Environmental Health PerspectivesVol. 9, pp. 1-32, 1974

Mycotoxins: Toxicity, Carcinogenicity, and theInfluence of Various Nutritional Conditions*

by Paul M. Newbernet

Toxicologic diseases of man and animals, associated with molds growing on foods, have beenrecognized for centuries. Only in recent years, however, have these mycotoxicoo reweived theattention of many laboratories and skilled scientists around the world in a broad inter-disciplinary effort.This review covers the literature on mycotoxicoses but centers on those about which most is

known, particularly the diseases associated with metabolites elaborated by some strains ofAspergilli, Penicillia, Fu.aria, Stachybotrys, and Claviceps.The ubiquitous nature of the aflatoxins, toxic metabolites produced by AspergUIiu flavu4,

make them important to public health, especially since it is now known that certain areas ofendemic liver disease coincide with consumption of aflatoxins and, often, malnutrition.The older disease of ergotism, the scourge of Europe for centuries, is considered in detail.

Alimentary toxic aleukia, which has caused enormous suffering in Russian human and animalpopulations, is better understood as a result of relatively recent experimental investigations.Stachybotryotoxicosis, a disease previously considered to be of significance only to man hasnow been identified in domestic animals

Finally, Japanese studies have clearly revealed the hepatotoxicity of certain metabolites ofPenicillium molds.

Factors that influence susceptibility to mycotoxins and the hazards they present to man arealso reviewed.

IntroductionMetabolites of certain molds growing on

foodstuff have caused toxic diseases in man andanimals throughout recorded history; but an un-derstanding of the relationship between themolds, the food, and the diseases has begun todevelop only recently.

Since the turn of the century, it has beenknown that certain fungi produce toxic

*Preparation of this state-of-the-art review was spon-sored by the Toxicology Information Program of theNational Library of Medicine, National Institutes of Health.

t Department of Nutrition and Food ScienRce,Massachusetts Institute of Technology, Cambridge, Mass.,02193. Consultant during the preparation of this manuscriptto the Environmental Information System Office, Oak RidgeNational Laboratory, operated by Union Carbide Corpora-tion Nuclear Division for the U.S. Atomic Energy Commis-sion, Oak Ridge, Tenn., 37830.

metabolites eliciting biologic responses in bothman and animals. As early as the 19th century,a disease associated with the consumption ofyellow, discolored rice was recognized in Japanand was established as a toxicologic entity.Similarly, alimentary toxic aleukia (ATA),associated with overwintered wheat, affectedboth man and animals in Russia and was knownfor many decades. Centuries prior to theseobservations, ergot poisoning through ingestionof flour and bread contaminated with a funguscreated widespread epidemics of ergotism inEurope-known to the ancients as St. Anthony'sfire. Thus, ergot poisoning was recognizedretrospectively as the first of many mycotoxi-coses.

Despite the early recognition of ergot poison-ing and the toxicoses associated with yellow riceand overwintered grain, the mycotoxicoses

December 1974 I

remained generally neglected diseases untilabout 1960. A serious outbreak of a toxic turkeydisease in England precipitated scientific in-vestigations which delineated the nature of thedisease presently known as aflatoxicosis. Thisincident led to the realization that mycotoxins,and especially aflatoxins, presented a seriousthreat to public health as well as to animaleconomy; large numbers of investigators andvast resources were drawn into investigation ofthe problem; and resultantly, during the pastdecade an enormous amount of literature in thefield has accumulated. This large volume ofliterature is somewhat misleading by subtlysuggesting a long-standing knowledge; actuallythe field of mycotoxin research is barely out ofits infancy.Surveys of foods and feeds around the world

have revealed that the problem of mycotoxicosisis not limited to any one geographic area but is areal or potential problem in all areas wheremolds grow (1). In fact, virtually all staple foodproducts consumed throughout the world aresubject to contamination by mold toxins. Obser-vations that some of the mycotoxins are car-cinogenic in certain animal species and further-more that they are associated with a high in-cidence of liver cancer in some human pop-ulations have added considerable impetus toresearch efforts.The huge amount of literature on mycotoxi-

coses cannot be covered in a relatively shortreview; therefore, this treatise centers on thesalient features of the diseases associated with afew of the most commonly encounteredmolds-Aspergilli, Penicillia, Fusaria,Stachobotrys, and Claviceps. Exclusion of othermolds and even of some of the toxins producedby those listed above does not imply that theyare not important, but rather that limitation ofboth time and space prevent their inclusion. Inaddition to pointing out the major toxic frac-tions that are known to be produced by themolds, other relevant information concerningthe mold metabolites will be presented. Further-more, factors that may influence biologicresponse to the toxins will be discussed, par-,ticularly emphasizing nutrition. With the excep-tion of aflatoxin research, investigation ofnutritional influences consists to a large extentonly of clinical observations.

Ergot

During the Middle Ages, large-scaleepidemics of a toxic disease occurred in middleand western Europe (2). Although the causativeagent was not known, it was recognized that thedisease was associated with rye used for makingbread. Two characteristic forms were described:gangrenous and convulsive ergotism. There aresome hints in the older literature that ergot mayhave been used as a medicinal; its actual use as adrug was described for the first time in 1582 (3).

Ergot is composed of several true alkaloids,some of which have been thoroughly in-vestigated. The best-known genus of fungicapable of forming ergot alkaloids is Clavicepspurpurea, one of the first species known toproduce mycotoxins; it is most often found asa parasitic fungus on rye and several wildgrasses, causing the disease ergotism (4).

In about 1875, the first crystalline alkaloidpreparation was isolated,from ergot by a Frenchpharmacist, and in 1918, Stoll described the firsthomogeneous, chemically pure alkaloid whichexhibited the typical biologic properties of ergotand which was designated as ergotamine (3).The highly variable composition of the ergot

alkaloid mixture and the complex chemicalstructure of the bases impeded the furtherevaluation and elucidation of ergot alkaloids (5).The ergot alkaloids are 3,4-substituted indolederivatives, for example, ergoline (I), lysergicacid derivatives (II), and clavine alkaloids (III).Many ergot alkaloids can be derived fromlysergic acid, but the main alkaloids of C. pur-purea are peptides. On hydrolysis, these pep-tides (IV) decompose into lysergic or isolysergicacid, ammonia, a keto acid, and two aminoacids. Proline is the amino acid common to allpeptide alkaloids; the various alkaloids differfrom each other mainly in the type of the secondamino acid obtained upon hydrolytic cleavage.There are many additional chemical compoundsderived from alkaloids which cannot be coveredin this review; for further information, thereader is referred to Groger (3).During recent years, the biosynthesis of ergot

alkaloids has been under intensive investiga-tion. The ergoline ring system in ergot fungi aswell as in other plants is constructed from tryp-tophan and mevalonic acid. The N-methyl group

Environmental Health Perspectives2

R CH2R

R N-CH3

HN

Lysergic acid derivatives

R = Tripeptide or smaller unitsI

Clavine alkaloids

R = H = OH.m

R = NH2: Ergine

R - NH* HICH3 Lysergic acidR=H * methylcarbinolamide

R=H,CH3 . Ergometrine

NHC H2 (ergobasine)

H= HCH(CH3)2 Lysergic acid-R = NHC

OCl L-valinemethylesterCOOCH3

is derived from methionine by a transmethyla-tion reaction. L-Tryptophan is the immediateprecursor of the ergoline nucleus. During the in-corporation of tryptophan into ergoline at thealpha carbon atom of the side chain, an inver-sion of configuration occurs; an isoprene unitalso may participate in the formation of thealkaloid-ergoline. The complicated chemistryof the alkaloids has been described in greatdetail by Gr6ger (3, 5).The particular interest in chemical assays for

ergot alkaloids and their derivatives arises fromtheir therapeutic and toxic properties. Manymethods have been described for the qualitativeand quantitative assessment of the compounds.Lysergic acid derivatives are unstable, usuallydecomposing upon exposure to light or oxidizingagents; the decomposition to isolysergic acidderivatives destroys the pharmacological effect,complicating assays of the active principles incrude drugs. Characteristic color reactions occurwhen the ergot alkaloids are exposed to sulfuricacid or p-dimethylaminobenzaldehyde in sul-furic acid. Through the addition of ferricchloride, this color reaction-known as the vanUrk reaction-has been improved and standard-ized for the quantitative measurements of

ergot alkaloids (6). Derivatives of lysergic acid,isolysergic acid, and some other compoundsproduce an intense blue color in these tests.Ergolines are also quantitatively assessed bytitration, spectrophotofluorometric methods,and by ultraviolet absorbance (7). Spectral dataabout infrared radiation and neutron magneticresonance (NMR) are known for many of thealkaloids. Thin-layer chromatography as well aspaper chromatographic methods have been usedfor further separation of the various alkaloids(8, 9).The ergot alkaloids have been used as

therapeutic agents for many years (8). Accor-ding to the sites of action-periphery,neurohumoral system, central nervoussystem-the six principal effects of all naturalergot alkaloids can be categorized as follows.Vasoconstriction and contraction of the uterusare perhaps the most important peripheraleffects; the classic use of ergot alkaloids inobstetrics arises from this influence. Principalneurohumoral effects appear as antagonisms toserotonin and adrenaline. Effects of ergot on thecentral nervous system are many and varied, in-cluding a depression of the vasomotor centerand a stimulation of sympathetic structures in

December 1974

10> ,5

12 _11 C 4

11

HN 2

Ergoline

I

H COR

9 -CH3I H

HN

JI

3

the midbrain, particularly in the hypothalamus.Slight modifications in the chemical structure

of an alkaloid can produce pronounced changesin biological activity. Thus, certain compoundsare used therapeutically in peripheral andcerebral vascular disorders, while others areused in essential hypertension.The interest and the amount of research

associated with lysergic acid diethylamide(LSD) is reflected by more than 2000 scientificand popular publications during the last severalyears (2, 10). As the prototype of hallucinogens,LSD has been an object of recent fascination(10).The ergot alkaloids reportedly influence

pregnancy (11), perhaps by affectingprogesterone metabolism; because of its poten-tial application to contraception, this area iscurrently under intensive investigation.

