O6-Pyridyloxobutylguanine Adducts Contribute to the Mutagenic

Sep 20, 2005 - Milton S. Hershey Medical Center, Pennsylvania State University ... Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware...
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Chem. Res. Toxicol. 2005, 18, 1619-1625

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O6-Pyridyloxobutylguanine Adducts Contribute to the Mutagenic Properties of Pyridyloxobutylating Agents Rene´e S. Mijal,§,⊥ Natalia A. Loktionova,† Choua C. Vu,⊥ Anthony E. Pegg,† and Lisa A. Peterson*,§,⊥ Division of Environmental Health Sciences and The Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, and Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Received May 27, 2005

The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are potent carcinogens in animal models and likely human carcinogens. Both NNK and NNN can be activated to a pyridyloxobutylating agent. This alkylating agent contributes to the carcinogenic effects of NNK and NNN via the formation of miscoding DNA adducts. One of these adducts, O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG) has been characterized as a mutagenic adduct which is a substrate for the repair protein O6alkylguanine-DNA alkyltransferase (AGT). Repair of O6-alkylguanine adducts by AGT protects cells from the mutagenic and carcinogenic effects of alkylating agents and is likely to play a similar role in shielding cells from the adverse effects of pyridyloxobutylating agents. Therefore, we examined the mutagenicity of the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), in Salmonella typhimurium YG7108 expressing hAGT. Expression of hAGT protected cells from NNKOAc-induced mutagenicity. Interestingly, hAGT did not shield cells from the toxicity of this agent. To confirm that the repair of O6-pobG was increased in the bacteria expressing hAGT, we measured levels of this adduct in NNKOAc-treated cultures. The levels of O6-pobG were lower in DNA from bacteria expressing hAGT. This work establishes an important role for O6-pobG in mediating the mutagenic, and possibly carcinogenic, effects of pyridyloxobutylating compounds.

Introduction The tobacco-specific nitrosamines 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK)1 and N′-nitrosonornicotine (NNN) are potent animal carcinogens and likely human carcinogens (1, 2). NNK produces pulmonary tumors in mice, rats, and hamsters independent of route of administration (1). NNN induces nasal cavity, esophageal, and respiratory cancers in rodent models (1). Both NNK and NNN require metabolic activation to exert their mutagenic and carcinogenic effects (1). Alphahydroxylation of both NNN and NNK can result in the formation of a pyridyloxobutylating intermediate (Scheme 1). The pyridyloxobutylation pathway contributes to the carcinogenic activity of both agents (1, 3-5). Pyridyloxobutylating agents are mutagenic in bacterial and mammalian systems. In the Ames assay, these compounds * To whom requests for reprints should be addressed at The Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware St. S.E., Minneapolis, MN 55455. Phone, 612-626-0164; fax, 612-6265135; e-mail, [email protected]. § Division of Environmental Health Sciences, University of Minnesota. ⊥ The Cancer Center, University of Minnesota. † Pennsylvania State University College of Medicine. 1 Abbreviations: AGT, O6-alkylguanine-DNA alkyltransferase; DTT, dithiothreitol; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; hAGT, human wild-type O6-alkylguanine-DNA alkyltransferase; MNNG, N-methylN′-nitro-N-nitrosoguanidine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone; NNKOAc, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1butanone; NNN, N′-nitrosonornicotine; O6-meG, O6-methylguanine; O6pobG, O6-[4-oxo-4-(3-pyridyl)butyl]guanine; O6-pobdG, O6-[4-oxo-4-(3pyridyl)butyl]-2′-deoxyguanosine.

cause mutations at GC (6-9) and AT base pairs (9), as well as frameshift mutations (8, 9). Mutations caused by pyridyloxobutyl adducts likely play an important role in cancer development, as tumors from A/J mice treated with the pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) exhibit a high frequency of GC f AT transitions and GC f TA transversions at the 12th codon of K-ras (10). The mutagenicity and carcinogenicity of alkylating agents is linked to the formation of mutagenic adducts at nucleophilic sites in DNA (1, 3, 4, 11). DNA pyridyloxobutylation produces adducts at O2 thymidine, O2 cytosine, and N2, 7, and O6 guanine positions in DNA, as well as the phosphodiester backbone of DNA (12-15). The particular adduct(s) responsible for the mutagenic activity of pyridyloxobutylating agents have not been identified. Only O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6pobG) has been tested for its mutagenic properties (16). It is highly mutagenic in bacteria and human cells. In Escherichia coli, O6-pobG produces only GC f AT transitions. In human cells, GC f AT transitions predominate, but other mutations are observed. These include GC f TA transversions, compound mutations (those comprised of a transition or transversion mutation at the site once containing the adduct and a deletion of one of the adjacent bases), deletions, and remote mutations (16). Repair of O6-alkylguanine adducts protects organisms from the mutagenic and carcinogenic effects of alkylating agents (17-19). O6-Alkylguanine adducts are repaired by

10.1021/tx050139t CCC: $30.25 © 2005 American Chemical Society Published on Web 09/20/2005

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Mijal et al.

