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Casarett & Doull's
Essentials of Toxicology
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Casarett & Doull’s
Essentials of Toxicology
Third Edition

Editors

Curtis D. Klaassen, PhD

University Distinguished Professor
Division of Gastroenterology
Department of Internal Medicine
University of Kansas Medical Center
Kansas City, Kansas

John B. Watkins III, PhD

Associate Dean and Director
Professor ofPharmacology and Toxicology
Medical Sciences Program
Indiana University School of Medicine
Bloomington, Indiana

beeMedical
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Milan NewDelhi SanJuan Seoul Singapore Sydney ‘Toronto
Casarett & Doull’s Essentials of Toxicology, 3ed.

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Casarett and Doull’s essentials of toxicology
Essentials of toxicology
Includes index.
ISBN 978-0-07-184708-7 (alk. paper) — ISBN 0-07-184708-1 (alk. paper)
I. Klaassen, Curtis D., editor. II. Watkins, John B. (John Barr), II, editor. III. Title: Casarett and Doull’s essentials of
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Contents
Contributors vii
Preface xiii

Cntr | unit 4
GENERAL PRINCIPLES OF TARGET ORGAN TOXICITY 163
TOXICOLOGY 1
11. Toxic Responses of the Blood
1. History and Scope of Toxicology John C. Bloom, Andrew E. Schade,
Michael A. Gallo 1 and John T. Brandt 163

2. Principles of Toxicology 12. Toxic Responses of the Immune System

David L. Eaton and Steven G. Gilbert 5 Barbara L.F. Kaplan, Courtney E. W. Sulentic,
Michael P. Holsapple, and Norbert E.
3. Mechanisms of Toxicity Kaminski 177
Zoltan Gregus 21
13. Toxic Responses of the Liver
4. Risk Assessment Hartmut Jaeschke 195
Elaine M. Faustman and Gilbert S.Omenn 49
14. Toxic Responses of the Kidney
Rick G. Schnellmann 209
Wh AN! ICae 2
15. Toxic Responses of the Respiratory System
DISPOSITION OF TOXICANTS 61 George D. Leikauf 223

5. Absorption, Distribution, 16. Toxic Responses of the Nervous System

and Excretion of Toxicants Virginia C. Moser, Michael Aschner,
Lois D. Lehman-McKeeman 61 Rudy J. Richardson, and Martin A. Philbert 237

6. Biotransformation of Xenobiotics 17. Toxic Responses of the Ocular

Andrew Parkinson, Brian W. Ogilvie, David B. and Visual System
Buckley, Faraz Kazmi, Maciej Czerwinski, Donald A. Fox and William K. Boyes 255
and Oliver Parkinson 79 18. Toxic Responses of the Heart
7. Toxicokinetics and Vascular System
Y. James Kang 271
DannyD. Shen 109
19. Toxic Responses of the Skin
Robert H. Rice and Theodora M. Mauro 291
UsNe Ir 3
20. Toxic Responses of the Reproductive System
NONORGAN-DIRECTED TOXICITY 121 Paul M.D. Foster and L. Earl Gray Jr. 303
8. Chemical Carcinogenesis 21. Toxic Responses of the Endocrine System
JamesE. Klaunig 121 Patricia B. Hoyer and Jodi A. Flaws 319
9. Genetic Toxicology
R. Julian Preston and George R. Hoffmann +135
10. Developmental Toxicology
John M. Rogers 149
Vi CONTENTS

unit 5 UN TE 7

TOXIC AGENTS 333 APPLICATIONS OF TOXICOLOGY 441

22. Toxic Effects of Pesticides 30. Ecotoxicology

Lucio G. Costa 333 Richard T. Di Giulio and MichaelC. Newman 441

23. Toxic Effects of Metals aL, Food Toxicology

Erik J. Tokar, Windy A. Boyd, Jonathan H. Frank N. Kotsonis and George A. Burdock 453
Freedman, and Michael P Waalkes 347
32. Analytical and Forensic Toxicology
24. Toxic Effects of Solvents and Vapors Bruce A. Goldberger and Diana G. Wilkins 463
James V. Bruckner, S. Satheesh Anand,
33. Clinical Toxicology
and D. Alan Warren 361
Louis R. Cantilena Jr. 471
25. Toxic Effects of Radiation and Radioactive
34. Occupational Toxicology
Materials
Peter S. Thorne 481
David G. Hoel 373

26. Toxic Effects of Plants and Animals

John B. Watkins, II 381 Answers to Chapter Questions 491
Index 495
27. Toxic Effects of Calories
Martin J. Ronis, Kartik Shankar,
and Thomas M. Badger 401

unit 6
ENVIRONMENTAL TOXICOLOGY 411
28. Nanotoxicology
Gunter Oberdorster, Agnes B. Kane,
Rebecca D. Kapler, and Robert H. Hurt 411
29. Air Pollution
Daniel L. Costa and Terry Gordon 425
Contributors

S. Satheesh Anand, PhD, DABT William K. Boyes, PhD

Senior Research Toxicologist Neurotoxicology Branch
Haskell Global Centers for Health and Environmental Toxicity Assessment Division
Sciences National Health and Environmental Effects Research
Newark, Delaware Laboratory
Chapter 24 Office of Research and Development
US Environmental Protection Agency
Michael Aschner, PhD
Research Triangle Park, North Carolina
Professor
Chapter 17
Department of Pediatrics
Vanderbilt University Medical Center John T. Brandt, MD
Nashville, Tennessee Eli Lilly & Co. (retired)
Chapter 16 Indianapolis, Indiana
Chapter 11
Thomas M. Badger, PhD
Distinguished Faculty Scholar James V. Bruckner, PhD
Professor Professor of Pharmacology & Toxicology
Departments of Pediatrics and Physiology/Biophysics Department of Pharmaceutical & Biomedical
University of Arkansas for Medical Sciences Sciences
Director College of Pharmacy
Arkansas Children’s Nutrition Center University of Georgia
Little Rock, Arkansas Athens, Georgia
Chapter 27 Chapter 24

John C. Bloom, VMD, PhD David B. Buckley, PhD

President Chief Scientific Officer
Bloom Consulting Services, LLC XenoTech, LLC
Special Government Employee Lenexa, Kansas
FDA Chapter 6
Adjunct Professor of Pathology
George A. Burdock, PhD, DABT, FACN
Schools of Veterinary Medicine
President
University of Pennsylvania and Purdue University
Burdock Group Consultants
Indianapolis, Indiana
Orlando, Florida
Chapter 11
Chapter 31
Windy A. Boyd, PhD
Louis R. Cantilena Jr., MD, PhD
Biologist
Professor, Medicine and Pharmacology
Biomolecular Screening Branch
Department of Medicine
National Toxicology Program Division
Uniformed Services University
National Institute of Environmental Health Sciences, NIH
Bethesda, Maryland
Research Triangle Park, North Carolina
Chapter 33
Chapter 23

Vii
Vili CONTRIBUTORS

Daniel L. Costa, PhD Paul M.D. Foster, PhD

Office of Research and Development Chief
National Program Director for Air, Climate, and Energy Toxicology Branch
Research Program Division of the National Toxicology Program
US Environmental Protection Agency National Institute of Environmental Health Sciences
Research Triangle Park, North Carolina Research Triangle Park, North Carolina
Chapter 29 Chapter 20

Lucio G. Costa, PhD Donald A. Fox, PhD

Professor Professor of Vision Sciences
Department of Environmental and Occupational Biology and Biochemistry, Pharmacology, and Health and
Health Sciences Human Performance
School of Public Health University of Houston
University of Washington Houston, Texas
Seattle, Washington Chapter 17
Chapter 22
Jonathan H. Freedman, PhD
Maciej Czerwinski, PhD Laboratory of Toxicology and Pharmacology
Principal Scientist National Institute of Environmental Health Sciences
XenoTech, LLC Research Triangle Park, North Carolina
Lenexa, Kansas Chapter 23 .
Chapter 6
Michael A. Gallo, PhD
Richard T. Di Giulio, PhD Environmental and Occupational Health Sciences
Professor Institute
Nicholas School of the Environmental Rutgers-The State University of New Jersey
Duke University UMDNJ-Robert Wood Johnson Medical School
Durham, North Carolina Piscataway, New Jersey
Chapter 30 Chapter |

David L. Eaton, PhD Steven G. Gilbert, PhD

Professor Director
Department of Environmental and Occupational Institute of Neurotoxicology & Neurological Disorders
Health Sciences Seattle, Washington
Associate Vice Provost for Research Chapter 2
University of Washington
Bruce A. Goldberger, PhD
Seattle, Washington
Professor and Director of Toxicology
Chapter 2
Departments of Pathology and Psychiatry
Elaine M. Faustman, PhD University of Florida College of Medicine
Professor Gainesville, Florida
Institute for Risk Analysis and Risk Communication Chapter 32
Department of Environmental and Occupational
Terry Gordon, PhD
Health Sciences
Professor
School of Public Health
University of Washington Department of Environmental Medicine
NYU School of Medicine
Seattle, Washington
Tuxedo, New York
Chapter 4
Chapter 29
Jodi A. Flaws, PhD
L. Earl Gray Jr., PhD
Professor
Department of Comparative Biosciences Reproductive Toxicology Branch
University of Illinois United States Environmental Protection Agency
Urbana, Illinois Adjunct Professor
Chapter 21 North Carolina State University
Raleigh, North Carolina
Chapter 20
 CONTRIBUTORS

Zoltan Gregus, MD, PhD, DSc, DABT Norbert E. Kaminski, PhD

Professor Professor
Department of Pharmacology and Therapeutics Department of Pharmacotherapy and Toxicology
Toxicology Section Director
University of Pecs Center for Integrative Toxicology
Medical School Michigan State University
Pecs, Hungary East Lansing, Michigan
Chapter 3 Chapter 12
David G. Hoel, PhD Agnes B. Kane, MD, PhD
Principal Scientist Professor
Exponent, Inc Department of Pathology and Laboratory Medicine
Alexandria, Virginia Brown University
Distinguished University Professor Providence, Rhode Island
Department of Medicine Chapter 28
Medical University of South Carolina
Y. James Kang, DVM, PhD, FATS
Charleston, South Carolina
Chapter 25
Professor and Distinguished University Scholar
Department of Pharmacology and Toxicology
George R. Hoffmann, PhD University of Louisville School of Medicine
Professor Louisville, Kentucky
Department of Biology Chapter 18
College of the Holy Cross
Barbara L.F. Kaplan, PhD
Worcester, Massachusetts
Assistant Professor
Chapter 9
Center for Integrative Toxicology
Michael P. Holsapple, PhD, ATS Department of Pharmacology and Toxicology and
Senior Research Leader Neuroscience
Systems Toxicology Program
Health and Life Sciences Global Business Michigan State University
Battelle Memorial Institute East Lansing, Michigan
Columbus, Ohio Chapter 12
Chapter 12
Faraz Kazmi, BS
Patricia B. Hoyer, PhD Senior Scientist
Professor XenoTech, LLC
Department of Physiology Lenexa, Kansas
College of Medicine Chapter 6
The University of Arizona
Rebecca D. Kapler, PhD
Tucson, Arizona
School of Freshwater Sciences
Chapter 21
University of Wisconsin- Milwaukee
Robert H. Hurt, PhD Milwaukee, Wisconsin
Professor Chapter 28
School of Engineering
James E. Klaunig, PhD, ATS, [ATP
Director
Professor
Institute for Molecular and Nanoscale Innovation
Environmental Health
Brown University
Indiana University
Providence, Rhode Island
Bloomington, Indiana
Chapter 28
Chapter 8
Hartmut Jaeschke, PhD, ATS
Frank N. Kotsonis, PhD
Professor and Chair
Retired Corporate Vice President
Department of Pharmacology, Toxicology & Therapeutics
Worldwide Regulatory Sciences
University of Kansas Medical Center
Monsanto Corporation
Kansas City, Kansas
Skokie, Illinois
Chapter 13
Chapter 31
x CONTRIBUTORS

Lois D. Lehman-McKeeman, PhD Gilbert S. Omenn, MD, PhD

Distinguished Research Fellow Professor of Internal Medicine, Human Genetics
Discovery Toxicology and Public Health
Bristol-Myers Squibb Company Director
Princeton, New Jersey Center for Computational Medicine and Bioinformatics
Chapter 5 University of Michigan Department of Computational
Medicine and Bioinformatics
George D. Leikauf, PhD
Ann Arbor, Michigan
Professor
Chapter 4
Department of Environmental and Occupational Health
Graduate School of Public Health Oliver Parkinson, PhD
University of Pittsburgh XPD Consulting, LLC
Pittsburgh, Pennsylvania Shawnee, Kansas
Chapter 15 Chapter 6

Theodora M. Mauro, MD Andrew Parkinson, PhD

Professor and Vice-Chair CEO
Dermatology Department XPD Consulting, LLC
University of California, San Francisco Shawnee, Kansas
Service Chief Chapter 6
Dermatology
Martin A. Philbert, PhD
San Francisco Veterans Medical Center
San Francisco, California
Professor of Toxicology and Dean ,
Chapter 19 School of Public Health
University of Michigan
Virginia C. Moser, PhD, DABT, FATS Ann Arbor, Michigan
Toxicologist Chapter 16
Toxicity Assessment Division
R. Julian Preston, MA, PhD
National Health and Environmental Effects Research
Laboratory Associate Director for Health
US Environmental Protection Agency National Health and Environmental Effects Research
Research Triangle Park, North Carolina Laboratory
Chapter 16 US Environmental Protection Agency
Research Triangle Park, North Carolina
Michael C. Newman, MS, PhD Chapter 9
A. Marshall Acuff Jr. Professor
Robert H. Rice, PhD
Virginia Institute of Marine Science
College of William & Mary Professor
Gloucester Point, Virginia Department of Environmental Toxicology
Chapter 30 University of California
Davis, California
Gunter Oberdoérster, DVM, PhD Chapter 19
Professor
Department of Environmental Medicine Rudy J. Richardson, ScD, DABT
University of Rochester Toxicology Program
School of Medicine & Dentistry University of Michigan School of Public Health
Rochester, New York Neurology Department
Chapter 28 University of Michigan School of Medicine
Ann Arbor, Michigan
Brian W. Ogilvie, BA Chapter 16
Principal Scientist
XenoTech, LLC
Lenexa, Kansas
Chapter 6
 CONTRIBUTORS

John M. Rogers, PhD Peter S. Thorne, MS, PhD

Toxicity Assessment Division Professor and Head
National Health and Environmental Effects Research Department of Occupational and Environmental Health
Laboratory College of Public Health
Office of Research and Development The University of Iowa
United States Environmental Protection Agency Iowa City, lowa
Research Triangle Park, North Carolina Chapter 34
Chapter 10
Erik J. Tokar, PhD
Martin J. Ronis, BA, MA, PhD Biologist
Professor Inorganic Toxicology Group
Department of Pharmacology & Toxicology Division of the National Toxicology Program
College of Medicine National Toxicology Program
University of Arkansas for Medical Sciences National Institute of Environmental Health Sciences
Associate Director for Basic Research Research Triangle Park, North Carolina
Arkansas Children’s Nutrition Center Chapter 23
Arkansas Children’s Hospital Research Institute
Michael P. Waalkes, PhD
Little Rock, Arkansas
Chapter 27 Chief
National Toxicology Group
Andrew E. Schade, MD, PhD Division of the National Toxicology Program
Senior Director National Toxicology Program
Clinical Diagnostics Laboratory National Institute of Environmental Health Sciences
Diagnostics Research and Development Research Triangle Park, North Carolina
Eli Lilly and Co. Chapter 23
Indianapolis, Indiana
D. Alan Warren, MPh, PhD
Chapter 11
Program Director
Rick G. Schnellmann, PhD Environmental Health Science
Professor and Chair University of South Carolina Beaufort
Department of Pharmaceutical and Biomedical Sciences Beaufort, South Carolina
Medical University of South Carolina Chapter 24
Charleston, South Carolina
John B. Watkins, ITI, PhD
Chapter 14
Associate Dean and Director
Kartik Shankar, PhD, DABT Professor of Pharmacology and Toxicology
Arkansas Children’s Nutrition Center Medical Sciences Program
Department of Pediatrics Indiana University School of Medicine
University of Arkansas for Medical Sciences Bloomington, Indiana
Little Rock, Arkansas Chapter 26
Chapter 27
Diana G. Wilkins, MS, PhD
Danny D. Shen, PhD Director
Professor Center for Human Toxicology
Departments of Pharmaceuticals and Pharmacy Research Associate Professor
School of Pharmacy Department of Pharmacology and Toxicology
University of Washington University of Utah
Seattle, Washington Salt Lake City, Utah
Chapter 7 Chapter 32

Courtney E.W. Sulentic, PhD

Associate Professor
Department of Pharmacology & Toxicology
Boonshoft School of Medicine
Wright State University
Dayton, Ohio
Chapter 12
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Preface

This updated full-color edition of Essentials of Toxicology dis- (6) Environmental Toxicology; and (7) Applications of Toxi-
tills the major principles and concepts of toxicology that were cology. A summary of key points is included at the beginning
described in detail in the eighth edition of Casarett & Doull’s of each chapter, and a set of review questions is provided at the
Toxicology: The Basic Science of Poisons. We are grateful to the end of each chapter. We invite readers to send us suggestions
authors who contributed to the eighth edition of Casarett & of ways to improve this text and we appreciate the thoughtful
Doull’s Toxicology: The Basic Science of Poisons; their chapters recommendations that we received on the last edition.
in the parent text provided the foundation for the chapters in We would like to acknowledge all individuals who were
this edition of Essentials of Toxicology. involved in this project. We particularly give a heartfelt and sin-
Essentials of Toxicology concisely describes the expansive cere thanks to our families for their love, patience, and support
science of toxicology, and includes important concepts from during the preparation of this book. We especially appreciate
anatomy, physiology, and biochemistry to facilitate the under- Richard J. Batka and Alyssa Shapiro who provided invaluable
standing of the principles and mechanisms of toxicant action assistance on this project. The capable advice, guidance, and
on specific organ systems. We trust that this book will assist assistance of the McGraw-Hill staff is gratefully acknowledged.
students in undergraduate and graduate courses in toxicology, Finally, we thank our students for their enthusiasm for learning
as well as students from other disciplines, to develop a strong and what they have taught us during their time with us.
foundation in the concepts and principles of toxicology.
The book is organized into seven units: (1) General Princi-
ples of Toxicology; (2) Disposition of Toxicants; (3) Nonorgan- Curtis D. Klaassen
directed Toxicity; (4) Target Organ Toxicity; (5) Toxic Agents; John B. Watkins III

xiii
 UNIT 1 GENERAL PRINCIPLES OF TOXICOLOGY

Cn A. Pe Bk

History and Scope

of Toxicology
Michael A. Gallo

HISTORY OF TOXICOLOGY 20™ CENTURY TOXICOLOGY: THE AWAKENING OF

Antiquity UNDERSTANDING
Middle Ages
AFTER WORLD WAR II
Renaissance
Age of Enlightenment 215’ CENTURY TOXICOLOGY

KEY POINTS

« Toxicology is the study of the adverse effects of xenobiot- « Toxicology applies safety evaluation and risk assessment
ics on living systems. to the discipline.
« Toxicology assimilates knowledge and techniques from
biochemistry, biology, chemistry, genetics, mathematics,
medicine, pharmacology, physiology, and physics.

HISTORY OF TOXICOLOGY Antiquity

Modern toxicology goes beyond the study ofthe adverse effects Knowledge of animal venoms and plant extracts for hunting,
of exogenous agents by assimilating knowledge and techniques warfare, and assassination presumably predate recorded his-
from most branches of biochemistry, biology, chemistry, genet- tory. One of the oldest known writings, the Ebers Papyrus (circa
ics, mathematics, medicine, pharmacology, physiology, and 1500 B.c.), contains information pertaining to many recognized
physics and applies safety evaluation and risk assessment to the poisons, including hemlock, aconite, opium, and metals such as
discipline. In all branches of toxicology, scientists explore the lead, copper, and antimony. The Book of Job (circa 1400 B.c.)
mechanisms by which chemicals produce adverse effects in bio- speaks of poison arrows (Job 6:4) and Hippocrates (circa
logical systems. Activities in these broad subjects complement 400 B.c.) added a number of poisons and clinical toxicology
toxicologic research. principles pertaining to bioavailability in therapy and
="
f UNIT 1 General Principles of Toxicology

overdosage. Theophrastus (370-286 B.c.), a student ofAristotle, Come bitter pilot, now at once run on
included numerous references to poisonous plants in De The dashing rocks thy seasick weary bark!
Historia Plantarum. Dioscorides, a Greek physician in the court Here’s to my love! O true apothecary!
Thy drugs are quick. Thus with a kiss I die.
of the Roman emperor Nero, made the first attempt at classify-
Romeo and Juliet, act 5, scene 3
ing poisonsas plant, animal, and mineral in his book De Materia
Medica, which contains reference to some 600 plants. Although Ellenbog (circa 1480) warned of the toxicity of
One legend tells of Roman King Mithridates VI of Pontus, mercury and lead from goldsmithing and Agricola published a
who was so fearful of poisons that he regularly ingested a mix- short treatise on mining diseases in 1556, the major work on
ture of 36 ingredients as protection against assassination. On the subject, On the Miners’ Sickness and Other Diseases of
the occasion of his imminent capture by enemies, his attempts Miners (1567), was published by Paracelsus. This treatise
to kill himself with poison failed because of his successful anti- addressed the etiology of miners’ disease, along with treatment
dote concoction. This tale leads to use of the word mithridatic and prevention strategies. Occupational toxicology was fur-
as an antidote or protective mixture. Because poisonings in ther advanced by the work of Bernardino Ramazzini when he
politics became so extensive, Sulla issued the Lex Cornelia published in 1700 his Discourse on the Diseases of Workers,
(circa 82 B.c.), which appears to be the first law against poison- which discussed occupations ranging from miners to mid-
ing and later became a regulatory statute directed at careless wives and including printers, weavers, and potters. Percival
dispensers of drugs. Pott’s (1775) recognition of the role of soot in scrotal cancer
among chimney sweeps was the first report of polyaromatic
hydrocarbon carcinogenicity. These findings led to improved
Middle Ages medical practices, particularly in prevention.
The writings of Maimonides (Moses ben Maimon, a.p. 1135-
1204) included a treatise on the treatment of poisonings from
insects, snakes, and mad dogs (Treatise on Poisons and Their
Age of Enlightenment
Antidotes, 1198). Maimonides described the subject of bioavail- Experimental toxicology accompanied the growth of organic
ability, noting that milk, butter, and cream could delay intestinal chemistry and developed rapidly during the nineteenth cen-
absorption. In the early Renaissance and under the guise of tury. Magendie (1783-1885), Orfila (1787-1853), and Bernard
delivering provender to the sick and the poor, Catherine de (1813-1878) laid the groundwork for pharmacology, experi-
Medici tested toxic concoctions, carefully noting the rapidity of mental therapeutics, and occupational toxicology.
the toxic response (onset of action), the effectiveness of the com- Orfila, a Spanish physician in the French court, used autopsy
pound (potency), the degree of response of the parts of the body material and chemical analysis systematically as legal proof of
(specificity and site of action), and the complaints of the victim poisoning. His introduction of this detailed type of analysis
(clinical signs and symptoms). survives as the underpinning of forensic toxicology. Orfila
published a major work devoted expressly to the toxicity of
natural agents in 1815. Magendie, a physician and experimen-
Renaissance tal physiologist, studied the mechanisms of action of emetine
and strychnine. His research determined the absorption and
All substances are poisons; there is none that is not a poison. The right
distribution of these compounds in the body. One of
dose differentiates a poison from a remedy.
Paracelsus Magendie’s more famous students, Claude Bernard, contrib-
uted the classic treatise, An Introduction to the Study of
Philippus Aureolus Theophrastus Bombastus von Hohenheim- Experimental Medicine.
Paracelsus (1493-1541) was pivotal, standing between the phi- German scientists Oswald Schmiedeberg (1838-1921) and
losophy and magic of classic antiquity and the philosophy and Louis Lewin (1850-1929) made many contributions to the sci-
science willed to us by figures of the seventeenth and eighteenth ence of toxicology. Schmeideberg trained approximately
centuries. Paracelsus, a physician-alchemist, formulated many 120 students who later populated the most important laborato-
revolutionary views that remain integral to the structure of ries of pharmacology and toxicology throughout the world.
toxicology, pharmacology, and therapeutics today. He focused Lewin published much of the early work on the toxicity of nar-
on the primary toxic agent as a chemical entity, and held that cotics, methanol, glycerol, acrolein, and chloroform.
(1) experimentation is essential in the examination of responses
to chemicals, (2) one should make a distinction between the
therapeutic and toxic properties of chemicals, (3) these proper- 20™ CENTURY TOXICOLOGY: THE
ties are sometimes but not always indistinguishable except by AWAKENING OF UNDERSTANDING
dose, and (4) one can ascertain a degree of specificity of chemi-
cals and their therapeutic or toxic effects. These principles led Toxicology has drawn its strength and diversity from its pro-
Paracelsus to articulate the dose-response relation as a bulwark clivity to borrowing from almost all the basic sciences to test its
of toxicology. hypotheses. This fact, coupled with the health and occupational
 CHAPTER 1 History and Scope of Toxicology 3

regulations that have driven toxicology research since 1900, has were founded. Cellular and molecular toxicology developed as
made this discipline exceptional in the history of science. a subdiscipline, and risk assessment became a major product of
With the advent of anesthetics and disinfectants in the late toxicologic investigations.
1850s, toxicology as it is currently understood began. The prev- Currently, many dozens of professional, governmental, and
alent use of “patent” medicines led to several incidents of poi- other scientific organizations with thousands of members and
sonings from these medicaments, which, when coupled with over 120 journals are dedicated to toxicology and related disci-
the response to Upton Sinclair's exposé of the meatpacking plines. In addition, the International Congress of Toxicology is
industry in The Jungle, culminated in the passage of the Wiley composed oftoxicology societies from Europe, South America,
Bill in 1906, the first of many U.S. pure food and drug laws. Asia, Africa, and Australia, which brings together the broadest
During the 1890s and early 1900s, the discovery of radioac- representation of toxicologists.
tivity and the vitamins, or “vital amines,’ led to the use of the
first large-scale bioassays (multiple animal studies) to deter-
mine whether these “new” chemicals were beneficial or harm- 215" CENTURY TOXICOLOGY
ful to laboratory animals.
The sequencing of the human genome and that of several other
One of the first journals expressly dedicated to experimental
organisms has markedly affected all biological sciences, includ-
toxicology, Archiv fiir Toxikologie, began publication in Europe
ing toxicology. Genetically modifying organisms is now com-
in 1930. That same year the National Institutes of Health (NIH)
monplace and those possessing orthologs of human genes (e.g.,
was established in the United States. As a response to the tragic
zebrafish [Danio rerio], roundworms [Caenorhabditis elegans],
consequences of acute kidney failure after taking sulfanilamide
and fruit flys [Drosophila melanogaster]) are widely used in
in glycol solutions, the Copeland bill was passed in 1938. This
toxicology. Deeper understanding of epigenetics has provided
was the second major bill involving the formation of the U.S.
novel approaches to studying the fetal origin of adult diseases
Food and Drug Administration (FDA). The first major U.S.
including cancers, diabetes, and neurodegenerative diseases
pesticide act was signed into law in 1947. The significance of
and disorders.
the initial Federal Insecticide, Fungicide, and Rodenticide Act
Toxicology has an interesting and varied history. Perhaps asa
was that for the first time in U.S. history a substance that was
science that has grown and prospered by borrowing from many
neither a drug nor a food had to be shown to be safe and effica-
disciplines, it has suffered from the absence of a single goal, but
cious for approval.
its diversification has allowed for the interspersion of ideas and
concepts from higher education, industry, and government.
This has resulted in an exciting, innovative, and diversified field
AFTER WORLD WAR II that is serving science and the community at large. Few disci-
plines can point to both basic sciences and direct applications
You too can be a toxicologist in two easy lessons, each of ten years.
at the same time. Toxicology—the study of the adverse effects
Arnold Lehman (circa 1955)
of xenobiotics—may be unique in this regard.
The mid-1950s witnessed the strengthening of the U.S. FDA
commitment to toxicology. The U.S. Congress passed and the
president of the United States signed the additives amendments BIBLIOGRAPHY
to the Food, Drug, and Cosmetic Act. The Delaney clause Bryan CP: The Papyrus Ebers. London: Geoftrey Bales, 1930.
(1958) of these amendments stated broadly that any chemical Carson R: Silent Spring. Boston, MA: Houghton Mifflin, 1962.
found to be carcinogenic in laboratory animals or humans Gunther RT: The Greek Herbal of Dioscorides. New York: Oxford
could not be added to the U.S. food supply. Delaney became a University Press, 1934.
battle cry for many groups and resulted in the inclusion at a new Guthrie DA: A History of Medicine. Philadelphia, PA: Lippincott,
level of biostatisticians and mathematical modelers in the field 1946.
of toxicology. Shortly after the Delaney amendment, the first Hays HW: Society of Toxicology History, 1961-1986. Washington, DC:
American journal dedicated to toxicology, Toxicology and Society of Toxicology, 1986.
Munter S (ed.): Treatise on Poisons and Their Antidotes. Vol. II of the
Applied Pharmacology, was launched. The founding of the
Medical Writings of Moses Maimonides. Philadelphia, PA: Lippincott,
Society of Toxicology followed shortly afterward.
1966.
The 1960s started with the tragic thalidomide incident, in
Pagel W: Paracelsus: An Introduction to Philosophical Medicine in the
which several thousand children were born with serious birth Era of the Renaissance. New York: Karger, 1958.
defects, and the publication of Rachel Carson's Silent Spring Thompson CJS: Poisons and Poisoners: With Historical Accounts of
(1962). Attempts to understand the effects of chemicals on the Some Famous Mysteries in Ancient and Modern Times. London:
embryo and fetus and on the environment as a whole gained Shaylor, 1931.
momentum. New legislation was passed, and new journals http://www.toxipedia.org/display/toxipedia/History+of+Toxicology
 UNIT 1 General Principles of Toxicology

QUESTIONS

Which one ofthe following statements regarding toxicol- The art of toxicology requires years of experience to
ogy is true? acquire, even though the knowledge base of facts may be
a. Modern toxicology is concerned with the study of the learned more quickly. Which modern toxicologist is cred-
adverse effects of chemicals on ancient forms of life. ited with saying that “you can be a toxicologist in two easy
b. Modern toxicology studies embrace principles from lesions, each of 10 years?”
such disciplines as biochemistry, botany, chemistry, a. Claude Bernard.
physiology, and physics. b. Rachel Carson.
c. Modern toxicology has its roots in the knowledge of c. Upton Sinclair.
plant and animal poisons, which predates recorded d. Arnold Lehman.
history and has been used to promote peace. e. Oswald Schmiedeberg.
d. Modern toxicology studies the mechanisms by which
inorganic chemicals produce advantageous as well as Which of the following statements is correct?
deleterious effects. a. Claude Bernard was a prolific scientist who trained
e. Modern toxicology is concerned with the study of over 120 students and published numerous contribu-
chemicals in mammalian species. tions to the scientific literature.
b. Louis Lewin trained under Oswald Schmiedeberg
Knowledge of the toxicology of poisonous agents was and published much of the early work on the toxicity
published earliest in the: of narcotics, methanol, and chloroform.
a. bers papyrus. c. An Introduction to the Study ofExperimental Medicine
De Historia Plantarum. was written by the Spanish physician Orfila.
De Materia Medica. d. Magendie used autopsy material and chemical analy-
Lex Cornelia. sis systematically as legal proof of poisoning.
some Treatise on Poisons and Their Antidotes. e. Percival Potts was instrumental in demonstrating the
chemical complexity of snake venoms.
Paracelsus, a physician-alchemist, formulated many revo-
lutionary views that remain integral to the structure of
toxicology, pharmacology, and therapeutics today. He
focused on the primary toxic agent as a chemical entity
and articulated the dose-response relation. Which one of
the following statements is not attributable to Paracelsus?
a. Natural poisons are quick in their onset of actions.
b. Experimentation is essential in the examination of
responses to chemicals.
c. One should make a distinction between the thera-
peutic and toxic properties of chemicals.
d. ‘These properties are sometimes but not always indis-
tinguishable except by dose.
e. One can ascertain a degree of specificity of chemicals
and their therapeutic or toxic effects.
 (Covel
yu 1g Me els

Principles of Toxicology
David L. Eaton and Steven G. Gilbert

INTRODUCTION TO TOXICOLOGY Assumptions in Deriving the Dose-Response

Different Areas of Toxicology Relationship

Toxicology and Society Evaluating the Dose-Response Relationship

Comparison of Dose—Responses
General Characteristics of the Toxic Response
Therapeutic Index
CLASSIFICATION OF TOXIC AGENTS Margins of Safety and Exposure
Potency versus Efficacy
SPECTRUM OF UNDESIRED EFFECTS
Allergic Reactions VARIATION IN TOXIC RESPONSES
Idiosyncratic Reactions Selective Toxicity
Immediate versus Delayed Toxicity Species Differences
Reversible versus Irreversible Toxic Effects Individual Differences in Response
Local versus Systemic Toxicity
DESCRIPTIVE ANIMAL TOXICITY TESTS
Interaction of Chemicals
Acute Toxicity Testing
Tolerance
Skin and Eye Irritations
CHARACTERISTICS OF EXPOSURE Sensitization
Route and Site of Exposure Subacute (Repeated-dose Study)
Duration and Frequency of Exposure Subchronic
Chronic
DOSE-RESPONSE RELATIONSHIP
Other Tests
Individual, or Graded, Dose-Response Relationships
Quantal Dose-Response Relationships TOXICOGENOMICS
Shape of the Dose-Response Curve Genomics
Essential Nutrients Epigenetics
Hormesis Transcriptomics and Proteomics
Threshold
Nonmonotonic Dose-Response Curves
6 UNIT 1 General Principles of Toxicology

w A poison is any agent capable of producing a deleterious form of life even though the two may exist in intimate
response in a biological system. contact.
« A mechanistic toxicologist identifies the cellular, bio- « The individual or “graded” dose-response relationship
chemical, and molecular mechanisms by which chemi- describes the response of an individual organism to
cals exert toxic effects on living organisms. varying doses of a chemical.
u Toxicogenomics permits mechanistic toxicologists to = A quantal dose-response relationship characterizes the
identify and protect genetically susceptible individuals distribution of responses to different doses in a popula-
from harmful environmental exposures, and to custom- tion of individual organisms.
ize drug therapies based on their individual genetic « Hormesis, a “U-shaped” dose-response curve, results
makeup. with some xenobiotics that impart beneficial or stimula-
a A descriptive toxicologist is concerned directly with tox- tory effects at low doses but adverse effects at higher
icity testing, which provides information for safety eval- doses.
uation and regulatory requirements. = Descriptive animal toxicity testing assumes that the
= A regulatory toxicologist both determines from available effects produced by a compound in laboratory animals,
data whether a chemical poses a sufficiently low risk to when properly qualified, are applicable to humans, and
be marketed for a stated purpose and establishes stan- that exposure of experimental animals to toxic agents in
dards for the amount of chemicals permitted in ambient high doses is a necessary and valid méthod of discover-
air, industrial atmospheres, and drinking water. ing possible hazards in humans.
e Selective toxicity means that a chemical produces injury
to one kind of living matter without harming another

INTRODUCTION TO TOXICOLOGY from harmful environmental exposures, and to customize drug

therapies based on their individual genetic makeup. Numerous
Toxicology is the study of the adverse effects of chemicals on genetic tests can identify susceptible individuals in advance of
living organisms. A toxicologist is trained to examine the nature pharmacological treatment.
of those effects (including their cellular, biochemical, and A descriptive toxicologist is concerned directly with toxicity
molecular mechanisms of action) and assess the probability of testing, which provides information for safety evaluation and
their occurrence. Fundamental to this process is characterizing regulatory requirements. Toxicity tests (described later in this
the relation of exposure (or dose) to the response. The variety chapter) in experimental animals are designed to yield infor-
of potential adverse effects from the abundant diversity of mation that can be used to evaluate risks posed to humans and
chemicals upon which our society depends often demands spe- the environment by exposure to specific chemicals.
cialization in one area of toxicology. A regulatory toxicologist has the responsibility for deciding,
on the basis of data provided by descriptive and mechanistic
toxicologists, whether a drug or another chemical poses a suf-
Different Areas of Toxicology ficiently low risk to be marketed for a stated purpose.
A mechanistic toxicologist identifies the cellular, biochemical, Regulatory toxicologists are involved in the establishment of
and molecular mechanisms by which chemicals exert toxic standards for the amount of chemicals permitted in foods,
effects on living organisms (see Chapter 3 for a detailed discus- drugs, ambient air, industrial atmospheres, and drinking water
sion of mechanisms oftoxicity). Mechanistic data may be useful (see Chapter 4).
in the design and production of safer chemicals and in rational Forensic toxicology is a hybrid of analytic chemistry and fun-
therapy for chemical poisoning and treatment of disease. In risk damental toxicologic principles that focuses primarily on the
assessment, mechanistic data may be very useful in demon- medicolegal aspects of the harmful effects of chemicals on
strating that an adverse outcome observed in laboratory ani- humans and animals (see Chapter 31).
mals is directly relevant to humans. Toxicogenomics permits the Clinical toxicology is concerned with disease caused by or
application of genomic, transcriptomic, proteomic, and metab- uniquely associated with toxic substances (see Chapter 32).
olomic technologies to identify descriptive and mechanistic Environmental toxicology focuses on the impacts of chemi-
information that can protect genetically susceptible individuals cal pollutants in the environment on biological organisms,
 CHAPTER 2 Principles of Toxicology 4

specifically studying the impacts of chemicals on nonhuman TABLE 2-1 Approximate acute LD., of some
organisms such as fish, birds, terrestrial animals, and plants. representative chemical agents.
Ecotoxicology, a specialized area within environmental toxi-
cology, focuses specifically on the impacts of toxic substances
on population dynamics in an ecosystem (see Chapter 29). Ethyl alcohol 10000
Developmental toxicology is the study of adverse effects on
the developing organism that may result from exposure to Sodium chloride 4000
chemical or physical agents before conception (either parent), Ferrous sulfate 1500
during prenatal development, or postnatally until the time of
puberty. Teratology is the study of defects induced during thorphine sulfate 900
development between conception and birth (see Chapter 10). Phenobarbital sodium 150
Reproductive toxicology is the study of the occurrence of
adverse effects on the male or female reproductive system that Picrotoxin sg
may result from exposure to chemical or physical agents (see Strychnine sulfate pe
Chapter 20).
Nicotine 1

Tubocurarine 0.5
Toxicology and Society
Hemicholinium-3 0.2
Knowledge about the toxicologic effect of a compound affects
consumer products, drugs, manufacturing processes, waste Tetrodotoxin 0.10
cleanup, regulatory action, civil disputes, and broad policy Dioxin (TCDD) 0.001
decisions. The expanding influence of toxicology on societal
issues is accompanied by the responsibility to be increasingly Botulinum toxin 0.00001

sensitive to the ethical, legal, and social implications of toxico- *LD., is the dosage (mg/kg body weight) causing death in 50% of exposed animals.
logic research and testing.
There are several ethica] dilemmas in toxicology. First, expe-
rience and new discoveries in the biological sciences have
emphasized the need for well-articulated visions of human,
animal, and environmental health. Second, experience with CLASSIFICATION OF TOXIC AGENTS
the health consequences of exposure to such things as lead,
asbestos, and tobacco has precipitated many regulatory and Toxic agents are classified depending on the interests and needs
legal actions and public policy decisions. Third, we have an of the classifier. These agents may be discussed in terms of their
increasingly well-defined framework for discussing our social target organs, use, source, and effects. The term toxin generally
and ethical responsibilities. Fourth, all research involving refers to toxic substances that are produced by biological sys-
humans or animals must be conducted in a responsible and tems such as plants, animals, fungi, or bacteria. The term toxi-
ethical manner. Fifth, the uncertainty and biological variabil- cant is used in speaking of toxic substances that are produced
ity inherent in the biological sciences requires decision mak- by or are a by-product of human activities. Toxic agents may be
ing with limited or uncertain information. classified in terms of their physical state, chemical stability or
reactivity, general chemical structure, or poisoning potential.
No single classification is applicable to the entire spectrum of
toxic agents and, therefore, a combination of classifications
General Characteristics of the
is needed to provide the best characterization of a toxic
Toxic Response substance.
Virtually every known chemical has the potential to produce
injury or death if it is present in a sufficient amount. Table 2-1
shows the wide spectrum of dosages needed to produce death SPECTRUM OF UNDESIRED EFFECTS
in 50% of treated animals (lethal dose 50, LD.,). Chemicals
producing death in microgram doses are often considered The spectrum of undesired effects of chemicals is broad. In
extremely poisonous. Note that measures of acute lethality such therapeutics, e.g., each drug produces a number of effects, but
as LD., may not accurately reflect the full spectrum of toxicity, usually only one effect is associated with the primary objective
or hazard, associated with exposure to a chemical. For example, of the therapy; all the other effects are referred to as undesirable
some chemicals with low acute toxicity may have carcinogenic or side effects. However, some of these side effects may be
or teratogenic effects at doses that produce no evidence of acute desired for another therapeutic indication. Some side effects of
toxicity. For a given chemical, each of the various effects that drugs are always deleterious to the well-being of humans.
may occur in a given organism will have their own dose- These are referred to as the adverse, deleterious, or toxic effects
response relationship. of the drug.
8 UNIT 1 General Principles of Toxicology

Allergic Reactions whether the effect is reversible or irreversible. Liver tissue has
high regeneration ability and most injuries are, therefore,
Chemical allergy is an immunologically mediated adverse reac-
reversible. However, CNS injury is largely irreversible because
tion to a chemical resulting from previous sensitization to that
its cells are differentiated and cannot be replaced. Carcinogenic
chemical or to a structurally similar one. The terms hypersensi-
and teratogenic effects of chemicals, once they occur, are usu-
tivity, allergic reaction, and sensitization reaction are used to
ally considered irreversible toxic effects.
describe this situation (see Chapter 12). Once sensitization has
occurred, allergic reactions may result from exposure to rela-
tively very low doses of chemicals. Importantly, for a given aller- Local versus Systemic Toxicity
gic individual, allergic reactions are dose-related. Sensitization
Another distinction between types of effects is made on the
reactions are sometimes very severe and may be fatal.
basis of the general site of action. Local effects occur at the site
Most chemicals and their metabolic products are not suffi-
of first contact between the biological system and the toxicant.
ciently large to be recognized by the immune system as a for-
In contrast, systemic effects require absorption and distribution
eign substance and thus must first combine with an endogenous
of a toxicant from its entry point to a distant site, at which del-
protein to form an antigen (or immunogen). Such a molecule is
eterious effects are produced. Most substances, except for
called a hapten. The hapten-protein complex (antigen) is then
highly reactive materials, produce systemic effects. Some mate-
capable of eliciting the formation of antibodies. Subsequent
rials can produce both effects.
exposure to the chemical results in an antigen-antibody inter-
Most chemicals that produce systemic toxicity usually elicit
action, which provokes the typical manifestations of an allergy
their major toxicity in only one or two organs, which are
that range in severity from minor skin disturbance to fatal ana-
referred to as the target organs of toxicity of a particular chemi-
phylactic shock.
cal. Paradoxically, the target organ of toxicity is often not the
site of the highest concentration of the chemical.
Idiosyncratic Reactions Target organs in order of frequency of involvement in sys-
temic toxicity are the CNS; the circulatory system; the blood
Chemical idiosyncrasy refers to a genetically determined abnor-
and hematopoietic system; visceral organs such as the liver,
mal reactivity to a chemical. The response observed is usually
kidney, and lung; and the skin. Muscle and bone are seldom
qualitatively similar to that observed in all individuals but may
target tissues for systemic effects.
take the form of extreme sensitivity to low doses or extreme
insensitivity to high doses of the chemical. For example, some
individuals are abnormally sensitive to nitrites and other sub- Interaction of Chemicals
stances capable of oxidizing the iron in hemoglobin. This pro-
duces methemoglobin, which is incapable of binding and Chemical interactions can occur via various mechanisms, such
transporting oxygen to tissues. Consequently, they may suffer as alterations in absorption, protein binding, and the biotrans-
from tissue hypoxia after exposure to doses of methemoglobin- formation and excretion of one or both of the interacting toxi-
producing chemicals, whereas normal individuals would be cants. In addition to these modes of interaction, the response of
unaffected. It is now recognized that many idiosyncratic drug the organism to combinations of toxicants may be increased or
reactions are due to the interplay between an individual’s ability decreased because of toxicologic responses at the site of action.
to form a reactive intermediate, detoxify that intermediate, An additive effect, most commonly observed when two
and/or mount an immune response to adducted proteins. chemicals are given together, occurs when the combined effect
Specific genetic polymorphismsin drug-metabolizing enzymes, of two chemicals is equal to the sum of the effects of each agent
transporters, or receptors are responsible for many of these given alone (e.g.: 2 + 3 = 5). A synergistic effect occurs when
observed differences. the combined effects of two chemicals are much greater than the
sum of the effects of each agent given alone (e.g.: 2 + 2 = 20).
Potentiation occurs when one substance does not have a toxic
immediate versus Delayed Toxicity effect on a certain organ or system but when added to another
Immediate toxic effects occur or develop rapidly after a single chemical makes that chemical much more toxic (eg.:0+2= 10).
administration of a substance, whereas delayed toxic effects Isopropanol, e.g., is not hepatotoxic, but when it is adminis-
occur after the lapse of some time. Most substances produce tered in addition to carbon tetrachloride, the hepatotoxicity
immediate toxic effects. However, carcinogenic effects of chem- of carbon tetrachloride is much greater than that when it is
icals usually have long latency periods, often 20 to 30 years after given alone.
the initial exposure, before tumors are observed in humans. Antagonism occurs when two chemicals administered
together interfere with each other’s actions or one interferes
with the action of the other (e.g.: 4 + 6 = 8; 4 + (—4) = 0;
Reversible versus Irreversible Toxic Effects 4 + 0 = 1). There are four major types of antagonism: func-
Some toxic effects of chemicals are reversible, and others are tional, chemical, dispositional, and receptor. Functional antago-
irreversible. Ifa chemical produces pathological injury to a tis- nism occurs when two chemicals counterbalance each other by
sue, the ability of that tissue to regenerate largely determines producing opposite effects on the same physiologic function.
 CHAPTER 2 Principles of Toxicology »)

For example, the marked fall in blood pressure during severe detoxified in the liver, would be expected to be less toxic when
barbiturate intoxication can be effectively antagonized by the given orally than when inhaled, because the oral route requires
intravenous administration of a vasopressor agent such as that nearly all of the dose pass through the liver before reaching
norepinephrine or metaraminol. Chemical antagonism or the systemic circulation and then the CNS.
inactivation is simply a chemical reaction between two com-
pounds that produces a less toxic product. For example, chela-
tors of metal ions decrease metal toxicity and antitoxins Duration and Frequency of Exposure
antagonize the action of various animal toxins. Dispositional Toxicologists usually divide the exposure of experimental ani-
antagonism occurs when the absorption, biotransformation, mals to chemicals into four categories: acute, subacute, sub-
distribution, or excretion of a chemical is altered so that the chronic, and chronic. Acute exposure is defined as exposure to
concentration and/or duration of the chemical at the target a chemical for less than 24h. While acute exposure usually
organ are diminished. Receptor antagonism occurs when two refers to a single administration, repeated exposures may be
chemicals that bind to the same receptor produce less of given within a 24-h period for some slightly toxic or practically
an effect when given together than the addition of their sepa- nontoxic chemicals. Acute exposure by inhalation refers to con-
rate effects (e.g.: 4 + 6 = 8) or when one chemical antagonizes tinuous exposure for less than 24h, most frequently for 4h.
the effect of the second chemical (e.g.: 0 + 4 = 1). Receptor Repeated exposure is divided into three categories: subacute,
antagonists are often termed blockers. subchronic, and chronic. Subacute exposure refers to repeated
exposure to a chemical for 1 month or less, subchronic for 1 to
3 months, and chronic for more than 3 months.
Tolerance In human exposure situations, the frequency and duration of
Tolerance is a state of decreased responsiveness to a toxic effect exposure are usually not as clearly defined as in controlled ani-
of a chemical resulting from prior exposure to that chemical or mal studies, but many of the same terms are used to describe
to a structurally related chemical. Two major mechanisms are general exposure situations. Thus, workplace or environmental
responsible for tolerance: one is due to a decreased amount of exposures may be described as acute (occurring from a single
toxicant reaching the site where the toxic effect is produced (dis- incident or episode), subchronic (occurring repeatedly over
positional tolerance) and the other is due to a reduced respon- several weeks or months), or chronic (occurring repeatedly for
siveness of a tissue to the chemical. many months or years).
For many agents, the toxic effects that follow a single expo-
sure are quite different from those produced by repeated expo-
CHARACTERISTICS OF EXPOSURE sure. Acute exposure to agents that are rapidly absorbed is
likely to produce immediate toxic effects but also can produce
Toxic effects in a biological system are not produced by a delayed toxicity that may or may not be similar to the toxic
chemical agent unless that agent or its metabolic breakdown effects of chronic exposure. Conversely, chronic exposure to a
(biotransformation) products reach appropriate sites in the toxic agent may produce some immediate (acute) effects after
body at a concentration and for a length of time sufficient to each administration in addition to the long-term, low-level, or
produce a toxic manifestation. Whether a toxic response chronic effects of the toxic substance. The other time-related
occurs is dependent on the chemical and physical properties of factor that is important in the temporal characterization of
the agent, the exposure situation, how the agent is metabolized repeated exposures is the frequency of exposure. The relation-
by the system, and the overall susceptibility of the biological ship between elimination rate and frequency of exposure is
system or subject. shown in Figure 2-1. A chemical that produces severe effects
with a single dose may have no effect if the same total dose is
given in several intervals. For the chemical depicted by line B
Route and Site of Exposure in Figure 2-1, in which the half-life for elimination (time nec-
The major routes (pathways) by which toxic agents gain access essary for 50% of the chemical to be removed from the blood-
to the body are the gastrointestinal tract (ingestion), lungs stream) is approximately equal to the dosing frequency, a
(inhalation), skin (topical, percutaneous, or dermal), and other theoretical toxic concentration of 2 U is not reached until the
parenteral (other than intestinal canal) routes. Toxic agents fourth dose, whereas that toxic concentration is nearly reached
generally produce the greatest effect and the most rapid response with only two doses for chemical A, which has an elimination
when given directly into the bloodstream (the intravenous rate much slower than the dosing interval (time between each
route). An approximate descending order of effectiveness for repeated dose). Conversely, for chemical C, where the elimina-
the other routes would be inhalation, intraperitoneal, subcuta- tion rate is much shorter than the dosing interval, a toxic con-
neous, intramuscular, intradermal, oral, and dermal. The “vehi- centration at the site of toxic effect will never be reached
cle” (the material in which the chemical is dissolved) and other regardless of how many doses are administered. Of course, it is
formulation factors can markedly alter absorption. In addition, possible that residual cell or tissue damage occurs with each
the route of administration can influence the toxicity of agents. dose even though the chemical itself is not accumulating. The
For example, an agent that acts on the CNS, but is efficiently important consideration, then, is whether the interval between
10 UNIT 1 General Principles of Toxicology

Repeated doses

Concentration range of toxic response

oe ~ meoe ie
me

om a meage
-

Concentration
site
target
at

Time Time

FIGURE 2-1 Diagrammatic view of the relationship between dose and concentration at the target site under different conditions
of dose frequency and elimination rate. Line A. A chemical with very slow elimination (e.g., half-life of 1 year). Line B. A chemical with a rate of
elimination equal to frequency of dosing (e.g., 1 day). Line C. Rate of elimination faster than the dosing frequency (e.g., 5h). Purple shaded area is
representative of the concentration of chemical at the target site necessary to elicit a toxic response.

doses is sufficient to allow for complete repair oftissue damage. Individual, or Graded, Dose-Response
Chronic toxic effects may occur, therefore, if the chemical
Relationships
accumulates in the biological system (rate of absorption
exceeds the rate of biotransformation and/or excretion), if it Individual dose-response relationships are characterized by a
produces irreversible toxic effects, or if there is insufficient dose-related increase in the severity of the response. For exam-
time for the system to recover from the toxic damage within ple, Figure 2-2 shows the dose-response relationship between
the exposure frequency interval. For additional discussion of different dietary doses of the organophosphate insecticide
these relationships, consult Chapters 5 and 7. chlorpyrifos and the extent of inhibition of two different
enzymes in the brain and liver: acetylcholinesterase and carbo-
xylesterase. In the brain, the degree of inhibition of both
DOSE-RESPONSE RELATIONSHIP enzymes is clearly dose-related and spans a wide range, although
the amount of inhibition per unit dose is different for the two
The characteristics of exposure and the spectrum of effects enzymes. From the shapes of these two dose-response curves,
come together in a correlative relationship customarily referred it is evident that, in the brain, cholinesterase is more easily
to as the dose-response relationship. Whatever response is inhibited than carboxylesterase. The toxicologic response that
selected for measurement, the relationship between the degree results is directly related to the degree of cholinesterase enzyme
of response of the biological system and the amount of toxicant inhibition in the brain. It should be noted that most toxic sub-
administered assumes a form that occurs so consistently as to be stances have multiple sites or mechanisms of toxicity, each with
considered the most fundamental and pervasive concept in its own “dose-response” relationship and subsequent adverse
toxicology. effect. When these dose-response data are plotted using a loga-
From a practical perspective, there are two types of dose-
rithmic scale for the dose, the data “fit” a straight line.
response relationships: (1) the individual dose-response rela-
tionship, which describes the response of an individual
organism to varying doses of a chemical, often referred to as a Quantal Dose-Response Relationships
“graded” response because the measured effect is continuous In contrast to the “graded” or continuous-scale dose-response
over a range of doses, and (2) a quantal dose-response rela- relationship that occurs in individuals, the dose-response rela-
tionship, which characterizes the distribution of responses to tionships in a population are by definition quantal—or “all or
different doses in a population ofindividual organisms. none’—in nature; that is, at any given dose, an individual in the
 CHAPTER 2 Principles of Toxicology 11

100

15
Cholinesterase

50

Inhibition
%
25
Carboxylesterase frequency
Response
(%)

100
0 25 5 7.5 10
Dose (mg/kg) 80

60

75 40

50 (%) 20
Cumulative
response

Inhibition
% 7.0
25

6.0

0 2 See Ss O77 Ie
5.0
Dose (mg/kg)

FIGURE 2-2 Dose-response relationship between different 4.0

doses of the organophosphate insecticide chlorpyrifos and
(Probit
Response
units)
esterase enzyme inhibition in the brain. Open circles and blue lines scale)
(Probit
Response
%
represent acetylcholinesterase activity and closed circles represent 3.0
carboxylesterase activity in the brains of pregnant female Long-Evans 10 20 50 100 200 800
rats given 5 daily doses of chlorpyrifos. A.Dose-response curve
Dose (mg/kg)
plotted on an arithmetic scale. B. Same data plotted on a semi-log
scale. (Data from LassiterTL, et al.: Gestational exposure to chlorpyrifos: FIGURE 2-3 Diagram of a quantal dose-response
dose response profiles for cholinesterase and carboxylesterase activity, relationship. The abscissa is a log dosage of the chemical. In the top
Toxicol Sci, 1999 Nov;52(1):92—100.) panel the ordinate is response frequency, in the middle panel the
ordinate is percent response, and in the bottom panel the response is
in probit units (see text).

population is classified as either a “responder” or a “nonre-

sponder.’ Although these distinctions of “quantal population”
and “graded individual” dose-response relationships are use- animals that responded at each dose minus the percentage that
ful, the two types of responses are conceptually identical. The responded at the immediately lower dose. One can clearly see
ordinate in both cases is simply labeled the response, which may that only a few animals responded to the lowest dose and the
be the degree of response in an individual or system or the frac- highest dose. Larger numbers of animals responded to doses
tion of a population responding, and the abscissa is the admin- intermediate between these two extremes, and the maximum
istered dose range. frequency of response occurred in the middle portion of the
The effective dose (ED) is a widely used statistical approach dose range. Thus, we have a bell-shaped curve known as a nor-
for estimating the response of a population to a toxic expo- mal frequency distribution. The reason for this normal distribu-
sure. Generally, the 50% response level is used (ED), although tion is that there are differences in susceptibility to chemicals
any response level, such as an ED, ED,), or ED, could be among individuals. Animals responding at the left end of the
chosen. 2 curve are referred to as hypersusceptible, and those at the right
The top panel of Figure 2-3 shows that quantal dose- end of the curve are called resistant. If the numbers of individuals
responses exhibit a normal or Gaussian distribution, The fre- responding at each consecutive dose are added together, a cumu-
quency histogram in this panel also shows the relationship lative, quantal dose-response relationship is obtained. When
between dose and effect. The bars represent the percentage of sufficient doses are used with a large number of animals per
12 UNIT 1 General Principles of Toxicology

dose, a sigmoid dose-response curve is observed, as depicted in a oO

the middle panel of Figure 2-3. With the lowest dose (6mg/kg),
1% of the animals respond. A normally distributed sigmoid 9) ro)
curve such as this one approaches a response of 0% as the dose
is decreased and approaches 100% as the dose is increased,
but—theoretically—it never passes through 0% and 100%.
However, the minimally ED of any chemical that evokes a
stated all-or-none response is called the threshold dose even wn
— oO
=>

though it cannot be determined experimentally. units)

(Probit
Response (Probit
Response
scale)
%
The sigmoid curve has a relatively linear portion between 16%
SS ro}
and 84%, These values represent the limits of 1 standard devia-
tion (SD) of the mean (and the median) in a population with A 3 A S10) 20 30 60
truly normal distribution. Thus, the mean +1 SD represents Dose (mg/kg)
68.3% of the population, the mean +2 SD represents 95.5% of
FIGURE 2-4 Comparison of dose-response relationship
the population, and the mean +3 SD equals 99.7% of the popula-
for two different chemicals, plotted on a log dose-probit scale.
tion. One can convert the percent response to units of deviation
Note that the slope of the dose-response relationship is steeper for
from the mean or normal equivalent deviations (NEDs). Thus, chemical B than for chemical A. Dotted lines represent the confidence
the NED for a 50% response is 0; an NED of +1 is equated with limits for chemical A.
an 84.1% response. Units of NED can be converted by the addi-
tion of 5 to the value to avoid negative numbers and be called
probit units (from the contraction of probability unit). In this significant change in resporise will be observed. However, com-
transformation, a 50% response becomes a probit of 5, a +1 devi- pound B exhibits a “steep” dose-response curve, where a rela-
ation becomes a probit of 6, and a —1 deviation is a probit of 4. tively small change in dosage will cause a large change in
The data given in the top two panels of Figure 2-3 are replot- response. The ED., for both compounds is the same (8 mg/kg);
ted in the bottom panel with the mortality plotted in probit units however, the slopes of the dose-response curves are quite differ-
to form a straight line. In essence, what is accomplished in a pro- ent. At one-half of ED.) of the compounds (4 mg/kg), less than
bit transformation is an adjustment of quantal data to an 1% of the animals exposed to compound B would respond but
assumed normal population distribution, resulting in a straight 20% of the animals given compound A would respond.
line. The ED., is obtained by drawing a horizontal line from the Allometry studies the relationship of body size to shape, and
probit unit 5, which is the 50% response point, to the dose-effect allometry is often expressed as a scaling exponent based on
line. At the point of intersection, a vertical line is drawn, and this body mass or body length. If allometric principles are consid-
line intersects the abscissa at the ED, point. In addition to the ered in dosage determination, then viewing dosage on the basis
ED,,, the slope of the dose-response curve can also be obtained. of body weight would be considered less appropriate than if
Figure 2-4 demonstrates the dose-response curves of two com- based on surface area, which is approximately proportional to
pounds. Compound A exhibits a “flat” dose-response curve, 10.5 x (body weight)*, where x = 2/3 or 3/4. In Table 2-2,
showing that a large change in dosage is required before a selected values are given to compare the differences in dosage

TABLE 2-2 Allometric scaling of dose across different species.

Fold Difference, Relative to Humans, Normalized by ©
: os Body Weight _
Species Weight (kg) Surface Area (cm2)* mg/kg — (BW)?8 . : - (ew

Mouse 0.02 103 1 13.0 | 7.0

Rat 0.2 365 1 6.9 4.3

Guinea pig 0.4 582 1 Sy) 3.6

Rabbit 1.5 1410 1 35 2.6

Cat 2 1710 1 322) 2.4

Monkey ee 2720 1 2.6 2.0

Dog {2 5680 1 1.8 Was)

Human 70 18500 1 1.0 1.0

*Surface area of animals is closely approximated by the formula SA = 10.5 x (body weight [in grams])”.
 CHAPTER 2 Principles of Toxicology 13

by the two alternatives. If a scaling factor of (body weight)?” is dose-related (curve A, Figure 2-6). However, there is substan-
used, then the dose would be approximately 13 times greater in tial clinical and epidemiologic evidence that low to moderate
mice than if that dosage were expressed per surface area consumption of alcohol reduces the incidence of coronary
(mg/cm’). However, not all toxic responses will necessarily heart disease and stroke (curve B, Figure 2-6). Thus, when all
scale across species in the same way. responses are plotted on the ordinate, a U-shaped dose-
response curve is obtained (curve C, Figure 2-6).

Shape of the Dose-Response Curve Threshold— Another important aspect of the dose-response
Essential Nutrients—The shape of the dose-response rela- relationship at low doses is the concept of the threshold, that is
tionship has many important implications in toxicity assess- some dose below which the probability of an individual
ment, e.g., for substances that are required for normal responding is zero. For the individual dose-response relation-
physiologic function and survival (e.g., vitamins and essential ship, thresholds for most toxic effects certainly exist, although
trace elements such as chromium, cobalt, and selenium), the interindividual variability in response and qualitative changes
shape of the “graded” dose-response relationship in an indi- in response pattern with dose make it difficult to establish a true
vidual over the entire dose range is actually U-shaped “no effects” threshold for any chemical. In the identification of
(Figure 2-5). That is, at very low doses (or deficiency), there
is a high level of adverse effect, which decreases with an increas-
ing dose. As the dose is increased to a point where the deficiency
no longer exists, no adverse response is detected and the organ-
ism is ina state of homeostasis. However, as the dose is increased
A Effect A—Adverse
to abnormally high levels, an adverse response (usually qualita-
tively different from that observed at deficient doses) appears
and increases in magnitude with increasing dose.
A
Response
Hormesis—Some nonnutritional toxic substances may also
impart beneficial or stimulatory effects at low doses but, at
higher doses, they produce adverse effects. This concept of
“hormesis” may also result ina U-shaped dose-response curve. B
~~ Effect B—Protective
For example, chronic alcohol consumption is well recognized
to increase the risk of esophageal cancer, liver cancer, and cir-
rhosis of the liver at relatively high doses, and this response is

<——_ Death B
Response

Threshold for adverse response

Region of
Response
Homeostasis
effect
Overall

<—____ Sal
Dose (mg/kg/day)
Deficiency Toxicity
re ee ee ee SS
FIGURE 2-6 Hypothetical dose-response relationship
Dose
depicting characteristics of hormesis. Hormetic effects of a
FIGURE 2-5 Individual dose-response relationship for substance are hypothesized to occur when relatively low doses result
an essential substance such as a vitamin or trace element. It in the stimulation of a beneficial or protective response (B), such
is generally recognized that, for most types of toxic responses, a as induction of enzymatic pathways that protect against oxidative
threshold exists such that at doses below the threshold, no toxicity stress. Although low doses provide a potential beneficial effect, a
is evident. For essential substances, doses below the minimum daily threshold is exceeded as the dose increases and the net effects will be
requirement, as well as those above the threshold for safety, may be detrimental (A), resulting in a typical dose-related increase in toxicity.
associated with toxic effects. The purple-shaded region represents The complete dose-response curve (C) is conceptually similar to the
the “region of homeostasis’—the dose range that results in neither individual dose-response relationship for essential nutrients shown
deficiency nor toxicity. in Figure 2-5.
14 UNIT 1 General Principles of Toxicology

“safe” levels of exposure to a substance, it is important to deter- observations are collected from onset of exposure to the toxi-
mine the absence or presence of a threshold. cant to the end of the observation period. An acute toxicity
In evaluating the shape of the dose-response relationship in study ordinarily is supported by histologic examination of
populations, it is realistic to consider inflections in the shape of major tissues and organs for abnormalities. From these obser-
the dose-response curve rather than absolute thresholds. That vations, one can usually obtain more specific information
is, the slope of the dose-response relationship at high doses about the events leading to the lethal effect, the target organs
may be substantially different from the slope at low doses, usu- involved, and often a suggestion about the possible mechanism
ally because of dispositional differences in the chemical. of toxicity.
Saturation of biotransformation pathways, protein-binding
sites or receptors, and depletion of intracellular cofactors rep-
resent some reasons why sharp inflections in the dose- Evaluating the Dose-Response
response relationship may occur. Relationship
Comparison of Dose-Responses—Figure 2-7 illustrates a
Nonmonotonic Dose-Response Curves—Some chemi- hypothetical quantal dose-response curve for a desirable
cals, especially the endocrine disruptors, may exert effects at effect of achemical ED such as anesthesia, a toxic dose (TD)
low doses that are not evident at high doses. These agents pro- effect such as liver injury, and the lethal dose (LD). Even
duce the so-called nonmonotonic dose-response curves. These though the curves for ED and LD are parallel, the mechanism
curves may result from upregulation of some receptors at low by which the drug works is not necessarily that by which the
doses with downregulation of those receptors at higher doses. lethal effects are caused. The same admonition applies to any
The chemical may also act on different molecular pathways with pair of parallel “effect” curves or any other pair of toxicity or
common endpoints but opposite effects. Bisphenol A is one lethality curves. |
chemical that shows nonmonotonic dose response curves.
Therapeutic Index—The hypothetical curves in Figure 2-7
illustrate two other interrelated points: the importance of the
Assumptions in Deriving the Dose- selection of the toxic criterion and the interpretation of com-
parative effect. The therapeutic index (T1) is defined as the ratio
Response Relationship of the dose required to produce a toxic effect and the dose
A number of assumptions must be considered before dose- needed to elicit the desired therapeutic response. Similarly, an
response relationships can be used appropriately. The first is index of comparative toxicity is obtained by the ratio of doses of
that the response is due to the chemical administered, a cause- two different materials to produce an identical response or the
and-effect relationship. ratio of doses of the same material necessary to yield different
The second assumption is that the magnitude ofthe response toxic effects.
is in fact related to the dose. This assumes that there is a molec- The most commonly used index of effect, whether beneficial
ular target site (or sites) with which the chemical interacts to or toxic, is the median dose—that is, the dose required to result
initiate the response, which is related to the concentration of in a response in 50% of a population (or to produce 50% of a
the agent at the target site, which, in turn, is related to the dose
administered.
The third assumption in using the dose-response relation-
7.0 98
ship is that there exists both a quantifiable method of measur-
ing and a precise means of expressing the toxicity. A given 90
chemical may have a family of dose-response relationships, 80
on io
one for each toxic endpoint. For example, a chemical that pro- 70
duces cancer through genotoxic effects, liver damage through ED 60
inhibition of a specific enzyme, and CNS effects via a different nn oO
fo)
mechanism may have three distinct dose-response relation- TD on
40
ships, one for each endpoint. 30
With a new substance, the customary starting point is a sin- 3o 20
gle dose acute toxicity test designed to provide preliminary (Probit
Response
units)
10
identification of target organ toxicity. Studies specifically scale)
(Probit
Responding
%
nn
designed with lethality as an endpoint are no longer recom- 3.0
N
mended by U.S. or international agencies. Data from acute
1OR20R> Omen 00 200 800
studies provide essential information for choosing doses for
repeated dosing studies, as well as choosing specific toxicologic Dose (mg/kg)
endpoints for further study. From these studies, clues as to the FIGURE 2-7 Comparison of effective dose (ED), toxic
direction of further studies come about in two important ways. dose (TD), and lethal dose (LD). The plot is of log dosage versus
Detailed physiologic measurements and __ behavioral percentage of population responding in probit units.
 CHAPTER 2 Principles of Toxicology 15

maximal response). The TI of a drug is an approximate state- thus refers to the range of doses over which a chemical produces
ment about the relative safety of a drug expressed as the ratio of increasing responses. Maximal efficacy reflects the limit of the
the TD (historically the LD) to the therapeutic dose: dose-response relationship on the response axis to a certain
chemical. Chemicals A and B have equal maximal efficacy,
n-™s EDs5o
whereas the maximal efficacy ofC is less than that of D.

From Figure 2-7, one can approximate a TI by using these VARIATION IN TOXIC RESPONSES
median doses. The larger the ratio is, the greater the relative
safety. The ED,, is approximately 20, and the TD., is about 60; Selective Toxicity
thus, the TI is 3, a number indicating that reasonable care in Selective toxicity means that a chemical produces injury to one
exposure to the drug is necessary to avoid toxicity. However, kind of living matter without harming another form of life even
median doses tell nothing about the slopes of the dose- though the two may exist in intimate contact. By taking advan-
response curves for therapeutic and toxic effects. tage of biological diversity, it is possible to develop agents that are
lethal for an undesired species and harmless for other species.
Margins of Safety and Exposure—One way to overcome Such selective toxicity can be due to differences in distribution
this deficiency is to use the ED, for the desired effect and the (absorption, biotransformation, or excretion) or to differing
LD, for the undesired effect. These parameters are used to cal- biochemical processing of the toxicant by different organisms.
culate the margin of safety:

LD, Species Differences

Margin of safety = ED
99 Although a basic tenet of toxicology is that “experimental
results in animals, when properly qualified, are applicable to
For nondrug chemicals, the term margin of safety is an indi- humans, it is important to recognize that both quantitative and
cator of the magnitude of the difference between an estimated qualitative differences in response to toxic substances may
“exposed dose” to a human population and the no observable occur among different species. Identifying the mechanistic
adverse effect level (NOAEL) determined in experimental basis for species differences in response to chemicals establishes
animals. the relevance of animal data to human response.

Potency versus Efficacy—To compare the toxic effects of

two or more chemicals, the dose-response to the toxic effects of Individual Differences in Response
each chemical must be established. The potency and maximal Even within a species, large interindividual differences in
efficacy of the two chemicals to produce a toxic effect can be response to a chemical can occur because of subtle genetic dif-
explained by reference to Figure 2-8. Chemical A is said to be ferences referred to as genetic polymorphisms. These may be
more potent than chemical B, and C is more potent than D, responsible for idiosyncratic reactions to chemicals and for
because of their relative positions along the dosage axis. Potency interindividual differences in toxic responses.

(Probit
Response
units)
scale)
(Probit
Responding
%

1 243 at 6 8 10 1 2 On ot 6
Dosage (mg/kg) (Log scale)

FIGURE 2-8 Schematic representation of the difference in the dose-response curves for four chemicals (A-D), illustrating the
difference between potency and efficacy (see text).
16 UNIT 1 General Principles of Toxicology

DESCRIPTIVE ANIMAL TOXICITY TESTS substance is applied and covered for 24h, and then removed.
The skin is cleaned and the animals observed for 14 days to
Two main principles underlie all descriptive animal toxicity calculate LD.». Acute inhalation studies are performed that are
testing. The first is that the effects produced by a compound in similar to other acute toxicity studies except that the route of
laboratory animals, when properly qualified, are applicable to exposure is inhalation for 4h.
humans. The second principle is that exposure of experimental Acute lethality studies are essential for characterizing the
animals to toxic agents in high doses is a necessary and valid toxic effects of chemicals and their hazard to humans. The most
method ofdiscovering possible hazards in humans because the meaningful scientific information derived from acute lethality
incidence of an effect in a population is greater as the dose or tests comes from clinical observations and postmortem exami-
exposure increases. Obtaining statistically valid results from nation of animals rather than from the specific LD,, value.
the small groups of animals used in toxicity testing requires the
use of relatively large doses so that the effect will occur fre-
quently enough to be detected. However, the use of high doses Skin and Eye Irritations
can create problems in interpretation if the response(s) obtained For the dermal irritation test (Draize test), the skin of rabbits is
at high doses does not occur at low doses. shaved, the chemical applied to one intact and two abraded sites
Toxicity tests are not designed to demonstrate that a chemi- and covered for 4h. The degree of skin irritation is scored for
cal is safe but to characterize the toxic effects a chemical can erythema (redness), eschar (scab), edema (swelling) formation,
produce. There are no set toxicology tests that have to be per- and corrosive action. These dermal irritation observations are
formed on every chemical intended for commerce. Depending repeated at various intervals after the covered patch has been
on the eventual use of the chemical, the toxic effects produced removed. To determine the degree of ocular irritation, the
by structural analogs of the chemical, as well as the toxic effects chemical is instilled into one eye of each test rabbit. The contra-
produced by the chemical itself, contribute to the determina- lateral eye is used as the control. The eyesof the rabbits are then
tion ofthe toxicology tests that should be performed. examined at various times after application.
Alternative in vitro models, including epidermal keratino-
cyte and corneal epithelial cell culture models, have been
Acute Toxicity Testing developed for evaluating cutaneous and ocular toxicity of
The first toxicity test performed ona new chemical is acute tox- substances.
icity, which is determined from the administration of a single
exposure. The LD, and other acute toxic effects are determined
after one or more routes of administration (one route being oral Sensitization
or the intended route of exposure) in one or more species, usu- Information about the potential of a chemical to sensitize skin
ally the mouse and rat, but sometimes the rabbit and dog. Daily is needed in addition to irritation testing for all materials that
examination of the animals for signs of intoxication, lethargy, may repeatedly come into contact with the skin. In general, the
behavioral modifications, food consumption, etc., and tabula- test chemical is administered to the shaved skin of guinea pigs
tion of the number of animals that die in a 14-day period after topically, intradermally, or both, over a period of 2 to 4 weeks.
a single dosage occurs. Acute toxicity tests (1) give a quantita- About 2 to 3 weeks after the last treatment, the animals are chal-
tive estimate of acute toxicity (LD.,), (2) identify target organs lenged with a nonirritating concentration of the test substance
and other clinical manifestations of acute toxicity, (3) identify and the development of erythema is evaluated.
species differences and susceptible species, (4) establish the
reversibility of the toxic response, and (5) provide dose-ranging
guidance for other studies. Subacute (Repeated-dose Study)
Determination ofthe LD. has become a public issue because Subacute toxicity tests are performed to obtain information on
of increasing concern for the welfare and protection of labora- the toxicity of a chemical after repeated administration for typ-
tory animals. Because LD,, is not a constant and many vari- ically 14 days and as an aid to establish doses for subchronic
ables influence its estimation, for most purposes it is only studies.
necessary to characterize the LD., within an order of magni-
tude range (e.g., 5 to 50 and 50 to 500 mg/kg).
If there is a reasonable likelihood of substantial exposure to Subchronic
the material by dermal or inhalation exposure, acute dermal Subchronic exposure usually lasts for 90 days. The principal
and acute inhalation studies are performed. When animals are goals of the subchronic study are to establish a “lowest observed
exposed acutely to chemicals in the air they breathe or the adverse effect level” (LOAEL) and a NOAEL, and to further
water they (fish) live in, the lethal concentration 50 (LC.,) is identify and characterize the specific organ or organs affected
usually determined for a known time of exposure, that is, the by the test compound after repeated administration.
concentration of chemical in the air or water that causes death A subchronic study is usually conducted in two species (rat
to 50% of the animals. The acute dermal toxicity test is usually and dog for FDA; mouse and rat for EPA) by the route of
performed in rabbits. The site of application is shaved, and the intended exposure. At least three doses are employed (a high
 CHAPTER 2 Principles of Toxicology U7

dose that produces toxicity but less than 10% fatalities, a low introduced in Chapter 8. Mutagenicity is discussed in detail in
dose that produces no apparent toxic effects, and an intermedi- Chapter 9. Information on methods, concepts, and problems
ate dose). Animals should be observed once or twice daily for associated with inhalation toxicology is provided in Chapters 15
signs of toxicity. All premature deaths should be recorded and and 28. A discussion of neurotoxicity and behavioral toxicology
necropsied. Severely moribund animals should be terminated can be found in Chapter 16. Immunotoxicity assessment is men-
immediately to preserve tissues and reduce unnecessary suf- tioned in Chapter 12.
fering. At the end of the 90-day study, all the remaining ani-
mals should be terminated and blood and tissues should be
collected for further analysis. The gross and microscopic con- TOXICOGENOMICS
ditions of the organs and tissues are recorded and evaluated.
Hematology, blood chemistry, and urinalysis measurements Toxicogenomics defines the interaction between genes and
are usually done before, in the middle of, and at the termina- toxicants in toxicity etiology. Transcript, protein, and metabo-
tion of exposure. Hematology measurements usually include lite profiling is combined with conventional toxicology. The
hemoglobin concentration, hematocrit, erythrocyte counts, human genome consists of approximately 3 billion base pairs of
total and differential leukocyte counts, platelet count, clotting deoxyribonucleotides. The differential expression of genes in a
time, and prothrombin time. Clinical chemistry determina- given cell is largely responsible for the diverse function of the
tions commonly include glucose, calcium, potassium, urea thousands of different cells, tissues, and organs that constitute
nitrogen, alanine aminotransferase (ALT), serum aspartate an individual organism. Experimental data on how a toxicant
aminotransferase (AST), gamma-glutamyltranspeptidase affects gene expression (transcriptomics), protein production
(GGT), sorbitol dehydrogenase, lactic dehydrogenase, alkaline (proteomics), and small molecule metabolism and function
phosphatase, creatinine, bilirubin, triglycerides, cholesterol, (metabolomics) from a test species (rat/mouse, etc.) can be
albumin, globulin, and total protein. Urinalysis includes deter- combined with those of humans and analyzed with the compu-
mination of specific gravity or osmolarity, pH, proteins, glu- tational tools of bioinformatics to ascertain unique or predic-
cose, ketones, bilirubin, and urobilinogen as well as tive patterns of toxicity.
microscopic examination of formed elements. If humans are
likely to have significant exposure to the chemical by dermal
contact or inhalation, subchronic dermal and/or inhalation Genomics
experiments may also be required. The identification and characterization of various genetic vari-
ants will aid understanding of interindividual differences in
susceptibility to chemicals or other environmental factors and
Chronic
the complex interactions between the human genome and the
Long-term or chronic exposure studies are performed similarly environment. How chemicals affect genomic DNA, mRNA,
to subchronic studies except that the period of exposure is usu- small interfering RNA (siRNA), etc. is of particular importance
ally for 6 months to 2 years. Chronic toxicity tests are often to toxicogenomics.
designed to assess both the cumulative toxicity and the carcino-
genic potential of chemicals. Both gross and microscopic path-
ological examinations are made not only on animals that survive
Epigenetics
the chronic exposure, but also on those that die prematurely.
Dose selection is critical to ensure that premature mortality Toxicants may also act on areas “above or in addition” to genes.
from chronic toxicity does not limit the number of animals Epigenetics concerns a mitotically or meiotically heritable
that survive to a normal life expectancy. Most regulatory change in gene expression that occurs independently of an
guidelines require that the highest administered dose be the alteration in DNA sequence. Changes in DNA methylation or
estimated maximum tolerable dose (MTD), that is, the dose histone acetylation may suppress, silence, or activate gene
that suppresses body weight gain slightly in a 90-day sub- expression without altering the DNA sequence. There is evi-
chronic study. Generally, one or two additional doses, usually dence in some animal models that epigenetic changes may be
one-half and one-quarter MTD, and a control group are tested. transgenerational thereby influencing toxicological assess-
Chronic toxicity assays commonly evaluate the potential ment. Subtle epigenetic changes resulting from environmental
oncogenicity of test substances. Both benign and malignant exposures may not produce cytotoxicity or mutation or may
tumors must be reported. Properly designed chronic oncoge- lead to cancer, neurodevelopment disorders, autoimmune dis-
nicity studies require a concurrent control group matched for eases, metabolic disorders, asthma, or neurologic/behavioral
disorders (Figure 2-9).
variables such as age, diet, and housing conditions.

Other Tests Transcriptomics and Proteomics

The effects of chemicals on reproduction and development are The transcriptome contains all mature mRNA species in the cell
discussed in Chapters 10 and 20. Oncogenicity bioassays are at a given time. It is dynamic and represents the steady state
18 UNIT 1 General Principles of Toxicology

2
i
Omics database(s) 35
%
Treatment

Gene, protein, or “Sequence anchoring” Literature

metabolite-expression of molecular expression mining
profiles

Histopathology

Identify gene/protein
functional groups,
Clinical chemistry pathways, and networks

Q) “Phenotypic anchoring ”
of molecular expression
Weight, physiology

Integrated
Absorbtion, distribution, systems
toxicology

ei —
metabolism, excretion

- Toxicology database(s)

FIGURE 2-9 Conceptual approach for incorporating “omics” technologies and resulting large databases into toxicological
evaluation. Data from experiments that evaluate the effects of a chemical on global patterns of gene expression (transcriptomics), protein
content (proteomics), and small molecules/metabolites (metabonomics/metabolomics), combined with genomic information from both the
test species (e.g., rats, mice) and the target species of interest (e.g., humans), are analyzed by computational tools (bioinformatics) for unique
or potentially predictive patterns of toxicity. Essential to the use of omics data for predictive toxicology/safety assessment is the ability to
reliably tie observed omics patterns to traditional measures of toxicity, such as histopathology and clinical chemistry (phenotypic anchoring).
(Reproduced with permission from Waters MD and Fostel JM. Toxicogenomics and systems toxicology: aims and prospects. Nat Rev Genet, 2004
Dec;5(12):936-948.)

between synthesis (transcription) and degradation of mRNA. expression that may be predictive of early toxicity or subse-
Northern blots, reverse transcriptase polymerase chain reac- quent disease development.
tion, and microarray technologies permit determination of
effects of chemical exposure on gene expression. One of the
major challenges in toxicogenomics is the recognition that BIBLIOGRAPHY
transcriptional regulation is highly dynamic as gene expression Boverhof DR, Gollapudi BB: Applications of Toxicogenomics in Safety
profiles can change dramatically with both dose and time. Evaluation and Risk Assessment. Hoboken, NJ: John Wiley & Sons,
Alterations in gene expression often contribute to phenotypic 2011.
changes that occur, but the transcriptome is somewhat removed Eaton DL: Scientific judgment and toxic torts: a primer in toxicology
from the ultimate biochemical functions that dictate the actual for judges and lawyers. J Law Policy 12:5-12, 2003.
biologic function ofthe cell. Hayes AW, Kruger CL (eds): Principles and Methods of Toxicology,
6th ed. Boca Raton, FL: CRC Press, 2014.
The proteome is the entire complement of proteins that are
Rosenfeld CS: Animal models to study environmental epigenetics.
present in a cell or tissue at a specific time point. Unambiguous
Biol Reprod 82:473-488, 2010.
protein identification is difficult and generally requires separa- Walsh CT, Schwartz-Bloom RD, Levine RR: Levine’ Pharmacology:
tion techniques (gel electrophoresis or high pressure liquid Drug Actions and Reactions, 7th ed. London: Taylor & Francis, 2005.
chromatography) followed by tandem mass spectrometry. Waters MD, Fostel JM: Toxicogenomics and systems toxicology: aims
Proteomics can potentially identify unique patterns of protein and prospects. Nat Rev Genet 5:936-948, 2004.
 CHAPTER 2 Principles of Toxicology 19

QUESTIONS

Five identical experimental animals are treated with 1 mg Toxic chemicals are most likely to be biotransformed in
of one of the following toxins. The animal treated with which of the following organs?
which toxin is most likely to die? a. central nervous system.
ethyl alcohol (LD, = 10,000 mg/kg). b. heart.
botulinum toxin (LD. = 0.00001 mg/kg). c. lung.
nicotine (LD,, = 1 mg/kg). d. pancreas.
ferrous sulfate (LD,, = 1500 mg/kg). e. _ liver.
®
oF
oanpicrotoxin (LD, = 5 mg/kg).
When chemicals A and B are administered simultane-
Place the following mechanisms of toxin delivery in order ously, their combined effects are far greater than the sum
from most effective to least effective—1: intravenous; of their effects when given alone. The chemical interaction
2: subcutaneous; 3: oral; 4: inhalation; 5: dermal. between chemicals A and B can be described as which of
pe Sy PEAS S the following?
be 454172) 3:5. a. potentiative.
Cy IA Apay ee b. additive.
re Bieter hieep c. antagonistic.
@:. Rls Aya 255: d. functionally antagonistic.
e. synergistic.
A toxin with a half-life of 12h is administered every 12h.
Which of the following is true? With respect to dose-response relationships, which of the
a. The chemical is eliminated from the body before the following is true?
next dose is administered. a. Graded dose-response relationships are often
b. The concentration of the chemical in the body will referred to as “all or nothing” responses.
slowly increase until the toxic concentration is b. Quantal dose-response relationships allow for the
attained. analysis of a population’s response to varying dosage.
c. A toxic level will not be reached, regardless of how c. Quantal relationships characterize the response of an
many doses are administered. individual to varying dosages.
d. Acute exposure to the chemical will produce imme- d. A quantal dose-response describes the response of an
diate toxic effects. individual organism to varying doses of a chemical.
e. The elimination rate of the toxin is much shorter than e. The dose-response always increases as the dosage is
the dosing interval. increased.

Urushiol is the toxin found in poison ivy. It must first When considering the dose-response relationship for an
react and combine with proteins in the skin in order for essential substance:
the immune system to recognize and mount a response a. there are rarely negative effects of ingesting too much.
against it. Urushiol is an example of which of the b. the curve is the same for all people.
following? c. adverse responses increase in severity with increasing
a. antigen. or decreasing dosages outside of the homeostatic range.
b. auto-antibody. the relationship is linear.
c. superantigen. e. deficiency will never cause more harm than over-
d. hapten. ingestion.
e. cytokine.
20 UNIT 1 General Principles of Toxicology

The therapeutic index ofa drug: 10. Penicillin interferes with the formation of peptidoglycan
a. is the amount of a drug needed to cure an illness. cross-links in bacterial cell walls, thus weakening the cell
b. is lower in drugs that are relatively safer. wall and eventually causing osmotic death of the bacte-
Cc. describes the potency of a chemical in eliciting a rium. Which of the following is true?
desired response. a. Treatment with penicillin is a good example of selec-
describes the ratio of the toxic dose to the therapeutic tive toxicity.
dose of a drug. b. Penicillin interferes with human plasma membrane
explains the change in response to a drug as the dose structure.
is increased. Penicillin is a good example of a drug with a low
therapeutic index.
Penicillin is also effective in treating viral infections.
Penicillin is completely harmless to humans.
 Crh
AreP sete bak

Mechanism: Loxicity
Zoltan Gregus

STEP 1—DELIVERY: FROM THE SITE OF EXPOSURE Toxic Alteration of Cellular Maintenance
TO THE TARGET Impairment of Internal Cellular Maintenance:
Absorption versus Presystemic Elimination Mechanisms ofToxic Cell Death
Absorption Depletion of ATP
Presystemic Elimination Sustained Rise of Intracellular Ca?*
Distribution to and away from the Target Interplay between the Primary Metabolic Disorders
Mechanisms Facilitating Distribution to a Target Spells Cellular Disaster
Mechanisms Opposing Distribution to a Target Mitochondrial Permeability Transition (MPT) and
Excretion versus Reabsorption the Worst Outcome: Necrosis
Excretion An Alternative Outcome of MPT: Apoptosis
Reabsorption What Determines the Form of Cell Death?
Toxication versus Detoxication Induction of Death by Unknown Mechanisms
Toxication Impairment of External Cellular Maintenance
Detoxication STEP 4—REPAIR OR DYSREPAIR
STEP 2—REACTION OF THE ULTIMATE TOXICANT WITH Molecular Repair
THE TARGET MOLECULE Repair of Proteins
Attributes of Target Molecules Repair of Lipids
Types of Reactions Repair of DNA
Noncovalent Binding Cellular Repair: A Strategy in Peripheral Neurons
Covalent Binding Autophagy of Damaged Cell Organelles
Hydrogen Abstraction Regeneration of Damaged Axons
Electron Transfer Tissue Repair
Enzymatic Reactions Apoptosis: An Active Deletion of Damaged Cells
Effects of Toxicants on Target Molecules Proliferation: Regeneration of Tissue
Dysfunction of Target Molecules Side Reactions to Tissue Injury
Destruction of Target Molecules Mechanisms of Adaptation
Neoantigen Formation When Repair and Adaptation Fail
Toxicity Not Initiated by Reaction with Adaptation
Target Molecules Toxicity Resulting from Inappropriate Repair
and Adaptation
STEP 3—CELLULAR DYSFUNCTION AND
Tissue Necrosis
RESULTANT TOXICITIES
Fibrosis
Toxicant-induced Cellular Dysregulation Carcinogenesis
Dysregulation of Gene Expression
Dysregulation of Ongoing Cellular Activity CONCLUSIONS

a
22 UNIT 1 General Principles of Toxicology

« Toxicity involves toxicant delivery to its target or targets » Sustained elevation of intracellular Ca** is harmful
and interactions with endogenous target molecules that because it can result in (1) depletion of energy reserves
may trigger perturbations in cell function and/or struc- by inhibiting the ATPase used in oxidative phosphoryla-
ture or that may initiate repair mechanisms at the molec- tion, (2) dysfunction of microfilaments, (3) activation of
ular, cellular, and/or tissue levels. hydrolytic enzymes, and (4) generation of reactive oxy-
e Biotransformation to harmful products is called toxica- gen and nitrogen species (ROS and RNS).
tion or metabolic activation. » Cell injury progresses toward cell necrosis (death) if
« Biotransformations that eliminate the ultimate toxicant molecular repair mechanisms are inefficient or the
or prevent its formation are called detoxications. molecular damage is not readily reversible.
« Apoptosis, or programmed cell death, is a tightly con- = Chemical carcinogenesis involves insufficient function
trolled, organized process whereby individual cells break of various repair mechanisms, including (1) failure of
into small fragments that are phagocytosed by adjacent DNA repair, (2) failure of apoptosis (programmed cell
cells or macrophages without producing an inflamma- death), and (3) failure to terminate cell proliferation.
tory response.

‘
\

An understanding of the mechanisms of toxicity provides a with the endogenous target molecule or critically alters the bio-
rational basis for interpreting descriptive toxicity data. The cel- logical environment, initiating structural and/or functional
lular mechanisms that contribute to the manifestation of toxi- alterations that result in toxicity. The ultimate toxicant can be
cities are overviewed by relating a series of events that begins with the original chemical to which the organism is exposed (parent
exposure, involves a multitude of interactions between the invad- compound), a metabolite, or a reactive oxygen or nitrogen spe-
ing toxicant and the organism, and culminates in a toxic effect. cies (ROS or RNS) generated during the biotransformation of
As a result of the huge number of potential toxicants and the the toxicant, or an endogenous molecule.
multitude of biological structures and processes that can be The concentration of the ultimate toxicant at the target mol-
impaired, there are a tremendous number of possible pathways ecule depends on the relative effectiveness of the processes that
that may lead to toxicity (Figure 3-1). Commonly, a toxicant is increase or decrease its concentration at the target site
delivered to its target, reacts with it, and the resultant cellular (Figure 3-2). Increased concentration is facilitated by absorp-
dysfunction manifests itself in toxicity. Sometimes a xenobi- tion, distribution to the site of action, reabsorption, and toxica-
otic does not react with a specific target molecule but rather tion, while presystemic elimination, distribution away from
adversely influences the biological environment, causing the site of action, excretion, and detoxication will decrease the
molecular, organellar, cellular, or organ dysfunction leading to toxicant concentration at its target.
deleterious effects.
The most complex path to toxicity involves more steps
Absorption versus Presystemic Elimination
(Figure 3-1). First, the toxicant is delivered to its target or targets
(step 1), interacting with endogenous target molecules (step 2a) Absorption—Transfer ofa chemical from the site of exposure,
or altering the environment (step 2b), triggering perturbations in usually an external or internal body surface, into the systemic
cell function and/or structure (step 3), which initiate repair mech- circulation is called absorption. Transporters contribute to gas-
anisms at the molecular, cellular, and/or tissue levels (step 4). trointestinal (GI) absorption of some chemicals; however, the
When the perturbations induced by the toxicant exceed repair vast majority of toxicants traverse epithelial barriers via diffu-
capacity or when repair becomes malfunctional, toxicity occurs. sion. Factors that influence absorption include concentration,
Tissue necrosis, cancer, and fibrosis are examples of chemically surface area of exposure, characteristics of the epithelial layer
induced toxicities that follow this four-step course. through which the toxicant is being absorbed, and, usually most
important, lipid solubility because lipid-soluble molecules are
absorbed most easily into cells.
STEP 1—DELIVERY: FROM THE SITE OF
EXPOSURE TO THE TARGET Presystemic Elimination—During transfer from the site of
exposure to the systemic circulation, toxicants may be elimi-
Theoretically, the intensity of a toxic effect depends on the con- nated. This is common for chemicals absorbed from the gastro-
centration and persistence of the ultimate toxicant at its site of intestinal (GI) tract because they must first pass through the GI
action. The ultimate toxicant is the chemical species that reacts mucosal cells, into the liver (enterohepatic circulation), and then
 CHAPTER3 Mechanisms ofToxicity 23

Absorption iGoPresystemic—
e |
| velimination
Distribution ‘Distribution
towardtarget__ \\ away fromtarget
Reabsorption ~ Excretion |
\
| \. Detoxication |

jl
Interaction / Alteration \
with target < of biological »;
molecule “environment ”

7 Target molecule
(Protein, lipid, nucleic acid |
Lr» O
macromolecular complex) L
y >
dy sfunction,
injury
.——~>
X
X | FIGURE 3-2 The process of toxicant delivery is the first step
in the development of toxicity. Delivery—that is, movement of the

C toxicant from the site of exposure to the site of its action in an active
form—is promoted by the processes listed on the left and opposed
by the events indicated on the right.
i
T
Inappropriate
repair and
adaptation particular type of cell. Chemicals also may be distributed to the
site or sites of toxication, usually an intracellular enzyme, where
Y the ultimate toxicant is formed through biotransformation.

FIGURE 3-1 Potential stages in the development of toxicity Mechanisms Facilitating Distribution to a Target
after chemical exposure.
Porosity of the Capillary Endothelium—‘here are three types
of capillaries (continuous, fenestrated, and sinusoidal), each
lung (pulmonary circulation) before being distributed to the rest with varying degrees of porosity. Endothelial cells in the hepatic
of the body (systemic circulation). The GI mucosa and the liver sinusoids and in the renal peritubular capillaries have large
may eliminate a significant fraction of a toxicant during its pas- fenestrae (50 to 150 nm in diameter) that permit passage of even
sage through these tissues. Presystemic or first-pass elimination protein-bound xenobiotics. This relatively free filtration pro-
generally reduces the toxic effects of chemicals that reach their motes the accumulation of chemicals in the liver and kidneys.
target sites by way of the systemic circulation, but may contribute
to injury of the digestive mucosa, the liver, and the lungs because Specialized Transport across the Plasma Membrane—
these processes necessitate toxicant delivery to those sites. Specialized ion channels and membrane transporters can
- contribute to the intracellular delivery of toxicants, making
those cells targets. Na*,K*-ATPase, voltage-gated Ca** chan-
Distribution to and away from the Target nels, carrier-mediated uptake, endocytosis, and membrane
Toxicants exit the blood during the distribution phase, enter the recycling are some examples of methods that facilitate the entry
extracellular space, and reach their site or sites of action, usually of toxicants into specific cells. Further, endocytosis of some
a macromolecule on either the surface or the interior of a toxicant-protein complexes also occurs in some cells.
24 UNIT 1 General Principles of Toxicology

Accumulation in Cell Organelles—Amphipathic xenobiotics There are no efficient elimination mechanisms for nonvola-
with a protonatable amine group and lipophilic character accu- tile, highly lipophilic chemicals. If they are resistant to bio-
mulate in lysosomes as well as mitochondria. Lysosomal accu- transformation, such chemicals are eliminated very slowly and
mulation occurs by pH trapping, that is, diffusion of the amine tend to accumulate in the body on repeated exposure. Three
in unprotonated form into the acidic interior of the organelle, rather inefficient processes are available for the elimination of
where the amine is protonated, preventing its efflux, so that it such chemicals: (1) excretion from the mammary gland in
impairs phospholipid degradation. Mitochondrial accumula- breast milk, (2) excretion in bile, and (3) excretion into the
tion takes place electrophoretically. The amine is protonated in intestinal lumen from blood. Volatile, nonreactive toxicants
the intermembrane space and then sucked into the matrix space such as gases and volatile liquids diffuse from pulmonary cap-
by the strong negative potential (—220 mV), where it may impair illaries into the alveoli and are exhaled.
8-oxidation of fatty acids and oxidative phosphorylation.
Reabsorption—Toxicants in the blood are filtered at the
Reversible Intracellular Binding—Chemicals such as organic glomerulus into the renal tubules. These filtered toxicants may
and inorganic cations and polycyclic aromatic hydrocarbons reenter the blood by diffusing through peritubular capillaries.
accumulate in melanin-containing cells by binding to melanin. This reentry is facilitated by tubular fluid reabsorption which
increases intratubular fluid concentration and residence time of
Mechanisms Opposing Distribution to a Target non-reabsorbed chemical by slowing urine flow.
Binding to Plasma Proteins—Hydrophobic xenobiotics gener- Reabsorption by diffusion is dependent on the lipid solubil-
ally bind proteins or lipoproteins in the plasma. In order to leave ity of the chemical and inversely related to the extent of ioniza-
the blood and enter cells, these xenobiotics must dissociate tion, because the nonionized molecule is more lipid soluble.
from these proteins. Therefore, strong binding to plasma pro- Therefore, pH of the tubular fluid affects reabsorption such
teins delays xenobiotics movement across membranes and pro- that acidification favors excretion of weak organic bases and
longs their effects and elimination. alkalinization favors the elimination of weak organic acids.
Toxicants delivered to the GI tract by biliary, gastric, and
Specialized Barriers—Brain capillaries lack fenestrae and are intestinal excretion and secretion by salivary glands and exo-
joined by extremely tight junctions, preventing the access of crine pancreas may be reabsorbed by diffusion across the intes-
hydrophilic chemicals to the brain except by active transport. tinal mucosa. Reabsorption of compounds excreted into bile is
The spermatogenic cells are supported by Sertoli cells that are possible only if they are sufficiently lipophilic or are converted
tightly joined to form the blood-testis barrier. Transfer of hydro- to more lipid-soluble forms in the intestinal lumen.
philic toxicants across the placenta is also restricted. However,
none of these barriers are effective against lipophilic substances.
Toxication versus Detoxication
Distribution to Storage Sites—Some chemicals accumulate in Toxication—A number of xenobiotics are directly toxic,
tissues (i.e., storage sites) where they do not exert significant whereas other xenobiotics exert a toxic effect through their
effects. Such storage decreases toxicant availability for their metabolites. Biotransformation to harmful products is called
target sites. toxication or metabolic activation. With some xenobiotics, toxi-
cation confers physicochemical properties that adversely alter
Association with Intracellular Binding Proteins—Binding to the microenvironment of biological processes or structures.
nontarget intracellular sites, such as metallothionein, tempo- Occasionally, chemicals acquire structural features and reactiv-
rarily reduces the concentration of toxicants at the target site. ity by biotransformation that allows for a more efficient inter-
action with specific receptors or enzymes. Most often, however,
Export from Cells—Intracellular toxicants may be transported toxication renders xenobiotics and occasionally other mole-
back into the extracellular space. Some ATP-dependent mem- cules in the body, such as nitric oxide, indiscriminately reactive
brane transporters, also known as the multidrug-resistance toward endogenous molecules with susceptible functional
(mdr) proteins, extrude chemicals from cells. groups. This increased reactivity may be due to conversion
into (1) electrophiles, (2) free radicals, (3) nucleophiles, or
(4) redox-active reactants.
Excretion versus Reabsorption Electrophiles are molecules that contain an electron-
Excretion—Excretion is the removal of xenobiotics from deficient atom with a partial or full positive charge that allows
blood and their return to the external environment. Excretion it to react by sharing electron pairs with the electron-rich
is a physical mechanism, whereas biotransformation is a chem- atoms in nucleophiles. A free radical is a molecule or molecu-
ical mechanism for eliminating the toxicant. lar fragment that contains one or more unpaired electrons.
The route and speed of excretion depend largely on the phys- One of the more biologically relevant free radicals is superox-
icochemical properties of the toxicant. The major excretory ide anion (O,*~), which is formed both endogenously and
organs—the kidney and the liver—efficiently remove highly exogenously. The immune system produces O,*~ and trans-
hydrophilic chemicals such as organic acids and bases. forms it into hypochorous acid (aka bleach, HOCI) through a
 CHAPTER3 Mechanisms ofToxicity 25

series of reactions in order to combat pathogens. The most including oxidation by flavin-containing monooxygenases and
reactive metabolites are electron-deficient molecules and oxidation to carboxylic acids, as is the case with ethanol.
molecular fragments such as electrophiles and neutral or cat-
ionic free radicals. Some nucleophiles are inherently reactive Detoxication of Electrophiles—Generally, detoxication of elec-
(e.g., HCN and CO); however, many are activated by conver- trophilic toxicants involves conjugation with the nucleophile,
sion into electrophiles. glutathione. This reaction may occur spontaneously or can be
facilitated by glutathione S-transferases. Covalent binding of
Detoxication—Biotransformations that eliminate the ulti- electrophiles to proteins can be regarded as detoxification, pro-
mate toxicant or prevent its formation are called detoxications. vided that the protein has no critical function and does not
In some cases, detoxication may compete with toxication. become a neoantigen or otherwise harmful.

Detoxication of Toxicants with No Functional Groups—In Detoxication of Free Radicals—Detoxication and elimination
general, chemicals without functional groups, such as benzene of O,*~ isimportant because it can be converted into much more
and toluene, are detoxicated in two phases. Initially, a func- reactive compounds (Figure 3-3) such as the hydroxyl radical
tional group such as hydroxyl or carboxyl is introduced into the (HO®), nitrogen dioxide (*NO,), and the carbonate anion
molecule, most often by cytochrome P450 enzymes. Next, an radical (CO,°~). Superoxide dismutases (SODs), located in the
endogenous acid, such as glucuronic acid, sulfuric acid, or an cytosol (Cu, Zn-SOD) and the mitochondria (Mn-SOD),
amino acid, is added to the functional group by a transferase. convert O,* to hydrogen peroxide (HOOH) (Figure 3-4).
With some exceptions, the final products are inactive, highly Subsequently, HOOH is reduced to water by cytosolic glutathi-
hydrophilic organic acids that are readily excreted. one peroxidase or peroxisomal catalase (Figure 3-4). Noenzyme
eliminates HO* owing to its extremely short half-life (10~°s).
Detoxication of Nucleophiles—Nucleophiles generally are The only effective protection against HO* is to prevent its forma-
detoxicated by conjugation of a functional group to the nucleo- tion by converting its precursor, HOOH, to water (Figure 3-4).
philic atom. Sulfonation, glucuronidation, methylation, and Peroxynitrite (ONOO), like HOOH, is an intermediate of
acetylation are common reactions. Conjugation prevents O,*” toxication and is not a free radical oxidant itself. It is sig-
peroxidase-catalyzed conversion of the nucleophiles to free radi- nificantly more stable than HO*, and rapidly reacts with CO, to
cals and biotransformation of phenols, aminophenols, catechols, form the reactive free radicals, *NO, and CO,°~ (Figure 3-3).
and hydroquinones to electrophilic quinines and quinoneimines. Glutathione peroxidase can reduce ONOO‘ to nitrite (ONO),
Alternative mechanisms of nucleophile detoxication exist, thereby preventing free radical production. In addition,

Fe(II), Cu(l), Mn(Il), Cr(V), Ni(II)

COs Fenton reaction

Fe(III), Cu(ll), Mn(Ill), Cr(VI), Ni(Il)

ONOOCO; [HOOH]”

HO’ | OH

FIGURE 3-3 Two pathways for toxication of superoxide anion radical (O,"-) via nonradical products (ONOO— and HOOH) to radical
products (*NO,, CO,°-, and HO’). In one pathway, conversion of (O,*") to HOOH is spontaneous or is catalyzed by SOD. Homolytic cleavage of
HOOH to hydroxyl radical and hydroxyl ion is called the Fenton reaction and is catalyzed by the transition metal ions shown. Hydroxyl radical
formation is the ultimate toxication for xenobiotics that form O,°~ or for HOOH, the transition metal ions listed, and some chemicals that form
complexes with these transition metal ions. In the other pathway, O,*" reacts avidly with nitric oxide (*NO), the product of *NO synthase (NOS),
forming peroxynitrite (ONOO). Spontaneous reaction of ONOO- with carbon dioxide (CO,) yields nitrosoperoxy carbonate (ONOOCO,-) that
is homolytically cleaved to nitrogen dioxide (*NO,) and carbonate anion radical (CO,*-). All three radical products indicated in this figure are
oxidants, whereas °NO, is also a nitrating agent.
26 UNIT 1 General Principles of Toxicology

2GSH
oe
GSSG
A
STEP 2—REACTION OF THE
"a ee
\ GPX /
ee) OS
ULTIMATE TOXICANT WITH THE
TARGET MOLECULE
O5 O> Prx(SH)> PrxS>
\ A Toxicity is typically mediated by a reaction of the ultimate toxi-
O5
; _\S0D HOOH a
ee ae
eee oe 2HOH cant witha target molecule (step 2a in Figure 3-1). Subsequently,
| a series of secondary biochemical events occur, leading to dys-
2H* | function or injury that is manifest at various levels of biological
organization, such as at the target molecule itself, cell organelles,
Ne eas =SS 2HOH

/ CAT cells, tissues and organs, and even the whole organism.
| ¥
HOOH O>
Attributes of Target Molecules
FIGURE 3-4 Detoxication of superoxide anion radical (O,°-)
by superoxide dismutase (SOD), glutathione peroxidase (GPX),
Practically all endogenous compounds are potential targets for
and catalase (CAT). toxicants. The most prevalent and toxicologically relevant
targets are nucleic acids (especially DNA), proteins, and mem-
branes. The first target for reactive metabolites is often the
ONOO* reacts with oxyhemoglobin, heme-containing peroxi- enzyme responsible for their production or the adjacent intra-
dases, and albumin, all of which could be important binding cellular structures. Not all targets .for chemicals contribute
sites for ONOO~. Furthermore, elimination of the two harmful effects. Covalent binding to proteins without adverse
ONOO-X precursors—that is, *NO by reaction with oxyhemo- consequences may even represent a form of detoxication by
globin and O,*~ by SODs—is a significant mechanism in pre- sparing toxicologically relevant targets. ‘Ihus, to conclusively
venting ONOO™ buildup. identify a target molecule as being responsible for toxicity, it
Peroxidase-generated free radicals are eliminated by elec- should be demonstrated that the ultimate toxicant (1) reacts
tron transfer from glutathione. This results in the oxidation of with the target and adversely affects its function, (2) reaches an
glutathione, which is reversed by NADPH-dependent glutathi- effective concentration at the target site, and (3) alters the target
one reductase (Figure 3-5). Thus, glutathione plays an impor- in a way that is mechanistically related to the observed toxicity.
tant role in the detoxication of both electrophiles and free
radicals.
Types of Reactions
Detoxication of Protein Toxins—Extra- and intracellular pro- The ultimate toxicant may bind to the target molecules nonco-
teases are involved in the inactivation of toxic polypeptides. valently or covalently and may alter it by hydrogen abstraction,
Several toxins found in venoms, such as @- and B-bungarotoxin, electron transfer, or enzymatically.
erabutoxin b, and phospholipase, contain intramolecular
disulfide bonds that are required for their activity. These pro- Noncovalent Binding—Hydrophobic interactions, hydro-
teins become inactivated by the enzyme thioredoxin, which gen bonding, and ionic bonding are forms of noncovalent bind-
reduces the essential disulfide bond. ing through which a toxicant can interact with targets such as
membrane receptors, intracellular receptors, ion channels, and
When Detoxication Fails—Detoxication may be insufficient certain enzymes. Noncovalent binding usually is reversible
for several reasons: (1) the toxicant overwhelms the detoxica- because of the comparatively low bonding energy.
tion processes, (2) a reactive toxicant inactivates a detoxicating
enzyme, (3) the detoxication is reversed after transfer to other Covalent Binding—Being practically irreversible, covalent
tissues, or (4) harmful by-products are produced by the detoxi- binding permanently alters endogenous molecules. Covalent
cation process. adduct formation is common with electrophilic toxicants such

HOOH «7 2GS' —> GSSG | NADPH + Ht

Va
VV

GR
yi
t J
eGS; Se 2GSH “ “ NADPt
Zt

FIGURE 3-5 Detoxication of peroxidase (POD)-generated free radicals such as chlorpromazine free radical (CPZ**) by glutathione
(GSH). The by-products are glutathione thiyl radical (GS*) and glutathione disulfide (GSSG), from which GSH is regenerated
by glutathione
reductase (GR).
 CHAPTER3 Mechanisms of Toxicity 27

as nonionic and cationic electrophiles and radical cations. These Other target molecules are susceptible to spontaneous deg-
toxicants react with nucleophilic atoms that are abundant in radation after chemical attack. Free radicals such as Cl,COO*
biological macromolecules, such as proteins and nucleic acids. and HO* can initiate peroxidative degradation of lipids by
Neutral free radicals such as HO®, *NO,, and Cl,C® also can bind hydrogen abstraction from fatty acids. This not only destroys
covalently to biomolecules. Nucleophilic toxicants are, in prin- lipids in cellular membranes but also generates endogenous
ciple, reactive toward electrophilic endogenous compounds. toxicants, free radicals, and electrophiles, which can go on to
However, such reactions are infrequent due to the rarity of elec- harm adjacent molecules (e.g., membrane proteins) or more
trophilic biomolecules. Carbon monoxide, cyanide, hydrogen distant molecules (e.g., DNA). Several forms of DNA frag-
sulfide, and azide are examples of nucleophiles that form coor- mentation can be caused by toxicants, including imidazole
dinate covalent bonds with iron in various heme proteins. ring-opening on purines, imidazole ring-contraction on
pyrimidines, single-strand breaks (SSBs), phosphodiester
Hydrogen Abstraction—Neutral free radicals can readily bond cleavage, and double-strand breaks (DSBs).
abstract H atoms from endogenous compounds, subsequently
converting those compounds into radicals. Radicals can also Neoantigen Formation—Covalent binding of xenobiotics
remove hydrogen from methylene groups (CH,) of free or their metabolites to proteins may evoke an immune response
amino acids or from amino acid residues in proteins and convert (Chapter 12). Some chemicals (e.g., dinitrochlorobenzene,
them to carbonyls (C=O), forming cross-links with DNA or penicillin, and nickel) bind to proteins spontaneously.
other proteins. Others may obtain reactivity by autooxidation to quinones
(e.g., urushiols, the allergens in poison ivy) or by enzymatic
Electron Transfer—Chemicals can exchange electrons to biotransformation.
oxidize or reduce other molecules, leading to formation of
harmful by-products. For example, chemicals can oxidize Fe(II)
in hemoglobin to Fe(III), producing methemoglobinemia. Toxicity Not Initiated by Reaction with
Target Molecules
Enzymatic Reactions—A few toxins act enzymatically on
Some xenobiotics alter the biological microenvironment (see
specific target proteins. For example, diphtheria toxin blocks
step 2b in Figure 3-1), leading to atoxic response. Included here
the function of elongation factor 2 in protein synthesis and
are (1) chemicals that alter H* ion concentrations in the aque-
cholera toxin activates a G protein through such a mechanism.
ous biophase, (2) solvents and detergents that physicochemi-
In summary, most ultimate toxicants act on endogenous cally alter the lipid phase of cell membranes and destroy
molecules on the basis of their chemical reactivity. Those with
transmembrane solute gradients, and (3) xenobiotics that cause
more than one type of reactivity may react by different mecha-
harm merely by occupying a site or space.
nisms with various target molecules.

STEP 3—CELLULAR DYSFUNCTION AND

Effects of Toxicants on Target Molecules
RESULTANT TOXICITIES
Dysfunction of Target Molecules—Some toxicants activate
protein target molecules, mimicking endogenous ligands. More Reaction of toxicants with a target molecule may result in
commonly, chemicals inhibit the function of target molecules impaired cellular function as the third step in the development
by blocking neurotransmitter receptors or ion channels, inhib- of toxicity (Figures 3-1). Each cell in a multicellular organism
iting enzymes, and interfering with cytoskeleton dynamics. carries out defined programs, some of which determine whether
Protein function is impaired when conformation or struc- cells undergo division, differentiation, or apoptosis. Other pro-
ture is altered by interaction with the toxicant. Many proteins grams control the ongoing (momentary) activity of differenti-
possess critical moieties that are essential for catalytic activity ated cells, determining whether they secrete more or less of a
or assembly to macromolecular complexes. Covalent and/or substance, whether they contract or relax, and whether they
oxidative modification of these moieties by xenobiotics can transport and metabolize nutrients at higher or lower rates. For
cause aberrant signal transduction and/or impaired mainte- regulation of these cellular programs, cells possess signaling
nance of the cell’s energy and metabolic homeostasis. Toxicants networks that can be activated and inactivated by external sig-
may also interfere with the template function of DNA. The naling molecules.
covalent binding of chemicals to DNA causes nucleotide As outlined in Figure 3-6, the nature of the primary cellular
mispairing during replication. dysfunction caused by toxicants, but not necessarily the ulti-
mate outcome, depends on the role of the target molecule
Destruction of Target Molecules—In addition to adduct affected. The reaction of a toxicant with targets serving external
formation, toxicants alter the primary structure of endogenous functions can influence the operation of other cells and inte-
molecules by means of cross-linking and fragmentation. Cross- grated organ systems. However, if the target molecule is involved
linking imposes both structural and functional constraints on predominantly in the cell’s internal maintenance, the resultant
the linked molecules. dysfunction can ultimately compromise survival of the cell.
 UNIT 1 General Principles of Toxicology

The target molecule The effect

as determinant of the effect
Inappropriate
of gene + Cell division —> neoplasia, teratogenesis
expression - Apoptosis —> tissue involution, teratogenesis
« Protein synthesis —> e.g., peroxisome proliferation |

| Dysregulation _ For example, Inappropriate neuromuscular activity

| of ongoing - e Tremor, convulsion, spasm, cardiac arrythmia
cell function

Impaired | Impaired
_ internal — + ATP synthesis
maintenance | + Ca?* regulation Cell
i eee ae ¢ Protein synthesis injury/death |
¢ Microtubular function :
¢ Membrane function .

__ Impaired Impaired function of integrated systems

/ external —— «Hemostasis —>bleeding
_ maintenance ee
tele ili. Ge ee le : : 7

FIGURE 3-6 The third step in the development of toxicity: alteration of the regulatory or maintenance function of the cell.

Toxicant-induced Cellular Dysregulation expression may occur at elements that are directly responsible
for transcription, at components of the intracellular signal-
Cells are regulated by signaling molecules that activate specific
transduction pathway, and at the synthesis, storage, or release
cellular receptors linked to signal-transducing networks that
ofthe extracellular signaling molecules.
transmit the signals to the regulatory regions of genes and/or
functional proteins. Receptor activation may ultimately lead to
altered gene expression and/or a chemical modification of spe- Dysregulation of Transcription—Transcription of genetic
cific proteins, typically by phosphorylation. Programs control- information from DNA to mRNA is controlled largely by inter-
ling the destiny of cells primarily affect gene expression, whereas play between transcription factors (TFs) and the regulatory or
those regulating the ongoing activities primarily influence the promoter region of genes. By binding to distinctive nucleotide
activity of functional proteins. However, one signal often evokes sequences in the promoter or regulatory regions, TFs can facil-
both responses because of branching and interconnection of itate or impede formation of the preinitiation complex, thereby
signaling networks. either promoting or repressing transcription of the adjacent
gene. Xenobiotics may interact with the promoter region of the
Dysregulation of Gene Expression—Gene expression is gene, the TFs, or other components of the transcription initia-
the process by which information from a gene is used to syn- tion complex. However, altered activation of TFs appears to be
thesize a functional gene product. The central dogma of the most common modality.
molecular biology is that information from DNA is tran- Several endogenous compounds, such as hormones, and
scribed into messenger RNA (mRNA), which is then trans- vitamins, influence gene expression by binding to and activat-
lated into a protein product. Genes that are transcribed into ing TFs or intracellular receptors; xenobiotics may mimic these
other types of RNA but not into proteins are called nonpro- natural ligands. Either natural or xenobiotic ligands may cause
tein-coding genes and they are one source of post- toxicity when present at extreme doses or during critical peri-
transcriptional control of protein synthesis. Among the ods of organism development. In addition to altering the fate of
alternative RNA types is the recently discovered small silen- specific cells, compounds that act on ligand-activated TFs can
cing RNA, called microRNA (miRNA), which can repress also evoke changes in the metabolism of endogenous and for-
translation of mRNA into proteins. Dysregulation of gene eign substances by inducing overexpression of relevant
 CHAPTER3 Mechanisms ofToxicity 29

enzymes. The effects of endobiotics and xenobiotics that act on Aberrant phosphorylation of proteins may result from
TFs may also be mediated by transcriptional up- or down- decreased dephosphorylation by phosphatases or by increased
regulation of protein-coding genes (i.e., genes transcribed into phosphorylation by kinases. Inhibition of phosphatases
mRNA) and/or nonprotein-coding genes (i.e., genes tran- appears to be the underlying mechanism of the mitogenic
scribed into miRNA). Xenobiotics may also dysregulate tran- effect of various chemicals, oxidative stress, and ultraviolet
scription by altering the regulatory gene regions and the (UV) irradiation. Soluble protein phosphatase 2A (PP2A) in
promoter methylation pattern. cells is likely responsible for reversing the growth factor-
induced stimulation of MAPK, thereby controlling the extent
Dysregulation of Signal Transduction—Extracellular signal- and duration of MAPK activity. PP2A also removes an activat-
ing molecules, such as growth factors, cytokines, hormones, ing phosphate from a mitosis-triggering protein kinase. Several
and neurotransmitters, can ultimately activate TFs by utilizing natural toxins are extremely potent inhibitors of PP2A, includ-
cell surface receptors and intracellular signal-transducing net- ing the blue-green algae poison microcystin-LR and the dino-
works. Figure 3-7 depicts such networks and identifies some flagellate-derived okadaic acid.
important signal-activated TFs that control transcriptional Apart from phosphatases, there are also other inhibitory
activity of genes that influence cell cycle progression and thus binding proteins that can keep signaling under control. For
determine the fate of cells. An example is the c-Myc protein, example, IKB binds to NF-KB, subsequently preventing its
which, on dimerizing with Max protein and binding to its cog- transfer into the nucleus and its function as a TF (Figure 3-7).
nate nucleotide sequence, transactivates cyclin D and E genes. Upon phosphorylation, IKB becomes degraded and NF-kB is
The cyclins, in turn, accelerate the cell-division cycle by activat- set free. NF-KB is an important contributor to proliferative and
ing cyclin-dependent protein kinases, which are involved in prolife signaling, as well as the acute and chronic inflammatory
regulating the cell cycle. Mitogenic signaling molecules thus response. IKB degradation which leads to NF-«B activation
induce cellular proliferation. can also be induced by oxidative stress.
The signal from the cell surface receptors to the TFs is relayed
by successive protein-protein interactions and protein phos- Chemically Altered Signal Transduction with Antiproliferative
phorylations, that is, a signal molecule phosphorylates another Effect: Downturning of increased proliferative signaling after cell
protein like mitogen-activated protein kinase (MAPK), which injury may compromise replacement of injured cells (follow the
activates that protein to phosphorylate and activate another. path in Figure 3-7: inhibition of Raf — diminished degradation
For example, ligands induce growth factor receptors (item 4 in of IKB — diminished binding of NF-KB to DNA — diminished
Figure 3-7) on the surface of all cells to self-phosphorylate, and expression of c-Myc mRNA). Down-regulation of a normal mito-
these phosphorylated receptors then bind to adapter proteins genic signal is a step away from survival and toward apoptosis.
through which they activate Ras. The active Ras initiates the
MAPK cascade, involving serial phosphorylations of protein Dysregulation of Extracellular Signal Production—Hormones
kinases, which finally reaches the TFs. These signal transduc- of the anterior pituitary exert mitogenic effects on endocrine
ers are typically, but not always, activated by phosphorylation, glands in the periphery by acting on cell surface receptors.
which is catalyzed by protein kinases, and are usually inacti- Pituitary hormone production is under negative feedback con-
vated by dephosphorylation, which is carried out by protein trol by hormones of the peripheral glands. Perturbation of this
phosphatases. circuit adversely affects pituitary hormone secretion and, in
Chemicals most often cause aberrant signal transduction by turn, the peripheral glands. Decreased secretion of pituitary
altering protein phosphorylation, and occasionally by interfer- hormone produces apoptosis followed by involution of the
ing with the GTPase activity or signal termination activity of peripheral target gland.
G proteins (e.g., Ras), disrupting normal protein-protein
interactions, establishing abnormal ones, or by altering the Dysregulation of Ongoing Cellular Activity—Toxicants
synthesis or degradation of signaling proteins. Such interven- can adversely affect ongoing cellular activity in specialized cells
tions may ultimately influence cell cycle progression. by disrupting any step in signal coupling.

Chemically Altered Signal Transduction with Proliferative Effect: Dysregulation of Electrically Excitable Cells—Many xenobi-
Xenobiotics that facilitate phosphorylation of signal transduc- otics influence cellular activity in excitable cells, such as neu-
ers often promote mitosis and tumor formation. For example, rons, skeletal, cardiac, and smooth muscle cells. Release of
the phorbol esters and fumonisin B activate protein kinase C neurotransmitters and muscle contraction are controlled by
(PKC) by mimicking diacylglycerol (DAG), one of the physio- transmitters and modulators synthesized and released by adja-
logic activators of PKC (item 6 in Figure 3-7). The other physi- cent neurons. Chemicals that interfere with these mechanisms
ologic PKC activator, Ca**, is mimicked by Pb**. Activated PKC are listed in Table 3-1.
promotes mitogenic signaling by starting a cascade that acti- Perturbation of ongoing cellular activity by chemicals may
vates other kinases and allows certain TFs to bind to DNA. be due to an alteration in (1) the concentration of neurotrans-
Protein kinases may also be activated by interacting with pro- mitters, (2) receptor function, (3) intracellular signal transduc-
teins that have been altered by a xenobiotic. tion, or (4) the signal-terminating processes.
30 UNIT 1 General Principles of Toxicology

o eo 8 oO 5] 4) 6 Oo (3)
IL-6 TNF IL-1 EGF ECM EGF PGF2, TSH TGF-8
Ligands

Membrane
receptors

iF Signal-
activated
transcription
factors

x Mediators of acute phase

protein expression
Cell cycle progression
t Mediates ECM formation mitosis
in ECM-producing cells

FIGURE 3-7 Signal-transduction pathways from cell membrane receptors to signal-activated nuclear transcription factors that
influence transcription of genes involved in cell-cycle regulation. The symbols of cell membrane receptors are numbered 1 to 8 and some
of their activating ligands are indicated. Circles represent G proteins, oval symbols protein kinases, rectangles transcription factors, wavy lines
genes, and diamond symbols inhibitory proteins, such as protein phosphatases (PTP and PP2A) and the lipid phosphatase PTEN, the GTPase-
activating protein GAP, and the inhibitory binding protein IKB. Arrowheads indicate stimulation or formation of second messengers (e.g.,
DAG, IP, PIP;, CAMP, and Ca**), whereas blunt arrows indicate inhibition. Phosphorylation and dephosphorylation are indicated by +P and -P,
respectively. Abbreviations for interfering chemicals are printed in black (As = arsenite; CALY = calyculin A; FA = fatty acids; FB1 = fumonisin B;
MC-LR = microcystin-LR; OKA = okadaic acid; MMS = methylmethane sulfonate; PMA = phorbol miristate acetate; ROS = reactive oxygen
species; SHR = SH-reactive chemicals, such as iodoacetamide; STAU = staurosporin).
In the center of the depicted networks is the pathway activated by growth factors, such as EGF, that acts on a tyrosine kinase receptor (#6), which
uses adaptor proteins (Shc, Grb2, and SOS; not shown) to convert the inactive GDP-bound Ras to active GTP-bound form, which in turn activates
the MAP-kinase phosphorylation cascade (Raf, MAPKK, and MAPK). The phosphorylated MAPK moves into the nucleus and phosphorylates
transcription factors, thereby enabling them to bind to cognate sequences in the promoter regions of genes to facilitate transcription. There are
numerous interconnections between the signal-transduction pathways. Some of these connections permit the use of the growth factor receptor
(#6)-MAPK “highway” for other receptors (e.g., 4, 5, and 7) to send mitogenic signals. For example, receptor (#4) joins in via its G protein B/y
subunits and tyrosine kinase Src; the integrin receptor (#5), whose ligands are constituents of the extracellular matrix (ECM), possibly connects via
G-protein Rho (not shown) and focal adhesion kinase (FAK); and the G-protein-coupled receptor (#7) via phospholipase C (PLC)-catalyzed formation
of second messengers and activation of protein kinase C (PKC). The mitogenic stimulus relayed along the growth factor receptor (#6)—MAPK axis
can be amplified by, e.g., the Raf-catalyzed phosphorylation of KB, which unleashes NF-kB from this inhibitory protein, and by the MAPK-catalyzed
inhibitory phosphorylation of Smad that blocks the cell-cycle arrest signal from the TGF-6 receptor (#9). Activation of protein kinases (PKC, CaMK,
and MAPK) by Ca** can also trigger mitogenic signaling. Several xenobiotics that are indicated in the figure may dysregulate the signaling network.
Some may induce cell proliferation by either activating mitogenic protein kinases (e.g., PKC) or by inhibiting inactivating proteins, such as protein
phosphatases (PTP and PP2A), GAP, or IKB. Others, e.g., inhibitors of PKC, oppose mitosis and facilitate apoptosis.
This scheme is oversimplified and tentative in several details. Virtually all components of the signaling network (e.g., G proteins, PKCs, and
MAPKs) are present in multiple, functionally different forms whose distribution may be cell specific. The pathways depicted are not equally
relevant for all cells. In addition, these pathways regulating gene expression not only determine the fate of cells, but also control certain aspects
of the ongoing cellular activity.
 CHAPTER 3 Mechanisms ofToxicity 31

TABLE 3-1 Agents acting on signaling systems for neurotransmitters and causing dysregulation of the
momentary activity of electrically excitable cells such as neurons and muscle cells.*

Receptor/Channel/Pump Agonist/Activator Antagonist/Inhibitor

Name Location Agent Effect Agent Effect

1. Acetyl-choline Skeletal muscle Nicotine Muscle fibrillation, Tubocurarine, Muscle paralysis

nicotinic and then paralysis lophotoxin
receptor
Anatoxin-a a-Bungarotoxin
Cytisine a-Cobrotoxin
Ind: ChE inhibitors a-Conotoxin
Erabutoxin b
Ind: botulinum toxin
Neurons See above Neurona! activation Pb?*, general Neuronal inhibition
anesthetics

2. Glutamate CNS neurons N-Methyl-p-aspartate Neuronal activation Phencyclidine Neuronal inhibition —

receptor — convulsion, anesthesia
neuronal injury
(“excitotoxicity”)
Kainate, domoate Ketamine Protection against
Quinolinate General anesthetics “excitotoxicity”

Quisqualate
Ind: hypoxia, HCN —
glutamate release

3. GABA, receptor CNS neurons Muscimol, Neuronal inhibition Bicuculline Neuronal activation —
Avermectins, — sedation, tremor, convulsion
Sedatives general anesthesia,
(barbiturates, coma, depression
benzodiazepines) of vital centers
General anesthetics Picrotoxin
(halothane) Pentylenetetrazole
Alcohols (ethanol)
Cyclodiene
insecticides
Lindane, TCAD
Ind: isoniazid

4. Glycine receptor CNS neurons, Avermectins (?) Inhibition of motor Strychnine Disinhibition of motor
motor neurons neurons — neurons — tetanic
paralysis convulsion
General anesthetics Ind: tetanus toxin

5. Acetylcholine Cardiac muscle Ind: ChE inhibitors Decreased heart rate Belladonna alkaloids Increased heart rate
M, muscarinic and contractility (e.g., atropine),
receptor atropine-like
drugs (e.g., TCAD)

6. Opioid receptor CNS neurons, Morphine and Neuronal inhibition Naloxone Antidotal effects in opiate
visceral congeners (é.g., — analgesia, intoxication
neurons heroin, meperidine) central respiratory
depression,
constipation, urine
retention

Ind: clonidine

7. Voltage-gated Neurons, muscle Aconitine, veratridine Neuronal activation Tetrodotoxin, Neuronal inhibition >
Na* channel cells, etc. — convulsion saxitoxin paralysis, anesthesia
Grayanotoxin [1-Conotoxin Anticonvulsive action
Batrachotoxin Local anesthetics
Scorpion toxins Phenytoin
Ciguatoxin Quinidine
DDT, pyrethroids

(Continued)
 [Se)bo UNIT 1 General Principles of Toxicology

TABLE 3-1 Agents acting on signaling systems for neurotransmitters and causing dysregulation of the
momentary activity of electrically excitable cells such as neurons and muscle cells.* (Continued)

Receptor/Channel/Pump Agonist/Activator Antagonist/Inhibitor

Name Location Agent Effect Agent Effect

8. Voltage-gated Neurons, muscle Maitotoxin (?) Neuronal/muscular w-Conotoxin Pb?* Neuronal inhibition —
Ca?* channel cell, etc. activation, cell paralysis
injury
Atrotoxin (?)
Latrotoxin (?)

9. Voltage/Ca?*- Neurons, smooth Pb?* Neuronal/muscular Ba?*, apamin Neuronal/muscular

activated K* and skeletal inhibition (bee venom), activation —
channel muscle, cardiac dendrotoxin, convulsion/spasm
muscle 20-HETE, hERG vasoconstriction,
inhibitors PMV tachycardia
(e.g., cisapride, (torsade de pointes)
terfenadine)

10. Nat,Kt-ATPase Universal Digitalis glycosides Increased cardiac

contractility,
excitability
Oleandrin, Increased neuronal
Chlordecone excitability —
tremor
\

11. Acetylcholine Smooth muscle, Ind: ChE inhibitors Smooth muscle Belladonna alkaloids Smooth muscle
M, muscarinic glands spasm (e.g., atropine) relaxation >
receptor intestinal paralysis,
decreased
salivation, decreased
perspiration
Salivation, lacrimation Atropine-like drugs
(e.g., TCAD)
Acetylcholine CNS neurons Oxotremorine Neuronal activation See above
M, muscarinic = convulsion
receptor
Ind: ChE inhibitors

12. Adrenergic a, Vascular smooth (Norepinephrine Vasoconstriction Prazosin Antidotal effects in

receptor muscle — ischemia, intoxication with
hypertension Q,-receptor agonists
Ind: cocaine,
tyramine,
amphetamine,
TCAD

13. 5-HT, receptor Smooth muscle Ergot alkaloids Vasoconstriction Ketanserine Antidotal effects in ergot
(ergotamine, — ischemia, intoxication
ergonovine) hypertension

14. Adrenergic 8, Cardiac muscle (Nor)epinephrine Increased cardiac Atenolol, metoprolol Antidotal effects in
receptor contractility and intoxication with 8,-
excitability receptor agonists
Ind: cocaine,
tyramine,
amphetamine,
TCAD

“Numbering of the signaling elements in this table corresponds to the numbering oftheir symbols in Figure
3-7. This tabulation is simplified and incomplete. Virtually all
receptors and channels listed occur in multiple forms with different sensitivity to the agents. The reader
should consult the pertinent literature for more detailed information.
CNS, central nervous system; ChE, cholinesterase; Ind, indirectly acting (i.e., by altering neurotransmitter level) ; 20-HETE,
20-hydroxy-5,8,11,14-eicosatetraenoic acid; PMV,
polymorphic ventricular; TCAD, tricyclic antidepressant.
The ? indicates there is some uncertainty regarding this action.
 CHAPTER3 Mechanisms ofToxicity 33

Alteration in Neurotransmitter Levels: Chemicals may alter syn- Dysregulation of the Activity of Other Cells—Whereas many
aptic levels of neurotransmitters by interfering with their syn- signaling mechanisms operate in nonexcitable cells, such as
thesis, storage, release, or removal from the vicinity of the exocrine secretory cells, Kupffer cells, and pancreatic beta cells,
receptor. Most pharmaceuticals employ this strategy, including disturbance ofthese processes is usually less consequential.
antidepressants, antiseizures, antipsychotics, etc.

Toxicant-Neurotransmitter Receptor Interactions: Some Toxic Alteration of Cellular Maintenance

hemicals interact directly with neurotransmitter receptors, Impairment of Internal Cellular Maintenance:
ncluding (1) agonists that associate with the ligand-binding Mechanisms of Toxic Cell Death—For survival, all cells
ite on the receptor and mimic the natural ligand, (2) antago- must synthesize endogenous molecules; assemble macromo-
nists that occupy the ligand-binding site but cannot activate lecular complexes, membranes, and cell organelles; maintain
the receptor, (3) activators, and (4) inhibitors that bind to a the intracellular environment; and produce energy for opera-
site on the receptor that is not directly involved in ligand tion. Agents that disrupt these functions jeopardize survival
binding. In the absence of other actions, agonists and activa- and may cause toxic cell death. There are three critical bio-
tors mimic, whereas antagonists and inhibitors block, the chemical disorders that chemicals inflicting cell death may
physiologic responses characteristic of endogenous ligands. initiate, namely, ATP depletion, sustained rise in intracellular
Because there are multiple types of receptors for each neu- Ca’*, and overproduction of ROS and RNS.
rotransmitter, these receptors may be affected differentially
by toxicants. Depletion of ATP—ATP plays a central role in cellular main-
tenance both as a chemical for biosynthesis and as the major
Toxicant-Signal Transducer Interactions: Many chemicals source of energy. ATP is utilized in numerous biosynthetic
alter neuronal and/or muscle activity by acting on signal- reactions, and is incorporated into cofactors as well as nucleic
transduction processes. Voltage-gated Na* channels, which acids. It is required for muscle contraction and polymerization
transduce and amplify excitatory signals generated by ligand- of the cytoskeleton, fueling cellular motility, cell division, vesic-
gated cation channels, are activated or inactivated by several ular transport, and the maintenance of cell morphology. ATP
toxins (see Table 3-1). drives ion transporters (e.g., Na*,K*-ATPase) that maintain
conditions essential for various cell functions.
Toxicant-Signal Terminator Interactions: The cellular signal Chemical energy is released by hydrolysis of ATP to ADP or
enerated by cation influx is terminated by removal of the cations AMP. The ADP is rephosphorylated in the mitochondria by
through channels or by transporters. Inhibition of cation export ATP synthase (Figure 3-8) via a process that couples oxidation
may prolong excitation. of hydrogen to water and is termed oxidative phosphorylation.

Substrates: Glucose Fattyacids O2 P; ADP

Products: |

Pyruvate Fatty acyl-CoA

it
‘ : Acetyl-CoA

= + Citrate
2 NADH + Ht
z;suited cycl

+ +
Electron 4-
A Ht @& transport chain
4Ht aa
2 ATP
‘SYN

FIGURE 3-8
° Inhibitors: > arar
ATP synthesis (oxidative phosphorylation) in mitochondria. Arrows with letters A-D point to the ultimate sites of action
of four categories of agents that interfere with oxidative phosphorylation (Table 3-2). For simplicity, this scheme does not indicate the outer
mitochondrial membrane and that protons are extruded from the matrix space along the electron transport chain at three sites. BOX = bets-
oxidation of fatty acids; e~ = electron; P; = inorganic phosphate; ANT = adenine nucleotide translocator; ATP SYN = ATP synthase (F,F,ATPase).
 34 UNIT 1 General Principles of Toxicology

Oxidative phosphorylation also requires several steps, each of cytochrome oxidase. Chemicals in class D inhibit oxidative
which can be interfered with by toxins, as described in phosphorylation by (1) direct inhibition of ATP synthase, (2)
Table 3-2. Impairment of oxidative phosphorylation is detri- interference with ADP delivery, (3) interference with inorganic
mental to cells because failure of ADP rephosphorylation phosphate delivery, and (4) deprivation of ATP synthase from its
results in the accumulation of ADP and its breakdown prod- driving force (i.e., the controlled influx of protons into the
ucts, as well as depletion of ATP. matrix space). Finally, chemicals causing mitochondrial DNA
Chemicals that impede oxidative phosphorylation are divided injury, thereby impairing synthesis of specific proteins encoded
into five groups (Figure 3-8; Table 3-2). Substances in class A by the mitochondrial genome, are listed in group E.
interfere with the delivery of hydrogen to the electron transport
chain. Class B chemicals inhibit the transfer of electrons along Sustained Rise of Intracellular Ca?*—Intracellular Ca**
the electron transport chain to oxygen. Class C agents interfere levels are highly regulated and maintained by the impermeabil-
with oxygen delivery to the terminal electron transporter, ity of the plasma membrane to Ca** and by transport

TABLE 3-2 Agents impairing mitochondrial ATP synthesis.*

A. Inhibitors of hydrogen delivery to the electron transport chain acting on/as

1. Glycolysis (critical in neurons): hypoglycemia; iodoacetate, koningic acid, and NO* at GAPDH
2. Gluconeogenesis (critical in renal tubular cells): coenzyme A depletors (see below)
3. Fatty acid oxidation (critical in cardiac muscle): hypoglycin, 4-pentenoic acid, 4-ene-valproic acid
4. Pyruvate dehydrogenase: arsenite, DCVC, p-benzoquinone
5. Citrate cycle
(a) Aconitase: fluoroacetate, ONOO-
(b) Isocitrate dehydrogenase: DCVC EN
(c) Succinate dehydrogenase: malonate, DCVC, PCBD-Cys, 2-bromohydroquinone, 3-nitropropionic acid, cis-crotonalide fungicides
6. Depletors of TPP (inhibit TPP-dependent PDH and a-KGDH): ethanol (when chronically consumed)
7. Depletors of coenzyme A (CoA)
(a) Thiol-reactive electrophiles: 4-(dimethylamino)phenol, p-benzoquinone
(b) Drugs enzymatically conjugated with CoA: salicylic acid (the metabolite of aspirin), valproic acid
8. Depletors of NADH
9. Alloxan, t-BHP, NAPBQI, fatty acid hydroperoxides, menadione
10. Activators of poly(ADP-ribose) polymerase: agents causing DNA damage (e.g., MNNG, hydrogen peroxide, ONOO-)

B. Inhibitors of electron transport acting on/as

1. Inhibitors of electron transport complexes
(a) NADH-coenzyme Q reductase (complex |): rotenone, amytal, MPP*, paraquat
(b) Coenzyme Q-cytochrome c reductase (complex III): antimycin-A, myxothiazole
(c) Cytochrome oxidase (complex IV): cyanide, hydrogen sulfide, azide, formate, *NO, phosphine (PH;)
(d) Multisite inhibitors: dinitroaniline and diphenylether herbicides, ONOO-
2. Electron acceptors: CCl,, doxorubicin, menadione, MPP+

C. Inhibitors of oxygen delivery to the electron transport chain

1. Chemicals causing respiratory paralysis: CNS depressants (e.g., opioids), convulsants

2. Chemicals impairing pulmonary gas exchange: CO,, “deep pulmonary irritants” (e.g., NO,, phosgene, perfluoroisobutene)
3. Chemicals inhibiting oxygenation of Hb: carbon monoxide, methemoglobin-forming chemicals
4. Chemicals causing ischemia: ergot alkaloids, cocaine

D. Inhibitors of ADP phosphorylation acting on/as

ATP synthase: oligomycin, cyhexatin, DDT, chlordecone

Adenine nucleotide translocator: atractyloside, DDT, free fatty acids, lysophospholipids
Phosphate transporter: N-ethylmaleimide, mersalyl, p-benzoquinone
Ra Chemicals dissipating the mitochondrial membrane potential (uncouplers)
ore
SS
ad
(a) Cationophores: pentachlorophenol, dinitrophenol-, benzonitrile-, thiadiazole herbicides, salicylate, CCCP, cationic amphiphilic drugs
(bupivacaine, perhexiline), valinomycin, gramicidin, calcimycin (A23187)
(b) Chemicals permeabilizing the mitochondrial inner membrane: PCBD-Cys, chlordecone
5. Multisite inhibitor drugs: phenformin, propofol, salicylic acid (when overdosed)

E. Chemicals causing mitochondrial DNA damage and/or impaired transcription of key mitochondrial proteins

1. Antiviral drugs: zidovudine, zalcitabine, didanosine, fialuridine

2. Antibiotics: chloramphenicol (when overdosed), linezolid
3. Ethanol (when chronically consumed)

*The ultimate sites of action of these agents are indicated in Figure 3-8.
CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCVC, dichlorovinyl-cysteine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase;
a-KGDH, a-ketoglutarate
dehydrogenase; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; MPP*, 1-methyl-4-phenylpyridinium; PCBD-Cys,
pentachlorobutadienylcysteine; PDH, pyruvate dehydrogenase;
TPP, thyamine pyrophosphate.
 CHAPTER3 Mechanisms ofToxicity 39

TABLE 3-3 Agents causing sustained elevation of There are at least three mechanisms by which sustained ele-
cytosolic Ca?*. vations in intracellular Ca** levels influence the cellular energy
balance. First, high cytoplasmic Ca** levels cause increased
A. Chemicals inducing Ca?* influx into the cytoplasm mitochondrial Ca** uptake by the Ca** “uniporter,’ which, like
|. Via ligand-gated channels in neurons ATP synthase, utilizes the inside negative mitochondrial mem-
1. Glutamate receptor agonists (“excitotoxins”): glutamate, kainate, brane potential as the driving force. Consequently, mitochon-
domoate
drial Ca** uptake dissipates the membrane potential and
2. TRPV1 receptor (capsaicin receptor) agonists: capsaicin,
resiniferatoxin inhibits the synthesis of ATP. Moreover, agents that oxidize
3. TRPA1 receptor agonists: SH-reactive electrophiles, such as mitochondrial NADH activate a transporter that extrudes
lacrimators (e.g., chlorobenzalmalonitrile), acrolein, methy| Ca’* from the matrix space. The ensuing continuous Ca**
isocyanate, phosgene, chloropicrin
Il. Via voltage-gated channels: maitotoxin (?), HO*
uptake and export (“Ca** cycling”) by the mitochondria fur-
Ill. Via“newly formed pores”: maitotoxin, amphotericin B, chlordecone, ther compromise oxidative phosphorylation.
methylmercury, alkyitins Second, an uncontrolled rise in cytoplasmic Ca** causes cell
IV. Across disrupted cell membrane injury by microfilamental dissociation. An increase of cyto-
1. Detergents: exogenous detergents, lysophospholipids, free fatty
acids plasmic Ca** causes dissociation of actin filaments from pro-
2. Hydroiytic enzymes: phospholipases in snake venoms, teins that promote anchoring of the filament to the plasma
endogenous phospholipase A, membrane, predisposing the membrane to rupture.
3. Lipid peroxidants: carbon tetrachloride Third, high Ca’* levels may lead to activation of hydrolytic
4. Cytoskeletal toxins (by inducing membrane blebbing):
cytochalasins, phalloidin enzymes that degrade proteins, phospholipids, and nucleic
V. From mitochondria acids. Many integral membrane proteins are targets for Ca’*-
1. Oxidants of intramitochondrial NADH: alloxan, t-BHP, NAPBQI, activated neutral proteases, or calpains. Indiscriminate activa-
divicine, fatty acid hydroperoxides, menadione, MPP+
tion of phospholipases by Ca** causes membrane breakdown
2. Others: phenylarsine oxide, gliotoxin, "NO, ONOO—
Vi. From the endoplasmic reticulum directly and by the generation of detergents. Activation of a
1. IP; receptor activators: y-HCH (lindane), IP; formed during Ca’**—Mg’*-dependent endonuclease causes fragmentation of
“excitotoxicity” the chromatin form of DNA.
2. Ryanodine receptor activators: 6-HCH

B. Chemicals inhibiting Ca?* export from the cytoplasm Overproduction of ROS and RNS—A number of xenobiotics
(inhibitors of Ca2*+-ATPase in cell membrane and/or can directly generate ROS and RNS, such as the redox cyclers
endoplasmic reticulum) and transition metals (Figure 3-3). Overproduction of ROS
|. Covalent binders: acetaminophen, bromobenzene, CCl,, chloro- and RNS can be secondary to intracellular hypercalcemia, as
form, DCE Ca** helps generate ROS and/or RNS by activating dehydroge-
ll. Thiol oxidants: cystamine (mixed disulfide formation), diamide,
nases in the citric acid cycle, leading to increased activity in the
t-BHP, O,° -, and HOOH generators (e.g., menadione, diquat)
Ill. Others: vanadate, Cd?*, thapsigargin (specific SERCA inhibitor) electron transport chain and increased formation of O,*~ and
IV. Chemicals impairing mitochondrial ATP synthesis (see Table 3-2) HOOH, and by activating nitric oxide synthase, which leads to
formation of ONOO-.
DCE, 1,1-dichloroethylene; t-BHP, t-butyl hydroperoxide; HCH,
hexachlorocyclohexane; MPP*, 1-methyl-4-phenylpyridinium; NAPBQI, N-acety|-p-
benzoquinoneimine; SERCA, sarco/endoplasmic reticulum calcium ATPase.
Interplay between the Primary Metabolic Disorders
Spells Cellular Disaster—The primary derailments in cel-
mechanisms that remove Ca** from the cytoplasm. Ca’* is lular biochemistry discussed above may interact and amplify
actively pumped from the cytosol across the plasma membrane each other in a number ofways:
to the extracellular space, and is also sequestered from the cyto- 1. Depletion of cellular ATP reserves deprives the endoplas-
sol into the endoplasmic reticulum and mitochondria. mic and plasma membrane Ca’* pumps of fuel, causing
Toxicants induce elevation of cytoplasmic Ca’* levels by pro- elevation of Ca** in the cytoplasm. With the influx of Ca’*
moting Ca’* influx into or inhibiting Ca’* efflux from the cyto- into the mitochondria, the mitochondrial membrane
plasm (Table 3-3). Opening of the ligand- or voltage-gated potential declines, hindering ATP synthase.
Ca?* channels or damage to the plasma membrane causes Ca** 2. Intracellular hypercalcemia facilitates formation of ROS
to move down its concentration gradient from extracellular and RNS, which oxidatively inactivates the Ca** pump
fluid to the cytoplasm. Toxicants also may increase cytosolic and exacerbates the hypercalcemia.
Ca’* inducing its leakage from the mitochondria or the endo- 3. ROS and RNS can also drain the ATP reserves. *NO is a
plasmic reticulum. They also may diminish Ca”* efflux through reversible inhibitor of cytochrome oxidase, NO* (nitroso-
inhibition of Ca** transporters or depletion of their driving nium cation, a product of *NO) inactivates glyceraldehyde
forces. Sustained elevation of intracellular Ca’* is harmful 3-phosphate dehydrogenase and impairs glycolysis, whereas
because it can result in (1) depletion of energy reserves by ONOO- irreversibly inactivates several components of the
inhibiting the ATPase used in oxidative phosphorylation, (2) electron transport chain, inhibiting cellular ATP synthesis.
dysfunction of microfilaments, (3) activation of hydrolytic 4. Furthermore, ONOO™ can induce DNA single-strand
enzymes, and (4) generation of ROS and RNS. breaks, which activate poly(ADP-ribose) polymerase
36 UNIT 1 General Principles of Toxicology

(PARP). As part of the repair strategy, activated PARP trans- Death receptor
fers multiple ADP-ribose moieties from NAD* to nuclear Lostimulation —
proteins and PARP itself. Because consumption of NAD*
severely compromises ATP synthesis (see Figure 3-8) and
resynthesis of NAD* consumes ATP, a cellular energy
deficit occurs as a major consequence of DNA damage
“a cae |
va ~ ps3stabilization "C8 jctivation |

by ONOO-.
TRO(N)S | Bax, Puma, Noxa | | Bid |
Mitochondrial Permeability Transition (MPT) and the
Worst Outcome: Necrosis— Mitochondrial Ca’* uptake,
ra = a
decreased mitochondrial membrane potential, generation of MPT/MOMP
ROS and RNS, depletion of ATP, and consequences ofthe pri- g Cyt c, Smac, AIF eD
mary metabolic disorders (e.g., accumulation of inorganic
phosphate, free fatty acids, and lysophosphatides) are all con- |
sidered as causative factors of an abrupt increase in the mito- Ina = ae
more In all
chondrial inner-membrane permeability, termed MPT. This lee ee mitochondria
is believed to be caused by the opening of a proteinaceous
pore that spans both mitochondrial membranes and is per-
eal of ae ATP
meable to solutes of 1500 Da. This opening permits free influx
mitochondria activation depletion
into the matrix space of protons, causing rapid and complete
dissipation of the membrane potential, cessation of ATP syn-
thesis, and the osmotic influx of water causing mitochondrial Cell : :
swelling. Ca’* accumulated in the matrix space effluxes Apoptosis Necrosis

through the pore, flooding the cytoplasm. Such mitochondria

are not only incapable of synthesizing ATP, but also even FIGURE 3-9 “Decision plan’
on the fate of injured cell.
waste the remaining sources because depolarization of the See the text for details. MOMP = mitochondrial outer membrane
inner membrane forces the ATP synthase to operate in the permeabilization; MPT = mitochondrial permeability transition;
reverse mode, as an ATPase, hydrolyzing ATP. Then glycolysis Puma = p53-upregulated modulator of apoptosis; RO(N)S = reactive
oxygen or nitrogen species.
may become compromised by the insufficient ATP supply to
the glycolytic enzymes that require ATP (hexokinase and
phosphofructokinase). A complete bioenergetic catastrophe
ensues in the cell if the metabolic disorders evoked by the As cyt cis the penultimate link in the mitochondrial electron
toxicant (such as those listed in Tables 3-2 and 3-3) are so transport chain, its loss will block ATP synthesis, increase for-
extensive that most or all mitochondria undergo MPT, caus- mation of O,°-, and potentially thrust the cell toward necrosis.
ing depletion of cellular ATP, and culminating in cell lysis or Simultaneously, the unleashed cyt c represents a signal or an
necrosis (see Figure 3-9). initial link in the chain of events directing the cell to the apop-
totic path (Figure 3-10). On binding, together with ATP, to an
adapter protein, cyt c can induce proteolytic cleavage of pro-
An Alternative Outcome of MPT: Apoptosis—Chemicals teins called caspases or cysteine proteases that cleave cytoplas-
that adversely affect cellular energy metabolism, Ca** homeo- mic proteins into fragments, beginning apoptosis. Some
stasis, and redox state and ultimately cause necrosis may also caspases (e.g., 2, 8, and 9) activate procaspases. These signaling
induce apoptosis. While the necrotic cell swells and lyses, the caspases carry the activation wave to the so-called effector cas-
apoptotic cell shrinks; its nuclear and cytoplasmic materials pases (e.g., 3, 6, and 7), which activate or inactivate specific cel-
condense, and then it breaks into membrane-bound fragments lular proteins. It is the hydrolysis of these specific proteins that
(apoptotic bodies) that are phagocytosed. accounts (directly or indirectly) for the morphological and
In contrast to the random sequence of multiple metabolic biochemical alterations in apoptotic cells.
defects that a cell suffers on its way to necrosis, the routes to The decisive mitochondrial events of cell death are con-
apoptosis are ordered, involving cascade-like activation of cat- trolled by the Bcl-2 family of proteins, which includes mem-
abolic processes that finally disassemble the cell. Many details bers that facilitate (e.g., Bax, Bad, and Bid) and those that
of the apoptotic pathways are presented schematically in Figure inhibit (e.g., Bcl-2 and Bcl-XL) these processes. Death-
3-10. It appears that most, if not all, chemical-induced cell promoting members can oligomerize and form pores in the
deaths will involve the mitochondria, and that MPT is a crucial mitochondrial outer membrane. By doing so, these may facili-
event. Another related event is release into the cytoplasm of tate release of cyt c and other intermembrane proapoptotic
cytochrome c (cyt c), a small hemeprotein that normally resides proteins. This release of cyt c can be caused by toxic insult of
in the mitochondrial intermembrane space attached to the sur- the mitochondria leading to the MPT or the mitochondrial
face of inner membrane. outer membrane permeabilization (MOMP) induced by Bax
 CHAPTER3 Mechanisms of Toxicity evs

Initiator caspases

FIGURE 3-10 Apoptotic pathways initiated by mitochondrial insult, nuclear DNA insult, and Fas or TNF receptor-1 stimulation. The
figure is a simplified scheme of three pathways to apoptosis. (1) Mitochondrial insult ultimately opens the permeability transition pore spanning
both mitochondrial membranes and/or causes release of cytochrome c (cyt c) from the mitochondria. Cyt c release is facilitated by Bax or Bid
proteins and opposed by Bcl-2 protein. (2) DNA insult, especially double-strand breaks, activates p53 protein, which increases the expression
of Bax (that mediates cyt c release) and the membrane receptor protein Fas. (3) Fas ligand or tumor necrosis factor binds to and activates their
respective receptor, Fas and TNF1 receptor. These ligand-bound receptors and the released cyt c interact with specific adapter proteins (i.e., FADD,
RAIDD, and Apaf-1) through which they proteolytically activate procaspases (PC) to active caspases (C). The latter in turn cleave and activate other
proteins (e.g., the precursor of Bid, P-Bid) and PC-3, a main effector procaspase. The active effector caspase-3 activates other effector procaspases
(PC-6 and PC-7). Finally, C-3, C-6, and C-7 clip specific cellular proteins, whereby apoptosis occurs. These pathways are not equally relevant in all
types of cells and other pathways, such as those employing TGF-6 as an extracellular signaling molecule and ceramide as an intracellular
signaling molecule. DFF = DNA fragmentation factor; FAK = focal adhesion kinase; PARP = poly(ADP-ribose) polymerase; SREBP = sterol
regulatory element binding protein.

and its congeners. The death-suppressing family of proteins The proapoptotic Bax and Bid proteins also represent links
can dimerize with the death-inducing counterparts and neu- whereby death programs, initiated by DNA damage in the
tralize them. Thus, the relative amount of these antagonistic nucleus or by stimulation of death receptors at the cell surface
proteins functions as a regulatory switch between cell survival (e.g., TNF-R1 and FasR), can trigger the mitochondria into the
and death. apoptotic process (Figure 3-10). DNA damage induces
38 UNIT 1 General Principles of Toxicology

stabilization and activation of p53 protein, which increases Molecular Repair

expression of the proapoptotic Bcl-2 family of proteins. DNA Damaged molecules may be repaired in different ways. Some
damage is potentially mutagenic and carcinogenic and apopto- chemical alterations, such as oxidation of protein thiols and
sis of cells possessing DNA that is damaged beyond repair is an methylation of DNA, are simply reversed. Hydrolytic removal
important self-defense against oncogenesis. Stimulation of of the molecule’s damaged unit or units and insertion ofa newly
death receptors leads to the activation of caspase 8, setting the synthesized unit or units often occur with chemically altered
caspase cascade into motion. Caspase-8 can activate Bid, DNA and peroxidized lipids. In some instances, the damaged
another member ofthe Bcl-2 family (Figure 3-10). Thus, apop- molecule is totally degraded and resynthesized.
tosis can be executed via multiple pathways; the preferred route
will depend on the initial insult as well as on the type and state
of the cell. Repair of Proteins—Thiol groups are essential for the func-
tion of numerous proteins. Oxidation of protein thiols can be
reversed by enzymatic reduction that is catalyzed by thiore-
What Determines the Form of Cell Death?—The mode of
doxin and glutaredoxin. Once oxidized, the catalytic thiol
cell death (ie. necrosis or apoptosis) is not trivial with respect
groups in these proteins are recycled by reduction with NADPH.
to the fate of surrounding cells, a topic that will be discussed
Soluble intracellular proteins are typically folded into a glob-
later. Toxicants tend to induce apoptosis at low exposure levels
ular form with their hydrophobic amino acid residues hidden
or early after exposure at high levels, whereas they cause necro-
inside, and their hydrophilic residues located externally.
sis later at high exposure levels. Recent research suggests a
Physical or chemical insults may lead to unfolding of the pro-
larger toxic insult causes necrosis rather than apoptosis because
tein (denaturation) or its aggregation. Molecular chaperones,
it incapacitates the cell such that it is unable to undergo apopto-
such as the heat-shock proteins, can prevent unfolding by
sis. Three causatively-related cellular events may lead to this
“clamping down” onto the exposed hydrophobic region and
incapacitation, namely increasing number of mitochondria
utilizing ATP hydrolysis to change that protein’s conformation.
undergoing MPT, depletion of ATP, and failed activation of
Proteins denatured beyond repair are ubiquinated multiple
caspases. When few mitochondria undergo MPT, they are
times, which targets that protein for degradation in the protea-
removed by selective autophagy and the cell survives. This
some. However, oligomerization and aggregation of damaged
autophagic mechanism can become overwhelmed and lead to
and unfolded proteins preclude the proteasome from degrad-
caspaseactivationas the degree of mitochondrial MPT increases.
ing them, and some can even trap the proteasomes and render
If all mitochondria undergo MPT, cell lysis occurs. ATP is cru-
them nonfunctional.
cial for executing the apoptotic program and depletion of ATP
can prevent caspase activation. Finally, toxicants can directly act
Repair of Lipids—Peroxidized lipids are repaired by a com-
on caspases and impede the cell’s apoptotic ability.
plex process involving a series of reductants, glutathione per-
oxidase, and glutathione reductase. NADPH is needed to
Induction of Death by Unknown Mechanisms— recycle the reductants that are oxidized in the process.
Mitochondrial integrity and intracellular Ca2+ homeostasis
are not the only mechanisms by which toxicants induce cell Repair of DNA—Despite its high reactivity with electrophiles
death. Toxicants that affect plasma membranes, lysosomal and free radicals, nuclear DNA is remarkably stable, in part
membranes, cytoskeletal components, protein phosphatase because it is packaged in a condensed form, called chromatin,
inhibitor, protein synthesis, and cholesterol-lowering drugs and because several repair mechanisms are available to correct
(statins) also lead to cell death. The exact mechanism subse- alterations. Mitochondrial DNA, however, is not condensed
quent to target damage remains unknown, however it is likely and lacks efficient repair mechanisms; therefore, mitochondrial
that cell death is mediated by the modes described above. DNA is more prone to damage. Different types of damages are
corrected by specialized mechanisms, each employing a differ-
Impairment of External Cellular Maintenance—Toxicants ent set of proteins.
may also interfere with cells that are specialized to provide sup-
port to other cells, tissues, or the whole organism. Chemicals act- Direct Repair—Certain covalent DNA modifications are
ing on the liver invoke this type of toxicity. directly reversed by enzymes such as DNA photolyase, which
cleaves adjacent pyrimidines dimerized by UV light. This
chromophore-equipped enzyme functions only in light-exposed
STEP 4—REPAIR OR DYSREPAIR cells. Minor adducts, such as methyl groups, that are attached to
the O®* position of guanine may be removed by specialized
The fourth step in the development of toxicity is inappropriate enzymes, such as O°-alkylguanine-DNA-alkyltransferase.
repair (Figure 3-1). Many toxicants alter macromolecules,
which, if not repaired, cause damage at higher levels of the bio- Excision Repair—Base excision and nucleotide excision are
logical hierarchy in the organism and influence the progression two mechanisms for removing damaged bases from DNA
of toxicity. (Chapters 8 and 9). Lesions that do not cause major distortion
 CHAPTER3 Mechanisms ofToxicity 39

of the helix are removed by base excision, in which the altered Autophagy of Damaged Cell Organelles—Cells suffering
base is recognized by a relatively substrate-specific DNA glyco- mild injury may repair themselves by removing and degrading
sylase that hydrolyzes the N-glycosidic bond, releasing the damaged components, such as organelles and protein aggre-
modified base and creating an apurinic or apyrimidinic (AP) gates, in a process called autophagy. This process is particularly
site in the DNA. The AP site is recognized by the AP endonucle- important in terminally differentiated cells, such as neurons,
ase, which hydrolyzes the phosphodiester bond adjacent to the cardiac myocytes, and skeletal myocytes, because renewal by
abasic site. After its removal, the abasic sugar is replaced with cell replication is not possible.
the correct nucleotide by a DNA polymerase and is sealed in In autophagy, an “isolation membrane” engulfs the cytoplas-
place by a DNA ligase. mic material and then encapsulates it in a double-membrane
Bulky lesions are removed by nucleotide-excision repair. An vesicle, called an autophagosome. This vesicle moves along
ATP-dependent nuclease recognizes the distorted double helix microtubules, driven by dynein motors, to the lysosome where
and excises a number of intact nucleotides on both sides of the it fuses to form an autolysosome. Contents of the autolysosome
lesion. The excised section ofthe strand is restored by insertion are degraded into amino acids, lipids, nucleosides, and carbo-
of nucleotides into the gap by DNA polymerase, using the hydrates, which are then transported to the cytosol for further
complementary strand as a template. DNA ligase then forms a metabolism.
continuous strand.
PARP appears to be an important contributor in excision
Regeneration of Damaged Axons—Peripheral neurons
repair. On base damage or single-strand break, PARP binds to
with axonal damage can regenerate their axons with the assis-
the injured DNA and becomes activated. The active PARP
tance of macrophages and Schwann cells. Macrophages remove
cleaves NAD* to use the ADP-ribose moiety ofthis cofactor for
debris by phagocytosis and produce cytokines and growth fac-
attaching long chains of polymeric ADP-ribose to nuclear pro-
tors which activate Schwann cells to proliferate and transdif-
teins. This causes the DNA to unwind, giving access to the
ferentiate from myelinating mode into growth-support mode.
repair enzymes and allowing the broken DNA to be fixed.
Schwann cells play an indispensable role in promoting axonal
regeneration by facilitating membrane construction, produc-
Nonhomologous End Joining—This process repairs DSBs that
ing neurotrophic factors, and physically guiding the axon
may be formed when two SSBs occur in close proximity, or
toward its target cell.
when DNA with SSBs undergoes replication. This repair system
In the mammalian central nervous system, axonal regrowth
directly ligates broken strands without the need for a homolo-
is prevented by growth inhibitory glycoproteins and chondroi-
gous template (as is the case with nucleotide excision repair).
tin sulfate proteoglycans produced by the oligodendrocytes
Nonhomologous end joining (NHE)J) is more error prone than
and by the scar produced by astrocytes. Although damage to
other types of DNA repair; however, it is unique in that it can
central neurons is irreversible, the large number of reserve
operate in any phase of the cell cycle. It is also the mechanism of
nerve cells can partly compensate by taking over the functions
DSB repair in terminally differentiated cells such as neurons.
of lost neurons.
Recombinational (or Postreplication) Repair—Recombina-
tional repair is a mechanism that fixes DSBs with higher fidelity
Tissue Repair
than NHEJ because it requires a template from sister chroma-
tids and, therefore, can function only after replication (in S and In tissues with cells capable of multiplying, damage is repaired
G2 phases). Recombinational repair can also fix postreplication by apoptosis or necrosis ofthe injured cells and regeneration of
gaps which may occur for a variety of reasons, such as when the tissue by proliferation.
excision ofa bulky adduct or intrastrand pyrimidine dimer fails
to occur before DNA replication begins. This is a complex pro- Apoptosis: An Active Deletion of Damaged Cells—
cess in which the two sister chromatids ultimately exchange Apoptosis initiated by cell injury can be regarded as tissue
DNA and represents the “crossing over” that occurs during repair. A cell undergoing apoptosis shrinks as its nuclear and
meiosis. Bifunctional electrophiles (e.g., nitrogen mustard- cytoplasmic materials condense, and then it breaks into
type drugs, and the cancer drug, cisplatin) produce interstrand membrane-bound fragments (apoptotic bodies) that are
cross-links that are fixed by a combination of excision and phagocytosed without inflammation. Also, apoptosis may
recombinational repair. intercept the process leading to neoplasia by eliminating the
cells with potentially mutagenic DNA damage.
Apoptosis of damaged cells may serve as a tissue restoration
Cellular Repair: A Strategy in Peripheral process only for tissues that are made up of constantly renew-
Neurons ing cells (e.g., the bone marrow, the respiratory and GI epithe-
Autophagic removal of damaged cell organelles may be viewed lium, and the epidermis of the skin), or of conditionally
as a universal mechanism of cellular repair, whereas clearance dividing cells (e.g., hepatic and renal parenchymal cells),
and regeneration of damaged axons is a mechanism specific for because the apoptotic cells can be replaced. The value of apop-
neurons. tosis as a tissue repair strategy is markedly lessened in organs
40 UNIT 1 General Principles of Toxicology

containing nonreplicating and nonreplaceable cells, such as hepatocytes). Activation of resting stellate cells is mediated
the neurons, cardiac muscle cells, and female germ cells. chiefly by two growth factors, platelet-derived growth factor
(PDGE) and transforming growth factor-3 (TGF-3), that may
Proliferation: Regeneration of Tissue—Tissues are com- be released from platelets accumulating and degranulating at
posed of various cells and the extracellular matrix. Tissue ele- sites of injury and later from the activated stellate cells them-
ments are anchored to each other by transmembrane proteins. selves. Proliferation of stellate cells is induced by the potent
Cadherins allow adjacent cells to adhere to one other, whereas mitogen PDGF, whereas TGF-£ acts on the stellate cells to stim-
connexins connect neighboring cells internally by association of ulate the synthesis of extracellular matrix components, includ-
these proteins into gap junctions. Integrins link cells to the extra- ing collagens, fibronectin, tenascin, and proteoglycans. TGF-6
cellular matrix. Therefore, repair of injured tissues involves both also plays a central role in extracellular matrix formation in
regeneration of lost cells and the extracellular matrix and reinte- other tissues.
gration of the newly formed elements into tissues and organs.
Side Reactions to Tissue Injury—In addition to mediators
Replacement of Lost Cells by Mitosis—Soon after injury, cells that aid in the replacement of lost cells and the extracellular
adjacent to the damaged area enter the cell-division cycle. matrix, resident macrophages and endothelial cells activated by
Quiescent cells residing in G, enter G, and progress to mitosis (M). cell injury also produce inflammation, altered production of
Sequential changes in gene expression occur in the cells that acute-phase protein, and generalized reactions such as fever.
are destined to divide. Early after injury, intracellular signaling
turns on, and expression of numerous genes is increased. Inflammation 4
Among these so-called immediate-early genes are those that Cells and Mediators: Alteration of the microcirculation and
code for TFs that amplify the initial gene-activation process by accumulation of inflammatory cells are largely initiated by resi-
stimulating other genes directly or through cell surface recep- dent macrophages secreting cytokines such as TNF-a and IL-1
tors and their coupled transducing networks. A few hours later, in response to tissue damage. These cytokines, in turn, stimu-
the so-called delayed-early genes are expressed whose products late neighboring stromal cells, such as the endothelial cells and
regulate the cell-division cycle. Genes for the cell cycle accelera- fibroblasts, to release mediators that induce dilation of the local
tor proteins and also genes whose products decelerate the cell microvasculature and cause permeabilization of capillaries.
cycle become temporarily overexpressed, suggesting that this Activated endothelial cells also facilitate the egress of circulat-
duality keeps tissue regeneration precisely regulated. Thus, ing leukocytes into the injured tissue by releasing chemoattrac-
genetic expression is reprogrammed so that DNA synthesis and tants and expressing cell-adhesion molecules. Subsequently a
mitosis gain priority over specialized cellular activities. stronger interaction (adhesion) is established between the
The regenerative process is probably initiated by the release of endothelial cells and leukocytes with participation of intercel-
chemical mediators from damaged cells. Nonparenchymal cells, lular adhesion molecules (e.g., ICAM-1) and leukocytes are
such as resident macrophages and endothelial cells, are recep- able to enter the tissues by crossing the endothelial layer. This is
tive to these chemical signals and produce a host of signaling facilitated by gradients of chemoattractants, including chemo-
molecules that promote and propagate the regenerative process. tactic cytokines, platelet-activating factor (PAF) and leukotri-
The cytokines tumor necrosis factor-a (TNF-a) and interleu- ene B4, that induce expression of leukocyte integrins.
kin-6 (IL-6) promote transition of the quiescent cells into cell
cycle and makes them receptive to growth factors (“priming”). Inflammation Produces Reactive Oxygen and Nitrogen Species:
Growth factors, especially the hepatocyte growth factor (HGF) Macrophages as well as leukocytes recruited to a site of injury
and transforming growth factor-a (TGF-q), initiate the pro- undergo a respiratory burst, producing free radicals and acti-
gression of the “primed” cells in the cycle toward mitosis. vated enzymes. Membrane-bound NAD(P)H oxidase, activated
Besides mitosis, cell migration also significantly contributes in both macrophages and granulocytes, produces O,*~ from
to restitution of certain tissues. In the mucosa of the GI tract, molecular oxygen, which can give rise to HO® (Figure 3-3).
cells of the residual epithelium rapidly migrate to the site of Macrophages, but not granulocytes, generate another cyto-
injury as well as elongate and thin to reestablish the continuity toxic free radical, *NO, from arginine by nitric oxide synthase:
of the surface even before this could be achieved by cell replica-
tion. Mucosal repair is dictated by growth factors and cyto- L-Arginine + O, — L-Citrulline + *NO
kines operative in tissue repair elsewhere and also by specific
peptides associated with the mucous layer of the GI tract that Subsequently, O,*~ and *NO, both of which are products of
become overexpressed at sites of mucosal injury. activated macrophages, can react with each other, yielding per-
oxynitrite anion; on reaction with carbon dioxide, this decays
Replacement of the Extracellular Matrix—The extracellular into two radicals, nitrogen dioxide and carbonate anion radical
matrix is composed of proteins, glycosaminoglycans, and the (Figure 3-3).
glycoprotein and proteoglycans glycoconjugates. In the liver, Granulocytes, but not macrophages, discharge the lysosomal
these molecules are synthesized by stellate or fat-storing cells enzyme myeloperoxidase into engulfed extracellular spaces, the
located in the space of Disse (between hepatic sinusoids and phagocytic vacuoles. Myeloperoxidase catalyzes the formation
 CHAPTER 3 Mechanisms ofToxicity 4]

of hypochlorous acid (aka bleach, HOCI) from hydrogen altered gene expression. For example, Figure 3-11 illustrates
peroxide and chloride ion: many genes that code for (1) enzymes that detoxify xenobiotics,
(2) enzymes that eliminate ROS, (3) proteins that detoxify
HOOH 4-H 'Cl- > HOH 4 HOC! heme, (4) enzymes involved in glutathione homeostasis, and (5)
transporters that pump xenobiotics and their metabolites out of
Like HOOH, HOCI can form HO*® as a result of electron cells.
transfer from Fe’* or from O,*~ to HOCI:

HOC O; <= > Oo ael= 1 HO? When Repair and Adaptation Fail
Although operating at molecular, cellular, and tissue levels,
All the above reactive chemicals, as well as the discharged repair mechanisms often fail to provide protection against
lysosomal proteases, are destructive products of inflammatory injury. DNA repair mechanisms do not have absolute fidelity,
cells. Although these chemicals exert antimicrobial activity at meaning that some lesions may be overlooked or erroneously
the site of microbial invasion, they can damage adjacent fixed. Repair fails most typically when the damage overwhelms
healthy tissues at the site of toxic injury and contribute to prop- the repair mechanisms as when necessary enzymes or cofactors
agation of tissue injury. In some cases, the chemical alone is are consumed. Sometimes the toxicant-induced injury
harmless, while the immune response it invokes is the primary adversely affects the repair process itself. Finally, some types of
cause of injury. toxic injuries cannot be repaired effectively, as occurs when
xenobiotics are covalently bound to proteins.
Altered Protein Synthesis: Acute-phase Proteins—Cytokines It is also possible that repair contributes to toxicity, as when
released from macrophages and endothelial cells of injured tis- excessive amounts of NAD* are cleaved by PARP when this
sues, IL-6, IL-1, and TNE, act on cell surface receptors to increase enzyme assists in repairing broken DNA strands, or when too
or decrease the transcriptional activity of genes encoding cer- much NAD(P)H is consumed for the repair of oxidized proteins
tain proteins, called positive and negative acute-phase proteins. and endogenous reductants. Either event can compromise oxi-
Positive acute-phase proteins may play roles in minimizing dative phosphorylation, which is also dependent on the supply
tissue injury and facilitating repair. For example, many of them of reduced cofactors (see Figure 3-8), thus causing or aggravat-
inhibit lysosomal proteases released from the injured cells and ing ATP depletion that contributes to cell injury. However,
recruited leukocytes. repair also may play an active role in toxicity. This is observed
Because negative acute-phase proteins, including albumin, after chronic tissue injury, when the repair process goes astray
several biotransformation enzymes, and membrane transport- and leads to uncontrolled proliferation instead of tissue remod-
ers, play important roles in the toxication and detoxication of eling. Such proliferation of cells may yield neoplasia, whereas
xenobiotics, the disposition and toxicity of chemicals may be overproduction of extracellular matrix results in fibrosis.
altered markedly during the acute phase of tissue injury.
Adaptation— Although adaptation mechanisms boost the
Generalized Reactions—Cytokines released from activated capacity of the organism to withstand toxicant exposure and
macrophages and endothelial cells at the site of injury also may damage, excessive exposure can overwhelm these protective
evoke neurohormonal responses. Thus, IL-1, TNF, and IL-6 responses. Toxicants may impair the adaptive process itself,
alter the temperature set point of the hypothalamus, triggering while some adaptive mechanisms may be harmful under
fever. In addition, IL-1 and IL-6 act on the pituitary to induce extreme conditions. For example, chronic inflammation, tissue
the release of ACTH, which in turn stimulates the secretion of injury, or cancer may lead to iron deficiency and anemia because
cortisol from the adrenals. This represents a negative feedback IL-6 reduces iron absorption from the GI tract.
loop because corticosteroids inhibit cytokine gene expression.

Mechanisms of Adaptation Toxicity Resulting from Inappropriate

Adaptation may be defined as a harm-induced capability of the Repair and Adaptation
organism for increased tolerance to the harm itself. It involves Like repair, dysrepair occurs at the molecular, cellular, and tis-
responses acting to preserve or regain the biological homeosta- sue levels. Some toxicities involve dysrepair at an isolated level,
sis in the face of increased harm from a noxious stimulus. Adap- such as a specific enzyme or process, or at different levels, such
tation of toxicity may result from biological changes causing as tissue necrosis, fibrosis, and chemical carcinogenesis.
(1) diminished delivery of the toxicant to the target, (2) decreased
size Or susceptibility of the target, (3) increased capacity of the Tissue Necrosis—Several mechanisms that can lead to cell
organism to repair itself, and (4) strengthened mechanisms death may involve molecular damage that is potentially revers-
to compensate the toxicant-inflicted dysfunction. Adaptation ible by repair mechanisms. Cell injury progresses toward cell
involves sensing the noxious chemical and/or the initial dam- necrosis if molecular repair mechanisms are inefficient or the
age or dysfunction, anda response that typically occurs through molecular damage is not readily reversible.
 UNIT 1 General Principles of Toxicology

SH
Electrophiles E> ‘ <a Oxidants
H H

HO-1
> Freres

Z|Dict
8k a “soD1 | | Tx.
AR |Catalase | { ferritin GGT Mrp3 | TrxR
EWI | § GPX2 I || | Mrp4 | proteosome,
CES | | Sol | |
GST | LGR ||

Tee : fncoe ue 7 1 oe
detoxication — pede bg “a synnhe
(in aes see a ee
oo

FIGURE 3-11 Signaling by Keap1/Nrf2 mediates the electrophile stress response. Normally NF-E2-related factor 2 (Nrf2) is kept
inactive and at a low intracellular level by interacting with Keap1 that promotes its proteasomal degradation by ubiquitination. Electrophiles
covalently bind to, whereas oxidants oxidize the reactive thiol groups of Keap1, causing Keap1 to release Nrf2. Alternatively, Nrf2 release may
follow its phosphorylation by protein kinases. After being released from Keap1, the active Nrf2 accumulates in the cell, translocates into the
nucleus, and forms a heterodimer with small Maf proteins to activate genes that contain electrophile response element (EpRE) in their promoter
region. These include enzymes, binding proteins, and transporters functioning in detoxication and elimination of xenobiotics, ROS, and
endogenous reactive chemicals, as well as some proteins that can repair or eliminate oxidized proteins. Induction of such proteins represents
an electrophile stress response that provides protection against a wide range of toxicants. Nrf1, a transcription factor structurally related to
Nrf2, also interacts with Keap1 and Maf proteins as well as EpRE and its role is partially overlapping with that of Nrf2. Abbreviations: AR, aldose
reductase; CES carboxylesterase; EH1, microsomal epoxide hydrolase; GCL, glutamate-cysteine ligase; GGT, gamma-glutamy| transpeptidase;
GPX2, glutathione peroxidase 2; GR, glutathione reductase; GST, glutathione S-transferase; HO-1, heme oxygenase 1; NQO1, NAD(P)H:quinone
oxidoreductase; Mrp2, Mrp3, and Mrp4, multidrug resistance protein 2, 3, and 4; SOD1, superoxide dismutase 1; Srx1, sulfiredoxin 1; UGT, UDP-
glucuronosyltransferase; Trx, thioredoxin; TrxR, thioredoxin reductase.

Progression of cell injury to tissue necrosis can be inter- The efficiency of repair is also an important determinant of
cepted by two repair mechanisms working in concert: apopto- the dose-response relationship for toxicants that cause tissue
sis and cell proliferation. Injured cells can initiate apoptosis, necrosis. Tissue necrosis is caused by a certain dose of a toxi-
which counteracts the progression of the toxic injury by pre- cant not only because that dose ensures sufficient concentra-
venting necrosis of injured cells and the consequent inflamma- tion of the ultimate toxicant at the target site to initiate injury,
tory response. but also because that quantity of toxicant causes a degree of
Another important repair process that can halt the propaga- damage sufficient to compromise repair, allowing for progres-
tion of toxic injury is proliferation of cells adjacent to the sion of the injury. Tissue necrosis occurs because the injury
injured cells. Initiated soon after cellular injury, this early cell overwhelms and disables the repair mechanisms, including
division is thought to be instrumental in the rapid and com- (1) repair of damaged molecules, (2) elimination of damaged cells
plete restoration of the injured tissue and the prevention of by apoptosis, and (3) replacement of lost cells by cell division.
necrosis. The sensitivity of a tissue to injury and the capacity of
the tissue for repair are apparently two independent variables, Fibrosis—Fibrosis is a pathologic condition characterized by
both influencing whether tissue restitution ensues with excessive deposition of an extracellular matrix of abnormal
survival or tissue necrosis occurs with death. composition and is a specific manifestation of dysrepair of the
 CHAPTER3 Mechanisms of Toxicity 43

chronically injured tissue. As discussed above, cellular injury Transient increases in the production or activity of proto-
initiates a surge in cellular proliferation and extracellular matrix oncogene proteins are required for regulated growth, as during
production, which normally ceases when the injured tissue is embryogenesis, tissue regeneration, and stimulation of cells by
remodeled. If increased production of extracellular matrix is growth factors or hormones. In contrast, permanent activation
not halted, fibrosis develops. and/or overexpression of these proteins favor neoplastic trans-
TGF-8 appears to be a major mediator of fibrogenesis. The formation. One mechanism whereby genotoxic carcinogens
increased expression of TGF-8 is a common response mediat- induce neoplastic cell transformation is by producing an acti-
ing regeneration of the extracellular matrix after an acute vating mutation of aproto-oncogene. The altered gene (called
injury. Normally, TGF-8 production ceases when repair is an oncogene) encodes a permanently active protein that forces
complete. Failure to halt TGF-8 overproduction, which leads the cell into the division cycle.
to fibrosis, could be caused by continuous injury or a defect in An example of mutational activation of an oncogene protein
the regulation of TGF-8. is that of the Ras proteins. Ras proteins are localized on the
The fibrotic action of TGF-( is due to (1) stimulation of the inner surface of the plasma membrane and function as crucial
synthesis of individual matrix components by specific target mediators in responses initiated by growth factors (see
cells and (2) inhibition of matrix degradation. Interestingly, Figure 3-7). Ras serves as a molecular switch, being active in
TGF-8 induces transcription of its own gene in target cells, the GTP-bound form and inactive in the GDP-bound form.
suggesting that the TGF-8 produced by these cells can amplify Some mutations ofthe Ras gene dramatically lower the GTPase
in an autocrine manner the production of the extracellular activity of the protein, which, in turn, locks Ras in the perma-
matrix. This positive feedback may facilitate fibrogenesis. nently active GTP-bound form. Continual, rather than signal-
Fibrosis involves not only excessive accumulation of the dependent, activation of Ras can lead eventually to
extracellular matrix, but also changes in its composition. uncontrolled proliferation and transformation.
Basement membrane components, such as collagens and lam-
inin, increase disproportionately during fibrogenesis. Mutation of Tumor Suppressor Genes: Tumor-suppressor genes
ncode proteins that inhibit the progression of cells in the division
ycle, or promote DNA repair or apoptosis upon irreparable DNA
Carcinogenesis—Chemical carcinogenesis involves inap-
damage. Some examples include cyclin-dependent protein
propriate function of various repair mechanisms, including
kinase nhibitors, TFs that transactivate genes encoding cyclin-
(1) failure of DNA repair, (2) failure of apoptosis, and (3) failure
dependent protein kinase inhibitors, and proteins that block TFs
to terminate cell proliferation.
involved in NA synthesis and cell division (Figure 3-12).
The p53 tumor-suppressor gene encodes a 53-kDa protein
Failure of DNA Repair: Mutation, the Initiating Event in with multiple functions. Acting as a TF, the p53 protein trans-
Carcinogenesis—Chemical and physical insults may induce activates genes whose products arrest the cell cycle, repair
neoplastic transformation of cells by genotoxic and nongeno- damaged DNA, or promote apoptosis. It also activates miRNA-
toxic mechanisms. Chemicals that react with DNA may cause coding genes whose products repress translation of mitogenic
damage such as adduct formation, oxidative alteration, and TFs and cell cycle accelerator proteins, and the genes that
strand breakage. If these lesions are not repaired or injured cells encode cell cycle accelerators or antiapoptotic proteins. DNA
are not eliminated, a lesion in the parental DNA strand may damage activates protein kinases to phosphorylate and stabi-
induce a heritable alteration, or mutation, in the daughter lize the p53 protein, which causes it to accumulate and either
strand during replication. The most unfortunate scenario for induce cell cycle arrest or apoptosis. In addition to aberrations
the organism occurs when the altered genes express mutant in critical protein-coding genes, damage in genes coding for
proteins that reprogram cells for multiplication. When such miRNA may also contribute to carcinogenesis.
cells undergo mitosis, their descendants also have a similar pro-
pensity for proliferation. Moreover, because enhanced cell divi- Epigenetic Mechanisms in Carcinogenesis: Inappropriate
sion increases the likelihood of mutations, these cells eventually Activation or Responsiveness of the Regulatory Region of
acquire additional mutations that may further increase their Critical Genes—Some chemicals cause cancer by reacting with
growth advantage over their normal counterparts. The final DNA and inducing a mutation, whereas others that do not damage
outcome of this process is a tumor consisting of transformed, DNA yet still induce cancer after prolonged exposure are desig-
rapidly proliferating cells. nated nongenotoxic (or epigenetic) carcinogens. Five examples
include (1) xenobiotic mitogens that promote proliferative
Mutation of Proto-oncogenes: Proto-oncogenes are highly con- signaling, (2) endogenous mitogens such as growth factors,
served genes encoding proteins that stimulate progression of (3) toxicants that cause sustained cell injury, (4) xenobiotics that
cells through the cell cycle or oppose apoptosis. The products are display differing carcinogenicity between species, and (5) ethi-
of proto-oncogenes that accelerate the cell cycle include onine and diethanolamine, which interfere with formation of the
(1) growth factors; (2) growth factor receptors; (3) intracellular endogenous methyl donor S-adenosyl-methionine (SAM).
signal transducers such as G proteins, protein kinases, cyclins, Nongenotoxic chemicals eventually influence the expression
and cyclin-dependent protein kinases; and (4) nuclear TFs. of proto-oncogenes and/or tumor suppressor genes by
44 UNIT 1 General Principles of Toxicology

Cell cycle accelerators Cell cycle decelerators

Oncogene proteins Tumor suppressor proteins
and some influencing factors and some influencing factors

ATX, BP, UV

Myc

GFs -> Ras ->( Raf )--»y 4

~cD< cdk4/6

° > Inhibition
by binding

P Inhibition by
| phosphorylation

fe Activation by
phosphorylation
Transcriptional
=. = ame # _¥v activation
cE( |DHFR || TK |} TS || POL |} E2F |} cA<« cdki >| cde25|| Overexpression
i Yr +S yy SUC” )6—hlU YUL he or activation
beees || | <M Activating
| mutation
Gi > S Gi
Inactivating
TRANSITION mutation

FIGURE 3-12 Key regulatory proteins controlling the cell-division cycle with some signaling pathways and xenobiotics affecting
them. Proteins on the left, represented by blue symbols, accelerate the cell cycle and are oncogenic if permanently active or expressed at high
level. In contrast, proteins on the right, represented by salmon symbols, decelerate or arrest the cell cycle and thus suppress oncogenesis, unless
they are inactivated (e.g., by mutation).
Accumulation of cyclin D (cD) is a crucial event in initiating the cell division cycle. cD activates cyclin-dependent protein kinases 4 and 6
(cdk4/6), which in turn phosphorylate the retinoblastoma protein (pRb) causing dissociation of pRb from transcription factor E2F. Then the
unleashed E2F is able to bind to and transactivate genes whose products are essential for DNA synthesis, such as dihydrofolate reductase (DHFR),
thymidine kinase (TK), thymidylate synthetase (TS), and DNA polymerase (POL), or are regulatory proteins, such as cyclin E (cE), cyclin A (cA),
and cyclin-dependent protein kinase 1 (cdk1), which promote further progression of the cell cycle. Expression of cD is increased, e.g., by growth
factorssignaling through Ras proteins and the MAPK pathway as well as by Wnt and Hedgehog (Hh) ligands that ultimately signal through B-cat
and Gli transcription factors, respectively. Some carcinogens, e.g., benzpyrene (BP) and reactive oxygen species (ROS), and diethylnitrosamine
(DENA) may cause mutation of the Ras or Raf gene that results in permanently active mutant Ras or Rab protein, but BP as well as TCDD may also
induce simple overexpression of normal Ras protein.
Cell cycle progression is counteracted, e.g., by pRb (which inhibits the function of E2F), by cyclin-dependent protein kinase inhibitors (such
as p15, p16, and p21), by p53 (which transactivates the p27 gene), and by ARF (also called p14 that binds to mdm2, thereby neutralizing the
antagonistic effect of mdm2 on p53). Signals evoked by DNA damage and TGF-8 will ultimately result in accumulation of p53 and p15 proteins,
respectively, and deceleration ofthe cell cycle. In contrast, mutations that disable the tumor suppressor proteins facilitate cell cycle progression
and neoplastic conversion and are common in human tumors. Aflatoxin B, (ATX), BP, and UV light cause such mutations of the p53 gene, whereas
pRb mutations occur invariably in methylcholanthrene (MC)-induced transplacental lung tumors in mice.
 CHAPTER3 Mechanisms of Toxicity 45

increasing synthesis of normal proto-oncogene proteins and/or cancer by promoting carcinogenesis initiated by genotoxic
repressing normal tumor suppressor genes. This is in contrast to agents or spontaneous DNA damage.
genotoxic chemicals, which induce the synthesis of permanently According to an emerging theory, cancers may form by
active mutant proto-oncogene proteins or permanently inactive genotoxic and/or epigenetic mechanisms in pluripotent stem
mutant tumor suppressor proteins. Secondarily, nongenotoxic cell populations. Such cells are characterized by quiescence,
carcinogens may also increase mutation of critical genes, which self-renewal, and conditional immortality, and thus would
is initiated by genotoxic agents or spontaneous DNA damage. potentially supply a lifelong, latent neoplastic population after
Spontaneous DNA damage commonly occurs in normal human carcinogenic attack. Finally, further changes in gene expres-
cells at a rate of 1 out of 10° to 10’° base pairs. Nongenotoxic car- sion may occur in these proliferating cells, making them capa-
cinogens increase the frequency of spontaneous mutations ble of invading other tissues (metastasis).
through a mitogenic effect and by inhibiting apoptosis, thereby
increasing the number of cells with DNA damage and mutations.
CONCLUSIONS
Failure of Apoptosis: Promotion of Mutation and Clonal Growth:
Preneoplastic cells, or cells with mutations, have much higher Selective or altered toxicity may be due to different or altered
apoptotic activity than do normal cells. Therefore, apoptosis (1) exposure; (2) delivery, thus resulting in a different concentra-
counteracts clonal expansion of the initiated cells and tumor tion of the ultimate toxicant at the target site; (3) target mole-
cells. Facilitation of apoptosis can induce tumor regression, cules; (4) biochemical processes triggered by the reaction of the
whereas inhibition of apoptosis is detrimental because mutations chemical with the target molecules; (5) repair at the molecular,
and clonal expansion of preneoplastic cells are facilitated. cellular, or tissue level; or (6) mechanisms suchas circulatory and
thermoregulatory reflexes by which the affected organism can
Failure to Terminate Proliferation: Promotion ofMutation, Proto- adapt to some of the toxic effects. Although a simplified scheme
oncogene Expression, and Clonal Growth: Transformation of outlines the development of toxicity (Figure 3-1), the route to
normal cells with controlled proliferative activity to malignant toxicity can be considerably more diverse and complicated. An
cells with uncontrolled proliferative activity is driven by three organism has mechanisms that (1) counteract the delivery of
major factors: (1) accumulation of genetic damage in the form toxicants, such as detoxication; (2) reverse the toxic injury, such
of mutant proto-oncogenes and mutant tumor suppressor as repair mechanisms; and (3) offset some dysfunctions, such as
genes, (2) increased transcription and/or translation of normal adaptive responses. Thus, toxicity is not an inevitable conse-
proto-oncogenes, and (3) silencing of normal tumor suppressor quence of toxicant exposure because it may be prevented,
genes at the transcriptional and/or translational level. reversed, or compensated for by such mechanisms. Toxicity
Uncontrolled proliferation results from an imbalance between develops ifthe toxicant exhausts or impairs the protective mech-
mitosis and apoptosis. anisms and/or overrides the adaptability of biological systems.
1. Enhanced mitotic activity increases the probability of
mutations. With activation of the cell-division cycle, a sub-
stantial shortening of the G1 phase occurs, and less time is BIBLIOGRAPHY
available for the repair of injured DNA before replication. Cribb AE, Peyrou M, Muruganandan S, Schneider L: The endoplas-
2. Enhanced mitotic activity may compromise DNA meth- mic reticulum in xenobiotic toxicity. Drug Metab Rev 37:405-442,
2005.
ylation, which occurs early in the postreplication period.
Giordano A: Cell Cycle Control and Dysregulation Protocols: Cyclins,
DNA cytosine methyltransferases (DNMTs) copy the
Cyclin-dependent Kinases, and Other Factors. Totowa, NJ: Humana
methylation pattern of the parental DNA strand to the Press, 2004.
daughter strand. Limitations of DNMTs by shortened Hancock JT: Cell Signalling, 3rd ed. New York: Oxford University
G2 phase or by the presence of other transacting factors Press, 2010.
might impair methylation and contribute to overexpres- Hansen JM, Go Y-M, Jones DP: Nuclear and mitochondrial compart-
sion of proto-oncogenes. mentation of oxidative stress and redox signaling. Annu Rev Phar-
3. Cell-to-cell communication through gap junctions and macol Toxicol 46:215-234, 2006.
intercellular adhesion through cadherins are temporarily Leung L, Kalgutkar AS, Obach RS: Metabolic activation in drug-
disrupted during proliferation, which contributes to the induced liver injury. Drug Metab Rev 44:18-33, 2012.
Liu X, Van Fleet T, Schnellmann RG: The role of calpain in oncotic cell
invasiveness of tumor cells.
death. Annu Rev Pharmacol Toxicol 44:349-—370, 2004.
4. Proliferation also promotes carcinogenesis through clonal
Orrenius S, Nicotera P, Zhivotovsky B: Cell death mechanisms and
expansion of the initial cells to form nodules (foci) and their implications in toxicology. Tox Sci 119:3-19, 2011.
tumors. , Pober JS, Min W, Bradley JR: Mechanisms of endothelial dysfunction,
injury and death. Annu Rev Pathol 4:71-95, 2009.
Nongenotoxic Carcinogens: Promoters of Mitosis and Inhibitors Wallace KB: Mitochondrial off targets of drug therapy. Trends Phar-
ofApoptosis: Many chemicals do not alter DNA or induce muta- macol Sci 29:361-—366, 2008.
tions yetinduce cancer after chronicadministration. Designated Yokoi T, Nakajima M: Toxicological implications of modulation of
nongenotoxic or epigenetic carcinogens, these chemicals cause gene expression by microRNAs. Tox Sci 123:1-14, 2011.
46 UNIT 1 General Principles of Toxicology

QUESTIONS

1. The severity of a toxin depends, in large part, on the con- 6. Which of the following proteins functions to prevent the
centration of the toxin at its site of action. Which of the progression of the cell cycle?
following will decrease the amount of toxin reaching its a. NF-KB.
site of action? b. MAPK.
a. absorption across the skin. Cue CREB:
excretion via the kidneys. d. c-Myc.
c. toxication. e. IKB.
d. reabsorption across the intestinal mucosa.
e. discontinuous endothelial cells of hepatic sinusoids. Which of the following would have the largest negative
impact on intracellular ATP levels?
Toxication (or metabolic activation) is the biotransfor- a. moderately decreased caloric intake.
mation of a toxin to a more toxic and reactive species. b. interference with electron delivery to the electron
Which of the following is not a reactive chemical species transport chain.
commonly formed by toxication? 9 inability to harvest ATP from glycolysis.
a. _electrophiles. d. increased synthesis of biomolecules.
b. nucleophiles. e. active cell division.
c. superoxide anions.
d. hydroxy radicals. What happens when a toxin induces elevation of cyto-
e. hydrophilic organic acids. plasmic calcium levels?
a. Mitochondrial uptake of calcium dissipates the elec-
Which of the following is not an important step in detoxi- trochemical gradient needed to synthesize ATP.
cation of chemicals? b. Formation of actin filaments increases the strength
a. formation of redox-active reactants. and integrity of the cytoskeleton.
b. reduction of hydrogen peroxide by glutathione c. It decreases the activity of intracellular proteases,
peroxidase. nucleases, and phospholipases.
c. formation of hydrogen peroxide by superoxide d. The cell becomes dormant until the calcium is
dismutase. actively pumped from the cell.
d. reduction of glutathione disulfide (GSSG) by gluta- e. The generation of reactive oxygen species slows
thione reductase (GR). because of calcium-induced decrease in activity of
e. conversion of hydrogen peroxide to water and the TCA cycle.
molecular oxygen by catalase.
Cytochrome cis an important molecule in initiating apop-
Regarding the interaction of the ultimate toxicant with its tosis in cells. All of the following regarding cytochrome c
target molecule, which of the following is false? are true EXCEPT:
a. ‘Toxins often oxidize or reduce their target molecules, a. ‘The release of cytochrome c into the cytoplasm is an
resulting in the formation of a harmful by-product. important step in apoptosis initiation.
b. The covalent binding of a toxin with its target mol- b. The loss of cytochrome c from the electron trans-
ecule permanently alters the target’s function. port chain blocks ATP synthesis by oxidative
c. The noncovalent binding of a toxin to an ion channel phosphorylation.
irreversibly inhibits ion flux through the channel. c. Loss of cytochrome c from the inner mitochondrial
d. Abstraction of hydrogen atoms from endogenous membrane results in increased formation of reactive
compounds by free radicals can result in the forma- oxygen species.
tion of DNA adducts. Bax proteins mediate cytochrome ¢ release.
e. Several toxins can act enzymatically on their specific e. Caspases are proteases that increase cytoplasmic
target proteins. levels of cytochrome c.

All of the following are common effects of toxicants on

target molecules EXCEPT:
a. blockage of neurotransmitter receptors.
b. interference with DNA replication due to adduct
formation.
c. cross-linking of endogenous molecules.
opening of ion channels.
e. mounting of an immune response.
 CHAPTER3 Mechanisms of Toxicity 47

10. All of the following regarding DNA repair are true 11. Apoptosis can serve as a tissue repair process in a number
EXCEPT: of cell types. In which of the following cell types would
a. In a lesion that does not cause a major distortion of this be a plausible mechanism of tissue repair?
the double helix, the incorrect base is cleaved and the female germ cells.
correct base is inserted in its place. gastrointestinal epithelium.
Base excision repair and nucleotide excision repair neurons.
are both dependent on a DNA polymerase and a retinal ganglion cells.
DNA ligase. oP
caocardiac muscle cells.
In nucleotide excision repair, only the adduct
is cleaved, and the gap is then filled by DNA WP, Which of the following is NOT associated with
polymerase. carcinogenesis?
Pyrimidine dimers can be cleaved and repaired a. mutation.
directly by DNA photolyase. b. normal p53 function.
Recombinational repair requires that a sister c. Ras activation.
strand serve as a template to fill in missing d. inhibition of apoptosis.
nucleotides. © DNA repair failure.
 C He sAS"P or ER

Risk Assessment
Elaine M. Faustman and Gilbert S. Omenn

INTRODUCTION AND HISTORICAL CONTEXT Statistical or Probability Distribution Models

Models Derived from Mechanistic Assumptions
DEFINITIONS
Toxicologic Enhancements of the Models

DECISION MAKING EXPOSURE ASSESSMENT

HAZARD IDENTIFICATION RISK CHARACTERIZATION

Assessing Toxicity of Chemicals—Introduction Variation in Susceptibility
Assessing Toxicity of Chemicals—Methods
INFORMATION RESOURCES
Structure/Activity Relationships (SARs)
In Vitro and Short-term Tests RISK PERCEPTION AND COMPARATIVE
Animal Bioassays ANALYSES OF RISK
Use of Epidemiologic Data in Risk Assessment
Integrating Qualitative Aspects of Risk Assessment EMERGING CONCEPTS

DOSE-RESPONSE ASSESSMENT PUBLIC HEALTH RISK MANAGEMENT

Integrating Quantitative Aspects of Risk Assessment
SUMMARY
Threshold Approaches
Nonthreshold Approaches

KEY POINTS

= Risk assessment is the systematic scientific charac- ew Risk is defined as the probability of an adverse outcome
terization of potential adverse health effects res- under specified conditions.
ulting from human exposures to hazardous agents or a Risk management refers to the process by which policy
situations. actions are chosen to control hazards.

INTRODUCTION AND analysis, and characterization ofrisks in Risk Assessment in the

HISTORICAL CONTEXT Federal Government: Managing the Process (widely known as
The Red Book). The scheme shown in Figure 4-1 provides a
Toxicologic research and toxicity testing conducted and inter- consistent framework for risk assessment across agencies with
preted by toxicologists constitute the scientific core of an bidirectional arrows showing an ideal situation where mecha-
important activity known as risk assessment for chemical expo- nistic research feeds directly into risk assessments and critical
sures. In 1983, the National Research Council detailed the steps data uncertainty drives research. Often, public policy objectives
of hazard identification, dose-response assessment, exposure require extrapolations that go far beyond the observation of

49
50 UNIT 1 General Principles of Toxicology

!
1

Laboratory and field Hazard identification _ _ Risk '

I
Development of
measurements of _ characterization !
1
regulatory options
exposures. Evaluation Does the agent cause 1

adverse effects? + What is the nature 1 + Control

of exposed populations 1
+ Structure activity analysis and estimated 1 - Substitute
and observation of 1
+ In vitro tests incidence of adverse «Inform
adverse effects. !

+ Animal bioassays effects ina given :

+ Epidemiology population? 1
!
+ How robust is the 1 =
| Dose-response assessment evidence? Evaluation of public
New mechanistic - How certain is the ; health, economic,
What is the relationship between evaluation? : social, political
oer iad ae a dose and response? f context for risk
- Are susceptible
: Mi + Susceptibility 1
management options
populations 1
I
Age characterized? I
Gene-environment «ls there a relevant
!
— Pere !
mode ofaction? 1
per EoL use assessment I
!
1
What types, levels, and I
Vv genomic : 1
News duration of exposures 1
information
are experienced or
anticipated?
Policy decisions
and actions

FIGURE 4-1 Risk assessment/risk management framework. This framework shows in blue the four key steps of risk assessment: hazard
identification, dose-response assessment, exposure assessment, and risk characterization. It shows an interactive, two-way process where
research needs from the risk assessment process drive new research, and new research findings modify risk assessment outcomes. (Adapted with
permission from Risk Assessment in the Federal Government: Managing the Process, Washington, DC: National Academies Press; 1983.)

actual effects and reflect different tolerances for risks, generat- assessment of the exposures, host susceptibility factors, and
ing controversy. potential magnitude of the hazard—and then a description of
A comprehensive framework that applies two crucial con- the uncertainties in the estimates and conclusions. The objec-
cepts: (1) putting each environmental problem or issue into tives of risk assessment are outlined in Table 4-1.
public health and/or ecological context and (2) proactively The phrase characterization of risk reflects the combination
engaging the relevant stakeholders, affected or potentially of qualitative and quantitative analyses. Unfortunately, many
affected community groups, from the very beginning of the users tend to equate risk assessment with quantitative risk
six-stage process shown in Figure 4-2. Particular exposures assessment, generating a number for an overly precise risk esti-
and potential health effects must be evaluated across sources mate, while ignoring crucial information about the uncertain-
and exposure pathways and in light of multiple end points, and ties of risk assessment, mode of action (MOA), and type of
not the current general approach of evaluating one chemical in effect across species or context.
one environmental medium (air, water, soil, food, and prod- Risk management refers to the process by which policy
ucts) for one health effect at a time. actions are chosen to control hazards identified in the risk
assessment/risk characterization stage of the framework
DEFINITIONS (Figure 4-2). Risk managers consider scientific evidence and
risk estimates—along with statutory, engineering, economic,
Risk assessment is the systematic scientific evaluation of poten- social, and political factors—in evaluating alternative options
tial adverse health effects resulting from human exposures to and choosing among those options.
hazardous agents or situations. Risk is defined as the probability Risk communication is the challenging process of making
of an adverse outcome based on the exposure and potency of the risk assessment and risk management information compre-
hazardous agent(s). The term hazard refers to intrinsic toxic hensible to community groups, lawyers, local elected officials,
properties, whereas exposure becomes an essential consider- judges, business people, labor, environmentalists, ete. A cru-
ation along with hazard for risk determination. Risk assessment cial, too-often neglected requirement for communication is
requires qualitative information about the strength of the evi- listening to the fears, perceptions, priorities, and proposed
dence and the nature of the outcomes—as well as quantitative remedies of these “stakeholders.”
 CHAPTER4 Risk Assessment mil

TABLE 4-1 Objectives of risk assessment.

1. Protect human and ecological health

Toxic substances

2. Balance risks and benefits

Drugs
Pesticides

3. Set target levels of risk

Food contaminants
Water pollutants

Engage 4. Set priorities for program activities

Stakeholders Regulatory agencies
Manufacturers
Environmental/consumer organizations

5. Estimate residual risks and extent of risk reduction after steps are
taken to reduce risks

attitudes toward local polluters, other responsible parties, and

relevant government agencies can greatly influence the com-
munication process and the choices for risk management.

HAZARD IDENTIFICATION
FIGURE 4-2 Risk management framework for environmental Assessing Toxicity of Chemicals—
health from the U.S. Commission on Risk Assessment and Risk
Management. The framework comprises six stages: (1) formulating
Introduction
the problem in a broad public health context, (2) analyzing In order to assess toxicity of chemicals, information from four
risks, (3) defining options, (4) making risk-reduction decisions, types of studies is used: structure-activity relationships (SAR), in
(5) implementing those actions, and (6) evaluating the effectiveness vitro or short-term studies, in vivo animal bioassays, and infor-
of the taken actions. Interactions with stakeholders are critical and mation from human epidemiologic studies. In many cases, tox-
thus have been put at the center of the framework. icity information for chemicals is limited; however, recent efforts
to mitigate this gap in understanding have been successful.

DECISION MAKING Assessing Toxicity of Chemicals—Methods

Risk management decisions are reached under diverse statutes Structure/Activity Relationships (SARs)—Given the cost
in the United States and many other countries. Some statutes of $2 to $4 million and the 3 to 5 years required for testing a
specify reliance on risk alone, whereas others require a balanc- single chemical in a lifetime rodent carcinogenicity bioassay,
ing of risks and benefits of the product or activity (Table 4-1). initial decisions on whether to continue development of a
Risk assessments provide a valuable framework for priority set- chemical, submit a premanufacturing notice, or require addi-
ting within regulatory and health agencies, in the chemical tional testing may be based largely on SARs and limited short-
development process within companies, and in resource alloca- term assays. A chemical’s structure, solubility, stability, pH
tion by environmental organizations. Currently, there are sig- sensitivity, electrophilicity, volatility, and chemical reactivity
nificant efforts toward a global harmonization of testing can be important information for hazard identification.
protocols and the assessment of risks and standards. SARs have been used for assessment of complex mixtures of
A major challenge for risk assessment, risk communication, structurally related compounds. However, it is difficult to pre-
and risk management is to work across disciplines to demon- dict activity across chemical classes and especially across
strate the biological plausibility and clinical significance of the multiple toxic end points using a single biological response.
conclusions from studies of chemicals thought to have potential Pharmaceutical companies are now using computerized com-
adverse effects. Biomarkers of exposure, effect, or individual binatorial chemistry and three-dimensional (3D) molecular
susceptibility can link the presence of a chemical in various modeling approaches to design new drugs (ligands) that can
environmental compartments to specific sites of action in target sterically fit into the “receptors of interest.” However, comput-
organs and to host responses. Individual behavioral and social erized SAR methods have given disappointing results because
risk factors may be critically important to both the characteriza- it is rare for environmental pollutants to exhibit selective
tion of risk and the reduction of risk. Finally, public and media ligand-receptor binding.
nni) UNIT 1 General Principles of Toxicology

In Vitro and Short-term Tests— The next approach for haz- Lifetime bioassays have been enhanced with the collection of
ard identification comprises using tests ranging from in vitro additional mechanistic data and with the assessment of multi-
bacterial mutation assays to more elaborate short-term tests ple noncancer end points. It is feasible and desirable to inte-
such as skin-painting studies in mice or altered rat liver-foci grate such information together with data from mechanistically
assays conducted in vivo, as well as other assays that evaluate oriented short-term tests and biomarker and genetic studies in
developmental, reproductive, neuro-, and immunotoxicity. epidemiology. Such approaches may allow for an extension of
Short-term assay validation and application is particularly biologically observable phenomena to doses lower than those
important to risk assessment because such assays can provide leading to frank tumor development and help to address the
information about mechanisms of effects while being faster issues of extrapolation over multiple orders of magnitude to
and less expensive than lifetime bioassays. Validation requires predict response at environmentally relevant doses.
determination of their sensitivity (ability to identify true car- In an attempt to improve the prediction of cancer risk to
cinogens), specificity (ability to recognize noncarcinogens as humans, transgenic mouse models have been developed as
noncarcinogens), and predictive value for the toxic end point possible alternatives to the standard 2-year cancer bioassay. By
under evaluation. Considerable effort to improve the utility of using mice that incorporate or eliminate a gene that is linked to
these tests is continually expended due to their value in provid- human cancer, these transgenic models have the power to
ing chemical-specific mechanistic information. improve the characterization of key cellular processes and the
mode of action of toxicological responses. It is suggested that
Animal Bioassays— Animal bioassay data are key compo- these models currently should not replace the 2-year assay, but
nents of the hazard identification process. A basic premise of should be used in conjunction with other types ofdata to assist
risk assessment is that chemicals that cause tumors in animals in the interpretation of additional toxicological and mechanis-
can cause tumors in humans. All human carcinogens that have tic evidence.
been adequately tested in animals produce positive results in at
least one animal model. Although this association cannot Use of Epidemiologic Data in Risk Assessment— The
establish that all agents and mixtures that cause cancer in exper- most convincing line of evidence for human risk is a well-
imental animals also cause cancer in humans, nevertheless, in conducted epidemiologic study in which a positive association
the absence of adequate data on humans, it is biologically plau- between exposure and disease has been observed. Table 4-2
sible and prudent to regard agents and mixtures for which there shows examples of epidemiologic study designs and provides
is sufficient evidence of carcinogenicity in experimental ani- clues on types of outcomes and exposures evaluated. There are
mals as if they presented a carcinogenic risk to humans—a important inherent limitations in epidemiologic studies. When
reflection of the “precautionary principle.” In general, the most the study is exploratory, hypotheses are often weak. Exposure
appropriate rodent bioassays are those that test exposure path- estimates are often crude and retrospective, especially for con-
ways of most relevance to predicted or known human exposure ditions with long latency before clinical manifestations appear.
pathways. Bioassays for reproductive and developmental toxic- Generally, there are multiple exposures, especially when a life-
ity and other noncancer end points have a similar rationale. time is considered. There is always a trade-off between detailed
Consistent features in the design of standard cancer bioas- information on relatively few persons and very limited infor-
says include testing in two species and both sexes, with mation on large numbers of persons. Contributions from life-
50 animals per dose group and near-lifetime exposure. style factors, such as smoking and diet, are a challenge to sort
Important choices include the strains of rats and mice, the out. Humans are highly outbred, so the method must consider
number of doses, and dose levels (typically 90%, 50%, and 10% to variation in susceptibility among those who are exposed.
25% of the maximally tolerated dose [MTD]), and the details Nevertheless, human epidemiology studies provide very
of the required histopathology (number of organs to be exam- useful information for hazard identification and sometimes
ined, choice of interim sacrifice pathology, etc.). Positive evi- quantitative information for data characterization. Three
dence of chemical carcinogenicity can include increases in major types of epidemiology study designs are available: cross-
number of tumors at a particular organ site, induction of rare sectional studies, cohort studies, and case-control studies
tumors, earlier induction (shorter latency) of commonly (Table 4—2). Cross-sectional studies survey groups of humans
observed tumors, and/or increases in the total number of to identify risk factors (exposure) and disease but are not use-
observed tumors. ful for establishing cause and effect. Cohort studies evaluate
Critical problems exist in using the hazard identification individuals selected on the basis of their exposure to an agent
data from rodent bioassays for quantitative risk assessments. under study. These prospective studies monitor over time indi-
This is because of the limited dose-response data available viduals who initially are disease-free to determine the rates at
from these rodent bioassays and nonexistent response infor- which they develop disease. In case-control studies, subjects
mation for environmentally relevant exposures. Results thus are selected on the basis of disease status: disease cases and
have traditionally been extrapolated from a dose-response matched cases of disease-free individuals. Exposure histories
curve in the 10% to 100% biologically observable tumor of the two groups are compared to determine key consistent
response range down to 10~° risk estimates (upper confidence features in their exposure histories. All case-control studies are
limit) or to a benchmark or reference dose-related risk. retrospective studies.
 CHAPTER4 Risk Assessment 33

TABLE 4-2 Attributes of three types of epidemiologic study designs.

Type of Study
Methodological
Attributes Cohort y - Case-Control _ Cross-sectional

Initial classification Exposure—nonexposure Disease—-nondisease Either one

Time sequence Prospective Retrospective Present time

Sample composition Nondiseased individuals Cases and controls Survivors

Comparison Proportion of exposed with disease Proportion of cases with exposure Either one

Rates Incidence Fractional (percent) Prevalence

Risk index Relative risk—attributable risk Relative odds Prevalence

Advantages Lack of bias in exposure, yields Inexpensive, small number of subjects, Quick results
rates of incidence and risk rapid results, suitable for rare diseases,
no attrition

Disadvantages Large number of subjects required, Incomplete information, biased recall, Cannot establish causation
long follow-up, attrition, change in problem in selecting control and matching, (antecedent consequence),
time of criteria and methods, costly, yields only relative risk—cannot establish population of survivors,
inadequate for rare diseases causation, population of survivors inadequate for rare diseases

Epidemiologic findings are judged by the following criteria: determination of the adequacy of the experiments to consistently
strength of association, consistency of observations (reproduc- detect the adverse end points of interest. Many agencies use simi-
ibility in time and space), specificity (uniqueness in quality or lar evidence classification for both animal and human studies.
quantity of response), appropriateness of temporal relationship These classifications include levels of sufficient, limited, inade-
(did the exposure precede responses?), dose-responsiveness, quate, no evidence, or evidence suggesting lack of carcinogenic-
biological plausibility and coherence, verification, and analogy ity. An overall weight-of-evidence approach to carcinogenicity
(biological extrapolation). In addition, epidemiologic study uses these evidence classifications, and considers the quality and
designs should be evaluated for their power of detection, quantity of data as well as any underlying assumptions.
appropriateness of outcomes, verification of exposure assess-
ments, completeness of assessing confounding factors, and
general applicability of the outcomes to other populations at DOSE-RESPONSE ASSESSMENT
risk. Power of detection is calculated using study size, variabil-
ity, accepted detection limits for end points under study, and a integrating Quantitative Aspects
specified significance level. of Risk Assessment
Recent advances from the human genome project, increased Quantitative considerations in risk assessment include dose-
sophistication of molecular biomarkers, and improved mecha- response assessment, exposure assessment, variation in suscep-
nistic bases for epidemiologic hypotheses have allowed epi- tibility, and characterization of uncertainty.
demiologists to expand our understanding of biological The fundamental basis of the quantitative relationships
plausibility and clinical relevance. “Molecular epidemiology” between exposure to an agent and the incidence ofan adverse
with improved molecular biomarkers of exposure, effect, and response is the dose-response assessment. Analysis of dose-
susceptibility has allowed investigators to more effectively link response relationships must start with the determination of the
molecular events in the causative disease pathway. The range of critical effects to be quantitatively evaluated. It is usual practice to
biomarkers has grown dramatically and includes identification choose the data sets with adverse effects occurring at the lowest
of single nucleotide polymorphisms (SNPs), genomic profil- levels of exposure from studies using the most relevant exposure
ing, transcriptome analysis, and proteomic analysis. routes. The “critical” adverse effect is defined as the significant
adverse biological effect that occurs at the lowest exposure level.

Integrating Qualitative Aspects Threshold Approaches—Threshold dose-response rela-

of Risk Assessment tionship characterization includes identification of“no or low-
Qualitative assessment of hazard information should include est observed adverse effect levels’ (NOAELs or LOAELs). On
consideration of the consistency and concordance of findings, the dose-response curve illustrated in Figure 4-3, the thresh-
including a determination of the consistency of the toxicologi- old, indicated as T, represents the dose below which no addi-
cal findings across species and target organs, an evaluation of tional increase in response is observed. The NOAEL is identified
consistency across duplicate experimental conditions, and a as the highest nonstatistically significant dose tested; in this
54 UNIT 1 General Principles of Toxicology

an agent that is assumed to be without adverse health impact in

humans. ADI values may be defined as the daily intake of chem-
ical during an entire lifetime, which appears to be without
appreciable risk on the basis of all known facts at that time. RfDs
and ADI values typically are calculated from NOAEL values by
dividing by uncertainty (UF) and/or modifying factors (MF):

_ NOAEL
UF x MF
Response
%
_ NOAEL
AD) =a
UF X MF

Tolerable daily intakes (TDIs) can be used to describe

intakes for chemicals that are not “acceptable” but are “tolera-
ble” as they are below levels thought to cause adverse health
ey ilae s 3 4 5
effects. These are calculated in a manner similar to ADI. In
Dose (mg/kg BW) principle, dividing by these factors allows for interspecies
FIGURE 4-3 Dose-response curve. This figure is designed to (animal-to-human) and intraspecies (human-to-human)
illustrate a typical dose-response curve with points E to | indicating variability with default values of 10 each. An additional UF can
the biologically determined responses. Statistical significance of be used to account for experimental inadequacies—e.g., to
these responses is indicated with a“*” symbol. The threshold is extrapolate from short-exposure-duration studies to a
shown by T, a dose below which no change in biological response situation more relevant for chronic study or to account for
occurs. Point E represents the point of departure (POD), the dose inadequate numbers of animals or other experimental limita-
near the lower end of the observed dose-response range, below tions. If only a LOAEL value is available, then an additional
which extrapolation to lower doses is necessary. Point F is the highest
10-fold factor commonly is used to arrive at a value more com-
nonstatistical significant response point; hence, it is the “no observed
parable to a NOAEL. Traditionally, a safety factor of 100 would
adverse effect level” (NOAEL) for this example. Point G is the “lowest
observed adverse effect level” (LOAEL) for this example. Curves A to D
be used for RfD calculations to extrapolate from a well-
show some options for extrapolating the dose-response relationship conducted animal bioassay (10-fold factor animal-to-human)
below the range of biologically observed data points and POD. and to account for human variability in response (10-fold
factor human-to-human variability).
MF can be used to adjust the UF if data on mechanisms,
example it is point F, at 2mg/kg body weight. Point G is the pharmacokinetics, or relevance of the animal response to
LOAEL (~2.3 mg/kg body weight), as it is the lowest dose tested human risk justify such modification.
with a statistically significant effect. Lines A to D represent pos- Recent efforts have focused on using data-derived and
sible extrapolations below the point of departure (POD), which chemical-specific adjustment factors to replace the 10-fold UF
is represented on this figure as a square and is labeled as traditionally used in calculating RfDs and ADIs. Such efforts
point E. POD is used to specify the estimated dose near the have included reviewing the human pharmacologic literature
lower end of the observed dose range, below which extrapola- from published clinical trials and developing human variabil-
tion to lower exposures is necessary. ity databases for a large range of exposures and clinical condi-
In general, animal bioassays are constructed with sufficient tions. Intra- and interspecies UF have two components:
numbers of animals to biological responses at the 10% response toxicokinetic and toxicodynamic aspects; Figure 4-4 shows
range. Significance usually refers to both biological and statisti- these distinctions. This approach provides a structure for
cal criteria and is dependent on the number of dose levels tested, incorporating scientific information on specific aspects of the
the number of animals tested at each dose, and background overall toxicologic process into the RfD calculations; thus, rel-
incidence of the adverse response in the nonexposed control evant data can replace a portion of the overall “uncertainty”
groups. Ihe NOAEL should not be perceived as risk-free. surrounding these extrapolations.
As described in Chapter 2, approaches for characterizing NOAEL values have also been utilized for risk assessment by
dose-response relationships include identification of effect evaluating a “margin of exposure” (MOE), where the ratio of
levels such as LD., (dose producing 50% lethality), LC; (con- the NOAEL determined in animals and expressed as mg/kg
centration producing 50% lethality), ED,, (dose producing per day is compared with the level to which a human may be
10% response), as well as NOAELs. exposed. Low values of MOE indicate that the human levels of
NOAELs have traditionally served as the basis for risk assess- exposure are close to levels for the NOAEL in animals. Unlike
ment calculations, such as reference doses (RfDs) or acceptable R£D and R{C, there is usually no factor included in this calcula-
daily intake (ADI) values. RfDs or concentrations (RfCs) are tion for differences in human or animal susceptibility or
estimates of a daily exposure (oral or inhalation, respectively) to animal-to-human extrapolation. Thus, MOE values of less
 CHAPTER 4 Risk Assessment 55

Species Human
differences variability

FIGURE 4-4 Toxicokinetic (TK) and toxicodynamic (TD) considerations inherent in interspecies and interindividual extrapolations.
Toxicokinetics refers to the processes of absorption, distribution, elimination, and metabolism of a toxicant. Toxicodynamics refers to the actions
and interactions of the toxicant within the organism and describes processes at organ, tissue, cellular, and molecular levels. This figure shows
how uncertainty in extrapolation both across and within species can be considered as being due to two key factors: a kinetic component and a
dynamic component. Refer to the text for detailed explanations.

than 100 have been used by regulatory agencies as flags for which experimentally observed data are available, the choice of
requiring further evaluation. models to generate curves in this region has received lots of
The NOAEL approach has been criticized on several points, attention. For nonthreshold responses, methods for dose-
including that (1) the NOAEL must, by definition, be one of the response assessments have also utilized models for extrapola-
experimental doses tested; and (2) once this is identified, tion to de minimus (10~‘ to 10~°) risk levels at very low doses,
the rest of the dose-response curve is ignored. Because ofthese far below the biologically observed response range and far
limitations, an alternative to the NOAEL approach, the bench- below the effect levels evaluated for threshold responses.
mark dose (BMD) method, was proposed. In this approach,
the dose-response is modeled and the lower confidence bound Statistical or Probability Distribution Models—Two
for a dose at a specified response level (benchmark response general types of dose-response models exist: statistical (or
[BMR]) is calculated. The BMR is usually specified at 1%, 5%, probability distribution models) and mechanistic models.
or 10%. The BMD, (with x representing the percent BMR) is The distribution models are based on the assumption that
used as an alternative to the NOAEL value for reference dose each individual has a tolerance level for a test agent and that
calculations. Thus the RfD would be: this response level is a variable following a specific probability
distribution function. These responses can be modeled using
BMD, a cumulative dose-response function. However, extrapola-
Rip ==
UF
x ME tion of the experimental data from 50% response levels to a
“safe,” “acceptable,” or “de minimus” level of exposure—e.g.,
The proposed values to be used for the UF and MF for BMDs one in a million risk above background—illustrates the
can range from the same factors as for the NOAEL to lower huge gap between scientific observations and highly protec-
values due to increased confidence in the response level and tive risk limits (sometimes called virtually safe doses, or those
increased recognition of experimental variability owing to use corresponding to a 95% upper confidence limit on adverse
of a lower confidence bound on dose. response rates).
Advantages of the BMD approach can include (1) the ability
to take into account the full dose-response curve; (2) the inclu- Models Derived from Mechanistic Assumptions—
sion of ameasure of variability (confidence limit); and (3) the This modeling approach designs a mathematical equation
use of a consistent BMR level for RfD calculations across stud- to describe dose-response relationships that are consistent
ies. Obviously, limitations in the animal bioassays in regard to with postulated biological mechanisms of response. These
minimal test doses for evaluation, shallow dose-responses, models are based on the idea that a response (toxic effect) in
and use of study designs with widely spaced test doses will limit a particular biological unit (animal or human) is the result of
the utility of these assays for any type of quantitative assess- the random occurrence of one or more biological events
ments, whether NOAEL- or BMD-based approaches. (stochastic events).
Radiation research has spawned a series of “hit models”
Nonthreshold Approaches—As Figure 4-3 shows, numer- for cancer modeling, where a hit is defined as a critical cellu-
ous dose-response curves can be proposed in the low-dose lar event that must occur before a toxic effect is produced.
region of the dose-response curve if a threshold assumption is The simplest mechanistic model is the one-hit (one-stage)
not made. Because the risk assessor generally needs to extra- linear model in which only one hit or critical cellular inter-
polate beyond the region of the dose-response curve for action is required for a cell to be altered. As theories of
56 UNIT 1 General Principles of Toxicology

cancer have grown in complexity, multi-hit models have RISK CHARACTERIZATION

been developed that can describe hypothesized single-target
multi-hit events, as well as multi-target, multi-hit events in Variation in Susceptibility
carcinogenesis. Toxicology has been slow to recognize the marked variation
among humans. Generally, assay results and toxicokinetic
Toxicologic Enhancements of the Models— Three exem- modeling utilize means and standard deviations to measure
plary areas of research that have improved the models used in variation, or even standard errors of the mean, thereby ignoring
risk extrapolation are time to tumor information, physiologi- variability in response due to differences in age, sex, health sta-
cally based toxicokinetic modeling (described in Chapter 7), tus, and genetics.
and biologically based dose-response (BBDR) modeling. The One key challenge for risk assessment will be interpretation
BBDR model aims to make the generalized mechanistic mod- and linking of observations from highly sensitive molecular
els discussed in the previous section more clearly reflect spe- and genome-based methods with the overall process of toxic-
cific biological processes. Measured rates are incorporated into ity. Biomarkers of early effects, like. frank clinical pathology,
the mechanistic equations to replace default or computer- arise as a function of exposure, response, and time. Early, sub-
generated values. tle, and possibly reversible effects can generally be distin-
Development of BBDR models for end points other than guished from irreversible disease states.
cancer is limited; however, several approaches have been The challenge for interpretation of early and highly sensitive
explored in developmental toxicity utilizing mode of action response biomarkers is made clear in the analysis of data from
information on cell cycle kinetics, enzyme activity, litter effects, gene expression arrays. Because oursrelatively routine ability to
and cytotoxicity as critical end points. Approaches have been monitor gene responses has grown exponentially in the last
proposed that link pregnancy-specific toxicokinetic models decade, the need for toxicologists to interpret such observa-
with temporally sensitive toxicodynamic models for develop- tions for risk assessment and the overall process of toxicity has
mental impacts. Unfortunately, the lack of specific, quantita- increased with equal or greater intensity.
tive biological information for most toxicants and for most end Microarray analysis for risk assessment requires sophisti-
points limits study and utilization of these models. cated analyses to arrive at a functional interpretation and link-
age to a conventional toxicologic end point. Because of the vast
number of measured responses with gene expression arrays,
EXPOSURE ASSESSMENT pattern analysis techniques are being used. The extensive data-
bases across chemical classes, pathological conditions, and
The primary objectives of exposure assessment are to deter- stages of disease progression that are essential for these analy-
mine source, type, magnitude, and duration of contact with the ses are being developed.
agent ofinterest. Obviously, a critical element ofthe risk assess-
ment process requires recognition that hazard does not occur in
the absence of exposure. However, exposure data are frequently INFORMATION RESOURCES
identified as the key area of uncertainty in overall risk determi-
nation. The primary goal of using exposure information in Though numerous information resources are available for risk
quantitative risk assessment is not only to determine the type assessment, a few are listed below in order to provide the reader
and amount of total exposure, but also to find out specifically with examples of risk assessment resources and databases. The
how much may be reaching target tissues. A key step in making Toxicology Data Network (TOXNET) from the National
an exposure assessment is determining what exposure Library of Medicine (http://toxnet.nlm.nih.gov/) provides
pathways are relevant for the risk scenario under development. access to databases on toxicology, hazardous chemicals, and
The subsequent steps entail quantitation of each pathway related areas. These information sources vary in the included
identified as a potentially relevant exposure and then summa- level of assessment, ranging from just listings of scientific
rizing these pathway-specific exposures for calculation of references without comment to extensive peer-reviewed risk
overall exposure. assessment information. The World Health Organization
Additional considerations for exposure assessments include (http://who.int/) provides chemical-specific information through
how time and duration of exposures are evaluated in risk the International Programme on Chemical Safety (http://who.
assessments. In general, estimates for cancer risk use averages int/pcs/IPCS/index.htm) criteria documents and health and
over a lifetime. In a few cases, short-term exposure limits safety documents. The International Agency for Research on
(STELs) are required and characterization of brief but high lev- Cancer (IARC) provides data on specific classes of carcinogens
els of exposure is significant. In these cases exposures are not as well as individual agents. The National Institute of
averaged over the lifetime and the effects of high, short- Environmental Health Sciences (NIEHS) National Toxicology
term doses are estimated. With developmental toxicity, a Program provides technical reports on the compounds tested as
single exposure can be sufficient to produce an adverse a part of this national program (http://ntp.niehs.nih.gov/).
developmental effect if exposures occur during a window of Recently, new toxicogenomic databases that identify and, in
developmental susceptibility; thus, daily doses are used, rather some cases, provide characterization of chemicals have
than lifetime weighted averages. become available. The National Center for Biotechnology
 CHAPTER4 Risk Assessment Sy

Information (NCBI) provides access to an enormous set of stimulating constructive risk communication and evaluating
biomedical and genomic information which can be valuable potential risk management options. In a classic study, students,
for risk assessment, and they have worked to incorporate League of Women Voters members, active club members, and
toxicologically relevant end points. ACToR (http://actor.epa. scientific experts were asked to rank 30 activities or agents in
gov/actor/faces/ACToRHome.jsp), the EPA’s online database order of their annual contribution to deaths. Club members
on chemical toxicity data and potential chemical risks to ranked pesticides, spray cans, and nuclear power as safer than did
human health and the environment, is another useful resource other lay persons. Students ranked contraceptives and food pre-
for risk assessments. The Comparative Toxicogenomics Database servatives as riskier and mountain climbing as safer than did oth-
(http://ctd.mdibl.org/) includes data describing cross-species ers. Experts ranked electric power, surgery, swimming, and X-rays
chemical-gene-protein interactions and chemical-gene- as more risky and nuclear power and police workas less risky than
disease relationships which illuminate molecular mechanisms did lay persons. There are also group differences in perceptions of
underlying variable susceptibility and environmentally risk from chemicals among toxicologists, correlated with their
induced diseases. Although these databases provide useful employment in industry, academia, or government.
hazard identification and mechanistic information, there is lit- Psychological factors such as dread, perceived uncontrolla-
tle emphasis on exposure data. bility, and involuntary exposure interact with factors that rep-
resent the extent to which a hazard is familiar, observable, and
“essential” for daily living. Figure 4-5 presents a grid on the
RISK PERCEPTION AND COMPARATIVE parameters controllable/uncontrollable and observable/not
ANALYSES OF RISK observable for a large number of risky activities; for each of the
two paired main factors, highly correlated factors are described
Individuals respond very differently to information about haz- in the boxes.
ardous situations and products, as do communities and whole Public demand for government regulations often focuses on
societies. Understanding these behavioral responses is critical in involuntary exposures (especially in the food supply, drinking

@ DNA technology
Microwave ovens @
@ Electric fields
Water fluoridation @ @ DES
Saccharin @ Nitrites @
Water chlorination e Polyvinyl chloride © @ Radioactive waste

Oral contraceptives @ . Nuclear reactor accidents @

Diagnostic X-rays @ Uranium mining
e Pesticides Nuclear weapons
e IUDs A
5 Antibiotics
@ Mercury@ PCBs * fallout A
a . BED
5
eR «© Aspirin Lead eee a ® Satellite crashes
be ae Asbestos insulation
oe Tey nealepaly catastrophic \ _ @Lead pain @ Coal-burning pollution
figsie hee FCO!ee isla
CS I Sd Ne

CT @Skateboards Carbon monoxide

Be Con tions
CaaS ae 3re not fatal ————- autos) @ Liquified natural \
RE AS eee cut‘<ss= Smoking-related ness ® Blacklung @ gas storage
Snowmobiles and transport
Sere oe ng Power mowers @ Large dams
@ Tractors e Nerve gas
@ Skyscraper fires
accidents
Chain saws @

: @ Elevators ; Nuclear war @

e@ Underwater construction
Home swimming pools @
@ Downhill skiing @ Parachuting @ Coal mining
@ General aviation
Recreational boating @
: Motorcycles @ @ High construction
Bicycles @ @ Train wrecks
Alcohol-related accidents @ @ Commercial aviation
Fireworks ® @ Auto accidents » Auto racing

@ Handguns
OBSERVABLE
@ Dynamite
+ Risks are known to those
exposed
+ Effect is immediate
¢ Risk is “old,” familiar
+ Risk is known to science

FIGURE 4-5 Perceptions of risk illustrated using a “risk space” axis diagram. Risk space has axes that correspond roughly to a hazard’s
perceived “dreadedness” and to the degree to which it is familiar or observable. Risks in the upper right quadrant of this space are most likely to
provoke calls for government regulation.
58 UNIT 1 General Principles of Toxicology

water, and air) and unfamiliar hazards, such as radioactive concepts of capacity assessment, vulnerability, and impact
waste, electromagnetic fields, asbestos insulation, and geneti- assessment are included. In this context, vulnerability assess-
cally modified crops and foods. Many people respond very ment would include consideration of exposure and susceptibil-
negatively when they perceive that information about hazards ity as part of the vulnerability assessment. Hazard analysis refers
or even about new technologies without reported hazards has to both hazard identification and probability-based frequency
been withheld by the manufacturers (genetically modified of anticipated events. Capacity assessment has been used for
foods) or by government agencies (HIV-contaminated blood identifying strengths and resiliency of a system to impact.
transfusions in the 1980s; extent of hazardous chemical or
radioactive wastes).
Most people regularly compare risks of alternative activities— SUMMARY
on the job, in recreational pursuits, in interpersonal interac-
Risk assessment objectives vary with the issues, risk manage-
tions, and in investments. Determining how best to conduct
ment needs, and statutory requirements. Hence, setting the con-
comparative risk analyses has proved difficult due to the great
text and problem formation for risk evaluation is essential. The
variety of health and environmental benefits, the gross uncer-
frameworks are sufficiently flexible to address various objectives
tainties of dollar estimates of benefits and costs, and the differ-
and to accommodate new knowledge while providing guidance
ent distributions ofbenefits and costs across the population.
for priority setting in industrial, environmental, governmental,
and public health agencies. Risk assessment analyzes the science,
EMERGING CONCEPTS identifies uncertainty and provides approaches for decisions.
Toxicology, epidemiology, exposure assessment, and clinical
There is a need to ensure that the risk question(s) is(are) observations can be linked with biomarkers, cross-species inves-
succinctly framed to answer questions in the real world. tigations of mechanisms of effects, and systematic approaches to
Environmental health is very dynamic and many divergent risk assessment, risk communication, and risk management.
emerging environmental challenges such as climate change, Advances in toxicology are certain to improve the quality of risk
energy shortages, and engineered nanoparticles will require an assessments as scientific findings substitute data for assumptions
expansion of our context well beyond single-chemical, single- and help to describe and model uncertainty more credibly.
exposure scenarios. In order to accomplish this goal, global and
international thinking will be required.
Well-being is increasingly being used to describe human health BIBLIOGRAPHY
and the goal of sustainable environmental risk management. Costa L, Eaton D (eds.): Gene-Environment Interactions: Fundamental
Well-being goes beyond “disease-free” existence to freedom from of Ecogenetics. Hoboken, NJ: John Wiley & Sons, 2006.
want (including food and water security) and fear (personal FDA US: Critical Path Initiative. Science & Research. Silver
safety) and sustainable futures. Recognition that environmental Spring, MD: US Food and Drug Administration; 2011.
problems are global is essential to how we manage risks and Available at: http://www.fda.gov/ScienceResearch/SpecialTopics/
address sustainability. Research and development efforts must CriticalPathInitiative/default.htm.
examine chemical safety for sustainable and healthy communi- Hood R (ed.): Developmental and Reproductive Toxicology: A Practical
Approach. 3rd ed. Boca Raton, FL: CRC Press, 2011.
ties with safe and sustainable water, air, and energy resources.
Hsieh A: A nation’s genes for a cure to cancer: evolving ethical, social
and legal issues regarding population genetic databases. ColumbiaJ
Law Soc Probl 37:359-411, 2004.
PUBLIC HEALTH RISK MANAGEMENT
NRC: Science and Decisions: Advancing Risk Assessment. Washington,
Associated with concepts of well-being and sustainability is a DC: National Academies Press; 2009.
public health orientation to use toxicological tests to identify NTP: National Toxicology Program. 2011. Available at: http://ntp.
niehs.nih.gov/.
and characterize potential health risks and to prevent the unsafe
Ryan PB: Exposure assessment, industrial hygiene, and environmen-
use of such agents. There are three stages of prevention: primary,
tal management. In: Frumkin H, ed. Environmental Health: From
whose goal is prevention and risk or hazard avoidance; second-
Global to Local. 2nd ed. San Francisco, CA: Jossey-Bass; 2010.
ary, whose goal is mitigation or preparedness including risk or Sahu SC (ed.): Toxicology and Epigenetics. New York: John Wiley &
vulnerability reduction and risk transfer; and tertiary, where Sons; 2012.
prompt response or recovery is an approach for decreasing US EPA: Ecological Risk Assessments. Pesticides: Environmental
residual risk or risk reduction. Figure 4-5 shows an overview of Effects. 2011. Available at: http://www.epa.gov/pesticides/
risk assessment and management for public health where ecosystem/ecorisk.htm.
 CHAPTER 4 Risk Assessment 59

QUESTIONS

1. Which of the following is NOT important in hazard Which of the following types of epidemiologic study is
identification? always retrospective?
a. structure-activity analysis. a. cohort.
b. _ in vitro tests. b. cross-sectional.
c. animal bioassays. c. case-control.
d. susceptibility. d. longitudinal.
e. epidemiology. e. exploratory.

