BIOTECHNOLOGY AND BIOENGINEERING VOL. X, PAGES 469482 (1968)

Toxic Metabolites Produced by Fungi Implicated in Mycotoxicoses

C . J. ILIIROCHA, C. IrI. CHRISTEXSEN, and G. H. NELSOS, University of Minnesota, St. Paul, Minnesota 55101

Mycotoxicoses are diseases of animals and humans caused by toxins produced by fungi that have grown in feeds or foods or in the grains of other ingredients of which the feeds or foods were made and then consumed by humans or animals.

Our own work on mycotoxins began in 1963 as a result of common interest in the problem by researchers in the Diagnostic Laboratories of the College of Veterinary Medicine and the Department of Plant Pathology of the Institute of Agriculture. The Diagnostic Labora- tories w-ork closely with practicing veterinarians throughout Minne- sota and parts of neighboring states. When these veterinarians encounter, in more than a few animals, illness or death that cannot be attributed to a known and definite cause, the ill or recently dead animals are likely to be sent to the Diagnostic Laboratories for thor- ough examination and diagnosis.

Many cases of illness or death in herds of swine or cattle and in flocks of chickens and turkeys as well as sudden drop in egg produc- tion by laying hens, cannot be attributed to diseases caused by bacteria, viruses, or other known agents. One reasonable possibility is that at least some of these diseases of unknown cause are due to something in the feed, such as mycotoxins. The nature of the work now in progress can best be illustrated by outlining a few of the problems.

Our major interest has been to determine the extent to which mycotoxicoses might be involved in the health of domestic animals in Minnesota and to determine where and when toxin-producing fungi might be encountered. We have been especially concerned uith the estrogenic syndrome in swine and cattle, with the hemorrhagic

469

470 MIROCHA, CHRISTENSEN, NELSON

syndrome in chickens and turkeys, and with polioencephalomalacia and other diseases of unknown etiology in cattle.

Our approach and procedures have been as follows : Samples of grains and prepared foods have been cultured on agar media. From 5 to 20 isolates of each of the more common fungi that grew- from each sample so cultured were inoculated into sterile soil for stock cultures, and into autoclaved moist corn, or corn-rice mixture, for feeding tests. The fungi on corn or on corn-rice were incubated for about 2 weeks at 22-25OC, followed by 3 weeks at 12”C, then were dried, ground, and fed to pairs of white rats as their sole ration. Those which resulted in death, within 7-10 days, of both rats to which they were fed, are referred to in the following tables as “lethal.” Some lots of fungus-invaded grain were fed to chicks or turkey poults, or both, as indicated in the tables.

Of 969 isolates of fungi tested over the past 2 years, about 50% of them were found toxic to rats. Of 396 isolates from peanuts, 157 of them proved lethal whereas of 573 isolates obtained from corn, feeds, and foods, 331 w-ere lethal to rats (Table I). Of the genera of fungi isolated from peanuts Penicillium spp were found most frequently and contained the greatest percentage of toxic isolates. In order of frequency of isolation, Penicillium led followed by Fusarium, Asper- gillus, Chaetomium, and Alternaria (Table 11). When the toxicity of the most numerous genera of fungi isolated from corn, feeds, and foods was tested on chicks, ducklings, turkey poults, or rats, Aspergillus, Penicillium, and Chaetomium spp. proved most important (Table 111).

To further exemplify the prevalence of toxic fungi in food or feed, a study of the microflora found in black pepper was undertaken.’ Under the direction of Professor Dr. C. RI. Christensen, samples of black pepper were collected from India, Mexico, US., Poland, and

TABLE I The Number of Fungi Isolated from Various Sources of Animal Feed and Number

Found Toxic to Rats during 1965-1967 at the University of Minnesota

Source No. tested Xo. lethal yo Lethal

Peanuts 396 157 40 Corn, feeds, and foods ~ 573 331 58 Total 969 488 50

~ -

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TOXIC METABOLITES IN MYCOTOXICOSES 47 1

TABLE I1 Toxicity to Rats of the Most Xumerous Genera of Fungi Isolated from Peanuts

Genus KO. tested No. lethal

Al temr ia 23 12 Aspergillus 37 25 Chaetumium 26 11 Fusarium 65 28 PeniciUium 80 40

~ ~

Total 231 116 = 50%

TABLE I11 Toxicity of Most Numerous Genera of Fungi Isolated from Corn, Feeds, and Foods

Genus Test' No. isolates

animal tested Lethal % hthal

AEtentaria R 60 53 88 Aspergillus R-C-D 159 60 38 Chaetumium R 18 15 83 Clados porium R 41 19 46 Fusarium R-T 87 65 75

46 PaiciUium R-T 116 53 Total 481 265 55

__ ~ -

*C = Chick; D = Duckling; T = Turkey poult; R = Rat.

