Human Cytochromes P450 1A1 and 1B1 Catalyze Ring Oxidation but

Chem. Res. Toxicol. 2004, 17, 1077-1085

1077

Human Cytochromes P450 1A1 and 1B1 Catalyze Ring Oxidation but Not Nitroreduction of Environmental Pollutant Mononitropyrene Isomers in Primary Cultures of Human Breast Cells and Cultured MCF-10A and MCF-7 Cell Lines Yuan-Wan Sun,† F. Peter Guengerich,‡ Arun K. Sharma,† Telih Boyiri,† Shantu Amin,† and Karam El-Bayoumy*,† Division of Cancer Etiology and Prevention, Institute for Cancer Prevention, Valhalla, New York 10595, and Department of Biochemistry & Center for Molecular Toxicology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232 Received April 20, 2004

The position of the nitro group determines the relative carcinogenic activities of mononitropyrene isomers (mono-NPs) in the rat mammary gland. To determine whether the results obtained in rodents treated with these environmental pollutants can be applicable to humans, we examined their metabolic activation in primary cultures of human breast cells derived from reduction mammoplasty, as well as in the cultured human breast cancer cell line MCF-7 and the immortalized human mammary epithelial cell line MCF-10A. Primary cultures as well as cell lines were competent in metabolizing all three isomers via both ring oxidation and nitro reduction pathways. Qualitatively similar metabolic patterns were observed but quantitative differences were evident. On the basis of cochromatography with synthetic standards in two HPLC systems, metabolites of 1-NP were identified as 1-OH-Py, 3-, 6-, and 8-OH-1-NP and 1-AP. In the case of 2-NP, 6-OH-2-NP and 2-AP were identified. 4-NP was metabolized to 9,10-DHD-4-NP, Py-4,5-Q, 9,10-Q-4-NP, 9/10-OH-4-NP, 6/ 8-OH-4-NP, and 4-AP. Varying degrees of sulfate and glucuronide conjugation of mono-NP metabolites were detected. In MCF-7 cells, we found that 1-, 2-, and 4-NP bind to DNA at levels of 68, 17, and 132 pmol/mg DNA, respectively. Following HPLC analysis of the DNA hydrolysates, we detected multiple DNA adducts including those derived from nitro reduction of 2- and 4-NP; however, none was detected in the case of 1-NP. To determine the P450 enzymes responsible for the metabolic activation of these carcinogens, we incubated [3H]mono-NPs with recombinant human P450 1A1 or 1B1. Metabolites identified were primarily derived from ring oxidation; both P450s 1A1 and 1B1 yielded similar metabolic profiles. This is the first report demonstrating that human breast (target organ) cells, immortalized human mammary epithelial cell line MCF-10A, and breast cancer cell line MCF-7 are capable of activating mono-NPs to metabolites that can damage DNA.

Introduction Breast cancer is the most frequent malignancy in American women. Epidemiological studies of migrants and regional differences in breast cancer incidence suggest that environmental factors and lifestyle play an important role in breast cancer etiology (1-3). Ubiquitous environmental pollutants [e.g., nitropolycyclic aromatic hydrocarbons (NO2-PAH)1], that are known inducers of mammary cancer in rodents (4-6), must be regarded as * To whom correspondence should be addressed. Tel: 914-789-7176. Fax: 914-592-6317. E-mail: [email protected]. † Institute for Cancer Prevention. ‡ Vanderbilt University. 1 Abbreviations: NO -PAH, nitropolycyclic aromatic hydrocarbons; 2 NP, nitropyrene; [3H]1-NP, [3H]2-NP, and [3H]4-NP, 1-nitro[4,5,9,103H]pyrene, 2-nitro[G-3H]pyrene, and 4-nitro[G-3H]pyrene, respectively; 1-OH-Py, 1-hydroxypyrene; 3-, 6-, and 8-OH-1-NP, 3-hydroxy-1nitropyrene, 6-hydroxy-1-nitropyrene, and 8-hydroxy-1-nitropyrene; 6-OH-2-NP, 6-hydroxy-2-nitropyrene; 9,10-DHD-4-NP, 9,10-dihydro9,10-dihydroxy-4-nitropyrene; 9,10-Q-4-NP, 4-NP-9,10-dione; Py-4,5Q, pyrene-4,5-dione; AP, aminopyrene.

potential human risk factors. There are three isomers of mono-nitropyrene (mono-NPs) (Chart 1), of which 1-nitropyrene (1-NP) is the most prevalent isomer detected in urban air, diesel engine emissions, and certain grilled foods (7-9). Unlike 1-NP, 2-NP is detected only in the atmospheric environment and is believed to be formed by a free radical process (10). 4-NP has been detected in both urban air and diesel engine emissions (8). In the ambient atmosphere, the levels of 2- and 4-NP are comparable but much lower than that of 1-NP (7). Despite their structural similarity, mono-NP isomers exhibit distinctly different mutagenic and carcinogenic activities (11-16). Imaida et al. (15) have shown that 4-NP is the most potent mammary carcinogen among the three isomers when these are administered intraperitoneally or by direct injection into the mammary pads of weanling female CD rats. 4-NP was also more tumorigenic than 1-NP in newborn female rats by subcutaneous injection (14) and more active in the livers and lungs of

10.1021/tx049889d CCC: $27.50 © 2004 American Chemical Society Published on Web 07/21/2004

