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MYCOTOXINS Introduction Fungal diseases are common place in plants and animals. In such diseases, the fungi are actively growing on and invading the body of their hosts. There is another means by which fungi can cause harm without invading our bodies. When fungi grow on a living organism or on stored food material that we consume, they may produce harmful metabolites that diffuse into their food. It is believed that fungi evolved these metabolites as a means of protecting their food supply by preventing other organisms from eating it. These metabolites are referred to as mycotoxins, which literally mean "fungus poisons". Fungi that produce mycotoxins do not have to be present to do harm. If a fungus is growing for example in a grain storage silo, the environment may have become unsuitable for the fungus and it dies. Even though the fungus is no longer alive, while it was growing, if it produced a mycotoxin, it will have poisoned the grains. A mycotoxin is derived from Greek words ‘mykes’, ‘mukos’ means "fungus" and from Latin word ‘toxicum’ means "poison". It is a toxic secondary metabolite produced by an organism of the fungus kingdom, including mushrooms, molds, and yeasts. The term 'mycotoxin' is usually reserved for the relatively small (MW ~700), toxic chemical products formed as secondary metabolites by fungi that readily colonize crops in the field or after harvest.

Toxicology ins

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MYCOTOXINS

Introduction

Fungal diseases are common place in plants and animals. In such diseases, the

fungi are actively growing on and invading the body of their hosts. There is another

means by which fungi can cause harm without invading our bodies. When fungi grow on

a living organism or on stored food material that we consume, they may produce harmful

metabolites that diffuse into their food. It is believed that fungi evolved these metabolites

as a means of protecting their food supply by preventing other organisms from eating it.

These metabolites are referred to as mycotoxins, which literally mean "fungus poisons".

Fungi that produce mycotoxins do not have to be present to do harm. If a fungus is

growing for example in a grain storage silo, the environment may have become

unsuitable for the fungus and it dies. Even though the fungus is no longer alive, while it

was growing, if it produced a mycotoxin, it will have poisoned the grains.

A mycotoxin is derived from Greek words ‘mykes’, ‘mukos’ means "fungus" and

from Latin word ‘toxicum’ means "poison". It is a toxic secondary metabolite produced

by an organism of the fungus kingdom, including mushrooms, molds, and yeasts. The

term 'mycotoxin' is usually reserved for the relatively small (MW ~700), toxic chemical

products formed as secondary metabolites by fungi that readily colonize crops in the

field or after harvest.

Most fungi are aerobic (use oxygen) and are found almost everywhere in

extremely small quantities due to the minute size of their spores. They consume organic

matter wherever humidity and temperature are sufficient. One mold species may produce

many different mycotoxins and/or the same mycotoxin as another species.

Growth of fungi on animal hosts produces the diseases collectively called

mycoses, while dietary, respiratory, dermal, and other exposures to toxic fungal

metabolites produce the diseases collectively called mycotoxicoses.

The production of toxins is dependant on the surrounding intrinsic and extrinsic

environments and the toxins vary greatly in their severity, depending on the organism

infected and its susceptibility, metabolism, and defense mechanisms.

Mycotoxins can appear in the food chain as a result of fungal infection of crops,

either by being eaten directly by humans, or by being used as livestock feed. Mycotoxins

greatly resist decomposition or being broken down in digestion, so they remain in the

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food chain in meat and dairy products. Even temperature treatments, such as cooking and

freezing, do not destroy mycotoxins.

Mycotoxins and Other Fungal Metabolites

While all mycotoxins are of fungal origin, not all toxic compounds produced by

fungi are called mycotoxins. The target and the concentration of the metabolite are both

important. Fungal products that are mainly toxic to bacteria (such as penicillin) are

usually called antibiotics. Fungal products that are toxic to plants are called phytotoxins

by plant pathologists.

Mycotoxins are made by fungi and are toxic to vertebrates and other animal

groups in low concentrations. Other low molecular weight fungal metabolites such as

ethanol that are toxic only in high concentrations are not considered mycotoxins. Finally,

although mushroom poisons are definitely fungal metabolites that can cause disease and

death in humans and other animals, they are rather arbitrarily excluded from discussions

of mycotoxicology. Molds (i.e., micro-fungi) make mycotoxins; mushrooms and other

macroscopic fungi make mushroom poisons.

The distinction between a mycotoxin and a mushroom poison is based not only

on the size of the producing fungus, but also on human intention. Mycotoxin exposure is

almost always accidental. In contrast, with the exception of the victims of a few

mycological accomplished murderers, mushroom poisons are usually ingested by

amateur mushroom hunters who have collected, cooked, and eaten what is misidentified

as a delectable species.

Mycotoxicoses

Mycotoxicoses is the term used for poisoning associated with exposures to mycotoxins.

The symptoms of a mycotoxicoses depend on: -

The type of mycotoxin

The concentration and length of exposure

Age, health, and sex of the exposed individual

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The synergistic effects associated with several other factors such as genetics, diet,

and interactions with other toxics also have been studied. Therefore it is possible that

vitamin deficiency, caloric deprivation, alcohol abuse, and infectious disease status can

all have compounded effects with mycotoxins. In turn, mycotoxins have the potential for

both acute and chronic health effects via ingestion, skin contact, and inhalation. These

toxins can enter the blood stream and lymphatic system; they inhibit protein synthesis,

damage macrophage systems, inhibit particle clearance of the lung, and increase

sensitivity to bacterial endotoxin.