Fusaria and Alimentary Toxic Aleukia

Probably since the 19th century, outbreaks ofan enigmatic toxic disease occurred in EasternSiberia and the Amur region. As a result, theSoviet government established the Institute ofEpidemiology and Microbiology of the U.S.S.R.within the Ministry of Health, including a spe-cial laboratory for investigating all aspects ofthe disease (12, 13). Starting during the springof 1932, when the same disease reappearedsuddenly in endemic form throughout severaldistricts of Western Siberia, the laboratory con-ducted comprehensive studies of the disease, itsetiologic agent(s), and its control.The signs and symptoms of the disease, called

alimentary toxic aleukia (ATA), have beendescribed repeatedly in the Russian literature.The signs include fever and hemorrhagic rash aswell as bleeding from gums, nose, and throat;signs and symptoms also comprise necroticangina, extreme leukopenia, agranulocytosis,and an exhaustion of the bone marrow withgeneral sepsis. Mortality has always been high.Mostly occurring in agricultural areas, the dis-ease severely affected whole families and evenentire villages.Although the disease initially received many

names, including septic angina, alimentarymycotoxicosis, alimentary hemorrhagic aleukia,aplastic anemia, hemorrhagic aleukia, andagranulocytosis, a committee of the Soviet

Health Ministry concluded that the most ap-propriate term was alimentary toxic aleukia(ATA).At first the disease was considered to be infec-

tious, but neither bacteriological studies norepidemiologic investigations confirmed thehypothesis. Alternatively, the disease was con-sidered a vitamin deficiency or a poisoningthrough bacterially contaminated food; neitherof these hypotheses was substantiated. Thevarious false concepts delayed recognition of thetrue nature of ATA. Eventually it was realizedthat the disease stemmed from an ingestion ofoverwintered grains infested by toxic fungi.These grains formed the staple diet of the peas-ant population in agricultural areas of Russia.ATA occurred with special severity during

World War II, reaching a peak in 1944, when thepopulation of the Orenburg district alone suf-fered an alarming number of casualties (12).The morbidity among the population in this dis-trict exceeded 10% and a high mortality oc-curred in 9 of the 50 counties. Occurrence of thedisease was related to the particular situationprevailing in some parts of the Soviet Union atthat time; because of famine, the populationwas driven to collect grains that had been left inthe field under the thick snow cover of winter.Under ordinary circumstances the wheat wouldhave been gathered earlier; but the shortage ofnvanpower occasioned by the war preventedharvesting at the proper time.The role of toxic fungi as an etiologic agent in

ATA was examined extensively in the Instituteof Epidemiology and Microbiology in Moscow.From more than 1000 samples of overwinteredgrains, the institute isolated over 3500 fungalcultures belonging to 42 genera and 200 species.Among the isolates, 61 showing Fusarium poaeand 57 showing Fusarium sporotrichioides weretoxic to animals. Numerous investigations indifferent laboratories supported these results;between 1943 and 1949, trial plot experimentsdetermined the most favorable conditions fortoxin production in overwintered wheat.The extensive work conducted in the Soviet

Union established that the disease is caused bytoxic metabolites stemming from those speciesof Fusaria which grow on grains that are har-vested after the snow has melted in the spring(Table 1); evidence suggests that the toxin isproduced during the spring thaws. The follow-

Environmental Health Perspectives4

Table 1. Toxicity of Fusarium fungi isolated from overwintered cereals, summer-harvested cereals, and their soils.a

Number of isolates

Overwintered cereals: Summer-harvested cereals:grains and vegetative parts -Soil grains and vegetative parts

Fungus Toxic Mildly Non- Total Toxic Mildly Non- Total Toxic Mildly Non- TotaltOXiC toxic toxic tOxic tOxic tOxicFusa7ium arthro- - 1 7 8 - - 2 2 - - 5 5

sporioides Sherb.

F. avenaceum (Fr.) 3 3 26 32 - - 10 10 - - 3 3Sacc.

F. culmorum (W. G. 2 1 13 16 - - - - - - - -Sm.) Sacc.

F. equiseti (Cda.) 7 3 41 51 - 1 9 10 - - 27 27Sacc.

F. graminearum - 1 2 3 - - - - - - - -Schw.

F. javanicum - - 8 8 - - - - - - 5 5Koord.

F. kihni(Fuck.) - 1 9 10 - - - - - -Sacc.

F. late7itum 2 2 24 28 - 1 3 4 - - - -Nees

F. moniliforme 1 3 22 26 - 1 10 11Sheld.

F. nivale (Fr.) - 2 11 13 - - - - - - - -Ces.

F. oxysporum Schl. 1 2 16 19 - 1 13 14 - - 2 2

F. poae (Pk.) Wr. 44 17 2 63 2 3 - 5 - - - -

F. redolens Wr.Wr. 1 - 5 6 - - 2 2

F. sambucinum 1 1 14 16 - - - - - - - -Fuck.

F. semitectum Berk. 2 2 23 27 - - - - - - - -et Rav.

F. solani (Mart.) - 3 16 19 - - 5 5 - - - -app. et Wr.

F. sporotrichioides 42 15 4 61 2 2 - 4 - - 2 2Sherb.

F. tricinctum (Cda.) 2 1 19 22 - - 5 5 - - - -Sacc.

aFrom Joffe (13).

December 1974 5

ing specific case serves as a typical example.When harvested in autumn and winter beforethe snow melted, grains were either nontoxic oronly slightly toxic; when harvested after arelatively mild winter with abundant snowfollowed by frequent spring freezing and thaw-ing, often grains were highly toxic. The diseaseusually appeared after victims had eaten only 2kg of overwintered grain, and death resultedfrom the eating of as little as 6 kg. Signs andsymptoms generally appeared within 2 to 3weeks after ingestion; death occurred 6 to 8weeks after the uptake of large toxin amounts.

Populations receiving balanced diets weremuch less sensitive to the toxins than pop-ulations subsisting mainly on overwinteredcereals (13). Additional factors influencing theresponse to the toxins included the kind of cerealwhich was ingested, the altitude, and thethorough washing of grains in boiling waterbefore grinding. Prosomillet and wheat were thecereals most likely to be toxic (Table 2); highaltitude appeared to decrease the incidence ofthe disease in several population groups; and thedegree of toxicity was considerably diminishedthrough thorough washing of the grains in boil-ing water which, as shown by later studies,removed some of the toxins.

Biological and chemical properties of thefungi responsible for ATA are known. Theevidence points conclusively to the Fusariummolds as primary agents, but climatic con-ditions also enter the toxicological problem. Anexcellent and comprehensive discourse on ATAis presented by Joffe (12, 13).

Data on the relative toxicities of barley,millet, and wheat during several years followingthe serious outbreaks in the early 1940s are com-piled in Table 2. Although wheat was most toxic,millet caused a higher incidence of disease;millet was consumed by a much larger numberof people, and it was widely grown in areaswhere other conditions contributed to a high in-cidence. Furthermore, the millet ripened toolate to be harvested before winter and earlyspring.

Several other conditions were studied indetail: persistence of toxicity in stored grain,role of the soil in the process of toxin formation,toxicity of both vegetable cereal parts and soil,meteorological conditions, seasonal effects, aswell as a comparison between toxicity of fungalcultures and cereal samples.A particularly interesting result of the

research on Fusaria is the immunizationassociated with toxic cultures. If investigationsconfirm the hypothesis that an immunity can beconferred on individuals, an exciting new areawill be opened to research in the field ofmycotoxicoses.Evidence from extensive investigations clearly

demonstrates that the strains of Fusaria varygreatly in their capacity to produce toxicmaterials (14, 15). Among the many factors in-fluencing toxicity are ecological conditions, par-ticularly the substrate on which the fungusgrows. The best nutrient sources for fungalgrowth are carbohydrates, including starch andglucose, along with peptone and asparagine assuppliers of nitrogen. Ammonium sulfate and

Table 2. Toxicity of samples of various cereal crops, 1944-1949, from the Orenburg District.a

Millet Wheat BarleyDegree of Number of Number of Number oftoxicity samples S samples S samples %

Toxic 11 2.6 19 4.6 5 1.9

Slightly toxic 22 5.2 19 4.6 14 5.5

Nontoxic 387 92.2 377 90.8 236 92.6

Total number ofsamples examined 420 415 255

a From Joffe (13).

Environmental Health Perspectives6

sodium nitrate also enhance the production oftoxins. The acidity of the medium, too, is impor-tant; the most suitable pH values lie between 4.6and 5.4.The toxins of Fusarium cultures produce both

localized and general 'effects (12). Localizedeffects, as evidenced by skin tests, are restrictedto necrosis of the skin; these confined effectsprovide a convenient biologic assay. Generaleffects comprise defective blood production,hyperemia of the digestive tract, and acuteorgan degeneration. Under practical conditions,skin testing is now used as a reliable techniquefor toxicity determination' of overwinteredgrain's.Guinea pigs and rabbits are most useful for

bioassays. In addition to skin responses, theseanimals also exhibit a severe depression ofleukocytic bone marrow elements together witha general depression of the hemopoietic system.The clinical picture of ATA parallels striking-

ly the signs and symptoms as well' as the bonemarrow depression observed in stachybotrystoxicity. The first, second, and third stages ofATA in human patients'closely resemble thefirst, second, and third stages of stachybo-tryotoxicosis in animals. Local manifestationsare burning sensations in mouth, esophagus,and stomach, General manifestations, are vomi-ting and depression of leukopoiesis, erythro-poiesis, and thrombopoiesis. Although ATA de-presses the hemopoietic system, the bone mar-row tends to remain viable. Based on theseobservations, several investigators assume thatthe toxins associated with overwintered cerealsdo not act primarily on the bone marrow but onother tissues which regulate the hematopoieticsystem, the autonomic nervous system, and theendocrine system.

In ATA, several investigators observedchanges of the nervous system such as impairedreflexes, general hyperesthesia, cerebralhemorrhages, encephalitis, and destructivelesions in the sympathetic ganglia.The only prophylactic measure against ATA

consists in the elimination of toxic grain fromfood.

