(3-pyridyl)-1-butanone (NNK - American Chemical Society

Rebecca Ziegel,† Anthony Shallop,‡ Roger Jones,‡ and Natalia Tretyakova*,†,§. Department of Medicinal Chemistry, University of Minnesota Scho...
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Chem. Res. Toxicol. 2003, 16, 541-550

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K-ras Gene Sequence Effects on the Formation of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-DNA Adducts Rebecca Ziegel,† Anthony Shallop,‡ Roger Jones,‡ and Natalia Tretyakova*,†,§ Department of Medicinal Chemistry, University of Minnesota School of Pharmacy, Minneapolis, Minnesota 55455, Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854, and University of Minnesota Cancer Center, Minneapolis, Minnesota 55455 Received September 17, 2002

The tobacco specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically activated to electrophilic species that form methyl and pyridyloxobutyl adducts with genomic DNA, including O6-methylguanine, N7-methylguanine, and O6-[4-oxo4-(3-pyridyl)butyl]guanine. If not repaired, these lesions could lead to mutations and the initiation of cancer. Previous studies used ligation-mediated polymerase chain reaction (LMPCR) in combination with PAGE to examine the distribution of NNK-induced strand breaks and alkali labile lesions (e.g., N7-methylguanine) within gene sequences. However, LMPCR cannot be used to establish the distribution patterns of highly promutagenic O6-methylguanine and O6-[4-oxo-4-(3-pyridyl)butyl]guanine adducts of NNK. We have developed methods based on stable isotope labeling HPLC-electrospray ionization tandem mass spectrometry (HPLCESI MS/MS) that enable us to accurately quantify NNK-induced adducts at defined sites within DNA sequences. In the present study, the formation of N7-methylguanine, O6-methylguanine, and O6-[4-oxo-4-(3-pyridyl)butyl]guanine adducts at specific positions within a K-ras genederived double-stranded DNA sequence (5′-G1G2AG3CTG4G5TG6G7CG8TA G9G10C-3′) was investigated following treatment with activated NNK metabolites. All three lesions preferentially formed at the second position of codon 12 (GGT), the major mutational hotspot for GfA and GfT base substitutions observed in smoking-induced lung tumors. Therefore, our data support the involvement of NNK and other tobacco specific nitrosamines in mutagenesis and carcinogenesis.

Introduction Cigarette smoke is a chemically complex mixture containing more than 60 known carcinogens. Among these is NNK1, a tobacco specific N-nitrosamine that induces lung tumors in rats, mice, and hamsters (1-4). NNK is produced through the nitrosation of nicotine and is suggested to be involved in the induction of smokingrelated pulmonary adenocarcinoma, the leading lung cancer type in the United States (3-5). Metabolic activation of NNK to DNA reactive species proceeds by hydroxylation of the carbons adjacent to the N-nitroso group, producing pyridyloxobutyl- and methyldiazohydroxide, respectively (Scheme 1). These metabolites can pyridyloxobutylate and methylate guanine * To whom correspondence should be addressed. † University of Minnesota School of Pharmacy. ‡ Rutgers University. § University of Minnesota Cancer Center. 1 Abbreviations: AGT, O6-alkylguanine-DNA-alkyltransferase; HPLCESI MS/MS, HPLC-electrospray ionization tandem mass spectrometry; MNNG, N-methyl-N′-nitro-nitrosoguanidine; MNU, N-nitroso-N-methylurea; N7-Me-G, N7-methylguanine; NDMAOAc, N-nitroso(acetoxymethyl)methylamine; DMS, dimethyl sulfate; NNK, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone; NNKOAc, 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone; O6-Me-G, O6-methylguanine; O6-MedG, O6-methyl-2′-deoxyguanosine; O6-POB-G, O6-[4-oxo-4-(3-pyridyl)butyl]guanine; O6-POB-dG, O6-[4-oxo-4-(3-pyridyl)butyl]-2′-deoxyguanosine; SPE, solid phase extraction; SRM, selected reaction monitoring.

