Toxicological Characterization of a Novel in Vivo Benzo[a]pyrene

Nutrition, Karolinska Institutet, Huddinge University Hospital, NOVUM, S-141 ... Robert E. Carroll , Robert A. Goodlad , Aleksandra J. Poole , Ang...
2 downloads 0 Views 105KB Size
Download PDF
1274

Chem. Res. Toxicol. 2002, 15, 1274-1280

Toxicological Characterization of a Novel in Vivo Benzo[a]pyrene Metabolite, 7-Oxo-benz[d]anthracene-3,4-dicarboxylic Acid Anhydride Mirjam M. Nordling,† Jonas Nygren,‡ Jan Bergman,‡ Kathrin Sundberg,§ and Joseph J. Rafter*,† Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, NOVUM, S-141 86 Huddinge, So¨ derto¨ rns ho¨ gskola, Box 4101, S-141 04 Huddinge and Department of Biosciences, Novum, Karolinska Institutet, S-141 57 Huddinge, and Department of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm Received April 30, 2002

Recently, we described a new in vivo pathway in the metabolism of benzo[a]pyrene (BP) that involves an opening of the aromatic ring system. One of the products of this pathway, isolated from rat urine, was the anhydride of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid (ABADA). We have now investigated the effect of ABADA on several cellular targets, known to be important in tumor formation. ABADA was as efficient as BP-7,8-diol-9,10-epoxide in inducing direct strand breaks but not alkali labile sites in DNA in HT-29 cells and exhibited weak mutagenic activity in Salmonella typhimurium strain TA 102. The cytotoxicity of ABADA to HCT 116 cells appeared to be due to apoptosis, as caspase-3 activity and poly-ADP-ribose polymerase (PARP) cleavage was observed. COX-2 promoter activity was induced by ABADA in HCT 116 cells. In conclusion, this novel metabolic pathway may also be contributing to the carcinogenicity of BP.

Introduction Polycyclic aromatic hydrocarbons (PAHs)1 constitute a large group of chemicals produced during the incomplete combustion of organic matter. The occurrence of PAHs is widespread and they can, for example, be found in tobacco smoke, coal, wood, food, water, plants, and soil (1), making exposure to these substances almost impossible to avoid. Benzo[a]pyrene (BP), one of the most studied PAHs, becomes after metabolic activation a powerful carcinogen and has in various in vivo and in vitro experimental systems been shown to induce mutations, chromosomal aberrations, and other genotoxic effects (2). BP is presently believed to be activated by the following mechanisms: monooxygenation to yield bayregion diol epoxides, e.g., BP-7,8-diol-9,10-epoxide (BPDE), and one-electron oxidation to form radical cations (3). The latter pathway is catalyzed by cytochrome P450 or peroxidases producing a radical cation at C-6 that can covalently bind to DNA. The C-6 cation-radical can also undergo nucleophilic attack by water to form 6-hydroxyBP. This phenol is unstable and is rapidly oxidized to a 6-oxo-BP radical that can form BP-1,6, -3,6-, and -6,12* To whom correspondence should be addressed. Phone: +46-8-585 837 17. Fax: +46-8-711 66 59. E-mail: [email protected]. † Department of Medical Nutrition. ‡ So ¨ derto¨rns ho¨gskola and Department of Biosciences. § Department of Environmental Medicine. 1 Abbreviations: PAHs, polycyclic aromatic hydrocarbons; BP, benzo[a]pyrene; ABADA, anhydride of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid; BPDE, BP-7,8-diol-9,10-epoxide; COX-2, cyclooxygenase2; DCA, deoxycholic acid; DMEM, Dulbecco’s modified eagle medium; FCS, fetal calf serum; DMSO, dimethyl sulfoxide; Fpg, E. coli formamidopyrimidine DNA glycosylase; PARP, poly(ADP-ribose) polymerase.

quinones (4). There is also evidence for an additional activation mechanism for BP involving the conversion of BP-7,8-diol to reactive and redox active ortho-quinones by dihydrodiol dehydrogenase. These ortho-quinones can damage DNA either directly, by forming stable and/or depurinating DNA adducts, or indirectly by ROS generation (5). Recently, we described a novel quantitatively important metabolic pathway for BP in the rat, which involved ring opening of the BP moiety (6). Two metabolites, which result from this pathway, were isolated from urine, 7-oxobenz[d]anthracene-3,4-dicarboxylic acid, and the anhydride of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid (ABADA). We suggested that the formation of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid proceeds via 1,6- or 3,6-quinones of BP and that ABADA is spontaneously formed from the dicarboxylic acid (Figure 1). In our previous study (6), we reported that ABADA could induce DNA strand breaks, using the COMET assay, a sensitive and rapid technique enabling the detection of DNA strand breaks in individual cells (7, 8). In the present paper, we have extended our studies on the toxic potential of this novel BP metabolite by further studying its genotoxicity and comparing it to that of its postulated precursors, using the COMET assay. We have also investigated its mutagenicity, using the Ames test and its ability to form DNA adducts. In addition, we have studied the cytotoxic and apoptotic effects of ABADA and its ability to activate transcription of cyclooxygenase-2 (COX-2), an enzyme that contributes to the formation of toxic derivatives of BP.

