Human CYP1B1-dependent genotoxicity of dioxin-like polychlorinated biphenyls in mammalian cells

Journal Pre-proof Human CYP1B1-dependent genotoxicity of dioxin-like polychlorinated biphenyls in mammalian cells Yuting Chen, Yifan Wu, Weiwei Xiao, Hansi Jia, Hansruedi Glatt, Ming Shi, Yungang Liu

PII:

S0300-483X(19)30286-0

DOI:

https://doi.org/10.1016/j.tox.2019.152329

Reference:

TOX 152329

To appear in:

Toxicology

Received Date:

30 July 2019

Revised Date:

12 November 2019

Accepted Date:

12 November 2019

Please cite this article as: Chen Y, Wu Y, Xiao W, Jia H, Glatt H, Shi M, Liu Y, Human CYP1B1-dependent genotoxicity of dioxin-like polychlorinated biphenyls in mammalian cells, Toxicology (2019), doi: https://doi.org/10.1016/j.tox.2019.152329

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Human

CYP1B1-dependent

genotoxicity

of

dioxin-like

polychlorinated biphenyls in mammalian cells

Yuting Chena,1, Yifan Wua,1, Weiwei Xiaob,1, Hansi Jiaa, Hansruedi Glattc,d, Ming Shie, and Yungang Liua* a

Department of Toxicology, bBiosafety Level-3 Laboratory, School of Public Health, Southern

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Medical University (Guangdong Provincial Key Laboratory of Tropical Disease Research), 1023 S. Shatai Road, Guangzhou 510515, China cDepartment

of Nutritional Toxicology, German Institute of Human Nutrition (DIfE), Arthur-

Scheunert- Allee 114-116, D-14558 Nuthetal, Germany

of Food Safety, Federal Institute for Risk Assessment (BfR), Max-Dohrn-Straße 8-10,

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dDepartment

D-10589 Berlin, Germany

of Environmental and Occupational Health, School of Public Health, Guangdong

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eDepartment

Medical University, Dongguan 523808, China

authors contributed equally to this work.

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1These

*Corresponding author at: Department of Toxicology, School of Public Health, Southern Medical

61648324.

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University, 1023 S. Shatai Road, Guangzhou 510515, China. Tel: +86-20-61648554; Fax: +86-20-

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Email address: [email protected] (Y. Liu)

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Abstract Polychlorinated biphenyls (PCBs) are persistent organic pollutants and human carcinogens. It was reported that rat CYP1A1 and catfish CYP1A can hydroxylate 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77), while potential roles of other CYP1 enzymes in the metabolism of dioxin-like (DL) PCBs remain unconfirmed. In this study, three representative DL-PCBs, i.e., PCB 77, PCB 126, and 3,4,4',5-tetrachlorobiphenyl (PCB 81), were investigated on their genotoxicity in Chinese hamster V79-derived cell lines genetically engineered for the expression of human CYP1A1, 1A2 and 1B1, and in the human hepatoma C3A cell line, which

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endogenously expresses various CYPs. Under both 6 h/18 h and 18 h/6 h (exposure/recovery) regimes, PCB 77 and 81 induced micronuclei in V79-hCYP1B1 cells at micromolar levels, with

slightly higher potency in the latter regime, while they were inactive in the parental V79-Mz cells

and the V79-derived cell lines expressing human CYP1A1 and 1A2. However, PCB 126 was

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negative in each cell line. Likewise, PCB 77 and 81 induced micronuclei formation in C3A cells,

which expressed CYP1B1. This effect was blocked by co-exposure to tetramethoxystilbene (30 nM),

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a selective CYP1B1 inhibitor. Immuno-fluorescent staining of centromere protein B in the micronuclei in PCB-treated cultures showed a predominance of centromere-negative micronuclei,

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which indicated a clastogenic effect. Moreover, all three PCBs elevated the level of γ-H2AX protein (indicating DNA double-strand breaks) in C3A cells, and these effects were blocked by tetramethoxystilbene (10 nM). This study demonstrates that some DL-PCBs are clastogenic in

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Keywords:

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mammalian cells following metabolic activation by human CYP1B1.

