Toxic Effects of Methylated Benzo[a]pyrenes in Rat Liver Stem-Like

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Toxic Effects of Methylated Benzo[a]pyrenes in Rat Liver Stem-Like Cells Lenka Trilecova,† Simona Krckova,† Sona Marvanova,† Katerina Pencíkova,† Pavel Krcmar,† Jirí Neca,† Petra Hulinkova,† Lenka Palkova,† Miroslav Ciganek,† Alena Milcova,‡ Jan Topinka,‡ Jan Vondracek,†,§ and Miroslav Machala*,† †

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, AS CR, Vídenska 1083, 14220 Prague, Czech Republic § Department of Cytokinetics, Institute of Biophysics, AS CR, Kralovopolska 135, 61265 Brno, Czech Republic ‡

ABSTRACT:

The methylated benzo[a]pyrenes (MeBaPs) are present at significant levels in the environment, especially in the sediments contaminated by petrogenic PAHs. However, the existing data on their toxic effects in vitro and/or in vivo are still largely incomplete. Transcription factor AhR plays a key role in the metabolic activation of PAHs to genotoxic metabolites, but the AhR activation may also contribute to the tumor promoting effects of PAHs. In this study, the AhR-mediated activity of five selected MeBaP isomers was estimated in the DR-CALUX reporter gene assay performed in rat hepatoma cells. Detection of other effects, including induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs, DNA adduct formation, production of reactive oxygen species, oxidation of deoxyguanosine, and cell cycle modulation and apoptosis, was performed in the rat liver epithelial WB-F344 cell line, a model of liver progenitor cells. We identified 1-MeBaP as the most potent inducer of AhR activation, stable DNA adduct formation, checkpoint kinase 1 and p53 phosphorylation, and apoptosis. These effects suggest that 1-MeBaP is a potent genotoxin eliciting a typical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), and cell death. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in the angular ring, elicited only low levels DNA adduct formation and apoptosis. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. MeBaPs induced a weak production of ROS, which did not lead to significant oxidative DNA damage. Importantly, 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their tumor promotion and carcinogenicity.

’ INTRODUCTION The methylated derivatives of polycyclic aromatic hydrocarbons (PAHs) are present at significant levels in the environment.1,2 Relatively high levels of monomethylbenzo[a]pyrenes (MeBaPs) (Figure 1), identified in automobile exhaust, cigarette smoke, coal tar, and especially in the sediments contaminated by petrogenic PAHs, have recently led to a renewed interest in their toxic action.3 MeBaPs are derivatives of benzo[a]pyrene (BaP), a potent genotoxic PAH, which has been classified as a human carcinogen.4,5 Both genotoxic and nongenotoxic modes of action of BaP have been extensively studied in vitro and in vivo.612 The substitution of the methyl group may significantly alter BaP mutagenicity in bacterial assays, as well as its tumor-initiating r 2011 American Chemical Society

activity and carcinogenicity in mouse skin. Although it has been demonstrated that different positions of the methyl groups in MeBaPs may lead to significant differences in their respective biological activities,1316 the existing data on the effects of MeBaPs in vitro and/or in vivo are still largely insufficient and incomplete. Several MeBaPs are complete sarcomagens;16 however, their tumor promoting activities are poorly understood, despite the fact that PAHs may exhibit toxic modes of action possibly contributing to tumor promotion.17 Many PAHs, including BaP, are relatively efficient ligands of the aryl hydrocarbon receptor Received: January 31, 2011 Published: May 23, 2011 866

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at passages 15 to 24 were used throughout the study. The rat hepatoma H4IIEGud.Luc1.1 cells (BioDetection Systems, Amsterdam, The Netherlands) were grown in Dulbecco’s modified Eagle’s medium (Invitrogene, Carlsbad, CA), 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 AhR-mediated activity in the DR-CALUX assay.27 The assays were performed in 96-well cell culture plates. The cells were grown for 24 h to 90100% confluency and then 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 luciferase was extracted with the low salt lysis buffer (10 mM Tris, 2 mM DTT, 2 mM 1,2-diamin cyclic hexane-N,N,N0 ,N0 -tetraacetic acid, pH 7.8). The plates were frozen at 80 °C, and luciferase expression was then measured on a microplate luminometer using Luciferase Assay Kit (BioThema, Handen, Sweden). Real-Time 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 a 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 50 -end, and with the Black Hole 1 (BH 1) fluorescent quencher dye on the 30 -end. The sequences of primers and probes have been published previously.23 The amplifications were run on LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) using the conditions described previously.28 Gene expression for each sample was expressed in terms of the threshold cycle (Ct), normalized to housekeeping gene porphobilinogen deaminase (ΔCt). ΔCt values were then compared between control samples (0.1% DMSO) and samples treated with PAHs to calculate ΔΔCt (ΔCt [control] ΔCt [xPAH]). The final comparison of transcript ratios between samples is given as 2ΔΔCt.29 Determination of PAH-DNA Adducts. WB-F344 cells in nearly confluent state (seeded at an initial density 23,000 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 Eppendorf tubes, and centrifuged, and cell pellets were stored at 80 °C. The 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 the phenol/chloroform/isoamylalcohol procedure as previously described.30 The DNA concentration was estimated spectrophotometrically by measuring the UV absorbance at 260 nm. DNA samples were kept at 80 °C until analysis. 32P-postlabeling analysis was performed as previously described.31,32 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 10 cm PEI-cellulose plates. Solvent systems used for TLC were the following: 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 to 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 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

Figure 1. Molecular structure of BaP with indicated positions of methyl substitutions in derivatives that were investigated in the present study.

