Induction Of Micronuclei And Cell Cycle Arrest By Some Non-coplanar Tri-and Tetrachlorobiphenyls In Mammalian Cells Deficient In Xenobiotic-metabolizing Enzymes

Polychlorinated biphenyls(PCBs)are a large group of environmental persistent organic pollutants,with biological toxicity,bio-accumulation,resistance to degradation and long-distance mobility,thus they are still subject to widespread concerns.PCBsmay be contributable toneurological damage,impared immune functions,hepatic injury,and cardiotoxic responses;in 2013,they have been classified into group 1(human)carcinogens by the International Agency for Research on Cancer(IARC).Differing in the number and position of chlorines,PCBs have 209 congeners,nominated in abbreviationas from PCB 1 to PCB 209.Most congeners have been detected in various environmental samples.The lower chlorinated biphenyls(containing 1-4 chlorines per molecule)are supposed to be mutagenic following metabolic activation.However,in a preliminary study we recently observed induction of micronuclei by several PCBs in a subclone of Chinese hamster V79 cell line,V79-Mz,which is deficient in xenobiotic-metabolizing enzyme activities,includingCYPs,UDP-glucuronosyl transferases(UGTs),and sulfotransferases(SULTs).This result indicates that some PCBs compounds(rather than their metabolites)might posess genetic toxicity,however,this possibility has to be further confirmed.In this study,six trichlorobiphenyls and four tetrachlorobiphenyls were investigatedfor theiractivities to induce micronucleiin V79 and V79-Mz cells.Two representative compounds were further observedfor their effects on cell proliferation activity,micronuclei formation,Hprtmutants formed,and the mitotic indexin V79 and V79-Mz cells(both being deficient in p53);moreover,induction of micronuclei by representative PCBswas further verified in human hepatotoma(HepG2)cells(proficient in p53),and their activity to inducecell cycle disturbance was analyzed in V79,V79-Mz,and HepG2 cells;lastly,the effects of representative PCBs on the levels of expression ofseveral proteins with relevance to cell cycle controlwere analyzed in HepG2 cells.Regarding the four tetrachlorobiphenyls,PCB 74 and PCB 66 are structurally similar to PCB 77 and PCB 81,however,only the former two compounds possess achlorine substitution at an ortho-position(the latter two have no ortho-substitution);PCB 74 and 66demonstrated induction of micronuclei in V79-Mz cells(PCB 74 with greater potency),while PCB 77 and PCB 81 were inactive.As for the six trichlorobiphenyls,PCB 20 and PCB 22 appeared to be potent inducers of micronuclei in V79-Mz and V79 cells at the concentration of 10μM and 20μM,respectively;these activities were stronger than the other trichlorobiphenyls,i.e.,PCB 16,PCB 17,PCB 18 and 28.Therefore,we chose PCB 20 and PCB 22 as representative PCBsfor conducting further investigations.Theresults of CCK-8 assays indicated that both PCB 20 and 22 started to be cytotoxic in V79-Mz cells at 10μM,while in HepG2 cells at 80μM.In V79-Mz cells,PCB 20 and 22 started to markedly change the morphology of V79-Mz at 10μM and that of V79 cells at 20μM.Micronuclei test results indicated thatPCB 20 and 22induced concentration-dependent formation of micronuclei in V79,V79-Mz and HepG2 cells(positive results in p53-proficient HepG2 cells may help exclude the possibility of false positive results in p53-deficient V79 and V79-Mz cells),with greatest potency in V79-Mz cells;however,these two compoundsdid not induce gene mutations in V79-Mz,up to the concentration of 16 p.M,a level with severe cytotoxicity.PCB 20 and 22 inducedmitotic arrest in V79 and V79-Mz cells,as indicated by the elevatedfrequency of mitotic cells,V79-Mz cells beinga little more sensitive than V79 cells.PCB 20 and 22 also induced G2/M arrest at varying potencies and efficiencies in V79-Mz,V79,and HepG2 cells,with V79-Mz being the most sensitive cells(with a threshold of 10μM)and HepG2 cells as relatively resistant cells(slightly increased proportion of cells at the G2/M phase with PCB 20 at200μM).Preliminary results of western blotting assay of cell cycle-related proteins in HepG2 cells indicated that PCB 20 may increase the expression of Cyclin B1 and CDK1,whilemay decrease the expression of p27.In summary,the present study indicates that some tri-and tetrachlorobiphenyls,of featured molecular structures,may induce cytotoxic responses,micronuclei,and G2/M arrest in mammalian cells,V79-Mz cells being particularly sensitive.Induction of cytotoxic responses and micronuclei by PCBs is structure-dependent:the 2,3,3’-and 2,3,4’-trichlorination is important for a high potency,and a non-coplanar configuration of tetrachlorobiphenyls is required for the effects.Moreover,PCB 20 may slightly arrest HepG2 cells at the G2/M phase,increase the expression of Cyclin B1 and CDK1 while decrease that of p27,proteins supposed to be cell cycle controllers.The observed effects may be independent of metabolism of PCBs,as the cell lines used are all deficient in activities of CYPs,especially CYP2E1 which is a major activating enzyme for non-coplanar PCBs.