Comparative Tumorigenicity of the Environmental Pollutant 6

6-NC and its metabolites in the rat mammary gland by intramammary ... results, the carcinogenic potency toward the mammary gland is ranked in the foll...
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Chem. Res. Toxicol. 2002, 15, 972-978

Comparative Tumorigenicity of the Environmental Pollutant 6-Nitrochrysene and Its Metabolites in the Rat Mammary Gland Karam El-Bayoumy,* Dhimant Desai, Telih Boyiri, Jose Rosa, Jacek Krzeminski, Arun K. Sharma, Brian Pittman, and Shantu Amin American Health Foundation, 1 Dana Road, Valhalla, New York 10595 Received March 4, 2002

Human exposure to the class of nitropolynuclear aromatic hydrocarbons is via inhalation and/or ingestion. Therefore, one of the goals of this study was to determine the propensity of the environmental contaminant 6-nitrochrysene (6-NC) for inducing mammary cancer following its oral administration to female CD rats. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), an established mammary carcinogen in the same animal model, was used as a positive control and trioctanoin as a negative control. Thirty-day-old female CD rats were gavaged once weekly for 8 weeks with 6-NC at 50, 25, or 12.5 µmol/rat or PhIP at 50 µmol/rat in 500 µL of trioctanoin. Twenty-three weeks after the last carcinogen administration, rats were decapitated, necropsied, and evaluated histologically. The most common mammary tumors were adenocarcinomas, followed by adenomas and fibroadenomas. The incidence and multiplicity (mean ( standard deviation) of mammary adenocarcinomas induced by these two carcinogens at the highest dose (6-NC: 90%, 3.73 ( 2.74; PhIP: 83%, 2.62 ( 2.58) were significantly higher than those in control rats (10%, 0.10 ( 0.31). However, there were no statistically significant differences between groups treated with 6-NC and PhIP or among groups receiving various doses of 6-NC. Following its metabolic activation, 6-NC is known to bind covalently to DNA; however, it remains to be determined whether it can also induce DNA base oxidation. Thus, employing the same route of administration, our studies revealed no effect of 6-NC on the basal level of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the mammary gland in tests at 6, 24, and 48 h after 6-NC treatment and at termination of the carcinogenesis assay in the normal, noninvolved tissue and in mammary tumors. This result suggests that covalent DNA binding of 6-NC metabolites is important in the induction of mammary cancer in rats. Therefore, the other goal of this study was to compare the tumorigenic activities of 6-NC and its metabolites in the rat mammary gland by intramammary administration. This route has also been used in our laboratory to induce mammary cancer in the rat by 6-NC and is employed here to avoid systemic effects and to determine the role of the mammary gland in the metabolic activation of 6-NC and its metabolites. Toward this end, a new method was developed to obtain ample materials of trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene (1,2DHD-6-AC); other metabolites were synthesized as reported previously. On the basis of the results, the carcinogenic potency toward the mammary gland is ranked in the following order: 6-NC > 1,2-DHD-6-NC > 6-AC > 6-NCDE > 1,2-DHD-6-AC. Among the metabolites tested, 1,2-DHD-6-NC was the most potent carcinogen. It was significantly more active than its reduced product 1,2-DHD-6-AC. However, the potency of 1,2-DHD-6-NC was not significantly different from 6-AC, a metabolite derived from simple nitroreduction, or from 6-NCDE. Collectively, these results suggest that metabolites derived from both ring-oxidation and nitroreduction contribute to the overall carcinogenicity of 6-NC in the rat mammary gland.

Introduction Ubiquitous environmental agents that induce certain cancers in rodents must be regarded as potential human risk factors and need to be evaluated more closely. The risk associated with human exposure to nitropolynuclear aromatic hydrocarbons (NO2-PAHs)1 has not been clearly defined, even though these agents are widely spread throughout the environment and possibly involved in the etiology of some human cancers (1-4). Many NO2-PAHs * To whom correspondence should be addressed at the Division of Cancer Etiology and Prevention, American Health Foundation, 1 Dana Rd., Valhalla, NY 10595.

are mutagens; some of them are also carcinogenic in laboratory animals (1, 5-7). Although 6-nitrochrysene (6NC) is less abundant than other NO2-PAHs in the environment, it is the most active “parent” compound ever tested in the newborn mouse assay; it is much more active than chrysene, and any of the other mononitro1 Abbreviations: NO -PAH, nitropolycyclic aromatic hydrocarbons; 2 6-NC, 6-nitrochrysene; 6-AC, 6-aminochrysene; 1,2-DHD-6-NC, trans1,2-dihydroxy-1,2-dihydro-6-NC; 1,2-DHD-6-AC, trans-1,2-dihydroxy1,2-dihydro-6-AC; 6-NCDE, 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro6-nitrochrysene; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; DMBA, 7,12-dimethylbenz[a]anthracene; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; HPLC-EC, high-performance liquid chromatography with electrochemical detection.

