Chem. Res. Toxicol. 1994, 7,690-695
690
In Vitro and In Vivo Metabolism of the Carcinogen 4-Nitrop yrene Pramod Upadhyaya,S Linda S. Von Tungeln,# Peter P. Fu,# and Karam El-Bayoumy*?* American Health Foundation, Valhalla, New York 10595,and Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas 72079 Received May 18, 1994@
The in vitro and in vivo metabolism of the potent mutagen and carcinogen, 4-nitropyrene, was studied. 4-Aminopyrene, 4-(acetylamino)pyrene, 9,10-epoxy-9,10-dihydro-4-nitropyrene, cis- and truns-9,10-dihydro-9,10-dihydroxy-4-nitropyrene, 9- and lO-hydroxy-4-nitropyrene, and 9- and lO-hydroxy-4-(acetylamino)pyrenewere synthesized to serve a s markers for the identification of 4-nitropyrene metabolites. Initially, 4-nitropyrene was metabolized by rat liver microsomes, or rat liver 9OOOg supernatant, to yield primarily two metabolites; one of these was identified as 4-nitropyrene-9,lO-dione.The major metabolite of 4-nitropyrene in the presence of 3,3,3-trichloropropylene-1,2-oxidewas 9,10-epoxy-9,10-dihydro-4-nitropyrene. In parallel studies, oral administration of 58 mg (0.3 mCi/mmol)/kg body weight of 13H14nitropyrene to female Sprague-Dawley rats, which are susceptible to mammary carcinogenesis by this agent, yielded 32% and 30.6% of the dose after 48 h a s urinary and fecal excretion products, respectively. Excretion of the radioactivity remained slightly higher in the urine than in feces throughout 168 h after administration. Some of the fecal metabolites (isolated amounts expressed a s % of dose) were identified a s 4-aminopyrene (5.41, 9(10)-hydroxy-4(acety1amino)pyrene (3.31, and unmetabolized 4-nitropyrene (2.4). Sulfates (3.3) and glucuronides (2.4) of 9(lO)-hydroxy-4-(acetylamino)pyrenewere identified in the urine. This study indicates that nitroreduction and ring oxidation are metabolic pathways of 4-nitropyrene in vivo; similar findings were obtained previously with its structural isomers 1-and 2-nitropyrene. However, the pattern of excretion of 4-nitropyrene is different; the significance of this observation in relation t o tumor induction is discussed.
Introduction Nitropolynuclear aromatic hydrocarbons (N02-PAHI' represent a class of environmental carcinogens t o which humans are exposed in certain occupational environments, in ambient air, and through food intake (1-3). Several members of this class of compounds are mutagenic in bacterial and mammalian systems and tumorigenic in rodents (4-6). It has been demonstrated that the mutagenic and carcinogenic potencies of NOzPAH are strongly dependent on their structure. Among the three isomers of mononitropyrene that occur in urban air (7,8),the levels of 2-nitropyrene (2-NP) and 4-NP are about the same; however, there is far less of these than of 1-NP and the latter is the only isomer detected in combustion materials (1). The three isomers of NP have distinctly different mutagenic and tumorigenic potencies (9-11). The mu*To whom correspondence and requests for reprints should be addressed, at the American Health Foundation, 1Dana Rd., Valhalla, NY 10595. f American Health Foundation. 0 National Center for Toxicological Research. Abstract published in Advance ACS Abstracts, September 1,1994. Abbreviations: N02-PAH, nitropolynuclear aromatic hydrocarbons; 1-,2-, and 4-NP, 1-, 2-, and 4-nitropyrene; 4-AP, 4-aminopyrene;THP, 4,5,9,10-tetrahydropyrene;9(10)-OH-4-NP, 9(10)-hydroxy-4-nitropyrene; 9(10bOH-PAAP,9(1O)-hydroxy-4-(acetylamino)pyrene;9(1O)-Ac4-AAP, 9(10)-acetoxy-4-(acetylamino)pyrene; 4-AAP, 4-(acetylaminolpyrene; 4-NP-9,10-dione,4-nitropyrene-9,lO-dione; 4-NP-g,lO-oxide, 9,10-epoxy-9,10-dihydro-4-nitropyrene; cis- and trans-g,lO-DHD-4-NP, cis- and trans-9,10-dihydro-9,1O-dihydroxy-4-nitropyrene; 3-MC, 3methylcholanthrene; ODQ, 2,3-dichloro-5,6-dicyano-l,4-benzoquinone; TCPO, 3,3,3-trichloropropylene1,a-oxide;9(10)-Ac-4-NP,9(10)-acetoxy4-nitropyrene.
