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Chem. Res. Toxicol. 2005, 18, 224-231

Effect of a Standardized Complex Mixture Derived from Coal Tar on the Metabolic Activation of Carcinogenic Polycyclic Aromatic Hydrocarbons in Human Cells in Culture Brinda Mahadevan,†,‡ Charis P. Marston,†,‡ Wan-Mohaiza Dashwood,† Yonghai Li,§ Clifford Pereira,§ and William M. Baird*,† Departments of Environmental and Molecular Toxicology and Statistics, Oregon State University, Corvallis, Oregon 97331 Received August 27, 2004

A complex mixture of polycyclic aromatic hydrocarbons (PAH) extracted from coal tar, standard reference material (SRM) 1597, has been shown to initiate tumor formation in mouse initiation-promotion assays in our laboratory [(2001) Carcinogenesis 22 (7), 1077-1086]. To determine the effects of SRM 1597 on PAH activation in human cells, we investigated the PAH-DNA adduct formation in the human mammary carcinoma-derived cell line MCF-7. We examined the effects of SRM 1597 on the metabolic activation to DNA binding derivatives of two carcinogenic PAHs, the bay region containing benzo[a]pyrene (B[a]P) and the more carcinogenic fjord region containing dibenzo[a,l]pyrene (DB[a,l]P). PAH-DNA adduct analysis by 33P-postlabeling and reversed phase high-performance liquid chromatography revealed a significant decrease in the levels of both B[a]P and DB[a,l]P DNA adduct formation on cotreatment with SRM 1597 in comparison to cells exposed to B[a]P or DB[a,l]P alone. However, the inhibition of PAH-DNA adduct formation only occurred within the first 48 h of exposure in cells cotreated with SRM 1597 and B[a]P. In contrast, SRM 1597 significantly inhibited the level of DB[a,l]P DNA adducts throughout the 120 h of exposure. Induction of human cytochrome P450 (P450) enzymes 1A1 and P4501B1 on treatment with SRM 1597 was observed by immunoblots. These results suggest that the important factors in determining the carcinogenic activity of PAH within a complex mixture would depend on the ability of other components of the mixture to promote or inhibit the activation of carcinogenic PAH by the induction of P450 enzymes followed by the formation of DNA adducts.

Introduction Humans are environmentally exposed to complex mixtures of polycyclic aromatic hydrocarbons (PAHs)1 in the atmosphere from combustion sources such as cigarette smoking and vehicle emissions as well as industrially through coal tar production (1-3). Coal tar, a byproduct of the gasification process, is a complex mixture of hundreds of different compounds, many of which are PAHs. Coal tar has been found to initiate tumor formation (4-8) and lead to DNA binding (7-15) when applied topically to mouse skin. Recent epidemiological studies have shown evidence of an elevated risk for lung cancer among asphalt workers (16, 17). Many individual PAHs have also been shown to be potent carcinogens in rodents, i.e., benzo[a]pyrene (B[a]P), 7,12dimethylbenz[a]anthracene (DMBA), and dibenzo[a,l]* To whom correspondence should be addressed. Tel: 541-737-1886. Fax: 541-737-4371. E-mail: [email protected]. † Department of Environmental and Molecular Toxicology. ‡ These authors contributed equally to this work. § Department of Statistics. 1 Abbreviations: B[a]P, benzo[a]pyrene; DB[a,l]P, dibenzo[a,l]pyrene; DB[a,l]PDE, dibenzo[a,l]pyrene-(11S,12R)-dihydrodiol (13S,14R)epoxide(s); DMBA, 7,12-dimethylbenz[a]anthracene; MCF-7, mammary carcinoma cells; P450, cytochrome P450; PAH, polycyclic aromatic hydrocarbon(s); SRM, standard reference material; NIST, National Institute of Standards and Technology; DMSO, dimethyl sulfoxide; PMSF, phenylmethylsulfonyl fluoride.