Centromere protein B

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CYP1B1 γ-H2AX

Micronuclei

Polychlorinated biphenyls

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1. Introduction Polychlorinated biphenyls (PCBs) are persistent organic pollutants with various health effects, such as endocrine disruption, oxidative stress, immune dysfunction, and carcinogenesis observed in animal models (Faroon & Ruiz 2015). PCBs were widely used in industrial products for approximately 50 years until 1980, when they were banned from production and restricted in use in most countries. However, PCBs are still present in the environment globally, owing to their resistance to degradation, food chain biomagnification, unintentional production from burning of chlorine-containing organic materials (Grossman 2013), manufacture of paint pigment products (Hu

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and Hornbuckle 2010), and wrong disposal of PCB-containing products, such as illegal e-waste disassembling activities in some Asian and African countries (Zhao et al. 2009; Tue et al. 2016). PCBs were classified by the International Agency of Research on Cancer (IARC) as human (group

1) carcinogens (IARC 2016). The toxicity of coplanar (also termed dioxin-like, DL) PCBs is

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supposed to be largely dependent on their high-affinity binding to, and sustained activation of, the

aryl hydrocarbon receptor (AHR) (Hennig et al. 2002). On the contrary, lower chlorinated and non-

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coplanar (non-dioxin-like, NDL) PCBs have to undergo metabolic activation before exerting most of their toxic effects (Ludewig and Robertson 2013; Grimm et al. 2015). Indeed, human CYP2B6

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and rat CYP2B1 have been observed to hydroxylate some NDL-PCBs to mono- and dihydroxylated metabolites (Warner et al. 2009; Lu et al. 2013). The most probable ultimate carcinogens of these

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PCBs are quinones, formed oxidatively from dihydroxylated metabolites (McLean et al. 1996; Zettner et al. 2007; Ludewig and Robertson 2013). The hydroxylated intermediates tend to undergo sulfo- and glucurono-conjugation, catalyzed by sulfotransferases (SULTs) and UDP-

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glucuronosyltransferases (UGTs) (Liu et al. 2006; Sacco et al. 2008; Grimm et al. 2017), presumably leading to detoxification. Recently, we have reported potent mutagenicity of a series of NDL-PCBs

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(PCB 20, 22, 32, 52, 56 and 74) in mammalian cells with human CYP2E1 activity (Liu et al. 2017; Chen et al. 2018). Thereafter, we were interested in the possibility for DL-PCBs to be promutagens metabolically activated by relevant biotransformation enzymes. DL-PCBs include 12 congeners, that bear 4 to 7 chlorine substituents per molecule (PCB 77, 81, 126, 169, 105, 114, 118, 123, 156, 157, 167, and 189). These chemicals are structurally similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent ligand of AHR, in the sense of a common coplanar conformation. Like TCDD, DL-PCBs bind to AHR (effective at sub- or low 3

nanomolar concentrations), activate it and then trigger multiple downstream cellular responses (Hestermann et al. 2000), including the induction of CYP1 (e.g., CYP1A1, 1A2 and 1B1) enzymes (Schmitz et al. 1995; Kakutani et al. 2014). Frequently, the inducers of biotransformation enzymes are also substrates for the induced enzymes. As previous studies suggested, DL-PCBs are likely to be metabolized by CYP1 enzymes, including CYP1A1 and 1A2, which are expressed in hepatocytes. For example, rat CYP1A1 catalyzed the metabolism of PCB 126 (3,3’,4,4’,5-pentachlorobiphenyl) to 4-OH-3,3’,4’,5-tetrachlorobiphenyl and 4-OH-3,3’,4’,5, 5’-pentachlorobiphenyl, though human CYP1A1 did not metabolize PCB 126 (Yamazaki et al. 2011). Molecular docking investigation of

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these orthologous CYP1A1 proteins with PCB 126 revealed differences in the size and shape of the cavities formed by the amino acid residues, where only rat CYP1A1 allows binding of PCB 126 in

a pattern permitting electron transfer. That report suggests that there might be important species differences in the metabolism of DL-PCBs by human CYP1 enzymes as compared with its

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homologues in rodent species. CYP1B1, which is primarily expressed at various extrahepatic sites

rather than in hepatocytes, metabolizes many of the same, mostly planar, substrates as CYP1A1 and

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CYP1A2 (Shimada et al. 1997). Therefore, it may also metabolize DL-PCBs (IARC 2016). It might be advantageous for the investigation of metabolism-dependent PCB toxicity to adopt human

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enzymes, e.g. subcellular fractions containing human CYP enzymes, human cells, or mammalian cells genetically engineered for expression of human CYP enzymes.

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We hypothesized that human CYP1 enzymes (i.e. CYP1A1, 1A2 and/or 1B1) are capable of activating some coplanar PCB compounds to genotoxic metabolites. To test this hypothesis, three representative

coplanar

PCBs,

i.e.,

3,3’,4,4’-tetrachlorobiphenyl

(PCB

77),

3,4,4’,5-

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tetrachlorobiphenyl (PCB 81) and PCB 126, were investigated in Chinese hamster V79-derived cell lines expressing human CYP1A1, CYP1A2, or CYP1B1, for the induction of micronuclei. V79 cells

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are deficient in p53, in which some genotoxicants are occasionally tested with false positive results, especially when chromosomal damage (as with the micronucleus test) is used as the endpoint (Fowler et al. 2012). Therefore, a human hepatoma cell line (C3A, a derivative from the HepG2 cell line) that is p53-proficient with endogenous expression of various CYP enzymes to some extent, was employed in the micronucleus test to verify the results in V79-derived cell lines. In both V79derived cells and C3A cells the origin of micronuclei induced by test chemicals was explored, by classifying them as chromosomal breakage (clastogenicity) or loss (aneugenicity) using immuno4

fluorescent analysis of the centromere protein B (CENP-B) in the micronuclei. Moreover, γ-H2AX, a phosphorylated product of histone protein H2AX formed and released from the nucleosome complex subsequent to double DNA breaks, was analyzed by Western blot assay in C3A cells treated with DL-PCBs.

