ht-2-toxin and scirpentriol

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitatively analyzing T-2 toxin and its major metabolites (HT-2 toxin and T-2 triol) in swine biological samples. For all matrices, liquid-liquid extraction (ethyl acetate or acetonitrile) and Varian Bond-Elut MycoSep cartridges for solid phase extraction were used for sample preparation. The analytes were separated via a Zorbax XDB-C18 column and were detected using LC-MS/MS with an electrospray ionization interface in positive ion mode. The resulting calibration curves offered satisfactory linearity (r(2)>0.992) within the test range. The limits of quantification for T-2 toxin, HT-2 toxin, and T-2 triol were 1ng/mL (μg/kg), 2ng/mL (μg/kg), and 5ng/mL (μg/kg), respectively. The recovery rates in different matrices ranged from 74.3% to 102.4%, and the interday and intraday precisions were all less than 10.2% for the target analytes. The developed method was successfully applied to toxicokinetics, tissue distribution, and excretion studies of T-2 toxin and its major metabolites after intravenous (i.v.) administration in pigs. The results provide important information for evaluating and controlling human exposure to residual T-2 toxin and its major metabolites in animal-derived food.

Topics: Animals; Chromatography, Liquid; Limit of Detection; Liquid-Liquid Extraction; Mycotoxins; Swine; T-2 Toxin; Tandem Mass Spectrometry; Tissue Distribution; Toxicokinetics

T-2 toxin belongs to the large group of trichothecene mycotoxins synthesized by various Fusarium molds which can infect raw agriculture materials. Among the trichothecenes, T-2 toxin is one of the most potent mycotoxins and poses a potential health risk in human nutrition. Several acute and chronic toxic effects were observed in humans after consumption of contaminated food. Due to the rapid metabolism of T-2 toxin by esterases, several metabolites can be found in food and also in vivo after ingestion. The aim of this work was to determine the effects of T-2 toxin and of several of its metabolites, namely HT-2 toxin, neosolaniol, T-2-triol and T-2 tetraol, on two human cells in primary culture: human renal proximal tubule epithelial cells (RPTEC) and normal human lung fibroblasts (NHLF). Concerning the cytotoxicity of T-2 toxin and its metabolites, different studies were performed with animal cells and cell lines but there are only little data about cytotoxic effects in human cells. The use of human cells in primary culture gives a good completion of the already known data because these might be limited due to the disadvantages of cell lines (e.g., immortalization, tumor derivation, longtime cultivation). In order to study the cytotoxicity and mode of cell death, the parameters cell viability, caspase-3-activity and LDH-release were measured after exposure to T-2 toxin and several of its metabolites. With IC(50) values of 0.2 and 0.5 microM T-2 toxin showed the strongest cytotoxic effect in both cells with triggering apoptosis as kind of cell death starting at a concentration of 100nM. The metabolites HT-2 toxin and neosolaniol revealed weaker cytotoxic effects (IC(50): 0.7-3.0 microM) and induced apoptosis at higher concentrations (>1 microM). The other metabolites were less cytotoxic (IC(50): 8.3-25.1 microM) and did not activate caspase-3. In addition to the analysis of cytotoxic effects, we also studied the metabolism of T-2 toxin in these cells in primary culture. Using LC-ESI-MS/MS we could demonstrate that both cells are able to transform T-2 toxin into HT-2 toxin. Further metabolic activity could only be observed in renal proximal tubule (RPTEC) cells by forming neosolaniol as a second metabolite.

