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Human CYP2E1-dependent mutagenicity of mono- and dichlorobiphenyls in Chinese hamster (V79)-derived cells
Chemosphere 144 (2016) 1908–1915
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Human CYP2E1-dependent mutagenicity of mono- and dichlorobiphenyls in Chinese hamster (V79)-derived cells Chiteng Zhang a,1, Yanmei Lai a,1, Guifang Jin a, Hansruedi Glatt b,c, Qinzhi Wei a, Yungang Liu a,∗ a b c
Department of Toxicology, School of Public Health and Tropical Medicine, Southern Medical University, 1023 S. Shatai Road, Guangzhou 510515, China Department of Nutritional Toxicology, German Institute of Human Nutrition (DIfE), Arthur-Scheunert- Allee 114-116, D-14558 Nuthetal, Germany Department of Food Safety, Federal Institute for Risk Assessment (BfR), Max-Hohrn-Straße 8-10, D-10589 Berlin, Germany
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
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A series of dichlorobiphenyls are mutagenic in V79-derived cells. • The mutagenicity of dichlorobiphenyls is dependent on human CYP2E1. • Human CYP2E1 is not active in activating monochlorobiphenyls to mutagens.
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Article history: Received 23 August 2015 Received in revised form 19 October 2015 Accepted 20 October 2015 Available online 11 November 2015 Handling editor: David C. Volz Keywords: CYP2E1 Gene mutations Micronuclei Polychlorinated biphenyls Sulfotransferase 1A1
a b s t r a c t Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants with confirmed carcinogenicity to humans. Metabolic activation of lower chlorinated PCBs to genotoxic metabolites may involve hydroxylation and further oxidation, and some hydroxylated metabolites may be sulfo-conjugated. However, the genotoxicity of individual PCB compounds is largely unknown. In this study, 15 mono- and dichlorobiphenyls were investigated for genotoxicity using the micronucleus and Hprt mutagenicity assays in a Chinese hamster V79-derived cell line expressing both human cytochrome P450 (CYP) 2E1 and human sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1). All tested compounds were inactive in both assays in V79 control cells. However, eight dichlorobiphenyls strongly induced micronuclei and other congeners were weakly positive for this endpoint in V79-hCYP2E1-hSULT1A1 cells. The effects of each PCB in V79-hCYP2E1-hSULT1A1 cells were abolished or reduced in the presence of a CYP2E1 inhibitor (1aminobenzotriazole), or enhanced by pretreatment of the cells with (CYP2E1-inducing) ethanol, while the genotoxicity was not significantly affected by a SULT1 inhibitor (pentachlorophenol). As representative dichlorobiphenyls, PCB 5, 10, 8 and 11 (2,3-, 2,5-, 2,4ʹ- and 3,3ʹ-dichlorobiphenyl, respectively) strongly induced Hprt gene mutations in V79-hCYP2E1-hSULT1A1 cells in a concentration-dependent manner. This is the first indication that human CYP2E1 is capable of converting a series of dichlorobiphenyls to strong mutagens. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction ∗
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Corresponding author. E-mail address: [email protected] (Y. Liu). These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.chemosphere.2015.10.083 0045-6535/© 2015 Elsevier Ltd. All rights reserved.
Polychlorinated biphenyls (PCBs) are a big group of persistent organic pollutants. They had been commercially produced in large
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quantities and applied commonly all over the world from 1929 to around 1980, when they were banned from production and restricted in use. Nevertheless, they are still present universally on the globe, due to their resistance to degradation, biomagnifications through food chains, ever-lasting unintentional production through burning or heating of chlorine-containing organic materials (Grossman, 2013), and regional pollution at some e-waste disassembling sites in various Asian and African countries in the recent decade (Zhao et al., 2009; Eguchi et al., 2013; Asante et al., 2011). In particular, as unintentional by-products of manufacturing certain pigments (used in inks, dyes and paints), PCB congeners other than the historical, later banned commercial PCB products, which are known as non-legacy PCBs, continue to be detected in water, sediments and air in various sites (Grossman, 2013). Here, lower chlorinated PCB by-products are associated with yellow pigments, while highly chlorinated PCBs are present in blue and green pigments. As a marker of non-legacy PCB contamination, PCB 11 is produced through the manufacture of diarylide yellows in the amount of 1.5 metric tons in the world annually (Rodenburg, 2010). Indeed, in some yellow pigments the PCBs detected at relatively high levels include predominantly dichlorobiphenyls, such as PCB 4, 6, 8, and 11 (Grossman, 2013). These lower chlorinated congeners are more volatile than those contained in blue and green pigments, thus they can readily move out of the pigments into the air. In general, lower chlorinated PCBs (molecules containing 1 to 4 chlorine substitutions) may move between atmosphere, water and soil through their vaporization and precipitation of rainfalls. Actually, lower chlorinated PCBs are important components among airborne PCBs detected in various locations, e.g., in urban Chicago air, where PCB 4, 8, 11, 18, 28 and 52 are included (Zhao et al., 2010). Di- and monochlorobiphenyls such as PCB 11, 14, 8, 3, 4 and 15 have also been detected in human serum (Koh et al., 2013; Grimm et al., 2015). Since lower chlorinated PCBs may also be metabolized (and covalently bound to biomacromolecules) in the human body more efficiently than highly chlorinated ones, the relatively low concentrations or absence of these compounds in human tissues compared to that of highly chlorinated congeners may underestimate the real exposure levels of these compounds. Additionally, Lower chlorinated PCBs may affect organisms by mechanisms different from dioxin-like PCBs (non-ortho-substituted tetraand more chlorinated biphenyls), which are sustained activators of the aryl hydrocarbon receptor (AHR) (Lauby-Secretan et al., 2013); most of the biologic effects of the former chemicals may depend on and be relatively susceptible to metabolic activation, and this may explain, at least partially, the relatively low concentrations of lower chlorinated biphenyls and their hydroxylated metabolites in the blood (Grimm et al., 2015). PCBs may have various health effects, such as endocrine disruption, developmental impairment, reproductive dysfunction, and carcinogenesis (Faroon and Ruiz, 2015). In 2013, PCBs have been classified by the International Agency for Research on Cancer (IARC) as human (group 1) carcinogens (IARC, 2013). Lower chlorinated PCBs are supposed to exert their carcinogenic potentials through their bio-reactive metabolites, formed under the action of biotransformation enzymes (Lauby-Secretan et al., 2013). It is suggested that some cytochrome P450 (CYP) enzymes should be responsible for the metabolic activation of PCBs to mutagenic metabolites (Robertson and Ludewig, 2011; Ludewig and Robertson, 2013). Investigation of the mutagenicity of various classic PCB mixtures in Chinese hamster (V79) cells resulted in negative results (Hattula, 1985). This is probably due to the lack of appropriate biotransformation enzymes in the cells. Using PCB 3 as a model compound for semi-volatile PCBs, it has been observed that some quinoid and dihydroxylated metabolites of PCB 3 strongly induced micronuclei in V79 cells, whereas the parent PCB 3 was negative in the test (Zettner et al., 2007). Meanwhile, it has been re-
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ported that some hydroxylated PCBs are substrates for sulfotransferases (SULTs), such as rat SULT1A1 (Liu et al., 2009) and human SULT2A1 (Liu et al., 2006), which implies a potential role of SULTs in modulating the formation of active metabolites of PCBs. Through a quantitative structure–activity relationship analysis of the genotoxic potentials of PCBs, it is proposed that mono- and dichlorinated PCBs and their metabolites may possess the highest genotoxic activities among PCBs chlorinated to different degrees (Ruiz et al., 2008); however, direct experimental evidence supporting this proposal is missing. Up to now, a systematic observation of the genotoxicity of individual PCB congeners is lacking, probably due to the fact that no biotransformation enzymes (e.g., CYPs) have been identified for activating PCBs to genotoxic metabolites. According to our recent finding that human CYP2E1 activates hydroquinone, catechol and 1,2,4-trihydroxybenzene to more genotoxic metabolites (Jiang et al., 2014), we hypothesized that human CYP2E1 may also activate some lower chlorinated PCBs to genotoxicants. Therefore, in this pilot study we investigated the induction of micronuclei by each of the 15 possible mono- and dichlorobiphenyls in a V79-derived cell line expressing both human CYP2E1 and human SULT1A1 (V79-hCYP2E1-hSULT1A1); modulation of each enzyme was employed to identify its role in activating or inactivating a test compound. Representative congeners were additionally investigated for induction of gene mutations at the Hprt locus.
2. Materials and methods 2.1. Chemicals PCB 3 and 15 were generous gifts from Dr. Yang Song (Chongqing, China); all the other 13 PCBs (i.e., PCB 1, 2, and 4 through 14) were purchased from AccuStandard Inc. (New Haven, CT, USA). Dimethylsulfoxide (DMSO), 1-aminobenzotriazle (ABT), N-nitrosodiethylamine (NDEA) and absolute ethanol were purchased from Sigma Aldrich (St. Louis, MO, USA). Ethyl methanesulfonate (EMS) and 1-methylpyrene (1-MP) were from J & K Chemical Ltd (Suzhou, China). Disulfonated tetrazolium salt (more commonly known as cell counting kit-8, CCK-8) was from Dojindo Laboratories (Kumamoto, Japan). Pentachlorophenol (PCP) was from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Prior to treatment of the cells with test compounds, pure water was used to dissolve EMS and NDEA, while DMSO served as the solvent for each PCB and 1-MP.
2.2. Cell lines V79 cell line was purchased from Shanghai Fuxiang Biotech. Co., LTD (Shanghai, China). V79-hCYP2E1-hSULT1A1 cell line was constructed from a V79-derived cell line expressing human CYP2E1 (Schmalix et al., 1995) through transfection of wild-type SULT1A1 (Glatt et al., 2005; Liu and Glatt, 2010). The parental V79-Mz cell line was completely deficient in the expression of CYPs, SULTs and UGTs (Glatt et al., 1990), in particular, no expression of CYP2E1 was observed in this cell line by an immunoblotting assay (Schmalix et al., 1995). V79-hCYP2E1-hSULT1A1 cells have performed well in both Hprt mutagenicity and micronucleus tests for the investigation of enzyme-dependent genotoxic responses (Glatt et al., 2005; Liu and Glatt, 2008; Jiang et al., 2015). Cells were cultured in Dulbecco’s modification of Eagle’s medium (Gibco) supplemented with 7% fetal bovine serum (Gibco), 100 IU/mL penicillin G, and 100 μg/mL streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2 .
