Activation of 1-nitropyrene by nitroreductase increases the DNA adduct level and mutagenicity Hideki Arimochi, Takemi Kinouchi, Keiko Kataoka, Tomomi Kuwahara and Yoshinari Ohnishi Department of Bacteriology, The University of Tokushima School of Medicine, Tokushima, Japan. Abstract:1-Nitropyrene (1-NP) is a mutagenic nitro compound in the environment. We studied correlations between the mutagenicity of 1-NP for three strains of Salmonella typhimurium, the activity of bacterial nitroreductases and the amount of 1-NP-derived DNA adducts. Bacterial strains used in this study were S. typhimurium strains TA98, nitroreductase-less mutant TA98NR and YG1021 carrying a nitroreductase-producing plasmid. The mutagenicity of 1-NP was measured using the Ames assay, and the nitroreductase activities of these strains were assayed by quantification of1-aminopyrene produced from1-NP. The DNA adducts were measured by the 32P-postlabeling method. Among the three bacterial strains, strain YG1021 was the highest in mutagenicity of1-NP, the nitroreductase activity and the DNA adduct level. However, S. typhimurium strain TA98NR had the lowest values of these three parameters. Nitroreductase activity, DNA adduct level and mutagenicity were strongly correlated with each other. These results indicate that bacterial nitroreductase plays an important role in forming the DNA adducts, and that the higher the adduct level the higher the level of mutagenicity. J. Med. Invest. 44:193-198, 1998 Keywords:Salmonella typhimurium, 1-nitropyrene, nitroreductase activity, DNA adduct, mutagenicity INTRODUCTION We are exposed to many nitro compounds in our daily life by smoking or by inhalation of polluted air. Nitro compounds exhibit mutagenicity following the formation of an active N-hydroxy arylamine intermediate produced by reduction of their nitro residues (1). 1-Nitropyrene (1-NP) is one of the nitro compounds found in airborne particles, diesel exhaust, emissions from kerosene heaters, waste water from gasoline stations, used crankcase oil, tea and grilled chicken (2). This compound has been shown to be mutagenic in bacteria and mammalian cells and carcinogenic for animals (2-4). Reductively activated1-NP forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP) (5). The major adduct causes –2 deletion of a GC or CG pair within a CGCGCGCG hot-spot sequence upstream of the hisD3052 mutation in Salmonella typhimurium strain TA98(6, 7). Howard et al. (5) demonstrated a linear correlation between the concentration of dG-C8-AP in S. typhimurium DNA and the number of revertant colonies. Watanabe et al. showed that new S. typhimurium strains, which possessed50-fold higher nitroreductase activity than the original strains, were more sensitive to the mutagenicity of1-NP than the original strains (8). Thus, nitroreduction of1-NP by nitroreductase is considered to be an essential step for expression of the mutagenic activity. However, there are few studies which demonstrate the correlation between the nitroreductase activity, the amount of DNA adducts and the mutagenicity of1-NP in the same system. The present study was initiated to elucidate the cor-relation between bacterial nitroreductase activity, the amount of a major DNA adduct generated from1-NP and the mutagenicity of the compound in bacteria using three strains of S. typhimurium. Strain TA98 has been used as a standard strain for detection of frameshift mutations (3). Strain TA98NR is resistant to the mutagenicity of nitrofuran because this strain does not produce a nitroreductase which can metabolize this mutagen (3). Strain YG1021 contains a plasmid carrying the nitroreductase-encoding gene (8, 9). MATERIALS AND METHODS Materials. 