Reprinted from Veterinary and Human Toxicology, Vol. 32, Supplement 1990, pp. 63-70 Purchased by U.S. Department of Agriculture for official use

Additional Mycotoxins of Potential Importance to Human and Animal Health*

JL Richard. PhD US Department of Agriculture, Agricultural Research Service National Animal Disease Center, PO Box 70, Ames, IA 50010

To expand the list of mycotoxins, that have already been The three mycotoxins or groups of mycotoxins that are discussed, to include all of those of potential significance included herein were selected because they are repre­ in human and animal disease, might require more insight sentatives within two or more of the above stated criteria. into the future than one's crystal ball may allow. However, in the selection of additions to the list, there should be some justification for their inclusion in such a presenta- CITREOVIRIDIN tion other than the biases of this author. Perhaps, justi- fication could be made for claiming the potentiality of any Historical Aspects A disease of humans occurred for three fungal secondary metabolite as important in human and centuries in Japan and Asian countries that was charact­ animal disease but, I believe that some criteria should be erized by convulsions, paralysis, and respiratory arrest. established by the author before proceeding. Therefore, The disease known as cardiac beriberi or "shoshin kakke" mycotoxins to be included as additions to the list are those in Japan was investigated from the standpoint of being that (a) frequently occur in commodities, (b) cause natural either an infection, an avitaminosis, or an intoxication. disease, (c) are produced by common food or feed inhabit­ Discovery of the etiology of the disease began when Sakaki ing fungi, (d) are produced by human and animal patho­ (1891) (cited in I) demonstrated that an ethanol extract of genic fungi, or (e) are known immunosuppressive agents. naturally contaminated rice caused neurotoxic signs in One additional important criterion is that any mycotoxin mice, frogs, and rabbits. Later, a toxic fungus was isolated included in such a list should elicit its toxic response when from "yellow rice" and named Penicillium toxicarium given by a natural route. (Miyake & Igaku, 1943) (cited in I), but later the name was changed to P. citreoviride (Naito 1964) (cited in I). Sub­ sequent studies on toxic metabolites of P. citreoviride * No endorsements are implied herein. yielded extracts that produced signs in animals similar to Vet Hum Toxicol 32 (Supplement) 1990 63 those in humans affected with cardiac beriberi. The yel­ thetic liquid media whereby toxin was demonstrated in the low pigment, named citreoviridin, was isolated from cul­ liquid broth as well as in the extracts of dried mycelium ture extracts of the fungus by Hirata (2) and the structure (420 mg from 127 g of dried mycelium)., (Fig. 1) was elucidated by Sakabe et al, (3). The causal relationship of citreoviridin to cardiac beriberi was finally demonstrated by Ueno and Ueno (4). Although cardiac beriberi is not an important disease in these Citreov.iridin was isolated from pecan fragments that were modern times because of improved inspection for rice contammated with an organism identified at that time as P. charlesii (11) [later they apparently decided it was P. quality and the vitamin enriched diet of the consumer, citreoviride (7)]. [Note: Pitt (6) considers P. charlesii to be c~treoviridin still remains as a potential causal agent of a synonym of P. fellutanum.] The concentration of disease because of its recognized occurrence in com and citreoviridin in pecans was 168.9 mg/kg. other food and feedstuffs.

Toxicity Because of the ubiquitous nature and occurrence Occurrence The fungi that have been reported to produce in agricultural products of Penicillium spp that produce citreoviridin are: Aspergillus terreus (5), P. toxicarium, P. citreoviridin, knowledge of the toxic nature of the com­ citreoviride, Eupenicillium ochrosalmoneum, (P. ochro­ pound is important. salmoneum, anamorph), P. pulvillorum, and P. felluta- num. However, P. toxicarium and P. citreoviride are ~ishie ~n? c.owor~ers considered to be synonymous with P. citrenigrum, and P. . (12) have evaluated the toxicity of pulvillorum is synonymous with P. simplicissimum (6). cltreOVlOdm m mice and rabbits, and reported LD50 values Generally, all of the species are soil borne organisms, as shown in Table 1. The major effects of this mycotoxin in although P. simplicissimum and P. fellutanum are seldom these sp.ecies were catalepsy, hypothermia, dyspnea, and isolated from that source. Most isolates of these species are hypotensIOn. They reported that male mice were more also obtained from contaminated grains or seeds of various susceptible to this toxin than female mice. Results of their plant species. toxicity studies indicated that the targets of the toxin ap­ pear to be the respiratory and cardiovascular systems. The organism described as P. citreoviride has been isolated Near lethal doses given to mice on day 4 or 5 of pregnancy from rice on numerous occasions and was detected in had no effect on rates of pregnancy, ova implantation, and wheat flour and the Japanese food "miso" (1). However, e:nbry~n~l. resorption in survivors. Doses of 0.5 mg/kg there have been limited chemical surveys for the toxin, cltreoVlfldm given IV to rabbits caused decreased blood citreoviridin, in foods and feeds contaminated with species pressure and produced irregular pulses and electrocardio- of fungi known to produce the compound. grams along with increased respiration. Rabbits given let~al doses of citreoviridin stopped respiration, and then Using an HPLC method of analysis (7), Wicklow and co­ then electroencephalograms showed inactivity followed workers (8) examined maize infected with E. ochrosalmo­ by nonresponsive electrocardiograms. No effects of citre­ neum and found concentrations of citreoviridin in bulk oviridin were demonstrated on body weight gains and his­ samples from five of eight Georgia fields, and the amounts topathologic changes in liver, adrenal, or kidney tissues. found varied from 19 to 2,790 ug/kg. However, when yellow kernels were picked from bulk samples and Earlier work on toxicity of citreoviridin or extracts con­ analyzed, six of eight samples were positive for citreo­ taining citreoviridin demonstrated that affected animals viridin, and the concentrations ranged from 53,800 to had ascending paralysis, dyspnea, Cheyne Stokes res­ 759,900 ug/kg. In maize that was wound inoculated with E. piration, and cardiovascular disturbance (4, 10, 13, 14). ochrosalmoneum, they found that citreoviridin was located primarily in the damaged kernels, probably the reason for The teratologic effects of citreoviridin were studied in their earlier finding of citreoviridin in insect damaged, Fisher rats (14). Rats were given doses of 0, 5, 10, or 15 mg unharvested com in Georgia (9). citreoviridin/kg by gastric intubation on either days 8 to 11 or days 12 to 15 of pregnancy. Although there were A note of interest is that aflatoxin occurred in all samples developmental defects (cleft palates and dilated lateral of Georgia com examined by Wicklow and coworkers (8) ventricles), postimplantation loss, and skeletal retardation, and allows for possible interaction of aflatoxin and citreo­ these changes did not occur in the absence of maternal viridin in producing animal disease. toxicity. No studies have been conducted whereby animals were fed the toxin at other times of or throughout the The conditions for production of citreoviridin have been gestation period. investigated with P. citreoviride grown on rice (10). Maxi- mum yields of toxin were obtained on this substrate at low Cats given a total IV dose of 15.4 mg/kg of citreoviridin developed ascending paralysis followed by loss of eyesight ~emperature (12 to 220 C) and high relative humidity. The at several days after dosing (1). Importance of low temperature was confirmed using syn-

