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.     

Bioconversion of alpha-[14C]zearalenol and beta-[14C]zearalenol into [14C]zearalenone by Fusarium roseum 'Gibbosum'

Cultures of Fusarium roseium 'Gibbosum' on rice were treated with [14C]zearalenone, alpha[14C]zearalenol, or beta-[14C]zearalenol to determine whether a precursor-product relationship exists among these closely related fungal metabolites. Culture extracts were purified by silica gel column chromatography and fractionated by high-pressure liquid chromatography, and the level of radioactivity was determined. Within 7 days, the beta-[14C]zearalenol was converted to zearalenone, and no residual beta-[14C]zearalenol was detectable. Most of the alpha-[14C]zearalenol added was also converted into zearalenone with 14 days. In cultures treated with [14C]zearalenone, no radioactivity was noted in any other components.     

Bioconversion of alpha-[14C]zearalenol and beta-[14C]zearalenol into [14C]zearalenone by Fusarium roseum 'Gibbosum'.

Cultures of Fusarium roseium 'Gibbosum' on rice were treated with [14C]zearalenone, alpha[14C]zearalenol, or beta-[14C]zearalenol to determine whether a precursor-product relationship exists among these closely related fungal metabolites. Culture extracts were purified by silica gel column chromatography and fractionated by high-pressure liquid chromatography, and the level of radioactivity was determined. Within 7 days, the beta-[14C]zearalenol was converted to zearalenone, and no residual beta-[14C]zearalenol was detectable. Most of the alpha-[14C]zearalenol added was also converted into zearalenone with 14 days. In cultures treated with [14C]zearalenone, no radioactivity was noted in any other components.     

Fermentation of wort containing deoxynivalenol and zearalenone

Wort containing deoxynivalenol and zearalenone, each added at a level of 1.9 μg/mL, was fermented by 3 strains ofSaccharomyces cerevisiae for 7 or 9 days to make beer. Analysis showed that deoxynivalenol was stable during this process. The major metabolite of zearalenone was β - zearalenol, which formed in up to 69% of the initial zearalenone concentration, while up to 8.1% of the initial zearalenone was converted to α - zearalenol. The major part of the metabolism of zearalenone occurred by 1 – 2 days. Control experiments, where the yeasts were omitted and deoxynivalenol, zearalenone and α - and β - zearalenol were added, showed good recovery and stability of the mycotoxins over the 7–9 day time period. No deoxynivalenol, zearalenone, α-zearalenol or β-zearalenol was detected in control yeast fermentations where they were not added to the wort.     

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.     

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.     

Excretion kinetics and metabolism of zearalenone in broilers in dependence on a detoxifying agent

Two experiments were carried out with male broilers to examine excretion kinetics of zearalenone (ZON) and its metabolites and their occurrence in blood plasma and bile fluid after a single oral dose of ZON (approximately 6 μg/kg BW) from naturally contaminated wheat (406 μg ZON per kg). In addition, this ZON bolus was administered either in the absence or presence of a detoxifying agent (Mycofix®‐Plus, Biomin GmbH, Herzogenburg, Austria). Specimens were sampled after administration of the zearalenone bolus at different times of up to 48 h.

Excretion of zearalenone and α‐zearalenol as the only detectable metabolite of ZON peaked at approximately 6.5 h after administration of the bolus. Cumulative excretion of both substances amounted to approximately 58% of ZON intake after 48 h, when a plateau was achieved. The incomplete recovery could have been due to a partial total degradation of ZON in the digestive tract, undetected sulfate conjugates of ZON or its metabolites, to other unknown and undetected metabolites or to incomplete analytical recovery from the matrix, and needs to be examined further.

Peak concentrations of zearalenone and a‐zearalenol in bile were detected in the time period of approximately 2 to 6 h after bolus, whereas ZON and metabolite concentrations in blood plasma were around or lower than the detection limits. Mycofix®‐Plus supplementation seemed to have only minor or no effects on the parameters examined.     

