Biological and chemical characterization of metabolites of Fusarium moniliforme isolates.

Metabolites of fusarium moniliforme isolates from different types of corn were characterized biologically and chemically. The biological assays included rat feeding, rat dermal tests and inoculation of embryonated eggs. Thin-layer chromatography, gas liquid chromatography, and mass spectrophotometry were used to isolate and determine their chemical identities. The metabolites were identified as diacetoxyscripenol, ipomeanol, ipomeanine and diplodiatoxin. The biological tests revealed significant weight loss in rats fed the contaminated corn for 5 w. Hemorrhages and edema in the brain and intestine were detected in all the groups of rats.

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material.

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (1:1) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate amino-transferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/methanol extraction. Fumonisins B1 and B2 were extracted from the CM by water, but not chloroform/methanol, and were present in the toxic diets at concentrations of 93-139 and 82-147 ppm, respectively. Nontoxic diets contained less than or equal to 22 ppm fumonisin B1 and less than or equal to 65 ppm fumonisin B2.

Criteria for determining purity of Fusarium mycotoxins.

Physical and chemical properties that may be used to determine the purity of several Fusarium mycotoxins have been investigated. A combination of analytical procedures, which include high performance thin-layer chromatography (HPTLC), liquid chromatography (LC), gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), ultraviolet spectrometry (UV), and nuclear magnetic resonance (NMR) spectrometry have been used to examine mycotoxin standards obtained from commercial sources and from laboratory fermentations. Results of this investigation indicate that commercially available standards are greater than 90% pure, but the label weight of purchased reference standards in individual containers should be verified. Mycotoxin standards, determined to be greater than 98% pure by HPTLC, LC, and GC/MS, were examined by UV spectrometry and the coefficients of extinction were determined. An interlaboratory study, involving 5 collaborators who determined coefficients of extinction (in methanol) for identical samples, gave the following results: alpha-zearalenol (lambda 236 = 28 538 +/- 558); beta-zearalenol (lambda 238 = 24 963 +/- 747); deoxynivalenol (lambda 219 = 6395 +/- 349, lot 1), (6020 +/- 228, lot 2); and T-2 toxin (lambda 202 = 3681 +/- 255). UV maxima and coefficients of extinction are also reported for HT-2 toxin (lambda 202 = 1959), diacetoxyscirpenol (lambda 203 = 2487), neosolaniol (lambda 203 = 2644), nivalenol (lambda 220 = 5142), and fusarenon-X (lambda 217 = 5997).

Monoclonal antibody-based enzyme linked immunosorbent assay of aflatoxin B1, T-2 toxin, and ochratoxin A in barley.

Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1, 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile-0.5% KCl-6% H2SO4 (89 + 10 + 1) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonitrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCl buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1, 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were less than 12% for B1 and OA but as high as 17% for T2.

A survey on the occurrence of Fusarium mycotoxins in Bavarian cereals from the 1987 harvest.

Bavarian cereals and wheat flour from the 1987 harvest were analysed for nivalenol (NIV) and deoxynivalenol (DON) using high performance liquid chromatography (HPLC) and for T-2 toxin and zearalenone (ZEA) by enzyme-linked-immunosorbent assay (ELISA). The study included 190 field samples of wheat, barley, rye and oat with visibly damaged ears, 45 samples of wheat intended for feed production and two series of wheat flour (type 550) and whole wheat flour collected in October 1987 and June 1988. The field samples examined showed a high DON contamination of wheat (87%) with an average of 3.96 mg/kg and a maximum of 43.8 mg/kg. Mean levels between 0.33 mg/kg and 0.27 mg/kg DON could be detected in barley, rye and oat. Of the wheat samples, 58% contained ZEA with a maximum of 1.560 mg/kg. The highest levels of ZEA were detected in samples which also showed high concentrations of DON. The NIV and T-2 toxin levels were comparatively low. Thirty percent of the samples showed NIV concentrations between 0.04 mg/kg and 0.29 mg/kg and 38% contained between 0.005 and 0.60 mg/kg of T-2. In the wheat samples for feed production, only DON was detected with an average of 0.190 mg/kg and a maximum of 0.75 mg/kg. The highest DON levels (0.58 mg/kg) from October 1987 were found in the wheat flour samples which were lower than the highest DON concentration (3.24 mg/kg) detected in the samples collected during June 1988. This fact was probably due to a substantial amount of non-contaminated wheat from 1986. The toxin concentrations in the whole wheat flour were not higher than in the type 550 flour.(ABSTRACT TRUNCATED AT 250 WORDS)

