In vitro metabolism of T-2 toxin in rats.

T-2 toxin was rapidly converted in the 9,000 X g supernatant fraction of rat liver homogenate into HT-2 toxin, T-2 tetraol, and two unknown metabolites designated as TMR-1 and TMR-2. TMR-1 was characterized as 4-deacetylneosolaniol (15-acetoxy-3 alpha, 4 beta, 8 alpha-trihydroxy-12,13-epoxytrichothec-9-ene) by spectroscopic analyses. Since the same metabolites were also obtained from HT-2 toxin used as substrate, it was concluded that T-2 toxin was hydrolyzed preferentially at the C-4 position to give HT-2 toxin, which was then metabolized to T-2 tetraol via 4-deacetylneosolaniol. In addition to HT-2 toxin, 4-deacetylneosolaniol and T-2 tetraol, a trace amount of neosolaniol was transformed from T-2 toxin by rat intestinal strips. In vitro metabolic pathways for T-2 toxin in rats are proposed.     

Identification of various T-2 toxin metabolites in chicken excreta and tissues

Gas chromatography-mass spectrometry was used to identify various T-2 toxin metabolites in chicken excreta and organs 18 h after intraperitoneal injection of the toxin. No trichothecenes were detected in the heart and kidneys, and only trace amounts were detected in the lungs. Most of the T-2 metabolites were found in the excreta, although considerable amounts were also found in the liver. In addition to the previously identified T-2 metabolites in chicken excreta (HT-2 toxin, 15 acetoxy T-2 tetraol, and T-2 tetraol), we found 3'-hydroxy HT-2 toxin (the major metabolite in excreta and organs), 3'-hydroxy T-2 toxin, 4-acetoxy T-2 tetraol, and trace amounts of 8-acetoxy T-2 tetraol, 3-acetoxy-3'hydroxy HT-2 toxin, and T-2 triol. Unmetabolized T-2 toxin and an unidentified isomer of T-2 tetraol monoacetate were also detected in the excreta. Most of the metabolites in the chicken are similar to those encountered in cultures of fungal species producing T-2 toxin. A comparison with T-2 toxin metabolism in the cow is also reported.     

Structures of deepoxytrichothecene metabolites from 3'-hydroxy HT-2 toxin and T-2 tetraol in rats

3'-Hydroxy HT-2 toxin and T-2 tetraol, in vivo metabolites of T-2 toxin, were orally administered to Wistar rats, and four metabolites having a trichothec-9,12-diene nucleus, which were termed deepoxytrichothecenes, were newly found in the excreta. Their structures were confirmed as 3'-hydroxy-deepoxy HT-2, 3'-hydroxy-deepoxy T-2 triol, 15-acetyl-deepoxy T-2 tetraol, and deepoxy T-2 tetraol on the basis of mass and nuclear magnetic resonance spectroscopy. Resolution of T-2 metabolites and corresponding deepoxytrichothecenes by gas-liquid and thin-layer chromatography was also described.     

Identification of various T-2 toxin metabolites in chicken excreta and tissues.

Gas chromatography-mass spectrometry was used to identify various T-2 toxin metabolites in chicken excreta and organs 18 h after intraperitoneal injection of the toxin. No trichothecenes were detected in the heart and kidneys, and only trace amounts were detected in the lungs. Most of the T-2 metabolites were found in the excreta, although considerable amounts were also found in the liver. In addition to the previously identified T-2 metabolites in chicken excreta (HT-2 toxin, 15 acetoxy T-2 tetraol, and T-2 tetraol), we found 3'-hydroxy HT-2 toxin (the major metabolite in excreta and organs), 3'-hydroxy T-2 toxin, 4-acetoxy T-2 tetraol, and trace amounts of 8-acetoxy T-2 tetraol, 3-acetoxy-3'hydroxy HT-2 toxin, and T-2 triol. Unmetabolized T-2 toxin and an unidentified isomer of T-2 tetraol monoacetate were also detected in the excreta. Most of the metabolites in the chicken are similar to those encountered in cultures of fungal species producing T-2 toxin. A comparison with T-2 toxin metabolism in the cow is also reported.     

Structures of deepoxytrichothecene metabolites from 3'-hydroxy HT-2 toxin and T-2 tetraol in rats.

