Binding characteristics of zearalenone analogs to estrogen receptors.

The estrogenic effect of zearalenone derivatives was investigated for their binding characteristics to cytosol and nuclear receptors in the uterus. Competition with 17beta-estradiol at the cytosol receptor sites was observed in four of the six derivatives tested, namely trans- and cis-zearalenone, zearalenol, and zearalanol. The other two, 8'-hydroxyzearalenone and 6'-aminozearalene, lacked the binding ability to receptors and were biologically inactive. trans-Zearalenone, like 17beta-estradiol, could elicit an immediate translocation of cytosol-receptor complexes into the uterine nuclei. However, it differs from either 17beta-estradiol or antiestrogens (tamoxifen) in three aspects: (a) a second wave of translocation occurred 6 to 12 hr following zearalenone injection; (b) there was a much longer nuclear retention (over 24 hr) than in the case of 17beta-estradiol; and (c) following a depletion of cytosol receptors, trans-zearalenone induced an overreplenishment by 24 hr, whereas tamoxifen is reported to suppress the replenishment.

Structure-activity relationships for human estrogenic activity in zearalenone mycotoxins.

Zearalenones are estrogenic Fusarium mycotoxins consisting of a resorcinol moiety fused to a 14-member macrocyclic lactone. Using an improved MCF7 human breast cell proliferation assay, we have compared the estrogenicity of 17 chromatographically-homogeneous zearalenones. Both similarities and substantial differences from published results in intact animal systems were observed. Substantial human estrogenicity was retained even in analogs lacking hydroxylation on the aromatic and macrocyclic rings.

Acute pathological effects on rats of orally administered wortmannin-containing preparations and purified wortmannin from Fusarium oxysporum.

The pathological effects in rats of orally administered wortmannin, a mycotoxin produced by Fusarium oxysporum, were studied. Weanling rats were fed a wortmannin-containing fungal culture for 4 or 5 days, or were given by intragastric gavage a single, lethal dose of extracts of a wortmannin-containing fungal culture or pure toxin. Haemoglobinuria, necrosis of lymphoid tissues and death occurred in rats fed the fungal culture. Administration by gavage of extracts of the wortmannin-containing culture and of purified wortmannin produced gastric and myocardial haemorrhage. Major microscopic lesions in gavaged rats were haemorrhage in the myocardium and gastric submucosa and necrosis of lymphocytes in the thymus, spleen and gut-associated lymphoid tissue. Necrosis of gastro-intestinal epithelium was not observed. Myocardial haemorrhage was severe, often transmural, and is an unusual lesion that may aid in the diagnosis of wortmannin toxicosis.

Biosynthesis of fumonisin and aal derivatives by Alternaria and Fusarium in laboratory culture.

Cultures of Fusarium moniliforme and Alternaria alternata f. sp. lycopersici were grown in the laboratory and analyzed for various fumonisin derivatives. Analyses were made by continuous flow fast atom bombardment and ionspray mass spectrometry interfaced to microcapillary HPLC. Besides FB1, FB2 and FB3 derivatives, two isomers of the one-armed FB1 (protonated molecular ion at m/z 564) and two isomers of the one-armed FB2 (m/z 548) were found. Two different isolates of A. alternata when grown in culture yielded FB1, FB2 and FB3. One of them also yielded the one-armed FB1 which was identical to that metabolite found in Fusarium and naturally infected corn. FB1, FB2 and FB3 have been found in 2 different isolates of Alternaria alternata, both obtained from tomato.

Diacetoxyscirpenol toxicity in pigs.

Purified diacetoxyscirpenol (DAS) mycotoxin fed to growing pigs at 0, 2, 4, 8, and 9 ppm for as long as nine weeks caused several lesions at all levels. The pigs developed multifocal, proliferative, gingival, buccal and lingual lesions. The small intestine had both glandular and mucosal cell hyperplasia. No other lesions were seen at necropsy or in any of the 19 tissues examined microscopically using haematoxylin and eosin stain. A one-way analysis of variance revealed statistically significant (P less than 0.05) decreased ration consumption and weight gain at all levels of DAS. Total ration refusal occurred at 10 ppm of DAS. There was no effect on the packed cell volume, haemoglobin concentration, total red blood cell count, total white blood cell count, 300 cell differential, terminal bone marrow smears or on the serum levels of aspartate transaminase, serum glutamic pyruvic transaminase and lactic dehydrogenase at any level of DAS. Based on the gross and microscopic lesions, decreased ration consumption and decreased weight gain the no-effect level was less than 2 ppm of DAS in the ration.

