Toxicants Occurring Naturally in Foods. (1966)

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C. L. COMAR Natural Radioactivity in the Biosphere and Foodstufts Although it is known that large amounts of radioactivity deposited in the body can produce harm, there is no evidence of deleterious effects from the amounts of radioactive materials that occur naturally in the diet and in the body. For this reason, and because of difficulties of measurement of such small amounts, there have been relatively few data accumulated. Within recent years, however, there has been the added incentive of the need to know more about the natural background of radiation so as to permit assessment of the effects of man-made additions arising from nuclear weapons tests and expanded peacetime uses of atomic energy. The references listed in the bibliography will serve to document all statements made. In particular, the report of the United Nations Scientific Committee on the Effects of Atomic Radiation presents a detailed and comprehensive coverage of radiation exposure to the human population from all sources. Since the discovery of polonium and radium by the Curies before the turn of the twentieth century, over forty naturally occurring radio- nuclides have been identified and characterized. Most are elements of high atomic weight, with atomic numbers greater than 81, and they fall into three distinct series, each of which begins with a very long-lived radionuclide and ends with a stable isotope of lead. These are known as the uranium series, the thorium series, and the actinium series; a listing is presented in Table 1. | Also in existence are a few naturally occurring radionuclides that are not members of the above series. These are listed in Table 2. As indi- cated, some of them have half-lives sufficiently long to have enabled 117

118 Cc. L. COMAR them to exist from the time of formation of the earth’s crust. On the other hand, others must be produced continuously. The latter are formed by nuclear reactions between components of cosmic radiation and stable nuclei. Carbon-14 is formed by the action of neutrons upon the nitrogen of the atmosphere; tritium is formed by several different reactions. Other radionuclides, known to be formed naturally from the interaction of cosmic-ray neutrons with the atmosphere include 7Be, 22Na, 32P, and 35S. Of the radionuclides listed in Table 2, 4C and 4°K are of greatest biological interest. TABLE 1 Naturally Occurring Radioactive Nuclides of the Uranium, Thorium, and Actinium Series OLDER COMMON GENERIC TYPE OF ELEMENT SYMBOL NAME SYMBOL RADIATION HALF-LIFE URANIUM SERIES Uranium 238) Uranium I UI a,y 4.5 X 1@ yr Thorium 24Th Uranium Xi UX B,y 24.1 days Protactinium 234Pa Uranium X2 UX2 B, y 1.17 mo Uranium 24 Uranium 0 Un a,y 2.5 XK 105 yr Thorium 20Th lonium Io a, 8.0 X 104 yr Radium 26Ra $$ Radium a,y 1620 yr Radon 22Rn Radon Em a, 3.82 days Polonium 218Po Radium A RaA a, B 3.05 mo Lead 214Ph Radium B RaB B, 26.8 mo Bismuth 214Bi Radium C RaC a, B, Y 19.7 mo Polonium 214Po Radium C’ RaC’ @ 1.6 X 10 sec Thallium 210T Radium C” RaC” B.Y 1.32 mo Lead 210Pb Radium D RaD B, 19.4 yr Bismuth 210Bi Radium E RaE a, B 5.0 days Polonium 210Po Radium F RaF a, 7 138 days Lead 26Pbh Radium G RaG None Stable THORIUM SERIES Thorium 232Th Thorium a, ¥ 1.39 X 10! yr Radium 228Ra Mesothorium: MsTh 8 6.7 yr Actinium 228Ac Mesothorium 1 MsTh2~ 8, y 6.13 hr Thorium 28Th §Radiothorium RdTh a,y 1.90 yr Radium 24Ra = Thorium X ThxX a,y 3.64 days Radon 20Rn Thoron Tn a 54.5 sec Polonium 216Po Thorium A ThA a 0.158 sec Lead 212Ph Thorium B ThB B 10.6 hr Bismuth 212Bi Thorium C ThC a, B, Y 60.5 mo Polonium 22P0 Thorium C’ ThC’ a 3 X 10-7 sec Thallium 208T] Thorium C’”’ ThC” BY 3.1 mo Lead 28Pbh $$ Thorium D ThD None Stable