2. The probability of an adverse outcome is defined as: . Which ofthe following is defined as the highest nonstatis-
a. hazard. tically significant dose tested?
b. exposure ratio. a, I)Dxe
Ce tisk: b. EDoo
d. susceptibility. c. NOAEL.
e. epidemiology. d. “ADI
emrecOArL
3. The systematic scientific characterization of adverse health
effects resulting from human exposure to hazardous Which of the following represents the dose below which
agents is the definition of: no additional increase in response is observed?
risk. as ID
hazard control. b. LD»
risk assessment. CoaeRiG.
risk communication. d. threshold.
a= risk estimate.
CROCE e. significance level.

4. Which of the following is not an objective of risk Which of the following is NOT needed to calculate the
management? reference dose using the BMD method?
setting target levels for risk. a. ME
balancing risks and benefits. b. percent benchmark response.
calculating lethal dosages. ¢. NOAEL.
setting priorities for manufacturers. dur
oS estimating residual risks.
Cao e. benchmark dose.

5. Which of the following is NOT a feature in the design of 10. Virtually safe doses are described at which confidence
standard cancer bioassays? level?
more than one species. a. 90%.
both sexes. b. 95%.
near lifetime exposure. ©, SEM:
approximately 50 animals per dose group. d. 99.9%,
Cr
i
fos same dose level for all groups. e, 99199%:
 et we are :
i"
Boe
i.
‘Sess sth oot ei < gree: Sgt a
be ee a i
 UNIT 2. DISPOSITION OF TOXICANTS |

Ga HT AS Pel aE eR

Absorption, Distribution,
and Excretion of Toxicants
Lois D. Lehman-McKeeman

INTRODUCTION Plasma Proteins as Storage Depot

Liver and Kidney as Storage Depots
CELL MEMBRANES Fat as Storage Depot
Passive Transport Bone as Storage Depot
Simple Diffusion Blood-Brain Barrier
Filtration Passage of Toxicants across
Special Transport the Placenta
Facilitated Diffusion Redistribution of Toxicants
Active Transport
Xenobiotic Transporters EXCRETION
Additional Transport Processes Urinary Excretion
Fecal Excretion
ABSORPTION
Nonabsorbed Ingesta
Absorption of Toxicants by the Gastrointestinal Tract
Biliary Excretion
Absorption of Toxicants by the Lungs
Exhalation
Gases and Vapors
Other Routes of Elimination
Aerosols and Particles
Cerebrospinal Fluid
Absorption of Toxicants through the Skin
Milk
Absorption of Toxicants after Special Routes
Sweat and Saliva
of Administration

DISTRIBUTION CONCLUSION

Volume of Distribution
Storage of Toxicants in Tissues
-

61
62 UNIT 2 Disposition of Toxicants

= Absorption is the transfer of a chemical from the site of » Excretion is the removal of xenobiotics from the blood
exposure, usually an external or internal body surface, and their return to the external environment via urine,
into the systemic circulation. feces, exhalation, etc.
= Toxicants are removed from the systemic circulation by
biotransformation, excretion, and storage at various sites
in the body.

INTRODUCTION slow rate with obvious consequences for the concentration

and, thus, the toxicity at the target site; and (4) the more rapidly
The disposition of a chemical or xenobiotic is defined as the a chemical is eliminated from an organism, the lower will be its
composite actions ofits absorption, distribution, biotransforma- concentration and hence its toxicity in target tissues. Ifa chem-
tion, and elimination. The quantitative characterization of ical is distributed to and stored in fat, its elimination is likely to
xenobiotic disposition is termed pharmacokinetics or toxicoki- be slow because very low plasma levels preclude rapid renal
netics (see Chapter 7). clearance or other clearances.
The toxicity of asubstance depends on the dose. The concen- The skin, lungs, and alimentary canal,are the main barriers
tration of achemical at the site of action is usually proportional that separate higher organisms from an environment contain-
to the dose, but the same dose of two or more chemicals may ing a large number of chemicals. Toxicants must cross one or
lead to vastly different concentrations in a particular target several of these incomplete barriers to exert deleterious effects.
organ of toxicity owing to differences in the disposition of the Only chemicals that are caustic and corrosive agents (acids,
chemicals. Various factors affecting disposition are depicted in bases, salts, and oxidizers) and act topically at the point of con-
Figure 5-1, such as (1) if the fraction absorbed or the rate of tact are exceptions. A chemical absorbed into the bloodstream
absorption is low, a chemical may never attain a sufficiently through any of these three barriers is distributed throughout the
high concentration at a potential site of action to cause toxicity; body, including the site where it produces damage, the target
(2) the distribution ofatoxicant may be such that it is concen- organ or target tissue. A chemical may have one or several target
trated in a tissue other than the target organ, thus decreasing organs, and, in turn, several chemicals may have the same
toxicity; (3) biotransformation of a chemical may result in the target organ(s). Because several factors other than the concen-
formation of less toxic or more toxic metabolites at a fast or tration influence the susceptibility of organs to toxicants, the

Ingestion Inhalation Dermal

| | Intravenous
| | | | Subcutaneous
| Intramuscular
| Intraperitoneal
Gastrointestinal
tract

_ Extracellular uid
eee E
Other organs |
A|

Soft tissues, bone

FIGURE 5-1 Routes of absorption, distribution, and excretion of toxicants in the body. Black lines represent routes of absorption into
the blood stream; blue lines designate distribution; green lines identify pathways offinal excretion; red lines show enterohepatic circulation.
 CHAPTERS Absorption, Distribution, and Excretion of Toxicants 63

organ or tissue with the highest concentration of a toxicant is exits from cells. A toxicant may pass through a membrane by
not necessarily the site of toxicity. It is important to note that either (1) passive transport, in which the cell expends no
the processes comprising xenobiotic disposition are interre- energy or (2) specialized transport, in which the cell provides
lated and influence each other (Figure 5-1). energy to translocate the toxicant across its membrane.

CELL MEMBRANES Passive Transport

Simple Diffusion— Most toxicants cross membranes by sim-
All processes of toxicant distribution involve passage across
ple diffusion, following the principles of Fick’s law, which estab-
biological membranes. Toxicants usually pass through the
lishes that chemicals move from regions of higher concentration
membranes of a number of cells, such as the stratified epithe-
to regions of lower concentration without any energy expendi-
lium ofskin, the thin cell layers of lungs or gastrointestinal tract,
ture. Small hydrophilic molecules (up to a molecular weight of
the capillary endothelium, and the cells of the target organ or
about 600 daltons [Da]) permeate membranes through aque-
tissue; the plasma membranes surrounding all these cells are
ous pores, in a process termed paracellular diffusion. In con-
remarkably similar.
trast, hydrophobic molecules diffuse across the lipid domain of
The basic unit of the cell membrane is a lipid bilayer, com-
membranes, in a process called transcellular diffusion. The
posed primarily of phospholipids, glycolipids, and cholesterol.
smaller a hydrophilic molecule is, the more readily it traverses
Phospholipids are amphiphilic, consisting of a hydrophilic
membranes by simple diffusion, and, consequently, a small
polar head and a hydrophobic lipid tail. In membranes, the
water-soluble compound such as ethanol is rabidly absorbed
polar head groups are oriented toward the outer and inner sur-
and distributed.
faces of the membrane, whereas the hydrophobic tails are ori-
For larger organic molecules with differing degrees of lipid
ented inward and face each other to form a continuous
solubility, the rate of transport across membranes correlates
hydrophobic inner space. Hydrophobic interaction between
with lipophilicity. Their rate of transport across membranes
these fatty acids is the major driving force for the formation of
correlates with their lipid solubility, which is determined by the
membrane lipid bilayers. Numerous proteins are inserted or
octanol/water partition coefficient, P, which is defined as the
embedded in the bilayer, and some transmembrane proteins
ratio of the concentration of neutral compound in organic and
traverse the entire lipid bilayer, functioning as important bio-
aqueous phases under equilibrium conditions. The octanol/
logical receptors or allowing the formation of aqueous pores,
water partition coefficient is usually expressed in log form, and
ion channels, and transporters (Figure 5-2). Fatty acids of the
is an informative physicochemical parameter relative to assess-
phospholipids and glycolipids do not have a rigid crystalline
ing potential membrane permeability, with positive values
structure, but are semifluid at physiological temperatures.
associated with high lipid solubility.
Many factors influence this fluid character, including degree of
Many chemicals are weak organic acids or bases, which are
unsaturation (lack of double bonds), the presence of choles-
ionized in solution according to Arrhenius theory. The ionized
terol, and temperature.
form of weak organic acids or bases usually has low lipid solu-
A key characteristic of plasma membranes is their ability to
bility and does not permeate readily through the lipid domain
be differentially permeable, which regulates what enters into or
of a membrane. In contrast, the nonionized form is more lipid
soluble and diffuses across membranes at a rate that is propor-
tional to its lipid solubility. The pH at which a weak organic
The cell membrane acid or base is 50% ionized is called its pK, or pK,. Values for
Extracellular fluid pK, relay the relative strength or weakness of the acid such that
a low values indicate a strong acid and high values indicate a
weak acid; the opposite is true for bases. Both pK, and pK, are
x " aiian defined as the negative logarithm of the ionization constant of
a weak organic acid or base. Knowing pK,, one can calculate
@l i}. ally
pK, for weak organic bases with the equation pK, = 14 — pK,.
Knowledge of the chemical structure is required to distinguish
between organic acids and bases, as the numerical value of pK,
does not indicate this characteristic.
Cytoplasm
The degree of ionization of a chemical depends on its pK,
<<
and on the pH of the solution. The relationship between
g ; 7 pK, and pH is described by the Henderson-Hasselbalch
(] ; $ ° equations:
Phospholipid Intergral proteins Cholesterol lonchannel Ligand
(Receptors,
Transporters)
For acids: pK, — pH = jog Momionized|
FIGURE 5-2 Schematic model of abiological membrane.
[ionized]
64 UNIT 2 Disposition ofToxicants

e transport except that the substrate is not moved against an

For bases pK, — pH = log e
electrochemical or concentration gradient and the transport
process does not require the input of energy. Because this pro-
The effect of pH on the degree of ionization of an organic cess is energy-independent, metabolic poisons do not interfere
acid (benzoic acid) and an organic base (aniline) is illustrated with this type of transport, as they would with active transport.
in Figure 5-3. According to Bronsted-Lowry acid-base theory,
an acid is a proton (H*) donor and a base is a proton acceptor. Active Transport—Active transport is characterized by
Thus, the ionized and nonionized forms of an organic acid rep- (1) movement of chemicals against electrochemical or concen-
resent an acid-base pair, with the nonionized moiety being the tration gradients, (2) saturability at high substrate concentra-
acid and the ionized moiety being the base. tions, thus exhibiting a transport maximum (T;,,), (3) selectivity
for certain structural features of chemicals, (4) competitive
Filtration—When water flows in bulk across a porous inhibition by chemical antagonists or compounds that are car-
membrane, any solute small enough to pass through the pores ried by the same transporter, and (5) requirement for expendi-
flows with it. Passage through these channels is called filtration. ture of energy (often in the form of ATP), so that metabolic
One ofthe main differences between various membranes is the inhibitors block the transport process.
size of these channels. In the renal glomeruli, a primary site of
blood filtration and subsequent urine formation, these Xenobiotic Transporters— Around 5% of all human genes
pores are relatively large and allow molecules smaller than are transporter related, indicating the importance of transport
albumin (approximately 60 kDa) to pass through. The channels function in normal biological and toxicological outcomes.
in most cells are much smaller, permitting substantial passage Transporters mediate the influx (uptake) and efflux of xenobi-
of molecules with molecular weights of no more than a few hun- otics and can be divided into two categories determined by
dred daltons. whether they employ active transport ar facilitative diffusion
(Tables 5-1 and 5-2).
Special Transport Energy-dependent xenobiotic transporters are part of a large
There are numerous compounds whose movement across superfamily known as ATP-binding cassette (ABC) transport-
membranes cannot be explained by simple diffusion or filtra- ers, and seven subfamilies (classified A to G) have been now
tion. Some compounds are too large to pass through aqueous identified. Many of these transporters play key roles in the
pores or too lipid-insoluble to diffuse across the lipid domain of homeostasis of numerous endogenous substances, including
plasma membranes. Nevertheless, these molecules are still absorption from the GI tract and maintenance of the blood-
transported, often very rapidly, across plasma membranes and brain barrier (BBB); mutations can lead to multidrug resis-
even against concentration gradients. Specialized transport tance (MDR). A notable example is from the B subfamily,
systems have been identified to explain these phenomena, and called MDR1 (ABCB1) that, in cancerous cells, exudes cyto-
identifying such transporters and their dysfunctions is a devel- toxic drugs out of the tumor cells thereby protecting the
oping area of toxicology. cell from drug-mediated destruction. The C subfamily of
ABC transporters is also known as the multidrug resistance-
Facilitated Diffusion—Facilitated diffusion is carrier- associated protein (MRP) family, and they are also involved
mediated transport that exhibits the properties of active in efflux of chemicals from cells.

Acidic pH Neutral pH
[Nonionized]
[lonized]

COOH

5
pk,~4

1 1 1
NH," 10000 1000 100

pk,~5 las ee

FIGURE 5-3 Effect of pH on the ionization of benzoic acid (pK, = 4) and aniline (pK, = 5).
 CHAPTERS Absorption, Distribution, and Excretion of Toxicants 65

TABLE 5-1 Human ABC transporters: gene family overview and major transporters involved in xenobiotic
disposition.

ABC Subfamily Genes in Family Gene Symbols

A 12 ABCA1-10, 12, 13

B 11 ABCB1-11

Cc 13 ABCC1-13*

D 4 ABCD 1-4

BE 1 ABCE1

E 3 ABCF 1-3

G 5 ABCG1, 2, 4, 5,8

Bolded subfamily designations are those with a major role in xenobiotic disposition.
*ABCC73 is reported to be a pseudogene.

Gene Symbol Common Name General Function

ABCB1 Multidrug resistant protein/P-glycoprotein (MDR) Efflux from gut, brain, placenta; biliary excretion

ABCB11 Bile salt export pump (BSEP) Bile salt transport

ABCC1 Multidrug resistance—associated protein 1 (MRP1) Multidrug resistance in many tissues; export pump

Organic anion efflux, glucuronide and glutathione conjugates,

ABCC2 Multidrug resistance—associated protein 2 (MRP2)
biliary excretion

ABCC3 Multidrug resistance—associated protein 3 (MRP3) Organic anion efflux, glucuronide and glutathione conjugates

ABCC4 Multidrug resistance—associated protein 4 (MRP4) Nucleoside transport and organic anion efflux

ABCC5 Multidrug resistance—associated protein 5 (MRP6) Mainly nucleoside transport

ABCC6 Multidrug resistance—associated protein 6 (MRP6) Some glutathione conjugates

ABCC10 Multidrug resistance—associated protein 7 (MRP7)* Organic anions, vinca alkaloids

ABCCT1 Multidrug resistance—associated protein 8 (MRP8)* Cyclic nucleotides and organic anions

ABCC12 Multidrug resistance—associated protein 9 (MRP9)* Not defined

ABCG2 Breast cancer resistance protein (BCRP) Organic anion efflux, many sulfate conjugates, biliary excretion

*There is little functional information available for MRP7, MRP8, and MRP9. A single nucleotide polymorphism in MRP8 is known to determine wet or dry earwax.

The second major class of xenobiotic transporters is the sol- 8-lactam antibiotics. Finally, the multidrug and toxin extru-
ute carriers (SLCs), which predominantly function through sion (MATE) transporters are a unique gene family of SLCs
facilitative diffusion. There are 43 SLC gene families identified, expressed predominantly in liver and kidney that function spe-
and many of the nearly 300 genes comprising the 43 distinct cifically as cation efflux pumps.
SLC families play important roles in the disposition of endog-
enous compounds, including glucose, neurotransmitters, Additional Transport Processes—Other forms of special-
nucleotides, essential metals, and peptides. Additionally, there ized transport (e.g., phagocytosis and pinocytosis used by cell
are several families that are vital to xenobiotic disposition, reg- membranes to engulf particles) have been proposed, but their
ulating the movement of many diverse organic anions and cat- overall importance is not as well established as that of active
ions across cell membranes. Organic-anion transporting transport and facilitated diffusion.
peptides (OATPs, SLCO family) are one of the major SLCs
involved in xenobiotic disposition in humans. OATPs are of ABSORPTION
particular importance in the liver and kidneys and mediate the
sodium-independent transport of a wide range of compounds, The process by which toxicants cross body membranes and
including organic bases, acids, and neutral compounds. enter the bloodstream is referred to as absorption. There are no
Peptide transporters (PEPTs) are responsible for the transport specific systems or pathways for the sole purpose of absorbing
of di- and tri-peptides as well as drugs and toxicants such as the toxicants, and xenobiotics penetrate membranes in the same
Oo UNIT 2 Disposition of Toxicants

TABLE 5-2 Major members of the human solute carrier transporter families involved in xenobiotic disposition.
2 ea a LS a ee ee |

Transporter Gene Family Human Proteins Gene Name Function

Organt- anion transporting —-SCO OATPIA2 SLCO1A2 Transport of organic anions, cations, and
polypeptide (QATP) neutral compounds
OATP1B1 SLCO1B1

OATP IBS SLCO1B3

OATPIC] SLCO1CI

OATP2A1 SLCO2A1

OATP2B81 SLCO2B1

OATP3A1I SLCO3A1

OATPAAI SLCO4A1

OATPACI SLCO4C1

OATPSAI SLCOSA1

OATPGAI SLCOGA1 \
‘

Organic-cation SLC22 oem] SLC22A1 Transport of organic cations

transporter (OCT) ‘
OCcT2 SLC22A2 \

OCcT3 SLC22A3

Organic-cation/carnitine StC22 OCTN1 SLC22A4 Organic cations;

transporter (OCTN)
OCTN2 SLC22A5 OCTN2 specific for carnitine

Organic-anion SLC22 OATI SLC22A6 Transport of organic anions

transporter (OAT)
OAT2 SLC22A7

OATS SLC22A8

OAT4 SLC22A11

OATS SLC22A10

Peptide transporter (PEPT) SUCTS PEPT1 SLCI5A1 Transport of di- and tripeptides, some
xenobiotics
PEPT2 SLC1ISA2

Multidrag and toxin SLEAZ MATE1 SLCA7A1 Efflux of organic cations;

extrusion transporter
(MATE) MATE2K SLC47A2 MATE2K localized to kidney

way as biologically essential substances such as oxygen and absorption is also particularly relevant to toxicologists because
toodstutis. The main sites of absorption are the gastrointestinal accidental ingestion is the most common cause of uninten-
(GD tract, lungs, and skin. Enteral administration includes all tional exposure to a toxicant (especially for children) and inten-
routes pertaining to the alimentary canal (sublingual, oral, and tional overdoses most frequently occur via the oral route.
rectal), whereas parenteral administration involves all other The GI tract may be viewed as a tube traversing the body.
routes (intravenous, intraperitoneal, intramuscular, subcuta- Although within the body, GI contents can be considered exte-
— a. )
neouy, etc, }. rior to the body. Unless a noxious agent has caustic or irritating
properties, poisons in the GI tract usually do not produce sys-
temic injury to an individual until they are absorbed into the
Absorption of Toxicants by the blood stream,
Gastrointestinal
Tract Absorption of toxicants can take place along the entire GI
Many environmental toxicants enter the food chain and are tract, even in the mouth and rectum. Ifa toxicant is an organic
absorbed together with food from the GI tract. This site of acid or base, it tends to be absorbed by simple diffusion in the
 CHAPTER 5 Absorption, Distribution, and Excretion of Toxicants 67

part of the GI tract in which it exists in the most lipid-soluble nanomaterials that may be used in a variety of chemica) and
(nonionized) form. Recall that the degree of ionization is, in biological processes (Chapter 28).
part, determined by the pH of its solution according to the Overall, the absorption of a toxicant from the GI trac
Henderson-Hasselbalch equation. Consequently, the lipid sol depends on its physical properties, including lipid solubility
ubility of weak organic acids or bases can differ markedly in the and its dissolution rate. An increase in lipid solubility typically
GI tract because gastric juice is acidic (pH ~ 2) and the intesti increases the absorption of chemicals and the dissolution rate
nal contents are nearly neutral (pH ~ 7). Other factors that is inversely proportional to particle size.
influence the absorption of weak organic acids or bases include In addition to the characteristics of the cormpounds them
the mass action law, intestinal surface area, and blood flow rate. selves, there are numerous additional factors relating to the GI
Continuous circulation of blood will help keep blood concen tract itself that influence the absorption of xenobiotics, incdudin ae
trations low, thereby maintaining a concentration gradient pH, the presence of food, digestive enzymes, bile acids, bacteria)
favoring absorption. The villi and microvilli of the small intes microflora, and the motility and permeability of the GI tract.
tine contribute to an approximate 600-fold increase in surface Chemical resistance or lack of resistance to alteration by the
area, which greatly facilitates absorption. acidic pH of the stomach, enzymes of the stornach or intestine,
The mammalian GI tract has specialized transport systems or the intestinal microflora are extremely important. For exam
(carrier mediated) for the absorption of nutrients and electro ple, a toxicant may be hydrolyzed by stomach acid, biotrans-
lytes (Table 5-3). The number oftoxicants actively absorbed by formed by enzymes in the GI tract, or modified by the resident
the GI tract is low; most enter the body by simple diffusion. microflora to new compounds with a toxicity different from
Lipid-soluble substances are absorbed by this process more that of the parent compound. Simple diffusion is proportional
rapidly and extensively than are water-soluble substances. not only to surface area and permeability but also to the resi
Particulate matter can also be absorbed by the GI epithe dency time within various segments of the GI tract suc pn; that
lium. In this case, particle size determines absorption rate, longer residencies lead to increased absorption and vice versa.
whereas factors such as lipid solubility and ionization charac Experiments have shown that the oral toxicity of some
teristics are less important. Particle size is inversely related to chemicals is increased by diluting the dose. This phenomenon
absorption rate such that absorption increases with decreasing may be explained by more rapid stomach emptying induced by
particle diameter. Large particles enter the GI epithelium by increased dosage volume, which in turn leads to more rapid
pinocytosis. There is increasing interest in particles of very absorption in the duodenum because of the larger surface area
small diameter (less than 100nm) called nanoparticles or there,

TABLE 5-3 Site distribution of specialized transport systems in the intestine of man and animals.
Location ofAbsorptive Capacity in Small Intestine
Substrates Upper Middle Lower Colon

Sugar (glucose, galactose, etc.) +4 +44 ++ 0

Neutral amino acids ++ +f + ++ 0

Basic amino acids +b 4 ++ ++ 7

Gamma globulin (newborn animals) 4 ++ +++ 7

Pyrimidines (thymine and uracil) 4. 4 7 7

Triglycerides apf ++ + rs

Fatty acid absorption and conversion to triglyceride ++ + + +4 + 0

Bile salts 0 4 Peake

Vitamin B,, 0 4 +++ 0

Na* + ++ +++ +++

H* (and/or HCO; secretion) 0 + +++ oe

Cat fat +4 + 7
Fe2* © he +4 + ]

cr +44 ++ a 0
os UNIT 2 Disposition of Toxicants

[he amount of a chemical entering the systemic circulation varies with partial vapor pressure, the ratio of the concentra
atter oral administration depends on the amount absorbed tion in the blood to the concentration in the alveolar space is
into the Gl cells, biotranstormation by the GI cells, and extrac constant, For example, the atmospheric pressure is much
tion by the liver into bile (with or without biotranstormation). lower at the peak of Mt. Everest (~ 252mm Hg) than itis at sea
Transporters can intluence this amount by atfecting the uptake level (760mm Hg), The partial vapor pressure of oxygen is
or ettlux trom the cells. This phenomenon of the removal of likewise lower atop Mt. Everest (~53 mm Hg) than at sea level
chemicals before entrance into the systemic circulation is (160 mm Hg), but the blood-to-gas partition coetticient would
referred to as presystemic elimination, or first-pass effect. remain the same regardless of elevation, This solubility ratio is
Chemicals that have a high tirst-pass effect will appear to have called the blood-to-gas partition coefficient and it is unique tor
a lower absorption because they are eliminated as quickly as each gas,
they are absorbed, Absorption of gasses in the lungs differs from intestinal and
A number of other factors have been shown to alter absorp percutaneous absorption in that the ionization state and the
tion. For example, heavy metal ions such as lead are not readily lipid solubility of molecules are less important factors in pul
absorbed trom the GI tract. However, EDTA and other chela monary absorption. This is because dittusion through cell
tors increase the lipid solubility of heavy metals and, thus, membranes is not normally rate-limiting in the pulmonary
absorption ot complexed ions. Consumption of grapetruit absorption of gases. The rate of absorption of gases in the
juice can also intluence GI absorption through the actions of lungs is variable and depends on a toxicant’s solubility ratio
naringin, a tlavonoid that can increase absorption (though (concentration in blood/concentration in gas phase) at equi
inhibition of MOR1) or decrease absorption (through inhibi librium. A gas with a high solubility ratio (e.g., >1) is readily
tion of OATPs) of numerous pharmaceutical agents, transterred to the blood Uuring each respiratory cycle so that
little if any remains in the alveoli just betore the next inhala
tion. Conversely, a gas with a low Sobubility ratio (e.g. <1)
Absorption of Toxicants by the Lungs means the blood is quickly saturated with this gas and a
Yoxicants absorbed by the lungs are usually gases, vapors of higher concentration of the gas remains in the alveolar
volatile or volatilizable liquids, and aerosols. Anatomical and space. Gases with high solubility ratios are said to be
physiologic details of the respiratory system are described in ventilation-limited because the rate and depth of respiration
Chapter 15, determine the extent of distribution of the gas. The rate of
blood tlow is the primary determinant of distribution for
Gases and Vapors— \ vapor is the gas form of a substance gasses with low solubility ratios and they are, therefore,
that can also exist as a liquid or a solid at atmospheric pressure considered pertusion-limited.,
and normal temperature, Most organic solvents evaporate and The blood carries dissolved gas molecules to the rest of the
produce vapors, and some solids can sublimate directly into a body, In each tissue, gas molecules are transferred from the
gaseous form, Vapor pressure is that exerted by a vapor above its blood to the tissue until equilibrium is reached. After releasing
own liquid in a closed system, such that liquids that have a high part of the gas to tissues, blood returns to the lungs to take up
vapor pressure have a higher tendency to evaporate. A toxicant more of the gas. The process continues until a gas reaches equi
witha high vapor pressure at room temperature is considered to librium between blood and each tissue. At this time, no net
be volatile. absorption of gas takes place as long as the exposure concen-
The absorption of inhaled gases takes place mainly in the tration remains constant. Of course, if biotransformation and
lungs. However, before a gas reaches the lungs, it passes excretion occur, alveolar absorption will continue until a cor-
through the nase, Because the mucosa of the nose is covered by responding steady state is established. The lung can also poten-
a film of fluid, gas molecules can be retained by the nose and tially contribute to the biotransformation or elimination of
not reach the lungs if they are very water soluble or react with chemicals before their entrance into the systemic circulation.
cell surface components. Therefore, the nose acts as a “scrub
ber” for water-soluble gases and highly reactive gases. Aerosols and Particles—<Acrosols are a colloid of solid
Although this may serve to reduce systemic exposure or to pro particles and liquid droplets in air. The major characteristics
tect the lungs, it also increases the risk that the nose could be that atfect absorption after exposure to aerosols are the aerosol
adversely affected. sive and water solubility of any chemical present in the aerosol
When a gas is inhaled into the lungs, gas molecules ditfuse (Chapter 15). The site of deposition of aerosols depends largely
trom the alveolar space into the blood until equilibrium is on the size of the particles. In general, the smaller the particle,
reached (i.e, no net movement of inhaled gas between the the turther into the respiratory tree the particle will deposit.
alveolar space and blood). At equilibrium, the concentration Particles 5 um or larger usually are deposited in the nasopha-
of gas in the alveolar space can be, and often is, different from tyngeal region (Figure 5-4) and are removed by nose wiping,
the concentration of gas in the blood. This difference is blowing, or sneezing. The mucous blanket of the ciliated nasal
accounted for by Henry's law, which states the concentration surtace propels insoluble particles by the movement of the cilia.
in the blood (or any fluid) is directly proportional to the par These particles and particles inhaled through the mouth are
tial vapor pressure of the inhaled gas. While the concentration swallowed within minutes. Soluble particles may dissolve in the
 CHAPTERS Absorption, Distribution, and Excretion of Toxicants 69

Removal or absorption of particulate matter from the alveoli

appears to occur by three major mechanisms. First, particles
may be removed from the alveoli by a physical process. It is
thought that particles deposited on the fluid layer ofthe alveoli
are aspirated onto the mucociliary escalator of the tracheo-
bronchial region. From there, they are transported to the
mouth and may be swallowed. Second, particles from the alve-
oli may be removed via phagocytosis by the alveolar macro-
Sea ea as phages. These cells are found in large numbers in normal lungs
_ Tracheobronch and contain many phagocytized particles of both exogenous
and endogenous origin. They migrate to the distal end of the
mucociliary escalator and are cleared and eventually swal-
lowed. Third, removal may occur via the lymphatics, although
particulates may remain in lymphatic tissue for long time
periods.
In general, the overall removal of particles from the alveoli is
relatively inefficient. The rate of clearance by the lungs can be
predicted by a compound's solubility in lung fluids such that
lower solubility means lower clearance rate. Thus, removal of
particles that enter the alveoli is largely due to dissolution and
vascular transport. Some particles may remain in the alveoli
indefinitely, as is the case when alveolar macrophages phago-
cytose indigestible dust particles.

Absorption of Toxicants through the Skin

Skin is the largest body organ and provides a relatively good
barrier for separating organisms from their environment.
Human skin comes into contact with many toxic chemicals,
but exposure is usually limited by its relatively impermeable
nature. However, some chemicals can be absorbed by the skin
FIGURE 5-4 Schematic diagram of the absorption and
in sufficient quantities to produce systemic effects.
translocation of chemicals by lungs.
The skin comprises two major layers, the epidermis and der-
mis (Figure 5-5). The epidermis is composed of four or five lay-
mucus and be carried to the pharynx or may be absorbed ers (called strata), depending on location. The stratum corneum
through the nasal epithelium into blood. is the outermost layer and is unique in that it represents the
Particles approximately 2.5m are deposited mainly in the single most important barrier to preventing fluid loss from the
tracheobronchiolar regions of the lungs, from which they are body while also serving as the major barrier to prevent absorp-
cleared by retrograde movement of the mucus layer in the cili- tion of xenobiotics into the body. The dermis is situated beneath
ated portions of the respiratory tract. Particles eventually may the epidermis and consists primarily of fibroblasts, which are
be swallowed and absorbed from the GI tract. Toxicants or cells responsible for synthesizing the collagen and extracellular
viral infections that damage cilia may impair the efficiency of matrix components of the dermis. A vascular network that pro-
this process. vides both the dermis and epidermis with blood supply is also
Particles 1 um and smaller penetrate to the alveolar sacs of contained within the dermis. Although the stratum corneum is
the lungs. Ultrafine or nanoparticles, particularly those that are the major barrier to absorption, compounds may also be
approximately 10 to 20 nm in size, have the greatest likelihood absorbed through dermal appendages (sweat glands, sebaceous
of depositing in the alveolar region. They may be absorbed into glands, and hair follicles) found within the dermis.
blood or cleared through the lymphatics after being scavenged To be absorbed through the skin, a chemical first pass
by alveolar macrophages. through the stratum corneum and then traverse the other six
As particle size decreases, the number of potential particles layers of the skin. All toxicants move across the stratum cor-
in a unit of space increases along with the total surface area of neum by passive diffusion. Lipophilic compounds are gener-
the particles. This relationship indicates that nanoparticles ally absorbed quickly and in a manner proportional to their
have the propensity to deliver a high amount of particulate to lipid solubility, but inversely related to molecular weight.
the lungs. However, it appears that the surface properties of Hydrophilic compounds are absorbed more slowly across the
nanoparticles may be more important determinants of toxic stratum corneum, and they are, therefore, more likely to pene-
potential than their size or surface area. trate through dermal appendages.
70 UNIT 2 Disposition of Toxicants

ij an Disjunction
corneum | Conjunction
Stratum
granulosum
ree
wees
Stratum
Epidermis
——
spinosum

e Stratum
germinativum

Sweat duct
Sebaceous
gland
Sweat gland
4 Blood vessel
Dermis Connective ae
tissue

Fat

Hair follicle

L Capillary

FIGURE 5-5 Diagram of a cross-section of human skin.

The permeability of the skin depends on both the coefficient

Absorption of Toxicants after Special
of diffusion (“diffusivity”) and the thickness of the stratum cor-
neum. The second phase of absorption consists of diffusion Routes of Administration
through the lower layers of the epidermis and dermis and sub- Besides absorption through the skin, lungs, or GI tract, chemi-
sequent entry into systemic circulation through the vascula- cal agents can be administered through other routes, including
ture of the dermis. The rate of diffusion here primarily depends (1) intravenous, (2) intraperitoneal, (3) subcutaneous, and
on blood flow and interstitial fluid movement. (4) intramuscular. The intravenous route introduces the toxi-
Several factors that influence the absorption of toxicants cant directly into the bloodstream, eliminating the process of
through the skin include (1) the integrity of the stratum absorption. Intraperitoneal injection results in rapid absorp-
corneum, (2) the hydration state of the stratum corneum, tion of xenobiotics because of the rich blood supply and the
(3) ambient temperature, (4) solvents as carriers, and relatively large surface area of the peritoneal cavity. Intra-
(5) molecular size. Absorption is increased by decreasing size, peritoneally administered compounds are absorbed primarily
and it is generally recognized that compounds above 400 Da through the portal circulation and therefore must pass through
exhibit poor dermal absorption. Paradoxically, the overall the liver before reaching other organs by way of systemic circu-
absorption of most nanoparticles is relatively low. lation. Subcutaneous and intramuscular injections are usually
Dermal absorption has been studied in most laboratory ani- absorbed at slower rates but enter directly into the general
mals, including rats, mice, rabbits, guinea pigs, primates, and circulation.
pig, and it has been found to vary widely between species. For The toxicity of a chemical may or may not depend on the
instance, dermal absorption across rodent skin is greater than route of administration. For example, if a toxicant is injected
human skin, whereas the cutaneous permeability characteris- intraperitoneally, the compound may be completely extracted
tics of guinea pigs, pigs, and monkeys are more similar to those and biotransformed by the liver with subsequent excretion into
observed in humans. Inter-species differences in dermal the bile without gaining access to the systemic circulation. Any
absorption of xenobiotics result from variance in (1) the com- toxicant displaying the first-pass effect with selective toxicity
position and thickness of the stratum corneum along with the for an organ other than the liver and GI tract is expected to be
nature of dermal appendages, (2) cutaneous blood flow, less toxic when administered intraperitoneally than when
(3) biotransformation reactions, and (4) the levels and patterns injected intravenously, intramuscularly, or subcutaneously
of xenobiotic transporters. because of extraction in the liver.
 CHAPTERS Absorption, Distribution, and Excretion of Toxicants 71

DISTRIBUTION constituents of the body. As depicted in Figure 5-6, albumin is

the major protein in plasma and it binds many different com-
After gaining entry into the bloodstream, regardless of route of pounds compared to other proteins, such as globulins, lipopro-
exposure, a toxicant may distribute to tissues throughout the teins, and glycoproteins.
body. The rate of distribution to organs or tissues is determined Protein-ligand interactions occur primarily as a result of
primarily by blood flow and the rate of diffusion out of the capil- hydrophobic forces, hydrogen bonding, and van der Waals
lary bed into the cells of aparticular organ or tissue. The final forces. Because of their high molecular weight, plasma proteins
distribution depends largely on the affinity of a xenobiotic for and the toxicants bound to them cannot cross capillary walls.
various tissues. Consequently, the fraction of toxicant bound to plasma pro-
teins is not immediately available for distribution into the
Volume of Distribution extravascular space or filtration by the kidneys. However, the
A key concept in understanding the disposition of a toxicant is interaction of a chemical with plasma proteins is a reversible
its volume of distribution (Vd), which is defined as the volume in process. As unbound chemical diffuses out of capillaries,
which the amount of drug would need to be uniformly dissolved bound chemical dissociates from the protein until the free
in order to produce the observed blood concentration. The fraction reaches equilibrium between the vascular space and
total water in one’s body accounts for approximately 60% of the extravascular space. In turn, diffusion in the extravascular
body weight and is partitioned into two main compartments: space to sites more distant from the capillaries continues, and
(1) intracellular water and (2) extracellular water. Extracellular the resulting concentration gradient causes continued dissoci-
water is further divided into interstitial water and plasma water. ation of the bound fraction in plasma.
If a chemical distributes only to the plasma compartment (no The binding of toxicants to plasma proteins is an important
tissue distribution), it has a high plasma concentration and a concept in toxicology for two reasons. First, toxicity is typically
low Vd. In contrast, if a chemical distributes throughout the manifested by the amount of a xenobiotic that is unbound.
body (into both compartments), it has a low plasma concentra- Therefore, a compound with a high degree of plasma protein
tion and a high Vd. The distribution of toxicants is more com- binding may not show toxicity when compared to one that is
plex than this, however, and strongly influenced by factors such less extensively bound to plasma proteins. Severe toxic reac-
as binding to and/or dissolution in fat, liver, and bone. tions can occur if a toxicant is displaced from plasma proteins
Some toxicants do not readily cross cell membranes and by another agent, increasing the free fraction of the toxicant in
therefore have restricted distribution, whereas other toxicants plasma. This will result in an increased equilibrium concentra-
rapidly pass through cell membranes and are distributed tion of the toxicant in the target organ, with the potential for
throughout the body. Some toxicants selectively accumulate in toxicity. Xenobiotics can also compete with and displace endog-
certain parts of the body as a result of protein binding, active enous compounds that are bound to plasma proteins, which
transport, or high solubility in fat. The target organ for toxicity can allow the endogenous compound to exert a toxic effect.
may be the site of accumulation ofa toxicant, but this is not always Plasma protein binding can also give rise to observed species
the case. If a toxicant accumulates at a site other than the target differences in the disposition of toxicants. Factors that
organ or tissue, the accumulation may be viewed as a protective
process in that plasma levels and consequently the concentration ff Gaia Gus 2n2+

ofa toxicant at the site of action are diminished. However, because Bilirubin
Uric acid
any chemical in a storage depot is in equilibrium with the free Vitamin C
Adenosine
fraction (unbound) of toxicant in plasma, it is released into the Tetracyclines
circulation as the unbound fraction of toxicant is eliminated. Chloramphenicol
Zn?*, lipids Digitonin
Cholesterol Fatty acids
Storage of Toxicants in Tissues Vitamins A, K, D, E
——_|
Suramin
Quinocrine
Since only the free fraction (unbound) of a chemical is in equi- \
]
Penicillin
Salicylate
librium throughout the body, binding to or dissolving in certain Cu** (Ceruloplasmin)
Para-aminosalicylate
Sulfonamides
body constituents greatly alters the distribution of a xenobiotic. Lithium carmine
Streptomycin
Hemoglobin
Toxicants are often concentrated in a specific tissue, called a __ (Haptoglobin)
Acid dyes
Phenol red
storage depot, which may or may not be their site of toxic action. Histamine
Toxicants in storage depots are always in equilibrium with the Triiodothyronine
Thyroxine
free fraction in plasma, so that as a chemical is biotransformed B> Q Oy Albumin \ L Barbiturates

or excreted from the body, more is released from the storage

Fe2+ I Steroid hormones
site. Asa result, the biological half-life of stored compounds can
(transferrin) (transcortin)
be very long. Vitamin B,>
Sialic acid
Thyroxine
Plasma Proteins as Storage Depot—Several plasma
proteins bind xenobiotics as well as some endogenous FIGURE 5-6 Ligand interactions with plasma proteins.
NIiw) UNIT 2 Disposition of Toxicants

influence plasma protein binding across species include differ- and 5 are located on the blood side of the capillary endothe-
ences in the concentration of albumin, binding affinity, and/or lium function to move xenobiotics back into the blood and
competitive binding of endogenous substances. hence limit their distribution into the brain.
The blood cerebrospinal fluid (CSF) barrier is found between
Liver and Kidney as Storage Depots—The liver and the circulating blood and the circulating CSF in the brain.
kidney have a high capacity for binding a multitude of che- Certain areas (the choroid plexus, the arachnoid membrane,
micals. These two organs probably concentrate more toxicants and the area postrema) are more permeable on the blood
than do all the other organs combined. In most cases, binding side, whereas the epithelium on the CSF side is a barrier. In
to tissue components is likely to be involved. addition, efflux transporters contribute to xenobiotic removal
from the CSF thereby protecting against toxicant distribution
Fat as Storage Depot— Many highly lipophilic toxicants into the CNS.
with a high lipid/water partition coefficient are distributed and In general, only the free unbound toxicant equilibrates rap-
concentrated in body fat. Storage lowers the concentration of idly with the brain. Lipid solubility and the degree of ionization
the toxicant in the target organ; therefore, the toxicity of such a are important determinants of the rate of entry of a compound
compound can be expected to be less severe in an obese person into the CNS. Increased lipid solubility enhances the rate of
than in a lean individual. However, the possibility of a sudden penetration of toxicants into the CNS, whereas ionization
increase in the concentration of a chemical in the blood and greatly diminishes it. A few xenobiotics appear to enter the brain
thus in the target organ of toxicity when rapid mobilization of by carrier-mediated processes. Some lipophilic compounds
fat occurs must be considered. Several studies have shown that may enter the brain, but are so efficiently removed by these
signs of intoxication can be produced by short-term starvation transporters that they never reach appreciable concentrations.
of experimental animals that were previously exposed to persis- The BBB is not fully developed at birth, and this is one reason
tent organochlorine insecticides. why some chemicals are more toxic tonewborns than to adults.

Bone as Storage Depot—Skeletal uptake of xenobiotics is

essentially a surface chemistry phenomenon, with exchange Passage of Toxicants across the Placenta
taking place between the bone surface of hydroxyapatite crys- The term “placental barrier” has been associated with the con-
tals and the extracellular fluid in contact with it. Deposition and cept that the main function of the placenta is to protect the fetus
reversible storage of toxicants in bone is dynamic and may or against the passage of noxious substances from the mother.
may not be detrimental. For instance, lead is not toxic to bone, However, the placenta is a multifunctional organ that also pro-
but the chronic effects of fluoride deposition (skeletal fluorosis) vides nutrition, exchanges maternal and fetal blood gases, dis-
and radioactive strontium (osteosarcoma and other neoplasms) poses of fetal excretory material, and maintains pregnancy
are well documented. through complex hormonal regulation. Placental structure and
function show more species differences than any other mam-
malian organ.
Blood-Brain Barrier Most vital nutrients for fetal development, including vita-
Access to the brain is restricted by the presence of two barriers: mins, amino acids, essential sugars, iron, and calcium, are
the blood-brain barrier (BBB) and the blood-cerebrospinal transported by active transport systems from mother to fetus.
fluid barrier (BCSFB). Although neither represents an absolute Many foreign substances can cross the placenta, and the same
barrier to the passage of toxic chemicals into the central ner- factors that dictate the passage of xenobiotics across other bio-
vous system (CNS), many toxicants do not enter the brain in logical membranes are important determinants of placental
appreciable quantities compared to other body tissues. transfer. These include previously discussed attributes includ-
There are four major anatomical and physiologic reasons ing the degree of ionization, lipophilicity, protein binding,
why some toxicants do not readily enter the CNS. First, the molecular weight, blood flow, and the concentration gradient
capillary endothelial cells of the CNS are tightly joined, leaving across the placenta. Among the substances that cross the pla-
few or no pores between the cells, which prevents diffusion of centa by passive diffusion, more lipid-soluble substances attain
polar compounds through paracellular pathways. Second, the a maternal-fetal equilibrium more rapidly. Under steady-state
capillaries in the CNS are to a large extent surrounded by glial conditions, the concentrations of a toxic compound in the
cell processes (astrocytes), which secrete chemical factors that plasma of the mother and fetus are usually the same. The con-
modulate endothelial permeability. Third, the protein concen- centration in the various tissues of the fetus depends on the
tration in the interstitial fluid of the CNS is much lower than ability of fetal tissue to concentrate a toxicant. Differential body
that in other body fluids, limiting the movement of water- composition between mother and fetus may be another reason
insoluble compounds by paracellular transport, which is pos- for an apparent placental barrier. For example, fetuses have
sible in a largely aqueous medium only when such compounds very little fat; hence, they do not accumulate highly lipophilic
are bound to proteins. Fourth, ATP-dependent transporters chemicals.
include members of both ABC and SLC families (Tables 5-1 Besides chemicals, viruses (e.g., rubella virus), cellular
and 5-2). Efflux transporters MDR1, BCRP, and MRP1, 2, 4, pathogens (e.g., syphilis spirochetes), and globulin antibodies
 CHAPTERS Absorption, Distribution, and Excretion of Toxicants 73

can traverse the placenta. In this regard, the placental barrier is metabolism from the body: glomerular filtration, tubular excre-
not as precise an anatomical unit as the BBB. Anatomically, the tion by passive diffusion, and active tubular secretion. (See
placental barrier consists of a number of cell layers—at most Chapter 14 for greater discussion of renal anatomy and phy-
six—interposed between the fetal and maternal circulations. siology). Compounds up to a molecular weight of about 60 kDa
Active transport systems and biotransformation enzymes are are filtered at the glomeruli. The degree of plasma protein binding
differentially expressed through the cell layers. These help pro- affects the rate of filtration, because protein-xenobiotic com-
tect the fetus from some xenobiotics while regulating the plexes are too large to pass through the pores of the glomeruli.
movement of essential nutrients. A toxicant filtered at the glomeruli may remain in the
tubular lumen and be excreted with urine or may be reab-
sorbed across the tubular cells of the nephron back into the
Redistribution of Toxicants
bloodstream. Toxicants with a high lipid/water partition coef-
The most critical factors that affect the distribution of xenobiot- ficient are reabsorbed efficiently, whereas polar compounds
ics are the organ blood flow and its affinity for a xenobiotic. The and ions are excreted with urine. The pH ofurine may vary but
initial phase of distribution is determined primarily by blood is usually slightly acidic (~6.0 to 6.5). Just as the Henderson-
flow to the various parts of the body. Therefore, a well- Hasselbalch calculations determine the absorption of nonion-
perfused organ such as the liver may attain high initial concen- ized compounds from the GI tract, they also determine urinary
trations of a xenobiotic. However, chemicals may have a high excretion. In this case, urinary excretion of the ionized moiety
affinity for a binding site (e.g., intracellular protein or bone is favored, such that bases are excreted to a greater extent at
matrix) or toa cellular constituent (e.g., fat), and, with time, will lower pH whereas excretion of acids predominates at higher
redistribute to these high-affinity sites. urinary pH.
Toxic agents can also be excreted from plasma into urine by
passive diffusion through the tubule. This process is probably
EXCRETION
of minor significance because filtration is much faster than
Toxicants are eliminated from the body by several routes. Many excretion by passive diffusion through the tubules, providing a
xenobiotics, though, have to be biotransformed to more water- favorable concentration gradient for reabsorption rather than
soluble products before they can be excreted into urine excretion.
(Chapter 6). All body secretions appear to have the ability to Xenobiotics can also be excreted into urine by active secre-
excrete chemicals; toxicants have been found in sweat, saliva, tion. This process involves the uptake of toxicants from blood
tears, and milk. into the cells of the renal proximal tubule, with subsequent
efflux from the cell into the tubular fluid from which urine is
formed. Figure 5-7 illustrates the various families of transport-
Urinary Excretion ers expressed in the human kidney that are directly involved in
Toxic compounds are excreted into urine by the same mecha- xenobiotic disposition. There are numerous other transporters
nisms the kidney uses to remove end products of intermediary such as specific glucose transporters or nucleotide transporters

Excretion Reabsorption

filtrate
Glomerular

FIGURE 5-7 Schematic model showing the transport systems in the proximal tubule of the kidney. The families of transporters are
organic-anion transporters (OAT), organic-cation transporters (OCT), multidrug-resistant protein (MDR), multidrug resistance-associated
protein (MRP), peptide transporters (PEP), and urate transporter (URAT).
74 UNIT 2 Disposition ofToxicants

that play a role predominantly in the flux of endogenous liver or passage into the systemic circulation. The liver can
substances that are not presented here. Transporters may be extract compounds from blood and prevent their distribution
expressed on the apical cell membrane where efflux pumps to other parts of the body. Furthermore, the liver is the main site
contribute to tubular secretion and influx pumps are impor- of biotransformation of toxicants, and the metabolites thus
tant for reabsorption. Transporters localized to the basolateral formed may be excreted directly into bile or into the hepatic
membranes serve to transport xenobiotics to and from the sys- venous blood for systemic distribution. Xenobiotics and/or
temic circulation or the renal tubular cells and also contribute their metabolites entering the intestine with bile may be
to reabsorptive and excretory processes. Specific transporters excreted with feces or undergo an enterohepatic circulation.
expressed on the basolateral side of renal tubules in humans Figure 5-8 illustrates the many transporters localized on
include OATs, OCTs, OATP4C1, and a subset of MRPs. Brush hepatic parenchymal cells that move foreign substances from
border transporters include MRPs, MDRs, MATEs, URATs, plasma into liver and from liver into bile. Biliary excretion is
PEPTs, and OAT4. regulated predominantly by xenobiotic transporters present
Because many functions of the kidney are incompletely on the canalicular membrane. Sodium-dependent taurocho-
developed at birth, some xenobiotics are eliminated more late peptide (ntcp) present on the sinusoidal side of the paren-
slowly in newborns than in adults and therefore may be more chymal cell transports bile acids such as taurocholate into the
toxic to newborns. For example, the clearance of penicillin by liver, whereas the bile salt excretory protein (bsep) transports
premature infants is only about 20% of that observed in older bile acids out of the liver cell into the bile canaliculi. The sinu-
children. The renal proximal tubule reabsorbs small plasma soidal membrane of the hepatocyte has a number of transport-
proteins that are filtered at the glomerulus. A toxicant binding ers including organic-anion transporting polypeptide (oatp) 1
those small proteins can be carried into the proximal tubule and 2, and oct that move xenobiotics into the liver. Once
cells and exert toxicity. inside the hepatocyte, the xenobiotic itself can be transported
Species differences in urinary excretion can be explained by into the blood or bile, or be biotransformed by phase | and II
variance in the pH urine, differences in plasma protein binding, drug-metabolizing enzymes to more water-soluble products
and xenobiotic transporter expression, regulation, and function. that are then transported into the bile or back into the blood.
Multidrug-resistant protein one (mdrl) and multidrug
resistance-associated protein two (mrp2) are responsible for
Fecal Excretion transporting xenobiotics into bile, whereas mrp3 and mrp6
Fecal excretion is the second major pathway for the elimination transport xenobiotics back into the blood.
of xenobiotics from the body. Many chemicals in feces directly An important concept relating to biliary excretion is the
transfer from blood into the intestinal contents by passive dif- phenomenon of enterohepatic circulation. Once a compound
fusion. In some instances, rapid exfoliation of intestinal cells is excreted into bile and enters the intestine, it can be reab-
may contribute to the fecal excretion of some compounds. sorbed or eliminated with feces. Many organic compounds are
Intestinal excretion is a relatively slow process that is a major conjugated before excretion into bile. Such polar metabolites
pathway of elimination only for compounds that have low rates are not sufficiently lipid soluble to be reabsorbed. However,
of biotransformation and/or low renal or biliary clearance.

Nonabsorbed Ingesta—In addition to indigestible material, Hepatocyte

varying proportions of nutrients and xenobiotics that are pres-
ent in food or are ingested voluntarily (drugs) pass through the
alimentary canal unabsorbed, contributing to fecal excretion.
Mucosal biotransformation and reexcretion into the intesti-
cs
nal lumen occur with many compounds. It has been estimated )
that 30% to 42% of fecal dry matter originates from bacteria.
Moreover, a considerable proportion of fecally excreted xeno-
biotic is associated with excreted bacteria. However, chemicals
may be profoundly altered by bacteria before excretion with
feces. It seems that biotransformation by intestinal flora favors
reabsorption rather than excretion. Nevertheless, there is evi-
dence that in many instances xenobiotics found in feces derive
from bacterial biotransformation.

Biliary Excretion— The biliary route of elimination is perhaps

FIGURE 5-8 Schematic model showing the transport
the most important contributing source to the fecal excretion of systems in the liver. OATP = organic-anion transporting polypeptide,
xenobiotics and their metabolites. Hepatic anatomy and physi- OCT = organic-cation transporter, BSEP = bile salt excretory protein,
ology and bile formation are discussed in greater detail in MDR = multidrug-resistant protein, MRP = multidrug resistance-
Chapter 13. To summarize, nutrients and xenobiotics in portal associated protein, BCRP = breast cancer resistance protein, and
venous blood from the GI tract are available for uptake by the NTCP = sodium-dependent taurocholate peptide.
 CHAPTERS Absorption, Distribution, and Excretion of Toxicants 7

intestinal microflora may hydrolyze glucuronide and sulfate Chemical

conjugates, making them sufficiently lipophilic for reabsorp- since:
tion and enterohepatic cycling. This principle has been utilized Pharmacological
in the treatment of dimethylmercury poisoning; ingestion of a effect
polythiol resin binds the mercury and thus prevents its reab-
sorption and cycling. Pathological Repair

DNA Repair
Exhalation injury ee

Substances that exist predominantly in the gas phase at body

temperature and volatile liquids are eliminated mainly by the
lungs. Because volatile liquids are in equilibrium with their gas uoljedijday
phase in the alveoli, they may also be excreted via the lungs. The
amount of liquid eliminated via the lungs is proportional to its Altered DNA

vapor pressure. A practical application ofthis principle is seen

FIGURE 5-9 Schematic representation of the disposition and
in the breath analyzer test for determining the amount of etha-
toxic effects of chemicals.
nol in the body.
No specialized transport systems have been described for
the excretion of toxic substances by the lungs. Some xenobiotic CONCLUSION
transporters, including MRP1 and MDR1, have been identified
in the lung, but overall, compounds seem to be eliminated by Humans are in continuous contact with toxic agents.
simple diffusion. Elimination of gases is roughly inversely Depending on their physical and chemical properties, toxicants
proportional to the rate of their absorption. The rate of elimi- may be absorbed by the GI tract, the lungs, and/or the skin. The
nation of a gas with low solubility in blood is perfusion- body has the ability to biotransform and excrete these com-
limited, whereas that of a gas with high solubility in blood pounds into urine, feces, and air. However, when the rate of
is ventilation-limited. absorption exceeds the rate of elimination, toxic compounds
may accumulate, reaching a critical concentration ata target site,
and toxicity may ensue (Figure 5-9). Whether a chemical elicits
Other Routes of Elimination toxicity depends not only on its inherent potency and site speci-
ficity, but also on how an organism can dispose of that toxicant.
Cerebrospinal Fluid—All compounds can leave the CNS
Many chemicals have very low inherent toxicity but have to
with the bulk flow of cerebrospinal fluid (CSF) through arach-
be activated by biotransformation into toxic metabolites and
noid villi, which allow fluid to flow from CSF to the venous sys-
the toxic response depends on the rate of production of toxic
tem. In addition, lipid-soluble toxicants also can exit at the site
metabolites. Alternatively, a very potent toxicant may be detox-
of the BBB. Active transport using the transport systems pres-
ified rapidly by biotransformation. The fundamental and over-
ent in the BCSFB can also remove toxicants.
arching concept is that adverse toxic effects are related to the
unbound concentration of “toxic chemical” at the site of action
Milk—tThe secretion of toxic compounds into milk is extre- (in the target organ), whether a chemical is administered or
mely important because (1) a toxicant may be passed with milk generated by biotransformation in the target tissue or at a dis-
from the mother to the nursing offspring and (2) compounds tant site. Accordingly, the toxic response exerted by chemicals
can be passed from cows to humans by way of dairy products. is critically influenced by the rates of absorption, distribution,
Toxic agents are excreted into milk by simple diffusion. Because biotransformation, and excretion.
milk is more acidic (pH ~ 6.5) than plasma, basic compounds
may be concentrated in milk, whereas acidic compounds may
attain lower concentrations in milk than in plasma. About 3%
BIBLIOGRAPHY
to 4% of milk consists of lipids, and the lipid content of milk
Anzai N, Kanai Y, Endou H: Organic anion transporter family: cur-
after parturition is even higher. Importantly, lipid-soluble rent knowledge. J Pharmacol Sci 100:411-426, 2006.
xenobiotics diffuse along with fats from plasma into the mam- Goodman J: Goodman and Gilman’s The Pharmacological Basis of
mary gland and are excreted with milk during lactation. Therapeutics, 12th ed. New York: McGraw-Hill, 2011.
Klaassen CD, Aleksunes LM: Xenobiotic, bile acid, and cholesterol
transporters: function and regulation. Pharmacol Rev 62:1-96, 2010.
Sweat and Saliva— The excretion of toxic agents in sweat and
Lin JH: Tissue distribution and pharmacodynamics: a complicated
saliva is quantitatively of minor importance. Again, excretion
relationship. Curr Drug Metab 7:39-65, 2006.
depends on the diffusion of the nonionized, lipid-soluble form Myllynen P, Pasanen M, Pelkonen O: Human placenta: a human organ
of an agent. Toxic compounds excreted into sweat may produce for developmental toxicology research and biomonitoring.
dermatitis (inflammation of the skin). Substances excreted in Placenta 26:361-371, 2005.
saliva enter the mouth, where they are usually swallowed to Zhai H, Wilhelm KP, Maibach HI (eds.): Marzulli and Maibach’s Der-
become available for GI absorption. matotoxicology, 7th ed. Boca Raton, FL: CRC Press, 2008.
 UNIT 2 Disposition of Toxicants

QUESTIONS

Biotransformation is vital in removing toxins from the Which of the following most correctly describes the first-
circulation. All of the following statements regarding bio- pass effect?
transformation are true EXCEPT: a. The body is most sensitive to a toxin the first time
a. Many toxins must be biotransformed into a more that it passes through the circulation.
lipid-soluble form before they can be excreted from b. Orally administered toxins are partially removed by
the body. the GI tract before they reach the systemic circulation.
b. The liver is the most active organ in the biotransfor- c. It only results from increased absorption of toxin by
mation of toxins. GI cells.
c. Water solubility is required in order for many toxins It is often referred to as “postsystemic elimination.”
to be excreted by the kidney. e. A majority of the toxin is excreted after the first time
d. The kidney plays a major role in eliminating toxi- the blood is filtered by the kidneys.
cants from the body.
e. The lungs play a minor role in ridding the body of Which of the following is an important mechanism of
certain types of toxins. removing particulate matter from the alveoli?
a. coughing.
Which of the following statements about active transport b. sneezing.
across cell membranes is FALSE? c. blowing one’s nose. ;
a. Unlike simple or facilitated diffusion, active trans- d. absorption into the bloodstream, followed by excre-
port pumps chemicals against an electrochemical or tion via the kidneys.
concentration gradient. e. swallowing. \
b. Unlike simple diffusion, there is a rate at which
active transport becomes saturated and cannot move For a toxin to be absorbed through the skin, it must pass
chemicals any faster. through multiple layers in order to reach the systemic circu-
c. Active transport requires the expenditure of ATP in lation. Which of the following layers is the most important
order to move chemicals against electrochemical or in slowing the rate of toxin absorption through the skin?
concentration gradients. stratum granulosum.
d. Active transport exhibits a high level of specificity for stratum spinosum.
the compounds that are being moved. stratum corneum.
e. Metabolic inhibitors do not affect the ability to per- stratum basale.
form active transport. mat dermis.
ei

Which of the following might increase the toxicity of a A toxin is selectively toxic to the lungs. Which of the fol-
toxin administered orally? lowing modes of toxin delivery would most likely cause
a. increased activity of the mdr _ transporter the LEAST damage to the lungs?
(p-glycoprotein). a. intravenous.
b. increased biotransformation of the toxin by gastroin- b. intramuscular.
testinal cells. c. intraperitoneal.
c. increased excretion of the toxin by the liver into bile. d. subcutaneous.
increased dilution of the toxin dose. e. inhalation.
e. increased intestinal motility.
 CHAPTERS Absorption, Distribution, and Excretion of Toxicants 77

Which of the following is NOT an important site of toxi- 10. Which ofthe following will result in DECREASED excre-
cant storage in the body? tion of toxic compounds by the kidneys?
adipose tissue. a. a toxic compound with a molecular weight of
bone. 25,000 Da.
plasma proteins. b. increased activity of the multidrug-resistance (mdr)
muscle. protein.
ge
enoliver. increased activity of the multiresistant drug
protein (mrp).
Which of the following regarding the blood-brain barrier increased activity of the organic cation transporter.
is TRUE: increased hydrophilicity of the toxic compound.
a. The brains of adults and newborns are equally sus-
ceptible to harmful blood-borne chemicals.
b. The degree of lipid solubility is a primary determi-
nant in whether or not a substance can cross the
blood-brain barrier.
Astrocytes play a role in increasing the permeability
of the blood-brain barrier.
Active transport processes increase the concentra-
tion of xenobiotics in the brain.
The capillary endothelial cells of the CNS possess
large fenestrations in their basement membranes.
 Che Hin AviePa ach GER,

Biotransformation
of Xenobiotics
Andrew Parkinson, Brian W. Ogilvie, David B. Buckley,
Faraz Kazmi, Maciej Czerwinski, and Oliver Parkinson

GENERAL PRINCIPLES Dihydrodiol Dehydrogenase

Molybdenum Hydroxylases
HYDROLYSIS, REDUCTION, AND OXIDATION
Xanthine Oxidoreductase
Hydrolysis Aldehyde Oxidase
Carboxylesterases, Cholinesterases, and Monoamine Oxidase
Paraoxonase Peroxidase-dependent Cooxidation
Prodrugs and Alkaline Phosphatase Flavin Monooxygenases
Peptidases Cytochrome P450
Epoxide Hydrolase Activation of Xenobiotics by Cytochrome P450
Reduction Inhibition of Cytochrome P450
Azo- and Nitro-reduction Induction of Cytochrome P450
Carbonyl Reduction
CONJUGATION
Disulfide Reduction
Sulfoxide and N-Oxide Reduction Glucuronidation
Quinone Reduction Sulfonation
Dehalogenation Methylation
Oxidation Acetylation
Alcohol Dehydrogenase Amino Acid Conjugation
Aldehyde Dehydrogenase Glutathione Conjugation

KEY POINTS

« Biotransformation is the metabolic conversion of endog- group (—OH, —NH,, —SH, or —COOH), and usually
enous and xenobiotic chemicals to more water-soluble result in only a small increase in hydrophilicity.
compounds. Phase II biotransformation reactions include glucuroni-
» Xenobiotic biotransformation is accomplished by a lim- dation, sulfonation (more commonly called sulfation),
ited number of enzymes with broad substrate specificities. acetylation, methylation, and conjugation with glutathi-
Phase I reactions involve hydrolysis, reduction, and oxi- one (mercapturic acid synthesis), which usually result in
dation. These reactions expose or introduce a functional increased hydrophilicity and elimination.

79
80 UNIT 2 Disposition of Toxicants

The enzymes that catalyze xenobiotic biotransformation are xenobiotic tend to proceed quicker at each subsequent step,
often called drug-metabolizing enzymes. The acronym ADME which prevents the accumulation ofintracellular metabolites.
stands for absorption, distribution, metabolism, and elimina- Were it not for the low catalytic turnover of CYP (one
tion. This acronym is widely used in the pharmaceutical indus- molecule of which may take several seconds or minutes to
try to describe the four main processes governing drug oxidize a single drug molecule), it would not be possible
disposition. The acronym is sometimes extended to include to achieve the once-a-day dosing characteristic of a large
drug transport (AMDET) or drug toxicity (ADME-Tox). This number ofdrugs.
chapter describes some fundamental principles of xenobiotic Point 5 Hydrolysis, reduction, and oxidation expose or
biotransformation, and describes the major enzyme systems introduce a functional group (such as -OH, —NH,, —SH,
involved in the biotransformation (or metabolism) of drugs or —COOH) that can be converted to a water-soluble conju-
and other xenobiotics. gate. The functional group introduced or exposed by hydroly-
sis, reduction, or oxidation must be nucleophilic (in the case of
glucuronidation, sulfonation, methylation, acetylation, and
GENERAL PRINCIPLES
conjugation with glycine or taurine) or electrophilic (in the
The following points, which might be considered principles or case of glutathionylation). The first three reactions (hydrolysis,
rules, apply in the majority of cases: reduction, and oxidation) are often called Phase 1 reactions,
Point 1 Xenobiotic biotransformation or drug metabolism is and the conjugation reactions are often called Phase 2
the process of converting lipophilic (fat-soluble) chemicals, reactions.
which are readily absorbed from the gastrointestinal tract and Point 6 Oxidation, reduction, hydrolysis, methylation, and
other sites, into hydrophilic (water-soluble) chemicals, which acetylation generally cause a modest increase in the water solu-
are readily excreted in urine or bile. There are exceptions even bility of a xenobiotic, whereas glucuronidation, sulfonation,
to this most basic rule. For example, acetylation and methyla- glutathionylation, and amino acid conjugation generally cause
tion are biotransformation reactions that can actually decrease a marked increase in hydrophilicity.
the water solubility of certain xenobiotics. Point 7 Xenobiotics can undergo biotransformation both by
Point 2 The biotransformation of xenobiotics is catalyzed by enzymes that normally participate in intermediary (endobi-
various enzyme systems that can be divided into four catego- otic) metabolism and by enzymes within gut microflora.
ries based on the reaction they catalyze: (1) hydrolysis (e.g., Point 8 Just as some xenobiotics are biotransformed by the
carboxylesterase); (2) reduction (e.g., carbonyl reductase); so-called endobiotic-metabolizing enzymes (Point 7), certain
(3) oxidation (e.g., cytochrome P450 [CYP]); and (4) conjuga- endobiotics are biotransformed by the so-called xenobiotic-
tion (e.g., UDP-glucuronosyltransferase [UGT]). The mamma- metabolizing enzymes. For example, the same CYP enzymes
lian enzymes involved in the hydrolysis, reduction, oxidation, implicated in xenobiotic biotransformation also contribute to
and conjugation of xenobiotics are listed in Table 6-1, together the hepatic catabolism of steroid hormones, and the same
with their principal subcellular location. UGTs that conjugate xenobiotics also glucuronidate biliru-
Point 3 In general, individual xenobiotic-biotransforming bin, thyroid hormones, and steroid hormones. On a case-by-
enzymes are located in a single organelle. In Table 6-1, some case basis, there is often no clear-cut distinction between
enzymes are listed with two or more subcellular locations. endobiotic- and xenobiotic-biotransforming enzymes.
Point 4 In general, xenobiotic biotransformation is accom- Point 9 Several xenobiotic-biotransforming enzymes are
plished by a limited number of enzymes with broad substrate inducible, meaning their expression can be increased (upreg-
specificities. In humans, e.g., 2 CYP enzymes—namely, ulated) usually in response to exposure to high concentra-
CYP2D6 and CYP3A4—metabolize over half the orally effec- tions of xenobiotics. Xenobiotics can act as ligands for certain
tive drugs in current use. Many of the enzymes involved in receptors (so-called xenosensors). Activated xenosensors
xenobiotic biotransformation are arranged in families and sub- (i.e., those bound to xenobiotics) interact with DNA-binding
families and named according to nomenclature systems based proteins which upregulate the transcription of various genes
on the primary amino acid sequence of the individual enzymes. encoding for xenobiotic-biotransforming enzymes. The
The convention of using italic and regular letters to distinguish major xenosensors are aryl hydrocarbon receptor (AhR),
between the gene and gene products (mRNA and protein), which induces CYP1 enzymes, the constitutive androstane
respectively, and the convention of using lower case letters to receptor (CAR) and the pregnane X receptor (PXR), which
designate mouse genes and gene products is not followed in induce CYP2B, 2C, and 3A enzymes, and the peroxisome
this chapter. proliferator—activated receptor-a (PPARQ), which induces
The structure (i.e., amino acid sequence) ofa given xenobiotic- CYP4 enzymes.
biotransforming enzyme may differ among individuals, which Certain xenosensors are activated by endogenous ligands
can give rise to differences in rates of drug metabolism. The (e.g., bilirubin, bile acids, and fatty acids activate CAR, PXR,
broad substrate specificity of xenobiotic-biotransforming and PPARQ, respectively). Induction is a reversible, adaptive
enzymes makes them catalytically versatile but slow compared response to xenobiotic exposure. Induction is also a pleiotropic
with most other enzymes (with the exception of hydrolytic reac- response: activation of AhR, CAR, PXR, PPARa, and Nrf2 all
tions). The sequential oxidation, conjugation, and transport ofa results in alterations in the expression of numerous genes.
 CHAPTER 6 Biotransformation of Xenobiotics 81

TABLE 6-1 General pathways of xenobiotic biotransformation and their major subcellular location.

Reaction Enzyme or Specific Reaction Localization

Hydrolysis Carboxylesterase Microsomes, cytosol, lysosomes, blood

Butyrylcholinesterase Plasma and most tissues

Acetylcholinesterase Erythrocytes and most tissues

Paraoxonases Plasma, microsomes, inner mitochondrial membrane

Alkaline phosphatase Plasma membrane

Peptidase Blood, lysosomes

8-Glucuronidase Microsomes, lysosomes, microflora

Epoxide hydrolase Microsomes, cytosol

Reduction Azo- and nitro-reduction Microflora

Carbonyl (aldo-keto) reduction Cytosol, microsomes, blood

Disulfide reduction Cytosol

Sulfoxide reduction Cytosol

Quinone reduction Cytosol, microsomes

Dihydropyrimidine dehydrogenase Cytosol

Reductive dehalogenation Microsomes

Dehydroxylation (mARC)* Mitochondria

Dehydroxylation (aldehyde oxidase) Cytosol

Oxidation Alcohol dehydrogenase Cytosol

Aldehyde dehydrogenase Mitochondria, cytosol

Aldehyde oxidase Cytosol

Xanthine oxidase Cytosol

Class |amine oxidases

Monoamine oxidase-A and B Inner mitochondrial membrane, platelets

Class Il amine oxidases (CuAQOs)

Diamine oxidase Microsomes, extracellular matrix

Peroxidase Microsomes, lysosomes, saliva

Flavin-monooxygenases Microsomes

Cytochrome P450 Microsomes, mitochondria

Conjugation . UDP-glucuronosyltransferase Microsomes

Sulfotransferase Cytosol

Glutathione transferase Cytosol, microsomes, mitochondria

Amino acid transferase Mitochondria, microsomes

N-Acetyltransferase Mitochondria, cytosol

Methyltransferase Cytosol, microsomes, blood

*mARC, mitochondrial amidoxime-reducing component.

82 UNIT 2 Disposition ofToxicants

Suppression (down-regulation) of drug-metabolizing enzymes qualitative and quantitative aspects of xenobiotic biotranstor-
is often associated with inflammatory diseases (such as arthritis), mation and toxicity.
cancer, infectious diseases (both bacterial and viral), vaccination, Point 17 In sexually mature rats and, to a lesser extent, mice
and treatment with certain proinflammatory biologics (thera- there are marked gender differences in the expression of cer-
peutic proteins). These disease processes activate nuclear factor- tain xenobiotic-biotransforming enzymes (both oxidative and
kappa B (NF-KB) and other nuclear receptors, which suppress the conjugating enzymes). In other species, including humans,
expression and induction of CYP and other xenobiotic- gender differences either do not exist or generally represent
metabolizing enzymes. This is because activated NF-KB sup- less than a twofold difference.
presses all four xenosensors (AhR, CAR, PXR, and PPAR) as Point 18 Large interindividual differences in pharmacokinetic
well as several other nuclear receptors. By reversing the disease parameters upon administration or exposure to a chemical can
process—such as lessening the inflammation associated with reflect genetically determined differences in the activity of
rheumatoid arthritis—some biologics (large drug molecules such xenobiotic-biotransforming enzymes or transporters (genetic
as monoclonal antibodies and other types of therapeutic pro- polymorphisms) or environmental factors, such as drug—drug
teins) can reverse the suppression of drug-metabolizing enzymes interactions. The study of the causes, prevalence, and impact of
and restore their activity to normal (pre-disease) levels. heritable differences in xenobiotic-biotransforming enzymes
Point 10 Xenobiotic biotransformation can alter the bio- is known as pharmacogenetics.
logical properties of a xenobiotic. The biotransformation of Point 19 Stereochemical aspects can play an important role
drugs can result in (1) a loss of pharmacological activity, in the interaction between a xenobiotic and its biotransform-
(2) no change in pharmacological activity, or (3) an increase in ing enzyme (from both a substrate and an inhibitor perspec-
pharmacological activity. tive), and xenobiotic-biotransforming enzymes can play a key
Point 11 The toxicity and potential carcinogenicity of electro- role in converting one stereoisomer to another, a process
philic metabolites produced by CYP and other xenobiotic- known as mutarotation or inversion af configuration.
biotransforming enzymes is reduced and often altogether elimi- Point 20 Mass spectrometry is widely used to characterize
nated by their conjugation with reduced glutathione (GSH). the structure of metabolites. Certain xenobiotic reactions are
Point 12 The biotransformation of some xenobiotics results associated with discrete changes in mass: the loss of 2 atomic
in the production of reactive oxygen species (ROS), which can mass units (amu) signifies dehydrogenation, whereas the loss
cause cell toxicity (including DNA damage) through oxidative of 14 amu usually signifies demethylation (—CH,). Several
stress and lipid peroxidation. GSH, GSTs, and glutathione per- reactions result in an increase in mass, including reduction
oxidases (GPXs) all limit the toxic effects of ROS just as they (+2 amu = 2H), methylation (+14 amu = CH,), oxidation
limit the toxicity of reactive metabolites formed directly from (+16 amu = O), hydration (+18 amu = H,O), acetylation
xenobiotics. Oxidative stress and the formation of electrophilic (+42 amu = C,H,O), sulfonation (+80 amu = SO,), gluc-
metabolites reduce GSH levels and thus result in the concur- uronidation (+176 amu = C,H,O,), carbamoyl glucuronida-
rent oxidation of KEAP-1, which then releases Nrf2, which in tion (+220 amu = C,H,O,), and conjugation with GSH
turn upregulates the expression of enzymes that detoxify elec- (+305 amu = C,,H,;N,O0,S). Conjugation of acidic drugs with
trophilic metabolites (e.g., epoxides) and those metabolites CoA (to form acyl-CoA thioesters) increases mass by 749 amu,
that generate ROS (e.g., quinones). but these conjugates are not transported out ofcells and, hence,
Point 13 The balance between activation and detoxication are not detected in blood, bile, or urine.
by xenobiotic-biotransforming enzymes is often a key deter-
minant of chemical toxicity, and is often the basis for organ or
species differences in toxicity. HYDROLYSIS, REDUCTION,
Point 14 Exposure to xenobiotics (especially drugs) is largely AND OXIDATION
through oral ingestion, and the small intestine and liver are
highly developed to limit systemic exposure to orally ingested Hydrolysis
xenobiotics, a process known as first-pass elimination (or pre- Carboxylesterases, Cholinesterases, and Paraoxonase—
systemic elimination). The enterocytes at the tips of the small The hydrolysis of carboxylic acid esters, amides, and thioesters
intestinal villi express the efflux transporters P-glycoprotein is largely catalyzed by carboxylesterases and by two cholines-
(ABCB1 or MDR1) and BCRP (ABCG2), which serve to limit terases: true acetylcholinesterase in erythrocyte membranes
xenobiotic absorption. Enterocytes and hepatocytes express and pseudocholinesterase, which is also known as butyrylcho-
high levels of certain CYP and UGT enzymes, which biotrans- linesterase and is located in serum. Phosphoric acid esters
form a wide variety of xenobiotics. are hydrolyzed by paraoxonase, a serum enzyme also known
Point 15 Although the small intestine and liver contain the as aryldialkylphosphatase. Phosphoric acid anhydrides are
highest concentrations, xenobiotic-biotransforming enzymes hydrolyzed by a related organophosphatase.
are nevertheless widely distributed throughout the body. Carboxylesterases in serum and tissues and serum cholines-
Point 16 Species differences in xenobiotic-biotransforming terase collectively determine the duration and site of action of
enzymes are often the basis for species differences in both the certain drugs. The hydrolysis of xenobiotic esters and amides
 CHAPTER 6 Biotransformation of Xenobiotics 83

in humans is largely catalyzed by just two carboxylesterases Epoxide hydrolase is one of the several inducible enzymes in
called hCE1 and hCE2. liver microsomes. Induction of epoxide hydrolase is invariably
Carboxylesterases are glycoproteins that are present in associated with the induction of cytochrome P450.
serum and most tissues. Carboxylesterases hydrolyze numerous
endogenous lipid compounds and generate pharmacologically
active metabolites from several ester or amide prodrugs. In Reduction
addition, carboxylesterases may convert xenobiotics to toxic Certain metals and xenobiotics containing an aldehyde, ketone,
and tumorigenic metabolites. disulfide, sulfoxide, quinone, N-oxide, alkene, azo, or nitro
Cholinesterases play an important role in limiting the toxic- group are often reduced in vivo. The reaction may proceed
ity of organophosphates, which inhibit acetylcholinesterase enzymatically or nonenzymatically by interaction with reduc-
and thus the termination of acetylcholine action. Factors that ing agents, such as the reduced forms of glutathione (GSH),
decrease esterase activity potentiate the toxic effects of organo- FAD, FMN, and NADP. Likewise, enzymes, such as alcohol
phosphates, whereas factors that increase serine esterase activ- dehydrogenase (ADH), aldehyde oxidase, and cytochrome
ity have a protective effect. P450, can catalyze both reductive and oxidative reactions
Paraoxonases, calcium-dependent enzymes containing a depending on the substrate and conditions.
critical sulfhydryl group, catalyze the hydrolysis of a broad
range of organic compounds, including lactones. Thus, “lac- Azo- and Nitro-reduction—Azo- and nitro-reduction are
tonase’ is a more encompassing name for this group of enzymes. catalyzed by intestinal microflora and under certain conditions
(i.e., low oxygen tension), by two liver enzymes: cytochrome
Prodrugs and Alkaline Phosphatase—Many drugs are P450 and NADPH-quinone oxidoreductase (also known as
designed as prodrugs, meaning biotransformation is required DT-diaphorase). The reactions require NADPH and are
to produce the active species. Hydrolytic enzymes such as car- inhibited by oxygen. The anaerobic environment of the lower
boxylesterases, cholinesterases, and alkaline phosphatase are gastrointestinal tract is well suited for azo- and nitro-reduction.
commonly invoked for this purpose. Potent anticancer agents
exist that are highly selective for certain sites and only release Carbonyl Reduction— The reduction of certain aldehydes to
active drug in the vicinity of tumor cells. primary alcohols and of ketones to secondary alcohols is cata-
lyzed by NAD(P)H-dependent reductases belonging to one of
Peptidases—Numerous human peptides and several recom- the two superfamilies, the aldo-keto reductases (AKRs) and the
binant peptide hormones, growth factors, cytokines, soluble short-chain dehydrogenases/reductases (SDRs). AKRs are
receptors, and monoclonal antibodies are used therapeutically. members of a superfamily of cytosolic enzymes that reduce
These peptides are hydrolyzed in the blood and tissues by a vari- both xenobiotic and endobiotic compounds. SDR carbonyl
ety of peptidases, which cleave the amide linkage between adja- reductases are monomeric enzymes, present in blood and the
cent amino acids. cytosolic fraction of various tissues. Hepatic carbonyl reductase
activity is present mainly in the cytosolic fraction, with a differ-
Epoxide Hydrolase—Epoxide hydrolase catalyzes the ent carbonyl reductase present in the microsomes.
trans-addition of water to alkene epoxides and arene oxides,
and is present in virtually all tissues. It plays an important Disulfide Reduction—Disulfide reduction by glutathione
role in detoxifying electrophilic epoxides that might other- is a three-step process, the last step of which is catalyzed by
wise bind to proteins and nucleic acids and cause cellular glutathione reductase. The first steps can be catalyzed by
toxicity and genetic mutations. There are five distinct forms GST, or they can occur nonenzymatically.
of epoxide hydrolase in mammals: microsomal epoxide
hydrolase (mEH), soluble epoxide hydrolase (sEH), choles- Sulfoxide and N-Oxide Reduction—Thioredoxin-dependent
terol epoxide hydrolase, LTA, hydrolase, and hepoxilin enzymes in liver and kidney cytosol can reduce sulfoxides,
hydrolase. The latter three enzymes appear to hydrolyze which were formed by cytochrome P450. Under reduced oxy-
endogenous epoxides exclusively and have virtually no gen tension, the NADPH-dependent reduction of N-oxides in
capacity to detoxify xenobiotic oxides. liver microsomes may be catalyzed by cytochrome P450 or
In contrast to the high degree of substrate specificity dis- NADPH-cytochrome P450 reductase.
played by the cholesterol, LTA,, and hepoxilin epoxide hydro-
lases, the mEH and sEH hydrolyze many alkene epoxides and Quinone Reduction—Quinones can be reduced to hydroqui-
arene oxides. Generally, these two forms of epoxide hydrolases nones by two cytosolic flavoproteins, NQO1 and NQO2, with-
and cytochrome P450 enzymes, which are often responsible out oxygen consumption. NADPH-quinone oxidoreductase-1
for producing the toxic epoxides, have a similar cellular local- (DT-diaphorase) and NADPH-quinone oxidoreductase-2
ization that presumably ensures the rapid detoxication of have different substrate specificities. The two-electron reduc-
alkene epoxides and arene oxides generated during the oxida- tion of quinones also can be catalyzed by carbonyl reductase.
tive biotransformation of xenobiotics. This pathway of quinone reduction is essentially nontoxic and
84 UNIT 2 Disposition of Toxicants

is not associated with oxidative stress because no oxygen are cytosolic, NADPH-requiring oxidoreductases that oxidize
is used. various polycyclic aromatic hydrocarbons to potentially toxic
The second pathway of quinone reduction catalyzed by metabolites.
microsomal NADPH-cytochrome P450 reductase results in
the formation of asemiquinone free radical by a one-electron Molybdenum Hydroxylases—Two major molybdenum
reduction of the quinone. The oxidative stress associated with hydroxylases or molybdozymes participate in the biotransfor-
autooxidation of a semiquinone free radical, which produces mation of xenobiotics: aldehyde oxidase and xanthine oxidore-
superoxide anion, hydrogen peroxide, and other active oxygen ductase (also known as xanthine oxidase [XO]). Sulfite oxidase,
species, can be extremely cytotoxic. a third molybdozyme, oxidizes sulfite, an irritating air pollut-
The properties of the hydroquinone determine whether, ant, to sulfate, which is relatively innocuous. All three molybdo-
during the metabolism of quinine-containing xenobiotics, zymes are flavoprotein enzymes. During substrate oxidation,
NQO functions as a protective antioxidant or a prooxidant aldehyde oxidase and XO are reduced and then reoxidized by
activator leading to the formation of reactive oxygen species molecular oxygen. The oxygen incorporated into the xenobiotic
and reactive semiquinone free radicals. is derived from water rather than oxygen, which distinguishes
the oxidases from oxygenases. Xenobiotics that are good sub-
Dehalogenation—There are three major mechanisms for strates for molybdozymes tend to be poor substrates for cyto-
removing halogens (F, Cl, Br, and I) from aliphatic xenobiotics: chrome P4590, and vice versa.
(1) reductive dehalogenation involves replacement of a halogen
with hydrogen, (2) oxidative dehalogenation replaces a halogen Xanthine Oxidoreductase— Xanthine dehydrogenase (XD)
and hydrogen on the same carbon atom with oxygen, and and XO are two forms of,the same enzyme involved in purine
(3) double dehalogenation involves the elimination of two halo- degradation that differ in the electron acceptor used in the final
gens on adjacent carbon atoms to forma carbon-carbon double step of catalysis. In the case of XD, the final electron acceptor is
bond. A variation of this third mechanism is dehydrohalogena- NAD*, whereas in the case of XO the final electron acceptor
tion, in which a halogen and hydrogen on adjacent carbon is oxygen. XD is converted to XO by oxidation of cysteine resi-
atoms are eliminated to form a carbon-carbon double bond. dues and/or proteolytic cleavage. The conversion of XD to XO
in vivo may be important in ischemia-reperfusion injury,
lipopolysaccharide-mediated tissue injury, and alcohol-induced
Oxidation hepatotoxicity. XO contributes to oxidative stress and lipid
Alcohol Dehydrogenase— ADH is a cytosolic enzyme present peroxidation because the oxidase activity of XO involves
in several tissues including the liver, which has the highest levels, reduction of molecular oxygen, which can lead to the
the kidney, the lung, and the gastric mucosa. There are five major formation of ROS.
classes of ADH. The class |ADH isozymes (a@-ADH, 3-ADH, and Allopurinol and other xanthine oxidoreductase inhibitors
7-ADH) are responsible for the oxidation of ethanol and other are being evaluated for the treatment of various types of
small aliphatic alcohols. Class I] ADH (7t-ADH) is primarily ischemia-reperfusion and vascular injury that appear to be
expressed in liver where it preferentially oxidizes larger aliphatic mediated, at least in part, by xanthine oxidoreductase.
and aromatic alcohols. Long-chain alcohols (pentanol and larger)
and aromatic alcohols are preferred substrates for class II] ADH Aldehyde Oxidase—The molybdozyme aldehyde oxidase
(x-ADH). Class IV ADH (o-ADH or p-ADH), which is not exists only in the oxidase form. Cytosolic aldehyde oxidase
expressed in liver, is the most active of the medium-chain ADHs transfers electrons to molecular oxygen, which can generate
in oxidizing retinol. Class V ADH has no subunit designation. reactive oxygen species and lead to lipid peroxidation.
Aldehyde oxidase plays an important role in the catabolism
Aldehyde Dehydrogenase—Aldehyde dehydrogenase of biogenic amines and catecholamines.
(ALDH) oxidizes aldehydes to carboxylic acids with NAD* as
the cofactor. The enzymes also have esterase activity. The Monoamine Oxidase—Monoamine oxidases (MAO) are
19 identified ALDHs differ in their primary amino acid involved in the oxidative deamination of primary, secondary,
sequences and in the quaternary structure. In contrast and tertiary amines, including serotonin and a number of xeno-
to ALDH1A1 and ALDH2, which specifically reduce NAD*, biotics. Oxidative deamination of a primary amine produces
ALDH3A1 reduces both NAD* and NADP*. ammonia and an aldehyde, whereas oxidative deamination
As shown in Figure 6-1, ALDH2 is a mitochondrial enzyme of asecondary amine produces a primary amine and an alde-
that, by virtue of its high affinity, is primarily responsible for hyde. The aldehydes formed by MAO are usually oxidized
oxidizing simple aldehydes, such as acetaldehyde. Genetic further by other enzymes to the corresponding carboxylic
deficiencies in other ALDHs impair the metabolism of other acids. MAO is located throughout the brain and in the outer
aldehydes. membrane of mitochondria of the liver, kidney, intestine, and
blood platelets.
Dihydrodiol Dehydrogenase—The AKR superfamily The substrate is oxidized by MAO, which itself is reduced
includes several forms of dihydrodiol dehydrogenases, which using FAD. The oxygen incorporated into the substrate is
 CHAPTER 6 Biotransformation of Xenobiotics 85

—
SS
Endoplasmic reticulum | _
cakevi
NADP* + H,0 CH;CH
N Mitochondria

NADPH + H*
+O,

ve
CH,CH,OH NADH + H*
CH3C
ye
Ethanol
Acetaldehyde

l : CH3C
Peroxisames ‘oH

H Acetic acid

‘ H,0, 2H,0
wound
FIGURE 6-1 Oxidation of ethanol to acetaldehyde by alcohol dehydrogenase (ADH), cytochrome P450 (CYP2E1), and catalase.
Note: The oxidation of ethanol to acetic acid involves multiple organelles.

derived from water, not molecular oxygen. The catalytic cycle of the peroxide oxygen to the xenobiotic, as shown in Figure 6-2
is completed by reoxidation of the reduced enzyme (FADH, — for the conversion of substrate X to the oxidized product XO.
FAD) by oxygen, which generates hydrogen peroxide. Xenobiotics that serve as electron donors, such as amines
Semicarbazide-sensitive amine oxidase (SSAO) is a copper- and phenols, can also be oxidized to free radicals during the
containing enzyme that catalyzes fundamentally the same reduction of a hydroperoxide by peroxidases. In this case, the
reaction as MAO. It can be distinguished from MAO by its sen- hydroperoxide is still converted to the corresponding alcohol,
sitivity to inhibitors and presence in plasma and various cell but the peroxide oxygen is reduced to water instead of being
surfaces, whereas MAO is found in mitochondria. incorporated into the xenobiotic. For each molecule of hydro-
peroxide reduced (which is a two-electron process), two mole-
Peroxidase-dependent Cooxidation—Oxidative bio- cules of xenobiotic can be oxidized (each by a one-electron
transformation of xenobiotics by peroxidases couples the reduc- process). Many of the metabolites produced are reactive elec-
tion of hydrogen peroxide and lipid hydroperoxides to the trophiles that can cause tissue damage.
oxidation of other substrates via a process known as cooxidation. PHS2 may play at least two distinct roles in tumor forma-
An important peroxidase is prostaglandin H synthetase (PHS), tion: it may convert certain xenobiotics to DNA-reactive
which possesses two catalytic activities: a cyclooxygenase that metabolites and initiate tumor formation, and it may promote
converts arachidonic acid to prostaglandins and a peroxidase subsequent tumor growth, perhaps through formation of
that converts the hydroperoxide to the corresponding alcohol growth-promoting eicosanoids.
PGH,. PHS has two forms (PHS1 and PHS2) that are better PHS is unique among peroxidases because it can both gener-
known as two forms of cyclooxygenase, namely, COX1 and ate hydroperoxides and catalyze peroxidase-dependent reac-
COX2. PHS peroxidases are important in the activation of xeno- tions, as shown in Figure 6-2. Xenobiotic biotransformation
biotics to toxic or tumorigenic metabolites, particularly in by PHS is controlled by the availability of arachidonic acid,
extrahepatic tissues that contain low levels of cytochrome P450. whereas conversion by other peroxidases is controlled by the
Oxidation of xenobiotics by peroxidases involves direct transfer availability of hydroperoxide substrates.
86 UNIT 2 Disposition of Toxicants

NADP* NADPH
H,0 FMO +H" RSS
| FAD
|
{ |

|Arachidonic acid ]

ao Cyclooxygenase
: 07+ 0)
| |
| FMO
|
| FMO
FADHOOH FADH,
| NADP* NADP*

|
| xO ‘ per
=O)
OO

| FMO
3 FADHOOH |
‘ NADP*
(PHS)
synthase
Prostaglandin
H
FIGURE 6-3 Catalytic cycle of flavin monooxygenase (FMO).
[___»i p eroxidase
id es
X and XO are the xenobiotic substrate and oxygenated product,
respectively. The C(4a)-hydroperoxyflavin and C(4a)-hydroxyflavin of
XO or 2X+ + H,O FAD are depicted as FADHOOH and FADHOH, respectively.

NADP‘. This final step is rate-limiting, and it occurs after

substrate oxygenation.

Cytochrome P450—The cytochrome P450 (CYP) system

ranks first in terms of catalytic versatility and the sheer number
of xenobiotics it detoxifies or activates. The highest concentra-
tion of CYP enzymes involved in xenobiotic biotransformation
is found in hepatic endoplasmic reticulum (microsomes), but
CYP enzymes are present in virtually all tissues. All CYP
enzymes are heme-containing proteins that catalyze the mono-
oxygenation of one atom of oxygen into a substrate, and the
other oxygen atom is reduced to water with reducing equiva-
Prostaglandins Thromboxane A, lents derived from NADPH.
(PGD, PGE, PGF,.)
During catalysis, CYP does not interact directly with
NADPH or NADH. In the endoplasmic reticulum, electrons
FIGURE 6-2 Cooxidation of xenobiotics (X) during the are relayed from NADPH to cytochrome P450 via a flavopro-
conversion of arachidonic acid to PGH, by prostaglandin H synthase. tein called NADPH-cytochrome P450 reductase. In mito-
chondria, electrons are transferred from NADPH to CYP via
ferredoxin and ferredoxin reductase.
Flavin Monooxygenases—Liver, kidney, intestine, brain, There are notable exceptions to the principle that cyto-
and lung contain one or more FAD-containing monooxygen- chrome P450 requires a second enzyme (i.e., a flavoprotein) for
ases (FMO) that oxidize the nucleophilic nitrogen, sulfur, and catalytic activity. One exception applies to thromboxane A syn-
phosphorus heteroatom of various xenobiotics. The mamma- thase (CYP5A1) and prostaglandin I, synthase (prostacyclin
lian FMO gene family comprises five microsomal enzymes that synthase or CYP8A1), which are involved in the conversion of
require NADPH and O,, and many ofthe reactions catalyzed by arachidonic acid to eicosanoids. In both cases, cytochrome
FMO can also be catalyzed by cytochrome P450. P450 functions as an isomerase and catalyzes a rearrangement
The mechanism of catalysis by FMO is depicted in of the oxygen atoms introduced into arachidonic acid by cyclo-
Figure 6-3. After the FAD moiety is reduced to FADH, by oxygenase. The second exception involves two CYP enzymes
NADPH, the oxidized cofactor NADP* remains bound to the expressed in the bacterium Bacillus megaterium. These CYP
enzyme. FADH, then binds oxygen to produce a relatively sta- enzymes are considerably larger than most CYP enzymes
ble peroxide. During the oxygenation of xenobiotics, the flavin because the P450 moiety and oxidoreductase flavoprotein are
peroxide oxygen is transferred to the substrate (depicted as expressed in a single protein encoded by a single gene.
X — XO in Figure 6-3). The final step in the catalytic cycle Cytochrome P450 and NADPH-cytochrome P450 reduc-
involves restoration of FAD to its oxidized state and release of tase are embedded in the phospholipid bilayer of the
 CHAPTER 6 Biotransformation of Xenobiotics 87

endoplasmic reticulum, which facilitates their interaction. As Cytochrome P450 catalyzes the following types of oxidation
shown in Figure 6-4, the first part of the catalytic cycle involves reactions:
the activation of oxygen, and the final part involves substrate 1. hydroxylation of an aliphatic or aromatic carbon
oxidation, which entails the abstraction of a hydrogen atom or . epoxidation of a double bond
an electron from the substrate followed by oxygen rebound . heteroatom (S-, N-, and I-) oxygenation and N-hydroxylation
(radical recombination). Following the binding of substrate to . heteroatom (O-, S-, N-, and Si-) dealkylation
the CYP enzyme, the heme iron is reduced from the ferric . oxidative group transfer
(Fe**) to the ferrous (Fe**) state by the addition of a single elec- . cleavage of esters
tron from NADPH-cytochrome P450 reductase. Release of the WS
NW
WW. dehydrogenation
oxidized substrate returns cytochrome P450 to its initial state. Liver microsomes from all mammalian species contain
If the catalytic cycle is interrupted, oxygen is released as super- numerous P450 enzymes, each with the potential to catalyze
oxide anion (O,~) or hydrogen peroxide (H,O,). the various reactions shown in Figures 6-5 to 6-12. In general,

]
Product (ROH)
@) Substrate (RH)

@©|Por “+e, Fe’ IV =O

—
co |:
RH

®) |“Cys —Fel'0OH
RH

Other reactions

One-electron reduction C (Cys—Fe"RH) A (Cys—Fel" + RH*)

Superoxide anion production D (Cys—Fe"'O5 RH) B (Cys—Fel" RH) + OF

Hydrogen peroxide production E (~Cys—Fe!'O> RH) + 2H* B (Cys—Fel"RH)+H,O0,

Hydrogen peroxide shunt B (Cys—Fe""! RH) +H 0, F (~Cys—Fel'OOH RH) +H* —

| Peroxide shunt to form

' Compound |
B (Cys—Fe! RH) + XOOH G (Por’*Fe'’ =O RH) + XOH

FIGURE 6-4 Catalytic cycle of cytochrome P450. Cytochrome P450 is represented as Cys-Fe""", where Cys represents the fifth ligand
(a cysteine thiolate) to the ferric heme iron. RH and ROH represent the substrate and product (hydroxylated metabolite), respectively. The
intermediates in the catalytic cycle are as follows: A, ferric resting state; B, substrate bound; C, ferrous intermediate; D, ferrisuperoxo anion
intermediate; E, ferriperoxo intermediate with an electron delocalized over the Cys thiolate bond; F, ferrihydroperoxy intermediate (with a
negative charge on the Cys thiolate bond); G, compound I, an iron'’-oxo porphyrin cation, which is responsible for most substrate oxidation
reactions; H, enzyme in its resting state prior to the release of product formed by hydrogen abstraction followed by oxygen rebound. Fe", Fe",
Fe’, and Fe” refer to iron in the ferrous, ferric, ferryl, and perferryl state, respectively. It should be noted that although it is written as por**Fe=O,
compound | is in the highly oxidized perferryl (Fe’) state when the oxidation state of the porphyrin ring is also taken into account.
88 UNIT 2 Disposition ofToxicants

O O O O
H I I ii

S Ni GNA Hct, ())$- Wee = SS

HI CYP2C9
O — O
Tolbutamide Hydroxymethyltolbutamide

OH
w-1 Hydroxylation

COOH CYP4A 11-Hydroxylauric acid

LOLI LEE
; OO OO OE, COOH
Lauric acid
wHydroxylation 12-Hydroxylauric acid

OH
OH

CYP3A4
=

0 O
OH
Testosterone 63-Hydroxytestosterone
——

FIGURE 6-5 Examples of reactions catalyzed by cytochrome P450: hydroxylation of aliphatic carbon.
»
\

The levels and activity of each CYP enzyme vary from one indi-
HO O vidual to the next, due to environmental and/or genetic factors.
Oo CYP2E1
> oH =————> Decreased CYP enzyme activity can result from (1) a genetic
‘a N a N mutation that either blocks the synthesis of a CYP enzyme or
Chlorzoxazone 6-Hydroxychlorzoxazone leads to the synthesis of a catalytically compromised, inactive,
or unstable enzyme, which gives rise to the poor and intermediate
metabolizer genotypes; (2) exposure to an environmental factor
(such as an infectious disease or an inflammatory process) that

tL
O O HO O O
CYP2A6 suppresses CYP enzyme expression; or (3) exposure to a xenobi-
ZB otic that inhibits or inactivates a preexisting CYP enzyme. By
Coumarin 7-Hydroxycoumarin inhibiting cytochrome P450, one drug can impair the biotrans-
formation of another, which may lead to an exaggerated pharma-
cologic or toxicologic response to the second drug. Increased
CH CH,
ro) / CYP enzyme activity can result from (1) gene duplication leading
Na
ec
O N
to overexpression of a CYP enzyme, (2) exposure to drugs and
ee uk =e HeC tin,
other xenobiotics that induce the synthesis of cytochrome P450,
N N
H CYP2€19 or (3) stimulation of preexisting enzyme by a xenobiotic.
H

Induction of cytochrome P450 by xenobiotics increases CYP

enzyme activity. By inducing cytochrome P450, one drug can
HO
stimulate the metabolism of a second drug and thereby
(S)-Mephenytoin 4 -Hydroxy-(S)-mephenytoin decrease or ameliorate its therapeutic effect. Allelic variants,
which arise by point mutations in the wild-type gene, are
FIGURE 6-6 Examples of reactions catalyzed by cytochrome another source of interindividual variation in CYP activity.
P450: hydroxylation of aromatic carbon. Environmental factors known to affect CYP levels include
medications, foods, social habits (e.g., alcohol consumption
and cigarette smoking), and disease status (diabetes, inflam-
CYP enzymes are classified into subfamilies based on amino mation, viral and bacterial infection, hyperthyroidism, and
acid sequence identity. hypothyroidism). When environmental factors influence CYP
The function and regulation of CYP1A1, CYP1A2, CYP1B1, enzyme levels, considerable variation may be observed during
GYP2EL CYPQRig CYP2SI CY P2U lands CY YP2Wilware repeated measures of xenobiotic biotransformation (e.g., drug
highly conserved among mammalian species and these proteins metabolism) in the same individual. Due to their broad sub-
have the same names in all mammalian species. In most other strate specificity, it is possible that two or more CYP enzymes
cases, the CYP enzymes are named in a species-specific manner. can contribute to the metabolism of a single compound.
 CHAPTER6 Biotransformation of Xenobiotics 89

The pharmacologic or toxic effects of certain drugs are exag- human liver microsomes. Inhibitors of cytochrome CYP
gerated in a significant percentage of the population due to a must be used cautiously because most of them can inhibit
heritable deficiency in a CYP enzyme. Inasmuch as the biotrans- more than one CYP enzyme.
formation of a xenobiotic in humans is frequently dominated by 3. Antibody inhibition determines the effects of inhibitory
a single CYP enzyme, the considerable effort in identifying antibodies against selected CYP enzymes on the bio-
which CYP enzyme or enzymes are involved in eliminating transformation of a xenobiotic by human liver micro-
the drug is known as reaction phenotyping or enzyme mapping. somes. This method alone can potentially establish which
Four approaches to reaction phenotyping are as follows: human CYP enzyme is responsible for biotransforming a
1. Correlation analysis involves measuring the rate of xenobi- xenobiotic.
otic metabolism by several samples of human liver micro- 4. Biotransformation by purified or recombinant human
somes and correlating reaction rates with the variation in CYP enzymes establishes whether a particular CYP
the level or activity of the individual P450 enzymes in the enzyme can or cannot biotransform a xenobiotic, but it
same microsomal samples. does not address whether that CYP enzyme contributes
2. Chemical inhibition evaluates the effects of known CYP substantially to reactions catalyzed by human liver
enzyme inhibitors on the metabolism of a xenobiotic by microsomes.

cl

OH
ee

H
ortho-Hydroxylation
2,3-oxide
cl

Direct insertion ——————>

OH
Chlorobenzene meta-Hydroxylation

i}

oO
OH
3,4-oxide para-Hydroxylation

oe N
ee
]
N
]
ies
Oo
O NH, O NH, ae
OH,
Carbamazepine Carbamazepine-10,11-epoxide Carbamazepine-2,3-epoxide
(stable epoxide) (unstable arene oxide)

Onno One® OH

Cs Z OBE O
H
ae er
CH,CHO
+ CO,

H
Coumarin Coumarin-3,4-epoxide ortho-hydroxyphenylacetaldehyde
ee
5 “~ COOH
CYP1A1
CcS56 CONICHS)2
ae)
Verlukast Verlukast epoxide

FIGURE 6-7 Examples of reactions catalyzed by cytochrome P450: epoxidation.

90 UNIT 2 Disposition of Toxicants

S-Oxygenation
N || ors 8
CH,O Nl ‘
CH,
Omeprazole
Sulfoxidation
CYP3A4
fe)

CLAN A oe
10)
H
N | CH3 Sulfone

N Note; The sulfoxide in omeprazole and

NS lansoprazole is a chiral center.
Lansoprazole Each drug is a racemic mixutre.

N-Oxygenation

ie;

ON O ON O 4
X i Yi \ II /] \
a (CH,)3 ——C > a (Chi) iG
HC ae H3C =
4-(MethyInitrosamino)-1-(3-pyridyl)butan-1-one (NNK) NNK N-oxide
(A tobacco-specific nitrosamine)

CH,O3 SN oe
W :
| CH,0 : SK ae
ZA
CH3;0 =
3 CHO Ze
CH, < \-Cl

6,7-Dimethoxy-4-(4'-chlorobenzyl)isoquinoline
(muscle relaxant)

FIGURE 6-8 Examples of reactions catalyzed by cytochrome P450: heteroatom oxygenation.

O-Dealkylation N-Dealkylation

iG—oO e) fe) HO O O
CYPIA2
ie wae Cee:
CYP2C19
7-Ethoxyresorufin CH3CHO Resorufin CYP3A4 \
cl q
O—CH, OH ie HCHO
CYP2D6
Diazepam Nordiazepam

O Se
H3C —N HCHO H3C —N
N,-demethylation —»
HN l
q>
Dextromethorphan Dextrorphan (CYP2E1) ace N
|
S-Dealkylation CH3
——! §—CH, SH Theobromine

NZ N {O] NZ N
O 3 O Hs
Re te Ie H3C ~
N~ ~N NZ N N ~~ N
H H ae ii > N;-demethylation —» Be ‘i
HCHO
6-Methylmercaptopurine 6-Mercaptopurine fo) N N (CYP1A2) Oo N N
| H
, HCHO
Si-Dealkylation ae Paraxanthine
Caffeine
CH a is
CH 3 O
H.C H
i Si 3* Nn N
CH; j els CH; } \ chs i N,-demethylation ——» ot | >
(CYP2E1) O N N
CH3 pa
H
eae aN CH; \ CH; TAOe 1EN | CH;
CH;
fa~ HCHO REN
CH, CH; CH; CH; Theophylline

Octamethylcyclotetrasiloxane (D,)

FIGURE 6-9 Examples of reactions catalyzed by cytochrome P450: heteroatom dealkylation.

 CHAPTER 6 Biotransformation of Xenobiotics 9]

Oxidative Deamination Examples of substrates, inhibitors, and inducers for each

: [O] : CYP enzyme in human liver microsomes are given in
Gi SCel iti ==> CH, —C =O +NH,
| | Table 6-2. Because reaction phenotyping in vitro is not always
CH, CH,
carried out with toxicologically relevant substrate concentra-
Amphetamine Phenylacetone
tions, the CYP enzyme that appears responsible for biotrans-
Oxidative Desulfuration
forming the drug in vitro may not be the CYP enzyme
S (@)
responsible for biotransforming the drug in vivo.
C,H,O —P — OCH, as C,H,O —P —OC,H,
{O} {S]
Activation of Xenobiotics by Cytochrome P450— The
role ofhuman CYP enzymes in the activation of procarcinogens
NO, NO, and protoxicants and some cytochrome P450-dependent
Parathion Paraoxon
reactions are summarized in Table 6-3. Many of the chemicals
i GH f G 2ns
H
listed in Table 6-3 are also detoxified by cytochrome P450
205
CH, CH,
a CH” A CHO by conversion to less toxic metabolites. In some cases, the same
oN ‘GH ee ‘GH CYP enzyme catalyzes both activation and detoxication reac-
On mNi Ome ea fo) 5} © NO pe
H H tions. For example, CYP3A4 activates aflatoxin B, to the hepa-
Thiopental Pentobarbital totoxic and tumorigenic 8,9-epoxide, but it also detoxifies
aflatoxin B, by 3-hydroxylation to aflatoxin Q,. Complex factors
FIGURE 6-10 Examples of reactions catalyzed by determine the balance between xenobiotic activation and
cytochrome P450: oxidative group transfer. detoxication.

S 5—o
SO)
| I
C,H,O —p — OCH, CHO =p— 0cH: |
C,H,O =p — OCH,
| | v\
10) 0 O OH

P450 OH" ®
ae ee
(0) 6

NO, NO, Oxidative NO,

Parathion desulfuration
S i) +H*
Il
C,H,O — p — OC,H, Paraoxon
OH
OH == (0)
Diethylphosphorothiolate Ne
O
C5H.O | OCHABD
I 2m OH
C,H,O — p — OCH, NO,

OH 4-Nitrophenol
Diethylphosphate

O O O
I | P450 |
C,H,O —C ¢ — OGH, C,H,O —C COOH

S
| CH,CHO
a
S
H3C N CH, 5 H3C N Ge

P450 (CYP3A4)

CH,CHO + CO,
Zz c
CH-
maynts
ome Ra

Loratadine Desloratadine

FIGURE 6-11 Examples of reactions catalyzed by cytochrome P450 that resemble hydrolytic reactions: cleavage of a thiophosphate
(parathion), a carboxylic acid ester (2,6-dimethyl-4-phenyl-3,5-pyridinecarboxylic acid diethyl ester), and a carbamate (loratadine).
92 UNIT 2 Disposition of Toxicants

0 O
ae on
G 6) ixSO ee
Haye Nice Digitoxin(dt)
|
CYP2ZEl
ters7 eae
CYP1A2 .we
CYP3A4 HC 0
HO.
H,C2 0
OH

OH ee O fe)
N-Acetylbenzoquinoneimine O
OH H3C OH
| = y [Digitoxigenin|
Digitoxigenin, ie) SES
NO, NO, ft Cy es aL z HO {== Digitoxoside
/ eG Se
|
O | Digitoxoside | O
O
il
O
HI ie
CYP3A4 O
iI
O
mn Ome aeSar Te Se O
H,CO—C GSOGH? I KCOC | me GOGH, | Digitoxoside | 3
ood ‘ 15'-Dehydro-dt, 9/-Dehydro-dt,
Hee NCH, Hece INC CH,
digitoxosyl bedigitoxosyl
Nifedipine Dehydronifedipine cleavage cleavage
Digitoxigenin Digitoxigenin
bisdigitoxoside (dt,) monodigitoxoside (dt,)
N
OH
SUN

N , CYP3A4
= —
i OH a a :
Sparteine N
‘6-Rehydrotestosterone
5-Dehydrosparteine

oe
Aldehyde
_CYP2A6 ‘g oxidase fo) onyEa
1, Testosterone
cH, ~ye281

)-Nicotine )-Nicotine Mi o -iminium ion Cotinine Androstenedione

COOH CYP2A9 COOH 8-oxidation COSCOA as HO. OH

a eee i +10 :
Valproic acid CYP2B1
4-Ene valproic acid A

e O
aa
2,4-Diene valproic acid
(CoA thioester)
Epi-testosterone gem-diol

FIGURE 6-12 Examples of reactions catalyzed by cytochrome P450: dehydrogenation.

Inhibition of Cytochrome P450—Inhibition of CYP is a major Induction of Cytochrome P450— Xenosensors The induc-
cause of drug—drug interactions and may cause the withdrawal of tion (upregulation) of xenobiotic-biotransforming enzymes
regulatory approval. The magnitude of the drug—drug interaction and transporters is a receptor-mediated, adaptive process that
depends on the degree of CYP inhibition by the perpetrator drug augments xenobiotic elimination during periods of high xeno-
(those xenobiotics that inhibit or induce the enzyme that is respon- biotic exposure. Itis nota toxicological or pathological response,
sible for clearing a victim drug) and the fractional metabolism of but enzyme induction is often associated with liver enlargement
the victim drug (xenobiotic whose clearance is largely determined (due to both hepatocellular hypertrophy and hyperplasia), and
by a single route of elimination, such as a single CYP) by the it may be associated with toxicological and pharmacological
affected enzyme. Inhibitory drug interactions generally fall into consequences, especially for the safety evaluation of drug can-
two categories: direct inhibition (which can be competitive, non- didates in laboratory animals and for clinical practice in
competitive, and uncompetitive) and metabolism-dependent humans. In animals and humans, enzyme induction may be
inhibition (which can be irreversible or quasi-irreversible). Direct associated with pharmacokinetic tolerance, whereby the xeno-
inhibition can be subdivided into two types. The first involves biotic induces its own elimination.
competition between two drugs that are metabolized by the same Inducers of cytochrome P450 increase the rate of xenobiotic
CYP enzyme. The second is also competitive in nature, but the biotransformation. Some of the CYP enzymes in human liver
inhibitor is not a substrate for the affected CYP enzyme. microsomes are inducible (Table 6-2). P450 induction typi-
Metabolism-dependent inhibition occurs when cytochrome P450 cally lowers blood levels, which compromises the therapeutic
converts a xenobiotic to a metabolite that is a more potent inhibi- goal of drug therapy but does not cause an exaggerated
tor, either reversible or irreversible, than the parent compound. response to the drug.
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CHAPTER 6 Biotransformation of Xenobiotics

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UNIT 2 Disposition of Toxicants

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 CHAPTER 6 Biotransformation of Xenobiotics 95

TABLE 6-3 Examples of xenobiotics activated by human P450.

CYP1A2 CYP2D6 CYP2E1

Acetaminophen Acetaminophen
_ NNK
2-Acetylaminofluorene Acrylonitrile
4-Aminobiphenyl CYP2F1 Benzene
2-Aminofluorene 3-Methylindole Carbon tetrachloride
2-Naphthylamine Acetaminophen Chloroform
NNK Valproic acid Dichloromethane
Amino acid pyrolysis products (DiMeQx, 1,2-Dichloropropane
CYP1A1 and 1B1
MelQ, MelQx, Glu P-2, 1Q, PhIP, Trp P-1, Ethylene dibromide
Trp P-2) Benzo[a]pyrene and other polycyclic
Ethylene dichloride
Tacrine aromatic hydrocarbons
Ethyl carbamate
CYP2A6 and 2A13 CYP3A4
Halothane
NNK and bulky nitrosamines Acetaminophen N-Nitrosodimethylamine
N-Nitrosodiethylamine Aflatoxin B1 and G1 Styrene
Aflatoxin B1 6-Aminochrysene Trichloroethylene
CYP2B6 Benzo[a]pyrene Le aly died) Vinyl chloride
Cyclophosphamide CYP4B1
6-Aminochrysene ostarmide
Cyclophosphamide 1-Nitropyrene |pomeanol

ifosfamide Sterigmatocystin 2 Meroindels

Senecionine 2-Aminofluorene
CYP2C8, 9, 18, 19
Tienilic acid Tris(2,3-dibromopropyl) phosphate
Phenytoin
Valproic acid

NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific nitrosamine.

Data from Guengerich FP, Shimada T: Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem Res Toxicol, 1991 Jul-Aug;4(4):391-407.

Although induction of cytochrome P450 may increase the acti- halves of a clothes peg coming together to form a functional
vation of procarcinogens to DNA-reactive metabolites, there is unit); (3) translocation of the functional receptor heterodimer
little evidence from either human epidemiologic studies or ani- from the cytoplasm to the nucleus; (4) binding of the functional
mal experimentation that P450 induction enhances the incidence receptor heterodimer to discrete regions of DNA (response ele-
or multiplicity of tumors caused by known chemical carcinogens. ments) that are typically located in the 5’-promoter region of
In fact, most evidence points to a protective role of enzyme induc- the gene (which is analogous to a clothes peg being fastened to
tion against chemical-induced neoplasia. Cytochrome P450 a clothes line); (5) recruitment of other transcription factors
induction can cause pharmacokinetic tolerance whereby larger and coactivators (such as histone and RNA methyltransferases,
drug doses must be administered to achieve therapeutic blood histone and chromatin deacetylases, and histone remodeling
levels due to increased drug bio-transformation. helicases) and RNA polymerase to form a transcription com-
CYP induction is mediated by four ligand-activated recep- plex; and (6) gene transcription, which leads to increased levels
tors, namely, AhR, CAR, PXR, and PPARa (Table 6-4). These of CYP mRNA and protein (as well as other xenobiotic-bio-
so-called xenosensors resemble other nuclear receptors, such transforming enzymes and transporters). As is the case with all
as steroid and thyroid hormone receptors, with cross-talk nuclear receptors, the details of the process of activating a
among xenosensors and cross-talk between xenosensors and xenosensor to its transcriptionally active form are complex and
other nuclear receptors. Xenosensors have a ligand-binding multifaceted.
domain (LBD) and a DNA-binding domain (DBD). In general,
CYP induction involves the following steps (with steps 2 and 3 CONJUGATION
reversed in the case of AhR): (1) binding of ligand (xenobiotic)
to the receptor, which triggers conformational changes that Conjugation reactions include glucuronidation, sulfonation
promote its dissociation from accessory proteins (such as core- (often called sulfation), acetylation, methylation, conjugation
pressors, chaperones, and cytoplasm retention proteins) and with glutathione (mercapturic acid synthesis), and conjugation
promote its association with coactivators; (2) dimerization of with amino acids (such as glycine, taurine, and glutamic acid).
the ligand-bound receptor with a partner protein to form a The cosubstrates for these reactions, which are shown in
DNA-binding heterodimer (which is analogous to the two Figure 6-13, react with functional groups that are either present
 96 UNIT 2 Disposition of Toxicants

TABLE 6-4 Receptors mediating the induction (or suppression) of cytochrome P450 enzymes and other
xenobiotic-biotransforming enzymes and transporters.

Response oes So
Nuclear Receptor Element(s) — _ Receptor Activators *

AhR XRE PAHs, TCDD (other PHAHs), 3-naphthoflavone, indigoids, CYP1A1, 1A2, 1B1, 2S1, UGT1A1, UGT1A6,
tryptophan metabolites, omeprazole, lansoprazole AKR1A1, AKR1C1-4

CAR DR-3 Phenobarbital, phenytoin, carbamazepine, CYP2A6, 2B6, 2C8, 2C9, 2C19, 3A4,
CITCO (human), TCPOBOP (mouse), clotrimazole, (Many PXR UGT1A1, SULT1A1, AKR1D1, ALAS,
DR-4 agonists are also CAR agonists, and vice versa) MRP2, MRP3, MRP4

ER-6

PXR DR-3 Amprenavir, avasimibe, bosentan, bile acids, carbamazepine, CYP2A6, 2B6, 2C8, 2C9, 2C19, 3A4, 3A7,
clindamycin, clotrimazole, cortisol, cyproterone acetate, 4F12, 7A11, CES2, SULT2A1, UGT1A1,
DR-4 dicloxacillin, efavirenz, etoposide, dexamethasone, 1A3, 1A4, 1A6, GSTA1, AKR1D1, PAPSS2,
griseofulvin, guggulsterone, hyperforin (SJW), indinavir, ALAS, MDR1, MRP2, AhR
ER-6 lovastatin, mifepristone, nafcillin, nelfinavir, nifedipine,
omeprazole, paclitaxel, PCBs, phenobarbital, phthalate
ER-8 monoesters, 58-pregnane-3,20-dione, rifabutin, rifampin,
ritonavir, saquinavir, simvastatin, spironolactone,
sulfinpyrazone, TAO, tetracycline, topotecan, transnonachlor, .
troglitazone, verapamil, vitamin E, vitamin K, ‘

PPARa DR-1 Fibrates, WY-14643, perfluorodecanoic acid CYP4A, UGT1A9, 2B4

y
\

Nrf2 ARE 8-Naphthoflavone, oltipraz, phenolic antioxidants (e.g., BHA NQO1, mEH, AKR7A, UGTs, GSTA1, y-GCL,
and BHT), phenylisothiocyanate, diethyl maleate, phorone MRP1

GR GRE Glucocorticoids (e.g., dexamethasone) CYP2C9, 2B6, 3A4, 3A5, CAR, PXR

FXR IR-1 Bile acids, GW4064, AGN29, AGN31 BSEP, I-BABP, MDR3, UGT2B4, SULT2A1,
OATP1B3, PPARa, SHP

LXRa DR-4 GW3965, 10901317, paxiline, F,methylAA,t acetylpodocarpic LRH1, SHP, CYP7A, LXRa, CYP3A4 ||, 2B6 |
dimer (APD)

VDR DR-3 1a,25-Dihydroxyvitamin D;, lithocholate CYP2B6, 209, 3A4, SULT2A1

ER-6

IR-O

HNFla* OATP1B1, OATP1B3, CYP7A1, UGT1A6, 1A8,

1A9, 1A10, HNF4q, PXR, kidney-specific
expression of OAT1, OAT3, URAT1

HNF4q DR CYP2A6, 2B6, 2C9, 2D6, 3A4, DD4, MDR1,

PXR, CAR, FXR, PPAR@, HNF1a

LRH-1 DR-4 CYP7A, ASBT

SHP None Targets of PPARa |, AhR |, PXR |, CAR |,
LRH-1 |, HNF4a |, LXRa |, GR |

“A downward arrow indicates downregulation (suppression). All others are upregulated (induced).
"(3-Chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenylacetic acid].
+The HNF 1a consensus sequence is GTTTAATNATTAAC.

on the xenobiotic or are introduced or exposed during acids or glutathione involves reactions with activated xeno-
oxidation, reduction, or. hydrolysis. With the exception of biotics. Except for the glucuronosyltransferases, most conjuga-
methylation and acetylation, conjugations result in a large tion enzymes are mainly located in the cytosol (Table 6-1).
increase in xenobiotic hydrophilicity, which greatly facilitates
excretion of foreign chemicals. Glucuronidation, sulfation, Glucuronidation
acetylation, and methylation involve reactions with activated or Glucuronidation requires the cosubstrate uridine diphosphate-
“high-energy” cosubstrates, whereas conjugation with amino glucuronic acid (UDP-glucuronic acid), and the reaction is
 CHAPTER 6 Biotransformation of Xenobiotics 97

Glucuronidation 6 Sulfonation (Sulfation) H5N

—= N
COO™ HN N

O O N
OH I I 2 i. r " N
oe pesae! —O—CH, es aes
HO i
Ox Og O On
OH

OH OH O OH
D |
PO;

Uridine-5’-diphospho-a-p-glucuronic acid (UDPGA) 3’-Phosphoadenosine-5’-phosphosulfate (PAPS)

if af

Acetylation H5N Methylation

H>N
==N :

i? ;pw
N IN|

O CH, O O ‘ i
| | | I| CH N
NHi=—€—GH alas —=© F O ; O—CH, COC l 3 N
i: | O
CH, OH CH, Der Oo ple NCH ise ee
| HN
a
ie O 7 OH
|
i= Chi=—— CH) = seers POs OHH
Acetyl-coenzyme A S-Adenosylmethionine (SAM)

Glutathione conjugation Amino acid conjugation

SS
COO™ O O GN
| II II es Cae -COO HN
Gan) 500
fumarase ora bee NH GH COO” 5 .
NH, 6 Glycine ie

ath CH,
|
SH C
SS i Gin). GH aoe
>-glutamic acid cysteine glycine =. OCHS ~SO3 -

Glutathione Taurine Glutamine

Formation of acyl-CoA thioesters

0 0
(#5)a 3a we
of}
Sp
H H
OH
O

Coenzyme A

FIGURE 6-13 Structures of cofactors for phase II biotransformation. The functional group that reacts with or is transferred to the
xenobiotic is shown in red.
98 UNIT 2 Disposition of Toxicants

O-Glucuronides (ethers) UGTA

GE
OH
OH C3
Ne Fis3 Gon Nay

OH
‘a | UGT1A3
SS
UGTIA1 |

HO
e
26,26,26,27,27,27,-hexafluoro-
Ces: N

s
Gr

P tri itamin D3 : .
10,23,25-trihydroxyvitamin Trifluoperazine
17 8 -Estradiol

UGT2B15
UGT1A6 UGT1A9 O we

| | "OH

OH CH, OH CH, UGT2B7 UGT2B7

Cela . “ v
‘

1-Naphthol Propofol Morphine S-Oxazepam

Other Examples mn
Acetaminophen Estrone Naloxone Progesterone Testosterone
Codeine Hexobarbital 4-Nitrophenol Propranolol Thyroxine
Dextrophan Methylphenylcarbinol Phenolphthalein Temazepam Trichloroethanol
Diethystilbestrol

N-Glucuronides Acyl-glucuronides
O-Glucuronides (esters)

NH, Hy CH;
O Ne
Ga
OH

_ neres
CH

HC

Aniline Tripelennamine Tolmetin

Other Examples Other Examples

Benoxaprofen
Amitryptyline Cyproheptadine Meprobamate Bilirubin Etodolac Naproxen
Benzidine N-Hydroxyarylamines Sulfadimethoxine Diclofenac Gemfibrozil Suprofen
Cyclopiroxolamine Imipramine Sulfathiazole Diflunisal Ketoprofen Valproic acid
Cyclobenzaprine Lamotrigine Sulfisoxazole Trifluoperazine lopanoic acid Zomepirac

$-Glucuronides SH C-Glucuronides

GHs = cA
SH <——
N-C.
Chie mS)
Phenylbutazone R=C,Hy R
Diethyldithiocarbamate Thiophenol Sulfinpyrazone R= (CH,),SOC.H, UGT1A9
Other Examples: Disulfiram Methimazole Other Examples: Suxibuzone Ethchlorvynol A®-THC

FIGURE 6-14 Examples of xenobiotics and endogenous substrates that are glucuronidated.
The arrow indicates the site of
glucuronidation, with the UGT enzyme if selective.

catalyzed by UDP-glucuronosyltransferases (UGTs). Examples aromatic and aliphatic amines, and free sulfhydryl groups.
of xenobiotics that are glucuronidated are shown in Endogenous substrates for glucuronidation include bilirubin,
Figure 6-14. The site of glucuronidation is generally an electron- steroid hormones, and thyroid hormones.
rich nucleophilic heteroatom (O, N, or S) as found in aliphatic Glucuronide conjugates of xenobiotics and endogenous
alcohols and phenols, carboxylic acids, primary and secondary compounds are polar, water-soluble metabolites. Whether
 CHAPTER6 Biotransformation of Xenobiotics 99

glucuronides are excreted from the body in bile or urine depends Methylation
on the size of the aglycone (parent compound or unconjugated
Methylation, a minor pathway of biotransformation, generally
metabolite). The carboxylic acid moiety ofglucuronic acid, which
decreases the water solubility of xenobiotics and masks func-
is ionized at physiologic pH, promotes excretion because (1) it
tional groups that might otherwise be conjugated by other
increases the aqueous solubility of the xenobiotic and (2) it is rec-
enzymes. Methylation can also lead to increased toxicity. The
ognized by the biliary and renal organic anion transport systems,
cosubstrate for methylation is S-adenosylmethionine (SAM)
which enables glucuronides to be secreted into urine and bile.
(Figure 6-13). The methyl group bound to the sulfonium ion in
Glucuronides of xenobiotics are substrates for (-glucuronidase
SAM is transferred to xenobiotics and endogenous substrates
present in the intestinal microflora. The intestinal enzyme can
by nucleophilic attack from an electron-rich heteroatom
release the aglycone, which undergoes enterohepatic circulation
(O, N, or S) leaving S-adenosylhomocysteine. Examples of
delaying elimination of the xenobiotic.
xenobiotics and endogenous substrates that undergo O-, N-,
Cofactor availability can limit the rate of glucuronidation of
or S-methylation are shown in Figure 6-16.
drugs that are administered in high doses and are conjugated
The O-methylation of phenols and catechols is catalyzed by
extensively, such as aspirin and acetaminophen.
two different enzymes known as phenol O-methyltransferase
(POMT) in microsomes and catechol-O-methyltransferase
Sulfonation (COMT) in cytosol and microsomes. In rats and humans, COMT
is encoded by a single gene with two different promoters and
Many xenobiotics and endogenous substrates undergo sulfo-
transcription initiation sites. Transcription at one site produces a
nation. Sulfate conjugation is catalyzed by sulfotransferases, a
cytosolic form of COMT, whereas transcription from the other
multigene family of enzymes that generally produces a highly
site produces a membrane-bound form by adding a 50-amino
water-soluble sulfuric acid ester. The cosubstrate for the reaction is
acid segment that targets COMT to the endoplasmic reticulum.
3’-phosphoadenosine-5’-phosphosulfate (PAPS; see Figure 6-13).
Substrates for COMT include several catecholamine neurotrans-
Sulfate conjugation involves the transfer of sulfonate, not
mitters and catechol drugs, such as L-DOPA and methyldopa.
sulfate (i.e., SO, -, not SO, ) from PAPS to the xenobiotic. (The
Several N-methyltransferases have been described in humans
commonly used terms sulfation and sulfate conjugation are
and other mammals. Phenylethanolamine N-methyltransferase
used here, even though sulfonation and sulfonate conjugation
catalyzes the N-methylation of the neurotransmitter norepi-
are more appropriate descriptors.) Table 6-4 lists examples of
nephrine to form epinephrine in the adrenal medulla and in
xenobiotics and endogenous compounds that are sulfonated
certain regions of the brain, and is of minimal significance in
without prior biotransformation by oxidation enzymes. An
xenobiotic biotransformation. However, histamine and nico-
even greater number of xenobiotics are sulfated after a hydroxy]
tine N-methyltransferases expressed in liver, intestine, and/or
group is exposed or introduced during oxidative or hydrolytic
kidney do methylate xenobiotics.
biotransformation.
S-Methylation is an important pathway in the biotransfor-
Sulfate conjugates of xenobiotics are excreted mainly in urine.
mation of sulfhydryl-containing xenobiotics. In humans,
Sulfatases present in the endoplasmic reticulum and lysosomes
S-methylation is catalyzed by thiopurine methyltransferase in
primarily hydrolyze sulfates of endogenous compounds. Some
cytosol and thiol methyltransferase in microsomes.
sulfate conjugates are substrates for further biotransformation.
PAPS is synthesized from inorganic sulfate (SO,’) and ATP
in a two-step reaction. The major source of sulfate required for Acetylation
the synthesis of PAPS appears to be derived from cysteine N-Acetylation is a major route of biotransformation for xeno-
through a complex oxidation sequence. The low cellular con- biotics containing an aromatic amine (R—NH,) ora hydrazine
centration of PAPS (~75 uM versus ~350 uM UDP-glucuronic group (R—NH—NH,), which are converted to aromatic
acid and ~10 mM glutathione) limits the capacity for xenobiotic amides (R—NH—COCH,) and hydrazides (R—NH—NH—
sulfonation. COCH,), respectively. N-Acetylation masks an amine with a
Multiple sulfotransferases have been identified in all mamma- nonionizable group, so that many N-acetylated metabolites
lian species examined. There are two major enzyme classes: are less water soluble than the parent compound. Nevertheless,
membrane-bound enzymes are found in the Golgi apparatus and N-acetylation of certain xenobiotics, such as isoniazid, facili-
soluble enzymes are located in the cytoplasm. Sulfotransferases tates their urinary excretion.
are arranged into gene families (SULT1 to SULTS) that share at Xenobiotic N-acetylation catalyzed by cytosolic N-
least 45% amino acid sequence identity, and are further subdi- acetyltransferases requires the cosubstrate acetyl-coenzyme A
vided into several subfamilies. Each family appears to work on a (acetyl-CoA; Figure 6-13). The two-step reaction involves
specific functional group (i.e., phenols, alcohols, and amines) (1) transfer of the acetyl group from acetyl-CoA to an active
(Table 6-5). site cysteine residue within the enzyme with release of coen-
In general, sulfonation is an effective means of decreasing the zyme A and (2) subsequent transfer of the acetyl group from
pharmacologic and toxicologic activity of xenobiotics. However, the acylated enzyme to the amino group of the substrate with
as shown in Figure 6-15, sulfonation has a role in the activation regeneration of the enzyme.
of aromatic amines, methyl-substituted polycyclic aromatic NAT1 and NAT2, the two acetyltransferases existing in
hydrocarbons, and safrole to tumorigenic metabolites. humans, are 79% to 95% identical in amino acid sequence with
 100 UNIT 2. Disposition of Toxicants

TABLE 6-5 Properties of the human cytosolic sulfotransferases (SULTs).

Human Sult Polymorphic? Tissue Distribution Major Substratest

SULT1A1 Yes Liver (very high), platelets. placenta, adrenals, 4-Nitrophenol, 4-ethylphenol, 4-cresol,
endometrium, colon, jejunum, leukocytes, brain 2-naphthol, other phenols, acetaminophen,
ial ets (cerebellum, occipital and frontal lobes) minoxidil, N-hydroxy-PhIP, T2, T3,
178-estradiol (and other phenolic
steroids), dopamine, benzylic alcohols,
2-nitropropane, aromatic amines,
hydroxylamines, hydroxamic acids,
apomorphine, troglitazone, genestein,
epinephrine

SULT1A2 Yes Liver, kidney, brain, Gl tract, bladder tumors 4-Nitrophenol, N-hydroxy-2-
acetylaminofluorene, 2-naphthol, various
ole Gs aromatic hydroxylamines and hydroxamic
acids

SULT1A3 Yes Jejunum and colon mucosa (very high), liver (low), Dopamine, 4-nitrophenol,
platelets, placenta, brain (superior temporal gyrus, 1-hydroxymethylpyrene, norepinephrine,
pene hippocampus, and temporal lobe), leukocytes, salbutamol, dobutamine, vanillin, albuterol
fetal liver

SULT1A4 Liver, pancreas, colon, brain Not characterized. Likely similar to SULT1A3
‘

SULT1B1 Colon (highest), liver, leukocytes, small intestine 4-Nitrophenol, T2, T3, r-T3, T4, dopamine,
benzylic alcohols
\

SULT1C2 Yes Fetal lung and kidney, kidney, stomach, thyroid gland 4-Nitrophenol, N-hydroxy-2-AAF, aromatic
hydroxylamines, thyroid hormones
*4-*5

SULT1C4 Kidney, ovary, spinal cord, fetal kidney, fetal lung 4-Nitrophenol, N-hydroxy-2-AAF, 173-estrone,
(highest) bisphenol-A, 4-octylphenol, nonylphenol,
diethylstilbestrol, 1-hydroxymethylpyrene

SULT1E1 Liver (highest), endometrium, jejunum, adrenals, 17B-Estradiol, estrone, ethinyl estradiol,
mammary epithelial cells, fetal liver, fetal lung, 17B-estrone, equilenin, 2-hydroxy-estrone,
fetal kidney 2-hydroxy-estradiol, 4-hydroxy-estrone,
4-hydroxy-estradiol, diethylstilbestrol,
tamoxifen, thyroid hormones,
4-hydroxylonazolac, pregnenolone,
dehydroepiandrosterone, 1-naphthol,
naringenin

SULT2A1 Yes Liver (highest), adrenals, ovaries, prostate, jejunum, Dehydroepiandrosterone (DHEA),
oe kidney, brain 1-hydroxymethylpyrene,
Jee 6-hydroxymethylbenzo[a]-pyrene,
hycanthone, bile acids, pregnenolone,
testosterone, androgens, estrone,
176-estradiol, other hydroxysteroids,
budesonide
SULT2B1a Placenta (highest), prostate, trachea, skin Dehydroepiandrosterone, pregnenolone,
(SULT2B_v1) oxysterols, other hydroxysteroids
SULT2B1b Lung, spleen, thymus, kidney, prostate, ovary, Cholesterol, pregnenolone,
(SULT2B_v2) adrenal gland, liver (low), Gl tract (low) dehydroepiandrosterone, other
hydroxysteroids
SULT4A1a Brain: cortex, globus pallidus, islands of Calleja, Endogenous: 4 unidentified compounds from
(SULT4A_v1) septum, thalamus, red nucleus, substantia nigra and mouse brain homogenate
pituitary
Other: T3, T4, estrone, 4-nitrophenol,
2-naphthylamine, 2-naphthol
SULT4A1b
(SULT4A_v2)

SULT6B1 Testis

tT4 is thyroxine. T2 and T3 are diiodothyronine and triiodothyronine. r-T3 is reverse triiodothyronine.
 CHAPTER 6 Biotransformation of Xenobiotics 101

| 2-Acetylaminofluorene (2-AAF) | | Safrole | 7-12-Dimethylbenz[aJanthracene (DMBA)

3
N-Hydroxylation (P450) ~ ili
1/-Hydroxylation (P450)__
(P450) 7-Methyl-hydroxylation (P450)

OH

‘~ |

7 LT cena
i 0 CH; @

N-Hydroxy-2-AAF ea |
OH
ol
SULT
PAPS
1/-Hydroxysafrole
ie
OH

PAPS
SULT
|Nn a2)
©
PAP
2-2
3 PAP

H5C LOGE Oe O Coe

ee)
: |
S OSO;
S07- CHa
1/-Sulfoxysafrole
S oso;

SOZ” SOzs 4

Nitrenium ion
2 ? nee
Carbonium ion

Carbonium ion

; Ve er
DNA binding and
tumor formation
Carbonium ion

FIGURE 6-15 Role of sulfonation in the generation of tumorigenic metabolites (nitrenium or carbonium ions) of
2-acetylaminofluorene, safrole, and 7,12-dimethylbenz[a]Janthracene (DMBA).

an active site cysteine residue in the N-terminal region. Genetic polymorphisms for N-acetylation have been docu-
Although encoded by genes on the same chromosome, NAT1 mented in humans, hamsters, rabbits, and mice. Polymor-
is expressed in most tissues of the body, whereas NAT2 is phisms in NAT2 have a number of pharmacologic and
mainly expressed only in liver and intestine. Most (but not all) toxicologic consequences: slow NAT2 acetylators are predis-
of the tissues that express NAT1 also appear to express low lev- posed to drug toxicities, including excessive hypotension from
els of NAT2, at least at the level of mRNA. NAT1 and NAT2 hydralazine, peripheral neuropathy from isoniazid and dap-
also have different but overlapping substrate specificities. sone, systemic lupus erythematosus from hydralazine and pro-
Examples of drugs that are N-acetylated by NAT] and NAT2 cainamide, and the toxic effects of coadministration of the
are shown in Figure 6-17. anticonvulsant phenytoin with isoniazid.
102 UNIT 2 Disposition of Toxicants

Preferred NAT1 substrates Preferred NAT2 substrates

O-Methylation COO’ , COO”
CH=CH & Gira Gnky COOH O
NH, NH, i}
SAM C —-NH —NH, <——

HO CH,0
Zen
S
OH OH NH, <—— N
L-Dopa 3-O-Methyl-L-dopa 4-Aminobenzoic acid Isoniazid

N-Methylation CH,CH,NH, CH,CH,NH, CH;

N=
then
DN
SAMO, >
neuen
¢ ha
etd SO, —NH — y
OH N
Histamine N-Methylhistamine CH,

NH, = Fee NH, oe —

ag N SAM as N 4-Aminosalicylic acid

NS | 2 ~ | Sulfamethazine
N CH; No CH,
|
CH, CH,
Nicotine N-MethylInicotinium ion
SO, —NH re SO, Nm,
N .
S-Methylation OH ‘
SH Se Chis

NZ x SAM NZ NXY
al as corer
ie ‘

u v NH, <—— NH, <——

|

N Sulfamethoxazole Dapsone
4
6-Mercaptopurine 6-Methylmercaptopurine

FIGURE 6-17 Examples of substrates for human

FIGURE 6-16 Examples of compounds that undergo O-, N-, N-acetyltransferases, NAT1, and the highly polymorphic NAT2.
or S-methylation.

The N-acetyltransferases detoxify aromatic amines by Substrates for amino acid conjugation are restricted
converting them to the corresponding amides that are less to certain aliphatic, aromatic, heteroaromatic, cinnamic, and
likely to be activated to DNA-reactive metabolites. However, arylacetic acids. The ability of xenobiotics to undergo amino
N-acetyltransferases can activate aromatic amines if they are acid conjugation depends on steric hindrance around the
first N-hydroxylated by cytochrome P450. The acetoxy esters of carboxylic acid group, and by substituents on the aromatic
N-hydroxyaromatic amines, like the corresponding sulfonate ring or aliphatic side chain. Amino acid conjugates of
esters (Figure 6-15), can break down to form highly reactive xenobiotics are eliminated primarily in urine. The acceptor
nitrenium and carbonium ions that bind to DNA. Whether fast amino acid used for conjugation is both species- and
acetylators are protected from or predisposed to the cancer- xenobiotic-dependent.
causing effects of aromatic amines depends on the nature of the Amino acid conjugation of N-hydroxy aromatic amines
aromatic amine and other risk modifiers. (hydroxylamines) is an activation reaction producing N-esters
that can degrade to form electrophilic nitrenium and carbo-
nium ions. Conjugation of hydroxylamines with amino acids is
Amino Acid Conjugation catalyzed by cytosolic aminoacyl-tRNA synthetases and
Two principal pathways by which xenobiotics are conjugated requires ATP (Figure 6-18).
with amino acids are illustrated in Figure 6-18. The first involves
conjugation of xenobiotics containing a carboxylic acid group
with the amino group of amino acids such as glycine, glutamine, Glutathione Conjugation
and taurine (see Figure 6-13). After activation of the xenobiotic Conjugation of xenobiotics with glutathione includes an enor-
by conjugation with CoA, the acyl-CoA thioether reacts with the mous array of electrophilic xenobiotics, or xenobiotics that can
amino group of an amino acid to form an amide linkage. The sec- be biotransformed to electrophiles. The tripeptide glutathione
ond pathway involves conjugation of xenobiotics containing an comprises glycine, cysteine, and glutamic acid (Figure 6-13).
aromatic hydroxylamine with the carboxylic acid group of such Glutathione conjugates are thioethers, which form by nucleo-
amino acids as serine and proline. This pathway involves activa- philic attack of glutathione thiolate anion (GS~) with an elec-
tion of an amino acid by aminoacyl-tRNA synthetase, which trophilic carbon, oxygen, nitrogen, or sulfur atom in the
reacts with an aromatic hydroxylamine to forma reactive N-ester. xenobiotic. This conjugation reaction is catalyzed by a family
 CHAPTER 6 Biotransformation of Xenobiotics 103

Amino acid conjugation of Amino acid conjugation

carboxylic acids of hydroxylamines

Benzoic acid

N-Hydroxy-4-aminoquinoline-1-oxide
ATP CoAR st GOGH
(Acetyl CoA) NH

ATP -O0OC =(Gh> = CH,OH

Acyl-CoA synthetase (serine)

Seryl-tRNA synthetase

AMP
zs OH
re) PPi
||
(Coy Sy GYAN
OQ NH
tie = at
H Ov Ce Chie CreOn
‘\ N va

SS

Benzoyl-CoA | ee
N

NH,CH,COO y
(glycine) O

Acyl-CoA: amino acid

N-acyltransferase

CoA — SH bess Nt

O | =
| y
CoN ia =H COO N

<<
(e)

Hippuric acid Reactive nitrenium ion

FIGURE 6-18 Conjugation of xenobiotics with amino acids.

104 UNIT 2 Disposition of Toxicants

of glutathione S-transferases that are present in most tissues, As shown in Figure 6-19, substrates for glutathione conjuga-
where they are localized in the cytoplasm (>95%) and endo- tion can be divided into two groups: those sufficiently electro-
plasmic reticulum (<5%). philic to be conjugated directly and those that must first be
Substrates for glutathione S-transferase are commonly biotransformed to an electrophilic metabolite prior to conju-
hydrophobic, contain an electrophilic atom, and react nonen- gation. The conjugation reactions themselves can be divided
zymatically with glutathione at some measurable rate. The into two types: displacement reactions, in which glutathione
mechanism by which glutathione S-transferase increases the displaces an electron-withdrawing group, and addition reac-
rate of glutathione conjugation involves deprotonation of GSH tions, in which glutathione is added to an activated double
to GS~. The concentration of glutathione in liver is extremely bond or strained ring system.
high (~5 to 10 mM); hence, the nonenzymatic conjugation The displacement of an electron-withdrawing group by
of certain xenobiotics with glutathione can be significant. glutathione typically occurs when the substrate contains
However, some xenobiotics are conjugated with glutathione halide, sulfate, sulfonate, phosphate, or a nitro group
stereoselectively, indicating that the reaction is largely cata- (i.e., good leaving groups) attached to an allylic or benzylic
lyzed by glutathione S-transferase. Like glutathione, the gluta- carbon atom.
thione S-transferases are themselves abundant cellular The addition of glutathione to a carbon-carbon double bond
components, accounting for up to 10% ofthe total cellular pro- is also facilitated by the presence of a nearby electron-
tein. These enzymes bind, store, and/or transport a number of withdrawing group; hence, substrates for this reaction typically
compounds that are not substrates for glutathione conjuga- contain a double bond attached to —CN, —CHO, —COOR,
tion. The cytoplasmic protein formerly known as ligandin, or —COR. ‘
which binds heme, bilirubin, steroids, azo-dyes, polycyclic Glutathione can also conjugate xenobiotics with an electro-
aromatic hydrocarbons, and thyroid hormones, is an alpha- philic heteroatom (O, N, and S). In each of the examples shown
class GST. in Figure 6-20, the initial conjugate formed between

| Direct conjugation by displacement of an electron-withdrawing group Conjugation of a strained

ring system (oxirane)
cl cl formed metabolically
. GS cl
SG
cl

oe
;
NO, NO,
1,2-Dichloro-4-nitrobenzene Chlorobenzene
NO,
SG

NO; P450

Za
~ (2)
O~<—Z

x}
4-Nitroquinoline-1-oxide zE

Direct conjugation by addition of glutathione

ag
O
I GS", H" GS I 3,4-Oxide
CH— C —OC,H, \
I | oe
CHC -OGH; CH, — C—OGH, ee c0
I i
O O
Diethyl maleate

g GS”, Ht O
CH, —C 7 I
] ] GS — CH,— CH,—C—OH H
CH, —O SG
B-Propiolactone

FIGURE 6-19 Examples of glutathione conjugation of xenobiotics with an electrophilic carbon. GS- represents the anionic form of
glutathione.
 CHAPTER 6 Biotransformation of Xenobiotics 105

glutathione and the heteroatom is cleaved by a second mole- site. Numerous subunits have been cloned and sequenced and
cule of glutathione to form oxidized glutathione (GSSG). The differ in substrate specificity, tissue location, and cellular
initial reactions are catalyzed by glutathione S-transferase, location. Conjugation with glutathione represents an impor-
whereas the second reaction (which leads to GSSG formation) tant detoxication reaction because electrophiles are poten-
generally occurs nonenzymatically. tially toxic species that can bind to critical nucleophiles, such
Glutathione conjugates formed in the liver can be effluxed as proteins and nucleic acids, causing cellular damage and
into bile and blood, and they can be converted to mercapturic genetic mutations (see Chapter 8 for more information).
acids in the kidney and excreted in urine. As shown in Glutathione is also a cofactor for glutathione peroxidase,
Figure 6-21, the conversion of glutathione conjugates to mer- which is important in protecting cells against lipid and hemo-
capturic acids involves the sequential cleavage of glutamic acid globin peroxidation.
and glycine from the glutathione moiety, followed by In some cases, conjugation with glutathione enhances the
N-acetylation of the resulting cysteine conjugate. toxicity of a xenobiotic. Glutathione conjugates of various
Glutathione S-transferases are dimers composed ofidenti- compounds can activate xenobiotics to become toxic by releas-
cal subunits, although some forms are heterodimers. Each ing a toxic metabolite, being inherently toxic itself, or being
subunit contains 199 to 244 amino acids and one catalytic degraded to a toxic metabolite.

=|
CH, -O-—NO, = g¢s- CH, —O—SG GS” Ht CH, —OH
| | : |
CH, — O—NO, ae CH, — O—NO, CH, —O—NO,
| : | |
CH, — O—NO, J CH, —O—NO, CH, — O—NO,
NO;
Trinitroglycerin Nitrite Dinitroglycerin

NO GSSG

Nitric oxide Oxidized glutathione

HO aa
SO GS, H"
i COOH
A
HO A GSOH
Glutathionesulfenic acid
15-Hydroperoxy-PGF,,, 6s ut PGF,,,

HO GSSG
Oxidized glutathione

= Sa
ay ee ep
O O O
II GS, H" i GS), H I
N — C —N(CH,) NH — C—N(CH,), NH — C—N(CH;),
{I 3/2 ee a | |

N — C—N(CH,), _N — C=NICH,), NH — C—N(CH,)5

I GS : GSSG I
O 2 fe)
Diamine Oxidized glutathione

GS” Gs”, H"

R — SCN =a R554 8 RSH

Alkylthiocyanate CN GSSG

Cyanide Oxidized glutathione

FIGURE 6-20 Examples of glutathione conjugation of electrophilic heteroatoms.

106 UNIT 2 Disposition of Toxicants

BIBLIOGRAPHY
(Glutamic acid») Coleman MD: Human Drug Metabolism: An Introduction, 2nd ed.
Substrate Hs cS Phi Hoboken, NJ: Wiley-Blackwell, 2010.
(RX)
Lee PW, Aizawa H, Gan L, Prakash C, Zhong D (eds.): Handbook of
Metabolic Pathways of Xenobiotics. Hoboken, NJ: John Wiley &

eee
Glutathione transferase
Sons, 2014.
Nassar AF: Biotransformation and Metabolite Elucidation of
0)
I
, COO” Xenobiotics: Characterization and Identification. Hoboken, NJ:
_NH-C—CH) — CH, — CH John Wiley & Sons, 2010.
Rs ch cH Yan Q: Pharmacogenomics in Drug Discovery and Development,
"eS NH= Che — coor 2nd ed. Totowa, NJ: Humana Press, 2014.
I
O
+-Glutamyltransferase (GGT1)

© Glutamic acid >

/NH2
R-S—CH,—CH
“C —NH- CH, — COO
i
O

ies sebeteta Ritaked eer

Vv

Alanylaminopeptidase (ANPEP)
H,0 le

NH
GH A
R= S— Gh
COO

Cysteine conjugate

Beta lyase
Acetyl-CoA |

N-Acetyltransferase
CH3COCOO™ 244
(Pyruvate)
CoA
O
NH3 -
PNG Gis
R-S—CH,—CH
COO R— SH

Mercapturic acid
v
| Methylation
se Seen Glucuronidation
Excretion in urine

FIGURE 6-21 Glutathione conjugation and mercapturic acid

biosynthesis.
 CHAPTER 6 Biotransformation of Xenobiotics 107

QUESTIONS

Xenobiotic biotransformation is performed by multiple . All of the following statements regarding cytochrome
enzymes in multiple subcellular locations. Where would P450 are true EXCEPT:
one of these enzymes most likely NOT be located? a. Poor metabolism or biotransformation of xenobiotics
a. cytosol. is often due to a genetic deficiency in cytochrome
b. Golgi apparatus. P450.
c. lysosome. b. Cytochrome P450 can be inhibited by both competitive
d. mitochondria. and noncompetitive inhibitors.
e. microsome. c. Certain cytochrome P450 enzymes can be induced
by one’s diet.
All of the following statements regarding hydrolysis, reduc- d. Increased activity of cytochrome P450 always slows
tion, and oxidation biotransformations are true EXCEPT: the rate of xenobiotic activation.
The xenobiotic can be hydrolyzed. e. Induction of cytochrome P450 can lead to increased
The xenobiotic can be reduced. drug tolerance.
There is a large increase in hydrophilicity.
f
ao” The reactions introduce a functional group to the . Which of the following statements regarding phase II
molecule. biotransformation (conjugation) reactions is true?
e. The xenobiotic can be oxidized. a. Phase II reactions greatly increase the hydrophilicity
of the xenobiotic.
Which of the following is often conjugated to xenobiotics b. Phase II reactions are usually the rate-determining
during phase IT biotransformations? step in the biotransformation and excretion of
a. alcohol group. xenobiotics.
b. sulfhydryl group. c. Carboxyl groups are very common additions of
c. sulfate group. phase II reactions.
d. aldehyde group. Most phase II reactions occur spontaneously.
e. carbonyl group. e. Increased phase II reactions result in increased xeno-
biotic storage in adipocytes.
Which of the following is a true statement about the
biotransformation of ethanol? . Where do most phase II biotransformations take place?
a. Alcohol dehydrogenase is only present in the liver. a. mitochondria.
b. Ethanol is reduced to acetaldehyde by alcohol b. ER.
dehydrogenase. c. blood.
c. Ethanol and hydrogen peroxide combine to form d. nucleus.
acetaldehyde with the aid of catalase. e. cytoplasm.
d. In spite of its catalytic versatility, cytochrome P450
does not aid in ethanol oxidation. 10. Which of the following is not an important cosubstrate
e. Acetaldehyde is oxidized to acetic acid in the mito- for phase II biotransformation reactions?
chondria by aldehyde dehydrogenase. UDP-glucuronic acid.
3’-phosphoadenosine-5’-phosphosulfate (PAPS).
Which of the following enzymes is responsible for the S-adenosylmethionine (SAM).
biotransformation and elimination of serotonin? N-nitrosodiethylamine.
a. cytochrome P450. fe acetyl CoA.
Oo
Sr
b. monoamine oxidase.
c. flavin monooxygenase.
d. xanthine oxidase.
e. paraoxonase.

Which of the following reactions would likely NOT be

catalyzed by cytochrome P450?
dehydrogenation.
oxidative group transfer.
epoxidation.
reductive dehalogenation.
go
ae
len
igh ester cleavage.
 FE - by
posses 5to

a
rea a 3

|
= fs

|
 Ci 4B eA gia (AGE aR

Toxicokinetics
Danny D. Shen

INTRODUCTION PHYSIOLOGIC TOXICOKINETICS

Basic Model Structure
CLASSIC TOXICOKINETICS
Compartments
One-compartment Model
Parameters
Two-compartment Model
Anatomical
Elimination
Physiologic
Apparent Volume of Distribution Thermodynamic
Clearance
Transport
Relationship of Elimination Half-life to Clearance Perfusion-limited Compartments
and Volume
Diffusion-limited Compartments
Absorption, Bioavailability, and Metabolite
Specialized Compartments
Kinetics
Lung
Saturation Toxicokinetics
Liver
Accumulation during Continuous or
Blood
Intermittent Exposure
Conclusion CONCLUSION

KEY POINTS

= Toxicokinetics is the study of the modeling and mathe- w Clearance describes the rate of chemical elimination
matical description of the time course of disposition from the body in terms of volume of fluid containing
(absorption, distribution, biotransformation, and excre- chemical that is cleared per unit of time.
tion) of xenobiotics in the whole organism. a ‘The half-life of elimination (T,,.) is the time required for
w Theapparent volume of distribution (V,) is the space into the blood or plasma chemical concentration to decrease
which an amount of chemical is distributed in the body by one-half.
to result in a given plasma concentration.

INTRODUCTION more compartments that may have no apparent physiologic

or anatomical reality. An alternate and newer approach,
Toxicokinetics is the study of the modeling and mathematical physiologically based toxicokinetic modeling, attempts to
description of the time course of disposition (absorption, portray the body as an elaborate system of discrete tissue or
distribution, biotransformation, and excretion) of xenobiot- organ compartments that are interconnected via the circula-
ics in the whole organism. In the classic model, chemicals are tory system. There is no inherent contradiction between the
said to move throughout the body as if there were one or classic and physiologically based approaches, yet certain
109
 110 UNIT 2 Disposition of Toxicants

assumptions differ between the two models. Ideally, physio- the time course of chemical in plasma and tissues and the extent
logic models can predict tissue concentrations, whereas of chemical accumulation with multiple doses, and determining
classic models cannot. effective dose and dose regimens in toxicity studies.

CLASSIC TOXICOKINETICS One-compartment Model

The simplest toxicokinetic analysis entails measurement of the
The least invasive and simplest method to gather information on
plasma concentrations of a xenobiotic at several time points
absorption, distribution, metabolism, and elimination ofa com-
after the administration ofa bolus intravenous injection. If the
pound is by sampling blood or plasma over time. Assuming that
data obtained yield a straight line when they are plotted as the
the concentration of a compound in blood or plasma is in equi-
logarithm of plasma concentrations versus time, the kinetics of
librium with concentrations in tissues, then changes in plasma
the xenobiotic can be described with a one-compartment
toxicant concentrations should reflect changes in tissue toxicant
model (Figure 7-2). Compounds whose toxicokinetics can be
concentrations. Compartmental pharmacokinetic models con-
described with a one-compartment model rapidly equilibrate,
sist of a central compartment representing plasma and tissues
or mix uniformly, between blood and the various tissues rela-
that rapidly equilibrate with chemical, connected to one or more
tive to the rate of elimination. The one-compartment model
peripheral compartments that represent tissues that more slowly
depicts the body as a homogeneous unit. This does not mean
equilibrate with the chemical (Figure 7-1). Chemical is admin-
that the concentration of a compound is the same throughout
istered into the central compartment and distributes between
the body, but it does assume that the changes that occur in the
central and peripheral compartments. Chemical elimination
plasma concentration reflect proportional changes in tissue
occurs from the central compartment, which is assumed to con-
chemical concentrations.
tain rapidly perfused tissues capable of eliminating the chemical
In the simplest case, a curve of this type can be described by
(e.g., kidneys, lungs, and liver). Compartmental pharmacoki-
the following expression:
netic models require no information on tissue physiology or
anatomical structure, and they are valuable in predicting the
plasma chemical concentrations at different doses, establishing G= Cente

where C is the blood or plasma chemical concentration over

_One-compartment model time t, C, the initial blood concentration at time t= 0, and k, the
first-order elimination rate constant with dimensions of recip-
rocal time (e.g., f”'). ky represents the overall elimination of the
chemical, which includes biotransformation, exhalation, and/
or excretion pathways.
el

Two-compartment Model
After the rapid intravenous administration of some chemicals,
the semilogarithmic plot of plasma concentration versus time
yields a curve rather than a straight line, which implies that
there is more than one dispositional phase. In these instances, the
chemical requires a longer time for tissue concentrations to reach
equilibrium with the concentration in plasma, and a multicom-
partmental analysis of the results is necessary (Figure 7-2).
Peripheral A multiexponential mathematical equation then best character-
(2) izes the elimination of the xenobiotic from the plasma.
Generally, a curve of this type can be resolved into two
monoexponential terms (a two-compartment model) and is
described by:

FIGURE 7-1 Compartmental pharmacokinetic models. k, is

C= Ae“ + Be#
the first-order extravascular absorption rate constant into the central
compartment (1), k,, is the first-order elimination rate constant from
where A and B are proportionality constants and a@ and 3 the
the central compartment (1), and k,, and k,, are the first-order rate first-order distribution and elimination rate constants, respec-
constants for distribution of chemical into and out of the peripheral tively (Figure 7-2). During the distribution (a) phase, concen-
compartment (2) in a two-compartment model, whereas k,, is the trations of the chemical in the plasma decrease more rapidly
first-order elimination rate constant from the central compartment in than they do in the postdistributional elimination (3) phase.
a two compartment model. The distribution phase may last for only a few minutes or for
 CHAPTER7 Toxicokinetics aL

One-compartment Model

Conc Conc

Time Time Time

Two-compartment Model

we Central
Y uy Y . ~ Tissue
3 ° ro)
Oo
Fe ~
ia] U

=
Peripheral
Tissue

Time Time Time

FIGURE 7-2 Plasma concentration versus time curves of toxicants exhibiting kinetic behavior conforming to a one-compartment
model (top row) and a two-compartment model (bottom row) following intravenous bolus injection. Left and middle panels show
the plots on a rectilinear and semilogarithmic scale, respectively. Right panels illustrate the relationship between tissue (dashed lines) and
plasma (solid line) concentration over time. The right panel for the one-compartment model shows a typical tissue with a higher concentration
than plasma. Note that tissue concentration can be higher, nearly the same, or lower than plasma concentration. Tissue concentration peaks
almost immediately, and thereafter declines in parallel with plasma concentration. The right panel for the two-compartment model shows
typical tissues associated with the central (1) and peripheral (2) compartments as represented by short-and-long dash lines, respectively. For
tissues associated with the central compartment, their concentrations decline in parallel with plasma. For tissues associated with peripheral
compartment, toxicant concentration rises, while plasma concentration declines rapidly during the initial phase; it then reaches a peak and
eventually declines in parallel with plasma in the terminal phase. Elimination rate constant k,, for one-compartment model and the terminal
exponential rate constant 3 are determined from the slope of the log-linear concentration versus time curve. Half-life (7,,.) is the time required
for plasma toxicant concentration to decrease by one-half. C, is the concentration of a toxicant for a one-compartment model at t = 0
determined by extrapolating the log-linear concentration time curve to the Y-axis.

hours or days. The equivalent of k, in a one-compartment elimination rate constant (k,,) can be determined from the
model is 3 in a two-compartment model. slope of the log C versus time plot (i.e., k,, = 2.303 slope).
The first-order elimination rate constants, k,, and G, have
units of reciprocal time (e.g., min” 'and h~') and are indepen-
Elimination dent of dose.
Elimination includes biotransformation, exhalation, and excre- Mathematically, the fraction of dose remaining in the body
tion. The elimination of a chemical from the body whose dispo- over time (C/C,) is calculated using the elimination rate con-
sition is described by a one-compartment model usually occurs stant by rearranging the equation for the monoexponential
through a first-order process; that is, the rate of elimination at function and taking the antilog to yield:
any time is proportional to the amount of the chemical in the
body at that time. First-order reactions occur at chemical con-

45}
G
centrations that are not sufficiently high to saturate elimination —
C — Antilo g
processes. 0

The equation for a monoexponential model, C = Coe“, can

be transformed to a logarithmic equation that has the general
form ofa straight line, y = mx + b: Apparent Volume of Distribution
: i In a one-compartment model, all chemical is assumed to dis-
tribute and equilibrate into plasma and tissues instantaneously.
logC = |—— |t + logC
“e een a The apparent volume of distribution (V,) is a proportionality
constant that relates the total amount of chemical in the body to
where logC, represents the y-intercept or initial concentra- its concentration in plasma, and typically has units of liters or
tion and (k,/2.303) the slope of the line. The first-order liters per kilogram of body weight. V, is the apparent space into
WIZ UNIT 2 Disposition of Toxicants

which an amount ofchemical is distributed in the body to result inflow that is irreversibly removed. After bolus intravenous
ina given plasma concentration. The apparent volume ofdistri- administration, total body clearance is defined as:
bution ofa chemical in the body is determined after intravenous
bolus administration, and is mathematically defined as the quo- Dose,,
C—.——
tient of the amount of chemical in the body and its plasma con- AUC}
centration. V, is calculated as follows:
Clearance can also be calculated if the volume ofdistribution
_ Dose, and elimination rate constants are known, and can be defined
da
Bx AUC® E as Cl = Vk, for a one-compartment model and Cl = V, for a
two-compartment model.
where Dose,, is the intravenous dose or known amount of
chemical in body at time zero, @ the elimination rate constant,
and AUC? the area under the chemical concentration versus
Relationship of Elimination Half-life to
time curve from time zero to infinity. The product, 8 x AUC>, Clearance and Volume
is the concentration of xenobiotic in plasma. The half-life of elimination (T,,,) is the time required for the
For a one-compartment model, V, can be simplified by the blood or plasma chemical concentration to decrease by one-
following equation: half, and is dependent on both volume ofdistribution and clear-
ance. T,,, can be calculated from V, and Cl:
Dose,,
=
Cy \ 0.693V;
Lae Ae
where C, is the concentration of chemical in plasma at time \
‘

zero. Cy is determined by extrapolating the plasma disappear- Because of the relationship T,,. = 0.693k,, the half-life of a
ance curve after intravenous injection to the zero time point compound can be calculated after k, (or 3) has been deter-
(Figure 7-2). V, is called the apparent volume of distribution. mined from the slope of the line that designates the elimina-
The magnitude of the V, term is chemical-specific and repre- tion phase on the log C versus time plot. The T\,. can also be
sents the extent of distribution of chemical out of plasma and determined by means of visual inspection of the log C versus
into other body tissues. Thus, a chemical with high afhnity for time plot, as shown in Figure 7-2. For compounds eliminated
tissues willalso havealarge volume of distribution. Alternatively, by first-order kinetics, the time required for the plasma con-
a chemical that predominantly remains in the plasma will have centration to decrease by one-half is constant. After seven
a low V, that approximates the volume of plasma. Once the V, half-lives, 99.2% of a chemical is eliminated, which can be
for a chemical is known, it can be used to estimate the amount practically viewed as complete elimination. The half-life of a
of chemical remaining in the body at any time if the plasma chemical obeying first-order elimination kinetics is indepen-
concentration at that time is also known by the relationship dent of the dose, and does not change with increasing dose.
X. = V,C,, where X, is the amount of chemical in the body and
C, the plasma chemical concentration.
Absorption, Bioavailability, and
Clearance Metabolite Kinetics
Clearance describes the rate of chemical elimination from the For most chemicals in toxicology, exposure occurs by extravas-
body in terms of volume of fluid containing chemical that is cular routes (inhalation, dermal, or oral), and absorption is
cleared per unit of time. Thus, clearance has the units of flow often incomplete. The extent of absorption of a xenobiotic can
(mL/min). A clearance of 100 mL/min means that 100 mL of be experimentally determined by comparing the plasma AUC?
blood or plasma containing xenobiotic is completely cleared of after intravenous and extravascular dosing. The resulting index
the substance each minute. quantifies the fraction of dose absorbed systemically and is
The overall efficiency of the removal of a chemical from the called bioavailability (F). Bioavailability can be determined by
body can be characterized by clearance. High values of clear- using different doses, provided that the compound does not dis-
ance indicate efficient and generally rapid removal, whereas play dose-dependent or saturable kinetics. Pharmacokinetic
low clearance values indicate slow and less efficient removal of data following intravenous administration are used as the
a xenobiotic from the body. Total body clearance is defined as reference from which to compare extravascular absorption
the sum of clearances by individual eliminating organs: because all of the chemical is delivered to the systemic circula-
tion (100% bioavailable). For example, bioavailability following
Cl a Clea ar Cle eae aE Cle OG
an oral exposure can be determined as follows:

Each organ clearance is determined by blood perfusion flow _ AUC./ Dose,

through the organ and the fraction of toxicant in the arterial ~ Dose,,/AUC,,
 CHAPTER7 Toxicokinetics 113

where AUC,,, AUC,, Dose,,, and Dose,, are the respective area distribution (V,), clearance (Cl), and half-life (T,,,) are inde-
under the plasma concentration versus time curves and doses pendent of dose; and (4) the concentration of the chemical in
for oral and intravenous administration. Bioavailabilities for plasma and other tissues decreases similarly by some constant
various chemicals range in values between 0 and 1. Complete fraction per unit oftime, the elimination rate constant (k, or (3).
availability of chemical to systemic circulation is demonstrated
by F = 1. When F < 1, less than 100% of the dose reaches sys-
temic circulation. The fraction of a chemical that reaches the Accumulation during Continuous or
systemic circulation is of critical importance in determining Intermittent Exposure
toxicity. Several factors can greatly alter this systemic Chronic exposure to a chemical leads to its cumulative intake
availability, including (1) limited absorption after oral dosing, and accumulation in the body. At a fixed level of continuous
(2) intestinal first-pass effect, (3) hepatic first-pass effect, and exposure, accumulation of a toxicant in the body eventually
(4) mode of formulation, which affects, e.g., dissolution rate or reaches a point when the intake rate of the toxicant equals its
incorporation into micelles (for lipid-soluble compounds). elimination rate, the steady state.
The toxicity of a chemical is in some cases attributed to Accumulation can also occur with intermittent exposure.
its biotransformation product(s). Hence, the formation and For a chemical with a relatively short half-life compared with
disposition kinetics of a toxic metabolite is of considerable the interval between episodes of exposure, little accumulation
interest. As expected, the plasma concentration of ametabolite is expected. In contrast, for a chemical with an elimination half-
rises as the parent drug is transformed into the metabolite. A life approaching or exceeding the between-exposure interval,
biologically active metabolite assumes toxicologic significance progressive accumulation is expected over the intervals.
when it is the major metabolic product and is cleared much less
efficiently than the parent compound.
Conclusion
Saturation Toxicokinetics For many chemicals, blood or plasma chemical concentration
versus time data can be adequately described by a one- or two-
As the dose of a compound increases, its volume of distribution
compartment, classic pharmacokinetic model when basic
or its rate of elimination may change, owing to saturation
assumptions are made (e.g., instantaneous mixing within com-
kinetics. Biotransformation, active transport processes, and
partments and first-order kinetics). In some instances, more
protein binding have finite capacities and can be saturated.
sophisticated models with increased numbers of compartments
When the concentration of a chemical in the body is higher
will be needed to describe blood or plasma toxicokinetic data.
than the K,,, (chemical concentration at one-half V,,,,, the maxi-
Knowledge of toxicokinetic data and compartmental modeling
mum metabolic capacity), the rate of elimination is no longer
are useful in deciding what dose or dosing regimen of chemical
proportional to the dose. The transition from first-order to sat-
to use in the planning of toxicology studies (e.g., targeting a
uration kinetics is important in toxicology because it can lead to
toxic level of exposure), in choosing appropriate sampling
prolonged residency time of a compound in the body or
times for biological monitoring, and in seeking an understand-
increased concentration at the target site of action, which may
ing of the dynamics ofa toxic event (e.g., what blood or plasma
result in increased toxicity.
concentrations are achieved to produce a specific response,
Nonlinear toxicokinetics are indicated by the following:
how accumulation of a chemical controls the onset and degree
(1) the decline in the levels of the chemical in the body is not
of toxicity, and the persistence of toxic effects following
exponential, (2) AUC? is not proportional to the dose, (3) Va, Cl,
termination of exposure).
k. (or 3), or T,,, changes with increasing dose, (4) the composi-
tion of excretory products changes quantitatively or qualitatively
with the dose, and (5) dose-response curves show a nonpropor- PHYSIOLOGIC TOXICOKINETICS
tional change in response to an increasing dose, starting at the
dose level at which saturation effects become evident. In classic kinetics, the rate constants are defined by the data
Important characteristics of zero-order processes are as and these models are often referred to as data-based. In physiologi-
follows: (1) an arithmetic plot of plasma concentration versus cally based models, the rate constants represent known or hypoth-
time yields a straight line, (2) the rate or amount of chemical esized biological processes. The advantages of physiologically
eliminated at any time is constant and is independent of the based models are that (1) these models can provide the time
amount of chemical in the body, and (3) a true T\,, or k,, does course of distribution of xenobiotics to any organ or tissue,
not exist, but differs depending on dose. (2) they allow estimation of the effects of changing physiologic
By comparison, the important characteristics of first-order parameters on tissue concentrations, (3) the same model can pre-
elimination are (1) the rate at which a chemical is eliminated at dict the toxicokinetics of chemicals across species by allometric
any time is directly proportional to the amount of that chemical scaling, and (4) complex dosing regimens and saturable processes
in the body at that time; (2) a semilogarithmic plot of plasma such as metabolism and binding are easily accommodated.
concentration versus time yields a single straight line; (3) the The disadvantages are that (1) much more information is
elimination rate constant (k, or @), apparent volume of needed to implement these models compared with classic
114 UNIT 2 Disposition of Toxicants

models, (2) the mathematics can be difficult for many toxicolo-

Vascular space
gists to handle, and (3) values for parameters are often poorly Q: X Cin Qt Cout
defined in various species and pathophysiologic states. Interstitial space
Nevertheless, physiologically based toxicokinetic models are
conceptually sound and are potentially useful tools for gaining Intracellular space
rich insight into the kinetics of toxicants beyond what classic z
~
toxicokinetic models can provide. ri Rie ee oe ;
“<<
ae * Binding sites es " 4
=—1 a ~ “/ Pps = Cee

Basic Model Structure FIGURE 7-3 Schematic representation of a lumped

Physiologic models often look like a number of classic one- compartment in a physiologic model. The blood capillary
compartment models that are linked together by the circulatory and cell membranes separating the vascular, interstitial, and
intracellular subcompartments are depicted in black. The vascular
system. The actual model structure, or how the compartments
and interstitial subcompartments are often combined into a
are linked together, depends on both the chemical and the
single extracellular subcompartment. Q, is blood flow, C;, is
organism being studied. It is important to realize that there is
chemical concentration into the compartment, and C,,, is chemical
no generic physiologic model. Models are simplifications of concentration out of the compartment.
reality and ideally should contain elements believed to be
important in describing a chemical’s disposition.
Physiologic modeling has enormous potential predictive (1) the vascular space through which the compartment is per-
power compared with classic compartmental modeling. Because fused with blood, (2) the interstitial space that surrounds the cells,
the kinetic constants in physiologic models represent measur- and (3) the intracellular space consisting of the cells in the tissue.
able biological or chemical processes, the resultant physiologic As shown in Figure 7-3, the toxicant enters the vascular sub-
models have the potential for extrapolation from observed compartment at a certain rate in mass per unit of time (e.g.,
data to predicted situations. mg/h). The rate of entry is a product of the blood flow rate to
One of the best illustrations of the predictive power of physi- the tissue (Q,, L/h) and the concentration of the toxicant in the
ologic models is their ability to extrapolate kinetic behavior blood entering the tissue (C;,, mg/L). Within the compart-
from laboratory animals to humans. Simulations are the out- ment, the toxicant moves from the vascular space to the inter-
comes or results (such as a chemical’s concentration in blood stitial space at a certain net rate (Flux,) and from the interstitial
or tissue) of numerically integrating model equations over a space to the intracellular space at a different net rate (Flux,).
simulated time period, using a set ofinitial conditions (such as Some toxicants can bind to cell components; thus, within a
intravenous dose) and parameter values (such as organ weights compartment there may be both free and bound toxicants. The
and blood flow). Whereas the model structures for the kinetics toxicant leaves the vascular space at a certain venous concen-
of chemicals in rodents and humans may be identical, the tration (C,,.)- Cou is equal to the concentration of the toxicant
parameter values, such as organ weight, heart beat rate, and in the vascular space.
respiration rate, for rodents and humans are different. Other
parameters, such as solubility in tissues, are similar in the
rodent and human models because the composition of tissues Parameters
in different species is similar. Because the parameters underly- The most common types of parameters, or information required,
ing the model structure represent measurable biological and in physiologic models are anatomical, physiologic, thermo-
chemical determinants, the appropriate values for those dynamic, and transport.
parameters can be chosen for each species, forming the basis
for successful interspecies extrapolation. Because physiologic Anatomical— Anatomical parameters are used to physically
models represent real, measurable values, such as blood flows describe the various compartments. The size of each of the com-
and ventilation rates, the same model structure can resolve partments in the physiologic model must be known. The size is
such disparate kinetic behaviors among species. generally specified as a volume (milliliters or liters) because a
unit density is assumed even though weights are most fre-
quently obtained experimentally. If a compartment contains
Compartments subcompartments such as those in Figure 7-3, those volumes
The basic unit of the physiologic model is the lumped compart- also must be known. Volumes of compartments often can be
ment (Figure 7-3), which is a single region of the body with a obtained from the literature or from specific toxicokinetic
uniform xenobiotic concentration. A compartment may be a experiments.
particular functional or anatomical portion of an organ, a single
blood vessel with surrounding tissue, an entire discrete organ Physiologic—Physiologic parameters encompass various
suchas the liver or kidney, or a widely distributed tissue type such processes including blood flow, ventilation, and elimination.
as fat or skin. Compartments consist of three individual well- The blood flow rate (Q,, in volume per unit time, such as
mixed phases, or subcompartments. These subcompartments are mL/min or L/h) to individual compartments must be known.
 CHAPTER7 Toxicokinetics 115

Additionally, information on the total blood flow rate or cardiac Figure 7-3) or across the cell membrane (Flux, in Figure 7-3).
output (Q.) is necessary. If inhalation is the route for exposure Flux refers to the rate of transfer of a chemical across a boundary.
to the xenobiotic or is a route of elimination, the alveolar venti- For simple diffusion, the net flux (mg/h) from one side of a
lation rate (Q,) also must be known. Blood flow rates and venti- membrane to the other is described as Flux = permeability
lation rates can be taken from the literature or obtained coefficient x driving force, or:
experimentally. Parameters for renal excretion and hepatic
metabolism are another subset of physiologic parameters that Flux = [PA](C, -C,) = [PA]C, - [PA]C,
are required if these processes are important in describing the
elimination of a xenobiotic. The term PA is often called the permeability-area product for
the membrane or cellular barrier in flow units (e.g., L/h), and is
Thermodynamic— Thermodynamic parameters relate the a product of the barrier permeability coefficient (P in velocity
total concentration of a xenobiotic in a tissue (C,) to the concen- units, e.g., m/h) for the toxicant and the total barrier surface
tration of free xenobiotic in that tissue (C,). Two important area (A, in um’). The permeability constant takes into account
assumptions are that (1) total and free concentrations are in the rate of diffusion ofthe specific xenobiotic and the thickness
equilibrium with each other and (2) only free xenobiotic can be of the cell membrane. C, and C, are the free concentrations of
exchanged between the tissue subcompartments. Whereas total xenobiotic on each side of the membrane.
concentration is measured experimentally, it is the free concen- For any given xenobiotic, thin membranes, large surface
tration that is available for binding, metabolism, or removal areas, and large concentration differences enhance diffusion.
from the tissue by blood. The extent to which a xenobiotic parti- Membrane transporters offer an additional route of entry into
tions into a tissue is directly dependent on the composition of cells, and allow more effective tissue penetration for toxicants
the tissue and independent of the concentration of the xenobi- that have limited passive permeability. Alternately, the pres-
otic. Thus, the relationship between free and total concentra- ence of efflux transporters at epithelial or endothelial barriers
tion becomes one of proportionality: total = free x partition can limit toxicant penetration into critical organs, even for
coefficient, or C, = C,P,. Knowledge of the value of P,, a partition highly permeable toxicants. There are two limiting conditions
or distribution coefficient, permits an indirect calculation ofthe for the uptake of a toxicant into tissues: perfusion-limited and
free concentration of xenobiotic or C;= C,/P.. diffusion-limited.
Table 7-1 compares the partition coefficients for a number
of toxic volatile organic chemicals. The larger values for the fat/
blood partition coefficients compared with those for other tis- Perfusion-limited Compartments
sues suggest that these chemicals distribute into fat to a greater A perfusion-limited compartment is also referred to as blood
extent than they distribute into other tissues. flow-limited, or simply flow-limited. A flow-limited compart-
A more complex relationship between the free concentra- ment can be developed if the cell membrane permeability coef-
tion and the total concentration of a chemical in tissues occurs ficient [PA] for a particular xenobiotic is much greater than the
when the chemical may bind to saturable binding sites on tis- blood flow rate to the tissue (Q,). In this case, uptake of xenobi-
sue components. In these cases, nonlinear functions relating otic by tissue subcompartments is limited by the rate at which
the free concentration in the tissue to the total concentration the blood containing a xenobiotic arrives at the tissue and not
are necessary. by the rate at which the xenobiotic crosses the cell membranes.
In most tissues, transport across vascular cell membranes is
Transport— The passage ofa chemical across a biological mem- perfusion-limited. In the generalized tissue compartment in
brane is complex and may occur by passive diffusion, carrier- Figure 7-3, this means that transport of the xenobiotic through
mediated transport, facilitated transport, or a combination of the loosely knit blood capillary walls of most tissues is rapid
processes. The simplest of these processes—passive diffusion—is compared with delivery of the xenobiotic to the tissue by the
a first-order process described by Fick’s law. Diffusion of xenobi- blood. Asa result, the vascular blood is in equilibrium with the
otics can occur across the blood capillary endothelium (Flux, in interstitial subcompartment and the two subcompartments are
usually lumped together as a single compartment that is often
called the extracellular space.
TABLE 7-1 Partition coefficients for four volatile As indicated in Figure 7-3, the cell membrane separates the
organic chemicals in several tissues. extracellular compartment from the intracellular compart-
ment. The cell membrane is the most important diffusional
Chemical Blood/Air Muscle/Blood Fat/Blood barrier in a tissue. Nonetheless, for molecules that are very
Isoprene 3 0.67 24 small (molecular weight < 100) or lipophilic, cellular permea-
bility generally does not limit the rate at which a molecule
Benzene 18 0.61 28
moves across cell membranes. For these molecules, flux across
Styrene 40 1 50 the cell membrane is fast compared with the tissue perfusion
rate (PA, > Q,), and the molecules rapidly distribute through-
Methanol 1,350 3 11
out the subcompartments. In this case, free toxicant in the
116 UNIT 2 Disposition ofToxicants

intracellular compartment is always in equilibrium with the assumption that significantly reduces the number of parame-
extracellular compartment, and these tissue subcompartments ters required in the physiologic model.
can be lumped as a single compartment. This flow-limited tis-
sue compartment is shown in Figure 7-4. Movement into and Diffusion-limited Compartments
out of the entire tissue compartment can be described by a
When uptake ofa toxicant into a compartment is governed by its
single equation: diffusion or transport across cell membrane barriers, the model
is said to be diffusion-limited or barrier-limited. Diffusion-
dc, limited uptake or release occurs when the flux, or the transport
(rR = OG. i, Ca)
of a toxicant across cell barriers, is slow compared with blood
flow to the tissue. In this case, the permeability—area product is
where V, is the volume of the tissue compartment, C, the con- small compared with blood flow, that is, PA < Q,. Figure 7-5
centration of free xenobiotic in the compartment (V,C, equals shows the structure of such a compartment. The toxicant con-
the amount of xenobiotic in the compartment), V,(dC,/dt) the centrations in the vascular and interstitial spaces are in equilib-
change in the amount of xenobiotic in the compartment with rium and make up the extracellular subcompartment, where
time expressed as mass per unit of time, Q, the blood flow to uptake from the incoming blood is flow-limited. The rate of toxi-
the tissue, C,, the xenobiotic concentration entering the com- cant uptake across the cell membrane from the extracellular
partment, and C,,, the xenobiotic concentration leaving the space into the intracellular space is limited by membrane
compartment. Equations of this type are called mass-balance permeability. Two mass-balance differential equations are nec-
differential equations. Differential refers to the term dC//dt. essary to describe the events in these two subcompartments:
Mass balance refers to the requirement that the rate of change
in the amount of toxicant in a compartment equals the differ- 1. Extracellular space:
ence in the rate of entry via arterial inflow and the rate of 2
\

departure via venous outflow. dC,,

In the perfusion-limited case, the concentration of toxicant Vo = Q (Cin Caw) pa, |S
pa,
dt tl t2
in the venous drainage from the tissue is equal to the concentra-
tion of toxicant in the tissue when the toxicant is not bound to
blood constituents (i.e., Cou. = C, = C;). As was noted previ- 2. Intracellular space:
ously, when there is binding of toxicant to tissue constituents, C;
(or C,,,) can be related to the total concentration of toxicant in
the tissue through a simple linear partition coefficient, C,.. =
va 8S = aS]
on|
C; = C/P,. In this case, the differential equation describing the
rate of change in the amount of a toxicant in a tissue becomes:
Here, Q, is blood flow and C the free xenobiotic concentration
in entering blood (in), exiting blood (out), extracellular space
de G (1), or intracellular space (2). Both equations contain terms for
V,—
di =
al
| Ge —
|
— flux, or transfer across the cell membrane [PA](C, — C,).

Specialized Compartments
In a flow-limited compartment, the assumption is that the
concentrations of a xenobiotic in all parts of the tissue are in Lung— The inclusion of a lung compartment in a physiologic
equilibrium. Additionally, estimates of flux are not required to model is an important consideration because inhalation is a
develop the mass balance differential equation for the com- common route of exposure to many toxic chemicals. The
partment. Given the challenges in measuring flux across the
vascular endothelium and cell membrane, this is a simplifying
Extracellular space
pa apenes hes = to
a *
4 : 6 fee enmnes7
oth he wr _
Intracellular space—e
a
Oe) Ss eer
® s
oD th eee

FIGURE 7-5 Schematic representation of a compartment

FIGURE 7-4 schematic representation of a compartment that is membrane-limited. Perfusion of blood into and out
that is blood flow-limited. Rapid exchange between the of the extracellular compartment is depicted by thick arrows.
extracellular space (salmon) and intracellular space (bisque) maintains Transmembrane transport (flux) from the extracellular to the
the equilibrium between them as symbolized by the dashed line. Q, is intracellular subcompartment is depicted by thin double arrows. Q, is
blood flow, C,, is chemical concentration into the compartment, and blood flow, C;, is chemical concentration into the compartment, and
Cour is Chemical concentration out of the compartment. Cour is chemical concentration out of the compartment.
 CHAPTER7 Toxicokinetics ly,

assumptions inherent in this compartment description are as The lung is viewed here as a portal of entry and not as a target
follows: (1) ventilation is continuous, not cyclic; (2) conducting organ, and the concentration of a xenobiotic delivered to other
airways (nasal passages, larynx, trachea, bronchi, and bronchi- organs by the blood, or the arterial concentration of that xeno-
oles) function as inert tubes, carrying the vapor to the alveoli biotic, is of primary interest. The assumptions of continuous
where gas exchange occurs; (3) diffusion of vapor across the ventilation, rapid equilibration with arterial blood, and no
alveolar epithelium and capillary walls is rapid compared with hold-up in lung tissues have proved applicable with many vola-
blood flow through the alveolar region; (4) all chemicals disap- tile organics. The use of these assumptions simplifies and
pearing from the inspired air appears in the arterial blood (i.e., speeds up model calculations and may be entirely adequate for
there is no hold-up of chemical in the lung tissue and insignifi- describing the toxicokinetic behavior of relatively inert vapors
cant lung mass); and (5) vapor in the alveolar air and arterial with low water solubility.
blood within the lung compartment are in rapid equilibrium.
In the lung compartment depicted in Figure 7-6, the rate Liver—The liver is almost always featured as a distinct com-
of inhalation of xenobiotic is controlled by the ventilation partment in physiologic models because biotransformation is
rate (Q,) and the inhaled concentration (C,,,). The rate of an important route of elimination for many toxicants and the
exhalation of axenobiotic is a product of the ventilation rate liver is considered the major organ for biotransformation of
and the xenobiotic concentration in the alveoli (C,,). xenobiotics. A simple compartmental structure for the liver is
Xenobiotic also can enter the lung compartment via venous one where uptake into the liver compartment is assumed to be
blood returning from the heart, represented by the product flow-limited. This liver compartment is similar to the general
of cardiac output (Q.) and the concentration of xenobiotic in tissue compartment in Figure 7-4, except that the liver
venous blood (C,,,,). Xenobiotic leaving the lungs via the compartment contains an additional process for metabolic
blood is a function of both cardiac output and the concentra- elimination. Under first-order elimination, the rate of hepatic
tion of xenobiotic in arterial blood (C,,,). Putting these four metabolism (R) by the liver can be presented as:
processes together, a mass balance differential equation can
be written for the rate of change in the amount of xenobiotic R=Cl1,+C;
in the lung compartment (L):
where C; is the free concentration of toxicant in the liver
dL (mg/L), and Cl, is the clearance of free toxicant within the
dt = Q:(Gan a Ce) Ug Ci Gre ak Ca) liver (L/h).
In the case ofa single enzyme mediating the biotransforma-
tion and Michaelis-Menten kinetics are obeyed, Ci, is related to
During continuous exposure at steady state, the rate of the maximum rate of metabolism V,,,, (in mg/h) and the
change in the amount of xenobiotic in the lung compartment Michaelis constant Ky, (in mg/L). Asa result, the rate of hepatic
becomes equal to zero (dL/dt = 0). C,, can be replaced by metabolism can be expressed in terms of the Michaelis
C,,,/P,j. and the differential equation can be solved for the parameters:
arterial blood concentration:

Vie

C =a Q,Cinh = ORG
KGa
pi Q. a6 (Q,/Poia)

Under nonsaturating or first-order condition (i.e., C; < Ky),

Cl, becomes equal to the ratio of V,,.,/Ky. Because many toxi-
cants at high exposure levels display saturable metabolism, the
Q, X Cinh
above equation is often invoked for simulation of toxicant dis-
position across a wide range of doses.
Other, more complex expressions for metabolism also can
Q-X Cart 1 Q.X Cyen be incorporated into physiologic models. Bi-substrate sec-
ond-order reactions, reactions involving the destruction of
FIGURE 7-6 Simple model of gas exchange in the alveolar enzymes, inhibition of enzymes, or the depletion ofcofactors,
region of the respiratory tract. Rapid exchange in the lumped lung have been simulated using physiologic models. Metabolism
compartment between the alveolar gas (blue) and the pulmonary blood
can be also included in other compartments in much the same
(salmon) maintains the equilibrium between them as symbolized by
way as described for the liver.
the dashed line. Q, is alveolar ventilation (L/h); Q. is cardiac output (L/h);
Gyn is inhaled vapor concentration (mg/L); C,,, is concentration of vapor
in the arterial blood; C,,, is concentration of vapor in the mixed venous Blood—In a physiologic model, the tissue compartments are
blood. The equilibrium relationship between the chemical in the alveolar linked together by the circulatory network. The decision to rep-
air (C,,) and the chemical in the arterial blood (C,,,) is determined by the resent blood as an explicit physiologic compartment depends
blood/air partition coefficient P,, €.9., Cay = Gor/Pova- on the role the blood plays in disposition and the type of
118 UNIT 2 Disposition of Toxicants

application. If the toxicokinetics after intravenous injection is Although simpler elements of physiologic models and
to be simulated or if binding to or metabolism by blood compo- the important assumptions that underlie model structures
nents is suspected, a separate compartment for the blood that are presented, toxicologists are developing increasingly more
incorporates these additional processes is required. A blood sophisticated applications. Three-dimensional visualizations
compartment is obviously needed if the model were developed of xenobiotic transport, physiologic models of aparent chem-
to explain a set of blood concentration-time data for a toxicant. ical linked in series with one or more active metabolites,
However, if blood is simply a conduit to the other compart- models describing biochemical interactions among xenobiot-
ments, as in the case for inhaled volatile organics, an algebraic ics, and more biologically realistic descriptions oftissues pre-
solution is acceptable. viously viewed as simple lumped compartments are just a few
of the more sophisticated applications. Finally, physiologi-
CONCLUSION cally based toxicokinetic models are now being linked to bio-
logically based toxicodynamic models to simulate the entire
Biological monitoring or biomonitoring is defined as the sys- exposure — dose — response paradigm that is basic to the
tematic sampling of body fluids, and at times body tissue, for the science oftoxicology.
purpose of estimating an individual’s internal dose from expo-
sure to chemicals in the workplace or assessing the range of
internal exposure within a select population to environmental
pollutants. The advantages of biomonitoring over traditional BIBLIOGRAPHY
environmental monitoring, such as ambient or personal air Andersen ME: Toxicokinetic modeling and its applications in chemi-
sampling or dermal dosimetry, include the accounting of other cal risk assessment. Toxicgl Lett 138:9-27, 2003.
unanticipated routes of exposure, individual differences in toxi- Esteban M, Castano A: Non-invasive matrices in human biomonitor-
ing—a review. Environ Int 35:438-449, 2009.
cant absorption and disposition, and critical personal or life-
Fowler BA (ed.): Computational Toxicology: Methods and Applications
style variables, such as body size and composition, workload
for Risk Assessment. New York: Academic Press/Elsevier, 2013.
that affects pulmonary ventilation, or cigarette smoking that
Lipscomb JC, Ohanian EV: Toxicokinetics and Risk Assessment. New
could affect the metabolic status of an individual. Linking envi- York: Informa Healthcare, 2007.
ronmental exposure or dose to measurements of concentration Rowland M, Tozer TN: Clinical Pharmacokinetics and
of the parent chemical or its metabolite(s) in a biological sample Pharmacodynamics—Concepts and Applications. Philadelphia, PA:
is essentially an exercise in toxicokinetics. Wolters Kluwer/Lippincott, Williams & Wilkins, 2011.
 CHAPTER7 Toxicokinetics 119

QUESTIONS

Regarding the two-compartment model of classic toxico- 6. With respect to first-order elimination, which of the
kinetics, which of the following is true? following statements is FALSE?
a. ‘There is rapid equilibration of chemical between a. The rate of elimination is directly proportional to the
central and peripheral compartments. amount of the chemical in the body.
b. The logarithm of plasma concentration versus time b. A semilogarithmic plot of plasma concentration
data yields a linear relationship. versus time shows a linear relationship.
c. ‘There is more than one dispositional phase. c. Half-life (T\,.) differs depending on the dose.
d. It is assumed that the concentration of achemical is Clearance is dosage-independent.
the same throughout the body. e. ‘The plasma concentration and tissue concentration
e. It is ineffective in determining effective doses in decrease similarly with respect to the elimination rate
toxicity studies. constant.

When calculating the fraction of a dose remaining in the The toxicity of a chemical is dependent on the amount of
body over time, which of the following factors need not be chemical reaching the systemic circulation. Which of the
taken into consideration? following does NOT greatly influence systemic availability?
half-life. a. absorption after oral dosing.
initial concentration. b. intestinal motility.
time. c. hepatic first-pass effect.
present concentration. d. intestinal first-pass effect.
Bd
TS elimination rate constant. e. incorporation into micelles.

All of the following statements regarding apparent . Which of the following is NOT an advantage of a
volume of distribution (V,) are true EXCEPT: physiologically based toxicokinetic model?
a. V, relates the total amount of chemical in the body to a. Complex dosing regimens are easily accommodated.
the concentration of chemical in the plasma. b. ‘The time course of distribution of chemicals to any
b. V, is the apparent space into which an amount of organ is obtainable.
chemical is distributed in the body to result in a given c. ‘The effects of changing physiologic parameters on
plasma concentration. tissue concentrations can be estimated.
c. A chemical that usually remains in the plasma has a The rate constants are obtained from gathered data.
low Vj. e. The same model can predict toxicokinetics of chemi-
d. V, will be low for a chemical with high affinity for cals across species.
tissues.
e. V,can be used to estimate the amount of chemical in . Which ofthe following will not help to increase the flux of
the body if the plasma concentration is known. a xenobiotic across a biological membrane?
decreased size.
Chemical clearance: decreased oil:water partition coefficient.
a. is independent of Vj. increased concentration gradient.
b. is unaffected by kidney failure. increased surface area.
c. is indirectly proportional to V4. oP decreased
cao membrane thickness.
d. is performed by multiple organs.
e. is not appreciable in the GI tract. 10. Which of the following statements is true regarding
diffusion-limited compartments?
A chemical with which of the following half-lives (T,,.) a. Xenobiotic transport across the cell membrane is
will remain in the body for the longest period of time limited by the rate at which blood arrives at the tissue.
when given equal dosage of each? b. Diffusion-limited compartments are also referred to
a. 7), = 30 min. as flow-limited compartments.
b. T,,. = 1 day. c. Increased membrane thickness can cause diffusion-
Cm lea limited xenobiotic uptake.
d. 7T,,=120s d. Equilibrium between the extracellular and intracel-
é, I, = ! month: lular space is maintained by rapid exchange between
the two compartments.
e. Diffusion of gases across the alveolar septa of a
healthy lung is diffusion-limited.
 riya + aahar fon ay TF

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crate x ap
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Bate | ewrts
TOS tO nee-
Neue Ries wie ¥.) a

wire)
nevy lhe tha a
— lhe
avd rng ii 1B a
Te 7 cP Or a Gg
; Ls
j rf

1 se (hem in ; x : Gmte TA Pat _

5 wT " * Ste , b ‘ta =) & (ie tiiw
OU TE ly Tavis opr dibity :
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cal 4?

i= }
 UNIT 3 NONORGAN-DIRECTED TOXICITY

Gal SAS Peeler

Chemical Carcinogenesis
James E. Klaunig

OVERVIEW DNA Methylation and Carcinogenesis

Definitions Oxidative Stress and Chemical Carcinogenesis
Gap Junctional Intercellular Communication and
MULTISTAGE CARCINOGENESIS Carcinogenesis
Initiation Modifiers of Chemical Carcinogenic Effects
Promotion Polymorphisms in Carcinogen Metabolism and
Progression DNA Repair
Proto-oncogenes and Tumor-suppressor Genes
MECHANISMS OF ACTION OF CHEMICAL
Retroviruses
CARCINOGENS
DNA Viruses
Genotoxic/DNA-Reactive Carcinogens Proto-oncogenes
Direct-acting (Activation-independent) Tumor-suppressor Genes
Carcinogens
Hormesis, Dose Response, and Carcinogenesis
Indirect-acting Genotoxic Carcinogens
Chemoprevention
Mutagenesis
Damage by Alkylating Electrophiles TEST SYSTEMS FOR CARCINOGENICITY ASSESSMENT
DNA Repair Short-term Tests for Mutagenicity
DNA Repair Mechanisms In Vitro Gene Mutation Assays
Mismatch Repair of Single-base Mispairs In Vivo Gene Mutation Assays
Excision Repair Chromosomal Alterations
End-joining Repair of Nonhomologous DNA DNA Damage
Classes of Genotoxic Carcinogens Transformation Assays
Polyaromatic Hydrocarbons Chronic Testing for Carcinogenicity
Alkylating Agents Chronic (2-Year) Bioassay
Aromatic Amines and Amides Organ-specific Bioassays and Multistage
Inorganic Carcinogens Animal Models
Nongenotoxic (Epigenetic) Carcinogens Transgenic Animals in Carcinogenicity Assessment
Cytotoxicity
CHEMICAL CARCINOGENESIS IN HUMANS
Receptor Mediated
Classification Evaluation of Carcinogenicity in Humans
Hormonal Mode of Action

121
L22 UNIT 3 Nonorgan-Directed Toxicity

= The term cancer describes a subset of neoplastic lesions. = Initiation requires one or more rounds of cell division for
« A neoplasm is defined as a heritably altered, relatively the “fixation” of the DNA damage.
autonomous growth of tissue with abnormal regulation » Promotion results from the selective functional enhance-
of gene expression. ment of the initiated cell and its progeny by the continu-
« Metastases are secondary growths of cells from the ous exposure to the promoting agent.
primary neoplasm. = Progression is the transition from early progeny of initi-
» A carcinogen is an agent whose administration to previ- ated cells to the biologically malignant cell population of
ously untreated animals leads to a statistically significant the neoplasm.
increased incidence of neoplasms of one or more histo-
genetic types as compared with the incidence in appro-
priate untreated animals.

OVERVIEW TABLE 8-1 Terminology.

’

Cancer is a disease ofcellular mutation, proliferation, and aber- Neoplasia New growth or autonomous growth of tissue
rant cell growth. It ranks as one of the leading causes of death in Neoplasm The lesion resulting from the neoplasia
the world. Multiple causes of cancer have been either firmly
established or suggested, including infectious agents, radiation, Benign Lesions characterized by expansive growth,
frequently exhibiting slow rates of proliferation
and chemicals. Estimates suggest that 70% to 90% of all human that do not invade surrounding tissues
cancers have a linkage to environmental, dietary, and behav-
ioral factors. Malignant Lesions demonstrating invasive growth, capable of
metastases to other tissues and organs

Metastases Secondary growths derived from a primary malignant

Definitions neoplasm
Table 8-1 lists definitions of terms commonly used in discuss- Tumor Lesion characterized by swelling or increase in size,
ing chemical carcinogenesis. For benign neoplasms, the tissue may or may not be neoplastic
of origin is frequently followed by the suffix “oma”; e.g., a
Cancer Malignant neoplasm
benign fibrous neoplasm would be termed fibroma, and
a benign glandular epithelium termed an adenoma. Malignant Carcinogen A physical or chemical agent that causes or induces
neoplasia
neoplasms from epithelial origin are called carcinomas,
whereas those derived from mesenchymal origin are referred Genotoxic Carcinogens that interact with DNA resulting in
to as sarcoma. Thus, a malignant neoplasm of fibrous tissue mutation

would be a fibrosarcoma, whereas that derived from bone Nongenotoxic Carcinogens that modify gene expression but do not
would be an osteosarcoma. damage DNA
Carcinogens may be genotoxic, meaning that they interact
physically with DNA to damage or change its structure. Other
carcinogens may change how DNA expresses information TABLE 8-2 Features of genotoxic and nongenotoxic
without modifying or directly damaging its structure, or may carcinogens.
create a situation in a cell or tissue that makes it more suscepti-
Genotoxic carcinogens
ble to DNA damage from other sources. Chemicals belonging » Mutagenic
to this latter category are referred to as nongenotoxic carcino- * Can be complete carcinogens
gens. Common features of genotoxic and nongenotoxic car- + Tumorigenicity is dose responsive
cinogens are shown in Table 8-2. * No theoretical threshold

Nongenotoxic carcinogens
* Nonmutagenic
MULTISTAGE CARCINOGENESIS * Threshold, reversible
+ Tumorigenicity is dose responsive
The carcinogenesis process involves a series of definable and * May function at tumor promotion stage
reproducible stages. Operationally, these stages have been * No direct DNA damage
+ Species, strain, tissue specificity
defined as initiation, promotion, and progression (Figure 8-1).
 CHAPTER 8 Chemical Carcinogenesis 123

Normal Initiated Focal Cancer

cell cell lesion

Repair Apoptosis Apoptosis

pop o®
fe
O ——_ >
e) —————
>_> iS

DNA damage Proliferation Proliferation i

Initiation Promotion Progression

FIGURE 8-1 Multistage model of carcinogenesis.

The defining characteristics of each of these stages are outlined Once initiated cells are formed, their fate has multiple poten-
in Table 8-3. tial outcomes: (1) the initiated cell can remain in a static nondi-
viding state; (2) the initiated cell may possess mutations
incompatible with viability or normal function and be deleted
Initiation through apoptotic mechanisms; or (3) the cell may undergo cell
The first stage of the cancer process involves initiation, a process division resulting in the proliferation of the initiated cell. Besides
that is defined as a stable, heritable change. This stage is a rapid, the production of an initiated cell through carcinogen binding
irreversible process that results in a carcinogen-induced muta- and misrepair, additional evidence has come forth showing that
tional event. Chemical and physical agents that interact with induction of continual stress, resulting in continual cell prolifer-
cellular components at this stage are referred to as initiators or ation, can also produce new mutated, initiated cells.
initiating agents. Initiating agents lead to genetic changes
including mutations and deletions. Chemical carcinogens that
covalently bind to DNA and form adducts that result in muta- Promotion
tions are initiating agents. The initiating event becomes “fixed” The second stage of the carcinogenesis process involves the
when the DNA damage is not correctly or completely repaired selective clonal expansion of initiated cells to produce a preneo-
prior to DNA synthesis and cell division. plastic lesion. This is referred to as the promotion stage of the
carcinogenesis process. Both exogenous and endogenous
agents that operate at this stage are referred to as tumor promot-
ers. Tumor promoters are not mutagenic and generally are not
TABLE 8-3 Characteristics of the stages of able to induce tumors by themselves; rather they act through
carcinogenesis process.
several mechanisms involving gene expression changes that
Initiation result in sustained cell proliferation through increases in cell
DNA modification proliferation and/or the inhibition of apoptosis. Promotion is
Mutation reversible upon removal of the promoting agent, and the focal
Genotoxic
cells may return to single initiated cell thresholds. In addition,
One cell division necessary to lock-in mutation
Modification is not enough to produce cancer these agents demonstrate a well-documented threshold for
Nonreversible their effects—below a certain dose or frequency of application,
Single treatment can induce mutation tumor promoters are unable to induce cell proliferation. Tumor
Promotion
promoters generally show organ-specific effects, e.g., a tumor
No direct DNA modification promoter of the liver, such as phenobarbital, will not function
Nongenotoxic as a tumor promoter in the skin or other tissues.
No direct mutation
Multiple cell divisions necessary
Clonal expansion of the initiated cell population
Increase in cell proliferation or decrease in cell death (apoptosis)
Progression
Reversible Progression involves the conversion of benign preneoplastic
Multiple treatments (prolonged treatment) necessary lesions into neoplastic cancer. In this stage, due to an increase in
Threshold
DNA synthesis and cell proliferation in the preneoplastic
Progression ‘ lesions, additional genotoxic events may occur, resulting in fur-
DNA modification ther DNA damage including chromosomal aberrations and
Genotoxic event
translocations. The progression stage is irreversible in that neo-
Mutation, chromosome disarrangement
Changes from preneoplasia to neoplasia benign/malignant - plasm formation, whether benign or malignant, occurs. With
Irreversible the formation of neoplasia, an autonomous growth and/or lack
Number of treatments needed with compound unknown (may of growth control is achieved. Spontaneous progression can
require only single treatment)
occur from spontaneous karyotypic changes that occur in
124 UNIT 3 Nonorgan-Directed Toxicity

mitotically active initiated cells during promotion. An accumu- TABLE 8-4 Proposed modes of action for selected
lation of nonrandom chromosomal aberrations and karyotypic nongenotoxic chemical carcinogens.
instability are hallmarks ofprogression.
Mode of Action . . Example -
Cytotoxicity Chloroform
MECHANISMS OF ACTION OF Melamine
CHEMICAL CARCINOGENS Q),-Globulin-binding D-limonene, 1,4-dichlorobenzene

The formation of a neoplasm is a multistage, multistep process Receptor-mediated CAR Phenobarbital

that involves the ultimate release of the neoplastic cells from nor-
PPAR Trichloroethylene
mal growth control processes and creating a tumor microenvi- Perchloroethylene
ronment. The eight properties of carcinogenesis are listed in Diethylhexylphthalate
Table 8-4. An important concept is that tumors are not just a col- Fibrates (e.g., clofibrate)
lection of clonal neoplastic cells but a complex tissue with multi- AhR TCDD
ple cell populations that interact with one another and function Polychlorinated biphenyls (PCBs)
as a unique tissue. This tumor microenvironment involves the Polybrominated biphenyls (PBBs)
recruitment of normal stromal and inflammatory cells that con- Hormonal Biogenic amines
tribute to the growth the development of the neoplasm. Steroid and peptide hormones
DES
Phytoestrogens (bisphenol-A)
Genotoxic/DNA-Reactive Carcinogens ‘ Tamoxifen
Phenobarbital
Genotoxic compounds directly interact with the nuclear DNA
Altered methylation Phenobarbital
of a target cell. If this damage is unrepairable, DNA damage is
Choline deficiency
inherited in subsequent daughter cells. DNA reactive car- Diethanolamine
cinogens can be further subdivided according to whether they
Oxidative stress inducers Ethanol
are active in their parent form (i.e., direct-acting carcinogens—
TCDD
agents that can directly bind to DNA without being meta- Lindane
bolized) and those that require metabolic activation Dieldrin
(i.e, indirect-acting carcinogens—compounds that require Acrylonitrile
metabolism in order to react with DNA).

Direct-acting (Activation-independent) Carcinogens—

Direct-acting carcinogens are highly reactive electrophilic mol- results in mutation and neoplastic transformation. It is impor-
ecules that can interact with and bind to nucleophiles, such as tant to note that besides activation of the procarcinogen to a
cellular macromolecules, including DNA without needing to be DNA reactive form, detoxification pathways may also occur
biotransformed into a reactive toxicant. Generally, these highly resulting in inactivation of the carcinogen.
reactive chemicals frequently result in tumor formation at the Indirect-acting genotoxic carcinogens usually produce their
site of chemical exposure. neoplastic effects at the target tissue where the metabolic acti-
The relative carcinogenic strength of direct-acting carcino- vation of the chemical occurs and not at the site of exposure (as
gens depends in part on the relative rates of interaction seen with direct-acting genotoxic carcinogens).
between the chemical and genomic DNA, as well as competing
reactions with the chemical and other cellular nucleophiles.
Chemical stability, transport, and membrane permeability Mutagenesis
determine the carcinogenic activity of the chemical. Direct- Effects of mutations depend on when in the cell cycle the
acting carcinogens are typically carcinogenic at multiple sites adducts are formed, where the adducts are formed, and the type
and in all species examined. of repair process used in response to the damage. Mutagenesis
may result from misread DNA (through transitions or transver-
Indirect-acting Genotoxic Carcinogens— The majority of sions), frame-shifting, or broken DNA strands.
DNA reactive carcinogens are found as parent compounds, or
procarcinogens, chemicals that require subsequent metabolism
to be carcinogenic. Terms have been coined to define the parent Damage by Alkylating Electrophiles
compound (procarcinogen) and its metabolite form, either As noted above, most chemical carcinogens require metabolic
intermediate (proximate carcinogen) or final (ultimate carcino- activation to exert a carcinogenic effect. The ultimate carcino-
gen), that reacts with DNA. The ultimate form of the carcinogen genic forms of these chemicals are frequently strong electro-
may not be known or may be several forms depending on meta- philes (Figure 8-2) that can readily form covalent adducts with
bolic pathway, but it is most likely the chemical species that nucleophilic targets. In general, the stronger electrophiles
 CHAPTER 8 Chemical Carcinogenesis 125

1.Carbonium ions 2.Nitrenium ions 3. Free radicals important determinant of carcinogenicity. Differences in sus-
ceptibility to carcinogenesis are likely the result of anumber of

R-cC@
i Rae i
R= GS
factors, including DNA replication within a tissue and repair of
l H l a DNA adduct. The development of cancer following exposure
R R to chemical carcinogens is a relatively rare event because of a
cell’s ability to recognize and repair damaged DNA. The DNA
4. Diazonium ions 5. Epoxides 6. Aziridinium ions region containing the adduct is removed and a new patch of
DNA is synthesized, using the opposite intact strand as a tem-
@
R—N=N©OH aa Pe plate. The new DNA segment is then spliced into the DNA mol-
R @ ~R ecule in place of the defective one. To be effective in restoring a
cell to normal, repair of DNA must occur prior to cell division.
7.Episultonium ions 8. Strained lactones 9. Sulfonates

DNA Repair Mechanisms

Nee alle RSO,0CH; Although cells possess mechanisms to repair many types of
DNA damage, these are not always completely effective, and
residual DNA damage can lead to the synthesis of altered pro-
10. Halo ethers 11.Enals
tein. Mutations in an oncogene, tumor-suppressor gene, or
O gene that controls the cell cycle can result in a clonal cell popula-
mae tion with a survival advantage. The development of a tumor
CICH,OCH,CI RCH=CHC 8
requires many such events, occurring over a long period of
FIGURE 8-2 Structures of reactive electrophiles. time, and for this reason human cancer induction often takes
place within the context of chronic exposure to chemical
carcinogens.
Cells have several mechanisms for repairing DNA damage.
display a greater range of nucleophilic targets, whereas weak Repair of DNA damage does not always occur prior to cell rep-
electrophiles are only capable of alkylating strong nucleophiles. lication, and repair of DNA damage by some chemicals is rela-
An important and abundant source of nucleophiles is con- tively inefficient.
tained not only in the DNA bases, but also in the phosphodies-
ter backbone. Different electrophilic carcinogens will often Mismatch Repair of Single-base Mispairs— Many sponta-
display different preferences for nucleophilic sites in DNA and, neous mutations are point mutations, a change in a single-base
thus, a different spectra of damage. pair in the DNA sequence. Depurination is a fairly common
Another common modification to DNA is the hydroxylation occurrence anda spontaneous event in mammals, and results in
of DNA bases. Oxidative DNA adducts have been identified in the formation of apurinic sites. All mammalian cells possess
all four DNA bases. The source of oxidative DNA damage is apurinic endonucleases that function to cut DNA near apurinic
typically formed from free radical reactions that occur endog- sites. The cut is then extended by exonucleases, and the result-
enously in the cell or from exogenous sources. ing gap repaired by DNA polymerases and ligases.
Methylation of DNA results in heritable expression or
repression of genes, with hypomethylation associated with Excision Repair—DNA regions containing chemically modi-
active transcription of genes, whereas hypermethylated genes fied bases, or DNA chemical adducts, are typically repaired by
tend to be rarely transcribed. Chemical carcinogens may excision repair processes. Proteins that slide along the surface
inhibit DNA methylation by forming covalent adducts, single- of adouble-stranded DNA molecule recognize irregularities in
strand breaks in the DNA, alteration of methionine pools, and the shape of the double helix, and induce repair ofthe lesion.
inactivation of the DNA methyltransferase responsible for
methylation. Whether a particular DNA adduct will result in End-joining Repair of Nonhomologous DNA—A cell that
mutation depends in part on the process of DNA replication has double-strand breaks can be repaired by joining the free
and in part on DNA repair. DNA ends. The joining of broken ends from different chromo-
somes, however, will lead to the translocation of DNA pieces
from one chromosome to another, translocations that have the
DNA Repair potential to enable abnormal cell growth. Homologous recom-
Following the formation of a carcinogen-DNA adduct, the per- bination is one of two mechanisms responsible for the repair of
sistence of the adduct is a major determinant of the outcome. double-strand breaks. In this process, the double-strand break
This persistence depends on the ability of the cell to repair the on one chromosome is repaired using the information on the
altered DNA. However, the presence of a DNA adduct is not homologous, intact chromosome.
sufficient for the carcinogenesis process to proceed. The relative The predominant mechanism for double-stranded DNA
rates or persistence of particular DNA adducts may be an repair in multicellular organisms is nonhomologous repair,
126 UNIT 3 Nonorgan-Directed Toxicity

which involves the rejoining of the ends of the two DNA mole- induction of cytotoxicity with compensatory hyperplasia may
cules. Although this process yields a continuous double- contribute to the observed tumorigenicity of many carcino-
stranded molecule, several base pairs are lost at the joining genic chemicals at high doses.
point. This type of deletion may produce a potentially muta-
genic coding change. Receptor Mediated
P450 Inducers: Phenobarbital-like Carcinogens—Phenobarbital
is a commonly studied non-DNA reactive compound that is
Classes of Genotoxic Carcinogens known to cause tumors by a nongenotoxic mechanism involv-
Polyaromatic Hydrocarbons—Polyaromatic hydrocarbons ing liver hyperplasia. The induction of CYP2B by phenobarbital
such as benzo(a)pyrene are found at high levels in charcoal is mediated by activation of the constitutive androstane recep-
broiled foods, cigarette smoke, and in diesel exhaust. tor (CAR), a member of the nuclear receptor family. Other
CAR-dependent phenobarbital responses that are critical for
Alkylating Agents— Whereas some alkylating chemicals are tumor formation include increased cell proliferation, inhibi-
direct-acting genotoxic agents, many require metabolic activa- tion of apoptosis, inhibition of gap junctional communication,
tion to produce electrophilic metabolites that can react with hypertrophy, and development of preneoplastic focal lesions in
DNA. Alkylating agents readily react with DNA at more than the liver.
12 sites. The N’ position of guanine and the N° position of ade-
nine are the most reactive sites in DNA for alkylating chemicals. Peroxisome Proliferator-activated Receptor-a (PPARa)—
Various chemicals are capable of increasing the number and
Aromatic Amines and Amides—Aromatic amines and volume of peroxisomes in the cytoplasm of cells. These
amides encompass a class of chemicals with varied structures. so-called peroxisome proliferators include chemicals such as
Classically, exposure to these chemicals was through the dye herbicides, chlorinated solvents (e-g.,,trichloroethylene and
industry, although exposure still occurs through cigarette smoke perchloroethylene), plasticizers (e.g., diethylhexylphthalate
and other environmental sources. The aromatic amines undergo and other phthalates), lipid-lowering fibrate drugs (e.g., cipro-
phase-I (hydrolysis, reduction, and oxidation) and phase-II (con- fibrate and clofibrate), and natural products. The currently
jugation) metabolism. Phase-I reactions occur mainly by accepted mode of action for this class of chemicals involves
cytochrome P450-mediated reactions, yielding hydroxylated agonist binding to the nuclear hormone receptor, PPARQ.
metabolites that are often associated with adduct formation in pro- PPARa is highly expressed in cells that have active fatty acid
teins and DNA, and produce liver and bladder carcinogenicity. oxidation capacity. PPARa plays a central role in lipid metabo-
lism and acts as a transcription factor to modulate gene exp-
Inorganic Carcinogens ression following ligand activation.
Several metals exhibit carcinogenicity in experimental animals
and/or humans, including arsenic, beryllium, cadmium, chro- Hormonal Mode of Action—Hormonally active chemicals
mium, nickel, and lead. The carcinogenic manifestations include biogenic amines, steroids, and peptide hormones that
are varied as well and include increased risk for skin, lung, and cause tissue-specific changes through interaction with a receptor.
liver tumors. Additional discussion of selected metals is in A number of non-DNA-reactive chemicals induce neoplasia
Chapter 23. through receptor-mediated mechanisms, and/or perturbation
of hormonal balance. Trophic hormones are known to induce
cell proliferation at their target organs. This action may lead to
Nongenotoxic (Epigenetic) Carcinogens the development of tumors when the mechanisms of hormonal
The targets induced by nongenotoxic carcinogens are often in control are disrupted and some hormone shows persistently
tissues where a significant incidence of background, spontane- increased levels.
ous tumors is seen in the animal model. Prolonged exposure to Estrogenic agents can induce tumors in estrogen-dependent
relatively high levels of chemicals is usually necessary for the tissue. Individuals with higher circulating estrogen levels and
production of tumors. Examples of the diverse modes of action those with exposure to the potent estrogenic agent diethylstil-
for non-DNA-reactive carcinogens are listed in Table 8-4. bestrol (DES) are at increased risk of cancer development. DES
has been causally linked to the higher incidence of adenocarci-
Cytotoxicity—Chemicals that function through this mecha- nomas of the vagina and cervix in daughters of women treated
nism produce sustained cell death that is accompanied by per- with the hormone during pregnancy. The effects of steroidal
sistent regenerative growth. This results in the potential for the chemicals on the cell cycle and on microtubule assembly may
acquisition of “spontaneous” DNA mutations and allowing be important in the aneuploidy inducing effects of some hor-
mutated cells to accumulate and proliferate. This process then monal agents.
gives rise to preneoplastic focal lesions that, upon expansion, A number of chemicals that reduce thyroid hormone con-
can lead to tumor formation. The induction of cytotoxicity centrations (T4 and/or T3) and increase thyroid-stimulating
may be observed with many carcinogens both genotoxic and hormone (TSH) have been shown to induce neoplasia in the
nongenotoxic when high toxic exposures occur. Thus, the rodent thyroid. TSH demonstrates proliferative activity in the
 CHAPTER 8 Chemical Carcinogenesis 127

thyroid, with chronic drug-induced TSH increases leading to nuclear DNA, the mitochondrial genome is relatively suscep-
progression of follicular cell hypertrophy, hyperplasia, and tible to oxidative base damage due to (1) close proximity to
eventually neoplasia. the electron transport system, a major source of reactive
oxygen species; (2) mitochondrial DNA is not protected by
DNA Methylation and Carcinogenesis—Post-DNA syn- histones; and (3) DNA repair capacity is limited in the
thetic methylation ofthe five position on cytosine is a naturally mitochondria.
occurring modification to DNA in higher eukaryotes that influ- Aside from oxidized nucleic acids, oxygen radicals can dam-
ences gene expression. Under normal conditions, DNA is age cellular biomembranes resulting in lipid peroxidation.
methylated symmetrically on both strands. Immediately fol- Peroxidation of biomembranes generates a variety of products
lowing DNA replication, the newly synthesized double- including reactive electrophiles such as epoxides and alde-
stranded DNA contains hemimethylated sites that signal for hydes, including malondialdehyde.
DNA maintenance methylases to transfer methyl groups from
S-adenosylmethionine to cytosine residues on the new DNA Oxidative Stress and Cell Growth Regulation—Reactive
strand. The degree of methylation within a gene inversely oxygen species production and oxidative stress can affect
correlates with the expression of that gene. Several chemical both cell proliferation and apoptosis. It has been demon-
carcinogens are known to modify DNA methylation, methyl- strated that low levels of reactive oxygen species influence
transferase activity, and chromosomal structure. During car- signal transduction pathways and alter gene expression. Many
cinogenesis, both hypomethylation and hypermethylation xenobiotics, by increasing cellular levels of oxidants, alter
of the genome have been observed. Tumor-suppressor genes gene expression through activation of signaling pathways
have been reported to be hypermethylated in tumors. including cAMP-mediated cascades, calcium-calmodulin
Hypomethylation has been associated with increased mutation pathways, transcription factors such as AP-1 and NF-kB, as
rates because many oncogenes are hypomethylated and well as signaling through mitogen-activated protein (MAP)
their expression is amplified. kinases. Activation of these signaling cascades ultimately
Reactive oxygen species have also been shown to modify leads to altered gene expression for a number of genes
DNA methylation by interfering with the ability of methyl- including those affecting proliferation, differentiation, and
transferases to interact with DNA; the resulting hypomethyl- apoptosis.
ation allows for the expression of normally quiescent genes.
Also, the abnormal methylation pattern observed in cells
transformed by chemical oxidants may contribute to an overall Gap Junctional Intercellular
aberrant gene expression and promote tumorigenesis. Communication and Carcinogenesis
Cells within an organism communicate in a variety of ways
Oxidative Stress and Chemical Carcinogenesis—Oxygen
including through gap junctions, which are aggregates of con-
radicals can be produced by both endogenous and exogenous
nexin proteins that form a conduit between two adjacent cells.
sources and are typically counterbalanced by antioxidants.
Gap junctional intercellular communication appears to play an
Antioxidant defenses are both enzymatic (e.g., superoxide dis-
important role in the regulation of cell growth and cell death, in
mutase, glutathione peroxidase, and catalase) and nonenzy-
part through the ability to exchange small molecules (<1 kDa)
matic (e.g., vitamin E, vitamin C, (3-carotene, and glutathione).
between cells. If cell communication is blocked between tumor
Endogenous sources of reactive oxygen species include oxida-
and normal cells, the exchange of growth inhibitory signals
tive phosphorylation, P450 metabolism, peroxisomes, and
from normal cells to initiated cells is prevented, thus allowing
inflammatory cell activation. Through these or other currently
the potential for unregulated growth and clonal expansion of
unknown mechanisms, a number of chemicals that induce can-
initiated cell populations.
cer (e.g., chlorinated compounds, radiation, metal ions, barbi-
turates, and some PPAR agonists) induce reactive oxygen
species formation and/or oxidative stress.
Modifiers of Chemical Carcinogenic
Oxidative DNA Damage and Carcinogenesis—Reactive oxy- Effects
gen species left unbalanced by antioxidants can result in dam- Genetic and environmental factors have a significant impact on
age to cellular macromolecules. In DNA, reactive oxygen the way in which individuals and/or organisms respond to car-
species can produce single- or double-stranded DNA breaks, cinogen exposure. As with most genes, enzymes that metabolize
purine, pyrimidine, or deoxyribose modifications, and DNA carcinogens are expressed in a tissue-specific manner. Within
crosslinks. Although many pathways exist that enable the for- tissues, the enzymatic profile can vary with cell type or
mation of oxidative DNA damage, mammalian cells also pos- display differential localization within cells. Further, carcinogen
sess specific repair pathways for the remediation of oxidative metabolizing enzymes are differentially expressed among spe-
DNA damage. ; cies. These differences may represent an underlying factor
Mutations and oxidative damage to mitochondrial DNA explaining the differential responses to chemical carcinogens
have been identified in a number of cancers. Compared to across species.
128 UNIT 3 Nonorgan-Directed Toxicity

can result in altered cell proliferation, differentiation, and/or

Polymorphisms in Carcinogen
survival of cancer cells.
Metabolism and DNA Repair
Genetic polymorphisms arise from human genetic variabil-
Retroviruses—The Rous sarcoma virus (RSV) is capable of
ity. A genetic polymorphism is whena gene has more than one
transforming a normal cell and producing sarcomas. The
allele. In assessing variability in the human genome project it
genome of RSV and other retroviruses consists of two identi-
was found that base variations occurred at approximately
cal copies of mRNA, which is then reverse transcribed into
once in every 1 000 base pairs. Therefore, there may be over
DNA and incorporated into the host-cell genome. Oncogenic
one million genetic variations between any two individuals. A
transforming viruses like RSV contain the v-src gene, a gene
single nucleotide polymorphism (SNP) is a variant in DNA
required for cancer induction. Normal cells contain a gene
sequence found in greater than 1% ofthe population. Thus, by
closely related to v-sre in RSV. This discovery showed that
definition, changes in DNA sequence go from mutation to
cancer may be induced by the action of normal, or nearly
polymorphism when a unique genotype is seen in over 1% of
normal, genes.
the population. Over three million candidate SNPs have been
identified to date with up to 10 million being estimated to be
present within the human genome. In carcinogenesis, genetic DNA Viruses— Unlike retroviral oncogenes, which are derived
polymorphisms may account for the susceptibility of some from normal cellular genes and have no function for the virus,
individuals to certain cancers. In carcinogenesis, genetic the known oncogenes of DNA viruses are integral parts of the
polymorphisms may account for the susceptibility of some viral genome required for viral replication. Infection by small
individuals to certain cancers. A number of polymorphisms DNA viruses is lethal to most non-host animal cells; however, a
have been described in carcinogen-metabolizing enzymes, small proportion integrates the viral DNA into the host-cell
with certain alleles linked to altered risk of selective cancers. genome. The cells that survive infection become permanently
Glutathione S-transferases (GSTs) are highly polymorphic in transformed due to the presence of one dr more oncogenes in the
humans. The GSTM1 isoform is particularly important in viral DNA. Papilloma viruses can infect and cause tumors in
carcinogenesis, because of its high reactivity toward humans. Some examples of oncogenic DNA viruses include
epoxides. human papilloma viruses, Epstein-Barr virus, hepatitis B virus,
Carcinogenic risk depends on both exposure (dose and and herpes viruses.
duration) as well as genetic susceptibility. For example, if the
genetic susceptibility is high, then exposure to a chemical car- Proto-oncogenes—An oncogene encodes a protein that is
cinogen will result in a higher risk for cancer development. capable of transforming cells in culture or inducing cancer in
animals. Of the known oncogenes, the majority appear to have
been derived from normal genes (i.e., proto-oncogenes), and
Proto-oncogenes and Tumor- are involved in cell signaling cascades. Because most proto-
suppressor Genes oncogenesare essential for maintaining viability, they are highly
Proto-oncogenes and tumor-suppressor genes encode a wide conserved. Activation of proto-oncogenes to oncogenes arises
array of proteins that function to control cell growth and through mutational events occurring within proto-oncogenes.
proliferation. Common characteristics of oncogenes and It has been recognized that a number of chemical carcinogens
tumor-suppressor genes are shown in Table 8-5. Mutations in are capable of inducing mutations in proto-oncogenes.
both oncogenes and tumor-suppressor genes contribute to the Oncogene products can operate at multiple levels of signaling
progressive development of human cancers. Accumulated cascades, including ligand, receptor, second messengers, and
damage to multiple oncogenes and/or tumor-suppressor genes transcription factor stages of transduction.

TABLE 8-5 Characteristics of proto-oncogenes, cellular oncogenes, and tumor-suppressor genes.

_Proto-oncogenes _
Dominant Dominant Recessive

Broad tissue specificity for cancer Broad tissue specificity for cancer Considerable tissue specificity for cancer
development development development

Germ line inheritance rarely involved Germ line inheritance frequently involved Germ line inheritance frequently involved
in cancer development in cancer development in cancer development

Analogous to certain viral oncogenes No known analogs in oncogenic viruses No known analogs in oncogenic viruses

Somatic mutations activated during all Somatic mutations activated during all stages of Germ line mutations may initiate, but mutation to
stages of carcinogenesis carcinogenesis neoplasia occurs only during progression stage
 CHAPTER 8 Chemical Carcinogenesis 129

Tumor-suppressor Genes—In contrast to oncogenes, the Wilms’ Tumor Gene (WT1)—Wilms tumor occurs in the
proteins encoded by most tumor-suppressor genes act as inhib- developing kidney at a rate of approximately one per 10000
itors of cell proliferation or cell survival (Table 8-6). The proto- children. The WT] gene is believed to be responsible for tumor
type tumor-suppressor gene, Rb, was identified by studies of development and is thought to function as a transcription
inheritance of retinoblastoma. factor.

Retinoblastoma (Rb) Gene—Loss or mutational inactivation p16 Gene—The group ofproteins that function as cyclin-kinase
of Rb contributes to the development ofa wide variety of human inhibitors play an important role in cell cycle regulation.
cancers. In its unphosphorylated form, Rb binds to the E2F Mutations, especially deletions of the p16 gene that inactivate
transcription factors preventing E2F-mediated transcriptional the ability of p16 to inhibit cyclin D-dependent kinase activity,
activation of anumber of genes whose products are required for are common in several human cancers including a high per-
DNA synthesis. Rb becomes phosphorylated during the late centage of melanomas. Loss of p16 would mimic cyclin D1
Gl, causing dissociation from E2F—a process that allows E2F overexpression, leading to Rb overactivation and release of
to induce synthesis of DNA replication enzymes, resulting in a active E2F transcription factor. Thus, p16 normally acts as a
commitment to the cell cycle. tumor suppressor. As with the BRCAI gene, relatively few
mutations have been found in this gene, and some researchers
p53 Gene—The p53 protein is a tumor-suppressor gene that is have speculated that epigenetic mechanisms such as gene silen-
essential for checkpoint control and arrests the cell cycle in cells cing by DNA methylation may occur during tumorigenesis.
with damaged DNA in G1. Cells with functional p53 arrest in
G1 when exposed to DNA damaging agents, whereas cells lack-
ing functional p53 are unable to block the cell cycle. p53 is Hormesis, Dose-Response,
activated by a wide array of stressors including ultraviolet and Carcinogenesis
light, irradiation, heat, and several carcinogens. Hormesis is defined as a dose-response curve in which a U-, J-,
In most cells, accumulation of p53 also leads to induction of or inverted U-shaped dose-response is observed, with low-dose
proteins that promote apoptosis, and therefore would prevent exposures often resulting in beneficial rather than harmful
proliferation of cells that are likely to accumulate multiple effects. Adaptive responses have been proposed to explain the
mutations. When the p53 checkpoint control does not operate hormetic effects observed by chemical carcinogens. These
properly, damaged DNA can replicate, producing mutations responses usually involve actions of the chemical on cellular sig-
and DNA rearrangements that contribute to the development naling pathways that lead to changes in gene expression, result-
of transformed cells. ing in enhanced detoxification and excretion of the chemical, as
well as preserving the cell cycle and programmed cell death. It
BRCAI1 Gene—Genetic analysis of breast tumors has revealed has been proposed that following very low doses of chemicals,
a hereditary predisposition for breast cancer linked to BRCA1, the upregulation of these mechanisms overcompensates for cell
a tumor-suppressor gene. Mutation of a single BRCA1 allele injury such that a reduction in tumor promotion and/or tumor
results in a 60% probability of developing breast cancer by age development is seen, and would explain the U- or J-shaped
50. A number of investigators have shown that germ line muta- response curves obtained following carcinogen exposure. A
tions lead to loss of function of the BRCA1 gene. However, no common feature of chemical carcinogens for which hormetic
mutations have been observed in sporadic breast cancer, sug- effects have been proposed is the formation of reactive oxygen
gesting that BRCAI gene silencing may occur through non- species and the induction of cytochrome P450 isoenzymes.
mutational mechanisms.

Chemoprevention
The study of chemicals that prevent, inhibit, or slow down the
TABLE 8-6 Examples of tumor-suppressor genes process of cancer is referred to as chemoprevention. A number
and cancer association. of chemicals, including drugs, antioxidants, foodstuffs, and
vitamins, have been found to inhibit or retard the components
Tumor Suppressor Disorder Neoplasm of the cancer process in both in vitro and in vivo models. A basic
Rb1 Retinoblastoma Small-cell lung assumption in chemoprevention is that treating early stages of
carcinoma malignant process will halt or delay the progression to neopla-
sia. Chemopreventive agents may function as inhibitors of car-
p53 Li-Fraumeni syndrome Breast, colon,
lung cancers cinogen formation, blocking agents, and/or suppressing agents.
Blocking agents serve to prevent the metabolic activation of
BRCA1 Unknown Breast carcinoma genotoxic or nongenotoxic carcinogens by either inhibiting its
WT-1 Wilms’ tumor Lung cancer metabolism or by enhancing the detoxification mechanisms.
Suppressing agents induce tissue differentiation, may counter-
p16 Unknown Melanoma act oncogenes, enhance tumor-suppressor gene activities,
130 UNIT 3 Nonorgan-Directed Toxicity

inhibit proliferation of premalignant cells, or modify the effect genotoxicity test systems will fail to identify nongenotoxic/
of the carcinogen on the target tissue. non-DNA reactive compounds.

Chromosomal Alterations—Chromosomal alterations are

TEST SYSTEMS FOR CARCINOGENICITY quite common in malignant neoplasms. Both in vivo and in
vitro assays are available to assess chromosomal alterations. To
ASSESSMENT
assess induction of chromosomalalterations, cells are harvested
A number of in vivo and in vitro experimental systems are avail- in their first mitotic division after the initiation of chemical
able to assess the potential carcinogenicity of chemicals. The exposure. Cells are stained with Giemsa and scored for com-
types of tests available to identify chemicals with carcinogenic pleteness of karyotype (21 +2 chromosomes). The classes of
potential can be classified into general categories, based on the aberrations recorded include breaks and terminal deletions,
duration required to conduct the test. Short-term tests are typi- rearrangements and translocations, as well as despiralized
cally of the duration of days to a few weeks, intermediate-term chromosomes, and cells containing 10 or more aberrations.
tests last from weeks up toa year, whereas chronic long-term tests Sister chromatid exchanges (SCEs) are a measure of DNA
usually encompass six months to two years exposure to a chemi- damage events that are associated with mutation induction and
cal. These bioassays use bacterial and mammalian targets. cancer. SCEs area reflection ofan interchange of DNA between
different chromatids at homologous loci within a replicating
chromosome. Second-division metaphase cells are scored to
Short-term Tests for Mutagenicity determine the frequency of SCE/cell for each dose level.
Short-term tests for mutagenicity were developed to identify Disruption of theDNA replication process or damage to chro-
potentially carcinogenic chemicals based on their ability to mosomes by chemicals can alter the genetic material distrib-
induce mutations in DNA either in vivo or in vitro. The majority uted to either of the two daughter nuclei. When this occurs, the
of these tests measure the mutagenicity of chemicals as a sur- genetic material that is not incorporated into a new nucleus
rogate for carcinogenicity. Although usually very predictive of may form its own “micronucleus,” which is clearly visible with
indirect action and direct action (if a metabolic source is a microscope. For this assay, animals are exposed to chemicals
provided), these tests routinely fail to detect nongenotoxic and the frequency of micronucleated cells is determined at
carcinogens. some specified time after treatment.