1-NP and1-aminopyrene (1-AP) were purchased from Aldrich Chemical Co., Milwaukee, WI, and 1-NP was purified by silica gel column chromatography with benzene as the elutant. The purity of1-NP was determined by high-performance liquid chromatography (10) and was found to be more than 99.99%. Nutrient broth No.2 (Oxoid) was from Unipath Ltd., Hampshire, England. Proteinase K and hypoxanthine were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Silica gel, xanthine oxidase, calf thymus DNA, glucose6-phosphate (G6-P), spermidine and apyrase were pur-chased from Sigma Chemical Co., St. Louis, MO. Reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH) and glucose 6-phosphate dehydrogenase (G6-PDH) were obtained from Oriental Yeast Co., Ltd., Tokyo, Japan. Lysozyme was obtained from Seikagaku Kogyo Co. Ltd., Tokyo. Micrococcal nuclease and spleen phosphodiesterase were purchased from Worthington Biochemical Co., Freehold, NJ. T4 polynucleotide kinase and nucleoside 3,5-bisphosphate of every base were purchased from Pharmacia Biotech, Uppsala, Sweden. [γ-32P] adenosine 5'-triphosphate (7,000 Ci/mmol) was obtained from ICN Radiochemicals, Irvine, CA. Polyethyleneimine (PEI)-cellulose sheet (Polygram CEL300PEI) was purchased from Machery-Nagel GmbH, Duren, Germany. X-ray film was obtained from Fuji Photo Film Co. Ltd., Kanagawa, Japan. All other chemicals used were reagent or higher grade. Mutagenicity Test. The Ames assay was performed once for each experiment with two plates per sample by the preincubation method (11) to determine the mutagenicity of1-NP in the three S. typhimurium strains. The numbers of sponta-neous revertants (31, 11 and 40 for strains TA98, TA98NR and YG1021, respectively) were subtracted. Preparation of Cell-free Extracts of S. typhimurium Bacterial cells were harvested by centrifugation from an overnight culture, washed with saline three times and suspended in saline. The cells were disrupted by sonic oscillation (Kubota Insonator Model200) at 200 watts for5min seven times in an ice bath. Cell debris was removed by centrifugation at 9,000x g for 20min at 4°C and the supernatant was used as an enzyme source for the nitroreductase assay. The protein concentration of the supernatant was determined by the method of Lowry et al. (12) with bovine serum albumin as a standard. Nitroreductase Activity The reaction mixture (total volume1ml) consisted of50mM sodium phosphate buffer (pH7.4), 1mM NADH, 1mM NADPH, 2mM G6-P, 3mM MgCl2, 1U of G6-PDH per ml and60μM1-NP. The mixture was purged with argon gas for 30min in an ice bath. The enzyme solution was added to the mixture, which was then incubated anaerobically at 37°C for 30 min. Extraction with the same volume of dichloromethane was performed three times and the combined organic phase was evaporated. The extract was dissolved in 1.5ml of methanol and the amount of1-AP produced from1-NP was determined by measuring the fluorescence (emission420nm, excitation365nm) with an auto fluorescence spectrometer (Hitachi F3010). The specific activity of the nitroreductase was expressed as nmol of1-AP produced per minute per mg of protein. DNA Adduct Formation and DNA Extraction from S. typhimurium. 1-NP dissolved in 25ml of DMSO, 25ml of the culture of each S. typhimurium strain and125ml of0.1M sodium phosphate buffer (pH7.4) were mixed, then incubated with shaking at 37°C for20min. The concentrations of1-NP were10μg/ml for strain YG1021 and500μg/ml for strains TA98 and TA98NR. The cells were collected and suspended in 6ml of 0.15M NaCl-0.1M EDTA (pH8.0)containing lysozyme at a final concentration of2.0mg/ml. After additional incubation at 37°C for20min, 6.0ml of0.1M Tris-1% sodium dodecyl sulfate-0.1M NaCl was added to the mixture. Then, phenol extraction and ethanol precipitation were performed to obtain bacterial DNA. The DNA solution was treated with200μg of RNase A and33.4U of RNase T1 per ml at 37°C for30min and further incubated with500μg of proteinase K per ml for30min. After phenol extraction and ethanol precipitation, the DNA yielded was used for the 32P-postlabeling assay. DNA Adduct Formation of1-NP by Xanthine Oxidase. A reaction mixture consisting of 50mM potassium phosphate buffer (pH5.8), 500μg of hypoxanthine per ml, 2mg of calf thymus DNA per ml and40μM1-NP was purged with argon gas for30min in an ice bath. After the addition of xanthine oxidase at a final concentration of0.1U/ml, the mixture was incubated at 37°C for1hr under anaerobic conditions. The modified DNA for the 32P-postlabeling assay was precipitated after treatment with RNase A, RNase T1and proteinase K as described above. 