Studies On Metabolic Activation And Genotoxicity Of Mono-and Dichlorinated Biphenyls And1-hydroxymethylpyrene

BackgroundPolychlorinated biphenyls (PCBs) are worldwide environmental pollutants that constitute a group of209different congeners. PCBs have been extensively used in the electrical utility industries in capacitors, transformers, hydraulic fluid, paints, plasticizers, and a variety of other commercial products, because of their extreme physico-chemical persistence. The global accumulation of PCBs production is about1million tons from late1929s to1970s and more than half of them have entered the dump or landfills. In China, PCBs have been produced about10,000tons from1965to1974in various products, which were distributed throughout the country. These PCBs-containing products have inevitably polluted the human environment. Although PCBs have been legally banned from production and transportation in most countries for more than30years, now they still exist at varied levels all over the word due to their chemical stability. Moreover, recycling of PCB-containing products, such as e-wastes, may also give rise to human tissue burdens of PCBs through occupational and environmental exposures.Before the national legislation that have banned PCBs business applications, PCBs were discharged to the environment with more than dozens of PCBs at varying chlorination degrees and patterns as PCB mixtures collectively. The animal carcinogenicity of PCBs has been confirmed for many years, nor is its mechanism for mutagenic potentials. Recently, PCBs as a class of chemicals and12individual dioxin-like PCBs have been classified by the International Agency for Research on Cancer (IARC) as human (group1) carcinogens. Sustained activation of aryl hydrocarbon receptor (AHR) by dioxin-like PCBs with subsequent signal cascades has been proposed to be the major mechanism for dioxin-like PCB carcinogenesis. On the other hand, lower chlorinated PCBs are more likely metabolized at substantial rates. Meanwhile, a variety of lower chlorinated PCBs have been identified in the air and water at varying concentrations in the domestic and foreign environment. By structure-activity modeling of the existing experimental data of PCBs and their metabolites in animal and cell culture systems, American Academy of Environmental Sciences speculated that mono-and dichlorinated biphenyls are most probably mutagenic after metabolic activation by human biotransformation enzymes. However, there have been no direct or systemic evidences to support this hypothesis.1-Hydroxymethylpyrene (1-HMP) is a benzylic alcohol derived from hydroxymethyl polycyclic aromatic hydrocarbons and a major metabolite formed in vitro from the hepatocarcinogen1-methypyrene (1-MP).1-HMP is genotoxic under further metabolism by various sulfitransferase (SULT) isoforms, strongest by SULT1A1. Recently,1-sulfooxythylpyrene has been confirmed in rat in vivo as the ultimate toxicant from1-MP. However, the effect of1-MP or its metabolites on mammalian chromosomes, especially in the presence of human metabolic enzymes, have never been reported.ObjectivesThis study is proposed according to our speculation that human CYP2E1may potentially be involved in the metabolic activation of lower chlorinated biphenyls, in that PCBs are structurally closer to benzene and phenol (substractes for CYP2E1) than to benzo(a)pyrene and other polycyclic aromatic hydrocarbons (substractes for CYP1A1), and hydroxylation of benzene and phenol may lead to mutagenic activation. As the initial part of our PCB bioactivation project, this study was focused on investigation of the role of human CYP2E1in the bioactivation of mono-and dichlorinated biphenyls, and the influence of sulfotransferase1A1on the effect of active metabolite from each PCB. The overall purpose of this study is to identify the genotoxicity of lower chlorinated biphenyls. Additionally, as the latter part of this thesis, human SULT1A1-catalyzed activation of1-HMP and the relevant genotoxic outcomes (both micronuclei formation and gene mutations) were explored. Methods1. The tested chemicals included3monochlorinated biphenyls (2-chlorobiphenyl,3-chlorobiphenyl and4-chlorobiphenyl),10dichlorinated biphenyls (2,2’-dichlorobiphenyl,2,3-dichlorobiphenyl,2,4-dichlorobiphenyl,2,4’-dichlorobiphenyl,2,5-dichlorobiphenyl,2,6-dichlorobiphenyl,3,3’-dichlorobiphenyl,3,4-dichlorobiphenyl,3,5-dichlorobiphenyl,4,4’-dichlorobiphenyl), and1-HMP.2. Cytotoxicity of each PCB was determined by the reduction of MTT to formazan. The V79cell line and a V79-derived cell line expressing both human CYP2E1and human SULT1A1(V79-hCYP2E1-hSULTlA1) were treated with each tested chemical at different concentrations for12h, and for another12h cells were cultured for cell expansion.3.In the micronucleus test in vitro, V79control and V79-hCYP2E1-hSULT1A1cells were treated with each tested chemical at varying concentrations for12h and for another12h cells were cultured in the absence of tested chemicals. Frequency of micronucleated cells was scored based on observation of4,000cells in each treatment (two cultures).4.1n the Hprt locus mutagenicity method, V79control cells and V79-hCYP2E1-hSULT1A1cells were treated with each tested chemical at varying concentrations for1d, then cultured in the absence of chemicals for another2d, Subsequently, cells were subcultured and cultured for another3d, afterwards, cells were again subcultured for testing colony-forming efficeincy and frequency of mutants resistant to6-thioguanine.5.For observation of the role of each expressed enzyme in the observed effects,1-aminobenzotriazole (ABT, CYP2E1inhibitor) and pentachlorophenol (PCP, SULT1A1inhibitor) were added in each culture2h prior to the addition of a tested chemical, and continued to be present in the cultures till the end of cell culturing or subculturing (12h in the micronucleus test and3d in the mutagenicity test).6. For an easy and clear structure-activity estimation with the tested PCBs, the efficiency of each dichlorobiphenyl was calculated as the highest ratio of the frequency of micronucleated cells (%o) to the corresponding concentration (μM), representing the genotoxic potential of each dichlorinated biphenyls.7. Statistical analysis:The relative cell viability and growth in the presence of each PCB or1-HMP (by MTT assay, n=6) was compared to the negative control using ANOVA (n=6). Comparison of the frequency of micronucleated cells in each treatment with the control was performed by χ2analysis, and that of the mutant frequency was performed by the exact probability (Fisher) method.Results1. Cytotoxicity of tested chemicals and the metabolic enzyme dependence1.1Cytotoxicity of mono-and dichlorinated biphenylsNone of the13PCBs induced cytotoxicity in V79cells at the concentrations ranging from10to100μM. However, in V79-hCYP2E1-hSULTlA1cells, PCB4, PCB5and PCB11induced reduction in cell viability and growth (p<0.05, compared with the control). PCB10did not show any cytotoxicity in V79-hCYP2E1-hSULTlA1cells, but potentinated by PCP. Pretreatment of the cells with0.2%ethanol led to mildly potentiated cytotoxicity of the above mentioned PCBs.1.2Cytotoxicity of1-HMP1-HMP was not cytotoxic in V79cells up to8μM. However, in V79-hCYP2E1-hSULTlA1cells1-HMP reduced cell viability/growth (p<0.05) at the highest concentration (8μM). This effect was alleviated by PCP, indicating the involvement of human SULT1A1in activation of1-HMP.2. Induction of micronuclei by test chemical and its metabolic enzyme dependence2.1None of the PCBs induced micronuclei in V79cellsExcept for PCB1,all the other12PCBs induced micronucleiin V79-hCYP2E1-hSULTlA1cells (p<0.05for at least one treatment). Meanwhile,7PCBs demonstrated concentration-dependent induction of micronuclei; concentration-dependent responses were observed with PCB4, PCB5, PCB7, PCB8, PCB10, PCB11. Co-exposure of the cells to ABT(20μM) led to blockage or significant redution in each PCB-induced micronucleation(p<0.05). Co-exposure of the cells to PCP (10μM) elevated (PCB8) or did not change the induction of micronuclei by the rest12PCBs.2.2Induction of micronuclei by1-HMP inV79-hCYP2E1-hSULT1A1cells1-HMP was inactive toward V79cells (1—8μM). However, it induced micronuclei apparently in V79-hCYP2E1-hSULTlA1cells in a concentration-dependent manner. Co-exposure of the cells with PCP almost completely prevented the induction of micronuclei by1-HMP.3. Gene mutations (at the Hprt locus) induced by PCBs and1-HMP3.1Mutagenicity of four representative dichlorinated biphenylsPCB5, PCB8, PCB10, PCB11, active in CYP2E1-dependent induction of micronuclei, were chosen as representing dichlorinated biphenyls in the mutagenicity assay. All these PCBs induced gene mutations in V79-hCYP2E1-hSULTlAl cells co-exposed to PCP for SULT1A1inhibition), in a concentration-dependent manner. Among them, PCB5and PCB10showed stronger levels of mutagenic responses than the other two PCBs.3.2Induction of gene mutations by1-HMP1-HMP did not elevate the mutant frequency of V79cells (p>0.05); however, it induced mutagenic response in V79-hCYP2E1-hSULTlA1cells in a concentration-dependent manner, and PCP co-exposure apparently reduced the frequency of gene mutations. The results of cytotoxicity in the cell lines were consistent with those of the mutagenicity assay:No cytotoxic reponse in V79control cells, while in V79-hCYP2El-hSULTlA1cells,1-HMP demonstrated obvious cytotoxicity at the highest concentration (4μM), which seemed to be reduced by PCP. Collectively, the results indicated human SULT1A1-dpendent mutagenicity of1-HMP in mammalian cells.3.3Structure-activity relationships for dichlorinated biphenyls-induced micronuclei in V79-hCYP2E1-hSULT1A1cells Among the10tested dichloribiphenyls, PCB4, PCB5, PCB7, PCB8, PCB10, PCB11showed relatively high potency and/or efficacy in micronuclei induction. Apart from PCB11, all the above strong micronuclei-inducers are structured with one or two chlorine residues at the2-or2’-position (ortho-to the single bond connecting the two rings). Most of the other4PCBs, weak inducers of micronuclei, bear chlorine substitutions at positions other than the ortho-position (except for PCB9). This suggests that ortho-(2-or6-) positionof chlorine substitution may be an important pattern for CYP2E1-dependent activation of dichlorinated biphenyls leading to strong induction of micronuclei in mammalian cells.Conclusion1. For the first time human CYP2E1has been suggested to be the specific enzyme for the bioactivation of PCBs, and human SULT1A1may not be important in detoxifying the reactive metabolites formed from PCBs, at least those of mono-and dichlorinated biphenyls.2. Human CYP2E1-catalyzed activation of mono-and dichlorinated biphenyls may induce chromosome aberration and gene mutations in mammalian cells.3. Human CYP2E1may not be able to activate monochlorinated biphenyls to strong micronuclei inducers, while many dichlorinated biphenyls are CYP2E1-dependent strong genotoxicants.4. Relatively high efficiency of human CYP2E1-dependent induction of micronuclei may be related to the presence of ortho-positioned chlorination of dichlorinated biphenyls..5. Human SULT1A1may activate1-HMP for mutagenic and chromosome-damaging activities in mammalian cells.