AFLATOXINS: Their Origin, Biosynthesis, Association with

Cancer and Regulations

Mekelle University
College of Health Sciences
School of Pharmacy
Department of Pharmacognosy

Tesfay H. January, 2018

 Objective
General objective
 To synthesize the current information on aflatoxins (AFTs), their origin,

biosynthesis, association with cancer and regulations

Specific objectives
 To review information provided by relevant articles about

 Aflatoxins and their origin

 Chemistry and biosynthesis
 Association of aflatoxins with cancer
 Different regulatory strategies of different organizations and countries and aflatoxin
determination techniques
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 Methodology
Electronic databases used Commonly used search terms
• aflatoxins, mycotoxins, health impacts of aflatoxins
 PubMed • history of aflatoxins, sources of aflatoxins
• chemistry of aflatoxins, biosynthesis of aflatoxins
 Science Direct • aflatoxins and cancer, association of aflatoxins and cancer,
• determination of aflatoxins, and regulation of aflatoxin
 Research Gate contamination
• Aflatoxin prevalence in Ethiopia, Regulation of aflatoxins in
 Google Scholar Ethiopia

 Searches were limited to articles written in English published by peer reviewed

journals and international organization.

 The search results were assessed for relevance by comparing the tittle, abstract

and the full text.

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 Outline
 Background, classification and health consequences of aflatoxins

 Origin of aflatoxins (historical background and potential sources)

 Chemistry and biosynthesis of aflatoxins

 Aflatoxins as liver carcinogens

 Regulations of aflatoxins

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 INTRODUCTION

• What are Aflatoxins?

• Classification of Aflatoxins
• Health Consequences of Aflatoxins

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 Aflatoxins?
What are aflatoxins?
 Group of secondary metabolites derived from polyketides produced by fungal

species such as:

 Aspergillus flavus
 A. parasiticus
 rarely A. nomius (Payne and Brown, 1998).

 Under humid conditions, these fungi are produced in

 Groundnuts, corn/ maize, sorghum, rice, spices and other agricultural products
 Livestock feeds
 Medicinal herbs
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 Aflatoxins?...
Aflatoxin contamination

can occur at any stage of food production

 from pre-harvest to storage stages of the food chain

 Aflatoxin accumulation is dependent on

 Environmental factors such as:

 moisture
 temperature
 plant density

 poor harvest practices and

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 Improper grain storage
 Aflatoxins?...
 The removal of aflatoxins is very difficult due to their

stability and thermal resistance in dried products

melting points > 250oC
stable at a pH range of 3 to 10.
resistant to food processing
 Thus remain unchanged throughout the food chain.

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 Classification of Aflatoxins
 There are more than 20 known aflatoxins, but the four main ones are

aflatoxin B1 (AFT-B1)
 AFTs B series are produced by
aflatoxin B2 (AFT-B2)  A. flavus, A. parasiticus and A. nomius
aflatoxin G1 (AFT-G1)  AFTs G series are produced by

aflatoxin G2 (AFT-G2)  A. parasiticus and A. nomius

 Named based on their

 blue (B) or green (G) fluorescence under UV light

 relative mobility, 1 and 2 (higher and lower respectively), by TLC

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 Classification of Aflatoxins…
 Other members of the aflatoxin family

 AFT-M1 and AFT-M2

 are oxidative forms of AFT-B1

 modified in the digestive tract of animals

 isolated from milk, urine and feces

 The level of toxicity associated with aflatoxin varies with the types present

 AFT-B1 > AFT-G1 > AFT-B2 > AFT-G2

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 Health Consequences of Aflatoxins
 Aflatoxins are of great concern to public health because of their ability to

accumulate in the body

found in edible tissues of animals, such as liver and muscles
found in animal food products such as milk and eggs
found in human maternal breast milk and maternal cord blood

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 Health Consequences…
 Exposure to aflatoxin can lead to several health-related conditions including

 acute and chronic aflatoxicosis

 aflatoxin-related immune suppression
 liver disease (liver cancer and liver cirrhosis)
 adverse pregnancy outcomes, including
 intrauterine growth restriction
 premature delivery
 pregnancy loss

 nutrition-related problems in children such as stunted growth

8  mutagenesis
 ORIGIN OF AFLATOXINS

• Historical Background of Aflatoxins

• Potential Sources of Aflatoxins

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Historical Background of Aflatoxins
Late 1950s and early 1960s
• Turkey “X” disease in the UK, more than 100, 000 died
• Resulted from A. flavus highly infested peanut feed
• Understanding towards AFTs came to light
• Name was given by designating ‘‘A’’ for the genus Aspergillus and ‘‘FLA’’ for the species flavus and
toxins

