Mycotoxins are secondary metabolites of fungi in the plants before or after harvest, which are capable of producing acute or chronic toxic effects (e.g. carcinogenic, mutagenic, and teratogenic) on animals and probably on humans at the levels of exposure. Several mycotoxins in agricultural products cause health hazards to people and animals and economical problem. Dangerous mycotoxins are naturally present in foods, feeds and our environment. They are pathologically classified as hepatotoxins, nephrotoxins, vomitoxin and neuromuscular toxin, some of which are potentially carcinogenic and mutagenic. Aflatoxin, for example, is the most potent hepatocarcinogen and mutagen among mycotoxins. Modern mycotoxicology began with the discovery of Aflatoxin in the early 1960s as the chemical compound responsible for causing “Turkey X” disease. Over 100,000 turkeys died in the United Kingdom after ingesting feed containing contaminated peanut meal from Brazil. The disaster concerned also ducklings, calves, and pigs. Toxic syndromes, resulting from the intake of Mycotoxins by man and animals, are known as mycotoxicosis. Although mycotoxicosis caused by mould Claviceps purpurea have been known for a very long time.

1. MYCOTOXINS

2. GUJARAT FORENSIC SCIENCE UNIVERSITY

3. Foodborne Diseases
Infections
Poisonous
Animal
Tissues
Poisonous
Plant
Tissues
Chemical
Poisoning
Intoxications
Microbial
Intoxicati
ons
Other Neurotoxins Enterotoxins
Toxic infection Invasive Infection
Intestinal
Mucosa
Systemic
Other Tissues or
Organs
(Muscle, Liver, Joints,
Fetus, Other)
Mycotoxins
(Fungal Toxins) Algal Toxins Bacterial Toxins
Diarrhogenic Emetic Enterotoxins Neurotoxins Other

4. What are Mycotoxins?
Mycotoxins are secondary metabolites of fungi in
the plants before or after harvest, which are
capable of producing acute or chronic toxic
effects (e.g. carcinogenic, mutagenic, and
teratogenic) on animals and probably on humans
at the levels of exposure.
.
• Mould growth in foods is very common, especially in warm and humid climates. It can
occur in fields or in storage after harvest. Mould infection of foods such as grains, seeds
and nuts is often localized in pockets, especially in bulk storage and warehouses.
• Currently a few hundred Mycotoxins are known, often produced by genre, Aspergillus,
Penicillium and Fusarium.
• The fungi are heterotrophic and feed by absorption of soluble nutrients and although many
fungi can metabolize complex insoluble materials, such as lignocellulose, these materials
have to be degraded by the secretion of appropriate enzymes outside the wall.
• A number of fungi are parasitic on both animals, plants and other fungi, and some of these
parasitic associations have become very complex and even obligate.
Introduction

5. HISTORY
• Modern mycotoxicology began with the discovery of Aflatoxin in the early 1960s as
the chemical compound responsible for causing “Turkey X” disease. Over 100,000
turkeys died in the United Kingdom after ingesting feed containing contaminated
peanut meal from Brazil. The disaster concerned also ducklings, calves, and pigs.
• Aspergillus flavus was established as the responsible organism and aflatoxin as the
causative substance.
• Toxic syndromes, resulting from the intake of Mycotoxins by man and animals, are
known as mycotoxicosis. Although mycotoxicosis caused by mould Claviceps
purpurea have been known for a very long time.
• Although the name implies a disease such as a viral infection, it was shown that the
birds had been poisoned by a contaminant in the groundnut meal used as a protein
supplement in the pelleted feed.
• The contaminant, which was called aflatoxins, fluoresces intensely under ultra-
violet light and was shown to be produced by the mould Aspergillus flavus growing
on the groundnuts.
• Mycotoxins remained neglected until the discovery of Aflatoxins in 1960.

