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.     

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.     

Different sample treatment approaches for the analysis of T-2 and HT-2 toxins from oats-based media

A LC-DAD method is proposed for the determination of the T-2 and HT-2 toxins in cultures of Fusarium langsethiae in oat-based and other in vitro media. Test media consisted of freshly prepared milled oats to which T-2 and HT-2 toxin stock solutions were added. Different mixtures of extraction solvent (acetonitrile:water and methanol:water), extraction times (30′, 60′ or 90′) and drying methods were investigated. Results showed that extraction with methanol:water (80:20, v/v) for 90 min, drying with N₂ and subsequent analysis by LC-DAD was the fastest and most user friendly method for detecting HT-2 and T-2 toxins production by F. langsethiae strains grown on oat-based media at levels of 0.459 and 0.508 mg of toxin/kg of agar, respectively. The proposed method was used to investigate toxin production of 6 F. langsethiae strains from northern Europe and provided clear chromatograms with no interfering peaks in media with and without glycerol as water activity modifier.     

Mechanisms involved in the induction of apoptosis by T-2 and HT-2 toxins in HL-60 human promyelocytic leukemia cells

T-2 and HT-2 toxins belong to a group of mycotoxins that are widely encountered as natural contaminants known to elicit toxic responses in hematopoietic cells. In the present study, HL-60 cells were used to characterize the apoptotic effects of T-2 and a major metabolite, HT-2, and to examine the mechanisms involved. Apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation, by flow cytometric analysis, and by DNA gel electrophoresis. T-2 and HT-2 induced concentration-dependent apoptosis after 24 h in HL-60 cells, starting at concentrations of 3.1 and 6.25 ng/ml respectively. An increased number of apoptotic cells could be observed 4–6 h after exposure to 12.5 ng/ml of toxin. Little cytotoxicity (plasma membrane damage) was observed even after exposure to concentrations of toxins (25–50 ng/ml) inducing apoptosis in 60–100% of the cells. The apoptotic process was almost completely blocked in the presence of the general caspase inhibitor zVAD.fmk. In contrast, no or only minor effects were observed with the more specific caspase inhibitors DEVD.CHO, IETD.fmk, and DEVD.fmk. As judged by Western blotting, the levels of several procaspases (-3, -7, -8, -9, but not -12) were reduced 3–6 h after exposure to toxin. Substantial increases in the presumed active form(s) of caspase-8 and -9 were observed. Furthermore, poly(ADP-ribose) polymerase (PARP) was already markedly cleaved 3 h after toxin treatment, indicative of active caspase-3 and -7. No or only minor changes in Bcl-2, Bcl-XL and Bax levels were observed. BAPTA-AM and ZnCl₂ blocked the degradation of procaspases, the fragmentation of PARP, and the induction of apoptosis. In summary, both T-2 and HT-2 induced apoptosis, with T-2 being somewhat more potent than HT-2. The divalent calcium concentration, [Ca²⁺], appears to be involved in the activation of several caspases, resulting in DNA fragmentation, chromosomal condensation, and nuclear fragmentation.     

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.     

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.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

Vorkommen von Deoxynivalenol, Zearalenon und HT-2/T-2 Toxin in Getreide und Getreideerzeugnissen

Since February 2004 in Germany maximum limits forFusarium toxins do exist, while harmonised legislation within the EU was recently published and will come into force in July 2006. Meanwhile, the problematic nature ofFusarium mycotoxins is perceived by all participants of the processing chain of cereals. In this study the presence of deoxynivalenol and zearalenone in comparison to the rarely investigated type A-trichothecenes (HT-2, T-2 toxin) in different cereal-products is discussed. About 1000 cereal-based samples have been analysed using a recently developed multitoxin method based on HPLC-MS/MS technique. Despite, up to now no concrete limit for HT-2/T-2 toxin is discussed, the degree of contamination is of special concern for food products dedicated to be placed on the market, to avoid possible risks for consumers. The used method proved to be extremely sensitive for T-2 toxin with a LOD below 1 μg/kg, therefore a comprehensive data set was achieved.     

