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).     

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.     

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.     

Trichothecene mycotoxins produced byFusarium sporotrichioides strain P-11

A highly toxic strain ofFusarium sporotrichioides Sherb. (P-11) isolated from wheat in Poland produced on rice culture up to 11 trichothecenes, which are: T-2 toxin (750 ppm), neosolaniol (300 ppm), HT-2 toxin (75 ppm), acetyl T-2 toxin (35ppm), 3′-hydroxy-T-2 (20ppm), T-2 triol (12.5ppm), 3′-hydroxy-HT-2 (1.2ppm), 4-acetoxy-T-2 tetraol (1.1 ppm), 15-acetoxy-T-2 tetraol (0.65 ppm), 8-acetoxy-T-2 tetraol (0.45 ppm), and T-2 tetraol (0.2 ppm). The presence of most of these trichothecenes, including the 3′-hydroxy-derivatives, in the excreta of animals treated with T-2 toxin indicates the existence of some correlation between T-2 toxin metabolism in animals and microorganisms, respectively.     

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.     

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.     

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.     

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.     

Trichothecene Production in Liquid Stationary Cultures of Fusarium tricinctum NRRL 3299 (Synonym: F. sporotrichioides): Comparison of Quantitative Brine Shrimp Assay with Physicochemical Analysis

Stationary liquid cultures of Fusarium tricinctum NRRL 3299 (synonym: F. sporotrichioides) produce T-2 toxin, neosolaniol, diacetoxyscirpenol, and HT-2 toxin when cultured on peptone-enriched Czapek Dox medium. At 15 and 27°C, maximum T-2 toxin yield (265 and 50 μg/ml) was found after 10 to 14 and 7 days, respectively. The T-2 toxin in the culture medium was metabolized rapidly at 27°C and slowly at 15°C. Addition of 0.025% (wt/vol) sorbic acid to the medium resulted in an increased production of trichothecenes at 15°C (400 μg of T-2 per ml after 14 days). Trichothecenes in the culture liquid were determined by the brine shrimp bioassay and physicochemical analysis. The brine shrimp assay was improved by using modern bioassay equipment, including tissue culture trays and multipipettes, and by a standardized approach with positive and negative controls. The physicochemical analysis was based on adsorption of the trichothecenes onto Amberlite XAD-2 columns, derivatization with trifluoroacetic anhydride followed by capillary gas chromatography, and identification by mass spectrometry (as many as 17 trichothecenes were detected in the culture medium). The brine shrimp assay offers an interesting monitoring system for the quantitation of T-2 toxin and should be useful for studies on production of this toxin in culture. Specific information on less toxic trichothecenes, however, requires a more time-consuming chemical analysis.     

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.     

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.     

Production of antibody against T-2 toxin.

Antibody against T-2 toxin was obtained after immunization of rabbits with bovine serum albumin-T-2 hemisuccinate conjugate. The antibody had greatest binding efficiency for T-2 toxin, less efficiency for HT-2, and least for T-2 triol. Cross-reaction of antibody with neosolaniol, T-2 tetraol, and 8-acetyl-neosolaniol was very weak. Diacetoxyscirpenol, trichodermin, vomitoxin, and verrucarin A essentially gave no cross-reaction with the antibody. The sensitivity of the binding assay for T-2 toxin detection was in the range of 1 to 20 ng per assay. Detailed methods for the preparation of the conjugate and the production of immune serum and methods for antibody determination are described.     

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.     

Isolation and characterization of two new trichothecenes from Fusarium sporotrichioides strain M-1-1.

Two new trichothecenes were isolated along with T-2 toxin, neosolaniol, and HT-2 toxin from the culture filtrate of Fusarium sporotrichioides strain M-1-1. The structures of the new toxins were characterized to be 4 beta, 8 alpha-diacetoxy-12,13-epoxytrichothec-9-ene-3 alpha, 15-diol (designated NT-1) and 4 beta-acetoxy-12,13-epoxy-trichothec-9-ene-3 alpha,8 alpha,15-triol (designated NT-2).     

