Studies On Human P450 Enzyme-activated Genotoxicity Of 2,2’,4’4’-tetrabromminated Biphenyl Ether (BDE-47)

BackgroundPolybrominated diphenyl ethers(PBDEs)are a category of flame retardants and new type of organic pollutants with a structure of brominated diphenyl ethers.PBDEs are environmentally persistent,biologically accumulating,which demonstrate endocrinal,neuro-,reproductive and immunotoxicity.Whether it is genotoxic is still controversial.2,2’,4,4’-Tetrabrominated diphenyl ether(BDE-47),a unique member of PBDEs,is contained in human plasma at particularly high levels and with strong toxicity.PBDEs can be metabolized by cytochrome P450(CYP)enzyme system and transformed to hydroxylated products,however,whether it can be activated by CYP enzyme for mutagenic activities remains unclear.ObjectiveTo explore the metabolism-related genotoxicity(DNA and chromosomal changes)of BDE-47 in mammalian cells and the mechanism of chromosomal damage.MethodsBy molecular docking analysis the energy of BDE-47 for binding to human CYP1A1,CYP1A2,CYP2B6,CYP2E1 and CYP3A4,and the substrate potential of BDE-47 for each enzyme was investigated.Based on the prediction of BDE-47-CYP isoform substrate possibility,experimental studies using Chinese hamster lung(V79)-derived cell lines genetically engineered for the expression of the relevant CYP enzymes,and a human hepatoma(HepG2)cell line,were employed in the micronucleus tests with BDE-47 as the test compound enzyme cells,the parental V79-Mz line,which was lack of the activities of CYPs,sulfotransferase and UDP-glucoronosyl transferases,served as the control.CCK-8 assay,a method to reflect the viability of cells,was used to determine the cytotoxicity of the test compound,while induced formation micronuclei was indicative of chromosome damage(either breakage or loss).The regimes used for chemical exposure included both short exposure/long recovery and long exposure/no recovery ones.In order to distinguish clastogenicity from aneugenicity,immunofluorescent visualization of centromere protein B(CENPB)was combined with the traditional micronuclei test.In HepG2 cells pretreated with ethanol or rifampicin.Western blot assay was used to analyze the content of y-H2AX(whose elevation indicates double-strand DNA breaks).The morphological changes of mitotic spindle and centrioles in V79-hCYP3A4-hOR and V79-Mz were analyzed by immunofluorescent staining withβ-tubulin and γ-tubulin.ResultsThe results of molecular docking showed that the binding energies of BDE-47 for the active centers of CYP3A4,CYP2E1,CYP2B6,CYP1A1 and CYP1A2 were similarly negative,and the distances between the ligand(BDE-47)and the heme each enzyme was always lower than 6(?),a condition permitting electron transfer(as required for CYP-catalyzed metabolism),which suggested substrate potential of BDE-47 for all test enzymes.Following the regime of 6 h(exposure)/18 h(recovery),BDE-47 elevated the frequency of micronucleated cells in V79-derived cell lines expressing human CYP3A4,2E1 and 2B6(V79-hCYP3A4-hOR,V79-hCYP2El-hSULT1A1,and V79-hCYP2B6,respectively)at concentrations≥ 40μM,while it was inactive in the parental V79-Mz and V79-derived cells expressing human CYP1A1 or 1A2.The micronuclei-inducing effect of BDE-47 was blocked or significantly reduced by 1-aminobenzotriazole(120 μM,CYPs inhibitor).Extended exposure(24 h,without recovery)of V79-hCYP3A4-hOR and V79-hCYP2E1-hSULT1A1 cells to BDE-47 caused micronuclei induction at increased potency(threshold as 20 μM)with concentration-dependence.Moreover,in HepG2 cells pretreated with ethanol[0.2%(v:v),CYP2E1 inducer]and rifampicin(10 μM,CYP3A4 inducer)for 16 h,BDE-47(48 h/0 h)significantly induced micronuclei formation(threshold being at 20μM).BDE-47 strongly induced micronuclei formation(threshold being 6 μM)in HepG2 cells pretreated with bisphenol AF(BPAF,0.1 μM,inducer of multiple CYP enzymes),and this effect was blocked by either trans-1,2-dichloroethylene(CYP2E1 inhibitor)or ketoconazole(CYP3A4 inhibitor).Moreover,BDE-47 induced DNA damage in HepG2 cells,which was further enhanced by pretreatment of the cells with BPAF.The micronuclei formed by BDE-47 in V79-hCYP3A4 cells were predominantly centromere positive(>60%),while in HepG2 cells pretreated with ethanol and rifampicin the frequency of only centromere-positive micronuclei was increased(that of centromere-negative micronuclei remained unaffected).Moreover,BDE-47 induced abnormal morphology and structure of spindle and centrioles in V79-hCYP3A4-hOR cells at concentrations≥4 μM.ConclusionBDE-47 is an aneugen requiring metabolic activation by multiple human CYP enzymes,primarily CYP3A4 and 2E1;it may also induce mitotic abnormalities.