Mutagenicity Of Non-Coplanar PCBs

Polychlorinated biphenyls(PCBs)are a group of persistent organic pollutants,which have been classified as group 1(human)carcinogens by the International Agency of Research on Cancer(IARC).Although PCBs have been banned for production and commercial use for more than 30 years,they still exist in the environment,due to their chemical stability and accumulation through food chains.As global pollutants,PCBs are very hazardous to human health,causing endocrinal dysfunction,precancerous and cancerous damage,hepatic injury,reproductive disturbance,immune system impairment,and growth and developmental obstacles.Therefore,studies about PCBs are very necessary.PCBs can be divided into two categories:coplanar(dioxin-like,DL)and non-coplanar(non-dioxin-like,NDL)PCBs.DL-PCBs may cause various biological and pathological changes[including carcinogenesis and potent induction of cytochrom P450(CYP)1 enzymes)],through persistent activation of aromatic hydrocarbon receptor(AHR);contrastively,NDL-PCBs may be activated by CYP2E1 to mutagens.There are totally 12 congeners as DL-PCBs,including PCB 77 and PCB 81,etc.;while among more congeners as NDL-PCBs,the 7 so-called indicator PCBs(including PCB 52 and PCB 28,etc.)are most noticeable due to their relatively high burdens in the human body.Regarding the mutagenicity of PCBs(a major mechanism of carcinogenesis),NDL-PCBs are currently supposed to be primarily involved.Our recent studies suggest that human CYP2E1 may activate a series of dichlorobiphenyls to mutagens.Our more recent results indicated that PCB 22(a trichlorobiphenyl)is more mutagenic than all(12)dichlorobiphenyl compounds.Therefore,this study is focused on PCB 22 and PCB 52(the latter is one of the indicator PCBs)(both being NDL-PCBs)for their activities to induce micronuclei and gene mutations in mammalian cells,wherein the relative contributions of human CYP2E1,1A1,1A2,1B1,and 3A4 to the metabolic activation were investigated.Objectives1.Verification of the expression of biotransformation enzymes(human CYP1A1,1B1,1A2,3A4,and 2E1)in genetically engineered V79-derived cell lines as indicated by the presence of enzymatic proteins and the mutagenic responses to known promutagens,in comparison to the parental cell line V79-Mz which is deficient in the activities of CYPs,UDP-glucuronosyl transferases(UGTs)and sulfotransferases(SULTs).2.Investigation of the metabolic activation and related mutagenicity of PCB 22 and PCB 52 in the presence of human CYP1A1,1A2,1B1,3A4,and 2E1 expressed intracellularly,with the capability of various CYP enzymes to activate the chemicals being compared to each other.3.Provide a rank of order of the mutagenicity of NDL-PCBs(with the reported dichlorobiphenyls included)and the structure-toxicity relationships,in order to provide a reference for the health risk analysis of PCBs.Methods1.SDS-polyacrylamide gel electrophoresis and immune-blot(Western blot)analysisWestern blot analysis was employed to identify the expression of individual CYP enzymes in various V79-derived cell lines,including V79-hCYP1A1,V79-hCYP1A2,V79-hCYP1B1,V79-hCYP3A4-hOR,and V79-hCYP2E1-hSULT1A1 cell lines.2.Anlysis of cell growth/survival using CCK-8 assayPCB 22,PCB 52,ethylmethanesulfonate(EMS),N-nitrosodimethylamine(NDMA),2-nitropropane(2-NP),benzo(a)pyrene(B(a)P),and aflatoxin B1(AFB1),each in a series of concentrations,were used to expose V79-Mz and V79-derived cells expressing various metabolic enzymes,with water or DMSO as appropriate at the level of<20‰(v:v)employed as the negative control,and 6 replicates were set up for each treatment.Treatment schedules(exposure/recovery)of 12 h/12 h and 24 h/48 h were used as corresponding to micronucleus test and mutagenicity assay,respectively.The standard assay procedure of CCK-8 assay was followed.Eventually the optical density at 450 nm was determined,with the percent of value relative to the negative control defined to represent the cell growth/survival level.A series of concentrations of each test compound giving 60%and higher cell growth/survival levels were chosen for the next micronucleus test.3.Micronucleus testMicronucleus test is a genetic toxicity assay aimed to detect chromosomal and mitotic apparatus damages.The six V79 or V79-derived cell lines described above were individually used in the micronucleus tests with PCB 22,PCB 52,PCB 20 and the known promutagens used as reference compounds.A 12 