Mycotoxins, Aflotoxin, Patulin, Ochratoxin, alkaloids, Aspergillus,

1. Mycotoxins
Dr. Gayathri Devaraja
Professor,
Department of Microbiology,
Davangere University,
Davangere-577007, India.
gayathridevaraja@gmail.com

2. Mycotoxins
• The term 'mycotoxin' is usually reserved for the toxic chemical products
produced by fungi.
• A mycotoxin from Greek (mukos) "fungus" and Latin (toxicum)
"poison"
• The fungus consume organic matter wherever humidity and
temperature are sufficient.
• The reason for the production of mycotoxins is not yet known; they are
neither necessary for growth nor the development of the fungi.

3. Aflatoxins
• are a type of mycotoxin
• produced by Aspergillus ignuf fo seiceps,
• such as A. flavus dna A. parasiticus .
• A. flavus common in Asia & Africa.
• A. parasiticus common in America.
• Among various mycotoxins, aflatoxins have
assumed significance due to their deleterious
effects on human beings, poultry and
livestock.

4. Aspergillus

5. Historical view
• The aflatoxin problem was first recognized in
1960, when there was severe outbreak of a
disease referred as "Turkey 'X' Disease" in UK,
in which over 100,000 turkey poults were
died.
• The cause of the disease was shown due to
toxins in peanut meal infected with Aspergillus
flavus and the toxins were named as
aflatoxins.

6. From the economic point of view
• Aflatoxins influence:
• Health of human being.
• Health of livestock and poultry.
• The marketability of medicinal herbs
• Agricultural products.

7. Natural occurrence
• Agricultural products contaminated with aflatoxins
include:
• Cereals (maize, rice, wheat).
• Oil seeds (groundnut, soybean, sunflower, cotton).
• Spices (black pepper, coriander, turmeric, zinger).
• Tree nuts (almonds, pistachio, walnuts, coconut).
• Milk.

8. Properties
• 18 different types of aflatoxins were identified.
• The major members are aflatoxin B1, B2, G1 and G2.
• Based on their fluorescent color when exposed to
ultraviolet light.
• (B series = blue fluorescence)
• (G series = yellow-green fluorescence).
• The aflatoxins fluoresce strongly in UV (365 nm).
• Aflatoxin M1and M2 are major metabolites of
aflatoxin B1 and B2 respectively, found in milk of
animals that have consumed feed contaminated with
aflatoxins.

9. Properties (cont.)
• Aflatoxins M1, M2 may be found in the absence of
other aflatoxins.
• Aflatoxin B1 (AFB1) is normally predominant in
cultures as well as in food products.
• Aflatoxins are slightly soluble in water.
• Soluble in moderately polar organic solvents.
• Insoluble in non polar solvents.
• Aflatoxins decompose at their melting points, which
are between 237°C (G1) and 299°C (M1),
• Not destroyed under normal cooking conditions.

10. Structure
• Aflatoxins are normally refers to the
• group of difuranocoumarins
• coumarin nucleus fused to a difuran units
•
coumarin nucleus furan unit

11. Structure
• They are classified in two broad groups:
• 1- Difurocoumarocyclopentenone Series.
• (B series) (AFB1 , AFB2 , AFB2A , AFBM1
, AFBM2, AFBM2A and aflatoxicol).
• ِِAFB1

12. • 2- Difurocoumarolactone series .
• (G series) (AFG1 , AFG2 , AFG2A , AFGM1
AFGM2 , AFGM2A).
AFG1
O O
O
O
O
O
OCH3

13. Detection and estimation of
aflatoxins
• Analytical methods ( TLC & HPLC ).
• Immunological methods.

14. Immunological methods
The Immunological method is a biochemical
technique.
• Can be performed to evaluate either the
presence of antigen or the presence of antibody.
• Radio Immuno Assay (RIA)
• Enzyme Immuno Assay (EIA)
or
• Enzyme-Linked Immuno Sorbent Assay
(ELISA)

15. Immunological methods (cont.)
• RIA and EIA are used for estimation and
detection of many drugs, (barbiturates,
digoxin, LSD, morphine, nicotine, THC,
aflatoxins…………

16. Immunological methods (cont.)
• In the RIA radio isotopes are used.
• Radiolabelled ( tagged )drug or antigen.
• Drug-protein complex (the immunogen).
• The antibody (antiserum)

17. Immunological methods (cont.)
• AFB1 – BSA = a conjugate between the
aflatoxin and bovine serum albumin and this is
required to induce antibodies.
• To render the small molecules drugs
immunogenic.