2. Materials and methods 2.1 Chemicals and materials PCB 77, PCB 81, PCB 126 and benzo[a]pyrene (BP) were purchased from AccuStandard Inc. (New

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Haven, CT, USA). Dimethylsulfoxide (DMSO) was purchased from Sigma Aldrich (St. Louis, MO, USA). Ethylmethane sulfonate (EMS) and aflatoxin B1 (AFB1) were from J & K Chemical Ltd

(Suzhou, China). Vincristine sulfate (VCR) was from Selleck Chemicals (Houston, TX), disulfonated tetrazolium salt (cell counting kit-8, CCK-8) from Dojindo Laboratories (Kumamoto,

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Japan), (E)-2,3',4,5'-tetramethoxystilbene (TMS) from Abcam (Cambridge, UK), paraformaldehyde

from Biosharp (Hefei, China), and antifade (with DAPI) from Yeasen Biotech Co., Ltd. (Shanghai,

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China). EMS was dissolved in water prior to exposing cells, while DMSO was used as the vehicle for BP, AFB1, VCR, and the PCBs, with its final concentration limited to 0.2% (v:v).

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Antibodies used in this study: antiserum raised in rabbit recognizing human CYP1A1, CYP1A2 and CYP1B1, and the HRP-conjugated goat anti-rabbit IgG antibodies, were all purchased

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from Abcam (Cambridge, UK). The antiserum raised in mouse recognizing human phosphoS139H2AX was purchased from Abcam (Cambridge, UK), and the goat anti-mouse IgG H&L (HRP) antibodies were purchased from Yeasen Biotech Co., Ltd (Shanghai, China). The antiserum in rabbit

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recognizing human CENP-B and that in mouse recognizing β-tubulin were purchased from Abcam (Cambridge, UK) and Sigma-Aldrich (St. Lous, MO), respectively, while the FITC-conjugated goat

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anti-rabbit IgG and Chromeo™ -conjugated goat anti-mouse IgG antibodies were both from Abcam (Cambridge, UK).

2.2 Cell lines The V79-Mz cell line did not show any expression of CYPs, SULTs and UGTs (Glatt et al. 1990). The V79-derived cell lines expressing various biotransformation enzymes were generous gifts from Prof. Johannes Doehmer (Munich, Germany), which had been established by transfecting 5

appropriate eukaryotic expression vectors containing the full-length cDNAs encoding human CYP1A1, 1A2 and 1B1 individually into V79-Mz cells (Schmalix et al. 1993; Wölfel et al. 1992; Luch et al. 1998), with protein expression and enzyme activity being determined in individual cell clones, resulting in cell lines V79-hCYP1A1 , V79-hCYP1A2 , and V79-hCYP1B1 . These cell lines were further characterized for the expression of the enzymes by use of appropriate promutagens in the micronucleus test and mutagenicity assay (Liu et al. 2017). The cells were cultured in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 7% fetal bovine serum, 100 IU/mL penicillin G, and 100 μg/mL streptomycin, at 37 °C in a humidified

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atmosphere containing 5% CO2. The population-doubling time of V79-Mz and its derivative lines was about 12 h.

The C3A cell line, a subclone of the HepG2 line, was obtained from Shanghai Fuxiang Biotech Co., Ltd. (Shanghai, China). CYP1B1, whose activity usually appears in some extrahepatic tissues

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rather than liver, is expressed together with other CYP enzymes, such as CYP1A1, 1A2, 2E1, and

3A4, in C3A cells (Zhang et al. 2017). C3A cells were cultured in DMEM supplemented with 10%

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2.3 Cell survival assay

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fetal bovine serum. The population-doubling time of C3A cells was around 36 h.

The CCK-8 assay was employed as a measure of the relative level of cell growth and viability, thus

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indirectly reflecting the cytotoxicity of each test compound. In this assay the cellular content of NADH is indicated chromogenically as the optical density at 450 nm (OD450) (Ishiyama et al. 1997). As reported previously, linear regression of cell counts with CCK-8 assay results yielded a

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correlation coefficient of 0.993 (p < 0.001), which suggests that the CCK-8 assay effectively represents cell number (division) and/or viability changes induced by chemicals (Jiang et al. 2015).