(AhR). The AhR plays a key role in the metabolic activation of PAHs to reactive metabolites, which are able to form DNA adducts.6,7,18 However, AhR activation may also contribute to the tumor promoting effects of PAHs,19 such as deregulation of cell cycle control.10,2022 Other tumor promoting effects of PAHs, probably not directly related to AhR activation, involve an acute inhibition of gap junctional intercellular communication23,24 or perturbation of intracellular signal transduction,9,11,25 which may further contribute to the disruption of cell proliferation/apoptosis balance.12,20 Presently, there are no data available on the AhR-mediated or other tumor promoting effects of BaPs, as the original studies were mostly devoted to their mutagenic and tumor initiating action. For the present study, we selected five MeBaPs (Figure 1) with different mutagenic/carcinogenic potencies based on the results of their mutagenic activity analysis in Salmonella typhimurium14 and the data from the mouse skin two-stage tumor initiation assay.15 We used an established model of the rat liver epithelial stem-like WB-F344 cell line26 in order to study the toxic effects of MeBaPs associated with DNA damage response, apoptosis, and cell survival (activation of AhR-dependent CYP1 gene expression, formation of DNA adducts, oxidative stress, induction of checkpoint kinase 1 (Chk1) and p53 protein phosphorylation, disruption of cell cycle control, and induction of apoptosis). The AhR-mediated activity of MeBaPs was estimated in the rat hepatoma cell line stably transfected with the luciferase reporter gene (DR-CALUX assay).27 Our present data seem to suggest that genotoxicity plays a prominent role in the toxic effects of tested MeBaPs in vitro. Nevertheless, given the substantial AhRmediated activity of especially 1- and 3-MeBaP, their contribution to nongenotoxic effects mediated by this transcription factor should also be considered, when evaluating their toxicity.

’ EXPERIMENTAL PROCEDURES Chemicals. BaP was purchased from Supelco (Bellefonte, PA), and MeBaPs were obtained from Midwest Research Institute (Kansas City, MO); the purities of all compounds were higher than 99.9%. 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); DAPI, ribonuclease A and T1, proteinase K, micrococcal nuclease, nuclease P1, R-methylcinnamic acid, and protein assay kit were from Sigma-Aldrich; polyethylene-imine cellulose TLC plates (0.1 mm) were from Macherey-Nagel (D€uren, Germany); T4 polynucleotide kinase was from USB (Cleveland, OH); γ-32P-ATP (3000 Ci/mmol, 10 μCi/μL) was from GE Healthcare (Little Chalfont, UK); and propidium iodide was from AppliChem GmbH (Darmstadt, Germany). Cells. The rat liver epithelial WB-F344 cells (kindly provided by James E. Trosko, MSU, East Lansing, MI) 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 867

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cell numbers (estimation of cell proliferation).10 The medium with test compounds was changed daily. 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.28 Cells were then analyzed on FACSCalibur, using a 488-nm (15 mW) air cooled argon-ion laser for propidium iodide excitation, and CELLQuest software, version 5.1.1 for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15,000 events was collected per sample. Data were analyzed using ModFit LT, version 3.0 software (Verity Software House, Topsham, ME). The cell numbers (data not shown) were determined as previously reported.10 Detection of Cell Death. Confluent WB-F344 cells were exposed to the test compounds for 48 h including a change of the fresh medium and the compound after 24 h. Early stages of apoptosis are 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, Mannheim, Germany) in combination with propidium iodide (40 μg/mL), in order to distinguish the cells with permeabilized and intact plasma membrane. Cells were harvested and stained according to 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 1020 μL of MOWIOL solution (10% MOWIOL 4-88 was prepared in 25% glycerol and 100 mM Tris-HCl, pH 8.5) and mounted for observation under a fluorescence microscope. A minimum of 300 nuclei were counted per sample.23 Western Blotting. Confluent WB-F344 cells (seeded at an initial density of 30,000 cells/cm2 and grown for 72 h) were exposed for 24 h to test compounds and/or DMSO as the solvent control. Camptothecin (0.3 μM) was used as a positive control. After exposure, cells were harvested into the lysis buffer (1% SDS, 10% glycerol, 100 mM Tris, and protease inhibitors), and the lysates were sonicated. Protein concentrations were determined using bicinchonic acid and copper sulfate (SigmaAldrich). For Western blot analyses, equal amounts of total protein lysates were separated by SDSpolyacrylamide gel electrophoresis on 8.5% gel and electrotransferred onto polyvinylidene fluoride membrane Hybond-P (GE Healthcare, Little Chalfont, UK). Prestained molecular weight markers (Bio-Rad, Hercules, CA, USA) were run in parallel. The blotted membranes were blocked and incubated with primary antibody against p53 phosphorylated at Ser15 (#9284, Cell Signaling Technology, Danvers, MA, USA) or total p53 (#sc-1313, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h at room temperature or primary antibody against Chk1 phosphorylated at Ser345 (#2341, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washing in TBS with 0.1% Tween 20, horseradish peroxidase-conjugated antirabbit IgG (#NA934, GE Healthcare, Little Chalfont, UK) or horseradish peroxidase-conjugated antimouse IgG (A9044, Sigma-Aldrich Corp., St. Louis, MO, USA) were used as secondary antibodies. Expression of β-actin was used to verify equal loading; monoclonal anti-β-actin antibody, clone AC-15 (A1978, Sigma-Aldrich Corp., St. Louis, MO, USA) was diluted in 2.5% milk in TBS and incubated for 2 h at room temperature; horseradish peroxidaseconjugated antimouse IgG (A9044, Sigma-Aldrich Corp., St. Louis, MO, USA) was used as a secondary antibody. To visualize peroxidase activity, ECL Plus reagents (GE Healthcare, Little Chalfont, UK) were used according to the manufacturer’s instructions. Short Interfering RNA (siRNA) Transfections. Transfections of cells with siRNA directed against p53 or with control, nonspecific siRNA (targeting Discosoma dsRED) were performed either in 12-well plates (Western blotting) or in 6-well plates (apoptosis detection). Cells were seeded at 20,000 cell/cm2 density and cultured for 24 h to reach 5070% confluency. Transfections were performed in a total volume of 1 mL (for 12-well plate) and 2.5 mL (for 6-well plate) of transfection