18. Immunological methods (cont.)
• Disadvantages of RIA
• Restricted to license (approved) laboratories.
• Problems of radioactive waste.
• Scintillation counter is very expensive.
• EIA or ELISA is an alternative method enjoying
the sensitivity and selectivity of RIA without any
disadvantages.

19. Immunological methods (cont.)
• In EIA the tagged drug or antigen is an enzyme
- labeled antigen rather than a radioactive
labeled antigen.
• The enzymatic activity is indicated by adding a
suitable substrate capable of producing optical
signals.
• The enzyme reaction can be measured
photometrical ( the measurement of the
intensity of light ).

20. Toxicity
• Aflatoxins are carcinogenic
• ( cause hepatocellular carcinoma )
• teratogenic
• mutagenic
• Immunosuppressive. (suppresses the immune
system).
• The aflatoxins display potency of toxicity,
carcinogenicity, mutagenicity in the order of
AFB1 > AFG1 > AFB2 > AFG2.

21. Carcinogenicity
• Aflatoxins are known to be human carcinogens
(hepatocellular carcinoma, or primary liver-
cell cancer).
• The risk of liver cancer increased significantly
with increasing aflatoxin consumption.
• Biomarkers for aflatoxin exposure (aflatoxin
metabolites in the urine and aflatoxin albumin
adducts in the blood)

22. Preventing exposure to aflatoxin
• The traditional approach to preventing
exposure to aflatoxin has been to ensure that
foods consumed have the lowest practical
aflatoxin concentrations .
• Chemoprotection and enterosorption to limit
biologically effective exposure .

23. Aflatoxin B1

24. Aflatoxin B1: Background
• Hepatotoxic mycotoxin (toxin from a
fungus) produced by the fungi
Aspergillus flavus and Aspergillus
parasiticus
• Causes aflatoxicosis and liver disease
• Aspergillus spp. grow ubiquitously on plants
and crops from tropical and subtropical areas:
peanuts, figs, spices, corn, maize, Brazil nuts,
pecans, walnuts, soybeans, pistachios, wheat
and grains

25. Background
• The carcinogenic metabolite of Aflatoxin B1 is found
in the milk of mammals who consume contaminated
crops
• Aspergillus growth and Aflatoxin B1 production is
dependant upon the temperature, humidity, host
plant type, and the strain of fungus; high humidity
usually required for growth
• Outbreaks more common in
developing countries due to
improper drying and storage of
crops and a lack of food
inspections

26. Background
• Outbreaks occur in groups because of a
shared contaminated food supply and the
optimal weather conditions for Aspergillus
growth
• First recorded outbreak was in England in
1960, where 100000 turkeys died.

27. Background
• In 1974 in clustered villages in India, 397 recorded
cases with 108 deaths; this was preceded by an
outbreak in the local dogs
• In Kenya in 2004, maize contaminated with Aflatoxin
B1 caused liver disease and jaundice in 317 people;
125 people died
The use of antifungal agents is often too costly for farmers in
developing countries

28. Toxicity Parameters
• Exposure to 2-6 mg of aflatoxin by consumption daily
for one month is cited to have toxic effects (David et al.
1994)
• Consumption of 55 µg/ kg of aflatoxin daily for an
unknown period of time will be fatal (FDA 1992)
• However, levels found to be toxic are inconsistent
between various studies.
•Reasons for this include:
1) most studies were done in African countries where
prevalence of Heptatitis B is very high
2) Active HepB virus is known to be synergistic with
aflatoxin in causing toxicities and hepatocellular
carcinoma