Topics: Cell Line, Transformed; Cell Survival; Cells, Cultured; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Epithelial Cells; Fibroblasts; Humans; Indicators and Reagents; Kidney; Kidney Tubules, Proximal; Lung; Molecular Structure; T-2 Toxin; Tetrazolium Salts; Trichothecenes

Contamination of feed with trichothecenes, a group of Fusarium mycotoxins, leads to losses in performance due to their immunosupressive effects and the negative effect on the gastrointestinal system in animal production. A possible way of detoxification is microbial degradation, which was the focus of this study. A bacterial strain--BBSH 797--which can degrade some mycotoxins of the trichothecene group, has already been isolated. It transforms deoxynivalenol (DON) into its metabolite DOM-1, the non-toxic deepoxide of DON. Analogous to the microbial degradation of DON, the transformation of six different type A trichothecenes was observed. The metabolites appearing were characterized by GC-MS after derivatization with TRI-SIL TBT. Two metabolites were additionally, identified by liquid chromatography-mass spectrometry with particle beam interface (LC-PB-MS) with electron impact (EI)-ionization mode. The major finding was that scirpentriol was completely transformed into its non-toxic metabolite deepoxy scirpentriol, while the mycotoxin T-2 triol underwent a more complicated metabolism. According to the study, T-2-triol was degraded into its non-toxic deepoxy form and into T-2 tetraol, which was then further metabolized to deepoxy T-2 tetraol. GC-MS after derivatization with TRI-SIL TBT was suitable for the structural characterization of trichothecenes and their degradation products. Besides the mass spectra of already known degradation products, spectra of new metabolites could be recorded by LC-PB-MS.

Topics: Animals; Chromatography, Liquid; Food Contamination; Gas Chromatography-Mass Spectrometry; Gram-Positive Asporogenous Rods, Irregular; Inactivation, Metabolic; T-2 Toxin; Trichothecenes

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 micrograms/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 and 4,15-diacetoxyscirpenol were formed in variable amounts. Neither nivalenol, 4- or 15-acetylnivalenol or 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.

Topics: Edible Grain; Fusarium; Norway; Species Specificity; T-2 Toxin; Trichothecenes

HT-2 toxin was the sole metabolite formed when T-2 toxin was treated with homogenate from brain without its blood content. Homogenate from brain with its full blood content produced--besides HT-2 toxin--T-2 triol, neosolaniol, 4-deacetylneosolaniol and T-2 tetraol, i.e. the same metabolites formed by incubation of T-2 toxin with whole rat blood.

Topics: Animals; Brain; Hydrolases; Hydrolysis; Male; Rats; Substrate Specificity; T-2 Toxin; Trichothecenes

Mycotoxicoses have been recognized worldwide to cause problems in animal production. Trichothecene mycotoxins, for the most part produced by Fusarium spp., have obtained particular importance. Between 1982 and 1984, 295 samples of feedstuffs (cereals and mixed feeds) have been analyzed on natural occurrence of type-A trichothecenes. The skin toxicity test with guinea pigs was used as a screening method. Analysis of trichothecenes by capillary gaschromatography with flame ionization detection was complicated by interfering substances from the complex matrix of the sample-material. Definite results were only obtained by the use of gaschromatography-mass spectrometry. In comparison to the electron impact ionization the chemical ionization technique showed to be advantageous. Forty-two of the 295 samples analyzed were found to be positive in the biological assay. Nine of these positive samples contained trichothecenes as determined by mass spectrometry: T-2 toxin in mixed feed (65 micrograms/kg); oats (80 and 86 micrograms/kg) and wheat (100 micrograms/kg); diacetoxyscirpenol in mixed feed (125 micrograms/kg) and wheat (50 micrograms/kg); neosolaniol in oats (310 and 350 micrograms/kg); HT-2 toxin in oats (700 micrograms/kg). Oats proved to be contaminated more frequently as compared to the other cereals and the analyzed mixed feeds.

Topics: Animal Feed; Animals; Biological Assay; Chemical Phenomena; Chemistry; Flame Ionization; Gas Chromatography-Mass Spectrometry; Guinea Pigs; Sesquiterpenes; Skin; T-2 Toxin; Trichothecenes

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.

Topics: Bacteria; Biotransformation; Kinetics; Sesquiterpenes; T-2 Toxin; Trichothecenes