Human CYP1 Enzymes-mediated Genotoxic Effects Of Coplanar PCBs In Mammalian Cells

Polychlorinated biphenyls(PCBs)are a class of persistent environmental organic pollutants with carcinogenicityin humans.PCBs has been banned for many years,however,they are still persistedin the environment,and theyalso cause regional high level pollution with human exposure,due to the resistance of these compounds to degradation and new pollution.PCBs are divided into coplanar(dioxin-like)PCBs and non-coplanar plane(non-dioxin-like)PCBs according to the spatial relation of the two rings of each PCB.The toxicity of PCBs of the former category is mainly through activating the aromatic hydrocarbon receptor(AHR),while the those of the latter category exert their toxicity primarily through their active metabolites.Coplanar PCBs arepotentinducers of CYP1 enzyme,moreover,3,3',4,4',5-pentachlorobiphenyl(PCB 126)and 3,3',4,4'-tetrachlorobiphenyl(PCB 77)as coplanar compounds can both be transformed by rat CYP1A1 and catfish CYP1A to4-hydroxylated metabolites.However,the potential of other CYP1 enzymes in the metabolism/activation of coplanar PCBs is unclear.Objectives1.To observe the genotoxic effects of different coplanar PCBs(PCB 77,PCB 81,PCB 126)under metabolic activation bydifferent CYP enzymes(CYPlA1,CYP1A2 and CYP1B1)in mammaliancells.2.Using immunofluorescence staining of centromere protein B(CENP-B),the micronuclei formed by test compounds were analyzed for the presence of centromere,in order to uncoverthe mechanism of micronuclei formation,i.e.,to discriminate aneugenic from clastogenic action.Methods1.Cytotoxicity(CCK-8)assayThe CCK-8 assay was employed to determine the cytotoxicity of each test compound in the Chinese hamster lung(V79)cell line,the V79-derivedcell lines genetically enjineered for the expression of human CYP1A1,CYP1B1,and CYP1A2 enzyme,and human hepatoma(C3A)cell line.2.Micronuclei testThe standard in vitromicronucleus test was used to observe the activity of each test compound to induce micronuclei in each of the cell lines described above,two exposure/recovery schedules,i.e.,6 h/18 h and 18 h/6 h,were employed.3.Hprt mutagenicity assayThe standard procedures of Hprt mutagenicity assay were used to determine the activity of each test compound to induce gene mutations in each V79-derivedcell line.4.Determination of mitotic index and cell cycle distributionThe mitotic index of cells of each line with and without each test compound wasscored after fixation and staining of harvested cells by microscopy,and the effect of test compounds on the distribution of cell cyclewas analyzed after dyeing of the cells with PI by flow cytometry.5.Immunofluorescent staining of centromere with micronucleiImmunofluorescence assay with anti-centromere protein B(CENP-B)antibodies was employed to discriminate the micronuclei induced by a representative PCB for the presence or absence of centromeres.6.Statistical analysisThe results of CCK-8 assays were analyzed by ANOVA;the duplicate data resulted from the micronucleus test or mutagenicity assay were combined to become quantal data and then analyzed by using x 2 analysis;and the triplicate data of CENP-B determination with immunofluorescence assay were examined by the Student's t test.Results1.Each of the coplanar PCB 77,PCB 81,PCB 126 at concentrations ranging from 5 ?mol/L to 40 ?mol/L(approaching its solvent)did not induce micronuclei in V79-Mz cells.However,under the 6 h/18 h exposure/recovery regime PCB 81 elevated the frequency of micronucleated cells in V79-hCYP1B1,V79-hCYPlAl,V79-hCYP1A2 cells.Under the other,i.e.,18 h/6 h,regime all three test compounds induced micronuclei in V79-hCYP1A2,while inactive in cells of the other lines.2.None of the three test PCBs,at concentrations ranging from 5 ?mol/L to 40?mol/L,induce Hprt gene mutations in any of the cell lines(V79-Mz,V79-hCYP1A1,V79-hCYP1A2,or V79-hCYP1B1),with experiments performed under the satandard procedures for the mutagenicity assay.The paralleling positive controls,benzo(a)pyrene(a promutagen dependent on CYP1A1 or 1B1)for V79-hCYP1A1 and V79-hCYP1B1,and aflatoxin B1(a promutagen activated by CYP1A2 or 3A4)for V79-hCYP1A2,also strongly elevated the frequency of mutants.This indicates that the CYP enzymes expressed in V79-derived cell lines have specific metabolizing functions,and thus the negative results with test PCBs should be valid.3.As a representative coplanar PCB compound,PCB 81 induced micronuclei mostly of CENP-B-negative,using immunofluorescence assayin combination with the nucleus test under both 6 h/18 h and 18 h/6 h regimes,with centromere-free micronuclei taking the proportion of 74%and 65%,respectively.Meanwhile,ethyl methyl sulfonate(EMS,a known clastogen)and vincristine(VCR,a known aneugen)induced micronuclei with CENP-B-positivity as 22%and 61%,respectively,which are consistent with previous reports.Therefore,it is valid toapplyanti CENP-B antibodies in an immunofluorescence assay to analyze the presence of centromeres withmicronuclei(as an alternative to the classicalCREST assay).ConclusionsThe three test coplanar PCBs could be activated by human CYP1B 1,CYP1A2 and CYP1A1 to micronuclei-inducing metabolites,under varied exposureregimes.Most significanteffect was observed with PCB81 activated by CYP1B1 enzyme,with micronuclei formed through a clastogenic action.The test compounds are incapable of inducing Hprt gene mutations in the V79-derived cells expressing each CYP1 enzyme.Taken together,the abovecoplanar PCBs can be genotoxic in mammalian cells expressing human CYP1 enzymes with limited efficacy and experimental endpoints.