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.     

Large-scale production of selected type A trichothecenes: the use of HT-2 toxin and T-2 triol as precursors for the synthesis of <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-T-2 and <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-HT-2 toxin

The type A trichothecenes T-2 and HT-2 toxins are toxic secondary metabolites produced by fungi of the Fusarium genus. Their occurrence in cereals, especially in oats, implies health risks for the consumer. Therefore, it is an important task to develop selective and sensitive methods for the analysis of T-2 and HT-2 toxins, and to undertake further studies on their stability and toxicity. Although most toxins are commercially available, their high prices are the limiting factor on the realization of these experiments. Thus, we developed a method for large-scale production of T-2 and HT-2 toxin as well as T-2 triol and T-2 tetraol. T-2 toxin was obtained in gram quantities by biosynthetic production with cultures of F. sporotrichioides. As HT-2 toxin was only formed as a by-product, and T-2 triol and T-2 tetraol were not generated, these compounds were produced by alkaline hydrolysis of T-2 toxin. Separation and isolation of crude toxins was achieved by fast centrifugal partition chromatography (FCPC), which is an efficient tool for the large-scale purification of natural products. Using this fast and yield effective technique, several hundred milligrams of HT-2 toxin, T-2 triol, and T-2 tetraol were obtained. Subsequent, HT-2 toxin and T-2 triol were used for the large-scale synthesis of isotope-labeled T-2 and HT-2 toxin, respectively. Using these standards, an isotope dilution-(ID)-HPLC-MS/MS method for the quantification of T-2 and HT-2 toxin in different matrices was developed.     

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.     

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.     

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.     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil.

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

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.     

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.     

Assay and Relationship of HT-2 Toxin and T-2 Toxin Formation in Liquid Culture

Both T-2 toxin and HT-2 toxin can be conveniently quantitated in crude extracts by using a combination of thin-layer chromatography and fluorodensitometry. This technique was used to follow the production of these toxins by liquid cultures of Fusarium poae (NRRL 3287). T-2 toxin was produced prior to HT-2 toxin and hexadeuterio-T-2 toxin was converted by the culture to trideuterio-HT-2 toxin.     

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.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides.

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Effects of T-2 toxin and its congeners on membrane functions of cultured human fibroblasts

Cytotoxicity of T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, and T-2 tetraol was compared between normal human fibroblasts and mutant I-cell human fibroblasts, which only produce 10 to 15% of lysosomal hydrolases present in normal fibroblasts. Both cleavage of 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and cell count by hemocytometer were used for evaluations. For all toxins, dose-related effects on both types of cultures were evident. Cytotoxicity of the above mycotoxins on both cell lines were similar, indicating that lysosomal enzymes were not involved in the toxicity of T-2 toxin and its congeners. An inhibitor of lysosomal cysteine proteases (E-64) did not alter the cytotoxicity of T-2 toxin. The decreasing order of toxicity was T-2 toxin, HT-2 toxin, neosolaniol, acetyl T-2 toxin, and T-2 tetraol in both cell lines. When normal human fibroblasts were loaded with the fluorescent dye Lucifer yellow CH (LY), a subsequent treatment of T-2 toxin did not disrupt lysosomal membranes. The uptake of LY was not affected by T-2 toxin, which indicated that T-2 toxin did not interfere with the endocytic pathway. Results indicate that T-2 toxin and its congeners do not exert their primary toxic effect through lysosomal enzymes, membranes, or via the endocytic pathway.     

A monoclonal antibody-based enzyme immunoassay for the detection of T-2 toxin at picogram levels

Thirteen monoclonal antibodies reactive with HT-2 were prepared by using a HT-2 hemisuccinate coupled to human serum albumin as antigen for the immunization of BALB/c mice. In a competitive enzyme immunoassay on a double antibody solid phase using HT-2 hemisuccinate coupled to horseradish peroxidase as enzyme linked toxin all antibodies reacted much better with T-2 toxin and acetyl T-2 than with HT-2. Eleven antibodies showed almost the same sensitivity and specificity, and one of these, designated 3E2, is extensively described. Its cross-reactivities with HT-2, T-2 toxin, acetyl T-2, iso T-2, T-2 tetraol tetraacetate and T-2 triol were 1·0, 140·2, 161·2, 0·32, 0·14 and 0·016, respectively. Two other antibodies, designated 2A4 and 2A5, behaved quite differently. The cross-reactivities of antibody 2A4 with these toxins were: 1·0, 113·9, 374·4, 1·35, 0·34 and 0·023, respectively; for antibody 2A5 they were 1·0, 46·1, 155·4, 8·31, 0·9 and 0·08, respectively. All antibodies proved to be IgGl. By using the antibody 3E2 a highly sensitive and very specific enzymc immunoassay for the detection of T-2 toxin was developed. The detection limit for T-2 toxin was 5 pg/ml (0·25 pg/assay).     

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.     

The enzymes of the metabolism of exogenous compounds in the comparative assessment of the toxicity of trichothecene mycotoxins

Moderate clinical, biochemical and hematologic signs of intoxication were observed in mice after single administration of HT-2 toxin (deacetylated derivative of T-2 toxin) in LD50 of 12.75 mg/kg or in 1/5 of LD50 for 7 days. The toxin produced no toxic effect when 1/10 and 1/50 of LD50 were administered for 14 days. With the dose exceeding 1/50 of LD50 a reduction in cytochrome P-450 content, carboxylesterase activity and increased activity of UDP-glucuronosyltransferase and glutathione transferase were recorded. T-2 toxin produced a more pronounced toxic effect, inhibited UDP-glucuronosyltransferase and insignificantly stimulated glutathione transferase activity. Lower HT-2 toxin toxicity is believed to depend on its ability to activate conjugation reactions to a greater extent than T-2 toxin.     

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.