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2.3. Cytotoxicity (CCK-8) assay Cytotoxicity of each test compound was determined by CCK8 assays, in which CCK-8 was used as a chromogenic indicator for NADH and cell viability (Ishiyama et al., 1997). The experimental procedures were adapted according to the exposurerecovery schedule for either the micronucleus or the mutagenicity assay (Jiang et al., 2015). Briefly, V79 or V79-hCYP2E1hSULT1A1 cells were inoculated on 96-well plates at the density of 6.8 × 103 cells/well (with 100 μL medium per well). At 24 h, each PCB at varying concentrations was added, 6 wells for each treatment; at 36 h each culture was changed with fresh culture medium; at 48 h, each culture received 10 μL of CCK-8 solution, and after being incubated for 2 h, the optical density at 450 nm (OD450 ) was measured on a microplate reader (BioRad Model 680, Hercules, CA). For the inhibition of CYP2E1 or SULT1A1 activity in V79-hCYP2E1-hSULT1A1 cells, ABT (60 μM) or PCP (10 μM) (Liu and Glatt, 2008) was present from 22 to 48 h. DMSO as the vehicle was limited to the final concentration of 0.15% (v:v). For cytotoxicity tests reflecting the exposure conditions of the Hprt mutagenicity assay, cells were inoculated at the density of 2 × 103 cells/well, and exposed to each of the four representative PCBs (PCB 5, 8, 10 and 11) at 24 h (day 1), six cultures for each treatment; at 48 h each culture was changed with fresh medium and further incubated for 2 days. At day 4, each culture was stained with CCK-8, and the OD450 was measured as described above. In V79-hCYP2E1hSULT1A1 cells, ABT and PCP at the indicated concentrations were present from 22 h to day 4 for observation of the influence of each enzyme on the cytotoxicity. 2.4. Micronucleus test The micronucleus test was conducted exactly as recently described (Jiang et al., 2015). Briefly, cells of either V79 or V79hCYP2E1-hSULT1A1 were inoculated at the density of 3 × 105 per 12.5-cm2 flask containing 3 mL medium. At 24 h, cells were treated with each PCB compound at varying concentrations for 12 h, and further cultured in fresh medium for another 12 h. At 48 h, cells in each culture were harvested by trypsinization for preparation of micronuclei-scored slides. In V79-hCYP2E1hSULT1A1 cells, SULT1A1 and CYP2E1 were inhibited, when indicated, by PCP (10 μM) and ABT (60 μM), respectively, from 22 h to 48 h; ethanol (0.2%, v:v) pretreatment from 6 h to 22 h (for inducing and stabilizing CYP2E1) (Liu and Glatt, 2008) was applied for tests with weakly active or inactive compounds (PCB 2, 3, 9, 12, 14, 15 and PCB 1). 1-MP (20 μM), bio-activated by a combination of CYP2E1 with SULT1A1 (Jiang et al., 2015), served as the positive control in V79-hCYP2E1-hSULT1A1 cells. EMS (5 mM), a direct mutagen, was used as the positive control in V79 cells. Two cultures were set up for each treatment. Micronucleated cells in 2000 qualified, randomly encountered cells from each culture were scored microscopically by an experienced experimenter. 2.5. Hprt mutagenicity assay This assay detects gene mutations as 6-thioguanine-resistant colonies, i.e., forward mutations at the Hprt locus (localized on the X chromosome). Mutagenicity of the PCB compounds in V79 and V79-hCYP2E1-hSULT1A1 cells was determined as described recently (Jiang et al., 2015) with minor modification. Briefly, 1.5 × 106 cells were inoculated in 30 mL medium into each 250cm2 flask. At 24 h, cells were treated with each representative PCB compound for 24 h with the flask caps screwed tight (for minimizing the loss of semi-volatile PCB compounds); from day 2 to day 4, cells were cultured in fresh medium, then subcultured for
another 3 days. EMS (1.6 mM) and NDEA (200 μM) (test concentrations determined by pre-test results) served as positive controls for V79 and V79-hCYP2E1-hSULT1A1 cells, respectively. PCP (10 μM) was present in the V79-hCYP2E1-hSULT1A1 cultures from 22 h to day 4 for avoidance of an influence of SULT1A1. Two cultures were set up for each treatment. At day 7, cells in each culture were harvested for the determination of colony-forming efficiency (serving as a measure of the cell survival) and 6-thioguanineresistant colonies, both of which were used in calculating the frequency of Hprt mutants. The averaged value of four paralleling 6thioguanine-containing cultures referred to each initial culture. 2.6. Statistical analysis The results of cytotoxicity assay were statistically analyzed by using ANOVA, with the relative OD450 value in each treatment compared with the control group. For the micronucleus and mutagenicity assays, data from duplicate experiments were expressed as means and ranges of variations, however, when applied in a χ 2 test they were combined to become quantal data. 3. Results 3.1. Cytotoxicity of PCBs Under the exposure/recovery (12 h/12 h) conditions, none of the 15 tested mono- or dichlorobiphenyls showed cytotoxicity as determined by the CCK-8 assay, in either V79 or V79-hCYP2E1hSULT1A1 cells, at concentrations ranging from 3 to 40 μM (PCB 5) or from 3 (or 10) to 100 μM (remaining PCBs tested), as applied in the micronucleus test with the corresponding compounds. Co-exposure of ABT (60 μM) or PCP (10 μM) did not affect the above results. Under the exposure/recovery (1 day/2 days) conditions of the Hprt mutagenicity assay, the cytotoxicity of four dichlorobiphenyls, i.e., PCB 5, 8, 10 and 11, were investigated in both V79 and V79hCYP2E1-hSULT1A1 cells by the CCK-8 assay. With each compound at concentrations ranging from 10 to 80 (or 100) μM, in V79 cells only PCB 5 (at the concentration of 100 μM) decreased the cell viability/growth, while all the four tested compounds showed some moderate cytotoxicity in V79-hCYP2E1-hSULT1A1 cells, as shown in Fig. 1. 3.2. Induction of micronuclei by dichlorobiphenyls and the impact of enzyme modulators None of the 12 dichlorobiphenyls induced the formation of micronuclei in V79 cells (Fig. 2), while EMS (5 mM) as a positive control elevated the frequency of micronucleated cells from 6‰ to around 50‰. As shown in Fig. 2, PCB 5, 6, 4, 8 (upper panel), 10, 11 and 13 (lower panel) induced micronuclei strongly (>3fold increase over control) and in a concentration-dependent manner in V79-hCYP2E1-hSULT1A1 cells; PCB 7 elevated the frequency of micronucleated cells starting from 10 μM and continuously at higher concentrations, however, a clear concentration dependence was absent in this range; the remaining dichlorobiphenyls (i.e., PCB 9, 12, 14 and 15) only marginally elevated the frequency of micronucleated cells at high concentrations (weakly positive results). As a positive control, 1-MP elevated the frequency of micronucleated cells to around 50‰, consistent with our recent report on this compound (Jiang et al., 2015). As shown in Fig. 3, PCB 7 (panel A) and PCB 10 (panel B) elevated the frequency of micronucleated cells in V79-hCYP2E1hSULT1A1 cells with clear concentration dependence, and the effects of both compounds were significantly reduced by ABT (60 μM), while PCP (10 μM) did not show significant modulation
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Fig. 1. Cytotoxicity of PCB 5 (circles), 8 (triangles), 10 (squares) and 11 (diamonds) in V79 control cells (empty symbols) or V79-hCYP2E1-hSULT1A1 cells (solid symbols) as detected by CCK-8 assay under the exposure/recovery schedule of 1 d/2 d (conditions of the Hprt assay). Pentachlorophenol (10 μM) was present in V79-hCYP2E1-hSULT1A1 cells for inhibition of SULT1A1. Data are means and standard deviations of six replicates; ∗ p < 0.05, ∗ ∗ p < 0.01, by ANOVA, compared with the negative control.