Occurrence of Zearalenols (Diastereomeric Mixture) in Corn Stalk Rot and Their Production by Associated Fusarium Species

Zearalenol was extracted from Fusarium-infected stems of corn from southern Italy. The toxin, which appeared as a single compound in various thin-layer chromatography systems, was resolved by high-pressure liquid chromatography into two components. A gas chromatography-mass spectrometry examination of a purified fraction confirmed the natural occurrence of zearalenol as a diastereomeric mixture and led to the identification of alpha (56 ng/g) and beta (27 ng/g) isomers. Among nine Fusarium species found associated with stalk rot in corn, only Fusarium culmorum (F. roseum `Culmorum') and F. equiseti (F. roseum `Gibbosum') produced zearalenol and always produced it in a diastereomeric mixture of alpha and beta isomers.     

P alpha-chiral phosphorothioate analogues of bis(5''-adenosyl)tetraphosphate (Ap4A); their enzymatic synthesis and degradation.

Synthesis of Sp and Rp diastereomers of Ap4A alpha S has been characterized in two enzymatic systems, the lysyl-tRNA synthetase from Escherichia coli and the Ap4A alpha, beta-phosphorylase from Saccharomyces cerevisiae. The synthetase was able to use both (Sp)ATP alpha S and (Rp)ATP alpha S as acceptors of adenylate thus yielding corresponding monothioanalogues of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S. No dithiophosphate analogue was formed. Relative synthetase velocities of the formation of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S were 1:0.38:0.15, and the computed Km values for (Sp)ATP alpha S and (Rp)ATP alpha S were 0.48 and 1.34 mM, respectively. The yeast Ap4A phosphorylase synthesized (Sp)Ap4A alpha S and (Rp)Ap4A alpha S using adenosine 5'-phosphosulfate (APS) as source of adenylate. The adenylate was accepted by corresponding thioanalogues of ATP. In that system, relative velocities of Ap4A, (Sp)Ap4A alpha S and (Rp)Ap4A alpha S formation were 1:0.15:0.60. The two isomeric phosphorothioate analogues of Ap4A were tested as substrates for the following specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow lupin (Lupinus luteus) seeds hydrolyzed each of the analogues to AMP and the corresponding isomer of ATP alpha S; (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from E. coli produced ADP and the corresponding diastereomer of ADP alpha S; and Ap4A phosphorylase (EC 2.7.7.53) from S. cerevisiae cleaved the Rp isomer only at the unmodified end yielding ADP and (Rp)ATP alpha S whereas the Sp isomer was degraded non-specifically yielding a mixture of ADP, (Sp)ADP alpha S, ATP and (Sp)ATP alpha S. For all the Ap4A-degrading enzymes, the Rp isomer of Ap4A alpha S appeared to be a better substrate than its Sp counterpart; stereoselectivity of the three enzymes for the Ap4A alpha S diastereomers is 51, 6 and 2.5, respectively. Basic kinetic parameters of the degradation reactions are presented and structural requirements of the Ap4A-metabolizing enzymes with respect to the potential substrates modified at the Ap4A-P alpha are discussed.     

Nachweis von Zearalenon-4-β-D-Glucopyranosid in Weizen

Maize (Zea mays) cell cultures were used for the production of zearalenone-4-β-D-glucopyranoside as standard compound. Wheat samples were extracted with acetonitrile: water, applied to a florisil column and eluted with methanol:ethyl acetate. For determination and quantification of zearalenone-4-β-D-glucopyranoside and zearalenone a LC-MS method was developed. A concentration of 10 μg/kg zearalenone-4-β-D-glucopyranoside and zearalenone was detectable. The recovery rates were calculated to be 69% and 89% at a concentration level of 100 μg/kg for zearalenone-4-β-D-glucopyranoside and zearalenone, respectively.24 Bavarian wheat samples from harvest 1999 were analyzed. Zearalenone was present in 22 out of 24 field samples, the levels ranged from 11–860 μg/kg. Zearalenone-4-β-D-glucopyranoside was found in 10 out of the zearalenone positive samples (42%) at levels ranging from 17 to 104 μg/kg. The amounts of zearalenone-4-β-D-glucopyranoside were correlated to those of zearalenone (r2=0,86; b=0,10).     