Quality Parameters in Small Grains from Minnesota Affected by Fusarium Head Blight.

Recent epidemics of Fusarium head blight (FHB) severely damaged the hard red spring wheat and barley crops in Minnesota. Samples of commercial grain were analyzed in 1993 and 1994 to determine the effects of FHB on several quality parameters. Wheat test weight (TW) averaged 832 kg m-3 (55.4 lb/bu), thousand kernel weight (TKW) averaged 27.4 g, and the proportion of visually scabby kernels (VSK) averaged 11.0%. Deoxynivalenol (DON) was detected in 493 of 500 samples (98.6%). The mean concentration was 8.3 µg/g (range = 0.0 to 44.7 µg/g). Scab in wheat could rapidly be estimated using easy-to-prepare visual comparison standards. Scores of percent VSK were correlated with DON concentration at r = 0.897 and 0.908 in 1993 and 1994, respectively. TW and TKW were less effective estimators of DON (r = -0.622 and -0.550, respectively). DON was detected in 100 of 100 six-row barley samples collected during the survey and averaged 10.4 µg/g (range = 0.5 to 39.7 µg/g). DON concentration in barley could not be effectively estimated with grading parameters including TW, TKW, percent plump kernels, or a visual index of kernel discoloration. In 28 samples of oats, DON averaged 1.4 µg/g (range = 0.0 to 6.4 µg/g). Nivalenol was not detected in any of the 628 samples analyzed during the two-year study.

Biosynthesis of Deoxynivalenol in Spikelets of Barley Inoculated with Macroconidia of Fusarium graminearum.

This research examined the biosynthesis of deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-ADON) in barley spikelets inoculated with macroconidia of Fusarium graminearum (Group-II). Investigations were conducted to determine if these toxins were present in macroconidia of the pathogen prior to inoculating barley spikelets. Extracts of macroconidia cultured from mung bean agar were analyzed using gas chromatography-mass spectrometry. Neither DON or 15-ADON was detected in three isolates' macroconidia when compared with macroconidia-DON-matrix standards adjusted to 100, 200, 300, and 400 ng/g with a detection limit of 100 ng/g. Mean recovery of DON that was added to macroconidia was 89.5%. The same isolates were pathogenic on barley cultivars Robust (moderately susceptible) and Chevron (moderately resistant) and produced DON (0 to 3.69 ng/g) and 15-ADON (detected but not quantified) when grown in rice culture. Greenhouse experiments were performed to determine when DON and 15-ADON were detectable after inoculation and to quantify their amount in inoculated barley spikelets. The three isolates of F. graminearum were separately inoculated to a central spikelet on heads of barley cultivars Robust and Chevron. Both toxins were detected in spikelets 48 h postinoculation (PI). DON increased dramatically after 72 h and did not diminish thereafter. Accumulation of 15-ADON peaked at 72 to 120 h and decreased by 240 h PI. There were no statistical differences between cultivars or among fungal isolates for accumulation of either toxin when averaged over the time intervals. Differences of toxin accumulation at each sampling interval were significant (P < 0.0001) when averaged over isolates and cultivars. Spikelets of six cultivars and lines were sampled at inoculation and 18, 36, 54, 72, and 90 h PI. DON and 15-ADON were detected at 36 h PI, but differences among the cultivars and lines were not significant. Yield of DON in inoculated spikelets of 31 barley cultivars and lines at 72 h PI ranged from 0.14 to 1.26 µg per spikelet, and differences among the cultivars and lines were significant (P < 0.002). The data demonstrate a useful range of variability for toxin accumulation in inoculated spikelets among germ plasm in the Minnesota breeding program. Macroconidia with no detectable DON or 15-ADON could be used for in vitro studies of toxin biosynthesis. Establishing when DON and 15-ADON are synthesized facilitates studying the effects of promising fungicides, biocontrol organisms, and new or novel genetic resistance mechanisms and if or how they may prevent or delay the biosynthesis of toxins.

