Chem. Res. Toxicol. 2008, 21, 503–512
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Toxic Effects of Methylated Benz[a]anthracenes in Liver Cells Sonˇa Marvanová,† Jan Vondrácˇek,†,‡ Katerˇina Peˇncˇíková,† Lenka Trilecová,† Pavel Krcˇmárˇ,† Jan Topinka,§ Zuzana Nováková,§ Alena Milcová,§ and Miroslav Machala*,† Department of Chemistry and Toxicology, Veterinary Research Institute, HudcoVa 70, 62100 Brno, Department of Cytokinetics, Institute of Biophysics, AS CR, KráloVopolská 135, 61265 Brno, and Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, AS CR, V.V.i., Vídenˇská 1083, 14220 Prague, Czech Republic ReceiVed August 28, 2007
Monomethylated benz[a]anthracenes (MeBaAs) are an important group of methylated derivatives of polycyclic aromatic hydrocarbons (PAHs). Although the methyl substitution reportedly affects their mutagenicity and tumor-initiating activity, little is known about the impact of methylation on the effects associated with activation of the aryl hydrocarbon receptor (AhR)-dependent gene expression and/or toxic events associated with tumor promotion. In the present study, we studied the effects of a series of MeBaAs on the above-mentioned end points in rat liver cell lines and compared them with the effects of benz[a]anthracene (BaA) and the potent carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Methyl substitution enhanced the AhR-mediated activity of BaA derivatives determined in a reporter gene assay, as the induction equivalency factors (IEFs) of all MeBaAs were higher than that of BaA. IEFs of 6-MeBaA and 9-MeBaA, two of the most potent MeBaAs, were more than two orders of magnitude higher than the IEF of BaA. Correspondingly, all MeBaAs induced higher levels of cytochrome P450 1A1 mRNA. Both BaA and MeBaAs had similar effects on the expression of cytochrome P450 1B1 or aldo-keto reductase 1C9 in rat liver epithelial WB-F344 cells. In contrast to genotoxic DMBA, MeBaAs induced low DNA adduct formation. Only 10-MeBaA induced apoptosis and accumulation of phosphorylated p53, which could be associated with the induction of oxidative stress, similar to DMBA. With the exception of 10-MeBaA, all MeBaAs induced cell proliferation in contact-inhibited WB-F344 cells, which corresponded with their ability to activate AhR. 1-, 2-, 8-, 10-, 11-, and 12-MeBaA inhibited gap junctional intercellular communication (GJIC) in WB-F344 cells. This mode of action, like disruption of cell proliferation control, might contribute to tumor promotion. Taken together, these data showed that the methyl substitution significantly influences those effects of MeBaAs associated with AhR activation or GJIC inhibition. Introduction 1
Monomethylated derivatives of benz[a]anthracene (MeBaAs) are an important group of methylated polyaromatic hydrocarbons (PAHs) occurring in the environment. They have been identified in various environmental compartments, such as in river sediments, where they can be found at varying concentrations of up to hundreds of nanograms per gram of sediment dry weight (1, 2). They are also significant cigarette smoke constituents (3). The parental compound of MeBaAs, benz[a]anthracene (BaA), is classified as a probable human carcinogen (4). Importantly, its dimethylated derivative 7,12-dimethylbenz[a]anthracene (DMBA) is one of the most potent mutagenic * To whom correspondence should be addressed. Tel: +420-533331813. Fax: +420-541211229. E-mail:
[email protected]. † Veterinary Research Institute. ‡ Institute of Biophysics. § Institute of Experimental Medicine. 1 Abbreviations: AhR, aryl hydrocarbon receptor; AKR, aldo-keto reductase; BaA, benz[a]anthracene; BPDE, benz[a]pyrene dihydrodiol epoxide; DAPI, 4′-6-diamidine-2-phenyl indole; DBalP, dibenzo[a,l]pyrene; DCF, dichlorofluorescein; DCFH-DA, dichlorofluorescein diacetate; DMBA, 7,12-dimethylbenz[a]anthracene; DMSO, dimethylsulfoxide; DR-CALUX, dioxin responsive chemical-activated luciferase expression; DRE, dioxin responsive element; GJIC, gap junctional intercellular communication; IEF, induction equivalency factor; MeBaA, methylated benz[a]anthracene; PAH, polyaromatic hydrocarbon; REP, relative potency; ROS, reactive oxygen species; RT-PCR, reverse trancription polymerase chain reaction; TBS, Trisbuffered saline; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.
