Studies On CYP2E1-dependent Genotoxicity Test Models And Their Use In Exploring The Mutagenicity Of Polychorinated Biphenyls

Most organic carcinogens exhibit no mutagenic or carcinogenic effects before being metabolically activated.In vitro genotoxicology tests are frequently used for investigation of chemical mutagenicity.Due to the lack of a relevant metabolic activity,assays with promutagens in common cell lines often give rise to false-negative results.Rat liver S9 mixture and other in vitro metabolic activation systems may increase the detectability of promutagens while minimize false negative results.However,some important biotransformation enzymes,such as cytochrome P450(CYP)1B1,were not expressed in hepatocytes,but in some extrahepatic tissues.Additionally,some CYP enzymes,like CYP2E1,is unresponsive to Aroclor 1254 and other commercially applied inducers,which may lead to false negative results even in the presence of rat liver S9 mixture.Using recombinant cell lines expressing particular CYP enzymes as a genotoxicity experimental system can greatly improve the detection premutagens.CYP2E1 is an important metabolic enzyme which activates some lipophilic small molecule compounds.Various cell lines expressing CYP2E1 have been used to study the genotoxicity of premutagens.However,there has been no report on the advantages of intracellular CYP2E1-activating mutagenicicity assays as compared with the presence of rat liver S9 mix.N-nitrosodiethlamine(NDEA),an important food contaminant and hepatocarcinogen,can induce gene mutations at very high concentrations in bacteria or mammalian cell models in the presence of rat S9 mixture,while it is inactive in the experimental conditions deficient in metabolic activities.Therefore,in this study recombinant V79 cells expressing CYP2E1,and human hepatocyte(L-02)cells,were used to study the genotoxicity of NDEA and the dependence on CYP2E1 activity;the observed effects were then compared with results obtained in metabolic enzyme-deficient V79-Mz cells in the presence of rat liver S9 mix,to show the degree of advantages of the intracellular activation system.Substrates of CYP2E1 are usually organic compounds,which need to be dissolved in organic solvents before application in genotoxicity assays.Dimethylsulfoxide(DMSO),the most commonly used organic solvent,has been observed to inhibit CYP-catalyzed biochemical reactions,with particularly potent effects on the activity of CYP2E1.Many other organic solvents(such as methanol,chloroform,acetone,etc.)are CYP2E1 substrates,which are unsuitable to serve as solvents in relevant genotoxicity assays.Whether DMSO affects the genotoxicity of precarcinogens dependent on activation by human CYP2E1 has not been reported so far.Considering that the concentration of DMSO may exert important influence on CYP2E1-activated toxic responses,NDEA,which is soluble in both water and organic phase,was chosen as a model premutagen to investigate the effects of DMSO on CYP2E1-dependent genotoxicity.The results of this study will provide a reliable basis for optimizing the experimental conditions of CYP2E1-dependent genotoxicity assays.Polychlorinated biphenyls(PCBs),a group of persistent organic pollutants still present in the environment,also have strong carcinogenic effects.However,the metabolic activating enzymes and related mutagenicity of PCBs have not been fullly understood.Our research team has observed in recent years that mono-and dichlorinated biphenyls can induced micronuclei and gene mutations in mammalian cells engineered for human CYP2E1.The concentrations of some tri-and tetrachlorinated biphenyls in the air and human tissues are higher than mono-and dichlorinated biphenyls,thus the former compounds may have greater potential to exert health effects,however,their genotoxicity data are lacking.Therefore,the present study was focused on the mutagenicity of tri-and tetrachlorinated biphenyls under metabolic activation of human CYP2E1,using human CYP2E1-expressing V79-derived cells,based on which e the spectrum of PCBs-induced Hprt mutations was also investigated.Objectives1.This study was conducted to confirm the genotoxic effects of NDEA under metabolic activation by CYP2E1 and the advantage of intracellular CYP2E1 activation system over the traditional S9 mix activating system,evaluate the validity of DMSO applied as an organic solvent in CYP2E1-activated genotoxicity assays,propose a concentration limit of DMSO without disturbing the experimental results,for the purpose of optimizing the experimental