Japan. Dilution cultures of 30 samples of ground black pepper yielded an average of 39,000 colonies of fungi per gram. Total number of colonies of bacteria from 11 samples averaged 194,000,000 per gram. Among the fungi from both black and red pepper were Aspergillus pavus and A . Ochtaceus some isolates of which, when grown for 8-10 days on moist autoclaved corn and fed to white rats or to 2-day old Pekin ducklings, were rapidly lethal to them. Afla- toxin BI was isolated from one of the samples of corn on which A . J%WS from black pepper was grown.

The consumption of black pepper by the consumer over a period of many years and the effect on his health is a question that as yet has not been answered but an answer must be found.

472 MIROCHA, CHRISTENSEN, NELSON

TOXICITY TO RATS OF CORN INVADED BY CEAETOMIUM GLOBOSUM

Death of rats used as test animals resulted from consumption of corn inoculated with isolates of C. globosum from corn and from commercially manufactured feed. Several isolates caused death within 4-5 days to both rats to which they were fed, after consump- tion of less than 5 g of feed per animal.

As early as the second day, many of the rats fed on corn invaded by C. globosum developed symptoms of disturbance of the Central nervous system, such as excitability, photophobia, and loss of equi- librium. After 1-2 days more, the affected animals went into coma and soon thereafter died. Gross necropsy lesions, consisted of sub- dural hemorrhage, hemorrhagic enteritis, and hemoglobinuria. Prothrombin time of triplicate subsamples of blood of two rats just before death resulting from ingestion of corn invaded by C. globosum was more than 5 min, a more than 20-fold increase over that of rats used as controls.

I n contradistinction to the symptoms incited in rats, the Chaeto- mium toxin was apparently harmless to pigs. When Chaetomium- invaded corn was fed ad libitum to seven boars, 6 weeks old, as their sole ration the boars consumed it with relish and no symptoms of illness or distress appeared in any of them.

The toxic compound or compounds were soluble in methylene chloride, chloroform, and acetone, but not petroleum ether (b.p. 3040°C or 6O-7O0C), methyl Cellosolve, or water (Table IV). Pre- extraction with petroleum ether (b.p. 6O-7O0C), followed by extrac- tion with either acetone or methylene chloride, resulted in higher yield of toxic materials, as judged by mortality of the rats, than did extraction with either of the solvents alone. Nore pigments, but also more of the toxic materials, were extracted by acetone than by methylene chloride.

The extract was further purified on a silica gel column, bed volume 2 X 15 in., and eluted with various solvents. Feeding tests indicated that the toxic material was eluted off the column in the acetone frac- tion (Table V). The toxin was further purified on a similar silica gel column, by use of the gradient-elution method, and was found to elute off in a 5% acetone-in-chloroform fraction. In further frac- tionation on a gradient column, the toxin was found in the 2% acetone-

BIOTECHSOLOGY AND BIOENGINEERING. VOL. X. ISSUE 4

TOXIC METABOLITES IS MYCOTOXICOSES 473

in-chloroform fraction. Sufficient material for chemical charac- terization of the compound is not as yet available. The toxic material produced by C. globosum appears to differ from other mycotoxins known to us.

TABLE IV Extraction of a Toxin Produced by C . globosum

Treatment ilmount feed

Lethal effects consumed,. g

None-sound control corn Xone Petroleum ether (b.p. 60-70°C) None Petroleum ether (b.p. 30-60"C) None Petroleum ether (b.p. 30-60"C) after

40 32 48

pre-extraction with pet. ether b.p. 60-70°C) Kone 48

Acetone Death (10 days) 14 Acetone after pre-extraction with pet.

ether (b.p. 60-70°C) Death (6 days) 6 Methylene chloride Death (10 days) 17 Methylene chloride after pre-extraction

with pet. ether (b.p. 60-70°C) Death (6 days) 18

* Average amount of food consumed by each of two rats. 500 g of feed were ex- tracted with the various solvents or sequence of solvents and incorporated onto 100 g of sound corn. 50 g were given to each rat.