and carcinogenic PAHs (5), which can be used as an experimental inducer of tumors in both skin and mammary tissues (6). DMBA is a highly potent experimental carcinogen, thus suggesting that methyl substitution can significantly modify the carcinogenicity of BaA. The position of the methyl group has been reported to strongly affect metabolic activation, mutagenicity, tumor-initiating ability, and carcinogenicity of MeBaAs, in both bacterial and rodent skin experimental models (7–11). In these studies, 7-, 6-, 8-, and 12-MeBaA have been identified as the most potent mutagenic and tumor-initiating derivatives of BaA. Major attention has been paid to 7-MeBaA, which shares one position of methyl substitution with DMBA. 7-MeBaA has been found to induce DNA adduct formation in adult mouse epidermis (12). Several early studies have indicated that MeBaAs are complete carcinogens in newborn or adult mice and rats (7, 9, 13). However, in contrast to tumor-initiating events, very little interest has been paid so far to the effects of methyl substitution on the tumor-promoting ability of BaA derivatives and related events. It has been hypothesized that at least some PAHs may have tumor-promoting, nongenotoxic effects, which could be induced by both parental compounds and their metabolites (14). One of the mechanisms possibly participating in tumor promotion induced by PAHs is the activation of the aryl hydrocarbon receptor (AhR) (15). A number of PAHs are AhR agonists (16–18), and this ligand-activated transcription factor regulates
10.1021/tx700305x CCC: $40.75 2008 American Chemical Society Published on Web 01/19/2008
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Figure 1. Molecular structure of benz[a]anthracene.
the expression of cytochromes P4501A1, 1A2, and P4501B1, metabolizing PAHs to reactive dihydrodiol epoxides, which are able to form adducts with DNA (18–20). Apart from being involved in the metabolization of PAHs to their ultimate genotoxic metabolites, AhR might also participate in other toxic effects of PAHs, such as deregulation of cell cycle control (21–24). Other mechanisms, which have been linked to tumorpromoting effects of PAHs, include the inhibition of gap junctional intercellular communication (GJIC) (25–27) and perturbation of cell signaling (28–30). Our previous studies have suggested that the ultimate impact of PAHs on cell fate may reflect both genotoxic and nongenotoxic events, leading to cytotoxicity and/or induction of apoptosis on one side and to an increased cell proliferation and/or disruption of cell-to-cell communication on the other side (21, 31). The BaA methylation might thus differentially affect this balance of genotoxic and nongenotoxic events. Given that the information about the effects of methyl substitution on toxic events associated with tumor promotion is very limited, we used an established model of rat liver progenitor cells, the rat liver epithelial WB-F344 cell line, to study both the genotoxic effects of MeBaAs (the formation of DNA adducts, phosphorylation of p53 tumor suppressor, and induction of apoptosis) and the toxic modes of action associated with tumor promotion (AhR activation, disruption of cell cycle control, and inhibition of GJIC), as well as additional mechanisms, which might contribute to both types of carcinogenic effects s induction of enzymes involved in metabolic activation of MeBaAs and generation of oxidative stress. Our data suggested that genotoxic effects play only a minor role in the toxicity of monomethylated BaAs to liver cells and that methyl substitution had a significant impact on both AhR-dependent and -independent nongenotoxic effects of MeBaAs.
Experimental Procedures Chemicals. Dibenzo[a,l]pyrene (DBalP) and DMBA were purchased from Promochem GmBH (Wesel, Germany); 2-, 11-, and 12-MeBaA were from Midwest Research Institute (Kansas City, MO); 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, and 10-MeBaA (Figure 11) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (Prague, Czech Republic); benz[a]anthracene was from Supelco (Bellefonte, PA). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was from Cambridge Isotope Laboratories (Andover, MA). Spleen phosphodiesterase was purchased from ICN Biomedicals, Inc. (Irvine, CA); 4′-6-diamidine-2-phenyl indole (DAPI), ribonuclease A and T1, proteinase K, micrococcal nuclease, nuclease P1, and protein assay kit were from Sigma-Aldrich; polyethylene-imine cellulose TLC plates (0.1 mm) were from Macherey-Nagel (Düren, Germany); T4 polynucleotide kinase was from USB (Cleveland, OH); and γ-32P-ATP (3000 Ci/mmol, 10 µCi/µL) was from GE Healthcare (Little Chalfont, United Kingdom); propidium iodide was from AppliChem GmbH (Darmstadt, Germany). Cells. WB-F344 rat liver epithelial cells (kindly provided by James E. Trosko, MSU, East Lansing) were grown in Dulbecco’s modified Eagle’s medium (Invitrogene, Carlsbad, CA) supplemented with 25 mM sodium bicarbonate, 10 mM HEPES, and 5% heatinactivated fetal bovine serum (PAA, Pasching, Austria). Only the cells at passages 15-24 were used throughout the study. The rat