10.1021/tx020019a CCC: $22.00 © 2002 American Chemical Society Published on Web 06/19/2002

Mammary Cancer Induction by 6-NC and Metabolites

chrysene isomers and benzo[a]pyrene (B[a]P) (5-9). We demonstrated by intramammary administration that 6-NC is a powerful mammary carcinogen (10); again, it was more potent than the ultimate carcinogen, the bay region diol epoxide of B[a]P (11). The carcinogenic activity of 6-NC in the mammary gland (10, 11), the colon of rats (12), and the lung and skin of mice (5-9, 13) and its environmental presence (14-17), as well as the ability of human liver and lung to convert 6-NC into genotoxic metabolites (18), suggest its potential importance with regard to human cancer development. In fact, a report by Zwirner-Baier and Neumann demonstrated the presence of hemoglobin adducts derived from several NO2PAHs, including 6-NC in humans (19). Human exposure to NO2-PAHs is via inhalation and/ or ingestion. Therefore, in the present study, we evaluated the carcinogenicity of 6-NC, upon oral administration, toward the mammary glands of rats. 2-Amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant of the mutagenic and carcinogenic heterocyclic amines present in cooked meat and fish, served as the positive control (20, 21). Metabolism and DNA binding studies in mice and rats have indicated that ring oxidation and nitroreduction are involved in the metabolic activation of 6-NC (22). Although, upon activation, 6-NC is known to covalently bind to DNA in vivo (22), its effect on levels of 8-hydroxy2-deoxyguanosine (8-OHdG), an oxidative DNA damage biomarker, in the rat mammary glands has not been reported. The lack of effect of 6-NC on levels of 8-OHdG, as demonstrated in this report, suggests that covalent DNA binding of 6-NC metabolites is important in the induction of mammary cancer in rats. Toward this end, metabolites were synthesized as reported (23), and a new method was developed to obtain ample materials of a potential proximate carcinogenic metabolite of 6-NC, namely, 1,2-DHD-6-AC, and the potency to induce mammary cancer in the rat was ranked by intramammary administration. This route has been used in our laboratory to induce mammary cancer in the rat and is employed here to avoid systemic effects and to determine the role of the mammary gland in the metabolic activation of 6-NC and its metabolites. Collectively, the results of the bioassay indicate that metabolites derived from ring oxidation and nitroreduction contribute to the overall carcinogenicity of 6-NC in the rat mammary gland.

Materials and Methods Melting points were recorded on a Fischer-Johnson melting point apparatus and are uncorrected. Unless stated otherwise, proton NMR spectra were recorded using a Bruker AM 360WB NMR spectrometer in CDCl3 with tetramethylsilane (TMS) as internal standard. Chemical shifts were recorded in ppm downfield from the internal standard. MS were run on a Hewlett-Packard model 5988A instrument. Thin-layer chromatography (TLC) was done on aluminum-supported precoated silica gel plates (EM Industries, Gibbstown, NJ). All starting materials were obtained from Aldrich Chemical Co. (Milwaukee, WI). 6-NC and PhIP were obtained as described previously (6, 20). 1,2-DHD-6-NC and 6-NCDE were synthesized as reported previously (23). 1,2-DHD-6-AC can be obtained by nitroreduction of 1,2-DHD-6-NC; however, this approach does not yield the desired product in satisfactory yield (24). Therefore, we developed a new method (Scheme 1) for the synthesis of 1,2-DHD6-AC. 1-(4-Bromonaphthyl)-2-(3-methoxyphenyl)ethylene (2). A solution of 1-bromo-4-bromomethylnaphthalene (33.0 g, 0.11