TABLE V Further Purification of the Chaetomium Toxin Eluted Off the Silica Gel Column

in the Acetone Fraction

Elutant' Amount of elutant, ml Lethal effects

Chloroform 1500 None 5 7 , Acetone in CHCls 1500 Death 10% Acetone in CHCls 1250 None 2070 Acetone in CHCl, 1500 Xone 50% Acetone in CHCla 1000 Sone Acetone 500 Xone Water 500 Xone

a The eluant was concentrated and incorporated unto 100 g of carrier corn, 50 g of which was fed to each rat.

474 MIROCHA, CHRISTEISSEK, NELSON

HEMORRHAGIC SYNDROME

Toxins produced by one or more species of the fungus Penicillium cause what is known as the “hemorrhagic syndrome” in chicks, duck- lings, and turkey poults. This condition is characterized by pro- found anemia and widespread hemorrhage due to bone marrow depression and not uncommonly resulting in death. Penicillium is common in some lots of corn, peanuts, and other grains and, in our experience, is one of the more common and abundant fungi in manu- factured feeds. h’umerous isolates of Penicillium, when grown on autoclaved moist, corn and fed to rats in our tests have caused death within 3 days, accompanied by hemorrhage in the intestinal lumen and in the subdural tissues of the brain.

The toxin or toxins are extracable with petroleum either (b.p. 30- 60°C) or methyl alcohol but appear to be labile. They are sparingly soluble in petroleum either (b.p. 60-70°C) and acetone but insoluble in methylene chloride, chloroform, and ethyl alcohol.

ESTROGENIC SYNDROME

The estrogenic syndrome in swine involves development of a swollen, edematous vulva in females, shrunken testes in young males, enlarged mammary glands in the young of both sexes, and possibly abortion in pregnant gilts or sows. The major causes of this disease is a toxin produced by the fungus Fusarium graminearum, and per- haps other species in the Gibberella zeae complex, when growing in corn stored in cribs. The toxin has been found in autoclaved corn inoculated with Fusarium, in samples of corn feed from farms in Minnesota reporting the estrogenic symptoms in their swine herds, as well as in commercially prepared pelleted feed.

The early work on the chemical isolation and structure of the estrogenic metabolite was reported by F. N. Andrews and 11. Stob (Belgian Pat. 611630, 1961, US. Pat. 3,196,019, 1965) and its partial characterization was reported by Christensen et al.293 Recent infor- mation on the chemical structure of the estrogen reveals it to be one of the enantiomorphs of 6-( lO-hydroxy-6-oxo-trans-l-undecenyl(-~- resorcylic acid l a ~ t o n e . ~ The estrogenic factor herein described has been called various trivial names such as “F-2” by the Minnesota group,* “RAL” by Commercial Solvents Corporation, and more recently “Zearalenone. ”4

BIOTECHXOLOGY A K D BIOENGINEERING, VOL. X, ISSUE 4

TOXIC METABOLITES IS MYCOTOXICOSES 475

Compounds similar in structure, but perhaps not in biological activity, to the estrogenic factor have been reported before, e.g., cumlar in , produced by the fungi Curvularia sp., Penicillium steckii Zaleski, and P. expansum Link,j and radicicol and monorden, pro- duced, respectively, by Nectria radicicola6 and Monosporium sp.’

BIOLOGICAL SPECTRUM OF “F-2” ACTIVITY

Estrogenic Response in Rats and Mice

When the purified F-2 in solution was injected or the crystal fed ad libitum to white weanling virgin female rats, a typical estrogenic response such as that reflected in the increase in fresh weight of the uterus was found. A dosage response curve (Fig. l a ) was obtained when a cumulative dose ranging from 20 to 650 pg was administered over a period of 7 days. There was a linear response in the increase

c .. 0

c

Fig. 1. (a) Effect of different dosages of F-2 on the weight of the rat uterus ( b ) Effect of the same concentra- when administered by intramuscular injection.

tions on the body weight.