RADIOACTIVITY TABLE 1—continued OLDER COMMON GENERIC TYPE OF ELEMENT SYMBOL NAME SYMBOL RADIATION HALF-LIFE ACTINIUM SERIES Uranium 235U Actinouranium AcU a, y 7.07 X 108 yr Thorium 231Th Uranium Y UY BY 25.6 hr Protactinium 23!Pa Protactinium Pa a, 7 3.4 X 10 yr Actinium 227Ac Actinium Ac a, B 21.6 yr Thorium 27Th Radioactinium RdAc_ a,8 18.1 days Francium 223Fr Actinium K Fr B 21 mo Radium 23Ra =s- Actinium X AcX a,y 11.7 days Radon 29Rn ~=Actinon An a, y 3.92 sec Polonium 215Po Actinium A AcA a 1.83 * 10-3 sec Lead 211Pb Actinium B AcB B, 36.1 m Astatine 21SAt Astatine a about 10 sec Bismuth 21 Bi Actinium C AcC a, B,y 2.16 mo Polonium 211Po Actinium C’ AcC’ a 0.5 sec Thallium 207T] Actinium C” AcC” By 4.76 mo Lead 207Pb Actinium D None Stable TABLE 2 Naturally Occurring Nonseries Radioactive Nuclides TYPE OF HALF-LIFE ELEMENT SYMBOL RADIATION (YEARS) FORMED WITH EARTH’S CRUST Beryllium 10Be B 2.5 X 106 Potassium 40K 8 Y 1.31 K 10 Rubidium S7Rb BY 6.0 < 1010 Indium 115In B 10!4 (2) Tellurium 130Te B 102 (2) Lanthanum 138L_q B 7 X 1010 Samarium 147§m a 7 X 1010 Lutecium 176. y BY 4.6 X 1010 Rhenium 187Re B 4X 102 Bismuth 209Bi @ 2.7 X 1017 PRODUCED CONTINUOUSLY Tritium 3H B 12.4 Carbon 4C B 5,570 Beryllium 7Be 7 53 days Sodium 22Na B, 2.6 Phosphorus 32P By 14 days Sulfur 35§ B 87 days

120 C. L. COMAR NATURAL RADIOACTIVITY IN THE CRUST AND LOWER ATMOSPHERE OF THE EARTH Naturally occurring radioactive elements are widely distributed throughout the earth’s crust. The more important of these radionuclides are long-lived 28U,22Th, and 226Ra (plus their shorter-lived daughter products), 4°K, and the cosmic-ray produced nuclides, '4C and 3H. Natural radionuclides are generally found to be more concentrated in granitic than in basaltic rocks. Limestones and sandstones are generally low in radionuclide content, but certain shales may be more radioactive, especially those containing organic matter. Marine sedi- ments appear to contain more radioactivity than do nonmarine or estuarian deposits. In various countries there exist large quantities of uranium ore and large deposits of monazite, the principal thorium- bearing mineral. . Potassium is relatively abundant in nature. Radioactive 4°K consti- tutes 0.0118 percent of the total potassium. The potassium content of soils is estimated to range from about 10-3 to 3 & 10-2 g of K per gram of soil, which represents 1 to 30 pCi of 4°K per gram of soil.* 226Ra, one of the daughter products of 28U, has been found in soil in the United States to vary between 0.09 and 0.8 pCi per gram of soil. Wherever there are uranium or thorium minerals in soil, their respective short-lived gaseous daughter products, 222Rn and 22°Rn, accumulate in soil and rocks. They diffuse into the air at a variable rate depending upon many factors of soil and climatic conditions. Although these gaseous radionuclides can deliver a radiation dose directly to the lungs and other tissues, they do not make a significant contribution via the food chain. In contrast, 14C, which is produced in the atmosphere, is incorporated into all living things on earth. Experimental determinations of the specific activity of natural '4C in the biosphere have ranged from 12.9 to 15.3 disintegrations per minute per gram of carbon. Likewise, tritium, 3H, is continuously produced in the atmosphere and enters the bio- sphere. The tritium content of molecular hydrogen in the troposphere of the middle latitude regions of the northern hemisphere in 1949-1951 _ * Amounts of radioactive material are expressed in units related to the curie (Ci), where 1 Ci represents an amount of a radionuclide that undergoes 3.7 x 10!0 disintegrations per second. 1 curie (Ci) = 1000 millicuries (mCi). 1 millicurie (mCi) = 1000 microcuries (Ci). 1 microcurie (wCi) = 106 picocuries (pCi).

RADIOACTIVITY 121 was about 30 pCi per gram of hydrogen. Nuclear weapons tests have added both !4C and 3H to the atmosphere. The other radionuclides produced in the atmosphere (see Table 2) are present in much lower amounts and contribute no significant radiation dose to the human population. NATURAL RADIOACTIVITY IN WATER The natural radioactivity in various types of water is highly variable depending upon origin and treatment. Certain natural springs in areas of high soil levels of uranium and thorium have high levels of radio- activity originating from these elements. Public water supplies are of interest as a possible source of radio- activity for the population. Table 3 presents some data on the concen- trations of 226Ra and 222Rn in various public water supplies. Drinking water supplies may also contain 22Th and its decay products. In a comparison of well waters from Illinois, it was found that 226Ra con- centrations ranged from 3 to 36 pCi/liter, whereas 228Ra ranged from 0.9 to 7.9 pCi/liter. Table 4 presents some values for concentrations of 238U, 226Ra, and 222Rn in various natural waters and springs. Spring waters may have high concentrations of 222Rn, but, since this has only a mean life in the body of about 1 hour, the resulting radiation dose is small. TABLE 3 2Ra and 222Rn in Public Water Supplies 226Ra 222Rn (pCi/liter) (pCi/liter) Austria (Bad Gastein) 0.6 — Germany 0.03-0.3 Up to 220 Sweden 0.2-1 — UK, ground and surface water Up to 0.7 Up to 200 Cornish waters Up to 2.4 Up to 3,000 Devon waters — Up to 13,000 USA, tap water Up to 0.2 — deep sandstone well Up to 37 —_ surface water <0.2 — USSR, fresh water 1

