Metabolic Activation Of Lower Chlorinated Biphenyls By Mouse CYP2E1 And Related Genotoxicity

As a big group of persistent organic pollutants and human(class 1)carcinogens,polychlorinated biphenyls(PCBs)are still widely distributed due to their extensive use during the period from 1930 to 1980 and the lack of appropriate degradation technology.According to the number of chlorine substituents on the benzene rings of PCBs,PCBs can be classified as lower(chlorine atoms<4)and highly chlorinated(chlorine atoms>4)congeners.The levels of lower chlorinated biphenyls in the air of some regions could be quite high,and they can be readily absorbed through inhalation,and more easily biotransformed to metabolites than the highly chlorinated congeners.Our research group has recently confirmed that human CYP2E1 can specifically activate PCBs,particularly lower chlorinated congeners,for potent mutagenicity.However,as a common species used in toxicological research models,mouse CYP2E1 has never been studied for the possibility to activate PCBs.Objectives1.To observe the formation of micronuclei in hepatocytes of NIH mice exposed to 2,3,4'-trichlorobiphenyl(PCB 22),a potent promutagen activated by human CYP2E1 as observed in our recent report.2.To predict the substrate activities of lower chlorinated biphenyls toward mouse CYP2E1,through docking of these compounds into the active site of mouse CYP2E1 individually,as compared with its human homologous protein(human CYP2E1),based on which some congeners evaluated as being highly affinic for mouse CYP2E1 will be used in the genotoxicity assays.3.To evaluate the mutagenicity of selected PCB compounds,2-mono-,2,3-di-,2,5-dicholorobiphenyls(PCB 1,5,and 9,respectively),PCB 22,and N-dimethylnitrosamines(NDMA),in V79-derived cells expressing mouse CYP2E1,as indicated by the formation of micronuclei and induction of Hprt mutations,in comparison with their effects in V79-derived cells expressing human CYP2E1,thus potentially appropriate PCB compounds may be suggested for application in intact animal(mouse)studies.Methods1.Hepatocyte micronucleus assay in NIH miceNIH mice were exposed to PCB 22 at three different doses,twice within 24 h,by intraperitoneal injection,with corn oil used as the solvent control,and N-nitrosodiethylamine serving as the positive control.9 or 10 replicates were set up for each group.Four days after chemical exposure,the mice were killed,hepatocytes were isolated for AO/DAPI staining.After smearing and mounting slides with the cells,2000 randomly encountered cells from each mouse were observed for the presence of micronuclei,under fluorescent microscopy.2.Homology modeling and molecular dockingThe three-dimensional structure of mouse CYP2E1 was constructed by using SWISS-MODEL platform with human CYP2E1 as a template.All mono-and dichlorobiphenyls and a collection of trichlorobiphenyls were docked into the active site of mouse CYP2E1 as potential ligands,by using Autodock 4.2.PCBs with relatively sufficient affinity to mouse CYP2E1 were applied in follow-up genotoxicity assays.3.In vitro cytotoxicity and genotoxicity assaysThe cytotoxicity of testing compounds(PCB 1,PCB 5,PCB 9 and PCB 22)and NDMA(positive control)in V79-mCYP2E1 and the control V79-Mz cells were determined using the Cell Counting Kit-8(CCK-8)assay,under exposure/recovery regimes corresponding the the relevant micronucleus test and Hprt mutagenicity assay.The micronucleus test was performed under a 3 h/21 h regime while 1 d/2 d regime was applied in the Hprt mutagenicity assay.4.Western blot assayThe expression of CYP2E1 in tested cell lines was determined by Western blot assay,with a series of steps such as SDS-PAGE,blotting of proteins,membrane blocking,incubating membrane in antibodies recognizing CYP2E1 and HRP-conjugated secondary antibodies sequentially,and detection with an enhanced chemiluminescence system.Results1.Based on our previous observation that PCB 22 has the strongest genotoxicity among various PCB compounds which is dependent on human CYP2E1 activity,the hepatocyte micronucleus test in NIH mouse in vivo was used to analyze the geno toxicity of PCB 22.The results showed that PCB 22 was inactive at doses ranging from 170 to 2000 mg/kg bodyweight(given intraperitoneally),while N-nitrosodiethylamine as the positive control elevated the frequency of micronucleated cells significantly.2.Molecular docking of 23 PCBs containing 1-3 chlorine substituents to the active site of mouse and human CYP2E1 indicated that 9 and 17 PCB compounds fitted valid with mouse and human CYP2E1,respectively,as potential substrates;the resultssuggested that mouse CYP2E1 may be a weaker activator of lower chlorinated biphenyls as compared with the human homologous enzyme.3.PCB 1,5 and 9,which might be substrates of mouse CYP2E1 as suggested by molecular docking analysis,and PCB 22,which showed binding with mouse CYP2E1 at a site outside of the active center,were tested for relevant cytogenetic toxicity.The results showed that PCB 9 induced both micronuclei and Hprt gene mutations potently in V79-mCYP2E1 cells,while inactive in V79-Mz cells;PCB 1 and 5 were both non-mutagenic in each cell line,but induced micronuclei at high concentrations in both cell lines,with similar intensity between the two cell lines.PCB 22 weakly induced gene mutations in V79-mCYP2El cells,while inactive in V79-Mz;it induced micronuclei in both cell lines,with a potency in each cell line similar to the other.Conclusions1.There seem to be significant species differences existing between mouse and human CYP2E1 in activating lower chlorinated biphenyls:mouse enzyme being largely weaker.Therefore,the mouse may not be suitable as an experimental animal model for CYP2E1-activated PCB toxicity.2.Relevant cytotoxicity and molecular docking between compounds and the target activating enzymes had better be carried out,in order to clarify the difference of enzymes between species in metabolic activation of test compounds,to screen for candidate compounds for experimental confirmation.In this way unnecessary waste of animal resources can be avoided.