Chem. Res. Toxicol. 1992,5, 863-869
863
In Vitro Metabolism and DNA Adduct Formation from the Mutagenic Environmental Contaminant 2-Nitrofluoranthene Diogenes Herreno-Saenz,t Frederick E. Evans,t Thomas Heinze,t Joellen Lewtas,l and Peter P. Fu*9t National Center for Toxicological Research, Jefferson, Arkansas 72079, a n d Genetic Toxicology Division, Environmental Protection Agency, Research Triangle Park, North Carolina 27711 Received July 17, 1992
The metabolism and DNA adduct formation by the mutagenic environmental contaminant 2-nitrofluoranthene (2-NFA) were studied. Incubation under aerobic conditions with liver microsomes of rats pretreated with 3-methylcholanthrene yielded trans-7,8-dihydroxy-7,8dihydro-2-nitrofluoranthene,trans-9,10-dihydroxy-9,10-dihydro-2-nitrofluoranthene, and 7-, 8-, and 9-phenolic metabolites. When the epoxide hydrolase inhibitor 3,3,3-trichloropropylene was present in the incubation, only phenolic metabolites were detected. Under hypoxic conditions, 2-aminofluoranthene was obtained, together with a trace of the ring-oxidized metabolites. The was prepared in situ and reacted with activated metabolite, N-hydroxy-2-aminofluoranthene, calf thymus DNA. Upon enzymatic hydrolysis of the DNA and purification by HPLC, a C8substituted deoxyguanosine adduct, N- (deoxyguanosin-8-yl)-2-aminofluoranthene, was identified by mass and proton NMR spectral analysis. This adduct was also formed a t a level of 10 pmol/mg of DNA when 2-NFA was metabolized by xanthine oxidase, 6 pmol/mg of DNA from incubation with liver microsomes of rats pretreated with 3-methylcholanthrene, and 3-pmol/mg of DNA from metabolism by liver microsomes of rats pretreated with phenobarbital.
Introduction Nitro polycyclic aromatic hydrocarbons (nitro-PAHs)l are widespread genotoxic environmental pollutants (17). Among these compounds, 2-nitrofluoranthene (2-NFA) has been found to be the most abundant nitro-PAH in ambient particulate organic matter (POM) at various locations in the U.S.and Europe. It contributes up to 5 % of the overall direct-acting mutagenicity of ambient POM in the Salmonella typhimurium test (8-12). The formation of 2-NFA in the environment is thought to occur during source-receptor transport of adsorbed or gas-phase fluoranthene with oxides of nitrogen, nitric acid, and other related species (9,10,12,13). Its formation is via a free radical mechanism, which is different from that of other nitro-PAHs, such as 3-NFA and 1-nitropyrene,which are formed during the combustion process and have an ionic mechanism (6,7,12,13).Thus, it is interesting to compare metabolic activation and DNA adduct formation between the isomeric nitro-PAHs formed from different mechanisms. Such studies would provide information on how a nitro group situated at different geometric locations can affect enzymatic activation. For example,while metabolic activation of 1-nitropyrene produces N-(deoxyguanosin8-yl)-l-aminopyreneas the predominant DNA adduct (14,
* To whom correspondence and reprint requeata should be addressed at the National Center for Toxicological Research, Jefferson, AR 72079. + National Center for Toxicological Research. 2 Environmental Protection Agency. 1 Abbreviations: nitro-PAHs,nitro polycyclic aromatichydrocarbons; 2-NFA,2-nitrofluoranthene;3-NFA,3-nitrofluoranthene;2-NFA,trans7,gdihydrodiol,trans-7,8dihydroxy-7,8dihydro-2-nitrofluoranthene (other trans-dihydrodiolaare similarly designated);2-AFA, 2-aminofluoranthene; 3-MC, 3-methylcholanthrene;3-MC-microsomes,liver microsomes of rata pretreated with 3-MC; TCPO, 3,3,3-trichloropropylene;POM, particulate organic matter; DMF, dimethylformamide;BSTFA, N,Obis(trimethylsily1)trifluoroacetamide;MSTFA, N-methyl-N-(trimethylsily1)trifluoroacetamide;PB, phenobarbital; Pd/C, palladium on carbon; N-dG-2-AFA,N-(deoxyguanosin-&yl)-2-aminofluoranthene; NCTR, National Center for Toxicological Research. This article not subject to U.S.Copyright.
