers, students, school health personnel, school
administrators, and appropriate community representatives involved in planning,
implementing, and assessing programs and policies to prevent skin cancer?
8. Does the skin cancer prevention program encourage and support sun-safety efforts by
students and school staff?

CONCLUSION
Schools can play a substantial role in protecting students from unnecessary exposure to
UV, thereby reducing their future risk for skin cancer. A comprehensive school approach to
skin cancer prevention includes policies, environmental change, educational curricula, family
involvement, professional development, integration with health services, and evaluation. The
exposure of youth to harmful UV radiation today contributes to their risk for skin cancer later
in life. Unlike many diseases, skin cancer is primarily preventable. Schools, in partnership
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1638 Karen Glanz, Mona Saraiya and Howell Wechsler

with community groups and other national, federal, state, and voluntary agencies, can
develop, implement, and promote initiatives that help protect youth from UV exposure [144,
145]. These guidelines serve as a framework for such initiatives.

APPENDIX A. PUBLIC HEALTH ACTION STEPS FROM THE

INTERNATIONAL AGENCY FOR RESEARCH ON CANCER
1. Protection of the skin from solar damage ideally involves various actions that include
wearing tightly woven protective clothing that adequately covers the arms, trunk, and
legs and a hat that provides adequate shade to the whole of the head; seeking shade
whenever possible; avoiding outdoor activities during periods of peak insolation; and
using sunscreens. Sunscreens should not be used as the sole agent for protection
against the sun.
2. Sunscreens should not be used as a means of extending the duration of solar exposure
(e.g., prolonging sunbathing) and it should not be used as a substitute for clothing on
sites that are usually unexposed (e.g., the trunk and buttocks).
3. Daily use of sunscreen with a high sun protection factor (>15) on exposed skin is
recommended for residents of areas of high insolation who work outdoors or enjoy
regular outdoor recreation. Daily use of a sunscreen can reduce the cumulative solar
exposure that causes actinic keratoses and squamous cell carcinoma.
4. Adequate solar protection is more important during childhood than any other time in
life, and parents and school managers should assiduously apply the first two
recommendations.
Source: The International Agency for Research on Cancer Working Group on the Evaluation of Cancer-
Preventive Agents. Sunscreens. In: IARC Handbooks of Cancer Prevention. Vol 5. Lyon, France:
International Agency for Research on Cancer, 2001.

APPENDIX B. SUNSCREEN: HOW TO SELECT, APPLY,

AND USE IT CORRECTLY

When to Apply Sunscreen

 Apply sunscreen approximately 30 minutes before being in the sun (for best results)
so that it can be absorbed by the skin and less likely to wash off when you perspire.
 Remember to reapply sunscreen after swimming or strenuous exercise.
 Apply sunscreen often throughout the day if you work outdoors, and wear hats and
protective clothing.

How to Apply Sunscreen

 Shake well before use to mix particles that might be clumped up in the container.
Consider using the new spray-on or stick types of sunscreen.

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 Guidelines for School Programs to Prevent Skin Cancer 1639

 Be sure to apply enough sunscreen. As a rule of thumb, use an ounce (a handful) to

cover your entire body.
 Use on all parts of your skin exposed to the sun, including the ears, back, shoulders,
and the back of the knees and legs.
 Apply thickly and thoroughly.
 Be careful when applying sunscreen around the eyes.

What to Look for When You Buy Sunscreen

 Pick a broad-spectrum sunscreen that protects against UV-A and UV-B rays and has
a sun protection factor (SPF) of at least 15.
 Read product labels. Look for a waterproof brand if you will be sweating or
swimming. Buy a nonstinging product or one specifically formulated for your face.
 Buy a brand that does not contain para-aminobenzoic acid (PABA) if you are
sensitive to that ingredient.
 Try a sunscreen with different chemicals if your skin reacts badly to the one that you
are using. Not all sunscreens have the same ingredients.
 Use a water-based sunscreen if you have oily skin or are prone to acne.
 Be aware that more expensive does not mean better. Although a costly brand might
feel or smell better, it is not necessarily more effective than a cheaper product.
 Be aware of the expiration date because some sunscreen ingredients might degrade
over time.

APPENDIX C. SKIN CANCER EDUCATION RESOURCES

Skin cancer information and resources are available from various governmental agencies,
voluntary organizations, medical associations, and corporations. Information is often
available in your state or local area. At the national level, information is available from the
sources listed below. The Internet address links take you directly to each organization’s skin
cancer information section.

American Academy of Dermatology

930 North Meacham Road
P.O. Box 681069
Schaumburg, IL 60173-4965
Phone: 847-330-0230
http://www.aad.org/skincnrUpdates.html

AMC Cancer Research Center

Phone: 800-321-1557
http://www.amc.org/html/market/h_market_sunnydays.html
email: [email protected]
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1640 Karen Glanz, Mona Saraiya and Howell Wechsler

American Cancer Society

1599 Clifton Road, N.E.
Atlanta, GA 30329
Phone: 800-227-2345
http://www3.cancer.org/cancerinfo

Anti-Cancer Council of Victoria

100 Drummond Street
Carlton Victoria 3053 Australia
Phone: 61-3-9635-5152
Fax: 61-3-9635-5260
http://www.sunsmart.com.au

CDC
National Center for Chronic Disease Prevention and Health Promotion
Division of Cancer Prevention and Control
4770 Buford Highway, N.E.; Mailstop K57

Atlanta, GA 30341-3724
Phone: 770-488-4751
http://www.cdc.gov/cancer/nscpep

National Cancer Institute

Cancer Information Service
Building 31, Room 10A16
31 Center Drive MSC-2580
Bethesda, MD 20892-2580
Phone: 800-422-6237
http://www.cancer.gov

National Council on Skin Cancer Prevention

http://www.skincancerprevention.org

Norris Cotton Cancer Center

The Sun Safe Project
Dartmouth Medical School
Department of Community and Family Medicine
7250 Strasenburgh
Hanover, NH 03755
Phone: 603-650-1566
http://www.dartmouth.edu/dms/sunsafe

U.S. Environmental Protection Agency

Sun Wise School Program
EPA Stratospheric Ozone Information
401 M Street SW (6205J)

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 Guidelines for School Programs to Prevent Skin Cancer 1641

Washington, DC 20460
Phone: 800-296-1996
http://www.epa.gov/sunwise

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[90] Chapman, S; Marks, R; King, M. Trends in tans and skin protection in Australian
fashion magazines 1982 through 1991. Am J Public Health, 1992, 82, 1677–80.
[91] Johnson, EY; Lookingbill, DP. Sunscreen use and sun exposure: trends in a white
population. Arch Dermatol, 1984, 120, 727–31.
[92] Banks, BA; Silverman, RA; Schwartz, RH; Tunnessen, WW, Jr. Attitudes of teenagers
toward sun exposure and sunscreen use. Pediatrics, 1992, 89, 40–2.
[93] Lescano, CM; Rodrique, JR. Skin cancer prevention behaviors among parents of young
children. Children’s Health Care, 1997, 26, 107–14.
[94] Hall, HI; May, DS; Lew, RA; Koh, HK; Nadel, M. Sun protection behaviors of the U.S.
white population. Prev Med, 1997, 26, 401–7.
[95] Hall, HI; Rogers, JD. Sun protection behaviors among African Americans. Ethn Dis,
1999, 9, 126–31.
[96] Marks, R. Role of childhood in the development of skin cancer. Aust Paediatr J, 1988,
24, 337–8.
[97] Mermelstein, RJ; Riesenberg, LA. Changing knowledge and attitudes about skin cancer
risk factors in adolescents. Health Psychol, 1992, 11, 371–6.
[98] Reynolds, KD; Blaum, JM; Jester, PM; Weiss, H; Soong, SJ; Diclemente, RJ.
Predictors of sun exposure in adolescents in southeastern U.S. population. J Adolesc
Health, 1996, 19, 409–15.
[99] Robinson, JK; Rigel, DS; Amonette, RA. Trends in sun exposure knowledge, attitudes,
and behaviors: 1986 to 1996. J Am Acad Dermatol, 1997, 37, 179–86.
[100] Jorgensen, CM; Wayman, J; Green, C; Gelb, CA. Using health communications for
primary prevention of skin cancer: CDC’s Choose Your Cover campaign. J Womens
Health Gend Based Med, 2000, 9, 471–5.
[101] Hall, HI; Jones, SE; Saraiya, M. Prevalence and correlates of sunscreen use among US
high school students. J Sch Health, 2001, 71, 453–7.
[102] Glanz, K; Lew, R; Song, V; Ah, Cook, VA. Factors associated with skin cancer
prevention practices in a multiethnic population. Health Educ Behav, 1999, 26, 344–59.
[103] Maducdoc, LR; Wagner, RF, Jr; Wagner, KD. Parents’ use of sunscreen on beach-
going children: the burnt child dreads the fire. Arch Dermatol, 1982, 128, 628–9.
[104] Grob, JJ; Guglielmina, C; Gouvernet, J; Zarour, H; Noe, C; Bonerandi, JJ. Study of
sunbathing habits in children and adolescents: application to the prevention of
melanoma. Dermatology, 1993, 186, 94–8.
[105] Vail-Smith, K; Watson, CL; Felts, WM; Parrillo, AV; Knight, SM; Hughes JL.
Childhood sun exposure: parental knowledge, attitudes, and behaviors. J Health Educ,
1997, 28, 149–55.
[106] Olson, AL; Dietrich, AJ; Sox, CH; Stevens, MM; Winchell, CW; Ahles, TA. Solar
protection of children at the beach. Pediatrics, 1997, 99, E1.
[107] Hall, HI; McDavid, K; Jorgensen, CM; Kraft, JM. Factors associated with sunburn in
white children aged 6 months to 11 years. Am J Prev Med, 2001, 20, 9–14.
[108] Robinson, JK; Rigel, DS; Amonette, RA. Summertime sun protection used by adults for
their children. J Am Acad Dermatol, 2000, 42, 746–53.

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[109] Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Institute
of Medicine. Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D,
and fluoride. Washington, DC: National Academy Press, 1997.
[110] Vieth, R. Vitamin D supplementation, 25-hydroxyvitamin D concentrations and safety.
Am J Clin Nutr, 1999, 69, 842–56.
[111] American Academy of Pediatrics. Vitamins: vitamin D. In: Kleinman RE, ed. Pediatric
nutrition handbook, 4th ed. Elk Grove Village, IL: American Academy of Pediatrics,
1998, 275–7.
[112] Joint Committee on National Health Education Standards. National health education
standards: achieving health literacy—an investment in the future. Atlanta, GA:
American Cancer Society, 1995.
[113] McKenzie, FD; Richmond, JB. Linking health and learning: an overview of coordinated
school health programs. In: Marx E, Wooley SF, eds. Health is academic: a guide to
coordinated school health programs. New York, NY: Teachers College Press, 1998.
[114] Carlyon, P; Carlyon, W; McCarthy, AR. Family and community involvement in school
health. In: Marx E, Wooley SF, eds. Health is academic: a guide to coordinated school
health programs. New York, NY: Teachers College Press, 1998.
[115] Glanz, K; Lewis, FM; Rimer, BK; eds. Health behavior and health education: theory,
research and practice. 2nd ed. San Francisco, CA: JosseyBass Inc, 1997.
[116] Henderson, A; Rowe, DE. A healthy school environment. In: Marx E, Wooley SF, eds.
Health is academic: a guide to coordinated school health programs. New York, NY:
Teachers College Press, 1998.
[117] Glanz, K; Lankenau, B; Foerster, S; Temple, S; Mullis, R; Schmid, T. Environmental
and policy approaches to cardiovascular disease prevention through nutrition:
opportunities for state and local action. Health Educ Qtly, 1995, 22, 512–27.
[118] Sallis, JF; Owen, N. Ecological models. In: Glanz K, Lewis FM, Rimer BK, eds. Health
behavior and health education: theory, research and practice. 2nd ed. San Francisco,
CA: Jossey-Bass Inc, 1997, 403–24.
[119] Queensland, Cancer, Fund. Working towards a SunSmart Queensland. Queensland,
Australia: Queensland Cancer Fund, 1997.
[120] Schofield, MJ; Edwards, K; Pearce, R. Effectiveness of two strategies for dissemination
of sun-protection policy in New South Wales primary and secondary schools. Aust N Z
J Public Health, 1997, 21, 743–50.
[121] Glanz, K; Carbone, E; Song, V. Formative research for developing targeted skin cancer
prevention programs for children in multiethnic Hawaii. Health Education Research,
1999, 14, 155–66.
[122] Glanz, K; Lew, RA; Song, V; Murakami-Akatsuka, L. Skin cancer prevention program
in outdoor recreation settings: effects of the Hawaii SunSmart Program. Effective
Clinical Practice, 2000, 3, 53–61.
[123] Wolf, SM; Swanson, LA; Manning, R. PROJECT SPF (Sun Safety, Protection and
Fun): Arizona Department of Health Services Early Childhood Skin Cancer Prevention
Education Program. Health Educ Behav, 1999, 26, 301–5.
[124] United States Environmental Protection Agency. The SunWise School Program Guide.
Available at http://www.epa.gov/sunwise/guide.pdf.
[125] Buller, DB; Borland, R. Skin cancer prevention for children: a critical review. Health
Educ Behav, 1999, 26, 317–43.
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[126] Buller, MK; Loescher, LJ; Buller, DB. Sunshine and Skin Health: a curriculum for skin
cancer prevention education. J Cancer Educ, 1994, 9, 155–62.
[127] Buller, MK; Goldberg, G; Buller, DB. Sun Smart Day: a pilot program for
photoprotection education. Pediatric Dermatol, 1997, 14, 257–63.
[128] Thornton, CM; Piacquadio, DJ. Promoting sun awareness: evaluation of an educational
children’s book. Pediatrics, 1996, 98, 52–5.
[129] Buller, DB; Buller, MK; Beach, B; Ertl, G. Sunny days, healthy ways: evaluation of a
skin cancer prevention curriculum for elementary schoolaged children. J Am Acad
Dermatol, 1996, 35, 911–22.
[130] Girgis, A; Sanson-Fisher, RW; Tripodi, DA; Golding, T. Evaluation of interventions to
improve solar protection in primary schools. Health Educ Qtly, 1993, 20, 275–87.
[131] Fork, HE; Wagner, RF; Wagner, KD. The Texas peer education sun awareness project
for children: primary prevention of malignant melanoma and nonmelanocytic skin
cancers. Cutis, 1992, 50, 363–4.
[132] Reding, DJ; Fischer, V; Gunderson, P; Lappe, K. Skin cancer prevention: a peer
education model. Wisc Med J, 1995, 94, 75–9.
[133] Hornung, RL; Lennon, PA; Garrett, JM; DeVellis, RF; Weinberg PD, Strecher, VJ.
Interactive computer technology for skin cancer prevention targeting children. Amer J
Prev Med, 2000, 18, 69–76.
[134] Cantor, MA; Rosseel, K. The United States Environmental Protection Agency SunWise
School Program. Health Educ Behav, 1999, 26, 303–4.
[135] Perry, CL. Creating health behavior change: how to develop community-wide programs
for youth. Thousand Oaks, CA: Sage, 1999.
[136] Mayer, JA; Slymen, DJ; Eckhardt, L; et al. Reducing ultraviolet radiation exposure in
children. Prev Med, 1997, 26, 516–22.
[137] Parrott, R; Duggan, A; Cremo, J; Eckles, A; Jones, K; Steiner, C. Communicating about
youth’s sun exposure risk to soccer coaches and parents: a pilot study in Georgia.
Health Educ Behav, 1999, 26, 385–95.
[138] Dietrich, AJ; Olson, AL; Sox, CH; et al. A community-based randomized trial
encouraging sun protection for children. Pediatrics, 1998, 102, E64.
[139] Dietrich, AJ; Olson, AL; Sox, CH; Tosteson, TD; Grant-Petersson, J. Persistent
increase in children’s sun protection in a randomized controlled community trial. Prev
Med, 2000, 31, 569–74.
[140] Miller, DR; Geller, AC; Wood, MC; Lew, RA; Koh, HK. The Falmouth Safe Skin
Project: evaluation of a community program to promote sun protection in youth. Health
Educ Behav, 1999, 26, 369–84.
[141] Arthey, S; Clarke, VA. Suntanning and sun protection: a review of the psychological
literature. Soc Sci Med, 1995, 40, 265–74.
[142] Glanz, K; Maddock, J; Lew, RA; Murakami-Akatsuka, L. A randomized trial of the
Hawaii SunSmart program’s impact on outdoor recreation staff. J Am Acad Dermatol,
2001, 44, 973–8.
[143] Easton, AN; Price, JH; Boehm, K; Telljohann, SK. Sun protection counseling by
pediatricians. Arch Pediatr Adolesc Med, 1997, 151, 1133–8.
[144] Green, LW; Kreuter, MW. Health promotion planning: an educational and ecological
approach. 3rd ed. Mountain View, CA: Mayfield Publishing, 1999.