StachybotryotoxicosisA disease of then unknown etiology, but

apparently of a toxic nature, affected horses

throughout the Ukraine in 1931 (16). The diseasewas characterized by an unusually high mortali-ty rate and had clinical and pathologicalhistories which were unlike any disease entitiespreviously reported. The early studies ofDrobotko and colleagues (17) established thatthe disease was neither contagious nor infec-tious; unaffected horses were stabled adjacentto sick ones without contracting the disease, andattempts to transmit the disease by othermethods failed also. Review of many casesrevealed a relationship between straw used asroughage or bedding and the appearance of thetoxic disease. It required several years and thecombined efforts from several medical dis-ciplines to establish the causative agents asmetabolites of the fungus Stachybotrys alter-nans. Working together, veterinarians andmicrobiologists established the etiology,described the clinical symptoms in detail, andrecommended therapeutic measures as well asprocedures for preventing the disease.Conclusive evidence of the toxicity of thematerial produced by the fungu's was establish-ed by culturing the mold on artificial media andfeeding it to horses. After being fed sufficientamounts of this material, the horses developedsigns, symptoms, and lesions which were iden-tical to those observed in the field. Typicallythere was an inflammatory response in the oralmucosa along with edema of the lips; leukopeniawas always observed.

Following these studies, the disease appearedin several other areas of Russia and in parts ofeastern Europe as well. Over a 10-year period,the toxicoses attained enzootic proportions andthen seemed to subside, with only sporadic casesappearing in those areas where it had previouslybeen enzootic. Reports of the disease were notavailable during World War II, but since theWar, occasional outbreaks have been reported inseveral regions (18).The Russian literature included statements

that the horse was the only large animal suscep-tible to Stachybotrys toxin; however, in theUnited States, Forgacs and co-workers observedthe toxicosis in swine, sheep, and calves as wellas in horses (19). 'A natural outbreak ofstachybotryotoxicosis in cattle has beenreported; it was associated with straw con-taminated by the fungus (20). More recently thedisease has been produced experimentally in

December 1974 7

laboratory animals including dogs, rabbits,guinea pigs, and mice (21); chicks, too, were sen-sitive to Stachybotrys toxin. In Russian ex-periments, human volunteers were exposed toaerosols containing toxic strains of S. alternansor substrate infected with these strains; thepatients developed systemic and localized toxicmanifestations (20).

Studies on the epizootiology of the disease in-dicate that the first cases in horses usuallyappear in the fall when the animals are stabledand when fodder, hay, or straw make up a con-siderable portion of the diet. The number ofaffected animals increases as winter passes on,usually reaching a peak about February andMarch. The disease generally subsides when thehorses are turned out to pasture. As with othermycotoxicoses, there is no immunity conferredupon the host; when the disease occurs on a par-ticular farm, it is likely to recur, even in thesmaller animals. When the disease appears on afarm, the number of affected horses quicklyrises within one week to 50% or more of the en-tire stock; during the second week usually all ofthe animals are sick. There is also a peracuteform of the disease in which entire herds can beviolently affected within 6 or 8 hr; such casesare exceptional, however. The toxin does notappear to be transmitted through the milk ofthe mother, since colts nursing affected maresdo not develop the disease. Age, breed, sex,physical condition, work load, and other factorsapparently have no influence on the severity orcourse of the disease. Although mortality maybe moderate in certain cases, it usually is high;the highest rate appears in the peracute forms,in the chronic form, and in animals sufferingfrom various degrees of malnutrition.The typical form of the disease (18) occurs in

animals with a continuous exposure to lowlevels of the toxin. The initial manifestationappears as stomatitis; necrosis is obvious aboutthe mouth, particularly in the wrinkles of theskin at the mucocutaneous junction. The stoma-titis then progresses to bleeding, swelling ofboth upper and lower lips, and excessive saliva-tion along with enlargement of the sub-maxillary lymph nodes. Temperatures may in-crease only 1-2°C, and the blood may show atransitory neutrophilia. The duration of thisearly stage generally varies from 1 to 3 weeks;despite continued ingestion of sublethal

amounts of the toxin, the local lesions thengradually subside and the animals enter an ap-parent period of clinical remission. During thisperiod, however, thrombocytes decreasemarkedly, clot retraction time increases, andthe blood ultimately fails to coagulate. Duringthe same period, leukopenia and agranulo-cytosis develop, the leukocyte count dropping to2000/cm3 of whole blood or less. Intestinal dis-turbances of various degrees, including atony,generally appear during this the second phase ofthe disease. The third stage is characterized byrising body temperatures, leukopenia, andsevere thrombocytopenia. The leukocyte countoften decreases to 100 white cells/cm3 of wholeblood. The animal develops a weak pulse andoften arrythmic heartbeat, along with distur-bances of the alimentary tract. Pathologicchanges are characteristic: necrotic areasappear on the mucous membranes of cheek,gum, tongue, soft palate, and lips; the bloodglucose level drops to about 50% of normal; theserum bilirubin level increases; and the in-organic phosphorus content declines by a factorof 10 or more. This final stage lasts from 1 to 6days and usually terminates in death.An atypical form, having-many of the signs,

symptoms, and lesions characterizing thetypical form, also occurs. The main syndromecomprises nervous disorders; loss of reflexes,hyperirritability, hyperesthesia, loss of vision,and inability to move about. Despite anorexia,the horse continues to drink large quantities ofwater even though it swallows with con-siderable difficulty. Death often ensues fromrespiratory failure. In the majority of animalswith the atypical form, no blood changes areobserved.

Thus, the pathology of stachybotryotoxicosispresents a characteristic set of lesions, includingprofuse hemorrhage and necrosis in such variedtissues as skeletal muscle, subcutaneous tissues,serous and mucous membranes, and severalparenchymatous organs. Hemorrhages occur inthe diaphragm, mesentery, large intestine,lymphatic nodes, lung, liver, brain, spinal cord,and adrenal glands. In stachybotryotoxicosis,unlike other diseases causing necrosis of themucous membranes, the necrotic focal areas arenot surrounded by reactive cells; the afflictedanimal appears to be incapable of providing in-flammatory reactive cells which form a zone

Environmental Health Perspectives8

demarcating the lesions.The signs, symptoms, and lesions in other

farm animals generally resemble those de-scribed in the equine disease (18).

Russian investigators also described stachy-botryotoxicosis in man; where the disease is en-zootic in the horse, it frequently occurs inhuman populations. Several cases have been de-scribed in which patients used contaminatedstraw for fuel or for sleeping mattresses. As anoccupational malady, the disease afflicted peo-ple who were exposed to dust aerosols heavilyladen with a variety of mold spores. These oc-cupations comprised work in cottonseed oil proc-essing plants, grain elevators, textile mills, andin plants processing various grains including themalt of breweries. In all reported cases, a rapidrecovery ensued after the individuals wereremoved from the source of toxins. Reexposureto toxins caused much more serious sequelae inhumans than in animals.Human patients generally develop a der-

matitis which usually involves the scrotal andaxillary regions; sometimes, however, handsand other parts of the body are also afflicted.The dermatitis leads initially to hyperemia,then to serum exudation, encrustations, and ne-crosis. In addition to dermatitis, the patientsdevelop catarrhal angina, bloody rhinitis, cough,and pains in throat and chest. Leukocytosis isfollowed by leukopenia with a sharp drop inwhite cell counts occurring in the majority ofpatients.The toxin produced experimentally by patho-

genic strains of Stachybotrys is formed in mediawithin 10 days and attains its highest level inabout 20 days (18); subsequently, the toxin con-centration falls but toxin is still detectable aftermore than 400 days. The toxin is soluble invarious organic fat solvents; anhydrous ethylether appears to be the best one. It has beensuggested that ingested toxin reacts with gastricjuice and is absorbed in a water-soluble form.The material can be extracted from infectedstraw within about 3 hr after exposure to themold. Russian investigators claim (17) to haveisolated the crystalline toxin deciphering an em'-pirical formula of C25H3406. They suggest thatthe toxin is representative of a newly dis-covered group of naturally occurring cardiactoxins, but the precise chemical structure hasnot been reported. The material is resistent to

sunlight, ultraviolet light, and x-rays; it is ther-mostable and withstands temperatures of 120°Cfor at least 1 hr. It is unaffected by 2% concen-trations of inorganic or organic acids but isreadily destroyed by alkaline materials.Based on these findings, Soviet scientists used

sodium, potassium, calcium, and ammoniumhydroxide, as well as ammonia and chlorine gas,to detoxify roughage intended for animal con-sumption.Although members of the genus Stachybdtrys

have been isQlated in almost all areas of theworld where it has been sought, there have beenno documented cases of toxic disease associatedwith it in the United States. We should bear inmind, however, that these fungi are present inthe United States and that, under propet con-ditions, they have created public health hazardsin other parts of the world.

Penicillium ToxinsYellowed-Rice Toxins

Shortly after World War II, mold metabolitescapable of inducing liver tumors in animalswere found by the Japanese in domestic rice im-ported from Spain, Egypt, Thailand, Burma,Italy, and the United States (22). Several ship-ments were contaminated with a strain of Peni-cillium islandicum Sopp, the mretabolite4 ofwhich proved to be highly toxic, with" liverdamage as the major manifestation.Although more than 15 kinds of fungi have

been incriminated in moldy or yellowed rice,this review covers only the most important orthe best know types: P. islandicum Sopp, Peni-cillium citrinum Thom, and Penicillium citreo-viride Biourge (Penicillium toxica7ium Miyacke).The Japanese isolated P. islandicum Sopp in

1948 (23). Tsunoda observed postnecrotic cir-rhosis of the liver in rats fed for one month orless on rice contaminated with P. islandicnum.Voluminous literature recounts the work thathas since been done with P. islandicum by chem-ists, pathologists, clinicians, pharmacologists,mycologists, and others (24). Oral admin-istration of a methanol extract taken from thefungus mat which had been cultured on Czapeksolution for 14 days induced in mice severe liverdamage, mainly centrolobular necrosis and fattydegeneration. Further investigations revealedthat the mold metabolites caused chronic