10.1021/tx025549l CCC: $22.00 © 2002 American Chemical Society Published on Web 09/27/2002

New Aspects of Benzo[a]pyrene Toxicity

Chem. Res. Toxicol., Vol. 15, No. 10, 2002 1275

Figure 1. Metabolites of benzo[a]pyrene (I) formed by ring opening: 7-oxo-benz[d]anthracene-3,4-dicarbolic acid (II) and the anhydride of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid (ABADA) (III).

Materials and Methods Caution. Benzo[a]pyrene and its derivatives are hazardous and should be handled carefully. All laboratory procedures involving these chemicals should be performed using safety gloves and where possible in a well-ventilated fume hood. Chemicals. The anhydride of 7-oxo-benz[d]anthracene-3,4dicarboxylic acid (ABADA) was synthesized by ozonization of BP as described by Moriconi et al. (9). BP-7,8-diol-9,10-epoxide (BPDE), BP-1,6-quinone, BP-3,6-quinone and BP-6,12-quinone were purchased from Midwest Research Institute (Kansas, MO). Deoxycholic acid (DCA) was purchased from Sigma Chemical Company (St. Louis, MO). Cells and Culture. HT-29 and HCT 116 cell lines were purchased from ATCC, Rockville, MD, and used between passages 3 and 25 for all experiments. The HT-29 cell line is derived from a moderately well differentiated grade II human adenocarcinoma and is epithelial-like. It harbors a mutated APC and expresses two truncated APC proteins (10). HCT 116 cells are epithelial-like and derived from a human carcinoma and harbor a normal APC gene (10, 11). Both cell lines were grown as monolayers in Dulbecco’s modified eagle medium (DMEM), 4500 mg/L glucose (Gibco, Life Technologies Ltd, U.K.) with 10% fetal calf serum (FCS), 2 mmol/L L-glutamine, 60 units/mL penicillin and 60 µg/mL streptomycin, in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Detection of DNA Damage (COMET Assay). The method was basically that of Olive et al. (12) with minor modifications. Briefly, HT-29 cells were incubated with ABADA, BP-1,6quinone, BP-3,6-quinone, BP-6,12-quinone (dissolved in dimethyl sulfoxide, DMSO), or BPDE (dissolved in THF) for 2 h at 37 °C in a 5% CO2 atmosphere. Ten microliters ((0.5-1) × 106 cells/mL) of cell suspension was mixed with 150 µL of LMP agarose (0.75% in PBS), distributed on precoated slides, and left to set on an ice tray. After solidification, the slides were treated in one of two ways. For low alkali conditions, the slides were immersed in room-temperature lysing solution (0.03 M NaOH, 1 M NaCl, 1 mM EDTA, and 0.5% sodium N-lauroylsarcosinate) in darkness for 1 h. The slides were then treated with 0.03 M NaOH and 1 mM EDTA for 45 min. Electrophoresis was performed at room temperature, in darkness, in a Bio-Rad subcell GT unit containing the same buffer, for 15 min using 20 V (0.67 V/cm). For high alkali conditions, lysis was performed in darkness for 1 h with an ice-cold freshly prepared solution containing 2 M NaCl, 25 mM EDTA, 20 mM Tris, and 0.5% Triton X-100 (pH 10). The slides were then placed in an electrophoresis buffer (0.3 M NaOH and 1 mM EDTA) in darkness at room temperature for 45 min. Electrophoresis was then carried out at room temperature, for 30 min, as above. After electrophoresis, the slides were neutralized and fixed. The slides were stained with ethidium bromide and examined in a fluorescence microscope, using the program Komet from Kinetic images. Images of 50 randomly selected cells were analyzed from each sample, and tail moment (TM) as defined by Olive et al. (13) was determined. TM ) I × L, where I is the fractional amount of DNA in the comet tail and L is the distance from the center of the comet head to the center of the tail distribution. Experiments were also conducted using the Escherichia coli formamidopyrimidine DNA glycosylase (Fpg, Trevigen Inc., Gaithersburg, MD). Teflon-coated slides with 3 × 14 mm Teflonfree areas were used. The HT-29 cells were incubated with