postlabeling experiment to check for interassay variability and to normalize the calculated DNA adduct levels.

Detection of Generation of Reactive Oxygen Species (ROS). Confluent WB-F344 cells were exposed to the test compounds for 24 h. Hydrogen peroxide (exposure 3 min) was used as a positive control. In some experiments, 25 μM R-methylcinnamic acid, an inhibitor of aldoketo reductases (AKR), was added to the cells for 30 min before exposure to the tested compounds. After the exposure the cells were washed twice by PBS, trypsinised, centrifuged, and resuspended with Hank's balanced salt solution (PAN BioTech GmbH, Aidenbach, Germany) with 5% heat-inactivated fetal bovine serum. The cell suspension was incubated for 15 min with the fluorogenic probe dichlorofluorescein 20 ,70 -diacetate (Sigma-Aldrich), at a final concentration of 20 μM. The cells were washed once again, centrifuged, and cooled on ice. Fluorescence of dichlorofluorescein was analyzed by FACSCalibur, using the CellQuest software (Becton Dickinson).33 Detection of 8-OHdG/dG. Confluent WB-F344 cells were exposed to test compounds for 6 h. After exposure, cells were washed twice by ice-cold PBS and harvested in 1 mL of PBS. The cell pellets were frozen at 20 °C. DNA was isolated from cells by a method based on the binding of DNA with diatom in guanidine thiocyanate (GuSCN) (ref 34, modified by ref 35). The samples were transferred to 2-mL test tubes and mixed with 1 mL of lysis buffer (5 M GuSCN; 0.05 M Tris-HCl, pH 6.4; 0.02 M EDTA, pH 8.0; 1.3% Triton X-100), and 2.5-mm glass beads were added. The mixture was homogenized in TissueLyser II (Qiagen GmbH, Germany) at a frequency of 30 s1 for 40 s. The mixture was then centrifuged (14,000g, 5 min), and 700 μL of the supernatant was transferred into a 1.5-mL test tube, mixed with 500 μL of lysis buffer and 40 μL of a diatom suspension (prepared on the preceding day by mixing 100 mg of Celite with 500 μL of water and 5 μL of 32% HCl), and vortexed for 15 s. The mixture was incubated at room temperature for 10 min and centrifuged (14,000g, 1 min), and then the supernatant was decanted. The diatom pellet was washed with 500 μL of washing buffer (5 M GuSCN; 0.05 M Tris-HCl, pH 6.4), with 500 μL of 70% ethanol, and with 500 μL of acetone. The content of the uncovered test tube was dried in an incubator at 56 °C for 15 min, mixed with 70 μL of tempered (56 °C) water, incubated at room temperature for 2 min, and centrifuged (14,000g, 1 min), and 40 μL of the supernatant was transferred to a 0.5-mL test tube. DNA in the samples was denaturated by heating, and the test tubes were then cooled down quickly. Denaturated samples were incubated with 4 μL of Nuclease P1 (1U/1 μL, Sigma-Aldrich) for 1 h at 37 °C and then with 2 μL of alkaline phosphatase (3.5 mg/30ul, Sigma-Aldrich) for 1 h at 37 °C. The reaction was stopped by the addition of 8 μL of 3 M sodium acetate (pH 5). The samples were frozen at 80 °C. The ratio of 8-OHdG/dG was determined by using liquid chromatographytandem mass spectrometry (LC/MS-MS). An Agilent 1200 chromatographic system (Agilent Technologies, Germany), which consisted of binary pump, vacuum degaser, autosampler, UV detector, and thermostatted column compartment was employed. Separation was carried out using a ZORBAX Eclipse Plus C18, 2.1 150 mm, 3.5 μm particle size column (Agilent, USA). The analytes were eluted isocratically with the mixture of water/methanol (90/10, v/v). The flow rate of the mobile phase was 0.2 mL/min, the column temperature was set at 45 °C. dG was detected with UV detector at λ = 260 nm. A triple quadrupole mass spectrometer Agilent 6410 Triple Quad LC/MS (Agilent Technologies, USA) with an electrospray interface (ESI) was used for detection of 8-HO-dG. The mass spectrometer was operated in the positive ion mode. Multiple reaction monitoring (MRM) was used for quantification, the mass transitions used were m/z 284.1fm/z 168.

