Review pubs.acs.org/JAFC
A Concise History of Mycotoxin Research John I. Pitt† and J. David Miller*,§ †
CSIRO Agriculture and Food, P.O. Box 52, North Ryde, New South Wales 1670, Australia Department of Chemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada
§
ABSTRACT: Toxigenic fungi and mycotoxins entered human food supplies about the time when mankind first began to cultivate crops and to store them from one season to the next, perhaps 10,000 years ago. The storage of cereals probably initiated the transition by mankind from hunter-gatherer to cultivator, at the same time providing a vast new ecological niche for fungi pathogenic on grain crops or saprophytic on harvested grain, many of which produced mycotoxins. Grains have always been the major source of mycotoxins in the diet of man and his domestic animals. In the historical context, ergotism from Claviceps purpurea in rye has been known probably for more than 2000 years and caused the deaths of many thousands of people in Europe in the last millennium. Known in Japan since the 17th century, acute cardiac beriberi associated with the consumption of moldy rice was found to be due to citreoviridin produced by Penicillium citreonigrum. This toxin was believed to be only of historic importance until its reemergence in Brazil a few years ago. Other Penicillium toxins, including ochratoxin A, once considered to be a possible cause of Balkan endemic nephropathy, are treated in a historical context. The role of Fusarium toxins in human and animal health, especially T-2 toxin in alimentary toxic aleukia in Russia in the 1940s and fumonisins in equine leucoencephalomalasia, is set out in some detail. Finally, this paper documents the story of the research that led to our current understanding of the formation of aflatoxins in grains and nuts, due to the growth of Aspergillus flavus and its role, in synergy with the hepatitis B virus, in human liver cancer. During a period of climate change and greatly reduced crop diversity on a global basis, researchers tasked with monitoring the food system need to be aware of fungal toxins that might have been rare in their working careers that can reappear. KEYWORDS: mycotoxin, history, ergotism, cardiac beriberi, ochratoxin, deoxynivalenol, alimentary toxic aleukia, fumonisin
■
INTRODUCTION Essentially all of the agriculturally important fungal toxins were first recognized as animal diseases, and often there was a long journey before their significance in humans was adequately understood. In most cases, the idea that fungal toxins were important was discounted. Mycotoxins have always been “black swans”. Romans assumed that black swans did not exist, something that would have surprised the Maori and Australian aboriginal peoples. For >1500 years, the black swan existed in the European imagination as a metaphor for that which could not exist. The term black swan has come to signify the role of high-impact, hard-to-predict, and rare events that are beyond the realm of normal expectations and the psychological biases that make people individually and collectively blind to uncertainty. In the second decade of the 21st century, it is relatively easy to identify the principal mycotoxins that affect food and feed. The use of next-generation sequencing combined with alpha taxonomy and reliable sequence databases inform our perspective on the presence of fungi on crops. The genes responsible for the agriculturally important mycotoxins have been identified in full or in part, and thus their presence can be rapidly assessed in new species. For example, this led to the understanding that, although first thought to be confined to a number of species of Fusarium, some fumonsins can be produced by Aspergillus niger. FB2 and FB4 have been found in raisins and other dried fruits and in wine.1 Untargeted analytical methods allow samples to be retrospectively analyzed for unanticipated fungal toxins in samples.2 In consequence, the focus of modern researchers is primarily on the management of known compounds emerging or re-emerging in new parts of © 2016 American Chemical Society
the world because of climate variability and changes in agronomic practices.3 In addition, genetic change in the principal toxigenic fungi including Fusarium graminearum and Aspergillus flavus is an increasing threat.3−5 The principal small grains, wheat, barley, and rye, arose from North Africa and the Near East.6 This enabled the appearance of the first agricultural settlements around 9000 B.C. along the fertile crescent between the Euphrates and Tigris Rivers.7 Thus began the two important drivers of mycotoxin problems: the need to store crops and the movement of crops outside their areas of adaptation. The storage of cereals probably initiated the transition by mankind from hunter-gatherer to cultivator, at the same time providing a vast new ecological niche for fungi pathogenic on grain crops or saprophytic on harvested grain many of which we now know produce mycotoxins. The fact that insects and fungi grew on stored seeds was clearly recognized as an issue for the rulers of the larger settlements and towns. The rise of the Egyptian empire required good grain stores to feed cities and for famine. Passmore8 noted that “Joseph [was] the pioneer in famine administration, who fed the people of Egypt during seven lean years on grain stored through seven years of plenty.” Earthenware barrels used during the Minoan civilization in 2000 B.C. can still be seen in their Special Issue: Public Health Perspectives of Mycotoxins in Food Received: Revised: Accepted: Published: 7021