History of Mycotoxins

The existence of mycotoxins was not documented until 1960. However, the

concept that moldy food could lead to illness in people or domestic animals was long

suspected before their existence was demonstrated by science. Long ago, before there

was adequate means of long term storage for perishable goods, food was normally

consumed a short time after it was acquired, but as the world has become more

industrialized and technological advanced, storage of food has become more of an issue.

Food is now commonly stored for long periods of time, giving fungi a greater

opportunity to contaminate our food.

Before 1900, in Italy, researchers there believed consumption of moldy corn by

children led to the development of illness. Some experiments, done at that time, included

the isolation, and growth of the suspected fungus in pure culture, and isolation of toxic

compounds from the fungus that the researchers believed to be the cause of the illness.

However, since the compound was not identified and was not actually isolated from the

moldy corn, it could not be concluded that this compound was the cause of the illness or

that the compound in question was even present on the moldy corn.

There was an extensive outbreak of moldy corn disease in the southeastern

United States in the early 1950's where hundreds of wild pigs foraging in cultivated corn

fields became ill and many died.

It would not be until 1960, when approximately 100,000 turkeys and a lesser

number of other domestic birds died in England, causing losses of approximately several

hundred thousand dollars, before the first mycotoxin was isolated and identified.

Initially, the disease was thought to be caused by a virus and the syndrome was named

"turkey-X disease". The "X" here indicated that the cause of the disease was unknown.

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After a great deal of research work, from their isolations, the scientists identified

Aspergillus flavus.

Chemists were also employed in this investigation, and they were able to isolate

and identify the toxin from the oil cake feed. The mycotoxin isolated was named

aflatoxin, the "a" from Aspergillus and "fla" from flavus. Feeding test of food containing

aflatoxin, with various laboratory animals, demonstrated that to varying degrees, all

animals tested were sensitive to aflatoxin. Even consumption of extremely small

amounts of aflatoxin damaged various internal organs and could induce development of

cancer to the liver.

The period between 1960 and 1975 has been termed the mycotoxin gold rush

because so many scientists joined the well-funded search for these toxigenic agents.

Some 300 to 400 compounds are now recognized as mycotoxins, of which approximately

a dozen groups regularly receive attention as threats to human and animal health.

Bioterrorism

Mycotoxins can be used as chemical warfare agents. There is considerable

evidence that Iraqi scientists developed aflatoxins as part of their bio weapons program

during the 1980s. Toxigenic strains of Aspergillus flavus and Aspergillus parasiticus

were cultured, and aflatoxins were extracted to produce over 2,300 liters of concentrated

toxin. The majority of this aflatoxin was used to fill warheads; the remainder was

stockpiled. Aflatoxins seem a curious choice for chemical warfare because the induction

of liver cancer is “hardly a knockout punches on the battlefield”. Even so, the

repugnance caused by the use of chemical and biological weapons is the kind of

emotional response that terrorists seek to elicit. Furthermore, if used against enemies, the

long-term physical and psychological results could be devastating.

Unlike the aflatoxins, trichothecenes can act immediately upon contact, and

exposure to a few milligrams of T-2 is potentially lethal. In 1981, then Secretary of State

Alexander Haig of the United States accused the Soviet Union of attacking Hmong

tribesman in Laos and Kampuchea with a mysterious new chemical warfare agent,

thereby violating the 1972 Biological Weapons Convention. The symptoms exhibited by

purported victims included internal hemorrhaging, blistering of the skin, and other

clinical responses that are caused by exposure to trichothecenes. The purported chemical

warfare agent came to be known as yellow rain.

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Mycotoxins and Human Health

Toxicologists tend to concentrate their efforts on hazardous chemicals such as

polyaromatic hydrocarbons, heavy metals, and organic pesticides and they have devoted

less effort to natural products. On the other hand agriculturalists, chemists,

microbiologists, and veterinarians, who are often unfamiliar with the basic principles of

toxicology, have conducted most of the mycotoxin research.

Human exposure to mycotoxins is further determined by environmental or

biological monitoring. In environmental monitoring, mycotoxins are measured in food,

air, or other samples and in biological monitoring, the presence of residues adducts, and

metabolites are assayed directly in tissues, fluids, and excreta.

In general, mycotoxin exposure is more likely to occur in parts of the world

where poor methods of food handling and storage are common, where malnutrition is a

problem, and where few regulations exist to protect exposed populations.

Food Safety and Regulations

Mycotoxin-producing mold species are extremely common, and they can grow on

a wide range of substrates under a wide range of environmental conditions. For

agricultural commodities, the severity of crop contamination tends to vary from year to

year based on weather and other environmental factors. Aflatoxin, for example, is usually

worst during drought years; the plants are weakened and become more susceptible to

insect damage. Mycotoxins occur, with varying severity, in agricultural products all

around the world. The estimate usually given is that one quarter of the world’s crops are

contaminated to some extent with mycotoxins.