Assessment of Cell Cycle Distribution and Proliferation. The confluent cells were exposed to test compounds dissolved in DMSO (maximal concentration 0.1%, v/v) for 72 h. The exposure time has been previously shown to be optimal for the determination of total 868



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Figure 2. AhR-mediated activity of MeBaPs. The AhR-mediated induction of luciferase reporter gene in H4IIEpGudLuc1.1 rat hepatoma cell line induced by MeBaPs or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (positive control) was determined after 24-h exposure. The data shown here are representative of the means ( SD of three independent experiments. The luciferase activity of the reporter gene was expressed as the percentage of maximum activity induced by TCDD.

Table 1. Values of EC50 and Relative Potencies of MeBaPs to Activate AhR (DR-CALUX Assay, Exposure 24 h) EC50 (nM)

IEF (TCDD 24 h)a

BaP

89

2.3 105

1-MeBaP

30

3.6 104

3-MeBaP 6-MeBaP

60 840

10-MeBaP

120

11-MeBaP

2820

MannWhitney U-test was used. A p-value of less than 0.05 was considered significant.

’ RESULTS Induction of the AhR-Dependent Gene Expression by MeBaPs. Transactivation of transcription factor AhR is a key

4

1.5 10 9.7 106 7.0 10

event, which controls both the induction of PAH metabolizing enzymes and several processes associated with tumor promotion.21,36,37 Therefore, we have first examined the effects of selected MeBaPs on AhR activation. For detection of the AhRmediated activity, we used the DR-CALUX assay, which employs the H4IIEGud.Luc cell line stably transfected with the luciferase reporter gene under the control of dioxin responsive elements (DREs).27 On the basis of our previous studies, we have determined the AhR-mediated activity of MeBaPs relative to the reference compound TCDD after 24 h of exposure and expressed them in induction equivalency factors (IEFs), which were based on their EC50 values.23,38,39 The full doseresponse curves for all compounds under study are shown in Figure 2. The EC50 and IEF values for individual MeBaPs are summarized in Table 1. 6-, 10-, and 11-MeBaP exhibited similar or lower potency to induce the AhR-mediated activity than BaP, while the relative potencies of 1- and 3-MeBaP were approximately one order of magnitude higher than that of the parent compound (Table 1). Next, we determined the AhR-dependent induction of xenobiotic metabolizing enzymes CYP1A1, CYP1B1, and AKR1C9 at the mRNA level in rat liver epithelial WB-F344 cells treated with individual MeBaPs. These enzymes participate in the formation of dihydrodiol epoxide metabolites of PAHs or in the production of reactive PAH o-quinones, which contribute to oxidative stress generation and/or further DNA damage.8,40 In WB-F344 cells, 1 μM concentration of BaP is able to induce CYP1A1 and CYP1B1 mRNA expression approximately to the same extent as the maximum effective concentration of the model AhR agonist, TCDD.26 As shown in Figure 3, all MeBaPs induced a significant increase of CYP1A1 mRNA expression, with 1-MeBaP being the most potent inducer. The doseresponses after MeBaP exposure

5

2.9 106

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 MeBaP inducing the 50% of maximum TCDD-induced luciferase activity. All experiments were performed independently three times in triplicate. medium containing 50 nmol siRNA and 1 μL of Lipofectamine2000 (Invitrogene, Carlsbad, CA), according to the manufacturer's instructions. After 9 h of incubation, transfection mixtures were replaced with fresh DMEM with antibiotics and 5% heat-inactivated fetal bovine serum. After 24 h, cells were exposed for 24 h to 0.1% DMSO, 10 μM 1-MeBaP, or to 0.3 μM camptothecin for 24 h. Apoptosis was determined after staining with Annexin V/PI by flow-cytometric analysis, as described above. In order to confirm efficient p53 knock-down at the protein level, whole cell extracts were separated by SDSPAGE and subjected to Western blot analysis, using a primary antibody against p53 (Santa Cruz Biotechnology). The p53 siRNA (directed against rat tumor protein 53 mRNA sequence; GenBank accession number NM_ 030989.3) was supplied by Thermo Scientific Dharmacon (Lafayette, CO, USA; Cat. No. J-080060-05-0010), and control siRNA20 were supplied by Ambion (Austin, TX, USA). Statistical Analysis. All experiments (with the exception CYPs and AKR1C9 mRNA detection) were performed independently at least three times, and the data were expressed as the means ( SD and analyzed by Student’s t test or ANOVA, followed by Tukey's range test. When the variances were not homogeneous, nonparametric 869



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Figure 3. Induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs as determined by real-time RT-PCR following 24 h exposure to MeBaPs or to 1 nM TCDD as a model AhR ligand (positive control). Isolation of total mRNA and RT-PCR was performed as described in Experimental Procedures. The results are expressed as the mean ( SD of two independent experiments.