29. Toxicity cont’d
3) most studies were done on rats, and it can be difficult to
extrapolate these results to humans
4) There is a double standard for accepted aflatoxin levels due to
economic stress:
• food intended for human consumption must have less
than 30ppb, and animal feed may be as high as 30ppb in
America

30. Mechanism of Action
• AFB1 is oxidized by CYT P450 in the liver
into AFB1-8,9-epoxide which is the major
metabolite that exerts hepatotoxic effects
• The 8,9 epoxide is neutralized by
conjugation in the liver with GSH
(glutathione) by glutathione-S-transferase,
an enzyme abundant in some animals
(mice), but which rats and humans are
relatively deficient in.

31. Mechanism of Action
• The 8,9 epoxide binds preferentially to mitochondrial
DNA hindering ATP production and FAD/NAD-linked
enzymatic functions. The result is reduced
mitochondrial function and increased prevalence of
mitochondrial directed apoptosis
• GSH has many roles in membrane maintenance and
stability as well as reducing oxidative stress. Its
reduction further enhances the damage to critical
cellular components (DNA, lipids, proteins) by the 8,9
epoxides

32. Mechanism of Action
• Abnormally high levels of ROS are found in
cells affected by aflatoxin due to uncoupling of
metabolic processes resulting from the lack of
GSH for GSH-peroxidase catalysis of O2· to
H2O2 leading to lipid peroxidation and
compromised cell membranes

33. • One of the most serious effects of the AFB1-
8,9-epoxide metabolite is it reacts with amino
acids in DNA and forms an adduct.
• This adduct is fairly resistant to DNA repair
processes and thus this gene mutation may
cause carcinoma of the liver.
• CYP 3A4 is the major CYP 450 enzyme
responsible for activation of AFB1 into the
epoxide form.
• However, the liver can detoxify AFB1 by
oxidizing it to other metabolites such as AFQ1
which has very little cancer-causing potential.
These are usually excreted in urine with little
effect on the body.
AFB1-8,9-epoxide Is Cancer-
causing!!
(Novoa and Diaz 2006)

34. When the epoxide combines with DNA or RNA, it forms what is
known as an adduct

35. How Does Aflatoxin Cause
Cancer?
•Inactivation of the p53 tumor suppressor gene enables
mutation of Codon 249 (primary gene mutation caused
specifically by AFB1)
•This shuts off the gene and potentially allows for
uncontrolled cell proliferation.
• But exposure to aflatoxin does not necessarily
predispose you to liver cancer.
•The human body is capable of genetic repairs and
some people are more resistant to cancer than others

36. Other Adverse Effects of AFB1-
8,9-epoxide
- Lipid accumulation in the liver due to decreased lipid
transport and reduced oxidation
-Symptoms of liver failure occur with acute aflatoxicosis:
-Jaundice
-ascites (fluid build up)
-portal hypertension
-necrosis of the liver

37. Other Adverse Effects of AFB1-8,9-
epoxide
• The binding of the epoxide
with proteins may:
– Inhibit the protein from
performing its enzymatic
functions
– Create a reservoir of the toxin
in proteins, prolonging
exposure

38. Metabolism
• Both metabolized and unmetabolized aflatoxin is
excreted mostly in urine. It is also excreted in milk,
stool, feces, and saliva (which may be swallowed
and re-enter the GI tract)

39. Other Chronic Effects of Aflatoxin
• Immunological Suppression
– Using animal models, AFB1 has been shown to
impair normal immune function either by
reducing phagocytic activity or reduce T cell
number and function.
• Nutritional Interference
– Aflatoxin is shown to have a dose response
relationship between exposure to aflatoxin and
rate of growth among small children. In addition,
it also interferes in nutrient modification such as
Vitamin A or D in animal models.