(panel A, in the case of PCB 10), or marginally potentiated the effect (without statistical significance) (for PCB 7, panel B). 3.3. Induction of micronuclei by monochlorobiphenyls and the effect of pretreatment with ethanol PCB 1, 2 and 3 did not induce micronuclei in V79 cells at concentrations ranging from 10 to 100 μM (data not shown). In V79-hCYP2E1-hSULT1A1 cells, PCB 1, 2 and 3 weakly elevated the frequency of micronucleated cells; however, with the cells pretreated with 0.2% (v:v) ethanol, the induction of micronuclei by each monochlorobiphenyl was potentiated, as shown by the presence of concentration dependence of the micronuclei induction by each compound (Fig. 4). 3.4. Induction of gene mutations at the Hprt locus in V79-hCYP2E1-hSULT1A1 cells by four representative dichlorobiphenyls As shown in Fig. 5, none of the four representative dichlorobiphenyls induced gene mutations in V79 cells up to the concentration of 80 or 100 μM, however, they all elevated the frequency of gene mutations in V79-hCYP2E1-hSULT1A1 cells strongly in a concentration-dependent manner. The relative efficiency of each compound in mutagenic activity towards these cells followed the order: PCB 5 ≈ PCB 10 >> PCB 8 ≈ PCB 11. In the meantime, these compounds were non- or mildly cytotoxic in the corresponding CCK-8 assays, and there were no obvious differences between results in the two cell lines (data not shown). 4. Discussion None of the 15 mono- and dichlorobiphenyls displayed cytotoxicity in V79 or V79-hCYP2E1-hSULT1A1 cells under a 12 h/12 h (exposure/recovery) schedule by CCK-8 assay. However, in an extended exposure and recovery (1 day/2 days) condition (in accordance to the Hprt mutagenicity assay), each of the four representative dichlorobiphenyls decreased cell viability/growth in V79hCYP2E1-hSULT1A1 cells, while in V79 cells only PCB 5 demonstrated some cytotoxicity. It appears that reduced cell density
upon inoculation and/or longer exposure and recovery time is favorable for the expression of cytotoxicity of PCBs. Moreover, the more active responses to the representative PCBs in V79-hCYP2E1hSULT1A1 cells (with SULT1A1 inhibited by PCP) than in V79 cells suggest that human CYP2E1 may activate the PCBs to cytotoxic metabolites. In the present study, some dichlorobiphenyls induced gene mutations and/or micronuclei strongly in V79-hCYP2E1-hSULT1A1 cells. The remaining dichlorobiphenyls and the three monochlorobiphenyls (PCB 1, 2 and 3) induced micronuclei in V79-hCYP2E1hSULT1A1 cells marginally. However, all compounds were nongenotoxic in V79 cells. These results indicate that metabolic activation is required for the genotoxicity of these compounds. Since induction of CYP2E1 by pretreatment of V79-hCYP2E1-hSULT1A1 cells with ethanol enhanced the formation of micronuclei by the monochlorobiphenyls (this effect was less pronounced for dichlorobiphenyls, data not shown), and inhibition of CYP2E1 by ABT drastically decreased the induction of micronuclei by some dichlorobiphenyls (e.g., PCB 7 and 10, shown in Fig. 3), human CYP2E1 may be responsible for the activation of these PCBs to genotoxic metabolites. In addition, as inhibition of SULT1A1 by PCP had no or little impact on the genotoxic response in V79-hCYP2E1-hSULT1A1 cells to the PCBs (shown in Fig. 3), human SULT1A1 may not be involved in the activation of these PCBs, nor has it significant detoxifying activity toward the active metabolites. The active metabolites responsible for CYP2E1-mediated genotoxicity of the tested PCBs in this study have not yet been directly identified. Previously, it has been demonstrated that incubation of PCB 3 with hepatic microsomes from rats treated with phenobarbital and 3-methylcholanthrene gives rise to the formation of a group of metabolites: 4-OH PCB 3 as the major metabolite, and the following in a decreasing order: 3 ,4 -(OH)2 PCB 3, 2 ,3 (OH)2 PCB 3, 3 -OH PCB 3, and 2 ,5 -(OH)2 PCB 3 (McLean et al., 1996). Dihydroxylated PCBs may further be activated to quinonic metabolites, which are highly bioreactive towards DNA and protein molecules (Amaro et al., 1996). The rate of metabolism of other PCBs may be determined by (1) the number and position of chlorines in the biphenyl core, and (2) the availability of vici-
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Fig. 2. Induction of micronuclei by each dichlorobiphenyl in V79 (empty symbols) and V79-hCYP2E1-hSULT1A1 cells (solid symbols). Cells were treated with each PCB at various concentrations from 24 h to 36 h, and harvested at 48 h. Each tested PCB did not show significant cytotoxicity (as determined by the CCK-8 assay) in the concentrations applied in the micronucleus test (data not shown). Data are means from duplicate determinations; ranges of variation and statistical analysis results are not shown here for a clear presentation of the mean values of multiple compounds in a single panel.
nal free (unsubstituted) carbon-hydrogen positions (Grimm et al., 2015). Formation of transient epoxides may be another pathway of metabolic activation of PCBs, which may result in adducts with biomacromolecules; epoxides may also isomerize to hydroxylated PCBs, hydrolyzed to dihydrodiol metabolites, or conjugated with glutathione (Grimm et al., 2015). OH-PCBs may undergo sulfo- and glucuronosyl-conjugation, probably inactivating reactions. For example, 4-OH PCB 8 is a substrate for rat SULT1A1 (Liu et al., 2009). However, in this study human SULT1A1 did not significantly modulate the CYP2E1-dependent genotoxicity of PCBs. This lack of effect may owe to a different specificity of human SULT1A1 towards its OH-PCB substrates than its rat counterpart, or the hydroxylated metabolite was formed intracellularly at a concentration inappropriate for a sulfo-conjugation rate detectable through the micronucleus test. Hydroxylated metabolites of highly chlorinated congeners have been identified in human blood (Bergman et al., 1994; Guvenius et al., 2002). These hydroxylated metabolites of PCBs may be accumulated in some solid tissues, such as the liver, as compared with their concentrations in the blood (Guvenius et al. 2002). The absence of hydroxylated mono- and dichlorobiphenyls
on the list of detected OH-PCBs may be due to their susceptibility to further metabolism or binding to macromolecules such as DNAs and proteins. Anyway, understanding of the hydroxylated metabolites of the dichlorobiphenyls that showed strong CYP2E1dependent genotoxicity in this study will be interesting, and addressed in future studies. With the data of CYP2E1-dependent induction of micronuclei by each of the twelve dichlorobiphenyl compounds (shown in Fig. 2), it is plausible to give an assessment of a potential structure–activity relationship. Since the para- and meta-positions are more readily oxidized, it is suggested that 2,3-, 2,5-, and 2,6dicholobiphenyls (PCB 5, 9 and 10, respectively) may be the most easily hydroxylated congeners among various dichlorobiphenyls (Grimm et al., 2015). Indeed, in this study PCB 5 and PCB 10 were the most mutagenic dichlorobiphenyls in V79-hCYP2E1-hSULT1A1 cells; on the contrary, PCB 9 was almost inactive. Actually, among the seven dichlorobiphenyls with one or two chlorine substitutions on the ortho-position, most (PCB 4, 5, 6, 7, 8 and 10) were strong inducers of micronuclei in V79-hCYP2E1-hSULT1A1 cells, with PCB 9 being the only exception. PCB 9 is unique, as the
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Fig. 3. Induction of micronuclei in V79-hCYP2E1-hSULT1A1 cells by PCB 7 (A) and PCB 10 (B) and the impact of 1-aminobenzotriazole (60 μM) and pentachlorophenol (10 μM). See legend of Fig. 2. Ethanol (0.2%, v:v) was present prior to the addition of modulator and test compounds for the induction and stabilization of CYP2E1, but absent from cultures to be co-exposed to 1-aminobenzotriazole. Effect of PCB 7 or 10 alone was symbolized with empty circles, and that in combination with 1-aminobenzotriazole and pentachlorophenol was symbolized with solid triangles and squares, respectively. Statistical analysis was performed by χ 2 examination following combining the duplicate data to become quantal data, ∗ p < 0.05, ∗ ∗ p < 0.01, compared with the control (no test compound); # p < 0.01, ## p < 0.01, comparison was made between a PCB treatment with and without a modulator.
two chlorine substitutions stand opposite (para-) to each other on a ring, and this constellation may cause an offset of the electron-attracting effect of each chlorine; thus, the polarity of the three carbon-hydrogen single bonds on the ring will not be influenced by the chlorines, which is otherwise favorable for an oxidation reaction (through an enhanced electron-deficiency of a hydrogen atom). It seems that a non-coplanar configuration [endowed by ortho-substitution with chlorine(s)] of the biphenyl core is favorable for the metabolic activation of dichlorobiphenyls, except for a unique structure like PCB 9. As for the five dichlorobiphenyls without an ortho-chlorination, two of them (PCB 11 and 13) were strong CYP2E1-dependent genotoxicants while the other three were nearly inactive (PCB 12, 14 and 15). There seem to be no obvious structural feature accounting for their distinct differ-
ences in CYP2E1-activated genotoxicity. Multiple factors, such as the availability of para- and meta-unsubstituted carbon atoms, increased polarity of the C–H single bonds, the steric and electrostatic fitfulness of each PCB molecule to the active center of the enzyme, and the bio-reactivity of the metabolites formed, may determine the structure–activity relationship for the human CYP2E1dependent genotoxicity of PCBs. A molecular docking-based threedimensional quantitative structure–activity relationship study may be useful for a more accurate assessment of the interaction of the compounds with the enzyme. In the present study, dichlorobiphenyls demonstrated stronger mutagenicity than monochlorobiphenyls. Then, what will be the CYP2E1-dependent mutagenicity of important tri- and tetrachloro-
Fig. 4. Induction of micronuclei in V79-hCYP2E1-hSULT1A1 cells by each monochlorobiphenyl and the effect of pretreatment of the cells with ethanol (0.2%, v:v). See legend of Fig. 2. Data are means and ranges of variation. Empty and solid symbols represent experiments without and with ethanol pretreatment, respectively. ∗p < 0.05, ∗∗p < 0.01, compared with the control (no test compound); ##p < 0.01, comparison was made between a PCB treatment with and without ethanol pretreatment.
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Fig. 5. Induction of gene mutations at the Hprt locus by PCB 5 (circles), PCB 8 (triangles), PCB 10 (squares) and PCB 11 (diamonds) in V79 (empty symbols) and V79-hCYP2E1hSULT1A1 cells (solid symbols). Pentachlorophenol (10 μM) was present in V79-hCYP2E1-hSULT1A1 cells for inhibition of SULT1A1. NDEA (200 μM), a CYP2E1-dependent promutagen serving as the positive control in each experiment, elevated the mutant frequency to about 350 mutants per 106 cells. Data from duplicate experiments were combined to become quantal data for χ 2 analysis; ∗p < 0.05, ∗∗p < 0.01, compared with the negative control.