nucleobases in DNA, producing primarily N7- and O6guanine lesions (Chart 1) (4). Both O6-Me-dG and O6POB-dG primarily give rise to GfA transition mutations (6-9), while O6-POB-dG also induces a small number of GfT transversions (9). In addition, POB adducts (e.g., O6-POB-dG) interact with AGT, inhibiting AGT-mediated repair of O6-Me-dG DNA lesions (10-12). N7-Me-G adducts are hydrolytically unstable, undergoing spontaneous depurination to produce abasic sites and single strand breaks in DNA (13). If unrepaired, abasic sites can potentially cause mutations in mammalian cells (1416). Previous studies have observed a correlation between the formation of O6-Me-dG adducts and lung tumor multiplicity in NNK-treated laboratory animals, supporting the role of NNK-induced DNA damage in tobacco mutagenesis (17, 18). A 24-56% amount of human primary adenocarcinomas contain genetic changes within codon 12 of the K-ras protooncogene (19-24). These mutations include both GfT transversions (GGTfTGT, GTT) and GfA transitions (GGTfGAT), which have been shown to activate the K-ras protooncogene, leading to uncontrolled cell growth and a loss of cell differentiation (19-22, 24). K-ras mutations are more common in smokers than in nonsmokers, suggesting a role for tobacco smoke components in the induction of these mutations. K-ras codon 12 mutations are also observed in mouse lung tumors

10.1021/tx025619o CCC: $25.00 © 2003 American Chemical Society Published on Web 03/22/2003

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Scheme 1. Metabolic Activation of NNK to DNA Reactive Species

induced by NNK or its reactive metabolites (25). The goal of the present work was to establish to what extent the patterns of NNK-induced adduct formation are reflected in the K-ras mutational spectra characteristic for smoking-induced lung adenocarcinoma. The distribution of alkylguanine adducts in DNA treated with acetylated precursors to electrophilic metabolites of NNK and other “SN1 type” alkylating agents is known to be sequence-dependent (26-33). Local DNA sequence may also affect the rates of lesion repair (26, 34-36). In the case of O6-guanine alkylation, the 5′flanking base appears to have the most profound influence on adduct yields. Guanine bases with a 5′-flanking purine form significantly higher amounts of O6-alkylguanine adducts than those with 5′-flanking pyrimidines (26-28). In addition, both N7- and O6-guanine alkylation are increased at the central guanine in a run of three or more guanines (29, 32-33). N7-Me-G lesions form more readily at the second G of 5′-GG-3′ dinucleotides (29, 33). Although several studies have examined the distribution of nucleobase damage within gene sequences following DNA treatment with methylating and pyridyloxobu-

tylating agents, these reports have been largely limited to single strand breaks and alkali labile lesions (29, 30, 32, 33). One exception is the paper by Richardson et al. who used [3H] radioactive labeling to determine the relative yields of O6-Me-dG in a DNA sequence derived from a region of the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (31). However, the patterns of formation of biologically relevant O6-Me-dG and O6- POB-dG lesions within the K-ras gene have not been previously reported. Our laboratory has developed a stable isotope labeling HPLC-ESI MS/MS approach that allows us to determine the extent of DNA lesion formation at specific sites within a DNA sequence (37). We have recently reported the use of this method for mapping (+)anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-induced DNA lesions (37). In the present study, the new methodology was used to follow the formation of NNK-induced O6-methyl, N7methyl-, and O6-pyridyloxobutylguanine adducts at specific positions within a K-ras exon 1-derived DNA sequence. The need for these studies is justified by the ample evidence for the involvement of O6-alkylguanines

Chart 1

K-ras Gene Sequence Effects on Adduct Formation

in nitrosamine mutagenesis and carcinogenesis (4, 6-9, 25) and the lack of information on the distribution of these lesions within critical genes. Together, the results of this study demonstrate a strong effect of sequence context on the formation of biologically relevant O6-Me-G and O6-pyridyloxobutylguanine lesions, with a good correlation between the patterns of adduct formation and K-ras mutational spectra observed in NNK-induced tumors.