Table 1 HH:H L D 5 0 values of citreoviridin in mice and rabbits CHtO~CH3 [adapted from Ueno, 1974 (1) and Nishie et aI, 1988 (12)] o An al Sex Route LD50(mg/kg) CH3 H Mouse M SC 9.6, 11 Mouse M IP 7.5 Mouse F SC 11.8, 3.6 Citreovirid in Mouse M PO 29 Rabbit M IP >40 Rabbit M IV ;::5 Fig. 1. Structure of citreoviridin 64 Vet Hum Toxicol 32 (Supplement) 1990 Other than those episodes that were mentioned in the first part of this discussion on citreoviridin, there are no known natural intoxications of animals. Moreau (15) reported a paralysis in sheep in France, where moldy chestnuts were naturally contaminated with P. ochro- salmoneum and fed to the sheep. Although he indicated that he was doing additional investigations into this toxic OH syndrome, no further reports were found. H The development and use of newer analytical procedures for citreoviridin such as the liquid chromatographic method of Stubblefield and coworkers (7) may increase the H H incidence of detection of this mycotoxin and allow for identification of this compound in cases of mycotoxicosis of uncertain cause. These methods may be useful in detecting Penitrem C; (5) R=CI small amounts of the toxin or its metabolites in animal tissues. Veno (10) showed that liver contained the greatest Penitrem D; (6) R=H concentration of citreoviridin in dosed rats. Low recover­ ies of cirteoviridin were found in feces, and none was detected in urine using spectrophotometric and TLC Penitrems C and 0 methods.

Fig. 3. Structure of penitrems PENITREMS

Historical Aspects Within a large group of chemically re­ tremors, polypnea, hyperkinesia, ataxia, and clonic lated fungal secondary metabolites known as tremorgens seizures with intermittent opisthotonos. Sodium pento- (named because they cause tremors or similar nervous barbital treatment was followed by recovery in approxi- signs when administered to or ingested by animals) is a mately 12 hr. Two other similar cases (20, 21) of toxicosis group of toxic compounds known as penitrems (Figs. 2 and in dogs consuming moldy walnuts and a hamburger bun, 3). respectively, have been reported.

The first isolation of penitrems was reported in 1968 from isolates of P. cyclopium from feeds that were lethal to Occurrence Penitrems are produced only by members of sheep, moldy horse feed, and peanuts (16). Subsequently, the fungal genus, Penicillium. Currently there are at least penitrems were implicated in naturally occurring disease, three taxonomic treatments of this genus resulting in some but a causal relationship was not established. In one case, confusion among investigators that are unfamiliar with a moldy commercial feed sample was suspected of being this group of organisms. An examination of the literature involved in mortalities of dairy cattle and a penitrem­ reveals the following species as producers of penitrem: P. producing isolate of P. palitans was obtained from the feed canescens, P. clavigerum, P. cyclopium, P. palitans, P. (17). Similarly, Dorner and coworkers (18) isolated a peni­ crustosum, P. nigricans, P. puberulum, P. commune, and P. trem producing isolate of P. crustosum from moldy corn lanosocoeruleum. However, because of synonymy of some involved in the intoxication of cattle in Michigan. How­ species by some taxonomists the reader is referred to Table ever, analytical tests of the feed did not yield penitrem. 2. The most commonly encountered species capable of The confirmed cases of penitrem toxicoses have all occur­ producing penitrems is P. crustosum, the species involved red in dogs. The first case was reported by Arp and in all confirmed cases of penitrem toxicosis. In a survey of Richard in 1979 (19), and involved two dogs that had con­ 1,400 isolates of Penicillium, it was the only species that sumed moldy cream cheese. They developed an acute produced penitrem (22). neurologic episode characterized by severe muscle The fungi that produce penitrems have been isolated from a wide variety of foods and feeds as well as from soil. Fol­ lowing isolation of tremorgenic fungi from soil, it was suggested that close grazing by livestock could potentially include ingestion of these fungi (23). Although it has not been demonstrated for penitrem, an intriguing concept is the production of toxin in soil by soil fungi followed by uptake and translocation of toxin by plants, a situation demonstrated with verruculogen (24). In confirmed cases H of penitrem toxicoses, the compound was isolated from cheese, walnuts, and bread. A listing of sources from which penitrem-producing species of Penicillium were isolated is in Table 3.