[Study on toxicological mechanism of moniliformin].

A mycotoxin was extracted and purified from a strain of Fusarium moniliforme var. subglutinans isolated from mouldy corn seeds harvested in a serious Keshan disease region in Shaanxi Province. The purification procedure involves water extraction, ion exchange chromatography, desalination and crystallization. Its UV spectrum, IR spectrum and NMR are identical with that of the moniliformin. The toxin is highly toxic to young Beijing ducklings. The electrocardiogram (ECG) of the duckling was changed immediately after feeding with moniliformin. The cardiac muscle cells of Beijing ducklings were injured by the toxin. Toxicity may be alleviated to certain extent by applying adequate dosage of Selenium (3.18 X 10(-10) mol/L of H2SeO4) prior to moniliformin treatment. The permeability of the cardic muscle cells of the rats and young ducklings was damaged posterior to the injection of the toxin as tested by extracellular marcromolecular tracer (HRP) method. The relationship between moniliformin and Keshan disease was discussed. We consider that the moniliformin may be the causal agent of the disease.

Ultrastructural study of galacturonic acid distribution in some pathogenic fungi using gold-complexed Aplysia depilans gonad lectin.

Aplysia gonad lectin, isolated from the mollusc Aplysia depilans, was successfully conjugated to colloidal gold and used for ultrastructural detection of galacturonic acids in some pathogenic fungi. These sugar residues were found to occur in the fibrillar sheath surrounding hyphal cells of Ascocalyx abietina and in intravacuolar dense inclusions of this fungus spores. In hyphae and spores of Ophiostoma ulmi, galacturonic acids were detected mainly in the outermost wall layers. In contrast, these saccharides appeared associated with the innermost wall layers and especially the plasma membrane of Verticillium albo-atrum cells. Galacturonic acids were found to be absent in cells of Fusarium oxysporum f.sp. radicis-lycopersici and Candida albicans. These cytochemical data indicate therefore that a heterogeneity in wall composition exists between ascomycete fungi. The significance of the presence of galacturonic acids in the cell walls of certain fungi is still open to question.

Hepatotoxicity and renal toxicity in rats of corn samples associated with field cases of equine leukoencephalomalacia.

Currently there is no convenient bioassay to determine the potential toxicity of corn naturally contaminated with Fusarium moniliforme. A short-term bioassay would be useful for future investigations aimed at isolating as yet unidentified toxins produced by this fungus. Two groups of five male Sprague-Dawley rats were each fed one of two F. moniliforme contaminated corn samples, designated CS-1 and CS-2, that were associated with separate field cases of equine leukoencephalomalacia (ELEM). A control group, also consisting of five male rats, was fed uncontaminated seed corn. All animals survived to the end of the study and there were no apparent differences in appearance or behaviour among groups. Weight loss and irregular food consumption occurred in all groups and probably resulted from nutritional deficiencies inherent in the corn diets. Hepatocellular degeneration, necrosis and hyperplasia as well as biliary hyperplasia were found in the test groups only and were attributed to F. moniliforme. Serum transaminase and alkaline phosphatase activities in animals fed CS-1 and CS-2 for 4 wk were significantly increased compared with the controls, while serum bilirubin concentration was increased only in the CS-1 group. Tubular nephrosis was also present in the renal cortex of all animals fed CS-1 and CS-2. These effects may have been related to fumonisins B1 and B2, recently discovered metabolites of F. moniliforme, that were found in both CS-1 and CS-2. Short-term studies of this type may be useful in screening naturally-contaminated grains and other materials for hepatotoxic metabolites produced by F. moniliforme.