Materials and Methods Caution: NDMAOAc and NNKOAc are carcinogenic in laboratory animals and should be handled with caution. A lab coat, eye protection, and impervious gloves should be worn at all times (38). Materials. O6-POB-dG was provided by Prof. Lisa Peterson at the University of Minnesota Cancer Center. NNKOAc was synthesized by Dr. Pramod Upadhyaya (University of Minnesota Cancer Center). NDMAOAc was obtained from the NCI Chemical Carcinogen Repository (Midwest Research Institute). N7Me-G and O6-Me-dG standards were purchased from Sigma (St. Louis, MO). DNA octadecamers of the sequence 5′-G1G2AG3CTG4G5TG6G7CG8TAG9G10C-3′ containing codons 10-15 of the K-ras protooncogene were prepared by standard phosphoramidite chemistry using a DNA synthesizer at the University of Minnesota Microchemical Facility. The 15N3-dG phosphoramidite was prepared at Rutgers University as described previously (39). In each oligomer, 15N3-dG was placed at G3, G4, G5, G6, or G7 of the above sequence. The complementary DNA strand 5′-GCCTACGCCACCAGCTCC-3′ and the unlabeled 18-mer representing K-ras codons 10-15, 5′-GGAGCTGGTGGCGTAGGC3′, were synthesized using standard phosphoramidites. Sodium citrate was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). EDTA was obtained from Avocado Research Chemicals Limited (Heysham, Lancashire). Porcine esterase and alkaline phosphatase were from Sigma Chemical Company. Ethanol was obtained from AAPER Alcohol and Chemical Company (Shelbyville, KY). Tris-HCl was purchased from EM Science (Cincinnati, OH). Magnesium chloride and Ultrapure Tris were purchased from ICN Biomedicals, Inc. (Aurora, OH). DNase I, phosphodiesterase (PDE) I, PDE II, and micrococcal nuclease were bought from Worthington Biochemical Corporation (Lakewood, NJ). Calcium chloride, ammonium acetate, acetonitrile, and methanol were obtained from Fisher Scientific (Fair Lawn, NJ). Sodium succinate was purchased from Alfa Aesar (Ward Hill, MA). DNA Purification and Annealing. All 15N-labeled DNA oligodeoxynucleotides were purified by HPLC as described elsewhere (37). An Agilent Technologies 1100 HPLC system was used. A Supelcosil LC-18-DB column (4.6 mm × 150 mm, 5 µm, Supelco, Bellefonte, PA) was maintained at 40 °C and eluted at a flow rate of 1 mL/min. The HPLC solvents were 100 mM triethylammonium acetate, pH 7 (A), and 25% acetonitrile in 100 mM triethylammonium acetate, pH 7 (B), with a gradient of 25-46% B in 40 min. Fractions corresponding to the full length oligomers were collected and concentrated under vacuum. DNA purity and amounts were established by HPLC with UV detection using a Supelcosil LC-18-DB column (2.1 mm × 250 mm, 5 µm). The solvent consisted of 150 mM ammonium acetate buffer, pH 6.5 (A), and acetonitrile (B) at a gradient of 5-12% B in 40 min. Purified DNA was quantified by HPLC using standard curves generated by injecting known amounts of unlabeled DNA strands of the same sequence. The DNA was considered pure if the impurity peaks in the HPLC traces constituted less than 2% of the total area. To obtain doublestranded DNA, equimolar amounts of the two complementary DNA strands were combined in a buffer containing 10 mM Tris/ 50 mM NaCl, pH 8.0, at a concentration of 182 µM. The solution was heated to 10 °C above the melting point and slowly cooled to room temperature.