6. GENERAL ACTION MECHANISM
• The filamentous fungi grow over and through their substrate by processes of hyphae tip
extension, branching and anastomosis leading to the production of an extensive
mycelium.
• Some species have been especially successful in growing at relatively low water activities
which allows them to colonize commodities, such as cereals, which should otherwise be
too dry for the growth of micro-organisms.
• The formation of an epoxide could well be the key to both acute and chronic toxicity and
those animals which fail to produce it are relatively resistant to both. Those animals which
produce the epoxide, but do not effectively metabolize it further, may be at the highest
risk to the carcinogenic activity of aflatoxin B1 because the epoxide is known to react with
DNA. Those animals which not only produce the epoxide but effectively remove it with a
hydrolase enzyme, thus producing a very reactive hydroxyacetal, are most sensitive to the
acute toxicity. The hydroxyacetal is known to react with the lysine residues in proteins.
• It is now known that aflatoxin B1 epoxide reacts rather specifically with guanine residues
of DNA at a number of hot spots, one of which is codon 249 of the p53 gene. The product
of this gene is involved in processes which normally protect against cancer and it is known
that the hepatitis B virus binds to the p53 gene product. Thus with aflatoxin B1 and
hepatitis B interacting with p53 in different ways it is easy to see that they could act
synergistically.

9. The chemical structures of some important
Mycotoxins are shown as below:

11. AFLATOXINS
• Aflatoxins are probably the most common and widely known Mycotoxin
contaminant.
• Aflatoxins (AT) are structurally related coumarin derivatives, altogether at least
13 types, the molecules of which contain a domain of condensed dihydrofuran.
• It is produced by the moulds, Aspergillus flavus and Aspergillus parasiticus.
• Develop at high temperatures and humidity levels.
• Foods Affected: Nuts, especially groundnuts, tree nuts such as pistachio and
Brazil nuts, cottonseed, copra, Almond, Paprika, rice, maize, wheat, sorghum,
pulses , figs and oilseed cakes.
• Unrefined vegetable oils made from contaminated seeds or nuts usually contain
Aflatoxins. However Aflatoxin is destroyed in the refining process so that refined
oils are safe.
• Types: Aflatoxin B1, B2, G1,G2, M1, M2.
 M1 & M2 as metabolites in the milk of animals who have been fed
contaminated feed & have been metabolized within the body of a lactating
animal.
 G1, G2, B1 & B2 occurs in foods (Most toxic, carcinogenic & causes cirrhosis of

12. • Caused by the postharvest storage of foodstuffs in improper conditions (above all, at a high
temperature and water activity).
• An earlier contamination of plant material with aflatoxins is also possible.
• ATM is stable in raw milk, milk products, pasteurized milk, cheese, yogurt (During the preparation
process of a fermented milk product, AFM1 concentrates in curds).
• ATB1 is partly metabolized in the liver into a number of highly reactive compounds such as ATB1-
8,9-epoxide .
• ATB1-8,9-epoxide capable of conjugating with a guanidine base of DNA. It leads to the breaking of
the DNA chain, replacement of base pairs, transversion, or frame shift mutations thus can be
mutagenic.
• In the mammals, the conjugation reaction is fortunately counterbalanced with detoxication, for
example, via non enzymatic conjugation of the epoxide with glutathione.
• Symptoms of chronic Aflatoxixosis: cholangial proliferation, periportal fibrosis, jaundice, liver
cirrhosis, weight loss, and an elevated sensibility to illnesses. Long-term exposure of animals to low
doses of ATB1 may lead to the formation of hepatome, cholangial, or hepatocellular carcinoma, and
other malignant tumors.
• In addition, ATB1 inhibits the synthesis of DNA, activity of the DNA dependent RNA polymerase, and
hence the synthesis of mRnA and protein molecules.
• Results can be
1. The formation of the so-called fatty liver connected with a loss of ability to remove fats from the liver
2. Coagulopathy caused by the inhibition of prothrombin synthesis
3. Reduced immuno function
4. Aflatoxins are well known as potent human hepatocarcinogens.

