Establishment of pantropic spotted dolphin (Stenella attenuata) fibroblast cell line and potential influence of polybrominated diphenyl ethers (PBDEs) on cytokines response

Abstract

The presence of polybrominated diphenyl ethers (PBDEs) in the aquatic environment is an issue of major concern which may be a cause of increasing prevalence and severity of diseases in marine mammals. Although, cell culture model development and in vitro investigation approach is a prime need of time to progress immunotoxic research on aquatic mammals. In this study, we stablished fibroblast cell line (pantropic spotted dolphin) to assess the potential effects of PBDEs on cytokines response. Cells were grown in 6 well cell culture plate and complete media (DMEM and Ham's F12 nutrient mixture, fetal bovine serum, antibiotic and essential amino acids) was provided. The primary culture of (PSP-LWH) cells identification was achieved by vimentin (gene and protein) expressions. Karyotyping revealed pantropic spotted dolphin chromosomes 20 pairs with XX. Transfection was achieved by SV40 LT antigen and transfected cells were expended for passages. Stability of cell line was confirmed at various passages intervals using RT-PCR, western blotting and immunofluorescence methods. After confirmation, cell line was exposed to BDE-47 (250 ng/ml), BDE-100 (250 ng/ml) and BDE-209 (1000 ng/ml), with control group (PBS), positive control DMSO (0.1%) and negative control LPS (500 ng/ml) for 24 h. The ELISA results showed significant increase in IL-6 in BDE- 100 and BDE-209 while IL-1β and IL-8 were found higher in BDE-47 and BDE-100. TNFα and IL-10 secretion was noted higher in control and positive control groups. Altogether, these results emphasize importance of transfected (PSP-LWHT) cell line in aquatic research and potential effects of PBDEs on fibroblast provides evident to understand immune modulating effects of PBDEs in marine mammals. The impact of PBDEs on dolphin’s fibroblast cells immune response and altered cytokine response have been presented for the first time.