ABADA as described earlier. Thirty microliters of 0.75% LMP made in PBS and kept at 37 °C was mixed with 4 µL of cell suspension ((0.5-1) × 106 cells/mL) and distributed on the pretreated slides. The slides were lysed as for high alkali conditions as described above. The slides were kept in 25 mM EDTA in PBS overnight at 4 °C and thereafter transferred to an ice-cold enzyme buffer (pH 7.5) containing 20 mM Tris, 100 mM NaCl, 1 mM EDTA, and 0.1 mg/mL bovine serum albumin for 1 h. Twenty microliters of Fpg protein (1 µg/mL) or enzyme buffer was distributed to the slides, which were thereafter put on a chilled plate for 30 min and in a humidified chamber at 37 °C for 30 min. The slides were treated with a low alkali solution and electrophoreses were performed under conditions described before. The slides were protected from light during the experiment, as far as possible. Determination of DNA Adducts. DNA adduct formation was studied by incubating oligonucleotides, 50 nmol of 5′[d(CG)]6 or 50 nmol 5′-[d(AT)]6, in water with a 4-fold excess of the ABADA (200 nmol) overnight at room temperature. Excess ABADA and eventual hydrolysis products were removed by extractions with water-saturated ethyl acetate (four times) and with water-saturated diethyl ether (two times). Following evaporation of the diethyl ether by N2, the reaction mixtures were analyzed by HPLC using a Dynamax RPC 5 µm, C18, 300 Å pore size 4.6 × 250 mm (Varian Associates, Inc., CA). The column was kept at 37 °C. The solvent system used was 0.1 M triethylammoniumacetate, pH 7.0 (solvent A), and 25% triethylammoniumacetate in acetonitrile (solvent B) delivered at 1.5 mL/min. The samples were eluted with the following gradients (10 to 25% B for 10 min, 25% B for 10 min, 25 to 30% B for 5 min, 30 to 60% B for 10 min, and 60 to 100% B for 2 min). The effluent was monitored by UV at 260 nm and by fluorescence (λexcitation ) 425 nm, λemission ) 510 nm). Assay of Mutagenicity (Ames test). ABADA dissolved in DMSO (0.5 mg/mL) was tested with the Salmonella typhimurium tester strains TA 98 and TA 102, obtained from Professor B. Ames, University of California, Berkley, CA, and the National Collections of Type Cultures and Pathogenic Fungi, Public Health Laboratory Service, London, England. Broth cultures were grown in Oxoid Nutrient Broth No. 2 and incubated on a mixing board at 37 °C until a density of about 109 bacteria/mL was reached. Bacteria suspension (0.1 mL, 109 bacteria/mL) was mixed with 100 µL of ABADA and 500 µL of S-9 mix or phosphate buffer (pH 7.4). The S-9 mix was composed of 4 mM NADP, 5 mM glucose-6-phosphate, salt solution (33 mM KCl and 8 mM MgCl2), 100 mM sodium phosphate buffer (pH 7.4) and 5% S-9 fraction prepared from livers of SPF Wistar rats of the strain Mol:WIST. The positive control plates with S-9 mix contained 2-aminoanthracene (4 µg/plate for TA 102 and 2 µg/ plate for TA 98). Cumene hydroperoxide (25 µg/plate for TA 102) and 2-nitrofluorene (1 µg/plate for TA 98) were used as positive control agents without S-9 mix. After 1 h incubation at 37 °C with gentle shaking, 2 mL of molten agar was added to the tubes and the mixture was poured on selective agar plates. The plates were incubated for 72 h at 37 °C, and the number of revertant colonies was counted. All samples were tested in triplicate plates. Assays for Cytotoxicity and Proliferative Activity. The ALMAR BLUE proliferation kit (Almar, Sacramento, CA) was used to measure cytotoxic and proliferative effects in HT-29 and

1276

Chem. Res. Toxicol., Vol. 15, No. 10, 2002

HCT 116 cells. For the cytotoxicity assay, the cells (30 × 103 cells/well) were seeded onto a 96-multiwell plate together with DMEM (10% FCS) and incubated overnight at 37 °C at 5% CO2. The day after, the cells were washed with PBS and incubated with the test substances, diluted in DMEM (0.1% FCS), for 24 h at 37 °C in a 5% CO2 atmosphere. Every experiment was performed in octuple and 500 µM DCA was used as a positive control. Almar blue dye (10%) was added and the cells were placed in the same incubator. After 5-6 h, the plates were read at 570 nm in a microplate spectrophotometer (SPECTRAmax 250) (Molecular Devices Corporation, California). Cell survival was expressed as the percentage absorbance of the mean absorbance of the negative control (DMEM 0.1% FCS). The proliferation assay (20 × 103 cells/well) was performed in a manner similar to the above, with the exception that the cells were starved in DMEM containing 0.1% FCS for 24 h prior to a 48-h treatment with the test substance. Caspase-3 Assay. HCT 116 cells were grown to near confluence in a 75 cm2 bottle. Cells were washed with PBS, trypsinised and centrifuged. The pellet was resuspended in DMEM (0.1% FCS) and transferred to eppendorf tubes together with the test substances. DCA (500 µM) was used as a positive control. After 30 min incubation on a shaker at 37 °C, the cells were centrifuged, and the pellets were lysed with a cell lysis buffer (pH 7.5) containing 10 mM TRIS-HCl, 10 mM NaH2PO4/ NaHPO4, 130 mM NaCl, 1% Triton X-100, and 10 mM NaPPi for 15 min on ice. Forty microliters of cell extract from each treatment was added to eppendorf tubes containing 250 µL of HEPES buffer (pH 7.5) (40 mM HEPES, 20% glycerol, 4 mM DTT) and 2.5 µL fluorogenic marker, DEVD-AMC [N-acetylAsp-Glu-Val-Asp-AMC (7-amino-4-methylcoumarin), Pharmingen, San Diego, CA]. After 1 h incubation at 37 °C, the fluorescence of the mixture was measured in a spectroflurometer at 380 nm excitation and 420-460 nm emission. Values obtained were adjusted to protein concentration of the cell extracts measured using Bio-Rad protein assay. Western Blotting. HCT 116 cells were treated with ABADA (66 µM) for different times. Cell lysates were prepared by treating cells with a lysis buffer (pH 7.8) containing 25 mM Tris acetate EDTA, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 2 mM DTT, and 0.2 mM phenylmethyl sulfonyl fluoride for 15 min on ice. Lysates were thereafter sonicated and centrifuged at 13 500 rpm for 10 min at 4 °C. Protein concentration was measured using the Bio-Rad protein assay and the supernatants were stored at -20 °C until use. SDS-PAGE was performed with 40 µg of protein under reducing conditions on a 7.5% polyacrylamide gel as described by Laemmli (14). The resolved proteins were transferred onto a nitrocellulose membrane as detailed by Towbin et al. (15). The membrane was probed with the mouse monoclonal poly(ADP-ribose) polymerase (PARP) antibody (Pharmingen, San Diego, CA), diluted 1:2000 for 1 h and then with the corresponding secondary antibody to IgG conjugated to horseradish peroxidase (DAKO A/S, Denmark). The ECL+ plus detection system (Amersham Pharmacia Biotech UK Limited, England) was used according to the manufacturer’s instructions. Plasmid Preparation. The COX-2 promoter constructs (-1432/+59, -327/+59) were a kind gift from Drs. Inoue and Tanabe (National Cardiovascular Center Research Institute, Osaka, Japan) (16). The plasmid DNA was purified from the bacteria cultures grown overnight in Luria broth medium containing 50 µg/mL Ampicillin (AstraZenica, So¨derta¨lje, Sweden) by using the Jetstar plasmid maxi kit 20 (GENOMED GmbH, Bad Oeynhausen, Germany). After centrifugation, the pellet was resuspended in a buffer containing 50 mM Tris and 10 mM EDTA (pH 8.0). The cells were lysed (200 mM NaOH and 1% SDS) in room temperature for 5 min, neutralized with 3.1 M potassium acetate (pH 5.5), and thereafter centrifuged at 20 °C and 15000g for 10 min. The supernatant was loaded unto a column and the plasmid DNA was eluted with 1250 mM NaCl and 100 mM Tris (pH 8.5). The DNA was precipitated with 2-propanol and centrifuged at 4 °C and 15000g for 30 min.

