Mycotoxins Presentation

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 SUBJECT FOOD TOXICOLOGY SUBMITTED BY MANSI LANGALIA MAHIMA MARU

3. Foodborne Diseases Infections Poisonous Animal Tissues Poisonous Plant Tissues Chemical Poisoning Intoxications Microbial Intoxications 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.

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

8. 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 the liver .)

9. 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 The formation of the so-called fatty liver connected with a loss of ability to remove fats from the liver Coagulopathy caused by the inhibition of prothrombin synthesis Reduced immuno function Aflatoxins are well known as potent human hepatocarcinogens .

10. 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.

11. 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.

12. 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.

13. STERIGMATOCYSTIN A precursor in the biosynthesis of Aflatoxins B1. P roduced 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.

14. 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 ( OT α), 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: P rimary 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 .

15. 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.

16. Zearalenone / z earalenol O estrogenic 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. interaction of zearalenone with estrogen receptors. transportation of the estrogen –receptor complex into the cellular nucleus conjugation with chromatin receptors selective transcription of RNA 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.

17. 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 .

18. PATULIN Produced by several species of Penicillium, Aspergillus and Byssochlamys but is especially associated with P. expansum . S table 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.

19. 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 .

20. 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.

21. 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.

22. 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 .

23. 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).

24. 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.

25. 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

26. 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 .

27. Quick Overview Trichothecenes Trichothecenes Zearalenone , T-2 toxin Many Ochratoxin A, Citrinin

28. 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.