122 Cc. L. COMAR TABLE 4 238U, 226Ra, and 222Rn in Natural Waters and Springs 238UJ 226Ra 222Rn (pCi/liter) (pCi/liter) (pCi/liter) Germany, river water — 0.07-0.8 — UK, river water — 0.01 0.2-0.3 USA, river water 0.005-0.01 0.03 — lake water 1.7 — ground water Up to 40 Up to 22 — Austria, springs Up to 4 — Up to 105 France, springs — Up to 139 Up to 105 Germany, springs — 0.07-18 Up to 103 Japan, springs Up to 0.3 — Up to7 x 105 Lebanon, springs —_ —_ Up to 6 x 103 UK, springs — Up to 12 Up to7 X 10? USA, springs — — Up to 3 x 105 USSR, springs and brooks Up to 3 — — NATURAL RADIOACTIVITY IN FOODSTUFFS Natural radioactivity in soil and water reaches man by way of the food chain. There is little or no information on the routes and mechanisms of the transfer of naturally occurring radionuclides through the various steps of the food chain. Knowledge is limited almost entirely to a few observations of concentrations of some of these substances in various food products. Data are available on the total alpha activity of foods; values varied from less than | to 1.7 X 104 pCi per kilogram of food. In general, values were low for milk products, fruits, and vegetables but high for cereals and nuts. Total intakes vary according to proportions of various food components that are eaten; however, it has been estimated that an adequate Western diet 1s not likely to contain less than 2 to 5 pCi of alpha activity per day. Values for the 226Ra content of foodstuffs collected in New York City, Chicago, and San Francisco are given in Table 5. It was esti- mated that the average daily intake of 226Ra from all foodstuffs includ- ing water in these three cities in 1962 was about 2.3, 2.1, and 1.7 pCi, respectively. Studies of infant diets in New York City suggest that the intake by infants during their first year of life would be about 0.6 pCi per day, of which about one third comes from milk and one half from cereals. The 225Ra contents of a wide variety of foods in Germany have

RADIOACTIVITY 123 been reported to range from 0.1 to 6 pCi/kg, which is in general agreement with the data of Table 5 for the United States. No evidence has been found for the presence in foodstuffs of 22Th, the long-lived parent of the thorium chain; nevertheless, 228Th, a daughter in the series, has been found. Since the metabolic uptake of thorium is known to be poor, it is postulated that 228Ra, a continuously produced daughter of 22Th, is taken up metabolically, and in the biological system it subsequently produces the 228Th and other daugh- ters. In a series of food samples, the ratio of 228Th/226Ra was found to be 0.9. The alpha activity observed in certain samples of grass has been found mainly to be due to 2!°Po accompanied by its parent, 2!9Pb. In all natural materials, 4°K is present as a constant percentage (0.0118%) of total potassium. The 4°K contents of food and the relative contribution of various foods to the dietary 4°K can thereby be estimated from data on total potassium. The “°K intake for the United States has been estimated as about 2300 pCi/day. Atmospheric !4C is almost exclusively in the form of carbon dioxide and is utilized by plants in photosynthesis to become incorporated in all living things. Little attention has been given to the levels of !*C in foodstuffs because, essentially, the specific activity of !*C in foods and even in the population (except for lag times of up to a year) will be the same as that existing in the atmosphere. The specific activity of tropo- spheric carbon dioxide is estimated to have been about 14.5 disintegra- tions per minute per gram of carbon (6.6 pCi/g) before nuclear testing. TABLE 5 26Ra in Foodstuffs in U.S. Diet (1963) NEW YORK CITY CHICAGO SAN FRANCISCO (pCi/yr) (pCi/yr) (pCi/yr) Milk 50.8 46.4 46.4 Cereal, whole grain and 206.0 276.9 186.6 bakery products Eggs, meat, poultry, and 140.6 134.2 102.0 fish Vegetables 263.2 181.3 160.6 Fruits 176.9 102.4 109.8 Water 5.5 10.0 4.0 TOTAL 843.0 751.2 609.4

124 Cc. L. COMAR IMPORTANCE TO MAN A major interest is the relative contribution of natural radioactive materials to the radiation dosage received by the human population. Such estimates represent a summing of extent of occurrence, transport through the food chain, and retention in the body. Table 6 presents a summary of radiation dosages from both internal and external sources. TABLE 6 Radiation Dosages from Internal and External Sources DOSE RATE IN mrem/yr GONAD BONE MARROW EXTERNAL IRRADIATION Cosmic rays 50 50 Terrestrial radiation 50 50 INTERNAL IRRADIATION “OK 20 15 226Ra and decay products 0.5 0.6 228Ra and decay products 0.8 1.0 210Pb and decay products 0.3 0.4 4C 0.7 1.6 222Rn 3 3 TOTAL 125.3 121.6 It is noted that irradiation from internally deposited sources con- tributes about 20 percent or about 25 mrem/year out of a total of 125 mrem/year.* Of the internal emitters, 4°K is most important, with lesser contributions made by 222Rn, 4C, and 2Ra, 228Ra, and 2!°Pb plus their decay products. * Rem, roentgen equivalent man, is that quantity of any type of ionizing radiation which, when absorbed by man, produces an effect equivalent to the absorption of 1 roentgen of x or gamma radiation.