13. DIFFERENCES & ANALYSIS
• Letters B—blue and G—green indicate the color of the
respective aflatoxin band at the TLC plate irradiated by
ultraviolet (UV).
• Aflatoxin B - Both A. flavus and A. parasiticus
• Aflatoxin G - A. parasiticus
• The only structural difference between B and G toxins is the
inclusion of an oxygen in the cyclopentanone ring.
• Structural difference between B and M is the addition of the
hydroxyl group.

14. • Typical symptoms of sub chronic intoxications by aflatoxins are jaundice, hypo
protrombinemia, hematoms, and gastroenteritis.
• Limit: limit set by the US FDA is 100–300 ppb for corn and other feed items, and 0.5 ppb
(µg/kg) for milk. For other items of food, the action limit has been set to 20 ppb.
• EU Commission Directive on July 16, 1998 fixed the maximum aflatoxin content in
commodities, anticipated for human food to 2 µg/kg. For famine mitigation programs, this
limit has been raised to 30 µg/kg by FAO and WHO.
• Toxicity of aflatoxins can be reduced by the dietary modulation of the biotransformation
and genotoxicity of aflatoxins either by the induction of detoxification enzymes such as
glutathione S-transferases or the inhibition of enzymes (CYP1A2) converting ATB1 into the
genotoxic metabolite AFBO.

15. Detection Methods:
• Aflatoxins are subject to light degradation. Therefore, all analytical materials must be adequately
protected from light and standard Aflatoxin solutions stored using amber colored vials or aluminium
foil.
Extraction of Raw Materials:
•Typical solvents such as acetone, chloroform, or methanol and
their mixtures are used
Purification and concentration of the extract:
•Carried out by SPE on micro columns filled with C18 or
immunoaffinity sorbent
Identification and quantitation:
• Chromatographic (HPLC and TLC)
•Immunochemical (ELISA, RIA, and ICA)
Detection:
• UV or fluorescence
• MS/MS detector

16. STERIGMATOCYSTIN
• A precursor in the biosynthesis of Aflatoxins B1.
• Produced by A. flavus, A. versicolor, A. sydowi, A. nidulans, Bipolaris spp., Chaetomium
spp., and Emericella spp.
• The toxin was first detected in brown rice that was stored at warehouse conditions .
• Sources: Cheeses of the Edam and Gouda type (grow only on the surface), wheat, green
coffee beans, corn.
• It is mutagen and a teratogen, and a hepatic carcinogen.
• It has been estimated that the carcinogenicity of sterigmatocystin is 10 times lower than
the same characteristic of aflatoxin B1.
• In this case, Sterigmatocystin does not penetrate beyond the first few millimetres below
the surface.
• Grows in damp environment & due to storage in warehouses for a long period of time.
• Analysis: Preliminary screening through TLC & HPLC.
• Most sensitive is an LC-MS method anticipated for the sterigmatocystin analysis in cheese,
bread, and corn products.
• Symptoms in Animals include: Pulmonary cancer of mice, and renal and hepatic cancer in
the case of cynomolgus, rhesus, and African green monkeys.

17. OCHRATOXIN
• Ochratoxins (OT) are a group of four moderately stable derivatives of isocoumarin linked via a amide bond to
the amino acid phenylalanin.
• Ochratoxins include ochratoxin A (OTA), ochratoxin B (OTB), ochratoxin C (OTC), and ochratoxin α (OTα), of
which OTA is the most abundant and also of the highest toxicity.
• First isolated from Aspergillus ochraceus in South Africa.
• Penicillium verrucosum can also produce.
• Potential nephrotoxic & cause cancer of the kidneys.
• Sources: Barley, corn, wheat, oats, rye, green coffee beans, peanuts, grape juice and wine, beer, cocoa, dried
fruits, and spices.
• Levels: EU, the maximum residue limit (MRL) of OTA in food cereals and grain products is 5 µg/kg
• Effects: described as nephrotoxic, carcinogenic, teratogenic, immunotoxic, and hepatotoxic & nephropathies
and urothelial tumors in humans.
• Symptoms: Primary symptoms of poisoning, such as fatigue, tiredness, anorexia, diffuse abdominal pain, and
severe anemia, are followed by the symptoms of renal damage such as a sequentially reduced concentration
ability, reduced intrarenal bloodstream, reduced glomerular filtration accompanied by general and
microscopic alterations of the kidneys, including necrosis, fibrosis, glomerular hyalinization, and interstitial
sclerosis, followed by death via uremia.
• Cellular toxicity is connected with the enzymes of glucose metabolism (reduced gluconeogenesis) and of
anion transport, leading to intercellular alkalinization.