1078 Chem. Res. Toxicol., Vol. 17, No. 8, 2004 Chart 1. Structures of 1-, 2-, and 4-Nitropyrenes

newborn male mice when administered by ip injection (16). Earlier studies have been conducted to provide insight into the varied carcinogenicity of mono-NPs (12, 17, 23). It was found that mono-NPs are metabolized through either nitro reduction or ring oxidation, or a combination of both pathways, to generate a complex array of metabolites (12, 13, 17-23). Metabolism studies of monoNPs by human liver and lung microsomes have revealed that the nitro reduction metabolite aminopyrene (AP) occurred only in the incubation of 4-NP, but not in that of 1- and 2-NP (23). While the structures of 4-NP-DNA adducts remain to be characterized, 1- and 2-NP-DNA adducts derived from the nitro reduction pathway in different biological systems were identified as N-(deoxyguanosin-8-yl)-1-aminopyrene, 6-/ 8-(deoxyguanosin-N2yl)-1-aminopyrene (24-26) and N-(deoxyguanosin-8-yl)2-aminopyrene, and N-(deoxyadenosin-8-yl)-2-aminopyrene (27-29), respectively. We reported that nitro reduction of 4-NP is responsible for DNA adducts detected in the rat mammary gland (30). 4-NP metabolism has also led to a greater level of DNA binding in the rat mammary gland than that of the other two isomers (17). These results may, in part, account for the higher tumorigenic activity of 4-NP. Mono-NPs can be metabolically activated in the liver whereby active metabolites can be transported to extrahepatic tissues, including the mammary gland, or the breast tissue in situ, or both organs. Recent research has suggested that the breast is the principal site responsible for metabolic activation of some carcinogens (31, 32). In breast tissues, P450s 1A1 and 1B1 are two major cytochrome P450 enzymes that metabolize PAHs and estrogens (33-37). In cell cultures and in breast tissues obtained from reduction mammoplasty, P450 1B1 is expressed constitutively; however, P450 1A1 was virtually not expressed in the absence of inducers. It has been shown that both P450s 1A1 and 1B1 are inducible by the aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in experimental animals as well as in human breast cancer cells and this regulation is highly tissue- and cell-specific (37-39). To determine whether the results observed in rodents treated with mono-NPs can be extrapolated to humans, we examined and compared the metabolic activation of these three isomers in human breast tissues obtained from reduction mammoplasty, in the immortalized human mammary epithelial cell line MCF-10A, and in the human breast cancer cell line MCF-7. Studies were also performed to precisely determine the contribution of recombinant human P450s 1A1 and 1B1 in metabolizing these environmental carcinogens.

Experimental Procedures Chemicals, Reagents, and Enzymes. [4,5,9,10-3H]1-NP, [G-3H]2-NP, and [G-3H]4-NP were purchased from Chemsyn

Sun et al. Science Laboratories (Lenexa, KS) and purified by HPLC prior to use (>98% radiochemically pure according to HPLC) to obtain specific activities of 6.58 Ci/mmol, 117 mCi/mmol, and 915 mCi/ mmol, respectively. [3H]1-NP and [3H]4-NP were diluted with the corresponding unlabeled compounds to obtain a specific activity identical to that of [3H]2-NP. 1-NP, ethyl acetate (EtOAc), and methanol (MeOH) were purchased from Aldrich Chemical Co. (Milwaukee, WI). 2-NP and 4-NP were synthesized as reported previously (12, 17-18). Mono-NP metabolite standards were synthesized as described previously (5, 12, 13, 27, 40, 41). β-Glucuronidase, arylsulfatase, saccharic acid 1, 4-lactone, proteinase K, bacterial alkaline phosphatase, snake venom phosphodiesterase I, deoxyribonuclease I, nuclease P1, glucose6-phosphate, glucose-6-phosphate dehydrogenase, NADP, and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO); collagenase A was obtained from Roche (Indianapolis, IN). Dulbecco’s modified Eagle’s medium (DMEM), (DMEM)/F-12, Iscove’s modified Dulbecco’s medium (IMDM), fungizone, penicillin-streptomycin, phosphate buffered saline (PBS), trypsin-EDTA, horse serum, cholera toxin, hydrocortisone, epidermal growth factor (EGF), and insulin were obtained from Bio-Whittaker (Walkersville, MD); fetal bovine serum (FBS) (heat inactivated) was bought from Gemini Bio-Products (Woodland, CA). Recombinant human P450s 1A1 and 1B1 were prepared as described (42, 43) and used in membranes (from Escherichia Coli) in which the P450s were coexpressed with human NADPHP450 reductase. Cell Culture. MCF-10A and MCF-7 cells were obtained from ATCC (Manassas, VA) and grown in 75-cm2 cell culture flasks at 37 °C in a humidified atmosphere with 5% CO2 in air. MCF-7 cells were maintained in IMDM, supplemented with 10% FBS and penicillin/streptomycin (50 µg/mL). MCF-10A cells were maintained in DMEM/F-12 (1:1 mixture) supplemented with 5% horse serum, penicillin/streptomycin (50 µg/mL), insulin (10 µg/ mL), hydrocortisone (500 ng/mL), EGF (20 ng/mL), and cholera toxin (100 ng/mL). Organ Culture. Normal mammary tissues from reduction mammoplasty were obtained from the National Disease Research Interchange (Philadelphia, PA). They were processed within 2-3 days after surgery according to previously published procedures (44, 45). Approximately 106 cells/well were seeded in 6-well plates and maintained in DMEM [supplemented with 5% FBS, penicillin/streptomycin (100 IU/mL), and fungizone (2.5 µg/mL)] for 3-4 days before treatment with [3H]mono-NPs. Metabolism of Mono-NPs by Mammary Cells and Cell Lines. Experimental protocols were similar to those employed previously (45). Preliminary incubations of [3H]1-, 2-, and 4- NPs with MCF-7 and MCF-10A cells were performed at different concentrations (5, 10, and 20 µM) and for various lengths of time (6, 12, 24, and 48 h). The optimal metabolic profiles of the three isomers in both cell lines were observed after 24 h with substrate concentrations of 10 µM. Therefore, the results described here are limited to a substrate concentration of 10 µM and an incubation period of 24 h. Briefly, breast cells obtained from mammoplasty or cell lines were seeded into 6-well plates and incubated for at least 24 h at 37 °C in a humidified atmosphere with 5% CO2 in air until reaching 60-70% confluence. Prior to the addition of mono-NPs, the medium was aspirated and fresh medium (2 mL) was added. Each of the [3H]mono-NP isomers was dissolved in DMSO and the solvent concentration in the culture medium was 0.1% (v/v). Both control (treated with 0.1% DMSO, v/v) and treated cells were incubated at 37 °C for 24 h and the incubations were performed in triplicate. At the end of the incubation period, the medium was transferred into a scintillation vial and the cells were washed with 1 mL PBS (2×). The collected solutions were extracted with twice their volumes of EtOAc (3×). The EtOAc was evaporated and the residue was dissolved in 200 µL of MeOH. An aliquot of the solution (100 µL) was analyzed by HPLC. Cell viability was determined by trypan blue exclusion.

