BOLETIM No. 27 -

Fungal Toxins Found in Foods

Fungi produce a dazzling array of chemical structures that have widely varying biological activity. Certain fungal metabolites are highly desired components in some foods, such as cheese, while others serve as valuable antibiotics. Some fungal metabolites have been used in chemotherapy and a few even cure the common cold.

Mycology is the study of fungi. An infection by a fungus is called a mycosis. A toxin produced by a mold or mildew is called a mycotoxin (only if the fungal metabolite causes disease or death in man or animals is it considered toxic). Illness (intoxication) caused by a mycotoxin is called a mycotoxicosis. Mushrooms and morels are fungi, but their toxins are not commonly referred to as mycotoxins. For all fungi - mushrooms, toadstools, molds and mildews - the body of the fungus is called the mycelium (also called the spawn by commercial mushroom growers). The mycelium is made up a mass of stringlike cells called hyphae. The conidia in general terms is defined as spores borne externally on hyphae or specialized hyphal branches called conidiophores. Conidia are asexual reproductive structures. A mushroom is the fruiting body of a fungus. Although a lot of people like to use the word toadstool to refer to poisonous mushrooms, the term toadstool applies to any mushroom that has a defined stalk and cap - not just poisonous ones.

Mycotoxins in Food

Mycotoxins comprise a structurally diverse family of over 300 naturally occurring, fungal-elaborated poisons, many of which frequently occur as contaminants of food and feed and have resulted in substantial economic losses to agriculture. Although rigorous guidelines have been established for the preventive management of mycotoxins in crops (i.e., recommended practices for growing, harvesting, handling, and storage), significant mycotoxin contamination still occurs. Consequently there is a growing awareness of the potential hazards of these substances as contaminants of food and feed. Mycotoxins commonly occur as mixtures that may potentially provoke complex interactions and toxic synergy.

Ergots. Man has been aware of mycotoxins since the beginning of recorded history. Records from ancient cuneiform clay plates describe ergotism and the use of ergots for medicine. The ancient Chinese, Greeks, and Romans also used ergots for medicinal purposes. Ergots are indole alkaloids that produce almost instantaneous central nervous system (CNS) effects. Ergots are strong serotonin antagonists and powerful vasoconstrictors. Almost all hallucinogens are serotonin antagonists. Many but not all ergots are hallucinogens with the most universally recognized of these being lysergic acid diethyl amide or LSD. The CNS effects caused by ergot intoxication or ergotism include visual disturbances (hallucinations), strong vasoconstriction (that can lead to gangrene), dry mouth, numbness, tingling, excitability, irritability, and smooth muscle contraction. Ergots are produced by Claviceps purpurea and are most common on rye, barley, and grasses (including Dallas grass and wild rice). It is so common that rye is routinely tested for its presence. The ancients recognized this association and used this in their medicine. A tea brewed from rusty rye has been used to stop post-partum hemorrhage, to stop bleeding of severe wounds, and to cause abortion. Ergots are still used (ergotamine tartrate and D.H.E. 45) as highly effective treatments of migraines. Historically ergotism has caused an affliction called St. Anthony's fire or "holy fire". In this the limbs (especially the fingers and toes) became swollen, inflamed, subject to sensations of extreme heat and cold, followed by gangrene and the loss of the limb. St. Anthony's fire was so common that the Order of St. Anthony was established to provide hospitals for those suffering from this "holy fire." Dr. Mary Matossian has published several papers which propose that the general unrest and excitability leading to the French Revolution was caused by ergotism. Matossian and others have implicated ergotism with the Salem witch trials of 1692.

