| BACKGROUNDPolychlorinated biphenyls(PCBs)are a group of persistent organic pollutants and human carcinogens,with metabolic activation by cytochrome P450(CYPs)enzyme being required for the carcinogenicity of most congeners.However,their mutagenicity and structure-activity relationships remain mostly unknown.Due to the close relevance of mutagenesis to carcinogenesis,the analysis of their mutagenicity is important for risk assessment of PCBs.Based on the pattern of chlorine substitution,PCBs are classified into co-planar and non-coplanar PCBs:similar to 2,3,7,8-tetrachlorodibenzodioxin(TCDD)the former are ligands for the aromatic hydrocarbon receptor(AhR),therefore they are also termed dioxin-like(DL-)PCBs;the latter do not have affinity toward TCDD and thus also termed non-dioxin-like(NDL-)PCBs.Recently,our research team have reported that a group of NDL-PCBs are activated by human CYP2E1,thus they demonstrate mutagenicity in mammalian cells;Single ortho-chlorine structure of PCBs seems to favor their potency/efficacy mutagenicity at most,yet there have been no systemic observations on the structure-activity relationships.Moreover,human CYP2E1 is incapable of activating DL-PCBs for mutagenic effects,however,whether other CYPs activate them for genotoxicity is still unclear.PCBs may modulate the expression of CYPs through acting on various nuclear receptors,and DL-PCBs may upregulate CYP1 enzymes by activating AhR,while NDL-PCBs may induce CYP2B and CYP3A by activating constitutive androstane receptor(CAR)and pregnane X receptor(PXR).Aflatoxin B1(AFB1)is a human carcinogen dependent on the metabolic activation by CYP1A2 and 3A4,however,whether and how its toxicity is enhanced by PCBs through a combined effect has not been evidenced.OBJECTIVE(1)To explore the structure-activity relationships for human CYP2E1-activated mutagenicity of NDL-PCBs,di-,tr-,and tetrachlorobiphenyls with varying number of ortho-chlorine were tested for the induction of micronuclei and Hprt mutations in a Chinese hamster V79-derived cell line expressing both human CYP2E1 and sulfotransferase(SULT)1A1(V79-hCYP2E1-hSULTlAl).(2)To test our hypothesis that DL-PCBs may be activated by CYP1 enzymes for genotoxic potentials,V79-derived cell lines expressing human CYP1A1,1A2,and 1B1,as well as a human hepatoma cell(C3A)line which endogenously expresses various CYP enzymes,were used in genotoxicity assays.(3)To investigate the impact of NDL-PCBs on the expression of nuclear receptors and several CYP enzymes,and the genotoxicity of AFB1,in a human hepatocyte(L-02)line.METHODSV79-Mz(control cell line),V79-dervied cell lines expressing human CYP2E1 and SULT1A1,CYP1A1,1A2 or 1B1(V79-hCYP2E1-hSULT1A1,V79-hCYP1A1,V79-hCYP1A2,V79-hCYP1B1,respecitively),and L-02 and C3A cell lines,were used for the determination of cytotoxicity(CCK-8 assay)and genotoxicity of test compounds,such as micronuclei induction and Hprt mutations;centromere protein B(CENP-B)with micronuclei was detected using immunofluorescence assay(to discriminate clastogenesis from aneugenesis);Western blot was used to analyze the levels of expression of various proteins.RESULTS(1)After treatment of V79-Mz and V79-hCYP2E1-hSULT1A1 cells with each tri-and tetrachlorobiphenyl following a 6 h/18 h(exposure/recovery)regime,induction of micronuclei in the latter cells was significantly more potent and efficacious than the former cells;in the other endpoint,i.e.,Hprt gene mutations,all test compounds were negative in V79-Mz cells,while most of them were positive.Taking other data regarding the effects of some other PCBs in these cell lines,which have recently been reported by our research group,into account,it was noticeable that a structure with single ortho-chlorine conferred a PCB compound the most potent mutagenicity,and along with the increase in the number of ortho-chlorine the mutagencity of PCBs were decreasing;this tendency was positively related with the degree of chlorination of compounds:dichlorobiphenyls were not significantly,while trichlorobiphenyls were obviously,and tetrachlorobiphenyls were greatly influenced(the mutagenicity of tetrachlorobiphenyls with 3 or 4 ortho-chlorines have become undetectable).Of the micronuclei induced by 2,4',6-Trichlorobiphenyl(PCB 32)and 2,3,3',4'-tetrachlorobiphenyl(PCB 56)(representative compounds)60%to 70%were CENP-B positive.