Studies On Metabolic Activation Of Various Environmental Chemicals By Human CYP2E1

There are millions of xenobiotics in the global environment and some of them have been proved to be human mutagens or carcinogens. Most of these compounds showed their mutagenicity subsequent to metabolism by specific biotransformation enzymes in the biologic body. These xenobiotics are called indirect mutagens or carcinogens. The mechanisms of the mutagenesis by some indirect mutagens have been fully elucidated, while those of others remain unclear. Poly chlorinated biphenyls (PCBs) are a class of environmental persistent organic pollutants (POPs). Although they have been legally banned in most countries for commercial production and usage for more than 30 years, PCBs are still produced in some manufactures and indoor-combustion. Abundant studies have proved that PCBs are involved in disruption of neurodevelopment, disturbed endocrine systems and development of cancers. PCBs have been categorized by the International Agency for Research on Cancer (IARC) as "human (Group 1) carcinogens" in 2013.Hydroxylation of PCBs is an important step leading to the production of mutagenic metabolites of PCBs, which is catalyzed by CYPs. However, the particular isoforms of CYPs responsible for this reaction are not well understood.. It has been established that dioxin-like PCBs (DL-PCBs) can efficiently induce the expression of some CYPs of the CYP1 family (i.e., CYP1A1,1A2 and 1B1) though activation of aromatic hydrocarbon receptor (AHR), thus these CYPs may be supposed to be potential metabolizing enzymes for DL-PCBs. Our recent studies indicate that the human CYP2E1 is capable of activating some non-dioxin-like PCBs (NDL-PCBs) to mutagenic metabolites. However, whether other isoforms of CYPs also contribute to activation of PCBs is never evidenced. Thus, an experimental model with multiple CYPs (especially those important in metabolism of xenobiotics) expressed is wanted. Human hepatocyte (L-02) cell line is a non-tumor immortalized cell line endogenously expressing various CYPs, at least CYP1A1,1A2,1B1,2E1,3A4. In this study two representative PCBs recently identified by our group as potent CYP2E1-dependent promutagen, PCB 22 and PCB 20, were chosen as test compounds in the micronucleus test with L-02 cells, wherein selective CYP2E1 inhibitors were used as modulators; known indirect mutagens metabolically dependent on other isoforms than CYP2E1 were also included were also tested for micronuclei induction in L-02 cells for identification of their involvement in the activation of PCBs. Meanwhile, we constructed a V79-derived cell line stably expressing human CYP1A1 (representative of the CYP isoenzymes inducible through AHR activation.)Benzene is a classical indirect mutagen, which is widely used in manufactures and human life. It has been classified as human carcinogen by IARC in 1982. Benzene is firstly metabolized by CYP2E1 to its hydroxylated metabolites (phenol, hydroquinone and catechol), which are then transferred though blood stream to bone marrows. There hydroquinone is conversed to 1,4-benzoquinone (highly electrophilic and mutagenic) by myeloperoxidase, and this has been suggested as a key step for benzene carcinogenicity (e.g., development of leukemia). Unlike benzene (the smallest aromatic compound), 1-methylpyrene(1-MP) is a poly cyclic aromatic hydrocarbons consisting of four benzoic cycles and a methyl group. It is produced from various human activities, including cigarette smoke, automobile exhausts and pyrolysis of hydrocarbons. Although it has been proved that 1-MP can be transformed into 1-hydroxymethylpyrene (1-HMP) by CYPs and further activated into 1-sulfooxymethylpyrene(1-SMP), which is proposed ultimate carcinogen of 1-MP, those studies were performed using only 1-HMP as an intermediate metabolite from 1-MP, without testing of toxicity end points