October 8, 2016 December 12, 2016 December 14, 2016 December 14, 2016 DOI: 10.1021/acs.jafc.6b04494 J. Agric. Food Chem. 2017, 65, 7021−7033
Review
Journal of Agricultural and Food Chemistry
mushroom toxins and compounds toxic to only lower animals such as insects or microorganisms.21−23
ancient capital, Knossos, on Crete, today. The design of Roman granaries included raised floors that allowed aeration to keep the grains dry and cool as well as treatments for insect infestations9 that would reduce the risk of fungal damage. The grain silos were built in walled enclosures, carefully plaster coated on the inside and whitewashed outside. To store the grain, the workers had to climb stairs to a small window near the top of the cone, carrying baskets. Through a little door at the bottom corn could be taken out. The small grains, emmer, wheat, barley, and oats, were moved from the Middle East and the Nile Delta across Europe over a period of 10,000 years. The climate in sites of origin of these crops is dry, but as they moved east and north, the seasons were shorter, cooler, and damper. In the British Isles, the dominant crops in the Neolithic age were (in order) emmer, naked barley, spelt, and wheat. By Roman times, this had changed to hulled barley, oats, emmer, wheat, and rye.10 Rye was an important crop from the 7th century11 such that by the Middle Ages, rye became dominant, and the way was opened for ergot of rye to become a serious problem. Although ergot is mentioned in the Old Testament and during Roman times, epidemics of ergotism were reported in Western Europe only from about 800 A.D.12 The toxicity associated with ergot sclerotia in bread was not proven until 1630,1 after which efforts were made to promote the sieving of sclerotia from grain used for breadmaking. The first pure ergot alkaloid was not reported for another 250 years.13 Wheat became more important through the 17th and 18th centuries. Cullen14 noted that “Wheat is the farinaceous food most generally used by the better sort of people over the whole of Europe, excepting the very northern parts in which it cannot be produced; but even there it is imported for the use of persons of condition. It has this advantage, that it can be formed into a more perfect kind of bread than any other of the Cerealia....” Mortality rates in England declined in the second half of the 18th century. On the basis of a careful study of population data from that period, Matossian15 has suggested that this decline coincided with the change from a rye-based diet to a wheat-based diet. The disease known then as “slow nervous fever”, which exhibits the symptoms of ergotism, declined in importance as the 18th century progressed. The sharp upsurge in human population, which began at about 1750, due to reduced mortality, may well have resulted from a change in diet from rye to wheatnot because of nutritional factors, but because of a reduction in the ingestion of a potent mycotoxin. A more modern example of dietary shifts has been seen in Africa. In 1948, few African people obtained their carbohydrate calories from corn.16 Calories came from starch from cassava, sorghum, and millet, which are much less prone to aflatoxin contamination.17 At that time, peanuts were the most important source of aflatoxin. The massive increase in corn production in Africa in the past 50 years has resulted in greatly increased exposure to aflatoxin, as well as fumonisin, in many African diets.18 Against this background, it might be considered a mystery that the term “mycotoxicosis” was first used just over six decades ago.19 The center of opinion in the food science world was that “there is very little evidence that moldy food causes illness”.20 Mycotoxins were defined21 as “fungal metabolites which when ingested, inhaled or absorbed through the skin cause lowered performance, sickness or death in man or domestic animals, including birds.” By convention, this excludes
■
ERGOTISM The association of one human illness with a fungus has been known for a long time, probably even by the Greeks and Romans. There are clear references to ergotism from the Middle Ages mainly in France but throughout continental Europe, the United Kingdom, Scandinavia, and Russia.24 Following the increased use of rye in central Europe in the Middle Ages, outbreaks of fatal ergotism were common. Although the secondary literature suggests that epidemics involved tens of thousands of people, this appears to be confusion with bubonic plague. However, documented examples of villages and surrounding areas with 100−1000 deaths in the period 900−1800 A.D. are found throughout the literature.24 These regions also experienced reduced fertility in villages where ergotism was common. This has been attributed to the reproductive toxicity of sublethal exposure to ergot.15,24 