Scheme 1. Activation Pathways for NNK, NNN, and the Model Alkylating Compounds NNKOAc and MNNG

the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) via a direct, stoichiometric, and errorproof repair mechanism (17). The alkyl group is transferred from the O6 position of guanine to the active site cysteine, inactivating the protein and signaling its degradation (17, 20). A variety of O6-alkylguanine adducts are substrates for AGT, including O6-methylguanine (O6meG) and O6-pobG (14, 17). Repair of the mutagenic O6-meG adduct by AGT protects against mutagenesis (21-26) and carcinogenesis (24, 27-30) caused by methylating agents. Human AGT protects against the mutagenic effects of O6-pobG when site-specifically incorporated into DNA (16), suggesting that AGT may play a key role in protecting cells against the mutagenicity of pyridyloxobutylating agents. In vitro experiments demonstrate that site-specifically incorporated O6-pobG is mutagenic, consistent with the hypothesis that O6-pobG contributes to the mutagenic effects of pyridyloxobutylating agents in cells and in vivo. However, the importance of this adduct to the total mutagenic activity of pyridyloxobutylating agents has not been established. To this end, we present this report on the results of experiments evaluating the mutagenicity of the model pyridyloxobutylating agent NNKOAc in Salmonella typhimurium expressing wild-type human O6-alkylguanine-DNA alkyltransferase (hAGT).

Experimental Procedures Materials. Lysozyme and proteinase K were purchased from Amersham Biosciences (Piscataway, NJ). Alkaline phosphatase, RNase A, and RNase T1 were obtained from Sigma Aldrich Chemical Co. (Milwaukee, WI). Phosphodiesterase II and micrococcal nuclease were purchased from Worthington Biochemical Corporation (Lakewood, NJ). Strata-X SPE cartridges were obtained from Phenomenex (Torrence, CA). 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), [5-3H]NNKOAc, and O6-[1,2,2-2H3-4-oxo-4-(3-pyridyl)butyl]-2′-deoxyguanosine ([2H3]O6-pobG) were prepared as described previously (31-33). All other reagents were obtained from Sigma Aldrich Chemical Co. (Milwaukee, WI) or Fisher Scientific (Fairlawn, NJ). Salmonella Strains. The S. typhimurium strain YG7108 (a derivative of strain TA1535) (34) that lacks both the ada and ogt genes (35) was transformed with plasmid expressing hAGT (pIN-hAGT) or an empty vector (pIN) (36, 37).