in mammalian cells. Our group have found that HQ can be further activated to BQ by human CYP2E1, and 1-MP is mutagenic (indicated by induction of micronuclei and gene mutations at the Hprt locus) after consecutive metabolic reactions catalyzed by human CYP2E1 and human SULT1A1. In this study, we repeated the cytotoxicity assays with 1-MP,1-HMP, benzene and its various metabolites in V79-hCYP2E1-hSULT1A1 and V79 cells, with and without enzyme inhibitors, to improve the clarity of results replacing former ones with unsatisfactory quality, particular for confirming the impact of each enzyme inhibitor.MethodThe effects of 2,3,3;-trichlorinated biphenyl (PCB 20) and 2,3,4’-trichlorinated biphenyl on the survival and growth of L-02 cells were determined by performing cell counting kit 8 (CCK-8) assays, with 6 repeats in each group. According to the cytotoxicity assay results the concentration ranges for micronucleus test was determined (with cell survival above 60%), and the frequencies of micronucleated cells in the presence or absence of PCB 20 and PCB 22 were observed. Selective CYP2E1 inhibitors, i.e., trans-1,2-dichloroethane and dimethyl sulfoxide (DMSO) were used as modulators in the tests for identifying the role of CYP2E1. With the activity of CYP2E1 prohibited (in the presence of 2%o of DMSO, v:v), the specific metabolic activating activities of other CYPs (CYP1A1,1B1,1A2 and 3A4) were demonstrated by the induction of micronuclei with benzo(a)pyrene and aflatoxin B1 (AFB1). Three repeats were set up for each treatment.V79-derived cells stably expressing human CYP1A1 was constructed by transfection of an mammalian expressing vector encoding the cDNA of wild-type human CYP1A1 and subsequent selection in the medium supplemented with zeocin. The immunity and enzymatic activity of the expressed protein were examined by Westernblot and micronuclei induction by benzo(a)pyrene (a CYP1A1-dependent promutagen). Then, cytotoxicity and micronucleus tests were performed with PCB 52, 74,77 and 81 in V79-hCYP2E1 and V79-hCYP1A1 cells, with PCB 20 and benzo(a)pyrene as the positive controls in the cell lines, respectively.MTT and CCK-8 assays were employed for the determination of the influences of benzene and its hydroxylated metabolites,1-MP and 1-HMP on the survival and growth of V79-hCYP2E1-hSULT1A1 cells (V79-derived cell line genetically engineered for stable expression of both human CYP2E1 and SULT1A1). The roles of CYP2E1 and SULT1A1 on the observed effects were analyzed by the influences of coexposure of their specific inhibitors,1-aminobenzotriazole for CYP2E1 and pentachlorophenol for SULT1A1. Six repeats were set up for each treatment.Statistical analysis The results of MTT, CCK-8 and micronucleus test were expressed as means±S.D., and the comparison among individual treatments was performed by ANOVA, with p< 0.05 as the standard for a statistical significance.Results1. Induction of micronuclei in L-02 cells by representative PCBs and known promutagensBoth PCB 20 and 22 were mildly cytotoxic at 10 μmol/L and higher concentrations, with the cell survival greater than 70%. Coexposure with DCE (1000 μmol/L) led to marginal reduction of cytotoxicity of these two PCBs. DMSO inhibited the induction of micronuclei by PCB 22 in L-02 cells, with a complete inhibition by DMSO at the concentration of 1.6%o (v:v). In the presence of DMSO at 2%o (v:v) (sufficient for complete CYP2E1 inhibition), benzo(a)pyrene and AFB1 still induced micronuclei in L-02 cells in a concentration-dependent manner; this indicated that the genotoxicity of PCBs in L-02 cells may depend primarily on human CYP2E1, while the other CYPs required for activation of benzo(a)pyrene and AFB1 may not be significant for that of PCBs.2. Stable expression of human CYPlA1 in V79 cells and induction of micronuclei by PCB 52,74,77 and 81 in V79-hCYP2E1 and V79-hCYP1A1 cellsUnlike V79 