Chem. Res. Toxicol., Vol. 15, No. 7, 2002 973 mol) and triphenylphosphine (39.3 g, 0.15 mol) in 300 mL of benzene was heated under reflux for 3 h. The reaction mixture was cooled, and the phosphonium salt 1 (53.1 g, 86%) was isolated by filtration. To a stirred solution of the salt 1 (5.0 g, 8.9 mmol) and m-anisaldehyde (1.21 g, 8.9 mmol) in 50 mL of CH2Cl2 was added a solution of NaOH (0.39 g, 9.7 mmol) in 1 mL of H2O. The mixture was stirred vigorously for 3 h, diluted with CH2Cl2, washed with water twice, dried (MgSO4), and concentrated. The crude product was purified by flash chromatography on silica gel (eluent: hexane:CH2Cl2, 80:20) to give olefin 2 (3.0 g, 99%) as a mixture of cis and trans isomers (37: 63): 1H NMR δ 3.47 (s, 1.89H, trans-OCH3), 3.88 (s, 1.11H, cisOCH3), 6.55-7.35 (m, 6H), 7.51-7.83 (m, 4H), 8.08 (d, 0.63H, trans-olefinic, J ) 8.2 Hz), 8.19-8.22 (m, 0.37H, cis-olefinic), 8.27-8.31 (m, 1H); MS (m/e, relative intensity) 340 (M+, 85%), 338 (M+, 80), 259 (M+ - Br, 47). 2-Methoxy-6-bromochrysene (3). A solution of 2 (3.0 g, 8.85 mmol) and iodine (5.0 mg) in dry benzene (1 L) was stirred and irradiated with a Pyrex-filtered Hanover 450 W mediumpressure UV lamp, while air was bubbled through the solution. The progress of the reaction was followed by TLC (hexane). After 5 h, 70-80% of the olefin was cyclized. Removal of the solvent gave a crude mixture of 2-methoxy-6-bromochrysene (3) and 4-methoxy-6-bromochrysene (4). This mixture was separated by chromatography on silica gel. Elution with hexane/CH2Cl2 (70: 30) gave early eluting 4-methoxy-6-bromochrysene (4) (0.63 g, 21%), mp 134-136 °C: 1H NMR δ 4.19 (s, 3H, OCH3), 7.19 (dd, 1H, H3, J3,2 ) 7.2 Hz, J3,1 ) 1.6 Hz), 7.55-7.62 (m, 2H, H1 and H2), 7.71-7.76 (m, 2H, H8 and H9), 7.99 (d, 1H, H12, J12,11 ) 8.9 Hz), 8.41-8.45 (m, 1H, H7), 8.72 (d, 1H, H11, J11,12 ) 8.9 Hz), 8.78-8.82 (m, 1H, H10), 10.17 (s, 1H, H5); MS (m/e, relative intensity) 338 (M+, 70), 336 (M+, 74). Further elution with hexane/CH2Cl2 (1:1) gave pure 2-methoxy 6-bromochrysene (3) (1.4 g, 47%), mp 211-212 °C: 1H NMR δ 4.00 (s, 3H, OCH3), 7.32-7.38 (m, 2H, H1 and H3), 7.68-7.77 (m, 2H, H8 and H9), 7.94 (d, 1H, H12, J12,11 ) 9.2 Hz), 8.42 (d, 1H, H7, J7,8 ) 7.6 Hz), 8.59 (d, 1H, H4, J4,3 ) 8.9 Hz), 8.65 (d, 1H, H11, J11,12 ) 9.2 Hz), 8.76 (d, 1H, H10, J10,9 ) 7.9 Hz), 8.97 (s, 1H, H5); MS (m/e relative intensity) 338 (M+, 92), 336 (M+, 100). 2-Hydroxy-6-bromochrysene (5). To a stirred solution of 3 (4.09 g, 12.1 mmol) in CH2Cl2 (200 mL) at 0 °C under an N2 atmosphere was added a 1 M solution of boron tribromide (35 mL, 35 mmol) in CH2Cl2 over a period of 10 min. After 12 h stirring at room temperature, the reaction mixture was poured into ice-cold H2O, the organic layer was washed with H2O (2 × 50 mL) and dried (MgSO4), and the solvent was removed to yield crude 5, which was recrystallized from CH2Cl2 (3.38 g, 85%), mp 216-218 °C: 1H NMR δ 7.28-7.33 (m, 2H, H1 and H3), 7.68-7.77 (m, 2H, H8 and H9), 7.88 (d, 1H, H12, J12,11 ) 9.2 Hz), 8.42 (d, 1H, H7, J7,8 ) 7.9 Hz), 8.60 (d, 1H, H4, J4,3 ) 9.2 Hz), 8.63 (d, 1H, H11, J11,12 ) 9.2 Hz), 8.74 (d, 1H, H10, J10,9 ) 7.9 Hz), 8.95 (s, 1H, H5); for MS GC, TMS derivative of 5 was prepared: MS (m/e, relative intensity) 396 (M+, 100%), 394 (M+, 96). 6-Bromochrysene-1,2-dione (6). To a stirred solution of 5 (1.60 g, 4.95 mmol) in 430 mL of C6H6/CH2Cl2/THF (16:5:1) were added 10 drops of Adogen 464 and a solution of Fremy’s salt (4.0 g, 14.9 mmol) in 250 mL of 0.17 M KH2PO4. Stirring was continued for 18 h at room temperature, and the organic layer was collected. The aqueous phase was extracted with benzene. Combined organic extracts were washed with water, dried (Na2SO4), and evaporated to dryness. The dark residue was recrystallized from CH2Cl2 to yield 1.25 g (75%) of dione 6, mp 236-239 °C (dec): 1H NMR δ 6.64 (d, 1H, H3, J3,4 ) 10.5 Hz), 7.80-7.86 (m, 2H, H8 and H9), 8.33 (d, 1H, H4, J4,3 ) 10.5 Hz), 8.55 (d, 1H, H12, J12,11 ) 8.5 Hz), 8.41-8.45 (m, 1H, H7), 8.52 (s, 1H, H5), 8.71-8.75 (m, 1H, H10), 8.82 (d, 1H, H11, J11,12 ) 8.5 Hz); MS (m/e, relative intensity) 338 (M+, 97), 336 (M+, 100). (()-trans-1,2-Dihydroxy-1,2-dihydro-6-bromochrysene (7). To a stirred suspension of 6 (1.36 g, 4.04 mmol) in absolute ethanol (500 mL) was added NaBH4 (4.1 g, 108 mmol) in portions. The mixture was stirred for 72 h while open to the

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El-Bayoumy et al.