MarVanoVá et al. hepatoma H4IIEGud.Luc1 cells, licensed from BioDetection Systems (Amsterdam, The Netherlands), were grown in Dulbecco’s modified Eagle’s medium (Invitrogene), supplemented with 10% of heat-inactivated fetal bovine serum. Cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C. Cells were routinely maintained in 75 cm2 flasks and subcultured twice a week. Detection of AhR-Mediated Activity. The rat hepatoma H4IIEGud.Luc1.1 cell line, stably transfected with a luciferase reporter gene under the control of dioxin responsive elements, was used to detect the AhR-mediated activity in the dioxin responsive chemical-activated luciferase expression (DR-CALUX) assay (32). The assays were performed in 96 well cell culture plates. The cells were grown for 24 h to 90–100% confluency and exposed to the test or reference compounds (TCDD) dissolved in DMSO (maximum concentration 0.4%, v/v) for 24 h. The medium was removed, cells were washed with PBS, and the luciferase was extracted with the low salt lysis buffer (10 mM Tris, 2 mM DTT, 2 mM 1,2diamin cyclic hexane-N,N,N′,N′-tetraacetic acid, pH 7.8). The plates were frozen at -80 °C, and the luciferase expression was then measured on a microplate luminometer using the Luciferase Assay Kit (BioThema, Handen, Sweden). Detection of GJIC. The modified scrape loading/dye transfer assay was performed as described previously (25). The confluent WB-F344 cells, grown in 24 well plates, were exposed to test compounds (up to 50 µM concentration), 12-O-tetradecanoylphorbol-13-acetate (TPA) (20 nM, positive control), or DMSO (negative control) for 30 min. After the exposure, the cells were washed twice with 0.5 mL of PBS; fluorescent dye was added (Lucifer Yellow 0.05% w/v in PBS), and the cells were scraped using a surgical blade. After 4 min, the cells were washed twice by 0.5 mL of PBS and fixed with 4% formaldehyde (v/v), and the migration of the dye was evaluated using an epifluorescence microscope. The distance of the dye migration from a scrape line was measured at six randomly chosen spots per scrape, using Lucia image analysis software (Laboratory Imaging, Prague, Czech Republic). Three independent experiments were carried out in duplicate, and at least three scrapes per well were evaluated. Real-Time Reverse Trancription Polymerase Chain Reaction (RT-PCR). Total RNA was isolated from cells using the NucleoSpin RNA II kit (Macherey-Nagel). The amplifications of the samples were carried out using QuantiTect Probe RT-PCR kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s specifications. All probes were labeled with the fluorescent reporter dye 6-carboxyfluorescein (FAM) on the 5′-end and with the Black Hole 1 (BH 1) fluorescent quencher dye on the 3′-end. The sequences of primers and probes have been published previously (33). The amplifications were run on the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) using the conditions described previously (33). Gene expression for each sample was expressed in terms of the threshold cycle (Ct), normalized to housekeeping gene porphobilinogen deaminase (∆Ct). ∆Ct values were then compared between control samples (0.1% DMSO) and samples treated with PAHs to calculate ∆∆Ct (∆Ct[control] ∆Ct[xPAH]). The final comparison of transcript ratios between samples is given as 2–∆∆Ct (34). Assessment of Cell Proliferation and Cell Cycle Distribution. The proliferative effects of BaA, 12 MeBaAs, and DMBA on confluent WB-F344 cells were determined as described previously (23). The confluent cells were exposed to test compounds dissolved in DMSO (maximal concentration, 0.1% v/v) for 72 h. The medium with test compounds was changed daily. Following the treatment, the medium was removed, and cells were harvested by trypsinization and counted with a Coulter Counter (model ZM, Coulter Electronics, Luton, United Kingdom). Cells were then washed with PBS and fixed in 70% ethanol at 4 °C overnight. Fixed cells were washed once with PBS and stained with propidium iodide as described previously (33). Cells were then analyzed on FACSCalibur, using 488 nm (15 mW) air-cooled argon-ion laser for propidium iodide excitation and CellQuest software ver. 5.1.1 for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15000 events
Toxic Effects of Methylated Benz[a]anthracenes was collected per sample. Data were analyzed using ModFit LT version 2.0 software (Verity Software House, Topsham, ME). Detection of PAH-DNA Adducts. WB-F344 cells in a nearly confluent state (seeded at an initial density 23000 cells/cm2 in 60 cm2 plates, grown for 48 h) were exposed for 24 h to test compounds and DMSO as a solvent control (0.1%). After exposure, cells were washed with cold PBS, scraped into the Eppendorf tubes, and centrifuged, and the cell pellets were stored at -80 °C. The cell pellets were homogenized in a solution of 10 mM Tris-HCl, 100 mM EDTA, and 0.5% SDS, pH 8.0. DNA was isolated using RNase A and T1 and proteinase K treatment followed by phenol/chloroform/isoamylalcohol procedure as previously described (35). The DNA concentration was estimated spectrophotometrically by measuring the UV absorbance at 260 nm. DNA samples were kept at -80 °C until analysis. 