Metabolism of Benzo[c]chrysene and Comparative Mammary Gland

the in vitro metabolism of BcC. ... we compared the carcinogenicity of the bay region (()-anti-1,2-dihydroxy-3,4-epoxy-1,2,3 .... on the metabolic act...
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Chem. Res. Toxicol. 2003, 16, 227-231

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Metabolism of Benzo[c]chrysene and Comparative Mammary Gland Tumorigenesis of Benzo[c]chrysene Bay and Fjord Region Diol Epoxides in Female CD Rats Shantu Amin,* Jyh-Ming Lin, Jacek Krzeminski, Telih Boyiri, Dhimant Desai, and Karam El-Bayoumy American Health Foundation Cancer Center, Institute For Cancer Prevention, 1 Dana Road, Valhalla, New York 10595 Received October 11, 2002

Benzo[c]chrysene (BcC), an environmental pollutant, is a unique polycyclic aromatic hydrocarbon that possesses both a bay region and a fjord region in the same molecule. We previously demonstrated that both bay region and fjord region terminal rings are involved in the in vitro metabolism of BcC. In the present investigation, we prepared [14-3H]BcC and tested the hypothesis that BcC can be activated to both bay region and fjord region diol epoxides in female CD rats. At 6 weeks of age, rats were gavaged with a single dose of [14-3H]BcC (5 mg/rat; specific activity, 6.7 Ci/mmol) in 0.5 mL of trioctanoin. During the first 48 h, 20.3% of the dose was eliminated in the feces and 2.8% was eliminated in the urine. After 1 week, cumulatively, 23.2 and 3.5%, respectively, were eliminated. 3-Hydroxybenzo[c]chrysene, 10hydroxybenzo[c]chrysene, and trans-7,8-dihydroxy-7,8-dihydrobenzo[c]chrysene were the major fecal metabolites. In urine, trans-1,2-dihydroxy-1,2-dihydrobenzo[c]chrysene, 2-hydroxybenzo[c]chrysene, (()-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrobenzo[c]chrysene, and (()-9,t-10,t-11,c-12-tetrahydroxy-9,10,11,12-tetrahydrobenzo[c]chrysene were detected, primarily as glucuronic acid and sulfate conjugates. The identification of the two tetraols clearly indicates that both bay region and fjord region diol epoxides are formed as intermediates in the metabolism of BcC in vivo. The second goal of this study was to test the hypothesis that the location of the epoxide moiety (fjord vs bay region) determines the carcinogenic activity. Thus, we compared the carcinogenicity of the bay region (()-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4tetrahydrobenzo[c]chrysene and the fjord region (()-anti-9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetahydrobenzo[c]chrysene in the rat mammary gland. The results clearly showed that the fjord region diol epoxide is a potent mammary carcinogen, while the bay region diol epoxide lacks activity in this model assay. This is the first report on a comparison of mammary cancer induction by fjord and bay region diol epoxides derived from the same molecule. It further supports previous observations that fjord region diol epoxides are more carcinogenic than structurally related bay region diol epoxides.

Introduction It is well-established that exposure to exogenous carcinogens causes several types of cancer in humans. A better understanding of the ability of chemicals to induce mammary tumors in rodents may provide important leads toward elucidating their role in the etiology of human breast cancer (1). This is very important because breast cancer is second only to lung cancer as the leading cause of death from cancer among American women (1, 2). Human exposures to environmental carcinogens include PAHs,1 nitro-substituted PAHs (NO2-PAHs), aromatic amines, and heterocyclic amines, some of which are established mammary carcinogens in laboratory animals (3). The sources of these pollutants include cooked foods, various combustion processes, and polluted air. In fact, several studies have detected various DNA adducts in human breast tissue (4-8). The precise nature of these adducts remains largely unknown. The identification of relevant environmental pollutants that may * To whom correspondence should be addressed. Tel: (914)789-7162. E-mail: [email protected].