In Vitro Gene Mutation Assays—The most widely used DNA Damage—Primary DNA damage represents possible
short-term test is the Ames assay. Salmonella typhimurium pre-mutational events that can be detected using mammalian
strains deficient in DNA repair and unable to synthesize histi- cells in culture or using rodent tissue. Unscheduled DNA syn-
dine are treated with several dose levels of the test compound, thesis (UDS) isa commonly used assay that measures the ability
after which reversion to the histidine-positive phenotype is of achemical to induce DNA lesions by measuring the increase
ascertained. in DNA repair. Among the available techniques is the measure-
The mouse lymphoma assay is a mutagenicity assay used to ment of DNA strand breaks both in vivo and in vitro.
determine whether a chemical is capable of inducing mutation
in eukaryotic cells. The ability of the cell cultures to acquire Transformation Assays—Various in vitro test systems have
resistance to trifluorothymidine (the result of forward mutation been developed to assess the carcinogenic potential of chemi-
at the thymidine kinase locus) is quantified. Another mamma- cals. The C3H/10T"” cell line has been widely used in the trans-
lian cell mutation assay, the Chinese hamster ovary (CHO) test, formation assays. It was originally derived from fibroblasts
is also commonly used to assess the potential mutagenicity of taken from the prostate of aC3H mouse embryo. The cells are
chemicals. This assay uses the hypoxanthine-guanine phospho- approximately tetraploid but the chromosome number in the
ribosyltransferase (HGPRT) gene as the end point. cells varies widely. As such, these cells are chromosomally
abnormal and have already passed through some of the stages
in Vivo Gene Mutation Assays—The in vivo tests have that might be involved in the production of a cancerous cell. On
advantages over the in vitro test systems in that they take into plating these cells, they will stop growing when their density is
account the whole animal processes such as absorption, tissue sufficiently high (contact growth inhibition). However, the con-
distribution, metabolism, and excretion of chemicals and their tact inhibition can fail, resulting in cell piling forming a trans-
metabolites. The commonly used in vivo models include trans- formed colony. Therefore, following exposure to xenobiotics,
genic rodent mutation assay systems based on the genes of the this assay assesses carcinogenic potential based on the percent-
lac operon, Muta™Mouse, and Big Blue’. age of colonies that are transformed.
To detect mutations following exposure of mice to test The most frequently used end point for cell transformation is
chemicals, mutations are analyzed in high molecular weight morphological transformation of mammalian cell fibroblasts
DNA isolated from the tissue under investigation. The ratio of in culture. Transformation assays using Syrian hamster embryo
mutants to the total population will provide a mutation fre- (SHE) cells are available for the assessment of the carcino-
quency for each chemical and each organ tested. In vivo genic potential of chemicals. The SHE cell assay measures
 CHAPTER 8 Chemical Carcinogenesis 131

carcinogenic potential of xenobiotics by assessing transformed developed. The available systems include animal models
colonies based on morphological criteria. directed at examining carcinogenicity in the lung, kidney, blad-
der, pancreas, stomach, colon, small intestine, and oral cavity.
These models vary in the initiating carcinogen used, and fre-
Chronic Testing for Carcinogenicity quency, duration, and site of application, as well as the duration
Chronic (2-Year) Bioassay— Two-year studies over the lifes- of promoting chemical exposure.
pan of rodents remain the primary method by which chemicals
or physical agents are identified as having the potential to be
hazardous to humans. In the chronic bioassay, two or three dose
Transgenic Animals in Carcinogenicity
levels (up to the maximum tolerated dose, MTD) ofa test chem- Assessment
ical and a vehicle control are administered to 50 males and 50 Animal models with genetic alterations that invoke a suscepti-
females (mice and rats), beginning at 8 weeks of age, continuing bility to carcinogenesis by chemical agents include Tg.AC and
throughout their lifespan. During the study, food consumption rasH2 transgenic mice, and p53*/ and XPA“ knockout mice.
and bodyweight gain are monitored, and the animals are Recently, the feasibility of the use of these animal models as
observed clinically ona regular basis, and at necropsy the tumor alternative assays for the 2-year chronic bioassay was assessed
number, location, and pathological diagnosis for each animal by the Health and Environmental Sciences Institute branch of
are thoroughly assessed. the International Life Sciences Institute. The conclusions drawn
from the scientific review suggested that these models appear to
Organ-specific Bioassays and Multistage Animal have usefulness as screening models for assessment of chemical
Models— Many tissue-specific bioassays have been developed carcinogenicity; however, they do not provide definitive proof
with the underlying goal being to produce a sensitive and reli- of potential human carcinogenicity. Further, the scientific panel
able assay that could be conducted in a time frame shorter in suggested that these models could be used in place of the mouse
duration than the 2-year chronic bioassay. These assays are 2-year bioassay. Coupled with information on genotoxicity,
commonly used to detect carcinogenic activity of chemicals in particularly DNA reactivity, structure—-activity relationships,
various target organs. results from other bioassays, and the results of other mechanis-
tic investigations including toxicokinetics, metabolism, and
Carcinogenicity Testing in the Liver—The liver represents a mechanistic information, these alternate mouse models for car-
major target organ for chemical carcinogens. It has been esti- cinogenicity appear to be useful models for assessing the carci-
mated that nearly half of the chemicals tested in the 2-year nogenicity of chemical agents.
chronic bioassay by the National Toxicology Program showed
an increased incidence of liver cancer. Liver carcinogenesis
assays have been developed to study and distinguish chemicals CHEMICAL CARCINOGENESIS
that affect the initiation or promotion stage of hepatocarcino- INHUMANS
genesis. The ability of the test chemical to promote the growth
of preneoplastic lesions can be assessed. Many factors have been implicated in the induction of cancer in
humans. Infectious agents, lifestyle, medical treatments, and
Carcinogenicity Testing in the Skin—The mouse skin model has environmental and occupational exposure account either
been used to dissect mechanisms of carcinogenesis and also is directly or indirectly for the majority of cancers seen in humans.
purported to be a useful intermediate-term cancer bioassay. This Of these, the component that contributes the most to human
model exploits many of the unique properties of the mouse skin, cancer induction and progression is lifestyle: tobacco use, alco-
one major advantage being that the development of neoplasia can hol use, and poor diet (Table 8-7). Tobacco usage either through
be followed visually. In addition, the number and relative size of
papillomas and carcinomas can be quantified as the tumors prog- TABLE 8-7 Carcinogenic factors associated
ress. Both initiating and promoting activities of chemical carcino- with lifestyle.
gens can be assessed using this model. Grossly, initiated cells of
the skin appear identical to normal skin. Because the terminally Chemical(s) Neoplasm(s)
differentiated cells in the skin are no longer capable of undergoing Alcohol beverage Esophagus, liver, oropharynx,
cell division, only initiated cells retain their proliferative capacity and larynx
and thus represent the cell populations that give rise to tumors.
Aflatoxins Liver
On repeated application of tumor promoters, selective clonal
expansion of initiated keratinocytes occurs, resulting in skin pap- Betel chewing Mouth
illomas, which over time can progress to carcinomas.
Dietary intake (fat, protein, calories) Breast, colon, endometrium,
gallbladder
Carcinogenicity Testing in Other Organs—Test systems to
examine the ability of a chemical to promote neoplastic devel- Tobacco smoking Mouth, pharynx, larynx, lung,
esophagus, bladder
opment at organ sites other than liver and skin have also been
132 UNIT 3 Nonorgan-Directed Toxicity

TABLE 8-8 Occupational human carcinogens.

Neen eee eee SEATS SSS SL LL ELAS LLL

Agent Industrial Process Neoplasms

Asbestos Construction, asbestos mining Peritoneum, bronchus

Arsenic Mining and smelting Skin, bronchus, liver

Alkylating agents (mechloroethamine hydrochloride Chemical manufacturing Bronchus

and bis[chloromethyl]ether)

Benzene Chemical manufacturing Bone marrow

Benzidine, beta-naphthylamine Dye and textile Urinary bladder

Chromium and chromates Tanning, pigment making Nasal sinus, bronchus

Nickel Nickel refining Nasal sinus, bronchus

Polynuclear aromatic hydrocarbons Steel making, roofing, chimney cleaning Skin, scrotum, bronchus

Vinyl chloride monomer Chemical manufacturing Liver

Wood dust Cabinet making Nasal sinus

Beryllium Aircraft manufacturing, electronics Bronchus

Cadmium Smelting Bronchus

Ethylene oxide Production of hospital supplies Bone marrow

Formaldehyde Plastic, textile, and chemical Nasal sinus, bronchus

Polychlorinated biphenyls Electrical-equipment production and maintenance Liver

smoking tobacco, chewing tobacco, or tobacco snuff-type secondary to selective diseases such as acquired immune defi-
products is estimated to be responsible for 25% to 40% of ciency syndrome (AIDS) result in an increase in a variety of
all human cancers. In particular, a strong correlation between different neoplasms. These results further support the role of
tobacco usage and mouth, larynx, lung, esophageal, and blad- the immune system in identifying and removing early preneo-
der cancer exists. It has been estimated that 85% to 90% of all plastic cells from the body.
lung cancer cases in the United States are a direct result
of tobacco use. The induction ofpancreatic cancer also appears
to havea linkage to tobacco use. Alcohol consumption contrib-
utes anywhere from 2% to 4% ofcancers ofthe esophagus, liver, TABLE 8-9 Human carcinogenic chemicals
and larynx. associated with medical therapy and diagnosis.
Poor diets, occupational exposures, and chemotherapeutic Chemical or Drug Associated Neoplasms
therapy account for many human cancers. High-fat and high-
Alkylating agents Bladder, leukemia
calorie diets have been linked to breast, colon, and gallbladder
(cyclophosphamide,
cancer in humans (see Chapter 27). Diets poor in antioxidants melphalan)
and/or vitamins such as vitamin A and vitamin E probably
also contribute to the onset of cancer. The method of cooking Azathioprine Lymphoma, reticulum cell sarcoma,
skin, Kaposi's sarcoma (?)
may also influence the production of carcinogens produced in
the cooking process. Carcinogenic heterocyclic amines and Chloramphenicol Leukemia
polycyclic aromatic hydrocarbons are formed during broiling
Diethylstilbestrol Vagina (clear cell carcinoma)
and grilling of meat. Acrylamide, a suspected human carcino-
gen, has been found in fried foods at low concentrations. A Estrogens Liver cell adenoma, endometrium, skin
number of occupations associated with the development of
Phenacetin Renal pelvis (carcinoma)
specific cancers are listed in Table 8-8. A number of medical
therapeutic and diagnostic tools have also been linked to Phenytoin Lymphoma, neuroblastoma
the induction of human cancer (Table 8-9). Therapeutic
Thorotrast Liver (angiosarcoma)
immunosuppression given to transplant patients or arising
 CHAPTER 8 Chemical Carcinogenesis 133

TABLE 8-10 IARC classification of the evaluation of epidemiological and experimental animal and in vitro data uti-
carcinogenicity for human beings. lizing assays as described earlier in this chapter. One ofthe first
devised schemes for the classification of an agent's carcinoge-
nicity was devised by the International Agency for Research on
1. Agent is carcinogenic to Human data strong Cancer (IARC) (Table 8-10). The IARC approach assigns the
humans Animal data strong chemical or mixture to one of five groupings based on strength
of evidence for the agent’s possible, probable, or definite carci-
2A. Agent is probably Human epidemiology data
carcinogenic to humans — suggestive — nogenicity to humans. Similar classifications exist for the U.S.
SA res aieEP th a me Animal data positive EPA, the U.S. Food and Drug Administration, and the
European Community (EC). The classification of agents with
2B. Agent is possibly Human epidemiology data weak
carcinogenic to humans Animal data positive
regard to human carcinogenicity can be very difficult, in par-
ticular when animal data and/or epidemiological data in
3. Agentisnot classifiable as to Human and animal data humans are inconclusive or confounded.
arcinogenicitytohumans ——_—sinadequate
4. Agent is probably not Human and animal data negative
carcinogenic to humans
BIBLIOGRAPHY
Gelmann EP, Sawyers CL, Rauscher FJ, (eds.): Molecular Oncology:
Causes of Cancer and Targets
for Treatment. New York: Cambridge
University Press, 2014.
Classification Evaluation of Penning TM, (ed.): Chemical Carcinogenesis. New York: Springer,
2011.
Carcinogenicity in Humans Shields PG, (ed.): Cancer Risk Assessment. Boca Raton, FL: Taylor &
The assessment and designation of a chemical or a mixture of Francis, 2005.
chemicals as carcinogenic in humans is evaluated by various Tannock IF, Hill RP, Bristow RG, Harrington L, (eds.): The Basic
agencies worldwide. The evaluation usually encompasses both Science of Oncology, 5th ed., New York: McGraw-Hill, 2013.
 UNIT 3 Nonorgan-Directed Toxicity

QUESTIONS

There is evidence that certain dietary components are 6. Tumor suppressor genes are mutated in a majority of
carcinogenic. Which of the following is NOT tabbed as a cancers. Which of the following is NOT a characteristic of
dietary carcinogen? a tumor suppressor gene?
a. excessive caloric intake. a. A mutation in a tumor suppressor gene is dominant.
excessive alcohol consumption. b. Germ line inheritance of amutated tumor suppressor
aflatoxin B1 (a food contaminant). gene is often involved with cancer development.
insufficient caloric intake. c. There is considerable tissue specificity for cancer
gemo
nitrites (found in some lunchmeats). development.
d. The p53 gene is a tumor suppressor gene that also
Which of the following statements regarding mechanisms acts as a transcription factor.
of chemical carcinogenesis is FALSE? e. Mutations in tumor suppressor genes can result in
a. Procarcinogens require metabolism in order to exert loss of cell cycle control.
their carcinogenic effect.
b. Free radicals are highly reactive molecules that have a Which of the following molecules does NOT play an
single, unpaired electron. important role in cell cycle regulation?
c. DNA adducts interfere with the DNA replication a: = pos.
machinery. b. cyclin-D.
d. Mutations in the DNA and failure to repair those cs.) (MAPK: ‘
mutations can be highly carcinogenic. d. MHC.
e. Biological reduction of molecular oxygen is the only Cre eZE: ae
way free radicals can be formed.
. Which of the following environmental factors is
In addition to being necessary for transcription to occur, proportionally responsible for the LEAST amount of
which of the following transcription factors also plays a cancer deaths?
crucial role in nucleotide excision repair? a. tobacco.
a. TPIIA. b. infection.
b. TFIIB. c. diet.
Ce LD: d. sexual behavior.
d. TFIF e. alcohol.
oe ABE
The evidence of the carcinogenicity of dietary intake
Which of the following statements regarding DNA repair is sufficient to include one’s diet as associated with
is true? neoplasms of all of the following EXCEPT:
a. Base excision repair requires the removal of a a. colon.
longer piece of DNA in comparison with nucleotide b. _ breast.
excision repair. c. pancreas.
b. ‘The repair of double-stranded DNA breaks is more d. endometrium.
prone to error than is base excision repair. e. gallbladder.
c. Dimerization of pyrimidines is repaired via base
excision repair. 10. Which of the following is the correct definition of acom-
d. Mismatch repair can only recognize normal nucleotides plete carcinogen?
that are paired with a noncomplementary nucleotide. a. achemical capable only of initiating cells.
e. Nucleotide excision and base excision are tolerance b. a chemical possessing the ability of inducing cancer
mechanisms used to respond to DNA damage. from normal cells, usually possessing properties of
initiating, promoting, and progression agents.
Which of the following statements is a characteristic of c. a chemical capable of converting an initiated cell
the initiation stage of carcinogenesis? or a cell in the stage of promotion to a potentially
a. Initiation is reversible in viable cells. malignant cell.
b. The dose-response exhibits an easily measurable d. a chemical capable of causing the expansion of
threshold. initiated cell clones.
c. Cell division is required for the fixation of the process. e. a chemical that will cause cancer 100% of the time
d. All initiated cells survive over the lifespan of the that it is administered.
organism.
e. Spontaneous initiation of cells is a rare occurrence.
 CP eA Peel RE eR

Genetic Toxicology
R. Julian Preston and George R. Hoffmann

HEALTH IMPACT OF GENETIC ALTERATIONS DNA Damage and Repair Assays

Somatic Cells Gene Mutations in Prokaryotes
Germ Cells Genetic Alterations in Nonmammalian
Eukaryotes
CANCER AND GENETIC RISK ASSESSMENTS Gene Mutations and Chromosome
Cancer Risk Assessment Aberrations
Genetic Risk Assessment Mitotic Recombination
Gene Mutations in Mammals
MECHANISMS OF INDUCTION OF GENETIC ALTERATIONS
Gene Mutations In Vitro
DNA Damage Gene Mutations In Vivo
lonizing Radiations Transgenic Assays
Ultraviolet Light Mammalian Cytogenetic Assays
Chemicals Chromosome Aberrations
Endogenous Agents Micronuclei
DNA Repair Sister Chromatid Exchange
Base Excision Repair Aneuploidy
Nucleotide Excision Repair Germ Cell Mutagenesis
Double-strand Break Repair Gene Mutations
Mismatch Repair Chromosomal Alterations
O%-Methylguanine-DNA Methyltransferase Repair Dominant Lethal Mutations
Formation of Gene Mutations Development of Testing Strategies
Somatic Cells
HUMAN POPULATION MONITORING
Germ Cells
Formation of Chromosomal Alterations
NEW APPROACHES FOR GENETIC TOXICOLOGY
Somatic Cells
Advances in Cytogenetics
Germ Cells
Molecular Analysis of Mutations
ASSAYS FOR DETECTING GENETIC ALTERATIONS and Gene Expression

Introduction to Assay Design CONCLUSION

Structural Alerts and In Silico Assays
136 UNIT 3 Nonorgan-Directed Toxicity

= Genetic toxicology assesses the effects of chemical and = Genetic toxicology assays serve to identify mutagens for
physical agents on the hereditary material (DNA) and on purposes of hazard identification, and to characterize
the genetic processes of living cells. dose-response relationships and mutagenic mechanisms.
= Oncogenes are genes that stimulate the transformation of = A broad range of short-term assays for genetic toxicology
normal cells into cancer cells. serve to identify many mutagens and address the rela-
tionship between mutagens and cancer-causing agents.

Genetic toxicology assesses the effects of chemical and physical Germ Cells
agents on both DNA and on the genetic processes ofliving cells. The relevance of gene mutations to health is evident from the
This chapter addresses the assays for qualitative and quantita- many disorders often caused by base-pair substitutions or small
tive assessment of cellular changes induced by chemical and deletions that are inherited as simple Mendelian characteristics.
physical agents, the underlying molecular mechanisms for Many genetic disorders (e.g., cystic fibrosis, phenylketonuria,
these changes, and how such information can be incorporated and Tay-Sachs disease) are caused by the expression of reces-
in risk assessments. sive mutations. These mutations are mainly inherited from pre-
vious generations and are expressed when an individual inherits
the mutant gene from both parents.
HEALTH IMPACT OF GENETIC Besides causing diseases that exhibit Mendelian inheritance,
ALTERATIONS gene mutations undoubtedly contribute to human disease
through the genetic component of disorders with a complex eti-
Somatic Cells ology such as heart disease, hypertension, and diabetes. Refined
Mutational alteration of proto-oncogenes can lead to over- cytogenetic methods have led to the discovery of minor varia-
expression of their growth-stimulating activity, whereas tions in chromosome structure that have no apparent effect.
mutations that inactivate tumor-suppressor genes, which Nevertheless, other chromosome aberrations cause fetal death
normally restrain cellular proliferation, free cells from their or serious abnormalities. Aneuploidy also contributes to fetal
inhibitory influence. The action of oncogenes is genetically deaths and causes disorders such as Down syndrome. Much of
dominant in that a single active oncogene is expressed even the effect of chromosomal abnormalities occurs prenatally.
though its normal allele is present in the same cell. Among Among the abnormalities, aneuploidy is the most common, fol-
chromosomal alterations that activate proto-oncogenes, lowed by polyploidy. Structural aberrations constitute about 5%
translocations are especially prevalent. A translocation can of the total. Most chromosomal anomalies detected in new-
activate a proto-oncogene by moving it to a new chromo- borns arise de novo in the germ cells of the parents.
somal location with a more active promoter, where its
expression is enhanced. Unlike oncogenes, the cancer-
causing alleles that arise from tumor-suppressor genes are CANCER AND GENETIC RISK
typically recessive in that they are not expressed when they ASSESSMENTS
are heterozygous.
Six acquired characteristics are essential for the formation of Cancer Risk Assessment
all tumors irrespective of tumor type and species. These include Cancer risk assessment involves investigation of sensitivity of
(1) self-sufficiency in growth signals, (2) insensitivity to anti- different species and subpopulations to tumor induction by a
growth signals, (3) evasion of apoptosis, (4) limitless replica- chemical and development of a dose-response curve of muta-
tive potential, (5) sustained angiogenesis, and (6) tissue tions to a chemical.
invasion and metastasis. It seems probable that there is no spe-
cific order for obtaining these characteristics.
Gene mutations, chromosome aberrations (morphologic Genetic Risk Assessment
abnormality), and aneuploidy (abnormal number of chromo- To investigate genetic risk, the frequency ofgenetic alteration
somes) are all implicated in the development of cancer. Many in human germ cells is estimated by extrapolation from data
mutagens and clastogens (chromosome-breaking agents) from rodent germ cells and somatic cells. For a complete esti-
contribute to carcinogenesis as initiators; however, mutagens, mate of genetic risk, it is necessary to obtain an estimate of
clastogens, and aneugens also may contribute to multiple the frequency of genetic alterations transmitted to the off-
genetic alterations. spring (Figure 9-1).
 CHAPTER9 Genetic Toxicology 137

(adducts). Alkylated bases can also lead to base loss from DNA,
which leaves an apurinic or apyrimidinic site, commonly called
an AP site. The insertion ofincorrect bases into AP sites causes
mutations. Bulky DNA adducts are recognized by the cell in a
similar way to UV damages and are also repaired similarly. Such
adducts can also hinder polymerases and cause mutation as a
consequence oferrors that they trigger in replication.

Endogenous Agents—Endogenous agents are responsible

for several hundred DNA damages per cell per day. The major-
ity of these damages are altered DNA bases (e.g., 8-oxoguanine
and thymine glycol) and AP sites. The cellular processes
that can lead to DNA damage are the formation of reactive
active oxygen species and deamination of cytosines and
S-methylcytosines leading to uracils and thymines, respec-
tively. The process of DNA replication itself is error-prone, and
an incorrect base can be added by the polymerase.

FIGURE 9-1 Parallelogram approach for genetic risk DNA Repair

assessment. Data obtained for genetic alterations in rodent somatic Two processes enable the cell to cope with the DNA damage that
and germ cells and human somatic cells are used to estimate the it sustains. With extensive damage, the cell can undergo apop-
frequency of the same genetic alterations in human germ cells. The tosis. If the damage is less severe, cells have developed a range of
final step is to estimate the frequency of these genetic alterations repair processes that return the DNA to its undamaged state
that are transmitted to offspring.
(error-free repair) or to an improved but still altered state
(error-prone repair). The basic principles underlying most
repair processes (but not translesion synthesis) are damage
recognition, followed by either direct reversal of the damage
(e.g., sealing of strand breaks or cleavage of pyrimidine
MECHANISMS OF INDUCTION OF dimers) or removal of the damage, repair DNA synthesis,
GENETIC ALTERATIONS and ligation.

DNA Damage Base Excision Repair— The major pathways by which DNA
The types of DNA damage range from single- and double- base damages are repaired involve removal of the damaged
strand breaks in the DNA backbone to cross-links between base. The resulting gap can be filled by a DNA polymerase, fol-
DNA bases and between DNA bases and proteins and chemical lowed by ligation to the parental DNA. Sites of oxidative dam-
addition to the DNA bases (adducts) (Figure 9-2). age, either background or induced, are important substrates for
base excision repair.
lonizing Radiations—lIonizing radiations such as x-rays,
‘-rays, and a particles produce DNA single- and double- Nucleotide Excision Repair— The nucleotide excision repair
strand breaks anda broad range of base damages from oxidative (NER) system provides the cell’s ability to remove bulky lesions
processes. Multiple damaged sites or cluster lesions appear to be from DNA. NER removes a damage-containing oligonucle-
more difficult to repair. These multiple lesions can be formed in otide from DNA by damage recognition, incision, excision,
DNA from the same radiation energy deposition event. The repair synthesis, and ligation. The DNA damage in actively
relative proportions of these different classes of DNA damage transcribing genes, and specifically the transcribed strand, is
vary with type of radiation. preferentially and more rapidly repaired than the DNA damage
in the rest of the genome. Thus, the cell protects the integrity of
Ultraviolet Light— Ultraviolet light (a nonionizing radia- the transcription process.
tion) induces two predominant lesions, cyclobutane pyrimi-
dine dimers and 6,4-photoproducts. These lesions can be Double-strand Break Repair—Cell survival is seriously
quantitated by chemical and immunologic methods. compromised by the presence of broken chromosomes.
Unrepaired double-strand breaks trigger one or more DNA
Chemicals—Chemicals can produce DNA alterations either damage response systems to either check cell-cycle progression
directly (DNA-reactive) as adducts or indirectly by intercala- or induce apoptosis. There are two general pathways for repair
tion of a chemical between the base pairs. Many electrophilic of DNA double-strand breaks: homologous recombination and
chemicals react with DNA, forming covalent addition products nonhomologous end-joining (NHEJ).
138 UNIT 3 Nonorgan-Directed Toxicity

< Apurinic site

(monofunctional
alkylating agents)
Alkylation
(monofunctional \ < Intercalations (acridines)
alkylating agents)

Radical formation
(BrdU + light, x-rays)
Apyridmidinic site >,
(monofunctional f Single-strand breaks
alkylating agents) (x-rays, UV, etc)

¥ Adduct of a bulky molecule

(eg, benzo(a)pyrene)

Pyrimidine dimers
CH; (UV)
‘4

Phosphotriesters
(monofunctional
alkylating agents)
< Base damage (x-rays)

Double-strand breaks
(ionizing radiation)

Interstrand cross-links
(bifunctional alkylating agents)

DNA-protein cross-links Intrastrand cross-links

(x-rays, polyfunctional (Polyfunctional alkylating agents)
alkylating agents)

FIGURE 9-2 Spectrum of DNA damage induced by physical and chemical agents.

Homologous Recombination—The repair of double-strand Nonhomologous End-joining—This type of recombination

breaks (and single-strand gaps) uses the following basic steps. requires the production of double-strand breaks, recombina-
The initial step is the production of a 3’-ended single-stranded tion of DNA pieces, and subsequent religation. A major
tail by exonucleases or helicase activity. Through strand inva- component of the NHEJ repair complex is a DNA-dependent
sion, whereby the single-stranded tail invades an undamaged protein kinase (DNA-PK). This protein probably functions to
homologous DNA molecule, together with DNA synthesis, a align the broken DNA ends to facilitate their ligation or to
so-called Holliday junction DNA complex is formed. By cleav- serve as a signal molecule for recruiting other repair proteins.
age of this junction, two DNA molecules are produced (with or
without a structural crossover), neither of which now contain a Mismatch Repair—The principal steps of DNA mismatch
strand break. repair are damage recognition bya specific protein that binds to
 CHAPTER9 Genetic Toxicology 139

the mismatch, stabilization of the binding by the addition of one and a stem cell. This stem cell can accumulate genetic damage
or more proteins, cutting the DNA at a distance from the mis- from chronic exposures.
match, excision past the mismatch, resynthesis, and ligation. In oogenesis, the primary oocyte arrests prior to birth, and
there is no further S phase until the zygote. For this reason, the
O°-Methylguanine-DNA Methyltransferase Repair—The oocyte is resistant to the induction of gene mutations by most
enzyme O°-methylguanine-DNA methyltransferase (MGMT) chemicals.
protects cells against the toxic effects of simple alkylating agents
by transferring the methyl group from O%-methylguanine in
DNA to a cysteine residue in MGMT. The adducted base is Formation of Chromosomal Alterations
reverted to a normal one by the enzyme, which is itself inacti- Somatic Cells
vated by the reaction. Structural Chromosome Aberrations—There are common
components between the formation of chromosome aberrations,
Formation of Gene Mutations sister chromatid exchanges (SCEs; the apparently reciprocal
Somatic Cells—Gene mutations, considered to be small exchange between the sister chromatids of a single chromo-
DNA-sequence changes confined to a single gene, are substitu- some), and gene mutations. In particular, damaged DNA serves
tions, small additions, and small deletions. Base substitutions as the substrate leading to chromosomal aberrations. However,
are the replacement of the correct nucleotide by an incorrect chromosome aberrations induced by ionizing radiations are gen-
one; they can be further subdivided as transitions, where the erally formed by errors of DNA repair, whereas those produced
change is purine for purine or pyrimidine for pyrimidine, and by nonradiomimetic chemicals are generally formed by errors of
transversions where the change is purine for pyrimidine or DNA replication on a damaged DNA template.
vice versa. Frameshift mutations are the addition or deletion The DNA repair errors that lead to the formation of chromo-
of one or a few base pairs (not in multiples of 3) in protein- some aberrations following ionizing radiation exposure arise
coding regions. from misligation of double-strand breaks or interaction of
The great majority of so-called spontaneous (background) coincidentally repairing regions during nucleotide excision
mutations arise from replication of an altered template. These repair of damaged bases. Incorrect rejoining of chromosomal
DNA alterations are either the result of oxidative damage or pieces during repair leads to chromosomal exchanges within
produced from the deamination of 5-methyl cytosine to and between chromosomes. Failure to rejoin double-strand
thymine at CpG sites resulting in G:C — A:T transitions. breaks or to complete repair of other types of DNA damage
Mutations induced by ionizing radiations tend to be deletions leads to terminal deletions.
ranging in size from a few bases to multilocus events. The failure to incorporate an acentric fragment into a daugh-
Gene mutations produced by a majority of chemicals and ter nucleus at anaphase/telophase, or the failure of a whole
nonionizing radiations are base substitutions, frameshifts, and chromosome to segregate to the cellular poles at anaphase, can
small deletions. Of these mutations, most are produced by result in the formation of a micronucleus that resides in the
errors of DNA replication on a damaged template. The relative cytoplasm. Errors of DNA replication on a damaged template
mutation frequency will be the outcome of the race between can lead to a variety of chromosomal alterations. The majority
repair and replication, that is, the more repair that takes place of these involve deletion or exchanges of individual chromatids
prior to replication, the lower the mutation frequency for a but some can involve both chromatids.
given amount of induced DNA damage. Significant regulators
of the race are cell-cycle checkpoint genes (e.g., P53) because if Numerical Chromosome Changes—Numerical changes (e.g.,
the cell is checked from entering the S phase at a G,/S check- monosomies, trisomies, and ploidy changes) can arise from
point, then more repair can take place prior to the cell starting errors in chromosomal segregation due to any of the numerous
to replicate its DNA. possible impairments of mitotic control processes. Alteration
of various cellular components can result in failure to segregate
Germ Cells—The mechanism of production of gene mutations the sister chromatids to separate daughter cells or in failure to
in germ cells is basically the same as in somatic cells. Ionizing segregate a chromosome to either pole.
radiations produce mainly deletions via errors of DNA repair;
the majority of chemicals induce base substitutions, frame- Sister Chromatid Exchange—SCEs are produced during
shifts, and small deletions by errors of DNA replication. S phase and are presumed to be a consequence of errors in the
An important consideration for assessing gene mutations replication process.
induced by chemicals in germ cells is the relationship between
exposure and the timing of DNA replication (i.e., if there is Germ Cells—The formation of chromosomal alterations in
damage, is it able to be repaired before replication’). The sper- germ cells is basically the same as that for somatic cells, namely,
matogonial stem cell is the major contributor to genetic risk via misrepair for ionizing radiations and radiomimetic chemi-
assessment because it is present generally throughout the repro- cals for treatments in G, and G,, and by errors of replication for
ductive lifetime of an individual. Each time a spermatogonial all radiations and chemicals for DNA damage present during
stem cell divides, it produces a differentiating spermatogonium the S phase.
140 UNIT 3 Nonorgan-Directed Toxicity

The types ofaberrations formed in germ cells are the same as Structural Alerts and In Silico Assays
those formed in somatic cells. The specific segregation of chro- The first indication that a chemical is a mutagen often lies in
mosomes during meiosis influences the probability of recovery chemical structure. Potential electrophilic sites in a molecule
of an aberration, particularly a reciprocal translocation, in the serve as an alert to possible mutagenicity and carcinogenicity
offspring of a treated parent. because such sites confer reactivity with nucleophilic sites in
DNA. Developmental work to formalize the structural predic-
tion through automated computer programs has not yet led to
ASSAYS FOR DETECTING GENETIC an ability to predict mutagenicity and carcinogenicity of new
ALTERATIONS chemicals with great accuracy. These computer-based systems
for predicting genotoxicity based on chemical properties are
Introduction to Assay Design sometimes called in silico assays. These assays include compu-
Genetic toxicology assays serve two interrelated but distinct tational and structural programs and the modeling of quantita-
purposes in the toxicologic evaluation of chemicals: (1) identi- tive structure-activity relationships. Although there is much
fying mutagens for purposes of hazard identification and skepticism that such approaches can replace biological testing,
(2) characterizing dose-response relationships and mutagenic they hold promise of improving the efficiency of testing strate-
mechanisms. gies and reducing current levels of animal use.
Table 9-1 lists many of the assays employed in genetic toxi-
cology. Some assays for gene mutations detect forward muta-
tions, whereas others detect reversion. Forward mutations are DNA Damage and Repair Assays
genetic alterations in a wild-type gene and are detected by a Some assays measure DNA damage itself rather than muta-
change in phenotype caused by the alteration or loss of gene tional consequences of DNA damage. They may do so directly,
function. In contrast, a back mutation or reversion is a muta- through such indicators as chemical adducts or strand breaks
tion that restores gene function in a mutant and thereby brings in DNA, or indirectly, through measurement of biological repair
about a return to the wild-type phenotype. The simplest gene processes. Adducts in DNA can be detected by *P-postlabeling,
mutation assays rely on selection techniques to detect muta- high-performance liquid chromatography (HPLC), fluorescence-
tions. By imposing experimental conditions under which only based methods, mass spectrometry, immunological methods
cells or organisms that have undergone mutation can grow, using antibodies against specific adducts, isotope-labeled
selection techniques greatly facilitate the identification of rare DNA binding, and electrochemical detection.
cells that have experienced mutation among the many cells A rapid method of measuring DNA damage is the comet
that have not. assay. In this assay, cells are incorporated into agarose on slides,
Studying mutagenesis in intact animals requires more com- lysed so as to liberate their DNA, and subjected to electropho-
plex assays, which range from inexpensive short-term tests resis. The DNA is stained with a fluorescent dye for observa-
that can be performed in a few days to complicated assays for tion and image analysis. Because broken DNA fragments
mutations in mammalian germ cells. Typically, there remains a migrate more quickly than larger pieces of DNA, a blur of frag-
gradation in which an increase in relevance for human risk ments (a “comet”) is observed when the DNA is extensively
entails more elaborate and costly tests. damaged. The extent of DNA damage can be estimated from
Many compounds that are not themselves mutagenic or car- the length and other attributes of the comet tail. The comet
cinogenic can be activated into mutagens and carcinogens by assay appears to be a sensitive indicator of DNA damage with
mammalian metabolism. Such compounds are called promu- broad applicability among diverse species, including plants,
tagens and procarcinogens. The most widely used metabolic worms, mollusks, fish, and amphibians.
activation system in microbial and cell culture assays is a post- The occurrence of DNA repair can serve as a readily mea-
mitochondrial supernatant from a rat liver homogenate, along sured indicator of DNA damage. A common excision repair
with appropriate buffers and cofactors. Most of the short-term assay in mammalian cells measures unscheduled DNA synthe-
assays in Table 9-1 require exogenous metabolic activation to sis (UDS). The occurrence of UDS indicates that the DNA had
detect promutagens. Exceptions are those in intact mammals. been damaged. The absence of UDS, however, does not provide
Despite their usefulness, in vitro metabolic activation sys- evidence that DNA has not been damaged because some classes
tems cannot mimic mammalian metabolism perfectly. There of damage are not readily excised, and some excisable damage
are differences among tissues in reactions that activate or inac- may not be detected as a consequence of assay insensitivity.
tivate foreign compounds, and organisms of the normal flora
of the gut can contribute to metabolism in intact mammals.
Agents that induce enzyme systems or otherwise alter the Gene Mutations in Prokaryotes
physiological state can also modify the metabolism of toxi- The most common means of detecting mutations in microor-
cants, and the balance between activation and detoxication ganisms is selecting for reversion in strains that have a specific
reactions in vitro may differ from that in vivo. nutritional requirement differing from wild-type members of
 CHAPTER9Y Genetic Toxicology 141

TABLE 9-1 Overview of genetic toxicology assays.

Assays

|. Prediction of genotoxicity C. Transgenic assays

A. Interpretation of chemical structure Mutations in the bacterial lac! gene in “Big Blue” mice and rats
Structural alerts to genotoxicity Mutations in the bacterial lacZ gene in the “Muta Mouse”
B. In silico predictive models Mutations in the phage cl// gene in lacl or lacZ transgenic mice
Computational and structural programs: MCASE, Point mutations and deletions in the lacZ plasmid mouse
TOPKAT, DEREK Point mutations and deletions in delta gpt mice and rats
Quantitative structure—activity relationship (QSAR) modeling Forward mutations and reversions in ®X174 transgenic mice
Inversions and deletions arising in pKZ1 mice by
ll. DNA damage and repair assays intrachromosomal recombination
A. Direct detection of DNA damage
Alkaline elution assays for DNA strand breakage Vi. Mammalian cytogenetic assays
in hepatocytes A. Chromosome aberrations
Comet assay (single-cell gel electrophoresis) for DNA strand Metaphase analysis in cultured Chinese hamster or
breakage human cells
Comet-FISH assay for region-specific DNA damage and repair Metaphase analysis of rodent bone marrow or lymphocytes
Nonmammalian comets in ecotoxicology in vivo
Assays for chemical adducts in DNA Chromosome painting and other FISH applications in vitro
B. DNA repair, recombination, and genotoxic stress responses as and in vivo
indicators of damage B. Micronuclei
Differential killing of repair-deficient and wild-type bacteria Cytokinesis-block micronucleus assay in human lymphocytes
Induction of the bacterial SOS system Micronucleus assay in mammalian cell lines
“Green Screen” forGADD45a gene induction in In vivo micronucleus assay in rodent bone marrow or blood
TK6 human cells In vivo micronucleus assay in tissues other than marrow
Unscheduled DNA synthesis (UDS) in isolated rat hepatocytes or blood
or rodents in vivo C. Sister chromatid exchange
Induction of mitotic recombination SCE in human cells or Chinese hamster cells
SCE in rodent tissues, especially bone marrow
lll. Prokaryote gene mutation assays
D. Aneuploidy in mitotic cells
A. Bacterial reverse mutation assays Hyperploidy detected by chromosome counting or FISH in
Salmonella/mammalian microsome assay (Ames test)
cell cultures or bone marrow
E. coli WP2 tryptophan reversion assay Micronucleus assay with centromere/kinetochore labeling in
Salmonella-specific base-pair substitution assay
cell cultures
(Ames II assay)
Altered parameters in flow-cytometric detection of
E. coli lacZ-specific reversion assay micronuclei in CHO cells
B. Bacterial forward mutation assays Mouse bone marrow micronucleus assay with centromere
E. coli lacl assay labeling
Resistance to toxic metabolites or analogs in Salmonella
VIL. Germ cell mutagenesis
IV. Assays in nonmammalian eukaryotes
A. Measurement of DNA damage
A. Fungal assays
Molecular dosimetry based on mutagen adducts in
Forward mutations, reversion, and small deletions
reproductive cells
Mitotic crossing over, gene conversion, and homology-
UDS in rodent germ cells
mediated deletions in yeast
Alkaline elution assays for DNA strand breaks in rodent testes
Genetic detection of mitotic and meiotic aneuploidy in yeast
Comet assay in sperm and gonadal tissue
B. Plant assays
B. Gene mutations
Gene mutations affecting chlorophyll in seedlings, the waxy
Mouse specific-locus test for gene mutations and deletions
locus in pollen, or Tradescantia stamen hair color
Mouse electrophoretic specific-locus test
Chromosome aberrations and micronuclei in mitotic and
Dominant mutations causing mouse skeletal defects or
meiotic cells of corn, Tradescantia, and other plants
cataracts
C. Drosophila assays
ESTR assay in mice
Sex-linked recessive lethal test in germ cells
Germ cell mutations in transgenic assays
Heritable translocation assays
C. Chromosomal aberrations
Mitotic recombination and LOH in eyes or wings
Cytogenetic analysis of oocytes, spermatogonia,
V. Mammalian gene mutation assays spermatocytes, or zygotes
A. In vitro assays for forward mutations Direct detection in sperm by FISH
tk mutations in mouse lymphoma or human cells Micronuclei in mouse spermatids
hprt or xprt mutations in Chinese hamster or human cells Mouse heritable translocation test
CD59 mutations in CHO-human hybrid AL cells D. Dominant lethal mutations
B. In vivo assays for gene mutations in somatic cells Mouse or rat dominant lethal assay
Mouse spot test (somatic cell specific-locus test) E. Aneuploidy
hprt mutations (6-thioguanine-resistance) in rodent Cytogenetic analysis for aneuploidy arising by nondisjunction
lymphocytes Sex chromosome loss test for nondisjunction or breakage
Pig-a mutations (immunological detection of mutations Micronucleus assay in spermatids with centromere labeling
FISH with probes for specific chromosomes in sperm
142 UNIT 3 Nonorgan-Directed Toxicity

the species; such strains are called auxotrophs. In the Ames tissues. Mutations may be detected either in somatic cells or in
assay, one measures the frequency of histidine-independent germ cells.
bacteria that arise in a histidine-requiring strain in the The mouse spot test is a traditional genetic assay for gene
presence or absence of the chemical being tested. Auxotrophic mutations in somatic cells. Visible spots of altered phenotype
(nutrient-deficient) bacteria are treated with the chemical of in mice heterozygous for coat color genes indicate mutations in
interest and plated on medium that is deficient in histidine; if the progenitor cells of the altered regions.
the colony survives, it must have a reversion mutation that Besides determining whether agents are mutagenic, muta-
allows it to survive without exogenous histidine. tion assays also provide information on mechanisms of
The development of specific reversion assays of histidine mutagenesis. Base-pair substitutions and large deletions can
mutations in Salmonella strains and of lacZ mutations in be differentiated through the use of probes for the target
Escherichia coli has made the identification of specific base-pair gene and Southern blotting, in that base substitutions are
substitutions more straightforward. Bacterial forward mutation too subtle to be detectable on the blots. Gene mutations have
assays, such as selections for resistance to arabinose or to purine been characterized at the molecular level by DNA-sequence
or pyrimidine analogs in Salmonella, are also used in research analysis both in transgenic rodents and in endogenous
and testing, although less extensively than reversion assays. mammalian genes.

Transgenic Assays—Transgenic animals are products of

Genetic Alterations in Nonmammalian DNA technology in which the animal contains foreign DNA
Eukaryotes sequences that have been added tq the genome and are trans-
Gene Mutations and Chromosome Aberrations—
The mitted through the germ line. The foreign DNA is therefore
fruit fly, Drosophila, has long occupied a prominent place in represented in all the somatic cells of the animal.
genetic research because of the sex-linked recessive lethal Mice that carry lac genes from E. coli se either acl or lacZ as
(SLRL) test. The SLRL test permits the detection of recessive a target for mutagenesis. After mutagenic treatment of the
lethal mutations at 600 to 800 different loci on the X chromo- transgenic animals, the lac genes are recovered from the ani-
some by screening for the presence or absence of wild-type mal, packaged into phage 4, and transferred to E. coli for muta-
males in the offspring of specifically designed crosses. A sig- tional analysis. Mutant plaques are identified on the basis of
nificant increase over the frequency of spontaneous SLRLs in phenotype, and mutant frequencies can be calculated for dif-
the lineages derived from treated males indicates mutagene- ferent tissues of the treated animals.
sis. The SLRL test yields information about mutagenesis in
germ cells, which is lacking in microbial and cell culture
systems.
Mammalian Cytogenetic Assays
Genetic and cytogenetic assays in plants continue to find use Chromosome Aberrations—Genetic assays without DNA
in special applications, such as in situ monitoring for mutagens sequencing are indirect, in that one observes a phenotype and
and exploration of the metabolism of promutagens by agricul- reaches conclusions about genes. In contrast, cytogenetic
tural plants. In situ monitoring entails looking for evidence of assays use microscopy for direct observation of the effect of
mutagenesis in organisms that are grown in the environment interest. In conventional cytogenetics, metaphase analysis is
of interest. used to detect chromosomal anomalies. Cells should be treated
during a sensitive period of the cell cycle (typically S), and
Mitotic Recombination— Assays in nonmammalian eukary- aberrations should be analyzed at the first mitotic division after
otes are important for the study of induced recombination. treatment. Examples of chromosome aberrations are shown in
Recombinogenic effects in yeast have long been used as a general Figure 9-3.
indicator of genetic damage. The best characterized assays for It is essential that sufficient cells be analyzed because a nega-
recombinogens are those that detect mitotic crossing over and tive result in a small sample is equivocal and inconclusive.
mitotic gene conversion in the yeast Saccharomyces cerevisiae. Results should be recorded for specific classes of aberrations,
not just as an overall index of aberrations per cell.
In interpreting results on the induction of chromosome
Gene Mutations in Mammals aberrations in cell cultures, questionable positive results have
Gene Mutations In Vitro— Mutagenicity assays in cultured been found at highly cytotoxic doses, high osmolality, and pH
mammalian cells have some of the same advantages as micro- extremes. Although excessively high doses may lead to artifac-
bial assays with respect to speed and cost, and they follow quite tual positive responses, the failure to test sufficiently high doses
similar approaches. The most widely used assays for gene muta- also undermines the utility of a test; therefore, testing should
tions in mammalian cells detect forward mutations that confer be conducted at an intermediate dose and extended to a dose at
resistance to a toxic chemical. which some cytotoxicity is observed.
In vivo assays for chromosome aberrations involve treat-
Gene Mutations In Vivo—In vivo assays involve treating ing intact animals and later collecting cells for cytogenetic
intact animals and analyzing genetic effects in appropriate analysis. The main advantage of in vivo assays is that they
 CHAPTER9 Genetic Toxicology 143

RE ce
. X ee
o # :
FIGURE 9-3 Chromosome aberrations induced by x-rays in Chinese hamster ovary (CHO) cells. A. A chromatid deletion (P). B. A
chromatid exchange called a triradial (»). C. A small interstitial deletion (Bb) that resulted from chromosome breakage. D. A metaphase with more
than one aberration: a centric ring plus an acentric fragment (®) and a dicentric chromosome plus an acentric fragment (—).

include mammalian metabolism, DNA repair, and pharma-

codynamics. The target is a tissue from which large numbers
of dividing cells are easily prepared for analysis such as
bone marrow.
In interphase cell analysis by fluorescence in situ hybridiza-
tion (FISH; Figure 9-4), a nucleic acid probe is hybridized to
complementary sequences in chromosomal DNA. The probe is
labeled with a fluorescent dye so that the chromosomal loca-
tion to which it binds is visible by fluorescence microscopy;
often, probes are used that cover the whole chromosome,
called “chromosome painting.”
Chromosome painting facilitates cytogenetic analysis,
because aberrations are easily detected by the number of fluo-
rescent regions in a painted metaphase. FISH permits the scor-
ing of stable aberrations, such as translocations and insertions,
which are not readily detected in traditional metaphase analy-
sis of unbanded chromosomes.

Micronuciei— Micronuclei are membrane-bounded struc-

tures that contain chromosomal fragments, or sometimes
whole chromosomes, that were not incorporated into a daugh-
ter nucleus at mitosis. Micronuclei usually represent acentric FIGURE 9-4 Chromosome aberrations identified by FISH.
chromosomal fragments, and they are commonly used’as sim- Human breast cancer cell with aneuploidy for some chromosomes
ple indicators of chromosomal damage. Micronuclei in a binu- and with reciprocal translocations identified by color switches along
cleate human lymphocyte are shown in Figure 9-5. a chromosome.
144 UNIT 3 Nonorgan-Directed Toxicity

kinetochores, and the observation of abnormal spindles or

spindle-chromosome associations in cells in which spindles
and chromosomes have been differentially stained.
The presence of the spindle attachment region ofachromo-
some (kinetochore) in a micronucleus can indicate that it con-
tains a whole chromosome. Aneuploidy may therefore be
detected by means ofantikinetochore antibodies with a fluores-
cent label or FISH with a probe for centromere-specific DNA.
Frequencies of micronuclei ascribable to aneuploidy and to
clastogenic effects may therefore be determined concurrently
by tabulating micronuclei with and without kinetochores.

Germ Cell Mutagenesis

Gene Mutations— Mammalian germ cell assays provide the
best basis for assessing risks to human germ cells. Mammalian
assays permit the measurement of mutagenesis at different
germ cell stages. Late stages of spermatogenesis are often found
to be sensitive to mutagenesis, but spermatocytes, spermatids,
and spermatozoa are transitory. Mutagenesis in stem cell sper-
FIGURE 9-5 Micronucleus in a human lymphocyte. The
cytochalasin B method was used to inhibit cytokinesis that resulted in matogonia and resting oocytes is of special interest in genetic
a binucleate nucleus. The micronucleus (arrow) resulted from failure risk assessment because of the persistence of these stages
of an acentric chromosome fragment or a whole chromosome being throughout reproductive life.
included in a daughter nucleus following cell division. (Used with
permission of James Allen, Jill Barnes, and Barbara Collins.) Chromosomal Alterations— Knowledge of the induction of
chromosome aberrations in germ cells is important for assessing
Sister Chromatid Exchange—SCE, in which apparently risks to future generations. A germ cell micronucleus assay has
reciprocal segments have been exchanged between the two been developed, in which chromosomal damage induced in
chromatids of a chromosome, is visible cytologically through meiosis is measured by observation of rodent spermatids.
differential staining of chromatids (Figure 9-6). SCE assays are Aneuploidy originating in mammalian germ cells may be
general indicators of mutagen exposure, rather than measures detected cytologically through chromosome counting for hyper-
of amutagenic effect. ploidy or genetically in the mouse sex-chromosome loss test.
Besides cytological observation, indirect evidence for chro-
Aneuploidy— Assays for aneuploidy include chromosome mosome aberrations is obtained in the mouse heritable translo-
counting, the detection of micronuclei that contain cation assay, which measures reduced fertility in the offspring of

FIGURE 9-6 Sister chromatid exchanges (SCEs) in human lymphocytes. A. SCEs in untreated cell. B. SCEs in cell exposed to ethyl
carbamate. The treatment results in a very large increase in the number of SCEs. (Used with permission of James Allen and Barbara Collins.)
 CHAPTER9 Genetic Toxicology 145

treated males. This presumptive evidence of chromosomal rear- The size of each study group should be sufficiently large to
rangements can be confirmed through cytogenetic analysis. avoid any confounder having undue influence. Certain charac-
teristics should be matched among exposed and unexposed
Dominant Lethal Mutations—The mouse or rat dominant groups. These include age, sex, smoking status, and general
lethal assay offers an extensive database on the induction of dietary features. Study groups of 20 or more individuals can be
genetic damage in mammalian germ cells. Commonly, males used as a reasonable substitute for exact matching because con-
are treated on an acute or subchronic basis with the agent of founders will be less influential on chromosome alteration or
interest and then mated with virgin females. The females are mutation frequency in larger groups. In some instances, it
killed and necropsied during pregnancy so that embryonic might be informative to compare exposed groups with a his-
mortality, assumed to be due to chromosomal anomalies, may torical control, as well as to a concurrent control.
be characterized and quantified. Reciprocal translocations are transmissible from cell genera-
tion to generation, and frequency can be representative of an
Development of Testing Strategies accumulation over time of exposure. The importance of this is
that stable chromosome aberrations observed in peripheral
Concern about adverse effects of mutation on human health, lymphocytes exposed in vivo, but assessed following in vitro
principally carcinogenesis and the induction of transmissible culture, are produced in vivo in hematopoietic stem cells or
damage in germ cells, has provided the impetus to identify envi- other precursor cells of the peripheral lymphocyte pool.
ronmental mutagens. Genetic toxicology assays may be used to
screen chemicals to detect mutagens and to obtain information
on mutagenic mechanisms and dose-responses that contribute NEW APPROACHES FOR
to an evaluation of hazards. Besides testing pure chemicals, GENETIC TOXICOLOGY
environmental samples are tested because many mutagens exist
in complex mixtures. The analysis of complex mixtures often The ability to manipulate and characterize DNA, RNA, and pro-
requires a combination of mutagenicity assays and refined ana- teins has been at the root of the advance in our understanding of
lytical methods. basic cellular processes and how they can be perturbed. However,
Assessment of a chemical’s genotoxicity requires data from the development of sophisticated molecular biology does not in
well-characterized genetic assays. Sensitivity refers to the pro- itself imply a corresponding advance in the utility of genetic
portion of carcinogens that are positive in the assay, whereas toxicology and its application to risk assessment. Knowing the
specificity is the proportion of noncarcinogens that are nega- types of studies to conduct and knowing how to interpret the
tive. Sensitivity and specificity both contribute to the predic- data remain as fundamental as always. There is a need for genetic
tive reliability of an assay. Assays are said to be validated when toxicology to avoid the temptation to use more and more sophis-
they have been shown to perform reproducibly and reliably ticated techniques to address the same questions and in the
with many compounds from diverse chemical classes in sev- end make the same mistakes as have been made previously.
eral laboratories.
Rather than trying to assemble batteries of complementary Advances in Cytogenetics
assays, it is prudent to emphasize mechanistic considerations Conventional chromosome staining with DNA stains such as
in choosing assays. Such an approach makes a sensitive assay Giemsa or the process of chromosome banding requires con-
for gene mutations (e.g., the Ames assay) and an assay for clas- siderable expenditure of time and a rather high level of exper-
togenic effects in mammals pivotal in the evaluation of geno- tise. Chromosome banding does allow for the assessment of
toxicity. Beyond gene mutations, one should evaluate damage transmissible aberrations such as reciprocal translocations and
at the chromosomal level with a mammalian in vitro or in vivo inversions with a fairly high degree of accuracy. Stable aberra-
cytogenetic assay. Other assays offer an extensive database on tions are transmissible from parent to daughter cell, and they
chemical mutagenesis (Drosophila SLRL), a unique genetic end represent effects of chronic exposures. The more readily ana-
point (i.e., aneuploidy; mitotic recombination), applicability to lyzed but cell-lethal, nontransmissible aberrations such as
diverse organisms and tissues (i.e, DNA damage assays, such dicentrics and deletions reflect only recent exposures and then
as the comet assay), or special importance in the assessment of only when analyzed at the first division after exposure.
genetic risk (i-e., germ cell assays). Specific chromosomes, specific genes, and chromosome
alterations can be detected readily since the development of
FISH. In principle, the technique relies on amplification of
HUMAN POPULATION MONITORING
DNA from particular genomic regions such as whole chromo-
For cancer risk assessment considerations, the human data uti- somes or gene regions and the hybridization of these amplified
lized most frequently, in the absence of epidemiologic data, are DNAs to metaphase chromosome preparations or interphase
those collected from genotoxicity/mutagenicity assessments in nuclei. Regions of hybridization can be determined by the use
human populations. The studies conducted most frequently are of fluorescent antibodies that detect modified DNA bases
for chromosome aberrations, micronuclei, and SCEs in periph- incorporated during amplification or by incorporating fluor-
eral lymphocytes. escent bases during amplification. The fluorescently labeled,
146 UNIT 3 Nonorgan-Directed Toxicity

hybridized regions are detected by fluorescence micros- The move in the field of genetic toxicology is away from the
copy. Alterations in tumors can also be detected on a whole- “yes/no” approach to hazard identification and much more
genome basis. Comparative genomic hybridization (CGH) has toward a mechanistic understanding of how a chemical or
allowed an accurate and sensitive assessment of chromosomal physical agent can produce adverse cellular and tissue
alterations present in tumors. CGH is adapted for automated responses. In turn such knowledge can be used for the develop-
screening approaches using biochips. ment of informative bioindicators representing the key events
The types of data collected will affect our understanding of along the pathway from initial interactions with cells to adverse
how tumors develop. Data on the dose-response characteris- outcome. The move is clearly toward analysis at the whole
tics for a specific chromosomal alteration as a proximate genome level and away from single gene responses.
marker of cancer can enhance the cancer risk assessment pro-
cess by describing effects of low exposures that are below those
for which tumor incidence can be reliably assessed. Cytogenetic CONCLUSION
data can also improve extrapolation of data generated with lab-
Genetic toxicology demonstrated that ionizing radiations and
oratory animals to humans.
chemicals could induce mutations and chromosome alterations
in plant, insect, and mammalian cells. Various short-term
Molecular Analysis of Mutations and assays for genetic toxicology identified many mutagens and
Gene Expression address the relationship between mutagens and carcinogens.
Failure of the assays to be completely predictive resulted in the
With technological advances, the exact basis of a mutation at
identification of nongenotoxic carcinogens. Key cellular pro-
the level of the DNA sequence can be established. With hybrid-
cesses related to mutagenesis have been identified, including
ization of test DNAs to oligonucleotide arrays, specific genetic
multiple pathways of DNA repair, cell-cycle controls, and the
alterations or their cellular consequences can be determined
role of checkpoints in ensuring that the cell cycle does not pro-
rapidly and automatically. cDNA microarray technologies
ceed until the DNA and specific cellular structures are checked
allow the measurement ofchanges in expression of hundreds or
for fidelity. Recent developments in genetic toxicology have
even thousands of genes at one time. The level of expression at
improved our understanding of basic cellular processes and
the mRNA level is measured by amount of hybridization of iso-
alterations that can affect the integrity of the genetic material
lated cDNAs to oligonucleotide fragments from known genes
and its functions. The ability to detect and analyze mutations in
or expressed sequence tags (EST) ona specifically laid out grid.
mammalian germ cells continues to improve and contribute to
This technique holds great promise for establishing a cell’s
a better appreciation for the long-term consequences of muta-
response to exposure to chemical or physical agents in the con-
genesis in human populations.
text of normal cellular patterns of gene expression.
These microarray-based techniques are now being replaced
by massively parallel sequencing or ultrahigh throughput
sequencing approaches that can quantitatively assess gene BIBLIOGRAPHY
expression changes in response to exposures. Such sequencing- Bansbach CE, Cortez D: Defining genome maintenance pathways
based techniques have the great advantage that they are based using functional genomic approaches. Crit Rev Biochem Mol Biol
49:327-341, 2011
on molecule counting approaches rather than on hybridiza-
Barile FA: Principles of Toxicology Testing. Boca Raton, FL: CRC Press,
tion, thereby making them more quantitative and able to detect 2013.
very low level transcripts. There are parallel efforts in the area Mahadevan B, Snyder RD, Waters MD, et al.: Genetic toxicity in the
of proteomics and metabolomics whereby changes in a broad 21st century: reflections and future directions. Environ Mol
range of cellular proteins can be assessed in response to endog- Mutagen 52:339-364, 2011.
enous or exogenous factors, potentially leading to the develop- Semizarov D, Blomme E: Genomics in Drug Discovery and
ment of biomarkers of effect. Development. Hoboken, NJ: John Wiley & Sons, 2009.
 CHAPTER9 Genetic Toxicology 147

QUESTIONS

Oncogenes: 6. All of the following statements regarding nondisjunction

a. maintain normal cellular growth and development. during meiosis are true EXCEPT:
b. exert their action in a genetically recessive fashion. a. Nondisjunction events can happen during meiosis I
c. are often formed via translocation to a location with or meiosis II.
a more active promoter. b. All gametes from nondisjunction events have an
can be mutated to form proto-oncogenes. abnormal chromosome number.
e. include growth factors and GTPases, but not c. Trisomy 21 (Down syndrome) is a common example
transcription factors. of nondisjunction.
d. Ina nondisjunction event in meiosis I, homologous
Which of the following is NOT one of the more common chromosomes fail to separate.
sources of DNA damage? e. The incorrect formation of spindle fibers is a common
ionizing radiation. cause of nondisjunction during meiosis.
UV light.
electrophilic chemicals. Which of the following diseases does NOT have a reces-
DNA polymerase error. sive inheritance pattern?
ea x-rays.
eRe
Pen phenylketonuria.
cystic fibrosis.
Which of the following pairs of DNA repair mechanisms Tay-Sachs disease.
is most likely to introduce mutations into the genetic sickle cell anemia.
composition of an organism? se Huntington's disease.
cao
a. nonhomologous end-joining (NHEJ) and base
excision repair. What is the purpose of the Ames assay?
b. nonhomologous end-joining and homologous a. to determine the threshold of UV light that bacteria
recombination. can receive before having mutations in their DNA.
c. homologous recombination and nucleotide excision b. to measure the frequency of aneuploidy in bacterial
repair. colonies treated with various chemicals.
nucleotide excision repair and base excision repair. c. to determine the frequency of a reversion mutation
e. homologous recombination and mismatch repair. that allows bacterial colonies to grow in the absence
of vital nutrients.
Which of the following DNA mutations would NOT be d. to measure rate of induced recombination in mutagen-
considered a frameshift mutation? treated fungi.
insertion of 5 nucleotides. e. to measure induction of phenotypic changes in
insertion of 7 nucleotides. Drosophila.
deletion of 18 nucleotides.
deletion of 13 nucleotides. In mammalian cytogenic assays, chromosomal aberra-
O
CEE
Sa deletion of 1 nucleotide. tions are measured after treatment of the cells at which
sensitive phase of the cell cycle?
Which of the following base-pair mutations is properly a. interphase.
characterized as a transversion mutation? b. M phase.
Aneel: c. S phase.
DAG: d. Gl.
G Gov e. G2.
Gh; “I 2 UE
Q N=axG 10. Which of the following molecules is used to gauge the
amount of a specific gene being transcribed to mRNA?
a. protein.
b. mRNA.
¢. DNA.
d. cDNA.
© CGH.
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min rhe ie jiia a vie o ee Pe itt hh W vay Peek
i pnt)
: ‘ up
<i Ae
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 Cu Ele A a Pestle Shite R

Developmental Toxicology
John M. Rogers

SCOPE OF PROBLEM—THE HUMAN EXPERIENCE Maternal Factors Affecting Development

Thalidomide Genetics
Diethylstilbestrol Disease
Ethanol Nutrition

Tobacco Smoke Stress

Cocaine Placental Toxicity

Retinoids Maternal Toxicity
Antiepileptic Drugs DEVELOPMENTAL TOXICITY OF ENDOCRINE-DISRUPTING
Angiotensin Converting Enzyme (ACE) Inhibitors CHEMICALS
and Angiotensin Receptor Antagonists
Laboratory Animal Evidence
PRINCIPLES OF DEVELOPMENTAL TOXICOLOGY Human Evidence
Critical Periods of Susceptibility and End Points Impact on Screening and Testing Programs
of Toxicity MODERN SAFETY ASSESSMENT
Dose-Response Patterns and the Threshold Concept
Regulatory Guidelines for In Vivo Testing
MECHANISMS AND PATHOGENESIS OF Multigeneration Tests
DEVELOPMENTAL TOXICITY Children’s Health
Advances in the Molecular Basis of Dysmorphogenesis Alternative Testing Strategies
Epidemiology
PHARMACOKINETICS AND METABOLISM IN PREGNANCY
Concordance of Data
RELATIONSHIPS BETWEEN MATERNAL AND Elements of Risk Assessment
DEVELOPMENTAL TOXICITY PATHWAYS TO THE FUTURE

KEY POINTS

= Developmental toxicology encompasses the study of Gametogenesis is the process of forming the haploid
pharmacokinetics, mechanisms, pathogenesis, and out- germ cells: the egg and the sperm.
comes following exposure to agents or conditions lead- Organogenesis is the period during which most bodily
ing to abnormal development. structures are established. This period of heightened
m Developmental toxicology includes teratology, or the susceptibility to malformations extends from the third
study of structural birth defects. to the eighth week of gestation in humans.

149
150 UNIT 3 Nonorgan-Directed Toxicity

SCOPE OF PROBLEM—THE HUMAN TABLE 10-1 Human developmental toxicants.

EXPERIENCE Radiation
° Atomic fallout
Successful pregnancy outcome in the general population occurs ° Radioiodine
at a surprisingly low frequency. Estimates of adverse outcomes Therapeutic
include postimplantation pregnancy loss, 31%; major birth
Infections
defects, 2% to 3% at birth and increasing to 6% to 7% at 1 year Cytomegalovirus
as more manifestations are diagnosed; minor birth defects, Herpes simplex virus 1 and 2
14%; low birth weight, 7%; infant mortality (prior to 1 year of Parvovirus B-19 (erythema infectiosum)
age), 1.4%; and abnormal neurologic function, 16% to 17%. Rubella virus
Syphilis
Thus, less than half ofall human conceptions result in the birth Toxoplasmosis
of a completely normal, healthy infant. Many hundreds of Varicella virus
chemicals are teratogens; most of them produce birth defects by Venezuelan equine encephalitis virus
an unknown mechanism. However, Table 10-1 lists chemicals, Maternal trauma and metabolic imbalances
chemical classes, or conditions known to alter prenatal devel- Alcoholism
opment in humans. Amniocentesis, early
Chorionic villus sampling (before day 60)
Cretinism
Thalidomide Diabetes
Folic acid deficiency .
In 1960, a large increase in newborns with rare limb malforma-
Hyperthermia \
tions of amelia (absence of the limbs) or various degrees of pho- Phenylketonuria
comelia (reduction ofthe long bones ofthe limbs) was recorded Rheumatic disease and congenital heart block
in West Germany. Congenital heart disease; ocular, intestinal, Sjogren's syndrome :
Virilizing tumors
and renal anomalies; and malformations of the external and
inner ears were also involved. Thalidomide, identified as the Drugs and chemicals
causative agent, was used throughout much of the world as a ° Aminoglycosides
sleep aid and to ameliorate nausea and vomiting in pregnancy. Androgenic hormones
Angiotensin converting enzyme inhibitors: captopril,
It had no apparent toxicity or addictive properties in adult enalapril
humans or rodents at therapeutic exposure levels. Angiotensin receptor antagonists: sartans
As a result of this catastrophe, regulatory agencies developed Anticonvulsants: diphenylhydantoin, trimethadione,
valproic acid, carbamazepine
requirements for evaluating the effects of drugs on pregnancy
Busulfan
outcomes. Carbon monoxide
Chlorambucil
Diethylstilbestrol Cocaine
Coumarins
Diethylstilbestrol (DES) is a synthetic nonsteroidal estrogen Cyclophosphamide
widely used from the 1940s to the 1970s in the United States to Cytarabine
Diethylstilbestrol
prevent threatened miscarriage. It was soon linked to clear cell Danazol
adenocarcinoma of the vagina. Maternal use of DES prior to the Ergotamine
18th week of gestation appeared to be necessary for induction of Ethanol
the genital tract anomalies in offspring; the overall incidence of Ethylene oxide
Fluconazole
noncancerous alterations in the vagina and cervix was esti-
Folate antagonists: aminopterin, methotrexate
mated to be as high as 75%. In male offspring of exposed preg- lodides
nancies, a high incidence of reproductive tract anomalies along Lead
with low ejaculated semen volume and poor semen quality were Lithium
Mercury, organic
observed. The realization of the latent and devastating manifes- Methimazole
tations of prenatal DES exposure has broadened the magnitude Methylene blue
and scope of potential adverse outcomes of intrauterine expo- Misoprostal
sures. A recent study in mice suggests that the increased suscep- Penicillamine
Polychlorobiphenyls
tibility to abnormalities conferred by DES exposure may be Quinine (high dose)
passed on to future generations of exposed mothers. Retinoids: accutane, isotretinoin, etretinate, acitretin
Tetracyclines
Thalidomide
Ethanol Tobacco smoke
Although the developmental toxicity of ethanol can be traced to Toluene
Vitamin A (high dose)
biblical times (e.g., Judges 13:3-4), only since the description of
the Fetal Alcohol Syndrome (FAS) in 1971 has a clear acceptance
 CHAPTER 10 Developmental Toxicology 151

of alcohol’s developmental toxicity occurred. FAS comprises Antiepileptic Drugs

craniofacial dysmorphism, intrauterine and postnatal growth
Clinical management of women ofchildbearing age who have
retardation, retarded psychomotor and intellectual develop-
epilepsy is difficult. Although control ofseizures during preg-
ment, and other nonspecific major and minor abnormalities.
nancy is crucial, most current antiepileptic drugs (AEDs)
In utero exposure to lower levels of ethanol than those that
have been shown to carry risk of developmental toxicity
produce full-blown FAS has been associated with a wide range
including birth defects, cognitive impairment, and fetal death.
of effects, including isolated components of FAS and milder
As a class, including phenytoin, carbamazepine, and valproic
forms of neurologic and behavioral disorders that have been
acid, AEDs are considered human teratogens. Studies to date
termed fetal alcohol spectrum disorder (FASD). Alcohol con-
suggest that newer AEDs such as gabapentin, lamotrigine,
sumption can affect birth weight in a dose-related fashion.
oxcarbazone, topiramate, and zonizamide may be safer than
the older AEDs.
Tobacco Smoke
Prenatal and early postnatal exposure to tobacco smoke or its
constituents may well represent the leading cause of environ- Angiotensin Converting Enzyme (ACE)
mentally induced developmental disease and morbidity today. Inhibitors and Angiotensin
Approximately 25% of women in the United States continue to Receptor Antagonists
smoke during pregnancy, despite public health programs aimed The renin-angiotensin system is a key controller of blood
at curbing this behavior. The consequences of developmental pressure. The active signaling messenger of this system is
tobacco smoke exposure include spontaneous abortions, perina- angiotensin II, which binds to angiotensin II (AT1) receptors to
tal deaths, increased risk of sudden infant death syndrome cause vasoconstriction and fluid retention, resulting in eleva-
(SIDS), increased risk of learning, behavioral, and attention dis- tion of blood pressure. ACE inhibitors and angiotensin receptor
orders, and lower birth weight. One component of tobacco blockers are widely prescribed and, when used in the second
smoke, nicotine, is a known neuroteratogen in experimental ani- half of pregnancy, are known to cause oligohydramnios (low
mals and can by itself produce many of the adverse developmen- amniotic fluid volume), fetal growth retardation, pulmonary
tal outcomes associated with tobacco smoke. Perinatal exposure hypoplasia, joint contractures, hypocalvaria, neonatal renal
to tobacco smoke can also affect branching morphogenesis and failure, hypotension, and death. Some studies suggest that
maturation of the lung, leading to altered physiologic function. exposure in the first trimester should be avoided.
Environmental (second-hand) tobacco smoke also represents a
significant risk to the pregnant nonsmoker and her baby, and
exposure to second-hand smoke has been associated with many PRINCIPLES OF DEVELOPMENTAL
of the effects caused by active maternal smoking. TOXICOLOGY
Some basic principles of teratology put forth by Jim Wilson in
Cocaine
1959 are listed in Table 10-2; they are still valid today.
Cocaine is a local anesthetic with vasoconstrictor properties.
Effects on the fetus are complicated and controversial and dem-
onstrate the difficulty of monitoring the human population for TABLE 10-2 Wilson’s general principles of teratology.
adverse reproductive outcomes. Accurate exposure ascertain-
|. Susceptibility to teratogenesis depends on the genotype of the
ment is difficult, as many confounding factors including socio- conceptus and the manner in which this interacts with adverse
economic status and concurrent use of cigarettes, alcohol, and environmental factors
other drugs of abuse may be involved. In addition, reported
Il. Susceptibility to teratogenesis varies with the developmental stage
effects on the fetus and infant (neurologic and behavioral changes)
at the time of exposure to an adverse influence
are difficult to identify and quantify. Nevertheless, adverse effects
reliably associated with cocaine exposure in humans include Ill. Teratogenic agents act in specific ways (mechanisms) on
abruptio placentae, premature labor and delivery, microcephaly, developing cells and tissues to initiate sequences of abnormal
developmental events (pathogenesis)
altered prosencephalic development, decreased birth weight,
SIDS, and a neonatal neurologic syndrome of abnormal sleep, IV, The access of adverse influences to developing tissues depends
tremor, poor feeding, irritability, and occasional seizures. on the nature of the influence (agent)

V. The four manifestations of deviant development are death,

Retinoids malformation, growth retardation, and functional deficit

Vitamin A (retinol) exposure can cause malformations of Vl. Manifestations of deviant development increase in frequency
the face, limbs, heart, central nervous system, and skeleton. and degree as dosage increases, from the no effect to the totally
Spontaneous abortion, live-born infants having at least one major lethal level

malformation, and numerous exposed children having full-scale Data from Wilson JG: Environment
and Birth Defects. New York, NY: Academic Press/
IQ scores below 85 at age 5 years have been documented. Elsevier; 1973.
152 UNIT 3 Nonorgan-Directed Toxicity

Critical Periods of Susceptibility and End Epigenetic changes include DNA methylation, histone modifi-
cations, and expression of microRNAs. There are specific stages
Points of Toxicity of the life cycle during which epigenetic marks may be erased
Development is characterized by various changes that are and reestablished, including two periods of development dur-
orchestrated by a cascade of factors regulating gene transcrip- ing which large-scale demethylations of the genome are known
tion throughout development. Intercellular and intracellular to occur. One is during migration and proliferation of the pri-
signaling pathways essential for normal development rely on mordial gem cells in which imprinted genes are demethylated,
transcriptional, translational, and posttranslational controls. with remethylation occurring in a gender-specific manner
The rapid changes occurring during development alter the during gametogenesis in the offspring. The other period of
nature of the embryo/fetus as a target for toxicity. Timing of widespread epigenetic reprogramming occurs shortly after
some key developmental events in humans and experimental formation of the zygote and in the early embryo, with total
animal species is presented in Table 10-3. genomic methylation reaching a nadir at the blastocyst stage.
Gametogenesis is the process of forming the haploid germ Following fertilization, the embryo moves down the fallo-
cells: the egg and the sperm. These gametes fuse in the process pian tube (oviduct) and implants in the wall of the uterus. The
of fertilization to form the diploid zygote, or one-celled embryo. preimplantation period comprises mainly an increase in cell
Gametogenesis and fertilization are vulnerable to toxicants. It number through a rapid series of cell divisions with little
is now known that the maternal and paternal genomes are not growth in size (cleavage of the zygote) and cavitation of the
equivalent in their contributions to the zygotic genome. The embryo to form a fluid-filled blastocoele. This stage, termed
process of imprinting (which involves cytosine methylation the blastocyst, contains cells destined to give rise to the embryo
and changes in chromatin conformation) occurs during game- proper and other cells that give rise to extraembryonic mem-
togenesis, conferring to certain allelic genes a differential branes and support structures.
expressivity depending on whether they are of maternal or Toxicity during preimplantatiort is generally thought to
paternal origin. result in no or slight effect on growth (because of regulative
Epigenetics refers to the biochemical changes in chromatin growth) or in death (through overwhelming damage or failure
that lead to changes in conformation and gene expression.

TABLE 10-3 Timing of key developmental events in some mammalian species.*

Rat Rabbit Monkey Human

Blastocyst formation 3-5 2.6-6 4-9 4-6

Implantation 5-6 6 3) 6-7

Organogenesis sil 6-18 20-45 21-56

Primitive streak 9 6.5 18-20 16-18

Neural plate 9.5 = 2 18-20

First somite 10 a oa 20-21

First branchial arch 10 — = 20

First heartbeat 10.2 baat a 22

10 somites 10-11 9 23-24 25-26

Upper limb buds 10.5 10.5 25-26 29-30

Lower limb buds die 11 26-27 31-32

Testes differentiation 14.5 20 os 43

Heart septation 15:5 o as 46-47

Palate closure 16-17 19-20 45-47 56-58

Urethral groove closed — see = 90

in male

Length of gestation 21-22 31-34 166 267

*Developmental ages are days of gestation.

Data from Shepard TH: Catalog of Teratogenic Agents, 9th ed. Baltimore, MD: The Johns Hopkins University Press; 1998.
 CHAPTER 10 Developmental Toxicology £53

to implant). Because ofthe rapid mitoses occurring during the Another key element of the dose-response relationship is
preimplantation period, chemicals affecting DNA synthesis/ the shape of the dose-response curve at low exposure levels.
integrity or those affecting microtubule assembly would be Because of the high restorative growth potential of the
expected to be particularly toxic if given access to the embryo. mammalian embryo, cellular homeostatic mechanisms, and
Following implantation the embryo undergoes gastrulation, maternal metabolic defenses, mammalian developmental tox-
the process of formation of the three primary germ layers—the icity has generally been considered a threshold phenomenon.
ectoderm, mesoderm, and endoderm. During gastrulation, cells Assumption of a threshold means that there is a maternal dos-
migrate through a midline structure called the primitive streak, age below which an adverse response is not elicited because
and their movements set up basic morphogenetic fields in the some repair or defense system is able to combat the exposure.
embryo. As a prelude to organogenesis, the period of gastrula-
tion is quite susceptible to teratogens. A number of toxicants
administered during gastrulation produce malformations of MECHANISMS AND PATHOGENESIS
the eye, brain, and face. These malformations are indicative of OF DEVELOPMENTAL TOXICITY
damage to the anterior neural plate, one of the regions defined
by the cellular movements of gastrulation. The term mechanisms refers to cellular-level events that initiate
The formation of the neural plate in the ectoderm marks the the process leading to abnormal development. Pathogenesis
onset of organogenesis, during which the rudiments of most comprises the cell-, tissue-, and organ-level sequelae that ulti-
bodily structures are established. This period of heightened mately manifest in abnormality. Mechanisms of teratogenesis
susceptibility to malformations extends from approximately include mutations, chromosomal breaks, altered mitosis,
the third to the eighth week of gestation in humans. The altered nucleic acid integrity or function, diminished supplies
rapid changes of organogenesis require cell proliferation, cell of precursors or substrates, decreased energy supplies, altered
migration, cell-cell interactions, and morphogenetic tissue membrane characteristics, osmolar imbalance, and enzyme
remodeling. Within organogenesis, there are periods of peak inhibition. Although these cellular insults are not unique to
susceptibility for each forming structure. The peak incidence development, they may trigger unique pathogenetic responses
of each malformation coincides with the timing of key in the embryo, such as reduced cell proliferation, cell death,
developmental events in the affected structure. altered cell-cell interactions, reduced biosynthesis, inhibition
The end of organogenesis marks the beginning of the fetal of morphogenetic movements, or mechanical disruption of
period, which is characterized primarily by tissue differentiation, developing structures.
growth, and physiologic maturation. All organs are present and Cell death plays a critical role in normal morphogenesis. The
grossly recognizable, although not yet completely developed. term programmed cell death refers specifically to apoptosis,
Exposure during the fetal period is most likely to result in which is under genetic control in the embryo. Apoptosis is
effects on growth and functional maturation. Functional anom- necessary for sculpting the digits from the hand plate and for
alies of the central nervous system and reproductive organs— assuring appropriate functional connectivity between the cen-
including behavioral, mental, and motor deficits as well as tral nervous system and distal structures. Cell proliferation
decreases in fertility—are among the possible adverse outcomes. rates change both spatially and temporally during ontogenesis.
Over the past two decades, the concept of “developmental There is a delicate balance between cell proliferation, cell dif-
programming” has emerged, in which the developmental envi- ferentiation, and apoptosis in the embryo. DNA damage might
ronment is thought to influence the metabolic parameters of lead to cell cycle perturbations and cell death.
the offspring that will persist throughout life and may affect As discussed in Chapter 9, DNA damage can inhibit cell
lifelong risk of disease. Much of the work on fetal program- cycle progression at the G,-S transition, through the S phase,
ming has focused on the role of maternal nutrition, and there is and at the G,-M transition. If DNA damage is repaired, the cell
a paucity of data concerning the long-term effects of chemical cycle can return to normal, but if damage is too extensive or
exposure during the fetal and early postnatal periods. Some cell cycle arrest too long, apoptosis may be triggered. The
effects could require years to become apparent (such as those relationship between DNA damage and repair, cell cycle pro-
noted above for DES), and others may even result in the pre- gression, and apoptosis is depicted in Figure 10-1. From the
mature onset of senescence and/or organ failure late in life. multiple checkpoints and factors present to regulate the cell
cycle and apoptosis, it is clear that different cell populations
may respond differently to a similar stimulus, in part because
Dose-Response Patterns and the cellular predisposition to apoptosis can vary.
Threshold Concept Besides affecting proliferation and cell viability, molecular
The major effects of prenatal exposure, observed at the time of and cellular insults can alter cell migration, cell-cell interac-
birth in developmental toxicity studies, are embryo lethality, tions, differentiation, morphogenesis, and energy metabolism.
malformations, and growth retardation. For some agents, these Although the embryo has compensatory mechanisms to offset
end points may represent a continuum of increasing toxicity, such effects, production of a normal or malformed offspring
with low dosages producing growth retardation and increasing will depend on the balance between damage and repair at each
dosages producing malformations and then lethality. step in the pathogenetic pathway.
154 UNIT 3 Nonorgan-Directed Toxicity

PHARMACOKINETICS AND
METABOLISM IN PREGNANCY
The extent and the form in which chemicals reach the concep-
tus are important determinants of whether the agent can impact
development. The maternal, placental, and embryonic com-
partments comprise independent, yet interacting, systems that
undergo profound changes throughout the course of preg-
DNA damage Activation of nancy. Alterations in placental physiology can have significant
signal DNA repair impact on the uptake, distribution, metabolism, and elimina-
tion of xenobiotics. For example, decreases in intestinal motil-
ity and increases in gastric emptying time result in longer
Growth factor|
retention of ingested chemicals im the upper gastrointestinal
_-7 | deprivation
Protein synthesis tract in the mother. Cardiac output increases by 50% during the
/
first trimester in humans and remains elevated throughout
pregnancy, whereas blood volume increases and plasma
proteins and peripheral vascular resistance decrease. The rela-
tive increase in blood volume over red cell volume leads to bor-
Growth factors
derline anemia and a generalized edema with a 70% elevation
Bcl-2/Bax
of extracellular space. Thus, the volume of distribution of a
chemical and the amount bound by plasma proteins may
change considerably during pregnancy: Other changes occur in
FIGURE 10-1 Relationships between DNA damage and
the renal, hepatic, and pulmonary systems as well. Clearly,
the induction of cell cycle arrest or apoptosis. DNA damage can
maternal handling of a chemical influences the extent of
signal inhibition of the cell cycle between G, and S, in S phase, or
between G, and mitosis. The signal(s) can also activate DNA repair embryotoxicity.
mechanisms and synthesis of proteins, including p53, that can initiate The placenta also influences embryonic exposure by helping
apoptosis. Growth factors and products of the proto-oncogene c-myc to regulate blood flow, offering a transport barrier, and metab-
and the Bcl-2/Bax gene family, as well as differentiation state and cell olizing chemicals. The placenta acts as a lipid membrane that
cycle phase, are important determinants of the ultimate outcome of permits bidirectional transfer of substances between maternal
embryonal DNA damage. and fetal compartments. It is important to note that virtually
any substance present in the maternal plasma will be trans-
ported to some extent by the placenta. The passage of most
Advances in the Molecular Basis drugs across the placenta seems to occur by simple passive dif-
fusion. Important modifying factors to the rate and extent of
of Dysmorphogenesis transfer include lipid solubility, molecular weight, protein
Advances in gene targeting and transgenic strategies now allow binding, the type of transfer (passive diffusion, and facilitated
modification of gene expression at specific points in develop- or active transport), the degree of ionization, and placental
mentand in specific cell types. Conditional knockouts (cKO) or metabolism. Blood flow probably constitutes the major rate-
knockins (cKI), inducible gene expression, and other tech- limiting step for more lipid-soluble compounds.
niques are being used to study the effects ofspecific gene prod- Maternal metabolism of xenobiotics is an important and
ucts on development in great detail. The use of synthetic variable determinant of developmental toxicity. As for other
antisense oligonucleotides allows temporal and spatial restric- health end points, the field of pharmacogenomics offers hope
tion of gene ablation by hybridizing to mRNA in the cell, for increasing our ability to predict susceptible subpopulations
thereby inactivating it. In this way, gene function can be turned based on empirical relationships between maternal genotype
off at specific times. RNA interference is a more recent gene and fetal phenotype.
knockdown technique, exploiting the discovery of the RNA
interference pathway. Small interference (si)RNA, plasmid-,
and virus-encoded small RNAs can be used to down-regulate RELATIONSHIPS BETWEEN MATERNAL
the expression of specific genes posttranscriptionally.
AND DEVELOPMENTAL TOXICITY
Gain of gene function can also be studied by engineering
genetic constructs with an inducible promoter attached to the Although all developmental toxicity must ultimately result
gene of interest. Ectopic gene expression can be made ubiqui- from an insult to the conceptus at the cellular level, the insult
tous or site-specific depending on the choice of promoter to may occur through a direct effect on the embryo/fetus, indi-
drive expression. Transient overexpression of specific genes rectly through toxicity of the agent to the mother and/or the
can be accomplished by adding extra copies using adenoviral placenta, or a combination of direct and indirect effects.
transduction. Maternal factors known to affect fetal development include
 CHAPTER 10 Developmental Toxicology 55

Toxicant —
exposure

Stress ~~
Nutritiona| Stat Maternal
susceptibility
O Mer exposures 4
factor
Pai SS |

Anemia or toxemia
Endocrine imbalance
Placental
eg Placenta Nutritional deficit —
toxicity | Potential
Electrolyte imbalance
Placental maternal
Acid-base disturbance
transfer? | effects
Decreased uterine blood flow
Altered organ function .
Direct Decreased milk quantity/quality
developmental 'Indirect
Placental insufficiency toxicity |developmental
« Reduced size 1toxicity
« Reduced blood flow
- Altered transport
« Altered metabolism Abnormal |
development

FIGURE 10-2 interrelationships between maternal susceptibility factors, metabolism, induction of maternal physiologic
or functional alterations, placental transfer and toxicity, and developmental toxicity. A developmental toxicant can cause abnormal
development through any one or a combination of these pathways. Maternal susceptibility factors determine the predisposition of the
mother to respond to a toxic insult, and the maternal effects listed can adversely affect the developing conceptus. Most chemicals traverse
the placenta in some form, and the placenta can also be a target for toxicity. In most cases, developmental toxicity is probably mediated
through a combination of these pathways.

genetics, disease, nutrition, stress, placental toxicity, and maternal toxicity and elucidation of the association with devel-
maternal toxicity. Some conditions that may adversely affect opmental effects is needed before one can begin to address the
the fetus are depicted in Figure 10-2. relevance of the observations to human safety assessment.
The distinction between direct and indirect developmental
toxicity is important for interpreting safety assessment results
in pregnant animals, as the highest dosage level in these experi- Maternal Factors Affecting Development
ments is chosen based on its ability to produce some maternal Genetics—The genetic makeup of the pregnant female has
toxicity (e.g., decreased food or water intake, weight loss, and been well documented as a determinant of developmental out-
clinical signs). However, maternal toxicity defined only by such come in both humans and animals. The incidence of cleft lip
crude manifestations gives little insight to the toxic actions of a and/or palate [CL(P)], which occurs more frequently in whites
xenobiotic. When developmental toxicity is observed only in than in blacks, has been investigated in offspring of interracial
the presence of maternal toxicity, the developmental effects may couples in the United States. Offspring of white mothers had a
be indirect (i.e., caused by an inappropriate growing condition higher incidence of CL(P) than offspring of black mothers after
because of an altered maternal environment rather than by a correcting for paternal race, whereas offspring of white fathers
direct interaction of the fetus with the toxin). Greater under- did not have a higher incidence of CL(P) than offspring of black
standing of the physiologic changes underlying the observed fathers after correcting for maternal race.
156 UNIT 3 Nonorgan-Directed Toxicity

Disease—Chronic hypertension in the mother, uncontrolled Phenytoin, an anticonvulsant, can affect maternal folate
maternal diabetes mellitus, and certain infections in the mother metabolism in experimental animals, and these alterations
(ie. cytomegalovirus and Toxoplasma gondii) are leading may play a role in the teratogenicity of this drug. A mechanism
causes ofseveral types ofdefects in the fetus. Exposure to hyper- of teratogenesis was proposed relating depressed maternal
thermia (such as febrile illness in the mother) is also implicated heart rate and embryonic hypoxia. Supporting studies have
in neural defects in the fetus. demonstrated that hyperoxia reduces the teratogenicity of
phenytoin in mice.
Nutrition—A wide spectrum of dietary insufficiencies
ranging from protein-calorie malnutrition to deficiencies of
vitamins, trace elements, and/or enzyme cofactors is known to DEVELOPMENTAL TOXICITY
adversely affect pregnancy. In fact, folate supplementation by OF ENDOCRINE-DISRUPTING
pregnant women can reduce neural tube defect recurrence by
CHEMICALS
over 70%.
There is the growing concern that exposure to chemicals that
Stress— Diverse forms of maternal toxicity may have in com- can interact with the endocrine system may pose a serious
mon the induction of a physiologic stress response. Various health hazard. An “endocrine disruptor” has been broadly
forms of physical stress have been applied to pregnant animals defined as an exogenous agent that interferes with the produc-
in attempts to isolate the developmental effects of stress. Noise tion, release, transport, metabolism, binding, action, or elimi-
stress of pregnant rats or mice throughout gestation can nation of natural hormones respansible for the maintenance
produce developmental toxicity. Restraint stress produces of homeostasis and the regulation of developmental processes.
increased fetal death in rats, and malformations of cleft palate, Due to the critical role of hormones in directing differentia-
fused and supernumerary ribs, and encephaloceles in mice. tion in many tissues, the developing organism is particularly
There is a positive correlation in humans between stress and vulnerable to fluctuations in the timing or intensity of expo-
adverse developmental effects, including low birth weight and sure to chemicals with hormonal or antihormonal activity.
congenital malformations. Various chemical classes induce developmental toxicity via at
least three modes of action involving the endocrine system:
Placental Toxicity—The placenta is the interface between (1) by serving as ligands ofsteroid receptors, (2) by modifying
the mother and the conceptus, providing attachment, nutri- steroid hormone metabolizing enzymes, and (3) by perturb-
tion, gas exchange, and waste removal. The placenta also pro- ing hypothalamic-pituitary release of trophic hormones.
duces hormones critical to the maintenance of pregnancy, and Interactions with the functions of estrogens, androgens, and
it can metabolize and/or store xenobiotics. Placental toxicity thyroid hormones have been the most studied, but the under-
may compromise these functions. Known placental toxicants lying principles apply to other hormones also.
include cadmium, arsenic or mercury, cigarette smoke, etha-
nol, cocaine, endotoxin, and sodium salicylate.
Laboratory Animal Evidence
Maternal Toxicity—A retrospective analysis of relation- Estrogenic or antiestrogenic developmental toxicants include
ships between maternal toxicity and specific types of prenatal DES, estradiol, antiestrogenic drugs such as tamoxifen and clo-
effects found species-specific associations between maternal miphene citrate, and some pesticides and industrial chemicals.
toxicity and specific adverse developmental effects. Various The pattern of outcomes is generally similar across different
adverse developmental outcomes include increased intrauter- estrogens. Female offspring are generally more sensitive to
ine death, decreased fetal weight, supernumerary ribs, and these toxicants than males, and altered pubertal development,
enlarged renal pelvises. reduced fertility, and reproductive tract anomalies are common
A number of studies directly relate specific forms of mater- findings.
nal toxicity to developmental toxicity, including those in which Antiandrogens represent another major class of endocrine-
the test chemical causes maternal effects that exacerbate the disrupting chemicals. Principal manifestations of develop-
agent’s developmental toxicity. However, clear delineation of mental exposure to an antiandrogen are generally restricted to
the relative role(s) of indirect maternal and direct embryo/fetal males, and include hypospadias, retained nipples, reduced
toxicity is difficult. testes and accessory sex gland weights, and decreased sperm
Diflunisal, an analgesic and anti-inflammatory drug, causes production.
axial skeletal defects in rabbits. Developmentally toxic dosages Hypothyroidism during pregnancy and early postnatal devel-
resulted in severe maternal anemia and depletion of erythro- opment causes growth retardation, cognitive defects, delayed
cyte ATP levels. Teratogenicity, anemia, and ATP depletion eye-opening, hyperactivity, and auditory defects. Polychlorinated
were unique to the rabbit. The teratogenicity of diflunisal in biphenyls (PCBs) may act at several sites to lower thyroid hor-
the rabbit was probably due to hypoxia resulting from maternal mone levels during development, and cause these developmen-
anemia. tal abnormalities.
 CHAPTER 10 Developmental Toxicology 157

Human Evidence then used in the risk assessment process to assess the likelihood
Whether human health is being adversely impacted from of effects in humans given certain exposure conditions.
exposures to endocrine disruptors present in the environment
is equivocal. Reports in humans are of two types: Multigeneration Tests
1. Observations of adverse effects on reproductive system
Information pertaining to developmental toxicity can also
development and function following exposure to chemi-
be obtained from studies in which animals are exposed to
cals with known endocrine activities that are present in
the test substance continuously over one or more generations.
medicines, contaminated food, or the workplace. These
For additional information on this approach, see Chapter 20.
have tended to involve relatively higher exposure to
chemicals with known endocrine effects.
2. Epidemiologic evidence of increasing trends in reproduc- Children’s Health
tive and developmental adverse outcomes that have an
Infants and children differ both qualitatively and quantita-
endocrine basis. For example, secular trends have been
tively from adults in their exposure to pesticide residues in
reported for cryptorchidism, hypospadias, semen quality,
food because of different dietary composition, intake pat-
and testicular cancer, but due to the lack of exposure
terns, and different activities, such as crawling on the floor or
assessment, such studies provide limited evidence of a
ground, putting their hands and foreign objects in their
cause and effect relationship.
mouths, and raising dust and dirt during play. Even the level
of their activity (ie., closer to the ground) can affect their
Impact on Screening and Testing Programs exposure to some toxicants. In addition to exposure differ-
ences, children are growing and developing, which makes
The findings of altered reproductive development following
them more susceptible to some types ofinsults. Effects of early
early life-stage exposures to endocrine-disrupting chemicals
childhood exposure, including neurobehavioral effects and
helped prompt revision of traditional safety evaluation tests.
cancer, may not be apparent until later in life. Debate contin-
These include assessments of female estrous cyclicity, sperm
ues over the approach to be used in risk assessment in consid-
motility, and sperm morphology in both parental and F1 gen-
eration of infants and children.
erations, the age at puberty in the F1s, histopathology of target
organs, anogenital distance in the F2s, and primordial follicu-
lar counts in the parental and F1 generations. For the new pre- Alternative Testing Strategies
natal developmental toxicity test guidelines, one important Various alternative test systems have been proposed to refine,
modification aimed at improved detection of endocrine dis- reduce, or replace the standard regulatory mammalian tests for
ruptors was the expansion ofthe period of dosing from the end assessing prenatal toxicity (Table 10-5). These can be grouped
of organogenesis (i.e., palatal closure) to the end of pregnancy into assays based on cell cultures, cultures of embryos in vitro
in order to include the developmental period of urogenital (including submammalian species), and short-term in vivo
differentiation. tests. It was initially hoped that the alternative approaches
would become generally applicable to all chemicals, and help
MODERN SAFETY ASSESSMENT prioritize full-scale testing; this has not yet been accomplished.
Indeed, given the complexity of embryogenesis and the multi-
Experience with chemicals that have the potential to induce ple mechanisms and target site of potential teratogens, it
developmental toxicity indicates that both laboratory animal was perhaps unrealistic to have expected a single test, or evena
testing and surveillance of the human population (i.e., epide- small battery, to accurately prescreen the activity of chemicals
miologic studies) as well as alert clinical evaluation after poten- in general.
tial exposure are all necessary to provide adequate public health An exception to the poor acceptance of alternate tests for
protection. Laboratory animal investigations are guided by prescreening for developmental toxicity is the Chernoft/
both regulatory requirements for drug or chemical marketing Kavlock in vivo test. In this test, pregnant females are exposed
and the need to understand mechanisms of toxicity. during the period of major organogenesis to a limited number
of dosage levels near those inducing maternal toxicity, and
offspring are evaluated over a brief neonatal period for external
Regulatory Guidelines for In Vivo Testing malformations, growth, and viability. It has proven reliable
New and internationally accepted testing protocols rely on the over a large number of chemical agents and classes.
investigator to meet the primary goal of detecting and bringing
to light any indication of toxicity to reproduction. Key elements
of various tests are provided in Table 10-4. The general goal of Epidemiology
these studies is to identify the NOAEL, which is the highest dos- Reproductive epidemiology studies associations between
age level that does not produce a significant increase in adverse specific exposures of the father or pregnant woman and her
effects in the offspring or juvenile animals. These NOAELs are conceptus and the outcome of pregnancy. The likelihood of
158 UNIT 3 Nonorgan-Directed Toxicity

TABLE 10-4 Summary of in vivo regulatory protocol guidelines for evaluation of developmental toxicity.
Study Exposure End Points Covered ae Comments =

Segment I: fertility Males: 10 weeks prior Gamete development, fertility, pre and Assesses reproductive capabilities of male
and general to mating postimplantation viability, parturition, and female following exposure over one
reproduction study Females: 2 weeks prior lactation complete spermatogenic cycle or several
to mating estrous cycles

Segment Il: Implantation (or mating) Viability, weight, and morphology Shorter exposure to prevent maternal
teratogenicity test through end of (external, visceral, and skeletal) of metabolic adaptation and to provide
organogenesis (or term) conceptuses just prior to birth high exposure to the embryo during
gastrulation and organogenesis. Earlier
dosing option for bioaccumulative agents
or those impacting maternal nutrition. Later
dosing option covers male reproductive
tract development and fetal growth and
maturation

Segment Ill: Last trimester of pregnancy Postnatal survival, growth, and Intended to observe effects on development
perinatal study through lactation external morphology of major organ functional competence
during the perinatal period, and thus may
be relatively more sensitive to adverse
effects at this time

ICH 4.1.1: Males: 4 weeks prior Males: Reproductive organ weights Improved assessment of male reproductive
fertility protocol to mating and histology, sperm counts, and end points; shorter treatment duration than
Females: 2 weeks prior motility Segment! ,
to mating Females: viability of conceptuses
at midpregnancy or later

ICH 4.1.2: effects Implantation through end Relative toxicity to pregnant versus
on prenatal of lactation nonpregnant female; postnatal
and postnatal viability, growth, development, and
development, functional deficits (including behavior,
including maternal maturation, and reproduction)
function

ICH 4.1.3: effects Implantation through end Viability and morphology (external, Similar to Segment II study. Usually conducted
on embryo/fetal of organogenesis visceral, and skeletal) of fetuses just in two species (rodent and nonrodent)
development prior to birth

OECD 414: prenatal Implantation (or mating) Viability and morphology (external, Similar to Segment II study. Usually conducted
developmental through day prior to visceral, and skeletal) of fetuses just in two species (rodent and nonrodent)
cesarean section prior to birth

linking a particular exposure with a series of case reports denominator is the number of live births rather than total
increases with the rarity of the defect, the rarity of the exposure pregnancies.
in the population, a small source population, a short time span Other issues particularly relevant to reproductive epidemi-
for study, and biological plausibility for the association. In ology include homogeneity, recording proficiency, and con-
other situations, such as occurred with ethanol and valproic founding. Homogeneity refers to the fact that a particular
acid, associations are sought through either a case-control or a outcome may be described differently by various recording
cohort approach. Both approaches require accurate ascertain- units and that there can be multiple pathogenetic origins
ment of abnormal outcomes and exposures, and a large enough for a given specific outcome. Recording difficulties relate to
effect and study population to detect an elevated risk. Another inconsistencies of definitions and nomenclature, and to
challenge to epidemiologists is the high percentage of human difficulties in ascertaining or recalling outcomes as well as
pregnancy failures related to a particular exposure that may go exposures. For example, birth weights are usually accurately
undetected in the general population. With the availability of determined and recalled, but spontaneous abortions and
prenatal diagnostic procedures, additional pregnancies ofmal- certain malformations may not be. Last, confounding by
formed embryos (particularly neural tube defects) are elec- factors such as maternal age and parity, dietary factors, dis-
tively aborted. Thus, the incidence of abnormal outcomes at eases and drug usage, and social characteristics must be con-
birth may not reflect the true rate of abnormalities, and the term sidered in order to control for variables that affect both
prevalence, rather than incidence, is preferred when the exposure and outcome.
 CHAPTER 10 Developmental Toxicology

TABLE 10-5 Brief survey of alternative test methodologies for developmental toxicity.
—— Brief Description and End Points Evaluated
Mouse ovarian tumor Labeled mouse ovarian tumor cells added to culture dishes with concanavalin
A-coated disks for 20 min. End Point is inhibition of attachment of cells
to disks

Human embryonic palatal Human embryonic palatal mesenchyme cell line grown in attached culture.
mesenchyme Cell number assessed after 3 days

Micromass culture Midbrain or limb bud cells dissociated from rat embryos and grown in
micromass culture for 5 days. Cell proliferation and biochemical markers of
differentiation assessed

Mouse embryonic stem cell Mouse embryonic stem cells and 373 cells in 96-well plates assessed for
test (EST) viability after 3 and 5 days. Embryonic stem cells grown for 3 days in
hanging drops form embryoid bodies which are plated and examined after
10 days for differentiation into cardiocytes

Chick embryo neural retina Neural retinas of day 6.5 chick embryos dissociated and grown in rotating
cell culture suspension culture for 7 days. End points include cellular aggregation,
growth, differentiation, and biochemical markers

Drosophila Fly larvae grown from egg disposition through hatching of adults. Adult flies
examined for specific structural defects (bent bristles and notched wing)

Hydra Hydra attenuata cells are aggregated to form an “artificial embryo”

and allowed to regenerate. Dose response compared to that for adult
Hydra toxicity

FETAX Midblastula stage Xenopus embryos exposed for 96 h and evaluated for
viability, growth, and morphology

Rodent whole embryo Postimplantation rodent embryos grown in vitro for up to 2 days and
culture evaluated for growth and development

Zebrafish Zebrafish eggs or blastulae exposed to chemical in water (can be in multiwell

plates) for up to 4 days and evaluated for growth, development, and (in
some cases) gene expression

Chernoff/Kavlock assay Pregnant mice or rats exposed during organogenesis and allowed to deliver.

Epidemiologic studies of abnormal reproductive outcomes Concordance of Data

are usually undertaken with three objectives in mind: the
Studies of the similarity of responses of laboratory animals and
first is scientific research into the causes of abnormal birth
humans for developmental toxicants support the assumption
outcomes and usually involves analysis of case reports or clus-
that results from laboratory tests are predictive of potential
ters; the second objective is prevention and is targeted at
human effects. Concordance is strongest when there are positive
broader surveillance of trends by birth defect registries
data from more than one test species. Humans tend to be more
around the world; and the last objective is informing the
sensitive to developmental toxicants than is the most sensitive
public and providing assurance. Cohort studies, with their
test species.
prospective exposure assessment and ability to monitor both
adverse and beneficial outcomes, may be the most method-
ologically robust approach to identifying human develop- Elements of Risk Assessment
mental toxicants. Extrapolation of animal test data for developmental toxicity
Information on differential genetic susceptibility to birth follows two basic directions, one for drugs where exposure is
defects continues to accrue. This new knowledge promises to voluntary and usually to high dosages and the other for
elucidate links between genetics and disease susceptibility. environmental agents where exposure is generally involuntary
Understanding the genetic basis of vulnerability to environ- and to low levels. For drugs, a use-in-pregnancy rating is
mentally induced birth defects will allow more inclusive risk utilized, wherein the letters A, B, C, D, and X are used to
assessments and a better appreciation of the mechanisms of classify the evidence that a chemical poses a risk to the human
action of developmental toxicants. conceptus. For example, drugs are placed in category A if
160 UNIT 3 Nonorgan-Directed Toxicity

adequate, well-controlled studies in pregnant humans have TABLE 10-6 Seventeen intercellular signaling
failed to demonstrate a risk, and in category X (contraindicated pathways used in development by most metazoans.
for pregnancy) if studies in animals or humans, or investiga-
tional or postmarketing reports, have shown fetal risk that
clearly outweighs any possible benefit to the patient. The default
category C (risks cannot be ruled out) is assigned when there Before organogenesis; 1. Wingless-Int pathway
later for growth 2. Transforming growth factor 3 pathway
is a lack of human studies and animal studies are either lacking 3. Hedgehog pathway
and tissue renewal
or are positive for fetal risk, but the benefits may justify the 4. Receptor tyrosine kinase pathway
potential risk. Categories B and D represent areas of relatively 5. Notch-Delta pathway
lesser or greater concern for risk, respectively. 6. Cytokine pathway (STAT pathway)
For environmental agents, the purpose of the risk assess- Organogenesis and 7. \Interleukin-1-toll nuclear factor-kappa
ment process for developmental toxicity is generally to define cytodifferentiation; B pathway
the dose, route, timing, and duration of exposure that induces later for growth 8. Nuclear hormone receptor pathway
effects at the lowest level in the most relevant laboratory animal and tissue renewal 9. Apoptosis pathway
10. Receptor phosphotyrosine phosphatase
model. The exposure associated with this “critical effect” is pathway
then subjected to a variety of safety or uncertainty factors in
order to derive an exposure level for humans that is presumed Larval and adult 11. Receptor guanylate cyclase pathway
physiology 12. Nitric oxide receptor pathway
to be relatively safe. In the absence of definitive animal test 13. G-protein-coupled receptor (large
data, certain default assumptions are generally made: G proteins) pathway
1. An agent that produces an adverse developmental effect 14. Integrin pathway
in experimental animals will potentially pose a hazard to 15. Cadherin pathway
16. Gap junction pathway
humans following sufficient exposure during development. 17. Ligand-gatedication channel pathway
2. All four manifestations of developmental toxicity (death,
structural abnormalities, growth alterations, and func-
tional deficits) are of concern.
3. The specific types of developmental effects seen in animal
studies are not necessarily the same as those that may be of these and other animal species, as well as in humans
produced in humans. (Table 10-6). The conserved nature of these key pathways
4, The most appropriate species is used to estimate human provides a strong scientific rationale for using these animal
risk when data are available (in the absence of such data, models to advantage for developmental toxicology. These
the most sensitive species is appropriate). organisms have well-known genetics, embryology, and rapid
5. In general, a threshold is assumed for the dose-response generation times, and they are also amenable to genetic manip-
curve for agents that produce developmental toxicity. ulation to enhance the sensitivity of specific developmental
Two approaches to aid defining developmental risk include pathways or to incorporate human genes to answer questions of
the benchmark-dose approach and biologically based dose- interspecies extrapolation.
response modeling. The use of uncertainty factors applied to Increased understanding of human genetic polymorphisms
an experimentally derived NOAEL to arrive at a presumed safe and their contribution to susceptibility to birth defects, use of
level of human exposure assumes that a threshold for develop- sensitized animal models for high- to low-dose extrapolation,
mental toxicity exists. The available USEPA’s Benchmark Dose use of stress/checkpoint pathways as indicators of develop-
Software is helping to make this approach a method of choice mental toxicity, implementation of bioinformatic systems to
for many risk assessment organizations. The biologically based improve data archival and retrieval, and increased multidisci-
dose-response model integrates pharmacokinetic data on plinary education and research on the causes of birth defects
tissue dosimetry with molecular, cellular and tissue response, will aid assessment of the developmental risk of toxicants.
and developmental toxicity.

BIBLIOGRAPHY
PATHWAYS TO THE FUTURE Harris C, Hansen JM: Developmental Toxicology: Methods and
Protocols. New York: Humana Press, 2012.
There are several mechanisms of normal development that are
Hood RD: Developmental and Reproductive Toxicology: A Practical
conserved in diverse animals, including the fruit fly, round- Approach. New York: Informa Healthcare, 2012.
worm, zebrafish, frog, chick, and mouse. Seventeen conserved Robinson JF, Pennings JL, Piersma AH: A review of toxicogenomic
intercellular signaling pathways are described that are used approaches in developmental toxicology. Methods Mol Biol
repeatedly at different times and locations during development 889:347-371, 2012.
 CHAPTER 10 Developmental Toxicology 161

QUESTIONS

Diethylstilbestrol (DES): Which of the following stages of the cell cycle are
a. was used to treat morning sickness from the 1940s to important in monitoring DNA damage and inhibiting
the 1970s. progression of the cell cycle?
b. was found to affect only female offspring in exposed a. G,-S, anaphase, M-G,.
pregnancies. G,-S, S, G,-M.
c. greatly affects the development of the fetal brain. S, prophase, G,.
d. exposure increases the risk of clear cell adenocarci- G,-M, prophase.
noma of the vagina. ce
aos
M-G,, anaphase.
e. is now used to treat leprosy patients.
Which of the following molecules is NOT important in
Early (prenatal) exposure to which of the following determining the ultimate outcome of embryonal DNA
teratogens is most often characterized by craniofacial damage?
dysmorphism? aa! Poo:
a. thalidomide. b. Bax.
b. retinol. c. Bel-2.
c. ethanol. d. c-Myc.
d. tobacco smoke. e. NE-KB.
e. diethylstilbestrol (DES).
Which of the following is NOT a physiologic response
The nervous system is derived from which of the to pregnancy?
following germ layers? a. increased cardiac output.
ectoderm. increased blood volume.
mesoderm. increased peripheral vascular resistance.
epidermal placodes. decreased plasma proteins.
paraxial mesoderm. semo increased extracellular space.
Cae endoderm.
Oe
Eas
Ce
All of the following statements are true EXCEPT:
Toxin exposure during which of the following periods is a. Offspring of white mothers have a higher incidence
likely to have the LEAST toxic effect on the developing of cleft lip or palate than do black mothers, after
fetus? adjusting for paternal race.
gastrulation. b. Cytomegalovirus (CMV) is a common viral cause of
organogenesis. birth defects.
preimplantation. c. Folate supplementation during pregnancy decreases
third trimester. the risk of neural tube defects.
Go
oe
Sp first trimester. d. Cigarette smoke and ethanol are both toxic to the
placenta.
Regarding prenatal teratogen exposure, which of the e. In humans, there is a negative correlation between
following statements is FALSE? stress and low birth weight.
a. Major effects include growth retardation and
malformations. 10. Which of the following is NOT a mechanism involving
b. Exposure to teratogens during critical developmental the endocrine system by which chemicals induce devel-
periods will have more severe effects on the fetus. opmental toxicity?
c. There is considered to be a toxin level threshold a. acting as steroid hormone receptor ligands.
below which the fetus is capable of repairing itself. b. disrupting normal function of steroid hormone
d. The immune system of the fetus is primitive, so the metabolizing enzymes.
fetus has little to no ability to fight off chemicals and c. disturbing the release of hormones from the
repair itself. hypothalamus.
e. Embryo lethality becomes more likely as the toxic d. disturbing the release of hormones from the pituitary
dose is increased. gland.
e. elimination of natural hormones.
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 UNIT 4 TARGET ORGAN TOXICITY

6/01 ipRR, es Se rd oe 4

Toxic Responses
of the Blood
John C. Bloom, Andrew E. Schade, and John T. Brandt

BLOOD AS A TARGET ORGAN LEUKEMOGENESIS AS A TOXIC RESPONSE

Human Leukemias
HEMATOPOIESIS
Mechanisms of Toxic Leukemogenesis
TOXICOLOGY OF THE ERYTHRON Leukemogenic Agents

The Erythrocyte TOXICOLOGY OF PLATELETS AND HEMOSTASIS

Alterations in Red Cell Production Toxic Effects on Platelets
Alterations in the Respiratory Function of Hemoglobin The Thrombocyte
Homotropic Effects Thrombocytopenia
Heterotropic Effects Toxic Effects on Platelet Function
Alterations in Erythrocyte Survival Toxic Effects on Fibrin Clot Formation
Nonimmune Hemolytic Anemia Coagulation
Immune Hemolytic Anemia Decreased Synthesis of Coagulation Proteins
TOXICOLOGY OF THE LEUKON Increased Clearance of Coagulation Factors
Toxicology of Agents Used to Modulate Hemostasis
Components of Blood Leukocytes
Oral Anticoagulants
Evaluation of Granulocytes
Heparin
Toxic Effects on Granulocytes
Fibrinolytic Agents
Effects on Proliferation
Inhibitors of Fibrinolysis
Effects on Function
Idiosyncratic Toxic Neutropenia RISK ASSESSMENT
Mechanisms ofToxic Neutropenia
164 UNIT 4 Target Organ Toxicity

= Hematotoxicology is the study of adverse effects of exog- « Leukemias are proliferative disorders of hematopoietic
enous chemicals on blood and blood-forming tissues. tissue that originate from individual bone marrow cells.
» Direct or indirect damage to blood cells and their precur- = Xenobiotic-induced thrombocytopenia may result from
sors includes tissue hypoxia, hemorrhage, and infection. increased platelet destruction or decreased platelet pro-
= Xenobiotic-induced aplastic anemia is a life-threatening duction, which lead to decreased platelet aggregation
disorder characterized by peripheral blood pancytope- and bleeding disorders.
nia, reticulocytopenia, and bone marrow hypoplasia. = Blood coagulation is a complex process involving a num-
s Idiosyncratic xenobiotic-induced agranulocytosis may ber of proteins whose synthesis and function can be
involve a sudden depletion of circulating neutrophils altered by many xenobiotics.
concomitant with exposure that persists as long as the
agent or its metabolites are in the circulation.

BLOOD AS ATARGET ORGAN nodes, while the primary location in adults is the bone marrow
of the axial skeleton and’ proximal limbs. Two types of bone
Hematotoxicology is the study of adverse effects of exogenous marrow exist: (1) red marrow, which is active in hematopoiesis,
chemicals on blood and blood-forming tissues. The delivery of and (2) yellow marrow, which is called so because it turns fatty
oxygen to tissues throughout the body, maintaining vascular as it ceases participation in hematopoiesis.
integrity and providing the many affector and effector immune Whereas the central function of bone marrow is hematopoi-
functions necessary for host defense, requires a prodigious pro- esis and lymphopoiesis (production of a subset of white blood
liferative and regenerative capacity. Each of the various blood cells), bone marrow is also one of the sites of the mononuclear
cells (erythrocytes, granulocytes, and platelets) is produced at phagocyte system (MPS), contributing monocytes that differ-
a rate of approximately 1-3 million/s in a healthy adult; this entiate into phagocytic cells in other tissues. A complex inter-
characteristic makes hematopoietic tissue a particularly sensi- play of developing cells with stromal (connective tissue) cells,
tive target for cytoreductive or antimitotic agents, such as those extracellular matrix components, and cytokines makes up the
used to treat cancer, infection, and immune-mediated disor- hematopoietic inductive microenvironment. Each lineage is sup-
ders. This tissue is also susceptible to secondary effects of toxic ported within a specific niche, and an array of cytokines and
agents that affect the supply of nutrients, such as iron; the clear- chemokines directs a particular progenitor cell to the appro-
ance of toxins and metabolites, such as urea; or the production priate niche.
of vital growth factors, such as erythropoietin (EPO) and gran-
ulocyte colony-stimulating factor (G-CSF). The consequences
of direct or indirect damage to blood cells and their precursors TOXICOLOGY OF THE ERYTHRON
are predictable and potentially life-threatening. They include
hypoxia, hemorrhage, and infection. The Erythrocyte
Hematotoxicity may be regarded as primary toxicity, where
Erythrocytes (red blood cells [RBCs]) comprise 40% to
one or more blood components are directly affected, or sec-
45% of the circulating blood volume and serve as the principal
ondary, where the toxic effect is a consequence of other tissue
vehicle for transportation of oxygen from the lungs to periph-
injury or systemic disturbances. Primary toxicity is regarded
eral tissues and of carbon dioxide from tissues to the lung.
as among the serious effects of xenobiotics, particularly drugs.
Erythrocytes are also involved as a carrier and/or reservoir for
Secondary toxicity is exceedingly common, due to the propen-
drugs and toxins. Xenobiotics may affect the production, func-
sity of blood cells to reflect various local and systemic effects of
tion, and survival of erythrocytes. These effects most frequently
toxicants on other tissues.
manifest as a change in the circulating red cell mass, usually
resulting in a decrease (anemia). Occasionally, agents that affect
HEMATOPOIESIS the oxygen affinity of hemoglobin lead to an increase in the red
cell mass (erythrocytosis), but this is distinctly less common.
The production of blood cells, or hematopoiesis, is a highly Shifts in plasma volume can alter the relative concentration of
regulated sequence of events by which blood cell precursors erythrocytes (and, therefore, hemoglobin concentration) and
proliferate and differentiate. The location of hematopoiesis can be confused with true anemia or erythrocytosis.
changes throughout one’s life. For instance, fetal hematopoiesis Two general mechanisms that lead to true anemia are either
is located in the liver, spleen, bone marrow, thymus, and lymph decreased production or increased destruction of erythrocytes.
 CHAPTER 11 Toxic Responses of the Blood 165

|! Hemoglobin

Fett

Coproporphyrinogen Ill

/ Hydroxymethylbilane —

Glycine+ Succinyl-CoA LL
ILL
ET
EAL
EET
ER
OSS
ESOS
SEA
SETI
SELES
TES
TITIES
OOD
LE
DELLE
ELE
IEC
ES
TEESE
ALE
LOSER
LEEEETESS
SEE
EDS
ELA
LEE
SRE

FIGURE 11-1 Hemeand hemoglobin synthesis. The initial step in heme synthesis is the mitochondria synthesis of 6-aminolevulinic acid,
a step that is commonly affected by xenobiotics, including lead. Ferrochelatase catalyzes the incorporation of ferrous iron into the tetrapyrrole
protoporphyrin IX. Inhibition of the synthetic pathway leading to protoporphyrin IX, as occurs in the sideroblastic anemias, can cause an imbalance
between iron concentration and ferrochelatase activity, resulting in iron deposition within mitochondria. Mitochondrial accumulation of iron is the
hallmark lesion of the sideroblastic anemias.

The usual parameters of a complete blood count (CBC), includ- between a- and (3-chain production is the basis of congenital
ing RBC count, hemoglobin concentration, and hematocrit thalassemia syndromes and results in decreased hemoglo-
(also referred to as packed cell volume [PCV]) can establish the bin production and microcytosis. Xenobiotics can affect glo-
presence of anemia. Two additional parameters that are help- bin chain synthesis and alter the composition of hemoglobin
ful in classifying an anemia are the mean corpuscular volume within erythrocytes.
(MCV) and the reticulocyte count. Increased destruction is Synthesis of heme requires incorporation ofiron into a por-
usually accompanied by an increase in reticulocytes (young phyrin ring (Figure 11-1). Iron deficiency is usually the result
erythrocytes containing residual RNA). Two related processes of dietary deficiency or increased blood loss. Any drug that
contribute to the increased number of reticulocytes in humans. contributes to blood loss may potentiate the risk of developing
First, increased destruction is accompanied by a compensatory iron deficiency anemia. Defects in the synthesis of porphyrin
increase in bone marrow production, with an increase in the ring of heme can lead to sideroblastic anemia, with its charac-
number of cells being released from the marrow into the cir- teristic accumulation of iron in bone marrow erythroblasts.
culation. Second, during compensatory erythroid hyperplasia, The accumulated iron precipitates within mitochondria caus-
the marrow releases reticulocytes earlier in their life span and ing injury. A number ofxenobiotics (Table 11-1) interfere with
thus the reticulocytes persist for a longer period in the periph-
eral blood.

Alterations in Red Cell Production TABLE 11-1 Xenobiotics associated with

sideroblastic anemia.
Erythrocyte production is a continuous process that is depen-
dent on frequent cell division and a high rate of hemoglobin Chloramphenicol Isoniazid
synthesis. Adult hemoglobin (hemoglobin A) is a tetramer
Copper chelation/deficiency Lead intoxication
composed of two a-globin chains and two (-globin chains,
each with a heme residue. . Cycloserine Pyrazinamide
Abnormalities that lead to decreased hemoglobin synthesis
Ethanol — Zinc intoxication
are relatively common (e.g., iron deficiency). An imbalance
166 UNIT 4 Target Organ Toxicity

one or more steps in erythroblast heme synthesis and result in TABLE 11-2 Xenobiotics associated with
sideroblastic anemia. megaloblastic anemia.
Hematopoiesis requires active DNA synthesis and frequent
mitoses. Folate and vitamin B,, are necessary to maintain syn-
B,, Deficiency FolateDeficiency __ .
thesis of thymidine for incorporation into DNA. Deficiency Antimetabolites Neomycin
of folate and/or vitamin B,, results in megaloblastic anemia,
p-Aminosalicylic acid Omeprazole
a result of improper cell division. Xenobiotics that may con-
tribute to a deficiency of vitamin B,, and/or folate are listed in Carbamazepine Phenobarbital
Table 11-2.
Cholestyramine Phenytoin
Many antiproliferative agents used in the treatment of
malignancy predictably inhibit hematopoiesis, including Colchicine Primidone
erythropoiesis. The resulting bone marrow toxicity may be
Ethanol Sulfasalazine
dose-limiting. Drugs, such as amifostine, have been devel-
oped that may help protect against the marrow toxicity of Fish tapeworm Triamterine
these agents.
Hemodialysis Zidovudine
Drug-induced aplastic anemia may represent either a
predictable or idiosyncratic reaction to a xenobiotic. This Malabsorption syndromes
life-threatening disorder is characterized by peripheral blood
pancytopenia, reticulocytopenia, and bone marrow hypo-
plasia. Agents associated with the development of aplastic
anemia are listed in Table 11-3. Pure red cell aplasia is a syn-
Alterations in the Respiratory
drome that may be due to genetic defects, infection, immune- Function of Hemoglobin —
mediated injury, myelodysplasia, drugs, or other toxicants, Hemoglobin transports oxygen and carbon dioxide between
in which the decrease in marrow production is limited to the the lungs and tissues. The individual globin units show
erythroid lineage. cooperativity in the binding of oxygen, resulting in the

TABLE 11-3 Drugs and chemicals associated with the development of aplastic anemia.

Allopurinol Diclofenac Penicillin

Amphotericin B Dinitrophenol Phenylbutazone

Azidothymidine Ethosuximide Potassium perchlorate

Benzene Felbamate Propylthiouracil

Bismuth Gold Pyrimethamine

Carbamazepine Indomethacin Quinacrine

Carbimazole Isoniazid Streptomycin

Carbon tetrachloride Mefloquine Sulfamethoxypyridazine

Carbutamide Mepazine Sulfisoxazole

Chloramphenicol Meprobamate Sulfonamides

Chlordane Mercury Tetracycline

Chlordiazepoxide Methazolamide Thiocyanate

Chlorphenothane Methicillin Ticlopidine

Chlorpropamide Methylphenylethylhydantoin Tolbutamide

Chlorpromazine Methylmercaptoimidazole Trifluoroperazine

Chlortetracycline Metolazone Trimethadione

Cimetidine Organic arsenicals Tripelennamine

D-Penicillamine Parathion
SOIC ANNONA NENTS
 CHAPTER 11 Toxic Responses ofthe Blood 167

familiar sigmoid shape to the oxygen dissociation curve Heterotropic Effects— There are three major heterotropic
(Figure 11-2). (extrinsic) effectors of hemoglobin function: pH, erythro-
cyte 2,3-bisphosphoglycerate (2,3-BPG, formerly desig-
Homotropic Effects—One of the most important homo- nated 2,3-diphosphoglycerate [2,3-DPG]) concentration,
tropic (intrinsic) properties of oxyhemoglobin is the slow and temperature. A decrease in pH (e.g., lactic acid and car-
but consistent oxidation of heme iron to the ferric state to bon dioxide) lowers the afhinity of hemoglobin for oxygen
form methemoglobin, which is not capable of binding and causing a right shift in the oxygen dissociation curve and
transporting oxygen. The presence of methemoglobin in a facilitating the delivery of oxygen to tissues (Figure 11-2).
hemoglobin tetramer results in a leftward shift of the oxy- As bicarbonate and carbon dioxide equilibrate in the lung,
gen dissociation curve (Figure 11-2). The combination of the hydrogen ion concentration decreases, which results in
decreased oxygen content and increased affinity may sig- increased affinity of hemoglobin for oxygen and facilitated
nificantly impair delivery of oxygen to tissues, as the oxy- oxygen uptake.
gen will not be readily released from hemoglobin in the Binding of 2,3-BPG to deoxyhemoglobin results in reduced
periphery. oxygen affinity (a shift to the right of the oxygen dissociation
The normal erythrocyte has metabolic mechanisms for curve), which promotes oxygen delivery to peripheral tissues.
reducing heme iron back to the ferrous state. Failure of these The conformational change induced by binding of oxygen to
control mechanisms leads to increased levels of methemo- hemoglobin alters the binding site for 2,3-BPG and results in
globin, or methemoglobinemia. Various chemicals that cause release of 2,3-BPG from hemoglobin. This facilitates uptake
methemoglobinemia are shown in Table 11-4. Most patients of more oxygen in the lungs for delivery to tissues. The con-
tolerate low levels (< 10%) of methemoglobin without clinical centration of 2,3-BPG increases whenever there is tissue
symptoms. Higher levels lead to tissue hypoxemia that is even- hypoxemia but may decrease in the presence of acidosis or
tually fatal. hypophosphatemia.
The oxygen affinity of hemoglobin decreases as the body tem-
perature increases. This facilitates delivery of oxygen to tissues
during periods of extreme exercise and febrile illnesses asso-
Increased O5 ciated with increased temperature. Correspondingly, oxygen
affinity affinity increases and delivery decreases during hypothermia.

Decreased O>
affinity
TABLE 11-4 Environmental and therapeutic agents
associated with methemoglobinemia.

Aminobenzenes Nitrobenzenes

Amy| nitrate Nitroethane

(%)
saturation
Oxygen
Aniline dyes and aniline Nitvoahveerin
derivatives gy
— PO, in venous blood
Benzocaine Nitrotoluenes

Beta-naphthol disulfonate ortho-Toluidine

Oxygen tension (mm Hg)
Butyl nitrite para-Toluidine
FIGURE 11-2 Hemoglobin-oxygen dissociation curves. The
Dapsone Potassium chlorate
normal oxygen dissociation curve (solid line) has a sigmoid shape
due to the cooperative interaction between the four globin chains Flutamide Prilocaine
in the hemoglobin molecule. Fully deoxygenated hemoglobin has
a relatively low affinity for oxygen. Interaction of oxygen with one Gasoline additives Primaquine
heme-iron moiety induces a conformational change in that globin
Isobutyl nitrite Phenacetin
chain. Through surface interactions, that conformational change
affects the other globin chains, causing a conformational change Lidocaine Phenazopyridine
in all of the globin chains that increases their affinity for oxygen.
Methylene blue Quinones
Homotropic and heterotropic parameters also affect the affinity of
hemoglobin for oxygen. An increase in oxygen affinity results in a shift Nitrates Silver nitrate
to the left in the oxygen dissociation curve. Such a shift may decrease
oxygen delivery to the tissues. A decrease in oxygen affinity results in Nitric oxide Sulfonamide
a shift to the right in the oxygen dissociation curve, facilitating oxygen
Nitrites Trinitrotoluene
delivery to the tissues.
168 UNIT 4 Target Organ Toxicity

The respiratory function of hemoglobin may also be impaired Infectious Diseases—Infectious diseases may be associated
by blocking the ligand binding site with other substances. with significant hemolysis, by either direct effect on the erythro-
Carbon monoxide has a relatively low rate of association with cyte or an immune-mediated hemolytic process. Erythrocytes
deoxyhemoglobin but shows high affinity once bound, and parasitized in malaria and babesiosis may undergo destruction.
causes a left shift in the oxygen dissociation curve, further Clostridial infections are associated with release of hemolytic
compromising oxygen delivery to the tissues. Nitric oxide, an toxins that enter the circulation and lyse erythrocytes.
important vasodilator that modulates vascular tone, binds
avidly to heme iron. Erythrocytes can influence the availability Oxidative Hemolysis—Molecular oxygen is a reactive and
of nitric oxide in parts of the circulation because the nitric oxide potentially toxic chemical species; consequently, the nor-
is bound to erythrocyte hemoglobin. mal respiratory function of erythrocytes generates oxidative
stress on a continuous basis. There are several mechanisms
that protect against oxidative injury in erythrocytes including
Alterations in Erythrocyte Survival NADH-diaphorase, superoxide dismutase, catalase, and the
The normal survival of erythrocytes in the circulation is about glutathione pathway.
120 days. During this period, erythrocytes are exposed to Xenobiotics capable of inducing oxidative injury in eryth-
a various oxidative injuries and must negotiate the tortuous rocytes are listed in Table 11-5. These agents appear to
passages of the microcirculation and the spleen. This requires potentiate the normal redox reactions and are capable of over-
a deformable cell membrane and energy to maintain the whelming the usual protective mechanisms. The interaction
sodium-potassium gradients and repair mechanisms. Very between these xenobiotics and hemoglobin leads to the for-
little protein synthesis occurs during this time, as erythrocytes mation of free radicals that denature critical proteins, includ-
are anucleate when they enter the circulation and residual ing hemoglobin, thiol-dependent enzymes, and components
mRNA is rapidly lost over the first 1 to 2 days in the circu- of the erythrocyte membrane. Significant oxidative injury
lation. Consequently, senescence occurs over time until the usually occurs when the concentration of the xenobiotic is
aged erythrocytes are removed by the spleen, where the iron high enough to overcome the normal protective mechanisms,
is recovered for reutilization in heme synthesis. Any insult that or, more commonly, when there is an underlying defect in the
increases oxidative injury, decreases metabolism, or alters the protective mechanisms.
membrane may cause a decrease in erythrocyte concentration The most common enzyme defect associated with oxidative
and a corresponding anemia. hemolysis is glucose-6-phosphate dehydrogenase (G-6-PD)
deficiency, a sex-linked disorder characterized by diminished
Nonimmune Hemolytic Anemia G-6-PD activity. It is often clinically asymptomatic until the
Microangiopathic Anemias—Intravascular fragmentation erythrocytes are exposed to oxidative stress from the host
of erythrocytes gives rise to the microangiopathic hemolytic response to infection or exposure to xenobiotics.
anemias. The hallmark ofthis process is the presence ofschis-
tocytes (fragmented RBCs) in peripheral blood. The forma- Nonoxidative Chemical-induced Hemolysis—Exposure
tion of fibrin strands in the microcirculation is a common to some xenobiotics is associated with hemolysis without
mechanism for RBC fragmentation. This may occur in the
setting of disseminated intravascular coagulation, sepsis,
hemolytic-uremic syndrome (HUS), and thrombotic throm- TABLE 11-5 Xenobiotics associated with oxidative
bocytopenic purpura (TTP). The erythrocytes are essentially injury.
sliced into fragments by the fibrin strands that extend across
Acetanilide Phenacetin
the vascular lumen and impede the flow of erythrocytes
through the vasculature. Excessive fragmentation can also Aminosalicylic acid Phenol
be seen in the presence of abnormal vasculature. The high
shear associated with malignant hypertension may also lead Chlorates Phenylhydrazine

to RBC fragmentation. Dapsone Primaquine

Other Mechanical Injuries—RBC destruction can also Furazolidone Phenazopyridine

occur as a result of mechanical stress. For instance, march Hydroxylamine Sodium sulfoxone
hemoglobinuria is an episodic anemia resulting from
mechanical trauma to the feet from prolonged activity. Methylene blue Sulfamethoxypyridazine
Major thermal burns are also associated with a hemolytic Nalidixic acid Sulfanilamide
process. The erythrocyte membrane becomes unstable as
temperature increases to the point where small RBC frag- Naphthalene Sulfasalazine

ments break off and the membrane reseals. Consequently, Nitrofurantoin Toluidine blue
these abnormal cell fragments are removed by the spleen,
Nitrobenzene
leading to anemia.
 CHAPTER 11 Toxic Responses of the Blood 169

significant oxidative injury. For example, inhalation of gaseous granulocytes may be seen in peripheral blood. In certain con-
arsenic hydride (arsine) can result in severe hemolysis, with ditions, neutrophils may show morphological changes indica-
anemia, jaundice, and hemoglobinuria. Several elements are tive of toxicity.
known to cause hemolysis in the absence of oxidative damage,
namely, lead, copper, and chromium. Additionally, significant
hemolysis may occur with biologic toxins found in insect and Toxic Effects on Granulocytes
snake venoms. Effects on Proliferation—The high rate of proliferation of
neutrophils makes their progenitor and precursor granulo-
immune Hemolytic Anemia—Immunologic destruction of cyte pool particularly susceptible to inhibitors of mitosis. Such
erythrocytes is mediated by the interaction of IgG or IgM anti- effects by cytotoxic drugs are generally nonspecific as they
bodies with antigens expressed on the surface of the erythro- similarly affect cells of the dermis, gastrointestinal tract, and
cyte. In the case of autoimmune hemolytic anemia, the antigens other rapidly dividing tissues. Agents that affect both neutro-
are intrinsic components of the patient’s own erythrocytes. phils and monocytes pose a greater risk for toxic sequelae, such
A number of mechanisms have been implicated in xenobi- as infection. Such effects tend to be dose-related, with mono-
otic-mediated antibody binding to erythrocytes. Some drugs, nuclear phagocyte recovery preceding neutrophil recovery.
of which penicillin is a prototype, appear to bind to the sur- Myelotoxicity is commonly seen with cytoreductive can-
face of the cell, with the “foreign” drug acting as a hapten and cer chemotherapy agents, which often act to inhibit DNA
eliciting an immune response. The antibodies that arise in synthesis or directly attack its integrity through the forma-
this type of response only bind to drug-coated erythrocytes. tion of DNA adducts or enzyme-mediated breaks. However,
Other drugs, of which quinidine is a prototype, bind to compo- this is changing, as more cancer cell-targeted, normal-tissue-
nents of the erythrocyte surface and induce a conformational sparing anticancer agents are being developed. The toxicity
change in one or more components of the membrane. A third associated with cytotoxic drugs, however, remains important
mechanism, for which @-methyldopa is a prototype, results in in that it is often dose-limiting (even with some of the newer
production ofadrug-induced autoantibody that cannot be dis- drugs) with serious manifestations that include febrile neu-
tinguished from the antibodies arising in idiopathic autoim- tropenia associated with life-threatening infections. While
mune hemolytic anemia. these drugs can be toxic to both resting and actively dividing
cells, nonproliferating cells such as metamyelocytes, bands,
and mature neutrophils are relatively resistant. Because stem
TOXICOLOGY OF THE LEUKON cells cycle slowly, they are minimally affected by a single
administration of a cytotoxic drug. Sustained exposure to
Components of Blood Leukocytes drugs affecting stem cells is believed to cause more prolonged
The leukon consists of leukocytes, or white blood cells, includ- myelosuppression.
ing granulocytes, which may be subdivided into neutrophils, Two innovations have had a dramatic impact on cancer
eosinophils, and basophils; monocytes; and lymphocytes. chemotherapy and the dose-limiting myelotoxicity associ-
Granulocytes and monocytes are nucleated ameboid cells that ated with these drugs: (1) the development of drugs with
are phagocytic. They play a central role in the inflammatory cancer cell-specific molecular targets that are relatively bone
response and host defense. Unlike the RBC, which resides marrow sparing, such as those that target aberrant growth
exclusively within blood, granulocytes and monocytes merely factor receptor signaling, apoptosis, angiogenesis, and other
pass through the blood on their way to extravascular tissues, metabolic, immune, inflammatory, and mutation-promoting
where they reside in large numbers. pathways that selectively advantage tumor cells, and (2) cotreat-
Granulocytes are defined by the characteristics of their cyto- ment with hematopoietic growth factors mitigates or success-
plasmic granules as they appear on a blood smear. Neutrophils, fully rescues patients from the effects of myelosuppression.
the largest component of blood leukocytes, are highly special- Cytokine-induced differentiation therapy of leukemias is
ized in the mediation of inflammation and the ingestion and another exciting treatment modality. The prospect of exagger-
destruction of pathogenic microorganisms. Eosinophils and ated pharmacology and off-target effects of these sophisticated
basophils modulate inflammation through the release of vari- interventions should provide the preclinical toxicologist and
ous mediators. oncologist with interesting hematotoxicologic challenges.

Effects on Function—While there are a variety of disorders

Evaluation of Granulocytes associated with defects in the parameters of neutrophil func-
In the blood, neutrophils are distributed between circulating tion discussed above, demonstrable in vivo effects associated
and marginated pools, which are of equal size in humans and with drugs and nontherapeutic chemicals are surprisingly few.
in constant equilibrium. A blood neutrophil count assesses Examples include ethanol and glucocorticoids, which impair
only the circulating pool, which remains remarkably constant phagocytosis and microbe ingestion. Iohexol and ioxaglate,
(1800 to 7500 uL~') in a healthy adult human. During inflam- components of radiographic contrast media, have also been
mation, an increased number of immature (non-segmented) reported to inhibit phagocytosis. Superoxide production,
170 UNIT 4 Target Organ Toxicity

required for microbial killing and chemotaxis, is reportedly the risk of infection is successfully managed during the leuko-
reduced in patients using parenteral heroin as well as in for- penic episodes.
mer opiate abusers on long-term methadone maintenance.
Chemotaxis is also impaired following treatment with zinc Mechanisms of Toxic Neutropenia—In immune-mediated
salts in antiacne preparations. neutropenia, antigen-antibody reactions lead to destruction
ofperipheral neutrophils, granulocyte precursors, or both. As
Idiosyncratic Toxic Neutropenia—Of greater concern are with RBCs, an immunogenic xenobiotic can act as a hapten,
agents that unexpectedly damage neutrophils and granulocyte where the agent must be physically present to cause cell dam-
precursors and induce agranulocytosis, which is character- age, or alternatively, may induce immunogenic cells to pro-
ized by a profound depletion in blood neutrophils to less than duce antineutrophil antibodies that do not require the drug to
500 pL~!. Such injury occurs in specifically conditioned indi- be present.
viduals, and is therefore termed idiosyncratic. Non-immune-mediated toxic neutropenia often shows a
Idiosyncratic xenobiotic-induced agranulocytosis may genetic predisposition. Direct damage may cause inhibition of
involve a sudden depletion of circulating neutrophils con- granulopoiesis or neutrophil function. Some studies suggest
comitant with exposure, which may persist as long as the agent that a buildup of toxic oxidants generated by leukocytes can
or its metabolites persist in the circulation. Hematopoietic result in neutrophil damage.
function is usually restored when the agent is detoxified or Examples of agents associated with immune and nonim-
excreted. Toxicants affecting uncommitted stem cells induce mune neutropenia/agranulocytosis are listed in Table 11-6.
total marrow failure, as seen in aplastic anemia. After agents
that affect more differentiated precursors, surviving uncom-
mitted stem cells eventually produce recovery, provided that
LEUKEMOGENESIS ASA
TOXIC RESPONSE
Human Leukemias
TABLE 11-6 Examples of toxicants that cause Leukemias are proliferative disorders of hematopoietic
immune and nonimmune idiopathic neutropenia. tissue that are monoclonal and originate from individual
bone marrow cells. Historically they have been classified
Drugs Associated with Drugs NotAssociated _ as myeloid or lymphoid, referring to the major lineages for
WBC Antibodies withWBC Antibodies _
erythrocytes, granulocytes, thrombocytes, or lymphocytes,
Aminopyrine Allopurinol respectively. Poorly differentiated phenotypes have been
designated as “acute,” including acute lymphoblastic leuke-
Ampicillin Ethambutol
mia (ALL) and acute myelogenous leukemia (AML), whereas
Aprindine Flurazepam well-differentiated ones are referred to as “chronic” leuke-
mias, which include chronic lymphocytic leukemia (CLL),
Azulfidine Hydrochlorothiazide
chronic myelogenous leukemia (CML), and the myelodys-
Chlorpropamide Isoniazide plastic syndromes (MDS).
There is considerable evidence supporting the notion that
Clozapine Phenothiazines
leukemogenesis is a multievent progression, which suggest
CPZ/phenothiazines Rifampicin that factors involved in the regulation of hematopoiesis also
influence neoplastic transformation. Such factors include
Dicloxacillin
cellular growth factors (cytokines), proto-oncogenes, and
Gold other growth-promoting genes, as well as additional genetic
and epigenetic factors that govern survival, proliferation, and
Levamisole
differentiation.
Lidocaine Secondary leukemia is a term used to describe patients with
AML or MDS who have a history of environmental, occupa-
Methimazole
tional, or therapeutic exposure to hematotoxins or radiation.
Metiamide It also includes patients with AML evolving from antecedent
myelodysplastic or other myeloid stem cell disorders. Therapy-
Phenytoin
related AML and MDS is a term applied to the former group;
Procainamide both are used to distinguish from features of AML that arise de
novo, Various cytogenetic findings have been associated with
Propylthiouracil
prognosis and response to therapy. It has been suggested that
Quinidine secondary leukemias be redefined as any leukemia with a spe-
cific cytogenic or molecular poor prognostic feature due to a
Tolbutamide
presumed predisposing factor.
 CHAPTER 11 Toxic Responses of the Blood Wl

Mechanisms of Toxic Leukemogenesis binding of von Willebrand factor (vWF) with the platelet
Ib/IX/V (GP Ib/IX/V) receptor complex. Activation ofa path-
AML is the dominant leukemia associated with drug or chemi-
way of several factors permits fibrinogen and other multivalent
cal exposure, followed by MDS. This represents a continuum of
adhesive molecules to form cross-links between nearby plate-
one toxic response that has been linked to cytogenetic abnor-
lets, resulting in platelet aggregation. Xenobiotics may inter-
malities, particularly the loss of all or part of chromosomes 5
fere with the platelet response by causing thrombocytopenia
and 7. Remarkably, the frequency of these deletions in patients
(low platelet levels) or interfering with platelet function.
who develop MDS and/or AML after treatment with alkylat-
ing or other antineoplastic agents ranges from 67% to 95%,
Thrombocytopenia—Like anemia, thrombocytopenia may
depending on the study. Some of these same changes have been
be due to decreased production or increased destruction of
observed in AML patients occupationally exposed to benzene,
platelets. Thrombocytopenia is a common side effect of inten-
who also show aneuploidy with a high frequency of involve-
sive chemotherapy, due to the predictable effect of antiprolifera-
ment of chromosome 7. The relatively low frequency of dele-
tive agents on hematopoietic precursors. Thrombocytopenia is
tions in chromosomes 5 and 7 in de novo as compared with
a clinically significant component of idiosyncratic xenobiotic-
secondary AML suggests that these cytogenetic markers can
induced aplastic anemia. Indeed, the initial manifestation of
be useful in discriminating between toxic exposures and other
aplastic anemia may be mucocutaneous bleeding secondary to
etiologies of this leukemia.
thrombocytopenia.
Exposure to xenobiotics may cause increased immune-
Leukemogenic Agents mediated platelet destruction through any one of the several
Most alkylating agents used in cancer chemotherapy can cause mechanisms. Penicillin is an example of a drug that functions
MDS and/or AML. Of the aromatic hydrocarbons, only ben- as a hapten, which is a small molecule that only produces a
zene has been proven to be leukemogenic. Treatment with specific immune response if bound to a protein carrier. The
the topoisomerase II inhibitors, etoposide and teniposide, can responding antibody then binds to the hapten on the plate-
induce AML. let surface, leading to removal of the antibody-coated platelet
Exposure to high-dose - or x-ray radiation has long been
from the circulation.
A second mechanism of immune thrombocytopenia is initi-
associated with ALL, AML, and CML, as demonstrated in sur-
ated by a change in a platelet membrane glycoprotein caused
vivors of the atom bombings of Nagasaki and Hiroshima. Less
by the xenobiotic. This elicits an antibody response, with the
clear is the association of these diseases with low-dose radia-
responding antibody binding to this altered platelet antigen in
tion secondary to fallout or diagnostic radiographs. Other
controversial agents include 1,3-butadiene, nonionizing radia- the presence of drug, resulting in removal of the platelet from
the circulation by the mononuclear phagocytic system.
tion (electromagnetic, microwave, infrared, visible, and the
Thrombocytopenia is an uncommon, but serious, compli-
high end of the ultraviolet spectrum), cigarette smoking, and
cation of drugs that inhibit the platelet glycoprotein IIb/IIla
formaldehyde.
receptor. Inhibitors like abciximab can change the confor-
mation of this receptor, causing exposure of certain peptides
TOXICOLOGY OF PLATELETS (called neoepitopes because they are newly exposed to the
AND HEMOSTASIS immune system) on the factors that react with endogenous
antibodies. This leads to phagocytosis of the platelets associ-
Hemostasis, the stoppage of bleeding or blood flow through an ated with these factors. Thus, exposure of epitopes that react
organ, is a multicomponent system responsible for preventing with naturally occurring antibodies represents a third mecha-
the loss of blood from sites of vascular injury and maintaining nism of immune-mediated platelet destruction.
circulating blood in a fluid state. Loss of blood is prevented by Heparin-induced thrombocytopenia (HIT) represents a
formation of stable hemostatic plugs. The major constituents of fourth mechanism of immune-mediated platelet destruction.
the hemostatic system include circulating platelets, a variety of When heparin (an anticoagulant) binds to certain clotting
plasma proteins, and vascular endothelial cells. Alterations in factors, a neoepitope is exposed, and an immune response is
these components or systemic activation of this system can lead mounted against the neoepitope. This results in platelet activa-
to the clinical manifestations of deranged hemostasis, includ- tion and aggregation instead of heparin’s normal function of
ing excessive bleeding and thrombosis. The hemostatic system preventing clot formation, which can lead to a risk of thrombo-
is a frequent target of therapeutic intervention as well as inad- sis (pieces of clots falling off and lodging in microvasculature,
vertent expression of the toxic effect of a variety of xenobiotics. impairing circulation).
- Thrombotic thrombocytopenic purpura (TTP) is a syn-
drome characterized by the sudden onset of thrombocytope-
Toxic Effects on Platelets nia, a microangiopathic hemolytic anemia, and multisystem
The Thrombocyte—Platelets are essential for formation of a organ failure. The syndrome tends to occur following an infec-
stable hemostatic plug in response to vascular injury. Platelets tious disease but may also occur following administration of
initially adhere to the damaged blood vessel wall through some drugs. The pathogenesis of TTP appears to be related
172 UNIT 4 Target Organ Toxicity

to the ability of unusually large vWf multimers to activate TABLE 11-7 Conditions associated with abnormal
platelets, even in the absence of significant vascular damage. synthesis of vitamin K-dependent coagulation factors.
Acquired TTP is associated with the development of an anti-
Warfarin and analogs Intravenous a-tocopherol
body that inhibits the protease responsible for processing very
Rodenticides (e.g., Dietary deficiency
large vWf multimers into smaller multimers; the large multi- brodifacoum) Cholestyramine resin
mers persist in circulation and inappropriately activate the Broad-spectrum antibiotics Malabsorption syndromes
platelets. The organ failure and hemolysis in TTP is due to the N-Methyl-thiotetrazole
formation of platelet-rich microthrombi throughout the cir- cephalosporins

culation. The development of TTP or TTP-like syndromes has

been associated with drugs such as ticlopidine, clopidogrel,
cocaine, mitomycin, and cyclosporine. may be used to screen for liver dysfunction and a decrease in
Hemolytic uremic syndrome (HUS) is a disorder with clini- clotting factors.
cal features similar to those of TTP, but with less severe neu- Factors II, VII, IX, and X are dependent on vitamin K for
rologic complications and predominant renal failure. Sporadic their complete synthesis. Anything that interferes with absorp-
HUS cases have been linked to Escherichia coli infection, but tion of vitamin K from the intestine or with the reduction of
HUS can also occur during therapy with some drugs, including vitamin K epoxide may lead to a deficiency of these factors and
mitomycin. Unlike TTP, the vWf-cleaving protease is normal a bleeding tendency (Table 11-7).
and the pathogenesis is thought to be related to endothelial
cell damage with subsequent platelet activation and thrombus Increased Clearance of Coagulation Factors—Idiosyn-
formation. cratic reactions to xenobiotics include the formation of antibod-
ies that react with coagulation proteins, forming an immune
Toxic Effects on Platelet Function—Platelet function is
complex that is rapidly cleared from the circulation resulting in
dependent on the coordinated interaction of a number of bio-
deficiency of the factor. The factors that are most often affected
chemical response pathways. Major drug groups that affect
by xenobiotics are listed in Table 11-8. In addition to causing
platelet function include nonsteroidal anti-inflammatory drugs
increased clearance from the circulation, these antibodies often
(NSAIDs), (-lactam-containing antibiotics, cardiovascular
inhibit the function of the coagulation factor. Other antibod-
drugs (particularly 8-blockers), psychotropic drugs, anesthetics,
ies have catalytic activity, resulting in proteolysis of the target
antihistamines, and some chemotherapeutic agents.
coagulation factor.
Xenobiotics may interfere with platelet function through a
Lupus anticoagulants are antibodies that are directed against
variety of mechanisms. Some drugs inhibit the phospholipase
phospholipid binding proteins like prothrombin, can potenti-
A,/cyclooxygenase pathway and synthesis of thromboxane A,
ate procoagulant mechanisms and interfere with the protein C
(e.g., NSAIDs). Other agents appear to interfere with the inter-
system, increasing the risk of thrombosis. The development of
action between platelet agonists and their receptors (e.g., anti-
lupus anticoagulants has been seen in association with chlor-
biotics, ticlopidine, and clopidogrel). As the platelet response is
promazine, procainamide, hydralazine, quinidine, phenytoin,
dependent on rapid increase in cytoplasmic calcium, any agent
and viral infections.
that interferes with translocation of calcium may inhibit plate-
let function (e.g., calcium channel blockers). Occasionally,
drug-induced antibodies will bind to a critical platelet receptor Toxicology of Agents Used to
and inhibit its function.
Modulate Hemostasis
Oral Anticoagulants—Oral anticoagulants (e.g., warfarin)
Toxic Effects on Fibrin Clot Formation interfere with vitamin K metabolism by preventing the reduc-
Coagulation—Fibrin clot formation results from sequential tion of vitamin K epoxide, resulting in a functional deficiency
activation of a series of serine proteases that culminates in the of reduced vitamin K. These drugs are widely used for prophy-
formation of thrombin. Thrombin is a multifunctional enzyme laxis and therapy of venous and arterial thrombosis. The thera-
that converts fibrinogen to fibrin; activates factors V, VIII, XI, peutic window for oral anticoagulants is relatively narrow, and
XIII, protein C, and platelets; and interacts with a variety of there is considerable interindividual variation in the response
cells (e.g., leukocytes and endothelial cells), activating cellular to a given dose. A number of factors, including concurrent
signaling pathways. medications and genetics, affect the individual response to oral
anticoagulants. For these reasons, therapy with these drugs
Decreased Synthesis of Coagulation Proteins—Most must be routinely monitored to maximize both safety and
proteins involved in the coagulation cascade are synthesized efficacy. This is routinely performed with the PT, with results
in the liver. Therefore, any agent that impairs liver function expressed in terms of the international normalized ratio (INR).
may cause a decrease in production of coagulation factors. The A number ofxenobiotics, including foods, have been found
common tests of the coagulation cascade, the prothrombin to affect the response to oral anticoagulants. Mechanisms for
time (PT) and activated partial thromboplastin time (aPTT), interference with oral anticoagulants include induction or
 CHAPTER 11 Toxic Responses ofthe Blood 173

TABLE 11-8 Relationship between xenobiotics Heparin—Heparin is widely used for both prophylaxis and
and the development of specific coagulation factor therapy of acute venous thromboembolism. The major com-
inhibitors. plication associated with heparin therapy is bleeding, which is
a direct manifestation of its anticoagulant activity. The aPTT
Coagulation Factor - - Xenobiotic
is commonly used to monitor therapy with unfractionated
Thrombin Topical bovine thrombin heparin, a naturally occurring polysaccharide. Long-term
Fibrin glue administration of heparin is associated with an increased risk
Factor V Streptomycin
of clinically significant osteoporosis.
Penicillin
Gentamicin Fibrinolytic Agents—Fibrinolytic agents dissolve patho-
Cephalosporins genic thrombi by converting plasminogen, an inactive zymo-
Topical bovine thrombin
gen, to plasmin, an active proteolytic enzyme. Plasmin is
Factor VIIl Penicillin normally tightly regulated and is not freely present in the
Ampicillin circulation. However, administration of fibrinolytic agents
Chloramphenicol
regularly results in the generation of free plasmin leading to
Phenytoin
Methyldopa systemic fibrin(ogen)olysis, which is characterized by prolon-
Nitrofurazone gation of the PT, aPTT, and thrombin time. All of these effects
Phenylbutazone increase the risk of bleeding. Platelet inhibitors and heparin
Factor Xill Isoniazid are commonly used in conjunction with fibrinolytic therapy to
Procainamide prevent recurrent thrombosis.
Penicillin Streptokinase is a protein derived from group C 8-hemolytic
Phenytoin
streptococci that is antigenic in humans. Allergic reactions to
Practolol
the protein can result from streptococcal infection or from
von Willebrand factor Ciprofloxacin exposure to streptokinase-containing fibrinolytic drugs. Acute
Hydroxyethyl starch allergic reactions may occur in 1% to 5% of patients exposed
Valproic acid
Griseofulvin
to streptokinase. Allergic reactions also occur with other
Tetracycline fibrinolytic agents containing streptokinase (e.g., anisoylated
Pesticides plasminogen-streptokinase complex, alteplase) or streptokinase-
derived peptides.

Inhibitors of Fibrinolysis—Antifibrinolytics are com-

monly used to control bleeding in patients with congenital
inhibition of biotransformation; interference with absorp-
abnormalities of hemostasis, such as von Willebrand dis-
tion of warfarin from the gastrointestinal tract; displace-
ease. Tranexamic acid and €-aminocaproic acid are small
ment of warfarin from albumin in plasma, which temporarily
molecules that block the binding of plasminogen and plas-
increases the bioavailability of warfarin until equilibrium is
min to fibrin. Although relatively well tolerated, there is
reestablished; diminished vitamin K availability; and inhibi-
some evidence that administration of these chemicals may
tion of the reduction of vitamin K epoxide, which potentiates
increase the risk of thrombosis due to the inhibition of the
the effect of oral anticoagulants. Additionally, administration
fibrinolytic system.
of oral anticoagulants may affect the activity or the half-lives
Aprotinin is a naturally occurring polypeptide inhibitor
of other medications.
of serine protease clotting factors that is immunogenic when
Oral anticoagulants have been associated with warfa-
administered to humans.
rin-induced skin necrosis, which is due to development of
microvascular thrombosis in skin. This uncommon effect
occurs most commonly in patients deficient in proteins C RISK ASSESSMENT
or S or in patients administered high doses of warfarin too
rapidly. | Assessing the risk that exposure to new chemical products
Vitamin K is also necessary for the synthesis of osteocalcin, a poses to humans—in terms of significant toxic effects on
major component of bone. Long-term administration of war- hematopoiesis and the functional integrity of blood cells and
farin has been associated with bone demineralization. hemostatic mechanisms—is challenging. This is due in part
Administration of warfarin, particularly during the first to the complexity of hematopoiesis and the range of impor-
12 weeks of pregnancy, is associated with congenital anomalies tant tasks that these components perform. Risk assessment
in 25% to 30% of exposed infants. Many of the anomalies are includes preclinical testing of animals and clinical trials in
related to abnormal bone formation. It is thought that warfarin humans. It is hoped that in preclinical trials, the test animals
may interfere with synthesis of additional proteins critical for will react similarly to humans on exposure to the xenobiotic,
normal structural development. and the animals are examined in detail for signs of toxicity.
174 UNIT 4 Target Organ Toxicity

TABLE 11-9 Examples of problem-driven tests used parameters (RBC, hemoglobin, PCV, MCV, and MCHC), leu-
to characterize hematologic observations in preclinical kocyte parameters (WBC and absolute differential counts),
toxicology. thrombocyte counts, coagulation tests (PT and aPTT),
peripheral blood cell morphology, and bone marrow cyto-
Reticulocyte count logic and histologic examinations. Additional tests should be
Heinz body preparation employed in a problem-driven fashion as required to better
characterize hematotoxicologic potential. Examples of these
Cell-associated antibody assays (erythrocyte, platelet, neutrophil) tests are listed in Table 11-9.
Erythrocyte osmotic fragility test Patient- or population-related risk factors include pharma-
cogenetic variations in drug metabolism and detoxification
Erythrokinetic/ferrokinetic analyses that lead to reduced clearance of the agent or production of
Cytochemical/histochemical staining novel intermediate metabolites, histocompatibility antigens,
interaction with drugs or other agents, increased sensitivity
Electron microscopy of hematopoietic precursors to damage, preexisting disease
In vitro hematopoietic clonogenic assays of the bone marrow, and metabolic defects that predispose to
oxidative or other stresses associated with the agent.
Platelet aggregation A central issue in drug and nontherapeutic chemical devel-
Plasma fibrinogen concentration opment is the predictive value of preclinical toxicology data
and the expansive but inevitably limited clinical database for
Clotting factor assays the occurrence of signiftcant hematotoxicity on broad expo-
Thrombin time sure to human populations.

Bleeding time

BIBLIOGRAPHY
Evans GO: Animal Hematotoxicology: A Practical Guide for
Toxicologists and Biomedical Researchers. Boca Raton, FL: CRC
Subsequent clinical trials are conducted in humans and mea-
Press, 2008.
sure myriad parameters of potential toxicity to determine the
Hillman R, Ault KA, Rinder HM: Hematology in Clinical Practice: A Guide
relative safety or toxicity of the test substance. to Diagnosis and Management, 4th ed. New York: McGraw-Hill, 2005.
Tests used to assess blood and bone marrow in preclini- Kaushansky K, Lichtman MA, Beutler E, Kipps TJ, Seligsohn U,
cal toxicology studies should provide information on the Prchal JT (eds.): Williams Hematology, 8th ed. New York: McGraw-
effects of single- and multiple-dose exposure on erythrocyte Hill, 2010.
 CHAPTER 11 Toxic Responses of the Blood ND

QUESTIONS

Which of the following statements is FALSE regarding All of the following statements regarding oxidative
true anemia? hemolysis are true EXCEPT:
a. Alterations of the mean corpuscular volume are a. Reactive oxygen species are commonly generated by
characteristic of anemia. RBC metabolism.
b. Increased destruction of erythrocytes can lead to b. Superoxide dismutase and catalase are enzymes that
anemia. protect against oxidative damage.
c. Decreased production of erythrocytes is not a com- c. Reduced glutathione (GSH) increases the likelihood
mon cause of anemia because the bone marrow is of oxidative injuries to RBCs.
continuously renewing the red blood cell pool. d. Glucose-6-phosphate dehydrogenase deficiency is
d. Reticulocytes will live for a longer period of time in commonly associated with oxidative hemolysis.
the peripheral blood when a person is anemic. e. Xenobiotics can cause oxidative injury to RBCs by
e. The main parameters in diagnosing anemia are RBC overcoming the protective mechanisms of the cell.
count, hemoglobin concentration, and hematocrit.
Which of the following sets of leukocytes is properly
Which of the following types of anemia is properly paired characterized as granulocytes because of the appearance
with its cause? of cytoplasmic granules on a blood smear?
iron deficiency anemia—blood loss. neutrophils, basophils, and monocytes.
sideroblastic anemia—vitamin B,, deficiency. basophils, eosinophils, and lymphocytes.
megaloblastic anemia—folate supplementation. eosinophils, neutrophils, and lymphocytes.
aplastic anemia—ethanol. basophils, eosinophils, and neutrophils.
se
caomegaloblastic anemia—lead poisoning. TES lymphocytes, neutrophils, and basophils.
CPEO

The inability to synthesize the porphyrin ring of hemoglo- All of the following statements are true EXCEPT:
bin will most likely result in which of the following? a. Xenobiotics can greatly slow down the proliferation
iron deficiency anemia. of neutrophils and monocytes, increasing the risk of
improper RBC mitosis. infection.
inability to synthesize thymidine. b. Ethanol and cortisol decrease phagocytosis and
accumulation of iron within erythroblasts. microbe ingestion by the immune system.
Tas bone marrow hypoplasia.
OR
Oat
OF c. Agranulocytosis is predictable and can be caused by
exposure to a number of environmental toxins.
Which of the following will cause a right shift in the d. Heroin and methadone abusers have reduced ability
oxygen dissociation curve? to kill microorganisms due to drug-induced reduc-
increased pH. tion in superoxide production.
decreased carbon dioxide concentration. e. Toxic neutropenia may be mediated by the immune
decreased body temperature. system.
increased 2,3-BPG concentration.
ose
caofetal hemoglobin.
All of the following statements regarding erythrocytes are
true: EXCEPT:
a. Aged erythrocytes are removed by the liver, where the
iron is recycled.
b. Erythrocytes have a life span of approximately
120 days.
c. Red blood cells generally lose their nuclei before
entering the circulation.
d. Reticulocytes are immature RBCs that still have a
little RNA.
e. Persons with anemia have a higher than normal
reticulocyte:erythrocyte ratio.
176 UNIT 4 Target Organ Toxicity

9. Leukemias: 10. Regarding platelets and thrombocytopenia, which of the

a. are often due to cytogenic abnormalities, particularly following statements is FALSE?
damage to or loss of chromosomes 8 and 11. a. Platelets can be removed from the circulation through
b. are rarely caused by agents used in cancer chemotherapy. a hapten-mediated pathway that is induced by drugs
c. originate in circulating blood cells. or chemicals.
d. are characterized as “acute” if their effects are short- Cortisol decreases platelet activity by inhibiting
lived and severe. thromboxane prostaglandin synthesis.
have long been associated with exposure to x-ray Toxins can induce a change in a platelet membrane
radiation. glycoprotein, leading to recognition and removal of
the platelet by phagocytes.
Heparin administration can result in platelet aggrega-
tion and cause thrombocytopenia.
Thrombotic thrombocytopenic purpura is most com-
monly caused by infectious disease, but can also be
associated with administration of pharmacologic
agents.
 CUM ASSP ST ER

Toxic Responses of the

Immune System
Barbara L.E. Kaplan, Courtney E.W. Sulentic,
Michael P. Holsapple, and Norbert E. Kaminski

THE IMMUNE SYSTEM IMMUNE MODULATION BY XENOBIOTICS

Antigen Recognition Halogenated Aromatic Hydrocarbons
Immunity Pesticides
Antigen Metals
Antibody Solvents and Related Chemicals
Complement Mycotoxins
Antigen Processing Natural and Synthetic Hormones
Innate Immunity Therapeutic Agents
Cellular Components: Neutrophils, Immunosuppressive Agents
Macrophages, Natural Killer Cells, NKT, AIDS Therapeutics
and y6T Cells Biologics
Soluble Factors Anti-inflammatory Agents
Acquired (Adaptive) Immunity Drugs of Abuse
Cellular Components: APCs, B Cells, and T
Cells Inhaled Substances
Humoral and Cell-mediated Immunity Ultraviolet Radiation
Inflammation
XENOBIOTIC-INDUCED HYPERSENSITIVITY
Cellular Components: Macrophages, Neutrophils,
AND AUTOIMMUNITY
andT Cells
immune-mediated Disease Hypersensitivity
Hypersensitivity Metals
Autoimmunity Drugs

Developmental Immunology Latex

Neuroendocrine Immunology Food and Genetically Modified Organisms
Formaldehyde
ASSESSMENT OF IMMUNOLOGIC INTEGRITY
Autoimmunity
Methods to Assess Immunocompetence Therapeutic Agents
General Assessment Methyldopa
Functional Assessment Hydralazine, Isoniazid, and Procainamide
Molecular Biology Approaches to Halothane
Immunotoxicology Vinyl Chloride
Mechanistic Approaches to Mercury
Immunotoxicology Silica
Regulatory Approaches to the Assessment Hexachlorobenzene
of Immunotoxicity
Animal Models in Immunotoxicology NEW FRONTIERS AND CHALLENGES IN
Evaluation of Mechanisms of Action IMMUNOTOXICOLOGY

177
178 UNIT 4 Target Organ Toxicity

» Immunity is a series of delicately balanced, complex, faster response to a future infection by the same agent
multicellular, and physiologic mechanisms that allow (memory).
an individual to distinguish foreign material from “self” = Autoimmunity occurs when the reactions of the immune
and to neutralize and/or eliminate that foreign matter. system are directed against the body’s own tissues, result-
= Innate immunity, which eliminates most potential ing in tissue damage and disease.
pathogens before significant infection occurs, includes u Hypersensitivity reactions require prior exposure lead-
physical and biochemical barriers both inside and out- ing to sensitization in order to elicit a reaction on subse-
side of the body as well as immune cells designed for quent challenge.
specific responses. » Xenobiotics that alter the immune system can upset the
« Acquired immunity involves producing a_ specific balance between immune recognition and destruction of
immune response to each infectious agent (specific- foreign invaders and the proliferation of these microbes
ity) and remembering that agent so as to mount a and/or cancer cells.

Immunity is a homeostatic process, a series of delicately bal- Antigen Recognition :

anced, complex, multicellular, and physiologic mechanisms Immunity— Mammalian immunity can be classified into two
that allow an individual to distinguish foreign material from functional divisions: innate immunity and acquired (adaptive)
“self” and to neutralize and/or eliminate the foreign matter. immunity. Innate immunity is a nonspecific, first-line defense
Decreased immunocompetence (immunosuppression) may
response with no associated immunologic memory. The innate
result in repeated, more severe, or prolonged infections as
immune response to a foreign organism is the same for a sec-
well as the development of cancer. Imnmunoenhancement may ondary or tertiary exposure as it is for the primary exposure.
lead to immune-mediated diseases such as hypersensitivity
Acquired immunity is characterized by both specificity and
responses, and if some integral bodily tissue is not identified as
memory, resulting in a much greater immune response on
self, an autoimmune disease may be the end result.
secondary challenge.

THE IMMUNE SYSTEM Antigen— The primary determinant in either type of immune
response is the ability of the immune system components to
The immune system comprises numerous lymphoid organs recognize self versus non-self. A broad definition of non-self
and different cellular populations with a variety of functions. is anything other than that encoded in one’s own germline
The bone marrow and the thymus support the production of genome, which includes foreign DNA, RNA, protein, carbo-
mature T and B lymphocytes and myeloid cells, such as macro- hydrates, and even mutated self-proteins. A non-self substance
phages and polymorphonuclear cells (PMN), and are referred that can be recognized by the immune system is called an anti-
to as primary lymphoid organs. gen, immunogen, or allergen. Antigens are usually (but not
Within the bone marrow, the cells of the immune system absolutely) biological molecules that can be cleaved and rear-
developmentally “commit” to either the lymphoid or myeloid ranged for presentation to other immune cells. Generally, anti-
lineages. Cells of the lymphoid lineage make a further commit- gens are at least 10 kDa in size. Smaller antigens are termed
ment to become either T cells or B cells. T-cell precursors are “haptens” and must be conjugated with carrier molecules
programmed to leave the bone marrow and migrate to the thy- (larger antigens) in order to elicit a specific response.
mus, where they differentiate further.
Mature naive or virgin lymphocytes (those T and B cells that Antibody— Antibodies are produced by B cells and are func-
have never undergone antigenic stimulation) are first brought tionally defined both by the antigen with which they react and
into contact with exogenously derived antigens within the their subtype, termed “isotypes” (e.g., IgM, IgG, IgE, IgD, and
spleen and lymph nodes, otherwise known as the secondary IgA). Antibodies of a known specificity are labeled as such,
lymphoid organs. e.g., an IgM antibody against sheep red blood cells (sRBCs)
Lymphoid tissues associated with the skin and the mucosal is called an anti-sRBC IgM. Antibodies of unknown specific-
lamina propria of the gut, respiratory tract, and genitourinary ity are referred to as immunoglobulin (Ig) until they can be
tract can be classified as tertiary lymphoid tissues. Tertiary lym- defined by their specific antigen.
phoid tissues are primarily effector sites where memory and effec- The basic components of an Ig are the same regardless of iso-
tor cells exert immunologic and immunoregulatory functions. type, namely, heavy chains, light chains, constant regions (Fc),
 CHAPTER 12 Toxic Responses of the Immune System 79

Heavy a manner similar to the blood-clotting cascade (Figure 12-2).

Light Chain
The final components that can enter the membrane and disrupt
chain
its integrity are termed the membrane attack complex (MAC).
Three pathways have been identified in the activation of the
complement cascade, the classical pathway (antibody:antigen-
dependent), the alternative pathway (spontaneous activation
of C3), and the mannin-binding lectin pathway (a hepatogenic
molecule that binds to pathogens).

Antigen Processing—To elicit an acquired immune

response to a particular antigen, that antigen must be taken up
and processed by accessory cells for presentation to lympho-
cytes. Accessory cells that perform this function are termed
antigen-presenting cells (APCs) and include macrophages, B
cells, and dendritic cells (DCs). Of these, the most proficient
APC is the DC. There are several subtypes of DC, including
plasmacytoid DCs (pDCs), conventional DCs (cDCs), special-
FIGURE 12-1 Ig structure. igs are composed of two heavy ized DCs in the skin called Langerhans cells, and follicular DCs
chains and two light chains, which are connected by disulfide bonds. in germinal centers within secondary lymphoid organs.
Orange areas are variable regions and green areas at top are antigen In most tissues, DCs are in an immature state in which they
recognition regions. capture antigens through phagocytosis, pinocytosis, or receptor-
mediated endocytosis. DCs then process the antigen (intra-
and variable regions (Fab). The general structure of antibodies cellular denaturation and catabolism) and display fragments of
are also conserved across isotypes (Figure 12-1). There are two it on the extracellular side of their cell membrane through direct
genes coding for light chains (V and J) and three genes coding association with special cell surface molecules (major histo-
for heavy chains (V, D, and J). Isotype is determined by which compatibility complex [MHC] classes MHCI and MHCI)).
Fe of the heavy chain is transcribed and translated (heavy chain Antigen presentation of the kind discussed here primarily
genes |, , €, 6, or & encode for the IgM, IgG, IgE, IgD, or IgA occurs via MHCII (Figure 12-3), although certain types of
heavy chain proteins, respectively). antigens are processed and presented via MHCI. The path-
The immune system generates antibodies to thousands of anti- way of presentation between these two classes have similari-
gens with which the host may or may not ever contact. During ties, but the major differences between the MHCI and MHCII
this process called somatic recombination, V and J segments for pathways are (1) antigens processed and presented via MHCI
light chains as well as V, D, and J segments for heavy chains are are not limited to professional APC; (2) all nucleated cells
combined, within the B cell, to form an Ig. The Fab regions of express MHC]; (3) the mechanisms by which the antigen is
both the heavy and light chains determine antibody specificity processed and loaded onto MHC] are slightly different than
and interact directly with antigen. In addition to isotype deter- MHCIH; (4) the MHCI antigenic peptides are usually smaller,
mination, the Fc region is also responsible for the various effector often 8 to 10 amino acids in length; (5) the MHCI antigens to
functions, such as complement activation (e.g., IgM and some be processed are usually aberrantly expressed proteins, such
subclasses of IgG) and phagocyte binding (via Fc receptors). as viral-associated proteins or mutated proteins; (6) MHCI
Antibodies possess several functions: (1) opsonization, facilitates antigen presentation to CD8* T cells, whereas
which is coating of a pathogen with antibody to enhance Fc MHCII facilitates antigen presentation to CD4* T cells.
receptor-mediated endocytosis by phagocytic cells; (2) ini- MHC antigen processing and presentation is the major path-
tiation of the classic pathway of complement-mediated lysis; way by which virally infected cells are detected and killed by
(3) neutralization of viral infection by binding to viral particles the acquired immune system.
and preventing further infection; and (4) enhancement of the Regardless of the MHC utilized to present antigens to lym-
specificity of effectors of cell-mediated immunity (CMI) by phocytes, T cells are able to recognize antigen in the context of
binding to specific antigens on target cells, which are then rec- MHC with their T-cell receptor (TCR). Similar to Ig, the abil-
ognized and eliminated by effector cells such as natural killer ity of T cells to specifically recognize thousands of antigens is
(NK) cells or cytotoxic T lymphocytes (CTLs). due to somatic recombination. All TCRs are composed of two
different subunits, each encoded from a distinct gene (most
Complement—The complement system is a series of about abundant T-cell population expresses a3, but 6 also exist).
30 serum proteins whose primary functions are the destruction All TCR subunits are made up of constant and variable regions.
of membranes of infectious agents, opsonization to facilitate For & subunits, two separate gene segments (V and J) are com-
phagocytosis, and the promotion of an inflammatory response bined to form the variable region, which is then joined to one
(see the “Inflammation” section). Complement activation constant region. For 8 subunits, three separate gene segments
occurs with each component sequentially acting on others, in (V, D, and J) are combined to form the variable region, which
180 UNIT 4 Target Organ Toxicity

See,
‘Classical pathway —Se : ee
Lectin pathway‘ Alternative pathway —%
activatedby SRS os activateddby eae activated by Siege at as
Clq binding to antigen-antibody s MBL bindingto
| oe oie spontaneous breakdown :
‘compline Re oihiechete
esa

(6 ae eae tay php

7 Ae3bBb3sb
aa

C3a,C4a,andCSaare
proinflammatory

Opsonization mediated by C5b6789n

|
(G3 _ membrane attack complex (MAC)

FIGURE 12-2 Thecomplement cascade.

The complement cascade can be activated in three different ways. Cytolysis occurs via generation of
the MAC. Various complement proteins generated along the pathway are either pro-inflammatory mediators (C3a, C5a) or result in opsonization (C3b).

is then joined to one of the two constant regions. Similar to the Ig receptor for B cells, (2) cellular activation and initiation of
Ig genes, there are several light chain V and J genes, and several intracellular signaling cascades that contribute to production
heavy chain V, D, and J genes. and release of cytokines and other cellular mediators, (3) clonal
With regard to T and B cells, key events that occur following expansion (proliferation) of antigen-specific cells, and (4) dif-
antigen encounter are (1) specific antigen recognition either ferentiation of antigen-stimulated lymphocytes into effector
in the context of MHCI or MHCII for T cells or through the and memory cells.
 CHAPTER 12 Toxic Responses of the Immune System 181

_.. Antigen ¢ deposited on the surface of the foreign target. They are also
important in the induction ofan inflammatory response.
Macrophages are terminally differentiated monocytes that are
i a Antigen developed from the myeloid lineage of HSCs. Upon exiting the
~.¥ . uptake
bone marrow, monocytes circulate within the bloodstream for
\ Antigen om about one day. At that time, they begin to distribute to the vari-
.| * wProcessing & |
*
ay ous tissues where they can then differentiate into macrophage
ee \ ‘ye z, : subsets. Tissue-specific macrophages include Kupffer cells
— / Peptide (liver), alveolar macrophages (lung), microglial cells (CNS),
: binds MHCII
and peritoneal and splenic macrophages. Macrophages can be
_“e MHC i & Sy
transport = gg classified as classically activated macrophages (M1), which are
. = vesicle i pro-inflammatory and participate in antigen presentation, or
Endosome alternatively activated macrophages (M2), which do not present
antigen well, but are efficient in apoptotic cell removal. Based on
this classification, M1 macrophages are also APCs.
FIGURE 12-3 Antigen processing by the MHCII pathway. Like other immune cells, natural killer (NK) cells are derived
Antigen is engulfed by an APC (DC, macrophage, or B cell), degraded, from a HSC. The two major functions of NK cells are cytokine
and loaded onto MHCIl. The MHCIl-peptide complex is then expressed (soluble protein messenger) production and cytolysis. NK cells
on the surface of the APC for presentation to CD4* Th cells. are the predominant producer of the cytokine, IFN-y, which
promotes DC maturation. In this way, NK cells serve as a bridge
between innate and adaptive immunity. NK cells also mediate
both antibody-independent and antibody-dependent cellular
Innate Immunity cytotoxicity (ADCC) using a variety of mechanisms, including
Innate immunity acts as a first line of defense against infectious perforin and granzyme, Fas L, TRAIL, and TNF-a. Antigen-
agents, eliminating most potential pathogens before significant dependent cell-mediated cytotoxicity occurs via the FcyRIIIA,
infection occurs. It includes physical and biochemical bar- which is present on most NK cell subsets. NKT cells are one
riers both inside and outside of the body as well as immune such subset of NK cells that express both NK- and T-cell mark-
cells designed for specific responses. There is no immunologic ers, which allows the NKT cell to present self and exogenous
memory associated with innate immunity. antigenic glycolipids.
Externally, the skin provides an effective barrier, as most Another cell type that has recently been shown to facilitate
organisms cannot penetrate intact skin. Most infectious agents the innate-adaptive immunity bridge is the 76 T cell. These
enter the body through the respiratory system, gut, or genito- cells migrate predominantly to “exposed” tissues, including
urinary tract. Innate defenses present to combat infection from skin, lung, gut, and reproductive organs, and are also expressed
pathogens entering through the respiratory system include highly in the liver. In part through the expression of TLRs, y6 T
mucus secreted along the nasopharynx, the presence of lyso- cells can acquire effector functions similar to those of NK cells.
zyme in most secretions, and cilia lining the trachea and main There is also a subpopulation of B cells that also bridge innate
bronchi. In addition, reflexes such as coughing, sneezing, and and adaptive immunity. Unlike the conventional B-2 cells, B-1
elevation in body temperature are also a part of innate immu- cells predominate in embryonic life and are later found mostly
nity. Pathogens that enter the body via the digestive tract are in the peritoneal and pleural cavities. B-1 cells are self-renewing
met with severe changes in pH (acidic) within the stomach and and spontaneously produce polyspecific IgM antibodies (i.e.,
a host of microorganisms living in the intestines. natural antibodies) independent of T-cell help.
Historically, innate immunity was defined as nonspecific.
Cellular Components: Neutrophils, Macrophages, It is now clear that innate cells express receptors that respond
Natural Killer Cells, NKT, and 75 T Cells—Neutrophils to soluble components (e.g., Fc or complement receptors) or
(also known as polymorphonuclear cells [PMNs]) are phago- to certain antigenic motifs. Pattern recognition receptors are
cytic cells that develop from the myeloid lineage of HSCs. They a family of receptors that recognize pathogen-derived mol-
enter the bloodstream where they circulate for about 10 h, after ecules or cell-derived molecules produced in response to cel-
which they enter tissues and perform effector functions for one lular stress (“danger” molecules). Receptors that recognize
or two days. Neutrophils are a primary line of defense because pathogen-associated molecular patterns (PAMPs) are toll-like
of their ability to pass between the endothelial cells of blood ves- receptors (TLRs); receptors that recognize danger-associated
sels (termed extravasation) and access peripheral tissues. They molecular patterns (DAMPs) include NOD-like receptors
are excellent phagocytic cells and can eliminate most microor- (NLR) and RIG-like receptors (RLR). TLRs are expressed both
ganisms through the release of various reactive oxygen species extracellularly and intracellularly in endosomes, which confers
(ROS), such as superoxide, singlet oxygen, ozone, hydrogen the ability to respond to a variety of pathogenic components,
peroxide, and hydroxyl radicals. Their phagocytic activity is such as bacterial cell wall lipids, single- and double-stranded
greatly enhanced by the presence of complement and antibody nucleic acids, or fungal and parasitic products. Functional
182 UNIT 4 Target Organ Toxicity

consequences of TLR engagement on cells include expression migration, trafficking, or effector functions (Table 12-1).
of adhesion molecules, chemokines, or cytokines to stimulate Although some of these molecules might be constitutively
T- or B-cell differentiation, enhance phagocytosis, or facilitate expressed, most are inducible in response to antigens, cellu-
maturation of DCs. lar stressors, or other cytokines. Thus, many cytokines, che-
mokines, and IFNs are not stored in the cell, but rather are
Soluble Factors—A common effector mechanism for many tightly regulated, often at the transcriptional level, so that they
immune cells, is cytokine, chemokine, or interferon (IFN) are quickly generated on demand. Also, many cytokines share
production. The primary function of cytokines, chemokines, common receptor subunits. This means if a particular subunit
and IFNs include cellular activation, initiation or termination of one receptor is affected by an immunotoxic agent, all others
of intracellular signaling events, proliferation, differentiation, that share this subunit are also affected.

TABLE 12-1 Cytokines: sources and functions in immune regulation.

Cytokine Source Physiologic Actions

IL-1 Macrophages Activation and proliferation of T cells

Epithelial cells Pro-inflammatory
Induces fever and acute-phase proteins
Induces synthesis of pro-inflammatory cytokines

IL-2 T cells Primary T-cell growth factor .

Growth factor for B and NK cells,
IL-4 Th2 cells Proliferation of activated T cells and B cells
Mast cells B-cell differentiation and IgE isotype switching functions
Antagonizes IFN-y
Inhibits Th1 responses

IL-5 Th2 cells Proliferation and differentiation of eosinophils

Mast cells
IL-6 Macrophages Enhances B-cell differentiation and immunoglobulin secretion
Th2 cells Induction of acute-phase proteins by liver
B cells Pro-inflammatory
Endothelial cells Proliferation ofTcells and increased IL-2 receptor expression
Synergizes with IL-4 to induce secretion of IgE

IL-10 Tregs Inhibits T-cell and macrophage responses

Bregs

IL-12 DCs Activates BK cells

Macrophages Induces TH1 responses

IL-13 Th2 cells Stimulates B-cell growth

Inhibits Th1 responses
IL-17 Th17 cells Pro-inflammatory
NK cells Inhibits Tregs
OT cells
Neutrophils

Interferon-a/B (IFN-c/®) (type1 IFN) Leukocytes Induction of MHC class | expression

DCs Antiviral activity
Fibroblasts Stimulation of NK cells

Interferon-y (IFN-7) T cells Induction of MHCI and MHCII

NK cells Activates macrophages

Transforming growth factor-B (TGF-3) Macrophages Enhances monocyte/macrophage chemotaxis

Megakaryocytes Enhances wound healing: angiogenesis, fibroblast proliferation, deposition
T cells of extracellular matrix
InhibitsT-and B-cell proliferation
Inhibits antibody secretion
Primary inducer of isotype switch to IgA

GM-CSF T cells Stimulates growth and differentiation of monocytes and granulocytes

Macrophages
Endothelial cells
Fibroblasts
 CHAPTER 12 Toxic Responses of the Immune System 183

Other soluble components of innate immunity include the This rigorous selection process produces T cells that can
complement cascade (discussed earlier), acute-phase pro- recognize MHC plus foreign peptides and eliminates auto-
teins, granzyme and perforin, and various cytokines, chemo- reactive T cells.
kines and IFNs. Complement is important in innate immunity Additionally, expression of CD4 or CD8 will determine to
because of its activation through the mannin-binding lectin which MHC class the a8 TCR will bind. CD4 will facilitate
pathway. Furthermore, C3a and C5a, which are chemokines binding to MHCII expressed on APCs; T cells expressing CD4
generated during the cascade, recruit phagocytic cells to the (helper T cells, Th) help activate other cells of the adaptive
site of complement activation. Acute-phase proteins, such as immune response. CD8 will facilitate binding to MHCI, which
serum amyloid A, serum amyloid P, and C-reactive protein, is expressed on all nucleated cells; generally, T cells expressing
participate in an acute-phase response to infection by binding CD8 mediate cell killing (CTL). The y6 T cells do not express
bacteria and facilitating complement activation. Granzyme CD4 or CD8 and therefore do not interact with MHC and do
and perforin work in conjunction, with perforin disrupting the not undergo positive or negative selection. Since 6 T cells are
target cell membrane, allowing granzyme to enter and mediate not negatively selected for autoreactivity, these cells may be
cell lysis by several mechanisms. associated with the development of hypersensitivity and auto-
immunity (see subsequent sections).
Mature T cells are found in the lymph nodes, spleen, and
Acquired (Adaptive) Immunity peripheral blood. Upon binding of the TCR to MHC plus
If the primary defenses against infection (innate immunity) antigen, the mature T cell becomes activated and, after pro-
are breached, the acquired arm of the immune system is acti- liferation, undergoes differentiation into either an effector
vated and produces a specific immune response to each infec- cell or a memory T cell. There are many subsets of effector
tious agent. This branch of immunity can protect the host Th cells and two subsets, Thl and Th2, dictate whether CMI
from future infection by the same agent. Two key features that or humoral immunity will predominate, respectively. Th1
distinguish acquired immunity are specificity and memory. cells predominantly express IL-2, IFN-7y, and lymphotoxin,
Acquired immunity may be further subdivided into humoral which promote CMI and humoral defense against intracel-
and cell-mediated immunity (CMI). Humoral immunity lular invaders. Th2 cells predominantly express IL-3, IL-4,
is directly dependent on the production of antigen-specific IL-5, IL-6, IL-10, and IL-13, which promote humoral defense
antibody by B cells and involves the coordinated interaction against extracellular invaders. Although the two popula-
of APCs, T cells, and B cells. CMI is that part of the acquired tions are not mutually exclusive, they do negatively regulate
immune system in which effector cells, such as phagocytic each other, such that a strong Thl response suppresses a Th2
cells, helper T cells (Th cells), T-regulatory cells (Tregs), APCs, response and vice versa.
CTLs, or T memory cells, play the critical role(s) without The ability of APCs, B cells, and T cells to communicate with
antibody involvement. each other is dependent on a variety of receptor-ligand inter-
actions between cell types. These interactions also help dic-
Cellular Components: APCs, B Cells, and T Cells— tate the type of immune response (i.e., humoral versus CMI)
APCs, which have been discussed previously in the “Antigen and the magnitude of the immune response. The duration
Processing” section, include professional APCs such as B cells, and extent of an acquired immune response is also controlled
macrophages, and DCs. Although all cells may act as APCs by specialized regulatory cells found in both the T-cell and
with internal antigen processing through the MHCI path- B-cell lineages.
way, what distinguishes a professional APC from the others is For the T-cell lineage, there is a small population of CD4+
the ability to internalize external antigens and process them cells that develop into T-regulatory cells (Tregs), which help to
through the MHCII pathway for presentation to T cells. control various immune responses, including those directed
Besides serving as APCs, B lymphocytes are also the effector against self. The mechanisms by which Tregs suppress immune
cells of humoral immunity, producing a number of Ig isotypes responses involve direct Treg-cell contact. Several subsets of
with varying specificities and affinities. Upon antigen binding regulatory B cells have already been identified, but more are
to surface Ig (part of the B-cell receptor [BCR]), the mature being discovered recently. These cells generally play a suppres-
B cell becomes activated and, after proliferation, undergoes sive role in hypersensitivity and autoimmune diseases. The
differentiation into either a memory B cell or an antibody- regulatory T- and B-cell subsets also appear to reciprocally
forming cell ([AFC]; also known as a plaque-forming cell activate or suppress each other and may cooperatively control
[PFC]) that actively secretes antigen-specific antibody. immune responses.
T-cell precursors migrate from the bone marrow to the
thymus where, in a manner analogous to B cells, they begin Humoral and Cell-mediated Immunity—Humoral
to rearrange their TCRs. These immature cells then undergo immunity is that part of the acquired immune system in which
positive and negative selection to (1) eliminate cells that do antibody is involved, and CMI is that part of the immune sys-
not produce a functional TCR or produce TCRs with no affin- tem in which various effector cells perform a wide variety of
ity for self- MHC (positive selection) or (2) eliminate cells that functions to eliminate invaders. These two branches are often
strongly bind MHC plus self-peptide (negative selection). coordinated, as depicted in Figure 12-4.
184 UNIT 4 Target Organ Toxicity

Ww &cD40 \- Xe a
; B B cBne Memory IFN- + Memory
Antigen B
Differentiation L
processing& \L-2
Proliferation
activation if:4
Differentiation Activation
aa Antigen
Proliferation
; Differentiation
Antigen
Activation
Proliferation
Differentiation TCR/
(T cell help) CD28
B7,
MHCII + CD8 L / IFNy
peptide L Perforin
IL-12 a Granzyme
pc @ MHC! + ig
Antigen-specific Bee =)
antibodies
he -infected Lysed
FIGURE 12-4 Cellular interactions in the humoral immune cell cell
response. Antigen is engulfed by an APC (usually DC) and the
antigenic peptide is presented to CD4* T cells in the context of MHCII. FIGURE 12-5 Cellular interactions in the CTL response.
CD4*T cells then become activated, proliferate, and differentiate Antigen is engulfed by an ARC (usually DC) and the antigenic peptide
into Th cells, which release cytokines to help B cells that had also is presented to CD4* and CD8* T cells in the context of MHCIl and |,
encountered the same antigen. B cells then become activated, respectively. CD4* T cells then become activated, proliferate, and
proliferate, and differentiate into memory B cells or antigen-producing differentiate intoTh cells, which release cytokines to help CD8* T cells
plasma cells. that had also encountered the same antigen. Especially in the
presence of IL-12 produced by the DC, CD8* T cells become activated,
proliferate, and differentiate into CTL that can kill other antigen-
infected cells.
In general, B cells produce antibodies specific to an anti-
gen, which may act to opsonize or neutralize the invader, or
the antibodies act to recruit other factors, such as the comple-
ment cascade. The production ofantigen-specific IgM requires inflammation
3 to 5 days after the primary (initial) exposure to antigen. Inflammation refers to a complex reaction to injury, irrita-
Upon secondary antigenic challenge, the B cells undergo iso- tion, or foreign invaders characterized by pain, swelling, red-
type switching, producing primarily IgG antibody, which is of ness, and heat. Inflammation involves various stages, including
higher affinity for the activating antigen. In addition, there is a release of chemotactic factors following the insult, increased
higher serum antibody titer associated with a secondary anti- blood flow, increased capillary permeability allowing for cellu-
body response. lar infiltration, followed by either an acute resolution of tissue
CMI functions include delayed-type hypersensitivity damage or persistence of the response that might contribute to
(DTH) and cell-mediated cytotoxicity. Cell-mediated cyto- fibrosis or subsequent organ failure. It is important to empha-
toxicity responses may occur in numerous ways: (1) MHC- size that while inflammation is a natural reaction to repair tis-
dependent recognition of specific antigens (such as viral sue damage or attack foreign invaders, the process often results
particles or tumor proteins) by CTL (Figure 12-5); (2) indirect in destruction of adjacent cells and/or tissues. Thus, there is
antigen-specific recognition by the binding of Fc receptors on overwhelming evidence that inflammation plays a critical role
NK cells to antibodies coating target cells; and (3) receptor- in many diseases, including asthma, multiple sclerosis, car-
mediated recognition of complement-coated foreign targets diovascular disease, Alzheimer’s disease, bowel disorders, and
by macrophages. cancer. In addition, inflammation exacerbates idiosyncratic
In cell-mediated cytotoxicity, the CTL or NK effector cell reactions to drugs and other chemicals.
binds in a specific manner to the target cell. The majority of
CTLs express CD8 and recognize either foreign MHCI on the Cellular Components: Macrophages, Neutrophils, and
surface of allogeneic cells or antigen in association with self- T Cells—Many of the cellular components described in the
MHCI (e.g., viral particles). Once the CTL or NK cells interact sections above are critical to initiation and maintenance of an
with the target cell, the effector cell releases granules contain- inflammatory response. Major cellular contributors to an inflam-
ing perforin and other enzymes. This degranulation damages matory response are macrophages, neutrophils, and T cells.
the target cell, after which the effector cell can release and Neutrophils are often the first, and most numerous, responders
attack other target cells In addition, CTLs induce the target to to sites of insult. In response to either host- or pathogen-derived
undergo apoptosis through activation of the Fas and cytotoxic signals, neutrophils secrete chemotactic factors to recruit other
cytokine (i.e., TNF and lymphotoxin) pathways. pro-inflammatory cells, such as macrophages, to the area.
 CHAPTER 12 Toxic Responses of the Immune System 185

Macrophages can be activated by a variety of mechanisms at Gell and Coombs

the site of insult, such as activation via toll-like receptor, pro- classification

inflammatory cytokines, or recognition of opsonized particles IgE |

by Fe receptors or complement receptors. Macrophages and
neutrophils also induce apoptosis of cells in the insult area
through the release of nitric oxide and other ROS, resulting TCR/ Th2 IgM, |gG II
in disruption of extracellular structures that compromise CD28
tissue structure and function. Both neutrophils and macro-
B7, CD4 IgM, |gG Ill
phages are phagocytic cells and can contribute to clearing of MHCII + |
apoptotic cells. peptide ial “=
Later in the inflammatory response, T cells are critical for Bei 0 ce git Thl1 —~ Tcells IV
» DC @
generating an adaptive immune response. T cells are attracted ee y i b ae

to the insult area by adhesion molecules and integrins, and are

activated in response to antigen presented in the context of
ya
MHC, often by a DC. Depending on the signals that the T cell Antigen
receives from the cytokine milieu, distinct subpopulations of
T cells are induced. FIGURE 12-6 Overview of classification of hypersensitivity
reactions. Hypersensitivity reactions are mediated viaT cells and
antibody production.
immune-mediated Disease
While the primary purpose of the immune system is to
results in binding to IgE on local mast cells and degranulation
preserve the integrity of the individual from disease states,
with the release of preformed mediators and cytokines which
situations arise in which the individual’s immune system
recruit and activate circulating eosinophils, basophils, macro-
responds in a matter producing tissue damage, resulting in
phages, and neutrophils leading to the synthesis and release of
self-induced disease. These disease states fall into two catego-
more cytokines and of leukotrienes and thromboxanes. These
ries: (1) hypersensitivity, or allergy, and (2) autoimmunity.
mediators promote vasodilation, bronchial constriction, and
inflammation. Clinical manifestations can vary from urti-
Hypersensitivity
carial skin reactions (wheals and flares) to signs of hay fever,
Classification of Hypersensitivity Reactions—There are four
including rhinitis and conjunctivitis, to more serious diseases,
types of hypersensitivity reactions as classified by Coombs and
such as asthma and potentially life-threatening anaphylaxis.
Gell. One characteristic common to all four types of hyper-
sensitivity reactions is the necessity of prior exposure leading
to sensitization in order to elicit a reaction upon subsequent
challenge. In the case of types I, IJ, and III, prior exposure to
Carrier
antigen leads to the production of allergen-specific antibodies Conjugated HN s ra protein
(IgE, IgM, or IgG) and, in the case of type IV, the generation penicilloic acid Ae —e *
HN e
of allergen-specific memory T cells. Figure 12-6 illustrates the
mechanisms of hypersensitivity reactions.

Type I (Immediate or IgE-mediated Hypersensitivity): Using peni-

cillin as an example, Figure 12-7 depicts the major events involved
in a type I hypersensitivity reaction (what most people think of
as “allergy” and is clinically referred to as “atopy”). Sensitization
occurs as the result of dermal exposure to antigens or by exposure
to antigens through the respiratory or gastrointestinal tract. Most °
people would mount an IgM, IgG, or IgA immune response to IgEboundto =
Fc receptors
these antigens and clear them without causing any allergic symp-
toms. It is unclear why these antigens become allergens in certain
individuals who respond by mounting an IgE immune response
Mast cell ©
instead, but appears to involve genetic and/or environmental
determinants and likely some type of triggering event (e.g., acute
pathogen exposure and emotional stress). FIGURE 12-7 Typethypersensitivity reaction. Metabolized
Once produced, soluble IgE not only binds to local tissue penicillin isa hapten that conjugates with a protein. The conjugated
mast cells, but also enters the circulation, where it binds to cir- hapten cross-links IgE antibodies on mast cells. IgE cross-linking
culating mast cells, basophils, and tissue mast cells at distant causes mast-cell degranulation and releasing histamine and other
sites. Once an individual is sensitized, reexposure to the antigen pro-inflammatory mediators.
 186 UNIT 4 Target Organ Toxicity

These responses may begin within minutes of reexposure to location is the vascular endothelium in the lung, joints, and
the offending antigen; therefore, type I hypersensitivity is often kidneys. The skin and circulatory systems may also be involved.
referred to as immediate hypersensitivity. Pathology results from the inflammatory response initiated by
the activation of complement. Macrophages, neutrophils, and
Type II (Antibody-dependent Cytotoxic Hypersensitivity): Type I] platelets attracted to the deposition site contribute to the tissue
hypersensitivity is lgG- or lgM-mediated. The antibody response damage. As with type II hypersensitivity, responses similar to
may be mediated by a foreign antigen attached to the surface of type III hypersensitivity can be induced in autoimmune dis-
a cell or tissue. Conversely, an antibody response could be medi- eases due to autoantibodies directed against soluble antigens
ated by an autoantibody due to a breakdown in tolerance and the such as double-stranded DNA or small nuclear proteins as seen
resulting response would be part of an autoimmune disease (e.g., with systemic lupus erythematosus (SLE).
autoimmune hemolytic anemias and Goodpasture’s syndrome).
Figure 12-8 shows the mechanisms of action for complement- Autoimmunity—Autoimmune disease occurs when the
independent and complement-dependent cytotoxic reactions. reactions of the immune system are directed against the body’s
Tissue damage may result from the direct action of cytotoxic own tissues and is characterized by a genetic susceptibility.
cells, such as macrophages, neutrophils, or eosinophils, linked These diseases can be either tissue-specific or nonspecific, and
through the Fe receptor to antibody-coated target cells (com- the targets from the perspective of the primary sites of tissue
plement-independent) or by antibody-induced activation of the damage in autoimmune disease are many and varied. Both
classic complement pathway. humoral immunity and CMI can be involved as effector mech-
anisms in causing the damage in autoimmune conditions.
Type III (Immune Complex-mediated Hypersensitivity): Type Although the resulting pathology may be the same for auto-
III hypersensitivity reactions also involve IgG or IgM. The dis- immune reactions and hypersensitivity, mechanisms of true
tinguishing feature of type III is that, unlike type II, in which autoimmune disease are distinguished from hypersensitivity.
Ig production is against specific cellular or tissue-associated In cases of autoimmunity, self-antigens are the target, and in
antigen, Ig production is against soluble antigen in the serum the case of chemical-induced autoimmunity, the disease state
(Figure 12-9). This allows for the formation of circulating is induced by a modification of host tissues or immune cells by
immune complexes composed of a lattice of antigen and Ig, the chemical and not the chemical acting as an antigen/hapten
which may result in widely distributed tissue damage in areas as in hypersensitivity reactions.
where immune complexes are deposited. The most common
Mechanisms of Autoimmunity—The rearrangement and
recombination of the genes that comprise Ig and TCR result

Complement-independent Complement-dependent
cytolysis cytolysis
IgG produced Platelets Antigen
against antigen : -

Antigen-antibody
complexes
Antigen + Antigen e deposited in
attached attached tissue
to normal to normal Neutrophil
cell cell Platelets interact
with complexes + Inflammation,
IgG ‘e tissue destruction
Complement gets
IgG directed Cytolysis | directed activated, which
against antigen ae against destroys cells,
antigen releasing
chemotactic Activation of
we)
Cytotoxic C3a, C5a bind C3a,C5a factors complement
cell binds complement me cascade bylgG Macrophage
to IgG via receptors Activation of Neutrophils and
complement macrophages
Fc receptor
cascade by IgG produce inflammation
and tissue destruction
FIGURE 12-8 Typell hypersensitivity reactions. In
complement-independent cytolysis, antigen becomes attached toa FIGURE 12-9 Type lil hypersensitivity reactions. IgG is
normal cell, which can be recognized by IgG. A cell capable of cytolysis produced against an antigen and antigen-antibody complexes form,
(CTL, NK cell) binds to IgG via its Fc receptor and kills the antigen-coated which can become deposited in tissue. Complement gets activated
cell. In complement-dependent cytolysis, antigen becomes attached by the classical pathway (antigen-antibody complexes), and platelets
to anormal cell, which can be recognized by IgG. Complement gets also interact with complexes. Following complement-mediated
activated by the classical pathway (antigen-antibody complexes) and cytolysis, released chemotactic factors attract neutrophils and
C3a and C5a bind complement receptors. macrophages, causing additional inflammation and tissue damage.
 CHAPTER 12 Toxic Responses of the Immune System 187

in tremendous diversity in the potential antigen recogni- depends on the specific parameter being measured, and varies
tion of B cells and T cells, respectively. Ideally, during devel- across species with striking differences noted between rodents
opment those lymphocytes recognizing self-antigens will and humans.
largely be deleted by negative selection as central tolerance Exposure to specific antigens during the perinatal period
is established. Autoreactive clones that escape central toler- results in a rapidly expanding accumulation of lymphocyte
ance and migrate to the periphery are normally controlled by specificities in the pool of memory cells in secondary lymphoid
peripheral tolerance mediated by various mechanisms that tissues. As thymic function wanes and thymocytes are no lon-
ultimately induce anergy or clonal deletion. For autoimmune ger produced in that tissue, it is this pool of memory B and
disease to occur, an autoreactive clone must escape central T cells that maintains immune competence for the life of the
tolerance, pass into the periphery, and bind with specificity individual. Senescence of immunity is associated with reduc-
to its self-antigen, then mechanisms of peripheral tolerance tions in both innate and acquired immune responses to anti-
must fail and the autoreactive clone must induce a detrimen- gens during the last quartile of life. This failure of the immune
tal immunological response. response is due, in part, to a continual reduction in the pro-
Autoimmunity is multifaceted and has been associated with duction of newly formed cells, and to the decreased survival of
several mechanisms primarily related to insufficient periph- long-lived memory cells in lymphoid tissues.
eral tolerance, while defects in central tolerance are rarely One feature of the developing immune system that clearly
encountered. Several mechanisms have been associated with distinguishes it from the mature immune system, especially
the breakdown of peripheral tolerance and prime events for during gestation, is the role played by organogenesis. Defects
the onset of autoimmune disease. These mechanisms include in the development of the immune system due to heritable
inflammation; molecular mimicry by pathogen antigens; changes in the lymphoid elements have provided clinical and
inherent defects in T or B cells including regulatory subsets, experimental examples of the devastating consequences of
APCs, cytokines, or complement; and epitope spreading. impaired immune development. Therefore, the effects of chem-
Effector mechanisms in autoimmune disease can be the same icals on the genesis ofcritical immune organs in the developing
as those described earlier for types II and III hypersensitivity fetus may be more important than effects on these tissues after
or, in the case of pathology associated with solid tissues, they having been populated by hematopoietic and lymphoid cells.
may involve CD8* cytotoxic T lymphocytes. Tissue damage Interestingly, immune organs, such as the thymus, spleen, and/
associated with CTL may be the result of direct cell mem- or bone marrow, are not typically assessed in routine develop-
brane damage and lysis, or the result of cytokines produced mental and reproductive toxicology studies.
and released by the T cell. TNF-( has the ability to kill suscep-
tible cells, and IFN-y may increase the expression of MHCI
on cell surfaces, making them more susceptible to CD8* Neuroendocrine Immunology
cells. Cytokines may also be chemotactic for macrophages, Cytokines, neuropeptides, neurotransmitters, and hormones
which can cause tissue damage directly or indirectly through (as well as their receptors) are an integral and interregulated
the release of pro-inflammatory cytokines. As is the case with part of the central nervous system, the endocrine system, and
hypersensitivity reactions, autoimmune disease is often the the immune system. Because receptors for neuropeptides, neu-
result of more than one mechanism working simultaneously. rotransmitters, and hormones are present on lymphoid cells,
some chemicals may exert their immunomodulatory effects
indirectly on the immune system by modulating the activity of
Developmental Immunology the nervous or endocrine systems. In addition, immune cells
A sequential series of carefully timed and coordinated devel- are capable of secreting peptide hormones and neurotransmit-
opmental events, beginning early in embryonic/fetal life and ters, which can have autocrine (immune system) and paracrine
continuing through the early postnatal period, is required to (endocrine and nervous systems) effects.
establish a functional immune system in all mammals. The
immune system develops initially from a population of plurip-
otent HSCs that gives rise to all circulating blood cell lineages, ASSESSMENT OF IMMUNOLOGIC
including cells of the immune system. The bone marrow and INTEGRITY
thymus are the primary sites of lymphopoiesis and appear to be
unique in providing the microenvironment factors necessary Xenobiotics can have significant effects on the immune system.
for the development of functionally competent immune cells. Among the unique features of immune cells is their ability to be
Immune system development does not cease at birth, but removed from the body and to function in vitro. This unique
continues to develop until 5 to 12 years of age in humans. After quality offers the toxicologist an opportunity to comprehen-
birth, immunocompetent cells continue to be produced from sively evaluate the actions of xenobiotics on the immune system.
proliferating progenitor cells in the bone marrow and thymus. Many medical devices may have intimate and prolonged
These cells subsequently migrate via the blood to the second- contact with the body. Possible immunologic consequences of
ary immune organs: spleen, lymph nodes, and mucosal lym- this contact could be envisioned to include immunosuppres-
phoid tissues. The onset of functional immune competence sion, immune stimulation, inflammation, and sensitization.
188 UNIT 4 Target Organ Toxicity

Methods to Assess Immunocompetence for sRBC-specific IgM or IgG to bind. After incubation, an
enzyme-conjugated monoclonal antibody (the secondary
General Assessment—All studies of immunocompetence
antibody) against IgM (or IgG) is added. This antibody recog-
should include toxicologic studies (such as organ weights,
nizes the IgM (or IgG) and binds specifically to that antibody.
serum characteristics, hematologic parameters, and bone mar-
Then, the enzyme substrate (chromogen) is added. When the
row function) to investigate the effects of immune modulation
substrate comes into contact with the enzyme on the second-
on other body organs. Histopathology of lymphoid organs
ary antibody, a color change occurs that can be detected by
also may provide insight into potential immunotoxicants.
measuring absorbance with a plate reader.
Moreover, use of fluorescently labeled monoclonal antibod-
ies to cell surface markers in conjunction with a flow cytom-
Acquired Immunity: Cell-mediated—Of numerous assays of
eter enables accurate enumeration of lymphocyte subsets and
CMI, three routinely performed tests are the cytotoxic T lym-
whether the xenobiotic may affect maturation.
phocyte (CTL) assay, the delayed hypersensitivity response
Functional Assessment (DHR), and the T-cell proliferative responses to antigens.
Innate Immunity—Innate immunity encompasses all those The CTL assay measures the in vitro ability of splenic T cells
immunologic responses that do not require prior exposure to to recognize allogeneic or antigenically distinct target cells by
an antigen and that are nonspecific in nature. These responses evaluating the ability of the CTLs to proliferate and then lyse
include recognition of tumor cells by NK cells, phagocytosis of the target cells. CTLs are incubated with target cells that have
pathogens by macrophages, and the lytic activity of the com- been treated so that they cannot themselves proliferate. CTLs
plement system. recognize the target cells and proliferate until they are har-
To evaluate phagocytic activity, macrophages are placed in vested. Then, they are incubated with radiolabeled target cells.
culture plates and incubated with radiolabeled red blood cells. CTLs that have acquired memory recognize the foreign MHC
Those cells that are not bound by the macrophages are removed, class I on target cells and lyse them.» ,
as are the cells that are bound but not phagocytized. The mac- The DHR evaluates the ability of memory T cells to recog-
rophages are then lysed to determine the amount of cells that nize foreign antigen, proliferate and migrate to the site of the
were phagocytized. This test provides information about both antigen, and secrete cytokines in vivo. Mice are sensitized by a
the binding and phagocytizing activity of the macrophages and subcutaneous injection of the chemical. Radiolabeled iodine is
can also be performed in vivo by measuring the uptake of the allowed to be incorporated into the mouse’s mononuclear cells
radiolabeled red blood cells by certain tissue macrophages. by injecting it into the mouse’s bloodstream. Then, some ofthe
Another method to evaluate phagocytosis in vitro is to eval- sensitizing chemical is injected into the ear, and, after eutha-
uate the uptake of latex spheres by macrophages. Evaluation of nizing the mouse, the ear is evaluated for the presence ofradio-
the ability of NK cells to lyse tumor cells is achieved by incu- labeled mononuclear cells.
bating radiolabeled target cells with NK cells and measuring Several mechanisms exist to evaluate proliferative capac-
the amount of radioactivity released into solution from the ity of T cells in CMI. The mixed lymphocyte response (MLR)
target cells. measures the ability of T cells to recognize foreign MHC class I
and undergo proliferation.
Acquired Immunity: Humoral—The plaque (antibody)-forming
cell (PFC or AFC) assay tests the ability of the host to mount Flow Cytometric Analysis—Flow cytometry employs light
an antibody response to a specific antigen, which requires scatter, fluorescence, and absorbance measurements to ana-
the coordinated interaction of several different immune cells: lyze large numbers of cells on an individual basis. Usually,
macrophages, T cells, and B cells. Therefore, an effect on any fluorochrome-conjugated monoclonal antibodies raised
of these cells (e.g., antigen processing and presentation, cyto- against a specific protein are employed for detection. This
kine production, proliferation, or differentiation) can have a approach can be used to provide insight into which specific
profound impact on the ability of B cells to produce antigen- T-cell subsets are targeted after exposure to a xenobiotic, and
specific antibody. to identify putative effects on T-cell maturation.
A standard PFC assay involves immunizing mice with sRBC.
The antigen is taken up in the spleen and an antibody response Molecular Biology Approaches to lmmunotoxicology—
occurs. Four days after immunization, spleens are removed Proteomics (the study of all expressed proteins in a particular
and splenocytes are mixed with RBCs, complement, and agar, cell, and thus the functional expression of the genome) and
the mixture plated, and incubated until the B cells secrete anti- genomics (the study of all genes encoded by an organisms
SRBC IgM antibody. This antibody then coats the surrounding DNA), combined with bioinformatics, facilitate the evaluation
sRBCs, and areas of hemolysis (plaques) can be seen. of xenobiotic-induced alterations in the pathways and signal-
The PFC assay can be evaluated in vivo using serum from ing networks of the immune system.
peripheral blood of immunized mice and an enzyme-linked
immunosorbent assay (ELISA). Serum from mice immu- Mechanistic Approaches to Immunotoxicology—Once
nized with sRBCs is incubated in microtiter plates that have an agent has been identified as beingan immunotoxicant,
been coated with sRBC membranes to serve as the antigen it may be necessary to further characterize its mechanism.
 CHAPTER 12 Toxic Responses of the Immune System 189

A general strategy involves the following steps: (1) identifying in lymphoid organs or in serum. Xenobiotics may exert an
the cell type(s) targeted by the agent, (2) determining whether indirect action on the immune system as well. They may be
the effects are mediated by the parent compound or by a metabolically activated to their toxic metabolites, and may also
metabolite of the parent, (3) determining whether the effects have effects on other organ systems (e.g., liver damage) that
are mediated directly or indirectly by the xenobiotic, and then impact the immune system.
(4) elucidating the molecular events responsible for altered
leukocyte function.
IMMUNE MODULATION
Regulatory Approaches to the Assessment BY XENOBIOTICS
of Immunotoxicity
The NTP Tier Approach—tThe National Toxicology Program The expansive and versatile nature of the immune system
screens for potential immunotoxic agents using a tier approach. renders it susceptible to modulation by a wide variety of
Tier I provides assessment of general toxicity (immunopathol- xenobiotics (Table 12-2). Many xenobiotics exhibit immu-
ogy, hematology, and body and organ weights) as well as end- nosuppressive actions, whereas some are immunomodula-
line functional assays (proliferative responses, PFC assay, and tory, meaning they might produce immune suppression and
NK assay). Tier II was designed to further define an immu- immune enhancement. Regardless of the end effect (immune
notoxic effect and includes tests for CMI (CTL and DHR), suppression, immune enhancement, hypersensitivity, or auto-
secondary antibody responses, enumeration of lymphocyte immunity) of a particular xenobiotic on the immune system,
populations, and host resistance models. several common themes exist regarding the mechanisms by
which these chemicals act. First, the mechanisms by which a
Health Effects Test Guidelines—Guidelines for functional xenobiotic affects immune function are likely to be multifac-
immunotoxicity assessments in regulatory studies recommend eted, involving several proteins, signaling cascades, or receptors.
conduct of three tests. Assessment of immunotoxicity begins In fact, there is evidence to suggest that immune system effects
by exposure for a minimum of 28 days to the chemical followed for some xenobiotics are both xenobiotic-specific receptor-
by assessment of humoral immunity (PFC assay or anti-sRBC dependent and independent. Second, whether a xenobiotic
ELISA). Ifthe chemical produces significant suppression of the produces a particular immune effect might depend on the con-
humoral response, surface marker assessment by flow cytome- centration or dose of the xenobiotic, the mode and/or magni-
try may be performed. Ifthe chemical produces no suppression tude of cellular stimulation, and the kinetic relationship between
of the humoral response, an assessment of innate immunity exposure to the xenobiotic and exposure to the immune stimu-
(NK assay) may be performed. lant (i.e., antigen, mitogen, and pharmacological agent). Third,
xenobiotic exposures rarely occur in one chemical at a time;
thus, the effects and/or mechanisms observed might be attrib-
Animal Models in Immunotoxicology utable to several chemicals or classes of chemicals. Finally,
determination of immune system effects and/or mechanisms
Rats and mice have been the animals of choice for studying the
actions of xenobiotics on the immune system because (1) there
by xenobiotics in humans might be further confounded by the
physiological or immunological state of the individual.
is a vast database available on the immune system, (2) rodents
are less expensive to maintain than larger animals, and (3) a
wide variety of reagents (cytokines, antibodies, etc.) are avail- Halogenated Aromatic Hydrocarbons
able. Many reagents that are available for studying the human Few classes of xenobiotics have been as extensively studied for
immune system can also be used in rhesus and cynomolgus immunotoxicity as the halogenated aromatic hydrocarbons
monkeys. Chicken and fish are being used to evaluate the (HAHs). The majority of the biochemical and toxic effects pro-
immunotoxicity of xenobiotics as alternative animal models duced by the HAHs are mediated via HAH binding to the cyto-
with heightened environmental consciousness. solic aromatic hydrocarbon receptor (AHR). Binding of HAH
The manipulation of the embryonic genome, creating trans- to AHR ultimately results in upregulation of certain proteins
genic and knockout mice, may allow complex immune responses with a net immunosuppressive effect. Interestingly, the degree
to be dissected into their components. In this way, the mecha- of immunosuppression is positively correlated with the bind-
nisms by which immunotoxicants act can be better understood. ing affinity of the HAH for the AHR.
Severe combined immunodeficient (SCID) mice have been used
to study immune regulation, hematopoiesis, hypersensitivity,
and autoimmunity. Pesticides
-
Pesticides include all xenobiotics whose specific purpose is
to kill another form of life, including insects (insecticides),
Evaluation of Mechanisms of Action small rodents (rodenticides), or even vegetation (herbicides).
Direct effects on the immune system may include chemical Exposure to pesticides occurs most often in occupational set-
effects on immune function, structural alterations in lymphoid tings, in which manufacturers, those applying the pesticides,
organs or on immune cell surfaces, or compositional changes or those harvesting treated agricultural products, are exposed.
190 UNIT 4 Target Organ Toxicity

TABLE 12-2 Xenobiotics capable of immunosuppression.

hl ay ae A Gna Oe aah ae Re ee Se ee ee
Halogenated aromatic hydrocarbons Aromatic hydrocarbons

Polychlorinated biphenyls Carbon tetrachloride

Polybrominated biphenyls Ethylene glycol monomethy! ether
Polychlorinated dibenzodioxins 2-Methoxyethanol
Polychlorinated dibenzofurans
Mycotoxins
Polycyclic aromatic hydrocarbons
Aflatoxin
Nitrosamines Ochratoxin
Tricothecenes
Pesticides Vomitoxin

Organophosphate pesticides Natural and synthetic hormones

Organochlorine pesticides
Organotin pesticides Estrogens
Carbamate pesticides Androgens
Pyrethroids Glucocorticoids

Metals Therapeutics

Arsenic AIDS therapeutics

Beryllium Biologics :
Cadmium Anti-inflammatory agents
Chromium
Cobalt Immunosuppressive drugs ae
Gold
Lead Azathioprine
Mercury Cyclophosphamide
Nickel Cyclosporin A
Platinum Leflunomide
Rapamycin
Inhaled substances Stavudine (2’,3’-didehydro-2',3-dideoxythymidine)
Videx (2’,3’-dideoxyinosine; ddl)
Asbestos Zalcitabine (2’3’-dideoxycytidine; ddC)
Ethylenediamine Zidovudine (3’-azido-3’-deoxythymidine; AZT)
Formaldehyde
Silica Drugs of abuse
Tobacco smoke
Urethane Cannabinoids
Cocaine
Oxidant gases Ethanol
Opioids: heroin and morphine
Ozone (O;)
Nitrogen dioxide (NO,)
Sulfur dioxide (SO,)
Phosgene

Pesticides act through a variety of mechanisms and can be both from paint). Many metals are immunotoxic and the interested
immunosuppressive and immunoenhancing (see Chapter 22). reader is referred to Chapter 23.

Metals Solvents and Related Chemicals

Generally speaking, metals target multiple organ systems and There is limited, but substantive, evidence that exposure to
exert their toxic effects via an interaction of the free metal organic solvents and their related compounds can produce
immune suppression. Chemicals in this category are aromatic
with targets, such as enzyme systems, membranes, or cellu-
hydrocarbons, such as benzene, haloalkanes and haloalkenes,
lar organelles. In considering their immunotoxicity, metals at
glycols and glycol ethers, and nitrosamines. The interested
high concentrations usually exert immunosuppressive effects;
however, at lower concentrations, immune enhancement is reader is referred to Chapter 24.
often observed. Furthermore, as with most immunotoxicants,
exposures to metals are likely not single exposures, although Mycotoxins
one metal might dominate depending on the exposure condi- Mycotoxins are structurally diverse secondary metabolites of
tions (e.g., high levels of mercury in fish or high levels of lead fungi (see Chapter 26). This class of chemicals comprises such
 CHAPTER 12 Toxic Responses of the Immune System 19]

toxins as aflatoxin, ochratoxin, and the trichothecenes, nota- Biologics—Biologics refers to those therapies that are derived
bly T-2 toxin and deoxynivalenol (vomitoxin). As a class, these in some manner from living organisms and include monoclo-
toxins can produce cellular depletion in lymphoid organs, nal antibodies, recombinant proteins, and adoptive cell thera-
alterations in T- and B-lymphocyte function, suppression of pies. By its very nature, the immune system is often both the
antibody responses, suppression of NK cell activity, decreased intended therapeutic target and unintended toxicological tar-
DTH responses, and an apparent increase in susceptibility to get of various biologics. Overall, manifestations of toxicity may
infectious disease. include exaggerated pharmacology, effects due to biochemical
cross-talk, and disruptions in immune regulation by cytokine
networks. Monoclonal antibodies can bind normal as well as
Natural and Synthetic Hormones targeted tissues, and any foreign protein may elicit the produc-
It is well established that a sexual dimorphism exists in the tion of neutralizing antibodies against the therapeutic protein
immune system. Females have higher levels ofcirculating Igs, (i.e., the therapeutic protein may be immunogenic).
a greater antibody response, and a higher incidence of auto-
immune disease than males. Males appear to be more suscep- Anti-inflammatory Agents—Anti-inflammatory agents
tible to the development of sepsis and the mortality associated include nonselective and_ selective nonsteroidal anti-
with soft tissue trauma and hemorrhagic shock. Specific inflammatory drugs (NSAIDs), which suppress the production
natural sex hormones in this dichotomy have been impli- of pro-inflammatory soluble factors, such as prostaglandins
cated. Immune effects of androgens and estrogens appear to and thromboxanes. Nonselective NSAIDs are a large class of
be very tightly controlled within the physiological range of drugs that reversibly inhibit both isoforms of cyclooxygenase
concentrations, and profound changes in immune activity (COX-1 and COX-2). The COX-2 enzyme, in particular, is
can result from very slight changes in concentrations of hor- induced in response to inflammatory cytokines and media-
mones. The interested reader is referred to Chapters 20 and tors and, therefore, represents an attractive target to combat
21 for more detailed discussion of estrogens, androgens, and inflammatory diseases. Aspirin, like nonselective NSAIDs,
glucocorticoids. inhibits COX-1 and COX-2 enzymes, but inhibition is irrevers-
ible due to covalent binding ofaspirin by acetylation to a serine
residue in the COX enzyme. Aspirin is an especially effective
Therapeutic Agents antiplatelet agent since platelets possess little biosynthesizing
Historically speaking, very few drugs used today as immuno- capacity and, therefore, aspirin will inhibit COX for the life of
suppressive agents were actually developed for that purpose. the platelet (8-11 days).
In fact, nearly all therapeutic agents possess some degree of
immunomodulatory activity at some dose.
Drugs of Abuse
Immunosuppressive Agents—Several immunosuppres- Drug abuse is a social issue with extensive pathophysiological
sive drugs are efficacious simply due to their ability to impair effects on the abuser. Drugs of abuse exhibit immunosuppres-
cellular proliferation, since proliferation is required for lym- sive actions, and in fact it has been suggested that in addi-
phocyte clonal expansion and, subsequently, differentiation. tion to the direct risk of HIV contraction via needle sharing
Other drugs inhibit specific intracellular proteins that are or judgment lapses, abuse of some drugs has been associated
critical in the activation of the immune response. with disease progression to AIDS. Several classes of drugs
are included in this category, such as cannabinoids, opioids,
AIDS Therapeutics—Traditionally, antiviral therapies have cocaine, methamphetamine, and ethanol.
not been extremely successful in their attempt to rid the host
of viral infection because these pathogens target the DNA of
the host. Eradication of the infection means killing infected Inhaled Substances
cells, which for HIV are primarily CD4* T cells. Numerous Pulmonary defenses against inhaled gases and particulates are
strategies have been developed to combat HIV, including dependent on both physical and immunological mechanisms.
targeting viral reverse transcriptase (nucleoside and non- Immune mechanisms primarily involve the complex interac-
nucleoside reverse transcriptase inhibitors), viral protease, tions between neutrophils and alveolar macrophages and their
viral fusion and entry, virus-T-cell interaction proteins, and abilities to phagocytize foreign material and produce cyto-
stimulating immune responses. The multidrug therapy used kines, which not only act as local inflammatory mediators, but
currently is referred to as highly active antiretroviral therapy also serve to attract other cells into the airways.
(HAART). However, eradication of this virus, and subse-
quently AIDS, remains a challenge because the very nature
of the infection has significant immunosuppressive con- Ultraviolet Radiation
sequences. In addition, some of the current therapies also Ultraviolet radiation (UVR), especially midrange UVB
exhibit immunosuppressive actions. One such antiviral drug (290-340 nm), is an important environmental factor affecting
is zidovudine (Retrovir). human health with both beneficial effects including vitamin D
192 UNIT 4 Target Organ Toxicity

production, tanning, and adaptation to UV, and adverse Food and Genetically Modified Organisms— Awareness
effects including sunburn, skin cancer, and ocular damage. It of hypersensitivity reactions to foods and genetically modi-
is important to emphasize that all humans encounter lifetime fied organisms (or crops; GMOs) has increased in the last
exposure to this ubiquitous environmental immunotoxicant. several years. The most common food allergens are milk, egg,
While UV-induced immunomodulation has been shown to peanuts and other tree nuts, fish, shellfish, soy, and wheat.
have some beneficial effects on some skin diseases, such as Hypersensitivity to peanuts occurs primarily via a type I reac-
psoriasis, and has been demonstrated to impair some aller- tion, and the IgE responses may include shortness of breath,
gic and autoimmune diseases in both animals and humans. asthma, and anaphylaxis.
UV-induced immunomodulation can also lead to several
adverse health consequences, including a pivotal role during Formaldehyde—Formaldehyde is used as a preservative,
sterilant, and fumigant. Additional exposures come from the
the process ofskin carcinogenesis.
textile industry, where it is used to improve wrinkle resistance,
and in the furniture, auto upholstery, and resins industries.
XENOBIOTIC-INDUCED Occupational exposure to formaldehyde has been associated
both with the occurrence of asthma and increased respiratory
HYPERSENSITIVITY AND allergic responses to other stimuli.
AUTOIMMUNITY
When an individual’s immune system responds in a manner Autoimmunity
producing tissue damage, it could result in hypersensitivity or There are numerous reports of xenobiotics that have been
autoimmunity, which could be exacerbated, or even induced, associated with autoimmunity; however, firm evidence for
by another xenobiotic. their involvement is difficult to obtain. Presently, there are
very few instances of human autoimmune diseases for which
an environmental trigger has been definitely identified. These
Hypersensitivity relationships may be causative through direct mechanisms,
Numerous xenobiotics illicit hypersensitivity reactions. or they may be indirect, acting as an adjuvant. In the area
Polyisocyanates, and toluene diisocyanate in particular, used of xenobiotic-induced autoimmunity, exact mechanisms of
in the production of adhesives and coatings are known to action are not always known. Chemical exposure may also
induce the full spectrum of hypersensitivity responses, types I serve to exacerbate a preexisting autoimmune state.
to IV, as well as nonimmune inflammatory and neuroreflex
reactions in the lung. Inhaled acid anhydrides, which are used
in the manufacturing of paints, varnishes, coating materials, Therapeutic Agents
adhesives, and casting and sealing materials, may conjugate Methyldopa—Methyldopa is a centrally acting sympa-
with serum albumin or erythrocytes leading to type I, I, or III tholytic drug that has been widely used for the treatment of
hypersensitivity reactions on subsequent exposure. essential hypertension, but with the advent of newer antihy-
pertensive drugs, the use of methyldopa has declined. Platelets
Metals—Metals and metallic substances, including metal- and erythrocytes are targeted by the immune system in indi-
lic salts, are responsible for producing contact and pulmonary viduals treated with this drug, resulting in thrombocytopenia
hypersensitivity reactions. Platinum, cobalt, chromium, nickel, and hemolytic anemia.
and beryllium are commonly implicated.
Hydralazine, Isoniazid, and Procainamide—Hydralazine
Drugs—Hypersensitivity responses to drugs are among the is a direct-acting vasodilator drug used in the treatment of
major types of unpredictable drug reactions. Drugs are designed hypertension. Isoniazid is an antimicrobial drug used in the
to be reactive in the body and multiple treatments are common. treatment of tuberculosis. Procainamide is a drug that selec-
This type of exposure is conducive to producing an immuno- tively blocks sodium channels in myocardial membranes,
logic reaction. Immunologic mechanisms of hypersensitivity making it useful in the treatment of cardiac arrhythmias. All
reactions to drugs include types I to IV. Penicillin is the most three drugs produce autoimmunity, which is manifested as a
common agent involved in drug allergy. systemic lupus erythematosus-like syndrome.

Halothane—Halothane, one of the most widely studied of

Latex—Natural rubber latex is used in the manufacture of
the drugs inducing autoimmunity, is an inhalation anesthetic
over 40000 products from balloons to surgical gloves. Allergic
that can induce autoimmune hepatitis. The pathogenesis of the
reactions to natural rubber latex products have become an
hepatitis results from the chemical altering a specific liver pro-
important occupational health concern with increased use of
tein to such a degree that the immune system recognizes the
universal precautions, particularly latex gloves, to combat the
altered protein and antibodies are produced.
spread of bloodborne pathogens. Hypersensitivity to latex usu-
ally occurs via a type I or type IV reaction. Dermatologic reac- Vinyl Chioride—Vinyl chloride is used in the plastics
tions to latex include irritant dermatitis and contact dermatitis. industry as a refrigerant and in the synthesis of organic
 CHAPTER 12 Toxic Responses of the Immune System 193

chemicals. Although the exact mechanism whereby this chem- cytometry, have already and will continue to greatly facili-
ical produces autoimmunity is unclear, it is presumed that tate the application of human primary leukocytes in studies
vinyl chloride acts as an amino acid and is incorporated into of immunotoxicology.
protein. Because this would produce a structurally abnormal A second area that will see significant changes will be the
protein, which would be antigenic, an immune response would application of human-derived cell lines, and validation of
be directed against tissues with the modified protein present. assays using these cell lines for the purpose of evaluating and
screening potential immunotoxicants. A significant driver for
Mercury—This widely used metal is known to have several broader employment of cell lines in immunotoxicity testing
target systems, including the CNS and renal system. Mercury are cost, ethical considerations to reduce the use of animals in
also has two different actions with respect to the immune toxicity testing, and a fundamental belief that toxicants which
system. The first action is direct injury, and the second action alter one or more of the major signaling pathways regulating
is mercury-induced autoimmune disease that is manifested cell function can be identified in this manner.
as glomerular nephropathy. Antibodies produced to laminin The third area of emphasis in immunotoxicology will be
are believed to be responsible for damage to the basement the application of computational biology to better understand
membrane of the kidney. and describe the underlying molecular mechanisms by which
an immunotoxicant alters immune function. Computational
Silica—Crystalline silica (silicon dioxide) is a primary source biology has tremendous potential in estimating the potential
of elemental silicon and is used commercially in large quan- risk from exposure to immunotoxicants as well as predicting
tities as a constituent of building materials, ceramics, con- the risk associated with exposure to multiple immunotoxicants
cretes, and glasses. Experimental animals as well as humans simultaneously.
exposed to silica may have perturbations in the immune sys- The last area on the forefront of immunotoxicology is
tem. Depending on the length of exposure, dose, and route of increased use of transcriptome analysis. This change will be
administration of silica, it may kill macrophages or may act primarily driven by major advancements and applications of
as an immunostimulant. Silica has been shown to be associ- next generation sequencing which will likely make microar-
ated with an increase in scleroderma in silica-exposed work- rays obsolete due to the significantly greater sensitivity of this
ers. Adjuvancy as a mechanism of causing autoimmunity has technology, decreased cost, and open platform (i.e., capable
been implicated with a number of other chemicals, including of quantifying the entire transcriptome and not restricted
paraffin and silicones. to the DNA tiled on a chip). Moreover, the applications of
next generation sequencing beyond studies of the transcrip-
Hexachlorobenzene—Hexachlorobenzene is a low molecu- tome are considerable and span uses such as identification of
lar weight compound that was used in the past as a fungicide single-nucleotide polymorphisms associated with sensitive
for seed grains. After exposure to hexachlorobenzene, its depo- subpopulations and applications in personalized medicine to
sition can directly induce cell damage or elicit damage by inter- identification and analysis of DNA methylation for studies of
fering with the integrity of cell membranes due to its lipophilic epigenetics.
nature. Ultimately, hexachlorobenzene exposure triggers pro- In spite of these advances, significant challenges remain
inflammatory mediators, such as TNF-a, IL-1, IL-6, ROS, to be addressed within the discipline of immunotoxicology
and chemokines. These pro-inflammatory mediators serve as and include (1) how to interpret the significance of minor or
adjuvant signals that induce a systemic inflammatory response moderate immunotoxic effects in animal models in relation to
with influxes of neutrophils and macrophages into various human risk assessment; (2) how to better integrate a consider-
nonimmune and immune organs. Subsequently, this leads to ation of exposure, especially to multiple agents simultaneously,
polyclonal activation of T and B cells, eosinophilia, and even- into immunotoxicologic risk assessment; (3) how to design
tually to visible clinical effects. better human studies to assess the impact on the immune
system in the species of greatest interest in the context of risk
assessment; (4) how to identify and establish sensitive human
NEW FRONTIERS AND CHALLENGES biomarkers of immunotoxicity; and (5) how to gain a better
IN IMMUNOTOXICOLOGY understanding of the role of genetics in identifying sensitive
subpopulations to immune-altering agents.
There are several specific areas within the subdiscipline of
immunotoxicology that are currently on the forefront, but
will likely see significant advancements and changes. The first BIBLIOGRAPHY
will be the continued evolution and application of human pri-
Corsini E, Van Loveren H: Molecular Immunotoxicology, Weinheim:
mary leukocytes in mechanistic studies of immunotoxicol- Wiley-VCH, 2015.
ogy. In spite of the similarities between the human immune Hayes AW Kruger CL: Hayes’ Principles and Methods of Toxicology,
system and that of other animal species, there is an increasing 6th ed. Boca Raton, FL: CRC Press/Taylor & Francis, 2014.
appreciation that often subtle but potentially important dif- Nijkamp FP, Parnham MJ (eds.): Principles ofImmunopharmacology,
ferences exist. Advancements in technology, especially flow 3rd ed., Basel: Springer, 2011.
194 UNIT 4 Target Organ Toxicity

QUESTIONS

iF Which of the following cells or substances is NOT part of The delayed hypersensitivity response (DHR) test does NOT:
the innate immune system? a. evaluate memory T-cells’ ability to recognize a foreign
a. lysozyme. antigen.
b. monocytes. evaluate memory T-cells ability to secrete cytokines.
c. complement. OS: evaluate memory T-cells’ ability to proliferate.
d. antibodies. d. evaluate memory T-cells’ ability to lyse foreign target
e. neutrophils. cells.
e. evaluate memory T-cells’ ability to migrate to the site
Myeloid precursor stem cells are responsible for the of foreign antigen.
formation of all of the following EXCEPT:
a. platelets. The number of alveolar macrophages in smokers is greatly
b. lymphocytes. increased relative to nonsmokers. What is a characteristic
c. basophils. of the alveolar macrophages found in smokers?
d. erythrocytes. They are in an inactive state.
e. monocytes. They are far larger than normal.
They have increased phagocytic activity.
When an Rh— mother is exposed to the blood of an Rh+ They are incapable of producing cytokines.
baby during childbirth, the mother will make antibodies ne
aI They have decreased bactericidal activity.
against the Rh factor, which can lead to the mother attack-
ing the next Rh+ fetus. This is possible because of which Which of the following is NOT. characteristic of a type I
antibody’s ability to cross the placenta. hypersensitivity reaction?
a. IgM. a. Itis mediated by IgE.
b. IgE. b. It involves immune complex deposition in peripheral
can IgG: tissues.
d. IgA. © It involves mast-cell degranulation.
e. IgD. d. Anaphylaxis is an acute, systemic, and very severe
type I hypersensitivity reaction.
Which of the following statements is FALSE regarding e. It is usually mediated by preformed histamine,
important cytokine function in regulating the immune prostaglandins, and leukotrienes.
system?
a. IL-1 induces inflammation and fever. Which of the following types of hypersensitivity is NOT
b. IL-3 is the primary T-cell growth factor. mediated by antibodies?
c. IL-4 induces B-cell differentiation and isotype a. typel.
switching. b. type II.
d. ‘Transforming growth factor-8 (TGF-8) enhances c. type IIL.
monocyte/macrophage chemotaxis. d. type IV.
e. Interferon gamma (IFN-gamma) activates macrophages. em type,

Which of the following is NOT a step performed during 10. Which of the following is NOT a common mechanism of
an enzyme-linked immunosorbent assay (ELISA)? autoimmune disorders?
A chromogen is added and color is detected. a. subjection to positive selection in the thymus.
The antigen of interest is fixed to a microtiter plate. b. anergic T cells become activated.
Radioactively labeled cells are added to the solution. c. interference with normal immunoregulation by
Enzyme-tagged secondary antibodies are added. CD8* suppressor T cells.
oD Test sera are added.
cao lack of subjection to negative selection in the thymus.
e. decreased self-tolerance.
 Co jHe AGP Ts tieek

Toxic Responses
of the Liver
Hartmut Jaeschke

INTRODUCTION Disruption of the Cytoskeleton

Fatty Liver
LIVER PHYSIOLOGY
Fibrosis and Cirrhosis
Hepatic Functions Tumors
Structural Organization Critical Factors in Toxicant-induced Liver Injury
Bile Formation Uptake and Concentration

LIVER PATHOPHYSIOLOGY Bioactivation and Detoxification

Regeneration
Mechanisms and Types of Toxicant-induced
Inflammation and Immune Responses
Liver Injury
Activation of Sinusoidal Cells
Cell Death
Mitochondrial Damage
Canalicular Cholestasis
Idiosyncratic Liver Injury
Bile Duct Damage
Sinusoidal Damage FUTURE DIRECTIONS

KEY POINTS

w The liver’s strategic location between intestinal tract = Cholestasis is either a decrease in the volume of bile
and the rest of the body facilitates its maintenance of formed or an impaired secretion of specific solutes into
metabolic homeostasis in the body. bile, which results in elevated serum levels of bile salts
The liver extracts ingested nutrients, vitamins, metals, and bilirubin.
drugs, environmental toxicants, and waste products of Hepatocytes have a rich supply of phase I enzymes
bacteria from the blood for catabolism, storage, and/or that often convert xenobiotics to reactive electrophilic
excretion into bile. metabolites and of phase II enzymes that add a polar
Formation of bile is essential for uptake of lipid nutrients group to a molecule and thereby enhance its removal
from the small intestine, protection of the small intestine from the body. The balance between phase I and phase II
from oxidative insults, and excretion of endogenous and reactions determines whether a reactive metabolite will
xenobiotic compounds. initiate liver cell injury or be safely detoxified.