Toxicity and Mutagenicity Studies. An overnight bacterial culture was inoculated in 10 mL of fresh LB medium and grown to an OD600 of 0.6. Cells were washed in 1× M9 salts and resuspended in 1 mL of 1× M9 salts. Aliquots (100 µL) of cell suspension were treated with different concentrations of NNKOAc (0.35 M stock solution in DMSO) or N-methyl-N′-nitroN-nitrosoguanidine (MNNG) at 37 °C for 30 min. For cell survival estimation, cells were washed with 1× M9 salts and diluted 1:106; 10-100 µL of each diluted sample was plated on nonrestrictive LB medium supplemented with ampicilin and kanamycin (50 µg/mL each). For the mutation assay, 10 µL of culture was diluted in 14 mL soft agar and plated over VogelBonner minimal medium with excess biotin and trace L-histidine (34). The mutation frequency (hisG+) was calculated by dividing the number of colonies grown on the selective medium by the number of survivors grown on the nonselective medium. Adduct Level Measurements. The hydrolysis of [5-3H]NNKOAc was measured via HPLC to determine the extent of NNKOAc conversion occurring during the 3-h treatment period. S. typhimurium pIN-hAGT and S. typhimurium pIN were grown at 37 °C in LB plus kanamycin and ampicillin (0.01 mg/mL) to a final OD600 of 0.6-0.7. One hundred milliliters of culture was pelleted and re-suspended in 11 mL of M9 salts media for carcinogen treatment. [5-3H]NNKOAc (0.42 mmol, 0.23 µCi) was dissolved in 1.2 mL of DMSO and added to the bacterial suspension to give a final concentration of 0 or 20 µM NNKOAc (∼0.5 µCi/mmol). Cells were treated for 3 h at 37 °C in a shaking water bath. Aliquots (100 µL) were removed periodically throughout the incubation to monitor the conversion of NNKOAc to its hydrolysis product, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). At the end of the 3 h incubation, the cultures were centrifuged to pellet cells, and 500 µL of the supernatant was used to measure the hydrolysis of [5-3H]NNKOAc. All aliquots were spiked with unlabeled NNKOAc and HPB standards, and the hydrolysis reaction was monitored via HPLC with radioflow detection as previously described (38). Briefly, [5-3H]NNKOAc (retention time ) 11.5 min) and its metabolite [5-3H]HPB (retention time ) 21 min) were separated over a Phenomenex Prodigy ODS3 column (150 mm × 4.6 mm; Torrance, CA) using a linear gradient from 20 mM sodium phosphate containing 15% methanol to 20 mM sodium phosphate containing 45% methanol over 15 min (flow, 1 mL/min). More than 80% of the [5-3H]NNKOAc was hydrolyzed within 3 h. Thus, the bacteria were treated with NNKOAc for 3 h prior to DNA isolation. To obtain DNA for adduct level measurements, S. typhimurium pIN-hAGT and S. typhimurium pIN bacteria were grown as described. NNKOAc was dissolved in DMSO to a concentration of 24 mM. Bacterial cultures (11 mL) were incubated with 0, 20, 95, or 380 µM NNKOAc for 3 h in a 37 °C shaking water

Mutagenicity of O6-Pyridyloxobutylguanine Adducts bath. Treatments were performed in triplicate or quadruplicate. After carcinogen treatment, cells were washed in M9 salts media, treated with lysozyme for 20 min (in 10 mL 50 mM Tris, 10 mM EDTA, and 0.25 mg/mL lysozyme, pH 7.2), and lysed by the addition of SDS (e0.01% final concentration). Lysates were extracted twice with an equal volume of 24:1 chloroform/ isoamyl alcohol prior to precipitation of the DNA with an equal volume of ethanol. DNA was redissolved in 5 mL of TE (50 mM Tris, 5 mM EDTA, and e0.01% SDS, pH 7.2) for treatment with RNase (0.68 mg RNase A, 2200 U RNase T1) at 37 °C for 3045 min. Proteinase K (0.8-1.5 mg) was added, and samples were incubated an additional 45-60 min. The DNA was extracted two additional times and precipitated with ethanol. DNA was washed in ice-cold 80% ethanol before drying under N2. Levels of O6-pobdG in DNA from NNKOAc-treated cells were measured using a previously described LC-MS/MS assay (32). To prepare DNA for analysis, DNA (0.1-0.5 mg) was dissolved in 10 mM sodium succinate and 5 mM CaCl2, pH 7, and solutions were spiked with 0.25-0.5 pmol [2H3]O6-pobdG standard (total volume 0.65 mL). Samples were heated at 100 °C for 10 min prior to the addition of enzymes to remove adducts that inhibit enzymatic digestion of the DNA. After digesting sequentially with micrococcal nuclease and phosphodiesterase II (22 U/mg DNA and 400 mU/mg DNA for at least 5 h) and alkaline phosphatase (150 U/mg DNA overnight), hydrolysates were applied to Strata-X solid-phase extraction (SPE) columns (Phenomenex, Torrance, CA) to remove proteins and to separate O6-pobdG from unmodified nucleosides. Columns were sequentially eluted with water (1 mL), 10% methanol (1 mL), and 100% methanol (2 × 1 mL). The 100% methanol fractions containing O6-pobdG were dried under pressure to 100 µL, at which point 100 mM ammonium acetate was added (50 µL). Samples were further concentrated to 20-30 µL. In some cases, methanol (10% of total volume) was added to the samples prior to injection to improve solubility of O6-pobdG. Mass spectrometric analyses were performed on a TSQ Quantum Ultra AM, TSQ Discovery, or TSQ (Thermo Electron, Bellafonte, PA) interfaced with an Agilent 1100 series capillary HPLC (Agilent Technologies, Palo Alto, CA). The previously reported LC conditions and mass spectrometer settings were used for adduct analyses (32). Briefly, hydrolysate mixtures were separated over a Zorbax C-18 column with a linear gradient using 15 mM ammonium acetate and containing 10% methanol to 15 mM ammonium acetate containing 50% methanol over 24 min. O6-pobG eluted around 25 min. Two different SRM methods were used for quantitative analyses of O6-pobdG levels. The first quadripole was set to isolate O6-pobdG (m/z ) 415) and [2H3]O6-pobdG (m/z ) 418) for both analyses. For reaction 1, the collision-induced dissociation energy was 11 V and the Ar gas pressure was 1.3 mTorr. For reaction 2, the collision energy was 25 V and Ar gass pressure was 0.8 Torr. The third quadripole was set to (M - dR + 2H) O6-pobdG (m/z ) 299) and [2H3]O6-pobdG (m/z ) 302) for reaction 1. For reaction 2, the third quadripole was set to (M - dG + 2H) O6pobdG (m/z ) 148) and [2H3]O6-pobdG (m/z ) 151). Accurate measurements of O6-pobdG/[2H3]O6-pobdG standards were obtained by monitoring both reactions. Furthermore, adduct levels measured using the two reactions showed good agreement. Finally, to confirm the identity of adduct observed in Salmonella DNA, multiple reaction monitoring was used. Quadripole 1 was set to O6-pobdG (m/z ) 415) and [2H3]O6-pobdG (m/z ) 418). The collision-induced dissociation energy was 11 V and the pressure was 1.3 mTorr. The third quadripole was set to the monitor the fragments resulting from the loss of 2′-deoxyribose and 2′-deoxyguanosine as described above (reactions 1 and 2). In addition, quadripole 3 was set to monitor O6-pobdG and [2H3]O6-pobdG (M - pyridyloxobutyl + H) (m/z ) 268). A 0.2 s scan time was used for monitoring multiple reactions. Guanine concentrations were determined as previously described using a Keystone Scientific Hypersil ODS column (5 uM, 4.6 mm × 250 mm, Bellafonte, CA) (32). Nucleosides were separated using a linear gradient from 100% 100 mM am-