be involved in human breast cancer constitutes an active area of research in our laboratories (9). Extensive studies have demonstrated that the structure of PAHs is an important determinant of their biological activities (10). In living cells, PAHs are metabolized by cytochrome P450 enzymes (11-14) to highly reactive electrophiles such as PAH diol epoxides that can 1 Abbreviations: PAH, polycyclic aromatic hydrocarbons; BcC, benzo[c]chrysene; BcC-1,2-dihydrodiol, trans-1,2-dihydroxy-1,2-dihydrobenzo[c]chrysene; BcC-9,10-dihydrodiol, trans-9,10-dihydroxy-9,10-dihydrobenzo[c]chrysene; BcC-1,2-diol-3,4-epoxide, (()-anti-1,2-dihydroxy3,4-epoxy-1,2,3,4-tetrahydrobenzo[c]chrysene; BcC-9,10-diol-11,12epoxide, (()-anti-9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetahydrobenzo[c]chrysene; 2-HO-BcC, 2-hydroxybenzo[c]chrysene; 3-HO-BcC, 3-hydroxybenzo[c]chrysene; 10-HO-BcC, 10-hydroxybenzo[c]chrysene; BcC7,8-dihydrodiol, trans-7,8-dihydroxy-7,8-dihydrobenzo[c]chrysene; BcC1,2,3,4-tetraol, (()-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrobenzo[c]chrysene; BcC-9,10,11,12-tetraol, (()-9,t-10,t-11,c-12-tetrahydroxy-9,10,11,12-tetrahydrobenzo[c]chrysene; BcPDE, (()-anti-3,4-dihydroxy1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene; BaP, benzo[a]pyrene; BaPDE, (()-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BgCDE, (()-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrobenzo[g]chrysene; DB[a,l]PDE, (()-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene; 6-NC, 6-nitrochrysene.

10.1021/tx0200921 CCC: $25.00 © 2003 American Chemical Society Published on Web 01/25/2003

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compare the carcinogenic potency of the two (()-anti-diol epoxides derived from the same molecule (BcC) in the rat mammary gland; 6-NC, an environmental pollutant and a potent mammary carcinogen in this animal model, was employed as a positive control (26, 27).

Materials and Methods

Figure 1. Structures of 6-NC, BcC, and the corresponding bay and fjord region diol epoxide.

bind covalently to exocyclic amino groups of guanine and adenine, forming stable adducts within DNA (15, 16). Many of these reactive metabolites are mutagenic in bacterial and human cells and are carcinogenic in laboratory animals (17). In general, the tumorigenic activities of fjord region PAHs are remarkably higher than those of structurally related bay region PAHs. For example, BcPDE is a more potent carcinogen than BaPDE on mouse skin, newborn mouse lung, and rat mammary gland; it has also been shown that BgCDE and DB[a,l]PDE are more potent carcinogens than BaPDE in newborn mouse lung and in the rat mammary gland (1821). Although these previous observations clearly support the contention that the nonplanar, sterically hindered fjord region diol epoxides are more carcinogenic than planar bay region diol epoxides, these diol epoxides were derived from different precursors. Ideally, one would therefore compare the activities of a bay region and a fjord region diol epoxide that reside in the same PAH. BcC (Figure 1) is a candidate that satisfies this criterion. BcC is an environmental pollutant detected in coal tar and crude oil (22), which has been shown to be a weak carcinogen on mouse skin (23). Giles et al. reported that BcC is metabolically activated in mouse skin to fjord region diol epoxides and an unidentified metabolite, possibly a bay region diol epoxide (24). In a previous in vitro study with rat liver S9 fraction, we identified both BcC-9,10-dihydrodiol and BcC-1,2-dihydrodiol as metabolites of BcC (25); these diols are the precursors of BcC9,10-diol-11,12-epoxide (fjord region) and BcC-1,2-diol3,4-epoxide (bay region). There is a paucity of information on the metabolic activation of BcC in vivo (24). In general, human exposure to PAHs occurs mainly via inhalation and/or ingestion. Therefore, we examined whether fjord and bay region diol epoxides are potential intermediates in the metabolism of BcC when administered orally to female CD rats. The results provided evidence that tetraols derived from both bay region and fjord region diol epoxides were formed in vivo although to different extents. Therefore, the second goal of this study was to