476 MIROCHA, CHRISTENSEN, NELSON

in the weight of the uterus with increase of F-2 concentration. The opposite was true however, u-hen the body weight of the animals was averaged. Those rats treated with the highest concentration of F-2 (Fig. l b ) showed no increase in body weight when compared with the control rats, whereas the lowest concentration caused a relative increase in weight.

Similarly, estrogenic activity was detected when the mouse uterine assay was used (Tables VI and VII). When the mouse uterus assay

TABLE VI “F-2” Mouse Estrogen Assay (Subcutaneous Injection)’

Material Total dose, Mean uterine administered Pi3 No. of mice ratio f S.E.

Control 0 10 0.75 f 0.19

Estrone 0.05 10 1.48 f 0.09 0.1 10 2.66 f 0.36 0.2 10 3.37 f 0.32 0 .4 10 3.60 f 0.18

F-2 5 10 0.72 f 0.05 20 10 0.88 f 0.03 80 9 1.41 f 0.11

___

a Data of Dr.Ralph 1.Dorfman of Syntex. F-2 - 0.00062 X as active as estrone.

TABLE V I I “F-2” Mouse Estrogen Assay (Gavage)”

Material Total dose, Mean uterine administered Pg No. of mice ratio f S.E.

Control

Estrone

F-2

0

0.5 1 2 4

12.5 25 50

100

10 0.76 f 0.06

10 1.21 f 0.12 9 1.19 f 0.10 9 2.12 f 0.23

10 2.93 4~ 0.21

10 1.11 f 0.04 10 1.48 f 0.18 10 1.80 f 0.12 9 2.35 f 0.17

* Data of Dr. Ralph I. Dorfman of Syntex F-2 N 0.016X as active as estrone. BIOTECHNOLOGY Ah-D BIOENGINEERING, VOL. X, ISSUE 4

TOXIC METABOLITES I X MYCOTOXICOSEE 477

was used and F-2 administered by subcutaneous injection, it was found to be 0.00063 times as active as the proprietary compound, Estrone (Table VI). When administered by gavage, it was 0.01 times as active as Estrone (Table VII). It appears that this com- pound is much more active when administered by the oral route (data of Dr. R. I . Dorfman of Syntex).

When F-2 was tested for its myotrophic activity in the mouse, a stimulation in grou-th of the perineal muscle was noted (Table VIII). The activity was not as great as that of the proprietary compound Stilbestrol but rather appeared to reach a plateau of activity which did not increase with further concentration (data of R. I. Dorfman of Syntex). This is similar to that noted in studies m-ith rats and plant tissue culture.

TABLE VIII Stimulation of Growth of Perineal Muscle"

Material Total dose, Mean muscle administered fa No. of animals ratio f S.E.

Control 0 10 0.60 f 0.03

Diethylstilbestrol 0.33 9 0.80 f 0.06 1 .o 10 1.04 f 0.04 3.0 10 1.19 f 0.06 60 9 0.99 + 0.09 540 10 1.03 f 0.05

Data of Dr. Ralph I. Dorfman of Syntex.

Effect on Growth of Plant Tissue Culture

A standard bioassay with tobacco pith callus tissue was employed in studying the activity of F-2 in promoting growth in cells of higher plants. The tissue, prior to testing, was grown in darkness at 28°C and 75% relative humidity.

F-2 was treated in concentrations between 0.02 and 25 pmoles/l in the presence of 2.0 mg/l3-indoleacetic acid and 0.2 mg/l of Kinetin. F-2 was dissolved in 1 ml purified 100% ethanol, diluted with warm double-distilled water to the desired volume, and added to the media before autoclaving.

478 MIROCHA, CHRISTENSEN, NELSOX

Under the conditions tested, it appears that F-2 has a slight growth-stimulating effect in the concentration range between 0.02 and 1.0 pmolell (Table IX). It inhibits growth at 2.5 pmole/l which corresponds to less than a part per million.

TABLE I X

Stimulation of Growth of Tobacco Callus Tissue by “F-2” in the Presence of IAA and Kinetin (Growth Period, 7 weeks).