Chem. Res. Toxicol., Vol. 18, No. 10, 2005 1621 monium acetate to 92% ammonium acetate containing 8% acetonitrile over 30 min, monitoring at 254 nm. Adduct levels were expressed as fmol O6-pobdG/µmol dG. Comparisons of adduct level used unpaired t-tests assuming unequal variances (Microsoft Excel, 1998). Reported p values are two-sided.

Results and Discussion S. typhimurium YG7108 expressing AGT were treated with NNKOAc to determine the contribution of O6-pobG and its repair to the mutagenicity and toxicity of pyridyloxobutylating agents. The YG7108 strain is a derivative of S. typhimurium TA1535 cells lacking the ogt and ada alkyltransferase genes. These cells, which detect mutations at GC base pairs, were transfected with either hAGT contained in a pIN expression plasmid (pIN-hAGT) or pIN vector alone. Cells were transfected with the human protein because it, unlike the bacterial proteins, repairs O6-pobG (39). The mutagenicity of NNKOAc was evaluated in the presence and absence of hAGT (Figure 1A). The mutation frequency induced by NNKOAc in YG7108 pIN was comparable to that previously reported for MMNG in this strain at similar levels of toxicity (40). The concentrations of NNKOAc required to produce mutagenic effects were higher than those reported for MNNG (35, 40). However, it is known that pyridyloxobutylating agents are less efficient at alkylating DNA in bacteria than methylating compounds (9). Poor alkylating efficiency can result from inefficient conversion of NNKOAc to the reactive intermediate within the bacterial cell or rapid reaction of the intermediate with water. Expression of hAGT reduced the mutagenicity of NNKOAc 3-4-fold in treated cells. This finding clearly demonstrates that hAGT protects cells against the mutagenic effects of pyridyloxobutylating agents just as it does for methylating (21-24), ethylating (41-43), propylating (44), and butylating agents (40, 45). Given that AGT also blocks the carcinogenic effects of methylating (24, 27-30) and ethylating agents (46), AGT likely shields against the carcinogenic effects of the pyridyloxobutylating compounds, NNN and NNK. The toxicity of NNKOAc was also evaluated and compared to that of the methylating agent, MNNG. YG7108 cells transfected with pIN and YG7108 cells transfected with pIN-hAGT were equally sensitive to the toxic effects of NNKOAc (Figure 1B). In contrast, MNNG was more toxic to YG7108pIN than to YG7108 pIN-hAGT (Figure 1C). These results suggest a fundamental difference in the nature of the AGT-repairable O6-alkylguanine adducts formed by methylating and pyridyloxobutylating agents. It is well-established that O6-meG is toxic as well as mutagenic (17, 28, 47-51). AGT significantly reduces both the mutagenicity and toxicity of methylating agents by removing O6-meG from DNA (17, 47, 48, 50). The marked protection against the toxicity of MNNG in YG7108 pIN-AGT is consistent with the role of O6-meG adducts in mediating the toxic effects of methylating compounds. In contrast, the expression of AGT did not modify the toxicity of pyridyloxobutylating compounds. These results indicate that O6-pobG differs from O6-mG in that it is mutagenic but not toxic. This difference may have important toxicological consequences; the competing effects of toxicity and mutagenicity may limit the mutagenic potential of O6-meG, whereas nontoxic O6-pobG