Chemicals and Reagents. [14-3H]BcC was synthesized as described below. 14-Bromobenzo[c]chrysene was prepared by photochemical cyclization of 2-(2-naphthyl)-1-(4-bromonaphthyl)ethylene according to a literature procedure (28); upon tritium exchange (Amersham Pharmacia Biotech, Piscataway, NJ), it yielded [14-3H]BcC. BcC-1,2-diol-3,4-epoxide and BcC9,10-diol-11,12-epoxide were synthesized by epoxidation of the corresponding dihydrodiol, using m-chloroperbenzoic acid according to an established method (25, 28). All other metabolites were prepared as described (25). 6-NC was obtained as described (26), and purities of all chemicals employed here were ascertained to be >99% on the basis of HPLC analysis. All other chemicals and enzymes were purchased from Sigma Chemical Co. (St. Louis, MO). Metabolism of [14-3H]BcC in Female CD Rats. Female CD rats [Crl:CD(SD)BR], purchased from Charles River Breeding Laboratories, Inc. (Kingston, NY), were fed AIN-76A diet and water ad libitum and were acclimated for 2 weeks prior to the administration of BcC. They were maintained on tap water and AIN-76 semipurified high-fat diet and kept under standard conditions (23 ( 1 °C, 50% humidity, 12 h light/dark cycle). Three rats were housed in cages (one rat per cage) designed for collection of urine and feces. At 6 weeks of age, rats were gavaged with a single dose of [14-3H]BcC (5 mCi/ 5 mg/rat; specific activity, 6.7 Ci/mmol) in 500 µL of trioctanoin. The urine and feces were collected every 24 h for 1 week; each urine sample was adjusted with deionized water to a final volume of 5.0 mL. From each sample, a 5 µL aliquot was removed and radioactivity was measured. Each fecal sample was processed according to a literature procedure (29). Ten microliter aliquots from each of the EtOAc fecal extracts were counted for radioactivity. HPLC analyses of urinary and fecal metabolites were carried out with a reverse phase Vydac C18 column (10 µm, 4.6 mm × 250 mm) and the following program: 50% MeOH in water for 10 min, followed by a linear gradient to 100% MeOH over 60 min at a flow rate of 1 mL/min. Radioactive metabolites were detected by a Beta-Ram Flow-Through Monitor (IN/US System, Fairfield, NJ). To analyze urinary metabolites, aliquots (0.5-1.0 mL) that had been extracted three times with EtOAc were then treated with arylsulfatase (10 units) in the presence of saccharic acid 1,4-lactone (3 mg) or with β-glucuronidase (1000 units). After it was incubated for 6 h at 37 °C, the solution was extracted with EtOAc and then analyzed by reverse phase HPLC under the conditions described above. Both tetraols were inseparable under reverse phase conditions; thus, effluents were collected between 10.5 and 15.2 min and were then further analyzed on a normal phase HPLC column as described below. We employed an EM-Hibar Lichrosorb Si 60 column (5 µm, 4.6 mm × 250 mm), from which metabolites were eluted with a 1:1 mixture of Hex/EtOAc at a flow rate of 1.2 mL/min. The eluant was collected at 30 s intervals, and the radioactivity was determined by a Beckman LS 9800 Liquid Scintillation Counter. Carcinogenicity Assay. One hundred female CD rats (21 days old) [Crl:CD(SD)BR] from Charles River Breeding Laboratories were maintained on tap water and AIN-76A semipurified high-fat diet and housed under standard conditions as described above. They were randomly assigned to four groups of 25 rats each. When they were 30 days old, each rat was given three injections of 0.1 mL of dimethyl sulfoxide (DMSO) containing 2.04 µmol of 6-NC, BcC-1,2-diol-3,4-epoxide, or BcC9,10-diol-11,12-epoxide. Control rats received 0.1 mL of DMSO alone. The test solutions were injected into the mammary tissue under each of the three left thoracic nipples. The three thoracic