Penitrem A; (1) R 1 = cr, R 2 =OH Toxicity In an animal with natural or experimental intoxi­ Penitrem 8; (2) R 1 ,R 2 =H cations with penitrem, there is a fine tremor produced that Penitrem E; (3) R1 =H, R 2 =OH is initiated within a few minutes to an hr after ingestion of Penitrem F; (4) R1 = CI, R2 =H toxin. The tremor may be noted first peripherally in the ears, tail, and other appendages; subsequently, whole body Penitrems A, B, E, and F tremors are noted, and are occasionally interrupted by periods of extensor rigidity, opisthotonos, and eventually the animal becomes recumbent with paddling of the legs. Often animals may be incoordinate, which may over­ Fig. 2. Structure of penitrems shadow the tremorgenic response (25) or the tremor may Vet Hum Toxicol 32 (Supplement) 1990 65 Table 2 Table 4 Classifications of penitrem-producing species of Toxicity of penitrems A and B to mice and chickens Penicillium [adapted from Richard et al, 1986 (33)] [adapted from Richard et ai, 1986 (33)]

Raper K, LD~.Q.rmg/kg) TD.iQ.(mg/kg) Thom C, 1949 Pitt, n, 1979 Ramirez C, 1982 Penitrem Mice Chickens Mice

P. canescens P. canescens P. canescens A 1. 1(IP) P. c1avigerum P. duclauxii P. clavigerum 10 (PO) P. cyclopium P. crustosum P. verrucosum 42 (PO) 0.19 (IP) var. cyclopium B 5.8 (IP) P. paiitans P. crustosum P. verrucosum var. cyclopium P. crustosum P. crustosum P. verruccsum var. cyclopium creatInIne phosphokinase, lactic dehydrogenase, aspartic P. puberulum P. puberulum P. verrucosum aminotransferase, and alanine aminotransferase (29, 30). var. cyclopium The increase in these enzymes in calves was considered to P. commune P. puberulum P. commune be due to increased muscle activity, as were increases in P. nigricans P. janczewskii P. nigricans plasma potassium and lactic and pyruvic acid concentra­ P. lanoso- P. aurantiogriseum P. verrucosum tions. In guinea pigs given penitrem A, there were no coeruleum var. cyc.iopium significant changes in liver-specific enzymes. Hayes and coworkers (31) did demonstrate an hepatic effect of peni­ ------trem in mice based on decreased liver glycogen and DNA. Notably, penitrem A was not metabolized by the liver of be increased by forced movement of the animal (26). sheep or by rat liver homogenates (32). Large doses of penitrem may produce a rapid convulsive response followed by death in intoxicated animals. Pathologic changes have not been detected in tissues of Chickens did not exhibit tremors in response to penitrem any animals experimentally intoxicated with penitrems unless they tried to move or hold their head erect (27). The (33). The mode of action of penitrem is not completely severity of response to penitrem diminished with succes­ understood, however, Stem (34) described tremors as sive dosing in guinea pigs (28), and the severity of the caused by an inhibition of the inhibitory interneurons. tremorgenic response at initial dosing was dose-related Glycine, known as a neurotransmitter of inhibitory and the response, even though diminished, was quite not­ neurons in the CNS of vertebrates (35), was reduced in able in guinea pigs given 0.75 mg penitrem every third concentration in brain tissues of penitrem A-treated mice day until seven total doses were given. (36). Substances that increase glycine levels in the CNS such as mephenesin and nalorphine, abolished tremors The amount of toxin producing a tremorgenic response due to penitrem A in mice. Thus, changes induced by varies from species to species, and there is animal variat­ penitrem appear to be chemical (glycine reduction) rather ion within species as well. Some of the LD50 values for than morphological. mice and chickens are given in Table 4. Sheep given single doses of penitrem did not respond unless they were given 15 mg of toxin (25). In sheep given repeated doses of GUOTOXIN approximately 2 mg penitrem/kg, there was an increase in the number of sheep responding as the number of doses Historical Aspects In 1932, Weindling (38) noted the anti­ increased. fungal activity of Trichoderma lignosum, a parasitic soil fungus. He continued to evaluate the antibiotic nature of Increased concentrations of serum enzymes were similar this fungus and, in 1937, reported that the fungus he W;lS" in both dogs and calves given penitrem A and included working with was a species of Gliocladium rather thari Trichoderma (39). The toxic substance isolated from cul­ ture filtrates of the fungus thus became known as glio­ Table 3 toxin (Fig. 4). By 1942, had attracted much atten­ Penicillium species producing penitrems [adaPted from tion because of its antimicrobial properties (40), and pro­ Richard et ai, 1986 (33)] duction studies were undertaken to increase the yields of =--:------_._-- this potentially important antibiotic (41). Gliotoxin was Penitrem A Source