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In accordance to the micronucleus assays under varied exposure/recovery schedules, cells of V79Mz and each V79-derived line were used to determine the cytotoxicity of test compounds, according to our published procedure (Liu et al. 2017). The cytotoxicity assay in C3A cells was adapted according to the doubling time (36 h). Thus, 18 h/54 h (short exposure followed by extended recovery) and 54 h/18 h (long exposure and short recovery) regimes were employed, corresponding to the relevant micronucleus tests.

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2.4 Micronucleus test The micronucleus test was conducted as described previously (Jia et al. 2016). Briefly, cells of V79Mz and V79-derived lines were inoculated at the density of 3 × 105 per 12.5-cm2 flask. At 24 h, cells were treated with the test compound according to the guidelines for micronucleus tests in vitro, which require a treatment regime of a short exposure (≤0.5 cycling time) and a long recovery period (≥1.5 cycling time). At least in case of negative or equivocal results, an additional experiment with a long exposure/no or short recovery regime is further required (Kirsch-Volders et al. 2003). Our tests in V79-Mz and V79-derived cell lines followed two different regimes, i.e., 6 h/18 h (typical

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for detecting clastogens), and 18 h/6 h (with extended exposure, potentially favourable for reactive metabolites to build up). At the end of the recovery, cells were harvested by trypsinization, and slides were prepared for scoring micronuclei. In V79-Mz cells, EMS (5 mM, a direct mutagen)

served as a positive control. BP (10 μM), a CYP1A1- and CYP1B1-dependent promutagen

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(Schmalix et al. 1993; Crespi et al. 1997; Glatt et al. 2002), was the positive control in both V79hCYP1A1 and V79-hCYP1B1 cells. AFB1 (0.2 μM), which is activated by CYP1A2 (Crespi et al.

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1991), was the positive control in V79-hCYP1A2 cells. Two or three cultures were set up for each treatment, in accordance to the most recent OECD guideline for the in vitro mammalian cell

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micronucleus test, which proposed that “either replicate or single treated cultures may be used at each concentration tested” (OECD 2016). Micronucleated cells in 2000 qualified, randomly

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encountered cells from each culture were scored microscopically by an experienced experimenter. In C3A cells, the micronucleus tests were conducted under the 18 h/54 h and 54 h/18 h regimes, as adapted from the regimes of V79-derived cells to C3A cells in accordance to the 3-fold extended

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doubling time (36 h). To determine the contribution of CYP1B1 to an effect, cells were co-exposed to TMS (30 nM), from 2 h prior to the addition of the test compound to the end of cell recovery

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period. TMS is a potent and selective inhibitor of human CYP1B1 (IC50 6 nM), compared to the values for CYP1A1 (300 nM) and 1A2 (3 μM) (Chun et al. 2001).

2.5 Western blot analyses of CYPs and γ-H2AX C3A cells were treated with PCBs for 54 h (for analysis of each CYP protein) or 12 h (for detection of γ-H2AX), then lysed with cell lysis buffer (Cell Signaling Technology, Danver, MA) supplemented with a protease and phosphatase inhibitor cocktail tablet (Roche Diagnostics, 7

Indianapolis, IN) following manufacturer's protocol, three separate cultures for each treatment. An aliquot of 30 μg of cell lysate protein was loaded onto 10% denaturing SDS-polyacrylamide gel (CoWin Biosciences, Taizhou, China), and subjected to electrophoresis and Western blot assay of each protein (CYP1A1, 1A2, 1B1, γ-H2AX, or GAPDH as a reference), according to the procedure described in our recent report (Chen et al. 2019) with minor modification. Antisera raised in rabbits against human CYP1A1, CYP1A2, CYP1B1 or GAPDH, and those raised in mouse against human phosphoS139-H2AX were used as primary antibodies, followed by the treatment with appropriate secondary antibodies. The gray values of protein bands were evaluated using an ImageQuant TL1D

2.6 Immuno-fluorescent detection of CENP-B in micronuclei

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analysis tool (GE Healthcare, Pittsburgh, PA).

To distinguish micronuclei formed through breakage versus loss of chromosomes, staining of

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CENP-B in the micronuclei was performed by using anti-CENP-B antibodies (Song et al. 2013) raised in rabbit, according to an established method (Dorn et al. 2008) with minor modification

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(Chen et al. 2018). Briefly, V79-hCYP1B1 cells were treated with PCB 77 (40 μM), PCB 81 (40 μM), PCB 126 (20 μM), or VCR (3 nM) under the 18 h/6 h regime. The test conditions were

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consistent with those described in the standard micronucleus test. The primary and secondary antibodies were the same as in our recent report as mentioned above.

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The corresponding experiments in C3A cells were performed with a modified treatment regime, i.e., 54 h/18 h, for the exposure to PCB 77 and 81 (each 40 μM) and EMS (1.6 mM).

