Isolated from Fusarium;

Transcription

1 APPLIED MIOBIOLOGY, Sept., 1965 Copyright 1965 American Society for Microbiology Vol. 13, No. 5 Printed in U.S.A. Effect on the White Rat Uterus of a Toxic Substance Isolated 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 ABSTRACT CHRISTENSEN, 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: Eighty-five fungi isolated from prepared feed and from corn collected on farms were grown separately in moist autoclaved corn. The corn was fed to virgin weanling rats for 5 to 12 days; the rats were then killed, and their uteri were removed and weighed. Twelve isolates of Fusarium from corn and one from poinsettias caused increases of five to eight times in weight of the uterus 'as compared with controls that were fed sound corn. The greatest increase in weight of the uterus was caused by corn inoculated with Fusarium No. 5 incubated for 21 days at 2 to 25 followed by 14 days at 12 C. Extraction of this corn with methylene chloride, separation into fractions by means of a silicic acid column, and further purification by thin-layer chromatography yielded a compound with ultraviolet-absorption maxima at 314, 274, and 236 m,u. Over the past 3 years or more, a number of workers in several countries have implicated Fusarium as a common or occasional cause of toxicoses of fairly serious nature (Dounin, 1926; Christensen and Kernkamp, 1936; Forgacs and Carill, 1962; Joffee, 1963). A recent report by Stob et al. (1962) indicated that Gibberella zeae, the perfect stage of one or more species of Fusarium, produces a compound(s) with marked uterotrophic activity in swine. Their work was undertaken evidently as a result of their having observed vulvar hypertrophy during 1957 and 1958 in a number of swine herds that had been fed on moldy corn. A similar disease of swine has been observed in Minnesota, of which the gross symptoms are tumefaction of the vulva (Jubb and Kennedy, 1963), prolapsed vagina, and hypertrophy of the mammae; in these cases, we thought that microflora in the feed, or in the grain from which the feed was made, might be involved. This was suggested not only by the work of Stob et al. (1962), but also by the syndrome observed in two herds of swine described below. In the summer of 1963, young swine in a herd near St. Paul, Minn. developed the symptoms listed above when fed a pelleted feed. A sample of this feed was obtained and fed to guinea pigs, all of which developed greatly enlarged uteri. It was then fed to virgin weanling white rats, and all of 1 Paper 5564, Scientific Journal Series, Minnesota Agricultural Experiment Station. them developed greatly enlarged uteri within 5 to 7 days. In January, 1964, this same syndrome appeared in a herd of swine near Buffalo, Minn. When feeding with the lot of corn on which they had been fed up to that time was discontinued, the tumefaction and mammary enlargement rapidly disappeared. The grain on which they had been fed when these symptoms developed consisted of approximately 3% corn that had been heavily invaded and decayed by fungi and 7% corn that appeared sound. According to the owner of the farm, that portion of the corn heavily invaded by fungi was of the 1962 crop, and had been stored on the cob in a crib, exposed to the weather, from harvest time in 1962 until late in 1963, when it was shelled, mixed with sound corn, and fed to the pigs. Several bushels of the mixture of sound and fungus-invaded corn fed to the herd of swine on the farm were purchased. A portion of this corn was fed to an immature gilt weighing approximately 1 lb (45.5 kg). After 1 days, the animal was killed and necropsied. During this 1-day period, the gilt developed enlarged mammae and a swollen, edematous vulva. The uterus was greatly enlarged (Fig. 1). Several hundred grams of kernels heavily invaded by fungi were picked from this corn, ground, and fed for 5 to 7 days to virgin weanling white rats; the rats were then killed and necropsied. Their uteri also were grossly enlarged. 653

2 654 CHRISTENSEN, NELSON, AND MIROCHA APPL. MICROBIOL. TIJ '--.::.... :... FIG. 1. Uterus, greatly enlarged, from 1-lb gilt fed for 1 days on moldy corn from farm where the estrogenic syndrome developed in swine herd fed on same corn. These two herds were fairly typical of a number encountered. Because of the economic importance of the disease and the need for more information on this and other mycotoxicoses, the investigations here reported were undertaken. MATERIALS AND METHODS Isolation of fungi from feeds and grains. Pellets of prepared feed were scattered on a variety of agar media in petri dishes, and were incubated at 12 to 3 C until the fungi that grew out could be identified and transferred. Dilution cultures were