CYP Enzyme-activated Mutagenicity Of Polychlorinated Biphenyls And Preliminary Evaluation On The Predictive Value Of Relavent Molecular Simulation

BackgroundPolychlorinated biphenyls(PCBs)are a class of persistent organic pollutants which persist in the environment worldwide and can be bioconcentrated and biomagnified through the food chain.PCBs enter the human body via a variety of pathways,including air,food,and water.Some of them can be catalyzed by phase I metabolic enzymes[such as cytochrome P450 enzyme(CYP)]and the metabolites may have greater bioreactivity and mutagenicity than their prototypes.Persistent exposure to PCBs can cause a variety of health problems and has been classified by the International Agency of Research on Cancer(IARC)as carcinogenic to humans(Group 1).Among the subfamilies of CYPs,the CYP2 family has an extensive substrate profile,including a lot of environmental compounds.We previously found that human CYP2E1 activates some of the low-chlorinated PCBs,however,it is unclear that whether high-chlorinated PCBs can be activated by CYP2E1 and whether other CYP2 subtypes are involved in PCBs metabolism.In addition,as PCBs have 209 congeners depending on the number and location of chlorine substitutions,it is time-and labor-consuming to cover the toxicity evaluation of all PCBs with classical experimental studies.The use of molecular simulation to predict the potential of PCBs as the substrate of CYP2 enzymes can significantly reduce the blindness of the experimental design,save experimental costs and shorten the study cycle if it can act as a primary screening for subsequent experimental studies.Therefore,the hypothesis in this study is that the prediction of the potential of PCBs as the substrate of CYP enzyme proteins by molecular simulations has the guiding significance for the design of compounds and their related enzymes in mutagenesis tests.Objective(1)The effectiveness of molecular simulation in predicting the potential of compounds as the substrate of human CYP2E1 enzymes in guiding the selection of relevant subjects for mutagenic test is investigated by applying dioxin-like polychlorinated biphenyls(DL-PCBs)as an example;(2)The effect of the number of ortho-chlorine substitution on the interaction of non-dioxin-like PCBs(NDL-PCBs)with human CYP2B6 enzymes is investigated by using molecular simulation and experiments;(3)Both the published data(about the mutagenic effects of various aromatic compounds dependent on human CYP enzyme activation)and the interaction of tested compounds with the corresponding enzymes(analyzed by molecular docking)are taken into account to summarize the level of compliance and uncertainty of molecular simulation in predicting mutagenic effects of CYP-remediated compounds.MethodThe affinity and substrate potential of the chemical-enzyme protein were analyzed by molecular docking and molecular dynamics methods;Chinese hamster lung fibroblast(V79)cell line,recombinant V79-derived cell line expressing different human CYP enzymes and human hepatocellular tumor(C3A)cell line were used as test cells;V79-hCYP2B6 cells were constructed by liposome method(the expression of CYP2B6 and its activity were verified at DNA,mRNA,protein level and related indirect mutagenic effects);cell number was indirectly reflected by CCK-8 cell viability test to detect cytotoxicity;and the mutagenic effects of test compounds were analyzed by micronucleus test for V79 cells and both micronucleus test and PIG-A mutation assay for C3A cells.Results(1)the potentials of DL-PCBs as human CYP2E1 substrates were firstly predicted by using molecular simulations.It showed that 2,3,3',4,4'-pentachlorobiphenyl(PCB 105)and 2,3',4,4',5-pentachlorobiphenyl(PCB 118)may be the proper substrates of the human CYP2E1,while the remaining 10 compounds were all more than 6.0 A away from the cofactor ferric ions at the active site,which is unfavorable for electron transfer.3,3',4,4'-,3,4,4',5-tetrachlorobiphenyl(PCB 77,PCB 81),PCB 105,118,and 3,3',4,4',5-pentachlorobiphenyl(PCB 126)were chosen as test compounds.Micronucleus experiments(6 h exposure/18 h recovery)showed that both PCB 105 and PCB 118 significantly induced micronucleus formation in V79-hCYP2E1 cells and this effect was inhibited by 1-aminobenzotriazole(a CYP inhibitor),whereas it is negative(PCB 1 18)or weakly positive(PCB 105)in V79-Mz cells.On the contrary,PCB 77,PCB 81,and PCB 126 did not induce micronucleation in either cell line.PCB 105 and PCB 118 also induced micronucleus formation and PIG-A gene mutations in C3A cells;both effects were blocked by trans-1,2-dichloroethylene(a selective CYP2E1 inhibitor).