Scheme 1. Synthesis of trans-1,2-Dihydroxy-1,2-dihydro-6-aminochrysene

air and then poured into H2O and extracted with EtOAc (3 × 100 mL). The organic phase was washed with H2O, dried (Na2SO4), and concentrated. The resulting brown diol was recrystallized from acetone/hexane (0.98 g, 71%), mp 216-218 °C: 1H NMR (CD3COCD3) δ 4.48-4.52 (m, 1H, H2), 4.92 (dd, 1H, H1, J1,2 ) 11.1 Hz, J1,OH ) 5.2 Hz), 6.28 (dd, 1H, H3, J3,4 ) 10.2 Hz, J3,2 ) 2.1 Hz), 7.35 (dd, 1H, H4, J4,3 ) 10.2 Hz, J4,2 ) 2.2 Hz), 7.76-7.84 (m, 2H, H8 and H9), 8.07 (d, 1H, H12, J12,11 ) 8.6 Hz), 8.33-8.37 (m, 1H, H7), 8.58 (s, 1H, H5), 8.81 (d, 1H, H11, J11,12 ) 8.6 Hz), 8.87-8.91 (m, 1H, H10). 1,2-Bis(tert-butyldimethylsilyloxy)-1,2-dihydro-6-bromochrysene (8). To a solution of diol 7 (1.45 g, 4.26 mmol) in 6 mL of dry pyridine at 0 °C was added tert-butyldimethylsilyl trifluoromethanesulfonate (3.3 g, 12.6 mmol) during a period of 5-10 min. The resulting reaction mixture was stirred under N2 at room temperature for 3 h. The mixture was concentrated in vacuo and coevaporated with benzene, and the residue was dissolved in EtOAc (200 mL). The solution was washed with NaHCO3 solution and H2O, dried (Na2SO4), and concentrated in vacuo. The residue was chromatographed on a silica gel column (flash) with hexane/2.5% EtOAc to give 1.84 g (76%) of 8, mp123-125 °C: 1H NMR δ 0.09 (s, 3H, SiCH3), 0.12 (s, 3H, SiCH3), 0.17 (s, 3H, SiCH3), 0.23 (s, 3H, SiCH3), 0.94 [s, 9H, SiC(CH3)3], 1.02 [s, 9H, SiC(CH3)3], 4.51-4.57 (m, 1H, H2), 4.94 (d, 1H, H1, J1,2 ) 9.9 Hz), 6.21 (dd, 1H, H3, J3,4 ) 10.2 Hz and J3,2 ) 2.6 Hz), 7.16 (d, 1H, H4, J4,3 ) 10.2 Hz), 7.65-7.73 (m, 2H, H8 and H9), 7.84 (d, 1H, H11, J11,12 ) 8.4 Hz), 8.32-8.36 (m, 1H, H7), 8.40 (s, 1H, H5), 8.59 (d, 1H, H12, J12,11 ) 8.4 Hz), 8.68-8.71 (m, 1H, H10); MS (m/e, relative intensity) 569 (M+, 2), 439 (M+ - OSiMe2CMe3, 5), 437 (M+ - OSiMe2CMe3, 5), 308 [M+ - 2(OSiMe2CMe3), 20], 307 [M+ - 2(OSiMe2CMe3), 20]. (()-trans-1,2-Dihydroxy-1,2-dihydro-6-aminochrysene (10). To a solution of 8 (0.38 g, 0.667 mmol) in 10 mL of freshly distilled THF at -78 °C was added dropwise a 2.5 M solution of n-BuLi in hexane (0.4 mL, 1.0 mmol), and the mixture was stirred under N2 for 1 h. Diphenylphosphoryl azide (DPPA) (0.25 mL, 1.16 mmol) was then added and the stirring continued for the next 3 h. Then, the temperature was raised during the period of 25 min and kept at -25 °C for 2 h. It was again brought down to -78 °C, and Red-AL [sodium bis(2-methoxyethoxy)aluminum hydride (65% in toluene, 1.5 mL, 0.97 g, 4.8 mmol)]