Isolation and characterization of trichothecin from corn cultures of Fusarium graminearum MRC 1125.

Trichothecin was isolated and purified from corn cultures of a toxic strain of Fusarium graminearum. This strain, designated MRC 1125, was obtained from corn in southern Africa. The brine shrimp toxicity assay was used throughout the isolation procedure to monitor the toxicity of the fractions. The compound was characterized by detailed 1H (500-MHz) and 13C (125-MHz) nuclear magnetic resonance spectroscopy and mass spectrometry. This is the first report of the production of trichothecin by a Fusarium species.

[Hyperestrogenism in swine due to natural poisoning with zearalenone]. / Hiperestrogenismo en cerdas por intoxicación natural con zearalenona.

A field case is described in which all prepuberal swine of a group of 20 pigs and 11 sows showed marked estrogenic effects. These consisted of enlarged mammary glands, swelled tumefacient vulva, and greatly enlarged internal reproductive organs. The corn used to feed these animals was found to contain 56 ppm zearalenone. Deoxynivalenol (4.9 ppm) was found in the corn; T-2 toxin, nivalenol, fusarenon-X, diacetoxyscirpenol, aflatoxins and ochratoxins were absent. Identity of Z was confirmed by TLC in four solvent systems, behavior of the suspected spots under UV light of different wavelengths, change of fluorescence from green to blue after spraying with 5% AlCl3 in alcohol and heating at 110 degrees C during 5 minutes, and by its UV spectrum. A zearalenone producing strain of Fusarium oxysporum was isolated from the suspected grain. Histopathology of uterine tissue showed typical changes produced by zearalenone: hyperplasia, hypertrophy, and metaplasia of the myometrium. Feeding of the grain to a prepuberal sow under controlled conditions reproduced all the effect found in the farm animals. This is the first field case of zearalenone poisoning reported in Argentina.

Fumonisins--novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme.

Cultures on corn of Fusarium moniliforme MRC 826 are known to cause leukoencephalomalacia in horses and to be toxic and hepatocarcinogenic in rats. Culture material of this F. moniliforme isolate has also been shown to exhibit cancer-promoting activity in a short-term cancer initiation-promotion bioassay with diethylnitrosamine-initiated rats and the induction of gamma-glutamyl-transpeptidase-positive (GGT+) foci as an endpoint after 4 weeks of promotion. This bioassay was used as a monitoring system to isolate cancer-promoting compounds from cultures of F. moniliforme MRC 826. Culture material was successively extracted with ethyl acetate and CH3OH-H2O (3:1). Most of the cancer-promoting activity was recovered in the CH3OH-H2O extract and remained in the aqueous phase following partitioning of this extract between CH3OH-H2O (1:3) and CHCl3. The CH3OH-H2O fraction was chromatographed on an Amberlite XAD-2 column, and the active fraction was eluted with CH3OH. This fraction was chromatographed on a silica gel column with CHCl3-CH3OH-CH3COOH (6:3:1) as eluent and further purified on a C18 reverse-phase column. Two pure compounds were isolated, and these have been chemically characterized and given the trivial names fumonisin B1 and B2. At least 2 g of the major compound fumonisin B1 was purified from 1 kg of culture material. Fumonisin B1 in the diet (0.1%) significantly (P less than 0.001) induced the formation of GGT+ foci in the livers of initiated as well as noninitiated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and purification of a hemorrhagic factor (wortmannin) from Fusarium oxysporum (N17B).

