Chem. Res. Toxicol. 1995,8, 269-277
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Identification of Two A@-DeoxyguanosinylDNA Adducts upon Nitroreduction of the Environmental Mutagen 1-Nitropyrene Diogenes Herreno-Saenz, Frederick E. Evans, Frederick A. Beland, and Peter P. Fu* National Center for Toxicological Research, Jefferson, Arkansas 72079 Received July 5, 1994@ 1-Nitropyrene, the most abundant nitro-polycyclic aromatic hydrocarbon in the environment, is a known mammalian and bacterial mutagen and a tumorigen in animals. Early studies on DNA adduct characterization for 1-nitropyrene identified N-(deoxyguanosin-8-y1)-1-aminopyrene as the major product from the modification of calf thymus DNA with N-hydroxy-1-aminopyrene, the activated metabolite from nitroreduction of 1-nitropyrene. I n this paper, we report the identification of two W-deoxyguanosinyl adducts, in addition to N-(deoxyguanosin-8-y1)-1aminopyrene, formed from the reaction of N-hydroxy-1-aminopyrene, prepared in situ, with calf thymus DNA. These DNA adducts were identified as 6-(deoxyguanosin-W-yl)-l-aminopyrene and 8-(deoxyguanosin-W-y1)-1-aminopyrene. The two An-deoxyguanosinyl adducts were also identified in an ascorbic acid-catalyzed activation of 1-nitrosopyrene and in the mammary gland of female Sprague-Dawley rats administered 1-nitropyrene. The DNA adducts were also formed when 1-nitropyrene was metabolized by xanthine oxidase in the presence of calf thymus DNA, and when 1-nitropyrene was activated by rat liver microsomes and cytosols, as well as from DNA isolated from Salmonella typhimurium suspension cultures incubated with 1-nitropyrene.
Introduction Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs)l are a class of genotoxic environmental contaminants ( I 6). 1-Nitropyrene (1-NP) is the most abundant nitro-PAH detected in the environment. It has been found in diesel exhaust particulates, urban air particulates, coal fly ash, certain gnlled foods, and emissions from wood stoves, fire places, kerosene heaters, and gas burners (2, 4 ) . 1-NP has been shown to be a direct-acting bacterial mutagen ( I ) , to be a mutagen in mammalian systems (7, 8), and to induce sister chromatid exchanges in Chinese hamster ovary cells (9). 1-NP is also tumorigenic in animals (1013). As with the majority of nitro-PAHs, 1-NP has to be metabolized to a reactive electrophile in order to exert its biological effects ( I -6, 13-29]. Nitroreduction has been shown t o be an important route for bioactivation of 1-NP to its activated metabolite N-hydroxy-l-aminopyrene (N-hydroxy-1-AP) (14,30-32), which has the capability to bind DNA (14). A DNA adduct from xanthine oxidase-catalyzed nitroreduction of 1-NP has been identi-
* Please address correspondence to this author at the following address: HFT-110, National Center for Toxicological Research, Jefferson, Arkansas 72079. Abstract published in Advance ACS Abstracts, January 15, 1995. Abbreviations: nitro-PAHs, nitro-polycyclic aromatic hydrocarbons; 1-NP, 1-nitropyrene;N-hydroxy-1-AP,N-hydroxy-1-aminopyrene; l-AP, 1-aminopyrene; N-dG1-AP, N-(deoxyguanosin-8-yl)-l-aminopyrene; 6-dG-l-AP,6-(deoxyguanosin-iV-yl)-l-aminopyrene; 8-dG-l-AP, 8-(deoxyguanosin-N2-yl)-l-aminopyrene; l-nitro-H1-BaP, l-nitro-7,8,9, 10-tetrahydrobenzo[alpyrene; 3-nitro-&-BaP, 