was added. The mixture was warmed to 0 °C for 15 min, and stirred for another 45 min at that temperature. It was then stirred at 20 °C for 30 min, and 1 mL of MeOH, followed by 5 mL of H2O was added. After dilution with H2O, the mixture was filtered, dried (MgSO4), and evaporated to dryness to yield the crude 1,2-bis(tert-butyldimethylsilyloxy)-1,2-dihydro-6-aminochrysene (9): 1H NMR δ 0.06 (s, 3H, SiCH3), 0.09 (s, 3H, SiCH3), 0.12 (s, 3H, SiCH3), 0.19 (s, 3H, SiCH3), 0.90 [s, 9H, SiC(CH3)3], 0.95 [s, 9H, SiC(CH3)3], 4.38-4.42 (m, 1H, H2), 4.80 (d, 1H, H1, J1,2 ) 8.2 Hz), 5.86 (s, 2H, NH2), 6.10 (dd, 1H, H3, J3,4 ) 10.2 Hz and J3,2 ) 2.9 Hz), 7.13 (d, 1H, H4, J4,3 ) 10.2 Hz), 7.21 (s, 1H, H5), 7.38 (d, 1H, H12, J12,11 ) 8.5 Hz), 7.597.68 (m, 2H, H8 and H9), 8.19 (d, 1H, H7, J7,8 ) 7.9 Hz), 8.55 (d, 1H, H11, J11,12 ) 8.5 Hz), 8.76 (d, 1H, H10, J10,9 ) 8.5 Hz); MS (m/e, relative intensity) 507 (M+ + 1, 91), 491 (M+ - NH2, 97). The crude 9 was dissolved in 10 mL of THF, and a solution of tetrabutylammonium fluoride (1.0 M in THF 3.7 mL, 3.7 mmol) was added. The mixture was stirred for 1.5 h at room temperature, 5 mL of MeOH was added, and the solvents were evaporated. Purification on a Florisil (30-60 mesh) column using gradient of MeOH (0.5-5%) in CH2Cl2/Et3N 0.5% gave 10 (74 mg, 40%), mp 184-186 °C (dec): 1H NMR (CD3COCD3) δ 4.28 (d, 1H, 2-OH, JOH,2 ) 4.9 Hz), 4.42-4.49 (m, 1H, H2), 4.55 (d, 1H, 1-OH, JOH,1 ) 5.5 Hz), 4.83 (dd, 1H, H1, J1,2 ) 11.4 Hz, J1,OH ) 5.5 Hz), 5.34 (br s, 2H, NH2), 6.12 (dd, 1H, H3, J3,4 ) 10.2 Hz, J3,2 ) 2.2 Hz), 7.13 (dd, 1H, H4, J4,3 ) 10.2 Hz, J4,2 ) 2.4 Hz), 7.39 (s, 1H, H5), 7.61-7.74 (m, 3H, H8, H9, H12), 8.19 (d, 1H, H7, J7,8 ) 7.9 Hz), 8.59 (d, 1H, H11, J11,12 ) 8.5 Hz), 8.79 (dd, 1H, H10, J9,10 ) 7.6 Hz, J8,10 ) 1.3 Hz). The compound was derivatized with TMS; MS of the silylated compound (m/e, relative intensity) 421 (M+, 33) 331 (M+ HOSiMe3, 37). Animals. Outbred female CD rats [CD(SD)IGS:Crl BR] (Charles River Breeding Labs, Kingston, NY) were maintained on a high-fat AIN-76A diet to mimic the Western dietary pattern; tap water was offered ad libitum. The animals were kept at controlled room temperature (22 ( 2 °C), relative humidity (55 ( 15%), and in a 12 h light/dark cycle. They were housed three to a cage in solid-bottomed polycarbonate cages containing hardwood chips as bedding. All diet ingredients were

Mammary Cancer Induction by 6-NC and Metabolites

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Table 1. Mammary Tumors in CD Rats Treated with 6-NC or PhIPa compound (total dose, µmol) 1. PhIP (400) 2. 6-NC (400) 3. 6-NC (200) 4. 6-NC (100) 5. control trioctanoin

number of rats with mammary tumors mammary tumor multiplicity no. of animals fibroadenoma adenoma adenocarcinoma others fibroadenoma adenoma adenocarcinoma 29

1 (3)b

0 (0)

24 (83)

0 (0)

30

3 (10)

1 (3)

27 (90)

2 (7)e

30

3 (10)

3 (10)

25 (83)

1 (3)g

30

5 (17)

0 (0)

25 (83)

0 (0)

30

0 (0)h

0 (0)

3 (10)i

0 (0)

0.03 ( 0.19 (1)c 0.30 ( 1.06 (9) 0.13 ( 0.43 (4) 0.37 ( 0.93 (11) 0(0 (0)

0(0 (0) 0.03 ( 0.18 (1) 0.10 ( 0.31 (3) 0(0 (0) 0(0 (0)