biphenyls (e.g., PCB 20, 28, 52 and 77) (Persoon et al., 2010; Marek et al., 2013)? Continued experimental studies in the cell model used in this study have to be performed for a more comprehensive knowledge on the genotoxicity of PCBs. It will be interesting to learn whether other CYP forms are also involved in the activation of PCBs to genotoxic metabolites. This can be achieved by using cell lines expressing other CYP forms or human hepatocytes endogenously expressing multiple CYPs with modulators specific for a particular CYP form. Of the strongly genotoxic PCBs identified in this study, at least PCB 4, 8 and 11 have been detected in the atmospheric environment (Hu et al., 2008; Zhao et al., 2010), PCB 11 has also been detected in a relatively high level in human bodies (Marek et al., 2013). It is true that the effective concentrations of these PCBs observed in this study must be many times higher than their levels in human bodies, however, the tiny or undetectable levels of dichlorobiphenyls in human tissues do not necessarily mean they can be ignored, since these lower chlorinated PCBs may be metabolized at significantly enhanced rates (to bioreactive metabolites) compared to the highly chlorinated congeners (Schnellmann et al., 1984; Grimm et al., 2015), thus may not be allowed for high internal levels. Furthermore, a potential effect of persistent exposure of humans to low level dichlorobiphenyls may be missing in the classic short-term genotoxicity assays, as applied in the present study. Therefore, this study may represent a starting point toward a comprehensive structure–activity relationship for the mutagenicity of PCBs and the application in the relevant environmental health risk analysis. 5. Conclusion The present study has connected a CYP enzyme (human CYP2E1) with PCB-induced transmittable genetic changes in mammalian cells with solid evidence for the first time. Our study has also revealed varied mutagenicity of individual mono- and dichlorobiphenyls, which are differentially structured. Mutagenicity of other PCBs, the possibility of the involvement of other CYP enzymes in the activation of PCBs, and structure of the reactive metabolites responsible for the mutagenicity of the PCBs deserve
further investigation. Acknowledgment This work was supported by the Education Department of Guangdong Province in China under an Academic Talentsintroducing Program [Y. Liu, Grant code: YueCaiJiao 2010(143)], and a project supported by the Natural Science Foundation of Guangdong Province, China (Y. Liu, Grant code: S2012010008922). References Amaro, A.R., Oakley, G.G., Bauer, U., Spielmann, H.P., Robertson, L.W., 1996. Metabolic activation of PCBs to quinones: reactivity toward nitrogen and sulfur nucleophiles and influence of superoxide dismutase. Chem. Res. Toxicol. 9, 623–629. Asante, K.A., Adu-Kumi, S., Nakahiro, K., Takahashi, S., Isobe, T., Sudaryanto, A., Devanathan, G., Clarke, E., Ansa-Asare, O.D., Dapaah-Siakwan, S., Tanabe, S., 2011. Human exposure to PCBs, BPDEs, and HBCDs in Ghana: temporal variation, sources of exposure estimation of daily intakes by infants. Environ. Int. 37, 921–928. Bergman, A., Klasson-Wehler, E., Kuroki, H., 1994. Selective retention of hydroxylated PCB metabolites in blood. Environ. Health Perspect. 102, 464–469. Eguchi, A., Nomiyama, K., Devanathan, G., Subramanian, A., Bulbule, K.A., Parthasarathy, P., Takahashi, S., Tanabe, S., 2013. Different profiles of anthropogenic and naturally produced organohalogen compounds in serum from residents living near a coastal area and e-waste recycling workers in India. Environ. Int. 47, 8–16. Faroon, O., Ruiz, P., 2015. Polychlorinated biphenyls: new evidence from the last decade. Toxicol. Ind. Health doi:10.117710748233715587849. Glatt, H., Gemperlein, I., Setiabudi, F., Platt, K.L., Oesch, F., 1990. Expression of xenobiotic-metabolizing enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds. Mutagenesis 5, 241–249. Glatt, H., Schneider, H., Liu, Y., 2005. V79-hCYP2E1-hSULT1A1, a cell line for the sensitive detection of genotoxic effects induced by carbohydrate pyrolysis products and other food-borne chemicals. Mutat. Res. 580, 41–52. Grimm, F.A., Hu, D., Kania-Korwel, I., Lehmler, H.J., Ludewig, G., Hornbuckle, K.C., Duffel, M.W., Bergman, A., Robertson, L.W., 2015. Metabolism and metabolites of polychlorinated biphenyls. Crit. Rev. Toxicol. 45, 245–272. Grossman, E., 2013. Nonlegacy PCBs: pigment manufacturing by-products get a second look. Environ. Health Perspect. 121, A86–A93. Guvenius, D.M., Hassanzadeh, P., Bergman, A., Noren, K., 2002. Metabolites of polychlorinated biphenyls in human liver and adipose tissue. Environ. Toxicol. Chem. 21, 2264–2269. Hattula, M.L., 1985. Mutagenicity of PCBs and their pyrosynthetic derivatives in cellmediated assay. Environ. Health Perspect. 60, 255–257. Hu, D., Martinez, A., Hornbuckle, K.C., 2008. Discovery of non-Aroclor PCB (3,3’dichlorobiphenyl) in Chicago air. Environ. Sci. Technol. 42, 7873–7877.
C. Zhang et al. / Chemosphere 144 (2016) 1908–1915 International Agency for Research on Cancer (IARC), 2013. Agents Classified by the IARC Monographs, vol. 1–109 150 Cours Albert Thomas, 69372 Lyon CEDEX 08, France. Available. http://monographs.iarc.fr/ENG/Classification/ (accessed 30.10.13.). Ishiyama, M., Miyazono, Y., Sasamoto, K., Ohkura, Y., Ueno, K., 1997. A highly watersoluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta 44, 1299–1305. Jiang, H., Lai, Y., Hu, K., We, Q., Liu, Y., 2014. Human CYP2E1-dependent and human sulfotransferase 1A1-modulated induction of micronuclei by benzene and its hydroxylated metabolites in Chinese hamster V79-derived cells. Mutat. Res. 770, 37–44. Jiang, H., Lai, Y., Hu, K., Chen, D., Liu, B., Liu, Y., 2015. Genotoxicity of 1methylpyrene and 1-hydroxymethylpyrene in V79-hCYP2E1-hSULT1A1 cell line. Environ. Mol. Mutagen 56, 404–411. Koh, W.X., Horbuckle, K.C., Thorne, P.S., 2013. Human serum from urban and rural adolescents and their mothers shows exposure to polychlorinated biphenyls not found in commercial mixtures. Environ. Sci. Technol. 49, 8105–8112. Lauby-Secretan, B., Loomis, D., Grosse, Y., E.I. Ghissassi, F., Bouvard, V., BenbrahimTallaa, L., Guha, N., Baan, R., Mattock, H., Straif, K., 2013. Carcinogenicity of polychlorinated biphenyls and polybrominated biphenyls. Lancet Oncol. 14, 287– 288. Liu, Y., Glatt, H., 2008. Mutagenicity of N-nitrosodiethanolamine in a V79-derived cell line expressing two human biotransformation enzymes. Mutat. Res. 643, 64–69. Liu, Y., Glatt, H., 2010. Human cytochrome P450 2E1 and sulfotransferase1A1 coexpressed in Chinese hamster V79 cells enhance spontaneous mutagenesis. Environ. Mol. Mutagen 51, 23–30. Liu, Y., Apak, T.I., Lehmler, H.J., Robertson, L.W., Duffel, M.W., 2006. Hydroxylated polychlorinated biphenyls are substrates and inhibitors of human hydroxysteroid sulfotransferase SULT2A1. Chem. Res. Toxicol. 19, 1420–1425. Liu, Y., Smart, J.T., Song, Y., Lehmler, H.J., Robertson, L.W., Duffel, M.W., 2009. Structure-activity relationships for hydroxylated polychlorinated biphenyls as substrates and inhibitors of rat sulfotransferases and modification of these relationships by changes in thiol status. Drug Metab. Dispos. 37, 1065–1072. Ludewig, G., Robertson, L.W., 2013. Polychlorinated biphenyls (PCBs) as initiating agents in hepatocellular carcinoma. Cancer Lett. 334, 46–55.
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Marek, R.F., Thorne, P.S., Wang, K., DeWall, J., Keri, K.C., 2013. PCBs and OH-PCBs in serum from children and mothers in urban and rural U.S. communities. Environ. Sci. Technol. 47, 3353–3361. McLean, M.R., Bauer, U., Amaro, A.R., Robertson, L.W., 1996. Identification of catechol and hydroquinone metabolites of 4-monochlorobiphenyl. Chem. Res. Toxicol. 9, 158–164. Persoon, C., Peters, T.M., Kmumar, N., Hornbuckle, K.C., 2010. Spatial distribution of airborne polychlorinated biphenyls in Cleveland, OH and Chicago, IL. Environ. Sci. Technol. 44, 2797–2802. Robertson, L.W., Ludewig, G., 2011. Polychlorinated biphenyl carcinogenicity with special emphasis on airborne PCB. Gefahrst. Reinhalt. Luft 71, 25–32. Rodenburg, L.A., 2010. Evidence for unique and ubiquitous environmental sources of 3,3ʹ-dichlorobiphenyl (PCB 11). Environ. Sci. Technol. 44, 2816–2821. Ruiz, P., Faroon, O., Moudgal, C.T., Hansen, H., Rosa, C.T.D., Mumtaz, M., 2008. Prediction of the health effects of polychlorinated biphenyls (PCBs) and their metabolites using quantitative structure–activity relationship (QSAR). Toxicol. Lett. 181, 53–65. Schmalix, W.A., Barrenscheen, M., Landsiedel, R., Janzowski, C., Eisenbrand, G., Gonzalez, F., Eliasson, E., Ingelman-Sundberg, M., Perchermeier, M., Greim, H., Doehmer, J., 1995. Stable expression of human cytochrome P450 2E1 in V79 Chinese hamster cells. Eur. J. Pharmacol. 293, 123–131. Schnellmann, R.G., Volp, R.F., Putnam, C.W., Sipes, I.G., 1984. The hydroxylation, dechlorination, and glucuronidation of 4,4ʹ-dichlorobiphenyl (4-DCB) by human hepatic microsomes. Biochem. Pharmacol. 33, 3503–3509. Zettner, M.A., Flor, S., Ludewig, G., Wagner, J., Robertson, L.W., Lehmann, L., 2007. Quinoid metabolites of 4-monochlorobiphenyl induce gene mutations in cultured Chinese hamster V79 cells. Toxicol. Sci. 100, 88–98. Zhao, G., Wang, Z., Zhou, H., Zhao, Q., 2009. Burdens of PBBs, PBDEs, and PCBs in tissues of the cancer patients in the e-waste disassembly sites in Zhejiang, China. Sci. Total Environ. 407, 4831–4837. Zhao, H.X., Adamcakova-Dodd, A., Hu, D., Hornbuckle, K.C., Just, C.L., Robertson, L.W., Thorne, P.S., Lehmler, H.J., 2010. Development of a synthetic PCB mixture resembling the average polychlorinated biphenyl profile in Chicago air. Environ. Int. 36, 819–827.