P. canescens Soil P. clavigerum Soil P. cyclopium Pelleted horse feed Sheep feed (moldy corn) Peanuts P. palitans Commercial feed P. crustosum Cream cheese English walnuts Bread P. puberulum Silage P. commune Cottonseed P. nigricans Soil P. lanoso-coeruleum Weevil-damaged pecans P. palitans Commercial feed (Penitrems B and C) P. crustosum Moldy peanuts Gliotoxin (Penitrems B-F) Fig. 4. Structure of gliotoxin 66 Vet Hum Toxicol 32 (Supplement) 1990 active against a wide range of Gram positive bacteria (42), produce gliotoxin at body temperatures. and the relative activity of gliotoxin and its structural ana­ logues against Bacillus subtilis were subsequently studied Most of the fungi that produce gliotoxin are soil-borne (43). Although some studies continued with gliotoxin, fungi. The ability of isolates to produce gliotoxin is consid­ interest in this compound as an antibiotic waned when it ered by some (49) as a means to compete with and displace was reported to be toxic in mammals (41), and its toxicity other organisms. has precluded its use clinically. However, there has been a renewed interest in gliotoxin after discovery of an immu­ Gliotoxin is a member of a group of fungal secondary meta­ nomodulating compound (later shown to be gliotoxin) bolites known as epipolythiodioxopiperazines and, thus, is produced by a fungus, identified as A. fumigatus, that had closely related to the mycotoxin sporidesmium, the agent of contaminated laboratory cell cultures (44). facial eczema in New Zealand. The latter compound is pro­ duced by Pithomyces chartarum, and is contained within the conidia of the fungus (50). However, we could not find Occurrence Subsequent to the finding that Gliocladium any evidence of gliotoxin from 2.5 g of A. fumigatus was capable of gliotoxin production by Weindling (38), conidia analyzed by an HPLC method (45). several species among four genera of fungi have been shown to be capable of producing gliotoxin. Although there is disagreement among taxonomists about classifi­ Toxicitv and Biological Effects Gliotoxin has varying ef­ cation of some of these organisms, the species that have fects on a number of biological systems including the anti­ been described as gliotoxin producers are listed in Table 5. biotic effects already mentioned. The earliest known ef­ fects of this mycotoxin was associated with its antifungal Only recently has a sensitive method been developed effects (41), and the antibacterial nature of gliotoxin was whereby gliotoxin can be analyzed by HPLC (45). There­ reviewed by Boutibonnes and coworkers (51). However, fore, to date, gliotoxin has not been found to occur in com­ the major effects of gliotoxin on B. subtilis was an exten­ modities or other matrices except under culture conditions. sion of the growth lag phase (43).

Because both pathogenic and saprophytic fungi are cap­ Antiviral activity of gliotoxin was reported in 1964 and able of gliotoxin production, interaction of this mycotoxin 1965 (52, 53) and this subject has been thoroughly in pathogenesis of certain mycotic diseases, as well as glio­ reviewed (40, 54). toxin functioning as the etiologic agent in toxic disease, is potentially important. Gliotoxin is known to interact with nucleic acids as deter­ mined by blocking of viral RNA synthesis (55) and modi­ Richard and coworkers (45) found that all nine isolates of fication of B. subtilis DNA as measured by a "Rec" assay for A. fumigatus tested were capable of gliotoxin production. genotoxicity (51). The compound is capable of both plas­ Similarly, they have found that 13 of 15 isolates of A. fumi­ mid (Escherichia coli) and cellular (mouse) DNA damage in gatus, from cases of avian aspergillosis, produced gliotoxin a cell-free system. In DNA, there was a dis­ (unpublished). crete fragmentation pattern caused by gliotoxin and this characteristic, along with morphologic changes in the In 1930, Henrici (46) wrote in his classic tome entitled cellular chromatin, suggested similarities with cellular "Molds, Yeasts and Actinomycytes", "It is not clearly under- changes associated with apoptosis (56, 57). stood how the pathogenic fungi injure the tissues. Al- though fibrosis and giant cell reactions about some lesions Interest in the immunosuppression related actIvity of glio­ bear a resemblance to a foreign body reaction, the exten­ toxin came from the discovery that it inhibited macro- sive necrosis and suppuration which occur in the center phage adherence to plastic (44, 58); a phenomenon related of most lesions cannot be readily explained in this way. to phagocytic capacity of these cells. Gliotoxin also inhi­ Moreover, the experimental lesions produced with freshly bited mitogenic stimulation of lymphocytes in vitro (59) isolated and highly virulent strains of some species, as and this inhibition occurred with mature hematopoietic Aspergillus fumigatus and Candida albicans, are so acute as cells, but pluripotent stem cells still responded (50, 60). to suggest that these diseases may be caused by the same The nature of this response may be related to observed mechanisms as those found in bacterial infections." cellular changes associated with those of apoptosis; how­ During the ensuing 50 years, this concept has received ever, another mechanism of action of gliotoxin would little attention. However, Eichner and Mullbacher (47) appear to be initiated by blocking of membrane thiol hypothesized that gliotoxin may be produced during the groups (49). This may be involved particularly in inhi- pathogenic state of A. fumigatus, and they subsequently biting adherence of , as it is known that demonstrated the fungus and gliotoxin in peritoneal fluid cellular adherence is inhibited by thiol reagents (61). from infected mice (48). Although Richard et al, (45) did Waring and coworkers (56) noted that disulphide forms of not find gliotoxin production by a pathogenic isolate of A. epipolythiodioxopiperazines (e.g., gliotoxin) were required fumigatus on rice at temperatures above 300 C, perhaps the for antiphagocytic activity of macrophages, but not for nutrient state could influence the ability of isolates to DNA fragmentation.

