Establishment Of Ovary Cell Line From Barfin Flounder, Verasper Moseri, And Their Toxicity Treated By Endocrine Disruption Chemicals

Ocean environmental pollution is one of the major issues of global concern. A large number of chemical pollutants enter the ocean each year from various sources. These pollutants, including endocrine disruptors, are known to be potentially cytotoxic to biota and present a health threat to the public. Endocrine disruption chemicals (EDCs) have been confirmed to be a group of particular environmental contaminants, including some natural and artificial compound. It's extremely harmful to the reproduction and inheritance of organisms, though the content is very low in environment. The pollution of EDCs has raised increasing concern from every walk of life. It is required to establish a sensitive biological monitoring system for early detection and ecotoxicological evaluation. Cultured cells, as a sensitive bioassay, are relatively rapid, cost-effective and readily reproducible. It can be easily adapted to automated high-throughput screening technologies. Establishment of more and more cell lines from marine and mammalian species has promoted the rapid development of cell lines in assessing the toxicological risk associated with chemical pollutants worldwide.Although many fish cell lines have been established, few cell lines have been established from commercial marine fishes. This study was conducted to establish ovary cell line from barfin flounder, Verasper moseri, and to verify its application in the detection of EDCs toxicity.Ovary tissue from V. moseri was digested with 0.25% hyaluronidase and 0.2% collagenaseⅡto initiate the primary cell culture in Dulbecco's Modified Eagle Medium:Ham's Nutrient F-12(1:1) (DMEM/F12) medium supplemented with 20% fetal bovine serum(FBS), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and carboxymethyl-chitooligosaccharide at 22℃. The ovary cell in primary culture was in fibroblastic morphology and proliferated to confluence within 15 days. Growth property studies indicated that the ovary cell had population doubling time of 59.7 hours at passage 60. Karyotype analysis showed that the ovary cell exhibited chromosomal aneuploidy with a modal chromosome number of 46 and typical characteristics of the normal diploid karyotype (44t+2sm), which implied that the ovary cell was indeed from V. moseri. To date, barfin flounder ovary cell has been subcultured up to passage 65 and still grows in a good proliferating status. The continuous barfin flounder ovary cell line named BFO cell line has been successfully established.In order to research the cytotoxicity and oxidative damage to ovary cell from EDCs, cadmium chloride(CdCl2), lead chloride(PbCl2) and polychorinated biphenyls(Aroclor 1254), were chosen to treat BFO cells with different concentrations. The acute cytotoxic effects of EDCs were evaluated by Morphological observation and MTT assay. The results showed that 10μmol/L CdCl2 and PbCl2 were toxic to BFO cells in a concentration-dependent manner. While only high concentration of Aroclor 1254 (1000μg/L) was toxic to BFO cells. After treated by EDCs, BFO cells became round and lysed, and began to detach and finally died within several hours.After treated with the 40μmol/L CdCl2,80μmol/L PbCl2 and high level Aroclor 1254 (1000ug/L), the activities of superoxide dismutase(SOD), glutathione peroxidase (GSH-Px) and malondialdehyde(MDA) in BFO cells were influenced in a concentration-dependent manner. It suggested that endocrine disruptors cause oxidative damage of BFO cells.In order to detect the effects of EDCs (CdCl2 and PbCl2) on the apoptosis of BFO cells, further research was carried out by AO/EB double-fluorescent staining and DNA electrophoresis. After treated with 40-160μmol/L of CdCl2 and 80-320μmol/L of PbCl2 respectively for 24h, cultured BFO cells underwent apoptosis, the respective apoptosis rates are 30%-85% and 20%-70%. The apoptotic extent was in a concentration-dependent manner.In order to detect the genotoxicity of EDCs, the DNA damage caused by CdCl2 to BFO cells were determined by single cell gel electrophoresis (SCEG) assay. After treated with 20μmol/L of CdCl2 for 24h, cultured BFO cells showed DNA damage, and the extent was in a concentration-dependent manner. It suggested that CdCl2 have intensive genotoxicity to BFO cells.Through the detection of reproductive toxicity and genotoxicity of EDCs to the established ovary cell line from barfin flounder, we can conclude that the cell line can be used as a useful tool for the research of environmental toxicology.