Aflatoxin. Aflatoxins are probably the most studied of all mycotoxins. In 1957, all of the guinea pigs in the United Kingdom died mysteriously. This was considered to be an obscure disease and was not studied. In 1958 however, all of the turkey poults in the U.K. died - this was an economic disaster. The disease was found to be noninfectious, and to be associated with the groundnut (peanut) meal from Brazil. Samples were shipped to Drs. Buchi, Tannenbaum, and Wogan at MIT. Buchi and Wogan found that the toxicity was associated with several brightly fluorescent bands. These scientists also found that the peanut (groundnut) meal was contaminated with the mold Aspergillus flavus. The toxins were named aflatoxins from Aspergillus flavus (+ toxin). Aflatoxin B1 was named because it was the first blue spot (B1), aflatoxin G1 was the first grey (or blue-green, or green - this is viewer defined - spot). Aflatoxin B1 is approximately 3 times more toxic than aflatoxin G1. Aflatoxins have since been found to be produced primarily by both A. flavus and A. parasiticus although other fungi also produce aflatoxins. Aflatoxins are extremely toxic with the target organ being the liver. Chronic toxicity causes the liver to become yellowish in color, fatty in appearance, and highly fibrous (to touch), needless to say, normal liver functions are strongly impaired. Aflatoxin B1 is considered to be the most potent naturally occurring heptocarcinogen (liver carcinogen) known. It is known that over half of liver tumors examined in one study in China had the same point mutation at the p53 gene in DNA. Aflatoxin B1 is known to add to guanine, which happens to be found atthe p53 gene. Because of its toxicity and carcinogenicity, the action level for aflatoxins (total) in food destined for human (or lactating dairy animal) use was set at its detection level which was 20 ppb shortly after it was discovered. This limit has not been lowered as the detection limits have dropped (to less than 1 ppb today). Aflatoxin is most prevlent in oil seeds and nuts although it can occur in anything. It has been found in corn, soybeans, green coffee beans and coffee, nuts, chili powder, paprika, wheat, rice, cottonseed, cheese, and sorghum to name just a few commodities. It is impossible to get coconut without a preservative even at a health food store (unless you fix it fresh yourself) because of the danger of mold growth and mycotoxin formation. When cows consume feed contaminated with aflatoxin, they can excrete aflatoxin M1 in their milk. Of the aflatoxins, only aflatoxin B1 is converted to aflatoxin M1. Cows secrete between 1-3% of the aflatoxin B1 they consume as aflatoxin M1 in the milk. The majority of the aflatoxin M1 (over 95%) is excreted in the urine. The action limit for aflatoxin M1 in fluid milk is 0.5 ppb. The limit is other dairy products is set by how much the milk is concentrated. In some cheeses, 1 oz. of cheese requires 8 oz. of milk to produce the cheese can "legally" contain up to 4.0 ppb of aflatoxin M1. Conceivably this could go up higher although the maximum limit for aflatoxins in human food is set at 20 ppb. Aside from liver, aflatoxin is only found in the breast meat of chickens fed very high levels of aflatoxin.

There has been considerable legal wrangling over the legality of these action limits. At present virtually all corn and corn products on the market contain detectable levels of aflatoxin (usually around 5 ppb). It is routinely considered that the average individual consumes 0.002 ppb of aflatoxin daily in the diet. In the Southeast, this level is at least 10 times higher (0.02 ppb). It would be expected that the Southeast would have a higher incidence of liver cancer than elsewhere in the U.S. This is not the case, the Northeast with the lowest consumption of aflatoxin has the highest incidence of liver cancer and the Southeast actually has the lowest liver cancer rate. Liver cancer is the most prevalent cancer worldwide and yet is the rarest in the U.S. There are scientists who point to this epidemiological data and state that the action levels are safe, there are those who point to the Delaney clause which forbids carcinogens in the diet and aflatoxin is a recognized carcinogen and demand that there be zero tolerance or that the action level be lowered to the detection limit (currently less than 1 ppb for food and 0.1 ppb in milk). A big portion of this debate is never stated - if the action limit were drastically lowered, U.S. agriculture would suffer dramatic losses - most US corn has detectable levels of aflatoxin. There is debate that the action limits set by other countries are actually methods to prevent import of US grain and not because the grain produced in that country has less aflatoxin. The debate still rages.

