Highly sensitive protein translation assay for trichothecene toxicity in airborne particulates: comparison with cytotoxicity assays.
Screening assays for environmental mycotoxins in bulk samples currently use cytotoxicity in cell cultures, but their application to air particulate samples often lacks sensitivity and specificity for fungal spores. An assay based on inhibition of protein synthesis using translation of firefly luciferase in a rabbit reticulocyte system has been developed for the detection of trichothecene mycotoxins found in the spores of toxigenic fungi. Ethanol extracts of air particulates trapped on polycarbonate filters are ultrafiltered and applied at several dilutions to a translation reaction mixture. The activity of translated luciferase is measured directly in a luminometer, eliminating the need for radioisotopes and time-consuming sample processing. Parallel standard curves using a commercially available trichothecene provide for expression of the results in T-2 toxin equivalents per cubic meter of air. The assay can be completed in 2 h and is readily applicable to multiple samples. Comparison to the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide cytotoxicity assay indicates a 400-fold increase in sensitivity of trichothecene detection in addition to a much higher specificity for these toxins. Initial field testing indicates a strong correlation between the measured level of toxicity and the presence of toxigenic fungi detected with microbiological methods. In conclusion, this luciferase translation assay offers a rapid and highly sensitive and specific method for quantitative detection of trichothecene mycotoxin activity in air particulate samples.
Yike
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Department of Pediatrics, Division of Pediatric Pulmonology, Rainbow Babies and Childrens Hospital, Case Western Reserve University, Cleveland, Ohio 44106-6006, USA.
Allan
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Sorenson
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Dearborn
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DG
D000393:Air Pollutants / Q000032:analysis / Q000633:toxicity*; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D005069:Evaluation Studies as Topic; D008156:Luciferases / Q000235:genetics; D009183:Mycotoxins / Q000032:analysis / Q000633:toxicity*; D014176:Protein Biosynthesis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012680:Sensitivity and Specificity; D013172:Spores, Fungal / Q000737:chemistry; D014255:Trichothecenes / Q000032:analysis / Q000633:toxicity*
D000393:Air Pollutants; D009183:Mycotoxins; D014255:Trichothecenes; D008156:Luciferases