Chem. Res. Toxicol., Vol. 16, No. 4, 2003 543 DNA Treatment with NDMAOAc and NNKOAc. NDMAOAc stock solution was prepared by combining 5 µL of NDMAOAc with 995 µL of H2O, followed by UV spectrophotometry to confirm the concentration (226 ) 7580). NNKOAc was dissolved in 15 mM sodium citrate/1 mM EDTA buffer, pH 7.0, and the stock solution concentration was determined by UV spectrophotometry (230 ) 13 600). Methylation and pyridyloxobutylation of DNA for our dose-response studies were performed by adding NDMAOAc (0.5-50 mM) or NNKOAc (0.1-10 mM, respectively) to double-stranded unlabeled oligonucleotides (5 nmol) dissolved in 15 mM, pH 7.0, sodium citrate buffer containing 1 mM EDTA (final DNA concentration ) 182 µM). The treatment of 15N3-labeled oligomers was carried out in a similar manner using 2 mM NDMAOAc or 10 mM NNKOAc. The reaction mixtures were incubated in the presence of porcine esterase (0.022 mg/mL) at room temperature for 90 min (NDMAOAc) or 18 h (NNKOAc). The reactions were terminated by DNA precipitation with cold ethanol. Neutral Thermal Hydrolysis of NDMAOAc-Treated DNA. NDMAOAc-treated DNA (1 nmol) was dissolved in 20 µL of distilled water and heated at 95 °C for 30 min to release N7Me-G. The DNA backbone was precipitated with cold ethanol, and the supernatant was dried under vacuum for HPLC-ESI MS analysis as described below. Enzymatic Digestion of NDMAOAc-Treated DNA to 2′Deoxynucleosides. NDMAOAc-treated DNA (4 nmol in 20 µL of 10 mM Tris-HCl/15 mM MgCl2 buffer, pH 7.0) was incubated with DNase I (30 units) in the presence of 1 mM CaCl2, pH 7.0 (30 min at 37 °C). This was followed by further digestion in the presence of PDE I (100 mU) and alkaline phosphatase (8 U) in 50 mM Tris/15 mM MgCl2, pH 9.3 buffer, for 18 h at 37 °C. O6-Me-dG adducts were isolated by SPE using 50 mg C18 Waters Sep-Pak cartridges (Waters Associates, Milford, MA). O6-Me-dG eluted in the 20% methanol fraction. This fraction was dried under vacuum, redissolved in 25 µL of 15 mM ammonium acetate, pH 5.5, and analyzed by HPLC-ESI MS/ MS as described below. Enzymatic Digestion of NNKOAc-Treated DNA. NNKOAc-treated DNA oligomers (5 nmol) were dissolved in 20 µL of 10 mM sodium succinate/5 mM calcium chloride buffer, pH 6.0, and incubated with micrococcal nuclease (1.2 U) and PDE II (22 mU) for 6 h at 37 °C. This was followed by further digestion with alkaline phosphatase (8.2 U) in the presence of 50 mM Tris/15 mM MgCl2 at pH 9.3 (18 h at 37 °C). O6-POBdG was purified by SPE using 50 mg C18 Waters Sep-Pak cartridges. O6-POB-dG eluted in the 50% methanol fraction. These fractions were dried under vacuum and analyzed by HPLC-ESI MS/MS as described below. HPLC-ESI MS/MS Analysis. 1. N7-Me-G Adducts. An Agilent 1100 Capillary LC-Ion Trap MS system was used for these analyses. Chromatographic separation was achieved using a capillary Zorbax SB-C18 column (150 mm × 0.5 mm, 5 µm, Agilent Technologies) eluted at a flow rate of 15 µL/min. The HPLC solvents were 15 mM ammonium acetate, pH 5.5 (A), and 100% acetonitrile (B). The mobile phase was held at 0% B for the first 3 min, changed to 20% B over the next 13.5 min, and further increased to 40% B over the remaining 2.5 min. The mass spectrometer was operated in the positive ion mode with nitrogen as a nebulizing and drying gas (15 psi, 5 L/min). The drying gas temperature was set to 200 °C. The instrument was tuned to maximum sensitivity by direct infusion of N7-Me-G standard solution. Quantitative analysis of N7-Me-G was achieved by full scan HPLC-MS with a scan range of m/z ) 140-180 and a target ion abundance value of 50 000. The maximum accumulation time was 50 ms. Extracted ion chromatograms (m/z ) 166.0 for N7-Me-G and m/z ) 169.0 for [15N3]-N7-Me-G) were obtained from the resulting total ion current HPLC-MS chromatograms. Standard solutions containing known amounts of unlabeled and D3-labeled N7-Me-G were injected prior to sample analyses to confirm that the ratios of HPLC-MS peak areas accurately reflected the relative concentrations of the two isotopomers.