In 1974
• Outbreak of hepatitis due to aflatoxin in the states of Gujrat and Rajasthan, and North west
part of India
• About 106 deaths
• Aflatoxin contained in the staple food prepared by maize

In 1981
• Major Aflatoxin outbreak occurred in East Province of Kenya
• Occurred because of consumption of home grown maize contaminated with
molds
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Historical Background…
In 1963
Structural characterization and synthesis of the major AFTs

In 1968-1985
Evaluation of the association of AFTs ingestion and HCC incidence in humans

Since 1985
Different organizations and countries set regulatory strategies
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 Potential Sources of Aflatoxins
 The major sources of aflatoxins are fungi such as

 Aspergillus flavus
 Aspergillus parasiticus the most commonly found in several foods

 Aspergillus nomius

 Taxonomically, these fungal species are from:

 Phylum: Ascomycota
 Class: Eurotiomycetes
 Order: Eurotiales
 Family: Trichocomaceae
10 (French Agency for Food, Environmental and Occupational Health & Safety, 2012)
 Potential Sources…
 The fungal species infect many crops in the field, during harvest, in storage,

and during processing.

A. flavus is dominant in corn, cottonseed, and tree nuts

A. parasiticus is dominant in peanuts.

A. nomius has been reported from tree nuts, sugarcane and insects such

as alkali bees

 They can grow at temperatures ranging between 12 and 48oC and 40

degrees latitude north and south

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 BIOSYNTHESIS OF AFLATOXINS

• Chemistry of Aflatoxins
• Biosynthetic Pathway of Aflatoxins
• Factors affecting Aflatoxin Synthesis

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 Chemistry of Aflatoxins
 AFTs are difuranocoumarin derivatives

 in which a bifuran group is attached at one side of the coumarin nucleus.

 AFTs B series

 pentanone ring is attached to the other side

 AFTs G series

 a six-membered lactone ring is attached(Kumar et al. 2016)

 AFTs M1 and M2 (milk toxins)

 bear a hydroxy group at the junction of the two furan rings (Bräse, 2013)

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 Chemistry…
 Structures were revealed by Büchi

 In 1963 (AFT-B1 and G1)

 In 1965 (AFT-B2 and AFT-G2 )

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 Chemistry…

Physicochemical properties of aflatoxins

Form colourless to pale-yellow crystals

Intensely fluorescent in ultraviolet light

Slightly soluble in water (10–30μg/mL)

Insoluble in non-polar solvents

Freely soluble in moderately polar organic solvents

 Examples: methanol and especially in dimethyl sulfoxide (IARC, 2002).

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 Biosynthesis of Aflatoxins
 The biosynthesis of AFTs takes place by a polyketide metabolic pathway

Consists at least 18 multienzymatic conversion reactions

As many as 30 genes are also potentially involved

A positive regulatory gene, aflR,

 required for transcriptional activation of most of the aflatoxin structural

genes
 Biosynthesis starts from a hexanoyl-CoA starter and seven malonyl-CoA

extender units
 Formation of a poly-β-keto chain

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 Biosynthesis…
NADPH
acetyl-CoA + 2 ×
malonyl-CoA

23 versicolorin B
 Biosynthesis…

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 Factors Affecting Aflatoxin Biosynthesis
The major factors that influence aflatoxin biosynthesis

 Nutritional factors

 Including carbon and nitrogen sources

 Environmental factors

 Including water activity and temperature

 Developmental factors

 Including sporulation and sclerotial formation

 Plant metabolites

 Including n-decyl aldehyde, Octanal, hexanal, 13(S)-hydroperoxide derivative of linoleic

15 acid
 Factors …
I. Nutritional factors
 Simple sugars such as glucose, sucrose, fructose, and maltose

support aflatoxin formation

while peptone, sorbose, and lactose do not
 The sources of nitrogen such as

 Asparagine, aspartate, alanine, ammonium nitrate, ammonium nitrite,

ammonium sulfate, glutamate, glutamine, and proline containing media
 support aflatoxin production
 while sodium nitrate and sodium nitrite containing media do not
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 Factors…
II. Environmental factors
 The fungi are capable of growing over a wide pH range (Yu, 2012).

 AFTs synthesis optimally occurs in the pH range of 3.4–5.5.

 The optimum temperature for AFTs production is between 24° and 30°C

 The higher the water activity, the better are the conditions for fungal

growth and toxin synthesis (Caira et al. 2012).