Nordling et al.

Figure 2. Induction of single strand breaks in HT-29 cells by ABADA ([), BP-1,6-quinone (9), BP-3,6-quinone (2), BP-6,12quinone (X) and BPDE (b), analyzed by the COMET assay. Tail moment is defined under Materials and Methods. Values are mean of two to seven experiments ( standard deviation. ABADA was tested at higher concentrations because it is more soluble than the other compounds. Transfection and Luciferase Assay. HCT 116 cells (120 × 103 cells/well) were seeded onto a 24-well plate with DMEM (10% FCS) and incubated for 24 h at 37 °C at 5% CO2. The cells were washed with PBS and incubated for 6 h with DMEM containing 2 µg/mL plasmid DNA and 10 µg/mL lipofectin reagent (Gibco, Life Technologies Ltd, U.K.). The transfection mixture was removed and replaced with DMEM (0.1% FCS) for 24 h. The cells were then incubated with the test substances, diluted in DMEM (0.1% FCS). After 16 h, the cells were washed with PBS, and lysed with 100 µL of lysis buffer (pH 7.8) containing 25 mM Tris acetate EDTA, 10% glycerol, 1% Triton X-100, 1 mM EDTA ,and 2 mM DTT for 30 min at 4 °C. The cell lysates were transferred onto a nontransparent 96-multiwell plate (20 µL/well) together with 100 µL of luciferin mix (GenGlow, BioOrbit, Turku, Finland). The luciferase activity was measured using the luminometer Lucy 1 (Anthos Labtec Instruments, Salzburg, Austria) according to the manufacturer’s instructions. Luciferase activity was expressed per microgram of protein in the cell lysate. The luciferase activity of the untreated control cells in each experiment was set to 100%, and the resulting activities for the test agents were calculated in relation (%) to the control cells. Statistical Analyses. To analyze changes in cytotoxicity, proliferation, caspase-3 induction, and COX-2 promoter activity, Student’s t-test (two-tailed) was used. The tests were performed with the software package STATISTICA 5.0 (Statsoft, Tulsa, OK). Ames data was analyzed using the analysis of variance test and Dunnett’s test. The analyses were performed with SAS 8.1 (SAS Institute Inc., Cary, NC). Statistically significant differences are represented as follows: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.

Results DNA Strand Break Induction. Figure 2 shows induction of DNA damage by ABADA, BP-1,6-quinone, BP-3,6-quinone, BP-6,12-quinone, and BPDE in HT-29 cells analyzed using the COMET assay. These experiments were conducted using 0.03 M NaOH alkali electrophoresis solution that enables detection of direct strand breaks. After treatment, a dose-dependent increase in DNA damage was observed for all substances tested and ABADA and BPDE exerted greater effects than the quinones. Similar results were obtained in HCT 116 cells (data not shown). By using a stronger alkali electrophoresis solution (0.3 M NaOH), in the COMET assay, it is possible to detect alkali labile sites, e.g., damaged bases, in addition to the direct strand breaks. Results from the assay, run under these conditions, are shown in Figure 3. It can be mentioned that, in this assay, the high alkali electrophoresis conditions give a

New Aspects of Benzo[a]pyrene Toxicity

Figure 3. Induction of single strand breaks and alkali labile sites in HT-29 cells by ABADA ([), BP-1,6-quinone (9), BP3,6-quinone (2), BP-6,12-quinone (×), and BPDE (b), analyzed by the COMET assay. Values are mean of two to five experiments ( standard deviation. ABADA was tested at higher concentrations because it is more soluble than the other compounds.