December 1974 9

liver damage including cirrhosis and tumors, inrats as well as mice; the end result depended onthe amount of moldy rice which had been con-sumed. According to severity of intoxication,the liver lesions were categorized as acute, sub-acute, and chronic (25, 26).Acute intoxication with atrophy of the liver is

caused by high levels of toxin given over a shortperiod of time. Animals fed these high levelsbecome inactive, progressively lose bothmuscular and cutaneous tone, and finally dieafter a prolonged comatose state similar tohepatic coma in man. Clinical pathology studiesreveal several signs of liver damage. In humanpatients, histopathologic studies show mainlyfatty degeneration and hemorrhagic cen-trolobular necrosis of the liver.The subacute and subchronic intoxications,

induced by lower concentrations of mold toxinsover a longer period of time, cause moderatecentrolobular necrosis with subsequent collapseof the stroma. These processes lead to fibrosis,liver atrophy, and proliferation of epithelialcells lining bile ducts in the periportal regions. Ifthe animal survives for a few weeks, the livermay show signs of regeneration.Chronic intoxication develops in mice fed

small to medium doses of moldy rice or moldmetabolites. These mice survive the early stageof intoxication without showing any signs orsymptoms; they usually have a life span of 6months or more. Post-mortem examinationyields a wide range of liver damage fromcirrhosis and cancer to slight fibrosis and cellpleomorphism. As with most other toxic con-ditions, a broad spectrum of clinical andhistopathologic responses is observed.The liver tumors have been described by Saito

et al. (26) and by Enomoto (27). Histologically,the changes range from mild parenchymal cellhyperplasia to differentiated and undifferen-tiated liver cell carcinoma. Although there is ahigh incidence of liver injury, the incidence ofmalignant parenchymal tumors is relativelylow, indicating a low carcinogenic potential formetabolites of P. islandicum Sopp.Among laboratory animals, rabbits were the

most susceptible species in regard tometabolites of P. islandicum Sopp (24); when fed1-5% moldy rice, they died within a few days.Those that survived for longer periods developedpostnecrotic cirrhosis as soon as 90 days after

the initiation of feeding studies. Althoughrhesus monkeys also showed acute toxic damageof the liver, they developed neither cirrhosis nortumors as end results of intoxication withmetabolites of P. islandicum.As shown by several investigators (24), diet

profoundly affects the responses of animals totoxins of P. islandicum. Both male and femalemice develop acute toxic effects in a short periodof time when fed rice infected with P. islan-dicum, but the response is greatly enhanced bylow protein intake, as illustrated by the follow-ing example. The death rate of mice duringthree weeks of feeding with an 11% protein dietcontaining 3% moldy rice was about 44% inmales and about 25% in females. The death rateof mice fed the same percentage of moldymaterial with a 34% protein diet was 28% inmales and 5% in females. As toward many otherliver toxins and carcinogens, the male of thespecies was considerably more sensitive.Organs other than the liver are also injured

by exposure to toxic metabolites of P. islan-dicum (28, 29). The pathological-findings includeatrophy of the thymus, spleen, and fat tissues.Fatty degeneration of the tubular epithelium ofthe kidney occurs, and pancreatic cirrhosis oc-casionally develops in mice and rats. In addi-tion, various tumors originate in tissues otherthan the liver.One of the toxic agents isolated from the

media in which P. islandicum Sopp was grownreceived the name luteoskyrin (30, 31). Thematerial was obtained from the media as one ofseven pigments, including rugulosin andcyclochlorotine (24). To isolate pure luteoskyrinfrom culture medium, a relatively simplemethod was described by Tatsuno (32).

Several physical and chemical properties ofluteoskyrin VI and its related compound,rugulosin (V) are compiled in Table 3. Thechemical structure of luteoskyrin was deter-mined with data from color tests, ultravioletabsorption spectra, infrared absorption spectra,chemical reactions, NMR spectra, and x-ray dif-fraction. These various tests were previouslydescribed (24).One outstanding characteristic of luteoskyrin

is its extreme sensitivity to sunlight, leading tophotochemical changes of luteoskyrin in severaldifferent organic solvents. The product ofphotodecomposition is lumiluteoskyrin.

Environmental Health Perspectives10

R = OHRubroskyrin

R = H Rugulosin (3)R = OH Luteoskyrin (I

Table 3. Physiochemical properties of luteoskyrinand rugulosin.

Property Luteoskyrin Rugulosin

Molecular formula C3oH2U0Z C3oH2oOio

Molecular weight 574 542

Melting point, °C 287 (dec) 290 (dee)

[ap, -880 (acetone) +492 (dioxane)Infrared (Nujol), cm-'

CO 1623 1690, 1620OH 3378 3450

aDataof Saito et al. p4).

Luteoskyrin appears to have antimicrobial ac-tivities against various microorganisms, andthis characteristic has made possible a bioassay

based on the sensitivity of a mutant ofE,scherichia coli Q-11.As revealed by extensive studies (2$), the

biosynthesis of luteoskyrin depends on en-vironmental factors. For instance, toxicity tendsto decrease when the mold is grown on variousgrains in the followiing order: rice, barley,wheat, and corn. The addition of certain aminoacids to the culture medium, includingasparagine, glutamine, and malonate, appearsto increase the production of luteoskyrin and ofother pigments as well. As shown by labelingstudies, these amino acids are incorporated intothe luteoskyrin molecule and into the chemicalstructure of the related pigments (33).Luteoskyrin, rugulosin, and their associatedpigments are produced under similar cir-cumstances, but the two series of compoundsare derived along pathways which diverge at anearly stage of biosynthesis.

December 1974 11

The biological effects of luteoskyrin include aswelling of the mitochondria and an inihibitionof oxygen uptake by homogenates of rat liver,kidney, and heart muscle. The major site of in-hibition apparently involves the mitochondrialphosphorylation reaction. Luteoskyrin binds toDNA in M'tro. Studies on liver, microsomalpreparations, and supernatant indicate that thetoxin evokes a high, prolonged incorporation ofcystine into the liver which impairs the transferof sulfur-containing amino acids into protein.Thus, to varying degrees and by differentmechanisms, luteoskyrin apparently damagesseveral intracellular granules.

Toxicity of luteoskyrin varies with the routeof administration (24). In mice, the LDso (mg/kgbody weight) is as follows: intravenous, 6.65; in-traperitoneal, 40.8; subcutaneous, 147; and oral,221. Repeated subcutaneous injection of lessthan 1/10 of the LDso over several days producesthe same lethal effect as a single subcutaneousLDso but requires a longer time. Very youngmice are more sensitive than older ones, andmales are more sensitive than females.The pathologic effect of luteoskyrin resides

primarily in the liver, being similar in rats,mice, rabbits, and monkeys. The microscopicchanges of yellow discolored liver can be seenwithin 24 hr after exposure. Marked cen-trolobular necrosis and fatty degeneration oc-cur, with some nuclear pleomorphism andhyperchromatosis. Followinng prolonged ex-posure, mice develop liver tumors but notcirrhosis.The pathologic response to rugulosin

reportedly is -almost identical with that toluteoskyrin.

Besides luteoskyrin, rugulosin, andassociated pigments, a chlorine-containing pep-tide and another toxin, referred to as islan-dotoxin, have been isolated from the culturefiltrate of P. islandicu.m. The chlorine-containing peptide is termed cyclochlorotine; itsstructure is unknown; its empirical formula isC2UH3UNOsCI2, and its melting point is 2510C.The chemical structure VII has been suggestedfor islandotoxin, but the precise structure is stillnot clearly established. Although both chlorine-containing peptide and islandotoxin share cer-tain characteristics, they apparently are notidentical.

vff

Cyclochlorotine, a rapidly acting hepatotoxin,causes the disappearance of membrane-boundribosomes and glycogen granules from injuredliver cells within a very short time. Thus it dis-turbs both protein and carbohydratemetabolisms. In mice, it causes an initialhyperglycemia followed by hypoglycemia; italso causes a disturbance in the number of liverenzymes which accelerates glycogen catabolismand inhibits glycogen neogenesis.

Pathologic effects of cyclochlorotine are large-ly restricted to the liver comprising an in-terference with circulation and an increasedpermeability of the capillaries. (When the pep-tide is introduced into the skin of dogs, a similarvascular effect occurs locally and leads tonecrosis of the epidermis and leukocytic in-filtration.) In addition, vacuolation and hyalinedroplet formation occur in the parenchymalcells, particularly in the perilobular areas.Acute toxicity produces also a proliferation ofthe smooth endoplasmic reticulum.Chronic exposure of mice to the peptide

results in cirrhosis and liver cell carcinomaalong with tumors of the reticuloendothelialsystem. Rats develop peritoneal hemorrhage asthe result of acute pancreatic necrosis; thesecomplications, however, usually occur only afterlong-term exposure.Although the toxins luteoskyrin, islandotoxin,

and cyclochlorotine share many pathologicaleffects, their biological effects are quitedifferent. Luteoskyrin and islandotoxin causeliver damage characterized by centrolobularnecrosis. Cyclochlorotine, on the other hand,causes damage in the peripheral zone,characterized by vacuolation of liver and en-dothelial cells and appearance of hyaline

Environmental Health Perspectives12

droplets in the cytoplasm. A similar cytotoxiceffect results from cysteine deficiency or fromallyl formate administration.Long-term feeding studies with

cyclochlorotine and luteoskyrin in mice haveshown that luteoskyrin has a hepatotoxic as wellas a possible carcinogenic action, but it is lesspotent than aflatoxin. The chlorine-containingpeptide is a cirrhogenic agent and may be car-cinogenic, although this aspect requires furtherstudy; in limited experiments, a few mice havedeveloped liver tumors.An additional toxin isolated from P. islan-

dicum Sopp has been designated erythroskyrine(34); it has not yet been as well characterized asthe already discussed toxins.

In 1953, Japanese investigators discoveredthat some rice imported from Thailand was con-taminated with Penicillium citrinum Thom, aproducer of potent toxins (22). The mold hassince been found in all rice-producing areas ofthe world, including Japan, Burma, Italy,Egypt, the United States, and more recently inthe People's Republic of China. Mice and dogsfed rice contaminated with P. citrinum dis-played enlarged kidneys which, onhistopathologic examination, revealed markeddegeneration and dilatation of the lowernephrons beneath Henle's loop (24, 35). Aflattening and desquamation of tubularepithelium obstructed the lumen at the cor-ticomedullary junction, partially accounting forthe renal lesion. More prolonged feeding of in-fested rice to mice resulted in glomerular lesionssuch as adhesion of the tuft to the capsule. Therenal lesions resembled glomerulonephrosis, apicture often seen in toxic nephrosis.