195
196 UNIT 4 Target Organ Toxicity

INTRODUCTION kill a considerable number of cells and when chronic insult

leads to replacement ofcell mass by nonfunctional scar tissue.
The liver is the main organ where exogenous chemicals are
metabolized and eventually excreted. As a consequence, liver
cells are exposed to significant concentrations of these chemi- Structural Organization
cals, which can result in liver dysfunction, cell injury, and even Two concepts exist for organization of the liver into operational
organ failure. The liver, with its multiple cell types and numer- units, namely, the lobule and the acinus. Classically, the liver
ous functions, can respond in many different ways to acute is divided into hexagonal lobules oriented around terminal
and chronic insults. To recognize potential liver cell dysfunc- hepatic venules (also known as central veins). At the corners
tion and injury, it is necessary to have a general knowledge of of the lobule are the portal triads (or portal tracts), containing
basic liver functions, the structural organization of the liver, a branch of the portal vein, a hepatic arteriole, and a bile duct
the processes involved in hepatic excretory functions, and (Figure 13-1). Blood entering from the portal vein and hepatic
mechanisms of cell and organ injury. artery mixes in the penetrating vessels, enters the sinusoids,
percolates along the cords of parenchymal cells (hepatocytes),
flows into terminal hepatic venules, and exits the liver via the
LIVER PHYSIOLOGY hepatic vein. The lobule is divided into three regions known as
centrolobular, midzonal, and periportal. The preferred concept
Hepatic Functions of a functional hepatic unit is the acinus. The base of the aci-
The liver’s strategic location between intestinal tract and the nus is formed by the terminal branches of the portal vein and
rest of the body facilitates the performance ofits enormous task hepatic artery, which extend out from the portal tracts. The aci-
of maintaining the metabolic homeostasis of the body. Venous nus has three zones: zone | is closest to the entry of blood, zone
blood from the stomach and intestines flows into the portal 3 abuts the terminal hepatic vein, and,zone 2 is intermediate.
vein, through the liver, and then enters the systemic circula- The three zones of the acinus roughly coincide with the three
tion. The liver is the first organ to encounter ingested nutrients, regions ofthe lobule (Figure 13-1).
vitamins, metals, drugs, and environmental toxicants as well Acinar zonation is of considerable functional consequence
as waste products of bacteria that enter portal blood. Efficient regarding gradients of components both in blood and in hepa-
scavenging or uptake processes extract these absorbed mate- tocytes. Blood entering the acinus consists of oxygen-depleted
rials from the blood for catabolism, storage, and/or excretion blood from the portal vein (60% to 70% of hepatic blood
into bile. flow) plus oxygenated blood from the hepatic artery (30% to
All of the major functions of the liver can be detrimentally 40%). En route to the terminal hepatic venule, oxygen rapidly
altered by acute or chronic exposure to toxicants (Table 13-1). leaves the blood to meet the high metabolic demands of the
When toxicants inhibit or otherwise impede hepatic transport parenchymal cells. Hepatocytes in zone 3 are exposed to sub-
and synthetic processes, dysfunction can occur without appre- stantially lower concentrations of oxygen than hepatocytes
ciable cell damage. Loss of function also occurs when toxicants in zone 1. In comparison to other tissues, zone 3 is hypoxic.

TABLE 13-1 Major functions of liver and consequences of impaired hepatic functions.
: Type of Function Examples Consequences of Impaired Functions

Nutrient homeostasis Glucose storage and synthesis Hypoglycemia, confusion

Cholesterol uptake Hypercholesterolemia

Filtration of particulates Products of intestinal bacteria (e.g., endotoxin) Endotoxemia

Protein synthesis Clotting factors Excess bleeding

Albumin Hypoalbuminemia, ascites
Transport proteins (e.g., very low-density Fatty liver
lipoproteins)

Bioactivation and detoxification Bilirubin and ammonia Jaundice, hyperammonemia-related coma

Steroid hormones Loss of secondary male sex characteristics
Xenobiotics Diminished drug metabolism
Inadequate detoxification

Formation ofbile and biliary secretion Bile acid—dependent uptake ofdietary lipids Fatty diarrhea, malnutrition, vitamin E
and vitamins deficiency
Bilirubin and cholesterol Jaundice, gallstones, hypercholesterolemia
Metals (e.g., Cuand Mn) Mn-induced neurotoxicity
Xenobiotics Delayed drug clearance
 CHAPTER 13 Toxic Responses ofthe Liver 197

Lobule particulate matter. Also, Kupffer cells are a major source of

cytokines and eicosanoids and can act as antigen-presenting
Bile duct ——> & cells (APCs). Ito cells (also known asfat-storing cells and stel-
Terminal
Hepatic artery ——» late cells) are located between endothelial cells and hepato-
hepatic cytes. Ito cells synthesize collagen and are the major storage site
vein for vitamin A in the body.

Bile Formation
Bile contains bile acids, glutathione, phospholipids, cholesterol,
bilirubin, and other organic anions, proteins, metals, ions, and
xenobiotics. Formation of this fluid is a specialized function
of the liver. Adequate bile formation is essential for uptake of
lipid nutrients from the small intestine (Table 13-1), protec-
tion of the small intestine from oxidative insults, and excre-
tion of endogenous and xenobiotic compounds. Hepatocytes
begin the process by transporting bile acids, glutathione, and
Acinus other solutes including xenobiotics and their metabolites into
the canalicular lumen (the space formed by specialized regions
FIGURE 13-1 Schematic of liver operational units, the classic of the plasma membrane between adjacent hepatocytes). The
lobule and the acinus. The lobule is centered around the terminal canaliculi are separated from the intercellular space between
hepatic vein (central vein), where the blood drains out of the lobule.
hepatocytes by tight junctions, which form a barrier permeable
The acinus has as its base the penetrating vessels, where blood
only to water, electrolytes, and to some degree to small organic
supplied by the portal vein and hepatic artery flows down the acinus
past the cords of hepatocytes. Zones 1, 2, and 3 of the acinus represent
cations. These canaliculi form channels between hepatocytes
metabolic regions that are increasingly distant from the blood supply. that connect to a series of larger and larger channels or ducts
within the liver. The large extrahepatic bile ducts merge into
the common bile duct. Bile can be stored and concentrated in
Well-documented acinar gradients exist for bile salts, biliru- the gallbladder before its release into the first segment of the
bin, and many organic anions as well. small intestine, the duodenum.
Heterogeneities in protein levels of hepatocytes along the The major driving force of bile formation is the active trans-
acinus generate gradients of metabolic functions. Hepatocytes port of bile salts and other osmolytes into the canalicular lumen.
in the mitochondria-rich zone 1 are predominant in fatty acid Most conjugated bile acids (taurine and glycine conjugates)
oxidation, gluconeogenesis, and ammonia detoxification to and some unconjugated bile acids are transported into hepato-
urea. Gradients of enzymes involved in the bioactivation and cytes by sodium-dependent transporters. Sodium-independent
detoxification of xenobiotics have been observed along the uptake of conjugated and unconjugated bile acids is performed
acinus by immunohistochemistry (exploiting the immune by members of the organic anion-transporting polypeptides
system's specificity to stain tissue). Notable gradients for hepa- (OATPs). OATPs also transport numerous drugs and hepato-
totoxicants are the higher levels of glutathione in zone 1 and toxicants. Lipophilic cationic drugs, estrogens, and lipids are
the greater amounts of cytochrome P450 proteins in zone 3, exported by the canalicular multiple-drug resistance (MDR)
particularly the CYP2E1 isozyme inducible by ethanol. P-glycoproteins, one of which is exclusive for phospholip-
Hepatic sinusoids are the channels between cords of hepato- ids. Conjugates of glutathione, glucuronide, and sulfate are
cytes where blood percolates on its way to the terminal hepatic exported by multidrug resistance—associated protein 2 (MRP2).
vein. The three major types of cells in the sinusoids are endo- The many different transporters are shown in Figure 13-2.
thelial cells, Kupffer cells, and stellate (Ito) cells. Sinusoids are Metals are excreted into bile by a series of processes that
lined by thin, discontinuous endothelial cells with numer- include (1) uptake across the sinusoidal membrane by facili-
ous fenestrae (or pores) that allow molecules smaller than tated diffusion or receptor-mediated endocytosis; (2) storage
250kDa to cross the interstitial space (known as the space of in binding proteins or lysosomes; and (3) canalicular secretion
Disse) between the endothelium and hepatocytes. The numer- via lysosomes, a glutathione-coupled event, or use of a specific
ous fenestrae and the lack of basement membrane facilitate canalicular membrane transporter, e.g., MRP2. Biliary excre-
exchanges of fluids and molecules, such as albumin, between tion is important in the homeostasis of metals, notably copper,
the sinusoid and hepatocytes, but hinder movement of parti- manganese, cadmium, selenium, gold, silver, and arsenic.
cles larger than chylomicron remnants. Inability to export Cu into bile is a central problem in Wilson’s
Kupffer cells, the resident macrophages of the liver, consti- disease, a rare genetic disorder characterized by accumulation
tute approximately 80% of the fixed macrophages in the body. of Cu in the liver and then in other tissues.
Kupffer cells are situated within the lumen of the sinusoid. Canalicular lumen bile is propelled forward into larger
The primary function of Kupffer cells is to ingest and degrade channels by dynamic, ATP-dependent contractions of the
198 UNIT 4 Target Organ Toxicity

OATP1B1

_ocnt| OATP1B3

OATP2B1

Hepatocyte

Cholehepatic
shunt pathway

Cholangiocyte

FIGURE 13-2 Transport proteins in human hepatocytes and cholangiocytes. Efflux transporters (blue ovals with blue arrows): BSEP,
bile salt export pump; MDR, multidrug resistance protein; MRP, multidrug resistance-associated protein; ABCG5/8, heterodimeric ATP binding
cassette transporter G5/G8; BCRP, breast cancer resistance protein; Osta/OstB, heterodimeric organic solute transporter alpha and beta. Uptake
transporters (green circles with red arrows): ASBT, apical sodium-—dependent bile salt transporter; NTCP, sodium taurocholate cotransporting
polypeptide; OATP, organic anion-transporting polypeptide; OCT, organic cation transporter; OAT, organic anion transporter. Transporters localized
to the sinusoidal membrane extract solutes from the blood. Exporters localized to canalicular membrane move solutes into the lumen of the
canaliculus. Exporters of particular relevance to canalicular secretion of toxic chemicals and their metabolites are the canalicular multiple organic
anion transporter (MOAT) system and the family of multiple-drug resistant (MDR) P-glycoproteins. MDR3 (ABCB4) flops phosphotidylcholine from
the inner to the outer leaflet of the canalicular membrane. ATP8B1 flips phosphatidylserine from the outer to inner membrane to maintain the
lipid asymmetry of the canalicular membrane. (Reproduced with permission from Pauli-Magnus C, Meier PJ: Hepatobiliary transporters and drug-
induced cholestasis, Hepatology, 2006 Oct;44(4):7 78-787.)

pericanalicular cytoskeleton. Bile ducts modify bile by absorp- neonates exhibit delayed development of bile salt synthesis
tion and secretion of solutes. Biliary epithelial cells also express and the expression of sinusoidal and canalicular transporters.
a variety of phase I and phase II enzymes, which may contrib- Neonates are more prone to develop jaundice when treated
ute to the biotransformation of toxicants present in bile. with drugs that compete with bilirubin for biliary clearance.
Secretion into biliary ducts is usually, but not always, a
prelude to toxicant clearance by excretion in feces or urine.
Exceptions occur when compounds are repeatedly delivered LIVER PATHOPHYSIOLOGY
into the intestinal lumen via bile, efficiently absorbed from
the intestinal lumen, and then redirected to the liver via portal
Mechanisms and Types of
blood, a process known as enterohepatic cycling. Toxicant-induced Liver Injury
Toxicant-related impairments of bile formation are more likely Hepatic response to insults by chemicals (Table 13-2) depends
to have detrimental consequences in populations with other on the intensity of the insult, the population of cells affected,
conditions where biliary secretion is marginal. For example, and whether the exposure is acute or chronic.
 CHAPTER 13 Toxic Responses ofthe Liver 199

TABLE 13-2 Types of hepatobiliary injury. of specific solutes into bile, cholestasis is characterized bio-
chemically by elevated serum levels of compounds normally
Type of Injury or Damage Representative Toxins concentrated in bile, particularly bile salts and bilirubin.
Fatty liver Amiodarone, CCI,, ethanol, fialuridine, When biliary excretion of the yellowish bilirubin pigment is
tamoxifen, valproic acid impaired, this pigment accumulates in the skin and eyes, pro-
ducing jaundice, and spills into urine, which becomes bright
Hepatocyte death Acetaminophen, allyl alcohol, Cu,
dimethylformamide, ethanol yellow or dark brown. Toxicant-induced cholestasis can be
transient or chronic; when substantial, it is associated with cell
Immune-mediated response Diclofenac, ethanol, halothane, swelling, cell death, and inflammation. Many different types of
tienilic acid
chemicals cause cholestasis (Table 13-2).
Canalicular cholestasis Chlorpromazine, cyclosporin A, The molecular mechanisms of cholestasis are related to
1,1-dichloroethylene, estrogens, expression and function of transporter systems in the basolat-
Mn, phalloidin
eral and canalicular membranes. An increased hepatic uptake,
Bile duct damage Alpha-naphthylisothiocyanate, decreased biliary excretion, and increased biliary reabsorption
amoxicillin, methylene dianiline, (cholehepatic shunting) of adrug may contribute to its accu-
sporidesmin mulation in the liver.
Sinusoidal disorders Anabolic steroids, cyclophosphamide, Bile formation is vulnerable to toxicant effects on the func-
microcystin, pyrrolizidine alkaloids tional integrity of sinusoidal transporters, canalicular export-
ers, cytoskeleton-dependent processes for transcytosis, and
Fibrosis and cirrhosis CCI,, ethanol, thioacetamide,
vitamin A, vinyl chloride
the contractile closure of the canalicular lumen (Figure 13-3).
Changes that weaken the junctions that form the structural bar-
Tumors Aflatoxin, androgens, arsenic, thorium rier between the blood and the canalicular lumen allow solutes
dioxide, vinyl chloride
to leak out of the canalicular lumen. These paracellular junc-
tions provide a size and charge barrier to the diffusion of solutes
Ceil Death—Based on morphology, liver cells can die by two between the blood and the canalicular lumen while water and
different modes, necrosis or apoptosis. Necrosis is character-
ized by cell swelling, leakage, nuclear disintegration (karyolysis),
and an influx ofinflammatory cells. When necrosis occurs in Diminished contractility
hepatocytes, the associated plasma membrane leakage can Impaired uptake of canaliculus

| ( | |
)
be detected biochemically by assaying plasma (or serum) for
liver cytosol-derived enzymes such as aspartate or alanine
aminotransferases (AST or ALT) or y-glutamyltranspeptidase '
ean we
: As)
|
(GGT). In contrast, apoptosis is characterized by cell shrinkage, } b

nuclear fragmentation, formation of apoptotic bodies, and a

Leaky paracellular
lack of inflammation. It is always a single cell event with the Diminished transcytosis junctions
main purpose of removing cells no longer needed during ————>"_, °

development or eliminating aging cells. ae ee re

Hepatocyte death can occur in a focal, zonal, or panacinar -+- | | $ .% 8 |

(widespread) pattern. Focal cell death is characterized by the

randomly distributed death of single hepatocytes or small clus- feltA,pat ope
ters of hepatocytes. Zonal necrosis is death to hepatocytes in
Concentration of
certain functional regions. Panacinar necrosis is massive death
Impaired secretion reactive species
of hepatocytes with only a few or no remaining survivors.
Mechanisms of toxicant-induced injury to liver cells include
lipid peroxidation, binding to cell macromolecules, mitochon-
drial damage, disruption of the cytoskeleton, and massive
calcium influx. Independent of the initial insult, the mitochon-
drial membrane permeability transition pore opens causing
collapse of the membrane potential and depletion of cellular FIGURE 13-3 Schematic of six potential mechanisms for
ATP, and necrotic cell death. The loss of ATP inhibits the ion cholestasis. Inhibited uptake, diminished transcytosis, impaired
pumps in the plasma membrane, which results in the loss of secretion, diminished canalicular contractility, leakiness of the junctions
cellular ion homeostasis and causes the characteristic swelling that seal the canalicular lumen from the blood, and detrimental
of oncotic necrosis. consequences of high concentrations of toxic entities in the
pericanalicular area are possible. Note that impaired secretion across
Canalicular Cholestasis—Defined physiologically as a the canalicular membrane can result from inhibition ofatransporter or
decrease in the volume of bile formed or an impaired secretion retraction of a transporter away from the canalicular membrane.
200 UNIT 4 Target Organ Toxicity

small ions diffuse across these junctions. One hepatotoxicant biliary proliferation and fibrosis resembling biliary cirrhosis.
that causes tight-junction leakage is a-napthylisothiocyanate. A rare response is the loss of bile ducts, a condition known as
Compounds that produce cholestasis do not necessarily vanishing bile duct syndrome. This persisting problem has been
act by a single mechanism or at just one site. Chlorpromazine reported in patients receiving antibiotics, anabolic steroids,
impairs bile acid uptake and canalicular contractility. Multiple contraceptive steroids, or the anticonvulsant carbamazepine.
alterations have been well documented for estrogens, a well-
known cause of reversible canalicular cholestasis. Estrogens Sinusoidal Damage—The sinusoid is, in effect, a special-
and progestins decrease bile salt uptake by effects at the sinu- ized capillary with numerous fenestrae for high permeability.
soidal membrane including a decrease in the Na*,K*-ATPase Functional integrity of the sinusoid can be compromised by
necessary for Na-dependent transport of bile salts across the dilation or blockade ofits lumen or by progressive destruction
plasma membrane and changes in lipid component of this of its endothelial cell wall. Dilation of the sinusoid will occur
membrane. At the canalicular membrane, estrogens diminish whenever efflux of hepatic blood is impeded. Blockade will
the transport of glutathione conjugates and reduce the number occur when red blood cells become caught in the sinusoids.
of bile salt transporters. Such changes have been illustrated after large doses of the drug
An additional mechanism for canalicular cholestasis is con- acetaminophen. A consequence of extensive sinusoidal block-
centration of reactive forms of chemicals in the pericanalicular ade is that the liver becomes engorged with blood cells and the
area (Figure 13-3). Most chemicals that cause canalicular cho- rest of the body goes into shock.
lestasis are excreted in bile. Therefore, the proteins and lipids in Progressive destruction of the endothelial wall of the sinu-
the canalicular region encounter a high concentration of these soid will lead to gaps and then ruptures of its barrier integrity,
chemicals. Observations consistent with this concentration with entrapment of red blood cells. These disruptions of the
mechanism have been reported for Mn, reactive thioether glu- sinusoid are considered the early structural features of the vas-
tathione conjugates of 1,1-dichloroethylene, and sporidesmin. cular disorder known as veno-occlusive disease, which occurs
Although no case of drug toxicity has been reported in after exposure to pyrrolizidine alkaloids, which may be found
response to modifications of basolateral uptake, OATPs can con- in some herbal teas and chemotherapeutic agents.
tribute to the liver injury potential of toxicants. The hepatotoxic-
ity of phalloidin, microcystin, and amanitin is facilitated by the Disruption of the Cytoskeleton—Phalloidin (from a mush-
uptake through OATPs. Furthermore, there is a growing list of room) and microcystin (from blue-green algae) disrupt the integ-
drugs including rifampicin, bosentan, and troglitazone, which rity of hepatocyte cytoskeleton by affecting proteins that are vital
are known to directly inhibit bile salt export pump (BSEP). to its dynamic nature, preventing disassembly of actin filaments.
Estrogens and progestins inhibit BSEP from the canalicular side Phalloidin uptake into hepatocytes leads to an accentuated actin
after excretion by MRP2. A substantial inhibition of bile salt web of cytoskeleton and the canalicular lumen dilates.
excretion can lead to accumulation of these compounds in hepa- Microcystin uptake into hepatocytes leads to hyperphos-
tocytes and may directly cause cell injury. However, more recent phorylation of cytoskeletal proteins. Reversible phosphoryla-
findings indicate that most of the bile acids accumulating in the tions of cytoskeletal structural and motor proteins are critical
liver after obstructive cholestasis are nontoxic and instead of cell to the dynamic integrity of the cytoskeleton. As depicted in
death cause proinflammatory gene expression in hepatocytes. Figure 13-4, extensive hyperphosphorylation produced by
While liver injury after obstructive cholestasis is produced large amounts of microcystin leads to marked deformation of
mainly by inflammatory cells, compensatory mechanisms hepatocytes due to a unique collapse of the microtubular actin
within the hepatocyte itself can limit this potential injury. Bile scaffold into a spiny central aggregate. Lower doses of micro-
acids are substrates for the nuclear farnesoid X receptor (FXR), cystin interfere with vesicle transport by hyperphosphorylat-
down-regulate NTCP and limit bile acid uptake. In addition, ing the transport protein dynein.
FXR activation causes increased expression of transporters on Dynein is a mechanicochemical protein that drives vesicles
the canalicular and basolateral membranes, which all work to along microtubules using energy from ATP hydrolysis; central to
limit the amount of bile acid accumulation. the hydrolysis ofthe dynein-bound ATP isa cycle of kinase phos-
phorylation and phosphatase dephosphorylation. Thus, hyper-
Bile Duct Damage—Damage to the intrahepatic bile ducts phosphorylation of dynein freezes this motor pump. Chronic
(which carry bile from the liver to the GI tract) is called exposure to low levels of microcystin has raised new concerns
cholangiodestructive cholestasis. A useful biochemical index about the health effects of this water contaminant. Specifically,
of bile duct damage is a sharp elevation in serum alkaline low levels of microcystin promote liver tumors and kill hepato-
phosphatase activity. In addition, serum levels of bile salts cytes in the zone 3 region, where microcystin accumulates.
and bilirubin are elevated, as observed with canalicular cho-
lestasis. Initial lesions following a single dose of cholangiode- Fatty Liver—Fatty liver (steatosis) is defined biochemically
structive agents include swollen biliary epithelium, debris of as an appreciable increase in the hepatic lipid (mainly triglyc-
damaged cells within lumens of the biliary tract, and inflam- eride) content, which is <5 wt% in the normal human liver.
matory cell infiltration of portal tracts. Chronic administra- Currently, the most common cause of hepatic steatosis is insu-
tion of chemicals that cause bile duct destruction can lead to lin resistance due to central obesity and sedentary lifestyle.
 CHAPTER 13 Toxic Responses ofthe Liver 201

Microcystin

Bile salts pi
Organic anions

Microtubular
Deformation
proteins
8)
5
~~ of hepatocyte
F
°
E. oS)
2
%,
@ 2%,
v2)
|
©

Microtubular
proteins(P)
Cytoskeleton
eee
Microtubular
dynein
2)Microcystin

oe. Diminished
ynein secretion
& export
—_—

FIGURE 13-4 Schematic of events in the mechanism by which microcystin damages the structural and functional integrity of
hepatocytes. Microcystin is taken up exclusively into hepatocytes by a sinusoidal transporter in a manner inhibitable by bile salts and organic
anions. Then microcystin inhibition of protein phosphatases leads to hyperphosphorylation of cytoskeletal proteins whose dynamic functions are
dependent on reversible phosphorylations. Extensive hyperphosphorylation of microtubular proteins leads to a collapse of the microtubular actin
filament scaffold into a spiky aggregate that produces a gross deformation of hepatocytes. More subtle changes in microtubule-mediated transport
activities have been linked to hyperphosphorylation of dynein, a cytoskeletal motor protein.

However, acute exposure to hepatotoxicants like carbon tet- patients have not been identified, the emerging evidence sug-
rachloride and some drugs can induce steatosis. Ethanol is by gests that the excessive burden of fatty acids in the liver from
far the most relevant drug or chemical leading to steatosis in either inappropriate lipolysis in adipose tissue or synthesis in
humans and in experimental animals. the liver may cause liver injury.
Often, drug-induced steatosis is reversible and does not lead This change, also known as steatosis, is a buildup of lipids in
to death of hepatocytes. The metabolic inhibitors ethionine, the hepatocyte. Fatty liver can stem from disruptions in lipid
puromycin, and cycloheximide cause fat accumulation with- metabolism. Steatosis isa common response to acute exposure
out causing cell death. Although steatosis alone may be benign, to many hepatotoxicants. Often, chemical-induced steatosis is
it can develop into steatohepatitis (alcoholic or nonalcoholic), reversible and does not lead to death of hepatocytes. Ethanol is
which is associated with significant liver injury. Livers with by far the most relevant drug or chemical leading to steatosis in
steatosis can be more susceptible to additional insults such as humans. The metabolic inhibitors ethionine, puromycin, and
hepatotoxicants or hepatic ischemia. cycloheximide cause fat accumulation without causing death
The previously preferred hypothesis of nonalcoholic ste- of cells. Many other conditions besides toxicant exposure, such
atohepatitis (NASH) considered triglyceride accumulation in as insulin resistance due to central obesity, are associated with
hepatocytes as the initial pathological event causing steatosis, marked fat accumulation in the liver.
with any additional stress (e.g., oxidant stress or lipid peroxi-
dation) causing progression to steatohepatitis. This thinking Fibrosis and Cirrhosis—Hepatic fibrosis (scarring) is char-
has recently been overturned and a new hypothesis postulates acterized by the accumulation of extensive amounts of col-
that nonalcoholic fatty liver disease (NAFLD) is mainly caused lagen fibers, in response to direct injury or to inflammation.
by lipotoxicity of non-triglyceride fatty metabolites. Although With repeated chemical insults, destroyed hepatic cells are
the specific fatty acids or their metabolites causing NAFLD in replaced by fibrotic scars. With continuing collagen deposition,
202 UNIT 4 Target Organ Toxicity

TABLE 13-3 Factors inthe site-specific injury of representative hepatotoxicants.

enn a ae een eee seen eet Ee ee ee
Site Representative Toxicants Potential Explanation for Site Specificity

Zone 1 hepatocytes (versus zone 3) Fe (overload) Preferential uptake and high oxygen levels

Allyl alcohol Higher oxygen levels for oxygen-dependent bioactivation

Zone 3 hepatocytes (versus zone 1) (ia, More P450 isozyme for bioactivation

Acetaminophen More P450 isozyme for bioactivation and less GSH for detoxification

Ethanol More hypoxic and greater imbalance in bioactivation/detoxification

reactions

Bile duct cells Methylene dianiline, sporidesmin Exposure to the high concentration of reactive metabolites in bile

Sinusoidal endothelium (versus Cyclophosphamide, monocrotaline Greater vulnerability to toxic metabolites and less ability to
hepatocytes) maintain glutathione levels

Kupffer cells Endotoxin, GdCl, Preferential uptake and then activation

Stellate cells Vitamin A Preferential site for storage and then engorgement

Ethanol (chronic) Activation and transformation to collagen-synthesizing cell

Benin

‘
’

the architecture of the liver is disrupted by interconnecting fundamental questions is incomplete. Influences of several
fibrous scars. When the fibrous scars subdivide the remaining factors are of obvious importance (Table 13-3). Location and
liver mass into nodules of regenerating hepatocytes, fibrosis specialized processes for uptake and biliary secretion produce
has progressed to cirrhosis and the liver has limited residual higher exposure levels in the liver than in other tissues of the
capacity to perform its essential functions. The primary cause body and strikingly high levels within certain types of liver cells.
of hepatic fibrosis/cirrhosis in humans worldwide is viral Then the abundant capacity for bioactivation reactions influ-
hepatitis. However, biliary obstruction and, in particular, alco- ences the rate of exposure to proximate toxicants. Subsequent
holic and NASH are of growing importance for the develop- events in the pathogenesis appear to be critically influenced by
ment of hepatic fibrosis. In addition, fibrosis can be induced responses of sinusoidal cells and the immune system.
by chronic exposure to drugs and chemicals especially ethanol A number of experimental systems are useful for defining
and heavy metals. Cirrhosis is not reversible, has a poor prog- factors and mechanisms of liver injury. In vitro systems using
nosis for survival, and is usually the result of repeated exposure the isolated perfused liver, isolated liver cells, and cell fractions
to chemical toxicants. allow observations at various levels of complexity without the
confounding influences of other systems. Models using cocul-
Tumors—Chemically induced neoplasia can involve tumors tures or agents that inactivate a given cell type can document
that are derived from hepatocytes, bile duct progenitor cells, the the contributions and interactions between cell types. Whole-
ductular “bipolar” progenitor cells, and the periductular stem animal models are essential for assessment of the progression
cells. The rare, highly malignant angiosarcomas are derived of injury and responses to chronic insult. Application of gene
from sinusoidal lining cells. Hepatocellular cancer has been transfection or repression attenuates some of these interpretive
linked to abuse of androgens, alcohol, and a high prevalence of problems. Knockout animals are extremely useful models for
aflatoxin-contaminated diets. studying complex aspects of hepatotoxicity.
Thorotrast (radioactive thorium dioxide used as a con-
trast medium for radiology) accumulates in Kupffer cells and Uptake and Concentration—Lipophilic drugs and envi-
emits radioactivity throughout its very extended half-life, thus ronmental pollutants readily diffuse into hepatocytes because
increasing the risk for developing gallbladder cancer about the fenestrated epithelium of the sinusoid enables close
14-fold and over 100-fold for liver cancers. Multiple types of contact between circulating molecules and hepatocytes. The
liver tumors are linked to thorium dioxide exposure. membrane-rich liver concentrates lipophilic compounds.
Other toxicants are rapidly extracted from blood because
they are substrates for sinusoidal transporters. Phalloidin and
Critical Factors in Toxicant-induced
microcystin are illustrative examples of toxicants that target
Liver Injury the liver as a consequence of extensive uptake into hepatocytes
Why is the liver the target site for so many chemicals of diverse by sinusoidal transporters. Vitamin A hepatotoxicity initially
structure? Why do many hepatotoxicants preferentially affects the sinusoidal stellate cells, which actively extract and
damage one type of liver cell? Our understanding of these store this vitamin. Cadmium hepatotoxicity becomes manifest
 CHAPTER 13 Toxic Responses of the Liver 203

when cells exceed their capacity to complex cadmium with the by metabolic activation and triggers sophisticated signaling
metal-binding protein metallothionein. mechanisms culminating in cell death (Figure 13-5). Second,
Hepatocytes contribute to the homeostasis of iron by the multitude of events following the initial stress offers many
extracting this essential metal from the sinusoid by a receptor- opportunities for therapeutic interventions at later time points.
mediated process and maintaining a reserve of iron within Because these events are not occurring in all cells to the same
the storage protein ferritin. Acute iron toxicity is most com- degree and at the same time, delayed interventions may not
monly observed in young children who accidently ingest iron completely prevent cell damage but limit the area of necrosis
tablets. The cytotoxicity of free iron is attributed to its func- enough to prevent liver failure.
tion as an electron donor for the formation of reactive oxygen
species, which initiate destructive oxidative stress reactions. Ethanol—Morbidity and mortality associated with the con-
Accumulation of excess iron beyond the capacity for its safe sumption of alcohol is mainly caused by the toxic effects of
storage in ferritin leads to liver damage. Chronic hepatic accu- ethanol on the liver. This targeted toxicity is due to the fact that
mulation of excess iron in cases of hemochromatosis is asso- >90% of a dose of ethanol is metabolized in the liver. Three
ciated with a spectrum of hepatic disease including a greater principal pathways of ethanol metabolism are known. In the
than 200-fold risk for liver cancer. primary pathway, ethanol is bioactivated by alcohol dehydro-
genase to acetaldehyde, a reactive aldehyde, which is subse-
Bioactivation and Detoxification— Hepatocytes have very quently detoxified to acetate by aldehyde dehydrogenase. Both
high constitutive activity of the phase I enzymes that often enzymes exhibit genetic polymorphisms that result in higher
convert xenobiotics to reactive electrophilic metabolites. Also, concentrations of acetaldehyde—a “fast” activity isozyme of
hepatocytes have a rich collection of phase II enzymes that add alcohol dehydrogenase [ALD2*2] and a physiologically very
a polar group to a molecule and thereby enhance its removal “slow” mitochondrial isozyme of aldehyde dehydrogenase
from the body. Phase II reactions usually yield stable, nonreac- [ALDH2*2]. Approximately 50% of Asian populations but vir-
tive metabolites. In general, the balance between phase I and tually no Caucasians have the slow aldehyde dehydrogenase;
phase II reactions determines whether a reactive metabolite alcohol consumption by people with this slow polymorphism
will initiate liver cell injury or be safely detoxified. Because the leads to uncomfortable symptoms of flushing and nausea due
expression of phase I and II enzymes and of the hepatic trans- to high systemic levels of acetaldehyde.
porters can be influenced by genetics (e.g., polymorphism of The second major pathway involves the alcohol-inducible
drug-metabolizing enzymes) and lifestyle (e.g., diet, consump- enzyme CYP2E1, which oxidizes ethanol to acetaldehyde. The
tion of other drugs and alcohol), the susceptibility to potential enzyme is located predominantly in hepatocytes of the centri-
hepatotoxicants can vary markedly between individuals. lobular region and requires oxygen and NADPH. Due to the
nature of the enzyme, this reaction is most relevant for high
Acetaminophen—One of the most widely used analgesics doses of ethanol and for chronic alcoholism. The third path-
acetaminophen (APAP) is a safe drug when used at therapeuti- way involves catalase in peroxisomes. In this reaction, ethanol
cally recommended doses. Overdose can cause severe hepato- functions as an electron donor for the reduction of hydro-
toxicity, and certain acquired factors (e.g., diet, drugs, diabetes, gen peroxide to water. Thus, the capacity of this pathway is
and obesity) can enhance hepatotoxicity. Typical therapeutic limited due to the low levels of hydrogen peroxide. It is esti-
doses of acetaminophen are not hepatotoxic, because most of mated that <2% of an ethanol dose is metabolized through
the acetaminophen gets glucuronidated or sulfated with little this pathway.
drug bioactivation. Injury after large doses of acetaminophen
is enhanced by fasting and other conditions that deplete gluta- Allyl Alcohol—An industrial chemical used in the production
thione and is minimized by treatments with N-acetylcysteine of resins, plastics, and fire retardants, allyl alcohol is also used
that enhance hepatocyte synthesis of glutathione. as a model hepatotoxicant due to its preferential periportal
Alcoholics are vulnerable to the hepatotoxic effects of (zone 1) hepatotoxicity. The alcohol is metabolized by alcohol
acetaminophen at dosages within the high therapeutic range. dehydrogenase to acrolein, a highly reactive aldehyde, which
This acquired enhancement has widely been attributed to is then further oxidized by aldehyde dehydrogenase to acrylic
accelerated bioactivation of acetaminophen to the electro- acid. The fact that the toxicity depends on depletion of hepatic
philic N-acetyl-p-benzoquinone imine (NAPQI) intermediate glutathione levels is prevented by inhibitors of alcohol dehy-
by ethanol induction of CYP2E1. Inducers of CYP3A includ- drogenase but enhanced by inhibitors of aldehyde dehydro-
ing many drugs and dietary chemicals potentially influence genase suggests that acrolein formation is the critical event in
acetaminophen toxicity. liver injury. Age and gender differences in allyl alcohol hepato-
Although many ofthe details of the mechanism for APAP- toxicity can be explained by variations in the balance between
induced hepatotoxicity remain to be elucidated, newly gained alcohol dehydrogenase and aldehyde dehydrogenase expres-
insight into signaling events in response to APAP overdose sion. The preferential occurrence of allyl alcohol injury in zone
suggests two fundamentally new developments. First, necrotic 1 hepatocytes is caused by the predominant uptake ofallyl alco-
cell death is in most cases not caused by a single catastrophic hol in the periportal region and the oxygen dependence of the
event but can be the result of a cellular stress, which is initiated toxicity. Although protein binding of the reactive metabolite
204 UNIT 4 Target Organ Toxicity

See Nucleus _

Joana
yYvy :
—
NAPQI

o: Gaines ee hentation > ae

‘a
ot

Oxidative stress a HOR EO

Nitric NR
y = \

F id oxggre ‘ AIF — Endonuclease G

uperoxide 7% ui/ \
7 = Peroxy= H— GSH Supplementation \
|SOD2 anItteS Nitrate i ATP.
RES 1 \y
g SOD2 1 / production
i
\ Hydrogen : en :

\ perogrs ae V7, Loss of

Xe _membrane
Oxidative stress Pysential
wee
panne
NAPQI ROS ince
\ F ROS >; \ = Nitrosative =
ee e: Z GN Py i 3 stress a ee
uy
JNK-GSTpi GSK-38 y
F 7 { MnAnnes

; activation ye , =—
GSTpi Fe ,
J H,0, HO
eNO Ey Lee Ts eax

fi ASKI AKI
\ ie
\ 2
\ /
we |

ary 5 ASK1
SS INC = = INI

FIGURE 13-5 Acetaminophen-induced mitochondrial oxidant stress and its influence on cellular signaling. Metabolism of APAP
results in the generation of the reactive intermediate, NAPQI, which forms protein adducts and induces mitochondrial oxidative stress. The
increased generation of superoxide and its reaction with NO results in the production of peroxynitrite. The superoxide can be scavenged by
SOD2 and converted into hydrogen peroxide, although the generation of peroxynitrite can interfere in this process by the nitration of SOD2.
Mitochondrial oxidative stress and hydrogen peroxide can also activate the mitogen-activated protein kinase, JNK, by multiple pathways, resulting
in its phosphorylation and translocation to the mitochondria. This then amplifies the mitochondrial oxidant stress, which, subsequently, leads
to activation of the mitochondrial permeability transition, and translocation of mitochondrial proteins, such as AIF and endonuclease G, to the
nucleus. This results in DNA fragmentation and, finally, oncotic necrosis. (Reproduced with permission from Jaeschke H, McGill MR, Ramachandra
A: Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity,
Drug Metab Rev, 2012 Feb;44(1):88-106.)

acrolein and subsequent adduct formation appears to be the indicates that CCl, also induces significant mitochondrial
main cause of liver cell death, lipid peroxidation can become a damage, which is dependent on lipid peroxidation events and
relevant mechanism of cell injury under conditions of a com- on CYP2E] activity. In addition, the «CCl, radical can directly
promised antioxidant status. Lipid peroxidation is caused by a bind to tissue macromolecules and some of the lipid peroxi-
reductive stress where the excessive NADH formation leads to dation products are reactive aldehydes, e.g., 4-hydroxynon-
mobilization of redox-active iron from storage proteins. enal, which can form adducts with proteins. These events
also cause the immune system to be involved, which can con-
Carbon Tetrachloride—Cytochrome P450-dependent con- tribute to liver injury. Conditions in which cytochrome P450
version of CCl, to eCCl, and then to CCl,OO¢ is the classic is depleted lead to decreased liver damage when exposed
example of xenobiotic bioactivation to a free radical that initi- to CCl.
ates lipid peroxidation by abstracting a hydrogen atom from
the polyunsaturated fatty acid of a phospholipid. Metabolic Regeneration—The liver has a high capacity to restore
activation of CCl, primarily involves CYP2E1. CCl,-induced lost tissue and function by regeneration. Loss of hepatocytes
lipid peroxidation increases the permeability of the plasma due to hepatectomy or cell injury triggers proliferation of all
membrane to Ca’*, leading to severe disturbances of the cal- mature liver cells. This process is capable of restoring the orig-
cium homeostasis and necrotic cell death. Recent research inal liver mass. However, regeneration is not just a response
 CHAPTER 13 Toxic Responses ofthe Liver 205

to cell death, but a process that actively determines the final but the mechanisms are not well understood. Two proposed
injury after exposure to hepatotoxic chemicals. Stimulation mechanisms of immune-mediated liver injury are the hapten
of repair by exposure to a moderate dose of a hepatotoxicant hypothesis and the danger hypothesis (Figure 13-7).
strongly attenuates tissue damage of a subsequent high dose The hapten hypothesis assumes that a reactive metabolite
of the same chemical. Tissue repair is dose-responsive up to a covalently binds to cellular proteins and the drug-modified
threshold, after which the injury is too severe and cell prolif- protein is taken up by APCs, cleaved to peptide fragments,
eration is inhibited. which are then presented within the major histocompatibility
complex (MHC) to T cells. This hypothesis does not explain,
Inflammation and Immune Responses—The activation however, why other drugs (e.g., APAP), which also form reac-
of resident macrophages (Kupffer cells), NK and NKT cells, tive metabolites and drug-modified proteins, do not trigger an
and the migration of activated neutrophils, lymphocytes, and immune response. The danger hypothesis (Figure 13-8) postu-
monocytes into regions of damaged liver are a well-recognized lates that damaged cells release danger signals, which induce
feature of the hepatotoxicity produced by many chemicals. The the upregulation of a peripheral protein B7 on activated anti-
main reason for an inflammatory response is to remove dead gen presenting cells (APCs), which when paired with CD28 on
and damaged cells. However, under certain circumstances, T cells generates a costimulatory signal. A cytotoxic immune
these inflammatory cells can aggravate the existing injury response occurs only when the T-cell receptor stimulation
by release of directly cytotoxic mediators or by formation of with the antigen is accompanied by an independent costimu-
pro- and anti-inflammatory mediators (Figure 13-6). lation of the T cell. In the absence of this costimulatory sig-
In addition to the activation of an inflammatory response, nal, the antigens derived from drug-modified proteins induce
immune-mediated reactions may also lead to severe liver immune tolerance.
injury. Drugs and chemicals that have been suggested to cause
immune-mediated injury mechanisms in the liver include Activation of Sinusoidal Cells—Four kinds of observa-
halothane, tienilic acid, and dihydralazine. A delay in onset of tions, collectively, indicate roles for sinusoidal cell (immune
the injury or the requirement for repeated exposure to the drug cells present in the liver sinusoids) activation as primary or
and the formation of antibodies against drug-modified hepatic secondary factors in toxicant-induced injury to the liver:
proteins are characteristic features of immune reactions, 1. Kupffer and Ito cells exhibit an activated morphology after
acute and chronic exposure to hepatotoxicants.
2. Pretreatments that activate or inactivate Kupffer cells
appropriately modulate the extent of damage produced
Chemical or Ischemic stress Direct cell injury by classic toxicants. Kupffer cell activation by vitamin A
profoundly enhances the acute toxicity of carbon tetra-
CD4'T cell Q chloride; this enhancement did not occur when animals
were also given an inactivator of Kupffer cells.
O in Leelee HMGB1
7, 3. Activated Kupffer cells secrete appreciable amounts of
\— ‘Complement | 0
TNF-o Wee. sp activation | soluble cytotoxins, including reactive oxygen and nitro-
: 5 — = [aad gen species.
Kupffer cell_ i190 ~C5a__—- Hepatocytes 4. Acute and chronic exposure to alcohol directly or
NADPHif 1
vy.
os
IL-13
oxidase \\
indirectly affects sinusoidal cells.
y , LY VPN Neutrophil Figure 13-8 summarizes information presented in this and
earlier sections of this chapter about the multiplicity of toxi-
GSH ROS 7 TNF-a, IL-1. ——>
=
|
TNF-a, IL-1 cant-induced interactions with and between various liver cells.
The effect on a given cell type can be direct or may result from a
ae Proteases f
a cascade of signals and responses between cell types.
Kupffer
HO) NF-KB->ICAM-1 HOCI <l
GSH _e Mitochondrial Damage—Mitochondrial DNA codes for
ck GSH >| |
\ several proteins in the mitochondrial electron transport chain.
“oOes ©.. ___ HMGB1 Nucleoside analog drugs for the therapy of hepatitis B and
CT adducts aati AIDS infections cause mitochondrial DNA damage directly,
when incorporation of the analog base leads to miscoding or
FIGURE 13-6 Self-perpetuating inflammatory response early termination of polypeptides. The severe hepatic mito-
after chemical or ischemic stress. C5aR, Coa complement receptor;
chondrial injury produced by the nucleoside analog fialuridine
CT, chlorotyrosine protein adducts; GSH, reduced glutathione;
is attributed to its higher affinity for the polymerase responsi-
HMGB1, high-mobility group box-1; HMPs, hypochlorous acid modified
proteins; HNE, hydroxynonenal; HOCI, hypochlorous acid; ICAM-1,
ble for mitochondrial DNA synthesis than for the polymerases
intercellular adhesion molecule-1; IFN-y, interferon-y; IL-1, interleukin-1, responsible for nuclear DNA synthesis. Mitochondrial DNA
LPS, lipopolysaccharide; NF-KB, nuclear factor-KB; ROS, reactive oxygen is also more vulnerable to miscoding (mutation) due to its
species; TLR4, toll-like receptor-4;
TNF, tumor necrosis factor. limited capacity for repair.
206 UNIT 4 Target Organ Toxicity

Reactive Covalent binding

—_ DGS CES metabolite to protein
(haptenization)

Vv
) Cell stress
; > Cell death (mild)

i]
; 1

= Antigen- Costimulation (danger) v

presenting Cytokines
cell
5

Costimulatory ¢ Ps \1
MHCII SS
oie — Bacteria
a. oe Viruses (HIV, HBV)
\O d ) HelperT cell
Inflammation

(Q) CytotoxicT cell B cells

ND

| Antihapten and Moan

autoantibodies
Allergic NaiveT cells
hepatotoxicity

FIGURE 13-7 The danger hypothesis for immune-mediated idiosyncratic hepatotoxicity. Hapten formation leading to major
histocompatibility complex class II (MHCII) presentation of haptenized peptide by antigen-presenting cells (APCs) along with costimulation of
APC signaling molecules by mild injury, inflammation, or infection promotes helper T-cell activation leading to T-cell responses to the antigen.
The cytotoxicT cells are then targeted against hepatocytes that express haptenized protein or MHCI presentation of haptenized peptides on the
cell surface. Antibody to haptenized protein or concomitant autoantibodies could theoretically mediate and promote antibody-dependent
cell-mediated hepatotoxicity. (Reproduced with permission from Kaplowitz N: Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov, 2005
June;4(6):489-499.)

FIGURE 13-8 Schematic depicting the complex cascade of toxicant-evoked interactions between hepatocytes and sinusoidal cells.
Sinusoidal cell responses to toxicants can lead to either injury or activation. A scenario could involve (1) toxicant injury to hepatocytes, (2) signals
from the injured hepatocyte to Kupffer and Ito cells, followed by (3) Kupffer cell release of cytotoxins, and (4) Ito cell secretion of collagen. Activation
of Kupffer cells is an important factor in the progression of injury evoked by many toxicants. Stimulation of collagen production by activated Ito
cells is a proposed mechanism for toxicant-induced fibrosis.
 CHAPTER 13 Toxic Responses ofthe Liver 207

TABLE 13-4 Examples of drugs with known most frequent reason for posting warnings, restricting use, or
idiosyncratic hepatotoxicity. even withdrawal of the drug from the market (Table 13-4). In
addition, idiosyncratic hepatotoxicity is observed after con-
A. Immune-mediated (allergic) idiosyncratic hepatotoxicity sumption of herbal remedies and food supplements. Because
* Diclofenac (analgesic)
+ Halothane (anesthetic)
idiosyncratic hepatotoxicity is a rare event for most drugs, it
« Nitrofurantoin (antibiotic) is likely that a combination of gene defects and adverse events
¢ Phenytoin (anticonvulsant) need to be present simultaneously in an individual to trigger
* Tienilic acid (diuretic)
the severe liver injury. A detailed genomic analysis of patients
B. Nonimmune-mediated (nonallergic) idiosyncratic with idiosyncratic responses to drug exposure may give addi-
hepatotoxicity tional insight as to what gene expression profile renders a
- Amiodarone (antiarrhythmic) patient susceptible.
+ Bromfenac (analgesic)—withdrawn from market
+ Diclofenac (analgesic)
« Disulfiram (alcoholism)
+ Isoniazid (antituberculosis) FUTURE DIRECTIONS
* Ketoconazole (antifungal)
Rifampicin (antimicrobial) Continued progress in the understanding of drug- and
¢ Troglitazone (antidiabetes)—withdrawn from market
chemical-induced hepatotoxicity will depend on the use of
* Valproate (anticonvulsant)
relevant in vivo and in vitro models including human hepato-
cytes and analysis of human liver tissue. Traditional mechanis-
tic investigations in combination with genomic and proteomic
Alcohol abuse causes mitochondrial injury by shifting the
approaches have the greatest potential to yield important
bioactivation/detoxification balance for ethanol, leading to an
new insight into pathophysiologic mechanisms. Progress in
accumulation of its reactive acetaldehyde metabolite within
the understanding of the liver’s response to known hepato-
mitochondria, because mitochondrial aldehyde dehydroge-
toxicants and other adverse conditions will not only aid in
nase is the major enzymatic process for detoxification of acet-
the development of therapies to limit and reverse acute and
aldehyde. Bioactivation of large amounts of ethanol by alcohol
chronic liver injury, but also improve the predictability of the
dehydrogenase hampers the detoxification reaction, since
potential hepatotoxicity of new drugs and other chemicals.
the two enzymes require the common, depletable cofactor
nicotinamide adenine dinucleotide (NAD). Any type of etha-
nol-induced change that enhances the leakiness of the mito-
chondrial transport chain would lead to an increased release of BIBLIOGRAPHY
reactive oxygen species capable of attacking nearby mitochon- Boyer TD, Manns MP, Sanyal AJ (eds.): Zakim and Boyer’s Hepatology:
drial constituents. A Textbook of Liver Disease. 6th ed. Philadelphia, PA: Saunders and
Elsevier, 2012.
Crawford JM: The Liver and the Biliary Tract. In Kumar V, Abbas AK,
Idiosyncratic Liver Injury—Idiosyncratic drug hepato-
Fausto N, Aster JC (eds.): Robbins and Cotran: Pathologic Basis of
toxicity is a rare but potentially serious adverse event, which Disease. 8th ed. Philadelphia, PA: Saunders, 2010.
is not clearly dose-dependent, is at this point unpredictable, Kaplowitz N, Deleve LD (eds.): Drug-induced Liver Disease. 3rd ed.
and affects only very few of the patients exposed to a drug Waltham, MA: Academic Press/Elsevier, 2013.
or other chemicals. However, idiosyncratic toxicity is a lead- Sahu S: Hepatotoxicity: From Genomics to In Vitro and In Vivo Models.
ing cause for failure of drugs in clinical testing and it is the Hoboken, NJ: John Wiley, 2007.
 UNIT 4 Target Organ Toxicity

QUESTIONS

The impairment of hepatic function can have numerous A patient suffering from canalicular cholestasis would
negative consequences. Which of the following. is likely NOT be expected to exhibit which of the following?
NOT caused by impaired hepatic function? a. increased bile salt serum levels.
a. jaundice. b. jaundice.
b. hypercholesterolemia. c. increased bile formation.
c. hyperammonemia. d. dark brown urine.
d. hyperglycemia. e. vitamin A deficiency.
e. hypoalbuminemia.
Which of the following statements regarding liver injury
All of the following statements regarding the liver are true is FALSE?
EXCEPT a. Large doses of acetaminophen have been shown to
a. The major role of the liver is to maintain metabolic cause a blockade of hepatic sinusoids.
homeostasis of the body. b. Hydrophilic drugs readily diffuse into hepatocytes
b. The liver encounters ingested nutrients before the because of the large sinusoidal fenestrations.
heart does. c. There are sinusoidal transporters that take toxicants
c. Hepatic triads contain a branch of the hepatic portal up into hepatocytes.
vein, a branch ofthe hepatic artery, and a bile ductule. d. Hepatocellular cancer has been associated with
The liver manufactures and stores bile. androgen abuse.
e. ‘The large fenestrae of hepatic sinusoids facilitate e. In cirrhosis, excess collagen is laid down in response
exchange of materials between the sinusoid and the to direct injury or inflammation.
hepatocyte.
The inheritance of a “slow” aldehyde dehydrogenase
Activation of which of the following cell types can result enzyme would result in which of the following after the
in increased secretion of collagen scar tissue, leading ingestion of ethanol?
to cirrhosis? a. high ethanol tolerance.
a. hepatocyte. b. _ little response to low doses of ethanol.
b. Ito cell. c. lowserum levels of acetaldehyde.
c. Kupffer cell. d. nausea.
d. endothelial cell. e. increased levels of blood ethanol compared to an
e. (-cell. individual with a normal aldehyde dehydrogenase.

Wilson's disease is a rare genetic disorder characterized Which of the following is not a common mechanism of
by the failure to export which of the following metals hepatocellular injury?
into bile? deformation of the hepatocyte cytoskeleton.
a. iron. mitochondrial injury.
Ds j2zine. cholestasis.
c. silver. interference with vesicular transport.
d. lead. ae
Coe increased transcytosis between hepatocytes.
e. copper.
10. Ethanol is not known to cause which of the following
Which of the following is NOT characteristic of apoptosis? types of hepatobiliary injury?
cell swelling. a. fatty liver.
nuclear fragmentation. hepatocyte death.
lack of inflammation. fibrosis.
programmed death. immune-mediated responses.
ge
oe
TENDchromatin condensation. sono canalicular cholestasis.
 COVTipe
Aa Paces Eek

Toxic Responses
of the Kidney
Rick G. Schnellmann

FUNCTIONAL ANATOMY BIOCHEMICAL MECHANISMS/MEDIATORS OF RENAL

Renal Vasculature and Glomerulus CELL INJURY
Proximal Tubule Cell Death
Loop of Henle Mediators ofToxicity
Distal Tubule and Collecting Duct Cellular/Subcellular and Molecular Targets

PATHOPHYSIOLOGIC RESPONSES OF THE KIDNEY SPECIFIC NEPHROTOXICANTS

Acute Kidney Injury Heavy Metals
Adaptation Following Toxic Insult Mercury
Chronic Kidney Disease Cadmium
Chemically Induced a,,-Globulin Nephropathy
SUSCEPTIBILITY OF THE KIDNEY TO
TOXIC INJURY Halogenated Hydrocarbons
Chloroform
Incidence and Severity of Toxic Tetrafluoroethylene
Nephropathy
Bromobenzene
Reasons for the Susceptibility of the Kidney
Mycotoxins
to Toxicity
Therapeutic Agents
Site-Selective Injury Rcetamincohen

elas a) , Nonsteroidal Anti-inflammatory Drugs

Proximal Tubular Injury
Aminoglycosides
Loop of Henle/Distal Tubule/Collecting
Amphotericin B
Duct Injury
Cyclosporine
Papillary Injury
Cisplatin
ASSESSMENT OF RENAL FUNCTION Radiocontrast Agents

209
210 UNIT 4 Target Organ Toxicity

a The kidney contributes to total body homeostasis via its » Renal transport, accumulation, and biotransformation
role in the excretion of metabolic wastes, the synthesis of xenobiotics contribute to the susceptibility of the
and release of renin and erythropoietin, and the regula- kidney to toxic injury.
tion of extracellular fluid volume, electrolyte composi- » Numerous nephrotoxicants cause mitochondrial dys-
tion, and acid-base balance. function via compromised respiration and ATP
» Xenobiotics in the systemic circulation will be delivered production, or some other cellular process, leading to
to the kidney in relatively high amounts. either apoptosis or necrosis.
a The processes that concentrate urine also serve to
concentrate potential toxicants in the tubular fluid.

The functional integrity of the mammalian kidney is vital to glomerular plasma flow rate. These arterioles are innervated by
total body homeostasis because the kidney plays a principal role the sympathetic nervous system and respond to nerve stimu-
in the excretion of metabolic wastes and in the regulation of lation, angiotensin II, vasopressin (also called arginine vaso-
extracellular fluid volume, electrolyte composition, and acid- pressin [AVP], anti-diuretic hormone {[ADH]), endothelin,
base balance. In addition, the kidney synthesizes and releases adenosine, and norepinephrine. The efferent arterioles drain-
hormones, such as renin and erythropoietin, and metabolizes ing the cortical glomeruli branch into a peritubular capillary
vitamin D, to the active 1,25-dihydroxyvitamin D, form. A network, whereas those draining the juxtamedullary glomeruli
toxic insult to the kidney therefore could disrupt any or all of forma capillary loop, called the vasa recta (literally, straight ves-
these functions and could have profound effects on total body sels), supplying the medullary structures. These postglomerular
metabolism. capillary loops provide delivery of nutrients to the postglo-
merular tubular structures, delivery of wastes to the tubule for
excretion, and return of reabsorbed electrolytes, nutrients, and
FUNCTIONAL ANATOMY water to the systemic circulation.
Gross examination of a sagittal section of the kidney reveals The glomerulus is a complex, specialized capillary bed com-
three clearly demarcated anatomical areas: the cortex, medulla, posed primarily of endothelial cells that are characterized by
and papilla (Figure 14-1). The cortex constitutes the major an attenuated and fenestrated cytoplasm, visceral epithelial
portion of the kidney and receives a disproportionately higher cells characterized by a cell body (podocyte) from which many
percentage (90%) of blood flow compared with the medulla trabeculae and pedicles (foot processes) extend, and a glomer-
(~6% to 10%) or papilla (1% to 2%). Thus, when a bloodborne ular basement membrane (GBM), which is a trilamellar struc-
toxicant is delivered to the kidney, a high percentage of the ture sandwiched between the endothelial and epithelial cells
material will be delivered to the cortex and will have a greater (Figure 14-2). A portion of the blood entering the glomerular
opportunity to influence cortical rather than medullary or pap- capillary network is fractionated into a virtually protein-free
illary functions. However, medullary and papillary tissues are and cell-free ultrafiltrate, which passes through Bowman’s
exposed to higher luminal concentrations of toxicants for pro- space and into the tubular portion of the nephron. The for-
longed periods of time, a consequence of the more concentrated mation of such an ultrafiltrate is the net result of the balance
tubular fluid and the more sluggish flow of blood and filtrate in between transcapillary hydrostatic pressure and colloid
these regions. oncotic pressure. An additional determinant of ultrafiltration
The functional unit of the kidney, the nephron, may be con- is the effective hydraulic permeability of the glomerular cap-
sidered in three portions: the vascular element, the glomeru- illary wall, in other words, the ultrafiltration coefficient (K;),
lus, and the tubular element. which is determined by the total surface area available for fil-
tration and the hydraulic permeability of the capillary wall.
Consequently, chemically induced decreases in glomerular
Renal Vasculature and Glomerulus filtration rate (GFR) may be related to decreases in transcapil-
The renal artery branches successively into interlobar, arcu- lary hydrostatic pressure and glomerular plasma flow due to
ate, interlobular arteries and afferent arterioles that supply increased afferent arteriolar resistance or to decreases in the
the glomerulus (Figure 14-1). Blood then leaves the glomeru- surface area available for filtration, resulting from decreases in
lar capillaries via the efferent arterioles. Both the afferent and the size and/or number of endothelial fenestrae or detachment
efferent arterioles control glomerular capillary pressure and or effacement of foot processes.
 CHAPTER 14 Toxic Responses ofthe Kidney Zit

such as inulin (MW ~5500), are freely filtered, whereas large

molecules, such as albumin (MW 56000-70000), are restricted.
Filtration of anionic molecules tends to be restricted compared
Interlobar
arteries
to that of neutral or cationic molecules of the same size; this is
primarily due to the charge-selective properties of the GBM
Arcuate
(Figure 14-2).
Renal ;
arteries
artery ‘

Proximal Tubule
Segmental The proximal tubule consists of three discrete segments: the
arteries Interlobular S, (pars convoluta), S, (transition between pars convoluta and
arterioles pars recta), and S, (pars recta) segments. The formation ofurine
is a highly complex and integrated process in which the vol-
ume and composition of the glomerular filtrate is progressively
altered as fluid passes through each ofthe different tubular seg-
ments. The proximal tubule is the workhorse of the nephron,
as it reabsorbs approximately 60% to 80% of solute and water
filtered at the glomerulus, mostly by numerous transport sys-
tems capable of driving the concentrative transport of many
metabolic substrates. Toxicant-induced injury to the proximal

Efferent Proximal Cortical

arteriole ~Glomerulus tubule collecting
Juxtaglomerular tubule
apparatus

Afferent
arteriole

Afferent
Bowman’s i
arteriole
space
Bowman's Efferent
capsule Bowman's arteriole
Arcuate Capsule
artery

Arcuate vein

Slit pores
Peritubular
capillaries
Epithelium

Collecting
Basement
duct
membrane

FIGURE 14-1 Schematic of the human kidney showing

the major blood vessels and the microcirculation and tubular Endothelium
components of each nephron. (Reproduced with permission
from Guyton AC, Hall JE: Textbook of Medical Physiology. 9th edition. B Fenestrations
Philadelphia, PA: Saunders/Elsevier; 1996.)
FIGURE 14-2 Schematic of the ultrastructure of the
glomerular capillary (A); cross section of the glomerular capillary
Although the glomerular capillary wall permits a high rate of membrane with the capillary epithelium, basement membrane
fluid filtration (~20% of blood entering the glomerulus is filtered and epithelium podocytes. (Reproduced with permission from
in a single pass), it provides a significant barrier to the trans- Guyton AC, Hall JE: Textbook of Medical Physiology. 9th edition.
glomerular passage of macromolecules. Thus, small molecules, Philadelphia, PA: Saunders/Elsevier; 1996)
212 UNIT 4 Target Organ Toxicity

tubule therefore will have major consequences to water and sol- late distal tubule and cortical collecting tubule. The combina-
ute balance. tion of medullary and papillary hypertonicity generated by
The proximal tubule also reabsorbs virtually all the filtered countercurrent multiplication and the action of ADH serves to
low-molecular-weight proteins by specific endocytotic pro- enhance water permeability of the medullary collecting duct.
tein reabsorption processes. An important excretory func- Chemicals that interfere with ADH action impair the concen-
tion of the proximal tubule is secretion of weak organic anions trating ability of the distal nephron.
and cations by specialized transporters that drive concentra-
tive movement of these ions from postglomerular blood into
proximal tubular cells, followed by secretion into tubular fluid. PATHOPHYSIOLOGIC RESPONSES
Toxicant-induced interruptions in the production of energy OF THE KIDNEY
for any of these active transport mechanisms or the function
of critical membrane-bound enzymes or transporters can pro- Acute Kidney Injury
foundly affect proximal tubular and whole-kidney function. One of the most common manifestations of nephrotoxic dam-
age is acute renal failure (ARF) or acute kidney injury (AKI).
AK is characterized by an abrupt decline in GFR with resulting
Loop of Henle azotemia, or a buildup of nitrogenous wastes in the blood. AKI
The thin descending and ascending limbs and the thick ascend- describes the entire spectrum of the disease and is defined as
ing limb of the loop of Henle are critical to the processes involved a complex disorder that comprises multiple causative factors
in urinary concentration (Figure 14-1). Approximately 25% with clinical manifestations ranging from minimal elevation in
of the filtered Na* and K* and 20% of the filtered water are serum creatinine to anuric renal failure.
reabsorbed by the segments of the loop of Henle. The tubular Any decline in GFR is complex and may result from prerenal
fluid entering the thin descending limb is iso-osmotic to the factors (renal vasoconstriction, intravascular volume depletion,
renal interstitium; water is freely permeable and solutes, such and insufficient cardiac output), postrenal factors (ureteral or
as electrolytes and urea, may enter from the interstitium. In bladder obstruction), and intrarenal factors (glomerulonephri-
contrast, the thin ascending limb is relatively impermeable to tis, tubular cell injury, death, and loss resulting in back-leak;
water and urea, and Na* and Cl are reabsorbed by passive renal vasculature damage; interstitial nephritis). Figure 14-3
diffusion. The thick ascending limb is impermeable to water, illustrates the pathways that lead to diminished GFR following
and electrolytes are reabsorbed by the active Na*/K*/2Cl- chemical exposure. Table 14-1 provides a partial list of chemi-
cotransport mechanism, with the energy provided by the cals that produce AKI through different mechanisms.
Na*,K*-ATPase. The maintenance of tubular integrity is dependent on cell-
to-cell and cell-to-matrix adhesion (Figure 14-4). It has been
hypothesized that after a chemical or hypoxic insult, adhesion
Distal Tubule and Collecting Duct of nonlethally damaged, apoptotic, and oncotic cells to the base-
The macula densa comprises specialized cells located between ment membrane is compromised, leading to gaps in the epithe-
the end of the thick ascending limb and the early distal tubule, lial cell lining, potentially resulting in back-leak of filtrate and
in close proximity to the afferent arteriole (Figure 14-1). Under diminished GFR. These detached cells may aggregate in the
normal physiologic conditions, increased solute delivery or tubular lumen (cell-to-cell adhesion) and/or adhere or reattach
concentration at the macula densa triggers a signal resulting to adherent epithelial cells downstream, resulting in tubular
in afferent arteriolar constriction leading to decreases in GFR obstruction.
(and hence decreased solute delivery). Thus, increases in fluid/ Extensive evidence supports the idea that inflammatory cells
solute out of the proximal tubule, due to impaired tubular reab- play a role in ischemia-induced AKI. Injury to the renal vascu-
sorption, will activate this feedback system, referred to as tubu- lature endothelium results in chemokine and proinflammatory
loglomerular feedback (TGF) and resulting in decreases in the cytokine production and neutrophil adhesion, but the specific
filtration rate of the same nephron. This regulatory mechanism role of each inflammatory cell remains to be elucidated.
is a volume-conserving mechanism, designed to decrease GFR
and prevent massive losses of fluid/electrolytes due to impaired
tubular reabsorption. Humoral mediation of TGF by the renin- Adaptation Following Toxic Insult
angiotensin system has been proposed, and evidence suggests The kidney has a remarkable ability to compensate for a loss
that other substances may be involved. The early distal tubule in renal functional mass. Following a unilateral nephrectomy,
reabsorbs most of the remaining intraluminal Na*, K*, and Cl- GER of the remnant kidney increases by approximately 40% to
but is relatively impermeable to water. 60%. Compensatory increases in single-nephron GFR are
The late distal tubule, cortical collecting tubule, and medul- accompanied by proportionate increases in proximal tubular
lary collecting duct perform the final regulation and fine-tun- water and solute reabsorption; glomerulotubular balance (i.e.,
ing of urinary volume and composition. The remaining Na* constant fractional reabsorption of GFR by all segments of the
is reabsorbed in conjunction with K* and H* secretion in the nephron) is therefore maintained and overall renal function
 CHAPTER 14 Toxic Responses of the Kidney 218

A Normal

Afferent arteriole Efferent arteriole

Glomerular
plasma flow -
7
Glomerular .
a
hydrostatic -
pressure Glomerular
filtration

Intratubular
pressure

Afferent
B : C
arteriolar
y constriction Back-leak

| Glomerular Obstructing Leakage of

pressure cast filtrate

FIGURE 14-3 Mechanisms of reduction of GFR. (A) GFR depends on adequate blood flow to the glomerulus, adequate glomerular
filtration pressure, glomerular permeability and low intratubular pressure. (B) Afferent arteriolar constriction decreases GFR by reducing blood
flow, resulting in diminished capillary pressure. (C) Obstruction of the tubular lumen by cast formation increases tubular pressure; when tubular
pressure exceeds glomerular capillary pressure, filtration decreases or ceases. (D) Back-leak occurs when the paracellular space between cells
increases and the glomerular filtrate leaks into the extracellular space and bloodstream. (Reproduced with permission from Schrier RW: Atlas of
Diseases ofthe Kidney. Philadelphia, PA: Current Medicine; 1999.)

appears normal by standard clinical tests. Consequently, chem- may undergo cell repair and/or adaptation, which contrib-
ically induced changes in renal function may not be detected ute to the structural and functional recovery of the nephron
until these compensatory mechanisms are overwhelmed by (Figure 14-5). In addition, there is a population of uninjured
significant nephron loss and/or damage. cells that may undergo compensatory hypertrophy, cellular
There are a number of cellular and molecular responses adaptation, and cellular proliferation. Tubular epithelial cells
to a nephrotoxic insult. After a population of renal cells are are primarily responsible for the structural and functional
exposed to a toxicant, a fraction of the cells will be severely recovery of the nephron following injury by replacing dead
injured and undergo cell death by apoptosis or oncosis and detached cells through de-differentiation, proliferation,
(necrotic cell death). Those cells with nonlethal injuries migration, and re-differentiation.

TABLE 14-1 Mechanisms of chemically induced acute kidney injury.

Endothelial
RESORTS Interstitial
Injury - Glomerulopathy Nephritis
Diuretics Nonsteroidal anti- Sulfonamides Aminoglycosides Cyclosporine Gold Antibiotics
Angiotensin inflammatory Methotrexate Cisplatin Mitomycin C Penicillamine Nonsteroidal anti-
receptor drugs Acyclovir Vancomycin Tacrolimus Nonsteroidal anti- inflammatory
antagonists Radiocontrast Triamterene Pentamidine Cocaine inflammatory drugs
Angiotensin agents Ethylene glycol Radiocontrast agents | Conjugated drugs Diuretics
converting Cyclosporine Protease Heavy metals estrogens
enzyme Tacrolimus inhibitors Haloalkane- and Quinine
inhibitors Amphotericin B Haloalkene-
Antihypertensive cysteine
agents conjugates
214 UNIT 4 Target Organ Toxicity

Loss of polarity, tight junction

integrity, cell-substrate adhesion,
Intact tubular epithelium simplification of brush border

Toxic Cell
injury death
> |Apoptosis

Necrosis

Sloughing of viable
and nonviable cells
with intraluminal
cell-cell adhesion

RBSRB Cytoskeleton
Ge

{__} Extracellular matrix

Cast formation
‘ Nat,Kt-ATPase
and tubular
<= 3, Integrin
obstruction
RGD peptide

FIGURE 14-4 After injury, alterations can occur in the cytoskeleton and in the normal distribution of membrane proteins such
as Na*,K*t-ATPase, and (31 integrins in sublethally injured renal tubular cells. These changes result in loss of cell polarity, tight-junction
integrity, and cell-substrate adhesion. Lethally injured cells undergo oncosis or apoptosis, and both dead and viable cells may be released into
the tubular lumen. Adhesion of released cells to other released cells and to cells remaining adherent to the basement membrane may result
in cast formation, tubular obstruction, and further compromise the GFR. (Reproduced with permission from Schrier RW: Atlas of Diseases ofthe
Kidney. Philadelphia, PA: Current Medicine; 1999.)

Two of the most notable cellular adaptation responses are lithium, and cyclosporine). Following nephron loss, adap-
metallothionein induction and stress protein induction. The tive increases in glomerular pressures and flows increase the
distribution of individual heat-shock proteins (Hsps) and single-nephron GFR of remnant viable nephrons, which serve
glucose-regulated proteins (Grps) are two examples of stress to maintain whole-kidney GFR. With time, these alterations
protein families that are induced in response to a number of are maladaptive, and focal glomerulosclerosis eventually devel-
pathophysiologic states such as heat shock, anoxia, oxidative ops that may lead to tubular atrophy and interstitial fibrosis.
stress, toxicants, heavy metal exposure, and tissue trauma. Compensatory increases in glomerular pressures and flows of
The distribution of Hsps and Grps varies between different the remnant glomeruli may result in mechanical damage to the
cell types in the kidney and within subcellular compartments. capillaries, leading to altered permeabilities.
‘These proteins play important roles in protein folding, translo-
cation of proteins across organelle membranes, prevention of
aggregation of damaged proteins, and repair and degradation
SUSCEPTIBILITY OF THE KIDNEY
of damaged proteins, and thereby provide a defense mechanism
against toxicity and/or for the facilitation of recovery and repair. TO TOXIC INJURY
Incidence and Severity of Toxic
Chronic Kidney Disease Nephropathy
Progressive deterioration of renal function may occur with A wide variety of drugs, environmental chemicals, and met-
long-term exposure to various chemicals (e.g., analgesics, als can cause site-specific nephrotoxicity (Table 14-1). The
 CHAPTER 14 Toxic Responses of the Kidney Zils

declines markedly and AKI ensues due to the unopposed

actions of vasoconstrictors. Another example of predisposing
risk factors relates to the clinical use of angiotensin convert-
= Uninjured cells LL Injured cells a Cell death |
ing enzyme (ACE) inhibitors. Glomerular filtration pressure is
dependent on angiotensin II-induced efferent arteriolar con-
striction. ACE inhibitors block this vasoconstriction, resulting

f
NX

Cellular Cellular Cellular in a precipitous decline in filtration pressure and AKI.

hypertrophy adaptation proliferation repair

Re-epithelialization Cellular adaptation Site-Selective Injury

Many nephrotoxicants have their primary effects on discrete
Differentiation segments or regions of the nephron. The reasons underlying
this site-selective injury are complex but can be attributed in
part to site-specific differences in blood flow, transport and
Structural and functional recovery of the nephron
accumulation of chemicals, physicochemical properties of the
epithelium, reactivity of cellular/molecular targets, balance of
FIGURE 14-5 The response of the nephron to a nephrotoxic
insult. After a population of cells is exposed to a nephrotoxicant,
bioactivation/detoxification reactions, cellular energetics, and/
the cells respond; ultimately the nephron recovers function or, if cell or regenerative/repair mechanisms.
death and loss are extensive, nephron function ceases. Terminally
injured cells undergo cell death through oncosis or apoptosis. Cells
injured sublethally undergo repair and adaptation in response to
Glomerular Injury
the nephrotoxicant. Cells not injured and adjacent to the injured The glomerulus is the initial site of chemical exposure within
area may undergo dedifferentiation, proliferation, migration or the nephron, and a number of nephrotoxicants produce struc-
spreading, and differentiation. Cells not injured may also undergo tural injury to this segment. In certain instances, chemicals alter
compensatory hypertrophy in response to the cell loss and injury. glomerular permeability to proteins by altering the size- and
Finally the uninjured cells also may undergo adaptation in response charge-selective functions.
to a nephrotoxicant exposure. (Reproduced with permission from Cyclosporine, amphotericin B, and gentamicin impair glo-
Schrier RW: Atlas of Diseases of the Kidney. Philadelphia, PA: Current
merular ultrafiltration without significant loss of structural
Medicine; 1999.)
integrity and decrease GFR. Amphotericin B decreases GFR by
causing renal vasoconstriction and decreasing the glomerular
capillary ultrafiltration coefficient (K,). Gentamicin interacts
consequences of AKI vary from recovery to permanent renal with the anionic sites on the endothelial cells, decreasing K;
damage, which may require dialysis or renal transplantation. and GFR. Finally, cyclosporine not only causes renal vasocon-
striction and vascular damage, but is also injurious to the glo-
merular endothelial cell.
Chemically induced glomerular injury may also be mediated
Reasons for the Susceptibility of by extrarenal factors. Circulating immune complexes may be
the Kidney to Toxicity trapped within the glomeruli (as could be the case in a type 3
Although the kidneys constitute only 0.5% of total body mass, hypersensitivity reaction). Neutrophils and macrophages are
they receive about 20% to 25% of the resting cardiac output. commonly observed within glomeruli in membranous glo-
Consequently, any drug or chemical in the systemic circulation merulonephritis, and the local release of cytokines and reac-
will be delivered to these organs in relatively high amounts. The tive oxygen species (ROS) may contribute to glomerular
processes involved in forming concentrated urine also serve to injury. Heavy metals, hydrocarbons, penicillamine, and cap-
concentrate potential toxicants in the tubular fluid, thereby driv- topril can produce this type of glomerular injury. A chemical
ing passive diffusion of toxicants into tubular cells. Therefore, a may function as a hapten attached to some native protein or
nontoxic concentration of a chemical in the plasma may reach as a complete antigen and elicit an antibody response. Anti-
toxic concentrations in the kidney and its tubules. Finally, renal body reactions with cell-surface antigens (e.g., GBM) lead to
transport, accumulation, and metabolism of xenobiotics contrib- immune deposit formation within the glomeruli, mediator
ute significantly to the susceptibility of the kidney to toxic injury. activation, and subsequent injury to glomerular tissue.
In addition to intrarenal factors, the incidence and/or sever-
ity of chemically induced nephrotoxicity may be related to the
sensitivity of the kidney to circulating vasoconstrictors (angio- Proximal Tubular Injury
tensin IJ or ADH), whose actions are normally counterbal- The proximal tubule is the most common site of toxicant-
anced by the actions of increased vasodilatory prostaglandins. induced renal injury. The reasons for this relate in part to the
When prostaglandin synthesis is suppressed by nonsteroidal selective accumulation of xenobiotics into this segment of the
anti-inflammatory drugs (NSAIDs), renal blood flow (RBF) nephron. The proximal tubule has a leaky epithelium, favoring
216 UNIT 4 Target Organ Toxicity

the flux of compounds into proximal tubular cells. More impor- often lacking in such an assessment, urinalysis provides a rela-
tantly, tubular transport of organic anions and cations, low- tively easy and noninvasive assessment of overall renal func-
molecular-weight proteins and peptides, GSH conjugates, and tional integrity and can provide some insight into the nature
heavy metals is localized primarily if not exclusively to the prox- of the nephrotoxic insult. The simultaneous analysis of cellular
imal tubule. Thus, transport of these molecules will be greater in metabolites in sera and urine using nuclear magnetic analy-
the proximal tubule than in other segments, resulting in proxi- sis (metabonomics) has matured over the past few years and
mal tubular accumulation and toxicity. Although correlations may provide an additional technology to identify and monitor
between proximal tubular transport, accumulation, and toxic- nephrotoxicity.
ity suggest that the site of transport is a crucial determinant of Chemically induced increases in urine volume accompanied
the site of toxicity, transport is unlikely to be the sole criterion. by decreases in osmolality may suggest an impaired concentrat-
In addition to segmental differences in transport, segmen- ing ability, possibly via a defect in ADH synthesis, release, and/
tal differences in cytochrome P450 and cysteine conjugate or action. Glucosuria may reflect chemically induced defects
8-lyase activity also are contributing factors to the enhanced in proximal tubular reabsorption of sugars or be secondary to
susceptibility of the proximal tubule. Both enzyme systems hyperglycemia. Urinary excretion of high-molecular-weight
are localized almost exclusively in the proximal tubule, proteins, such as albumin, is suggestive of glomerular dam-
with negligible activity in the glomerulus, distal tubules, age, whereas excretion of low-molecular-weight proteins,
or collecting ducts. Thus, nephrotoxicity requiring P450 and such as 3,-microglobulin, suggests proximal tubular injury.
3-lyase-mediated bioactivation will most certainly be local- Urinary excretion of enzymes localized in the brush border
ized in the proximal tubule. (e.g., alkaline phosphatase and +glutamyl transferase) may
Finally, proximal tubular cells appear to be more suscepti- reflect brush-border damage, whereas urinary excretion of
ble to ischemic injury than distal tubular cells. Therefore, the other enzymes (e.g., lactate dehydrogenase) may reflect more
proximal tubule will likely be the primary site of toxicity for generalized cell damage. Enzymuria is often a transient phe-
chemicals that interfere with RBF, cellular energetics, and/or nomenon, as chemically induced damage may result in an early
mitochondrial function. loss of most of the enzyme available. Thus, the absence of enzy-
muria does not necessarily reflect an absence of damage.
GFR can be measured directly by determining creatinine or
Loop of Henle/Distal Tubule/Collecting inulin clearance, both of which are essentially freely filtered
Duct Injury and not reabsorbed or secreted. Therefore, the clearance ofcre-
Functional abnormalities at distal nephron sites manifest pri- atinine or inulin is about the same as the GFR. Creatinine is an
marily as impaired concentrating ability and/or acidification endogenous compound released from skeletal muscle. Inulin
defects. Amphotericin B, cisplatin, and methoxyflurane induce is an exogenous compound. Creatinine or inulin clearance is
an ADH-resistant polyuria, suggesting that the concentrating determined by the following formula:
defect occurs at the level of the medullary thick ascending limb
and/or the collecting duct. Inulin clearance (mL/min) =
Inulin concentration in urine (mg/L) X Urine volume (mL/min)
Inulin concentration in serum (mg/L)
Papillary Injury
The renal papilla is susceptible to the chronic injurious effects of Indirect markers of GFR are serial blood urea nitrogen
abusive consumption ofanalgesics. The initial target of abusive (BUN) and serum creatinine concentrations. However, a
consumption of analgesics is the medullary interstitial cells, 50% to 70% decrease in GFR must occur before increases in
followed by degenerative changes in the medullary capillaries, serum creatinine and BUN develop. Chemically induced
loops of Henle, and collecting ducts. High papillary concentra- increases in BUN and/or serum creatinine may not neces-
tions of potential toxicants and inhibition of vasodilatory pros- sarily reflect renal damage, but rather may be secondary to
taglandins compromise RBF to the renal medulla/papilla and dehydration, hypovolemia, and/or protein catabolism. Serum
result in tissue ischemia. cystatin C levels may be more sensitive as a marker of mildly
impaired GFR.
Histopathologic evaluation of the kidney following treat-
ASSESSMENT OF RENAL FUNCTION ment is crucial in identifying the site, nature, and severity of
the nephrotoxic lesion. Assessment of chemically induced
Both in vivo and in vitro methods are available for evaluation nephrotoxicity therefore should include urinalysis, serum
of the effects of a chemical on kidney function. Initially, neph- clinical chemistry, and histopathology to provide a reasonable
rotoxicity can be assessed by evaluating serum and urine chem- profile of the functional and morphologic effects of a chemical
istries following treatment with the chemical in question. The on the kidney. Site-specific biomarkers for common nephro-
standard battery of noninvasive tests includes measurement toxicants are shown in Figure 14-6.
of urine volume and osmolality, pH, and urinary composition Various in vitro techniques may be used to elucidate under-
(e.g., electrolytes, glucose, and protein). Although specificity is lying mechanisms of chemically induced nephrotoxicity.
 CHAPTER 14 Toxic Responses of the Kidney 217

Thrombotic microangiopathy Glomerular markers Hemodynamic alteration Distal tubule injury

Calcineurin inihibitors Collagen IV ACE-|, ARB Amphotericin B
Clopidogrel Cystatin C Amphotericin B Calcineurin inhibitors
Cocaine Total protein Calcineurin inhibitors Lithium
Mitomycin Diuretics Sulfadiazine
Quinine NSAIDs
Radiocontrast agents Distal tubule markers _
Clusterin
H-FABP
NGAL
Osteopontin
T-GST

J
Proximal tubule injury Tubular obstruction oa

Acylovir Acylovir
Aminoglycosides Indinavir
Cadmium Methotrexate
Cidofovir Sulfonamides
Cisplatin
Foscarnet Collecting duct markers
Lead Calbindin d28
Mercuric chloride RPA-1

al-microglobulin
82-microglobulin
Clusterin Interstitial nephritis
Cystatin-C Allopurinol
HGF Aristolochic acid
KIM-1 Cephalosporins
L-FABP Ciprofloxacin
Microalbumin Diuretics
NAG Macrolides
Netrin1 NSAIDs
NHE3 Pencillins
NGAL Phenytoin
Osteopontin PPls
RBP
Loop
of Henle markers
NHE3
Osteopontin

FIGURE 14-6 Site-specific biomarkers, common nephrotoxicants, and mechanisms of injury. (Reproduced with permission from
McQueen CA, Schnellmann (eds): Comprehensive Toxicology. Oxford, UK: Elsevier; 2010.)

Freshly prepared isolated perfused kidneys, kidney slices, and

renal tubular suspensions and cells exhibit the greatest degree BIOCHEMICAL MECHANISMS/
of differentiated functions and similarity to the in vivo situa- MEDIATORS OF RENAL CELL INJURY
tion, but these models have limited life spans of 2 to 24h. In
contrast, primary cultures of renal cells and established renal Cell Death
cell lines exhibit longer life spans (> 2 weeks). Once a mecha- Cell death may occur through either oncosis or apoptosis.
nism has been identified in vitro, the postulated mechanism Apoptosis is a tightly controlled, organized process that usually
must be tested in vivo. Thus, appropriately designed in vivo affects scattered individual cells, which break into small frag-
and in vitro studies should provide a complete characterization ments that are phagocytosed by adjacent cells or macrophages
of the biochemical, functional, and morphologic effects of a without producing an inflammatory response. In contrast,
chemical on the kidney and an understanding of the underly- oncosis often affects many contiguous cells; the cells rupture,
ing mechanisms in the target cell population(s). releasing cellular contents and inflammation follows. With
218 UNIT 4 Target Organ Toxicity

many toxicants, lower but injurious concentrations produce produced by mitochondrial inhibition and hypoxia. Mito-
cell death through apoptosis. As the concentration of the toxi- chondria are known to accumulate Ca’* in lethally injured
cant increases, oncosis plays a predominant role. cells through a low-affinity, high-capacity Ca** transport sys-
tem. Although this system plays a minor role in normal cellular
Ca** regulation, under injurious conditions the uptake of Ca**
Mediators of Toxicity may facilitate ROS formation and damage.
A chemical can initiate cell injury by various mechanisms Signaling kinases such as protein kinase C, mitogen-acti-
(Figure 14-5). The chemical may initiate toxicity due to its vated protein kinases (e.g., ERK1/2, p38, JNK/SAPK), protein
intrinsic reactivity with cellular macromolecules, may require kinase B (Akt), src, and phosphoinositide-3-kinase phosphor-
renal or extrarenal bioactivation to a reactive intermediate, or ylate other proteins and, thereby, alter their activity, expres-
may initiate injury indirectly by inducing oxidative stress via sion, or localization. Numerous recent studies reveal critical
increased production of ROS, such as superoxide anion, hydro- roles for signaling kinases in renal cell death and in the recov-
gen peroxide, and hydroxyl radicals. ROS and reactive nitro- ery of renal cells after toxicant injury.
gen species from nitric oxide, such as peroxynitrite (ONOO), Cell volume and ion homeostasis are tightly regulated and
can attack proteins, lipids, and DNA to induce cellular injury are critical for the reabsorptive properties of the tubular epi-
and death. thelial cells. Toxicants generally disrupt cell volume and ion
homeostasis by either increasing ion permeability or inhibit-
ing energy production. Loss of ATP results in the inhibition of
Cellular/Subcellular and Molecular Targets membrane transporters that maintain the internal ion balance.
A number of cellular targets have been identified to play a role
in cell death. It is generally thought that an intracellular inter-
action (e.g., an alkylating agent or ROS with a macromolecule) SPECIFIC NEPHROTOXICANTS
initiates a sequence ofevents that leads to cell death. In the case
of oncosis, a “point ofno return” is reached in which the cell will Heavy Metals
die regardless of any intervention. The idea of asingle sequence Many metals—including cadmium, chromium, lead, mer-
of events is probably simplistic for most toxicants, given the cury, platinum, and uranium—are nephrotoxic. The nature
extensive number of targets available for alkylating species and and severity of metal nephrotoxicity varies with respect to its
ROS. Rather, multiple pathways, with both distinct and com- form. In addition, different metals have different primary tar-
mon sequences ofevents, may lead to cell death. gets within the kidney. Metals may cause renal cellular injury
Many cellular processes depend on mitochondrial ATP and, through their ability to bind to sulfhydryl groups of critical pro-
thus, become compromised simultaneously with inhibition of teins within the cells and thereby inhibit their normal function.
respiration. Conversely, mitochondrial dysfunction may be
a consequence of some other cellular process altered by the Mercury—Humans and animals are exposed to elemental
toxicant. Numerous nephrotoxicants cause mitochondrial dys- mercury vapor, inorganic mercurous and mercuric salts, and
function in different ways. Whether toxicants target mitochon- organic mercuric compounds through the environment.
dria directly or indirectly, it is clear that mitochondria play a Administered elemental mercury is rapidly oxidized in eryth-
critical role in determining whether cells die by apoptosis or rocytes or tissues to inorganic mercury, and thus the tissue dis-
oncosis. It is thought that the mitochondrial permeability tribution of elemental and inorganic mercury is similar. Due to
transition (MPT) occurs during cell injury and ultimately pro- its high affinity for sulfhydryl groups, virtually all of the Hg’*
gresses to apoptosis if sufficient ATP is available, or to oncosis found in blood is bound to albumin, other sulfhydryl-containing
if ATP is depleted. Further, the release of apoptotic proteins, proteins, glutathione, and cysteine.
such as apoptosis inducing factor, cytochrome c, Smac/Diablo, The kidneys are the primary target organs for accumulation
Omi, and Endonuclease G following MPT play a key role in of Hg’*, and the S, segment of the proximal tubule is the initial
activating downstream caspases and executing apoptosis. site of toxicity, but the S, and S, segments may become affected
Ca** is a second messenger and plays a critical role in a as dose or duration increases. Renal uptake of Hg’* is very
variety of cellular functions. Sustained elevations or abnor- rapid with as much as 50% of a nontoxic dose of Hg’* found
mally large increases in cytosolic free Ca** can exert a number in the kidneys within a few hours of exposure. Considering the
of detrimental effects on the cell. For example, an increase in fact that virtually all of the Hg’* found in blood is bound to an
cytosolic free Ca** can activate a number of degradative Ca’*- endogenous ligand, it is likely that the luminal and/or basolat-
dependent enzymes, such as phospholipases and proteinases eral transport of Hg** into the proximal tubular epithelial cell is
(e.g., calpains), and can produce aberrations in the structure through cotransport of Hg’* with an endogenous ligand such
and function of cytoskeletal elements. Release of endoplasmic as glutathione, cysteine, or albumin, or through some plasma
reticulum (ER) Ca’* stores may be a key step in initiating the membrane Hg’*-ligand complex.
injury process and increasing cytosolic free Ca?* concentra- The acute nephrotoxicity induced by HgCl, is character-
tions, because prior depletion of ER Ca’* stores protects renal ized by proximal tubular necrosis and AKI within 24 to 48 h
proximal tubules from extracellular Ca** influx and cell death after administration. Early markers of HgCl,-induced renal
 CHAPTER 14 Toxic Responses ofthe Kidney 219

dysfunction include an increase in the urinary excretion of with acute and chronic exposure to halogenated hydrocarbons,
brush-border enzymes such as alkaline phosphatase and including nephrotoxicity.
y-glutamyl transferase. Subsequently, when tubular injury
becomes severe, intracellular enzymes, such as lactate dehy- Chloroform—The primary cellular target of chloroform is the
drogenase and aspartate aminotransferase, increase in the proximal tubule, with no primary damage to the glomerulus or
urine. As injury progresses, tubular reabsorption of solutes and the distal tubule. Proteinuria, glucosuria, and increased BUN
water decreases and there is an increase in the urinary excre- levels are all characteristic of chloroform-induced nephrotoxic-
tion of glucose, amino acids, albumin, and other proteins. Also ity. The nephrotoxicity produced by chloroform is linked to its
associated with the increase in injured proximal tubules is a metabolism by renal cytochrome P450, which biotransforms
decrease and progressive decline in the GFR. chloroform to trichloromethanol, which is unstable and releas-
Changes in mitochondrial morphology and function are es HCl to form phosgene, which injuriously reacts with cellular
very early events following HgCl, administration, support- macromolecules.
ing the hypothesis that mitochondrial dysfunction is an early
and important contributor to inorganic mercury-induced cell Tetrafluoroethylene— Tetrafluoroethylene is conjugated with
death along the proximal tubule. glutathione in the liver, and the GSH conjugate is secreted into
the bile and small intestine where it is degraded to the cysteine
Cadmium—Chronic exposure of nonsmoking humans and ani- S-conjugate (TFEC), reabsorbed, and transported to the kid-
mals to cadmium is primarily through food and results in neph- ney. Although several metabolites are formed, the cysteine
rotoxicity. In the workplace, inhalation of cadmium-containing S-conjugate is the penultimate nephrotoxicant. Following
dust and fumes is the major route of exposure. Cadmium has a transport into the proximal tubule, the cysteine S-conjugate is a
half-life of greater than 10 years in humans and thus accumulates substrate for the cytosolic and mitochondrial forms of the
in the body over time. Approximately 50% of the body’s burden of enzyme cysteine conjugate B-lyase. The products of the reac-
cadmium can be found in the kidney. Cadmium produces proxi- tion are ammonia, pyruvate, and a reactive thiol that is capable
mal tubule dysfunction (S, and S, segments) and injury that may of binding covalently to cellular macromolecules causing
progress to a chronic interstitial nephritis. cellular damage. Functionally, increases in urinary glucose,
protein, cellular enzymes, and BUN are noted.

Chemically Induced a,,-Globulin

Bromobenzene—Biotransformation of bromobenzene and
Nephropathy other halogenated benzenes is critical for their nephrotoxicity.
A diverse group of chemicals, including unleaded gasoline, jet Hepatic cytochrome P450 metabolizes bromobenzene and
fuels, p-limonene, 1,4-dichlorobenzene, decalin, tetrachloro- conjugates it to glutathione, and releases it as a form that can
ethylene, and lindane, causes Q,,-globulin nephropathy or hya- cause nephrotoxicity. The diglutathione conjugate of the hydro-
line droplet nephropathy in male rats. Binding to a,,-globulin quinone is approximately 1000-fold more potent than bromo-
decreases lysosomal proteases breakdown of q,,-globulin. benzene in producing nephrotoxicity, producing the same
Chronic exposure to these compounds results in progression of pathologic changes in the S; segment, and increasing the
these lesions and ultimately in chronic nephropathy. amount of protein, glucose, and cellular enzymes in the urine.
Humans are not at risk because (1) humans do not synthe-
size Q,-globulin; (2) humans secrete less proteins in general
and in particular less low-molecular-weight proteins in urine
Mycotoxins
than the rat; (3) the low-molecular-weight proteins in human
urine are either not related structurally to @,,-globulin, do not Mycotoxins are products of molds and fungi, and a number of
bind to compounds that bind to a,,-globulin, or are similar mycotoxins produce nephrotoxicity. Three examples of neph-
to proteins in female rats, male Black Reiter rats, rabbits, or rotoxic mycotoxins will be discussed. Citrinin nephrotoxicity is
guinea pigs that do not exhibit ,,-globulin nephropathy; and characterized by decreased urine osmolality, GFR and RBE, gly-
(4) mice excrete a low-molecular-weight urinary protein that is cosuria, and increased urinary enzyme excretion. Interestingly,
90% homologous to Q,,-globulin, but they do not exhibit ,,,- the location of citrinin-induced tubular vacuolization and
globulin-nephropathy and renal tumors following exposure to necrosis (proximal, distal) varies among species. Whereas the
Q,,-globulin-nephropathy-inducing agents. mechanism of citrinin toxicity to the tubules remains unre-
solved, citrinin enters the cells through the organic anion trans-
porter and causes mitochondrial dysfunction.
Halogenated Hydrocarbons Fumonisins B, and B, are commonly found on corn and corn
Halogenated hydrocarbons are a diverse class of compounds products and they are known to produce nephrotoxicity in rats
and are used extensively as chemical intermediates, solvents, and rabbits. Histologic examination of the kidney revealed dis-
and pesticides. Consequently, humans are exposed to these ruption of the basolateral membrane, mitochondrial swelling,
compounds not only in the workplace but also through the increased numbers of clear and electron-dense vacuoles, and
environment. Numerous toxic effects have been associated apoptosis in proximal tubular cells at the junction ofthe cortex
220 UNIT 4 Target Organ Toxicity

and medulla. Changes in renal function included increased joined in a glycosidic linkage to a central hexose nucleus.
urine volume, decreased osmolality, and increased excretion Although they are drugs of choice for many gram-negative
of low- and high-molecular-weight proteins. The toxicity of infections, their use is primarily limited by their nephrotoxicity.
fumonisins may be through increased sphinganine, reactive Renal dysfunction by aminoglycosides is characterized by a
oxygen species, and apoptosis. nonoliguric renal failure with reduced GFR, an increase in
Aristolochic acids (AAs) and aristolactams are natural prod- serum creatinine and BUN, and polyuria. Within 24 h, increases
ucts found in the Aristolochia and Asarum genera. Despite the in urinary brush-border enzymes, glucosuria, aminoaciduria,
extensive use ofAristolochia as a herbal remedy for thousands and proteinuria are observed. Histologically, lysosomal altera-
of years, recent reports of its human toxicity include tubular tions are noted initially, followed by damage to the brush border,
dysfunction, proteinuria, and interstitial fibrosis. AAs form ER, mitochondria, and cytoplasm, ultimately leading to tubular
covalent DNA adducts, and are genotoxic and carcinogenic. cell necrosis. Interestingly, proliferation of renal proximal tubule
Renal uptake of the penultimate toxicant, AA-I, involves cells can be observed early after the onset of nephrotoxicity.
mOat-mediated transport, and it is bioactivated through nitro- The earliest lesion observed following clinically relevant
reduction to produce DNA and protein adducts. doses of aminoglycosides is an increase in the size and number
of lysosomes, which contain phospholipids. The renal phos-
pholipidosis produced by the aminoglycosides is thought to
Therapeutic Agents
occur through their inhibition of lysosomal hydrolases, such as
Acetaminophen— Acetaminophen (APAP) nephrotoxicity is sphingomyelinase and phospholipases.
characterized by proximal tubular necrosis with increases in
BUN and plasma creatinine, decreases in GFR and clearance
Amphotericin B—Amphotericin B is an effective antifungal
of para-aminohippurate, increases in the fractional excretion of
agent, causing nephrotoxicity charaeterized by ADH-resistant
water, sodium, and potassium, and increases in urinary glucose,
polyuria, renal tubular acidosis, hypokalemia, and either acute
protein, and brush-border enzymes. Although renal cytochrome
or chronic renal failure. The functional integrity of the glom-
P450 plays a role in APAP activation and nephrotoxicity, gluta-
erulus and ofthe proximal and distal portions of the nephron is
thione conjugates of APAP may also contribute to APA P-induced
impaired, leading to decreases in RBF and GFR secondary to
nephrotoxicity.
renal arteriolar vasoconstriction or activation of tubuloglomer-
Nonsteroidal Anti-inflammatory Drugs—NSAIDs such as ular feedback.
aspirin, ibuprofen, naproxen, indomethacin, and cyclooxygenase-
2 inhibitors (e.g., celecoxib) are extensively used as analgesics Cyclosporine—Cyclosporine is an important immunosup-
and anti-inflammatory drugs and produce their therapeutic ef- pressive agent and is widely used to prevent graft rejection in
fects through the inhibition of prostaglandin synthesis. At least organ transplantation. Cyclosporine is a fungal cyclic polypep-
three different types of nephrotoxicity have been associated with tide and acts by selectively inhibiting cyclophylin and, in turn,
NSAID administration. AKI may occur within hours ofa large calcineurin and T-cell activation. Nephrotoxicity is a critical side
dose ofa NSAID, is usually reversible on withdrawal of the drug, effect of cyclosporine, with nearly all patients who receive the
and is characterized by decreased RBF and GFR and by oliguria. drug exhibiting some form of nephrotoxicity. Cyclosporine-
When normal production of vasodilatory prostaglandins (e.g., induced nephrotoxicity may manifest as (1) acute reversible renal
PGE,, PGI.) is inhibited by NSAIDs, vasoconstriction induced dysfunction, (2) acute vasculopathy, and (3) chronic nephropa-
by circulating catecholamines and angiotensin II is unopposed, thy with interstitial fibrosis. Acute renal dysfunction is character-
resulting in decreased RBF and ischemia. ized by dose-related decreases in RBF and GFR and increases in
In contrast, chronic consumption of NSAIDs and/or APAP BUN and serum creatinine. The decrease in RBF and GER is
(> 3 years) results in an often irreversible nephrotoxicity that related to marked vasoconstriction induced by cyclosporine.
is known as analgesic nephropathy. The primary lesion is pap- Acute vasculopathy or thrombotic microangiopathy follow-
illary necrosis with chronic interstitial nephritis. The mecha- ing cyclosporine treatment affects arterioles and glomerular
nism by which NSAIDs produce analgesic nephropathy is not capillaries, without an inflammatory component. The lesion
known but may result from chronic medullary/papillary isch- consists of fibrin-platelet thrombi and fragmented red blood
emia, secondary to renal vasoconstriction, or genesis of areac- cells occluding the vessels.
tive intermediate that, in turn, initiates an oxidative stress or Long-term treatment with cyclosporine can result in chronic
binds covalently to critical cellular macromolecules. nephropathy with interstitial fibrosis and tubular atrophy.
The third albeit rare type of nephrotoxicity associated with Modest elevations in serum creatinine and decreases in GFR
NSAIDs is an interstitial. nephritis. Patients normally present occur along with hypertension, proteinuria, and tubular dys-
with elevated serum creatinine and proteinuria. If NSAIDs are function. Histologic changes are profound; they are charac-
discontinued, renal function improves in 1 to 3 months. terized by arteriolopathy, global and segmental glomerular
sclerosis, striped interstitial fibrosis, and tubular atrophy. These
Aminoglycosides—The aminoglycoside antibiotics are so lesions may not be reversible if cyclosporine therapy is discon-
named because they consist of two or more amino sugars tinued and may result in end-stage renal disease.
 CHAPTER 14 Toxic Responses ofthe Kidney Vn

Cisplatin—Cisplatin is a valuable drug in the treatment of activates numerous pathways in the mitogen-activated protein
solid tumors, with nephrotoxicity limiting its clinical use. The kinase family.
kidney is not only responsible for the majority of cisplatin ex-
creted but is also the primary site of accumulation. Cisplatin Radiocontrast Agents—lIodinated contrast media used for the
nephrotoxicity includes acute and chronic renal failure, renal imaging of tissues have a very high osmolality (> 1200 mOsm/L)
magnesium wasting, and polyuria. Patients treated with cispla- and are potentially nephrotoxic, particularly in patients with
tin regimens permanently lose 10% to 30% of their renal existing renal impairment, diabetes, or heart failure or who are
function. receiving other nephrotoxic drugs. The newer nonionic contrast
The nephrotoxicity of cisplatin can be grouped as (1) tubu- agents (e.g., iotrol and iopamidol) have lower nephrotoxicity. The
lar toxicity, (2) vascular damage, (3) glomerular injury, and nephrotoxicity of these agents is due to both hemodynamic altera-
(4) interstitial injury. Early effects of cisplatin are decreases tions (vasoconstriction) and tubular injury (via ROS).
in RBF and polyuria that is concurrent with increased elec-
trolyte excretion. GFR is produced by vasoconstriction and
is followed by tubular injury with enzymuria. Although the BIBLIOGRAPHY
primary cellular target associated with AKI is the proximal Brenner BM, Rector FC (eds.): Brenner and Rectors The Kidney, 9th
tubule S, segment in the rat, in humans the S, and S, segments, ed. Philadelphia, PA: Saunders Elsevier, 2011.
distal tubule, and collecting ducts can also be affected. Cispla- Fogo AB, Kashgarian M: Diagnostic Atlas of Renal Pathology: Expert
tin may produce nephrotoxicity through its ability to inhibit Consult, 2nd ed. Philadelphia, PA: Elsevier Saunders, 2011.
DNA synthesis as well as transport functions. In addition, Tarloff JB, Lash LH (eds.): Toxicology of the Kidney, 3rd ed. Boca
cisplatin is known to induce mitochondrial dysfunction and Raton, FL: CRC Press, 2005.
in)bo UNIT 4 Target Organ Toxicity

QUESTIONS

The kidney is responsible for all of the following EXCEPT: All of the following statements regarding toxicity to the
synthesis of renin. kidney are true EXCEPT:
acid-base balance. a. Concentration of toxins in tubular fluid increase the
reabsorption of electrolytes. likelihood that the toxin will diffuse into tubular cells.
regulation of extracellular fluid. b. Drugs in the systemic circulation are delivered to the
ma release of angiotensin.
ont kidneys at relatively high amounts.
c. The distal convoluted tubule is the most common site
Which of the following does NOT contribute to filtrate of toxicant-induced renal injury.
formation in the nephron? d. Immune complex deposition within the glomeruli
a. capillary hydrostatic pressure. can lead to glomerulonephritis.
b. positive charge of glomerular capillary wall. e. Antibiotics and/or antifungal drugs affect the func-
c. hydraulic permeability of glomerular capillary wall. tioning of the nephron at multiple locations.
d. colloid oncotic pressure.
e. size of filtration slits. Which of the following test results is NOT correctly
paired with the underlying kidney problem?
Which of the following is NOT a characteristic of the loop a. increased urine volume—defect in ADH synthesis.
of Henle? b. glucosuria—defect in reabsorption in the proximal
a. There is reabsorption of filtered Nat and K*. convoluted tubule.
b. Tubular fluid in the thin descending limb is iso- c. proteinuria—glomerular damage.
osmotic to the renal interstitium. proteinuria—proximal tubular injury.
c. Water is freely permeable in the thin ascending limb. e. brush-border enzymuria—glomerulonephritis.
Na* and Cl are reabsorbed in the thin ascending limb.
e. The thick ascending limb is impermeable to water. Renal cell injury is NOT commonly mediated by which of
the following mechanisms?
Although the kidneys constitute 0.5% of total body mass, a. loss of membrane integrity.
approximately how much of the resting cardiac output do impairment of mitochondrial function.
they receive? increased cytosolic Ca** concentration.
a. 0.5% to 1%. increased Na*,K*-ATPase activity.
b. 5%. scao
caspase activation.
ComeOCos
d. 20% to 25%. 10. Which of the following statements is FALSE with respect
e. 50% to 60%. to nephrotoxicants?
a. Mercury poisoning can lead to proximal tubular
Which of the following is most likely to occur after a toxic necrosis and acute renal failure.
insult to the kidney? b. Cisplatin may cause nephrotoxicity because of its
a. GFR will decrease in the unaffected kidney. ability to inhibit DNA synthesis.
b. Tight-junction integrity will increase in the nephron. c. Chronic consumption of NSAIDs results in nephro-
c. The unaffected cells will undergo atrophy and toxicity that is reversible with time.
proliferation. d. Amphotericin B nephrotoxicity can result in
d. Clinical tests will likely show normal renal function. ADH-resistant polyuria.
e. Glomerulotubular balance is lost. e. Acetaminophen becomes nephrotoxic via activation
by renal cytochrome P450.
Chronic renal failure does not typically result in:
decrease in GFR ofviable nephrons.
glomerulosclerosis.
tubular atrophy.
increased glomerular pressures.
az altered capillary permeability.
Seen
 Cael Aw DSi EER:

Toxic Responses of the

Respiratory System
George D. Leikauf

RESPIRATORY TRACT STRUCTURE AND FUNCTION ACUTE RESPONSES OF THE LUNG TO INJURY
Oronasal Passages Trigeminally Mediated Airway Reflexes
Structure Bronchoconstriction, Airway Hyperreactivity, and
Sensory Functions Neurogenic Inflammation
Irritant, Thermosensory, and Mechanosensory Acute Lung Injury (Pulmonary Edema)
Functions
CHRONIC RESPONSES OF THE LUNG TO INJURY
Conducting Airways
Chronic Obstructive Pulmonary Disease
Structure
Lung Cancer
Mucociliary Clearance and Antimicrobial
Functions Asthma
Gas Exchange Region Pulmonary Fibrosis

Structure AGENTS KNOWN TO PRODUCE LUNG INJURY INHUMANS

Function
EVALUATION OF TOXIC LUNG DAMAGE
BIOTRANSFORMATION IN THE RESPIRATORY TRACT
Human Studies
GENERAL PRINCIPLES IN THE PATHOGENESIS OF LUNG Animal Studies
DAMAGE CAUSED BY CHEMICALS Inhalation Exposure Systems
Toxic Inhalants, Gases, and Dosimetry Pulmonary Function Tests in Experimental Animals
Regional Particle Deposition Morphological Techniques
Deposition Mechanisms Pulmonary Lavage and Pulmonary Edema
Particle Clearance In Vitro Studies
Nasal Clearance Isolated Perfused Lung
Tracheobronchial Clearance Airway Microdissection and Organotypic Tissue
Alveolar Clearance Culture Systems
Lung Cell Culture
 4
NOin) UNIT 4 Target Organ Toxicity

» Inhaled xenobiotics can affect lung tissues directly or a Asthma is characterized by increased reactivity of the
distant organs after absorption. bronchial smooth muscle in response to exposure to
= Water solubility is a decisive factor in determining how irritants.
deeply a given gas penetrates into the lung. « In emphysema, destruction of the gas-exchanging
w Particle size is usually the critical factor that determines surface area results in a distended, hyperinflated lung
the region of the respiratory tract in which a particle or that no longer effectively exchanges oxygen and carbon
an aerosol will deposit. dioxide.
a Thelung contains most of the enzymes involved in xeno-
biotic biotransformation that have been identified in
other tissues.

Toxic substances can disrupt the respiratory system and dis- Chemosensory function of the nasal passages is accom-
tant organs after chemicals enter the body by means of inha- plished by a wide variety of specialized receptors in major
lation. Pathological changes in the respiratory tract also can subtypes including (1) olfactory receptors, (2) trace amine-
be a target of blood-borne agents. Inhalation toxicology refers associated receptors (TAARs), (3) membrane guanylyl cyclase
to the route of exposure, whereas respiratory toxicology refers GC-D receptors, (4) vomeronasal receptors, and (5) formyl
to target organ toxicity. Lung tissue can be injured directly or peptide receptors (FPRs).
secondarily by metabolic products from organic compounds. The olfactory epithelium contains specialized chemosen-
However, the most important effect of many toxic inhalants is sory olfactory neurons located in the superior portion ofthe
to place an undue oxidative burden on the lungs. nasal passage. Airflow in this region of the nasal passage is
typically low, thus sniffing can increase perception. TAARs
detect trace amines, with fishy or putrid odor, that are found
RESPIRATORY TRACT STRUCTURE in foods and can also be generated during fermentation
AND FUNCTION or decay. GC-D receptors are located in the cilia of olfac-
tory sensory neurons and detect the natriuretic peptides,
Oronasal Passages uroguanylin (found in urine) and guanylin. In rodents,
Structure—The respiratory tract is divided into the upper these receptors detect carbon dioxide, which is odorless
respiratory tract (extrathoracic airway passages above the in humans and other primates. Vomeronasal receptors are
neck) and lower respiratory track (airway passages and lung separate from, but adjacent to, olfactory neurons. They
parenchyma below the pharynx) (Figure 15-1). The upper can detect higher molecular weight stimuli, including non-
respiratory track reaches from the nostril or mouth to the volatile chemicals. FPRs are also a part of the vomeronasal
pharynx and functions to conduct, heat, humidify, filter, and system and detect bacterial or mitochondrial formylated
chemosense incoming air. Leaving the nasal passage, air is peptides, which are thought to identify pathogens or patho-
warmed to about 33°C and humidified to about 98% water sat- genic states.
uration. Air is filtered in the nasal passages with highly water-
soluble gases being absorbed efficiently. The nasal passages also Irritant, Thermosensory, and Mechanosensory
filter particles, which may be deposited by impaction or diffu- Functions—In addition to the detection of odor, the detec-
sion on the nasal mucosa. tion of irritant chemicals, cold and hot temperatures, or
mechanical stress can be a protective mechanism that may
Sensory Functions—In addition to conducting, condition- limit exposure. The main nerve endings that perceive irri-
ing, and filtering air to the lower respiratory tract, a major func- tants, the chemical nociceptors also discern temperature and
tion of the oronasal passage is chemosensory. Nasal epithelia mechanical stress. Two protein families, the transient receptor
can metabolize many foreign compounds by cytochrome P450 potential (TRP) channels and the taste (TAS) receptors, per-
and other enzymes. Humans can distinguish between more form these functions in the upper respiratory tract. TRP chan-
than 5000 odors. The detection of odor can be protective and nels are ion channels that are permeable to cations, including
can induce avoidance behaviors. Odorant can be added to the calcium, magnesium, and sodium. These receptors are sensi-
otherwise colorless and almost odorless gas used by consumers tive to a variety of natural ingredients, pain stimuli, and heat.
(e.g., mercaptans to methane), to assist in detecting leaks and Taste buds, which contain TAS, determine salt, sour, sweet,
thereby preventing fires or explosions. umami (glutamate and nucleotides), and bitter.
 CHAPTER 15 Toxic Responses ofthe Respiratory System 225

1.0
S Nasal pharyngeal, laryngeal
< 0.8 \
is \ {~™.
S 0.6 \\ !! /
a \ | /
J) \ It f
LO '
ioe} |
Nasal airway 5
(0)?
J)
oc i
0.0
0.0001 0.001 0.01 0.1 ] 10 100
Pharynx \ Diameter (jum)
1.0
< Tracheobronchial
= 0:8
ne) |
ca |
Trachea So 0.6
Lymph nodes J)
oe S04
Bronchihi oee ©
Xe 2 02
/ \e Pulmonary &
Nonrespiratory |/ / arteries 0.0
bronchioles on) Bagh 0.0001 0.001 0.01 0.1 1 10 100
We: / ¥ = 3 q Diameter (1m)
Respiratory N Sexi 4 Pulmonary S Ee Aiveolae
bronchioles LX | in || : s I
ay, r N veins 0.8 I
sek IA c 0.
Alveolar a , s
ducts x ue
Alveolar ow fed
; a Cj |
Alveolar “Seu 2
(e)
sacs re) 0.2
ac
0.0
0.0001 0.001 001 o1 1 10 ~=100
Diameter (jum)

FIGURE 15-1 Predicted fractional deposition ofinhaled particles in the nasopharyngeal, tracheobronchial, and alveolar region of
the human respiratory tract during nose breathing. (Used with permission of J. Harkema.) (Reproduced with permission from Oberdorster G,
Oberdorster E, OberdorsterJ:Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles, Environ Health Perspect, 2005
Jul;113(7):823-839.)

Conducting Airways Mucociliary Clearance and Antimicrobial Functions—

Structure—At the beginning of the lower respiratory track In humans, the proximal airway and a portion of the nasal
is the larynx, which is responsible for speech (phonation). passage are covered by a pseudostratified respiratory epithe-
The conducting airways of the lower respiratory tract can be lium that contains a number of specialized cells including
divided into proximal (trachea and bronchi) and distal regions ciliated, mucous, and basal cells (Figure 15-2). These cells
(bronchioles). Conducting airways have a bifurcating struc- work together to form a mucous layer that traps and removes
ture, with successive airway generations containing about inhaled material via mucociliary clearance. For mucociliary
twice the number of bronchi progressively decreasing in inter- clearance in the airways to function optimally, regulation of
nal diameter. Eventually a transition zone is reached where ion transport, fluid, and mucus must be coordinated. Chloride
cartilaginous bronchi give way to noncartilaginous bronchi- ion channels and the cystic fibrosis transmembrane regulator
oles, which in turn give way to gas exchange regions, respira- are needed to move fluid into the airway lumen and sodium
tory bronchioles, and alveoli. In the bronchiolar epithelium, channels are needed to move water out of the lumen.
mucus-producing cells and glands give way to bronchiolar Ciliated cells have microtubule-based protrusions, cilia, of
secretoglobin cells (BSCs). One way in which airflow is altered which there are two types: motile and primary. Motile cilia
is by smooth muscle that surrounds the airways and is under exert mechanical force through continuous motion to propel
autonomic innervation via the vagus nerve. harmful inhaled material out of the nose and lung. Motile cilia
 6
i)i) UNIT 4 Target Organ Toxicity

FIGURE 15-2 Pseudostratified respiratory epithelium lines

the nasal cavity, trachea, and bronchi.
The surface includes mainly
ciliated epithelial cells that may or may not touch the basement
membrane, (arrow) surface mucous (goblet) cell, and (arrowhead)
basal cells. (Modified with permission from the Human Protein Atlas
(www.proteinatlas.org).)

also exhibit mechanosensory and chemosensory functions and

can respond to mechanical stress, heat, acidic pH, and endog-
enous and synthetic agonists. Primary cilia often serve as sen-
sory organelles.
Serous cells contain and secrete a less viscous fluid, and are
also enriched in antimicrobial proteins including lysozyme
and lactotransferin. These cells also contain the antimicro- FIGURE 15-3 Centriacinar region (ventilatory unit) of the
bial protein, BPIF2 (aka SPLUNC2). Secretory leukocyte lung. An airway (AW) and a blood vessel (BV) (arteriole) are in close
proteinase inhibitor (SLPI) is a serine proteinase inhibitor proximity to the terminal bronchiole (TB). The terminal bronchiole
that is produced locally in the lung by cells of the submuco- leads to the bronchiole—alveolar duct junction (BADJ) and the alveolar
sal bronchial glands and by nonciliated epithelial cells. The duct (AD). A number ofthe (arrows) alveolar septal tips close to the
main function of SLPI is the inhibition of neutrophil elas- BADJ are thickened after a brief (4-h) exposure to asbestos fibers,
tase and other proteinases, and may also have antimicrobial indicating localization offiber deposition. Other inhalants, such as
functions. ozone, produce lesions in the same locations. (Used with permission of
Another airway secretory cell is the bronchiolar secreto- Dr Kent E. Pinkerton, University of California, Davis.)
globin cell (BSC), previously called the Clara cell. The role of
secretoglobins is not fully understood in the lung, but they are
known to inhibit phospholipase A2 and limit inflammation. In
humans, BSCs are found mainly in the distal airways and can ~95% of the alveolar surface and therefore are susceptible
act as tissue stem cells. to damage by noxious agents that penetrate to the alveolus.
Alveolar type I cells have an attenuated cytoplasm to enhance
gas exchange. Alveolar type II cells produce and secrete surfac-
Gas Exchange Region tant, a mixture of lipids, and four surfactant associated proteins
Structure—The gas exchange region consists of terminal and can undergo mitotic division and replace damaged type I
bronchioles, respiratory bronchioles, alveolar ducts, alve- cells. Surfactant protein B and C are amphipathic and aid in
oli, blood vessels, and lung interstitium (Figure 15-3). Gas spreading secreted lipids which form a monolayer that reduces
exchange occurs in the alveoli, which comprise ~85% of the surface tension. Surfactant proteins Al, A2, and D are mem-
total parenchymal lung volume. Adult human lungs contain an bers of the subfamily of C-type lectins called collectins, which
estimated 300 to 500 million alveoli. Capillaries, blood plasma, defend against pathogens. Surfactant proteins Al and A2 do
and formed blood elements are separated from the air space not alter lipid structure but do bind lipopolysaccharides (LPS)
by a thin layer of tissue formed by epithelial, interstitial, and and various microbial pathogens, enhancing their clearance
endothelial components. from the lung. Surfactant protein D is also necessary in the
The alveolar epithelium consists of two cells, the alveolar suppression of pulmonary inflammation and in host defense
type I and type II cell (Figure 15-4). Alveolar type I cells cover against viral, fungal, and bacterial pathogens.
 CHAPTER 15 Toxic Responses ofthe Respiratory System oor

Liters }

Total
6 lung
capacity
Vital
capacity

+ 4

re

ee ae Functional = A
Se < stills residual —_—Residual
capacity volume
FIGURE 15-4 Alveolar region of the lung. The (A) alveolus is
separated by the thin air-to-blood tissue barrier of the alveolar septal iB Me Pe be oe 8 et Eviowibias uM
wall, which is composed offlat alveolar type | cells and occasional
FIGURE 15-5 Aspirometer reading of lung volumes. The total
rounded (Il) alveolar type Il cells. A small interstitial space separates the
lung capacity is the total volume ofair in an inflated human lung. After
epithelium and endothelium that form the (C) capillary wall. During
a maximum expiration, the lung retains a small volume ofair, which
lung injury the interstitial space enlarges and interferes with gas
is the residual volume.
The air volume moved into and out ofthe lung
exchange. (Used with permission of Dr Kent E. Pinkerton, University of
during maximal inspiratory and expiratory movement, which is called
California, Davis.)
the vital capacity.
The tidal volume is typically moved into and out of
the lung during each breathe. The functional residual capacity and
residual volume cannot be measured with spirometer.

Function
Ventilation—The principal function of the lung is gas Spirometry is a test in which an individual inhales maxi-
exchange, which consists of ventilation, perfusion, and dif- mally and then exhales as rapidly as possible. The volume of
fusion. During inhalation, fresh air is moved into the lung air expired in one second, called the forced expiratory volume
through the upper respiratory tract and conducting airways 1 second (FEV1), and the total amount expired, forced vital
and into the terminal respiratory units when the thoracic cage capacity (FVC), and the ratio of FEV1/FVC, are good measures
enlarges and the diaphragm moves downward; the lung pas- of the recoil capacity and airway obstruction of the lung. In a
sively follows this expansion. After diffusion of oxygen into healthy individual the FEV1/FVC = ~80%.
the blood and that of CO, from the blood into the alveolar
spaces, the air (now enriched in CO,) is expelled by exhala- Perfusion—tThe lung receives the entire output from the right
tion. Relaxation of the chest wall and diaphragm diminishes ventricle, ~75 mL of blood per heartbeat. Blood with high CO,
the internal volume of the thoracic cage, the elastic fibers and low O, travels to the lung via the pulmonary artery and
of the lung parenchyma recoil, and air is expelled from the leaves the lung with high O, and low CO, via the pulmonary
alveolar zone through the airways. Any interference with vein. The bronchi also have independent circulation with O,-
the elastic properties of the lung, e.g., the alteration of elastic enriched blood supplied by an artery. Substantial amounts of
fibers that occurs in emphysema, adversely affects ventila- toxic chemicals carried in the blood can be delivered to the
tion, as do the decrease in the diameters of, or blockage of, the lung. A chemical placed onto or deposited under the skin (sub-
conducting airways, as in asthma. cutaneous injection) or introduced directly into a peripheral
Lung function changes with age and disease and can be mea- vein (intravenous injection) travels through the venous system
sured with a spirometer (Figure 15-5). The total lung capac- to the right ventricle and comes into contact with the lungs
ity (TLC) is the total volume of air in an inflated human lung, before distribution to other organs or tissues in the body.
4 to 5L (women) and 6 to 7 L (men). After a maximum expira-
tion, the lung retains 1.1 L (women) and 1.2 L (men), which is Diffusion—Gas exchange takes place across the entire alveo-
the residual volume (RV). The vital capacity is the air volume lar surface, meaning contact with an airborne toxic chemical
moved into and out of the lung during maximal inspiratory occurs over an area of ~140 m7’. This surface area is second
and expiratory movement and typical is 3.1L (women) and only to the small intestine (~250 m’) and is considerably larger
4.8L (men). Only a small fraction of the VC, the tidal volume than the skin (~2 m’). Oxygen normally diffuses, unhindered,
(TV), is typically moved into and out of the lung during quiet across the pulmonary capillary and into erythrocytes. Acute
breathing. In resting humans, the TV measures ~0.5L with events that can disrupt this process may include collection
each breath. The respiratory frequency is 12 to 20 breaths per of liquid in the alveolar or interstitial space and disruption of
minute (thus the resting ventilation is about 6-8 L/min). the pulmonary surfactant system. Chronic toxicity can impair
Nwi)CO UNIT 4 Target Organ Toxicity

diffusion due to abnormal alveolar architecture or abnormal concentration of mg/m? or ug/m’. For reference, 1m* is
formation and deposition of extracellular substances such as 1000L. For gases, concentration may also be expressed as
collagen in the interstitium. volume to volume of air, that is, parts per million (ppm) or
parts per billion (ppb). This can be calculated from the mass
per unit air by using the ideal gas law to determine the gas's
BIOTRANSFORMATION IN THE volume. It is important to note that exposure does not equate
RESPIRATORY TRACT to dose (compound mass per unit), which requires a measure
of organ, cell, or subcellular target.
Often overlooked as an organ involved in metabolism of chem-
The sites of deposition of gases in the respiratory tract
icals, in favor of the liver, the lung has substantial capabilities
define the pattern of toxicity of those gases. Solubility, dif-
for biotransformation (see Chapter 6). Total lung cytochrome
fusivity, and metabolism/reactivity in respiratory tissues and
P450 (CYP) activity is roughly one-tenth to one-third of that
breathing rate are the critical factors in determining how
in the liver. However, when specific activity in a few cell types
deeply a given gas penetrates into the lung. Highly soluble
is considered, the difference is only twofold for many enzymes,
gases such as SO, or formaldehyde do not penetrate farther
and in the case of nasal mucosa, higher enzyme activity is
than the nose (during nasal breathing) unless doses are very
reported per cell. Metabolic competence in the lung and nasal
high, and are therefore relatively nontoxic to the lung of rats
tissues is concentrated in a few cell types and these have a
defined, and sometimes limited, distribution in the respiratory (which are obligatory nasal breathers). Relatively insoluble
tract that can vary substantially by species.
gases such as ozone and NO, penetrate deeply into the lung
The CYP monooxygenase system is concentrated into a few and reach the smallest airways ard the alveoli (centriacinar
lung cells: BSCs, alveolar type II cells, macrophages, and endo- region), where they can élicit toxic responses. Very insoluble
thelial cells. Of these cell types, BSCs have the most CYP, fol- gases such as CO and HS efficiently pass through the respi-
lowed by the type II cells. The total amount of total lung CYP ratory tract and are taken up by the pulmonary blood sup-
contributed by BSCs is species-dependent, as are the CYP iso- ply to be distributed throughout the body. Mathematical
forms present and their location along the respiratory tract. models of gas entry and deposition in the lung predict sites
Most species have CYPs in nasal tissue and some are predom- of lung lesions fairly accurately. These models may be use-
inantly expressed in the olfactory mucosa, which may play a ful for extrapolating findings made in laboratory animals
role in providing or preventing access of inhalants directly to to humans.
the brain.
Phase II enzymes include glutathione S-transferases (GSTs) Regional Particle Deposition
(alpha, mu, and pi), glucuronosyl transferases, and sulfotrans- Particle size is a critical factor in determining the region of the
ferases (SULTs). GSTs (and glutathione) play a major role in respiratory tract in which a particle will be deposited. In respi-
the modulation of both acute and chronic chemical toxicity ratory toxicology, aerosols (solid or liquid particles dispersed
in the lung. These enzyme systems work in concert with one into air) include dusts, fumes, smoke, mists, fog, or smog
another and it is the combined action of all these enzymes that (ranging from = 1.0,1m for dusts to = 0.01-50 [um for smog).
determines toxicity. The regulation of many of these enzymes is Smaller aerosols include submicrometer particles, nanometer
under coordinated control of the transcription factor nuclear particles or nanoparticles. All these distinguishing forms are
factor, erythroid-derived 2, -like 2 (aka Nrf2). included in the term “aerosol” or “particle.”
A major determinant of the potential for detoxification may The upper respiratory tract is very efficient in remoy-
also be the cellular localization of, and the ability to synthe- ing particles that are very large (> 101m) or very small
size, glutathione in the lung. The distribution of GST isoforms (<0.01j1m) (Figure 15-1). During nasal breathing, 1 to
varies by lung region and their activity is 5% to 15% of that 101m particles are usually deposited in the upper naso-
in the rodent liver and about 30% of that in the human liver. pharyngeal region or the first five generations of large con-
Polymorphisms in glutathione transferase genes have been ducting airways. During oral breathing, deposition of these
associated with a possible increase in risk of developing lung particles can increase in the tracheobronchial airways and
cancer, particularly in smokers. alveolar region. Smaller particles (0.001-0.1 [1m) can also be
deposited in the tracheobronchial region. Particles ranging
GENERAL PRINCIPLES IN THE from 0.003 to 5 jum can be transported to the smaller airways
and deposited in the alveolar region.
PATHOGENESIS OF LUNG DAMAGE Patterns of breathing can change the site of deposition of a
CAUSED BY CHEMICALS particle ofa given size. The size of a particle may change during
inspiration before deposition in the respiratory tract. Materials
Toxic Inhalants, Gases, and Dosimetry that are hygroscopic (i.e., those that readily absorb moisture),
In inhalation toxicology, exposure is measured as a con- such as sodium chloride, sulfuric acid, and glycerol, take on
centration (compound mass per unit of air). Typically water and grow in size in the warm, saturated atmosphere of
highly toxic compounds can produce adverse effects in a the upper and lower respiratory tract.
 CHAPTER 15 Toxic Responses of the Respiratory System 229

Deposition Mechanisms
In the respiratory tract, particles deposit by impaction, inter-
ception, sedimentation, diffusion, and electrostatic deposition
(for positively charged particles only) (Figure 15-6). Impaction
occurs in the upper respiratory tract and large proximal air-
ways where the airflow is faster than in the small distal airways
because the cumulative diameter is smaller than in the proxi-
mal airways. In humans, most > 10 tm particles are deposited Electrostatic
in the nose or oral pharynx and cannot penetrate tissues distal attraction

to the larynx. For 2.5 to 101m particles, impaction continues

to be the mechanism of deposition in the first generations of
the tracheobronchial region.
Interception occurs when the trajectory of a particle brings
it near enough to a surface so that an edge of the particle con-
tacts the airway surface. Although fiber diameter determines
the probability of deposition by impaction and sedimentation,
interception is dependent on fiber length. Thus, a fiber with
a diameter of 1 {1m and a length of 200 1m will be deposited
in the bronchial tree primarily by interception rather than FIGURE 15-6 Mechanism of particle deposition in the
impaction. Interception is also important for submicrometer respiratory tract. |mpaction occurs in the upper respiratory tract
particles in the tracheobronchial region where inertial airflow and large proximal airways where fast airflow imparts momentum
directs a disproportionately large fraction of the flow volume to the inhaled particle. The particle's inertia causes it to continue
toward the surface of small airway bifurcations. to travel along its original path and deposit on the airway surface.
Sedimentation controls deposition in the smaller bronchi, the Interception occurs when the trajectory of a particle brings it near
bronchioles, and the alveolar spaces, where the airways are small enough to a surface so that an edge ofthe particle contacts the airway
and the velocity of airflow is low. Air resistance and buoyancy surface. Sedimentation controls deposition in the smaller bronchi, the

act on a particle in an upward direction while gravity acts on a bronchioles, and the alveolar spaces, where the airways are small and
the velocity ofairflow is low. Sedimentation is dependent on the time
particle in a downward direction. These forces eventually bal-
a particle is ina compartment (i.e.,an alveolus) and can be increased
ance as a particle travels through air, causing the particle to set-
by breath holding. Diffusion is an important factor in the deposition
tle. Sedimentation is not a significant route of particle deposition of submicrometer particles. Electrostatic deposition is a minor
when the aerodynamic diameter is <0.5|1m. Sedimentation is deposition mechanism for positively charged particles. The surface
dependent on the time a particle is in a compartment (i.e., an of the airways is negatively charged and attracts positively charged
alveolus) and can be increased by breath holding. particles. (Adapted with permission from Lippmann M (ed):
Diffusion ofa particle within the air is an important factor in Environmental toxicants human exposures and their health effects. 3rd
the deposition of submicrometer particles. The distance a par- edition. New York, NY: Wiley; 2009.)
ticle travels within a gas depends upon the ratio of the particle’s
mass to the momentum of the colliding gas molecules such
that large particles are hardly moved and nanoparticles are impaction in the large airways and sedimentation and diffu-
moved extensively. Diffusion is an important deposition mech- sion in the smaller airways and alveoli increase. Breath holding
anism in the nose, airways, and alveoli for particles < 0.5 [um. also increases deposition from sedimentation and diffusion.
Nanometer particles (0.1{12m and smaller) are also trapped Cigarette smoke is hydroscopic aerosol of nicotine-laden parti-
relatively efficiently in the upper airways by diffusion. Particles cles that grow to a median diameter of about 0.5 to 1.0 um. Thus,
that penetrate beyond the upper airways are available to be a smoker's respiratory pause at the end of inhalation increases
deposited in the bronchial region and the deep-lying airways. alveolar sedimentation and thereby nicotine delivery to the alve-
Electrostatic deposition is a minor deposition mechanism olar surface and to the blood upon absorption.
for positively charged particles. The surface of the airways is Factors that modify the diameter of the conducting airways
negatively charged and attracts positively charged particles. can alter particle deposition. In patients with chronic bronchi-
Freshly fractured mineral dust particles and laboratory- tis or pneumonia, the airway lining fluid can greatly thicken
generated aerosols from evaporation of aqueous droplets can and may partially block the airways in some areas. Sonic jets
have substantial electrostatic mobilities. (e.g., during wheezing and rales) formed by high air flowing
During quiet breathing, in which the TV is only two to three through such partially occluded airways have the potential to
times the volume of the anatomic dead space (i.e., the volume of increase the deposition of particles by impaction and diffusion
the conducting airways where gas exchange does not occur), a in the small airways. Irritant materials that produce broncho-
large proportion of the inhaled particles may be exhaled. During constriction tend to increase the proximal tracheobronchial
exercise, when larger volumes are inhaled at higher velocities, deposition ofparticles.
 0
Nwee) UNIT 4 Target Organ Toxicity

Particle Clearance the respiratory tract. The epithelial barrier in the alveolar zone,
after a latency period ofseveral hours, begins to leak, flooding
Lung defense is dependent on particle clearance, wherein
the alveoli and producing a delayed pulmonary edema that is
rapid removal lessens the time available to cause damage to the
often fatal.
pulmonary tissues or permit local absorption. However, it is
A different pathogenetic mechanism is typical of highly
important to remember that particle clearance is not equiva-
reactive molecules such as ozone. It is unlikely that ozone as
lent to clearance from the body. such can penetrate beyond the layer of fluid covering the cells
ofthe lung. Instead, ozone lesions are propagated by a cascade
Nasal Clearance— Particles deposited in the anterior portion
of secondary reaction products and by reactive oxygen species
of the nose are removed by extrinsic actions such as wiping
and blowing. Particles deposited in the posterior portion of the arising from free radical reactions.
nose are removed by mucociliary clearance that propels mucus
toward the glottis, after which the particles are swallowed. Bronchoconstriction, Airway
Soluble particles may dissolve and enter the epithelium and/or Hyperreactivity, and Neurogenic
blood before they can be mechanically removed.
Inflammation
Tracheobronchial Clearance—Particles deposited in the Large diameter airways are surrounded by bronchial smooth
tracheobronchial tree are also removed by mucociliary clear- muscles, which help maintain airway tone and diameter during
ance. In addition to deposited particles, particle-laden macro- expansion and contraction ofthe lung. Bronchial smooth mus-
phages are also moved upward to the oropharynx, where they cle tone is normally regulated by the autonomic nervous system.
are swallowed. Bronchoconstriction can be provoked by irritants (acrolein),
cigarette smoke, air pollutants, cholinomimetic drugs (acetyl-
Alveolar Clearance—Particles deposited in the alveolar choline), histamine, various prostaglandins and leukotrienes,
region are removed by specialized cells, the alveolar macro- substance P, and nitric oxide. Bronchoconstriction causes a
phage. Lung defense involve both the innate and adaptive and decrease in airway diameter and a corresponding increase in
immune systems (see Chapter 12). Macrophages are the pri- resistance to airflow. Characteristic symptoms include wheez-
mary effector of innate lung immunity and their ability to ing, coughing, a sensation of chest tightness, and dyspnea.
accomplish phagocytosis depends on the recognition of foreign Exercise potentiates these problems. Because the major com-
or damage cells by a variety of macrophage surface macromol- ponent of airway resistance usually is contributed by large
ecules and receptors. Phagocytosis requires (1) particle binding bronchi, inhaled chemicals that cause reflex bronchoconstric-
to the membrane specifically via recognition molecule-receptor tion are generally irritant gases with moderate solubility.
interactions or nonspecifically by electrostatic forces (inert
materials), (2) receptor activation that initiates cell signaling,
(3) actin polymerization and coordinated cytoskeletal move- Acute Lung Injury (Pulmonary Edema)
ments that leads to extension of membranes, and (4) vesicular Acute lung injury (adult or infant respiratory distress syn-
membrane closure closely apposed to the particle or the fiber drome) is marked by alveolar epithelial and endothelial cell
ingested forming a phagosome shaped by the material ingested. perturbation and inflammatory cell influx that leads to sur-
factant disruption, pulmonary edema, and atelectasis. Toxic
pulmonary edema represents an acute, exudative phase of
ACUTE RESPONSES OF THE lung injury that alters ventilation—perfusion relationships and
LUNGTO INJURY limits diffusive transfer of O, and CO, even in otherwise struc-
turally normal alveoli. Acrolein, HCl, NO,, NH;, or phosgene
Trigeminally Mediated Airway Reflexes may compromise alveolar barrier function several hours after
exposure to low concentrations, and immediate alveolar dam-
Certain gases and vapors stimulate nerve endings in the nose,
age and death with high concentrations.
particularly those of the trigeminal nerve. The result is hold-
ing of the breath or changes in breathing patterns, to avoid or
reduce further exposure. Transient receptor potential channel
receptors may be activated by many irritants causing tickling, CHRONIC RESPONSES OF THE
itching, and painful nasal sensations. Subfamily A receptors LUNGTO INJURY
are activated by several irritants including acrolein, allyl iso-
thiocyanate (wabasi), allicin (garlic), cinamaldehyde, chlorine, Chronic Obstructive Pulmonary Disease
ozone, and hydrogen peroxide. Characterized by a progressive airflow obstruction, chronic
If continued exposure cannot be avoided, many acidic or obstructive pulmonary disease involves airway (bronchitis)
alkaline irritants produce cell necrosis and increased perme- and alveolar pathology. Chronic bronchitis is defined by the
ability of the alveolar walls. Other inhaled agents can be more presence of sputum production and cough for at least three
insidious; inhalation of high concentrations of HCl, NO,, NH,, months. In emphysema, destruction of the gas-exchanging
or phosgene may at first produce very little apparent damage in surface area results in a distended, hyperinflated lung that no
 CHAPTER 15 Toxic Responses ofthe Respiratory System 231

longer effectively exchanges oxygen and carbon dioxide as

a result of both loss of tissue and air trapping (Figure 15-7).
The major cause of human emphysema is, by far, cigarette
smoke inhalation, although other toxicants also can elicit this
response. A feature of toxicant-induced emphysema is severe
or recurrent inflammation.
The pathogenesis of emphysema involves aproteinase-
antiprotease imbalance that leads to the remodeling of the
supportive connective tissue in the parenchyma and separate
lesions that coalesce to destroy lung tissue. Alpha,-antiprotease
(also called alpha,-antitrypsin) is one of the body’s main
defenses against uncontrolled proteolytic digestion by this
class of elastolytic enzymes, which includes elastase. Studies
in smokers led to the hypothesis that neutrophil (and perhaps
alveolar macrophage) elastases can break down lung elastin
and thus cause emphysema; these elastases usually are kept in
check by alpha,-antiprotease that diffuses into the lung from
the blood. As the individual ages, an accumulation of random
elastolytic events can cause the emphysematous changes in the
lungs that are normally associated with aging. Toxicants that
cause inflammatory cell influx and thus increase the burden of
neutrophil elastase can accelerate this process.

Lung Cancer
Lung cancer is now the leading cause of death from cancer
among men and women. Retrospective and prospective epide-
miologic studies unequivocally show an association between
tobacco smoking and lung cancer. Average smokers have a
10-fold and heavy smokers a 20-fold increased risk of devel-
oping lung cancer compared with nonsmokers. Many other
agents also cause lung cancer (see Table 15-1).
Human lung cancers may have a latency period of 20
to 40 years, making the relationship to specific exposures dif-
ficult to establish. Two major forms are non-small-cell lung
cancer, which accounts for about 85% of all lung cancers, and
may be characterized as squamous cell carcinoma, adenocar-
cinoma, and large-cell lung cancer. Small-cell lung cancers
account for about 15% of lung cancers. Compared with can-
cer in the lung, cancer in the upper respiratory tract is less
common.
The potential mechanisms of lung carcinogenesis center
on damage to DNA. An activated carcinogen or its metabolic
product may interact with DNA. DNA damage caused by
active oxygen species is another potentially important mecha-
nism. Ionizing radiation leads to the formation of superoxide.
Cigarette smoke contains high quantities of active oxygen
species and other free radicals. Critical genetic and epigen-
etic changes include DNA mutations, loss of heterozygosity,
FIGURE 15-7 Airspace enlargement induced by tobacco
smoke and pulmonary fibrosis induced by asbestos in rat lung.
and promoter methylation. Global transcriptome changes can
Top panel: Normal rat luhg. Middle panel: Extensive distention of the include stimulation of mitogenic pathways and suppression of
alveoli (emphysema) in rat lung following inhalation of tobacco apoptosis.
smoke (90 mg/m’ oftotal suspended particulate material). Bottom
panel: Lung ofakat one year ai ee to chrysotile asbestos. Asthma
Note accumulation of connective tissue around blood vessel
and airways (fibrosis). Bar length: 100 jum. (Used with permission of Asthma is characterized clinically by attacks of shortness of
Dr Kent E. Pinkerton, University of California, Davis.) breath, which is caused by narrowing of the large conducting
Zoe
S UNIT 4 Target Organ Toxicity

TABLE 15-1 Agents that produce lung injury and disease.

Toxicant Disease Exposure Acute Effect Chronic Effect

Acrolein Acute lung injury, chronic Biomass or hot oil cooking, fire Cough, shortness of Chronic obstructive
obstructive pulmonary fighters, environmental tobacco breath, extreme oronasal pulmonary disease,
disease smoke, biocide water treatment irritation, pulmonary possibly asthma or lung
edema, airway cancer
hyperreactivity

Aluminum abrasives Shaver disease, Abrasives manufacturing, smelting Alveolar edema Interstitial fibrosis,
corundum smelter's emphysema
lung, bauxite lung

Aluminum dust Aluminosis Aluminum, firework, ceramic, paint, Cough, shortness of breath Interstitial fibrosis
~
electrical good, and abrasive
manufacturing

Ammonia Farming, refrigeration operations, Oronasal and bronchial Acute lung injury, chronic
ammonia, fertilizer, chemical, irritation, pulmonary bronchitis
and explosive manufacturing edema

Arsenic Pesticide, pigment, glass, and alloy Bronchitis Laryngitis, bronchitis, and
manufacturing lung cancer

Asbestos Asbestosis Mining, construction, shipbuilding, Fibrosis, pleural

brake repair, vermiculite , Calcification, lung cancer,
contaminant mesothelioma

Aspergillus Framer lung, composte Working with moldy hay, compost, Bronchoconstriction, Extrinsic allergic alveolitis
lung, malt worker's or barley cough, chest tightness (hypersensitivity
lung pneumonitis)

Avian protein Bird fancier’s lung Bird handling and farming with Bronchoconstriction, Extrinsic allergic alveolitis
exposure to bird droppings cough, chest tightness (hypersensitivity
pneumonitis)

Beryllium Berylliosis Mining, alloy, and ceramic Pulmonary edema, Interstitial granulomatosis,
manufacturing, Milling beryllium pneumonia progressive dyspnea, cor
pulmonarle, fibrosis, and
lung cancer

Cadmium Welding, smelting, and electrical Cough, pneumonia Emphysema, cor pulmonale
equipment, battery, alloy, and
pigment manufacturing

Carbides of Hard metal disease Metal cutting and manufacturing Bronchial epithelial hyper- Peribronchial and
tungsten, and metaplasia perivascular fibrosis
titanium, or
tantalum

Chlorine Paper, plastics, chlorinated product Cough, hemoptysis,

manufacturing dyspnea, bronchitis,
pneumonia

Chromium (VI) Chromium compound, paint, Oronasal and bronchial Fibrosis, lung cancer
pigment, chromite ore reduction irritation
manufacturing

Coal dust Coal worker's Coal mining Fibrosis with emphysema

pneumoconiosis

Cotton dust Byssinosis Textile manufacturing Chest tightness, wheezing, Restrictive lung disease,
dyspnea chronic bronchitis
Hydrogen fluoride Chemical, photograph film, solvent Airway irritation,
and plastic manufacturing hemorrhagic pulmonary
edema
 CHAPTER 15 Toxic Responses of the Respiratory System pagsFe)

TABLE 15-1 Agents that produce lung injury and disease. (Continued)
ae

7
ae et
i ANE ee
AeA e
a“ =
= Disease 4 2 2 Exposure Acute Effect Chronic Effect
= OO
Oo

Iron oxides Siderotic lung disease, Welding, steel and jewelry Cough Silver finisher’s lung
silver finisher’s lung, manufacturing, foundry work, with subpleural and
hematite miner's lung, hematite mining perivascular macrophage
arc welder’s lung aggregates; hematite
miner’s lung with
diffuse fibrosis-like
pneumoconiosis; arc
welder’s lung with
bronchitis

Isocyanates Auto painting, and plastic and Airway irritation, Asthma

chemical manufacturing cough, dyspnea

Kaolin Kaolinosis Pottery making Fibrosis

Manganese Manganese pneumonia Chemical and metal manufacturing Acute pneumonia Recurrent pneumonia
(often fatal)

Nickel Nickel mining, smelting, Delayed pulmonary Acute lung injury, chronic
electroplating, battery edema, skin allergy bronchitis, non- small-cell
manufacturing, fossil fuel lung cancer, nasal cancer
combustion

Nitrogen oxides Silo-filler’s diseases Silo filling, welding, explosive Immediate or delayed Bronchiolitis obliterans,
manufacturing pulmonary edema emphysema in
experimental animals

Nontuberculous Metalworking fluid Working with metal cutting fluid Bronchoconstriction, Extrinsic allergic alveolitis
mycobacteria hypersensitivity contain water and contaminated cough, chest tightness (hypersensitivity
with mycobacteria pneumonitis)

Organic (sugar cane) Bagassosis Sugarcane and molasses Bronchoconstriction, Extrinsic allergic alveolitis
dust (possibly manufacturing (bagasse is the cough, chest tightness (hypersensitivity
contaminated fibrosis residue from sugar pneumonitis)
with thermophilic extraction)
actinomycete)

Ozone Welding, photocopying, bleaching Substernal pain, Fibrosis (including airways)

flour, water treatment, exacerbation of asthma,
deordorizing bronchitis, pulmonary
edema

Perchloroethylene Dry cleaning, metal degreasing, Edema Hepatic and lung cancer
grain fumigation

Phosgene Plastic, pesticide, and chemical Severe pulmonary Bronchitis and fibrosis
manufacturing edema

Silica Silicosis, pneuMOcoOniosis Mining, stone cutting, sand Acute silicosis Fibrosis, silicotuberculosis
blasting, farming, quarry mining, (inflammation)
tunneling

Sulfur dioxide Chemical manufacturing, Bronchoconstriction, Chronic bronchitis

refrigeration, bleaching, cough, chest tightness
fumigation

Talc Talcosis Mining, rubber manufacturing, Cough Fibosis

cosmetics

Thermophilic Farmer’s lung, mushroom Farming (hay or grain degradation) Bronchoconstriction, Extrinsic allergic alveolitis
actinomycete - worker's lung, penguin cough, chest tightness (hypersensitivity
humidifier lung pneumonitis)

Tin Mining, tin processing Widespread mottling in

chest X-ray often without
clinical impairment

Vanadium Metal cutting and manufacturing, Airway irritation and Chronic bronchitis
specialty steel manufacturing mucus production
234 UNIT 4 Target Organ Toxicity

airways (bronchi). The clinical hallmark of asthma is increased arterial and venous blood, distribution ofventilation, and lung
airway reactivity of the bronchial smooth muscle in response and chest wall compliance.
to exposure to irritants. There may be common mechanisms Diffusion defects (i.e., defects in gas exchange across the pul-
between asthma and pulmonary fibrosis, with regard to the monary capillary) can be evaluated by measuring the arterial
role of recurrent or chronic inflammation in disease pathogen- partial pressure of both oxygen and CO,, In general, blood gas
esis. Agents that can induce asthma are listed in Table 15-1. analysis is a comparatively insensitive assay for disturbed ven-
tilation because ofthe organisms’ buffering and reserve capaci-
ties, but may be a useful tool in clinical medicine. Measurement
Pulmonary Fibrosis of diffusion capacity with CO, a gas that binds with 250 times
Fibrotic lungs from humans with acute or chronic pulmo- higher affinity to hemoglobin than does oxygen, is more sen-
nary fibrosis contain increased amounts of collagen. In lungs sitive. Proper lung function in humans can be evaluated with
damaged by toxicants, the response resembles adult or infant several additional techniques, including computed tomogra-
respiratory distress syndrome. Excess lung collagen is usu- phy (CT), molecular content analysis, fiberoptic bronchoscopy.
ally observed not only in the alveolar interstitium, but also
throughout the alveolar ducts and respiratory bronchioles
(Figure 15-7). Animal Studies
Types I and III collagen are major interstitial compo- The toxicology of inhaled materials has been and continues
nents and are found in an approximate ratio of 2:1. There to be extensively studied in experimental animals. Obviously,
is an increase in type I collagen relative to type HI collagen selecting animals with a respiratory system similar to that of
in patients with idiopathic pulmonary fibrosis and patients humans is particularly ‘desirable (e.g., monkey). However,
dying of acute respiratory distress syndrome. It is not known rodents are widely used despite fundamental differences to
whether shifts in collagen types, compared with absolute combat cost and ethical considerations’
increases in collagen content, account for the increased
stiffness of fibrotic lungs. Because type III collagen is more Inhalation Exposure Systems—In inhalation studies,
compliant than type I, increasing type I relative to type I animals are kept within a chamber that is ventilated with a
collagen may result in a stiffer lung. Changes in collagen defined test atmosphere. Generation of such an atmosphere is
cross-linking in fibrotic lungs also may contribute to the comparatively easy for gases that are available in high purity
increased stiffness. in a compressed tank (e.g., SO,, O,, NO). Gas concentration
within the chamber is measured continuously, and is usually
within 5% of the targeted concentration. More challenging is
AGENTS KNOWN TO PRODUCE LUNG
the generation of particles or complex mixtures (e.g., tobacco
INJURY INHUMANS smoke, diesel, and gasoline exhaust or residual oil fly ash),
There are over 7 900 unique chemicals that are commonly used particularly because of the possibility of interactions between
in industry, many of which represent hazards to the respira- individual mixture constituents and the possibility of forma-
tory tract. Exposure prevention is one of the most effective tion of artifacts.
approaches to prevent lung injury and disease, and many val-
ues and exposure limits exist to aid prevention. Nonetheless, Pulmonary Function Tests in Experimental Animals—
given the large morbidity and mortality associated with current Conducting pulmonary function tests in experimental ani-
acute and chronic lung disease, a great need exists to develop mals poses distinct challenges, especially in small rodents.
additional preventative and therapeutic strategies based on the Experimental animals cannot be made to maximally inhale
knowledge of the cellular and molecular events that determine or exhale at the investigator’s will, for instance. Analysis
lung injury and repair. Table 15-1 lists a portion ofthe respira- of pressure-volume curves, which provides an indication of
tory toxicants that can produce acute and chronic lung injury lung compliance, is comparatively easy to perform in animals
in humans. in that it does not require a specialized apparatus. Another
pulmonary function test is the analysis of airway resistance,
which can be measured via restrained plethysmography, unre-
EVALUATION OF TOXIC LUNG DAMAGE strained video-assisted plethysmography, or unrestrained
acoustic plethysmography. Analysis of breathing pattern can
Human Studies also be used and may differentiate between upper airway and
Although the lung is susceptible to multiple toxic injuries, it lower airway irritants. In rodents, upper airway (“sensory”)
is also amenable to a number of tests that allow evaluation of irritants produce a breathing pattern of decreased respira-
proper functioning. Commonly used tests include measure- tory frequency with increased tidal volume, whereas lower
ment of FEV1, FVC, and airway resistance. Additional tests airway (“pulmonary”) irritants produce a breathing pattern of
evaluate the maximal flow rates and different lung volumes, increased respiratory frequency and decreased minute volume
diffusion capacity, oxygen, and carbon dioxide content of the (i.e., the total volume of air breathed in 1 minute).
 CHAPTER 15 Toxic Responses of the Respiratory System 235

Morphological Techniques—The pathology of acute and (e.g., mouse, rat, guinea pig, or rabbit). The lung is perfused
chronic injury may be examined by gross inspection and under with blood or a blood substitute through the pulmonary
the microscope and should include the nasal passages, larynx, arterial bed. At the same time, the lung is actively (through
major bronchi, and the lung parenchyma. rhythmic inflation-deflation cycles with positive pressure)
Regional distribution of lesions in nasal passages can be or passively (by creating negative pressure with an artifi-
assessed after fixation and decalcification. Various regions of the cial thorax in which the lung is suspended) ventilated. Toxic
nasal passages can then be examined by obtaining cross sections agents can be introduced into the perfusate or the inspired air.
at multiple levels, staining the tissue to highlight particular struc- Repeated sampling of the perfusate allows one to determine
tures, and examining the tissue under a microscope. This per- the rate of metabolism of drugs and the metabolic activity
mits semiquantitative or quantitative measurements to be made. of the lung.
Additional tools for the study of toxic lung injury include
immunohistochemistry, in situ hybridization, and analysis of Airway Microdissection and Organotypic Tissue
cell kinetics. Transcriptome, proteome, and metabolome pro- Culture Systems—Many inhalants act in specific regions of
filing are additional valuable tools to assess the lung in health the respiratory tract. Microdissection of the nasal passage and
and disease. airways consists of stripping away surrounding tissue or paren-
chyma while maintaining the airway structure and expos-
Pulmonary Lavage and Pulmonary Edema—Pulmonary ing the epithelium. Microdissected airways can be studied in
edema and/or pulmonary inflammation are early events in culture for up to one week, can be used to study site-specific
acute and chronic lung injury. The fluid lining the pulmonary gene expression, morphological changes in toxicant injury
epithelium can be recovered by the medical procedure, bron- and repair, or can be used for biochemical analyses including
choalveolar lavage. Analysis of the lavage fluid is a useful tool to enzyme activity measurements and determination of antioxi-
detect respiratory tract toxicity. Influx of neutrophils or other dant concentrations (such as glutathione).
leukocytes such as lymphocytes or eosinophils into the lavage Tissue culture systems have been developed in which epi-
fluid is the most sensitive sign of inflammation. Measurements thelial cells maintain their polarity, differentiation, and normal
of lung injury include total protein and/or albumin. Additional function similar to what is observed in vivo. Epithelial cell sur-
measurements include secretory products of macrophages and faces are exposed to air (or a gas phase containing an airborne
epithelial cells include fribronectin, chemokines, and other toxic agent), while the basal portion is bathed by a tissue cul-
cytokines (e.g., TNF or IL1B). Reduced glutathione levels may ture medium.
be an indicator of oxidative stress. Lactate dehydrogenase activ-
ity (and its substituent isozymes), N-acetylglucosaminidase, Lung Cell Culture— Many lung-specific cell types have been
acid or alkaline phosphatase, other lysosomal hydrolases, and isolated and can be maintained as cell culture. Human and
sialic acid add additional information. In addition pulmonary animal alveolar or interstitial macrophages can be obtained
edema can be assessed by determining lung wet:dry ratio or from lavage or lung tissue. Their function can be examined in
injection of Evan blue dye albumin. vitro with or without exposure to appropriate toxic stimuli.
Type II alveolar epithelial cells can be isolated and primary
cell cultures maintained in culture for short periods. Direct
In Vitro Studies isolation oftype I epithelial cells has also been successful.
In vitro systems with materials originally obtained from
either human tissues or experimental animals are particularly
suited for the study of mechanisms that cause lung injury. The BIBLIOGRAPHY
methods include isolated perfused lung, microdissection/ Gardner DE (ed): Toxicology of the Lung, 4th ed. Boca Raton, FL: CRC
organotypic tissue culture systems, and cell type-specific cell Press/Taylor & Francis, 2006.
culture. Morris JB, Shusterman DJ: Toxicology of the Nose and Upper Airways,
New York: Informa, 2010.
Isolated Perfused Lung—The isolated perfused lung Salem H, Katz SA: Inhalation Toxicology, 3rd ed., Boca Raton, FL:
method is applicable to lungs from many laboratory species CRC Press, 2015.
booS) UNIT 4 Target Organ Toxicity

QUESTIONS

Which ofthe following statements is FALSE regarding the All of the following statements regarding particle deposi-
role of mucus in the conducting airways? tion and clearance are true EXCEPT:
a. Pollutants trapped by mucus can be eliminated via a. One of the main modes of particle clearance is via
expectoration or swallowing. mucociliary escalation.
Mucus is of a basic pH. b. Diffusion is important in the deposition of particles
The beating of cilia propels mucus out ofthe lungs. in the bronchial regions.
Mucus plays a role promoting oxidative stress. c. Larger volumes of inspired air increase particle depo-
a Free radical scavenging is believed to be a role of sition in the airways.
mucus. Sedimentation results in deposition in the bronchioles.
e. Swallowing is an important mechanism of particle
Respiratory distress syndrome sometimes affects pre- clearance.
mature neonates due to lack of surfactant production by
which of the following cell types? Which ofthe following is not a common location to which
a. lung fibroblasts. particles are cleared?
type II pneumocytes. a. stomach.
endothelial cells. b. lymph nodes.
alveolar macrophages. c. pulmonary vasculature. »
cans
type I pneumocytes. d. _ liver. :
e: Glitract:
In a situation where there is an increased metabolic
‘
\

demand for oxygen, which of the following volume Pulmonary fibrosis is marked by which of the following?
measurements will greatly increase? a. increased type I collagen.
a. total lung capacity (TLC). b. decreased type III collagen.
b. residual volume (RV). c. increased compliance.
c. functional residual capacity (FRC). d. elastase activation.
d. tidal volume (TV). e. decreased overall collagen levels.
e. vital capacity (VC).
Activation of what enzyme(s) is responsible for emphysema?
The free radicals that inflict oxidative damage on the lungs a. antitrypsin.
are generated by all of the following EXCEPT: b. epoxide hydrolase.
a. tobacco smoke. c. elastase.
b. neutrophils. d. hyaluronidase.
c. ozone. e. nonspecific proteases.
d. monocytes.
en SOr 10. Which of the following measurements would NOT be
expected from a patient with restrictive lung disease?
Which of the following gases would most likely pass all a. decreased FRC.
the way through the respiratory tract and diffuse into the b. decreased RV.
pulmonary blood supply? c. increased VC.
a. O, (ozone). d. decreased FEV,.
b. NO.. e. impaired ventilation.
, lal(O)
dvEGO:
® GO),
 Cate
Aap OMT as Baek

Toxic Responses of the

Nervous System
Virginia C. Moser, Michael Aschner, Rudy J. Richardson,
and Martin A. Philbert

OVERVIEW OF THE NERVOUS SYSTEM Myelinopathies

Blood-Brain Barrier Hexachlorophene

Energy Requirements Tellurium
Axonal Transport Lead
Axonal Degeneration Astrocytes
Myelin Formation and Maintenance Ammonia
Neurotransmission Nitrochemicals
Development of the Nervous System Methionine Sulfoximine
Factors Relevant to Neurodegenerative Diseases Fluoroacetate and Fluorocitrate
Neurotransmission-associated Neurotoxicity
FUNCTIONAL MANIFESTATIONS OF NEUROTOXICITY RCOHine

MECHANISMS OF NEUROTOXICITY CESSES OTUIST NS

Excitatory Amino Acids
ee oneparnies Models of Neurodegenerative Disease
Doxorubicin MPTP
ety pore) Manganese

Ula cauayln Developmentally Neurotoxic Chemicals

Axonopathies
Gamma-diketones CHEMICALS THAT INDUCE DEPRESSION OF NERVOUS
Carbon Disulfide SYSTEM FUNCTION

Pe ee eu ey) IN VITRO AND OTHER ALTERNATIVE APPROACHES

pyle ge TO NEUROTOXICOLOGY
Organophosphorus Compounds
Pyridinethione
Microtubule-associated Neurotoxicity
 238 UNIT 4 Target Organ Toxicity

KEY POINTS

w The central nervous system (CNS) is protected from w Neuronopathy is the toxicant-induced irreversible loss
the adverse effects of many potential toxicants by an of neurons, including its cytoplasmic extensions, den-
anatomical blood-brain barrier. drites, and axons, and the myelin ensheathing the axon.
» Neurons are highly dependent on aerobic metabolism a Neurotoxicants that cause axonopathies cause axonal
because this energy is needed to maintain proper ion degeneration, and loss of the myelin surrounding that
gradients. axon; however, the neuron cell body remains intact.
s Individual neurotoxic compounds typically target the » Numerous naturally occurring toxins as well as synthetic
neuron, the axon, the myelinating cell, or the neurotrans- chemicals may interrupt the transmission of impulses,
mitter system. block or accentuate transsynaptic communication, block
reuptake of neurotransmitters, or interfere with second-
messenger systems.

OVERVIEW OF THE NERVOUS SYSTEM Blood-Brain Barrier

es \

Several generalities that allow a basic understanding of the The NS is protected from the adverse effects of many potential
actions of neurotoxicants include (1) the privileged status of
toxicants by an anatomical barrier between the blood and the
brain, or a “blood-brain barrier” (BBB). Most ofthe brain, spi-
the nervous system (NS) with the maintenance of a biochemi-
nal cord, retina, and peripheral NS (PNS) maintain this barrier
cal barrier between the brain and the blood; (2) the importance
of the high energy requirements of the brain; (3) the spatial with the blood, with selectivity similar to the interface between
extensions of the NS as long cellular processes and the require- cells and the extracellular space. To gain entry to the NS, mol-
ments of cells with such a complex geometry; (4) the mainte- ecules must pass into the cell membranes of endothelial cells of
nance of an environment rich in lipids; (5) the transmission of
the brain rather than between endothelial cells, as they do in
information across extracellular space at the synapse; (6) the other tissues (Figure 16-1). The principal basis of the blood-
distances over which electrical impulses must be transmitted, brain barrier is thought to be specialized endothelial cells in
coordinated, and integrated; and (7) development and regen- the brain’s microvasculature, aided, at least in part, by inter-
erative patterns of the NS. actions with glia. In addition to this interface with blood, the

Systemic capillaries Brain capillaries

Glial cells
Pericyte E>

Endothelial cell

Vascular Vascular
lumen lumen

Pinocytosis Lipid soluble

Fenestration Active transport Lipid soluble
FIGURE 16-1 Schematic diagram of the blood-brain barrier. Systemic capillaries are depicted with intercellular gaps, or fenestrations,
which
permit the passage of molecules incapable ofcrossing the endothelial cell. There is also more abundant pinocytosis
in systemic capillaries, in addition
to the transcellular passage of lipid-soluble compounds. In brain capillaries, tight junctions between endothelial
cells and the lack of pinocytosis limit
transport to compounds with active transport mechanisms or those that pass through cellular membranes
by virtue oftheir lipid solubility.
 CHAPTER 16 Toxic Responses of the Nervous System 239

brain, spinal cord, and peripheral nerves are also completely Axonal Transport
covered with a continuous lining of specialized cells that limits
Impulses are conducted over great distances at rapid speed,
the entry of molecules from adjacent tissue. In the brain and
providing information about the environment to the organism
spinal cord, this is the meningeal surface; in peripheral nerves,
in a coordinated manner that allows an organized response to
each fascicle of nerve is surrounded by perineurial cells.
be carried out at a specific site. However, the intricate organiza-
Among the unique properties of endothelial cells in the NS is
tion of such a complex network places an unparalleled demand
the presence of tight junctions between cells. Thus, molecules
on the cells of the NS. Single cells, rather than being spheri-
must pass through membranes of endothelial cells, rather than
cal and a few micrometers in diameter, are elongated and may
between them, as they do in other tissues.
extend over 1m in length. Two immediate demands placed
The blood-brain barrier also contains xenobiotic transport-
on the neuron are the maintenance of a much larger cellular
ers that transport some xenobiotics that have diffused through
volume, requiring more protein synthesis, and the transport
endothelial cells back into the blood. If not actively transported
of intracellular materials over great distances using various
into the brain, the penetration of toxicants or their metabolites
mechanisms. These demands require ATP.
is largely related to their lipid solubility and to their ability to
Axonal transport moves protein products from the cell body
pass through the plasma membranes of cells forming the bar-
to the appropriate site in the axon. Fast axonal transport car-
rier. However, spinal ganglia, autonomic ganglia, and a small
ries a large number of proteins from their site of synthesis in
number of other sites within the brain are not protected by
the cell body into the axon. Many proteins associated with
blood-tissue barriers. This discontinuity of the barrier is the
vesicles migrate through the axon at a rate of 400 mm/day
basis for the selective neurotoxicity of some compounds. The
(Figure 16-2). This process is dependent on microtubule-
blood-brain barrier is incompletely developed at birth and
associated ATPase activity and the microtubule-associated
even less so in premature infants. This predisposes the prema-
motor proteins (kinesin and dynein) that provide both
ture infant to brain injury by toxicants that are excluded from
the mechanochemical force in the form of a microtubule-
the NS later in life.
associated ATPase and the interface between microtubules
as the track and vesicles as the cargo. Vesicles are transported
Energy Requirements rapidly in an anterograde direction by kinesin, and they are
transported in a retrograde direction by dynein. This mech-
Neurons (and cardiac myocytes) are highly dependent on aer-
anism of cytoplasmic transport is amplified within the NS,
obic metabolism because they must use this energy to main-
compared with other cells, by the distances encompassed by
tain proper ion gradients. The brain is extremely sensitive to
the axonal extensions of neurons.
even brief interruptions in the supply of oxygen or glucose.
The transport of some organelles, including mitochondria,
Exposure to toxicants that inhibit aerobic respiration (e.g.,
constitutes an intermediate component of axonal transport,
cyanide) or to conditions that produce hypoxia (e.g., CO poi-
moving at 50 mm/day. The slowest component of axonal
soning) leads to early signs of neuronal dysfunction. Damage
transport represents the movement of the cytoskeleton itself
to the NS under these conditions is a combination of direct
(Figure 16-2). The cytoskeleton is composed of microtu-
toxic effects on neurons and secondary damage from systemic
bules formed by the association of tubulin subunits and
hypoxia or ischemia.

Vesicles and kinesin

Fast transport
400 mm/day

Hi

SCa
1 mm/day

FIGURE 16-2 Schematic diagram ofaxonal transport. Fast axonal transport is depicted as spherical vesicles moving along microtubules
with intervening microtubule-associated motors. The slow component A (SCa) represents the movement ofthe cytoskeleton, composed of
neurofilaments and microtubules. Slow component B (SCb) moves at a faster rate than SCa and includes soluble proteins, which are apparently
moving between the more slowly moving cytoskeleton.
240 UNIT 4 Target Organ Toxicity

neurofilaments formed by the association of three neurofila-

ment protein subunits.
Neurofilaments and microtubules move at a rate of approxi-
mately 1 mm/day and make up the majority of SCa, which is
the slowest moving component of axonal transport. Moving at
only a slightly more rapid rate of 2 to 4 mm/day in an antero-
grade direction is SCb, which is composed of many proteins. IOI,
LETS

Included in SCb are several structural proteins, such as the com-

ponent of microfilaments (actin) and several microfilament- ><

associated proteins (M2 protein and fodrin), as well as clathrin

and many soluble proteins. Se,

This continual transport of proteins from the cell body

through the various components of anterograde axonal trans-
port is the mechanism through which the neuron provides the
distal axon with its complement of functional and structural
proteins. Some vesicles are also moving in a retrograde direc-
tion and undoubtedly provide the cell body with information
6
concerning the status ofthe distal axon. ST

| I

}\ SR,
ASE
Ty
Stay U 4/ |\
Axonal Degeneration AN eo eo **@
When the neuronal cell body has been lethally injured, it
degenerates, in a process called neuronopathy. This is charac- FIGURE 16-3 Patterns of neurotoxic injury. (A) Normal neuron
terized by the loss of the cell body and all of its processes, with showing (1) cell body and dendrites, (2) myelinating cells, encircling
no potential for regeneration. However, when the injury is at the (3) axon, and (4) synapse. (B) A neuronopathy resulting from the
the level of the axon, the axon may degenerate while the neu- death of the entire neuron. Astrocytes often proliferate in response
ronal cell body continues to survive, a condition known as an to the neuronal loss, creating both neuronal loss and gliosis. (C) An
axonopathy. In this setting, there is a potential for regeneration axonopathy occurs when the axon is the primary site of injury, the axon
and recovery from the toxic injury as the axonal stump sprouts degenerates, and the surviving neuron shows only chromatolysis with
margination of its Niss] substance and nucleus to the cell periphery.
and regenerates (Figure 16-3).
(D) Myelinopathy resulting from disruption of myelin or selective injury
The result of axotomy (transection of an axon) is that the
to the myelinating cells.Toprevent cross-talk between adjacent axons,
distal axon is destined to degenerate, a process known as
myelinating cells divide and cover the denuded axon rapidly; however,
axonal degeneration, which is unique to the NS. The cell body the process of remyelination is much less effective in the CNS than
of the neuron responds to the axotomy as well and undergoes in the PNS. (E) Some forms oftoxicity are due to interruption of the
a process of chromatolysis. The sequence of events that occurs process of neurotransmission, either through blocking excitation
in the distal stump of an axon following transection is referred or by excessive stimulation, rather than actual cell death.
to as Wallerian degeneration. Because the axonal degeneration
associated with chemicals and some disease states is thought to
occur through a similar sequence of events, it is often referred In the PNS, Schwann cells respond to loss of axons by
to as Wallerian-like axonal degeneration. decreasing synthesis of myelin lipids, down-regulating genes
Following axotomy, there is degeneration of the distal encoding myelin proteins, and dedifferentiating to a premy-
nerve stump, followed by generation of a microenvironment elinating mitotic Schwann cell phenotype. The proliferat-
supportive of regeneration and involving the distal axon, ing Schwann cells create a tubular structure around the axon
ensheathing glial cells and the blood nerve barrier. Initially (referred to as a band of Bungner), providing physical guidance
there is a period during which the distal stump survives and for regenerating axons. These tubes also provide trophic sup-
maintains relatively normal structural, transport, and conduc- port from nerve growth factor (NGF), brain-derived neuro-
tion properties. The duration of survival is proportional to the trophic factor, insulin-like growth factor, and corresponding
length of the axonal stump, and this relationship appears to be receptors produced by the associated Schwann cells. Resident
maintained across species. macrophages distributed along the endothelium within the
Terminating the period of survival is an active proteolysis endoneurium and the denervated Schwann cells assist in clear-
that digests the axolemmia and axoplasm, leaving only a myelin ing myelin debris, but the recruitment of hematogenous mac-
sheath surrounding a swollen degenerate axon. Digestion of rophages accounts for the removal of the majority of myelin.
the axon appears to be an all-or-none event effected through Another essential role of recruited, circulating macrophages is
endogenous proteases that are activated through increased the production ofinterleukin-1 (IL-1), which is responsible for
levels of intracellular free Ca’*. stimulating production of NGF by Schwann cells.
 CHAPTER 16 Toxic Responses of the Nervous System 241

A critical difference exists between axonal degeneration surface of their membranes to form the major dense line of
in the CNS compared with that in the PNS: peripheral axons myelin. In a similar process, the extracellular space is reduced
can regenerate, whereas central axons cannot. Main factors on the extracellular surface of the bilayers, and the lipid mem-
contributing to the inability of the CNS to regenerate include branes stack together.
inhibitory factors secreted by oligodendrocytes, astrocyte scar- The maintenance of myelin is dependent on a number of
ring, and glial interference. Interestingly, experiments involv- membrane-associated proteins and on metabolism of specific
ing cellular transplants of Schwann cells to the CNS or CNS lipids present in myelin bilayers. Some toxic compounds inter-
neurons to the PNS show that the regenerative capability of fere with this complex process of the maintenance of myelin
CNS neurons depends on both the microenvironment and the and result in the toxic “myelinopathies” (Figure 16-3). In
properties of mature neurons. general, the loss of myelin with the preservation of axons is
Wallerian degeneration was long thought to be a passive pro- referred to as demyelination.
cess that proceeded inexorably after separating the axon from
the trophic support provided by the cell body. However, we
Neurotransmission
now know from several lines of evidence that Wallerian degen-
eration is an active process mediated by the axon itself, and Intercellular communication is achieved in the NS through the
that it is possible to slow or even halt its progression. Moreover, synapse. Neurotransmitters released from one neuron act as
although axonal degeneration can be initiated by many different the first messenger. Binding of the transmitter to the postsyn-
means, including physical, genetic, or toxic, the mechanisms of aptic receptor is followed by modulation of an ion channel or
degeneration converge into common regulated pathways that activation of asecond-messenger system, leading to changes in
are potentially subject to pharmacological intervention. the responding cell. Various therapeutic drugs and toxic com-
pounds impact the process of neurotransmission.
Neurotoxicity expresses itself in terms of altered conduction
Myelin Formation and Maintenance and propagation of nerve impulses and changes in functions
Myelin is formed in the CNS by oligodendrocytes and in the such as behavior, performance, and conditioning. Chemicals
PNS by Schwann cells. Both of these cell types form concentric acting on neurotransmission may interrupt the transmission
layers of lipid-rich myelin by the progressive wrapping of their of impulses, block or accentuate transsynaptic communica-
cytoplasmic processes around the axon in successive loops tion, block reuptake of neurotransmitters or precursors, or
(Figure 16-4). These cells exclude cytoplasm from the inner interfere with second-messenger systems.
In terms of toxicity, many side effects of neurological drugs
may be viewed as short-term interactions that are reversible
Repetitive =
with time or that may be counteracted by the use of appropri-

oe = 0 SO
KE Schwann cell wrapping Z =—
7, ~~ enclosure AN of axon s/ \\i\; ate antagonists. However, some of the toxicity associated with
long-term exposures may be irreversible. Excessive stimula-
* =e =e SSS tion of neurotransmitter systems may also have long-term
Schwann cell consequences; e.g., excitatory system (e.g., glutamate) pro-
duces excitotoxicity that is manifest as CNS diseases and nerve
cell death.
Compaction
Lipid Major Intraperiod of myelin
bilayers denseline line Development of the Nervous System
The NS begins development during gestation and continues
through adolescence. Proliferation, migration, differentiation,
synaptogenesis, apoptosis, and myelination are the basic pro-
cesses that underlie development of the NS, and these occur in
a tightly choreographed sequence that depends on the region,
cell type, and neurotrophic signals. The proliferation and
migration of neurons and glia occur in waves that are specific
for brain regions, but in general, the brain develops in a caudal
FIGURE 16-4 Process of myelination. Myelination begins to rostral direction (with cerebellar development being a nota-
when a myelinating cell encircles an axon, either Schwann cells
ble exception). During differentiation (phenotype expression)
in the peripheral nervous system or oligodendrocytes in the CNS.
and synaptogenesis (formation of functional synaptic connec-
Simple enclosure ofthe axon persists in unmyelinated axons. Myelin
tions), the circuitry of the NS is established. Chemicals such as
formation proceeds by a progressive wrapping of multiple layers of the
myelinating cell around the axon, with extrusion of the cytoplasm and
nerve growth factors, adhesive molecules, and neurotransmit-
extracellular space to bring the lipid bilayers into close proximity. The ters serve as morphogenic signals; neurotransmitter develop-
intracellular space is compressed to form the major dense line of myelin, mental signals are separate from their synaptic transmission
and the extracellular space is compressed to form the intraperiod line. function. Selected cells are also removed during ontogeny via
 4
i) bo UNIT 4 Target Organ Toxicity

apoptosis (programmed cell death), which results in the appro- fully appreciated. Emerging studies also suggest that miRNAs
priate cell types in the correct regions. The glial supportive cells may be targeted by neurotoxicants, thus potentially affecting
develop last, and myelination is protracted. a broad spectrum of functions, encompassing cell differentia-
The immature NS is especially vulnerable to certain agents tion and migration, neurogenesis, as well as synaptic function,
and there are several factors that make the developing NS to name a few.
uniquely susceptible. Cell sensitivity differs with the devel-
opmental stage, leading to critical windows of vulnerability.
Chemicals that alter the timing and formation of neural con- FUNCTIONAL MANIFESTATIONS
nections could result in permanent malformations, the conse- OF NEUROTOXICITY
quences of which may be quite unlike the chemical’s effects in
Functions of the NS include motor, sensory, autonomic, and
the adult NS. Furthermore, while synaptogenesis can continue
cognitive capabilities. Functional assessment uses a bat-
throughout life, proliferation cannot; therefore, the CNS is
tery of tests as a means for screening potentially neurotoxic
unique in that damaged neural cells are not readily replaced.
compounds. Specific behavioral methods include functional
Finally, there are physiological and kinetic differences in the
observational batteries (FOBs), Irwin screens, tests of motor
developing organism that may profoundly influence its sen-
activity, and expanded clinical observations. These tests have
sitivity, including the slow formation of the blood-brain bar-
the advantage over biochemical and pathological measures in
rier and lack of key metabolic enzymes to protect the brain and
that they permit evaluation of a single animal over longitudi-
eliminate toxicants.
nal studies to determine the onset, progression, duration, and
reversibility of a neurotoxic injury.
Factors Relevant to Neurodegenerative Some functional tests are more specific than observations and
Diseases motor activity, and many of these functions have a clinical or
A classic example of toxicant-induced neurodegeneration behavioral correlate in humans. Electrophysiological tests pro-
is exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine vide sensory-specific information on nerve conduction veloc-
(MPTP), which is a by-product of the opioid analgesic, MPPP. ity and integrity, and have been used to complement behavioral
Exposure to a sufficient amount of MPTP can lead to immedi- evaluations. Measures of sensory function tap specific neuronal
ate parkinsonism, a disease in which dopaminergic neurons of pathways that govern stimuli-dependent reflexes. Autonomic
the substantia nigra are lost. Exposure to an amount of MPTP function includes evaluations of cardiovascular status and
insufficient to cause immediate parkinsonism leads to early cholinergic/adrenergic balance.
signs of the disease years later. It does not seem likely that an Deficits in cognitive function, especially in the context of
early sublethal injury to dopaminergic neurons later becomes developmental toxicity, represent an end point of great public
lethal. Rather, smaller exposures to MPTP may cause a decre- concern and rhetoric. In most cases, deficits in human cognitive
ment in the population of dopaminergic neurons and leave the function may be detected in laboratory animals as well, although
individual vulnerable to further loss of dopaminergic neurons. the affected cognitive domain may vary. Ultimately, neurotoxi-
Epidemiological studies also implicate exposure to herbi- cants identified by behavioral methods are also evaluated at a
cides, pesticides, and metals as risk factors for Parkinson's dis- cellular and molecular level to provide an understanding of the
ease (PD). Several studies suggest that dithiocarbamates also events in the NS that cause the neurological dysfunction.
play an important role. Interestingly, some studies suggest that
cigarette smoking may have a protective effect against both
MECHANISMS OF NEUROTOXICITY
Alzheimer’s disease and PD.
Environmental chemicals may cause heritable alterations in Individual neurotoxic compounds typically have one of four
gene expression in the absence of changes in genome sequences. targets: the neuron, the axon, the myelinating cell, or the neu-
The study of epigenetics has established two categories of rotransmitter system.
mechanisms affecting gene expression: DNA methylation and
histone posttranslational modifications. In most instances,
methylation of the promotor region results in transcriptional Neuronopathies
repression of the gene. Histone posttranslational modifica- Certain toxicants are specific for neurons, resulting in their
tions are characterized by lysine acetylation, arginine and lysine injury or death. Neuron loss is irreversible and includes degen-
methylation, serine phosphorylation, lysine ubiquitylation, etc. eration of all of its cytoplasmic extensions, dendrites and
Finally, it is necessary to recognize that microRNAs (miR- axons, and the myelin ensheathing the axon (Figure 16-3).
NAs) provide regulatory. control over gene expression. mRNAs Unique features of the neuron that place it at risk for the action
can control developmental timing, cell proliferation, cell death, of cellular toxicants include a high metabolic rate, a long cel-
and patterning of the NS, thus providing extensive regulatory lular process that is supported by the cell body, and an excitable
networks with a complexity comparable to that of transcrip- membrane that is rapidly depolarized and repolarized.
tion factors. More than 250 miRNAs have been already iden- Although a large number of compounds are known to result
tified, but their mRNA targets and functions have yet to be in toxic neuronopathies (Table 16-1), all of these toxicants
 CHAPTER 16 Toxic Responses of the Nervous System 243

TABLE 16-1 Chemicals associated with neuronal injury (neuronopathies).

Neurotoxicant Neurologic Findings Cellular Basis of Neurotoxicity

Aluminum Dementia, encephalopathy (humans), learning Spongiosis cortex, neurofibrillary aggregates, degenerative
deficits changes in cortex

6-Amino-nicotinamide Not reported in humans; hind limb paralysis Spongy (vacuolar) degeneration in spinal cord, brainstem,
(experimental animals) cerebellum; axonal degeneration ofthe peripheral
nervous system (PNS)

Arsenic Encephalopathy (acute), peripheral neuropathy Brain swelling and hemorrhage (acute); axonal
(chronic) degeneration in PNS (chronic)

Azide Insufficient data (humans); convulsions, ataxia Neuronal loss in cerebellum and cortex
(primates)

Bismuth Emotional disturbances, encephalopathy, Neuronal loss, basal ganglia, and Purkinje cells of
myoclonus cerebellum

Carbon monoxide Encephalopathy, delayed parkinsonism/dystonia Neuronal loss in cortex, necrosis of globus pallidus,
focal demyelination; blocks oxygen-binding site of
hemoglobin and iron-binding sites of brain

Carbon tetrachloride Encephalopathy (secondary to liver failure) Enlarged astrocytes in striatum, globus pallidus

Chloramphenicol Optic neuritis, peripheral neuropathy Neuronal loss (retina), axonal degeneration (PNS)

Cyanide Coma, convulsions, rapid death; delayed Neuronal degeneration, cerebellum, and globus pallidus;
parkinsonism/dystonia focal demyelination; blocks cytochrome oxidase/ATP
production

Doxorubicin Insufficient data (humans); progressive ataxia Degeneration ofdorsal root ganglion cells, axonal
(experimental animals) degeneration (PNS)

Ethanol Mental retardation, hearing deficits (prenatal Microcephaly, cerebral malformations

exposure)

Lead Encephalopathy (acute), learning deficits (children), Brain swelling, hemorrhages (acute), axonal loss in PNS
neuropathy with demyelination (rats) (humans)

Manganese Emotional disturbances, parkinsonism/dystonia Degeneration of striatum, globus pallidus

Mercury, inorganic Emotional disturbances, tremor, fatigue Insufficient data in humans (may affect spinal tracts;
cerebellum)

Methanol Headache, visual loss or blindness, coma (severe) Necrosis of putamen, degeneration of retinal ganglion cells

Methylazoxymethanol Microcephaly, retarded development (rats) Developmental abnormalities offetal brain (rats)
acetate (MAM)

Methyl bromide Visual and speech impairment; peripheral Insufficient data

neuropathy

Methyl mercury (organic Ataxia, constriction ofvisual fields, paresthesias Neuronal degeneration, visual cortex, cerebellum, ganglia
mercury) (adult) Spongy disruption, cortex, and cerebellum
Psychomotor retardation (fetal exposure)

1-Methyl-4-phenyl- Parkinsonism, dystonia (acute exposure) Neuronal degeneration in substantia nigra

1,2,3,6-tetrahydropyridine Early onset parkinsonism (late effect of acute Neuronal degeneration in substantia nigra
(MPTP) exposure)

3-Nitropropionic acid Seizures, delayed dystonia/grimacing Necrosis in basal ganglia

Phenytoin (dipheny|-hydantoin) Nystagmus, ataxia, dizziness Degeneration of Purkinje cells (cerebellum)

Quinine e Constriction ofvisual fields Vacuolization of retinal ganglion cells

Streptomycin (aminoglycosides) Hearing loss Degeneration of inner ear (organ of Corti)

Thallium Emotional disturbances, ataxia, peripheral Brain swelling (acute), axonal degeneration in PNS
neuropathy

Trimethyltin Tremors, hyperexcitability (experimental animals) Loss of hippocampal neurons, amygdala pyriform cortex
244 UNIT 4 Target Organ Toxicity

share certain features. Each toxic condition is the result of a limbic-cerebellar syndrome in humans and similar behav-
cellular toxicant that has a predilection for neurons. The initial ioral changes in primates. Trimethyltin gains access to the NS
injury to neurons is followed by apoptosis or necrosis, leading where, by an undefined mechanism, it leads to diffuse neuronal
to permanent loss of the neuron. These agents tend to be dif- injury. Several hypotheses are suggested for the mechanism of
fuse in their action, although they may show some selectivity trimethyltin neurotoxicity, however, including energy depriva-
in the degree of injury of different neuronal subpopulations. tion and excitotoxic damage.
The expression ofthese cellular events is often a diffuse enceph-
alopathy, with global dysfunctions.
Axonopathies
Doxorubicin—Doxorubicin (Adriamycin), a quinone- The neurotoxic disorders termed axonopathies are those in which
containing anthracycline antibiotic, is one of the most effective the primary site of toxicity is the axon itself. The axon degener-
antimitotics in cancer chemotherapy. Unfortunately, clinical ates, and with it the myelin surrounding that axon; however,
application of doxorubicin is greatly limited by its acute and the neuron cell body remains intact (Figure 16-5). The toxicant
chronic cardiotoxicity. Doxorubicin injures neurons in the results in a “chemical transection” of the axon at some point along
PNS, specifically those of the dorsal root ganglia and auto- its length, and the axon distal to the transection degenerates.
nomic ganglia by intercalating with DNA and interfering A critical difference exists in the significance of axonal
with transcription. Other important mechanisms of action degeneration in the CNS compared with that in the PNS:
of doxorubicin include its interaction with topoisomerase IJ, peripheral axons can regenerate, whereas central axons can-
which forms a DNA-cleavable complex and generation of reac- not. In the PNS, glial cells and macrophages support axonal
tive oxygen species (ROS) by enzymatic electron reduction of regeneration. In the CNS, release of inhibitory factors from
doxorubicin by variety of oxidases, reductases, and dehydro- damaged myelin and astrocyte scarring actually interferes with
genases. The vulnerability of sensory and autonomic neurons regeneration. The clinical relevance of the disparity between
appears to reflect the lack of protection of these neurons by a the CNS and PNS is that partial to complete recovery can occur
blood-tissue barrier within ganglia. after axonal degeneration in the PNS, whereas the same event
is irreversible in the CNS.
Methyl Mercury—Methyl mercury (MeHg) exposure occurs Axonopathies can be considered to result from a chemical
primarily from eating fish in which the substance has accu- transection of the axon. The number of axonal toxicants is large
mulated. In addition, mercury is a common pollutant in haz- and increasing in number (Table 16-2). As the axons degener-
ardous waste sites in the United States. The clinical picture ate, sensations and motor strength are first impaired in the most
of MeHg poisoning varies with both the severity of exposure distal extent of the axonal processes (e.g., the hands and feet),
and the age of the individual at the time of exposure. In adults, resulting in a “glove-and-stocking” neuropathy. With time and
the most dramatic sites of injury are the neurons of the visual continued injury, the deficit progresses to involve more proxi-
cortex and the small internal granular cell neurons of the cer- mal areas of the body and the long axons of the spinal cord.
ebellar cortex, whose massive degeneration results in blindness
and marked ataxia. In children, developmental disabilities, Gamma-diketones—Humans develop a progressive senso-
retardation, and cognitive deficits occur. It has been suggested rimotor distal axonopathy when exposed to high concentra-
that these differences are caused by an immature blood-brain tions of a simple alkane, n-hexane, day after day in work settings
barrier causing a more generalized distribution of mercury or after repeated intentional inhalation of hexane-containing
in the developing brain. Recent studies in rats show that the glues. An identical axonopathy can be produced by methyl-n-
neurons that are most sensitive to the toxic effects of MeHg butyl ketone (2-hexanone).
are those that reside in the dorsal root ganglia, perhaps again The W-1 oxidation of n-hexane results in the y-diketone,
reflecting the vulnerability of neurons not shielded by blood- 2,5-hexanedione (HD), which reacts with amino groups in all
tissue barriers. The mechanism of MeHg toxicity has been tissues to form pyrroles that derivatize and cross-link neurofila-
the subject of intense investigation and it remains unknown ments, leading to development of neurofilament aggregates of
whether the ultimate toxicant is MeHg itself or the liberated the distal, subterminal axon (Figure 16-5). The neurofilament-
mercuric ion. A variety of aberrations in cellular function have filled axonal swellings distort nodal anatomy and impair axonal
been noted, including impaired glycolysis, nucleic acid biosyn- transport. The pathologic processes of neurofilament accumu-
thesis, aerobic respiration, protein synthesis, and neurotrans- lation and degeneration of the axon are followed by the emer-
mitter release. In addition, there is evidence for enhanced gence of a clinical peripheral neuropathy.
oxidative injury and altered calcium homeostasis. Exposure to
MeHg leads to widespread neuronal injury and subsequently Carbon Disulfide—The most significant exposures of
to a diffuse encephalopathy. humans to CS, have occurred in the vulcan rubber and viscose
rayon industries. High-level exposures of humans to CS, cause
Trimethyltin—Organotins are used industrially as plasti- a distal axonopathy that is identical pathologically to that caused
cizers, antifungal agents, or pesticides. Intoxication with tri- by hexane. Covalent cross-linking of neurofilaments also
methyltin has been associated with a potentially irreversible occurs and it is known that CS, is itself the ultimate toxicant.
 CHAPTER 16 Toxic Responses of the Nervous System 245

Dimethyl
Normal Hexanedione hexanedione IDPN Pyridinethione

Neuron

Nissl
substance

|
ii
f
Myelinating )
cell ~ \ia

eee

ee

FIGURE 16-5 Diagram of axonopathies. Whereas 2,5-hexanedione results in the accumulation of neurofilaments in the distal regions
of the axon, 3,4-dimethyl-2,5-hexanedione results in identical accumulation within the proximal segments. These proximal neurofilamentous
swellings are quite similar to those that occur in the toxicity of 8,8 ’-iminodipropionitrile (IDPN), although the distal axon does not degenerate in
IDPN axonopathy but becomes atrophic. Pyridinethione results in axonal swellings that are distended with tubulovesicular material, followed by
distal axonal degeneration.

The clinical effects of exposure to CS, in the chronic setting swelling without degeneration in most animals. These neu-
are very similar to those of hexane exposure, with the devel- rofilament swellings are similar to those observed in carbon
opment of sensory and motor symptoms occurring initially in disulfide or y-diketone toxicity.
a glove-and-stocking distribution. In addition to this chronic Repeated exposure to IDPN leads to demyelination and
axonopathy, CS, can also lead to aberrations in mood and signs onion bulb formation (Figure 16-5), and eventually can pro-
of diffuse encephalopathic disease. duce distal axonal atrophy due to a reduction in anterograde
neurofilament transport to the distal axon. This impairment of
8,8’-Iminodipropionitrile (IDPN)—IDPN is a synthetic, axonal transport results from the disruption of the association
bifunctional nitrile that causes a bizarre “waltzing syndrome” between microtubules and neurofilaments by IDPN, causing
in rats and other mammals, although human exposure has neurofilament accumulation. This leads to complete distur-
never been documented. Features of this syndrome include bance of the cytoskeleton of the axon.
excitement, circling, head twitching, and overalertness, which
appears to result from degeneration of the vestibular sensory Acrylamide—Acrylamide is a man-made vinyl mono-
hair cells. In addition, administration of IDPN leads to accu- mer used widely in water purification, paper manufactur-
mulation of neurofilaments in the proximal axon, leading to ing, mining, and waterproofing. It is also used extensively in
246 UNIT 4 Target Organ Toxicity

TABLE 16-2 Chemicals associated with axonal injury (axonopathies).

i ene ea en nas ee See ee ee a,
Neurotoxicant Neurologic Findings Basis of Neurotoxicity

Acrylamide Peripheral neuropathy (often sensory) Axonal degeneration, axon terminal affected in earliest stages

p-Bromophenylacetyl urea Peripheral neuropathy Axonal degeneration in the peripheral nervous system (PNS)
and central nervous system (CNS)

Carbon disulfide Psychosis (acute), peripheral neuropathy (chronic) Axonal degeneration, early stages include neurofilamentous
swelling

Chlordecone (Kepone) Tremors, in coordination (experimental animals) Insufficient data (humans); axonal swelling and degeneration

Chloroquine Peripheral neuropathy, weakness Axonal degeneration, inclusions in dorsal root ganglion cells;
also vacuolar myopathy

Clioquinol Encephalopathy (acute), subacute myelooptic Axonal degeneration, spinal cord, PNS, optic tracts
neuropathy (subacute)

Colchicine Peripheral neuropathy Axonal degeneration, neuronal perikaryal filamentous

aggregates; vacuolar myopathy

Dapsone Peripheral neuropathy, predominantly motor Axonal degeneration (both myelinated and unmyelinated axons)

Dichlorophenoxyacetate Peripheral neuropathy (delayed) Insufficient data ,

Dimethylaminopropionitrile Peripheral neuropathy, urinary retention Axonal degeneration (both myelinated and unmyelinated axons)
a
\

Ethylene oxide Peripheral neuropathy Axonal degeneration

Glutethimide Peripheral neuropathy (predominantly sensory) Insufficient data

Gold Peripheral neuropathy (may have psychiatric Axonal degeneration, some segmental demyelination
problems)

n-Hexane Peripheral neuropathy, severe cases Axonal degeneration, early neurofilamentous swelling,
have spasticity PNS, and spinal cord

Hydralazine Peripheral neuropathy Insufficient data

3,8 ’-Iminodipropionitrile No data in humans; excitatory movement Proximal axonal swellings, degeneration ofolfactory
disorder (rats) epithelial cells, vestibular hair cells

Isoniazid Peripheral neuropathy (sensory), ataxia Axonal degeneration

(high doses)

Lithium Lethargy, tremor, ataxia (reversible) Insufficient data

Methyl n-butyl ketone Peripheral neuropathy Axonal degeneration, early neurofilamentous swelling,
PNS, and spinal cord

Metronidazole Sensory peripheral neuropathy, ataxia, seizures Axonal degeneration, mostly affecting myelinated fibers;
lesions of cerebellar nuclei

Misonidazole Peripheral neuropathy Axonal degeneration

Nitrofurantoin Peripheral neuropathy Axonal degeneration

Organophosphorus compounds Abdominal pain (acute); peripheral neuropathy Axonal degeneration

(NTE inhibitors)

Paclitaxel (taxoids) Delayed peripheral neuropathy (motor), spasticity Axonal degeneration (delayed after single exposure),
PNS, and spinal cord

Platinum (cisplatin) Peripheral neuropathy Axonal degeneration; microtubule accumulation in early stages

Pyridinethione (pyrithione) Movement disorders (tremor, choreoathetosis) Axonal degeneration (variable)

Vincristine (vinca alkaloids) Cranial (most often trigeminal) neuropathy Insufficient data
Peripheral neuropathy, variable autonomic Axonal degeneration (PNS), neurofibrillary changes
symptoms (spinal cord, intrathecal route)
 CHAPTER 16 Toxic Responses of the Nervous System 247

biochemical laboratories, and is present in many foods pre- produce axonal degeneration in the CNS and PNS albeit in a
pared at high temperatures. Although it can be dangerous different form than classical OPIDN.
if not handled carefully, most toxic events in humans have
been observed as peripheral neuropathies in factory workers Pyridinethione—This compound is a chelating agent that
exposed to high doses. is usually encountered as the zinc complex, called zinc pyridi-
Studies of acrylamide neuropathy revealed a distal axo- nethione (ZPT), which has antibacterial and antifungal prop-
nopathy characterized by multiple axonal swellings. A single erties and is a component of shampoos that are effective in the
large dose is sufficient to produce these swellings; however, treatment of seborrhea and dandruff. Although the compound
repeated dosing results in a more proximal axonopathy, in a is applied to the human scalp in antidandruff shampoos, der-
“dying back” process. These changes are caused by accumula- mal absorption of ZPT is minimal and exposure primarily
tions of neurofilaments at the nerve terminal. Recently it has occurs orally. Only the pyridinethione moiety is absorbed fol-
been observed that nerve terminal degeneration occurs prior lowing ingestion, with the majority of zinc eliminated in the
to development of axonopathy, suggesting that this degenera- feces. Pyridinethione appears to interfere with the fast axonal
tion is the primary lesion. transport systems, impairs the turnaround of rapidly trans-
ported vesicles, and slows the retrograde transport of vesicles.
Organophosphorus Compounds—Organophosphorus Aberration of the fast axonal transport systems most likely con-
(OP) compounds are used as insecticides, chemical warfare tributes to the accumulation of tubular and vesicular structures
agents, chemical intermediates, flame retardants, fuel addi- in the distal axon (Figure 16-5). As these materials accumulate
tives, hydraulic fluids, lubricants, pharmaceuticals, and plas- in one region of the axon, the axon degenerates in its more distal
ticizers. The OP insecticides and nerve agents are designed regions beyond the accumulated structures. The earliest signs
to inhibit AChE, thereby causing accumulation of acetyl- are diminished grip strength and changes of the axon terminal,
choline in cholinergic synapses resulting in cholinergic toxi- leading to a peripheral neuropathy.
city and death. Some OP compounds, such as tri-o-cresyl
phosphate (TOCP), can cause a severe sensorimotor central Microtubule-associated Neurotoxicity—A number of
peripheral distal axonopathy called OP compound-induced plant alkaloids alter the assembly and depolymerization of
delayed neurotoxicity (OPIDN) without inducing choliner- microtubules in nerve axons, causing neurotoxicity. The oldest
gic poisoning. known of these are colchicine and the vinca alkaloids, which
Many OP compounds are lipophilic and readily enter the bind to tubulin and cause depolymerization of microtubules.
NS, where they can phosphorylate neural target proteins. Colchicine is an alkaloid pharmaceutical used in the treatment
When the principal target is AChE, cholinergic toxicity can of gout, familial Mediterranean fever, and other disorders.
ensue, either because of suprathreshold levels of inhibition Vincristine and vinblastine are two vinca alkaloids used as che-
or inhibition plus aging. When aging of inhibited AChE also motherapeutic agents. Both colchicine and the vinca alkaloids
occurs (i.e., net loss of a ligand from the phosphorus of the produce a similar peripheral axonal neuropathy. Hallmarks of
OP-enzyme conjugate, leaving a negatively charged phospho- this neuropathy include paresthesia (tingling) of the fingers,
ryl moiety attached to the active site), the qualitative nature generalized weakness, and clumsiness.
of the toxicity does not change. Instead, the inhibited AChE Paclitaxel (Taxol), another plant alkaloid, has become a
becomes intractable to reactivation. When the principal tar- popular chemotherapeutic drug used to treat a variety of neo-
get is neuropathy target esterase (neurotoxic esterase, NTE), plasms. However, side effects include a predominantly sensory
OPIDN can result only if both suprathreshold (>70%) inhibi- neuropathy, beginning in the hands and feet. Like colchicine
tion occurs and the inhibited enzyme undergoes aging. Thus, and the vinca alkaloids, paclitaxel binds to tubulin; however,
in the case of NTE and OPIDN, inhibition alone is insufficient instead of leading to depolymerization, it promotes the forma-
to precipitate toxicity. Neuropathic (aging) inhibitors of NTE tion of microtubules. Once formed, these microtubules remain
include compounds from the phosphate, phosphonate, and stabilized by paclitaxel even in conditions that normally lead to
phosphoramidate classes of OP compounds. dissociation of tubulin subunits, including cold temperatures
Axonal degeneration does not commence immediately after or the presence of calcium.
acute exposure to a neuropathic OP compound but is delayed The pathologies of the axon induced by these drugs are dif-
for at least eight days between the acute high-dose exposure ferent. Although colchicine leads to atrophy of the axon and a
and clinical signs of axonopathy. Some effective regenera- decrease in the number of microtubules, paclitaxel causes the
tion of axons occurs in the PNS while axonal degeneration is aggregation to form a matrix that may inhibit fast axonal trans-
progressive and persistent in the long tracts of the spinal cord. port, which has been demonstrated with both colchicine and
Human cases of OPIDN are now rare and usually arise from paclitaxel.
intentional ingestion of massive doses of OP insecticides in
suicide attempts. Nevertheless, the fact remains that OPIDN
is a debilitating and incurable condition. While the preceding Myelinopathies
discussion was limited to organic compounds of pentacovalent Myelin provides electrical insulation of neuronal processes,
phosphorus, organic compounds of trivalent phosphorous also and its absence leads to a slowing of conduction and aberrant
248 UNIT 4 Target Organ Toxicity

conduction of impulses between adjacent processes. Toxicants accumulate in Schwann cells, which eventually lose their abil-
exist that result in the separation ofthe myelin lamellae, termed ity to maintain myelin in the PNS.
intramyelinic edema, and in the selective loss of myelin, termed
demyelination. Intramyelinic edema may be caused by altera- Lead—Lead exposure in animals results in a peripheral neu-
tions in the transcript levels of myelin basic protein mRNA, ropathy with prominent segmental demyelination. In young
and early in its evolution is reversible. Demyelination may children, acute massive exposures to lead result in severe cere-
result from progressive intramyelinic edema or from direct bral edema, perhaps from damage to endothelial cells. Children
toxicity to the myelinating cell. Remyelination in the CNS absorb lead more readily, and the very young do not have the
occurs to only a limited extent after demyelination. However, protection of the blood-brain barrier. Chronic lead intoxica-
Schwann cells in the PNS are capable of remyelinating the tion in adults results in peripheral neuropathy, gastritis, col-
axon. All the compounds in Table 16-3 lead to a myelinopathy. icky abdominal pain, anemia, and the prominent deposition
of lead in particular anatomical sites, creating lead lines in the
Hexachlorophene—Hexachlorophene, or 2,2’-methylenebis- gums and in the epiphyses of long bones in children. Lead in
(3,4,6-trichlorophenol), caused neurotoxicity when newborn the peripheral nerve of humans slows nerve conduction. The
infants were bathed with the compound to avoid staphylococ- basis of lead encephalopathy is unclear, although an effect on
cal skin infections. Following skin absorption of this hydro- the membrane structure of myelin and myelin membrane fluid-
phobic compound, hexachlorophene enters the NS and results ity has been shown.
in intramyelinic edema, which leads to the formation of vacu-
oles creating a “spongiosis” of the brain. Hexachlorophene
causes intramyelinic edema that leads to segmental demy- Astrocytes
elination. Swelling of the brain causes increased intracranial Astrocytes perform and regulate a wide range of physiologic
pressure, axonal degeneration, along with degeneration of functions in the CNS. The astrocyte appears to be a primary
photoreceptors in the retina. Humans exposed acutely to hexa- means of defense in the CNS following exposure to neurotoxi-
chlorophene may have generalized weakness, confusion, and cants, as a spatial buffering system for osmotically active ions,
seizures. Progression may occur, to include coma and death. and as a depot for the sequestration and metabolic processing
of endogenous molecules and xenobiotics.
Tellurium—Although exposures have not been reported in
humans, the neurotoxicity of tellurium in young rats alters the Ammonia—At high CNS concentrations, ammonia produces
synthesis of myelin lipids in Schwann cells, because of vari- seizures, resulting from its depolarizing action on cell mem-
ous lipid abnormalities. As biochemical changes occur, lipids branes, whereas at lower concentrations, ammonia produces

TABLE 16-3 Chemicals associated with injury of myelin (myelinopathies).

Neurotoxicant -—NeurologicFindings Basis of Neurotoxicity _
Acetylethyltetramethy! Not reported in humans; hyperexcitability, Intramyelinic edema; pigment accumulation in neurons
tetralin (AETT) tremors (rats)

Amiodarone Peripheral neuropathy Axonal degeneration and demyelination; lipid-laden lysosomes

in Schwann cells

Cuprizone Not reported in humans; encephalopathy Status spongiosis of white matter, intramyelinic edema
(experimental animals) (early stages); gliosis (late)

Disulfiram Peripheral neuropathy, predominantly sensory Axonal degeneration, swellings in distal axons

Ethidium bromide Insufficient data (humans) Intramyelinic edema, status spongiosis of white matter

Hexachlorophene Irritability, confusion, seizures Brain swelling, intramyelinic edema in CNS and PNS, late axonal
degeneration

Lysolecithin Effects only on direct injection into PNS or CNS Selective demyelination
(experimental animals)

Perhexilene Peripheral neuropathy Demyelinating neuropathy, membrane-bound inclusions

in Schwann cells

Tellurium Hydrocephalus, hind limb paralysis (experimental Demyelinating neuropathy, lipofuscinosis (experimental
animals) animals)

Triethyltin Headache, photophobia, vomiting, paraplegia Brain swelling (acute) with intramyelinic edema, spongiosis
(irreversible) of white matter
soba ANON RUN A RONOASetUi ance NAN CencsinnNtaoNeE
 CHAPTER 16 Toxic Responses of the Nervous System 249

stupor and coma, consistent with its hyperpolarizing effects. glutamine synthase. Ingestion of large amounts of MSO leads
Ammonia intoxication is associated with astrocytic swelling to neuronal cell loss in the hippocampal fascia dentata and
and morphological changes. Increased intracellular ammonia pyramidal cell layer, in the short association fibers and lower
concentrations have also been implicated in the inhibition of layers of the cerebral cortex, and in cerebellar Purkinje cells.
neuronal glutamate precursor synthesis, resulting in dimin- MSO also leads to large increases of glycogen levels, primarily
ished glutamatergic neurotransmission, changes in neurotrans- within astrocytic cell bodies, as well as swollen and damaged
mitter uptake (glutamate), and changes in receptor-mediated astrocytic mitochondria.
metabolic responses of astrocytes to neuronal signals.
Fluoroacetate and Fluorocitrate—
The Krebs cycle inhibi-
Nitrochemicals—Organic nitrates are used for peripheral tor fluorocitrate (FC) and its precursor fluoroacetate (FA) are
vasodilatation and reduction of blood pressure (nitroglycer- preferentially taken up by glia. FA occurs naturally in a num-
ine) in treatment of cardiovascular disease. The dinitroben- ber of plants, and is available commercially as a rodenticide
zenes are important synthetic intermediates in the industrial (Compound 1080). Exposure to FA may also occur via expo-
production of dyes, plastics, and explosives. The neurotoxic sure to the anti-cancer drug 5-fluorouracil. Ingestion of large
compound, 1,3-dinitrobenzene (DNB), produces gliovascu- amounts of FA results in ionic convulsions, with onset of sei-
lar lesions that specifically target astrocytes in the periaque- zures within minutes of consumption; those surviving these
ductal gray matter of the brainstem and deep cerebellar roof episodes frequently die later on due to respiratory arrest or
nuclei. Metronidazole, a 5-nitroimidazole [1-(2-hydroxyethyl)- heart failure. The actions of FC and FA have been attributed
2-methyl-5-nitroimidazole], is an antimicrobial, antiproto- both to the disruption of carbon flux through the Krebs cycle
zoal agent that is commonly used for the treatment of a wide and to impairment of ATP production.
variety of infections. Prolonged treatment with metronida-
zole is associated with a peripheral neuropathy characterized
Neurotransmission-associated
by paraesthesias, dysaesthesias, headaches, glossitis, urticaria,
and pruritus in addition to other somatosensory disorders. Neurotoxicity
A wide variety ofnaturally occurring toxins, as well as synthetic
Methionine Sulfoximine— Methionine sulfoximine (MSO) chemicals, alter specific mechanisms of intercellular commu-
is an irreversible inhibitor of the astrocyte-specific enzyme, nication (Table 16-4). Although neurotransmitter-associated

TABLE 16-4 Chemicals associated with neurotransmitter-associated toxicity.

Neurotoxicant Neurologic Findings Basis of Neurotoxicity

Amphetamine and Tremor, restlessness (acute); cerebral infarction Bilateral infarcts of globus pallidus, abnormalities in
methamphetamine and hemorrhage; neuropsychiatric disturbances dopaminergic, serotonergic, cholinergic systems
Acts at adrenergic receptors (PNS)

Atropine Restlessness, irritability, hallucinations Blocks cholinergic receptors (anticholinergic)

Cocaine Increased risk of stroke and cerebral atrophy Infarcts and hemorrhages; alteration in striatal dopamine
(chronic users); increased risk of sudden neurotransmission
cardiac death; movement and psychiatric
abnormalities, especially during withdrawal
Decreased head circumference (fetal exposure) Structural malformations in newborns

Domoic acid Headache, memory loss, hemiparesis, Neuronal loss, hippocampus and amygdala, layers 5
disorientation, seizures and 6 of neocortex
Kainate-like pattern of excitotoxicity

Kainate Insufficient data in humans; seizures in animals Degeneration of neurons in hippocampus, olfactory cortex,
(selective lesioning compound in neuroscience) amygdala, thalamus
Binds AMPA/kainate receptors

8-N-Methylamino-t-alanine Weakness, movement disorder (monkeys) Degenerative changes in motor neurons (monkeys)
(BMAA) Excitotoxic probably via NMDA receptors

Muscarine (mushrooms) Nausea, vomiting, headache Binds muscarinic receptors (cholinergic)

-

Nicotine Nausea, vomiting, convulsions Binds nicotinic receptors (cholinergic) low-dose stimulation;
high-dose blocking

B-N-Oxalylamino-t-alanine Seizures Excitotoxic probably via AMPA class of glutamate receptors

(BOAA)
 0
boSp) UNIT 4 Target Organ Toxicity

actions may be well understood for some agents, the specificity 5-HT axons and axon terminals. The result is a distal axotomy
of the mechanisms should not be assumed. of DA and 5-HT neurons.
The exact mechanism of amphetamine neurotoxicity is still
Nicotine—Widely available in tobacco products and in cer- unknown, but it seems that oxidative stress plays a key role. DA
tain pesticides, nicotine has diverse pharmacological actions is oxidized to produce free radicals, and chronic use can affect
and may be the source of considerable toxicity. Nicotine exerts superoxide dismutase (SOD) and catalase balance in rodents.
its effects by binding to a subset of nicotinic cholinergic recep- In support of this hypothesis, studies have shown amphetamine
tors. Smoking and “pharmacologic” doses ofnicotine accelerate neurotoxicity is attenuated by antioxidants.
heart rate, elevate blood pressure, and constrict blood ves-
sels within the skin as a result of stimulation of the ganglionic Excitatory Amino Acids—Glutamate and certain other
sympathetic NS. acidic amino acids are excitatory neurotransmitters. The
The rapid rise in circulating levels of nicotine after acute toxicity of glutamate can be blocked by certain glutamate
overdose leads to excessive stimulation of nicotinic receptors, antagonists, and the concept has emerged that the toxicity of
a process that is followed rapidly by ganglionic paralysis. Initial excitatory amino acids may be related to such conditions as
nausea, rapid heart rate, and perspiration are followed shortly hypoxia, epilepsy, and neurodegenerative diseases.
by marked slowing of heart rate with a fall in blood pressure. Glutamate is the main excitatory neurotransmitter of the
Somnolence and confusion may occur, followed by coma; if brain, and its effects are mediated by several subtypes of
death results, it is often the result of paralysis of the muscles receptors (Figure 16-6) called excitatory amino acid recep-
of respiration. tors (EAARs). The two major subtypes of glutamate receptors
Acute poisoning with nicotine fortunately is uncommon; are those that are ligand-gated directly to ion channels (iono-
however, exposure to lower levels for longer duration is very tropic) and those that are coupled with G proteins (metabo-
common. In humans, it has been difficult to separate the tropic). Ionotropic receptors may, be further subdivided by
effects of nicotine from those of other components ofcigarette their specificity for binding kainate, quisqualate, a-amino-3-
smoke. The complications of smoking include cardiovascular hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and
disease, cancers (especially malignancies of the lung and upper N-methyl-p-aspartate (NMDA). The entry of glutamate into
airway), chronic pulmonary disease, and attention deficit dis- the CNS is regulated at the blood-brain barrier, and glutamate
orders in children of women who smoke during pregnancy. exerts its effects in the circumventricular organ of the brain in
An increased propensity for platelets to aggregate is seen in which the blood-brain barrier is least developed. Within this
smokers, and this platelet abnormality correlates with the level site of limited access, glutamate injures neurons, apparently by
of nicotine. Nicotine also places an increased burden on the opening glutamate-dependent ion channels, ultimately leading
heart through its acceleration ofheart rate and blood pressure, to neuronal swelling and neuronal cell death. The only known
suggesting that nicotine may play a role in the onset of myo- related human condition is the “Chinese restaurant syndrome,’
cardial ischemia. In addition, nicotine also inhibits apoptosis
and may play a direct role in tumor promotion and tobacco-
related cancers.

Cocaine and Amphetamines—Cocaine blocks the reup-

take of dopamine (DA), norepinephrine (NE), and serotonin
(5-HT) at the nerve terminal in the CNS, and also causes
release of DA from storage vesicles. The primary event respon-
sible for the addictive properties and euphoric feeling when
intoxicated is a block on the DA reuptake transporter (DAT).
Cocaine abuse also puts individuals at risk for cerebrovas-
cular defects, cerebral atrophy, stroke, and intracranial hem-
orrhage. Cerebrovascular resistance has also been found to
be higher in cocaine abusers. In chronic cocaine users, neu- Synapse
rodegenerative disorders have been observed, similar to those
observed with amphetamine use.
Amphetamines affect catecholamine neurotransmission in
the CNS and have the potential to damage monoaminergic cells
directly. Amphetamines, including methylenedioxymetham- Calcium influx NMDA receptor
phetamine (MDMA, or “ecstasy”), have become popular with FIGURE 16-6 Schematic diagram of a synapse. Synaptic
young adults in recent decades due to the belief that it is a “safe” vesicles are transported to the axonal terminus, and released across
drug, and its ability to increase energy and sensation in adults. the synaptic cleft to bind to the postsynaptic receptors. Glutamate,
Similar to cocaine, the most pronounced effect of amphet- as an excitatory neurotransmitter, binds to its receptor and opens a
amines is on the DAergic neurons, but they can also damage calcium channel, leading to the excitation of the postsynaptic cell.
 CHAPTER 16 Toxic Responses of the Nervous System pay |

in which consumption of large amounts of monosodium gluta- patients who died within 4 months of intoxication showed
mate (MSG) as a seasoning may lead to a burning sensation in neurodegeneration that was most prominent in the hippocam-
the face, neck, and chest. pus and amygdala.
The cyclic glutamate analog kainate, isolated from a seaweed
in Japan, is extremely potent as an excitotoxin, being a 100-
fold more toxic than glutamate, and is selective at a molecular Models of Neurodegenerative Disease
level for the kainate receptor. Like glutamate, kainate selec- MPTP—A contaminant formed during meperidine synthe-
tively injures dendrites and neurons and shows no substantial sis, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
effect on glia or axons. Injected into a region ofthe brain, it can (Figure 16-7), produces over hours to days the signs and symp-
destroy the neurons of that area without disrupting all of the toms of irreversible Parkinson's disease. Autopsy studies have
fibers that pass through the same region. Kainate has become demonstrated marked degeneration of dopaminergic neurons
a tool for neurobiologists to explore the anatomy and function in the substantia nigra, with degeneration continuing many
of the NS. Kainate, through its selective action on neuronal cell years after exposure. It appears that MPTP is metabolized by
bodies, has provided a greater understanding of the functions two 2-electron oxidation reactions to the pyridinium ion,
of cells within a specific region of the brain, whereas previous MPP-+, which enters the dopaminergic neurons of the substan-
lesioning techniques addressed only regional functions. This tia nigra, resulting in their deaths by blocking mitochondrial
void in understanding and the epidemiologic evidence that respiration at complex I. Although not identical, MPTP neu-
some neurodegenerative diseases may have environmental rotoxicity and Parkinson’s disease produce symptomatology of
contributors inspire a heightened desire to appreciate more masked facies, difficulties in initiating and terminating move-
fully the effects of elements of our environment on the NS. ments, resting “pill-rolling” tremors, rigidity, and bradykinesias.
Development of permanent neurologic deficits occurred in
individuals accidentally exposed to high doses of the EAAR Manganese—As an essential trace metal that is found in
agonist domoic acid, an analog of glutamate. The acute illness all tissues, manganese (Mn) is required for normal metabo-
most commonly presented as gastrointestinal disturbance, lism of amino acids, proteins, lipids, and carbohydrates, act-
severe headache, and short-term memory loss. A subset of ing as a cofactor of synthesis enzymes. Excessive exposure to
the more severely afflicted patients had chronic memory defi- Mn produces neurotoxicity. The most common commercial
cits and motor neuropathy. Neuropathologic investigation of sources of Mn include the fuel additive methylcyclopentadienyl

Blood-brain
barrier
Astrocyte

MAO-B = NZ
on
, NZ
eh
Bane
OH

MPDP+

Dopaminergic
neuron

FIGURE 16-7 MPTP toxicity. MPP*, either formed elsewhere in the body following exposure to MPTP or injected directly into the blood, is
unable to cross the blood-brain barrier. In contrast, MPTP gains access and is oxidized in situ to MPDP* and MPP*. The same transport system that
carries dopamine into the dopaminergic neurons also transports the cytotoxic MPP”.
 2
iw)Nn UNIT 4 Target Organ Toxicity

manganese tricarbonyl (MMT), pesticides such as Maneb, steel offspring; however, the specific outcomes depend on the
factories, welding, and mining plants. Occupational exposure congener or mixture tested as well as the timing of exposure.
to toxic levels of Mn in industrial workers results in psychologic Changes in estrogen or thyroid hormone, neurotransmitter
and neurologic disturbances, including delusions, hallucina- function, and second messenger systems have been proposed
tions, depression, disturbed equilibrium, compulsive or violent as cellular bases for PCB toxicity. Another persistent class of
behavior, weakness, and apathy, followed by extrapyramidal hydrocarbons, polybrominated diphenyl ethers (PBDEs), have
motor system defects such as tremors, muscle rigidity, ataxia, shown similarities in altering thyroid hormone metabolism
bradykinesia, and dystonia. Mn toxicity causes a loss of DA and cholinergic function, and it has thus been proposed that
neurons in the substantia nigra, and as in Parkinsons disease, this chemical class would also be developmentally neurotoxic.
oxidative stress appears to play a significant role in the disorder.
CHEMICALS THAT INDUCE DEPRESSION
Developmentally Neurotoxic Chemicals OF NERVOUS SYSTEMFUNCTION
Replication, migration, differentiation, myelination, and syn-
Generalized depression of CNS function is produced by a
apse formation are the basic processes that occur in specific spa-
variety of volatile solvents, including ethanol, organics, and
tial and temporal patterns and underlie development of the NS.
anesthetics. These solvents include several chemical classes—
There are a variety of insults known to disrupt NS development,
aliphatic and aromatic hydrocarbons, halogenated hydro-
the outcomes of which may be very different depending on the
carbons, ketones, esters, alcohols, and ethers—that are small,
time of exposure, including exposures to certain metals, sol-
lipophilic molecules. They are widely found in industry, medi-
vents, antimetabolites, persistent organic pollutants, pesticides,
cine, and commercial products. Human exposure ranges from
pharmaceuticals, and ionizing radiation. Multiple mechanisms
chronic low level to occupational to high levels occurring with
of action may be present, producing a wide array of effects in
solvent abuse. Recent research has implicated interactions
the offspring. The impact on the developing NS may be very dif-
with ligand-gated ion channels as well as voltage-gated calcium
ferent, and often cannot be predicted, from effects observed in
channels as the mechanism of generalized depression.
adults. A number of neurodevelopmental disorders have been,
at least partially, attributed to exposures to neurotoxicological
agents during the fetal, infant, or childhood periods. IN VITRO AND OTHER ALTERNATIVE
Ethanol exposure during pregnancy can result in abnormali- APPROACHES TO NEUROTOXICOLOGY
ties in the fetus, including abnormal neuronal migration and
facial development, and diffuse abnormalities in the develop- The goal for future studies of neurotoxicology is to replace stan-
ment of neuronal processes, especially the dendritic spines. dard in vivo assessments with high-throughput in vitro assays
The clinical result of fetal alcohol exposure is often men- and quantitative structure—activity relationships (QSARs) to
tal retardation, with malformations of the brain and delayed predict adverse outcomes. The use of tiered testing schemes has
myelination of white matter. been proposed, where the first tiers rely on high-throughput
MeHg exposure leads to developmental disabilities, includ- methods that test for chemical actions on key biological recep-
ing cerebral palsy, mental retardation, and seizures, in many tors that initiate pathways of changes that lead to adverse out-
children at birth. Children exposed to MeHg in utero show comes, in order to identify chemicals for future testing. Second
widespread neuronal loss, disruption of cellular migration, tier tests could involve the use of alternative species, such as
profound mental retardation, and paralysis. small fish or invertebrate species, that will allow more moder-
There is considerable evidence that chronic exposure to nic- ate throughput, but in an intact or developing NS. Chemicals
otine has effects on the developing fetus. Along with decreased identified as having neurotoxic properties could then be tested
birth weights, attention deficit disorders are more common in in intact mammalian models as necessary. The extraordinary
children whose mothers smoke cigarettes during pregnancy, conservation of both genomic/epigenomic elements and dif-
and nicotine has been shown to lead to analogous neurobehay- ferentiation processes between mammals and nonmammals,
ioral abnormalities in animals exposed prenatally to nicotine. which has been revealed during the last two decades, makes
Cocaine is able to cross the placental barrier and the fetal more feasible the use of these alternative models.
blood-brain barrier, and also causes reduced blood flow in
the uterus. In severe events at large doses taken by the mother,
the fetus may develop hypoxia, leading to a higher rate of birth BIBLIOGRAPHY
defects. Maternal cocaine use is associated with low-birth weight Berent S, Albers JW: Neurobehavioral Toxicology: Neuropsychological
and Neurological Perspectives. New York: Taylor & Francis, 2005.
and behavioral defects, including a decreased awareness of the
Dobbs MR, Rusyniak DE: Frontiers in Clinical Neurotoxicology,
surroundings and altered response to stress and pain sensitivity.
an Issue of Neurologic Clinics. 1st ed. Philadelphia, PA: Saunders
Several epidemiological studies have reported deficits in
Elsevier, 2011.
neurodevelopment and psychological performance in children Harry GJ, Tilson HA: Neurotoxicology. 3rd ed. New York: Informa,
exposed to polychlorinated biphenyls (PCBs) and/or dioxins. 2010.
These persistent pollutants produce endocrine disruptions, Webster LR: Neurotoxicity Syndromes. New York: Nova Biomedical,
cognitive deficits, and changes in activity levels in exposed 2012"
 CHAPTER 16 Toxic Responses of the Nervous System Dey)

QUESTIONS

Which of the following statements regarding axons and/or Which of the following statements regarding the PNS and
axonal transport is FALSE? . the CNS is TRUE?
a. Single nerve cells can be over 1 m in length. a. Nerve impulse transduction is much faster in the
b. Fast axonal transport is responsible for movement of CNS than in the PNS.
proteins from the cell body to the axon. b. PNS axons can regenerate, whereas CNS axons
c. Anterograde transport is accomplished by the protein cannot.
kinesin. c. Remyelination does not occur in the CNS.
d. ‘The motor proteins, kinesin and dynein, are associ- Oligodendrocytes perform remyelination in the PNS.
ated with microtubules. e. Inthe CNS, oligodendrocyte scarring interferes with
e. A majority of the ATP in nerve cells is used for axonal axonal regeneration.
transport.
Platinum (cisplatin) results in which of the following
Which of the following statements is not characteristic of neurologic problems?
Schwann cells in Wallerian degeneration? a. peripheral neuropathy.
a. Schwann cells provide physical guidance needed for b. trigeminal neuralgia.
the regrowth of the axon. c. spasticity.
b. Schwann cells release trophic factors that stimulate d. gait ataxia.
growth. e. tremor.
c. Schwann cells act to clear the myelin debris with the
help of macrophages. Which of the following is NOT characteristic of
d. Schwann cells increase synthesis of myelin lipids in axonopathies?
response to axonal damage. a. ‘There is degeneration ofthe axon.
e. Schwann cells are responsible for myelination of b. The cell body of the neuron remains intact.
axons in the peripheral nervous system. c. Axonopathies result from chemical transaction of
the axon.
Prenatal exposure to ethanol can result in mental retar- A majority of axonal toxicants cause motor deficits.
dation and hearing deficits in the newborn. What is the e. Sensory and motor deficits are first noticed in the
cellular basis of the neurotoxicity? hands and feet following axonal degeneration.
neuronal loss in cerebellum.
acute cortical hemorrhage. All of the following statements regarding lead exposure
microcephaly. are true EXCEPT:
loss of hippocampal neurons. a. Lead exposure results in peripheral neuropathy.
OB degeneration of the basal ganglia. b. Lead slows peripheral nerve conduction in humans.
c. Lead causes the transection of peripheral axons.
Which of the following characteristics is LEAST likely d. Segmental demyelination is a common result of lead
to place a neuron at risk of toxic damage? ingestion.
a. high metabolic rate. e. Lead toxicity can result in anemia.
b. ability to release neurotransmitters.
c. long neuronal processes supported by the soma. 10. Regarding excitatory amino acids, which of the following
d. excitable membranes. statements is FALSE?
e. large surface area. a. Glutamate is the most common excitatory amino acid
in the CNS.
The use of meperidine contaminated with MPTP will b. Excitotoxicity has been linked to conditions such as
result in a Parkinson's disease-like neurotoxicity. Where is epilepsy.
the most likely site in the brain that MPTP exerts its toxic c. Overconsumption of monosodium glutamate (MSG)
effects? can result in a tingling or burning sensation in the
cerebellum. face and neck.
cerebral cortex. d. An ionotropic glutamate receptor is coupled to a
brainstem. G protein.
substantia nigra. e. Glutamate is toxic to neurons.
go
Cr
festa hippocampus.
ee:
 Cer tsrAvssP
ss Labi Rk

Toxic Responses of the

Ocular and Visual System!
Donald A. Fox and William K. Boyes

INTRODUCTION TO OCULAR AND VISUAL SYSTEM TARGET SITES AND MECHANISMS OF ACTION: RETINA
TOXICOLOGY
Retinotoxicity of Systemically Administered
Therapeutic Drugs
EXPOSURE TO THE EYE AND VISUAL SYSTEM
Cancer Chemotherapeutics
Ocular Pharmacodynamics and Pharmacokinetics Chloroquine and Hydroxychloroquine
Nanoparticles and Ocular Drug Delivery Digoxin and Digitoxin
Ocular Drug Metabolism ; Indomethacin
Central Visual System Pharmacokinetics Sildenafil Citrate
Light and Phototoxicity Tamoxifen
EVALUATING OCULAR TOXICITY AND VISUAL FUNCTION Vigabatrin
Retinotoxicity of Known Neurotoxicants
Evaluation of Ocular Irritancy and Toxicity
Inorganic Lead
Ophthalmologic Evaluations
Methanol
Electrophysiologic Techniques
Organic Solvents
Behavioral and Psychophysical Techniques
Color Vision Testing TARGET SITES AND MECHANISMS OF ACTION: OPTIC
NERVE AND TRACT
TARGET SITES AND MECHANISMS OF ACTION: CORNEA
Acrylamide
Acids
Carbon Disulfide
Bases or Alkalies
Ethambutol
Organic Solvents
Surfactants TARGET SITES AND MECHANISMS OF ACTION: THE
CENTRAL VISUAL SYSTEM
TARGET SITES AND MECHANISMS OF ACTION: LENS
Lead
Corticosteroids
Methyl Mercury
Naphthalene
Phenothiazines

'This chapter has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. EPA, and approved for publication.

BS
256 UNIT 4 Target Organ Toxicity

» Toxic chemicals and systemic drugs can affect all parts of = Most electrophysiologic or neurophysiologic procedures
the eye, including cornea, iris, ciliary body, lens retina, for testing visual function after toxicant exposure involve
and optic nerve. stimulating the eyes with visual stimuli and electrically
« Ophthalmologic procedures for evaluating the health recording potentials generated by visually responsive
of the eye include routine clinical screening evaluations neurons.

using a slit-lamp biomicroscope and ophthalmoscope,

and an examination of the pupillary light reflex.

INTRODUCTION TO OCULAR AND water, collagen, and glycosaminoglycans, which permits hydro-
philic chemicals to easily dissolve in this thick layer. The inner
VISUAL SYSTEM TOXICOLOGY edge of the corneal stroma is bounded by a thin basement mem-
Environmental and occupational exposure to toxic chemicals, brane, called Descemet’s membrane, which is secreted by the
gases, and vapors as well as side effects resulting from therapeu- corneal endothelium. The innermost layer of the cornea, the
tic drugs frequently result in structural and functional altera- corneal endothelium, is composed of a single layer of cells that
tions in the eye and central visual system. The retina and central are surrounded by lipid membranes. The permeability of the
visual system are especially vulnerable to toxic insult. corneal endothelial cells to ionized chemicals is relatively low.
There are two separate vascular systems in the eye: (1) the
uveal blood vessels, which include the vascular beds of the iris,
EXPOSURE TO THE EYE AND ciliary body, and choroid, and (2) the retinal vessels. In the ante-
VISUAL SYSTEM rior segment of the eye, there is a blood-aqueous barrier that
has relatively tight junctions between the endothelial cells of the
Ocular Pharmacodynamics and iris capillaries and nonpigmented cells of the ciliary epithelium.
Pharmacokinetics The major function of the ciliary epithelium is to produce aque-
Toxic chemicals and systemic drugs can affect all parts of the ous humor from the plasma filtrate present in the stroma of the
eye (Figure 17-1; Tables 17-1 and 17-2). Factors determining ciliary processes.
whether a chemical can reach a particular ocular site of action In humans and several widely used experimental animals
include physiochemical properties of the chemical, concentra- (e.g., monkeys, pigs, dogs, rats, and mice), the retina has a dual
tion and duration of exposure, and movement across ocular circulatory supply: choroidal and retinal. The retina consists
compartments and barriers. The cornea, conjunctiva, and eye- of the outer plexiform layer (OPL), inner nuclear layer (INL),
lids are often exposed directly to chemicals, gases, drugs, and inner plexiform layer (IPL), and ganglion cell layer (GCL). The
particles. The first site of action is the tear film, a three-layered endothelial cells of capillaries of the retinal vessels have tight
structure with both hydrophobic and hydrophilic properties. junctions forming the blood-retinal barrier. However, at the
The outermost thin tear film layer is secreted by the meibo- level of the optic disk, the blood-retinal barrier is lacking and
mian (sebaceous) glands. This superficial lipid layer protects thus hydrophilic molecules can enter the optic nerve (ON) head
the underlying thicker aqueous layer that is produced by the by diffusion from the extravascular space and cause selective
lacrimal glands. The third layer is the very thin mucoid layer damage at this site of action. The outer or distal retina, which
that is secreted by the goblet cells of the conjunctiva and acts as consists of the retinal pigment epithelium (RPE), rod and cone
an interface between the hydrophilic layer of the tears and the photoreceptor outer segments (ROS and COS) and inner seg-
hydrophobic layer of the corneal epithelial cells. ments (RIS and CIS), and the photoreceptor outer nuclear layer
The avascular cornea is considered the external barrier to (ONL) are avascular. These areas of the retina are supplied by
the internal ocular structures. Greater systemic absorption the choriocapillaris: a dense, one-layered network of fenestrated
occurs through contact with the vascularized conjunctiva vessels formed by the short posterior ciliary arteries and located
(Figure 17-2). The human cornea has several distinct layers next to the RPE. Consistent with their known structure and
through which a chemical must pass in order to reach the ante- function, these capillaries have loose endothelial junctions and
rior chamber. The first is the corneal epithelium of stratified abundant fenestrae; they are highly permeable to large proteins.
squamous, nonkeratinized cells with tight junctions. The per- Following systemic exposure to drugs and chemicals by the
meability of the corneal epithelium is low and only lipid-soluble oral, inhalation, dermal, or parenteral route, these compounds
chemicals readily pass through this layer. Bowman’s membrane are distributed to all parts of the eye by the blood in the uveal
separates the epithelium from the stroma. The corneal stroma blood vessels and retinal vessels (Figure 17-3). Most chemi-
comprises 90% of the corneal thickness and is composed of cals rapidly equilibrate with the extravascular space of the
 CHAPTER 17 Toxic Responses of the Ocular and Visual System 297,

The cornea The Iris and ciliary body

ceeeeeeeereeed wa ny

Cornea

Schlemm’s canal

|(a° Ss S iy e. <9

_ Trabecular =)
Ciliary
|_ meshwork muscle

Vitreous face

i
Ciliary processes
.
Descemet’s
layer
|
Sees
cecers eee Endothelium LL
Cornea ~ Anterior —
chamber

Posterior
chamber

7
e es 8
heer ee
oe
ee,
ee
ee

Retina i
Optic NFL
nerve GCL
head IPL
[ieee

Nuclei
INL

Wye fibers |
OPL
ONL

RIS and CIS

Epithelium \y W\wWayyy ~~ Epithelial
AWWA bow | ROS and COS”
Lens NRA
capsule | RPE
L ee S E vise
Lens at the equatorial border Cross-section of retina

FIGURE 17-1 Diagrammatic horizontal cross-section of the eye, with medium-power enlargement of details for the cornea, iris and
ciliary body, lens, and retina. The morphologic features, their role in ocular pharmacodynamics, pharmacokinetics, drug metabolism, and the
adverse effects of drugs and chemical agents on these sites are discussed in the text.
 8
nNnn UNIT 4 Target Organ Toxicity

TABLE 17-1 Ocular and central visual system sites of action of selected xenobiotics following systemic exposure.
Outer Outer Retina: Inner Retina: RGCs, Optic LGN, Visual
Xenobiotic Cornea Lens Retina: RPE Rods and Cones BCs, ACs, IPCs Nerve or Tract Cortex

Acrylamide = — ++ ++
ab
Amiodarone

Carbon disulfide + ~ ++ +

Chloroquine + +

Chlorpromazine A,

ae
Corticosteroids cleats

Digoxin and digitoxin ++ + +

Ethambutol a ++

Hexachlorophene + + +

Indomethacin oH \

Isotretinoin

Lead ++ + zee a +

Methanol ++ - ++ +

Methyl mercury, mercury + - - ++

n-Hexane + ~

Naphthalene a

Organic solvents ul +

Organophosphates + a +

Styrene ae

Tamoxifen + af +

Vigabatrin + + + -

RPE, retinal pigment epithelium; BC, bipolar cell; AC, amacrine cell; IPC, interplexiform cell; RGC, retinal ganglion cell; LGN, lateral geniculate nucleus.
“+"and"—"indicate that this site of action was cited as being positively affected or not affected by the exposure to the toxicant.

choroid where they are separated from the retina and vitreous Intraocular melanin plays a special role in ocular toxicol-
body by the RPE and endothelial cells of the retinal capillaries, ogy. First, it is found in several different locations in the eye:
respectively. Hydrophilic molecules with molecular weights pigmented cells of the iris, ciliary body, RPE, and uveal tract.
less than 200 to 300 Da can cross the ciliary epithelium and Second, it has a high binding affinity for polycyclic aromatic
iris capillaries and enter the aqueous humor. Thus, the corneal hydrocarbons, electrophiles, calcium, and toxic heavy metals
endothelium—the cells responsible for maintaining normal such as aluminum, iron, lead, and mercury. Although this ini-
hydration and transparency of the corneal stroma—could be tially may play a protective role, the excessive accumulation,
exposed to chemical compounds by the aqueous humor and long-term storage, and slow release of numerous drugs and
limbal capillaries. Similarly, the anterior surface of the lens also chemicals from melanin can influence toxicity.
can be exposed as a result of its contact with the aqueous humor.
The most likely retinal target sites following systemic drug and
chemical exposure appear to be the RPE and photoreceptors, Nanoparticles and Ocular Drug Delivery
because the endothelial cells of the choriocapillaris are perme- The main ocular target sites of importance for disease treatment
able to proteins smaller than 50 to 70 kDa. However, the cells and neuroprotection are the anterior segment and posterior ret-
of the RPE are joined on their basolateral surface by tight junc- ina. As noted above, there are numerous barriers that restrict bio-
tions that limit the passive penetration of large molecules into availability, decrease therapeutic efficacy, and increase side effects.
the neural retina. Development of nanoscale preparations for drug delivery is a new
 CHAPTER 17 Toxic Responses of the Ocular and Visual System 259

TABLE 17-2 Common signs and symptoms ofvisual system dysfunction.