Synthesis and biological properties of 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl- and 16alpha-[125I]iodo-estradiols

The synthesis, receptor binding affinity, estrogenic potency and tissue distribution of the 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl-(CIVE) and 16alpha-[125I]iodo-estradiols (CIE) are reported. The iodovinyl derivatives were prepared via the (17alpha,20E/Z)-tri-n-butylstannyl intermediates, derived from the addition of tri-n-butyl tin hydride to the 17alpha-ethynyl group of the 7alpha-cyano-17alpha-ethynylestradiol, using triethylborane as a catalyst. The no-carrier-added [125I]-CIVE isomers were prepared via the same stereospecific reaction. [125I]-CIE was prepared from 7alpha-cyano-16beta-bromoestradiol via halogen exchange with Na125I. Addition of the 7alpha-cyano group to 16alpha-iodoestradiol did not affect estrogen receptor binding affinity (RBA of CIE is 115). However the estrogenic potential of CIE, as measured by the capacity to stimulate the expression of the pS2 gene, was reduced to 1% as compared to that of estradiol. Addition of a 7alpha-cyano group to the (17alpha,20E/Z)-IVE isomers reduced the RBA to 21 and 36, respectively, while the estrogenic potential was reduced to 2-3% of that of estradiol. Uterus uptake in immature rats of the 125I-labeled CIVE 20E-isomer and the 16alpha-iodo CIE peaked at 0.5h post injection while the (17alpha,20Z)-CIVE isomer showed a maximum only past 5h post injection. Uptake of all three 125I-labeled 7alpha-cyanoestrogens was suppressed by the co-injection of non-radioactive estradiol confirming the role of estrogen receptors in the localization process. Uterus retention pattern differ substantially from those of the analogues 7alpha-methylestrogens, which were previously shown to give high maximum 125I-uptake values at 2h post injection. Overall our data indicate that addition of a 7alpha-cyano group to 123I-labeled estrogens does not improve their potential to serve as SPECT agents for the imaging of estrogen receptor densities in breast cancer.     

Effect of zearalenone on female White Leghorn chickens

Acute toxic effects of purified zearalenone were studied in growing female White Leghorn chickens. In the first experiment, zearalenone in gelatin capsules was administered to 10 chickens (zearalenone-treated chickens [ZC]) in a single oral dose of 15.0 g/kg. Another 10 control chickens (CC) received empty gelatin capsules. All chickens survived the 10-day experiment and did not show any noticeable gross or histopathological lesions. There were no differences between CC and ZC in weight gain, oviduct, comb and liver weights, hematological parameters, and serum cholesterol. ZC had significantly less (P less than 0.05) serum calcium but significantly greater (P less than 0.01) serum phosphorus than CC. In the second experiment, zearalenone was administered orally or intramuscularly (pectoral muscle) at levels of 0, 50, 200, 400, and 800 mg/kg for 7 consecutive days. The oviduct weight increased with increasing toxin levels in both orally (OZC) and intramuscularly (IZC) administered groups: there were more pronounced effects in the IZC. The liver weight increased and comb weight decreased in IZC. The relative estrogenic biopotency of zearalenone in IZC, using estradiol dipropionate as a standard, was 1.37%. The results of this experiment demonstrate that chickens are highly tolerant to zearalenone and that the estrogenic effects of the toxin are greater when it is administered in multiple doses than in a single dose and in IZC than in OZC.     

Effect of zearalenone on female White Leghorn chickens.