3-nitro-7,8,9,10-tetrahydrobenzo[alpyrene; 6-dG-l-AP-3", 6-(deoxyguanosin-~-yl)-l-aminopyrene 3'-monophosphate; 8-dG-l-AP-3", 8-(deoxyguanosin-iV-y1)-1aminopyrene 3'-monophosphate; N-dG-l-AP-ZP, N-(deoxyguanosin8-y1)-1-aminopyrene 3'-monophosphate; poly(dA).poly(dT),polydeoxyadenylic-polythymidylic acid; poly(dG>poly(dC),polydeoxyguanylicpolydeoxycytidylic acid; 3-MC microsomes, liver microsomes of rats treated with 3-methylcholanthrene; DMSO, dimethyl sulfoxide; NOE, nuclear Overhauser effect; NCTR, National Center for Toxicological Research. @
fied as N-(deoxyguanosin-8-y1)-1-aminopyrene (N-dG-1-
AP) (14). The same adduct was formed in Salmonella typhimurium treated with 1-NP (14). I t has been reported that mice and rats treated with 1-NP or l-nitrosopyrene form multiple DNA adducts, of which N-dG-1AP was the only identified DNA adduct ( 1 5 , 1 7 , 2 1 , 2 3 , 24,28). We recently reported that reaction of N-hydroxy-laminobenzo[alpyrene and N-hydroxy-3-aminobenzo[alpyrene with calf thymus DNA generated 6-(deoxyguanosinW-yl)-1-aminobenzo[alpyrene and 6-(deoxyguanosin-Wyl)-3-aminobenzo[a]pyrene,respectively, as predominant DNA adducts (33,341. The unique characteristic of these DNA adducts is that their site of substitution is remote from the N-hydroxyamino group. Under similar reaction conditions, the same kind of W-deoxyguanosinyl adducts were identified from the reaction of calf thymus DNA with the N-hydroxyarylamines of l-nitro-7,8,9,10-tetrahydrobenzo[alpyrene (l-nitro-%-BaP) and 3-nitro-7,8,9,10-tetrahydrobenzo[alpyrene (3-nitro-H4-BaP) (34) for which the aromatic moiety is 1-nitropyrene. In the present study, we report the characterization of two W deoxyguanosinyl adducts derived from the nitroreduction of 1-NP in addition to the N-dG-1-AP adduct that had been previously identified. These two W-deoxyguanosinyl adducts were also identified in the mammary gland of rats treated with 1-NP in vivo and in several enzymatic systems in vitro.
Materials and Methods Materials. Caution: 1-NP has been determined to be highly mutagenic in Salmonella. Therefore, appropriate safety procedures should be followed when working with this compound. 1-NP,1-aminopyrene (1-AP),hydrazine monohydrate (98% pure), 5% palladium on carbon, and 3-methylcholanthrene (3MC) were obtained from Aldrich Chemical Co. (Milwaukee,
This article not subject to U.S. Copyright. Published 1995 by the American Chemical Society
270 Chem. Res. Toxicol., Vol. 8, No. 2, 1995 WI). l-Nitr0[4,5,9,10-~~C]pyrene (specific activity 58.8 mCi/ mmol) and l-nitros~[G-~H]pyrene (specific activity 89.5 mCi/ mmol) were obtained from Chemsyn Science Laboratories (Lenexa, KS). Polydeoxyadenylic-polythymidylic acid (poly(dA>poly(dT)),polydeoxyguanylic-polydeoxycytidylic acid (poly(dG)poly(dC)), and diglyme were obtained from Sigma Chemical Co. (St. Louis, MO). All solvents used were HPLC grade. Male Sprague-Dawley rats (150-200 g body weight), obtained from the NCTR breeding colony, received intraperitoneal injections of 3-MC (25 m a g body weight) on 3 consecutive days before sacrifice. Liver microsomes were prepared as previously described (35). The protein content was assayed according to established procedures (36). Mammary gland epithelial cells of Sprague-Dawley rats treated with 1-NP were obtained as reported in Smith et al. (24).
