APPLIED MIOBIOLOGY, Sept., 1965Copyright © 1965 American Society for Microbiology

Vol. 13, No. 5Printed in U.S.A.

Effect on the White Rat Uterus of a Toxic SubstanceIsolated from Fusarium;

C. M. CHRISTENSEN, G. H. NELSON, AND C. J. MIROCHA

Institute of Agriculture, and College of Veterinary Medicine, University of Minnesota,St. Paul, Minnesota

Received for publication 4 February 1965

ABSTRACTCHRISTENSEN, C. M. (University of Minnesota, St. Paul), G. H. NELSON, AND C. J.

MIROCEIA. Effect on the white rat uterus of a toxic substance isolated from Fusarium.Appl. Microbiol. 13:653-459. 1965.-Eighty-five fungi isolated from prepared feed andfrom corn collected on farms were grown separately in moist autoclaved corn. The cornwas fed to virgin weanling rats for 5 to 12 days; the rats were then killed, and theiruteri were removed and weighed. Twelve isolates of Fusarium from corn and one frompoinsettias caused increases of five to eight times in weight of the uterus 'as comparedwith controls that were fed sound corn. The greatest increase in weight of the uteruswas caused by corn inoculated with Fusarium No. 5 incubated for 21 days at 20 to 25followed by 14 days at 12 C. Extraction of this corn with methylene chloride, separationinto fractions by means of a silicic acid column, and further purification by thin-layerchromatography yielded a compound with ultraviolet-absorption maxima at 314, 274,and 236 m,u.

Over the past 30 years or more, a number ofworkers in several countries have implicatedFusarium as a common or occasional cause oftoxicoses of fairly serious nature (Dounin, 1926;Christensen and Kernkamp, 1936; Forgacs andCarill, 1962; Joffee, 1963). A recent report byStob et al. (1962) indicated that Gibberella zeae,the perfect stage of one or more species of Fu-sarium, produces a compound(s) with markeduterotrophic activity in swine. Their work wasundertaken evidently as a result of their havingobserved vulvar hypertrophy during 1957 and1958 in a number of swine herds that had beenfed on moldy corn. A similar disease of swine hasbeen observed in Minnesota, of which the grosssymptoms are tumefaction of the vulva (Jubband Kennedy, 1963), prolapsed vagina, andhypertrophy of the mammae; in these cases, wethought that microflora in the feed, or in the grainfrom which the feed was made, might be involved.This was suggested not only by the work of Stobet al. (1962), but also by the syndrome observedin two herds of swine described below.

In the summer of 1963, young swine in a herdnear St. Paul, Minn. developed the symptomslisted above when fed a pelleted feed. A sample ofthis feed was obtained and fed to guinea pigs, allof which developed greatly enlarged uteri. It wasthen fed to virgin weanling white rats, and all of

1 Paper 5564, Scientific Journal Series, Minne-sota Agricultural Experiment Station.

them developed greatly enlarged uteri within 5 to7 days.

In January, 1964, this same syndrome appearedin a herd of swine near Buffalo, Minn. Whenfeeding with the lot of corn on which they hadbeen fed up to that time was discontinued, thetumefaction and mammary enlargement rapidlydisappeared. The grain on which they had beenfed when these symptoms developed consisted ofapproximately 30% corn that had been heavilyinvaded and decayed by fungi and 70% corn thatappeared sound. According to the owner of thefarm, that portion of the corn heavily invaded byfungi was of the 1962 crop, and had been storedon the cob in a crib, exposed to the weather, fromharvest time in 1962 until late in 1963, when itwas shelled, mixed with sound corn, and fed to thepigs.

Several bushels of the mixture of sound andfungus-invaded corn fed to the herd of swine onthe farm were purchased. A portion of this cornwas fed to an immature gilt weighing approxi-mately 100 lb (45.5 kg). After 10 days, the animalwas killed and necropsied. During this 10-dayperiod, the gilt developed enlarged mammae anda swollen, edematous vulva. The uterus wasgreatly enlarged (Fig. 1). Several hundred gramsof kernels heavily invaded by fungi were pickedfrom this corn, ground, and fed for 5 to 7 days tovirgin weanling white rats; the rats were thenkilled and necropsied. Their uteri also weregrossly enlarged.

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FIG. 1. Uterus, greatly enlarged, from 100-lb gilt fed for 10 days on moldy corn from farm where theestrogenic syndrome developed in swine herd fed on same corn.