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The extent of N7-Me-G formation at the [15N3]-labeled guanine (X) was calculated from the following equation:

% reaction at X ) A 15N-MeG / (A 15N-MeG + AMe-G)

Scheme 2. Approach Used to Quantify NNK-Induced Lesions at Specific Guanines within a Region of the K-ras Gene

(1)

where A15N-MeG and AMe-G are the areas under the extracted ion chromatogram peaks corresponding to [15N3]-labeled and unlabeled N7-Me-G, respectively. Quantitative analysis of N7-Me-G in double-stranded unlabeled DNA was accomplished using calibration curves constructed by plotting the values of HPLC-MS peak areas (extracted ion m/z ) 166) as a function of N7-Me-G amounts, followed by regression analysis. Equations for the calibration curves analyzed concurrently with each data set were used to calculate N7-Me-G amounts in methylated DNA samples. 2. O6-Me-dG Adducts. A Finnigan TSQ 7000 mass spectrometer (ThermoQuest, San Jose, CA) interfaced with an Agilent 1100 capillary HPLC system was used for these analyses. Chromatographic separation was achieved with the same method as described above for N7-Me-G. The mass spectrometer was operated in the positive ion mode with nitrogen (60 psi) as a nebulizing and drying gas. The temperature of the heated capillary was set to 210 °C, and the spray voltage was 4.5 kV. The instrument was tuned to maximum sensitivity while infusing a standard solution of O6-Me-dG. O6-Me-dG was analyzed in SRM mode. The [M + H]+ions of O6-Me-dG (m/z ) 282.1) and [15N3]-O6-Me-dG (m/z ) 285.1) were isolated in the first quadrupole (Q1). Fragmentation of the ions was achieved in Q2 at a collision energy of 15.0 V and gas pressure of 1.9 mT (Ar). The third quadrupole was set to analyze the product ions corresponding to the loss of deoxyribose (MdR + 2H)+ (m/z ) 166.1 for O6-Me-dG and m/z ) 169.1 for [15N3]O6-Me-dG). The isolation width was set to 0.7 amu, and the scan time was 1 s. Standard solutions containing known amounts of unlabeled and D3-labeled O6-Me-dG were injected prior to sample analyses to confirm that the ratios of HPLC-MS peak areas accurately reflected the relative concentrations of the two isotopomers. The extent of O6-Me-dG formation at [15N3]-labeled guanine (X) was calculated from the areas of the corresponding HPLCESI MS/MS peaks as described above for N7-Me-G (eq 1). Quantitative analysis of O6-Me-dG in our dose-response studies was performed with the aid of standard curves. The calibration curves were obtained by injecting known amounts of O6-MedG, followed by a least-squares linear regression analysis of HPLC-ESI MS/MS peak area (m/z 282.1f166.1) vs O6-Me-dG concentration. 3. O6-POB-dG Adducts. An Agilent 1100 Capillary LC-Ion trap mass spectrometer in conjunction with an Agilent 1100 capillary HPLC system was used to analyze O6-POB-dG. Chromatographic separation was achieved using a Zorbax SBC18 column (150 mm × 0.5 mm, 5 µm, Agilent Technologies) eluted at a flow rate of 15 µL/min. The HPLC solvents were 15 mM ammonium acetate (A) and 100% acetonitrile (B) with a 3 min wash period (100% A), followed by a gradient from 0 to 35% B in 15 min. The mass spectrometer was operated in the positive ion ESI mode with nitrogen as a nebulizing (15 psi) and drying gas (5 L/min, 200 °C). The instrument was tuned to maximize sensitivity by directly infusing O6-POB-dG standard. Quantitative analysis of O6-POB-dG was achieved by isolating the [M + H]+ ions of O6-POB-dG (m/z ) 415.1) and [15N3]-O6POB-dG (m/z ) 418.1). The target ion abundance value was set to 30 000, the maximum accumulation time was 300 ms, and three scans were taken per average. Fragmentation amplitude was set to 0.55 V, with a scan width of 1.0 amu. Fragment ions corresponding to the loss of deoxyribose from the [M + H]+ ions (m/z ) 299.1 for O6-POB-dG and m/z ) 302.1 for [15N3]-O6-POB-dG) were monitored. Standard solutions containing known amounts of unlabeled and D3-labeled O6-POB-dG were injected prior to sample analyses to confirm that the ratios of HPLC-MS/MS peak areas accurately reflected the relative concentrations of the two isotopomers. The extent of O6-POB-