also made, to determine the number of viable spores per gram of feed. Seeds of corn from various farms were cultured without surface disinfection and with surface disinfection in 2% sodium hypochlorite for 1 min followed by a sterile water rinse. Since the embryos of many seeds were more heavily invaded and decayed by fungi than the endosperm, and an individual embryo might harbor several fungi, many kernels after surface disinfection were split and placed on agar with the cut surface of the embryo upward, so that fragments of mycelium could be transferred as soon as they became visible. Fungi were transferred to plates of acid-potato-dextrose-agar (APDA); as soon as their purity was assured, they were transferred to autoclaved moist soil in perfume bottles. After a few days at room temperature to allow mycelium to grow, the bottles were stored at 5 C to serve as inoculum. When a small portion of this soil later was scattered sparsely on agar, mycelium of the fungus with which it had been inoculated grew from almost every particle, and a small amount of such inoculated soil introduced into autoclaved corn in 1-gal jugs or 5-gal paint cans served to inoculate the corn thoroughly. Production of test lots of feed invaded by fungi. Test lots were produced by several methods, including cultures in Czapek medium in 1-liter Florence flasks incubated on a shaker, and cultures in autoclaved corn meal, in autoclaved corn meal plus corn oil, and in moist whole autoclaved- corn kernels. As a result of preliminary feeding tests, and because our major interest was in production of possibly estrogenically active feed under conditions that might prevail on farms, it was decided to use whole corn. Sound corn was put in 1-gal glass jugs, or in 5-gal metal paint cans, and water added to bring the moisture content of the grain to ca. 45%; the mixtures were autoclaved for 1 hr on each of 2 successive days, cooled, and inoculated with cultures of the fungi growing in soil, as described above. Various incubation times and temperatures were tested, but incubation at 2 to 25 C for 3 weeks, followed by incubation at 12 C for 2 weeks, was

3 ATOL. 13, 1965 FUSARIUM-PRODUCED SUBSTANCE IN RATS 655 TABLE 1. Fungi which produced no enlargement of uteri when grown in autoclaved moist corn for 3 weeks at 2 to 25 C and 2 weeks at 12 C, andfedfor 7 days to virgin white rats 21 days old at start of test No. of isolates Fungus Source tested Aspergillus glaucus.pelleted feed 8 A. candidus... Pelleted feed 9 A. ochraceus... Pelleted feed 5 A. flavus... Pelleted feed 7* Botryotrichum... Straw 1 Fusarium... Moldy corn 28 Memnoniella... American Type 1 Culture Collection Microascus... Sound corn 1 Mucor Pelleted feed 1 Nigrospora Moldy corn 1 Papulospora... Moldy corn 12 Sordaria... MMoldy corn 1 Total 75 * Isolates of A. flavus from pelleted feed have caused death of several rats to which they were fed, and the symptoms suggest that a toxin other than the described aflatoxins may be involved. adopted more or less as a standard. The containers of inoculated corn were shaken every few days to keep the kernels from matting together. At the end of the incubation period, the corn, heavily overgrown by fungus mycelium, was poured into a pan,. dried overnight at 45 C with a fan blowing over it to aid the drying, and then ground in a hand mill. Feeding tests. Pairs of virgin female weanling white rats 21 days old were fed ad libitum for 5 to 21 days (most tests were for 7 days), and then killed; the uteri were removed and weighed immediately, and samples were taken for later study of hematological and histopathological changes. Isolation and characterization of uterotropic compounds. The following solvents were tested: petroleum ether (bp, 6 to 7 C or 3 to 6 C), acetone, methylene chloride, chloroform, methanolchloroform (1:2), methyl ethyl ketone, methanol, diethyl ether, ethyl alcohol-water, isopropyl alcohol, n-butyl alcohol, and methyl cellosolve. Lots of 5 g of corn that had been inoculated with pure cultures of fungi and which in feeding iests had produced an increase in weight of rat uteri were dried, finely ground, and extracted for 24 hr in large flasks or for 12 hr in a Soxhlet extractor. After preliminary trials, most of the extractions were made with methylene chloride because of its low boiling point and the solubility of the toxin in it. After extraction, the solvent was concentrated in a flash evaporator to ca. 5 ml, applied to 1 g of clean ground carrier corn, dried under an infrared lamp to remove the solvent, and fed to virgin weanling rats. The control consisted of corn to which an equal amount of solvent had been applied 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 inoculated with Fusarium No. 5.