Oxidation of Mononitropyrenes For the analysis of glucuronide and sulfate conjugates, the H2O layer which had been exhaustively extracted with EtOAc was dried, reconstituted in 500 µL H2O, and then treated with β-glucuronidase (104 units) or arylsulfatase (63 units) in the presence of saccharic acid 1, 4-lactone (20 mg). The incubation was carried out at 37 °C for 6 h. Following enzymatic hydrolysis, the incubation mixture was extracted with twice its volume of EtOAc (3×) and the solvent was removed under nitrogen. The dry residue was reconstituted in MeOH and then analyzed by HPLC. Additional experiments found the presence of sulfate conjugate but not glucuronide conjugate in the organic layer. Protein Quantification. Proteins from cell lines and normal mammary cells were extracted with lysis buffer, consisting of 50 mM Tris (pH 7.4), 1 mM EDTA, 1% Triton X-100 (v/v), and 1% Tween 20 (v/v). Briefly, 1 mL of ice-cold lysis buffer was added to each well and cells were scraped and transferred to Eppendorf tubes. The samples were incubated on ice for 20 min and centrifuged at 103 × g in a microfuge centrifuge. The protein concentration in the supernatant was determined with Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL). Metabolism of Mono-NPs by Recombinant Human P450s 1A1 and 1B1. Incubations were conducted with E. Coli membranes containing both the P450 and NADPH-P450 reductase as previously described by Shimada et al. (35) with some modifications. The incubation mixture contained 100 mM potassium phosphate buffer (pH 7.4), 5 mM MgCl2, 5 mM glucose6-phosphate, 0.5 U/mL of yeast glucose-6-phosphate dehydrogenase, 0.5 mM NADP, and 20 pmol of P450 1A1 or 1B1 (containing an equivalent amount of NADPH-P450 reductase) in a final volume of 1 mL. After preincubation at 37 °C for 2 min, the reaction was initiated by adding [3H]mono-NP substrate at a final concentration of 10 µM. Substrates were dissolved in DMSO, yielding a final DMSO concentration of 0.1% (v/v) in the reaction mixture. After 30 min, the incubations were terminated by adding 1 volume ice-cold MeOH. Mono-NPs and their metabolites were extracted with 2 volumes of EtOAc/ CH2Cl2 (1:1) three times. The organic solvent extracts were combined and dried under nitrogen. The residue was dissolved in MeOH and the sample was analyzed by HPLC. HPLC Analysis of Mono-NP Metabolites. HPLC analysis of mono-NPs and their metabolites was performed with a system consisting of two Waters Model 501 solvent delivery pumps (Waters Associates, Milford, MA). Absorbance was monitored at 254 nm with a Waters Model 440 multiwavelength detector. A C18 Vydac analytical column (10 µm, 0.46 × 25 cm; Separation Group, Hesperia, CA) was used with two gradient systems: system 1, a linear gradient from 30% MeOH in H2O to 100% MeOH over 60 min at a flow rate of 1 mL/min; system 2, a linear gradient from 45% MeOH in H2O to 100% MeOH over 100 min at flow rate 1.5 mL/min. Radioactivity was monitored every 1 s by β-Ram Radio-HPLC Detector (IN/US Systems, Tampa, FL). The identification of metabolites was based on cochromatography with synthetic standards. Metabolites were quantified on the basis of the specific activity of [3H]mono-NPs. Isolation, Hydrolysis, and Analysis of Mono-NPs-DNA Adducts in MCF-7 Cells. Genomic DNA was isolated and analyzed according to published procedures (17, 45). Briefly, approximately 90 µg of DNA was obtained from 2 × 107 MCF-7 cells treated with 10 µM [3H]mono-NPs using a QIAGEN Genomic-tip 100/G and QIAGEN cell culture DNA kit (QIAGEN, Valencia, CA). Radioactivity associated with DNA was determined and then DNA was hydrolyzed by the sequential addition of DNase I, nuclease P1, phosphodiesterase I, and alkaline phosphatase. The DNA hydrolysate was extracted with H2Osaturated 1-butanol (3 × 5 mL). The extract was evaporated under reduced pressure, and the resulting residue was dissolved in a minimal volume of 10 mM Tris buffer (pH 7.4). This DNA solution was loaded onto a Sep-Pak C18 cartridge (Waters Associates, Milford, MA) and the MeOH elute was analyzed by HPLC using a Vydac C18 analytical column (10 µm, 0.46 × 25 cm; Separation Group, Hesperia, CA) with a linear gradient from 20% to 80% MeOH in H2O over 45 min, followed by 80%

Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1079

Figure 1. Representative HPLC traces of 1-, 2-, and 4-NP metabolites formed after 24-h incubation of 10 µM [3H]1-NP (A), [3H]2-NP (B), and [3H]4-NP (C) at 37 °C with breast cells (HPLC system 1). MeOH in H2O to 100% MeOH over 10 min at a flow rate of 1.5 mL/min (system 3). HPLC eluates were collected every 1 min for radiochromatography. Radioactivity was counted using a TriCarb 1900 CA liquid scintillation analyzer (Packard Instruments Co., Meriden, CT).

Results Metabolism of Mono-NPs by MCF-7, MCF10A, and Normal Mammary Cells. Initially, different concentrations (5, 10, and 20 µM) of mono-NPs were incubated with MCF-7 and MCF-10A cells for various lengths of time (6, 12, 24, and 48 h). Optimal levels of metabolites derived from [3H]1-, 2-, and 4-NP in MCF-7 and MCF-10A cells were observed after a 24 h incubation at a substrate concentration of 10 µM. Thus, the comparisons described here were established only under these conditions for MCF-7 and MCF-10A and for cultured normal breast cells derived from reduction mammoplasty of three women. Representative HPLC radiochromatograms (system1) of EtOAc-extracted metabolites of mono-NPs obtained from incubations with mammary cells are shown in Figure 1. Identification of metabolites was based on cochromatography with synthetic standards using two different HPLC conditions (system 1 and system 2); additionally, the identity of the 1-OH-Py metabolite was further confirmed by comparing its UV spectrum with that of a synthetic standard. There was no significant difference in cell viability between control (treated with 0.1% DMSO (v/v)) and treatment groups under the experimental conditions used in this study.