15), metabolic activation of the isomeric 2-nitropyrene generates both N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene(16, 17). Metabolism of 3-NFA with liver microsomes of several species has been studied by Howard et al. (18). From xanthine oxidase-catalyzed reduction of 3-NFA in vitro, N-( deoxyguanosin-8-yl)-3-aminofluoranthenewas formed in a yield of 2.4 adducts/105 nucleosides (19). Earlier studies of the metabolism of 2-NFA by liver S9 of Aroclor 1254-treated rats have shown that several ring-oxidized metabolites were formed (20-22). In this study, we report the results of metabolism from 2-NFA by rat liver microsomes and the characterization of the carcinogenDNA adduct formed by reaction of N-hydroxy-2-AFA, prepared in situ, with calf thymus DNA.
Materials and Methods Materials. 2-NFA and [GJHIP-NFA (specific activity 347 mCi/mmol; radiochemical purity >99%) were obtained from Chemsyn Science Laboratories (Lenexa, KS). Hydrazine monohydrate (98% pure) and 5 % palladium on carbon were purchased from Aldrich Chemical Co. (Milwaukee,WI). Liver microsomes and cytosols of male Sprague-Dawleyrata (150-200 g body wt, obtained from the NCTR breeding colony) pretreated with 3-methylcholanthrene (3-MC) or with phenobarbital were prepared as described previously (23). Protein concentration was determined by the Lowry method (24). In Vitro Metabolism of 2-NFA with Rat Liver Microsomes. (i) In vitro metabolism of 2-NFA was performed aerobically with shaking at 37 "C for 1 h in a 200-mL reaction mixture containing 10 mmol of Tris-HC1 (pH 7.4), 0.2 mmol of NADP+, 0.6 mmol of magnesium chloride, 0.4 mmol of glucose 6-phosphate (type XII, Sigma,St. Louis, MO), 20unita of glucose6-phosphate dehydrogenase, 200 mg of 3-MC-microsomes, and 8 pmol of 2-NFA (dissolvedin 2 mL of acetone). Incubation was terminated with the addition of 100 mL of ice-cold acetone, and the incubationmixture was extracted with 300mL of ethyl acetate. The organic phase was evaporated under reduced pressure, and
Published 1992 by the American Chemical Society
864 Chem. Res. Toxicol., Vol. 5, No. 6, 1992 the residue was redissolved in methanol for HPLC separation. The recovered substrate and ita metabolites were separated on a DuPont Zorbax ODS (9.4 X 250 mm) using a 30-min linear gradient of 60-100% methanol with a flow rate of 3.0 mL/min. (ii) Aerobicmetabolism of 2-NFAin the presence of the epoxide hydrolase inhibitor TCPO (60 pmo1/100-mLincubation volume) was similarly performed in a 100-mL incubation volume. (iii) Hypoxic metabolism was conducted in a 100-mLreaction mixture as described above, except that the reaction mixture was argon-purged40 min prior to the addition of 2-NFA. Analysis of the gaseous phase in the incubation vessel, performed using a Dorhmann Model 15C-3gas chromatograph, indicated less than 2% of molecular oxygen was present. For determination of reaction rates, incubation of [G-3H]2NFA was conducted aerobicallyunder conditions described above for the unlabeled 2-NFA, except that reactions were conducted for 10 min in a 1-mL incubation volume containing 80 nmol of substrate and 100 pg of 3-MC-microsomes. Acid-Catalyzed Dehydration of the 2-NFA trans-Dihydrodiol Metabolites. 2-NFA trans-7,8-dihydrodiol and 2-NFA trans-9,lO-dihydrodiol(ca l00pg each),obtained from incubation of 2-NFA with 3-MC-microsomes described above, were dehydrated in 200 pL of tetrahydrofuran, 1mL of glacial acetic acid, and two drops of hydrochloric acid a t 60 "C for 30 min. After extraction with ethyl acetate, the resulting phenolic products were separated by reversed-phase HPLC under conditions described above. Chemical Reduction of 2-NFA and Reaction with Calf Thymus DNA. N-Hydroxy-2-AFA and [G-3HlN-hydroxy-2AFA, synthesized in situ from 2-NFA and [G-3H]2-NFA, respectively, were reacted