Acute toxic effects of purified zearalenone were studied in growing female White Leghorn chickens. In the first experiment, zearalenone in gelatin capsules was administered to 10 chickens (zearalenone-treated chickens [ZC]) in a single oral dose of 15.0 g/kg. Another 10 control chickens (CC) received empty gelatin capsules. All chickens survived the 10-day experiment and did not show any noticeable gross or histopathological lesions. There were no differences between CC and ZC in weight gain, oviduct, comb and liver weights, hematological parameters, and serum cholesterol. ZC had significantly less (P less than 0.05) serum calcium but significantly greater (P less than 0.01) serum phosphorus than CC. In the second experiment, zearalenone was administered orally or intramuscularly (pectoral muscle) at levels of 0, 50, 200, 400, and 800 mg/kg for 7 consecutive days. The oviduct weight increased with increasing toxin levels in both orally (OZC) and intramuscularly (IZC) administered groups: there were more pronounced effects in the IZC. The liver weight increased and comb weight decreased in IZC. The relative estrogenic biopotency of zearalenone in IZC, using estradiol dipropionate as a standard, was 1.37%. The results of this experiment demonstrate that chickens are highly tolerant to zearalenone and that the estrogenic effects of the toxin are greater when it is administered in multiple doses than in a single dose and in IZC than in OZC.     

E-Screen evaluation of sugar beet feedstuffs in a case of reduced embryo transfer efficiencies in cattle: the role of phytoestrogens and zearalenone

The E-Screen assay was used to evaluate the estrogenicity of sugar beet by-products obtained from a dairy farm experiencing low success rates of embryo transfer. The beet tailings had ~3-fold the estradiol equivalents of the pelleted beet pulp (3.9 and 1.2 μg estradiol equivalents or E₂Eq/kg dry matter, respectively). Whole sugar beets, sugar beet pellets, and shreds from several Midwest US locations were also evaluated by E-Screen. All pellets examined were found to have some estrogenic activity (range ~0.1–2.0 μg E₂Eq/kg DM) with a mean of 0.46 μg/kg dry matter and median of 0.28 μg/kg dry matter. Relative E₂Eq ranked as follows: pellets?>?shreds?>?most unprocessed roots. Using recommended feeding levels and conservative absorption estimates (10%), the estrogenic activity in the original samples could result in blood estradiol equivalents?≥?those found at estrus (10 pg/mL, cows). Chemical analyses revealed no known phytoestrogens, but the estrogenic mycotoxin, zearalenone, was found in 15 of 21 samples. Of significance to those using the E-Screen are our findings that contradict previous reports: ß-sitosterol has no proliferative effect and genistein’s glucuronidated form—genistin—is equal to genistein in proliferative effect. The latter is the result of deconjugation of genistin to genistein in the presence of fetal bovine serum (determined by LC MSMS). These data show the usefulness and caveats of the E-Screen in evaluation of feedstuffs, and indicate a potential for sugar beet by-products to contain zearalenone at concentrations that may impact reproduction.     

Isolation and characterization of zearalenone sulfate produced by Fusarium spp.

A water-soluble compound related to zearalenone was isolated from a culture of Fusarium graminearum 30 grown in rice. The structure of the novel metabolite was determined to be zearalenone-4-sulfate on the basis of fast-atom-bombardment mass spectrometry, proton nuclear magnetic resonance, UV spectroscopy, and by chemical and enzymatic reactions. Strains representing Fusarium equiseti, Fusarium sambucinum, and Fusarium roseum produced the sulfate conjugate as well. In the rat uterus enlargement bioassay, the metabolite or its hydrolysis product was found to retain the estrogenic activity characteristic of zearalenone. Natural occurrence of this novel metabolite might be significant because analytical methods devised for zearalenone in grain cannot detect the conjugate but the conjugate retains the biological properties of the mycotoxin when ingested by animals.     

Isolation and characterization of zearalenone sulfate produced by Fusarium spp.

A water-soluble compound related to zearalenone was isolated from a culture of Fusarium graminearum 30 grown in rice. The structure of the novel metabolite was determined to be zearalenone-4-sulfate on the basis of fast-atom-bombardment mass spectrometry, proton nuclear magnetic resonance, UV spectroscopy, and by chemical and enzymatic reactions. Strains representing Fusarium equiseti, Fusarium sambucinum, and Fusarium roseum produced the sulfate conjugate as well. In the rat uterus enlargement bioassay, the metabolite or its hydrolysis product was found to retain the estrogenic activity characteristic of zearalenone. Natural occurrence of this novel metabolite might be significant because analytical methods devised for zearalenone in grain cannot detect the conjugate but the conjugate retains the biological properties of the mycotoxin when ingested by animals.     