Chem. Res. Toxicol., Vol. 15, No. 10, 2002 1277

Figure 5. Mutagenicity of ABADA toward S. typhimurium tester strains TA 98 and TA 102, with and without S-9 mix activation. 2-Nitroflourene (1 µg, 730 revertant colonies/plate) was used as a positive control for TA 98 without S-9 mix and 2-aminoanthracene (2 µg, 831 revertant colonies/plate) with S-9 mix. For TA 102, cumene hydroperoxide (25 µg, 1140 revertant colonies/plate) was used without S-9 mix and 2-aminoanthracene (4 µg, 811 revertant colonies/plate) with S-9 mix. Values are mean of three plates ( standard deviation. (**) P < 0.01 compared with control.

Figure 4. Determination of the degree of oxidative DNA base damage in HT-29 cells, by the use of Fpg-enzyme in the COMET assay. ABADA + Fpg ([), ABADA - Fpg (]). Values are mean of three to four experiments ( standard deviation.

lower tail moment than low alkali electrophoresis conditions, at a certain number of strand breaks. The ratio between strand breaks (0.03 M NaOH) and strand breaks + alkali labile sites (0.3 M NaOH) gives an indication of the amount of alkali labile base damage relative to strand breaks. These results indicated that, in contrast to BPDE, the majority of DNA damage caused by ABADA is due to direct strand breaks and not alkali labile sites. The results also indicate that BP-3,6-quinone is giving rise to a significant number of alkali labile sites. Both BP1,6-quinone and BP-6,12-quinone yielded little DNA damage. By using the Fpg protein, in combination with the COMET assay, it is possible to measure oxidative DNA damage such as 8-oxoguanine and other oxidized purines (17). The results presented in Figure 4 indicate that a large part of the damage, induced by ABADA, is due to such oxidative damage. None of the vehicles used, DMSO or THF, had any effect on tail moment. Using the assay for DNA adduct formation (described in the Materials and Methods), ABADA was shown to produce no DNA adducts (data not shown). Effects on Mutagenicity. The mutagenic potential of ABADA was tested using S. typhimurium strains TA 98 and TA 102 with and without S-9 activation (Figure 5). ABADA did not exhibit toxic effects in either tester strain at the dose levels tested. At the higher doses tested, ABADA showed a statistically significant increase in the number of revertant colonies in TA 102, both with and without S-9 mix, and in TA 98 in the absence of S-9 mix. These increases reached a maximum of 1.5-fold higher than the negative control value in TA 102 with S-9 mix at a concentration of 62 µM.

Figure 6. (A) Effect of ABADA on cytotoxicity in HT-29 cells ([) and HCT 116 cells (]). (B) Effect of BPDE (O) and BP-3,6quinone (4) on cytotoxicity in HT-29 cells (filled symbols) and HCT 116 cells (unfilled symbols). Results represent mean values (n ) 8 × 3 experiments) ( standard deviation, in relation to the untreated cells, which were set to 100%. DCA (500 µM) was used as a positive control: % cell survival, 42% (HT-29 cells), 17% (HCT 116 cells).

Effects on Cytotoxicity and Cell Proliferation. Using the ALMAR BLUE proliferation kit, under conditions to detect cell toxicity, it was found that both ABADA and BPDE induced a dose-dependent cytotoxic effect in HCT 116 cells, with ABADA exerting the greater cytotoxicity, and little or no cytotoxic effect in HT-29 cells (Figure 6). On the other hand, BP-3,6-quinone did not exert any cytotoxicity in either cell line (Figure 6B). When the above kit was used, under conditions to detect cell proliferative effects, a significant induction of cell proliferation after treatment with ABADA was observed in HT-29 cells but not in HCT 116 cells (Figure 7). No effects on cell proliferation were observed, in either cell line, after treatment with BPDE or BP-3.6-quinone (data not shown).

1278

Chem. Res. Toxicol., Vol. 15, No. 10, 2002

Figure 7. Effect of ABADA on cell survival in HT-29 cells ([) and HCT 116 cells (]). Results represent mean values (n ) 8 × 3 experiments) ( standard deviation, in relation to the untreated cells, which were set to 100%. DCA (500 µM) was used as a positive control: % cell survival, 39% (HT-29 cells), 14% (HCT 116 cells).

Nordling et al.

Figure 10. Induction of COX-2 promotor activity in HCT 116 cells by ABADA (10-20 µM). TPA (160 nM) and FCS (10%) were used as positive controls. Diagram bars represent mean values (n ) 7-11) ( standard deviation in relation to untreated cells (control), which were set to 100%. (***) P < 0.001 compared with control.

treatment with 10% FCS and TPA (160 nM) (positive controls) caused a marked increase in COX-2 promoter activity. It was also clear that ABADA, at concentrations 10, 15, and 20 µM, significantly induced promoter activity (Figure 10). When the cells were transfected with the shorter construct (-327/+59), the results were of the same magnitude as those given in Figure 10 (data not shown). Figure 8. Caspase-3 activity in HCT 116 cells treated with ABADA for 30 min. Values are mean of three experiments ( standard deviation, calculated in relation to the control cells, which were set to 100%. DCA (500 µM) was used as a positive control (1800%).