Fusarium Head Blight of Cultivated and Natural Wild Rice (Zizania palustris) in Minnesota Caused by Fusarium graminearum and Associated Fusarium spp.

Symptoms of Fusarium head blight on diseased wild rice seed from both cultivated fields and natural stands are shrunken, light weight seeds discolored light tan to light brown with infrequent light pink due to mycelial growth of Fusarium spp. F. graminearum was the predominant species isolated from whole seed at all growth stages, and from shattered seed gathered from four fields in 1994 to 1995. F. anthophilum and F. subglutinans were also frequently isolated at most growth stages, whereas F. acuminatum, F. culmorum, F. solani, and F. semitectum were infrequently isolated at one or more growth stages, and F. camptoceras was isolated only from shattered seed. F. graminearum was the only species isolated from processed seed, although rarely. Fusarium spp. were isolated at the highest percentage from shattered seed. The highest percentage of total Fusarium spp. isolated during seed development was at the milk and dough stages in a cultivated field and at the milk stage in a natural stand; the percentage then declined until the ripe stage in seed from both sites. There were no significant differences in the percentages of Fusarium spp. isolated at growth stages between seed from the cultivated field and from the natural stand. Fusarium spp. were isolated most frequently from whole seed grown in three cultivated fields, compared with the palea and lemma, and caryopsis. F. graminearum and F. anthophilum were frequently isolated from whole seed and all seed structures, whereas F. culmorum and F. sporotrichioides were isolated only from whole seed, F. moniliforme from whole seed, palea and lemma, and caryopsis, and F. subglutinans from whole seed and the palea and lemma only. Deoxynivalenol and nivalenol were identified in three isolates of F. graminearum; however, none of the seed samples from which the isolates were obtained yielded either mycotoxin. Survival of Fusarium spp. in diseased seed was similar from both cultivated and natural sources and occurs in whole seed not immersed in water but not in seed immersed in water.Survival was better in seed stored at 4°C than at -20°C. F. graminearum was reisolated from 81% of seed from inoculated plants but not from seed of noninoculated plants. Pathogenicity of other Fusarium spp. remains to be demonstrated.

Effects of the mycotoxin zearalenone on swine reproduction.

Concentrations of 25, 50, or 100 ppm of 95% purified zearalenone fed to groups of healthy, multiparous sows during preestrus or throughout the gestation period (or both) produced multiple reproductive deficiencies. These reproductive disorders included infertility, constant estrus, pseudopregnancy, diminished fertility, reduced litter size, smaller offspring, malformation, juvenile hyperestrogenism, and probably fetal resorption. Gross and histologic examinations of sows revealed lesions in the reproductive organs. Marked epithelial changes characterized by squamous metaplasia were noticed in the uterus, uterine duct, cervix, vagina, and mammary glands.

Effect of zearalenone on dairy cows.

Doses of 99% pure zearalenone (0.0, 31.25, 62.5, 125.0, 250.0, or 500.0 mg) in gelatin capsules were given once a day per os to 18 nonpregnant, nonlactating, multiparous dairy cows for 2 consecutive estrous cycles. There was no effect (P less than 0.10) on serum progesterone concentrations, RBC, WBC, PCV, hemoglobin, and estrous cycle length. Differential cell counts, clinical health, and sexual behavior were not affected by the zearalenone. One cow from each of the groups given zearalenone and a control were euthanatized at the end of the study. The zearalenone had no effect on the terminal bone marrow smears and did not induce any gross lesions discernible at necropsy or any microscopic lesions in representative samples of 30 tissues/cow. Rectal palpation of the reproductive tracts once a week indicated that the corpora lutea were small in cows given zearalenone. There was a general trend to increased hemoglobin concentrations in cows given the larger doses of zearalenone. Zearalenone of and by itself does not seem to be an important factor in dairy cow health.

Effect of zearalenone on the fertility of virgin dairy heifers.