tagenicity of 4-NP exceeds that of 2-NP, which, in turn, exceeds that of 1-NP (11).In the liver and lung of male newborn mice, 4-NP was more tumorigenic than 1-NP in this animal model (10).Direct administration of each isomer into the mammary pads of weanling female CD rats revealed that 4-NP is also the strongest tumorigen of the three isomers (9);similar observations were made when each isomer was administered by ip injection. Among the three isomers, only 1-NP was tested by oral administration, and it has been shown to induce mammary tumors (12). Thus, the present study was conducted to test the hypothesis that differences in the metabolism may, in part, account for the observed variations of the tumorigenic activities of these isomers in the rat (9). Several investigators have reported on the metabolism of 1-NP; however, far fewer studies have been done on 2-NP (13), and virtually nothing is known about the metabolism of 4-NP. Therefore, pilot experiments were carried out toward identifying 4-NP metabolites in vitro using rat liver microsomes and were followed by assays in vivo under conditions similar to those described for its other isomers (13,14).
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Materials and Methods Instrumentation. HPLC was performed with a Waters Associates System (Millipore; Waters Division, Milford, MA) equipped with a Model 510 solvent delivery system, a Model U6K septumless injector, a Model 440 UV-visible detector, and a Model 680 Waters automated gradient controller, as well as with a Beckman system consisting of two Model lOOOA pumps, a Model 210 injector, and a Model 420 solvent programmer, and
0893-228x/94/2707-0690$04.50/00 1994 American Chemical Society
Metabolism of 4-Nitropyrene equipped with a Waters Associates Model 440 absorbance detector, operated at 254 nm. HPLC analyses of the metabolites were performed with the following systems: System 1: A linear gradient from 60% CH30H-HzO to 100% CH30H in 30 min a t a flow rate of 2.8 m u m i n using a DuPont Zorbax ODS column (0.94 x 25 cm). System 2: A linear gradient from 30% CH~OH-HZOto 100% CH30H in 60 min at a flow rate of 1m u m i n using a Vydac c i s 10-pm reverse-phase column (0.46 x 25 cm, Separation Group, Hesperia, CAI. System 3: A linear gradient from 100% HzO to 100% methanol in 60 min a t a flow rate of 1m u m i n using the column described in System 2. System 4: A normal-phase HPLC analysis under isocratic conditions using 25% THF in hexane a t a flow rate of 1 mL/ min and a 5;um Lichrosorb column (0.46 x 25 cm, Si 60, Merck, Darmstadt, Germany). System 5: Isocratic conditions using the same column as in System 4 and elution with 5% THF in hexane a t a flow rate of 2 mumin. Radioactivity was measured with a Beckman Model LS9800 scintillation counter. For radiochromatography, a radioactive flow detector (Radiomatic Instruments and Chemical Co., Inc., Tampa, FL) was used. Mass spectra were obtained on a Hewlett-Packard Model HP5988A dual-source mass spectrometer (Hewlett-Packard Co., Palo Alto, CA) and on a Finnigan Model 1015 system, using electron impact with a solid probe a t 70 eV. W-visible spectra were prepared with a Beckman Model 25 spectrophotometer. lH NMR analyses were performed with a Bruker AM-360 Wl3 spectrometer and with a Bruker WM 500 (USA Bruker Instruments, Inc., Manning Park, Billerica, MA); chemical shifts are reported in ppm using TMS as an internal standard. Preparative TLC was carried out with silica gel 60F254s-coated plates (20 x 20 x 1 mm) with a preconcentration zone (4 x 20 mm, EM Science, Gibbstown, NJ).