Mycotoxins can enter the food chain in the field, during storage, or at later

points. Mycotoxin problems are exacerbated whenever shipping, handling, and storage

practices are conducive to mold growth. The end result is that mycotoxins are commonly

found in foods. Scientists rank mycotoxins as the most important chronic dietary risk

factor, higher than synthetic contaminants, plant toxins, food additives, or pesticide

residues.

The economic consequences of mycotoxin contamination are profound. Crops

with large amounts of mycotoxins often have to be destroyed. Alternatively,

contaminated crops are sometimes diverted into animal feed. Giving contaminated feeds

to susceptible animals can lead to reduced growth rates, illness, and death. Moreover,

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animals consuming mycotoxin contaminated feeds can produce meat and milk that

contain toxic residues and biotransformation products. Thus, aflatoxins in cattle feed can

be metabolized by cows into aflatoxin M1, which is then secreted in milk. The ability to

diagnose and verify mycotoxicoses is an important forensic aspect of the mycotoxin

problem.

Many mycotoxins survive processing into flours and meals. When mold-damaged

materials are processed into foods and feeds, they may not be detectable with out special

assay equipment. It is important to have policies in place that ensure that such “hidden”

mycotoxins do not pose a significant hazard to human health.

Since it is normally impracticable to prevent the formation of mycotoxins, the

food industry has established internal monitoring methods. Similarly, government

regulatory agencies survey for the occurrence of mycotoxins in foods and feeds and

establish regulatory limits.

Considerable research has been devoted to developing analytical methods for

identifying and quantifying mycotoxins in food and feeds. The chemical diversity of

mycotoxins and the equally diverse substrates in which they occur pose challenges for

analytical chemistry. Each group of compounds and each substrate have different

chemical and physical properties, so the methods for the separation of toxins from

substrates must be developed on a case-by-case basis. It is, for example, quite a different

matter to assay aflatoxin from peanut butter than it is to identify T-2 toxin from corn.

Mycotoxins are often produced in trace concentrations, so the sensitivity of the detection

systems is also essential.

Complete elimination of any natural toxicant from foods is an unattainable

objective. Therefore, naturally occurring toxins such as mycotoxins are regulated quite

differently from food additives. The Codex Alimentarious Commission, U.S. Food and

Drug Administration, the European Union, the Institute of Public Health in Japan, and

many other governmental agencies around the world test products for aflatoxins and

other mycotoxins and have established guidelines for safe doses.

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CLASSIFICATION OF MYCOTOXINS

Mycotoxins are not only hard to define, they are also challenging to classify. Due

to their diverse chemical structures and biosynthetic origins, their myriad biological

effects, and their production by a wide number of different fungal species, classification

schemes tend to reflect the training of the person doing the categorizing.

Clinicians often arrange them by the organ they affect. Thus, mycotoxins can be

classified as hepatotoxins, nephrotoxins, neurotoxins, immunotoxins, and so forth. Cell

biologists put them into generic groups such as teratogens, mutagens, carcinogens, and

allergens. Organic chemists have attempted to classify them by their chemical structures

(e.g., lactones, coumarins); biochemists according to their biosynthetic origins

(polyketides, amino acid-derived, etc.); physicians by the illnesses they cause (e.g., St.

Anthony’s fire, stachybotryotoxicosis), and mycologists by the fungi that produce them

(e.g., Aspergillus toxins, Penicillium toxins).

Some 300 to 400 mycotoxins are now recognized, of which the following groups

regularly receive attention as threats to human and animal health.

1. Aflatoxin

2. Citrinin

3. Ochratoxin

4. Ergot Alkaloids

5. Patulin

6. Fumonisins

7. Trichothecenes

8. Zearalenone

1) AFLATOXIN

Aflatoxins are naturally occurring mycotoxins that are produced by many species

of Aspergillus, such as Aspergillus flavus and A. parasiticus. A. bombycis, A.

ochraceoroseus, A. nomius, and A. pseudotamari are also aflatoxin-producing species,

but they are encountered less frequently. But only about half of A. flavus strains produce

aflatoxins.

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Aflatoxins are potent toxic, carcinogenic, mutagenic, immunosuppressive agents,

produced as secondary metabolites by the fungus. After entering the body, aflatoxins are

metabolized by the liver to a reactive intermediate, aflatoxin M1, an epoxide. Aflatoxins

are soluble in methanol, chloroform, actone, acetonitrile.

There are 18 different types of aflatoxins that are identified. Aflatoxin B1 is

considered the most toxic a potent carcinogen. It has been directly correlated to adverse

health effects, such as liver cancer. The presence of Aspergillus in food products does

not always indicate that harmful levels of aflatoxin are also present; it does imply a

significant risk in consumption.