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Mijal et al. Scheme 2. O6-pobdG Transitions Monitored Using LC-MS/MSa

a Expected masses for fragmentation of O6-pobdG (m/z 415) are presented in the figure. For [2H3]O6-pobdG (m/z 418), the expected masses are Reaction 1 (the loss of 2′-deoxyribose), m/z 418 f 302; Reaction 2 (the loss of 2′-deoxyguanosine), m/z 418 f 151; and Reaction 3 (the loss of the pyridyloxobutyl group), m/z 418 f 268. Reactions 1 and 2 were used to measure adduct levels in S. typhimurium DNA. The quantitation limit in DNA for Reaction 1 was approximately 150 fmol O6-pobdG/µmol dG. Reaction 2 was a more sensitive reaction, having a limit of detection in pure standard in the sub-attomole to attomole range and quantitation limit in DNA ≈15-20 fmol O6-pobdG/µmol dG.

Figure 1. Mutagenicity and toxicity of alkylating agents to YG7108 pIN and YG7108 pIN-AGT. (A) The mutagenicity of NNKOAc to cells, with the spontaneous mutation rates of 16.5 ( 7.5 and 9.6 ( 3.4 per million cells in YG7108pIN and YG7108 pIN-AGT, respectively. (B and C) The toxicity of NNKOAc (panel B) and MNNG (panel C) to cells. The results shown in panel C are representative of five separate experiments, while those presented in panels A and C represent two experiments. For each experiment, three estimates were made for each point. Error bars represent the standard deviation.

should persist in DNA and exert a stronger mutagenic effect on a per adduct basis. Since previous experiments demonstrated that O6pobG is mutagenic and that hAGT protects cells from the mutagenic effects of this adduct contained in plasmid DNA (16), we hypothesized that O6-pobG was the mutagenic adduct repaired by hAGT in cells treated with pyridyloxobutylating agents. Therefore, we measured levels of O6-pobG in DNA from NNKOAc-treated YG7108 cells via LC-MS/MS. Adduct levels were measured by monitoring one of two O6-pobG transitions (Scheme 2). Initially, adduct levels were measured while monitoring reaction 1, the loss of 2′-deoxyribose from O6-pobdG. Samples with adduct levels at or below the limit of detection were remeasured using the more sensitive reaction representing the loss of 2′-deoxyguanosine (reac-

tion 2). In addition, we confirmed the identity of the peak coeluting with labeled internal [2H3]O6-pobdG standard in DNA hydrolysates by simultaneously monitoring for three expected O6-pobdG fragments (Figure 2). Adduct levels were consistently higher in YG7108 lacking hAGT as compared to repair-proficient bacteria (Figure 3). At the 0.38 mM concentration, adduct levels in YG7108 pIN were 2710 ( 510 fmol O6-pobdG/µmol dG as compared to 630 ( 170 fmol O6-pobdG/µmol dG in YG7108 pIN-hAGT (p < 0.0017). The same trend was observed at lower concentrations. Levels of O6-pobdG adducts correlated with mutation frequency in Salmonella pIN and pIN-hAGT (Figures 1A and 3). These results demonstrate that (1) O6-pobG is an important mutagenic adduct formed by pyridyloxobutylating compounds and (2) AGT protects against the mutagenicity of these compounds by repairing O6-pobG. Furthermore, these findings are consistent with the known mutagenic activity of O6-pobG at GC base pairs (16), with the ability of mammalian AGTs to repair O6-pobG (14, 39, 52, 53) and the ability of hAGT to protect cells from the mutagenic effects of O6-pobG (16). While hAGT protects against mutagenesis in S. typhimurium exposed to NNKOAc, the protection it confers is not complete. The depletion of AGT is unlikely to account for this observation. There are likely sufficient levels of AGT present to repair all of the O6-pobG formed; the level of AGT expressed in YG7108 pIN-hAGT cells is 17.2 pmol/mg protein. In addition, we observed a constant level of protection against mutagenesis over the range of NNKOAc concentrations tested. If the AGT activity was exhausted, the protection would be lower at higher concentrations. Other explanations for the inability of AGT to completely protect against mutagenesis are more plausible. First, low levels of O6-pobG adducts remaining in the