with calf thymus DNA. Typically, 2-NFA (10 mg) was dissolved in 5 mL of diglyme, and 5 mg of 5% palladium on carbon was added, followed by addition of 25 p L of hydrazine monohydrate. The reaction, which was monitored by TLC (developed with toluene/ethanol, 17:3 v/v), was complete in 2 h. The resulting reaction product was filtered through Celite directly into an argon-purged solution of the untreated calf thymus DNA (100 mg, type I, Sigma) dissolved in 100 mL of 10 mM sodium citrate buffer (pH 5.0). After the reaction mixture was incubated overnight at 37 "C, it was extracted with HPLC-grade methylene chloride (3 x 1volume) andethyl acetate (3 X 1volume). The aqueous layer was extracted three times with 100 mL of phenol saturated with 50 mM BisTris buffer (pH 8), twice with equal volumes of phenol/ chloroform/isoamyl alcohol (2524:l v/v), and twice with chloroform/isoamyl alcohol (241 v/v). Ten milliliters of 5 N sodium chloride was added to the aqueous phase, and the DNA was precipitated by adding 100 mL of ice-cold ethanol (25). The modified DNA was redissolved in 5 mM Bis-Tris (pH 7.1), at a concentration of 1mg/mL, and 5 mM MgC12. The DNA concentration was determined spectrophotometrically by measuring the UV absorbance a t 260 nm (1 mg/mL DNA = 20 absorbance units). The DNA was treated with DNase I (0.1 mg/ mg of DNA, type IV, Sigma) for 3 h a t 37 "C, followed by incubation with nuclease P1 (from Penicillium citrinum,6 units/ mg of DNA, Sigma), acid phosphatase (0.3 unit/mg of DNA, type 11, from potato, Sigma), and alkaline phosphatase (0.6 unit/mg of DNA, type 1114, Sigma) a t 37 OC for 18h (19). The hydrolysis mixture was extracted three times with an equal volume of watersaturated 1-butanol, and the 1-butanol extracts were combined and washed once with 1-butanol-saturated water. The 1-butanol phase was taken to dryness under reduced pressure, and the residue was redissolved in DMSO for HPLC analysis on a pBondpak C18 analytical column with a 30-min linear gradient of 40-100% methanol at 1.5 mL/min. Derivatization of the N-Hydroxy-2-AFA-Deoxyribonucleoside Adduct. For determination of the molecular weight by electron impact mass spectrometry, the purified N-hydroxy2-AFA-deoxyribonucleoside adducts obtained above were derivatized by trimethylsilylation according to published procedure with modification (26). The nucleoside adduct (15pg) in a 1-mL reaction vial was predried in a desiccator for 2 h and was treated with 15 pL each of DMF, BSTFA, and MSTFA. The resulting
Herreno-Saenz et al. mixture was incubated at 37 "C for 1 hand used for direct mass spectral measurement. Incubation of [G-3H]2-NFA with Xanthine Oxidase and Reaction with Calf Thymus DNA. Hypoxic incubation of [G-3H12-NFAwith xanthine oxidase in the presence of calf thymus DNA was conducted at 37 "C for 4 h, as described by Howard et al. (14) and Djuric et al. (25). Xanthine oxidase (0.1 unit/mL) was added to an argon-purged incubation solution of 50 mM potassium phosphate buffer (pH 5.8) which contained 20 pM [G-3H12-NFA(4 mM in DMSO), 0.5 mg/mL hypoxanthine, and 2 mg/mL calf thymus DNA. After incubation and solvent extraction, the DNA was precipitated and was hydrolyzed enzymatically to nucleosides for analysis by HPLC using on a 5-rm reversed-phase Vydac CISanalytical column (the Separations Group, Hesperia, CA) eluted with a 20-min linear gradient of 50-100% methanol at a flow rate of 1.5 mL/min, or on a pBondpak CISreversed-phase analytical column (Waters Associates) using a 30-min linear gradient of 40-100% methanol at a flow rate of 1.5 mL/min. Hypoxic Incubation of [G-W]2-NFA with Rat Liver Microsomes in the Presence of Calf Thymus DNA. Similar to the incubation conditions described above for hypoxic metabolism of 2-NF, a 2-mLincubation was conducted under hypoxic conditions (