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 Guidelines for School Programs to Prevent Skin Cancer 1649

[145] Hill, D; Dixon, H. Promoting sun protection in children: rationale and challenges.
Health Educ Behav, 1999, 26, 409–17.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 74

SHADE PLANNING FOR

AMERICA’S SCHOOLS *

Centers for Disease Control and Prevention

WHAT IS THE PURPOSE OF THIS MANUAL?

In 2002, the Centers for Disease Control and Prevention (CDC) published the Guidelines
for School Programs to Prevent Skin Cancer, which outlines steps that school communities
can take to develop a comprehensive approach to reducing the risk for skin cancer among
students, teachers, staff, and visitors. The guidelines include the following recommendations:

 Establish policies that reduce exposure to solar ultraviolet (UV) radiation.

 Provide and maintain physical and social environments that support sun safety.
 Provide opportunities for students to gain the knowledge, develop the attitudes, and
practice the skills needed to prevent skin cancer.
 Involve family members in skin cancer prevention efforts.
 Provide pre-service and in-service skin cancer prevention education for school
administrators, teachers, coaches, school nurses, and other professionals who work
with students.
 Support sun-safety policies, sun-safe environments, and skin cancer prevention
education with school health services.
 Evaluate the implementation of policies, environmental change, education, family
involvement, professional development, and health services.

This manual has been created to support school communities in their implementation of
the Guidelines for School Programs to Prevent Skin Cancer and, specifically, to help schools

*
This is an edited, reformatted and augmented version of a report prepared under contract for the Centers for
Disease Control and Prevention, National Center for Chronic Disease Prevention and Health, Division of
Cancer Prevention and Control, 2008.
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1652 Centers for Disease Control and Prevention

create and maintain a physical environment that supports sun safety by ensuring that school
grounds have adequate shade.

Between 68% and 90% of all melanomas result from exposure to ultraviolet
radiation.1

Why Should Schools Care About Skin Cancer?

Cancer of the skin is the most commonly diagnosed cancer in the United States and
perhaps the most preventable. Melanoma and non-melanoma cancers, including basal cell
cancer and squamous cell cancer, account for as much as 50% of all cancers. Because the
reporting of non-melanoma cancers to cancer registries is not required, the exact number of
non-melanoma cancer cases is not known. However, estimates indicate that as many as 1
million cases of basal cell and squamous cell skin cancer occur each year.2 Melanoma, which
accounts for only about 5% of skin cancer cases, also accounts for 79% of skin cancer deaths.
From 1973 and through the early eighties, the incidence rate of melanoma among white men
and women in the United States increased by about 6% per year. Since the early eighties, the
increase has been around 3% annually. Approximately 55,100 new melanomas were
diagnosed in the United States in 2004, and about 7,910 people died of melanoma that same
year.3
Almost all of these cancers are preventable.4 In most cases, exposure to solar UV
radiation is the cause of the cancer. Using multiple methods for estimating the incidence of
melanoma that might be attributable to exposure to the sun, Armstrong and Kricker, reporting
in Melanoma Research, suggest that between 68% and 90% of all melanomas result from
exposure to UV radiation.5

SOLAR RADIATION ADDED TO THE LIST OF

“KNOWN” CARCINOGENS
The federal government’s 11th edition of the Report on Carcinogens listed the sun and
any other source of broad spectrum ultraviolet radiation as “known” causes of cancer.
“The report cites data indicating a cause-and-effect relationship between this radiation
and skin cancer, cancer of the lip and melanoma of the eye. The report goes on to say that
skin cancers are observed with increasing duration of exposure and for those who
experience sunburn.”

From National Institutes of Health News Release dated December 11, 2002

Why Shade?

There are many reasons that a school might want to improve the quality and increase the
amount of accessible shade on school grounds. The most obvious and one of the most
important reasons is that shade provides protection from solar UV radiation. Due to the

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scheduling complexities of physical education classes, sporting events, and other outdoor
activities, students are often exposed to solar UV radiation during the peak sun hours of the
day—between 10:00 a.m. and 4:00 p.m. For some schools and for some students, using sun
protective methods, such as hats or sunscreen, or implementing policy changes could prove to
be problematic. Providing shade in areas where students already participate in outdoor
activities can afford passive protection from the sun’s damaging rays.

What Are the Additional Benefits of Shade?

Extending the Classroom

Schools are often looking for ways to extend their classrooms. Two strategies for
increasing shade on school grounds can also help schools create novel classroom experiences
for their students. These strategies may be employed independently or in concert. The first is
to modify existing structures or build new ones to provide shade where students play and
socialize. The second calls for the strategic planting of additional shade-producing trees,
vines, and shrubs. Structures built to provide shade can also be designed as covered outdoor
learning areas, thereby extending the classroom beyond the school walls. Planting shade-
producing vegetation affords schools the opportunity to create and maintain natural outdoor
classrooms where students can enjoy hands-on experiences in the natural world. Both
strategies could potentially provide teachers with new ideas for curricula and new reasons to
take their students outdoors.

Extended Periods of Physical Activity

In adults, regular physical activity is linked to enhanced health and reduced risk for the
development of many chronic diseases. Lifelong physical activity patterns are often
developed in childhood and adolescence. In the section on preventing physical activity-related
injuries in CDC’s Guidelines for School and Community Programs to Promote Lifelong
Physical Activity Among Young People, the use of shaded spaces or indoor facilities to reduce
the incidence of heat-related illnesses is recommended. Not all schools have indoor facilities
designed for active play; however, providing shade on existing outdoor play areas could
reduce the temperature in those areas by as much as 10° to 20 °, increasing the period of time
that students could engage in active outdoor play.

School Grounds Aesthetics

All too often, school grounds are an environment of concrete, asphalt, steel, turf grass,
and chain link fences. In planning strategies to provide or increase shade on school grounds,
schools have a second chance to improve the aesthetics of the school property, making the
grounds more inviting to students, teachers, staff, parents, and visitors. A well-planned shade
implementation project engages the entire school community in making the school a more
pleasant place to learn.
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1654 Centers for Disease Control and Prevention

Who Should Read This Manual?

A school includes not just the principal, teachers, students, and staff, but also key
stakeholders and decision-makers that comprise the school community. In addition to school
officials, the school community includes parents, neighbors, and members of the broader
community, all of whom have a stake in helping to protect the community’s children and
adolescents from skin cancer. This manual was written as a reference tool for the entire
school community, encompassing both the school district and the individual school.

How Can This Manual Be Used?

Section 1 School board members, superintendents, principals, and school

health advisory councils can use this manual to acquaint
themselves with issues relating to the damaging effects of solar
UV radiation, skin cancer prevention, and planning for shade
implementation at their schools. The first section gives
information on both the short- and long-term effects of UV
radiation on health and provides a rationale for developing sun-
safe policies in schools.
Section 2 The second section addresses strategies for providing shade at
schools and includes some of the advantages and disadvantages
of each strategy. School board members, superintendents,
principals, school health advisory councils, and school shade
planning teams will find this information useful in determining
the strategies that will work best at their school.
Section 3 The third section presents an overview of the process of
planning a shade implementation project. School board
members, superintendents, principals, and school health
advisory councils can use it as a brief overview of the process.
School shade planning teams can refer to this section for an
introduction to the process and to sections 4 and 6 for more
detailed information to guide them through the steps of shade
planning.
Section 4 The fourth section presents information about schools and
school districts that have engaged in shade planning projects
and reveals some successful strategies. The needs of every
school differ; there is no one-size-fits-all solution to providing
shade at schools. This section offers a glimpse of shade
strategies that others have employed.
Section 5 The fifth section gives the reader a basic introduction, or
reintroduction, to solar geometry and the relationship between
the Earth and Sun. Illustrations are included showing the
effects of daily and seasonal changes in solar angles on the
length and direction of shadows.

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 Shade Planning for America’s Schools 1655

Section 6 The sixth section guides shade planning team members through
the process of conducting a shade audit. The shade audit allows
planning teams to consider the needs of the school in relation to
the quantity and quality of shade already available to students,
teachers, and staff.

SECTION 1. WHAT IS UV RADIATION?

Ultraviolet (UV) radiation is one component of a broad spectrum of electromagnetic
radiation emitted by the sun. The spectrum also includes visible light, which we see, and
infrared radiation, which we feel as heat.
The ultraviolet area of the spectrum can be further divided into three bands, ultraviolet A
(UVA), ultraviolet B (UVB), and ultraviolet C (UVC). All UVC radiation and almost all
UVB radiation are absorbed in the ozone layer of the atmosphere. UVA radiation penetrates
the atmosphere unimpeded and, until recently, had been considered innocuous.

What Are the Factors That Affect UV Radiation Levels?

Time of Day
At solar noon, the sun is at its highest point of the day. As much as 30% of total UV
radiation is received in the hour before and the hour after solar noon, and as much as 75% is
received during the 3 hours before and the 3 hours after solar noon.6

Time of Year
Because the angles of the sun change throughout the year, the intensity of UV radiation
changes as well. In the northern hemisphere, UV radiation tends to be greater in the summer
months.
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1656 Centers for Disease Control and Prevention

SHADE PLANNING TOOLBOX

Q: What time is solar noon?
A: That depends on the day of the year, your latitude and longitude,
and your location within your time zone.
On July 4, solar noon is at:
12:48 (EDT) in Boston
1:40 (EDT) in Knoxville
12:51 (CDT) in Nashville
12:53 (PDT) in San Diego
On February 1, solar noon is at:
11:57 (EST) in Boston
12:49 (EST) in Knoxville
12:00 (CST) in Nashville
12:02 (PST) in San Diego

To find out what time solar noon occurs on any day of the year at
any location, visit the National Oceanic and Atmospheric
Administration (NOAA) Surface Radiation Research Branch web site at
http://www.srrb.noaa.gov/ highlights/sunrise/sunrise.html.

Geographical Latitude
UV radiation decreases as the distance from the equator increases.

Altitude
Because the atmosphere is thinner at higher altitudes and less able to absorb UVB, total
UV radiation is greater at higher altitudes.

Weather Conditions
Although clouds reduce the full spectrum of solar radiation, they do not reduce UV
radiation to the same extent that they reduce visible light and infrared radiation. Clouds may
make us feel cooler and block our view of the sun, but they do not fully protect us from UV
radiation.

Atmospheric Ozone
The stratosphere’s ozone layer provides us with an enormous amount of protection
against the damaging effects of UV radiation. Unfortunately, in certain areas, ozone has been
depleted to a dangerous extent, primarily due to the release of chlorofluorocarbons (CFCs)
and other ozone-depleting chemicals, such as carbon tetrachloride and methyl chloroform,
into the atmosphere. Both carbon tetrachloride and methyl chloroform are solvents that have
been used in industrial applications, and CFCs have, in the past, been used as refrigerants and
aerosol propellants. The use of all three of these chemicals has since been restricted or
prohibited.7 Nonetheless, much of the damage that has been done remains.

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What Are Direct and Indirect UV Radiation?

Direct UV radiation, or UV radiation that travels from the sun in a straight line, may pose
the greatest risk to our health, but we are also at risk from exposure to indirect (scattered and
reflected) UV radiation. Scattered UV radiation results from being bounced around by
atmospheric dust and water droplets in clouds. Throughout the day, the level of indirect UV
radiation varies, as does the level of direct UV radiation. In the early morning and late
evening when the sun is low on the horizon, indirect UV radiation may exceed direct.
Likewise, on a cloudy day, UV radiation scattered by atmospheric particles may result in
greater exposure to indirect than to direct UV radiation.
UV radiation can also be reflected off buildings and the terrain. Smoother surfaces, such
as concrete or asphalt, whether they are dark in color or not, typically result in greater
reflectance of UV radiation than irregular surfaces. Surface irregularities, such as that found
in grass or bark nuggets, reduce the level of reflectance, thereby reducing exposure to
reflected UV radiation. One exception, however, is water. Smooth water absorbs almost all
UV radiation, whereas the irregular surface of choppy water reflects a considerable amount of
UV radiation. The following table lists surfaces and terrains commonly encountered on
school grounds and their UV radiation reflectance. Materials with a lower reflectance are
more desirable.

Ultraviolet Radiation Reflectance for School Grounds Surfaces

Surface UV Radiation Reflectance8,9,10

Grass 1% – 4%
Still water 3% – 8%
Soil 4% – 6%
Asphalt 4% – 9%
Concrete 7% – 12%
Choppy water 8% – 13%
Dry sand 15% – 18%
Fresh snow 85% – 88%

How is UV Radiation Measured?

In the past, different countries measured and reported solar UV radiation intensity in
different ways. One common way of reporting UV radiation intensity was in the form of
estimated “burn time” or “time to burn,” expressed as the number of minutes of solar
exposure required for the reddening of a fair-skinned person’s exposed skin, assuming a clear
sky. Although there may be some advantages to this method of reporting UV radiation
intensity, there are a number of disadvantages. In 1994, international agreement was reached
on standardizing the measure of UV radiation intensity.
Revised by the World Health Organization in 2002 and adopted by the Environmental
Protection Agency and the National Oceanic and Atmospheric Administration’s National
Weather Service in 2004, the UV Index is the internationally accepted system for reporting
the intensity of UV radiation. Although the mathematical model developed to determine the
UV Index might be complicated, the measurement is easy to understand. The UV Index is a
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1658 Centers for Disease Control and Prevention

measure of the amount of damaging UV radiation that reaches the earth’s surface at noon on a
given day and at a given location, expressed as a risk scale. It is predicted daily on a scale of 0
to 11+, where 0 represents a minimal risk of overexposure to UV radiation and any number
higher than 11 represents an extreme risk of overexposure to UV radiation.

SHADE PLANNING TOOLBOX

Q: Where do I find the UV Index for tomorrow?
A: In most communities, the UV Index is reported in newspapers
and on television with the daily weather forecast.

Every day at approximately 1:30 PM Eastern, the National Oceanic

and Atmospheric Administration (NOAA) and the Environmental
Protection Agency (EPA) post the UV Index for the next day at the
EPA’s Website, http://www.epa.gov/sunwise/ uvindex.html.

What Are the Health Effects of Exposure to UV Radiation?

Few people would dispute the beneficial effects of solar radiation. The sun warms the
earth, fuels photosynthesis, and ensures the continued existence of life on earth. Many of us
enjoy the warmth of the sun on our skin. But at some point in our lives, most of us have
experienced the painful effect of too much sun exposure in the form of sunburn. Having
experienced sunburn, many would agree that there must be some negative health effect to
exposure to the sun. Although many different conditions occur as a normal response to
exposure to UV radiation, they all fall into one of two classifications, acute or chronic. Acute
effects of UV radiation exposure usually have a rapid onset and are of short duration, such as
sunburn, tanning, and synthesis of vitamin D3. Chronic effects of UV radiation exposure
usually have a gradual onset and are of long duration, such as skin cancer and photoaging.

Sunburn
Sunburn is an acute injury resulting from excessive exposure to the sun. The redness
associated with sunburn results from the dilation of superficial blood vessels in the skin.
Redness usually appears within 4 hours of exposure, reaches a maximum within 12 hours,
and fades after a few days. High doses of UV radiation can result in blistering and peeling.
Skin color, hair color, eye color, and freckles all are characteristics that help predict an
individual’s susceptibility to sunburn. Individuals are typically grouped into one of six sun-
reactive types, ranging from those with blue or green eyes and very light skin that never tans
to those with very dark hair and eyes and dark skin that almost never burns. Although
sunburn is not as common among blacks, compared to whites, blacks are susceptible.
Approximately 15% of the African American respondents to a national health survey reported
experiencing mild to severe sunburns.11 Differences also exist in the sun-sensitivity of
different parts of the body. The face, neck, and trunk are two to four times more sensitive than
the limbs.12

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Tanning
Tanning, or melanin pigmentation, is a consequence of overexposure to the sun that many
people find desirable. UVA exposure results in the skin’s production of more melanin, the
substance responsible for skin’s pigmentation. There are two ways in which our skin can tan,
by immediate tanning and by delayed tanning. Immediate tanning occurs as quickly as 5 to 10
minutes after exposure to the sun and will last as long as 2 hours. One’s ability to exhibit
immediate tanning is directly related to the genetically determined pigmentation of the skin.
Delayed tanning, which is the more familiar form of tanning, is noticeable 1 to 2 days after
exposure, increases for several days, and lasts for weeks or months. Although having a tan
provides some degree of protection from UVB, melanin is not an effective sunscreen for
Caucasian skin.13

Photosynthesis of Vitamin D3
The only positive health effect associated with exposure to solar UV radiation is the
synthesis of vitamin D3. Without UVB, the body cannot synthesize vitamin D3, which is
essential for regulating calcium metabolism. Few studies have examined the effect of sun
avoidance or the use of sunscreen on the production of vitamin D3in children or adults.
Therefore, the American Academy of Pediatrics has recommended that infants, children, and
adolescents who do not consume at least 500 mL (16.9 oz) of vitamin D-fortified milk or
formula daily should take one of the many available daily multivitamin supplements that
contain 400 IU of vitamin D3.14

Photoaging of the Skin

Exposure to UVA and UVB radiation, and perhaps to radiation in the infrared range as
well,13 causes photoaging, a process in which the skin’s elastic fibers break down leading to
wrinkled and leathery-looking skin. Dryness, deep wrinkles, sagging, loss of elasticity, and
mottled pigmentation are all photoaging symptoms.