pyrene (DB[a,l]P) (18-20), and several PAH mixtures have been implicated in the induction of human cancer (1-3). Carcinogenic PAHs such as B[a]P (bay region containing molecule) are metabolically activated to B[a]P7,8-diol-9,10-epoxide (B[a]PDE) (21) and have been identified as a component of coal tar (22). DB[a,l]P (fjord region containing molecule) is the most potent carcinogenic PAH identified in rodent bioassays (18-20) and has been identified as a component in complex mixtures of smoky coal tar (23) and air particulate (24). Metabolism of both B[a]P and DB[a,l]P to its ultimate carcinogenic metabolites occurs primarily through oxidation by cytochrome P450 (P450) enzymes. The long-term exposure to low levels of complex mixtures of PAH greatly complicates the task of predicting relative risk that PAH mixtures pose to humans. Elucidating the mechanisms of carcinogenesis by PAH mixtures is further complicated by numerous findings that weakly or noncarcinogenic PAHs present in these complex mixtures can act as co- or anticarcinogens by enhancing or decreasing the carcinogenic activity of known carcinogens (25-29). Consequently, the analysis of the levels of certain carcinogenic PAHs present in complex mixtures such as B[a]P is not sufficient to allow prediction of the risk that exposure to that mixture poses toward humans. It is also necessary to take into account

10.1021/tx0497604 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/12/2005

Coal Tar Affects Metabolic Activation of PAH

the modifiers within the mixture that may alter the carcinogenic potency of the carcinogenic PAH present. An understanding of the mechanisms by which co- and anticarcinogens modify carcinogenic PAH metabolism, metabolic activation, and the induction of biological effects is necessary to determine the relative carcinogenic potential of complex PAH mixtures. The National Institute of Standards and Technology (NIST) has characterized the standard reference material (SRM) with respect to both PAH composition (30) and mutagenic activity (31). Recently, Schubert et al. (32) determined the PAH with molecular weights 300 and 302 in the environmental matrix of SRMs including SRM 1597. The studies described here were conducted with SRM 1597, which is a natural, combustion-related complex mixture of PAHs extracted from a medium crude coke oven coal tar and dissolved in toluene (33). To determine how the concentration of carcinogenic PAH within the coal tar mixture affects DNA binding in human cells, we treated the cells with SRM 1597 with additional amounts of B[a]P or DB[a,l]P. We then compared DNA binding to that of cells treated with the same concentration of the carcinogenic PAHs alone. Here, we report the effects of SRM 1597 on the metabolic activation and PAH-DNA binding of B[a]P and DB[a,l]P on mammary carcinoma (MCF-7) cells in culture.

Experimental Procedures Caution: DB[a,l]P and B[a]P are potent carcinogenic agents and should be handled according to the NIH guidelines for the use of carcinogens. SRM 1597 is also a suspected human carcinogen and should be handled with the same precautions. Chemicals. B[a]P and DB[a,l]P were purchased from Chemsyn Science Laboratories (Lenexa, KS). Nuclease P1 (EC 3.1.30.1; from Penicillium citrinum), human prostatic acid phosphatase (EC 3.1.3.2; from human semen), apyrase (EC 3.6.1.5; from Solanum tuberosum), phosphodiesterase I (EC 3.1.4.1; from Crotalus atrox), and proteinase K (EC 3.4.21.64; from Tritirachium album) were purchased from Sigma Chemical Co. (St. Louis, MO). RNase T1 (EC 3.1.21.3; from Asperigillus oryzae) and RNase (DNase free, a heterogeneous mixture of ribonucleases from bovine pancreas) were obtained from Boehringer Mannheim (Indianapolis, IN). Unequilibrated phenol and cloned T4 polynucleotide kinase were purchased from United States Biochemical (Cleveland, OH). [γ-33P]ATP (1 mCi) was purchased from Amersham (Arlington Heights, IL). Cell Culture and Treatment. The human mammary carcinoma-derived MCF-7 cell line was obtained from the Michigan Cancer Foundation (Karmanos Cancer Center, Detroit, MI). The cells were cultured in a 75 cm2 flask (Corning, Corning, NY) in a 1:1 mixture of F-12 Nutrient Mixture (Gibco-Invitrogen, Carlsbad, CA) and Dulbecco’s Modified Eagle Medium (GibcoInvitrogen). The medium was supplemented with 10% fetal calf serum (Intergen, Purchase, NY), containing 15 mM HEPES buffer and antibiotics (200 units/mL penicillin, 200 µg/mL streptomycin, and 25 µg/mL ampicillin) at 37 °C with 5% CO2. Cell cultures were subcultured at a ratio of 1:4 once the surface of the flask was completely covered. All cell culture flasks, in which cells covered approximately 90% of the surface, were replenished with fresh medium (20 mL) 24 h prior to treatment. SRM 1597 was obtained from NIST (Gaithersburg, MD) and had a concentration of 8 g of coal tar sample per liter of toluene. Before treatment with SRM 1597, toluene was removed via evaporation under nitrogen gas and the residue was reconstituted in dimethyl sulfoxide (DMSO). SRM 1597 contained a certified concentration of 95.8 + 5.8 mg of B[a]P per kg or 82.9 ( 5.3 µg/mL (33). A detailed description and chemical composition of SRM 1597 are available under http://patapsco.nist.gov/srmcatalog/certificates/1597.pdf. SRM