Chemicals and Enzymes. 1,2,3,6,7&Hexahydropyrene, 3,3,3-trichloropropylene-1,2-oxide (TCPO), 4-(dimethylamino)pyridine, m-chloroperbenzoicacid, and 2,3-dichloro-5,6-dicyano1,Cbenzoquinone (DDQ)were purchased from Aldrich Chemical Co. (Milwaukee, WI). 4-Nitr0[G-~H]pyrene(2.09 CVmmol, 98%) was acquired from Chemsyn Science Laboratories (Lenexa, KS). Enzymes and other biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO); these included arylsulfatase (from limpets, type V), /?-glucuronidase (from E.coli, type E), D-saccharic acid 1,4lactone, glucose-6-phosphate dehydrogenase, glucose 6-phosphate, and NADP+. Synthesis of 4-Nitropyrene (4."). 4-NP was prepared with nitric acid in by nitration of 1,2,3,6,7,8-hexahydropyrene acetic anhydride at ambient temperature for 48 h t o afford 4-nitro-1,2,3,6,7,8-hexahydropyrene (15)which was dehydrogenated by DDQ in benzene (16).The crude product was purified on Florisil by eluting with benzene and was then recrystallized from benzene a s yellowish needles: mp 197-198 "C [lit. (15) mp 196-197.5 "Cl; MS (70 eV): m l z 247; 500-MHz lH NMR (acetone-&): 6 8.25 (t, l H , H7), 8.27 (t, l H , Hz), 8.30 (AB pattern, 2H, H ~ J o )8.50 , (d, l H , HI), 8.55 (d, l H , Hs), 8.62 (d, l H , &), 8.81 (d, l H , H3), 9.05 (s, l H , H5); Ji,z = J6,7 = J7,a = 7.7, 5 2 , s = 8.2, J g ~ = o 9.0 Hz. Synthesis of 4-(Acetylamino)pyrene (4-AAP). 4-Aminopyrene (4-AP) (2.3 mg, 0.01 mM), prepared by reduction of 4-NP with Zn/NH&l, was dissolved in 20 mL of dry EtOAc, and acetic anhydride (1.0 mL) and 4-(dimethylamino)pyridine(20 mg) were added. After stirring overnight, the reaction mixture was washed successively with saturated NaHC03 and HzO. The EtOAc extract was dried over MgS04. 4-AAP was obtained by HPLC (System 2, retention time 36 min, 2 mg, 77% yield): MS (relative intensity): m l z 259 (M+, 46), 217 (loo), 189 (48); lH NMR (CDC13) 6 2.45 ( s , 3H, CH3), 8.0-8.25 (m, 8H, aromatic), 8.65 (s, l H , H5). Synthesis of 9,10-Epoxy-9,10-dihydro-4-nitropyrene (4NP-9,lO-oxide).4-NP-9,lO-oxidewas synthesized by treatment of 4-NP with m-chloroperoxybenzoic acid as described for the synthesis of l-NP-4,5-oxides and -9,lO-oxides(17,181.The pure product was collected by HPLC; it eluted after 30 min using
Chem. Res. Toxicol., Vol. 7, No. 5, 1994 691 System 2. MS (relative intensity): m l z 263 (M+, 80),235 (lo), 217 (401,205 (60), 189 (100); lH NMR (acetone-&): 6 5.02 (AB, 2H, Hg,io), 7.95 (t, l H , H7), 7.98 (dd, l H , Hz), 8.26 (d, l H , Hs), 8.30 (dd, lH, Hi), 8.39 (dd, l H , Ha), 8.51 (d, lH, H3), 8.80 (s, 1H, H5); J1,2 = 7.3, 52,s = 8.5, J6,7 = 57,s = 8.0, J 6 , a = 1.0 Hz.