RADIOACTIVITY 125 BIBLIOGRAPHY R. A. Dudley, “Natural and Artificial Radiation Background of Man,” in Low- Level Irradiation, A. M. Brues, ed., American Association for the Advancement of Science, Washington, D.C. (1959). S. Glasstone, ed., Sourcebook on Atomic Energy, Van Nostrand, Princeton, N.J. (1958). “‘Health and Safety Laboratory, United States Atomic Energy Commission, Total Diets,” in Contributions to the Study of Fallout in Food Chains, HASL 147 (July 1, 1964). R. E. Lapp and H. L. Andrews, Nuclear Radiation Physics, Prentice-Hall, Englewood Cliffs, N.J. (1963). *‘United Nations Scientific Committee on the Effects of Atomic Radiation, Report,” General Assembly, 17th Session, Suppl. No. 16 A/5216, United Na- tions, New York (1962).

BENJAMIN J. WILSON Fungal Toxins It is probable that toxic diseases suspected of having mold origins have been known for many centuries. However, with the exception of ergotism, which has a long and well-documented history, the myco- toxicoses caused by filamentous fungi (molds) have been neglected insofar as systematic research endeavor is concerned. Scattered scientific reports tending to implicate common molds as causative factors in toxic diseases of livestock date back to the last century.! The first half of the present century produced only limited and somewhat sporadic studies on mold toxins in the United States,2-5 although concentrated effort was devoted to the problem by scientists in the Soviet Union, where both human and livestock populations in certain rural areas have been affected by diseases caused by moldy food.6-9 In America, the first definitive demonstration of saprophytic species as causative agents in toxic disease of animals was reported in 1950.!0 Since that time there has been an increasing awareness of the problem by veterinarians and farmers. The recent discovery of an important hepatotoxic-carcinogenic substance in peanuts from various world sources has focused wide attention on the possible hazards of fungus contamination to food materials consumed by man as well as by animals.!! Most species of fungi associated with toxic disease are saprophytes, which are widely distributed in nature. Fortunately, only certain strains of specified organisms have demonstrated toxigenicity on food materials. Some, however, may have the potential for elaborating more than one toxin, depending on the substrate and various physical factors.!2 126

FUNGAL TOXINS | 127 Perhaps the most decisive factor determining whether normally dry foods will become contaminated with fungus growth, and thus be potentially toxigenic, is the amount of available moisture. Moisture content above 10 percent, especially in the range of 13 to 18 percent, predisposes susceptible foods to attack by potentially toxigenic orga- nisms.!3 Most fungi are able to grow, over a wide range of temperature and pH, on a diversity of substrates. It is the purpose of this review to summarize available information on the filamentous fungi known to be capable of toxin production on food materials. Accordingly, each section, except the last, treats one genus of organisms and the diseases attributed to its toxigenic members. With few notable exceptions the applicability of data on animal mycotoxicoses to human diseases is not known, since, usually, only livestock are involved. The etiological information in many early reports is sketchy and quite empirical, but has pointed to the need for further careful research in this heretofore neglected area. In view of the large number of micro- organisms already recognized as sources of toxic antibiotics under experimental conditions,'* the probability exists that additional food toxigens will be discovered as the search for such contaminants is intensified. Claviceps purpurea and Ergotism Claviceps purpurea 1s a parasitic fungus that commonly infects seed grains of rye and, less often, other cereals such as wheat, barley, and oats. The fungus forms a structure known as a sclerotium, which is readily recognized as an enlarged, purple, spur-shaped body among the other seeds on the diseased rye plant. Mature sclerotia constitute the commercial source of the drug ergot, a composite of several pharmacologically active compounds.!5 Ergot has been used therapeutically for more than two centuries, especially in connection with strengthening of uterine tone in childbirth and control of postpartum hemorrhage.!* In modern medical practice the appropriate purified alkaloid derivatives are now preferred for more predictable therapeutic action. The toxicity of ergot, whether used medicinally or unintentionally ingested with food made from infected grain, has also been documented for several centuries.!7 Apparently, human ergotism due to toxic food is practically nonexistent at the present time, although an outbreak occurred in France as recently as 1951.!8-21 Epidemics of poisonings