The great mycologist Louis Tulasne recognized ergot sclerotia as the result of infection by the fungus Claviceps purpurea.25 During milling, ergot sclerotia are not readily separated from sound grain, but become fragmented and dispersed throughout the flour. The earliest reports of ergotism described two types: convulsive ergotism and gangrenous ergotism. The former “seized upon men with a twitching and kind of numbness in the hands and feet, sometimes on one side, and sometimes on both. Hence a convulsion invaded men on a sudden when they were about their daily employments, and first the fingers and toes were troubled, which convulsion afterwards came to the arms, knees, shoulders, hips, and indeed the whole body, until the sick would lie down, and roll up their bodies round like a ball, or else stretch out themselves straight at length. Terrible pains and visions accompanied this evil, and great clamours and screeching did the sick make.” Gangrenous ergot was described as “a plague of invisible fire broke out, cutting off limbs from the body and consuming many in a single night. The cries of those in pain and the shedding of burned up limbs alike excited pity; the stench of rotten flesh was unbearable.”24,26 In 1630, Dr. Thuillier, physician to the then Prime Minister of France, the Duke of Sully, was the first to prove that consumption of ergoty rye caused ergotism. He observed that the intensity of the malady was in proportion to the amount of ergoty grain consumed and that people with more diverse diets suffered less. He fed ergot sclerotia to a variety of domestic animals and they all died. This led to a recommendation to the King and Clergy to advocate removal of the sclerotia by sieving. More than a century after this, L’Abbé Tessier proposed cultivation of potatoes instead of rye, improved soil drainage, and the enforced cleaning of grains.1 Removing sclerotia from grain was promoted from the 17th century but often remained ineffective well into the 20th century in Europe.15,24,27 Sieving the sclerotia from small grains in the United States and Canada was enforced in grain quality standards from the turn of the 20th century. As noted, the first structures of ergot alkaloids were reported in the 19th century and are derivatives of lysergic acid.13 The mechanism of action of ergot alkaloids is now well understood, and some are used pharmaceutically. The last reported outbreak of ergotism in Europe that affected consumers occurred in the French village of Pont St Esprit in 1954. “Bread of madness” was sold to many in the town.28 This resulted in more than 200 people becoming ill, 4 7022
DOI: 10.1021/acs.jafc.6b04494 J. Agric. Food Chem. 2017, 65, 7021−7033
Review
Journal of Agricultural and Food Chemistry of whom died from cardiovascular collapse.29,30 More recently, occupational exposures to ergot during milling have resulted in severe toxicosis of the miller.31 In the past decade, ergot has become more common in parts of Europe32 and North America,33 and preventing contamination, especially in animal feed, will require vigilance.
Other Yellow Rice Toxins in Japan. After World War II, exhaustion of food production in Japan forced the government to import large quantities of rice from all over the world. The Rice Utilization Institute was re-established as the Food Control Bureau in 1947.37 The Bureau soon identified other sources of toxic yellow rice. One fungal species, identified as Penicillium islandicum, was found to produce the toxic chemicals luteoskyrin and cyclochlorotine.37 Both were hepatotoxic, and luteoskyrin was also reported to induce hepatic cancer at 5 mg/ kg bw, a dose that is close to half the LD50.44 The presence of P. citrinum and citrinin in discolored rice also caused concern for Japanese authorities in postwar years.37 Citrinin has low acute toxicity.45 Arai and Hibino46 reported that feeding male F344 rats 70 mg/kg bw of citrinin for up to 80 weeks resulted in kidney damage; however, no effects were seen in a 90-day study with Wistar rats fed 0.2 and 20 μg/kg bw citrinin per day.47 Penicillium Toxins in Corn. In 1913, a Penicillium species isolated from moldy corn in Nebraska was reported to produce a compound that was toxic to animals when injected at levels of 200−300 mg/kg body weight.48 The isolate was identified as Penicillium puberulum Bainier, and the toxin was named penicillic acid. These authors cited work by an Italian investigator, Prof. B. Gosio, from 1896. He reported that a Penicillium isolated from corn was toxic to various laboratory animals. A compound was crystallized from liquid culture filtrate with the empirical formula C9H10O3, similar to the known formula for penicillic acid. Gosio’s work is likely the first reliable account of toxin production by a Penicillium species. In the end, penicillic acid is not notably toxic.49 In the major growing areas in the United States, corn was stored primarily in cribs and shelled just before use.50 During the 1940s, producers began changing to harvesting systems that shell corn at harvest, so that by 1998, only 7% of the U.S. corn crop was stored unshelled in cribs.51 The growth of Penicillium species on corn stored on the husk was a