Suitability of the Neuro-2a cell line for the detection of palytoxin and analogues (neurotoxic phycotoxins)

Palytoxin and related compounds are neurotoxic phycotoxins produced by benthic microalgae belonging to the genus Ostreopsis. For several years this family of phycotoxins has been posing a threat to human health since they can bioaccumulate in shellfish. With the aim of replacing current biological assays, such as the mouse or hemolytic assays, we investigated using the Neuro-2a neuroblastoma cell line to detect palytoxin and related compounds. Cell death induced by the effects of PlTX and analogues on Na+, K+-ATPase were measured using the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay for mitochondrial reductase activity as a surrogate for cell number. The specificity of the Neuro-2a cell-based assay for palytoxin detection was confirmed by using ouabain, which also acts on Na+, K+-ATPase. Pre-treatment of the Neuro-2a cells with ouabain minimizes the effects of palytoxin. The specificity of the Neuro-2a assay was confirmed by the finding that cell death was not detected when Neuro-2a cells were exposed to other phycotoxins with unrelated cellular targets. When the Neuro-2a assay was used to detect palytoxin in mussel extracts spiked with levels of palytoxin around the proposed regulatory value of 250 μg palytoxin/kg shellfish, a good correlation was observed between the levels found and the expected values.We conclude by proposing an experimental design for functional assays using the Neuro-2a cell line for the specific detection of four neurotoxic phycotoxin families: saxitoxins, brevetoxins, ciguatoxins and palytoxins.