Eighteen cycling, virgin, Holstein heifers daily were given 250 mg of 99% purified zearalenone in a gelatin capsule orally, and 18 controls were given an empty gelatin capsule once a day. The study lasted through 1 non-breeding estrous cycle and the next 2 consecutive estrous cycles during which the 36 heifers were bred, using artificial insemination. Serum concentrations of progesterone and complete blood cell counts were determined throughout the study. The treated and control heifers had conception rates of 62% and 87%, respectively. There was no effect (P less than 0.05) on the serum concentration of progesterone or the complete blood cell counts. Three heifers, bred but not pregnant by the end of the study, were euthanatized and necropsied. The treated heifer did not have any zearalenone-attributable lesions, and there was no effect seen in the bone marrow smears. The remaining 33 heifers were sold as a herd, and the 31 pregnant heifers calved normally. There was no effect (P less than 0.05) on the sex ratio of the offspring, which were all clinically healthy. Zearalenone did lower the conception rate of the treated heifers (P less than 0.065).

In vitro stimulation of bovine peripheral blood lymphocytes: suppression of phytomitogen and specific antigen lymphocyte responses by aflatoxin.

The effects of aflatoxin B1 on responses of the peripheral blood lymphocytes (PBL) of 2 normal animals to phytohemagglutinin, concanavalin-A, and pokeweed mitogen and of the PBL of 2 Mycobacterium bovis-infected animals to phytohemagglutinin and purified protein derivative of M bovis (PPD) were studied. Aflatoxin concentrations of greater than or equal to 10 microgram/ml significantly suppressed the lymphocyte response of normal animals to the phytomitogens. Lymphocyte response of M bovis-infected animals to specific antigen PPD was significantly suppressed at aflatoxin concentration of 0.5 microgram/ml. Fifty- to 100-fold higher concentrations of aflatoxin were required to produce 50% suppression of lymphocyte response to phytomitogens, as compared with that produced to PPD.

Necrotic oral lesions in chickens fed diacetoxyscirpenol, T-2 toxin, and crotocin.

One-day-old broiler chicks were fed a diet containing either 5 ppm diacetoxyscirpenol (DAS), 5 ppm T-2 toxin, 10 ppm crotocin, or a control diet for 3 weeks. Chicks fed the diet containing DAS and T-2 toxin showed yellowish plaque-type lesions on the beak, tongue, and angle of the mouth. Crotocin did not cause such lesions nor any apparent clinical signs. Chicks fed DAS had the lowest weight gain followed by the T-2, crotocin, and control group in that order; however, chicks fed crotocin had the poorest feed conversion ratio.

Metabolism of high concentrations of dietary zearalenone by young male turkey poults.

Six male turkey poults (3 weeks of age) were fed a starter ration artificially contaminated with 800 mg zearalenone/kg for a 2-week period to examine zearalenone metabolism and residues in various tissues, excreta, and blood plasma. Zearalenone had no effect on either feed consumption or body weight gain. All the birds fed zearalenone frequently showed strutting behavior, displayed an increased size and coloration of caruncles and dewlaps, and had swollen vent tissue. None of these signs were seen among six control birds fed uncontaminated starter feed. Hormone analysis, however, revealed that the testosterone concentrations in blood plasma were the same in both controls and treated birds. Analysis after 14 days of feeding showed that most of the dietary zearalenone had been metabolized into alpha-zearalenol. Levels of zearalenone and alpha-zearalenol were: blood plasma 66 +/- 27 and 194 +/- 80 ng/ml, excreta 182 +/- 33 and 644 +/- 86 micrograms/g, lung 56 +/- 45 and 202 +/- 161 ng/g, heart 57 +/- 40 and 238 +/- 121 ng/g, kidney 122 +/- 25 and 477 +/- 53 ng/g, and liver 276 +/- 54 and 2715 +/- 590 ng/g, respectively. Only traces of beta-zearalenol could be detected in plasma, excreta, and the various tissues. The percentage alpha-zearalenol of total zearalenone plus alpha-zearalenol rose significantly in both blood plasma and excreta during the experimental period. Almost all zearalenone and alpha-zearalenol was found conjugated in blood plasma, and the conjugates consisted of both glucuronides and sulfate conjugates. Approximately 65% of all zearalenone and alpha-zearalenol in excreta was found to be conjugated.

