Microbial and chemical transformations of some 12,13-epoxytrichothec-9,10-enes.
Resting cells of Streptomyces griseus, Mucor mucedo, and a growing culture of Acinetobacter calcoaceticus when mixed with compounds related to 12,13-epoxytrichothec-9-ene-4beta,15-diacetoxy-3alpha-ol(anguidine) produced a series of derivatives that were either partially hydrolyzed or selectively acylated. These derivatives showed marked differences in activities as assayed by antifungal and tissue culture cytotoxicity tests. (+info)
Biosynthesis of 3-acetyldeoxynivalenol and sambucinol. Identification of the two oxygenation steps after trichodiene.
The first two oxygenation steps post-trichodiene in the biosyntheses of the trichothecenes 3-acetyldeoxynivalenol and sambucinol were investigated. The plausible intermediates 2-hydroxytrichodiene (2alpha- and 2beta-) and 12,13-epoxytrichodiene and the dioxygenated compounds 12,13-epoxy-9,10-trichoene-2-ol (2alpha- and 2beta-) were prepared specifically labeled with stable isotopes. They were then fed separately and/or together to Fusarium culmorum cultures, and the derived trichothecenes were isolated, purified, and analyzed. The stable isotopes enable easy localization of the labels in the products by 2H NMR, 13C NMR, and mass spectrometry. We found that 2alpha-hydroxytrichodiene is the first oxygenated step in the biosynthesis of both 3-acetyldeoxynivalenol and sambucinol. The stereoisomer 2beta-hydroxytrichodiene and 12,13-epoxytrichodiene are not biosynthetic intermediates and have not been isolated as metabolites. We also demonstrated that the dioxygenated 12, 13-epoxy-9,10-trichoene-2alpha-ol is a biosynthetic precursor to trichothecenes as had been suggested in a preliminary work. Its stereoisomer was not found in the pathway. A further confirmation of our results was the isolation of both oxygenated trichodiene derivatives 2alpha-hydroxytrichodiene and 12,13-epoxy-9, 10-trichoene-2alpha-ol as natural metabolites in F. culmorum cultures. (+info)
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. (+info)
Overt signs of toxicity to dogs and cats of dietary deoxynivalenol.
Studies were conducted to determine the dietary amounts of deoxynivalenol (DON; vomitoxin) in dog and cat food that are required to produce overt signs of toxicity (e.g., vomiting or reduced food intake). Wheat naturally contaminated with 37 mg of DON/kg was used to manufacture pet foods containing 0, 1, 2, 4, 6, 8, and 10 mg of DON/kg. Deoxynivalenol concentration in pet food following manufacture was unchanged, indicating that the toxin was stable during conventional extrusion processing. Dogs previously fed DON-contaminated food were able to preferentially select uncontaminated food. Dogs not previously exposed to DON-contaminated food consumed equal quantities of contaminated and uncontaminated food. There was no effect of 6 mg of DON/kg on dog food digestibility. Food intake of dogs was significantly reduced by DON concentrations greater than 4.5 +/- 1.7 mg/kg, and DON greater than 7.7 +/- 1.1 mg/kg reduced cat food intake. Vomiting by dogs and cats was commonly observed at the 8 and 10 mg DON levels. (+info)
Trichothecene mycotoxins trigger a ribotoxic stress response that activates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase and induces apoptosis.
The trichothecene family of mycotoxins inhibit protein synthesis by binding to the ribosomal peptidyltransferase site. Inhibitors of the peptidyltransferase reaction (e.g. anisomycin) can trigger a ribotoxic stress response that activates c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases, components of a signaling cascade that regulates cell survival in response to stress. We have found that selected trichothecenes strongly activate JNK/p38 kinases and induce rapid apoptosis in Jurkat T cells. Although the ability of individual trichothecenes to inhibit protein synthesis and activate JNK/p38 kinases are dissociable, both effects contribute to the induction of apoptosis. Among trichothecenes that strongly activate JNK/p38 kinases, induction of apoptosis increases linearly with inhibition of protein synthesis. Among trichothecenes that strongly inhibit protein synthesis, induction of apoptosis increases linearly with activation of JNK/p38 kinases. Trichothecenes that inhibit protein synthesis without activating JNK/p38 kinases inhibit the function (i.e. activation of JNK/p38 kinases and induction of apoptosis) of apoptotic trichothecenes and anisomycin. Harringtonine, a structurally unrelated protein synthesis inhibitor that competes with trichothecenes (and anisomycin) for ribosome binding, also inhibits the activation of JNK/p38 kinases and induction of apoptosis by trichothecenes and anisomycin. Taken together, these results implicate the peptidyltransferase site as a regulator of both JNK/p38 kinase activation and apoptosis. (+info)
Identification of mimotope peptides which bind to the mycotoxin deoxynivalenol-specific monoclonal antibody.
Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON. (+info)
Development and use of a reverse transcription-PCR assay to study expression of Tri5 by Fusarium species in vitro and in planta.
The Tri5 gene encodes trichodiene synthase, which catalyzes the first reaction in the trichothecene biosynthetic pathway. In vitro, a direct relationship was observed between Tri5 expression and the increase in deoxynivalenol production over time. We developed a reverse transcription (RT)-PCR assay to quantify Tri5 gene expression in trichothecene-producing strains of Fusarium species. We observed an increase in Tri5 expression following treatment of Fusarium culmorum with fungicides, and we also observed an inverse relationship between Tri5 expression and biomass, as measured by beta-D-glucuronidase activity, during colonization of wheat (cv. Avalon) seedlings by F. culmorum. RT-PCR analysis also showed that for ears of wheat cv. Avalon inoculated with F. culmorum, there were different levels of Tri5 expression in grain and chaff at later growth stages. We used the Tri5-specific primers to develop a PCR assay to detect trichothecene-producing Fusarium species in infected plant material. (+info)
Toxic effects of mycotoxins in humans.
Mycotoxicoses are diseases caused by mycotoxins, i.e. secondary metabolites of moulds. Although they occur more frequently in areas with a hot and humid climate, favourable for the growth of moulds, they can also be found in temperate zones. Exposure to mycotoxins is mostly by ingestion, but also occurs by the dermal and inhalation routes. Mycotoxicoses often remain unrecognized by medical professionals, except when large numbers of people are involved. The present article reviews outbreaks of mycotoxicoses where the mycotoxic etiology of the disease is supported by mycotoxin analysis or identification of mycotoxin-producing fungi. Epidemiological, clinical and histological findings (when available) in outbreaks of mycotoxicoses resulting from exposure to aflatoxins, ergot, trichothecenes, ochratoxins, 3-nitropropionic acid, zearalenone and fumonisins are discussed. (+info)