2.62d ( 2.58 (76) 3.73f ( 2.74 (112) 2.73d ( 2.24 (82) 3.03f ( 2.72 (91) 0.10 ( 0.31 (3)

others 0(0 (0) 0.07 ( 0.25 (2) 0.03 ( 0.18 (1) 0(0 (0) 0(0 (0)

a Thirty-day-old female CD rats were fed a high-fat (HF) diet (AIN-76A, 23.5% corn oil) and gavaged with 6-NC (50, 25, and 12.5 µmol rat-1 week-1) or PhIP (50 µmol rat-1 week-1) in trioctanoin once weekly for 8 weeks. Rats were sacrificed 23 weeks after the last carcinogen dose. b Numbers in parentheses represent percent. c Numbers are mean ( standard deviation; numbers in parentheses represent the total number of mammary tumors. d p < 0.001 compared to the control. e Two rats had fibroma. f p < 0.0001 compared to the control. g One rat had adenolipoma. h Unadjusted p ) 0.02 compared to group 4 (not significant after adjustment for multiple comparisons using the Bonferroni criterion). i Adjusted p < 0.0001 compared to groups 1-5.

obtained from Dyets, Inc. (Bethlehem, PA), mixed in our laboratory, and fed in powdered form. The diet was composed of casein, 23.5%; DL-methionine, 0.3%; corn starch, 32.0%; dextrose, 8.3%; corn oil, 23.5%; Alphacel, 5.9%; mineral mix (AIN-76), 4.22%; vitamin mix (AIN-76A), 1.8%; and choline bitartrate, 0.24%. Carcinogenicity Assay No. 1. Thirty-day-old female rats [Crl: CD(SD)BR, 78 ( 5 g] were set up in groups and treated by gavage once weekly for 8 weeks at three dose levels with 6-NC (50, 25, and 12.5 µmol/rat/week) and with PhIP at 50 µmol/ rat/week in 500 µL of trioctanoin. Control rats received trioctanoin only. The rats were palpated for mammary tumors, and body weights were recorded weekly until termination. Twentythree weeks after the last carcinogen dose, the rats were decapitated. All animals were necropsied; all organs, especially the mammary glands, were examined macroscopically for any gross lesions or abnormalities. The mammary tissues were fixed in 10% buffered formalin solution, processed for paraffin sections, and stained with H&E. Histological diagnosis of mammary tumors was based on criteria outlined by Russo et al. (25). Portions of mammary tumors, noninvolved tissues, and normal tissues were stored at -80 °C for the analysis of 8-OHdG as described in a later section. Carcinogenicity Assay No. 2. Thirty-day-old female CD rats were randomly assigned to 6 treatment groups of 25 rats each, as shown in Table 2. The first injections of DMSO or test compound in DMSO (2.04 µmol/100 µL) were given at 30 days of age. The second injections were administered on the following day. The protocol was identical to that previously described (10). The mammary tissue underneath each of the three left thoracic nipples was injected with 100 µL of one of the solutions. The corresponding areas of the three nipples on the right side were treated with DMSO only. On the second day, the inguinal nipple areas received the same treatments. The total dose of 6-NC or its metabolites was 12.3 µmol per rat. Body weights were measured prior to carcinogen administration, weekly during the first month and then monthly until termination of the experiment 36 weeks after the second injection; all rats survived the course of the experiment. Beginning approximately 3 months after carcinogen treatment, animals were palpated every 2 weeks. The location of palpable tumors was recorded and monitored throughout the remainder of the experiment. At necropsy, all organs and especially the mammary glands were examined macroscopically for any gross lesion or abnormalities. The tissues were processed and examined as described above. Effect of Orally Administered 6-NC on the Basal Levels of 8-OHdG in Rat Mammary Gland. As described above (Carcinogenicity Assay No. 1), portions of mammary tumors, noninvolved tissues, and normal tissue were saved for the analysis of 8-OHdG. In addition, a short-term study was performed to determine whether a single dose of 6-NC can induce DNA base oxidation in the target organ; the dose of 6-NC

used here is half of the lowest dose used in Carcinogenicity Assay No. 1. Thus, female CD rats were set up in 2 groups of 24 rats each. One group received a single oral dose of 6-NC (50 µmol/rat) in trioctanoin, and the other group received trioctanoin only. Rats were sacrificed 6, 24, and 48 h after carcinogen treatment. Mammary fat pads from Carcinogenicity Assay No. 1 and those obtained from a short-term study were excised and stored at -80 °C until analysis. DNA isolation and analysis of 8-OHdG were carried out as described in the literature (26, 27). Statistical Methods. Tumor incidence among the groups was compared with the chi-square test. Multiplicity among the groups was compared using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Body weight gain was analyzed among the groups using one-way ANOVA with repeated measures. For both tumor incidence and body weight gain, the p-values for all pairwise comparisons were adjusted for multiple comparisons using the Bonferroni criterion (28).