Citrinin is the major yellowed-rice toxinproduced by P. citrinum; its chemical structure(VIII) has been established (36, 37) and itsbiosynthesis has been partially determined.

OH

HOOC

CH3

The LD5o of citrinin has been reported formice, rats, rabbits, and guinea pigs; the sub-cutaneous LD5o varies among these species from35 to 67 mg/kg body weight. Intraperitoneally,the LD5o is 30 mg/kg in mice and about 50mg/kg in rabbits.Besides renal damage, citrinin causes

acetylcholine or pilocarpine-like responses, in-cluding vasodilation, constriction of the bronchi,and increased muscular tone.One additional mold associated with yellowed

rice was isolated several years ago from ricecollected in Taiwan and Japan. Designated asPenicillium toxicarium Miyake, it was latershown to be identical with Penicilliumcitreoviride Biourge, which had been describedearlier by two laboratories (24). Feeding studiesproved the contaminated rice to be toxic to rats.Rice infested with P. citreoviride was studiedfor its toxicologic effects; more recently, an ac-tive fraction has been isolated, chemically iden-tified '(24, 38), and designated as citreoviridin(IX).

The toxicology of citreoviridin is known to alimited extent for several mammalian species. Itcauses a typical acute poisoning that ischaracterized by an early onset of progressiveparalysis in the hindlegs, vomiting, convulsions,and respiratory disorder. At an advanced stage,cardiovascular disturbances, flaccid paralysis,and hypothermia occur along with dyspnea, gas-ping, and coma; respiratory arrest and death

OCHs HO OH

H3C H

December 1974 13

follow. The symptoms in animals resembleclosely those in human patients suffering fromacute cardiac beriberi-also called shoshin-kakke-a common disease throughout Asiancountries in the past. The cause of this type ofberiberi remains unknown; there appears to beample thiamine in the tissues of patients. Inboth man and animals, the disease is charac-terized primarily by ascending progressiveparalysis.

Despite the very severe clinical symptoms at-tributable to citreoviridin, histopathologicalchanges are minimal or not detectable at all.

The Rubratoxins

The rubratoxins are a group of metabolitesproduced by Penicillium rubrum Stoll. Growingon feeds, some strains produce large quantitiesof rubratoxins which create an actual hazard tolivestock and a potential hazard to man (39). Themold grows on a large variety of feedstuffs, andduring the past decade some information on thegrowth and toxicity of crude compounds has ac-cumulated.

In the earliest accounts describing a disease incattle and swine, Sippel et al. (40) isolated 13different molds from toxin 'corn. Only two ofthese, A. flavus and P. rubrum caused illnessand death when fed to laboratory experimentalanimals. Further studies showed that the isolateof P. rubrum was considerably more toxic thanthat of A. flvus. When fed to pigs o-ve'r a periodof about 5 days, a total dose of 7-8 lb of corncontaminated with A. flavus resulted in death;however, a single dose of only 1/2 lb of corn con-taminated with P. rubrum resulted in death,usually'within 24 hr.

Burnside et al. (41) reported moldy cornpoisoning in cattle; they isolated -two organisms,Aspergillus flavus Link and P. rubrum Stoll,from corn associated with the disease. Whengrown in proper media, these cultures produceda material that was lethal to mice, horses, pigs,and chick embryos.Although P. rubrum is probably the most

prominent mold associated with this type of tox-icosis,' other genera, including Fusaria andAspergilli, produce toxins when grown on cornproducts; in fact, the naturally occurring dis-ease probably results more often than not fromthe interaction of several toxins produced by dif-

ferent molds. Seibold and Bailey (42) de-scribed a form of toxicosis in dogs, designatedhepatitis X, in the early 1950s. Following detail-ed experimental studies, they concluded thathepatitis X in dogs and moldy corn toxicosis inpigs shared the same etiology.

After aflatoxins were dis'covered and chem-ically identified in the 1960s, it was realizedthat they acted synergistically with the tox-ins produced by P. rubrum; although aflatoxinalone induced many of the manifestationsobserved in field cases of hepatitis X, only thesynergistic action of aflatoxin Bi and rubratox'inB provoked the full spectrum of manifestationscharacterizing the disease (43).Forgacs and Carll (44) described a

hemorrhagic disease related to contaminatedpoultry feed. They subsequently demonstratedthat P. rubrum and the closely related speciesPenicillium purpurogenum caused good grain tobecome toxic to chicks. With the toxic feed, theyreproduced the hemorrhagic disease seen infield cases.

Isolation of the two principal toxins,rubratoxin A and rubratoxin B, was 'achieved byinvestigators who worked with a partiallypurified substance that was produced by moldsgrown in pure culture. The toxins 'have beenchemically characterized and their structuresproposed (X and XI) (45-48).

Rubratoxin B (XI) is clearly the principal tox-in produced by toxigenic strain's of P. rubrum,but there is still very little known about its dis-tribution in feeds and foods. Furthermore, ex-cept for the report of Wogan et al. (49), little in-formation is available on toxicity of the purifiedcompounds studied under controlled conditions.

I

Environmental Health Perspectives14

Although it is difficult to assess the role of P.rubrum in naturally occurring diseases, there islittle doubt that strains of this mold can producelarge quantities of toxic metabolites underlaboratory conditions and that the mold is oftenisolated from feeds collected during outbreaksof disease in animals. The magnitude of theproblem remains to be elucidated.

Both growth and toxin production of P.rubrum vary considerably, apparently depend-ing upon the composition of the media uponwhich it is cultured. Complex substrates sup-port considerably greater growth and toxinproduction than do synthetic media.Pure rubratoxins A and B are soluble in

acetone, moderately soluble in alcohols and es-ters, and minimally soluble in water. The twocompounds are completely insoluble in non-poplar solvents. Rubratoxin A (X) is much moresoluble in ethyl alcohol, however, than isrubratoxin B; conversely, rubratoxin B is by farthe more soluble in ethyl acetate. Thesecharacteristics have been used to separate thecompounds individually from the culturemedium. Thin-layer chromatography is usefulfor this purpose. From mixtures of benzene andethyl acetate, pure rubratoxin B crystallizes aslong lathes with the geometry of hexagonalplates extended along one axis in the plane ofthe hexagon. In diethyl ether, pure rubratoxin Bcrystallizes as rosettes. It may crystallize asregular hexagonal plates, however, from'solvents such as amyl acetate. The mass spectraof both rubratoxin A and B are known, as arethe ultraviolet, infrared, and other usefulanalytical spectra.Forgacs and Carll (50) described hemorrhage

and congestion of the breast, lung, kidney,spleen, and many other organs and tissues ofchicks feeding on grain contaminated with P.purpurogenum and P. rubrum. Extracts from P.rubrum grown bn cracked corn were lethal toguinea pigs, mice, rabbits, and dogs. Accordingto Wilson and Wilson (45),.the manifestations ofthe disease closely resembled those described byother investigators using moldy feeds.Sprague-Dawley rats injected with a sub-

lethal dose of crude rubratoxin developed asevere fatty infiltration of the liver (51). Despitevisible damage to the mitochondria, no signifi-cant impairment of mitochondrial function wasshown by in vitro tests. Townsend et al. (46)gave rubratoxins to mice in doses so small thatmicroscopic lesions were absent. Nonetheless,metabolic processes in the livers of these micewere adveirsely affected; after a standard dose ofphenobarbital, the sleepink time was increased.The liver is clearly the major site of injury in-

flicted by rubratoxins; this is not surprisingbecause the liver plays a central role in detox-ification.

Recently, Wogan et al. (49) investigated theacute and subacute toxicities of rubratoxin B;they administered the material by variousroutes to several species of animals. Theydemonstrated an interaction between rubratox-in B and aflatoxin Bi. In all of the tested species,the most important lesion was, in the liver, com-prising congestion, hemorrhagic degeneration,and necrosis. There was also general depletionof lymphocytes in the spleen and the lymphnodes along with frequent hemorrhages at thesesites. Of the various species, cats were the mostsensitive, developing hemorrhages and massiveascites in addition to the lesions. Following asublethal dose of rubratoxin, the livers of guineapigs, ducklings, mice, and rats regenerated overa period of 7 days. Occasionally, mild renaldamage was observed microscopically, but thefinding was not consistent among the variousspecies or within one particular species.Chronic toxicity studies yielded a con-

siderable body of important information (49).Very low doses of rubratoxin B administeredover long periods of time were not carcinogenic,although they caused severe liver damage(Tables 4 and 5). Given a maximum toleratedlevel of rubratoxin B, none of the 60-week sur-vivors showed any evidence of neoplastic or even

December 1974 15

Table 4. Lethal potency ofrubratoxin B in several species.a

Weight, No. of Dosing LD5o,Species Sex g animals route b Vehicle C mg/kg

Rat M 58 60 IP PG 0.36 (0.27-0.49)

Rat F 60 50 IP PG 0.36 (0.28-0.46)

Rat F 59 70 IP DMSO 0.35 (0.28-0.45)

Rat M 60 25 po e DMSO ca. 400

Rat F 58 25 PO DMSO ca. 450

Mouse F 25 25 IP DMSO 0.27 (0.22-0.34)

Mouse F 25 30 IP PG 2.6 (2.0-3.1)

Guinea pig M 565 18 IP DMSO 0.48 (0.41-0.56)

Cat M 3000 3 IP DMSO ca. 0.2

Cat M, F 3000 8 IP PG 1.0-1.5

Dog M 3000 7 IP PG >5.0

Chicken M 500 6 IP PG >4.0

a Data of Wogan et al. (49).b Dosing routes: IP, intraperitoneal; PO, by mouth.I Vehicles: PG, propylene glycol; DMSO, dimethyl sulfoxided 95% confidence interval.e By stomach tube.