Immunochemical analysis of trichothecenes produced by various fusaria

The production of type A trichothecene mycotoxins by 19 Fusaria, including 12Fusarium sporotrichioides, 4F. chlamydosporum and 3F. graminearum at 15°C and 25°C over a 35-day period was analyzed by ELISA using antibodies cross-reactive with most type A trichothecenes after conversion to T-2 tetraol tetraacetate. The toxin production peaked at 20–25 days of incubation with maximum yield between 4–6 mg type A trichothecene/ml of culture medium for 5F. sporotrichioides cultures and between 1 to 2 mg/ml for 6F. sporotrichioides cultures. OneF. sporotrichioides produced 700 µg type A trichothecenes/ml of culture medium. Detectable type A trichothecene was also found in the culture extracts ofF. chlamydosporum andF. graminearum, but the yield was very low (less than 100 µg/ml). Quantitative determination of individual trichothecenes was achieved by separation of different toxin in HPLC and followed by ELISA analysis. Eight to 10 immunoreactive peaks, corresponding to various type A trichothecenes, were detected in all the fungal extracts. T-2 tetraol (T-2-4ol), 4-acetyl-T-2 tetraol (4-Ac-T-2-4ol), neosolaniol (NEOS), diacetoxyscirpenol (DAS), HT-2 and T-2 toxin accounted for more than 85% of the total toxins. In general, low temperature was preferred for total type A trichothecene production. More T-2-4ol, 4-Ac-T-2-4ol, HT-2 and DAS were produced at 25°C. In contrast, more T-2 toxin and NEOS were produced at 15°C. Transformation of T-2 toxin and NEOS to polar metabolites such as T-2-4ol, 4-acetyl-T-2-4ol and HT-2 by various strains were observed at both temperatures after 25 days incubation.     

T-2 toxin metabolism by ruminal bacteria and its effect on their growth.

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.     

Determination of T-2 toxin,HT-2 toxin,and three other type A trichothecenes in layer feed by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)—comparison of two sample preparation methods

A sensitive method for the simultaneous determination of T-2 toxin, HT-2 toxin, neosolaniol, T-2 triol, and T-2 tetraol in layer feed using high-performance liquid chromatography coupled to triple quadrupole mass spectrometry in the positive ionization mode (LC-ESI-MS/MS) is described. Two fast and easy clean-up methods—with BondElut Mycotoxin and MycoSep 227 columns, respectively—were tested. The separation of the toxins was conducted on a Pursuit XRs Ultra 2.8 HPLC column using 0.13 mM ammonium acetate as eluent A and methanol as eluent B. Detection of the mycotoxins was carried out in the multiple reaction monitoring (MRM) mode using ammonium adducts as precursor ions. Quantification of all analytes was performed with d₃-T-2 toxin as an internal standard. The clean-up method with MycoSep 227 columns gave slightly better results for layer feed compared to the method using BondElut Mycotoxin columns (MycoSep 227: recovery between 50 and 63 %, BondElut Mycotoxin: recovery between 32 and 67 %) and was therefore chosen as the final method. The limits of detection ranged between 0.9 and 7.5 ng/g depending on the mycotoxin. The method was developed for the analysis of layer feed used at carry-over experiments with T-2 toxin in laying hens. For carry-over experiments, it is necessary that the method includes not only T-2 toxin but also the potential metabolites in animal tissues HT-2 toxin, neosolaniol, T-2 triol, and T-2 tetraol which could naturally occur in cereals used as feed stuff as well.     

Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay

A colorimetric MTT (tetrazolium salt) cleavage test was used to evaluate cytotoxicity of twenty-three Fusarium mycotoxins on two cultured human cell lines (K-562 and MIN-GL1) as well as their inhibitory effect on proliferation of phytohemagglutinin-stimulated human peripheral blood lymphocytes. The values of 50% inhibition of lymphocyte blastogenesis were very close to the 50% cytotoxic doses observed with the more sensitive cell line (MIN-GL1). T-2 toxin was the most cytotoxic with CD₅₀ and ID₅₀ values less than 1 ng/ml. Type A trichothecenes were the most cytotoxic followed by the type B trichothecenes; the non-trichothecenes were the least cytotoxic. The MTT cleavage test, in conjunction with cell culture, is a simple and rapid bioassay to evaluate cytotoxicity and immunotoxicity of Fusarium mycotoxins.Abbreviations Ac acetyl - ACU acuminatin - DAS diacetoxyscirpenol - DON deoxynivalenol - FUS fusarenon-X - HT-2 HT-2 toxin - MC mononuclear cell - MTT 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide - NEO neosolaniol - NIV nivalenol - NT-1 4,8-diacetoxy T-2 tetraol - PBS phosphate buffered saline - TAT-2T tetraacetoxy T-2 tetraol - T-2 T-2 toxin