Table 5 Additional immunosuppressive actIvity of gliotoxin has Species of fungi known to produce gliotoxin been demonstrated by its ability to inhibit allograft re- jection. Although in early studies murine lymphosarcoma Trichoderma viride Weindling, 1936 (33) cells exposed to gliotoxin and transplanted into syngeneic recipient mice failed to grow (62), recent studies deter­ GIiocladium fimbriatum Johnson et ai, 1943 (41) mined that thyroid tissue from mice placed in media con­ G. deliquescens Kirby et ai, 1980 (65) taining 1 Jl.M gliotoxin for 16 hr and then ttansplanted in Penicillium cinerascens Bracken and Raistrick, 1947(66) an allogeneic strain of mice, resulted in a 60-70% graft P. terlikowskii Johnson et ai, 1953 (67) P. obscurum Mull et aI, 1945 (68) success rate (63). Such activity could be important in Aspergillus fumigatus Glister and Williams, 1944 (69) transplant technology, because the recipient or host need A. terreus Miller et al, 1968 (55) not be placed at an increased risk because of immunosup­ A. chevalieri Johnson et ai, 1943 (41) pressive therapy. Thermoascus crustaceus Waring et aI, 1987 (70) Toxicity of gliotoxin to animals has been demonstrated.

Vet Hum Toxicol 32 (Supplement) 1990 67 Mice given 50 mg gliotoxin!kg either orally or intraperi­ 10. Ueno Y: Production of citreoviridin, a neurotoxic mycotoxin of toneally died within 24 hr (41). Similar results were ob­ Penicillium citreoviride Biourge. In Symposium on mycotoxins in tained with similar dosages in the rat, and a rabbit given Human Health. Ed Purchase IFH, McMillan, Pretoria, pp 115-132, an IV injection of 45 mg gliotoxin!kg died in 4 hr. Mon­ 1971. keys tolerated 0.2 mg!kg dosages of gliotoxin given intra­ muscularly for 15 days (52). Since that time, there has 11. Cole RD, Dorner JW, Cox RH, Hill RA, Cutler HG, Wells JM: Isolation been little information available in the literature con­ of citreoviridin from Penicillium charlesii cultures and molded pecan cerning the toxicity of gliotoxin in vivo. Recently, we fragments. Appl Environ MicrobioI42:677-681, 1981. detennined that an oral dosage of 7.5 mg gliotoxin/kg in 12. Nishie K, Cole RJ, Dorner JW: Toxicity of citreoviridin. Res Comm day-old turkey poults caused 100% mortality within 24 hr; Chem Pathol PharmacoI59:31-52, 1988. only one of 8 poults given 5 mg!kg died. Also, Frame and Carlton (64) have detennined that 25 and 35 mg glio­ 13. Uraguchi, K: Citreoviridin. In Microbial Toxins, Vol. VI. Ciegler A, toxin!kg given as single oral dosages to hamsters caused Kadis S, Ajl SJ, ed. Academic Press, New York, pp 367-375,1971. 100% mortality in 72 hr. 14. Morrisey RE, Visonder RF: Teratogenic potential of the mycotoxin citreoviridin in rats. Fd Chem Toxic 24:1315-1320,1986. SOME ADDITIONAL CONSIDERATIONS 15. Moreau C: Le Penicillium ochrosalrnoneum udagawa moisissure des Obviously, the list of additional mycotoxins that have po­ chataignes et la neurotoxicose des moritons in ardeche. Bull Soc tential for involvement in human and animal health could Myc Fr 91 :413-421, 1975. be expanded beyond these mycotoxins, but I believe citreo­ viridin, penitrems, and gliotoxin would be near the top of 16. Wilson BJ, Wilson CA, Hayes AW: Tremorgenic toxin from Penicil­ any prioritized listing. Additionally, in considering lium cyclopium grown on food materials. Nature 220:77-78, 1968. mycotoxins of potential importance in health, we should look further into the activity of those compounds that de­ 17. Ciegler A: Tremorgenic toxin from Penicillium palitans. Appl monstrate some capacity to be used as therapeutic agents of MicrobioI18:128-129,1969. disease. I alluded only briefly to this aspect of gliotoxin. 18. Dorner JW, Cole RJ, Hill RA: Tremorgenic mycotoxins produced by If one reexamines the criteria, from my introductory com­ Aspergillus fumigatus and Penicillium crustosum isolated from molded corn implicated in a natural intoxication of cattle. J Agric Fd mentary, for including the mycotoxins discussed herein Chem 32:411-413,1984. and attempted to list other mycotoxins, the following would likely be included: ergot , sporidesmins, cyto­ 19. Arp LH, Richard JL: Intoxication of dogs with the mycotoxin chalasins, , slaframine, swainsonine, rubratoxins, penitrem A. J Am Vet Med Assoc 175:565-566,1979. and phomopsins. Unfortunately, neither time nor space would allow for adequate discussion of these mycotoxins. 20. Richard JL, Bacchett P, Arp LH: Moldy walnut toxicosis in a dog, caused by the mycotoxin, penitrem A. Mycopathologia 76:55-58, Finally, the reader should be aware that, although certain 1981. mycotoxins are included in such a discussion as being potentially important in human or animal health, some are 21. Hocking AD, Holds K, Tobin NF: Intoxication by tremorgenic demonstrably involved in disease, while the involvement mycotoxin (penitrem A) in a dog. Aust Vet J 65:82-85, 1988. of others is only conjectural. 22. EI-Banna AA, Pitt JI, Leistner L: Production of mycotoxin by Penicillium species. System Appl Microbiol 10:42-46, 1987.