Chemical Reduction of 1-Nitropyrene and Reaction with Calf Thymus DNA. N-Hydroxy-1-aminopyrene (Nhydroxy-1-AP) was synthesized in situ from nitroreduction of 10 mg of 1-NP, dissolved in 5 mL of diglyme, with 25 pL of hydrazine monohydrate and 5 mg of 5% palladium on carbon as described previously (37). The N-hydroxy-1-AP reaction product was filtered and incubated 18 h a t 37 "C in an argonpurged solution of calf thymus DNA (1 mg/mL, type I, Sigma) dissolved in 100 mL of 10 mM sodium citrate buffer (pH 5.0). The reaction mixture was extracted with ethyl acetate (5 x 100 mL). Ten milliliters of 5 N sodium chloride was added to the aqueous phase, and the DNA was precipitated with 100 mL of ice-cold 95% ethanol. The DNA concentration was determined spectrophotometrically by measuring the U V absorbance at 260 n m (1 mg/mL DNA = 20 absorbance units). The modified DNA was redissolved in 5 mM Bis-Tris, pH 7.1 (1mg/mL), and 5 mM MgC12, and hydrolyzed with DNase I (0.1 mg/mg of DNA, type IV, Sigma)for 3 h at 37 "C, followed by incubation with phosphodiesterase I (0.01 uniumg of DNA, type VII: from Crotalus atrox venom, Sigma) and alkaline phosphatase (0.6 unit/mg DNA, type 1113, Sigma) at 37 "C for 18 h. The hydrolysis mixture was extracted with water-saturated 1-butanol (3 x 1 volume) and washed once with 1-butanol-saturated water. The l-butanol phase was taken to dryness under reduced pressure, and the residue was redissolved in methanol for HPLC analysis on a pBondpack C18 (Waters Millipore, Milford, MA) or a Vydac C18 (The Separation Group, Hespera, CA) analytical column with a 30-min linear gradient of 40% methanol in water to methanol at a flow rate of 1 m u m i n . Before the adducts were separated by HPLC for NMR analysis, each sample was applied to a Sep-Pak C18 (Waters Millipore) cartridge prewashed with 10%methanol in water. The adduct mixture was eluted from the cartridge with 5 mL of methanol. The methanol volume was reduced to 500 pL for HPLC analysis. The major adduct (N-dG-1-AP) (14)was separated from the two minor products using a Zorbax ODS column (9.4 x 250) mm with a 15 min linear gradient of 70% methanol in water to methanol at a flow rate of 1.5 m u m i n . The two minor adducts were then separated with the same column isocratically with 65% methanol in water at 1.5 mumin. For mass and NMR spectral analysis, the purity of each adduct was verified using the pBondpack C18 column with the elution conditions described above. Reaction of [G-3H]1-Nitrosopyrenewith DNA Catalyzed by Ascorbic Acid. Calf thymus DNA (1mg/mL) dissolved in 5 mL of argon-purged 10 mM sodium citrate buffer (pH 5.0) was treated overnight at 37 "C with 0.67 pmol of [G-3H]l-nitrosopyrene (in 168 pL of DMSO, specific activity 89.5 mCi/mmol) and 3.2 pmol of ascorbic acid (in 300 pL of water). After incubation and solvent extraction, the DNA was precipitated, hydrolyzed enzymatically, and analyzed by HPLC as described above. Reaction of Polydeoxyguanylic-Polydeoxycytidylic
Acid (Poly(dG).Poly(dC))and Polydeoxyadenylic-Polythymidylic Acid (poly(dA)*Poly(dT)) with [G-3H11-Nitrosopyrene in the Presence of Ascorbic Acid. An argonpurged solution of polydeoxyguanylic-polydeoxycytidylic acid (poly(dG>poly(dC))(5 units/mL) in 10 mM sodium citrate buffer (pH 5.0) was reacted with 0.67 pmol of [G-3H]1-nitrosopyrene
Herreno-Saenz et al. (in 50 pL of DMSO, specific activity 89.5 mCi/mmol) in the presence of 2.5 pmol of ascorbic acid (in 300 pL of 10 mM sodium citrate buffer, pH 5.0) at 37 "C for 18 h. After extraction with ethyl acetate (7 x 1volume), the modified polydeoxynucleotides were precipitated by adding 100 pL of 5 N NaCl and 2 volumes of cold 95% ethanol, and the reaction mixture was kept at 4 "C overnight. The samples were centrifuged at l200g at 0 "C for 20 min, and the ethanol phase was discarded. The deoxypolynucleotide mixture was dissolved in 1 mL of 5 mM Bis-Tris buffer (pH 7.1) for hydrolysis to nucleosides as described above. Samples were dissolved in methanol for HPLC analysis. Reaction of polydeoxyadenylic-polythymidylic acid (poly(dA)poly(dT)) with [G-3H11-nitrosopyrene was similarly conducted.
Incubation of 1-NP with Xanthine Oxidase and Reaction with Calf Thymus DNA. Hypoxic incubation of [4,5,9, 10-l4C11-NP with xanthine oxidase in the presence of calf thymus DNA was conducted following the procedure described by Howard et al. (15).Xanthine oxidase (0.1 uniUmL) was added t o an argon-purged incubation solution containing 10 pM [4,5,9, 10-14C11-NP(specific activity 58.8 mCi/mmol) in 20pL of DMSO, 0.5 mg/mL hypoxanthine, 10 pg/mL catalase, and 2 mg/mL of calf thymus DNA in 50 mM potassium phosphate buffer (pH 5.8). After 4 h of reaction a t 37 "C followed by solvent extraction, the DNA was precipitated and analyzed by HPLC with the conditions as described above.