Effects of fusarium cultures, T-2 toxin, and zearalenone on reproduction of turkey females.

Nicholas Large White turkey hens in egg production (10 per treatment) were individually fed cultures of Fusarium roseum 'Gibbosum' to provide 100 ppm zearalenone, Fusarium tricinctum at a level of .1% of the diet, Fusarium roseum Alaska at a level of 2% of the diet, 100 ppm purified zearalenone, and 5 ppm purified T-2 toxin for 8 weeks. The following 4 weeks the birds were fed a control diet. Hens were inseminated every 2 weeks with .05 ml of pooled semen from males fed a control diet. After 30 days of toxin feeding, hens were innoculated with a killed Newcastle disease virus preparation. Blood samples were obtained periodically. Egg fertility and titers to Newcastle disease virus were unaffected by treatment. Egg weight was reduced by F. roseum 'Gibbosum'. F. roseum 'Gibbosum' and F. tricinctum caused decreases in feed consumption, body weight, and egg production. Egg production was decreased by zearalenone and T-2 toxin. Hens fed F. roseum 'Gibbosum', F. tricinctum, and T-2 toxin exhibited mouth lesions that healed rapidly upon withdrawing toxic feed. Hatchability of fertile eggs was reduced by feeding F. roseum 'Gibbosum', F. tricinctum and F. roseum Alaska to 28, 78, and 49%, respectively, of control values by the end of the 8 week test period. Upon removal of toxic feed, hatchability rapidly returned to control levels. Embryo mortality occurred mainly in the first 10 days of incubation for F. roseum Alaska and the last 18 days for F. roseum 'Gibbosum' and F. tricinctum fed hens. It appears that mycotoxins other than zearalenone and T-2 toxin are responsible for reduced hatchability from feeding Fusarium cultures.

The effect of a purified water-soluble fraction of a Fusarium roseum 'Graminearum' culture on reproduction of white Leghorn females.

White Leghorn females (36 weeks old) in egg production were individually fed a purified water-soluble fraction residue remaining from the water extract and a crude culture of Fusarium roseum 'Graminearium'. Each fraction was fed at 3% of the diet for 6 weeks followed by 2 weeks with a control diet. Hens were inseminated weekly with .05 ml of pooled semen from males given control diets. The purified water-soluble fraction increased feed consumption during the test periods. During the posttest period, hens fed test diets consumed less feed than those fed a control diet. All test diets did not affect body weight change during the test periods. There was a significant increase in body weight of hens on the diet containing 3% crude culture and a marked decrease in body weight of hens fed the purified water-soluble fraction during the posttest period. Egg production and egg weight were not affected by treatments during the test and posttest periods. Fertility was reduced by the crude culture of F. roseum 'Graminearum' during the 6-week test. Hatchability of fertile eggs was significantly reduced by the purified water-soluble fraction and the crude culture of F. roseum 'Graminearum'. Hatchability rapidly increased when these toxic diets were replaced with control diets. The majority of embryo mortality occurred during 5 days of incubation. The major mycotoxins responsible for reduced hatchability of fertile eggs appeared to be water soluble components of F. roseum 'Graminearum' and not trichothecenes or zearalenone.

Effects on laying hens of feeding corn invaded by two species of Fusarium and pure T-2 mycotoxin.

Balanced rations containing 2.5 and 5.0% of corn invaded by Fusarium tricinctum, (with 8 and 16 p.p.m. of T-2 toxin) were fed to White Leghorm laying hens. The ration containing 5% of the fungus-invaded corn resulted in reduced feed intake and reduction in weight gain and in egg production. Rations containing 2.5 and 5.0% of corn invaded by F. roseum 'Gibbosum' (with 25 and 50 p.p.m., respectively, of monoacetoxyscirpenol) resulted in an abrupt decrease in feed intake to 10-20% of normal, subsequent loss in weight, and cessation of egg production. Purified T-2 toxin consumed at the rate of 16 p.p.m. in the ration resulted in loss of body weight and decreased egg production; lesser amounts of T-2 toxin resulted in lessened but still detectable injurious effects. Mouth lesions developed in the birds fed these rations, their severity being proportional to the amount of toxin present.