Chem. Res. Toxicol., Vol. 18, No. 2, 2005 225 1597 treatments in cell culture were performed using a dose of 400 µg in 50 µL of DMSO, containing 4.1 µg of B[a]P. The cells were treated with either DMSO (75 µL) as solvent control, SRM 1597 alone (400 µg), B[a]P (20.2 µg), SRM 1597 (400 µg) plus B[a]P (20.2 µg), DB[a,l]P (0.2 µg), or SRM 1597 (400 µg) plus DB[a,l]P (0.2 µg). The concentrations were based on previous studies in our laboratory using 1 mg/mL B[a]P and 8 mg/mL SRM 1597 (9, 34). We treated with 100-fold higher concentration of B[a]P in comparison to DB[a,l]P based upon their relative carcinogenic potency in mouse skin assays (9). The cells were harvested after 6, 12, 24, 48, 72, 96, 120, 144, 168, and 192 h of continuous exposure. The cells were harvested at appropriate exposure times after treatment and washed with phosphatebuffered saline (PBS), and the cell pellet was stored at -80 °C. DNA Isolation. A standard DNA isolation protocol was used (35). Briefly, cell pellets were homogenized in a glass homogenizer with EDTA-SDS buffer [10 mM Tris, 1 mM Na2EDTA, 1% SDS (w/v), pH 8]. The homogenates were treated with DNase-free RNase, (50 U/mL) and RNase T1 (1000 U/mL) (Boehringer-Mannheim) at 37 °C for 1 h, followed by treatment with proteinase K (500 µg/mL) (Sigma) at 37 °C for 1 h. The DNA was extracted with equal volumes of Tris-equilibrated phenol (Boehringer-Mannheim) followed by extraction with 1:1 volume of Tris-equilibrated phenol and chloroform:isoamyl alcohol (24:1) and then with equal volumes of chloroform:isoamyl alcohol (24:1). The aqueous layer was treated with a 1/10 volume of 5 M NaCl and twice the volume of cold 100% ethanol to precipitate the DNA, which was then dissolved in doubledistilled water. The concentration was determined by UV absorbance at 260 nm. 33P-Postlabeling of DNA Adducts. Postlabeling was carried out as described previously (36). Briefly, 10 µg of DNA isolated from treated MCF-7 cells was digested with nuclease P1 and prostatic acid phosphatase, postlabeled with [γ-33P]ATP (3000 Ci/mmol), cleaved to adducted mononucleotides with snake venom phosphodiesterase I, and prepurified with a Sep-Pak C18 cartridge (Waters, Milford, MA). Subsequent separation by analytical HPLC (HPLC-Water system equipped with two pumps; Waters) was carried out using a C18 reverse phase column (5 µm Ultrasphere ODS, 4.6 mm × 250 mm). For the determination of the effects of SRM 1597 on B[a]P-DNA binding, adducts were resolved by elution at 1 mL/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A), and 100% HPLC grade methanol (solvent B). The elution gradient was as follows: 44-60% solvent B over 40 min, 60-80% solvent B over 10 min, isocratic elution at 80% solvent B over 10 min, and 80 to 44% solvent B over 5 min. For the determination of the effects of SRM 1597 on DB[a,l]P-DNA binding, adducts were resolved by elution at 1 mL/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A), and 90% HPLC grade methanol/10% acetonitrile (solvent B). The elution gradient was as follows: 44-55% solvent B for 50 min, 55-80% solvent B over 10 min, and 80 to 44% solvent B over 10 min. The radiolabeled nucleotides were detected by an on-line radioisotope flow detector (Packard Instruments, Downers Grove, IL), and the level of DNA binding was calculated based on the labeling efficiency of a [3H]B[a]P7,8-dihydrodiol 9,10-epoxide standard (37). Three independent sets of the postlabeling reaction were carried out for every sample treated, to determine the total PAH-DNA adduct levels.