substantial problem in the cribs used prior to the 1970s.52−54 Corn damaged by Penicillium was associated with animal toxicosis primarily in pigs and poultry. Penicillium viridicatum cultured on autoclaved corn produced the toxic naphthaquinones xanthomegnin and viomellein55,56 and citrinin.57 Modern harvesting systems and grain storage have virtually eliminated this problem. A converse trend has been the increase in silage fed to dairy cattle. In the 1960s, severe toxicoses in cows (e.g., fatalities, bovine abortion. and placental retention) were associated with the growth of Penicillium crustosum, Penicillium roqueforti, and related species in stored corn silage in Japan58,59 and in the northern United States.3,60 General ill-thrift mainly in cows was also reported.61,62 In North America, P. roqueforti and Penicillium paneum isolated from silage produce structurally diverse metabolites including roquefortine, PR toxin, penitrem A, marcfortines A, B, and C, andrasins A and B, patulin, and mycophenolic acid.63,64 P. roqueforti, which uniquely produces PR toxin, is associated with more severe toxicosis. Festuclavine, produced by Penicillium paneum and associated with ill-thrift, is similar to the alkaloids found in cool-season fescues colonized by endophytes that are known to cause ill-thrift in grazing animals.64 This problem has reappeared in cooler dairy areas around the Great Lakes and eastern Canada and the northeastern United States and coincided with the cultivation of short-season hybrid corn. Much of this crop is used to produce silage. In Canada, the use of ensiled corn has increased 120 times in the past 25 years.65 Surveys in the northeast
■
PENICILLIUM TOXINS IN RICE Cardiac Beriberi. “Heart-attacking paralysis”, also known as acute cardiac beriberi, was reported in a number of areas from the 17th century. Apparently this was a quite common disease in Japan, beginning in the second half of the 19th century and continuing into the 20th century34 and was notable for primarily affecting young healthy males.35 The first symptoms of cardiac beriberi are heart distress and palpitation, with rapid breathing. After a few hours, breathing becomes labored, nausea and vomiting are experienced, and within 2−3 days, anguish, pain, restlessness, and unusual behavior occur. In extreme cases, progressive paralysis leading to respiratory failure may cause death. In 1881, Dr. Junjiro Sakaki reported toxicity studies on this type of moldy rice. Using ethanol extracts, he elicited symptoms in experimental animals similar to those of this disease in humans, leading him to conclude that moldy rice could cause this type of paralysis. As a result, in 1910, a government inspection scheme dramatically reduced the sale of moldy rice in Japan, and the incidence of cardiac beriberi suddenly decreased.35 Similar observations were reported by a British doctor working in the jungles in Borneo. Dr. Hose36 reported that Chinese laborers suffered from beriberi and that the rice in their bags often became moldy. He carried out experimental feeding studies with monkeys and chickens. He observed that animals exposed to the moldy rice demonstrated a lack of energy and paralytic symptoms. He thus concluded that the principal cause of beriberi in Borneo was the consumption of moldy rice. The study of acute cardiac beriberi was continued by Miyake at the Rice Utilization Institute in Japan. In 1937, a Penicillium species, subsequently described as Penicillium toxicarium, was isolated from yellow Taiwanese rice imported into Japan. The species was later shown to be synonymous with the earlier species Penicillium citreoviride. After World War II, a mycotoxin was isolated, characterized, and named citreoviridin by Yoshimasa Hitata.37 Using high doses of pure citreoviridin, the symptoms of acute cardiac beriberi were reproduced in experimental animals.35 Using CF1 mice, Nishie et al.38 showed that near-lethal doses (∼5 mg/kg bw) of citreoviridin decreased motor activities and body temperature and had cataleptic effects. Intravenous lethal doses resulted in erythema and shallow breathing. Death was due to heart failure precipitated by respiratory arrest. P. citreoviride was renamed P. citreonigrum, a still earlier name, by Pitt.39 It appears to be a very rare species even in rice,40,41 and until very recently, cardiac beriberi was considered to be a historical disease. However, the disease reappeared in 2006 in northern Brazil, where more than 1000 cases and more than 40 deaths occurred.42 The presence of P. citreonigrum and citreoviridin was confirmed. The disease occurred among subsistence farmers growing rice crops on recently cleared land, and it was more common in young healthy males. Subsequent work showed that the rice was often also contaminated by low levels of aflatoxin and trichothecenes; however, symptoms of the disease indicated death from cardiac beriberi.43 7023
DOI: 10.1021/acs.jafc.6b04494 J. Agric. Food Chem. 2017, 65, 7021−7033
Review
Journal of Agricultural and Food Chemistry
Figure 1. Structures of the agriculturally important mycotoxins discussed: ochratoxin A, 1; deoxynivalenol, 2; zearalenone, 3; T-2 toxin, 4; fumonisin B1, 5; and aflatoxin B1, 6.