Some important types of aflatoxins are: -

Aflatoxin B1 & B2 are produced by Aspergillus flavus and A. parasiticus

Aflatoxin G1 & G2 are produced by Aspergillus parasiticus

Aflatoxin M1 is metabolite of aflatoxin B1 in humans and animals

Aflatoxin M2 is metabolite of aflatoxin B2 in milk of cattle fed on contaminated

foods

Aflatoxicol is produced by A. flavus

Aflatoxin GM1 is also produced by A. flavus

Parasiticol is also produced by A. flavus

Substrates

Aflatoxin producing members of Aspergillus are common and widespread in

nature. They can colonize and contaminate grains before harvest or during storage. Host

crops are particularly susceptible to infection by Aspergillus following prolonged

exposure to a high humidity environment or damage from stressful conditions such as

drought, a condition which lowers the barrier to entry.

The native habitat of Aspergillus is in soil, decaying vegetation, hay, and grains

undergoing microbiological deterioration and it invade all types of organic substrates

whenever conditions are favorable for its growth. Favorable conditions include 7%

moisture content and high temperature.

Aflatoxins are largely associated with commodities produced in the tropics and

subtropics include cereals (maize, sorghum, pearl millet, rice, wheat), oilseeds (peanut,

soybean, sunflower, cotton), spices (peppers, black pepper, coriander, turmeric, ginger),

and tree nuts (almond, pistachio, walnut, coconut).

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Aflatoxins M1, M2 are originally discovered in the milk of animals which are fed

contaminated feed. They metabolically bio-transform aflatoxin B1 into a hydroxylated

form called aflatoxin M1. All sources of commercial peanut butter also contain minute

quantities of aflatoxin.

Mode of Action

Biological activity of aflatoxins in liver cell is represented as: -

1. Interaction with DNA and inhibition of the polymerase responsible

for DNA and RNA synthesis

2. Suppression of DNA synthesis: The inhibition of nucleic acid

biosynthesis may be by inactivation of the synthetic enzyme systems

or because the DNA molecules present in the injured cell no longer

act as suitable models for their duplication.

3. Reduction of RNA synthesis and inhibition of messenger RNA: By

interacting in the first place with DNA, aflatoxins may also affect

the biosynthesis of RNA by preventing the transcription of DNA by

RNA polymerase.

4. Reduction of protein biosynthesis

Due to the biological activity of aflatoxins in liver cell, results can be:

Formation of the so-called fatty liver, connected with the loss of

ability to remove fats from the liver

Coagulopathy caused by inhibition of prothrombin synthesis

Reduced immuno function

Toxicity

The diseases caused by aflatoxin consumption are loosely called aflatoxicoses.

High level aflatoxin exposure produces an acute hepatic necrosis, resulting later in

cirrhosis or carcinoma of the liver. Acute hepatic failure is manifested by hemorrhage,

edema, mental changes and coma. It also results in alteration in digestion, absorption and

metabolism of nutrients. It may also result in death of the infected individual.

In 2004 in Kenya 125 people died and nearly 200 others were treated after eating

aflatoxin contaminated maize. The deaths were mainly associated with homegrown

maize that had not been treated with fungicides or properly dried before storage.

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It is observed in male rats that LD50 of aflatoxin B1 is 5.55 mg/kg. It is also noted

that female rat is less sensitive than male rat. LD50 of aflatoxin B1 for female rat is 7.44

mg/kg.

Acceptable Level in Food

The Codex Alimentarious Commission regulates the aflatoxin concentration in

food and feed because of their toxic effects. They proposed that in peanuts maximum

limit of aflatoxin should be 15 µg / kg. The level of aflatoxin should not exceed from 20

ppb (ug/kg) in food commodities. The maximum limit of aflatoxin M1 in milk should be

0.5 µg / kg.

2) CITRININ

Citrinin is a mycotoxin that was first isolated from Penicillium citrinum prior to

World War II, but has also been identified in over a dozen species of Penicillium and

several species of Aspergillus (Aspergillus terreus and Aspergillus niveus). Some of

these species are used to produce human foodstuffs such as cheese (Penicillium

camemberti), sake, miso, and soy sauce (Aspergillus oryzae).

Substrates

It is associated with many other foods such as wheat, rice, corn, barley, oats, rye,

etc. More recently, citrinin has also been isolated from Monascus ruber and Monascus

purpureus, industrial species used to produce red pigments.

Toxicity

Citrinin has been implicated as a contributor to porcine nephropathy. Citrinin acts

as a nephrotoxin in all animal species tested, but its acute toxicity varies in different

species. The LD50 for ducks is 57 mg/kg; for chickens it is 95 mg/kg; and for rabbits it is

134 mg/kg. Rats are killed by parental administration of 35-58 mg/kg of citrinin. Oral

LD50 in rats is 50 mg/kg. Citrinin can also act synergistically with ochratoxin A to

depress RNA synthesis in marine kidneys.

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Citrinin is also associated with yellow rice disease in Japan. Although citrinin is

regularly associated with human foods, its full significance for human health is

unknown.

3) OCHRATOXIN

Ochratoxin was discovered as a metabolite of Aspergillus ochraceus in 1965

during a large screen of fungal metabolites that was designed specifically to identify new

mycotoxins. Shortly thereafter, it was isolated from a commercial corn sample in the

United States and recognized as a potent nephrotoxin. Ochratoxin is a mycotoxin that

comes in three secondary metabolite forms, A, B, and C.