Eye Damage
Eye damage from solar radiation is a risk factor for developing a number of eye disorders
including cataracts, skin cancer around the eyes and degeneration of the macula. Although the
evidence is not conclusive, such damage appears to have a dose-response relationship and to
be cumulative; that is, the more unprotected solar radiation exposure to the eyes over an entire
lifetime, the greater one’s risk for developing cataracts.13, 15, 16

Basal Cell Cancer

Typically occurring on the most sun-damaged parts of the body, basal cell carcinoma is a
slow-growing cancer that begins as a raised lump on the skin and eventually breaks open to
form an exposed sore. Although most of these types of cancers are colorless, some are dark in
color. Like other skin cancers, basal cell cancer usually appears in middle age, as a result of
UV radiation exposure during childhood or adolescence.
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1660 Centers for Disease Control and Prevention

SUN PROTECTION IS THE KEY

The vast bulk of skin cancers in the U.S. are due to excessive skin
exposure to UV radiation from the sun, so sun protection is the key to
preventing the disease.

Martin Weinstock, MD, PhD Director of Dermatoepidemiology at Brown University and

Chair of the American Cancer Society (ACS) Skin Cancer Advisory Board

Squamous Cell Cancer preventing the disease.

Squamous cell cancer is a more aggressive form of skin cancer that ultimately basal cell
cancer in appearance. It often follows a pre-cancerous condition called actinic keratosis,
which is a dry and crusty area on the skin. Both basal cell and squamous cell carcinoma
usually result from chronic exposure to UV radiation over a period of years.

Melanoma
By far, the most serious consequence of exposure to UV radiation is malignant
melanoma. Unlike the other two types of skin cancer, basal cell and squamous cell,
melanomas involve the dark pigmented cells of the skin, the melanocytes. A growing body of
evidence indicates that intermittent sun exposure, as opposed to chronic sun exposure, causes
this most deadly of skin cancers. Of particular concern, findings from certain studies point to
childhood exposure to sunlight, especially severe childhood sunburn, as an indicator for
melanoma as an adult.17, 18, 19, 20

Where Can I Find More Information?

Section 5, “The Earth-Sun Relationship,” provides more information on seasonal sun

angles and their effects on shade design. On the following pages are internet links to more
information on skin cancer and its prevention as well as to sun-safety curricula.

Information on Skin Cancer and its Prevention

The Centers for Disease Control and Prevention

www.cdc.gov/ca CDC provides leadership for nationwide efforts to reduce illness
ncer/nscpep/ and death caused by skin cancer. Although these efforts comprise
skin.htm a variety of approaches and strategies, their common focus is
education and prevention. CDC’s Web site describes
programmatic approaches to skin cancer prevention and education.
The National Cancer Institute
www.nci.nih.gov The National Cancer Institute (NCI) is a component of the
/cancertopics/typ National Institutes of Health (NIH) and is the federal
es/ skin government’s principal agency for cancer research and training.
Articles related to the causes of cancer, diagnosis, prevention,
treatment, and the most current cancer statistics are available at
NCI’s Web site.

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The United States Environmental Protection Agency

www.epa.gov/eb The Environmental Protection Agency’s mission is to protect
tpages/ human health and to safeguard the natural environment. The
humasunprotecti agency’s Web site contains information about the health risks
on.html posed by UV radiation and describes the steps people can take to
protect themselves from overexposure to the sun.
The National Council on Skin Cancer Prevention
www.skincancer The mission of the National Council on Skin Cancer Prevention is
prevention.org to facilitate national skin cancer awareness and prevention efforts
through education and promotion of sun-safe behaviors. The
council, comprising of 30 separate organizations, increases
awareness and prevention behaviors among all populations by
providing special programs addressing high-risk populations,
including infants, children, young adults, parents, educators,
outdoor workers, and athletes. Back issues of NEWSLINK, the
council’s quarterly electronic newsletter, are available at the
ogranization’s Web site.
The American Cancer Society
www.cancer.org The American Cancer Society is the nationwide community-based
voluntary health organization dedicated to eliminating cancer as a
major health problem by preventing cancer through research,
education, advocacy, and service. The organization’s Web site
provides the latest research , information about activities and
resources at the local level, and educational and advocacy
materials.
The Skin Cancer Foundation
www.skincancer. The Skin Cancer Foundation is the only national and international
org organization that is concerned exclusively with the world’s most
common malignancy—cancer of the skin. The foundation’s Web
site offers information on the three types of skin cancer;
information on skin cancer prevention; news of local and national
events; and public information posters, pamphlets, and brochures.

Sun Safety Curricula

National Safety Council—Environmental Health Center

1025 Connecticut The National Safety Council’s Enviromental Health Center
Avenue, NW Suite developed the Sun Safety Activity Guide for elementary
1200 Washington, DC school representatives who would like to incorporate sun
20036 safety into their school curricula. The guide includes cross-
www.nsc.org/ehc/sunw curriculum classroom activities and background information
ise/ activity.htm packaged as a 1-hour “core” sun-safety lesson. The core is
divided into three 20-minute units, including the effects of
UV, risk factors for overexposure to the sun, and sun
protection habits. Included in the guide are developmentally
appropriate activities for primary (grades K through 2) and
intermediate (grades 3 through 6) learning levels.
Project S.A.F.E.T.Y.
The University of Project S.A.F.E.T.Y. (Sun Awareness for Educating Today’s
Texas M. D. Anderson Youth) is a cross-curricular, multimedia skin cancer
Cancer Center with prevention program for grades 4 through 9. It is available
Texas Cancer Council free of charge to any school in Texas. It is also available to
www.mdanderson.org/ schools outside of Texas for a minimal cost.
departments/
projectsafety/
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(Continued)

The SHADE Foundation

10510 N. 92nd Street The mission of The SHADE Foundation, a non-profit
Suite 100 Scottsdale, organization, is to eradicate melanoma through the education
AZ 85258 of children and the community in the prevention and
www.shadefoundation. detection of skin cancer and the promotion of sun safety. In
org collaboration with the Environmental Protection Agency
(EPA), the Foundation has developed partnerships with
schools which implement the EPA SunWise School Program
and, in turn, are awarded shade structures to the schools.
Sunny Days, Healthy Ways
Klein Buendel, Inc. Sunny Days, Healthy Ways is a sun-safety curriculum that
14023 Denver West uses a comprehensive, cross-curricular approach to teaching
Parkway Suite No. 190 skin cancer prevention skills to children in grades K through
Golden, CO 80401 5. The curriculum provides an average of 8 hours of sun-
(877) 258-2915 safety instruction per grade that can be tailored to the
[email protected]/ teacher’s time frame and needs. The curriculum includes
prepared lesson plans, student activity sheets, experiment
materials, story books, and assessments.
The SunSafe Project
Norris Cotton Cancer The SunSafe intervention aims to enhance and promote sun
Center Dartmouth protection of children ages 2 to 9 years through the delivery
Medical School One of a multicomponent intervention in three settings:
Medical Center Drive elementary schools and day care centers, town beach areas,
Lebanon, NH 03756 and primary care practices. The school/day care component
(603) 650-8254 consists of an age-specific (2 to 9 years old) and grade-
http://sunsafe.dartmout specific curriculum promoting sun protection. Child-care
h.edu providers and elementary school teachers need 2 theme days
or 2 class periods to deliver SunSafe materials. Ongoing
reminder activities are suggested as a means for reinforcing
the SunSafe message.
The United States Environmental Protection Agency
SunWise School The SunWise School Program is an environmental and
Program health education program that aims to teach children and
www.epa.gov/sunwise/ their caregivers how to protect themselves from
overexposure to the sun. Using classroom-based, school-
based, and community-based components, the SunWise
School Program seeks to develop sustained sun-safe
behaviors in schoolchildren.
The program’s learning components build on a solid
combination of traditional and innovative education practices
already in use in many U.S. elementary and middle schools.
Through the program, students and teachers will increase
their awareness of simple steps that they can take to protect
themselves from overexposure to the sun. Students will
demonstrate the ability to practice health-enhancing
behaviors and reduce health risks. Children also will acquire
scientific knowledge and develop an understanding of
environmental concepts related to sun protection.
Currently, more than 12,000 schools are registered for the
SunWise program, representing all 50 states, the District of
Columbia, and Puerto Rico.
Commercial Products Disclaimer
The list of product manufacturers and retailers is provided for general information purposes only
and does not represent an inclusive list of vendors.

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 Shade Planning for America’s Schools 1663

Furthermore, reference to any specific commercial products or services by trade name, trademark,
manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation or
favoring by the Centers for Disease Control and Prevention (CDC).
The list of product manufacturers and retailers contains URL addresses to Web sites and
information created and maintained by private organizations. These links are provided for
convenience of reference only. CDC does not control or guarantee the accuracy, relevance,
timeliness, or completeness of this outside information. The inclusion of URL addresses to
particular Web sites is not intended to reflect their importance, nor is it intended to endorse any
views expressed or products or services offered by the author of the site or by the organization
operating the server on which the site is maintained.

SECTION 2. STRATEGIES FOR PROVIDING SHADE

A number of strategies for providing shade on school grounds are available; however, no
single approach is best for all schools. This section introduces three strategies for providing
shade on school grounds: solid roof structures, shade cloth structures, and natural shade.
Some of the advantages and disadvantages of each approach are discussed. This information
will assist schools in determining how best to provide shade for their students, teachers, staff,
and visitors. Regional differences in vegetation, the need for winter warmth, playground
usage patterns, and seasonal weather threats to playground structures factor into the decision
process of determining the best approach for each school. For many schools, a combination of
strategies that capitalizes on the advantages of several approaches will be the most effective.

Solid Roof Structures

Solid roof structures are permanent structures that provide protection from the sun’s
harmful rays and can be designed to serve a multitude of purposes. Typically, the structures
are designed to be open on at least three sides and often include furniture that can be moved
around. To maximize flexibility, the design can include lighting and plumbing.

Advantages

 Provides “all-weather” protection.

 Provides additional classroom space.
 Provides exercise space during inclement weather.
 Provides flexibility of design.
 Can be used as a lunch or picnic area.
 Has a long life span.

Disadvantages

 Requires drainage and guttering.

 Can be more expensive than other strategies.
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Outdoor classroom at Poplar Creek Elementary School—Siler, Kentucky.

With little more than a few hundred dollars, the support of local
businesses, and a State environmental education grant, Principal Tom
Shelly and the teachers at Poplar Creek Elementary School, along with the
students and their parents, were able to fund the construction of this
outdoor classroom and nature trail on their school grounds that otherwise
would have cost as much as $30,000.

Considerations

 Schools considering any type of construction project will need to determine which of
their local building codes and fire codes are applicable to their project.
 Careful planning will result in the positioning of the structure so that it creates shade
at the right place, at the right time of day, throughout the year.
 Schools located in areas that experience heavy snowfall will need to consider the
snow load when designing the roof of the structure.
 Likewise, schools located in areas that experience high winds will need to design
accordingly.
 Exposed roof supports may be attractive nesting sites for birds. Strategies to deter
this should be incorporated into the building’s design.
 Lighting will allow for evening use of the building.
 To provide additional light during daytime hours, polycarbonate panels can be
incorporated into the roof design and provide a great deal of light while blocking up
to 99% of ultraviolet (UV) radiation.

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Outdoor classroom at Hermantown School Duluth, Minnesota.

Constructed by the Duluth Skyline Rotary Club as a gift to the

Hermantown School District, the outdoor classroom is directly behind the
Hermantown Elementary School. Besides serving as an outdoor
classroom, the building is accessible to elementary school students during
recess periods. According to Fred Majeski, the Superintendent of the
Hermantown School District, the building, with its metal framing and roof
and concrete floor, would have cost the school well over $25,000 to build,
had it not been donated by the Rotary Club.

 No matter what type of design is selected, all buildings require maintenance. A

maintenance schedule and estimated costs should factor into the design selection
process.
 In the design of the structure, efforts should be made to close off the view of the sky
by extending the eaves as far as possible. If the sky can be seen by people under the
structure, they are at risk for exposure to indirect UV radiation.
 The design of any structure should ensure access for people with disabilities.
 If the structure is to be used as a classroom or meeting room, the acoustics of the
building should also be addressed.

Shade Cloth Structures

Another strategy for providing shade on school grounds is the use of shade cloth or
structural fabric supported by a framework or poles. This strategy often is used when the goal
is to cover large play areas without employing extensive structural support. Shade cloth is
typically a knitted or woven fabric that is rated as to how much sun is blocked. Transmission
of the sun’s rays through the fabric depends on the tightness of the weave or knit, with more
densely woven or knitted fabric blocking out more of the sun’s radiation. Fabrics with a
looser weave transmit between 50% and 80% of the sun’s harmful rays and are typically
designed for horticultural applications.
Shade cloth that blocks 80% of solar radiation provides the approximate protective
equivalent of sunscreen with a sun protection factor (SPF) of 6.7, whereas shade cloth that
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1666 Centers for Disease Control and Prevention

blocks 94% of solar radiation provides the approximate protective equivalent of sunscreen
with an SPF rating of 15. Shade cloth rated to block 94% of solar radiation is the minimum
that schools should consider.

Sun Protection Factor (SPF)

Advantages

 Can be relatively inexpensive to construct.

 Generally requires minimal upkeep.

Disadvantages

 Provides varying UV radiation protection.

 Can be susceptible to weather damage.
 Has a shorter life span than solid roof structures.

Considerations

 Because the UV protection qualities of shade cloth and other structural fabrics vary
widely, care should be taken in determining the most appropriate fabric.
 Care must be taken in the positioning of supporting posts so that they do not create a
danger to children at play.
 As with any other structure, one of shade cloth should be positioned for maximum
sun protection.
 The structural integrity of a fabric structure is related to its curvature and positioning.
Structures must be designed to withstand the snow and wind loads that can be
expected in their locations.
 The design and installation of these structures should be left to specialists. Names
and contact information of organizations that design and install fabric shade
structures can be found at the end of this section.

WHAT DOES SPF MEAN?

The common interpretation is how much longer skin covered with
sunscreen takes to burn as compared to unprotected skin. Sunscreens can
be rated for their protective factor against either UVA or UVB radiation or
both. The thickness and thoroughness of application, the type of sunscreen,
and the frequency of reapplication factor into whether or not sunscreen
delivers the protection for which it is rated.

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Natural Shade

Incorporating natural shade into the overall design offers several advantages. The best
approach to creating shade is one that provides protection from the sun’s harmful radiation
during the spring, summer, and fall, yet does not completely block the sun’s warmth during
winter months. Incorporating deciduous trees, shrubs, and vines into the design provides the
seasonal variation in protection that structures alone cannot provide. Likewise, evergreen
trees, vines, and shrubs planted alongside structures can serve to block wind in the winter and
provide protection from scattered UV radiation during the rest of the year.

Advantages

 Reduces the ambient temperature more so than structures.

 Provides seasonal sun protection.
 Provides low-cost alternatives.
 Improves the aesthetics of the school grounds.
 Provides an opportunity for students to learn about nature.

Disadvantages

 Vegetation takes time to grow.

 Trees can create litter, such as leaves, nuts, and fruits.

Considerations

 There are regional differences in vegetation. Plants native to a particular region have
evolved to thrive in the conditions prevalent there. Whenever possible, locally grown
native varieties of trees, shrubs, and vines should be used in the design. The United
States Department of Agriculture (USDA) Cooperative Extension Service agent in
your area can help determine the best species for your design.
 The effectiveness of trees and vines in providing UV radiation protection is directly
related to the density of the plant’s foliage.
 As a rule of thumb, trees should be planted to the south and west of where you want
to shade so they can provide it during the midday and afternoon hours. Section 5,
“The Earth-Sun Relationship,” gives information on creating shade in the right place
at the right time.
 Some plants are poisonous or cause allergic reactions. Other plants can attract bees or
have dangerous spikes or thorns. One needs to become familiar with the possible
harmful effects of the species that are being considered.
 Trees may interfere with a school’s electrical service, plumbing, and drainage
systems. Care should be taken that vegetation is not planted where it might later
present a threat to the school’s utility systems.
 Many trees will not tolerate root compaction, which occurs when foot traffic
compacts the soil around the roots of a maturing tree. Several strategies will prevent
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1668 Centers for Disease Control and Prevention

it, including a temporary fence around the maturing tree or specially designed pavers
to absorb the impact of busy feet.
 A short-term or transitional structure can be built to provide shade while the
vegetation is maturing.
 The first year after planting vegetation is the most important for ensuring the survival
of trees, shrubs, and vines. Plants must be watered at regular intervals if rainfall is
inadequate. The USDA Cooperative Extension Service agent in your area can help
you determine an appropriate watering schedule.
 Some communities have restrictions on water use that might affect decisions on
which plants would be most appropriate.