Chinese herbal medicine inadvertently containing the weed Aristolochia, which produced aristolochic acid. This is a potent renal toxicant, an IARC Class 1 human carcinogen.86,87 Aristolochia was shown to occur in the region, seeding just before wheat harvest and then contaminating flour used for breadmaking.88,89 Exposure to aristolochic acid may result in tumors with the characteristic genetic signature reported in some BEN patients.90,91 A recent analysis of the weight of evidence concluded that aristolochic acid is the probable cause of BEN.92
United States report pervasive contamination of silage by toxins from P. roqueforti and related species.66
■
OCHRATOXIN A Ochratoxin A (OTA, 1; Figure 1) was originally described as a metabolite of Aspergillus ochraceus from laboratory experiments on fungal toxicity in South Africa.67 OTA was not reported in crops until 1969.68 In the same year, the first report of OTA from a Penicillium species was published.69 Soon after, more species of Aspergillus and Penicillium were shown to produce OTA. In post-World War II Scandinavia, porcine nephropathy was common and was subsequently linked to moldy grain and then to a fungus identified as Penicillium viridicatum.70 A representative isolate was shown to produce citrinin and OTA.71 The major source of the toxin in Denmark in swine diets was shown to be barley,72 and the fungus was later correctly identified as Penicillium verrucosum.73 OTA was characterized as a potent kidney toxin.74 An OTA serum adduct was found to be pervasive in the blood of pigs at slaughter in the region75,76 and later in humans.77 Although now seldom seen, Balkan endemic nephropathy (BEN) was a kidney disease with a long history in certain parts of Bulgaria, Yugoslavia, and Romania, all within the lower Danube basin. Whole families were affected, resulting in towns with houses boarded up because people could not be induced to occupy them after the mysterious deaths of the original inhabitants. At least one small town in Bulgaria was moved to a new location.78,79 Many etiological agents were suggested, including plant and fungal toxins, viruses, lead, uranium, and silica.80,81 Danish researchers strongly promoted the hypothesis that BEN was caused by exposure to OTA82−85 to the exclusion of competing ideas. Historically, exposure to OTA was common in parts of Eastern Europe where BEN occurred, but it has been shown more recently that OTA is present in the blood of most Northern and Eastern European people, in the absence of symptoms of BEN. A new hypothesis arose from the discovery that symptoms similar to those of BEN occurred in people consuming a
■
FUSARIUM TOXINS IN CEREALS Red Mold DiseaseScab. Human toxicosis from consuming grain damaged by what we now call Fusarium head blight (FHB) occurred in southern Japan from about 1890. Symptoms, including nausea, vomiting, diarrhea, abdominal pain, fever, and throat irritation, were also reported in humans in China and Korea.93−95 Mycological surveys of suspect grain revealed infections by Fusarium graminearum and related species. In the mid-1950s, feeding trials with rodents using naturally contaminated grain in Japan showed signs of toxicosis that we now associate with deoxynivalenol (2; Figure 1).94 Probably because the toxin associated with F. graminearum was ultimately discovered in Japan, the literature has tended to overlook human cases elsewhere. Tsarist Russia also experienced serious problems with FHB. Cases of human toxicosis associated with consumption of moldy grains (“intoxicating bread”) were reported in Russian literature abstracted in English by 1917.96 This was described by Donuin:97 “In connection with the extraordinary prevalence of Fusarium roseum Lk. on cereals, it was observed that the bread became poisonous (inebriant bread). People who ate it suffered from weakness, vertigo, headache, nausea, and vomition.” American studies on FHB were reported from the mid-19th century.98 By the 1920s, it was known that grain infected by F. graminearum was toxic, especially to pigs.96,99 German farmers complained about feed refusal and illness in swine after feeding grain from a shipment from the 1927 harvest in the 7024
DOI: 10.1021/acs.jafc.6b04494 J. Agric. Food Chem. 2017, 65, 7021−7033
Review
Journal of Agricultural and Food Chemistry