3'-Hydroxy HT-2 toxin and T-2 tetraol, in vivo metabolites of T-2 toxin, were orally administered to Wistar rats, and four metabolites having a trichothec-9,12-diene nucleus, which were termed deepoxytrichothecenes, were newly found in the excreta. Their structures were confirmed as 3'-hydroxy-deepoxy HT-2, 3'-hydroxy-deepoxy T-2 triol, 15-acetyl-deepoxy T-2 tetraol, and deepoxy T-2 tetraol on the basis of mass and nuclear magnetic resonance spectroscopy. Resolution of T-2 metabolites and corresponding deepoxytrichothecenes by gas-liquid and thin-layer chromatography was also described.     

Inhibition of mitogen-induced blastogenesis in human lymphocytes by T-2 toxin and its metabolites

Concentrations of T-2, HT-2, 3'-OH T-2, 3'-OH HT-2, T-2 triol, and T-2 tetraol toxins which inhibited [3H]thymidine uptake in mitogen-stimulated human peripheral lymphocytes by 50% were 1.5, 3.5, 4.0, 50, 150, and 150 ng/ml, respectively. The results suggested that the initial hydrolysis of T-2 toxin and the hydroxylation of T-2 toxin to 3'-OH T-2 toxin did not significantly decrease the immunotoxicity of the parent molecule, whereas further hydrolysis to T-2 triol and T-2 tetraol toxins or hydroxylation to 3'-OH HT-2 toxin decreased in vitro toxicity for human lymphocytes.     

Inhibition of mitogen-induced blastogenesis in human lymphocytes by T-2 toxin and its metabolites.

Concentrations of T-2, HT-2, 3'-OH T-2, 3'-OH HT-2, T-2 triol, and T-2 tetraol toxins which inhibited [3H]thymidine uptake in mitogen-stimulated human peripheral lymphocytes by 50% were 1.5, 3.5, 4.0, 50, 150, and 150 ng/ml, respectively. The results suggested that the initial hydrolysis of T-2 toxin and the hydroxylation of T-2 toxin to 3'-OH T-2 toxin did not significantly decrease the immunotoxicity of the parent molecule, whereas further hydrolysis to T-2 triol and T-2 tetraol toxins or hydroxylation to 3'-OH HT-2 toxin decreased in vitro toxicity for human lymphocytes.     

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).     

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.     

Preparation and characterization of the deepoxy trichothecenes: deepoxy HT-2, deepoxy T-2 triol, deepoxy T-2 tetraol, deepoxy 15-monoacetoxyscirpenol, and deepoxy scirpentriol.

The production of deepoxy metabolites of the trichothecene mycotoxins T-2 toxin and diacetoxyscirpenol, including deepoxy HT-2 (DE HT-2), deepoxy T-2 triol, deepoxy T-2 tetraol, deepoxy 15-monoacetoxyscirpenol, and deepoxy scirpentriol is described. The metabolites were prepared by in vitro fermentation with bovine rumen microorganisms under anaerobic conditions and purified by normal and reverse-phase high-pressure liquid chromatography. Capillary gas chromatographic retention times and mass spectra of the derivatized metabolites were obtained. The deepoxy metabolites were significantly less toxic to brine shrimp than were the corresponding epoxy analogs. Polyclonal and monoclonal T-2 antibodies were examined for cross-reactivity to several T-2 metabolites. Both HT-2 and DE HT-2 cross-reacted with mouse immunoglobulin monoclonal antibody 15H6 to a greater extent than did T-2 toxin. Rabbit polyclonal T-2 antibodies displayed greater specificity to T-2 toxin compared with the monoclonal antibody, with relative cross-reactivities of only 17.4, 14.6, and 9.2% for HT-2, DE HT-2, and deepoxy T-2 triol, respectively. Cross-reactivity of both antibodies was weak for T-2 triol, T-2 tetraol, 3'OH T-2, and 3'OH HT-2.     

