Natural toxins最新文献

Fumonisins are mycotoxins produced by the maize pathogen Gibberella fujikuroi mating population A and frequently contaminate maize. Wild-type G. fujikuroi produces four B-series fumonisins, FB1, FB2, FR3 and FB4. These toxins are identical in structure except for the number and positions of hydroxyls along their linear carbon backbone. To elucidate the genetic and biosynthetic relationships among these fumonisins, we conducted meiotic and biochemical analyses of G. fujikuroi mutants with altered fumonisin production that resulted from defective alleles at three loci, Fum1, Fum2 and Fum3. These mutants produced either no fumonisins, only FR2 and FB4, or only FR3 and FR4. Genetic analyses revealed the orientation of the Fum loci along linkage group 1 of the fungus. The mutants were grown together in pair-wise combinations to determine if their fumonisin production phenotypes could be complemented. When FR3- and FB2-producing mutants were grown together, complementation occurred. However, when a nonproducing mutant was grown with a FR2- or FB3-producing mutant, complementation did not occur or was incomplete. When purified FR2, FR3, or FB4 was fed to mutant cultures, FR4 was converted primarily to FR2, FR3 was converted to FB1 and FB2 was not converted. The results from these assays suggest a previously unrecognized branch in the fumonisin biosynthetic pathway.

Like all eucaryotic cells, yeasts are sensitive to trichothecenes, especially T-2 toxin and verrucarin A. Based on this sensitivity, a yeast bioassay was developed to evaluate the toxicity of corn samples. The bioassay was optimized using spiked maize extracts. The toxicity of samples was defined as toxicity equivalent to a certain concentration of T-2 toxin standards. The assay can be performed on crude extracts, but the results are more precise after column clean-up. The test can also be used for the screening of trichothecene toxicity in general. The relative standard deviation (RSD) at 85 % growth inhibition (EC85) was 4.5% for the T-2 toxin standards (n = 8). This corresponds to an initial T-2 toxin concentration of approximately 58 ppb in the corn sample. Samples containing 188 and 113 ppb T-2 toxin caused a growth inhibition higher than 85%, whereas samples with toxin concentrations of 56 and 19 ppb had a growth inhibition less than 85%. Therefore the test can be used for the qualitative evaluation of corn samples up to a level of 58 ppb +/- 2.8 ppb. The bioassay is easy to perform with minimum requirements for equipment. Results can be obtained within 24 h and a large number of samples can be analysed daily. The costs are low and the results obtained are repeatable. With some modifications this test can be used for toxicity studies on trichothecene metabolites as well as for extracts with unknown compounds with properties similar to trichothecenes.

A negative mode liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) method was developed to detect low concentrations of the diarrhetic shellfish poisoning (DSP) toxins okadaic acid and dinophysistoxin-1 (DTX-1). Detection relies upon monitoring the transition of negative precursor ions [M - H]- to a common fragment ion of m/z 255. Our limit of detection for okadaic acid with this method is 0.5 pg on column. LC-SRM MS has allowed us to detect persistent, low concentrations of DSP toxins from Singapore shellfish.

Evanescent wave-based fiber-optic immunosensors were studied for the detection of fumonisins and aflatoxins in maize. Two formats, competitive and non-competitive, were used. A competitive format was used to measure fumonisin B1 (FB1) in both spiked and naturally contaminated maize samples. Fumonisin monoclonal antibodies were covalently coupled to an optical fiber and the competition between FB1 and FB1 labeled with fluorescein (FB1-FITC) for the limited number of binding sites on the fiber was assessed. The signal generated in the assay was inversely proportional to the FB1 concentration. For samples, the concentration causing an inhibition of binding by 50% (IC50) was dependent upon the clean-up procedure used. Simple dilution of methanolic maize extracts yielded an assay with an IC50 equivalent to 25 microg FB1 g(-1) maize with a limit of detection of 3.2 microg g(-1) maize. Affinity column clean-up yielded an assay with an IC50 equivalent to 5 microg FB1 g(-1) maize (limit of detection 0.4 microg FB1 g(-1)). An HPLC method and the immunosensor method agreed well for naturally contaminated maize samples except when large amounts of other fumonisins that cross-react with the immunosensor were present. The second sensor format, for the mycotoxin aflatoxin B1 (AFB1), was a non-competitive assay using the native fluorescence of this mycotoxin. Because the fluorescence of AFB1 itself was detected, the response of the sensor was directly proportional to the toxin concentration. The sensor, while capable of detecting as little as 2 ng ml(-1) of AFB1 in solution was technically not an immunosensor, since the attachment of aflatoxin specific antibodies was not required. Sensors of the formats described have the potential to rapidly screen individual maize samples but require coupling with a clean-up technique to be truly effective.

Fumonisins are mycotoxins of world-wide distribution in maize infected by the fungus Fusarium verticillioides. They are highly toxic to certain livestock and are potential carcinogens. Exophiala spinifera, a black yeast fungus found on moldy maize kernels, was identified previously as capable of growing on fumonisin B1 as a sole carbon source and thus is a potential source for fumonisin detoxifying enzymes. Pure cultures of E. spinifera transform fumonisin B(1) to the amino polyol AP(1) plus free tricarballylic acid through the activity of a soluble extracellular esterase, and further transformation is evidenced by accumulation in culture supernatant of a less polar compound(s) lacking a fluorescamine-reactive amino group. A free amine is thought to be critical for biological activity of FB(1) or AP(1). As a first step towards characterizing this amine-modifying activity, we investigated the biotransformation of AP(1) by E. spinifera liquid cultures that had been previously grown in liquid medium containing AP(1) as a sole carbon source. Accumulation of AP(1)-derived metabolites was monitored by thin-layer chromatography of culture supernatants, and product metabolites were purified and evaluated by mass spectrometry and nuclear magnetic resonance. Two products of treatment of purified AP(1) with cultures of E. spinifera are shown to be N-acetyl AP(1) and a new compound, 2-oxo-12,16-dimethyl-3,5,10, 14,15-icosanepentol hemiketal (or 2-OP(1) hemiketal).