Common Signs and Symptoms Possible Pathophysiological Basis Examples of Chemicals Producing this Effect

Cornea

Pigment deposits in corneal epithelium Intralysosomal accumulation of lipids Amiodarone, chloroquine, clofazimine,
(verticillate keratopathy) phenothiazines, suramin

Lens

Cataracts: anterior cortical (AC), posterior Chemical deposition, photochemical oxidation Long-term systemic use of phenothiazine (AC),
subcapsular (PSC) corticosteroids (PSC)

Pupil

Pupil constriction (miosis) Anticholinesterases Organophosphate and carbamate insecticides,

Pupil dilation (mydriasis) and photophobia nerve gas agents such as sarin, soman, and
tabun
Cholinergic antagonists Atropine or belladonna alkaloids
Adrenergic agonists Amphetamine, cocaine, phenylephrine

Ocular motility

Diplopia (double vision), nystagmus Oculomotor impairment, damage or Acute alcohol intoxication, barbiturate toxicity,
dysfunction in vestibular/oculomotor acute solvent exposure
reflex pathways

Retinal pigment epithelium

Loss of central vision (central scotoma) Degeneration of retinal pigment epithelium and Carbon disulfide, chloroquine
underlying photoreceptors

Retina

Poor night (scotopic) vision and impaired Damage to and apoptosis of rod Lead, methyl mercury, vigabatrin
dark adaptation photoreceptors
Acetylcholinesterase inhibitors Organophosphate and carbamate insecticides,
nerve gas agents
Altered color perception, central scotoma Inhibition of cone photoreceptor Digitalis/digitoxin
sodium-pumps
Altered color perception Inhibition of cone photoreceptor Sildenafil and tadalafil
cGMP-phosphodiesterase
Impaired color discrimination (blue/yellow) Damage to cone photoreceptors and inner Chronic exposure to styrene and organic solvents,
retina trimethadione, chronic high-dose antibiotics
Impaired color discrimination (red/green) Acquired damage to cone photoreceptors, Higher level chronic exposure to organic solvents,
neural retina, and/or afferent visual pathway carbon disulfide or hexane, chronic carbon
monoxide, chronic alcoholism, ethambutol
Loss of peripheral vision (tunnel vision, Degeneration of peripheral retina and nerve Methyl mercury, vigabatrin
peripheral scotoma, visual field fiber layer
constriction)
Reduced contrast sensitivity and Degeneration ofthe retinal ganglion cells and Acrylamide, carbon disulfide
visual acuity optic tract, microaneurysms and retinal
vasculopathy

Optic nerve and optic tract

Reduced contrast sensitivity and visual Optic neuritis and/or degeneration of the optic Higher level chronic exposure to organic solvents
acuity tract, generally affecting mitochondrial ATP such as carbon disulfide or hexane, ethambutol,
production ethylene glycol, isoniazid, linezolid and
chloramphenicol, methanol, vigabatrin
Monocular and/or binocular visual loss Nonarteretic anterior ischemic optic neuropathy Amiodarone, sildenafil and tadalafil

Lateral geniculate and visual cortex

+
Central scotoma Degeneration of calcarine fissure of Methyl mercury
visual cortex
Visuomotor deficits and reduced contrast Visual and motor cortex dysfunction Lead, chronic exposure to carbon disulfide, hexane
sensitivity and other solvents, toluene

Inclusion in this table indicates that this drug, chemical, or toxicant was cited in one or more case reports, review articles, or clinical or animal studies. The pathophysiological causes and
chemicals listed are provided as examples and are not exhaustive.
260 UNIT 4 Target Organ Toxicity

and chemical
oe exposure :

| Aqueous |
Cornea
humor |

Nasolacrimal Vitreous
Conjunctiva
| duct J humor

Digestive
system

Optic nerve, brain &

other organs

FIGURE 17-2 Ocular absorption and distribution of drugs and chemicals following the topical route of exposure. The details for
movement of drugs and chemicals between compartments ofthe eye and subsequently to the optic nerve, brain, and other organs are discussed
‘\
in the text. \

approach to drug delivery which can substantially enhance pen- Ocular Drug Metabolism
etration from the cornea, deliver a wide variety of drugs and mol-
Metabolism of xenobiotics occurs in all compartments of the
ecules, and increase the concentration and contact time of drugs
eye by well-known phase I and II xenobiotic-biotransforming
with these tissues. A wide variety of nanoformulations have been
enzymes. Drug-metabolizing enzymes that are present in the
considered including solid lipid nanoparticles containing lipids,
tears, iris—ciliary body, choroid, and retina of many different
phospholipids, and/or metals; liposomes; nanosuspensions; and
species are listed in Table 17-3. Whereas the activity of these
emulsions; and the use of biocompatible coatings such as chito-
enzymes varies between species and ocular tissues, the whole
san. Metallic particles that enable remote magnetic targeting of
lens has low biotransformational activity.
drug delivery also are under development.

Retinal =
_ choroid — [Opticnerve, brain& |
| otherorgans

Corneal
endothelium
Aqueous
humor
Iris &
ciliary body
Vitreous
humor
i
FIGURE 17-3 Distribution of drugs and chemicals in the anterior and posterior segments of the eye, optic nerve, brain, and other organs
following the systemic route of exposure. The details for movement of drugs and chemicals between compartments of the eye are discussed in the
text. The solid and dotted double lines represent the different blood-tissue barriers present in the anterior segment of
the eye, retina, optic nerve, and
brain. The solid double lines represent tight endothelial junctions, whereas the dotted double lines represent loose endothelial junctions.
 CHAPTER 17 Toxic Responses of the Ocular and Visual System 261

TABLE 17-3 Distribution of ocular xenobiotic-biotransforming enzymes.

Enzymes Tears Cornea lris/Ciliary Lens Retina Choroid

Phase | reactions

Acetylcholinesterase (AChE) ae + uy ue

Alcohol dehydrogenase + = 4 an

Aldehyde dehydrogenase + + a a

Aldehyde reductase + ah 4 aie SL

Aldose reductase + a5 4

Carboxylesterase ar a + + zis

Catalase - + Ah ae + aie

Cu/Zn superoxide dismutase aF aR —/+ +

CYP1A1 or CYP1A2 aE ae + = + +

CYP1B1 ae hh BL ae

CYP2B1 or CYP2B2 oe 4

CYP2C11 -
CYP3A1 4 as

CYP4A1 or CYP4B2 + a ae

CYP27A1 ais

MAO-A or MAO-B ak f 4 +

Phase il reactions

Glutathione peroxidase - am + + + +

Glutathione reductase + + +

Glutathione S-transferase + + se se

Sulfotransferases 4 ae

UDP-glucuronosyltransferases =f +

N-Acetyltransferase ar + ae aR +

“+"and"—"indicate that the enzyme was present (localized by immunohistochemistry, immunogold electron microscopy, Western blot, or gene expression) or absent, respec-
tively, in human, monkey, or rodent tissues.

Central Visual System Pharmacokinetics which could cause this part of the central visual system to be
vulnerable to exposures that do not affect much of the
The penetration of potentially toxic compounds into visual
remainder ofthe brain.
areas of the central nervous system (CNS) is governed by the
blood-brain barrier (Figure 17-3), which is differentially
permeable to compounds depending on their size, charge,
and lipophilicity. Compounds that are large, highly charged, Light and Phototoxicity
or otherwise not very lipid soluble tend to be excluded from The most important oxidizing agents are visible light and
the brain, whereas smaller, uncharged, and lipid-soluble UV radiation, particularly UV-A (320 to 400nm) and
compounds more readily penetrate into the brain tissue. UV-B (290 to 320 nm), and other forms of electromagnetic
In some cases, toxic compounds may be actively trans- radiation. Light- and UV-induced photooxidation leads to
ported into the brain by mimicking the natural substrates generation of reactive oxygen species (ROS), and oxida-
of active transport systems. One area of the brain lacking tive damage that can accumulate over time. Higher energy
a blood-brain barrier is the ON near the lamina cribrosa, UV-C (100 to 290 nm) is even more damaging. At sea level,
262 UNIT 4 Target Organ Toxicity

the atmosphere filters out virtually all UV-C and all but a The Draize test has been criticized on several grounds,
small fraction of UV-B derived from solar radiance. The including high interlaboratory variability, the subjective nature
cornea absorbs about 45% of light with wavelengths below of the scoring, poor predictive value for human irritants, and
280 nm, but only about 12% between 320 and 400nm. The for causing undue pain and distress to the tested animals. These
lens absorbs much of the light between 300 and 400 nm and criticisms have spawned development of alternative methods
transmits 400 nm and above to the retina. Absorption of or strategies to evaluate compounds for their potential to cause
light energy in the lens triggers a variety of photoreactions, ocular irritation.
including the generation of fluorophores and pigments that
lead to the yellow-brown coloration of the lens. Sufficient
exposure to infrared radiation, as occurs to glassblowers,
Ophthalmologic Evaluations
or microwave radiation will also produce cataracts through There are many ophthalmologic procedures for evaluating the
direct heating of the ocular tissues. health of the eye. Procedures available range from fairly routine
Drugs and other chemicals can mediate photo-induced toxic- clinical screening evaluations to sophisticated techniques for
ity in the cornea, lens, or retina. This occurs when the chemical targeted purposes. Examination of the adnexa includes evalu-
structure allows absorption of light energy and the subsequent ating the eyelids, lacrimal apparatus, and palpebral (covering
generation of activated intermediates, free radicals, and ROS. the eyelid) and bulbar (covering the eye) conjunctiva. The
The propensity of chemicals to cause phototoxic reactions can anterior structures or anterior segment include the cornea, iris,
be predicted using photophysical and in vitro procedures. lens, and anterior chamber. The posterior structures, referred
The phototoxic properties of chemicals are being exploited to as the ocular fundus, include the retina, retinal vasculature,
for photodynamic therapies where photoactive chemicals choroid, ON, and sclera. The adnexa and surface of the cornea
are delivered to pathological tissues. Wavelength-specific can be examined initially with the naked eye, a hand-held light,
light is introduced to the tissue causing the photoactive or a slit-lamp biomicroscope, usingamydriatic drug (which
chemical to activate thereby initiating a free-radical cascade causes pupil dilation) if the lens is to be observed. The width
that kills the pathological tissues. These agents also are being ofthe reflection ofathin beam oflight projected from the slit-
developed to utilize long wavelengths near the red/infrared lamp is an indication of the thickness of the cornea and may
end of the spectrum where the irradiation penetrates deeper be used to evaluate corneal edema. Lesions of the cornea can
into the tissue. be better visualized with the use of fluorescein dye, which is
retained where there is an ulceration of the corneal epithelium.
Examination of the fundus requires use of a mydriatic drug
EVALUATING OCULAR TOXICITY and a direct or an indirect ophthalmoscope.
AND VISUAL FUNCTION An examination of the direct pupillary reflex involves shin-
ing a bright light into the eye and observing the reflexive pupil
Evaluation of Ocular Irritancy and Toxicity constriction in the same eye. The consensual pupillary reflex
The so-called Draize test, with some additions and revisions, is observed in the eye not stimulated. Both the direct and con-
has formed the basis of standard procedures employed for eval- sensual pupillary light reflexes are dependent on function of
uating ocular irritation and safety evaluations. Traditionally, a reflex arc involving cells in the retina, which travel through
albino rabbits are the subjects evaluated in the Draize test. The the ON, optic chiasm, and optic tract (OT) to project to neu-
procedure involves instillation of 0.1 mL of a liquid or 100mg rons in the pretectal area. The absence of a pupillary reflex is
ofa solid into the conjunctival sac of one eye and then gently indicative of damage somewhere in the reflex pathway, and dif-
holding the eye closed for 1 s. The untreated eye serves as a ferential impairment of the direct or consensual reflexes can
control. Both eyes are evaluated at 1, 24, 48, and 72h after treat- indicate the location of the lesion. The presence of a pupillary
ment. If there is evidence of damage in the treated eye at 72h, light reflex, however, is not synonymous with normal visual
the examination time may be extended. The cornea, iris, and function. Pupillary reflexes can be maintained even with sub-
conjunctiva are evaluated and scored according to a weighted stantial retinal damage. In addition, lesions in visual areas out-
scale. The cornea is scored for both the degree of opacity and side of the reflex pathway, such as in the visual cortex, may also
area of involvement, with each measure having a potential leave the reflex function intact.
range from 0 (none) to 4 (most severe). The iris receives a sin-
gle score (0 to 2) for irritation, including degree of swelling,
congestion, and degree of reaction to light. The conjunctiva is Electrophysiologic Techniques
scored for the redness (0 to 3), chemosis (swelling 0 to 4), and Most electrophysiologic or neurophysiologic procedures for
discharge (0 to 3). The individual scores are then multiplied testing visual function in a toxicologic context involve stimu-
by a weighting factor: 5 for the cornea, 2 for the iris, and 5 for lating the eyes with visual stimuli and electrically recording
the conjunctiva. The results are summed for a maximum total potentials generated by visually responsive neurons. The most
score of 110. In this scale, the cornea accounts for 73% of the commonly used procedures are the flash-evoked electroretino-
total possible points, in accordance with the severity associated gram (ERG), visual-evoked potentials (VEPs), and, less often,
with corneal injury. the electrooculogram (EOG).
 CHAPTER 17 Toxic Responses of the Ocular and Visual System 263

ERGs are typically elicited with a brief flash of light and cones. Congenital loss of short-wavelength cones, resulting in
recorded from an electrode placed in contact with the cor- a blue-yellow confusion (tritanopia, or type III), is extremely
nea. A typical ERG waveform includes an a-wave that reflects rare. Most acquired color vision deficits, such as those caused
the activation of photoreceptors and a b-wave that reflects the by drug and chemical exposure, begin with a reduced ability to
activity of retinal bipolar cells (BC) and associated membrane perform blue-yellow discriminations. With increased or pro-
potential changes in Miller cells (MC). A standard set of ERG longed low-level exposure, the color confusion can progress to
procedures includes the recording of (1) a response reflective of the red-green axis as well. Because of the rarity of inherited
only rod photoreceptor function in the dark-adapted eye, (2) the tritanopia, it is generally assumed that blue-yellow deficits,
maximal response in the dark-adapted eye, (3) a response devel- when observed, are acquired deficits. Generally, disorders of
oped by cone photoreceptors, (4) oscillatory potentials, and the outer retina produce blue-yellow deficits, whereas disor-
(5) the response to rapidly flickered light. ders of the inner retina and ON produce red-green perceptual
Flash-elicited VEPs are recorded from electrodes overlying deficits. Bilateral lesions in the visual cortex can also lead to
visual (striate) cortex, and they reflect the activity of the ret- color blindness.
inogeniculostriate pathway and the activity of cells in the visual Assessment of color vision in human toxicologic evaluations
cortex. Pattern-elicited VEPs (PEPs), which are widely used in includes the Farnsworth-Munson 100 Hue (FM-100) test and
human clinical evaluations, have diagnostic value. the simplified 15-chip tests using either the saturated hues of
The EOG is generated by a potential difference between the the Farnsworth D-15 or the desaturated hues of the Lanthony
front and back of the eye, which originates primarily within Desaturated Panel D-15. The Farnsworth-Munson procedure
the RPE. The magnitude of the EOG is a function of the involves arrangement of 85 chips in order of progressively
level of illumination and health status of the RPE. Electrodes changing color. The relative chromatic value of successive
placed on the skin on a line lateral or vertical to the eye mea- chips induces those with color perception deficits to abnor-
sure potential changes correlated with eye movements as the mally arrange the chips. The pattern is indicative of the nature
relative position of the ocular dipole changes. Thus, the EOG of the color perception anomaly. The FM-100 is considered
finds applications in assessing both RPE status and measur- more diagnostically reliable but takes considerably longer to
ing eye movements. The EOG is also used in monitoring eye administer than the similar but more efficient Farnsworth and
movements during the recording of other brain potentials, so Lanthony tests. The desaturated hues of the Lanthony D-15 are
that eye movement artifacts are not misinterpreted as brain- designed to better identify subtle acquired color vision deficits.
generated electrical activity.

TARGET SITES AND MECHANISMS OF

Behavioral and Psychophysical Techniques
ACTION: CORNEA
Behavioral testing procedures typically vary the parameters
of the visual stimulus and then determine whether the subject The cornea provides three essential functions. First, it pro-
can discriminate or perceive the stimulus. Contrast sensitivity vides a clear refractive surface and the curvature of the cor-
refers to the ability to resolve small differences in luminance nea must be correct for the visual image to be focused at the
contrast, such as the difference between subtle shades of gray retina. Second, the cornea provides tensile strength to main-
or a series of visual patterns that differ in pattern size, or the tain the appropriate shape of the globe. Third, the cornea pro-
luminance changes across the pattern in a sinusoidal profile. tects the eye from external factors, including potentially toxic
Contrast sensitivity functions depend primarily on the neu- chemicals.
ral as opposed to the optic properties of the visual system. The The cornea is transparent to wavelengths of light ranging
assessment of visual acuity and contrast sensitivity has been between 310 nm (UV) and 2500 nm (IR). Exposure to UV light
recommended for field studies of humans potentially exposed below this range can damage the cornea. It is most sensitive to
to neurotoxic substances. wavelengths of approximately 270 nm. Excessive UV exposure
leads to photokeratitis and corneal pathology, the classic exam-
ple being welder’s-arc burns. Also, the cornea can be damaged
Color Vision Testing by topical or systemic exposure to chemicals.
Color vision deficits are either inherited or acquired. Hereditary Direct chemical exposure to the eye requires emergency
red-green color deficits occur in about 8% of males (X-linked) medical attention. Products at pH extremes < 2.5 or = 11.5
whereas only about 0.5% of females show similar congeni- can cause severe ocular damage and permanent loss of vision.
tal deficits. Inherited color deficiencies take two common Damage that extends to the corneal endothelium is associated
forms: protan, a red-green confusion caused by abnormal- with poor repair and recovery. The most important therapy
ity or absence of the long-wavelength (red) sensitive cones is immediate and adequate irrigation with large amounts of
(L-type cones); and deutan caused by abnormality or absence water or saline. The extent of damage to the eye and the ability
of the middle-wavelength sensitive (green) cones (M-type to achieve a full recovery depend on the nature of the chemical,
cones). Dutanopes demonstrate a concomitant confu- the concentration and duration of exposure, and the speed and
sion of red-green and blue-yellow due to the lack of M-type magnitude of the initial irrigation.
264 UNIT 4 Target Organ Toxicity

Acids and fibroblasts and the release of proteolytic enzymes. Stromal

ulceration usually stops when the corneal epithelium is restored.
Among the most significant acidic chemicals in terms of the
tendency to cause clinical ocular damage are hydrofluoric
acid, sulfurous acid, sulfuric acid, and chromic acid, followed Organic Solvents
by hydrochloric and nitric acid and finally acetic acid. Injuries When organic solvents are splashed into the eye, the result is
may be mild if contact is with weak acids or with dilute solu- typically a painful immediate reaction. Exposure of the eye to
tions of strong acids. Compounds with a pH between 2.5 and solvents should be treated rapidly with abundant water irriga-
7 produce pain or stinging, but with only a brief contact, they tion. Highly lipophilic solvents can damage the corneal epithe-
will cause no lasting damage. Following mild burns, the cor- lium and produce swelling of the corneal stroma. Most organic
neal epithelium may become turbid as the corneal stroma solvents cause minimal chemical burns to the cornea. In most
swells (chemosis). Mild burns are typically followed by rapid cases, the corneal epithelium will be repaired over the course
regeneration of the corneal epithelium and full recovery. In of a few days and there will be no residual damage. Exposure to
more severe burns, the epithelium of the cornea and conjunc- solvent vapors may produce small transparent vacuoles in the
tiva become opaque and necrotic and may disintegrate over the corneal epithelium, which may be asymptomatic or associated
course of a few days. In severe burns, there may be no sensation with moderate irritation and tearing.
of pain because the corneal nerve endings are destroyed.
Acid chemical burns of the cornea occur through hydro-
gen ion-induced denaturing and coagulation of proteins. As Surfactants
epithelial cell proteins coagulate, glycosaminoglycans precipi- These compounds have water-soluble (hydrophilic) proper-
tate and stromal collagen fibers shrink causing the cornea to ties at one end of the molecule and lipophilic properties at the
become cloudy. The protein coagulation and shrinkage of the other end that help to dissolve fatty substances in water and
collagen is protective in that it forms a barrier and reduces fur- also serve to reduce water surface tension. The widespread use
ther penetration of the acid. The collagen shrinkage, however, of these agents in soaps, shampoos, detergents, cosmetics, and
contracts the eye and can lead to a dangerous acute increase in similar consumer products leads to abundant opportunities for
intraocular pressure. exposure to ocular tissues. Many of these agents may be irritat-
ing or injurious to the eye. In general, cationic surfactants tend
to be stronger irritants and more injurious than the other types,
Bases or Alkalies
and anionic compounds more so than neutral ones. Because
Compounds with a basic pH are potentially more damaging to these compounds are soluble in both aqueous and lipid media,
the eye than are strong acids. Among the compounds of clini- they readily penetrate the sandwiched aqueous and lipid barri-
cal significance in terms of frequency and severity of injuries ers of the cornea.
are ammonia or ammonium hydroxide, sodium hydroxide
(lye), potassium hydroxide (caustic potash), calcium hydrox-
ide (lime), and magnesium hydroxide. One reason that caustic TARGET SITES AND MECHANISMS OF
agents are so dangerous is their ability to rapidly penetrate the ACTION: LENS
ocular tissues. The toxicity of these substances is a function of
their pH, being more toxic with increasing pH values. Rapid The lens of the eye plays a critical role in focusing the visual
and extensive irrigation after exposure and removal of parti- image on the retina. The lens is a biconvex transparent body,
cles, if present, is the immediate therapy of choice. encased in an elastic capsule, and located between the pupil
Caustic burns differ from acid burns in that two phases of and the vitreous humor (Figure 17-1). The mature lens has
injury may be observed with caustic burns. There is an acute a dense inner nuclear region surrounded by the lens cortex.
phase from time of exposure up to 1 week. Depending on the The high transparency of the lens to visible wavelengths of
extent of injury, direct damage from exposure is observed light is a function of its chemical composition, approxi-
in the cornea, adnexia, and possibly the iris, ciliary body, and mately two-thirds water and one-third protein, and the
lens. Strong alkali substances attack membrane lipids, causing special organizational structure of the lenticular proteins.
necrosis, hydration of the collagen matrix, and corneal swell- Nutrients provided from the aqueous and vitreous fluids
ing. Intraocular pressure may increase. Conversely, if the alkali are transported into the lens substance through a system of
burn extends to involve the ciliary body, the intraocular pres- intercellular gap-type junctions. The lens is a metabolically
sure may decrease due to reduced formation of aqueous humor. active tissue that maintains careful electrolyte and ionic bal-
The acute phase of damage is typically followed by initiation of ance. The lens continues to grow throughout life, with new
corneal repair. The repair process may involve corneal neovas- cells added to the epithelial margin of the lens as the older
cularization along with regeneration of the corneal epithelium. cells condense into a central nuclear region. The dramatic
Approximately 2 to 3 weeks after alkali burns, however, dam- growth ofthe lens is illustrated by its increasing weight, from
aging ulceration of the corneal stroma often occurs as a result approximately 150 mg at 20 years of age to approximately
of inflammatory infiltration of polymorphonuclear leukocytes 250 mg at 80 years of age.
 CHAPTER 17 Toxic Responses ofthe Ocular and Visual System 265

Cataracts are decreases in the optic transparency of the lens demonstrates a dose-related increase in the risk of cataracts
that ultimately can lead to functional visual disturbances. from use of nonantipsychotic phenothiazines.
Cataracts can occur at any age; they can also be congenital.
Risk factors for the development of cataracts include aging,
TARGET SITES AND MECHANISMS OF
diabetes, low antioxidant levels, and exposure to a variety of
environmental factors, including exposure to UV radiation ACTION: RETINA
and visible light, trauma, smoking, and exposure to a large The adult mammalian retina is a highly differentiated tissue
variety of topical and systemic drugs and chemicals. containing eight distinct layers plus the RPE, 10 major types
Several different mechanisms have been hypothesized to of neurons, and a Miller glial cell ({MGC] Figure 17-1). The
account for the development of cataracts. These include the eight layers of the neural retina, which originate from the cells
disruption of lens energy metabolism, hydration and/or elec- of the inner layer of the embryonic optic cup, are the nerve fiber
trolyte balance, oxidative stress due to the generation of free layer (NFL), ganglion cell layer (GCL), inner plexiform layer
radicals and ROS, and the occurrence of oxidative stress due (IPL), inner nuclear layer (INL), outer plexiform layer (OPL),
to a decrease in antioxidant defense mechanisms such as glu- outer nuclear layer (ONL), rod and cone photoreceptor inner
tathione, superoxide dismutase, catalase, ascorbic acid, or segment layer (RIS, CIS), and the rod and cone photoreceptor
vitamin E. The generation of ROS leads to oxidation of lens outer segment layer (ROS, COS). The retinal pigment epithe-
membrane proteins and lipids. A critical pathway is oxidation lium (RPE) is a single layer of cuboidal epithelial cells that lies
of protein thiol groups, particularly in methionine or cyste- on Bruch’s membrane adjacent to the vascular choroid. Between
ine amino acids, leading to the formation of polypeptide links the RPE and photoreceptor outer segments lies the subretinal
through disulfide bonds, and in turn, high-molecular-weight space, which is similar to the brain ventricles. The 10 major
protein aggregates. These large aggregations of proteins can types of neurons are the rod and cone photoreceptors, (depo-
attain a size sufficient to scatter light, thus reducing lens trans- larizing) ON-rod and ON-cone bipolar cells, (hyperpolarizing)
parency. Oxidation of membrane lipids and proteins may also OFF-cone bipolar cells, horizontal cells, numerous subtypes
impair membrane transport and permeability. of amacrine cells, an interplexiform cell, and ON-RGCs and
OFF-RGCs. The MGC is the only glial cell in the retina. The
Corticosteroids somas of the MGCs are in the INL. The end feet of the MGCs
in the proximal or inner retina along with a basal lamina form
There are two proposed mechanisms by which systemic treat-
the internal limiting membrane of the retina, which is similar
ment with corticosteroids may cause cataracts. Corticosteroids
to the pial surface of the brain. In the distal retina, the MGC end
alter lens epithelium electrolyte balance, which disrupts the
feet join with the photoreceptors and zonula adherens to form
normal lens epithelial cell structure causing gaps to appear
the external limiting membrane, which is located between the
between the lateral epithelial cell borders. Another theory is
ONL and RIS/CIS.
that corticosteroid molecules react with lens crystallin pro-
The mammalian retina is highly vulnerable to toxicant-
teins, producing corticosteroid-crystallin adducts that would
induced structural and/or functional damage due to (1) the
be light-scattering complexes.
highly fenestrated choriocapillaris that supplies the distal or
outer retina as well as a portion of the inner retina; (2) the very
Naphthalene high rate of oxidative mitochondrial metabolism, especially that
Accidental exposure to naphthalene results in cortical cata- in the photoreceptors; (3) high daily turnover of rod and cone
racts and retinal degeneration. The metabolite 1,2-dihydro- outer segments; (4) high susceptibility of the rod and cones to
1,2-dihydroxynaphthalene (naphthalene dihydrodiol) is the degenerate due to inherited retinal dystrophies as well as associ-
cataract-inducing agent instead of naphthalene itself. Subse- ated syndromes and metabolic disorders; (5) presence of spe-
quent studies showed that aldose reductase in the rat lens is cialized ribbon synapses and synaptic contact sites; (6) presence
the enzyme responsible for the formation of naphthalene dihy- of numerous neurotransmitter and neuromodulatory systems,
drodiol, and that treatment with aldose reductase inhibitors including extensive glutamatergic, GABAergic, and glycinergic
prevents naphthalene-induced cataracts. systems; (7) presence of numerous and highly specialized gap
junctions used in the information signaling process; (8) pres-
ence of melanin in the choroid and RPE and also in the iris
Phenothiazines and pupil; (9) a very high choroidal blood flow rate, as high as
Schizophrenics receiving phenothiazine drugs develop pig- 10 times that of the gray matter of the brain; and (10) the addi-
mented deposits in their eyes and skin. The phenothiazines tive or synergistic toxic action of certain chemicals with ultra-
combine with melanin to form a photosensitive product that violet and visible light.
reacts with sunlight, causing formation of the deposits in lens Each of the retinal layers can undergo specific or general
and cornea. The amount of pigmentation is related to the toxic effects. These alterations and deficits include, but are
dose of the drug, with the annual yearly dose being the most not limited to, visual field deficits, scotopic vision deficits
predictive dose metric. More recent epidemiologic evidence such as night blindness and increases in the threshold for
266 UNIT 4 Target Organ Toxicity

dark adaptation, cone-mediated (photopic) deficits such as visual system abnormalities include decreased vision, flicker-
decreased color perception, decreased visual acuity, macular ing scotomas, and altered color vision. Digoxin produces more
and general retinal edema, retinal hemorrhages and vasocon- toxicity than digitoxin due to its greater volume of distribu-
striction, and pigmentary changes. tion and plasma protein binding. The most frequent visual
complaints are color vision impairments and hazy or snowy
vision, although complaints of flickering light, colored spots
Retinotoxicity of Systemically surrounded by bright halos, blurred vision, and glare sensi-
Administered Therapeutic Drugs tivity also are reported. Photoreceptors are the primary site of
toxicity, with cone photoreceptors being more susceptible to
Cancer Chemotherapeutics—Ocular toxicity is a com-
the effects than rod photoreceptors. The retina has the highest
mon side effect of cancer chemotherapy, resulting in blurred
number of Na*,K*-ATPase sites of any ocular tissue, which are
vision, diplopia, decreased color vision and visual acuity,
potently inhibited by digoxin and digitoxin.
optic/retrobular neuritis, transient cortical blindness, and
demyelination of the ONs. The retina, due to its high meta-
bolic activity and choroidal circulation (vide infra), appears to Indomethacin—Indomethacin is a nonsteroidal anti-
be particularly vulnerable to numerous cytotoxic drugs such inflammatory drug with analgesic and antipyretic properties
as the alkylating agents cisplatin, carboplatin, and carmustine; that is frequently used for the management of arthritis, gout,
the antimetabolites cytosine arabinoside, 5-fluorouracil, and and musculoskeletal discomfort. Chronic administration of
methotrexate; and the mitotic inhibitors such as docetaxel. The 50 to 200 mg/day of indomethacin for 1 to 2 years has been
ocular toxicity of different drugs is dependent upon the dose, reported to produce corneal opacities, discrete pigment scat-
duration of dosage, and route of administration. If not detected tering of the RPE perifoveally, paramacular depigmentation,
at an early stage of toxicity, the ocular complications are often decreases in visual acuity, altered visual fields, increases in
irreversible even after chemotherapy is discontinued. the threshold for dark adaptation, blue-yellow color deficits,
and decreases in ERG and EOG amplitudes. Decreases in the
Chloroquine and Hydroxychloroquine—Chloroquine (Ara- ERG a- and b-wave amplitudes, with larger changes observed
len) and hydroxychloroquine (Plaquenil) are 4-aminoquinoline under scotopic dark-adapted than light-adapted conditions,
derivatives used as antimalarial and anti-inflammatory drugs that have been reported. On cessation of drug treatment, the ERG
can cause irreversible loss of retinal function. Chloroquine, its waveforms and color vision changes return to near normal,
major metabolite desethylchloroquine, and hydroxychloroquine although the pigmentary changes are irreversible. The mecha-
have high affinity for melanin, which results in these drugs accu- nism of retinotoxicity is unknown; however, it appears likely
mulating in the choroid and RPE, ciliary body, and iris during and that the RPE is a primary target site.
following drug administration. Prolonged exposure of the retina
to these drugs, especially chloroquine, may lead to an irreversible Sildenafil Citrate—Sildenafil citrate (Viagra) is a cGMP-
retinopathy. Doses of hydroxychloroquine less than 400 mg/day specific phosphodiesterase (PDE) type 5 inhibitor that is uti-
appear to produce little or no retinopathy even after prolonged lized in the treatment of erectile dysfunction. Sildenafil is also
therapy. a weak cGMP PDE type 6 inhibitor, which is present in rod and
The clinical findings accompanying chloroquine retinopa- cone photoreceptors. Transient visual symptoms such as a blue
thy can be divided into early and late stages. The early changes tinge to vision, increased brightness of lights and blurry vision,
include (1) the pathognomonic “bull’s-eye retina” visualized as well as alterations in scotopic and photopic ERGs have been
as a dark, central pigmented area involving the macula, sur- reported.
rounded by a pale ring of depigmentation, which, in turn, is
surrounded by another ring of pigmentation; (2) a diminished Tamoxifen—Tamoxifen (Nolvadex, Tamoplex), a triphenyl-
EOG; (3) possible granular pigmentation in the peripheral ethylene derivative, is a nonsteroidal antiestrogenic drug that
retina; and (4) visual complaints such as blurred vision and competes with estrogen for its receptor sites. It is a highly effec-
problems discerning letters or words. Late-stage findings, tive antitumor agent used for the treatment of metastatic breast
which can occur during or even following cessation of drug carcinoma in postmenopausal women. Chronic high-dose
exposure, include (1) a progressive scotoma, (2) constriction of
therapy (180-240 mg/day for ~2 years) produces widespread
the peripheral fields commencing in the upper temporal quad- axonal degeneration in the macular and perimacular areas.
rant, (3) narrowing of the retinal artery, (4) color and night
Clinical symptoms include a permanent decrease in visual
blindness, (5) absence ofa typical retinal pigment pattern, and acuity and abnormal visual fields, as the axonal degeneration
(6) very abnormal EOGs and ERGs. These late-stage symptoms is irreversible. Chronic low-dose tamoxifen (20 mg/day) can
are irreversible. result in a small increase in the incidence of keratopathy, with
minimal alterations in visual function. Following cessation of
Digoxin and Digitoxin—The cardiac glycosides digoxin low-dose tamoxifen therapy, most of the keratopathy and reti-
and digitoxin are used in the treatment of congestive heart nal alterations, except the corneal opacities and retinopathy,
disease and in certain cardiac arrhythmias. Digitalis-induced are reversible.
 CHAPTER 17 Toxic Responses of the Ocular and Visual System 267

Vigabatrin— Vigabatrin, an inhibitor of GABA-transaminase, consisting of formic acidemia, uncompensated metabolic

is used to treat refractory complex partial seizures and infantile acidosis, ocular and visual toxicity, coma, and possibly death.
spasms. Retinopathy induced by vigabatrin is characterized by Acute methanol poisoning results in profound and permanent
irreversible bilateral, concentric peripheral visual constriction, structural alterations in the retina and ON, and visual impair-
and decreased retinal nerve fiber thickness. Onset of the visual ments ranging from blurred vision to decreased visual acuity
field loss has been observed after six weeks of exposure, but and light sensitivity to blindness. Formate is directly toxic to
generally requires a couple of years. Rod and cone ERGs as well Miller glial cell function as well as rod and cone photorecep-
as flicker responses are altered, indicating that retinal damage tors. The mechanism of formate toxicity appears to involve a
also occurs. This drug is now recommended only for epileptic disruption in oxidative phosphorylation in photoreceptors,
patients with no alternative choices. Miller glial cells, and ON.

Organic Solvents—Organic solvents produce structural

Retinotoxicity of Known Neurotoxicants alterations in rods and cones as well as functional alterations
Inorganic Lead—Lead poisoning (mean blood lead [BPb] such as color vision deficits, decreased contrast sensitivity, and
= 80 g/dL) in humans produces amblyopia, blindness, optic altered visuomotor performance. Dose-response color vision
neuritis or atrophy, peripheral and central scotomas, paraly- loss and decreases in the contrast sensitivity function occur in
sis of eye muscles, and decreased visual function. Moderate- workers exposed to organic solvents such as trichlorethylene,
to high-level lead exposure produces scotopic and temporal alcohols, xylene, toluene, n-hexane, styrene, mixtures of these,
visual system deficits in occupationally exposed factory work- and others. Adverse effects usually occur only at concentra-
ers, and developmentally lead-exposed monkeys and rats. This tions above the occupational exposure limits.
lead exposure dosage produces irreversible retinal deficits in
the experimental animals.
Occupational lead exposure produces concentration- and TARGET SITES AND MECHANISMS OF
time-dependent alterations in the retina such that higher levels ACTION: OPTIC NERVE AND TRACT
of lead directly and adversely affect both the retina and ON,
whereas lower levels of lead appear to primarily affect the rod The ON consists primarily of RGC axons carrying visual infor-
photoreceptors and the rod pathway. ‘These retinal and oculo- mation from the retina to several distinct anatomical destina-
motor alterations are, in most cases, correlated with blood lead tions in the CNS. Disorders of the ON may be termed optic
levels and occurred in the absence of observable ophthalmo- neuritis, optic neuropathy, or ON atrophy, referring to inflam-
logic changes, CNS symptoms, and abnormal performance test mation, damage, or degeneration, respectively, of the ON.
scores. Thus, these measures of temporal visual function may Retrobulbar neuritis refers to inflammation or involvement of
be among the most sensitive for the early detection of the neu- the orbital portion of the ON posterior to the globe. Among
rotoxic effects of inorganic lead. the symptoms of ON disease are reduced visual acuity, contrast
sensitivity, and color vision. Toxic effects observed in the ON
Methanol— Methanol is a low-molecular-weight (32 Da), col- may originate from damage to the ON fibers themselves or to
orless, and volatile liquid that is readily and rapidly absorbed the RGC somas that provide axons to the ON. A number of
from all routes of exposure (dermal, inhalation, and oral), eas- toxic and nutritional disorders can adversely affect the ON.
ily crosses all membranes, and thus is uniformly distributed Deficiency of thiamine, vitamin B,,, or zinc results in degener-
to organs and tissues in direct relation to their water content. ative changes in ON fibers. A condition referred to as alcohol-
Following different routes of exposures, the highest concentra- tobacco amblyopia or simply as toxic amblyopia is observed in
tions of methanol are found in the blood, aqueous, and vitre- habitually heavy users of these substances and is associated
ous humors, and bile as well as the brain, kidneys, lungs, and with nutritional deficiency.
spleen. In the liver, methanol is oxidized sequentially to form-
aldehyde by alcohol dehydrogenase in human and nonhuman
primates or by catalase in rodents and then to formic acid. It Acrylamide
is excreted as formic acid in the urine or oxidized further to Acrylamide monomer is used in a variety of industrial and
carbon dioxide and then excreted by the lungs. Formic acid is laboratory applications, where it serves as the basis for the pro-
the toxic metabolite of methanol that mediates the metabolic duction of polyacrylamide gels and other polyacrylamide prod-
acidosis as well as the retinal and ON toxicity observed in ucts. Exposure to acrylamide produces a distal axonopathy in
humans, monkeys, and rats with a decreased capacity for folate large-diameter axons of the peripheral nerves and spinal cord
metabolism. that is well documented in humans and laboratory animals.
Human and nonhuman primates are highly sensitive to In contrast, middle diameter axons of optic tract are affected,
methanol-induced neurotoxicity due to their limited capacity specifically, RGCs that project to the parvocellular layers of the
to oxidize formic acid. The toxicity occurs in several stages. It LGN. Why the axons of the optic nerve and tract show a dif-
first occurs as a mild CNS depression, followed by an asymp- ferent size-based pattern of vulnerability than do axons of the
tomatic 12 to 24h latent period, followed by a syndrome peripheral nerve and spinal cord is not understood.
268 UNIT 4 Target Organ Toxicity

Carbon Disulfide the central fovea is proportionately larger than the peripheral
retina in order to accommodate a proportionately larger need
Carbon disulfide (CS,) is used in industry to manufacture vis-
for neural image processing. The magnocellular and parvocel-
cose rayon, carbon tetrachloride, and cellophane. CS, damages
lular pathways project differently to the histologically defined
both the PNS and CNS, and has profound effects on vision. In
layers of primary striate visual cortex and then to extrastriate
the visual system, workers exposed to CS, experience loss of
visual areas. The receptive fields of neurons in the visual cortex
visual function accompanied by observable lesions in the reti-
are more complex than the circular center-surround arrange-
nal vasculature. Central scotoma, depressed visual sensitivity
ment found in the retina and LGN. Cortical cells respond bet-
in the peripheral visual field, optic atrophy, pupillary distur-
ter to lines ofaparticular orientation than to simple spots. The
bances, blurred vision, and disorders of color perception have
receptive fields of cortical cells are thought to represent com-
all been reported. The retinal and ON pathologies produced by
putational summaries of a number of simpler input signals.
CS, are likely a direct neuropathologic action and not the indi-
As the visual information proceeds from area V1 to extrastri-
rect result of vasculopathy.
ate visual cortical areas, the representation of the visual world
reflected in the receptive fields of individual neurons becomes
Ethambutol progressively more complex.
The dextro isomer of ethambutol is widely used as an antimy-
cobacterial drug for the treatment of tuberculosis. Ethambutol Lead
produces dose-related alterations in the visual system, such as
In addition to the retinal effects of lead (see above), lead expo-
blue-yellow and red-green dyschromatopsias, decreased con-
sure during adulthood or perinatal development produces
trast sensitivity, reduced visual acuity, and visual field loss. The
structural, biochemical, and functional deficits in the visual
earliest visual symptoms appear to be a decrease in contrast
cortex of humans, nonhuman primates, and rats. Quantitative
sensitivity and color vision. Impaired red-green color vision
morphometric studies in monkeys exposed to high levels of
is the most frequently observed and reported complaint. The
lead from birth or infancy to 6 years of age revealed a decrease
symptoms are primarily associated with one of the two forms
in visual cortex (areas V1 and V2), cell volume density, and a
of retrobulbar neuritis (i.e., optic neuropathy). The most com-
decrease in the number of initial arborizations among pyrami-
mon form, seen in almost all cases, involves the central ON
dal neurons. These alterations could partially contribute to the
fibers and typically results in a central or paracentral scotoma
alterations in the amplitude and latency measures of the flash-
in the visual field and is associated with impaired red-green
evoked and pattern-reversal-evoked potentials in lead-exposed
color vision and decreased visual acuity, whereas the second
children, workers, monkeys, and rats, and the alterations in
form involves the peripheral ON fibers and typically results in
tasks assessing visual function in lead-exposed children.
a peripheral scotoma and visual field loss.

TARGET SITES AND MECHANISMS OF Methyl Mercury

ACTION: THE CENTRAL VISUAL SYSTEM Methyl mercury—poisoned individuals experience a striking
and progressive constriction of the visual field (peripheral sco-
Many areas of the cerebral cortex are involved in the percep- toma). The narrowing of the visual world gives impression of
tion of visual information. The primary visual cortex (V1), looking through a long tunnel, hence the term tunnel vision.
Brodmann area 17, or striate cortex receives the primary The damage is most severe in the regions of primary visual
projections of visual information from the lateral geniculate cortex subserving the peripheral visual field, with relative spar-
nucleus (LGN) and also from the superior colliculus. Neurons ing of the cortical areas representing the central vision. Methyl
from the LGN project to the visual cortex maintaining a topo- mercury—poisoned individuals also experience poor night
graphic representation of the receptive field origin in the retina. vision that is also attributable to peripheral visual field losses.
The receptive fields in the left and right sides of area 17 reflect
the contralateral visual world and representations of the upper
and lower regions of the visual field are separated below and
above, respectively, the calcarine fissure. Cells in the posterior
BIBLIOGRAPHY
Bartlett JD, Jaanus SD: Clinical Ocular Pharmacology, 5th ed. Boston,
aspects of the calcarine fissure have receptive fields located in
MA: Butterworth-Heinemann, 2008.
the central part of the retina. Cortical cells progressively deeper
Fraunfelder FT, Fraunfelder FW, Chambers WA: Clinical Ocular
in the calcarine fissure have retinal receptive fields that are Toxicology: Drugs, Chemicals, and Herbs. Philadelphia, PA: Elsevier
located more and more peripherally in the retina. The central Saunders, 2008.
part of the fovea has tightly packed photoreceptors for resolu- Weir AB, Collins M: Assessing Ocular Toxicology in Laboratory
tion of fine detailed images, and the cortical representation of Animals. New York: Springer Humana, 2013.
 CHAPTER 17 Toxic Responses of the Ocular and Visual System 269

QUESTIONS

In which of the following locations would one NOT find Which of the following statements regarding color vision
melanin? deficits is FALSE?
iris. a. Inheritance ofablue-yellow color deficit is common.
ciliary body. b. Bilateral deficits in the visual cortex can lead to color
retinal pigment epithelium (RPE). blindness.
uveal tract. c. Disorders of the outer retina produce blue-yellow
sp
enosclera. deficits.
d. Drug and chemical exposure most commonly results
Systemic exposure to drugs and chemicals is most likely to in blue-yellow color deficits.
target which of the following retinal sites? e. Disorders of the optic nerve produce red-green deficits.
RPE and ganglion cell layer.
optic nerve and inner plexiform layer. A substance with which of the following pH values would
RPE and photoreceptors. be most damaging to the cornea?
photoreceptors and ganglion cell layer. ale!
Sa inner plexiform layer and RPE.
EOS
OF b. 3.0.
©, FAV.
Which of the following structures is NOT part of the d. 10.0.
ocular fundus? @ IO.
a. retina.
Which of the following statements concerning the lens is
b. lens.
FALSE?
c. choroid.
a. UV radiation exposure is a common environmental
d. sclera.
risk factor for developing cataracts.
e. optic nerve.
b. Cataracts are opacities of the lens that can occur at
any age.
Drugs and chemicals in systemic blood have better access
c. ‘The lens continues to grow throughout one’s life.
to which of the following sites because of the presence of
d. Naphthalene and organic solvents both can cause
loose endothelial junctions at that location?
cataracts.
retinal choroid.
e. Topical treatment with corticosteroids can cause
inner retina.
cataracts.
optic nerve.
iris. Which of the following is NOT a reason why the retina is
FEN
i)
ee
@ ciliary body. highly vulnerable to toxicant-induced damage?
presence of numerous neurotransmitter systems.
All of the following statements regarding ocular irritancy presence of melanin in the RPE.
and toxicity are true EXCEPT: high choroidal blood flow rate.
a. The Draize test involves instillation of a potentially high rate of oxidative mitochondrial metabolism.
toxic liquid or solid into the eye. ee
te
SN lack of gap junctions.
b. The effect of the irritant in the Draize test is scored
on a weighted scale for the cornea, iris, and 10. A deficiency in which of the following vitamins can result
conjunctiva. in degeneration of optic nerve fibers?
c. The Draize test usually uses one eye for testing and a. vitamin A.
the other as a control. b. vitamin B,.
The Draize test has strong predictive value in humans. c. vitamin C.
e. The cornea is evaluated for opacity and area of d. vitamin B,,.
involvement in the Draize test. e. vitamin E.
 a os | at

ei AoA &
eter diet
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 Cer
Ave PS laa BR

Toxic Responses of the

Heart and Vascular System
Y. James Kang

INTRODUCTION CARDIAC TOXIC CHEMICALS

Alcohol and Alcoholic Cardiomyopathy
OVERVIEW OF THE HEART
Pharmaceutical Chemicals
Overview of Cardiac Structural and Physiologic Natural Products
Features
Environmental Pollutants and Industrial Chemicals
Review of Cardiac Structure
Electrophysiology OVERVIEW OF VASCULAR SYSTEM
Contractility Vascular Physiology and Structural Features
Electrotonic Cell-to-Cell Coupling Arterial System and Physiologic Function
Electrocardiogram Capillaries and Microcirculation
Neurohormonal Regulation Venous System and Physiologic Function
Cardiac Output Lymphatic System and Physiologic Function
Regulatory Mechanisms ofthe Vascular System
CARDIAC TOXIC RESPONSES
Neurohormonal Regulation
Basic Concepts and Definitions
Local Metabolic Regulation
Myocardial Degeneration and Regeneration
Myocardial Degenerative Responses VASCULAR SYSTEM TOXIC RESPONSES
Toxic Effect on Myocardial Regeneration Mechanisms of Vascular Toxicity
Myocardial Cell Death and Signaling Pathways Responses ofVascular Endothelial Cells to
Apoptosis and Necrosis Toxic Insults
Mitochondrial Control of Cell Death Responses of Smooth Muscle Cells to Toxic Insults
Death Receptors and Signaling Pathways Oxidative Stress and Vascular Injury
Mitochondrial Dynamics and Autophagy Inflammatory Lesions
Cardiac Hypertrophy and Heart Failure Toxic Responses of Blood Vessels
Adaptive and Maladaptive Responses Hypertension and Hypotension
Hypertrophic Signaling Pathways Atherosclerosis
Transition from Cardiac Hypertrophy Hemorrhage
to Heart Failure Edema
QT Prolongation and Sudden Cardiac Death
VASCULAR SYSTEM TOXIC CHEMICALS
Molecular Basis of QT Prolongation
Torsade De Pointes and Sudden Pharmaceutical Chemicals
Cardiac Death Sympathomimetic Amines

Parameters Affecting QT Prolongation Nicotine

and forsadogenesis Cocaine
Biomarkers for Cardiac Toxicity Psychotropic Agents
bo“INw UNIT 4 Target Organ Toxicity

Antineoplastic Agents Vitamin D

Analgesics and Nonsteroidal Anti-Inflammatory B-Amyloid
Agents Environmental Poliutants and Industrial Chemicals
Oral Contraceptives Carbon Monoxide
Natural Products Carbon Disulfide
Bacterial Endotoxins 1,3-Butadiene
Homocysteine Metals and Metalloids
Hydrazinobenzoic Acid Aromatic Hydrocarbons
T-2 Toxin Particulate Air Pollution

KEY POINTS

= Typical chemical-induced disturbances in cardiac func- in systolic and diastolic function that reflect specific
tion consist of effects on heart rate (chronotropic), con- alterations in ventricular function and abnormalities in
tractility (inotropic), conductivity (dromotropic), and/ a variety of subcellular processes.
or excitability (bathmotropic). « Acute cardiac toxicity occurs after a single exposure to
= Cardiomyopathy includes morphologic and functional a high dose of cardiotoxic chemicals and may be mani-
alterations induced by toxic exposure, leading to decreased fested by arrhythmia and can involve apoptosis.
cardiac output and peripheral tissue hypoperfusion. » Chronic cardiac toxicity, which results from long-term
= Concentric cardiac hypertrophy is an increased size of exposure to chemicals, is often manifested by cardiac
cardiac myocytes in which new contractile-protein units hypertrophy and the transition to heart failure.
are assembled in parallel, resulting in a relative increase » Any xenobiotic that disrupts ion movement or homeo-
in the width of individual cardiac myocytes. stasis may induce a cardiotoxic reaction composed prin-
« Eccentric cardiac hypertrophy is an increased size of car- cipally of disturbances in heart rhythm.
diac myocytes in which new contractile-protein units a All toxicants absorbed into the circulatory system con-
are assembled in series, resulting in a relatively greater tact vascular cells before reaching other sites in the body.
increase in the length than in the width of individual a Common mechanisms of vascular toxicity include
myocytes. (1) alterations in membrane structure and function,
« Heart failure is the inability of the heart to maintain (2) redox stress, (3) vessel-specific bioactivation of pro-
cardiac output sufficient to meet the metabolic and oxy- toxicants, and (4) preferential accumulation of the active
gen demands of peripheral tissues, including changes toxin in vascular cells.

INTRODUCTION This chapter is divided into two parts: the heart and the vas-
cular system. The manifestations of toxicologic response of the
Cardiovascular toxicology is concerned with the adverse heart include cardiac arrhythmia, hypertrophy, and overt heart
effects of extrinsic and intrinsic stresses on the heart and vas- failure. The responses of the vascular system include changes
cular system. Extrinsic stress involves exposure to therapeutic in blood pressure and lesions in blood vessels in the form of
drugs, natural products, and environmental toxicants. Intrinsic atherosclerosis, hemorrhage, and edema.
stress refers to exposure to toxic metabolites derived from non-
toxic compounds such as those found in food additives and
supplements. The intrinsic exposures also include second- OVERVIEW OF THE HEART
ary neurohormonal disturbance such as overproduction of
inflammatory cytokines derived from pressure overload of the Overview of Cardiac Structural
heart and counter-regulatory responses to hypertension. These and Physiologic Features
toxic exposures result in alterations in biochemical pathways, The main purpose of the heart is to pump blood to the lungs and
defects in cellular structure and function, and pathogenesis of the systemic arteries so as to provide oxygen and nutrients to all
the affected cardiovascular system. body tissues. Figure 18-1 illustrates the basic anatomy of the heart.
 CHAPTER 18 Toxic Responses of the Heart and Vascular System 278

Atrioventricular Aorta
node 3
Pulmonary
Sinoatrial veins
Heart
node Left
tri
Right atrium

atrium

Cardiac muscle
Left tissue
ventricle

Right Purkini
ventricle SS
fibers

FIGURE 18-1 Diagram illustrating the basic anatomy

Myosin
y ,
of the heart.
ATP
iy)
Review of Cardiac Structure—The primary contractile
unit within the heart is the cardiac muscle cell, or cardiac
myocyte. Cardiac myocytes are composed of several major
structural features and organelles, as illustrated in Figure 18-2. Actin Tropomyosin «~
A primary component is the contractile elements known as the
myofibril. Each myofibril consists of a number of smaller fila-
ments (the thick and thin myofilaments). The thick filaments
are special assemblies of the protein myosin, whereas the thin Thin filament Thick filament
filaments are made up primarily of the protein actin. (actin) (myosin)
Cardiac and skeletal muscle share many similarities, includ-
ing contractile elements and sarcomere structure. However, a FIGURE 18-2 Structural organization of cardiac muscle tissue.
major difference lies in the organization of cardiac myocytes
into a functional syncytium; cardiac myocytes are joined end-
to-end by dense structures known as intercalated disks. Within transporters (pumps) on the membrane of cardiac myocytes.
these, there are tight gap junctions that facilitate action poten- Through the movement of these ions across the cell membrane,
tial propagation and intercellular communication. an action potential is generated and propagated from one cell to
Cardiac myocytes are the largest cells in the heart and con- another, so that electric conductance is produced in the heart.
tribute to the majority of cardiac mass. However, cardiac
myocytes are only about 25% of the total number of cells. Action Potential—Cardiac myocytes produce an action poten-
Approximately 90% of the non-muscle cells are cardiac fibro- tial when activated by pacemaker cells and other stimuli.
blasts, with vascular cells, Purkinje cells, and other connective A sudden depolarization changes the membrane potential from
tissue cells comprising the remaining 10%. negative inside to positive inside, followed by a repolarization
The heart normally undergoes significant increase in size to reset the resting potential. The process of an action poten-
and mass throughout organism growth, primarily through tial from depolarization to the completion of repolarization is
enlargement (hypertrophy) of the preexisting cardiac myo- divided into five phases in cardiac Purkinje fibers as shown in
cytes. Pathologic conditions, including exposure to toxicants, Figure 18-3. Phase 0 represents a rapid depolarization due to
also results in hypertrophy of surviving cardiac myocytes. the inward current of Na‘. Phase 1 is associated with an imme-
Cardiac fibroblasts may continue to proliferate after birth, diate rapid repolarization, during which the Na* inward current
but promote fibrosis and scarring of injured cardiac tissue is inactivated and a transient K* outward current is activated,
in response to myocardial injuries. The limited proliferative followed by an action potential plateau or phase 2, which is
capacity of cardiac myocytes and propensity of scar formation dominated by slowly decreasing inward Ca** current and a slow
by cardiac fibroblasts make the heart vulnerable to injury. activation of an outward K* current. Phase 3 reflects a fast K*
outward current and inactivation of the plateau Ca** inward
Electrophysiology—Bioelectricity is the result of charge current, and phase 4 is the diastolic interval for the resetting of
generated from the Movement of positively and negatively the resting potential.
charged ions in tissues. In cardiac myocytes, three major
positively charged ions make a significant contribution to the Automaticity—A group of specialized cells in the heart are
bioelectricity of the heart: calcium (Ca**), sodium (Na*), and capable of repetitively spontaneous self-excitation, which gen-
potassium (K+). Each of the ions has specific channels and erate and distribute each impulse through the heart in a highly
274 UNIT 4 Target Organ Toxicity

Superior vena
cava

Left atrium

Sinoatrial
Bundle ofHis
node
Bundle
Right atrium branches
Left ventricle
+20
Purkinje fibers
0 Atrioventricular
20 node

~40

-60 Right ventricle Papillary muscle Purkinje fibers

-80

-—100
a
\

FIGURE 18-3 Characteristic cardiac action potential recorded from sinoatrial node and Purkinje fibers as indicated. (Reproduced
with permission from Berne RM, Levy MN (eds): Physiology. St. Louis, MO: Mosby/Elsevier; 1983.)

Phase 1 pacemaker cells of automaticity. In pacemaker cells, phase 0 is

(Cat* in; Nat channel closed; Kt out) mediated almost entirely by increased conductance of Ca’* ions.

Phase 0 Phase 3 Contractility—Cardiac myocytes, like other muscle cells,

Nat j (K* out) have a unique functional feature called contractility. Myocyte
ery Phase 2 contraction occurs when an action potential causes the release
hace dia (Ca** in) Phase 4 of Ca’* from the sarcoplasmic reticulum as well as the entry of
(Resting)
(Resting) extracellular Ca** into the cell. This action potential-triggered
Ca’* increase in the plasma and myocyte contraction is called
excitation—contraction coupling. The strength of contraction is
P: Atrial <— QRS: Ventricular directly proportional to the concentration of Ca** ions such
depolarization depolarization
that a large amount of ions will cause a strong contraction.
’ T
An increase in intracellular Ca** concentrations allows Ca’*
to bind to troponin C, which moves tropomyosin thereby
exposing a site of interaction between actin and myosin.
QS_ T: Ventricular Binding of ATP to the myosin head and its subsequent hydroly-
repolarization sis causes the myosin head to bend in a ratchet-like fashion. This
FIGURE 18-4 Characteristic cardiac action potential action increases the overlap of the actin and myosin filaments,
and electrocardiogram (ECG). resulting in shortening of the sarcomeres and contraction of
the myocardium.

coordinated manner to control the normal heartbeat. The sinus Electrotonic Cell-to-Cell Coupling—Myocardium as a
node P cells or pacemaker cells have three distinct phases of whole has to synchronize the contraction and relaxation of
action potential (Figure 18-4): phase 0, rapid depolarization; individual myocytes in order to perform its pump function.
phase 3, plateau and repolarization; and phase 4, slow depolar- This is achieved by a special structural feature of cell-to-cell
ization or often referred to as pacemaker potential. It is the pace- interaction, electrotonic cell-to-cell coupling via the gap junc-
maker potential that brings the membrane potential to a level tion. Through the gap junction, major ionic fluxes between
near the threshold for activation of the inward Ca’* current, adjacent cardiomyocytes are spread, thus allowing electrical
which triggers the phase 0 rapid depolarization and makes the synchronization of contraction.
 CHAPTER 18 Toxic Responses of the Heart and Vascular System PAS

Electrocardiogram—
The electrocardiogram (ECG) records failure reflect myocardial functional alterations resulting from
electrical currents generated during depolarization and both acute and chronic cardiac toxicity.
repolarization. On the ECG shown in Figure 18-5, deflections
(or waves) are recorded that correspond to atrial depolariza-
tion (P wave), ventricular depolarization (QRS complex), and CARDIAC TOXIC RESPONSES
ventricular repolarization (T wave); however, atrial repo-
larization is not normally observed on the ECG because it is Basic Concepts and Definitions
obscured by the large QRS complex. The interplay between environmental factors, genetic suscep-
Useful intervals noted on the ECG include the following. The tibility, and myocardial pathogenesis is critical in the study
PR interval corresponds primarily to the speed of conduction of cardiac toxicity. A triangle model of cardiac toxicity is pre-
through the AV node. The QRS complex represents ventricular sented in Figure 18-6, which highlights the complexity of the
depolarization. The ST segment is the interval during which interaction between environmental stresses and the heart, and
the entire ventricular myocardium is depolarized. The QT the balance between myocardial protection and deleterious
interval corresponds to ventricular depolarization and repo- dose and time effects are considered. First, it is important to
larization, which reflects the action potential duration. The QT recognize that chemicals can lead to heart failure without heart
interval prolongation is recognized as a major life-threatening hypertrophy. Second, a chemical can lead to activation of both
factor of drug cardiac toxicity. protective and destructive responses in the myocardium. Third,
long-term toxicologic responses often result in maladaptive
Neurohormonal Regulation—Although the heartbeat hypertrophy, which primes the heart for malignant arrhythmia,
is governed by the automaticity of the sinus node P cells, leading to sudden cardiac death or transition to heart failure.
neurohormonal regulation of cardiac electrophysiology and
contraction controls cardiac function under normal and
abnormal conditions. Toxicants often exert their effects on the
Myocardial Degeneration
cardiac system through interference with neurohormonal reg- and Regeneration
ulation, and there are many neurohormonal systems that have Myocardial degeneration is the ultimate response of the heart
significant impact on the heart. to toxic exposure, which can be measured by both morpho-
logic and functional degenerative phenotypes. The heart was
Cardiac Output—The primary indicator of cardiac func- previously considered incapable of regenerating. However,
tion is cardiac output, which is the volume of blood pumped evidence now indicates myocardial regeneration and recovery
by the ventricles per minute. Cardiac output is dependent on from cardiomyopathy is possible in some instances. Cardiac
heart rate and stroke volume (the amount of blood ejected toxic responses or damage are now divided into reversible and
by the ventricles during systole). Normal cardiac output at irreversible.
rest is approximately 5 L/min in an average adult human, and
this value may increase three- to fourfold during strenuous
exercise. Toxicants may alter cardiac output through numer-
ous mechanisms and effects on the heart, vasculature, and/or
nervous system. Cardiac arrhythmia, hypertrophy, and heart i‘ : Gellideath
Fetal gene ppepiore Dilation
expression <——
Survival (gp130)

Rees ee ANP, ET-1, TNF

hypertrophy |apoptosis —> Dilation

FIGURE 18-6 Triangle analytical model of cardiac responses

to drugs and xenobiotics. Drugs or xenobiotics can directly cause
both heart failure and heart hypertrophy. Under severe acute toxic
insults, myocardial cell death becomes the predominant response
leading to cardiac dilation and heart failure. In most cases, myocardial
survival mechanisms can be activated so that myocardial apoptosis
is inhibited.The survived cardiomyocytes often become hypertrophy
through activation of calcium-mediated fetal gene expression and
other hypertrophic program. If toxic insult continues, the counter-
regulatory mechanisms against heart hypertrophy such as activation
of cytokine-medicated pathways eventually lead to myocardial cell
FIGURE 18-5 Atypical electrocardiogram (ECG) with the death through apoptosis or necrosis, dilated cardiomyopathy, and
illustration of important deflections and intervals. heart failure.
276 UNIT 4 Target Organ Toxicity

Myocardial Degenerative Responses—Myocardial cell myocardial infarction and the cardiomyopathy induced by
death, fibrosis (scar tissue formation), and contractile dys- environmental toxicants and pollutants.
function are considered as degenerative responses, which can
result in cardiac arrhythmia, hypertrophy, and heart failure. If Mitochondrial Control of Cell Death—Mitochondrial
acute cardiac toxicity does not affect the capacity of myocar- control of cell death is an important topic of apoptotic
dial regeneration, the degenerative phenotype is reversible. research. Factors affecting mitochondrial control of cell death
Both acute and chronic toxic stresses can lead to irreversible are covered in Chapter 3.
degeneration, depending on whether or not the cardiac repair
mechanisms are overwhelmed. Cell death is the most com- Death Receptors and Signaling Pathways—The death
mon phenotype of myocardial degeneration. Both apoptosis receptor-mediated apoptotic signaling pathway can be
and necrosis occur in the process of myocardial cell death. triggered by cytokines (see Chapter 12) and is one focus of
Myocardial cell death is also accompanied by hypertrophy of cardiotoxicity research. Tumor necrosis factor-a (TNF-q) is
the remaining cardiac myocytes. the most studied cytokine in myocardial cell death signaling
Myocardial fibrosis results from excess accumulation of pathways. This pathway is mediated by TNF receptors (TNFRI
extracellular matrix (ECM), which is mainly composed of col- and TNFR2).
lagens. The net accumulation of ECM results from enhanced Briefly, binding of TNF-a to TNFRs leads to activation of
synthesis or diminished break down of the matrix, or both. caspase 8, which in turn cleaves BID, a BH3 domain-containing
Collagens, predominately types I and III, are the major fibrous pro-apoptotic Bcl2 family member. The truncated BID is
proteins in ECM and their synthesis may increase in response translocated from cytosol to mitochondria, inducing first the
to toxic insults. Degradation of ECM is dependent on the clustering of mitochondria around the nuclei and release of
activity of matrix metalloproteinases (MMPs), which fall into cytochrome c, and then the loss of mitochondrial membrane
five categories based on substrate specificity and organ. The potential, cell shrinkage, and nuclear condensation, that is,
activity of MMPs is altered under toxic stress conditions such apoptosis. Caspase 8 also directly activates caspase-3, lead-
that enhanced fibrogenesis and excess collagen accumulation ing to apoptosis. Fas ligand is also able to induce apoptosis of
(i.e., fibrosis) occurs. cardiomyocytes through the death receptor-mediated signal-
ing pathway.
Toxic Effect on Myocardial Regeneration— The recent
discovery of cardiac progenitor cells has challenged the view
that all myocardial degeneration is permanent. These cells Mitochondrial Dynamics and Autophagy
possess the fundamental properties of stem cells and can The importance of mitochondria in cardiac response to toxic
make myocytes and vascular structures. Myocardial vascular- insults and in the process of toxicologic cardiomyopathy is
ization is required for myocardial regeneration. Many toxic related not only to the control of cell death, but also to autoph-
insults affect the capacity of angiogenesis in the myocardium, agy, the tightly regulated cellular “housekeeping” process
so that cardiac ischemia occurs. The combination of cardiac responsible for the degradation of damaged and dysfunctional
ischemia and the direct toxic insults to cardiomyocytes con- cellular organelles and protein aggregates.
stitute synergistic damage to the heart. During regeneration, Autophagy occurs in all eukaryotic cells under the stress
coronary arterioles and capillary structures are formed to of starvation, hypoxia, and toxic insults, as well as under
bridge the dead tissue (scar tissue) and supply nutrients for physiologic stimulation such as hormones and developmen-
the survival of the regenerated cardiomyocytes. There is an tal signals. Selective autophagy of mitochondria is termed
orderly organization of myocytes within the myocardium mitophagy, which is triggered by mitochondrial perme-
and a well-defined relationship between the myocytes and the ability transition pore opening and loss of mitochondrial
capillary network. This proportion is altered under cardiac membrane potential. In cardiomyocytes and other termi-
toxic conditions. nally differentiated cells, mitophagy is a continuous process
of mitochondrial turnover, but the rate of this turnover is
influenced by stresses that make a critical contribution to
Myocardial Cell Death and
myocardial pathogenesis. Nonselective autophagy has been
Signaling Pathways observed in response to nutrient starvation; the degradation
Apoptosis and Necrosis—Toxic insults trigger a series of of cytosolic components including mitochondria via autoph-
reactions in cardiac cells leading to measurable changes. Mild agy provides amino acids and lipid substrates for intermedi-
injuries can be repaired. However, severe injuries will lead to ate metabolism.
cell death in the modes of apoptosis and necrosis. If the cell
survives the insults, structural and functional adaptations will
take place. Cardiac Hypertrophy and Heart Failure
Apoptosis is an important mode of myocardial cell loss Adaptive and Maladaptive Responses—Myocardial
that has been demonstrated in heart failure and myocardial adaptation refers to the general process by which the ven-
infarction patients. Necrosis is also important in patients with tricular myocardium changes in structure and function. This
 CHAPTER 18 Toxic Responses of the Heart and Vascular System oUF,

process is often referred to as “remodeling.” In response to Alterations of biochemical reactions in the myocardium are
pathologic stimuli, such as exposure to environmental toxi- often seen soon after exposure to environmental toxicants.
cants, myocardial remodeling is adaptive in the short term, These include alterations in ionic homeostasis, such as changes
but is maladaptive in the long term, and often results in fur- in intracellular calcium concentrations, which occur in most
ther myocardial dysfunction. The central featureofmyocardial exposures to environmental toxicants. Aberrant energy metab-
remodeling is an increase in myocardial mass associated with a olism is another early response to environmental toxicants in
change in the shape of the ventricle. the heart, resulting in decreased production and/or enhanced
At the cellular level, the increase in myocardial mass is consumption of ATP. Alterations in enzymatic reactions are
reflected by cardiac myocyte hypertrophy, which is character- also often observed in cardiac toxic responses.
ized by enhanced protein synthesis, heightened organization Physiologic alterations occur both as early responses to
of the sarcomere, and the eventual increase in cell size. At the environmental toxicants and as subsequent events in the late
molecular level, the phenotypic changes in cardiac myocytes development of cardiomyopathy. The most obvious myocardial
are associated with reintroduction of the so-called fetal gene dysfunction that occurs in the early responses to toxicants is
program, characterized by the patterns of gene expression cardiac arrhythmia. Arrhythmia often results from the changes
mimicking those seen during embryonic development. These in intracellular calcium concentrations and other biochemical
cellular and molecular changes are observed in both adaptive alterations, leading to miscommunication between cells and
and maladaptive responses, thus distinguishing adaptive from misconduction of electricity.
maladaptive responses is difficult. Changes in myocardial morphology take place when
There are both physiologic hypertrophy and pathologic extensive toxic insults are imposed on the heart and/or toxic
hypertrophy of the heart. Physiologic hypertrophy is consid- exposures persist. Cardiac hypertrophy is often observed
ered an adaptive response, which is an adjustment of cardiac as a consequence of long-term toxic insults. From cardiac
function for an increased demand of cardiac output. One hypertrophy to heart failure, activation of compensatory
example of adaptive hypertrophy is the increase in cardiac mass mechanisms, including the sympathetic nervous system and
in response to exercise. The heart often increases its mass in the renin-angiotensin system, occurs. The compensatory
response to toxicologic stresses, but this is generally viewed as response in turn activates counter-regulatory mechanisms
maladaptive. An important distinction between adaptive and such as upregulation of ANP expression and increases in
maladaptive hypertrophy is whether the hypertrophy is neces- cytokines, such as TNF-a production. Extensive biochemi-
sary for the compensatory function of the heart under physi- cal, physiologic, and molecular changes result in myocardial
ologic and pathologic stress conditions. Cardiac hypertrophy remodeling and remarkable cell death, ultimately leading to
in response to extrinsic and intrinsic stresses is not a compen- heart failure.
satory response and actually increases the risk for malignant
arrhythmia and heart failure.
QT Prolongation and Sudden
Hypertrophic Signaling Pathways—Extrinsic and intrin- Cardiac Death
sic stresses activate signaling transduction pathways leading A simple definition for QT prolongation is that the length
to fetal gene program activation, enhanced protein synthesis of QT interval observed from a typical electrocardiogram
of adult cardiomyocytes, and the eventual hypertrophic phe- is prolonged. Clinically, long QT syndrome is defined when
notype. The signaling pathways include several components: the QT interval is longer than 460 ms. However, torsades de
G-protein-coupled receptors, protein kinases including MAPK, pointes (TdP) occurs with an average increase in QT inter-
PKC, and AMPK, calcium and calcineurin, and phosphoinosit- val by approximately 200 ms (a normal QT interval is about
ide 3-kinase (PI3K)/glycogen synthase kinase 38 (GSK3), and 300 ms). In general, the long QT syndrome can be divided
transcription factors. Activation of each of the components is into two classes: congenital and acquired. Congenital long
sufficient to induce myocardial hypertrophic growth. These QT syndrome is rare and acquired is the major concern
components also affect each other through cross-talk. of drug cardiac toxicity in pharmaceutical discovery and
development.
Transition from Cardiac Hypertrophy to Heart
Failure—The critical cellular event of the transition from Molecular Basis of QT Prolongation—The longer QT
cardiac hypertrophy to heart failure is myocardial apopto- interval on the electrocardiogram is caused by prolongation
sis triggered by inflammatory cytokines, such as TNF-a. of the action potential of ventricular myocytes. The duration
This transition can also be triggered by neurohormonal fac- of the QT interval is related to the length of the ventricular
tors, such as atrial natriuretic peptide (ANP), which leads to action potentials. A reduction in net outward current and/or
dilated cardiomyopathy and deterioration of cardiac function. an increase in inward current are potential contributors to the
Toxicologic exposures may cause dilated cardiomyopathy prolongation of cardiac action potential, thereby QT prolon-
or heart failure without an intermediate hypertrophic stage. gation on the electrocardiogram. Although many channels
Myocardial cell death also plays an essential role in direct are potentially involved in the prolongation of the cardiac
cardiac dilation pathogenesis. action potential, current studies have identified sodium inward
278 UNIT 4 Target Organ Toxicity

channels and potassium outward channels (Jk, and I,) as Myocardial Ischemic Injury—Acute myocardial ischemia
important players in the plateau phase (phase 2) ofthe cardiac can cause immediate arrhythmia due to disturbance in ionic
action potential. homeostasis. Acute ischemia can also induce myocardial
infarction that can lead to the block of cardiac conductance.
Torsade De Pointes and Sudden Cardiac Death—The After the myocardial infarction, the areas separated by the
trigger for arrhythmia in the long QT syndrome is believed scar tissue would be uncoupled, making the differences in
to be spontaneous secondary depolarization that arises dur- the duration of action potential of myocardial cells in different
ing or just following the plateau of the action potential. This regions apparent.
small action potential is the so-called “early afterdepolariza-
tion.” When the spontaneous depolarization is accompanied Cardiac Hypertrophy—The normal distribution of Purkinje
by a marked increase in dispersion of repolarization, the like- fibers in the myocardium is proportional to the mass of the
lihood to trigger an arrhythmia is increased. Once triggered, heart. Cardiac hypertrophy would lead to unbalanced distribu-
the arrhythmia is maintained by a regenerative circuit ofelec- tion of Purkinje fibers in the remodeling heart. The conduction
trical activity around relatively inexcitable tissue, a phenom- of pacemaker potentials would thus be interrupted.
enon knownas reentry. The development of multiple reentrant
circuits within the heart causes ventricular arrhythmia, or
TdP, leading to sudden cardiac death. Drugs causing TdP are Myocardial Fibrosis—Dilated cardiomyopathy in alcoholics
considered severe cardiac toxic agents. Several drugs that were often involves myocardial fibrosis, which simulates the effect of
removed from the market due to their TdP effect include the myocardial infarction on the electrical conduction in the heart
cyclooxygenase-2 (COX-2) inhibitors rofecoxib (Vioxx) and and block of cardiac conductance.
valdecoxib (Bextra).
Heart Failure—Most individuals with failing hearts die
Parameters Affecting QT Prolongation and suddenly of cardiac arrhythmias. In human heart failure,
Torsadogenesis—Many factors affect the clinical manifes- selective down-regulation of two potassium channels, /,,, and
tations of QT prolongation and torsadogenesis. Genetic poly- Ix;, has been shown to be involved in action potential pro-
morphisms and female gender are two distinct risk factors. The longation. The J,,, current is involved in phase 1 ofthe action
mechanism of the polymorphisms and the rationale for high potential and opposes the depolarization. The increase in
susceptibility of females to QT prolongation and torsadogenesis depolarization may be adaptive in the short term because
are yet to be determined. it provides more time for excitation—contraction coupling,
mitigating the decrease in cardiac output. However, down-
Drugs and Environmental Toxicants—Drug-induced QT regulation of potassium channels becomes maladaptive in
prolongation is a major acquired long QT syndrome. Selective the long term because it predisposes the individual to early
blockers of potassium channels, including the so-called afterdepolarization, inhomogeneous repolarization, and
class III antiarrhythmic drugs, have been developed for the polymorphic ventricular tachycardia.
treatment of various atrial arrhythmias. Environmental
exposure to particulate matter in air is a risk factor for QT
prolongation in the elderly, children, and individuals with Biomarkers for Cardiac Toxicity
compromised hearts.
Myocardial injury can be divided into two major classes: struc-
tural and nonstructural injuries. The structural damage of the
Disturbances in Ion Homeostasis—Hypokalemia in combi- heart includes cell death and the associated histopathologic
nation with torsadogenic drugs is a recognized risk factor for changes such as myocardial infarction. Functional deficits
QT prolongation and TdP. It is also known that sodium supple- often accompany the structural injury. Nonstructural dam-
mentation can diminish the long QT syndrome due to the gain- age represents functional deficits without apparent structural
of-function mutations in sodium channels. Stress-induced alterations. Myocardial structural changes and functional
Ca’* overload in myocardial cells increases the likelihood alterations can be indirectly measured by echocardiography
of arrhythmia. and electrocardiogram in combination with stress testing.
These measurements can be considered in a broad sense as
Abnormal Gap Junction—Gap junction-mediated intercel- biomarkers. However, in clinical practice and experimental
lular communication is essential in the propagation of electri- approach, biomarkers are referred to as indexes of myocardial
cal impulses in the heart. Under normal conditions, the gap injury measured from blood samples. The fundamental prin-
junction electrotonic current flow attenuates the differences ciple of the biomarkers is that molecules that are released from
in action potential duration of myocardial cells. Toxicologic the myocardium under various injury conditions are readily
exposures cause damage to constituents of gap junctions, lead- detectable from blood samples. Biomarkers that are currently
ing to disruption ofelectrotonic cell-to-cell coupling. available in a clinical setting are listed in Table 18-1.
 CHAPTER 18 Toxic Responses ofthe Heart and Vascular System 279

TABLE 18-1 Biomarkers for cardiac toxicity. The pharmaceutical chemicals that cause cardiac toxic
responses can be simply classified as drugs that are used
: - Proposed Cardiac _ to treat cardiac disease, and others that are used to treat
oe Tissue Abnormality Indicated
_ Biomarker Location _byElevatedLevels noncardiac disease. For drugs used to treat cardiac disease,
cardiac toxicity is often produced by overexpression of the
Creatine kinase
principal pharmaceutical effects. Although overdosing
CK-MM Skeletal muscle, —
myocardium — of these drugs can be a major factor for untoward effects,
CK-BB Brain, kidney cardiac toxicity is often inevitable for this group of drugs.
CK-MB Myocardium Acute myocardial infarction; Table 18-2 summarizes key pharmaceutical agents with their
peak values observed prominent cardiotoxic effects and proposed mechanisms
18-24 h after infarction
of toxicity.
Myoglobin Allmuscle types, | Acute myocardial infarction; Drugs used to treat cardiac disease such as digitalis, quini-
including peak values observed 1-4h dine, and procainamide often cause acute cardiac toxicity in
myocardium after infarction
the form of arrhythmia, which is reversible upon cessation
B-type Ventricular Volume pressure overload; of their use. Other cardiac drugs may cause cardiotoxicity by
natriuretic myocardium ventricular wall tension; mechanisms different from that of the therapeutic action. For
peptide (BNP) chronic heart failure
instance, catecholamines may cause cardiac toxicity through
C-reactive Liver Systemic and vascular oxidative stress, rather than by their pharmaceutical action on
protein (CRP) inflammation the sympathetic nervous system.
The other category is noncardiac drugs that produce cardiac
Cardiac Cardiomyocytes Irreversible myocardial injury
troponins (i.e., myocardial infarction) toxicity. For instance, anthracyclines, such as adriamycin, are
effective anticancer drugs, but their ability to produce severe
cardiac toxicity limits their use in cancer patients.
CARDIAC TOXIC CHEMICALS
Many substances can cause cardiac toxic responses directly or Natural Products
indirectly. However, only chemicals that primarily act on the Natural products include naturally occurring catecholamines,
heart or whose cardiac toxicity is the primary concern should hormones, and cytokines, as well as animal and plant toxins.
be categorized as cardiotoxic chemicals. Many of these products have been shown to cause cardiac toxic
responses. It is difficult to define whether or not the cardiac
toxicity results directly from the action of these products in
Alcohol and Alcoholic Cardiomyopathy vivo, although these products cause deleterious effects on cul-
Clinically, the most recognized toxicologic cardiomyopathy is tured cardiomyocytes. However, Table 18-3 summarizes the
often referred to as alcoholic cardiomyopathy (ACM), which cardiotoxicity of various naturally occurring substances, and
is characterized by an increase in myocardial mass, dilation proposed mechanisms of toxicity.
of the ventricles, wall thinning, ventricular dysfunction, and
heart failure. While ACM has been recognized for a long time,
its pathogenesis incompletely understood. However, the dura- Environmental Pollutants and
tion of heavy alcohol use in patients is a critical factor. Clinical Industrial Chemicals
data have shown that ACM typically is seen after a long term of
There are many chemicals classified in this category that cause
consistent consumption of at least 80 g of alcohol per day. Also,
cardiac toxicity. Metals and metalloids can be found both in
a combination of multiple factors is involved, including malnu-
environmental pollutants and industrial chemicals. Some
trition, cigarette smoking, systemic hypertension, and beverage
heavy metals, such as cadmium, block calcium channels that
additives, in addition to a long-term consumption of alcohol in
affect cardiac rhythm leading to arrhythmia, others such as
the ACM patients. The generation of reactive oxidative metabo-
arsenic have high affinity for sulfhydryl groups, and inter-
lites from the biotransformation of ethanol has been suggested
fere with sulfhydryl-containing proteins, such as receptors,
to be a major contributing factor for ACM, because these
regulatory proteins, and transporters. During the last decade,
metabolites lead to lipid peroxidation of cardiac myocytes or
epidemiologic and experimental studies have identified an
oxidation of cytosolic and membraneous protein thiols.
association of air pollution of particulate matter and cardiac
-
toxicity; however, mechanistic insights into cardiac toxic-
ity induced by particulate matter remain elusive. Table 18-4
Pharmaceutical Chemicals provides a summary of selected industrial agents with their
Cardiotoxicity of pharmaceutical chemicals is a major prominent cardiotoxic effects and proposed mechanisms of
problem in drug development and their clinical application. cardiotoxicity.
280 UNIT 4 Target Organ Toxicity

TABLE 18-2 Cardiotoxicity of key pharmaceutical agents.

Facesbe eg se a ag a a
Agents Cardiotoxic Manifestations Proposed Mechanisms of Cardiotoxicity
EHOISIMS tins SRS See ORCS OS OS A
Ethanol | Conductivity (acute) Acetaldehyde (metabolite)
Cardiomyopathy (chronic) Altered [Ca?*]; homeostasis
Oxidative stress
Mitochondrial injury

Antiarrhythmic drugs

Class | (disopyramide, encainide, flecainide, lidocaine, mexiletine, { Conduction velocity Nat channel blockade
moricizine, phenytoin, procainamide, propafenone, quinidine, Proarrhythmogenic
tocainide)

Class Il (acebutolol, esmolol, propranolol, sotalol) Bradycardia, heart block B-Adrenergic receptor blockade

Class Ill (amiodarone, bretylium, dofetilide, ibutilide, quinidine, T Action potential duration K+ channel blockade
sotalol) QTc interval prolongation
Proarrhythmogenic

Class IV (diltiazem, verapamil) { AV conduction Ca?* channel blockade

Negative inotropic effect
Negative chronotropic effect
Bradycardia

Inotropic drugs and related agents

Cardiac glycosides (digoxin, digitoxin) Action potential duration Inhibition of Na*,K*-ATPase, {[Ca?*],
AV conduction
Parasympathomimetic (low doses)
Sympathomimetic (high doses)

Ca?*-sensitizing agents (adibendan, | Diastolic function? | Ca** sensitivity

levosimendan, pimobendan) Proarrhythmogenic Inhibition of phosphodiesterase

Other Ca?*-sensitizing agents (allopurinol, oxypurinol) ? Inhibition of xanthine oxidase

Catecholamines (dobutamine, epinephrine, isoproterenol, Tachycardia B,-Adrenergic receptor activation

norepinephrine) Cardiac myocyte death Coronary vasoconstriction
Mitochondrial dysfunction
J [Ca**],
Oxidative stress
Apoptosis

Bronchodilators (albuterol, bitolterol, fenoterol, formeterol, Tachycardia Nonselective activation of 8,-adrenergic

metaproterenol, pirbuterol, procaterol, salmeterol, terbutaline) receptors

Nasal decongestants (ephedrine, ephedrine alkaloids, ma huang, Tachycardia Nonselective activation of a,-adrenergic
phenylephrine, phenylpropanolamine, pseudoephedrine) receptors

Appetite suppressants (amphetamines, fenfluramine, phentermine) Tachycardia { Serotonin?

Pulmonary hypertension Na* channel blockade?
Valvular disease

Antineoplastic drugs

Anthracyclines (daunorubicin, doxorubicin, epirubicin) Cardiomyopathy Altered [Ca2*], homeostasis

Heart failure Oxidative stress
Mitochondrial injury
Apoptosis

5-Fluorouracil Proarrhythmogenic Coronary vasospasm?

Cyclophosphamide Cardiac myocyte death 4-Hydroxycyclophosphamide (metabolite)

Altered ion homeostasis

Antibacterial drugs

Aminoglycosides (amikacin, gentamicin, kanamycin, netilmicin, Negative inotropic effect 1 [Ca?*],

streptomycin, tobramycin)

Macrolides (azithromycin, clarithromycin, dirithromycin, { Action potential duration K* channel blockade

erythromycin) QTc interval prolongation
Proarrhythmogenic
 CHAPTER 18 Toxic Responses of the Heart and Vascular System 281

TABLE 18-2 Cardiotoxicity of key pharmaceutical agents. (Continued)

Cardiotoxic Manifestations Proposed Mechanisms of Cardiotoxicity

Fluoroquinolones (grepafloxacin, moxifloxacin, | Action potential duration K* channel blockade

sparfloxacin) QTc interval prolongation
Proarrhythmogenic

Tetracycline Negative inotropic effect J [Ca**],

Chloramphenicol Negative inotropic effect J [Ca**];
Antifungal drugs

Amphotericin B Negative inotropic effect Ca** channel blockade?

Na* channel blockade?
| Membrane permeability?

Flucytosine Proarrhythmogenic 5-fluorouracil metabolite

Cardiac arrest Coronary vasospasm?

Antiviral drugs

Nucleoside analog reverse transcriptase inhibitors Cardiomyopathy Mitochondrial injury

(stavudine, zalcitabine, zidovudine) Inhibition of mitochondrial DNA polymerase
Inhibition of mitochondrial DNA synthesis
Inhibition of mitochondrial ATP synthesis

Centrally acting drugs

Tricyclic antidepressants (amitriptyline, desipramine, ST segment elevation Altered ion homeostasis

doxepin, imipramine, protriptyline) QTc interval prolongation Ca** channel blockade
Proarrhythmogenic Na* channel blockade
Cardiac arrest Kt channel blockade

Selective serotonin reuptake inhibitors (fluoxetine) Bradycardia Ca** channel blockade

Atrial fibrillation Na* channel blockade

Phenothiazine antipsychotic drugs (chlorpromazine, Anticholinergic effects Ca?* channel blockade?

thioridazine) Negative inotropic effect
QTc interval prolongation
PR interval prolongation

Other antipsychotic drugs (clozapine) Blunting of Twaves

ST segment depression

General inhalational anesthetics (enflurane, desflurane, Negative inotropic effect Ca?* channel blockade
halothane, isoflurane, methoxyflurane, sevoflurane) Decreased cardiac output Altered Ca** homeostasis
Proarrhythmogenic B-Adrenergic receptor sensitization

Other general anesthetics (propofol) Negative inotropic effect Ca** channel blockade
Altered Ca** homeostasis
B-Adrenergic receptor sensitization

Local anesthetics

Cocaine Sympathomimetic effects Na* channel blockade

Ischemia/myocardial Coronary vasospasm, infarction
Proarrhythmogenic Altered Ca** homeostasis
Cardiac arrest Mitochondrial injury
Cardiac myocyte death Oxidative stress
Apoptosis

Other local anesthetics (bupivacaine, etidocaine, Decreased excitability Na* channel blockade
lidocaine, procainamide) | Conduction velocity
Proarrhythmogenic

Antihistamines (astemizole, terfenadine) { Action potential duration K* channel blockade

-
QTc interval prolongation
Proarrhythmogenic

Immunosuppressants (rapamycin, tacrolimus) Cardiomyopathy Altered Ca** homeostasis

Heart failure

(continued)
282 UNIT 4 Target Organ Toxicity

TABLE 18-2 Cardiotoxicity of key pharmaceutical agents. (Continued)

Agents Cardiotoxic Manifestations Proposed Mechanisms of Cardiotoxicity

Miscellaneous drugs

Cisapride { Action potential duration K* channel blockade

QTc interval prolongation
Proarrhythmogenic

Methylxanthines (theophylline) | Cardiac output Altered Ca?* homeostasis

Tachycardia Inhibition of phosphodiesterase
Proarrhythmogenic

Sildenafll ? Inhibition of phosphodiesterase

Radiocontrast agents (diatrizoate meglumine, iohexol) Proarrhythmogenic Apoptosis?

Cardiac arrest

TABLE 18-3 Cardiotoxicity of naturally occurring substances.

Agents Cardiotoxic Manifestations Proposed Mechanisms of Cardiotoxicity
ee

Estrogens
Natural estrogens (178-estradiol, estrone, estriol) QTc interval prolongation? Gender differences in K* channel expression?
Synthetic estrogens (diethylstilbestrol, equilin, ethinyl Cardioprotection? Antiapoptotic effécts?
estradiol, mestranol, quinestrol)
Nonsteroidal estrogens (bisphenol A, diethylstilbestrol, Antioxidant activity?
DDT, genistein) T Na*,K*-ATPase activity?
Ca?* channel blockade?
Other mechanisms?

Progestins (desogestrel, hydroxyprogesterone, Enhanced toxicity of cocaine? Mechanisms?

medroxyprogesterone, norethindrone, norethynodrel,
norgestimate, norgestrel, progesterone)

Androgens
Natural androgens (androstenedione, dehydroepiandrosterone, Myocardial infarction Mitochondrial injury?
dihydrotestosterone, testosterone) Cardiac hypertrophy Altered Ca?* homeostasis?
Synthetic androgens (boldenone, danazol, fluoxymesterone, Other mechanisms?
methandrostenolone, methenolone, methyltestosterone,
nandrolone, oxandrolone, oxymetholone, stanozolol)

Glucocorticoids
Natural glucocorticoids (corticosterone, cortisone, hydrocortisone) Cardiac hypertrophy Increased collagen expression
Cardiac fibrosis Other mechanisms?
Synthetic glucocorticoids (e.g., dexamethasone,
methylprednisolone, prednisolone, prednisone)
Mineralocorticoids (aldosterone) Cardiac fibrosis Increased collagen expression
Heart failure Other mechanisms?

Thyroid hormones (thyroxine, triiodothyronine) Tachycardia Altered Ca2* homeostasis

Positive inotropic effect
Increased cardiac output
Cardiac hypertrophy
Proarrhythmogenic

Cytokines
Interleukin-13 Negative inotropic effect T Nitric oxide synthase expression
Cardiac myocyte death Apoptosis
Interleukin-2 Negative inotropic effect T Nitric oxide synthase expression
Interleukin-6 Negative inotropic effect T Nitric oxide synthase expression
Interferon-y Cardiomyopathy T Nitric oxide synthase expression
Tumor necrosis factor-a Proarrhythmogenic Altered ion homeostasis
Negative inotropic effect T Nitric oxide synthase expression
Cardiac myocyte death T Sphingosine production
| Ca?* transients
Apoptosis
 CHAPTER 18 Toxic Responses ofthe Heart and Vascular System 283

TABLE 18-4 Cardiotoxicity of selected industrial agents.

Agents Cardiotoxic Manifestations Proposed Mechanisms of Cardiotoxicity

Solvents
Toluene (paint products) Proarrhythmogenic | Parasympathetic activity
T Adrenergic sensitivity
Altered ion homeostasis

Halogenated hydrocarbons (carbon tetrachloride, chloroform, Proarrhythmogenic | Parasympathetic activity

chloropentafluoroethane, 1,2-dibromotetra-fluoromethane, Negative inotropic effect T Adrenergic sensitivity
dichlorodifluoromethane, cis-dichloroethylene, trans- Decreased cardiac output Altered ion homeostasis
dichloroethylene, dichlortetrafluorethane, difluoroethane, Altered coronary blood flow
ethyl bromide, ethyl chloride, fluorocarbon 502, heptafluoro-
1-iodo-propane, 1,2-hexafluoroethane, isopropyl chloride,
methyl bromide, methyl chloride, methylene chloride,
monochlorodifluoroethane, monochlorodifluoromethane,
octafluorocyclobutane, propyl chloride, 1,1,1-trichloroethane,
trichloroethane, trichloroethylene, trichlorofluoromethane,
trichloromonofluoroethylene, trichlorotrifluoroethane,
trifluoroiodomethane, trifluorobromomethane)

Ketones (e.g., acetone, methyl ethyl ketone) Proarrhythmogenic | Parasympathetic activity

T Adrenergic sensitivity
Altered ion homeostasis

Heavy metals Negative inotropic effect Complex formation

(Cadmium, cobalt, lead) Cardiac hypertrophy Altered Ca?* homeostasis
Proarrhythmogenic
(Barium, lanthanum, manganese, nickel) Proarrhythmogenic Ca** channel blockade

OVERVIEW OF VASCULAR SYSTEM

Vascular Physiology and
Structural Features
The vascular system consists of blood vessels of varying size
and different cellular composition. Blood vessels can be
divided into arterial, venous, and capillary systems. In addi-
tion, the lymphatic system belongs to the vascular system, but
it only carries plasma. The main function of the vascular system
is to provide oxygen and nutrients to and remove carbon diox-
ide and metabolic products from organ systems (Figure 18-7).
In addition, the vascular system is a conduit that delivers hor-
mones and cytokines to target organs. The vascular system also Mesenteric artery
has regulatory functions to manipulate organ system responses
under certain toxicologic conditions. |
Arterial System and Physiologic Function—The arterial
system is composed of the aorta, major arteries, and small arte-
rioles. The aorta and major arteries are thick-walled structures Peritubular Glomeruli
with vascular smooth muscle, elastic, and connective tissues capillaries
(Figure 18-8). Blood flow within the arterial system is initiated FIGURE 18-7 Schematic diagram of vascular supply to
by contraction of the heart and begins at the ascending aorta. selected organs.
The capillary beds are represented by a meshwork
The ascending aorta receives all of the output of the heart with connnecting the arteries (right) with the veins (left); the distribution
the exception of the coronary blood flow. Blood is distributed of the vasculature in several organs (liver, kidney, lung) indicates the
to the organ systems of the body through the major.arteries importance ofthe vascular system in toxicology.
284 UNIT 4 Target Organ Toxicity

Endothelial cells proteins that have not been reabsorbed by the venous system.
In general, in all organ systems with the exception of the CNS,
more fluid is filtered than reabsorbed by the venous system.
Therefore, removal of the excess fluid as well as plasma proteins
that diffuse into the interstitial spaces by the lymphatic system
Tunica intima is essential. All the lymphatics ultimately drain into the vena
cava. Toxic insults to the lymphatic system can lead to elevated
Tunica media interstitial pressures and subsequent edema.

Tunica adventitia
Regulatory Mechanisms of the
FIGURE 18-8 Cross-sectional representation of the vascular Vascular System
wall of large- and medium-size blood vessels. The tunica intima Is
The vascular system includes conduits and microcircula-
composed of endothelial cells, facing the vessel lumen, which rest ona
tion. The mechanisms controlling vascular physiology can be
thin basal lamina. The tunica media consists mainly of vascular smooth
muscle cells interwoven with collagen and elastin. The tunica adventia
divided into neural, hormonal, and local controls that function
is a layer offibroblasts, collagen, elastin, and glycosaminoglycans. in an integrated way as each of the three mechanisms affects
the other two.

Neurohormonal Regulation—Most arteries, arterioles,

that branch from the aorta. All these arteries further branch to venules, and veins, with the exception of those of the external
give rise to smaller arteries and become arterioles that connect genitalia, receive sympathetic innervation only. The catechol-
to capillaries for the delivery of oxygen and nutrients to tar- amine, norepinephrine, is the usual neurotransmitter and
get tissues. The arterioles are composed of a tube of endothelial binding to receptors on vascular smooth muscle cells causes
cells, surrounded by connective tissue basement membrane, a their contraction. Epinephrine is another catecholamine which
single or double layer of vascular smooth muscle cells, and a acts on vascular smooth muscle cells to cause relaxation and
thin outer adventitial layer. The vascular smooth muscle cells vasodilation. In addition, the blood vessels of skeletal muscles
are critical for the regulation of vascular resistance and, there- receive sympathetic cholinergic innervation in addition to
fore, blood pressure. their sympathetic adrenergic innervation, whose activation
leads to vascular smooth muscle relaxation and vasodilation.
Capillaries and Microcirculation—Capillaries directly There are many other hormones that control the vascular sys-
connect to the distal portion of the arterioles serving as the tem, including renin-angiotensin-aldosterone, antidiuretic
communication site between blood and tissues, and constitute hormone (ADH), and atrial natriuretic peptide (ANP).
the major part of the microcirculation where nutrients, water,
gases, hormones, cytokines, and waste products are exchanged The Renin-Angiotensin-Aldosterone System—Renin is released
between blood and tissues. Capillaries are only one cell layer from the kidney in response to reduced arterial pressure and
thick. The passage of molecules through the capillary wall can volume and catalyzes the conversion of a plasma protein angio-
occur both between and through the endothelial cells. Lipid- tensinogen to angiotensin I. Angiotensin I is further con-
soluble molecules such as oxygen and carbon dioxide readily verted to angiotensin II by an angiotensin-converting enzyme.
pass through the endothelial cell membranes. Water-soluble Angiotensin II is a powerful arteriolar vasoconstrictor and
molecules diffuse between endothelial cells. also causes the release of aldosterone from the adrenal cor-
tex. Aldosterone reduces renal sodium excretion, resulting in
Venous System and Physiologic Function—Blood flow retention ofwater and increased blood volume.
in the venous system starts from the thin-walled venules that
have a relatively large surface area facilitating the reabsorption ADH—ADH is a vasoconstrictor released from the posterior
of filtered plasma from the tissue. The venules merge to veins pituitary gland in response to volume-depleting conditions,
and eventually drain into the vena cava, returning blood to the such as hemorrhage. ADH increases water retention by the
heart. The important physiologic function of the venous system kidney, and thus increases blood volume. Atrial natriuretic
is collecting blood from organ systems of the body and return- peptide (ANP), a hormone with actions opposing ADH, is
ing the blood to the heart. Large veins contain vascular smooth released from atrial muscle cells in volume-overload states and
muscle cells which can increase return of blood to the heart by results in increased excretion of sodium and water, thereby
constricting. Xenobiotics can exert adverse effects on the vas- decreasing blood volume.
cular smooth muscle cells and compromise this function.
Local Metabolic Regulation—The local regulation of the
Lymphatic System and Physiologic Function—Lymphatic vascular system is primarily referred to as the control of micro-
vessels are endothelial tubes within tissues. This is a low- circulation. Oxygen is a major regulator of microcirculation
pressure system that collects excess tissue water and plasma which must be replenished constantly from the blood flow.
 CHAPTER 18 Toxic Responses of the Heart and Vascular System 285

Therefore, a change in the metabolic rate of an organ requires a secrete cytokines, such as TGF-, which lead to a cascade of
parallel change in oxygen supply. While vascular smooth mus- signaling transduction and a series of injurious responses
cle cells cannot respond to oxygen tension under normal con- including deposition ofcollagen.
ditions, reduced oxygen tension causes the release of adenine
nucleotides, free adenosine, and Krebs cycle intermediates; all Responses of Smooth Muscle Cells to Toxic Insults—
these cause vasodilation. The consequence of damage to vascular smooth muscle cells
Nitric oxide (NO) is an important mediator of local micro- involves changes in the vascular tone and atherosclerosis.
circulation regulation. NO is generated from arginine by nitric Activation of receptors localized in the plasma membrane of
oxide synthase (NOS), and ultimately leads to relaxation of smooth muscle cells leads to increased intracellular calcium,
vascular smooth muscle cells, suppression of platelet activa- which initiates contraction of the affected vessels. Toxic sub-
tion, and reduction of leukocyte adhesion. stances can also influence calcium homeostasis in other ways,
including disruption of calcium-binding proteins and calcium-
activated proteins.
VASCULAR SYSTEM TOXIC RESPONSES Proliferation and migration of medial smooth muscle cells
are primarily responsible for the formation of sclerosis. Under
Mechanisms ofVascular Toxicity certain circumstances, smooth muscle cells lose most of their
All chemicals, after absorption, contact the vascular system. contractility. In most cases, this transformation is revers-
Vascular endothelial cells are the immediate targets of the chem- ible. This new form of smooth muscle cells synthesizes colla-
icals and are of the most frequent risk for toxic insults. These gen, accumulates low-density lipoproteins, and decreases the
cells are the major component of the microcirculation system. number of myofilaments. This phenotypic transformation of
smooth muscle cells occurs in atherosclerosis.
Responses of Vascular Endothelial Cells to Toxic
Insults—Vascular endothelial cells play a critical role in both Oxidative Stress and Vascular Injury—Both endothelial
vascular protection from toxic insults and triggering detri- and smooth muscle cells are capable of producing ROS and
mental cascade in response to toxic insults. In response to toxic subsequent oxidative injury by enzymatic and nonenzymatic
insults, production of NO and reactive oxygen species (ROS) mechanisms. Enzymes involved in the generation of ROS in
increases in endothelial cells. Substances mimicking agonists vascular cells include amine oxidase, cytochrome P450 mono-
activate the receptors on the endothelial cells and trigger intra- oxygenases, and prostaglandin synthetase. These enzymes use
cellular signaling transduction, leading to activation of nuclear a diversity of substrates to produce ROS. The nonenzymatic
factor kappa-B (NFKB) and MAPK activity. The downstream reaction involves free iron and copper in the circulation sys-
signaling transduction pathways triggered by NFKB, MAPK, tem, which catalyze the Fenton reaction to produce ROS.
NO, and ROS then activate gene expression and regulate post-
translational modification of proteins leading to cytoprotec- Inflammatory Lesions—Inflammatory lesions of the vas-
tive action against toxic insults, or the production of cytokines, cular system, termed vasculitis, are a common response of the
chemokines, and adhesion molecules to protect the circulatory vascular system. The causes of many types of vasculitis are still
system and the affected organ systems. unknown despite much research on the subject. The initial
Angiogenesis is an adaptive response to damages that follow injury to endothelial cells and the release of chemicals from the
toxic insults. Vascular endothelial cells are both central to ini- injured cells are responsible for the initiation of the inflamma-
tiating and promoting the formation of new blood vessels and tory response, including recruitment of inflammatory cells to
essential for blood vessel formation by forming initial tube- the injured site. Cytokines released from the activated inflam-
like structures. Xenobiotics can both promote and suppress matory cells further propagate the inflammatory response
angiogenesis, and the primary target is the vascular endothe- leading to the eventual lesion or vasculitis.
lial cell. Apoptosis is a major mechanism for cell death of the
vascular endothelial cells and mechanisms and molecular sig-
naling pathways leading to apoptosis are basically the same as Toxic Responses of Blood Vessels
described for cardiomyocytes. Hypertension and Hypotension—Vasculature pressure
Lesions to endothelial cells can result in atherosclerosis. change is a major phenotype of vascular injury. Hypertension
Injury to endothelial cells results in increased production of results from excessive constriction of the arterial vasculature
endothelin-1 (ET-1) and increased release of prostacyclins. and/or increased resistance of the microcirculation system.
ET-1 secreted by endothelial cells is a major mediator of vas- However, the primary problem of sustained hypertension is
cular toxicity and also contributes to the pathogenesis of myo- an elevated vascular resistance in all organs. Once hyperten-
cardial disease. ET-1 is a potent vasoconstrictor that plays an sion is established, it becomes a disease of the microvascula-
important role in the maintenance of vascular tone and blood ture, particularly the arteriolar microvasculature. An increased
pressure in healthy subjects. incidence of temporary or, in some cases, permanent closure
Endothelial cells are also involved in the recruitment of of small arterioles is associated with increased resistance of
inflammatory cells to the lesion site. Activated lymphocytes the end organs. The vascular smooth muscle cells become
 6
i)(oe) UNIT 4 Target Organ Toxicity

hypertrophied, and vascular smooth muscle cells become ultimately drains into the vena cava. Xenobiotics can change
exceptionally responsive to norepinephrine. the pressure gradients such that there is even more filtration
Toxic substances may directly or indirectly affect the sympa- than reabsorption than normal. Further, toxic insults to the
thetic nervous system or alter the turnover of catecholamines lymphatic system can lead to elevated interstitial pressures and
in the circulation, resulting in hypertension. However, sus- subsequent tissue edema.
tained hypertension by xenobiotics may involve more compli-
cated metabolic changes in the end organs and thus changes in
microcirculation also take place. For example, chemicals may VASCULAR SYSTEM TOXIC CHEMICALS
enhance the renin-angiotensin system as well as renal toxicity,
Like cardiac toxicants, those that cause vascular toxicity can
which may cause hypertension.
include pharmaceutical chemicals, natural products, and envi-
Hypotension is practically defined as the symptoms caused
ronmental pollutants and industrial chemicals. Although blood
by low blood pressure. Baroreceptors, volume receptors,
vessels are the primary target of these chemicals, some affect
chemoreceptors, and pain receptors are all involved in the
the heart as well. For instance, blood vessels in the heart belong
integrated regulatory action to maintain adequate blood pres-
to the vascular system, so that the toxicity of vascular toxic
sure. During chemical exposure, these mechanisms may be
chemicals may express their toxicity in the form of cardiac toxic
affected individually or jointly resulting in a disturbance in
manifestations. Endothelial cells are major target cells of the
the integration of the regulatory mechanisms. Both transient
chemicals affecting the vascular system, which are also found in
and sustained hypotension can be produced by xenobiotics.
the heart and make a contribution to cardiac toxicity. This same
The most common adverse effect of antihypertensive drugs
principle applies to other organ systems. Due to the distribu-
is hypotension. Other major causes of hypotension include
tion of the vascular system in the end organs, vascular toxicity
hemorrhage and alcohol overdose.
affects the organs in which the vessels are localized and is often
accompanied with functional defects of the organ.
Atherosclerosis—The most frequent vascular structural
injury is atherosclerosis. The classic definition of athero-
sclerotic plaque is a combination of changes in the intima of
Pharmaceutical Chemicals
arteries consisting of local accumulation of lipids, complex
carbohydrates, blood and blood products, fibrous tissue, Sympathomimetic Amines— The sympathomimetic amines,
and calcium deposits. However, some advanced atheroscle- including epinephrine, norepinephrine, dopamine, and isopro-
rotic plaques can invade the media and produce bulging or terenol, can damage the arterial vasculature by various mecha-
enlarged arteries, cellular infiltration, and neovascularization. nisms. Large or repeated doses of catecholamines produce
The primary problem is the mechanical occlusion of the blood toxic effects on the endothelium, such as unusual endothelial
vessels so that blood flow is inadequate for the metabolic cytoarchitecture and atherosclerotic lesions in several animal
demands of the organs. species. Thus, the formation of arteriosclerotic lesions in cer-
Activation of vascular smooth muscle cells is critically tain forms of hypertension may be initiated and/or potentiated
involved in atherosclerosis. Once stimulated, the vascular by high levels of circulating catecholamines.
smooth muscle cells proliferate, migrate to the lesion site,
undergo phenotype transformation and increase the produc- Nicotine—Nicotine is an alkaloid found in various plants
tion of type I and II collagen, dermatan sulfate, proteoglycan, that mimics the actions of acetylcholine at nicotinic recep-
and stromelysins. In addition, the smooth muscle cells produce tors throughout the body. At pharmacologic concentrations,
cytokines including macrophage colony-stimulating factor, nicotine increases heart rate and blood pressure as a result of
TNF-a, and monocyte chemoattractant protein-1. The recruit- stimulation of sympathetic ganglia and the adrenal medulla.
ment of inflammatory cells to the lesion site is the perpetuation Epidemiologic and experimental studies have suggested that
process of atherosclerosis. nicotine is a causative or aggravating factor in myocardial and
cerebral infarction, gangrene, and aneurysm.
Hemorrhage—A direct mechanical injury to blood vessels
causes hemorrhage (i.e., bleeding), while chemical-induced Cocaine—The central actions of cocaine are to increase the
hemorrhages are seen when damage to capillaries takes place. circulating levels of catecholamines and cause a generalized
Toxic effects on blood clotting also increase the probability of state of vasoconstriction. Hypertension and cerebral strokes are
hemorrhage. common vascular complications. In pregnant women, cocaine-
induced vascular changes have been associated with abor-
Edema—Edema is defined as excess fluid in the interstitial tions and abruptio placentae. Cocaine also enhances leukocyte
space. The capillary exchange of fluid is bidirectional, meaning migration across the cerebral vessel wall during inflammatory
the balance between hydrostatic and colloid osmotic pressure conditions. This effect is exerted through a cascade of aug-
can drive fluid both out of and into a capillary. Under normal mented expression of inflammatory cytokines and endothelial
physiologic conditions, more fluid is filtered than reabsorbed. adhesion molecules and may in fact underlie the cerebrovascu-
This excess fluid is removed via the lymphatic system, which lar complications associated with cocaine abuse.
 CHAPTER 18 Toxic Responses of the Heart and Vascular System 287

Psychotropic Agents—Trifluoperazine and chlorproma- tumors in the aorta and large arteries of mice when adminis-
zine have been shown to cause intracellular cholesterol accu- tered over the life span of the animals.
mulation in cultured cells of the aortic intima. Aside from the
atherogenic effects, postural hypotension has been identified T-2 Toxin—Trichothecene mycotoxins, commonly classified
as the most common cardiovascular side effect of tricyclic as tetracyclic sesquiterpenes, are naturally occurring cytotoxic
antidepressants. metabolites of Fusarium species. These mycotoxins, including
T-2 toxin, are major contaminants of foods and animal feeds
Antineoplastic Agents—The vasculotoxic responses elic- and may cause illness in animals and humans. Intravenous
ited by antineoplastic drugs range from asymptomatic arterial infusion of T-2 toxin in rats causes an initial decrease in heart
lesions to thrombotic microangiopathy. Pulmonary venooc- rate and blood pressure, followed by tachycardia and hyper-
clusive disease has been reported after the administration of tension and finally by bradycardia and hypotension. Acute
various drugs, including 5-fluorouracil, doxorubicin, and mito- T-2 toxin exposure causes extensive destruction of myocardial
mycin. Cyclophosphamide causes cerebrovascular and viscero- capillaries, while repeated dosing promotes thickening oflarge
vascular lesions, resulting in hemorrhages. coronary arteries.

Analgesics and Nonsteroidal Anti-inflammatory Vitamin D—The toxic effects of vitamin D may be related
Agents— Aspirin can produce endothelial damage as part of to its structural similarity to 25-hydroxycholesterol, a potent
a pattern of gastric erosion. Regular use of analgesics contain- vascular toxin. The manifestations of vitamin D hypervitamin-
ing phenacetin has been associated with an increased risk of osis include medial degeneration, calcification of the coronary
hypertension and cardiovascular morbidity. NSAIDs may arteries, and smooth muscle cell proliferation in laboratory
induce glomerular and vascular renal lesions. animals.

Oral Contraceptives—Oral contraceptive steroids can pro- 8-Amyloid—Accumulation of 8-amyloid is a major lesion
duce thromboembolic disorders. Epidemiologic studies have in the brain of Alzheimer’s patients. Studies have shown that
shown that oral contraceptive users have an increased risk of administration of 8-amyloid produces extensive vascular dis-
MI relative to nonusers, a correlation that is markedly exacer- ruption, including endothelial and smooth muscle damage,
bated by smoking, and increased risk of cerebral thrombosis, and adhesion and migration of leukocytes across arteries and
hemorrhage, venous thrombosis, and pulmonary embolism. venules. Most importantly, the vascular actions of 8-amyloid
appear to be distinct from the neurotoxic properties of the
peptide. It appears that vascular toxicity of B-amyloid makes
Natural Products contributions to Alzheimer’s dementia.
Natural products that cause vascular toxicity include those
discussed for drugs causing cardiotoxicity. In addition, many
Environmental Pollutants and
other drugs also cause vascular lesions and toxicity such as
bacterial endotoxins and homocysteine, which have unique Industrial Chemicals
vascular toxic effects. The environmental pollutants and industrial chemicals dis-
cussed in the cardiotoxicity section all have toxic effects on
Bacterial Endotoxins—Bacterial endotoxins are potent the vascular system. The cardiac effect of some of these agents
toxic agents to the vascular system. These toxins are known and pollutants actually may result primarily from the vascu-
to cause thickening of endothelial cells and the formation of lar effect. The by-products of vascular tissue damage or the
fibrin thrombi in small veins. The terminal phase of the effects secreted substances, such as cytokines derived from vascular
of endotoxin on the systemic vasculature results in marked injury, can affect the heart either directly because of the vascu-
hypotension. The action of these agents is somehow related lar system in the heart or indirectly through blood circulation.
to oxidative stress mechanisms, as evidenced by the ability of
vitamin E to prevent some of the toxin-induced damage. Carbon Monoxide—Carbon monoxide induces focal inti-
mal damage and edema in laboratory animals at a concen-
Homocysteine— Moderately elevated levels of homocysteine tration (180 ppm) to which humans may be exposed from
have been associated with atherosclerosis and venous throm- environmental sources such as automobile exhaust, tobacco
bosis. Toxicity may involve oxidative injury to vascular endo- smoke, and fossil fuels. Short-term exposure to carbon mon-
thelial and/or smooth muscle cells, leading to deregulation of oxide is associated with direct damage to vascular endo-
vascular smooth muscle growth, synthesis and deposition of thelial and smooth muscle cells. The toxic effects of carbon
matrix proteins, and adverse effects on anticoagulant systems. monoxide have been attributed to its reversible interaction
with hemoglobin. As a result of this interaction, carboxyhe-
Hydrazinobenzoic Acid—This nitrogen-nitrogen bonded moglobin decreases the oxygen-carrying capacity of blood,
chemical is present in the cultivated mushroom Agaricus eventually leading to functional anemia. In addition, carbon
bisporus. This hydrazine derivative causes smooth muscle cell monoxide interacts with cellular proteins such as myoglobin
288 UNIT 4 Target Organ Toxicity

and cytochrome c oxidase and elicits a direct vasodilatory with and block calcium channels. Intracellular calcium-binding
response ofthe coronary circulation. proteins, such as calmodulin, are biologically relevant targets of
heavy metals, including cadmium, mercury, and lead, although
Carbon Disulfide—Carbon disulfide (dithiocarbonic anhy- the contribution of this mechanism to the toxic effects of metals
dride) occurs in coal tar and crude petroleum and is commonly is not fully understood.
used in the manufacture of rayon and soil disinfectants. This
chemical has been identified as an atherogenic agent in labora- Aromatic Hydrocarbons— Aromatic hydrocarbons, includ-
tory animals. The mechanism for carbon disulfide-atheroma ing polycyclic aromatic hydrocarbons and polychlorinated
production may involve direct injury to the endothelium cou- dibenzopdioxins, are persistent toxic environmental contami-
pled with hypothyroidism, because thiocarbamate (thiourea), nants. Aromatic hydrocarbons have been identified as vascular
a potent antithyroid substance, is a principal urinary metab- toxins that can initiate and/or promote the atherogenic process
olite of carbon disulfide. Carbon disulfide also modifies low- in experimental animals. The atherogenic effect is associated
density lipoprotein in vitro and enhances arterial fatty deposits with cytochrome P450-mediated conversion of the parent
induced by a high-fat diet in mice. compound to toxic metabolic intermediates, but aromatic
hydrocarbons can also initiate the atherogenic process.
1,3-Butadiene—Studies have shown that 1,3-butadiene,
a chemical used in the production of styrene—butadiene,
Particulate Air Pollution—Recent epidemiologic studies
increases the incidence of cardiac hemangiosarcomas, which
have provided a strong body of evidence that elevated lev-
are tumors of endothelial origin. Although hemangiosarco-
els of ambient particulate air pollution are associated with
mas have also been observed in the liver, lung, and kidney,
increased cardiovascular and respiratory morbidity and mor-
cardiac tumors are a major cause of death in animals exposed
tality. Vascular effects of inhaled ambient particles include
to this chemical. The toxic effects of 1,3-butadiene depend on
endothelial dysfunction and promotion of atherosclerotic
its metabolic activation by cytochrome P450 to toxic epoxide
lesions. Importantly, these lesions lead to release or secretion
metabolites.
of cytokines and chemokines, worsening cardiac complica-
tions (discussed previously).
Metals and Metalloids—The vascular toxicity of food- and
water-borne elements (selenium, chromium, copper, zinc,
cadmium, lead, and mercury) as well as airborne elements
(vanadium and lead) involves reactions of metals with sulfhy- BIBLIOGRAPHY
dryl, carboxyl, or phosphate groups. Metals such as cobalt, mag- Acosta D (ed.): Cardiovascular Toxicology, 4th ed. New York: Informa
nesium, manganese, nickel, cadmium, and lead also interact Healthcare, 2008.
 CHAPTER 18 Toxic Responses of the Heart and Vascular System 289

QUESTIONS

In which of the following locations would one NOT find Which of the following statements regarding the cardio-
spontaneous depolarization? toxic manifestations of ethanol consumption is FALSE?
a. SAnode. a. Acute ethanol toxicity causes decreased conductivity.
b. myocardium. b. Chronic alcohol consumption is associated with
c. AV node. arrhythmias.
d. bundle of His. c. Acute ethanol toxicity causes an increased threshold
e. Purkinje fibers. for ventricular fibrillation.
Chronic ethanol toxicity can result in cardiomyopathy.
Which of the following scenarios would increase contrac- e. Acetaldehyde is a mediator of cardiotoxicity.
tility of the myocardium?
a. increased activity of the Na*/K*-ATPase. Cardiac glycosides:
b. increased activity of sacroplasmic reticulum Ca’* a. increase the activity of the Na*/K*-ATPase.
ATPase. b. make the resting membrane potential more negative.
c. decreased activity of sacroplasmic reticulum Ca’* c. can have sympathomimetic and parasympathomi-
ATPase. metic effects.
decreased intracellular calcium levels. decrease ventricular contractility.
e. increased intracellular K* levels. e. increase AV conduction.

All of the following statements regarding abnormal Which of the following is NOT a common cardiotoxic
cardiac function are true EXCEPT: manifestation of cocaine abuse?
a. Ventricular arrhythmias are generally more severe parasympathomimetic effects.
than atrial arrhythmias. myocardial infarction.
b. Ventricular hypertrophy is a common cause of cardiac myocyte death.
ventricular arrhythmias. ventricular fibrillation.
c. Coronary artery atherosclerosis is a major cause of ge
iS
fon
To ischemia.
ischemic heart disease.
Right-sided heart failure results in pulmonary edema. Using high doses of anabolic—androgenic steroids is NOT
e. Tachycardia is classified as a rapid resting heart rate likely associated with which ofthe following?
(> 100 beats/min). an increase in LDL.
cardiac hypertrophy.
Ion balance is very important in maintaining a normal myocardial infarction.
cardiac rhythm. Which of the following statements is increased nitric oxide synthase expression.
TRUE? ts
ee
yg)a decrease in HDL.
a. Blockade of K* channels decreases the duration of the
action potential. 10. Which of the following is NOT a common mechanism of
b. Blockade of Ca** channels has a positive inotropic vascular toxicity?
effect. membrane disruption.
c. Inhibition of Na* channels increases conduction oxidative stress.
velocity. bioactivation of protoxicants.
Blockage of the Na*/K*-ATPase increases contractility. reduction and accumulation of LDL in endothelium.
e. Calciumis transported into the cell viaa Ca**-ATPase. Oe
CRS
Cue accumulation oftoxin in vascular cells.

Which of the following is most likely NOT a cause of

myocardial reperfusion injury?
cellular pH fluctuations.
damage to the sarcolemma.
generation of toxic oxygen radicals.
Ca’* overload.
ees inhibition of the electron transport chain.
 Cy) HeprAt
Ps ale oe SR,

Toxic Responses
of the Skin
Robert H. Rice and Theodora M. Mauro

SKIN AS A BARRIER Photosensitivity

Skin Histology Phototoxicity
Percutaneous Absorption Photoallergy
Transdermal Drug Delivery ACNE
Measurements of Penetration
fe ; Chloracne
Biotransformation
PIGMENTARY DISTURBANCES
CONTACT DERMATITIS
Irritant Dermatitis URTICARIA
Chemical Burns
TOXIC EPIDERMAL NECROLYSIS
Allergic Contact Dermatitis
Diagnosis and Testing SKIN CANCER

GRANULOMATOUS REACTIONS Radiation

UV-induced Skin Cancer
PHOTOTOXICOLOGY
Polycyclic Aromatic Hydrocarbons
Adverse Responses to Electromagnetic Mouse Skin Tumor Promotion
Radiation Arsenic

KEY POINTS

« The skin participates directly in thermal, electrolyte, a Irritant dermatitis is a nonimmune-related response
hormonal, metabolic, and immune regulation. caused by the direct action of an agent on the skin.
= Percutaneous absorption depends on the xenobiotics = Allergic contact dermatitis represents a delayed (type IV)
hydrophobicity, which affects its ability to partition into hypersensitivity reaction, whereby minute quantities of
epidermal lipid, and rate of diffusion through this barrier. material elicit overt reactions.
= The cells of the epidermis and pilosebaceous units
express biotransformation enzymes.

AS)
292 UNIT 4 Target Organ Toxicity

SKIN AS A BARRIER glands, and eccrine glands) span the epidermis and are embed-
ded in the dermis. In thickness, the dermis makes up approxi-
The skin protects the body against external insults in order to mately 90% of the skin and has largely a supportive function.
maintain internal homeostasis. It participates directly in ther- Separating the dermis from underlying tissues is a layer of
mal, electrolyte, hormonal, metabolic, and immune regulation. adipocytes, whose accumulation of fat has a cushioning action.
Rather than merely repelling noxious physical agents, the skin The blood supply to the epidermis originates in the capillar-
may react to them with various defensive mechanisms that ies located in the rete ridges at the dermal-epidermal junc-
serve to prevent internal or widespread cutaneous damage. If tion. Capillaries also supply the bulbs of the hair follicles and
an insult is severe or intense enough to overwhelm the protec- the secretory cells of the eccrine (sweat) glands. The ducts from
tive function ofthe skin, acute or chronic injury becomes read- these glands carry a dilute salt solution to the surface of the
ily manifest. The specific presentation depends on a variety of skin, where its evaporation provides cooling.
intrinsic and extrinsic factors including body site, duration of The interfollicular epidermis is a stratified squamous epi-
exposure, and other environmental conditions (Table 19-1). thelium consisting primarily of keratinocytes, which are
tightly attached to each other and to the basement membrane.
Melanocytes are distributed sparsely in the dermis, with occa-
Skin Histology sional concentrations beneath the basal lamina and in the
The skin consists of two major components: the outer epi- papillae of hair follicles. In the epidermis, these cells are stim-
dermis and the underlying dermis, which are separated by a ulated by ultraviolet light to produce melanin granules. The
basement membrane (Figure 19-1). The junction ordinar- granules are extruded and taken up by the surrounding kerati-
ily is not flat but has an undulating appearance (rete ridges). nocytes, which thereby become pigmented. Migrating through
In addition, epidermal appendages (hair follicles, sebaceous the epidermis are numerous Langerhans cells (LCs), which
‘
\

TABLE 19-1 Factors influencing cutaneous responses.

Variable Comment

Body site

Palms/soles Thick stratum corneum—good physical barrier

Common site of contact with chemicals
Occlusion with protective clothing

Intertriginous areas (axillae, groin, neck, finger webs, umbilicus, genitalia) Moist, occluded areas
Chemical trapping
Enhanced percutaneous absorption

Face Exposed frequently

Surface lipid interacts with hydrophobic substances
Chemicals frequently transferred from hands

Eyelids Poor barrier function—thin epidermis

Sensitive to irritants

Postauricular region Chemical trapping

Occlusion

Scalp Chemical trapping

Hair follicles susceptible to metabolic damage

Predisposing cutaneous illnesses—atopic dermatitis Increased sensitivity to irritants

Impaired barrier function

Psoriasis Impaired barrier function

Genetic factors Predisposition to skin disorders

Variation in sensitivity to irritants
Susceptibility to contact sensitization

Temperature Vasodilation—improved percutaneous absorption

Increased sweating—trapping

Humidity Increased sweating—trapping

Season Variation in relative humidity

Chapping and wind-related skin changes
 CHAPTER 19 Toxic Responses ofthe Skin 293

The viable layer of epidermis provides a much less effective

barrier, because hydrophilic agents readily diffuse into the
intercellular water, whereas hydrophobic agents can partition
( Stratum corneum into cell membranes, and each can diffuse readily to the blood
Stratum granulosum supply in the rete ridges of the dermis.
4 Stratum spinosum
The stratum corneum prevents water loss from underlying tis-
Stratum
Epidermis sues by evaporation. Its hydrophobic character reflects the lipid
(.germinativum
content of the intercellular space. The lipids, a major component
( Sweat duct being sphingolipids, have a high content of long-chain cerami-
Sebaceous gland i
isge
des, removal of which seriously compromises barrier function as
ees‘es
seer measured by transepidermal water loss. The stratum corneum is
Sweat gland wees
Ee ordinarily hydrated (typically 20% water), the moisture residing
4 | Blood vessel in corneocyte protein, but it can take up a great deal more water
€
& Connective tissue Eg on prolonged immersion, thereby reducing the effectiveness of
Fat the barrier to agents with a hydrophilic character. Indeed, occlu-
Hair follicle g
sion of the skin with plastic wrap, permitting the retention of
perspiration underneath, is a commonly employed technique to
\ Capillary
enhance uptake of agents applied to the skin surface. Penetration
from the air is generally too low to be of concern.
Uptake through the skin is now incorporated in pharmaco-
FIGURE 19-1 Diagram of across-section of human skin. kinetic modeling to estimate potential risks from exposures.
The degree of uptake depends on the details of exposure con-
ditions, being proportional to solute concentration (assuming
are important participants in the immune response of skin to it is dilute), time, and the amount of skin surface exposed. In
foreign agents. addition, two intrinsic factors contribute to the absorption
Keratinocytes of the basal layer make up the germinative rate of a given compound: its hydrophobicity, which affects its
compartment. When a basal cell divides, one of the progeny ability to partition into epidermal lipid, and its rate of diffu-
detaches from the basal lamina and migrates outward. As cells sion through this barrier. A measure of the first property is the
move toward the skin surface, they undergo a remarkable pro- commonly used octanol/water partitioning ratio (K,,,). This is
gram of terminal differentiation. They gradually express new particularly relevant for exposure to contaminated water, such
protein markers and accumulate keratin proteins. At the gran- as occurs during bathing or swimming. However, partitioning
ular layer, the cells become flattened and increase in volume of an agent into the skin is greatly affected by its solubility in or
nearly 40-fold. Lipid granules fuse with the plasma membrane, adhesion to the medium in which it is applied (including soil).
replacing the aqueous environment in the intercellular space Similarly, very hydrophobic compounds, once in the stratum
with their contents. Meanwhile, the plasma membranes of these corneum, may diffuse only very slowly into less hydrophobic
cells become permeable and cell organelles are degraded, while regions below. The second property is an inverse function of
a protein envelope is synthesized immediately beneath the molecular weight (MW) or molecular volume. Thus, hydro-
plasma membrane. The membrane is altered characteristically phobic agents of low MW permeate the skin better than those
by the loss of phospholipid and the addition of sphingolipid. of high MW or those that are hydrophilic. For small molecules,
This program of terminal differentiation, beginning as kera- hydrophobicity is a dominant factor in penetration.
tinocytes leave the basal layer, produces the outermost layer of Diffusion through the epidermis is considerably faster at
the skin, the stratum corneum. No longer viable, the mature some anatomical sites than others. A list in order of decreasing
cells (called corneocytes) are ~80% keratin in content. They are permeability under steady-state conditions gives the following
gradually shed from the surface and replaced from beneath. hierarchy: foot sole > palm > scrotum > forehead > abdomen.
The process typically takes 2 weeks for basal cells to reach the Absorption through the epidermal appendages is generally
stratum corneum and another 2 weeks to be shed from the sur- neglected, despite the ability of agents to bypass the stratum cor-
face. In instances in which the outer layer is deficient due to neum by this route, because the combined appendageal surface
area is a small fraction of the total available for uptake. However,
disease or physical or chemical trauma, the barrier to the envi-
ronment that the skin provides is inferior to that provided by penetration through the appendages can be appreciable.
normal, healthy skin.
Transdermal Drug Delivery—Specially designed patches
are currently in use to deliver drugs such as clonidine, estra-
Percutaneous Absorption diol, testosterone, nitroglycerin, scopolamine, fentanyl, and
The stratum corneum is the primary barrier to percutaneous nicotine for therapeutic purposes. Advantages of this approach
absorption. Diseases (e.g., psoriasis) or other conditions (e.g., over oral dosing include providing a steady infusion for
abrasion and wounding) that compromise this barrier can per- extended periods (typically 1 to 7 days) thereby avoiding large
mit greatly increased uptake of poorly permeable substances. variations in plasma concentration, preventing exposure to the
294 UNIT 4 Target Organ Toxicity

acidic pH of the stomach, and avoiding biotransformation in give an adverse reaction to anyone if the concentration is high
the gastrointestinal tract or from first-pass removal by the liver. enough and the exposure time long enough. Certain chemicals
at sufficient concentration produce an acute irritation, some-
Measurements of Penetration—For risk assessment and times called a second-degree chemical burn, that can even
pharmaceutical design, the most useful subject for experimen- result in scarring in serious cases. Strong acids, alkalies, and
tation is human skin. Volunteers are dosed, plasma and/or urine powerful oxidizing or reducing agents can substantially disrupt
concentrations are measured at suitable intervals, and amounts the cornified layer, producing cytotoxicity directly. Contact
excreted from the body are estimated. For in vitro work, excised with a variety ofplants can also have irritant effects, resulting in
split-thickness skin can be employed in special diffusion cham- the production of pro-inflammatory cytokines (IL1-a, IL1-(,
bers, though care is needed to preserve the viability of the liv- and TNF-q) from keratinocytes. Exposure is more commonly
ing layer of epidermis. The agent is removed for measurement the result of chronic cumulative irritation from repeated expo-
from the underside by a fluid into which it partitions, thereby sures to mild irritants such as soaps, detergents, solvents, and
permitting continued penetration. A simpler setup commonly cutting oils. Chronic exposure in the occupational setting often
employed uses cadaver skin with the lower dermis removed. elicits a process of “hardening.”
This lacks biotransformation capability but retains the barrier Response to exposure varies depending on the sensitivity of
function of the stratum corneum. To simplify determination of the anatomic site. The eyelids are quite sensitive, e.g., and the
penetration kinetics, skin flaps may be employed and the cap- back is more sensitive than the forearm. Individuals also vary
illary blood flow monitored to measure penetration. For this greatly in sensitivity to irritant dermatitis. Atopic individuals
purpose, pig skin has particular utility. A promising variation are the most sensitive to irritants.and exhibit a propensity to
minimizing species differences is to use skin grafts on experi- produce specific IgE antibodies to allergens and typically suffer
mental animals for these measurements. Human skin persists from hay fever. These individuals usually have a poorer prog-
well on athymic mice and retains its normal barrier properties. nosis than nonatopics and have a Higher frequency of persis-
tent dermatitis. The best preventive measure for atopics and
others is to avoid exposure to contact irritants.
Biotransformation
Information on the irritancy of chemicals toward human
The ability of the skin to metabolize agents that diffuse through skin may be obtained as part of differential diagnosis by patch
it contributes to its barrier function. This influences the poten- testing for allergic response. The skin of laboratory animals
tial biological activity of xenobiotics and topically applied (mice, rats, rabbits, and guinea pigs) can be used for testing,
drugs, leading to their degradation or their activation as skin but it is thinner and more sensitive than human skin to irri-
sensitizers or carcinogens. The epidermis and pilosebaceous tants. For development of new pharmaceuticals, cosmetics,
units are the major sites of such activity in the skin. Enzymes and other consumer products, a great need exists for an in vitro
participating in biotransformation that are expressed in skin system to determine the potential for irritant responses. Use of
include multiple forms of cytochrome P450, epoxide hydrolase, human epidermal cell cultures has been increasing as recon-
UDP-glucuronosyltransferase, quinone reductase, and gluta- structed epidermal and skin models come closer to the native
thione transferases. Other metabolic enzyme activities detected differentiated state.
in human epidermal cells include sulfatases, 8-glucuronidase,
N-acetyltransferases, esterases, and reductases. The intercellu-
lar region of the stratum corneum has catabolic activities (e.g., Chemical Burns
proteases, lipases, glycosidases, and phosphatase). Extremely corrosive and reactive chemicals may produce
immediate coagulative necrosis that results in substantial tissue
CONTACT DERMATITIS damage, with ulceration and sloughing. Sometimes referred to
as a third-degree chemical burn, the damage does not have a
Of all occupational skin diseases, contact dermatitis accounts primary inflammatory component and thus may not be clas-
for over 90% of reported causes. Contact dermatitis falls into sified as an irritant reaction. In addition to the direct effects
the two major categories of irritant and allergic forms. Both of the chemical, necrotic tissue can act as a chemical reservoir
involve inflammatory processes and can have indistinguish- resulting in either continued cutaneous damage or percutane-
able clinical characteristics of erythema (redness), induration ous absorption and systemic injury after exposure. Table 19-2
(thickening and firmness), scaling (flaking), and vesiculation lists selected corrosive chemicals that are important clinically.
(blistering) on areas directly contacting the chemical agent.
Figure 19-2 shows examples of many types of contact dermati-
tis as a result of occupational skin toxicity. Allergic Contact Dermatitis
Allergic contact dermatitis is a delayed (T-cell mediated) hyper-
sensitive reaction (see Chapter 12). To induce sensitiza-
Irritant Dermatitis tion, chemical haptens must penetrate the skin and become
Irritant dermatitis is the condition that arises from the direct con- attached to carrier proteins. Complete antigens are processed
tact of agents on the skin and accounts for nearly 80% of contact by Langerhans cells and presented to type 1 T-helper cells in
dermatitis cases. A chemical in this category is anticipated to regional lymph nodes. Memory T cells are produced over a
 CHAPTER 19 Toxic Responses of the Skin 295

eal | aie (svanlannttt

| lf hytty
LH Hid TULL
amTUUNVUVOQOQNNVOOOIIE

FIGURE 19-2 Examples of occupational skin toxicity. The panels, available at the NIOSH website (http://www.cdc.gov/niosh/topics/skin/
occderm-slides/ocderm1.html), are a small selection from the 140-slide NIOSH program “Occupational Dermatoses—A Program for Physicians”
prepared by Drs. E Shmunes, MM Key, JB Lucas, and JS Taylor. (A. Eczema from cutting oil. B. Atopic irritant dermatitis. C. Burn from ethylene oxide.
D. Burn from alkali exposure. E. Sensitization to dichromate. F. Beryllium granulomas. G. Phototoxicity from lime juice. H. Acne from cutting oil.
!, Leukoderma from rubber antioxidants.J.Hyperpigmentation from mercaptobenzothiazole.)

1- to 3-week period and enter the circulation. Subsequent effect. In addition, the dose required to elicit a reaction is lower
exposure to the same antigen results in an amplified immune after sensitization with a higher dose.
response characterized by dermal infiltration and spongiosis.
Thousands of chemicals have been reported to give rise to Diagnosis and Testing—In order to find the responsible
allergic contact dermatitis, many across a variety of occupa- chemical causing allergic contact dermatitis, patch testing is
tions and consumer products (Table 19-3). Because most commonly employed. On the washed backs of patients, patches
chemicals in the chemical universe are only weakly active or are placed containing a small amount of a potential allergen.
infrequently encountered, much effort has focused on finding Diagnostic patch testing utilizes standardized concentrations
the major allergens in the population by systematic patch test- of material dissolved or suspended in petrolatum or water that
ing of dermatology patients. Although not measuring sensitiv- are placed on stainless steel chambers adhering to acrylic tape.
ity in the population at large, the results are quite useful. The After two to three days, during which time a maximal reac-
panel of chemicals tested can vary with geographic location to tion usually develops, the patches are removed and sites of
accommodate local usage, or it can be directed to specific ana- exposure are scored for degree of response. Relevance to the
tomic sites such as the foot. patient’s actual environment must be considered so that expo-
Unlike contact irritants, where the response is generally sure in daily life can be minimized to appropriate chemicals.
proportional to the applied dose and time, contact allergens Interpretation of the results and environmental modification
can elicit reactions at very small doses. Nevertheless, a higher should take into account the phenomenon of cross-sensitivity,
dose confers a greater likelihood of sensitization and that doses where reactivity to a compound may be evident if it shares
below a threshold for sensitization can have a cumulative functional groups that have provoked sensitization in another
296 UNIT 4 Target Organ Toxicity

TABLE 19-2 Selected chemicals causing skin burns.

ee LL CC“ ‘te ~_ sn

Chemical Comment

Ammonia Potent skin corrosive

Contact with compressed gas can cause frostbite

Calcium oxide (CaO) Severe chemical burns

Extremely exothermic reaction—dissolving in water can cause heat burns

Chlorine Liquid and concentrated vapors cause cell death and ulceration

Ethylene oxide Solutions and vapors may burn

Compressed gas can cause frostbite

Hydrogen chloride (HCl) Severe burning with scar formation

Hydrogen fluoride (HF) Severe, painful, slowly healing burns from high concentration
Lower concentration causes delayed cutaneous injury
Systemic absorption can lead to electrolyte abnormalities and death
Calcium-containing topical medications and quaternary ammonium compounds are used to limit damage

Hydrogen peroxide High concentration causes severe burns and blistering

Methyl! bromide Liquid exposure produces blistering, deep burns :

Nitrogen oxides Moist skin facilitates the formation of nitric acid causing severe yellow-colored burns
SN
Phosphorus White phosphorus continues to burn on skin in the presence ofair

Phenol Extremely corrosive even in low concentrations

Systemic absorption through burn sites may result in cardiac arrhythmias, renal disease, and death

Sodium hydroxide High concentration causes deep burns, readily denatures keratin

Toluene diisocyanate Severe burns with contact

Skin contact rarely may result in respiratory sensitization

compound. Common cross-reacting chemicals are listed produce granulomatous reactions, including silica, talc, paraf-
in Table 19-4. fin or mineral oil, beryllium, and gadolinium. Metallic mercury
In animal testing, a chemical is applied to intact or abraded and zirconium compounds, formerly used in deodorants, and
skin or through intradermal injection with or without adju- tattoo dyes (containing cobalt, chromium, mercury, lead, iron,
vant. The skin reaction to a subsequent challenge with the cadmium, and manganese compounds) can also induce granu-
chemical is observed and graded, in an attempt to identify lomatous reactions that, in rare cases, can be induced by intense
causative agents. Increasing emphasis on reducing or eliminat- light treatment.
ing animal use in toxicity testing, driven in part by regulatory
initiatives, has stimulated development of integrated testing
strategies, where predictions of toxic effects such as skin sensi- PHOTOTOXICOLOGY
tization include physical, chemical, and structural analysis and The ultraviolet and visible spectra of solar radiation reaching
in vitro testing. the earth extend from 290 to 700 nm. Wavelengths beyond this
range are either filtered by the earth’s atmosphere or are insuf-
GRANULOMATOUS REACTIONS ficiently energetic to cause cutaneous pathology. Adequate
doses ofartificially produced UV-C (< 290 nm) or X-rays can
A granulomatous reaction to a foreign body is one in which produce profound physical and toxicological skin changes.
invading substances that cannot be readily removed are conse- The protective skin pigment melanin, synthesized in melano-
quently isolated. These occur infrequently toward a variety of cytes, absorbs a broad range of radiation from UV-B (290 to
agents introduced into the skin through injection or after lac- 320nm) through the visible spectrum. Other chromophores
eration or abrasion. Persistent lesions with abundant inflam- in the skin include amino acids, primarily tryptophan and toa
matory cells can be produced, resembling chronic infectious lesser extent tyrosine, and their breakdown products (e.g., uro-
conditions (e.g., tuberculosis, leprosy, leishmaniasis, and syphi- canic acid), which absorb light in the UV-B range. Biologically,
lis) and present diagnostic challenges. Many substances can the most significant chromophore is DNA, since damage from
 CHAPTER 19 Toxic Responses of the Skin 297.

TABLE 19-3 Common contactallergens.

Common Allergens
Topical medications/hygiene products Antibiotics Therapeutics
Bacitracin Benzocaine
Neomycin
Polymyxin Idoxuridine
Aminoglycosides a-Tocopherol (vitamin E)
Sulfonamides Corticosteroids

Preservatives Others
Benzalkonium chloride Cinnamic aldehyde
Formaldehyde Ethylenediamine
Formaldehyde releasers Lanolin
Quaternium-15 p-Phenylenediamine
Imidazolidinyl urea Propylene glycol
Diazolidinyl urea Benzophenones
DMDM hydantoin Fragrances
Methylchloroisothiazolinone Thioglycolates

Plants and trees Abietic acid Pentadecylcatechols

Balsam of Peru Sesquiterpene lactone
Rosin (colophony) Tuliposide A

Antiseptics Chloramine Glutaraldehyde

Chlorhexidine Hexachlorophene
Chloroxylenol Thimerosal (Merthiolate)
Dichlorophene Mercurials
Dodecylaminoethyl glycine HCI Triphenylmethane dyes

Rubber products Diphenylguanidine Resorcinol monobenzoate

Hydroquinone Benzothiazolesulfenamides
Mercaptobenzothiazole Dithiocarbamates
p-Phenylenediamine Thiurams

Leather Formaldehyde Potassium dichromate

Glutaraldehyde

Paper products Abietic acid Rosin (colophony)

Formaldehyde Triphenyl phosphate
Nigrosine Dyes

Glues and bonding agents BisphenolA Epoxy resins

Epichlorohydrin p-(t-Butyl)formaldehyde resin
Formaldehyde Toluene sulfonamide resins
Acrylic monomers Urea formaldehyde resins
Cyanoacrylates

Metals Chromium Mercury

Cobalt Nickel

radiation can have lasting effects on the genetic information in from injured keratinocytes, and may be responsible for several
target cells. of the systemic symptoms associated with sunburn, such as
fever, chills, and malaise. Environmental conditions that affect
UV-induced injury include duration of exposure, season, alti-
Adverse Responses to Electromagnetic tude, body site, skin pigmentation, and previous exposure.
Radiation UV-B (290 to 320nm) is the most effective solar band to
After exposure, the most evident acute feature of UV radia- cause erythema in human skin. A substantially greater dos-
tion exposure is erythema (redness or sunburn). The minimal age of UV-A (320 to 400 nm) reaches the earth compared with
erythema dose (MED), the smallest dose of UV light needed UV-B (up to 100-fold); however, its efficiency in generating
to induce an erythematous response, varies greatly from per- erythema in humans is about 1000-fold less than that of UV-B.
son to person. Vasodilation responsible for the color change UV-A is likely more responsible for long-term UV effects such
is accompanied by significant alterations in inflammatory as wrinkling, skin atrophy, and easy bruisability. Overt pigment
mediators released from local inflammatory cells as well as darkening is another typical response to UV exposure. This may
298 UNIT 4 Target Organ Toxicity

TABLE 19-4 Common cross-reacting chemicals. neoantigens on malignant cells and thus allow such a transfor-
mation to proceed unabated. Exposures to ionizing radiation
Chemical Cross-reactor may produce a different spectrum of disease depending upon the
Abietic acid Pine resin (colophony) dose delivered. Large acute exposures will result in local redness,
blistering, swelling, ulceration, and pain. After a latent period or
Balsam of Peru Pine resin, cinnamates, benzoates following subacute chronic exposures, characteristic changes
Bisphenol A Diethylstilbestrol, hydroquinone such as epidermal thinning, freckling, telangiectasias, and non-
monobenzyl ether healing ulcerations may occur. Also, a variety of skin malignan-
cies have been described years after skin exposure to radiation.
Canaga oil Benzyl salicylate
Aside from the toxic nature of electromagnetic radiation, nat-
Chlorocresol Chloroxylenol ural and environmental exposures to certain bands of light are
vital for survival. Ultraviolet radiation is critical for the conver-
Diazolidinyl urea Imidazolidinyl urea, formaldehyde
sion of 7-dehydrocholesterol to previtamin D,, a required pre-
Ethylenediamine di-HCI Aminophylline, piperazine cursor for normal endogenous production of vitamin D. Blue
light in the 420- to 490-nm range can photoisomerize bilirubin
Formaldehyde Arylsulfonamide resin, chloroallyl-
hexaminium chloride
(a red blood cell breakdown product) in the skin, rendering uri-
nary excretion of this neurotoxic metabolite by infants with ele-
Hydroquinone Resorcinol vated serum bilirubin. In addition, the toxic effects of UV light
Parabens, hydroquinone monobenzy!
have been exploited for decades through artificial light sources
Methyl hydroxybenzoate
ether for treatment of hyperproliferative skin disorders like psoriasis.

p-Aminobenzoic acid p-Aminosalicylic acid, sulfonamide ‘

\

Phenylenediamine Parabens, p-aminobenzoic acid

Photosensitivity
Anabnormal sensitivity to UV and visible light, photosensitiv-
Propyl hydroxybenzoate Hydroquinone monobenzyl ether
ity may result from endogenous or exogenous factors. Various
Phenol Resorcinol, cresols, hydroquinone genetic diseases, such as xeroderma pigmentosum, and the
autoimmune disease lupus erythematosus impair the cell's
Tetramethylthiuram Tetraethylthiuram mono- and disulfide
ability to repair UV light-induced damage. In hereditary or
disulfide
chemically induced porphyrias, enzyme abnormalities disrupt
the biosynthetic pathways producing heme, leading to accu-
mulation of porphyrin precursors or derivatives throughout
be accomplished by enhanced melanin production by melano- the body. These compounds in general fluoresce when exposed
cytes or by photooxidation of melanin. Tanning or increased to light of 400 to 410nm (Soret band), and in this excited
pigmentation usually occurs within 3 days of UV light expo- state interact with cellular macromolecules or with molecu-
sure, whereas photooxidation is evident immediately. The tan- lar oxygen to generate toxic-free radicals. A “constitutional”
ning response is most readily produced by exposure to UV-B sensitivity to light (porphyria cutanea tarda) can be precipi-
and may be induced, along with erythema and DNA repair, by tated by alcohol, estrogens, or certain antibiotics in individu-
DNA damage. The tanning response serves to augment the pro- als with hereditary abnormalities in porphyrin synthesis, and
tective effects of melanin in the skin. However, the immediate an “acquired” sensitivity in general by hexachlorobenzene and
pigment-darkening characteristic after exposure to UV-A and mixtures of polyhalogenated aromatic hydrocarbons.
to visible light does not confer improved photoprotection.
Chronic exposure to radiation induces a variety of character- Phototoxicity—Phototoxic reactions from exogenous chem-
istic skin changes. For ultraviolet light, these changes acceler- icals may be produced by systemic or topical administration
ate or mimic aging, but the rate depends greatly on the baseline or exposure. In acute reactions, the skin may appear red and
skin pigmentation of the individual. Lighter skinned people blister within minutes to hours after ultraviolet light exposure.
suffer from chronic skin changes with greater frequency than Chronic phototoxic responses may result in hyperpigmenta-
darker individuals. Pigmentary changes such as freckling and tion and thickening of the affected areas. UV-A (320 to 400 nm)
hypomelanotic areas, wrinkling, telangiectasias (fine super- is the most commonly responsible; UV-B (290 to 320 nm) may
ficial blood vessels), actinic keratoses (precancerous lesions), occasionally be involved.
and malignant skin lesions such as basal and squamous cell Agents most often associated with phototoxic reactions are
carcinomas and malignant melanomas are all consequences of listed in Table 19-5. These chemicals readily absorb UV light and
chronic exposure to ultraviolet light exposure. assume a higher energy excited state. The oxygen-dependent
One significant pathophysiological response of chronic expo- photodynamic reaction is the most common as these excited
sure to ultraviolet light is the pronounced decrease of epider- molecules return to the ground state. Here, excited triplet-
mal Langerhans cells. Chronically sun exposed skin may have state molecules transfer their energy to oxygen, forming sin-
up to 50% fewer of these compared to photoprotected areas. glet oxygen, or become reduced and form other highly reactive
This decrease may result in lessened immune surveillance of free radicals. These reactive products are capable of damaging
 CHAPTER 19 Toxic Responses of the Skin 299

TABLE 19-5 Selected phototoxic chemicals. a potential photosensitizing chemical into a hapten that elicits
an allergic response. Photoallergy generally is distinguishable
Furocoumarins from phototoxicity because the former results from delayed
8-Methoxypsoralen
5-Methoxypsoralen i
hypersensitivity, and amounts of chemical too low to give a
Trimethoxypsoralen toxic response still suffice to elicit allergy.
Diagnosis is best performed by patch testing with and with-
Polycyclic aromatic hydrocarbons
out light exposure of the treated surface to distinguish photo-
Anthracene
Fluoranthene contact from contact allergy. Because the offending chemical
Acridine may not be obvious from the patient history due to the delay
Phenanthrene between exposure to the chemical and sunlight and the symp-
Drugs toms, a panel of test chemicals may include some 50 common
Tetracyclines photoallergens as well as the patient’s own sunscreen and per-
Sulfonamides sonal care products. To assist in predicting risks of photoal-
Sulfonylureas
lergy, efforts have been made to derive important chemical
Nalidixic acid
Thiazides features among existing photoallergens that account for their
Phenothiazines reactivity toward proteins. This information, coupled with
Nonsteroidal anti-inflammatory drugs assessment of physical properties such as aqueous/lipid parti-
Dyes
tioning, is anticipated to streamline testing of new products.
Disperse blue 35
Eosin
Acridine orange
ACNE
Porphyrin derivatives Acne is a pleomorphic disease with a multifactorial etiology.
Hematoporphyrin The influence of sebum, hormones, bacteria, genetics, and
environmental factors is well known. In many situations, one
of these factors has an overwhelmingly greater influence in the
genesis oflesions than the others.
cellular components and macromolecules and causing cell Comedogenic chemicals induce comedone lesions, which
death. The resulting damage elaborates a variety of immune may be open or closed (blackhead or whitehead, respectively,
mediators from keratinocytes and local white blood cells that in the vernacular). Additionally papules, pustules, cysts, and
recruit more inflammatory cells to the skin, and thus yield the scars may complicate the process. Hair follicles and associated
clinical signs of phototoxicity. sebaceous glands become clogged with compacted keratino-
Nonphotodynamic mechanisms have been described in the cytes that are bathed in sebum. The pigmentary change most
pathogenesis of phototoxicity, with psoralens (furocoumarins) evident in open comedones is from melanin.
being prime examples. On entering the cell, psoralens intercalate
with DNA. Subsequent excitation with UV-A provokes a photo-
Chloracne
chemical reaction that ultimately results in a covalently linked
cycloadduct between the psoralen and pyrimidine bases. ‘This Chloracne, one of the most disfiguring forms of acne in
substantially inhibits DNA synthesis and repair, resulting in clin- humans, is caused by exposure to polyhalogenated aromatic
ical phototoxic reactions. Psoralens may be found in sufficiently hydrocarbons. Chloracne is a relatively rare disease; however,
high concentrations in limes and celery to cause a significant blis- its recalcitrant nature and preventability make it an important
tering eruption called phytophotodermatitis. Psoralen-induced occupational and environmental illness. Typically, comedones
phototoxicity may be harnessed and controlled pharmacologi- and straw-colored cysts are present behind the ears, around
cally. Topically and orally administered psoralens are used thera- the eyes, and on the shoulders, back, and genitalia. In addition
peutically to enhance the effects of controlled delivery of UV-A. to acne, hypertrichosis (increased hair in atypical locations),
Psoralens plus UV-A (PUVA) is administered to control kerati- hyperpigmentation, brown discoloration of the nail, conjunc-
nocyte and lymphocyte hyperproliferative diseases such as pso- tivitis, and eye discharge may be present.
riasis, eczema, and cutaneous T-cell lymphomas.
PIGMENTARY DISTURBANCES
Photoallergy—In contrast to phototoxicity, photoallergy
is a type IV delayed hypersensitivity reaction, leading typi- Several factors influence pigmentation of the skin. Melanin
cally to eczema. Hence, photoallergy requires prior sensitiza- is produced through a series of enzymatic pathways begin-
tion to the chemical. Induction and subsequent elicitation of ning with tyrosine. Errors in this pathway or exposure to
reactions may result from topical exposure as in photocontact tyrosine analogs may result in abnormal pigmentation.
dermatitis or from systemic photoallergy. Generally, the mech- Hyperpigmentation results from increased melanin produc-
anisms of photocontact dermatitis and even that of systemic tion or deposition of endogenous or exogenous pigment in the
photoallergy are the same as that described above for allergic upper dermis. Exogenous hyperpigmentation can arise from
contact dermatitis. However, UV light is necessary to convert deposition of metals and drugs in dermal tissue. Conversely,
300 UNIT 4 Target Organ Toxicity

TABLE 19-6 Selected causes of cutaneous TABLE 19-7 Selected substances reported to elicit
pigmentary disturbances. contact urticaria.

Chemicals — Foods — oo
1. Hyperpigmentation
Ultraviolet light exposure
Anhydrides Animal viscera
Postinflammatory changes (melanin and/or hemosiderin
Methylhexahydrophthalic Apple
deposition)
Hexahydrophthalic Artichoke
Hypoadrenalism
Maleic Asparagus
Internal malignancy
Chemical exposures
Antibiotics Beef
Coal tar volatiles
Bacitracin Beer
Anthracene
Streptomycin Carrot
Picric acid
Cephalosporins Chicken
Mercury
Penicillin Deer
Lead
Rifamycin Egg
Bismuth
Furocoumarins (psoralens)
Benzoic acid Fish
Hydroquinone (paradoxical)
Drugs Cobalt chloride Lamb
Chloroquine
Amiodarone Butylhydroxyanisol (BHA) Mustard
Bleomycin
Zidovudine (AZT) Butylhydroxytoluene (BHT) Paprika
Minocycline
Carboxymethylcellulose * Potato
Il. Hypopigmentation/depigmentation/leukoderma
Postinflammatory pigmentary loss Cyclopentolate hydrochloride Pork .
Vitiligo
Chemical leukoderma/hypopigmentation Diphenyl| guanidine Rice
Hydroquinone
Monobenzyl, monoethyl, and monomethyl ethers of hydroquinone Epoxy resin Strawberry
p-(t-Butyl) phenol
Mercaptoamines Formaldehyde Turkey
Phenolic germicides
p-(t-Butyl)catechols Fragrances
Butylated hydroxytoluene Balsam of Peru
Cinnamic aldehyde

Isocyanates
hypopigmentation is a loss of pigmentation from melanin loss, Diphenylmethane-4,4-diisocyanate
melanocyte damage, or vascular abnormalities. Leukoderma
Menthol
(vitiligo) and depigmentation denote complete loss of mela-
nin from the skin, imparting a porcelain-white appearance. Plants, woods, trees, and weeds
Table 19-6 lists chemicals capable of altering pigmentation. Latex

Phenylmercuric acetate
URTICARIA
For those allergens to which IgE antibodies have been elic-
ited by previous or ongoing exposure, subsequent contact can
lead to development of utricaria (hives), typically in minutes, common in atopic individuals. Among the numerous occu-
through an immediate type I hypersensitivity reaction (see pations where this response occurs include hairdressers and
Chapter 12). Hives are raised wheals that usually itch or sting those involving routine handling of food, plant, or animal
and may appear reddish. They generally disappear within products. Healthcare is an occupation in which allergic contact
hours and rarely lasting longer than a day or two. dermatitis to latex rubber is a common problem. Latex proteins
Food allergies and pharmaceuticals are major causes of acute have a propensity to induce immediate type I hypersensitive
urticaria, but many other causes are known. Certain food aller- reactions, where the response can range from a mild skin reac-
gies (e.g., to nuts, fish, and shellfish) are capable of producing tion to anaphylaxis and death. Some substances that have been
the life-threatening response, anaphylactic shock. Some agents reported to cause contact urticaria are listed in Table 19-7.
(e.g., opiates) can bring about direct release of histamine from
mast cells without antibody mediation, while others (nonste-
TOXIC EPIDERMAL NECROLYSIS
roidal anti-inflammatories) may do so through effects on ara-
chidonic acid metabolism or by uncertain mechanisms. Toxic epidermal necrolysis (TEN) represents one of the most
Contact urticaria in an occupational setting can arise life-threatening dermatologic diseases that is caused by drugs
from exposure to plant or animal proteins and appears more and chemicals. At the most severe end of a spectrum, TEN
 CHAPTER 19 Toxic Responses of the Skin 301

involves detachment of => 30% of the epidermal surface from not cause cancer in normal individuals, sun exposure leads to
the dermis, commonly accompanied by severe erosions of premature aging ofthe skin. For this reason, sunbathing is dis-
mucous membranes, and has a fatality rate ~30%. TEN com- couraged and the use of sun-block lotions is encouraged.
monly resembles an upper respiratory tract infection in the
first several days (fever, cough, sore throat, and malaise), but
prompt diagnosis when the cutaneous lesions become evident Polycyclic Aromatic Hydrocarbons
several days later improves survival chances. Substances rich in polycyclic aromatic hydrocarbons (coal tar,
Nearly 200 drugs have been reported to cause this syndrome creosote, pitch, and soot) are skin carcinogens in humans and
with major contributors being anticonvulsants, nonsteroidal animals. Oxidative biotransformation of polycyclic aromatic
anti-inflammatories, antibacterial sulfonamides, allopurinol, compounds produces electrophilic epoxides that can form DNA
and nevirapine. Mechanisms leading to this idiosyncratic drug adducts. Phenols, produced by rearrangement of the epoxides,
reaction are under scrutiny and current hypotheses identify can be oxidized further to quinones, yielding active oxygen spe-
HLA genotype and ethnic background as contributing factors. cies, and they are also toxic electrophiles. Occupations at risk of
A characteristic feature of the syndrome is the large-scale skin cancer from exposure to these compounds (e.g., roofing)
apoptosis of epidermal keratinocytes. Candidates for medi- often involve considerable sun exposure, an additional risk factor.
ating apoptosis through cell surface death receptors include
tumor necrosis factor and FAS ligand, which appear elevated;
in addition, drug-sensitized natural killer and cytotoxic T lym- Mouse Skin Tumor Promotion
phocytes, secreting granulysin, and other components of the Mouse skin has been developed as an important target for car-
innate immune response may participate in inducing kerati- cinogenicity testing. The observed incidence of squamous cell
nocyte death. Effectiveness of treatments has been difficult to carcinomas in mouse skin is taken as evidence of a general car-
evaluate, but promising approaches involve immunosuppres- cinogenic risk for humans. Much has been learned about squa-
sion (cyclophosphamide, cyclosporine) or blockage of death mous cell carcinoma pathogenesis in mouse skin that does have
receptors using intravenous immunoglobulin therapy. general applicability to human squamous cell carcinomas. An
advantage of the mouse skin carcinogenesis model is the ability
to separate the neoplastic process into stages of initiation, pro-
SKIN CANCER motion, and progression depending on experimental design.

Radiation
Radiation from ionizing wavelengths to ultraviolet wavelengths Arsenic
has been shown to cause skin cancer. Shortly after the discovery Arsenic is an abundant element in the earth’s crust that is
of radioactive elements at the turn of the twentieth century, it encountered routinely in small doses in the air, water, and food.
was observed that X-rays could cause severe burns, squamous High exposures from smelting operations and from well water
cell carcinoma, and basal cell carcinomas. X-ray-induced non- derived from rock strata with a high arsenic content are asso-
melanoma skin cancers (NMSC) continued to be observed ciated with arsenical keratoses (premalignant lesions), black-
throughout the twentieth century, as X-rays were used thera- foot disease (a circulatory disorder reflecting endothelial cell
peutically until the mid-twentieth century for a variety of skin damage), and squamous cell carcinoma of the skin and several
diseases (acne, atopic dermatitis, psoriasis, and tinea). Although other organs (bladder, lung, and liver). Arsenite (+3 oxida-
NMSC from X-rays are now uncommon, dermal atrophy or tion state) avidly binds vicinal thiols and is thought to inhibit
sclerosis still is seen as sequelae of radiodermatitis, which some- DNA repair, whereas arsenate (+5 oxidation state) can replace
times develops after X-ray treatment of internal malignancies. phosphate in macromolecules such as DNA, but the resulting
esters are unstable. Arsenic also alters DNA methylation, sup-
presses keratinocyte differentiation markers, and enhances
UV-induced Skin Cancer growth factor secretion in the epidermis. Methylation has been
Most skin cancers in the United States now are UV-induced. considered the most likely detoxification method, because the
The most common UV-induced skin cancers are NMSC and observed mono- and dimethyl arsenate isolated in urine from
cutaneous malignant melanoma. UV-B (290 to 320nm) induces exposed humans and animals are indeed much less toxic.
pyrimidine dimers and 8-oxoguanine modifications, thereby
eliciting mutations in critical genes. The p53 tumor suppressor
gene has been targeted in nearly all squamous cell carcinomas. BIBLIOGRAPHY
Because the p53 protein arrests cell cycling until DNA damage Chilcott RP, Price S (eds.): Principles and Practice of Skin Toxicology.
is repaired and may induce apoptosis, its loss destabilizes the Hoboken, NJ: John Wiley & Sons, 2008.
genome of initiated cells and gives them a growth advantage. Monteiro-Riviere NA: Toxicology of the Skin. New York: Informa
UV light also has immunosuppressive effects that may help Healthcare, 2010.
skin tumors survive. Skin cancer incidence is highest in the Wilhelm KP, Zhai H, Maibach HI: Dermatotoxicology, 8th ed., New
tropics and in pale-complexioned whites. Even when it does York: Informa Healthcare, 2012.
 UNIT 4 Target Organ Toxicity

QUESTIONS

Which of the following statements is FALSE regarding Photoallergies:

skin histology? a. represent a form oftype III hypersensitivity reaction.
a. Blood supply to the epidermis originates in the b. can occur without exposure to UV radiation.
epidermal-dermal junction. c. are hapten-mediated
b. Melanin is made and stored by melanocytes. d. cannot be tested for as contact dermatitis allergies can.
c. The stratum corneum is made up of nonviable cells. e. often occur on first exposure.
d. It takes approximately 2 weeks for cells to be sloughed
off from the stratum corneum. Diffusion through the epidermis would occur most slowly
e. Stem cells in the basal layer replenish the keratino- across skin at which of the following locations?
cytes of the layers of epidermis. a. palm.
b. forehead.
Transdermal drug delivery does NOT: c. scrotum.
a. prevent drug exposure to low pH. d. foot sole.
b. avoid first-pass metabolism. e. abdomen.
c. provide steady infusion over an extended period of
time. Which of the following statements regarding photosensi-
avoid large variation in drug plasma concentration. tivity is FALSE?
e. increase safety of drug delivery. a. Porphyrias cayse light sensitivity because of the lack
of heme synthesis.
Irritant and contact dermatitis are marked by all of the b. Lupus patients are unable.to repair damage caused by
following characteristics EXCEPT: UV light.
a. softness. c. Chronic phototoxic responses often result in
b. erythema. hyperpigmentation.
c. flaking. d. Photoallergy represents a type IV hypersensitivity
d. induration. reaction.
e. blistering. e. UV radiation causes cycloadducts between pyrimi-
dine bases.
Nickel is a common cause of allergic contact dermatitis,
which is which type of hypersensitivity reaction? . Acne is caused by all of the following EXCEPT:
am etypel: a. clogged sebaceous glands.
b. type II. b. hormones.
cr typelil. c. viruses.
d. type IV. d. genetics.
e. type V. e. environmental factors.

All of the following statements regarding phototoxicology 10. All of the following statements regarding urticaria are true
are true EXCEPT: EXCEPI;
a. Melanin is primarily responsible for the absorption of a. Urticaria is a delayed-type hypersensitivity reaction.
UV-B radiation. b. Hives are mediated partly by histamine release from
b. UV-A is the most effective at causing sunburn in mast cells.
humans. 2 Latex is a common chemical cause of urticaria.
c. IL-1 release is responsible for systemic symptoms d. Select foods have been reported to elicit contact
associated with sunburn. urticaria.
d. Melanin darkening is a common response to UV e. Urticaria is mediated by IgE antibodies.
exposure.
e. UV radiation exposure causes thickening of the
stratum corneum.
 CAH SA Pal tee

Toxic Responses of the

Reproductive System
Paul M.D. Foster and L. Earl Gray Jr.

INTRODUCTION IMPLANTATION

THE REPRODUCTIVE CYCLE PLACENTA

REPRODUCTIVE DEVELOPMENT AND SEXUAL PREGNANCY

DIFFERENTIATION
PARTURITION
GAMETOGENESIS
LACTATION
NEONATAL DEVELOPMENT
SENESCENCE
INFANTILE DEVELOPMENT
ENDOCRINE DISRUPTION
PUBERTAL DEVELOPMENT Known Effects of EDCs in Humans and Animals
Rodent Models of Puberty Effects of Drugs on Human Sexual Differentiation
Known Effects of Plant and Fungal Products in
SEXUAL MATURITY
Animals and Humans
Hypothalamo-pituitary-Gonadal Axis Known Effects of Organochlorine Compounds in
Ovarian Function Humans
Oogenesis Occupational Exposures
Case Study: Busulfan Environmental Androgens
Ovarian Cycle Environmental Antiandrogens
Postovarian Processes Fungicides
Oviducts Linuron (Herbicide)
Uterus p,p’-DDE (Pesticide Metabolite)

TESTICULAR STRUCTURE AND FUNCTION Phthalates (Plasticizers)

Environmental Estrogens
Targets for Toxicity
EDC Screening Programs
Testicular Structure and Spermatogenesis
In Vivo Mammalian Assays
Posttesticular Processes
Alternative Screening Assays
Erection and Ejaculation
Case Studies for Effects on the Male TESTING FOR REPRODUCTIVE TOXICITY
m-Dinitrobenzene Screens and Multigeneration Studies
Ethylene Glycol Monomethyl Ether Testing for Endocrine-disrupting Chemicals
(EGME) Testing Pharmaceuticals

FERTILIZATION EVALUATION OF TOXICITY TO REPRODUCTION

303
304 UNIT 4 Target Organ Toxicity

s The gonads possess a dual function: an endocrine func- = Xenobiotics can act directly on the hypothalamus and the
tion involving the secretion of sex hormones and a non- adenohypophysis, leading to alterations in the secretion of
endocrine function relating to the production of germ hypothalamic-releasing hormones and/or gonadotropins.
cells (gametogenesis). « Steroid hormone biosynthesis can occur in several endo-
= Gametogenic and secretory functions of either the crine organs including the adrenal cortex, ovary, and the
ovary or testes are dependent on the secretion of follicle- testes.
stimulating hormone (FSH) and luteinizing hormone = Female reproductive processes of oogenesis, ovulation,
(LH) from the pituitary. the development of sexual receptivity, coitus, gamete and
a The blood-testis barrier between the lumen of an inter- zygote transport, fertilization, and implantation of the
stitial capillary and the lumen of a seminiferous tubule conceptus may be sites of xenobiotic interference.
impedes or prevents the free exchange of chemicals/ « Xenobiotics may influence male reproductive organ
drugs between the blood and the fluid inside the semi- structure, spermatogenesis, androgen hormone secre-
niferous tubules. tion, and accessory organ function.

1
\

INTRODUCTION differentiate, produce a placenta, and undergo normal embryo-

genesis and fetal development.
Chemicals can adversely affect reproduction in males and Acquisition of sexual maturity is marked by the generation
females. Recent trends in human fertility point to the poten- of gametes by the gonads. For parental animals, once their
tial for declines in normal human reproduction and suggest reproductive life span has finished, the process of reproductive
that exposure to environmental chemicals and drugs may con- senescence then occurs. These processes all involve complex
tribute to these declines. The reproductive cycle is outlined in interplay between tissues and cells, under hormonal control
Figure 20-1. that provides the critical signals and precise timing of these
events. All these processes can be targets for the action of spe-
cific agents that can disturb events leading to adverse effects
THE REPRODUCTIVE CYCLE on reproduction, such that the normal production of viable
Numerous complex processes are orchestrated in a precise and offspring cannot occur.
sequential order for optimal performance at different stages Any description of reproductive toxicity has to be in the con-
of the life cycle of animals and humans. Following fertiliza- text of the life stage of exposure and effect. Chemicals can have
tion of an egg by a sperm, the resulting zygote must be trans- different effects on reproduction at different life stages and via
ported along the oviduct while maturing into an early embryo. different modes of action/mechanisms. Indeed, it might be
This embryo must then implant in the uterus successfully, useful for this particular aspect of toxicity to modify the adage
of Paracelsus to “It is the timing of the dose that makes the poi-
son.” That is, the dose of the toxic chemical and its resultant
Sexual effects will be dependent on when in the life stage of the organ-
maturation Silas ism that the chemical is administered and evaluated.
Growth &
development production
& release
REPRODUCTIVE DEVELOPMENT
Lactation &
postnatal Fertilization AND SEXUAL DIFFERENTIATION
development
During the seventh week of human gestation, the male and
Zygote
female morphological characteristics begin to develop.
Parturition Gonadal differentiation depends on signals from the Y chro-
transport
Fetal mosome, which contains the genes necessary to induce tes-
development Implantation ticular morphogenesis. One of these signals is the SRY gene,
Embryogenesis
which is the sex-determining region on the short arm of the Y
chromosome and acts as a “switch” to initiate transcription of
FIGURE 20-1 Thereproductive cycle. other genes that contribute to testicular organogenesis. In the
 CHAPTER 20 Toxic Responses of the Reproductive System 305

Fetal testosterone level Miaienenal genital 7 until the 12th week of development. Development of the
external genitalia coincides with gonadal differentiation. Fetal
__ differentiation & growth
testicular androgens are responsible for the induction of
Genital tubercle formation |
masculinization of the androgynous external genitalia. Thus,
Leydig cell activity | Testisdescent | male, but not female, reproductive tract development is totally
Sertoli cell Ns Wolffian duct | hormonally dependent and inherently more susceptible to
endocrine disruption.
activity __ differentiation_
|Germ cell Mullerian duct |
migration? —_ regression |
GAMETOGENESIS
4 7 9 12 13 Se! ate
Gestation (weeks) The critical feature in the production of gametes is the process
of meiosis, in which there are two cell divisions with no inter-
FIGURE 20-2 Male sexual differentiation in humans
during gestation. (Reproduced with permission from Klonisch T, vening DNA replication. This results in four daughter cells that
Fowler PA, Hombach-Klonisch S: Molecular and genetic regulation of possess half of the chromosome complement ofthe parent cell.
testis descent and external genitalia development, Dev Biol, 2004 The mammalian oocyte (Figure 20-3) begins meiosis during
Jun1;270(1):1-18.) fetal development but arrests partway through meiosis I and
does not complete the first division until ovulation; the second
division is completed only if the egg is fertilized. In the males,
absence of the SRY protein, the gonad remains indifferent for a
meiosis begins at puberty and is a continuous process, with
short period of time before differentiating into an ovary.
spermatocytes progressing from prophase to the meiotic sec-
Interstitial Leydig cells produce the male sex hormone testos-
ond division in little more than a week. This difference in strat-
terone, which induces masculine differentiation of the Wolffian
egy has implications for the action of toxicants and critical time
duct (aka mesonephric duct) and external genitalia. Figure 20-2
periods when these cells may be vulnerable to attack. Critical to
provides a diagrammatic representation of sexual differentia-
this is the understanding that the complement ofoocytes avail-
tion in the human male. In rodent and human species, fetal tes-
able to the mammalian female is complete at birth, whereas in
ticular androgen production is necessary for proper testicular
the male there is significant stem cell (spermatogonial) renewal
development, normal male sexual differentiation, and differen-
to maintain the significantly higher number of germ cells avail-
tiation of the Wolfhan ducts into the epididymides, vasa defer-
able in males.
entia, and seminal vesicles.
Androgens derived from the Leydig interstitial cells stimu-
late the Wolfhan ducts to form the male genital ducts, while NEONATAL DEVELOPMENT
Sertoli cells produce Millerian-inhibiting substance (aka anti-
Miillerian hormone), which suppresses development of the Late in gestation and at birth, male rats display longer ano-
paramesonephric (Miillerian) ducts, or female genital ducts. genital distances (AGD) than do female rats, with neonatal
In the humans, the external genitalia are indistinguishable male AGD being more than twice as long as females. There are
until the ninth week of gestation, and not fully differentiated homologous sex differences in humans. In many mammalian

Spermatogenesis Oogenesis
Spermatogonium @)44xy 44xx @) Oogonium
Mitosis
Primary ae Primary
spermatocyte / oocyte

Secondary e We aes Secondary

spermatocyte
cane ») oocyte and
Le first polar body
22x 22a (22y 22y Fertilized
Spermatid = e@ oe), ©) 4 ~@ @©6¢4
eS)
ovum and
Bie S ce D2XENZ2X eX 2X polar bodies
Spermatozoon @ 22x @ 22x @22y | Metamorphosis 22x ory

FIGURE 20-3 Cellular replication (mitosis) and cellular reductive divisions (meiosis) involved in spermatogenesis, oogenesis, and
fertilization.
306 UNIT 4 Target Organ Toxicity

species, including humans and rats, males of the species gonadarche characterized by the onset of gonadal hormone
engage in more aggressive play than do females. Both AGD production. In females, secretion of androgens from theca cells
and behavior can be altered by exposure to hormonal and and estradiol from granulosa cells of maturing follicles prior
antihormonal agents. to ovulation is followed by secretion of progesterone from the
corpus luteum after ovulation. In males, LH stimulates tes-
ticular synthesis and secretion of androgens and insulin-like
INFANTILE DEVELOPMENT peptide 3 hormone from the Leydig cells of males.
Premature thelarche (secondary breast development) and
During the infantile period of development, emergence of the
premature adrenarche are often referred to as pseudopreco-
nipple buds and areolae in females as well as maturation of the
cious puberty when the full spectrum of pubertal changes does
hypothalamic-pituitary axis occurs. Emergence of the nipple
not occur. Premature thelarche in girls and gynecomastia in
buds is prevented in males by prenatal androgen-induced atro-
boys result from direct exposure to estrogen-containing per-
phy of the nipple anlagen. Prenatal androgen-treated females
sonal care and “natural” products. Untoward consequences of
may display reproductive tract malformations (retained male
these conditions may occur with prolonged exposure, includ-
tissues or vaginal agenesis).
ing shortened stature due to effects of estrogens on the growth
plates of the long bones and sexual-social behavior that is inap-
PUBERTAL DEVELOPMENT propriate for the chronological age of the child. Concerns have
also been expressed that premature thelarche may enhance
Puberty is initiated by activation of the hypothalamic- the likelihood of developing diseases like breast cancer and
pituitary-gonadal (HPG) and hypothalamic-pituitary—adrenal endometriosis. ;
(HPA) axes (see Figure 20-4). At the onset, the HPG axis The association of pubertal alterations with environmental
releases gonadotropin-releasing hormone (GnRH) pulses exposure to persistent halogenated organic chemicals such as
with increasing frequency and amplitude that induces comple- polychlorinated biphenyls (PCBs), brominated flame retar-
mentary pulsatile secretions of luteinizing hormone (LH) and dants, dioxin, hexachlorobenzene, endosulfan, and heavy met-
follicle-stimulating hormone (FSH) from the anterior pitu- als also has been studied but a consensus about the causative role
itary. In turn, LH and FSH stimulate the gonads inducing of these chemicals in altering puberty has not been achieved.

PRES CTOTS REND

| Sperm prod.
NER |
Ova prod.,
__ Menarche

Female
a
=
(Adrenal
Se cortex
Female/Male

Androgen+ Estrogen+ Androstenedione+

DHEA+
Inhibin-
activin+
Development of: Development of: Development of:
+ Penis « Breasts
» Pubic hair * Ovaries
(tPubichair
«Testes _ + Uterus

FIGURE 20-4 Endocrine control of puberty in males and females. Prod., production.
 CHAPTER 20 Toxic Responses of the Reproductive System 307

chemicals (EDCs). Also, onset of pubertal landmarks in rats

is delayed after peripubertal exposures to antiandrogenic
chemicals. Throughout puberty and into adulthood, the sex
‘acento > +/- |Hypothalamus |
so el a ea . accessory glands and other androgen-dependent tissues
(i.e., muscles and nervous system) continue to depend on tes-
! [enkH ! tosterone and 5a-dihydrotestosterone for maturation and
ee
L----> +/-| Anterior pituitary |
Be ct Saat maintenance of function.

PU ioaee ut SEXUAL MATURITY

Hypothalamo-pituitary—-Gonadal Axis
FSH and LH are glycoproteins synthesized and released from
the anterior portion of the pituitary gland (adenohypophysis).
Hypothalamic neuroendocrine neurons secrete specific releas-
ing or release-inhibiting factors into the hypophyseal portal sys-
tem, which carries them to the adenohypophysis, where they act
Oviduct to stimulate or inhibit the release of hormones. GnRH acts on
gonadotropic cells, thereby stimulating the release of FSH and LH.
FIGURE 20-5 Endocrine control of the female reproductive The neuroendocrine neurons have nerve terminals contain-
cycle. CNS, central nervous system; GnRH, gonadotrophin-releasing ing monoamines (norepinephrine, dopamine, and serotonin)
hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; that impinge on them. Reserpine, chlorpromazine, and mono-
Prl, prolactin;T,testosterone; E2, estradiol; P4, progesterone.
amine oxidase (MAO) inhibitors modify the content or actions
of brain monoamines that affect gonadotropin production.
In females (Figure 20-5), LH acts on thecal cells of the ovary
Rodent Models of Puberty to induce steroidogenesis, particularly the production of pro-
Rodents are important animal models in the study of the gesterone and androgens that are transferred to the granulosa
effects of toxicants on puberty. In the laboratory rats, the stan- cells that can be stimulated by FSH to produce estradiol. These
dard landmarks of puberty are the age of preputial separation steroids provide feedback on the hypothalamus and pituitary
(PPS) in the males, and the ages of vaginal opening (VO) and to regulate gonadotropin production.
first estrus in females. Similarly in the males (Figure 20-6), FSH acts primarily
Onset of pubertal landmarks in rats can be altered after acute in on the Sertoli cells, but it also appears to stimulate the mitotic
utero and/or lactational exposures to 2,3,7,8-tetrachlorodibenzo- activity of spermatogonia. LH stimulates steroidogenesis in the
p-dioxin (TCDD), busulfan, androgens, and endocrine-disrupting interstitial Leydig cells. A defect in the function of the testis

Vas ._ Urinary bladder pc

deferens 4
a tz»,
Seminal (
- a GF,
AG
vesicle * (OG

Ejaculatory :
duct sis: Prostate
g gland
Bulbourethral
gland

Penis

Epididymis

, i | Tubule Sperm Interstitial

Se wall tails cell

Seminiferous
tubule

FIGURE 20-6 Male reproductive system.

308 UNIT 4 Target Organ Toxicity

(in the production of spermatozoa or testosterone) will tend to as severe thrombocytosis, and polycythemia vera. Busulfan
be reflected in increased levels of FSH and LH in serum because causes ovarian failure and prevents or delays the onset of
ofthe lack of the negative feedback effect of testicular hormones. puberty in girls.
The HPG feedback system is a very delicately modulated In rodents, administration of busulfan specifically inhibits
hormonal process. Toxicants that alter the hepatic and/or renal germ cell development. Offspring display permanent reproduc-
biotransformation of endogenous sex steroids may be expected tive and CNS alterations. The most severely affected females do
to interfere with the pituitary feedback system. not display estrous cycles or spontaneous sexual behavior as a
consequence of this exposure. Even though the gonads of both
sexes are affected at similar dosage levels, fertility and gonadal
Ovarian Function hormone production are much more easily disrupted in female
Oogenesis— About 400000 follicles are present at birth in than male offspring, because the steroid-producing cells in
each human ovary. After birth, many undergo atresia (follicu- the ovary fail to differentiate in the absence of the oocyte.
lar death), and those that survive are continuously reduced in A diagrammatic representation of the sites of actions of female
number. Any chemical that damages the oocytes will acceler- reproductive toxicants is presented in Figure 20-7.
ate the depletion of the pool and can lead to reduced fertility
in females. About one-half of the numbers of oocytes present
at birth remain at puberty; the number is reduced to about Ovarian Cycle
25.000 by 30 years of age. About 400 primary follicles will yield The cyclic release of pituitary gonadotropins involving the
mature ova during a woman's reproductive life span. During secretion of ovarian progesterone and estrogen is depicted in
the approximately three decades of fecundity, follicles in vari- Figure 20-8. These female sex steroids determine ovulation
ous stages of growth can always be found. After menopause, and prepare the female accessory sex organs to receive the
follicles are no longer present in the ovary. male sperm. This axis can be disrupted, resulting in infertility
Although ovarian weight does not fluctuate during the at any level of the endocrine system. For example, chemicals
estrous cycle, ovarian weight and histology can provide very that block the LH surge transiently can prevent or delay ovula-
useful information about the effects of toxicants on the female tion, resulting in infertility or lower fecundity due to delayed
reproductive system. Ovarian weight can be reduced by either fertilization of ova.
depletion of oocytes or disruption of the HPG axis. Toxicants
induce various ovarian lesions, including polyovular follicles,
oocyte depletion, interstitial cell hyperplasia, corpora albani- Postovarian Processes
cans, and an absence of corpora lutea. Female accessory sex organs function to bring together the
ovulated ovum and the ejaculated sperm. The chemical com-
Case Study: Busulfan—Busulfan is an alkylating agent used position and viscosity of reproductive tract fluids, as well as the
to treat several diseases in humans, including chronic myelog- epithelial morphology of these organs, are controlled by ovar-
enous leukemia, certain myeloproliferative disorders such ian (and trophoblastic) hormones.

PR —
|

a ae DNere
| Site of action is |
development and/or altered |
| —> Mate . | corpus luteum function |
cyclicity tes? |

Count f PACE, See res 2 ths a aT ae |

implantation | Site of action is fertilization or

| maintenance of implantation |

| Decreased

Decreased
|—> fedite ae
ofactionisovaryor
|
corpora
lutea
_| hypothalamus/pituitary |

FIGURE 20-7 Sites of action for female reproductive toxicants.

 CHAPTER 20 Toxic Responses of the Reproductive System 309

Comparative endocrinology of menstrual and estrous cycles and early pregnancy

©0©O©
.
G&G (2)
cs Gy
4
«
Gi
\ is
as ‘
Q ¢

ie 2A Bi GE AE I IG See ee OLN 4 eS eel 2 Ore Ole 4S

Days from LH peak

Rat Ovulation
Ovulation and coitus

—~=_<-" = -

FIGURE 20-8 Comparison of the timing of the human and rat cycles. LH, leutinizing hormone; FSH, follicle-stimulating hormone;
PRL, prolactin; E,, estradiol; P,, progesterone; hCG, human chorionic gonadotropin; rCG, rat chorionic gonadotropin.

Oviducts—The oviducts provide conduit for, and aid the TESTICULAR STRUCTURE
movement of, gametes. Movement of both the oviducts and
AND FUNCTION
fimbriae (finger-like projections of the oviduct) are under the
influence of the autonomic nervous system (ANS). Therefore, The blood-testis barrier between the lumen of an interstitial
drugs known to alter the ANS may alter function and fertility. capillary and the lumen of a seminiferous tubule impedes or
Progression offertilized eggs through the oviduct and uterus is prevents the free exchange of chemicals/drugs between the
under hormonal regulation, and chemicals such as the estro- blood and the fluid inside the seminiferous tubules.
gens can stimulate oviductal transport and interfere with uter-
ine endometrial function, precluding implantation.
Targets for Toxicity
Uterus— Uterine endometrium reflects the cyclicity of the For an adult male, there are numerous potential targets for the
ovary as it is prepared to receive the conceptus. In the prolif- action of chemicals on the system (Figure 20-9). These would
erative stage, estrogens from the developing follicle increase range from the action of dopamine analogs on the hypothala-
the thickness of the endometrium and development of uterine mus interrupting the normal secretion of GnRH, the action of
glands. The secretory phase follows ovulation and is character- estrogens on the pituitary (and hypothalamus) to interfere with
ized by an edematous endometrium due to the action of the gonadotropin (LH and FSH) production through direct effects
uterine glands. Estrogen and progesterone from the corpus on spermatogenesis—where the vast majority of toxicants have
luteum guide the changes of this stage. If fertilization fails to their site of action. Perturbing the homeostasis of nutrients can
occur, the endometrium is shed (menstruation) and a new lead to direct effects on spermatogenesis and subsequent issues
cycle begins. Uterine weight and vaginal cytology change with fertility. Similarly, chemicals that have direct effects on
remarkably, but in a well-defined way during the estrous cycle. the liver (e.g., CCl,) can disturb the normal metabolism of sex
These measures can be used as end points in assessment of the steroids leading to changes in clearance (predominantly of
toxic effects of chemicals. glucuronide and sulfate conjugates of hydroxytestosterones
310 UNIT 4 Target Organ Toxicity

Potential target sites Posttesticular Processes

--€NS__| Dopamine antagonists
Following the release of mature spermatids from the seminifer-
ous epithelium, the extraneous cytoplasm and organelles form
the residual body that is phagocytosed by the Sertoli cell and
moves from the periphery of the tubule to its base. These non-
Testicular
Cd vasculature Melatonin motile sperm are moved along the tubules by a peristaltic-like
Spermatogenesis action of the myoepithelial cells of the tubule and eventually
empty into the rete testis. Sperm are then moved into the effer-
ent ducts that exit the testis and enter the head of the epididy-
VitA,Zn
mis. The sperm undergo maturation in the head and body and
begin to acquire motility, whereas the tail is principally used
for sperm storage. Most mammals possess seminal vesicles and
a prostate, which are secretory in nature and produce fluid for
Paternal developmental toxicity | Cyclophosphamide the ejaculation of sperm to survive within the female repro-
ductive tract. Any disturbance in these components may have
FIGURE 20-9 Potential target sites for male reproductive an effect on subsequent fertility.
toxicants. Examples of agents shown in italics.

Erection and Ejaculation

in the male), indirectly affecting the HPG axis and exerting These physiological processes are controlled by the CNS but are
effects on male reproduction. modulated by the autonomic nervaus system. Parasympathetic
The testis also has a finely tuned circulatory system in nerve stimulation results in dilation of the arterioles of the
mammals, termed the pampiniform plexus, designed to shunt penis, which initiates an erection. Ejaculation is a two-stage
the arterial venous blood supply and aid in scrotal cooling. spinal reflex involving emission and ejaculation. Emission
Some chemicals (e.g., cadmium) can actually target this is the movement of the semen into the urethra; ejaculation is
structure and the testicular circulatory system to induce the propulsion of the semen out of the urethra at the time of
ischemic shock to the testes, resulting in injury and reduced orgasm. Emission is a sympathetic response produced by con-
fertility. traction of the smooth muscle of the vas deferens and seminal
vesicles. Semen is ejaculated out of the urethra by contraction
ofthe bulbocavernosus muscle.
Testicular Structure and Spermatogenesis Little is known concerning the effects of chemicals on erec-
The overwhelming number of chemicals known to affect the tion or ejaculation. Pesticides, particularly the organophos-
male reproductive system appears to do so by a direct effect phates, are known to affect neuroendocrine processes involved
on the testis and interfere with the process of spermatogen- in erection and ejaculation. Many drugs acting on the auto-
esis. Spermatogenesis is an extremely ordered process in the nomic nervous system affect potency. Impotence, the failure
rat. The spermatogonia have populations that act as the stem to obtain or sustain an erection, is rarely of endocrine origin;
cells for the seminiferous tubules and a proportion of these cells more often, the cause is psychological.
then undergo a series of mitotic divisions to increase numbers Penile erection depends upon the relaxation of smooth mus-
and move into meiotic prophase, and are then committed to cles in the corpora cavernosa. In response to sexual stimuli,
becoming spermatozoa, which are released into the lumen of cavernous nerves and endothelial cells release nitric oxide,
the seminiferous tubule. which stimulates the formation of cyclic guanosine mono-
Different biochemical events can go on during the different phosphate (GMP) by guanylate cyclase. Sildenafil (Viagra)
stages and indeed this can provide clues as to potential modes selectively inhibits cCGMP-specific phosphodiesterase type 5 in
of action of chemicals that produce stage-specific lesions. Such cavernosal smooth muscle cells, thereby restoring the natural
occurrences do occur regularly with certain phthalate esters, erectile response.
glycol ethers, and antiandrogenic agents.
Once released into the seminiferous tubule lumen, sperm
proceed to the epididymis where they can also be the target of Case Studies for Effects on the Male
toxicant action. Chlorosugars and epichlorohydrin have both m-Dinitrobenzene—m-Dinitrobenzene (m-DNB) has been
been shown to inhibit energy metabolism in sperm, preventing extensively studied for its ability to produce a rapid deleterious
them from functioning normally. The number of environmen- effect on the rat testis. Testicular weight remained reduced for
tal chemicals that produce adverse responses in human males many weeks after the treatment period with significant dose-
is not large. All of these have been shown to induce effects in related effects on fertility, pregnancy rate, and implantation
rodents, and especially in rats, there may be differences in sen- success. Other studies have shown abnormal sperm function
sitivity based on dose. and failure of fertilization in rat in vitro fertilization (IVE)
 CHAPTER 20 Toxic Responses of the Reproductive System 311

studies. Detailed electron microscopic evaluation has shown progesterone dependent on the secretion of human chorionic
initial lesions to be present in the Sertoli cells of the testis, gonadotropin (hCG) by the trophoblast. Sufficient progester-
which results rapidly in germ cell apoptosis and death. one is produced by the trophoblast after 8 weeks of gestation in
humans to maintain pregnancy even in cases of ovariectomy.
Ethylene Glycol Monomethyl Ether (EGME)—EGME pro- Early in implantation the blastocyst contacts the endome-
duces testicular toxicity in various species. Sertoli cell vacuoles, trium and becomes surrounded by an outer layer (syncytiotro-
swollen germ cell mitochondria, and a breakdown of the mem- phoblast), which is a multinucleated mass of cells that erodes
brane between the Sertoli cell and the pachytene spermatocyte the endometrium, and the blastocyst implants. Placental circu-
have been described. Within hours, death of (probably) those lation is then established and trophoblastic tissue differentiates
pachytene spermatocytes follows. EGME is metabolized to into cytotrophoblast and syncytiotrophoblast cells. The syncy-
active intermediates methoxyacetaldehyde and methoxyacetic tiotrophoblast cells produce chorionic gonadotropin, chorionic
acid (MAA). Treating animals with MAA produces identical growth hormones, placental lactogen, estrogen, and progester-
testicular lesions as that of the parent compound. one, which are needed to achieve independence from the ovary
in maintaining the pregnancy. Shortly after implantation, the
syncytiotrophoblast becomes bathed by maternal venous blood,
FERTILIZATION which supplies nutrients and permits an exchange of gases.
Fertilization is the process whereby the genome from one gen- Generally, the placenta is quite impermeable to chemicals/
eration is passed to the next to begin the development of a new drugs with molecular weights of 1000 Da or more. Because
organism. In mammals, the oocyte is surrounded by two lay- most medications and xenobiotics have molecular weights of
ers: an outer layer of cumulus cells and an inner layer of extra- 500 Da or less, molecular size is rarely a factor in denying a
cellular matrix termed the zona pellucida. To reach the oocyte, drug’s entrance across the placenta and into the embryo/fetus.
the sperm must penetrate both layers requiring high motility, Placental permeability to a chemical is affected by placental
the release of sperm enzymes and the presence ofproteins that characteristics including thickness, surface area, carrier sys-
will facilitate binding of the sperm to the oocyte. Moreover, tems, and lipid-protein concentration of the membranes. The
once fertilization has occurred, mechanisms must be in place inherent characteristics of the chemical itself, such as its degree
to prevent the binding of further sperm to the fertilized oocyte. of ionization, lipid solubility, protein binding, and molecular
Sperm possess special enzymes to facilitate these activities. size, also affect its transport across the placenta.

IMPLANTATION PREGNANCY
Implantation is an intricately timed event that allows mammals Because the transition from early to midpregnancy in the rat
to nourish and protect their young during early development requires hormones from the feto-placental unit, if implanta-
and results from an intimate relationship between the devel- tion or uterine decidualization is blocked by a chemical, then
oping embryo and the differentiating uterus. Implantation the female would resume her estrous cycles and the corpora
can only occur when the embryo reaches the blastocyst stage lutea would regress. Chemicals that induce whole-litter loss
and gains implantation competency and the uterus, through at mid- to late pregnancy may cause abortions in some of the
steroid hormone-dependent changes, attains a receptive state. females, whereas others fail to deliver and appear pregnant for
This reciprocal interaction must occur between the blasto- an unusually long period of time.
cyst and uterus together with an increase in uterine vascular Many abortifacients induce pregnancy loss by reducing pro-
permeability at the site of blastocyst attachment. There are gesterone levels in the rat. Generally, reducing midpregnancy
four stages that comprise early implantation in mammals, (1) progesterone levels by half or more is sufficient to terminate
apposition and adhesion of the blastocyst to the uterine lumen, pregnancy.
(2) penetration of the epithelium, (3) decidualization of the
stromal cells, and (4) trophoblastic invasion into the stromal
vasculature. These four stages can vary in length and in precise PARTURITION
order in a species-dependent manner. Parturition is a complex process involving fetal, placental, and
maternal signals, and the precise molecular events controlling
PLACENTA this physiological process are not clear. Parturition is best to be
thought of as a release from the inhibitory effects of pregnancy
The placenta plays a key role in pregnancy, mediating exchanges on the myometrium of the uterus rather than an active pro-
between the mother and fetus and maternal tolerance of anti- cess, although the timing and order of the precise events are an
gens produced by the fetus. There are a huge number of dif- active process. For most mammals, the uterus is held in a qui-
ferent placental types across species. In humans, the pituitary escent state by high levels of progesterone and it is the decrease
gland is not required for the initiation and maintenance of of progesterone that provides the trigger for parturition, but
pregnancy, with maintenance of the corpus luteum to produce this does not appear to be the case in humans.
312 UNIT 4 Target Organ Toxicity

LACTATION between exposure to EDCs and adverse effects in several verte-

brate classes from fish to mammals.
The endocrine control oflactation is one of the most complex Reports of U-shaped (nonmonotonic), ultralow dose effects
physiological mechanisms. Mammogenesis, lactogenesis, and nonthreshold effects for EDCs are challenging some of
galactopoiesis, and galactokinesis are all essential to assure the basic assumptions of risk assessment for noncancer end
proper lactation. Prolactin is the key hormone oflactation and points. While the focus of this debate has centered on the low
seems to be the single most important galactopoietic (milk dose effects of bisphenol A, well-documented U-shaped dose
synthesis) hormone. Oxytocin, serotonin, opioids, histamine, response curves are known from many other in vitro and some
substance P, and arginine-leucine modulate prolactin release in vivo studies. Thus, for some EDCs the timing of exposure
by means of an autocrine/paracrine mechanism, whereas dictates not only the effect, but also whether the effects are
estrogen and progesterone hormones can act at the hypotha- adverse or beneficial. Even when administered during adult
lamic and adenohypophysial levels. Human placental lactogen life, drugs with EDC activity can simultaneously have a benefi-
and growth factors play an essential role to assure successful cial effect on one tissue and an adverse effect on another.
lactation during pregnancy with oxytocin being the most pow-
erful galactokinetic (milk ejection) hormone.
Known Effects of EDCs in
Humans and Animals
SENESCENCE The list of chemicals that are known to affect humans, domes-
Reproductive senescence is usually preceded by a dysregulation tic animals, and/or wildlife via functional developmental tox-
of the HPG axis. This dysregulation leads to alterations in serum icity or endocrine mechanisms includes 2,3,7,8-TCDD, PCBs
HPG hormones, accompanied an upregulation in GnRH, LH, and polychlorinated dibenzofurans (PCDFs), methylmercury,
and activin activities and a decrease in steroids in the brain. In ethinylestradiol, alkylphenols, plant sterols, fungal estrogens,
females, reproductive senescence (menopause) is associated with androgens, chlordecone, DBCP, dichlorodiphenyltrichloro-
a transition from regular to irregular estrus (menstrual) cycles ethane (DDT), and other organochlorine compounds. In addi-
leading to acyclicity and ultimately a loss of fertility. Perinatal tion to these xenobiotics, over 30 different drugs taken during
exposure to toxicants with estrogenic activity can defeminize pregnancy have been found to alter human development as
the HPG axis such that the female rats are acyclic and infertile, a consequence of endocrine disruption. These drugs are not
whereas less affected females display the “delayed anovulatory limited to estrogens, like diethylstilbestrol (DES). EDCs are
syndrome” and become anovulatory and acyclic at an early age. known to alter human development via several mechanisms
In males, a decrease in androgen is noted in around 20% besides the estrogen receptor (ER), including binding to reti-
of fit 60-year-old men, but the value of androgen supplementa- noic acid (RAR and RXR) receptors, and inhibiting synthesis
tion is not clear with regard to reproductive senescence. of steroidogenic enzymes or thyroid hormones. Findings on
the effects of background levels of PCBs on the neurobehav-
ioral development of the child have contributed to the con-
ENDOCRINE DISRUPTION cerns about the effects of EDCs on human health via alteration
of hormone function.
Currently, the potential effects of endocrine disrupting chemi-
cals (EDCs) on human health and the proven effects of EDCs Effects of Drugs on Human Sexual Differentiation—
on wildlife are a major focus among the scientific community. Exposure to hormonally active chemicals during sex differ-
It has been suggested that in utero exposure to environmen- entiation can produce pseudohermaphroditism. Androgenic
tal estrogens, antiandrogens, or chemicals like phthalates drugs like danazol and methyltestosterone can masculinize
or 2,3,7,8-TCDD could be responsible for the reported 50% human females (i.e., “female pseudohermaphroditism”). The
decline in sperm counts in some areas and the apparent increase drug aminoglutethimide, which alters steroid hormone syn-
in cryptorchid testes, testicular cancer, and hypospadias. thesis in a manner identical to many fungicides, also masculin-
Phthalate exposures have been associated with reduced ano- izes human females following in utero exposure.
genital distances (AGD) in boys and lower testosterone levels Transplacental exposure of the developing fetus to DES
in men. In females, exposure to EDCs during development causes clear cell adenocarcinoma of the vagina, as well as gross
could contribute to earlier age of puberty and to increased structural abnormalities of the cervix, uterus, and fallopian
incidences of endometriosis and breast cancer. Besides pes- tube. These DES-exposed women are more likely to have an
ticides and other toxic substances in the environment, many adverse pregnancy outcome, including spontaneous abortions,
compounds that are phytosterols, estrogens, antibiotics, beta- ectopic pregnancies, and premature delivery. Some of the path-
blockers, antiepileptics, and lipid-regulating agents have ological effects that develop in males following fetal DES expo-
significant endocrine-disrupting activity and are capable of sure appear to result from an inhibition of androgen action or
inducing reproductive toxicity. synthesis (underdevelopment or absence of the vas deferens,
In the area of wildlife toxicology and ecosystem health, it epididymis, and seminal vesicles) and anti-Miillerian duct fac-
is apparent that clear-cut cause and effect relationships exist tor (persistence of the Miillerian ducts).
 CHAPTER 20 Toxic Responses of the Reproductive System 313

Known Effects of Plant and Fungal Products in Animals were affecting gonadal differentiation and skewing sex ratios.
and Humans—Although most naturally occurring environ- Effluents from beef-cattle concentrated animal feeding opera-
mental estrogens are relatively inactive, the phytoestrogen miro- tions have been shown to display androgenicity.
estrol is almost as potent as estradiol in vitro and even more
potent than estradiol when administered orally. In addition,
many plant estrogens occur in such high concentrations that Environmental Antiandrogens
they induce reproductive alterations in domestic animals. Fungicides—Vinclozolin and procymidone are two mem-
“Clover disease,’ which is characterized by dystocia, prolapse bers of the dicarboximide fungicide class that act as AR antag-
of the uterus, and infertility, is observed in sheep that graze on onists. These pesticides, or their metabolites, competitively
highly estrogenic clover pastures. Permanent infertility can be inhibit the binding of androgens to AR, leading to an inhibi-
produced in ewes by much lower amounts of estrogen over a tion of androgen-dependent gene expression.
longer time period than are needed to produce “clover disease.” Administration of vinclozolin during sexual differentiation
demasculinizes and feminizes the male rat offspring such that
Known Effects of Organochlorine Compounds in treated males display female-like AGD at birth, retained nip-
Humans—Several pesticides and toxic substances have been ples, hypospadias, suprainguinal ectopic testes, a blind vaginal
shown to alter human reproductive function. An acciden- pouch, and small to absent sex accessory glands.
tal high-dose in utero exposure to PCBs and PCDFs has been Procymidone induces shortening of the AGD in male pups,
associated with reproductive alterations in boys, increased still- and older males display retained nipples, hypospadias, crypt-
births, low birth weights, malformations, and IQ and behavioral orchidism, cleft phallus, a vaginal pouch, and reduced sex
deficits. In addition to the effects associated with this inadver- accessory gland size. Fibrosis, cellular infiltration, and epithe-
tent exposure, subtle adverse effects were seen in infants and lial hyperplasia are noted in the dorsolateral and ventral pros-
children exposed to relatively low levels of PCBs and PCDFs. tatic and seminal vesicular tissues in adult offspring.
One metabolite of DDT (mitotane, 0,p’-DDD) was found to Prochloraz is a fungicide that disrupts reproductive devel-
alter adrenal function with sufficient potency to be used as a opment and function by inhibiting the steroidogenic enzymes
drug to treat adrenal steroid hypersecretion associated with 17,20-lyase and aromatase and it is an AR antagonist. Prenatal
adrenal tumors. In addition, lower doses of mitotane restored exposure to prochloraz reduces fetal testis testosterone and
menstruation in women with spanomenorrhea associated with increases progesterone production without affecting Leydig
hypertrichosis. cell insl3 mRNA levels. Also, prenatal prochloraz treatment
delayed parturition and altered reproductive development in
Occupational Exposures—Occupational exposure to the male offspring in a dose-related manner. Treated males dis-
pesticides and other toxic substances (ie., chlordecone and played reduced AGD and female-like areolas and high-dose
DBCP) in the workplace has been associated with reduced males displayed hypospadias, but the epididymides and guber-
fertility, lowered sperm counts, and/or endocrine alterations nacular ligaments were relatively unaffected.
in male workers. Workers exposed to high levels of chlorde-
cone, an estrogenic and neurotoxic organochlorine pesticide, Linuron (Herbicide)—This herbicide binds rat and human
displayed intoxication, severe neurotoxicity, and abnormal tes- AR and inhibits DHT-hAR-induced gene expression in vitro.
ticular function. Male workers involved in the manufacture of In utero linuron exposure produces male rats displaying epi-
4,4’-diaminostilbene-2,2’-disulfonic acid (DAS), a key ingredi- didymal and testicular abnormalities. In contrast to the effects
ent in the synthesis of dyes and fluorescent whitening agents, of vinclozolin and procymidone, malformed external genita-
had lower serum testosterone levels and reduced libido as com- lia and undescended testes were rarely displayed by linuron-
pared with control workers. Thus, it is surprising that occupa- exposed males. Interestingly, the syndrome of effects for linuron
tional exposures to potential EDCs at effective concentrations is atypical of an AR antagonist and more closely resembles those
have not been entirely eliminated from the workplace. seen with in utero to phthalates. Also, fetal testosterone produc-
tion is significantly reduced in linuron-treated fetal males.

Environmental Androgens p,p’-DDE (Pesticide Metabolite)—p,p’-DDE displays AR

Androgenic activity has been detected in several complex antagonism both in vivo and in vitro. In vitro, p,p’-DDE binds
environmental mixtures. Pulp and paper mill effluents (PME) to the AR and inhibits androgen-dependent gene expres-
include a chemical mixture that binds androgen receptors sion. In vivo, p,p’-DDE delays pubertal development in male
(AR) and induces androgen-dependent gene expression in rats by about 5 days at 100 mg/kg/day and inhibits androgen-
vitro. This mode of action is consistent with the masculinized stimulated tissue growth. p,p’-DDE administered male rats in
female mosquitofish (Gambusia holbrooki) collected from con- utero reduces AGD, induces nipples, and permanently reduces
taminated sites. Male-biased sex ratios of fish embryos have androgen-dependent organ weights.
been reported in broods of eelpout (Zoarces viviparus) in the
vicinity of a large kraft pulp mill on the Swedish Baltic coast, Phthalates (Plasticizers)—In utero, some phthalate esters
suggesting that masculinizing compounds in the effluent alter the development of the male rat reproductive tract at
314 UNIT 4 Target Organ Toxicity

relatively low dosages. Prenatal exposures to DBP, benzyl-butyl Hershberger Assay—The second in vivo assay in tier 1, the
phthalate (BBP), di-isononyl phthalate (DINP), and diethyl- Hershberger assay, detects antiandrogenic activity simply by
hexylphthalate (DEHP) cause a syndrome of effects, including weighing androgen-dependent tissues in the castrated male
underdevelopment and agenesis of the epididymis and other rat. In this assay, weights of the ventral prostate, Cowper's
androgen-dependent tissues and testicular abnormalities. The glands, seminal vesicle (with coagulating glands and fluids),
phthalates are unique in their ability to induce agenesis of the glans penis, and levator ani/bulbocavernosus muscles are mea-
gubernacular cords, a tissue whose development is dependent sured after 10 days of oral treatment with the test compound.
on the peptide hormone insulin-like peptide 3. This assay is very sensitive for detection of androgens and
antiandrogens.

Environmental Estrogens Pubertal Female Rat Assay—The third in vivo mammalian/rat

Methoxychlor is an estrogenic pesticide that produces assay in the screening battery is the pubertal female rat assay.
estrogen-like effects. This pesticide requires metabolic acti- Weanling female rats are dosed daily by gavage for 21 days
vation in order to display full endocrine activity in vitro. while the age at VO (puberty) is monitored. The females are
The active metabolites of methoxychlor activate estrogen- necropsied at about 42 days of age. This assay detects altera-
dependent gene expression in vitro and in vivo in the female tions in thyroid hormone status, HPG function, inhibition of
rats, thereby stimulating an uterotropic response, accelerat- steroidogenesis, estrogens, and antiestrogens, and has been
ing VO and inducing constant estrus, and reducing infertility. found to be highly reproducible and very sensitive to certain
In the ovariectomized female rat, methoxychlor also induces endocrine activities including estrogenicity, inhibition of ste-
estrogen-dependent reproductive and nonreproductive roidogenesis, and antithyroid activity.
behaviors, including female sex behaviors, running wheel
activity, and food consumption. Alternative Screening Assays— Alternative in vivo assays
When given to the dam during pregnancy and lactation, were also discussed by EDSTAC and are currently being evalu-
both male and female offspring are affected. Females display ated by the EPA. If they are of sufficient sensitivity, specificity,
irregular estrous cycles and reduced fecundity, whereas male and relevance, they might replace or augment current tier 1
fertility is unaffected at doses up to 200 mg/kg/day. assays.
Ethinylestradiol is a synthetic derivative of estradiol that is in
almost all modern formulations of combined oral contracep- Pubertal Male Rat Assay—The pubertal male rat assay detects
tive pills. This drug is found in many aquatic systems contami- alterations of thyroid function, HPG maturation, steroidogen-
nated by sewage effluents, originating principally from human esis, and altered steroid hormone function (androgen). Intact
excretion. Thus, ethinylestradiol plays a major role in causing weanling males are exposed to the test substance for approxi-
widespread endocrine disruption in wild populations of fish mately 30 days. The age at puberty is determined by measur-
species and other lower vertebrate species. ing the age at PPS, and reproductive tissues are evaluated and
serum taken for optional hormonal analyses.

EDC Screening Programs In Utero-Lactational Assay—The EDSTAC recommended

The Endocrine Disruptor Screening and Testing Advisory development of utero-lactational assays due to the unique sen-
Committee (EDSTAC) proposed (1) a process to prioritize sitivity of the fetal reproductive system to certain toxicants.
chemicals for evaluation and recommendations, for (2) screen- One version of the assay takes about 80 days and uses approxi-
ing (Tier 1), and for (3) testing (Tier 2) batteries for EDCs. The mately 10 litters per group (120-150 pups). In this protocol,
recommended screening battery was designed to detect altera- androgens and antiandrogens can be detected in approxi-
tions of HPG function; estrogen, androgen, and thyroid hor- mately 2 to 3 weeks, and EDCs with antithyroid activity can be
mone synthesis; and AR- and ER-mediated effects in mammals detected in infant or weanling offspring after four to five weeks
and other taxa. of maternal treatment.

In Vivo Mammalian Assays—EDSTAC recommended the

laboratory rat as the species of choice for the endocrine screen- TESTING FOR REPRODUCTIVE TOXICITY
ing and testing assays. The EDSTAC proposed three short-
term in vivo mammalian assays for the tier | screening battery: Screens and Multigeneration Studies
the uterotropic, Hershberger, and pubertal female rat assays. Significant attention has focused on the development of
“screens” for reproductive toxicity. The screens currently
Uterotropic Assay—Estrogen agonists and antagonists are employed have been developed to prioritize chemicals for
detected in a 3-day uterotropic assay using subcutaneous more comprehensive testing.
administration of the test compound. The selected uterotropic The most comprehensive assessment of reproductive toxic-
assays for estrogens and antiestrogens use either the intact juve- ity would be provided by a protocol that exposes the animal
nile or the castrated ovariectomized adult/juvenile female rat. model throughout the reproductive cycle (see Figure 20-1)
 CHAPTER 20 Toxic Responses of the Reproductive System 315

Multigeneration reproduction study because only these protocols expose the animals during all
critical stages of development and examine reproductive func-
Sexual tion of offspring after they mature.
maturation
Gamete Although the EPA multigenerational test provides for a com-
Growth &
development == production prehensive evaluation of the F, or parental generation, too few
& release F, animals (offspring with developmental exposure) are exam-
ined after maturity to detect anything but the most profound
Lactation &
postnatal Fertilization reproductive teratogens. F, animals within a dose group typi-
development cally respond in a similar fashion to the chemical exposure;
however, the response to toxicants in utero can vary greatly
Parturition Zygote even within a litter with only a few animals displaying severe
transport reproductive malformations in the lower dosage groups.
Fetal “Transgenerational” protocols typically use fewer litters
development Implantation (7 to 10 per dose group) but examine all of the animals in each
Embryogenesis litter. These protocols actually use fewer animals but provide
enhanced statistical power to detect reproductive effects in the
FIGURE 20-10 Multigeneration reproduction study. F, generation. The lifelong exposure of both males and females
in the F, generation, which allows one to detect effects induced
in utero, during lactation, or from direct exposure after
puberty, can confound the identification of when the effect
and assesses multiple end points at different life stages during was induced (i.e., during adulthood versus development) or of
this continuous exposure. The protocol and guideline com- which sex was affected.
ing closest to this ideal is the multigeneration reproduction Some EDCs disrupt pregnancy by altering maternal ovarian
study (Figure 20-10) used for the assessment of chemicals, hormone production in F, dams at dosage levels that appear
pesticides, and some food additives. Multigeneration stud- to be without direct effect on the offspring. In such cases, the
ies normally encompass detailed measurements of reproduc- standard EPA multigenerational protocol with minor enhance-
tive performance (number of pregnant females from number ments would be recommended, or a transgenerational protocol
of pairs mated, number of females producing a litter, litter with exposure continued after weaning. The transgenerational
size, and number of live pups with their birth weights and or in utero lactational protocols fill a gap in the testing program
sex). Measurement of growth and analysis of the reproductive for EDCs that should be used only on a case-by-case basis.
organs in the F, parental generation is conducted. Similar mea-
surements to those undertaken for the F, are made on the F,
parents, and the offspring are examined at birth (and sexually Testing Pharmaceuticals
dimorphic end points may be collected such as AGD), at wean- In the case of pharmaceuticals, it is rare for multigeneration
ing, and at puberty (particularly the assessment of VO and studies to be conducted, because it is not common for all the
time of first estrus in females and balanopreputial separation in population to use a specific drug and exposure to the drug is
males) in addition to the adult measurements of reproductive over many different life stages, and not necessarily chronic.
performance, organ weights, histology, etc. Typically three specific studies are undertaken:
1. A study of fertility and early embryonic development
(see Figure 20-11). Parental adults are exposed to the test
Testing for Endocrine-disrupting chemical for 2 weeks (females) or 4 weeks (males) prior
Chemicals to breeding and then during breeding. Females then con-
In the tiered screening and testing approach, only chemicals tinue their exposure through to implantation. Males can
that display positive reproducible responses in tier 1 screening be necropsied for the end points noted above for the mul-
(T1S) would continue evaluation in full-life cycle or multigen- tigeneration studies after pregnancy has been confirmed,
erational tests. In tier 2 testing (T2T), issues of dose-response, and for the pregnant females, necropsy takes place any
relevance of the route of exposure, sensitive life stages, and time after midgestation. Reproductive and target organs
adversity are resolved. are weighed and examined histologically, sperm param-
Data should be summarized in a manner that clearly delin- eters are assessed in males, and the uterine implantation
eates the proportion of animals that are affected. In teratology sites and ovarian corpora lutea are counted in females, as
studies, data are typically presented and analyzed in this man- well as live and dead embryos.
ner, indicating the number of malformed/number observed 2. A study of effects on pre- and postnatal development
on an individual and litter basis, whereas multigenerational including maternal function (see Figure 20-12). In this
studies are frequently presented and analyzed differently, even study, pregnant females are exposed from the time of implan-
when clear teratogenic and other developmental responses are tation until weaning of their offspring (usually PND 21
noted after birth. Multigenerational protocols are used in T2T in the rat). After cessation of exposure, selected offspring
316 UNIT 4 Target Organ Toxicity

Fertility and early embryonic Embryo-fetal development study

development study
Sexual
maturation Gamete
Sexual Growth & roduction
maturation Gamete development E
Growth & & release
roduction
development
& release Lactation &
postnatal Fertilization
Lactation &
development Assessment
postnatal Fertilization
development
ene

Parturition
Aerort
t

Assessment transp
Parturition
Zygote
transport Fetal
~-.-5- Implantati:on
development
Fetal
development implantation Embryogenesis
Embryogenesis FIGURE 20-13 Embryo-fetal developmental toxicity study
as used by FDA guidelines. Dosing starts at implantations and
FIGURE 20-11 Fertility and early embryonic study.
continues to closure of the hard palate with an assessment offetuses
just prior to parturition.

(one male and one female per litter) are raised to adult- One of the following three summary risk conclusions would
hood and then mated to assess reproductive competence. be applied to the drug label: (1) the drug is not anticipated to
These animals are observed for maturation and growth produce reproductive and/or developmental effects above the
(but are not exposed). Puberty indices, as employed in the background incidence for humans when used in accordance
multigeneration study, are measured. In addition, sensory with the dosing information on the product label; (2) the drug
function, reflexes, motor activity, learning, and memory may increase the incidence of adverse reproductive and/or
are also evaluated. developmental events; or (3) the drug is expected to increase
3. A study of embryo-fetal development (see Figure 20-13). the incidence of adverse reproductive and/or developmental
This study tests for enhanced toxicity relative to that noted effects in humans when used according to the product label.
in pregnant females and, unlike the previous two studies, An examination of the reproductive cycle in a comparison
is normally conducted in two species (typically the rat of these three most likely options for FDA studies indicates an
and rabbit). Exposure occurs between implantation and obvious gap in the exposure regime for the complete repro-
closure of the hard palate and females are killed just prior ductive cycle, namely exposure of weanlings through puberty
to parturition. At necropsy, dams are observed for any af- to adulthood. This exposure period has become of increas-
fected organs and corpora lutea are counted. Live and dead ing interest to many companies developing drugs for specific
fetuses are counted and examined for external, visceral, administration to infants and juveniles, and “bridging-type”
and skeletal abnormalities. protocols have been developed to specifically address toxicity
that may occur after exposure during this specific life stage.
Pre-and postnatal development study

Sexual
EVALUATION OF TOXICITY TO
maturation Shit REPRODUCTION
Growth &
development production
& release
There are a number of general points that the investiga-
tor should note in any estimation of potential reproductive
etere at j bi
Lactation & toxicity:
postnatal Fertilization ¢ Adequacy of experimental design and conduct. Was there
development
sufficient statistical power in the evaluation(s)?
¢ Occurrence of common versus rare reproductive deficits.
Parturition Zygote
Biological versus statistical significance.
transport
¢ Use of historical control data to place concurrent control
Fetal
development Implantation data into perspective and to estimate population back-
ground incidence of various reproductive parameters and
Embryogenesis
deficits.
FIGURE 20-12 Pre-and postnatal developmental toxicity « Known structure-activity relationships for inducing repro-
study. Dosing is from implantation until the litters are weaned. ductive toxicity.
 CHAPTER 20 Toxic Responses of the Reproductive System Bly

Concordance of reproductive end points. Did a decrease in may suggest developmental, as well as reproductive toxicity.
litter size relate to ovarian histology and changes in vaginal Likewise, findings in an F, generation animal may (or may
cytology? not) be reproduced in F, offspring. For example, effects in the
Did the reproductive deficits become more severe with F, generation on reproductive parameters may have resulted
increases in dose? Did histological changes at one dose level in the selection out of sensitive animals in the population,
become decrements in litter size and then reductions in thus not producing F, offspring for subsequent evaluation.
fertility at higher dose levels in any generation?
Did the reproductive deficits increase in prevalence
(more individuals and/or more litters) with dose level in any BIBLIOGRAPHY
generation? Diamanti-Kandarakis E, Gore AC: Endocrine Disruptors and Puberty.
Special care should be taken for decrements in reproductive New York: Humana Press, 2012.
parameters noted in the F, generation (and potentially later Gupta RC: Reproductive and Developmental Toxicology. Burlington,
generations) that were not seen in the F, generation, which MA: Academic Press, 2011.
 UNIT 4 Target Organ Toxicity

QUESTIONS

Which of the following cell types secretes anti-Miullerian 6. Reduction division takes place during the transition
hormone (AMH)? between which two cell types during spermatogenesis?
a. spermatogonium. spermatogonium and primary spermatocyte.
Leydig cell. primary spermatocyte and secondary spermatocyte.
Sertoli cell. secondary spermatocyte and spermatid.
primary spermatocyte. spermatid and spermatozoon.
a spermatid. tS
(oe
Qtekspermatozoon and mature sperm.

Penile erections are dependent on: Which of the following cell types is properly paired with
the CNS. the substance that it secretes?
sympathetic nerve stimulation. a. ovarian granulosa cells—progesterone.
helicine (penile) artery constriction. Leydig cells—ABP.
corpora cavernosa smooth muscle relaxation. ovarian thecal cells—estrogens.
oe
Oe
&
2 a spinal reflex arc. Sertoli cells—testosterone.
eB gonadotroph—LH.
ale
Se
The corpus luteum is responsible for the secretion of
which of the following hormones during the first part of 8. Which of the following .statements regarding male
pregnancy? reproductive capacity is FALSE?
a. estradiol and hCG. a. Klinefelter’s syndrome males are sterile.
b. progesterone and estradiol. b. FSH levels are often measured in order to determine
c. progesterone and hCG. male reproductive toxicity of a particular toxin.
d. FSHand LH. c. Divalent metal ions, such as An, Hg, and Cu, act
e. FSH and progesterone. as androgen receptor antagonists and affect male
reproduction.
All of the following statements regarding the hypothalamo- d. The number of sperms produced per day is approxi-
pituitary—gonadal axis are true EXCEPT: mately the same in all males.
a. FSH increases testosterone production by the Leydig e. ABP is an important biochemical marker for testicu-
cells. lar injury.
FSH and LH are synthesized in the anterior pituitary.
c. Estradiol provides negative feedback on the hypo- Reduction of sperm production can be caused by all of the
thalamus and the anterior pituitary. following diseases EXCEPT:
d. GnRH from the hypothalamus increases FSH and LH hypothyroidism.
release from the anterior pituitary. measles.
e. The LH spike during the menstrual cycle is respon- Crohn's disease.
sible for ovulation. renal failure.
le
I mumps.
Which of the following statements is FALSE regarding
gametal DNA repair? 10. Of the following, which is LEAST likely to be affected by
a. DNA repair in spermatogenic cells is dependent on estrogen?
the dose of chemical. nervous system.
b. Spermiogenic cells are less able to repair damage from musculoskeletal system.
alkylating agents. digestive system.
c. Female gametes have base excision repair capacity. cardiovascular system.
d. Meiotic maturation of the oocyte decreases its ability oS
>
o
fo urinary system.
to repair DNA damage.
e. Mature oocytes and mature sperm no longer have the
ability to repair DNA damage.
 Cos tis Ate Dasa
bark

Toxic Responses of the

Endocrine System
Patricia B. Hoyer and Jodi A. Flaws

INTRODUCTION Thyroid Hormone Clearance

Regulation of Thyroid Hormone Release
PITUITARY GLAND
Physiological Effects
Anatomy and Physiology Thyroid Toxicity
Pituitary Toxicity PCBs
PBDEs
ADRENAL GLANDS
Perchlorate
ADRENAL CORTEX Pesticides
Perfluorinated Chemicals
Steroidogenesis
Bisphenol A
Glucocorticoids
Phthalates
Adrenocortical Toxicity
In Vitro Toxicity PARATHYROID GLAND
Serum Binding Proteins
General Anatomy
Target Tissue Receptors
Parathyroid Toxicity
Neuroendocrine Regulation
PTH Structure and Synthesis
Mineralocorticoids
PTH Receptors
Fetal Adrenal
Physiological Effects
ADRENAL MEDULLA Regulation of PTH Release

Sympathetic Response ENDOCRINE PANCREAS

Catecholamines Role of the Liver in Glucose Production
Adrenergic Receptors
Pancreatic Hormones
General Toxicity Insulin
Pheochromocytoma
Glucagon
In Vitro Testing
Somatostatin
THYROID GLAND Interactions of Release
Metabolic Responses in Diabetes
General Anatomy
Pancreatic Toxicity
Thyroid Hormone Structure and Synthesis
Insulin Resistance
Thyroid Hormone Binding Proteins
In Vitro Testing
Thyroid Hormone Receptors
320 UNIT 4 Target Organ Toxicity

= Endocrine glands are collections of specialized cells that = Toxicants can influence the synthesis, storage, and
synthesize, store, and release their secretions directly release of hypothalamic-releasing hormones, adeno-
into the bloodstream. hypophyseal-releasing hormones, and the endocrine
= Each type of endocrine cell in the adenohypophysis is gland-specific hormones.
under the control of a specific releasing hormone from
the hypothalamus.

luteinizing hormone (LH), follicle-stimulating hormone (FSH),

INTRODUCTION
thyrotropic hormone (TTH), adrenocorticotropic hormone
Higher animals have developed the ability to regulate their (ACTH), and melanocyte-stimulating hormone (MSH). From
internal environment, independent of wide external fluctua- the pars nervosa, ADH enhances reabsorption of water by the
tions via the endocrine system. An endocrine system consists kidney and causes contraction of vascular smooth muscle,
of an endocrine gland that secretes a hormone, the hormone whereas oxytocin stimulates contraction of smooth muscle for
itself, and a target tissue that responds to the hormone. A parturition and milk let-down. These neurohypophyseal hor-
hormone is a chemical substance produced by a ductless mones are synthetized in the cell body of hypothalamic neu-
endocrine gland that is secreted into the blood. The hormone- rons, packaged in secretory granules, transported along the
producing glands include the pituitary, the thyroid and para- axon to terminal processes in the pars nervosa for release into
thyroids, the adrenals, the gonads, and the pancreas. There the blood.
are primarily three chemical classes of hormones: amino acid To maintain appropriate homeostasis, the endocrine organ
derivatives (catecholamines and thyroid hormones), peptide must constantly monitor systemic hormone concentrations
hormones (pancreatic), and steroids (derivatives of choles- accomplished in the form of negative feedback loops. For
terol). Endocrine glands are sensing and signaling devices that example, high circulating levels of cortisol will inhibit cortico-
are capable of responding to changes in the internal and exter- trophin-releasing hormone (CRH) release from the hypothala-
nal environments and coordinating multiple activities that mus, and the adrenocorticotropic hormone (ACTH) release
maintain homeostasis. from the pituitary.

PITUITARY GLAND Pituitary Toxicity

Studies consistently show that heavy metals may target
Anatomy and Physiology pituitary gland structure or function. Cadmium inhib-
The pituitary may be divided into two major subdivisions: the its prolactin, LH, and FSH secretion. Cadmium exposure
pars distalis and the pars nervosa (Figure 21-1). The pars dista- increases ACTH levels in rodents exposed during puberty
lis, adenohypophysis or anterior pituitary, is the largest subdi- and decreases ACTH levels in animals exposed during adult-
vision and it receives peptides from the hypothalamus through hood. Furthermore, studies indicate that acute exposure to
a capillary portal system (hypothalamo-hypophyseal vessels). cadmium decreases circulating GH levels, while longer period
The pars nervosa, neurohypophysis or posterior pituitary, has treatment increases circulating GH levels. Lead and mercury
its cell bodies in the hypothalamus with their axons stretching also decrease LH and FSH.
to the posterior lobe of the pituitary; therefore, functionally Environmental contaminants such as_ polychlorinated
and anatomically, the posterior pituitary is an extension of the biphenyls (PCBs) and polybrominated diphenylethers inhibit
hypothalamus. release of LH and FSH as well as TSH. The insecticide dimeth-
The releasing and release-inhibiting hormones are synthe- oate causes pituitary tumors in rats. Methoxychlor, dieldrin,
sized by neurons in the hypothalamus, transported by axo- and endosulfan increase prolactin and LH levels.
nal processes, and released into capillary plexus. They are Several phytoestrogens affect pituitary cells: coumestrol
transported to the adenohypophysis by the hypothalamic-— reduces pulsatile LH and suppresses the pituitary response to
hypophyseal portal system, where they interact with specific exogenous GnRH. Acute exposure to genistein or bisphenol A
populations of trophic hormone-secreting cells to govern the alter LH secretion as well.
rate of release of preformed hormones, such as growth hor- Industrial chemicalsalter pituitary structure orfunction. Flame
mone (GH), somatotropic hormone (STH), prolactin (PRL), retardants tetrabromo- and tetrachlorobisphenol A stimulate
 CHAPTER 21 Toxic Responses of the Endocrine System 324

Ill Ventricle

PRL-RH
PRL-RIF
(Dopamine)

LH (FSH)-RH

Optic
chiasm

Anterior Median
hypophyseal eminence
artery
Pars tuberalis

Pars distalis

GH-RH PRL-RH
PRL-RIF Pars
GH-RIH
(Dopamine) mn at _ nervosa
____ | Yo
Sees
“Chromophobes o ;

STH Prolactin LH FSH TTH ACTH 8-END MSH

(GH) (LTH) (a8)

FIGURE 21-1 Control of trophic hormone secretion from the adenohypophysis by hypothalamic-releasing hormones (RH)
and release-inhibiting hormones (RIH). The releasing and release-inhibiting hormones are synthesized by neurons in the hypothalamus,
transported by axonal processes, and released into capillary plexus in the median eminence. They are transported to the adenohypophysis by the
hypothalamic—-hypophyseal portal system, where they interact with specific populations of trophic hormone-secreting cells to govern the rate of
release of preformed hormones, such as growth hormone (GH), somatotropic hormone (STH), luteotropic hormone (LTH), luteinizing hormone
(LH), follicle-stimulating hormone (FSH), thyrotropic hormone (TTH), adrenocorticotropic hormone (ACTH), and melanocyte-stimulating hormone
(MSH). There are RIHs for those trophic hormones (e.g., prolactin and growth hormone) that do not directly influence the activity of target cells and
result in production of a final endocrine product (hormone) that could exert negative feedback control.

proliferation of a pituitary cell line. 2-Mercaptobenzothiazoleye ADRENAL GLANDS

Praiieinicutise duiecUDUe my Oc Veisecall cele ta cOMiACU Witt) |) SS
drinking water and cause pituitary tumors in chronically The adrenals are two small glands situated on the superior
exposed rats and mice. Finally, cynamide, a chemical used in _ poles of the kidneys. The major physiological role of the adre-
the treatment of alcoholics, increases the ACTH precursor nals is management of stress. Each adrenal gland is divided
mRNA in the anterior pituitary when co-administered with into two morphologically and functionally distinct regions: the
ethanol. outer cortex and the interior medulla. The adrenals have not
 ies)i)bo UNIT 4 Target Organ Toxicity

been as widely studied in toxicology as other endocrine glands, The outer region (cortex) synthesizes and secreted adreno-
even though it has been documented to be the most common corticosteroid hormones. The cortex consists of three zones
toxicological target of all. (Figure 21-2). The zona glomerulosa produces the miner-
alocorticoid aldosterone. The inner zones, fasciculata and
reticularis, produce glucocorticoids, corticosterone, and cor-
ADRENAL CORTEX tisol, as well as adrenal androgens. The inner region, medulla,
synthesizes and secretes catecholamines, epinephrine, and
The adrenal cortex regulates many physiological functions
norepinephrine.
such as the immune system, inflammation, water and electro-
lyte balance, carbohydrate and protein metabolism involving
such target organs as the liver, kidney, heart, bone, and nervous
system. The cortex is predisposed to the toxic effects of xeno- Steroidogenesis
biotic chemicals because many are lipophilic and the adrenal Adrenal steroids are synthesized from cholesterol and through
cortical cells contain large stores oflipids. the involvement of the mitochondria and endoplasmic

17a-Hydroxylase 17, 20-Lyase

Aceiead CH (P450c17) CHy .
i
C=0 eee c=0 : fe)
Cholesterol :
(ACTH?) a
NADPH 3
0;
! Dehydroepi-
Fsed
ohrelease androsterone
be ae CH, :
33-Dehydrogenase % : & |
AY’, A"-Isomerase@ ct=0 °
NAD*

‘ 4
Progesterone Arne:
3,17-dione
° CH
21f-Hydroxylase | —
(P4 1) 3 a _— a
SCROLLS Ss [>—

11-Deoxy-
corticosterone
: ¥ CH,OH
DO ee ee Se ee
(P450c11)
HO.
CHZ0OH
bee Corticosterone
o

Aldosterone
fo}
Mineralocorticoid Glucocorticoid Androgen and
pathway pathway estrogen pathway

Source: Katzung BG, Masters SB, Trevor AJ: Basic & Clinical Pharmacology, 12th edition:
www.accessmedicine.com
Copyright The McGraw-Hill Companies, Inc. All rights reserved.

FIGURE 21-2 Adrenocortical hormone pathway. A series of cytochrome P450 enzymes participate in the synthesis of aldosterone
(zona
gomerulosa), or cortisol and adrenal androgens (zonae fasciculata and reticularis).
The zona glomerulosa does not express CYP17A1; the zonae
fasciculata and reticularis do not express CYP11B2. (Modified with permission from Barrett KE, Boitano
S, Barman SM, et al.: Ganong's Review of
Medical Physiology, 24th edition. New York, NY: The McGraw-Hill Companies, Inc; 2012.)
 CHAPTER 21 Toxic Responses of the Endocrine System 2238

reticulum. The most common biosynthetic pathway from cho- Serum Binding Proteins
lesterol is the formation of pregnenolone, the basic precursor
Cortisol and corticosterone are transported in the blood by
for the three major classes of adrenal steroids. A series of cyto-
transcortin (corticosteroid binding globulin). When bound,
chrome P450 enzymes participate in synthesis of aldosterone
it is biologically inactive. Thus, a chemical affecting transcor-
(zona glomerulosa) or cortisol and adrenal androgens (zona
tin could alter the balance between free and bound hormone,
fasciculata and zona reticularis).
and impact its availability in target tissues. Nonsteroidal anti-
inflammatory drugs (NSAIDs) have been reported to decrease
its binding capacity.
Glucocorticoids
The physiological effects of glucocorticoids include hepatic
glucose production, gluconeogenesis, protein catabolism, Target Tissue Receptors
fat catabolism, increased bone resorption, altered mood, and
Adrenocortical steroids exert their effects through receptors in
increased gastric acidity. Therapeutically, the effects of cortisol
target tissues throughout the body that may be up- or down-
include prevention of vascular collapse during overwhelming
regulated by the action of xenobiotic compounds. For example,
stress, providing an anti-inflammatory effect, and invoking
spironolactone is an antisteroidal compound that competes
immunosuppression.
with the receptor sites.

Adrenocortical Toxicity Neuroendocrine Regulation

The zonae fasciculata and reticularis appear to be the prin-
The zonae fasciculata and reticularis are under tropic control
cipal targets of xenobiotic chemicals in the adrenal cortex
by ACTH which stimulates them to produce cortisol. Increased
leading to necrosis from things such as 7,12-dimethylbenz[a]
cortisol produced then provides negative feedback; however,
anthracene, acrylonitrile, thioacetamide, and basic polyglu-
stress can override the negative feedback control system and
tamic acid. Lipidosis inducers can cause accumulations of
stimulate cortisol secretion. Persistent exposure of the adrenal
fats which may be of sufficient quantity to cause a reduction
cortex to high levels of ACTH during chronic stress can result
or loss of organellar function and eventual cell destruction.
in adrenocortical hypertrophy.
Spironolactone, ketoconazole, and various PCBs directly
A one month toxicology study of corticosterone admin-
target glucocorticoid secretion. A wide range of lesions may
istration in rats observed reduced body weight gain, and
be produced that may be classified as follows: endothelial
lower thymus, adrenal, prostate, and seminal vesicle weight.
damage, mitochondrial damage, endoplasmic reticulum
The body and thymus weight effects were attributed directly
disruption, lipid aggregation, and lysosomal phospholipid
to high corticosterone, and reduced prostate and seminal
aggregation.
vesicle weights to the inhibition of LH and testosterone by
Biologically active cationic amphiphilic compounds pro-
corticosterone.
duce a generalized phospholipidosis that involves primarily
the zonae fasciculata and reticularis and produce microscopic
phospholipid-rich inclusions. The compounds that affect the
functional integrity of lysosomes include chloroquin, tripara- Mineralocorticoids
nol, and chlorphentermine. The adrenals are essential to life, mainly because of the aldo-
Adrenocortical toxicity can also involve increased secre- sterone promotion of sodium reabsorption and increased
tion of endogenous glucocorticoids due to compounds such excretion of potassium and hydrogen ions by the kidney.
as ethanol, cannabinoids, cocaine, and cytotoxic anticancer Loss of mineralocorticoid production by the cortex results in
drugs. Furthermore, pharmacological treatment with gluco- a life-threatening retention of potassium and hypovolemic
corticoid agonists that have been widely used as anti-inflam- shock associated with excessive urinary loss of sodium, chlo-
matory agents can produce symptoms that resemble Cushing's ride, and water. Chemicals that target steroidogenic enzymes
syndrome. (CYPI1A1, CYP21, CYP11B1) in the glucocorticoid or aldo-
sterone pathways could affect corticosteroid and/or aldoste-
rone production.
In Vitro Toxicity
Of particular usefulness for in vitro testing has been the human
adrenocortical carcinoma-derived NCI-H295R cell line. It Fetal Adrenal
has proven useful for identification of specific steroidogenic A specialized fetal adrenal cortex exists in primates during
enzymes that are targeted by xenobiotics. Because it is derived late gestation that is critical for the normal development of the
from a human source, it is also worthwhile for hazard risk fetus. After birth, there is a rapid regression, apoptosis, and
assessment. lysis of the fetal cortex with dilation of cortical capillaries and
324 UNIT 4 Target Organ Toxicity

replacement by the typical three cortical zones. It is important preventing catecholamine release. The most common patho-
not to misinterpret this as a lesion in neonatal primates because logical changes seen in the adrenal medulla in toxicological
it represents physiological replacement of the fetal cortex with studies involve proliferative lesions classified as nodular hyper-
postnatal adrenal cortex. plasia, although degenerative changes can also occasionally be
observed.