Figure 9. Western blot, showing PARP protein (116 kDa) from cell extracts (HCT 116 cells) treated with ABADA (66 µM) for 1-20 h. Lane 1, untreated 20 h; lane 2, 1 h treatment; lane 3, 2 h; lane 4, 4 h; lane 5, 5 h; lane 6, 6 h; lane 7, 18 h; lane 8, 20 h. The lower band appearing in lanes 7 and 8 represent cleaved PARP (85 kDa).

Effects on Caspase-3 Activation and PARP Cleavage. In view of the ability of ABADA to induce DNA strand breaks and its cytotoxicity in HCT 116 cells, we considered it of interest to examine the effect of ABADA on apoptosis-related parameters in HCT 116 cells. A dose-dependent induction of caspase-3 activity by ABADA was observed already after 30 min (Figure 8). The induction of caspase-3 activity remained at the level seen at 30 min, for up to 4 h (data not shown). To investigate the degradation of PARP (a protein substrate for caspase-3) western blots were performed. Protein extracts from HCT 116 cells, treated with ABADA for up to 20 h, were electroblotted and probed with a PARP monoclonal antibody that recognizes the 116 kDa intact form as well as an 85 kDa fragment. ABADA was shown to be able to induce PARP cleavage after 18 h of treatment (Figure 9). Effects on COX-2 Promoter Activity. To investigate the ability of ABADA to modulate the expression of COX2, transient transfections were performed in HCT 116 cells with a human full-length COX-2 promoter-luciferase construct (-1432/+59). As is evident from Figure 10,

Discussion We recently reported on a quantitatively important new pathway in the metabolism of BP in the rat that involves an opening of the aromatic ring system (6). We also demonstrated, in preliminary experiments using the COMET assay, that one of the products of this pathway, i.e., the anhydride of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid (ABADA), isolated from urine, exhibited genotoxic activity. In view of the much studied toxicity of BP, any metabolic route resulting in products with biological activity deserves further scrutiny. In the present study, we have carried out a more thorough characterization of the biological activity/toxicity of ABADA. It can be mentioned that the concentrations used in the experiments discussed below were in the range of the concentration of ABADA found in rat urine (∼67 µM) in our previous studies. First, we extended our studies utilizing the COMET assay and were able to show that ABADA and the extensively studied genotoxic BPDE had a similar ability to induce direct strand breaks in DNA in HT-29 colonic cells. The potential precursors of ABADA, the 1,6- and 3,6-quinones of BP, were considerably less genotoxic in the assay. The results also showed that ABADA was not as efficient as BPDE at inducing alkali labile sites in DNA (base adducts). This is in line with our finding that ABADA did not give rise to DNA adducts in the present study. However, it must be pointed out that the approach used to assay for DNA adducts in this study has limitations, in terms of both sensitivity and dynamic range, and thus final conclusions regarding DNA adduct formation by ABADA must await further study. Interestingly, the BP-3,6-quinone appeared to be an efficient inducer of alkali labile sites. In addition, by using the Fpg protein in the COMET assay, that nicks the DNA at sites of oxidative damage (17), we were able to establish more firmly our earlier preliminary finding that this type of oxidative damage is important in the

New Aspects of Benzo[a]pyrene Toxicity

case of ABADA. The reasons for the use of colonic cells in the above genotoxicity assay included the interest of this laboratory in the role of environmental factors in tumor formation in the colon and the fact that BP, administered to rats, is predominantly excreted via the feces (18). Studies are underway to determine whether 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid and ABADA are excreted in feces, in addition to urine, after administration of BP to rats. It can also be mentioned that the type of genotoxic activity that is detected by the COMET assay, precedes tumor formation in the colon of rats that were treated with chemical carcinogens (19). It is also interesting that components of human feces have been shown to induce this type of DNA damage in colonic cells (20) and that diet can influence the capacity of feces to induce this damage (21). The mutagenic potential of ABADA was also checked in the standard Ames test. Small, statistically significant increases in the number of revertant colonies were observed in tester strain TA 102 (detects oxidative mutagens) at the highest dose levels both in the presence and absence of S-9 mix, and in strain TA 98 (detects frameshift mutagens) at the highest dose level in the absence of S-9 mix. Since these increases reached a maximum of 1.5-fold higher than the negative control value only in TA 102, we conclude only that ABADA showed weak mutagenic activity in this strain, which would be in line with its ability to induce oxidative DNA damage as discussed above. It is clear that these initial results on the mutagenicity of ABADA must be followed up by a more thorough study of this effect. In view of the DNA damaging effects, discussed above, we considered it of interest to determine whether these compounds were also inducing cytotoxicity. BPDE and to a greater extent ABADA were cytotoxic in HCT 116 cells, whereas little or no effect was observed in HT-29 cells. BP-3,6-quinone induced little or no cytotoxicity in both cell lines. What is evident from Figure 6 is that the HCT 116 cells were markedly more sensitive to the cytotoxic effects of all three compounds than the HT-29 cells. It is interesting to note that HCT 116 cells harbor normal APC and p53 genes (10, 22), both of which are mutated and nonfunctional in HT-29 cells (10, 22). Both of these genes are important for the initiation of the apoptosis program and may contribute to explain the difference observed between the two cell lines in the cell death response (23, 24). Finally, not only did ABADA not induce cytotoxicity in the HT-29 cells, it induced a proliferative effect. Any significance that this finding has for tumorigenesis requires further work. We then considered it of interest to determine whether the cell death, induced by ABADA, was due to apoptosis. This appeared to be in fact the case as this BP metabolite induced caspase-3 activity and PARP cleavage, two hallmarks of the apoptotic process. The fact that we did not see PARP cleavage earlier than 18 h was surprising in view of the observation that caspase-3 activity began to increase already after 30 min. However, we did not assay caspase-3 activity at time points greater than 4 h, and it cannot be ruled out that one would have seen even more marked increases in enzyme activity at later time points. A later caspase-3 induction (12-24 h) has actually been reported when HeLa cells are exposed to BP (25). On the basis of our results, we cannot say that the DNA damage, being induced by ABADA, is initiating the apoptotic process but it is a possibility.