Effects of T-2 toxin on reproductive performance and health of laying hens.

Purified T-2 toxin was fed to S.C.W.L. hens at levels of 0 (control), 0.5, 1.0, 2.0, 4.0 and 8.0 p.p.m. of an otherwise balanced diet. Feed consumption, egg production and shell thickness were significantly (P less than 0.05) decreased in hens fed 8 p.p.m. as compared with control hens. The fertility and progeny performance were not depressed by feeding T-2 toxin, but the hatchability of fertile eggs of hens fed 2 and 8 p.p.m. was significantly (P less than 0.05) lower than that of hens fed the control diet. The weights of liver, heart, gizzard and spleen were not influenced by T-2 toxin. Serum levels of alkaline phosphatase, LDH and uric acid of hens fed high concentrations of T-2 toxin were greater than those of control hens. SGPT in hens fed 8.0 p.p.m. was lower when compared with control hens. No outward changes in hematocrit, hemoglobin, erythrocyte, leukocyte and differential leukocyte counts were noted with feeding T-2 toxin. Most hens fed T-2 toxin developed oral lesions: circumscribed proliferative yellow caseous plaques at the margin of the beak, mucosa of the hard palate and angle of the mouth, and tongue. The incidence and severity of lesions were proportional to the dietary level of T-2 toxin. The only other lesion observed in necropsy examination at the end of the experiment was the small mucosal ulcer in the anterior portion of the gizzard in hens fed high levels of T-2 toxin. Microscostrointestinal tract, etc.) revealed no significant pathological change except the necrotic lesions in the gizzard and crop.

The effect of a Fusarium roseum culture and diacetoxyscirpenol on reproduction of White Leghorn females.

White Leghorn females in egg production (36 weeks old) were fed a culture of Fusarium roseum containing 15 ppm diacetoxyscirpenol (DAS) and other unidentified toxins at culture levels of 0, 1, and 2% of the diet for 8 weeks. Following this, all hens were placed on control (0% toxins) feed for 6 weeks. Birds were inseminated weekly with .05 ml of pooled semen from males given normal diets. The F. roseum had no significant effect on body weight change or egg weights. During the initial 8 weeks, egg production was significantly depressed by both the 1 and 2% levels whereas feed consumption, fertility, and hatchability of fertile eggs was reduced only by the 2% level of F. roseum. Moreover, the majority of embryo mortality occurred prior to the 7th day of incubation. All production levels returned to normal when the toxins were removed during the final 6 weeks. In a second experiment, control (0%), .5 ppm purified DAS, and 3% F. roseum culture were fed to White Leghorn females (50 weeks old) for 4 weeks followed by a 2-week withdrawal period when all birds were given control diets. In 4 weeks, hatchability of fertile eggs was reduced 24% by DAS and 99% by the culture of F. roseum but returned to normal after the toxins were removed. Other production indices were unaffected by dietary treatment. The DAS appears to be only partially responsible for the reduced hatchability; the major toxicant has not been identified.

Effects of T-2 toxin on brain catecholamines and selected blood components in growing chickens.

Four-week-old male broiler chickens were intubated with a single dose of purified T-2 toxin at 2.5 mg/kg body weight. The brain concentrations of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) and selected blood components were determined in T-2 toxin treated and control chickens at 4, 12, 24, and 48 hr after the toxin treatment. The brain DA concentration of T-2 treated chickens was significantly greater at 12 and 24 hr, whereas brain NE was lower at 24 and 48 hr after toxin treatment as compared with controls. The brain 5-HT level was not altered by T-2 toxin. Serum cholesterol was increased at 4 and 12 hr after T-2 treatment. The serum LDH and GOT activities were not changed by T-2 toxin. T-2 treated chickens had greater packed cell volume and hemoglobin than controls at 24 hr after dosing. Red blood cell counts were not affected but white blood cell counts were decreased during the 12 to 48 hr period after T-2 treatment. The results of this study suggest that T-2 toxin influences brain catecholamines and blood components and thereby possibly brain function in chickens.