18. Methods of Analysis:
• Precautions: Products must be maintained at 5–10°C and packaged products at 15–
20°C and their water content must be under 0.75–0.80.
Extraction of Raw Materials:
• Mixtures of water with various organic solvents
Purification and concentration of the extract:
• immunoaffinity columns
Identification and quantitation:
• HPLC-FID or HPLC-MS, TLC
• mouse bioassay
• immunochemical methods
Detection:
• PCR methods

19. Zearalenone/zearalenol
• Oestrogenic mycotoxin , endocrine disrupters .
• Produced by Fusarium (mainly by F. roseum or F. graminearum and F. culmorum).
• Sources: Maize, wheat, barley, oats, cassava, soy, sorgo, bananas, and other fruits.
• Also known as F-2 toxin, has also been found in beer.
• Caused due to long-period low positive temperatures and oscillations between low and
medium temperatures.
• Symtoms/Effects: Hyperestrogenicity syndrome, infertility of both female and male
animals.
1. interaction of zearalenone with estrogen receptors.
2. transportation of the estrogen–receptor complex into the cellular nucleus
3. conjugation with chromatin receptors
4. selective transcription of RNA
5. Thus, an increase of the muscular content of water and a decrease of the lipid
content & enhancing the uterus permeability in relation to glucose, RnA, and
preproteins.
• The possible impact of combined exposure to zearalenone with other estrogenic
substances in food (such as phytoestrogens in soya) or the environment could be additive
or antagonistic.

20. FUMONISINS
• Produced by Fusarium (F. monoliforme and F.
proliferatum).
• Sources: Corn, sorghum, white beans, adzuki
beans, mung beans, wheat, barley, soybean,
asparagus spears, figs, black tea, and medicinal
plants.
• Three fumonisins—B1, B2, and B3— are the
best known; less known are B4 and fumonisins
of type A (1–3), C1, and P (1–3).
• Feed can be contaminated most by Fumonisin
B1.
• Effects/Symptoms: leukoencephalomalacia;
hepatic and renal toxicoses of horses, hogs,
and rats; and porcine pulmonary edema and
hepatic cancer of rats.
• High rate of esophageal cancer in the Transkei
region of South Africa as well as with endemic
hepatic cancer in some regions of China.
• Exposure to fumonisin increased lipid
peroxidation in the liver, which is one of the
main manifestations of oxidative damage that
has been found to play an important role in the
toxicity and carcinogenicity of fumonisins.

21. PATULIN
• Produced by several species of Penicillium, Aspergillus and Byssochlamys but is especially associated
with P. expansum.
• Stable at the relatively low pH, but can be destroyed while fermentation.
• Sources: Barley malt sprouts, Apples, plums, pears, apricots, cherries, and grapes, Juices, Cheese
• Causes rotting of apples and pathogenesis of many fruits and vegetables.
• Limit: 50ppb in 33 countries for fruit juices.
• Precautions: Outer Surface of fruits should be protected to minimise damage and reduce chances of
fungal growth.
• PAT is unstable in the presence of sulfhydrylic compounds, and sulfur dioxide, but resistant to
pasteurization.
• Up to 99% of PAT is destroyed during fermentation, for example, during the making of apple cider.
• Symptoms/effects: mutagenic, carcinogenic to rats, and teratogenic to hens, inflammations, ulcers,
and bleeding in the gastrointestinal tract.
• Mechanism of the adverse effect of PAT is the covalent binding to the cellular nucleophiles, particularly
proteins and SH-groups of glutathione.
• As a result, covalently cross-linked over thiol and amino groups, essentially denatured proteins such as
inhibited protein tyrosine phosphatase, are formed.
• In recent study with mice, genotoxic effects via DnA strand breaks and pro-oxidant effects, increasing
lipid peroxidation in several organs.