Synthesis of cis- and trans-9,lO-Dihydro-9,lO-dihydroxy. 4-nitropyrene (9,10-DHD-4-NP).cis- and trans-9,lO-DHD4-NP were obtained from the 4-NP-9,lO-oxide (2.6 mg, 0.01 mmol) by stirring with 5 mL of a mixture of acetic acid-H2O (1:3) a t room temperature for 30 min. The product was analyzed by HPLC, revealing a mixture of two peaks, presumably the cis and trans isomers. The major peak eluted after 36 min, the minor peak after 36.5 min, in System 3. However, a better separation was achieved using System 4. Approximately 1 mg (36%) of the major isomer (31 min) was collected. The spectral data for this major isomer are a s follows: MS (relative intensity): m l z 281 (M+, 1001, 263 (401, 246 (28), 217 (541, 189 (62); lH NMR (CDC13-DzO) 6 5.2 ( s ,2H, H ~ J O7.7-7.85 ), (m, 2H, H z , ~ ) , 7.9-8.1 (m, 3H, H1,6,8),8.58 (s, l H , H5), 8.50 (d, l H , H3, J = 8.5 Hz). The cis or trans configuration could not be determined since H9 and H10 appeared as a singlet. To remove the cis isomer, the crude reaction (0.5 mg) mixture was dissolved in 10 mL of acetone and an excess of anhydrous CuSO4 powder (20 mg) was added (19). The mixture was then heated a t reflux for 4 h, cooled, filtered, concentrated, and analyzed by HPLC. The disappearance of the minor isomer (cis) indicated that the major isomer is trans. Synthesis of 9- and 10-Hydroxy-4-nitropyrene(9- and 10-OH-4-NP). Stirring of 4-NP-9,lO-oxide(2.6 mg, 0.01 mmol) with 5 mL of acetic acid-Hz0 (1:3) a t room temperature as described above, but for 3 h, yielded a mixture of 9- and 10OH-4-NP. This mixture was analyzed by HPLC (System 31, yielding two peaks with retention times of 50 and 50.5 min, respectively (1.0 mg, 36% yield). Efforts to separate the two isomers in pure forms by reverse-phase HPLC were not successful; however, normal-phase HPLC analysis using System 5 yielded two peaks, eluting after 31 and 34 min; these were unequivocally assigned as 9-OH-4-NP and 10-OH-4-NP,respectively, based on lH NMR analysis, including decoupling experiments: 9-OH-4-NP (CDC13): 6 7.61 ( s , l H , Hlo), 8.14 (t, l H , Hz), 8.25 (dd, l H , Hi), 8.27 (t, l H , H7), 8.56 (dd, l H , Hs), 8.64 (dd, l H , Ha), 8.85 (dd, l H , H3), 9.01 (s,l H , H5);J i , z = 7.9, J i , 3 = 0.9, J 2 , 3 = 8.0,57,8 = 7.8, J6,s = 1.2 Hz. 10-OH-4-NP (CDC13): 6 7.61 (s, l H , Hg), 8.12 (t, l H , H7), 8.15 (t, l H , Hz), 8.31 (dd, 1H, Ha), 8.40 (d, 1H, H d , 8.83 (d, 2H, Hi,3), 9.0 (s,1H, H5); Ji,z = J z , ~ = 8.3, J6,7 = 7.5, 5 7 , s = 7.7, J s ,=~1.2 Hz. MS ofthe two isomers were similar: MS (relative intensity): m / z 263 (loo), 233 (81,217 (52), 187 (38).
Synthesis of 9- and 10-Acetoxy.4-nitropyrene (9-and 10Ac-4-NP). These compounds were synthesized from a mixture of 9- and 10-OH-4-NP (approximately 1.0 mg, 0.005 mmol), as described (13) using acetic anhydride (0.5 mL) and (dimethylaminolpyridine (10 mg) in 20 mL of EtOAc. The two isomers were obtained by HPLC (System 3, retention times 53 and 53.5 min, 80% yield); MS of the mixture (relative intensity): m l z 305 (M+, 4), 277 (221, 263 (821, 217 (42). Synthesis of 9- and lO-Acetoxy-4-(acetylamino)pyrene
(9- and 10-Ac-4-AAP)and Their Corresponding Phenols. A mixture of 9- and 10-Ac-4-NP (1.0 mg, 0.003 mmol) was reduced with ZdNH4Cl followed by acetylation (13). The two compounds were collected from HPLC (System 3, retention times 41.5 and 42 min, 80% yield); MS of the mixture (relative intensity): m l z 317 (M+, 26), 275 (701, 233 (loo), 204 (281, 176 (28). A mixture of 9- and 10-Ac-4-AAP was treated with CH30Na in CH30H to yield the corresponding phenols (13). Both phenols were collected as a mixture by HPLC in System 3 (retention times 35 and 35.5 min); MS (relative intensity): m l z 276 (M+, 401, 234 (9). Incubation of 4-NP with Rat Liver Microsomes. Male Sprague-Dawley rats (150-200 g) were pretreated with three daily ip injections of 3-methylcholanthrene (3-MC), 25 mgkg body weight, in corn oil; liver microsomes were prepared according t o Schenkman and Cinti (201,except that the livers were perfused with 0.15 M KCl before removal. 4-NP (40 mmol in 10 mL of acetone) was added to a 500-mL reaction mixture
Upadhyaya et al.