h/12h(exposure/recovery)regimen was employed in the test.After cell harvest,a series of treatments(hypotonic treatment,fixation,droping on slides,and staining in Giemsa stains)were performed,and eventually each slide was scored for the frequency of micronucleated cells,through observing randomLy encountered integrate(excluding apoptitic or broken cells)cells under microscopy at × 1000 magnification.Totally 2000 cells were observed in each cell smaple,with cells containing at least one micronucleus recorded and numbered.Finally the frequency of micronucleated cells in each group was expressed as a mean± variation range(n = 2).4.Hprt mutagenicity assayThe forward mutations of the hypoxanthine phosphoribosyltransferase(hprt)[as indicated by acquiring the resistance to 6-thioguanine(6-TG)]was used as the end point of this assay.At 1 d,cells of each line were treated with each chemical for 24 h,followed by subculturing for two times at 4 d and 7 d,respectively.At the second suculturing,cells were seeded for the assay of colony-forming efficiency in fresh medium(in three dishes)and that of 6-TG-resistant mutants(in four dishes).After 7 days,the colony-forming efficiency and the frequency of mutants were observed.5.Statistical analysisAVOVA was employed to analyze the data of CCK-8 assays.Hprt assay and micronucleus assay results were expressed as means ± range of variation(n = 2);however,for statistical analysis the duplicate data were combined to become quantal data,then analyzed using Fischer's and x2 analysis,respectively.Results1.No expression of any CYP protein in V79-Mz was observed using Western blot assay,on the contrary,each of CYP 1A1,1A2,1B1,3A4,and 2E1 proteins was clearly observed in V79-derived cell line V79-hCYPlA1,V79-hCYP1A2,V79-hCYP1B1,V79-hCYP3A4-hOR,and V79-hCYP2E1-hSULTlA1.2.The eytotoxic effect of each promutagen in V79-Mz and the relevant V79-derived cells was similar to each other,and so did that of PCB 22 or PCB 52.Based on the observed cytotoxic concentrations of each test compound,a series of concentrations corresponding to 60%and higher cell growth/viability levels were designed for the micronucleus teat and hprt mutagenicity assay(EMS,5 mM;NDMA,100 and 300 ?M;2-NP,2 and 4 mM;B(a)P,2,4,8 ?M;AFB1,0.05,0.1,0.2,0.4,and 0.8 pM).3.EMS(5 mM,a direct mutagen)induced both micronuclei and gene mutations in V79-Mz cells,while NDMA,2-NP,B(a)P and AFB1 were all inactive toward these cells.However,NDMA and 2-NP induced concentration-dependent gene mutations in V79-hCYP2E1-hSULT1A1 cells;B(a)P induced gene mutations(in a clear concentration-effect relationship)and micronuclei in both V79-hCYP1A1 and V79-hCYP1B1 cells;AFB1 induced both gene mutations and micronuclei in both V79-hCYP1A2 and V79-hCYP3A4-hOR cells,in a concentration-dependent manner.4.PCB 22,PCB 52 and PCB 20 both induced micronuclei in V79-hCYP2E1?hSULT1A1 cells in a concentration-dependent manner;however,they were both inactive in V79-Mz cells,and weakly positive toward V79-derived cells expressing other CYP enzymes(with statistically significant elevation of frequency of micronucleated cells only at 6 or 40 ?M,the highest concentration of PCB 22 and 52,respectively..5.PCB 22(1?10 ?M),PCB 52(10?40?M)and PCB 20(1?4 ?M)were both non-mutagenic in V79-Mz cells,however,they induced gene mutations potently in V79-hCYP2E1-hSULT1A1 cells,with a clear concentration-effect relationship.These two test compounds at the highest concentrations were slightly mutagenic in V79-hCYP1B1 cells,while completely negative in the other cell lines expressing other CYP enzymes(V79-hCYP1A1,V79-hCYP1A2,and V79-hCYP3A4-hOR).Conclusions1.V79-derived cell line V79-hCYP1A1,V79-hCYP1A2,V79-hCYP1B1,V79-hCYP3A4-hOR,and V79-hCYP2E1-hSULT1A1 obviously expressed the protein of human CYP1A1,CYP1A2,CYP1B1,CYP3A4,and CYP2E1,respectively.Moreover,the induction of micronuclei and genemutations by B(a)P,AFB1,NDMA and 2-NP in V79-derived cells expressing relevant enzyme(s)indicates that the combinantly expressed enzymes are capable of appropriate metabolic activation.2.Human CYP2E1 is capable of potently activating PCB 22,PCB 52 and PCB 20 to mutagenic metabolites,while other CYP enzymes are either negative or weakly positive in activating these two compounds.Human CYP2E1 may be critical in activating NDL-PCBs.3.It is worth considering that metabolism-dependent mutagenicity should be included into the endpoints for the health risk analysis of environmental PCB exposure.