dG formation at the [15N3]-labeled base was calculated as shown above for methylated adducts (eq 1). O6-POB-dG amounts in unlabeled double-stranded K-ras oligodeoxynucleotides following NNKOAc treatment were quantified by comparison to concurrently obtained standard curves. The calibration curves were generated using an authentic standard of O6-POB-dG by the least-squares linear regression analysis of HPLC-ESI MS/MS peak area (m/z 415.1f299.1) plotted against O6-POB-dG amounts. Statistical Analyses. All statistical analyses were performed by Susan Schulte and Shelby Li at the Biostatistics Core of the University of Minnesota Cancer Center using the ANOVA model. The reactivities of individual guanines were compared by t-test using the formula (µ1 - µ2)/(MSE(1/n1 + 1/n2))1/2 where µ is the mean reactivity at positions 1 and 2, respectively, and MSE is the mean squared error from ANOVA. The reactivity at specified guanine nucleobases was compared with the theoretical “random” reactivity value using the equation (µi - c)/ (MSE(1/ni))1/2 where µi is the mean reactivity at position i, c is the theoretical reactivity value, and MSE is the mean squared error from ANOVA.

Results Stable Isotope Labeling Approach. The present study has employed stable isotope labeling in combination with HPLC-ESI MS analyses to determine the patterns of NNK-DNA adduct formation in a critical region of the K-ras protooncogene containing codon 12. Our method centers on the incorporation of [15N]-labeled guanine nucleobases at specific positions within synthetic double-stranded DNA oligodeoxynucleotides representing the gene sequence of interest (Scheme 2) (37). The formation of guanine adducts at specific guanines following the treatment of these oligomers with the pyridyloxobutylating agent NNKOAc or the methylating agent NDMAOAc (Scheme 2) is established by mass spectral analyses of DNA hydrolysates. Because the molecular weight of any lesion originating from the [15N]-labeled guanine is increased by three mass units due to the presence of three 15N atoms in the molecule, they can be readily distinguished from the lesions formed at other nonlabeled guanines. Relative reactivity of a specific site within the sequence is calculated directly from the area ratios of the HPLC-ESI MS peaks corresponding to [15N]labeled and unlabeled adducts (Figures 1 and 2). The preparation of several oligomers of the same sequence, each containing an [15N3]-labeled guanine at a different position, makes it possible to determine the extent of

K-ras Gene Sequence Effects on Adduct Formation

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Figure 2. HPLC-ESI+ MS/MS analysis of O6-Me-dG and [15N3]O6-Me-dG in double-stranded DNA oligonucleotide: 5′-CCC CCG CCC XGC ACC CGC-3′ (X ) [15N3]dG) following NDMAOAc treatment (2 mM). Top panel: Analysis of all unlabeled O6-Me-dG: SRM of the transitions m/z 282.1 f 166.1. Bottom panel: Detection of O6-Me-dG adducts formed at the 15N3labeled guanine: SRM of m/z 285.1 f 169.1. HPLC: An Agilent 1100 capillary HPLC system (Agilent Technologies) was used. A Zorbax SB-C18 column (150 mm × 0.5 mm, 5 µm) was eluted at 15 µL/min with a gradient of 15 mM ammonium acetate, pH 5.5 (A) and 100% acetonitrile (B). Gradient: 0% B, 0-3 min; 0-20% B in 13.5 min; and 20-40% B in 2.5 min. MS: A Finnigan TSQ 7000 was operated in the ESI+ mode (spray voltage, 4.5 kV; heated capillary temperature, 210 °C; nitrogen as a nebulizing and drying gas at 60 psi; isolation width, 0.7 amu; collision gas pressure, 1.9 mT; collision energy, 15 V; electron multiplier, 1800 V; scan time, 1 s).