4 6- CHRISTENSEN, NELSON, AND MIROCHA APPL. MICROBIOL. TABLE 2. Weight of uteri of virgin weanling white rats, 21 days old at start of test, after feeding for 7 days on corn inoculated with various fungi* Inoculum Wt of uteri of two rats Fusarium # , 244 Fusarium # , 233 Fusarium # , 235 Fusarium WP , 22 Fusarium WP , 253 Fusarium YN , 28 Fusarium YN , 238 Fusarium YN , 216 Fusarium YN , 51 Fusarium YN , 32 Fusarium YN , 4 Ex poinsettia , 158 Nigrospora... 33, 38 Microascus... 48, 52 * Controls: the average weight of uteri of seven rats was 4 ; the uteri of three controls were abnormally heavy for immature rats, and if these are excluded the average weight of uteri was 29.6, with a range of 22 to 36 and a SD of 45. TABLE 3. Weight of uteri of virgin white rats, 21 days old at start of test, after feeding for 7 days on corn inoculated with Fusarium No. 5 and incubated for 2 to 12 weeks at 25 to 28 C Weeks of incubation Wt of uteri of two rats 2 42, , , , , , 46 Control* 26, 32 * Not inoculated. fied method of Davidoff and Korn (1963). Best results were obtained by use of a column with a final bed volume of 5 by 36 cm. The toxic material was eluted off the column with chloroform and separated from various pigments. The compound was further purified on a column of silicic acid (2 by 5 cm) with chloroform containing 1% methanol as eluant. The preparation was further resolved by separation on thin-layer chromatograms. Silica gel G (Merck & Co., Inc., Rahway, N. J.) was used as adsorbent in thin-layer chromatography and was prepared in the usual manner. The solvent mixtures found most useful, of several tested, were petroleum ether (bp, 6 to 7 C)-methanol (9:1, v/v) and chloroform-methanol (25:1, v/v). The spots were made visible with ultraviolet light or by spraying with a 5% solution of H2SO4 in methanol. RESULTS Fungi isolated from pelleted feed and from corn. Storage fungi, principally Aspergillus repens, A. candidus, A. ochraceus, and A. flavus, comprised the major fungus flora of the pelleted feed. This feed was compounded of 14 ingredients, of which several grains and seeds made up a major portion, and it is quite possible that some of the fungi present in the original grains no longer survived. Many genera of fungi were isolated from samples of moldy corn collected from farms in Minnesota, South Dakota, and Iowa, but Fusarium and Papulospora were the principal ones. Increased weight of uteri of white rats fed corn inoculated with fungi isolated from pelleted feed and from corn. Table 1 lists the fungi which, when incubated on autoclaved corn and fed to white rats, 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 sound corn were induced by 12 of 4 isolates of Fusarium; typical data are summarized in Table 2. Weight of uteri is an accepted measure of estrogenic activity (Dorfman, 1962). The isolates of Fusarium which resulted in increased weight of TABLE 4. Weight of uteri of virgin white rats, 21 days old at start of tests, after feeding for 7 days on corn inoculated with Fusarium No. 6 and incubated for 5 weeks at various temperatures Incubation Wt of uteri of two rats 2 to 3 C, 5weeks... 17, 3 Alternate days at 2 to 3 C and 25 to 28 C... 22, 35 3 weeks at 25 C,2 weeks at 2 to 3 C.. 54, 55 7 to 8 C, 5weeks... 29, 29 Alternate days at 7 to 8 C and 25 to 28 C... 28, 34 3 weeks at 25 to 28 C, 2 weeks at 7 to 8 C... 34, C, 5 weeks... 25, 35 Alternate days at 14 C and 25 to 28 C... 95, weeks at 25 to 28 C, 2 weeks at 14 C 123, C, 5 weeks... 34, 93 Alternate days at 2 C and 25 to 28 C... 37, 47 3 weeks at 25 to28 C, 2 weeks at 2 C 57, 129 Not inoculated, 5 weeks at 25 to 29 C... 28* * One control rat was found dead after 48 hr, and the necropsy revealed no visible lesions.