22. CYCLOPAZONIC ACID
• First isolated from Penicillium cyclopium (now known as P. aurantiogriseum)
• Subsequently been isolated from Aspergillus versicolor and A. flavus.
• Sources: In parts of India a disease known as kodua poisoning occurs following
the consumption of kodo millet (Paspalum scrobiculatum) which is both a staple
food and an animal feed.
• Aflatoxins are isolated from the millet which synthesis cyclopiazonic acid.
• Affected may show symptoms like: symptoms of nervousness, lack of muscle co-
ordination, staggering gait, depression and spasms and, in humans, sleepiness, tremors
and giddiness may last for one to three days
• In cattle, A.flavus produces complex Indole alkaloid metabolites like tremorgens &
aflatrem.

23. CITRININ AND
CITREOVIRIDIN
• Produced by P. citrinum.
• Citrinin is a polyketide mycotoxin, which is a secondary
metabolite of some fungi species.
• Can cause yellow rice disease.
• Citrinin was first recognized as a promissing antibiotic
but it was later found to cause kidney damage, retard
growth, and eventually cause death in animal.
• Citrinin was isolated in the 1930s and produced by
Penicillium citrinum; however, P. Verrucosum is also
known to produce the toxin.

24. ERGOT TOXINS
• The ergot alkaloids are mycotoxins produced by several species of
fungi in the genus Claviceps.
• Ergot poisoning in humans and domestic animals is known as
argotism.
• This disease may cause strange hallucinations, the feeling of itchy
and burning skin, gangrene, loss of hands and feet, and even death.
• Ergotism is one of the oldest known human disesdes caused by
mycotoxins.
• There are four main groups of ergot alkaloids:
1) The clavines,
2) The lysergic acids,
3) Amides, and
4) Ergopeptides.

25. TRICHOTHECENES
• The trichothecene mycotoxins are a group of toxins
produced by multiple genera of fungi. Some of these
substances may be present as contaminants from mold or
may occur naturally in foodstuffs or in livestock feeds.
• Symptoms may occur among exposed humans or animals.
• Hazardous concentrations of trichothecenes have been
detected in corn, wheat, barley, oats, rice, rye, vegetables,
and other crops.
• Diseases resulting from infection include seed rot, seedling
blight, root rot, stalk rot, and ear rot.
• Toxin production is greatest with high humidity and
temperatures of 6-24°C.
• Trichothecenes are a very large family of chemically related
mycotoxins produced by various species of Fusarium,
Myrothecium, Trichoderma, Trichothecium, Cephalosporium,
Verticimonosporium, and Stachybotrys.
• They are produced on many different grains like wheat, oats
or maize by various Fusarium species such as F.
graminearum, F. sporotrichioides, F. poae and F. equiseti.

26. COMBINED TOXICOLOGY OF MYCOTOXINS
• It is known for many years that several food items, derived from plants infected by fungi in the
field during growing of the plant or during harvest and storage of the food item, can contain
concomitantly different mycotoxins.
• As these combined mycotoxins occur simultaneously in the food item, consumption of the food
will lead to a combined intake depending on the absorption rates of the different mycotoxins.
• Therefore, the question is justified whether such a combined intake of mycotoxins would lead to
a possible higher risk for adverse health effects than the intake of one of these mycotoxins
alone.
• When the mycotoxins are of similar structure and of the same species, or of the same families, it
is likely to expect that the mode of action of the mycotoxins and or the toxicity profiles will be
quite similar. This indicates that such related mycotoxins are likely to exert only additive effects,
which is important to know.
• In terms of risk assessment, these mycotoxins could be dealt with by establishing a group daily
tolerable intake (TDI) or a provisional tolerable weekly intake (PTWI).