REFERENCES 23. DiMenna ME, Mantle PG, Mortimer PA: Experimental production of a staggers syndrome in ruminants by a tremorgenic Penicillium from 1. Ueno Y: Citreoviridin from Penicillium citreoviride Biourge. In soil. NZ Vet J 24:45-46, 1976. Mycotoxins. Ed Purchase IFH, Elsevier Scientific Publ Co, New York, pp 283-302, 1974. 24. Day JB, Mantle PG: Tremorgenic forage and rye grass staggers. Vet Rec 106:463-464, 1980. 2. Hirata Y: On the production of mould. I. Poisonous substance from mouldy rice (Part I). Extraction. J Chem Soc, Japan 68:63 69, 1949. 25. Penny RHC, O'Sulfivan BM, Mantle PG, Shaw BI: Clinical studies of tremorgenic mycotoxicoses in sheep. Vet Rec 105:392-393,1979. 3. Sakabe N, Goto T, Hirata Y: Structure of Citreoviridin, a toxic compound produced by P. citreoviride molded on rice. Tetrahedron 26. Peterson OW, Penny RHC, Day JB, Mantle PG: A comparative Lett 27:1825-1830, 1964. study of sheep and pigs given the tremorgenic mycotoxins verruculogen and penitrem A. Res Vet Sci 33:183-187, 1982. 4. Ueno Y, Ueno I: Isolation and acute toxicity of citreoviridin, a neurotoxic mycotoxin of Penicillium citreoviride Biourge. Jap J Exp 27. Wyatt RD, Hamilton PB, Colwell WM, Ciegler A: The effect of Med 42:91-105, 1972. tremortin A on chickens. Avian Dis 16:461-464, 1972.

5. Franck B, Gehrken HP: Citreoviridins from Aspergillus terreus. 28. Arp LH, Richard JL: Experimental intoxication of guinea pigs with Chem Int Ed Engl 19:461-462, 1980. multiple doses of the mycotoxin, penitrem A. Mycopathologia 73:109-113, 1981. 6. Pitt JI: The genus Penicillium and its telemorphic states Eupenicillium and Talaromyces. Academic Press, New York, pp 29. Cysewski SJ, Baetz AL, Pier AC: Penitrem A intoxication of calves: 219-276, 1979. Blood chemical and pathologic changes. Am J Vet Res 36:53-58, 1975. 7. Stubblefield RD, Greer JI, Shotwell OL: Uquid chromatographic method for determination of citreoviridin in corn and rice. JAOAC 30. Hayes AW, Presley DB, Neville JA: Acute toxicity of penitrem A in 71:721-724, 1988. dogs. Toxicol Appl Pharmacol 35:311-320, 1976.

8. Wicklow DT, Stubblefield RD, Horn BW, Shotwell OL: Citreoviridin 31. Hayes AW, Phillips RD, Wallace LC: Effect of penitrem A on mouse levels in Eupenicillium ochrosalmoneum infested maize kernels at liver composition. Toxicol 15:293-300, 1977. harvest. Appl Environ Microbiol 54:1096-1098, 1988. 32. Mantle PG: Metabolism and elimination of tremorgenic mycotoxins. 9. Wicklow DT, Cole RJ: Citreoviridin in standing corn infested by In Mycotoxins and Phycotoxins, Stern PS, Vleggaar R, ed. Elsevier Eupenicillium ochrasalmoneum. Mycologia 76:959-961, 1984. Science Publ, Amsterdam, pp 399-408, 1986. 68 Vet Hum loxicol 32 (Supplement) 1990 33. Richard JL, Peden WM, Thurston JR: Occurrence of penitrem 55. Miller PA, Milstrey KP, Trown PW: Specific inhibition of viral ribo­ mycotoxins and clinical manifestations of penitrem intoxications. In nucleic acid replication by gliotoxin. Science 159:431-432, 1968. Diagnosis of Mycotoxicoses, Richard JL, Thurston JR, ed. Martinus Nijhoff Publ, Dordrecht, pp 51-59, 1986. 56. Waring P, Eichner RD, Mullbacher A: The molecular mechanism of toxicity of gliotoxin and related epipoly1hiodioxopiperazines. In 34. Stern P: Pharmacological analysis of the tremor induced by cyclo­ Cellular and Molecular Mode of Action of Selected Microbial Toxins pium toxin. Yugoslav Physiol Pharmacol Acta 7:187-196, 1971. in Foods and Feeds, Pohland AE, Dowell VR, Richard JL, ed. Plenum Publ, New York, 1989. 35. Curtis DR, Duggan AW, Johnson GAR: The specificity of strychnine as a glycine antagonist in the mammalian spinal cord. Exp Brain Res 57. Waring P, Eichner RD, Mullbacher A, Sjaarda A: Gliotoxin induces 12:547-565, 1971. apotosis in macrophages unrelated to its antiphagocy1ic properties. J Bioi Chem (in press), 1989. 36. Catovic S, Filipovic N, Stern P: The effect of penitrem A upon the level of glycine in the CNS. Bull Scientifique 20:284-285, 1975. 58. Mullbacher A, Eichner RD: Immunosuppression in vitro by a metabolite of a human pathogenic fungus. Proc Nat! Acad Sci 37. Stern P, Catovic S: Mechanism of mephenesine action. Nauoryn 81 :3835-3837, 1984. Schniedebergs Arch Pharmacol 285:76R, 1974. 59. Mullbacher A, Hume 0, Braithwaite AW, Waring P, Eichner RD: 38. Weindling R: Trichoderma Iignorum as a parasite of other soil fungi. Selective resistance of derived hemopoietic progeni­ Phy10path 22:837-845, 1932. tor cells to gliotoxin. Proc Natl Acad Sci 84:3822-3825,1987.