1080 Chem. Res. Toxicol., Vol. 17, No. 8, 2004

Sun et al.

Figure 2. Levels of 1-, 2-, and 4-NP metabolites measured after 24-h incubations of 10 µM [3H]1-NP (A), [3H]2-NP (B), and [3H]4NP (C) with cultured MCF-7 (right diagonal stripes) and MCF-10A (horizontal stripes) cell lines (values represented as means ( SD, n)3) and with three primary culture of human breast tissues [tissue 1 (white bars), tissue 2 (left diagonal stripes), and tissue 3 (polka dots)].

Mono-NPs were metabolized by ring oxidation and nitro reduction pathways in MCF-7 and MCF-10A cells and the three samples of normal breast cells; conjugates derived from certain metabolites were also formed. Upon exhaustive extraction of the incubation medium with EtOAc, the aqueous layers were further treated with arylsulfatase and/or β-glucuronidase, followed by EtOAc extraction. HPLC analysis showed the presence of sulfate and glucuronide conjugates. Levels of metabolites derived from [3H]1-, 2-, and 4-NP incubated with MCF-7 and MCF-10A cells and three cultures of three sets of normal breast cells are shown (Figure 2). Although quantitative differences were evident, qualitatively similar metabolic profiles were observed. We found that MCF-7 cells

Table 1. Percentage of Metabolism of 1-, 2- and 4-NP in MCF-7, MCF-10, and Cultures Normal Human Breast Cells (Value Represented as Means ( SD, n ) 3) metabolic conversion (%) compound

MCF-7

MCF-10A

normal breast tissues

1-NP 2-NP 4-NP

29.6 ( 5.2 25.8 ( 5.7 24.3 ( 3.7

17.9 ( 3.7 6.8 ( 1.7 7.6 ( 1.6

22.9 ( 5.0 26.6 ( 17.1 18.1 ( 7.5

metabolized [3H]mono-NPs more efficiently than did MCF-10A cells (Table 1). However, the normal breast cells derived from reduction mammoplasty of three women exhibited varing degrees of metabolism of the three isomers (10-44%).

Oxidation of Mononitropyrenes

Following the incubations of [3H]1-NP with MCF-7, MCF-10A, and normal breast cells, metabolites eluting at tR 42.3, 43.5, 46, 47.4, and 49 min (system 1, Figure 1A) were identified as 1-AP, 1-OH-Py, 8-OH-1-NP, 6-OH-1-NP, and 3-OH-1-NP, respectively. 1-OH-Py and 1-AP were the most prevalent metabolites in all of the incubations. Analysis of conjugates showed that sulfate conjugates of 1-NP metabolites are mainly derived from phenolic metabolites (1-OH-Py and 3-, 6-, and 8-OH-1NP) and these accounted for >80% of the total metabolites. No sulfate conjugate of 1-AP was detected. However, most of the glucuronide conjugates are derived from 1-AP (60-75%) and only minor glucuronide conjugates derived from phenolic metabolites were detected (90%), and a minor 6-OH-2-NP glucuronide conjugate was detected (3-5%). In the case of 4-NP, using system 1 (Figure 1C), metabolites identified were 9,10-DHD-4-NP (tR 26.2 min), 9,10-Q-4-NP (31 min) and Py-4,5-Q (33 min), 4-AP (42 min), 9/10-OH-4-NP (43.5 min), and 6/8-OH-4-NP (45.4 min). The metabolite designated as Product y (38 min) was detected but not identified. Unlike 1- and 2-NP, considerable amounts of dihydrodiol (9,10-DHD-4-NP) and quinone (9,10-Q-4-NP and Py-4,5-Q) metabolites were detected in the incubations of 4-NP. In MCF-7 cells, the levels of 4-AP (232 ( 37 pmol/mg protein) were much higher than those of 1- and 2-AP (55 ( 23 and 21 ( 7 pmol/mg protein, respectively, Figure 2); in the case of MCF-10A cells, the amount of 1-AP (94 ( 10 pmol/mg protein) appeared to be higher than that of 4-AP (39 ( 13 pmol/mg); in turn, the level of 4-AP exceeded that of 2-AP (21 ( 7 pmol/mg protein). In three samples of normal breast cells, the levels of 1- and 4-AP were comparable but higher than that of 2-AP. Consistent with the results obtained using 1- and 2-NP, metabolites derived from ring oxidation (9,10-DHD-4-NP, 9,10-Q-4NP, Py-4,5-Q, and 6/8-OH-4-NP) constituted the majority (60-70%) of sulfate conjugates while 4-AP accounted for the majority of glucuronide conjugates (73-82%). Metabolism of Mono-NPs by Recombinant Human P450 Enzymes. “Bicistronic” recombinant human P450 1A1 or 1B1 membranes containing P450s and NADPH-P450 reductase metabolized all three monoNPs via ring oxidation (Figure 3); metabolites derived from nitroreduction were not detected under the experimental conditions employed here. Both P450s 1A1 and 1B1 produced qualitatively similar metabolic profiles; however, variations in levels of some metabolites were evident (Figure 4). Major metabolites of 1-NP formed by recombinant human P450 1A1 and 1B1 were identified as 1-OH-Py and 3-, 6-, and 8-OH-1-NP. With the exception of 8-OH-1-NP (245 ( 94 and 69 ( 25 pmol/ nmol P450/min for P450 1A1 and P450 1B1, respectively), the levels of other metabolites were comparable for both P450s 1A1 and 1B1. In the case of 2-NP, 6-OH-2-NP was identified as a major metabolite with both enzymes and

Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1081

Figure 3. Representative HPLC traces of 1-, 2-, and 4-NP metabolites formed after incubations of 10 µM [3H]1-NP (A), [3H]2-NP (B), and [3H]4-NP (C) with P450 1A1 or 1B1 (HPLC system 1) at 37 °C for 30 min. Asterisk indicates the elution time of APs.

at comparable levels. The other metabolite, designated as Product x, although minor, was not structurally identified. 4-NP was metabolized to 9,10-epoxy-4-NP (tR 40 min, system 1, Figure 3C) and to 6/8-OH-4-NP. Two metabolites, designated as Products y and z, were not identified. The level of 6/8-OH-4-NP was higher with P450 1A1 than with P450 1B1. P450 1A1 appeared to be more efficient than P450 1B1 in metabolizing 4-NP (28 ( 7% and 15 ( 3% conversion, respectively) while there were no significant differences between P450s 1A1 and 1B1 in metabolizing 1-NP (33 ( 11% vs 29 ( 3% conversion) and 2-NP (27 ( 8% vs 31 ( 10% conversion). DNA Adducts Obtained from the Incubation of Mono-NPs in MCF-7 Cells. We used the MCF-7 cell line since it was the most efficient in metabolizing monoNPs. On the basis of radioactivity measurements, the levels of DNA binding with 1-, 2-, and 4-NP in MCF-7 cells were 68, 17, and 132 (pmol/mg DNA), respectively. HPLC radiochromatograms (system 3) of enzymatic hydrolysates of DNA obtained from MCF-7 cells treated with mono-NPs are shown in Figure 5. We did not detect any radioactivity at a retention time corresponding to an N-(deoxyguanosin-8-yl)-1-aminopyrene adduct derived from nitroreduction of 1-NP; an early eluting peak accounting for most of the radioactivity injected into the HPLC was detected (Figure 5A). Multiple DNA adducts including N-(deoxyguanosin-8-yl)-2-aminopyrene (based on chromatographic characteristics, tR 23 min) were formed from 2-NP (Figure 5B) as observed in the reaction with calf thymus DNA catalyzed by xanthine oxidase

1082 Chem. Res. Toxicol., Vol. 17, No. 8, 2004

Sun et al.

Figure 4. Levels of 1-, 2-, and 4-NP metabolites measured after incubations of 10 µM [3H]1-NP (A), [3H]2-NP (B), and [3H]4-NP (C) with recombinant human P450 1A1 (black bars) or 1B1 (white bars) at at 37 °C for 30 min (values represented as means ( SD, n ) 3).

(17). In the case of 4-NP (Figure 5C), multiple DNA adducts were detected that were also observed in the DNA hydrolysates prepared from calf thymus DNA in the presence of xanthine oxidase (peaks 1, 2, and 3, based on the chromatographic characteristics) (17). Previous studies had shown that peak 1 (tR 22 min) was unstable and gradually decomposed to yield peak 2 (tR 31 min); the latter was identified as Py-4,5-Q. On the basis of elution time, peak 3 (tR 37 min) was tentatively identified as a deoxyinosine-derived 4-AP adduct (30).

Discussion Our study clearly demonstrates that human breast tissues obtained from reduction mammoplasty, as well as immortalized human mammary epithelial cells (MCF10A) and human breast cancer cells (MCF-7), can metabolize all three mono-NP isomers via ring-oxidation and nitro reduction pathways. Although quantitative differences were observed among different breast cells, qualitatively similar metabolic profiles were evident. Despite the relatively small number of normal human breast tissues samples employed here, our results, combined with observations from studies with other NO2-PAH (45), point to inter-individual variations in the metabolism of mono-NPs.

Figure 5. HPLC chromatograms of enzymatic hydrolysates of DNA obtained from MCF-7 cells treated with 10 µM [3H]1-NP (A), [3H]2-NP (B), and [3H]4-NP (C) for 24 h (HPLC system 3).

The carcinogenic potency of 4-NP in rodents is much greater than that of 1- and 2-NP (15), and literature data have suggested that nitro reduction is an important pathway in the bioactivation of mono-NPs (25-30). We detected varying degrees of nitro reduction by quantifying 1-, 2-, and 4-AP as mono-NP metabolites in all of the incubations with MCF-7, MCF-10A, and normal mammary cells. The level of 4-AP was highest from the three NPs when the three isomers were analyzed in MCF-7 cells. Thus, 4-NP can apparently be metabolized to yield active intermediate(s) (e.g., hydroxylamine derivatives) at levels higher than those formed from 1- and 2-NP. Recent studies have shown that P450s 1A1 and 1B1 are mainly expressed in extrahepatic organs, such as the mammary gland, and they are capable of activating many carcinogens to highly reactive metabolites that cause DNA damage (46). Thus, these two P450 enzymes may play major roles in susceptibility to breast cancer (36, 37). P450 1B1 was found to be responsible for catalyzing the aromatic hydroxylation of estrone and 17β-estradiol