Production and characterization of antibodies against HT-2 toxin and T-2 tetraol tetraacetate

Three new immunogens which were prepared by conjugation of the carboxymethyl oxime (CMO) derivatives of HT-2 toxin, T-2 tetraol (T-2 4ol), and T-2 tetraol tetraacetate (T-2 4Ac) to bovine serum albumin (BSA) were tested for the production of antibodies against the major metabolites of T-2 toxin. Antibodies against HT-2 toxin and T-2 4Ac were obtained from rabbits 5 to 10 weeks after immunizing the animals with CMO-HT-2-BSA and CMO-T-2 4Ac-BSA conjugates. Immunization with CMO-T-2 4ol-BSA resulted in no antibody against T-2 4ol. The antibody produced against HT-2 toxin had great affinity for HT-2 toxin as well as good cross-reactivity with T-2 toxin. The relative cross-reactivities of anti-HT-2 toxin antibody with HT-2 toxin, T-2 toxin, iso-T-2 toxin, acetyl-T-2 toxin, 3'-OH HT-2, 3'-OH T-2, T-2 triol, and 3'-OH acetyl-T-2, were 100, 25, 10, 3.3, 0.25, 0.15, 0.12 and 0.08%, respectively. Antibody against CMO-T-2 4Ac was very specific for T-2 4Ac and had less than 0.1% cross-reactivity with T-2 toxin, HT-2 toxin, acetyl-T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and deoxynivalenol triacetate as compared with T-2 4Ac. The detection limits for HT-2 toxin and T-2 4ol by radioimmunoassay were approximately 0.1 and 0.5 ng per assay, respectively.     

Production and characterization of antibodies against HT-2 toxin and T-2 tetraol tetraacetate.

Three new immunogens which were prepared by conjugation of the carboxymethyl oxime (CMO) derivatives of HT-2 toxin, T-2 tetraol (T-2 4ol), and T-2 tetraol tetraacetate (T-2 4Ac) to bovine serum albumin (BSA) were tested for the production of antibodies against the major metabolites of T-2 toxin. Antibodies against HT-2 toxin and T-2 4Ac were obtained from rabbits 5 to 10 weeks after immunizing the animals with CMO-HT-2-BSA and CMO-T-2 4Ac-BSA conjugates. Immunization with CMO-T-2 4ol-BSA resulted in no antibody against T-2 4ol. The antibody produced against HT-2 toxin had great affinity for HT-2 toxin as well as good cross-reactivity with T-2 toxin. The relative cross-reactivities of anti-HT-2 toxin antibody with HT-2 toxin, T-2 toxin, iso-T-2 toxin, acetyl-T-2 toxin, 3'-OH HT-2, 3'-OH T-2, T-2 triol, and 3'-OH acetyl-T-2, were 100, 25, 10, 3.3, 0.25, 0.15, 0.12 and 0.08%, respectively. Antibody against CMO-T-2 4Ac was very specific for T-2 4Ac and had less than 0.1% cross-reactivity with T-2 toxin, HT-2 toxin, acetyl-T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and deoxynivalenol triacetate as compared with T-2 4Ac. The detection limits for HT-2 toxin and T-2 4ol by radioimmunoassay were approximately 0.1 and 0.5 ng per assay, respectively.     

Metabolism of trichothecene mycotoxins. I. Microsomal deacetylation of T-2 toxin in animal tissues

In an attempt to elucidate the active form of T-2 toxin, one of trichothecene mycotoxins in vivo, the metabolism in animal tissues was studied in vitro by using gas liquid chromatography. T-2 toxin was selectively hydrolysed by the microsomal esterase at C-4, giving rise to HT-2 toxin as the only metabolite. This esterase activity was found mainly in the microsomes of liver, kidney, and spleen of laboratory animals. Since the enzymatic hydrolysis of T-2 toxin was inhibited by eserine, and diisopropylfluorophosphate, it is concluded that non-specific carboxyesterase [EC 3.1.1.1] of microsomal origin participates in this type of selective hydrolysis of T-2 toxin. The microsomal fraction from rabbit liver was proved to be a convinient material for the preparation of HT-2 toxin from T-2 toxin. From the evidence that the toxicity of HT-2 toxin is comparable to that of T-2 toxin and that the microsomal fraction of whole liver possesses the ability to biotransform the total lethal dose of T-2 toxin into HT-2 within a few minutes, T-2 toxin administered to animals is presumed to exhibit its toxicity partly as HT-2 toxin.     

