T-2 toxin degradation by micromycetes

The biodegradation of T-2 toxin was studied by strains of micromycetes which were isolated from the environment. The 26 tested strains were divided into three groups. Group contains strains which degraded T-2 toxin very fast. This toxin could not be chromatographically determined in the medium even after 48 hours of incubation and the antifungal activity of residua against Kluyveromyces fragilis CCY-51-1-2 was low or zero. There were strains of Alternaria sp., Ulocladium sp., Aspergillus candidus, Cladosporium cladosporioides, Rhodotorula sp., Aspergillus flavus and Cladosporium macrocarpum. Group II contains with a low activity and in group III the results were variable and non stable.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Characterisation of hemolysis induced by T-2 toxin

The erythrocyte constitutes a good model system for the study of membrane-associated toxicity events caused by the trichothecene mycotoxin, T-2. This study confirms that T-2 has a direct lytic effect on erythrocytes. Lysis of guinea pig red cells requires approx. 10(10) molecules/cell and reaches plateau values after 4-6 h. An activation energy, Ea approximately equal to 4.5 kcal was derived from the Arrhenius equation. By use of osmotic blockers of differing Stokes' radii, the functional size of the membrane lesion caused by T-2 toxin was shown to be smaller than 5.5 A. It is concluded that T-2 toxin may exert its toxic effects via the cell membrane.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid.

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Cross-reactivity of antibodies against T-2 with deepoxide T-2 toxin

Two types of antibodies raised against T-2 toxin, namely anti-T-2-HS-BSA and anti-3 -Ac -NEOS-HS -BSA, showed good cross-reactivity with deepoxy T-2 toxin. Our results indicate that the epoxide is not an important epitope for the production of antibody against T-2 toxin     

Detection of T-2 toxin by an improved radioimmunoassay

T-2 toxin in serum, urine, and saline was analyzed by a modified radioimmunoassay procedure. The specimens were added directly to the assay tubes without extraction steps. The reaction between antibody and ligands was optimal at 1 h. Albumin-coated charcoal was used to separate bound from free radioactivity. Quenching, which occurred with hemolyzed specimens, was corrected by a wet oxidation process with 60% perchloric acid and 30% hydrogen peroxide. The shorter incubation times resulted in an assay that takes less than 6 h to complete. The average affinity constant of the antibody (Km) was 1.75 X 10(10) liters/mol. The sensitivity was 1 ng per assay or 10 ng/ml. Among the other trichothecenes tested, only H-T-2 cross-reacted significantly (10.3%).     

T-2 toxin and diacetoxyscirpenol metabolism by Baccharis spp

Hybrids resulting from crosses between Baccharis sarothroides and B. pilularis (FS1), B. sarothroides (FS2) and B. megapotamica (FS3) were tested for their tolerance to trichothecenes as well as their ability to metabolize the toxins. B. sarothroides (desert broom) was placed in an aqueous solution containing 500 ppm of T-2 toxin and showed visible signs of toxicity on the twigs at 21 h after exposure but not at 6 h, indicating some resistance. Samples of the twigs harvested 6 and 21 h after treatment contained, respectively, T-2 (0.03 and 2.2 micrograms/g), HT-2 (0.09 and 7.6 micrograms/g), and T-2-tetraol (2.1 and 2.6 micrograms/g). The hybrid FS1 showed no signs of toxicity 6 h after treatment, and its twigs contained T-2 (0.8 micrograms/g), HT-2 (10.2 micrograms/g), and T-2-tetraol (10.8 micrograms/g). The leaves at 6 h contained 0.5 micrograms of T-2, 1.7 micrograms of HT-2, 0.01 microgram of 3'-hydroxy-HT-2, and 41 micrograms of T-2-tetraol per g. At 21 h, toxic signs were apparent and the twigs contained T-2 (39 micrograms/g), HT-2 (62 micrograms/g), 3'-hydroxy-HT-2 (0.8 microgram/g), and T-2-tetraol (22 micrograms/g).(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunopathological effect of the mycotoxins cyclopiazonic acid and T-2 toxin on broiler chicken