Oxidation of Mononitropyrenes

to 4-hydroxy catechol which could be further oxidized to o-quinones/semiquinone derivatives; these metabolites could damage DNA by inducing DNA oxidation or adduct formation (36, 37, 47-49). In this study, we determined the role of mammary P450s 1A1 and 1B1 in the activation of mono-NPs. Our results have shown that only ring oxidation metabolites of mono-NPs were formed. These oxidative metabolites are consistent with those obtained in the incubations with cells (1-OH-Py, 3-, 6-, and 8-OH-1-NP, 6-OH-2-NP, and 6/8-OH-4-NP) except that the metabolite 9,10-epoxy-4NP was detected in the incubations with P450s 1A1 and 1B1, but not with the cells. Consistent with our previous reports on the metabolism of 2-NP in numerous in vitro and in vivo studies (17, 18, 23), 6-OH-2-NP was the only phenolic metabolite detected in this study. However, 9,10DHD-4-NP and 9,10-Q-4-NP were found in the incubations of 4-NP with cells. Other studies have shown that most carcinogenic PAHs are activated by the combined action of P450s and epoxide hydrolase to highly reactive diol-epoxides (33). Therefore, we propose that in cells, upon P450-catalyzed formation of 9,10-epoxy-4-NP and subsequent hydrolysis to 9,10-DHD-4-NP by epoxide hydrolase, oxidation produces 9,10-Q-4-NP. The other quinone metabolite Py-4,5-Q was detected in the incubations of 4-NP with cells but not with the recombinant P450s. The formation of Py-4,5-Q may proceed via oxidation of 4-AP in the incubation mixture. This mechanism has been proposed previously to explain the formation of 5,6-chrysenedione in 6-nitrochrysene metabolism (50). Unlike the situation with 4-NP, no dihydrodiol or quinone metabolites were detected in the breast cells treated with 1-NP or 2-NP. It is possible that these quinone metabolites of 4-NP cause genotoxic effects in a manner similar to quinone metabolites of estrogens by increasing mutation rates (48, 49). This mechanism may be responsible, in part, for the mammary carcinogenicity induced by 4-NP. Considerable variation in the enzymes responsible for nitro reduction has been observed in different organisms. Nitro reduction of NO2-PAH is primarily catalyzed by cytosolic reductases, such as xanthine dehydrogenase/ oxidase, DT-diaphorase, and aldehyde oxidase (51). Nevertheless, P450-mediated nitro reduction (including that of 1-NP and 4-NP) has been reported (23, 52). In humans, xanthine oxidase and microsomal NADPH-cytochrome c have been identified as the enzymes involved in nitro reduction (51). Although xanthine dehydrogenase/oxidase is known to be expressed in mammary tissue (53), enzyme(s) involved in the nitro reduction of mono-NPs in breast cells were not investigated in this study. It appears from our results that recombinant human P450s 1A1 and 1B1 are only responsible for the formation of ring-oxidized metabolites, not for nitro reduction of monoNPs. Consistent with a previous report (54), 1-OH-Py was detected as a metabolite of 1-NP; however, the mechanisms that can account for its formation need to be re-evaluated (54). Since MCF-7 cells are more efficient in metabolizing mono-NPs than are MCF-10A cells, we determined the DNA binding levels and nature of DNA adducts in MCF-7 cells. Consistent with the levels of APs in MCF-7 and with a previous DNA binding study in rat mammary gland (17), 4-NP exhibited the highest DNA binding level among the three isomers. Although no 1-NP-DNA adduct was detected, we demonstrated that the major DNA

Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1083

adducts detected in [3H]2-NP and [3H]4-NP-treated MCF-7 cells are chromatographically similar to those obtained from the incubations of calf thymus DNA with [3H]2-NP and [3H]4-NP in the presence of xanthine oxidase (17), indicating that the nitro reduction pathway is responsible for the formation of DNA adducts derived from monoNPs in the MCF-7 cells. Moreover, the 4-NP-DNA adduct profile obtained in the MCF-7 cell line is chromatographically similar to that obtained from the liver and mammary gland of female CD rats following ip injection of 4-NP (17, 30). Although in this study the structures of 4-NP-DNA adducts were not fully characterized, previous studies have identified Py-4,5-Q (peak 2) as the decomposition product of an unstable adduct and another peak has been proposed to be a deoxyinosine adduct (peak 3) (30). In addition, they are not chromatographically identical with the adduct derived from nitro reduction of 4-NP (i.e., N-(deoxyguanosin-8-yl)-4-aminopyrene) nor with the adducts derived from incubation of 4-NP-9,10oxide with calf thymus DNA (30). Further structural elucidation of the DNA adducts derived from 2- and 4-NP in MCF-7 cells (or normal breast cells) is critical to our understanding of the role of mono-NP DNA adducts in mammary carcinogenesis. These adducts can be used as synthetic markers to assist in the identification of those DNA adducts that still remain undefined in human breast tissues (55, 56). In summary, our metabolism studies have shown that human breast cells are capable of metabolizing 1-, 2-, and 4-NP to genotoxic metabolites that can damage DNA. We have also demonstrated that the DNA adducts obtained from MCF-7 cells treated with 2- and 4-NP are primarily derived from the nitro reduction pathway. Moreover, 4-NP was found to bind to DNA in MCF-7 cells to a greater extent than the other two isomers. These results suggest that humans will be more susceptible to breast cancer from 4-NP than from the other two isomers.

Acknowledgment. We thank Mrs. Ilse Hoffmann for editing the manuscript. This work was supported by the National Institutes of Health Grants CA 35519 (K.E.) and R01 CA 90426 (F.P.G.).

References (1) Jemal, A., Tiwari, R. C., Murray, T., Ghafoor, A., Samuels, A., Ward, E., Feuer, E. J., and Thun, M. J. (2004) Cancer statistics, 2004. CA Cancer J. Clin. 54, 8-29. (2) WHO (1997) The World Health Report. World Health Organization, Geneva. (3) Kuller, L. H. (1995) The etiology of breast cancer-from epidemiology to prevention. Public Health Rev. 23, 157-213. (4) Hirose, M., Lee, M.-S., Wang, C. Y., and King, C. M. (1984) Induction of rat mammary gland tumors by 1-nitropyrene, a recently recognized environmental mutagen. Cancer Res. 44, 1158-1162. (5) El-Bayoumy, K., Rivenson, A., Johnson, B., DiBello, J., Little, P., and Hecht, S. S. (1988) Comparative tumorigenicity of 1-nitropyrene, 1-nitrosopyrene, and 1-aminopyrene administered by gavage to Sprague-Dawley rats. Cancer Res. 48, 4256-4260. (6) El-Bayoumy, K. (1992) Environmental carcinogens that may be involved in human breast cancer etiology. Chem. Res. Toxicol. 5, 585-590. (7) Korfmacher, W. A., Rushing, L. G., Arey, J., Zielinska, B., and Pitts, J. N., Jr. (1987) Identification of mononitropyrenes and mononitrofluoranthenes in air particulate matter via fused silica gas chromatography combined with negative ion atmospheric pressure ionization mass spectrometry. J. High Resolut. Chromatogr. Chromatogr. Commun. 10, 641-646. (8) Gallagher, J., Heinrich, U., George, M., Hendee, L., Phillips, D. H., and Lewtas, J. (1994) Formation of DNA adducts in rat lung