These two herds were fairly typical of a numberencountered. Because of the economic importanceof the disease and the need for more informationon this and other mycotoxicoses, the investiga-tions here reported were undertaken.

MATERIALS AND METHODS

Isolation of fungi from feeds and grains. Pelletsof prepared feed were scattered on a variety ofagar media in petri dishes, and were incubated at12 to 30 C until the fungi that grew out could beidentified and transferred. Dilution cultures werealso made, to determine the number of viablespores per gram of feed. Seeds of corn from variousfarms were cultured without surface disinfectionand with surface disinfection in 2% sodium hypo-chlorite for 1 min followed by a sterile water rinse.Since the embryos of many seeds were moreheavily invaded and decayed by fungi than the en-dosperm, and an individual embryo might harborseveral fungi, many kernels after surface disinfec-tion were split and placed on agar with the cut sur-face of the embryo upward, so that fragments ofmycelium could be transferred as soon as they be-came visible. Fungi were transferred to plates ofacid-potato-dextrose-agar (APDA); as soon astheir purity was assured, they were transferred toautoclaved moist soil in perfume bottles. After a

few days at room temperature to allow myceliumto grow, the bottles were stored at 5 C to serve asinoculum. When a small portion of this soil laterwas scattered sparsely on agar, mycelium of thefungus with which it had been inoculated grewfrom almost every particle, and a small amount ofsuch inoculated soil introduced into autoclavedcorn in 1-gal jugs or 5-gal paint cans served to in-oculate the corn thoroughly.

Production of test lots of feed invaded by fungi.Test lots were produced by several methods, in-cluding cultures in Czapek medium in 1-literFlorence flasks incubated on a shaker, and culturesin autoclaved corn meal, in autoclaved corn mealplus corn oil, and in moist whole autoclaved- cornkernels. As a result of preliminary feeding tests,and because our major interest was in productionof possibly estrogenically active feed under condi-tions that might prevail on farms, it was decidedto use whole corn. Sound corn was put in 1-galglass jugs, or in 5-gal metal paint cans, and wateradded to bring the moisture content of the grain toca. 45%; the mixtures were autoclaved for 1 hron each of 2 successive days, cooled, and in-oculated with cultures of the fungi growing in soil,as described above.

Various incubation times and temperatures weretested, but incubation at 20 to 25 C for 3 weeks, fol-lowed by incubation at 12 C for 2 weeks, was

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TABLE 1. Fungi which produced no enlargement ofuteri when grown in autoclaved moist corn for 3weeks at 20 to 25 C and 2 weeks at 12 C, andfedfor7 days to virgin white rats 21 days old at start oftest

No. ofisolates

Fungus Source tested

Aspergillus glaucus.Pelleted feed 8A. candidus ............. Pelleted feed 9A. ochraceus ............ Pelleted feed 5A. flavus ............. Pelleted feed 7*Botryotrichum ........... Straw 1Fusarium .............. Moldy corn 28Memnoniella .......... American Type 1

CultureCollection

Microascus ............. Sound corn 1Mucor Pelleted feed 1Nigrospora Moldy corn 1Papulospora ............ Moldy corn 12Sordaria ............. MMoldy corn 1

Total 75

* Isolates of A. flavus from pelleted feed havecaused death of several rats to which they werefed, and the symptoms suggest that a toxin otherthan the described aflatoxins may be involved.

adopted more or less as a standard. The containersof inoculated corn were shaken every few days tokeep the kernels from matting together. At theend of the incubation period, the corn, heavily

overgrown by fungus mycelium, was poured intoa pan,. dried overnight at 45 C with a fan blowingover it to aid the drying, and then ground in a handmill.Feeding tests. Pairs of virgin female weanling

white rats 21 days old were fed ad libitum for 5 to21 days (most tests were for 7 days), and thenkilled; the uteri were removed and weighed im-mediately, and samples were taken for later studyof hematological and histopathological changes.

Isolation and characterization of uterotropic com-pounds. The following solvents were tested: pe-troleum ether (bp, 60 to 70 C or 30 to 60 C), ace-tone, methylene chloride, chloroform, methanol-chloroform (1:2), methyl ethyl ketone, methanol,diethyl ether, ethyl alcohol-water, isopropyl alco-hol, n-butyl alcohol, and methyl cellosolve. Lotsof 500 g of corn that had been inoculated with purecultures of fungi and which in feeding iests hadproduced an increase in weight of rat uteri weredried, finely ground, and extracted for 24 hr inlarge flasks or for 12 hr in a Soxhlet extractor.After preliminary trials, most of the extractionswere made with methylene chloride because of itslow boiling point and the solubility of the toxin init.