(2)V79-derived cell lines expressing human CYP1A1,1A2,or 1B1 were treated with 3,3',4,4'-tetrachlorobiphenyl(PCB 77),3,4,4',5-tetrachlorobiphenyl(PCB 81)and 3,3',4,4',5-tetrachlorobiphenyl(PCB 126)under varied exposure/recovery regimes,then observed for micronuclei formation.The results showed that under the 6 h/18 h and 18 h/6 h regime,PCB 77 and PCB 81 at micromolar levels induced micronuclei in V79-hCYP1B1(more potent under the latter regime),with negative results in V79-Mz,V79-hCYP1A1 and V79-hCYP1A2 cells;while under both regimes,PCB 126 induced no change in the frequency of micronucleated cells in all cell lines.In C3A cells,PCB 77,81 and 126 exposure for 54 h elevated the levels of CYP1A1 and CYP1A2 protein,while CYP1B1 was unchanged;under a 54 h/18 h regime,PCB 77 and 81 induced micronuclei in C3A cells,in a concentration-dependent manner,and these effects were completely prohibited by the coexposure with(E)-2,3',4,5'-tetramethoxystilbene(TMS,30 nM),a selective CYP1B1 inhibitor.Immunofluorescent staining of CENP-B with the micronuclei formed with each PCB in V79-hCYPlB1 cells and C3A cells consistently showed the predomination of CENP-B negative micronuclei.In addition,after exposure of C3A cells to PCB 77,81 and 126 for 12 h,the level of y-H2AX was always significantly elevated(indicating DNA double-strand breaks),however,these effects were completely blocked by the coexposure of TMS(10 nM).(3)After the exposure of human hepatocyte(L-02)cells to three representative NDL-PCBs individually,i.e.,2,3,3-trichlorobiphenyl(PCB 20),2,2'5,5'-tetrachlorobiphenyl(PCB 52)and PCB 56 for 48 h,the levels of CAR,PXR,CYP1A1,1A2 and 3A4 protein were increased,while that of AhR protein was reduced,with thresholds at low micromolor concentrations.PCB 126 significantly induced all the above proteins starting from sub-nanomolar concentrations.Overexpression of CAR in L-02 cells increased CYP1A2 and 3A4 levels,which was further enhanced by the exposure of each NDL-PCB.Pretreatment of L-02 cells with PCB 126 and each NDL-PCB promoted both micronuclei formation and double-strand DNA breaks induced by AFB1,as indicated by threshold of AFB1 lowered from 40 to 5 or 10 ?M,PCB 126 being far more potent than each NDL-PCB in potentiating the effect of AFB1.CONCLUSIONS(1)Under metabolic activation by human CYP2E1,the mutagenicity of NDL-PCBs is featured by the following structure-activity relationships:the number of ortho-chlorine may influence their mutagenicity in such a way,that those have single ortho-chlorine are relatively potent and efficacious,while increasing numbers of ortho-chlorine lead to reduced mutagenicity;this impact is simultaneously determined by the degree of chlorination of the compound:tetrachlorobiphenyls are most sensitively,trichlorobiphenyls moderately,while dichlorobiphenyls mildly influenced.(2)Human CYP1B1 is capable of activating some DL-PCBs to clastogenic metabolites.(3)Varied spectra and potencies as well as a complex connection exist between NDL-PCBs and DL-PCB in modulating the protein expression of nuclear receptors and CYP enzymes in L-02 cells,nevertheless,both categories of PCBs potentiate the genotoxicity of AFB1 via induction of CYP1A2 and 3A4.Taken together,PCBs may have specific structure-activating enzyme-genotoxicity relationships;on the other hand,they may modulate the expression of various nuclear receptors and then induce CYPs including CYP1A2 and 3A4,and in such a way the genotoxic effect of AFB1 can be potentiated.The significance of these effects of PCBs in the carcinogenesis in humans deserves further investigation. |
PDF Full Text Request