1084 Chem. Res. Toxicol., Vol. 17, No. 8, 2004

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

following chronic inhalation of diesel emissions, carbon black and titanium dioxide particles. Carcinogenesis 15, 1291-1299. Kinouchi, T., Tsutsui, H., and Ohnishi, Y. (1986) Detection of 1-nitropyrene in yakitori (grilled chicken). Mutat. Res. 171, 105113. Ramdhal, T., Zielinska, B., Arey, J. A., Atkinson, R., Winer, A. M., and Pitts, J. N., Jr. (1986) Ubiquitous occurrence of 2-nitrofluoranthene and 2-nitropyrene in air. Nature 321, 425-427. Mermelstein, R., Kiriazides, D. K., Butler, M., McCoy, E. C., and Rosenkranz, H. S. (1981) The extraordinary mutagenicity of nitropyrenes in bacteria. Mutat. Res. 89, 187-196. Upadhyaya, P., Von Tungeln, L. S., Fu, P. P., and El-Bayoumy, K. (1994) In vitro and in vivo metabolism of the carcinogen 4-nitropyrene. Chem. Res. Toxicol. 7, 690-695. El-Bayoumy, K. and Hecht, S. S. (1983) Identification and mutagenicity of metabolites of 1-nitropyrene formed by rat liver. Cancer Res. 43, 3132-3137. Imaida, K., Lee, M. S., Land, S. J., Wang, C. Y., and King, C. M. (1995) Carcinogenicity of nitropyrenes in the newborn female rat. Carcinogenesis 16, 3027-3030. Imaida, K., Hirose, M., Tay, L., Lee, M. S., Wang, C. Y., and King, C. M. Comparative carcinogenicities of 1-, 2-, and 4-nitropyrene and structurally related compounds in the female CD rat. Cancer Res. 51, 2902-2907. Wislocki, P. G., Bagan, E. S., Lu, A. Y., Dooley, K. L., Fu, P. P., Han-Hsu, H., Beland, F. A., and Kadlubar, F. F. (1986) Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay. Carcinogenesis 7, 1317-1322. Chae, Y. H., Upadhyaya, P., Ji, B. Y., Fu, P. P., and El-Bayoumy, K. (1997) Comparative metabolism and DNA binding of 1-, 2-, and 4-nitropyrene in rats. Mutat. Res. 376, 21-28. Upadhyaya, P., Roy, A. K., Fu, P. P., and El-Bayoumy, K. (1992) Metabolism and DNA binding of 2-nitropyrene in the rat. Cancer Res. 52, 1176-1181. Howard, P. C., Flammang, T. J., and Beland, F. A. (1985) Comparison of the in vitro and in vivo hepatic metabolism of the carcinogen 1-nitropyrene. Carcinogenesis 6, 243-249. Ball, L. M., Kohan, M. J., Inmon, J. P., Claxton, L. D., and Lewtas, J. (1984) Metabolism of 1-nitro[14C]pyrene in vivo in the rat and mutagenicity of urinary metabolites. Carcinogenesis 5, 15571564. El-Bayoumy, K., and Hecht, S. S. (1984) Metabolism of 1-nitro[U-4,5,9,10-14C]pyrene in the F344 rat. Cancer Res. 44, 43174322. Dutcher, J. S., Sun, J. D., Bechtold, W. E., and Unkefer, C. J. (1985) Excretion and metabolism of 1-nitropyrene in rats after oral or intraperitoneal administration. Fundam. Appl. Toxicol. 5, 287-296. Chae, Y. H., Thomas, T., Guengerich, F. P., Fu, P. P., and ElBayoumy, K. (1999) Comparative metabolism of 1-, 2-, and 4-nitropyrene by human hepatic and pulmonary microsomes. Cancer Res. 59, 1473-1480. Smith, B. A., Korfmacher, W. A., and Beland, F. A. (1990) DNA adduct formation in target tissues of Sprague-Dawley rats, CD-1 mice and A/J mice following tumorigenic doses of 1-nitropyrene. Carcinogenesis 11, 1705-1710. Herreno-Saenz, D., Evans, F. E., Beland, F. A., and Fu, P. P. (1995) Identification of two N2-deoxyguanosinyl DNA adducts upon nitroreduction of the environmental mutagen 1-nitropyrene. Chem. Res. Toxicol. 8, 269-277. Beland, F. A., Smith, B. A., Thornton-Manning, J. R., and Heflich, R. H. (1992) Metabolic activation of 1-nitropyrene to a mammalian cell mutagen and a carcinogen. Xenobiotica 22, 1121-1133. Roy, A. K., Upadhyaya, P., Fu, P. P., and El-Bayoumy, K. (1991) Identification of the major metabolites and DNA adducts formed from 2-nitropyrene in vitro. Carcinogenesis 12, 475-479. Fu, P. P., Miller, D. W., Von Tungeln, L. S., Bryant, M. S., Lay, J. O., Jr., Huang, K., Jones, L., and Evans, F. E. (1991) Formation of C8-modified deoxyguanosine and C8-modified deoxyadenosine as major DNA adducts from 2-nitropyrene metabolism mediated by rat and mouse liver microsomes and cytosols. Carcinogenesis 12, 609-616. Yu, S., Heflich, R. H., Von Tungeln, L. S., El-Bayoumy, K., Kadlubar, F. F., and Fu, P. P. (1991) Comparative direct-acting mutagenicity of 1- and 2-nitropyrene: evidence for 2-nitropyrene mutagenesis by both guanine and adenine adducts. Mutat. Res. 250, 145-152. Chae, Y. H., Ji, B. Y., Lin, J. M., Fu, P. P., Cho, B. P., and ElBayoumy, K. (1999) Nitroreduction of 4-nitropyrene is primarily responsible for DNA adduct formation in the mammary gland of female CD rats. Chem. Res. Toxicol. 12, 180-186.