128 BENJAMIN J. WILSON have occurred in North America, and in Great Britain, Ireland, and other European countries.2? Ergotized plants are to be found in many parts of the world, and in such countries as the United States and Russia ergotlike diseases of certain grasses are still of economic sig- nificance to livestock producers.“ In man, two distinct forms of ergotism are usually seen—gangrenous and convulsive. In the former there is first an intense burning sensation in the extremities that was once given the name “Saint Anthony’s Fire.” If consumption of contaminated food continues, a progressive restriction in the blood supply to the limbs and feet may result in gangrenous necrosis and subsequent complete, but painless, severance of the distal portions of affected limbs. In the convulsive form, a variety of symptoms are experienced relating to neurological involvement. Hallucinogenic manifestations develop along with characteristic con- vulsive seizures which may terminate fatally. The major toxic alkaloidal components of ergot have been divided into three groups by Stoll and Hofmann:24 the ergotamine group con- sisting of the active Jevo- compounds, ergotamine and ergosine, and their less potent dextro-isomers; the ergotoxine group made up of ergocornine, ergocristine, and ergokryptine, and their corresponding isomers; and ergometrine (ergonovine) and its isomer. Ergotamine and the ergotoxine groups are polypeptide derivatives of lysergic acid; the lysergic acid nucleus of ergonovine differs in having an amino group attachment.'5 The varied symptomatology of ergot poisoning can be traced to the combined effects of the above-named lJevo- substances along with smaller amounts of other physiologically active entities such as histamine, tyramine, and acetylcholine, which are also com- ponents of the crude drug. Data on the stability of ergot are variable. Some reports, however, indicate that certain dry preparations may lose their potency slowly, and contaminated food preparations could remain potentially dan- gerous for a year or longer. The prescribed dose of ergot for oxytocic effect in man is 0.15 to 1.0 g.26 For some individuals this level may provide undesirable side effects of incipient intoxication. In a mild but extensive epidemic of ergotism among Jewish immigrants in Manchester, England, in 1927, incriminated rye meal was found to contain about 1 percent ergot. This meal had been employed in a 1:4 ratio with wheat flour for use in bread.27 In past decades, the tolerance limit for ergoty flour in endemic countries was 0.10 to 0.15 percent.28 With the consumption of 0.5 kg of

FUNGAL TOXINS 129 bread per day, the dose of drug would be 0.50 to 0.75 g, well within the usual therapeutic range. However, variations in individual tolerances and amounts of bread consumed daily, coupled with prolonged intake over a period of time, would tend to make even the above limit of doubtful value. The United States Department of Agriculture Grain Division classifies grain as “‘ergoty’’ when it contains 0.30 percent or more ergot as determined on a crude weight basis. Grain lots containing this much ergot are seldom encountered according to inspection officials at the Nashville, Tennessee, office of the USDA Grain Division. Growth of Claviceps purpurea is promoted by moist warm climatic conditions. Primary infection of plants is effected by wind dissemina- tion of ascospores, while secondary spread by asexual conidia is favored by the secretion by the fungus of a yellow nectar-like material called “honeydew” that serves to attract insect vectors. The recognition of Claviceps-parasitized grain as the cause of ergotism, followed by improved agricultural and milling practices, has tended to eliminate outbreaks of the disease from areas of the world where it once had devastating effects.29 Several other species of Claviceps, particularly C. paspali, have been responsible for widely scattered outbreaks of ergot-like disease of agricultural animals in countries where various grasses serve as specific hosts. Only rarely is a particular plant attacked by more than one species of the parasite. There is no evidence to indicate that these organisms constitute a source of contamination of human food, but their role in poisoning of grazing animals is still of concern. 3° Aspergillus Species Species of Aspergillus form a large portion of all mold colonies en- countered in cultural examination of world foodstuffs.3! 32 In Oriental countries, species of the A. flavus-oryzae group are widely used in the preparation of soybean sauces, sake, and various other products.334 Toxigenic strains have been isolated from several different food ma- terials, especially feeds causing animal diseases.35—40 Among the common disease features in different animals affected by certain aspergillus toxins are severe liver damage and hemorrhages throughout the body resulting in a high mortality rate.*! No human illnesses have yet been attributed to the poisonous sub- stances produced by aspergilli, but certain high-protein foods consumed by man, such as peanuts, frequently become contaminated with toxi- genic strains with resultant toxin formation.‘2-3

130 BENJAMIN J. WILSON Table 1 lists some of the numerous outbreaks of disease in animals attributed to strains of aspergilli in the United States and Great Britain. TABLE 1 Reports of Toxic Feed Disease Outbreaks Attributed to Aspergilli TOXIGENIC ANIMALS ORGANISM FEED INVOLVED DISEASE ISOLATED REFERENCES Feed pellets Calves Liver and kidney A. clavatus 35, 40 disease Food con- Cattle Hyperkeratosis A. chevalieri 36 centrate Soft corn Swine, cattle Hepatohemor- A. flavus 40, 44 rhagic syndrome (moldy corn disease) Laboratory Laboratory Hepatomas A. flavus 43, 45 rations rats containing peanut meal Peanut meal Various com- Hepatohemor- A. flavus 44, 46-51 mercial animals rhagic syndrome (i.e., turkey X disease) Worldwide interest was suddenly focused on the aspergillus myco- toxicoses in 1961 when English investigators reported the widespread incidence of fatal liver disease syndromes in various commercial animals (see Table 1 references). All had been fed a ration partly com- posed of peanut meal contaminated with A. flavus. Chromatographic analysis of the meal extracts revealed the presence of fluorescing sub- stances, later called aflatoxins, which were found to be responsible for the poisonings.°23 At present, there are four recognized compounds designated aflatoxin B;, Bz, G;, and G2, according to whether they fluoresce blue or green under ultraviolet light and to their respective Ry values on paper or thin-layer chromatograms.*455 The chemical structure of aflatoxin B; (I) was demonstrated by Asao et al. to be related to coumarin. B2 is apparently a dihydro derivative of B;. Aflatoxin G; (II) is less toxic than B,. Go, like Bo, lacks the double bond in the terminal dihydrofuran ring.5’