39. Weindling R, Emerson OH: The isolation of a toxic substance from 60. Mullbacher A, Waring P, Tiwari-Palni U, Eichner RD; Structural the culture filtrate of Trichoderma. Phytopath 26:1068-1070,1936. relationship of epipolythiodioxopiperazines and their immunomodulating activity. Molec ImmunoI23:231-235, 1986. 40. Taylor A: The toxicology of sporidesmins and other epipcly1hio­ dioxopiperazines. In Kadis S, Ciegler A, Ajl SJ, ed. Microbial Toxins, 61. Grinnell F, Srere PA: Inhibition of cellular adhesiveness by Vol. VII. Academic Press, New York, pp 337-376,1971. sulphydryl blocking agents. J Cell PhysioI78:153-158.

41. Johnson JR, Bruce WR, DutcherJD: Gliotoxin, the antibiotic 62. Mason JW, Kidd JG: Effects of gliotoxin and other sulfur-containing principles of Gliociadium fimbriatum. I. Production, physical and compounds on tumor cells in vitro, with observations on mechanism biological properties. JAm Chem Soc 65:2005-2009, 1943. of action of gliotoxin. J Immunol 66:99-106, 1951.

42. Waksman SA, Woodruff HB: Selective antibiotic action of various substances of microbial origin. J BacterioI44:373-384, 1942. 63. Mullbacher A, Moreland AF, Waring P, Sjaarda A, Eichner RD: Prevention of graft versus host disease by treatment of bone 43. Brewer 0, Hannah DE, Taylor A: The biological properties of 3, 6­ marrow with gliotoxin in fully allogeneic chimeras and their cytotoxic epidithiadiketopiperazines. Inhibition of growth of Bacillus subtilis T cell repertoire. Transplantation 46:120-126, 1988. by gliotoxins, sporidesmins, and chetomin. Can J Microbiol 12:1187-1195, 1966. 64. Frame R, Cartton WW: Acute toxicity of gliotoxin in hamsters. Toxicol Lett 40:269-274, 1988. 44. Mullbacher A, Waring P, Eichner RD: Identification of an agent in 65. Kirby GW, Robins OJ, Sefton MA, Talekar RR: Biosynthesis of cultures of Aspergillus fumigatus displaying antiphagocy1ic and immunomodulating activity in vitro. J Gen Microbiol131 :1251-1258, bisdethiobis (methylthio) gliotoxin, a new metabolite of Gliociadium 1985. deliquescens. J Chem Soc, Perkin Trans 1, 119-121, 1980.

45. Richard JL, Lyon RL, Fichtner RE, Ross PF: Use of thin layer 66. Bracken A, Raistrick H: Biochemistry of microorganisms. Dehydrocarolic acid, a metabolic product of Penicillium canescens. chromatography for detection and high periormance liquid Biochem J 41 :569-575, 1947. chromatography for quantitating gliotoxin from rice cultures of Aspergillus fumigatus Fresenius. Mycopathologia (in press), 1989. 67. Johnson JR, Kidwai AR, Wamer JS: Gliotoxin XI. A related antibiotic 46. Henrici AT: Molds, Yeasts and Actinomycetes. John Wiley and from Penicillium terlikowskii: gliotoxin monoacetate. J Am Chem Sons, 1930. Soc 75:2110-2112, 1953. 68. Mull RP, Townley RW, Scholz CR: Production of gliotoxin and a 47. Eichner RD, Mullbacher A: Hypothesis: Fungal toxins are involved in aspergillosis and AIDS. Aust J Exp Bioi Med Sci 62:479-484, 1984. second isolate by Penicillium obscurum Biourge. J Am Chem Soc 67:1626-1627, 1945.