28. SAFETY REQUIREMENTS FOR HANDLING MYCOTOXINS
• All food samples suspected of being contaminated with Mycotoxins must be handled with
care.
• Use disposable gloves and protective masks if grinding the food creates dust. Aflatoxins are
potent carcinogenic substances.
• While handling pure Aflatoxin reference material, extreme precautions must be taken as
they are electrostatic.
• All work must preferably be carried out in a hood.
• Swab any accidental spill of toxin with 1% sodium hypochlorite bleach (NaOCl), leave 10
minutes and then add 5 % aqueous acetone.
• Rinse all glassware exposed to Aflatoxin with methanol, add 1% sodium hypochlorite
solution and after 2 hours add acetone to 5 % of total volume. Let it react for 30 minutes
and then wash thoroughly.
• Use a laboratory coat or apron soaked in 5% sodium hypochlorite solution over night and
washed in water.
• Reactive vapours i.e. O2, SO2, HCl can affect adsorbents used in TLC as well as the stability of
adsorbed spots. TLC must, therefore, be performed only in a laboratory free of volatile
reagents.
• Always dry TLC plates thoroughly before exposure to UV light. UV light from sunlight or
fluorescent lamps can catalyse changes to compounds being examined when exposed on
adsorbent surface, particularly in the presence of solvent.

29. • Avoid exposure to UV light of underdeveloped spots and expose developed plates to UV light for the
minimum time needed for visualization
• Protect analytical material adequately from light and keep Aflatoxin standard solutions protected from
light by using amber vials or aluminium foil.
• Put a warning note on the label.
• Use of non acid washed glassware for Aflatoxin aqueous solutions may cause loss of Aflatoxin.
• Before use soak new glassware in dilute acid (carefully add 105 ml concentrated Sulphuric Acid to water
and make up to 1 litre) for several hours, then rinse extensively with distilled water to remove all traces
of acid. (Check with pH paper),
SAFETY REQUIREMENTS FOR HANDING MYCOTOXINS
• More susceptible to Drought region.
• Food and Agricultural Organization of the United nations (FAO) has estimated that 25% of the world’s
crops are contaminated with mycotoxins and certain diseases have just been linked to the ingestion of
food or feed contaminated with mycotoxins
• Newborn & Male are more prone to toxicity.
• Acute human toxicity of aflatoxin was reported in India in 1974 when a large outbreak of poisoning
occurred involving nearly 1000 people of whom nearly 100 died.
• Several studies have demonstrated that very young children may be exposed to aflatoxins even before
they are weaned because mothers, consuming aflatoxin in their food, may secrete aflatoxin M1 in
their milk. There is no doubt about the potential danger of aflatoxin in food and every effort should be
made to reduce or, if possible, eliminate contamination.
FACTS

31. WHAT CAN WE DO AS FOOD
TECHNOLPGY?
• Ensure adequate levels of vitamins (A,E,B-1) and
minerals (Se,Cu,Zn,Mn).
• Dilution is the solution.
• Reduce & Eliminate intake of contaminated feeds.
• Remove moldy layers of feed before feeding.
• Ensure that feed is dried sufficiently after harvesting.
• Have farmers select strains resistant to contamination.
• Scientists hope to genetically engineer plants resistant
to fungal infection.
• Use feed additives that sequester the toxins and
prevent absorption from the gastrointestinal tract.

32. Quick Overview
Organ System Affected
Vascular
Digestive
Respiratory
Nervous
Cutaneous
Urinary
Reproductive
Immune
Toxins Associated
Aflatoxin
Aflatoxin, T2 toxin, Vomit toxin
Trichothecenes
Trichothecenes
Trichothecenes
Zearalenone, T-2 toxin
Many
Ochratoxin A, Citrinin

34. What are some of the regulations
guiding the levels of toxins in food?
• Commission Regulation (EC) No 466/2001 of 8 March
2001
Setting maximum levels of certain contaminants in
foodstuffs.
• USFDA
• CODEX STAN 193-1995 (Rev.1-1997)
How to establish the maximum limit of toxins or
contaminants in foods.