Establishment Of Two Novel Cell Lines From Spotted Halibut (Verasper Variegates) And Barfin Flounder (Verasper Moseri), And Studies On The Cytotoxicities Of Tributyltin Oxide To The Cell Lines

In recent years, because of the industrialization and urbanization, the problems of environmental pollution have become increasingly serious. A large number of harmful substances were poured into the ocean, which led many marine fishes to death and caused great economic loss in aquaculture. These substances also posed the potential threat to our human health. Fish cell lines are important tools for studies on the detection of various environmental pollutants in the sea and on the evaluation of their toxicity in order to determine the critical concentrations that environmental pollutants can affect the marine fish. The cell lines are also used to filter out the appropriate Biometric indicators combined with physical and chemical testing methods and establish a system that can detect the toxicity of environmental pollutants rapidly, sensitively and effectively. However, most cell lines which have been established are freshwater fish and migratory fish, and marine fish cell lines are far from the needs of the study on cytotoxicities of pollutants in the ocean.Spotted halibut (Verasper variegates) and barfin flounder (Verasper moseri) belong to Verasper, Pleuronectidae, Pleuronectiformes, Osteicthys. They are two of the most important commercial fish which both live in cold water. Recently, the farming scales of the two fishes have been gradually expanding, which lead to enormous economic values in aquaculture. But with the increase of environmental pollution, they were deeply suffered with the pollutants. This study was conducted to establish two novel cell lines from spotted halibut and barfin flounder, and to apply them in the field of studies on environmental pollutants monitoring.To establish cell lines from the two fishes, the fin tissue of spotted halibut were digested with 0.5% hyaluronidase and 0.2% collagenaseⅡand the gill tissue of barfin flounder were digested with 0.25% tripsin. Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (1:1) powder (DMEM/F12) Medium were used to initiate the primary culture at 22℃, which was supplemented with 20% fetal bovine serum(FBS), 50μg/mL N-acetyl glucosamine hydrochloride,50μg/mL carboxymethyl chitosan, lOng/mL basic fibroblast growth factor (bFGF) and 40ng/mL I insulin-like growth factor (IGF-I). Seven days later, the cells of spotted halibut began to migrate from the fin tissues, which were in fibroblastic shape. The cultured cells formed to confluence at 20th day. Ten days after the initiation of primary culture, the cells of barfin flounder began to migrate from the gill tissues, which were also in fibroblastic morphology and proliferated to confluence at 30th day. The cells could be sub-cultured subsequently and maintained their original growth rate after cryopreservation and resuscitation. The population doubling time of the spotted halibut fin cell line and barfin flounder gill cell line were 41.5 hours and 54.4 hours at passage 60 respectively. Chromosome analysis showed that both of the cell lines have their Characteristic chromosome number in 46, although some cells are chromosomal aneuploidy. Karyotype analysis showed that the fin cells have a typical diploid karyotype(46t) and gill cells also have the typical character of the normal diploid karyotype(44t+2sm). To date, the fin cells have been sub-cultured up to passage 110 and gill cells have been sub-cultured up to passage 70. They still grow in a good proliferating status. The two continuous cell lines which named SHF01 and BFG01 respectively have been successfully established.To examine the cytotoxicity of tributyltin oxide(TBTO) on the two cell lines, we dealt the two cell lines with different concentrations of TBTO, and then detected in vitro cytotoxicities of TBTO by methods of MTT and determination of cell protein content. The results showed that,0.5ng/ml TBTO can be cytotoxic to the two cell lines, and the cytotoxicity increased with the concentrations of TBTO, which showed significant dose-effect dependence. The 48h-IC50 values of TBTO to fin cells were 41.58 and 44.49 ng/mL, and this values were 37.07and 39.48ng/mL in gill cells. After treated with TBTO, the fin and gill cells showed no significant morphological changes comparing with control. The result of enzyme activities experiment showed that the activities of SOD,GST and GSH-Px in barfin flounder gill cells treated with TBTO changed significantly at the concentration of 40ng/mL(p<0.05). It suggested that the activities of SOD,GST and GSH-Px in barfin flounder gill cells could be suitable Biometric indicators for the detection of TBTO. In summary, the two cell lines established in the present studies can be used as helpful tools for studies in the fields of environmental toxicology and cytotoxicology, which make great contributions to establish a system that can detect the toxicity of environmental pollutants rapidly, sensitively and effectively.