Results Mammary Cancer Induction by the Oral Administration of 6-NC to Rats. In group 1 (PhIP-treated rats), one rat died during the seventh gavage due to an accidental esophageal puncture (Table 1). Except in group 1, all animals survived until termination of the study. Mean body weights of the rats treated with 6-NC or PhIP were evaluated statistically (cf. Materials and Methods). We found the mean body weight of PhIP-treated rats to differ significantly from that of trioctanoin-treated rats, but no other differences were found (data not shown). The incidence of palpable mammary tumors is shown in Figure 1. Tumors were located on both thoracic and abdominal glands. Regression of palpable mammary tumors was not noted; increasing numbers of tumors developed in all groups as the assay progressed. Mammary tumors induced by 6-NC at all dose levels were observed 1 week earlier than those induced by PhIP. When the rats were killed, 23 weeks after the last dose of carcinogen administration, histologically, the most common mammary tumors were adenocarcinomas, followed by adenomas and fibroadenomas. As shown in Table 1, there were no significant differences among groups treated with carcinogens, but all carcinogentreated rats had significantly higher incidence than those treated with trioctanoin only. A comparison of mammary tumor multiplicity among groups yielded similar results. Effect of 6-NC on Basal Levels of 8-OHdG in Rat Mammary Tissue. Levels of 8-OHdG in rat mammary

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El-Bayoumy et al. Table 2. Mammary Tumor Induction by 6-NC and Its Metabolitesa

Figure 1. Percentile incidence of palpable mammary tumors in rats treated by gavage with 6-NC, PhIP, or trioctanoin.

group

tumor multiplicity (mean ( SD)

tumor incidence (%)

1. 6-NC 2. 6-AC 3. 1,2-DHD-6-NC 4. 1,2-DHD-6-AC 5. 6-NCDE 6. control DMSO

3.46 ( 1.86b 0.67 ( 1.09 1.40 ( 1.80d 0.20 ( 0.5e 0.44 ( 77f 0.00

95.8b 37.5c 48d 16e 28f 0

a Thirty-day-old female CD rats were fed a high-fat diet (AIN76A, 23.5% corn oil) and administered 6-NC or its metabolites at equimolar dose by intramammary injection; refer to Materials and Methods for doses and frequency of administration. b p < 0.001 compared to groups 2-6. c p < 0.004 compared to group 6. d p < 0.02 compared to group 4. e p < 0.001 compared to group 6. f p < 0.004 compared to group 6.

metabolite derived from simple nitroreduction or from the bay region diol epoxide 6-NCDE.

Discussion

Figure 2. Levels of 8-OHdG in the mammary gland at 6, 24, and 48 h after treatment with 6-NC (A), and those measured at termination (23 weeks after the last oral administration of 6-NC) in normal, noninvolved and in mammary adenocarcinoma tissues (B).

tissue were analyzed by HPLC-EC at various time points after 6-NC administration. There was no significant difference between 6-NC-treated and control (trioctanoin) rats at all time points after carcinogen treatment (Figure 2). Frozen normal mammary tissues, tumor tissues, and noninvolved tissues obtained from rats that were assigned to the Carcinogenesis Assay No. 1 were also analyzed for 8-OHdG. 6-NC had no effect on basal levels of 8-OHdG in normal, noninvolved or cancer tissues in the mammary gland (Figure 2). Mammary Cancer Induction by Intramammary Administration of 6-NC and Metabolites in the Rat. The body weights of rats treated with 6-NC or its metabolites were virtually identical to those of controls over the course of the experiment (data not shown). Survival was 100% in all groups. Consistent with our previous investigation (10), 6-NC induced mammary cancer predominantly at the site of injection. Similarly, intramammary administration of 6-NC metabolites resulted in the formation of mammary tumors at the site of injection. Table 2 summarizes the results. We only report adenocarcinoma, since it was the major form diagnosed histologically. On the basis of tumor incidence or tumor multiplicity, the potency is ranked in the following order: 6-NC > 1,2-DHD-6-NC > 6-AC > 6-NCDE > 1,2-DHD-6-AC. Statistical evaluation indicates that among all metabolites tested, 1,2-DHD-6-NC was significantly more active than its nitro-reduced product 1,2-DHD-6-AC. However, the potency of 1,2DHD-6-NC was not significantly different from 6-AC, a