Forty, newly hatched, unsexed broiler chicks were fed diets containing 10 ppm cyclopiazonic acid (CPA) and 1 ppm T-2 toxin (T₂) either individually or in combination for 28 days to study the immunopathological effects. Lymphoid organs revealed lymphocytolysis and lymphoid depletion in all toxin fed birds. Thymic and splenic CD⁺₄ and CD⁺₈ lymphocytes decreased significantly (p < 0.01) in toxin fed birds when compared to the control. Thymic CD⁺₈ lymphocytes of T₂ and CPA-T₂ showed significant (p < 0.01) decrease from that of CPA and control groups. Splenic CD⁺₄ and CD⁺₈ lymphocytes showed significant (p < 0.01) decrease in CPA and CPA-T₂ fed groups when compared to the control. The T₂ group did not differ significantly from that of control. The stimulation index (SI) of splenocytes to concavalin A revealed significant (p < 0.01) decrease in all toxin fed birds. Significant (p < 0.01) decrease were observed for the haemagglutination inhibition (HI) titres to Newcastle disease virus vaccine F strain (NDV) of birds fed CPA, T₂ and in combination. Significant (p < 0.01) interaction was found for lymphocyte subsets, SI and HI titres to NDV. The study indicated the immunosuppressive effect of these toxins either alone or in combination in broiler chicks.Forms part of M.V.Sc. thesis of the first author approved by the Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 051, India.     

Detection of T-2 toxin by an improved radioimmunoassay.

T-2 toxin in serum, urine, and saline was analyzed by a modified radioimmunoassay procedure. The specimens were added directly to the assay tubes without extraction steps. The reaction between antibody and ligands was optimal at 1 h. Albumin-coated charcoal was used to separate bound from free radioactivity. Quenching, which occurred with hemolyzed specimens, was corrected by a wet oxidation process with 60% perchloric acid and 30% hydrogen peroxide. The shorter incubation times resulted in an assay that takes less than 6 h to complete. The average affinity constant of the antibody (Km) was 1.75 X 10(10) liters/mol. The sensitivity was 1 ng per assay or 10 ng/ml. Among the other trichothecenes tested, only H-T-2 cross-reacted significantly (10.3%).     

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.     

T-2 metabolites in the excreta of broiler chickens administered 3H-labeled T-2 toxin.

A method for the detection of T-2 metabolites was developed and applied to analysis of metabolites in excreta of broiler chickens administered 3H-labeled T-2 toxin. The method used acetonitrile extraction and partitioning with petroleum ether followed by chromatography on Amberlite XAD-2, Florisil, and Sep-Pak C18. The recovery of T-2 toxin added to the chicken excreta was 73% at a concentration of 0.2 microgram/g. About 80% of orally administered 3H-labeled T-2 toxin was rapidly metabolized to more polar derivatives and eliminated in the excreta within 48 h. T-2 toxin, HT-2 toxin, neosolaniol, and T-2 tetraol were detected at 0.06 to 1.13% of the total dose, 48 h after administration. Eight unknown derivatives, named TB-1 to TB-8, were quantitatively more significant than the metabolites above. TB-3 and TB-9 represented about 12 and 25% of the total dose, respectively. One of the metabolites (TB-6), 1.5% of the total dose, was identified as 4-deacetylneosolaniol (15-acetyl-3 alpha, 4 beta, 8 alpha-trihydroxy-12, 13-epoxytrichothec-9-ene).     

Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylenediaminetetraacetic acid.

The thiobarbituric acid (TBA) reactivity of rat liver and brain homogenates was characterized to elucidate what kinds of aldehyde species contributed to the reactivity. Characteristic pH dependence of the reactivity with a maximum at around pH 3 and marked enhancement of the reactivity by t-butyl hydroperoxide (t-BuOOH) and ferric ion were similar to those of alkadienals. The amounts of aldehyde species, including alkadienals determined as 2,4-dinitrophenylhydrazones, were high enough to account for the enhanced reactivity. The reactivity was inhibited by ethylenediaminetetraacetic acid (EDTA) but not completely, suggesting the presence of malonaldehyde whose reactivity was not affected by EDTA. The amounts of malonaldehyde determined as 1-(2,4-dinitrophenyl)pyrazole could account for a part of the reactivity in the presence of EDTA. Hence, the TBA reactivity of liver and brain homogenates at around pH 3 in the presence of t-BuOOH and ferric ion may be accounted for by alkadienals and malonaldehyde and that in the presence of EDTA by malonaldehyde.     