40. Treatment
• Inactivation of toxin before ingestion:
– Synergistic combination of gamma radiation and
hydrogen peroxide degrades the toxin
– Treatment of corn with ammonia
– Dichlorvos inhibits aflatoxin B1 biosynthesis
– NaHSO3 soak for corn also shows beneficial
results

41. Treatment
• After ingestion
– Treatment with antibiotics or other drugs has little effect
– UV doses increased rate and extent of removal of aflatoxin B12 adducts from
DNA
– Chlorophyll intervention: supplement diets with foods rich in chlorophyll
• Blocks carcinogen bioavailability
– Certain types of flavanoids found in grapefruit stimulates the microsomal
activation of aflatoxin B1 to the exo-8,9-epoxide which is not harmful
– Administration of drugs that induce hepatic detoxification enzymes
(ethoxyquin, butylated hydroxyanisol, butylated hydroxytuluene,
phenobarbital)
– Oltipraz inhibits aflatoxin B1 mediated hepatocarcinogens in rats
– Induction of an antibody that would allow the body to identify and remove
the toxin upon exposure (aflatoxin B1- lysine adduct monoclonal antibody)

42. Patulin
• Produced by Aspergillus, Penicillium,
Byssochlayms. It is a genotoxin and also a
group 3 carcinogen.
• Found in grains, fruits, vegetables. Frequently,
found in apples and apple products,cherries,
blueberries, plums, bananas, strawberries,
and grapes.grains like barley, wheat, corn and
their processed products as well as in
shellfish.

43. Structure of patulin

44. • Immunotoxicity: Reduced cytokine secretion, oxidative burst in
macrophages, increased splenic T lymphocytes, and
increased neutrophil numbers.
• Patulin is toxic primarily through affinity to sulfhydryl groups (SH), which
results in inhibition of enzymes. Oral LD 50 in rodent models have ranged
between 20 and 100 mg/kg. Major acute toxicity findings include
gastrointestinal problems, neurotoxicity (i.e., convulsions), pulmonary
congestion, and edema.
• FDA has set an action limit of 50 ppb in cider due to its potential
carcinogenicity and other adverse effects.
• WHO concluded that patulin is genotoxic based on
variable genotoxicity data, however it is considered a group 3 carcinogen
by the International Agency for Research on Cancer(IARC).

45. • Reproduction studies: Patulin decreased sperm count and altered sperm
morphology in the rat. Also, it resulted in abortion of F1 litters in rats and
mice after i.p. injection.Embryotoxicity and teratogenicity were also
reported in chick eggs.
• To test for patulin contamination, a variety of methods and sample
preparation methods have been employed including thin layer
chromatography (TLC), gas chromatography (GC), high-performance
liquid chromatography (HPLC), capillary electrophoresis.

46. Ochratoxin
• Aspergillus species (mainly A. ochraceus, but
also by 33% of A. niger industrial strains) &
some Penicillium species,
especially P.verrucosum &P.carbonarius .
• Ochratoxin A is the most prevalent and
relevant fungal toxin of this group, while
ochratoxins B and C are of lesser importance.

47. Och.A
Och.B
Och.C

48. • Ochratoxin A is potentially carcinogenic to humans (Group 2B), and has
been shown to be weakly mutagenic, possibly by induction of oxidative
DNA damage.
• Ochratoxin A has a strong affinity for the brain, especially the cerebellum
(Purkinje cells), ventral mesencephalon, and hippocampal structures.The
affinity for the hippocampus could be relevant to the pathogenesis of
Alzheimer's disease, and subchronic administration to rodents induces
hippocampal neurodegeneration.
• Ochratoxin causes acute depletion of striatal dopamine, which constitutes
the bed of Parkinson's disease, but it did not cause cell death in any of brain
regions examined.
• The developing brain is very susceptible to ochratoxin, hence the need for
caution during pregnancy.

49. • Immunotoxicity: Ochratoxin A can
cause immunosuppression and immunotoxicity in animals. The
toxin's immunosuppressant activity in animals may include
depressed antibody responses, reduced size of immune organs
(such as the thymus, spleen, and lymph nodes), changes in
immune cell number and function, and
altered cytokine production.
• Immunotoxicity probably results from cell death
following apoptosis and necrosis, in combination with slow
replacement of affected immune cells due to inhibition of
protein synthesis.