FUNGAL TOXINS 131 Day-old ducklings are the most sensitive animals for toxicity mea- surements. Guinea pigs and laboratory rats are also susceptible, but mice and sheep are resistant to the toxin.55 The respective oral LDso values for ducklings of 50-g body weight according to Carnaghan et al5’ are: Bi, 18.2 mcg (5 percent fiduciary limits 14.0-23.8 mcg); Bo, 84.8 mcg (65-110 mcg); Gi, 39.2 mcg (27.1-56.7 mcg); and Ga, 172.5 mcg (158-188 mcg). Hepatic cell damage and bile duct proliferation are characteristic reactions in ducklings. Although rats are considerably more resistant than ducklings to acute effects, feeding for several weeks results in malignant hepatomas that may metastasize to the lungs.58 Hepatomas of trout caused by aflatoxin in the cottonseed meal portion of the diet have also recently been reported.59 Details of aflatoxin pathology are contained in reports by Butler and Barnes,©° Newberne ef ai.,5! and Dickens and Jones.® Reports from workers in several other countries suggest that aflatoxin contamination of foods may be a widespread occurrence. Studies con- ducted by British investigators indicate that contamination of peanuts occurs most often after nuts are lifted from the ground. Damage in harvesting or by termites, as well as improper drying and storage, are important factors favoring contamination and toxin production.® Experimentally, aflatoxins are synthesized by A. flavus in various foods including oats, soybeans, corn, rye, buckwheat, rice, and wheat. They have also been reported among metabolites of A. parasiticus® and Penicillium puberulum® growing on peanuts and wheat, respectively. Other workers have failed to confirm the aflatoxigenicity of P. puberu- lum, however. Administration of the purified compounds or naturally contaminated feed to cattle results in secretion of a somewhat modified (Ry value) B aflatoxin in the milk.§7 In addition to aflatoxins, A. flavus and closely related species are capable of forming other poisonous compounds on food materials under experimental conditions.39.6 Among these are oxalic and kojic acids, both of which have been known for many years.34.59—7! Recently,

132 BENJAMIN J. WILSON a tremorgenic substance was described as a metabolite of A. flavus grown experimentally on such food materials as oats, corn, rice, potato, and millet.72 In unpublished studies the author noted that a similar metabolite was produced by a species of Penicillium isolated from three food sources, two of which had caused fatal diseases in sheep and horses, respectively. Other toxic compounds are ascribed to A. flavus cultured in certain laboratory media. They are 8-nitropropionic acid,?3 aspergillic acid,’ hydroxyaspergillic acid,’5 flavacol,”6 and unidentified yellow crystalline toxins similar to aspergillic acid.77:78 Closely related chemically to aspergillic acid are neoaspergillic acid and neohydroxyaspergillic acid, metabolites of A. sclerotiorum,’9 and muta- aspergillic acid from A. oryzae.®° Aspergillic and neoaspergillic acids are hydroxamic acid derivatives of pyrazine. No reports have impli- cated any of these interesting metabolites in the mycotoxicoses of animals. A brief review of these toxins was presented in a recent symposium article by the author.®! Kinosita and Shikata, in a recent publication,®2 listed several other aspergilli and their previously described toxins. Several of the species have been isolated from moldy rice in Japan. Aspergillus niger and A. luchuensis are notable oxalate synthesizers on hay and other substrates.®833 The mycelia of both A. flavus and A. fumigatus are reported to contain endotoxins that elicit renal dam- age in injected animals.®4 85 The ubiquitous nature of aspergilli, combined with their remarkable ability to grow and form toxins on foods, portends discoveries of further involvement of these organisms in international food problems, and they may prove to be more important in food toxicology than has previously been thought. Penicillium Species Like the aspergilli, the penicillia are among the most common fungi.® Various species have been studied by Japanese investigators in con- nection with “yellowed rice” both of domestic origin and imported from various countries including the United States.87 The role of fungus-contaminated food in human disease in Japan is not well defined, although the toxic properties of various metabolites of peni- cillia have been studied for several years. The Japanese diet is made up largely of rice, and the average person consumes more than 150 kg per year. Concern has been expressed over a possible causative relation- ship between mold contamination of foods and liver carcinoma, cirrho- sis, and other liver diseases of people in the rice-eating countries.®8