Metabolic effects of trichothecene T-2 toxin

Cereals and other agricultural products contaminated with trichothecene mycotoxins are unfit for consumption. Until recently, the metabolic effects of T-2 toxin (T-2) were thought to reside in its ability to inhibit protein synthesis. It is now clear that trichothecenes have multiple effects, including inhibition of DNA, RNA, and protein synthesis in several cellular systems, inhibition of in vitro protein synthesis, inhibition of mitochondrial functions, effects on cell division, normal cell shape, and hemolysis of erythrocytes. It is argued that these effects are pleiotropic responses of the cell's biosynthetic network to protein synthesis inhibition. However, in studies with erythrocytes, which lack nuclei and protein synthesis, changes in cell shape and lytic response towards T-2 are observed. Susceptibility to lysis is species dependent and correlates with the presence of phosphatidylcholine. Owing to their amphipathic nature, T-2 and other trichothecenes could exert their cytotoxicity by acting on cell membranes. As for cell energetics, T-2 inhibits the mitochondrial electron transport system, with succinic dehydrogenase as one site of action. Although initial investigations of the metabolic effects of T-2 mediated cytotoxicity suggested the inhibition of protein synthesis as the principal site of action, current thought suggests that the effects of trichothecenes are much more diverse.     

In vitro formation of 3'-hydroxy T-2 and 3'-hydroxy HT-2 toxins from T-2 toxin by liver homogenates from mice and monkeys.

In vitro metabolism of T-2 toxin was studied in homogenates of mouse and monkey livers. In addition to several hydrolyzed products, including HT-2 toxin, neosolaniol, 4-deacetylneosolaniol, 15-deacetylneosolaniol, and T-2 tetraol, two metabolic products were isolated from the incubation mixture. Their structures were confirmed as 3'-hydroxy T-2 toxin and 3'-hydroxy HT-2 toxin on the basis of mass and nuclear magnetic resonance spectroscopy. The formation of these hydroxylated metabolites was found in the microsomes in the presence of NADPH, and the hydroxylation reaction was enhanced by treating mice with phenobarbital. The results suggest that a cytochrome P-450 is catalyzing the hydroxylation at the C-3' position of T-2 and HT-2 toxins. An in vitro metabolic pathway of T-2 toxin in the hepatic homogenates containing the NADPH-generating system is proposed.     

Modification of in vitro metabolism of T-2 toxin by esterase inhibitors

In vitro metabolism of T-2 toxin with S-9 fraction obtained from livers of phenobarbital-treated pigs and rats in the presence of different esterase inhibitors, including NaF, p-hydroxymercuribenzoate, phenylmethylsulfonyl fluoride, eserine sulfate, diisopropylfluorophosphate, and diethyl p-nitrophenyl phosphate, was studied. The metabolism was completely shifted to the hydroxylation at the C-3' position in the T-2 toxin molecule when esterase inhibitors were present. Diethyl p-nitrophenyl phosphate was found to be the most potent among six esterase inhibitors tested. In the presence of 10(-4) M diethyl p-nitrophenyl phosphate, 3'-hydroxy-T-2 toxin was the only metabolite detected. Similar results were obtained when other T-2-related metabolites were tested. The yield of conversion of T-2 toxin, acetyl T-2 toxin, HT-2 toxin and T-2 triol to their respective 3'-hydroxyl derivatives were 82, 73, 72, and 75%, respectively.     

T-2 toxin and diacetoxyscirpenol metabolism by Baccharis spp.