After extraction, the solvent was concentratedin a flash evaporator to ca. 50 ml, applied to 100 gof clean ground carrier corn, dried under an infra-red lamp to remove the solvent, and fed to virginweanling rats. The control consisted of corn towhich an equal amount of solvent had been ap-plied and evaporated under an infrared lamp.The estrogenic constituent was separated by

passage through a silicic acid column by the modi-

FIG. 2. Uteri of control rat (left) fed on sound corn and of rat (right) fed on corn inoculatedwith Fusarium No. 5.

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TABLE 2.rats, 217 days

Weight of uteri of virgin weanling whitedays old at start of test, after feeding foron corn inoculated with various fungi*

Wt of uteriInoculum of two rats

mg

Fusarium # 4 ................... 176, 244Fusarium #5 ................... 233, 233Fusarium #6 ................... 129, 235Fusarium WP-2 ................. 185, 220Fusarium WP-3 ................. 191, 253Fusarium YN-3 ................. 119, 208Fusarium YN-13 ................ 197, 238Fusarium YN-16 ................ 195, 216Fusarium YN-9................. 42, 51Fusarium YN-11 ................ 28, 32Fusarium YN-19 ................ 38, 40Ex poinsettia ................... 175, 158Nigrospora ...................... 33, 38Microascus ..................... 48, 52

* Controls: the average weight of uteri of sevenrats was 40 mg; the uteri of three controls wereabnormally heavy for immature rats, and if theseare excluded the average weight of uteri was 29.6mg, with a range of 22 to 36 mg and a SD of 45 mg.

TABLE 3. Weight of uteri of virgin white rats, 21days old at start of test, after feeding for 7 days oncorn inoculated with Fusarium No. 5 and in-cubated for 2 to 12 weeks at 25 to 28 C

Weeks of incubation Wt of uteri of two rats

mg2 42, 474 33, 456 70, 738 24, 2910 125, 17212 41, 46

Control* 26, 32

* Not inoculated.

fied method of Davidoff and Korn (1963). Best re-sults were obtained by use of a column with a finalbed volume of 5 by 36 cm. The toxic material waseluted off the column with chloroform and sepa-rated from various pigments. The compound wasfurther purified on a column of silicic acid (2 by 50cm) with chloroform containing 1% methanol aseluant. The preparation was further resolved byseparation on thin-layer chromatograms. Silicagel G (Merck & Co., Inc., Rahway, N. J.) was usedas adsorbent in thin-layer chromatography andwas prepared in the usual manner. The solventmixtures found most useful, of several tested, werepetroleum ether (bp, 60 to 70 C)-methanol (9:1,v/v) and chloroform-methanol (25:1, v/v). Thespots were made visible with ultraviolet light or

by spraying with a 50% solution of H2SO4 in meth-anol.

RESULTS

Fungi isolated from pelleted feed and from corn.Storage fungi, principally Aspergillus repens, A.candidus, A. ochraceus, and A. flavus, comprisedthe major fungus flora of the pelleted feed. Thisfeed was compounded of 14 ingredients, of whichseveral grains and seeds made up a major portion,and it is quite possible that some of the fungipresent in the original grains no longer survived.Many genera of fungi were isolated from samplesof moldy corn collected from farms in Minnesota,South Dakota, and Iowa, but Fusarium andPapulospora were the principal ones.

Increased weight of uteri of white rats fed corninoculated with fungi isolatedfrom pelleted feed andfrom corn. Table 1 lists the fungi which, whenincubated on autoclaved corn and fed to whiterats, did not cause increase in weight of uteri.Increases of five to eight times in weight of uteri(Fig. 2) above those of the control rats fed soundcorn were induced by 12 of 40 isolates of Fusa-rium; typical data are summarized in Table 2.Weight of uteri is an accepted measure of estro-genic activity (Dorfman, 1962). The isolates ofFusarium which resulted in increased weight of

TABLE 4. Weight of uteri of virgin white rats, 21days old at start of tests, after feeding for 7 dayson corn inoculated with Fusarium No. 6 and in-cubated for 5 weeks at various temperatures

