Preparation of conjugated antigens based on zearalenone carboxymethyloxime and their use in enzyme immunoassay

Zearalenone-6′-carboxymethyloxime was synthesized, and its conjugates to albumins and gelatin were prepared. Polyclonal rabbit antibodies against the conjugate with bovine serum albumin were shown to be highly specific to zearalenone and to have a lower cross-reactivity toward its structural analogues (a-zearalenol, 28%; ß-zearalenol, 6%; zearalanone, 12%; and α-zearalanol, 5%). The sensitivity of enzyme immunoassay with immobilized gelatin-based conjugates for determination of zearalenone in solution was 1 ng/ml, and this allowed us to determine this substance in feed at a threshold concentration of 200 μg/kg.     

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.     

Production and analytical properties of antibodies with high specificity to zearalenone

The time course of production, specificity, and analytical potential of anti-zearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; alpha-zearalenol, 0.15%; and beta-zearalenol, < 0.02%. With synthetic analogues: zearalanone, 31.7% and alpha-zearalanol, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.     

Heterologous expression of Arabidopsis UDP-glucosyltransferases in Saccharomyces cerevisiae for production of zearalenone-4-O-glucoside

Zearalenone, a secondary metabolite produced by several plant-pathogenic fungi of the genus Fusarium, has high estrogenic activity in vertebrates. We developed a Saccharomyces cerevisiae bioassay strain that we used to identify plant genes encoding UDP-glucosyltransferases that can convert zearalenone into zearalenone-4-O-glucoside (ZON-4-O-Glc). Attachment of the glucose moiety to zearalenone prevented the interaction of the mycotoxin with the human estrogen receptor. We found that two of six clustered, similar UGT73C genes of Arabidopsis thaliana encode glucosyltransferases that can inactivate zearalenone in the yeast bioassay. The formation of glucose conjugates seems to be an important plant mechanism for coping with zearalenone but may result in significant amounts of "masked" zearalenone in Fusarium-infected plant products. Due to the unavailability of an analytical standard, the ZON-4-O-Glc is not measured in routine analytical procedures, even though it can be converted back to active zearalenone in the digestive tracts of animals. Zearalenone added to yeast transformed with UGT73C6 was converted rapidly and efficiently to ZON-4-O-Glc, suggesting that the cloned UDP-glucosyltransferase could be used to produce reference glucosides of zearalenone and its derivatives.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone.

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

Production and Analytical Properties of Antibodies with High Specificity to Zearalenone

The time course of production, specificity, and analytical potential of antizearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; -zearalenole, 0.15%; and -zearalenole, <0.02%. With synthetic analogues: zearalanone, 31.7% and -zearalanone, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.     

Heterologous Expression of Arabidopsis UDP-Glucosyltransferases in Saccharomyces cerevisiae for Production of Zearalenone-4-O-Glucoside

Zearalenone, a secondary metabolite produced by several plant-pathogenic fungi of the genus Fusarium, has high estrogenic activity in vertebrates. We developed a Saccharomyces cerevisiae bioassay strain that we used to identify plant genes encoding UDP-glucosyltransferases that can convert zearalenone into zearalenone-4-O-glucoside (ZON-4-O-Glc). Attachment of the glucose moiety to zearalenone prevented the interaction of the mycotoxin with the human estrogen receptor. We found that two of six clustered, similar UGT73C genes of Arabidopsis thaliana encode glucosyltransferases that can inactivate zearalenone in the yeast bioassay. The formation of glucose conjugates seems to be an important plant mechanism for coping with zearalenone but may result in significant amounts of “masked” zearalenone in Fusarium-infected plant products. Due to the unavailability of an analytical standard, the ZON-4-O-Glc is not measured in routine analytical procedures, even though it can be converted back to active zearalenone in the digestive tracts of animals. Zearalenone added to yeast transformed with UGT73C6 was converted rapidly and efficiently to ZON-4-O-Glc, suggesting that the cloned UDP-glucosyltransferase could be used to produce reference glucosides of zearalenone and its derivatives.     

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.     

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.     

Group-specific antibodies against zearalenone and its metabolites and synthetic analogs

Formation kinetics, specificity, and analytical potential of polyclonal antibodies raised in rabbits against BSA conjugates of carboxymethyloxime-zearalanone (CMO-ZAN) and carboxymethyloxime-zearalenone (CMO-ZEN). Preparation of the conjugates involved conversion of CMO-ZAN and CMO-ZEN into activated esters and carbodiimide condensation. Two versions of a group-specific enzyme immunoassay (for zearalenone/alpha-zearalenone and zearalanol/alpha-zearalanol) based on heterologous combination of solid-phase antigens are described (sensitivity, 0.01 ng/ml).     