corresponded with the data obtained in the DR-CALUX assay and the relative efficiencies of MeBaPs to activate expression of AhR target genes were in the following order: 1-MeBaP > 3-MeBaP > 10-MeBaP > 6-MeBaP > 11-MeBaP. All MeBaPs also induced CYP1B1 and aldo-keto reductase 1C9 (AKR1C9) mRNAs in a manner similar to that of CYP1A1 (Figure 3). Formation of Stable DNA Adducts and Induction of Oxidative Stress by MeBaPs. The exposure to PAHs and/or their methylated derivatives often leads to the formation of stable PAH-DNA adducts and/or the generation of oxidative stress in target cells.8,40 Therefore, we next determined the DNA adduct formation in WB-F344 cells after 24 h exposure to MeBaPs or BaP as a standard. As shown in Figure 4, all MeBaPs efficiently

induced the formation of DNA adducts with 1-MeBaP being the most potent compound (1-MeBaP > 11-MeBaP > 6-MeBaP > BaP > 3-MeBaP > 10-MeBaP). Importantly, 1- and 11-MeBaP produced significantly higher levels of DNA adducts than their parent compound, thus suggesting that methyl substitution at positions 1 and 11 enhances the genotoxicity of the respective MeBaPs. As genotoxic PAHs have been reported to induce oxidative stress in target cells,8 we further evaluated the production of ROS by flow cytometry, using dichloroflurescein as a probe.33 As shown in Figure 5A, 24 h exposure to all MeBaPs resulted in a significant increase in the production of ROS, similar to that of BaP. 870



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Figure 4. Formation of stable DNA adducts in WB-F344 cells. Cells were exposed to DMSO (solvent control), 1 μM BaP (positive control), or 1 μM MeBaPs for 24 h, and DNA isolation followed by 32P-postlabeling was performed as described in Experimental Procedures. The duration of screen enhanced autoradiography was 24 h, and the numbers above each chromatograph represent the number of DNA adducts per 108 nucleotides ( SD. The data represent the means ( SD of three independent experiments.

6-MeBaP was the most potent inducer of ROS among the tested compounds. Therefore, we next investigated the impact of this methylated BaP derivative on oxidative DNA damage. We used an increased ratio between 8-oxo-7,8-dihydro-20 -deoxyguanosin (8-OH-dG) and deoxyguanosin (dG) as a marker of oxidative damage to DNA.41 We found that although 6-MeBaP elevated 8-OH-dG levels, this effect was not significant, and these results suggest that increased ROS levels only made minor contributions to the DNA damage in rat liver epithelial cells (Figure 5B). Accumulation of WB-F344 Cells in S-Phase of Cell Cycle and Induction of Apoptosis. PAHs induce cell cycle arrest and/ or apoptosis as a part of cellular defense to extensive DNA damage.30,42 In WB-F344 cells, strong genotoxins, such as BaP, induce both accumulation of cells in S-phase of cell cycle and apoptosis, while potent AhR agonists with relatively low genotoxic potencies, such as benzofluoranthenes, can increase cell number by AhR-dependent disruption of contact inhibition.10,43 Using Annexin V staining of the phosphatidylserine externalized on plasmatic membrane surface, all MeBaPs under study were found to be potent inducers of apoptosis after 48 h of exposure, with the exception of 10-MeBaP (Figure 6). Similar results were also obtained when we assessed the fragmentation of nuclei after the staining of fixed cells with DAPI (data not shown). The increased percentage of cells in S-phase largely corresponded with the apoptotic response, and with exception of 10-MeBaP, there were always over 20% of cells in S-phase (Figure 6A). Additionally, we found a decrease in cell numbers under the same experimental conditions (data not shown), which indicated that the observed increased percentage of cells in S phase was a result of genotoxic signaling and not a consequence of the AhRdependent disruption of contact inhibition.20 Activation of p53 and Chk1 Proteins after MeBaP Exposure. The formation of bulky PAH-DNA adducts leads to the activation of Chk1 and p53 proteins, key regulators of DNA damage response (including cell cycle delay, DNA repair, and/ or apoptotic signaling44,45). Phosphorylation of p53 protein at serine 15 and protein kinase Chk1 at serine 345 is indicative of their activation;44 levels of phosphorylated p53 and Chk1 were

determined after 24 h exposure to 10 μM MeBaPs by Western blotting (Figure 7). Camptothecin and BaP were used as positive controls. 1-MeBaP and 3-MeBaP were the most efficient inducers of phosphorylation of p53 and Chk1; a weak activation of both Chk1 and p53 was observed also after the exposure to 6- and 11-MeBaP. In order to confirm a direct link between activation of p53 and induction of apoptosis, we compared the percentage of apoptotic cells in wild type WB-F344 cells with the induction of apoptosis in cells with silenced p53 expression, using 1-MeBaP as the model MeBaP. The efficient p53 knock-down was confirmed by Western blotting (Figure 8a). We found a substantial suppression of apoptosis in the cells with silenced p53, which were treated either with 1-MeBaP or camptothecin (Figure 8b). Taken together, the above data suggested that, in a model of rat liver epithelial cells, genotoxic effects of MeBaPs prevailed associated with DNA damage induction, which prevailed over the AhRdependent nongenotoxic effects, including disruption of contact inhibition of growth.