5 VOL^. 13, 1965 FUSARIUM-PRODUCED SUBSTANCE IN RATS 657 TABLE 5. Weight of uteri of virgin weanling white rats fed materials extracted by different solvents from corn inoculated with Fusarium No. 5 Sample Wt of uteri of two rats Petroleumether, bp 6 to 7C 52, 43 Petroleum ether, bp 3 to 6 C... 22, 54 Acetone... 81, 69 Methylene chloride , 13 Chloroform... 91, 154 Methanol-chloroform (1:2)... 12, 188 Methylethyl ketone... 91, 1 Diethylether... 54, 37 Methanol... 74, 75 Ethyl alcohol... 8, 25 Isopropanol... 41, 15 n-butanol... 17, 172 Methyl cellosolve , 47 Water... 27, 47 Control corn (uninoculated)... 44, 26 Carrier corn... 26, 36 TABLE 6. Weight of uteri of virgin weanling white rats fed materials extracted by methylene chloride from corn inoculated with Fusarium No. 5 after the extract was separated into fractions by means of a silicic acid column Wt of Fraction Eluted with uterus 13-1 CHC1, 1, ml CHCla, 1,5 ml CHC13, 5 ml CHCl,, 5 ml CHCls, 5 ml CH,OH, 3 ml 29 Saturated with NaCl 13-7 Control corn 33 uteri were from three sanples of corn, one from the farm in Minnesota mentioned above, where the estrogenic syndrome appeared in the herd of swine fed with this corn, one from a crib of corn near Yankton, S.D. (the crib contained a fair amount of corn similar in condition to the sample taken), and one from an undesignated location in Iowa. Evidently strains of Fusarium that produce estrogens are rather common and widely distributed. All or most of the isolates of Fusarium tentatively appear to be F. culmorum or F. graminearum, both of which may be part of the Gibberella zeae complex; in preliminaxy studies, there have been no obvious cultural differences between isolates that produced estrogenic compounds and those which did not. Effect of time and temperature of incubation upon production of estrogenic compounds as evaluated by increase in weight of uteri of white rats. Moist autoclaved corn inoculated with Fusarium No. 5, an isolate which consistently produced marked increase in weight of uteri in white rats, was incubated at various temperatures and for different lengths of time, and was then dried, ground, and fed for 7 days to paired virgin weanling female white rats, after which the rats were killed and the uteri were removed and weighed. The results (Tables 3 and 4) suggest that time and temperature may greatly influence production of estrogenic compounds, even by a strain of the fungus known to be a rather potent producer of these compounds when incubated under suitable conditions. Forgacs and Caril (1962) cited extensive evidence from work in the USSR that alimentary toxic aleukia in man results from consumption of grain (proso millet) invaded by F. sporotrichioides during weather of frequent alternating freezes and thaws after a mild winter. The fact that the majority of isolates of Fusarium, and all but two of the other fungi, tested by us produced no obvious toxicosis in the rats to which they were fed does not mean that these fungi might not be harmful if incubated and tested under other cqnditions '" %F_ Nw WA%1Il WAVr.Lr.1%V1 F Nl OD - N in).t4tw (-,... tmy..j FIG. 3. Comparison of absorption spectra of ergosterol and F-i isolated from corn invaded with Fusarium No. 5. The absorption was measured in ethyl alcohol on a Beckman DB spectrophotometer. _ ERGO STEF OL