(2)Molecular simulations were used to analyze the interactions between 2,2',3,3'-,2,2',3,6'-,2,2',6,6'-and 2,3,3',4'-tetrachlorobiphenyl(PCB 40,PCB 46,PCB 54 and PCB 56 with different ortho-chlorine substitution)and human CYP2B6,suggesting that they all have substrate potential for CYP2B6.A V79 recombinant cell line(V79-hCYP2B6)stably expressing human CYP2B6 enzyme was subsequently constructed by gene transfection.Experimental studies showed that four PCBs had cytotoxic effects on V79-Mz cells under different exposure conditions.However,the cytotoxic effect was weakened or disappeared in V79-hCYP2B6 cells(suggesting the metabolic detoxification).PCB 56 induced micronucleus formation in V79-Mz cells,but not in V79-hCYP2B6 cells;the other three compounds had no effect on micronucleus formation in both cell lines.Since the above compounds did not show any genotoxic effect under metabolic activation,the present study further analyzed their effect on the genotoxic effect of aflatoxin B1,an indirect mutant known to be activated by CYP2B6 enzymes,in V79-hCYP2B6 cells.It was showed that aflatoxin B1 significantly induced micronucleus formation in V79-hCYP2B6 cells,and this effect was inhibited by PCB 46,PCB 54 and PCB 56,but not by PCB 40.A comparison of the conformation of PCB 56 in the active sites of human CYP2E1 or CYP2B6 revealed different potential hydroxylation sites,suggesting different structures metabolites,which may partly explain the discrepancy between the negative effect of PCB 56 in this study and the previous finding of strong mutagenic effect induced by human CYP2E1 activation.(3)Molecular docking analysis was used to predict the substrate potential of some aromatic compounds for the relevant CYP enzymes,which showed a high consistency between the molecular docking and the experimental results.However,for CYP2E1 proteins,flexible residues such as PHE 478 need to be set up to satisfy requirements of some relatively large molecules such as PCBs,1?methylpyrene,etc,for an enough cavity of the active site.A retrospective analysis of the consistency between PCBs and CYP enzyme docking results and the reported experimental data on relevant toxicity(mainly reported by our group)revealed that the results of metabolic activation toxicity were consistent with molecular docking findings in 36 of the 47 tests,accounting for 76.7%of the total.Conclusion1)For some PCB compounds,such as DL-PCBs,the substrate prediction based on the molecular simulation of PCBs and human CYP2E1 enzyme was significantly consistent with the relevant mutagenic experimental data,suggesting that the molecular simulation is promising to be a preliminary screening method for the genotoxicity test of metabolic activation of compounds.2)For other compounds,such as NDL-PCBs containing different numbers of ortho-chlorine substituents,the substrate prediction results(positive)of human CYP2B6 by molecular simulation were not supported by the relevant micronucleus test results(negative).Further experimental results supported the binding of most PCBs to human CYP2B6(determined by the inhibited micronuclei formation of AFB1 which is activated by this enzyme),suggesting that metabolic activation is not only way for PCBs binding to CYP enzyme.3)According to the confirmed evidences that 1-MP is activated by human CYP2E1,the setting of the flexibility of PHE 478 is essential to modulate the volume of the active center of human CYP2E1.Based on this setting,molecular docking was performed with some aromatic compounds which have been proved to be metabolized by particular CYP enzyme.Most results of molecular docking showed consistent with the experimental data.Therefore,as a screening method,it is worth further studying for the application of molecular simulation in genotoxicity tests which are involved in metabolic activation.