Metabolism of BcC and Rat Mammary Gland Carcinogenicity

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Table 1. Mammary Tumors in Rats Treated with 6-NC, BcC-1,2-diol-3,4-epoxide, and BcC-9,10-diol-11,12-epoxide no. of mammary tumors group 6-NC BcC-1,2-diol-3,4-epoxide BcC-9,10-diol-11,12-epoxide DMSO

total dose (mmol)

effective no. of rats

no. of rats with mammary tumors (%)

no. of tumors per rata

fibroadenoma or adenoma

adenocarcinoma

12.2 12.2 12.2

25 25 25 25

21 (84%) 4 (16%) 24 (96%) 0 (0%)

2.20 ( 1.53b 0.16 ( 0.38c 3.92 ( 1.73d,e 0

27 4 3 0

28 0 95 0

a Multiplicity was evaluated using analysis of variance followed by Tukey’s multiple comparisons procedure. b Significantly greater than DMSO; p < 0.0001. c Not significantly greater than DMSO. d Significantly greater than DMSO; p < 0.0001. e Significantly greater than BcC-1,2-diol-3,4-epoxide; p < 0.0001.

Figure 2. Urinary and fecal excretion of radioactivity following administration of [14-3H]benzo[c]chrysene ([14-3H]-BcC) to female CD rats by gavage. nipple areas on the right side were each injected with 0.1 mL of DMSO. On the following day, the tissues underlying each of the three left inguinal nipples were treated with the diol epoxides in DMSO or with DMSO alone in the control group and the three right nipple areas were treated with DMSO only. The total dose of each compound was 12.2 µmol/rat. All injections were given under light ether anesthesia. Body weights were measured weekly during the first month and then monthly until termination. Rats were inspected and palpated weekly for mammary tumors beginning 7 weeks after the last injections. Rats were sacrificed when large or ulcerated tumors developed or when they were moribund. The experiment was terminated 35 weeks after the injections.

Figure 3. (A) Reverse phase HPLC profile of fecal extract: peak A, BcC-7,8-dihydrodiol; peak B, 3-HO-BcC; peak C, 10HO-BcC; peak D, BcC. (B) Reverse phase HPLC profile of rat urine after hydrolysis with arylsulfatase or β-glucuronidase: peak E, BcC-1,2-dihydrodiol; peak F, 2-HO-BcC.

Results and Discussion The time courses of excretion of radioactivity in the urine and feces after oral administration of [14-3H]BcC to female CD rats are depicted in Figure 2. During the first 48 h, 20.3% of the radioactivity was eliminated in the feces and 2.8% was eliminated in the urine. Cumulative excretion after 1 week was 23.2% in feces and 3.5% in urine. Although the major portion of radioactivity remained unknown, it is clear that fecal elimination is the major route of excretion. Analysis of fecal extracts showed that 3-HO-BcC, 10HO-BcC, and BcC-7,8-dihydrodiol were the major metabolites on the basis of retention times (Figure 3A). However, HPLC analysis of urine showed that no detectable radioactivity eluted with any of the synthetic derivatives of BcC that are available in our laboratory; instead, we observed only polar compounds that could be conjugates of BcC metabolites. Thus, enzymatic digestion of the urine with arylsulfatase or β-glucuronidase was carried out. On the basis of HPLC retention times, we have identified 2-HO-BcC and BcC-1,2-dihydrodiol as metabolites (Figure 3B). Coinjection of synthetic BcC1,2,3,4-tetraol and BcC-9,10,11,12-tetraol indicated that