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2.7 Statistical analysis

The relative OD450 values from a CCK-8 assay (n = 6), the levels of CYP1A1, 1A2, 1B1, and γ-

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H2AX protein (n = 3) were expressed as means and S.D., and the value in each treatment was compared with that in the solvent control group by using one-way ANOVA. The frequency of cells with CENP-B-positive or negative micronuclei (n = 3) was expressed as means and S.D. However, data from the duplicate determinations in the micronucleus assays were expressed as means and half ranges of variation, combined to become quantal data, followed by a χ2 test.

3. Results 8

3.1 Induction of micronuclei by positive control compounds The validity of V79-Mz and V79-derived cells expressing each enzyme was verified in a micronucleus test by using appropriate positive controls. As indicated in Table 1, the direct acting mutagen EMS induced a significant increase in the frequency of micronucleated cells in V79-Mz cells. BP did not induce micronuclei in V79-Mz cells, but caused significant increases in the frequency of micronuclei in V79-hCYP1B1 and V79-hCYP1A1 cells. AFB1 was inactive in V79Mz, however, it greatly elevated the frequency of micronucleated cells in V79-hCYP1A2 cells. As BP is a CYP1A1- and CYP1B1-dependent promutagen, while AFB1 is activated by CYP1A2 for

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mutagenicity, these results provided further evidence for the expression of the relevant enzymes in cell lines V79-hCYP1A1, V79-hCYP1A2 and V79-hCYP1B1, in addition to the protein expression observed when the cell lines were established. The cell viability level (relative to the solvent control)

as determined by the CCK-8 assay with EMS in V79-Mz cells, with BP in V79-hCYP1A1 and V79-

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hCYP1B1 cells, and with AFB1 in V79-hCYP1A2 cells, at the indicated concentration (as used in

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the micronucleus test) was 94.8 ± 5.5, 76.9 ± 1.5, 91.9 ± 2.7, and 84.3 ± 6.4 %, respectively.

3.2 Influence of PCBs on the viability of V79-derived cell lines

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Under the 6 h/18 h regime, PCB 77 at the concentrations ranging from 5 to 80 μM was nearly noncytotoxic in V79-Mz, V79-hCYP1A1, V79-hCYP1B1 and V79-hCYP1A2, and PCB 126 was non-

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cytotoxic in all the above cell lines (Fig. 1, panels A1 and C1). In contrast, PCB 81 demonstrated concentration-dependent reduction of cell viability, with slightly higher potency in V79-hCYP1B1 than the other cell lines (Fig. 1, panel B1).

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Under prolonged exposure, i.e., the 18 h/6 h regime, PCB 77 and 81 decreased the cell viability more potently (Fig. 1, panels A3 and B3) than their effects under the 6 h/18 h regime, while PCB

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126 remained non-cytotoxic in each cell line (Fig. 1, panel C3).

3.3 Induction of micronuclei by PCBs in V79-derived cell lines PCB 77 at the highest concentration (80 μM) elevated the level of micronucleated cells in V79hCYP1B1, while it was inactive in the other V79-derived cell lines (Fig. 1, panel A2). Likewise, PCB 81 induced micronuclei in V79-hCYP1B1 line, with a slightly higher potency than PCB 77, while it was negative in the other cell lines (Fig. 1, panel B2). The third congener tested, PCB 126, 9

did not induce micronuclei in any of the V79-derived cell lines under this regime (Fig. 1, panel C2). Under the 18 h/6 h regime, PCB 77 and 81 were more potent in micronuclei induction in V79hCYP1B1 cells (Fig. 1, panels A4 and B4) as compared with their effects under the 6 h/18 h regime, while PCB 126 remained negative in the micronucleus tests in each cell line. As shown in Fig. 1 (panels A4 and B4), both PCB 77 and PCB 81 induced micronuclei in V79-hCYP1B1 cells, with PCB 81 being more potent. The highest test concentration of PCB 81 was limited to 40 μM since the cell viability at 80 μM was too low, <55%, for valid micronucleus tests. PCB 81 also elevated the frequency of micronucleated cells in V79-hCYP1A2, but only at the highest test concentration

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(40 μM).

3.4 Effects of PCBs on levels of CYP1A1, 1A2 and 1B1 protein, cell survival, and micronuclei formation in C3A cells

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After exposure of C3A cells to PCB 77, 81 and 126 for 54 h, the levels of CYP1A1 and 1A2 protein were significantly increased, while that of CYP1B1 was unchanged (Fig. 2, panels A1 and A2).

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Under the 54 h/18 h regime, PCB 77 and 126 at the concentrations up to 80 μM were noncytotoxic. PCB 81 was mildly cytotoxic, while in the presence of TMS (30 nM) that effect was

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reduced (Fig. 2, panel C1). PCB 77 and 81 induced micronuclei in C3A cells, with elevated frequency of micronucleated cells at the concentration of 40 and 80 μM (p < 0.01); these effects of

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were completely abolished by co-exposure with TMS (Fig. 2, panels B2 and C2). PCB 126 at the indicated concentrations did not induce micronuclei in C3A cells (Fig. 2, panel D2). Under the 18 h/54 h (short exposure/long recovery) regime, each PCB did not change the frequency of

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micronucleated cells in C3A cells (data not shown).

