Studies On The Mutagenicity Of Bisphenol Compounds,Their Impact On The Action Of Several Precarcinogens And The Relevance To Metabolic Enzymes

BACKGOUNDBisphenols(BPs)are a group of organic pollutants commonly exposing the environment and humans.However,little has been known regarding their metabolic activation,genotoxicity and mode of action.OBJECTIVESTo explore the influences of BPs on the expression of xenobiotic nuclear receptors(NRs)(involved in the regulation of CYP enzyme expression)[(such as the aryl hydrocarbon receptor(AhR),the pregnane X receptor(PXR),and the constitutive androstane receptor(CAR)]as well as cytochrome P450(CYP)enzymes,and to further analyze the mutagenicity of BPs,and their influence on the genotoxicity of several pro-cariconogens,thus to provide a scientific basis for the assessment of environmental health risk of BPs themselves and the complex exposures of both BPs and relevant procarcinogens.METHODSComputer-simulated molecular docking of BP-CYP enzyme protein(BPA,BPF and BPS being docked to the active site of CYP1A1,1A2,1B1,2B6,2E1 and 3A4)was performed to analyze the affinity and substrate potential,moreover,molecular kinetics was used to simulate the binding of each BP compound with AhR,PXR and CAR.Subsequently,experiments with cell models were employed to verify the molecular docking results,i.e.,cytotoxicity(CCK-8 assay)and chromosome damage(formation of micronuclei)by BPs under various regimes were conducted in cell lines V79-Mz(control line),V79-hCYP1A1,V79-hCYP1A2,V79-hCYP1B1,V79-hCYP2B6,V79-hCYP2E1,V79-hCYP3A4-hOR,and a human hepatoma(C3A)cell line(which endogenously expresses multiple CYP enzymes at substantial levels).Moreover,immunofluorescent staining of centromere protein(CENP)B was used to analyze the formed micronuclei by BPs for the presence of centromeres,so as to distinguish between clastogenic from aneugenic action.In addition,Western blot assay were adopted to examine the influence of BPA,BPF,BPS and BPAF on the levels of transcription and protein expression of NRs and CYP enzymes in a human hepatoma(HepG2,expression of CYPs at insignificant levels)cells;furthermore,HepG2 cells were pretreated with each BP compound at various concentrations for 48,followed by exposure to benzo(a)pyrene(BaP),aflatoxin B1(AFB1),benzene,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK),or a BP compound itself for another 48h for the observation of micronuclei formation.RESULTS(1)In cell lines V79-Mz,V79-hCYP1A1,V79-hCYP1A2,V79-hCYP1B1,V79-hCYP 1 E1,and V79-hCYP3 A4-hOR treated with BPA,BPF,and BPS under the regime of 9h/15h(exposure/recovery,two cell cycles in total),all the three compounds induced micronuclei formation in V79-hCYP1A1,they were either inactive or weakly positive in the other cell lines.In C3A cells exposed to each BP compound for 72h(covering two cell cycles as well,with no recovery)micronuclei formation started to be observed with each BP at rather low concentrations(2.5 to 5μM),with concentration-dependence,and the effects were blocked by 1-aminobenzotriazole(60μM,a broad-spectrum CYP inhibitor)or 7-hydroxyflavone(sective CYP1A1 inhibitor).Immunofluorescent assay demonstrated that BPs only elevated the frequency of centromere-free micronuclei in C3A cells(while that of centromere-containing micronuclei was unaffected).Coexposure of the cells to ketoconazole(10μM,inhibitor of UDP-glucoronosyltransferases)or pentachlorophenol(5μM,inhibitor of sulfotransferase 1)potentiated the formation of micronuclei by BPs to some extent.(2)After treatment of HepG2 cells with BPA,BPF,BPS,and BPAF at concentrations ranging from 1 to 100nM for 48h,the mRNA transcripts and protein expression of AhR,PXR,and CYP1A1,1A2,1B1,2E1 and 3A4 were all increased,while the level of transcripts of CAR was unchanged,and its protein level was decreased.All the four BPs at the above concentration range potentiated the induction of micronuclei formation by BaP,AFB1,benzene and NNK in HepG2 cells,with the threshold of each carcinogen being significantly lowered.(3)Through extension of exposure time from 48 to 72 or 96h,BPA,BPF,BPS,and BPAF,alone or in varying combinations,potentiated micronuclei formation in HepG2 cells was observed.Moreover,pretreatment of HepG2 cells with each BP at 100nM and higher concentrations significantly enhanced the induction of micronucleiformation by itself or each other.CONCLUSIONS(1)BPs may be activated by human CYP1A1 for chrosomosome breakage,where phase Ⅱ biotransformation enzymes may be involved for a detoxifying impact.(2)BPs at low concentrations comparable with the human internal exposure levels may significantly potentiate the genotoxicity of multiple procarcinogens(BaP,AFB1,benzene,and NNK)through induction of the proteins of AhR,PXR,and C YP1A1,1A2,1B1,2E1,and 3A4.(3)The genotoxicity of BPs could be significantly potentiated through extended exposure or combined exposure to multiple BP compounds.In summary,in condition that numerous organic compounds may complexly expose the biosphere in combination,BPs may potentiate the toxicity of important carcinogens through induction of human nuclear receptors and relevant CYP enzymes;persistent exposure and exposure of multiple BP congeners may magnify the genotoxicity of BPs themselves,thus their real hazardousness to human health might be more serious than our estimation up to now.