48. Eichner RD, Waring P, Gene AM, Braithwaite AW, Mullbacher A: 69. Glister GA, Williams TI: Production of gliotoxin by Aspergillus Gliotoxin causes oxidative damage to plasmid and cellular DNA. J fumigatus mut. helvola Yuill. Nature 153:651, 1944. Bioi Chem 263:3772-3777, 1988. 70. Waring P, Eichner RD, Tiwari-Palni U, Mullbacher A: Gliotoxin E: a 49. Jones RW, Hancock JG: Mechanism of gliotoxin action and factors new biologically active epipoly1hiodioxopiperazine isolated from mediating gliotoxin sensitivity. J Gen MicrobioI134:2067-2075. Penicillium terlikowskii. Aust J Chem 40:991-997, 1987. 50. Richard JL: Mycotoxin photosensitivity. J Am Vet Med Assoc 163:1298-1299, 1973.

51. Boutibonnes P, Auffray Y, Malherbe C, Kogbo W, Marias C: Proprietes antibacteriennes et genotoxiques de 33 mycotoxines. DISCUSSION Mycopathologia 87:43-49, 1984.

52. Larin NM, Copping T, Herbst-Laier RH, Roberts B, Wenham RB: Chair (Dr. Blodgett) We have another question: With respect to the Antiviral activity of gliotoxin. Chemotherapia 10:12-23, 1965. toxins that were discussed this afternoon, is there evidence that residues of these toxins in meat, milk or eggs could be 53. Rightsel WA, Schneider HGF, Sloan GS, Graf PR, Miller FA, Bartz OR, Ehrlich J, Dixon GJ: Antiviral activity of gliotoxin and gliotoxin immunosuppresive in the human consumers? . Nature 204:1333-1335, 1964.

54. Nagarajan R: Gliotoxin and epipolythiodioxopiperazines. In Dr. Richard: I think a general statement could be made that...and Mycotoxins Production, Isolation, Separation and Purification. it's not really answering this question either... that the Betina V, ed. Elsevier Science Publ, Amsterdam, pp 351-385, immunosuppresive effects are often found, generally (for 1984. mycotoxins that are immunosuppressive), at levels that are lower Vet Hum laxical 32 (Supplement) 1990 69 than you'd find with other toxic effects. So you'd have to take this consumed in toto, not individually in food, many of these individual on an individual basis, that is to say, what are the levels that occur in studies might not be able to list them. meat, milk or eggs, and are those levels immunosuppressive? And I think that if you looked at most of the literature on or less case reports, where they used the naturally contaminated feed immunosuppression, the work has been done at given levels, but which probably had several different mycotoxins in it, but usually they really haven't gone down to see how far they can go and still they don't elaborate on too many concentrations other than say, get immunosuppression. , and and Dr. Beasley would know more on the synergism in that aspect.

Dr. Finke-Gremmels: When you go down to low levels of ochratoxin-a or T-2 toxin, you easily come to a level where you Dr. Beasley: Well, I think there is probably not too much there as have immune stimulation, so that's one reason why we have so far as those two are concerned. Some of the work with aflatoxin much difficulty in an effort to interpret our results. and has indicated additive or less than additive toxicity. I haven't seen that much in the way of synergism. Anybody else have any experience? Dr. Richard: And it depends on what immunologic phenomena you happen to be looking at. Dr. Kuiper-Goodman: I think zearalenone is protective or antagonistic to the effect of ochratoxin. It is a species and sex Dr. Finke-Gremmels: It was IGM expression and the inhibition of related effect. the thymidine incorporation rate. And with the first test you can see easily that you can come to a level, to a very minimal dose, where you have immune stimulation. Dr. Robens: Bill Huff at the Veterinary Toxicology and Entomology Lab at College Station, Texas, has looked at a fair number of interactions in poultry. Its too bad that the Texas group Dr. Richard: You can see lymphoblastogenesis with T-2 toxin. could not be here (weather interfered with travel) because they could elaborate on that.

Audience: My comment is that it was shown with a few mycotoxins that you can have immunostimulation, but it depends on how long Dr. Chu: There are some earlier studies which show that aflatoxin you continue the experiment because in the same experiment after and rubratoxin have a synergistic effect and there are some reactions immunostimulation you can have immunosuppression. with the trichothecenes, DAS and DON in combination. I think some of your Canadian colleagues such as Dr. Schiefer's lab may have worked on that. Chair (Dr. Blodgett): Good comment. It all goes back to design then, how long, what doses. Another question for Dr. Richard: Do you see the possibility that gliotoxin can be used in humans for Dr. Richard: Ifyou recall, the slide that I showed earlier today with prevention of allograft rejection? respect to aflatoxin and rubratoxin in combination, there was synergism.

Dr. Richard: I think that is the premise under which the people in Australia are working. That's about all I can say. Audience: In our laboratory we have investigated interactions of ochratoxin and citrinin, and we found very weak interactions, but there was something there. Chair (Dr. Blodgett): Anybody want to tackle that one? Personally, I haven't read too many studies that have looked at the interactions Chair (Dr. Blodgett): So, I take it, most of the interactions have in dosed combinations. There have been studies which were more been with mycotoxins that effect the same target organ.

Chair (Dr. Blodgett): Thank you. Any questions from the Dr. Norred: We've done one study of rodents looking at the audience? combined effects of aflatoxin and cyclopiazonic acid. This was an acute study and we saw just additive effects without synergism. One reason that we don't see many of these types of studies is that Question from the audience: What is known about the synergism or they are very difficult studies to design especially with regard to antagonism among the various mycotoxins? Since they are being meaningful levels and meaningful dosing regimens.

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