Figure 4. Normal phase HPLC profile of the fraction eluting from 10.5 to 15.2 min under reverse phase conditions (see Figure 3, panel B).

these two tetraols were not separated by the HPLC system shown in Figure 3. Therefore, we collected the effluent that eluted between 10.5 and 15.2 min and rechromatographed it on a normal phase system. This led to the successful separation of BcC-1,2,3,4-tetraol and BcC-9,10,11,12-tetraol (Figure 4). The amount of BcC1,2,3,4-tetraol observed is 6-8 times greater than that of BcC-9,10,11,12-tetraol. In a previous study, several fjord region diol epoxides, including that derived from BcC, were shown to be more stable in physiological buffer at 37 °C (t1/2 > 2 h) than numerous bay region diol epoxides (t1/2 ) 0.011-1.2 h) (30). The relative stability of these diol epoxides may, in part, account for the lower level of fjord region tetraol than the bay region tetraol derived from BcC. Nevertheless, the presence of both the

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BcC-1,2,3,4-tetraol and the BcC-9,10,11,12-tetraol suggests that both the bay region and the fjord region terminal ring of BcC are involved in the metabolic activation and the corresponding bay region and fjord region diol epoxides are potential intermediates in the metabolism of BcC in the rat. Interestingly, using a 32Ppostlabeling approach, Giles et al. have reported the detection of DNA adducts derived from the BcC fjord region diol epoxides in mouse epidermis; however, DNA adducts derived from the bay region have not yet been confirmed (24). Furthermore, these investigators reported that the level of adducts in mouse epidermal DNA following topical treatment with BcC was still very low (0.89 fmol/µg DNA) by comparison to BgC (6.55 fmol/µg DNA) but similar to that of BcPh (24). In an effort to determine the level of DNA binding, we tried to measure the radioactivity of DNA isolated from rat mammary gland; yet, we did not succeed. The lack of detectable DNA adducts in the rat mammary gland could be due to several factors, including a low level of binding in this organ, comparable to that observed in mouse skin (24); such a low level would be undetectable with the specific activity of [14-3H]BcC used in this study. Currently, we are developing a 32P-postlabeling method employing DNA adduct markers derived from both diol epoxides to determine levels of DNA binding and the relative contribution of fjord and bay region diol epoxides to the total binding in the rat mammary gland. The detection of both tetraols as metabolites of BcC encouraged us to assess the carcinogenicity of the parent diol epoxides in the rat mammary gland model assay. The bioassay was carried out as described in the literature (26). Results are listed in Table 1. Consistent with previous studies, the positive control 6-NC induced both malignant and benign tumors in the mammary gland (26). Rats treated with BcC-9,10-diol-11,12-epoxide showed a significantly higher tumor incidence (96%) than rats treated with BcC-1,2-diol-3,4-epoxide (16%). At the same time, the tumor multiplicity was significantly higher in the BcC-9,10-diol-11,12-epoxide-treated group. In this bioassay, BcC-9,10-diol-11,12-epoxide induced a total of 95 adenocarcinomas and three benign tumors, while BcC1,2-diol-3,4-epoxide induced only four benign tumors and no malignant tumors were detected. The results are consistent with those obtained previously with BcPDE, a nonplanar fjord region diol epoxide, which is a more potent carcinogen than BaPDE, a planar bay region diol epoxide (20). In summary, we have shown that both fjord and bay region diol epoxides are potential intermediates in the metabolism of BcC in the rat. Furthermore, we have demonstrated that BcC-9,10-diol11,12-epoxide, a fjord region diol epoxide, is a more potent rat mammary gland carcinogen than BcC-1,2-diol-3,4epoxide, a bay region diol epoxide. These results are consistent with the general observations that fjord region diol epoxides are more potent mammary carcinogens than bay region diol epoxides. This is the first paper on the comparative carcinogenicity between a fjord region and a bay region diol epoxide derived from the same PAH.