’ DISCUSSION Methylated PAHs, including MeBaPs, belong among the important environmental pollutants. However, the studies describing their in vitro and in vivo toxic effects cover mostly their mutagenicity or tumor initiation properties, and they do not provide sufficient information regarding other possible toxic modes of action and toxic potencies, especially in important target organs such as the lung and the liver. Our recent two experimental studies covering the in vitro effects of methylated chrysenes and benz[a]anthracenes have shown that rat liver epithelial cell line WBF344 is a suitable model of hepatic progenitor cells with high metabolic activation capacity and intact p53 signaling pathway, allowing one to analyze a wide range of toxic effects of methylated PAHs.23,39 The same model allows one to also study nongenotoxic effects of PAHs, related to tumor promotion, namely, the inhibition of gap junctional intercellular communication23 or induced proliferation of confluent cells, which is the AhR-dependent process.20 In the present study, we selected five monomethylated 871



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Figure 5. (A) Production of reactive oxygen species (ROS) in cells treated with MeBaPs. Dichlorofluorescein fluorescence was detected by flow cytometry after 24 h of exposure of WB-F344 cells to 1 μM MeBaPs or BaP and expressed as the median fluorescence intensity (MFI). Hydrogen peroxide (250 μM, 5 min exposure) was used as a positive control. The results are expressed as the means ( SD of three independent experiments performed in triplicate. *, Significant difference between the control and treated samples (p < 0.05); **, significant difference between the control and treated samples (p < 0.01). (B) Detection of 8-OHdG/dG in cells treated with 6-MeBaP for 6 h. KBrO3 (2 mM) was used as a positive control. The results are the means of three independent experiments performed in triplicate. **, Significant difference between the control and treated samples (p < 0.01).

BaP derivatives, in order to study their adverse mechanisms in the same cellular model. The selection of MeBaP isomers was based on previously reported data on their mutagenicity in bacterial assays and tumor-initiation activity in the mouse skin two/stage carcinogenesis protocol.1315 Our principle aim was to evaluate and compare various genotoxic and nongenotoxic modes of action, which may contribute to DNA damage and/or to tumor promotion, and which are both related to the AhR-mediated activity of PAHs. The AhR activation is a key step in the toxic modes of action of many PAHs and PAH derivatives. The induction of AhR-dependent expression of CYP1 enzymes metabolizing PAHs to reactive intermediates, as well as an increased ROS production during the oxidative metabolism of PAHs, illustrate the prominent role of activated AhR in the genotoxicity of these compounds. The resulting DNA damage then leads to activation of cellular DNA damage response, including the activation (phosphorylation) of p53 tumor suppressor, cell cycle arrest/delay, activation of DNA repair mechanisms, or induction of apoptosis.42,46 Nevertheless, AhR activation may also significantly modulate cell survival and proliferation.2022 Therefore, in this study, we evaluated the AhR-mediated activity

Figure 6. Induction of cell cycle arrest and apoptosis by MeBaPs. (A) Percentage of cells in the S-phase of the cell cycle in WB-F344 after 72 h of exposure to MeBaPs was determined by flow cytometry using propidium iodide staining of DNA. BaP (10 μM) was used as a positive control. All data are expressed as the means ( SD of at least three independent experiments. *, Significant difference between the control and treated samples (p < 0.05); **, significant difference between the control and treated samples (p < 0.01). (B) Induction of cell death measured after annexin V/propidium iodide of cells by flow cytometry after 48 h exposure. The gray bars represent early apoptotic cells (annexin-V, positive; propidium iodide, negative), the open bars represent late apoptotic and necrotic cells (both annexin-V and propidium iodide, positive). BaP (10 μM) was used as a positive control. All data were expressed as the means ( SD of at least three independent experiments. *, Significant difference between the control and treated samples (p < 0.05); **, significant difference between the control and treated samples (p < 0.01).

of MeBaPs and the related genotoxic and nongenotoxic effects following the exposure of liver epithelial cells to MeBaPs. First, we determined relative potencies of individual MeBaP isomers to transactivate AhR in the established DR-CALUX 872