Where Can I Find More Information?

Section 5, “The Earth-Sun Relationship,” gives more information on providing shade at

the right place, at the right time, throughout the year. The following pages contain links to
information about selecting trees, vines, and shrubs; sources for plants and products related to
natural shade strategies; information on creating natural wildlife habitats; organizations that
manufacture and install fabric shade structures; and contact information for the USDA’s
Cooperative Extension Service.

WILDLIFE HABITAT CREATION

American Forests: A Tree for Every Child
www.americanforests.org/resources The “A Tree for Every Child” project is a hands-on, flexible
/kids/a_tree_ for_every_child environmental education program that allows students to see how
practical action can create a better world. The project allows
schools to teach students the benefits and rewards of planting
trees as part of American Forests’ Global ReLeaf 2000 campaign
to plant 20 million trees for the new millennium.
Acorn Naturalists
P.O. Box 2423 Tustin, CA 92781- This organization offers resources for science and environmental
2423 Toll Free: (800) 422-8886 educators. Offerings include educator guides, interpretative tools,
http://acornnaturalists.com/store and books on nature and the environment. An online catalog is
available.
Cornell Lab of Ornithology
159 Sapsucker Woods Road Ithaca, This site describes classroom projects and provides educational
NY 14850-1999 Toll Free: (800) materials that support habitat development. Projects include the
843-2473 http://birds.cornell.edu Great Backyard Bird Count (which is held every February) and
the Classroom Feeder Watch. The curriculum description for the
Classroom Feeder Watch can be found at
http://www.birds.cornell.edu/cfw/index.html
Project Wild
555 Morningside Drive, Suite 212 Project Wild provides instructional materials that can be adapted
Houston, TX 77005 Phone: (713) for academic disciplines ranging from science and environmental
520-1936 www.projectwild.org education to social studies, math, and language arts. Numerous
education materials, Web links, and guidebooks are available at
this site, including the publication Wild School Sites: A Guide to
Preparing for Habitat Improvement Projects on School Grounds.

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Kidsgardening.com
National Gardening Association This site is an online source of information and materials for
1100 Dorset Street South environmental science in the classroom. A keyword-searchable
Burlington, VT 05403 Toll Free: resources directory provides links to regional resources.
(800) 538-7476
www.kidsgardening. com

RESOURCES FOR NATURAL SHADE SOLUTIONS

United States Department of Agriculture
Cooperative State Research, Education, and Extension Service
www.csrees.usda.gov/qlinks/ This site provides links to the county offices of the Cooperative
partners/state_partners. Extension Services system in each state. The Cooperative
html Extension Services system has field agents assigned to each county.
That agent will be able to answer your questions regarding the best
species to plant for the application that you are considering;
provide you with sources for the trees, shrubs and vines; and offer
detailed instructions on the proper way to plant and care for them.
United States Department of Agriculture
U.S. Forest Service/Urban and Community Forestry
www.fs.fed.us/ucf/ The goal of the USDA Forest Service Urban and Community
Forestry Program is to provide technical and financial assistance to
help improve the livability of cities and communities by managing
urban forest resources to promote a healthy ecosystem. This Web
site provides links to the regional coordinators.

U.S. Department of Agriculture

Natural Resources Conservation Service
www.plants.usda.gov The PLANTS Database provides standardized information about
plants of the United States and its territories. The databse includes
names, distributional data, species abstracts, characteristics,
images, and links to related Web sites with information on the
region-specific culture for each species.
Treelink
www.treelink.org This Web site provides information, research, and networking for
people who work in urban and community forestry. Tips on grant
writing and fund-raising are also included.
The National Arbor Day Foundation
www.arborday.org The National Arbor Day Foundation helps individuals and groups
plant and care for trees and encourages the celebration of Arbor
Day to advance global environmental stewardship for the benefit of
this and future generations.
The International Society of Arboriculture
www.treesaregood.com The International Society of Arboriculture is a worldwide
professional organization dedicated to fostering a greater
appreciation for trees. The Web site was created to provide the
public with quality tree care-related information.
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U.S. Environmental Protection Agency

Landscaping with Native Plants
Ariel Rios Building 1200 This Environmental Protection Agency site promotes landscaping
Pennsylvania Avenue, N.W. with native plants and discusses the environmental benefits of using
Washington, DC 20460 native plant material. Topics discussed include attracting birds and
Phone: (202) 272-0167 butterflies and being considerate of local weed laws. The Wild Ones
www.epa.gov/glnpo/greenacr Handbook is a compendium of practical information for
es/ nativeplants/factsht.html landscaping with native plants.

SECTION 3. PLANNING FOR SHADE

Planning for shade requires the completion of a series of interrelated tasks. These include
convening a planning team, conducting a site audit to determine whether the existing level of
shade is adequate, determining the most appropriate strategies if more shade is required; and
developing a plan to increase the amount of shade accessible to students, teachers, staff, and
visitors. The process can be lengthy, taking as long as 1 year. This section briefly describes
each step. In section 6, “How to Conduct a Shade Audit,” the reader will find more detailed
information on the steps for shade planning.

The Shade Planning Team

It is important for any school undertaking a shade planning project to first identify the
stakeholder groups that may have an interest in, or be affected by, the resulting plan.
Representatives of these groups should be included on the planning team. For most schools,
the stakeholder list would include school administrators, the school nurse, coaches, teachers,
students, parents, groups that use the school grounds after hours, and neighbors living
adjacent to the school. In addition to stakeholder representatives, the planning team may need
to call on professions with expertise in horticulture, landscaping, and architecture. Although it
may not be necessary to include such individuals on the planning team, taking the time to
identify and recruit them during the earliest stages of the planning process will keep the
project moving when their expertise is required.

Designed by Shade ‘n’ Sails of Victoria, Australia.

Transitional shade.

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The process will be well served if the goals of the team, the roles and responsibilities of
its members, and a method for decision making are determined at the outset. In the course of
developing and proposing a shade plan, many decisions will need to be made. One method for
decision making that lends itself to a participatory process is decision by consensus.

The Shade Audit

Once a planning team has been assembled and its roles, goals, and procedures
determined, the group’s first major task will be to conduct a shade audit. The audit will help
the planning team determine how much shade is currently accessible on the school grounds
and if more is needed. The audit consists of a series of user interviews, behavioral
observations, and environmental observations. All of the information collected through the
audit will be used by the planning group to develop their recommendations.

Interviews
Although members of the planning team may be very familiar with their school, their
expertise may not be comprehensive. Any shade planning endeavor should begin by
interviewing several members of each of the identified stakeholder groups. In those
interviews, the planning team can collect important background information regarding:

 When and where outdoor activities occur.

 Which areas of the school grounds are off-limits.
 Any long-term plans for the school grounds, including new construction.
 Opinions regarding the adequacy of existing shade.
 Expectations regarding the plans for additional shade.

Section 6, “How to Conduct a Shade Audit,” contains sample interview questions for
school principals, teachers, and students. Planning teams will need to tailor interview
questions to issues and concerns specific to their school.
Prior to conducting stakeholder interviews, the planning team should secure a site plan.
This is a drawing of the school grounds and buildings that has been drafted to scale. Often site
plans are prepared by surveyors or architects, and may be available from the school’s
principal or the office of the superintendent of the school district. With a site plan,
interviewees can refer to activities in relation to the zones and features of the school grounds
and interviewers can record the information directly onto the plan.

Behavioral Observations
The next step in the planning process involves collecting data at the school site. Adequate
data collection will require several visits to the school. Initial visits will be to observe outdoor
activities conducted on the school grounds and document the usage patterns of students,
teachers, and staff. Knowing in advance at what times the students can be expected to be
outdoors will facilitate the process. Observers will want to document the types of activities
taking place, the location in which they are occurring, the number of students participating,
and their duration. Once again, it will be helpful for observers to have a site plan on which to
make notes regarding outdoor student and teacher activities.
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Environmental Observations
Other visits to the site are recommended in order to take measurements on school
grounds without interfering with the school’s day-to-day activities. On these visits, an
accurate site plan will be essential. If none is available, the planning team will need to draw a
freehand plan of the site, recording the distances between the various buildings and play
equipment. It might be helpful to name different zones if they do not already have names,
such as queuing area or passive play area. It is also important to document any significant
topographical features, such as low spots, slopes, or ravines, as these will influence decisions
about which shade planning strategies will be most appropriate. The site plan should indicate
the boundaries of the school’s property, which direction is north, and whether it is magnetic
north or true north. Often there is an appreciable difference between the two. Determining
true north will be important to ensure that shade is cast in the right place, at the right time of
day, at the right time of year.
It may also be important to mark the locations of important features outside of the school
boundaries, such as the neighboring homes or businesses.
Because ground and building surfaces can reflect ultraviolet (UV) radiation, the planning
team should make notes regarding the surfaces and finishes of each building and play area on
the school grounds.
It will be important for the planning team to also consider the school’s sports areas, such
as baseball diamonds, soccer fields, and basketball courts. In thinking about these features of
the school grounds, the planning team should take into account the shade needs of the
students and coaches who are participating and those of the spectators.
The next task will require some degree of horticultural expertise. The planning team
should inventory each tree and planted area on the school grounds. Trees should be numbered
on the site plan, and a separate set of notes should record the team’s findings for each tree,
including the following:

SHADE PLANNING TOOLBOX

When Is North Not Really North?
The short answer is "Almost always!" There is almost always a difference
between true north and magnetic north. Fluid motion in the outer core, which is
the molten metallic region of Earth, causes the magnetic field to change
unpredictably both over time and by location.
Magnetic declination is the measurement of the angle between magnetic
north and true north. For example, on July 4, 1955, the magnetic declination
for Washington, D.C., was 6 degrees west of true north. On the same day in
2003, the magnetic declination was 10 degrees west of true north.
To find out more about magnetic declination, visit The National
Geophysical Data Center at: http://www.ngdc.noaa.gov/seg/potfld/
declination.shtml

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 Species.
 Estimated height.
 Trunk diameter.
 Condition (e.g., broken branches, dead limbs), paying particular attention to any that
appear to be unhealthy.
 Estimated diameter of the tree’s canopy, that is, the upper part which includes the
branches and leaves.
 Density of the tree’s canopy.

Notes should also be made on the predominant vegetation for areas of densely planted
mixed species.
The final task of the shade audit is to estimate the amount of existing shade on the school
grounds. Measurements should be taken of all of the shade, regardless of whether it is in an
off-limits area. There are two methods for measuring shade, one of which is highly technical
and requires a detailed knowledge of sun projection techniques. The second method requires
only that the planning team mark the shade patterns on the ground at the times of day that
students are outdoors. The ground can be marked with chalk, rope, or baking flour, then
measured and marked to scale on the site plan. Measurements will need to be taken at several
times during the day and throughout the school year to ensure that seasonal changes in the
shade patterns are recorded.

Assessing the Findings

Having completed interviews with representatives of stakeholder groups, observed usage
patterns, and plotted the seasonal shade patterns at the school, the next step in the planning
process is to analyze the quantity and quality of shade that is accessible on school grounds,
and determine if and where additional shade is needed. The following questions will guide the
analysis:

 Will future growth of existing trees result in additional accessible shade?

 Are any areas currently off-limits that could provide additional shade if they were
accessible?
 Are any areas protected from direct UV radiation, but not protected from indirect
(reflected or diffuse) UV radiation?
 Are there future building plans that might be modified to provide additional shade?

Shade Design

Based on the shade audit, the planning team should present its recommendations in text
and graphic format. Recommendations should clearly state the shade goals for each specific
zone of the school property, such as bus queuing area, sports venues, active play areas, or
informal social gathering places, along with strategies for achieving those goals. The team
should consider the range of options at the same time that it is considering the nature of the
shade to be provided. Questions that the planning team should take into account when
developing a shade plan include:
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1674 Centers for Disease Control and Prevention

 Is there a need for protection from rain?

 What are the initial costs for each strategy considered?
 What are the long-term maintenance costs associated with each strategy?
 Is the strategy safe, considering the local weather conditions?
 Is there risk of vandalism, and how can that risk be minimized?

Consulting with knowledgeable architects, landscape architects, or horticulturists is

advisable at this point. Not only will they know the species of vegetation that will meet the
shade requirements for natural applications and the local building codes for any structural
applications, they also can advise the planning team on the potentially complicated tasks of
obtaining local building permits and contracting with builders and landscapers.

Funding

At the same time that the planning team is finalizing the shade design, team members can
explore potential funding sources and volunteer resources for the project. Several potential
sources for funding and hands-on participation are discussed further in section 4, “Case
Studies” and in the appendices of this manual. Some possibilities include:

 Contributions from local and national corporations, including in-kind contributions.

 State and federal grants.
 Volunteers and financial contributions from community service organizations.
 Local fund-raisers.
 Support from environmental organizations.
 Advice from local master gardeners associations and programs.
 Volunteer project work for Boy Scout or Girl Scout troops.
 Student class projects.

Where Can I Find More Information?

Section 6, “How to Conduct a Shade Audit,” provides more detailed information on the
steps of conducting such an audit, including examples of questions that would be appropriate
for interviews with stakeholders. On the following pages are resources for facilitating
participatory decision-making processes, funding, and working with volunteers.

Resources for Shade Planning Teams

Facilitators Guide to Participatory Decision-Making (1996)

Sam Kaner, with Lenny This guide is designed to help groups increase
Lind, Catherine Toldi, Sarah participation and collaboration; promote mutual
Fisk, and Duane Berger understanding; honor diversity; and make effective,
New Society Publishers inclusive, and participatory decisions.
Gabriola Island, BC

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Evergreen
355 Adelaide Street West, Evergreen is a Canadian non-profit environmental
Fifth Floor Toronto, Ontario organization with a mandate to bring nature to
M5V 1S2 Phone: (416) 596- Canadian cities through naturalization projects.
1495 www.evergreen.ca Evergreen motivates people to create and sustain
healthy, natural outdoor spaces and gives them the
practical tools to be successful. Following are several
publications available from Evergreen that should be of
interest to shade planning teams.
Hands for Nature: A Volunteer Management
Handbook This booklet provides practical tips and
ideas for working effectively with volunteers to create
and sustain greening projects. It includes many insights
and helpful statistics from the Community Greening
Volunteerism 2002 Survey as well as generous input
and discussions with experienced volunteer
coordinators and greening participants.
Design Ideas for the Outdoor Classroom: Dig it, Plant
it, Build it, Paint it!
This booklet is a collection of ideas and techniques for
creating native plant and vegetable gardens, and
includes a whole range of built and artistic features for
your school grounds.
All Hands in the Dirt: A Guide to Designing and
Creating Natural School Grounds
This manual will guide you through the planning
process, providing tips and templates for designing a
site that reflects your local natural environment and the
ideas of all involved.
Nature Nurtures: Investigating the Potential of
School Grounds
This report is a comprehensive review of the literature
pertaining to school ground naturalization. It examines
the work of some of the most advanced thinkers in the
fields of child development, education, and
environmental psychology, and it explores the web of
benefits that results when an entire school community
participates in creating more nurturing and diverse
environments for learning on the school grounds.

SECTION 4. CASE STUDIES

Schools and school districts across the United States have already begun the process of
shade planning. This section introduces three case studies. The first, Collier County, Florida,
demonstrates the power of a single individual to motivate a school board to erect shade
canopies over the playgrounds of all of the school district’s elementary schools. The second
case study, Pinellas County, Florida, demonstrates the fund-raising capacity of schools’
parent-teacher organizations (PTOs) and the effect that they can have on decisions of local
school boards. Lastly, the story of the collaboration between Shonda Schilling SHADE
Foundation and the United States Environmental Protection Agency’s SunWise School
Program demonstrates the great potential for partnerships that exists for schools and
organizations concerned with preventing skin cancer.
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Collier County, Florida

Located in Southwest Florida, Collier County encompasses 2,025 square miles and is
home to 296,678 residents, according to the U.S. Census Bureau (2004). The January average
high temperature is 77.6°, which is also the lowest average high throughout the year. The
county’s per capita income for the year 1999 was just over $31,000, approximately $3,000
above the U.S. average for that year, and 10.3% of the county’s population lived below the
U.S. Department of Health and Human Service’s poverty guidelines.
One reason for the county’s higher-than-average per capita income may be the large
number of retirees who have chosen to take up residence in this county. Even with the large
percentage of residents older than 65 years of age, almost 20% of Collier County’s residents
are 18 years old or younger.
The Collier County school district is home to 44 public schools including 2 charter
schools. The policy-making body for the school district is a five-member school board.