Aflatoxins have been proposed as the cause of the mysterious disappearances of civilizations in the American Southwest and Central and South America (prior to the appearance of Europeans). In nutritionally compromised individuals (famine conditions possibly brought on by drought), 400 ppb is lethal to humans. Horses are killed by 50 ppb. Rainbow trout are the most sensitive species with less than 1 ppb causing liver tumors.

Sterigmatocystin. This mycotoxin is produced by Aspergillus versicolor. It is also produced by toxigenic strains of A. flavus and A. parasiticus. Sterigmatocystin contains a xanthone nucleus and is considered to be a biosynthetic precursor of aflatoxin. Like aflatoxin, it contains the bis-dihydrofurofuran ring (the vinyl ether) and as would be expected is a carcinogen. The target organ for toxicity is the liver. Sterigmatocystin is found in commodities that are heavily contaminated with aflatoxin. Sterigmatocystin is also found in heavily molded rice, green coffee beans, beans, salami, home cured ham, cheese, and wheat.

Ochratoxin. Ochratoxin is a phenylalanine containing dihydroisocoumarin. Ochratoxin A also contains a chlorine on thearomatic ring which accounts for its toxicity. Ochratoxin is produced by both Aspergillus ocraceus and Penicillium viridicatum (among others). Ochratoxin A (OA) has been associated with a disease called Balkan nephropathy. OA has also been shown to be a human carcinogen. It preferentially grows on sorghum, corn, and cheeses. Unlike aflatoxin which is a warm weather toxin (formed only when temperatures exceed 80 F), ochratoxin formation is favored by colder temperature (below 50 F) - it has even been found growing on cheesecake. Because of this, ochratoxin has only been detected twice in Texas – once on Texas sorghum fed by a Texas swine operation and once in corn fed in a dairy operation. Pigs are not only very sensitive to ochratoxin A, they also can store ochratoxin A in edible tissue. The USDA is establishing action limits for ochratoxin in feed and in tissues. The target organ for ochratoxin A seems to be the kidney, followed by the liver. Ochratoxin B (OB) is nontoxic. When the phenylalanine is removed the ihydroisocoumarins of OA and OB are called ochratoxin and (O and O) respectively.OA is a potent teratogen. It also is a strong synergist - a trace of OA dramatically increases the toxicity of aflatoxins although kidney damage not liver damage occurs in these cases.

Cyclopiazonic acid. This is an unusual mycotoxin. Cyclopiazonic acid (CPA) is frequently found in association with aflatoxin. In a recent North Carolina survey 57 different types of A. flavus were found in 1 truckload of corn. Of these, 15 were found to produce aflatoxins (7 produced aflatoxin only), 27 strains were found to produce CPA. The disease that killed the U.K. turkeys was called turkey X disease. A similar disease called hepatitis-X in dogs was also associated with aflatoxin. It has been impossible to reproduce the symptoms of these diseases using diets containing only aflatoxin. It is now thought that both aflatoxins and CPA were responsible for the original outbreaks. CPA is associated with hemorrhagic lesions seen on poultry thighs and is thought to be responsible for many hemorrhagic syndromes. CPA is difficult to detect without extensive laboratory equipment. It is not known if this toxin is stored in edible tissues. CPA may cause cancer but is not thought to be a potent teratogen.

Alternariols. Alternariols are produced by Alternaria species. This is one of the most common mold spore encountered. This mold can grow on cement, wall paper, tile grout, leaves, onions, tomatoes, corn, peanuts, fruits, vegetables, grain, etc. It is similar in chemical structure to benza[a]pyrene, the carcinogen in tobacco smoke. The parent ring system in alternariol is a benza[a]pyrone. Its one claim to fame is that 1968 NCR report found that there was a 1:1 correlation between the number of Alternaria spores on tobacco in a country and that country's lung cancer incidence. It has been curious that all researchers who had done alternariol research and had found a link to cancer have never published on the subject again. Their next series of research articles were always on a completely different topic and were always funded by the American Tobacco Institute.