ADRENAL MEDULLA Pheochromocytoma

Because it is classified as a specialized postganglionic neu- Large benign adrenal medullary proliferative lesions are desig-
ron, the adrenal medulla is a functional extension ofthe ner- nated pheochromocytomas. They are composed of chromaffin
vous system. It is composed of chromaffin cells, which are cells with variable numbers of hormone-containing secretory
the site of catecholamine synthesis and secretion. These are granules. In humans, pheochromocytomas are uncommon
true neuroendocrine cells, which provide a direct interface except in patients with inherited clinical syndromes of mul-
between the two systems. That is, sympathetic, cholinergic tiple endocrine neoplasia (MEN). In rats, these tumors do not
stimulation of the cell bodies results in secretion of catechol- secrete excess catecholamines, whereas in humans they secrete
amines, which behave as hormones. Chromaffin cells also increased amounts leading to hypertension and other clinical
contain encephalin, neuropeptide Y, substance P, vasopres- disturbances.
sin, and oxytocin. Pheochromocytomas in rats differ from those in all other
species in that they are common, often bilateral, and can be
induced by many chemicals. Vitamin D is the most power-
Sympathetic Response ful mitogenic stimulus to cause chromaffin cell prolifera-
tion in the adrenal medulla in rats. Because the vitamin D
The general functions of the sympathetic nervous system
effect has been seen in vivo, but not in vitro, it is thought
are to ensure reciprocity to counteract and balance the tonic
to result from impaired calcium homeostasis, resulting in
effects of parasympathetic stimulation, assist in the mainte-
nance of steady state functions, and assist in the mobilization
hypercalcemia.
of body reserves to meet emergency situations— “fright, fight, The human adrenal medulla, as in mice, has a low sponta-
or flight.” neous incidence of proliferative lesions of chromaffin cells.
Human chromaffin cells also failed to respond to a variety of
mitogenic stimuli in culture. These findings and others sug-
gest that the rat represents an inappropriate model to assess the
Catecholamines
potential effects of xenobiotic chemicals on chromaffin cells of
The adrenal medulla is the major site of (nor)epinephrine pro- the human adrenal medulla.
duction with a tyrosine precursor and dopamine intermediate. A relationship exists between the adenohypophyseal hor-
Release of catecholamines is stimulated by acetylcholine from mones and the development of adrenal medullary prolif-
cholinergic preganglionic neurons. Physiological activators erative lesions. For example, the long-term administration
of release include decreased blood pressure, decreased blood of growth hormone is associated with an increased inci-
glucose, decreased oxygen availability, stress, anxiety, cold, dence of pheochromocytomas as well as the development
exercise, and postural hypotension. Catecholamines affect all of tumors at other sites. In long-term animal studies, pheo-
tissues but are most pronounced on the heart, liver, skeletal chromocytomas often are accompanied by tumors or toxic
muscle, adipocytes, vascular smooth muscle, and bronchial effects in other organs. They are often seen in cases involv-
smooth muscle. ing renal, lung, and hepatic toxicity, in addition to endo-
crine disturbances. They are also associated with hypoxia,
uncoupling of oxidative phosphorylation, disturbances of
Adrenergic Receptors calcium homeostasis, or disturbances of the hypothalamic
There are two major types of these receptors, known as alpha endocrine axis.
and beta adrenergic receptors with two subtypes of each.
Beta-2 receptors bind epinephrine 10 times greater than nor-
epinephrine. Therefore, the receptor type variation on target In Vitro Testing
tissues contributes to the diversity with which the sympathetic A commonly employed cell line used in neurobiology is the
response exerts its specific effects. PC12 pheochromocytoma line derived from a rat adrenal
medullary tumor. The PC12 cell line has been useful in deter-
mining intracellular mechanisms at the molecular level that
General Toxicity are involved in chromaffin cell signaling and proliferation.
Examples of specific chemicals that target chromaffin cells Substances that inhibit mitochondrial function (cyanide, rote-
include toxins that block voltage-gated ion channels and bacte- none) or uncouple oxidative phosphorylation (dinitrophenol)
rial toxins that block exocytosis of secretory granules, thereby stimulate catecholamine secretion.
 CHAPTER 21 Toxic Responses of the Endocrine System 825

THYROID GLAND activity. While the thyroid gland synthesizes and secretes both
T, and T,, it primarily releases T,. Figure 21-3 shows the struc-
General Anatomy tures required to make T; and T,. At the apical membrane of the
The thyroid gland consists of two lobes of endocrine tissue follicular cells, 1, combines with tyrosine residues on thyroglob-
located just below the larynx on each side of the trachea with ulin (TGB) to form monoiodotyrosine (MIT) and diiodotyro-
an isthmus connecting the two lobes. The thyroid secretes two sine (DIT). Coupling between MIT and DIT occurs such that
hormones known as thyroxine (T,) and triiodothyronine (T;), combined MIT and DIT forms T;, whereas combined DIT and
which are produced in the thyroid follicle (Figure 21-3). DIT forms T,. T, from the thyroid gland can be peripherally
T, and T; are important regulators of overall metabolism converted to T; (active hormone) or rT; (inactive metabolite),
with their primary target tissues including the liver, kidney, then successively diodinated by the monodeiodinases.
heart, brain, pituitary, gonads, and spleen. Some studies indi- Several studies indicate that xenobiotics can interfere with
cate that xenobiotics directly affect the structure of the thyroid the thyroid gland function by adversely affecting the process
gland. For example, heavy metals and red dye #3 are known to of thyroid hormone synthesis. For example, environmental
decrease the size of the colloid space within the follicle. This chemicals such as perchlorate, chlorate, and bromate inhibit
leads to an impaired ability of the thyroid gland to synthesize uptake of iodide and thus decrease thyroid hormone synthe-
and store thyroid hormones. sis. Other goitrogenic chemicals are indicated at the bottom of
Figure 21-3.

Thyroid Hormone Structure and Synthesis

Thryoid hormones are composed of two covalently linked Thyroid Hormone Binding Proteins
tyrosine amino acids. Both T, and T, contain iodides that are Once released into the blood, thyroid hormones are rapidly
derived from dietary intake and are required for biological bound to high-affinity serum binding proteins. Less than

Cerebral cortex v io — Influences from periphery

via nervous system

Hypothal
yp othalamus DF: aad a aac i a ie
[T, i— Ts]
ean 2
a Boal ak

5’ deiodinase (Type Il)

Adenohypophysis ie 1s (-)
(Thyrotrophs)
(1)TSH (+)

Thyroid gland Thyro-

peroxidase
Thyro- Thyroglobulin Coupling

a
peccaaeal 3: ORS
(0)
eee
Duby,
ers MIT +DIT—(T )
DIT +DIT —(T,)
|
|
(OG)| OG)|
a ae ers a I! [ TBG ~=srTTR
H i} ‘ i

Na/Isymporter | 2-delodinase
(Type l) | p if A
\ sare “Sea a nen
3
------ Deiodination --------~*-------
.
f ante

trapping Thyroperoxidase inhibition (|) Proteolysis

a he: . ie Ty,T3 release

form. Mae yon

Goitrogenic - Thiocyanate - Thiourea, PTU * lodide-excess
chemical - Perchlorate « Sulfonamides - Lithium
« Methimazole
« Aminotriazole
- Acetoacetamide

FIGURE 21-3 Mechanism of action of goitrogenic chemicals on thyroid hormone synthesis and secretion. (Reproduced with
permission from Dunlop RH, Malbert C, Capen CC, O’Brien TD: Pathophysiology of Endocrine Homeostasis: Examples IN Veterinary Pathophysiology,
Blackwell Publishing, 2004.)
326 UNIT 4 Target Organ Toxicity

1% of T; is free in circulation. Only this small-unbound fraction Regulation of Thyroid Hormone Release
has access to receptors in target cells. Environmental chemicals Thyroid hormone secretion is regulated by thyroid-stimulating
such as PCBs are known to displace thyroid hormones from hormone (TSH) from the anterior pituitary gland. The rate
serum binding proteins and lead to a rapid decline in serum
of release of TSH is under a hypothalamic-pituitary-thyroid
thyroid hormone levels. regulatory axis involving negative feedback. The hypothalamus
synthesizes and secretes thyroid-releasing hormone (TRH).
Thyroid Hormone Receptors TRH travels to the anterior pituitary via the portal plexus and
stimulates synthesis and secretion of TSH. TSH acts on the
Thyroid hormones act by binding to the thyroid hormone
receptors (TRs). Environmental chemicals can interfere with thyroid gland to stimulate production and/or release of T;
thyroid hormone binding to TRs and thyroid hormone-related and T,. These can then exert negative feedback control at the
transcription at multiple levels. Some can bind directly to TRs level of the anterior pituitary to inhibit further release of TSH.
and induce either agonistic or antagonistic effects. Others Chemicals such PBDEs may increase TSH levels, leading to
interfere with the thyroid hormone binding to receptors via increased levels of T; and T,.
indirect mechanisms. There are xenobiotics that can interfere
with cross-talk between TRs and other nuclear receptors.
Physiological Effects
Thyroid hormones influence nearly every tissue in the body
Thyroid Hormone Clearance with its primary function being the determination of metabolic
The main pathway for clearance of thyroid hormones from rate. In general, thyroid hormone’stimulates both anabolic and
the serum is via conjugation to glucuronic acid or sulfate catabolic biochemical pathways; however, its overriding effect is
(Figure 21-4). Studies indicate that some xenobiotics includ- catabolism. Thyroid hormone also produces significant effects
ing coplanar and noncoplanar congeners of PCBs may increase on growth and development of the CNS and skeleton early in life.
the clearance of thyroid hormones from the serum by inducing
glucuronosyltransferases and sulfotransferases. Others have
shown that xenobiotics such as rifampicin and phenobarbital Thyroid Toxicity
may decrease the transport of thyroid hormones into the brain Given the influence of thyroid hormones on numerous tis-
and liver by inhibiting transporters. sues in the body, it is not surprising that xenobiotics that affect

UDP-glucurony| Thyrotrophic
Hypothalamus
transferase area
Bile
T4-glucuronide
T4-glucuronide
(-) bd Pituitary gland
ee
IT; Decreased T,/T3_ Hypothalamic—
so
4 er
+ Synthesis/secretion pituitary-
ee 3 : " thyroid
(\)5’-deiodinase _ (1) Fuptake Pituitary— axis
(Tite _ ({)Thyroperoxidase | TSH thyroid
are -Binding/coupling axis
|(|) Proteolysis |

i
TSH
Neoplasia <— Hyperplasia Thyroid
(late) (early) gland
FIGURE 21-4 Multiple sites of disruption of the hypothalamic-pituitary-thyroid axis by xenobiotic chemicals. Chemicals can exert
direct effects by disrupting thyroid hormone synthesis or secretion and indirectly influence the thyroid through an inhibition of 5’-deiodinase or
by inducing hepatic microsomal enzymes (e.g., T,-UDP-glucuronyltransferase). All of these mechanisms can lower circulating levels
of thyroid
hormones (T, and/orT,), resulting in a release from negative feedback inhibition and increased secretion of thyroid-stimulating
hormone (TSH) by
the pituitary gland. The chronic hypersecretion of TSH predisposes the sensitive rodent thyroid gland to develop an increased incidence
of focal
hyperplastic and neoplastic lesions (adenomas) by a secondary (epigenetic) mechanism.
 CHAPTER 21 Toxic Responses of the Endocrine System O27,

thyroid hormone levels often cause symptoms of hypothyroid- Bisphenol A—BPA blocks T, action by antagonizing the
issn or hyperthyroidism, or lead to a significant impairment in binding of T, to its receptor. Further, some studies have shown
brain development and function. that BPA inhibits T,-mediated gene expression in cell lines. It
is suggested that BPA leads to symptoms of hypothyroidism or
PCBs—PCBs are some of the best characterized thyroid dis- thyroid resistance syndrome in animal models.
rupting chemicals. PCBs are known to interfere with the thy-
roid system in a manner that leads to serious neurocognitive Phthalates—To date, a few small human studies have shown
defect. Several studies indicate that PCBs decrease the level of that phthalate exposures may alter the levels of T, and T, in
thyroid hormone by inhibiting synthesis and/or increasing the adult men and pregnant women. They result in low thyroid
metabolism. Further, some studies indicate that they interfere hormone levels and to symptoms of hypothyroidism.
with thyroid hormone action by inhibiting the binding of thy-
roid hormones to binding proteins or blocking their ability to
1s 44. 7D-
pind to TRs. PARATHYROID GLAND

PBDEs—Polybrominated diphenyl ethers (PBDEs) are struc- General Anatomy

turally similar to that of PCBs. Thus, it is not surprising that Humans have four parathyroid glands that are embedded in
many of the toxic effects between the two are similar leading to the surface of the thyroid gland. They are composed of mainly
neurocognitive defects. chief cells that produce parathyroid hormone (PTH). The
parathyroid glands are critical for life largely because PTH
Perchlorate—A few studies indicate that perchlorate expo- helps maintain normal plasma calcium levels (Figure 21-5).
sure inhibits thyroid hormone levels, possibly leading to hypo- Calcium is required in optimal concentrations for processes
thyroid-like outcomes. such as fertilization, vision, locomotion-muscle contraction,
nerve conduction, blood clotting, exocytosis, cell division, and
the activity of anumber of enzymes and hormones. When the
Pesticides— Pesticide mixtures containing dichlorodiphen- parathyroids are removed or damaged, PTH levels drop, caus-
yitrichloroethane (DDT) have been shown to increase thyroid ing a major drop in circulating calcium levels. In turn, this can
volume and to induce antibodies that attack the thyroid gland, lead to tetanic convulsions and death.
resulting in autoimmune thyroid disease.

Perfluorinated Chemicals—Some studies have shown that Parathyroid Toxicity

perfluorooctane sulfonate and perfluorooctanoic acid decrease Xenobiotic exposures may alter the structure of the parathy-
T, and T, levels by potentially upregulating phase II enzymes roid gland. In some cases, chemicals cause death of the para-
in liver and deiodinases in the thyroid. thyroid cells resulting in a reduced size and limited release of

Dietary
Ca** HPO,2-
Bone fluid
compartment |
vy

Gastrointestinal 1,25(OH),VD;
lumen
(PTH)

Osteocyte-
osteoblast
“pum p”

Renal tubular
epithelium Glomerular filtrate
Osteoblast

Urine

FIGURE 21-5 interrelationship of parathyroid hormone (PTH), calcitonin (CT), and 1,25-dihydroxycholecalciferol (1,25(OH),VD,) in
the regulation of calciurn (Ca) and phosphorus in extracellular fluids. Receptors for PTH are on osteoblasts and for CT on osteoclasts in bone.
PTH and CT are antagonistic in their action on bone but synergistic in stimulating the renal excretion of phosphorous. Vitamin D exerts its action
prirnarily on the intestine to enhance the absorption of both calcium and phosphorus.
328 UNIT 4 Target Organ Toxicity

PTH. Other xenobiotic exposures have been shown to increase plasma proteins and bones. In turn, this increases circulating
the size of the parathyroid gland (lead, rotenone, malathion, levels of ionized calcium.
hexachlorobenzene) often leading to parathyroid cancer. CT reduces circulating calcium levels by reversing the action
of PTH on bone resorption. CT serves to prevent hypercalce-
mia by shutting down efflux ofcalcium from bone, and it nega-
PTH Structure and Synthesis tively regulates PTH to prevent kidney calcification. Vitamin D
PTH isa polypeptide hormone that is derived from a precursor also serves to inhibit PTH actions and build bone. Vitamin D,
molecule called preproparathyroid hormone (Figure 21-6). is essential for calcium absorption in the GI tract.
Xenobiotics may interfere with the normal synthesis of PTH. Some xenobiotics such as pesticides and fungicides can
Metals such as aluminum and cadmium have been shown to cause excessive PTH secretion by the parathyroid gland and
inhibit PTH secretion. Similarly, alcohol consumption has lead to hyperparathyroidism. Other xenobiotic exposures such
been shown to decrease PTH levels in pregnant rats. Lithium as those to heavy metals may cause low PTH secretion and lead
has been associated with a rise in PTH levels as well as abnor- to hypoparathyroidism.
mally high calcium levels.

Regulation of PTH Release

PTH Receptors When the calcium receptors in the parathyroid gland sense
The PTH receptor is a single G-protein-coupled receptor called low calcium levels, they stimulate the parathyroid gland to
PTHRI1. A study shows that xenobiotics may alter the expression release PTH.
of PTHR1. Specifically, studies have shown that binge alcohol
drinking significantly decreases expression of PTHR1 in male rats.
ENDOCRINE PANCREAS .
Physiological Effects Scattered among the pancreatic acini are the endocrine units of
The main physiological role of the parathyroid gland is to con- the pancreas, the Islets of Langerhans. The major physiologi-
trol circulating calcium levels (Figure 21-5). PTH works in cal function of the endocrine pancreas is to serve as the pri-
concert with calcitonin (CT) and vitamin D. PTH serves to mary homeostatic regulator of fuel metabolism, particularly
increase circulating calcium levels by increasing the release of circulating glucose. Islet cells are sensors of glucose homeo-
calcium from bone through demineralization. PTH also serves stasis that respond to changes in their nutrient and hormonal
to increase calcium levels by increasing the tubular reabsorp- environment.
tion of calcium by the kidney. Further, it inhibits the renal
reabsorption of phosphate, which aids in increasing the solu-
bility of calcium. PTH also enhances magnesium reabsorption, Role of the Liver in Glucose Production
inhibits bicarbonate ion reabsorption, and blocks exchange Energy for cellular metabolism can be derived from fatty acids
of sodium ions by the tubules. These actions of PTH result or glucose in the blood. The liver is the primary contributor to
in metabolic acidosis, which favors removal of calcium from increasing blood glucose levels.

Circulation

||
Direct secretion |
(“By-pass”)

-PTH (1-84)
PTH (1-84)
-PSP

GOUNS Ci ieee ~—
(min) 0 5

FIGURE 21-6 Biosynthesis of PTH. Active PTH is synthetized as a larger biosynthetic precursor (preproPTH) that undergoes rapid
posttranslational processing to proPTH prior to secretion as active PTH (aminoacids 1-84) from chief cells in the parathyroid glands.
 CHAPTER 21 Toxic Responses of the Endocrine System 329

Pancreatic Hormones Carbohydrate metabolism

diabetes mellitus
Insulin—The overall effects of insulin are to stimulate ana- Decreased Increased
bolic processes (energy storage). Specifically, insulin functions insulin glucagon
to lower blood levels of glucose, fatty acids, and amino acids
and to promote their conversion to the storage form of each:
Decreased Increased Glycogen
glycogen, triglycerides, and protein, respectively. glycogen glycogeno- = depletion-
synthesis lysis glucose in
Glucagon—Glucagon is the primary hormone with action blood
counterregulatory to insulin, because it stimulates catabolic
Lipid metabolism
processes to prevent hypoglycemia. The release of glucagon diabetes mellitus
is stimulated by epinephrine and norepinephrine, and by the Decreased Increased
amino acids, arginine, leucine, and alanine. Conversely, gluca- insulin glucagon
gon secretion is inhibited by insulin and somatostatin.
Decreased Increased Increased
Somatostatin—The role of somatostatin is its role in reg- lipid lipolysis m=» FFA glycerol
ulation of neuroendocrine function to inhibit secretion of synthesis ketones in
growth hormone in the anterior pituitary. The generalized blood
function of somatostatin appears to be as a hormone release Protein metabolism
inhibitor. diabetes mellitus
Decreased Increased
insulin glucagon
Interactions of Release |
Although glucagon and insulin exert opposing effects on car- Increased Protein loss; Increased
bohydrate metabolism, they act in concert to preserve normo- protein =! amino = gluconeo-
breakdown acids in genesis
glycemia in the face of perturbations that might tend to elevate
bood
or lower blood glucose. Insulin and glucagon exert opposing
effects on various metabolic processes. Therefore, many inves- FIGURE 21-7 Effects of diabetes mellitus on metabolism.
tigators like to think of the insulin-to-glucagon ratio in blood Decreased insulin (type 1) or insulin action (type 2) inhibits glycogen,
lipid, and protein synthesis. Increased glucagon stimulates glyco-
as an important determinant of the overall metabolic status.
genolysis, lipolysis, and protein breakdown. Glycogenolysis increases
When there is a high ratio of insulin to glucagon, a relative ana-
circulating glucose. Increased glycerol and amino acids serve as
bolic state exists. When the ratio of insulin to glucagon is low, a substrates for gluconeogenesis to further increase circulating glucose.
catabolic state exists.

widely used to generate animal models of diabetes are alloxan

Metabolic Responses in Diabetes and streptozotocin. A common target of these in pancreatic
Two major forms of diabetes mellitus result from either beta cells is DNA. There are data to support that DNA damage
decreased insulin production (type 1) due to autoimmune occurs, poly(ADP-ribose) synthetase is activated, polyadenyl-
destruction of pancreatic B cells or reduced insulin function ation increases, and NAD declines.
(type 2) owing to end organ insensitivity or resistance to insu-
lin. Insufficient insulin action leads to decreased glycogen,
lipid, and protein synthesis (Figure 21-7). Reduced removal of Insulin Resistance
glucose from the blood causes hyperglycemia and various met- Insulin resistance and defective function of pancreatic beta
abolic alterations. Increased action of the counter-regulatory cells usually occur sometime before the development of type
hormone glucagon stimulates glycogenolysis, lipolysis, and 2 diabetes. In a study investigating nondiabetic residents liv-
protein breakdown. Stimulation of glycogenolysis and gluco- ing near a deserted pentachlorophenol and chloralkali factory
neogenesis increases circulating glucose. in Taiwan, insulin resistance was associated with increasing
circulating levels of dioxins and mercury. In addition, BPA
exposure of pregnant mice resulted in increased insulin, leptin,
Pancreatic Toxicity triglyceride, and glycerol levels.
The insulin-secreting beta cells are particularly sensitive to
chemical attack. The clinical consequences of insulin defi-
ciency are physiologically more severe than those that would In Vitro Testing
result from glucagon deficiency because the other counterreg- Several cell lines are available for testing of insulin secretion.
ulatory hormones that oppose insulin action can compensate Pancreatic beta-cell-derived RINm5F cells were exposed to
for reduced glucagon regulation. Two chemicals that have been a combination of the cytokines, IL-18, TNF-a, and IFN-7y
330 UNIT 4 Target Organ Toxicity

to simulate type 1 diabetes mellitus conditions. This study BIBLIOGRAPHY

showed that hydrogen peroxide produced by these cytokines Eldridge JC, Stevens JT: Endocrine Toxicology, 3rd ed. London:
reacted in the presence of trace metal Fe** with nitric oxide Informa Healthcare, 2010.
to form highly toxic hydroxyl radicals. RINmSF cells were also Gardner DG, Shoback DM, Greenspan FS: Greenspan’ Basic and Clin-
used to investigate the role of oxidative stress in inorganic arse- ical Endocrinology. New York: McGraw-Hill, 2007.
nic exposure. A number of proapoptotic mitochondrial and Jameson JL: Harrison’s Endocrinology, 11th ed. New York: McGraw-
cytosolic markers were investigated and found to be elevated Hill, 2013.
during 8-cell toxicity.
 CHAPTER 21 Toxic Responses of the Endocrine System $31

QUESTIONS

The inability to release hormones from the anterior Chromaffin cells of the adrenal gland are responsible for
pituitary would NOT affect the release of- which of the secretion of which of the following?
following? a. aldosterone.
ae LH: b. epinephrine.
Dae RI c. corticosterone.
Coe ADET. d. testosterone.
dd ISH. e. estradiol.
Come AC TEL.
The parafollicular cells of the thyroid gland are responsible
Which of the following statements regarding pituitary for secreting a hormone that:
hormones is TRUE? a. increases blood glucose levels.
a. The hypothalamic-hypophyseal portal system trans- b. decreases plasma sodium levels.
ports releasing hormones to the neurohypophysis. c. increases calcium storage.
b. Dopamine enhances prolactin secretion from the d. decreases metabolic rate.
anterior pituitary. e. increases bone resorption.
c. Somatostatin inhibits the release of GH.
The function of chromophobes in the anterior Parathyroid adenomas resulting in increased PTH levels
pituitary is unknown. would be expected to cause which of the following?
e. Oxytocin and ADH are synthesized by hypothalamic a. hypocalcemia.
nuclei. b. hyperphosphatemia.
c. increased bone formation.
21-Hydroxylase deficiency causes masculinization of d. osteoporosis.
female genitals at birth by increasing androgen secretion e. rickets.
from which region of the adrenal gland?
zona glomerulosa. Which of the following vitamins increases calcium and
zona reticularis. phosphorus absorption in the gut?
adrenal medulla. a. vitamin D.
zona fasciculata. b. niacin.
sp
caochromaffin cells. c. vitamin A.
d. vitamin B,,.
Which of the following statements regarding adrenal tox- e. thiamine.
icity is TRUE?
a. ‘The adrenal cortex and adrenal medulla are equally 10. All of the following statements regarding glucose control
susceptible to fat-soluble toxins. are true EXCEPT:
b. Adrenal cortical cells lack the enzymes necessary to a. Glucagon stimulates glycogenolysis, gluconeogen-
metabolize xenobiotic chemicals. esis, and lipolysis.
c. Pheochromocytomas of the adrenal medulla can b. Insulin stimulates glycogen synthesis, gluconeogen-
cause high blood pressure and clammy skin due to esis, and lipolysis.
increased epinephrine release. c. Glucagon stimulates catabolic processes (mobilizes
Xenobiotics primarily affect the hydroxylase enzymes energy) to prevent hypoglycemia.
in the zona reticularis. Insulin promotes storage of glucose, fatty acids, and
e. Vitamin D is an important stimulus for adrenal cor- aminoacids by their conversion to glycogen, triglyc-
tex steroid secretion. erides, and protein, respectively.
e. Insulin and glucagon exert opposing effects on blood
Chemical blockage of iodine transport in the thyroid gland: glucose concentrations.
a. affects export of T; and T,.
b. prevents reduction to I, by thyroid peroxidase.
c. decreases TRH release from the hypothalamus.
d. interrupts intracellular thyroid biosynthesis.
e. mimics goiter.
 UNIT 5 TOXIC AGENTS

OO be, Ed A ed ss

Toxic Effects of Pesticides

Lucio G. Costa

INTRODUCTION Nicotine
Avermectins
ECONOMICS AND PUBLIC HEALTH
INSECT REPELLENTS
Use of Pesticides
Exposure Picaridin

Human Poisoning HERBICIDES

Regulatory Mandate
Chlorophenoxy Compounds
INSECTICIDES Bipyridil Compounds

Organophosphorus Compounds Chloroacetanilides

Biotransformation Triazines

Signs and Symptoms ofToxicity and Mechanism Phosphonomethyl Amino Acids

of Action Glyphosate
Treatment of Poisoning Glufosinate
The Intermediate Syndrome FUNGICIDES
Organophosphate-induced Delayed
Captan and Folpet
Polyneuropathy (OPIDP)
Dithiocarbamates
Long-term Toxicity
Inorganic and Organometal Fungicides
Carbamates
Pyrethroids RODENTICIDES
Signs and Symptoms ofToxicity and Mechanism Fluoroacetic Acid and Its Derivatives
of Action Anticoagulants
Organochlorine Compounds
DDT and Its Analogs FUMIGANTS
Hexachtorocyclohexanes and Cyclodienes Methyl Bromide
Other Old and New Insecticides 1,3-Dichloropropene
Rotenoids Sulfur

333
334 UNIT 5 Toxic Agents

u A pesticide may be defined as any substance or mixture « Chemical insecticides in use today poison the nervous
of substances intended for preventing, destroying, repel- systems of the target organisms.
ling, or mitigating any pest. « An herbicide is any compound that is capable of either
s Pesticide exposures include (1) accidental and/or suicidal killing or severely injuring plants.
poisonings; (2) occupational exposure (manufacturing, = A fungicide is any chemical capable of preventing growth
mixing/loading, application, harvesting, and handling and reproduction of fungi.
of crops); (3) bystander exposure to off-target drift from
spraying operations; and (4) the general public who con-
sume food items containing pesticide residues.

INTRODUCTION Use of Pesticides

In the past 20 years, use of pesticides (as amount of active ingre-
Pesticides can be defined as any substance or mixture of
dient) has plateaued due to the utilization of more efficacious
substances intended for preventing, destroying, repelling,
compounds, which require less active ingredient. Pesticides are
or mitigating pests. Pests can be insects, rodents, weeds, and
often, if not always, used as multiagent formulations, in which
a host of other unwanted organisms. Pesticides may be more
the active ingredient is present together with other ingredi-
specifically identified as insecticides (insects), herbicides
ents to allow mixing, dilution, application, and stability. These
(weeds), fungicides (fungi and molds), rodenticides (rodents),
other ingredients are lumped under the term “inert” or “other.”
acaricides (mites), molluscides (snails and other mollusks),
Though they do not have pesticidal action, such inert ingredi-
miticides (mites), larvicides (larvae), and pediculocides
ents may not always be devoid of toxicity.
(lice). In addition, for regulatory purposes, plant growth regu-
lators, repellants, and attractants (pheromones) often also fall
in this broad classification of chemicals. Exposure
Exposure to pesticides can occur via the oral or dermal routes
ECONOMICS AND PUBLIC HEALTH or by inhalation. High oral doses, leading to severe poisoning
and death, are achieved as a result of pesticide ingestion for
The use of pesticides must consider the balance of the benefits suicidal intent, or of accidental ingestion, commonly due to stor-
versus the possible risks of injury to human health or degrada- age of pesticides in improper containers. Chronic low doses,
tion of environmental quality. Pesticides play a major role in on the other hand, are consumed by the general population
the control of vector-borne diseases, which represent a major as pesticide residues in food or as contaminants in drinking
threat to the health of large human populations. When intro- water. Regulations exist to ensure that pesticide residues are
duced in 1942, DDT appeared to hold immense promise of maintained at levels below those that would cause any adverse
benefit to agriculture and public health by controlling vector- effects. Workers involved in the production, transport, mixing
borne diseases. However, because of its bioaccumulation in the and loading, and application of pesticides, as well as in har-
environment and its detrimental effects on bird reproduction, vesting of pesticide-sprayed crops, are at the highest risk for
DDT was eventually banned in most countries by the mid- pesticide exposure. Dermal exposure during normal handling
1970s. When DDT was banned in 1996 in South Africa, less or application of pesticides, or in case of accidental spillings,
than 10000 cases of malaria were registered in that country. By occurs in body areas not covered by protective clothing, such as
2000, the number of malaria cases had increased to 62 000, but the face or the hands, or by inhalation. Furthermore, pesticides
with the reintroduction of DDT at the end of that year, cases deposited on clothing may penetrate the skin and/or poten-
were down to 12500. tially expose others, if clothes are not changed and washed on
Excessive loss of food crops to insects or other pests contrib- termination of exposure.
utes to economic loss and possible starvation. In developed
countries, pesticides allow production of abundant, inex-
pensive, and attractive fruits and vegetables, as well as grains. Human Poisoning
Along with insecticides, herbicides and fungicides play a major Pesticides are not always selective for their intended target
role in this endeavor. species, and adverse health effects can occur in nontarget
 CHAPTER 22 Toxic Effects of Pesticides S 5)8)

TABLE 22-1 WHO-recommended classification of pesticides by hazard.

LD,, in Rat (mg/kg Body Weight)

- Oral Dermal

‘Class Solids Liquids Solids : Liquids

la: Extremely hazardous 5 orless 20 or less 10 or less 40 or less

Ib: Highly hazardous 5-50 20-200 10-100 40-400

Il: Moderately hazardous 50-500 200-2000 100-1000 400-4000

Ill: Slightly hazardous Over 500 Over 2000 Over 1000 Over 4000

V+: Unlikely to present hazard in normal use Over 2000 Over 3000 Over 4000 Over 6000

species, including humans. In the general population and in Other nations, such as Canada, Japan, and most European
occupationally exposed workers, concerns range from acute countries, have legislated similar procedures for pesticide reg-
human poisoning to a possible association between pesticide istration. The European Union (EU) has created a harmonized
exposure and increased risk of cancer, reproductive and devel- Union-wide framework for pesticide regulation. The WHO
opmental toxicity. provides guidance, particularly with the setting of acceptable
With several million poisonings causing hospital admis- daily intake (ADI) values for pesticides.
sion and a couple hundred thousand deaths, the World Health
Organization (WHO) has recommended a classification of
pesticides by hazard, where acute oral or dermal toxicities in TABLE 22-2 Basic toxicology testing requirements
rats were considered (Table 22-1). As a class, insecticides are for pesticide registration.
the most acutely toxic followed by herbicides and fungicides.
Test Animal Species*

Acute lethality (oral, dermal, Rat, mouse, guinea pig, rabbit

Regulatory Mandate inhalation)
In the United States, the Environmental Protection Agency
Dermal irritation Rabbit, rat, guinea pig
(EPA) regulates pesticide use under the Federal Insecticide,
Fungicide and Rodenticide Act and the Federal Food, Drug and Dermal sensitization Guinea pig
Cosmetic Act through registration for use and establishment of
Eye irritation Rabbit
maximum allowable levels of pesticide residues (tolerances) in
foods and animal feeds. Acute delayed neurotoxicity Hen
The Food Quality Protection Act gives EPA the mandate to
Genotoxicity studies (in vitro, Bacteria, mammalian cells,
assess risks of pesticides to infants and children based on dietary in vivo) mouse, rat, Drosophila
consumption patterns of children, possible susceptibility of
infants and children to pesticides, and cumulative effects of com- Teratogenicity Rabbit, rodent (mouse, rat,
hamster)
pounds that share the same mechanism of toxicity. Additional
regulations concerning pesticides are present in other laws, such 2- to 4-week toxicity study Rat, mouse
as the Safe Drinking Water Act or the Clean Air Act. (oral, dermal, inhalation)
All pesticides sold or distributed in the United States must
90-Day toxicity study (oral) Rat
be registered by the EPA. To register a pesticide or a for-
mulated product, a large number of studies (over 140) are Chronic toxicity study (oral; Rat, dog
6 months to 2 years)
required, a process that takes several years and costs between
$50 and $100 million. The database includes information on Oncogenicity study Rat, mouse
product and residue chemistry, environmental fate, toxicology,
biotransformation/degradation, occupational exposure and Reproductive/fertility study Rat

reentry protection, spray drift, environmental impact on non- Developmental neurotoxicity Rat
target species (birds, mammals, aquatic organisms, plants, and study
soil), environmental persistence and bioaccumulation, as well
*Substantial efforts are being devoted to develop alternative nonanimal test systems.
as product performance and efficacy. Table 22-2 lists basic Only one in vitro test for primary irritation has been validated and accepted by
toxicology data needed for new pesticide registration. regulatory bodies.
336 UNIT 5 Toxic Agents

INSECTICIDES the phosphorus with a double bond. Based on chemical dif-

ferences, OPs can be divided into several subclasses, which
Insecticides play a most relevant role in the control of insect include phosphates, phosphorothioates, phosphoramidates,
pests, particularly in developing countries. All of the chemical phosphonates, and others. Figure 22-1 shows the chemical
insecticides in use today are neurotoxicants, and act by poison- structures of some commonly used OPs.
ing the nervous systems of the target organisms (Table 22-3).
The central nervous system of insects is highly developed and Biotransformation—For all compounds that contain a
not unlike that of mammals. As a class, insecticides have high sulfur bound to the phosphorus, a metabolic bioactivation is
acute toxicity toward nontarget species compared with other necessary for their biological activity to be manifest, as only
pesticides. Some of them, most notably the organophosphates, compounds with a P=O moiety are effective inhibitors of
are involved in a great number of human poisonings and AChE. Oxidative desulfuration (leads to the formation of an
deaths each year. “oxon, or oxygen analog of the parent insecticide) and thio-
ether oxidation (formation ofa sulfoxide, S=O, followed by the
formation ofa sulfone, O=S=O) are catalyzed by cytochrome
Organophosphorus Compounds P450s. Catalytic hydrolysis by phosphotriesterases, known as
The general structure of organophosphorus (OP) insecticides A-esterases (which are not inhibited by OPs), plays an important
can be represented by: role in the detoxication of certain OPs. Noncatalytic hydrolysis
of OPs also occurs when these compounds phosphorylate serine
at vs or S)
esterases classified as B-esterases..

Signs and Symptoms of Toxicity and Mechanism of

aie as Action—OP insecticides have high acute toxicity, with oral

LD., values in rat often below 50 mg/kg. For several OPs, acute
dermal toxicity is also high. Inhibition of AChE by OPs causes
accumulation of acetylcholine at cholinergic synapses, with
where X is the so-called leaving group that is displaced when overstimulation of muscarinic and nicotinic cholinergic recep-
the OP phosphorylates acetylcholinesterase (AChE), and is tors. As these receptors are localized in most organs ofthe body,
the most sensitive to hydrolysis; R, and R, are commonly alkoxy a “cholinergic syndrome” ensues, which includes increased
groups (i.e. OCH; or OC,H;) or other chemical substitu- sweating, salivation, bronchial secretion, bronchoconstriction,
ents; either an oxygen or a sulfur (in this case the compound miosis, increased gastrointestinal motility, diarrhea, trem-
should be defined as a phosphorothioate) is also attached to ors, muscular twitching, and various central nervous system
effects (Table 22-4). Whereas respiratory failure is a hallmark
of severe OP poisoning, mild poisoning and/or early stages of
TABLE 22-3 Molecular targets of the major classes an otherwise severe poisoning may display no clear-cut signs
of insecticides. and symptoms.
Target Insecticide Effect OPs with a P=O moiety phosphorylate a hydroxyl group on
serine in the active (esteratic) site of the enzyme, impeding its
Acetylcholinesterase Organophosphates Inhibition
action on the physiological substrate. Phosphorylated AChE is
Carbamates Inhibition
hydrolyzed by water slowly, and the rate of “spontaneous reac-
Sodium channels Pyrethroids (types | and II) Activation tivation” depends on the chemical nature of the R substituents.
DDT Activation Reactivation of phosphorylated AChE does not occur once
Dihydropyrazoles Inhibition
the enzyme-inhibitor complex has “aged,” which occurs when
Nicotinic acetylcholine Nicotine Activation there is loss by nonenzymatic hydrolysis of one of the two alkyl
receptors Neonicotinoids Activation (R) groups. When phosphorylated AChE has aged, the enzyme
GABA receptor-gated Cyclodienes Inhibition
is considered to be irreversibly inhibited, and the only means
chloride channels Phenylpyrazoles Inhibition of replacing its activity is through synthesis of new enzyme, a
Pyrethroids (type Il) Inhibition process that may take days.
Glutamate-gated chloride | Avermectins Activation
channels” Treatment of Poisoning—Procedures aimed at decon-
tamination and/or at minimizing absorption depend on the
Octopamine receptors' Formamidines Activation
route of exposure. In case of dermal exposure, contaminated
Mitochondrial complex | Rotenoids Inhibition clothing should be removed, and the skin washed thoroughly
with alkaline soap. In case ofingestion, procedures to reduce
Ryanodine receptors Diamides
absorption from the gastrointestinal tract do not appear to
*Found only in insects. In mammals, avermectins activate GABA, receptors. be very effective. Atropine, a muscarinic receptor antago-
In mammals, formamidines activate alpha,-adrenoceptors. nist, prevents the action of accumulating acetylcholine on
 CHAPTER 22 Toxic Effects of Pesticides 337

(CH;0), —P —O NO,
Ny
Methylparathion Azinphos-methyl (Guthion)

S
| N
(C,H,O), —P —O | Sy— CH (CH3)2

|
Yn
Romans
2 SZ

CH;

Chlorpyrifos Diazinon

O
|
; CH, —C — OCH, O
| | I
(C,H,0)) —P —S —CH —C —OC,H, (CH;0),) —P —O—CH =CCl,
I
O
Malathion Dichlorvos

CH; Ta VA

CH,
TS NH,
Metamidophos Sarin

FIGURE 22-1 Structures of some organophosphorus insecticides and ofthe nerve agent sarin. Note that most commonly used compounds
are organophosphorothioates (i.e., have a P=S bond), but some, including sarin, have a P=O bond and do not require metabolic activation.

TABLE 22-4 Signs and symptoms of acute poisoning these receptors. The administration of pralidoxime (2-PAM)
with anticholinesterase compounds. early after OP exposure can help prevent AChE aging, but
its effectiveness is equivocal and harm may ensue. Diazepam
Site and Receptor may be used to relieve anxiety in mild cases, and to reduce
Affected Manifestations
muscle fasciculations and control convulsions in the more
Exocrine glands (M) Increased salivation, lacrimation, severe Cases.
perspiration

Eyes (M) Miosis, blurred vision The Intermediate Syndrome—A second distinct mani-
Gastrointestinal tract (M) Abdominal cramps, vomiting, diarrhea
festation of exposure to OPs is the so-called intermedi-
ate syndrome, which is seen in 20% to 50% of acute OP
Respiratory tract (M) Increased bronchial secretion, poisoning cases. The syndrome develops 1 to several days
bronchoconstriction
after the poisoning, during recovery from cholinergic mani-
Bladder (M) Urinary frequency, incontinence festations, or in some cases, when patients have completely
recovered from the initial cholinergic crisis. Prominent fea-
Cardiovascular system (M) Bradycardia, hypotension
tures include a marked weakness of respiratory, neck, and
Cardiovascular system (N) Tachycardia, transient hypertension proximal limb muscles. Mortality due to respiratory paraly-
sis and complications ranges from 15% to 40%, and recovery
Skeletal muscles (N) Muscle fasciculations, twitching, cramps,
in surviving patients takes up to 30 days. The intermediate
generalized weakness, flaccid paralysis
syndrome is not an effect of AChE inhibition, and its precise
Central nervous system Dizziness, lethargy, fatigue, headache, underlying mechanisms are unknown. The hypothesis that
(M, N) mental confusion, depression of muscle weakness may result from nicotinic receptor desen-
respiratory centers, convulsions, coma
sitization due to prolonged cholinergic stimulation remains
M, muscarinic receptors; N, nicotinic receptors. the most valid.
 8
OoLSS) UNIT 5 Toxic Agents

Organophosphate-induced Delayed Polyneuropathy Carbamates

(OPIDP)—A few OPs may cause OPIDP. Signs and symptoms Carbamate insecticides are derived from carbamic acid, and
include tingling of the hands and feet, followed by sensory most are N-methylcarbamates. Acute oral toxicity ranges from
loss, progressive muscle weakness and flaccidity of the distal moderate to low toxicity, such as carbaryl, to extremely high
skeletal muscles of the lower and upper extremities, and toxicity, such as aldicarb. Dermal skin penetration by carba-
ataxia. These may occur 2 to 3 weeks after a single exposure, mates is increased by organic solvents and emulsifiers present
when signs of both the acute cholinergic and the intermedi- in most formulations. Carbamates are susceptible to a vari-
ate syndromes have subsided. OPIDP can be classified as a ety of enzyme-catalyzed biotransformation reactions, and
distal sensorimotor axonopathy. Neuropathological studies in the principal pathways involve oxidation and hydrolysis. The
experimental OPIDP have evidenced that the primary lesion mechanism of toxicity of carbamates is by inhibition of AChE,
is a bilateral degenerative change in distal levels of axons and which is rapidly reversible.
their terminals, primarily affecting larger/longer myelinated The signs and symptoms of carbamate poisoning include
central and peripheral nerve fibers, leading to breakdown of miosis, urination, diarrhea, salivation, muscle fasciculation, and
neuritic segments and the myelin sheaths. Although several CNS effects (Table 22-4). Acute intoxication by carbamates is
epidemics of OPIDP have occurred in the past such as generally resolved within a few hours. The treatment of carba-
Ginger-Jake paralysis with tri-ortho-cresyl phosphate in the mate intoxication relies on the use of atropine. Carbamates can
1930s, its occurrence in humans is now rare. Athough past inhibit neuropathy target esterase (NTE), but because carba-
studies suggested that aging of neuropathy target esterase mylated NTE cannot age, they are thought to be unable to initiate
(NTE) was involved in OPIDP, the exact mechanisms involved OPIDP. Additionally, when given before a neuropathic organo-
in phosphorylation and aging of NTE and axonal degenera- phosphate, carbamates offer protection against OPIDP, but when
tion remain obscure. given after, they can promote OPIDP.
Long-term Toxicity—There is still controversy on possible Methylcarbamates are not mutagenic, and there is no
long-term effects of OPs. The fact that acute exposure to high evidence of carcinogenicity. Embryotoxicity or fetotoxicity
doses of OPs may result, in some cases, in long-lasting adverse is observed only at maternally toxic doses. Limited evidence
health effects (particularly in the CNS) has been shown in ani- suggests that carbamates (e.g., aldicarb) may be more acutely
mals, as well as humans. More controversial is the possibility toxic to young animals than to adults, possibly because of lower
that low exposure to OPs, at doses that produce no cholinergic detoxication.
signs, also may lead to long-term adverse health effects, partic-
ularly in the central and peripheral nervous systems. Chronic
Pyrethroids
exposure of animals to OPs, at doses that significantly inhibit
AChE but may not be associated with clinical signs, results in Pyrethrins were first developed as insecticides from extracts
the development of tolerance to their cholinergic effects (which of the flower heads of Chrysanthemum cinerariaefolium,
is mediated, at least in part, by down-regulation of choliner- whose insecticidal potential was appreciated in ancient
gic receptors), and has been associated with neurobehavioral China and Persia. Because pyrethrins decompose rapidly
abnormalities, particularly at the cognitive level. on exposure to light, the synthetic pyrethroid analogs were
developed. Because of their high insecticidal potency,
Developmental Toxicity and Neurotoxicity—Experimental relatively low mammalian toxicity, lack of environmental
data indicate that young animals are more sensitive to the persistence, and relatively low tendency to induce insect
acute toxicity of OPs. This increased sensitivity does not appear resistance, pyrethroids now account for 15% to 20% of the
to be due to intrinsic differences in AChE, but rather due to global insecticide market. The pyrethroids are used widely as
lower detoxication abilities of young animals. Accumulating insecticides both in the house and in agriculture, in medicine
recent evidence suggests that perinatal exposure to OPs may for the topical treatment of scabies and head lice, and in
cause developmental neurotoxicity. Studies in rodents indi- tropical countries in soaked bed nets to prevent mosquito
cate that OPs can affect various cellular processes (e.g., DNA bites. Pyrethroids alter the normal function of insect nerves
replication, neuronal survival, neurite outgrowth) and non- by modifying the kinetics of voltage-sensitive sodium chan-
cholinergic pathways (e.g., serotoninergic synaptic functions, nels, which mediate the transient increase in the sodium per-
the adenylate cyclase system), and cause various behavioral meability of the nerve membrane that underlies the nerve
abnormalities. Such effects are also seen at dose levels that pro- action potential.
duced no cholinergic signs of toxicity. These findings, together On absorption, pyrethroids are very rapidly metabolized
with results of biomonitoring studies that indicate exposure of through two major biotransformation routes: hydrolysis of
children, particularly in inner cities and farming communities, the ester linkage, which is catalyzed by hepatic and plasma
to OPs have led to regulatory restrictions on the use of certain carboxylesterases, and oxidation of the alcohol moiety by
OPs and to concerns for their potential neurotoxic effects in cytochrome P450s. These initial reactions are followed by
children. Furthermore, specific guidelines for developmental further oxidations, hydrolysis, and conjugation with sulfate
neurotoxicity have been implemented. or glucuronide.
 CHAPTER 22 Toxic Effects of Pesticides 339

Signs and Symptoms of Toxicity and Mechanism of as chlordane, aldrin, dieldrin, heptachlor, endrin, and toxa-
Action—Based on toxic signs in rats, pyrethroids have been phene; the hexachlorocyclohexanes, such as lindane; and the
divided into two types (Table 22-5). Type I compounds pro- caged structures mirex and chlordecone. Their acute toxicity
duce a syndrome consisting of marked behavioral arousal, is moderate (less than that of organophosphates), but chronic
aggressive sparring, increased startle response, and fine body exposure may be associated with adverse health effects
tremor progressing to whole-body tremor and prostration particularly in the liver and endocrine disruption of the repro-
(type I or T syndrome). Type II compounds produce profuse ductive system.
salivation, coarse tremor progressing to choreoathetosis, and
clonic seizures (type II or CS syndrome). The pyrethroids dis- DDT and Its Analogs—DDT is effective against a wide variety
rupt voltage-gated sodium channels in mammals and insects. of agricultural pests, as well as against insects that transmit some
Pyrethroids bind to the a subunit of the sodium channel and of the world’s most serious diseases, such as typhus, malaria,
slow the activation (opening), as well as the rate of inactivation and yellow fever. DDT has a moderate oral acute toxicity and
(closing), of the sodium channel, leading to a stable hyper- its dermal absorption is very limited. In humans, oral doses of
excitable state. The higher sensitivity of insects to pyrethroid 10 to 20 mg/kg produce illness, but doses as high as 285 mg/kg
toxicity, compared with mammals, is believed to result from a have been ingested accidentally without fatal results. Toxicity
combination of higher sensitivity of insect sodium channels, from dermal exposure in humans is also low, as evidenced by
lower body temperature (as pyrethroids show a negative tem- the lack of significant adverse health effects when thousands
perature coefficient of action), and slower biotransformation. of people were liberally dusted with this compound. On
Type I] pyrethroids bind to and inhibit GABA,-gated chlo- absorption, DDT distributes in all tissues, and the highest con-
ride channels at higher concentrations than those sufficient to centrations are found in adipose tissue. It is excreted through
affect sodium channels (10-7M versus 107!°M). This effect is the bile, urine, and milk.
believed to contribute to the seizures that accompany severe Acute exposure to high doses of DDT causes motor unrest,
type II pyrethroid poisoning. increased frequency of spontaneous movements, abnor-
On occupational exposure, the primary adverse effect mal susceptibility to fear, and hypersusceptibility to external
resulting from dermal contact with pyrethroids is paresthesia. stimuli (light, touch, and sound). This is followed by the
Symptoms include continuous tingling or pricking or, when development of fine tremors, progressing to coarse tremors,
more severe, burning. The condition reverses in about 24h, and and eventually tonic-clonic convulsions. Death is typically
topical application of vitamin E has been shown to be an effec- due to respiratory failure. In humans, the earliest symptom of
tive treatment. Paresthesia is presumably due to pyrethroid- poisoning by DDT is hyperesthesia of the mouth and lower
induced abnormal repetitive activity in skin nerve terminals. part of the face, followed by paresthesia of the same area and
Chronic studies with pyrethroids indicate that at high dose lev- of the tongue. Dizziness, tremor of the extremities, confusion,
els they cause slight liver enlargement often accompanied by and vomiting follow; convulsions occur only in severe poison-
some histopathologic changes. There is little evidence of tera- ing. Both in insects and in mammals, DDT interferes with the
togenicity and mutagenicity. An increased rate of lymphoma sodium channels in the axonal membrane by a mechanism
incidence in rodents has been reported for deltamethrin, but similar to that of type I pyrethroids.
the effect was not dose-dependent. An important target for chronic DDT exposure is the liver.
DDT and its breakdown product DDE increase liver weight
and cause hepatic cell hypertrophy and necrosis, and they are
Organochlorine Compounds potent inducers of cytochrome P450s, particularly CYP2B and
The organochlorine insecticides include the chlorinated ethane CYP3A. Both DDE and DDD, another breakdown product,
derivatives, such as DDT and its analogs; the cyclodienes, such are carcinogenic in rodents, causing primarily an increase in
hepatic tumors.
TABLE 22-5 Classification of pyrethroid insecticides Hexachlorocyclohexanes and Cyclodienes—These two
based on toxic signs in rats. families of organochlorine insecticides comprise a large num-
.Signs and Symptoms : :Examples _ . ber of compounds that share a similar mechanism of neuro-
toxic action. Lindane is the y isomer of benzene hexachloride
Type | (T syndrome) Aggressive sparring Allethrin
(BHC; 1,2,3,4,5,6-hexachlorocyclohexane). Cyclodiene com-
Increased sensitivity to Bioallethrin
external stimuli Resmethrin pounds include chlordane, dieldrin, aldrin (which is rapidly
Whole-body tremors Phenothrin metabolized to dieldrin), heptachlor, and endrin. Toxaphene
Prostration is a complex mixture of over 200 chlorinated bornanes and
Type Il (CS syndrome) Pawing and burrowing Deltamethrin
camphenes.
Profuse salivation Fenvalerate Lindane and cyclodienes have moderate to high acute oral
Coarse tremor Cypermethrin toxicity (Figure 22-2). However, in contrast to DDT, these
Choreoathetosis Cyhalothrin compounds are readily absorbed through the skin. The pri-
Clonic seizures
sneteveneneecencceecEs tCtCpUbeet NCDODiOLLeeT ACLRANELD EM A ELEN NONLIN ALATA CICERO CPLR mary target for their toxicity is the central nervous system.
340 UNIT 5 Toxic Agents

Approximate catecholamines. In contrast to cyclodienes, chlordecone does

Cl LDso (mg/kg) not cause seizures. Furthermore, chlordecone induces hepatic
Cl Cl drug-metabolizing enzymes, and causes hepatosplenomegaly
Lindane (y-BHC) 200 in rats and humans.

Cl Cl DDT and Public Health: Risk-Benefit Considerations—

Cl The Stockholm Convention on Persistent Organic Pollutants,
ale cl ratified in 2004 by 50 states, outlawed the use of 12 indus-
Cl trial chemicals (the “Dirty Dozen”), including DDT. Yet, an
Chlordane Cl 500
Wy Cl exemption clause allows malaria-endemic nations to continue
utilizing DDT for indoor residual wall spraying. The United
Cl
Nations Environment Program estimates that about 25 coun-
Cl, ra tries would use DDT under this exemption from its ban. This
situation is keeping the debate on the risks and benefits of
Aldrin Cl 50 DDT usage very much alive.
Be Cl

Cl Other Old and New Insecticides

Cl, CI Rotenoids—The roots of Derris elliptica and those of
O Lonchocarpus utilis and Lonchocarpus urucu in South America
Dieldrin Cl 50 contain at least six rotenoid esters. The most abundant is rote-
) Cl none, which is used as an agricultural insecticide/acaricide
particularly in organic farming. Toxicity of rotenone in target
cl
and nontarget species is due to its ability to inhibit, at nano-
Cl, Cl molar concentrations, mitochondrial respiration by blocking
electron transport at NADH-ubiquinone reductase, the energy-
Endrin Cl 20 conserving enzyme complex commonly known as complex I.
yy, Cl a Poisoning symptoms include initial increased respiratory and
Cl cardiac rates, clonic and tonic spasms, and muscular depres-
sion, followed by respiratory depression. Rotenone may play a
Cd, ¢ role in the etiology of Parkinson's disease.

Heptachlor Cl 150 Nicotine—Nicotine is an alkaloid extracted from the leaves

Cc Cl
of tobacco plants (Nicotiana tabacum and Nicotiana rustica),
Cl and is used as a free base or as the sulfate salt. Nicotine has a
high acute toxicity, and the signs and symptoms of poisoning
FIGURE 22-2 Structure and acute toxicity (oral LD., in rat) of
include nausea, vomiting, muscle weakness, respiratory effects,
selected organochlorine insecticides of different chemical classes.
headache, lethargy, and tachycardia. Most cases of poisoning
with nicotine occur after exposure to tobacco products, or gum
or patches. Workers who cultivate, harvest, or handle tobacco
Unlike DDT, tremor is essentially absent, but convulsions are a may experience green tobacco sickness, caused by dermal
prominent aspect of poisoning. Lindane and cyclodienes bind absorption ofnicotine.
to the picrotoxin binding site on the chloride channel, thereby
blocking its opening and antagonizing the inhibitory action of Avermectins— The avermectins are macrocyclic lactones that
GABA. Treatment of acute poisoning is symptomatic; pheno- are isolated from the fermentation broth of Streptomyces aver-
barbital and diazepam can be used as anticonvulsants. mitilis. This fungus synthesizes eight individual avermectins
that have antiparasitic activity. The semisynthetic derivatives
Chlordecone—Chlordecone (Kepone) has been the most of avermectin B,,, emamectin benzoate, and ivermectin are
studied because of one episode that involved 148 workers used as insecticides, and for parasite control in human and
in a chlordecone-producing factory in Hopewell, Virginia, veterinary medicine, respectively. Abamectin is used primar-
between 1973 and 1975. The primary manifestation of chlor- ily to control mites, whereas emamectin benzoate is effective at
decone toxicity is the presence of tremors, which are observed controlling lepidopterian species in various crops and emerald
in animals as well as in humans. The exact mechanism of ash borer in trees. Ivermectin is used as an antihelmintic and
chlordecone neurotoxicity has not been elucidated, but it antiparasitic drug in veterinary medicine, and in humans it has
is believed to involve inhibition of ATPases (both Na*,K’- proven to be an effective treatment for infection of intestinal
and Mg’*-ATPases), and ensuing inhibition of the uptake of threadworms, onchocerciasis (river blindness), and lymphatic
 CHAPTER 22 Toxic Effects of Pesticides 34]

filariasis. In insects and nematodes, avermectins exert their TABLE 22-6 Some mechanisms ofaction
toxic effects by binding to, and activating, glutamate-dependent of herbicides.
chloride channel. Signs and symptoms of intoxication include
hyperexcitability, tremors, and incoordination, followed by Mechanism Chemical Classes (Example)
ataxia and coma-like sedation. Inhibition of photosynthesis Triazines (atrazine), substituted
ureas (diuron), uracils (bromacil)

INSECT REPELLENTS Inhibition of respiration Dinitrophenols

Auxin growth regulators Phenoxy acids (2,4-D), benzoic

Insect-transmitted diseases remain a major source of illness and acids (dicamba), pyridine acids
death worldwide, as mosquitoes alone transmit disease to more (picloram)
than 700 million persons annually. DEET (N,N-diethyl-m-
Inhibition of protein synthesis Dinitroanilines
toluamide or N,N-diethyl-3-methylbenzamide) is very effective
at repelling insects, flies, fleas, and ticks, and protection time Inhibition of lipid synthesis Aryloxyphenoxypropionates
increases with increasing concentrations. Subchronic toxicity (diclofop)
studies in various species did not reveal major toxic effects and Inhibition of specific enzymes
no significant effects of DEET were seen in mutagenicity, repro- + Glutamine synthetase Glufosinate
ductive toxicity, and carcinogenicity studies. Acute and chronic + Enolpyruvylshikimate- Glyphosate
neurotoxicity studies also provided negative results. However, 3-phosphate synthetase
« Acetolactate synthase Sulfonylureas
in children DEET is possibly responsible for neurotoxic effects
and children should only be exposed to products with up to Cell membrane disruptors Bipyridyl derivatives (paraquat)
10% DEET.

Picaridin
kill all vegetation, whereas selective compounds are those used
Picaridin was developed as an alternative to DEET. Insect
to kill weeds without harming the crops.
repellent formulations (cream, aerosol, wipe) containing 5% to
A number of herbicides can cause dermal irritation and con-
20% picaridin are highly effective against a variety of arthro-
tact dermatitis, particularly in individuals prone to allergic
pod pests, especially mosquitoes, ticks, and flies. Its action in
reactions. Other compounds have generated much debate for
insects is believed to be due to the interaction with specific
their suspected carcinogenicity or neurotoxicity. The principal
olfactory receptors of the arthropod. In humans it is absorbed
classes of herbicides associated with reported adverse health
through the skin to a limited degree, and is metabolized via
effects in humans are discussed below.
hydroxylation and glucuronidation, before excretion in the urine.
‘The toxicological profile of picaridin is unremarkable. Acute der-
mal toxicity is low. There is no evidence of genotoxicity, carcino- Chlorophenoxy Compounds
genicity, teratogenicity, reproductive toxicity, or neurotoxicity.
Chlorophenoxy herbicides are chemical analogs of auxin, a
When used as directed, picaridin-containing formulations are
plant growth hormone, that produce uncontrolled and lethal
deemed to be safe and effective.
growth in target plants. Because the auxin hormone is crit-
ical to the growth of many broad-leaved plants, but is not
HERBICIDES used by grasses, chlorophenoxy compounds can suppress
the growth of weeds (e.g., dandelions) without affecting the
Herbicides are chemicals that are capable of either killing or grass. The most commonly used compound of this class is
severely injuring plants. Some of the various mechanisms by 2,4-dichlorophenoxyacetic acid (2,4-D).
which herbicides exert their biological effects are shown in Ingestion of 2,4-D has caused acute poisoning in humans,
Table 22-6, together with examples for each class. Another resulting in vomiting, burning of the mouth, abdominal pain,
method of classification pertains to how and when herbicides hypotension, myotonia, and CNS involvement including
are applied. Thus, preplanting herbicides are applied to the soil coma. Dermal exposure is the major route of unintentional
before a crop is seeded, preemergent herbicides are applied to exposure to 2,4-D in humans.
the soil before the time of appearance of unwanted vegetation, There are several case reports suggesting an association
and postemergent herbicides are applied to the soil or foliage between exposure to 2,4-D and neurologic effects like periph-
after the germination of the crop and/or weeds. Herbicides eral neuropathy, demyelination and ganglion degeneration in
are also divided according to the manner they are applied to the CNS, reduced nerve conduction velocity, myotonia, and
plants. Contact herbicides are those that affect the plant that behavioral alterations. 2,4-D does not appear to have genotoxic
was treated, whereas translocated herbicides are applied to the or carcinogenic properties in rats, mice, and dogs. ‘The chloro-
soil or to above-ground parts of the plant, and are absorbed phenoxy herbicides have attracted much attention because of an
and circulated to distant tissues. Nonselective herbicides will association between exposure and non-Hodgkin's lymphoma
342 UNIT 5 Toxic Agents

or soft-tissue sarcoma, found in a few epidemiological studies.

Nevertheless, 2,4-D is classified as a group D agent (not classifi-
@ NADPH NADP*
able as to human carcinogenicity).
part PQ”

Bipyridil Compounds
Paraquat is a fast-acting, nonselective contact herbicide, used
to control broad-leaved weeds and grasses in plantations and
fruit orchards, and for general weed control. Paraquat has one O° O,
of the highest acute toxicities among herbicides. On absorption,
independent of the route of exposure, paraquat accumulates in 4 2 2H*
the lung and the kidney. Paraquat is very poorly metabolized, 0, +0, — > H,0,
and is excreted almost unchanged in the urine. It has minimal @ Fet + 0, ———> Fe* + 0,
to no genotoxic activity, is not carcinogenic in rodents, has no
effect on fertility, is not teratogenic, and only produces fetotox- Fe** + H,O, ———» OH’ + OH + Fe™
icity at maternally toxic doses. The major toxicologic concerns
for paraquat are related to its acute systemic effects, particularly NADP* GSH
in the lung, and secondarily, the kidney.
Once paraquat enters a cell, it undergoes alternate reduc- : Vv
tion followed by reoxidation, a process known as redox cycling. \ Lipid peroxidation
Intracellular redox cycling of paraquat would also result in the
oxidation of NADPH, leading to its cellular depletion, which is
augmented by the detoxification of hydrogen peroxide formed
in the glutathione peroxidase/reductase enzyme system to v
Il death
regenerate GSH (Figure 22-3). NADPH core le | Bet
Damage to alveolar epithelial cells occurs within 24h after H,O0
acute exposure to lethal doses of paraquat. Damage progresses
in the following 2 to 4 days with loss of the alveolar epithelium, FIGURE 22-3 Mechanism of toxicity of paraquat. (1) Redox
alveolar edema, extensive infiltration of inflammatory cells cycling of paraquat utilizing NADPH; (2) formation of hydroxy
into the alveolar interstitium, and finally death due to severe radicals leading to lipid peroxidation (3); (4) detoxication of HO, via
anoxia. Survivors of this destructive first phase show exten- glutathione reductase/peroxidase couple, utilizing NADPH. (Modified
from Smith LL: Mechanism of paraquat toxicity in the lung and its
sive proliferation of fibroblasts in the lung. The second phase is
relevance to treatment, Hum Toxicol, 1987 Jan;6(1):31-36).
characterized by attempts by the alveolar epithelium to regen-
erate and restore normal architecture, and presents as an inten-
sive fibrosis. Individuals who survive the first phase may still
(corn, soybeans, and peanuts). Alachlor, acetochlor, and
die from the progressive loss of lung function several weeks
butachlor are probable human carcinogens (Group B2). The
after exposure.
discovery of alachlor in well water led to cancellation of its
The herbicide diquat presents a different toxicologic pro-
registration in some countries, and to its restriction in others.
file. Acute toxicity is somewhat lower. In contrast to paraquat,
Both are believed to be threshold-sensitive phenomena.
diquat does not accumulate in the lung, and no lung toxicity is
seen on acute or chronic exposure. On chronic exposure, target
organs for toxicity are the gastrointestinal tract, the kidney,
and particularly the eye. Like paraquat, diquat can be reduced Triazines
to form a free radical and then reoxidized in the presence of The family of triazine herbicides comprises several compounds
oxygen, with the concomitant production of superoxide anion. (atrazine, simazine, and propazine) that are extensively used
This process of redox cycling occurs in the eye and is believed for the preemergent control of broad-leaved weeds. Triazines
to be the likely mechanism of cataract formation. Human clini- have low acute oral and dermal toxicity, and chronic toxicity
cal symptoms include nausea, vomiting, diarrhea, ulceration of studies indicate primarily decreased body weight gain. There is
mouth and esophagus, decline in renal functions, and neuro- no evidence that triazines are teratogenic, genotoxic, or devel-
logic effects, but no pulmonary fibrosis. opmental or reproductive toxicants. However, a more recent
study has suggested a possible clastogenic effect. Though
exposure to atrazine through residues in food commodities is
Chloroacetanilides very low, contamination of ground water and drinking water
Representative compounds of this class of herbicides are ala- is common. Nevertheless, the known hormonal effects of tri-
chlor, acetochlor, and metolachlor, which are used to control azines call for careful evaluation of the endocrine-disrupting
herbal grasses and broad-leaved weeds in a number of crops effects of these herbicides.
 CHAPTER 22 Toxic Effects of Pesticides 343

Phosphonomethyl Amino Acids Captan and Folpet

The two compounds of this class are glyphosate (N-phos Captan and folpet are broad-spectrum protectant fungicides;
phonomethyl glycine) and glufosinate (N-phosphonomethyl together with captafol, they are called chloroalkylthio fungi-
homoalanine). Both are broad-spectrum nonselective systemic cides, due to the presence of side chains containing chlorine,
herbicides used for postemergent control of annual and peren- carbon, and sulfur. They are potent eye irritants, but only mild
nial plants. Though both compounds contain a P=O moiety, skin irritants. Dermal absorption is low. Captan and folpet, as
they are organophosphonates and do not inhibit AChE. well as thiophosgene, are mutagenic in vitro tests; however, in
vivo mutagenicity tests are mostly negative, possibly because
Glyphosate—Glyphosate exerts its herbicidal action by of the rapid degradation of these compounds. These fungi-
inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate cides induce the development of duodenal tumors in mice,
synthase, responsible for the synthesis of an intermediate in and on this basis, they are classified by the US EPA as probable
the biosynthesis of various amino acids. Although important human carcinogens. Because of their structural similarity to
in plant growth, this metabolic pathway is not present in the potent teratogen thalidomide, chloroalkylthio fungicides
mammals. It has no teratogenic, developmental, or reproductive have been extensively tested in reproductive/developmental
effects. Genotoxicity and carcinogenicity studies in animals studies in multiple species, but no evidence of teratogenicity
were negative. has been found.
Glyphosate is one of the most widely used herbicides, and the
development of transgenic crops that can tolerate glyphosate
treatment has expanded its utilization. Given its widespread Dithiocarbamates
use, including the home and garden market, accidental or The nomenclature of many of these compounds arises from
intentional exposure to glyphosate is inevitable. The most the metal cations with which they are associated; thus, there
widely used glyphosate product is Roundup® which is formu- are, e.g., Maneb (Mn), Ziram and Zineb (Zn), and Mancozeb
lated as a concentrate containing water, 41% glyphosate (as (Mn and Zn) (Figure 22-4). Thiram is an example of dithio-
isopropylamine salt), and 15% polyoxethyleneamine (POEA). carbamate without a metal moiety (Figure 22-4). The dithio-
Mild intoxication results mainly in transient gastrointestinal carbamates have low acute toxicity by the oral, dermal, and
symptoms. Moderate or severe poisoning presents with gastro- respiratory routes. However, chronic exposure is associated
intestinal bleeding, hypotension, pulmonary dysfunction, and with adverse effects that may be due to the dithiocarbamate
renal damage.

Glufosinate—Glufosinate is a nonselective contact herbi-

S
cide that acts by irreversibly inhibiting glutamine synthetase. |
Plants die as a consequence of the increased levels of ammonia. AG —WNial-—=C eS
<
Mammals have other metabolizing systems that can cope | Mn
we
with the effects on glutamine synthetase activity to a certain ne —INInl= = SS
limit. There is no evidence of genotoxicity or carcinogenicity, |
or direct effects on reproductive performance and fertility. S
Maneb
Developmental toxic effects were found in rabbits (premature
Manganese ethylenebisdithiocarbamate
deliveries, abortions, and dead fetuses). Humans experience
gastrointestinal effects, impaired respiration, neurologic dis-
S
turbance, and cardiovascular effects.
|
ne N= CS
SS
| Zn
FUNGICIDES
iG Ni Ge S
Fungal diseases are virtually impossible to control without |
S
chemical application. Fungicidal chemicals are derived from Zineb
a variety of structures, ranging from simple inorganic com- Zinc ethylenebisdithiocarbamate
pounds, such as copper sulfate, to complex organic compounds.
Most fungicides are surface or plant protectants, and are applied CH3 CH,
prior to potential infection by fungal spores, either to plants or Za
NC SS = C=N
to postharvest crops. Other fungicides can be used therapeuti- Gee Cuil | NH,
cally, to cure plants when an infestation has already begun. Still S S)
others are used as systemic fungicides that are absorbed and Thiram
Bis(diethylthio-carbamoyl)disulfide
distributed throughout the plant. With a few exceptions, fun-
gicides have low acute toxicity in mammals. Some fungicides FIGURE 22-4 Structures of three dithiocarbamate
have been associated with severe epidemics of poisoning. fungicides.
344 UNITS5 Toxic Agents

acid or the metal moiety. These compounds are metabolized Anticoagulants

to a common metabolite, ethylenethiourea, that is responsi-
In addition to their use as rodenticides, coumarin derivatives,
ble for the effects of dithiocarbamates on the thyroid, which
including warfarin itself, are used as anticoagulant drugs and
include hypertrophy and hyperplasia of thyroid follicular cells
have become a mainstay for prevention of thromboembolic
that progress to adenomas and carcinomas. Similarly, dithio-
disease. Coumarins antagonize the action of vitamin K in the
carbamates alter thyroid hormone levels, and cause thyroid synthesis of clotting factors (factors II, VII, IX, and X). Their
hypertrophy. Also, the structure of dithiocarbamate fungi- specific mechanism involves inhibition of vitamin K epoxide
cides resembles that of disulfiram, which inhibits aldehyde reductase, which regenerates the reduced vitamin K necessary
dehydrogenase and may, after ingestion of ethanol, lead to for sustained carboxylation and synthesis of relevant clot-
elevated acetaldehyde levels. ting factors. Human poisonings by these rodenticides are rare
because they are dispersed in grain-based baits. However, there
Inorganic and Organometal Fungicides are a significant number of suicide or homicide attempts or of
accidental consumption ofwarfarin.
Copper sulfate has overall low toxicity and remains one of
the most widely used fungicides. Triphenyltin acetate is used
as a fungicide, whereas tributyltin is utilized as an antifouling FUMIGANTS
agent. Triphenyltin has moderate to high acute toxicity, but
may cause reproductive toxicity and endocrine disruption. These agents are active toward insects, mites, nematodes, weed
Organic mercury compounds, such as methylmercury, were seeds, fungi, or rodents, and have in common the property of
used extensively as fungicides in the past for the prevention of being in the gaseous form at the time they exert their pesticidal
seed-borne diseases in grains and cereals. action. They can be liquids that readily vaporize (e.g., ethyl-
ene dibromide), solids that can release a toxic gas on reaction
with water (e.g., phosphine released by aluminum phosphide),
RODENTICIDES or gases (e.g., methyl bromide). For soil fumigation, the com-
Rats and mice can cause health and economic damages to pound is injected directly into the soil, which is then covered
humans. Rodents are vectors for several human diseases, with plastic sheeting, which is sealed. Compounds used as
including plague, endemic rickettsiosis, spirochetosis, and fumigants are usually nonselective, highly reactive, and cyto-
several others; they can occasionally bite people; they can toxic. They provide a potential hazard from the standpoint of
consume large quantities of postharvest stored foods, and can inhalation exposure, and to a minor degree for dermal expo-
contaminate foods with urine, feces, and hair. Rodenticides sure or ingestion, in case ofsolids or liquids.
play an important role in rodent control. To be effective, yet
safe, rodenticides must satisfy several criteria: (1) the poison
must be very effective in the target species once incorporated
Methyl Bromide
into bait in small quantity; (2) baits containing the poison Methyl bromide is a broad-spectrum pesticide, used for soil
must not excite bait shyness, so that the animal will continue fumigation, commodity treatment, and structural fumigation.
to eat it; (3) the manner of death must be such that survi- Acute exposure results in respiratory, gastrointestinal, and
vors do not become suspicious of its cause; and (4) it should neurologic symptoms; the latter include lethargy, headache,
be species-specific, with considerably lower toxicity to other seizures, paresthesias, peripheral neuropathy, and ataxia, and
animals. Toxicologic problems can arise from acute acciden- are considered to be more relevant than other toxic effects
tal ingestions or from suicidal and homicidal attempts. Every for human risk assessment. Acute and chronic neurotoxic-
year, thousands of accidental ingestions of rodenticide baits ity studies in rats have demonstrated behavioral effects and
by children occur, most of which resolve without serious morphological lesions, which were concentration- and time-
consequences. dependent. Methyl bromide is positive in some genotoxicity
tests, but it is listed in Group 3 as not classifiable as a human
carcinogen. Methyl bromide is an odorless and colorless
Fluoroacetic Acid and Its Derivatives gas, but chloropicrin, with a pungent odor and eye irrita-
Sodium fluoroacetate (Compound 1080) and _ fluoroacet- tion, is often used in conjunction with methyl bromide and
amide are white in color and odorless. Their high mammalian other fumigant mixtures, to warn against potentially harmful
toxicity limits use to trained personnel. The main targets of exposures.
toxicity are the central nervous system and the heart. Initial
gastrointestinal symptoms are followed by severe cardiovas-
cular effects (ventricular tachycardia, fibrillation, and hypo- 1,3-Dichloropropene
tension), as well as CNS effects (agitation, convulsions, and 1,3-Dichloropropene is a soil fumigant, extensively utilized
coma). Use of Compound 1080 in the United States is severely for its ability to control soil nematodes. It is an irritant, and
restricted primarily because of toxicity to nontarget animals, can cause redness and necrosis of the skin. It is extensively
such as dogs. metabolized, with the mercapturic acid conjugate being the
 CHAPTER 22 Toxic Effects of Pesticides 345

major urinary metabolite. Data on genotoxicity are contra- elemental sulfur is dermatitis. In ruminants, excessive sulfur
dictory, and carcinogenicity studies in rodents have found an ingestion can cause cerebrocortical necrosis (polioencephalo-
increase in benign liver tumors in rats but not in mice, after malacia), possibly due to its conversion by microorganisms in
oral administration. f the rumen to hydrogen sulfide.

Sulfur
BIBLIOGRAPHY
Elemental sulfur is an effective fumigant for the control of Davis FR: Banned: A History of Pesticides and the Science of Toxicology.
many plant diseases, particularly fungal diseases, and rep- New Haven, CO: Yale University Press, 2014.
resents the most heavily used crop protection chemical in Marrs TT, Ballantyne B: Pesticide Toxicology and International
the United States. Sulfur finds its major uses in grapes and Regulation. Hoboken NJ: John Wiley & Sons, 2004.
tomatoes, and can be used in organic farming. The primary Yu SJ: The Toxicology and Biochemistry of Insecticides, 2nd ed. Boca
health effect in humans associated with the agricultural use of Raton, FL: CRC Press/Taylor & Francis, 2014.
346 UNIT 5 Toxic Agents

QUESTIONS

1. Which of the following does NOT contribute to the Es Which of the following herbicides is NOT correctly paired
environmental presence of organochlorine insecticides? with its mechanism of action?
high water solubility. a. glufosinate—inhibition of glutamine synthetase.
low volatility. b. paraquat—interference with protein synthesis.
chemical stability. c. glyphosate—inhibition of amino acid synthesis.
low cost. d. chlorophenoxy compounds—growth stimulants.
OSslow rate of degradation.
ClG e. diquat—production of superoxide anion through
redox cycling.
2. All of the following are characteristic of DDT poisoning
EXCEPT: Captan:
paresthesia. a. isa herbicide that inhibits root growth.
hypertrophy of hepatocytes. b. is an insecticide that targets the reproductive organs.
increased potassium transport across the membrane. c. isa fungicide that could cause duodenal tumors.
slow closing of sodium ion channels. d. isa herbicide that stimulates growth.
te
Ne
DSU) dizziness. e. isa fungicide that is a known teratogen.

3. Anticholinesterase agents: What is a mechanism of action ofnicotine?

a. enhance the activity of AChE. a. Nicotine antagonizes ACh at the neuromuscular
b. increase ACh concentration in the synaptic cleft. junction.
c. only target the neuromuscular junction. b. Nicotine decreases the rate of repolarization of the
d. antagonize ACh receptors. axonal membrane.
e. cause decreased autonomic nervous system stimulation. c. Nicotine interferes with sodium permeability.
Nicotine acts as an ACh agonist in the synapse.
4. All of the following symptoms would be expected following e. Nicotine inhibits the release of neurotransmitter.
anticholinesterase insecticide poisoning EXCEPT:
a. bronchodilation. 10. Which of the following is the most characteristic of
b. tachycardia. warfarin poisoning?
c. diarrhea. a. diarrhea.
d. increased blood pressure. b. cyanosis.
e. dyspnea. c. decreased glucose metabolism.
d. hematomas.
5. Which of the following insecticides blocks the electron e.” “seizures:
transport chain at NADH-ubiquinone reductase?
a. nicotine.
b. carbamate esters.
c. nitromethylenes.
d. pyrethroid esters.
e. rotenoids.

6. What is the main mechanism of pyrethroid ester toxicity?

a. blockage of neurotransmitter release.
b. inhibition of neurotransmitter reuptake.
c. acting as a receptor agonist.
d. causing hyperexcitability of the membrane by inter-
fering with sodium transport.
e. interfering with Cl transport across the axonal
membrane.
 GocHe AsePee Tab AR

Toxic Effects of Metals

Erik J. Tokar, Windy A. Boyd, Jonathan H. Freedman,
and Michael P. Waalkes

INTRODUCTION Exposure
What Is a Metal? Toxicity
Metals as Toxicants Nickel
Movement of Metals in the Environment Toxicity
Chemical Mechanisms of Metal Toxicology Carcinogenicity
Factors Impacting Metal Toxicity ESSENTIAL METALS WITH POTENTIAL FOR TOXICITY
Biomarkers of Metal Exposure
Copper
Molecular Responses to Metal Exposure
Toxicity
Metal-binding Proteins and Metal Transporters
Hereditary Disease of Copper Metabolism
Pharmacology of Metals
lron
MAJOR TOXIC METALS Toxicity
Arsenic Zinc
Toxicokinetics Essentiality and Deficiency
Toxicity Toxicity
Carcinogenicity
METALS RELATED TO MEDICAL THERAPY
Treatment
Cadmium Aluminum

Exposure Toxicity
Toxicity
Lithium
Lead Toxicokinetics
Exposure Toxicity
Toxicity Platinum
Mercury Toxicity
Global Cycling and Ecotoxicology

KEY POINTS 2
= Persons at either end of the life span, young children a Chelation occurs when bidentate ligands form ring
or elderly people, are more susceptible to toxicity from structures that include the metal ion and the two ligand
exposure to a particular level of metal than most adults. atoms attached to the metal.
a Metals that provoke immune reactions include mercury, a Metal—-protein interactions include binding to numer-
gold, platinum, beryllium, chromium, and nickel. ous enzymes, the metallothioneins, nonspecific binding
= Complexation is the formation of a metal ion complex to proteins such as serum albumin or hemoglobin, and
in which the metal ion is associated with a charged or specific metal carrier proteins involved in the membrane
uncharged electron donor, referred to as a ligand. transport of metals.

347
348 UNIT 5 Toxic Agents

INTRODUCTION metals has influenced environmental levels of metals in air,

water, soil, and food. Human use of metals can also alter the
What Is a Metal? chemical form or speciation of an element and thereby impact
toxic potential. As elemental species, metals are nonbiode-
Metals are typically defined by physical properties of the ele-
gradable. This indestructibility combined with bioaccumula-
ment in the solid state. General metal properties include high
tion contributes to the high concern for metals as toxicants.
reflectivity (luster), high electrical conductivity, high thermal
conductivity, and mechanical ductility and strength. A toxi-
cologically important characteristic of metals is that they may
react in biological systems by losing one or more electrons to Movement of Metals in the Environment
form cations. Metals are redistributed naturally in the environment by both
Many metals have been reported to produce significant tox- geological and biological cycles. Rainwater dissolves rocks and
icity in humans. These include major toxic metals (e.g., lead and ores and transports materials, including metals, to rivers and
cadmium), essential metals (e.g., zinc and copper), medicinal underground water (e.g., arsenic), depositing and stripping
metals (e.g., platinum and bismuth), minor toxic metals includ- materials from adjacent soil and eventually transporting these
ing metals in emerging technology (e.g., indium and uranium), substances to the ocean to be precipitated as sediment or taken
toxic metalloids (e.g., arsenic and antimony), and certain non- up into forming rainwater to be relocated elsewhere. Biological
metallic elemental toxicants (e.g., selenium and fluoride). An cycles moving metals include biomagnification by plants and
overview of metal toxicology is shown in Figure 23-1. animals resulting in incorporation into food cycles. Human
industry greatly enhances metal distribution in the global envi-
ronment by dischargesto soil, water, and air. Reports of metal
Metals as Toxicants intoxication are common in plants, aquatic organisms, inverte-
Metals are unique among pollutant toxicants in that they are brates, fish, sea mammals, birds, and.\domestic animals. Not all
all naturally occurring and are already ubiquitous to some human toxicity occurs from metals deposited in the biosphere
level within the human environment. Regardless of how safely by human activity. For example, chronic arsenic poison-
metals are used in industrial processes or consumer products, ing from high levels of naturally occurring inorganic arsenic
some level of human exposure is inevitable. Metals differ from in drinking water is a major health issue in many parts of the
other toxic substances because, as elements, they are neither world. Endemic intoxication from excess fluoride, selenium, or
created nor destroyed by human endeavors. Human use of thallium can all occur from natural high environmental levels.

Eionpcute| cycling Ecotoxtcity

| oo a oe :
aes distribution
-Biotransformation
. So
a Biomagnification
a .|
oe
Occurrence

Natural
Anthropogenic

Chemistry \y Human exposure Toxicokinetics

Occupational oe
Chemical form : sorpuon
Environmental |
Speciation }
Distributi on.
Diietary
Essentiality Biotransformation
Medical g
Excretion

Mechanisms Host factors

Adventitious bind, mimicry
Age, gender, etc.
Oxidative stress
Adverse health effects Adaptive mechanisms
Enzyme inhibition
Metal transporters
DNA damage
_ Metal-binding proteins
Gene expression

FIGURE 23-1 Overview of metal toxicology.

 CHAPTER 23 Toxic Effects of Metals 349

Chemical Mechanisms of Metal Toxicology for biomarkers. Thus, in the case of chromium, DNA-protein
complexes may serve as a biomarker of both exposure and car-
Chemically, metals in their ionic form can be very reactive
cinogenic potential. Also, hair can be useful in assessing varia-
and can interact with biological systems in a large variety of
tions in exposure to metals over the period ofits growth.
ways. In this regard, a cell presents numerous potential metal-
binding ligands. The inhibition ofbiologically critical enzymes
is an important molecular mechanism of metal toxicology. Molecular Responses to Metal Exposure
The metals can show more specific forms of chemical attack
Exposure to elevated levels of nonessential and essential met-
through mimicry: acting as mimics of essential metals, they
als can induce intracellular damage. This damage includes
bind to physiological sites that normally are reserved for
oxidative stress, which can lead to lipid peroxidation, protein
an essential element. For example, mimicry for, and replace-
denaturation, DNA damage, and organelle dysfunction. In
ment of, zinc is a mechanism of toxicity for cadmium, copper,
addition, metals can disrupt the biological function/activity
and nickel.
of proteins by either directly binding to the protein or dis-
Another key chemical reaction in metal toxicology is metal-
placing metals within metalloproteins. The intended conse-
mediated oxidative damage. Many metals can directly act as
quence of metal activation of gene expression is to protect
catalytic centers for redox reactions with molecular oxygen or
the organism from metal-induced damage. Metal exposure
other endogenous oxidants, producing oxidative modification
is associated with increased expression of genes that encode
of biomolecules such as proteins or DNA. This may bea key step
proteins that (1) remove the metal from the cell via chela-
in the carcinogenicity of certain metals. Besides oxygen-based
tion or increased export, (2) reduce the level of oxidative
radicals, carbon- and sulfur-based radicals may also occur.
stress, and (3) repair the metal-induced intracellular damage.
Metals in their ionic form can be very reactive and form
However, the inappropriate activation of gene expression
DNA and protein adducts in biological systems. They can also
following metal exposure can be a contributing factor to a
induce an array of aberrant gene expression, which, in turn,
variety of human pathologies.
produces adverse effects. .

Factors Impacting Metal Toxicity Metal-binding Proteins and

Exposure-related factors include dose, route of exposure,
Metal Transporters
duration, and frequency of exposure. Host-based factors that Protein binding of metals is a critical aspect of essential and
can impact metal toxicity include age at exposure, gender, and toxic metal metabolism. Many different types of proteins play
capacity for biotransformation. For instance, it is quite clear roles in the disposition of metals in the body. Nonspecific bind-
that younger subjects are often more sensitive to metal intoxi- ing to proteins, like serum albumin or hemoglobin, acts in
cation, as, e.g., with the neurotoxicity of lead in children. The metal transport and tissue distribution. Proteins with specific
major pathway of exposure to many toxic metals in children metal-binding properties play special roles in the trafficking
is food, and children consume more calories per pound of of specific essential metals, and toxic metals may interact with
body weight than adults. Moreover, children have higher gas- these proteins through mimicry.
trointestinal absorption of metals, particularly lead. Elderly The metallothioneins are the best known example of metal-
persons are also believed to be generally more susceptible to binding proteins. These thiol ligands provide the basis for high-
metal toxicity than younger adults. Lifestyle factors such as affinity binding of several essential and toxic metals including
smoking or the composition of diet (i.e., alcohol ingestion) zinc, cadmium, copper, and mercury.
may have direct or indirect impacts on the level of metal Transferrin is a glycoprotein that binds most of the ferric
intoxication. iron in plasma and helps transport iron across cell membranes.
Adaptive mechanisms can be critical to the toxic effects of The protein also transports aluminum and manganese. Ferritin
metals, and organisms have a variety of ways in which they can is primarily a storage protein for iron. It has been suggested
adapt to otherwise toxic metal insults. Adaptation can be at that ferritin may serve as a general metal detoxicant protein,
the level of uptake, excretion, or long-term storage in a toxi- because it binds a variety of toxic metals including cadmium,
cologically inert form. Metal exposure can induce a cascade of zinc, beryllium, and aluminum.
molecular/genetic responses that may, in turn, reduce toxicity, Ceruloplasmin is a copper-containing glycoprotein oxidase
such as with metal-induced oxidative stress responses. in plasma that converts ferrous iron to ferric iron, which then
binds to transferrin. This protein also stimulates iron uptake by
a transferrin-independent mechanism.
Biomarkers of Metal Exposure
Biomarkers of exposure, toxicity, and susceptibility are impor-
tant in assessing the level of concern with metal intoxica- Pharmacology of Metals
tion. Exposure biomarkers, such as concentrations in. blood, Many metallic chemicals are valuable pharmacological tools in
urine, or hair, have long been used with metals. Techniques in the treatment of human disease, as exemplified by the highly
molecular toxicology have greatly expanded the possibilities effective use of platinum compounds in cancer chemotherapy.
350 UNIT 5 Toxic Agents

In addition, gallium and titanium complexes are promising after large doses and consisting of Wallerian degeneration of
metal compounds in cancer chemotherapy. Other medicinal axons. Anemia and leucopenia, particularly granulocytopenia,
metals used today include aluminum (antacids and buffered occur a few days following high-dose arsenic exposure and are
analgesics), bismuth (peptic ulcer and Helicobacter pylori- reversible.
associated gastritis), lithium (mania and bipolar disorders),
and gold (arthritis). Chronic Toxicity—The skin is a major target organ in chronic
Treatment of metal poisoning is sometimes used to prevent, inorganic arsenic exposure. Skin cancer is common with pro-
or even attempt to reverse, toxicity. The typical strategy is to tracted high-level arsenical exposure. Liver injury, character-
give metal chelators that will complex the metal and enhance istic of long-term or chronic arsenic exposure, manifests itself
its excretion. Such therapy can be used for many different met- initially as jaundice, abdominal pain, and hepatomegaly and
als including lead, mercury, iron, and arsenic. may progress to cirrhosis and ascites, even to hepatocellular
carcinoma.
Repeated exposure to low levels of inorganic arsenic can pro-
MAJOR TOXIC METALS duce peripheral neuropathy. This neuropathy usually begins
with sensory changes, such as numbness in the hands and
Arsenic feet, but later may develop into a painful “pins and needles”
The most common inorganic trivalent arsenic compounds sensation. Both sensory and motor nerves can be affected,
are arsenic trioxide and sodium arsenite, whereas common and dying-back axonopathy with demyelination may occur.
pentavalent inorganic compounds are sodium arsenate, arse- In addition, peripheral vascular disease has been observed
nic pentoxide, and arsenic acid. Important organoarsenicals in persons with chronic exposure to inorganic arsenic in the
include arsenilic acid, arsenosugars, and several methylated drinking water.
forms produced as a consequence ofinorganic arsenic biotrans-
formation in various organisms, including humans. Arsine Mechanisms of Toxicity—The trivalent compounds ofarsenic
(AsH,) is an important gaseous arsenical. are thiol-reactive, and thereby inhibit enzymes or alter pro-
Occupational exposure to arsenic occurs in the manufac- teins by reacting with proteinaceous thiol groups. Pentavalent
ture of pesticides, herbicides, and other agricultural products. arsenate is an uncoupler of mitochondrial oxidative phos-
High exposure to arsenic fumes and dusts may occur in smelt- phorylation, by a mechanism likely related to competitive
ing industries. Environmental arsenic exposure mainly occurs substitution (mimicry) of arsenate for inorganic phosphate
from arsenic-contaminated drinking water, which is often in the formation of adenosine triphosphate. Arsenic and its
from natural sources, and from the burning of coal containing metabolites have been shown to produce oxidants and oxi-
naturally high levels of arsenic. Food, especially seafood, may dative DNA damage, alteration in DNA methylation status
contribute significantly to daily arsenic intake. and genomic instability, impaired DNA damage repair, and
enhanced cell proliferation.
Toxicokinetics—Inorganic arsenic is well absorbed (80% to
90%) from the gastrointestinal tract, distributed through- Carcinogenicity— Arsenic is a known human carcinogen,
out the body, often metabolized by methylation, and then associated with tumors of the skin, lung, and urinary bladder,
excreted primarily in urine. Arsenic compounds of low solu- and possibly kidney, liver, and prostate. Arsenic-induced skin
bility are absorbed less efficiently after oral exposure. Arsenic cancers include basal cell carcinomas and squamous cell car-
has a predilection for skin and is excreted by desquama- cinomas, both arising in areas of arsenic-induced hyperkera-
tion of skin and in sweat, particularly during periods of pro- tosis. In humans, increased mortality occurs from lung cancer
fuse sweating. It also concentrates in forming fingernails and in young adults following in utero exposure to arsenic. Thus,
hair. Arsenic exposure produces characteristic transverse the developing fetus appears to be hypersensitive to arsenic
white bands across fingernails (Mees’ line), which appear carcinogenesis.
about six weeks after the onset of symptoms of arsenic
toxicity. Arsenic in the fingernails and hair has been used as a Treatment—For acute arsenic poisoning, treatment is symp-
biomarker for exposure. tomatic, with particular attention to fluid volume replacement
and support of blood pressure. The oral chelator penicillamine
Toxicity or succimer (2,3-dimercaptosuccinic acid, DMSA) is effective
Acute Poisoning—Ingestion of large doses (70 to 180 mg) of in removing arsenic from the body. The best strategy for pre-
inorganic arsenic can be fatal. Symptoms of acute intoxica- venting chronic arsenic poisoning is by reducing exposure.
tion include fever, anorexia, hepatomegaly, melanosis, cardiac
arrhythmia, and, in fatal cases, eventual cardiac failure. Acute
arsenic ingestion can damage mucous membranes of the gastro- Cadmium
intestinal tract, causing irritation, vesicle formation, and even About 75% of cadmium produced is used in batteries, espe-
sloughing. Sensory loss in the peripheral nervous system is the cially nickel-cadmium batteries. Because of its noncorro-
most common neurological effect, appearing at 1 to 2 weeks sive properties, cadmium has been used in electroplating or
 CHAPTER 23 Toxic Effects of Metals 351

galvanizing alloys for corrosion resistance. It is also used as a Toxicity— Acute toxicity from the ingestion of high concentra-
color pigment for paints and plastics. This metal is produced as tions of cadmium in the form of heavily contaminated beverages
a by-product of zinc and lead smelting. or food causes severe irritation to the gastrointestinal epithelium,
leading to nausea, vomiting, and abdominal pain. Inhalation of
Exposure—Food is the major source of cadmium for the cadmium fumes or other heated cadmium-containing materials
general population. Many plants readily accumulate cad- may produce acute pneumonitis with pulmonary edema.
mium from soil. Both natural and anthropogenic sources of The major long-term toxic effects of low-level cadmium expo-
cadmium contamination occur for soil, including fallout of sure are renal injury, obstructive pulmonary disease, osteoporo-
industrial emissions, some fertilizers, soil amendments, and sis, and cardiovascular disease. Cancer is primarily a concern
use of cadmium-containing water for irrigation, all resulting in occupationally exposed groups. The chronic toxic effects of
in a slow but steady increase in the cadmium content in veg- cadmium are clearly a much greater concern than the rare acute
etables over the years. Shellfish and animal liver and kidneys toxic exposures.
can accumulate relatively high levels of cadmium. Air can be
a significant source of direct exposure or environmental con- Nephrotoxicity—Cadmium is toxic to tubular cells and glom-
tamination. Cigarette smoking is a major nonoccupational eruli, markedly impairing renal function leading to protein-
source of cadmium exposure. uria. These lesions consist of initial tubular cell necrosis and
Inhalation is the dominant route of exposure in occupational degeneration, progressing to an interstitial inflammation and
settings. Occupations potentially at risk from cadmium expo- fibrosis. Because of the potential for renal toxicity, there is con-
sure include those involved with refining zinc and lead ores, siderable concern about the levels of dietary cadmium intake
iron production, cement manufacture, industries involving for the general population.
fossil fuel combustion, the manufacturing of paint pigments,
cadmium -nickel batteries, and electroplating. Chronic Pulmonary Disease—Cadmium inhalation is toxic to
Figure 23-2 illustrates that cadmium is protein-bound in the respiratory system in a fashion related to the dose and dura-
blood, is rapidly taken up by tissues, and is deposited in the tion of exposure. Cadmium-induced obstructive lung disease
liver and kidney. Cadmium stored in hepatocytes is bound to in humans can be slow in onset, and results from chronic bron-
metallothionine. This complex may be released and then travel chitis, progressive fibrosis of the lower airways, and accom-
to the kidney where the complex is reabsorbed, degraded, and panying alveolar damage leading to emphysema. Pulmonary
free cadmium is able to induce metallothionine synthesis or function is reduced with dyspnea, reduced vital capacity, and
cause toxicity. increased residual volume.

Kidney
Bil
Cd-Alb oils basolateral
< membrane
Cd-LMWPr
wo n-n----------
: Tubular
: fluid
‘ Sensitive site
Cd-GSH
Cd-Alb '
Cd-LMWPr —b> Cd-LMWPr

Lysosome

Cd-MT ——+—»> Cd-MT Cd-MT

y
To urine

y
Liver cell Kidney cell

Glomerular
membrane

FIGURE 23-2 Cadmium transport, protein binding, and toxicity. GSH, glutathione; MT, metallothionine; aa, amino acids; Cd-Alb,
cadmium-albumin; Cd-LMWPr, cadmium associated with low-molecular-weight proteins.
Bey UNIT 5 Toxic Agents

Other Toxicities—Cadmium toxicity affects calcium metab- TABLE 23-1 Summary of lowest observed effect
olism, and associated skeletal changes probably related to levels for lead-related health effects.
calcium loss include bone pain, osteomalacia, and/or osteo-
Blood Lead Levels, ug/dl. oS
porosis. Epidemiologic studies suggest that cadmium may be
an etiologic agent for hypertension. Epidemiologic studies Effect — “Adult Children
in humans have suggested a relationship between abnormal
Neurologic
behavior and/or decreased intelligence in children and adults
exposed to cadmium. In humans, occupational respiratory Encephalopathy (overt) 80-100 100-120

exposure to cadmium has been most clearly associated with Hearing deficits 20 _—
lung cancer.
IQ deficits 10-15 _—

In utero effects 10-15 —

Lead
Lead is a ubiquitous toxic metal and is detectable in practi- Nerve conduction velocity | AO 40
cally all phases of the inert environment and in all biological
Hematologic
systems. The phasing out of leaded gasoline and the removal
of lead from paint, solder, and water supply pipes have sig- Anemia 80-100 80-100
nificantly lowered blood lead levels in the general population.
U-ALA 7 40 40
Lead exposure in children still remains a major health concern.
Additionally, lead is not biodegradable and ecotoxicity of lead B-EP T ; LAAs 15
remains a concern.
ALA-D inhibition 10 10
N
Exposure—Lead-containing paint is a primary source of lead Renal -
exposure in children. Major environmental sources of lead
Nephropathy 40 40-60
for infants and toddlers up to four years of age are hand-to-
mouth transfer of lead-containing paint chips and dust from Vitamin D metabolism
floors of older housing. Lead in household dust can also come
from outside of the home (i.e., soil). A major route of expo-
sure for the general population is from food and water. Dietary
intake of lead has decreased dramatically in recent years. Other The most sensitive indicators of adverse neurologic out-
potential sources of lead exposure are recreational shooting, comes are psychomotor tests or mental development indices,
hand-loading ammunition, soldering, jewelry making, pottery and broad measures of IQ. Lead can affect the brain by multiple
making, gun smithing, glass polishing, painting, and stained mechanisms. Lead may act as a surrogate for calcium and/or dis-
glass crafting. rupt calcium homeostasis. The stimulation of protein kinase C
may result in alteration of the blood-brain barrier. Lead affects
Toxicity—The toxic effects of lead and the minimum blood virtually every neurotransmitter system in the brain, including
level at which an effect is likely to be observed are shown in glutamatergic, dopaminergic, and cholinergic systems. All these
Table 23-1. Lead can induce a wide range of adverse effects in systems play a critical role in synaptic plasticity and cellular
humans depending on the dose and duration of exposure. The mechanisms for cognitive function, learning, and memory.
toxic effects range from inhibition of enzymes to the produc-
tion of severe pathology or death. Children are most sensitive Neurotoxic Effects in Adults—Adults with occupational expo-
to effects in the central nervous system, whereas peripheral sure may demonstrate abnormalities in a number of measures
neuropathy, chronic nephropathy, and hypertension are con- in neurobehavior. Peripheral neuropathy is a classic manifes-
cerns in adults. Other target tissues include the gastrointesti- tation of lead toxicity in adults. Footdrop and wristdrop may
nal, immune, skeletal, and reproductive systems. Effects on the be observed in workers with excessive occupational exposure
heme biosynthesis provide a sensitive biochemical indicator to lead. Peripheral neuropathy is characterized by segmental
even in the absence of other detectable effects. demyelination and possibly axonal degeneration.

Neurologic, Neurobehavioral, and Developmental Effects Hematologic Effects—Lead has multiple hematologic effects,
in Children—Symptoms of lead encephalopathy begin with ranging from increased urinary porphyrins, coproporphy-
lethargy, vomiting, irritability, loss of appetite, and dizziness, rins, 6-aminolevulinic acid (ALA), and zinc-protoporphyrin
progressing to obvious ataxia, and a reduced level of con- to anemia. The heme biosynthesis pathway and the sites of
sciousness, which may progress to coma and death. Recovery lead interference are shown in Figure 23-3. The most sensi-
is often accompanied by sequelae including epilepsy, men- tive effects of lead are the inhibition of 6-aminolevulinic acid
tal retardation, and, in some cases, optic neuropathy and dehydratase (ALAD) and ferrochelatase. ALAD catalyzes the
blindness. condensation of two units of ALA to form porphobilinogen
 CHAPTER 23 Toxic Effects of Metals 359

Mitochondria

Ferrochelatase lron

é.
Heme Protoporphyrin 1X —————————> (Cytochromec

Glycine + ALA-synthetase
Heme oxidase Coproporphyrinnogen on Succinyl-CoA ALA ALA
(microsome) oxidase

Bilirubin
Coproporphyrinnogen

ALA
dehydrogenase

Coproporphyrinnogen <——— Uroporphyrinogen «———— Porphobilinogen

FIGURE 23-3 Leadinterruption of heme biosynthesis. ALA, 6-aminolevulinate; Pb, sites for lead effects.
The major lead inhibition sites are
ALA dehydrogenase and ferrochelatase.

(PBG). Inhibition of ALAD results in accumulation of ALA. exchange pump; and (4) a possible rise in endothelin and
Ferrochelatase catalyzes the insertion of iron into the pro- thromboxane.
toporphyrin ring to form heme. Inhibition of ferrochelatase
results in accumulation of protoporphyrin IX, which takes the Other Toxic Effects—As an immunosuppressive agent, lead
place of heme in the hemoglobin molecule and, as the erythro- decreases immunoglobulins, alters T-cell subpopulations,
cytes containing protoporphyrin IX circulate, zinc is chelated and reduces polymorphonuclear leukocyte chemotactic activ-
at the site usually occupied by iron. Anemia only occurs in very ity. Lead can affect bone by interfering with metabolic and
marked cases of lead toxicity. homeostatic mechanisms including parathyroid hormone,
calcitonin, vitamin D, and other hormones that influence cal-
Renal Toxicity—Acute lead nephrotoxicity consists of proxi- cium metabolism. Lead substitutes for calcium in bone. Lead
mal tubular dysfunction and can be reversed by treatment is known to affect osteoblasts, osteoclasts, and chrondrocytes
with chelating agents. Chronic lead nephrotoxicity consists and has been associated with osteoporosis and delays in frac-
of interstitial fibrosis and progressive nephron loss, azote- ture repair. Lead colic, although rare, is a major gastrointesti-
mia, and renal failure. Lead nephrotoxicity impairs the renal nal symptom of severe lead poisoning, and is characterized by
synthesis of heme-containing enzymes in the kidney, such as abdominal pain, nausea, vomiting, constipation, and cramps.
heme-containing hydroxylase involved in vitamin D metabo-
lism causing bone effects. Hyperuricemia with gout occurs
more frequently in the presence of lead nephropathy. Lead Mercury
nephropathy can be a cause of hypertension. Also called quicksilver, metallic mercury is in liquid state
at room temperature. Mercury vapor (Hg°) is much more
Effects on Cardiovascular System—The pathogenesis of lead- hazardous than the liquid form. Mercury binds to other ele-
induced hypertension is multifactorial including (1) inactiva- ments (such as chlorine, sulfur, or oxygen) to form inorganic
tion of endogenous nitric oxide and cGMP, possibly through mercurous (Hg*) or mercuric (Hg**) salts.
lead-induced reactive oxygen species; (2) changes in the renin-
angiotensin-aldoster6ne system, and increases in sympathetic Global Cycling and Ecotoxicology—Mercury exempli-
activity, important humoral components of hypertension; fies movement of metals in the environment (Figure 23-4).
(3) alterations in calcium-activated functions of vascular Atmospheric mercury, in the form of mercury vapor (Hg’), is
smooth muscle cells including contractility by decreasing derived from natural degassing of the earth’s crust and through
Na*t/K*-ATPase activity and stimulation of the Na*/Ca’* volcanic eruptions as well as from evaporation from oceans
Oo1a)> UNIT 5 Toxic Agents

KRas
ES
es
i ip
WOR? sel
SE wee:
a
eo=
ee
‘a

FIGURE 23-4 The movement of mercury in the environment. In nature, mercury vapor (Hg°), a stable gas, evaporates from the earth’s
surface (both soil and water) and is emitted by volcanoes. Anthropogenic sources include emissions from coal-burning power stations and
municipal incinerators. After ~1 year, mercury vapor is converted to soluble form (Hg**) and returned to the earth by rainwater. It may be
converted back to the vapor by microorganisms and reemitted into the atmosphere. Thus, mercury may recirculate for long periods. Mercury
attached to aquatic sediments is subjected to microbial conversion to methylmercury, starting with plankton, then herbivorous fish, and finally
ascending to carnivorous fish and sea mammals. This biomethylation and biomagnification result in human exposure to methylmercury through
consumption offish, and pose the health risk to humans, especially the developing fetus.

and soils. Anthropogenic sources have become a significant scientific instruments and electrical control devices, in den-
contributor to atmospheric mercury. These include emissions tistry where mercury amalgams are used in tooth restoration,
from metal mining and smelting (mercury, gold, copper, and and in the extraction of gold.
zinc), coal combustion, municipal incinerators, and chloralkali
industries. Methylmercury enters the aquatic food chain start- Accidental Exposure—Elemental mercury exposure can occur
ing with plankton, then herbivorous fish, and finally ascending from broken elemental mercury containers, medicinal devices,
to carnivorous fish and sea mammals. On the top of the food barometers, and melting tooth amalgam fillings to recover sil-
chain, tissue mercury can rise to levels 1800 to 80000 times ver. Inhalation of large amounts of mercury vapor can be deadly.
higher than levels in the surrounding water. This biometh-
ylation and bioconcentration result in human exposure to Toxicity
methylmercury through consumption of fish. Mercury Vapor—Inhalation of mercury vapor at extremely high
concentrations may produce an acute, corrosive bronchitis and
Exposure interstitial pneumonitis and, if not fatal, may be associated with
Dietary Exposure—Consumption of fish is the major route of central nervous system effects such as tremor or increased excit-
exposure to methylmercury. Inorganic mercury compounds ability. This condition has been termed the asthenic-vegetative
are also found in other foods. The source of inorganic mercu- syndrome or micromercurialism. Identification of the syndrome
rial is unknown but the amounts ingested are far below known requires neurasthenic symptoms and three or more of the fol-
toxic levels. Mercury in the atmosphere and in drinking wa- lowing clinical findings: tremor, enlargement of the thyroid,
ter is generally so low that it does not constitute an important increased uptake of radioiodine in the thyroid, labile pulse,
source of exposure to the general population. tachycardia, dermographism, gingivitis, hematologic changes, or
increased excretion of mercury in urine.
Occupational Exposure—Inhalation of mercury vapor may
occur from working in the chloralkali industry. Occupational Inorganic Mercury—The kidney is the major target organ for
exposure may occur during manufacture of a variety of inorganic mercury. Although a high dose of mercuric chloride
 CHAPTER 23 Toxic Effects of Metals B55

is directly toxic to renal tubular cells, chronic low-dose expo- nasal cancers among workers heavily exposed to nickel sulfide,
sure to mercury salts may induce an immunologic glomeru- nickel oxide, and metallic nickel.
lar disease. Exposed persons may develop proteinuria that is
reversible after they are removed from exposure.
ESSENTIAL METALS WITH POTENTIAL
Methylmercury—The major human health effect from expo- FOR TOXICITY
sure to methylmercury is neurotoxicity. Clinical manifesta-
tions of neurotoxicity include paresthesia (a numbness and This group includes eight metals generally accepted as essential:
tingling sensation around the mouth and lips) and ataxia, man- cobalt, copper, iron, magnesium, manganese, molybdenum,
ifested as a clumsy, stumbling gait, and difficulty in swallow- selenium, and zinc. All can produce some target organ toxicity
ing and articulating words. Other signs include neurasthenia (Table 23-2).
(a generalized sensation of weakness), vision and hearing loss,
and spasticity and tremor. These may finally progress to coma
and death. The overall acute effect is cerebral edema, but with
Copper
prolonged destruction of gray matter and subsequent gliosis, Food, beverages, and drinking water are major sources of
cerebral atrophy results. exposure in the general population. Copper exposure in indus-
try is primarily from inhaled particulates in mining or metal
Mechanism of Toxicity—High-affinity binding of divalent fumes in smelting operations, welding, or related activities.
mercury to sulfhydryl groups of proteins in the cells is an
important mechanism for producing nonspecific cell injury or Toxicity—The most commonly reported adverse health
even cell death. Other general mechanisms include an increase effects of excess oral copper intake are gastrointestinal distress.
in genes associated with oxidative stress, reduced glutathione Nausea, vomiting, and abdominal pain have been reported
levels, disruption of microtubules in neuritis, damage mito- shortly after drinking solutions of copper sulfate or beverages
chondria, and disrupt intracellular calcium homeostasis. stored in containers that readily release copper. Ingestion of
drinking water with > 3 mg Cu/L will produce gastrointes-
tinal symptoms. Ingestion of large amounts of copper salts,
Nickel most frequently copper sulfate, may produce hepatic necrosis
Nickel is used in various metal alloys, including stainless steels, and death.
and in electroplating. Occupational exposure to nickel occurs
by inhalation of nickel-containing aerosols, dusts, or fumes, or Hereditary Disease of Copper Metabolism
dermal contact in workers engaged in nickel production (min- Menkes Disease—This is a rare sex-linked genetic defect in
ing, milling, refinery, etc.) and nickel-using operations (melting, copper metabolism resulting in copper deficiency in male
electroplating, welding, nickel-cadmium batteries, etc.). Nickel infants. It is characterized by peculiar hair, failure to thrive,
is ubiquitous in nature, and the general population is exposed to severe mental retardation, neurological impairment, connec-
low levels of nickel in air, cigarette smoke, water, and food. tive tissue dysfunction, and death usually by three to five years
of age. The majority of the pathologies associated with Menkes
Toxicity disease can be linked to deficiencies in copper-containing pro-
Contact Dermatitis—Nickel-induced contact dermatitis is teins. Bones are osteoporotic with flared metaphases of the long
the most common adverse health effect from nickel exposure bones and bones of the skull. There is extensive degeneration of
and is found in 10% to 20% of the general population. It can the cerebral cortex and of white matter. The gene responsible
result from exposure to airborne nickel, liquid nickel solutions, for Menkes disease, ATP7A, belongs to the family of ATPases
or prolonged skin contact with metal items containing nickel, and is a copper transporter. Deficiency in this copper trans-
such as coins and jewelry. porter in Menkes disease blocks copper transportation across
the basolateral membrane of intestinal cells into the portal cir-
Nickel Carbonyl Poisoning—Metallic nickel combines with culation, resulting in accumulation of copper in the enterocytes
carbon monoxide to form nickel carbonyl (Ni{CO}],), which and systemic copper deficiency in the body.
decomposes to nickel and carbon monoxide on heating. Nickel
carbonyl is extremely toxic. Intoxication begins with headache, Wilson’s Disease—This is an autosomal recessive genetic dis-
nausea, vomiting, and epigastric or chest pain, followed by order of copper metabolism characterized by the excessive
cough, hyperpnea, cyanosis, gastrointestinal symptoms, and accumulation of copper in liver, brain, kidneys, and cornea.
weakness. The symptoms may be accompanied by fever and Serum ceruloplasmin is low and serum copper not bound to
leukocytosis. More sévere cases can progress to pneumonia, ceruloplasmin is elevated. Urinary excretion of copper is high.
respiratory failure, and eventually to cerebral edema and death. Clinical abnormalities of the nervous system, liver, kidneys,
and cornea are related to copper accumulation. Patients with
Carcinogenicity—Nickel is a respiratory tract carcinogen in Wilson's disease have impaired biliary excretion of copper,
nickel-refining industry workers. Risks are highest for lung and which is believed to be the fundamental cause of the copper
356 UNIT 5 Toxic Agents

TABLE 23-2 Toxicity of several metals or metalloids.

Neen eee ee eee ee ee ae ee PT aD TEE AEE EEE LEELA

Metal CNS Gl Tract Lung Kidney Liver Heart Blood Skin ©

Aluminum * i

Antimony i 2

Arsenic i i is se %

% 2
Beryllium

Bismuth e *

Cadmium = x * *

Chromium ‘ ” * *

Cobalt m = ts * *

a x
Copper

* * * x
Iron x

o ; x x
Lead cs

Lithium * “ s

Manganese % i ean

Mercury * = *

Nickel . * x

Selenium * x

* x
Silver

Zinc ts
*

overload. Reversal of abnormal copper metabolism is achieved collapse. Death may occur in severely poisoned children
by liver transplantation, confirming that the basic defect is in within 24h.
the liver. Clinical improvement can be achieved with chelation Chronic iron toxicity from iron overload in adults is a
therapy. relatively common problem. There are three basic ways in
which excessive amounts of iron can accumulate in the body:
(1) hereditary hemochromatosis due to abnormal absorption
Iron of iron from the intestinal tract, (2) excess intake via the diet
Iron is an essential metal for erythropoiesis and a key compo- or from oral iron preparations, and (3) repeated blood trans-
nent of hemoglobin, myoglobin, heme enzymes, metalloflavo- fusions for some form of refractory anemia (transfusional
protein enzymes, and mitochondrial enzymes. In biological siderosis). Increased body iron may play a role in the develop-
systems, iron mainly exists as the ferrous (+2) and ferric (+3) ment of cardiovascular disease. It is suspected that iron may
forms. Toxicologic considerations are important in terms of act as a catalyst to produce free radical damage resulting in
iron deficiency, accidental acute exposures, and chronic iron artherosclerosis and ischemic heart disease. Some neurode-
overload due to idiopathic hemochromatosis or as a conse- generative disorders associated with aberrant iron metabolism
quence of excess dietary iron or frequent blood transfusions. in the brain include neuroferritinopathy, aceruloplasminemia,
and manganism.
Toxicity—Acute iron poisoning from accidental ingestion
of iron-containing dietary supplements is the most com-
mon cause of acute toxicity. Severe toxicity occurs after the Zinc
ingestion of more than 0.5 g of iron or 2.5 g of ferrous sul- An essential metal, zinc deficiency results in severe health
fate. Toxicity occurs about | to 6 h after ingestion. Symptoms consequences. However, zinc toxicity is relatively uncommon
include abdominal pain, diarrhea, and vomiting. Ofparticular and occurs only at very high exposure levels. Zinc is present in
concern are pallor or cyanosis, metabolic acidosis, and cardiac most foodstuffs, water, and air. Occupational exposure to dusts
 CHAPTER 23 Toxic Effects of Metals 2)5)//

and fumes of metallic zinc occurs in zinc mining and smelting. METALS RELATED TO MEDICAL
The zinc content of substances in contact with galvanized cop-
per or plastic pipes may be high.
THERAPY
Metals that are used to treat a number of human illnesses,
Essentiality and Deficiency—More than 300 catalyti- including aluminum, bismuth, gold, lithium, and platinum,
cally active zinc metalloenzymes and 2000 zinc-dependent exert some toxicity (Table 23-2).
transcription factors exist. Zinc participates in a wide variety
of metabolic processes, supports a healthy immune system, Aluminum
and is essential for normal growth and development dur-
Chemical compounds of aluminum occur typically in the tri-
ing pregnancy, childhood, and adolescence. Zinc deficiency
valent valence state (Al°**). As a hard trivalent ion, aluminum
is related to poor dietary zinc intake, dietary phytate intake,
binds strongly to oxygen-donor ligands such as citrate and
chronic illness, or oversupplementation with iron or copper.
phosphate. Human exposure to aluminum comes primarily
Symptoms of zinc deficiency include growth retardation,
from food and secondarily from drinking water. The amount of
appetite loss, alopecia, diarrhea, impaired immune func-
aluminum in the food supply is small compared with pharma-
tion, cognitive impairments, dermatitis, delayed healing of
ceutical use of aluminum in antacids and buffered analgesics.
wounds, taste abnormalities, and impaired sexual function.
Occupational exposures to aluminum occur during mining
Therapeutic uses of zinc include the treatment of acute diar-
and processing, as well as in aluminum welding.
rhea in infants with severe zinc deficiency, the treatment of
common cold by its antiviral and immunomodulatory effects,
therapy for Wilson’s disease to help reduce copper burden and Toxicity— Most cases of aluminum toxicity in humans are
to induce metallothionein, and the prevention of blindness in observed in patients with chronic renal failure, or in persons
age-related macular degeneration. exposed to aluminum in the workplace, with the lung, bone,
and central nervous system as major target organs. Aluminum
can produce developmental effects.
Toxicity— Although uncommon, gastrointestinal distress and
diarrhea have been reported following ingestion of beverages
Lung and Bone Toxicity—Occupational exposure to alumi-
standing in galvanized cans. Following inhalation of zinc oxide
num dust can produce lung fibrosis in humans. Osteomalacia
the most common effect is “metal-fume fever” characterized
has been associated with excessive intake of aluminum-
by fever, chest pain, chills, cough, dyspnea, nausea, muscle
containing antacids in otherwise healthy individuals. This is
soreness, fatigue, and leukocytosis. Acute inhalation of high
assumed to be due to interference with intestinal phosphate
levels of zinc chloride as in the military use of “smoke bombs”
absorption. Osteomalacia also can occur in uremic patients
results in more pronounced damage to the mucous membrane
exposed to aluminum in the dialysis fluid. In these patients,
including interstitial edema, fibrosis, pneumonitis, bronchial
osteomalacia may be a direct effect of aluminum on bone min-
mucosal edema, and ulceration. Following long-term expo-
eralization as bone levels are high.
sure to lower doses of zinc, symptoms generally result from a
decreased dietary copper absorption, leading to early symp-
toms of copper deficiency, such as decreased erythrocyte Neurotoxicity—Aluminum is neurotoxic to experimental
number or decreased hematocrit. animals, with wide species and age variations. In susceptible
animals, such as rabbits and cats, the most prominent early
pathologic change is the accumulation of neurofibrillary tan-
Neuronal Toxicity—As an essential cofactor for numerous gles (NFTs) in large neurons, proximal axons, and dendrites
enzymes and proteins, zinc deficiency may alter an antioxidant of neurons of many brain regions. This is associated with loss
enzyme resulting in excess free radicals that are damaging to of synapses and atrophy of the dendritic tree. In other species,
cell membranes. Excess zinc released by oxidants can act as a impairment of cognitive and motor function and behavioral
potent neurotoxin, contributing to excitotoxic brain injury. abnormalities are often observed.
The release of excess, toxic free zinc could be a factor that sets
the stage for the later development of Alzheimer’s disease.
Dialysis Dementia—A progressive, neurologic syndrome has
been reported in patients on long-term intermittent hemo-
Pancreatic Toxicity—Because large amounts of zinc accu- dialysis for chronic renal failure. The first symptom in these
mulate in secretory granules of pancreatic islet B-cells, zinc patients is a speech disorder followed by dementia, convul-
released under certain conditions can affect the function or sions, and myoclonus. The disorder, which typically arises
survival of islet cells and cause B-cell death. Excess dietary zinc after 3 to 7 years of dialysis treatment, may be due to alumi-
is associated with damage to exocrine pancreas. A single, high- num intoxication. The aluminum content of brain, muscle, and
dose injection of zinc increases plasma Q-amylase activity and bone increases in these patients. Sources of the excess alumi-
can produce fibrosis and necrosis of pancreatic exocrine cells, num may be from oral aluminum hydroxide commonly given
but does not affect the islets of Langerhans cells. to these patients or from aluminum in dialysis fluid derived
358 UNIT 5 Toxic Agents

from the tap water used to prepare the dialysate fluid. The high remain within the therapeutic range. Chronic lithium-induced
serum aluminum concentrations may be related to increased neurotoxicity, nephritis, and thyroid dysfunction may occur,
parathyroid hormone levels that are due to low blood calcium especially in susceptible patients with nephrogenic diabetes
and osteodystrophy common in patients with chronic renal insipidus, older age, abnormal thyroid function, and impaired
disease. The syndrome may be prevented by avoiding the use of renal function. Acute lithium overdose produces neurological
aluminum-containing oral phosphate binders and by monitor- sequelae and cardiac toxicity, which can be fatal. The toxicity
ing of aluminum in the dialysate. may be treated by the administration of diuretics (amiloride)
and lowering of blood levels via hemodialysis.
Alzheimer’s Disease—A possible relationship between alumi-
num and Alzheimer’s disease has been a matter of speculation
for decades. Elevated aluminum levels in Alzheimer’s brains Platinum
may be a consequence and not a cause of the disease. The Platinum compounds are used as automobile catalysts, in jew-
reduced effectiveness of the blood-brain barrier in Alzheimer's elry, in electronics, and in dental alloys. Platinum coordination
might allow more aluminum into the brain. Also, recent stud- complexes are very important antitumor agents.
ies have raised the possibility that the staining methods in ear-
lier studies may have led to aluminum contamination. There Toxicity—Platinum can produce profound hypersensitivity
are conflicting conclusions from studies examining the role of reactions in susceptible individuals. The signs of hypersensitiv-
aluminum in Alzheimer’s disease. However, there is increasing ity include urticaria, contact dermatitis of skin, and respiratory
evidence suggesting a link between aluminum in the brain and distress, ranging from irritation to an asthmatic syndrome,
other neurodegenerative diseases. following exposure to platinum dust. The skin and respira-
tory changes, platinosis, are mainly confined to persons with
a history of industrial exposure to soluble compounds such as
Lithium sodium chloroplatinate.
Lithium is used in batteries, alloys, catalysts, photographic
materials, and the space industry. Lithium hydride produces Antitumor Effects of Platinum Complexes—The platinum-
hydrogen on contact with water and is used in manufactur- coordinated complexes are important antitumor agents,
ing electronic tubes, in ceramics, and in chemical analysis. including cisplatin, carboplatin, and oxaliplatin. They are rou-
Groundwater contamination with lithium from man-made tinely administered, often in combination with other antican-
waste disposal could be a risk factor for the aquatic environ- cer drugs, in the treatment ofawide spectrum ofmalignancies,
ment. Lithium carbonate and lithium citrate are widely used especially advanced testicular cancer and also cancers of head
for mania and bipolar disorders. and neck, bladder, esophagus, lung, and ovary.

Toxicokinetics—L ithium is readily absorbed from the gastro- Carcinogenic Effects of Platinum Complexes—Although cis-
intestinal tract. It is distributed to total body water with higher platin has antitumor activity in humans, it is considered to be
levels in kidney, thyroid, and bone as compared with other tis- a probable carcinogen in humans and is clearly carcinogenic in
sues. Excretion is chiefly through the kidneys with 80% of the rodents. In fact, in mice deficient in metallothionein, cisplatin
filtered lithium reabsorbed. Lithium can substitute for sodium can induce liver carcinoma at clinically relevant doses.
or potassium on several transport proteins.
Toxicities of Platinum Antitumor Complexes—Cisplatin pro-
Toxicity—Except for lithium hydride, no other salts are con- duces proximal and distal tubular cell injury, mainly in the cor-
sidered hazardous, nor is the metal very toxic itself. Lithium ticomedullary region, where the concentration of platinum is
hydride is intensely corrosive and may produce burns on the highest. Hearing loss can occur and can be unilateral or bilat-
skin because of the formation of hydroxides. Intoxications eral but tends to be more frequent and severe with repeated
related to lithium exposure are mainly related to its medicinal doses. Marked nausea and vomiting occur in most patients
uses. The toxic responses to lithium include neuromuscular receiving the platinum complexes but can be controlled with
changes (tremor, muscle hyperirritability, and ataxia), central ondansetron or high dose ofcorticosteroids.
nervous system disorders (blackout spells, epileptic seizures,
slurred speech, coma, psychosomatic retardation, and increased
thirst), cardiovascular disturbances (cardiac arrhythmia, hyper- BIBLIOGRAPHY
tension, and circulatory collapse), gastrointestinal symptoms
Hirner AV, Emons H (eds.): Organic Metal and Metalloid Species in
(anorexia, nausea, and vomiting), and renal damage (albumin- the Environment: Analysis, Distribution, Processes and Toxicological
uria and glycosuria). Evaluation. New York: Springer, 2010.
Chronic lithium nephrotoxicity and interstitial nephritis Nordberg GF, Fowler BA, Nordberg M (eds.): Handbook on the
may occur with long-term exposure even when lithium levels Toxicology ofMetals, 4th ed. Boston, MA: Academic Press, 2015.
 CHAPTER 23 Toxic Effects of Metals 359

QUESTIONS

Which of the following is NOT a major excretory pathway Which of the following statements regarding mercury
of metals? (Hg) toxicity is FALSE?
sweat. a. A major source of environmental mercury is
urine. rainwater.
respiration. b. Mercury vapor is much more dangerous than liquid
feces. mercury.
re
eanhair. c. Mercury vapor inhalation is characterized by fatigue
and bradycardia.
Metallothioneins: d. Microorganisms in bodies of water can convert
are responsible for metal transport in the bloodstream. mercury vapor to methylmercury.
are involved in the biotransformation of metals. e. Methylmercury is the most important source of
invoke hypersensitivity reactions. human mercury toxicity.
provide high-affinity binding of copper and mercury.
2
CLO are involved in extracellular transport of metals. Which of the following is a common symptom of nickel
exposure?
Which of the following metal-binding proteins is NOT a. renal failure.
correctly paired with the metal it binds? b. diarrhea.
transferrin—iron. c. hepatic cirrhosis.
ceruloplasmin—copper. d. contact dermatitis.
metallothioneins—zinc. e. tachycardia.
ferritin—lead.
ge
eanalbumin—nonspecific metal binding. Which of the following statements regarding Wilson's
disease is FALSE?
Which of the following groups is LEAST likely to chelate a. Serum ceruloplasmin is high.
metals? b. Urinary excretion of copper is high.
a. —COOH. c. ‘There is impaired biliary excretion of copper.
b. —Cl. d. ‘The disease can be treated with liver transplantation.
c. —NH. e. ‘This is an autosomal recessive disorder.
d. —OH.
e. —SH. 10. Which of the following statements regarding metals and
medical therapy is FALSE?
What is the mechanism of toxicity of arsenic (As)? a. There are elevated levels of aluminum in the brains of
a. inhibition of mitochondrial respiration. Alzheimer’s patients.
b. impairment of calcium uptake by membrane Lithium is used to treat depression.
transporters. c. Chronic nephrotoxicity is a common result of excess
c. accumulation in renal corpuscle. aluminum exposure.
abolition of sodium-—potassium gradient. d. Platinum is used as cancer treatment.
e. destruction of surfactant in the lungs. e. Platinum salts can cause an allergic dermatitis.

Lead’s toxicity is largely due to its ability to mimic and

interfere with normal functioning of which of the follow-
ing ions?
a, Nel.
bs) K’.
Cae lee
d. Fe**,
ex Ca
 a ; A. A ; - : +)
; be)ARR, Teh oy
OT apres
am
oI
shh

2 ees coke winches whet adaeey Iie

ecnhileet Pei
reeves
holebuhake
 Cee
ee PP ER

Toxic Effects of Solvents

and Vapors
James V. Bruckner, S. Satheesh Anand,
and D. Alan Warren

INTRODUCTION Tetrachloroethylene
Methylene Chloride
IS THERE A SOLVENT-INDUCED CHRONIC Carbon Tetrachloride
ENCEPHALOPATHY?
Chloroform

SOLVENT ABUSE AROMATIC HYDROCARBONS

Benzene
ENVIRONMENTAL CONTAMINATION
Toluene
TOXICOKINETICS Xylenes and Ethylbenzene
Absorption ALCOHOLS
Transport, Distribution, and Elimination
Ethanol
Metabolism
Methanol
Physiologic Modeling
GLYCOLS
POTENTIALLY SENSITIVE SUBPOPULATIONS
Ethylene Glycol
Endogenous Factors
Propylene Glycol
Children
Elderly GLYCOL ETHERS
Gender Reproductive Toxicity
Genetics Developmental Toxicity
Exogenous Factors Hematotoxicity
P450 Inducers and Inhibitors
AUTOMOTIVE GASOLINE AND ADDITIVES
Physical Activity
Diet CARBON DISULFIDE
CHLORINATED HYDROCARBONS
Trichloroethylene
Metabolism and Modes of Action
Liver Cancer
Kidney Cancer
Lung Cancer

361
 W 6 NO UNIT 5 Toxic Agents

KEY POINTS

w ‘The term solvent refers to a class of liquid organic chemi- » Solvents are readily absorbed from the gastrointestinal
cals of variable lipophilicity and volatility, small molecular tract and across the skin.
size, and lack of charge. « Most solvents produce some degree of CNS depression.
« Absorption of inhaled volatile organic compounds occurs
in the alveoli, with almost instantaneous equilibration
with blood in the pulmonary capillaries.

INTRODUCTION hydrocarbons, many of which are halocarbons, aromatic hydro-

carbons, alcohols, ethers, esters/acetates, amides/amines, alde-
The term solvent refers to a class of liquid organic chemicals hydes, ketones, and complex mixtures that defy classification.
of variable lipophilicity and volatility, small molecular size, The main determinants of a solvents inherent toxicity are
and lack of charge. Solvents undergo ready absorption across (1) its number of carbon atoms; (2) whether it is saturated or
the lung, skin, and gastrointestinal (GI) tract. In general, the has double or triple bonds between adjacent carbon atoms;
lipophilicity of solvents increases with increasing molecular (3) its configuration (i.e., straight chain, branched chain, or
weight, while volatility decreases. Solvents are frequently used cyclic); (4) whether it is halogenated; and (5) the presence of
to dissolve, dilute, or disperse materials that are insoluble in functional groups. Subtle differences in chemical structure can
water. Most solvents are refined from petroleum. Many, such translate into dramatic differences in solvent toxicity.
as naphthas and gasoline, are complex mixtures consisting of Nearly everyone is exposed to solvents during normal daily
hundreds of compounds. As such, they are widely employed as activities. Environmental exposures to solvents in air and
degreasers. groundwater use multiple exposure pathways (Figure 24-1).
Solvents are classified largely according to molecular struc- Though not reflected in Figure 24-1, household use of solvent-
ture or functional group. Classes of solvents include aliphatic contaminated water may result in solvent intake from inhalation,

«—— Prevailing wind direction

Transport medium (air)
Exposure eeer-= --. - pws
- =
point oa
ae Release mechanism
s, (volatilization)
s

. Exposure
Exposure Inhalation
point exposure
route
eee Kil __Gt,
Ingestion Release y
exposure
_ mechanism oe 4 Waste pile
route renmersarane
| (spill). ee ay (source)
Exposure “
Release mechanism
medium
(soil) (site leaching)

Water table
sa
aa
sepa
2

<— Transport medium

(groundwater)

FIGURE 24-1 Solvent exposure pathways and media. (Reproduced with permission from EPA Risk Assessment Guidance for Superfund.
Human Health Evaluation Manual PartA,Interim Final. Washington, DC: Office of Emergency and Remedial Response,
1989.)
 CHAPTER 24 Toxic Effects of Solvents and Vapors 363

and dermal and oral absorption. In many cases, environmental effects as extreme as unconsciousness. These can be breathed
risk assessment requires that risks be determined for physiologi- in through the nose or the mouth by “sniffing” or “snorting”
cally diverse individuals who are exposed to several solvents by vapors from containers, spraying aerosols directly into the
multiple exposure pathways. nose or mouth, “bagging” by inhaling vapors from substances
The Occupational Safety and Health Administration (OSHA) inside plastic or paper bags, or “huffing” from a solvent-soaked
has established legally enforceable Permissible Exposure Limits rag stuffed into the mouth. Solvents can be addicting and are
(PELs) for over 100 solvents. The majority of existing PELs were often abused in combination with other drugs. Solvents present
adopted from the list of Threshold Limit Values (TLVs) pub- in relatively inexpensive household and commercial products
lished by the American Conference of Governmental Industrial are readily available to children and adolescents. While intoxi-
Hygienists (ACGIH). Whereas the ACGIH’s TLVs for an 8-h cation may last only a few minutes, abusers frequently seek to
work day, 40-h work week are designed to be protective for a prolong the “high” by inhaling repeatedly over the course of
working lifetime, its Short-term Exposure Limits (STELs) and several hours. Death may occur as a result of cardiac arrhyth-
ceiling values are designed to protect against the acute effects mias, asphyxiation, and/or cachexia.
of high-level, short-term solvent exposures. If warranted, the
ACGIH will assign a skin notation to a solvent, indicating that
significant dermal exposure is possible. ENVIRONMENTAL CONTAMINATION
Most solvent exposures involve a mixture of chemicals, rather
Most solvents enter the environment through evaporation
than a single compound. Whereas the assumption is frequently
(Figure 24-1). The majority of the more volatile organic com-
made that the toxic effects of multiple solvents are additive,
pounds (VOCs) volatilize when products containing them
solvents may also interact synergistically or antagonistically.
(e.g., aerosol propellants, paint thinners, cleaners, and soil
Although some solvents are less hazardous than others, all
fumigants) are used as intended. Solvent loss into the atmo-
solvents can cause toxic effects. Most have the potential to
sphere also occurs during production, processing, storage,
induce narcosis and cause respiratory and mucous membrane
and transport activities, resulting in elevated concentrations
irritation. As with other chemicals, whether adverse health
in air in the proximity of point sources. Winds dilute and
effects occur from solvent exposure is dependent on several
disperse solvent vapors across the world. Atmospheric con-
factors: (1) toxicity/carcinogenicity ofthe solvent; (2) exposure
centrations of most VOCs are usually extremely low, though
route; (3) amount or rate of exposure; (4) duration of exposure;
higher concentrations have been measured in urban areas,
(5) individual susceptibility; and (6) interactions with other
around petrochemical plants, and in the immediate vicinity of
chemicals.
hazardous waste sites.
Solvent contamination of drinking water supplies is a major
IS THERE A SOLVENT-INDUCED health concern. Solvents spilled onto the ground may permeate
the soil and migrate until reaching groundwater or imperme-
CHRONIC ENCEPHALOPATHY?
able material. All solvents are soluble in water to some extent.
Considerable debate has examined whether chronic, low-level Concentrations diminish rapidly after VOCs enter bodies of
exposure to virtually any solvent or solvent mixture can pro- water, due primarily to dilution and evaporation. VOCs in sur-
duce a pattern of neurologic dysfunction referred to as painters face waters rise to the surface or sink to the bottom, according
syndrome, organic solvent syndrome, psychoorganic syndrome, to their density. VOCs on the surface will largely evaporate.
and chronic solvent encephalopathy (CSE). CSE is characterized VOCs on the bottom depend on solubilization in the water or
by nonspecific symptoms (e.g., headache, fatigue, and sleep mixing by current or wave action to reach the surface. VOCs
disorders) with or without changes in neuropsychological in groundwater tend to remain trapped until the water reaches
function. A reversible form of CSE, the neuroasthenic syn- the surface.
drome, consists of symptoms only. The “mild” and “severe”
forms are accompanied by objective signs of neuropsycho-
logical dysfunction that may or may not be fully reversible. TOXICOKINETICS
Well-designed and controlled clinical epidemiologic studies Toxicokinetic (TK) studies delineate the uptake and disposi-
are needed to resolve this controversy of CSE. tion of chemicals in the body. Toxicity is a dynamic process,
in which the degree and duration of injury of a target tissue
depends on the net effect of toxicodynamic (TD) and TK pro-
SOLVENT ABUSE
cesses including systemic absorption, metabolism, interaction
Inhalants are volatile substances that can be inhaled to induce a with cellular components, and tissue repair.
psychoactive or mind-altering effect with vapor concentrations Volatility and lipophilicity are two important properties of
high enough to produce effects that resemble alcohol intoxica- solvents that govern their absorption and deposition in the
tion and may lead to unconsciousness. Solvent abuse is a unique body. Lipophilicity also can vary from quite water soluble
exposure situation, in that participants repeatedly subject (e.g., glycols and alcohols) to quite lipid soluble (e.g., halo-
themselves to vapor concentrations high enough to produce carbons and aromatic hydrocarbons). Many solvents have a
364 UNIT5 Toxic Agents

relatively low molecular weight and are uncharged, enabling fraction of cardiac output (~3%) supplying fat depots. Body fat
passive diffusion through membranes from areas of high to low increases the volume of distribution and total body burden of
concentration. lipophilic solvents.

Absorption Metabolism
Most systemic absorption of inhaled VOCs occurs in the alve- Biotransformation can modulate the toxicities of solvents.
oli, with some absorption occurring in the upper respiratory Many solvents are poorly soluble in water and must be enzymat-
tract. Gases in the alveoli equilibrate almost instantaneously ically converted to relatively water-soluble derivatives, which
with blood in the pulmonary capillaries. Blood:air partition may be more readily eliminated in the largely aqueous urine
coefficients (PCs) of VOCs may be defined as the ratio of con- and/or bile. Some solvents can undergo bioactivation to pro-
centration of VOC achieved between two different media at duce reactive metabolites that are cytotoxic and/or mutagenic.
equilibrium. More hydrophilic solvents have relatively high Toluene, benzene, 1,1,1-trichloroethylene, hexane, and carbon
blood:air PCs, which favor extensive uptake. Because VOCs tetrachloride are examples of solvents that are metabolized to
diffuse from areas of high to low concentration, increases in toxic products. In particular, CYP2E1 catalyzes the oxidation
respiration (to maintain a high alveolar concentration) and in of halogenated and aromatic hydrocarbons, including benzene,
cardiac output/pulmonary blood flow (to maintain a large con- styrene, chloroform, and vinyl chloride to electrophilic metabo-
centration gradient by removing capillary blood containing lites capable of causing cytotoxicity and/or mutagenicity.
the VOC) enhance pulmonary absorption.
Solvents are well absorbed from the GI tract. Peak blood ‘
levels are observed within minutes of dosing, although the Physiologic Modeling
presence of food in the GI tract can delay absorption. It is Physiologically based toxicokinetic(PBTK) models are used to
usually assumed that 100% of an oral dose of most solvents is relate the administered dose to the tissue dose of a bioactive
absorbed systemically. The vehicle or diluent in which a solvent moiety or moieties. With knowledge of the physiology of the
is ingested can affect the absorption and TK of the compound. test animal and tissue, physiologically based toxicodynamic
Absorption of solvents through the skin can result in both (PBTD) models can be developed. PBTK/TD models are well
local and systemic effects. Lipophilic solvents penetrate the suited for species-to-species extrapolations, because human
stratum corneum by passive diffusion. Determinants of the physiologic and metabolic parameter values can be entered
rate of dermal absorption ofsolvents include the chemical con- and simulations of target tissue doses and effects in humans
centration, surface area exposed, exposure duration, integrity generated. Thus, solvent exposures necessary to produce the
and thickness of the stratum corneum, and lipophilicity and same target organ dose in humans as that found experimen-
molecular weight of the solvent. tally to cause an unacceptable cancer or noncancer incidence
in test animals can be determined in some cases with reason-
able certainty. In the limited number of cases where there may
Transport, Distribution, and Elimination be species differences in tissue sensitivity, PBTD models can
Solvents absorbed into portal venous blood from the GI tract are be used to forecast toxicologically effective target organ doses.
subject to uptake/elimination by the liver and exhalation by the
lungs during their first pass through the pulmonary circulation.
Those solvents that are well metabolized and quite volatile are POTENTIALLY SENSITIVE
most efficiently eliminated before they enter the arterial blood. SUBPOPULATIONS
Hepatic first-pass elimination depends on the chemical and the
rate at which it arrives in the liver. Pulmonary first-pass elimina- Endogenous Factors
tion, in contrast, is believed to be a zero-order process as a fixed Children—Limited information is available on the toxic
percentage of the chemical is thought to exit the pulmonary potential of solvents in children. Most age-dependent differ-
blood at each pass through the pulmonary circulation. ences are less than an order of magnitude, usually varying no
Solvents transported by the arterial blood are taken up more than two- to threefold. The younger and more immature
according to rate of tissue blood flow, mass, and the tissue:blood the subject, the more different is his or her response from that
PC of the solvent. Relatively hydrophilic solvents solubilize to of adults.
different extents in plasma. Lipophilic solvents do not bind to GI absorption of solvents varies little with age, because most
plasma proteins or hemoglobin, but partition into hydropho- solvents are absorbed by passive diffusion. Systemic absorp-
bic sites in the molecules. They partition into phospholipids, tion of inhaled VOCs may be greater in infants and children
lipoproteins, and cholesterol that are present in the blood. than in adults owing to the relatively high cardiac output and
Blood levels of solvents drop rapidly during the initial elimi- respiratory rates despite their lower alveolar surface area.
nation phase. This redistribution phase is characterized by Extracellular water, expressed as percentage of body weight, is
rapid diffusion of solvent from the blood into most tissues. highest in newborns and gradually diminishes through child-
Equilibration of adipose tissue is prolonged due to the small hood. Body fat content is high from ~1/2 to 3 years of age, and
 CHAPTER 24 Toxic Effects of Solvents and Vapors 365

then steadily decreases until adolescence, when it increases Physical Activity—Exercise increases alveolar ventilation
again in females. Lipophilic solvents accumulate in adipose and cardiac output/pulmonary blood flow. Polar solvents
tissue, so more body fat would result in greater body burdens with relatively high blood:air PCs (e.g., acetone, ethanol,
and slower clearance of the chemicals. , and ethylene glycol [EG]) are very rapidly absorbed into the
Changes in xenobiotic metabolism during maturation may pulmonary circulation. Alveolar ventilation is rate-limiting
impact susceptibility to solvent toxicity. P450 isoforms develop for these chemicals. In contrast, pulmonary blood flow and
asynchronously. Increased rates of metabolism, urinary excre- metabolism are rate-limiting for uptake of more lipophilic
tion, and exhalation by children should hasten elimination solvents. Heavy exercise can increase pulmonary uptake of
and reduce body burdens of solvents. However, the net effect relatively polar solvents as much as fivefold in human sub-
of immaturity on solvent disposition and toxicity is difficult jects. Light exercise doubles uptake of relatively lipid-soluble
to predict. solvents, but no further increase occurs at higher workloads.
Blood flow to the liver and kidneys diminishes with exercise,
Elderly— Age influences the distribution of xenobiotics in the which may diminish biotransformation of metabolized solvents
body as well as their metabolism and elimination. With aging, and urinary elimination.
body fat usually increases substantially at the expense of lean
mass and body water. Thus, relatively polar solvents tend to Diet— The mere presence offood in the stomach and intestines
reach higher blood levels during exposures. Relatively lipid- can inhibit systemic absorption of ingested chemicals by pre-
soluble solvents accumulate in adipose tissue and are released venting contact of the chemical with the GI epithelium. VOCs
slowly. Cardiac output and renal and hepatic blood flows are in the GI tract partition into dietary lipids, largely remaining
diminished in the elderly. there until the lipids are emulsified and absorbed. Food intake
The elderly, like infants and children, may be more or less results in increased splanchnic blood flow, which favors GI
sensitive to the toxicity of solvents than young adults. Greater absorption, hepatic blood flow, and biotransformation. Foods
organ system toxicity could be due to increased inflammatory may contain certain natural constituents, pesticides, and other
damage or to age-related dysregulation of cytokines. It must chemicals that may enhance or reduce solvent metabolism.
be taken into account that memory, attention, visual percep- In addition, fasting for 1 to 3 days results in decreased detoxi-
tion, and motor skills diminish with aging, even in the absence fication of electrophilic metabolites (especially since hepatic
of chemical exposure. Other major sources of variability and glutathione levels are decreased) and the formation of cyto-
complexity in geriatric populations include inadequate nutri- toxic, mutagenic, metabolites.
tion, the prevalence of disease states, and the concurrent use of Chronic consumption of ethanol potentiates the hepatic
multiple medications. or renal damage caused by hepatotoxic or renotoxic solvents,
such as CCL,, 1,1,1-trichloroethane, 1,1,2-trichloroethylene,
Gender—Physiologic and biochemical differences between or tetrachloroethylene. Many medications and nicotine and
men and women have the potential to alter tissue dosimetry other components of tobacco smoke can induce metabolism
and health effects of certain solvents. Whereas most predic- of solvents.
tive models suggest effects of toxicants are independent of sex, Disease can have an important influence on solvent toxicity.
physical differences, such as the tendency of men to have more Many illnesses impair hepatic metabolism and biliary and
lean body mass and a larger body size, could potentially cause renal elimination. Cirrhosis, hepatitis, chronic kidney disease,
physiologic differences. diabetes mellitus, and gram-negative infections that release
endotoxin may decrease solvent toxicokinetics and toxicity.
Genetics—Genetic polymorphisms for biotransforma-
tion occur at different frequencies in different ethnic groups.
Polymorphisms for xenobiotic-metabolizing enzymes may CHLORINATED HYDROCARBONS
affect the quantity and quality of enzymes and the outcomes of
exposures to solvents in different racial groups. Disentangling Trichloroethylene
the influences of genetic traits from those of socioeconomic 1,1,2-Trichloroethylene (TCE) is a widely used solvent for
status, lifestyles, and geographic setting is difficult. metal degreasing. Moderate to high doses of TCE, as with
other halocarbons, are associated with a number of noncancer
toxicities including autoimmune disorders, immune system
Exogenous Factors dysfunction, and potentially a male reproductive toxicant.
P450 Inducers and Inhibitors—Preexposure to chemicals Cancer remains the dominant issue for TCE.
that induce or inhibit biotransformation enzymes can potenti-
ate or reduce the toxicity/carcinogenicity of high doses of sol- Metabolism and Modes of Action—Toxicities associated
vents that undergo metabolism. Inhibitors would generally be with TCE are predominantly mediated by metabolites rather
anticipated to enhance the toxicity of solvents that are meta- than by the parent compound. Even the CNS-depressant effects
bolically inactivated and protect from solvents that undergo of TCE are due in part to the sedative properties of the metabo-
metabolic activation. lite trichloroethanol (TCOH). After either oral or inhalational
366 UNIT 5 Toxic Agents

absorption, most of the TCE undergoes oxidation via cyto- intermediate. The highest exposures usually occur in occupa-
chrome P450s, with a small proportion being conjugated with tional settings via inhalation. PERC is the third most frequently
glucuronic acid and excreted in the urine. These metabolic found chemical contaminant in groundwater at hazardous
pathways are implicated in the carcinogenicity of TCE: reactive waste sites in the United States.
metabolite(s) of the GSH pathway in kidney tumors in rats and The systemic disposition and metabolism of PERC and
oxidative metabolites in liver and lung tumors in mice. TCE are quite similar, although PERC is much less extensively
metabolized. Both chemicals are well absorbed from the lungs
Liver Cancer—TCE induces liver cancer in B6C3F1 mice but and GI tract, distributed to tissues according to their lipid con-
not in rats. This differential susceptibility is due to the greater tent, partially exhaled unchanged, and metabolized by P450s.
capacity of mice to metabolize TCE to an oxidative metabolite PERC is oxidized by hepatic P450s to a much lesser degree
that stimulates peroxisome proliferation. Propagation results than TCE, though trichloroacetic acid is a common major
in an increased potential for oxidative DNA damage, lipid metabolite. GSH conjugation is a minor metabolic pathway,
peroxidation, and decreased gap-junctional intercellular com- quantitatively, for TCE and PERC. The extent of GSH conjuga-
munication, all of which have been implicated in neoplastic tion of PERC increases when the oxidative pathway becomes
transformation. saturated at high exposure levels. Metabolic products are the
primary contributors to PERC-induced nephrotoxicity.
Kidney Cancer—TCE exposure by inhalation or the oral route PERC-induced hepatic injury is believed to be a consequence
results in kidney tumors in male but not female rats. The sus- of its intermediate metabolites: PERC oxide and trichloroacetyl
ceptibility of the male rat can be explained by its greater capacity chloride. The many epidemiologic studies of cancer incidence
for TCE metabolism via the GSH pathway. TCE-induced kidney and mortality in groups of persons occupationally exposed
tumors are believed to result from reactive metabolite(s) of this to PERC are equivocal and do not support a cause-and-effect
pathway alkylating cellular nucleophiles, including DNA. The relationship between either PERC 6r FCE and cancer. Cigarette
resulting DNA mutations lead to alterations in gene expression, smoking and alcohol consumption only partially account for
which in turn lead to neoplastic transformation and tumorigen- an increased rate of esophageal cancer. Kidney, liver, and lung
esis via a genotoxic pathway. cancer incidences did not appear to be elevated.
Alternatively, proximal tubular cell cytotoxicity and subse-
quent tumor formation via a nongenotoxic mode of action could
be induced by reactive metabolites that cause oxidative stress, Methylene Chloride
alkylation of cytosolic and mitochondrial proteins, marked Methylene chloride (dichloromethane, MC) is used widely as
ATP depletion, and perturbations in Ca** homeostasis. Tubular a solvent in industrial processes, food preparation, degreasing
necrosis ensues, with subsequent reparative proliferation that agents, aerosol propellants, and agriculture. The primary route
can alter gene expression and, in turn, alter the regulation of of exposure to this very volatile solvent is inhalation.
cell growth and differentiation. In fact, somatic mutations in the MC is rapidly absorbed and distributed throughout the body
von Hippel-Lindau (VHL) tumor suppressor gene might be a and has limited systemic toxicity potential. High, repeated inha-
specific and susceptible target of TCE. lation exposures produce slight, reversible changes in the livers
Chronic tubular damage may be a prerequisite to TCE- of rodents. Persons subjected to high vapor levels manifest kid-
induced renal cell cancer. Reactive metabolite(s) of the GSH
ney injury occasionally. Carbon monoxide that is formed from
pathway may have a genotoxic effect on the proximal tubule MC binds to hemoglobin to produce dose-dependent increases
of the human kidney, but full development of a malignant in carboxyhemoglobin. Residual neurologic dysfunction in
tumor requires a promotional effect such as cell proliferation in MC-exposed workers has been reported.
response to tubular damage. Occupational and environmental MC exposures are of con-
cern primarily because of MC’s carcinogenicity in rodents and
Lung Cancer—Inhaled TCE is carcinogenic to the mouse lung
its potential as a human carcinogen. Epidemiologic studies of
but not to that of the rat. Oral TCE is not carcinogenic to the lung,
employees exposed to MC have revealed that cancer risks from
probably due to hepatic metabolism that limits the amount of
occupational exposure to MC, if any, are quite small.
TCE reaching the organ. The primary target of TCE within the
mouse lung is the nonciliated Clara cell. Cytotoxicity to these cells
is characterized by vacuolization and increases in cell replication Carbon Tetrachloride
in the bronchiolar epithelium. Clara cells of the mouse efficiently
Carbon tetrachloride (CCl,) is a classic hepatotoxin, but
metabolize TCE to toxic metabolites, including chloral. In mouse
kidney injury is often more severe in humans. It also plays a
lung, Clara cells are more numerous and have a much higher
significant role in atmospheric ozone depletion.
concentration of metabolizing enzymes than rat lung.
Early signs of hepatocellular injury in rats include disso-
ciation of polysomes and ribosomes from rough endoplasmic
Tetrachloroethylene reticulum, disarray of smooth endoplasmic reticulum, inhibi-
Tetrachloroethylene (perchloroethylene, PERC) is commonly tion of protein synthesis, and triglyceride accumulation. CCl,
used as a dry cleaner, fabric finisher, degreaser, rug and uphol- undergoes metabolic activation, producing lipid peroxida-
stery cleaner, paint and stain remover, solvent, and chemical tion, covalent binding, and inhibition of microsomal ATPase
 CHAPTER 24 Toxic Effects of Solvents and Vapors 367

activity. Single cell necrosis, evident 5 to 6 h postdosing, pro- auto exhaust are the other key contributors to exposures ofthe
gresses to maximal centrilobular necrosis within 24 to 48 h. general populace.
Cellular regeneration is maximal 36 to 48 h postdosing. The The hematopoietic toxicity of chronic exposure to benzene
rate and extent of tissue repair are important determinants of may manifest initially as anemia, leukopenia, thrombocy-
the ultimate outcome ofliver injury. topenia, or a combination of these. Bone marrow depression
Perturbation of intracellular calcium (Ca’*) homeostasis appears to be dose-dependent in both laboratory animals and
appears to be part of CCl, cytotoxicity. Increased cytosolic humans. Continued exposure may result in marrow aplasia
Ca’** levels may result from influx of extracellular Ca’* due to and pancytopenia, an often fatal outcome. Survivors ofaplastic
plasma membrane damage and from decreased intracellular anemia frequently exhibit a preneoplastic state, termed myelo-
Ca’* sequestration. Elevation of intracellular Ca** in hepato- dysplasia, which may progress to myelogenous leukemia.
cytes can activate phospholipase A, and exacerbate membrane There is strong evidence from epidemiologic studies that
damage. Elevated Ca** may also be involved in alterations in high-level benzene exposures result in an increased risk of
calmodulin and phosphorylase activity as well as changes acute myelogenous leukemia (AML) in humans. Evidence
in nuclear protein kinase C activity. High intracellular Ca’* of increased risks of other cancers in such populations is
levels activate a number of catabolic enzymes including pro- less compelling.
teases, endonucleases, and phospholipases, which kill cells via Various potential mechanisms require the complementary
apoptosis or necrosis. Increased Ca** may stimulate the release actions of benzene and several of its metabolites for toxicity.
of cytokines and eicosanoids from Kupffer cells, inducing (1) A number of benzene metabolites bind covalently to GSH,
neutrophil infiltration and hepatocellular injury. CCl, hepato- proteins, DNA, and RNA. This can result in disruption of
toxicity is obviously a complex, multifactorial process. the functional hematopoietic microenvironment by inhibi-
tion of enzymes, destruction of certain cell populations, and
alteration of the growth of other cell types. Covalent binding
Chloroform of hydroquinones to spindle-fiber proteins will inhibit cell
Chloroform (CHCl,, trichloromethane) is used primarily replication. (2) Oxidative stress contributes to benzene toxicity.
in the production of the refrigerant chlorodifluoromethane As the bone marrow is rich in peroxidase activity, phenolic
(Freon 22). Measurable concentrations of CHCl, are found in metabolites of benzene can be activated there to reactive qui-
municipal drinking water supplies. CHCl, is hepatotoxic and none derivatives, which can cause DNA damage, leading to cell
nephrotoxic. It can invoke CNS symptoms at subanesthetic mutation or apoptosis. Modulation of apoptosis may lead to
concentrations similar to those of alcohol intoxication and can aberrant hematopoiesis and neoplastic progression.
sensitize the myocardium to catecholamines, possibly result-
ing in cardiac arrhythmias.
The metabolite phosgene covalently binds hepatic and renal Toluene
proteins and lipids, which damages membranes and other Toluene is present in paints, lacquers, thinners, cleaning
intracellular structures, leading to necrosis and subsequent agents, glues, and many other products. It is also used in the
reparative cellular proliferation that promotes tumor formation production of other chemicals. Gasoline, which contains
in rodents by irreversibly “fixing” spontaneously altered DNA 5% to 7% toluene (w/w), is the largest source of atmospheric
and clonally expanding initiated cells. The expression of certain emissions and exposure of the general populace. Inhalation
genes, including myc and fos, is altered during regenerative cell is the primary route of exposure, though skin contact occurs
proliferation in response to CHCl,-induced cytotoxicity. frequently. Toluene is a favorite of solvent abusers, who inten-
Although a rodent carcinogen, ingestion of CHCl, in small tionally inhale high concentrations of the VOC.
increments, similar to drinking water patterns of humans, fails Toluene is well absorbed from the lungs and GI tract. It rap-
to produce sufficient cytotoxic metabolite(s) per unit time to idly accumulates in the brain, and subsequently, is deposited
overwhelm detoxification mechanisms. Currently, CHCl, is in other tissues according to their lipid content, with adipose
classified as a probable human carcinogen (group B2). tissue attaining the highest levels. Toluene is well metabolized,
but a portion is exhaled unchanged.
The CNS is the primary target organ of toluene and other
AROMATIC HYDROCARBONS alkylbenzenes. Manifestations of exposure range from slight
dizziness and headache to unconsciousness, respiratory depre-
Benzene ssion, and death. Occupational inhalation exposure guidelines
Benzene is derived primarily from petroleum and is used in are established to prevent significant decrements in psycho-
the synthesis of other chemicals and as an antiknock agent in motor functions. Acute encephalopathic effects are rapidly
unleaded gasoline. Inhalation is the primary route of exposure reversible on cessation of exposure. Subtle neurologic effects
in industrial and in everyday settings. Cigarette smoke is the have been reported in some groups of occupationally exposed
major source of benzene in the home. Smokers have benzene individuals. Severe neurotoxicity is sometimes diagnosed
body burdens which are 6 to 10 times greater than those of in persons who have abused toluene for a prolonged period.
nonsmokers. Passive smoke can be a significant source of ben- Clinical signs include abnormal electroencephalographic
zene exposure to nonsmokers. Gasoline vapor emissions and (EEG) activity, tremors, and nystagmus, as well as impaired
368 UNIT 5 Toxic Agents

hearing, vision, and speech. Magnetic resonance imaging has utilizing H,O, supplied by the actions of NADPH oxidase
revealed permanent changes in brain structure, which cor- and xanthine oxidase, will normally account for more than
respond to the degree of brain dysfunction. These changes 10% of ethanol metabolism; (3) CYP2E1, which is the prin-
include ventricular enlargement, cerebral atrophy, and white cipal isoform of the hepatic microsomal ethanol oxidizing
matter hyperintensity, a characteristic profile termed toluene system (MEOS).
leukoencephalopathy. ALDH activity is usually sufficiently high to metabolize large
amounts of acetaldehyde to acetate. Caucasians, blacks, and
Asians have varying percentages of different ALDH isozymes,
Xylenes and Ethylbenzene which impact the efficiency of acetaldehyde metabolism. Some
Large numbers of people are exposed to xylenes and ethyl- 50% of Asians have inactive ALDH, and these persons may
benzene occupationally and environmentally. Xylenes and experience flushing, headache, nausea, vomiting, tachycardia,
ethylbenzene, like benzene and toluene, are major compo- and hyperventilation on ingestion of ethanol. Whereas this
nents of gasoline and fuel oil. The primary uses of xylenes syndrome offers protection against developing alcoholism, it
industrially are as solvents and synthetic intermediates. Most increases the risk of acetaldehyde-related cancers of the esoph-
of the aromatics released into the environment evaporate into agus, stomach, colon, lung, head, and neck.
the atmosphere. Gender differences in responses to ethanol are well recog-
Similar to toluene, xylenes and other aromatic solvents nized. Females exhibit slightly higher blood ethanol levels
are well absorbed from the lungs and GI tract, distributed than men following ingestion of equivalent doses. This phe-
to tissues according to tissue blood flow and lipophilicity, nomenon is due in part to more extensive ADH-catalyzed
exhaled to some extent, well metabolized by hepatic P450s, metabolism of ethanolsby the gastric mucosa of males and
and largely excreted as urinary metabolites. Acute lethality to the smaller volume of distribution in women for relatively
of hydrocarbons (i.e., CNS depression) varies directly with polar solvents such as alcohols. Alse, women are more suscep-
lipophilicity. There is limited evidence that chronic occu- tible to alcohol-induced hepatitis and cirrhosis.
pational exposure to xylenes is associated with residual Fetal alcohol syndrome (FAS) is the most common prevent-
neurologic effects. able cause of mental retardation. Diagnostic criteria for FAS
Xylenes and ethylbenzene have limited capacity to adversely include (1) heavy maternal alcohol consumption during gesta-
affect organs other than the CNS. Mild, transient liver and/ tion; (2) pre and postnatal growth retardation; (3) craniofacial
or kidney toxicity has been reported occasionally in humans malformations including microcephaly; and (4) mental retar-
exposed to high vapor concentrations of xylenes. The majority dation. Less complete manifestations of gestational ethanol
of alkylbenzenes do not appear to be genotoxic or carcinogenic. exposure are referred to as fetal alcohol spectrum disorder
Ethylbenzene and styrene are known animal carcinogens, but (FASD). Potential mechanisms causing FASD include (1) simple
there are limited human data. oxidative stress in fetal tissues, (2) alteration of neurotransmitter-
gated ion channels such as the NMDA receptor, (3) alterations
in the regulation of gene expression with reduced retinoic acid
ALCOHOLS signaling or variant DNA methylation, (4) interference with
mitogenic and growth factor responses involved in neural stem
Ethanol cell proliferation, (5) disturbances in molecules that mediate
Many humans experience greater exposure to ethanol (ethyl cell-cell interactions, and (6) derangements of glial prolifera-
alcohol and alcohol) than to any other solvent. Ethyl alcohol is tion, differentiation, and function. Overconsumption during
used as an additive in gasoline, as a solvent in industry, in many all three trimesters of pregnancy can result in particular mani-
household products and pharmaceuticals including hand sani- festations depending on the period of gestation during which
tizers, and in intoxicating beverages. Frank toxic effects are less insult occurs.
important occupationally than injuries resulting from psycho- Human CYP2E1 is effective in production of reactive oxy-
motor impairment. Driving under the influence of alcohol is the gen intermediates from ethanol that cause lipid peroxidation.
major cause of fatal auto accidents. Blood alcohol level and the Also, ethanol induces the release of endotoxin from gram-
time necessary to achieve it are controlled largely by the rapid- negative bacteria in the gut. The endotoxin is taken up by
ity and extent of ethanol consumption. Ethanol is distributed in Kupffer cells, causing the release of inflammatory mediators,
body water and to some degree in adipose tissue. The alcohol which are cytotoxic to hepatocytes and chemoattractants
is eliminated by urinary excretion, exhalation, and metabolism. for neutrophils.
The blood level in an average adult decreases by ~15 to 20 mg/dL Alcohol-induced tissue damage results from both nutri-
per hour. Thus, a person with a blood alcohol level of 120 mg/dL tional disturbances and direct toxic effects. Malabsorption
would require 6 to 8 h to reach negligible levels. of thiamine, diminished enterohepatic circulation of folate,
Ethanol is metabolized to acetaldehyde by three enzymes: degradation of pyridoxal phosphate, and disturbances in the
(1) alcohol dehydrogenase (ADH) catalyzes oxidation of most metabolism of vitamins A and D can occur. Prostaglandins
of the ethanol to acetaldehyde, which is rapidly oxidized by released from endotoxin-activated Kupffer cells may be
acetaldehyde dehydrogenase (ALDH) to acetate; (2) catalase, responsible for a hypermetabolic state in the liver. With the
 CHAPTER 24 Toxic Effects of Solvents and Vapors 369

increase in oxygen demand, the viability of centrilobular (THF)-dependent pathway. Susceptibility to methanol tox-
hepatocytes would be most compromised due to their rela- icity is dependent on the relative rate of formate clearance.
tively poor oxygen supply. Metabolism of ethanol via ADH Conversion of formate to CO, is slower in primates than in
and ALDH results in a shift in the redox state of the cell. The rodents. In fact, formate acts as a direct ocular toxin and the
resulting hyperlacticacidemia, hyperlipidemia, hyperurice- acidotic state potentiates formate toxicity because the inhibi-
mia, and hyperglycemia lead to increased steatosis and col- tion of cytochrome oxidase increases as pH decreases.
lagen synthesis.
Alcoholism can result in damage of extrahepatic tissues.
Alcoholic cardiomyopathy is a complex process that may GLYCOLS
result from decreased synthesis of cardiac contractile proteins,
attack of oxygen radicals, increases in endoplasmic reticulum Ethylene Glycol
Ca**-ATPase, and antibody response to acetaldehyde-protein Ethylene glycol (EG) (1,2-dihydroxyethane) is a major con-
adducts. Heavy drinking appears to deplete antioxidants and stituent of antifreeze, deicers, hydraulic fluids, drying agents,
increases the risk of both hemorrhagic and ischemic strokes. and inks, and is used to make plastics and polyester fibers. The
The brain and pancreas may be adversely affected in alcoholics. most important routes of exposure are dermal and accidental
The associations between alcohol and cancers came primarily or intentional ingestion. EG is rapidly degraded in environ-
from epidemiologic case-control and cohort studies. Ethanol mental media.
and smoking act synergistically to cause oral, pharyngeal, and Three clinical stages of acute poisoning entail (1) a period
laryngeal cancers. It is generally believed that alcohol induces of inebriation, the duration and degree depending on dose;
liver cancer by causing cirrhosis or other liver damage and/or (2) the cardiopulmonary stage 12 to 24 h after exposure, char-
by enhancing the bioactivation of carcinogens. acterized by tachycardia and tachypnea, which may progress
Chronic ethanol consumption may promote carcino- to cardiac failure and pulmonary edema; and (3) the renal
genesis by (1) production of acetaldehyde, a weak mutagen toxicity stage 24 to 72 h postexposure. Metabolic acidosis can
and carcinogen; (2) induction of CYP2E1 with conversion progress in severity during stages 2 and 3.
of procarcinogens to carcinogens; (3) depletion of SAM and, Absorption from the GI tract of rodents is very rapid and
consequently, global DNA hypomethylation; (4) increased virtually complete. Dermal absorption in humans appears to
production of inhibitory guanine nucleotide regulatory pro- be less extensive. EG is distributed throughout the total body
teins and components of extracellular signal-regulated kinase- water. As illustrated in Figure 24-2, EG is metabolized by
mitogen-activated protein kinase signaling; (5) accumulation NAD*-dependent ADH to glycolaldehyde and on to glycolic
of iron and associated oxidative stress; (6) inactivation of acid. Glycolic acid is oxidized to glyoxylic acid by glycolic
the tumor suppressor gene BRCAI and increased estrogen acid oxidase and lactic dehydrogenase. Glyoxylic acid may
responsiveness (primarily in the breast); and (7) impairment be converted to formate and CO,, or oxidized by glyoxylic acid
of retinoic acid metabolism. oxidase to oxalic acid. Metabolic acidosis in humans appears
to be due to accumulation of glycolic acid. Hypocalcemia can
result from calcium chelation by oxalic acid to form calcium
Methanol oxalate crystals. Deposition of these crystals in tubules of
Methanol (methyl alcohol and wood alcohol) is found in the kidney and small blood vessels in the brain is associated
a host of consumer products including windshield washer with damage of these organs. Acute renal failure may follow.
fluid, carburetor cleaners, and copy machine toner, and is Additionally, hippuric acid crystals and direct cytotoxicity by
used in the manufacture of formaldehyde and methyl tert- other metabolites may act as damaging agents to the kidney
butyl ether. Serious methanol toxicity is most commonly in EG exposure. EG appears to have limited chronic toxicity
associated with ingestion. Acute methanol poisoning in potential, exhibits no evidence of carcinogenicity, and does not
humans is characterized by an asymptomatic period of 12 to appear to be a reproductive toxicant.
24 h followed by formic acidemia, ocular toxicity, coma, and
in extreme cases death. Visual disturbances develop between
18 and 48 h after ingestion and range from mild photopho- Propylene Glycol
bia and blurred vision to markedly reduced visual acuity and Propylene glycol (PG) is used as an intermediate in the synthe-
complete blindness. sis of polyester fibers and resins, as a component of automotive
The target of methanol within the eye is the retina, specifically antifreeze/coolants, and as a deicing fluid for aircraft. As PG is
the optic disk and optic nerve. Miiller cells, rod, and cone cells “generally recognized as safe” by the FDA, it is a constituent of
are altered functionally and structurally, because cytochrome many cosmetics and processed foods. Furthermore, it serves
c oxidase activity in mitochondria is inhibited, resulting in a as a solvent/diluent for a substantial number of oral, dermal,
reduction in ATP. and intravenous drug preparations. The most important routes
Though metabolized in liver, intraretinal conversion of of exposure are ingesting and dermal contact. PG is readily
methanol to formaldehyde and formate is critical. Metabolism metabolized by ADH to lactaldehyde, which is then oxidized
of formate to CO, then occurs via a two-step, tetrahydrofolate by aldehyde dehydrogenase to lactate. Excessive lactate is
370 UNIT 5 Toxic Agents

HOi=— Chin Cho OF

Ethylene glycol

|Alcohol dehydrogenase

O
I
HO=CH, = C—aH
Glycoaldehyde
|Aldehyde dehydrogenase

O
|
HO — CH, —C —OH
Glycolic acid
Glycolate a-OH-3-keto adipate
oxidase
ane
1
Oxalate
crystals ehydrogenase
Ha=GSG= On 2-ox0-4-OH-glutarate ——» Malate

Glyoxylic acid
Calcium
oxalate
oxidase Bg Formate ‘

SS S6S SRS S985 5 G52 TSS aS SSS ie IWS <ESSeses

sess sess=s= Cialis —
XN

~Benzoate

FIGURE 24-2 Metabolic scheme for ethylene glycol in animals. Key metabolites that have been observed in vivo are highlighted in boxes.
Dashed lines are theoretical pathways that have not been verified in vivo or in vitro. (Adapted with permission from Corley RA, Bartels MJ, Carney
EW, et al.: Development of a physiologically based pharmacokinetic model for ethylene glycol and its metabolite, glycolic acid, in rats and humans.
Toxicol Sci, 2005 May;85(1):476-490.)

primarily responsible for the acidosis. PG has a very low order abortion, menstrual disturbances, and subfertility among
of acute and chronic toxicity. women employed in the semiconductor industry. Reversible
spermatotoxicity in males has been described for those
exposed to glycol ethers. Typical responses include testicular
GLYCOL ETHERS and seminiferous tubule atrophy, abnormal sperm head mor-
The glycol ethers include EG monomethyl ether, also called phology, necrotic spermatocytes, decreased sperm motility
and count, and infertility.
2-methoxyethanol (2-ME; CH3—O—CH,—CH,—OH),
EG dimethyl ether (CH3—O—CH,—CH,—O—CHS3),
2-butoxyethanol (2-BE; CH3—CH2—CH2—CH2—O—
CH,—CH,—OH), and 2-ME acetate (CH3—CO—O—CH,—
Developmental Toxicity
CH,—O—CH,). These solvents undergo rapid ester hydrolysis Developmental toxicity in rodents includes a variety of
in vivo, and exhibit the same toxicity profile as unesterified gly- minor skeletal variations, hydrocephalus, exencephaly, car-
cols. The glycol ethers are metabolized to alkoxyacetic acids, diovascular malformations, dilatation of the renal pelvis,
which are regarded as the ultimate toxicants. Their acetalde- craniofacial malformations, and digit malformations. There
hyde precursors have also been implicated. are significant associations for glycol ether exposure induc-
Like glycol ether metabolism, glycol ether toxicity varies ing cleft lip.
with chemical structure. With increasing alkyl chain length,
reproductive and developmental toxicity decrease, whereas
hematotoxicity increases. Hematotoxicity
Some glycol ethers are hemolytic to red blood cells. Typically,
the osmotic balance of the cells is disrupted, they imbibe water
Reproductive Toxicity and swell, their ATP concentration decreases, and hemolysis
Epidemiologic studies have reported associations between occurs. Humans are less susceptible than rodents to glycol
glycol ether exposure and increased risk for spontaneous ether-induced erythrocyte deformity and hemolysis.
 CHAPTER 24 Toxic Effects of Solvents and Vapors 371

AUTOMOTIVE GASOLINE metabolic pathways for CS, exist: (1) the direct interaction of
CS, with free amine and sulfhydryl groups of amino acids and
AND ADDITIVES
polypeptides to form dithiocarbamates and trithiocarbonates;
Gasoline is a mixture of hundreds of hydrocarbons predomi- and (2) microsomal metabolism of CS, to reactive sulfur inter-
nantly in the range of C, to C,,. Because its composition varies mediates capable of covalently binding tissue macromolecules.
with the crude oil from which it is refined, the refining process, The conjugation of CS, with sulfhydryls of cysteine or GSH
and the use of specific additives, generalizations regarding the results in the formation of2-thiothiazolidine-4-carboxylic acid
toxicity of gasoline must be made carefully. Experiments con- (TTCA), which is excreted in urine and has been frequently
ducted with fully vaporized gasoline may not be predictive of used as a biomarker of CS, exposure.
actual risk, because humans are exposed primarily to the more CS, is capable of targeting multiple organ systems includ-
volatile components in the range of C, to C;, which are gener- ing the cardiovascular system, CNS and PNS, male and female
ally less toxic than higher molecular-weight fractions. fertility, and eyes (retinal angiopathy and impairment of color
The most extreme exposures occur to those intention- vision). CS, toxicity requires frequent and prolonged exposures
ally sniffing gasoline for its euphoric effects. This dangerous in occupational settings. The most common neurotoxic effect
habit can cause acute and chronic encephalopathies that are is a distal sensorimotor neuropathy that preferentially affects
expressed as both motor and cognitive impairment. Ingestion long axons in the PNS and CNS (particularly the ascending and
of gasoline during siphoning events is typically followed by a descending tracks of the spinal cord and the visual pathways).
burning sensation in the mouth and pharynx, as well as nausea, Encephalopathy with motor and cognitive impairment has also
vomiting, and diarrhea resulting from GI irritation. Gasoline been reported following chronic, low-level exposure to CS,.
aspirated into the lungs may produce pulmonary epithelial The following clinical syndromes have been associated with
damage, edema, and pneumonitis. CS,: (1) acute and chronic encephalopathy (often with promi-
Oxygenated gasoline contains additives that boost its octane nent psychiatric manifestations), (2) polyneuropathy (both
quality, enhance combustion, and reduce exhaust emissions. peripheral and cranial), (3) Parkinsonism, and (4) asymptom-
Benzene and 1,3-butadiene are classified as known or probable atic CNS and PNS dysfunction. Pathological changes occur
human carcinogens. The co-exposure of ethanol and gasoline in both the CNS and PNS. CNS pathology consists of neuro-
shows additive and possibly synergistic toxic effects on growth, nal degeneration throughout the cerebral hemispheres, with
neurochemistry, and histopathology of the adrenal gland and maximal diffuse involvement in the frontal regions. Cell loss
respiratory tract. No significant epidemiologic association exists is also noted in the globus pallidus, putamen, and cerebellar
between methyl tertiary-butyl ether (MTBE) exposure and the cortex, with loss of Purkinje cells. Vascular abnormalities with
acute symptoms commonly attributed to MTBE, including endothelial proliferation of arterioles may be seen, sometimes
headache; eye, nose, and throat irritation; cough; nausea; diz- associated with focal necrosis or demyelination. PNS changes
ziness; and disorientation. Because three MTBE animal cancer consist primarily of myelin swelling and fragmentation and
bioassays indicate kidney and testicular tumors in male rats and large focal axonal swellings, characteristic of distal axonopathy.
liver adenomas, leukemia, and lymphoma in female rats, MTBE
is classified as a possible human carcinogen (group C).

BIBLIOGRAPHY
CARBON DISULFIDE Karch SB, Drummer O: Karch’s Pathology of Drug Abuse. 4th ed. Boca
Raton, FL: CRC Press, 2009.
The major uses of CS, are in the production of rayon fiber, Patnaik P: A Comprehensive Guide to the Hazardous Properties of
cellophane, and CCl, and as a solubilizer for waxes and oils. Chemical Substances. 3rd ed. Hoboken, NJ: John Wiley & Sons,
Human exposure is predominantly occupational. Two distinct 2007.
372 UNIT 5 Toxic Agents

QUESTIONS

Which of the following statements regarding solvents is Which of the following statements regarding benzene is
FALSE? FALSE?
a. Solvents can be absorbed from the GI tract and a. High-level exposure to benzene could result in acute
through the skin. myelogenous leukemia (AML).
b. Equilibration of absorbed solvents/vapors occurs b. Gasoline vapor emissions and auto exhaust are the
most quickly in the lungs. two main contributors to benzene inhalation.
c. Solvents are small molecules that lack charge. c. Benzene is used as an ingredient in unleaded gasoline.
Volatility of solvents increases with molecular weight. d. Benzene metabolites covalently bind DNA, RNA, and
e. Most solvents are refined from petroleum. proteins and interfere with their normal functioning
within the cell.
What is the route in which most solvents enter the e. Reactive oxygen species can be derived from benzene.
environment?
a. chemical spills. Which of the following is NOT a criterion for fetal alcohol
b. contamination of drinking water. syndrome diagnosis?
c. evaporation. maternal alcohol consumption during gestation.
d. improper waste disposal. pre and postnatal growth retardation.
e. wind. microcephaly.
ocular toxicity.
All of the following statements are true EXCEPT: ce
ge mental retardation.
aa
a. Most solvents can pass freely through membranes by ‘
\

diffusion. Which of the following is NOT an important enzyme in

b. A solvents lipophilicity is important in determining ethanol metabolism?
its rate of dermal absorption. a. alcohol dehydrogenase.
c. Hydrophilic solvents have a relatively low blood:air b. formaldehyde dehydrogenase.
partition coefficient. CowiGYP2EA:
d. Biotransformation ofa lipophilic solvent can result in d. catalase.
the production of a mutagenic compound. e. acetaldehyde dehydrogenase.
e. Hepatic first-pass metabolism determines the amount
of solvent absorbed in the GI tract. Which of the following is NOT associated with glycol
ether toxicity?
Which of the following statements regarding age solvent a. irreversible spermatotoxicity.
toxicity is TRUE? b. craniofacial malformations.
a. Gl absorption is greater in adults than it is in children. c. hematotoxicity.
b. Polar solvents reach higher blood levels in the elderly d. seminiferous tubule atrophy.
than they do in children. e., ‘cleit lip:
c. Children are always more susceptible to solvent
toxicity than are adults. 10. Which of the following statements regarding chlorinated
d. Increased alveolar ventilation increases uptake of hydrocarbons is FALSE?
lipid-soluble solvents to a greater extent than water- a. ‘Toxicities of trichloroethylene (TCE) are medi-
soluble solvents. ated mostly by reactive metabolites, not the parent
e. Increased body fat percentage increases clearance of compound.
solvent chemicals. b. Glutathione conjugation is an important metabolic
step of both trichloroethylene (TCE) and perchloro-
Huffing gasoline can result in which of the following ethylene (PERC).
serious health problems? c. Many chlorinated hydrocarbons are used as degreas-
a. renal failure. ing agents.
b. pneumothorax. d. Chloroform interferes with intracellular calcium
c. Hodgkin's disease. homeostasis.
d. encephalopathy. e. Carbon tetrachloride causes hepatocellular and
e. thrombocytopenia. kidney toxicity.
 Cr HH. Age Ps risEigek

Toxic Effects of Radiation 2 5

and Radioactive Materials
David G. Hoel

INTRODUCTION CANCER EPIDEMIOLOGY

RADIATION BACKGROUND PEATE TUE e

Nonoccupationally Exposed Groups
Types of lonizing Radiation
Radionuclides
Relative Biologic Effectiveness and
Radon
Quality Factors
Radium
Units of Radiation Activity and Dose
Plutonium
RADIOBIOLOGY Radioiodine

Nontargeted Radiation Effects NONCANCER EPIDEMIOLOGY

Bystander Effects
Cardiovascular Disease
Genomic Instability
Cataracts
Adaptive Response
Mental Effects
Gene Expression
Summary DISCUSSION

KEY POINTS

a The four main types of radiation are due to alpha parti- by the nucleus as photon or gamma-ray emission coinci-
cles, electrons (negatively charged beta particles or posi- dent with the ejection of the particle.
tively charged positrons), gamma-rays, and X-rays. a The Compton Effect occurs when a photon scatters at a
Alpha particles are helium nuclei (consisting of two pro- small angle from its original path with reduced energy
tons and two neutrons), with a charge of +2, that are because part of the photon energy is transferred to an
ejected from the nucleus of an atom. electron.
Beta particle decay occurs when a neutron in the nucleus s lonizing radiation loses energy when passing through
of an element is effectively transformed into a proton matter by producing ion pairs (an electron and a posi-
and an electron, which is ejected. tively charged atom residue).
Gamma-ray emission occurs in combination with m Radiation may deposit energy directly in DNA (direct
alpha, beta, or positron emission or electron capture. effect) or may ionize other molecules closely associated
Whenever the ejected particle does not utilize all the with DNA, hydrogen, or oxygen, to form free radicals
available energy for decay, the excess energy is released that can damage DNA (indirect effect).

373
374 UNIT 5 Toxic Agents

INTRODUCTION radiation may either be electrically charged (a, 8, proton) or

have no charge (neutron). Ionizing electromagnetic radiation
Ionizing radiations such as ‘y-rays and X-rays are radiations (photons) in the form of X-rays or y-rays has considerably
that have sufficient energy to displace electrons from mole- more energy than nonionizing radiation, such as ultraviolet
cules. These freed electrons then have the capability of dam- and visible light. Radionuclides (i-e., radioactive atoms), being
aging other molecules and, in particular, DNA. Atoms of the unstable, release both electromagnetic and particulate radia-
DNA target may be directly ionized or indirectly affected by tion during their radioactive decay. The radionuclides decay
the creation of a free radical that can interact with the DNA into either stable elements or through a decay chain of succes-
molecule. In particular, the hydroxyl radical is predominant sive radionuclides called decay daughters. The types of radia-
in DNA damage. Thus, the potential health effects of low tion emitted, its rate of decay, and the energies of the released
levels of radiation are important to understand in order to radiation are unique to each type of radionuclide. For example,
be able to quantify their effects. Cancer has been the major the uranium decay series is illustrated in Figure 25-2, with
adverse health effect of ionizing radiation. National Council specific details provided in Table 25-1.
on Radiation Protection (NCRP) Report 160 gives a summary The rate of energy dissipation by a single event is referred to
breakdown of exposure sources in Figure 25-1. as linear energy transfer (LET). The LET of a charged particle is
the average energy lost due to interactions per unit length of its
trajectory given as kiloelectron volts per micrometer (keV/[.m).
RADIATION BACKGROUND X-rays, y-rays, and @ particles of similar energies produce
sparse ionization tracks and are, classified as low-LET radia-
Types of lonizing Radiation tion. Particulate radiation (e.g., neutrons and a particles) causes
When ionizing radiation passes through matter, it has the interactions with large amounts of energy being dissipated
energy to ionize atoms so that one or more of its electrons can within short distances. a-Particles (helium nucleus), which
be dislodged and chemical bonds broken. Ionizing radiation is are released from the nucleus of some radionuclides, are slow-
of two types: particulate and electromagnetic waves. Particulate moving with a positive charge. Although they cannot penetrate a
piece of paper or skin, they are of concern if ingested or inhaled.
The most recognized example is the lung cancer risk from the
Terrestrial (background) (3%)
inhalation of radon (Rn 222) and its daughter products.
Internal (background) (5%)

(a Space (background) (5%) Relative Biologic Effectiveness and

Quality Factors
The various types of ionizing radiation have similar biologic
effects that occur because of the ionization of molecules.
a eg
x
A a
~
However, without knowing the type of radiation, one cannot
specify how much radiation is needed to produce a specific bio-
Computed (background) — logic effect. This is because a given absorbed dose (energy per
tomography Oe. |) unit mass) of X-rays does not have the same biologic effect as an
(medical) “cae
path EWS 33 identical dose of neutrons. The relative effectiveness of differ-
(24%) isk Haas
ie
ent types of radiation in producing biologic changes depends on
G
ae deposition of energy. The relative biologic effectiveness is numer-
Industrial (<0.1%) ically equal to the inverse of the ratio of absorbed doses of the two
Occupational (<0.1%)

7-
radiations required to produce equal biologic effects. The diffi-
Consumer (2%)
culty is that the relative biologic effectiveness may differ depend-
ing on the biologic end point and it may also be dose-dependent.
Conventional
radiography/fluoroscopy
(medical) (5%)
Units of Radiation Activity and Dose
Interventional fluoroscopy
The basic unit of radiation activity is the Becquerel (Bq), which
(medical) (7%)
is nuclear disintegrations per second. The older unit of activity
Nuclear medicine is the Curie (Ci), which corresponds to the number of disin-
(medical) (12%) tegrations in 1 s from 1 g of radium 226 or 1 Ci = 3.7 x 10”
FIGURE 25-1 Percent contribution of total effective dose decays per second; thus, 1 Bq = 2.7 X 107" Ci. The EPA con-
to individuals (Reproduced with permission from NCRP Report tinues to use the old unit of activity with regard to radon.
No. 160. lonizing Radiation Exposure of the Population ofthe United The basic unit of dose is the Gray (Gy), which is the amount
States. Bethesda, MD: National Council on Radiation Protection and of energy released in a given mass of tissue. One Gray is defined
Measurements; 2009. http://NCRPpublications.org). as | joule of energy released in 1 kg of tissue. The other common
 CHAPTER 25 Toxic Effects of Radiation and Radioactive Materials 375

234
Uranium Uranium
io
245,500 234
year

Protactinium
234
Qa

Thorium | ae
234

218
34 PO Polonium
3.1min ) 218 Oi 210

Bismuth

Lead
206

ee
ro
Sr Thallium | Thallium
210 ie | 206
Q iey,

FIGURE 25-2 Uranium decay chain.

measure is the Sievert (Sv), which is a dose equivalent; that is, the basic mode of action is linearly related to dose and that the
the dose in Gray multiplied by the appropriate quality factor. individual cell is the unit of risk. However, effects occurring in
nontargeted cells such as with induced genomic instability and
RADIOBIOLOGY bystander effects suggest that responses can occur nonuniformly
over time at the tissue level. Following irradiation, various pro-
Radiation biology has made significant progress in our under- tective cellular processes occur that depend on the degree of
standing of radiation effects at low doses. Currently radiation damage and the tissue type. These mechanisms include DNA
cancer risk extrapolations make two assumptions: namely that repair, intracellular metabolic oxidation/reduction reactions,
376 UNIT 5 Toxic Agents

TABLE 25-1 Radioisotopes in the uranium decay series.

Nuclide Decay Mode Half-Life (a = Year) Energy Released, MeV Product of Decay

238U) Ou 4.468 x 10°a 4.270 arta

24TH B- 24.10 days 0.273 2240

Pa

234mpq B— 99.84% 1.16 min 2.271 ee

IT 0.16% 0.074 24Pa

24D B- 6.70h 2.197 234)

234UJ a 245 500 a 4.859 220K

230Th Qa 75 380a 4.770 226Ra

26Ra a 1602a 4.871 22kni

22Rhy a 3.8235 days 5.590 218PO

218Pg 0199.98% 3.10 min GAS 2p D

B~ 0.02% 0.265 EIeAG

218At 1 99.90% 1.55 6.874 ANSBi

B- 0.10% 28837) 212Rn

218RH a 35 ms 7.263 we 2149

214Ph B- 26.8 min 1.024 24Bj

214B; B~ 99.98% 19.9 min S272 212Po

a 0.02% 5.617 ZA

214P9 a 0.1643 ms 7.883 210Pb

2107 iG 1.30 min 5.484 AN) 9)

210Ph B- 22.34 0.064 NOB i

2108; Bo 5.013 days 1.426 210P9

99.99987% 5.982 206T|
a 0.00013%

210Pg Qa 138.376 days 5.407 206P

206T| B- 4.199 min 1.533 206Pbh

206Ph Se Stable

cell cycle checkpoint controls, cellular signaling, senescence, also be observed in the progeny of an irradiated cell (genomic
and apoptosis. instability). Both targeted and nontargeted effects can result in
A study reviewed in detail the effects of DNA damage after DNA mutations, gene amplifications, chromosomal rearrange-
exposure to low doses ofionizing radiation. After an exposure ments, carcinogenesis, and cell death.
to 5 mGy of low-LET radiation (average background per year),
each cell nucleus is on average hit by one electron, resulting in Bystander Effects—Radiation-induced bystander effects are
5 to 10 damaged bases, 2.5 to 5 single-strand breaks and 0.25 those in which cells that have not been directly exposed to
double-strand breaks. ionizing radiation react as though they have been exposed by
receiving a biochemical signal from a radiation-exposed cell.
That is, they show chromosomal instability and other abnor-
Nontargeted Radiation Effects malities, or die. For high-LET radiation a bystander effect has
Exposure to ionizing radiation can result in direct damage been shown for inducing cell lethality, chromosome aber-
to the irradiated cells as well as producing effects in cells that rations, sister-chromatid exchanges, mutations, genomic
were not irradiated (bystander effects). These nontargeted instability, signal transduction pathways, and in vitro transfor-
effects can occur in the nonirradiated neighbors ofirradiated mation. For low-LET radiation, the bystander effect has been
cells and at sites distant from the irradiated cells. Effects can limited to cell lethality and lethal mutations.
 CHAPTER 25 Toxic Effects of Radiation and Radioactive Materials B77,

These bystander cells can be either adjacent or at some dis- response as they relate to the induction of chromosomal aber-
tance from the radiation-exposed cell. The important issue rations and gene mutations because cancer is believed to be
from a risk assessment view is whether bystander effects are associated with these cellular responses. The future of under-
beneficial (e.g., adaptive response and apoptosis. [removal of standing low-dose radiation cancer risks will depend on the
damaged cells]) or detrimental to the nonexposed cells, and continued advancement of molecular biology, gene expression
what impact they may have on dose response at low doses. It analysis, and computational biology.
should be noted that most observed effects are detrimental,
but beneficial effects are more difficult to measure. However,
bystander effects demonstrate that the organism and tissues CANCER EPIDEMIOLOGY
communicate and are responding as an organized structure Epidemiologic studies have been extensive and provide the
to radiation insult. Large DNA deletions are the major type of basis for our understanding of radiation-induced cancer
radiation-induced mutations. In bystander cells, however, the effects. Radiation cancer studies are no different from other
types of mutations are similar to those that occur spontane- types of occupational and environmental cancer studies in that
ously, with the majority being point mutations. radiation-induced cancers are not distinguishable pathologi-
cally, and there are usual issues of exposure levels and durations,
Genomic Instability— Genomic instability has been defined
long latencies (e.g., 10 to 20 years for solid tumors), and study
as the increase in rate of acquiring genetic change, and induced
size. Generally for acute exposures only epidemiologic studies
genomic instability can be observed in the progeny of irradi-
with exposures to relatively high doses of radiation (> 0.15 Sv)
ated cells and can persist for many generations. When a cell is
have shown such an excess of cancer. Because of these difficul-
saturated in repairing radiation damage it may change its gene-
ties, the most informative studies are those that involve a large
product profile without any specific genetic damage. This has
number of individuals with large radiation doses and follow-
been suggested as a cause of genomic instability, which is an
up of several decades. Table 25-2 lists radiation-linked human
anti-inflammatory response, and is a risk for malignancy.
carcinogenicity.
Adaptive Response—In cells that are exposed to a low
priming dose of radiation (e.g., 10 to 20 mGy) followed in a Occupational Studies
short time interval with a larger challenge dose (e.g., 1 Gy), There have been numerous studies over the years among
the frequency of chromosomal aberrations induced by the nuclear workers, primarily at governmental facilities. In most
challenge dose was found to be less than that from the chal- of these studies, mortality rates were compared with those in
lenge dose given alone. This effect is referred to as “adaptive the general population. In most cases, the cancer mortality
response.’ Studies have also shown that low doses of radia- rates were less than those for the general public, which may
tion may reduce the biologic background effect. This has been be due to the healthy worker effect. However, a series of analy-
shown for cell transformation and chromosomal damage. It ses of workers at the Russian nuclear facility at Mayak have
has also been observed that the normal rate of cell transfor- been published. The Mayak workers generally experienced
mation and chromosome damage can be decreased to below very high doses from both internal (plutonium, a particles)
the normal background level after an initial low-dose radia- and external radiation exposures. High levels of body burdens
tion exposure. Adaptive responses have been observed both of plutonium were found to have a relative risk ofliver cancer
in vitro and in vivo for both cancer and genetic effects, which and for bone cancer. Small nonsignificant increases were seen
suggests that low doses may decrease radiation risk. These at low doses.
adaptive responses suggest that enhancing normal repair or It is estimated that there are 2.3 million medical radia-
protective processes make it possible to decrease the risk for tion workers worldwide. Epidemiologic studies of exposures
low-dose radiation-induced cancer. of radiologists and radiologic technologists have been going
on for many years. These workers were some of the earliest
Gene Expression exposed to radiation with the first finding in 1902 that radia-
It has been shown that dose, dose rate, radiation quality, and tion can cause skin cancer. It was recognized in the 1940s that
time since exposure result in variations in the response of
radiologists had increased rates of leukemia.
genes, so that gene expression signatures may be markers of
The Chernobyl cleanup workers are of interest because of
radiation exposure. Using gene expression methods, scientists their higher exposures compared with other nuclear work-
have been able to distinguish a number of post-Chernobyl thy- ers. The excess relative risk for solid tumors was significant
roid tumors and postradiotherapy thyroid tumors from their and the increase was observed in the highest dose interval
with no increase in the lower-dose interval. Besides solid
sporadic counterparts:
tumors, a significant increase in leukemia incidence was
observed for those with increased exposures, compared with
Summary workers with lower exposures. Also, increases in the inci-
Cancer being the primary health concern from exposure to dence of cardiovascular disease were observed among those
ionizing radiation, there is a focus on mechanisms and dose at higher exposures.
 8
Oo“SI UNIT 5 Toxic Agents

TABLE 25-2 IARC:Tumor sites with sufficient evidence of human carcinogenicity.

epee es pr pean mga nee en enna pr err eres ie eae os De oa
Tumor Sites (and Types) on Which Sufficient
Radiation Type Major Study Populations Evidence Is Based

a-particle and B-particle emitters

Radon 222 and decay products General population (residential exposure), Lung
underground miners
Radium 224 and decay products Medical patients Bone
Radium 226, radium 228, and decay products Radium-dial painters Bone, paranasal sinus, and mastoid process
(radium 226 only)
Thorium 232 and decay products Medical patients Liver, extrahepatic bile ducts, gall bladder,
leukemia (excluding CLL)
Plutonium Plutonium-production workers Lung, liver, bone
Phosphorus 32 Medical patients Acute leukemia
Fission products, including strontium 90 General population, following nuclear reactor Solid cancers, leukemia
accident
Radioiodines, including iodine 131 Children and adolescents, following nuclear Thyroid
reactor accident

X-radiation or y-radiation Atomic-bomb survivors, medical patients; Salivary gland, esophagus, stomach, colon, lung,
in utero exposure (offspring of pregnant bone, skin (BCC), female breast, urinary bladder,
medical patients and of atomic-bomb brain and CNS, leukemia (excluding CLL),
survivors) thyroid, kidney (atomic-bomb survivors, medical
patients); multiple sites (in utero exposure)

Solar radiation General population Skin (BCC, SCC, melanoma)

UV-emitting tanning devices General population Skin (melanoma), eye (melanoma, particularly
choroid and ciliary body)

BCC, basal cell carcinoma; CLL, chronic lymphocytic leukemia; CNS, central nervous system; SCC, squamous cell carcinoma.

Nonoccupationally Exposed Groups cancer registry data. Previously the NCI analyzed cancer mortal-
ity rates in those counties with nuclear power reactors compared
Studies of the population living in the high background radia-
with control counties. Basically no differences were found; how-
tion areas in Yangjiang, China evaluated dose reconstruction
ever, the use of counties as the analysis unit is likely inappropriate
and noted a correlation between estimated radiation exposure
as a geographical area to detect any possible small effects.
and frequency of dicentric and ring chromosomes, which are
recognized as a good biomarker of radiation exposure. They
observed that for those in the high radiation background
area, the incidence of these markers agrees with what has been
Radionuclides
observed in other studies of radiation exposures and chromo- Radon—Radon is a natural radioactive gas produced by the
some aberrations. This result provides some evidence in sup- decay of uranium and thorium. Originally, exposures to radon
port of the program's exposure estimates. In conclusion, the and its daughter radionuclides among uranium miners and
high-background Chinese studies have not shown an increase some other groups of miners established that high exposures
in cancer incidence at low-dose and dose-rate exposures. were a Clear risk for lung cancer. The lung cancer risk was also
A study of childhood cancers in relation to natural back- significantly increased when the cases were restricted to expo-
ground radiation in Great Britain using the National Registry sures less than 200 Bq/m’. The lung cancer effects were also
of Childhood Tumours involved radiation exposures esti- consistent, with the risks projected downward from the higher
mated on the basis of the mother’s residence at the time of exposed uranium miners.
birth. The study found a significant increase in leukemias in
increased radiation exposure areas. Additionally, children who Radium—There are 25 isotopes of radium of which four occur
live within 5 km ofa nuclear facility are also at an increased risk naturally (radium 223, 224, 226, and 228); the others are man-
for the development of leukemia. The concept of population made or decay products of man-made radionuclides. Radium
mixing, which basically is the idea that workers arriving in a 226 with a half-life of 1601 years is by far the common natural
typically rural area bring foreign infectious agents that in turn form, followed by 228 with a half-life of 5.75 years. Radium 223
will affect local childhood leukemias, may explain increased and 224 have half-lives of only a few days. Except for radium
leukemia effects in children when there is no evidence of radia- 228, which is a 8 emitter, the other three are all a emitters.
tion exposures from the nuclear plants. The different isotopes have been used both occupationally as
The US National Research Council released recommendations luminescent paint on watches and instruments (radium 226
on how a study could be carried out in the United States using and 228) and in medical applications (radium 223 and 224).
 CHAPTER 25 Toxic Effects of Radiation and Radioactive Materials 379

These uses, as well as radium found environmentally in drinking the eye. There are basically three types of cataracts including
water, have provided material for many epidemiologic studies. nuclear or nuclear sclerosis, cortical, and posterior-subcapsular
Beginning in the 1920s, young women worked painting the (PS) cataracts. Each ofthese clinical types has its known risk fac-
dials of watches with paint containing radium 226 and 228. tors such as cigarette smoking for nuclear and possibly PS cata-
Many of them “pointed” the tips of their paintbrushes by mouth racts, while UV-B is a risk factor for cortical cataracts. Ionizing
resulting in ingestion of relatively large amounts of radium radiation is a risk factor for both cortical and PS cataracts but
for some of the women. Radium as a bone seeker resulted in not nuclear sclerotic cataracts. Also there is limited evidence
increases in bone cancer as well as paranasal sinus cancers. that those exposed at a younger age are at greater risk.
Bone sarcomas were also the major cancer effect among
patients with tuberculosis and ankylosing spondylitis who
were treated with high doses of radium 224 (mean bone sur-
Mental Effects
face dose of 30 Gy) in two cohort studies in Germany. There In the A-bomb survivor analyses, significant effects on the
were increases in bone cancer in both studies, but there were developing brain were observed among those exposed during
also some increases in other cancer sites. the period of the eighth week through the 25th week of gesta-
tion. During the most sensitive period of 8 to 15 weeks, there
Plutonium—Plutonium is used for nuclear weapons produc- was an increased frequency of severe mental retardation, a
tion, and in the production of mixed oxide fuels. Most of the diminution in IQ scores and school performances, as well as
exposure to plutonium is to workers involved in the process- an increase in the occurrence of seizures. During this sensitive
ing of plutonium in nuclear weapons (Pu 239) and in nuclear period there is a rapid increase in the number of neurons; they
power generation (Pu 238). The major exposure to plutonium migrate to the cerebral cortex where they lose their capacity to
is by inhalation and is retained primarily in the lung, liver, divide, becoming perennial cells.
and bone.

Radioiodine—Releases from nuclear facilities of fission- DISCUSSION

product radionuclides deposited in the environment as well as
The major issue in radiation health effects is the causation
internal doses from the ingestion of foods containing fission
of cancer. We now see noncancer effects such as CV disease
products have been the result of the Chernobyl and Fukushima
at low-dose and also low dose-rate exposures that occur at
accidents. The major observable health effect has been child-
environmental levels and in diagnostic medical screening.
hood thyroid cancer resulting from the 8 emitter iodine 131.
Currently the linear-no threshold (LNT) model is used to esti-
From studies of external radiation exposures in the A-bomb
mate these effects well below what can be observed in epide-
survivor studies as well as the children who were treated by
miologic studies. The simple defense of the LNT model is that
radiation for tinea capitis (ring worm present on the scalp), it
the physical energy deposition of ionizing radiation increases
is clear that radiation is a risk for thyroid cancer for exposures
cancer risk linearly with increasing dose, and that the carcino-
to adolescents. The risk of radiation-related thyroid cancer
genic effectiveness is constant independent ofdose.
was three times higher in iodine-deficient areas and the use
It is recognized that a cell is not passively affected by the
of potassium iodide as a supplement reduced this risk of
accumulation oflesions induced by ionizing radiation. The cell
radiation-related thyroid cancer by a factor of 3.
reacts through at least three main mechanisms: first by react-
ing against radiation-induced ROS; secondly by eliminating
NONCANCER EPIDEMIOLOGY damaged cells by either apoptosis or through cell death during
mitosis of unrepaired cells; and thirdly by immunosurveillance
Cardiovascular Disease systems that eliminate clones of transformed cells.
What is of particular interest is cardiovascular disease (CV dis-
ease) mortality, because although it may have a lower relative
risk from radiation exposure than solid tumors, it accounts for BIBLIOGRAPHY
more total background deaths. Atherosclerosis is an inflamma- Cember H, Johnson TE: Introduction to Health Physics. 4th ed. New
tory disease of the arteries, which can lead to ischemia of the York: McGraw-Hill Medical, 2009.
Dauer LT, Brooks AL, Hoel DG, Morgan WF, Stram D, Tran P: Review
heart. In a study of inflammatory biomarkers (TNF-a, IL-10,
and evaluation ofupdated research on the health effects associated with
IgM, IgA, and IFN-‘) as well as erythrocyte sedimentation
low-dose ionizing radiation. Radiat Prot Dosim 140:103-136, 2010.
rates among A-bomb survivors, it was shown that these mark- Hall EJ, Giaccia AJ: Radiobiologyfor the Radiologist. 7th ed. Philadelphia:
ers were associated with radiation exposure. Lippincott, Williams & Wilkins, 2012.
IARC. A review of human carcinogens: Part D radiation. IARC
Monogr Eval Carcinog Risks Hum 100D:1-362, 2009.
Cataracts Mettler F, Upton AC: Medical Effects of Ionizing Radiation. 3rd ed.
Cataracts were one of the earliest radiation-associated effects Philadephia: WB Saunders, 2008.
found after the discovery of X-rays. It has long been believed Stabin MG: Radiation Protection and Dosimetry: An Introduction to
that it results from only high doses of radiation to the lens of Health Physics. New York: Springer, 2010.
380 UNIT 5 Toxic Agents

QUESTIONS

1. Which ofthe following is NOT a main type of radiation? 6. Low-LET radiation:

a. alpha particles. a. causes large-scale ionizations throughout the cell.
b. microwaves. b. results from alpha particle emission.
c. beta particles. c. causes damage that is readily repaired by cellular
d. gamma-rays. enzymes.
e. X-rays. is also known as densely ionizing radiation.
e. usually causes irreparable cell damage.
2. Which of the following statements regarding alpha
particles is FALSE? Te What is the most common type of DNA damage caused by
a. Alpha particles are ejected from the nucleus of an low-LET radiation exposure?
atom. a. base damage.
b. The atomic number decreases by two after emission b. DNA protein cross-links.
of an alpha particle. c. single-strand breaks.
c. The atomic weight decreases by two after emission of d. double-strand breaks.
an alpha particle. e. thymine-dimer formation.
d. Energies of most alpha particles range between 4 and
8 MeV. 8. Which of the following statements regarding radon
e. Alpha particles are helium nuclei. exposure is FALSE?
a. Miners are exposed to increased environmental
3. Which of the following types of radiation is likely the radon levels. .
MOST energetic? b. Radon exposure has been linked to the development
a. alpha particles. of lung cancer.
beta particles. c. Smokers are ata higher risk from radon exposure.
positron emission. d. Radon levels are relatively higher in urban areas than
electron capture. in rural areas.
Cr
he photon emission. e. The use of open flames indoors increases radon
exposure.
4, Pair production and the Compton effect characterize
which type of radiation’s interaction with matter? o The largest dose of radiation is received from which of the
a. alpha particles. following sources?
b. _ beta particles. a. inhalation.
c. positron emission. b. in body.
d. electron capture. Ce COSInc:
e. photon emission. d. cosmogenic.
e. terrestrial.
5. Which of the following statements regarding radiation
DNA damage is FALSE? 10. The largest contributor to the effective dose of radiation in
a. lonizing radiation slows down by forming ion pairs. the U.S. population is which ofthe following?
b. A main form of radiation DNA damage occurs by the a. nuclear medicine.
production of free radicals. b. medical X-rays.
c. High-LET radiation causes more ionizations than Cre tentestrial:
does low-LET radiation. d. internal.
d. Most DNA damage caused by radiation happens Cnmeradome
directly.
e. Direct and indirect ionization cause similar damage
to DNA.
 Cah AL Peat SEe

Toxic Effects of Plants

and Animals
John B. Watkins, III

INTRODUCTION Parasympathetic Stimulation

Parasympathetic Block
INTRODUCTION TO PLANT TOXICITIES Sensory Neuron Block
Skeletal Muscle and Neuromuscular Junction
TOXIC EFFECTS BY ORGAN
Neuromuscular Junction
Skin
Skeletal Muscle Damage
“Irritant Contact Dermatitis
Bone and Tissue Calcification
Allergic Contact Dermatitis
Bone and Soft Tissue
Photosensitivity
Reproduction and Teratogenesis
Respiratory Tract
Abortifacients
Allergic Rhinitis
Teratogens
Cough Reflex
Gastrointestinal System CLINICAL STUDY OF PLANT POISONS
Direct Irritant Effects
INTRODUCTION TO ANIMAL VENOMS
Antimitotic Effects
Protein Synthesis Inhibition PROPERTIES OF ANIMAL TOXINS
Cardiovascular System
Cardioactive Glycosides ARTHROPODS
Actions on Cardiac Nerves
ARACHNIDA
Vasoactive Chemicals
Liver Scorpions
Hepatocyte Damage Spiders
Mushroom Toxins Agelenopsis Species (American Funnel
Mycotoxins Web Spiders)

Kidney and Bladder Latrodectus Species (Widow Spiders)

Loxosceles Species (Brown orViolin
Carcinogens
Spiders)
Kidney Tubular Degeneration
Steatoda Species
Blood and Bone Marrow
Cheiracanthium Species (Running
Anticoagulants
Spiders)
Bone Marrow Genotoxicity
Theraphosidae Species (Tarantulas)
Cyanogens
Ticks
Nervous System
Epileptiform Seizures CHILOPODA (CENTIPEDES)
Excitatory Amino Acids
Motor Neuron Demyelination DIPLOPODA (MILLIPEDES)

381
 8
1S.) ie) UNIT 5 Toxic Agents

INSECTA Snakes
General Information and Classification
Heteroptera (True Bugs)
Snake Venoms
Hymenoptera (Ants, Bees, Wasps, and Hornets)
Enzymes
Formicidae (Ants)
Polypeptides
Apidae (Bees)
Toxicology
Vespidae (Wasps)
Snakebite Treatment
Lepidoptera (Caterpillars, Moths, and Butterflies)
ANTIVENOM
MOLLUSCA (CONE SNAILS)

POTENTIAL CLINICAL APPLICATION

REPTILES
OF VENOMS
Lizards

= Different portions of the plant (root, stem, leaves, « Venomous animals produce poison in a highly devel-
seeds) often contain different concentrations of a toxic oped secretory gland or group of cells and can deliver
substance. their toxin during biting or stinging.
= The age ofa plant contributes to variability. Young plants s Poisonous animals are those whose tissues, either in
may contain more or less of some constituents than whole or in part, are toxic. Poisoning usually takes place
mature plants. through ingestion.
= Climate and soil influence the synthesis of some toxins. « The bioavailability of a venom is determined by its
s Plants contain substances that may exert toxic effects on composition, molecular size, amount or concentration
skin, lung, cardiovascular system, liver, kidney, bladder, gradient, solubility, degree of ionization, and the rate of
blood, nervous system, bone, and the endocrine and blood flow into specific tissues.
reproductive systems. a The distribution of most venom fractions is rather
= Contact dermatitis and photosensitivity are common unequal, being affected by protein binding, variations in
skin reactions with many plants. pH, and membrane permeability, among other factors.
« Gastrointestinal effects range from local irritation to = A venom may be metabolized in several or many differ-
emesis and/or diarrhea. ent tissues.
» Cardiac glycosides in plants may cause nausea, = Because of their protein composition, many toxins
vomiting, and cardiac arrhythmias in animals and produce an antibody response; this response is essential
humans. in producing antisera.

INTRODUCTION INTRODUCTION TO PLANT TOXICITIES

History is replete with stories of the earliest humans using The plant kingdom contains potentially 300 000 species, and
plant extracts and animal venoms for hunting, war, assassina- the toxic effects of plants serve primarily as defense mecha-
tion, and political intrigue for millennia. The toxic properties nisms against natural predators. Toxic effects on humans can
of plants and animals often enhance their ability to survive. range from simple hay fever caused by exposure to plant pollen
Some toxic compounds are used primarily to aid an animal in all the way to serious systemic reactions caused by ingestion
obtaining food while plants have developed toxic properties to of specific plants. Table 26-1 lists some of the poisoning syn-
specifically ward off being used as food. Toxins have been uti- dromes plants can produce. Many variables that can affect the
lized as tools to study human biochemistry and physiology in concentration of a plant’s toxin and that can be a major factor
order to pave the way for new pharmaceuticals. In fact, some in the severity of reaction one will experience on exposure
components are in active development for clinical use. include what part of the plant exposure is from, the age of the
 CHAPTER 26 Toxic Effects of Plants and Animals 383

TABLE 26-1 Poisoning syndromes caused by plants.

Syndrome Genera Mechanism(s)

Antimuscarinic Atropa, Datura, Hyoscyanmus, Solanum Blockade of muscarinic cholinoceptors

Cardiotoxic Adenium, Digitalis, Convallaria, Nerium Inhibition of cellular Na*,Kt-ATPase increases contractility, enhanced
vagal effect

Convulsants Anemone, Conium, Labrunum, Nicotinia, Ranunculus Blockade of gamma-aminobutyric acid (GABA) receptor on the neuronal
chloride channel, alteration of acetylcholine homeostasis, mimic
excitatory amino acids, sodium channel alteration, hypoglycemia

Cyanogenic Eriobotrya, Hydrangea, Prunus Gastric acid hydrolysis of cyanogenic glycosides releases cyanide

Dysrhythmia Acotinum, Rhododendron, Veratrum Sodium channel activation

Nicotinic Conium, Laburnum, Lobelia, Nicotinia Stimulation of nicotinic cholinoceptors

Pyrrolizidine Crotalaria, Heliotropium, Senecia Pyrroles injure endothelium of hepatic or pulmonary vasculature leading
to veno-occlusive disease and hepatic necrosis

Toxalbumin Abrus, Ricinus Protein synthesis inhibitors leading to multiple organ system failure

plant, amount of sunlight and soil quality that the plant has Mucinain, contained in the toxin, is the proteinase responsible
grown in, and genetic differences within a species. Also, plant for causing the pruritus.
toxins fall under a number of different chemical structures,
which is useful in understanding related toxins. Table 26-2 Allergic Contact Dermatitis—Many people have experi-
lists some of the common classifications. enced allergic dermatitis, most frequently from contact with
poison ivy. Allergic dermatitis is an actual allergic reaction
occurring within the skin as opposed to just a response to the
TOXIC EFFECTS BY ORGAN presence of an irritant. Due to this immunological component,
the severity of the reaction can range widely.
Skin Philodendron scandens (Araceae, arum family) and the toxico-
Irritant Contact Dermatitis—Plants that cause irritation dendron group of plants, which contain Rhus radicans (poison
of the skin on contact are rather common (Table 26-3). The ivy, Figure 26-2), Rhus diversiloba (poison oak), and Rhus vernix
trichomes, or barb-like hairs (Figure 26-1), found on stinging (poison sumac), are all known to cause allergic dermatitis. In the
nettles (Urtica species, Urticaceae) puncture skin on contact Rhus species the allergen is a fat-soluble substance called urushiol
and release an irritating sap containing a mixture of formic that can penetrate the stratum corneum where it then binds to
acid, histamine, acetylcholine, and serotonin. Mucuna pruriens Langerhans cells in the epidermis. These haptenated cells then
(cowhage), which also deploys its toxin via barbed trichomes migrate to lymph nodes, where T cells are activated resulting in
on contact, may cause pain, itching, erythema, and vesication. the allergic response.

TABLE 26-2 Chemical classification of plant toxins.

Chemical Category Genera Examples

Alkaloids Atropa, Senecio, Nicotinia, Coffea, Papaver, Tropines, pyrrolizidines, pyridines, purines, isoquinolines, steroids,
Solanum, Acotinum diterpines

Glycosides Digitalis, Aesculus Steroids, coumarins

Proteinaceous compounds Abrus, Amanitin, Lathyrus Toxalbumins (abrin, ricin), polypeptides (amatoxins, phallotoxins,
phalloidin), amines (aminopropionitrile)
o

Organic acids Caladium, Dieffenbachia, Rheum Oxalates

Alcohols Cicuta, Eupatorium Cicutoxin, tremetol

Resins and resinoids Cannabis, Rhus Tetrahydrocannabinol, urushiol

384 UNIT 5 Toxic Agents

TABLE 26-3 Selective plants producing contact dermatitis.

Neen ee re rar eaparnararraenssetns-assnsseasa sn sess Sesae AONEG ASSL LL LLL
Botanical Family Genus Species CommonName —

Amaryllidaceae Narcissus Narcissus

Apocynaceae Nerium oleander Oleander

Bromeliaceae Ananas comosus Pineapple

Asteraceae Ambrosia, Aster, Chrysanthemum, Rudbeckia hirta, Tagetes minuta Ragweed, aster, chrysanthemum, Blackeyed Susan,
Mexican marigold

Euphorbiaceae Ricinus communis Castor bean

Fumariaceae Dicentra spectabilis Bleeding heart

Ginkgoaceae Ginkgo biloba Ginkgo

Liliaceae Allium cepa Onion

Myrtaceae Eucalyptus globulus Eucalyptus

Pinaceae Abies balsamea Balsam fir

Saxifragaceae Hydrangea Hydrangea

Solanaceae Lycopersicon esculentum, Solanum carolinense, S. turerosum Tomato, horse nettle, potato
‘
\

Umbelliferae Daucus carota, Heracleum lanatum Carrot, cow parsnip

Urticaceae Urtica dioica, U. urens Stinging nettle

Photosensitivity—Dermatitis does not necessarily have to exposure to sunlight, edematous lesions form on areas of skin
be caused by skin contact. Consumption of Hypericum perfora- that are not protected by hair such as the nose and ears.
tum (St. John’s wort) by animals can lead to serious dermatitis
and even may be life threatening. The toxic agent is hypericin
(a bianthraquinone) that, once ingested and dispersed sys- Respiratory Tract
temically, causes photosensitization of the animal's skin. On Allergic Rhinitis—“Hay fever” or rhinitis from inhalation of
plant pollens is a seasonal problem for many individuals. Trees,
grasses, and weeds are all responsible to contributing to air-
borne pollen.

FIGURE 26-1 Stinging hairs of Urtica ferox (nettles). FIGURE 26-2 Toxicodendron radicans (poison ivy).
 CHAPTER 26 Toxic Effects of Plants and Animals 385

Grass species Poa and Festuca are major contributors along esculin. Ingestion by humans causes gastroenteritis, which
with pollen from several weed genera in the Asteraceae family increases in severity with the number of nuts consumed.
(e.g., mugwort, Artemisia vulgaris, in Europe, and ragweed,
Ambrosia sp., in North America). Antimitotic Effects—Podophyllotoxin is found in Podo-
phyllum peltatum (May apple, Berberidaceae) especially in its
Cough Reflex—Workers who process peppers have a sig- foliage and roots. In low doses, mild purgation occurs; however,
nificantly increased incidence of coughing when specifically overdose results in nausea and severe paroxysmal vomiting. By
handling Capsicum annuum (sweet pepper) and Capsicum fru- binding microtubules, podophyllotoxin blocks mitosis from
tescens (red pepper). These two types of peppers produce the proceeding. This has made podophyllotoxin of interest for treat-
major irritants capsaicin and dihydrocapsaicin. Specific nerves ment of cancer.
in the airway have been found to be capsaicin-sensitive, which
leads to the irritation and cough. Protein Synthesis Inhibition—The family Euphorbiaceae
contains several genera that are known to be very toxic. The castor
bean (Ricinus communis) is an ornamental plant that produces
Gastrointestinal System seeds that, if eaten by children or adults, causes no symptoms of
Direct Irritant Effects—Ingestion of a toxic plant can cause poisoning for several days after ingestion. Gradually, gastroen-
irritation of the gastrointestinal tract often resulting in nausea, teritis develops resulting in some loss of appetite, with nausea,
vomiting, and diarrhea (Table 26-4). Toxic quinolizidine alka- vomiting, and diarrhea and can be deadly. The toxic agents are
loids are found in buffalo beans. Ingestion by children causes two lectins found in the beans: ricin I and ricin I of which ricin
nausea, vomiting, dizziness, and abdominal discomfort. Also, II is more toxic. Ricin II is made up of an A-chain and a B-chain.
consumption by livestock of the mature plant with seeds has The B-chain is responsible for helping the A-chain get inside the
been reported to be fatal. cell. It binds to a terminal galactose residue on the cell mem-
Nuts from Aesculus hippocastanum (horse chestnut) and brane that then allows for the A-chain to be endocytosed. Once
Aesculus glabra (Ohio buckeye) contain a glucoside called inside, the A-chain inactivates the 60s ribosomal subunit of cells

TABLE 26-4 Selective plants causing gastrointestinal irritation.

‘Common Name Scientific Name - Toxic Part Toxin

Amaryllis Hippeastrum equestre Bulb Lycorine

Barberry Berberis vulgaris Root Protoberberine and other isoquinoline alkaloids

Boxwood Buxus sp. Leaves, stems Steroidal alkaloids

Buttercup Ranunculus sp. All parts Ranunculin, protoanemonin

Crown of thorns Euphorbia milii All parts Resiniferatoxin

Daffodil Narcissus All, especially bulb Lycorine, narcissin, phenanthridine alkaloids

English lvy Hedera helix All parts Hederin from hederagenin

Euonymus Euonymus sp. All parts Alkaloids

Hyacinth Hyacinthus orientalis Bulb Calcium oxalate, lycorine

Iris Iris Bulb Irritant resin

Mayapple Podophyllum peltatum Green fruit, roots Podophyllotoxin

Mistletoe Phoradendron flavescens Berries, other parts Phoratoxin

Pokeweed Phytolacca americana All parts Phytolaccatoxin, related triterpines

Purging nut Jatropha curcas Seeds Jatrophin (curcin) (toxalbumin)

Tung nut Aleurites fordii Nut Derivative of phorbol, saponins, toxalbumins

Wiseria Wisteria sinensis Pods Wistarine (glycoside)

satan eR AONORNRE NA
IPTRCCROEN ASE
386 UNIT 5 Toxic Agents

by catalytic depurination of an adenosine residue within the 28s

rRNA, thereby blocking protein synthesis.

Cardiovascular System
Cardioactive Glycosides— Various plants that contain car-
dioactive glycosides include Digitalis purpurea (Figure 26-3),
squill (Scilla maritima), which contains scillaren, lily of the
valley, milkweeds, and other species listed in Table 26-5.
The cardiac glycosides inhibit Na*, K*-ATPase.

Actions on Cardiac Nerves—Toxic alkaloids found in

Veratrum viride (American hellebore, Liliaceae), Veratrum
album (European hellebore), and Veratrum californicum cause
nausea, emesis, hypotension, and bradycardia on ingestion.
Veratrum album has been used for centuries to “slow and soften
the pulse.” The mixture of alkaloids includes protoveratrine,
veratramine, and jervine that affects the heart by causing a
repetitive response to a single stimulus resulting from prolon-
gation of the sodium current. Aconitum species causes not only
cardiac arrhythmias and hypotension, but ingestion causes
gastrointestinal upset and neurological symptoms. This works
by causing a prolonged sodium current with slowed repolar-
ization of cardiac muscle and nerve fibers. Grayanotoxins bind
to sodium channels in cardiac and muscle cells resulting in
increased sodium conductance.

Vasoactive Chemicals— Mistletoe produces a toxin (pho-

ratoxin and viscotoxin) that has marked effects on the cardio-
vascular system. It causes hypotension, vasoconstriction of the
FIGURE 26-3 Digitalis purpurea (common foxglove).
vessels in skin and skeletal muscle, and bradycardia resulting
from negative inotropic actions on heart muscle.
Ingestion of the fungus Claviceps purpurea (ergot), which Liver
grows on grains that are used for food, causes vasoconstric- Hepatocyte Damage—Ingestion of significant concentra-
tion. In extreme cases, the vasoconstriction was severe enough tions of pyrrolizidine alkaloids causes liver damage in the form
that gangrene would develop in the extremities. Abortion in of hepatic veno-occlusive disease associated with lipid peroxi-
pregnant women is also common after ingestion of ergot- dation. Cattle that graze on grass contaminated with Senecio
contaminated grains. have been found to develop hepatitis that can progress to death

TABLE 26-5 Selective plants causing cardiotoxicity.

‘CommonName _ ScientificName — Toxic Part Toxin

Azalea Rhodendron sp. All Grayanotoxins

Death camus Zigadenus All Zygadenine, veratrine

Foxglove Digitalis sp. Leaves, seeds Digitalis glycosides

Larkspur Delphenium ambiguum All Delphinine

Lily of the valley Convallaria majalis All Convallarin, convallamarin

Milkweed Asclepias sp. Leaves, stem (Hydroxycinnamoyl) desglucouzarin

Monkshood Aconitum sp. Leaves, roots, seeds Aconitine, aconine

Oleander Nerium oleander All Oleandrin, oleandrosine

ncacnyeennnntsnsensennenennuvinse anderencnseseevvwsvsininsnonnacevatsennoot annonce enanrinncnasennnwerennscsiannne ssnoeoiene tenance anes snnn enn etiaciettarmsncoosnutwseseunaannseeusvooinncmcennmtencnt
 CHAPTER 26 Toxic Effects of Plants and Animals 387

if allowed to continue grazing. Human deaths have also been to be carcinogenic in animals under natural feeding condi-
reported from consumption of contaminated wheat crops. tions. The commonest bladder tumors in cattle are epithelial
The liver damage caused by ingestion clinically appears to be and mesenchymal neoplasms. Ptaquiloside, a norsesquiter-
similar to cirrhosis and some hepatic tumors that can easily pene glucoside, is the known carcinogen present in the fern
be mistaken to be the source of the disease. ; and it has been found to alkylate adenines and guanines of
DNA. Bovine consumption of bracken fern has been shown to
Mushroom Toxins—Most nonedible mushrooms may cause significantly increase chromosomal aberrations.
mild discomfort and are not life threatening; however, repeated
ingestion of the false morel, Gyromitra esculenta, has been found Kidney Tubular Degeneration—Species of Xanthium
to cause hepatitis. Boiling generally inactivates the toxin gyromi- (cocklebur, Asteraceae) have been found to contain the toxin
trin. Most fatal poisonings related to wild mushrooms are from carboxyatractyloside, which causes microvascular hemorrhages
ingestion of different species within Amanita, Galerina, and in multiple organs. The toxin causes tubular degeneration and
Lepiota. Amanita phalloides (Figure 26-4) contains phalloidin necrosis in the kidney and centrilobular necrosis in the liver.
and amatoxins. Phalloidin is capable of binding actin in muscle Consumption of the mushroom species Cortinarius has been
cells; however, it is not readily absorbed during digestion, which found to cause acute kidney injury but different species vary
limits its harmful effects. The smaller a-, 8-, and y-amanitins widely in toxicity and, therefore, edibility.
are readily absorbed. Of the amatoxins, @-amanitin is the most
toxic as it inhibits protein synthesis in hepatocytes by binding to
RNA polymerase II. In addition to liver, intestinal mucosa and Blood and Bone Marrow
kidneys are also affected and serious clinical signs develop about Anticoagulants—Fungal infections in sweet clover (Melilotus
three days after ingestion. In cases of severe poisoning, a liver alba) have been found to produce dicumarol, a coumarin deriva-
transplant may be required. Amatoxin-q irreversibly inhibits tive that is a potent anticoagulant. Deaths in cattle have been
acetylcholinesterase. reported and are caused by hemorrhages.

Mycotoxins—Fumonisin toxins are produced by the fungus Bone Marrow Genotoxicity—Argemone (Papaveraceae), a
Fusarium that is known to grow on corn. Ingestion in humans species of poppy, produces sanguinarine, a benzophenanthridine
has been suggested to be associated with esophageal cancer. alkaloid that is known to intercalate DNA and have carcinogenic
potential.

Kidney and Bladder Cyanogens—Cyanogens are found in a wide variety of

Carcinogens—The bracken fern (P aquilinum), which is plants including the kernels of apples, cherries, and peaches.
extremely common worldwide, is the only higher plant known Metabolism of amygdalin in peaches releases hydrocyanic acid
that binds to the ferric ion in methemoglobin and cytochrome
oxidase system, which, if severe enough, results in cyanide
poisoning with death from asphyxiation.
Cassava produced from Manihot esculenta (Euphorbiaceae)
is a major food source for some regions of Africa. The raw root
contains a cyanogenic glucoside linamarin that must be removed
oh:
it
during processing of the root for human consumption.

Nervous System
Epileptiform Seizures—The common and scientific names
for selective plants that produce neurotoxins can be found in
Table 26-6. Within the family Apiaceae, which contains carrots,
the fleshy tubers of Cicuta maculata (water hemlock) produce
neurotoxic cicutoxin (a C17-polyacetylene). Consumption of
a single tuber can result in fatal poisoning, characterized by
tonic-clonic convulsions, owing to the cicutoxin binding to
GABA-gated chloride channels.
Members of the mint family (Labiatae) such as penny-
royal (Hedeoma), sage (Salvia), and hyssop (Hyssopus) are
well known for their essential oils containing monoterpenes.
Ingestion of these monoterpenes in concentrations much
higher than those used for flavoring can cause tonic-clonic
FIGURE 26-4 Amanita phalloides (death cap). convulsions.
388 UNIT 5 Toxic Agents

TABLE 26-6 Selective plants producing neurotoxicity.

ee —————————————— ee SS Eeeee error

Common Name Scientific Name Part Toxin Mechanism

Acacia tree Acacia willardiana Seeds Willardiine Glutamate receptor agonist

Alga Digenea simplex All Kainic acid Depolarization of glutamate-gated

Chondria armata Domoic acid channels

Betel nut Areca catechu Nut Guvacine GABA uptake inhibitor, anticonvulsant
Arecoline Stimulates muscarinic cholinoceptors; CNS
stimulation

Buckthorn; Coyotillo Karwinskia humboldtiana Seeds, leaves Tullidinol Demyelination of motor neurons leading
to paralysis

Chrysanthemum Chrysanthemum cinerarifolium Seeds Pyrethrins Stimulate sodium efflux from neurons in
insects

Deadly nightshade Atropa belladonna Berries Tropine alkaloid Blockade of muscarinic cholinoceptors

Fly agaric mushroom Amanita muscaria All Muscarine Stimulates muscarinic cholinoceptors; CNS
stimulation
Muscimol GABA receptor agonist

Rhododendron Rhododendron sp. Leaves Grayanotoxins , Stimulate sodium channel and membrane
depolarization

Ryania Ryania speciosa Stems Ryanodine Stimulates calcium channels and muscle
contraction

Poison nut tree Strychnos nux vomica All, especially seeds Strychnine Glycine receptor antagonist that produces
convulsions

Tobacco Nicotiana tabacum Leaves Nicotine Nicotinic cholinoceptor agonist (low

doses) or antagonist (high doses); CNS
stimulation

Excitatory Amino Acids—Red algae (Digenia simplex) Inhibition of catalase in peroxisomes has been proposed as the
under certain conditions can proliferate rapidly leading to mechanism of cell toxicity.
the notorious beach vacating “red tide” and producing kainic
acid. Kainic acid may be ingested by humans who eat filter- Parasympathetic Stimulation—Certain mushrooms ofthe
feeding mussels that have eaten red algae. Acute symptoms are genera Inocybe, Clitocybe, and Omphalatus contain significant
most notably gastrointestinal distress, headache, hemiparesis, amounts of muscarine, the principal neurotransmitter in the
confusion, and seizures. Severe exposure can result in severe
memory deficits and sensorimotor neuropathy.
The fungi Amanita muscaria (fly agaric, Figure 26-5) and
Amanita pantherian (panther agaric) produce the excitatory
amino acid ibotenic acid and its derivative muscimol that is
neurotoxic causing central nervous system depression, ataxia,
hysteria, and hallucinations. Myoclonic twitching and seizures
sometimes develop. Other genera of fungi have been marked
for their hallucinogenic actions, notably Psilocybe, which con-
tains psilocin and psilocybin.

Motor Neuron Demyelination— Karwinskia humboldtiana

produces anthracenones in its seeds. Both human and livestock
poisonings have been known to occur. Several days following
ingestion, ascending flaccid paralysis develops with demyelin-
ation of large motor neurons in the legs and eventually leads to
bulbar paralysis in fatal cases. In addition to neurotoxicity, the
anthracenones in Karwinskia, especially peroxisomicine A,,
cause lung atelectasis, emphysema, and massive liver necrosis. FIGURE 26-5 Amanita muscaria (fly agaric).
 CHAPTER 26 Toxic Effects of Plants and Animals 389

parasympathetic nervous system. Consumption of one of these Skeletal Muscle Damage—Certain species of Thermopsis
species results in extreme parasympathetic activation resulting produce seeds that contain quinolizidine alkaloids. Livestock
in urination, diarrhea, sweating, salivation, and lacrimation. grazing on Thermopsis montana (false lupine, mountain gold-
enbanner) develop locomotor depression and recumbency
Parasympathetic Block—Atropine, t-hyoscyamine, and due to areas of necrosis in skeletal muscle that have been found
scopolamine are belladonna alkaloids that can be found in on autopsy.
varying concentrations in several genera of Solanaceae, such
Consumption ofCassia obtusifolia (sicklepod, Leguminosae)
as Datura stramonium (jimson weed), Hyoscyamus niger (hen-
seeds by livestock causes a degenerative myopathy in cardiac
bane), Atropa belladonna (deadly nightshade, Figure 26-6), and
and skeletal muscle. Extracts of C. obtusifolia have been found
Duboisia myoporoides (pituri). These alkaloids all effectively
to inhibit NADH-oxidoreductase in bovine and swine mito-
block the muscarinic receptor, essentially turning off the para-
chondria in vitro.
sympathetic drive at the target organ. This explains why tachy-
cardia, dry mouth, dilated pupils, and decreased gastrointestinal
motility all occur on ingestion of these toxins. Bone and Tissue Calcification
Sensory Neuron Block—Capsaicin found in C. annuum Bone and Soft Tissue—Consumption of Solanum mala-
(sweet pepper) and C. frutescens (red pepper) causes a burn- coxylon (Solanaceae) by sheep and cows can cause a marked
ing sensation on vanilloid-type (VR1) sensory receptors. It decrease in bone calcium and calcification of the entire vascu-
also desensitizes the transient potential vanilloid 1 receptor lar system due to the presence of a water-soluble vitamin D-
(TRPV1) of sensory endings of C-fiber nociceptors to stimuli, like substance. In severe cases other organs can also be affected
a property that has therapeutic use in treating chronic pain. such as the lungs, joint cartilage, and kidney.
Capsaisin also can relax ileal smooth muscle.

Reproduction and Teratogenesis

Skeletal Muscle and Neuromuscular Abortifacients—Besides its actions on the nervous system,
Junction swainsonine, the active alkaloid in the legumes Astragalus and
Neuromuscular Junction—Anabasine, an isomer of nico- Oxytropus, also causes abortions in pregnant livestock that
tine, is present in Nicotiana glauca (tree tobacco, Solanaceae) accidentally ingest these weeds. Foliage and seeds of Leucaena
and produces prolonged depolarization of the junction after leucocephala, Leucaena glauca, and Mimosa pudica contain a
a period of excessive stimulation. Consumption of N. glauca toxic amino acid, mimosine, which on ingestion by cattle leads
leaves can result in flexor muscle spasm and gastrointestinal to uncoordinated gait, goiter, and reproductive disturbances
irritation, followed by severe, generalized weakness, and respi- including infertility and fetal death.
ratory compromise. Curare, which is used as a poison placed
on the tips of arrows, is a potent neuromuscular blocking agent Teratogens—lIngestion of V. californicum (California false
that is obtained from Strychnos toxifera and Chondrodendron hellebore, Liliaceae) by pregnant sheep is known to cause
tomentosum. Anabaena flosaquae, a species of alga, can pro- malformations in its offspring that can include cyclopia, exen-
duce under the right conditions a neurotoxin anatoxin A that, cephaly, and microphthalmia. The toxic alkaloid called jervine
when ingested by animals that drink pond water with the alga causes teratogenesis by blocking cholesterol synthesis that,
present, depolarizes and blocks the animal's nicotinic and among other things, prevents a proper response of fetal target
muscarinic acetylcholine receptors, which can cause death tissue to the sonic hedgehog gene (Shh). Shh has an important
from respiratory arrest within minutes to hours. role in proper developmental patterning of head and brain, and
blocking cholesterol synthesis has been shown experimentally
to cause a loss of midline facial structures.

CLINICAL STUDY OF PLANT POISONS

Old herbal remedies are a ripe field of study for many of their
effects that can be beneficial yet toxic at high enough concen-
trations. A goal for new research is to elucidate the mechanism
of action so that treatments can be tailored to the individual
needs and toxic effects can be avoided or interactions with con-
ventional drugs can be minimized. Recent research has shown
that Uzara root extract reduced chloride secretion by the gut
specifically by inhibiting Na*, K*-ATPase. This effect was seen
even in the presence of cholera toxin that causes potent diar-
FIGURE 26-6 Atropa belladonna (deadly nightshade). rhea by increasing chloride secretion in the gut. Interestingly,
390 UNIT5 Toxic Agents

anemonin, which is the active skin irritant produced by species can be pinpointed. Conversely, animal toxins must be studied
ofRanunculus (buttercup), has been found to show potent anti- in the context of the entire venom or poison that typically is
inflammation effects under certain conditions. The compound very complex and contains many individual toxic compounds
was found to reduce nitric oxide production that resulted in a and very large proteins that essentially work together to cause
lessened inflammatory response to inflammatory stimuli. their effects.