Intestinal metabolism of T-2 toxin in the pig cecum model

T-2 toxin, a toxic member of the group A trichothecenes, is produced by various Fusarium species that can potentially affect human health. As the intestine plays an important role in the metabolism of T-2 toxin for animals and humans, the degradation and metabolism of T-2 toxin was studied using the pig cecum in vitro model system developed in the author??s group. In order to study the intestinal degradation of T-2 toxin by pig microbiota, incubation was performed with the cecal chyme from four different pigs in repeat determinations. A large variation in the intestinal degradation of T-2 toxin was observed for individual pigs. T-2 toxin was degraded almost completely in one out of four pigs, in which only 3.0?±?0.1?% of T-2 toxin was left after 24?h incubation. However, in the other three incubations with pig cecal suspension, 54.1?±?11.7?C68.9?±?16.1?% of T-2 toxin were still detectable after 24?h incubation time. The amount of HT-2 toxin was increased along with the incubation time, and HT-2 toxin accounted for 85.2?±?0.7?% after 24?h in the most active cecum. HT-2 toxin was the only detectable metabolite formed by the intestinal bacteria. This study suggests that the toxicity of T-2 toxin for pigs is caused by the combination of T-2 and HT-2 toxins.     

Metabolism of T-2 toxin in Curtobacterium sp. strain 114-2.

The metabolic pathway of T-2 toxin in Curtobacterium sp. strain 114, one of the T-2 toxin-assimilating soil bacteria, was investigated by thin-layer and gas-liquid chromatographic analyses. T-2 toxin added to the basal medium as a single carbon and energy source was biotransformed into HT-2 toxin and an unknown metabolite. Infrared, mass spectrum, proton magnetic resonance, and other physico-chemical analyses identified this new metabolite as T-2 triol. T-2 toxin was first deacetylated by the bacterium into HT-2 toxin, and this metabolite was then biotransformed into T-2 triol without formation of neosolaniol and T-2 tetraol. No trichothecenes remained in the culture medium after prolonged culture. Some properties of T-2 toxin-hydrolyzing enzymes were observed with whole cells, the cell-free soluble fraction, and the culture filtrate. Besides T-2 toxin, trichothecenes such as diacetoxyscirpenol, neosolaniol, nivalenol, and fusarenon-X were also assimilated by this bacterium.     

Microbial acetyl conjugation of T-2 toxin and its derivatives.

The acetyl conjugation of T-2 toxin and its derivatives, the 12,13-epoxytrichothecene mycotoxins, was studied by using mycelia of trichothecene-producing strains of Fusarium graminearum, F. nivale, Calonectria nivalis, and F. sporotrichoides, T-2 toxin was efficiently converted into acetyl T-2 toxin by all strains except a T-2 toxin-producing strain of F. sporotrichoides, which hydrolyzed the substrate to HT-2-toxin and neosolaniol. HT-2 toxin was conjugated to 3-acetyl HT-2 toxin as an only product by mycelia of F. graminearum and C. nivalis, but was also resistant to conjugation by both F. nivale and F. sporotrichoides. Neosolaniol was also biotransformed selectively into 3-acetyl neosolaniol by F. graminearum. However, 3-acetyl HT-2 toxin was not acetylated by any of the strains under the conditions employed, but was hydrolyzed to HT-2 toxin by F. graminearum and F. nivale. This is the first report on the biological 3 alpha-O-acetyl conjugation of T-2 toxin and its derivatives.     

Structure-Function Analysis of Porcine Cytochrome P450 3A29 in the Hydroxylation of T-2 Toxin as Revealed by Docking and Mutagenesis Studies

T-2 toxin, one of the type A trichothecenes, presents a potential hazard to human and animal health. Our previous work demonstrated that porcine cytochrome P450 3A29 (CYP3A29) played an important role in the hydroxylation of T-2 toxin. To identify amino acids involved in this metabolic process, T-2 toxin was docked into a homology model of CYP3A29 based on a crystal structure of CYP3A4 using AutoDock 4.0. Nine residues of CYP3A29, Arg105, Arg106, Phe108, Ser119, Lys212, Phe213, Phe215, Arg372 and Glu374, which were found within 5 Å around T-2 toxin were subjected to site-directed mutagenesis. In the oxidation of nifedipine, the CL _int value of R106A was increased by nearly two-folds compared with the wild-type CYP3A29, while the substrate affinities and CL _int values of S119A and K212A were significantly reduced. In the hydroxylation of T-2 toxin, the generation of 3′-OH-T-2 by R105A, S119A and K212A was significantly less than that by the wild-type, whereas R106A slightly increased the generation of 3′-OH-T-2. These results were further confirmed by isothermal titration calorimetry analysis, suggesting that these four residues are important in the hydroxylation of T-2 toxin and Arg105 may be a specific recognition site for the toxin. Our study suggests a possible structure-function relationship of CYP3A29 in the hydroxylation of T-2 toxin, providing with new insights into the mechanism of CYP3A enzymes in the biotransformation of T-2 toxin.     