32Ppostlabeling analysis was performed as previously described (36, 37). Briefly, DNA samples (6 µg) were digested by a mixture of micrococcal endonuclease and spleen phosphodiesterase for 4 h at 37 °C. Nuclease P1 was used for adduct enrichment. The labeled DNA adducts were resolved by two-directional thin layer chromatography on 10 cm × 10 cm PEI-cellulose plates. Solvent systems used for TLC were as follows: D-1, 1 M sodium phosphate, pH 6.8; D-2, 3.8 M lithium formate and 8.5 M urea, pH 3.5; and D-3, 0.8 M lithium chloride, 0.5 M Tris, and 8.5 M urea, pH 8.0. Autoradiography was carried out at -80 °C for 1-24 h. The radioactivity of distinct adduct spots was measured by liquid scintillation counting. To determine the exact amount of DNA in each sample, aliquots of DNA enzymatic digest (1 µg of DNA hydrolysate) were analyzed for nucleotide content by reverse-phase HPLC with UV detection, which simultaneously allowed us to check the DNA purity. DNA adduct levels were expressed as adducts per 108 nucleotides. A benzo[a]pyrene dihydrodiol epoxide (BPDE)-DNA adduct standard was run in triplicate in each postlabeling experiment to check for interassay variability and to normalize the calculated DNA adduct levels. Detection of Phosphorylated Form of p53 Protein. WB-F344 cells in a confluent state (seeded at an initial density 30000 cells/ cm2, grown for 72 h) were exposed for 24 h to test compounds and maximum 0.5% DMSO as the solvent control. DBalP was used as a positive control. After the exposure, cells were harvested into the lysis buffer (1% SDS, 10% glycerol, 100 mM Tris, and protease inhibitors) and the lyzates were sonicated. Protein concentrations were determined using bicinchonic acid and copper sulfate (SigmaAldrich). For Western blot analyses, equal amounts of total protein lyzates were separated by SDS-polyacrylamide gel electrophoresis on 10% gel and electrotransferred onto a PVDF membrane Hybond-P (GE Healthcare). Prestained molecular weight markers (Bio-Rad, Hercules, CA) were run in parallel. The blotted membranes were blocked overnight at 4 °C and incubated with primary antibody against p53 phosphorylated on Ser15 for 2 h at room temperature (Cell Signaling Technology, Beverly, MA), diluted in 2.5% nonfat milk in Tris-buffered saline (TBS) with 0.1% Tween 20. After the membranes were washed in TBS with 0.1% Tween 20, peroxidase-conjugated swine antirabbit immunoglobulin antisera (Sevapharma, Prague, Czech Republic) were used as a secondary antibody. Expression of β-actin was used to verify equal loading; monoclonal anti-β-actin antibody, clone AC-15 (Sigma-Aldrich), was diluted in 2.5% milk in TBS and incubated for 2 h at room temperature; peroxidase-conjugated antimouse antibody (SigmaAldrich) was used as a secondary antibody. To visualize peroxidase activity, ECL Plus reagents (GE Healthcare) were used according to the manufacturer’s instructions. Detection of Cell Death. Confluent WB-F344 cells were exposed to the test compounds for 48 h including change of the fresh medium and the compound after 24 h. Early stages of apoptosis were characterized by translocation of phosphatidylserine from the inner part of the plasma membrane to the outer layer. The presence of phosphatidylserine at the cell surface was determined by staining with Annexin-V-Fluos (Roche Diagnostics) in combination with propidium iodide (40 µg/mL), to distinguish the cells with permeabilized and intact plasma membranes. Cells were harvested and
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 505 stained according the manufacturer’s protocol and analyzed by FACSCalibur with CellQuest software (Becton Dickinson). For DAPI staining, cells fixed in 70% ethanol were incubated with 1 µg/mL DAPI (final concentration) for 5 min at room temperature. After incubation, the cells were centrifuged and mixed with 10-20 µL of MOWIOL solution (10% MOWIOL 4-88 was prepared in 25% glycerol, 100 mM Tris-HCl, pH 8.5) and mounted for observation under a fluorescence microscope. A minimum of 300 nuclei were counted per sample. Detection of Reactive Oxygen Species (ROS). Confluent WBF344 cells were exposed to the test compounds for 24 h. Hydrogen peroxide (exposure 5 min) was used as a positive control. After the exposure, the cells were twice washed by PBS, trypsinised, centrifuged, and resuspended with Hank’s balanced salt solution (PANBioTech GmbH, Aidenbach, Germany) with 5% heatinactivated fetal bovine serum. The cell suspension was incubated for 15 min with the fluorescent probe dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich); the final concentration was 20 µM. The cells were washed once again, centrifuged, and cooled on ice (except hydrogen peroxide-exposed cells). The fluorescence of dichlorofluorescein (DCF) was analyzed by FACSCalibur with CellQuest software (Becton Dickinson) (38). Statistical Analysis. All experiments were performed independently at least three times, and the data were quantitatively expressed as means ( SD and analyzed by t test and ANOVA followed by Tukey’s range test. When the variances were not homogeneous, a nonparametric Mann–Whitney U test was used. A p value of less than 0.05 was considered significant.