INTRODUCTION TO ANIMAL VENOMS PROPERTIES OF ANIMAL TOXINS

Venomous animals are capable of producing a poison in a Venoms are very complex, containing polypeptides, high-
highly developed exocrine gland or group of cells and can deliver and low-molecular-weight proteins, amines, lipids, steroids,
their toxin during a biting or stinging act. Conversely, poisonous amino-polysaccharides, quinones, glucosides, and free amino
animals have no specific mechanism or structure for the deliv- acids, as well as serotonin, histamine, and other substances.
ery of their poisons, and poisoning usually takes place through Some venoms are known to consist of more than 25 proteins.
ingestion. Animal venom may play a role in offense (as in the The venom is a source of peptides and proteins that act on
capture and digestion of food), in the animal's defense (as in pro- myriad exogenous targets such as ion channels, receptors, and
tection against predators or aggressors), or in both functions. enzymes within cells and on cell membranes.
Venoms used in an offensive posture are generally associated Novel instrument developments have permitted the greater
with the oral pole, as in the snakes and spiders, while those used application of mass spectrometry, coupled with various sepa-
in a defensive function are usually associated with the aboral pole ration technologies, to tease out the complexity of natural
or with spines, as in the stingrays and scorpion fishes. venoms, thereby identifying the peptide and protein compo-
It is worth noting that animal and plant toxins tend to differ nents of venoms. Figure 26-7 demonstrates the application of
significantly in their chemical complexity, yet both are capable gel filtration and high-pressure liquid chromatography, as cone
of causing massive harm. Plant toxins tend to be smaller com- snail venom was fractionated into numerous peptides with
pounds or proteins and often times a single offending substance varying activities. Similar fractionations have been performed

Head swinging
Kicking on back, scratching
Circular motion
* Back legs drag Depressed activity
* Sleeper/climber Comatose t
Uncoordinated Paralysis t
Twisted jumping Depressed activity
Paralysis Trembling
Dragging t

alles Depressed, and then hyperactivity t

1
{
i] Normal
Scratching, convulsion t
1
I
!
{
*Convulsion, bleeding t
| Convulsion t
i
1 Normal
i
\
t
1
1
1
(
S fo) L '
i
i
t
Absorbance

o
=

4 8 12 16 20 24
Time (min)

FIGURE 26-7 Multiple biologically active components were obtained from Conus geographus venom by first subjecting the venom
to gel filtration on Sephadex G-25 into four fractions and then separation of fraction B (which contains the a-conotoxins) by high-
pressure liquid chromatography on a VYDAC C18 column using a trifluoroacetic acid-acetonitrile gradient. Various peak
fractions were then
injected intracerebrally into mice and different responses were noted. (t) The fraction was lethal in at least one injected animal. (Reproduced
with
permission from Olivera BM, Rivier J, Clark C, et al.: Diversity of Conus Neuropeptides. Science, 1990 Jul 20;249(4966):257-263.)
 CHAPTER 26 Toxic Effects of Plants and Animals 391

on many other types of venom to identify the individual com- TABLE 26-7 Location of some medically important
ponents. Unfortunately, studying the chemistry, pharmacology, scorpions.
and toxicology of venoms requires isolating and dismantling the
venoms and losing the synergy among multiple components. Genus Distribution
The bioavailability of a venom is determined by its com- Androctonus North Africa, Middle East, Turkey
position, molecular size, amount or concentration gradient,
solubility, degree of ionization, and the rate of blood flow Buthus France and Spain to Middle East and North Africa,
Mongolia, China
into that tissue, as well as the properties of the engulfing sur-
face itself. The venom can be absorbed by active or passive Buthotus Africa, Middle East, Central Asia
transport, facilitated diffusion, or pinocytosis, among other
Centruroides North, Central, South America
physiologic mechanisms. Besides the bloodstream, the lymph
circulation not only carries surplus interstitial fluid produced Heterometrus Central and Southeast Asia
by the venom but also transports larger molecular components
Leiurus North Africa, Middle East, Turkey
and other particulates back to the bloodstream. The site of
action and metabolism of venom is dependent on its diffusion Mesobuthus Turkey, India
and partitioning along the gradient between the plasma and
Parabuthus Southern Africa
the tissues where the components are deposited.
Tityus Central and South America

ARTHROPODS
Arthropods include the arachnids (scorpions, spiders, whip The symptoms and signs of scorpion envenomation differ
scorpions, solpugids, mites, and ticks), the myriapods (centipedes considerably depending on the species. Common offenders
and millipedes), the insects (water bugs, assassin bugs, and wheel are members of the family Vejovidae and their sting gives rise
bugs), beetles (blister beetles), Lepidoptera (butterflies, moths, to localized pain, swelling, tenderness, and mild paresthesia.
and caterpillars), and Hymenoptera (ants, bees, and wasps). Systemic reactions are rare, although weakness, fever, and
The number of deaths from arthropod stings and bites is muscle fasciculations have been reported.
unknown. Among the disease states that were confused with Envenomations by some members of the genus Centruroides
spider or arthropod bites or stings were erythema chronicum are clinically the most important. In children, their sting may
migrans, erythema nodosum, periarteritis nodosum, pyro- produce initial pain, although some children do not complain
derma gangrenosum, kerion cell-mediated response to a fun- of pain and are unaware of the injury. The child becomes tense
gus, Stevens—Johnson syndrome, toxic epidermal necrolysis, and restless and shows abnormal and random head and neck
herpes simplex, and purpura fulminans. Any arthropod may movements. Often the child will display roving eye movements.
bite or sting and not eject venom. Centruroides sculpturatus stings display visual signs, including
nystagmus roving eye and oculogyric movements. Tachycardia,
hypertension, and respiratory rates are increased. Fasciculations
ARACHNIDA may be seen and the child may display ataxia. The respiratory
distress may proceed to respiratory paralysis. As opposed to
Scorpions children, almost all adults complain of immediate pain after the
Of the more than 1000 species of scorpions, the stings of more sting. They become tense and anxious and develop tachycardia,
than 75 can be considered of sufficient importance to warrant hypertension, and increased respirations. Most adults become
medical attention. Some of the more important scorpions are asymptomatic within 12h.
noted along with their location in Table 26-7.
Many scorpion venoms contain low-molecular-weight pro-
teins, peptides, amino acids, nucleotides, and salts, among other Spiders
components. Short-chain toxins appear to affect potassium or Of the 30 000 or so species, at least 200 have been impli-
chloride channels, while the long-chain toxins affect mainly the cated in significant bites on humans. Spiders are predaceous,
sodium channels. The neurotoxic fractions are generally classi- polyphagous arachnids that generally feed on insects or other
fied on the basis of their molecular size; the short-chain toxins arthropods. Table 26-8 provides a short list of spiders with
are composed of 20 to 40 amino acid residues with 3 or 4 disul- their associated toxins and the targets of their toxins.
fide bonds and appear to affect potassium or chloride channels, All spiders except the Uloboridae family possess a venom
while the long-chain toxins have 58 to 76 amino acid residues apparatus that produces neurotoxins designed to paralyze or kill
(6500-8500 Da) with four disulfide bonds and affect mainly the prey. Spider venoms are complex mixtures of low-molecular-
sodium channels. The toxins can selectively bind to a specific weight components, including inorganic ions and salts, free
channel of excitable cells, thus impairing the initial depolariza- acids, glucose-free amino acids, biogenic amines and neu-
tion of the action potential in the nerve and muscle that results in rotransmitters, and polypeptide toxins. The acylpolyamines are
their neurotoxicity. voltage-dependent open-channel blockers (sodium, calcium,
392 UNIT 5 Toxic Agents

TABLE 26-8 Some significant spiders, their toxins,

and the targets of the toxins.

Spider Peptide Target

Acanthoscurria gomesiana Gomesin PLM

Agelenopsis aperta w-Afal-IVA Gaze

yt-Afatoxin 1-6 Nat

Grammostola spatula HaTx1,2 Ke

GsMTx2,4 MS
GSTxSIA Gat

Hadronyche versuta w-ACTX-Hv1a Gaze

w-ACTX-Hv2a Ca**
6-ACTX-Hv1a Nat

Heteroscodra maculate HmTx1,2 K*

Ornithoctonus huwena Huwentoxin | Car FIGURE 26-8 Latrodectus mactans (female black widow
Huwentoxin lV Na* spider).

Psalmopoeus cambridgei PcTx1 ASIC

Phrixotrichus auratus PaTx1,2 Kt stimulate massive release of neurotransmitters after binding to

Thrixopelma pruriens ProTxl,ll Na* specific neuronal receptors.
Additional species, their toxins, and their targets may be obtained from the article by
c-Latrotoxin is the most studied protein that is toxic only
Corzo G, Escoubas P: Pharmacologically active spider peptide toxins. Cell Mol Life Sci to vertebrates and not to insects or crustaceans. It is a pre-
2003;60:2409-2426. synaptic toxin that is said to exert its toxic effects on the
PLM, phospholipid membranes; Ca’*, K*, and Na*, calcium, potassium, and sodium ion
channels; MS, mechanosensitive ion channels; ASIC, acid-sensing ion channels. vertebrate central nervous system depolarizing neurons by
increasing intracellular [Ca**] and by stimulating exocytosis
of neurotransmitters from nerve terminals. a-Latrotoxin
and its mutants are versatile tools for the study of exocyto-
and potassium channels) and/or blockers of the ion channel sis. In particular, studies with this toxin have helped con-
associated with glutamate receptors. They also act on nicotinic firm the vesicular hypothesis of transmitter release, establish
acetylcholine receptors. the requirement of calcium ion for endocytosis, character-
ize individual neurotransmitter sites in the central nervous
Agelenopsis Species (American Funnel Web Spiders)— system, and identify two families of important neuronal cell
The American funnel web spider (Agelenopsis aperta) con- surface receptors.
tains three classes of agatoxins that target ion channels. The Bites by the black widow are described as sharp and
Q-agatoxins appear to be use-dependent, noncompetitive pinprick-like, followed by a dull, occasionally numbing pain
antagonists of the glutamate receptor channels. The p-agatoxins in the affected extremity and by pain and cramps in one or
cause increased spontaneous release of neurotransmitter from several of the large muscle masses. Muscle fasciculations
presynaptic terminals and repetitive action potentials in motor frequently can be seen, sweating is common, and lymphadeni-
neurons. In addition, the |t-agatoxins are specific for insect tis is frequently observed. Severe paroxysmal muscle cramps
sodium channels. The w-amatoxins are a structurally diverse may occur and hypertension is a common finding, particularly
group of peptides that are selective for voltage-activated cal- in the elderly after moderate to severe envenomations. Blood
cium channels. The four w-amatoxins can be distinguished by studies are usually normal.
sequence similarity and their spectrum ofaction against insect
and vertebrate calcium channels. The action of the a-agatoxins Loxosceles Species (Brown or Violin Spiders)—These
is synergized by the [t-agatoxins causing channels to open at primitive spiders are variously known in North America as
the normal resting potentials. the fiddle-back spider or the brown recluse (Figure 26-9). The
venom of Loxosceles spiders appears to contain phospholipase,
Latrodectus Species (Widow Spiders)—Found through- protease, esterase, collagenase, hyaluronidase, deoxyribonu-
out the world in all continents with temperate or tropical cli- clease, ribonuclease, dipeptides, dermonecrosis factors, and
mates, these spiders are commonly known as the black widow, sphingomyelinase D. The venom has coagulation and vasocon-
brown widow, or red-legged spider (Figure 26-8). The latro- striction properties and it causes selective vascular endothelial
toxins, a family of high-molecular-weight proteins that are damage. There are adhesions of neutrophils to the capillary
found in Latrodectus venoms, target different classes of animals wall with sequestration and activation of passing neutrophils
including vertebrates, insects, and crustaceans. All latrotoxins by the perturbed endothelial cells.
 CHAPTER 26 Toxic Effects of Plants and Animals 393

including severe local pain, nausea and vomiting, headache,

chest discomfort, severe pruritus, and shock.

Theraphosidae Species (Tarantulas)— True tarantulas are

members of the family Theraphosidae. Tarantulas are predators
and they feed on various vertebrate and invertebrate preys that
are captured after envenomation with venoms that act rapidly
and irreversibly on the central and peripheral nervous systems.
In humans, reported bites elicit mild to severe local pain, strong
itching, and tenderness that may last for several hours. Edema,
erythema, joint stiffness, swollen limbs, burning feelings, and
cramps are common.
Theraphosid spiders contain several toxins that are being
FIGURE 26-9 Loxosceles reclusa (male brown recluse spider) evaluated for development as antiarrhythmic or as antinoci-
with the violin pattern on the dorsal cephalothorax. ceptive drugs. In particular, Grammostola mechanotoxin 4
from Grammostola spatulata has considerable promise as an
antiarrhythmic. Protoxin I and I from Thrixopelma pruriens
have promise as analgesics because they inhibit the tetrodo-
The bite of this spider produces about the same degree of toxin-resistant sodium channels.
pain as does the sting of an ant, but sometimes the patient
may be unaware of the bite. Pruritis over the area often occurs
with reddening and elevated skin temperature at the lesion. Ticks
With significant envenomations, hemorrhages may develop Tick paralysis is caused by the saliva of certain ticks of the
throughout the area, lymphadenopathy is common, and families Ixodidae, Argasidae, and Nuttalliellidae. Ticks are
necrosis of the surrounding tissue may be visualized. Systemic known to transmit the organisms causing Lyme disease, Rocky
symptoms and signs include fever, malaise, stomach cramps, Mountain spotted fever, babesiosis, leptospirosis, Q fever,
nausea and vomiting, jaundice, spleen enlargement, hemoly- ehrlichiosis, typhus, tick-borne encephalitis, and others.
sis, hematuria, and thrombocytopenia. Fatal cases, while rare, Tick saliva contains a number of active constituents. For
usually are preceded by intravascular hemolysis, hemolytic example, saliva from Ixodes scapularis contains apyrase (ATP-
anemia, thrombocytopenia, hemoglobinuria, and renal failure. diphosphohydrolase), which hydrolyzes ADP that is released at
the bite site thereby inhibiting ADP-induced platelet aggregation;
Steatoda Species—These spiders are variously known as kininase (ACE-like protein or angiotensin-converting enzyme-
the false black widow, combfooted, cobweb, or cupboard spi- like protein), which hydrolyzes circulating kinins and reduces
ders. The venom of Steatoda paykulliana stimulates the release the host inflammatory response; glutathione peroxidase; serine
of transmitter substances similar to Latrodectus. The venom is protease inhibitors, which inhibit coagulation enzymes; an anti-
said to form ionic channels that are permeable for bivalent and complement protein that inhibits an enzyme in the alternative
monovalent cations, and the duration of time in the open state pathway for complement; an amine-binding protein that binds
depends on the membrane potential. S. paykulliana venom serotonin, histamine, and other biogenic amines.
induces strong motor unrest, clonic cramps, exhaustion, As tick bites are often not felt, the first evidence of enven-
ataxia, and then paralysis. omation may not appear until several days later, when small
macules 3 to 4 mm in diameter develop that are surrounded by
Cheiracanthium Species (Running Spiders)— erythema and swelling. The patient often complains of difficulty
Cheiracanthium punctorium, C. inclusum, C. mildei, C. diver- with gait, followed by paresis and eventually locomotor paresis
sum, and C. japonicum are often implicated in envenomations. and paralysis. Problems in speech and respiration may ensue and
Cheiracanthium tends to be tenacious and sometimes must lead to respiratory paralysis if the tick is not removed. The saliva
be removed from the bite area. For that reason there is a high of Ixodes holocyclus has yielded a peptide holocyclotoxin-1 that
degree of identification following the bite of these spiders. The may cause paralysis.
most toxic venom fraction is said to be a protein of 60 kDa, and
the venom is high in norepinephrine and serotonin.
CHILOPODA (CENTIPEDES)
The patient usually describes the bite as sharp and painful.
A reddened wheal with a hyperemic border develops. Small In the United States, the prevalent biting genus is a Scolopendra
petechiae may appear near the center of the wheal. Skin tem- species. The venom is concentrated within the intracellular
perature over the lesion is often elevated, but body temperature granules, discharged into vacuoles of the cytoplasm of the
is usually normal. Lymphadenitis and lymphadenopathy may secretory cells, and moved by exocytosis into the lumen of the
develop. C. japonicum produces more severe manifestations, gland; from thence ducts carry the venom to the jaws.
394 UNIT 5 Toxic Agents

Centipede venoms contain high-molecular-weight proteins, Apidae (Bees)—This family includes the bumble bees, hon-
proteinases, esterases, 5-hydroxytryptamine, histamine, lipids, eybees, carpenter bees, and yellow jackets. The commonest
and polysaccharides. The bite produces sharp pain, immediate stinging bees are Apis mellifera and the Africanized bee, Apis
bleeding, redness, and swelling. Localized tissue changes and mellifera adansonii, and the incidence of Hymenoptera poison-
necrosis have been reported, and severe envenomations may ings is increasing.
cause nausea and vomiting, changes in heart rate, vertigo, and The venom contains biologically active peptides, such as
headache. melittin, apamine, mast cell-degranulating peptide, and others,
as well as phospholipases A, and B, hyaluronidase, histamine,
dopamine, monosaccharides, and lipids. Melittin tetramers
DIPLOPODA (MILLIPEDES)
cause a breakdown of the resting potential and rapid depolariza-
The repellent secretions expelled from the sides of their bodies tion of nociceptors, which induces pain. Apamine is a blocker of
contain a toxin of benzoquinone derivatives plus a variety of calcium-dependent potassium channels and is thought to be the
complex substances such as iodine and hydrocyanic acid, “lethal factor”.
which the animal makes use of to produce hydrogen cyanide. Bee stings typically produce immediate, sharp or burning
Some species can spray these defensive secretions. The lesions pain, slight local erythema, and edema followed by itching. It
produced by millipedes consist of a burning or prickling is said that 50 stings can be serious and lead to respiratory dys-
sensation and development of a yellowish or brown-purple function, intravascular hemolysis, hypertension, myocardial
lesion; subsequently, a blister containing serosanguinous fluid damage, hepatic changes, shock, and renal failure. With 100 or
forms, which may rupture. Eye contact can cause acute con- more stings, death can occur.
junctivitis, periorbital edema, keratosis, and much pain; such
an injury must be treated immediately. Vespidae (Wasps)—This family includes wasps and hornets.
These venoms contain a high content of peptides, which
include mastoparan in wasps and hornets and crabolin from
INSECTA hornet venom. These peptides release histamine from mast
cells. Wasp kinins cause immediate pain, vasodilation, and
Heteroptera (True Bugs)
increased vascular permeability leading to edema. These ven-
The clinically most important of the true bugs are the oms also contain phospholipases and hyaluronidases, which
Reduviidae (the reduviids): the kissing bug, assassin bug, wheel contribute to the breakdown of membranes and connective
bug, or cone-nose bug of the genus Triatoma. The venom of these tissue to facilitate diffusion of the venom.
bugs appears to have apyrase activity and to lack 5-nucleotidase,
inorganic pyrophosphatase, phosphatase, and adenylate kinase
activities, but it is fairly rich in protease properties. It inhibits Lepidoptera (Caterpillars, Moths,
collagen-induced platelet aggregation. Three peptides isolated
from the saliva are calcium channel inhibitors. The bites of
and Butterflies)
Triatoma species are painful and give rise to erythema, pruri- The urticating hairs, or setae, of caterpillars are effective defen-
tus, increased temperature in the bitten part, localized swelling, sive weapons that protect some species from predators. The toxic
and—in those allergic to the saliva—systemic reactions such as material found in the venom glands contains aristolochic acids,
nausea and vomiting and angioedema. cardenolides, kallikrein, histamine and a fibrinolytic peptide.
The spicules of Thaumetopoea pityocampa contain a toxin that
is a strong dermal irritant and highly allergenic peptide. In some
Hymenoptera (Ants, Bees, Wasps, parts of the world the stings of several species of Lepidoptera give
and Hornets) rise to a bleeding diathesis, often severe and sometimes fatal.
Formicidae (Ants)— Most ants have stings, but those that lack
them can spray a defensive secretion from the tip of the gaster,
MOLLUSCA (CONE SNAILS)
which is often placed in the wound of the bite. Clinically impor-
tant stinging ants are the harvesting ants (Pagonomyrmex), fire Human interest in this group of mollusks has been due to the
ants (Solenopsis), and little fire ants (Ochetomyrmex). beautiful patterns on their shells. Cone snails have a venom
The venoms of the ants vary considerably. Formicinae ant duct for synthesis and storage of venom and hollow harpoon-
venom contains about 60% formic acid. Fire ant venoms are like teeth for injection of the venom. There are probably
rich in alkaloids. The sting of the fire ant gives rise to a painful over 100 different venom components per species known as
burning sensation, after which a wheal and localized erythema conotoxins. Molecular targets include G-protein-coupled
develop, forming a vesicle that becomes purulent and turns receptors, neuromuscular transporters, and ligand- or voltage-
into a pustle. The pustule may then break down, become a gated ion channels. Some components have enzymatic activity.
crust, or become a fibrotic nodule. In multiple stings there may Figure 26-10 provides an overview of peptidic Conus venom
be nausea, vomiting, vertigo, increased perspiration, respira- components, indicating gene superfamilies, disulfide bond
tory difficulties, cyanosis, coma, and even death. characteristics, and general targets.
 CHAPTER 26 Toxic Effects of Plants and Animals 395

z
2
_ Conopeptides

Non-disulfide-rich. Disulfide-rich

- Single S- _

“i
nn amily Contulakin Conopressin |Contryphan|

a Neurotensin NMDA Rfamide Vasopressin

4
oct receptor receptor receptor (?) receptor
(?) |

Superfamily

S-S motif CECeEEe

etre
CG-G-G-CE CcCc-C-C CC-C-C-C-C
4
CC-CC-C-C-C-C-C-C vit elke te lbs CCcc-cC-C-C

General Na K Ca Na nACh K nACh nACh K 5-HT (2) NE (2) K

target channel ||channel ||channel |}channel |}channel ||channel ||receptor ||receptor|| channel receptor : transporter ‘ Channels

Conotoxin \ I ie | | I
family WO te Ww Mm NY KM a aA KA oO xX

FIGURE 26-10 Organizational diagram for Conus peptides, indicating gene superfamilies, disulfide patterns, and known
pharmacologic targets. Only the superfamilies of the disulfide-rich peptides are shown. (Reproduced with permission from Terlau H, Olivera BM:
Conus venoms: A rich source of novel ion channel-targeted peptides. Physiol Rev, 2004 Jan;84(1):41-68.)

Cone snails could be called sophisticated practitioners of the base of the larger teeth of the lower jaw. The venom is then
combination drug therapy. After injection, multiple conopep- drawn up along grooves in the teeth by capillary action. The
tides act synergistically to affect the targeted prey. The term venom of this lizard has serotonin, amine oxidase, phospholipase
toxin cabal has been applied to this coordinated action of the A, a bradykinin-releasing substance, helodermin, gilatoxin, and
conopeptide mixture. The fish-hunting species Conus purpura- low-proteolytic as well as high-hyaluronidase activities. The clin-
scens apparently has two distinct cabals whose effects differ in ical presentation of a helodermatid bite can include pain, edema,
time and space. The “lightning-strike cabal” causes immediate hypotension, nausea, vomiting, weakness, and diaphoresis. No
immobilization of the injected prey because various venom antivenin is commercially available.
components inhibit voltage-gated sodium channel inactivation
and block potassium channels, resulting in massive depolariza-
tion of axons in the vicinity of the injection site and a tetanic
Snakes
state. The second physiologic cabal, the “motor cabal,’ acts General Information and Classification—Venomous
more slowly as conotoxins must be distributed throughout the snakes primarily belong to the following families: Viperidae
body of the prey. The overall result is total inhibition of neuro- (vipers), Elapidae, Atractaspididae, and Colubridae. Overall
muscular transmission. Various conopeptides inhibit presyn- the Colubridae are considered the largest venomous family,
aptic calcium channels that control neurotransmitter release, and are composed of nearly 60% ofall snakes.
the postsynaptic neuromuscular nicotinic receptors, and the
sodium channels involved in the muscle action potential. Snake Venoms—These venoms are complex mixtures:
proteins and peptides, consisting of both enzymatic and non-
enzymatic compounds. Snake venoms also contain inorganic
REPTILES cations such as sodium, calcium, potassium, magnesium, and
small amounts of zinc, iron, cobalt, manganese, and nickel.
Lizards The metals in snake venoms are likely catalysts for metal-based
The Gila monster (Heloderma suspectum) and the beaded liz- enzymatic reactions. For example, in the case of some elapid
ards (Heloderma horridum) are far less dangerous than is gener- venoms, zinc ions appear to be necessary for anticholinesterase
ally believed. Their venom is transferred from venom glands in activity, and calcium may play a role in the activation of phos-
the lower jaw through ducts that discharge their contents near pholipase A and the direct lytic factor. Some proteases appear
 396 UNIT 5 Toxic Agents

to be metalloproteins. Some snake venoms also contain carbo- ptosis; to ophthalmoplegia, flaccid facial muscle paralysis, and
hydrates (glycoproteins), lipids, and biogenic amines, such as inability to swallow; to paralysis of larger muscle groups; and
histamine, serotonin, and neurotransmitters (catecholamines finally to paralysis of respiratory muscles and death by asphyxi-
and acetylcholine) in addition to positively charged metal ation. Coagulants may have an initial procoagulant action that
ions. The complexity of snake venom components is illustrated uses up clotting factors leading to bleeding. Coagulants may
nicely in Figure 26-11. directly inhibit normal clotting at several places in the clotting
Actions of snake venoms can be said to be broad rang- cascade or via inhibition of platelet aggregation. In addition,
ing in several areas. A simplistic approach would group toxin some venom components may damage the endothelial lining
components as neurotoxins, coagulants, hemorrhagins, hemo- of blood vessels leading to hemorrhage. Bite victims may
lytics, myotoxins, cytotoxins, and nephrotoxins. Neurotoxins show bleeding from nose or gums, from the bite site, and in
produce neuromuscular paralysis ranging from dizziness to saliva, urine, and stools. Myotoxins can directly impact muscle

-Acetylcholinesterases (AChE)
-Aminotransferases
-ADPases and ATPases
-3-glucosaminidase
-CVF
-Catalases
-Phosphoesterases -Phosphomonoesterases
-Phosphodiesterases
-Enzymes
-PLA, (synovial and pancreatic-type)
-Hyaluronidases
-L-amino acid oxidases (LAO)
-NAD nucleosidases
-Aspartic/Thiol proteases (traces)
-Proteases {-Metalloprotease
-Serinoproteases
--C protein activators
Proteins --Growth factors (NGF, VEGF)
--Inhibitors of the prothrombinase complex formation
--Lectins (C-type lectins, galactose-binding lectins)
-Nonenzymatic
--Precursors of bioactive peptides
--von Willebrand factor-binding proteins
--Platelet GPlb-binding proteins
--CRISPs
-Enzymatic inhibitors
--Toxic (cytotoxic, cardiotoxic, myotoxic, neurotoxic)
--Disintegrins ¢ -RGD
-Non-RGD
--Natriuretic
-Peptides
--Waglerins
--Bradykinin potentiators {-ACE inhibitors (ACEI)
--Prokinecitin-like
--CRISPs
-Biogenic amines -Serotonin, histamine
-Amino acids
Organic compounds with
-Carbohydrates
low molecular mass
-Citrate
-Nucleosides
-Calcium
-Cobalt
-Cooper
-lron
Inorganic -Phosphorus
compounds -Potassium
-Magnesium
-Manganese
-Sodium
-Zinc

FIGURE 26-11 Components of snake venoms. ACE, angiotensin-converting enzyme; CRISP, cysteine-rich secretory protein; CVF, cobra
venom factor-like proteins; LAO, t-amino acid oxidase; PLA,, phospholipase A2; RGD, arginine—glyc
ine—aspartate. (Reproduced with permission
from Ramos OHP, Selistre-de-Araujo HS: Snake venom metalloproteases—structure and function
of catalytic and disintegrin domains. Comp
Biochem Physiol C Toxicol Pharmacol. 2006 Mar-Apr;142(3-4):328-346.)
 CHAPTER 26 Toxic Effects of Plants and Animals 397

contraction leading to paralysis or cause rhabdomyolysis or the ligand receptors. The small basic polypeptide myotoxins are
breakdown of skeletal muscle. Myoglobinuria, or a dark brown widely distributed in Crotalus snake venoms. The specific agent
urine, and hyperkalemia may be noted. Cytotoxic agents have crotamine from Crotalus durissus terrificus venom induces
proteolytic or necrotic properties leading to the breakdown skeletal muscle spasms and paralysis by changing the inactiva-
of tissue. Typical signs include massive swelling, pain, dis- tion process of sodium channels leading to depolarization of
coloration, blistering, bruising, and wound weeping. Finally, the neuromuscular junction.
nephrotoxins can cause direct damage to kidney structures
leading to bleeding, damage to several parts of the nephron, Toxicology—In general, the venoms of rattlesnakes and other
tissue oxygen deprivation, and renal failure. New World crotalids produce alterations in the resistances and
often in the integrity of blood vessels, changes in blood cells
Enzymes—At least 26 different enzymes have been isolated and blood coagulation mechanisms, direct or indirect changes
from snake venoms. No single snake venom contains all in cardiac and pulmonary dynamics, and—with crotalids such
26 enzymes and some important snake venom enzymes as C. durrissus terrificus and C. scutulatus—serious alterations
are shown in Figure 26-11. Proteolytic enzymes that cata- in the nervous system and changes in respiration. In humans,
lyze the breakdown of tissue proteins and peptides include the course of the poisoning is determined by the kind and
peptide hydrolases, proteases, endopeptidases, peptidases, amount of venom injected; the site where it is deposited; the
and proteinases. Collagenase is a specific kind of proteinase general health, size, and age of the patient; the kind of treat-
that digests collagen. This activity has been demonstrated in ment; and those pharmacodynamic principles noted earlier in
the venoms of a number of species of crotalids and viperids. this chapter. Death in humans may occur within less than 1 h
Hyaluronidase cleaves internal glycoside bonds in certain acid or after several days, with most deaths occurring between 18
mucopolysaccharides resulting in a decrease in the viscosity of and 32 h. Hypotension or shock is the major therapeutic prob-
connective tissues. The breakdown in the hyaluronic barrier lem in North American crotalid bites.
allows other fractions of venom to penetrate the tissues, caus-
ing hyaluronidase to be called “spreading factor.” Fibrin(ogen) Snakebite Treatment—The treatment of bites by venom-
olytic enzymes break down fibrin-rich clots and help to pre- ous snakes is now so highly specialized that almost every
vent further clot formation. An exciting development from envenomation requires specific recommendations. However,
the research on these enzymes is that one specific recombinant three general principles for every bite should be kept in mind:
fibrinolytic enzyme derived from fibrolase called alfimeprase is (1) snake venom poisoning is a medical emergency requir-
progressing through clinical trials for the treatment of periph- ing immediate attention and the exercise of considerable
eral arterial occlusions. Phosphodiesterase has been found in judgment; (2) the venom is a complex mixture of substances
the venoms of all families of poisonous snakes. It acts as an exo- of which the proteins contribute the major deleterious prop-
nucleotidase, attacking DNA and RNA. Acetylcholinesterase, erties, and the only adequate antidote is the use of specific or
found in the cobra, catalyzes the hydrolysis of acetylcholine to polyspecific antivenom; and (3) not every bite by a venomous
choline and acetic acid thereby facilitating tetanic paralysis and snake ends in an envenomation. Venom may not be injected.
capture of prey. Phospholipase A, is widely distributed in snake In almost 1000 cases of crotalid bites, 24% did not end in a
venoms, and this enzyme family interacts with other venom poisoning. The incidence with the bites of cobras and perhaps
components often resulting in synergistic reactions. other elapids is probably higher (see www.toxinology.com).
The snake venom metalloproteinases (SVMP) are enzymes
that disrupt the hemostatic system that blocks the function of
integrin receptors, a function that could alleviate a variety of ANTIVENOM
pathological conditions such as inflammation, tumor angio-
genesis and metastasis, and thrombosis. SVMPs degrade Antivenoms have been produced against most medi-
proteins such as laminin, fibronectin, type IV collagen, and cally important snake, spider, scorpion, and marine toxins.
proteoglycans from the endothelial basal membrane; degrade Antivenom consists of venom-specific antisera or antibodies
fibrinogen and von Willebrand factor enhancing the hemor- concentrated from immune serum to the venom. Antisera
rhagic action; and inhibit platelet aggregation and stimulate contain neutralizing antibodies: one antigen (monospecific) or
release of cytokines. several antigens (polyspecific). Monovalent antivenoms have
a high neutralization capacity, which is desirable against the
Polypeptides—Snake venom polypeptides are low-molecular- venom of a specific animal. Neutralization capacity of antive-
weight proteins that do not have enzymatic activity. More than nom is highly variable as there are no enforced international
80 polypeptides with pharmacologic activity have been isolated standards. Antivenom may cross-react with venoms from dis-
from snake venoms. Most of the lethal activity of the poison tantly related species and may not react with venom from the
of the sea snake Laticauda semifasciata involves erabutoxins. intended species. Nevertheless, in general, the antibodies bind
Erabutoxin-a and a-cobratoxin are curamimetic at the mam- to the venom molecules, rendering them ineffective.
malian neuromuscular junction. Disintegrins are a family of All antivenom products may produce hypersensitivity
short cysteine-rich polypeptides that exhibit affinity for many reactions. Type I (immediate) hypersensitivity reactions are
398 UNIT 5 Toxic Agents

caused by antigen cross-linking of endogenous IgE bound to as the snake venom thrombin-like enzymes are valuable tools
mast cells and basophils. Binding of antigen by a mast cell may in both research and therapeutic applications. Fibrin(ogen)
cause the release of histamine and other mediators, produc- olytic enzymes that break down fibrin-rich clots preventing
ing an anaphylactic reaction. Once initiated, anaphylaxis may further clot formation may be useful as controls in blood clot-
continue despite discontinuation of antivenom administra- ting research or to treat heart attacks and strokes.
tion. Type II hypersensitivity (serum sickness) may develop Animal venoms contain components that can reduce pain,
several days after antivenom administration. In these cases, can selectively kill specific cancers, may reduce the incidence
antigen-antibody complexes are deposited in different areas of stroke via effects on blood coagulability, and function as
of the body, often producing inflammatory responses in the antibiotics. Other venom components act as enzyme inhibi-
skin, joints, kidneys, and other tissues. Fortunately, these reac- tors. Finally, leeches, earthworms, helminths, snails, centipedes,
tions are rarely serious. The risks of anaphylaxis should always spiders, and ticks all produce substances with potential clinical
be considered when one is deciding whether to administer applications, such as osteoarthritis, deep vein thrombosis, anti-
antivenom. microbial action, inflammatory bowel disease, analgesia, and
hyperlipidemia. Blood from mongoose, hedgehog, and opos-
sum contains proteins that inhibit the hemorrhagins in snake
POTENTIAL CLINICAL APPLICATION venoms. These proteins may become valuable as agents of resis-
OF VENOMS tance to snakebites.

Toxin specificities for receptors and channels that facilitate

the interface and coordination of neuromuscular activity
are utilized and manipulated to study, model, diagnose, and BIBLIOGRAPHY
sometimes treat acute and degenerative conditions. On closer Auerbach PS (ed.): Wilderness Medicine, 6th ed., Philadelphia, PA:
examination of a-bungarotoxin and candoxin nicotinic acetyl- Mosby, 2012.
choline receptor specificity, plans are under way to utilize the Bingham J-P, Mitsunaga E, Bergeron ZL: Drugs from slugs—past,
reversible and irreversible receptor binding in muscular and present and future perspectives of conotoxin research. Chem Biol
Interact 183:1-18, 2010.
neuronal tissues, respectively, in Alzheimer patients. In addi-
Burrows GE, Tyrl RJ: Toxic Plants of North America, 2nd ed., Ames,
tion to treating neurological diseases, specific a-toxins (longer
Iowa: Wiley Blackwell, 2013.
chained) are also studied for their antiangiogenic capabilities
Mackessy SP: Handbook of Venoms and Toxins of Reptiles. Boca Raton,
in treating malignant tumor growth in patients suffering from FL: CRC Press/Taylor & Francis, 2010.
small-cell lung carcinoma. In cases such as this, there is an Mayer AMS, Glaser KB, Cuevas C, et al.: The odyssey of marine phar-
inherent trade-off between promoting some degree of neuro- maceuticals: a current pipeline perspective. Trends Pharmacol Sci
logical deficit in light of combating tumor growth. Toxins such 31:255-265, 2010.
 CHAPTER 26 Toxic Effects of Plants and Animals 399

QUESTIONS

1. All of the following statements regarding plant toxicity yh Which of the following plant toxins does NOT affect the
are true EXCEPT: - neuromuscular junction?
a. Genetic variability plays a role in the toxicity of a a. nicotine.
plant. b. anabasine.
b. Plant toxins are most highly concentrated in the ¢), curare.
leaves. d. anatoxin A.
c. Young plants may have a higher toxin concentration e. muscimol.
than older plants.
d. The weather can influence the toxicity of plants. 8. Which of the following statements regarding animal
e. Soil composition can alter a plant’s production of toxin. toxins is FALSE?
a. Animal venoms are strictly metabolized by the liver.
2. Contact with which of the following plant species would b. ‘The kidneys are responsible for the excretion of
be LEAST likely to cause an allergic dermatitis? metabolized venom.
a. Urtica. c. Venoms can be absorbed by facilitated diffusion.
b. Philodendron. d. Most venom fractions distribute unequally through-
c. Rhus. out the body.
d. Dendranthema. e. Venom receptor sites exhibit highly variable degrees
e. Hevea. of sensitivity.

3. Which of the following statements regarding lectin o: Scorpion venoms do NOT:

toxicity is FALSE? affect potassium channels.
a. Lectins have an affinity for N-acetylglucosamine on affect sodium channels.
mammalian neurons. affect chloride channels.
b. Consumption of lectins can cause severe gastrointes- affect calcium channels.
tinal disturbances. ge
caoaffect initial depolarization of the action potential.
c. The fatality rate after ingestion of a fatal dose is very
high. 10. Which of the following statements regarding widow
d. Some toxic lectins inhibit protein synthesis. spiders is TRUE?
e. Adiet high in some lectins has been linked to reduced a. Widow spiders are exclusively found in tropical regions.
weight gain. b. Both male and female widow spiders bite and enveno-
mate humans.
4. Colchicine, found in lily bulbs: c. The widow spider toxin decreases calcium concentra-
a. causes severe dehydration. tion in the synaptic terminal.
b. is sometimes used as a purgative. d. Alpha-latrotoxin stimulates increased exocytosis
c. causes a severe contact dermatitis. from nerve terminals.
d. inhibits sphingolipid synthesis. e. Asevere alpha-latrotoxin envenomation can result in
e. blocks microtubule formation. life-threatening hypotension.

5. Activation ofa vanilloid receptor is characteristic of which 1s Which of the following diseases is not commonly caused
of the following chemicals? by tick envenomation?
a. acetylandromedol. a. Rocky Mountain spotted fever.
b. capsaicin. b. Lyme disease.
c. colchicine. c. Q fever.
d. ergotamine. d. ehrlichiosis.
e. linamarin. e. cat scratch fever.

6. Which of the following plant species is known to cause 12; Which of the following is NOT characteristic Lepidoptera
cardiac arrhythmias on ingestion? envenomation?
Dieffenbachia. a. increased prothrombin time.
Phytolacca americana. b. decreased fibrinogen levels.
Digitalis purpurea. c. decreased partial thromboplastin time.
Pteridium aquilinum. d. increased risk of hemorrhaging.
oe Cicuta maculate.
cao e. decreased plasminogen levels.
400 UNIT 5 Toxic Agents

13. Which of the following animals has a venom containing 15. Which of the following statements regarding snakes is
histamine and mast cell-degranulating peptide that is FALSE?
known for causing hypersensitivity reactions? a. Inorganic anions are often found in snake venoms.
bees. b. About 20% of snake species are venomous.
ants. Ce Snake venoms often interfere with blood coagulation
snakes. mechanisms.
spiders. Proteolytic enzymes are common constituents of
ge reduviids.
pao snake venoms.
Snakebite treatment is often specific for each type of
14, Which of the following enzymes is not typically found in envenomation.
snake venoms?
a. hyaluronidase.
b. lactate dehydrogenase.
c. collagenase.
d. phosphodiesterase.
e. histaminase.
 Cer
Aa Pale kak

Toxic Effects of Calories

Martin J. Ronis, Kartik Shankar, and Thomas M. Badger

BIOLOGY OF EATING AND DIGESTION Ectopic Fat Deposition

Digestion of Foods Metabolic Syndrome
Integrated Fuel Metabolism Therapeutic Options for Managing Metabolic Syndrome
Set-Point Theory and Neural Control of Energy Balance Nonalcoholic Steatohepatitis (NASH)
Endocrine Dysfunction in Obesity, Metabolic Syndrome,
METHODS TO ASSESS ENERGY BALANCE and NAFLD
Assessing Caloric Intake Obesity and Cancer Risk
Assessing Caloric Content of Foods
HEALTH BENEFITS AND LIFE EXTENSION ASSOCIATED
Assessing Energy Expenditure
WITH CALORIC RESTRICTION
Assessing Body Composition
Anthropometric Analysis TREATMENT OF OBESITY
Hydrodensitometry Lifestyle Modification: Dieting and Exercise
Air Displacement Plesmography Toxic Effects of Dieting

Absorptiometry Drug Therapy for Weight Loss

Computerized Tomography
Nuclear Magnetic Resonance (NMR) ECONOMIC, SOCIOLOGIC, AND LEGAL ASPECTS OF THE
Electrical Impedance OBESITY EPIDEMIC
Total Body Water Health Insurance and Obesity
Assessing Physical Activity Changing the Environment: Family and Community
Approaches to Healthy Eating and Physical Activity
BIOLOGY OF OBESITY eReditabele
Obesity Risk: Genes and Fetal Environment Governmental and Corporate Issues

TOXICITY RELATED TO EXCESS CALORIC INTAKE/OBESITY

Adaptation of Liver and Adipose Tissue to Excess Calories

401
402 UNITS Toxic Agents

= Nutrients can broadly be defined as chemical substances « The “set-point” hypothesis proposes that food intake
found in food that are necessary for proper growth, and energy expenditure are coordinately regulated in the
development, reproduction, and repair. central nervous system to maintain a relatively constant
w Energy in the body is derived from three main nutrient level of energy reserve and body weight.
classes: carbohydrates, protein, and fat, which in turn = Dieting is defined as the use of a healthy, balanced
are made up of sugars, amino acids, and free fatty acids, diet that meets the daily nutritional needs of the body
respectively. and that reduces caloric intake with increased moderate
« Hormonal messages generated by the pancreas, adipose exercise.
tissue, and GI tract orchestrate multiple responses asso-
ciated with caloric intake and utilization.

BIOLOGY OF EATING AND DIGESTION metabolism in various tissues to be either oxidized to extract
energy or stored for future utilization.
All biotic organisms derive energy from food to sustain life.
‘
\

This energy “drives” various cellular functions, including

digestion, metabolism, pumping blood, and muscle contrac- Integrated Fuel Metabolism
tions. Nutrients can broadly be defined as chemical substances Energy in the body is derived from three main nutrient classes:
found in food that are necessary for proper growth and devel- carbohydrates, protein, and fat, which in turn are made up
opment, reproduction, and repair following injury. of sugars, amino acids, and free fatty acids, respectively. The
Because most bacteria and higher organisms cannot carry principal circulating fuels in the body, glucose and free fatty
out photosynthesis, they derive their energy by metabolism of acids, are stored as glycogen and triglycerides, respectively.
preformed organic molecules, such as carbohydrates. In gen- Triglycerides are stored in specialized cells (adipocytes) within
eral, bacteria utilize simpler organic molecules and animals large lipid droplets. Proteins are critical in maintaining struc-
and humans require more complex macronutrients (proteins, ture and function and are catabolized for energy only under
fats, and carbohydrates) to meet their needs. extreme conditions.
Maintaining a stable supply of substrate for utilization by the
brain is required because the brain has little to no stored energy
Digestion of Foods in the form of glycogen or triglycerides. Unlike the brain, the
The process of digestion is a remarkable orchestration of many heart and to some degree the liver and skeletal muscles derive
complex biochemical and physiologic events. Breakdown of most of their energy needs through the oxidation of fatty acids.
food begins in the mouth via the actions of enzymes in saliva. Hormonal messages generated by the endocrine cells of the
In the stomach, food is acted upon by gastric juices, which con- pancreas, adipose tissue (adipokines), and GI tract (gut neu-
tains high amounts of hydrochloric acid. Numerous enzymes ropeptides) are critical to orchestrating the multiple processes
supplied by the pancreas, liver, and gall bladder aid digestion in associated with fuel flux and metabolism. Insulin is the princi-
the small intestine. pal hormone required to manage nutrient fuels in both fed and
The latter parts of the small intestine, the jejunum and ileum, fasted states. A rise in glucagon and glucocorticoids (such as
are primary sites of nutrient absorption. The surface area of the cortisol) promote lipolysis and breakdown of glycogen.
intestinal mucosa available for absorption is greatly increased
due to acombination of folds called valvulae conniventes (folds
of Kerckring) and finger-like projections (villi) that are lined
Set-Point Theory and Neural Control
with enterocytes. Digestion of proteins begins in the stomach of Energy Balance
and continues in the lumen of the small intestine. The jeju- A number of redundant feedback mechanisms that maintain
num is the site of absorption of amino acids, dipeptides, and energy homeostasis in living systems regulate the balance
tripeptides by amino acid and peptide carriers in the entero- between food intake and energy expenditure to maintain fuel
cyte brush border. Lipids are hydrolyzed by pancreatic and reserves at preset levels. Under steady-state conditions, energy
intestinal lipases. Bile salts, along with phospholipids, facilitate is normally utilized to maintain basal metabolic rate and ther-
the absorption of lipids. Macronutrient molecules (proteins, mogenesis, and to carry out cellular processes, organ-specific
sugars, and fatty acids) that end up in the circulation undergo functions, and movement (muscle contractions). Excess fuels
 CHAPTER 27 Toxic Effects of Calories 403

are converted to triglycerides and stored in adipose tissues. consumed is maintained. Details may include portion sizes,
Because adipose tissue is the major depot of preserving energy, cooking methods, and patterns ofeating.
signals derived from the periphery communicate with regions
in the brain that coordinate energy balance. When total energy
consumed equals the total energy required to meet basal Assessing Caloric Content of Foods
metabolic needs, growth, thermogenesis, and physical activ- Accurate assessment of the caloric value of foods is essential
ity, the individual is in energy balance, and maintaining this for effective nutritional management in clinical and public
balance will result in relatively stable weight and healthy body policy arenas. The general calorie factors of 4, 9, and 4 for the
composition. major sources of energy—carbohydrate, fat, and protein—
One theory called the “set-point” hypothesis proposes that have been widely used. The heat released by combustion of a
food intake and energy expenditure are coordinately regulated food in a bomb calorimeter is a measure ofits gross energy. The
by defined regions in the central nervous system that signal to truly metabolizable energy can be derived by accounting for
maintain a relatively constant level of energy reserve and body energy lost in urine (mainly from nitrogen) and on the body
weight. Implicitly, the model requires the existence of four surface. Protein content is mainly determined via estimating
major components of an energy homeostasis system: (1) affer- nitrogen. Fat content can be assessed by measuring the sum of
ent signals relaying the levels of energy stores, (2) efferent pro- methanol-chloroform extractable total fatty acids that can be
cesses regulating energy storage and expenditure, (3) efferent expressed as triglyceride equivalents. Carbohydrate content is
mechanisms controlling ingestive behavior, and (4) integrative generally measured by difference as the remaining energy after
centers in the brain to coordinate these processes. Studies have accounting for protein, fat, alcohol, and ash.
shown that the hypothalamus plays a central role in the control
of energy balance, especially food intake. The hormone leptin,
which is secreted in proportion to body fat stores from the adi- Assessing Energy Expenditure
pose tissue, was the first signal to be identified to be a homeo- The total energy expenditure or metabolic cost for an aver-
static regulator of energy balance. age adult is primarily composed of three components:
Two populations of neurons involved in appetite control in (1) basal energy expenditure, (2) thermic effect of food, and
the brain are sensitive to the action of leptin and other neu- (3) energy expenditure associated with physical activity. Basal
ropeptides, including orexigenic peptides neuropeptide Y and energy expenditure, also called as resting energy expenditure,
agouti-related peptide and the anorexigenic peptides proopi- is the energy expended when the individual is lying down
omelanocortin and cocaine and amphetamine-regulated tran- and at complete rest, generally after sleep in the postabsorp-
script. Downstream projections from these neurons interact tive state. The energy expenditure from physical activity con-
with the melanocortin receptor neurons and the neurons in sists of expenditure related to exercise and nonexercise activity
the paraventricular nucleus of the hypothalamus. In addition thermogenesis.
to the hypothalamic control of appetite per se, reward and Components of energy expenditure can be measured using
hedonic processes of “liking” and “wanting” food occur in the either direct or indirect calorimetry. The basic principle in
ventral striatum of the midbrain in conjunction with the meso- direct calorimetry is to measure the actual heat produced by
limbic dopamine system. In addition, the corticolimbic system the organism in a highly controlled environment as an esti-
of reward is controlled by areas in the prefrontal cortex, which mate of energy expenditure. Most commonly used methods to
integrates sensory, emotional, and cognitive information to estimate energy expenditure involve indirect calorimetry. By
coordinate behavioral responses. Hence, the homeostatic con- using experimentally derived estimates for energy yields per
trol of energy balance fits into the larger decision scheme of mole of oxygen, heat production can be calculated based on the
choice behavior via a complex neural system. quantity of oxygen consumed.

METHODS TO ASSESS ENERGY Assessing Body Composition

BALANCE Body composition assessments permit describing the overall
mass of an individual organism in terms of water, fat mass, lean
Assessing Caloric Intake mass, protein, and minerals. In a simple two-compartment
In animal studies, caloric intake can be quantitatively moni- model of body composition assessment, total body mass is
tored by measuring the amount of food consumed by animals divided into fat mass (essential and nonessential fat) and fat-
in metabolic cages. Caloric intake can be derived by multiply- free mass (including lean mass and water). Lean mass in this
ing the quantity (g/day) of diets consumed with the caloric scenario includes protein, carbohydrate, and minerals.
density of the diet. A prospective method to collect informa-
tion about current intake is maintenance of food records. These Anthropometric Analysis—Although individuals with
are usually carried out for a specific duration of time (three greater body weight (mass) per height tend to have greater fat
to seven days, generally including both week and weekend mass, total body weight may also be determined by increased
days) during which a written record of all food and beverages muscle mass. The simplest indirect measure of body fatness is
404 UNIT 5 Toxic Agents

the relative proportion of body weight (in kilograms) to body BIOLOGY OF OBESITY
height squared (meters*), more commonly referred to as body
mass index (BMI). BMI, however, is only an estimate: BMI Obesity Risk: Genes and Fetal Environment
does not always reflect fat mass, and care must be taken when Historically, human life was marked by unpredictable access
using BMI as an index ofbody fat. to food. Fitness and survival of an individual were likely to be
closely related to the ability to maximally seek, acquire, con-
Hydrodensitometry— Using the density of the whole body sume, and store energy (as fat) when food was available, and to
and correcting for residual air in the lungs and GI tract, the select for mechanisms that reduce energy expenditure during
relative body fat can be estimated using derived equations. This times when food is scarce. The advent of agrarian lifestyle and
procedure is also known as underwater weighing. recent industrialization has meant that much of the developed
and emerging world now has a drastically altered environment.
Air Displacement Plesmography—This procedure employs Food is generally available for most people and our lifestyles
the same principles as underwater weighing described above, require less physical activity and exertion. Hence, our genetic
except rather than the body displacing water, it displaces air. This legacy in the context of caloric abundance acts as a powerful
is probably the most accurate, precise, and cost-effective measure engine for weight gain, obesity, and its associated metabolic
of total body fat, and is employed widely in clinical research in dysfunction. Natural variation and random mutation in genes
the United States. controlling hypothalamic energy balance set-points occurred
as human beings developed fire and social behaviors and were
Absorptiometry—In this technique imaging is performed released from risk of predation. The “drifty gene” hypoth-
throughout the entire body by a photon beam. This allows esis explains why even in societies where obesity is high, not
imaging of both soft tissues and bone. Percentage of body fat, everyone becomes obese. Obesity is a highly heritable trait and
lean tissue, and bone mineral density can be computed for the studies comparing monozygotic ‘with dizygotic twins indi-
whole body or specific sites based on the analysis of images. cate that 40% to 75% of the interindividual difference in trait
is accounted for by genetic variability. Several genes whose
Computerized Tomography— The ability to generate three- disruption causes severe monogenic forms of familial obesity
dimensional cross-sectional images allows regional localization have been described. Remarkably, most of these genes impair
of adipose tissues, muscles, and organs (e.g., liver). Using the central control of food intake. However, the genetic basis of
image data, percent body fat and lean mass can be calculated. non-syndromic (common) obesity has remained elusive.
The incidence of obesity continues to rise, including the
Nuclear Magnetic Resonance (NMR)—NMR works by prevalence among infants. As for many chronic diseases, it is
interpreting radio-frequency signals of excited nuclei in an now widely accepted that increased susceptibility to obesity
external magnetic field. The physical characteristics of the can be programmed in utero and early postnatal life. Another
hydrogen atom differ when the hydrogen is located on protein, important influence on risk of obesity in later life is maternal
fat, or water and this can be detected and quantitated to deter- body composition (fat mass) at conception and gestational
mine body composition. weight gain.

Electrical Impedance—Bioelectrical impedance analy-

sis and total body electrical conductivity measure total body TOXICITY RELATED TO EXCESS
composition based on measuring electrical impedance (the CALORIC INTAKE/OBESITY
inverse of conductance) of an electric current passed through
the body. Lean mass has more water and greater conductivity Many of the adaptive, physiologic responses to the positive
than fat mass and predictive equations are employed to derive energy balance produced as a result of overeating and inad-
fat and lean body mass. equate physical activity result in toxicity over the long term.
Short-term coordinated changes in metabolic pathways in
Total Body Water—Body fat and lean mass can be calcu- white adipose tissue in response to overfeeding result in excess
lated by estimating total body water using stable isotopes energy storage in the form of triglycerides, which leads to
(either deuterium or O'*). Whereas body water occupies 73% increased size of preexisting adipocytes (hypertrophy) and to
of lean mass, fat-free mass can be estimated using appropriate formation of new adipocytes (hyperplasia). Under conditions
assumptions. of chronic excess ingested energy, the efficiency of energy stor-
age in adipose tissue is decreased and the body stores energy in
ectopic sites. Triglycerides begin to accumulate in nonadipose
Assessing Physical Activity tissues such as liver, skeletal muscle, and the pancreas as lipid
Devices such as accelerometers and pedometers can be utilized droplets resulting in insulin resistance, inflammation, and tis-
to empirically estimate activity. An important challenge in uti- sue damage.
lizing accelerometers is to convert the count data into energy In addition, adipose tissue from obese individuals releases
expenditure, which is done using different regression models. chemokines and cytokines, the so-called adipokines, which
 CHAPTER 27 Toxic Effects of Calories 405

contribute to a state of “metabolic inflammation.” Non-esterified Ectopic Fat Deposition

fatty acids and the other factors released from adipose tissue
The major sites for ectopic fat deposition (i.e., extra-adipocyte)
contribute to the development of metabolic syndrome in some
are the liver and skeletal muscle. Correlation between cen-
overweight and obese individuals. This is a cluster of compo-
tral (visceral) adiposity, waist circumference, and ectopic fat
nents including insulin resistance, disruptions in lipid homeo-
deposition is better than that for BMI and is also highly cor-
stasis (dyslipidemia), and elevated blood pressure, all of which
related with progressive insulin resistance. In the liver, intra-
substantially increase the risk for development of cardiovascu-
hepatocellular lipid accumulation, also known as fatty liver,
lar disease and type 2 diabetes.
or steatosis, is defined as an increase in hepatic lipid content
above 5% by weight. To confirm that steatosis is actually pres-
Adaptation of Liver and Adipose Tissue ent, histochemical staining of frozen sections for triglyc-
to Excess Calories erides using stains such as Oil Red O is required. Abnormal
lipid accumulation in the liver in the absence of heavy alcohol
Triglycerides and glycogen are used by the body to store excess
usage is referred to as nonalcoholic fatty liver disease (NAFLD)
caloric energy. Although obesity is often associated with over-
and is associated with a wide spectrum of hepatic dysfunc-
consumption of high-fat diets, it can develop from excessive
tion. Simple steatosis is generally reversible with weight loss
caloric intake of any food energy source, including carbohy-
and/or lifestyle modification (e.g., diet and exercise). Hepatic
drates and proteins. Dietary carbohydrates are converted to
lipid accumulation can occur as the result of one or more of
monosaccharides, mainly glucose and fructose, which are
the following: (1) increased fatty acid supply to the liver and
further metabolized in the liver and peripheral tissues. Excess
increased fatty acid transporter expression, (2) increased de
glucose can be stored in the liver as the glucose polymer, glyco-
novo fatty acid and triglyceride synthesis, (3) decreased fatty
gen. However, the majority of excess hepatic glucose is shunted
acid oxidation, and (4) decreased synthesis and/or secretion
into de novo fatty acid and triglyceride synthesis.
of VLDL. Which of these processes predominates depends on
Recent DNA microarray analysis of gene expression in
the degree of obesity, total caloric intake, and diet composition.
human adipose tissue biopsies suggests that coordinated
Reduced serum concentrations of the adipokine, adiponectin,
upregulation of lipogenesis occurs in fat rapidly as a result of
that accompany development of obesity will result in increased
increased caloric intake. The increase in triglyceride synthe-
hepatic fatty acid synthesis and reduced fatty acid degradation
sis ultimately drives adipose tissue hypertrophy. In addition
and thus contribute to development of steatosis.
to adipocyte hypertrophy, excess calories also trigger prolif-
The other major site of ectopic fat deposition in obesity is
eration and differentiation of pre-adipocytes in adipose tissue
skeletal muscle in the form of intramyocellular lipid (IMCL).
depots into new adipocytes, a process known as hyperplasia. It
IMCL has been shown to positively correlate with visceral
is clear that there is a limit to which adipose tissue can expand
adiposity.
safely without damage to adipocytes and that when this limit is
reached toxicity results.
When fat mass increases excessively, adipose tissue undergoes Metabolic Syndrome
extensive structural remodeling. An extracellular matrix with Overnutrition and a sedentary lifestyle lead to a clustering
high concentrations of collagen fibrils and fibronectin appears of metabolic and physiologic components called the meta-
to be essential for maintenance of the structural integrity of adi- bolic syndrome. Central obesity (waist circumference), insu-
pocytes and for pre-adipocyte differentiation. At the point when lin resistance (increased fasting glucose above 100 mg/dL
adipocytes reach a certain size limit within a particular fat pad, and increased fasting insulin, as a result of impaired glucose
the transcription factor, hypoxia-inducible factor la (HIF-1a), uptake into skeletal muscle/fat and increased glucose output
is expressed. HIF-1a regulates inappropriate remodeling of the by the liver, resulting from end-organ insensitivity to insulin),
extracellular matrix and development of fibrosis in adipose tissue dyslipidemia (decreased serum HDL below 40 mg/dL in men
in response to hypoxia and obesity. Fibrosis can be analyzed by and 50mg/dL in women, and increased serum triglycerides
collagen fibril staining and by col6a3 gene expression. Secreted above 150mg/dL), and hypertension (blood pressure higher
protein acidic and rich in cysteine (SPARC), also known as than 130/85) may be evident.
osteonectin, is required for appropriate collagen synthesis dur- However, the relationship between obesity and whole-body
ing matrix remodeling. Both SPARC-/- mice and obesity-prone insulin resistance appears to be mediated through increased cir-
ob/ob mice where the collagen VI gene has been deleted display culating fatty acids and ectopic fat deposition in IMCL in skeletal
increased adipocyte and fat pad size, loose extracellular matrix muscle (Figure 27-2). Consumption of a very low calorie diet in
structure, and reduced inflammation and metabolic distur- obese subjects for as little as five days has been shown to produce
bances after high-fat feeding. The complex interactions between marked decreases in IMCL and enhanced insulin sensitivity
enlarging adipocytes arid the fibrotic extracellular matrix trigger without significant changes in body fat mass. Reductions in glu-
activation of MAP kinase pathways resulting in development of cose import into skeletal muscle with IMCL result from inhibi-
adipocyte insulin resistance, apoptosis, and necrosis, which in tion of translocation of the glucose transporter GLUT-4 from
turn results in activation of resident macrophages in the fat and cytosolic- to membrane-associated compartments through
an inflammatory response (Figure 27-1). the action of IMCL metabolites such as diacylglycerol (DAG),
406 UNIT 5 Toxic Agents

SPARC-/-, HIF-10 -/-, Coll 6-/- Mice

Lean fat Sees>

ran Soe Lean fat
CO)
las
Thiazolidinediones
Excess calories (PPAR activation) Excess calories

Triglyceride synthesis, Triglyceride synthesis,

hypertrophy hyperplasia, hypertrophy hyperplasia,
hypoxia, matrix production, blockage of hypoxia, or
macrophage activation matrix production

Obese fat Obese fat

NEFA
ainsi tam Chemokines (MIP-1)
is Cytokines
i (TNFa, IL- IL-6)

(Increased leptin, : f
Normal metabolism
resistin, decreased Increased weight gain
adiponectin)

Metabolic syndrome

Size limited adipocyte w Activated macrophage

surrounded by fibrous matrix

Enlarged adipocyte surrounded

by disorganized matrix

FIGURE 27-1 Effects of excess calories (energy) on fat morphology under conditions leading to metabolic syndrome (left) or
following stimulation of adipocyte differentiation and hyperplasia by thiazolidinedione treatment/in knockout mice incapable of normal
responses to hypoxia (HIF-1« -/-)/in knockout mice incapable of normal extracellular matrix production (SPARC-/-, Coll 6-/-) (right).

C-reactive protein VLDL => | HDL Hypertension

Dee
+

C7 crokines
Cytokines

NEFA
- A a
Liver / 2 va
seen a
Blood vessels
“ Cytokines i Insulin
ae Adipokines
* Obese fat
Glucose NEFA - i
|Cytokines Sympathetic
ia ~ Insulin yt te
Skeletal muscle Bg > x
+ Glucose ad
Pancreas

ie) Size limited adipocyte surrounded by fibrous matrix

w Activated macrophage © Lipid droplet

FIGURE 27-2 Pathogenesis of metabolic syndrome.

long-chain fatty acid CoAs, ceramides, and oxidized lipids. to further increase the release of nonesterified fatty acids. Insulin
Reduced glucose transport also occurs in insulin-resistant adi- resistance in liver leads to excess glucose production as the result
pose tissue itself and the negative effects of obesity are exacerbated of a reduced ability of insulin to suppress the gluconeogenic
because reduced insulin signaling in adipocytes also enhances enzyme phosphoenolpyruvate carboxykinase (PEPCK). This
expression of hormone-sensitive and adipose triglyceride lipases contributes to systemic hyperglycemia and increased pancreatic
 CHAPTER 27 Toxic Effects of Calories 407

insulin production. Insulin resistance and steatosis are strongly Treatment with insulin sensitizers, bariatric surgery, and life-
correlated and interventions that lead to lower plasma insulin style modification (e.g., diet and exercise) resulting in weight
levels also decrease liver triglyceride content. loss and reduction of hepatic fat content improve NASH liver
pathology.

Therapeutic Options for Managing

Metabolic Syndrome Endocrine Dysfunction in Obesity,
Lifestyle modifications in the obese, including diets produc- Metabolic Syndrome, and NAFLD
ing stable weight loss, long-term increased physical activity, Hyperglycemia resulting from systemic insulin resistance
or bariatric surgery (discussed below), are of benefit in treat- provokes a compensatory increase in insulin secretion from
ing all the components of metabolic syndrome, but suffer from the pancreas in obese individuals. Hyperinsulinemia then
limited compliance and significant risk of complications in the appears to have secondary effects on other endocrine systems.
case of surgery. Therefore, routine clinical management has For example, growth hormone (GH) secretion is dramatically
focused on pharmaceutical therapies for insulin resistance, suppressed by obesity in both adults and children, but can be
hyperglycemia, dyslipidemia, and hypertension to reduce the reversed by weight loss. Mechanisms appear to involve direct
risks of cardiovascular disease and type 2 diabetes. feedback effects of insulin on the pituitary and a reduction in
Several classes of drugs are used to target insulin resistance. secretion of the endogenous GH-releasing peptide ghrelin that
Dyslipidemia associated with metabolic syndrome is a major is produced by the stomach and hypothalamic centers.
modifiable risk factor for cardiovascular disease that may be Increased serum concentrations of free androgens appear
treated with the statins, which are inhibitors of 3-hydroxy-3- to explain, in part, why childhood obesity is associated with
methyl glutaryl coenzyme A reductase (HMG-CoA reduc- earlier pubertal development in girls. Hyperandrogenization
tase). These compounds improve overall lipid profiles by is involved in the increased incidence of polycystic ovary syn-
decreasing LDL concentration, increasing HDL, and decreas- drome in obese adolescent girls with metabolic syndrome,
ing triglycerides. anovulatory cycles, and subfertility in obese women of child-
bearing age. Increased adipose tissue mass also results in
increased estrogen production as a result of androgen aroma-
Nonalcoholic Steatohepatitis (NASH) tization in fat tissues. This may also contribute to accelerated
Ectopic fat deposition in the liver is strongly correlated with puberty in girls.
obesity and insulin resistance. The disease progression of non- In obese boys, data suggest that puberty may be delayed
alcoholic fatty liver disease (NAFLD) is first to NASH, which is owing to increased aromatization of androgens in adipose
characterized by cell death, inflammation, and fibrosis, then to tissue. Negative feedback of estrogens at the level of the
cirrhosis in which liver function is significantly impaired, and hypothalamic-pituitary axis may result in reduced luteinizing
ultimately to hepatocellular carcinoma (Figure 27-3). hormone secretion and reduced testosterone production.

Excess calories/high-fat diet

Paks
Bia
Insulin
resistance Obese fat
Gut
! \ Toll-like receptor =~
M | \ activation ~ ros

z = Pancreas | NEFA \
wits Se
||Adipokines\ Me
ere

SS |Cytokines he a
_~ Endotoxin
oe \

_ ===> Liver
cancer
ER stress
Normal liver Fatty liver NASH Cirrhosis

oO Size limited adipocyte surrounded by fibrous matrix

ww Activated macrophage © Lipid droplet = Collagen fiber

FIGURE 27-3 Progression of nonalcoholic fatty liver disease (NAFLD). ROS, reactive oxygen species; ER stress, endoplasmic
reticulum stress.
408 UNIT 5 Toxic Agents

TABLE 27-1 Estimated risk ratios* for cancer in age-related disease in comparison with ad libitum feeding in
relation to body mass index. a wide variety of organisms including yeast, nematodes, fruit
flies, fish, many rodent species, and dogs. Preliminary data
Cancer Type Men Women in humans suggest reproduction of many of the results from
Colon cancer 1.24 1.09 animal studies, including reduced fat and lean mass, reduced
insulin, reduced energy expenditure, lower core body tem-
Gallbladder cancer — 159 perature, and improved lipid profiles. It has been suggested
Leukemia 1.08 Welly that the increased health and longevity associated with CR is
related to reduced energy utilization, increased insulin sensi-
Malignant melanoma Te7 — tivity, and reduced inflammation.
Multiple myeloma tei el

Esophageal adenocarcinoma 152 1.51

TREATMENT OF OBESITY
Renal cancer 1.24 1.34
Lifestyle Modification: Dieting
Thyroid cancer 1:33 1.14
and Exercise
Prostate cancer 1.03 ==
As applied to body composition, dieting involves a plan or reg-
Postmenopausal breast cancer — Te2 imen to improve body composition. Trimming excess body fat
generally requires reductions in total caloric intake, increases
Endometrial cancer’ — 73
in the total energy expenditure (through exercise or physical
*Per increase in body mass index by 5 kg/m? (Roberts DL, Dive C, Renehan AG: activity), and modifications in diet composition. This com-
Biological mechanisms linking obesity and cancer risk: new perspectives. Annu Rev bined strategy is intended to reduce energy intake to a level
Med 61:301-316, 2010).
*For body mass indexes above 27 kg/m?. low enough to drive the body to utilize stored fat as an energy
source, thereby burning body fat.
Comprehensive lifestyle changes that promote a neutral
energy balance include two major and closely linked com-
In addition to suppression in GH and gonadotropin secre-
ponents: (1) learning to consume only the amount of calo-
tion, hypothyroidism is common in individuals with metabolic
ries from high-quality foods necessary to support basal body
syndrome. Reduced thyroid hormone concentrations may
energy needs plus energy needs to maintain physical activity
exacerbate NASH progression in the liver by increasing triglyc-
and (2) selecting a reasonable physical activity plan that fits
eride synthesis, reducing fatty acid oxidation, and increasing
into the dieters’ overall lifestyle patterns.
hepatic cholesterol concentrations by reducing conversion to
The energy in food eaten can be “cost accounted” roughly
bile acids.
as follows: (1) energy required to digest and absorb food,
(2) energy utilized to support basal functions such as pump-
Obesity and Cancer Risk ing blood and breathing, (3) energy for body functions other
than basal functions such as walking and playing golf, and
Increased BMI is well known to be associated with signifi-
(4) nonutilized food calories such as food components not
cantly increased risk of a number of cancers. These include sex
fully digested or absorbed.
steroid—dependent endometrial, breast, and prostate cancer, GI
Insulin has a major influence on carbohydrate, fat, and pro-
tract cancers such as esophageal adenocarcinoma, and colon
tein metabolism. Under conditions of adequate carbohydrate
cancer and renal cancer (Table 27-1). Additional potential
intake, insulin causes the sugar not utilized as fuel to be stored
associations between obesity and cancer may involve deple-
as fat and prevents utilization of fat as an energy source. Thus,
tion of cellular antioxidant systems as a result of the low-grade
a high-carbohydrate diet tends to induce insulin secretion,
chronic systemic inflammation that accompanies morbid obe-
which promotes carbohydrate energy storage as fat and tends
sity and the possibility that mesenchymal stromal cells aris-
to reduce the utilization of fat as an energy source.
ing from expanding white adipose tissue may be recruited to
tumors to promote angiogenesis and drive tumor progression.

Toxic Effects of Dieting

HEALTH BENEFITS AND LIFE If the diet does not include all the required nutrients (i.e., an
EXTENSION ASSOCIATED imbalanced diet), metabolism will suffer and, with time, this
WITH CALORIC RESTRICTION can result in health problems. Recalling the concept of hor-
mesis, this is true whether in the case of deficiency of specific
The opposite of overfeeding is caloric restriction (CR; also nutrients (deficiency disorders such as anemia or osteoporosis)
known as dietary restriction). Over the past two decades, CR or toxicity caused by excesses of a particular nutrient (such as
has been repeatedly shown to increase life span and reduce thyroid impairment, vitamin deficiencies, mental confusion).
 CHAPTER 27 Toxic Effects of Calories 409

Some popular diet plans call for excess intake of a particular is prevalent. Prior to 1970, the average BMI was 25.1 for men
food and these can not only alter metabolism, but also interfere and 24.9 for women in the United States. Physical education
with medications. (PE) classes were a regular feature of school curriculums; most
meals were prepared using fresh produce, meats, and dairy
products. It was common for children to walk or ride bicycles
Drug Therapy for Weight Loss to school and to participate in games requiring physical activ-
In addition to the diet plans described above, many over- ity during school recess and after school and on weekends. By
weight individuals turn to drug therapy to help lose body 2002, there were fewer schools with PE classes, consuming
weight. Appetite suppressants, e.g., sympathomimetics such electronic games and devices, fewer meals cooked from fresh
as diethylpropion, attempt to lessen the psychologic moti- components, more fast food, and a far more sedentary lifestyle
vation for food, usually by acting on central nervous system than that prior to 1970. BMI values had risen to 27.8 in US men
appetite control centers, such as those in the hypothalamus. and 28.1 in US women. One approach to fighting the obesity
Although sympathomimetics can be used for long periods of issue is to bring back many of those practices used in the past.
time, their appetite-reducing effects tend to decrease after a There are initiatives to establish community gardens, build
few weeks in many people. Thus, appetite suppressants are community walking and riding trails, and teach people cook-
often used in the early stages of aweight loss program. People ing and shopping skills that lead to healthier meal preparation.
are likely to lose weight while taking sympathomimetics, but School systems are starting to return to PE classes on a regular
the weight loss is generally temporary without modifications basis and remove high-density caloric foods and drinks from
in diet composition, eating behavior, and physical activity. vending machines.
Short-term use is usually accompanied by minor side effects
such as thirst, irritability, constipation, stomach pain, diz-
ziness, dryness of mouth, heightened sense of well-being, Food Labels
headache, irritability, nausea, nervousness or restlessness, The Food and Drug Administration (FDA) is responsible
trembling or shaking, and trouble sleeping. However, long- for assuring that foods sold in the United States are properly
term use of appetite suppressants often times leads to more labeled, regardless oforigin (i-e., domestic or foreign). Food
serious side effects: intracerebral hemorrhage, acute dysto- labels can be an important factor to help consumers in their
nia, myocardial injury, psychosis, cerebral arteritis, cardiac food choices that can help prevent obesity and other dis-
arrhythmias, heart valve damage, and even fatal pulmonary eases. Federal law requires that a minimal amount of infor-
hypertension. mation be listed on food packaging, including ingredients
and nutrition data.

ECONOMIC, SOCIOLOGIC,
AND LEGAL ASPECTS OF THE Governmental and Corporate Issues
OBESITY EPIDEMIC There are increasing pressures from local, state, and federal
governments in the United States to regulate various aspects
Health Insurance and Obesity of food production and marketing as a means of promoting
Obesity is not considered an illness for most insurance pur- health, reducing obesity, and its consequences. Food labeling
poses. However, obesity can affect the cost of health insurance is just one example of government intervention, whereby food
because as a group, obese people have a significantly greater processors and restaurants must provide a measure of nutrient
risk of cardiovascular disease, hypertension, type 2 diabe- and/or caloric content.
tes, and other health issues than lean people. Several health
insurance companies use BMI as a measure of obesity and
use BMI to compute disease risk and health insurance premi-
ums. Obesity can result in high premiums and in the case of
BIBLIOGRAPHY
Gropper SS, Smith JL (eds.): Advanced Nutrition and Human Metabo-
morbidly obese individuals, insurers may decline their applica- lism, 6th ed. Belmont, CA: Thomson Wadsworth, 2013.
tion. Obesity is also regarded by insurance companies as a sub- Kulie T, Slattengren A, Redmer J, et al.: Obesity and women’ health:
stantial risk for both life and disability policies. Clearly, costs an evidence-based review. ]Am Board Fam Med 24:75-85, 2011.
increase proportionally with the degree of obesity. Roberts DL, Dive C, Renehan AG: Biological mechanisms link-
ing obesity and cancer risk: new perspectives. Annu Rev Med 61:
301-316, 2010.
Changing the Environment: Family Smith DL, Nagy T, Allison DB: Calorie restriction: what recent
and Community Approaches to Healthy results suggest for the future of aging research. Eur JClin Invest 40:
440-450, 2010.
Eating and Physical Activity Stipanuk M (ed.): Biochemical, Physiological, Molecular Aspects
Basic practices that were common in past decades, when the of Human Nutrition. 3rd ed., St. Louis, MO: Saunders Elsevier,
general population was leaner, are lacking now when obesity 2012.
410 UNIT 5 Toxic Agents

QUESTIONS

1. Humans consume food to provide energy needed to hs Excess caloric intake

a. drive cellular functions including digestion, metabolism, a. maylead to nonalcoholic steatohepatitis.
pumping blood, nerve activity, and muscle contractions. b. is always correlated with obesity and insulin
promote photosynthesis. resistance.
synthesize oxygen in the lungs. c. is characterized by elevations of serum ALT concen-
prepare minerals for use in the body. trations in all cases.
a produce carbon dioxide to fuel body functions. d. leads to hepatic cirrhosis and liver cancer in almost all
cases.
2. Neural control of energy balance e. is readily reversible by dieting.
a. may be defined as the action of leptin on CNS
function. 8. Although dieting may effectively reduce body weight,
b. may be defined as the action of hypothalamic cholin- a. toxicity may result from stimulation of adipokine
ergic control of appetite and hedonic control. release.
c. may involve a balance between food intake and b. toxicity may result from inhibition of drug metaboliz-
energy expenditure. ing enzymes.
d. may involve a balance between leptin’s action on c. toxicity may result from a loss of required nutrients.
orexigenic versus anorexigenic peptide expression. toxicity may result from extreme mental illness.
e. may involve adrenocortical control of hepatic function. e. toxicity may result from weight cycling.

3. Body composition may be assessed by O), Body mass index oy,

a. electrical impedance because lean mass has more a. may be used as an indicator of sufficient caloric and
water and greater conductivity than fat mass. essential nutrient intake.
b. anthropometric analysis of the body mass index. b. may be defined as body height divided by body weight
c. hydrodensitometry, which uses the density of the squared.
whole body and corrects for residual air in the lungs c. has risen insignificantly over the past 30 years in the
and GI tract to determine relative body fat. United States.
d. nuclear magnetic resonance. d. may not be used in the estimation of cancer risk in
e. all ofthe above. humans.
e. may be defined as body weight divided by height
4. Ectopic fat deposition includes squared.
adipose tissue.
skeletal muscle. 10. Which of the following definitions is false?
lungs. a. ‘The set-point hypothesis proposes that food intake
heart. and energy expenditure are coordinately regulated by
go
se
oa
eteGI tract. defined regions in the brain that signal to maintain a
relatively constant level of energy reserve and body
5. Excess calories may be weight.
a. stored as glucose in adipose tissue. b. Hormonal messages generated by the endocrine
stored as triglycerides in CNS tissue. cells of the pancreas, adipose tissue, and GI tract are
stored as glycogen in CNS tissue. involved in orchestrating multiple responses associ-
stored as glycogen in the liver. ated with caloric intake and caloric utilization.
scao
stored as triglycerides in the GI tract. c. Caloric content of foods generally assumes fac-
tors of 4, 9, and 4 for carbohydrate, fat, and protein,
6. Metabolic syndrome is a constellation of actions including respectively.
a. typically results from elevated fasting glucose, d. The body mass index (BMI) is an accurate method for
increased HDL, and hypertension. assessing body composition.
b. typically results from elevated fasting glucose, e. Liver, adipose, muscle, and other tissues adapt to
increased LDL, and hypertension. excess caloric loads.
typically results from elevated fasting glucose, hyper-
triglyceridemia, and hypotension.
typically results from elevated fasting glucose,
hypotriglyceridemia, and truncal obesity.
typically results from elevated fasting glucose, hyper-
triglyceridemia, and truncal obesity.
 ON IV IS NG V. VA N KO ).€( 0( 0) KO Lc ) Gam
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CUT aan alee:

Nanotoxicology
Gunter Oberdorster, Agnes B. Kane, Rebecca D. Kapler,
and Robert H. Hurt

NANOMATERIAL BASICS Portals of Entry

Perspectives: Engineered Nanoparticles versus ambient Dosing of the Respiratory Tract
particulate matter Respiratory Tract Deposition
Properties and Behaviors of ENPs versus Larger Particles Respiratory Tract Clearance and Disposition of NP:
Classes of ENMs Nanomaterials
Physicochemical Properties of Nanomaterials Relevant Nanomaterials and the Brain
for Toxicity Elimination of Nanomaterials
Surface Area and Reactivity
CASE STUDY: MWCNTS
Surface Charge
Bolus-type Exposures
Surface Chemistry
Inhalation Studies
Unique Quantum and Magnetic Properties
Critical Appraisal of CNT In Vivo Studies
Geometry and Dimensions
Biologic Degradation of Carbon Nanomaterials
Biopersistence
TOXICITY TESTING
THE NANOMATERIAL BIOLOGIC INTERFACE
In Vitro Dosimetry
TOXICITY MECHANISMS Predictive Toxicology
Transition, Human Eco-nanotoxicology
CAVEATS IN NANOTOXICOLOGIC ASSAYS
ECOTOXICOLOGY OF ENMS
SAFETY CONSIDERATIONS IN NANOMATERIAL DESIGN
Environmental Uses and Exposures to Nanomaterials
CASE STUDY: DESIGNING SAFER SUNSCREENS Ecologic Risk Assessment of Manufactured Nanomaterials
Toxicity of Manufactured Nanomaterials
MAMMALIAN TOXICOLOGY Complications of Assays
Introduction Ecotoxicity of Nanomaterials
Concepts of Nanotoxicology Mechanisms ofToxicity
Dosemetrics

411
412 UNIT 6 Environmental Toxicology

« Nanotechnology is the understanding and control of = The respiratory tract is the major route for humans to
matter at nanoscale dimensions between approximately exposure of nanomaterials.
1 and 100nm, where unique phenomena enable novel w Surface properties are major determinants of biologic
applications. reactivity due to high surface area, surface charge,
s Nanotoxicology can be defined as the study of adverse hydrophobicity and partitioning into lipid mem-
effects of nanomaterials on living organisms and the branes, dissolution and release of metal ions, and redox
environment. activity.
= Surface properties are major determinants of biologic « Dosemetric defines a dose in terms of an inherent prop-
reactivity due to high surface area, surface charge, dissolu- erty (physical, chemical, reactivity, etc.).
tion and release of metal ions, and redox activity leading
to generation of ROS.

Nanotechnology has become a multibillion dollar industry diabetes), age (very young, elderly), or genetic background
worldwide, producing high volume, commercial nanoma- (polymorphism). :
terials including nanosilver, fullerenes, quantum dots, car-
\

bon nanotubes (CNTs), and metal oxide nanoparticles (NPs)

(Figure 28-1). Nanotoxicology seeks to identify and charac-
Properties and Behaviors of ENPs versus
terize the hazards of engineered nanomaterials (ENMs) for Larger Particles
purposes of risk assessment for humans and the environment, Table 28-1 contrasts differences between NPs (<100nm) and
which requires a highly multidisciplinary team approach larger particles (>500nm) in general characteristics, translo-
covering expertise in toxicology, biology, chemistry, physics, cation propensity, and cellular effects assuming inhalational
material science, geology, exposure assessment, physiologic- exposure. Biologic systems do not perceive a precise bound-
based pharmacokinetics (PBPK), and medicine. All these dis- ary at the 100 nm size threshold, but rather a gradual transition
ciplines are necessary to develop testing strategies, establish between nano- and larger-sized particles.
toxicity ranking, determine “safe” exposure levels, and derive Efficient mucociliary and alveolar macrophage-mediated
preventative exposure guidelines. elimination following deposition in the respiratory tract is
efhcient for both nano-and larger particles once they are
internalized by macrophages. NPs inhaled and deposited as
singlets are too small to be efficiently recognized and phago-
NANOMATERIAL BASICS cytized by alveolar macrophages—unless they aggregate or
agglomerate to form larger particles—and thus overall alveolar
Perspectives: Engineered Nanoparticles
macrophage-mediated clearance in the lung is poor. In contrast,
versus Ambient Particulate Matter uptake by epithelial cells and translocation into blood and lym-
Airborne ambient particulate matter (PM) can elicit adverse phatic circulation occur regularly for NPs and only under heavy
effects in the respiratory tract, in secondary organs and system- overload conditions for larger particles.
ically (see Chapter 29). The smallest fraction of PM, referred to
as ultrafine particulates (UFPs, <100 nm), has been associated
with effects in the cardiovascular and central nervous system Classes of ENMs
(CNS) as a consequence of their translocation to and distri- Manufactured nanomaterials have an enormous range of com-
bution via blood circulation and neurons. Natural sources position, geometry, and complexity ranging from simple iso-
of ultrafine and nanoparticles include gas to particle conver- metric forms (NPs), one-dimensional (1D) forms (fibers or
sions, forest fires, volcano eruptions, viruses, magnetotactic tubes), and two-dimensional (2D) forms (plate-like or disk-
bacteria, mollusks, arthropods, fish and birds. Unintentional like materials) as shown in Figure 28-2. A large fraction of
anthropogenic sources include internal combustion engines, the stable elements in the periodic table have now been cast
power plants, metal fumes from smelting and welding and into NPs. Nanomaterials may be applied to surfaces such as
heated surfaces. Engineered nanoparticles would represent biomedical implants to enhance their function and biocom-
intentional sources. Epidemiologic studies have demonstrated patibility, incorporated into nanostructured solids, or com-
that increased susceptibility to adverse effects from ambient posites to improve strength, conductivity, and durability. As
particulate air pollution includes preexisting disease (asthma, nanoscience progresses, there will be less emphasis on simple
 CHAPTER 28 Nanotoxicology 413

Nanoscale materials

Sodium Fructose Protein Virus Bacteria Macrophage Daphnia Baseball

molecule
: ey ae
© Agee
a

oo) ad e{ ; rs
re

10? 104 10° 10° 107 108

| l !

——— < Nanometers

ig ee
nm —|um variable

Bucky-ball Carbon Graphene ZnO nanorods Nano-onion

nanotube

FIGURE 28-1 Length scales for natural and synthetic structures (above) and some examples of engineered nanomaterials of varying
size and shape (below).

TABLE 28-1 Whatis different: nanoparticles versus larger particles (respiratory tract as portal of entry).
Nanoparticles (<100nm) Larger Particles (>500nm)

General characteristics
Ratio: number or surface area/volume or mass High Low
Agglomeration in air, liquids Likely (dependent on medium and surface) Less likely
Deposition mechanism in respiratory tract Diffusion; throughout resp. tract Sedimentation, impaction, interception;
throughout resp. tract
Protein/lipid adsorption in vitro Very effective and important Less effective
Some
Protein/lipid adsorption in vivo Yes

Translocation to secondary target organs: Yes Generally not (to liver under “overload”)
Clearance
« Mucociliary Probably yes Efficient
- Byalveolar macrophages Poor Efficient
« Into or across lung epithelium Yes Mainly under overload
« Lymphatic Yes Under overload
+ Blood circulation Yes Under overload
~ « Sensory neurons (uptake + transport) Yes Not likely

Cell entry/uptake Yes (caveolae; clathrin; lipid rafts; diffusion) Yes (primarily phagocytic cells)
« Mitochondria Yes No
« Nucleus Yes (<40nm) No

Effects (caveat: dose!):

At secondary target organs Yes (No)
At portal of entry (resp. tract) Yes Yes
« Inflammation Yes Yes
« Oxidative stress Yes Yes
* Activation of signaling pathways Yes Yes
« Genotoxicity, carcinogenicity Probably yes Some

Data from Oberdorster G, Elder A, et al.: “Nanoparticles and the Brain: Cause for Concern?” Journal of Nanoscience and Nanotechnology, 2009;9:4996-5007.
414 UNIT 6 Environmental Toxicology

Geometry

Isometric particles 1D; fibers/tubes 2D; plates, disks

Chemistry

Silver, gold nanoparticles Gold or platinum Silver nanoplates

Metals Iron, cobalt, nickel magnetic NPs nanowires
copper NP conducting inks
Plate-like
CdSe/ZnS quantum dots Si, ZnO semiconducting
iconductors semiconductor
pemicond (see example) nanowires, nanorods
nanocrystals

Zinc oxide, titanium dioxide Electrospun ceramic

nanofibers for Nanoclays
Ceramics pigments and sun screens,
cerium oxide catalysts composite fillers

Fullerenes, carbon black, Carbon nanotubes Graphene, graphene oxide

Carbons
carbon nanohorns Carbon nanofibers few-layer graphene
(example)
Biodegradable polymer Electrospun
Polymers nanobeads for medical polymer nanofibers
applications, branched dendrimers

v4, Semiconductor §
core

Examples:
2-121nm

Shell for
Organic caps efficiency
or ligands and stability

FIGURE 28-2 Classification of nanomaterials by geometry and chemistry.

The examples in this matrix illustrate the diversity in
engineered nanomaterials, a diversity that continues to increase as new nanomaterials are synthesized.

geometries and chemistries, and more emphasis on complex TABLE 28-2 Physicochemical NP properties relevant
material structures that combine nanoelements into active or to toxicology.
smart structures.
Size (aerodynamic, hydrodynamic)
Size distribution
Physicochemical Properties of Shape
Agglomeration/aggregation state
Nanomaterials Relevant for Toxicity Density (material, bulk)
Chemical composition and phase Properties can
Table 28-2 summarizes the nanomaterial properties thought + Crystallinity change
to be relevant to biologic responses. - Dissolution and toxicant (ion) release + With method
* Coatings and bioavailable of production,
contaminants r preparation
Surface Area and Reactivity—At the nanoscale, struc- + Biopersistence process, storage
tures have a high surface-to-volume or surface-to mass ratio. Surface properties « When introduced
This high surface area of NPs is responsible for increased sur- + Surface area (external, internal) into physiologic
- Electrical charge (zeta potential) media, organism
face reactivity, increased adsorption of chemicals and strong
+ Redox activity
catalytic activity. High surface reactivity is a desirable property » Hydrophobicity/hydrophilicity
for catalysis; however, the large number of exposed surface » Adsorptive capacity for biomolecules
molecules or atoms exposes surface defects, vacant sites, and Nanoscale quantum and magnetic
properties (?)
dangling chemical bonds that enhance chemical and redox
reactivity.
to add coatings that prevent particle—particle attachment or to
Surface Charge—NPs have a strong tendency to form aggre- impart an electrical charge that lead to particle-particle repul-
gates as a natural consequence of small size, which leads to sion. This is applicable to drug delivery whereby coating with
strong intermolecular attractive forces. To obtain stable disper- biocompatible surfactants stabilizes NPs. Figure 28-3 depicts
sions of unagglomerated nanomaterials, it is often necessary the characteristics of agglomerated and aggregated NPs.
 CHAPTER 28 Nanotoxicology 415

Dry powders O -
EA

Primary Agglomerates Aggregates Agglomerated

: Primary particles held by weak Primary particles held by strong
pa rticle van der Waals forces chemical bonds (sintered) Aggregates

Liquid dispersions

Electrical double layer Hydrodynamic

(thickness depends on
solution ionic strength) diameter

Repulsive forces dominant Weak repulsive forces in liquid

(high surface charge; thicker double layer; resulting in agglomeration
steric forces) (low surface charge; thinner double layer;
no steric forces)

~ Important parameters: Primary particle size (nm); hydrodynamic diameter (nm); zeta
potential (mV, measure of surface charge); double layer thickness (nm); steric forces

FIGURE 28-3 Agglomeration and aggregation of nanoparticles in liquids and as dry powders. (Modified with permission from
Jiang JG, Oberdorster E, et al.: Characterization of size, surface charge, and agglomeration state of nanoparticle disperisons for toxicological studies,
J Nanopart Res, 2009 Jan;11(1): 77-89.)

Surface Chemistry—High surface area and exposed surface Unique Quantum and Magnetic Properties—
atoms or molecules promote increased dissolution and release Ferromagnetic NPs less than 10nm in diameter respond to an
of ions from metallic or metal oxide NPs relative to bulk par- external magnetic field. This is exploited for contrast enhance-
ticles of the same chemical composition. Metal ions are toxic to ment in diagnostic MRI and for hyperthermia induced by an
bacteria and aquatic organisms by inhibition of enzymes and external magnetic field to kill tumors targeted by magnetic NPs.
transport proteins. For example, ZnO NPs are incorporated
into sunscreens where they absorb ultraviolet (UV) light; how- Geometry and Dimensions—These are important determi-
ever, in water, Zn’* ions are rapidly released and cause acute nants in cellular uptake, systemic translocation, and potential
toxicity. Surface hydrophilicity of charged NPs increases their toxicity. Nanomaterials can enter target cells by passive diffu-
ability to be suspended in water, whereas surface hydrophobic- sion, direct physical penetration, or active, receptor-mediated
ity of fullerenes or grapheme repels water and enables these uptake by endocytosis or phagocytosis depending on their size
hydrophobic nanomaterials to partition into lipid membranes and extent of agglomeration. Small NPs appear to enter cells and
and enter target cells. In addition, surface defects expose elec- organelles by passive diffusion. Single walled CNTs (SWCNTs)
tron active groups that donate an electron to molecular oxygen have been shown to directly puncture bacterial cells leading
which generates superoxide anions which are reactive oxygen to osmotic lysis and death. SWCNTs have also been reported
species (ROS). to translocate from the alveoli into the interstitium of the lung
CNTs are synthesized in the presence of metal catalysts that where they promote collagen deposition and interstitial fibrosis.
can undergo redox cycling and catalyze generation of highly
reactive hydroxyl radical groups. Nanomaterials with high Biopersistence—Biopersistence of ENMs is an impor-
surface area can adsorb organic molecules such as polycyclic tant factor in their environmental and biologic toxicity.
aromatic hydrocarbons that are potentially carcinogenic and Biopersistence is related to dissolution, which produces bio-
quinones that also participate in generation of free radicals and logically active ionic species as well as has the ability to degrade
redox cycling. Cationic NPs that have surface amide groups the particle and clear it from biologic tissue or the environ-
and cationic dendrimers are especially cytotoxic because ment. The rate of dissolution of metal oxides is increased by
they induce membrane damage, especially in lysosomes, that natural organic matter in the aqueous environment; therefore,
leads to accumulation of water and chloride ions and osmotic these NPs have low biodurability and it is predicted that they
rupture. would not bioaccumulate in the environment.
416 UNIT 6 Environmental Toxicology

Biopersistence in the lungs and pleural or peritoneal spaces TABLE 28-3 Invitro mechanisms of nanoparticle
is an important physicochemical characteristic of asbestos toxicity.
and man-made mineral fibers associated with carcinogenicity.
. Damage to cell wall and plasma membrane
Several recent studies have shown that carboxylated SWCNTs
. Interference with electron transport and aerobic respiration
do not undergo oxidative degradation in the presence of stim- . Induction of oxidant stress
ulant fluid that mimics the lysosomal compartment of mac- . Activation of cell signaling pathways
rophages. However, oxidatively degraded SWCNTs did not . Perturbed ion homeostasis
. Release of toxic metal ions from internalized nanoparticles
induce lung inflammation or toxicity following pharyngeal
. Disruption of lysosomal membrane integrity
aspiration in mice providing proof-of-principle for deliber- . Incomplete uptake orfrustrated phagocytosis
ate design of engineered CNTs that are biodegradable and less . Interference with cytoskeletal function
likely to induce disease following inhalation or injection for —_A . DNA and chromosomal damage
DOAN
NPWHN

tumor imaging or drug delivery.

holes in the plasma membrane resulting in extracellular release
THE NANOMATERIAL BIOLOGIC of cytoplasmic contents as assessed by efflux of ribosomal RNA
INTERFACE and decreased survival.
A wide variety of NPs have been designed to facilitate deliv-
The high surface area of NPs provides a platform for adsorp- ery of imaging agents, genes, proteins, and drugs into mam-
tion of a variety of biologic molecules including proteins, malian cells. NPs can also be designed to target specific cell
lipids, and nucleic acids. A “protein corona” exists on the NP and surface receptors triggering internalization. In order to facili-
governs its initial reaction with target cells. The interaction of tate delivery, NPs can be engineered to escape from endosomes
nanomaterials with blood plasma proteins has been highly inves- or lysosomes by coating with pH-sensitive polymers, viral cas-
tigated due to its importance in drug delivery, circulation time, pids, cations, or biodegradable carriers.
organ distribution, and clearance. The consequences of protein NPs that are recognized by surface receptors may activate
adsorption to NPs are not clear; although, depending on the NP cell type-specific signaling pathways leading to cell prolif-
surface, proteins may denature resulting in loss of normal struc- eration or death by apoptosis, stress-related signaling, or
ture and function, with altered enzyme activity or unfolding that calcium-mediated signal transduction events. Dysregulated
exposes new antigenic determinants. An important potential intracellular calcium ion homeostasis may be the consequence
pathologic consequence of serum protein adsorption to NPs is of influx across a damaged plasma membrane permeability
binding of fibrinogen leading to formation of blood clots. barrier or release of calcium ion from the major intracellular
NPs that are inhaled or ingested encounter a lipid mucus storage sites. Sustained elevation in intracellular calcium can
layer that provides a natural barrier to penetration of particu- cause cell death by necrosis.
lates and microorganisms. NPs may adhere to mucins caus- Macrophages are the initial cells to phagocytize inhaled par-
ing enlargement of pore size with increased susceptibility to ticulates deposited in the airways or alveoli. If they are longer
penetration of microorganisms. Smaller, charged NPs may than the diameter of macrophages, incomplete uptake occurs
be repelled by the hydrophilic domains and will not be able to with prolonged generation of ROS by the respiratory burst
penetrate the mucus layer. Aquatic organisms and bacterial mechanism of phagocytes and extracellular release of damag-
biofilms are similarly surrounded. ing lysosomal enzymes. In general, incomplete sequestration
ENPs also bind nucleic acids and have been proposed as of NPs that are too large within lysosomes results in physical
gene delivery devices. Small grapheme oxide nanosheets can interference with cytoskeletal function that can cause impaired
also intercalate into double-stranded DNA and induce DNA cell motility.
breaks in the presence of Cu’* ions.

CAVEATS IN NANOTOXICOLOGIC
TOXICITY MECHANISMS ASSAYS
The mechanistic pathways associated with toxicity are pre- Due to their high surface area and hydrophobicity, NPs
dictable based on the physicochemical properties of ENMs. can adsorb vital dyes, cell culture micronutrients, or released
Oxidative stress due to direct generation of ROS at the surface cytokines.
of NPs or indirectly by target cells following internalization of
NPs isa common mechanism responsible for toxicity of ENMs.
The most vulnerable subcellular organelles and physiologic SAFETY CONSIDERATIONS IN
functions that can be perturbed by exposure to ENPs are sum- NANOMATERIAL DESIGN
marized in Table 28-3.
The cell wall of bacteria and the plasma membrane of eukary- In principle, it should be possible to engineer NPs with desir-
otic cells are the initial barriers to penetration of NPs. Carbon able surface properties for commercial or biomedical appli-
nanomaterials are proposed to act as “nanodarts” creating cations. Capping or coating of NPs using antioxidants may
 CHAPTER 28 Nanotoxicology 417

decrease toxicity. Release of toxic metal ions from quantum Dosemetrics

dots and iron oxide NPs can be minimized using inorganic
Expressing dose-response relationships is most informative
shells or biocompatible polymers. In addition, there is some
utilizing surface area of the NP. Figure 28-4 shows the pulmo-
evidence that CNTs are less pathogenic if they-are shorter or
nary inflammatory dose-response relationship of two sizes
entangled to hide their fibrous nature.
of TiO, particles induced by intratracheal instillation in rates.
A significantly greater influx of inflammatory neutrophils into
the lung was induced by 25nm TiO, per unit mass than by
CASE STUDY: DESIGNING SAFER 250nm TiO,. The result is a very steep dose-response for the
SUNSCREENS nanosized TiO, and a flatter dose-response for the larger TiO,.
Likewise there was a clear separation of the dose-response
As previously mentioned, due to its rapid dissolution in water when based on the number as dosemetric; however, when
and release of Zn,, ions, ZnO NPs are considered as potential the same data was expressed based on particle surface area, a
toxicants. TiO, NPs are used in sunscreens as well. The poten-
tial of ZnO and TiO, NPs to induce photo toxicity and pen-
etrate into the dermis has been a major concern for human
A
safety of sunscreens. A series of skin penetration studies using
both ex vivo and in vivo models showed that these NPs do
not penetrate deeper than the outer most layer, stratum cor-
neum, of intact skin. Thus, it is suggested that the benefits of
protection against carcinogenic UV light radiation provided
by sunscreens formulated with ZnO or TiO, NPs outweigh the
minimal risks associated with phototoxicity, DNA damage,
and skin penetration. 0 === T T 1
0 500 1000 1500 2000
Mass, jg

MAMMALIAN TOXICOLOGY Byers

Sse
jo¥
ie)
Introduction 5
(eB)
6
os

CNTs are a prime example of the two opposing faces of nano- &
© 4
materials: Many highly desirable properties that are suitable &
for numerous beneficial applications contrast with reports of nD

Sue
serious adverse effects in experimental animals. For example, ic

the excitement of future use of CNTs for delivery of drugs, 2

= 0 a aa Cte T T Sa TS Se

genes, and biosensors is dampened by reports of inflamma- 108 Ww (° qo" foP i=

tory fibrogenic and even mesothliogenic effects in laboratory Number
rodents.
C 8

6
Concepts of Nanotoxicology
4
The shape, size, and size distribution are important deter-
minants for the deposition efficiency of inhaled materials
2
throughout the respiratory tract. Uptake into cells is influ-
enced by their surface charge, surface reactivity, the chemistry 0
of surface coatings, and also surface defects to the material as 0 50 100 150 5992008 250
synthesized or introduced during surface functionalization or Surface, cm?
processing.
Many ENPs are insoluble in the as-produced form and do 4 Fine TiO, (250nm) @ Ultrafine(25nm) TiO, —@ Saline
not undergo simple dissolution but can undergo chemical oxi-
FIGURE 28-4 Inflammatory cell response (neutrophil
dation in solution, tissue, or the environment to produce solu- number in lung lavage of rats 24 h after intratracheal instillation
ble species in a process that gradually degrades and eliminates of two sizes of TiO, particles expressed by different dosemetrics.
the particle state. Such NPs can act via a “Trojan Horse” mech- Particle-mass (A); -number (B); -surface area (C). (Reproduced
anism in that they are taken up into cells and subsequently dis- with permission from Oberdorster G, Oberdorster E, OberdorsterJ:
solve, thereby creating a very high intracellular, ionic metal Concepts of Nanoparticle Dose metric and Response Metric, Environ
concentration that is cytotoxic. Health Perspect, 2007 Jun;115(6):A290.)
418 UNIT 6 Environmental Toxicology

common dose-response relationship emerged. More specifi- Dose-rate-Response correlation:

Achieving same lung burden of 200 jug nano TiO, (25 nm)
cally, although the concept of particle surface area is plausible,
Pulmonary response 24 h after end of dosing
the biologically available surface area is of greater value for
defining a proper dosemetric.
30 4 @ Instill-0.5s 2%,
Volume of NPs has been suggested as another dosemetric.
Inhal.- 4h Ra
The “particle overload” hypothesis states as follows: When
® Inhal.-4
x 4h poe
the volume of phagocytized particles in alveolar macrophages poe

exceeds 6% of the normal macrophage volume, their physi-

=

S oe se
ologic clearance function becomes impaired; if the volume iS)
=]
230) | Ae
cD) ¢
reaches 60%, clearance no longer functions. This concept has
— a
f= ya

been applied to estimate certain human occupational expo- oO

> ¢
¢

sure limits. Dependent upon the situation, either surface &

x2 10 yr
oa

area or volume dosemetric may be used. It is also important iL?

to remember that chemical properties of NPs are critical Fle

Pee

determinants of effects resulting from NP-cell interactions i freee Control lO sh/s+0.26 0), alte. cate apes
(Table 28-2). 0 SSF ae T T T 1
107! 10° 10! 102 103 10* 10°
Dose rate (\1g/min)
Portals of Entry
The respiratory tract, the gastrointestinal (GI) tract, and skin FIGURE 28-5 Dose-rate-response correlation: Deposition
of 200 jug nano-TiO, in the lungs of rats either by instillation (high
are the main organs of direct exposure of ENM. For medical
dose rate) or by inhalation (low dose rate) induces widely differing
application, injection will also be an important entry route.
pulmonary inflammatory responses as determined by the appearance
Intake via the respiratory tract is the most prevalent exposure of inflammatory neutrophils in lung lavage. (Used with permission of
route for occupational exposures. Additives of ENM to food G. Oberd6rster).
and potential contamination of food result in exposure via
GI tract. Based on available data, translocation of nanomate-
rial in vivo across Gl-tract epithelial cells seems to be limited;
however, DNA damage has been found in bone marrow of cells in dose rate is significant with no response at the lowest dose
following very high gavage dosing of rats. Skin exposure via rate of inhalation. This supports that adaptive responses are an
cosmetic and skin-care products occurs, although penetration important physiologic protective mechanism, which need to
of healthy skin by NP has not been demonstrated. be considered when interpreting results of nanomaterial toxic-
ity testing. Despite the limitations of bolus-type delivery, they
may be viewed as “proof of principle” with the findings to be
Dosing of the Respiratory Tract confirmed by subsequent inhalation studies. The concept of
differential adsorption states that the physicochemical prop-
Dosing of the respiratory tract of laboratory rodents involve
erties of nanoparticles such as size, surface properties, shape,
the administration of materials as a bolus in a second or less.
dissolution, and others when in contact with media in the dif-
However, inhalation is the only physiologic method and
ferent body compartments, such as respiratory tract lining
should be considered the gold standard for exposure to air-
fluid, gastrointestinal secretions, etc., determine protein and
borne materials. Major differences between bolus-type and
lipid adsorption and thereby influence biodistribution across
inhalation exposures relate to the dose rate, use of anesthesia,
barriers and in target tissues and cells.
and the distribution of administered material within the respi-
ratory tract.
Bolus delivery occurs within a fraction of a second, whereas
inhalation at realistic concentration takes hours to months Respiratory Tract Deposition
of exposure in order to deposit the same does in the lung. Inhalation of ENMs results in significant deposition in the
Treating a dose delivered by bolus to be the same that has accu- three compartments of the respiratory tract: the nasopharyn-
mulated in the lung over a lifelong exposure is not justifiable. geal region from the nose/mouth to the larynx, the tracheo-
Inundating cells abruptly with an extraordinarily high dose bronchial region from the larynx to terminal bronchioles, and
overwhelms the cell’s defense mechanisms and leaves no time the alveolar region from the first generation of respiratory
for developing adaptive responses. Consequently, mechanisms bronchioles to the last generation of alveolar ducts. The depo-
of effects induced but unrealistic high doses are different from sition efficiency depends on particle characteristics, anatomi-
those induced by relevant dose and dose rates. cal structure of the airways, and breathing parameters. Particle
Figure 28-5 illustrates a tremendous difference of inducing size, size distribution, density, and shape are the most impor-
a pulmonary inflammation by either intratrachially instilling tant because they govern deposition in the respiratory tract by
200 ug of TiO, NP versus depositing the same dose by inhala- intertial impact, gravitational settling, and displacement
tion over a period of four hours for four days. The difference by diffusion.
 CHAPTER 28 Nanotoxicology 419

Studies have been performed involving nasal inhalation the interstitium and subsequently into blood and lymph cir-
in rats and humans. Obvious differences between rats and culation distinguishes NPs from microparticles. Figure 28-6
humans are the maximum size of particles that are respirable, depicts the blood compartment as a plenum from which any
that is, will reach the alveolar region. In rats this is about 5 um tissue or organ can be reached by circulating NP. However,
aerodynamic size, in humans about 15um. Although these the amount of NP translocating from the lung to the blood
sizes are outside the range ofsingle NP, airborne NP occurs for circulation and accumulation in secondary organs is very
the most part as agglomerates. low. Long-term retention studies with radioactive NPs have
It should be noted in a reminder that realistic in vivo doses shown that clearance in extrapulmonary organs following the
to cells of the respiratory tract are mostly orders of magnitude initial accumulation is very efficient, so after six months, with
lower than doses that are typically applied in vitro to lung the exception of liver and spleen, only minor amounts were
epithelial cell cultures. In the alveolar region where the airflow still present. Despite the low translocation rates, it has to be
is very low, no deposition hotspots for NPs exist. This nonho- considered that continuous exposure may result in significant
mogeneous deposition and formation of hotspots seem to cor- accumulation in some secondary organs.
relate with predilection sites for bronchial carcinoma, which
is further enhanced by less effective mucociliary clearance at
carnal ridges. Nanomaterials and the Brain
Organs with tight endothelial junctions, in particular the CNS,
will not likely accumulate blood-borne NPs, unless the tight
Respiratory Tract Clearance and blood-brain barrier is damaged or NP surface has been spe-
Disposition of NP: Nanomaterials cially modified. The most efficient pathway of NP transloca-
Once NPs are deposited in the respiratory tract they will tion to the CNS appears to be via olfactory sensory neurons
encounter clearance mechanisms. However, there are several from the nasal olfactory mucosa directly to the olfactory bulb.
differences that separate NP from larger particles. Alveolar Results of epidemiologic studies of impaired cognitive func-
macrophages generally are attracted to deposited particles tion and of neurodegenerative brain pathology associated with
by chemotactic signals generated at the site of deposition. exposure to traffic-related particles raised the question as to
NPs may be too small to generate such signals leading to whether ambient UFPs as constituents of urban air pollution
uptake into the pulmonary interstitium. Translocation into may be etiologically involved.

——>_ Confirmed routes Exposure and biokinetics of nanoparticles

----> Potential routes

|‘Exposure media. Air, water, clothes | Drug delivery

Deposition Inhalation Ingestion

ieee Respiratory tract _

Trach-

lee

(platelets, monocytes,
endothelial cells)

| Bone marrow Other sites

ee
SS
(eg, muscle,
--
-
--.
placenta) \
Pisa eat ape Ponca ants
—— Excretory \

“ Sweat/exfoliation Urine || Breast milk | Feces |

| pathways

FIGURE 28-6 Exposure and biokinetics of nanoparticle routes of exposure and biokinetics (uptake, distribution, elimination)
of nanomaterials. Translocation rates in general are very low (see text). (Reproduced with permission from Oberd6rster G, Oberdérster
E, OberdérsterJ:Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles, Environ Health Perspect, 2005
Jul;113(7):823-839.)
420 UNIT 6 Environmental Toxicology

Elimination of Nanomaterials to determine these properties, in particular as they appear in

the airborne state at sites of human exposures, at occupational
Elimination pathways for ENM from the body include mainly
sites, or for the consumer. Adding dispersants for testing pur-
feces and urine. Urinary excretion is restricted to nanostruc-
poses will change surface properties; conceptually, inhala-
tures <5.5nm in size for metal-based NP. Circulating fibrous
tion studies in experimental animals for purposes of hazard
structures of ENM, such as large MWCNTs, can collect in the
identification should mimic human exposure conditions with
urine of rats following intravenous application. This phenom-
regard to airborne size distribution. Of course, differences in
enon may be explained by a hydrodynamic lining of nanotubes
respirability between humans and rodents must be consid-
so they will pass through glomerular pores. The fecal excretory
ered and adjustments be made without use of surface altering
clearance pathway consists of several inputs: one is mucocili-
dispersants.
ary clearance of deposited particles from the airways into the
Appropriately designed multiconcentration, subchronic
GI tract; another is the hepatobiliary clearance of blood-borne
inhalation studies, including a longer recovery period, are essen-
ENM via liver and bile into the small intestine. This elimina-
tial for deriving no observed adverse effect levels (NOAELs);
tion pathway is also a well-known excretory path for heavy
results can be used as basis for deriving occupational exposure
metals in the blood.
levels (OELs) by applying rodent/human dosemetric adjust-
Another clearance pathway of deposited ENM in the lung
ments. Using results from bolus-type studies is difficult and
involves translocation via interstitium or lymph to the pleura
raises questions, although national institute for occupational
and subsequent elimination via lymphatic openings on the
safety and health has combined results of fibrotic responses
parietal pleura to mediastinal lymph nodes from where NP
from diverse bolus-type and inhalation studies to derive a pro-
may enter the blood circulation via the thoracic ducts. This
visional recommended exposure level of 7 g/m’. This REL is
pathway is of particular importance for fiber-shaped ENM
based on dose-response data from the available studies with
because the size of the parietal stomata prevents efficient
bolus-type and short-term inhalation exposures and a sub-
clearance of structures >10um in length. As a consequence,
chronic inhalation study.
the interaction of the retained fibers in the pleural cavity with
There has been no conclusive data regarding carcinogenic
mesothelial cells induce inflammatory and granulomatous
effects of realistic exposure to CNTs. Thus, exposure should be
responses and in long-term potentially mesothelioma.
avoided with appropriate measures (ventilation, filtration, per-
sonal protective equipment). There is an obvious and urgent
CASE STUDY: MWCNTS need to perform additional long-term inhalation studies to
assess carcinogenic potential.
Bolus-type Exposures
Bolus-type delivery of CNTs to the respiratory tract of rats and Biologic Degradation of Carbon Nanomaterials—
mice revealed induction of dose-dependent significant inflam- CNTs have been generally regarded as stable nondegradable
matory, granulomatous, and fibrogenic responses; they showed materials, which has important implications for long-term
also that MWCNTs can reach subpleural and intrapleural sites. health effects following inhalation into the lungs. Recently,
In addition, intraperitoneal injection studies clearly show the however, SWCNT degradation has been observed in acellular
potential of CNTs, specifically MWCNTs, to induce severe assays that stimulate the phagolysosome of macrophages, but
adverse length-dependent effects at mesothelial sites once they only if the tubes have been surface carboxylated, which intro-
reach the pleural cavity. duces collateral defects in the side walls. Graphene oxide is
also susceptible to oxidative attack by hydrogen peroxide and
horseradish peroxidase. These observations may enable design
Inhalation Studies of safer carbon materials that are potentially biodegradable in
Relatively few inhalation studies with CNTs in rodents have order to minimize adverse environmental and human health
been reported. The most meaningful and best justified for impacts. However, degradation of CNTs in vivo is still to
the risk assessment process would be a subchronic multicon- be confirmed.
centration study with sufficient postexposure observation.
However, short-term exposure to a relevant concentration is
useful for dosemetric purposes when determining the bio- TOXICITY TESTING
distribution from deposition sites in the lung to secondary
organs. Parameters varied, but within these variations out- In order to perform risk assessment, exposure and hazard
comes ranged from no significant effects to severe pulmonary data are required. To identify and characterize a hazard, in
inflammation/oxidative stress responses. vitro and in vivo studies will be useful, and results should be
derived via well-designed dose-response relationships. Key
considerations include physicochemical characterization of
Critical Appraisal of CNT In Vivo Studies the ENM to be tested, justification of the method(s) of dosing,
Given the importance of the physicochemical properties of selection of target cells, tissues, or animal species, and appro-
CNTs for inducing adverse effects, it is of utmost importance priate end points.
 CHAPTER 28 Nanotoxicology 421

Considering exposure and hazard for risk assessment

Long-term goal
Prediction
Extrapolation Prediction
Prediction Validation

Phys-chem. properties In vivo Phys-chem. properties In vitro

Target organs animals (inhal/Bolus) endpoints; ref. material Cells (bolus/ALI)
Workplace Respirability Biokinetics hi-lo dose; relevancy Target cells,
Laboratory (translocation; tissues
Consumer NOAELs; OELs; corona formation) Mechanisms dose-response
HECs Exposure- reproducibility
dose-response
Inhalation; oral; dermal Pristine; dispersed Long-term
Dosimetry Dosimetr eeedsaos5
” In silico |
|_models |
FS
Exposure Hazard (hazard scale)

_
Risk assessment

FIGURE 28-7 Concepts and goals of nanomaterial toxicity testing (see text).

In vitro studies should be stressed as far as uncovering Knowledge about the time to deposit a certain dose allows con-
underlying mechanisms of effects are concerned. They are also sideration dose rate as a determinant for responses.
useful for toxicity ranking of nanomaterials for the purpose of
hazard identification. In contrast, the design of in vivo stud-
ies allows the full evaluation of exposure dose-response rela- Predictive Toxicology
tionships, which is necessary for the process of risk assessment Critical elements for hazard identification based on toxicity
(Figure 28-7). testing of ENM are detailed information about their physico-
For medicinal applications, injection is an important route chemical properties prior to any experiments, the selection of
of exposure requiring specific awareness with respect to assur- appropriate target cells, validation of in vitro assays in terms
ing desired beneficial outcomes yet avoiding undesirable of correlation and relevancy to in vivo results, the inclusion
responses. For example, the desired pharmacologic target of biokinetics in the design of in vivo studies, and the inclu-
organelle for drug delivery by SWCNT are the lysosomes, sion of realistic doses in the design of dose-response in vitro
whereas the mitochondria are the target organelles for SWCNT and in vivo studies. Biokinetic information is crucial to iden-
toxicity. tify potential secondary target organs based on significant
accumulation of ENM. With real-world exposure scenarios
relationships can be established to both characterize a hazard
in Vitro Dosimetry and assess a risk. An awareness of dosimetry-related aspects
As most ENM toxicity studies are performed using in vitro is of highest importance (Figure 28-7) for the risk assessment
assays, which are generally short-term, dosing-related ques- process, which often gets lost or is ignored because of a mis-
tions are highly relevant. The dose received by the cell is a func- conception of risk being analogous to hazard. Risk is a func-
tion of colloidal dynamics in the culture medium governed by tion of hazard and exposure, and neither aspect alone can
diffusion and settling phenomena, which in turn is governed determine risk.
by particle and media properties that include particle size,
agglomeration state, shape, density, and charge. At equal mass
concentrations in the medium, the magnitude of the cellular Transition, Human-Eco-nanotoxicology
dose of ENM will differ significantly from those implied by the The goals of nanotoxicology are to identify and characterize a
media concentration. hazard of ENMs for purposes or risk assessment for humans
An in vitro sedimentation diffusion and dosimetry (ISDD) and the environment. Exposures throughout the life cycle of
model may be used to predict the in vitro behavior and cell ENMs, from their source to their disposal, have to be consid-
doses of particles. The value of this model lies in the clear sepa- ered for both. Dispersion and intermediate transformations
ration between exposure (concentration in the cell medium), in air, water, food, and soil are important modifiers of ENM-
the deposited dose on the cell surface, and the cellular dose. receptor interactions.
422 UNIT 6 Environmental Toxicology

ECOTOXICOLOGY OF ENMS change over time as particles are effectively removed from sus-
pension. Researchers have attempted to circumvent this issue by
Environmental Uses and Exposures to either changing the surface chemistry or by altering the expo-
sure conditions. Unfortunately, changing the surface chemistry
Nanomaterials
of a nanomaterial can also change its toxicity. Worse yet is that
As with many chemicals in the marketplace, it is estimated that many of the coatings can cause toxicity on their own regardless
a portion ofthe nanomaterials used in industry and consumer of whether it is attached to the nanomaterial.
products will enter the greater environment during some As part of determining the dose an organism actually encoun-
part of their life cycle, either through waste during produc- ters is determining how much of any nanomaterial actually
tion or through product use. The exponential increase in use reaches the organism and is taken up. There are several difficul-
of ENMs in a multitude of industries and consumer products ties in measuring uptake including identifying the nanomaterial
have been documented and shown exponential increase by the within the matrix of the organism versus inside the organism.
Woodrow Wilson Center's project on Emerging Technologies. Another complication is that of calculating the dose as a mass
The most common nanomaterials within cosmetics, cloth- versus surface area. The real adverse impacts of nanomaterials
ing, personal care products, and sporting goods are silver and may not be due to the ambient environmental concentrations
carbon. Other chemicals used in these products have been that arise but may be due to some subset of materials that are
found to wash into the wastewater treatment system and end persistent and biomagnify in the environment.
up in the aquatic environment. Despite removal potentials, it
has been shown that NPs can be emitted through the waste-
water process in the NP size range in significant quantities. Ecotoxicity of Nanomaterials
Nanomaterials directly applied to a particular ecosystem, such The studies on the toxicity of ENMs to date conclude that tox-
as those for cleanup of environmental toxicants or as part of a icity varies with the type of nanomaterial and is not universal
pesticide formulation, may also lead to exposure. across materials. Most studies find some degree of toxicity but
the concentrations of most nanomaterials that are needed to
kill half the sample population are in the mg/L range, which is
Ecologic Risk Assessment of Manufactured
far above the estimates of potential exposures. Silver nanoma-
Nanomaterials terials are some of the most widely used materials and appear
For nanomaterials most of the ecologic risk assessment to demonstrate the greatest toxicity of materials investigated
research has been conducted as the analysis ofeffects of a lim- in the literature. Silver in particular is toxic at [1g/L doses to
ited number of commercially available materials, using tra- a variety of organisms. Rather than creating a free radical in
ditional acute single-organism mortality end points of a few media, the impacts of metal nanomaterials may be due to metal
select species and with little information regarding sublethal imbalance in cells after uptake and accumulation leading to
types of effects or other end points of concern at the commu- apoptosis and cellular disregulation.
nity or ecosystem level. In addition, the concentration of expo- Nanosilver and possibly other nanomaterials based on soft
sures are much higher than what is considered to be a probably metals may react with environmental sulfides to produce silver
environmental level. Nanomaterials may also be transformed sulfide nanomaterials in which the silver bioavailability and
within the environment, and therefore the toxicity of the initial toxicity is much reduced.
nanomaterial may not provide a complete idea of the toxicity
over the lifetime of the material.
Mechanisms ofToxicity
As in mammalian toxicology studies, oxidative stress has been
Toxicity of Manufactured Nanomaterials implicated as a major way in which nanomaterials exert tox-
Complications of Assays—Traditionally, ecotoxicology icity either by generating free radicals within the suspension
assays follow standard protocols and involve a group of species media or by changing the chemistry of the cells in which they
that has been selected to be representative of various organ- come in contact. Metal oxide nanomaterials in particular have
isms in the environment including bacteria, fish, birds, and been found to generate oxidative stress with greater toxicity
insects. Some major issues in toxicology assays include delivery than their bulk counterparts. Metal nanomaterials have been
of nanomaterials in media and approximating environmental found to cause a suite of effects, which in fish include negative
conditions, characterizing exposures, maintaining exposures impacts on respiration, oxidative stress, and development, in
throughout an assay, and determining the state of exposure Caenorhabditis elegans increased mortality and decreased
throughout an assay. The fact that many nanomaterials are not reproduction and inhibit algal growth.
easily dispersible and aggregate substantially when introduced The toxicity of nanomaterials to aquatic organisms can be
into common exposure media causes several issues. First, aggre- greatly dependent upon the interaction of nanomaterials with
gated nanomaterials may no longer be in the nanosized range. the media to which they are introduced. Nanomaterials may
Second, nanomaterials as they aggregate settle out of suspension, also impact the bioavailability and toxicity of other contami-
so depending on the organism involved the actual exposure may nants in the environment.
 CHAPTER 28 Nanotoxicology 423

As the immune response is the first interaction of foreign sub- BIBLIOGRAPHY

stances with an organism, it has been shown that nanomaterials Houdy P, Lahmani M, Marano F (eds.): Nanoethics and Nanotoxicol-
are stimulatory to the immune system of fish in particular and ogy. New York: Springer, 2011.
have an effect that is equal to the response to bacterial cell com- Monteiro-Riviere NA, Tran CL (eds.): Nanotoxicology: Progress
ponents, which may indicate an eventual cost to the organism. toward Nanomedicine, 2nd ed., Boca Raton, FL: CRC Press, 2014.
Nanomaterials also have the potential to be mutagenic and in Sahu SC, Casciano DA (eds.): Handbook of Nanotoxicology,
fruit flies cause mutations that alter the phenotype significantly Nanomedicine and Stem Cell Use in Toxicology. West Sussex: John
into the second generation. Wiley & Sons, 2014.
 UNIT 6 Environmental Toxicology

QUESTIONS

Which ofthe following is not a nanoparticle? Nanoparticles can exert toxicity by all of the following
a. carbon nanotubes. mechanisms except:
bucky-ball. a. damage to DNA and chromosomes.
c. graphene. induction of oxidant stress.
d. zinc nanorods. interference with biotransformation enzyme activities.
e. bacteria. activation of signaling pathways.
oe release of toxic metal ions from internalized NPs.
eS.
Which of the following answers is not true regarding
nanoparticles? Biodistribution of nanoparticles may be influenced by
a. NPs can originate from natural sources including a. physicochemical properties such as plasma protein
forest fires, volcanoes, and viruses. and respiratory tract mucus.
b. NPs can originate from unintentional sources includ- b. physicochemical properties such as surface size and
ing internal combustion engines and electric motors. chemistry.
c. NPscan originate from unintentional sources includ- c. physicochemical properties such as the gastrointesti-
ing ferritin and magnetotactic bacteria. nal milieu.
d. NPs can originate from intentional sources including d. body compartment media including surface
carbon nanotubes and metal oxide nanoparticles. hydrophobicity.
e. NPs can originate from natural, and intentional and e. body compartment media including size.
unintentional anthropogenic sources.
Assays to determine the toxicity ofmanufactured nanopar-
In contrast to larger particles >500 nm, nanoparticles ticles suffer from all of the complications below except:
a. are highly likely to enter the body by dermal a. the nanomaterial aggregate may no longer be in the
absorption. nanosize range.
b. are highly likely to enter the body through the respi- b. aggregates of the nanoparticle may settle out of solu-
ratory tract. tion which may affect exposure dose.
c. are unlikely to adsorb to protein or lipid. c. alterations in surface chemistry to stabilize suspen-
d. are efficiently removed from the lungs via mucociliary sion may evoke other issues in toxicity assessment.
transport. d. coatings of particles may have their own toxicity.
e. are not likely to undergo uptake and transport in e. uptake of the nanoparticle into an organism is easily
sensory neurons. determined.

Which of the following statements is NOT true? The goals of nanotoxicology are
a. Nanomaterials may be classified by geometry and a. to identify and characterize hazards of engineered
chemistry. nanomaterials.
b. Engineered nanomaterials include quantum dots, b. to determine “safe” exposure levels.
C-nanofiber array, and few-layer grapheme. c. to determine biologic and biochemical actions.
c. Agglomerates include primary particles held together d. to determine manufacturing procedures and cost.
by weak van der Waals forces. e. to determine preventive exposure guidelines.
d. Aggregates include primary particles held by strong
chemical bonds.
e. Hydrodynamic diameter is unimportant in particle
interactions.
 Ga Hehe PER

Air Pollution*
Daniel L. Costa and Terry Gordon

AIR POLLUTION IN PERSPECTIVE Particulate Matter

A Brief History of Air Pollution and Its Regulation Metals
Gas-Particle Interactions
TOOLS TO ASSESS RISKS ASSOCIATED WITH Ultrafine Carbonaceous Matter
AIR POLLUTION
Chronic Effects and Cancer
Animal-to-Human Extrapolation: Issues and Mitigating Photochemical Air Pollution
Factors Chronic Exposures to Smog
OVERARCHING CONCEPTS Ozone
General Toxicology
What Is an Adverse Health Effect?
Pulmonary Function Effects
Susceptibility
Ozone Interactions with Copollutants
EXPOSURE Nitrogen Dioxide
Air Pollution: Sources and Personal Exposure General Toxicology
Indoor versus Outdoor Pulmonary Function Effects
Indoor Air in the Developing World Inflammation of the Lung and Host Defense
The Evolving Profile of Outdoor Air Pollution Other Oxidants
Aldehydes
EPIDEMIOLOGICAL EVIDENCE OF HEALTH EFFECTS
Formaldehyde
Outdoor Air Pollution Acrolein
Acute and Episodic Exposures Carbon Monoxide
Long-Term Exposures Hazardous Air Pollutants

POLLUTANTS OF OUTDOOR AMBIENT AIR THE MULTIPOLLUTANT REALITY OF AIR POLLUTION

Classic Reducing-Type Air Pollution
CONCLUSIONS
Sulfur Dioxide
Sulfuric Acid and Related Sulfates

*This chapter has been reviewed by the US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily
reflect the views and the policies of the Agency.

425
426 UNIT 6 Environmental Toxicology

= Reducing-type air pollution, characterized by SO, and » Indoor air can be even more complex than outdoor air,
smoke, is capable of producing deleterious human health and outdoor air can permeate the indoor environment in
effects. spite of the reduced air exchange in buildings.
a Photochemical air pollution arises from a series of com- s Sick-building syndrome may occur in new, poorly venti-
plex reactions in the troposphere close to the earth's lated, or recently refurbished office buildings due to the
surface and comprises a mixture of ozone, nitric oxides, outgasing of combustion products, volatile chemicals,
aldehydes, peroxyacetyl nitrates, and myriad reactive biological materials and vapors, and emissions from
hydrocarbon radicals. furnishings.

AIR POLLUTION IN PERSPECTIVE and even when the emissions were vented outdoors, they sim-
ply combined with those of the neighbors to settle around the
The second half of the twentieth century was marked by village on damp cold nights. With urbanization and a con-
remarkable changes in how the public viewed its relation- comitant decrease in forest wood as a source of fuel to heat and
ship to the environment. From expansive urban factories with cook, the need for energy led to the burning of easily acces-
smokestacks belching opaque dark clouds of industrial efflu- sible, dirty coal and the ambient release of sulfurous, sooty
ent into a neutral blue sky, regulation and cost-efficient inno- smoke. Industrialization brought kilns to make quicklime for
vations by the private sector have reduced emissions. Decades construction and metal smelters needed for the development
to come will see change in our energy portfolio that is driven of progressive “modern” cities, only to push smoke and chemi-
by cost and access, environmental impacts including climate cal emissions into the air. Unfortunately, the city dwellers who
change, and technological innovation. Nevertheless, so long worked near these industries had to endure the bad air, while
as organically derived fuel is combusted to derive energy, its those of wealth frequently had country homes to which they
potential for impact on air quality and on public health and could escape.
the environment will remain. As the developing world grows The accidental release of 30 tons of methyl isocyanate vapor
industrially, air pollution now is intercontinental with trans- into the air of the shanty village of Bhopal, India, on December 3,
port through the atmosphere via pathways close to the earth’s 1984, killed an estimated 3000 people within hours of the
surface as well as upper atmosphere. Air pollution now extends release, with several thousand delayed deaths and 200000
even into remote and wilderness areas, and significant damage injured or permanently impaired. The tragedy shocked the
to flora and crops can also occur. world, and raised the issue of hazardous air pollutants (HAPs)
Other issues facing many parts of the developing world tie to a new level of concern.
closely to domestic culture and economy, as well as to the level The HAPs have since garnered more public and policy atten-
of technological sophistication. Prime among these problems tion. There is concern for the acute effects ofaccidental releases
is exposure to carbon and soot from combustion of biomass of fugitive or secondary chemicals—such as phosgene, ben-
in cooking and heating in domestic stoves. Approximately zene, butadiene, and dioxin, into the air of populated industrial
three billion people worldwide use biomass for home cook- centers—and for potential chronic health effects, with cancer
ing in households with little ventilation. The World Health often being the focus of attention. While many of HAP chem-
Organization (WHO) estimates two million deaths per year as icals are now better controlled than in the past, residual risk
a result of these exposures, especially women who are exposed estimates are yet to be completed for many HAPs. The database
day in and day out over many years, often with their infant chil- from which these assessments are made is called the Integrated
dren by their sides. Understanding the intersection of techno- Risk Information System (IRIS, www.epa.gov/iris/index.html)
logical as well as socioeconomic and political challenges will be and currently contains 550 chemicals that have health data.
at the core ofany resolution to these issues. Internationally, the magnitude and control of air pollu-
tion sources vary considerably, especially among developing
nations, which often forgo concerns for health and welfare
A Brief History ofAir Pollution and
because of cost and the desire to achieve prosperity. Figure 29-1
Its Regulation illustrates the international variation in air pollution-related
For most ofhistory, air pollution has been a problem of micro- mortality (outdoor and indoor) based on economic groupings.
environments and domestic congestion. The smoky fires of It is clear that there are wide differences reflecting economic
early cave and hut dwellers choked the air inside their homes, imbalances—particularly prominent are the indoor particulate
 CHAPTER 29 Air Pollution 427

“international pollution.” Long-range transport of polluted

[) Urban ambient
air masses from one country to another has been a global issue
0 Urban indoor
for several years. The air mass transport of acid sulfates from
(J Rural indoor
industrial centers of the Midwestern United States to southern
Canada, and of NO, and airborne mercury from China to the
US are examples.
Ww

TOOLS TO ASSESS RISKS ASSOCIATED

WITH AIR POLLUTION

|
aN
thousands)
of
(hundreds
Mortality Risk assessment is a formalized process whereby toxicity, expo-
Ee i co | sure, and dose-dependent outcome data can be systematically
EME EE China India SEAWP EM AL SSA integrated to estimate risk to a population. Figure 29-2 out-
lines a paradigm for incorporating all available data and risk
FIGURE 29-1 Excess mortality due to outdoor and indoor
assessments to providing evidence of “accountability” of appli-
particulate matter in various international economic groupings.
Bottom and top of each bar represent the lower and upper estimates
cable regulations on public health. The health database for any
of mortality, respectively, computed using the methodology of air pollutant may comprise data from animal toxicology, con-
Schwela (2000): established market economies (EME), Eastern Europe trolled human studies, and/or epidemiology. But, because each
(EE), China, India, Southeast Asia/Western Pacific (SEAWP), Eastern of these research approaches has inherent strengths and limi-
Mediterranean (EM), Latin America (AL), and Sub-Saharan Africa (SSA). tations, an appropriate assessment of an air pollutant requires
(Modified with permission from Schwela D:Air Pollution in the Megacities the careful integration and interpretation of data from all three
of Asia—Seoul Workshop Report: Urban Air Pollution Management and methodologies.
Practice in Major and Megacities of Asia.) Epidemiological studies reveal associations between expo-
sure to a pollutant(s) and the health effect(s) in the community
levels in developing nations where biomass combustion is used or population of interest. Because data are garnered directly
for heating and cooking. These regions also contain many of under real-world exposure conditions and often involve large
the megacities of the world with major air pollution prob- numbers of people, the data are of direct utility to regulators
lems. In addition to local socioeconomic and political con- assessing pollutant impacts. With proper design and analy-
cerns, emissions of air pollutants have spawned problems of sis, studies can explore either acute or long-term exposures

Exposure Risk assessment Risk management

(Source to receptor) (Receptor response)

Biological Adverse
ff effects 4 health effects Legal considerations
Sources
\

WA
/

Health
assessment
Transport and Public health
transformation

|
considerations

Environmental
Social, Risk
concentration oa
Oe management
Exposure & political Ae
\ Exposures , decision
~ assessment factors /

x Risks es ; aS Saye Oe ANS

7
Mee
Sees
Ss
ae
|
ublic health improvement —
Bg RE REIS SESE Bas
Accountability

FIGURE 29-2 NRCrisk assessment paradigm. Components of risk assessment within the left circle provide data to development of risk
managementas depicted in the right circle, modified to include an “accountability” component as a means to address air quality management
impacts on the process risk reduction.
428 UNIT 6 Environmental Toxicology

and theoretically can examine patterns in mortality and mor- whenever possible, effects that are homologous and involve
bidity, both acute and chronic, especially if these responses the same mode of action between the study species and the
appear disproportionately in population subsets (i.e., sensitive human should guide the decision of the most appropriate test
groups). However, it is difficult to control confounding per- species. An essential, but often overlooked, part of response
sonal variables in the population, such as genetic diversity and extrapolation from species to species is knowledge of the rela-
lifestyle differences among individuals, and population mobil- tive dosimetry of the pollutant along the respiratory tract.
ity are difficult to control. Perhaps most problematic is the lack Significant advances in studies of the distribution of gaseous
of adequate exposure data—especially on a personal basis. and particulate pollutants have been made through the use
Studies that involve controlled human exposures are very valu- of empirical and mathematical models, the latter of which
able in assessing potential human risk, since they are derived incorporate parameters of respiratory anatomy and physiol-
from the species of concern and are rooted in well-established ogy, fluid dynamics, and physical chemistry into predictions
clinical knowledge and experience. of deposition and retention. Empirical models combined
Animal toxicology is used to predict or corroborate, through with theoretical models aid in relating animal toxicity data to
plausible mechanisms, suspected effects in humans. In the humans and help refine the study of injury mechanisms due
absence of human data, animal toxicology constitutes the to better estimates ofthe target dose.
essential first step of risk assessment: hazard identification.
Animal toxicology is often required before any controlled
human exposure can be conducted. It is particularly use- OVERARCHING CONCEPTS
ful in elucidating pathogenic mechanisms involved in toxic
injury or disease, providing basic knowledge that is critical to What Is an Adverse Health Effect?
extrapolating databases across species, estimating uncertain- When relating a health effect to an air pollutant, a response
ties, and determining the relevance of information to humans. must be appreciated at two levels—that of the individual and
Knowledge of the toxic mechanism(s) provides the underpin- that of the population. Clearly, an effect on an individual can
nings to the “plausibility” of findings in the human context and, be beyond an acceptable limit potentially putting that per-
under carefully defined and highly controlled circumstances, son’s overall health in jeopardy, but this response may be lost
may allow quantitative estimates ofrisk to human populations. in an index reflecting a population-based response. The risk
Animal toxicology studies have been used to investigate all of to a population reflects the averaging of individual responses
the criteria air pollutants (ozone, sulfur dioxide, nitrogen diox- or risks and may be measured as a shift in the normal distri-
ide, carbon monoxide, particulate matter and lead) and many bution of some index of response for that population. Hence,
of the HAPs (over 30 compounds listed) as well. The strength on average, the entire population may be judged to be at some
of this discipline is that it can involve methods that are not enhanced risk. These two forms of risk are clearly related, but
practical in human studies and can provide more rapid turn- most often in practice, the population risk is considered most
around of essential toxicity data under diverse exposure con- appropriate from a public health perspective. It is also gener-
centrations and durations. The minimization of uncontrolled ally most credibly quantifiable.
variables (e.g., genetic and environmental) may be the greatest Defining an air pollutant effect as “adverse” within the
strength of the animal bioassay. range of effects that may result from exposure is not always
Lastly, studies of botanical responses to air pollutants are straightforward. Clearly, in humans, some effects would pass
now appreciated more than ever. Not only are commercial uncontested as adverse, e.g., death, acute life-threatening dys-
and native vegetation affected by pollution but also some plant function or disease, irreversible impairments, and pain. In ani-
species are being exploited as sensitive “sentinels,” warning mal models, pathology has traditionally been the hallmark of
of the impacts of pollution on both human and environmen- an adverse effect. In either humans or animals, however, other
tal receptors. Interestingly, some basic mechanisms (e.g., the effects that reflect minor and temporary dysfunctions or dis-
involvement of antioxidants) between plants and animals have comfort could be argued as not warranting significant or costly
remarkable parallels. concern, especially if the effects are minor and transient with
no long-term untoward outcomes. This vein of thought would
simply attribute these effects to be within normal physiologic
Animal-to-Human Extrapolation: Issues
ranges and are readily compensated within functional or bio-
and Mitigating Factors chemical reserve. Thus, if one is to try to assess the impacts
The value of animal toxicology in inhalation studies is highly of air pollution on health, it is desirable that there exist some
dependent on the ability to extrapolate or relate empirical find- objective criteria to define what is indeed adverse based on
ings to real-world human scenarios. Several factors of study the nature and the magnitude of the effect under evaluation.
design play into the process of extrapolation (e.g., exposure Moreover, distinguishing an air pollution effect from other
concentration, duration, and patterns), but most important is adverse stimuli or disease processes can be complex and
the selection of the animal species that will serve as the toxico- fraught with confounding factors, such as smoking and nega-
logical model. Although cost and convenience are considered, tive lifestyle factors.
 CHAPTER 29 Air Pollution 429

TABLE 29-1 Factors that influence the response of

individuals or subpopulations to ambient air pollutants.

Susceptibility Factors Vulnerability/Exposure Factors

Preexisting cardiopulmonary Proximity to point source

disease

Genetic factors Proximity to high-traffic-volume

roadway
Ce

Age Occupation
ee re

biomarker
of
Level ers
oe
yee Gender Activity level
4 -F
=eie
Race/ethnicity Use ofair conditioning/building
leakiness

Obesity In utero exposure

Exposure dose of an air pollutant
Pregnancy Geographic location (e.g., East versus
West coast of the United States)
FIGURE 29-3 Schematic illustration of the elements of the
dose response to an air pollutant(s) of a susceptible versus a Diabetes Lower social economic status
healthy individual. The hypothetical susceptible individual may be
more sensitive or may have a loss ofreserve, either of which results in
an inability to maintain homeostasis. The leftward shift or increased slope
in the dose-response curve suggests an increase in responsiveness.
Either situation may contribute to sensitivity and the likelihood of once considered “outliers” in a controlled chamber study may
enhanced progression from subtle to severe outcomes. well be evidence of unusual responsiveness.

Susceptibility EXPOSURE
A common thread through these subject areas is the influential
Air Pollution: Sources and
role of susceptibility, which can take the form of hyperrespon-
siveness or loss of reserve. What is a minor reversible effect in Personal Exposure
the majority of individuals may be a dysfunction that cannot be Six major air pollutants (particulate matter (PM), O;, NO,
reversed or compensated in certain individuals (Figure 29-3). SO,, CO, and Pb) are considered ubiquitous to industrial-
Obvious examples would be cardiopulmonary-compromised ized communities and are thought to carry the greatest risk to
individuals who function with little or no reserve. As science human and environmental health. With the exception of O,,
continues to advance, especially in the realm of molecular these pollutants are emitted by anthropogenic combustion
biology where small signals can be detected that may fore- processes along with the myriad chemical compounds (mostly
cast an adverse effect or otherwise may identify individuals volatile organic compounds [VOCs]) considered under the
or groups at risk, the definition of adverse will certainly need category of HAPs. There are many natural sources of air pollut-
reexamination. ants as well (e.g., volcanoes, wildfires, windblown dust, natural
In actuality, there is no widely accepted definition for a biogenic vapors) but it is the anthropogenic sources that emit
“susceptible” individual and quite frequently the term is used pollutants, which concentrate where people live, that raise
interchangeably with “vulnerable.” However, “vulnerability” concerns about potential health impacts. These factors do not
refers to extrinsic nonbiological factors (e.g., an increased dismiss the significance of potential risks posed by the natural
exposure to ambient air pollutants because one’s school is emissions but put focus on the potential for human exposure
located adjacent to a high-traffic-volume roadway), whereas and risk.
“susceptibility” refers to intrinsic biological factors such as Assessing exposure to an air pollutant has long rested on
genetics, age, or preexisting disease. Factors that influence observational measures of what is in the air. Exposure science
susceptibility and vulnerability are listed in Table 29-1. is advancing rapidly and now utilizes approaches that range
Susceptible subpopulations that show exaggerated respon- from novel statistical treatments of traditional exposure met-
siveness to pollutants merit special mention. Some definable rics to sophisticated models that systematically involve aero-
subgroups that are considered inherently more susceptible dynamic and microenvironmental characteristics to estimate
include children, the elderly, and those with a preexisting dis- or predict exposures to individuals or populations. Typically,
ease (e.g., asthma, cardiovascular disease, lung disease). The inhalation studies of animals involve square-wave exposure
importance of susceptibility in air pollutant responses is gain- patterns, although it is well appreciated that human exposures
ing more and more attention as test subject responses that were vary spatially and temporally.
 430 UNIT 6 Environmental Toxicology

Indoor versus Outdoor—People in the United States (and legitimacy of studies that rely solely on outdoor monitoring
in most industrialized nations) spend in excess of 80% of their data. Nevertheless, the national monitoring network for the
time indoors while at work, school, and home or in an automo- criteria pollutants has been shown to reflect human exposure
bile. Perhaps the most significant risk factor in indoor air for reasonably well for some pollutants, especially those that are
children is the presence of asthma. With nearly 10% of children nonreactive. Indeed, outdoor air permeates the indoor envi-
with asthma, the risk of exposure to pollutants and allergens in ronment in spite of the reduced air exchange in most buildings.
concentrated form is particularly great. However, many variables determine how well components of
As to the issue of total exposure, children and outdoor the outdoor air infiltrate. The complexity of the multiple sources
workers are thought to be more likely to encounter outdoor underscores the importance of appreciating the total exposure
air pollution at its worst; in fact, the relatively high physical scenario if we are to understand the nature of air pollution and
activity levels of these subgroups leads to larger doses of any its potential effects on human health (Figure 29-4).
given pollutant being delivered to the lungs. Defining personal There remain two broadly defined illnesses that are largely
exposure can be extremely difficult, as personal monitoring is unique to the indoor building environment. The first is “sick-
tedious and expensive, and can sometimes be confounded by building syndrome” (SBS), which is a collection of ailments
other contributions to the indicator being monitored. Hence, defined by a set of persistent symptoms enduring for at least
exposure measures are typically drawn from ambient mea- two weeks (Table 29-2) and appears to occur in at least 20%
surements or derived from models developed from studies of those exposed. Frequently but not always, this syndrome
of groups of people carefully characterized across personal occurs in new, poorly ventilated, or recently refurbished office
exposure modifiers—exercise, personal lifestyles, etc. buildings. The suspected causes, include combustion prod-
It is clear that indoor air can at times be more complex ucts, cleaning chemicals, biological emissions from mold,
than outdoor air, a point often raised in challenging the and vapor emissions from furnishings frequently exacerbated
»
‘

_ Sunlight (photochemistry)

Transport medium (air)

<— Wind transport

ee

|
Inahalation
exposure
route

Release source
(site leaching)

Water table
\\ {
—\ ————— ee <a

a Transport medium
(groundwater)

FIGURE 29-4 illustration of contributors to the total personal exposure paradigm showing how these indoor and outdoor
factors interact.
 CHAPTER 29 Air Pollution 431

TABLE 29-2 Symptoms commonly associated with The classical types of air pollution were implicitly seasonal.
the sick building syndromes. Reducing-type air pollution occurred during winter peri-
ods of oil and coal combustion for heating and power coupled
Eyes, nose, and throat irritation with meteorological inversions, while the oxidant atmospheres
Headaches occurred during the warmer months of spring and summer,
when sunlight is most intense and can catalyze reactions among
Fatigue the constituents of auto exhaust. Today the urban distinctions
Reduced attention span between reducing and oxidant smogs have become largely an
academic exercise. As climate change progresses, there is expec-
Irritability tation that the underlying chemistry will change further and
Nasal congestion alter these patterns.
Many megacities remain plagued by the classic reducing and
Difficulty breathing oxidant forms of air pollution. Uncontrolled industrial and coal-
Nosebleeds fired power plant emissions surrounding cities such as Beijing
and the northern sectors of Mexico City are dominated by sul-
Dry skin furous, particulate emissions, whereas southern Mexico City,
Nausea Santiago, and Tokyo have substantially automobile-associated
oxidant smogs. Impacts on health, visibility, and general wel-
fare are clear and are bringing ever increasing public concern.
Urban air pollution is a worldwide problem, where the estimate
by discomfort. The perception of irritancy to the eyes, nose, of people exposed to O; at potentially harmful levels exceeds
and throat ranks among the predominant symptoms that can 480 million.
become intolerable with repeated exposures.
The second syndrome (building-related illnesses) is a group
of illnesses that consists of well-documented conditions with EPIDEMIOLOGICAL EVIDENCE
defined diagnostic criteria and generally recognizable etiology. OF HEALTH EFFECTS
These illnesses typically call for conventional medical treat-
ment strategies, because simply exiting the building where the Outdoor Air Pollution
illness was contracted may not readily reverse the symptoms. Acute and Episodic Exposures—A number of air pollution
Several biocontaminant-related illnesses include Legionnaires’ incidents have been documented where pollutant concentra-
disease, hypersensitivity pneumonitis, humidifier fever, as tions rose to levels that are clearly hazardous to human health.
do allergies to animal dander, dust mites, and cockroaches. Where a single chemical has been accidentally released (e.g.,
Medical treatment and mitigation of exposure (source elimi- methyl isocyanate in Bhopal, India), establishing the relation-
nation or personal protection) are generally needed to abate ship between cause and ill effect is straightforward. However,
symptoms. Some typical outdoor pollutants can also be prob- most air pollution situations involve complex atmospheres,
lematic indoors—CO from poorly vented heaters, NO,, and and establishing a specific cause other than the air pollution
many VOCs (passively emitted from new furniture or rugs, or incident itself can be difficult.
from molds in the ventilation system) including trichloroethyl- Of the many air pollution studies over the last 25 years, none
ene (a VOC common to the indoor air arising from chlorinated have had more impact on the perception of pollutant risk and
water or dry-cleaned clothes). the direction of research today than a series of epidemiological
studies that showed an association between PM mass concen-
Indoor Air in the Developing World—Pollution of the tration and daily mortality. Studies utilizing novel time-series
indoor environment in the developing world is a major issue. analyses that blunt the impact of weather, smoking, and other
Superimposed on the infiltration of ambient air pollutants are variables that might obscure patterns in health variables linked
indoor emissions from cooking practices, the cultural use of to the air monitoring data showed significant and consistent
incense, tobacco, and various other substances, such as per- associations between health outcomes of ambient PM at levels
fumes. In less developed communities, unvented or poorly previously thought to be safe. Time-series analyses are based
vented cookstoves that burn biomass are used much as they on Poisson regression modeling to distinguish changes in daily
have been for centuries. Chronic lung diseases, such as bron- death counts (or hospital admissions) associated with short-
chitis, emphysema, and cancer, are major killers of exposed term changes in air pollution. The statistical methodology
women while children suffer from bronchitis and various other applied in these time-series studies could detect short-term
infectious lung diseases. trends and minimized the effects of other pollutants and
potential confounders with longer time constants.
The Evolving Profile of Outdoor Air Pollution—Classically, PM stands as the preeminent air pollutant because of its
ambient air pollution was distinguished on the basis of the health impact as well as the pollutant that opened the door
chemical redox nature of its primary components SO, and NO,,. to unsuspected targets of injury. The major health outcome
432 UNIT 6 Environmental Toxicology

PM Exposure
4
q
Inflammation
4 ‘ 4 Eicosanoids
4 Ps
Cyto/Chemokines
Growth factors
Ventilation
Reactive O> & N5

Ate

Autonomic
dysfunction

Chronic impact
PM Dissolution — bio/chemical Lung remodeling
cancer
Interactions (endotoxin, metals, PAHs)
Systemic inflammation
Systemic oxidants (reactive O3/N>)
Clotting dysfunction >| Allergenic/lmmune reactions
Vascular injury/Atherosclerosis

FIGURE 29-5 Schematic of the multiple mechanisms thought to function in cardiopulmonary response($) to air pollutants—derived
from current hypothesized mechanisms for particulate matter.

revealed in the study of PM has been the involvement of the advantage of more precise control of confounding variables,
cardiovascular system as a prime target for adverse impact. such as the tracking of urinary cotinine as an index of tobacco
Both epidemiological and toxicological studies now point smoke exposure, but they can be very expensive and require
to major cardiac involvement in PM-associated mortality. substantial time and dedication on the part of both the inves-
Not surprisingly, effects are most apparent in subpopulations tigators and the study population. Depending on the study size
already compromised by cardiopulmonary and perhaps vascu- and design, exposure assessments can be complex, and the loss
lar diseases (e.g., diabetes). Several pathways have been pro- of subjects due to dropout is sometimes unpredictable.
posed that attempt to link exposure and cardiac effects that
may or may not include pulmonary mediation. These potential
mechanisms are illustrated in Figure 29-5. POLLUTANTS OF OUTDOOR
The association between PM and health outcomes appar-
ently is linked to particle composition rather than mass alone.
AMBIENT AIR
The actual “biochemical lesion” caused by PM is generally
Classic Reducing-Type Air Pollution
thought to involve oxidant mechanisms (generation of reactive
oxygen and perhaps nitrogen species) by constituents or attri- High concentrations of the reducing-type air pollution, char-
butes (e.g., reactive surface area) of the particles at the cell or acterized by SO, and smoke, are capable of producing dramatic
molecular level. human health effects. Empirical studies in human subjects and
animals have long stressed the irritancy of SO, and its role in
Long-Term Exposures—Epidemiological studies of the these incidents, while the full potential for interactions among
chronic effects of air pollution are difficult to conduct by the very the copollutants in the smoky, sulfurous mix has a mixed
nature of the goal: outcomes associated with long-term expo- record of replication in the human exposure laboratory. It is an
sures. Looking back in time with retrospective, cross-sectional irritant gas that has a toxicology of its own and, through atmo-
studies frequently were confounded with unknown variables spheric reactions, can transform photochemically into sulfites
and inadequate historical exposure data. A good example of or sulfates within a secondarily irritant particle.
the problem of confounding is cigarette smoking. Without
extensive information on both active and passive smoking, the Sulfur Dioxide
ability to discern the impact of an air pollution disease outcome General Toxicology—Sulfur dioxide is a water-soluble
such as chronic bronchitis and emphysema would be greatly irritant gas that is absorbed predominantly in the upper air-
impaired. Prospective studies, on the other hand, have the ways and that stimulates bronchoconstriction and mucus
 CHAPTER 29 Air Pollution 433

secretion in a number of species, including humans. Early narrowing, or bronchoconstriction, which impedes the flow of
studies with relatively high exposure concentrations of SO, air into and out of the lungs. This response might be thought
showed airway cellular injury and subsequent proliferation of as a defensive measure to limit the inhalation of air contain-
of mucus-secreting goblet cells. At concentrations <1 ppm, ing noxious gases, but this explanation may be more teleologi-
such as might be encountered in the polluted ambient air of cal than fact. The magnitude ofthe response is related to both
industrialized areas, long-term residents experience a higher acid concentration and particle size. The thicker mucus blan-
incidence of bronchitis. Other factors (diet, access to health ket of the nose may also blunt (by dilution or neutralization
care, other pollutants) have been involved in this reversal by mucus buffers) much of the irritancy of the deposited acid.
reductions in ambient smoke and SO, are generally thought to In contrast, the less shielded distal airway tissues, with higher
be the most important. receptor density, would be expected to be more sensitive to the
The penetration of SO, into the lungs is greater during acid particles reaching that area.
mouth as opposed to nose breathing. An increase in the air- Asthmatics appear to be somewhat more sensitive to the
flow during deep rapid breathing augments penetration of the bronchoconstrictive effects of sulfuric acid than are healthy
gas into the deeper lung. As a result, persons exercising would individuals, owing to hyperresponsive airways, so their tendency
inhale more SO, and, as noted with asthmatics, are likely to to constrict at low acid concentrations would be expected, just
experience greater irritation. Once deposited along the airway, as asthmatic airways are sensitive to nonspecific airway smooth
SO, dissolves into surface lining fluid as sulfite or bisulfite and muscle agonists (e.g., carbachol, histamine, exercise). The general
is readily distributed throughout the body. It is thought that the correlation between airway responsiveness and inflammation
sulfite interacts with sensory receptors in the airways to initiate that appears to be important in grading asthma severity and risk
local and centrally mediated bronchoconstriction. of negative clinical outcomes may also be predictive of responses
to environmental stimuli.
Pulmonary Function Effects—The basic pulmonary response
to inhaled SO, is mild bronchoconstriction, which is reflected Effects on Mucociliary Clearance and Macrophage Function—
as a measurable increase in airflow resistance due to narrowing Sulfuric acid alters the clearance of particles from the lung.
of the airways. Concentration-related increases in resistance Mucus clearance appears to vary directly with the acidity ({H*])
have been observed in guinea pigs, dogs, and cats as well as of the acid sulfate, with sulfuric acid having the greatest effect
humans. Airflow resistance increased more when the gas was and ammonium sulfate the smallest. Collectively, there seems to
introduced through a tracheal cannula than via the nose, since be coherence in the data to rank sulfate irritancy: sulfuric acid
nasal scrubbing of the water-soluble gas was bypassed. > ammonium bisulfate > ammonium sulfate. Acidity [H*]
appears to be the primary driver on most respiratory effects
Sulfuric Acid and Related Sulfates—The conversion of attributable to the acid sulfates even at the level of pulmonary
SO, to sulfate is favored in the environment with subsequent macrophages.
ammonia neutralization to ammonium sulfate [(NH,),SO,] or
ammonium bisulfate [NH,HSO,]. During oil and coal com- Chronic Effects—Not surprisingly, sulfuric acid induces
bustion or the smelting of metal ores, sulfuric acid condenses qualitatively similar effects in the airways as found at high
downstream of the combustion processes with available metal concentrations of SO). As a fine aerosol, sulfuric acid depos-
ions and water vapor to form submicrometer sulfuric acid its deeper along the respiratory tract, and its high specific
fume and sulfated fly ash. Photochemical reactions also pro- acidity imparts greater injury on phagocytes and epithelial
mote acid sulfate formation via both metal-dependent and cells. Thus, a primary concern with regard to chronic inhala-
independent mechanisms, but most of the oxidation of SO, tion of acidic aerosols is the potential for bronchitis, since
occurs within plumes as they disperse in the atmosphere. Stack this has been a problem in occupational settings in which
emissions may undergo long-range transport to areas dis- employees are exposed to sulfuric acid mists (e.g., battery
tant from the emission source, allowing considerable time for plants).
sunlight-driven chemistry. Studies conducted with sulfuric acid have demonstrated
that the airways of exposed animals become progressively
General Toxicology—Sulfuric acid irritates by virtue of its abil- more sensitive to challenge with acetylcholine, show a progres-
ity to protonate (H*) receptor ligands and other biomolecules. sive decrease in diameter, and experience an increase in the
This action can either directly damage membranes or activate number of secretory cells, especially in the smaller airways.
sensory reflexes that initiate inflammation. Unlike other irri- These studies have expanded our knowledge of the biological
tants, such as O; (see below), inhaled sulfuric acid does not response and its exposure-based relationship to sulfuric acid.
appear to stimulate a classic neutrophilic lung inflammation. It seems reasonable to postulate that chronic daily exposure
Rather, eicosanoid homeostasis appears to be disturbed result- of humans to ~100 g/m? sulfuric acid may lead to impaired
ing in macrophage dysfunction and altered host defense. clearance and mild chronic bronchitis. The possibility that
chronic irritancy may elicit bronchitis-like disease in suscep-
Pulmonary Function Effects—Sulfuric acid produces an tible individuals (perhaps over a lifetime or in children because
increase in flow resistance in guinea pigs due to reflex airway of dose differences) appears to be reasonable.
434 UNIT 6 Environmental Toxicology

Particulate Matter the atmosphere). Solubility appears to play a role in the toxicity
of many inhaled metals by enhancing metal bioavailability (e.g.,
PM in the atmosphere can be solid, liquid, or a combination of
nickel from nickel chloride versus nickel oxide), but insolubility
both with a mélange of organic, inorganic, and biological com-
can also be a critical factor in determining toxicity by increas-
pounds. The compositional matrix of PM can vary significantly
ing pulmonary residence time within the lung (e.g., insoluble
depending on the emission source and secondary transforma-
cadmium oxide versus soluble cadmium chloride). Moreover,
tions, many ofwhich involve gas to particle conversions. Long-
some metals, either in their soluble forms or when partially
range transport of emissions or transformation products can
coordinated on the surface of silicate or bioorganic materials,
contribute significantly to the regional matrix of PM. Particles
can promote electron transfer to form reactive oxidants.
of larger size tend to have more local sources, the reason being
that they are formed from dispersed dust and attrition of mate-
Gas-Particle Interactions—As already noted, these gas-
rials. Being of larger size, they tend to “fall out” or settle from
particle interactions can be extremely complex involving
the air due to gravity (although winds can in fact carry these
multiple components of the particles, gases/vapors, and sun-
particles great distances—e.g., Sahara desert particles have
light and lead to toxicity of either the particle or the gas. Metal
been found on the US East Coast). Particles in the range of 10 to
smelting or the combustion of coal can emit sulfuric acid
2.5 4m (PM,..;—coarse PM) are highly inhalable by humans.
that is physically associated with ultrafine metal oxide par-
In the urban setting there is considerable spatial and temporal
ticles. Complex chemistry also occurs within the effluent of
heterogeneity of coarse PM while PM, ;appears more homoge-
the combustion source. Similar interactions may result from
nous throughout a regional environment. The size designation
gaseous pollutants that impair the clearance of particles from
of fine and coarse PM is based on their relative respirability—
the lung or otherwise alter their metabolism. Studies focus-
those in the range of PM,, are inhalable into the larger thoracic
ing on irritancy and infectivity raise the prospect that realistic
airways while the PM,, is inhalable into the deeper reaches
exposure scenarios of gaseous and, particulate pollutants can
(gas exchange areas) ofthe lung (see Chapter 15).
interact through either chemical or physiologic mechanisms to
enhance health risks of complex polluted atmospheres.
Metals— There have been many standard acute and subchronic
rodent inhalation studies with specific metal compounds, often Ultrafine Carbonaceous Matter—Ultrafine carbon par-
as oxides, chlorides, or sulfates. These exposure studies relate ticles (often called black carbon) typically result from high-
most appropriately to occupational exposures. Metals may arise temperature pyrolysis or as the product of atmospheric
from natural as well as anthropogenic activities, and as a result transformation involving organic vapors and sunlight. The size
metals are a common constituent in ambient PM. The metal of these particles allows them to slip between gas molecules
profiles among regions differ appreciably in concentration and moving primarily by diffusion and principles of Brownian
type and they also differ by the size mode of PM. Coarse PM motion. Agglomeration on surfaces or other particles in the
(2.5-10|1m) arises largely from natural sources and thus has air is their primary mode of dissipation. When concentrations
prominent earthen metals such as iron, sodium, silica, and exceed ~1 million/cm’, they rapidly agglomerate with each
magnesium—usually in oxide forms. Combustion-derived other to form larger clumps or chains of ultrafine particles.
metals reflect the fuel source. For example, oil may have As an air pollutant, therefore, elemental carbon particles
vanadium, nickel, and perhaps zinc and iron, while coal may generally do not exist as singlets except near their emission
have zinc and selenium. Their chemical forms vary from water- points—e.g., traffic or other high-temperature sources. Fine
soluble salts to oxide and phosphate forms. Other metals are PM consists in part of agglomerates of carbonaceous organic
emitted from vehicles burning fuels to which metal compounds material that if partially oxidized may be somewhat soluble in
were added to alter functionality (e.g., lead, manganese, plati- water. Some organic materials, which exist in the vapor form,
num) or as engine wear and catalyst by-products. Similarly, condense on the ultrafine carbon (e.g., diesel PM). Estimates
metals may also derive from brake (copper, iron), tire (zinc), of the carbonaceous (including organic) content of ambient
and dispersed road (earthen silicates) wear. Metals have many fine PM vary considerably but are nominally considered to be
biological properties, some essential to life while others being about 10% to 60% of the total mass depending on the urban or
directly toxic to cells or act indirectly in a pro-oxidant toxic regional area.
fashion. Thus, metals have garnered considerable interest Diesel particles vary widely in the ratio of organic and ele-
regarding their role in PM toxicity. mental carbonaceous materials, which in empirical studies
Metal compounds can be separated nominally by physi- has been shown to influence toxic outcomes, such as to their
cochemical characteristics: those that are essentially water- inflammatory and carcinogenic potential. Diesel exhaust that
insoluble (e.g., metal oxides and hydroxides such as those that also contains significant amounts of gaseous pollutants: NO,,
might be released from high-temperature combustion sources CO, and SO, as well as various VOCs and carbony] irritants.
or derived from the geocrustal matrix) and those that are sol- Elemental carbon itself is generally considered to be of low tox-
uble or somewhat soluble in water (often chlorides or sulfates icity, although long-term, high-concentration exposure condi-
such as those that might form under acidic conditions in a tions in rats can lead to lung “overload” where there is evidence
smoke plume or leach from acid-hydrated silicate particles in of lung damage and carcinogenicity. In the environment, carbon
 CHAPTER 29 Air Pollution 435

has the potential to act as a carrier of certain irritant gases. it comprises a mixture of nitric oxides (NO,), aldehydes, per-
However, carbon in the ultrafine mode (<0.1[1m) has been oxyacetyl nitrates (PAN), and a myriad of aromatics and
suggested to be more toxic than the fine mode (2.5 |1m) form, alkenes along with analog reactive radicals. If SO, is pres-
perhaps due to enhanced surface reactivity or tissue penetra- ent, sulfates may also be formed and, collectively, they yield
tion. Composition of the ultrafine particle also contributes to its “summer haze.” Likewise, the complex chemistry can generate
effects and behavior. Ultrafine particles in the environment exist organic PM, nitric acid vapor, and various condensates.
in extremely high numbers but contribute negligibly to mass. From the point of view of the toxicology of photochemical
Recent commercial introduction of “engineered” nanoparticles air pollutant gases, O, is by far the toxicant of greatest concern.
brings many of the same concerns as ultrafines by virtue of their It is highly reactive and more toxic than NO,, and because
similar sizes. Additionally, being “engineered” particles, they its generation is fueled through cyclic hydrocarbon radicals,
may possess design features that “natural” combustion ultrafine it reaches greater concentrations than the hydrocarbon radi-
(or nano) particles do not. cal intermediates. Although O, is of toxicological importance
in the troposphere, in the stratosphere it plays a critical pro-
Chronic Effects and Cancer—The role of air pollution in tective role. About 10 to 50km above the earth’s surface, UV
human lung cancer is difficult to assess because the vast major- light directly splits molecular O, into atomic O*, which then
ity of respiratory cancers result from cigarette smoking. VOCs combines with O, to form O,. The O, also dissociates back
and nitrogen-containing and halogenated organics account but much more slowly. The result is an accumulation of O, to
for most of the compounds that are derived from combustion several ppm within a relatively thin strip of the stratosphere
sources ranging from tobacco to power plants to incinerators forming an effective “permanent” barrier by absorbing the
to motor vehicles with potential carcinogenic effects. Human short-wavelength UV in the chemical process. This barrier
exposure to airborne toxicants is highly complex composition- had in recent years been threatened by various anthropo-
ally as well as in its temporal and spatial heterogeneity. genic emissions (Cl, gas and certain chlorofluorocarbons) that
The lung cancer risk of any individual is some function of enhance O, degradation (creation of an “O; hole”), but recent
the carcinogenic nature of the substance, the amount of mate- restrictions on the use of these degrading chemicals seem to
rial deposited in the lungs, which is itself a function of the have been effective in reversing this process. The benefits are
concentration in the ambient air, the physical and chemical believed to be a reduction of excess UV light infiltration to the
properties of the inhalant that may determine deposition effi- earth's surface and reduced skin cancer risk.
ciency, and the cumulative volume of air inhaled. Of course, This protective issue is quite different in the troposphere,
the innate susceptibility of the individual (including genotype where accumulation of O,; serves no known purpose and poses
and environmental factors such as diet, etc.) is also likely to be a threat to the respiratory tract. Near the earth’s surface, NO,
important. The majority of lung cancer risk from ambient air arising from combustion processes efficiently absorbs longer-
pollution lies within the PM fraction, including the polycyclic wavelength UV light, from which a free O atom is cleaved,
organic chemicals, along with the less volatile (semivolatile) initiating the following simplified series of reactions:
nitroaromatics. These persistent organics associate with the
PM matrix and thus could have a prolonged residence time at NO, + hv (UV light) > O* + NO* (29-1)
deposition sites within the respiratory tract. Genetic bioassays
O° sr ©; ae OO} (@9=))
have revealed the potent mutagenicity, and presumably carci-
nogenicity, of various chemical fractions of ambient aerosols.
Some of these compounds require metabolic transformation to
activate their potency while others may be detoxified by their This process is inherently cyclic, with NO, regenerated by
metabolism. Carcinogenic vapors such as benzene are inhaled the reaction of the NO* and QO,. In the absence of unsaturated
but target the bone marrow producing leukemia. hydrocarbons (olefins and substituted aromatics) arising from
The cells lining the respiratory tract turn over relatively fuel vaporization or combustion, as well as biogenic terpenes,
quickly, since they interface with the ambient environment this series of reactions would approach a steady state with little
with every breath. Conceptually, their DNA would thus be buildup of O . The free electrons of the double bonds of unsat-
vulnerable to. carcinogenic or oxidant-induced replication urated hydrocarbons are attacked by free atomic O’, resulting
errors that, when fixed as mutations, could give rise to tumors. in oxidized compounds and radicals that react further with
Copollutants, such as irritant gases, that initiate inflamma- NO?’ to produce more NO,. Thus, the balance of the reactions
tion may promote carcinogenic activity by damaging cells and sequence shown in Eqs. (29-1) to (29-3) is tipped to the right,
further enhancing their turnover. leading to buildup of O,. This reaction is particularly favored
when the sun’s intensity is greatest at midday, utilizing the NO,
-
provided by morning rush-hour traffic. Carbonyl compounds
Photochemical Air Pollution (especially short-chained aldehydes) are also by-products
Photochemical air pollution (notably O,) arises secondarily of these reactions. Formaldehyde and acrolein account for
from a series of complex reactions in the troposphere activated about 50% and 5%, respectively, of the total aldehyde content
by the ultraviolet (UV) spectrum of sunlight. In addition to O,, in urban atmospheres. Peroxyacetyl nitrate (CH,COONO,),
436 UNIT 6 Environmental Toxicology

often referred to as PAN, and its homologs also arise in urban PUFA Ozone Trioxolane Carbonyl oxide Aldehyde

air, most likely from the reaction of the peroxyacyl radicals

O
with NO,,. san
RHC=CH + 0; —~> i RHC=O—O + RHC=O

Chronic Exposures to Smog

yeaa
Epidemiological studies in human populations as well as
empirical studies in laboratory animals have attempted to link =e
es =
degenerative lung disease with chronic exposure to photo- =O
chemical air pollution. Cross-sectional and prospective field \
ee Ghil RHC © ——> RCH=O + H,0,
studies have suggested an accelerated loss of lung function Yee OOH
in people living in areas of high pollution. However, as with
many studies of this type, there were problems with confound- Criegee ozonide — _Hydroxyhydroperoxide Aldehyde Hydrogen
ing factors (meteorology, imprecise exposure assessment, and peroxide
population variables). Studies have been conducted in children
FIGURE 29-6 Major reaction pathways of O, with lipids in
living in modern-day Mexico City, which has high oxidant and
lung lining fluid and cell membranes. (Adapted with permission
PM levels, noted severe epithelial damage and metaplasia as
from the Air Quality Criteria Document for Ozone and Photochemical
well as permanent remodeling of the nasal epithelium. When
Oxidants, U.S. EPA, 1996.)
children migrated into Mexico City from cleaner, nonurban
regions, even more severe damage was observed, suggesting
that the tissue remodeling in the permanent residents imparted consider the role of exercise-associated dosimetry in a study
some degree of incomplete adaptation. Because the children of O, or any inhalant before making cross-study comparisons,
were of middle-class origin, these observations were less likely especially if that comparison is across species.
confounded by socioeconomic variables. In fact, the epithelial As a powerful oxidant, O, seeks to extract electrons from
cell damage in the nasal cavity of Mexico City children was other molecules. The surface fluid lining the respiratory tract
inversely correlated with glutathione peroxidase, a marker of and the cell membranes that underlie the lining fluid con-
oxidative stress. tain a significant quantity of polyunsaturated fatty acids
(PUFA), either free or as part of the lipoprotein structures
of the cell. The double bonds within these fatty acids have a
Ozone labile, unpaired electron that is easily attacked by O; to form
General Toxicology—Ozone is the primary oxidant of con- ozonides that progress through a less stable zwitterion or tri-
cern in photochemical smog because of its inherent bioreac- oxolane (depending on the presence of water); these ultimately
tivity and its concentration relative to other reactive species. recombine or decompose to lipohydroperoxides, aldehydes,
Current mitigation strategies for O; have been only largely and hydrogen peroxide. These pathways are thought to initi-
unsuccessful owing to sustained population growth. With sub- ate propagation of lipid radicals and auto-oxidation of cell
urban sprawl and the downwind transport of air masses from membranes and macromolecules (Figure 29-6).
populated areas to more rural environments, the geographic
distribution of those exposed has also expanded, as has the Pulmonary Function Effects—Exercising human subjects
temporal profile of individual exposure. In other words, ambi- exposed for 2 to 3 h to 0.12 to 0.4 ppm O, experience reversible
ent O, exposures are no longer stereotyped as brief 1 to 2 h concentration-related decrements in forced exhaled volumes
peaks. Instead, there is more typically a prolonged period of (FVC and forced expiratory volume in one second [FEV,]). It
exposure of 6 h or more at or near the NAAQS level. is not clear what mechanisms underlie the altered lung func-
Ozone induces a variety of effects in humans and experi- tion (in terms of changes in FEV,) produced by O,. There is
mental animals at concentrations that occur in many urban also evidence that the decrements in lung function are vagally
areas. These effects include morphologic, functional, immu- mediated, and that the response can be abrogated by analge-
nologic, and biochemical alterations. Because of its low water sics, such as ibuprofen and opiates, which also reduce pain
solubility, a substantial portion of inhaled O, penetrates deep and inflammation. Thus, pain reflexes involving C-fiber net-
into the lung, but its reactivity is such that about 17% and 40% works may be important in the reduction in forced expiratory
are scrubbed by the nasopharynx of resting rats and humans, volumes along with changes in vagal reflexes that alter airway
respectively. Nevertheless, regardless of species, the region of reactivity and bronchoconstriction.
the lung that is predicted to have the greatest O, deposition Airway responsiveness to specific (e.g., allergen) and non-
(dose per surface area) is the centriacinar region, from the specific (e.g., cold air, inhaled methacholine) bronchoconstric-
terminal bronchioles to the alveolar ducts, also referred to as tion is another commonly used test of the pulmonary response
the proximal alveolar ductal region. Because O, penetration to inhaled pollutants such as O,. These types of tests are very
increases with increased tidal volume and flow rate, exercise important because airway hyperresponsiveness is a central
increases the dose to the target area. Thus, it is important to feature of asthma and asthmatics are a sizeable subpopulation
 CHAPTER 29 Air Pollution 437

(7% to 9% of the total population in the United States) that Pulmonary Function Effects—Exposure of normal human
may be particularly sensitive to the adverse respiratory effects subjects to concentrations of <4 ppm NO, for up to 3h pro-
of inhaled pollutants. duces no consistent effects on spirometry. A number of factors
appear to be involved (e.g., exercise, inherent sensitivity of the
Ozone Interactions with Copollutants—An approach asthmatic subject, exposure method).
simplifying the complexity of synthetic smog studies, yet
addressing the issue of pollutant interactions, involves the Inflammation of the Lung and Host Defense— Unlike O,,
exposure of laboratory animals or humans to binary or more NO, does not induce significant neutrophilic inflammation in
complex synthetic mixtures of pollutants that occur together humans at exposure concentrations encountered in the ambi-
in ambient air. The most frequent combination involves inter- ent outdoor environment. There is some evidence for bronchial
actions of O, and NO, or O, and PM (e.g., sulfuric acid or inflammation after 4 to 6h at 2.0 ppm, which approximates the
diesel particles). Not surprisingly, study design adds a level highest transient peak indoor levels of this oxidant. Exposures
of complexity in interpretation such that evidence exists sup- at 2.0 to 5.0ppm have been shown to affect T lymphocytes,
porting either augmentation or antagonism of lung function particularly CD8* cells and natural killer cells that function in
impairments, lung pathology, and other indices of injury. This host defenses against viruses. Although these concentrations
apparent conflict in the findings only emphasizes the need may be high, epidemiological studies variably show effects of
to carefully consider the myriad of factors that might affect NO, on respiratory infection rates in children, especially in
studies involving multiple determinants and the nature of the indoor environments.
exposure that is most relevant to reality.
As the number of interacting variables increases, so does
the difficulty in interpretation. Studies of complex atmo- Other Oxidants
spheres involving acid-coated carbon combined with O, at PAN is thought to be responsible for much of the eye-stinging
near-ambient levels also show varied evidence of interaction activity of smog. It is more soluble and reactive than O,, and
on lung function and macrophage receptor activity. The sta- hence rapidly decomposes in mucous membranes. The cornea
tistical separation of the interacting variables and responses is a sensitive target and is prominent in the burning/stinging
from the individual or combined components is difficult. discomfort often associated with oxidant smogs.
However, it is indeed the complex mixture to which people
are exposed that we wish to evaluate. Creative approaches
to understanding mixture responses must be addressed in Aldehydes
the future. Carbonyl compounds, notably short-chained (2-4C) alde-
hydes, are common photo-oxidation products of unsaturated
hydrocarbons. Two aldehydes are of major interest by virtue
Nitrogen Dioxide of their concentrations and irritancy: formaldehyde (HCHO)
General Toxicology—Nitrogen dioxide, like O,, is a deep and acrolein (H,C=CHCHO). They contribute to the odor as
lung irritant that can produce pulmonary edema if it is inhaled well as eye and sensory effects of smog. Formaldehyde accounts
at high concentrations. Potential life-threatening exposure for about 50% of the estimated total aldehydes in polluted air,
is a real-world problem for farmers, as near-lethal high levels while acrolein, the more irritating of the two, accounts for
of NO, can be liberated from fermenting fresh silage. Being about 5% of the total. Acetaldehyde (C; HCHO) and many
heavier than air, the generated NO, and CO, displace air and other longer-chain aldehydes make up the remainder, but they
oxygen at the base of silo and diffuse into closed spaces where are not as intrinsically irritating, exist at low concentrations,
workers can inadvertently get exposed to very high concen- and have less solubility in airway fluids.
trations perhaps with depleted oxygen. Typically, shortness of
breath rapidly ensues with exposures nearing 75 to 100 ppm
NO,, with delayed edema and symptoms of pulmonary dam- Formaldehyde
age. Not surprisingly, the symptoms are collectively termed Formaldehyde is a primary sensory irritant. Because it is very
“silo-filler’s disease.” Nitrogen dioxide is also an important soluble in water, it is absorbed in mucous membranes in the
indoor pollutant, especially in homes with unventilated gas nose, upper respiratory tract, and eyes. The dose-response
stoves or kerosene heaters or in developing countries with the curve for formaldehyde is steep: 0.5 to 1 ppm yields a detect-
unvented burning of biomass fuels. able odor, 2 to 3 ppm produces mild irritation, and 4 to 5 ppm
The distal lung lesions produced by acute NO, are similar is intolerable to most people. Formaldehyde is thought to act
among species. Theoretical dosimetry studies indicate that via sensory C-fibers that signal locally as well as through the
NO, is deposited along the length of the respiratory tree, with trigeminal nerve to reflexively induce bronchoconstriction
preferential deposition being in the distal airways. Damage is through the vagus nerve.
most apparent in the terminal bronchioles. At high concentra- Two aspects of formaldehyde toxicology have brought
tions, the alveolar ducts and alveoli are also affected, with type 1 it from relative obscurity to the forefront of attention in
cells again showing their sensitivity to oxidant challenge. recent years. One is its near ubiquitous presence in indoor
438 UNIT 6 Environmental Toxicology

atmospheres as an off-gassed product of construction mate- THE MULTIPOLLUTANT REALITY

rials such as plywood, furniture, or improperly polymerized
OF AIR POLLUTION
urea-formaldehyde foam insulation. In addition, the potential
carcinogenicity of formaldehyde is a concern. Formaldehyde Pollutants in the atmosphere of any community vary consider-
is a probable human carcinogen. There is epidemiological evi- ably in space and time, and are charged by the varied output
dence that formaldehyde causes nasopharyngeal cancer, strong from a wide range of sources, only to be transformed stoichio-
but not sufficient evidence of leukemia, and limited evidence of metrically by a patterned intensity of sunlight. The reductionist
sinonasal cancer. approach examining one pollutant at a time has been success-
ful in diminishing pollutants and improving public health. But
there are likely chemical and physiologic interactions between
Acrolein
and among pollutants that are of public health consequence
Because acrolein is an unsaturated aldehyde, it is more reactive that have not been appreciated. Parallel efforts within both
than formaldehyde. It penetrates a bit deeper into the airways the scientific and the regulatory/policy communities need to
and may not have the same degree of sensory irritancy but it advance methods for evaluating and managing the effects of air
may cause more damage. Concentrations below 1 ppm cause
pollution in a multipollutant manner.
irritation of the eyes and the mucous membranes of the respira-
tory tract. The mechanism ofincreased flow resistance appears
to be mediated through both a local C-fiber and centrally
mediated cholinergic reflexes. Ablation of the C-fiber network
CONCLUSIONS
and atropine (muscarinic blocker) block this response. The breadth and complexity of the problem of air pollution—
from the development of credible databases to supporting
Carbon Monoxide regulatory action and decision making—have been the theme
throughout. The classic and still most important air pollutants
Carbon monoxide is classed toxicologically as a chemical
provide a foundation for understanding and appreciating the
asphyxiant because its toxic action stems from its formation
nuances of the issues and strategies for air pollution control
of carboxyhemoglobin, preventing oxygenation of the blood
and protection of public health. The key role of the toxicolo-
for systemic transport (see Chapter 11). Motor vehicles still
gist is to develop sensitive methods to assay responses to low
account for two thirds of urban CO emissions. Other sources
pollutant concentrations, apply these methods to relevant
of CO include both main and sidestream tobacco smoke, and
exposure scenarios and test species, and develop paradigms
residential and commercial heating systems and mobile auxil-
to relate empirical toxicological data to real life through an
iary heating units. No overt clinical human health effects have
understanding of mechanism. Last, the toxicologist must con-
been demonstrated for COHb levels below 2%, while levels
tinually integrate laboratory data with those of epidemiology
above 40% cause fatal asphyxiation.
and clinical study to ensure their maximum utility.

Hazardous Air Pollutants

HAPs (so-called air toxics) represent an inclusive classification
for air pollutants of anthropogenic origin that are generally
BIBLIOGRAPHY
Foster WM, Costa DL (eds.): Air Pollutants and the Respiratory Tract,
of measurable quantity in the air, The HAPs include organic
2nd ed. Boca Raton, FL: Taylor & Francis, 2005.
chemicals like acrolein, benzene, minerals like asbestos, poly- Phalen RF, Phalen RN: An Introduction to Pollution Science: A Public
cyclic hydrocarbon such as benzo(a)pyrene, various metals and Health Perspective. Burlington, MA: Jones and Bartlet, 2013.
metal compounds like mercury and beryllium compounds, Vallero DA (ed.): Fundamental of Air Pollution, 5th ed. Waltham,
and pesticides such as carbaryl and parathion. MA: Elsevier, 2014.
 CHAPTER 29 Air Pollution 439

QUESTION

Which of the following compounds is NOT an oxidant- . Which ofthe following statements is NOT true?
type air pollutant? a. Ozone (O,) combines with a nitric oxide radical to
a. NO,. form NO,,.
b. SO;. O, combines with an oxygen radical to form ozone.
Cre O).: Os O,; can cause damage to the respiratory tract.
d. radical hydrocarbons. d. Accumulation of O, in the stratosphere is important
e. aldehydes. for protection against UV radiation.
e. Cl, gas is known to cause O, degradation.
Which of the following pollutants contributes most to
nontobacco-smoking lung cancer? Which of the following is NOT a likely symptom of NO,
a. asbestos. exposure?
b. vinyl chloride. increased secretion by Clara cells.
c. benzene. pulmonary edema.
d. products of incomplete combustion. shortness of breath.
e. formaldehyde. loss of ciliated cells in bronchioles.
ere
S&
2 decreased immune response.
Inhalants, such as NO, and trichloroethylene, can increase
proliferation of opportunistic pathogens in the lungs by:
Which of the following statements regarding aldehyde
destroying goblet cells in the respiratory tract.
exposure is FALSE?
damaging the alveolar septa.
a. ‘The major aldehyde pollutants are formaldehyde and
inactivating cilia in the respiratory tract.
acrolein.
killing alveolar macrophages.
b. Formaldehyde is found in tobacco smoke, but acro-
cane dampening the immune system.
lein is not.
Which of the following is NOT a characteristic of SO, c. Acrolein causes increased pulmonary flow resistance.
toxicology? Formaldehyde exposure induces bronchoconstriction.
a. SO, isa major reducing-type air pollutant. e. The water solubility of formaldehyde increases its
b. Increased airflow rate increases the amount of SO, nasopharyngeal absorption.
inhaled.
c. SO, inhalation causes vasoconstriction and increased 10. Carbon monoxide (CO) exerts its toxic effects via its inter-
blood pressure. action with which of the following?
d. SO, is predominately absorbed in the conducting a. DNA polymerase.
airways. b. actin.
e. SO, inhalation increases mucus secretion in humans. c. kinesin.
d. hemoglobin.
Which of the following would be MOST likely to occur on e. microtubules.
sulfuric acid exposure?
a. vasoconstriction.
b. decreased mucus secretion.
c. ananti-inflammatory response.
d. vasodilation.
e. bronchoconstriction.

All of the following statements regarding particulate

matter are true EXCEPT:
a. Metals are most commonly released into the environ-
ment during coal and oil combustion.
b. The interaction of gases and particles in the atmo-
sphere can create a more toxic product than the gas or
particle alone.
c. Solubility does not play a role in the bioavailability of
a metal.
d. The earth’ crust is an important source of -atmo-
spheric magnesium.
e. Diesel exhaust contains reducing- and oxidant-type
air pollutants.
 UNIT 7 APPLICATIONS OF sie). 4(er0) Kolen

Cla ALP? ST: GES ER

Ecotoxicology
Richard T. Di Giulio and Michael C. Newman

INTRODUCTION Disease Susceptibility

Behavior
SOME DISTINCT ASPECTS OF EXPOSURE @ance:

TOXICANT EFFECTS oe
Community
Molecular and Biochemical Effects
Ecosystem to Biosphere
Gene Expression and Ecotoxicogenomics
Estrogen Receptor APPROACHES
Aryl Hydrocarbon Receptor Toxicity Tests
Genomics and Ecotoxicogenomics Biomarkers
Protein Damage Population
Oxidative Stress Community and Ecosystem
DNA Damage Landscape to Biosphere
Cellular, Tissue, and Organ Effects ECOLOGIC RISK ASSESSMENT
Cells
Target Organs INTERCONNECTIONS BETWEEN ECOSYSTEM
Organismal Effects INTEGRITY AND HUMAN HEALTH
Mortality
Reproduction and Development

44]
442 UNIT 7 Applications of Toxicology

= Ecotoxicology is the study of the fate and effects of toxic a The biologic availability (or bioavailability) of a chemical
substances on an ecosystem. is the portion of the total quantity of chemical present
= Chemodynamics is, in essence, the study of chemi- that is potentially available for uptake by organisms.
cal release, distribution, degradation, and fate in the « Pollution may result in a cascade of events, beginning
environment. with effects on homeostasis in individuals and extend-
e Achemical can enter any of the four matrices: the atmo- ing through populations, communities, ecosystems, and
sphere by evaporation, the lithosphere by adsorption, the landscapes.
hydrosphere by dissolution, or the biosphere by absorp- s Terrestrial toxicology is the science of the exposure to
tion, inhalation, or ingestion (depending on the species). and effects of toxic compounds in terrestrial ecosystems.
Once in a matrix, the toxicant can enter another matrix = Aquatic toxicology is the study of effects of anthropogenic
by these methods. chemicals on organisms in the aquatic environment.

INTRODUCTION ecosystems, and other higher level entities has become increas-
ingly apparent, more cause-effect models relevant to these higher
Ecotoxicology is the study of contaminants in the biosphere and levels of biologic organization are added to the conventional set
their effects on constituents of the biosphere. It has an overarching of toxicology models applied by pioneering ecotoxicologists.
goal of explaining and predicting effect or exposure phenomena Contaminant chemical form, phase association, and movement
at several levels of biologic organization (Figure 30-1). Relevant among components of the biosphere are also central issues in
effects to nonhuman targets range from biomolecular to global. ecotoxicology because they determine exposure, bioavailability,
As the need to predict major effects to populations, communities, and realized dose.

Biosphere
SOME DISTINCT ASPECTS OF EXPOSURE
A ‘
Continent
i}
i}
Ecotoxicology commonly uses sparse information for a few
Landscape species to predict effects to many species and their interac-
1
'
tions. Relevant exposure routes are the conventional inges-
a > Ecosystem aaa aa,
J)
Aae : A\ tion, inhalation, and dermal absorption. But, unique features
es Community ' of exposure pathways must be accommodated for species
a
that ingest a wide range of materials using distinct feeding
g Population Habitat/environment
<< ; | mechanisms, breathe gaseous or liquid media using different
5 Organism
L \ ; ;
structures, and come into dermal contact with a variety of
=| Organ system Microenvironment
| |
gaseous, liquid, and solid media.
Organ ey Prediction of oral exposure can be limited because species feed
1 Phase association on different materials; however, conventional principles about
Cell/tissue
| i} oral bioavailability remain relevant. Many techniques applied to
Biomolecule Chemical form/species determining human oral bioavailability are available to the eco-
Biotic Biotic and abiotic Abiotic toxicologist. As an example, some birds are uniquely at high risk
of lead poisoning because they ingest and then use lead shot as
FIGURE 30-1 Ecologic scales relevant to ecotoxicology. grit. The birds grind shot in their gizzards under acidic condi-
Solely biologic scales relevant to ecotoxicology range from the tions, releasing significant amounts of dissolved lead.
molecular to the community levels: solely abiotic scales range from
Estimation of chemical speciation is central to predicting bio-
the chemical to the entire habitat. Biotic and abiotic components
availability of water-associated contaminants. Speciation can
are usually combined at levels above the ecologic community and
habitat. The ecologic community and physicochemical habitat
determine the bioavailability of dissolved metals. Movements
combine to form the ecosystem. Ecologic systems can be considered of nonionic and ionizable organic compounds across the gut or
at the landscape scale, that is, the combination of marine, freshwater, gills are strongly influenced by lipid solubility and pH partition-
and terrestrial systems at a river's mouth. Recently, the continental ing, respectively. Consequently, determination of a compound's
and biospheric scales have become relevant as in the cases of ozone lipophilicity or calculation of pH- and pK,-dependent ioniza-
depletion, acid precipitation, and global warming. tion facilitates some predictive capability for bioavailability.
 CHAPTER 30 Ecotoxicology 443

The free ion activity model (FIAM) states that uptake and toxic- response elements (ERE) of specific genes that contain one or
ity of cationic trace metals are best predicted from their free ion more EREs, thereby causing gene transcription. Genes regulated
activity or concentration, although exceptions exist. in this manner by E2-ER play various important roles in sexual
Bioavailability, bioaccumulation, or exposure concentra- organ development, behavior, fertility, and bone integrity.
tions for sediment-associated toxicants are also approached A number of chemicals can serve as ligands for ER; in
by considering chemical speciation and phase partitioning. most cases these “xenoestrogens” activate gene transcrip-
Metals in sediments are either incorporated into one of the tion acting as receptor agonists. Some ofthese xenoestrogens
many solid phases or dissolved in the interstitial waters sur- include diethylstilbestrol (DES), DDT, methoxychlor, endo-
rounding the sediment particles. Bioavailable metals have been sulfan, surfactants (nonyl-phenol), some PCBs, bisphenol
estimated by normalizing sediment metal concentrations to A, and ethinyl E2, a synthetic estrogen observed in munici-
easily extracted iron and manganese concentrations because pal effluents and surface waters. Environmental exposures
solid iron and manganese oxides sequester metals in poorly to these chemicals are sufficient to perturb reproduction or
bioavailable solid forms. development. Moreover, endocrine disruption by environ-
Another issue of importance to the ecotoxicologist is the pos- mental xenoestrogens appears to be stronger for wildlife than
sibility of biomagnification, the increase in contaminant con- for humans, likely due to instances of elevated exposures that
centration as it moves through a food web. Biomagnification are less prone to confounding factors than is typically the
can result in harmful exposures to species situated high in the case for human exposures. Egg-laying vertebrates provide a
food web such as birds ofprey. biomarker of estrogen exposure—vitellogenin production,
which is produced in the liver and transferred to the ovary
to become a key component of yolk protein. Increased vitel-
TOXICANT EFFECTS logenin production in males is useful biomarker of estrogenic
chemical exposures.
One approach to this complex topic of ecotoxicologic effects
is to organize effects according to biologic levels of organiza-
Aryl Hydrocarbon Receptor—The aryl hydrocarbon recep-
tion. One may consider effects, in ascending order, at the sub-
tor (AHR) is a member of the basic helix-loop-helix Per ARNT
cellular (molecular and biochemical), cellular, organismal,
Sim (bHLH-PAS) family of receptors/transcription factors that
population, community, and ecosystem levels of organization.
is involved in development, as sensors of the internal and exter-
Ecotoxicology deals with, theoretically at least, all species, and
nal environment in order to maintain homeostasis, and in estab-
in line with other aspects of natural resource management, the
lishment and maintenance of circadian clocks. Characterized
primary concern is one ofsustainability. The policies and regu-
genes that are upregulated by the AHR system code for enzymes
lations surrounding chemical effects in natural ecosystems
involved in the metabolism of lipophilic chemicals, includ-
are designed to protect ecologic features such as population
ing organic xenobiotics and some endogenous substrates such
dynamics, community structures, and ecosystem functions.
as steroid hormones. These enzymes include mammalian
CYP1AI1, 1A2, and 1B1 and their counterparts in other verte-
Molecular and Biochemical Effects brates, glutathione transferase, glucuronosyltransferase, alcohol
This lowest level of organization includes fundamental pro- dehydrogenase, and quinone oxidoreductase.
cesses associated with the regulation of gene transcription and Some ubiquitous pollutants that act as AHR ligands and
translation, biotransformation of xenobiotics, and the deleteri- markedly upregulate gene transcription via the AHR-ARNT
ous biochemical effects of xenobiotics on cellular constituents signaling pathway include the polycyclic aromatic hydrocar-
including proteins, lipids, and DNA. bons (PAHs) and the polyhalogenated aromatic hydrocarbons
(pHAHs). In general, pHAH-type AHR ligands are more potent
AHR ligands and enzyme inducers than PAHs.
Gene Expression and Ecotoxicogenomics Ethoxyresorufin O-deethylase (EROD) activity is often used
Xenobiotics can affect gene transcription through interac- as a biomarker for AHR-related changes. Elevated activities of
tions with transcription factors and/or the promoter regions hepatic EROD have been associated with exposures to PCBs,
of genes. In the context of environmental toxicology, perhaps dioxins, PAHs, and complex mixtures of these associated with
the most studied xenobiotic effects involve ligand-activated harbor sediments, municipal effluents, paper mill effluents,
transcription factors. These intracellular receptor proteins rec- refinery effluents, and oil spills.
ognize and bind specific compounds, thus forming a complex
that binds to specific promoter regions of genes, thereby acti- Genomics and Ecotoxicogenomics—Ecotoxicogenomics
vating transcription ef mRNAs, and ultimately translation of has great potential for elucidating impacts of chemicals of eco-
the associated protein. logic concern and ultimately for playing an important role in
ecologic risk assessments (ERAs) and regulatory ecotoxicol-
Estrogen Receptor—The dominant natural ligand for this ogy. Genome sequencing of many species has set the stage for
nuclear receptor is estradiol (E2). Binding of E2 with estrogen genome-wide analysis of gene expression (transcriptomics),
receptor (ER) produces a complex that can then bind to estrogen changes in protein production (proteomics), and metabolite
444 UNIT 7 Applications of Toxicology

profiles (metabolomics). Appropriate bioinformatic analy- human health issue is associated with cancer. Cancer is an
sis can help reveal biologically meaningful patterns of gene important health outcome associated with chemical exposures
expression after exposures to various toxicants. Specific areas to in wildlife, particularly for bottom-dwelling fishes. In the con-
which these emerging fields can contribute include prioritiza- text of ecotoxicology, the most widely studied form of damage
tion of chemicals investigated in ERAs, identification of modes has been the formation of stable DNA adducts, DNA strand
of action of pollutants, identification of particularly sensitive breaks, and oxidized DNA bases. Adduct formation is particu-
species, and effect prediction at higher levels of organization. larly common with exposure to PAHs. PAHs must be activated
to reactive metabolites to form these adducts.
Protein Damage—Acetylcholinesterase (AChE) degrades
the neurotransmitter acetylcholine, and controls nerve trans-
mission in cholinergic nerve tracts. The widely used organo- Cellular, Tissue, and Organ Effects
phosphate and carbamate classes of insecticides kill by Cells— Most free-living organisms routinely experience energy
inhibiting AChE, and this mechanism is operative for “nontar- deficits. For example, food resources are often scarce during the
get” organisms including invertebrates, wildlife, and humans. winter for many animals, which adapt by conserving energy
Of particular ecologic concern has been the ingestion of (by hibernating or lowering metabolism) or by storing energy
AChE-inhibiting insecticides with food items or granular for- beforehand (as is the case for many migratory birds). Thus, the
mulations (mistaken as seed or grit) by birds and aquatic animal effects of pollutants on mitochondrial energy metabolism can
exposures from agricultural run-off. Another example is the be ofparticular importance to wildlife.
inhibition of delta-aminolevulinic acid dehydratase after lead Lysosomes, which are involved in the degradation of dam-
exposure. Studies of this enzyme have been exploited as a bio- aged organelles and proteins, sequester many environmental
marker for lead exposure in humans and wildlife. In addition contaminants, including metals, PAHs, and nanoparticles.
to enzyme inhibition, chemicals can damage proteins in other The accumulation of xenobiotics by lysosomes can elicit
ways, including oxidative damage and the formation of stable membrane damage, which warns ofpathologic effects in both
adducts similar to those formed with DNA. invertebrates and vertebrates.
Chemical effects on nuclei have been examined in ecologic
Oxidative Stress—Oxidative stress has been defined as contexts. Micronuclei are chromosomal fragments that are
the point at which production of ROS exceeds the capacity of not incorporated into the nucleus at cell division, and chemi-
antioxidants to prevent damage. Numerous environmental cal exposures can markedly increase their frequency. Elevated
contaminants act as prooxidants and enhance production of micronuclei numbers have been observed in erythrocytes in
ROS. The resulting oxidative damage can account wholly or fish and in hemocytes in clams from a PCB-polluted harbor.
partially for toxicity. Mechanisms by which chemicals enhance
ROS production include redox cycling, interactions with elec- Target Organs—An important target organ in ecotoxicology
tron transport chains (notably in mitochondria, microsomes, of nonmammalian aquatic vertebrates and many invertebrates
or chloroplasts), and photosensitization. Redox cycling chemi- is the gill, which is the major site of gas exchange, ionic regula-
cals include diphenols and quinones, nitroaromatics and azo tion, acid-base balance, and nitrogenous waste excretion. Gills
compounds, aromatic hydroxylamines, paraquat, and certain are immersed in a major exposure medium for these animals
metal chelates, particularly of copper and iron. (surface water), so metabolically active epithelial cells are in
Photosensitization is an important mechanism in aquatic direct contact with this medium. They also receive blood sup-
systems. Ultraviolet (UV) radiation (specifically UV-B and ply directly from the heart. Common structural lesions in gills
UV-A) can penetrate surface waters to varying depths, depend- include cell death (via necrosis and apoptosis), rupture of the
ing on the wavelength of the radiation and the clarity of the epithelium, hyperplasia, and hypotrophy of various cell popu-
water. The UV radiation generates ROS and other free radicals lations that can lead to lamellar fusion, epithelial swelling, and
via excitation of photosensitizing chemicals, including com- lifting of the respiratory epithelium from the underlying tissue.
mon pollutants of aquatic systems. Chloride cells have a major role in ionic homeostasis, and they
ROS can drive redox status to a more oxidized state, potentially can be compromised after exposure to metals, such as cadmium,
reducing cell viability. These ROS-mediated impacts and others copper, lead, silver and zinc. In some cases, this may be due to
have been associated with several human diseases including ath- inhibition of ATPases and/or increased membrane permeability.
erosclerosis, arthritis, cancer, and neurodegenerative diseases
such as Alzheimer’s disease, Parkinson's disease, and amyo-
trophic lateral sclerosis. With the exception of cancer, the role of Organismal Effects
ROS in specific diseases in wildlife has received little attention. It Mortality—Chemical pollution of the environment does not
is reasonable to assume that oxidative stress accounts in part for generally attain levels sufficient to outrightly kill wildlife. The
the toxicity of diverse pollutants to free-living organisms. ecotoxicologic concerns are the long-term, chronic impacts of
chemicals on organismal variables such as reproduction and
DNA Damage—The importance of DNA as a molecular development, behavior, and disease susceptibility, and how
target was discussed in Chapter 9, and the most important such impacts parlay into effects at the population and higher
 CHAPTER 30 Ecotoxicology 445

levels of organization. However, mortality is an end point in feeding behaviors and on AChE inhibition in coho salmon
exposure studies. (Oncorhynchus kisutch). Mercury, particularly as methyl-
mercury, comprises another potent neurotoxin that has been
Reproduction and Development—Contaminant effects shown to perturb behavior in wildlife.
on development are often difficult to discern in field studies, Environmental contaminants not generally thought of as
due to the small size of embryos and the fact that developmen- neurotoxicants have also been shown to perturb behavior. For
tal impacts are generally either lethal or greatly reduce survival. example, cadmium and copper have been shown to impact olfac-
Because early life stages of most organisms are generally more tory neurons and associated behaviors (preference/avoidance
sensitive to xenobiotics than other life stages, developmental to chemicals, including pheromones) in several fish species.
impacts merit careful attention by ecotoxicologists. Copper exposure in zebrafish also led to loss of neurons in the
Chlorinated hydrocarbons continue to generate concerns peripheral mechanosensory system (“lateral line”), which could
although many (DDT and other insecticides, and PCBs) have lead to altered behaviors associated with schooling, predator
had their production and use sharply curtailed. The dioxins avoidance, and rheotaxis (physical alignment of fish in a cur-
(TCDD) and coplanar PCBs compromise cardiac development, rent). Clearly, numerous mechanisms of chemical toxicity can
among other effects in vertebrates, and these developmental result in behavioral impacts, including direct toxicity to neurons,
perturbations are largely receptor-mediated and dependent on alterations in hormones that modulate behaviors, and impaired
binding ofthe chemical (such as TCDD) with the AHR. energy metabolism. Also, impaired behavior may comprise a
Hydrocarbons, in large part PAHs, associated with oil spills, sublethal impact with substantive ecologic consequence.
contaminated sediments, paper mill effluents, and creosote
used for wood treatment have profound developmental effects Cancer—Beginning in the 1960s, numerous cases of can-
in fish embryos. In many cases, the effects observed visually cer epizootics in wildlife that are associated with chemi-
appear similar to those observed in fish embryos exposed to cal pollution, particularly in specific fish populations, have
dioxins and coplanar PCBs, and include malformed hearts been reported in North America and northern Europe. As in
(“tube hearts”), craniofacial deformities, hemorrhaging, and humans, cancer in these animals occurs largely in relatively
edema of the pericardium and yolk sac, the latter resulting in older age classes and therefore is oftentimes considered a dis-
a distended, faintly blue yolk sac that gives this syndrome the ease unlikely to directly impact population dynamics or other
name “blue sac disease.” ecologic parameters. However, this may not always be the case,
particularly in species that require many years to attain sexual
Disease Susceptibility—The potential impacts of envi- maturity and/or have low reproductive rates.
ronmental contaminants on immune systems that render Lifestyle is a major contributor to differential cancer sus-
organisms more susceptible to disease are of great concern. ceptibility; benthic (bottom-dwelling) species such as brown
Numerous laboratory studies have demonstrated chemical bullhead (Ameiurus nebulosus) and white sucker (Catostomus
impacts on immune systems in animals of ecologic relevance. commersoni) in freshwater systems, and English sole (Parophrys
These include pesticides in amphibians, PCBs in channel cat- vetulus) and winter flounder (Pseudopleuronectes americanus)
fish, heavy metals in rainbow trout, PAHs in bivalves, and flame in marine systems generally exhibit the highest cancer rates in
retardants (polybrominated diphenyl ethers) in American kes- polluted systems. The bulk of chemicals in these systems asso-
trels. The potential effects of chemicals on immune function ciated with cancer epizootics, such as PAHs, PCBs, and other
and disease susceptibility in wildlife is an important problem halogenated compounds, reside in sediments; benthic fish live
in ecotoxicology and future work with powerful genomic tools in contact with these sediments and prey in large measure on
will help make significant advances in our understanding. other benthic organisms. The molecular and biochemical path-
ways underlying chemical carcinogenesis, such as PAH metab-
Behavior—Relatively subtle effects on behaviors associ- olism, DNA damage, and effects on oncogenes are qualitatively
ated with mating and reproduction, foraging, predator-prey similar between most fish and mammalian species examined.
interactions, preference/avoidance of contaminated areas, and It is noteworthy that many reports of elevated cancer rates in
migration have potentially important ramifications for popu- free-living animals occur in fish, with few reports of potentially
lation dynamics. In some cases, the biochemical mechanisms chemically related cancers to our knowledge in other verte-
underlying behavioral effects have been elucidated, which may brates. It is likely that elevated exposures play an important role
assist our understanding of these issues and provide useful bio- in the relatively high frequency of reports of cancers in ben-
markers for behavioral toxicants in field studies. thic fish; relative inherent sensitivities among mammals, birds,
Chemicals causing behavioral effects in wildlife are known reptiles, and amphibians, and fish are unclear.
to be neurotoxicants. Behavioral effects of insecticides have
been observed in fish. For example, impacts of the organo-
phosphate diazinon on olfactory-mediated behaviors such as Population
the alarm response and homing in the Chinook salmon have A population is a collection of individuals of the same species
been observed, as well as similar thresholds for the effects of that occupy the same space and within which genetic informa-
another organophosphate (chlorpyrifos) on swimming and tion can be exchanged. Population ecotoxicology covers a wide
446 UNIT 7 Applications ofToxicology

TABLE 30-1 Asummary of one popular set of rules of thumb for assessing plausibility of a causal association in an
ecologic epidemiology.

Rule Description

1. Strength ofassociation How strong the association is between the possible cause and the effect, e.g., a very large relative risk

2. Consistency of association How consistently is there an association between the possible cause and the effect, e.g., consistent among
several studies with different circumstances

3. Predictive performance How good is the prediction of effect made from the presence/level of the possible cause

4. Monotonic trend How consistent is the association between possible cause and effect to a monotonic trend (i.e., either a
consistent increase or decrease in effect level/prevalence with an increase in exposure)

5. Inconsistent temporal sequence The effect, or elevated level of effect, occurs before exposure to the hypothesized cause

6. Factual implausibility The hypothesized association is implausible given existing knowledge

7. \Inconsistency
with replication Very poor reproducibility of association during repeated field assessments encompassing different
circumstances or repeated formal laboratory testing

range of topics with core research themes being (1) epidemiol- Genetic qualities are also used to infer past toxicant influence
ogy of chemical-related diseases, (2) effects on general popula- in an exposed population. Another piece of evidence demon-
tion qualities including demographics and persistence, and strating past toxicant influence on populations can be a change
(3) population genetics. in genetic diversity. A drop in genetic diversity in populations
The level of belief warranted for possible contaminant- is thought to be an adverse effect because genetic diversity is
related effects in nonhuman populations is assessed by apply- required in populations to evolutionarily adapt to environ-
ing routine epidemiologic methods. Rules of thumb for mental changes. Toxicants can influence genetic diversity by
gauging the level of belief warranted by evidence that emerged purely stochastic means.
from human epidemiology are also applied in population eco-
toxicology (Table 30-1).
Defining and predicting alterations in population size, Community
dynamics, and demographic composition owing to toxicant An ecologic community is an assemblage of populations
exposure are important. Some species populations fluctuate occupying a defined habitat at a particular time. Populations
within a range of densities. These fluctuations are characteristic
of the species’ strategy for maintaining itself in various types of
habitats and toxicant exposure could potentially change this
range. Combined with decreases in population densities driven
by external forces such as weather events, these toxicant-
induced modifications of the average population densities and
dynamics can increase the risk of a population’s density fall-
Inhabiting
ing so low that local extinction occurs. Toxicants can change a keystone
species population’ vital rates, such as age- and sex-dependent habitat Barrier to migration
death, birth, maturation, and migration rates. Combined, these
changes determine the population density and distribution of
individuals among ages and sexes during exposure.
Individuals of the same species often are grouped into
subpopulations within a habitat and all of these subpopula-
tions together comprise a metapopulation (Figure 30-2). Barrier
migration
to
Subpopulations in the metapopulation have different levels of
exchange and different vital rates that depend on the nature of
their habitat. Spatial distances and obstacles or corridors for FIGURE 30-2 Metapopulations are composed of
subpopulations that differ in their vital rates and tendency to
migration influence migration among patches; habitat quality
exchange individuals. In this illustration, subpopulation A occupies
determines vital rates.
a keystone habitat. The loss of subpopulation A would devastate
The genetics of exposed populations are studied to under- the metapopulation. Also, loss of the migration corridor between
stand changes in tolerance to toxicants and to document toxi- subpopulations A, B, and D would devastate the metapopulation.
cant influence on field populations. Some populations have the In contrast, the loss of subpopulation F would not influence the
capacity to become more tolerant of toxicants via selection. metapopulation to the same degree.
 CHAPTER 30 Ecotoxicology 447

in a community interact in many ways and, because these be completely understood in this framework, so a landscape
many interactions are complex, a community has properties scale might be chosen instead. As an example, acid precipita-
that are not predictable from those of its component popula- tion might be examined in the context of an entire watershed,
tions. Some species play a crucial role (keystone species) or mountain range, or even a continental region. Still other eco-
numerical dominance (dominants) and these are essential to toxicants require a global context in order to fully understand
maintaining community structure, which refers to the number their movements and accumulation. As an example, hexachlo-
of species present and the numbers of individuals present in robenzene concentration in tree bark collected worldwide
each of these species. Community may also refer to the dis- showed a clear latitudinal gradient.
tribution of species among different functional groups such as
decomposers, detritivores, primary producers, primary con-
sumers (herbivores), and secondary consumers (carnivores APPROACHES
that consume herbivores).
Communities take on characteristic structures as predicted Toxicity Tests
by the law of frequencies: the number of individual organisms Toxicity testing encompassing representative animals and
in a community is related by some function to the number of plants at different levels of organization offers a practi-
species in the community. Ecotoxicants can alter the result- cal approach to characterize chemical effects on biologic
ing community structure in predictable ways by either directly systems. Toxicity tests address the potential direct effects of
impacting the fitness of individuals in populations that make toxic substances on individual ecosystem components in a
up the community or by altering population interactions. controlled and reproducible manner. Ecotoxicology tests
Recently, structural and functional qualities in communi- feature a wide variety of aquatic (including algae, inverte-
ties have been combined to generate multimetric indices such brates, tadpoles, bivalves, shrimp, and fish), avian (quail and
as the Index of Biotic Integrity (IBI). Ecologic insight is used duck), and terrestrial (soil microorganisms, crops, honey
to select and then numerically combine community qualities bees, earthworms, and wild mammals) species. Species are
such as species richness, health of individual animals in a sam- selected based on their traditional use as laboratory animals,
ple, and the number of individuals in a sample belonging to but also on ecologic relevance, which further complicates
a particular functional group, such as number of piscivorous global harmonization of ecologic testing. In addition, test-
fish. The IBI score for a study site is calculated and compared ing of aquatic species requires monitoring of water quality,
with that expected for an unimpacted site in order to estimate investigation of the solubility and stability of the test sub-
its biologic integrity. stance under the conditions of testing, and determination
Another central theme in community ecotoxicology is toxi- of nominal versus measured concentrations. Testing can
cant transfer during trophic interactions. Toxicant concen- be conducted in aqueous systems without renewal of test
trations can decrease (biodiminution), remain constant, or substance (static), renewal at predetermined time intervals
increase (biomagnification) with each trophic transfer within (static renewal), or continuous flow oftest substance through
a food web. Metals that biomagnify are mercury and the alkali the test compartment (flow-through).
metals, cesium, and rubidium. Zinc, an essential metal that In acute toxicity testing, single species are exposed to vari-
is actively regulated in individuals, can exhibit biomagnifica- ous concentrations of the test agent. The most common end
tion or biominification depending on whether ambient levels point in acute tests is death. Abnormal behavior and other
are below or above those required by the organism to function gross observations are commonly noted, and nonlethal end
properly. points occasionally apply. Data from different test concentra-
Most individuals in a community can feed on different spe- tions are used to derive concentration-response curves. The
cies depending on their life stage, seasons, and relative abun- LC,, represents the concentration of test substance killing 50%
dances of prey species. These trophic interactions are best of the tested animals and EC., the concentration of test sub-
described as occurring in a trophic web, not a trophic chain. stance affecting 50% of the test population during a specified
period of time, such as growth; the IC, is the concentration
causing a 50% reduction in a nonquantal measurement (such
Ecosystem to Biosphere as movement) for the test population. Other quantitative val-
Ecosystems, the functional unit of ecology, are composed of ues are the lowest observed effect concentration (LOEC), that
the ecologic community and its abiotic habitat. The ecotoxi- is, the lowest concentration where an effect is observed, and
cologist is interested in understanding how toxicants dimin- the no observed effect concentration (NOEC), the highest
ish an ecosystem’s capacity to perform essential functions and concentration resulting in no adverse effects.
to understand toxicant movement within different ecosystem Short-term laboratory studies conducted with single species
components enough to assess exposure. are useful for rapid screening, provide information on thresh-
Conventional ecosystem studies involve descriptions of olds for effects, and selective and comparative toxicity, and
contaminant concentrations and movements in easily defined can be used as range finders to guide subsequent, often more
ecosystems such as lakes, forests, or fields. Some toxicants, involved, studies. Long-term and reproductive studies evaluate
especially those subject to wide dispersal by air or water, cannot the effects of substances on organisms over extended periods
448 UNIT 7 Applications of Toxicology

of time and/or sequential generations (chronic toxicity, life cleanups. However, careful case-specific thought is required
cycle, and reproduction). for the selection of biomarkers.
Unique to ecotoxicology are the more elaborate microcosm,
mesocosm, and field studies. Microcosms are representative
aquatic or terrestrial ecosystems created under laboratory con- Population
ditions that include a number of relevant species (such as pro- Demographic surveys or experiments can be conducted for
tozoa, plankton, algae, plants, and invertebrates). Simulated exposed populations. Some studies explore age-specific vital
field studies or mesocosms can be created in the laboratory rates but others are designed to explore vital rates for different
or in the field (e.g., artificial streams and ponds) or consist of ages such as nestling, fledgling, juvenile, and adult. Most result
enclosures of existing habitats, containing representative soil, in data sets that can be analyzed profitably using either a simple
water, and biota. Lastly, full-scale field studies (aquatic organ- life table or more involved matrix analysis. The matrix method
isms, terrestrial wildlife, and pollinators) evaluate the effects of allows one to describe the population state and also to under-
a substance on wildlife under real-life scenarios of actual use stand the sensitivity of the population to effects on vital rates
conditions of a product (e.g., pesticide field usage rate), and for various ages or stages. The value of such studies lies in the
thus are more complicated, subject to considerable variability, ability to integrate effects on several factors into a projection of
and require extensive knowledge of the local population and population consequences. Demographic studies are becoming
community dynamics. more common in ecotoxicology, especially with species ame-
Asa final point, plant studies are a significant component of nable to laboratory manipulation.
ecologic toxicity testing, particularly for pesticide registration, Conventional studies of increased tolerance after genera-
and involve tiered testing of both target area and nontarget ter- tions of exposure and molecular genetic surveys of exposed
restrial and aquatic plants. End points of phytotoxicity include populations are the primary approaches by which genetic con-
seedling emergence and growth, vegetative vigor, etc. Central sequences are assessed. Increased tolerance is usually detected
to the toxicity testing with plants are the substrate and environ- by subjecting individuals from the chronically exposed popula-
mental conditions, which greatly influence plant health. tion and a naive population to toxicant challenge and formally
testing for tolerance differences. Alternatively, a change associ-
ated with a tolerance mechanism might be examined in chron-
Biomarkers ically exposed and naive populations. Close examinations of
The term “biomarker” is most often employed to refer to mole- population genetics associated with contaminated habitats are
cular, physiologic, and organismal responses to contaminant also used to infer consequences of multigenerational exposure.
exposure that can be quantified in organisms inhabiting or
captured from natural systems. Biomarkers do not directly
provide information concerning impacts on the higher levels Community and Ecosystem
of organization that ecotoxicology ultimately endeavors to Most studies of community and ecosystem effects use modi-
discern. Nevertheless, biomarkers often provide important fied methods developed in community and systems ecology.
ancillary tools for discerning contaminant exposures and The approach affording the most control and ability to repli-
potential impacts of ecologic importance. Biomarkers can cate treatments involves laboratory microcosms. A microcosm
provide sensitive early warning signals of incipient ecologic is a simplified system that is thought to possess the commu-
damage. nity or ecosystem qualities of interest. The experimental con-
Chemical specificity among biomarkers is also highly vari- trol and reproducibility associated with microcosms come at
able and is imbued with trade-offs. Nonspecific biomark- the cost of losing ecologic realism. Gaining back some realism
ers may be preferred if complex mixtures are being studied. by giving up some degree of tractability, outdoor mesocosms
The larger the number of biomarkers, the more expensive are also applied to community and ecosystem ecotoxicology.
and time-intensive the study. Effects of environmental vari- Mesocosms are larger experimental systems, usually con-
ables such as temperature, time of day or year, salinity, and structed outdoors that also attempt to simulate some aspect
dissolved oxygen and physiologic variables including sex, of an ecosystem such as community species composition.
age, reproductive status, and nutritional status needed to be Terrestrial ecotoxicologists apply the term enclosure instead of
accounted for and controlled. Many biomarkers are inva- mesocosm for such experimental units. Terrestrial mesocosms
sive and require sacrifice of the organism in order to obtain can be pens, enclosures, or large soil plots depending on the
needed tissues. This can be problematic, particularly in cases effects being quantified. Field studies are the third means of
involving rare species or charismatic species such as marine exploring effects at the community or ecosystem level. The high
mammals. In such cases, and in others where feasible, the use realism of associated findings from field studies is balanced
of noninvasive biomarkers is either preferred or required. against the difficulty of achieving true replication and sufficient
Biomarkers can provide powerful tools as early warning sig- control of other factors influencing the system's response. Field
nals of ecologic damage, to assist in assessments of environ- studies can involve manipulations such as introducing toxicant
mental contamination, and in determining the effectiveness into replicate water bodies; however, the majority of field stud-
of various environmental management decisions such as ies involve biomonitoring of an existing, notionally impacted,
 CHAPTER 30 Ecotoxicology 449

community or ecosystem. Because mesocosm and field studies Planning with risk managers
involve data generation in the presence of many uncontrolled and interested parties
A
variables and poor replication or pseudoreplication, appropri-
ate multivariate statistical techniques for recognizing patterns v
among locations or through time are required. Establish
Assessment end point Problem
Conceptual model formulation
Landscape to Biosphere Analysis plan

Technologies for acquiring, processing, and analyzing large

amounts of information have been essential. Archived and new
imagery from satellites and high-altitude platforms are now Analysis
integrated with off-the-shelf geographic information systems Characterize
Ecosystem qualities
(GIS) software with affordable computers. Much of this imag- Receptor qualities Exposure Effects ==
ery is gathered with remote sensing technology, and arrays of Exposure profile
sensors are being assembled to enable real-time input. Remote Stressor-response profile
sensing data from satellites or aircraft provide information for
wide spatial areas and the rapidly emerging, ground- or water-
based observing system networks have begun to produce Summarize
extremely rich data streams. Risk estimation Risk
Risk description characterization
Uncertainties

ECOLOGIC RISK ASSESSMENT

v
ERA applies ecotoxicologic knowledge to support environ- Risk communication -------
mental decision making (Figure 30-3). A widely dispersed A
ecotoxicant such as acid precipitation or widely used prod- v
uct such as a herbicide might require assessment of risk at Risk management
a landscape or subcontinental scale. Ecotoxicants requiring FIGURE 30-3 The general form ofan ecologic risk
a global ERA might include greenhouse gases contributing assessment including problem formulation, analysis, and risk
to global warming, hydrofluorocarbons depleting the ozone characterization stages. Problem formulation is done in dialog with
layer, and persistent organic pollutants that accumulate to risk managers and stakeholders, and involves a clear statement ofthe
harmful concentrations in polar regions far from their release ecologic entity to be assessed, a conceptual model for the process,
point at industrialized latitudes. and a plan for conducting the assessment. The analysis stage involves
Adaptations are based on the context of an ERA. Some exposure and effect characterizations. Using the context developed
ERAs address existing situations. Considerable field infor- during problem formulation and information organized together in
mation might be available for such a retroactive ERA and the analysis stage, a statement of risk and associated uncertainties are
made in the risk characterization stage.
epidemiologic methods might be applied advantageously. In
contrast, predictive ERAs assess possible risk associated with
a future or proposed toxicant exposure.
Exposure characterization describes or predicts contact probability of a specified intensity of an adverse effect occur-
between the toxicant and the assessment end point. Depending ring to the assessment end point, generally only a qualitative
on the ERA context, this could involve a simple calculation of likelihood is expressed. Nevertheless, the risk characteriza-
average exposure, or a temporally and spatially explicit descrip- tion must provide details surrounding the statement, includ-
tion of amounts present in relevant media. Toxicant sources, ing important uncertainties.
transport pathways, kinds of contact, and potential costressors
are also defined.
Ecologic effect characterization describes the qualities INTERCONNECTIONS BETWEEN
of any potential effects of concern, the connection between ECOSYSTEM INTEGRITY AND
the potential effects and the assessment end point, and how HUMAN HEALTH
changes in the level of exposure might influence the effects
manifesting in the assessment end point. Normally, a state- It is important to consider interconnections between human
ment about the strength of evidence associated with the health and ecologic integrity, or health. By determining how
descriptions and uncertainties is presented in the ecologic chemicals and other anthropogenic stressors degrade ecosys-
effect characterization. tems and impact human health and well-being, and vice versa,
Risk characterization uses the analysis of exposure and a holistical understanding of the results of environmental
ecologic effects to address questions of risk. Although it contamination is obtained. For example, a conceptual model
is desirable to have an explicit statement of risk, that is, the attempts to elucidate the interconnections linking natural and
450 UNIT 7 Applications of Toxicology

social systems in a circular manner with continuous feedbacks. contamination of seafoods valued by humans is one example.
The natural system produces both positive outputs (such as Others are less clear but potentially very significant, such as
natural resources and raw materials) and negative outputs human impacts on aquatic systems that foster the propaga-
(such as hurricanes and disease vectors) to the social system. tion of human disease vectors, or human impacts on global
The culture and institution of the social system in turn trans- climate that may concomitantly impact humans and eco-
form the natural system outputs in various ways and subse- systems in varied and complex ways. Collaboration among
quently deliver various positive outputs (consumer goods biomedical, environmental, and social scientists and policy-
and conservation efforts) and negative outputs (pollution and makers will catalyze the integrated protection of human and
deforestation) to the natural system. These outputs influence ecosystem health.
the quantity and quality of life (human and nonhuman) of the
natural system, and the circular flow of resources continually
creates conditions that influence the well-being of individuals, BIBLIOGRAPHY
societies, and ecosystems. Newman MC (ed.): Fundamentals of Ecotoxicology: The Science of
This rather abstract model formalizes the interconnections Polution, 4th ed. Boca Raton, FL: CRC Press, 2014.
between human and ecologic health that most of us intui- Walker CH, Sibly RM, Hopkin SP, Peakall DB: Principles ofEcotoxicology,
tively sense. Some ofthese connections are obvious. Chemical 4th ed. Boca Raton, FL: CRC Press/Taylor & Francis, 2012.
 CHAPTER 30 Ecotoxicology 451

QUESTIONS

1. What is the mode by which a chemical enters the 6. Which of the following is FALSE regarding terrestrial
lithosphere? - ecotoxicology?
a. evaporation. a. ‘Terrestrial organisms are generally exposed to con-
b. adsorption. taminants via ingestion.
c. dissolution. b. Predation is an important confounder of measure-
d. absorption. ments in terrestrial toxicology field studies.
e. diffusion. c. Reproductive tests are not important in measuring
end points in toxicity tests.
2. ‘The bioavailability of contaminants in the hydrosphere is d. Enclosure studies are better able to control for envi-
directly related to: ronmental factors in field studies.
chemical concentration. e. Toxicity tests usually test the effects of an oral chemi-
amount of chemical. cal dose.
water solubility of chemical.
toxicity of chemical. An important type(s) of compound that is far more toxic
se
enomolecular size of chemical. in water than in air is/are:
a. organic compounds.
3. All of the following regarding biomarkers are true b. photochemicals.
EXCEPT: c. vapors.
a. Dermal absorption is considered an external dose. d. lipid-soluble xenobiotics.
b. Biomarkers of susceptibility are useful in extrapolat- e. metals.
ing wildlife disease to human diseases.
c. Induction of certain enzymes is an important Which ofthe following are used to record end point toxic-
biomarker. ity of aquatic toxicity tests?
d. The biologically effective dose is the amount ofinter- ADE unre WelDees
nal dose needed to elicit a certain response. LG, and EG...
e. ‘The effects of chemical exposure can be different reproductive tests.
across species. PID ana hCG.
oP Dy
cao and ECs.
4. Which of the following processes is LEAST likely to be
affected by endocrine-disrupting agents? Biologic availability is:
enzyme activity. a. the total amount of chemical within an organism.
transcription. b. the concentration of chemical in an environmental
hormone secretion. reservoir.
signal transduction. c. the threshold concentration of achemical needed for
Cae DNA replication.
OR
OS
Ce toxic effect.
the concentration of chemical within an organism.
5. Estrogen exposure has been shown to cause all of the e. the proportion of chemical potentially available for
following in wildlife species EXCEPT: uptake.
sexual imprinting.
altered sex hormone levels. . Chemodynamics does NOT study:
immune suppression. the fate of chemicals in the environment.
gonadal malformations. the rate at which chemicals are metabolized.
Er
@
(4 sex reversal. the distribution of chemicals in the environment.
the effects of toxic substances on the environment.
OTSthe
Cer release of chemicals into the environment.
 a
eS

Se
tae
 Gap mAsePrr TieEwoR

Food Toxicology
Frank N. Kotsonis and George A. Burdock

UNIQUENESS OF FOOD TOXICOLOGY Establishing Safe Conditions of Use for New Foods,
Nature and Complexity of Food Macroingredients and New Technologies

Importance of the Gastrointestinal Tract Transgenic Plant (and New Plant Varieties) Policy
Nanotechnology
SAFETY STANDARDS FOR FOODS, FOOD INGREDIENTS, Safety Requirements for Dietary Supplements
AND CONTAMINANTS Assessment of Carcinogens
The Food, Drug, and Cosmetic Act Carcinogenicity as a Special Problem
Methods Used to Evaluate the Safety of Foods,
ADVERSE REACTIONS TO FOOD OR FOOD INGREDIENTS
Ingredients, and Contaminants
Safety Evaluation of Direct Food and TOXIC SUBSTANCES IN FOOD
Color Additives
Metals, Hydrocarbons, N-Nitroso Substances
Exposure:
The Estimated Daily Intake
and Mycotoxins
Assignment of Concern Level (CL) and
Toxins in Fish, Shellfish, and Turtles
Required Testing
Microbiologic Agents
Safety Determination of Indirect Food
Bovine Spongiform Encephalopathy
Additives
Safety Requirements for GRAS Substances CONCLUSION

KEY POINTS

= Food is an exceedingly complex mixture of nutrient and be used and the concentration of the substance in that
nonnutrient substances. food.
z A substance listed as Generally Recognized as Safe = Food hypersensitivity (allergy) refers to a reaction involv-
(GRAS) achieves this determination on the adequacy of ing an immune-mediated response, including cutaneous
safety, as shown through scientific procedures or through reactions, systemic effects, and even anaphylaxis.
experience based on common use. = ‘The vast majority of food-borne illnesses in developed
= Anestimated daily intake (EDI) is based on two factors: countries are attributable to microbiologic contamination
the daily intake of the food in which the substance will of food.

UNIQUENESS OF FOOD TOXICOLOGY under strict quality controls and thus cannot meet the rigor-
ous standards of chemical identity, purity, and good manufac-
The nature of food is responsible for the uniqueness of food turing practice met by most consumer products. The fact that
toxicology. Food contains hundreds of thousands of sub- food is harvested from the soil, the sea, or inland waters or is
stances that have not been fully characterized or tested. Food derived from land animals subject to the unpredictable forces
cannot be commercially produced in a definable environment of nature makes the constancy of raw food unreliable. Food is

453
454 UNIT 7 Applications ofToxicology

more complex and variable in composition than all other sub- TABLE 31-2 Systems transporting enteric
stances to which humans are exposed, and humans are exposed constituents.
more to food than to any other chemicals!
System Enteric Constituent

Passive diffusion Water, chloride, fats (as micelles), short- and

Nature and Complexity of Food medium-chain fatty acids
Food is an exceedingly complex mixture whether it is consumed Fructose, D-xylose, 6-deoxy-1,5-anhydro-
Facilitated diffusion
in the “natural” (unprocessed) form or as a highly processed p-glucitol, glutamic acid, aspartic acid,
“Meal Ready to Eat” (MRE). Nonnutrient substances (substances short-chain fatty acids, glucose, galactose,
other than carbohydrates, proteins, fats, or vitamins/minerals) xenobiotics with carboxy groups, sulfates,
glucuronide esters, lead, cadmium, zinc
may be contributed by food processing, but nature provides the
vast majority of nonnutrient constituents. Table 31-1 indicates Active transport Cations, anions, sugars, vitamins, nucleosides
that natural, or minimally processed, foods contain far more (pyrimidines, uracil, and thymine,
which may be in competition with
nonnutrient than nutrient constituents.
5-fluorouracil and 5-bromouracil), cobalt,
Nonnutrient substances include plant hormones and natu- manganese (which competes for the iron
rally occurring pesticides, antinutrients such as lectins, sapo- transportation system)
nins, trypsin, and/or chymotrypsin inhibitors in soybeans,
Pinocytosis Long-chain lipids, vitamin B,, complex, azo
phytates that may bind minerals, antithiamines, and frankly dyes, maternal antibodies, botulinum
toxic constituents such as tomatine or cycasin. Some 7800 volatile toxin, hemagglutinins, phalloidins, E. coli
chemicals have been identified in food. Moreover, nonnutrient endotoxins, virus particles
substances may be classified as food additives. Approximately
200 flavoring ingredients, most already in food naturally, may ‘
\

be added to food, often in concentrations similar to that which

is found naturally. The constituents of food and other ingesta (e.g., drugs,
contaminants, and inhaled pollutants dissolved in saliva and
swallowed) are physicochemically heterogeneous, and the
importance of the Gastrointestinal Tract primary mechanisms for intestinal absorption are passive or
The gut is a large, complex, and dynamic organ with vast simple diffusion, active transport, facilitated diffusion, and
absorptive surface. The GI transit time provides for adequate pinocytosis. Each mechanism characteristically transfers a
exposure of ingesta to a variety of processing conditions includ- defined group of constituents from the lumen into the body
ing, but not limited to, variable pH, digestive acids and enzymes (Table 31-2).
(trypsin, chymotrypsin, etc., from the pancreas and carbohy- Apart from its duties of absorption and metabolism, the GI
drases, lipases, and proteases from the enterocytes), saponifica- tract is also the largest immunologic organ in the body and
tion agents (in bile), and a luxuriant bacterial flora (estimated is constantly exposed to a large number of antigens in food
to be 100 trillion organisms in adults). In addition, enterocytes (approximately 88kg of protein annually) and commensal and
possess an extensive capacity for the metabolism of xenobiotics ingested bacteria. One cell layer away from these antigens is
that may be second only to the liver, with a full complement of the lamina propria of the GI tract, which contains the muco-
phase (type) I and phase (type) II reactions present. sal-associated lymphoid tissue, composed of lymphocytes and
antigen-presenting cells, as well as unique dendritic cells, which
interact with dietary antigens and ultimately determine whether
TABLE 31-1 Nonnutrient substances in food. an antigen is tolerated or an immune response is launched.

Number of Identified
Food Nonnutrient Chemicals
SAFETY STANDARDS FOR FOODS, FOOD
Cheddar cheese 160 INGREDIENTS, AND CONTAMINANTS
Orange juice 250
The Food, Drug, and Cosmetic Act
Banana 325
The Food, Drug, and Cosmetic (FD&C) Act presumes that
Tomato 350 traditionally consumed foods are safe if they are free of
contaminants. To ban such foods, the FDA must have clear
Wine 475
evidence that death or illness can be traced to the consump-
Coffee ) 625 tion of a particular food. The FD&C Act permits the addition
of substances to food to accomplish a specific technical effect if
Beef (cooked) 625
the substance is determined to be Generally Recognized as Safe
Reproduced with permission from Smith RL: Does one man’s meat become another (GRAS). The act requires that scientific experts base a GRAS
man’s poison? Trans Med Soc Lond 1991-1992;108:6-17. determination on the adequacy of safety, as shown through
 CHAPTER 31 Food Toxicology 455

scientific procedures or through experience based on common of adults and about 6% of young children in the United States
use. If a food contains an unavoidable contaminant even with suffer from food allergies, and food allergies account for 35%
the use of current good manufacturing practices (CGMP), it to 50% of all cases of anaphylaxis.
may be declared unfit as food if the contaminant may render
the food injurious to health. Foods containing unavoidable
contaminants are not automatically banned, but the FDA has Methods Used to Evaluate the Safety of
regulatory tolerance levels or more informal action levels on Foods, Ingredients, and Contaminants
the tolerable quantity of unavoidable contaminants. Safety Evaluation of Direct Food and Color Additives—
The primary factors that must be considered in the evalu- The safety of any substance added to food must be established
ation of animal drugs are (1) consumption and absorption on the basis of specific intended conditions of use or uses in
by the target animal, (2) metabolism of the drug by the tar- food. Factors that need to be considered include (1) the purpose
get food animal, (3) excretion and tissue distribution of the for use of the substance, (2) the food to which the substance is
drug and its metabolites in food animal products and tissues, added, (3) the concentration level used in the proposed foods,
(4) consumption of food animal products and tissues by and (4) the population expected to consume the substance.
humans, (5) potential absorption of the drug and its metabo-
lites by humans, (6) potential metabolism of the drug and its Exposure:
The Estimated Daily Intake—Exposure is most
metabolites by humans, and (7) potential excretion and tissue often referred to as an estimated daily intake (EDI) and is based
distribution in humans of the drug, its metabolites, and the on two factors: the daily intake () of the food in which the sub-
secondary human metabolites derived from the drug and its stance will be used and the concentration (C) of the substance
metabolites. Thus, the pharmacokinetic and biotransforma- in that food. In estimates of consumption and/or exposure, one
tion characteristics of both the animal and the human must must also consider other sources of consumption for the pro-
be considered in an assessment of the potential human health posed intended use of the additive if it already is used in other
hazard of an animal drug. foods for another purpose, occurs naturally in foods, or is used
In addition to allowing GRAS substances to be added to in nonfood sources.
food, the act provides for a class of substances that are regu- Before approval, regulatory agencies require evidence that a
lated food additives, which must be approved and regulated food additive is safe for its intended use(s) and that the EDI is
for their intended use by the FDA. Two distinct types of color less than its acceptable daily intake (ADI). The ADI is generally
additives have been approved for food use: those requiring based on results from animal toxicology studies.
certification by FDA chemists and those exempt from certi-
fication. Most certified colors approved for food use bear the Assignment of Concern Level (CL) and Required
prefix FD&C (such as FD&C Blue No. 1). Orange B and Citrus Testing—Structures of functional groups in food additives are
Red No.2 are the two certified colors lacking the FD&C desig- assigned to categories (A, B, and C) based on their relative harm-
nation. Such color additives consist of structures that cannot ful nature (category A is least harmful and category C is most
be synthesized without a variety of impurities, and so must harmful). Based on structure assignment and calculated expo-
be carefully monitored and certified as safe before use in food sure, a CL for a certain additive can be assigned (Table 31-3). An
products. Food colors that are exempt from certification are additive with a higher CL (CLI) is more likely to be dangerous
derived primarily from natural sources. than one with a lower CL (CLI). Once the CL is established, a
The importance of using safety warning labeling is dem- specific test battery is prescribed, as shown in Table 31-4.
onstrated by the effort to protect particularly susceptible
consumers who have food allergies or food intolerance. Safety Determination of Indirect Food Additives—
Although accidental exposure is common, avoidance of the Indirect food additives are substances that are not added
offending foods is the only successful noninterventional directly to food but enter food by migrating from surfaces that
approach. Food allergy is the leading cause of anaphylaxis, contact food. These surfaces may be from packaging mate-
often requiring hospitalization. It is estimated that 3% to 4% rial (cans, paper, and plastic) or surfaces used in processing,

TABLE 31-3 Assignment of concern level.

