Study On Mechanism Of Bde-47 Induced Thyroid Toxicity Mediated By Pxr Receptor

Polybrominated Diphenyl Ethers(PBDEs) is a kind of new POPs,used in large quantities as flame retardants additives. Bioamplification and stability is the main enviromental characteris .It's harmful to human and high-grade creature, because it's hard to decompose, has long resistance time and high lipophilicity, and can amplify through food chains. The tetra-brominated diphenyl ethers(BDE-47) is the monomer that usually found and accumulated in human organism and liver. The toxic effect is endocrine disturbance toxicity, thyrotoxic, neurotoxicity and reproductive toxicity, especially its thyroid toxicity is dominant property, but the toxic mechanism is unclear. OBJECTIVE:To explore the mechanism that BDE-47 induced thyroid toxicity and the possibility mideated by the Pregnane X receptor (PXR) in vitro, whether BDE-47 can induce CYP3A4, UGT1A3, and SULT2A1 by activating PXR in vitro, and discuss the transcription effect to the two main hypotypes TRα1 and TRβ1 of the thyroid hormone nuclear receptors (TRs). To elucidate the mechanism of PXR in thyrotoxic caused by BDE-47, and provides basis experiments for other toxic mechanism. METHODS: After exposure to BDE-47, HepG2 cell toxic effect was analyzed by Cell Counting Kit-8(CCK-8) assay. The dual-luciferase reporter assay system was built by co-transfer the plasmids pGL-CYP3A4-Luc, pGL4.74[hRluc/TK] and pCI-hPXR-neo. and stable expression system with karyocyte HepG2 cell of hPXR gene, HepG2-pCI-hPXR-neo,were constructed by transfection and G418 selection in our lab, then the induction ability of BDE-47 on PXR was assessed, and CYP3A4, UGT1A3, and SULT2A1 expression in mRNA and protein level was investigated and analyzed in HepG2-pCI-hPXR-neo cell, so does the TRα1 and TRβ1. RESULTS: BDE-47 had significant cytotoxicity to HepG2 cells depending on time and dose(p<0.01). IC50 of BDE-47 to HepG2 was 110μmol/L and expressed the strongest toxicity effect at 48hrs exposure. Luciferase determination results suggested that BDE-47 could induce the expression of CYP3A4, and showed dose-time-effect relationship significantly at the dose of 1μmol/L~100μmol/L. Q-PCR and Western Blotting analysis showed that the mRNA and protein of CYP3A4, UGT1A3, SULT2A1 transcription expression could be significantly induced by BDE-47 depending on dose(p <0.01), and the mRNA and protein of TRα1 and TRβ1 transcription expression could be significantly inhibit by BDE-47 depending on dose(p<0.01). CONCLUSION: BDE-47 showed cytotoxicity toward HepG2 cells and can activate PXR, and induced the controlling gene CYP3A4 of PXR, also induced downstream gene including UGT1A3 and SULT2A1 elevated in mRNA and protein levels, and inhibited the mRNA and protein transcription expression of TRα1 and TRβ1. Thyrotoxic effect of BDE-47 may be mediated by PXR activation.