Getting Started
Teryl Brzeski is a skin cancer survivor. In 1986, at the age of 37, Ms. Brzeski was
diagnosed with melanoma, the most deadly form of skin cancer. Her diagnosis motivated her
to research the causes of skin cancer and inspired her to do all that she could to help prevent
others from developing the disease. “Having a deadly disease and being lucky enough to have
a full recovery is a wonderful thing. In my case, it has instilled a passion for being alive and a
desire to share cancer prevention information with others.”
Ms. Brzeski became especially concerned about her daughter and the other students at
Seagate Elementary School. Other Collier County parents were equally concerned about their
children’s exposure to ultraviolet (UV) radiation. In fact, during the late 1980s, Seagate’s
PTO raised the $30,000 necessary to erect a pavilion on the school grounds to provide a
shaded area for physical education classes. Other than the covered pavilion, however, much
of the playground was not protected from excessive solar radiation, and during recess,
children played in areas where there was no shade.
Determined to do something about the situation, Ms. Brzeski began her campaign for a
shaded playground by researching options for providing shade on school grounds, eventually
presenting her ideas to the Collier County School Board. Along with two dermatologists and
a representative from the American Cancer Society, she explained to the school board why it
was important to provide children, teachers, and staff with adequate protection from solar
radiation. Their presentation included an architectural drawing of their vision for a shaded
playground at Seagate. Informational packets were distributed to school board members,
making a clear and convincing medical case for providing sun protection on a daily basis. The
presenters told the school board that they were prepared to raise funds themselves, if
necessary, to have the shade structures built.

Approval and Building Costs

The school board not only approved the request for Seagate, but also approved the
construction of shade structures on the playgrounds of all Collier County elementary schools
at a cost of $2.1 million dollars. In June 2002, Seagate Elementary School became the first
county school to be fitted with a shade structure over the playground. By August 2002, before

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the school year began, 21 more schools in Collier County were fitted with shade structures.
Funding for the project was drawn from the school district’s capital budget.
Once the school board decided to build the structures, the project was outsourced to a
contractor, with specifications that 95% of the playground of each school should be covered
by the shade structure. At Seagate Elementary School, the structure covers 8,100 square feet
of the playground.
The shade structure comprises multiple canopies. Each canopy is made of a polyethylene
mesh fabric that is supported by steel cables and held up by galvanized steel poles, which are
secured to concrete pilings. The canopy over Seagate’s playground covers a swing set, three
slides, and other playground equipment. The polyethylene mesh fabric blocks 90% to 95% of
the sun’s UV rays. In addition, it allows heat to escape, promoting air circulation. As a result,
the temperature beneath the canopies is about 15° lower than that of the ambient air in the
middle of the day.

Seagate Elementary School.

Students, teachers, and staff at Seagate Elementary are now protected

from ultraviolet radiation during recess.

Maintenance
After the shade structures were built, several potential modifications were identified as
necessary to increase the longevity of the structures. For example, additional poles were
subsequently added to guard against severe windstorms, and the sails were redesigned and
restrung. In 2005 the county incurred additional expenses due to hurricane damage.
Consequently, when hurricanes are forecasted, maintenance staff take down the structures.
However, contractors must be hired to reassemble them. The county school board will
continue their cost benefit analysis and may consider other options in the future.

Continued Efforts
To date, 25 Collier County elementary schools have been outfitted with shade structures
over their playgrounds. The scope of the initial project was 22 schools, but since that time,
Collier County has built three more schools and has incorporated shade structures into their
design and construction.
Since the implementation of shade coverings in the Collier County school district, other
neighboring school districts have seen the utility and importance of providing students,
teachers, and staff with protection from UV radiation. For example, in the Lee County school
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1678 Centers for Disease Control and Prevention

district, Bonita Elementary School has begun fund-raising efforts to build a shade structure
for the school. The school’s parent-teacher organization has pledged to donate a portion of the
funds, and solicitations of businesses and service clubs are expected to raise more. The school
expects to pay $64,000 to install shade canopies over its two playgrounds.
Teryl Brzeski has won her battle with skin cancer and now fights every day to ensure that
her daughter and the children of Collier County will not have to face the same battle. Her
efforts to educate others on skin cancer and its prevention and to promote sun safety through
the construction of shade structures at elementary schools have motivated not only her county
to take action but have also influenced neighboring counties to take the same steps.

Pinellas County, Florida

Pinellas County, Florida, is located on a peninsula bordered by the Gulf of Mexico and
Tampa Bay. The 280-square-mile county is home to about 921,500 year-round residents and
welcomes an average of 4.5 million visitors each year. Unemployment in Pinellas County
tends to be lower than the Florida state average, which tends to be lower than the national
average. According to the U.S. Census Bureau, the per capita income for the year 1999 was
just under $24,000, and 12.8% of the county’s population lived below the U.S. Department of
Health and Human Service’s poverty guidelines.
With 114 elementary, middle, and secondary schools, the Pinellas County School District
is the 7th largest district in Florida and the 21st largest in the United States.

Image source: http://www.pinellascounty.org/.

Getting Started
By 1995, parents and teachers in several of Pinellas County’s 82 elementary schools had
become concerned about their schools’ lack of indoor gymnasium facilities. Physical
education classes had to be taught outdoors throughout the year and were canceled when
inclement weather required it. The Parent-Teacher Associations (PTA) in those few schools
took the initiative to conduct fund-raisers, such as bake sales and jog-a-thons, to collect the
$35,000 that each school would need to erect a weather-protected outdoor play area. The
schools tapped local expertise in determining the best design for the structures and for
executing the construction.

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In 2002, the local PTA efforts in Pinellas County paid off for all elementary school
children in the county, prompting the Pinellas County school board to determine that all 82
elementary schools in the county should have similar structures to protect students, teachers,
and staff. Walter Miller, associate superintendent of institutional services, cites health and
medical concerns as the rationale for the school board’s decision, “We ensure that the
students, faculty, and staff have a clean and safe environment inside of the school, so it’s only
right that we are concerned about the environment outside of the school as well. Children and
physical education teachers were exposed to extreme heat and put at risk for skin cancer,
which in Florida is a major concern, so the shade structures buy students and teachers the
opportunity to escape the heat and exposure to ultraviolet radiation.”

Building Costs
Funding for the construction has been supported through the school district’s capital
budget. The school district’s facilities department identified a general contractor to oversee
construction of the buildings, and the contractor received bids from a number of
manufacturers. A local firm was ultimately selected, because using a local builder would
reduce shipping and travel costs.
To reduce costs, a single design was used for all schools, although each school
determined the placement of its shade structure. The structures are 40-by-80 feet, roughly the
size of a tennis court, and are constructed of metal with concrete floors. The designers had to
consider the threat of strong winds associated with hurricanes or tornados, very real threats in
Florida. As a result, a larger foundation was incorporated into the design to create uplift
resistance in the event of a windstorm. Each structure approximately cost $60,000, which
according to the director of facilities was considerably lower than it would have been if each
building had been individually bid. Those schools that had begun fund-raising efforts for a
shade structure were allowed to add that money to the school board’s budget, and make
improvements to the original design, including a larger structure, if that was what the school
wanted.

Maintenance
As the oldest of the buildings begin to age, some of the maintenance requirements are
becoming more obvious. Most apparent is the need to paint the steel structures regularly to
prevent premature rusting. Structures that need repainting must first be sandblasted to remove
old paint and rust. In some cases, as more time passes, other maintenance issues will be
identified. Recently, some of the schools have experienced problems with birds nesting in the
metal framing that supports the roof. Modifications to the design of future buildings and to
those already constructed may solve that problem.

Continued Efforts
Currently, 66 of the 82 elementary schools in Pinellas County have shade structures. The
school district expects that all schools in the county will have these structures by 2007. This
tremendous accomplishment is the result of the efforts of just a few concerned parents,
teachers, and principals who recognized the importance of providing sun protection to their
students, teachers, and staff.
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If you would like to find out more about the efforts to prevent skin cancer at Pinellas
County Schools, feel free to contact:
Walter Miller, Associate Superintendent, Institutional Services in Pinellas County Phone:
(727) 547-7167
Jim Ewbank, Supervisor of Pre-K Physical Education Phone: (727) 588-6078

SHADE Foundation of America and the U.S. Environmental Protection

Agency’s SunWise School Program

SHADE Foundation of America

In February 2001, Shonda Schilling, a 33-year old mother of four young children and
wife of Boston Red Sox pitcher Curt Schilling, was diagnosed with melanoma. Having been a
lifelong sunbather, Ms. Shilling felt a need to inform the public about the dangers of exposure
to the sun’s UV rays. She learned that although Arizona has the highest melanoma rate in the
United States, the state had no organizations devoted to preventing the cancer. As a way to fill
that gap, Shonda founded the SHADE Foundation.
SHADE Foundation of America is a non-profit organization dedicated to the education,
prevention, and detection of skin cancer. Created in September 2002, the foundation’s goal is
to prevent the development of skin cancer through educational programs and free skin cancer
screenings.

The U.S. Environmental Protection Agency’s SunWise School Program

First piloted in May 1999, the SunWise School Program, developed by the United States
Environmental Protection Agency (EPA), is an environmental and health education program
designed to teach children and their caregivers how to protect themselves from overexposure
to the sun. The standards-based, cross-curricular lessons in the SunWise Tool Kit were
designed to foster sun-safe behaviors in children and to increase their knowledge and
appreciation of the environment. In addition, the program encourages schools to create shade
structures, adopt sun-safe policies, and develop additional community partnerships.
Since the year 2000, the K–8 curriculum has been available on a national basis, free of
charge, to any school registering at the EPA SunWise Web site, www.epa.gov/sunwise. To
participate in the SunWise program, schools must adopt a SunWise activity, which may
include implementing classroom lessons, collecting and reporting UV radiation data, adopting
school-wide sun-safe policies, or engaging in skin cancer prevention community outreach.
Schools must also participate in a program evaluation.

Creating a Collaboration
The development of the collaboration between the SHADE Foundation and the EPA
began in October 2002. Linda Rutsch, Director of the SunWise School Program at the EPA,
was visiting the Web site of a melanoma foundation in Nevada when she noticed a link to the
SHADE Foundation. “Usually I will check out various links in order to keep up with what’s
out there,” Ms. Rutsch recalls. She clicked on the SHADE Foundation’s link, learned about
the Foundation’s skin cancer prevention efforts in Arizona, and found a contact name. Ms.

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Rutsch called Sue Gorham, executive director of the SHADE Foundation, and told her, “I
would like to send you a SunWise Kit and tell you a little about our program.”
As luck would have it, the SHADE Foundation was in the process of searching for a
school curriculum on sun safety. Ms. Schilling and Ms. Gorham decided to develop a
program for schools that would allow them to acquire shade structures for their campuses
through the SHADE Foundation. The idea was to encourage schools to implement a sun-safe
curriculum and to develop sun-safe policies within the school.
The EPA’s SunWise curriculum was exactly what the SHADE Foundation was seeking,
and the partnership began there. As Ms. Rutsch remembers, “That was in October of 2002
when we first started corresponding. In January of 2003, we had our first face-toface meeting
where we talked about working together.”

How the Program Works

The SHADE SunWise School Program requires schools to qualify in two steps. The
first step is to request the EPA SunWise curriculum, usually via the EPA SunWise Web site.
Once a school has reviewed the curriculum and made the decision to implement it, the next
step is to apply to the SHADE Foundation to participate in the SHADE SunWise School
Program. The written grant statement must include a description of the policy and activities
the school proposes to adopt for skin cancer prevention. Upon demonstration of sustained
teaching activities and the implementation of sun protection policies, the Foundation gives
grants for the consideration of a shade structure.
The shade structures are an appropriate reward for the school’s achievements. Besides
protecting students, teachers, staff, and visitors from overexposure to UV radiation, the
structures contribute to a more comfortable environment for students to enjoy outdoor
physical activities. As Sue Gorham points out, “Here in Arizona, when children slide down
the sliding board, it is so hot that it could take the skin off their backs. Under these shade
structures, the temperatures will drop as much as 20 degrees.”

Building Costs
The cost for providing a school with the 24' x 35' shade structure is between $6,000 and
$10,000. The shade structures are of shade cloth, approximately 24' x 35' and hoisted on
metal poles. The shade cloth provides 98% protection from UV radiation. Schools have
flexibility in determining where on their grounds would be the most appropriate location for
their shade structure.
The SHADE Foundation assumes all costs of the shade structures, with money raised
through a number of fund-raising events, ranging from auctions to golf tournaments. The first
year of implementation was used to demonstrate both the program’s needs and SHADE’s
fiscal responsibility and ability to execute the managerial and administrative duties of the
project. Hoping to supplement its fund-raising efforts with grant money, the program began
applying for grants in 2004. In 2005, SHADE received its first grant of $25,000 from
Teammates for Kids. SHADE, now in its 4th year of giving grants, has funded approximately
50 structures across the nation at a cost of $267,000.
Though some schools have expressed concern over the required maintenance on the
structures, to date this has not been a great problem, because the structures have not required
much maintenance.
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Continued Efforts
To date, the SHADE Foundation has constructed 50 shade structures in U.S. schools and
community areas which benefit children. In the coming year, the Foundation plans to raise
funds to support 95% of grant applications.
The SHADE Foundation and EPA were recognized for their work by being awarded the
“2003 Excellence in Cancer Awareness Award,” presented by Congressional Families Action
for Cancer Awareness. This award is presented annually to the organization that best
exemplifies a total commitment and dedication to assisting others in preventing cancer. This
award honors the partnership between the SHADE Foundation and the EPA SunWise
program in addressing the issue of skin cancer prevention. “We are so proud of our
partnership receiving the award,” says Ms. Gorham. “We hope that through qualifying for
grants in the future, we will be able to carry on this work in all states.”

Cherokee Elementary school: the first school to graduate with the SHADE Foundation and SunWise
curriculum.

If you would like to find out more about the SHADE Foundation’s efforts to prevent skin
cancer, feel free to contact:

Sue Gorham Executive

Director of the SHADE Foundation
www.shadefoundation.org
Phone: (480) 614-2278
Fax: (480) 614-2279
E-mail: [email protected]
To find out more about the EPA’s SunWise School Program, contact:
Linda Rutsch
Director of the SunWise Program
www.epa.gov/sunwise
Phone: (202) 343-9924
Fax: (202) 343-2362
E-mail: rutsch. [email protected]

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KEY TERMS AND CONCEPTS

Latitude is a north-south measurement of any position on the Earth. Measured in
degrees, latitude is 0° at the equator and 90° at the North and South Poles. The line that
connects all locations of the same latitude is called a parallel. Shade planning teams will
need to know the latitude to determine where a tree or structure will cast its shadow
throughout the day.
Longitude is a west-east measurement of any position on the Earth. The line that
connects all locations of the same longitude is called a meridian. Longitude, like
latitude, is measured in degrees, with 0° occurring at the Greenwich Meridian or Prime
Meridian. Measurements of longitude range from Prime Meridian at 0° to 180° going
either west or east. The 180th meridian east is the International Date Line. Shade
planning teams will need to know their longitude to determine solar noon.
Solar Noon is the time of day when the sun is aligned with true north and true south
and is specific to each longitude. In the northern hemisphere, a shadow cast by a vertical
pole at solar noon will point toward true north. Solar noon is also the midpoint between
sunrise and sunset.

SECTION 5. THE EARTH-SUN RELATIONSHIP

For any shade planning team, the primary objective is to ensure that shade falls in the
right place, at the right time of day, throughout the year. A shade planning team should
include members with at least a working knowledge of the Earth’s relation to the sun. This
section is designed to:

 Graphically illustrate the effects of daily and seasonal changes in solar angles on the
length and direction of shadows.
 Provide a basic introduction, or reintroduction, to the Earth’s relationship to the sun
and solar geometry.
 Provide a list of resources that can assist the planning team in ensuring that shade
falls in the right place, at the right time of day, throughout the year.

The Sun’s Annual Path and the Creation of Shade

Shadows cast by the sun grow longer from June 21st until December 21st, and then grow
shorter from December 21st until June 21st. This is a result of annual changes in the solar
altitude angle. The change in shadow length is depicted graphically in the following
illustration. A 36-foot tall tree with a canopy spread of 48 feet planted at the geographic
center of the contiguous United States (Longitude=98°3'West; Latitude=39°0'North) tree
would cast shadows at solar noon throughout the year as follows:
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The Sun’s Daily Path and the Creation of Shade

Observing that same 36-foot tall tree, the shadows that are cast move from west to east
throughout the day in response to the sun’s east-to-west movement. On March 21st, the
shadows that would be cast throughout the school day would appear as follows:

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The Earth’s Rotation and Revolution

Every 24 hours, the Earth makes one rotation on its axis; every 365.24 days, the Earth
makes one revolution around the sun. If one were to view the Earth spinning on its axis, one
would note that the axis is not perpendicular to the Earth’s orbit around the sun, but is tilted at
an angle of 23.5°, with the North Pole always pointing directly at the North Star.
Furthermore, the Earth’s orbit around the sun is not circular, but elliptical, causing the Earth’s
distance from the sun to vary by as much as 3 million miles throughout the year. The annual
variation in the Earth’s distance from the sun affects the amount of solar radiation intercepted
by the Earth by as much as 7%. The changing distance from the sun, however, is not
responsible for the changes in seasons. The changes in season are caused instead by the
constant 23.5° tilt of the Earth and the Earth’s rotation around the sun.
Summer solstice, which is on or about June 21st, marks the beginning of summer in the
northern hemisphere. The Earth is positioned so that the North Pole is leaning toward the sun
at 23.5°. On summer solstice, the length of the day, from sunrise until sunset is greater than
12 hours for all latitudes north of the equator, and less than 12 hours for all latitudes south of
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the equator. On summer solstice, the center of the sun lines up with the latitude known as the
Tropic of Cancer, which is at 23.5° north.