Structure Category B Structure Category C Concern Level

< 0.05 ppm in the total diet < 0.025 ppm in the total diet < 0.0125 ppm in the total diet |
(< 0.0012 mg/kg per day) (< 0.00063 mg/kg per day) (< 0.00031 mg/kg per day)

> 0.05 ppm in the total diet > 0.025 ppm in the total diet > 0.0125 ppm in the total diet Il
(= 0.0012 mg/kg per day) (= 0.00063 mg/kg per day) (= 0.00031 mg/kg per day)

> 1 ppmin the total diet = 0.5 ppm in the total diet > 0.25 ppm in the total diet II
(= 0.025 mg/kg per day) (= 0.0125 mg/kg per day) (= 0.0063 mg/kg per day)
456 UNIT 7 Applications of Toxicology

TABLE 31-4 Summary of the toxicity tests regarded as GRAS is given in Table 31-5. It is important to
recommended for different levels of concern.* reemphasize that GRAS substances, though used like food
additives, are not food additives; this allows GRAS substances
Concern Levels to be exempt from the premarket clearance restrictions applied
Toxicity Studies* I rf lil to food additives.
Short-term tests for genetic toxicity x X X

Metabolism and pharmacokinetic studies xX X

Establishing Safe Conditions of
Use for New Foods, Macroingredients
Short-term (28-day) toxicity studies with rodents X#
and New Technologies
Subchronic (90-day) toxicity studies with rodents xt x Transgenic Plant (and New Plant Varieties) Policy—New
Subchronic (90-day) toxicity studies x? and novel foods or ingredients and new technologies pres-
with nonrodents ent new challenges and may require innovative methods for
determining safety. Scientists have employed biotechnology to
Reproduction studies with teratology phase x? x?
add one or more specific genes into crops like soybeans, corn,
One-year toxicity studies with nonrodents X cotton, and canola, to improve pest and disease management,
resulting in agronomic, economic, environmental, health, and
Carcinogenicity studies with rodents xs
social benefits for farmers. Irrespective of the breeding method
Chronic toxicity/carcinogenicity studies xed used to produce a new plant variety, tests must be done to
with rodents ensure that the levels of nutrients or toxins in the plants have
*http://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatory
not changed and that the food is still safe to consume. In par-
information/ingredientsadditivesgraspackaging/ucm2006826.htm ticular, tests on new plant varieties must demonstrate that any
*Not including dose range-finding studies, if appropriate. new proteins produced in the plant by genetic engineering are
*Including neurotoxicity and immunotoxicity screens.
5An in utero phase is recommended for one of the two recommended carcinogenicity nontoxic and nonallergenic.
studies with rodents, preferably the study with rats.
‘Combined study may be performed as separate studies.
Nanotechnology—Nanotechnology offers some distinct
advantages in delivery systems using micelles and liposomes
and other technologic advantages such as nanoemulsions
holding, or transporting food. The level of overall consump- (emulsion stability), biopolymeric nanoparticles (encapsulation
tion of these materials determines the testing required by the technology), and cubosomes (solubilized hydrophobic,
FDA to allow certain foods to be packaged in certain ways. hydrophilic, and amphiphilic molecules, among other uses).
Nanotechnology allows new and more efficient uses of old
Safety Requirements for GRAS Substances— The FD&C products by enhancing solubility, facilitating controlled release,
Act regards foods as GRAS when they are added to other food, improving bioavailability, and protecting labile substances
such as green beans in vegetable soup. It also regards a number (including micronutrients and bioactive substances) during
of food ingredients as GRAS. A list of examples of substances processing, storage, and distribution.

TABLE 31-5 Examples of GRAS substances and their functionality.

CFR Number Substance Functionality

Substances Generally Recognized as Safe 21 CFR 182

182.2122 Aluminum calcium silicate Anticaking agent
182.8985 Zinc chloride Nutrient supplement

Direct food substances affirmed as Generally Recognized as Safe 21 CFR 184

184.1005 Acetic acid Several
184.1355 Helium Processing aid

Indirect food substances affirmed as Generally Recognized as Safe 21 CFR 186

186.1025 Caprylic acid Antimicrobial
186.1374 Iron oxides Ingredient of paper and paperboard

Notified GRAS substances with “No Objection”

GRN 305 Carnobacterium maltaromaticum strain CB1 (viable and heat-treated) (Antimicrobial) inhibitor of Listeria monocytogenes
GRN 211 Xanthan gum (with reduced pyruvate) Stabilizer, emulsifier, thickener, suspending and bodying
agent, and foam enhancer

GRAS, Generally Recognized as Safe.

 CHAPTER 31 Food Toxicology 457

FDA has not yet promulgated specific guidelines for testing. TABLE 31-6 Symptoms of IgE-mediated food
However, particle behavior and characteristics at the nanoscale allergies.
enable applications that can affect safety, effectiveness, perfor-
mance, quality, and, where applicable, public health impact of Cutaneous Urticaria (hives), eczema, dermatitis, pruritus, rash

FDA-regulated products. Nanoparticle properties may be due Gastrointestinal Nausea, vomiting, diarrhea, abdominal cramps
to altered chemical, biologic, or magnetic properties, altered
electrical or optical activity, increased structural integrity, or Respiratory Asthma, wheezing, rhinitis, bronchospasm
other unique characteristics of nanoscale materials not nor- Other Anaphylactic shock, hypotension, palatal itching,
mally observed in their larger counterparts. Conversion of an swelling including that of tongue and larynx,
approved product (GRAS or food additive) to the nanoscale methemoglobinemia*
may well render the material unsafe. *An unusual manifestation ofallergy reported to occur in response to soy or cow milk
protein intolerance in infants.
Data from Murray KF, Christie, DL: Dietary protein intolerance in infants with transient
Safety Requirements for Dietary methemoglobinemia and diarrhea.
J Pediatr 122:90, 1993. Elsevier; Taylor SL,
Scanlan RA (eds.): Food Toxicology:APerspective on the Relative Risks. New York:
Supplements Marcel Dekker, 1989.

Dietary supplements have a special status within the law and

the regulations: supplements are regarded as foods or food
constituents and not food additives or drugs. The standard of symptoms associated with food allergy. Any protein in food
safety uses the concept of reasonable expectation of no harm. may act as an allergen; some of the allergenic components of
This is a lesser safety standard than the reasonable certainty common food allergens are listed in Table 31-7.
standard for substances added to foods. The basis for this ratio- Food idiosyncrasies are generally defined as quantitatively
nale is that consumption of a dietary supplement is by choice, abnormal responses to a food substance or additive. They may
not involuntary as for a food (i.e., food must have a presump- resemble hypersensitivity, but do not involve immune mecha-
tion of safety). Thus, there is a higher standard of safety for nisms. Examples of such reactions and the foods that are prob-
food. Also, because (1) there is a deliberate choice involved ably responsible are given in Table 31-8.
in consuming a dietary supplement and (2) the daily recom-
mended intake is clearly stated on the label, there is an implied
assumption of some risk on the part of the consumer.
TABLE 31-7 Knownallergenic food proteins.
Food Allergic Proteins
Assessment of Carcinogens
Cow’s milk Casein, B-lactoglobulin, a-lactalbumin
Carcinogenicity as a Special Problem—The Delaney
clause of the FD&C Act prohibits the approval of regulated Egg whites Ovomucoid, ovalbumin
food additives “found to induce cancer when ingested by man
Egg yolks Livetin
or animals.” It must be emphasized that the Delaney prohi-
bition applies only to the approval of food additives, color Peanuts Arah 2, peanut |
additives, and animal drugs; it does not apply to unavoidable
Soybeans 8-Conglycinin (7S fraction), glycinin
contaminants, GRAS substances, or ingredients sanctioned by
(11S fraction), Gly mlA, Gly mIB, Kunitz
the FDA or USDA before 1958. To be a carcinogen under the trypsin inhibitor
Delaney clause, a food or color additive must be demonstrated
to directly induce cancer when ingested by humans or animals. Codfish Gad cl
This is interpreted to mean that the findings of cancer must be Shrimp Antigen Il
clearly reproducible and that the cancers found are not second-
ary to nutritional, hormonal, or physiologic imbalances. This Green peas Albumin fraction

position allows the agency to argue that changing the level of Rice Glutelin fraction, globulin fraction
protein or fat in the diet does not induce cancer but simply
modulates tumor incidence. Cottonseed Glycoprotein fraction

Peach, guava, banana, 30 kDa protein

mandarin, strawberry
ADVERSE REACTIONS TO FOOD
Tomato Several glycoproteins
OR FOOD INGREDIENTS
Wheat Gluten, gliadin, globulin,
albumin
Food hypersensitivity (allergy) refers to a reaction involving an
immune-mediated response. An allergic reaction may be man- Okra Fraction |
ifested by one or more of the symptoms listed in Table 31-6. Data from Taylor SL, Scanlan RA (eds.): Food Toxicology:A Perspective on the Relative
Cutaneous reactions and anaphylaxis are the most common Risks. New York: Marcel Dekker, 1989.
458 UNIT 7 Applications of Toxicology

TABLE 31-8 Idiosyncratic reactions to foods.

ee ee ee a ee ee ee ee ee ee ee

Food Reaction Mechanism

Fava beans Hemolysis, sometimes accompanied by jaundice Pyramidene aglycones in fava bean cause irreversible oxidation of
and hemoglobinuria; also, pallor, fatigue, nausea, GSH in G-6-PD-deficient erythrocytes by blocking NADPH supply,
dyspnea, fever and chills, abdominal and dorsal resulting in oxidative stress of the erythrocyte and eventual
pain hemolysis

Chocolate Migraine headache Phenylethylamine-related

Beets Beetanuria: passage of red urine (often mistaken for Excretion of beetanin in urine after consumption of beets
hematuria)

Asparagus Odorous, sulfurous-smelling urine Autosomal dominant inability to metabolize methanthiol of asparagus
and consequent passage of methanthiol in urine

Red wine Sneezing, flush, headache, diarrhea, skin itch, Diminished histamine degradation: deficiency of diamine oxidase (?),
shortness of breath histamines present in wine

Choline- and Fish odor syndrome: foul odor of body secretions Choline and carnitine metabolized to trimethylamine in gut by
carnitine- bacteria, followed by absorption but inability to metabolize to
containing foods odorless trimethylamine N-oxide

Milk Abdominal pain, bloating, diarrhea Lactase deficiency .

Fructose-containing Abdominal pain, vomiting, diarrhea, hypoglycemia Reduced activity of hepatic aldolase B toward fructose-1-phosphate
foods ‘
\

TABLE 31-9 Anaphylactoid reactions to food.

Food Reaction Mechanism

Western Australian salmon Erythema and urticaria of the skin, facial flushing Scombroid poisoning; high histamine levels demonstrated
(Arripis truttaceus) and sweating, palpitations, hot flashes of the inthe fish
body, headache, nausea, vomiting, and dizziness

Fish (spiked with histamine) Facial flushing, headache Histamine poisoning; histamine concentration in plasma
correlated closely with dose ingested

Cape yellow tail (fish) (Seriola Skin rash, diarrhea, palpitations, headache, nausea Scombroid poisoning; treated with antihistamines
lalandii) and abdominal cramps, paresthesia, unusual
taste sensation, and breathing difficulties

Sulfite sensitivity Bronchospasm, asthma Sulfite oxidase deficiency to metabisulfites in foods and wine

Tuna, albacore, mackerel, Reaction resembling an acute allergic reaction Scombroid poisoning; treated with antihistamines and
bonito, mahimahi, and cimetidine
bluefish

Cheese Symptoms resembling acute allergic reaction Responds to antihistamines; histamine poisoning

Anaphylactoid reactions are historically thought of as eaten in excess or improperly processed. Specific examples are
reactions mimicking anaphylaxis (and other “allergic-type” provided in Table 31-10. The susceptible population exists as a
responses) through direct application of histamine. Ingestion of result of its own behavior, that is, the “voluntary” consumption
some types of fish that have been acted upon by certain micro- of food as a result of alimited food supply or an abnormal crav-
organisms to produce histamine may result in an anaphylactoid ing for a specific food.
reaction also called “scombrotoxicosis” (Table 31-9). Sulfite-
induced bronchospasm was first noticed as an acute sensitivity
to metabisulfites sprayed on restaurant salads and in wine. TOXIC SUBSTANCES IN FOOD
Also referred to as false food allergies, pharmacologic food
reactions are characterized by exaggerated responses to phar- Metals, Hydrocarbons, N-Nitroso
macologic agents in food and possibly due to receptor sensiti- Substances and Mycotoxins
zation. In contrast, metabolic food reactions differ from other Certain substances are unavoidable in food because of their
categories of adverse reactions in that the foods are more or widespread use; presence in the earth’s crust, which has resulted
less commonly eaten and demonstrate toxic effects only when in their becoming a persistent and/or ubiquitous contaminant
 CHAPTER 31 Food Toxicology 459

TABLE 31-10 Metabolic food reactions.

Food Reaction Mechanism

Lima beans, cassava roots, millet (sorghum) Cyanosis Cyanogenic glycosides releasing hydrogen cyanide on
sprouts, bitter almonds, apricot, and peach pits contact with stomach acid

Cabbage family, turnips, soybeans, radishes, Goiter (enlarged thyroid) Isothiocyanates, goitrin, or S-5-vinyl-thiooxazolidone
rapeseed, and mustard interferes with utilization of iodine

Unripe fruit of the tropical tree Blighia sapida, Severe vomiting, coma, and acute Hypoglycin A, isolated from the fruit, may interfere
common in Caribbean and Nigeria hypoglycemia sometimes resulting with oxidation offatty acids, so that glycogen stores
in death, especially among the have to be metabolized for energy, with depletion of
malnourished carbohydrates, resulting in hypoglycemia

Leguminosae, Cruciferae Lathyritic symptoms: neurologic .-2,4-Diaminobutyric acid inhibition of ornithine

symptoms of weakness, leg transcarbamylase ofthe urea cycle, inducing
paralysis, and sometimes death ammonia toxicity

Licorice (glycyrrhizic acid) Hypertension, cardiac enlargement, Glycyrrhizic acid mimicking mineralocorticoids
sodium retention

Polar bear and chicken liver Irritability, vomiting, increased Vitamin A toxicity
intracranial pressure, death

Cycads (cycad flour) Amyotrophic lateral sclerosis Cycasin (methylazoxymethanol); primary action is
(humans), hepatocarcinogenicity methylation, resulting in a broad range of effects from
(rats and nonhuman primates) membrane destruction to inactivation of enzyme
systems

in the environment; or presence as a product of normal food to contribute to human disease. Details of mycotoxins effects
processing. are listed in Table 31-11.
Among the natural elements, approximately 22 are known to
be essential nutrients of the mammalian body. These elements
are referred to as micronutrients and include iron, zinc, copper, Toxins in Fish, Shellfish, and Turtles
manganese, molybdenum, selenium, iodine, cobalt, and even Seafood toxins under FDA policy have a zero tolerance, with
aluminum and arsenic. However, lead, cadmium, and mercury any detectable level considered cause for regulatory action.
are familiar as contaminants (or at least have more specifica- Ciguatera, scaritoxin, and maitotoxin are neurotoxins (anticho-
tions setting their limits in food ingredients). The prevalence linesterase) found in 11 orders, 57 families, and over 400 spe-
of these elements as contaminants is not due so much to their cies of fish as well as in oysters and clams. Ciguatera is originally
ubiquity in nature but rather to their use by humans (see made by dinoflagellates and biotransformed into the active
Chapter 23 for the toxicology of these metals). form by fish; after consumption, humans experience gastroin-
Polychlorinated hydrocarbons have extensive use as pes- testinal disorders, neurologic symptoms, or death. Palytoxin is
ticides, solvents, and heat-transfer agents. As a result of their produced by the zoanthid soft coral of the genus Palythoa, and
facile nature, their resulting wide-range uses, and resistance to fish, crabs, and polychaete worms, living in close association
degradation (and ease of detection), chlorinated hydrocarbons with or eating this mass, may become contaminated with paly-
have been found in a wide variety of foods. Polybrominated toxin. The toxin has been reported in mackerel, parrotfish, and
biphenyls and polybrominated biphenyl ethers are used in several species of crabs. Victims report a bitter, metallic taste
electrical equipment, paint, and plastics. from the meat (most often muscle, liver, ovary, and digestive
Nitrogenous compounds such as amines, amides, guani- tract), followed immediately by nausea, vomiting, and diar-
dines, and ureas can react with oxides of nitrogen (NO,) to rhea. Within several hours, symptoms include myoglobinuria,
form N-nitroso compounds (NOCs). These compounds origi- a burning sensation around the mouth and extremities, mus-
nate from two sources: environmental formation and endog- cle spasms, dyspnea, and dysphonia. Death may result from
enous formation. Environmental sources have declined over myocardial injury. Brevetoxins, produced by dinoflagellates
the last several years but still include foods (e.g., nitrate-cured (Gymnodinium breve) and concentrated in filter-feeding organ-
meats) and beverages (e.g., malt beverages), cosmetics, occu- isms, bind to voltage-dependent sodium channels. Symptoms
pational exposure, and rubber products. after human consumption include nausea, tingling, and
Food-borne mycotoxins (toxins elaborated by fungi), such numbness of the oral area, loss of motor control, and severe
as the carcinogenic and hepatotoxic aflatoxins, and the hyper- muscular ache, all of which resolve in a few days. Saxitoxin is
estrogenic mycotoxin zearalenone have considerable potential found in shellfish feeding on dinoflagellates; blockade of ion
460 UNIT 7 Applications of Toxicology

TABLE 31-11 Selected mycotoxins produced by various molds: some oftheir effects and the commodities that
are potentially contaminated.

Mycotoxin Source Effect Commodities Contaminated

Aflatoxins B,, B,, G,, G, Aspergillus flavus, A. parasiticus Acute aflatoxicosis, carcinogenesis Corn, peanuts, and others

Aflatoxin M, Metabolite of AFB, Hepatotoxicity Milk

Fumonisins B,,B>,B3,B,,A,,A, Fusarium verticillioides Renal and liver carcinogenesis Corn

Trichothecenes (e.g., Fusarium and Myrothecium Hematopoietic toxicity, meningeal Cereal grains, corn
T-2, deoxynivalenol, hemorrhage of brain, “nervous”
diacetoxyscirpenol) disorder, necrosis of skin, hemorrhage
in mucosal epithelia of stomach and
intestine, emesis, feed refusal, immune
suppression

Zearalenones Fusarium Estrogenic effect Corn, grain

Cyclopiazonic acid Aspergillus, Penicillium Muscle, liver, and splenic toxicity Cheese, grains, peanuts

Kojic acid Aspergillus Hepatotoxic? Grain, animal feed

3-Nitropropionic acid Arthrinium sacchari, A. saccharicola, Central nervous system impairment Sugarcane
‘
A. phaeospermum

Citreoviridin Penicillium citreoviride, P. toxicarium Cardiac beriberi Rice ,

Cytochalasins E, B, F,H Aspergillus and Penicillium Cytotoxicity Corn, cereal grain

Sterigmatocystin Aspergillus versiolar Carcinogenesis Corn

Penicillinic acid Penicillium cyclopium Nephrotoxicity, abortifacient Corn, dried beans, grains

Rubratoxins A, B Penicillium rubrum Hepatotoxicity, teratogenic Corn

Patulin Penicillium patulatum Carcinogenesis, liver damage Apple and apple products

Ochratoxin Aspergillus ochraceus, A. carbonarius, Endemic nephropathy, carcinogenesis Grains, peanuts, grapes, green
Penicillium verrucosum coffee

Citrinin Aspergillus and Penicillium Nephrotoxicity Cereal grains

Penitrem(s) Aspergillus, Claviceps, and Penicillium Tremors, incoordination, bloody diarrhea, Moldy cream cheese, English
death walnuts, hamburger bun, beer

Ergot alkaloids Claviceps purpurea Ergotism Grains

channels in neural transmission at neuromuscular junctions diarrhea known as gempylid fish poisoning, gempylotoxism,
leads to paresthesia and muscular weakness. Domoic acid is or keriorrhea. The toxin consists of wax esters (C3, C34, Cy¢,
also found in shellfish; an analog of the neurotransmitter glu- and C;, fatty acid esters), the primary component of which
tamine, it leads to damage to the hippocampus and other brain is C,,H,,O,. Another innate toxin is tetramine. It is found in
areas, causing various neurologic symptoms. Tetrodotoxin the salivary glands of Buccinum, Busycon, or Neptunia spp., a
is consumed by humans by eating improperly prepared puff- type of whelk or sea snail that is distributed in temperate and
erfish. It causes paralysis of the central nervous system and tropic waters and has long been a food source for humans. This
peripheral nerves by blocking the movement of all monova- heat-stable neurotoxin, tetramine, which upon ingestion by
lent cations, leading to muscular paralysis, respiratory distress, humans causes, among other symptoms, eyeball pain, head-
and sometimes death. Chelonitoxin is found in sea turtles and ache, dizziness, abdominal pain, ataxia, tingling in the fin-
causes necrosis of the myocardium and pulmonary edema. gers, nausea, and diarrhea. Finally, the meat of the Greenland
There are naturally occurring toxins that are innate to a shark (Somniosus microcephalus) and the pacific sleeper shark
particular marine species, but do not involve marine algae or (Somniosus pacificus) contains trimethylamine oxide, which
other environmental influences. Escolar (Lepidocybium fla- breaks down to trimethylamine in the gut, probably by enteric
vobrunneum) and Oilfish or Cocco (Ruvettus pretiosus) con- bacteria. The neurotoxic trimethylamine produces ataxia in
tain a strong purgative oil, which when consumed can cause both humans and dogs.
 CHAPTER 31 Food Toxicology 461

Microbiologic Agents that is improperly handled. BSE manifests clinically as neuro-

logic deterioration leading to death.
Most U.S. food-related illness results from microbial contami-
nation. Botulism is due to toxin produced by C. botulinum and
C. butyricum in improperly canned foods. The toxin interferes CONCLUSION
with acetylcholine at peripheral nerve endings, leading to respi-
ratory distress and respiratory paralysis. C. perfringens food Food consists of myriad chemical substances in addition to the
poisoning occurs when meat has been contaminated with intes- macro- and micronutrients that are essential to life. There are
tinal contents at slaughter, and then roasted and inadequately two principal means by which a food can be toxic and still be
stored, allowing C. perfringens to grow and elaborate its toxin. considered a food: (1) an ordinarily nontoxic food has become
The toxin causes death of enterocytes and severe fluid loss as toxic (through some act of man or nature), if even for a small
diarrhea. Bacillus cereus makes two toxins; one causes vomit- subpopulation (e.g., allergy, intolerance); or (2) overconsump-
ing and is elaborated in improperly prepared rice, whereas the tion of a food not ordinarily considered toxic at historic levels
other causes vomiting and can be present in various foods. of use. The vast majority of food-borne illnesses are attribut-
Staphylococcus aureus, which is normal flora of human skin and able to microbiologic contamination of food. Thus, the over-
nasal discharge, produces a wide variety of endo- and exotox- whelming concern for food safety must be directed toward
ins. Foods are usually contaminated after cooking by persons preserving the microbiologic integrity of food.
handling them and then keeping the foods at room temperature
for several hours. Cattle are natural reservoirs of Escherichia
coli; outbreaks of E. coli are associated with improperly pre- BIBLIOGRAPHY
Barceloux DG: Medical Toxicology of Natural Substances: Foods,
pared beef as well as unpasteurized juices and raw vegetables
Fungi, Medicinal Herbs, Plants, and Venomous Animals. Hoboken,
from plants fertilized with manure.
NJ: John Wiley & Sons, 2008.
Kotsonis F, Mackey M (eds.): Nutritional Toxicology. New York:
Taylor & Francis, 2002.
Bovine Spongiform Encephalopathy Omaye ST: Food and Nutritional Toxicology. Boca Raton, FL: CRC
Bovine spongiform encephalopathy (BSE, or mad cow disease) Press, 2004.
is transmitted by an infectious protein called a prion. Present Pussa T: Principles of Food Toxicology, 2nd ed. Boca Raton, FL:
in diseased cows, prions are transmitted to humans in meat CRC Press, 2013.
462 UNIT 7 Applications of Toxicology

QUESTIONS

Which of the following statements regarding food com- Which ofthe following wheat proteins is famous for being
plexity is FALSE? allergenic?
a. Many flavor additives are nonnutrient substances. a. casein.
b. Foods are subjected to environmental forces that alter b. ovalbumin.
their chemical composition. c. livetin.
c. There are more nonnutrient chemicals in food than d. gluten.
nutrient chemicals. e. glycinin.
d. A majority of nonnutrient chemicals are added to
food by humans. Which of the following foods contains a chemical
e. Food is more variable and complex than most other that causes hypertension by acting as a noradrenergic
substances to which humans are exposed. stimulant?
a. cheese.
Which of the following foods contains the most nonnutri- b. peanuts.
ent chemicals? c. shrimp.
a. beef. d. chocolate.
b. banana. e. beets.
c. tomato.
d. orange juice. What is the mechanism of saxitoxin, found in shellfish?
e. Cheddar cheese. a. interference with ion channels.
b. direct neurotoxicity. ies
Which of the following is considered an indirect food c. interference with DNA replication.
additive? d. binding to hemoglobin.
a. nitrites. e. interference with a stimulatory G protein.
b. _ plastic.
c. food coloring. Which of the following foods can cause a reaction that
d EDTA. mimics iodine deficiency?
e. citric acid. a. chocolate.
b. shellfish.
Estimated daily intake (EDI) is based on which of the c. peanuts.
following? d. fava beans.
a. metabolic rate. e. cabbage.
b. daily intake.
c. substance concentration in a food item. 10. Improperly canned foods can be contaminated with which
d. body mass index. of the following bacteria, causing respiratory paralysis?
e. concentration of substance in a food item and daily a. C. perfringens.
intake. b. R. ricketsii.
c. S. aureus.
Which of the following is NOT characteristic of IgE- d. C. botulinum.
mediated food allergies? e. E.coli.
a. urticaria.
b. wheezing.
c. hypertension.
d. nausea.
e. shock.
 Céctive
Aye’ Te Ey R

Analytical and Forensic

Toxicology
Bruce A. Goldberger and Diana G. Wilkins*

ANALYTICAL TOXICOLOGY FORENSIC URINE DRUG TESTING

ROLE IN GENERAL TOXICOLOGY HUMAN PERFORMANCE TESTING

ROLE IN FORENSIC TOXICOLOGY COURTROOM TESTIMONY

TOXICOLOGIC INVESTIGATION OF A POISON DEATH ROLE IN CLINICAL TOXICOLOGY

Case History and Specimens
ROLE IN THERAPEUTIC MONITORING
Toxicologic Analysis
Interpretation of Analytical Results SUMMARY

CRIMINAL POISONING OF THE LIVING

KEY POINTS

« Analytic toxicology involves the application of the tools = ‘The toxicologic investigation of a poison death involves
of analytic chemistry to the qualitative and/or quanti- (1) obtaining the case history in as much detail as possible
tative estimation of chemicals that may exert adverse and gathering suitable specimens, (2) conducting suitable
effects on living organisms. toxicologic analyses based on the available specimens,
« Forensic toxicology involves the use of toxicology for the and (3) the interpretation of the analytic findings.
purposes of the law; by far the most common application a The toxicologist as an expert witness may provide two
is to identify any chemical that may serve as a causative objectives: testimony and opinion. Objective testimony
agent in inflicting death or injury on humans or in caus- usually involves a description of analytic methods and find-
ing damage to property. ings. When a toxicologist testifies as to the interpretation of
analytic results, that toxicologist is offering an “opinion”

With its roots in forensic applications, analytical toxicology humans, or in causing damage to property. There is no substi-
involves the application of the tools of analytical chemistry tute for the unequivocal identification of a specific chemical
to the qualitative and/or quantitative estimation of chemicals substance that is demonstrated to be present in tissues from the
that may exert effects on living organisms. Forensic toxicol- victim at a sufficient concentration to explain the injury with a
ogy involves the use of toxicology for the purposes of the law. reasonable degree of scientific probability or certainty. For this
The most common application is to identify any chemical that reason, forensic toxicology and analytical toxicology have long
may serve as a causative agent in inflicting death or injury on shared a mutually supportive partnership.

*Drs Goldberger and Wilkins acknowledge the contribution of Alphonse Poklis, PhD, who authored this chapter in previous editions of Casarett & Doull’s Toxicology.

463
464 UNIT 7 Applications of Toxicology

ANALYTICAL TOXICOLOGY tissue constituents. Chemical techniques can be applied to

detect these ions when they are in great excess over nor-
Forensic toxicologists learned long ago that when the nature mal concentrations.
of a suspected poison is unknown, a systematic, standardized . Metals—Metals are encountered frequently as occupa-
approach must be used to identify the presence of most com- tional and environmental hazards. Separation involves
mon toxic substances. An approach that was first suggested destruction of the organic matrix by chemical or thermal
by Chapuis in 1873 in Elements de Toxicologie is based on the oxidation.
origin or nature of the toxic agent. Such a system can be char- . Anions and nonmetals—These present an analytical chal-
acterized as follows: lenge as they are rarely encountered in an uncombined form.
Ile Gases—Gases are most simply measured by means of gas . Nonvolatile organic substances—These constitute the
chromatography. largest group of substances that must be considered by
Z, Volatile substances— These are generally liquids of various analytical toxicologists. This group includes drugs, pes-
chemical types that vaporize at ambient temperatures. Gas ticides, natural products, pollutants, and industrial com-
chromatography is the simplest approach for separation pounds. These substances are solids or liquids with high
and quantitation. boiling points. A scheme for analysis of these chemicals
. Corrosive agents—These include mineral acids and is illustrated in Figure 32-1. Separation procedures rely
bases. Many corrosives consist of ions that are normal on differential extractions of biologic tissues and fluids

Tissue sample -
(Minced)

Acidify & steam-distill

_Make basic&extract se-
Ulee Or eS TOO ERR NG eS

_with organic solvent _

Test for volatiles Test for metals

Color test Spectroscopy
GC

FIGURE 32-1 Ascheme of separation for poisons from tissues.

 CHAPTER 32 Analytical and Forensic Toxicology 465

and this process is often tedious and inefficient, with poor Case History and Specimens
recovery of the analyte. Immunoassay may permit avoid-
Today, thousands of compounds are readily available that are
ance of extractions and facilitate quantification.
lethal if ingested, injected, or inhaled. Usually, a limited amount
7. Miscellaneous—This category covers the large number
of specimen is available on which to perform analyses; therefore
of compounds that cannot be detected by routine appli-
it is imperative that before the analyses are initiated, as much
cation. Venoms and other toxic mixtures of proteins or
information as possible concerning the facts of the case be col-
uncharacterized constituents fall into this class.
lected. The age, sex, weight, medical history, and occupation
of the decedent as well as any treatment administered before
ROLE IN GENERAL TOXICOLOGY death, the gross autopsy findings, the drugs available to the
decedent, and the interval between the onset of symptoms and
It is universally acknowledged that the chemical under study death should be noted. In a typical year, a postmortem toxicol-
must be either pure or the nature of any contaminant well-char- ogy laboratory will perform analyses for such diverse poisons as
acterized to enable interpretation of the experimental results over-the-counter medications (e.g., analgesics, antihistamines),
with validity. Chemicals may degrade when in contact with prescription drugs (e.g., benzodiazepines, opioids), drugs of
air, by exposure to ultraviolet or other radiation, by interaction abuse (e.g., cocaine, marijuana, methamphetamine), and gases
with constituents of the vehicle or dosing solution, and by other (e.g., inhalants, carbon monoxide).
means. Developing an analytical procedure by which these Specimens of many different body fluids and organs are
changes can be recognized and corrected is essential in achiev- necessary, as drugs and poisons display varying affinities for
ing consistent and reliable results over the course of a study. body tissues. It is paramount that the handling of all speci-
Finally, analytical methods are necessary to determine the mens be authenticated and documented. Fluids and tissues
bioavailability of a compound that is under study. Some sub- should be collected before embalming, as this process will
stances with low water solubility are difficult to introduce dilute or chemically alter the poisons present, rendering their
into an animal, and a variety of vehicles may be investigated. detection difficult or impossible. Although forensic toxicology
However, a comparison of the blood concentrations for the laboratories typically receive blood, urine, liver tissue, and/or
compound under study provides a simple means of comparing stomach contents for identification of xenobiotics, they have
the effectiveness of vehicles. been increasingly called upon to meet the analytical challenges
of many alternative types of samples. Nontraditional matrices,
such as bone marrow, hair, vitreous humor, and nails, among
ROLE IN FORENSIC TOXICOLOGY others, may be submitted to the laboratory. For example, on
occasion, toxicologic analysis is requested for cases of burned,
The duties of a forensic toxicologist in postmortem investiga-
exhumed, putrefied, or skeletal remains. Finally, in severely
tions include the qualitative and quantitative analysis of drugs
decomposed bodies, the absence of blood and/or the scarcity
or poisons in biologic specimens collected at autopsy and the
of solid tissues suitable for analysis have led to the collection
interpretation of the analytical findings with respect to the
and testing of maggots (fly larvae) feeding on the body.
physiologic and behavioral effects of the detected chemicals on
the deceased at the time of injury and/or death. The cause of
death in cases of poisoning cannot be proved beyond conten-
Toxicologic Analysis
tion without toxicologic analysis that confirms the presence of
the toxicant in either body fluids or tissues of the deceased. Before the analysis begins, several factors must be considered,
Additionally, the results of postmortem toxicologic testing pro- including the amount of specimen available, the nature of the
vide valuable epidemiologic and statistical data. Forensic toxicolo- poison sought, and the possible biotransformation of the poi-
gists are often among the first to alert the medical community son. In cases involving oral administration of the poison, the
to new epidemics of substance abuse and the dangers of abus- gastrointestinal (GI) contents are analyzed first because large
ing over-the-counter drugs. Similarly, they often determine the amounts of residual unabsorbed poison may be present. The
chemical identity and toxicity of novel analogs of psychoactive urine may be analyzed next, as the kidney is the major organ
agents that are subject to abuse, including “designer drugs” such of excretion for most poisons and high concentrations of toxi-
as “china white” (methylfentanyl), “ecstasy” (methylenedioxy- cants and/or their metabolites often are present in urine. After
methamphetamine), and GHB (gamma-hydroxybutyric acid). absorption from the GI tract, drugs or poisons are carried
to the liver before entering the general systemic circulation;
therefore, the first analysis of an internal organ is conducted
TOXICOLOGIC INVESTIGATION on the liver.
A thorough knowledge of drug biotransformation is often
OF A POISON DEATH
essential before an analysis is performed. The parent com-
The toxicologic investigation of a poison death may be divided pound and any major pharmacologically active metabolites
into three steps: (1) obtaining the case history and suitable should be isolated and identified. Many screening tests, such
specimens, (2) the toxicologic analyses, and (3) the interpreta- as immunoassays, are specifically designed to detect not the
tion of the analytical findings. parent drug but its major urinary metabolite.
466 UNIT 7 Applications of Toxicology

The analysis may be complicated by the normal chemical A new extension of forensic toxicology is the analysis of
changes that occur during the decomposition of acadaver. The impurities of illicit drug synthesis in biologic specimens.
autopsy and toxicologic analysis should be started as soon after Many drugs of abuse, particularly methamphetamine, are
death as possible. However, many poisons—such as arsenic, illicitly manufactured in clandestine laboratories. There
barbiturates, mercury, and strychnine—are extremely stable are several popular methods of methamphetamine synthe-
and may be detectable many years after death. sis; when these are applied in clandestine laboratories, side
Forensic toxicology laboratories analyze specimens by reactions or incomplete conversion of the reactants yield an
using a variety of analytical procedures. Initially, nonspecific impure mixture of methamphetamine and synthetic impuri-
tests designed to determine the presence or absence of a class ties. These impurities can be characteristic ofaparticular syn-
or group of analytes may be performed directly on the speci- thetic method and suggest the synthetic method that was used
mens. Examples of tests used to rapidly screen urine are the to produce the drug; point to a possible common source of
FPN (ferric chloride, perchloric, and nitric acid) color test illicit production; and provide a link between manufacturers,
for phenothiazine drugs and immunoassays for the detection dealers, and users.
of amphetamines, benzodiazepines, and opiate derivatives,
among others. Today, gas chromatography-mass spectrom-
etry (GC-MS) and liquid chromatography-mass spectrometry CRIMINAL POISONING OF THE LIVING
(LC-MS) are the most widely applied methodology in toxicol-
Over the past few decades, forensic toxicologists have become
ogy and are generally accepted as unequivocal identification
more involved in the analysis of specimens obtained from
for all drugs.
living victims of criminal poisonings. Generally, this increase
in testing is a result of two types of cases: (1) administration of
Interpretation of Analytical Results drugs to incapacitate victims of kidnapping, robbery, or sexual
assault and (2) poisoning as a form of child abuse.
Once the analysis of the specimens is complete, the toxicolo-
While alcohol is still often a primary factor in cases of
gist must interpret his or her findings with regard to the physi-
alleged sexual assault, common drugs of abuse or other psy-
ologic or behavioral effects of the toxicants on the decedent at
choactive drugs are often involved (Table 32-1). Of particu-
the concentrations found. Specific questions may be answered,
lar concern are the many potent inductive agents medically
such as the route of administration, the dose administered, and
administered prior to general anesthesia. Many of these
whether the concentration of the toxicant present was sufh-
drugs, such as benzodiazepines and phenothiazines, are avail-
cient to cause death or alter the decedent's actions enough to
able today through illicit sources or legal purchase in foreign
cause his or her death. Assessing the physiologic or behavioral
meanings of analytical results is often the most challenging
aspect confronted by the forensic toxicologist.
In determining the route of administration, the toxicologist TABLE 32-1 Distribution of drugs of abuse
notes the results of the analysis of the various specimens. As a encountered in urine specimens in 1179 cases of alleged
general rule, the highest concentrations of a poison are found sexual assault.*
at the site of administration. Therefore, the presence of large
amounts of drugs and/or poisons in the GI tract and liver indi- Rank Drug/Drug Group | _ Incidence
cates oral ingestion, while higher concentrations in the lungs 1 No drugs found 468
than in other visceral organs can indicate inhalation or intra-
venous injection. 2 Ethanol 451

The physiologic effects of most drugs and poisons are gen- 3 Cannabinoids 218
erally correlated with their concentrations in blood or blood
fractions such as plasma and serum. The survival time between 4 Benzoylecgonine (cocaine metabolite) 97

the administration of a poison and death may be sufficiently 5 Benzodiazepines 97

long to permit biotransformation and excretion of the agent.
Blood values may appear to be nontoxic or consistent with 6 Amphetamines 51

therapeutic administration. Death from hepatic failure after 7 Gamma-hydroxybutyrate (GHB) 48

an acetaminophen overdose usually occurs at least three to
four days after ingestion. Postmortem acetaminophen con- 8 Opiates 25

centrations in blood may be consistent with the ingestion of 9 Propoxyphene 17.

therapeutic doses. Therefore, fatal acetaminophen overdose is
10 Barbiturates 12
determined by case history, central lobular necrosis of the liver,
and, if available, analysis of serum specimens collected from *Thirty-five percent of the drug-positive specimens were positive for more than one
the decedent when he or she was admitted to the emergency drug.
Data from ElSohly MA, Salamone SJ: Prevalence of drugs used in cases of alleged
department. sexual assault. JAnal Toxicol, 1999;23(3):141-146.
 CHAPTER 32 Analytical and Forensic Toxicology 467

countries. When administered surreptitiously, they cause or confirmatory drug test. Thus, FUDT laboratories now
sedation and incapacitate the victim while also producing routinely test not only for drugs of abuse, but also for a wide
amnesia in the victim as to the events while drugged, with- variety of chemical adulterants. In most instances, a positive
out causing severe central nervous system depression. These test result for adulteration has as serious a consequence as a
cases often present a difficult analytical challenge to the toxi- positive drug test.
cologist. Usually, the victim does not bring forth an allegation
of assault until 24 h to several days after the attack. Thus, the
intoxicating drug may have been largely eliminated or exten- HUMAN PERFORMANCE TESTING
sively metabolized such that extremely low concentrations of
drug or metabolites are present in the victim’s blood, urine, Forensic toxicology activities also include the determination
and/or hair specimens. of the presence of ethanol and other drugs and chemicals in
Poisoning as a form of child abuse involves the deliberate blood, breath, or other specimens and the evaluation of their
administration of toxic or injurious substances toa child, usually role in modifying human performance and behavior. The most
by a parent or other caregiver. Common agents used to inten- common application of human performance testing is to deter-
tionally poison children have included syrup of ipecac, table mine impairment while driving under the influence of ethanol
salt, laxatives, diuretics, antidepressants, sedative-hypnotics, or drugs. Several studies have demonstrated a relatively high
and narcotics. As in the case of sexual assault, sophisticated occurrence of drugs in impaired or fatally injured drivers.
MS testing methods may be required to detect such agents as These studies tend to report that the highest drug-use accident
emetine and cephaeline, the emetic alkaloids in syrup of ipecac. rates are associated with the use of such illicit or controlled
drugs as cocaine, benzodiazepines, marijuana, and phencycli-
dine. Before driving under the influence of drugs is as readily
accepted by the courts as ethanol testing, legal and scientific
FORENSIC URINE DRUG TESTING problems regarding drug concentrations and driving impair-
Concerns regarding the potentially adverse consequences of ment must be resolved.
substance abuse for the individual, the workplace, and soci-
ety have led to widespread urine analysis for controlled or
illicit drugs. Currently, such testing is conducted routinely COURTROOM TESTIMONY
by the military services, regulated transportation and nuclear The forensic toxicologist often is called upon to testify in
industries, many federal and state agencies, public utilities, legal proceedings as an “expert witness.” An expert witness
federal and state criminal justice systems, and numerous pri- may provide two types of testimony: objective testimony
vate businesses and industries. Significant ethical and legal and “opinion.” Objective testimony by a toxicologist usually
ramifications are associated with such testing. Those having involves a description of his or her analytical methods and
positive test results may not receive employment, be dis- findings. When a toxicologist testifies as to the interpretation
missed from a job, be court-martialed, or suffer a damaged of his or her analytical results or those of others, that toxicolo-
reputation. gist is offering an “opinion.” Whether a toxicologist appears in
Forensic urine drug testing (FUDT) differs from other areas criminal or civil court, workers’ compensation, or parole hear-
of forensic toxicology in which urine is the only specimen ana- ings, the procedure for testifying is the same: direct examina-
lyzed and testing is performed for a limited number of drugs tion, cross-examination, and redirect examination. Regardless
and metabolites. Under the federal certification program, anal- of which side has called for the expert witness, the toxicologist
yses are performed for a limited number of classes or drugs of should testify with scientific objectivity. An expert witness is
abuse. Initial testing is performed by immunoassays on rapid, called to provide informed assistance to the jury, not to judge
high-throughput chemistry analyzers. A confirmation analy- the case.
sis in FUDT-certified laboratories is performed by GC-MS
and LC-MS/MS. FUDT results are reported only as positive or
negative for the drugs sought. ROLE IN CLINICAL TOXICOLOGY
Many individuals who are subject to regulated urine test-
ing have devised techniques to mask their drug use either Analytical toxicology in a clinical setting plays a role very
by physiologic means such as the ingestion of diuretics or by similar to its role in forensic toxicology. As an aid in the diag-
attempting to adulterate the specimen directly with bleach, nosis and treatment of toxic incidents, as well as in monitor-
vinegar, or other products that interfere with the initial ing the effectiveness of treatment regimens, it is useful to
immunoassay tests. Thus, specimens are routinely tested for clearly identify the nature of the toxic exposure and measure
adulteration by checking urinary pH, creatinine, and specific the amount of the toxic substance that has been absorbed.
gravity and noting any unusual color or smell. Recently a Frequently, this information, together with the clinical state
mini-industry has developed to sell various products that are of the patient, permits a clinician to relate the signs and
alleged to “beat the drug test” by interfering with the initial symptoms observed to the anticipated effects of the toxic
468 UNIT 7 Applications of Toxicology

agent. This may permit a clinical judgment as to whether the TABLE 32-2 Drugs commonly indicated for
treatment must be vigorous and aggressive or whether simple therapeutic monitoring.
observation and symptomatic treatment of the patient are
Antiarrythmics
sufficient.
Digoxin
A cardinal rule in the treatment of poisoning cases is to Digitoxin
support vital cardiopulmonary function and to remove any Lidocaine
unabsorbed material, limit the absorption of additional poi- Procainamide and N-acetylprocainamide
son, and hasten its elimination. Although the instrumentation Quinidine

and the methodology used in a clinical toxicology laboratory Antibiotics

are similar to those utilized by a forensic toxicologist, a major Amikacin
difference between these two applications is responsiveness. In Chloramphenicol
Gentamicin
emergency toxicology testing, results must be communicated
Tobramycin
to the clinician within hours to be meaningful for therapy. Vancomycin
Primary examples of the usefulness of emergency toxicology
Anticancer
testing are the rapid quantitative determination of acetamino-
Methotrexate
phen, salicylate, alcohols, and glycol serum concentrations in
instances of suspected overdose. Anticonvulsants
Ethanol is the most common chemical encountered in Carbamazepine
Gabapentin
emergency toxicology. Although relatively few fatal intoxica- Lamotrigine
tions occur with ethanol alone, serum values are important Phenobarbital 4
in the assessment of behavioral, physiologic, and neurologic Phenytoin
function, particularly in trauma cases where the patient is Primidone
Topiramate
unable to communicate and surgery with the administration Valproic acid
of anesthetic or analgesic drugs is indicated. Intoxications Zonisamide
from accidental or deliberate ingestion of other alcohols or
Antidepressants
glycols—such as methanol from windshield deicer or paint
Amitriptyline/nortriptyline
thinner, isopropanol from rubbing alcohol, and ethylene Desipramine/imipramine
glycol from antifreeze—are often encountered in emer- Doxepin/nordoxepin
gency departments. Following ingestion of methanol or
Antipsychotics
ethylene glycol, patients often present with similar neuro- Clozapine
logic symptoms and severe metabolic acidosis due to the Pimozide
formation of toxic aldehyde and acid metabolites. A rapid
Bronchodilators
quantitative serum determination for these intoxicants will
Caffeine
indicate the severity of intoxication and the possible need for Theophylline
dialysis or therapy with an alcohol dehydrogenase inhibitor
(fomepizole). Immunosuppressants
Azathioprine
Cyclosporine
Mycophenolic acid
ROLE IN THERAPEUTIC MONITORING Sirolimus
Tacrolimus
Historically, the administration of drugs for long-term therapy
was based largely on experience. A dosage amount was selected Mood stabilizing
Lithium
and administered at appropriate intervals based on what the
clinician had learned was generally tolerated by most patients.
If the drug seemed ineffective, the dose was increased; if
toxicity developed, the dose was decreased or the frequency of
dosing was altered. At times, a different dosage form might be
substituted. Establishing an effective dosage regimen was par- one or more of these variables need to be identified and cor-
ticularly difficult in children and the elderly. rected. Drugs that are commonly monitored during therapy
are presented in Table 32-2.
The factors responsible for individual variability in responses
to drug therapy include the rate and extent of drug absorption,
distribution, and binding in body tissues and fluids, rate of SUMMARY
metabolism and excretion, pathologic conditions, and interac-
tion with other drugs. Monitoring of the plasma or serum con- The analytical techniques employed by forensic toxicologists
centration at regular intervals will detect deviations from the have continued to expand in complexity and improve in reliabil-
average serum concentration, which, in turn, may suggest that ity and sensitivity. Many new analytical tools have been applied
 CHAPTER 32 Analytical and Forensic Toxicology 469

to toxicologic problems in almost all areas of the field, and the analytical toxicologists will continue to play a substantial role
technology continues to open new areas of research. Forensic in the expansion of the discipline of toxicology.
toxicologists continue to be concerned about conducting
unequivocal identification of toxic substances in such a man-
ner that the results can withstand a legal challenge. The issues BIBLIOGRAPHY
of substance abuse, designer drugs, increased potency of thera- Levine B: Principles of Forensic Toxicology, 4th ed. Washington, DC:
peutic agents, and widespread concern about pollution, and the AACC Press, 2013.
safety and health of workers present challenges to the analyst's Negruz A, Cooper G: Clarke’ Analytical Forensic Toxicology. London:
knowledge, skills, and abilities. As these challenges are met, Pharmaceutical Press, 2013.
470 UNIT7 Applications of Toxicology

QUESTIONS

1. Which of the following is most commonly used as a drug I Which of the following is LEAST important in determin-
of sexual assault? ing variability in response to drug therapy?
a. narcotics. a. drug interactions.
b. amphetamines. distribution in body tissue.
c. benzodiazepines. body mass index.
d. ethanol. pathologic conditions.
e. antidepressants. cmos rate of metabolism.

2. Allof the following statements regarding analytic/forensic 8. Which of the following statements is FALSE regarding
toxicology are true EXCEPT: steady state?
a. Analytic toxicology uses analytic chemistry to char- a. Steady-state concentrations are proportional to the
acterize a chemical’s adverse effect on an organism. dose/dosage interval.
b. Medical examiners and coroners are most important b. Steady state is attained after approximately four
in determining cause of death. half-lives.
c. Tissues and body fluids are vital in forensic toxicology. c. The steady-state concentrations are proportional
Forensic toxicology is used for purposes of the law. to F/CL.
e. Chapuis first characterized a system for classifying d. Monitoring of steady-state drug concentration
toxic agents. assumes that an effective concentration is present.
e. Fluctuations in concentration are increased by slow
3. Which of the following criteria is NOT routinely used to drug absorption. Lies
check for adulteration of drug urine analysis?
a. urea. 9. Which of the following is an indirect method of measur-
b. pH. ing a chemical or its metabolite?
c. color. blood test.
d. specific gravity. hair sample.
e. creatinine. urinalysis.
hemoglobin adduct detection.
4. Which blood alcohol concentration (BAC) is most se breath analysis.
pao
commonly used as the statutory definition of DUI?
a. 0.04. 10. Which of the following statements regarding analytic/
b. 0.06. forensic toxicology is TRUE?
c. 0.08. a. Antidepressants are commonly used to incapacitate
dy 002: victims.
@ OLIN. b. It is easy to test for and prove that marijuana is a factor
in an automobile accident.
5. Which of the following drugs is NOT properly matched c. Heroin is the drug most commonly encountered in
with its most common analytic method? emergency toxicology.
benzodiazepines—GC/MS. d. Toxicologists can play an important role in court-
ibuprofen—TLC/HPLC. room testimonies.
amphetamines—immunoassays. e. Ethanol intoxication often results in death.
barbiturates—GC/immunoassays.
ge ethanol—immunoassays.
pao
6. For which of the following drugs is serum NOT used
during toxicology testing?
a. ethanol.
b. cocaine.
c. aspirin.
d. barbiturates.
e. ibuprofen.
 CigEl eve PPh
E SOR

Clinical Toxicology
Louis R. Cantilena Jr.

HISTORY OF CLINICAL TOXICOLOGY Prevention of Further Poison Absorption

Enhancement of Poison Elimination
INTRODUCTION OF THE POISON CONTROL CENTER
Use of Antidotes in Poisoning
Supportive Care of the Poisoned Patient
CLINICAL STRATEGY FOR TREATMENT
OF THE POISONED PATIENT CASE EXAMPLES OF SPECIFIC POISONINGS
Clinical Stabilization Acetaminophen
Clinical History in the Poisoned Patient Ethylene Glycol
Physical Examination Valproic Acid
Laboratory Evaluation
CONCLUSION
Radiographic Examination

KEY POINTS

s Clinical toxicology encompasses the expertise in the the patient, clinical evaluation (history, physical, labo-
specialties of medical toxicology, applied toxicology, and ratory, and radiology), prevention of further toxin
clinical poison information. absorption, enhancement of toxin elimination, admin-
« Important components of the initial clinical encoun- istration of antidote, and supportive care with clinical
ter with a poisoned patient include stabilization of follow-up.

HISTORY OF CLINICAL TOXICOLOGY patients with expert recommendations for medical treatment,
critical diagnostic and treatment information for health care
The history of poisoning and poisoners goes back to ancient professionals, education for health care professionals, and poi-
times. Formulas for creating poisonous and noxious vapors son prevention activities through public education. Poison
have been found in Chinese writings dating back to 1000 BC. control centers serve as a potential early-warning system for a
Documentation regarding the use of antidotes can be found in potential chemical or biologic terrorist attack.
Homer’s Odyssey and Shastras from 600 BC. Additional his-
tory is found in Chapter 1. CLINICAL STRATEGY FORTREATMENT
OF THE POISONED PATIENT
INTRODUCTION OF THE POISON
The following general steps represent important components
CONTROL CENTER
of the initial clinical encounter with a poisoned patient:
In the United States, poison control centers are staffed by a 1. Stabilization of the patient
medical director (medical toxicologist), administrator, spe- 2. Clinical evaluation (history, physical, laboratory, and
cialists in poison information, and educators for poison pre- radiology)
vention programs. Personnel provide direct information to 3. Prevention offurther toxin absorption
471
472 UNIT 7 Applications of Toxicology

TABLE 33-1 Clinical features of toxic syndromes.

Neen eee era arnmrmanncpiiioposasensoSaytsSPPSSSSSRGUSHGRERIESSSTLSUSSGASTERSS SRGSASS ESTAS EEE LS eT en TEES ETSY

_ Bloodpressure Pulse Temperature _ Pupils Lungs Abdomen Neurologic

Sympathomimetic Increase Increase Slightincrease Mydriasis NC NC Hyperalert, increased

reflexes

Anticholinergic Slight increase Increase — Increase Mydriasis NC © Decreased Altered mental status
or NC bowel sounds

Cholinergic Slight decrease Decrease NC Miosis Increased bronchial Increasedbowel Altered mental status
or NC sounds sounds

Opioid Decrease Decrease Decrease Miosis NC or rales (late) Decreased Decreased level
bowel sounds of consciousness

4. Enhancement of toxin elimination frequently the most difficult aspect of the clinical history in the
nn. Administration of antidote (if available) setting of treatment of the poisoned patient.
6. Supportive care and clinical follow-up Taking an accurate history in the poisoned patient can be
challenging and in some cases unsuccessful. When the history
is unobtainable, the clinical toxicologist is left without a clear
Clinical Stabilization picture of the exposure history. In this setting, the treatment
The first priority in the treatment of the poisoned patient is sta- proceeds empirically as an “unknown ingestion” poisoning.
bilization. Initial assessment of airway, respiration, and circu- »
\

lation is crucial. Some toxins or drugs can cause seizures early

in the course of presentation. The steps and clinical proce- Physical Examination
dures incorporated to stabilize a critically ill, poisoned patient A thorough physical examination is required to assess the
are numerous and include, if appropriate, support of ventila- patient’s condition, determine the patient’s mental status, and,
tion, circulation, and oxygenation. In critically ill patients, if altered, determine possible additional causes such as trauma
sometimes treatment interventions must be initiated before a or central nervous system infection. Whenever possible, the
patient is truly stable. patient's physical examination parameters are categorized
into broad classes referred to as toxic syndromes (toxidromes),
constellations of clinical signs that, taken together, are likely
Clinical History in the Poisoned Patient associated with exposure from certain classes of toxicologic
The primary goal of taking a medical history in poisoned agents. Categorization of the patient’s presentation into toxic
patients is to determine, if possible, the substance ingested or syndromes allows for the initiation of rational treatment based
the substance to which the patient has been exposed as well on the most likely category of toxin responsible, even if the
as the extent and time of exposure. In the setting of a suicide exact nature of the toxin is unknown. Table 33-1 lists clinical
attempt, patients may not provide any history or may give features of the major toxic syndromes. Occasionally a charac-
incorrect information so as to increase the possibility that teristic odor detected on the poisoned patient’s breath or cloth-
they will successfully bring harm to themselves. Information ing may point toward exposure or poisoning by a specific agent
sources commonly employed in this setting include family (Table 33-2).
members, emergency medical technicians who were at the
scene, a pharmacist who can sometimes provide a listing of
prescriptions recently filled, or an employer who can disclose TABLE 33-2 Characteristic odors associated with
what chemicals are available in the work environment. poisonings.
In estimating the level of exposure to the poison, one
generally should maximize the possible dose received. That Odor _ PotentialPoison
is, one should assume that the entire prescription bottle Bitter almonds Cyanide
contents were ingested, that the entire bottle of liquid was
consumed, or that the highest possible concentration of Eggs Hydrogen sulfide, mercaptans

airborne contaminant was present in the case of a patient Garlic As, organophosphates, DMSO, thallium
poisoned by inhalation.
With an estimate of dose, the toxicologist can refer to various Mothballs Naphthalene, camphor
information sources to determine what the range of expected Vinyl Ethchlorvynol
clinical effects might be from the exposure. The estimation
Wintergreen Methylsalicylate
of expected toxicity greatly assists with the triage of poisoned
patients. Estimating the timing of the exposure to the poison is DMSO, dimethyl sulfoxide.
 CHAPTER 33 Clinical Toxicology 473

Laboratory Evaluation TABLE 33-4 Differential diagnosis of metabolic

Table 33-3 lists drugs or other chemicals that are typically acidosis with elevated anion gap:“AT MUD PILES”.
available for immediate measurement in a hospital facility. As A Alcohol (ethanol ketoacidosis)
one can see, the number of agents for which detection is possi-
ble in the rapid-turnaround clinical setting is extremely limited T Toluene
compared with the number of possible agents that can poison M Methanol
patients. This further emphasizes the importance of recogniz-
ing clinical syndromes for poisoning and for the clinical toxi- U Uremia
cologist to initiate general treatment and supportive care for D Diabetic ketoacidosis
the patient with poisoning from an unknown substance.
For the substances that can be measured ona rapid-turnaround P Paraldehyde
basis in an emergency department setting, the quantitative mea- lron, isoniazid
surement can often provide both prognostic and therapeutic
guidance. L Lactic acid
Predictive relationships of drug plasma concentration and E Ethylene glycol
clinical outcome and/or suggested concentrations that require
therapeutic interventions are available for several agents includ- S Salicylate
ing salicylates, lithium, digoxin, iron, phenobarbital, and the-
ophylline. Some authors have identified “action levels” or toxic
threshold values for the measured plasma concentrations of
HCO, ion concentrations. A normal anion gap is <12. When
various drugs or chemicals. Generally, these values represent
there is laboratory evidence of metabolic acidosis, the finding
mean concentrations of the respective substance that have been
of an elevated anion gap would suggest systemic toxicity from a
retrospectively shown to produce a significant harmful effect.
relatively limited number of agents (Table 33-4).
Because of the limited clinical availability of “diagnostic”
The second calculated parameter from clinical chemistry
laboratory tests for poisons, toxicologists utilize specific, rou-
values is the osmol gap. The osmol gap is calculated as the
tinely obtained clinical laboratory data—especially the anion
numerical difference between the measured serum osmolality
gap and the osmol gap—to determine what poisons may have
and the serum osmolarity calculated from the clinical chemis-
been ingested. An abnormal anion or osmol gap suggests a dif-
try measurements of the serum sodium ion, glucose, and blood
ferential diagnosis for significant exposure. Both calculations
urea nitrogen (BUN) concentrations. The normal osmol gap is
are used as diagnostic tools when the clinical history suggests
<10mOsm. An elevated osmol gap suggests the presence of an
poisoning and the patient’s condition is consistent with expo-
osmotically active substance (methanol, ethanol, ethylene gly-
sure to agents known to cause elevations of these parameters
col, and isopropanol) in the plasma that is not accounted for by
(i.e., metabolic acidosis, altered mental status, etc.).
the sodium ion, glucose, or BUN concentrations.
The anion gap is calculated as the difference between the
Although calculation of both the AG and the osmol gap can
serum Na ion concentration and the sum of the serum Cl and
provide very useful information from readily available clinical
chemistry measurements, these determinations must be inter-
preted cautiously in certain clinical settings. For example, even
TABLE 33-3 List of tests that are commonly
though a patient may have ingested a large, significantly toxic
measured in a hospital setting on a stat basis.
amount of methanol, if measured late in the clinical course of
Acetaminophen Osmolality the exposure, the osmol gap may not be significantly elevated
as most of the osmotically active methanol has left the plasma
Acetone Phenobarbital
and has been biotransformed or cleared but is still producing
Carbamazepine Phenytoin serious clinical effects.

Carboxyhemoglobin Procainamide/NAPA

Digoxin : Quinidine
Radiographic Examination
The use of clinical radiographs to visualize drug overdose
Ethanol Salicylates or poison ingestions is relatively limited due to lack of radi-
Gentamicin Theophylline opacity. Generally, plain radiographs can detect a significant
amount of ingested oral medication containing ferrous or
lron 2 Tobramycin potassium salts. In addition, certain formulations that have an
Lithium Valproic Acid enteric coating or certain types of sustained release products
are radiopaque as well.
Methemoglobin
The most useful radiographs ordered in a case of overdose
NAPA, N-acetylprocainamide. or poisoning include the chest and abdominal radiographs
474 UNIT 7 Applications of Toxicology

and the computed tomography (CT) study of the head. The that the charcoal has been specially processed to be more effi-
abdominal radiograph has been used to detect recent lead cient at adsorbing toxins.
paint ingestion in children, and ingestion of halogenated The usefulness of whole bowel irrigation for a poisoned
hydrocarbons, such as carbon tetrachloride or chloroform, patient is very limited. Considerable absorption of the toxicant
that may be visualized as a radiopaque liquid in the gut lumen. can occur before the procedure “washes” the lumen of the GI
Abdominal plain radiographs have been helpful in the set- tract clear of unabsorbed material. The best evidence for eff-
ting where foreign bodies are detected in the gastrointestinal cacy of this procedure in the setting of poisoning is for removal
tract, such as would be seen in a “body packer,’ or one who of ingested packets of illegal drugs swallowed by people smug-
smuggles illegal substances by swallowing latex or plastic gling the material and hoping to avoid detection by concealing
storage vesicles filled with cocaine or some other substance. the agents in their intestines.
Occasionally these storage devices rupture and the drug is
released into the gastrointestinal tract, with serious and some-
times fatal results.
Enhancement of Poison Elimination
Plain radiography and other types of diagnostic imaging in There are several methods available to enhance the elimination
clinical toxicology can also be extremely valuable for the diag- of specific poisons or drugs once they have been absorbed into
nosis of toxin-induced pathology. For example, the detection the systemic circulation. The primary methods employed for
of drug-induced noncardiac pulmonary edema is associated this use today include alkalinization of the urine, hemodialysis,
with serious intoxication with salicylates and opioid agonists. hemoperfusion, hemofiltration, plasma exchange or exchange
Another example of the use of radiologic imaging in clinical transfusion, and serial oral activated charcoal.
toxicology is with CT of the brain. Significant exposure to car- The use of urinary alkalinization results in enhancement
bon monoxide (CO) has been associated with CT lesions of the of the renal clearance of weak acids. The basic principle is to
brain consisting of low-density areas in the cerebral white mat- increase the pH of urinary filtrate.toa level sufficient to ion-
ter and in the basal ganglia, especially the globus pallidus. ize the weak acid and prevent renal tubule reabsorption of the
molecule (ion trapping). Although there are potentially similar
advantages to be gained from acidification of the urine in order
Prevention of Further Poison Absorption to enhance the clearance of weak bases, this method is not used
During the early phases of poison treatment or intervention because acute renal failure and acid-base and electrolyte dis-
for a toxic exposure via the oral, inhalational, or topical route, turbances are associated with acidification.
a significant opportunity exists to prevent further absorption The dialysis technique, either peritoneal dialysis or hemo-
of the poison by minimizing the total amount that reaches the dialysis, relies on passage of the toxic agent through a semi-
systemic circulation. For toxins presented by the inhalational permeable dialysis membrane so that it can subsequently be
route, the main intervention used to prevent further absorp- removed. Hemodialysis incorporates a blood pump to pass
tion involves removing the patient from the environment blood next to a dialysis membrane, which allows agents perme-
where the toxin is found and providing adequate ventilation able to the membrane to pass through and reach equilibrium.
and oxygenation for the patient. For topical exposures, cloth- Some drugs are bound to plasma proteins and so cannot pass
ing containing the toxin must be removed and the skin washed through the dialysis membrane; others are distributed mainly
with water and mild soap taking care not to cause cutaneous to the tissues and so are not concentrated in the blood, making
abrasions that may enhance dermal absorption. dialysis impractical. Hemodialysis has been shown to be clini-
The four primary methods to prevent continued absorp- cally effective in the treatment of poisoning by the drugs and
tion of an oral poison are induction of emesis with syrup toxins shown in Table 33-5.
of ipecac, gastric lavage, oral administration of activated The technique of hemoperfusion is similar to hemodialysis
charcoal, and whole bowel irrigation. Although potentially except there is no dialysis membrane or dialysate involved in
indicated for individuals who are hours away from a medi- the procedure. The patient's blood is pumped through a per-
cal facility, syrup of ipecac use for induction of emesis in the fusion cartridge, where it is in direct contact with adsorptive
treatment of a potentially toxic ingestion has declined. Risk of material (usually activated charcoal). Protein binding does
cardio- and neurotoxicity and lower effectiveness at remov-
ing the toxicant than desired limit its use. Likewise, gastric
TABLE 33-5 Differential diagnosis of elevated
lavage, which involves placing an orogastric tube into the
osmol gap.
stomach and aspirating fluid, and then cyclically instilling
fluid and aspirating until the effluent is clear, is limited by the Methanol
risk of aspiration during the lavage procedure and evidence of
limited effectiveness. Ethanol

For many years, orally administered activated charcoal has Ethylene glycol
been routinely incorporated into the initial treatment of a
Isopropanol
patient poisoned by the oral route. The term activated means
 CHAPTER 33 Clinical Toxicology 475

not significantly interfere with removal by hemoperfusion. TABLE 33-6 Chemicals for which hemodialysis
Because of the more direct contact of the patient’s blood with has been shown effective as a treatment modality
the adsorptive material, the medical risks of this procedure for poisoning.
include thrombocytopenia, hypocalcemia, and leukopenia.
The technique of hemofiltration is relatively new in clini- Alcohols Meprobamate
cal toxicology applications. As in the case of hemodialysis, the Antibiotics Metformin
patient's blood is delivered through hollow fiber tubes and an
ultrafiltrate of plasma is removed by hydrostatic pressure from Boric acid Paraldehyde
the blood side of the membrane. The perfusion pressure for the Bromide Phenobarbital
technique is generated either by the patient’s blood pressure
(for arteriovenous hemofiltration) or by a blood pump (for Calcium Potassium
venovenous hemofiltration). Needed fluid and electrolytes Chloral hydrate Salicylates
removed in the ultrafiltrate are replaced intravenously with
sterile solutions. Fluorides Strychnine
The use of either plasma exchange or exchange transfusions lodides Theophylline
has been relatively limited in the field of clinical toxicology.
Although the techniques afford the potential advantage of Isoniazid Thiocynates
being able to remove high-molecular-weight and/or plasma Lithium Valproic acid
protein-bound toxins, their clinical utility in poison treat-
ment has been limited. Plasma exchange, or pheresis, involves
removal of plasma and replacement with frozen donor plasma,
albumin, or both with intravenous fluid. The risks and compli- The mechanism of action of various antidotes is quite differ-
cations of this technique include allergic-type reactions, infec- ent. For example, a chelating agent or Fab fragments specific
tious complications, and hypotension. Exchange transfusion to digoxin will work by physically binding the toxin, prevent-
involves replacement of a patient's blood volume with donor ing the toxin from exerting a deleterious effect in vivo, and,
blood. The use of this technique in poison treatment is uncom- in some cases, facilitating body clearance of the toxin. Other
mon and mostly confined to inadvertent drug overdose in a antidotes pharmacologically antagonize the effects of the toxin.
neonate or premature infant. Atropine, an antimuscarinic, anticholinergic agent, is used to
Serial oral administration of activated charcoal, also referred pharmacologically antagonize at the receptor level the effects
to as multiple-dose activated charcoal (MDAC), has been of organophosphate insecticides that produce lethal cholin-
shown to increase the systemic clearance of various drug sub- ergic, muscarinic effects. Certain agents exert their antidote
stances. The mechanism for the observed augmentation of effects by chemically reacting with biologic systems to increase
nonrenal clearance caused by repeated doses of oral charcoal is detoxifying capacity for the toxin. For example, sodium nitrite
thought to be transluminal efflux of the drug from the blood to is given to patients poisoned with cyanide to cause formation
the charcoal passing through the gastrointestinal tract. The acti- of methemoglobin, which serves as an alternative binding site
vated charcoal in the gut lumen serves as a “sink” for the toxin. A for the cyanide ion, thereby making it less toxic to the body.
concentration gradient is maintained and the toxin passes con-
tinuously into the gut lumen, where it is adsorbed to charcoal.
In addition, MDAC is thought to produce its beneficial effect Supportive Care of the Poisoned Patient
by interrupting the enteroenteric—enterohepatic circulation of The supportive care phase of poison treatment is very impor-
drugs. The technique involves continuing oral administration tant. Not only are there certain poisonings that have delayed
of activated charcoal beyond the initial dosage every 2 to 4 h. toxicity, but there are also toxins that exhibit multiple phases
An alternative technique is to give a loading dose of activated of toxicity. Close clinical monitoring can detect these later-
charcoal via an orogastric tube or nasogastric tube, followed by phase poisoning complications and allow for prompt medical
a continuous infusion intragastrically. A list of agents for which intervention.
MDAC has been shown to be an effective means of enhanced Another important component of the supportive care phase
body clearance is given in Table 33-6. of poison treatment is the psychiatric assessment. Generally, a
patient who has attempted suicide should be constantly moni-
tored until he or she has been evaluated by the psychiatric
Use of Antidotes in Poisoning consultant and judged to be at low risk for being without con-
A relatively small number of specific antidotes are available for stant surveillance. In many cases, it is not possible to perform
clinical use in the treatment of poisoning. The U.S. Food and a psychiatric interview of the patient during the early phases of
Drug Administration (FDA) has placed incentives for spon- treatment and evaluation. Once the patient has been stabilized
sors to develop drugs for rare diseases or conditions through and is able to communicate, a psychiatric evaluation should be
the Orphan Drug Act. obtained.
476 UNIT 7 Applications of Toxicology

CASE EXAMPLES OF SPECIFIC Acetaminophen in normal individuals is inactivated by

sulfation and glucuronide conjugation, with about 4% bio-
POISONINGS transformed by CYP2E1 to a toxic metabolite that is normally
detoxified by conjugation with glutathione and excreted as the
Acetaminophen
mercapturate. Patients who are concurrently using, or have
A 16-year-old female patient arrives in the ED by ambu- recently used, agents that induce CYP2E1 may produce more
lance after being found by a parent in what appeared to be an than 4% of the toxic metabolite. When there is evidence (medi-
intoxicated state with empty pill bottles scattered about her cal history) of concurrent chemicals that induce CYP2E1, the
room. The parent reports the patient was despondent recently treatment nomogram from acetaminophen should be modi-
after breaking up with her boyfriend. The patient is tearful fied to a lower threshold for treatment with NAC.
and reports abdominal pain and admits to drinking alcohol Follow-up liver biopsy studies of patients who have recov-
and taking over-the-counter (OTC) pills in an apparent sui- ered three months to a year after hepatotoxicity have demon-
cide attempt. The estimated time of ingestion is 6 h prior to strated no long-term sequelae or chronic toxicity. A very small
arrival in the ED. The patient does not use prescription, OTC percentage (0.25%) of patients in the national multiclinic study
medications, or dietary supplements and is not known to have conducted in Denver may progress to hepatic encephalopathy
a history of regular consumption of alcoholic beverages or use with subsequent death. The clinical nature of the overdose is
illicit drugs. one of a sharp peak of serum glutamic-oxaloacetic transami-
On physical examination the blood pressure was 118/80 mm Hg, nase (SGOT) by day 3, with recovery to less than 100IU/L
pulse 88/min and regular, respiratory rate 18/min, and tem- by day seven or eight. Patients with SGOT levels as high as
perature 37.0°C. She was awake and oriented, responded to 20000IU/L have shown complete recovery and no sequelae
questions appropriately with slightly slurred speech. Other one week after ingestion.
pertinent findings included normal bowel sounds with mild Laboratory evaluation of a potentially poisoned patient is
epigastric tenderness. The neurologic examination was only crucial in terms of both hepatic measures of toxicity and plasma
significant for slightly slurred speech. levels of acetaminophen. Accurate estimation of acetamino-
The patient was given 1.5 g/kg oral activated charcoal as a
phen in the plasma should be done on samples drawn at least
slurry in a sorbitol cathartic. Forty minutes later, the labo- 4h after ingestion, when peak plasma levels can be expected.
ratory results showed a mildly increased white blood cell
Once an accurate plasma level has been obtained, it should
count, liver transaminase values elevated to approximately
be plotted on the Rumack-Matthew nomogram to deter-
three times the upper limit of normal, and an acetamino-
mine if NAC therapy is indicated. This nomogram is based
phen concentration was 308 [jg/mL. Based on the Rumack-
on a series of patients with and without hepatotoxicity
Matthew nomogram, which plots acetaminophen plasma
and their corresponding measured plasma acetaminophen
concentration versus hours of after ingestion, one can discern
concentrations.
whether hepatic toxicity is probable. For example, a plasma
acetaminophen concentration of 308j1g/mL at approxima-
tely 6 h after ingestion was well within the “probable hepatic Ethylene Glycol
toxicity” range, and treatment with N-acetylcysteine NAC was A 37-year-old female was brought to the ED after being found
initiated. unresponsive in her home. At the scene, emergency medical
The patient received the first dose of [V NAC in the ED and personnel administered oxygen and naloxone and performed
was admitted to the medical ward to complete the treatment a finger stick for glucose (standard procedure for a person
course of IV NAC. Transient increases of hepatic transami- with altered mental status and suspected toxic ingestion),
nases were measured over the ensuing two days of the hospi- which showed a normal value of 95 mg/dL. The patient’s spouse
talization. The psychiatry consultation service determined she reported that she had been depressed and despondent with the
was not actively suicidal; she was discharged from the hospital recent loss of her job. No empty pill bottles or liquid containers
two days after admission with scheduled psychiatric and medi- were found with her at home.
cal follow-up appointments. Upon arrival to the hospital, she remained comatose. Her
The clinical presentation of patients poisoned with acet- vital signs were: blood pressure 105/65 mm Hg, pulse 78/min,
aminophen is sufficiently confusing in some cases; it is difficult respiratory rate elevated at 32/min, and her body tempera-
to estimate the time of ingestion. Due to the paucity of clini- ture was normal. The remainder of the physical examination
cal symptoms with acute overdose, most clinicians will request was significant as her pupils were 3mm and sluggishly reac-
an acetaminophen concentration be measured for any patient tive to light; the lung and heart examinations were normal;
suspected of having a toxic exposure to any substance. The the abdominal examination revealed diminished but present
paucity of signs and symptoms associated with an acetamino- bowel sounds, and no tenderness, organomegaly, or masses
phen overdose makes inadvertent missing of a potentially fatal were detected. The rectal examination was normal; the stool
overdose until the window for maximum antidote effective- was without detectable gross or occult blood. Neuro examina-
ness has passed. tion was nonfocal with a diminished gag reflex.
 CHAPTER 33 Clinical Toxicology 477

The patient was placed on a cardiac monitor, an IV line was dehydrogenase. The latter two acid metabolites are thought
started, clinical laboratory specimens were obtained, and she to be responsible for both the renal and the acid-base toxic-
was placed on oxygen, given naloxone, thiamine, and dex- ity observed during poisoning by ethylene glycol. If untreated
trose (50%) intravenously. Chest and abdominal radiography or treated too late, ethylene glycol poisoning can result in
was without abnormality. A 12-lead ECG was also normal. fatal cerebral edema with seizures as well as irreversible renal
Faced with the uncertainty of oral ingestion versus topical damage.
and inhalation exposure, a decision was made to proceed
with gastric decontamination. The patient was endotrache-
Valproic Acid
ally intubated to protect her airway before an orogastric tube
was placed. Gastric lavage was performed and no blood was A 33-year-old male was brought to the ED after being found
found. The fluid withdrawn from the stomach was bright yel- unresponsive with two empty prescription pill bottles of
low in appearance and slightly viscous. When a Wood’s lamp extended release valproic acid at his side. He was last seen 8h
illuminated this fluid in a darkened room, fluorescence was prior to being found unresponsive and was then in normal
observed. This finding suggests the presence of automotive health. The pharmacy confirmed that monthly prescrip-
antifreeze that contains ethylene glycol. Activated charcoal tions, each containing 30, 250 mg extended release valproic
(2.0 g/kg) was placed via the orogastric tube into the stomach acid tablets, had been dispensed within the preceding three
with a cathartic even though the efficacy for binding ethylene months.
glycol is limited; the use of activated charcoal here was for The patient was unresponsive to verbal or tactile stimula-
other, potentially unknown coingestants. Clinical laboratory tion. Vital signs were blood pressure 85/55mm Hg, pulse
results returned showing the following: 94/min, respiratory rate 20/min, and temperature 33.2°C.
Naloxone was administered without effect. A cardiac moni-
tor showed sinus rhythm. The physical examination showed
Na = 140 mEq/L K =3.1 mEq/L the patient to be without obvious signs of trauma; the skin
Cl= 94 mEq/L HCO, = 8 mEq/L was cool and without track marks; the pupils were 2mm and
poorly reactive to light; bowel sounds were diminished. The
BUN = 12 mg/dl Glucose = 100 mg/dl rectal examination was negative for occult blood. The neuro-
Arterial blood gas: logic examination revealed coma without focal motor abnor-
malities and an absent gag reflex.
pH =7.20; pCO, = 20 mm Hg; pO, = 98 mm Hg
Initial laboratories showed mild metabolic acidosis with
elevated serum lactate, an increased anion, slightly increased
The complete blood count was normal, the urine analysis serum ammonia, normal glucose, liver function tests, and
was normal, measured serum osmolarity was 330 mOsm/kg, renal function tests. The chest and abdominal radiographs
and acetaminophen and salicylate levels were below the limits were normal. The 12-lead ECG showed a prolonged QT inter-
of detection, and the urine toxicology screen was negative. val without arrhythmia. The patient was endotracheally intu-
The laboratory results were interpreted as follows: a meta- bated to protect his airway prior to gastric lavage that yielded
bolic acidosis with elevated AG (AG=38) and an elevated some pill fragments only. The patient was placed on a venti-
osmol gap (40 mOsm). These findings are consistent with either lator to support his respiration. Activated charcoal (1.5 g/kg)
methanol or ethylene glycol poisoning (Tables 33-4 and 33-5). was administered via the orogastric tube immediately fol-
The patient was treated with IV fomepizole (4-methylperazole), lowing the lavage procedure. The blood pressure continued
sodium bicarbonate was given intravenously for the profound to remain low despite IV fluid administration. A STAT val-
metabolic acidosis, and the patient underwent hemodialy- proic acid serum measurement showed the concentration was
sis. After 4 h of hemodialysis, the acid—base and electrolyte 572 ug/mL.
abnormalities were corrected but the patient remained coma- Blood pressure responded to low-dose vasopressors
tose. The patient underwent a second 4-h course of hemodi- (IV dopamine) with continued IV fluid administration. A
alysis 8 h later to again correct her metabolic acidosis with the repeat serum valproic acid concentration was 890 ug/mL at
appearance of minor renal injury (serum creatinine increased 2 h postadmission. Serial oral activated charcoal (every 4 h)
to 1.8mg/dL). She regained normal consciousness within was initiated via the orogastric tube and hemodialysis was
18 h and her renal function recovered completely within three started 3 h after admission. IV L-carnitine was given when a
days. Subsequently, the patient admitted that she intentionally repeat serum ammonia concentration was further elevated
drank more than half a container of antifreeze with the intent at 94 mg/dL. Subsequent measured plasma concentrations of
of harming herself. She was evaluated by the psychiatry consul- valproic acid gradually declined to <100 ug/mL over the next
tation service and transferred to their service for further care. 48 h after one additional hemodialysis session was conducted.
Ethylene glycol exerts primary toxicity after undergoing The patient regained consciousness 24 h after admission and
biotransformation by alcohol dehydrogenase to glycolic acid made a full recovery by the fourth hospital day. The psychi-
and then to glycolic and oxalic acid by the action of aldehyde atry consultation service accepted the patient in transfer to
478 UNIT 7 Applications of Toxicology

their inpatient service after he was medically cleared by the CONCLUSION

toxicology service.
The increasing plasma concentrations of the toxic substance Clinical toxicology encompasses the expertise in the special-
despite gastric decontamination procedures can occur after ties of medical toxicology, applied toxicology, and clinical
ingestion of an extended release formulation, which is phar- poison information specialists. The clinical science has signifi-
maceutically designed to slowly dissolve in the gastrointestinal cantly evolved to the present state of the discipline over the past
tract and provide for ongoing sustained release of the active 50 years or more. The incorporation of evidence-based, out-
drug product as opposed to immediate release of the agent. come-driven practice recommendations has significantly
Drug substances that demonstrate the “slow release” profile improved the critical evaluation of treatment modalities and
without having been formulated in a sustained release dosage methods for poison treatment. A careful diagnostic approach to
form include salicylates and barbiturates as well as formula- a poisoned patient is essential, as important medical history is
tions of iron supplements. The presence of a drug bezoar or often absent or unreliable. Skillful use of antidotes is an impor-
concretion can be dangerous because the treating team could tant component ofthe practice of medical toxicology. Continued
erroneously stratify a patient based on the initial measured research will increase the repertoire of effective treatments for
plasma concentration and be unprepared for severe toxicity or poisoning and ultimately improve clinical practice.
a prolonged toxicity time course.
Moderate to severe valproic acid intoxication leads to deple-
tion of L-carnitine, which may cause the observed hyper-
BIBLIOGRAPHY
Barile FA: Clinical Toxicology: Principles and Mechanisms, 2nd ed.
ammonemia. The FDA has recently approved the use of
New York: Informa Healthcare, 2010.
IV L-carnitine for the treatment of valproic acid poisoning in
Hoftman RS, Howland MA, Lewin NA, et al. (eds.): Goldfrank’s Toxi-
the setting of hepatotoxicity, hyperammonemia, large over- cologic Emergencies, 10th ed. New York: McGraw-Hill, 2014.
doses of valproate by history, or measured serum concentra- Tintinalli JE, Stapczynski JS, Ma OJ et al. (eds.): Emergency Medicine: A
tions of valproic acid exceeding 450 ug/mL. Comprehensive Study Guide, 7th ed. New York: McGraw-Hill, 2010.
 CHAPTER 33 Clinical Toxicology 479

QUESTIONS

What is the primary goal in taking a history in a poisoned Which is NOT included in the differential diagnosis of an
patient? ; elevated anion gap?
a. determining drug allergies. a. ethanol.
b. determining susceptibility to drug overdose. b. methanol.
c. determining likelihood of an attempted suicide. c. diabetes.
d. determining the ingested substance. d. ethylene glycol.
e. determining the motive behind the poisoning. e. diarrhea.

Who is most likely to give incorrect information while An elevated osmol gap might suggest which of the
taking a history of a poisoned patient? following?
a. patient. a. methanol poisoning.
b. EMT. b. chronic vomiting.
c. employer. c. lactic acidosis.
d. pharmacist. d. diabetic ketoacidosis.
e. family members. e chronic diarrhea.

Which of the following sets of clinical features character- Which of the following is LEAST likely to prevent further
izes an anticholinergic toxic syndrome? poison absorption?
a. increased blood pressure, decreased heart rate, a. induction of emesis.
decreased temperature. b. activated charcoal.
b. decreased blood pressure, increased heart rate, c. gastric lavage.
decreased temperature. d. syrup of ipecac.
c. increased blood pressure, increased heart rate, e. parasympathetic agonist.
increased temperature.
d. decreased blood pressure, decreased heart rate, Which of the following would NOT be used to enhance
decreased temperature. poison elimination?
e. increased blood pressure, decreased heart rate, a. oral activated charcoal.
increased temperature. b. hemoperfusion.
c. acidification of urine.
Which of the following sets of clinical features character- d. hemodialysis.
izes a sympathomimetic toxic syndrome? e. plasma exchange.
miosis, decreased bowel sounds, decreased alertness.
decreased heart rate, increased temperature, mydriasis. 10. Which of the following might be used as an antidote for
hyperalertness, decreased blood pressure, miosis. patients with cyanide poisoning?
increased temperature, increased heart rate, miosis. syrup of ipecac.
cp
aore
mydriasis, increased blood pressure, hyperalertness. atropine.
chelating agents.
Which of the following drugs CANNOT be tested for in a sodium nitrite.
hospital on a stat basis? iS
er
ie quinine.
a. ethanol.
b. cocaine.
c. aspirin.
d. phenytoin.
e. digoxin.
 Cockle
oP egies cEigeak:

Occupational Toxicology
Peter S. Thorne

INTRODUCTION Animal Toxicology Testing for Establishing Acceptable

Levels of Exposure
WORKPLACES, EXPOSURES, AND STANDARDS Worker Health Surveillance
Determinants of Dose Linkage of Animal Studies and Epidemiologic
Occupational Exposure Limits Studies

OCCUPATIONAL DISEASES EXPOSURE MONITORING

Routes of Exposure Environmental Monitoring for Exposure
Agents Associated with Diseases Assessment
Occupational Respiratory Diseases Biologic Monitoring for Exposure Assessment
Other Occupational Diseases
CONCLUSION
TOXICOLOGIC EVALUATION OF OCCUPATIONAL AGENTS
Evaluation of Occupational Risks
Establishing Causality

KEY POINTS

« Occupational toxicology is the application of the prin- = Occupational exposure limits do not correspond to the
ciples and methodology of toxicology toward chemical level of exposure below which the probability of impair-
and biologic hazards encountered at work. ing the health of the exposed workers is acceptable.
» In occupational environments, exposure is often used as a Diseases arising in occupational environments involve
a surrogate for dose. exposure primarily through inhalation, ingestion, or
dermal absorption.

INTRODUCTION environment often presents exposures to complex mixtures,

the occupational toxicologist must also recognize exposure
The work environment with its chemical and biologic hazards combinations that are particularly hazardous.
plays a role in the occurrence of adverse human health effects. It is often difficult to establish a causal link between a worker’s
Occupational toxicology is the application of the principles illness and job. First, the clinical expressions of occupation-
and methodology of toxicology toward chemical and biologic ally induced diseases are often indistinguishable from those
hazards encountered at work. The objective of the occupational arising from nonoccupational causes. Second, there may be a
toxicologist is to prevent adverse health effects in workers long interval between exposure and the expression of disease.
that result from their work environment. Because the work Third, diseases of occupational origin may be multifactorial
481
482 UNIT7 Applications of Toxicology

with personal or other environmental factors contributing in Figure 34-1, the dose is a function of exposure concentra-
to the disease process. Ongoing assessments of occupational tion, exposure duration, and exposure frequency. Individual
risk must occur as new hazards arise with the emergence of and environmental characteristics also can affect dose. Table
new technologies. 34-1 indicates determinants of dose for exposure via the inha-
lation and dermal routes. Personal protective equipment must
be used properly to maximize effectiveness.
WORKPLACES, EXPOSURES,
AND STANDARDS
TABLE 34-1 Determinants of toxicant dose.
Approximately 40% of the global work force works in agricul-
Inhalation exposure
tural production. The demographics of laborers in industrial
+ Airborne concentration
nations has shifted away from jobs in heavy industry toward + Particle size distribution
jobs in the service sector and high-technology industries. Respiratory rate
Tidal volume
+ Other host factors
Determinants of Dose Duration of exposure
Chemical, physical, or biologic properties of the hazardous agent
Dose is defined as the amount of toxicant that reaches the - Effectiveness of personal protective devices
target tissue over a defined time span. In occupational envi-
Dermal exposure
ronments, exposure is often used as a surrogate for dose. The * Concentration in air, droplets, or solutions
response to a toxic agent is dependent on both host factors + Degree and duration of wetness
and dose. Figure 34-1 illustrates the pathway from exposure + Integrity of skin
to subclinical disease or adverse health effect and suggests + Percutaneous absorption rate
« Region of skin exposed nee
that there are important modifying factors: contemporane- + Surface area exposed
ous exposures, genetic susceptibility, age, gender, nutritional « Preexisting skin disease
status, and behavioral factors. These modifying factors can + Temperature in the workplace
influence whether a worker remains healthy, develops subclini- Vehicle for the toxicant
+ Presence of other chemicals on skin
cal disease that is repaired, or progresses to illness. As illustrated

Genetic
Additive or susceptibility Age, gender,
synergistic nutrition,
coexposure behavior

Exposure
concentration

2 Screening ee
Exposure D wellness programs ~ Adverse
duration ee ( yeded
‘ ; effect
SS Surveillance mesh aNc

Exposure
frequency Occupational health
standards Progression

Personal protective
Subclinical
equipment
disease
Engineering and
administrative
controls
Repair

Healthy A
worker }

FIGURE 34-1 Pathway from exposure to disease, showing modifying factors and opportunities for intervention.
 CHAPTER 34 Occupational Toxicology 483

Occupational Exposure Limits responsible for the creation of the occupational regulatory
framework. Although death rates are fairly low, many expo-
Workplace exposure limits exist for chemical, biological,
sures result in debilitating illnesses. Many of the diseases
and physical agents in order to promote worker health and
listed in Table 34-2 are known by other names that refer
safety. For chemical and biological agents, exposure limits are
to a particular occupation or agent. One example is hyper-
expressed as acceptable ambient concentration levels (occu-
sensitivity pneumonitis, an allergic lung disease marked by
pational exposure limits [OELs]) or as concentrations of a
interstitial lymphocytic pneumonitis and granulomatous
toxicant, its metabolites, or a specific marker ofits effects (bio-
lesions. Hypersensitivity pneumonitis is also known as extrin-
logic exposure indices [BEIs]).
sic allergic alveolitis, farmer’s lung disease, bagassosis (sugar
OELs are established as standards by regulatory agen-
cane), humidifier fever, Japanese summer house fever, pigeon
cies or as guidelines by research groups or trade organiza-
breeder’s lung, and maple bark stripper’s lung, depending on
tions. In the United States, the Occupational Safety and Health
the occupational setting in which it arises. Although we often
Administration under the Department of Labor promulgates
think of these as the same disease, it is important to recognize
legally enforceable standards known as permissible exposure
that the exposures and physiologic responses they induce are
limits (PELs). The National Institute for Occupational Safety
complex and may differ in the manifestation ofthe disease.
and Health (NIOSH), under the Centers for Disease Control and
Toxic gas injuries are often characterized by leakage of both
Prevention, publishes recommended exposure limits that are
fluid and osmotically active proteins from the vascular tissue
frequently updated and are generally more stringent than PELs.
into the interstitium and airways. The vapors of anhydrous
The European Commission has established legally enforceable
ammonia combine with water in the tissues ofthe eyes, sinuses,
binding occupational exposure limit values (BOELVs) and bio-
and upper airways and form ammonium hydroxide, quickly
logic limit values for the protection of worker health and safety.
producing liquefaction necrosis. Chemicals with lower solubil-
The American Conference of Governmental Industrial
ity, such as nitrogen dioxide, act more on the distal airways and
Hygienists is a trade organization that annually publishes OELs
alveoli and take longer to induce tissue damage.
for chemicals and for physical agents. These take the form of
Occupational asthma occurs when airways restrict in
threshold limit values (TLVs) and BEls. They are developed as
response to some stimulus present in the workplace. In
guidelines and are not enforceable standards.
chemical-based industries, plastic and rubber polymer pre-
OELs correspond to the level of exposure below which the
cursors, diisocyanates, reactive dyes, and acid anhydrides are
probability of impairing the health of the exposed workers is
recognized low-molecular-weight sensitizing compounds.
acceptable. To determine that the risks from an occupational
Biocides and fungicides used in metal fabrication and machin-
hazard are acceptable, it is necessary to characterize the haz-
ing, custodial services, lawn and turf growing, and agriculture
ard, identify the potential diseases or adverse outcomes, and
are also chemicals associated with occupational asthma. A
establish the relationship between exposure intensity or dose
number of metals can induce sensitization and asthma, includ-
and the adverse health effects.
ing chromium, cobalt, nickel, platinum, and zinc. Enzymes
include a-amylase among bakery workers and subtilisin, a pro-
OCCUPATIONAL DISEASES tease used in laundry detergents.
Animal handlers, processors, and laboratory technicians
Routes of Exposure who work with animals can become immunologically sensi-
Diseases arising in occupational environments involve exposure tized to urine or salivary proteins in many vertebrates; pro-
primarily through inhalation, ingestion, or dermal absorption. teins in bat guano and bird droppings; animal dander; serum
Exposures leading to occupational infections may arise through proteins in blood products; dust from horns, antlers, and
inhalation or ingestion of microorganisms, from needle sticks tusks; or the shells of crustaceans. Very high rates of sensiti-
in health care workers, or from insect bites among those who zation can occur in shellfish processors. Arthropods such as
work outdoors. Additionally, poisonings from toxic plants or insect larvae, cockroaches, mites, or weevils are recognized
venomous animals can occur through skin inoculation (e.g., inducers of work-related asthma. Plants and plant products
zookeepers, horticulturists, or commercial skin divers). (e.g., soy flour, spices, and coffee beans) can also cause asthma
among workers. Exposure to fungi, especially of the genera
Agents Associated with Diseases Aspergillus, Penicillium, Rhizopus, Mucor, and Paecilomyces,
are associated with allergic rhinitis and asthma. These are
Table 34-2 outlines some major occupational diseases and
especially present in sawmills, woodchip handling, and com-
examples of toxicants that cause them. Table 34-3 lists known
posting facilities.
human carcinogens (group 1), for which there are extensive
An emerging area of concern is adverse effects of respira-
occupational exposufe.
tory exposures to manufactured nanomaterials. Occupational
exposures occur in the manufacture of the nanomaterials
Occupational Respiratory Diseases and in their use in fabricating materials and consumer prod-
Occupational lung diseases (such as coal workers’ pneumo- ucts. Exposures can also occur when nanomaterials are cut
coniosis, asbestosis, and occupational asthma) are largely or shaped and when product waste is discarded. Engineered
 484 UNIT 7 Applications ofToxicology

TABLE 34-2 Examples of occupational diseases and the toxicants that cause them.
Organ System or Disease Group Disease Causative Agent

Lung and airways Acute pulmonary edema, bronchiolitis Nitrogen oxides, phosgene, diacetyl
obliterans
Allergic rhinitis Pollens, fungal spores
Asphyxiation Carbon monoxide, hydrogen cyanide, inert gas dilution
Asthma Toluene diisocyanate, a-amylase, animal urine proteins
Asthma-like syndrome Swine barn environments, cotton dust, bioaerosols
Bronchitis, pneumonitis Arsenic, chlorine
Chronic bronchitis Cotton dust, grain dust, welding fumes
Emphysema Coal dust, cigarette smoke
Fibrotic lung disease Silica, asbestos
Hypersensitivity pneumonitis Thermophilic bacteria, avian proteins, pyrethrum,
Penicillium, Aspergillus
Metal fume fever Zinc, copper, magnesium
Mucous membrane irritation Hydrogen chloride, swine barn environments
Organic dust toxic syndrome “Moldy” silage, endotoxin
Upper respiratory tract inflammation Endotoxin, peptidoglycan, glucans, viruses

Cancer Acute myelogenous leukemia Benzene, ethylene oxide

Bladder cancer Benzidine, 2-naphthylamine, 4-biphenylamine
Gastrointestinal cancers Asbestos “
Hepatic hemangiosarcoma Vinyl chloride 4
Hepatocellular carcinoma Aflatoxin, hepatitis B virus
Mesothelioma, lung carcinoma Asbestos, arsenic, radon, bis-chloro methyl ether
Skin cancer Polycyclic aromatic hydrocarbons, ultraviolet irradiation

Skin Allergic contact dermatitis Natural rubber latex, isothiazolins, poison ivy, nickel
Chemical burns Sodium hydroxide, hydrogen fluoride
Chloracne TCDD, polychlorinated biphenyls
Irritant dermatitis Sodium dodecyl sulfate

Nervous system Cholinesterase inhibition Organophosphate insecticides

Neuronopathy Methyl mercury
Parkinsonism Carbon monoxide, carbon disulfide
Peripheral neuropathy N-Hexane, trichloroethylene, acrylamide

Immune system Autoimmune disease Vinyl chloride, silica

Hypersensitivity See entries for allergic rhinitis, asthma, hypersensitivity
pneumonitis, allergic contact dermatitis
Immunosuppression TCDD, lead, mercury, pesticides

Renal disease Indirect renal failure Arsine, phosphine, trinitrophenol

Nephropathy Paraquat, 1,4-dichlorobenzene, mercuric chloride

Cardiovascular disease Arrhythmias Acetone, toluene, methylene chloride, trichloroethylene

Atherosclerosis Dinitrotoluene, carbon monoxide
Coronary artery disease Carbon disulfide
Cor pulmonale Beryllium
Systemic hypotension Nitroglycerine, ethylene glycol dinitrate

Liver disease Fatty liver (steatosis) Carbon tetrachloride, toluene

Cirrhosis Arsenic, trichloroethylene
Hepatocellular death Dimethylformamide, TCDD
Reproductive system Male Chlordecone (Kepone), dibromochloropropane, hexane
Female Aniline, styrene
Both sexes Carbon disulfide, lead, vinyl chloride

Infectious diseases Arboviral encephalitides Alphavirus, Bunyavirus, Flavivirus

Aspergillosis Aspergillus niger, A fumigatus, A. flavus
Cryptosporidiosis Cryptosporidium parvum
Hepatitis B Hepatitis B virus
Histoplasmosis Histoplasma capsulatum
Legionellosis Legionella pneumophila
Lyme disease Borrelia burgdorferi
Psittacosis Chlamydia psittaci
Tuberculosis Mycobacterium tuberculosis hominis

TCDD, 2,3,7,8-tetrachlorodibenzo-para-dioxin.
 CHAPTER 34 Occupational Toxicology 485

TABLE 34-3 Occupational exposure agents classified by IARC as group 1 definite human carcinogens.
Agent - Industries and Occupations Where Some Workers May be Exposed

Particulate matter
Asbestos Miners, abatement workers, construction workers, sheet metal workers, steam fitters,
shipyard workers
Crystalline silica (quartz or cristobalite) Stone and ceramics industry, foundries, construction, abrasives manufacturing
Erionite Waste treatment workers, building materials manufacturing
Hematite Underground mining
Talc containing asbestiform fibers Ceramics industry
Wood dust Wood and wood-products industries, pulp and paper industry, wood working trades

Metals
Arsenic and arsenic compounds Miners, nonferrous metal smelting, arsenical pesticide manufacturers and applicators
Beryllium Specialty metallurgy workers, avionics, electronics, nuclear industry
Cadmium and cadmium compounds Cadmium smelting, battery production, dyes and pigment making, electroplating
Gallium arsenide Microelectronics manufacturing
Hexavalent chromium compounds Chromate production plants, dye and pigment making, welders, tanners
Nickel compounds* Nickel smelting, welding

Organic chemicals
Aflatoxin Animal feed industry, grain handling and processing
4-Aminobiphenyl Chemical industry, dyes and pigment manufacturing
Benzene Refineries, shoe industry, chemical, pharmaceutical and rubber industry, printing industry
Benzidine Chemical industry, dyes and pigment manufacturing
Benzo(a)pyrene Coke oven emissions, coal tar pitch volatiles, diesel exhaust, environmental tobacco smoke
Bis(chloromethyl) ether and chloromethyl ether Chemical industry, laboratory reagent, plastic manufacturing
(technical grade)
1,3-Butadiene Chemical industry, petrochemical plants, styrene-butadiene rubber manufacturing
Coal tars and pitches Coke production, coal gasification, refineries, foundries, road paving, hot tar roofing
Ethylene oxide Chemical industry, dry vegetable fumigation, hospital sterilizing
Formaldehyde Textiles, composite wood industry, chemical industry, medical laboratories
4,4’-Methylenebis(2-chloroaniline) Epoxy resin manufacturing, polyurethane product fabrication
Mineral oils, untreated and mildly treated Metal machining and honing, roll steel production, printing
2-Naphthylamine Chemical industry, dyestuffs, and pigment manufacturing
2,3,4,7,8-Pentachlorodibenzofuran Hazardous waste processing, chlorophenoxy herbicide production and use, pulp and
paper industry
3,4,5,3’,4’-Pentachlorobiphenyl Hazardous waste processing, waterway dredging, transformer handling, pulp and
paper industry
Shale oils or shale-derived lubricants Mining and processing, cotton textile industry
Soots Chimney sweeps, heating and ventilation contractors, firefighters, metallurgical workers
2,3,7,8-Tetrachlorobibenzo-para-dioxin (TCDD) Hazardous waste processing, chlorophenoxy herbicide production and use, pulp and
paper industry
Vinyl chloride Plastics industry, production of polyvinyl chloride products and copolymers

Other agents with occupational exposure

Environmental tobacco smoke Restaurant, bar and entertainment industry; other smoke-exposed workers
Occupational exposures as a painter Commercial painting
Leather dust Garment industry, auto seat fabrication, saddle and tack manufacturing
Magenta dye (rosaniline, pararosaniline) Dye manufacture, textile dying, commercial art and printing
Mustard gas Production, soldiers, some research laboratories
Exposures in the rubber industry Work in rubber manufacturing industries
Strong inorganic acid mists containing sulfuric acid Steel industry, petrochemical industry, fertilizer industry, pickling industry

Physical Agents
lonizing radiation* Radiology and nuclear medicine staff, nuclear workers, miners, hazardous waste workers
Solar radiation Farmers, gardeners and landscapers, lifeguards, construction workers

*Certain combinations ofnickel oxides and sulfides.

‘Includes X-rays, y-rays, neutrons, radon gas, and wand 6 particle-emitting substances internally deposited.

nanomaterials may be carbon-based, metal-based, or biologic

in nature. Inhaled nanomaterials may induce pulmonary tox- Other Occupational Diseases
icity or they can cause adverse effects in other tissues through Occupational toxicants may induce diseases in a variety of
adsorption and transport, generation of toxic substances by sites distant from the lung or skin. These include tumors aris-
their dissolution or degradation, or by crossing key physiologic ing in the liver, bladder, gastrointestinal tract, or hematopoi-
barriers, or cell and nuclear membranes. etic system and are attributable to a variety of chemical classes.
486 UNIT 7 Applications of Toxicology

Nervous system damage can be central, peripheral, or both. TOXICOLOGIC EVALUATION OF

It may be acute, as with some organophosphate exposures, OCCUPATIONAL AGENTS
or chronic, as with organomercury poisoning or acrylamide-
induced neuropathy. Immune system injury may arise from Evaluation of Occupational Risks
the immunosuppressive effects of chemicals or from hyper-
To recommend an acceptable exposure level to an industrial
sensitivity leading to respiratory or dermal allergy or systemic
chemical, one must attempt to define the risks associated with
hypersensitivity reactions. Autoimmune syndromes have been
adverse effects in the most sensitive exposed populations. It
associated with occupational exposures to crystalline silica and
then remains to decide what proportion of exposed subjects
vinyl chloride.
may still develop an adverse effect at the proposed acceptable
Occupational diseases of the cardiovascular system include
exposure level.
atherosclerosis, various arrhythmias, impaired coronary blood
supply, systemic hypotension, and right ventricular hypertro-
phy usually due to pulmonary hypertension. Liver diseases Establishing Causality—In complex occupational envi-
include carbon tetrachloride-induced fatty liver. Occupational ronments, it may be difficult to establish a causal relationship
diseases of the reproductive system can be gender- and organ- between a toxic substance and a disease. A matrix was devel-
specific, or may affect both sexes. Disease due to exposures to oped to evaluate the weight of evidence for a causal association
infectious agents occurs in such occupations as veterinarians, between a toxicant and an occupational disease (Figure 34-2).
health care workers, biomedical researchers, and farmers. Evidence from well-conducted in vitro studies, animal stud-
Both industrial and nonindustrial indoor environments may ies, human challenge studies (intentional clinical exposure
pose occupational hazards due to the presence of chemical or to humans), case reports, and epidemiologic investigations
biologic agents. Problems with ventilation and use of synthetic are evaluated. This evaluation is guided by seven criteria. If a
building materials have led to a rise in complaints associ- chemical were thoroughly studied in‘animals, humans, and in
ated with occupancy in buildings. Volatile and semivolatile vitro studies and produced clear and convincing evidence of
chemicals are released from process materials in manufactur- an exposure-response relationship in controlled studies that
ing, building materials, floor coverings, furniture, cleaning used appropriate models and relevant endpoints, then that
products, biocides, and microorganisms. In some cases, the would constitute compelling evidence of a causal relationship
occupied space of a building may be clean and dry, but local between that chemical and that disease.
amplification sites for molds, such as damp closets or sub- To evaluate with some degree of confidence, the level of
floors, may develop. Airborne viruses, bacteria, and fungi are exposure at which the risk of health impairment is accept-
responsible for a variety of building-related illnesses. able, a body of toxicologic information is required. Five

Assessment Consideration Evidence of a Consistent Objective Endpoints Appropriate

of exposure or control of dose — results from clinical data related to subjects or
to specific confounders response different human models
agents relationship studies pathology

In vitro
studies

Animal
studies

challenge
studies

Case studies

Epidemiology
studies

For each type of study listed in the first column weight the quality of data from existing studies based on the criteria listed in the column
headings as follows:
0 No evidence or condition is not met
1 Equivocal evidence or condition is partially met
2 Some evidence or condition is mostly met
3 Clear evidence or condition is convincingly met

FIGURE 34-2 Matrix for assessing the strength of an association between a toxicant and an occupational disease.
 CHAPTER 34 Occupational Toxicology 487

sources of data may be available to inform the occupational metabolism, the nature of its adverse effects, and how workers
risk-assessment process. These sources include in vitro assays, are exposed to the toxicant. Investigations that can make use of
animal toxicology studies, human challenge studies, case specific physiologic or biochemical tests, based on knowledge
reports, and epidemiology studies. of the principal target organ or function, produce highly valu-
able information and increase confidence in the OEL derived
from them.
Animal Toxicology Testing for Establishing
Acceptable Levels of Exposure
Animal studies provide valuable data from which to estimate Worker Health Surveillance
the level of exposure at which the risk of health impairment The primary objective of occupational toxicology is to pro-
is acceptable. Comparison of the animal studies with epidemi- vide both periodic screening of general health and wellness
ology testing is provided in Table 34-4. The duration of tests and health exposure monitoring tailored to recognized haz-
necessary to establish an acceptable level for occupational ards of the workplace. Monitoring of exposures to toxicants
exposure is primarily a function of the type of toxic action sus- in the workplace may be important in detecting excessive
pected. It is generally recognized that for systemically acting exposures before the occurrence of significant biologic dis-
chemicals, subacute and short-term toxicity studies are usu- turbances and health impairment. When a new chemical is
ally insufficient for proposing OELs. Subacute and short-term being used on a large scale, careful clinical surveillance of
toxicity tests are usually performed to find out whether the workers and monitoring of workplaces should be instituted.
compound exhibits immunotoxic properties and cumulative Evaluation of the validity of the proposed OEL derived from
characteristics. They also aid in selection of the doses for long- animal experiments through workplace surveillance is the
term exposures. Studies designed to evaluate reproductive major goal.
effects and teratogenicity should also be considered. Epidemiologic studies designed to assess exposure—response
Information derived from exposure routes similar to those relationships will have more validity if both the target dose
sustained by workers is clearly most relevant. The choice of and the critical biologic changes are monitored in exposure-
what studies to perform using which routes of administration response studies. Knowledge of the fate of the chemical in the
must be evaluated scientifically for each toxicant. Important organism and its mechanism of action are required. Because
considerations include its target sites and mechanism of action, early biomarkers of effect are subtle and individual variations

TABLE 34-4 Comparison of epidemiologic studies and experimental exposure studies.

Observational Epidemiologic Studies Experimental Animal Exposure Studies

Toxicant exposure Reflects true exposure among population at risk Controlled to represent major toxicant of interest
Character Complex and variable in space and time Usually one or two test compounds
May include nonoccupational exposures to toxicant May not reflect complexity of human exposures
or related compounds
Frequency and duration Work day, work week, and years in that job Acute, subacute, subchronic, chronic
May be task specific
Exposure route Inhalation, ingestion, percutaneous, or a combination Injection, inhalation, oral, or dermal. Rarely a
combination by design
Appropriateness of dose Reflect the actual range of exposure Often doses studied are far higher than human
exposures
Assessment Environmental sampling, or measurement of Measurement of administered dose with or without
biomarkers measurement of biomarkers
May be retrospective and based on employer records, Sampling of exposure chamber air for inhalation
group-based approaches, or questionnaires studies

Species considerations Humans—cohorts or cases and controls Laboratory animals, usually inbred strains of mice
or rats
Must protect the safety and confidentiality of subjects Must ensure proper care and use ofanimals
Representativeness May exist a selection bias such that the study population Experimental animal species may not represent
may not represent the occupational work force humans
Relevance to human health Directly relevant if appropriate outcomes are studied Relevant if species differences are known
ca
Of limited relevance if species or strain effects on
absorption, distribution, metabolism, and disease
are unknown

Analytical challenges Selection bias, misclassification, and confounding in Control of genetics, feeding, and housing between
characterization of outcomes exposed and contro! groups
Within- and between-subject variance may be high Low variance in outcomes
488 UNIT 7 Applications of Toxicology

exist in the response to a chemical insult, results generally EXPOSURE MONITORING

require a statistical comparison between a group of exposed
workers and a similar group of workers without the exposure Environmental Monitoring for
of interest. If exposure induces an adverse effect, it is expected Exposure Assessment
that these studies may permit establishment of the relation-
A critical element of establishing OELs is the accurate and
ship between integrated exposure (intensity < time) and fre-
uniform assessment of exposure. Methodology for exposure
quency of abnormal results and, consequently, a redefinition of
assessment must be specifically tailored to the agent under
the OEL.
study and the environment in which it appears. To assess air-
In cases where a surveillance program was not instituted
borne exposures, personal samples taken in the breathing
before the introduction of anew chemical, it is more difficult
zone are generally used. Repeated random sampling is usu-
to establish the efficacy of the exposure limit. In this situation,
ally the best approach to developing unbiased measures of
evaluation depends on retrospective cohort studies or case-
exposure. Recent studies have demonstrated that group-based
control studies or on cross-sectional studies on workers who
approaches, assessing exposures to groups rather than to indi-
have already sustained exposure. In fact, case reports of iso-
viduals, are more efficient in terms of measurement effort for
lated overexposures resulting from specific incidents such as
obtaining a desired level of accuracy.
containment breaches, chemical spills, or vessel or pipe rup-
Although one cannot assess dose directly through exposure
tures can provide useful information. Such observations may
monitoring, it has distinct advantages over biomonitoring,
indicate whether human symptomatology is similar to that
which cannot provide route-specific exposure data. Exposure
found in animals and may suggest functional or biologic tests
monitoring allows one ,to quantify workplace exposure by
that might prove useful for routine monitoring of exposed
route through selective air monitoring in the breathing zone
workers.
of the worker and dermal dosimetry using absorptive mate-
rial affixed to the workers’ skin or clothing. Environmental
Linkage of Animal Studies and monitoring techniques are generally less expensive and less
Epidemiologic Studies invasive than techniques involving the collection and analysis
of biologic samples such as blood or urine. Spatial, temporal,
In the field of occupational toxicology, close cooperation
and work practice associations can be established by air moni-
between those conducting animal studies and those conduct-
toring and can suggest better interventions and engineering
ing studies of workers is essential for examining risks associated
controls. Exposure monitoring allows one to quantify work-
with overexposure to chemicals and other toxicants. Several
place exposure by route through selective air monitoring in
occupational carcinogens have been identified clearly through
the breathing zone of the worker and dermal dosimetry using
combined epidemiologic and experimental approaches. For
absorptive material affixed to the workers’ skin or clothing.
example, the carcinogenicity of vinyl chloride was first dem-
A fully validated sampling and analysis method requires
onstrated in rats, and a few years later, epidemiologic studies
specification of the sampling methods; sample duration,
confirmed the same carcinogenic risk for humans. This obser-
handling, and storage procedures; the analytic method and
vation stimulated several investigations on the metabolism of
measurement technique; the range, precision, accuracy, bias,
vinyl chloride in animals and on its mutagenic activity in in
and limits of detection; quality assurance issues; and known
vitro systems, leading to a better understanding of its mecha-
interferences. It is also important to document intralabora-
nism of carcinogenicity.
tory and interlaboratory variability. Once a standard method is
Studies of the metabolic handling of occupational toxi-
established, it must be closely followed in every detail in order
cants in animals are instrumental in the characterization of
to assure consistency of results.
reactive intermediates and may suggest unsuspected risks or
indicate new methods of biologic monitoring. Conversely,
clinical observations on workers may stimulate studies of the
metabolism or the mechanism of toxicity of a toxicant in ani- Biologic Monitoring for
mals, thereby revealing the health significance of a biologic Exposure Assessment
disturbance. Biomonitoring consists of the measurement of toxicants,
Arsenic is one of the very few compounds for which there are their metabolites, or molecular signatures of effect in speci-
limited data of predictive value from animal studies to human mens from humans or animals, including urine, blood, feces,
health effects. Inorganic arsenic has been shown conclusively exhaled breath, hair, fingernails or toenails, bronchial lavage,
to cause human cancers of many organs, but not to cause can- breast milk, and adipose tissue. These may serve as biomark-
cer in animals. This demonstrates that the occupational toxi- ers of exposure, biologic effect, or susceptibility. Emerging
cologist cannot rely solely on animal or epidemiologic studies. technologies will allow measurement and monitoring of
A combined approach is necessary in order to identify, elu- chemicals in the body and transmission of the data from
cidate, and prioritize risks and to develop interventions and indwelling biosensors. Biomonitoring data provide a measure-
techniques for worker health surveillance. ment of exposure based on internalized dose, or the amount
 CHAPTER 34 Occupational Toxicology 489

of chemical stored in one or in several body compartments or that are biotransformed through identical pathways or that
in the whole body, and, thus, account for all exposures by all modify the activity of the biotransformation enzymes.
routes for the assessed analyte. Furthermore, metabolic interferences may occur between
The term internalized dose may have different meanings. occupational toxicants and alcohol, tobacco, food additives,
The measured biomarker may reflect the amount of chemical prescription drugs, natural product remedies, or recreational
absorbed shortly before sample collection, as with the concen- drugs. Changes in any of several biologic variables (weight,
tration of a solvent in exhaled air or in a blood sample obtained body mass, pregnancy, diseases, immune status, etc.) may
during the work shift. It may reflect exposure during the pre- modify the metabolism of an occupational chemical. These
ceding day, as with the measurement of a metabolite in blood factors have to be taken into consideration when the results
or urine collected after the end of exposure. For toxicants with of biomonitoring are interpreted. Whatever the parameter
a long biologic half-life, the measured parameter may reflect measured, whether it is the substance itself, its metabolite, or
exposure accumulated over a period of weeks or months, as an early biomarker of effect, the test must be sufficiently sen-
with arsenic in toenails. Internal dose may refer to the amount sitive and specific to provide meaningful data in the range of
of chemical stored in one or in several body compartments or workplace exposures.
in the whole body (the body burden).
The greatest advantage of using biologic measurements
is that the biologic parameter of exposure is more directly
CONCLUSION
related to the adverse health effects than environmental mea- In summary, environmental and biologic monitoring should
surements. It may offer a better estimate of the risk than can be regarded as complementary elements in an occupational
be determined from ambient monitoring. Biologic monitoring health and safety program. The working environment will
accounts for uptake by all exposure routes. always present the risk of overexposure of workers to vari-
Several factors can influence uptake. Personal hygiene hab- ous toxicants. Recognition of these risks should not wait until
its vary from one person to another, and there is some degree epidemiologic studies have defined hazardous levels. A com-
of individual variation in the absorption rate of a chemi- bined experimental, clinical, and epidemiologic approach is
cal through the lungs, skin, or gastrointestinal tract. Because most effective for evaluating the potential risks, promulgat-
of its ability to encompass and evaluate the overall exposure ing scientifically based occupational health standards, and
(whatever the route of entry), biologic monitoring also can be implementing workplace controls to ensure adherence to the
used to test the overall efficacy of personal protective equip- standards.
ment such as respirators, gloves, or barrier creams. Another
consideration with biologic monitoring is the fact that the
nonoccupational exposures (hobbies, residential exposures, BIBLIOGRAPHY
dietary habits, smoking, and second jobs) also may be expressed ACGIH: 2014 TLVs and BEIs: Threshold Limit Values for Chemical
in the biologic sample. Substances and Physical Agents and Biological Exposure Indices.
Relationships between air monitoring and biologic moni- Cincinnati, OH: American Conference of Governmental Industrial
toring may be modified by factors that influence the fate of Hygienists, 2014.
an occupational toxicant in vivo. Metabolic interactions can LaDou J, Harrison R: Current Occupational and Environmental
occur when workers are exposed simultaneously to chemicals Medicine, 5th ed. New York: McGraw-Hill, 2014.
490 UNIT 7 Applications of Toxicology

QUESTIONS

1. Which ofthe following is NOT a modifying factor that can Lyme disease is caused by which of the following?
influence the likelihood ofdisease? B. burgdorferi.
a. age. H. capsulatum.
b. dose. M. tuberculosis.
c. nutritional status. L. pneumophila.
d. gender. om
te
eS
gd C. psittaci.
e. genetic susceptibility.
Asbestos exposure is unlikely to cause:
2. Which of the following is LEAST likely to increase a. lung cancer.
occupational inhalation of a chemical? b. Gl cancer.
increased airborne concentration. c. emphysema.
increased respiratory rate. d. pulmonary fibrosis.
increased tidal volume. e. mesothelioma.
increased particle size.
eoincreased length of exposure.
SS Exposure to which of the following can cause autoimmune
disease?
3. Which would increase the likelihood of toxic dosage a. mercury.
through dermal exposure? b. nitrogen dioxide.
no preexisting skin disease. c. vinyl chloride.
toxic exposure to thick skin. d. lead. an
increased percutaneous absorption rate. e. flavivirus.
low surface area of exposure.
oe high epidermal intercellular junction integrity.
pao Which of the following might be linked to parkinsonism?
a. nitrogen dioxide.
4. Prolonged arsenic exposure could cause: b. zinc.
a. infertility. c. copper.
b. cirrhosis. d. magnesium.
c. cor pulmonale. e. carbon monoxide.
d. skin cancer.
e. nephropathy. 10. Which of the following infectious agents can cause
hepatocellular carcinoma?
5. Which of the following lung diseases has the highest flavivirus.
occupational death rate? bunyavirus.
a. asbestosis. alphavirus.
b. coal workers’ pneumoconiosis. hepatitis C virus.
c. _byssinosis. are hepatitis B virus.
One
Oss
OE
d. hypersensitivity pneumonitis.
e. silicosis.
 49]
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Index

NOTE: Pages in boldface refer to major discussions; page numbers followed by f indicate figures; those followed by t indicate tables.

1,1,2-trichloroethylene (TCE), 365-366 Acetylcholine M, muscarinic receptor, 31t

1,2-dihydroxyethane, 369 Acetylcholine M,; muscarinic receptor, 32t
1,3-butadiene, 288 Acetylcholine nicotinic receptor, 31
1,3-dichloropropene, 344-345 Acetylcholinesterase (AChE), 82, 336, 336t, 346, 444
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 242, Acetylethyltetramethyl tetralin (AETT), 248¢
DAS ZOOS AChE (acetylcholinesterase), 82, 336, 336t, 346, 444
2,3-bisphosphoglycerate (2,3-BPG), 167 Acid, 64
2,4-dichlorophenoxyacetic acid (2,4-D), 341-342 Acinar zonation, 196
2-mercaptobenzothiazole, 321 Acinus, 196
2-PAM, 337 ACM (alcoholic cardiomyopathy), 279
2-year chronic bioassay, 131 Acne, 299, 302
3-nitropropionic acid, 243t, 460t Acquired immunity, 178, 183-184, 188
3'-phosphoadenosine-5'phosphosulfate (PAPS), 97f, 99 Acrolein, 232t, 438
5-fluorouracil, 280t Acrylamide, 132, 245, 246t, 247, 267
5-HT, receptor, 32t ACTH (adrenocorticotropic hormone), 320, 321, 323
6-amino-nicotinamide, 243t Action potential, 273, 274f
21-hydroxylase deficiency, 331 Activated partial thromboplastin time (aPTT), 172, 173
Active transport, 64, 454t
ACTOR, 57
Acute cardiac toxicity, 272
A Acute exposure, 9
A-esterases, 336 Acute kidney injury (AKI), 212, 213t
AA (aristolochic acid), 220 Acute lung injury, 230
Abamectin, 340 Acute lymphoblastic leukemia (ALL), 170
ABC transporters/subfamilies, 64, 65t Acute myelogenous leukemia (AML), 170, 171, 367
Abortifacients, 389 Acute-phase proteins, 41, 183
Absorptiometry, 404 Acute renal failure, 212
Absorption, 22, 65-70 Acute toxicity testing, 16
defined, 65 Acyl-CoA thioesters, 97f
GI tract, 66-68 Adaptation, 41
lungs, 68-69 Adaptive response, 377
skin, 69-70 Addition reactions, 104
special routes of administration, 70 Additive effect, 8
Acacia tree, 388f Additives amendments (Food, Drug, and Cosmetic Act), 3
Acceptable daily intake (ADI), 54 Adenoma, 122
ACE inhibitors, 151, 215 ADH (alcohol dehydrogenase), 84, 368
Acetaldehyde, 203, 437 ADH (antidiuretic hormone), 284
Acetaldehyde dehydrogenase (ALDH), 368 ADI (acceptable daily intake), 54
Acetaminophen (APAP), 203, 220 ADM (anti-Miillerian hormone), 305, 318
Acetaminophen-inducéd mitochondrial oxidant stress, 203, 204f ADME, 80
Acetaminophen poisoning, 476 ADME- Tox, 80
Acetyl-coenzyme A (acetyl-CoA), 97f, 99 ADMET, 80
Acetylation, 97f, 99-102 Adrenal cortex, 322-324
Acetylcholine M, muscarinic receptor, 32t Adrenal glands, 321-322
496 INDEX

Adrenal medulla, 324 ALDH (aldehyde dehydrogenase), 84

Adrenal toxicity, 331 Aldo-keto- reductase (AKR), 83
Adrenergic receptor, 324 Aldosterone, 284
Adrenergic a, receptor, 32t Aldrin, 340f
Adrenergic 8, receptor, 32t Alga, 388t
Adrenocortical hormone pathway, 322f Aliphatic carbon hydroxylation, 88f
Adrenocortical toxicity, 323 Alkali, 263
Adrenocorticotropic hormone (ACTH), 320, 321, 323 Alkaline phosphatase, 83
Adriamycin, 244, 279 Alkylating agents, 126, 132
Adverse effects, 7, 428 Alkylating electrophiles, 124-125
AEDs (antiepileptic drugs), 151 ALL (acute lymphoblastic leukemia), 170, 171
Aerosols and particles, 68-69 Allergen, 178
AETT, 248t Allergenic food proteins, 457t
AFC assay, 187 Allergic contact dermatitis, 294-296, 383, 399
Afferent arteriole, 210, 211f Allergic idiosyncratic hepatotoxicity, 207t
Aflatoxins, 131t, 460¢ Allergic reactions, 8
Age of Enlightenment, 2 Allergic rhinitis, 384-385, 483
Agelenopsis species (American funnel web spiders), 392 Allometric scaling, 12t
Agranulocytosis, 170 Allopurinol, 84
Agricola, Georgius, 2 Alloxan, 329
AhR (aryl hydrocarbon receptor), 80, 96f, 124t, 443 Allyl alcohol, 203-204
AIDS therapeutics, 191 Alpha particles, 373, 374, 380
Air displacement plesmography, 404 Alpha,-antiprotease, 231
Air pollution, 425-439 Alpha,-antitrypsin, 231 .
acrolein, 438 a,,-globulin nephropathy, 219
adverse health effect, 428 Alternate pathway, 179, 180f
aldehydes, 437 Aluminosis, 232t
animal toxicology, 428 Aluminum, 243¢, 356t, 357-358
building-related illnesses, 431 Aluminum abrasives, 232t
carbon monoxide, 438 Aluminum dust, 232t
epidemiologic evidence of health effects, 431-432 Alveolar clearance, 230
formaldehyde, 437-438 Alveolar duct, 225f 226f
hazardous air pollutants (HAPs), 438 Alveolar epithelium, 226
historical overview, 426 Alveolar macrophages, 181, 191, 194
international considerations, 426-427, 427f Alveolar sac, 225f
IRIS, 426 Alveolar type I cells, 226
nitrogen dioxide, 437 Alveolar type II cells, 226, 228
ozone, 435, 436-437 Alveolus, 227f
PAN, 436, 437 Alzheimer’s disease, 242, 287, 358, 398
particulate matter (PM), 434-435 Amanita muscaria (fly agaric), 388f 388t
photochemical, 435-436 Amanita phalloides (death cap), 387f
reducing-type, 432-433 Amaryllis, 385¢
risk assessment, 427-428, 427f American Conference of Governmental Industrial
sick-building syndrome, 430, 431t Hygienists, 483
smog, 436 American funnel web spiders, 392
sources and personal exposure, 429-431 Ames assay, 130, 142, 147
susceptibility and vulnerability, 429, 429 Amides, 126
Airway microdissection, 235 Amino acid conjugation, 97f, 102, 103f
AKI (acute kidney injury), 212, 213 Aminoglutethimide, 312
AKR (aldo-keto- reductase), 83 Aminoglycosides, 220, 280t
AKR superfamily, 84 Amiodarone, 248t
Akylation, 138f AML (acute myelogenous leukemia), 170, 171, 367
Alcohol consumption, 131¢, 132 Ammonia, 232¢, 248-249, 296t
Alcohol dehydrogenase (ADH), 84, 368 Amphetamines, 250
Alcohol-tobacco amblyopia, 267 Amphotericin B, 220, 281t
Alcoholic cardiomyopathy (ACM), 279 Anabasine, 389
Alcoholism, 369 Anabolic-androgenic steroids, 289
Alcohols, 368-369 Analytical and forensic toxicology, 463-470
Aldehyde dehydrogenase (ALDH), 84 analytical toxicology, 463-464
Aldehyde oxidase, 84 courtroom testimony, 467
Aldehydes, 437, 439 definitions, 463
ALDH (acetaldehyde dehydrogenase), 368 drug testing, 467
 INDEX 497

Analytical and forensic toxicology (continued) Antigen-antibody interaction, 8

human performance testing, 467 Antigen-presenting cell (APC), 179, 183
investigation of poison death, 465-466 Antigen recognition, 178-180
living victims of poisoning, 466-467 Antihistamines, 281t
role in clinical toxicology, 467-468 Antimony, 356¢
role of forensic toxicologist, 464 Antineoplastic drugs, 280t, 287
sexual assault, 466, 466t Antipsychotic drugs, 281¢
therapeutic monitoring, 468, 468t Antiseptics, 297¢
Anaphylactoid reactions to food, 458, 458t Antivenom, 397-398
Anatomical parameters, 114 Antiviral drugs, 281t
Androgens, 191, 282t Ants, 394
Anemia, 164-169, 175 Aorta, 273f, 283
Anesthetics, 281t AP site, 137
Aneuploidy, 136, 141t, 144 APAP-induced hepatotoxicity, 203, 204f
Angiogenesis, 285 APC (antigen-presenting cell), 179, 183
Angiotensin, 284 Apidae (bees), 394
Angiotensin converting enzyme (ACE) inhibitors, 151, 215 Aplastic anemia, 166, 166t
Angiotensin receptor blockers, 151 Apoptosis, 36-38
Animal bioassay, 52, 131 active deletion of damaged cells, 39
Animal toxicology, 428 cell cycle arrest, 30f
Animals and animal venoms, 390-398 chapter-ending question, 47, 208
antivenom, 397-398 developmental toxicity, 153, 154f
arachnida, 391-393 failure of, 45
arthropods, 391 liver, 199
bioavailability of a venom, 391 myocardial cell loss, 277
chilopoda (centipedes), 393-394 Apparent volume of distribution (Vd), 111-112
clinical applications of venoms, 398 Appetite suppressants, 280t, 409
diplopodia (millipedes), 394 Aprotinin, 173
hypersensitivity reactions, 397-398 aPTT (activated partial thromboplastin time), 172, 173
insecta, 394 Aquatic toxicology, 442, 447, 451
lizards, 395 Aqueous humor, 257f
mollusca (cone snails), 394-395 Arachnida, 391-393
properties of animal toxins, 390-391 Aralen, 266
reptiles, 395-397 Arc welder’s lung, 233t
scorpions, 391, 391t Archiv fiir Toxikologie, 3
snakes, 395-397 Aristolochia, 220
spiders, 391-393 Aristolochic acid (AA), 220
ticks, 393 Aromatic amines, 126
Anion gap, 473, 479 Aromatic carbon hydroxylation, 88f
ANP (atrial natriuretic peptide), 277, 284 Aromatic hydrocarbons, 288, 367-368
Answers to end-of-chapter questions, 491-493 Arrhenius’ theory, 63
Antagonism, 8 Arrhythmia, 277, 289
Anthracyclines, 279, 280¢ Arsenic, 350
Anthropometric analysis, 403-404 chapter-ending question, 359
Anti-inflammatory agents, 191 lung injury, 232t
Anti-Miullerian hormone (ADM), 305, 318 neuronal injury, 243¢
Anti-sRBC ELISA, 188, 189 predictive value from animal studies, 488
Anti-sRBC IgM, 178 skin cancer, 301
Antiandrogens, 156 toxicity, 356t
Antiarrhythmic drugs, 280t Arsine, 350
Antibacterial drugs, 280t Arterioles, 284
Antibiotics, 172 Arthropods, 391
Antibodies, 178-179 Aryl hydrocarbon receptor (AhR), 80, 96t, 124, 443
Antibody inhibition, 89 Aryldialkylphosphatase, 82
Anticholinergic toxic syndrome, 472t, 479 Asbestos, 232t
Anticoagulants, 172-173, 344, 387 Asbestosis, 232t, 483
Antidiuretic hormone (ADH), 284 Ascending aorta, 283
Antidotes, 475 Aspergillus, 232t
Antiepileptic drugs (AEDs), 151 Aspirin, 287
Antifibrinolytics, 173 Assessing toxicity of chemicals, 51-53
Antifungal drugs, 281 Assignment of concern level, 455, 455t, 456t
Antigen, 8, 178 Asthenic-vegetative syndrome, 354
498 INDEX

Asthma, 231, 234, 483 §-N-methylamino-L-alanine (BMAA), 249¢

Astrocytes, 72, 248-249 §-N-oxalylamino-L-alanine (BOAA), 249t
AT MUD PILES, 473t Beta particle decay, 373
Atherosclerosis, 286, 289 8,B8-iminodipropionitrile (IDPN), 244-245, 246t¢
ATP-binding cassette (ABC) transporters, 64, 65t Betel chewing, 131t
ATP-dependent membrane transporters, 24 Betel nut, 388¢
ATP depletion, 33-34 Bifunctional electrophiles, 39
ATP synthesis, 33f 34t Bile duct, 197f
Atrial natriuretic peptide (ANP), 277, 284 Bile duct cells, 202t
Atrioventricular node, 273f Bile duct damage, 1992, 200
Atropa belladonna (deadly nightshade), 389f Bile formation, 197-198
Atropine, 2491, 336, 475 Bile salt exporter pump (BSEP), 65¢, 74f, 200
Autoimmunity, 186-187, 192 Biliary excretion, 74-75, 197
Automotive gasoline and additives, 371 Binding occupational exposure limit value (BOELV), 483
Autophagosome, 39 Bioavailability, 112-113, 442, 443, 451
Autophagy, 39, 276 Bioelectrical impedance analysis, 404
Avermectins, 340-341 Bioelectricity, 273
Avian protein, 232t Bioinformatics, 17, 18f
Axon regeneration, 39 Biologic availability, 442, 451
Axonal degeneration, 240 Biologic exposure index (BEI), 483
Axonal transport, 239-240, 253 Biological extrapolation, 53
Axonopathy/axonopathies, 240, 244-247, 253 Biological membrane, 63, 63f
Azalea, 386t Biologically based dose-response (BBDR) modeling, 56
Azathioprine, 132t Biologics, 191 . \
Azide, 243t Biomagnification, 443
Azinphos-methly (Guthion), 337f Biomarkers
Azo-reduction, 83 cardiac toxicity, 278, 279t
ecotoxicology, 448
kidney, 217f
metal exposure, 349
molecular epidemiology, 53
B cells, 184 Biomonitoring, 488-489
B-esterases, 336 Biosphere, 442. See also Ecotoxicology
B-type natriuretic peptide (BNP), 279t Biotransformation of xenobiotics, 79-107
Bacillus cereus, 461 conjugation. See Conjugation
Bacterial endotoxins, 287 defined, 80
Bacterial forward mutation assay, 141f, 142 general principles, 80-82
Bacterial reverse mutation assay, 141¢ hydrolysis, 81f, 82-83
Bagassosis, 233t, 483 overview, 81f
Band of Bungner, 240 oxidation. See Oxidation
Barberry, 385t reduction, 81f, 83-84
Base, 64, 263 respiratory system, 228
Base excision repair, 137 skin, 294
Base substitution, 139 Bipyridyl compounds, 342
Basophils, 169 Bird fancier’s lung, 232t
Bauxite lung, 232t Bismuth, 243, 356¢
BBB (blood-brain barrier), 72, 238-239, 238f Bisphenol A, 14, 327
BBDR (biologically based dose-response) modeling, 56 Black carbon, 434
BCRP (breast cancer resistance protein), 65t, 74f Black widow spider, 392f
BCSEB (blood-cerebrospinal fluid barrier), 72 Bladder, 307f
Beaded lizards, 395 Blastocyst, 152
Becquerel (Bq), 374 Blockers, 9
Bees, 394 Blocking agents, 129
BEI (biologic exposure index), 483 Blood, 163-176
Benchmark dose (BMD), 55 anemia, 164—169
Benchmark dose software, 160 anticoagulants, 172-173
Benchmark response (BMR), 55 erythrocytes, 164-165, 168-169
Benzene, 367, 372 fibrin clot formation, 172
Bernard, Claude, 2 granulocytes, 169-170
Beryllium, 232t, 356t hematopoiesis, 164
Berylliosis, 232t heme and hemoglobin synthesis, 165f
6-amyloid, 287 hemoglobin, 166-169
 INDEX 499

Blood (continued) Butterflies, 394

hemoglobin-oxygen dissociation curve, 167f Butyrylcholinesterase, 82
homeostasis, 171-173 Byssinosis, 232t
leukemia, 170-171 Bystander effects, 376-377
leukon, 169
platelets, 171-172
primary/secondary toxicity, 164
problem-driven tests, 174, 174t C
risk assessment, 173-174 C. perfringens food poisoning, 461
thrombocytopenia, 171-172 c-Myc protein, 29
toxic neutropenia, 170 C-reactive protein (CRP), 279t
Blood-brain barrier (BBB), 72, 238-239, 238f Ca**, 34-35, 218
Blood-cerebrospinal fluid barrier (BCSFB), 72 Cadherins, 40
Blood compartment, 117-118 Cadmium, 219, 232t, 320, 350-352, 356¢
Blood flow-limited compartment, 115 Cadmium hepatotoxicity, 202
Blood-testis barrier, 309 Calcium channel blockers, 172
Blood-to-gas partition coefficient, 68 Calcium oxide (CaO), 296t
Blue sac disease, 445 Caloric content of foods, 403
BMAA, 249t Caloric intake, 403. See also Food and nutrition
BMD (benchmark dose), 55 Caloric restriction (CR), 408
BMI (body mass index), 404, 408t, 409, 410 Canalicular cholestasis, 199-200, 199t, 208
BMR (benchmark response), 55 Canalicular lumen, 197
BNP (B-type natriuretic peptide), 279t Cancer
BOAA (8-N-oxalylamino-L-alanine), 249t defined, 122
Body burden, 489 development of. See Chemical carcinogenesis
Body composition, 403-404, 410 ecotoxicology, 444, 445
Body fat, 72 food toxicology, 457
Body mass index (BMI), 404, 408¢, 409, 410 genetic toxicology, 136
Body systems/organs. See Target organ toxicity hepatocellular, 202
BOELYV (binding occupational exposure limit value), 483 hit models, 55-56
Bone, 72 kidney, 366
Bone marrow, 164 liver, 366
Book of Job, 1 lung, 132, 231, 366, 435
Botulinum toxin, 7t obesity, 408
Botulism, 461 ocular and visual system, 266
Bovine spongiform encephalopathy (BSE), 461 occupational toxicology, 485t
Bowman's capsule, 211f pancreatic cancer, 132
Bowman's membrane, 256, 257f radiation and radioactive materials, 377-379
Bowman’ space, 211f skin, 301
Boxwood, 385t types of neoplasms, 122
BPIF2, 226 Cancer bioassay, 52
Brain capillaries, 24 Cancer chemotherapeutics, 266
BRCAI, 129, 129t Cancer risk assessment, 136
Breast cancer resistance protein (BCRP), 65t, 74f Capillaries, 284
Brevetoxins, 459 Capillary endothelium, 23
Bromobenzene, 219 Captan, 343, 346
Bronchi, 225f CAR (constitutive androstane receptor), 80, 96f, 124t, 126
Bronchiolar secretoglobin cell (BSC), 226, 228 Carbamates, 338
Bronchiole-alveolar duct junction, 226f Carbon disulfide (CS,), 244-245, 246f, 268, 288, 371
Bronchoconstriction, 230 Carbon monoxide (CO), 168, 243¢, 287, 438, 439
Bronchodilators, 280t Carbon nanotubes (CNTs), 412, 420
Bronsted-Lowry acid-base theory, 64 Carbon tetrachloride (CCl,), 204, 243t, 366-367
Brown recluse spider, 392, 393f Carbonyl reduction, 83
BSC (bronchiolar secretoglobin cell), 226, 228 Carboxylesterases, 82-83
BSE (bovine spongiform encephalopathy), 461 Carboxylic acid group, 102, 103f
BSEP (bile salt exporter pump), 65t, 74f, 200 Carcinogenesis, 43-45, 47. See also Chemical carcinogenesis
Buckthorn, 388t Carcinoma, 122
Building-related illnesses, 431 Cardiac arrhythmia, 277, 399
Bulky DNA adducts, 137 Cardiac glycosides, 280f, 289, 382, 386
Bull’s-eye retina, 266 Cardiac hypertrophy, 272, 276-277, 278
Busulfan, 308 Cardiac myocytes, 273
Buttercup, 385t Cardiac output, 275
500 INDEX

Cardiac remodeling, 277 testing for carcinogenicity, 130-131

Cardiac troponins, 279t tumor-suppressor genes, 1281, 129
Cardiomyopathy, 272 Chemical clearance, 112, 119
Cardiovascular toxicology, 272. See also Heart; Vascular system Chemical idiosyncrasy, 8
Case-control study, 52, 53¢ Chemical inactivation, 9
Cassava, 387 Chemical inhibition, 89
Cataracts, 265, 379 Chemodynamics, 442, 451
Catechol O-methyltransferase (COMT), 99 Chemokines, 182
Catecholamines, 280t, 324 Chemoprevention, 129-130
Caterpillars, 394 Chemotherapeutics, 266
CCl, (carbon tetrachloride), 204, 243t, 366-367 Chernoft/Kavlock assay, 159t
cDNA microarray technology, 146 Chick embryo neural retina cell culture, 159¢
Cell cycle accelerators/decelerators, 44f Chilopoda (centipedes), 393-394
Cell cycle arrest apoptosis, 30f Chinese hamster ovary (CHO) test, 130
Cell cycle progression mitosis, 30f Chloracne, 299
Cell-mediated immunity (CMI), 184 Chloramphenicol, 132¢, 243t, 281t
Cell membrane, 63, 63f Chlordane, 340f
Cellular dysfunction and resultant toxicities, 27-38 Chlordecone, 246¢, 340
Cellular repair, 39 Chlorinated hydrocarbons, 365-367, 372, 445
Centipedes, 393-394 Chlorine, 232t, 296t
Central visual system, 261, 268 Chloroacetanilides, 342
Cerebrospinal fluid (CSF), 75 Chloroform (CHCI,), 219, 367
Ceruloplasmin, 349 Chlorophenoxy herbicides, 341-342
CGH (comparative genomic hybridization), 146 Chloroquine, 246t, 266 oN
Chapter questions, answers, 491-493 Chlorpromazine, 287
Characterization of risk, 50 Chlorpyrifos, 337f
CHCI, (chloroform), 219, 367 CHO test, 130
Cheiracanthium species (running spiders), 393 Cholangiodestructive cholestasis, 200
Chelation, 347 Cholehepatic shunting, 199
Chelation therapy, 356 Cholestasis, 199-200, 199t
Chelonitoxin, 460 Cholinergic toxic syndrome, 472t
Chemical allergy, 8 Cholinesterases, 83
Chemical antagonism, 9 Choriocapillaris, 256
Chemical burns, 294, 296t Choroid, 257f
Chemical carcinogenesis, 43-45, 47, 121-134 Chromaffin cells, 324, 331
alkylating electrophiles, 124-125 Chromatin, 38
chemoprevention, 129-130 Chromium, 232t, 356t
classification of carcinogenic agents, 133 Chromosomal alterations, 130
definitions, 122 Chromosome painting, 143
DNA methylation, 127 Chronic (2-year) bioassay, 131
DNA repair, 125-126 Chronic bronchitis, 230
DNA virus, 128 Chronic cardiac toxicity, 272
gap junctional intercellular communication, 127 Chronic exposure, 9, 113
genotoxic carcinogens, 122¢, 124, 126 Chronic exposure study, 17
hormesis, 129 Chronic kidney disease, 214
humans, carcinogenesis in, 131-133 Chronic lymphocytic leukemia (CLL), 170
initiation, 123, 123t Chronic myelogenous leukemia (CML), 170, 171
inorganic carcinogens, 126 Chronic obstructive pulmonary disease (COPD), 230-231
mechanisms of action, 124-130 Chronic pulmonary disease, 351
modifiers of carcinogenic effects, 127 Chronic renal failure, 222
multistage model, 122-124, 123f Chronic solvent encephalopathy (CSE), 363
mutagenesis, 124 Chrysanthemum, 388t
nongenotoxic carcinogens, 122t, 126-127 Cigarette smoking, 131t, 132, 151. See also Nicotine
occupational human carcinogens, 132t Ciguatera, 459
oncogenes, 128, 128¢ Ciliary body, 257f
oxidative stress, 127 Ciliary epithelium, 256
polymorphisms, 128 Ciliated cells, 225-226
progression, 123-124, 123t Circulating pool, 169
promotion, 123, 123¢ Cirrhosis, 199¢, 201-202
proto-oncogenes, 128, 128t Cisapride, 282t
retrovirus, 128 Cisplatin, 221, 246t, 253, 358
 INDEX 501

Citreoviridin, 460t Compartments

Citrinin, 460t classical toxicokinetics, 110-111
Citrinin nephrotoxicity, 219 physiologic toxicokinetics, 114, 115-118
CK-BB, 279t Complement cascade, 180f
CK-MB, 279t Complement system, 179, 180f
CK-MM, 279t Complete blood count (CBC), 165
Clara cell, 226, 366 Complete carcinogen, 134
Classical pathway, 179, 180f Complex I, 340
Classical toxicokinetics, 110-113 Complexation, 347
Classification of toxic agents, 7 Composite lung, 232t
Clearance, 112, 119 Composting facilities, 483
Cleft lip/palate, 155 Compound 1080, 344
Clinical chemistry calculations, 17 Compton effect, 373, 380
Clinical toxicology, 471-479 Computational biology, 193
antidotes, 475 COMT (catechol O-methyltransferase), 99
case example (acetaminophen poisoning), 476 Concentric cardiac hypertrophy, 272
case example (ethylene glycol poisoning), 476-477 Conducting airways, 224-225, 236
case example (valproic acid poisoning), 477-478 Cone snails, 394-395
enhancement of poison elimination, 474-475 Confounding, 158
history taking, 472 Conjugated bile acids, 197
laboratory evaluation, 473 Conjugation, 25, 95-106
odors, 472, 472t acetylation, 97f, 99-102
physical examination, 472 amino acid, 97f, 102, 103f
poison control center, 471 glucuronidation, 96-99
prevention of further poison absorption, 474 glutathione, 97f, 102-105, 106f
radiographic evaluation, 473-474 methylation, 97f, 99
stabilization of patient, 472 overview, 97f
steps in clinical strategy, 471-472 sulfonation, 97f 99, 100t
supportive care of poisoned patient, 475 Conjunctiva, 257f
toxic syndromes, 472, 472t Constitutive androstane receptor (CAR), 80, 96t, 124t, 126
Clioquinol, 246t Contact dermatitis, 294-296, 355, 383, 384t
CLL (chronic lymphocytic leukemia), 170 Contact urticaria, 300, 300t
Clopidogrel, 172 Contraceptives, 287
Clostridial infections, 168 Contractility, 274, 289
Clot formation, 172 Contrast sensitivity, 263
Clover disease, 313 Cooper sulfate, 344
CMI (cell-mediated immunity), 184 Cooxidation, 85
CML (chronic myelogenous leukemia), 170, 171 COPD (chronic obstructive pulmonary disease), 230-231
CNT (carbon nanotube), 412, 420 Copeland bill (1938), 3
CO (carbon monoxide), 168, 243t, 287, 438, 439 Copper, 355-356, 356t
Coagulation, 172 Cornea, 256, 257f, 264-265
Coal dust, 232t Corneal endothelium, 256, 257f
Coal worker’s pneumoconiosis, 232¢, 483 Corneal epithelium, 256, 257f
Cobalt, 356t Corneal stroma, 256, 257f
Cocaine Corneocytes, 293
cardiotoxicity, 281t, 286 Corpus luteum, 311, 318
chapter-ending question, 289 Correlation analysis, 89
developmental toxicology, 151, 252 Corticosteroids, 265
neurotransmitter-associated toxicity, 249f, 250 Corundum smelter’s lung, 232¢
pregnancy, 252 Cotton dust, 232t
Cohort study, 52, 53t Cough reflex, 385
Colchicine, 246t, 247, 399 Coumarin derivatives, 344
Collagen, 234 Covalent binding, 26-27
Collecting duct, 211f 212 COX-1/COX-2 enzymes, 191
Collecting duct injury, 216 Coyotillo, 388¢
Collectins, 226 - Creatine kinase, 279t
Color vision, 263, 269 Creatinine, 216
Comet assay, 140 Criminal poisoning. See Analytical and forensic toxicology
Community ecotoxicology, 447 Cross-reacting chemicals, 298t
Comparative genomic hybridization (CGH), 146 Cross-sectional study, 52, 53¢
Comparative Toxicogenomics Database, 57 Cross-sensitivity, 295
502 INDEX

Crown of thorns, 385t Dehalogenation, 84

CRP (C-reactive protein), 279t Dehydrogenation, 92f
Crystalline silica, 193 Dehyrdrohalogenation, 84
CS, (carbon disulfide), 244-245, 246t, 268, 288, 371 Delaney amendment, 3, 457
CS syndrome, 339, 339t Delayed anovulatory syndrome, 312
CSE (chronic solvent encephalopathy), 363 Delayed hypersensitivity response (DHR), 188, 194
CSF (cerebrospinal fluid), 75 Delayed toxic effects, 8
CTL (cytotoxic T lymphocyte), 179, 184, 184f Deleterious effects, 7
CTL assay, 188 Demyelination, 241, 248
Cuprizone, 248¢ Dendritic cell (DC), 179
Curare, 389 Depigmentation, 300, 300t
Curie (Ci), 374 Dermal absorption, 69-70
Cyanamide, 321 Dermal irritation test (Draize test), 16
Cyanide, 243t Dermis, 69, 70f, 293f
Cyanide poisoning, 475, 479 DES (diethylstilbestrol), 126, 132t, 150, 312
Cyanogens, 387 Descemet’s membrane, 256, 257f
Cyclodienes, 339-340 Descriptive animal toxicity tests
Cyclophosphamide, 132t, 280t, 287 acute toxicity testing, 16
Cyclopiazonic acid, 460t long-term (chronic) exposure study, 17
Cyclosporine, 220 multistage animal models, 131
CYP induction, 92, 95, 96t other tests, 17
CYP inhibition, 92 sensitization, 16 ‘
CYP monooxygenase system, 227 skin and eye irritations, 16
CYP system. See Cytochrome P450 (CYP) system subacute tests, 16 ne
CYP2E1, 203, 204, 363, 364, 368 subchronic study, 16-17
Cysteine, 97f, 102 underlying principles, 16
Cytochalasins, 460t Descriptive toxicologist, 6
Cytochrome c (cyt c), 35, 46 Detoxication, 25-26
Cytochrome P450 induction, 92, 95, 96t Deutan, 263
Cytochrome P450 inhibition, 92 Development of toxicity. See Mechanisms of toxicity
Cytochrome P450 (CYP) system, 86-95, 228 Developmental immunology, 187
activation of xenobiotics, 91, 95t Developmental programming, 153
catalytic cycle of cytochrome P450, 87f Developmental toxicology, 6, 149-161
catalyzation, 87, 88f, 89f 90f 91f, 92f critical points of susceptibility, 152-153
inducers, 93-94t defined, 149
induction of cytochrome P450, 92, 95, 96t dose-response patterns, 153
inhibition of cytochrome P450, 92 endocrine-disrupting chemicals, 156-157
inhibitors, 93-94t epidemiology, 157-159
substrates, 93-94t future directions, 160
Cytokines, 182, 182, 282t human developmental toxicants, 150t
Cytometry, 193 intercellular signaling pathways, 160, 160t
Cytotoxic T lymphocyte (CTL), 179, 184, 184f maternal factors, 154-156
Cytotoxic T lymphocyte (CTL) assay, 188 mechanisms and pathogenesis, 153-154
Cytotoxicity, 124f, 126 pregnancy, 154
safety assessment, 157-160
scope of problem, 150-151
threshold concept, 153
D in vivo testing, 157, 158t
Daffodil, 385t Wilson’s principles of teratology, 151t
Danazol, 312 Developmentally neurotoxic chemicals, 252
Danger hypothesis, 205, 206f DHR (delayed hypersensitivity response), 188, 194
Data-based models, 113 Diabetes mellitus, 329, 329f
DBD (DNA-binding domain), 95 Dialysis dementia, 357
DC (dendritic cell), 179 Dialysis technique, 474
DDT, 339, 340, 346 Diazepam, 337
De Historia Plantarum (Theophrastus), 2 Diazinon, 337f
De Materia Medica (Dioscorides), 2 Dichloromethane, 366
Deadly nightshade, 388¢, 389f Dichlorophenoxyacetate, 246t
Death camus, 386t Dichlorovos, 337f
Death cap, 387f Dieldrin, 340f
DEET, 341 Diesel particles, 434
 INDEX 503

Diet, 1314, 132 flat/steep dose-response curve, 12

Dietary restriction, 408 graded, 10
Dietary supplements, 457 hormesis, 13
Diethylpropion, 409 individual, 10
Diethylstilbestrol (DES), 126, 132, 150, 312 nonmonotonic dose-response curve, 14
Dieting, 401, 408-409 quantal, 10-13
Diffusion, 229, 229f shape of dose-response curve, 13-14
Diffusion-limited compartments, 116, 119 sigmoid dose-response curve, 12
Diffusivity, 70 slope, 14
Diflunisal, 156 therapeutic index, 14-15
Digitalis-induced visual system abnormalities, 266 threshold, 13-14
Digitalis purpurea (common foxglove), 386 Dosimetrics, 417-418
Digitoxin, 266 Double dehalogenation, 84
Digoxin, 266 Double-strand break, 138f
Dihydrodiol dehydrogenase, 84 Double-strand break repair, 137-138
Dimethylaminopropionitrile, 246t Doxorubicin, 243t, 244
Dimethylmercury poisoning, 75 Draize test, 16, 262
Dioscorides, 2 Driving under the influence (DUI), 467, 470
Dioxin (TCDD), 7t Drosophila assay, 141t, 159t
Diplopodia (millipedes), 394 Drug-induced autoantibody, 169
Diquat, 342 Drug-induced QT prolongation, 278
Direct-acting carcinogens, 124 Drug-induced steatosis, 201
Direct repair (DNA), 38 Drug metabolism, 80. See also Biotransformation of xenobiotics
“Dirty Dozen,’ 340 Drugs of abuse, 190, 191
Discourse on the Diseases of Workers (Ramazzini), 2 DT-diaphorase, 83
Displacement reactions, 104 DUI (driving under the influence), 467, 470
Dispositional antagonism, 9 Dutanopes, 263
Dispositional tolerance, 9 Dynein, 200
Distal tubule, 212 Dysregulation of gene expression, 28-29
Distal tubule injury, 216 Dysregulation of signal transduction, 29
Distribution
blood-brain barrier (BBB), 72
placental transfer, 72-73
rate of, 71 E
storage of toxicants, 71-72 E. coli, 461
Vd, 71 Early afterdepolarization, 278
Disulfide reduction, 83 Ebers Papyrus, 1
Disulfiram, 248¢ ECs 447
Dithiocarbamate fungicides, 343f Eccentric cardiac hypertrophy, 272
DNA-binding domain (DBD), 95 ECG (electrocardiogram), 275, 275f
DNA cytosine methyltransferase (DNMT), 45 Ecologic community, 446
DNA damage, 130, 137, 138f 147, 154f 444 Ecological risk assessment (ERA), 449
DNA damage and repair assays, 140, 141t Ecotoxicology, 6, 441-451
DNA-dependent protein kinase (DNA-PK), 138 biomagnification, 443
DNA hydroxylation, 125 biomarkers, 448
DNA methylation, 125, 127 cancer, 444, 445
DNA-PK, 138 cellular, tissue, and organ effects, 444
DNA repair, 38-39, 47, 125-126, 134, 137-139, 147 community, 446-447, 448-449
DNA virus, 128 defined, 442
DNMT (DNA cytosine methyltransferase), 45 ecological scales, 442f
Domoic acid, 249#, 251, 460 free ion activity model (FIAM), 443
Dopamine, 250 gene expression and ecotoxicogenomics, 443-444
Dose, 482 interconnections between ecosystem and human health, 449-450
Dose-response assessment, 53-56 molecular and biochemical effects, 443
Dose-response curve, 54f organismal effects, 444-445
Dose-response models, 55-56 population, 445-446, 448
Dose-response relationship, 10-15 risk assessment, 449, 449f
assumptions, 14 toxicity tests, 447-448
comparison of dose responses, 14 Ectopic fat deposition, 405, 410
defined, 10 Ectopic gene expression, 154
essential nutrients, 13 EDatiMi2
504 INDEX

ED (effective dose), 11, 14f Environmental health, 58

EDCs (endocrine disrupting chemicals), 156, 157, 312, 314, 315 Environmental pollutants and industrial chemicals, 279, 283,
Edema, 286 287-288
EDI (estimated daily intake), 455, 462 Environmental toxicology
EDSTAC, 314 air pollution, 425-439
Effective dose (ED), 11, 14f nanotoxicology, 411-424
Effector cells, 183 Enzymatic reactions, 27
Efferent arteriole, 210, 211f Enzyme-linked immunosorbent assay (ELISA), 188, 194
Efficacy vs. potency, 15 Enzyme mapping, 89
EG (ethylene glycol), 369 EOG (electrooculogram), 262, 263
EGME (ethylene glycol monomethyl ether), 311 Eosinophils, 169
Ejaculation, 310 Epidemiological studies, 52-53, 53t
Electrical impedance, 404 Epidermis, 69, 70f, 293f
Electrocardiogram (ECG), 275, 275f Epididymis, 307f
Electrooculogram (EOG), 262, 263 Epigenetic carcinogens, 45
Electrophile, 24, 25 Epigenetic reprogramming, 152
Electrophile detoxication, 25 Epigenetics, 3, 17, 152
Electrophile stress response, 42f Epileptiform seizures, 387
Electrophilic carcinogens, 124-125, 125f Epinephrine, 284
Electrophilic heteroatoms, 105f Epoxdiation, 89f
Electroretinogram (ERG), 262, 263 Epoxide hydrolase, 83
Electrostatic deposition, 229, 229f e-aminocaproic acid, 173 °
Electrotonic cell-to-cell coupling, 274 ERA (ecological risk assessment), 449
Elements de Toxicologie (Chapuis), 464 Erection and ejaculation, 310 caer
Elimination, 62, 111. See also Excretion; Exhalation ERG (electroretinogram), 262, 263
ELISA (enzyme-linked immunosorbent assay), 188, 194 Ergot alkaloids, 460t
Ellenbog, Ulrich, 2 EROD (ethoxyresorufin O-deethylase), 443
Emamectin benzoate, 340 Erythrocytes, 164-165, 168-169, 175
Embryo-fetal developmental toxicity study, 316, 316f Erythrocytosis, 164
Emphysema, 230-231, 236 EST (expressed sequence tag), 146
End-of-chapter questions, answers, 491-493 Estimated daily intake (EDI), 455, 462
Endobiotic-metabolizing enzymes, 80 Estrogen exposure, 451
Endocrine disrupting chemicals (EDCs), 156-157, 312, 314, 315 Estrogen receptor, 443
Endocrine Disruptor Screening and Testing Advisory Estrogens, 132¢, 191, 282t, 318
Committee (EDSTAC), 314 ET-1 (endothelin-1), 285
Endocrine glands, 320 Ethambutol, 268
Endocrine pancreas, 328-330 Ethanol, 368-369
Endocrine system, 319-331 cardiotoxicity, 280t
adrenal cortex, 322-324 chapter-ending question, 208, 253, 289, 372
adrenal glands, 321-322 effects on function, 169
adrenal medulla, 324 FAS, 150-151
diabetes mellitus, 329, 329f forensic toxicology, 468
pancreas, 328-330 liver, 203
parathyroid gland, 327-328 neuronal injury, 243t
pheochromocytoma, 324 pregnancy, 252
pituitary gland, 320-321 Ethical dilemmas, 7
steroidogenesis, 322 Ethidium chloride, 248t
thyroid gland, 325-327 Ethinylestradiol, 314
Endogenous agents, 137 Ethyl alcohol, 7t
Endothelian cells, 285 Ethylbenzene, 368
Endothelin-1 (ET-1), 285 Ethylene glycol (EG), 369
Endrin, 340f Ethylene glycol monomethyl ether (EGME), 311
Energy expenditure, 403 Ethylene glycol poisoning, 476-477
Engineered nanomaterials (ENMs), 412, 422-423 Ethylene oxide, 246t, 296t
Engineered nanoparticles (ENPs), 412, 416, 417 Euonymus, 385t
English Ivy, 385t Excess caloric intake, 404, 410. See also Obesity
Enterohepatic circulation, 74-75, 99 Exchange transfusion, 475
Enterohepatic cycling, 198 Excision repair (DNA), 38-39, 125, 137
Environmental androgens, 313 Excitation-contraction coupling, 274
Environmental antiandrogens, 313 Excitatory amino acids, 250, 253, 388
Environmental estrogens, 314 Excretion, 24, 73-75
 INDEX 505

Exhalation, 75 FISH (fluorescence in situ hybridization), 143, 143f 145

Experimental animal exposure studies, 487t Flash-elicited VEPs, 263
Exposure Flat dose-response curve, 12
duration/frequency, 9-10 Flavin monooxygenase (FMO), 86, 86f
route/site, 9 Flow-limited compartment, 115
Exposure assessment, 56 Flucytosine, 281t
Expressed sequence tag (EST), 146 Fluorescence in situ hybridization (FISH), 143, 143f, 145
Extracellular matrix, 40, 276 Fluoroacetate (FA), 249
Extracellular space, 115, 116, 116f Fluoroacetic acid, 344
Extrinsic allergic alveolitis, 483 Fluoroquinolones, 281t
Flurocitrate (FC), 249
Flux, 115, 116f 119

F
Fly agaric mushroom, 388f, 388
FM-100 test, 263
F (bioavailability), 112-113 FMO (flavin monooxygenase), 86, 86f
Fab region, 179, 179f Focal cell death, 199
Facilitated diffusion, 64, 454t Follicle-stimulating hormone (FSH), 306, 307, 309
Factor V, 173t Folpet, 343
Factor VIII, 173t Fomepizole, 468
Factor XIII, 173t Food, Drug, and Cosmetic Act, 454-455
False food allergies, 458 Food additives, 455-456
Farmer's lung disease, 233t, 483 Food and nutrition, 401-410
Farnsworth-Munson procedure, 263 body composition, 403-404
FAS (fetal alcohol syndrome), 150-151, 368, 372 caloric content of foods, 403
FASD (fetal alcohol syndrome disorder), 151 caloric intake, 403
FASD (fetal alcohol spectrum disorder), 368 digestion of foods, 402
Fast axonal transport, 239 energy expenditure, 403
Fat-storing cells, 197 excess caloric intake, 404, 410
Fatty liver, 199#, 200-201 integrated fuel metabolism, 402
Fe region, 179f neural control of energy balance, 402-403, 410
Fecal excretion, 74 obesity. See Obesity
Federal Insecticide, Fungicide, and Rodenticide Act (1947), 3 physical activity, 404
Female pseudohermaphroditism, 312 set-point hypothesis, 403
Female reproductive cycle, 307, 307f. See also Reproductive system Food complexity, 454, 462
Ferrochelatase, 165f Food idiosyncrasies, 457, 458t
Ferrous sulfate, 7t Food labels, 409
Fertility and early embryonic study, 315, 316f Food Quality Protection Act, 335
Fertilization, 152, 311 Food toxicology, 453-462
Fetal adrenal, 323-324 adverse reactions to food, 457-458
Fetal alcohol spectrum disorder (FASD), 368 assignment of concern level, 455, 455, 456t
Fetal alcohol syndrome (FAS), 150-151, 368, 372 carcinogens, 457
Fetal alcohol syndrome disorder (FASD), 151 dietary supplements, 457
Fetal gene program, 277 estimated daily intake (EDI), 455
Fetal hematopoiesis, 164 Food, Drug, and Cosmetic Act, 454-455
Fetal period, 153 food additives, 455-456
FETAX assay, 159t GI tract, 454
FEV1/FVC, 227 GRAS substances, 456, 456t
FIAM (free ion activity model), 443 mad cow disease, 461
Fibrin clot formation, 172 microbial contamination, 461
Fibrinolytic agents, 173 nanotechnology, 456-457
Fibroblasts, 69 new and novel foods, 456
Fibroma, 122 nonnutrient substances in food, 454, 454t
Fibrosarcoma, 122 safety standards, 454-457
Fibrosis, 42-43, 199t, 201-202, 276, 278 seafood toxins, 459-460
Fick’s law, 63, 115 toxic substances in food, 458-461
Fiddle-back spider, 392 - Forensic toxicology, 463. See also Analytical and forensic toxicology
Field studies, 448 Forensic urine drug testing (FUDT), 467, 470
Filtration, 64 Formaldehyde, 192, 437-438
First-order elimination, 113, 119 Formate, 267
First-pass effect, 68 Formic acid, 267
First-pass elimination, 82 Formicidae (ants), 394
 506 INDEX

Formyl peptide receptor (FPR), 224 Germ cell mutagenesis, 141t, 144-145
Foxglove, 386, 386t Germ cells, 136, 139-140
FPN color test, 466 GER (glomerular filtration rate), 210, 212, 213f 216, 220
FPR (formyl peptide receptor), 224 GER reduction, 213f
Framer lung, 232t GI epithelium, 67
Frameshift mutation, 139, 147 GI tract, 66-68
Free ion activity model (FIAM), 443 Gila monster, 395
Free radical, 24, 231, 236 Glomerular capillary, 211, 211f
Free radical detoxication, 25-26 Glomerular filtration pressure, 215
Fruit fly, 3 Glomerular filtration rate (GFR), 210, 212, 213f, 216, 220
PSH (follicle-stimulating hormone), 306, 307, 309 Glomerulus, 210, 211f
FUDT (forensic urine drug testing), 467, 470 Glucagon, 329
Fumigants, 344-345 Glucocorticoids, 169, 282t, 323
Fumonisin toxins, 387, 460t Glucose-6-phosphate dehydrogenase (G-6-PD), 168
Fumonisins, 219-220 Glucose control, 331
Functional antagonism, 8 Glucose production, 328
Fungal assay, 141t Glucose-regulated proteins (Grps), 214
Fungicides, 313 Glucosuria, 216
Furocoumarins, 299t Glucuronidation, 96-99
FXR, 96t Glues and bonding agents, 297t
Glufosinate, 343
Glutamate, 250f .
Glutamate receptor, 31t
G Glutamic acid, 97f, 102 SSN
G-6-PD (glucose-6-phosphate dehydrogenase), 168 Glutamine, 97f, 102
GABA, receptor, 31 Glutathione, 26
Gametal DNA repair, 318 Glutathione conjugation, 97f, 102-105, 106f
Gametogenesis, 152, 305 Glutathione peroxidase, 25
Gamma-diketones, 244 Glutathione S-transferase (GST), 104, 105, 128, 228
Gamma-ray emission, 373 Glutethimide, 246t
Gap junctional intercellular communication, 127, 278 Glycine, 97f, 102
Gas chromatography-mass spectrometry (GC-MS), 466 Glycine receptor, 31t
Gas exchange region, 226-228 Glycogenolysis, 329f
Gases and vapors, 68 Glycol ethers, 370, 372
Gasoline, 371, 372 Glycols, 369-370
Gastrointestinal (GI) tract, 66-68 Glyphosate, 343
Gastrulation, 153 GM-CSF, 182t
GC-D receptors, 224 GnRH (gonadotropin-releasing hormone), 306, 307, 309
GC-MS (gas chromatography-mass spectrometry), 466 Gold, 246t
Gempylid fish poisoning, 460 Gonadotropin-releasing hormone (GnRH), 306, 307, 309
Gempylotoxism, 460 Gonads, 304
Gene knockdown techniques, 154 Goodpasture’s syndrome, 186
Generally recognized as safe (GRAS), 453, 456, 456t GR, 96t
Genetic polymorphism, 15, 128 Graded dose-response relationship, 10
Genetic risk assessment, 136, 137f Granulocytes, 169-170
Genetic toxicology, 135-147 Granulomatous reactions, 296
cancer risk assessment, 136 Granzyme, 183
DNA damage, 137, 138f GRAS substances, 456, 456t
DNA repair, 137-139 Gray (Gy), 374
formation of chromosomal alterations, 139-140 Grps (glucose-regulated proteins), 214
formation of gene mutations, 139 GS, 102, 104
genetic risk assessment, 136, 137f GSSG (oxidized glutathione), 105
germ cells, 136, 139-140 GST (glutathione S-transferase), 104, 105, 128, 228
human population monitoring, 145 Guthion, 337f
molecular analysis of mutations, 146 Gynecomastia, 306
new approaches, 145-146
somatic cells, 136, 139
testing for abnormalities, 140-145
Genetic toxicology assays, 140-145 H
Genomics, 17 HAH (halogenated aromatic hydrocarbon), 189, 190t
Genotoxic carcinogens, 122f, 124, 126 Half-life, 111f 112
 INDEX 507

Halogenated aromatic hydrocarbon (HAH), 189, 190t Hepatic steatosis, 200-201

Halogenated hydrocarbons, 219, 283t Hepatocellular cancer, 202
Halothane, 192 Hepatocellular injury, 208
HAPs (hazardous air pollutants), 438 Hepatocyte, 196, 197f 203
Hapten, 8, 178 Hepatocyte death, 199, 199t
Hapten hypothesis, 205 Heptachlor, 340f
Hapten-protein complex, 8 Herbicides, 341-343
Hard metal disease, 232t Hershberger assay, 314
Hardening, 294 Heteroatom dealkylation, 90f
Hay fever, 384-385 Heteroatom oxygenation, 90f
Hazard, 50 Heteropsia (true bugs), 394
Hazard identification, 51-53 Hexachlorobenzene, 193
Hazardous air pollutants (HAPs), 438 Hexachlorocyclohexanes, 339
Heart, 272-283. See also Vascular system Hexachlorophene, 248, 248t
action potential, 273, 274f HIF-1a (hypoxia-inducible factor 1a), 405
anatomical diagram, 273f Hippocrates, 1
automaticity, 273-274 Historical overview, 1-3
autophagy, 276 Age of Enlightenment, 2
biomarkers of cardiac toxicity, 278, 279t antiquity, 1-2
cardiac hypertrophy, 272, 276-277 Middle Ages, 2
cardiac output, 275 Renaissance, 2
contractility, 274 21st century, 3
ECG, 275, 275f 20th century, 2-3
electrophysiology, 273-274 HIT (heparin-induced thrombocytopenia), 171
electrotonic cell-to-cell coupling, 274 Hit models (cancer), 55-56
environmental pollutants and industrial chemicals, 279, 283t HnFla, 96¢
heart failure, 272, 277 Holliday junction DNA complex, 138
myocardial cell death and signaling pathways, 276 Homeostasis, 171-173
myocardial degeneration and regeneration, 275-276 Homocysteine, 287
natural products, 279, 282t Homogeneity, 158
neurohormonal regulation, 275 Homologous recombination, 138
pharmaceutical chemicals, 279, 280-282t HOOH, 25
plants and plant toxicities, 386, 386t Hormesis, 13, 129
QT prolongation, 277-278 Hormonally active chemicals, 126
radiation, 379 Hormone, 320
structural organization, 273, 273f Hornets, 394
sudden cardiac death, 278 HPG (hypothalamic-pituitary-gonadal) axis, 307-308, 318
toxic chemicals, 279-283 Hsps (heat-shock proteins), 214
triangle model of cardiac toxicity, 275, 275f Human ABC transporters, 64, 65¢
Heart failure, 272, 277, 278 Human body systems/organs. See Target organ toxicity
Heat-shock proteins (Hsps), 214 Human cytosolic sulfotransferases (SULTs), 100t
Heavy metals, 218-219. See also Metals Human developmental toxicants, 150t
Hematite miner's lung, 233¢ Human embryonic palatal mesenchyme, 159t
Hematology measurements, 17 Human epidemiological studies, 52-53, 53¢
Hematopoiesis, 164 Human genome, 17
Hematotoxicology, 164. See also Blood Human performance testing, 467
Heme and hemoglobin synthesis, 165f Human solute carrier transporter families, 65, 66t
Hemicholinium-3, 7t¢ Humidifier fever, 431, 483
Hemofiltration, 475 Humoral immunity, 183-184, 184f 188
Hemoglobin, 166-169 HUS (hemolytic uremic syndrome), 171
Hemoglobin-oxygen dissociation curve, 167f, 175 Hyacinth, 385¢
Hemolytic uremic syndrome (HUS), 171 Hydra assay, 159t
Hemoperfusion, 474-475 Hydralazine, 192, 246t
Hemorrhage, 286 Hydrazinobenzoic acid, 287
Henderson-Hasselbalch equations, 63-64, 73 Hydrodensitometry, 404
Henry’s law, 68 3 Hydrogen abstraction, 27
Heparin, 173 Hydrogen chloride (HCl), 296t
Heparin-induced thrombocytopenia (HIT), 171 Hydrogen fluoride, 232t, 296t
Hepatic artery, 197f Hydrogen peroxide, 296t
Hepatic fibrosis/cirrhosis, 201-202 Hydrolysis, 81f, 82-83
Hepatic sinusoids, 197, 200 Hydrolytic enzymes, 83
508 INDEX

Hydrophilic compounds, 69 testing for immunity, 187-189

Hydrophobic xenobiotics, 24 therapeutic agents, 192-193
Hydroxychloroquine, 266 xenobiotics, 189-193
Hydroxylamines, 102 Immunity, 178
Hydroxylation of aliphatic carbon, 88f Immunoenhancement, 178
Hydroxylation of aromatic carbon, 88f Immunogen, 8, 178
Hymenoptera (ants, bees, etc.), 394 Immunoglobulin (Ig), 178-179, 179f
Hyperglycemia, 407 Immunohistochemistry, 235
Hyperinsulinemia, 407 Immunosuppressants, 281
Hyperpigmentation, 298, 299, 300t Immunosuppression, 178
Hypersensitivity, 8, 185-186, 192 Immunosuppressive agents, 190t, 191
Hypersensitivity pneumonitis, 431, 483, 484t Immunotoxicology. See Immune system
Hypersusceptible, 11 Impaction, 229, 229f
Hypertension, 285-286 Implantation, 311
Hypertrophic signaling pathways, 277 Imprinting, 152
Hypokalemia, 278 In silico assay, 140, 141t¢
Hypomethylation, 127 In situ hybridization, 235
Hypopigmentation, 300, 300t In utero-lactational assay, 314
Hypotension, 286 In vitro bacterial mutation assay, 52, 130
Hypothalamic-pituitary-gonadal (HPG) axis, 307-308, 318 In vitro dosimetry, 421
Hypoxia-inducible factor la (HIF-1a), 405 In vivo gene mutation assay, 130
Index of biotic integrity (IB1), 447
Indirect-acting genotoxic carcinogens, 124
Indirect food additives, 455-456, 462 * \
Individual dose-response relationship, 10
IARC (International Agency for Research on Cancer), 56 Indomethacin, 266
IARC classification of carcinogenic agents, 133t Inducers (CYP system), 93-94f, 126, 365
IBI (index of biotic integrity), 447 Industrial chemicals/pollutants, 279, 283t, 287-288
ICopp 447 Infantile development, 306
Ideal gas law, 228 Inflammation, 40-41, 184-185
Idiosyncratic drug hepatotoxicity, 207 Inhalation exposure system, 234
Idiosyncratic reactions, 8 Inhalation toxicology, 224, 228
Idiosyncratic toxic neutropenia, 170 Inhalational anesthetics, 281¢
IDPN (6,6'-iminodipropionitrile), 244-245, 246t Inhaled nanomaterials, 485
IFN (interferon), 182 Inhaled substances, 1904, 191
Ig (immunoglobulin), 178-179, 179f Inhibitors (CYP system), 93-94, 365
IgE-mediated food allergies, 457t, 462 Initiation stage of carcinogenesis, 123, 123t, 134
IxB, 29 Innate immunity, 178, 181-183, 188
IL (interleukin), 182¢, 183 Inorganic arsenic, 488
IL-1, etc., 182¢, 183, 240 Inorganic carcinogens, 126
IMCL (intramyocellular lipid), 405 Inotropic drugs, 280
Immediate toxic effects, 8 INR (international normalized ratio), 171
Immune hemolytic anemia, 169 Insect repellents, 341
Immune-mediated idiosyncratic hepatotoxicity, 207t Insecta, 394
Immune-mediated neutropenia, 170 Insecticides, 336-341
Immune system, 177-194 avermectins, 340-341
acquired immunity, 183-184, 188 carbamates, 338
animal models, 189 DDT, 339, 340
antibodies, 178-179 intermediate syndrome, 337
antigen recognition, 178-180 molecular targets, 336t
autoimmunity, 186-187, 192 nicotine, 340
cell-mediated immunity (CMI), 184 OPIDP, 338
challenges, 193 organochlorine compounds, 339-340
complement system, 179, 180f organophosphorus (OP) compounds, 336-338
developmental immunology, 187 pyrethroids, 338-339
humoral immunity, 183-184, 184f, 188 rotenoids, 340
hypersensitivity, 185-186, 192 Insulin, 329, 408
inflammation, 184-185 Insulin resistance, 329
innate immunity, 181-183, 188 Integrated Risk Information System (IRIS), 426
neuroendocrine immunology, 187 Integrins, 40
new frontiers, 193 Interaction of chemicals, 8-9
 INDEX 509

Intercellular signaling pathways, 160, 160t

Interception, 229, 229f
K
Interferon (IFN), 182 K'-ATPase, 32t
Interferon-a/B (IFN-a/B), 182t Kainate, 249t, 251

Interferon-y (IFN-y), 182t Kaolin, 233t

Interfollicular epidermis, 292 Kaolinosis, 233t
Interleukin (IL), 182, 183, 240, 282t Kepone, 2461, 340

Intermediate syndrome, 337 Kerinatocytes, 293

Intermittent exposure, 113 Keriorrhea, 460
Internal dose, 489 Ketones, 283t
Internalized dose, 489 Kidney, 209-222
International Agency for Research on Cancer (IARC), 56 acute kidney injury, 212, 213t
International Congress of Toxicology, 3 a,,-globulin nephropathy, 219
International normalized ratio (INR), 171 anatomical diagrams, 211f
International pollution, 427 assessment of renal function, 216-217
International Programme on Chemical Safety (WHO), 56 cell death, 218
Interstitial nephritis, 358 cellular/subcellular and molecular targets, 218
Interstitial space, 114, 114f chronic kidney disease, 214
Interstrand cross-link, 138f collecting duct injury, 216
Intracellular space, 114, 114f 116, 116f distal tubule injury, 216
Intramuscular injection, 70 functional anatomy, 210-212
GFR reduction, 213f
Intramyelinic edema, 248
Intramyocellular lipid (IMCL), 405 halogenated hydrocarbons, 219
Intraocular melanin, 258 heavy metals, 218-219
Intraperitoneal injection, 70 incidence of severity of toxic nephropathy, 214-215
Intrastrand cross-link, 138f loop of Henle injury, 216
Intravenous bolus injection, 111f mechanisms ofinjury, 217f
mediators of toxicity, 218
Intravenous route, 70
mycotoxins, 219-220
Introduction to the Study of Experimental Medicine, An (Bernard), 2
Inulin clearance, 216 papillary injury, 216
Inversion of configuration, 82
plants and plant toxicities, 387
Iodinated contrast media, 221 proximal tubular injury, 215-216
Iodine deficiency, 462 site-selective injury, 215
Iohexol, 169 site-specific biomarkers, 217f
Ion balance, 289
therapeutic agents, 220-221
toxic insult, 212-214, 215f 222
Ionizing radiation, 137, 138f 374
Kidney cancer, 366
lopamidol, 221
Iotrol, 221
Klinefelter’s syndrome, 318
Kojic acid, 460¢
Toxaglate, 169
Kupffer cells, 197, 202, 205
Iris, 257f
IRIS (Integrated Risk Information System), 426
Iris (plant), 385¢
Iron, 356, 356t
Iron deficiency anemia, 165 L
Iron oxides, 233t Lactation, 312
Irritant dermatitis, 294, 383 Lactonase, 83
Islet cells, 328 Lanthony D-15, 263
Isocyanates, 233t Larkspur, 386t
Isolated perfused lung, 235 Larynx, 225f
Isoniazid, 192, 246t Lateral geniculate nucleus (LGN), 268
Isotope, 178, 179 Latex, 192
Ito cells, 205 Latrodectus mactans (female black widow spider), 392f
Ivermectin, 340 Latrodectus species (widow spiders), 392
LBD (ligand-binding domain), 95
e447,
LC-MS (liquid chromatography-mass spectrometry), 466
J D7 ).74
Japanese summer house fever, 483 LD (lethal dose), 14f
Jervine, 389 Lead, 352-353
Jungle, The (Sinclair), 3 chapter-ending question, 253, 359
Juxtaglomerular apparatus, 211f nervous system, 243t, 248
510 INDEX

Lead (continued) tumors, 199f, 202

ocular and visual system, 267, 268 uptake and concentration, 202-203
toxicity, 356t¢ Liver cancer, 366
Leather, 297t Liver compartment, 117
Leaving groups, 104 Lizards, 395
Lectin pathway, 179, 180f LNT (linear-no threshold) model, 379
Legionnaire’s disease, 431 LOAEL (lowest observed adverse effect level), 16, 54f
Lehman, Arnold, 3 Lobule, 196, 197f
Lens, 257f, 264-265, 269 Local effects, 8
Lepidoptera (caterpillars, moths), 394, 399 Local metabolic regulation, 284
Leptin, 403 LOEC (lowest observed effect concentration), 447
LET (linear energy transfer), 374 Long QT syndrome, 277
Lethal dose (LD), 14f Long-term (chronic) exposure study, 17
Lethal dose 50 (LD.,), 7, 7t Loop of Henle, 211f 212, 222
Leukemia, 170-171, 176 Loop of Henle injury, 216
Leukemogenesis, 170 Low-LET radiation, 374, 376, 380
Leukocytes, 169, 175 Lowest observed adverse effect level (LOAEL), 16, 54f
Leukoderma, 300, 300t Lowest observed effect concentration (LOEC), 447
Leukon, 169 Loxosceles reclusa (brown recluse spider), 392, 393f
Lewin, Louis, 2 Loxosceles species (brown/Vviolin spiders), 392-393
Lex Cornelia, 2 LRH-1, 96¢
LGN (lateral geniculate nucleus), 268 Lung, 68-69, 226f, 227, 227f
LH (luteinizing hormone), 306, 307, 309 Lung cancer, 132, 231, 366, 435, 439
Lifetime bioassay, 52 Lung cell culture, 235 Par
Ligand, 347 Lung compartment, 116-117
Ligand-binding domain (LBD), 95 Lung defense, 230
Ligandin, 104 Lung volumes, 227, 227f
Light and phototoxicity, 261-262 Luteinizing hormone (LH), 306, 307, 309
Lily bulbs, 399 LXRa, 96¢
Lily of the valley, 386t Lymph nodes, 225f
Lindane, 339, 340, 340f Lymphatic system, 284
Linear energy transfer (LET), 374 Lymphoid tissues, 178
Linear-no threshold (LNT) model, 379 Lysolecithin, 248¢
Linuron, 313 Lysosomal accumulation, 24
Lipid bilayer, 63
Lipid repair, 38
Lipophilic compounds, 69
Liquid chromatography-mass spectrometry (LC-MS), 466 M
Lithium, 246¢, 356¢, 358 m-Dinitrobenzene (m-DNB), 310-311
Liver, 195-208 M1 macrophages, 181
activation of sinusoidal cells, 205 M2 macrophages, 181
bile duct damage, 199¢, 200 MAC (membrane attack complex), 179
bile formation, 197-198 Macrolides, 280t
bioactivation and detoxification, 203-204 Macrophages, 39, 181, 185, 230
canalicular cholestasis, 199-200, 199t Mad cow disease, 461
cell death, 199 Magendie, Francois, 2
disruption of cytoskeleton, 200 Maimonides, 2
excess Calories, 405 Major compatibility complex (MHC), 179
factors in site-specific injury, 202t Malathion, 337f
fatty liver, 199t, 200-201 Male reproductive system, 307f. See also Reproductive cycle
fibrosis and cirrhosis, 199t, 201-202 Malt worker’s lung, 232t
functions, 196, 196t MAM (methylazoxymethanol), 243¢
future directions, 207 Mammalian cytogenic assays, 141t, 142-144, 147
idiosyncratic liver injury, 207, 207t Mammalian gene mutation assays, 141t, 142
inflammation and immune response, 205 Mammalian GI tract, 67, 67¢
mitochondrial damage, 205-207 Mancozeb, 343
plants and plant toxicities, 386-387 Maneb, 343f
regeneration, 204-205 Manganese, 233t, 243t, 251-252, 356t
sinusoidal damage, 199f, 200 Manganese pneumonia, 233t
structural organization, 196-197 Mannin-binding lectin pathway, 179, 180f
transport proteins, 198f Manufactured nanomaterials, 483-485
 INDEX onBa

MAO (monoamine oxidase), 84-85 Mercapturic acid biosynthesis, 106f

MAPK (mitogen-activated protein kinase), 29 Mercapturic acid synthesis, 95
Maple bark stripper’s lung, 483 Mercury, 353-355, 356t
March hemoglobinuria, 168 chapter-ending question, 359
Margin of exposure (MOE), 54 immune system, 193
Margin of safety, 15 kidney, 218-219
Marginated pool, 169 MeHg. See Methyl mercury (MeHg)
Mass spectrometry, 82 neuronal injury, 243¢
MATE (multidrug and toxin extrusion) transporters, 65, 66t Mesocosm, 448
Maternal toxicity, 156 Messenger RNA (mRNA), 28
Matrix metalloproteinase (MMP), 276 Metabolic acidosis, 473t
Maximum tolerable dose (MTD), 17 Metabolic activation, 24
Mayapple, 385t Metabolic food reactions, 458, 459t
MC (methylene chloride), 366 Metabolic kinetics, 113
MDAC (multiple-dose activated charcoal), 475 Metabolic syndrome, 405-407, 410
MDR (multidrug resistant protein), 24, 65t, 73f, 74f Metabonomics/metabolomics, 17, 18f
MDS (myelodysplastic syndrome), 170, 171 Metal-binding proteins, 349
Mechanisms of toxicity, 21-47 Metal transporters, 349
absorption, 22 Metallothioneins, 349, 359
adaptation, 41 Metals, 347-359
apoptosis, 36-38, 39, 45 aluminum, 357-358
ATP depletion, 33-34 arsenic, 350
Ca**, 34-35 biomarkers of metal exposure, 349
carcinogenesis, 43-45 cadmium, 350-352
cell cycle accelerators/decelerators, 44f cardiotoxicity, 279, 283t
cellular dysfunction and resultant toxicities, 27-38 contact allergens, 297t
cellular repair, 39 copper, 355-356
detoxication, 25-26 defined, 348
excretion, 24 immune system, 190, 190t
fibrosis, 42-43 iron, 356
inflammation, 40-41 kidney, 218-219
mitochondrial permeability transition (MPT), 36 lead, 352-353
mitosis, 40 lithium, 358
molecular repair, 38-39 mercury, 353-355
necrosis, 36 metal-binding proteins/metal transporters, 349
nongenotoxic carcinogens, 45 nickel, 355
overproduction of ROS and RNS, 35 particulate matter, 434
overview (key points), 22 pharmacology, 349-350
presystemic elimination, 22-23 platinum, 358
proliferation, 40, 45 toxicity/toxicology, 348, 348f 349, 356t
reabsorption, 24 ZING, SOO—Soy/
reaction of ultimate toxicant with target molecule, 26-27 Metalworking fluid hypersensitivity, 233t
stages in development of toxicity, 22, 23f Metamidophos, 337f
tissue necrosis, 41-42 Metastases, 122t
tissue repair, 39-41 Metastasis, 45
toxic alteration of cellular maintenance, 33-38 Methanol, 243¢, 267, 369
toxicant delivery, 22-26, 23f Methemoglobinemia, 167, 167t¢
toxicant-induced cellular dysfunction, 28-33 Methionine sulfoximine (MSO), 249
toxication, 24-25 Methoxychlor, 314
ultimate toxicant, 22 Methyl alcohol, 369
Mechanistic toxicologist, 6 Methyl bromide, 296t, 344
Median dose, 14 Methyl mercury (MeHg), 244, 252, 268, 344, 355
Medical toxicologist, 471 Methyl n-butyl ketone, 246t
Medici, Catherine, 2 Methyl tertiary-butyl ether (MTBE), 371
Megaloblastic anemia, 166, 166t Methylation, 97f, 99
Meiosis, 305f - Methylazoxymethanol (MAM), 243
Melanin, 258, 296, 299 Methylcarbamates, 338
Melphalan, 132t Methyldopa, 192
Membrane attack complex (MAC), 179 Methylene chloride (MC), 366
Menkes disease, 355 Methylparathion, 337f
Menopause, 312 Methyltestosterone, 312
 1
nan Nw INDEX

Methylxanthines, 282t MTBE (methy] tertiary-butyl ether), 371

Metronidazole, 246t, 249 MTD (maximum tolerable dose), 17
MF (modifying factor), 54 Mucosal repair, 40
MGC (Miller glial cell), 263 Mucus, 225, 236
MGMT (O°-methylguanine-DNA methyltransferase), 139 Miller glial cell (MGC), 263
MHCTI, 179 Multi-hit model, 56
MHCII, 179 Multi-walled carbon nanotube (NWCNT), 420
Michaelis-Menton kinetics, 117 Multidrug and toxin extrusion (MATE) transporters, 65, 66¢
Michaelis parameters, 117 Multidrug resistance-associated protein (MRP), 65t, 73f, 74f
Microangiopathic anemia, 168 Multidrug resistant protein (MDR), 24, 65t, 73f, 74f
Microcosm, 448 Multigenerational reproduction study, 315, 315f
Microcystin, 200, 202 Multiple-dose activated charcoal (MDAC), 475
Microdissected airway, 235 Muscarine, 249t
Micromass culture, 159¢ Mushroom toxins, 387
Micromercurialism, 354 Mushroom worker’s lung, 233t
Micronucleus, 130, 141¢, 143, 144f Mutagenesis, 124
Micronutrients, 459 Mutagenicity, 17
MicroRNA (miRNA), 28, 242 Mutarotation, 82
Microtubule-associated neurotoxicity, 247 MWCNT (multi-walled carbon nanotube), 420
Middle Ages, 2 Mycotoxins
Milk, 75 immune system, 190-191, 190t
Milkweed, 386¢ kidney, 219-220 :
Millipedes, 394 Myelin, 247
Mineralocorticoids, 282¢, 323 Myelin formation, 241 aa
Mismatch repair, 138-139 Myelination, 241f
Misonidazole, 246t Myelinopathies, 247-248, 248t
Mistletoe, 385t, 386 Myelodysplasia, 367
Mithridates VI, 2 Myelodysplastic syndrome (MDS), 170, 171
Mitochondrial accumulation, 24 Myeloid precursor stem cells, 194
Mitochondrial ATP synthesis, 33f, 34t Myelotoxicity, 169
Mitochondrial DNA damage, 205-207 Myocardial adaptation, 276-277
Mitochondrial permeability transition (MPT), 36, 38, 218 Myocardial cell death and signaling pathways, 276
Mitogen-activated protein kinase (MAPK), 29 Myocardial degeneration and regeneration, 275-276
Mitogenic signaling molecules, 29 Myocardial fibrosis, 276, 278
Mitophagy, 276 Myocardial ischemic injury, 278
Mitosis, 40, 305f Myocardial reperfusion injury, 289
Mixed lymphocyte response (MLR), 188 Myofibril, 273
MLR (mixed lymphocyte response), 188 Myoglobin, 279t
MMP (matrix metalloproteinase), 276
Modifying factor (MF), 54
MOE (margin of exposure), 54
Molecular epidemiology, 53 N
Molecular repair, 38-39 N-acetylation, 99-102
Mollusca (cone snails), 394-395 N-acetyltransferase, 102
Molybdenum hydroxylases, 84 n-hexane, 246t
Molybdozymes, 84 iNaiao2 6
Monkshood, 386t NADH-ubiquinone reductase, 340, 346
Monoamine oxidase (MAO), 84-85 NADPH-cytochrome P450 reductase, 83, 84, 86
Moths, 394 NADPH-quinone oxidoreductase, 83
Motile cilia, 225-226 NAFLD (nonalcoholic fatty liver disease), 201, 405, 407, 407f
Mouse embryonic stem cell test (EST), 159t Nanoparticles (NPs), 69, 412, 413t
Mouse lymphoma assay, 130 Nanosilver, 422
Mouse ovarian tumor assay, 159t Nanotoxicology, 411-424
Mouse skin model, 131 biopersistence, 415-416
Mouse skin tumor promotion, 301 brain, 419
MPT (mitochondrial permeability transition), 36, 38, 218 carbon nanotubes (CNTs), 420
MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), 242, 243t, classes/classification, 412, 414, 414f
Zole2oa3 defined, 412
mRNA (messenger RNA), 28 dosimetrics, 417-418
MRP (multidrug resistance-associated protein), 65t, 73f, 74f ecotoxicology of engineered nanomaterials (ENMs), 422-423
MSO, 249 elimination of nanomaterials, 420
 INDEX S13

Nanotoxicology (continued) Neuronopathy/neuronopathies, 240, 242-244

goals, 421 Neurotoxicity. See Nervous system
nanomaterial biologic interface, 416 Neurotransmitter-associated neurotoxicity, 249-251
nanoparticles vs. larger particles, 412, 413t Neutrophils, 169, 181, 184
nanotoxicologic assays, 416, 424 NF-«B, 29
physicochemical properties, 414-416 NHEJ (nonhomologous end joining), 39, 138
portals of entry, 418 Nickel, 233t, 355, 356t, 359
predictive toxicology, 421 Nickel carbonyl poisoning, 355
respiratory tract, 418-419 Nicotine
sunscreen, 417 cardiotoxicity, 286
surface properties, 413t, 414-415 chapter-ending question, 346
toxicity mechanisms, 416 UD st
toxicity testing, 420-421 neurotransmitter-associated toxicity, 249f, 250
in vitro dosimetry, 421 toxicity, 340
Naphthalene, 265 Nitric oxide, 168, 285
Nasal airway, 225f Nitro-reduction, 83
Nasal clearance, 230 Nitrofurantoin, 246¢
Nasal decongestants, 280t Nitrogen dioxide (NO,), 437, 439, 483
NASH (nonalcoholic steatohepatitis), 201, 407 Nitrogen oxides, 233t, 296t
NAT1/NAT2, 101 NK assay, 189
National Center for Biotechnology Information (NCBI), 56-57 NK (natural killer) cells, 179, 181
National Toxicology Program (NIEHS), 56 NMSC (non-melanoma skin cancer), 301
National Toxicology Program screens, 189 NO, (nitrogen dioxide), 437, 439, 483
Natural killer (NK) cells, 179, 181 No observable adverse effect level (NOAEL), 15, 54, 54f, 55, 157
Natural rubber latex, 192 No observed effect concentration (NOEC), 447
NCBI (National Center for Biotechnology Information), 56-57 NOAEL (no observable adverse effect level), 15, 54, 54f 55, 157
Necrosis, 36, 199, 276 NOEC (no observed effect concentration), 447
NED (normal equivalent deviation), 12 Nolvadex, 266
Negative acute-phase proteins, 41 Non-immune-mediated neutropenia, 170
Neoantigen formation, 27 Non-melanoma skin cancer (NMSC), 301
Neoepitopes, 171 Non-self, 178
Neonatal development, 305-306 Nonabsorbed ingesta, 74
Neoplasia, 122t, 126 Nonalcoholic fatty liver disease (NAFLD), 201, 405, 407, 407f
Neoplasm, 122t Nonalcoholic steatohepatitis (NASH), 201, 407
Nephron, 210, 222 Noncovalent binding, 26
Nephrotic insult, 212-214, 215f, 222 Nondisjunction (meiosis), 147
Nephrotoxic mycotoxins, 219-220 Nongenotoxic carcinogens, 45, 122¢, 126-127
Nephrotoxicants, 222 Nonhomologous end joining (NHEJ), 39, 138
Nephrotoxicity. See Kidney Nonimmune hemolytic anemia, 168-169
Nervous system, 237-253 Nonimmune-mediated idiosyncratic hepatotoxicity, 207t
astrocytes, 248-249 Nonionic contrast agents, 221
axonal degeneration, 240 Nonlinear toxicokinetics, 113
axonal transport, 239-240 Nonmonotonic dose-response curve, 14
axonopathies, 244-247 Nonoxidative chemical-induced hemolysis, 168-169
blood-brain barrier (BBB), 238-239, 238f Nonsteroidal anti-inflammatory drugs (NSAIDs)
depression of nervous system function, 252 adrenal cortex, 323
development of, 241-242 anti-inflammatory agents, 191
developmentally neurotoxic chemicals, 252 glomerular and vascular renal lesions, 287
energy requirements, 239 kidney, 220
functional manifestations of neurotoxicity, 242 platelet function, 172
myelin formation, 241 Nontuberculous mycobacteria, 233
myelinopathies, 247-248, 248t Norepinephrine, 284
neuronopathies, 242-244 Normal equivalent deviation (NED), 12
neurotransmission, 241 Normal frequency distribution, 11
neurotransmitter-associated neurotoxicity, 249-251 NPs (nanoparticles), 69, 412, 413¢
patterns of neurotoxie injury, 240f NQO1/NQ02, 83
plants and plant toxicities, 387-389 NRC risk assessment paradigm, 427f
tiered testing schemes, 252 Nrf2, 96t
Nettles, 384f NSAIDs. See Nonsteroidal anti-inflammatory agents (NSAIDs)
Neurasthenic syndrome, 363 NTCP (sodium-dependent taurocholate peptide), 74f
Neuroendocrine immunology, 187 NTP tier approach, 189
514 INDEX

Nuclear magnetic resonance (NMR), 404 OCTN (organic-cation/carnitine transporter), 66¢

Nucleophile, 25, 125 Ocular and visual system, 255-269
Nucleophile detoxication, 25 acid burns, 264
Nucleoside analog drugs, 205, 281 alkali burns, 264
Nucleotide excision repair (NER), 134, 137 anatomical diagrams, 257f
Numerical chromosome changes, 139 behavioral testing procedures, 263
cancer chemotherapeutics, 266
cataracts, 265
caustic burns, 264
O central visual system, 261, 268
O°-methylguanine-DNA methyltransferase (MGMT), 139 color vision, 263
OAT (organic-anion transporter), 66f, 73f cornea, 264-265
OATP (organic-anion transporting peptide), 65, 66t, 74f, 197, 200 corticosteroids, 265
Obesity, 404-409 Draize test, 262
adipose tissue, 405 electrophysiologic techniques, 262-263
cancer risk, 408 lens, 264-265
dieting, 408-409 light and phototoxicity, 261-262
ectopic fat deposition, 405 naphthalene, 265
endocrine dysfunction, 407-408 ocular drug delivery, 258-260
family and community interventions, 409 ocular drug metabolism, 260
food labels, 409 ocular irritancy and toxicity, 262, 269
genes and fetal environment, 404 ophthalmologic evaluations, 262
governmental and corporate issues, 409 optic nerve and tract, 267-268
health insurance, 409 organic solvents, 264, 267 as
lifestyle modification, 408 pharmacodynamics/pharmacokinetics, 256-262
liver, 405 phenothiazines, 265
metabolic syndrome, 405-407 retina/retinotoxicity, 265-267
NASH, 407 signs/symptoms of dysfunction, 259t
treatment, 408-409 surfactants, 264
Observational epidemiologic studies, 487t tunnel vision, 268
Occupational asthma, 483 Ocular fundus, 262, 269
Occupational exposure limit (OEL), 483 OEL (occupational exposure limit), 483
Occupational human carcinogens, 132t Oleander, 386t
Occupational lung diseases, 483 Olfactory receptors, 224
Occupational respiratory diseases, 483, 485, 485t “omics” technologies, 17, 18f
Occupational skin toxicity, 295f ON atrophy, 267
Occupational toxicology, 481-490 On the Miners’ Sickness and Other Diseases of Miners (Agricola), 2
animal toxicology testing, 487, 487 Oncogene, 43, 147
biomonitoring, 488-489 Oncogenes, 128, 128t
combined experimental, clinical, and epidemiologic Oncogenicity bioassay, 17
approach, 488, 489 One-compartment model, 110
defined, 481 One-hit (one-stage) linear model, 55
determinants of dose, 482, 482t ONOO,, 25, 26
difficulty establishing causal link, 481-482 Oogenesis, 308
establishing causality, 486-487 OP compound-induced delayed neurotoxicity (OPIDN), 247
experimental animal exposure studies, 487t OP (organophosphorus) compounds, 246t, 247
exposure monitoring, 488-489 OP (organophosphorus) insecticides, 336-338
human carcinogens, 485t OPIDN, 247
manufactured nanomaterials, 483-485 OPIDP, 338
objective, 481, 487 Opioid receptor, 31t
observational epidemiologic studies, 487t Opioid toxic syndrome, 472t
occupational diseases, 483-486 Opsonization, 179
respiratory diseases, 483, 485, 485t Optic nerve and tract, 267-268, 269
routes of exposure, 483 Optic neuritis, 267
sources of toxicologic information, 486-487 Oral anticoagulants, 172-173
worker health surveillance, 487-488 Oral contraceptives, 287
workplace exposure limits, 483 Orfila, Mathieu, 2
Ochratoxin, 460t Organ clearance, 112
OCT (organic-cation transporter), 66t, 73f 74f Organ-specific bioassay, 131
Octanol/water partition coefficient (P), 63 Organic-anion transporter (OAT), 66t, 73h,
 INDEX 515

Organic-anion transporting peptide (OATP), 65, 66t, 74f 197, 200 Panacinar necrosis, 199
Organic-cation/carnitine transporter (OCTN), 66t Pancreas, 328-330
Organic-cation transporter (OCT), 66t, 73f, 74f Pancreatic cancer, 132
Organic solvent syndrome, 363 Pancreatic hormones, 329
Organic solvents, 264, 267 Pancreatic toxicity, 329
Organochlorine compounds, 313 Paper products, 297t
Organochlorine insecticides, 339-340, 346 Papillary injury, 216
Organogenesis, 153, 187 PAPS, 97f, 99
Organophosphate-induced delayed polyneuropathy (OPIDP), 338 Paracellular diffusion, 63
Organophosphorus (OP) compounds, 246t, 247 Paracelsus, 2
Organophosphorus (OP) insecticides, 336-338 Parafollicular cells, 331
Organotypic tissue culture system, 235 Paraoxonase, 82, 83
Oronasal passages, 223 Paraquat, 342, 342f
Orphan Drug Act, 475 Parathyroid adenoma, 331
Osmol gap, 473, 474t, 479 Parathyroid gland, 327-328
Osteonectin, 405 Parathyroid hormone (PTH), 327, 328
Osteosarcoma, 122 Parathyroid toxicity, 327-328
Ovarian cycle, 308 Paresthesia, 339
Oviduct, 309 Parkinson's disease, 242, 252
Oxidation, 81t, 84-95 PARP (poly(ADP-ribose) polymerase), 35-36, 39
alcohol dehydrogenase (ADH), 84 Particle clearance, 230, 236
aldehyde dehydrogenase (ALDH), 84 Particle overload hypothesis, 418
aldehyde oxidase, 84 Particles, 68-69
cytochrome P450 (CYP) system. See Cytochrome P450 (CYP) system Particulate air pollution, 288
dihydrodiol dehydrogenase, 84 Particulate matter (PM), 431-432, 434-435, 439
flavin monooxygenase (FMO), 86, 86f Particulate radiation, 374
molybdenum hydroxylases, 84 Partition coefficient, 115, 115f
monoamine oxidase (MAO), 84-85 Parturition, 311
peroxidase-dependent cooxidation, 85 Passive diffusion, 115, 454t
xanthine oxidoreductase (XO), 84 Passive transport, 63-64
Oxidative dehalogenation, 84 Pattern-elicited VEPs, 263
Oxidative group transfer, 91f Patulin, 460t
Oxidative hemolysis, 168, 175 PBDEs (polybrominated diphenyl ethers), 252, 327
Oxidative phosphorylation, 33, 33f, 34 PC12 cell line, 324
Oxidative stress, 127, 285, 444 PCBs (polychlorinated biphenyls), 156, 252, 306, 327
Oxidative stress inducers, 124t PDGF (platelet-derived growth factor), 40
Oxidized glutathione (GSSG), 105 PEL (permissible exposure limit), 483
Oxygen dissociation curve, 167f, 175 Penguin humidifier lung, 233t
Oxyhemoglobin, 167 Penicillin, 20, 169, 192
Ozone, 233t, 435, 436-437, 439 Penicillinic acid, 460t
Penile erection, 310, 318
Penis, 307f
Penitrem(s), 460t
P Peptidase, 83
p16, 129, 129t Peptide transporter (PEPT), 65, 66¢, 73f
p53, 43, 129, 129f, 301 Perchlorate, 327
p-bromophenylacetyl urea, 246t Perchloroethylene, 233
P450 inducers, 93-94t, 126, 365 Perchloroethylene (PERC), 366
P450 induction, 92, 95, 96t Percutaneous absorption, 293-294
P450 inhibition, 92 Perfluorinated chemicals, 327
P450 inhibitors, 93-94t, 365 Perforin, 183
P450 substrates, 93-94t Perfusion-limited compartments, 115-116
Pacemaker potential, 274 Perhexiline, 248t
Paclitaxel, 246t, 247 Permeability-area product, 115
PAHs (polycyclic aromatic hydrocarbons), 299¢, 301, 443, 445 Permissible exposure limit (PEL), 483
Paint products, 283t - Peroxidase-dependent cooxidation, 85
Painter’s syndrome, 363 Peroxidase-generated free radicals, 26
Palytoxin, 459 Peroxisome proliferator-activated receptor-a (PPARa), 80, 96,
Pampiniform plexus, 310 124t, 126
PAN (peroxyacetyl nitrate), 436, 437 Peroxyacetyl nitrate (PAN), 436, 437
516 INDEX

Peroxynitrite (ONOO), 25, 26 Physiologically based models, 113, 364

Pesticides, 333-346 Picaridin, 341
defined, 334 Picrotoxin, 7t
erection and ejaculation, 310 Pigeon breeder's lung, 483
exposure, 334 Pigmentary disturbances, 299-300, 300t
fumigants, 344-345 Pinocytosis, 65, 454t
fungicides, 343-344 Pituitary gland, 320-321
herbicides, 341-343 Pituitary hormones, 331
immune system, 189, 190t Pituitary toxicity, 320-321
insect repellents, 341 pK,, 63
insecticides, 336-341 pK,, 63
registration, 335, 335t PKC (protein kinase C), 29
regulations, 335 Placenta, 72, 154, 311
rodenticides, 344 Placental barrier, 73
thyroid gland, 327 Placental toxicity, 156
types, 334 Placental transfer, 72-73
WHO-recommended classification, 335t Plant assay, 141t
PFC assay, 187, 189 Plants and plant toxicities, 382-390
PG (propylene glycol), 369-370 blood and bone marrow, 387
pH, 167 bone and tissue calcification, 389
Phagocytosis, 65, 230 cardiovascular system, 386, 386t
Phalloidin, 200, 202 chemical classification of plant toxins, 383t
Pharmaceutical chemicals, 279, 280-282t, 286-287 clinical study of plant poisons, 389-390
Pharmacogenetics, 82 gastrointestinal system, 385-386, 385t ,
Pharynx, 225f kidney and bladder, 387
Phenacetin, 132t liver, 386-387
Phenobarbital, 126 nervous system, 387-389
Phenobarbital-like carcinogens, 126 neuromuscular junction, 389
Phenobarbital sodium, 7t poisoning syndromes, 383t
Phenol O-methyltransferase (POMT), 99 reproduction and teratogenesis, 389
Phenothiazine drugs, 265, 281t respiratory tract, 384-385
Phenotypic anchoring, 18f skeletal muscle, 389
Phenytoin, 132t, 156, 243t skin, 383-384
Pheochromocytoma, 324 Plaquenil, 266
Pheresis, 475 Plasma exchange or exchange transfusion, 475
Phonation, 225 Plasma proteins, 71-72
Phosgene, 233t Plasmin, 173
Phospholipids, 63 Plasticizers, 313-314
Phosphonomethyl amino acids, 343 Platelet-derived growth factor (PDGF), 40
Phosphorus, 296t Platelets, 171-172
Photo-induced toxicity, 261-262 Platinum, 246¢, 253, 358
Photoallergy, 299, 302 Plutonium, 379
Photochemical air pollution, 435-436 PM (particulate matter), 431-432, 434-435, 439
Photosensitivity, 298-299, 302, 384 PMN (polymorphonuclear cell), 181
Photosensitization, 444 Pneumoconiosis, 233t
Phototoxicity, 298-299, 299t, 302 Point of departure (POD), 54, 54f
PHS1/PHS2, 85 Poison, 6
Phthalates, 313-314, 327 Poison control center, 471
Physical activity, 404 Poison death. See Analytical and forensic toxicology
Physiologic parameters, 114-115 Poison ivy, 384f
Physiological toxicokinetics, 113-118 Poison nut tree, 388¢
blood compartment, 117-118 Poisoned patient. See Clinical toxicology
compartments, 114 Pokeweed, 385t
diffusion-limited compartments, 116 Poly(ADP-ribose) polymerase (PARP), 35-36, 39
liver compartment, 117 Polyaromatic hydrocarbons, 126
lung compartment, 116-117 Polybrominated diphenyl ethers (PBDEs), 252, 327
model structure, 114 Polychlorinated biphenyls (PCBs), 156, 252, 306, 327
parameters, 114-115 Polycyclic aromatic hydrocarbons (PAHs), 299t, 301, 443, 445
perfusion-limited compartments, 115-116 Polyisocyanates, 192
specialized compartments, 116-118 Polymorphonuclear cell (PMN), 181
transport, 115 POMT (phenol O-methyltransferase), 99
 INDEX 517

Population ecotoxicology, 445-446 Propylene glycol (PG), 369-370

Porphyria cutanea tarda, 298 Prostaglandin H synthetase (PHS), 85
Porphyrin derivatives, 299t Prostate gland, 307f
Portal vein, 197f Protan, 263
Positive acute-phase proteins, 41 Protein kinase C (PKC), 29
Post-DNA methylation, 127 Protein-ligand interactions, 71
Postovarian processes, 308-309 Protein repair, 38
Postreplication repair, 39 Protein toxin detoxication, 26
Posttesticular processes, 310 Proteome, 18
Potency vs. efficacy, 15 Proteomics, 18, 18f
Potentiation, 8 Prothrombin time (PT), 172, 173
Pott, Percival, 2 Proto-oncogenes, 43, 128, 128t
p,p-DDE, 313 Proximal tubular injury, 215-216
PPARa (peroxisome proliferator-activated receptor-a), 80, 96t, Proximal tubule, 211-212
124t, 126 Proximate carcinogen, 124
PR interval, 275, 275f Pseudocholinesterase, 82
Pralidoxime (2-PAM), 337 Psychoorganic syndrome, 363
Pre- and postnatal developmental toxicity study, 315-316, 316f Psychotropic agents, 287
Precautionary principle, 52 PT (prothrombin time), 172, 173
Pregnancy, 154, 311 PTH (parathyroid hormone), 327, 328
Pregnane X receptor (PXR), 80, 96t PTHRI, 328
Preimplantation, 152 Pubertal development, 306-307
Premature thelarche, 306 Pubertal female rat assay, 314
Preneoplastic cells, 45 Pubertal male rat assay, 314
Presystemic elimination, 22-23, 68, 82. Public health risk management, 58
Primary cilia, 226 Pulmonary edema, 230, 235
Primary DNA damage, 130 Pulmonary fibrosis, 234, 236
Primary lymphoid organs, 178 Pulmonary function tests, 234
Primitive streak, 153 Pulmonary lavage, 235
Principles of toxicology, 5-20 Pupillary reflex, 262
allergic reactions, 8 Pure red cell aplasia, 166
classification of toxic agents, 7 Purging nut, 385t
dose-response relationship, 10-15 Purkinje fibers, 273f
duration and frequency of exposure, 9-10 PXR (pregnane X receptor), 80, 96t
idiosyncratic reactions, 8 Pyrethroids, 338-339, 346
immediate vs. delayed toxicity, 8 Pyridinethione, 246f, 247
interaction of chemicals, 8-9 Pyrithione, 246t¢
key points, 6
LD. 757t
local vs. systemic toxicity, 8
margin of safety, 15 Q
potency vs. efficacy, 15 QRS complex, 275, 275f
reversible vs. irreversible toxic effects, 8 QT interval, 275, 275f, 277
route and site of exposure, 9 QT prolongation, 277-278
tolerance, 9 Quantal dose-response relationship, 10-13
toxicity tests, 16-17 Quicksilver, 353
toxicogenomics, 17-18 Quinidine, 169
variation in toxic responses, 15 Quinine, 243t
Probability distribution model, 55 Quinone reduction, 83-84
Probit units, 12
Procainamide, 192
Procarcinogen, 124
Prochloraz, 313 R
Procymidone, 313 Radiation and radioactive materials, 373-380
Progestins, 282t adaptive response, 377
Programmed cell death; 153 bystander effects, 376-377
Progression stage of carcinogenesis, 123-124, 123t cancer epidemiology, 377-379
Prokaryote gene mutation assays, 140, 141f, 142 cardiovascular disease, 379
Prolactin, 312 cataracts, 379
Proliferation, 40, 45 Compton effect, 373
Promotion stage of carcinogenesis, 123, 123t gene expression, 377
518 INDEX

Radiation and radioactive materials (continued) neonatal development, 305-306

genomic instability, 377 oogenesis, 308
ionizing radiation, 374 ovarian cycle, 308
mental effects, 379 parturition, 311
nontargeted radiation effects, 376 placenta, 311
radiobiology, 375-378 postovarian processes, 308-309
radionuclides, 378-379 pregnancy, 311
types of radiation, 373 pubertal development, 306-307
units of radiation activity, 374-375 reproductive cycle, 304, 304f
uranium decay series, 375f, 376t senescence, 312
x-ray, 301 sexual differentiation, 304-305, 305f
Radiation cancer studies, 377-379 sexual maturity, 307-309
Radiation DNA damage, 380 spermatogenesis, 310
Radical formation, 138f testicular structure and function, 309-311
Radiobiology, 375-378 testing for reproductive toxicity, 314-317
Radiocontrast agents, 221, 282t Reproductive toxicology, 6
Radioiodine, 379 Reptiles, 395-397
Radionuclides, 378-379 Residual volume (RV), 227
Radium, 378-379 Resistant, 11
Radon, 374, 378, 380 Respiratory distress syndrome, 230, 236
Ramazzini, Bernardino, 2 Respiratory system, 223-236
Random sampling, 488 acute lung injury, 230°
Ras proteins, 43 agents that produce lung disease, 232-233, 234
Rb, 129, 129t asthma, 231, 234 oe
Reabsorption, 24 biotransformation, 228
Reaction phenotyping, 89 bronchoconstriction, 230
Reactive oxygen species (ROS), 82, 181, 444 chronic obstructive pulmonary disease (COPD), 230-231
Receptor antagonism, 9 conducting airways, 224-225
Recombinational repair, 39 disposition mechanisms, 229, 229f
Recording difficulties, 158 evaluation of lung damage, 234-235
Red blood cells (RBCs). See Erythrocytes gas exchange region, 226-228
Red Book, 49 lung cancer, 231
Red marrow, 164 lung defense, 230
Redistribution of toxicants, 73 oronasal passages, 223
Redox cycling, 342 particle clearance, 230
Reducing-type air pollution, 432-433 plants and plant toxicities, 384-385
Reduction, 81f, 83-84 pulmonary fibrosis, 234
Reduction of sperm production, 318 regional particle disposition, 228
Reductive dehalogenation, 84 toxic inhalants/gases, 228
Reference concentration (RfC), 54 trigeminally mediated airway reflexes, 230
Reference dose (RfD), 54 in vitro studies, 235
Regeneration of damaged axons, 39 Respiratory toxicology, 224
Regional particle disposition, 228 Restrictive lung disease, 232t, 236
Regulatory toxicologist, 6 Retina, 256, 257f, 265-267
Remodeling, 277 Retinal pigment epithelium (RPE), 256, 263, 265
Remyelination, 248 Retinoblastoma (Rb) gene, 129, 129t
Renaissance, 2 Retinoids, 151
Renal artery, 210, 211f Retrobulbar neuritis, 267, 268
Renal failure, 212. See also Kidney Retrovir, 191
Renal papilla, 216 Retrovirus, 128
Renin-angiotensin system, 151, 284 Reversible intracellular binding, 24
Reproductive cycle, 304, 304f RfC (reference concentration), 54
Reproductive epidemiology, 157-159 R£D (reference dose), 54
Reproductive system, 303-318 Rhododendron, 388t
endocrine disruption, 312-314 RINmSF cells, 329, 330
erection and ejaculation, 310 Risk, 50
fertilization, 311 Risk assessment, 49-59
gametogenesis, 305 assessing toxicity of chemicals, 51-53
implantation, 311 decision making, 51
infantile development, 306 definitions, 50
lactation, 312 dose-response assessment, 53-56
 INDEX alt)

Risk assessment (continued) SD (standard deviation), 12

dose-response models, 55-56 SDR (short-chain dehydrogenase/reductase), 83
exposure assessment, 56 SDR carbonyl reductase, 83
information resources, 56-57 Seafood toxins, 459-460
NOAEL, 54, 54f 55 Sebaceous gland, 70f, 293f
objectives, 51t Secondary leukemia, 170
public health risk management, 58 Secretory leukocyte proteinase inhibitor (SLPI), 226
public opinion, 51 Sedimentation, 229, 229f
qualitative assessment, 53 Selective serotonin reuptake inhibitors, 281t
risk assessment/risk management framework, 50f Selective toxicity, 15
risk characterization, 56 Selenium, 356t
risk perception, 57-58 Semicarbazide-sensitive amine oxidase (SSAO), 85
risk space axis diagram, 57f Senescence, 312
six-stage framework, 51f Sensitivity, 145
stages of prevention, 58 Sensitization, 16
variation in susceptibility, 56 Sensitization reaction, 8
well being/susceptibility, 58 Serial oral administration of activated charcoal, 475
Risk Assessment in the Federal Government: Managing the Process, 49 Serous cells, 226
Risk characterization, 56 Set-point hypothesis, 403
Risk communication, 50 Sex-linked recessive lethal (SLRL) test, 142
Risk management, 50 Sexual assault, 466, 466¢, 470
Risk perception, 57-58 Sexual differentiation, 304-305, 305f
Risk space axis diagram, 57f Sexual maturity, 307-309
RNA interference, 154 Shaver disease, 232t
Rodent whole embryo culture, 159t SHE cell assay, 130-131
Rodenticides, 344 Shellfish processors, 483
Romeo and Juliet (Shakespeare), 2 Shh, 389
ROS (reactive oxygen species), 82, 181, 444 Short-chain dehydrogenase/reductase (SDR), 83
Rotenoids, 340 Short-term assay validation, 52
Rotenone, 340 Short-term exposure limit (STEL), 56
Roundup, 343 SHP, 96t
Roundworms, 3 Sick-building syndrome, 430, 431t
Rous sarcoma virus (RSV), 128 Side effects, 7
RPE (retinal pigment epithelium), 256, 263, 265 Sideroblastic anemia, 165, 165t
RSV (rous sarcoma virus), 128 Siderotic lung disease, 233t
Rubber products, 297t Sievert (Sv), 375
Rubratoxins, 460t Sigmoid dose-response curve, 12
Running spiders, 393 Sildenafil, 266, 282¢, 310
Ryania, 388t Silent Spring (Carson), 3
Silica, 193, 233t
Silicon dioxide, 193
Silicosis, 233t
S Silver, 356t¢
S-adenosylmethionine (SAM), 97f 99 Silver finisher’s lung, 233t
S-methylation, 99 Silver nanomaterials, 422
Saliva, 75 Simple diffusion, 63
SAM (S-adenosylmethionine), 97f 99 Simulations, 114
SAR (structure-activity relationship), 51 Single nucleotide polymorphism (SNP), 128
Sarcoma, 122 Single-strand break, 138f
Sarin, 337f Single walled carbon nanotube (SWCNT), 415, 416, 420
Saturation toxicokinetics, 113 Sinoatrial node, 273f
Sawmills, 483 Sinusoid, 197, 200
Saxitoxin, 459-460, 462 Sinusoidal damage, 199t, 200
SCE (sister chromatid exchange), 130, 139, 141t, 144 Siol-filler’s disease, 233t
Schistocytes, 168 Sister chromatid exchange (SCE), 130, 139, 141t, 144
Schlemm’s canal, 257f ~ Skin, 69-70, 70f, 291-302
Schmiedeberg, Oswald, 2 acne, 299
Schwann cells, 39, 240 anatomical diagram, 293f
Sclera, 257f biotransformation, 294
Scombrotoxicosis, 458 chemical burns, 294, 296t
Scorpions, 391, 391, 399 contact dermatitis, 294-296
520 INDEX

Skin (continued) SPARC, 405

factors influencing cutaneous response, 292t Special transport, 64-65
granulomatous reactions, 296 Specificity, 145
percutaneous absorption, 293-294 Spermatogenesis, 310, 318
photosensitivity, 298-299 Spiders, 391-393
pigmentary disturbances, 299-300, 300t Spirometry, 227
plants and plant toxicities, 383-384 SPLUNC2, 226
skin cancer, 301 Spontaneous depolarization, 278, 289
toxic epidermal necrolysis (TEN), 300-301 Spontaneous mutation, 139
transdermal drug delivery, 293-294 Spontaneous progression, 123
urticaria, 300, 300 SRY gene, 304
UV radiation, 296-298 SSAO (semicarbazide-sensitive amine oxidase), 85
Skin and eye irritations, 16 ST segment, 275, 275f
Skin cancer, 301 Standard deviation (SD), 12
SLC gene families, 65, 66¢ Staphylococcus aureus, 461
SLE (systemic lupus erythematosus), 186 Statistical distribution model, 55
“Slow” aldehyde dehydrogenase, 203, 208 Steady-state concentrations, 470
SLPI (secretory leukocyte proteinase inhibitor), 226 Steatoda species (spiders), 393
SLRL test, 142 Steatosis, 200-201
Smog, 436 Steep dose-response curve, 12
Snake venom metalloproteinase (SVMP), 397 STEL (short-term exposure limit), 56 »
Snakes, 395-397, 400 Stellate cells, 197, 202t .
SNP (single nucleotide polymorphism), 128 Sterigmatocystin, 460t
SO, (sulfur dioxide), 2334, 432-433, 439 Steroid hormone biosynthesis, 304 Sx
Sodium chloride, 7t Steroidogenesis, 322
Sodium-dependent taurocholate peptide (NTCP), 74f Stockholm Convention on Persistent Organic Pollutants, 340
Sodium fluoroacetate, 344 Storage of toxicants, 71-72
Sodium hydroxide, 296t Stratum corneum, 69, 70f, 293, 293f
Sodium nitrate, 475 Stratum germinativum, 70f, 293f
Solute carriers (SLCs), 65, 66¢ Stratum granulosum, 70f 293f
Solvent abuse, 363 Stratum spinosum, 70f, 293f
Solvents and vapors, 361-372 Streptokinase, 173
adverse health effects, 363 Streptomycin, 243t
alcohols, 368-369 Streptozotocin, 329
aromatic hydrocarbons, 367-368 Stress proteins, 214
automotive gasoline and additives, 371 Structural chromosome aberration, 139
carbon disulfide, 371 Structure-activity relationship (SAR), 51
children, 364-365 Strychnine sulfate, 7t
chlorinated hydrocarbons, 365-367 Subacute exposure, 9-10
chronic encephalopathy?, 363 Subacute toxicity, 16
classes, 362 Subchronic exposure, 9, 16
defined, 362 Subcutaneous injection, 70
diet, 365 Substrates (CYP system), 93-94t
elderly persons, 365 Sudden cardiac death, 278
environmental contamination, 363 Sulfate conjugation, 99
exposure limits, 363 Sulfation, 99
gender, 365 Sulfite oxidase, 84
genetics, 365 Sulfonate conjugation, 99
glycol ethers, 370 Sulfonation, 97f 99, 100t
glycols, 369-370 Sulfotransferases (SULTs), 99, 100t
inherent toxicity, 362 Sulfoxide and N-oxide reduction, 83
P450 inducers and inhibitors, 365 Sulfur, 345
physical activity, 365 Sulfur dioxide (SO,), 233t, 432-433, 439
physiologic modeling, 364 Sulfuric acid, 433, 439
solvent abuse, 363 SULTs (sulfotransferases), 99, 100t
solvent exposure pathways, 362f “Summer haze,” 435
toxicokinetics, 363-364 Sunscreen, 417
Somatic cells, 136, 139 Superoxide anion radical, 24, 25f 26f
Somatic recombination, 179 Suppressing agents, 129
Somatostatin, 329 Surfactant, 264
Space of Disse, 197 Surfactant protein Al/A2, 226
 INDEX 521

Surfactant protein D, 226 Testicular structure and function, 309-311

Sustainability, 58 Testis, 307f
SVMP (snake venom metalloproteinase), 397 Tetrachloroethylene, 366
SWCNT (single walled carbon nanotube), 415, 416, 420 Tetracycline, 281t
Sweat, 75 Tetrafluoroethylene, 219
Sweat gland, 70f, 293f Tetramine, 460
Sympathomimetic toxic syndrome, 472t, 479 Tetrodotoxin, 7t, 460
Sympathomimetics, 409 TF (transcription factor), 28
Synapse, 250f TGF (tubuloglomerular feedback), 212
Synergistic effect, 8 TGF-6 (transforming growth factor-B), 40, 43, 182t
Synthetic antisense oligonucleotides, 154 Thalidomide, 150
Syrian hamster embryo (SHE) cell assay, 130-131 Thallium, 243¢
Systemic effects, 8 Theophrastus, 2
Systemic lupus erythematosus (SLE), 186 Theophylline, 282
Therapeutic agents
immune system, 192-193
kidney, 220-221
T Therapeutic index (TI), 14-15
Ty, (half-life), 111f 112 Therapeutic monitoring, 468, 468t
T;, 324 Theraphosid spiders, 393
Ty, 324 Therapy-related AML and MDS, 170
T-2 toxin, 287 Thermodynamic parameters, 115
licell, 179; 183,185 Thermophilic actinomycete, 233t
T-cell proliferative responses, 188 Thiourea, 288
T-cell receptor (TCR), 179, 183 Thiram, 343, 343f
T-regulatory cells (Tregs), 183 Thorotrast, 132, 202
T syndrome, 339, 339t Threshold, 13-14
TAAR (trace amine-associated receptor), 224 Threshold concept, 153
Talc, 233t Threshold dose, 12
Talcosis, 233t Threshold dose-response relationship, 53-55
Tamoplex, 266 Threshold limit value (TLV), 483
Tamoxifen, 266 Thrombin, 173
Target organ toxicity Thrombocyte, 171
blood, 163-176 Thrombocytopenia, 171-172
endocrine system, 319-331 Thrombotic thrombocytopenic purpura (TTP), 171-172, 176
heart and vascular system, 271-289 Thyroid gland, 325-327
immune system, 177-194 Thyroid hormone, 282t, 325, 326
kidney, 209-222 Thyroid hormone binding proteins, 325-326
liver, 195-208 Thyroid hormone clearance, 326
nervous system, 237-253 Thyroid hormone receptors, 326
ocular/visual system, 255-269 Thyroid-stimulating hormone (TSH), 126-127, 326
reproductive system, 303-318 Thyroid toxicity, 326-327
respiratory system, 223-236 Thyroxine (T;), 324
skin, 291-302 TI (therapeutic index), 14-15
Target organs, 8, 62 Ticks, 393, 399
Target tissue, 62 Ticlopidine, 172
TAS, 224 Tidal volume (TV), 227
Taste buds, 224 Tin, 233t
Taurine, 97f, 102 Tissue culture system, 235
Taxol, 247 Tissue necrosis, 41-42
TCE (1,1,2-trichloroethylene), 365-366 Tissue repair, 39-41
TCR (T-cell receptor), 179, 183 TLR (toll-like receptor), 181
TD (toxic dose), 14f TLV (threshold limit value), 483
Tear film, 256 TNF-a (tumor necrosis factor-a), 276, 277, 282t
Tellurium, 248, 248t TNF receptor (TNFR), 276
TEN (toxic epidermal nécrolysis), 300-301 Tobacco plant, 388
Teratogens, 389 Tobacco smoking, 131t, 132, 151. See also Nicotine
Teratology, 6 : Tolerance, 9
Terminal bronchiole, 226f Toll-like receptor (TLR), 181
Terminal hepatic vein, 197f Toluene, 367-368
Terrestrial toxicology, 442, 448, 451 Toluene diisocyanate, 192, 283t, 296t
 2
1| iw) INDEX

Toluene leukoencephalopathy, 368 genetic. See Genetic toxicology

Torsade de pointes (TdP), 277, 278 historical overview, 1-3
Total body clearance, 112 occupational. See Occupational toxicology
Total body water, 404 society, and, 7
Total lung capacity (TLC), 227 specialties, 6-7
Toxic agents terrestrial, 442, 448, 451
food and nutrition, 401-410 Toxicology and Applied Pharmacology, 3
metals, 347-359 Toxicology Data Network (TOXNET), 56
pesticides, 333-346 Toxidromes, 472, 472t
plants and animals, 381-400 Toxin, 7
radiation and radioactive materials, 373-380 TOXNET, 56
solvents and vapors, 361-372 Trace amine-associated receptor (TAAR), 224
Toxic alteration of cellular maintenance, 33-38 Trachea, 225f
Toxic amblyopia, 267 Tracheobronchial clearance, 230
Toxic dose (TD), 14f Tranexamic acid, 173
Toxic effects, 7 Transcription factor (TF), 28
Toxic epidermal necrolysis (TEN), 300-301 Transcriptome analysis, 193
Toxic inhalants/gases, 228 Transcriptomics, 17-18, 18f
Toxic neutropenia, 170 Transdermal drug delivery, 293-294, 302
Toxic response Transferrin, 349
individual differences, 15 Transformation assay, 130
selective toxicity, 15 Transforming growth factot-B (TGF-6), 40, 43, 182t
species differences, 15 Transfusional siderosis, 356
Toxic syndromes, 472, 472t Transgenic animals (carcinogenicity assessment), 131
Toxicant, 7 Transgenic assays, 141f, 142
Toxicant delivery, 22-26, 23f Transient receptor potential (TRP) channels, 224
Toxicant dose, 482, 482t Translocation, 136
Toxicant-induced cellular dysfunction, 28-33 Transmembrane flux, 115, 116f
Toxicant-neurotransmitter receptor interactions, 33 Transversion mutation, 139, 147
Toxicant-signal terminator interactions, 33 Treatise on Poisons and Their Antidotes (Maimonides), 2
Toxicant-signal transducer interactions, 33 Tregs, 183
Toxication, 24-25 Triangle model of cardiac toxicity, 275, 275f
Toxicity tests, 16-17 Triazine herbicides, 342
Toxicodendron radicans (poison ivy), 384f Trichloroethylene, 365-366, 431
Toxicodynamics, 55f Trichloromethane, 367
Toxicogenomic databases, 56-57 Trichothecenes, 460t
Toxicogenomics, 6, 17-18 Tricyclic antidepressants, 281t
Toxicokinetics, 55f, 109-119 Triethyltin, 248¢
accumulation, 113 Trifluoperazine, 287
bioavailability, 112-113 Trigeminally mediated airway reflexes, 230
classical model, 110-113 Triiodothyronine (T,), 324
clearance, 112 Trimethylamine, 460
defined, 109 Trimethyltin, 243, 244
elimination, 111 Triphenyltin, 344
half-life, 111f 112 Tritanopia, 263
metabolic kinetics, 113 Trophic hormones, 126
one-compartment model, 110 TRP channels, 224
physiologically based model, 113-118 True bugs, 394
saturation, 113 TSH (thyroid-stimulating hormone), 126-127, 326
two-compartment model, 110-111 TTP (thrombotic thrombocytopenic purpura), 171-172, 176
Vd, 111-112 Tube hearts, 445
Toxicologist, 6 Tubocurarine, 7t
Toxicology Tubuloglomerular feedback (TGF), 212
animal, 428 Tumor, 136, 199¢, 202
aquatic, 442, 447, 451 Tumor necrosis factor-a (TNF-a), 276, 277, 282t
clinical. See Clinical toxicology Tumor-suppressor genes, 43, 128f, 129, 134
defined, 6 Tung nut, 385t¢
developmental. See Developmental toxicology Tunica adventia, 284f
environmental. See Environmental toxicology Tunica intima, 284f
ethical dilemmas, 7 Tunica media, 284f
food. See Food toxicology Tunnel vision, 268
 INDEX 529

Two-compartment model, 110-111, 119 Vasculitis, 285

Type I hypersensitivity reaction, 185-186, 185f 194 Vd, 71, 111-112
Type II hypersensitivity reaction, 186, 186f VDR, 96t
Type III hypersensitivity reaction, 186, 186f Venous system, 284
Ventricular arrhythmia, 278, 289
VEPs (visual-evoked potentials), 262, 263
U Vespidae (wasps), 394
Viagra, 266, 310
UDP-glucuronic acid, 96
Vigabatrin, 267
UDP-glucuronosyltransferase (UGT), 98
Vinblastine, 247
UDS (unscheduled DNA synthesis), 130, 140
Vinca alkaloids, 246t, 247
UF (uncertainty factor), 54
Vinclozolin, 313
UGT (UDP-glucuronosyltransferase), 98
Vincristine, 246t, 247
Ultimate carcinogen, 124
Vinyl chloride, 192-193, 488
Ultimate toxicant, 22
Violin spiders, 392-393
Ultrafiltration coefficient (K;), 210
Virtually safe dose, 55
Ultrafine carbon particles, 434
Vision. See Ocular and visual system
Ultraviolet light, 137
Visual-evoked potentials (VEPs), 262, 263
Ultraviolet radiation (UVR), 191-192, 261-262, 296-298
Vital capacity (VC), 227
Uncertainty factor (UF), 54
Vitamin A hepatotoxicity, 202
Uncontrolled proliferation, 45
Vitamin D, 287
Undesirable effects, 7
Vitamin K, 173
Unscheduled DNA synthesis (UDS), 130, 140
Uranium decay series, 375f, 376t
Vitiligo, 300
Volatile organic compounds (VOCs), 363, 364, 429, 430, 435
Urate transporter (URAT), 73f
Voltage/Ca**-activated K* channel, 32t
Ureter, 307f
Voltage-gated Ca** channel, 32t
Urethra, 307f
Voltage-gated Na* channel, 31¢
Uridine diphosphateglucoronic acid (UDP-glucuronic acid), 96
Volume of distribution (Vd), 71
Urinalysis, 17
Vomeronasal receptors, 224
Urinary alkalinization, 474
von Willebrand factor (vWf), 173t
Urinary excretion, 73-74
Urtica ferox (nettles), 384f
Urticaria, 300, 300t, 302
Urushiol, 19 W
Uterotropic assay, 314
Wallerian degeneration, 240, 241, 253
Uterus, 309
Wallerian-like axonal degeneration, 240
UV-induced immunomodulation, 192
Warfarin/warfarin poisoning, 171, 173, 344, 346
UV radiation, 191-192, 261-262, 296-298
Wasps, 394
Well-being, 58
Wheat proteins, 457t, 462
V Widow spiders, 392, 399
Valproic acid poisoning, 477-478 Wiley Bill (1906), 3
Vanadium, 233t Wilms’ tumor gene (WT1), 129, 129t
Vanilloid receptor, 389, 399 Wilson’s disease, 197, 208, 355-356, 359
Vanishing bile duct syndrome, 200 Wilson's principles of teratology, 151t
Vapor, 68. See also Solvents and vapors Wiseria, 385t
Variation in susceptibility, 56 Wood alcohol, 369
Vas deferens, 307f Woodchip handling, 483
Vasa recta, 210 Work environment. See Occupational toxicology
Vascular endothelial cells, 285 Workplace exposure limits, 483
Vascular space, 114, 114f Wali 2 O29
Vascular system, 283-288. See also Heart
atherosclerosis, 286
edema, 286
hemorrhage, 286 X
hypertension/hypotension, 285-286 X-ray (radiation), 301
local metabolic regulation, 284-285 Xanthine dehydrogenase (XD), 84
mechanisms of vascular toxicity, 285 Xanthine oxidoreductase (XO), 84
neurohormonal regulation, 284 XD (xanthine dehydrogenase), 84
physiology and structural features, 283-284 Xenobiotic biotransformation. See Biotransformation of xenobiotics
toxic chemicals, 286-288 Xenobiotic-biotransforming enzymes, 80, 82
524 INDEX

Xenobiotic N-acetylation, 99-102 Z

Xenobiotic transport, 115
Zearalenones, 460t
Xenobiotic transporters, 64-65
Zebrafish, 3, 445
Xenosensors, 80, 95
Zebrafish assay, 159t
XO (xanthine oxidoreductase), 84
Zero-order processes, 113
Xylenes, 368
Zidovudine, 191
Zinc, 356-357, 356t, 447
Zinc pyridinethione (ZPT), 247
Y Zineb, 343f
y6 T cell, 181 ZPT, 247
Yellow marrow, 164 Zygote, 152
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