Mutagenicity Of Benzene And Its Hydroxylated Metabolites In V79-Derived Cells Genetically Engineered For Expression Of Human CYP2E1

Benzene is a common environmental pollutant and human carcinogen,which is most widely known as an inducer of aplastic anemia and acute myelogenous leukemia.Previous studies have shown that benzene's chronic toxicity(including carcinogenicity)is mediated by its active metabolites,i.e.,the biologically reactive products catalyzed by CYP2E1.However,there has been no evidence for the induction of gene mutations by benzene under the action of human CYP2E1 activity.In addition,our team has reported in recent years that benzene and its metabolites induced micronuclei in a Chinese hamster V79-derived cell line expressing both human CYP2E1 and human SULT(sulfotransferase,SULT)1A1,and benzene,phenol,hydroquinone,catechol and 1,2,4-trihydroxybenzene can all be activated by human CYP2E1,whiletheir effects can be attenuated by human SULT1A1.In this study,we have extended the above study by investigating the mutagenicity of benzene,its various hydroxylated metabolites,and its ultimate toxicant(1,4-benzoquinone)in V79-hCYP2E1-hSULT1A1 cells,moreover,the possibility of an involvement of reactive oxygen species(ROS)as a mechanism in the effects was also examined.Objectives1.Using induction of gene mutations as an end point of experimental observation,the intracellular metabolic activation of benzene and its hydroxylated metabolites,i.e.,phenol,hydroquinone,catechol,and trihydroxybenzene,by human CYP2E1 enzyme was investigated.2.The ROS formed from cells treated with benzene,a series of its hydroxylated metabolites and 1,4-benzoquinone was determined,for the purpose of exploring the relationship of metabolic activation of benzene and its metabolites with ROS and the involvement of ROS in the toxicity of each test compound.MethodsCytotoxicity was observed by CCK-8 assay under a 72 h/0 h or 24 h/48 h(exposure-recovery)regime;In the Hprt genotoxicity assay,the above treatment scheme was adopted and a standard experimental procedure was followed.Through fluocytometry with DCFH-DA used as a fluorescent probe,the intracellular production of reactive oxygen species(ROS)was determined.The cytotoxicity data and ROS data were both expressed as means ± standard deviation(n=6 and 4,respectively),and statistically analyzed by the ANOVA;the gene mutant frequency data were analyzed by the Fisher's Exact Test after combining the duplicate data(to transform them into quantal data).Results1.The results of CCK-8 assays indicated that each of the six test compounds demonstrated cytotoxicity in V79-hCYP2E1-hSULT1A1 and V79-Mz cells,with no obvious difference with each compound between the two cell lines.The potency of test compounds for induction of cytotoxic effects was in the following order:benzene<1,2,4-trihydroxybenzene<phenol<catechol<benzoquinone<hydroquinone.Except for benzene,all the other compounds showed concentration-dependent cytotoxic effects.2.Under the 72 h/0 h treatment schedule,benzene was completely inactive in the Hprt mutagenicity assay in both cell lines.However,using the 24 h/48 h regime benzene induced gene mutations in V79-hCYP2El-hSULTlA1 cells,while it was still inactive in V79-Mz cells.All the other five test compounds induced gene mutations inV79-hCYP2El-hSULTlA1 cells,as determined under the 72 h/0 h regime,in a concentration-dependent manner.The rank of mutagenicity potency of each compound in V79-hCYP2E1-hSULTlA1 cells was:benzene<phenol<1,2,4-trihydroxybenzene<catechol<hydroquinone<benzoquinone.On the contrary,in V79-Mz cells only 1,4-benzoquinone and hydroquinone were mutagenic,with the former being more potent;while the other five compounds were completely inactive.3.The results of ROS determinations indicated that only 1,2,4-trihydroxybenzene increased the intracellular level of ROS,with a little stronger effect in V79-hCYP2E1-hSULT1A1 than V79-Mz cells.None of the other compounds changed ROS in either cell line obviously.ConclusionsIn this study,we observed the mutagenic effect of benzene in mammalian cells for the first time,in which human CYP2E1 serves as an essential enzyme for activating benzene.Our experimental results also support that human CYP2E1 can further activate various hydroxylated metabolites from benzene,and confirm once again that 1,4-benzoquinone might be the ultimate mutagen of benzene.ROS may not be a major mediator of the mutagenic effect induced by benzene or its metabolites.