mouths, vomiting, weakness, fatigue, and tachycardia. After a period of time, affected individuals felt better, but there was a progressive leucopenia, anemia, and decreased platelet count, lowering “the resistance of the body to bacterial infection”. As consumption of toxic grains continued, petechial hemorrhages on the upper part of the body appeared together with necrotic lesions in the mouth and face. Bacterial infections were common. Patients who reached this stage almost always died.128,129 From 1942 to 1948 this disease resulted in largescale mortalities in the former Soviet Union, especially in the Orenburg district north of the Caspian Sea, but also throughout the southern and central regions of the U.S.S.R.126,127 In some localities, mortality was as high as 60% of those afflicted and up to 10% of the population.130 The metabolites identified by the Russian scientists were called poaefusarin from Fusarium poae and sporofusarin from Fusarium sporotrichiodes.128 Aware of the Soviet work, and working on Fusarium-contaminated grain that was acutely toxic to domestic animals, researchers at the University of Wisconsin isolated T-2 toxin (4; Figure 1).131,132 This fungus, strain “T-2” of F. sporotrichiodes, misidentified as F. tricinctum, was ultimately shown to produce a suite of potent trichothecenes, among other compounds.133 Prof. Chet Mirocha at the University of Minnesota established collaboration with Soviet scientists to resolve the toxin associated with ATA. He obtained a small sample of poaefusarin from early preparations and confirmed that this was T-2 toxin.134 In animals, T-2 toxin results in hemorrhagic lesions of the mouth, nose, and skin. Systemic exposure results in damage to white and red blood cells. The gross pathology and hematopoietic effects of ATA were recapitulated in cats.135 The toxicity of T-2 has been exhaustively studied136,137 and needs little comment here.
United States. Researchers in Germany isolated a number of fungi from the grain, including Fusarium. Culture filtrate of the Fusarium species fed by gavage produced feed refusal.100 Subsequent studies in the United States showed that damaged barley resulted in emesis in pigs.101 A water extract of barley contaminated by G. saubentii (a species concept that included F. graminearum) induced emesis in pigs by gavage.102 Experiments conducted in the mid-1960s with water and methanol extracts of cultures of F. graminearum resulted in toxicity in mice and pigs.103 Using strains isolated from cereals affected by FHB, Prentice et al.104 reported an emetic principle but were unable to determine its chemical structure.105 Deoxynivalenol. Japanese researchers solved the identity of the compound that caused feed refusal and emesis. Deoxynivalenol, 3-acetyl-DON, and 3,15-diacetyl-DON were identified as metabolites of F. roseum no. 117, an isolate from the 1970 epidemic in Japan.94,106 U.S. researchers later reported a partial structure of deoxynivalenol as “vomitoxin” from an isolate of F. graminearum from contaminated corn that had elicited emesis in pigs.107 As Japanese researchers had reported the full structure in an open meeting and in the first publication, the appropriate trivial name that should be used is deoxynivalenol.94 On the basis of the emetic response, humans are probably more sensitive to deoxynivalenol than the most sensitive domestic animal, pigs.108 The full toxigenic potential of F. graminearum109−112 and the existence of two important chemotypes, one producing deoxynivalenol via 15-acetyldeoxynivalenol and the other via 3-acetyldeoxynivalenol, were resolved by Canadian researchers.113 Fusarium head blight damaged grain and chronic human exposure to deoxynivalenol remain stubborn problems in much of the world.114 However, in contrast to 30 years ago, the toxicity of this mycotoxin is now well understood.95,115,116 Zearalenone. Corn contaminated by F. graminearum was occasionally associated with estrogenic symptoms, particularly in pigs in the 1920s.117 An active fraction was isolated from corn with a partial structure reported in 1962118 with the full structure some years later.119,120 Zearalenone (3; Figure 1) proved to be a major contaminant in corn in the United States and Canada, but the problem had largely disappeared by the early 1980s. This was due to a combination of warmer temperatures and the use of corn hybrids that matured earlier, both factors that reduced the accumulation of zearalenone.1 Zearalenone affects reproduction in female pigs at very low exposures, with a dietary no-effect level of