Table 5. Toxicity in male rats exposed simultaneously to rubratoxin B and aflatoxin Bi. a

No. ofTotal Body weight at preneoplastictoxin, end of dosing, liver lesions

Treatment regimen mg/rat % of control Mortality at 70-80 wks

Controls(DMSO 3x/wk for 5 wk) - 100 0/10 0/7

Rubratoxin B(25 mg/kg, 3x/wk for 5 wk) 39.7 95 0/10 0/7

Aflatoxin Bi(0.2 ppm in diet for 6 wk) 0.11 102 0/10 6/7

Rubratoxin B and aflatoxin Bisimultaneously - 86 9/20 5/8a~~~~~~~~~~~~~~~~~~~~~~~~~~~~

a Data of Wogan et al. (49).Data of Newberne and Wogan (52).

Environmental Health Perspectives16

preneoplastic lesions; several animals survivedas long as 87 weeks without any evidence ofneoplasia.The synergism between rubratoxin B and

aflatoxin is important from the standpoint ofpublic health. Rubratoxins, while not par-ticularly toxic by themselves, may well bepotentiating factors for other toxinssimultaneously occurring in moldy feeds andfoods. The interaction of the more prominentmycotoxins must be examined in detail to deter-mine their hazard to public health.

Other Penicillium ToxinsOther Penicillium toxins comprise patulin,

penicillic acid, and certain lactones; they areassociated with several species of Penicillia andAspergilli, including Penicillium urticae,Penicillium claviforme, Penicillium expansum,Aspergillus clavatus, Aspergillus giganteus, andAspergillus terreus. Although not studied as ex-tensively as others, the toxins induce significantbiological effects. In mice, they reduce thelymphocyte count in blood, increase vascularpermeability (resulting in edema), and suppressthe formation of urine. They also elevate theblood sugar level. Patulin has been usedtherapeutically in very low concentrations as anose and throat spray and as a treatment forcommon head cold.The molds and their metabolic products were

described in detail by Ciegler et al. (53).An additional class of compounds,

cyclopiazonic acid and related toxins, areproduced mainly by Penicillium cyclopiumWestling. This mold and its toxic metaboliteshave been isolated from peanuts and corn meal.The biologic effects of cyclopiazonic acid areonly partially known. When the toxin is givenorally to ducks, chicks, and rats, the animalsdisplay convulsions and die within a very shorttime. Other but less well-defined toxins are alsoassociated with P. cyclopium Westling.Miscellaneous Penicillium toxins were

described by Wilson (54); although some of thetoxic fractions have been isolated, they aregenerally not yet sufficiently characterized forinclusion in the discussion.

Aspergillis Toxins (Aflatoxins)Although Aspergillus flavus is associated

with most food and feed contaminations, only afew strains of A. flavus actually produceaflatoxins; however, these few strains are themost important producers of aflatoxin. OtherAspergilli, too, produce aflatoxins (55).Taber and Schroeder (56) found that

A spergillus flavus-oryzae, isolated frompeanuts grown in the United States, producedno aflatoxin Gi or G2, although several culturesproduced Bi. Likewise, the aflatoxinsoriginating from Penicillia (e.g., Penicilliumcitrinum, Penicillium frequentans, Penicilliumvariable, and Penicillium puberlum) were con-sidered a relatively minor hazard (57).

Aspergillus toxins other than aflatoxin werereviewed previously (57-63).

Early StudiesThe majority of the more than 1000

publications about aflatoxins appeared duringthe last 10 or 12 years, reflecting an intense in-terest in mycotoxins, and more specifically inaflatoxins, which was aroused by the outbreakof turkey X disease throughout England in 1960(64).During the early development of antibiotics, a

fundamental discovery was made that somematerials from antibiotic-producing molds weretoxic in animal trials. However, these moldswere usually discarded because they were poorproducers of antibiotics; little attention waspaid to their potential for producing toxins.Thus, the recognition of mycotoxins as a threatto public health was delayed for two or threedecades.

It has long been known that fungi growing onfoods and feeds are ubiquitous; they may con-taminate virtually every stable foodstuff ofeither plant or animal origin. The majoritygrows throughout extremely wide ranges of pHand temperature, making moisture conditionsthe primary environmental restriction. Oncereal grains and oil seeds, molds grow atmoisture levels which are commonly en-countered under storage conditions. Thesestorage molds mainly are species of Aspergilliand Penicillia. For instance, moldy corn tox-icosis in swine, occurring with considerablefrequency in the humid southeastern parts ofthe United States, is associated with bothAspergillus and Penicillium molds (41, 65).The small number of publications indicated

December 1974 17

clearly the lack of interest in mycotoxinresearch prior to the 1960 outbreak of turkeyXdisease. Several of the early reports (40, 66)associated liver disease in swine with a rationthat contained corn or peanuts. Aspergillusmolds were incriminated in the epizootics ofbovine hyperkeratosis, although later studies in-dicated that the disease was more complex; itprobably resulted from an interaction ofchlorinated cyclic hydrocarbons with mold tox-ins (67-69). Forgacs et al. (44, 50) isolatedseveral molds, most species of Aspergilli andPenicillia, from feeds that led to a hemorrhagicdisease in poultry. Furthermore, isolates fromthese molds on poultry feed were often capableof reproducing the hemorrhagic syndrome inbirds. These diseases were then designated asmoldy feed toxicoses. The English episode inturkeys brought together microbiologists andveterinarians who recognized that toxicmanifestations were caused by the ingestion ofcertain mold-contaminated feeds.During the period from 1960 to 1962, several

later reports (70- 72) about outbreaks of diseasein poultry and fish at diverse geographicallocations incriminated fungi in feeds as the like-ly etiologic agents. In avian species, the acutedisease resulted in loss of appetite, weakness ofwings, and lethargy. Histologic examination oftissues revealed an acute hepatic necrosis andusually a marked bile duct proliferation.Ultimately, the problem was traced to importedBrazilian peanut meal in the birds' rations. Itwas then discovered that ducklings, swine, andcattle had also been poisoned by similarly con-taminated lots of peanut meal. The Brazilianpeanut meal, however, was not alone in causingtoxicity. At about the time when the turkey Xdisease broke out, a similar disease, associatedwith peanuts processed in East Africa, affectedducklings in Kenya (73). The early discoverythat ducklings were particularly sensitive toaflatoxin, as evidenced by a rapid and extensivebile duct proliferation, was fortuitous; thisspecies became the major tool for biologic assay.Following the initial studies of peanut mealpoisoning in turkeys and ducklings, Lancaster etal. (74) reported that rats developed liver cellcarcinomas when toxic peanut meal was includ-ed in their diet for 30 weeks or longer. Sargeantet al. (75) and Nesbitt et al. (76) identified thetoxin-producing organism in the peanut meal asthe saprophytic mold Aspergillus flavus Link ex

Fries, a widely distributed organism that hasbeen isolated from virtually every staple foodproduct in the tropical and semitropical areas ofthe world (77).

While the turkey X disease was at its height,an epizootic of liver cancer afflicted hatchery-reared rainbow trout in Washington and Oregon(72, 78). Investigations then revealed that livercarcinoma occurred in fish at many differentlocations. Trout raised in hatcheries had formany years been fed a diet including vegetablesources of protein, with cottonseed meal as themajor source (79). As a result of many in-vestigations, the etiology of trout hepatomaswas finally traced to contaminants in the cot-tonseed meal. Furthermore, the investigatorsdiscovered the influence of nutritional factorson both the neoplastic processes (80) and theresistance to carcinogenic toxins (81).

In addition to toxicoses in various domesticanimals and liver carcinomas in trout, an obser-vation in laboratory rats further heightened theinterest in contaminated feed. During studiescomparing the effects of choline-deficient andadequate diets, Salmon and Newberne (82)observed liver carcinoma in rats were feddiets containing peanut meal. Within a relative-ly short time, several reports (83, 84) showedthat the contaminant in peanut meal was thesame as that produced by a strain of Aspergillusflavus which was isolated from toxic meal inEngland and identified as a complex ofmetabolites (73, 85, 86).

Identification and Characterization of Afla-toxins

The discovery that the toxins had acharacteristic fluorescence pattern on thin-layerchromatograms greatly facilitated their isola-tion and characterization, culminating in thedetermination of the molecular formulas of fourcomponents designated as aflatoxins Bi, B2, Gi,and G2; the four components were distinguishedby their blue or green fluorescence and by theirRf values on thin-layer chromatograms (87).More recently, related substances have beenisolated and chemically characterized, the mostimportant ones being Mi, M2, B2, and G2a(63).The Mi and M2 fractions, first isolated from themilk of cows feeding on aflatoxin-contaminatedfodder (88) were later found in the milk oflaboratory rats. Mi and M2 possessed the same

Environmental Health Perspectives18

toxicity as the aflatoxins from which they werederived (89). On the other hand, the hydrox-ylated B2a and G2a derivatives of B2 and G2described by Dutton and Heathcote (90) werevirtually nontoxic, suggesting that con-taminated foods may be detoxified by acid treat-ment.The isolation, purification, and identification

of the various fractions of aflatoxins aredescribed in detail by several authors 62,91-93). The structures are shown in Figure 1.Although not highly specific, quantitative

assays for aflatoxins are reasonably accurate.The current methodology relies on thin-layerchromatography, the use of known referencematerials, derivative formation, and bioassay.The same methodology, however, does not worksatisfactorily for all food products. For example,corn and corn products require modifications ofthe methods that are commonly used to assay

0 0 0 0

0 0

0 0 ~OCH, 0 0 OCH,

B, B,

for aflatoxin in cottonseed, cocoa, oats, andpeanuts (94).A biological test, using the duckling,

supplements the various chemical methods foranalysis (84). One-day-old ducklings are usuallyused, and the suspected toxin is dissolved ineither proplylene glycol, dimethyl sulfoxide, orsome other suitable solvent. The ducklings arethen dosed with varying amounts of thematerial, killed within 48-72 hr, and a sectionof each liver is examined for characteristichistologic alterations (Figure 2). The alterationsinclude periportal liver cell necrosis and bileduct hyperplasis, the latter being graded tomirror the concentration of administeredaflatoxin (Table 6). The histologic changes aredescribed by Newberne et al. (84) and therelative toxicities of the compounds are listed byseveral investigators (95, 96). The bile ducthyperplasia is not specific for the toxins, but

I-.MIIho.x-allatoxin U2 B2-M4ctioxy-aflatoxin B3

0 0

0

110

0'I 0 OCH,

0 0

HO 0

0 0 OCH,

0 0

ItO 0 OCH,

0 0

0

110 0 0 OCH,

0 0

00HO

0 0 OCII,

GM,B,,

0 0

( 0

0 I(I 1C0 0 (1

I-Ac.toxy-allaitoxin B2

FIGURE 1. Chemical structures of aflatoxins and metabolites.