KEY TERMS AND CONCEPTS

The Earth’s axis of rotation is not perpendicular to its orbit around the sun, but is
tilted at an angle of approximately 23.5°. Solar Declination is the angle that a given
hemisphere is tilted toward the sun on any given day. It is marked by the latitude on
the Earth where the location of the sun is directly overhead at solar noon. Because of
the 23.5° tilt, this location is always somewhere between 23.5° north and 23.5° south,
depending on the time of the year. Solar declination is 0° when the sun lines up with
the equator on the equinoxes.
Equinox is one of the two periods when the declination of the sun is 0°, or the
sun is lined up exactly with the equator. The autumnal equinox occurs on or about
September 21st, and the vernal equinox occurs on or about March 22nd. Only on
these 2 days are the hours of the day and night equal, and only on these 2 days does
the sun rise due east and set due west.
Solstice is either the longest or the shortest day of the year. In the northern
hemisphere, summer solstice is the longest day of the year and occurs on or about
June 21st. Winter solstice occurs on or about December 22nd.
True north, also known as geographic north, is the northernmost point on the
Earth as determined by the Earth’s rotation. This usually differs from what a compass
indicates as north. When a compass points to north, it is pointing toward magnetic
north, which in some locations in the United States, may be as far as 20° from true
north.

Image Source: National Geophysical Data Center.

At the winter solstice, on or about December 22nd, the Earth is positioned so that the
North Pole is leaning away from the sun, and all latitudes south of the equator experience
days longer than 12 hours and all latitudes north of the equator experience days shorter than
12 hours. This marks the beginning of summer in the southern hemisphere and the beginning
of winter in the northern hemisphere. On winter solstice, the center of the sun lines up with
the latitude known as the Tropic of Capricorn, which is at 23.5° south. On the equinoxes,
those being around September 21st and March 21st, the Earth is positioned so that the North
Pole points neither toward nor away from the sun. On those 2 days, the Earth’s equator lines

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up with the center of the sun, resulting in days that are exactly 12 hours long, regardless of
latitude.

KEY TERMS AND CONCEPTS

Solar azimuth angle is the angle on the horizontal plane between the point on the
horizon that is directly beneath the sun and true south. The azimuth angle determines the
direction of shadows.
Solar elevation angle or solar altitude angle is the angle that describes the height of
the sun in relation to the nearest point on the horizon. It varies according to the time of day
and the season and determines the length of shadows.

Axis Tilt and Solar Radiation

That the Earth’s axis is tilted affects the amount of solar radiation reaching the Earth in
three ways. First, the length of the day changes throughout the year. In the northern
hemisphere, beginning on the summer solstice, the sun’s daily path is increasingly lower in
the sky (making shadows longer) until the winter solstice, after which the days become
increasingly longer. As days grow longer, the risk of exposure to excessive ultraviolet (UV)
radiation is greater.
Second, when the sun is at a lower altitude, its rays are spread over a larger area, reducing
the intensity of the sun’s radiation, including UV radiation.
Third, levels of solar radiation are also affected by how much of the atmosphere the sun’s
rays must pass through. Solar radiation levels, including UV radiation, are greatest when the
sun is higher in the sky and solar radiation has a shorter path to travel through the atmosphere.
When the sun is lower in the sky, solar radiation has a longer path to travel through the
atmosphere, resulting in more of the sun’s radiation, including UV radiation, being absorbed
or scattered by the atmosphere.

Putting It All Together

In order to create shade that falls in the right place, at the right time of day, throughout
the year, it is essential that the planning team model the shade that their proposed buildings
and plantings will cast. To do that, the team will need to know:

 Longitude and latitude of the school’s location.

 Location of true north and true south.
 Size, shape, and orientation of proposed buildings.
 Growth patterns and locations of proposed plantings.
 Time of day that solar noon occurs throughout the year at that longitude.
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1688 Centers for Disease Control and Prevention

The more perpendicular a beam is to a surface, the brighter the beam will be on the surface.

Armed with that information and a working knowledge of solar geometry, it is possible to
use mathematical and geometrical procedures to model the shade that will be cast by
proposed structures and plantings. A number of computer software programs are available to
do that work for you. Following are descriptions of two such programs, one of which has
been developed for modeling sun and shade patterns for solar collectors, though it may well
fit into the budgets and serve the purposes of some schools. The other is a program that has
been designed specifically for shade planning.

Visual Sun Chart

www.visualsunchart.com/

Visual Sun Chart is a graphics program that was developed for visualizing solar shading
to maximize the efficiency of solar collectors. It allows the user to model trees and buildings
and, along with a free download, visualize the shade patterns that are cast. With Visual Sun
Chart, a user can:

 Model buildings and trees.

 Export scenes to POV Ray 3.5, a free download that models shade produced by
buildings and trees.
 Create reports which give information about positions of the sun, times of sunrise
and sunset, horizontal and vertical shadow angles, and angles of incidence.
 View and print 3D models of sun movements.

webShade
www.shadeaudit.com.au
www.webshade.com.au

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webShade is an interactive software package that allows users to prepare shade audits
without the need for technical skills or expertise. Users can either scan their site plan into the
computer or use a simple drawing program to create a 3D model of their site. On completion
of the shade audit with webShade, the program produces a printed site plan, complete with
shade solutions and simulated outcomes. Available as an internet download, a webShade user
can:

 Project existing summer and winter shade patterns.

 Model alternative shade strategies.
 Project the summer and winter shade patterns for those alternatives.
 Prepare presentation-ready materials, including site-specific UV radiation
information and design strategies.

Where Can I Find More Information?

To determine a location’s latitude and longitude:

 www.pbs.org/wgbh/nova/longitude/find.html
 http://zipinfo.com/search/zipcode.htm
 http://geonames.usgs.gov/pls/gnis/web_query.gnis_web_query_form
 http://tiger.census.gov/cgi-bin/mapbrowse-tbl

To determine the time of day that solar noon occurs:

 http://www.srrb.noaa.gov/highlights/sunrise/sunrise.html
 http://users.vei.net/pelican/sunrise.html
 http://www.spot-on-sundials.co.uk/calculator.html

For more information on solar geometry:

 http://www.geog.ouc.bc.ca/physgeog
 http://education.gsfc.nasa.gov/experimental/July61999siteupdate/inv99Project.Site/
Pages/solar.insolation.html

SECTION 6. HOW TO CONDUCT A SHADE AUDIT

The purpose of the shade audit is to document the usage patterns of the school grounds
and determine the amount and quality of existing shade. All of the information collected
through the shade audit will be used by the planning group to develop recommendations for
making the best use of existing shade and, if required, for creating additional shade. The final
product of a shade audit is a written report or presentation of the shade team’s findings and
recommendations to the school community.
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1690 Centers for Disease Control and Prevention

The process includes a series of interviews, behavioral observations, and environmental

observations followed by an assessment of the findings and the generation of prioritized
recommendations for achieving the goals. The entire process may take as long as 1 year. The
first step in the process is to conduct stakeholder interviews.

Stakeholder Interviews

Before the first interview is conducted, the shade planning team should produce a
comprehensive list of stakeholder groups that may have an interest in, or be affected by, the
resulting plan. The team should identify and invite representatives from each stakeholder
group to be interviewed. Ideally, each of the stakeholder groups will be represented on the
planning team; nonetheless, it will be necessary to interview members of all stakeholder
groups even if they are also represented on the team. For many schools the list would include:

 School administrators.
 Teachers, particularly the physical education teacher.
 Coaches.
 School nurse.
 Students.
 Building maintenance staff.
 Organizers of little league programs or others who may use the school grounds on
weekends.
 Residents of the neighborhood adjacent to the school.

Interview Guides

For each stakeholder group, the shade planning team should generate a list of appropriate
questions to explore in the interview process. Questions should cover topics with which the
interviewee is very familiar. Some may ask the interviewee to discuss outdoor activities that
they observe or in which they participate. In order to get the most out of the interviews,
interviewers should ask interviewees to reference the school’s site plan while describing those
activities. This is a scaled drawing of the school grounds and buildings. Often such plans are
prepared by surveyors or architects, and may be available from the school’s principal or the
office of the superintendent of the school district. Referring to a site plan during the interview
process allows interviewees to reference activities in relation to the zones and features of the
school grounds and allows interviewers to record the information directly onto the site plan.
Through the interview process, interviewees may identify school grounds users of whom the
shade planning team was not previously aware. Such newly identified stakeholders should be
added to the list of interviewees. Following are examples of questions that would be
appropriate for school principals, teachers, students, and other stakeholders. This is not meant
to be a comprehensive list, but instead gives samples of questions that might be most
appropriate for each stakeholder group. The planning team will need to tailor their questions
to the issues and concerns specific to their schools.

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Sample Questions for School Principals

 How many students attend the school?
 What is the age range and distribution of the students?
 Does the school have sun-safe policies in effect?
Hats?
Sunglasses?
Long-sleeved shirts?
Sunscreen?
 What are the outdoor activities in which students participate?
Recess?
Lunch?
Assemblies?
Physical education?
Educational activities?
Informal social gatherings?
Extended day activities?
Intermural and intramural athletic events?
 For each: Where do they occur? (Have interviewee refer to site plan.)
At what time of day do they occur?
What is the duration of the activity?
 Do you believe there is adequate accessible shade on the school grounds? If not,
where is additional shade most needed? (The interviewee should be asked to refer to
the site plan.)
 Is there inaccessible shade on the school grounds that could be made accessible?
 Is there a need for an area on the school grounds that is protected from precipitation?
 Are there plans for making improvements to the school, such as remodeling, adding
classroom space, or landscaping projects?
 Does the school currently have problems with, or has it recently experienced
problems with vandalism?
 Are there groups that have permission to use the school grounds after school hours
and on weekends?
 Are there groups that, without formal permission, use the school grounds after school
hours and on weekends?
 Can you think of any barriers to providing additional shade at this school?
 What type of shade do you think would work best at this school (solid-roof structure,
shade cloth structure, or natural shade)?

Sample Questions for School Teachers

 What age group do you teach and how many students are in your class?
 What are the outdoor activities in which your class participates?
Recess?
Lunch?
Assemblies?
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1692 Centers for Disease Control and Prevention

Physical education?
Educational activities?
Informal social gatherings?
Extended day activities?
Intermural and intramural athletic events?
 For each: Where do they occur? (Have interviewee refer to site plan.)
At what time of day do they occur?
What is the duration of the activity?
 Does the school have sun-safe policies in effect?
Hats?
Sunglasses?
Long-sleeved shirts?
Sunscreen?
 Do you believe there is adequate accessible shade on the school grounds? If not,
where is additional shade most needed? (The interviewee should be asked to refer to
the site plan.)
 Is there inaccessible shade on the school grounds that could be made accessible?
 Is there a need for an area on the school grounds that is protected from precipitation?
 Are there times that teachers are required to be outdoors, such as to monitor the
activities on the school grounds? If so, how often does this happen and for how long?
 If there were outdoor classroom space, would you use it? Do you think that other
teachers would?
 Can you think of any barriers to providing additional shade at this school?
 What type of shade do you think would work best at this school (solid-roof structure,
shade cloth structure, or natural shade)?

Sample Questions for Students

 How old are you?
 When you are outside, do you usually play/socailize in the sun or in the shade?
During recess?
During and after lunch?
During physical education class?
During extended day activities (if applicable)?
Intermural and intramural athletic events (if applicable)?
 For each, where do you play? (Have student refer to site plan.)
 Does the school have sun-safe policies in effect?
Hats?
Sunglasses?
Long-sleeved shirts?
Sunscreen?
 Do you believe there is adequate accessible shade on the school grounds? If not,
where is additional shade most needed? (The interviewee should be asked to refer to
the site plan.)
 Are there shaded areas on the playground that you would prefer not to use? Why?
 Are there areas on the school grounds where you are not allowed to play?

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Sample Questions for Building Maintenance Engineers

 Do you believe there is adequate accessible shade on the school grounds? If not,
where is additional shade most needed? (The interviewee should be asked to refer to
the site plan.)
 Is there inaccessible shade on the school grounds that could be made accessible?
 Is there a need for an area on the school grounds that is protected from precipitation?
 Are there plans for making improvements to the school, such as remodeling, adding
classroom space, or landscaping projects?
 How would a plan to plant trees or build a shade structure be affected by the school’s
utility services? (On the site plan, have the interviewee mark the service lines for the
school’s utility services.)
 Does the school currently have problems with, or has it recently experienced
problems with vandalism?
 Are there groups that have permission to use the school grounds after hours and on
weekends?
 Are there groups that, without formal permission, use the school grounds after school
hours and on weekends?
 Can you think of any barriers to providing additional shade at this school? For
example, would watering trees or removing tree litter be a problem?
 What type of shade do you think would work best at this school (solid-roof structure,
shade cloth structure, or natural shade)?

Sample Questions for Neighbors

 Do you believe there is adequate accessible shade on the school grounds? If not,
where do you believe that additional shade is most needed? (The interviewee should
be asked to refer to the site plan.)
 Do residents of the neighborhood utilize the school grounds for activities on
weekends or after school hours?
 In what types of activities have you seen neighbors participate on the school
grounds?
 For each: Where do they occur? (Have interviewee refer to site plan.)
On what days of the week and at what time of day do they occur? What is the
duration of the activity?
 Is there a need for a rain-protected area on the playground?
 Do you think that the school currently has problems with, or has recently experienced
problems with vandalism?
 Can you think of any barriers to providing additional shade at this school?
 If the school were to plant additional shade trees, are there areas on the school
grounds where planting them might create a problem for neighbors?
 If the school were to build a shade structure, would it create a problem for neighbors?
 Are there areas of the school grounds where a shade structure might create a problem
for neighbors?
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1694 Centers for Disease Control and Prevention

 What type of shade do you think would work best at this school (solid-roof structure,
shade cloth structure, or natural shade)?

Once all interviews have been conducted, the planning group should meet to discuss their
findings. Information collected from different stakeholder sources should be synthesized and
clarifications on, or answers to, any resulting questions should be sought by the group.

Behavioral Observations

The next step in conducting a shade audit involves observation of students, teachers,
staff, and visitors on school grounds. Visits to school grounds after hours and on weekends
will be necessary to confirm non-school–related activities that might be affected by the shade
plan. Information collected in the interview process should guide the behavioral observations.
Observers will want to document the types of activities, their locations, the number of
individuals who participate in each activity, and the duration of the activity. Once again, it
will be helpful for observers to make notes regarding all activities on a site plan. Participants
in this process should model good sun-protective behaviors by wearing sunglasses, hats, and
long-sleeved shirts. Observations should be made on several occasions to capture the many
activities that occur on school grounds and, as much as possible, observations should be made
unobtrusively. Observers should note whether activities are occurring in a particular location
because no other area will accommodate that activity, or if there are other locations where the
activity could take place, particularly a shaded area. Once again, upon completion of this step,
the planning team should meet to discuss findings. If there are discrepancies between the
information collected in the interview process and the behavioral observations, the team
should note the discrepancies and seek clarification.

Environmental Observations

Other visits to the site will need to be planned to take measurements on school grounds
without interfering with the day-to-day activities of the school. On these visits, having an
accurate site plan will be essential. If none is available, the planning team will need to draw a
freehand plan of the site, taking careful measurements of the buildings and recording the
location and size of each. The site plan should indicate the boundaries of the school’s
property, which direction is north, and indicate if it is magnetic or true north, since there is an
appreciable difference between the two. Determining true north will be important to ensure
that shade is cast in the right place, at the right time of day, throughout the year. (See text box
on page 22 of this manual.) It may also be important to mark the locations of important
features outside of the school boundaries, such as the location of neighboring homes or
businesses and any buildings not on school property that cast shadows or reflect solar
radiation onto the school grounds. Once at the site, the planning team should number the site
plan with all buildings and play equipment on the school grounds, recording the distances
between features. The planning team will need to estimate the height of each of the buildings
on the school grounds and record it on a separate set of notes, along with other characteristics
of the building. Attachment A is an example of a building description sheet. It may be helpful

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to name areas or zones of the site if they do not already have a name. Zones can be named
according to their use, such as “queuing area” or “passive play area.” It is also important to
document any significant topographical features, such as low spots, slopes, or ravines,
because they will influence decisions on which shade strategies are most appropriate.
Because ultraviolet (UV) radiation can be reflected off ground and building surfaces, the
planning team should make notes regarding the surfaces and finishes of each of the buildings
and play areas on the school grounds.