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Human CYP2E1-dependent mutagenicity of mono- and dichlorobiphenyls in Chinese hamster (V79)-derived cells Chiteng Zhang a,1, Yanmei Lai a,1, Guifang Jin a, Hansruedi Glatt b,c, Qinzhi Wei a, Yungang Liu a,∗ a b c
Department of Toxicology, School of Public Health and Tropical Medicine, Southern Medical University, 1023 S. Shatai Road, Guangzhou 510515, China Department of Nutritional Toxicology, German Institute of Human Nutrition (DIfE), Arthur-Scheunert- Allee 114-116, D-14558 Nuthetal, Germany Department of Food Safety, Federal Institute for Risk Assessment (BfR), Max-Hohrn-Straße 8-10, D-10589 Berlin, Germany
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
•
A series of dichlorobiphenyls are mutagenic in V79-derived cells. • The mutagenicity of dichlorobiphenyls is dependent on human CYP2E1. • Human CYP2E1 is not active in activating monochlorobiphenyls to mutagens.
a r t i c l e
i n f o
Article history: Received 23 August 2015 Received in revised form 19 October 2015 Accepted 20 October 2015 Available online 11 November 2015 Handling editor: David C. Volz Keywords: CYP2E1 Gene mutations Micronuclei Polychlorinated biphenyls Sulfotransferase 1A1
a b s t r a c t Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants with confirmed carcinogenicity to humans. Metabolic activation of lower chlorinated PCBs to genotoxic metabolites may involve hydroxylation and further oxidation, and some hydroxylated metabolites may be sulfo-conjugated. However, the genotoxicity of individual PCB compounds is largely unknown. In this study, 15 mono- and dichlorobiphenyls were investigated for genotoxicity using the micronucleus and Hprt mutagenicity assays in a Chinese hamster V79-derived cell line expressing both human cytochrome P450 (CYP) 2E1 and human sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1). All tested compounds were inactive in both assays in V79 control cells. However, eight dichlorobiphenyls strongly induced micronuclei and other congeners were weakly positive for this endpoint in V79-hCYP2E1-hSULT1A1 cells. The effects of each PCB in V79-hCYP2E1-hSULT1A1 cells were abolished or reduced in the presence of a CYP2E1 inhibitor (1aminobenzotriazole), or enhanced by pretreatment of the cells with (CYP2E1-inducing) ethanol, while the genotoxicity was not significantly affected by a SULT1 inhibitor (pentachlorophenol). As representative dichlorobiphenyls, PCB 5, 10, 8 and 11 (2,3-, 2,5-, 2,4ʹ- and 3,3ʹ-dichlorobiphenyl, respectively) strongly induced Hprt gene mutations in V79-hCYP2E1-hSULT1A1 cells in a concentration-dependent manner. This is the first indication that human CYP2E1 is capable of converting a series of dichlorobiphenyls to strong mutagens. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction ∗
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Corresponding author. E-mail address: [email protected] (Y. Liu). These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.chemosphere.2015.10.083 0045-6535/© 2015 Elsevier Ltd. All rights reserved.
Polychlorinated biphenyls (PCBs) are a big group of persistent organic pollutants. They had been commercially produced in large
C. Zhang et al. / Chemosphere 144 (2016) 1908–1915
quantities and applied commonly all over the world from 1929 to around 1980, when they were banned from production and restricted in use. Nevertheless, they are still present universally on the globe, due to their resistance to degradation, biomagnifications through food chains, ever-lasting unintentional production through burning or heating of chlorine-containing organic materials (Grossman, 2013), and regional pollution at some e-waste disassembling sites in various Asian and African countries in the recent decade (Zhao et al., 2009; Eguchi et al., 2013; Asante et al., 2011). In particular, as unintentional by-products of manufacturing certain pigments (used in inks, dyes and paints), PCB congeners other than the historical, later banned commercial PCB products, which are known as non-legacy PCBs, continue to be detected in water, sediments and air in various sites (Grossman, 2013). Here, lower chlorinated PCB by-products are associated with yellow pigments, while highly chlorinated PCBs are present in blue and green pigments. As a marker of non-legacy PCB contamination, PCB 11 is produced through the manufacture of diarylide yellows in the amount of 1.5 metric tons in the world annually (Rodenburg, 2010). Indeed, in some yellow pigments the PCBs detected at relatively high levels include predominantly dichlorobiphenyls, such as PCB 4, 6, 8, and 11 (Grossman, 2013). These lower chlorinated congeners are more volatile than those contained in blue and green pigments, thus they can readily move out of the pigments into the air. In general, lower chlorinated PCBs (molecules containing 1 to 4 chlorine substitutions) may move between atmosphere, water and soil through their vaporization and precipitation of rainfalls. Actually, lower chlorinated PCBs are important components among airborne PCBs detected in various locations, e.g., in urban Chicago air, where PCB 4, 8, 11, 18, 28 and 52 are included (Zhao et al., 2010). Di- and monochlorobiphenyls such as PCB 11, 14, 8, 3, 4 and 15 have also been detected in human serum (Koh et al., 2013; Grimm et al., 2015). Since lower chlorinated PCBs may also be metabolized (and covalently bound to biomacromolecules) in the human body more efficiently than highly chlorinated ones, the relatively low concentrations or absence of these compounds in human tissues compared to that of highly chlorinated congeners may underestimate the real exposure levels of these compounds. Additionally, Lower chlorinated PCBs may affect organisms by mechanisms different from dioxin-like PCBs (non-ortho-substituted tetraand more chlorinated biphenyls), which are sustained activators of the aryl hydrocarbon receptor (AHR) (Lauby-Secretan et al., 2013); most of the biologic effects of the former chemicals may depend on and be relatively susceptible to metabolic activation, and this may explain, at least partially, the relatively low concentrations of lower chlorinated biphenyls and their hydroxylated metabolites in the blood (Grimm et al., 2015). PCBs may have various health effects, such as endocrine disruption, developmental impairment, reproductive dysfunction, and carcinogenesis (Faroon and Ruiz, 2015). In 2013, PCBs have been classified by the International Agency for Research on Cancer (IARC) as human (group 1) carcinogens (IARC, 2013). Lower chlorinated PCBs are supposed to exert their carcinogenic potentials through their bio-reactive metabolites, formed under the action of biotransformation enzymes (Lauby-Secretan et al., 2013). It is suggested that some cytochrome P450 (CYP) enzymes should be responsible for the metabolic activation of PCBs to mutagenic metabolites (Robertson and Ludewig, 2011; Ludewig and Robertson, 2013). Investigation of the mutagenicity of various classic PCB mixtures in Chinese hamster (V79) cells resulted in negative results (Hattula, 1985). This is probably due to the lack of appropriate biotransformation enzymes in the cells. Using PCB 3 as a model compound for semi-volatile PCBs, it has been observed that some quinoid and dihydroxylated metabolites of PCB 3 strongly induced micronuclei in V79 cells, whereas the parent PCB 3 was negative in the test (Zettner et al., 2007). Meanwhile, it has been re-
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ported that some hydroxylated PCBs are substrates for sulfotransferases (SULTs), such as rat SULT1A1 (Liu et al., 2009) and human SULT2A1 (Liu et al., 2006), which implies a potential role of SULTs in modulating the formation of active metabolites of PCBs. Through a quantitative structure–activity relationship analysis of the genotoxic potentials of PCBs, it is proposed that mono- and dichlorinated PCBs and their metabolites may possess the highest genotoxic activities among PCBs chlorinated to different degrees (Ruiz et al., 2008); however, direct experimental evidence supporting this proposal is missing. Up to now, a systematic observation of the genotoxicity of individual PCB congeners is lacking, probably due to the fact that no biotransformation enzymes (e.g., CYPs) have been identified for activating PCBs to genotoxic metabolites. According to our recent finding that human CYP2E1 activates hydroquinone, catechol and 1,2,4-trihydroxybenzene to more genotoxic metabolites (Jiang et al., 2014), we hypothesized that human CYP2E1 may also activate some lower chlorinated PCBs to genotoxicants. Therefore, in this pilot study we investigated the induction of micronuclei by each of the 15 possible mono- and dichlorobiphenyls in a V79-derived cell line expressing both human CYP2E1 and human SULT1A1 (V79-hCYP2E1-hSULT1A1); modulation of each enzyme was employed to identify its role in activating or inactivating a test compound. Representative congeners were additionally investigated for induction of gene mutations at the Hprt locus.
2. Materials and methods 2.1. Chemicals PCB 3 and 15 were generous gifts from Dr. Yang Song (Chongqing, China); all the other 13 PCBs (i.e., PCB 1, 2, and 4 through 14) were purchased from AccuStandard Inc. (New Haven, CT, USA). Dimethylsulfoxide (DMSO), 1-aminobenzotriazle (ABT), N-nitrosodiethylamine (NDEA) and absolute ethanol were purchased from Sigma Aldrich (St. Louis, MO, USA). Ethyl methanesulfonate (EMS) and 1-methylpyrene (1-MP) were from J & K Chemical Ltd (Suzhou, China). Disulfonated tetrazolium salt (more commonly known as cell counting kit-8, CCK-8) was from Dojindo Laboratories (Kumamoto, Japan). Pentachlorophenol (PCP) was from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Prior to treatment of the cells with test compounds, pure water was used to dissolve EMS and NDEA, while DMSO served as the solvent for each PCB and 1-MP.
2.2. Cell lines V79 cell line was purchased from Shanghai Fuxiang Biotech. Co., LTD (Shanghai, China). V79-hCYP2E1-hSULT1A1 cell line was constructed from a V79-derived cell line expressing human CYP2E1 (Schmalix et al., 1995) through transfection of wild-type SULT1A1 (Glatt et al., 2005; Liu and Glatt, 2010). The parental V79-Mz cell line was completely deficient in the expression of CYPs, SULTs and UGTs (Glatt et al., 1990), in particular, no expression of CYP2E1 was observed in this cell line by an immunoblotting assay (Schmalix et al., 1995). V79-hCYP2E1-hSULT1A1 cells have performed well in both Hprt mutagenicity and micronucleus tests for the investigation of enzyme-dependent genotoxic responses (Glatt et al., 2005; Liu and Glatt, 2008; Jiang et al., 2015). Cells were cultured in Dulbecco’s modification of Eagle’s medium (Gibco) supplemented with 7% fetal bovine serum (Gibco), 100 IU/mL penicillin G, and 100 μg/mL streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2 .