692 Chem. Res. Toxicol., Vol. 7, No. 5, 1994 containing 25 mM Tris-HC1 buffer (pH 7.51, 1.5 mM MgC12, 50 units of glucose-6-phosphate dehydrogenase (type X I ) , 48 mg of NADP+, 280 mg of glucose 6-phosphate, and 500 mg of microsomal protein. This mixture was incubated under aerobic conditions a t 37 "C for 1 h. The incubation was quenched by adding 350 mL of acetone. 4-NP and its metabolites were then extracted with 1 L of EtOAc. The organic layer was removed and the solvent was evaporated under reduced pressure. The residue was extracted twice with 5 mL of acetone; the insoluble microsomal protein was removed by filtration. After removal of the acetone, the residue was dissolved in methanol for reverse-phase HPLC (System 1)separation of the metabolites. Metabolism of 4-NP was also studied in the presence of the epoxide hydrolase inhibitor TCPO (0.3 mM) under conditions similar to those described above, except that the incubation time was decreased to 30 min.
Metabolism of [3H]4-NP in Female Sprague-Dawley Rats. Female Sprague-Dawley rats (approximately 238 g, Charles River Breeding Laboratories, Kingston, NY) were acclimatized to the conditions of the test laboratory for 2 weeks before metabolism studies were begun. Urine and feces were collected from 3 rats each, housed in a metabolism cage (13, 14). Rats were fed NIH-07 diet and received water ad libitum. The metabolism cages were kept at 22 f 2 "C and 55 f 15% humidity in light-controlled rooms (12 h lighudark cycle). Each rat was given [3Hl4-NP by intragastric gavage in trioctanoin a t a dose of 58 mg (0.3 mCi/mmol)/kg body weight. This dose was comparable to those administered in previous metabolism studies with 1-NP and 2-NP (13, 14). Twenty-four-hour urine voids were collected a t 0-4 "C, and feces were collected at ambient temperature over a 7-day period. Urine and feces were stored below 0 "C until analysis. Analysis of Fecal and Urinary Metabolites. Each 24-h fecal sample was analyzed before and after acetylation as described previously (13, 14). Acetylation of fecal metabolites was performed to ensure higher recovery of unstable metabolites such as aminohydroxypyrenes that decompose rapidly ( 1 3 , 1 4 ) . Each 24-h urine sample was extracted with EtOAc. The extracts were dried (MgS04) and concentrated. The residues were dissolved in THF for HPLC (System 2) analysis. Radioactivity in each urine sample was measured before and after removal of the organic soluble metabolites by ethyl acetate extraction. An aliquot of the EtOAc extract of the combined 7-day urine samples was acetylated with acetic anhydridd4(dimethy1amino)pyridine. The acetylated metabolites were also analyzed by HPLC (System 2). To isolate HzO-soluble glucuronide and sulfate conjugates, aliquots of urine samples that had been exhaustively extracted with EtOAc were incubated in citrate buffer (pH 5.5) with ,&glucuronidase or arylsulfatase in the presence of saccharic acid 1,4-lactone at 37 "C for 6 h. In a typical experiment, either 10 000 units of &glucuronidase or 63 units of arylsulfatase were used for each 10 mL of pH-adjusted urine sample. When arylsulfatase was employed, 20 mg of saccharic acid l,4-lactone was included in the incubation mixture. Control experiments were performed as described above but without enzymes. The enzymatic reactions were quenched with 2 mL of ice-cold EtOAc, and the released metabolites were further extracted with 4 x 10 mL of EtOAc. The extract was dried and analyzed by HPLC (System 2) before and after acetylation.