Microsome Isolation. Microsomes were prepared as described by Otto et al. (38) with minor modifications. Briefly, cell culture samples were homogenized with a steel homogenizer containing microsomal homogenization buffer [0.25 M K2HPO4, 0.15 M KCl, 10 mM EDTA, and 0.25 mM phenylmethylsulfonylfluoride (PMSF)] and centrifuged at 15000g for 20 min at 4 °C. The supernatant was centrifuged at 100000g for 90 min at 4 °C, and the pellet was resuspended in microsome dilution buffer (0.1 M KH2PO4, 20% glycerol, 10 mM EDTA, 0.1 mM DTT, and 0.25 mM PMSF). The protein concentration was spectrophotometrically determined at 562 nm using the Bicinchoninic acid colorimetric assay (Pierce, Rockford, IL).

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Western Blot. Microsomal proteins were separated by SDSPAGE utilizing 8% acrylamide separating gel and a 2.5 cm 3% acrylamide stacking gel according to Laemmli (39). Microsomal proteins (50 µg) were diluted with sample buffer [0.625 M Trisbase (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromophenol blue] to a final volume ranging from 50 to 70 µL. Proteins were denatured by boiling for 3 min followed by rapid cooling on ice. Following gel electrophoresis at 20 mA for 3-4 h, the microsomal proteins were transferred to nitrocellulose using a TE50 Transfor apparatus (Hoeffer Scientific, San Francisco, CA) at 250 mA for 2 h. Uniformity of sample loading was confirmed by staining the nitrocellulose membrane with Ponceau S (Sigma). The membrane was washed three times with PBS-T [PBS with 0.3% (w/v) Tween-20] for 5 min each and blocked with 5% Carnation nonfat dry milk in 150 mL of PBS-T and 0.2% goat serum for 1 h on a shaker. After three further washes with PBS-T, the membrane was then incubated with the primary antibody for 2 h while shaking and washed again with PBS-T. Human P4501A1 was detected utilizing a rabbit polyclonal P4501A1 antibody (1:200 dilution) prepared against purified recombinant human P4501A1 protein. The antibody against human P4501A1 was a gift from Dr. F. P. Guengerich (Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN). Dr. C. Marcus (Department of Pharmacology and Toxicology, University of New Mexico, Albuquerque, NM) prepared and provided the human P4501B1 rabbit polyclonal antibody (1:200 dilution). The immunoreactive proteins were detected by incubating the membrane with peroxidase-conjugated anti-rabbit IgG (1:15000 dilution in PBS-T with 0.2% goat serum) for 30 min. After three washes with PBS-T, the immunoreactive proteins were observed utilizing an enhanced chemiluminescence detection method, as described by the manufacturer (Amersham Life Science). The positive control used for the detection of P4501A1 enzyme was Aroclor-induced rat liver S9 (Gentest, Bedford, MA). Microsomal protein isolated from 10 mM benz[a]anthracene-treated transformable mouse embryo C3H10T1/2 cells was used as the positive control for the detection of P4501B1 protein (40, 41). Statistical Analysis. At each time of exposure, the adduct levels were compared between treatments. To achieve homogeneity of variance, the adduct responses were log transformed. With the response being the average log(adduct) response over the replicate postlabelings within a set, the linear model was that for a randomized complete block design with three sets (blocks) and three treatments for each time of exposure. Carcinogen (B[a]P or DB[a,l]P) alone was compared to carcinogen plus SRM 1597 using a priori contrast. Additional modeling was done using a more complex mixed linear model accounting for unequal numbers of postlabelings per set, but the results were nearly identical to the simpler model. Analyses were conducted with the Mixed procedure in SAS version 9.1 (SAS Statistical System, Cary, NC).