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 Factors…
III. Developmental stage
 Spore formation and secondary metabolite formation occur at about the same

time.
 Some mutants that are deficient in sporulation

 Are unable to produce aflatoxins

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 Factors…
IV. Plant metabolites
Metabolites Fungal growth AFTs production
n-decyl aldehyde Reduces Reduces by 95%
Octanal Reduces by 60% Increases by 500%
Hexanal Reduces by 50% No effect
13(S)-hydroperoxide Reduces Reduces
derivative of linoleic
acid

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ASSOCIATION OF AFLATOXINS WITH CANCER

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 Association of Aflatoxins with Cancer
 The IARC (1993) classified AFTs as group I carcinogens

 The most potent naturally occurring human hepatocarcinogens

 AFT-B1 is the most potent
 Direct correlation b/n AFT degree of exposure and incidence of HCC
(with odds ratio 3.74:1.0 to 10.86:1.0) (Kew, 2013)

HCC
• Accounts for about 9.2% of all new cancers worldwide
• Approximately 84% of all new cases occur in resource limited regions
• Ranked third in annual cancer mortality rates
20 • About 93% of patients dying within 12 months of the onset of symptoms
 Association …
AFTs poisoning in the presence of HBV infection

 Increased risk of HCC development

 30X greater than the risk in individuals exposed to aflatoxin only

 AFTs with chronic HBV act as cofactor for HCC development

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 Association…
Mechanism of AFT induced carcinogenesis
 Hepatic enzymes metabolize AFTs, generating reactive epoxide species

 The epoxide species form a covalent bond with guanine

 Results in DNA adducts

 The adducts produce mutation in the p53 tumor-suppressor gene

 a G-to-T transversion at codon 249

 biomarker for aflatoxin contamination
 unscheduled DNA synthesis and chromosomal strand breaks in human cells

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 REGULATIONS OF AFLATOXINS

• Regulations of aflatoxins
• Determination of aflatoxins

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 Regulations of Aflatoxins
 When AFTs are confirmed to cause cancer, countries established various

regulations (>75 countries) (Ali et al. 2005).

Either total AFTs or AFT-B1
 In view of toxic and carcinogenic effects of aflatoxin contaminated foods,

 USDA and FDA set the tolerance limits for AFB1 and total AFTs in food

 AFT-B 5μg/kg
1-

 Total AFTs - 20μg/kg

 The Codex Alimentarius Commission, adopted a limit of

 15μg/kg for total AFTs

 10μg/kg total AFTs in ready-to-eat almonds, hazelnuts, and pistachios (Siddique
23
et al. 2013).
 Regulations…
 EU countries allow much lower permitted concentration of aflatoxins

 AFT-B1 - 2μg/kg
 Total AFTs - 4μg/kg

 AFTs remains largely unregulated throughout Africa.

 As of 2003, aflatoxin regulations existed for five countries (Van

Egmond and Jonker, 2004)
 Ethiopia has no aflatoxin and other mycotoxin regulation (Wolde, 2016).

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 Regulations…
Permissible limits of aflatoxins in different countries (Sarma et al. 2017)
Country Food items Limits(μg/kg)
USA Nuts, figs, and related products 2-12
UK Milk, dairy products, barley, cocoa, coffee, corn, 0.5-20
peanuts, oats, rice, sorghum, soy, tree nuts, and
wheat
EU Groundnut, peanuts, spices and other food stuff 2-12
Australia All the food stuff except peanuts 5
Peanuts and related products 15
India Nuts, spices, cereals, and all other food products 30
China Rice, sorghum, barley, nuts etc. 5-50
Japan All of the food stuffs 10
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 Determination of Aflatoxins
 The determination of AFTs has been carried out using

 TLC
 HPTLC
 HPLC
Designed to detect the possible presence of
 LC–MS
AFTs B1, B2, G1 and G2 (Wolde, 2017).
 immunological methods

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 Determination…
 The WHO quality control for herbal materials guideline (1998)

 Recommended to use HPLC system based tests

 Why HPLC is recommended?

 It does not require the use of toxic solvents

 such as chloroform and dichloromethane

 It uses a multifunctional column, which contains lipophilic and charged

active sites
 It uses fluorescence detection to determine aflatoxins B1, B2, G1 and G2

 It has high precision, high sensitivity, and high automation

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 Future directions
 A holistic and multidisciplinary approach is required to mitigate the exposure

to this toxin
 Increase awareness of AFTs and support risk mitigation practices

 Future epidemiologic studies of HCC should focus on good practices

 in food preparation
 food storage
 consumption of aflatoxin containing foods and milk

 In Ethiopia

 more emphasis to develop regulatory actions and preliminary prevention

40 strategies
 Conclusion
 AFTs are the most well-known and researched mycotoxins.

 Maize and groundnut are the most contaminated commodities

 Like the food items medicinal herbs can also be infected by these pathogens

 Contamination is more common in developing countries than the developed

countries
 AFT-B1 is the most potent AFT to cause HCC and other adverse health

effects to human and animal.

 Even though it is common in Ethiopia, the country has no aflatoxin and

other mycotoxins regulatory systems

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 References
Aflatoxins review (final).pdf

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THANK YOU!