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activity, similar to that of BaP. Correlation was found between the DR-CALUX data on the transactivation of AhR (Figure 2) and doseresponses of CYP1A1 mRNA induction in WB-F344 cells (Figure 3). MeBaPs were also able to induce CYP1B1 and AKR1C9 mRNAs in a similar manner. All MeBaPs under study induced significant formation of DNA adducts in liver epithelial WB-F344 cells (Figure 4). Nevertheless, with the exception of the most potent 1-MeBaP, there was no clear correlation between the AhR activation potencies of individual MeBaPs and the levels of stable DNA adduct formed. The order for relative potencies to induce AhR transactivation (1-MeBaP = 3-MeBaP > 10-MeBaP > BaP > 6-MeBaP > 11-MeBaP) differed from the ability of MeBaPs to form DNA adducts (1-MeBaP > 11-MeBaP > 6-MeBaP > BaP > 3-MeBaP . 10-MeBaP). Similar results have been observed previously for methylated chrysenes39 and benz[a]anthracenes.23 This suggests that although the role of AhR activation is substantial for the formation of genotoxic intermediates, the potencies of methylated PAHs to induce AhR-mediated activity (IEF values) do not also represent their genotoxic potentials. It is known from previous studies that DNA adduct formation does not depend only on the presence of bioactivating CYP1 enzymes but mainly on molecule structure and the persistency of the ultimate genotoxic intermediates.6,37,40 1-MeBaP has been previously selected, as the only methylated BaP, for the determination of its mutagenicity in mammalian in vitro systems.50 Its potency exceeded the mutagenicity of BaP, which corresponds with our results. However, it is necessary to stress that this particular mutagenicity assay may can pick up either true mutations or phenocopies produced by altered gene expression, an epigenetic event. In our study, 11-MeBaP, which is substituted in the vicinity of the bay region, was also a very potent inducer of DNA adducts, suggesting that this position of the methyl group more efficiently hindered the detoxification reactions. Similarly, 6-MeBaP (substituted in the peri position) produced high levels of stable DNA adducts (Figure 4). Angular ring dihydrodiol epoxides are ultimate genotoxic metabolites of many PAHs.6 Substitution of BaP in positions 7, 8, 9, or 10 by the methyl group led to suppression of genotoxicity, and MeBaP isomers substituted in the 7, 8, 9, or 10 positions have been reported to elicit very low or no mutagenic activity in bacterial assays,13,14 and they were inactive in the mouse skin tumor initiation assay.15 In this study, 10-MeBaP (in which the methyl group is placed directly in the position where the most effective metabolic activation occurs) had the lowest capacity to form stable DNA adducts. In general, MeBaPs substituted in positions 1, 3, and 11, as well as the parent compound, have shown a significant tumor-initiating activity in mouse skin,15 which corresponded with their high potencies to induce DNA adduct formation in liver cells (this study). Formation of high levels of DNA adducts in liver cells suggests that these compounds might have a systemic tumorinitiating activity. However, 6-MeBaP has been reported to produce only minimal level of papillomas in mice;51 therefore, the strong DNA adduct production in liver cells suggests that the 6-methyl derivative may be a tissue (liver)-specific genotoxin. Another consequence of the uptake and metabolism of MeBaPs might be the production of ROS and oxidative DNA damage. The ROS generate a large number of oxidative DNA modifications, including base oxidations and strand breaks.41,52 Apart from the CYP1 monooxygenase activities, the ROS can be generated via the alternative AKR metabolic pathway leading to the formation of PAH quinones.8 AKR1C9 (which seems to be

Figure 7. Induction of p53 (at Ser15) and Chk1 (at Ser345) phosphorylation in WB-F344 cells exposed to DMSO, 10 μM BaP, 10 μM MeBaPs, or 0.3 μM camptothecin (positive control) for 24 h. Whole cell lysates were prepared and analyzed by Western blotting as described in Experimental Procedures. The results are representative of three independent experiments; β-actin was used as a loading control.

Figure 8. (a) Levels of total p53 protein in WB-F344 cells exposed for 24 h to 0.1% DMSO, 10 μM BaP, 0.3 μM camptothecin, or UV. Whole cell lysates were prepared and analyzed by Western blotting as described in Experimental Procedures. R-19 was used as the primary antibody. (b) Detection of cell death measured after FITC-annexin V/propidium iodide of cells by flow cytometry after 24 h of exposure. The cells were transfected with dsRED (gray bars) or with siRNA against p53 (white bars) and exposed to vehicle (DMSO), 10 mM 1-MeBaP, or 0.3 mM camptothecin for 24 h. All data were expressed as a mean ( SD. *, Significant difference between the control (dsRED, nonspecific siRNA) and p53 siRNA-treated samples (p < 0.05).

assay, using the transgenic hepatoma H4IIE cell line. This assay has been successfully used both for the detection of dioxin-like toxicants in complex environmental mixtures47 as well as for the determination of AhR-inducing potencies of less persistent toxicants such as large series of PAHs and their mixtures.48,49 The relative potencies of individual MeBaPs, derived from full doseresponse experiments, were expressed as IEF values related to the reference toxicant TCDD (Figure 2 and Table 1). 1-MeBaP and 3-MeBaP were the most potent AhR agonists; however, the other MeBaP isomers also possessed a relatively high AhR-inducing 873


Chemical Research in Toxicology indirectly regulated by the AhR) and AKR1A1, another member of the AKR family responsible for ROS production,53 have been found in rat liver epithelial cells.54 A weak, but significant induction of ROS production was found in the cells exposed to all MeBaPs and BaP, with 6-MeBaP being the most potent inducer (Figure 5A). R-Methylcinnamic acid, an inhibitor of AKR1 enzymes,55 partly suppressed the ROS production induced by 6-MeBaP (data not shown), indicating that the AKR-dependent metabolic pathway may contribute to increased ROS concentration. Nevertheless, the oxidative damage to DNA, detected as the 8-OH-dG/dG ratio, was not significantly increased by 6-MeBaP, suggesting only a minor direct contribution of oxidative stress to the genotoxicity of MeBaPs (Figure 5B). Nevertheless, we cannot exclude that modulation of gene expression by low levels of ROS and related signaling may also contribute to some cellular effects of MeBaPs. 1-, 3-, 6-, and 11-MeBaPs induced the accumulation of cells in the S-phase of the cell cycle and apoptosis in rat liver epithelial cells (Figure 6). Apoptosis is considered to be an integral process reflecting various types of major DNA damage, as well as some additional mechanisms of toxicity; however, on the basis of the above data, we would conclude that the formation of high levels DNA adducts was the principle mechanism contributing to the observed induction of apoptosis by 1-, 3-, 6-, and 11-MeBaPs. This corresponds with the data obtained with highly genotoxic unsubsituted PAHs in the same cellular model.26 The MeBaP isomers with substitution in positions 1 or 3 were potent activators of Chk1 and p53 (Figure 7), suggesting that DNA damage response signaling through these particular pathways may contribute to induction of apoptosis. Although these proteins were to a certain extent also phosphorylated by other MeBaPs, this does not also exclude the participation of other signaling pathways contributing to apoptosis induction.56 Nevertheless, using siRNA-mediated p53 knock-down, we confirmed that p53 activation was a major mechanism linking DNA damage responses to apoptosis induction. Taken together, we identified 1-MeBaP to be the most potent inducer of AhR activation, stable DNA adduct formation, Chk1 and p53 phosphorylation, and apoptosis in liver epithelial cells. These effects suggested that this compound elicits a canonical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, followed by metabolic activation of a parent compound, which in turn leads to the formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), finally leading to elimination of cells suffering from substantial amount of DNA damage via apoptosis induction. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in an angular ring, formed only low levels DNA adducts, and it was a poor apoptosis inducer. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. Importantly, both 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their carcinogenicity. As the present study indicated that MeBaPs are potent toxicants in liver cells, future studies should further address their toxic mechanisms and potencies in major target organs of toxicants, including both liver and lung tissues, as the currently available data on their carcinogenicity have been almost exclusively obtained with skin models.