Yellowed rice toxins. In the 1950's to early '60's, it was thought to be unhealthy to eat yellowed rice because it was vitamin deficient due to mold growth. Beriberi, particularly cardiac beriberi, was associated with this vitamin B deficiency. It was only later that it became apparent that beriberi was easily treated with vitamin B injections. Cardiac beriberi caused by the yellowed rice did not respond to vitamin treatment. It was also noticed that this yellowed rice induced beriberi frequently was associated with staggering, neurological problems and occasionally even brain damage. Analysis led to the discovery of the causative agent, the mycotoxin citreoverdin. Citrioverdin is produced by Penicillium citreoverdi. There are other yellowed rice mycotoxins including islanditoxin and luteoskirin, these are also produced by various Penicillium species.

Another yellowed rice toxin is citrinin (produced by Penicillium citrinum). Citrinin is unusual in that it exists in as a pH dependent tautomer. In neutral conditions, and in membranes it exists as it is shown here. In alkaline conditions, it exits as the tautomeric phenol. Because of this dual personality, citrinin is locked in a vicious cycle of the body excreting the toxin, the toxin entering the urine (alkaline pH) and tautomerizing to the phenol which is specifically scavenged by the transport system and rapidly reabsorbed by the kidneys. At which time it tautomerizes back to the blood pH stable tautomer which is again excreted. This cycle leads to nephrotoxicity (kidney toxicity) and ultimately kidney damage and kidney failure. Citrinin is also a potent synergist for many toxins including OA. Like OA, citrinin is a powerful teratogen.

Patulin. The old wives tale (adage) of "an apple a day keeps the doctor away" is true and is due to the mycotoxin patulin. Patulin is a strong antiviral agent that is active against the common cold virus and is also active against cancer cells. It is produced by Penicillium patulinum and is commonly encountered in apples and grapes. Bruised fruit has higher levels of patulin than unblemished fruit. Unfortunately bruised fruit also makes the best tasting fruit juice. Also, bruised fruit is either thrown away (at a loss) or used for juicing (break even or make a profit) - needless to say a lot a bruised fruit finds its way to fruit juice. The World Health Organization has established an action limit of 50 ppb for patulin in apple juice. In addition to apples, patulin is found in all fruit juices, corn, and many berries.

Tremorgens. These mycotoxins cause "staggers" in man and animals. Staggers are defined as a neurological syndrome whose clinical signs include muscle tremor, uncoordinated movements and general weakness in the hind legs with stiff, stilted movements of the forelegs. These symptoms become more evident when the victim is disturbed or excited. Many but not all tremoragens also produce excessive salivation. These toxins are encountered in a variety of commodities including animal forage, grasses, and silage, corn, wheat, and nuts. Pecans have been noted to be infected with a tremorgenic mycotoxin called paxilline. Paxilline has been known to affect dogs as well as man.

Trichothecenes. The most toxic mycotoxins produced by Fusarium are the trichothecenes. These comprise a structurally diverse group of polyhydroxysesquiterpenes that may be divided into four family classes. One class consists of macrolides, the verracarins and the roridins - these are both rare and unbelievably toxic. The other three classes are more often encountered. The first of these are the T-2 toxins (T-2). The T-2 family is associated with alimentary toxic aleukia (ATA). This disease was first recognized in Russia in 1913. Subsequently it was seen in Russia in 1932 and during (and just after) World War II. It was associated with consumption of grain that had been overwintered in the field. Outbreaks of the disease are associated with 50% mortality. Contact with crystalline T-2 causes cellular death, blistering, and necrosis at the site of contact. Stage I symptoms following ingestion were a burning sensation in the mouth, throat, esophagus, and stomach. This is often followed by vomiting and diarrhea (both fluids are corrosively contaminated with T-2). This is associated with abdominal pain due to inflammation of the gastric and intestinal mucosae. Headache, dizziness, vertigo, fatigue, tachycardia, salivation, and fever are also frequently encountered. The victims endure this for 3 to 9 days. At this time (Stage II), the victim recovers slightly and may be able to perform normally (although red blood cell count is dropping dramatically). During this time, the victim is seriously immunosuppressed, general weakness, headache, and reduced blood pressure are encountered. If clean food can be obtained and the victim is hospitalized, chances of recovery are good. However, if contaminated food cannot be avoided, Stage III is entered. Stage III is characterized by hemorrhaging of the skin, mucous membranes (mouth, esophagus, stomach, and intestinal lining). These areas become necrotic. Bacterial infection of these areas usually leads to death. ATA is thought to be the cause of the fall of ancient Athens. In acute T-2 toxicosis, the victim begins to hemorrhage from every orifice, death occurs within minutes. T-2 is too toxic to usually be considered to be a teratogen - fetal death rather than survival is the outcome.