Acknowledgment. This study was supported by NCI Contract NO2-CB-77022-75 and Grant Nos. CA-35519 and CA-44377. We are grateful to Mrs. Ilse Hoffmann for editing the manuscript.

Amin et al.

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Metabolism of BcC and Rat Mammary Gland Carcinogenicity

(22) (23)

(24)

(25)

(26) (27)

cally hindered diol epoxides of benzo[g]chrysene, dibenzo[a,l]pyrene (dibenzo[def,p]chrysene), 4H-cyclopenta[def]chrysene, and fluoranthene. Carcinogenesis 16, 2813-2817. Harvey, R. G. (1991) Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity, Cambridge University Press, Cambridge, MA. Hartwell, J. L. (1951) Survey of Compounds Which Have Been Tested for Carcinogenic Activity. U. S. Public Health Service Publication No. 149, Superintendent of Documents, U. S. Government Printing Office, Washington, DC. Giles, A. S., Seidel, A., and Phillips, D. H. (1997) Covalent DNA adducts formed by benzo[c]chrysene in mouse epidermis and by fjord-region benzo[c]chrysene fjord-region epoxides reacted with DNA and polynucleotides. Chem. Res. Toxicol. 10, 1275-1284. Desai, D., Krzeminski, J., Lin, J.-M., Chadha, A., Miyata, N., Yagi, H., Jerina, D. M., and Amin, S. (1999) Synthesis and identification of benzo[c]chrysene metabolites. Polycyclic Aromat. Compd. 16, 255-264. El-Bayoumy, K., Rivenson, A., Upadhyaya, P., Chae, Y.-H., and Hecht, S. S. (1993) Induction of mammary cancer by 6-nitrochrysene in female CD rats. Cancer Res. 53, 3719-3722. El-Bayoumy, K., Desai, D., Boyiri, T., Rosa, J., Krzeminski, J., Sharma, A. K., Pittman, B., and Amin, S. (2002) Comparative

Chem. Res. Toxicol., Vol. 16, No. 2, 2003 231 tumorigenicity of the environmental pollutant 6-nitrochrysene and its metabolites in the rat mammary gland. Chem. Res. Toxicol. 15, 792-798. (28) The physical properties and spectral data of 14-bromobenzo[c]chrysene are listed as follows: 41% yield; mp 128-130 °C; 1H NMR (CDCl3): 7.64-7.81 (m, 4H, H2, H3, H10, H11), 7.87 (d, 1H, H7 or H8, J7,8 ) 8.5 Hz), 7.93 (d, 1H, H8 or H7, J8,7 ) 8.5 Hz), 8.05 (d, 1H, H9, J9,10 ) 7.9 Hz, J9,11 ) 1.3 Hz) 8.08 (d, 1H, H6, J5,6 ) 8.9 Hz), 8.43-8.48 (m, 1H, H1), 8.79 (d, 1H, H5, J5,6 ) 8.9 Hz), 8.82-8.84 (m, 1H, H4), 9.00 (d, 1H, H12, J11,12 ) 8.5 Hz), 9.39 (s, 1H, H13). MS m/z (relative intensity): 356 and 358 (M+, 50), 276 (M+-Br, 100), 248 (10), 138 (30). (29) Chae, Y.-H., Delclos, K. B., Blaydes, B., and El-Bayoumy, K. (1996) Metabolism and DNA binding of the environmental colon carcinogen 6-nitrochrysene in rats. Cancer Res. 56, 2052-2058. (30) Glatt, H., Piee, A., Pauly, K., Steinbrecher, T., Schrode, R., Oesch, F., and Seidel, A. (1991) Fjord- and bay-region diol-epoxides investigated for stability, SOS induction in Escherichia coli, and mutagenicity in Salmonella typhimurium and mammalian cells. Cancer Res. 51, 1659-1667.

TX0200921