Members of the ochratoxin family have been found as metabolites of many

different species of Aspergillus, including Aspergillus alliaceus, A. auricomus, A.

carbonarius, A. glaucus, A. melleus, and A. niger. It is also observed that Penicillium

verrucosum, a common contaminant of barley, is the only confirmed ochratoxin producer

in genus Penicillium.

The three forms differ in that Ochratoxin B (OTB) is a non-chlorinated form of

Ochratoxin A (OTA) and that Ochratoxin C (OTC) is an ethyl ester form Ochatoxin A.

Substrates

Aspergillus ochraceus is found as a contaminant of a wide range of commodities

including beverages such as beer and wine. A. carbonarius is the main species found on

vine fruit, which releases its toxin during the juice making process. Ochratoxin A has

been found in barley, oats, rye, wheat, coffee beans, and other plant products, with barley

having a particularly high likelihood of contamination. It can also be accumulated in the

meat of animals. Thus meat and meat products can be contaminated with this toxin.

The level of ochratoxin produced is influenced by the substrate on which the

molds grow as well as the moisture level, temperature, and presence of competitive

microflora.

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Mode of Action

Toxic effects of ochratoxin on the cellular level are connected with the enzymes

of glucose metabolism and of anion transport, leading to intercellular alkalinization.

Ochratoxin interferes with the enzyme phosphorylase causing an increase of glycogen in

the liver. This inhibitory effect is due to competition of the toxin with 3’, 5’ cyclic AMP.

Toxicity

Of the Aspergillus toxins, only ochratoxin is potentially as important as the

aflatoxins. The kidney is the primary target organ. Ochratoxin A is a nephrotoxin to all

animal species studied to date. In addition to being a nephrotoxin, animal studies indicate

that ochratoxin A is a liver toxin, an immune suppressant, a potent teratogen, and a

carcinogen. It causes tumors in the human urinary tract, although research in humans is

limited by confounding factors.

Ochratoxin has been detected in blood and other animal tissues and in milk,

including human milk. It is frequently found in pork intended for human consumption.

Ochratoxin is believed to be responsible for a porcine nephropathy that has been studied

intensively in the Scandinavian countries. In addition, ochratoxin is associated with

disease and death in poultry. Oral LD50 for rats is 59 mg/kg and for dogs it is 0.2 mg/kg.

Acceptable Level in Food

The Codex Alimentarious Commission proposes the maximum level ochratoxin

in wheat, barley, rye and derived products i-e 5 µg / kg. EU suggests a limit of 5 µg / kg

in raw cereals and a limit of 3 µg / kg for processed cereals and 10 µg / kg in dried vine

fruits.

4) ERGOT ALKALOIDS

The ergot alkaloids are among the most fascinating of fungal metabolites. These

compounds are produced as a toxic cocktail of alkaloids in the sclerotia (formation of

compact mass) of species of Claviceps, which are common pathogens of various grass

species. Ergotamine is the principal alkaloid produced by the ergot fungus, Claviceps

purpurea.

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Pharmaceutical Use

It is used medicinally for treatment of acute migraine attacks (sometimes in

combination with caffeine), and to induce childbirth and prevent post-partum

haemorrhage.

Mode of Action

Ergot alkaloids are known for their negative effects neuroreceptors. The primary

effect of ergot alkaloids is stimulation of smooth muscle. Ergot alkaloids bind alpha-

adrenoreceptors and further inhibit beta-adrenoreceptors which results in

vasoconstriction. Ergot alkaloids also have been shown to inhibit prolactin secretion in

humans and animals. This effect is attributed to stimulation of the dopamine receptors,

which regulate prolactin.

Toxicity

Human ergotism was common in Europe in the middle Ages in which slow

nervous fever usually occurred in the summer and fall after a severe winter. The

ingestion of ergot sclerotia from infected cereals, commonly in the form of bread

produced from contaminated flour, cause ergotism the human disease historically known

as St. Anthony’s fire.

Two forms of ergotism are usually recognized, gangrenous and convulsive. The

gangrenous form affects the blood supply to the extremities, while convulsive ergotism

affects the central nervous system. Other symptoms that are observed include vomiting,

diarrhea, abdominal distress, headache and general feeling of illness. Modern methods of

grain cleaning have significantly reduced ergotism as a human disease.

5) PATULIN

Patulin is produced by many different molds but was first isolated as an

antimicrobial active principle during the 1940s from Penicillium patulum (later called

Penicillium urticae, now Penicillium griseofulvum). The same metabolite was also

isolated from other species and given the names clavacin, claviformin, expansin, mycoin

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c, and penicidin. Patulin is a toxin produced by the P. expansum, Aspergillus,

Penicillium, and Paecilomyces fungal species.

Substarte

Penicillium expansum, the blue mold that causes soft rot of apples, pears,

cherries, and other fruits, is recognized as one of the most common offenders in patulin

contamination. Patulin is also regularly found in unfermented apple juice. It is destroyed

by the fermentation process and so is not found in apple beverages, such as cider.

Mode of Action

In patulin the mechanism of adverse effect is due to the covalent binding of

patulin to cellular nucleophiles, particularly proteins and SH-groups of glutathione. As a

result, covalently cross-linked, over thiol and aminogroups, essentially denatured

proteins such as inhibited protein tyrosin phosphatase are formed.