Biotransformation and detoxification of T-2 toxin by soil and freshwater bacteria

Bacterial communities isolated from 17 of 20 samples of soils and waters with widely diverse geographical origins utilized T-2 toxin as a sole source of carbon and energy for growth. These isolates readily detoxified T-2 toxin as assessed by a Rhodotorula rubra bioassay. The major degradation pathway of T-2 toxin in the majority of isolates involved side chain cleavage of acetyl moieties to produce HT-2 toxin and T-2 triol. A minor degradation pathway of T-2 toxin that involved conversion to neosolaniol and thence to 4-deacetyl neosolaniol was also detected. Some bacterial communities had the capacity to further degrade the T-2 triol or 4-deacetyl neosolaniol to T-2 tetraol. Two communities, TS4 and KS10, degraded the trichothecene nucleus within 24 to 48 h. These bacterial communities comprised 9 distinct species each. Community KS10 contained 3 primary transformers which were able to cleave acetate from T-2 toxin but which could not assimilate the side chain products, whereas community TS4 contained 3 primary transformers which were able to grow on the cleavage products, acetate and isovalerate. A third community, AS1, was much simpler in structure and contained only two bacterial species, one of which transformed T-2 toxin to T-2 triol in monoculture. In all cases, the complete communities were more active against T-2 toxin in terms of rates of degradation than any single bacterial component. Cometabolic interactions between species is suggested as a significant factor in T-2 toxin degradation.     

Biotransformation and detoxification of T-2 toxin by soil and freshwater bacteria.

Bacterial communities isolated from 17 of 20 samples of soils and waters with widely diverse geographical origins utilized T-2 toxin as a sole source of carbon and energy for growth. These isolates readily detoxified T-2 toxin as assessed by a Rhodotorula rubra bioassay. The major degradation pathway of T-2 toxin in the majority of isolates involved side chain cleavage of acetyl moieties to produce HT-2 toxin and T-2 triol. A minor degradation pathway of T-2 toxin that involved conversion to neosolaniol and thence to 4-deacetyl neosolaniol was also detected. Some bacterial communities had the capacity to further degrade the T-2 triol or 4-deacetyl neosolaniol to T-2 tetraol. Two communities, TS4 and KS10, degraded the trichothecene nucleus within 24 to 48 h. These bacterial communities comprised 9 distinct species each. Community KS10 contained 3 primary transformers which were able to cleave acetate from T-2 toxin but which could not assimilate the side chain products, whereas community TS4 contained 3 primary transformers which were able to grow on the cleavage products, acetate and isovalerate. A third community, AS1, was much simpler in structure and contained only two bacterial species, one of which transformed T-2 toxin to T-2 triol in monoculture. In all cases, the complete communities were more active against T-2 toxin in terms of rates of degradation than any single bacterial component. Cometabolic interactions between species is suggested as a significant factor in T-2 toxin degradation.     

Influence of T-2 and HT-2 Toxin on the Blood-Brain Barrier In Vitro: New Experimental Hints for Neurotoxic Effects

The trichothecene mycotoxin T-2 toxin is a common contaminant of food and feed and is also present in processed cereal derived products. Cytotoxic effects of T-2 toxin and its main metabolite HT-2 toxin are already well described with apoptosis being a major mechanism of action. However, effects on the central nervous system were until now only reported rarely. In this study we investigated the effects of T-2 and HT-2 toxin on the blood-brain barrier (BBB) in vitro. Besides strong cytotoxic effects on the BBB as determined by the CCK-8 assay, impairment of the barrier function starting at low nanomolar concentrations were observed for T-2 toxin. HT-2 toxin, however, caused barrier disruption at higher concentrations compared to T-2 toxin. Further, the influence on the tight junction protein occludin was studied and permeability of both toxins across the BBB was detected when applied from the apical (blood) or the basolateral (brain) side respectively. These results clearly indicate the ability of both toxins to enter the brain via the BBB.