Establishment Of Two Novel Cell Lines From Barfin Flounder (verasper Moseri) And Spotted Halibut (verasper Variegates), And Their Cytotoxicities Treated By Monocrotophos

Fish cell lines are ideal in vitro research systems on environmental toxicology, virology, genetics, immunology, Pharmacology, et al, and have the high applied value. Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegates) are new rarer species and have the high economic value for their fresh meats and rich nutrition. These two cell lines can be not only applied in the studies of controlling disease and breeding new species, but also be precious research materials for the studies of fish of cytotoxicity, virology, genomics, genetic breeding and preservation of germplasm resources. Among them the detection of marine pollutants is one of important applications.Recently with the insectpest happening frequently, various pesticides have been used a lot all over the world. Organophosphorus was over used for their obvious effects. It made seawater suffer from excess pesticide through entering rivers and underground water, which led to the death of fishes and threatened to human health. Fish cell lines can evaluate the toxicity of environmental pollutants rapidly and accurately at cell level in order to determine the critical concentration that environment pollutants can affect marine fish, which will provide basic materials for marine ecological and environment health evaluation standard.To establish fin cell line from Verasper moseri and gill cell line from Verasper variegates, fin tissues digested with 0.5%hyaluronidase and 0.2%collagenaseⅡand gill tissues digested with 0.25%trypsin were cultured in vitro respectively. The cultures in vitro in Dulbecco's Modified Eagle Medium:Ham's Nutrient F-12(1:1)(DMEM/F12), L15, M199 medium respectively supplemented with fetal bovine serum(FBS), basic fibroblast growth factor, insulin-like growth factor-I and carboxymethyl-chitooligosaccharide showed that DMEM/F12 was the optimal medium. The cultures in vitro in the optimal medium at 18℃,22℃,26℃showed 22℃was the optimal temperature. The primary cultures of two kinds of tissues initiated in the optimal system showed that, fin tissues had cells migrated on the 10th day and proliferated to confluence on the 20th day, gill tissues had cells migrated on the 9th day and proliferated to confluence on the 30th day. The fin and gill cells in primary culture were all in fibroblastic morphology and grew at a steady rate during subsequent subcultures. To date, fin cells have been subcultured up to passage 135 and gill cells have been subcultured up to passage 80. Barfin flounder continuous fin cell line(BFF) and spotted halibut continuous gill cell line(SHG) have been successfully established. Growth property studies of BFF and SHG at passage 60 indicated that they had population doubling times of 35.1 h and 44.2h, respectively. Chromosome analysis of BFF cells and SHG cells at passage 60 showed two kinds of cells both had 46 chromosomes. Although BFF cells and SHG cells all exhibited chromosomal aneuploidy, the cells with 46 chromosomes still accounted for 70 percent of the total number of cells. In addition, Karyotype analysis showed that the two kinds of cells both have normal diploid Karyotypes, which were 44t+2sm and 46t, respectively. The results showed that BFF cells were barfin flounder cells and SHG cells were spotted halibut cells indeed.Monocrotophos is one kind of organophosphorus and used frequently. BFF cells and SHG cells were used to evaluate the cytotoxic effects of monocrotophos by MTT assay and cell protein assay. The result showed that 1μg/mL monocrotophos was toxic to BFF cells and SHG cells in concentration-dependent manner. The 48h-IC50 values of monocrotophos to BFF cells were 40.78 and 40.93ug/mL, both of which were rather close, and the 48h-IC50 values of monocrotophos to SHG cells were 39.27 and 38.22ug/mL, both of which were also close. The result showed that the 48h-IC50 of two kinds of cells had no evident difference and SHG cells were more sensitive to monocrotophos. After treated by above 20ug/mL monocrotophos, BFF and SHG cells all became round and dropped at 96h, which showed monocrotophos can cause morphological changes of cells. In the 10ug/mL monocrotophos-treated BFF cells, the activities of glutathione S-transferase(GST) changed significantly (P<0.05). In the 10ug/mL monocrotophos-treated SHG cells, the activities of superoxide dismutase (SOD) changed significantly (P<0.05), too. It suggested that lOug/mL monocrotophos could affect these enzyme activities with marked changes. The GST activities of BFF cells and the SOD activities of SHG cells could be suitable biomarkers for the detection of monocrotophos.