Results DNA Binding of SRM 1597 in Human Cells in Culture. The effect of SRM 1597 on DNA adduct formation by itself and upon cotreatment with carcinogenic PAH, B[a]P, and DB[a,l]P was determined by 33Ppostlabeling and analyzed by HPLC. MCF-7 cells treated with DMSO (vehicle control) did not exhibit any DNA adduct formation. Representative HPLC elution profiles of the PAH-DNA adducts on exposure to SRM 1597 alone and on cotreatment with B[a]P or DB[a,l]P after 24 and 120 h are depicted in Figures 1A,B and 2A,B. The HPLC profile of PAH-DNA adducts revealed several peaks, with one major peak eluting at a retention time of 25 (Figure 1A,B) or 22 min (Figure 2A,B). This major peak corresponded with the (+)-anti-B[a]PDE-dG adduct peak of B[a]P eluting at a retention time of 25 min (Figure 1A,B). Also, this (+)-anti-B[a]PDE-dG ad-

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duct peak was the significant adduct resolved in the SRM plus B[a]P cotreatment (Figure 1A, lower panel). MCF-7 cells treated with DB[a,l]P exhibited four major dibenzo[a,l]pyrene-(11S,12R)-dihydrodiol (13S,14R)-epoxide(s) (DB[a,l]PDE)-DNA adducts with retention times of 32, 40, 42, and 50 min (Figure 2A,B). Upon cotreatment of SRM plus DB[a,l]P, although the (+)-anti-B[a]PDEdG adduct peak was detected, a reduction in the peak area (of the four major peaks) when cotreated with SRM 1597 and DB[a,l]P was observed (Figure 2A, lower panel). Total DNA Adducts. The levels of PAH-DNA adducts formed in MCF-7 cells treated with either SRM 1597, B[a]P, or cotreatment with SRM 1597 and B[a]P are graphically represented in Figure 3A. The maximum level of DNA adducts formed in the SRM 1597 treated cells was 11.6 pmol/mg of DNA detected at 144 h. The binding although was relatively constant from 48 to 192 h. The highest level of DNA binding observed in B[a]Ptreated cells was 102.3 pmol/mg of DNA after 48 h of exposure (Figure 3A). The binding although was relatively constant from 24 to 48 h. A significant decrease was observed in the total B[a]P-DNA adducts formed throughout the first 96 h on cotreatment with SRM 1597 and B[a]P. The maximum decrease in the level of DNA binding was identified at 24 h where SRM 1597 revealed a significant decrease (p ) 0.0002) in the total DNA adducts in cells treated with B[a]P alone (95.1 pmol/mg of DNA) in comparison to cotreatment with SRM 1597 plus B[a]P (9.7 pmol/mg of DNA). The levels of PAH-DNA adducts formed in MCF-7 cells treated with either SRM 1597, DB[a,l]P, or cotreatment with SRM 1597 and DB[a,l]P are graphically represented in Figure 3B. The maximum level of DNA adducts formed in the SRM 1597 treated cells was 10 pmol/mg of DNA detected at 72 h. The highest level of DNA binding observed in DB[a,l]P-treated cells was 25.5 pmol/mg of DNA after 48 h of exposure (Figure 3B). The binding although was relatively constant from 24 to 192 h. A significant decrease was observed in the total DB[a,l]P-DNA adducts formed throughout the 192 h cotreatment with SRM 1597 and DB[a,l]P. The maximum decrease in the level of DNA binding was identified at 12 h where SRM 1597 revealed a significant decrease (p ) 0.0005) in the total DNA adducts in cells treated with DB[a,l]P alone (14 pmol/mg of DNA) in comparison to cotreatment with SRM 1597 plus DB[a,l]P (1.2 pmol/mg of DNA). P450 1A1 and P4501B1 Protein Expression. Western analysis was performed to examine the ability of the complex mixture of PAH present in SRM 1597 to affect the level of human P4501A1 and P4501B1 proteins in MCF-7 cells in culture (Figure 4). In comparison to the DMSO treatment, SRM 1597 exhibited an increase in the induction levels of both P4501A1 (Figure 4A) and P4501B1 (Figure 4B) at 24 and 120 h. Both B[a]P treatment and cotreatment with SRM 1597 revealed an increase in the levels of P4501A1 and P4501B1 proteins after 24 h. However, by 120 h, the levels of P4501A1 and P4501B1 proteins in B[a]P treatment and P4501B1 in the SRM plus B[a]P cotreatment had markedly decreased (Figure 4A,B). In contrast to the finding with B[a]P, on treatment with SRM 1597 plus DB[a,l]P, although the induction of P4501A1 and P4501B1 proteins was observed, both proteins were not detected when treated with DB[a,l]P alone at either 24 or 120 h (Figure 4A,B).