Results Effects of MeBaAs on AhR Activation and Inhibition of GJIC. In our previous work, we determined induction equivalency factors (IEFs) of a large array of PAHs to activate AhR, using a single reporter gene assay using the H4IIEGud.Luc cell line, which is stably transfected with luciferase reporter gene under the control of dioxin responsive elements (DREs) (16, 39). These included both BaA and DMBA, which both had similar IEFs of 7.04 × 10-6 and 5.41 × 10-6, respectively (IEFs were derived from EC50 values of PAHs and the reference compound TCDD after 24 h of exposure). To determine whether a single methyl substitution may have an impact on AhR activation by MeBaAs, we used the same experimental settings to determine their IEF values. The results are summarized in Table 1. All 12 monomethylated BaA derivatives activated AhR, with EC50 values ranging from 2 to 229 nM. It should be stressed that these values were in each case lower than the EC50 of parental compound. The most potent AhR agonists were 6- and 9-MeBaA. Their IEFs were almost three orders of magnitude higher than those previously reported for BaA and DMBA. With the exception of 5-MeBaA, all MeBaAs with methyl groups at positions 4-9, including the K region, were the most potent AhR ligands. In contrast, both MeBaAs with methyl groups positioned within the bay region, 1- and 12-MeBaA, were the weakest AhR agonists among MeBaAs. It has been suggested that inhibition of GJIC is closely correlated with tumor-promoting activity, therefore being a suitable parameter reflecting possible promoting activity of studied chemicals (40). The closure of gap junctions by tumor-promoting chemicals may lead to a release of the cells, including genotoxically damaged cells, from the control of neighboring cells and consequently to the disruption of homeostasis and cell-to-cell communication (26). The rat liver epithelial WB-F344 cell line is an established model for the detection of effects of tumor-promoting agents on GJIC, where gap junctions are formed almost exclusively by
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Table 1. Values of EC50 and Relative Potencies of MeBaAs To Activate AhR (DR-CALUX Assay; Exposure 24 h) and To Inhibit GJIC in WB-F344 Cells (Exposure 30 min)a DR-CALUX 1-MeBaA 2-MeBaA 3-MeBaA 4-MeBaA 5-MeBaA 6-MeBaA 7-MeBaA 8-MeBaA 9-MeBaA 10-MeBaA 11-MeBaA 12-MeBaA BaA DMBA
inhibition of GJIC
EC50 (nM)
IEF (TCDD, 24 h)
IC50 (µM)
229 87 72 27 101 7 25 37 2 117 116 215 1551 2026
4.8 × 10-5 1.3 × 10-4 1.5 × 10-4 4.1 × 10-4 1.1 × 10-4 1.7 × 10-3 4.4 × 10-4 3.0 × 10-4 4.6 × 10-3 9.4 × 10-5 9.5 × 10-5 5.1 × 10-5 7.0 × 10-6 5.4 × 10-6
10 15 NIb NI NI NI NI 13 NI 25 14 10 NI 21
a The numbers represent induction equivalency factors (IEFs) calculated as the ratio between the 50% effective concentration (EC50) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the concentration of appropriate MeBaA inducing the 50% of maximum TCDD-induced luciferase activity. All experiments were performed independently three times in triplicate. b NI, not inhibiting GJIC up to 50 µM. All experiments were performed independently three times in triplicate.
connexin 43 protein (41). A number of PAHs have been shown to inhibit GJIC in this cell model. Importantly, methyl substitution might significantly affect the inhibitory activity of MeBaAs against GJIC, as DMBA has been found to be an inhibitor of GJIC, while BaA has not been able to fully suppress GJIC (25). Therefore, we next examined the effects of MeBaAs on GJIC, using a standardized scrape loading/ dye transfer assay. We found that 1-, 2-, 10-, 11-, and 12MeBaA, as well as 8-MeBaA, inhibited GJIC with similar IC50 values (Table 1), while other MeBaAs caused no inhibition of GJIC up to the 50 µM concentration. Interestingly, with the exception of 8-MeBaA, we observed an inverse relationship between AhR activation and GJIC inhibition, as the compounds inhibiting GJIC were less efficient AhR inducers. AhR-Dependent Activation of Xenobiotic-Metabolizing Enzymes, Induction of Cell Proliferation, and Cell Cycle Perturbations. Our previous studies have suggested that activation of AhR by PAHs might play a dual role in their effects on WB-F344 cells. Activated AhR may both induce expression of enzymes involved in metabolic activation and detoxification of PAHs and stimulate cell proliferation in contact-inhibited cells, that is, disrupt the cell cycle control (21, 23). As we observed differences in effects of MeBaAs on AhR activity, we next examined the ability of MeBaAs (i) to induce expression of P450 1A1, P450 1B1, and aldo-keto reductase 1C9 (AKR1C9); (ii) to change cell cycle distribution of contact-inhibited WBF344 cells; and (iii) to stimulate cell proliferation of contactinhibited WB-F344 cells. First, we employed real-time RT-PCR to determine the effects of MeBaAs, BaA, and DMBA on the expression of enzymes, which participate in the formation of either dihydrodiol epoxide metabolites forming stable DNA adducts or in the production of PAH quinones leading to oxidative stress and/or DNA damage (42, 43). As summarized in Figure 22, all MeBaAs were able to elicit maximal induction of P450 1A1 and P450 1B1 mRNAs, as well as to significantly increase AKR1C9 mRNA, when applied at a 1 µM concentration. MeBaAs induced higher levels of P450 1A1 mRNA than BaA, similar to their effects on the DRE-controlled luciferase reporter in H4IIE.Gud.Luc cells. The AhR-dependent gene expression in WB-F344 cells was observed from low nanomolar
Figure 2. Induction of P450 1A1, P450 1B1, and AKR1C9 mRNA determined by real-time PCR following 24 h of treatment with MeBaAs or BaA (1 µM), DMBA (100 nM), or with a prototypical AhR ligand, TCDD (1 nM). Data are representative for three independent experiments performed in duplicate (means ( SD).