control cells, cells after transfection of the mammalian expressing vector encoding human CYP1A1 grew to colonies in zeocin-supplemented (300 μg/L) selective medium (with a colony-forming efficiency of 25.3%). Lysate of the cells expanded from a zeocin-resistant colony demonstrated expression of human CYP1A1, as indicated by the Westernblot assay, while that of the V79 control cells did not show an obvious band of immuno-reactive protein. Benzo(a)pyrene (10 umol/L) induced elevation of micronucleated cells in the transfected cells, but not in the control cells. Based on the evidences of protein expression and enzyme-dependent genotoxicity of benzo(a)pyrene, it is concluded that a V79-derived cell line recombinantly expressing human CYP1A1 has been established.In V79-hCYP1A1 cells, PCB 74,77 and 81 weakly induced induction of micronuclei at the highest test concentration (40 μmol/L), and PCB 52 was inactive throughout the test concentrations. In V79-hCYP2E1 cells, PCB 52,74 and 81 elevated the frequency of micronucleated cells, with PCB 74 and 81 obviously induced micronuclei at the concentration of 20 μmol/L, seemingly more active than PCB 52. PCB 77 was inactive toward V79-hCYP2E1 cells.3. Cytotoxicity of benzene and its hydroxylated metabolites in V79-hCYP2E1-hSULTlAl cellsBenzene and phenol were non-cytotoxic in the cells at concentrations ranging from 10 to 300 μmol/L; 1,2,4-trihydroxybenzene decreased the level of cell survival/growth only at the highest tested concentration (320 μmol/L); hydroquinone, catechol and benzoquinone demonstrated cytotoxicity at 20 μmol/L and higher concentrations. CYP inhibitor ABT inhibited the cytotoxicity of hydroquinone, indicated by a blockage of effect of hydroquinone at 20 μmol/L by ABT. Inhibition of catechol toxicity by ABT was more obvious, as indicated by the apparently protective effect of ABT on the cytotoxic response to catechol at all tested concentrations. These results suggest that human CYP2E1 may activate hydroquinone and catechol to more cytotoxic metabolites.4. Induction of cytotoxic response in V79-hCYP2E1-hSULT1A1 cells by 1-MP and 1-HMPUnder the exposure conditions adapted for micronucleus test,1-MP was non-cytotoxic in both V79 and V79-hCYP2E1-hSULTlA1 cells. On the contrary, it demonstrated hormesis in both cell lines at the concentration of 5 μmol/L, with this effect in V79-hCYP2E1-hSULTlAl cells unaffected by each enzyme inhibitor. This indicated that the observed hormesis is independent of each enzyme. Similarly, but under the exposure condition adapted for mutagenicity assay,1-HMP also showed hormesis in V79-hCYP2El-hSULT1A1 rather than V79 cells at the concentrations of 0.5 and 1 μmol/L, and this effect was blocked by coexposure to PCP. Therefore, the hormesis observed with 1-HMP may actually be induced by the ultimate toxicant transformed from 1-HMP. Based on these array of experiments, concentration ranges of 0.5-4 μmol/L (for 1-HMP) and 2-20 μmol/L (for 1-MP) may be planned for the relevant micronucleus test and Hprt mutagenicity assay.Conclusion1. PCB 20 and PCB 22 are genotoxic to human cells under the metabolic activation by endogenously expressed human CYP2E1. On the other hand, human CYP1A1,1A2,1B1, and 3A4 may not be significant in activation of these PCBs. In this study, induction of chromosomal changes in human cells by PCBs was firstly observed.2. Human CYP2E1 is primarily involved in the activation of NDL-PCBs, with only weak ability to activate DL-PCBs. Human CYP1A1 is far weaker in activating PCBs for genotoxic response.3. Human CYP2E1 is not only able to activate benzene, but also (independent of myeloperoxidase) further activate the multi-hydroxylated metabolites of benzene to more genotoxic metabolites.4.1-MP and 1-HMP are both hormetic to mammalian cells under some conditions, however, only the effect of 1-HMP is dependent on metabolic activation by SULT 1A1.