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Figure 1. HPLC elution profiles of 33P-postlabeled DNA adducts formed in MCF-7 cells in culture after (A) 24 and (B) 120 h exposure to SRM 1597 (400 µg), B[a]P (20.2 µg), or SRM 1597 (400 µg) plus B[a]P (20.2 µg). The bottom panel indicates exposure to DMSO (control).

Discussion Environmental mixtures containing PAH have been implicated in the induction of human cancer (1-3, 42). Although human exposure generally occurs to mixtures of chemicals, limited toxicological information is available to characterize the potential interaction between the

various components of environmental mixtures. Nesnow et al. (42) demonstrated that a correlation exists between mouse skin tumor induction and the relative carcinogenicity of four complex mixtures of PAH, i.e., coke oven emissions, roofing tar emissions, diesel exhaust, and cigarette smoke condensate, in human lung. Culp et al.

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Figure 2. HPLC elution profiles of 33P-postlabeled DNA adducts formed in MCF-7 cells in culture after (A) 24 and (B) 120 h to SRM 1597 (400 µg), DB[a,l]P (0.2 µg), or SRM 1597 (400 µg) plus DB[a,l]P (0.2 µg). In comparison with elution times reported by Buters et al. (48) and Marston et al. (9), the DB[a,l]P peaks were labeled. Peak 2b resulted from reactions of (+)-syn diol epoxide with dA, and peak 4 resulted from reactions of (-)-anti diol epoxide with dA.

(6) have concluded from their studies that coal tar components other than B[a]P appear to induce tumors in the lung tissue. Recently, Mahadevan et al. (34) reported the formation of DNA adducts in MCF-7 cells on exposure to artificial mixtures of weak and noncarcinogenic PAHs present in coal tar, urban dust, and diesel exhaust particulate. A previous study employing mouse skin assays also indicated that SRM 1597 had no effect on the binding of B[a]P to DNA nor did it affect tumor formation (9). However, they reported that SRM 1597 revealed a significant decrease in both the level of DB[a,l]P-DNA adducts and the tumorigenic activity on cotreatment with SRM 1597 and DB[a,l]P (9). In our study, an extended length of exposure (192 h) was chosen to determine if the components in SRM 1597 were competing with the metabolic activation of the carcinogenic PAH on cotreatment with B[a]P or DB[a,l]P. The level of B[a]P-DNA adducts significantly decreased

by the presence of SRM 1597 throughout the first 48 h of exposure (Figure 3A). By 96 h of exposure, the levels of PAH-DNA adducts in the cotreatment as compared to the B[a]P treatment were quite similar, which may have occurred due to removal of DNA adducts by DNA repair and replication. However, SRM 1597 significantly decreased the level of DB[a,l]P DNA adducts throughout the entire 192 h exposure (Figure 3B) and SRM 1597 inhibited the metabolic activation of DB[a,l]P to DB[a,l]PDE DNA adducts on cotreatment. The long exposure (120 h) to SRM 1597 resulted in active metabolism of B[a]P present within the mixture that was revealed by the formation of the (+)-anti-B[a]PDE-dG adduct. These results suggest that there may be a selective metabolism of certain PAHs within complex mixtures, which may be influenced by important P450 enzymes involved in the metabolism of PAH.

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Figure 4. Induction of P4501A1 and P4501B1 in MCF-7 cells in culture. Microsomes were isolated from cells treated with SRM 1597 (400 µg), B[a]P (20.2 µg), or SRM 1597 (400 µg) plus B[a]P (20.2 µg), DB[a,l]P (0.2 µg), or SRM 1597 (400 µg) plus DB[a,l]P (0.2 µg) as described in the Experimental Procedures. Fifty micrograms of microsomal protein was loaded in each lane. (A) Human P4501A1 protein expression. Lane ) + control, represents 25 µg of Aroclor-induced rat liver S9. (B) Human P4501B1 protein expression. Lane ) + control represents 50 µg of microsomal protein isolated from benz[a]anthracenetreated C3H10T1/2 cells.