The use of nucleotide phosphorothioate diastereomers to define the structure of metal-nucleotide bound to GTP-AMP and ATP-AMP phosphotransferases from beef-heart mitochondria

The diastereomers of adenosine 5'-O-[1-thio]triphosphate (ATP[alpha S]) and adenosine 5'-O-[2-thio]triphosphate (ATP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5'-O-[thio]triphosphate (GTP[alpha S]) and guanosine 5'-O-[2-thio]triphosphate (GTP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5'-O-[1-thio]diphosphate (GDP-[alpha S]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221-227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109-115]. In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP [beta S] or GTP [beta S] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP [alpha S] or GTP[alpha S], or for GDP[alpha S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group. The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.     

Estrogen-controlled gene expression in tissue culture cells by zearalenone

In two estrogen-sensitive cell lines, Le42 and MCF-7, the estrogenic potential of the nonsteroidal mycotoxin zearalenone has been investigated. The chloramphenicol acetyltransferase (CAT) gene expression in Le42 cells is induced by zearalenone after transfection with a CAT-gene construct controlled by an estrogen responsive element [(1986) Cell 46, 1053-1061]. In MCF-7 cells zearalenone induces at least 2 exoproteins (52 and 160 kDa) which are estrogen-specific [(1980) Cell 20, 353-362). These data suggest that zearalenone acts by activating the estrogen receptor. Due to the high sensitivity of these cell lines for zearalenone both test systems are proposed as assays for a quantitative estimation of the biological (estrogenic) activity of this widespread mycotoxin.     

Experimental Determination and Theoretical Calculation of the Eutectic Composition of Cefuroxime Axetil Diastereomers

Cefuroxime axetil (CFA), an ester prodrug of cefuroxime exists as a pair of diastereoemers, namely isomer A and isomer B. To enable phase diagram construction, crystallization of the diastereomers of CFA from the commercially available amorphous drug substance was carried out. Isomer A was separated with a purity approaching 100% whereas the maximum purity of isomer B was 85% as confirmed by solution state proton NMR spectroscopy. The crystalline forms of isomer A and isomer B were confirmed as forms AI and BI, respectively, based on differential scanning calorimetry (DSC) analysis and powder X-ray diffraction. DSC analysis was used to observe the melting behavior of different diastereomer mixture compositions. The binary solid-liquid phase diagram for mixture compositions ranging from 0 to 85% w/w isomer B indicated the formation of a eutectic mixture having a melting temperature of 124.7?±?0.4°C and a composition of 75% w/w (+/?5% wt.) isomer B. The eutectic composition was calculated using an index based on the van’t Hoff equation for melting point depression and was found to be 75% isomer B and 25% isomer A. As CFA is present in commercial preparations as a mixture of diastereomers, the formation of a eutectic mixture between the diastereomers may impact the solubility and stability of the commercial product. Eutectic formation can be explained on the basis of the chemical similarity of diastereomers that favor miscibility in the liquid state.     

alpha-Zearalenol, a major hepatic metabolite in rats of zearalenone, an estrogenic mycotoxin of Fusarium species

The chemical and biological properties of the hepatic metabolite of zearalenone, an estrogenic and non-steroidal fungal toxin produced by Fusarium species, were investigated by employing TLC, GC/MS, high pressure liquid chromatography and fluorospectral analyses, as well as uterine weight bioassay in immature mice. All the chemical and physical data supported the view that the major metabolite, obtained by incubating zearalenone with S-9 and microsomes of rat liver in the presence of NADPH, is C-6'-alpha-hydroxylated zearalenone (alpha-zearalenol). The estrogenic activity of this metabolite was several times higher than that of the parent zearalenone, and the results of biological and toxicological evaluations of alpha-zearalenol are discussed.