A new simple screening method for the detection of cytotoxic substances produced by harmful red tide phytoplankton

A highly sensitive, but simple and quantitative, cytotoxic assay method for the detection of toxic substances produced by red tide phytoplankton was developed by utilizing Vero cells which were the most resistant to seawater among the six cell lines tested. Heterocapsa circularisquama, which is known to be highly toxic to shellfish, showed cytotoxicity to Vero cells in a cell-density dependent manner when Vero cells were directly exposed to the cell suspension of H. circularisquama in seawater-based plankton culture medium, whereas Heterocapsa triquetra, which is morphologically similar to H. circularisquama but non-toxic to shellfish, showed no cytotoxic effect. Since the potent cytotoxicity was also detected in the cell-free culture supernatant of H. circularisquama, it was suggested that a certain cytotoxic substance is extracellularly secreted by H. circularisquama. Furthermore, by this direct exposure method, we found that Alexandrium fraterculus, Alexandrium tamiyavanichii, Alexandrium tamarense, and Alexandrium affine but not Alexandrium taylorii and Alexandrium catenella cause toxic effect on Vero cells with different extent depending on species. By gel-filtration and subsequent two cytotoxicity assays using Vero and mouse neuroblastoma cell line (Neuro-2a), we found that high molecular weight cytotoxic substance distinct from paralytic shellfish poisoning toxins is present in the aqueous extract of A. tamarense. These results suggest that our 96-well microplate cytotoxicity assay using Vero cells is useful not only as a primary screening assay for the detection of potential toxic activity of harmful phytoplankton but also as a quantitative routine toxicity assay for following the active substances during the extraction and purification processes.