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3.5 Differentiation between CENP-B-positive and -negative micronuclei in cells treated with PCBs

The validity for the anti-CENP-B antibodies raised in rabbit (commercially available), used as an alternative to the traditional CREST serum in the micronucleus test in V79-derived cells for distinguishing chromosomal breakage from whole chromosome loss, has been validated in our recent report (Chen et al. 2018). Exemplary photographs of CENP-B-positive (CENP-B+) and －

negative (CENP-B ) micronuclei are shown in Fig. 3. 10

As indicated in Table 2, VCR significantly elevated the frequency of both CENP-B+ and CENP-B

－

micronuclei in V79-hCYP1B1 cells. However, most micronuclei (73%) were CENP-B+.

PCB 77 and PCB 81 elevated only the frequency of CENP-B

－

micronuclei in V79-hCYP1B1 cells,

suggesting a purely clastogenic effect. A similar result was observed in C3A cells: PCB 77 and 81 only elevated the frequency of CENP-B

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micronuclei, implying typical clastogenic effects of these

two compounds in C3A cells (Table 2).

3.6 γ-H2AX assay in C3A cells and an impact of TMS, a CYP1B1 inhibitor

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As shown in Fig. 4, PCB 77, 81 and 126 elevated the level of γ-H2AX protein in C3A cells (p < 0.01). The positive control, BP, also significantly increased the level of γ-H2AX. However, co-

exposure of C3A cells to TMS (10 nM) completely blocked the effect of each test compound, i.e., γ-H2AX protein was reduced to such a level that no statistical difference from the negative control

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was observed.

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4. Discussion

In the present study, dioxin-like compounds PCB 77 and 81 induced micronuclei in V79-hCYP1B1

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cells under the regime of 6 h/18 h and 18 h/6 h. This effect occurred in the absence of severe cytotoxicity, which would otherwise limit the validity of a micronucleus test (OECD 2016). PCB

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81 also elevated the frequency of micronucleated cells in V79-hCYP1A2 somewhat, otherwise negative results were observed in parental V79-Mz cells and other V79-derived cell lines. The results indicated that human CYP1B1 is specifically capable of activating DL-PCBs to genotoxic

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metabolites. Moreover, it appears that this activation proceeds slowly, as long exposure generally enhanced the genotoxic effect. Our results are in accordance to an early report that PCB 77 was

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clastogenic in cultured human lymphocytes (Sargent et al. 1989), though potential relevance of the effect to metabolic activities was not studied. Our continued study with C3A cells indicates that PCB 77 and 81 induced micronuclei under

the 54 h/18 h regime (with no or mild cytotoxicity), effects being more intensive than those observed in V79-hCYP1B1 cells under the 18 h/6 h regime. Induction of micronuclei in C3A cells by either PCB 77 or PCB 81 was blocked by the selective CYP1B1 inhibitor TMS (30 nM, a concentration 5-fold higher than its IC50 for CYP1B1). This finding is again strong evidence for the idea that the 11

genotoxicity of the PCBs tested in C3A cells is dependent on their metabolism catalyzed specifically by human CYP1B1. As the positive control BP significantly elevated the frequency of micronucleated cells in C3A cells; however, this effect was not clearly changed by co-exposure to TMS. It is known that both CYP1A1 and 1B1 are capable of activating BP (Glatt et al. 2002). Nevertheless, each test PCB induced CYP1A1 and 1A2, rather than CYP1B1. Immuno-fluorescent analysis of CENP-B in the micronuclei induced by PCB 77 and 81 in V79-hCYP1B1 and C3A cells consistently indicated clastogenic potential of the reactive metabolites formed from the PCBs. Moreover, PCB 77, 81 and 126 induced double strand DNA

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breaks, as indicated by the elevation of the level of γ-H2AX, in C3A cells after 12 h of exposure. This effect was again blocked by co-exposure to TMS (10 nM, a concentration nearly 2-fold higher

than its IC50 for human CYP1B1). Here, the elevation of γ-H2AX by BP was also completely inhibited by TMS, probably due to the short-term exposure, which might be insufficient for

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substantial induction of CYP1A1. Regarding the absence of micronuclei induction by PCB 126 in

cell lines even with CYP1B1 activities, maybe PCB 126 is insufficiently clastogenic for observable

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micronuclei induction.

