Study On Cell Bioassay For Diarrhetic Shellfish Poisoning

Diarrhetic shellfish poisoning (DSP) are a group of marine toxins, which usually coexist with shellfish and consist primarily of okadaic acid (OA) and its derivatives. The most commonly reported symptom of human intoxication was diarrhoea. OA has been reported as a tumor promoter and it may pose a threat to human health even at extremely low concentration, so the long term effects of OA must be attentioned. A variety of methods with both advantages and disadvantages have been developed for the detection of DSP, but a convenient, rapid and accurate routine method is not yet developed.HL-7702 Human liver cell line was employed to study the cell bioassay for DSP using OA standard solution. Firstly, the cytotoxic effect of OA on cell proliferation was investigated by cell counting kit-8, while the apoptosis induced by OA was examed by morphology and flow cytometry. Then, Images of HL-7702 was scanned by confocal laser scanning microscope after being treated with OA. Furthermore, TECAN Infiite M1000 was used for the quantitation of F-actin depolymerization and the cell F-actin assay was primarily developed for the detection of DSP. The feasibility of this assay was evaluated by comparative analysis of shellfish samples by the cell F-actin assay, mouse bioassay and ELISA. Main conclusions are as follow:(1) OA inhibited the proliferation of HL-7702 significantly in a time and dose dependent manner with IC50 of OA being 89.684 nmol/L when the exposure time is 24 h.(2) After 24 h of exposure, OA induced morphological changes in HL-7702 which consist with the characteristics of cell apoptosis. Then cell apoptosis was detected by flow cytometry after Annexin V-FITC and PI double staining. The result showed that OA can induce cell apoptosis when the concentration was as low as 5 nmol/L, and the early apoptotic ratio increased along with the increase of OA concentration.(3) After 24 h of exposure, OA induced depolymerization of F-actin in HL-7702 significantly, showing a linear relationship with the concentration of OA in the range of 2.5~40 nmol/L.(4) The methanolic extract obtained from shellfish sample should be dried under nitrogen, keeping the concentration of methanol below 4%.(5) The cell F-actin assay showed a specific, reproducible and accurate detection for DSP, having a detection limit of 2.5 nmol/L which correspond to 2.01μg/100 g.(6) 42 samples, previously detected positive by ELISA, were also submitted to the cell F-actin assay. The results obtained by cell F-actin assay correlated well with those obtained by ELISA. Compared with ELISA, the cell F-actin assay had lower detection limit, thus could assure the safety of shellfish more effectively.