Pharmaceutical Use

It is tested as both a nose and throat spray for treating the common cold and as an

ointment for treating fungal skin infections. It became apparent that, it shows

antibacterial and antiviral properties.

Toxicity

Patulin is also toxic to both plants and animals, precluding its clinical use as an

antibiotic. Although patulin has not been shown to be carcinogenic, it has been reported

to damage the immune system in animals.

Acceptable Level in Food

In 2004, the EU set limits to the concentrations of patulin in food products. They

currently stand at 50 μg / kg in all fruit juice concentrations, at 25 μg / kg in solid apple

products used for direct consumption and at 10 μg / kg for children's apple products,

including apple juice. Codex Alimentarious Commission suggests 50 μg / liter maximum

limit of patulin in apple juice and apple juice ingredients in ready made soft drinks.

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6) FUMONISINS

Fumonisins were first described and characterized in 1988. The most abundantly

produced member of the family is fumonisin B1. They are thought to be synthesized by

condensation of the amino acid alanine into an acetate-derived precursor. Fumonisins are

produced by over 50 species of Fusarium, notably Fusarium verticillioides, Fusarium

proliferatum, and Fusarium nygamai, as well as Alternaria alternata.

Some of the other major types of Fusarium toxins include: -

Beauvercin

Enniatins

Butenolide

Equisetin

Fusarins

Substrate

It infects the grain of developing cereals such as wheat and maize. It is present in

high levels in corn meal and corn grits.

Mode of Action

Fumonisins are both cytotoxic and carcinogenic to animals. The modes of such

actions, however, are not completely understood. However, it is demonstrated that

fumonisin B1 disrupts sphingolipid metabolism by inhibiting sphingosine N-

acyltransferase (ceramide synthase) in rat liver microsomes. It also has been shown that

fumonisin B1 inhibits other intracellular enzymes including protein phosphatases and

argino-succinate synthetase (Jenkins et al., 2000). Therefore, FB1 exerts its cytotoxicity

by inhibiting sphingolipid metabolism, protein metabolism, and the urea cycle. The

carcinogenic role of fumonisin B1 has been linked to the accumulation of sphingoid

bases that cause unscheduled DNA synthesis.

Toxicity

Fumonisins affect animals in different ways by interfering with sphingolipid

metabolism. They cause leuko-encephalomalacia (hole in the head syndrome) in equines

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and rabbit, pulmonary edema and hydro-thorax in swine, and hepatotoxic and

carcinogenic effects and apoptosis in the liver of rats. In humans, there is a probable link

with esophageal cancer. The occurrence of fumonisin B1 is correlated with the

occurrence of a higher incidence of esophageal cancer in regions of Transkei (South

Africa), China, and northeast Italy.

The dose of fumonisins in the range of 5-15 mg / kg cause severe inflammation

of the intestinal mucosa and fatty degeneration of the liver in albino rats.

Acceptable Level in Food

The maximum level fumonisins in cereals should be 5 µg / kg as proposed by

Codex Alimentarious Commission.

7) TRICHOTHECENES

The trichothecenes constitute a family of more than sixty metabolites produced

by a number of fungal genera, including Fusarium, Myrothecium, Phomopsis,

Stachybotrys, Trichoderma, Trichothecium, and others. The term trichothecene is

derived from trichothecin, which was the one of the first members of the family

identified. They are commonly found as food and feed contaminants. The trichothecenes

are extremely potent inhibitors of eukaryotic protein synthesis; different trichothecenes

interfere with initiation, elongation, and termination stages.

Diacetoxyscirpenol, deoxynivalenol, and T-2 are the best studied of the

trichothecenes produced by Fusarium species. The macrocyclic trichothecenes are

produced largely by Myrothecium, Stachybotrys, and Trichothecium species.

Substrate

Deoxynivalenol is one of the most common mycotoxins found in grains. It is the

most prevalent and is commonly found in barley, corn, rye, safflower seeds, wheat, and

mixed feeds.

Stachybotrys grows well on all sorts of wet building materials with high cellulose

content, for example, water-damaged gypsum board, ceiling tiles, wood fiber boards, and

even dust-lined air conditioning ducts.

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Mode of Action

Cytotoxicity of trichothecenes has been attributed to their potent inhibition of

protein, RNA, and DNA synthesis. When trichothecene binds to active polysomes and

ribosomes, the peptide linkages are interrupted, the initiation and termination sequences

are diminished, and the ribosomal cycle is disrupted.

Toxicity

It has been hypothesized that T-2 and diacetoxyscirpenol are associated with a

human disease called alimentary toxic aleukia. The symptoms of the disease include

inflammation of the skin, vomiting, and damage to hematopoietic tissues. The acute

phase is accompanied by necrosis in the oral cavity, bleeding from the nose, mouth, and

vagina, and central nervous system disorders.

When deoxynivalenol is ingested in high doses by agricultural animals, it causes

nausea, vomiting, and diarrhea. For this reason, deoxynivalenol is sometimes called

vomitoxin or food refusal factor. It is less toxic than many other major trichothecenes.