We demonstrated here for the first time that following its oral administration, 6-NC is capable of inducing a high incidence of mammary tumors in the rat. We selected 30day-old rats to determine the carcinogenic potency of 6-NC. Between 30 and 55 days of age, rats are highly susceptible to mammary cancer induction by chemical carcinogens. Russo et al. (29) attributed the high incidence of mammary tumors, obtained upon treatment of virgin rats (30-55 days of age) with carcinogens such as DMBA, to the existence of undifferentiated terminal end buds whose component cells exhibited a relatively high DNA labeling index. On the basis of our previous study with PhIP, B[a]P, and 1-NP (20), we selected a protocol of multiple gavaging with a total dose of 400 µmol/rat as the highest dose of 6-NC. To determine a dose-response relationship, 6-NC was also administered at 200 and 100 µmol/rat. The results clearly show that 6-NC is a potent mammary carcinogen and, consistent with our previous study, the positive control agent, PhIP, induced a high incidence of adenocarcinoma in the rat mammary gland (20). The lack of a dose-response may suggest that 6-NC is a powerful carcinogen in the rat mammary gland at lower dose levels than those employed in this study; future studies will address this question. It is intriguing that administration of 6-NC to CD rats by intraperitoneal (i.p.) injection elicited adenomas and adenocarcinomas in the colon (12) while, as shown in the present study, oral administration led to mammary tumors. Thus, organ specificity (mammary cancer vs colon cancer) is dependent on the route of administration (oral vs i.p.), among other factors, including the dose and the age of the rat. The exact mechanism that accounts for such specificity requires further investigation. Nevertheless, oral administration is more relevant in view of the route of exposure of humans to NO2-PAHs such as 6-NC. The remarkable carcinogenic activity of 6-NC and the detection of its hemoglobin adducts in humans (19) suggest additional chronic exposure and dose-response experiments to establish its carcinogenic activity at the low dose levels that may be encountered by humans. As shown previously, following its metabolic activation, 6-NC can bind covalently to DNA, forming lesions that can initiate the process of tumorigenesis in the mammary gland. However, it was not known whether this carcino-

Mammary Cancer Induction by 6-NC and Metabolites

gen can also induce DNA base oxidation. Therefore, one of our goals in this study was to determine the effect of 6-NC on the levels of 8-OHdG as a marker of oxidative DNA damage in the rat mammary glands. 8-OHdG induces G:C to T:A transversion unless repaired prior to DNA replication; it is considered to be a promutagenic DNA lesion produced by reactive oxygen species (30). In general, oxidative stress has been implicated to play an important role in several human diseases, including breast cancer (31, 32). Several investigators have examined oxidative DNA damage in human breast cancer tissue employing different analytical methods; some of their studies showed a higher level of 8-OHdG in noninvolved tissue in human breast cancer cases than in normal tissue, but others were not in agreement (3235). Independent of the rationale, for the lack of consistency provided by these researchers, we sought to determine the effect of multiple doses of 6-NC on the levels of 8-OHdG in a well-defined animal model in tumors, in noninvolved, and normal mammary gland at termination, and in normal mammary gland 6, 24, and 48 h after a single dose of 6-NC treatment. This investigation clearly showed that 6-NC has no effect on the basal level of 8-OHdG. However, we did not assess other oxidative base damage that may have occurred. An earlier report stated that exposure to 1,6dinitropyrene resulted in increased levels of 5-hydroxymethyl-2′-deoxyuridine but had no effect on 8-OHdG in the rat mammary gland (36). Mammary carcinogens such as DMBA and PhIP, but not 1-nitropyrene, are capable of inducing 8-OHdG in the mammary gland of the rat in vivo (37). The lack of an effect of 6-NC metabolism on 8-OHdG suggests that 6-NC-induced covalent DNA adducts are important in the induction of mammary cancer in the rat by this environmental pollutant. The results of our previous studies indicate that the ring oxidation and nitroreduction combination pathway is a requisite pathway for the activation of 6-NC in vivo and in vitro (22, 24, 38, 39). Therefore, to avoid systemic effects and to determine the role of the mammary gland in the metabolic activation of these agents, we compared the carcinogenic potency of 6-NC with its metabolites by intramammary administration in the rat. Therefore, ample materials were needed, and, thus, we developed a new strategy to synthesize 1,2-DHD-6-AC while other metabolites were synthesized using approaches reported previously in our laboratory (23). 6-NC was the most potent mammary carcinogen; however, our results clearly indicate that metabolites derived from ring oxidation (1,2DHD-6-NC and 6-NCDE), nitroreduction (6-AC), as well as 1,2-DHD-6-AC that derived from a combination of ring oxidation and nitroreduction were all more active than DMSO-treated rats and 1,2-DHD-6-NC appears to be the most active. To translate our ongoing studies of 6-NC in rodents, we are currently examining its metabolic activation in primary cultures of human breast tissue. These types of studies will assist in the identification of DNA adducts detected but remained unidentified in human breast tissue.

Acknowledgment. This work was supported by National Cancer Institute Grant CA 35519 and, in part, by National Cancer Institute Support Grant CA 17613. We thank the staff of the Research Animal Facility, Mrs. Ilse Hoffmann (editorial assistance), and Mrs. Patricia Sellazzo for preparing the manuscript.

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