Results In Vitro Metabolism of 4-NP.4-NP was incubated under aerobic conditions with liver microsomes prepared from rats pretreated with 3-MC. The organic solventextractable metabolites and unreacted substrate were separated in reverse-phase HPLC System 1(Figure 1A). Peak 3, which eluted at 33.5 min, was the recovered substrate. Peak 2 had a mass spectrum (Figure 2A) with a molecular ion at m l z 277 and fragments at m l z 249 (loss of CO), m l z 219 (loss of CO and NO), m / z 203 (loss of CO and NOz), and m l z 175 (loss of NO2 and two CO), suggesting that it was a dione of 4-NP. The structure of
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Figure 1. (A) Reverse-phase HPLC (System 1)profile of the organic solvent-extractable metabolites formed from aerobic metabolism of 4-nitropyrene by liver microsomes of rats pretreated with 3-MC; (B) reverse-phase HPLC profile of the organic-extractable metabolites formed from aerobic metabolism of 4-nitropyrene in the presence of TCPO by liver microsomes of rats pretreated with 3-MC. (M-CO-Nd'
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Figure 2. Mass spectra of the metabolite identified as 4-nitropyrene-9,lO-dione (A), and its lH NMR (B).
this metabolite was then determined by analysis of its high-resolution 500-MHz proton NMR spectrum (Figure 2B). The NMR resonance assignments were aided by comparisons with the NMR spectral parameters of 4-NP and by homonuclear decoupling and nuclear Overhauser enhancement experiments. On the basis of chemical shifts and coupling patterns, the metabolite contained in chromatographic peak 2 was identified as 4-nitropyrene-9,lO-dione (4-NP-9,lO-dione). lH NMR: (acetoneds): 6 7.83 (dd, 1, H,), 7.86 (dd, 1, Hz), 8.08 (d, 1, Hi), 8.10 (d, 1, H8),8.17 (d, 1, He), 8.32 (d, 1, H3), 8.69 (s, 1, H& J i , z 7.2, J2,3 = 8.4, J6,7 = 7.9, J7.8 = 7.3 Hz. Metabolites eluted after 4 min (peak 1) and those (minor) eluted between peaks 1 and 2 could not be purified in sufficient quantities for structural identification. The retention time on HPLC of peak 1 did not match any of the polar synthetic standards, including trans-9,lO-DHD-4-NP. The metabolic profile was similar
Metabolism of 4-Nitropyrene
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Chem. Res. Toxicol., Vol. 7, No. 5, 1994 693
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Figure 3. (A) Excretion of radioactivity following administration of [3H14-nitropyreneto F344 rats by intragastric gavage. (B)and (C) represent the corresponding data (cb refs 12 and 13)following the administration of 2-nitropyrene and l-nitropyrene under similar conditions.
to that obtained upon incubation of r3H]4-NPwith 3-MCinduced S9 fraction of the liver from female SpragueDawley rats; the further metabolism of 4-NP-9,10-dione, using S9 fraction, yielded a major metabolite which eluted after 4 min (data not shown). The metabolites obtained from incubating 4-NP with rat liver microsomes in the presence of TCPO were separated by reverse-phase HPLC (Figure 1B). The structural assignment of this compound was confirmed as 4-NP-9,lO-oxide by comparison of its MS, lH NMR, and chromatography with the synthetic standard (cf . Materials and Methods). In Vivo Metabolism of 4-NP. Following the oral administration of l3H14-NP to female Sprague-Dawley rats, 32% and 30.6% of the dose were excreted in the urine and feces, respectively, after 48 h. Slightly more radioactivity was excreted in the urine than in the feces throughout the 168 h of monitoring (Figure 3). Panels B and C of Figure 3 were included for comparison of the data with those for 1-NP and 2-NP (13, 14). On the basis of chromatographic characteristics (Figure 4), using System 2, chemical transformation, and, in some instances, mass spectral analysis, some of the fecal metabolites (expressed as percentage of dose) were identified as 4-AP (5.4) and 9(10)-hydroxy-4-(acetylamino)pyrene [9(10)-OH-4-AAPl (3.3); unmetabolized 4-NP accounted for 2.4% of the dose. Other metabolites were detected but were not chromatographically compatible with any of the standards synthesized in this study. However, of particular interest is the metabolite(s) that eluted between 4 and 5 min since, upon further in vitro metabolism, 4-NP-9,lO-dione yielded primarily one major
Figure 4. Radiochromatogram obtained upon HPLC (System 2) analysis of the ethyl acetate-ethanol extract of feces from rats treated with L3H14-nitropyrene.