It has been reported that catfish CYP1A enzymes may metabolize PCB 77 to mono- and

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dihydroxylated metabolites (Doi et al. 2006). However, in our study human CYP1A1 and 1A2 did not activate PCB 77 for a genotoxic response. In fact, both PCB 77 and 81 induced micronuclei in

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human CYP1B1-expressing V79-derived cells. Noticeably, major differences in the tissuedistribution and metabolism of a PCB mixture (Kanechlor 500) between animals of different species, i.e., the rat, hamster and guinea pig, have been observed. For example, 4-OH-PCB 107, 3-OH-PCB

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99 and dihydroxylated metabolites were identified as the major metabolites in rats, guinea pigs and hamsters, respectively, indicating varied activities of transporters or/and metabolic enzymes of

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different species for the same PCB mixture (Haraguchi, et al. 2005). Particularly for PCB 126, major differences in its metabolism by the rat and human CYP1A1 have been identified: the rat enzyme was active in hydroxylating PCB 126, while the human enzyme was inactive (Yamazaki et al. 2011), to which the absence of micronuclei induction by PCB 126 in V79-hCYP1A1 cells is in accordance. More importantly, our results suggest that PCB 77 and 81 are clastogenic in mammalian cells and human CYP1B1 is responsible for the activation of these PCBs. In mammals CYP1B1 is usually expressed in some extrahepatic tissues, such as the kidney, 12

colon, intestines, uterus, heart and brain (Shimada et al. 1996; Ding and Kaminsky 2003). While CYP1B1 is not constitutively expressed at detectable levels in other healthy tissues, it may be present at high levels in corresponding malignant tissues (Murray et al. 1997). In mice, CYP1B1 expression (mRNA and protein) can be largely induced by TCDD and PCB 77 via activation of AHR (Shimada et al. 2003). However, whether CYP1B1 specifically expressed in malignant cells can be induced through activation of AHR has not been reported. Our results suggest that expression of CYP1B1 in C3A hepatoma cells might be unresponsive, or less responsive than the expression of CYP1A1 and 1A2, to AHR activators.

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We have recently observed micronuclei formation and gene mutations in V79-hCYP1B1 cells by PCB 22 and 52, an ortho-chlorinated tri- and tetrachlorobiphenyl, respectively, though with far lower potency than in human CYP2E1-expressing cells (Liu et al. 2017). It seems that human

CYP1B1 may activate a series of lower chlorinated PCBs, particularly important for DL-PCBs,

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which cannot be activated by human CYP2E1. Further studies to identify the reactive metabolites

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formed with the relevant CYP enzymes may help to specify the genotoxicity of DL-PCBs.

Conclusion

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The present study demonstrates that human CYP1B1 is able to activate some DL-PCBs to clastogenic metabolites, as evidenced by the formation of centromere-free micronuclei and double

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strand DNA breaks in mammalian cells, though with lower potency/efficacy as compared with human CYP2E1-activated genotoxicity of NDL-PCBs.

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Conflict of interest

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The authors declare that they have no conflict of interest.

Acknowledgements This work was supported by a grant (Y. L., 21577054) from the National Science Foundation of China.

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Legends for Figures

Fig. 1 Cytotoxicity and micronuclei induction by PCB 77, 81 and 126 under varying regimes in V79-Mz

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(○), V79-hCYP1A1 (▲), V79-hCYP1A2 (■), and V79-hCYP1B1 cells (◇)

Cells were exposed to each PCB for 6 h, followed by 18 h of recovery (A1-C2), or under a different, i.e., 18 h/6 h, regime (A3-C4). Data are means ± S.D. (n = 6) for the cytotoxicity (CCK-

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8) assays, and means ± half range of variation for the micronucleus test (n = 2). The duplicate data were combined to form quantal data valid for a χ2 analysis; *p< 0.05, **p< 0.01, compared with the

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solvent control, with the data from CCK-8 assays by ANOVA and those from micronucleus tests by χ2 analysis. All experiments were repeated, and similar results were obtained. Since PCB 81 at the highest concentration, 80 µM, decreased the cell viability of some cell lines to such levels (< 55%) that the micronucleus test would be invalid, micronucleus tests with congener were only conducted with concentrations ≤ 40 µM.

19

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Fig. 2 Effects of PCB 77, 81 and 126 on the levels of CYP1A1, 1A2 and 1B1 protein, cell viability,

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and micronuclei formation in C3A cells, and the influence of the selective CYP1B1 inhibitor TMS C3A cells were treated with PCB 77, 81 or 126 for 54 h, followed by Western blot analysis of

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the levels of CYP1A1, 1A2 and 1B1 (A1 and A2) (the grey values of target bands being normalized with respect to that of GAPDH), or – after 18 h of incubation for recovery – determination of cell

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viability and the frequency of micronucleated cells, with (solid symbols) or without (empty symbols) co-exposure to TMS (30 nM) from 2 h prior to the addition of the test compound to the end of the culture (B1, B2, C1, C2, D1, and D2). As the positive control, BP (1 μM) alone elevated the frequency of micronucleated cells from 10.8 to 24.8 per 1000 cells (χ2 test, p < 0.01); in the presence of TMS (30 nM), the values in the control and BP group were 10.0 and 22.5 per 1000 cells, respectively (χ2 test, p < 0.01). * p < 0.05, ** p < 0.01, compared with the negative control, by ANOVA (panels A2, B1, C1, and D1) or χ2 test (panels B2, C2 and D2);

##

p < 0.01, comparison

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was made between a treatment with and without TMS (panels B2, C2 and D2).