Group-Specific Antibodies against Zearalenone and Its Metabolites and Synthetic Analogs

Formation kinetics, specificity, and analytical potential of polyclonal antibodies raised in rabbits against BSA conjugates of zearalanone carboxymethyloxime (CMO-ZAN) and zearalenone carboxymethyloxime (CMO-ZEN) have been studied. Preparation of the conjugates involved conversion of CMO-ZAN and CMO-ZEN into activated esters or carbodiimide condensation. Two versions of a group-specific enzyme immunoassay (for zearalenone/-zearalenol and zearalanone/-zearalanol) based on the heterologous combination of solid-phase antigens are described (sensitivity, 0.01 ng/ml).     

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.     

Effect of whole and fractionated dietary alfalfa meal on zearalenone toxicosis and metabolism in rats and swine

Experiments were conducted to determine the mechanism by which dietary alfalfa can protect against zearalenone toxicosis. Female weanling rats were fed semipurified diets containing whole alfalfa meal, fractionated alfalfa meal (fiber, solvent extract, and water extract), and purified components of alfalfa (coumestrol, saponin, lignin, coumestrol + lignin, and saponin + lignin) with and without 250 mg zearalenone/kg of diet. All ingredients were provided for 2 weeks at levels corresponding to those found in diets containing 15 and 25% alfalfa. Yorkshire gilts were fed 15 and 25% alfalfa meal with and without 10 mg zearalenone/kg of diet for 4 weeks. The feeding of zearalenone to rats reduced growth and food consumption but this was overcome by 25% alfalfa. Zearalenone also increased the activity of hepatic 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), the enzyme believed to metabolize zearalenone to alpha- and beta-zearalenols. Dietary alfalfa did not overcome this effect. Alfalfa fiber was the only fraction to partially overcome the growth-depressing effects of zearalenone while the other fractions had no beneficial effects and 3 alpha-HSD was not affected by diet. None of the purified components affected growth parameters or 3 alpha-HSD. The enzyme was also not affected by zearalenone or alfalfa in swine diets. Coumestrol, alpha-zearalenone, and beta-zearalenone were shown to be competitive inhibitors of 3 alpha-HSD in rat liver. It was concluded that the fiber fraction of alfalfa protects against zearalenone toxicity, and that this effect is not dependent on coumestrol or saponin and is not likely mediated through 3 alpha-HSD.     

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).     

Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of porcine antibodies

To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6'-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The porcine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.     

Role of zearalenone lactonase in protection of Gliocladium roseum from fungitoxic effects of the mycotoxin zearalenone

Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of < or =10 microg/ml. The fungitoxic effect declined in the order zearalenone > alpha-zearalenol > beta-zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.     

Radioimmunoassay for Zearalenone and Zearalanol in Human Serum: Production, Properties, and Use of Porcine Antibodies

To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6′-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The porcine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.     

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.     

Relation of 8-ketotrichothecene and zearalenone analog structure to inhibition of mitogen-induced human lymphocyte blastogenesis.

The 50% effective doses of fusarenon X, nivalenol, deoxynivalenol, and 15-acetyldeoxynivalenol required to reduce [3H]thymidine uptake in mitogen-stimulated human lymphocytes by 50% were 18, 72, 140, and 240 ng/ml, respectively. These results indicated that lymphotoxicity of 8-ketotrichothecenes decreased according to the C-4 substituent order acetyl greater than hydroxyl greater than hydrogen, whereas acetylation of position C-15 of deoxynivalenol caused a slight decrease in in vitro toxicity. The 50% effective doses for zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were 3,500, 6,300, 36,000, 3,750, and 33,000 ng/ml, respectively, suggesting that a keto group or alpha-hydroxyl at the position C-6' contributed to the lymphotoxicity of the parent molecule. The inhibitory effects of zearalenone analogs observed in the blastogenesis assay did not correlate with the estrogenic potencies of these compounds. All 8-ketotrichothecenes and zearalenone analogs tested were capable of inhibiting B- and T-cell subsets stimulated by a mitogen panel of leukoagglutinin, concanavalin A, and pokeweed mitogen.