50. • Impact on animal fodder industries: Ochratoxin-
contaminated feed has its major economic impact on
the poultry industry. Chickens, turkeys, and ducklings
are susceptible to this toxin. Clinical signs
of avianochratoxicosis generally involve reduction in
weight gains, poor feed conversion, reduced egg
production, and poor egg shell quality.
• Toxicity does not seem to constitute a problem in
cattle, as the rumen harbors protozoa that hydrolyze
OTA.

51. Fumonisin
• A fumonisin is a mycotoxin derived from Fusarium, produced primarily by
Fusarium verticillioides and Fusarium proliferatum. There are at least 28
different forms of fumonisins, most designated as A-series, B-series, C-
series, and P-series. Fumonisin B1 is the most common and economically
important form, followed by B2 and B3.
• fumonisin B1- hepato & nephrotoxin. Found in corn & other cereals.
• Because of their similarity, fumonisins are able to inhibit sphingosine-
sphinganin-transferases and ceramide synthases and are
therefore competitive inhibitors of sphingolipid biosynthesis and
metabolism.
• Neural tube defects, Esophageal cancer, Acute mycotoxicosis,

52. Structure of fumonisin

53. • Fumonisin B2 is fumonisin mycotoxin produced by the
fungi Fusarium verticillioides and Fusarium moniliforme. It is a structural
analog of fumonisin B1. Fumonisin B2 is more cytotoxic than fumonisin
B1. Fumonisin B2 inhibits sphingosine acyltransferase.
• Fumonisin B2 and other fumonisins frequently contaminate maize and
other crops.
• Tolerable daily intake (TDI) : The risks of fumonisin B1 have been
evaluated by The World Health Organization’s International Programme on
Chemical Safety(IPCS) and the Scientific Committee on Food(SCF) of the
European Commission. They determined a tolerable daily intake(TDI) for
FB1, FB2, FB3, alone or in combination of 2 µg/kg body weight.

54. Zearalenone
• Zearalenone (ZEA), also known as RAL and F-2 mycotoxin, is
a potent estrogenic metabolite produced by some Fusarium
and Gibberella species.
• Zearalenone is heat-stable and is found worldwide in a number
of cereal crops, such as maize, barley, oats, wheat, rice, and
sorghum
• infertility, abortion or other breeding problems, especially in
swine.
• Zearalenone can permeate through the human skin. However,
no significant hormonal effects are expected after dermal
contact in normal agricultural or residential environments.

55. Structure of zearalenone

56. • Toxicity: The most important effect of
zearalenone is on the reproductive system.
Animal studies show that zearalenone is fairly
• rapidly absorbed following oral administration
and can be metabolised by intestinal tissue in
pigs and possibly also in humans
• with the formation of a - and b -zearalenols,
which are subsequently conjugated with
glucuronic acid.

57. • Diet with 50 mg/kg of ZEA resulted in abortion and stillbirths in swine.
Where as 0.25 mg/kg reduced the litter size and weight of the piglets.
Calves show earlier sexual maturity,
• dairy cows reported to have vaginitis, prolonged oestrus and infertility and
sheep are reported to become sterile. The effective dose for sheep may be
approximately 1 mg/kg. 0.02 μg/kg of body weight per day suggesting a
significant margin of safety in humans based on current knowledge.
• TLC, HPLC, HPLC-MS are commonly used tests.

58. Trichothecenes
• Trichothecenes are a very large family of chemically related
mycotoxins produced by various species of Fusarium,
Myrothecium, Trichoderma, Trichothecium, Cephalosporium,
Verticimonosporium, and Stachybotrys.
• Trichothecenes belong to sesquiterpene compounds. The most
important structural features causing the biological activities of
trichothecenes are: the 12,13-epoxy ring, the presence of
hydroxyl or acetyl groups at appropriate positions on the
trichothecene nucleus and the structure and position of the side
chain.
• They are produced on many different grains like wheat, oats or
maize.