32P-Postlabeling Assay The experimental procedures of the 32P-postlabeling assay were according to the butanol extract method (13)with some modifications. 32P-labeled samples were applied on a PEI-cellulose sheet and developed overnight in0.9M sodium phosphate (pH6.8) to remove normal nucleotides. DNA adducts were developed with3.6M lithium formate-8.5M Urea (pH3.5), 1.2M LiCl-0.5M Tris-HCl-8.0M Urea (pH8.0) and 0.9M sodium phosphate (pH6.0) as D3, D4 and D5, respectively. To determine the total nucleotide number, labeled samples were chromatographed with 0.25M LiCl. Positions of adducts on the cellulose sheet were determined by exposure to an X-ray film. Radioactivity of adducts on the PEI-cellulose sheet was measured with a liquid scintillation counter (Aloka LSC-602) to calculate the relative DNA adduct level. Statistical Analysis The correlation coefficient was calculated by the formula of Pearson with Stat View4.5 (Abacus Concepts, Inc.). RESULTS Mutagenicity of1-NP The mutagenicity of purified 1-NP for the three S. typhimurium strains was examined using the Ames assay. The highest mutagenicity was observed in strain YG1021. In contrast, the mutagenic activity was very low in strain TA98NR (Fig1). The number of revertants increased dose-dependently in all three strains. The numbers of revertants of strains YG1021 and TA98NR were18.6- and0.18-fold higher, respectively, than strain TA98 (Table1). Nitroreductase Activity The amount of1-AP was measured with a fluorescence spectrophotometer after incubation of1-NP with a cell-free extract of each S. typhimurium strain (Fig2). The amount of1-AP produced by incubation with extracts from strains YG1021 and TA98 increased protein-dose dependently. Strain YG1021 possessed 31.8-fold higher specific activity than strain TA98 and the specific activity of strain TA98NR was1/10that of TA98 (Table1). The qualification and quantification of DNA adducts Figure3A shows an autoradiogram of 32P-postlabeled DNA adducts formed in the mixture of1-NP, calf thymus DNA and xanthine oxidase after anaerobic incubation. The major adduct was dG-C8-AP (5). Figures3B, C and D show autoradiograms of DNA adducts in the three S. typhimurium strains incubated with1-NP. The spots of DNA adducts from strains YG1021 (B) and TA98 (C) appeared at the same position as the DNA adduct, dG-C8-AP (A). The spot was not observed in strain TA98NR treated with1-NP after exposure for8hr at -80°C(D). The amount of dG-C8-AP adduct was97.2-fold more in strain YG1021 than in strain TA98 (Table1). The Correlation Coefficients Correlation coefficients between the mutagenicity and the nitroreductase activity, the mutagenicity and the DNA adduct level, and nitroreductase activity and the DNA adduct level were 0.999898, 0.999986 and 0.999873,respectively. Positive correlations were observed between the mutagenicity and the nitroreductase activity, between the mutagenicity and the DNA adduct level at P<0.01, and between the nitroreductase activity and the DNA adduct level at P<0.05. DISCUSSION 1-NP is a mutagenic and carcinogenic pollutant which is frequently found in the environment (2, 3). The metabolic pathway of1-NP in S. typhimurium cells has been studied extensively, and it has been shown that nitroreduction of1-NP is an essential step for expressing its mutagenicity(5, 14). However, there are few studies which demonstrate the correlation between the nitroreductase activity of S. typhimurium cells, the level of DNA adducts of 1-NP metabolites in the bacterial cells and the mutagenicity of1-NP for bacteria. Therefore, we initiated this study to clarify these correlations. S. typhimurium strain YG1021 was the most sensitive and strain TA98NR was the least sensitive to the mutagenicity of 1-NP among the three strains studied (Fig1, Table1). Tokiwa