FUNGAL TOXINS 133 Table 2, based on data from Japanese reports, lists some of the penicillia isolated from moldy foods and certain toxic substances produced by each. TABLE 2 Toxigenic Penicillium Isolates From Japanese Food Materials. ORGANISM FOOD TOXINS REFERENCES P. islandicum Rice Several toxic metabolites includ- 87, 88, 89 ing islandicin, luteoskyrin, and chlorine-containing peptide P. citrinum Rice Citrinin 90 P. urticae Malt cow feed _‘ Patulin (clavacin) 91 P. citreo-virede Rice Citreoviridin 92 P. rugulosun Importedrices Rugulosin 82 There are no data indicating the toxic levels of the above substances for man, but the marked hepatorenal damage and carcinogenic effects elicited in experimental animals suggest significant hazards to people consuming similarly contaminated food. Workers in the United States have also encountered P. citrinum as a feed contaminant.3943 One group demonstrated that foods such as corn and wheat may support production of significant quantities of the antibiotic citrinin under experimental conditions.39 The pharmacological responses to citrinin were detailed by American investigators several years ago.93 4 In the United States, Burnside and co-workers“ isolated a strain of P. rubrum (later designated P-13) from feed causing disease outbreaks in livestock.9 This strain produced a hepatotoxin on corn that was lethal for horses and other domestic animals. More recently, other investigators extracted from this isolate an acidic toxic material that brought about an acute hepatotoxic-hemorrhagic syndrome when administered to various laboratory animals including the dog.%97 The toxin was formed during growth of the fungus on moistened corn, rice, oats, rye seed, and timothy hay. Forgacs et al.98:99 demonstrated the toxigenicity of both P. rubrum and P. purpurogenum for the chicken. The frequency of food contamination with species of Penicillium points to a need for additional studies on these organisms in order to identify toxigenic strains and the possibility of toxin production on food substrates.

134 BENJAMIN J. WILSON Fusarium Species Different species of the genus Fusarium have been studied in connection with unusual human diseases noted principally in parts of the Soviet Union.7:100 One of these is alimentary toxic aleukia (ATA), attributed to contamination of cereal grain with F. sporotrichioides (F. sporo- trichiella var. sporotrichioides) and closely related fungi. ATA in man may be divided into four stages, progressing from local irritation of mucous membranes of the oral cavity and pharnyx, occurring shortly after eating contaminated cereal products, to a hemorrhagic diathesis with anemia and severe leukopenia, developing upon continued inges- tion of toxic foods. Grains, such as millet, wheat, oats, barley, rye, and buckwheat, that have overwintered under snow are often infected with different Fu- sarium species. However, not all the isolates are toxigenic. Joffe! found cultures of Fusariwn and Cladosporium to be most toxic at the time of abundant spore formation when grown at simulated winter temperatures of —2 to — 10°C. These fungi may develop on plant roots in the field and on the stored ears of grain. In past years, when average humidity was high, an in- creased quantity of toxic grain was observed in autumn. Grain stored for several years may remain toxic. A “‘lipotoxol” extracted from contaminated millet was reported to be a beta isomer of an alpha sapogenin, named ‘“‘sporofusariogenin” (empirical formula, C24H3;04). A related compound termed “poefu- sariogenin” (suggested formula, C24H2s0s) was obtained from F. sporotrichiella var. poae. Both compounds contained lactone rings and were Classed as phenanthrene compounds. The two produced a dermo- necrosis of unabraided rabbit skin and caused a persistent leukopenia when fed to experimental animals. Threshing, washing, and other processing in commercial mills were reported to be effective in de- creasing or eliminating toxicity of grain.100 The poisonous principle(s) in contaminated grain is reportedly destroyed only at temperatures above 200°C. Thus it remains un- affected in ordinary baking. Another important endemic disease attributed to varieties of F. sporo- trichiella growing on moistened grain is Urov disease, also termed Kaschin-Beck disease in honor of two Soviet investigators who re- ported their studies on it in some detail.!02 The disease is a distinctive manifestation of chondro-osteodystrophy, a disease of bones and joints that first develops in children of preschool

FUNGAL TOXINS 135 and school ages. The condition is characterized by a shortening of the long bones, deformity of the joints, flexor contractures, and muscular atrophy. Disability develops gradually with a chronic course until skeletal growth is completed. A marked reduction in work capacity ensues which remains throughout life.!% In the Soviet Union, the disease has been reported in the Transbaikal and Far East areas. In addition, some cases have been noted in near Baikal, in rayons of Irkutsk, in Vo!'ogodskaya, Pskovskaya, and Leningradskaya oblasts, and in Kiev. According to Russian authors, Urov disease has also been seen in northern Sweden and in Holland, and has been widespread in Korea and in northwest and northeast China. In China it is known as ‘‘ToKut-ze disease” or “Liu kuang-tz’u.”” The relationship of the etiological agent(s) of ATA to this condition is not clear. For example, it is possible that Urov disease may be caused by small but prolonged intake of the toxic substances that cause the ATA syndrome. However, in Kaschin-Beck disease the erythropoietic elements do not show any abnormalities, although there is a lympho- cytosis that parallels the stages of the disease. Strains of F. sporotrichiella are widespread in soil, but their geo- graphic distribution does not always parallel that of Urov disease. Screening studies indicate that perhaps relatively few isolates are capable of toxin production. In the mountain-taiga endemic areas of eastern Transbaikal, U.S.S.R., the climate is characterized by marked temperature changes during the day and by heavy precipitation in late summer and early autumn during harvest time for the grain. Soviet mycologists have demonstrated the importance of low to normal variations in culture temperatures of Fusarium in production of toxic substances on grain and these variations tend to simulate climatic conditions found in this endemic area of Urov disease.!°% Stachybotrys atra (alternans) Stachybotrys atra growing on various cereal grains or hay is capable of producing a toxin that may be lethal for certain farm animals, par- ticularly horses and cows. Stachybotryotoxicosis may develop in various stages ranging from a mild dermatitis and catarrhal inflam- mation of the mouth to a more severe form characterized by liver damage, anemia, leukopenia, and hemorrhages in various tissues and organs. The condition has been endemic for several years in the Soviet