Figure 3. Effect of exposure time on the level of PAH-DNA adducts in MCF-7 cells in culture. Data points indicate means with standard error bars for the log transformed data over n ) 3 separate experiments. Cells were exposed to either SRM 1597 (400 µg), B[a]P (20.2 µg), or SRM 1597 (400 µg) plus B[a]P (20.2 µg), DB[a,l]P (0.2 µg), or SRM 1597 (400 µg) plus DB[a,l]P (0.2 µg). A statistically significant difference at different exposure times between cotreatment of SRM 1597 and B[a]P or DB[a,l]P in comparison to B[a]P alone or DB[a,l]P alone is indicated with an asterisk below, categorized as follows: *p ) 0.055 (close to significance at the 0.05 level), **p < 0.05, ***p < 0.01, and ****p < 0.001. (A) SRM 1597 significantly decreased the level of B[a]P DNA binding after 6*, 12***, 24****, 48***, and 72** h, respectively. (B) SRM 1597 significantly decreased the level of DB[a,l]P DNA binding after 6**, 12****, 24***, and 48** h, respectively.

Therefore, we further investigated the expression of P450 enzymes on exposure to SRM 1597. P450 enzymes are inducible by xenobiotics such as PAH through the aryl hydrocarbon (Ah) receptor (43). Coal tars have been found to act as Ah receptor agonists (44). We hypothesized that the components within SRM 1597 may be affecting the activation of B[a]P and DB[a,l]P through the protein expression of P4501A1 and/or P4501B1 enzymes. Western analysis of microsomal proteins from MCF-7 cells demonstrated that SRM 1597 alone increased both P4501A1 and P4501B1 proteins (Figure 4A,B). Because the induction of P4501A1 and P4501B1 proteins was observed even after 120 h of exposure to SRM 1597, there is a probability that SRM 1597 was unable to block the bioactivation and inhibit P4501A1

and P4501B1 induction. It has been demonstrated that B[a]P is metabolized to its ultimate carcinogenic metabolite, (+)-anti-B[a]PDE, by P4501A1 more efficiently than P4501B1 (45). The maintained elevated P4501A1 protein levels even at 120 h of exposure to SRM 1597 may explain why B[a]P within SRM 1597 was actively metabolized with time. Also, the level of PAH-DNA adducts formed on exposure to SRM 1597 was substantially lower than the level of DNA adducts formed when cells were treated with either B[a]P or DB[a,l]P despite the increase of P4501A1 and P4501B1 proteins. This effect was also evident in the DNA adduct levels on cotreatment of SRM 1597 with B[a]P or DB[a,l]P. Although P4501A1 and P4501B1 proteins were both induced by SRM 1597, the PAH-DNA adduct analysis indicated that the effects of SRM 1597 on the metabolic activation of B[a]P and DB[a,l]P were quite different (Figure 3). This suggests that the two P450 enzymes may have different roles in the metabolic activation of certain carcinogenic PAHs found in environmental mixtures (46, 47). The results obtained in this study indicate that the metabolism of SRM 1597 in MCF-7 cells may have relied in part on the activation by P4501A1. The decrease in PAH-DNA adduct levels on cotreatment with SRM 1597 may have resulted due to an inhibitory effect of SRM 1597 on the P450 enzyme to metabolize the carcinogenic PAH. Future competitive inhibition studies are planned with V79 cells expressing human P4501A1 and P4501B1 to further understand the metabolism of PAHs present in mixtures by P450 enzymes. Overall, the results from this study on MCF-7 cells allow for the comparison of those results from a previous study on mouse epidermis (9). It also demonstrates that a similar phenomenon of inhibition of carcinogenic PAH activation was observed in coal tar treatment of human cells as in the case of mouse epidermis.

Acknowledgment. This work was supported by Grant CA 28825, DHHS, from the National Cancer Institute. B.M. was supported in part by Grant 5T32 ES07060-23 from the National Institute of Environmental Health Sciences (NIEHS). We acknowledge the Statistics Core Facility of the NIEHS at Oregon State Unviersity supported by Grant P30 ES00210. We thank

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Dr. George Bailey for his helpful discussions at the Carcinogenesis and Chemoprevention Research Core meetings of the Environmental Health Sciences Center at Oregon State University. We also thank Dan Albershardt for assistance with generation of graphs and Jennifer Atkin and Eric Brooks for organization of references.

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