concentrations of MeBaAs (data not shown), thus confirming the results of the DR-CALUX assay. Our previous studies suggest that a moderately increased percentage of S phase cells (5-10% increase) in contactinhibited WB-F344 cells is indicative of the proliferative effects of nongenotoxic AhR ligands, whereas strong genotoxins induce massive accumulation of cells in the S phase (21, 23, 39). In the present study, we observed a modest accumulation of cells in the S phase of the cell cycle after 72 h of exposure to MeBaAs at a micromolar range concentration similar to BaA with one notable exception. As outlined in Figure 3, 10-MeBaA induced a higher accumulation of cells in the S phase (>20% cells at 1 µM) than any other MeBaA. In Figure 3, the effects of 10MeBaA are compared with those of 7- and 12-MeBaA, which were selected as representative MeBaAs, based on their methyl groups being present in one of the positions substituted in DMBA and with DMBA itself. DMBA induced a much higher accumulation of cells in the S phase than any MeBaA, apparently due to its known genotoxicity. These results suggested that only 10-MeBaA might have genotoxic effects in WB-F344 cells. This was partially confirmed, when we found that all MeBaAs, with the exception of 10-MeBaA, significantly increased cell numbers upon 72 h of cultivation; the potent genotoxin DMBA caused a significant decrease in cell numbers (Figure 4). Effects of MeBaAs on DNA Adduct Formation, Oxidative Stress, and Apoptosis. The above data suggest that
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Figure 3. Percentage of cells in the S phase of the cell cycle in WB-F344 after 72 h of treatment by 7-, 10-, and 12-MeBaA and DMBA detected on a FACSCalibur flow cytometer using propidium iodide staining. The data are expressed as means ( SD of three independent experiments done in duplicate. *A significant difference between control and treated samples (p < 0.05); **a significant difference between control and treated samples (p < 0.01).
compounds found to be mutagenic in other models, such as 7-MeBaA (10, 11), are probably not genotoxic to liver epithelial WB-F344 cells, whereas 10-MeBaA, which is considered only a weak mutagen and tumor-initiating compound in skin or bacterial models (10, 11), had effects suggesting its possible genotoxicity in WB-F344 cells. Therefore, we next determined the DNA adduct formation in WB-F344 cells exposed to selected MeBaAs, BaA, or DMBA by 32P-postlabeling assay (Table 2). DMBA produced a high level of DNA adducts, confirming its strong genotoxic potency, whereas 10-MeBaA induced much lower number of DNA adducts, similar to 12MeBaA and higher than 7-MeBaA (Table 2). These results suggest that MeBaAs do not form stable DNA adducts in WBF344 cells, despite their ability to increase levels of enzymes involved in their metabolic activation. Genotoxic PAHs have been suggested to induce oxidative stress in target cells, which might contribute to the damage of DNA and other macromolecules (42). As the formation of DNA adducts could not fully explain the toxicity of 10-MeBaA, we determined the production of ROS by flow cytometry, using DCF as a probe (38). As outlined in Figure 5, 24 h of exposure to DMBA and 10-MeBaA resulted in a significant increase in ROS formation, whereas 7-MeBaA did not elicit a significant increase of ROS levels as compared to the control cells. Interestingly, 7-MeBaA was not able to induce either stable DNA adducts or oxidative damage to DNA in liver cells. The toxic effects of 10-MeBaA also corresponded with the induction of phosphorylation of tumor suppressor protein p53 on Ser15 residue after 24 h of exposure (Figure 6). No other MeBaA induced phosphorylation of this amino acid residue, which has been shown to play a significant role in p53 stabilization, up-regulation, and functional activation during stress induced by genotoxic insult, including some PAHs (44–46). These data also corresponded with the induction of apoptosis. All MeBaAs, which were found to increase cell
numbers, induced a slight increase in percentage of cells with fragmented nuclei after 48 h of exposure (Figure 7), as well as a slight increase of Annexin-V-positive cells, representing early apoptotic stages (data not shown). In contrast, 10-MeBaA induced a significantly higher number of Annexin-V-positive cells (Figure 8) and cells with fragmented nuclei (Figure 7), although lower than DMBA. DMBA induced both a high accumulation of phosphorylated p53 and more pronounced apoptotic effects than 10-MeBaA. The parental compound BaA had no effects on either accumulation of phosphorylated p53 (Figure 6) or on apoptosis (reported previously in ref 23).