Figure 1. ESI+ MS/MS spectra of O6-Me-dG (a) and O6-POBdG (b). A Finnigan TSQ 7000 triple quadrupole instrument was operated in the ESI+ mode (spray voltage, 4.5 kV; collision gas pressure, 1.9 mT; collision energy, 15 V; heated capillary, 210 °C; electron multiplier, 1800 V). O6-Me-dG or O6-POB-dG (∼10 µM in 3:1 water:methanol) were infused at a flow rate of 5 µL/ min.

adduct formation at each site of interest (Scheme 2). The use of synthetic DNA oligomers as a model for genomic DNA in these studies is justified by the available evidence that the patterns of DNA modification by tobacco carcinogens are controlled primarily by the local DNA sequence and endogenous cytosine methylation (29, 4042). In contrast, chromosomal structure impacts the overall yield of DNA modification, rather than affecting relative adduct yields at a given DNA base within sequence. Our initial studies attempted to use enzymatic DNA digestion to release both O6- and N7-Me-dG nucleosides. However, because N7-methyl-2′-deoxyguanosine partially depurinated during enzymatic digestion, the N7-guanine adducts existed in two forms: as the free base N7-Me-G and as the corresponding 2′-deoxynucleoside. Although the sensitivity of our HPLC-ESI MS/MS method was sufficient to quantify the N7-Me-dG lesions remaining in the DNA, we were concerned that the extent of depurination may be affected by the local sequence context, affecting our results. We thus chose to analyze N7-Me-G adducts as free bases following their quantitative release from the DNA backbone by neutral thermal hydrolysis. In the optimized version of our method (Scheme 2), an aliquot of NDMAOAc-treated DNA is removed and subjected to neutral thermal hydrolysis to quantitatively release N7-Me-G and [15N3]-N7-Me-G as free bases, followed by HPLC-ESI MS analysis. The

remaining DNA in each sample is digested enzymatically with DNase I, PDE I, and alkaline phosphatase to release O6-Me-dG nucleosides. To ensure complete digestion of DNA to nucleosides, small aliquots of enzymatic hydrolysates are taken and checked by HPLC with UV detection at 260 nm. The digests were considered complete if the only HPLC peaks observed were the signals of dA, dG, dC, and dT, and the molar amounts of released nucleosides were consistent with the number of nucleobases of each type in the sequence. An alternative digestion protocol was developed for pyridyloxobutylated DNA, since our previous experiments (43) have uncovered the ability of O6-POB-dG adducts to block the exonuclease activity of PDE I. Our optimized procedure for enzymatic digestion of pyridyloxobutylated DNA uses PDE II, an exonuclease previously demonstrated to be unaffected by O6-POB-dG lesions (43). SPE was used to remove the salts, the enzymes, and the bulk of unmodified nucleosides prior to HPLC-ESI MS/MS analysis of O6-Me-dG and O6-POB-dG adducts (Scheme 2). Our HPLC-ESI MS/MS method for O6-Me-dG and O6POB-dG adducts is based on the facile loss of deoxyribose (M ) 116) from pseudomolecular ions of the adducts under collision-induced dissociation conditions (Figures 1 and 2). O6-Me-dG (m/z ) 282.1, (M + H)+) fragments to m/z ) 166.1 (M + 2H-dR)+ (Figure 1a) while O6-POBdG (m/z ) 415.1, (M + H)+) produces the product ions with m/z ) 299.1 (M + 2H-dR)+ (Figure 1b). Because the [M + H]+ ions of the N7-Me-G bases are not easily fragmented under CID conditions, the best sensitivity is achieved in full scan HPLC-ESI MS mode. The quantitation of N7-Me-G and its [15N3] analogue is performed using extracted ion chromatograms of the appropriate [M + H]+ ions (m/z ) 166.1 and m/z ) 169.1, respectively). We determined that our HPLC-ESI+-MS detection limits for NNK-induced DNA adducts were in the low femto-

