Effects of growth temperature on toxicity of T-2 toxin and roridin A to yeast

In yeasts, growth temperature is known to affect the membrane phospholipid content. The effect of temperature on the growth inhibition of Kluyveromyces marxianus and Saccharomyces cerevisiae by the trichothecene mycotoxins, T-2 toxin and roridin A, was investigated. Examination of EC50 values for T-2 toxin and roridin A showed that these toxins were least inhibitory to both yeasts at 30 and 25 degrees C, respectively. Increasing or decreasing growth temperature from these temperatures gradually increased the inhibitory effect of the trichothecene mycotoxins. Temperature may affect the toxicity of the trichothecenes to the yeasts by regulating the composition of yeast cell membranes.     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

Spontaneous regression of pulmonary paracoccidioidomycosis

The Fusarium and Myrothecium mycotoxins roridin A, diacetoxyscirpenol, verrucarin A, T-2 toxin and zearalenone (10(-2) and 10(-3) mg/ml) inhibit the unspecific dehydrogenase activity of baker's yeast (Saccharomyces cerevisiae) in vivo. The action of these toxins is in the same order as that of aflatoxin B1. It is suggested that at least the trichothecenes decrease the dehydrogenase activity by an interaction with thiol groups of the active center of the enzymes.     

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.     

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.     

Correlation of Biological to Chromatographic Data for Two Mycotoxins Elaborated by Fusarium

Thirty-seven identified strains of Fusarium, most of them isolated from fescue grass, were tested for their ability to elaborate mycotoxins in laboratory culture. The presence of the toxins was determined by infrared light, thin-layer chromatography, mouse toxicity, fungistatic effects, and phytotoxic properties. A good correlation was demonstrated between T-2 toxin detection by thin-layer chromatography and inhibition of Rhodotorula rubra by culture extracts. All of the strains producing either butenolide or T-2 toxin were toxic to mice with but one exception; those producing T-2 toxin inhibited growth of the yeast.     

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.     

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.     

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.     

A Colorimetric Technique for Detecting Trichothecenes and Assessing Relative Potencies

We tested a novel colorimetric toxicity test, based on inhibition of β-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B₁, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 μg/ml, nor could aflatoxins B₁ and M₁ be detected at concentrations up to 25 μg/ml. This test should be useful for trichothecene detection and for studies of relevant interactions—both between trichothecenes themselves and between trichothecenes and other food constituents.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp.

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

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).     

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.     

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).     

The effect of combinations of Fusarium mycotoxins (deoxynivalenol, zearalenone and fumonisin B1) on growth of brewing yeasts

The interactive effect of combinations of the Fusarium mycotoxins deoxynivalenol (DON), zearalenone (ZEA) and fumonisin B1 (FB1) on growth of brewing yeasts was examined. Yeast growth was assessed by measurement of dry weight or relative growth, cell number, viability and conductance change of the growth medium using direct and indirect methods. The interactive effect of a combination of these mycotoxins was subject to the ratio of toxins in the mixture and the toxicity of individual toxins on yeast growth. When a combination of mycotoxins at low concentration was added into the growth medium, no significant inhibitory effect on growth was observed compared to controls. However, when a combination of high concentrations of DON and ZEA which individually inhibited yeast growth was examined, the interactive effect was shown to pass from antagonism to synergism depending on the ratio of the toxins in the mixture. As a synergistic interaction between these Fusarium mycotoxins was observed only at high concentrations, which were far higher than would be expected in good quality grain, they are not a concern when related to yeast growth under the brewing conditions studied.     

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.     

Chromatographic analysis of Fusarium toxins in grain samples

Summary A method for the analysis of zearalenone, T-2 toxin, neosolaniol and HT-2 toxin from the grains of barley, wheat and oats has been developed. Toxins are extracted with ethyl acetate, purified on a kieselgel TLC-plate and analysed on a HPTLC-plate. The limits of detection are 0.2 mg/kg for zearalenone and T-2 toxin and 5 mg/kg for neosolaniol and HT-2 toxin. For more accurate estimation the purified toxins are analysed as their trimethysilyl derivatives by gas chromatography, in which the detection limit for all toxins in 50 g/kg and the accuracy ±10%–30%. The percentage recovery in both methods is 80%.