Chem. Res. Toxicol., Vol. 15, No. 10, 2002 1279

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins. There are two isoforms of COX, designated COX-1 and COX-2. COX-1 is expressed constitutively in most tissues and appears to be responsible for housekeeping functions (26). In contrast, COX-2 is not detectable in most normal tissues but is induced by oncogenes, growth factors, tumor promoters, and carcinogens (27-29). Several lines of evidence support the idea that COX-2 is important in carcinogenesis (30). COX also contributes to the formation of toxic derivatives of BP, e.g., it catalyzes the conversion of BP7,8-diol to highly reactive BP diolepoxides that bind DNA (31). Thus, our results which indicate that ABADA can induce COX-2 transcription are interesting and may suggest a feedback mechanism capable of amplifying the toxic effects of BP. Our observation that activation of COX-2 promoter activity by ABADA using the shorter COX-2 promoter construct (-327/+59), containing the response elements, SP-1, NF-kB, AP-2, C/EBP, and c-AMP response element (CRE), gave similar results to using the full-length construct (-1432/+59) indicates that the above response elements are mediating this effect of ABADA. Overexpression of COX-2 in epithelial cells has been shown to inhibit apoptosis and increase the invasiveness of malignant cells, thus favoring tumorigenesis and metastasis (32-35). This is interesting in view of the effect of ABADA on apoptosis discussed above. However, it can be pointed out that the well-known tumor promoter, the bile acid deoxycholate, also induces the same panorama of effects in HCT 116 cells as ABADA, i.e., DNA strand breaks, apoptosis and COX-2 promoter activity. In conclusion, it is clear that ABADA, a recently discovered product of BP metabolism in vivo, has marked effects on several cellular targets known to be important in tumor formation. Although it is still too early to classify ABADA as a potential tumor initiator or tumor promoter, one might speculate that it has more potential as a promoter, based on the above results. Thus, our recently discovered pathway in the in vivo metabolism of BP, that involves an opening of the aromatic ring system, may be an additional route involved in the carcinogenicity of BP. Since ABADA was identified in urine, bladder cancer is a potential target as it is known to be related to tobacco smoking, a rich source of PAHs.

Acknowledgment. This work was supported by the Swedish Cancer Society.

References (1) Angerer, J., Mannschreck, C., and Gundel, J. (1997) Biological monitoring and biochemical effect monitoring of exposure to polycyclic aromatic hydrocarbons. Int. Arch. Occup. Environ. Health 70, 365-377. (2) Hollstein, M., McCann, J., Angelosanto, F. A., and Nichols, W. W. (1979) Short-term tests for carcinogens and mutagens. Mutat. Res. 65, 133-226. (3) Cavalieri, E. L., and Rogan, E. G. (1995) Central role of radical cations in metabolic activation of polycyclic aromatic hydrocarbons. Xenobiotica 25, 677-688. (4) Cavalieri, E. L., and Rogan, E. G. (1992) The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation. Pharmacol. Ther. 55, 183-199. (5) 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.