Hybrids resulting from crosses between Baccharis sarothroides and B. pilularis (FS1), B. sarothroides (FS2) and B. megapotamica (FS3) were tested for their tolerance to trichothecenes as well as their ability to metabolize the toxins. B. sarothroides (desert broom) was placed in an aqueous solution containing 500 ppm of T-2 toxin and showed visible signs of toxicity on the twigs at 21 h after exposure but not at 6 h, indicating some resistance. Samples of the twigs harvested 6 and 21 h after treatment contained, respectively, T-2 (0.03 and 2.2 micrograms/g), HT-2 (0.09 and 7.6 micrograms/g), and T-2-tetraol (2.1 and 2.6 micrograms/g). The hybrid FS1 showed no signs of toxicity 6 h after treatment, and its twigs contained T-2 (0.8 micrograms/g), HT-2 (10.2 micrograms/g), and T-2-tetraol (10.8 micrograms/g). The leaves at 6 h contained 0.5 micrograms of T-2, 1.7 micrograms of HT-2, 0.01 microgram of 3'-hydroxy-HT-2, and 41 micrograms of T-2-tetraol per g. At 21 h, toxic signs were apparent and the twigs contained T-2 (39 micrograms/g), HT-2 (62 micrograms/g), 3'-hydroxy-HT-2 (0.8 microgram/g), and T-2-tetraol (22 micrograms/g).(ABSTRACT TRUNCATED AT 250 WORDS)     

T-2 toxin inhibits mitochondrial function in yeast

T-2 toxin inhibits oxygen consumption of whole cells and purified mitochondria of Saccharomyces cerevisiae. Inhibition of mitochondrial respiration is not relieved by 2, 4-dinitrophenol, indicating that T-2 toxin inhibits mitochondrial function at the level of the electron transport chain. T-2 toxin inhibition of state 3 respiration (with succinate) is overcome by N, N, N', N'-tetramethyl-p-phenylenediamine, indicating inhibition of site II of the electron transport chain. T-2 toxin inhibits mitochondrial succinate dehydrogenase activity and increases mitochondrial NADH dehydrogenase activity.     

Production of T-2 toxin by a Fusarium resembling Fusarium poae

A Fusarium species with a micro morphology similar to F. poae and a metabolite profile resembling that of F. sporotrichioides has been identified. Like typical F. poae, the microconidia have a globose to pyriform shape, but the powdery appearance, especially on Czapek-Dox Iprodione Dichloran agar (CZID), less aerial mycelium and the lack of fruity odour on Potato Sucrose Agar (PSA) make it different from F. poae. The lack of macroconidia, polyphialides and chlamydospores differentiates it from F. sporotrichioides. All 18 isolates investigated, 15 Norwegian, two Austrian and one Dutch, produced T-2 toxin (25–400 μg/g) on PSA or Yeast Extract Sucrose agar (YES). In addition, neosolaniol, iso-neosolaniol, HT-2 toxin, 4- and 15-acetyl T-2 tetraol, T-2 triol and T-2 tetraol and4,15-diacetoxyscirpenol were formed in variable amounts. Neither nivalenol, 4- or 15-acetylnivalenolor 4,15-diacetylnivalenol were detected in any of the cultures, while these toxins were produced at least in small amounts by all the 12 typical F. poae isolates studied. The question of whether this Fusarium should be classified as F. poae or F. sporotrichioides or a separate taxon should be addressed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of antibody against T-2 toxin.

Antibody against T-2 toxin was obtained after immunization of rabbits with bovine serum albumin-T-2 hemisuccinate conjugate. The antibody had greatest binding efficiency for T-2 toxin, less efficiency for HT-2, and least for T-2 triol. Cross-reaction of antibody with neosolaniol, T-2 tetraol, and 8-acetyl-neosolaniol was very weak. Diacetoxyscirpenol, trichodermin, vomitoxin, and verrucarin A essentially gave no cross-reaction with the antibody. The sensitivity of the binding assay for T-2 toxin detection was in the range of 1 to 20 ng per assay. Detailed methods for the preparation of the conjugate and the production of immune serum and methods for antibody determination are described.