unknown that was also eluting between 4 and 5 min. Partitioning of the peak eluting between 32 and 34 min, using NaOH in the aqueous phase, suggested its phenolic nature. The EtOAc-soluble metabolites in urine that had been treated with either p-glucuronidase or arylsulfatase were analyzed by HPLC. On the basis of cochromatography (Systems 2 and 31, glucuronide (1.4% of the dose) and sulfate (2.5%) conjugates of 9(10)-OH-4-AAPwere identified. The structure of these metabolites was further confirmed by acetylation to the corresponding acetoxy derivatives and comparison to standards. Organicsoluble metabolites in urine were identified as 4-AP (0.3%),9(10)-OH-4-AAP (0.2%),and 9(10)-OH4NP (0.8%); unmetabolized 4-NP accounted for 0.2% of the dose.
Discussion This is the first report that describes the metabolism of 4-NP and synthesis of several of its derivatives and/or metabolites. Aerobic incubation of 4-NP with liver microsomes of rats pretreated with 3-MC generated 4-NP9,lO-dione as the predominant metabolite. The metabolism of 4-NP is markedly different from that of its structural isomers 1-NP and 2-NP, as the latter yield several phenolic derivatives and truns-dihydrodiols as major metabolites (21-23). In vitro metabolism of NO2PAH in general yields truns-dihydrodiols, phenolic derivatives, and tetrahydrotetrols as major metabolites (24-31). It is not known how 4-NP-9,lO-dione is formed; however, it is likely that hydrolysis of 4-NP-9,10-oxide, followed by oxidation of the truns-S,lO-DHD-4-NP intermediate through an enzymatic or a chemical reaction, is involved. However, chemical oxidation of truns-9,lODHD-4-NP to 4-NP-9,lO-dione using Mn02 (32)was not successful. This suggests that the oxidation of trum-9,lODHD-4-NP to the corresponding dione appears to be more facile in biological systems. Nitro-PAH diones have been suspected of being formed in the environment either from the reaction of PAH diones with NO,, both of which are present in air, or by photooxidation of nitro-PAHs (33,341. Interest in these compounds has been enhanced by the observation that a N02-PAH dione, 2-methyl-l-nitro8,l0-anthraquinone, induced tumors in rats and mice (35). Previously, we have synthesized l-nitropyrene-4,5-dione and l-nitropy-
694 Chem. Res. Toxicol.,Vol. 7,No.5, 1994
rene-9,lO-dione and found that both are mutagenic (32, 36). Taken together, these results suggest the significance of N02-PAH diones as potentially toxic derivatives (or metabolites) of N02-PAH. When TCPO was incorporated into microsomal incubation with 4-NP, 4-NP-9,lO-oxide was the major metabolite. Like the 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,lO-oxide formed in the metabolism of 1-nitropyrene (37) and the 1-nitrobenzo[e]pyrene 4,5-oxide formed in the metabolism of 1-nitrobenzo[e]pyrene (31), 4-NP-9,lOoxide is a somewhat stable compound and can be collected by reverse-phase HPLC. Genotoxicity of K-region oxides derived from 1-NP has been reported, and structures of their adducts with DNA in vitro have been elucidated (18,381. On the basis of chromatographic characteristics, it has been suggested that DNA adducts derived from K-region oxides of 1-NP in vivo may be contributing to the genotoxicity (39-41 ). Thus, involvement of 4-NP9,lO-oxide in the biological activity of 4-NP should not be underestimated. The in vivo metabolism of 4-NP was allowed to proceed under conditions similar to those reported previously for 1-NP and 2-NP ( 1 3 , 1 4 , 4 2 , 4 3 ) . In the study presented here, urinary elimination of 4-NP and its metabolites exceeded excretion via the gastrointestinal tract. This pattern of elimination and excretion is different from those reported in the metabolism of its structural isomers 1-NP and 2-NP. Excretion of 1-NP and 2-NP and their metabolites occurs mainly via the intestinal tract while urinary elimination was less than 20% of the dose; on the other hand, approximately 40% of the dose was found in the urine of animals treated with 4-NP during the course of experiment (cf:Figure 