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.     

Interacting climate change environmental factors effects on Fusarium langsethiae growth,expression of Tri genes and T-2/HT-2 mycotoxin production on oat-based media and in stored oats

This study examined the effect of climate change (CC) abiotic factors of temperature (20, 25, 30 °C), water activity (a_w; 0.995, 0.98) and CO₂ exposure (400, 1000 ppm) may have on (a) growth, (b) gene expression of biosynthetic toxin genes (Tri5, Tri6, Tri16), and (c) T-2/HT-2 toxins and associated metabolites by Fusarium langsethiae on oat-based media and in stored oats. Lag phases and growth were optimum at 25 °C with freely available water. This was significantly reduced at 30 °C, at 0.98 a_w and 1000 ppm CO₂ exposure. In oat-based media and stored oats, Tri5 gene expression was reduced in all conditions except 30 °C, 0.98 a_w, elevated CO₂ where there was a significant (5.3-fold) increase. The Tri6 and Tri16 genes were upregulated, especially in elevated CO₂ conditions. Toxin production was higher at 25 °C than 30 °C. In stored oats, at 0.98 a_w, elevated CO₂ led to a significant increase (73-fold) increase in T2/HT-2 toxin, especially at 30 °C. Nine T-2 and HT-2 related metabolites were detected, including a new dehydro T-2 toxin (which correlated with T-2 production) and the conjugate, HT-2 toxin, glucuronide. This shows that CC factors may have a significant impact on growth and mycotoxin production by F. langsethiae.     

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.     

The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.     

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).     

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.     

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).     

Intake estimates for trichothecene toxins of the population in Southwest Germany in 1998 and in 1999

The intake of theFusarium toxins deoxynivalenol (DON), nivalenol (NIV), HT-2 and T-2 toxin (HT-2, T-2), 3-, 15-acetyldeoxynivalenol (3-, 15- ADON), and fusarenon-X (FUS-X) was calculated for adults, children and babies, for an area of southwest Germany and two years (1998, 1999). Estimates were based on consumption data of bread and pasta by both adults and children and of infant food by babies, reported for the German population in a study on behalf of the European Union, and on toxin contents of a total of 208 samples of these commodities. No exceeding of the tolerable daily intake (TDI) of DON, NIV and the sum of HT-2 and T-2, as stated by the EU, was found for adults (70 kg body weight (BW)) and for babies (10 kg BW), independent of year and level of consumption. For children (20 kg BW) the intake of DON exceeded the TDI in 1998 for high, and in 1999 for both mean and high consumers. For both years the intake of the sum of HT-2 and T-2 was below the TDI following mean but above this value following high consumption. The intake of NIV was far below the TDI for both levels of ingestion. The daily intake of each of the three toxins 3-, 15- ADON and FUS-X was below 0.03, 0.11 and 0.05 μg/kg/BW for adults, children and babies, respectively. Presented at the 27^th Mykotoxin-Workshop, Dortmund. Germany, June 13–15, 2005     

Fusarium toxin contents of maize and maize products purchased in the years 2000 and 2001 in Germany

Samples (n=106) of maize and maize products were analysed for 13 trichothecene toxins and zearalenone (ZON). All 14 toxins examined were detected, although with varying frequency. Cooccurrence of two or more toxins was observed in 96% of samples. The toxins of the scirpenol group scirpentriol, 15-monoacetoxyscirpenol and diacetoxyscirpenol were detected in 14, 27 and 3% of the samples analysed, the toxins of the T-2 group T-2 toxin, HT-2 toxin, T-2 triol und T-2 tetraol were found in 33, 66, 2 and 7%. Toxin content was higher in feeds than in foods (semolina and flour). In food samples, the German regulatory level for DON (500 μg/kg) was not exceeded, three samples of maize flour contained ZON above the regulatory level (50 μg/kg). Presented at the 26^th Mykotoxin-Workshop in Herrching, Germany, May17–19, 2004