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2.3. Cytotoxicity (CCK-8) assay Cytotoxicity of each test compound was determined by CCK8 assays, in which CCK-8 was used as a chromogenic indicator for NADH and cell viability (Ishiyama et al., 1997). The experimental procedures were adapted according to the exposurerecovery schedule for either the micronucleus or the mutagenicity assay (Jiang et al., 2015). Briefly, V79 or V79-hCYP2E1hSULT1A1 cells were inoculated on 96-well plates at the density of 6.8 × 103 cells/well (with 100 μL medium per well). At 24 h, each PCB at varying concentrations was added, 6 wells for each treatment; at 36 h each culture was changed with fresh culture medium; at 48 h, each culture received 10 μL of CCK-8 solution, and after being incubated for 2 h, the optical density at 450 nm (OD450 ) was measured on a microplate reader (BioRad Model 680, Hercules, CA). For the inhibition of CYP2E1 or SULT1A1 activity in V79-hCYP2E1-hSULT1A1 cells, ABT (60 μM) or PCP (10 μM) (Liu and Glatt, 2008) was present from 22 to 48 h. DMSO as the vehicle was limited to the final concentration of 0.15% (v:v). For cytotoxicity tests reflecting the exposure conditions of the Hprt mutagenicity assay, cells were inoculated at the density of 2 × 103 cells/well, and exposed to each of the four representative PCBs (PCB 5, 8, 10 and 11) at 24 h (day 1), six cultures for each treatment; at 48 h each culture was changed with fresh medium and further incubated for 2 days. At day 4, each culture was stained with CCK-8, and the OD450 was measured as described above. In V79-hCYP2E1hSULT1A1 cells, ABT and PCP at the indicated concentrations were present from 22 h to day 4 for observation of the influence of each enzyme on the cytotoxicity. 2.4. Micronucleus test The micronucleus test was conducted exactly as recently described (Jiang et al., 2015). Briefly, cells of either V79 or V79hCYP2E1-hSULT1A1 were inoculated at the density of 3 × 105 per 12.5-cm2 flask containing 3 mL medium. At 24 h, cells were treated with each PCB compound at varying concentrations for 12 h, and further cultured in fresh medium for another 12 h. At 48 h, cells in each culture were harvested by trypsinization for preparation of micronuclei-scored slides. In V79-hCYP2E1hSULT1A1 cells, SULT1A1 and CYP2E1 were inhibited, when indicated, by PCP (10 μM) and ABT (60 μM), respectively, from 22 h to 48 h; ethanol (0.2%, v:v) pretreatment from 6 h to 22 h (for inducing and stabilizing CYP2E1) (Liu and Glatt, 2008) was applied for tests with weakly active or inactive compounds (PCB 2, 3, 9, 12, 14, 15 and PCB 1). 1-MP (20 μM), bio-activated by a combination of CYP2E1 with SULT1A1 (Jiang et al., 2015), served as the positive control in V79-hCYP2E1-hSULT1A1 cells. EMS (5 mM), a direct mutagen, was used as the positive control in V79 cells. Two cultures were set up for each treatment. Micronucleated cells in 2000 qualified, randomly encountered cells from each culture were scored microscopically by an experienced experimenter. 2.5. Hprt mutagenicity assay This assay detects gene mutations as 6-thioguanine-resistant colonies, i.e., forward mutations at the Hprt locus (localized on the X chromosome). Mutagenicity of the PCB compounds in V79 and V79-hCYP2E1-hSULT1A1 cells was determined as described recently (Jiang et al., 2015) with minor modification. Briefly, 1.5 × 106 cells were inoculated in 30 mL medium into each 250cm2 flask. At 24 h, cells were treated with each representative PCB compound for 24 h with the flask caps screwed tight (for minimizing the loss of semi-volatile PCB compounds); from day 2 to day 4, cells were cultured in fresh medium, then subcultured for
another 3 days. EMS (1.6 mM) and NDEA (200 μM) (test concentrations determined by pre-test results) served as positive controls for V79 and V79-hCYP2E1-hSULT1A1 cells, respectively. PCP (10 μM) was present in the V79-hCYP2E1-hSULT1A1 cultures from 22 h to day 4 for avoidance of an influence of SULT1A1. Two cultures were set up for each treatment. At day 7, cells in each culture were harvested for the determination of colony-forming efficiency (serving as a measure of the cell survival) and 6-thioguanineresistant colonies, both of which were used in calculating the frequency of Hprt mutants. The averaged value of four paralleling 6thioguanine-containing cultures referred to each initial culture. 2.6. Statistical analysis The results of cytotoxicity assay were statistically analyzed by using ANOVA, with the relative OD450 value in each treatment compared with the control group. For the micronucleus and mutagenicity assays, data from duplicate experiments were expressed as means and ranges of variations, however, when applied in a χ 2 test they were combined to become quantal data. 3. Results 3.1. Cytotoxicity of PCBs Under the exposure/recovery (12 h/12 h) conditions, none of the 15 tested mono- or dichlorobiphenyls showed cytotoxicity as determined by the CCK-8 assay, in either V79 or V79-hCYP2E1hSULT1A1 cells, at concentrations ranging from 3 to 40 μM (PCB 5) or from 3 (or 10) to 100 μM (remaining PCBs tested), as applied in the micronucleus test with the corresponding compounds. Co-exposure of ABT (60 μM) or PCP (10 μM) did not affect the above results. Under the exposure/recovery (1 day/2 days) conditions of the Hprt mutagenicity assay, the cytotoxicity of four dichlorobiphenyls, i.e., PCB 5, 8, 10 and 11, were investigated in both V79 and V79hCYP2E1-hSULT1A1 cells by the CCK-8 assay. With each compound at concentrations ranging from 10 to 80 (or 100) μM, in V79 cells only PCB 5 (at the concentration of 100 μM) decreased the cell viability/growth, while all the four tested compounds showed some moderate cytotoxicity in V79-hCYP2E1-hSULT1A1 cells, as shown in Fig. 1. 3.2. Induction of micronuclei by dichlorobiphenyls and the impact of enzyme modulators None of the 12 dichlorobiphenyls induced the formation of micronuclei in V79 cells (Fig. 2), while EMS (5 mM) as a positive control elevated the frequency of micronucleated cells from 6‰ to around 50‰. As shown in Fig. 2, PCB 5, 6, 4, 8 (upper panel), 10, 11 and 13 (lower panel) induced micronuclei strongly (>3fold increase over control) and in a concentration-dependent manner in V79-hCYP2E1-hSULT1A1 cells; PCB 7 elevated the frequency of micronucleated cells starting from 10 μM and continuously at higher concentrations, however, a clear concentration dependence was absent in this range; the remaining dichlorobiphenyls (i.e., PCB 9, 12, 14 and 15) only marginally elevated the frequency of micronucleated cells at high concentrations (weakly positive results). As a positive control, 1-MP elevated the frequency of micronucleated cells to around 50‰, consistent with our recent report on this compound (Jiang et al., 2015). As shown in Fig. 3, PCB 7 (panel A) and PCB 10 (panel B) elevated the frequency of micronucleated cells in V79-hCYP2E1hSULT1A1 cells with clear concentration dependence, and the effects of both compounds were significantly reduced by ABT (60 μM), while PCP (10 μM) did not show significant modulation
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Fig. 1. Cytotoxicity of PCB 5 (circles), 8 (triangles), 10 (squares) and 11 (diamonds) in V79 control cells (empty symbols) or V79-hCYP2E1-hSULT1A1 cells (solid symbols) as detected by CCK-8 assay under the exposure/recovery schedule of 1 d/2 d (conditions of the Hprt assay). Pentachlorophenol (10 μM) was present in V79-hCYP2E1-hSULT1A1 cells for inhibition of SULT1A1. Data are means and standard deviations of six replicates; ∗ p < 0.05, ∗ ∗ p < 0.01, by ANOVA, compared with the negative control.