Yield, g/flask F-2,

PM/l Fresh wt. Dry wt.

0 0.02 0 .1 0 .5 1 .o 2.5

12.5 25.0

3.79 f 0 . 2 0.23 4.17 f 0 .3 0.28 3.98 f 0.3 0.28 4.04 f 0.1 0.28 3.71 f 0 . 2 0.26 2.86 f 0 . 3 0.21 0.62 f 0.06 0.06 0.48 f 0.03 0.05

a Data of Dr. E. hl. Linsmaier-Bednar, University of Minnesota.

In tests where F-2 at increasing concentrations was tested together with increasing concentrations of kinetin, the data suggested that with high concentrations of added kinetin (0.2 mg/l) or high endog- enous cytokinin carry-over in the assay bissue, there is a greater tolerance of the callus tissue towards increasing concentrations of F-2. Tissues grown prior to assaying with low or zero concentrations of kinetin and assayed on a medium with kinetin at a concentration of 0.2 mg/l, counteracted the inhibitary effect of F-2 when grown at an otherwise inhibiting concentration of 25 pmoles/l.

Other experiments in which the activity of F-2 on organ induction in undifferentiated callus tissue was tested showed that this compound was able to stimulate shoot formation in vitro. Concomitant with the increase in shoot formation, there m-as a decrease in the amount of callus tissue.

Research concerning F-2 and plant-tissue culture was carried out by Dr. E. hf. Linsmaier-Bednar, University of Minnesota.

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TOXIC METABOLITES IN MYCOTOXICOSES 479

Effect on Microorganisms

Preliminary examinations suggest that F-2 has stimulatory effect in promoting the development of the sexual stage of Fusarium graminearum (data of hfiss Cesaria Eugenio, Univ. Minnesota). F-2 is produced by this species of Fusarium and hence is natural to this biological system. This hormone also affects the sexual cycle of other fungi however, which, as far as is known, do not have metabolic ability to synthesize this compound.

The enzymes responsible for the biosynthesis of F-2 are apparently induced at low temperature (12°C). When the organism is seeded unto sterile, moistened corn and allowed to incubate at room tempera- ture for 1 week until sufficient biomass develops and is then subjected to different temperatures, we find after 3.5 weeks of incubation at 12"C, 390 ppm of the estrogen and none at 25°C (Table X). The same pattern of synthesis continues at 7.5 and 11 weeks of incubation until a concentration of 4200 ppm is obtained at 12°C and none at 25°C. The pattern of ergosterol synthesis is the opposite of that obtained with F-2.

Once the F-2 synthesizing enzymes are formed or activated at 12"C, optimum production of F-2 can be attained at higher temperatures. After subjecting the culture to low temperature for a time sufficient to allow activation or induction of F-2 synthesizing enzymes, the cultures were subjected to temperatures of 12, 27, and 32°C for vary- ing amounts of time. In all cases, greatest production of F-2 occurred

TABLE X Temperature-Dependent Production of "F-2" by Fusarium graminearum

Culture age, Incubated at, F-2, Ergosterol, weeks "C PPm PPm

3 . 5 12 3 .5 25 7 . 5 12 i . 5 25

11.0 12 11 .o 25

390 0

0 528

2328 0 0 1680

4200 0 0 1596

11.0 25°C for 8 weeks and 12°C for 3 weeks 0 1824

480 MIROCHA, CHRISTENSEN, NELSON

TABLE XI F-2 Production after Induction of Enzymes at Low Temperature Followed by

Incubation at Higher Temperature

Temperature, Incubation time, F-2,’ “C days ppm

12

12 27 32

12 27

0 1834

3 2082 3 3619 3 1134

5 1810 5 651 1

~

32

12 27 32

12 27 32

5 3888

7 3111 7 7280 7 3056

11 2360 11 8375 11 4674

__

Average of four replicates.

at 27°C when incubated for 3, 5 , 7, and 11 days (Table XI). Yields as high as 8000 ppm resulted after 11 days of incubation at 27°C which is a 3.5-fold increase over that held at 12°C.