Discussion Methyl substitution has been shown to exert remarkable effects on the carcinogenicity of PAHs, such as DMBA. Monomethylated BaA derivatives can be found at significant levels in the environment or as cigarette smoke constituents (1–3). MeBaAs substituted in position 6, 7, 8, or 12 have been reported to be carcinogenic in both rat and mouse skin assays (7, 9), while other derivatives had only weak or no activity. The tumor-initiating activity of all 12 monomethylated benz[a]anthracenes has also been examined in the mouse skin model; 7-MeBaA was the most potent derivative, while all other MeBaAs elicited lower but significant tumor-initiating activity (11, 47). However, to this date, the carcinogenic or tumorinitiating activities of MeBaAs were studied almost exclusively in skin (7, 9, 11, 47), and little is known about their toxic or carcinogenic effects in other tissues or cellular models. In the present study, only one compound, 10-MeBaA, was found to be moderately genotoxic in liver epithelial WB-F344 cells, which could be, in part, also associated with the induction of oxidative stress in target cells. Nevertheless, although 10MeBaA induced the formation of DNA adducts, increased production of ROS, phosphorylation of p53 protein, apoptosis,
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Figure 4. Modulation of cell proliferation in WB-F344 assessed by counting cell numbers after 72 h of treatment by test compounds. The data are expressed as means ( SD of three independent experiments done in duplicate. *A significant difference between control and treated samples (p < 0.05); **a significant difference between control and treated samples (p < 0.01).
Table 2. DNA Adducts per 108 Nucleotides for Selected MeBaAs and DMBA as Determined by 32P-Postlabeling Method after 24 h of Exposure DNA adducts/108 nucleotides BaA (10 µM) 7-MeBaA (10 µM) 10-MeBaA (10 µM) 12-MeBaA (10 µM) DMBA (1 µM)
mean
SD
1.2 0.4 3.1 2.8 230
0.38 0.031 1.4 0.24 77
and increased percentage of cells in the S phase, all of these effects were markedly lower than genotoxic effects of DMBA, which was found be a powerful genotoxin in WB-F344 cells. Interestingly, 7-MeBaA did not form significant levels of stable DNA adducts and did not induce ROS formation, p53 phos-
phorylation, or apoptosis. This is in contrast with data obtained in bacterial mutagenicity assays or in rodent skin two-stage initiation-promotion assay. 7-MeBaA has been found to be significantly mutagenic in the Ames test with metabolic activation (10). It has also been the most potent derivative inducing sister chromatid exchanges (48). These results suggest that toxic effects of MeBaAs might be significantly different in organs such as the liver or lung. This fact should be taken into account in the risk assessment of MeBaAs based on bacterial mutagenicity or skin carcinogenicity data. In marked contrast to mutagenic and tumor-initiating effects of MeBaAs, other modes of toxic action, such as those contributing to tumor promotion, have received very little attention. Many PAHs are known to act as complete carcinogens, producing tumors following their repeated application, which
Toxic Effects of Methylated Benz[a]anthracenes
Figure 5. Fluorescence of dichlorofluorescein (DCF) following 24 h of treatment with selected MeBaAs and DMBA (10 µM) was detected as median fluorescence intensity (MFI). Hydrogen peroxide (250 µM, 5 min exposure) was used as a positive control. The results are expressed as means ( SD of results of three independent experiments done in triplicate. The data were analyzed statistically by Student’s t test. *Significant difference between control (0.1% DMSO) and treated samples (p < 0.05); **significant difference between control (0.1% DMSO) and treated samples (p < 0.01).
Figure 6. Induction of p53 phosphorylation at Ser15 after 24 h of exposure to the 10 µM MeBaAs or BaA as detected by Western blotting. DMBA was tested in concentrations of 0.1, 1, and 10 µM; the 10 µM concentration was used as a positive control on each blot. The detection of β-actin was used to confirm the equal loading.
suggests that either PAHs or their further active metabolites may act also as tumor promoters (14, 28). Although the mechanisms responsible for the tumor-promoting effects of PAHs are largely unknown, PAHs might activate several signaling pathways involved in the control of cell proliferation, differentiation, or apoptosis (28–30, 49). In the present study, we used liver epithelial cells as a model to study the impact of methyl substitution on AhR activation, deregulation of cell proliferation/apoptosis, and inhibition of GJIC. It has been proposed that AhR affinity may reflect the promoting effect of PAHs and that “initiation and promotion are provoked by different chemical species: reactive metabolites and the parent hydrocarbons, respectively” (15). However, there is only limited information on AhR-inducing potencies of MeBaAs. The AhR-binding and induction of EROD activities of seven MeBaAs in rat hepatoma cells have been reported, suggesting that these compounds have similar potencies to the parental BaA (17). On the other hand, Brack et al. (1) have reported that the relative potency (REP) of 9-MeBaA to induce EROD activity in a fish liver cell line could be two orders of