An isolate of Fusarium oxysporum Schlecht, emend. Synd. et Hans. N17B isolated from a grassy area in Lakselv, Norway (Arctic region) produced a toxin in culture when grown on rice in the laboratory. This new toxin, which was given the trivial name of H-1 (indicating hemorrhagic factor), caused toxic effects in rats, including food refusal, weight loss, hemorrhage in the stomach, intestines, heart, and thymus, and finally death. The UV spectrum of H-1 showed 210, 254, and 292 nm as absorption maxima. The infrared spectrum showed carbonyl groups at 1,675 and 1,750 cm-1 and an ether group at 1,215 cm-1. H-1 does not fluoresce under short- or long-wavelength UV light and exists as fluffy, white crystals that turn yellow when subjected to basic reagents such as ammonium hydroxide or tetraethylenepentamine. Elemental and accurate mass determinations in both electron impact and positive chemical ionization indicate an empirical formula of C23H24O8. Its mass spectra (electron impact, chemical ionization, and fast atom bombardment [FAB]) show a molecular ion of 428 and major fragments at m/z+ 386, 368, 355, and 295. H-1 was found to be identical to the antibiotic called wortmannin which is produced by Penicillium wortmannii and Myrothecium roridum. This is the first report of the synthesis of wortmannin by species of the genus Fusarium.

Production of moniliformin by Canadian isolates of Fusarium.

Twenty-eight Canadian isolates of Fusarium were tested for their ability to produce moniliformin in corn. Both F. moniliforme (2/6 isolates) and F. subglutinans (11/15 isolates) produced the mycotoxin, while F. graminearum did not. Field-corn inoculated with F. moniliforme M3783 was able to support production of both moniliformin and fusarin C.

N alpha-dimethylcoprogens. Three novel trihydroxamate siderophores from pathogenic fungi.

Three novel siderophores have been isolated from a highly pathogenic strain of Alternaria longipes (ATCC 26293). The compounds are N alpha-dimethylated analogs of coprogen, neocoprogen I and isoneocoprogen I. Structures of the compounds have been determined by 1H- and 13C-NMR, fast-atom-bombardment (FAB) mass spectroscopy and partial hydrolysis. One of the new compounds, N alpha-dimethylcoprogen, is also produced, as the major siderophore, in another fungus, Fusarium dimerum.

High-performance liquid chromatography of siderophores from fungi.

A reversed-phase HPLC separation of iron(III) chelates of 16 representative fungal siderophores including ferrichromes, coprogens and triacetylfusarinine C was established in order to investigate siderophore production of fungi. For comparison purposes, the widely used bacterial siderophore ferrioxamine B was included. Culture filtrates of the fungi Penicillium resticulosum, Fusarium dimerum, Aspergillus fumigatus and Neurospora crassa were quantitatively analyzed for the presence of known and unknown siderophores after growth in low-iron culture media and adsorption on XAD-2 columns using this HPLC separation system. Photodiode array detection allowed the distinction between siderophores and non-siderophores. According to their ultraviolet/visible spectra, a further classification of the siderophores into four types due to the number of anhydromevalonic acid residues per molecule (0-3) was possible.

Hiperestrogenismo en cerdas por intoxicación natural con zearalenona / Hyperestrogenism in swine due to natural poisoning with zearalenone

Se describe un caso de intoxicación de cerdas prepúberes con zearalenona (Z) al consumir una dieta conteniendo 56 ppm Z. En el maíz contaminado se encontró una cepa de Fusarium oxysporum que produjo Z en cultivo sobre ese grano. Se indicaron la evidencias de distinta índole que permitieron confirmar que los efecto observados fueron producidos por dicha micotoxina

Hiperestrogenismo en cerdas por intoxicación natural con zearalenona / Hyperestrogenism in swine due to natural poisoning with zearalenone

Se describe un caso de intoxicación de cerdas prepúberes con zearalenona (Z) al consumir una dieta conteniendo 56 ppm Z. En el maíz contaminado se encontró una cepa de Fusarium oxysporum que produjo Z en cultivo sobre ese grano. Se indicaron la evidencias de distinta índole que permitieron confirmar que los efecto observados fueron producidos por dicha micotoxina (AU)