(panel A, in the case of PCB 10), or marginally potentiated the effect (without statistical significance) (for PCB 7, panel B). 3.3. Induction of micronuclei by monochlorobiphenyls and the effect of pretreatment with ethanol PCB 1, 2 and 3 did not induce micronuclei in V79 cells at concentrations ranging from 10 to 100 μM (data not shown). In V79-hCYP2E1-hSULT1A1 cells, PCB 1, 2 and 3 weakly elevated the frequency of micronucleated cells; however, with the cells pretreated with 0.2% (v:v) ethanol, the induction of micronuclei by each monochlorobiphenyl was potentiated, as shown by the presence of concentration dependence of the micronuclei induction by each compound (Fig. 4). 3.4. Induction of gene mutations at the Hprt locus in V79-hCYP2E1-hSULT1A1 cells by four representative dichlorobiphenyls As shown in Fig. 5, none of the four representative dichlorobiphenyls induced gene mutations in V79 cells up to the concentration of 80 or 100 μM, however, they all elevated the frequency of gene mutations in V79-hCYP2E1-hSULT1A1 cells strongly in a concentration-dependent manner. The relative efficiency of each compound in mutagenic activity towards these cells followed the order: PCB 5 ≈ PCB 10 >> PCB 8 ≈ PCB 11. In the meantime, these compounds were non- or mildly cytotoxic in the corresponding CCK-8 assays, and there were no obvious differences between results in the two cell lines (data not shown). 4. Discussion None of the 15 mono- and dichlorobiphenyls displayed cytotoxicity in V79 or V79-hCYP2E1-hSULT1A1 cells under a 12 h/12 h (exposure/recovery) schedule by CCK-8 assay. However, in an extended exposure and recovery (1 day/2 days) condition (in accordance to the Hprt mutagenicity assay), each of the four representative dichlorobiphenyls decreased cell viability/growth in V79hCYP2E1-hSULT1A1 cells, while in V79 cells only PCB 5 demonstrated some cytotoxicity. It appears that reduced cell density
upon inoculation and/or longer exposure and recovery time is favorable for the expression of cytotoxicity of PCBs. Moreover, the more active responses to the representative PCBs in V79-hCYP2E1hSULT1A1 cells (with SULT1A1 inhibited by PCP) than in V79 cells suggest that human CYP2E1 may activate the PCBs to cytotoxic metabolites. In the present study, some dichlorobiphenyls induced gene mutations and/or micronuclei strongly in V79-hCYP2E1-hSULT1A1 cells. The remaining dichlorobiphenyls and the three monochlorobiphenyls (PCB 1, 2 and 3) induced micronuclei in V79-hCYP2E1hSULT1A1 cells marginally. However, all compounds were nongenotoxic in V79 cells. These results indicate that metabolic activation is required for the genotoxicity of these compounds. Since induction of CYP2E1 by pretreatment of V79-hCYP2E1-hSULT1A1 cells with ethanol enhanced the formation of micronuclei by the monochlorobiphenyls (this effect was less pronounced for dichlorobiphenyls, data not shown), and inhibition of CYP2E1 by ABT drastically decreased the induction of micronuclei by some dichlorobiphenyls (e.g., PCB 7 and 10, shown in Fig. 3), human CYP2E1 may be responsible for the activation of these PCBs to genotoxic metabolites. In addition, as inhibition of SULT1A1 by PCP had no or little impact on the genotoxic response in V79-hCYP2E1-hSULT1A1 cells to the PCBs (shown in Fig. 3), human SULT1A1 may not be involved in the activation of these PCBs, nor has it significant detoxifying activity toward the active metabolites. The active metabolites responsible for CYP2E1-mediated genotoxicity of the tested PCBs in this study have not yet been directly identified. Previously, it has been demonstrated that incubation of PCB 3 with hepatic microsomes from rats treated with phenobarbital and 3-methylcholanthrene gives rise to the formation of a group of metabolites: 4-OH PCB 3 as the major metabolite, and the following in a decreasing order: 3 ,4 -(OH)2 PCB 3, 2 ,3 (OH)2 PCB 3, 3 -OH PCB 3, and 2 ,5 -(OH)2 PCB 3 (McLean et al., 1996). Dihydroxylated PCBs may further be activated to quinonic metabolites, which are highly bioreactive towards DNA and protein molecules (Amaro et al., 1996). The rate of metabolism of other PCBs may be determined by (1) the number and position of chlorines in the biphenyl core, and (2) the availability of vici-
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Fig. 2. Induction of micronuclei by each dichlorobiphenyl in V79 (empty symbols) and V79-hCYP2E1-hSULT1A1 cells (solid symbols). Cells were treated with each PCB at various concentrations from 24 h to 36 h, and harvested at 48 h. Each tested PCB did not show significant cytotoxicity (as determined by the CCK-8 assay) in the concentrations applied in the micronucleus test (data not shown). Data are means from duplicate determinations; ranges of variation and statistical analysis results are not shown here for a clear presentation of the mean values of multiple compounds in a single panel.
nal free (unsubstituted) carbon-hydrogen positions (Grimm et al., 2015). Formation of transient epoxides may be another pathway of metabolic activation of PCBs, which may result in adducts with biomacromolecules; epoxides may also isomerize to hydroxylated PCBs, hydrolyzed to dihydrodiol metabolites, or conjugated with glutathione (Grimm et al., 2015). OH-PCBs may undergo sulfo- and glucuronosyl-conjugation, probably inactivating reactions. For example, 4-OH PCB 8 is a substrate for rat SULT1A1 (Liu et al., 2009). However, in this study human SULT1A1 did not significantly modulate the CYP2E1-dependent genotoxicity of PCBs. This lack of effect may owe to a different specificity of human SULT1A1 towards its OH-PCB substrates than its rat counterpart, or the hydroxylated metabolite was formed intracellularly at a concentration inappropriate for a sulfo-conjugation rate detectable through the micronucleus test. Hydroxylated metabolites of highly chlorinated congeners have been identified in human blood (Bergman et al., 1994; Guvenius et al., 2002). These hydroxylated metabolites of PCBs may be accumulated in some solid tissues, such as the liver, as compared with their concentrations in the blood (Guvenius et al. 2002). The absence of hydroxylated mono- and dichlorobiphenyls
on the list of detected OH-PCBs may be due to their susceptibility to further metabolism or binding to macromolecules such as DNAs and proteins. Anyway, understanding of the hydroxylated metabolites of the dichlorobiphenyls that showed strong CYP2E1dependent genotoxicity in this study will be interesting, and addressed in future studies. With the data of CYP2E1-dependent induction of micronuclei by each of the twelve dichlorobiphenyl compounds (shown in Fig. 2), it is plausible to give an assessment of a potential structure–activity relationship. Since the para- and meta-positions are more readily oxidized, it is suggested that 2,3-, 2,5-, and 2,6dicholobiphenyls (PCB 5, 9 and 10, respectively) may be the most easily hydroxylated congeners among various dichlorobiphenyls (Grimm et al., 2015). Indeed, in this study PCB 5 and PCB 10 were the most mutagenic dichlorobiphenyls in V79-hCYP2E1-hSULT1A1 cells; on the contrary, PCB 9 was almost inactive. Actually, among the seven dichlorobiphenyls with one or two chlorine substitutions on the ortho-position, most (PCB 4, 5, 6, 7, 8 and 10) were strong inducers of micronuclei in V79-hCYP2E1-hSULT1A1 cells, with PCB 9 being the only exception. PCB 9 is unique, as the
C. Zhang et al. / Chemosphere 144 (2016) 1908–1915
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Fig. 3. Induction of micronuclei in V79-hCYP2E1-hSULT1A1 cells by PCB 7 (A) and PCB 10 (B) and the impact of 1-aminobenzotriazole (60 μM) and pentachlorophenol (10 μM). See legend of Fig. 2. Ethanol (0.2%, v:v) was present prior to the addition of modulator and test compounds for the induction and stabilization of CYP2E1, but absent from cultures to be co-exposed to 1-aminobenzotriazole. Effect of PCB 7 or 10 alone was symbolized with empty circles, and that in combination with 1-aminobenzotriazole and pentachlorophenol was symbolized with solid triangles and squares, respectively. Statistical analysis was performed by χ 2 examination following combining the duplicate data to become quantal data, ∗ p < 0.05, ∗ ∗ p < 0.01, compared with the control (no test compound); # p < 0.01, ## p < 0.01, comparison was made between a PCB treatment with and without a modulator.
two chlorine substitutions stand opposite (para-) to each other on a ring, and this constellation may cause an offset of the electron-attracting effect of each chlorine; thus, the polarity of the three carbon-hydrogen single bonds on the ring will not be influenced by the chlorines, which is otherwise favorable for an oxidation reaction (through an enhanced electron-deficiency of a hydrogen atom). It seems that a non-coplanar configuration [endowed by ortho-substitution with chlorine(s)] of the biphenyl core is favorable for the metabolic activation of dichlorobiphenyls, except for a unique structure like PCB 9. As for the five dichlorobiphenyls without an ortho-chlorination, two of them (PCB 11 and 13) were strong CYP2E1-dependent genotoxicants while the other three were nearly inactive (PCB 12, 14 and 15). There seem to be no obvious structural feature accounting for their distinct differ-
ences in CYP2E1-activated genotoxicity. Multiple factors, such as the availability of para- and meta-unsubstituted carbon atoms, increased polarity of the C–H single bonds, the steric and electrostatic fitfulness of each PCB molecule to the active center of the enzyme, and the bio-reactivity of the metabolites formed, may determine the structure–activity relationship for the human CYP2E1dependent genotoxicity of PCBs. A molecular docking-based threedimensional quantitative structure–activity relationship study may be useful for a more accurate assessment of the interaction of the compounds with the enzyme. In the present study, dichlorobiphenyls demonstrated stronger mutagenicity than monochlorobiphenyls. Then, what will be the CYP2E1-dependent mutagenicity of important tri- and tetrachloro-
Fig. 4. Induction of micronuclei in V79-hCYP2E1-hSULT1A1 cells by each monochlorobiphenyl and the effect of pretreatment of the cells with ethanol (0.2%, v:v). See legend of Fig. 2. Data are means and ranges of variation. Empty and solid symbols represent experiments without and with ethanol pretreatment, respectively. ∗p < 0.05, ∗∗p < 0.01, compared with the control (no test compound); ##p < 0.01, comparison was made between a PCB treatment with and without ethanol pretreatment.
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Fig. 5. Induction of gene mutations at the Hprt locus by PCB 5 (circles), PCB 8 (triangles), PCB 10 (squares) and PCB 11 (diamonds) in V79 (empty symbols) and V79-hCYP2E1hSULT1A1 cells (solid symbols). Pentachlorophenol (10 μM) was present in V79-hCYP2E1-hSULT1A1 cells for inhibition of SULT1A1. NDEA (200 μM), a CYP2E1-dependent promutagen serving as the positive control in each experiment, elevated the mutant frequency to about 350 mutants per 106 cells. Data from duplicate experiments were combined to become quantal data for χ 2 analysis; ∗p < 0.05, ∗∗p < 0.01, compared with the negative control.