Hypoxic Incubation of 1-NPwith Rat Liver Microsomes and Cytosol in the Presence of Calf Thymus DNA. Hypoxic microsomal and cytosolic incubations were performed in duplicate at 37 "C for 4 h in a 2 mL reaction volume. The incubation mixtures (in 2 mL solution) contained 500 pL of 0.2 M TrisHC1 (pH 7.1),60pL of 1M MgC12,2pmol of glucose 6-phosphate, 250 nmol of NADP+, 4 units of glucose-6-phosphate dehydrogenase, 2 mg of liver microsomal or cytosolic protein of rats treated with 3-methylcholanthrene, 4 mg of DNA, and 120 nmol of 1-NP in 40 p L of DMSO. The incubation mixture was icecooled and extracted with buffer-saturated phenol (3 x 2 mL), twice with an equal volume of phenovchlorofordisoamyl alcohol (25:24:1 dv), and once with equal volume of chlorofordisoamyl alcohol (24:l v/v). The DNA was precipitated by the addition of 4 mL of ice-cold ethanol and 200 pL of 5 N NaC1. The DNA adducts were then analyzed by 32P-postlabeling/HPLC. DNA Adducts from the Incubation of S. typhimurium with 1-NP. Suspension cultures of S. typhmurium strain TA98 were treated with 20 pM of 1-NP for 4 h, and the DNA was isolated following the procedures previously described by Heflich et al. (38). The resulting DNA adducts were analyzed by 32Ppostlabeling/HPLC.
92P-PostlabelingMPLCIdentification of 3',5'-Bisphosphate Deoxyribonucleotides. The modified DNA (2 mg) from a n ascorbic acid-catalyzed reaction with 1-nitrosopyrene suspended in 4 mL of 5 mM Bis-Tris/O.l mM EDTA buffer (pH 7.1) was hydrolyzed to deoxyribonucleoside 3'-monophosphates at 37 "C for 18 h with 720 pL of a mixture containing micrococcal nuclease (125 unitsiml) and spleen phosphodiesterase (12.5 units/mL) in 20 mM sodium succinate and 10 mM calcium chloride (pH 6.0). The deoxyribonucleoside 3'-monophosphate mixture was separated by HPLC on a pBondpack C18 analytical column eluted at 1 m u m i n with a gradient as follows: 5% methanol in water for 10 min, a linear gradient to 30% methanol in water in 20 min, 30% methanol for 10 min, then a linear gradient to 100% methanol in 50 min. The HPLC eluent was collected at 1-min intervals. A 50-pL aliquot of the reaction mixture was further hydrolyzed to deoxyribonucleosides with 2 pL of alkaline phosphatase (type 111-S, Sigma) at 37 "C for 18 h. The sample was then extracted with water-saturated 1-butanol (2 x 100 pL). The pooled 1-butanol was dried, and the residue was dissolved in 300 pL of methanol for HPLC analysis. The deoxyribonucleoside 3'-monophosphate mixture and each of the purified deoxyribonucleoside 3'-monophosphates (ca. 5 pg) were dissolved in 25 pL of water and [5'-32Plphosphorylated with 5 pL of PNK mix containing 200 pCi of [y-32P]ATPand 1 unifffig of T4 polynucle-
Chem. Res. Toxicol., Vol. 8, No. 2, 1995 271
N2-Deoxyguanosinyl Adducts from 1-Nitropyrene otide kinase DNA a t 37 "C for 40 min. The residual [y-32PlATP was destroyed by the addition of 2 pL of potato apyrase (40 microunits), followed by incubation a t 37 "C for 30 min (39). To separate the modified 3',5'-bisphosphate deoxyribonucleotides from the unmodified nucleotides, thin-layer chromatography was performed using Machery-Nagel PEI cellulose plates (Alltech Associates, Deerfield, IL) with a 15-cm Whatman 1 paper wick that had been attached to the top of the plate and eluted with 0.65 M sodium phosphate (pH 6.8). The material from the origin of the plates was scraped, transferred to 1.5mL polypropylene microcentrifuge tubes, and extracted with 600 pL of pyridinium formate, pH 4.2, a t room temperature for 4 h with stirring on a mechanical mixer. After centrifugation for 10 min, the supernatant was collected and centrifuged again for 10 min to remove fine particles. The supernatants were dried under reduced pressure and redissolved in methanol for HPLC analysis, using a Radiomatic FLO-ONE\Beta A-515 system in line with the HPLC system, on a Vydac C18 reversedphase column, eluting at 1 m u m i n with a linear gradient as follows: 0.02 M sodium phosphate (pH 4.2) to 10% methanol in sodium phosphate in 20 min, to 50% methanol in sodium phosphate in 20 min, and to 100% methanol in 40 min.
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