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magnitude higher than the relative potency of BaA. This would suggest that MeBaAs could significantly contribute to the overall AhR-mediated activity of complex environmental mixtures of PAHs, despite being present at significantly lower levels than BaA itself (1, 2). Indeed, we found in the DR-CALUX assay that several MeBaAs were more potent AhR ligands than BaA. The most potent AhR ligands were those MeBaAs that do not possess methyl group within or in the vicinity of the bay region. 4-, 6-, 7-, 8-, and 9-MeBaA were efficient inducers of AhRmediated activity. This holds true especially for 6-MeBaA and 9-MeBaA, whose EC50 values were comparable to some of the most potent AhR agonists among PAHs, such as dibenzanthracenes or benzofluoranthenes (16). Correspondingly to AhR activation data, all MeBaAs were efficient inducers of expression of AhR-dependent enzymes, P450 A1 and 1B1, with induction of P450 1A1 being higher than in case of BaA, their parent compound. All MeBaAs also induced expression of AKR1C9 mRNA, which may also participate in the bioactivation of polyaromatic compounds (42). These data suggested that AhR activation by MeBaAs could play a significant role in the toxic effects of MeBaAs in liver cells. However, the potencies of MeBaAs to induce AhR-mediated activity in liver cells did not correspond either with their mutagenic activities in bacterial assays (7-, 12-, and 5-MeBaAs being the most potent mutagens) (10) or with their skin carcinogenicity (7-, 12-, 6-, and 8-methyl derivatives have been reported as the most effective) (11, 47). Our previous studies have suggested that activation of AhR by PAHs may lead to disruption of cell cycle control in contactinhibited cells, followed by enhanced cell proliferation (21, 23). This mode of action might contribute to tumor-promoting effects of PAHs. In contrast, we have found that strong genotoxins induce cell cycle arrest/delay in the S phase and apoptosis in liver epithelial cells, both events being linked to DNA damage (21, 23, 39). Therefore, we determined modulation of cell cycle, cell proliferation, and apoptosis, to discriminate between genotoxic and nongenotoxic effects of MeBaAs. In accordance with their AhR-mediated activities, all MeBaAs, with the exception of weakly genotoxic 10-MeBaA, induced cell proliferation in contact-inhibited WB-F344 cells in a dose-dependent manner. Disruption of contact inhibition has been suggested to participate in tumor promotion (50, 51), suggesting that deregulation of cell cycle control by MeBaAs might potentially contribute to carcinogenic effects of MeBaAs. In contrast to the AhR-dependent effects of MeBaAs, only the compounds with the methyl group located within or in the vicinity of bay region (1-, 2-, 10-, 11-, and 12-MeBaA) were effective inhibitors of GJIC. The exception from this rule was 8-MeBaA, which was the only compound that acted as a strong inhibitor of GJIC and potent AhR ligand. The IC50 values of GJIC inhibitory effect of MeBaAs were similar to those of the most potent inhibitors of GJIC among PAHs, such as fluoranthene (25). Importantly, these results also confirm that methyl substitution may significantly increase the GJIC inhibitory potency of BaA derivatives, as previously demonstrated for DMBA (25). As outlined above, methyl substitution significantly modulated both GJIC inhibition and AhR-mediated activity of MeBaAs. Various approaches have been previously employed to define structure–activity relationships for carcinogenic effects of PAHs. However, these are hampered by the fact that classical parameters, such as area/depth2, length/width, or electron density do not change significantly within a series of structurally similar MeBaAs (52, 53). Nevertheless, there are some indications of parameters that might define toxic activities of MeBaAs. It has
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Figure 7. Induction of late apoptosis following 48 h of treatment of WB-F344 cells to the test compounds. Late apoptosis was determined as a percentage of cells with fragmented nuclei using DAPI staining and microscopic evaluation. The data are expressed as means ( SD of three independent experiments done in duplicate. *A significant difference between control and treated samples (p < 0.05); **a significant difference between control and treated samples (p < 0.01).
Figure 8. Example of apoptosis induction measured on a FACSCalibur flow cytometer. R1, region 1 represents early apoptotic cells (annexin-V positive, propidium iodide negative); R2, region 2 represents viable cells (both annexin-V and propidium iodide negative); and R3, region 3 contains late apoptotic and necrotic cells (both annexin-V and propidium iodide positive).
been suggested that substitutions in the bay region lead to a distortion of planarity, whereas MeBaAs substituted in the K region and neighboring positions are nearly planar (54). As AhR binds preferentially planar ligands (55), this might explain why both 1- and 12-MeBaA were the least potent AhR agonists identified in the reporter gene assay. Taken together, the present data suggest that methyl substitution modulates both AhR-dependent and -independent toxic effects of BaA derivatives. The in vitro toxic potencies of MeBaAs determined in liver cells were different from tumorinitiating, carcinogenic, and mutagenic potencies found either in skin or in bacterial models. This indicates that more attention should be paid to the evaluation of toxic effects of MeBaAs and related compounds in cellular models derived from other tissues, such as liver or lung. Nongenotoxic modes of action of MeBaAs, particularly the AhR transactivation, appeared to be more important than their genotoxicity in liver cells. As the AhR-mediated activity of MeBaAs was mostly higher than that of BaA, these compounds might significantly contribute to the
toxicity of complex mixtures of PAHs, which are present in various environmental compartments. Acknowledgment. This work was supported by EU FP6 project MODELKEY (51112-GOCE), the Czech Ministry of Agriculture (Grant MZE0002716201), and the Academy of Sciences of the Czech Republic (Research Plan AV0Z50040702).
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