136 BENJAMIN J. WILSON Union where a considerable amount of research data has been pub- lished.6.8:105 Qutbreaks have also been reported in Hungary and Czechoslovakia. Fialkov and Serebryanyy!™ have reported isolation of a toxic principle from S. atra with the empirical formula of C25H340c or CxH3gO6. No detailed reviews of the work are noted in available scientific literature translated into English. Man is most likely to exhibit inflammation of the mouth and nasal passages as a result of contact with hay contaminated with the mold or the inhalation of aerosols generated by handling the hay. However, fatalities have been attributed to consumption of wheat or corn that overwintered in the field. The toxicity of fungus extracts may be determined by dermal appli- cation to susceptible rabbits or guinea pigs. In the United States, Forgacs and others have published results of studies on the experi- mental disease in various laboratory and commercial animals,!0-107.108 Pithomyces chartarum and Facial Eczema In New Zealand and parts of Australia a hepatic disease of sheep known as facial eczema has been recognized since the late 1800's. Similar clinical pictures have been seen in the state of Florida. Cattle are sometimes affected but to a lesser degree. In sheep, the disease is marked by a generalized illness with photosensitization and photo- phobia, caused by accumulation in the blood of phylloerythrin, a derivative of chlorophyll. This syndrome, which is due to acute obstruc- tive cholangitis, may mask a liver damage more severe than the external signs would suggest.109 The disease is now known to be caused by a compound, spori- desmin,!!0 which is a metabolite of Pithomyces chartarum (formerly erroneously identified as Sporidesmium bakeri)''! growing on dead pasture grasses. Spores of the fungus, which contain’ much of the toxin, are present year-round in pastures of endemic areas, but growth and toxin production are most favored under climatic conditions in which the relative humidity reaches nearly full saturation at a minimum temperature of 55°F. These are attained during rainy periods in the late summer or autumn in New Zealand, at which time fungus growth is abundant on pasture.!!2 Rye grass does not disintegrate readily after cutting and serves as an ideal fungus substrate.!09 At present, there is no indication that any food consumed by man contains this metabolite. However, worldwide distribution of the fungus and the fact that sporidesmin may be elaborated experimentally

FUNGAL TOXINS 137 on cooked vegetable substrates!!3 suggest consideration of this mold as a hazardous potential contaminant of foods. The fungus known as Sporidesmium bakeri has been isolated in various parts of Africa, Malaya, Mauritius, Jamaica, and Trinidad from plants such as rice, maize, sorghum, bananas, and tobacco. It has also been obtained from the air in Queensland, Australia,!!4 and, recently, toxigenic isolates were obtained in Great Britain.!45 North and Gwynne,!!! however, in assessing the possible human implications of facial eczema, concluded that outside the laboratory there is no health hazard to man, and that while slight dangers do exist in laboratory work with the fungus and its toxin, sporidesmin, these can be minimized with reasonable care. At least three toxic compounds called, respectively, sporidesmin, sporidesmin B, and sporidesmin B3 have been isolated from cultures of P. chartarum. The structure of sporidesmin (III) has been indicated by analytical data. Sporidesmin B lacks the secondary alcoholic group, and sporidesmin B3 is considered to be deoxysporidesmin.!10.116.117 ry Hoon CH30 ¥ N oS OAT Nn CHs pia Liver damage is evident in sheep following an oral dose of 0.5 mg of sporidesmin/kg of weight, while 3 mg/kg produces a moribund state. The pathological picture shows that, among other changes, injury to cell membranes and increased cell permeability are important aspects of sporidesmin intoxication.!"7 Using rats as experimental animals, Slater and Griffiths!!8 demonstrated a rapid decrease in bile flow rate in the common duct after intraperitoneal injection of sporidesmin. This was not the case, however, for oral administration. The transient diminution of flow was attributed to direct irritant action of the toxin on the biliary tree. Other Toxigenic Fungi Various other filamentous fungi have been isolated from toxic feed causing disease among livestock. In some instances the specific causa- tive relationships of isolates to the diseases have not been well defined, and additional screening work will be required to establish unequivocal roles for certain organisms mentioned in reports of apparent myco- toxicoses.