Wt of uteri ofIncubation two rats

mg2 to 3 C, 5weeks ................. 17, 30Alternate days at 2 to 3 C and 25 to28C .......................... 22, 35

3 weeks at 25 C,2 weeks at 2 to 3 C.. 54, 55

7 to 8 C, 5weeks ................. 29, 29Alternate days at 7 to 8 C and 25 to28 C .......................... 28, 34

3 weeks at 25 to 28 C, 2 weeks at 7to 8C .......................... 34, 37

14 C, 5 weeks ..................... 25, 35Alternate days at 14 C and 25 to

28 C ......................... 95, 1673 weeks at 25 to 28 C, 2 weeks at 14 C 123, 192

20 C, 5 weeks ..................... 34, 93Alternate days at 20 C and 25 to

28 C .......................... 37, 473 weeks at 25 to28 C, 2 weeks at 20 C 57, 129

Not inoculated, 5 weeks at 25 to29 C ........................... 28*

* One control rat was found dead after 48 hr,and the necropsy revealed no visible lesions.

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TABLE 5. Weight of uteri of virgin weanling whiterats fed materials extracted by different solventsfrom corn inoculated with Fusarium No. 5

Wt of uteri ofSample two rats

mgPetroleumether, bp 60 to 70C ... 52, 43Petroleum ether, bp 30 to 60 C...... 22, 54Acetone .......................... 81, 69Methylene chloride ............... 146, 130Chloroform ....................... 91, 154Methanol-chloroform (1:2)........ 120, 188Methylethyl ketone ............... 91, 100Diethylether ...................... 54, 37Methanol ......................... 74, 75Ethyl alcohol ..................... 80, 205Isopropanol ...................... 41, 105n-Butanol ........................ 170, 172Methyl cellosolve .................. 132, 47Water ................ 27, 47Control corn (uninoculated) ...... 44, 26Carrier corn ...................... 26, 36

TABLE 6. Weight of uteri of virgin weanling whiterats fed materials extracted by methylene chloridefrom corn inoculated with Fusarium No. 5 afterthe extract was separated into fractions by meansof a silicic acid column

Wt ofFraction Eluted with uterus

mg13-1 CHC1, 1,000 ml 1813-2 CHCla, 1,500 ml 14013-3 CHC13, 500 ml 2613-4 CHCl,, 500 ml 3613-5 CHCls, 500 ml 2713-6 CH,OH, 300 ml 29

Saturated with NaCl13-7 Control corn 33

uteri were from three sanples of corn, one fromthe farm in Minnesota mentioned above, wherethe estrogenic syndrome appeared in the herd ofswine fed with this corn, one from a crib of cornnear Yankton, S.D. (the crib contained a fairamount of corn similar in condition to the sampletaken), and one from an undesignated location inIowa. Evidently strains of Fusarium that produceestrogens are rather common and widely dis-tributed. All or most of the isolates of Fusariumtentatively appear to be F. culmorum or F. grami-nearum, both of which may be part of the Gibbe-rella zeae complex; in preliminaxy studies, therehave been no obvious cultural differences betweenisolates that produced estrogenic compounds andthose which did not.

Effect of time and temperature of incubation upon

production of estrogenic compounds as evaluated byincrease in weight of uteri of white rats. Moist auto-claved corn inoculated with Fusarium No. 5, an

isolate which consistently produced marked in-crease in weight of uteri in white rats, was incu-

bated at various temperatures and for differentlengths of time, and was then dried, ground, andfed for 7 days to paired virgin weanling femalewhite rats, after which the rats were killed andthe uteri were removed and weighed. The results(Tables 3 and 4) suggest that time and tempera-ture may greatly influence production of estro-genic compounds, even by a strain of the fungusknown to be a rather potent producer of thesecompounds when incubated under suitable con-

ditions. Forgacs and Caril (1962) cited extensiveevidence from work in the USSR that alimentarytoxic aleukia in man results from consumption ofgrain (proso millet) invaded by F. sporotrichioidesduring weather of frequent alternating freezesand thaws after a mild winter. The fact that themajority of isolates of Fusarium, and all but twoof the other fungi, tested by us produced no obvi-ous toxicosis in the rats to which they were feddoes not mean that these fungi might not beharmful if incubated and tested under othercqnditions.

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657VOL^. 13, 1965

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FIG. 4. Absorption spectrum of purified F-2 in n-

butanol. This compound was isolated from corn in-vaded by Fusarium No. 5 as well as from feeds thatcaused the estrogenic syndrome in swine.