Stachybotryotoxicosis was first described as an equine disease of high mortality

associated with moldy straw and hay. Until recently, human stachybotryotoxicosis is

considered a rare occupational disease limited largely to farm workers who handle moldy

hay. The presence of Stachybotrys has been associated with pulmonary bleeding in

infants.

Rats can tolerate the intravenous injection of 5 mg trichothecene per kg but are

affected by a dose of 12.5 mg / kg and killed by 500 mg / kg.

8) ZEARALENONE

Zearalenone is a secondary metabolite from Fusarium graminearum. The

reduced form of zearalenone, zearalenol, has increased estrogenic activity.

Substrate

This toxin is found almost entirely in grains and in highly variable amounts

ranging from a few nanograms per gram to thousands of nanograms per gram. The

appearance of mold on grain plants cannot be relied upon to warn of toxin production

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because Fusarium-infected grain does not necessarily appear visibly moldy in the

presence of high concentrations of mycotoxins

Pharmaceutical Use

Zearalenone has also been used to treat postmenopausal symptoms in women and

both zearelanol and zearalenone have been patented as oral contraceptives.

Mode of Action

Interaction of zearalenone with estrogen receptors is followed by transportation

of the estrogen-receptor complex into the cellular nucleus, conjugation with chromatin

receptors and a selective transcription of RNA. It results in a number of biochemical

effects like an increase of the muscular content of water and decrease of the lipid

content; enhancing of uterus permeability in relation to glucose, RNA and pre-proteins.

Toxicity

Genotoxicity is a reported concern with respect to zearalenone. Recently,

endocrine (hormone) disrupters have received a lot of public attention. Zearalenone, has

attracted recent attention due to concerns that environmental estrogens have the potential

to disrupt sex steroid hormone functions. Occasional outbreaks of zearalenone

mycotoxicosis in livestock are known to cause infertility.

Acceptable Level

The recommended safe human intake of zearalenone is estimated to be 0.05

µg/kg of body weight per day. Zearalenone levels in foodstuffs are not yet regulated

anywhere.

Other Mycotoxins

Penicillium roqueforti and Penicillium camemberti, species used to manufacture

mold-ripened cheeses, produce a number of toxic metabolites, including penicillin acid,

roquefortine, isoflumigaclavines A and B, PR toxin, and cyclopiazonic acid.

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The yellow rice toxins (citrinin, citreoviridin, luteoskyrin, rugulosin, rubroskyrin,

and related compounds) are believed to have exacerbated “Shoshin-kakke”, a particularly

malignant form of beriberi seen in Japan in the early 20th century.

A number of rare and obscure diseases have been hypothesized to be possible

mycotoxicoses, often on extremely meager evidence. These include Kashin-Beck disease

in Russia, mselini joint disease and onylalai in Africa, endemic familial arthritis of

Malnad in India, frontoethmoidal encephalomenin-gocele in Myanmar, sago hemolysis

in Papua New Guinea, and deteriorated sugar cane poisoning in China.

MYCOTOXIN CONTROL STRATEGIES

Methods for controlling mycotoxins are largely preventive. They include good

agricultural practice and sufficient drying of crops after harvest. There is considerable

on-going research on methods to prevent pre-harvest contamination of crops. These

approaches include developing host resistance through plant breeding and through

enhancement of antifungal genes by genetic engineering, use of bio control agents, and

targeting regulatory genes in mycotoxin development. As of now, none of these methods

has solved the problem. Because mycotoxins are “natural” contaminants of foods, their

formation is often unavoidable. Many efforts to address the mycotoxin problem simply

involve the diversion of mycotoxin contaminated commodities from the food supply

through government screening and regulation programs.

In order to control mycotoxin contamination of foods and food products the

following chemical and physical (processing) measures should be adopted.

1. GOOD AGRICULTURAL PRACTICES (GAPS) /

GOOD MANUFACTURING PRACTICES (GMPS)

The first line of defense against the introduction of mycotoxins is at the farm

level and starts with implementation of good agricultural practices to prevent infection.

Preventive strategies should be implemented from pre- through post harvest.

Pre-harvest strategies include maintenance of proper planting/growing conditions

(for example, soil testing, field conditioning, crop rotation, irrigation), antifungal

chemical treatments (for example, propionic and acetic acids), and adequate insect and

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weed prevention. Harvesting strategies include use of functional harvesting equipment,

clean and dry collection/transportation equipment, and appropriate harvesting conditions

(low moisture and full maturity).

Post harvest measures include use of drying as dictated by moisture content of the

harvested grain, appropriate storage conditions, and use of transport vehicles that are dry

and free of visible fungal growth. While implementation of these precautions goes a long

way toward reducing mycotoxin contamination of foods, they alone do not solve the

problem and should be an integral part of an integrated HACCP-based management

system.

2. HACCP

Inclusion of mycotoxin control in HACCP plans, an important aspect of an

overall management approach, should include strategies for prevention, control, and

quality from farm-to-fork. In the food industry, post harvest control of mycotoxins has

been addressed via HACCP plans, which include use of approved supplier schemes.