conditions of intracellular CYP2E1-activating genotoxicity assays.2.To explore the mutagenicity(at the Hprt locus)of non-coplanar(non-dioxin-like)PCBs containing 3 or 4 chlorine substituents(PCB 22,28,52 and 74)in mammalian cells enjineered for human CYP2E1 enzyme,and the changes of Hprt-encoding gene sequence in mutant cells.Methods1.Cell lines:Chinese hamster(V79)cell line(subclone V79-Mz),V79-derived cell lines genetically engineered for the expression of human CYP2E1(V79-hCYP2E1),both CYP2E1 and sulfotransferase(SULT)1A1(V79-hCYP2E1-hSULTlAl),and human hepatocyte(L-02)line which endogenously expresses various CYP enzymes including CYP2E1.2.Test compounds:NDEA,2,3,4’-trichlorobiphenyl(PCB 22),2,4,4’-trichlorobiphenyl(PCB 28),2,2’5,5’-tetrachlorobiphenyl(PCB 52),and 2,4,4’,5-tetrachlorobiphenyl(PCB 74).3.Assays:CCK-8 assay,micronucleus test and Hprt mutagenicity assay were used to investigate the cytotoxic and genotoxic effects of test compounds;Western-blot and Q-PCR were used for the detection of CYP2E1 protein and mRNA transcripts,respectively.Results1.Investigation on the experimental model of CYP2E1 enzyme-dependent genotoxicity detection(1)Mutagenicity of NDEA under metabolic activation by human CYP2E1 and the comparison of genotoxicity effect induced by NDEA in different experimental systems(D NDEA did not induce micronuclei or gene mutations in V79-Mz cells,however,it induced micronuclei and gene mutations potently in V79-hCYP2E1-hSULTlAl cells.The effect was blocked by ABT(a CYP enzyme inhibitor).NDEA induced micronuclei in L-02 cells and this effect was completely inhibited by 4-methylpyrazole,a selective CYP2E1 inhibitor.(2)The potency and efficacy of NDEA-induced micronuclei formation in V79-hCYPEl-hSULT1A1 cells were significantly higher than that in V79-Mz cells with rat liver S9 mix.(2)Exploration of appropriate DMSO concentration limits,an effort for optimizing the experimental conditions in CYP2E1-activated genotoxicity assays.①DMSO at concentrations ranging from 0.05 to 0.2%(v:v)did not show any influence on NDEA-induced micronuclei formation and gene mutations in V79-hCYP2E1-hSULT1A1 cells.DMSO at the concentration of 0.1%and 0.2%partially decreased NDEA-induced formation of micronuclei in L-02 cells and gene mutations in V79-hCYP2E1 cells(protein level of CYP2E1 in both two cell lines were significantly lower than in V79-hCYP2E1hSULT1 A1 cells).② DMSO(0.05-0.2%,v:v)did not affect the level of CYP2E1 protein in V79-hCYP2E1-hSULT1A1 cells or the level of CYP2E1 transcripts in each cell line.2.Mutagenicity of several tri-and tetrachlorobiphenyls in V79-hCYP2E1-hSULT1A1 cells(1)None of PCB 22,28,52 and 74)induced gene mutations in V79-Mz cells,however,they all induced gene mutations potently in V79-hCYP2E1-hSULT1A1 cells(SULT1A1 activity was inhibited by co-exposed pentachlorophenol),with a clear concentration-effect relationship.(2)The mutagenicity of PCB 74(tetra-chlorinated)was more potent and efficacious than PCB 28(tri-chlorinated,structurally similar to PCB 74);this result suggested that increased chlorination may not always lead to reduced mutagenicity,and on the contrary it may sometimes lead to enhanced effects.(3)Analysis of the DNA base sequence changes induced by PCB 28 and PCB 52,two representative PCBs,indicated that base substitutions and exon deletion were the most prevalent modes of mutations.NDEA,the positive control compound,induced Hprt mutations prevalent of base substitutions,most frequently A:T→G:C transitions.Conclusions1.The genetically engineered cell line expressing CYP2E1(V79-hCYP2E1-hSULT1A1)pertain significantly greater metabolic activating efficiency than L-02 cells and V79-Mz cell in the presence of S9 mix,thus the former cell line is clearly suitable for the study of CYP2E1-dependent promutagens as an experimental model;in this model,DMSO as a solvent may not influence the expression of mutagenic effect at the concentrations ranging from 0.05 to 0.2%(v:v),especially when the level of CYP2E1 protein is relatively abundant.2.Non-dioxin-like compounds PCB 22,PCB 28,PCB 52 and PCB 74 are capable of inducing gene mutations under metabolic activation by human CYP2E1.Increased chlorination may sometimes increase the mutagenicity of PCBs,such as PCB 22,PCB 52 and PCB 74 are more potently mutagenic than PCB 5,PCB 9 and PCB 28(structurally similar to the former ones,but less chlorinated),respectively.PCBs and positive control NDEA can induce mutations such as single base substitution and exon deletion,dominated by single base substitution.