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Figure 2. LC-MS/MS traces of (A) YG7108pIN-AGT treated with vehicle control (DMSO) monitoring the loss of 2′-deoxyribose, (B) YG7108 pIN treated with 380 µM NNKOAc monitoring the loss of 2′-deoxyribose, (C) YG7108 pIN treated with 380 µM NNKOAc monitoring the loss of the pyridyloxobutyl group, and (D) YG7108 pIN treated with 380 µM NNKOAc monitoring the loss of 2′deoxyguanosine. The top figure in each panel A-D shows the chromatogram for O6-pobdG, and the bottom figure is the chromatogram for the internal standard [2H3]-O6-pobdG.

Figure 3. The O6-pobdG levels measured in YG7108 pIN and YG7108 pIN-AGT treated with NNKOAc. Data points for YG7108 pIN and YG 7108 pIN-AGT (0 and 0.380 mM treatments) represent three or four independent measurements. Error bars indicate standard deviations. All other points represent two independent measures.

DNA may be responsible for mutagenesis. This is consistent with the known potency of O6-pobG adducts (16) and the sensitivity of O6-pobG repair to the effects of sequence context.2 Low levels of O6-pobG remaining in DNA may represent adducts formed in sequences that are resistant to repair. Second, there could be other mutagenic O6-pyridyloxobutylguanine adducts formed as a result of DNA pyridyloxobutylation. On the basis of the reaction of pyridyloxobutylating agents with various nucleophiles, we initially proposed that a cyclic O6pyridyloxobutylguanine adduct could be formed in pyridyloxobutylated DNA (14, 31, 54). However, this explanation is unlikely. Recent experiments suggest that the model nucleophiles used may not have been good models for reactions occurring at nucleophilic sites in DNA; cyclic adducts at the N2 of deoxyguanosine, an exocyclic heteroatom that has a nucleophilic character 2 Mijal, R. S., Kanugula, S., Vu, C. C., Fang, Q., Pegg, A. E., and Peterson, L. A., manuscript in preparation.

much like that of the O6 position, are not formed in pyridyloxobutylated DNA (12). Instead, it is more likely that other pyridyloxobutylguanine adducts contribute to the mutagenicity of NNKOAc at GC base pairs; possibilities include O2-cytidine and N2- and 7-guanine adducts (12, 13). In summary, this work establishes an important role for O6-pobG in the mutagenic activity of pyridyloxobutylating nitrosamines in biological systems. Furthermore, it highlights the importance of AGT repair in protecting cells from the effects of these agents. These findings support the hypothesis that O6-pobG contributes to the mutagenicity of pyridyloxobutylating agents and may be responsible, in part, for their carcinogenic properties. Further studies need to determine the overall importance of O6-pobG and its repair by AGT to the mutagenic and carcinogenic activities of pyridyloxobutylating agents such as NNN and NNK.

Acknowledgment. The authors would like to thank Nancy Fleisher for the preparation of O6-pobG and [2H3]O6-pobG standards, Nicole M. Thomson for her collaboration with 5-[3H]NNKOAc hydrolysis experiments, and Dr. Peter Villalta and Brock A. Matter for assistance with mass spectrometry experiments. This work is supported by NIH Grants CA-59887 (L.A.P.) and CA-18137 (A.E.P.). R.S.M. is supported by NIEHS training grant ES-10956.

References (1) Hecht, S. S. (1998) Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 11, 560-603. (2) Wogan, G., Hecht, S., Felton, J., Conney, A., and Loeb, L. (2004) Environmental and chemical carcinogenesis. Semin. Cancer Biol. 14, 473-486. (3) Staretz, M. E., Foiles, P. G., Miglietta, L. M., and Hecht, S. S. (1997) Evidence for an important role of DNA pyridyloxobutylation in rat lung carcinogenesis by 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone: effects of dose and phenethyl isothiocyanate. Cancer Res. 57, 259-266.

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