Sun et al. (31) Carmichael, P. L., Stone, E. M., Grover, P. L., Gusterson, B. A., and Phillips, D. H. (1996) Metabolic activation and DNA binding of food mutagens and other environmental carcinogens in human mammary epithelial cells. Carcinogenesis 17, 1769-1772. (32) Williams, J. A., and Phillips, D. H. (2000) Mammary expression of xenobiotic metabolizing enzymes and their potential role in breast cancer. Cancer Res. 60, 4667-4677. (33) Shimada, T., and Fujii-Kuriyama, Y. (2004) Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1. Cancer Sci. 95, 1-6. (34) Shimada, T., Oda, Y., Gillam, E. M., Guengerich, F. P., and Inoue, K. (2001) Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009. Drug Metab. Dispos. 29, 1176-1182. (35) Shimada, T., Gillam, E. M., Oda, Y., Tsumura, F., Sutter, T. R., Guengerich, F. P., and Inoue, K. (1999) Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and purified liver epoxide hydrolase. Chem. Res. Toxicol. 12, 623-629. (36) Hayes, C. L., Spink, D. C., Spink, B. C., Cao, J. Q., Walker, N. J., and Sutter, T. R. (1996) 17β-Estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc. Natl. Acad. Sci. U.S.A. 93, 9776-9781. (37) Spink, D. C., Spink, B. C., Cao, J. Q., DePasquale, J. A., Pentecost, B. T., Fasco, M. J., Li, Y., and Sutter, T. R. (1998) Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells. Carcinogenesis 19, 291-298. (38) Iscan, M., Klaavuniemi, T., Coban, T., Kapucuoglu, N., Pelkonen, O., and Raunio, H. (2001) The expression of cytochrome P450 enzymes in human breast tumors and normal breast tissue. Breast Cancer Res. Treat. 70, 47-54. (39) Larsen, M. C., Angus, W. G., Brake, P. B., Eltom, S. E., Sukow, K. A., and Jefcoate, C. R. (1998) Characterization of CYP1B1 and CYP1A1 expression in human mammary epithelial cells: Role of the aryl hydrocarbon receptor in polycyclic aromatic hydrocarbon metabolism. Cancer Res. 58, 2366-2374. (40) El-Bayoumy, K., Villucci, P., Roy, A. K., and Hecht, S. S. (1986) Synthesis of K-region derivatives of the carcinogen 1-nitropyrene. Carcinogenesis 7, 1577-1578. (41) Fifer, E. K., Howard, P. C., Heflich, R. H., and Beland, F. A. (1986) Synthesis and mutagenicity of 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, microsomal metabolites of 1-nitropyrene. Mutagenesis 1, 433-438. (42) Parikh, A., Gillam, E. M. and Guengerich, F. P. (1997) Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat. Biotechnol. 15, 784-788. (43) Shimada, T., Wunsch, R. M., Hanna, I. H., Sutter, T. R., Guengerich, F. P., and Gillam, E. M. (1998) Recombinant human cytochrome P450 1B1 expression in Escherichia coli. Arch. Biochem. Biophys. 357, 111-120. (44) Easty, G. C., Easty, D. M., Monaghan, P., Ormerod, M, G., and Neville, A. M. (1980) Preparation and identification of human breast epithelial cells in culture. Int. J. Cancer 26, 577-584. (45) Boyiri, T., Leszczynska, J., Desai, D., Amin, S., Nixon, D. W., and El-Bayoumy, K. (2002) Metabolism and DNA binding of the environmental pollutant 6-nitrochrysene in primary culture of human breast cells and in cultured MCF-10A, MCF-7 and MDAMB-435s cell lines. Int. J. Cancer 100, 395-400. (46) Shimada, T., Hayes, C. L., Yamazaki, H., Amin, S., Hecht, S. S., Guengerich, F. P, and Sutter, T. R. (1996) Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res. 56, 2979-2984. (47) Samuni, A. M., Chuang, E. Y., Krishna, M. C., Stein, W., DeGraff, W., Russo, A., and Mitchell, J. B. (2003) Semiquinone radical intermediate in catecholic estrogen-mediated cytotoxicity and mutagenesis: chemoprevention strategies with antioxidants. Proc. Natl. Acad. Sci. U.S.A. 100, 5390-5395. (48) Bolton, J. L. (2002) Quinoids, quinoid radicals, and phenoxyl radicals formed from estrogens and antiestrogens. Toxicology 177, 55-65. (49) Yue, W., Santen, R. J., Wang, J. P., Li, Y., Verderame, M. F., Bocchinfuso, W. P., Korach, K. S., Devanesan, P., Todorovic, R., Rogan, E. G., and Cavalieri, E. L. (2003) Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis. J. Steroid. Biochem. Mol. Biol. 86, 477-486. (50) El-Bayoumy, K., Delclos, K. B., Heflich, R. H., Walker, R., Shiue, G.-H., and Hecht, S. S. (1989) Mutagenicity, metabolism and DNA adduct formation of 6-nitrochrysene in Salmonella typhimurium. Mutagenesis 4, 235-240. (51) Fu, P. P. (1990) Metabolism of nitro-polycyclic aromatic hydrocarbons. Drug Metab. Rev. 22, 209-268.

Oxidation of Mononitropyrenes (52) Saito, K., Kamataki, T., and Kato, R. (1984) Participation of cytochrome P-450 in reductive metabolism of 1-nitropyrene by rat liver microsomes. Cancer Res. 44, 3169-3173. (53) Linder, N., Rapola, J., and Raivio, K. O. (1999) Cellular expression of xanthine oxidoreductase protein in normal human tissues. Lab. Invest. 79, 967-974. (54) Djuric, Z., Fifer, E. K., Howard, P. C., and Beland, F. A. (1986) Oxidative microsomal metabolism of 1-nitropyrene and DNAbinding of oxidized metabolites following nitroreduction. Carcinogenesis 7, 1073-1079.

Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1085 (55) Perera, F. P., Estabrook, A., Hewer, A., Channing, K., Rundle, A., Mooney, L. A., Whyatt, R., and Phillips, D. H. (1995) Carcinogen-DNA adducts in human breast tissue. Cancer Epidemiol. Biomarkers Prev. 4, 233-238. (56) Li, D., Wang, M., Dhingra, K., and Hittelman, W. N. (1996) Aromatic DNA adducts in adjacent tissues of breast cancer patients: clues to breast cancer etiology. Cancer Res. 56, 287-293.

TX049889D