6 658 CHRISTENSEN, NELSON, AND MIROCHA APPL. MICROBIOL. w z 49 D ) I'.1 1 o it OD NM N N,\ WAVELENGTH rij FIG. 4. Absorption spectrum of purified F-2 in n- butanol. This compound was isolated from corn invaded by Fusarium No. 5 as well as from feeds that caused the estrogenic syndrome in swine. Isolation of a compound causing increased weight of uteri of white rats. Table 5 gives the weight of uteri of rats fed the extracts obtained with different solvents, and Table 6 gives the weight of uteri of rats fed the fractions separated by means of the silicic acid column. Fraction 13-2 (Table 6) was the only one to induce a large increase in the weight of uteri of rats to which it was fed. These fractions were resolved into their components on thin-layer chromatoplates and were compared with comparable extracts from sound corn. The extracts from corn inoculated with Fusarium No. 5, which resulted in increased weight of uteri of rats to which it was fed, differed from the extract from sound corn in two compounds, one (designated F-1) having an RF value of.5 and the other (designated F-2) having an RF value of.12 in petroleum ether-methanol and.7 in chloroform-methanol solvent system. Compound F-1, after further purification by thinlayer chromatography, was identified as ergosterol. The comparison of spectra between an authentic sample of ergosterol (Nutritional BiochemicaJs Corp., Cleveland, Ohio) and F-1 is shown in Fig. 3. Further tests confirming the identity of F-1 were performed. A positive test resulted when F-1 was allowed to react with an alcoholic solution of digitonin. Digitonin forms a precipitate with crgosterol. The Rosenheim (1929) color test, specific for ergosterol, was also positive. Both ergosterol and F-1 had identical RF values when tested on thin-layer chromatograms with activated silica gel as adsorbent and chloroform as solvent. Both compounds showed identical fluorescent characteristics when viewed under ultraviolet light. Compound F-2 was purified by column chromatography followed by thin-layer chromatography. Its purity was checked by charring with H2SO4 after resolution on thin-layer chromatoplates in two different solvent systems. Only a single spot was found. Similar preparations were made, and compound F-2 was eluted and collected in a test tube. The absorption spectrum of F-2 in n- butanol is shown in Fig. 4. Its ultraviolet-absorption maxima corresponded to 314, 274, and 236 ma. The purified preparation of F-2 was dried under nitrogen, taken up in propylene glycol, and in jected into weanling rats. Two control and two F-2 treated rats were used. After 6 days, the weight of the uterus of the control rats averaged 53 (fresh weight) and that of the F-2 treated rats was 128. A significant response in size and weight of the uterus occurred in the F-2 treated rats (Fig. 2). Compound F-2 fluoresced bright blue when illuminated with ultraviolet light. It gave a positive test for a phenol when treated with FeCI3. When treated with base, F-2 showed a bathochromic shift to 256 m,u, and the weak band at 314 ma became broad and strong. The absorption bands at 236 and 276 m,u disappeared. This bathochromic shift reversed upon immediate acidification. Compound F-2 has an absorption spectrum identical to a uterotrophic compound isolated from corn infected with Giberella zeae, as reported by Stob et al (1962). LITERATURE CITED CHRISTENSEN, J. J., AND H. C. H. KERNKAMP Studies on the toxicity of blighted barley to swine. Minn. Univ. Agr. Expt. Sta. Tech. Bull DAVIDOFF, F., AND E. C. KORN Fatty acid and phospholipid composition of the cellular slime mold, Dictyostellum discoideum. J. Biol. Chem. 238: DORFMAN, R. D Mlethods in hormone research, vol. 2. Academic Press, Inc., New York. DOUNIN, M The fusariosis of cereal crops in European Russia in Phytopathology 16:

7 VOL FUSARIUM-PRODUCED SUBSTANCE IN RATS 659 FORGACS, J., AND W. T. CARILL Mycotoxicoses. Advan. Vet. Sci. 7: JOFFEE, A. J Toxicity of overwintered cereals. Plant Soil 18: JUIBB, K. V. F., AND P. C. KENNEDY Pathology of domestic animals, vol. 1. Academic Press, Inc., New York. ROSENHEIM, A specific color reaction for ergosterol. Biochem. J. 23:47. STOB, M., R. S. BALDWIN, J. TUITE, F. N. ANDREWS, AND K. G. GILLETTE Isolation of an anabolic, uterotrophic compound from corn infected with Gibberella zeae. Nature 196: 1318.