Fig. 3 Domination of centromere protein B-negative micronuclei induced by PCB 77 in V79hCYP1B1 and C3A cells, as determined by an immunofluorescent assay

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V79-hCYP1B1 cells were exposed to PCB 77 (40 μM) and VCR (3 nM) under the 18 h/6 h

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regime (A); C3A cells were exposed to PCB 77 (40 μM) and EMS (1.6 mM) under the 54 h/18 h regime (B). Then, cells were immune-stained for CENP-B (green) and β-tubulin (red). DNA was

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micronuclei, respectively.

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stained with DAPI (blue). The arrows and arrow heads mark CENP-B-positive and -negative

Fig. 4 Influence of PCBs on the level of γ-H2AX protein (a measure of DNA damage) in C3A cells Cells were exposed to PCB 77, 81, 126, each at 40 μM, or BP at 1 μM, for 12 h, with or without co-exposure to TMS (10 nM, added 2 h prior to the addition of each test compound), then cells of each treatment were examined for the expression of γ-H2AX by Western blot assays (values being normalized to that of GAPDH). Data are means ± S.D. (n = 3); ** p < 0.01, compared with the 21

solvent control by ANOVA. ## p < 0.01, comparison was made between a treatment with and without

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TMS.

22

Table 1 Influence of positive control compounds (EMS, BP and AFB1) on the cell viability and micronuclei formation in V79-Mz and V79-derived cells expressing various CYP1 enzymes Cell viability, % of control V79Mz

EMS

5000

BP

V79-

V79-

hCYP1A1

hCYP1A2

hCYP1B1

10

86

87 ±

0.2

±

7**

7** AFB1

V79-

86 ± 4**

81

±

9** 77

±

2**

94 ±

Micronucleated cells / 1000 cells

5

±

6*

92

±

±

V79-

V79-

hCYP1A

hCYP1A

hCYP1B

1

2

1

56 ± 4**

6 ± 2

94 ± 9

4**

V79-

42 ± 1**

3** 81

Mz

±

8** 88

101 ± 3

88

V79-

1.3** 14 ±

2**

6 ±

1

67 ±

74 ± 4**

4 ±

20 ±

ro of

Chemical, μM

0.5

5 ±

0.3

2**

61 ±

1**

2**

-p

Cells were treated with the chemical for 6 h, followed by a recovery period of 18 h. The relative cell

survival level (measured by a CCK-8 assay, representing cytotoxicity) was determined from the optical

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density at 450 nm (percent of control) in a treatment as divided by a value in the control, and expressed as means ± S.D. (n = 6). Data of the micronucleus test are means and half range of variation (n = 2). **p

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< 0.01, compared with the control (CCK-8 assay analyzed by ANOVA, those of micronucleus test by χ2

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test).

23

35 ±

Table 2 Immuno-fluorescent staining of CENP-B in the micronuclei induced by PCB 77, PCB 81 and positive control compounds in V79-hCYP1B1 and C3A cells Cell line / chemical

Concentration

Micronuclei / 1000 cells CENP-B

＋

CENP-B

－

＋

CENP-B , %

V79-hCYP1B1 4.0 ± 1.0

5.3 ± 0.3

43

PCB 77

40 μM

8.3 ± 0.3

14.8 ± 2.4**

36

PCB 81

40 μM

6.8 ± 2.0

14.5 ± 1.8**

32

VCR

3 nM

49.7 ± 3.8**

18.0 ± 3.8**

73

—

2.2 ± 1.0

PCB 77

40 μM

3.7 ± 1.3

PCB 81

40 μM

5.7 ± 1.8

EMS

1.6 mM

3.8 ± 0.3

None

15

21.3 ± 1.3**

15

22.5 ± 2.2**

20

30.2 ± 2.3**

11

V79-hCYP1B1 cells were treated with each chemical for 18 h, followed by 6 h of recovery; C3A cells

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a

12.7 ± 0.8

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C3A

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—

None

were treated for 54 h, followed by 18 h of recovery. Immuno-fluorescent staining of CENP-B in the

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micronuclei was employed to discriminate clastogenic from aneugenic effects. Data are means and S.D. (n = 3), which were combined to become quantal data, then statistically analyzed by a χ2 test. ** p < 0.01,

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compared with the negative (no chemical) control.

24