Biosynthesis of zearalenone: a simple and efficient method to incorporate [13C]acetate label by using solid cultures.

A suitable method was developed to efficiently incorporate 13C-labeled acetate into zearalenone by using solid cultures. Periodic feeding of the label during the zearalenone production phase significantly increased the label incorporation for the singly labeled acetate.

Biotransformation and elimination of 14C-flecainide acetate in humans.

The metabolism of 14C-flecainide acetate, a new antiarrhythmic, was investigated in four healthy human subjects, after a single, 200-mg oral dose. The cumulative recovery of radioactivity ranged from 81 to 90% (mean, 86%) in urine and from 4 to 6% (mean, 5%) in feces; thus, flecainide does not appear to undergo extensive biliary excretion, unless reabsorption occurs after biliary elimination. The cumulative excretion in urine of unchanged flecainide ranged from 35 to 50% (mean, 42%) of the dose and was essentially complete by 72 hr postdose. Peak plasma levels of radioactivity (524 to 848 ng eq/ml) and of unchanged flecainide (214 to 281 ng/ml) were attained at 2 to 3 hr postdose. The average half-life for the disappearance of unchanged flecainide from plasma was about 16 hr; disappearance of total metabolites from plasma was only slightly slower. Radiomonitored TLC analysis showed that urine contained two major metabolites and two or three minor ones; both major metabolites were extensively conjugated. By TLC, NMR, and mass spectral comparisons to reference compounds, the two major urinary metabolites were shown to be meta-O-dealkylated flecainide and the meta-O-dealkylated lactam of flecainide. Most of the radioactivity in urine was accounted for by flecainide and the two major metabolites.

Macrocyclic trichothecene toxins produced by a strain of Stachybotrys atra from Hungary.

A strain of Stachybotrys atra isolated from a field case of stachybotryotoxicosis in Hungary was cultured in Hungary. All of the compounds toxic to brine shrimp were separated from the culture extract by solvent partition, column chromatography, and preparative thin-layer chromatography. Two of the toxic compounds were identified as verrucarin J and satratoxin H by comparison with pure standards resolved by high-pressure liquid chromatography and characterized by mass spectrometry. Two other toxic components were identified as roriden E and satratoxin G on the basis of their mass spectra. The fifth toxic compound was identified as a macrocyclic trichothecene based on the following findings: a positive 4-(p-nitrobenzyl)pyridine color reaction, hydrolysis resulting in verrucarol verified by combined gas chromatography-mass spectrometry, and a characteristic trichothecene proton-nuclear magnetic resonance spectrum. This macrocyclic trichothecene has a molecular ion (528) identical to satratoxin H, and its mass spectrum is similar; however, its Rf value on Silica Gel G differs.

Biosynthesis of radiolabeled T-2 toxin by Fusarium tricinctum.

Incubation of Fusarium tricinctum NRRL 3299 on a solid rice medium in the presence of [1-14C]sodium acetate, [2-3H]mevalonic acid, [2-14C]mevalonic acid, or [5-3H]mevalonic acid yielded preparations of radiolabeled T-2 toxin with specific activities of 1.008, 1.64, 0.656, and 7.35 muCi/mmol, respectively.

Identification of the naturally occurring isomer of zearalenol produced by Fusarium roseum 'Gibbosum' in rice culture.

One diastereomer of trans-zearalenol [2,4-dihydroxy-6-(6,10-dihydroxy-trans-1-undecenyl)-benzoic acid-mu-lactone] was isolated from cultures of Fusarium roseum 'Gibbosum.' This strongly estrogenic metabolite was identified by analysis of its mass spectrum and its behavior in thin-layer, high-pressure liquid and gas-liquid chromatographic systems. The concentration of zearalenol in cultures was 563 mu g/g, or 7% of the 8,000-mu g/g zearalenone content, while the two diastereomers of 8'-hydroxyzearalenone each occurred at 3% of the zearalenone level. Of the two possible diastereomers of zearalenol, the one occurring in cultures was identical to the low-melting-point (171 degrees C) isomer (alpha) obtained by synthesis. In the rat uterus bioassay, the alpha zearalenol isomer was three times more estrogenic than zearalenone while the beta isomer was equal in activity in zearalenone. The two diastereomers of zearalenol can be distinguished from each other by the intensity of the m/e+ 302 fragment of the mass spectrum of the pure underivatized compound.

Mycotoxins - Their Biosynthesis in Fungi: Zearalenone Biosynthesis 1.

Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-ß-resorcylic acid lactone] is biosynthesized by Fusarium roseum by head-to-tail condensation of acetate units via the acetate-malonyl-CoA (polyketide) pathway. Incorporation and distribution of 1-[14C]acetate into zearalenone was demonstrated by degradation of the molecule into the aromatic ring, CO2, oxalic, succinic and glutaric acids. Incorporation and distribution of 1-[13C]acetate and 1,2-[13C]acetate was demonstrated by 13C-NMR and confirmed the conclusion reached by 14C-incorporation studies. CO2 is rapidly fixed by cultures of F. roseum , presumably by phosphoenolpyruvate carboxykinase and pyruvate carboxylase and incorporated into zearalenone. The wild-type isolates of F. roseum normally produce optimum amounts of zearalenone on a solid medium such as rice and at lower temperatures (10-14 C), although mutants are available that produce copious amounts in liquid medium. Some wild-type isolates also produce zearalenone optimally at 20-25 C.