Implementation at pre-harvest stages of the food system needs more attention. Such

action provides a critical front-line defense to prevent introduction of contaminants into

the food and feed supplies. Pre-harvest HACCP programs have been documented for

controlling aflatoxin in corn and coconuts in Southeast Asia, peanuts and peanut

products in Africa, nuts in West Africa, and patulin in apple juice and pistachio nuts in

South America.

3. BIOLOGICAL CONTROL MEASURES

Levels of mycotoxins have reduced in the field and in storage without

intervention. It includes: -

Degradation mechanisms resulting in reduced mycotoxin levels in the field.

Limited research on trichothecenes has suggested the possibility that the

mycotoxin may be metabolized by corn enzymes; and

Decline in trichothecenes levels in grains stored at −18°C to 4°C and

trichothecenes at temperatures greater than 0°C.

The potential for using microorganisms to detoxify mycotoxins has shown

promise. Exposure of trichothecenes to microbes contained in the contents of the large

intestines of chickens completely transformed it in vitro to de-epoxy-DON

(deoxynivalenol, type of trichothecenes), which is 24 times less toxic than DON itself.

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4. TRANSGENIC APPROACHES

Current research efforts are focusing on methods to prevent infection at the pre-

harvest stage with emphasis on mechanisms by which the affected plants may inhibit

growth of molds or destroy mycotoxins that they produce. Traditional grain-breeding

strategies to select for preferred genetic traits have been conducted for many years. There

has been limited success with this approach to Fusarium graminearum and Aspergillus

flavus. There are hybrids currently in use that limit mycotoxin production; however, the

potential to reach unacceptable levels remains. Fumonisin production has received less

attention from researchers.

Traditional methods are plagued by many hurdles, however, including

inconsistent, labor-intensive inoculation techniques, lack of single genes and resistant

control genotypes, and the financial implications of evaluating results.

Genetic modification of mold-susceptible plants holds great promise for

controlling this food safety issue. One approach involves increasing production of

compounds (for example, anti-fungal proteinsor secondary metabolites, such as

hydroxamic acids, phenolics, stilbenes) that reduce infection by the microorganism. This

may be accomplished by introducing a novel gene to express the target compound.

Another option is to enhance expression of such a compound by the existing

gene, there by capitalizing on the plant’s own defense mechanisms. For example,

enzymes that catalyze production of anti-fungal could be targeted for expression.

Alternatively, genetic engineering methods to increase production of enzymes that

degrade mycotoxins are also being pursued. Efforts are also under way to engineer plants

to produce compounds that disrupt mycotoxin synthesis. For example, enhanced

expression of α-amylase inhibitor in Aspergillus spp. could result in significantly reduced

aflatoxin levels.

Another avenue for reducing mycotoxin levels would be to reduce insect injury to

plant kernels. Insects play an important role in the proliferation of mold growth in the

field and in storage. Resistance developed through the use of several Bt (Bacillus

thermophilus) genes in corn, wheat, and other cereal grains to minimize insect damage

has led to effective reduction in Fusarium ear rot (F. verticillioides and F. proliferatum)

mycotoxin levels in grain.

CONCLUSION

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The variability in mycotoxin contamination and the potential for novel

mycotoxicoses to emerge make the prospects for ongoing significant human

mycotoxicoses likely, especially in low-income countries in which surveillance is less

available because of economical and technological constraints. The human health

consequences of acute aflatoxicosis alone range from death to exacerbated malnutrition,

devastating to the affected populations. Very little is known about the effects of long-

term low-level exposure, especially with regard to co-contamination with multiple

mycotoxins.

Thus, development of low-tech, inexpensive methods for mycotoxin surveillance

is a world health imperative. With several novel approaches being developed, such as

molecular imprint polymers and immuno-assays and bio-assays, adoption of such

methods is within reach. The prevention of mycotoxin contamination of human foods

could have a significant effect on public health in low-income countries, and deserves

significant attention. The food industry should take the lead in these efforts, because it

will lead to improved economic sustainability of the industry, enhanced food safety

efforts, enhanced international trade efforts, and improved public health.

REFERENCES

http://en.wikipedia.org

http://www.botany.hawaii.edu/faculty/wong/BOT135/lect11.htm

http://www.mold-survivor.com/symptoms.html

Bennett, J. W. and M. Klich. 2003. Mycotoxins. Clinical Microbiology Reviews,

16(3): 497-516.

Murphy, P. A., S. Hendrich, C. Landgren and C. M. Bryant. 2006. Food

Mycotoxins: An Update. Journal of Food Science, 71: 51-65.

Torsten Berg. 2003. How to establish international limits for mycotoxins in food

and feed? Food Control, 14: 219-224.

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Claude Moreau. 1979. Moulds, Toxins and Food. Wiley-Interscience Publication,

Paris, France, pp. 27-271.

Hussein S. Hussein, Jeffrey M. Brasel. 2001. Toxicity, metabolism, and impact of

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Haig AM Jr. Chemical Warfare in Southeast Asia and Afghanistan. Washington,

DC: US Government Printing Office; March 22, 1982. Report to the Congress.

Watson, S.A, C. J. Mirocha and A. W. Hayes. 1984 Analysis for trichothecenes

in samples from Southeast Asia associated with “Yellow Rain.” Fundam Appl

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