December 1974

Palr;siticol IB83)

I-Etlioxy-allatoxiii B2 -allato\ill ('02M2

G¢,

19

FIGURE 2. Typical bile duct hilowing exposure to aflatox

J3F

Effects of Aflatoxin on Various Species

Mollusk Eggs: Various biological assaymethods for aflatoxins have been proposed withthe goal of increasing sensitivity and speed ofdetection (101). Townsley and Lee (102) reportedthat aflatoxin Bi inhibits cell cleavage in fer-tilized eggs of the mollusk Bankia setacea butprevents neither fertilization nor nuclear divi-sion. It has been suggested that the eggs be usedas a bioassay system because their handling re-quires a minimum of technique and trainingand they are sensitive to aflatoxin concen-trations of 0.05 ,ug/ml. However, limitingfeatures include the availability of the mollusk,the requirement of sea water, and the need towork at low temperatures.Embryonated Eggs: Embryonated chicken

eggs are also sensitive to aflatoxin, but their useis limited by high death rates (103, 104). Thebiological effects are interesting, however, con-sisting of growth retardation, edema,hemorrhage, decreased brain development,skeletal defects, and beak anomalies. Their non-specific nature diminishes the usefulness forassays.

Tissue Culture: Tissue culture studies in-dicated that aflatoxin suppressed mitotic divi-sion in heteroploid and diploid embryonic lungcells (105). In concentrations from 0.5 to 1.0ppm, aflatoxin reduced growth of heteroploidembryonic lung cells of human origin (106); at5.0 ppm, the cells did not grow at all. Nucleolarvolume decreased 12 hr after chick embryoniccells were dosed with aflatoxin, and the nucleolidisappeared eventually (62).Plants: It was reported that aflatoxins

caused albinism in the leaves of young cress.(Lepidium stivum) that were subjected toaflatoxin concentrations ranging from 1 to 10,ug/ml (107). Seed germination was prevented byhigher doses; the authors suggested that theeffects could be elaborated into a simple test forthe detection of aflatoxin; however, thisproposal never was implemented.Microorganinus: In 1966, Burmeister and

Hesseltine (108) surveyed 325 microorganismsfor sensitivity to aflatoxin. Many of the variousorganisms (bacteria, fungi, algae, and protozoa)were affected, but the damage was not suf-ficiently specific to warrant further investiga-

tion. Clements (109) used similar methods butthe sensitivity was not sufficient for a usefultest.Domestic Animals: The carcinogenicity of

aflatoxin is well established. Fish, birds, ferrets,trout, pigs, sheep, and laboratory rats developliver carcinoma under appropriate conditions(52, 95, 110, 111). Although liver cell carcinomascomprise the majority, primary tumors alsoarise in other organs such as kidney, stomach,lung, salivary and lacrymal glands, colon, andskin.Although aflatoxin poisoning apparently has

long been a problem in animals throughout theworld (112), the majority of records and obser-vations appeared after 1960, as shown by thefollowing: Loosmore and Markson (85) observedthat the clinical signs and liver lesions in cattlesuffering from aflatoxin poisoning resembledthose produced by the pyrrolizidine alkaloids.The liver lesions produced in cattle by these twodifferent classes of toxins were, in fact, in-distinguishable. Furthermore, they resembledlesions observed in children with "childhoodcirrhosis." In 1963, Allcroft and Lewis (113)reported that calves suffering from aflatoxinpoisoning had a decreased rate of growth andsevere tenesmus. Post-mortem examinationsrevealed visceral edema, ascites, and fibrosis ofthe liver. Similarly, well-controlled experimentswith steers showed that aflatoxin significantlydecreased weight gains and feed efficienciesover a 133-day trial (114). Given large doses ofaflatoxins, milk cows showed decreased milkproduction and occasional weight loss. When fedaflatoxin, cattle produce milk containing the Miand M2 fractions' In most of the cattle studies,enzymes of serum and liver were affected, butthe changes were equivocal. As one importantobservation, aflatoxin-fed cattle showed a mark-ed decrease in serum vitamin A (115).Swine are very sensitive to aflatoxins (116);

clinical observations of aflatoxicosis in swinehave been recorded in detail. As with otherspecies, the age of the animal is important, theyounger being more sensitive. The marked liverdamage occurs mainly in the centrolobular zonebut may involve the entire lobule in cases of ex-posure to high concentrations (117). Serum en-zyme levels are increased, reflecting liverdamage; vitamin A concentrations in serum and

December 1974 21

liver are markedly decreased. Aflatoxin-inducedliver cancer in swine is unknown.Sheep apparently are least sensitive domestic

animals, although they excrete relatively largeamounts of aflatoxin metabolites in urine andmilk (118). Despite their relative insensitivity,sheep develop liver damage after exposure tohigh levels of aflatoxins; and if exposure con-tinues over a long period, they develop liver cellcarcinoma, nasal carcinoma, and chondroma(119).The early observations of a toxic disease in

dogs in the southeastern United States (42, 112)led to the discovery that the majority of toxicepisodes were associated with feed containingpeanut meal. The disease, referred to ashepatitis X, shared many characteristics withmoldy corn poisoning in swine, and in-vestigators finally concluded that thereprobably was a commnon etiologic agent. Dogssuffered hemorrhages in various tissues and theliver underwent cell necrosis, severe fattychange, cirrhosis, and fibrosis. Bile ductproliferation varied with the amount and dura-tion of exposure. The disease was reproduced indogs by aflatoxin Bi and by a combination ofaflatoxin Bi and rubratoxin B (43).

Poultry are highly susceptible to aflatoxins.Not only is the liver seriously injured, but thekidney also sustains damage (83). Ducklings,quail, turkeys, and chickens (in order of decreas-ing sensitivity to aflatoxin) respond with bileduct proliferation and various degrees of livercell damage (95). Fatty liver, common in allspecies, is most marked in the very young duck-ling, the condition partly resulting from residueof the yolk. Long-term studies (65, 120) revealthat the ducks often develop coexistent liver celland bile duct carcinomas as a consequence of ex-posure to aflatoxins for sufficiently longperiods. The dose to produce carcinoma is quitelow in comparison with carcinogenic doses forother species developing liver tumors.Monkeys: Madhavan et al. (121) presented

the pathologic features of lesions produced byaflatoxin in the livers and kidneys of monkeys.The administered doses were so high, however,that the animals did not survive very long; thus,lesions were primarily acute or subacute toxicresponses. The major findings were biliaryfibrosis associated with severe fatty change and

parenchymal necrosis. Determined chemically,liver fat was sharply increased.Cuthbertson et al. (122), conducting a long-

term experiment with cynomologous monkeys,observed that the feeding of aflatoxin Bi at alevel of 2 ,ug/kg body weight per day had verylittle influence on the monkeys; but if the dosagewas increased to 50 ,ug/kg body weight daily, themonkeys died in 1 to 2 months after initiation ofexposure.

In African monkeys receiving 0.10 to 1.0 mgaflatoxin per day, survival time ranged from 6to 22 days, generally depending on the dose(123). There was a consistent pattern of toxicityto the liver which was similar to that seen inman.During an extensive study of rhesus monkeys,

Deo et al. (124) observed that monkeys receivinga mixed aflatoxin preparation at a level of 1mg/kg body weight per day died within about 3weeks, with extensive hemorrhagic necrosis ofthe liver. Monkeys receiving 0.25 mg/kg twiceweekly or 62 mg/kg once weekly survived up to2 yr; all animals, even those at the lowest doselevel, had liver injury. Dietary protein levels didnot influence the response, perhaps because ofthe low toxin dosage.Svoboda et al. (125), investigating the cellular

damage of aflatoxin Bi to rat and monkey livers,demonstrated that the damage in rat liver wasusually periportal; the monkey liver showed anecrosis similar to that observed in human liverduring acute viral hepatitis. Svoboda's studieswere paralleled by Kelly et al. (126), who in-vestigated liver tumors induced in monkeys byN-nitrosodiethylamine; prior to tumor forma-tion, the pathologic changes resembled thoseobserved in the livers of animals suffering fromhepatitis.

Hazard to ManBased on studies of chemical carcinogenesis

in recent years, Boyland (127) suggests thatchemical compounds cause at least 90% ofcancer in man, and that naturally occurringsubstances induce 80% or more of the cancersfound in Western peoples. Various surveys ofworldwide mortality from cancer also indicatethat environmental factors exert considerableinfluence; mortality rates are not uniform,

Environmental Health Perspectives22

depending on the region and the type of popula-tion. The same applies to liver disease. Oettle(128) reviewed the earlier concepts concerningthe incidences of liver tumors in different ethnicgroups; malnutrition, liver parasites, viralhepatitis, and chronic alcoholism are possiblecausitive factors, along with senecio alkaloidsand several other environmental toxins.

Shortly after the aflatoxins were discovered,the question arose whether mycotoxins-andparticularly aflatoxin-might be associatedwith high incidences of liver disease, includingliver carcinoma. Typical for the investigativeapproach to the problem was the study ofpeanuts. Contamination of peanuts was foundin almost all regions of the world (1, 129) in-cluding the United States (56). Being a majorsource of protein in many African communities,peanuts were examined for contamination, es-pecially in view of the very high incidence ofliver cancer among several African populations.The Tropical Products Institute in Englandtested peanut samples from Africa, LatinAmerica, and Asia; less than 50% containedsignificant amounts of aflatoxin Bi. On theother hand, peanut meal and peanut cake werecontaminated to a higher extent, 42% containingover 0.25 ppm aflatoxin Bi (130). Purchased onthe open market in Uganda, about 15% of thepeanut samples were contaminated with morethan 1 ppm aflatoxin Bi (131). In South Africa,the yearly peanut crops often contained highlevels of aflatoxins (132) (Table 7).