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.

Uterotropic activity of cis and trans isomers of zearalenone and zearalenol.

The cis and trans isomers of zearalenone [2,4-dihyroxy-6-(10-hydroxy-6-oxo-1-undecenyl)-benzoic acid mu-lactone] and zearalenol [2,4-dihydroxy-6-(6,10-dihydroxy-1-undecenyl)-benzoic acid mu-lactone] were tested for uterotropic activity in the white rat. The metabolites were administered through the oral route (per os) and by topical application to the freshly shaven skin on the back. cis-Zearalenone was significantly more active than trans when fed orally to the rats in the diet or when applied topically by skin application. However, the cis isomer of zearalenol was not significantly different than its trans isomer. trans-Zearalenone was less active than trans-zearalenol.

Analysis of deoxynivalenol from cultures of Fusarium species.

Eight isolates of Fusarium roseum and three of Fusarium colmorum were found to produce deoxynivalenol in rice cultures. Deoxynivalenol was extracted with aqueous methanol (40%) and purified by partitioning with ethyl acetate and acetonitrile-petroleum ether (boiling point, 60--70 degrees C). The toxin was identified by gas chromatography/mass spectrometry and quantified by gas-liquid chromatography. High recoveries (80%) of deoxynivalenol were obtained from rice cultures, and as low as 0.250 microgram of the toxin per g was detected.

Metabolism of zearalenone by Fusarium roseum.

Twenty products associated with the metabolism of zearalenone by Fusarium roseum were analyzed with respect to time and culture conditions. By statistical analysis a set of six metabolites possibly related to zearalenone were selected. These products were characterized by gas chromatography--mass spectrometry and other methods. The chemical structures of these six confirmed the statistical relationships, and evidence for the formation of these materials from zearalenone is presented. A suggested scheme of metabolism of zearalenone by F. roseum includes the formation of the two isomers of 8'-hydroxyzearalenone, 6',8'-dihydroxyzearalene, 6-(carboxypentyl)-beta-resorcyclic acid, and phenylacetic acid derivatives.

Identification of mycotoxins produced by species of Fusarium and Stachybotrys obtained from Eastern Europe.

Isolates of Fusarium and Stachybotrys spp. and crude extracts from these fungi were obtained from Hungary and the U.S.S.R. and used for the evaluation of the mycotoxins they produced. The cultures were grown on millet and oats and extracted in Budapest, Hungary (Veterinary Medical Research Institute) and chemically analyzed at the University of Minnesota using thin-layer chromatography (TLC), gas-liquid chromatography (GLC), gas chromatograph-mass spectrometry (GC-MS), and the rat skin bioassay. Zearalenone was found in most of the Fusarium cultures, T-2 toxin, neosolaniol, T-2 tetraol, and HT-2 toxin were found in extracts of Fusarium poae and F. sporotrichioies. A special effort was made to isolate the steroid-like toxins reported in the early Russian literature as sporofusarin and poaefusarin. None of the extracts from the Fusarium species yielded poaefusarin or sporofusarin when analyzed by our chemical methods or by those of L.E. Olifson, S.M. Kenina, and V.L. Kartashova, 1972. We therefore accounted for the toxicity of the Fusarium extracts as due to the 12,13,epoxytrichothecenes. One culture of Stachybotrys alternans yielded a macrocyclic ester of 12,13-epoxytrichothecene which, upon hydrolysis, yielded verrucarol; a steroid-like molecule (SB-3) was also isolated. The former had skin-irritant activity but SB-3 did not; the latter exhibited cardiac activity on the heart of the cockroach.

Natural occurrence of Fusarium toxins in feedstuff.

The mycotoxins diacetoxyscirpenol, deoxynivalenol, and zearalenone, produced by Fusarium roseum, were found naturally occuring in mixed feed samples. In all cases analyzed, deoxynivalenol occurred together with zearalenone. The natural occurrence of zearalenone in sesame seed is reported for the first time. Strains of F. roseum isolated in various parts of the world form feed implicated in animal mycotoxicosis produced monoacetoxyscirpenol, diacetoxyscirpenol, deoxynivalenol, and zearalenone.

Substances interfering with the gas-liquid chromatographic determination of T-2 mycotoxin.

Two substances interfering with the gas-liquid chromatographic (GLC) detection of T-2 mycotoxin were identified as 1-glyceryl-monooleate and 1-glycerylmonolinoleate. These monoglycerides are natural products formed by species of Fusarium growing on cereal grains and are also additives contained in liquid vegetable and animal fats added to the feed mixture. The monoglycerides can be removed from the analytical sample by resolution by thin layer chromatography prior to separation by GLC. Trimethylsilyl ether derivatives of the monoglycerides and T-2 toxin have almost identical retention times on 3% OV-1 columns, whereas the trifluoroacetyl and pentafluoropropionyl derivatives give baseline separation on the same column. The monoglycerides can be misidentified as the T-2 toxin in analyses involbing GLC.