1280

Chem. Res. Toxicol., Vol. 15, No. 10, 2002

(6) Yang, Y., Griffiths, W. J., Nordling, M., Nygren, J., Moller, L., Bergman, J., Liepinsh, E., Otting, G., Gustafsson, J. A., Rafter, J., and Sjovall, J. (2000) Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway for formation of potentially genotoxic metabolites. Biochemistry 39, 15585-15591. (7) Fairbairn, D. W., Olive, P. L., and O’Neill, K. L. (1995) The comet assay: a comprehensive review. Mutat. Res. 339, 37-59. (8) Holz, O., Jorres, R., Kastner, A., Krause, T., and Magnussen, H. (1995) Reproducibility of basal and induced DNA single-strand breaks detected by the single-cell gel electrophoresis assay in human peripheral mononuclear leukocytes. Int. Arch Occup. Environ. Health 67, 305-310. (9) Moriconi, E. J., Rakoczy, B., and O’Connor, W. F. (1961) Ozonolysis of polycyclic aromates. VIII. Benzo[a]pyrene. J. Am. Chem. Soc. 83, 4618-4623. (10) Hsi, L. C., Angerman-Stewart, J., and Eling, T. E. (1999) Introduction of full-length APC modulates cyclooxygenase-2 expression in HT-29 human colorectal carcinoma cells at the translational level. Carcinogenesis 20, 2045-2049. (11) Kutchera, W., Jones, D. A., Matsunami, N., Groden, J., McIntyre, T. M., Zimmerman, G. A., White, R. L., and Prescott, S. M. (1996) Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect. Proc. Natl. Acad. Sci. U.S.A. 93, 4816-4820. (12) Olive, P. L., Wlodek, D., Durand, R. E., and Banath, J. P. (1992) Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp. Cell Res. 198, 259-267. (13) Olive, P. L., Banath, J. P., and Durand, R. E. (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “comet” assay. Radiat. Res. 122, 86-94. (14) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. (15) Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354. (16) Inoue, H., Nanayama, T., Hara, S., Yokoyama, C., and Tanabe, T. (1994) The cyclic AMP response element plays an essential role in the expression of the human prostaglandin-endoperoxide synthase 2 gene in differentiated U937 monocytic cells. FEBS Lett. 350, 51-54. (17) Collins, A. R., Dusinska, M., Gedik, C. M., and Stetina, R. (1996) Oxidative damage to DNA: do we have a reliable biomarker? Environ. Health Perspect. 104 (Suppl. 3), 465-469. (18) Yang, Y., Sjovall, J., Rafter, J., and Gustafsson, J. A. (1994) Characterization of neutral metabolites of benzo[a]pyrene in urine from germfree rats. Carcinogenesis 15, 681-687. (19) Pool-Zobel, B. L., Neudecker, C., Domizlaff, I., Ji, S., Schillinger, U., Rumney, C., Moretti, M., Vilarini, I., Scassellati-Sforzolini, R., and Rowland, I. (1996) Lactobacillus- and bifidobacteriummediated antigenotoxicity in the colon of rats. Nutr. Cancer 26, 365-380. (20) Venturi, M., Hambly, R. J., Glinghammar, B., Rafter, J. J., and Rowland, I. R. (1997) Genotoxic activity in human faecal water and the role of bile acids: a study using the alkaline comet assay. Carcinogenesis 18, 2353-2359. (21) Rieger, M. A., Parlesak, A., Pool-Zobel, B. L., Rechkemmer, G., and Bode, C. (1999) A diet high in fat and meat but low in dietary fibre increases the genotoxic potential of ‘faecal water’. Carcinogenesis 20, 2311-2316.

Nordling et al. (22) Chendil, D., Oakes, R., Alcock, R. A., Patel, N., Mayhew, C., Mohiuddin, M., Gallicchio, V. S., and Ahmed, M. M. (2000) Low dose fractionated radiation enhances the radiosensitization effect of paclitaxel in colorectal tumor cells with mutant p53. Cancer 89, 1893-1900. (23) Rich, T., Allen, R. L., and Wyllie, A. H. (2000) Defying death after DNA damage. Nature 407, 777-783. (24) Browne, S. J., Williams, A. C., Hague, A., Butt, A. J., and Paraskeva, C. (1994) Loss of APC protein expressed by human colonic epithelial cells and the appearance of a specific lowmolecular-weight form is associated with apoptosis in vitro. Int. J. Cancer 59, 56-64. (25) Yoshii, S., Tanaka, M., Otsuki, Y., Fujiyama, T., Kataoka, H., Arai, H., Hanai, H., and Sugimura, H. (2001) Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene. Mol. Cell Biol. 21, 6796-6807. (26) Smith, W. L., Garavito, R. M., and DeWitt, D. L. (1996) Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J. Biol. Chem. 271, 33157-33160. (27) Subbaramaiah, K., Telang, N., Ramonetti, J. T., Araki, R., DeVito, B., Weksler, B. B., and Dannenberg, A. J. (1996) Transcription of cyclooxygenase-2 is enhanced in transformed mammary epithelial cells. Cancer Res. 56, 4424-4429. (28) DuBois, R. N., Awad, J., Morrow, J., Roberts, L. J., 2nd, and Bishop, P. R. (1994) Regulation of eicosanoid production and mitogenesis in rat intestinal epithelial cells by transforming growth factor-alpha and phorbol ester. J. Clin. Invest. 93, 493498. (29) Kelley, D. J., Mestre, J. R., Subbaramaiah, K., Sacks, P. G., Schantz, S. P., Tanabe, T., Inoue, H., Ramonetti, J. T., and Dannenberg, A. J. (1997) Benzo[a]pyrene up-regulates cyclooxygenase-2 gene expression in oral epithelial cells. Carcinogenesis 18, 795-799. (30) Markowitz, S. D., Dawson, D. M., Willis, J., and Willson, J. K. V. (2002) Focus on colon cancer. Cancer Cell 1, 233-236. (31) Eling, T. E., and Curtis, J. F. (1992) Xenobiotic metabolism by prostaglandin H synthase. Pharmacol. Ther. 53, 261-273. (32) Boolbol, S. K., Dannenberg, A. J., Chadburn, A., Martucci, C., Guo, X. J., Ramonetti, J. T., Abreu-Goris, M., Newmark, H. L., Lipkin, M. L., DeCosse, J. J., and Bertagnolli, M. M. (1996) Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res. 56, 2556-2560. (33) Sheng, H., Shao, J., Morrow, J. D., Beauchamp, R. D., and DuBois, R. N. (1998) Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res. 58, 362-366. (34) Sheng, H., Shao, J., O’Mahony, C. A., Lamps, L., Albo, D., Isakson, P. C., Berger, D. H., DuBois, R. N., and Beauchamp, R. D. (1999) Transformation of intestinal epithelial cells by chronic TGF-beta1 treatment results in downregulation of the type II TGF-beta receptor and induction of cyclooxygenase-2. Oncogene 18, 855867. (35) Tsujii, M., and DuBois, R. N. (1995) Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 83, 493-501.

TX025549L