Isolation of a compound causing increased weightof uteri of white rats. Table 5 gives the weight of

uteri of rats fed the extracts obtained with differ-ent solvents, and Table 6 gives the weight ofuteri of rats fed the fractions separated by meansof the silicic acid column. Fraction 13-2 (Table 6)was the only one to induce a large increase in theweight of uteri of rats to which it was fed.These fractions were resolved into their com-

ponents on thin-layer chromatoplates and were

compared with comparable extracts from soundcorn. The extracts from corn inoculated withFusarium No. 5, which resulted in increasedweight of uteri of rats to which it was fed, differedfrom the extract from sound corn in two com-

pounds, one (designated F-1) having an RF valueof 0.5 and the other (designated F-2) having an

RF value of 0.12 in petroleum ether-methanol and0.7 in chloroform-methanol solvent system. Com-pound F-1, after further purification by thin-layer chromatography, was identified as ergos-terol. The comparison of spectra between an

authentic sample of ergosterol (Nutritional Bio-chemicaJs Corp., Cleveland, Ohio) and F-1 isshown in Fig. 3.

Further tests confirming the identity of F-1were performed. A positive test resulted when F-1

was allowed to react with an alcoholic solution ofdigitonin. Digitonin forms a precipitate withcrgosterol. The Rosenheim (1929) color test,specific for ergosterol, was also positive. Bothergosterol and F-1 had identical RF values whentested on thin-layer chromatograms with acti-vated silica gel as adsorbent and chloroform assolvent. Both compounds showed identicalfluorescent characteristics when viewed underultraviolet light.Compound F-2 was purified by column chroma-

tography followed by thin-layer chromatography.Its purity was checked by charring with H2SO4after resolution on thin-layer chromatoplates intwo different solvent systems. Only a single spotwas found. Similar preparations were made, andcompound F-2 was eluted and collected in a testtube. The absorption spectrum of F-2 in n-butanol is shown in Fig. 4. Its ultraviolet-absorp-tion maxima corresponded to 314, 274, and236 mA.The purified preparation of F-2 was dried under

nitrogen, taken up in propylene glycol, and injected into weanling rats. Two control and twoF-2 treated rats were used. After 6 days, theweight of the uterus of the control rats averaged53 mg (fresh weight) and that of the F-2 treatedrats was 128 mg. A significant response in sizeand weight of the uterus occurred in the F-2treated rats (Fig. 2).Compound F-2 fluoresced bright blue when

illuminated with ultraviolet light. It gave a posi-tive test for a phenol when treated with FeCI3.When treated with base, F-2 showed a batho-chromic shift to 256 m,u, and the weak band at314 mA became broad and strong. The absorptionbands at 236 and 276 m,u disappeared. This batho-chromic shift reversed upon immediate acidifi-cation.Compound F-2 has an absorption spectrum

identical to a uterotrophic compound isolatedfrom corn infected with Giberella zeae, as reportedby Stob et al (1962).

LITERATURE CITEDCHRISTENSEN, J. J., AND H. C. H. KERNKAMP.

1936. Studies on the toxicity of blighted barleyto swine. Minn. Univ. Agr. Expt. Sta. Tech.Bull. 113.

DAVIDOFF, F., AND E. C. KORN. 1963. Fatty acidand phospholipid composition of the celLularslime mold, Dictyostellum discoideum. J. Biol.Chem. 238:3199-3215.

DORFMAN, R. D. 1962. Mlethods in hormone re-search, vol. 2. Academic Press, Inc., NewYork.

DOUNIN, M. 1926. The fusariosis of cereal crops inEuropean Russia in 1923. Phytopathology 16:305-308.

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FORGACS, J., AND W. T. CARILL. 1962. Mycotoxi-

coses. Advan. Vet. Sci. 7:273-382.JOFFEE, A. J. 1963. Toxicity of overwintered

cereals. Plant Soil 18:31-44.JUIBB, K. V. F., AND P. C. KENNEDY. 1963. Pathol-

ogy of domestic animals, vol. 1. Academic Press,Inc., New York.

ROSENHEIM, 0. 1929. A specific color reaction forergosterol. Biochem. J. 23:47.

STOB, M., R. S. BALDWIN, J. TUITE, F. N.ANDREWS, AND K. G. GILLETTE. 1962. Isolationof an anabolic, uterotrophic compound fromcorn infected with Gibberella zeae. Nature 196:1318.

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