Diacetoxyscirpenol (DAS) also known as anguidine is another trichothecene. It is less acutely toxic and far more teratogenic than T-2. Like T-2 it is produced by Fusarium and prefers to grow on wheat and millet (including bread in the refrigerator or freezer). DAS has been used in conjunction with cis-platinin for ovarian chemotherapy. DAS is a potent teratogen. It is also a powerful synergistic agent.

Deoxynivalenol (DON) is the least toxic and most common of the last family of trichothecenes, the nivalenols. DON is also known as vomitoxin because it was thought to be responsible for emesis (vomiting) seen in DON intoxications. The USDA surveyed breakfast cereal and found DON present in detectable levels in over 65% of the samples tested (wheat makes up far less than 65% of breakfast cereals). It is also found in breads.

When DON is found in food, it is often found at relatively high levels making it the most easily detected trichothecene. It is not uncommon to detect all of the trichothecenes in a single sample. Therefore DON is considered to be the sentinel of the potential presence of other toxins.

Zearalenone. Zearalenone is a potent mycotoxin found in grains. Humans and swine are particularly sensitive to its action. A modified form of this is used as a growth hormone in cattle. Zearalenone is produced by Fusarium. It is associated with hyperestrogenism - strong feminization of the victim. In female swine, uterine prolapse is frequently encountered. In males, testicular atrophy is evident as is enhanced mammary development. Many countries have action levels for zearalenone. Puerto Rico in particular enacted an action level after young boys were observed with obvious breast development (this symptom was overlooked in young girls). Serious reproductive difficulties have also been associated with zearalenone. Zearalenone is found throughout the world (hot and cold climates). It is particularly a problem in swine operations that are feeding last year's feed (before the new crop comes in). Unlike the trichothecenes preference for wheat, zearalenone is found in all grains - particularly rapeseed (used for canola oil). At present there is a world wide outcry over the perceived increase in reproductive dysfuntion including sexual maturity at increasingly younger age, especially in young girls. There has been blame associated with US use of growth hormones in beef - it is interesting to note that for the most part these children have never tasted US beef.

Fumonisin B1. A South African scientist, Dr. Marassas, was investigating the high incidence of esophageal and stomach cancer found in certain tribes including the Zulus. He found Fusarium moniliforme was the predominant fungus present ans suspected a mycotoxin was involved. After purifying the fungus, he cultured the fungus, took the culture material and injected it i.v. into two 15-year old mares. After 2 weeks of receiving 5 liters of culture material a day, the two mares were unsteady on their feet. Upon autopsy, they had brain lesions similar to those seen in equine leukoencephalo-malatia (ELEM). This group then claimed that these mycotoxins were the causes of ELEM. A tremendous amount of research is currently being done on the fumonisins. There has been a concern that fumonisin is not responsible for LEM. This is because fumonisins are found in corn at incredibly high levels (ppm levels). This is a very high level to have no observed effect for most of the population – and at a few ppm more have toxic manifestations. Another reservation is that ELEM is unknown in South Africa - the country where the toxin was discovered. To date, no link has been found between the fumonisins and cancer.