F-2 is synthesized on moist autoclaved rice or corn with or without the addition of glucose. Yields thus far obtained have ranged be- tween 10,OOO and 15,000 ppm depending on length and temperature of incubation. No real difference has been obtained in yield of F-2 when either corn or rice is used as a substrate. When glucose is added to the corn or rice substrate and incubated at 12°C for 5, 6.5, and 8.0 weeks, there appears to be an inhibitory effect at concentra- tions of 2 and 47, which is more noticeable in the corn that the rice substrate. At a concentration of 6%, there is a stimulation in F-2 production in rice cultures but not in corn.

Recent findings emanating out of studies involving the biosynthesis of “F-2” reveal another naturally occurring but unidentified com- pound related to the latter which we call “F-3”. I t appears to be an intermediate in the biosynthesis of F-2 by Fusarium graminearum and is found in small amounts in cultures of the fungus. Perhaps of

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TOXIC METABOLITES I N MYCOTOXICOSES 48 1

more importance is the fact that this compound (F-3) has been found in feed samples suspected of causing abortion or infertility in dairy cattle in Minnesota. In contrast to the microflora found infesting corn implicated in causing abortion in swine (F . graminearum) another species of Fusarium ( F . monilijorme) has been found in the feed suspected of causing infertility in dairy cattle. Further, these isolates of F . monolijorme produce copious amounts of F-3 but no F-2. Finally, F-3 has been found naturally occurring in feed suspected of causing infertility problems in cattle.

Studies involving the chemical isolation and characterization of F-3 show it to be a highly labile compound subject to oxidation. It is readily extractable from biological material using the same methods and solvents as used with F-2. Its absorption spectrum in the ultra- violet is identical to that of F-2 except that it lacks an absorption maximum a t 314 mp. Unlike F-2, it does not have a ketonic group in the side ring as evidenced by its inability to react m-ith Girard’s reagent (trimethylaminoacetohydrazide chloride) and can be sepa- rated from F-2 using this method.

F-3 like F-2 reacts with silating agent (N,O-bis(trimethylsily1)- acetamide) to form the trimethylsilyl ether. The latter compound can then be separated on SE-30 GLC column; it has a retention time of about 2 min less than that of F-2 at about 260°C.

F-3 like F-2 can be separated from other biological constituents on a silica-gel column. When using common solvents of the eluotropic series, it comes off the column in the diethyl ether fraction.

Studies are being continued in an effort to isolate sufficient amounts of the compound so that they can be subjected to various spectro- scopic methods for functional group analysis.

The authors wish to acknowledge the cooperation of Dr. E. M. Linsmaier- Bednar and Miss Cesaria Eugenio, Departments of Horticulture and Plant Pathology, respectively, University of Minnesota; Dr. D. Huissingh, Depart- ment of Plant Pathology, University of North Carolina, Raleigh, North Carolina, and Dr. R. I. Dorfman, Institute of Hormone Biology, Syntex Research, Palo Alto, California.

References 1. C. M. Christensen, H. A. Fanse, G. H. Nelson, Mrs. F. Bates, and C. J.

2. C. M. Christensen, G. H. Nelson, and C. J. Mirocha, -4ppl . Microbiol., 13, Mirocha, A p p l . Microhbl., 15, 622426 (1967).

653-659 (1965).

482 MIROCHA, CHRISTENSEN, NELSON

3. C. J. Mirocha, C. M. Christensen, and G. H. Nelson, A p p l . Microbwl., 15,

4. W. H. Urry, H. L. Wehrmeister, E. B. Hodge, and P. H. Hidy, Tetrahedron

5. S. Shibata, S. Natori, and S. Udagana, List of Fungal Products, Univ. of

6. B. N. Mirrington, E. Ritchie, C. W. Shoppee, W. C. Taylor, and S. Stern-

7. F. McCapra, A. I. Scott, P. Delmotte, J. Delmotte-Placequee, and N. S.

8. C. M. Christensen, G. H. Xelson, C. J. Mirocha, F. Bates, and C. E. Dor-

497-503 (1967).

Letters, No. 27, 310S3114 (1966).

Tokyo Press, Tokyo, 1964.

hell, Tetrahedron Letters, S o . 7, 365-370 (1964).

Bhacca, Tetrahedron Letters, No. 15, 869-875 (1964).

worth, A p p l . Microbiol., 14, 774-777 (1966).

Received November 21, 1967

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