RESUMEN
Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone; however, an approximate 1000-fold increase in sensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts.
Asunto(s)
Intoxicación por Ciguatera/metabolismo , Ciguatoxinas/química , Citometría de Flujo/métodos , Músculo Esquelético/química , Saxitoxina/química , Canales de Sodio/química , Animales , Automatización , Línea Celular Tumoral , Peces Planos , Análisis de los Alimentos , Músculo Esquelético/metabolismo , NeuroblastomaRESUMEN
By virtue of their ability to block depolarization of nerve cells, the saxitoxins exert the toxic effects associated with paralytic shellfish poisoning and allow for their detection through various methodologies. When veratridine-induced depolarization is followed using voltage-sensitive fluorescent dyes, the presence of these toxic blocking agents can be observed as a decrease in fluorescence of dye-treated nerve cells. Detection using flow cytometry provides for selection of the most responsive population of cultured mouse neuroblastoma (Neuro 2a) cells thereby enhancing assay sensitivity and this approach can be accomplished in real time. The method is demonstrated in preliminary studies using saxitoxin and crude shellfish extracts.