3). These observations clearly indicate that the position of the nitro group on the pyrene moiety affects the excretion pattern. Differences in elimination and excretion patterns may in part account for the varied tumorigenicity of the three N P isomers toward the mammary gland in the rat (9). Conjugates other than glucuronides and sulfates were not identified in this study. However, from previous studies with 1-NP (44, 451, it is apparent that some of the unknown metabolites may occur in the urine as glutathione conjugates. In contrast to the in vitro findings, the in vivo study shows that 4-NP can be metabolized in the rat via both nitroreduction and ring oxidation. Both pathways are involved also in the in vivo metabolism of 1-NP and 2-NP (13, 14, 42, 43). Numerous previous studies have also shown that certain N02-PAH can be metabolically activated by nitroreduction and ring oxidation (46),and we have demonstrated that such pathways are not limited to adult animals, but metabolites derived from both routes have also been detected in newborn animals after treatment with representative N02-PAH (39, 47). On the basis of the in vitro results, we initially proposed that 4-NP-9,lO-dione or its precursors could be formed in vivo after 4-NP administration. Although the peak labeled 9(10)-OH-4-AAP(cf:Figure 4) also coeluted with the synthetic trans-g,lO-DHD-4-NP,only the latter and not this peak was amenable to dehydration with p-toluenesulfonic acid to yield the corresponding phenolic derivatives (data not shown). In addition, the fate of r3H14-NP-9,10-dione was investigated following nitroreduction by ZnfNH&l and reduction of the quinone moiety by NaBH4. In each case, the resulting radioactivity was eluted between 4 and 5 min when analyzed by HPLC (System 2) using conditions described in Figure 4; the nature of this material was not characterized, and we
Upadhyaya et al.
did not recover any starting material. Therefore, it is likely that further reduction of 4-NP-9,lO-dione or its precursors may be responsible for some of the unidentified metabolites eluted between 4 and 5 min (cf: Figure 4) in this study. Nitroreduction of 4-NP to 4-AP suggests the formation of the corresponding hydroxylamine. The formation of hydroxylamines of numerous compounds by enzymatic or chemical catalysis in the presence of DNA has been shown to result in DNA adduct formation in vitro. For example, nitroreduction of 1-NP and 2-NP in the presence of DNA resulted in the formation of CSdeoxyguanosine and C8-deoxyadenosine adducts ( 2 2 , 4 8 , 4 9 ) . The identification of these adducts in vivo following the oral administration of 1-NP and 2-NP to rats was reported (13,39,40,50,51).Hydroxylamine formation from ringoxidized metabolites of 4-NP may also contribute to its biological activity. Thus, further studies are in progress to determine the critical metaboliteb) that are involved in DNA adduct formation in the target organ leading to the initiation of carcinogenesis by 4-NP. In summary, like 1-NP and 2-NP, 4-NP is metabolized in vivo by nitroreduction, ring oxidation, and a combination of both pathways. The contribution of each pathway to DNA adduct formation in the target organ and to mammary tumor induction needs to be examined. The elimination and excretion pattern of 4-NP is different from those of its structural isomers. More metabolites of 4-NP in the urine would reflect more in the blood circulation, and consequently, more metabolites will be delivered to the mammary tissues; this may, in part, be responsible for the potent tumorigenic activity of 4-NP. Thus, structural elucidation of DNA adducts combined with detailed pharmacokinetic studies are underway to fully delineate why 4-NP is such a powerful carcinogen in the mammary glands of rats.
Acknowledgment. We thank Mrs. Patricia Sellazzo for preparing the manuscript and Mrs. Ilse Hoffmann for editing the manuscript. This study was supported by National Cancer Institute Grant CA-35519.
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