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’ AUTHOR INFORMATION Corresponding Author

*Tel: þ420-533331813. Fax: þ420-541211229. E-mail: machala@ vri.cz. Funding Sources

This work was supported by the Czech Science Foundation project No. 525/08/1590. The institutional support was provided by the Czech Ministry of Agriculture (MZE0002716202) and the Academy of Sciences of the Czech Republic (Research Plans AV0Z50040702 and AV0Z50390512).

’ ABBREVIATIONS AhR, aryl hydrocarbon receptor; AKR, aldo-keto reductase; BaP, benzo[a]pyrene; BPDE, benz[a]pyrene dihydrodiol epoxide; Chk1, checkpoint kinase 1; CYP, cytochrome P450; DAPI, 40 ,6-diamidine2-phenyl indole; DMSO, dimethylsulfoxide; DR-CALUX, dioxin responsive chemical-activated luciferase expression assay; DRE, dioxin responsive element; DTT, dithiothreitol; LC/MS-MS, high performance liquid chromatographymass spectrometry; IEF, induction equivalency factor; MeBaP, methylbenzo[a]pyrene; 8-OH-dG, 8-oxo-7,8-dihydro-20 -deoxyguanosin; PAHs, polycyclic aromatic hydrocarbons; ROS, reactive oxygen species; RT-PCR, reverse trancriptionpolymerase chain reaction; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TBS, tris-buffered saline; TLC, thin layer chromatography. ’ REFERENCES (1) Means, J. C. (1998) Compound-specific gas chromatographic mass spectrometric analysis of alkylated and parent polycyclic aromatic hydrocarbons in waters, sediments, and aquatic organisms. J. AOAC Int. 81, 657–672. (2) Zakaria, M. P., Takada, H., Tsutsumi, S., Ohno, K., Yamada, J., Kouno, E., and Kumata, H. (2002) Distribution of polycyclic aromatic hydrocarbons (PAHs) in rivers and estuaries in Malaysia: a widespread input of petrogenic PAHs. Environ. Sci. Technol. 36, 1907–1918. (3) Avila, B. M., Pereira, R., Gomes, A. O., and Azevedo, D. A. (2011) Chemical characterization of aromatic compounds in extra heavy gas oil by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry. J. Chromatogr., A 1218, 3208–3216. (4) IARC (2010) Air Pollution, Part 1, Some Non-Heterocyclic Polycyclic Aromatic Hydrocarbons and Some Related Industrial Exposures, Vol. 92, IARC, Lyon, France. (5) WHO (1998) Non-Heterocyclic Polycyclic Aromatic Hydrocarbons, WHO, Geneva, Switzerland. (6) Baird, W. M., Hooven, L. A., and Mahadevan, B. (2005) Carcinogenic polycyclic aromatic hydrocarbon-DNA adducts and mechanism of action. Environ. Mol. Mutagen. 45, 106–114. (7) Shimada, T., Inoue, K., Suzuki, Y., Kawai, T., Azuma, E., Nakajima, T., Shindo, M., Kurose, K., Sugie, A., Yamagishi, Y., FujiiKuriyama, Y., and Hashimoto, M. (2002) Arylhydrocarbon receptordependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice. Carcinogenesis 23, 1199–1207. (8) Penning, T. M., Burczynski, M. E., Hung, C. F., McCoull, K. D., Palackal, N. T., and Tsuruda, L. S. (1999) Dihydrodiol dehydrogenases and polycyclic aromatic hydrocarbon activation: generation of reactive and redox active o-quinones. Chem. Res. Toxicol. 12, 1–18. (9) Burdick, A. D., Davis, J. W., 2nd, Liu, K. J., Hudson, L. G., Shi, H., Monske, M. L., and Burchiel, S. W. (2003) Benzo(a)pyrene quinones increase cell proliferation, generate reactive oxygen species, and transactivate the epidermal growth factor receptor in breast epithelial cells. Cancer Res. 63, 7825–7833. 874



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Toxic Effects of Methylated Benzo[a]pyrenes in Rat Liver Stem-Like

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