Creating a Tree Inventory

The next task will require a degree of horticultural expertise. The planning team should
inventory each tree and planted area on the school grounds, noting for each:

 Species.
 Estimated height.
 Trunk diameter.
 Condition (e.g., broken branches, dead limbs), paying particular attention to any that
appear to be unhealthy.
 Estimated diameter of the tree’s canopy, that is, the upper part which includes the
branches and leaves.
 Density of the tree’s canopy.

Trees should be numbered on the site plan and a separate set of notes should record the
team’s findings regarding each. Where densely planted areas of mixed species exist, notes
should be made on which ones predominate. Attachment B is an example of a tree inventory
data sheet.

Estimating the Height and Trunk and Canopy Diameters of a Tree

There are many ways to estimate the height and canopy diameter of a tree. Following is
one method:

a. One member of the planning team first measures his or her own height in inches or
feet.
b. The team member then stands next to the tree while another team member stands
about 20 paces away.
c. With one eye closed, the second team member holds a pencil vertical at arm’s length
and covers part of the pencil so that the visible part is the same apparent length as the
team member standing next to the tree.
d. Still keeping one eye closed, the second team member then moves the pencil up the
tree and measures how many times taller the tree is than the team member standing
next to it.
e. Multiply that number by the first team member’s height and the result is a good
estimate of the tree’s height.
f. To estimate the canopy diameter, the second team member again closes one eye and
holds the pencil at arm’s length, covering part of the pencil so that the visible part is
the same apparent length as the tree height.
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g. The pencil is then turned horizontal and, measuring at the canopy’s widest point,
used to determine how many times wider the canopy is than the tree’s height.

To determine the diameter of a tree, simply measure the tree’s circumference, usually at
about 3 feet above the ground, and divide it by 3.14.

Describing a Tree’s Canopy Density

The task of describing a tree’s canopy density is somewhat subjective since there is no
common metric. For the purposes of shade planning one could describe the density of a tree’s
canopy by rating it as “open,” “moderate,” or “dense.” Standing beneath the tree and looking
through its branches, if over 90% of the sky is blocked by the tree’s canopy, it can be
described as “dense.” If between 50% and 90% of the sky is blocked by the canopy, it can be
described as “moderate,” and if less than 50% of the sky is blocked by the canopy, it can be
described as “open.” An open canopy provides little UV radiation protection.

Measuring Existing Shade

The final task of the shade audit is to estimate the amount of existing shade on the school
grounds. Measurements should be taken of all shade, regardless of whether it is off-limits.
There are two methods for measuring shade, one of which is highly technical and requires a
working knowledge of both solar geometry and computer-assisted design software. The
second method requires only that the planning team mark the shade patterns on the ground at
the times of day that the school grounds are used. The ground can be marked with chalk, rope,
or baking flour, then measured and marked to scale on the site plan. Measurements at several
times during the day and throughout the school year will be neccesary to ensure that adequate
shade is provided at the right time of day, throughout the year.
If the sky can be seen by those under a tree or structure, they are at risk for
exposure to indirect UV radiation.

Considering Potential Shade Strategies

Having collected information regarding the activity patterns of the different users of the
school grounds and the shade patterns cast by trees and buildings, it is now time for the
planning team to assess their findings and make recommendations to the school community.
The planning team might find that the school grounds provide adequate protection from both
direct and indirect UV radiation; however, if not, the team will need to make
recommendations for making more shade accessible to students, teachers, staff, and visitors.
The team should first consider strategies that increase the amount of accessible shade at very
low or no additional cost to the school. These might include revising school policies to allow
access to off-limits shaded areas or relocating playground equipment or picnic tables to areas
of the school grounds where shade already exists. The planning team will also need to
consider reflected UV radiation in their recommendations. These may include modifying
ground and building surfaces to reduce their reflectivity. The planning team might determine
that climbing vines would be the best solution for the indirect UV radiation reflected off a
smooth wall or that artificial turf would be most appropriate to reduce the UV radiation

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reflected from a concrete playground. The team should engage in a process to help them
examine the cost effectiveness of strategies that could be employed as they are evaluating
whether or not a particular strategy will accomplish the intended goal. More information
about this is provided below.

The Shade Planning Matrix

The Shade Planning Matrix is a tool that can assist the planning team with comparing
potential strategies for achieving their goals while examining and comparing the cost
effectiveness of each of the strategies. The team’s stated shade goals must be specific, both at
this stage of planning and when it is time to present recommendations to the school
community. The planning team should also clearly state any goals that are ancillary to
providing shade, such as the construction of an outdoor classroom or the creation of a natural
wildlife habitat. Matrices should be developed for each area that is being considered.
Information for completing the Shade Planning Matrix may come from a variety of
sources. It could be necessary to request an informal quote from a contractor if the planning
team is considering a solid-roof structure. Many vendors will provide prices over the internet
if the team believes that a shade cloth structure would be an effective strategy. If a natural
solution is being considered, the team will want to enlist the help of their United States
Department of Agriculture County Extension Service agent, who can provide information on
the most appropriate trees, shrubs, and vines for each geographical area, and the rates at
which the vegetation is expected to grow. The Shade Planning Matrix is Attachment C.

Making Recommendations

When the team has considered potential strategies for achieving their shade goals and has
arrived at a consensus on a set of recommendations, their final task is to formalize them in a
report to the school community. Across schools and shade planning teams, considerable
variability is likely in the report format. However, several components are essential:

The Rationale
Besides the most important reason, that shade provides protection from the harmful
effects of solar UV radiation, the rationale should include any additional benefits that could
be accrued through the strategies that the shade planning team has recommended. Those
benefits might include the creation of an outdoor classroom and sheltered area for active play
during inclement weather, provision of shade for athletic event spectators, or the creation of a
wildlife habitat.

Statement of Goals
With as much specificity as possible, the statement of goals should include a physical
description of the location, the amount of shade needed, and a description of the activities that
occur. One example is the following:
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The planning team has determined that there is a need to increase the amount of shade
over the area of the playground bounded to the north by the south wall of the main building,
to the east by the chain-link fence, to the south by the edge of the playground pavement, and
to the west by the basketball courts. Based on patterns of use by the students, it is estimated
that there is a need for a total of 2,500 square feet of shade. This section of the playground is
home to the jungle gym and is popular for passive play as well. Because it provides an
unobstructed view of the entire playground, this is where the playground monitors usually
stand.

Strategies for Achieving the Goals

Strategies for achieving the goals could include moving playground equipment to a
shaded area of the playground, revising school policy to make off-limit areas accessible, or
building structures or planting trees to provide additional shade. The planning team should
present their recommendations, but should also be prepared to discuss alternative plans.
If the team is recommending the creation of new shade, it should be prepared to discuss
the performance characteristics of the materials providing shade, including:

 Where the tree(s) or shade structure will be located.

 The amount of shade that will be provided.
 Whether the need is for seasonal or year-round shade.
 The costs associated with providing the shade, including both initial and annual
maintenance costs.
 The estimated lifespan of the building or tree(s) providing the shade.

Approaches for Achieving the Goals

The cost of providing shade can range from an afternoon of volunteer labor to the
expense of building a large outdoor classroom with electricity and plumbing. The school
district’s budget for the project often cannot be known until the planning team presents their
recommendations. For this reason, modifiable plans and alternate funding sources should be
explored. While members of the planning team are completing the shade audit, other
members can be exploring potential funding sources and volunteer resources. As the planning
team presents their recommendations, they should also be prepared to present the options for
advancing their plan. Options could include fund-raising efforts by the parent-teacher
organization, donation of building materials by a local builder’s supply store, or a tree
planting project by a local Boy Scout troop.

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 Attachment A: Building Description

School: ______________ Date: ______________

Building Name of Building Height at Eaves Height at Ridge Wall Material Wall Color
Number

Attachment B: Tree Inventory

School: ______________ Date: ______________

Tree Species Height Trunk Canopy Canopy Age Condition

Number Diameter Diameter Density
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Attachment C: Shade Planning Matrix

Area to be Addressed: __________ Recommended Total _______

Square Footage of Shade:
Current Status: ____________ Goal: ____________

Immediate Estimated Total Life Expectancy

Initial Total Five- Total Shade at Total Ten-
Strategy 1: Amount of Annual Shade at and Average
Investment Year Cost Five Years Year Cost
Additional Shade Cost Ten Years Annual Cost

Advantages: Disadvantages:
Immediate Estimated Total Life Expectancy
Initial Total Five- Total Shade at Total Ten-
Strategy 2: Amount of Annual Shade at and Average
Investment Year Cost Five Years Year Cost
Additional Shade Cost Ten Years Annual Cost

Disadvantages:
Advantages:
Immediate Estimated Total Life Expectancy
Initial Total Five- Total Shade at Total Ten-
Strategy 3: Amount of Annual Shade at and Average
Investment Year Cost Five Years Year Cost
Additional Shade Cost Ten Years Annual Cost

Advantages: Disadvantages:
Immediate Estimated Total Life Expectancy
Initial Total Five- Total Shade at Total Ten-
Strategy 4: Amount of Annual Shade at and Average
Investment Year Cost Five Years Year Cost
Additional Shade Cost Ten Years Annual Cost

Advantages: Disadvantages:

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End Notes
1
MMWR 2002; 51(RR04):1-16. Available at http://www.cdc.gov/mmwr/review/mmwrhtm/ rr5104a1.htm
2
American Cancer Society. What Are The Key Statistics For Nonmelanoma Skin Cancer? Available at
http://www.cancer.org/docroot/CRI/content/CRI_2_4_1X_What_are_the_key_statistics_for_skin_cancer_51.
asp?sitearea=
3
American Cancer Society. What Are The Key Statistics For Melanoma Skin Cancer? Available at
http://www.cancer.org/docroot/CRI/content/CRI_2_4_1X_What_are_the_key_statistics_for_melanoma_50.
asp?sitearea=
4
American Academy of Dermatology. Skin Cancer. Available at http://www.aad.org/pamphlets/ skincan.html
5
Armstrong BK, Kricker A. How much melanoma is caused by sun exposure? Melanoma Res, 1993; 3: 395-401.
6
Diffey, BL. Solar ultarviolet radiation effects on biological systems. Physiology Medica; Biology 1991;
36(3):299-328.
7
United Nations Development Programme, Montreal protocol on substances that deplete the ozone layer. Available
at http://www.undp.org/seed/eap/montreal.htm.
8
Williams ML, Pennella R. Melanoma, melanocytic nevi, and other melanoma risk factors in children. J Pediatr
1994; 124:833-45
9
Moore LA. Ocular protection from solar ultraviolet radiation (UVR) in sport: factors to consider when
prescribing. The South African Optometrist 2003; 62(2):72-79.
10
Sliney, DH. Physical factors in cataractogenesis: ambient ultraviolet radiation and temperature. Invest
Ophthalmol Vis Sci 1986; 27(5):781-790.
11
Hall HI, Rogers JD. Sun protection behaviors among African Americans. Ethnicity and Disease 1999;9(1):126-
31.
12
Olson RL, Sayre RM, Everett M A. Effect of anatomic location and time on ultraviolet erythema. Arch Dermatol
1996; 93(2): 211-15.
13
Diffey BL. Ultraviolet radiation and human health. Clinics in Dermatology 1998;16:83-9.
14
Gartner LM, Greer FR. Prevention of rickets and vitamin D deficiency: new guidelines for vitamin D intake.
Pediatrics 2003;111(4 Pt 1): 908-10.
15
West SK, Duncan DD, Munoz B, Rubin GS, Fried LP, Bandeen-Roche K, et al. Sunlight exposure and risk of
lens opacities in a population-based study: the Salisbury Eye Evaluation project. JAMA 1998;280:714-8.
16
Rosmini F, Stazi MA, Milton RC, Sperduto RD. Pasquini P, Maraini G. A dose-response effect between a
sunlight index and age-related cataracts. Italian-American Cataract Study Group.[comment]. Annals of
Epidemiology. 4(4):266-70.
17
Westerdahl J, Olsson H, Ingvar C. At what age do sunburn episodes play a crucial role for the development of
malignant melanoma. Eur J Cancer 1994; 30A(11):1647-54.
18
Zanetti R, Franceschi S, Rosso S, Colonna S, Bidoli, E. Cutaneous melanoma and sunburns in childhood in a
southern European population. Eur J Cancer 1992;28A(6-7):1172-76.
19
Elwood JM, Whitehead SM, Davison J, Stewart M, Galt M. Malignant melanoma in England: risks associated
with naevi, freckles, social class, hair colour, and sunburn. Int J Epidemiol 1990;19(4):801-10.
20
Gandini S, Sera F, Cattaruzza MS, et al. Meta-analysis of risk factors for cutaneous melanoma: II. Sun exposure.
Eur J Cancer 2005;41:45-60.
In: Encyclopedia of Dermatology (6 Volume Set) ISBN: 978-1-63483-326-4
Editor: Meghan Pratt © 2016 Nova Science Publishers, Inc.

Chapter 75

SUN SAFETY FOR AMERICA’S

YOUTH TOOLKIT *

Centers for Disease Control and Prevention

1. ABOUT THE SUN SAFETY FOR AMERICA’S

YOUTH TOOLKIT
Since 1998, the National Comprehensive Cancer Control Program (NCCCP) has
provided funding and technical support to develop and implement comprehensive cancer
control (CCC) plans to “reduce cancer incidence, morbidity, and mortality through
prevention, early detection, treatment, rehabilitation and palliation.”[1,2] Currently the
Centers for Disease Control and Prevention (CDC) Division of Cancer Prevention and
Control (DCPC) “funds CCC programs in all 50 states, the District of Columbia, 7 tribes and
tribal organization and 7 U.S. territories” [3].
The CCC program in each state/tribe/territory/jurisdiction has a mandate to develop a
plan that addresses a wide variety of cancer prevention and control priorities. This often
includes skin cancer prevention. The Sun Safety for America’s Youth Toolkit is designed as a
resource for state/tribe/territory/ jurisdiction CCC programs interested in engaging schools
and other education partners in sun safety efforts to reduce their state/tribe/territory/
jurisdiction’s incidence of skin cancer. Since a majority of sun exposure occurs during
childhood and early adulthood[4] and key sun protective behaviors can most easily be
established at this time, addressing sun safety for young people is an important cancer control
objective.
There is a wide variety of sun safety programs, materials, and resources available to
organizations interested in implementing sun safety efforts, many of which are described in
this toolkit and the accompanying reference documents. This toolkit builds upon lessons
learned from CDC’s long history of sun safety and skin cancer prevention. In 2002, CDC
released the Guidelines for School Programs to Prevent Skin Cancer [5]. The Guidelines

*
This is an edited, reformatted and augmented version of a document issued by the Centers for Disease Control and
Prevention, July 2009.
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1704 Centers for Disease Control and Prevention

provide resources and suggestions for schools to improve sun safety practices in seven major
areas: policy, environmental change, education, family involvement, professional
development, health services, and evaluation. To foster implementation of these guidelines,
DCPC, in partnership with the Division of Adolescent and School Health (DASH), made
funds available to states with Coordinated School Health Programs (CSHPs) to conduct pilot
skin cancer prevention activities. In 2003, through the Skin Cancer Priority Supplement to
PA03004—Improving the Health, Education, and Well-Being of Young People through
Coordinated School Health Programs—three states (Colorado, Michigan, and North Carolina)
were awarded funds to pilot activities to address the school skin cancer guidelines. From late
2003 through early 2007, the Department of Education in each of the three funded states
received funds to implement their pilot sun safety initiatives. Each state was required to
develop an annual work plan that would guide their efforts to address CDC’s skin cancer
guidelines and develop a partnership with their state CCC program.
This toolkit draws from these efforts and is designed to provide CCC programs with
resources and information that will help them to understand the burden of skin cancer within
their state/tribe/territory/jurisdiction, assess current state/ tribe/territory/jurisdiction-level sun
safety interest and activity, engage in implementation of sun safety efforts with schools and
key education partners, and evaluate their efforts.
The toolkit consists of four key steps that will help CCC programs move through a
logical process for engaging and implementing sun safety efforts for young people:

 Step I: Identify and Recruit Sun Safety Partners

 Step II: Assess and Understand Sun Safety Needs and Resources in Your
State/Tribe/Territory/Jurisdiction
 Step III: Plan and Implement Sun Safety Activities
 Step IV: Evaluate Sun Safety Efforts

Step I describes the numerous organizations and individuals CCC programs may want to
engage in sun safety planning and implementation. These partners represent both
state/tribe/territory/jurisdiction and local organizations that can play an important role in
reaching schools and young people to implement and enhance effective sun safety strategies.
Step II provides recommendations for understanding the current state skin cancer burden
and how to utilize that information to inform the development and targeting of sun safety
efforts. To understand the current level of sun safety activity within the
state/tribe/territory/jurisdiction, recommendations and resources are provided for conducting
a sun safety program and resource inventory at the state/tribe/territory/jurisdiction level.
Resources are also provided to help your CCC program understand the current legal and/or
policy issues related to sun safety and how sun safety may already be integrated into existing
school resources and tools.
Step III outlines a process for conducting a Strengths, Weaknesses, Opportunities and
Threats (SWOT) analysis related to implementation of sun safety activities.
Recommendations and resources on selection of sun safety activities are provided as well as
examples of activities implemented using the CDC Guidelines for School Programs to
Prevent Skin Cancer.