59. There are 60 known types of trichothecene mycotoxins. Some of them are:
• Deoxynivalenol
• Diacetoxyscirpenol
• HT-2 mycotoxins
• Neosolaniol
• Nivalenol
• Satratoxin-H
• T-2 mycotoxins
• Verrucarin A
• Vomitoxin

60. Trichothecene Mycotoxin
Symptoms
Exposure to trichothecene mycotoxins can cause these symptoms
in people:
• Dry eyes
• Tiredness, fatigue
• General discomfort
• Vomiting
• Diarrhea
• Abdominal pain
• Mental impairment
• Rash
• Bleeding

61. • Trichothecenes are powerful inhibitors of protein synthesis mainly
interacting with ribosomes.
• Unlike aflatoxin the trichothecenes do not appear to require metabolic
activation to exert their biological activity. After direct dermal application
or oral ingestion, the trichothecene mycotoxins can cause rapid irritation to
the skin or intestinal mucosa.
• In cell-free systems or singlecells in culture, these mycotoxins cause a
rapid inhibition of protein synthesis and polyribosomal disaggregation.
• Regulatory issues: When it comes to animal and human food, type A
trichothecenes (e.g. T-2 toxin, HT-2 toxin, diacetoxyscirpenol)are of special
interest because they are more toxic than the other foodborne
trichothecenes i.e. type B group (e.g.deoxynivalenol, nivalenol, 3- and 15-
acetyldeoxynivalenol). However, deoxynivalenol is of concern as it is the
mostprevalent trichothecene in Europe.

62. • Trichothecene is amongst the most toxic types of mycotoxins.
The LD50 rate (the dosage level that caused 50% of the group
to die) for laboratory mice given trichothecene mycotoxins is
between 1 and 7 mg/kg depending on the specific type of
trichothecene and the method of exposure.
• T-2 mycotoxins are also the only substances used in biological
warfare that can be absorbed through a person's skin.
• These trichothecene mycotoxins have the advantages of being
highly stable in the air, not degrading under ultraviolet light
and being able to withstand heat.
• They are also used because they can be produced relatively
easily and cheaply and they are extremely toxic with no
antidote or vaccine available.

63. • The United States military is reportedly doing 90% of its
current biological weapons research in T-2 mycotoxins. The
Yellow Rain biological attacks used in Vietnam and
Afghanistan were concentrated T-2 mycotoxins and Gulf War
syndrome is believed to be caused by American soldiers'
exposure to T-2 mycotoxins during biological attacks in Desert
Storm.

64. Ergot alkaloids
• Ergot or ergot fungi refers to a group of fungi of the genus
Claviceps. The most prominent member of this group is
Claviceps purpurea ("rye ergot fungus"). This fungus grows on
rye
• and related plants, and produces alkaloids that can cause
ergotism in humans and other mammals who consume grains
contaminated with its sclerotium.
• It is common to rye, barley, pearl millet.
Ergot alkaloids can be classified into two classes:
• 1. derivatives of 6,8-dimethylergoline and
• 2. lysergic acid derivatives.

65. • The ergot sclerotium contains high concentrations (up to 2% of
dry mass) of the alkaloid ergotamine, a complex molecule
consisting of a tripeptide-derived cyclol-lactam ring connected
via amide linkage to a lysergic acid (ergoline) moiety, and
other alkaloids of the ergoline group that are biosynthesized by
the fungus.
• Ergot alkaloids have a wide range of biological activities
including effects on circulation and neurotransmission.
Symptoms:
• Severe burning sensation in limbs due to effects of ergot
alkaloids on the vascular system due to vasoconstriction,
sometimes leading to gangrene and loss of limbs due to
severely restricted blood circulation.

66. • The neurotropic activities of the ergot
alkaloids may also cause hallucinations and
attendant irrational behaviour, convulsions,
and even death. Other symptoms include
strong uterine contractions, nausea, seizures,
and unconsciousness.

67. Benefits from ergots
• In pharmaceutical prepartions like Cafergot
(containing caffeine and ergotamine or
ergoline) to treat migraine headaches, and
• In ergometrine used to induce uterine
contractions and to control bleeding after
childbirth.
• Tolerable daily intake (TDI): 0.6μg/kg of body
weight.

68. THANK YOU