Studies On The Toxic Effects And Mechanism Of 2,2’,4,4’-Tetra-Bromodiphenyl Ether(BDE-47) On Human Embryonic Kidney Cells(HEK293)

Because of good flame retardancy and thermal stability, polybrominated diphenyl ethers(PBDEs) are extensively used as brominated flame retardants in products of daily life. However, PBDEs can release from products easily and persist in various environmental media for a long time. Furthermore, high liposolubility contributes to accumulation of PBDEs in organisms, and subsequent biomagnification allows PBDEs to be transferred to human through food chain and exert adverse effects on human health. Previous studies have revealed that PBDEs could cause various toxic effects, including developmental neurotoxicity, hepatotoxicity and endocrine disruption. Kidney is an important excretive organ, and renal cell injury may influence renal function. Therefore, it is necessary to investigate toxic effects and toxicity mechanism of PBDEs on kidney cell lines.In the present study, one of predominant PBDE congeners in human body-2,2’,4,4’-tetra-bromodiphenyl ether(BDE-47) was chosen to study its toxic effects and mechanism in human embryonic kidney 293 cells(HEK293). Multiple techniques were applied in this study, such as spectroscopy, flow cytometry, quantitative real-time PCR(q RT-PCR), western blotting and NMR-based metabolomics, etc. The results were supposed to provide scientific evidences for toxicological study and ecological risk assessment of PBDEs. The major results were summarized as follows.(1). Toxic effects were induced by the concentration range of BDE-47(10-6- 10-4 M) on HEK293 cells, including toxic and metabolic responses. The results of toxic responses showed that BDE-47 caused hormesis effect in HEK293 cells from 10-6 to 10-4 M. Cell apoptosis and ROS overproduction detected at 10-5 M of BDE-47 indicated the correlation between them. Metabolic response revealed that BDE-47 induced dose-responsive effects on metabolite variations of HEK293 cells at selected concentrations(10-6- 10-4 M). In details, the metabolic responses indicated the disturbance in energy metabolism, stimulation of protein catabolism, intracellular oxidative stress status and stress-response of cellular membrane caused by BDE-47 exposures in HEK293 cells.(2). Toxic mechanism of HEK293 cells induced by the concentration range of BDE-47(10-6- 10-4 M) were studied at transcription and translation levels.a. Firstly, the expression levels of apoptosis-related genes suggested that BDE-47 could raise the expression levels of p53, Bcl-2 family-encoding genes(Bcl-2, Bax, Bad and Hrk) and Caspases family-encoding genes(Caspase-8) at concentrations of 10-5 and 10-4 M. The results revealed that BDE-47 caused HEK293 cells apoptosis through death receptor and mitochondrial pathways. p53 and Bcl-2 family mainly played regulatory roles in mitochondrial pathway. Caspase-8 could be involved in both death receptor pathway and mitochondrial pathway. In the executional stage, HEK293 cells apoptosis might be induced by Caspases-independent pathway.b. Secondly, the expression levels of proteins related to cell proliferation(SFRS3), oxidative stress(APE1) and cell apoptosis(p53) suggested there was consistency between SFRS3 protein expression and cell proliferation ratio as the concentration of BDE-47 increased. The expression level of APE1 protein was also consistent with the intracellular ROS level. The results revealed that SFRS3 was closely associated with the alteration of HEK293 cell proliferation caused by BDE-47. APE1 might be an important mediator between oxidative stress and cell apoptosis by inducing high expression of p53.c. Lastly, the spectral changes of the promoter region segment of the p53 gene(p53-DNA) showed that as the concentration of BDE-47 increased(10-6- 10-5 M), p53-DNA produced a hyperchromic effect and the fluorescence system of EB-p53-DNA had a static quenching. Furthermore, the melting point of p53-DNA increased by 0.5 oC in the presence of 10-5 M of BDE-47. Spectroscopic results suggested that groove binding was the major binding mode of BDE-47 with p53 gene causing DNA damage.