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 Sun Safety for America’s Youth Toolkit 1705

Step IV highlights the importance of evaluation of state/tribe/territory/ jurisdiction sun

safety efforts and offers some examples of how to evaluate sun safety efforts locally and at
the state/tribe/territory/jurisdiction level. We also provide suggestions for modification of
state surveillance systems.
Within each step, we provide examples from state sun safety efforts to help CCC
programs understand how these recommendations, tools, and resources have been utilized by
other states.
The toolkit also includes an extensive sun safety resource list, which highlights potential
sun safety partners at the state/tribe/territory/jurisdiction and national levels, and other sun
safety programs and materials that are currently available.

2. WHY IS SUN SAFETY IMPORTANT FOR YOUNG PEOPLE?

Skin cancer is the most common type of cancer and is thought to account for half of all
cancers [6, 7]. About one million cases of basal cell or squamous cell cancers, the two most
common types of skin cancer, are diagnosed each year. Melanoma, the third most common
type of skin cancer [5], accounts for less than 5% of skin cancer cases but causes a majority of
skin cancer deaths [6, 7].
Although children are not commonly diagnosed with skin cancer, it is during childhood
that much of one’s lifetime sun exposure occurs and when important protective behaviors can
be established. Approximately 65%–90% of melanomas are caused by ultraviolet (UV )
radiation [5], and because a substantial percentage of lifetime sun exposure occurs before age
20 [4], UV radiation exposure during childhood and adolescence plays an important role in
the development of skin cancer [8]. Persons with a history of more than one blistering
sunburn during childhood or adolescence are at a greater risk for developing basal cell
carcinoma [9] and are two times more likely to develop melanoma than those without such
exposures [5].

Why Is It Important to Work with Schools?

Sun exposure preventive behaviors can yield the most positive effects if they are initiated
early and established as healthy and consistent patterns throughout life [5]. In 2003, the U.S.
Community Preventive Services on Reducing Exposure to Ultraviolet Light recommended
that primary schools implement educational and policy strategies to improve behaviors that
reduce exposure to UV light by covering exposed skin and therefore preventing skin cancer
[10]. Because much time during childhood and adolescence is spent at school, schools
provide a favorable environment in which to teach and model healthy behaviors.
There are approximately 50 million students attending more than 98,000 schools
nationwide [11]. Schools therefore provide the most far-reaching access to young people.
Schools can play a critical role in educating young people about their health and ensuring that
they have a healthy environment when they are at school and engaged in school activities.
However, it is important to acknowledge that schools are faced with the challenge of
maintaining high academic standards and addressing a wide variety of health priorities with
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1706 Centers for Disease Control and Prevention

limited time and resources. Therefore, identifying opportunities to integrate sun safety into
existing resources, activities, and curricula is critical to successful implementation and
sustainability and to reducing burden on school partners.
In addition to schools, there are numerous other community organizations that provide
services to young people and thus have an opportunity to educate them about the importance
of sun safety. These organizations include parks and recreation departments, summer camps,
museums, health education centers, and others. Because of the important role these
organizations and groups serve, they have been included as potential partners for any
state/tribe/ territory/jurisdiction sun safety effort.

3. STEP I: IDENTIFY AND RECRUIT SUN SAFETY PARTNERS

When preparing to engage in a sun safety effort, CCC programs can complement their
own cancer control expertise by identifying and engaging additional partners who are
knowledgeable and skilled in working with schools and young people. Such partnerships
represent an opportunity for experts in cancer control and education to bring their respective
areas of expertise together to identify strategies that can be implemented in a manner that
reduces the burden on the educational system. These partners can facilitate sun safety
activities in a number of ways, including

 building upon existing relationships to reduce potential barriers and reluctance by

schools administrators and teachers to add sun safety to an already full agenda,
 serving as critical links to educate and improve school awareness and knowledge of
the importance of sun safety,
 identifying opportunities to integrate sun safety into existing school programs and
curriculum, and
 serving as sun safety advocates within their own organizations.

Develop Partnerships to Facilitate Sun Safety Planning and Implementation

In 2008, DCPC developed a Partnership Tool Kit: Program Version[12] that helps
programs navigate the process of

 determining the need for a partnership,

 developing a partnership,
 evaluating a partnership, and
 sustaining a partnership.

This tool is recommended to CCC programs to help engage partners in skin cancer
prevention efforts. Key aspects of this tool are included in this toolkit, but programs are
encouraged to refer to the Partnership Tool Kit: Program Version for additional details and
guidance.

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 Sun Safety for America’s Youth Toolkit 1707

When determining the need for a partnership, programs should consider the following
questions:

 What specific outcomes or products does your program hope to achieve through
partnerships?
 In what ways do you need partnerships to achieve the identified out- comes or
products?
 How would partnerships provide additional expertise rather than duplicating
expertise in your program?

Programs can then move toward developing a new partnership. Potential questions to
consider at this stage include the following:

 What potential partners has your program identified?

 In what ways could nontraditional partners be helpful in a new partnership?
 What does your program aim to achieve by working with these potential partners?
 How will these potential partners complement and strengthen your program?
 What might be some potential drawbacks to working with these potential partners?
 How would these potential partners help your program better achieve your goals and
objectives?
 What might your program and the potential partners gain through this partnership?
 What resources does your program have available to contribute to new partnerships?
 Is there an existing state-level school health coordinating committee?

Identify Other State/Tribe/Territory/Jurisdiction Partners to Engage in Sun

Safety Planning

After assessing the need for additional partners and partnerships to promote a
state/tribe/territory/ jurisdiction sun safety effort, CCC programs can identify a number of
other state government agencies and programs that may serve as critical stakeholders and
resources. The list below includes some examples of possible state-level partners that could
be engaged in sun safety efforts. This list is not exhaustive, so each CCC program should
identify other resources and partners specific to their state/tribe/territory/jurisdiction.

State Department of Education: The State Department of Education is an important

stakeholder when addressing any issue that impacts young people. Twenty-two states and one
tribal government are funded to build the capacity of their school systems through CDC’s
Coordinated School Health Program (CSHP) [13]. The CSHP model consists of eight key
components for a comprehensive health program: health education, physical education, health
services, nutrition services, counseling and psychological services, healthy school
environment, health promotion for staff, and family/community involvement. State health and
education agencies are encouraged to develop an ongoing partnership to facilitate the
implementation of effective policies and practices to promote the health and well-being of
students and staff.
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1708 Centers for Disease Control and Prevention

With or without funding for a CSHP, the State Department of Education represents a key
partner in sun safety for young people and can provide valuable linkages, insight, and
resources for engaging schools in sun safety. While health education classes are perhaps the
most traditional mechanism for providing education on a particular topic, they are not the
only avenues to be explored. Department experts or leaders in physical education, physical
activity/wellness, school health services, and after-school activities are natural partners as
they can incorporate sun safety into physical education classes and school sports activities.
Sun safety can also be incorporated into a number of other academic areas, such as science
and math (see the Guidelines for School Programs to Prevent Skin Cancer for additional
information on incorporation of sun safety into various curricula); thus, it is important to try
to identify partners beyond just the health education curriculum.
State Agency that Regulates Commercial Tanning Facilities: Every state has a state
agency that is responsible for oversight, certification, and regulations related to indoor
tanning/commercial tanning facilities (i.e., tanning beds). While not directly related to the
schools, these organizations provide oversight to ensure that tanning facility staff are trained
and that any state laws to protect minors from tanning are adhered to. Representatives from
these agencies can be valuable partners for enforcement of existing state tanning laws or for
development of such legislation if it does not already exist. A list of all state agencies
responsible for commercial tanning facilities oversight can be found at
http://www.tanningtraining.com/reginfo/state.html.

Note: State Education Agencies (SEA) that receive DASH funding are shaded. The tribal government
from Nez Perce also receives CSHP funding. Source: CDC Coordinated School Health Program
http://www.cdc.gov/healthyyouth/partners/ funded/cshp.htm.

Figure 1. Coordinated School Health Programs (CSHP).

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 Sun Safety for America’s Youth Toolkit 1709

State Medical Associations: Clinical partners can play an important role in advocating
for sun safe behaviors with adults and young people.*

 The National Association of School Nurses has affiliate school nurse organizations in
49 states (there is no affiliate in Hawaii) and the District of Columbia. State affiliate
information can be found at http://www.nasn.org/Default.aspx?tabid=60.* School
nurses play an important role in training teachers and students about practicing sun
safe behavior and, in some cases, may have the responsibility of administering
sunscreen to students.
 The American Academy of Dermatology (AAD) has affiliate State Dermatology
Society organizations that can provide resources and support to sun safety efforts.
AAD offers the Seal of Recognition Program and the Skin Cancer Awareness:
Intervention Plan for Tomorrow (SCRIPT ) Plan. The SCRIPT Plan includes a shade
structure grant program, free skin cancer screenings, advocacy for federal and state
legislation to regulate indoor tanning, paid and public service advertising, and sun
safety resource materials. More information on AAD and its resources can be found
at http://www.aad.org/index.html.*
 The American Academy of Pediatrics (AAP) has state chapters across the United
States. AAP endorses a policy statement titled Ultraviolet Light: A Hazard to
Children. Pediatricians can provide valuable information to children and parents on
sun safety and can be a valuable advocate for passing state laws addressing skin
cancer and skin cancer prevention. A list of state chapters can be found at
http://www.aap.org/member/ chapters/chapters.htm.*

State Athletic and Coaching Associations: Young people are exposed to the sun during
a variety of outdoor activities, including participation in school or community athletic
activities. Including partners who represent state athletic and coaching associations provides a
valuable resource for educating adults who oversee thousands of children during outdoor
sports and athletic events about the importance of sun safety while on the field.

 The National Federation of State High School Associations includes member

associations in each of the 50 states and the District of Columbia. A list of member
associations and their contact information can be found at
http://www.nfhs.org/stateassociations.aspx.*

Colleges and Universities: State colleges and universities often have a variety of
experts, resources, and programs addressing sun safety, including

*
Links to non-Federal organizations found in this document are provided solely as a service to our users. These
links do not constitute an endorsement of these organizations or their programs by CDC or the Federal
government, and none should be inferred. CDC is not responsible for the content of the individual organization
web pages found at these links.
*
Links to non-Federal organizations found in this document are provided solely as a service to our users. These
links do not constitute an endorsement of these organizations or their programs by CDC or the Federal
government, and none should be inferred. CDC is not responsible for the content of the individual organization
web pages found at these links.
 Free ebooks ==> www.Ebook777.com
1710 Centers for Disease Control and Prevention

 Cooperative Extension Services,

 schools of medicine,
 schools of nursing, and
 schools of public health.

American School Health Association (ASHA): ASHA has state constituent chapters in
12 states and has passed a resolution supporting sun safety school policies and education to
prevent skin cancer. A list of member associations can be found at
http://www.ashaweb.org/i4a/pages/ index.cfm?pageid=3317.*
Parent Teacher Association (PTA): The national PTA places an emphasis on healthy
youth, and state PTA chapters can be valuable partners in implementing sun safety activities.
State PTA chapters have access to local PTA chapters whose members can be educated to
become suns safety advocates within their own school chapters. State PTAs can also pass
resolutions and position statements in support of sun safety. State and local PTA chapters can
be found at http://pta .org/jp find your pta .html.

Identify Local Partners to Engage in Sun Safety Planning

Depending on the sun safety approach and activities to be implemented, many CCC
programs may also choose to engage local partners in their sun safety planning and activities.
In many cases, local partners may be members or affiliates of state-level organizations that
have already become engaged as sun safety partners. Local PTA chapters and individual
health care providers are two examples of local organizations/members that may be closely
affiliated with state-level organizations. There are a number of other local partners that can be
engaged in sun safety planning and implementation.
Schools: Public, private, and alternative schools are perhaps the most obvious partners to
engage in sun safety efforts, but also the most challenging. Learning how schools work is an
important first step to working effectively with schools [14]. Because many policies
impacting schools are implemented at the school level, it is often critical to work directly with
schools and school administrators to integrate sun safety into school policies, curricula, and
activities. Schools are faced with the task of addressing numerous student health issues while
meeting or maintaining high academic standards. Gaining access to schools is often difficult
and is most easily done by individuals and organizations that have preexisting relationships
with schools and school administrators. While many schools are familiar with student health
issues such as tobacco and drug use, obesity, physical activity, nutrition, and asthma, most
schools are less familiar with the issue of sun safety. Engaging schools often requires time
and resources to educate school decision makers on the importance of sun safety for their
students and the approaches that a school can implement to protect students from the sun
while in their care. Many schools have school health teams, and districts have school health
councils that meet regularly to discuss school health problems and plans for improvement.
While the resources may not always be available, it is beneficial when even relatively
small amounts of funding can be provided to schools interested in making improvements in
sun safety policies, erecting or planting shade structures, or conducting education and
awareness events and activities.

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 Sun Safety for America’s Youth Toolkit 1711

Local Education Agencies (LEAs): LEAs reside between individual schools and the
Department of Education at the state level. LEAs are often considered school districts and
provide administrative oversight to public schools in a particular geographic area. Like
individual schools, LEAs can serve to provide leadership and guidance on school sun safety
policies, shade planning, and curricula.
County or Area Parks and Recreation Organizations: While not directly related to
schools, area parks and recreation organizations serve youth across the country, providing
recreational sports activities, parks, and summer camps. Parks and recreation staff and the
young people they oversee can benefit from educational and policy sun safety efforts. The
National Recreation and Park Association was a sponsor of the Pool Cool sun safety effort
aimed at improving sun safety behaviors of pool users. More information on Pool Cool can be
found at http://www .poolcool .org/*
Health Education Centers: The National Association of Health Education Centers seeks
to support and promote organizations that provide health education programs. Thirty-eight
member organizations can be found in 22 states and the District of Columbia. A list of health
education centers can be found at http://www .nahec .org/documents/2007AnnualReport .pdf
.*

LESSONS FROM THE FIELD

 Recognize that cancer control and education organizations have different
structures, resources, and sometimes languages. It is important to take the
time to learn what they are and how they can be used most effectively to
promote sun safety efforts.
 Nurture the partnership through communication, development of goals,
and setting timelines.
 Include partners who are familiar with education agencies at the local level
so they can share what might and might not work with local sun safety
efforts. These relationships can also help engage key school personnel
who may serve as advocates or potential gatekeepers.
 Work with partners to create a plan of action, rather than approaching
partners once a plan has been developed.
 Many potential partners and stakeholders are not knowledgeable about the
issue of sun safety. There needs to be an effort to educate and improve
awareness to recruit partners before many will be willing to engage in
activities.
 Plan for sustainability from the beginning. Look closely at what kinds of
activities and partnerships can help ensure financial and programmatic
longevity.

*
Links to non-Federal organizations found in this document are provided solely as a service to our users. These
links do not constitute an endorsement of these organizations or their programs by CDC or the Federal
government, and none should be inferred. CDC is not responsible for the content of the individual organization
web pages found at these links.
 Free ebooks ==> www.Ebook777.com
1712 Centers for Disease Control and Prevention

Parent Teacher Organizations (PTOs): Many schools have parent groups that are not
affiliated with the national PTA; these groups are often called PTOs. There is no national or
state PTO, but each PTO is independently organized and operated.
Other Community-based Organizations: Many other types of organizations that serve
young people can be included in sun safety planning and program implementation, including
the following:

 YMCA: http://www .ymca .net/*

 YWCA: http://www .ywca .org/
 Boys/Girls Clubs: http://www .bgca .org/*
 Boy Scouts: http://www.scouting .org/*
 Girl Scouts: http://www.girlscouts .org/*
 Museums, especially those with outdoor activities: http://www.muse-
umca.org/usa/states.html
 Zoos, aquariums, and amusement and water parks:
http://www.themeparkcity.com/USA_index.htm*
 Minor league ball parks: http://www.littleballparks .com/*
 Public or community pools

4. STEP II. UNDERSTAND SUN SAFETY NEEDS AND RESOURCES IN

YOUR STATE/TRIBE/ TERRITORY/JURISDICTION
Once major partners are identified, CCC programs should undergo a process for
understanding the sun safety needs, skin cancer burden, and existing sun safety resources
within their state/tribe/territory/jurisdiction. Every state/tribe/territory/jurisdiction will have
diffe