biphenyls (e.g., PCB 20, 28, 52 and 77) (Persoon et al., 2010; Marek et al., 2013)? Continued experimental studies in the cell model used in this study have to be performed for a more comprehensive knowledge on the genotoxicity of PCBs. It will be interesting to learn whether other CYP forms are also involved in the activation of PCBs to genotoxic metabolites. This can be achieved by using cell lines expressing other CYP forms or human hepatocytes endogenously expressing multiple CYPs with modulators specific for a particular CYP form. Of the strongly genotoxic PCBs identified in this study, at least PCB 4, 8 and 11 have been detected in the atmospheric environment (Hu et al., 2008; Zhao et al., 2010), PCB 11 has also been detected in a relatively high level in human bodies (Marek et al., 2013). It is true that the effective concentrations of these PCBs observed in this study must be many times higher than their levels in human bodies, however, the tiny or undetectable levels of dichlorobiphenyls in human tissues do not necessarily mean they can be ignored, since these lower chlorinated PCBs may be metabolized at significantly enhanced rates (to bioreactive metabolites) compared to the highly chlorinated congeners (Schnellmann et al., 1984; Grimm et al., 2015), thus may not be allowed for high internal levels. Furthermore, a potential effect of persistent exposure of humans to low level dichlorobiphenyls may be missing in the classic short-term genotoxicity assays, as applied in the present study. Therefore, this study may represent a starting point toward a comprehensive structure–activity relationship for the mutagenicity of PCBs and the application in the relevant environmental health risk analysis. 5. Conclusion The present study has connected a CYP enzyme (human CYP2E1) with PCB-induced transmittable genetic changes in mammalian cells with solid evidence for the first time. Our study has also revealed varied mutagenicity of individual mono- and dichlorobiphenyls, which are differentially structured. Mutagenicity of other PCBs, the possibility of the involvement of other CYP enzymes in the activation of PCBs, and structure of the reactive metabolites responsible for the mutagenicity of the PCBs deserve
further investigation. Acknowledgment This work was supported by the Education Department of Guangdong Province in China under an Academic Talentsintroducing Program [Y. Liu, Grant code: YueCaiJiao 2010(143)], and a project supported by the Natural Science Foundation of Guangdong Province, China (Y. Liu, Grant code: S2012010008922). References Amaro, A.R., Oakley, G.G., Bauer, U., Spielmann, H.P., Robertson, L.W., 1996. Metabolic activation of PCBs to quinones: reactivity toward nitrogen and sulfur nucleophiles and influence of superoxide dismutase. Chem. Res. Toxicol. 9, 623–629. Asante, K.A., Adu-Kumi, S., Nakahiro, K., Takahashi, S., Isobe, T., Sudaryanto, A., Devanathan, G., Clarke, E., Ansa-Asare, O.D., Dapaah-Siakwan, S., Tanabe, S., 2011. Human exposure to PCBs, BPDEs, and HBCDs in Ghana: temporal variation, sources of exposure estimation of daily intakes by infants. Environ. Int. 37, 921–928. Bergman, A., Klasson-Wehler, E., Kuroki, H., 1994. Selective retention of hydroxylated PCB metabolites in blood. Environ. Health Perspect. 102, 464–469. Eguchi, A., Nomiyama, K., Devanathan, G., Subramanian, A., Bulbule, K.A., Parthasarathy, P., Takahashi, S., Tanabe, S., 2013. Different profiles of anthropogenic and naturally produced organohalogen compounds in serum from residents living near a coastal area and e-waste recycling workers in India. Environ. Int. 47, 8–16. Faroon, O., Ruiz, P., 2015. Polychlorinated biphenyls: new evidence from the last decade. Toxicol. Ind. Health doi:10.117710748233715587849. Glatt, H., Gemperlein, I., Setiabudi, F., Platt, K.L., Oesch, F., 1990. Expression of xenobiotic-metabolizing enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds. Mutagenesis 5, 241–249. Glatt, H., Schneider, H., Liu, Y., 2005. V79-hCYP2E1-hSULT1A1, a cell line for the sensitive detection of genotoxic effects induced by carbohydrate pyrolysis products and other food-borne chemicals. Mutat. Res. 580, 41–52. Grimm, F.A., Hu, D., Kania-Korwel, I., Lehmler, H.J., Ludewig, G., Hornbuckle, K.C., Duffel, M.W., Bergman, A., Robertson, L.W., 2015. Metabolism and metabolites of polychlorinated biphenyls. Crit. Rev. Toxicol. 45, 245–272. Grossman, E., 2013. Nonlegacy PCBs: pigment manufacturing by-products get a second look. Environ. Health Perspect. 121, A86–A93. Guvenius, D.M., Hassanzadeh, P., Bergman, A., Noren, K., 2002. Metabolites of polychlorinated biphenyls in human liver and adipose tissue. Environ. Toxicol. Chem. 21, 2264–2269. Hattula, M.L., 1985. Mutagenicity of PCBs and their pyrosynthetic derivatives in cellmediated assay. Environ. Health Perspect. 60, 255–257. Hu, D., Martinez, A., Hornbuckle, K.C., 2008. Discovery of non-Aroclor PCB (3,3’dichlorobiphenyl) in Chicago air. Environ. Sci. Technol. 42, 7873–7877.
C. Zhang et al. / Chemosphere 144 (2016) 1908–1915 International Agency for Research on Cancer (IARC), 2013. Agents Classified by the IARC Monographs, vol. 1–109 150 Cours Albert Thomas, 69372 Lyon CEDEX 08, France. Available. http://monographs.iarc.fr/ENG/Classification/ (accessed 30.10.13.). Ishiyama, M., Miyazono, Y., Sasamoto, K., Ohkura, Y., Ueno, K., 1997. A highly watersoluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta 44, 1299–1305. Jiang, H., Lai, Y., Hu, K., We, Q., Liu, Y., 2014. Human CYP2E1-dependent and human sulfotransferase 1A1-modulated induction of micronuclei by benzene and its hydroxylated metabolites in Chinese hamster V79-derived cells. Mutat. Res. 770, 37–44. Jiang, H., Lai, Y., Hu, K., Chen, D., Liu, B., Liu, Y., 2015. Genotoxicity of 1methylpyrene and 1-hydroxymethylpyrene in V79-hCYP2E1-hSULT1A1 cell line. Environ. Mol. Mutagen 56, 404–411. Koh, W.X., Horbuckle, K.C., Thorne, P.S., 2013. Human serum from urban and rural adolescents and their mothers shows exposure to polychlorinated biphenyls not found in commercial mixtures. Environ. Sci. Technol. 49, 8105–8112. Lauby-Secretan, B., Loomis, D., Grosse, Y., E.I. Ghissassi, F., Bouvard, V., BenbrahimTallaa, L., Guha, N., Baan, R., Mattock, H., Straif, K., 2013. Carcinogenicity of polychlorinated biphenyls and polybrominated biphenyls. Lancet Oncol. 14, 287– 288. Liu, Y., Glatt, H., 2008. Mutagenicity of N-nitrosodiethanolamine in a V79-derived cell line expressing two human biotransformation enzymes. Mutat. Res. 643, 64–69. Liu, Y., Glatt, H., 2010. Human cytochrome P450 2E1 and sulfotransferase1A1 coexpressed in Chinese hamster V79 cells enhance spontaneous mutagenesis. Environ. Mol. Mutagen 51, 23–30. Liu, Y., Apak, T.I., Lehmler, H.J., Robertson, L.W., Duffel, M.W., 2006. Hydroxylated polychlorinated biphenyls are substrates and inhibitors of human hydroxysteroid sulfotransferase SULT2A1. Chem. Res. Toxicol. 19, 1420–1425. Liu, Y., Smart, J.T., Song, Y., Lehmler, H.J., Robertson, L.W., Duffel, M.W., 2009. Structure-activity relationships for hydroxylated polychlorinated biphenyls as substrates and inhibitors of rat sulfotransferases and modification of these relationships by changes in thiol status. Drug Metab. Dispos. 37, 1065–1072. Ludewig, G., Robertson, L.W., 2013. Polychlorinated biphenyls (PCBs) as initiating agents in hepatocellular carcinoma. Cancer Lett. 334, 46–55.
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Marek, R.F., Thorne, P.S., Wang, K., DeWall, J., Keri, K.C., 2013. PCBs and OH-PCBs in serum from children and mothers in urban and rural U.S. communities. Environ. Sci. Technol. 47, 3353–3361. McLean, M.R., Bauer, U., Amaro, A.R., Robertson, L.W., 1996. Identification of catechol and hydroquinone metabolites of 4-monochlorobiphenyl. Chem. Res. Toxicol. 9, 158–164. Persoon, C., Peters, T.M., Kmumar, N., Hornbuckle, K.C., 2010. Spatial distribution of airborne polychlorinated biphenyls in Cleveland, OH and Chicago, IL. Environ. Sci. Technol. 44, 2797–2802. Robertson, L.W., Ludewig, G., 2011. Polychlorinated biphenyl carcinogenicity with special emphasis on airborne PCB. Gefahrst. Reinhalt. Luft 71, 25–32. Rodenburg, L.A., 2010. Evidence for unique and ubiquitous environmental sources of 3,3ʹ-dichlorobiphenyl (PCB 11). Environ. Sci. Technol. 44, 2816–2821. Ruiz, P., Faroon, O., Moudgal, C.T., Hansen, H., Rosa, C.T.D., Mumtaz, M., 2008. Prediction of the health effects of polychlorinated biphenyls (PCBs) and their metabolites using quantitative structure–activity relationship (QSAR). Toxicol. Lett. 181, 53–65. Schmalix, W.A., Barrenscheen, M., Landsiedel, R., Janzowski, C., Eisenbrand, G., Gonzalez, F., Eliasson, E., Ingelman-Sundberg, M., Perchermeier, M., Greim, H., Doehmer, J., 1995. Stable expression of human cytochrome P450 2E1 in V79 Chinese hamster cells. Eur. J. Pharmacol. 293, 123–131. Schnellmann, R.G., Volp, R.F., Putnam, C.W., Sipes, I.G., 1984. The hydroxylation, dechlorination, and glucuronidation of 4,4ʹ-dichlorobiphenyl (4-DCB) by human hepatic microsomes. Biochem. Pharmacol. 33, 3503–3509. Zettner, M.A., Flor, S., Ludewig, G., Wagner, J., Robertson, L.W., Lehmann, L., 2007. Quinoid metabolites of 4-monochlorobiphenyl induce gene mutations in cultured Chinese hamster V79 cells. Toxicol. Sci. 100, 88–98. Zhao, G., Wang, Z., Zhou, H., Zhao, Q., 2009. Burdens of PBBs, PBDEs, and PCBs in tissues of the cancer patients in the e-waste disassembly sites in Zhejiang, China. Sci. Total Environ. 407, 4831–4837. Zhao, H.X., Adamcakova-Dodd, A., Hu, D., Hornbuckle, K.C., Just, C.L., Robertson, L.W., Thorne, P.S., Lehmler, H.J., 2010. Development of a synthetic PCB mixture resembling the average polychlorinated biphenyl profile in Chicago air. Environ. Int. 36, 819–827.