and Ohnishi (2) found that the numbers of revertant colonies from strains TA98and TA98NR were 467±25 and 87±52 per nmol of1-NP, respectively, without S9 mix. Watanabe et al. (8) reported that the mutagenicity of1-NP for strain YG1021without liver S9 mix was 24,700 revertants per nmol of1-NP. Einisto et al., working with the same three strains as in this study, showed that the numbers of revertants per nmol of 1-NP for strains TA98, TA98NR and YG1021without S9 mix were514, 12and12,172, respectively (15). Although the number of revertant colonies in these reports were different from those obtained in this experiment, the tendency of sensitivity of the three strains to1-NP was similar. These differences may result from different conditions of preincubation of these tester strains and their cultures (16). The nitroreductase activity of S. typhimurium strain YG1021 was the highest and that of strain TA98NR was the lowest among these three strains (Fig2). The high nitroreductase activity of strain YG1021was caused by the overproduction of this enzyme in response to plasmid pYG216 (8), which was made by recombination of the S. typhimurium strain TA1538 nitroreductase gene with the plasmid pBR322. A large number of copies (15to20per cell) of this plasmid (17) may result in the overproduction of nitroreductase. However, the low nitroreductase activity of strain TA98NR may be related to the resistance to nitrofuran, which shows its mutagenicity after nitroreduction, like1-NP(3). Byrant et al. found that in S. typhimurium strain TA98NR a "classical nitroreductase", which is one of the two nitroreductases of S. typhimurium strain TA98, was deleted (18). The correlation coefficient between the mutagenicity and the nitroreductase activity was very high. These results indicate that nitroreductase plays an important role in expressing the mutagenicity of1-NP in bacterial cells. This indication is in accordance with the findings of Oda et al. (19), who demonstrated high sensitivity of a nitroreductase-overexpressing strain to1-NP using the umu test. We used the 32P-postlabeling method to detect DNA adducts derived from1-NP in S. typhimurium strains TA98,TA98NR and YG1021 because this method is highly sensitive for detecting DNA adducts of non-radiolabeled mutagens or carcinogens even in a small quantity of DNA(20). In strains TA98 and YG1021, the DNA adduct spots were detected at the same position, by thin layer chromatography, as dG-C8-AP, which was made by in vitro incubation of1-NP with xanthine oxidase and calf thymus DNA (Fig3A, B, C). No DNA adduct spot was detected after exposure for 8 hr at -80°C in strain TA98NR (Fig3D). The DNA adduct level was highest in strain YG1021 and lowest in strain TA98NR among the three strains (Table1). These results indicate that larger amounts of 1-NP are transformed to dG-C8-AP in S. typhimurium cells producing larger amounts of nitroreductase. Correlation coefficients between the nitroreductase activity and the DNA adduct level and between the mutagenicity and the DNA adduct level were also very high. These results suggest that the nitroreductase activity is important for formation of the DNA adduct, and that the DNA adduct plays an important role in expression of the mutagenicity. Salmeen et al. reported that the amount of 1-AP and the number of revertants were higher under anaerobic conditions than under aerobic conditions and that for strain TA98 the ratio of mutagenicities (anaerobic/aerobic) was comparable with the ratio of the 1-NP reduction rates under anaerobic and aerobic incubation(14). Similarly, Lee et al. demonstrated that benzo[a]pyrene inhibited the nitroreduction of 1-NP to N-hydroxy-1-AP and the formation of dG-C8-AP and exhibited a more antagonistic effect on the mutagenicity of 1-NP in strain TA98 than in strain TA98NR (21). These findings are consistent with our suggestion that nitroreductase is correlated with the DNA adduct formation and expression of the mutagenicity of1-NP. In this study, we demonstrated strong correlations between the