Aflatoxins were isolated from virtually everystaple food consumed by man (77, 133, 134), andcomprehensive studies confirmed the presence

of aflatoxins in virtually every area of theworld, as discussed in the section onepidemiology of Mycotoxins in Human Health(1).The toxins are most often found in areas like

southeast Asia or parts of Africa where climaticconditions are especially conducive to moldgrowth and where the methods for harvestingand storage are rather primitive. Now it appearscertain that the regional incidences ofhepatomas are related to tropical climate,malnutrition, and the consumption of foods con-taminated with aflatoxin and other substances(135).

Several investigators (89) presented evidencethat the correlation was not totally valid inevery instance and pointed out that the evidencesuggesting an etiological relationship betweenmold metabolites and cancer was, at best,presumptive. The most useful studies,therefore, are those dealing with specific pop-ulations where a possible relationship existedbetween aflatoxin consumption and liver dis-ease.For example, Campbell and Salamat (136)

reported' high consumption of aflatoxins frompeanuts and other foods in the Philippines(Table 8). Studying many members of a popula-tion that manifested an apparent correlationbetween a high incidence of liver disease andconsumption of aflatoxin-contaminated peanutbutter, Campbell et al. (137) found that about 15,ug of aflatoxin Bi had to be ingested in order forMi to be detected in a 24-hr urine sample. Inthese studies, neither aflatoxin nor itsmetabolites were detected in human milk.

Table 7. Incidence of Aspergillus flavus and of aflatoxin above 0.05 ppm in foodstuffs of Swaziland. a

Frequency of Frequency ofNo. of A. flavus, samples with

Foodstuffs samples % aflatoxin, %

Maize 256 53.5 1.6

Groundnuts 180 49.4 11.1

Groundnut meal 48 50.0 8.3

Sorghum 39 33.3 7.7a Data of Martin et al. (132).

December 1974 23

Table 8. Aflatoxin analyses of Philippine foods (1967-1969). a

No. greater Median (and highest)No. of than 30 value of samples,

Food samples ,ug/kg ,g/kgb

P.0-MII-1+0 xsnhnIn P71 r, 1 F7 {1 AA'II

Peanut butter,Philippine (1967-68)

Peanut butter,imported from U.S.A

Other peanutproducts

Nuts and seeds

Tubers

Beans

Soybean products

29

3

32

23

59

29

24

29 155 (8600)

0

11 37 (220)

1 38 (64)

6 68 (440)

2 45 (86)

0 16 (16)

Rice andrice products

Maize products(1967-68)

Maize products(1969)

Cocoa

Livestock feeds

Fish products

Cocoanut products

Cooking oil

Mangoa Data of Campbell and Salamat (136).b Values above 10 ,g/kg.

Robinson (138) reviewed the clinical history ofinfantile cirrhosis in India and discussed severalpossible etiologic factors including mycotoxins,microbial infections, viral hepatitis, and inges-tion of various toxins. He examined 43 samples,of milk from mothers of cirrhotic children anddiscovered that a significant number hadfluorescent spots and Rf values that indicatedcontamination and aflatoxin Bi. In addition, 18of 50 urine samples from cirrhotic children were

positive for Bi.Recent publications by Shank et al. (129,

139-145) provide compelling evidence thataflatoxins play a role in both chronic and acuteliver disease in populations in southeast Asia.Particularly convincing is the evidence inchildren dying of an acute encephalopathy andfatty degeneration of the liver and kidney;significant amounts of aflatoxin are present inthe tissues of these patients at autopsy (145).

Environmental Health Perspectives

1 (IUU)

72 1

14

16 (33)

1

27

11

12 (39)

14

11

0

47 (400)

27

8

19 (29)

7

0

74 (103)

16

0

12

21 (26)

0

0

reanuLs, wnoie 0

24

Influence of Nutrition on Response to Afla-toxin

The influence of nutrition is particularly in-teresting, because malnutrition, aflatoxins, anda high incidence of liver diseases, including livercell cancer, coexist in many populations.The influence of dietary protein was in-

vestigated by many laboratories, but the resultswere not consistent. Madhavan et al. (121)speculated that undernourishment and proteindeficiency, causing kwashiorkor in children,might be a basis for the induction of infantilecirrhosis when aflatoxins were superimposed.To test the effect of dietary protein, they design-ed experiments in monkeys and observed thatreduced protein intake significantly increasedthe susceptibility of monkeys to aflatoxin. Whendose levels of 100 ,ug/day were administered toboth protein-deficient and control animals, allmembers of the protein-deficient group died,whereas those receiving adequate protein sur-vived. This implied that, conversely, a high-protein diet in the monkeys would precludesevere injury from aflatoxin. Although the con-trol animals on a normal protein diet showed noeffect after one month's exposure to 100 or 250,Ag aflatoxin per day, the monkeys showedperiportal fatty degeneration of the liver after a6-month exposure. Rhesus monkeys kept oneither high or low protein diets and exposed to500 ,g aflatoxin daily developed fatty livers andbiliary fibrosis in 16-30 days. Monkeys on low-protein diets which received only 100 ,g of toxindaily showed similar liver changes, again in-dicating that the protein level of the diet mightsignificantly influence the response to aflatoxin.

Similarly, Newberne et al. (146, 147), workingwith rats given a total dose of 375 ,ug aflatoxinBi over 3 weeks, observed that animals givendiets containing 9% protein suffered a higher in-cidence of liver tumors in a shorter period oftime than did rats receiving diets containing22% protein. Madhavan and Gopalan (148)reported that rats fed a low-protein diet (5%)were more sensitive to the toxic effects ofaflatoxin but less sensitive to the hepatocar-cinogenic effects than those fed a 20% proteindiet.Foy et al. (149) discussed the similarity

between carcinoma in Africans and theaflatoxin-induced hepatic cirrhosis in baboonssuffering from pyridoxine deficiency; in addi-

tion to bile duct hyperplasia, the pyridoxine-deficient baboons also revealed disturbances inthe liver content of lipid, glycogen, RNA, andDNA. The changes resembled those induced byaflatoxin. Thus, these investigators suggestedthat the very high incidence of liver cirrhosisand liver carcinoma in certain African pop-ulations might result from diets that weredeficient in pyridoxine and contained aflatoxinfrom contaminated food; the local beer wasoften contaminated with aflatoxin.

Aflatoxin together with pyridoxine deficiencyappear to induce liver damage. It seemsprobable that the reduction in pyridoxine im-pairs the capacity of liver cells to carry outmany of the reactions essential to normalhomeostasis, particularly transamination reac-tions; these matabolic changes may sensitize theliver to the toxic effects of aflatoxin.Newberne et al. (146) determined the in-

fluence of cirrhosis induced by lipotrope deficien-cy on the sensitivity of rats to low levels ofaflatoxin Bi. The concurrent application ofdietary and toxic injuries increased tumor in-cidence. Animals returned to a normal diet aftercirrhosis induction and then exposed to aflatox-in at a level of 1 ppm developed tumors at aneven higher incidence. This seemed to implythat, once the initial biochemical lesion was in-duced by the toxin, a return to normal diet ac-tually speeded up the carcinogenic process.Other forms of injury in this study, such asrepeated biopsy, did not affect the induction ofliver cell carcinoma.The populations in several areas with a high

incidence of liver carcinoma also suffer frommarginal lipotrope - methionine and vitaminB12- deficiencies. Laboratory studies indicatethat marginal deficiencies of lipotropes maysignificantly influence the response to aflatoxin.Rogers and Newberne (150, 151) showed that

rats with marginal lipotrope deficiency wereprotected against the acute effects of a singledose of aflatoxin Bi (Table 9); they speculatedthat this protection resulted from the lowresting level of drug-metabolizing enzymesobserved in the animals. However, if this sametotal amount of aflatoxin Bi was administeredin several daily doses, the rats were con-siderably more sensitive (Table 10). Further-more, deficient animals treated with car-cinogenic doses of aflatoxin Bi developed livertumors in a higher incidence and in a much

December 1974 25

Table 9. Effects of diet on acute toxicity of a single dose of aflatoxin B1.a

2-weekDosage, Route of mortality

Animal mg/kg b administration Diet No. of rats %

Sprague-Dawley rats 7 Intragastric Control

Marginallipotrope

9

7

Intragastric Control

Marginallipotrope

Intraperitoneal Control

Marginallipotrope

Fischer rats 7 Intragastric Control

Marginallipotrope

3/5 60

0/5

4/5 80

0/10

5/5 100

0/5

10/10 100

0/10

a Data of Newberne, and Rogers (152).b Aflatoxin Bi dissolved in 0.1 ml DMSO.

Table 10. Effect of lipotropes on parenchymal hyperplasia and tumor incidence induced by aflatoxin B,.a

Tumor incidence atvarious times after exposure

Appearance of to aflatoxin, %Dietary hyperplastic cell 6 9 12treatment clusters months months months

Control 6 months 0 30 71

Severe deficiency none 0 50 60

Marginal deficiency 3 weeks 40 95 95

a Data of Newberne and Rogers (152).b Rats were given 15 daily doses, 25 Ag, each, of aflatoxin Bi.

shorter period of time than did controls. Possi-ble mechanisms for these interactions triggeredan ongoing intensive investigation. tNewberne and Rogers (153) reported that low

dietary vitamin A apparently decreased the in-duction of liver tumors. Despite this protectionfrom the carcinogenic effects of aflatoxin,however, the vitamin A-deficient rats had a

much higher mortality rate and their growthrates differed somewhat from animals receivingadequate vitamin A. Furthermore, colon car-cinomas were observed in a significant numberof deficient rats. Clearly, the influence ofvitamin A on aflatoxin-induced tumors posed animportant problem requiring further investi-gation.

Environmental Health Perspectives

0

0

26

Influence of Factors Other Than Diet

Goodall and Butler (154) studied the effects ofhypophysectomy on rats fed a diet containing 4ppm aflatoxin Bi. All of the control animalsdeveloped liver tumors in 49 weeks, whereasnone of the hypophysectomized animalsdeveloped tumors in the same period of time.Hypophysectomized rats, however, grew little ifany over the period of the experiment, whereasthe other animals grew normally. This dis-crepancy rendered the interpretation of resultsexceedingly difficult. Similar observations,however, were made under analogous condit