bacterial nitroreductase activity, the DNA adduct level and the mutagenicity of 1-NP in bacteria, suggesting that nitroreductase is important for the formation of dG-C8-AP and that the adduct generated leads to the expression of the mutagenicity of 1-NP in bacterial cells. ACKNOWLEDGEMENTS S. typhimurium strain YG1021 was kindly provided by Drs. M. Watanabe and T. Nohmi, National Institute of Health Sciences, Tokyo. REFERENCES 1. Weisburger JH, Weisburger EK:Biochemical formation and pharmacological, toxicological, and pathological properties of hydroxylamines and hydroxamic acids. Pharmacol Rev 25:1-66, 1973 2. Tokiwa H, Ohnishi Y:Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment. CRC Crit Rev Toxicol17:23-60, 1986 3. Rosenkranz HS, Mermelstein R:Mutagenicity and genotoxicity of nitroarenes. All nitro-containing chemi-cals were not created equal. Mutat Res114:217-267, 1983 4. Wislocki PG, Bagan ES, Lu AY, Dooley KL, Fu PP, Han Hsu H, Beland FA, Kadlubar FF:Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay. Carcinogenesis7:1317-1322, 1986 5. Howard PC, Heflich RH, Evans FE, Beland FA: Formation of DNA adducts in vitro and in Salmonella typhimurium upon metabolic reduction of the environ-mental mutagen1-nitropyrene. Cancer Res43:2052-2058, 1983 6. Bell DA, Levine JG, DeMarini DM:DNA sequence analysis of revertants of the hisD3052 allele of Salmonella typhimurium TA98 using the polymerase chain reaction and direct sequencing:application to1-nitropyrene-induced revertants. Mutat Res252:35-44, 1991 7. Malia SA, Vyas RR, Basu AK:Site-specific frame-shift mutagenesis by the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene located in the (CG)3 sequence:effects of SOS, proofreading, and mismatch repair. Biochemistry35:4568-4577, 1996 8. Watanabe M, Isidate M Jr, Nohmi T:A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100.Mutat Res216:211-220, 1989 9. Watanabe M, Nohmi T, Isidate M Jr:New tester strains of Salmonella typhimurium highly sensitive to mutagenic nitroarenes. Biochem Biophys Res Commun147:974-979, 1987 10. Kinouchi T, Manabe Y, Wakisaka K, Ohnishi Y:Bio-transformation of 1-nitropyrene in intestinal anaerobic bacteria. Microbiol Immunol26:993-1005, 1982 11. Maron DM, Ames BN:Revised methods for the Salmonella mutagenicity test. Mutat Res113:173-215, 1983 12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem193:265-275, 1951 13. Gupta RC:Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts. Cancer Res45:5656-5662, 1985 14. Salmeen I, Zacmanidis P, Ball J : 1-Nitropyrene reduction by Salmonella typhimurium, V79 Chinese hamster and primary rat liver cells. Mutat Res122:23-28, 1983 15. Einisto P, Watanabe M, Ishidate M Jr, Nohmi T: Mutagenicity of 30chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities. Mutat Res 259:95-102, 1991 16. Mermelstein R, Kiriazides DK, Butler M, McCoy EC, Rosenkranz HS:The extraordinary mutagenicity of nitropyrenes in bacteria. Mutat Res89:187-196, 1981 17. Maniatis T, Fritsh EF, Sambrook J : Molecular Cloning. Plasmid vectors. Cold Spring Harbor Laboratory Press, New York, 1982 18. Bryant DW, McCalla DR, Lultschik P, Quilliam MA, McCarry BE:Metabolism of 1, 8-dinitropyrene by Salmonella typhimurium. Chem Biol Interact49:351-368, 1984 19. Oda Y, Yamazaki H, Watanabe M, Nohmi T, Shimada T:Highly sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM3009 having high O-acetyltransferase and nitro-reductase activities. Environ Mol Mutagen21:357-364, 1993 20. Gupta RC, Reddy MV, Randerath K:32P-postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis3:1081-1092, 1982 21. Lee H, Cherng SH, Liu TY:Bacterial mutagenicity, metabolism, and DNA adduct formation by binary mixtures of benzo[a]pyrene and 1-nitropyrene. Environ Mol Mutagen24:229-234, 1994 Received for publication December 10, 1997 ; accepted December 22, 1997. 1 Address correspondence and reprint requests to Yoshinari Ohnishi, M.D., Ph.D., Department of Bacteriology, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima770-8503, Japan and Fax:+81-886-33-7069.