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Figure 3. Dose-response analyses of methyl- and pyridyloxobutylguanine adducts in double-stranded DNA 18-mer (GGAGCTGGTGGCGTAGGC, 5 nmol) following its treatment with either NDMAOAc (0-50 mM) or NNKOAc (0-5 mM). (a) HPLCESI MS analysis of N7-Me-G was performed with full scan HPLC-MS using extracted ion chromatograms for m/z ) 166.0. HPLC: Same as described in Figure 2 for O6-Me-dG analysis. MS: An 1100 series Ion Trap MS system (Agilent Technologies) was operated in positive ion mode; scan range, m/z ) 140-180; nebulizing gas (N2), 15 psi; drying gas (N2), 5 L/min; drying gas temperature, 200 °C; target ion abundance, 50 000; maximum accumulation time, 50 ms. See Figure 2 for HPLC-ESI MS/MS details for the analysis of O6-Me-dG. (b) HPLC-ESI MS/MS analysis of O6-POB-dG was performed by monitoring the transition m/z ) 415.1 f 299.1. HPLC: A Zorbax SB-C18 (150 mm × 0.5 mm, 5 µm) was eluted at 15 µL/min with a gradient of 15 mM ammonium acetate (A) and 100% acetonitrile (B) on an Agilent 1100 capillary HPLC system. Gradient: 0% B, 0-3 min followed by 0-35% B in 15 min. MS: An Agilent 1100 Series Ion Trap MS system was operated in positive ion mode: nebulizing gas (N2), 15 psi; drying gas (N2), 5 L/min; drying gas temperature, 200 °C; target ion abundance value, 30 000; maximum accumulation time, 300 ms; three scans per average; fragmentation amplitude, 0.55; scan width, 1.0 amu.

mole range (1 fmol for O6-POB-dG, 25 fmol for N7-MeG, and 5 fmol for O6-Me-dG, S/N ) 3). The calibration curves for HPLC-ESI MS analysis were linear from 0.10 to 100 pmol of the adducts, and the typical R2 values were 0.9964 (N7-Me-G), 0.9986 (O6-Me-dG), and 0.9938 (O6POB-dG). To establish optimal DNA amounts and carcinogen concentrations for use in our stable isotope labeling HPLC-ESI MS/MS experiments, dose-response analyses were conducted for the formation of O6-Me-dG, N7-MeG, and O6-POB-dG in unlabeled, double-stranded oligomers representing K-ras codons 10-15 (Figure 3). Ideally, the overall adduct numbers should be high enough to allow accurate measurement of the lesions originating from a specific guanine, while retaining sequence selectivity (37). Dose-response analysis of O6-Me-dG and N7Me-G formation in NDMAOAc-treated double-stranded DNA is shown in Figure 3a. The amounts of both adducts gradually increased between NDMAOAc doses 0.5-10 mM and began to plateau above 10 mM NDMAOAc

Ziegel et al.

(Figure 3a). The leveling off of the adduct yields above 10 mM NDMAOAc may be due to a decrease in pH (from 7.0 to ∼5.5) at high NDMAOAc concentrations. Consistent with the previous reports (13), N7-Me-G adduct amounts were approximately 10-fold higher than that of O6-Me-dG (Figure 3a). Using the concentration-dependent curves and detection limit information, we estimated that when starting with 5 nmol of DNA per sample, a dose of 2 mM NDMAOAc was sufficient for our isotope labeling experiments. Dose-response analysis of O6-POB-dG in NNKOActreated DNA was performed in a similar manner (Figure 3b). The amounts of pyridyloxobutylated lesions in NNKOAc-treated DNA were 32-50 pmol/106 G following treatment with 0.1-1 mM NNKOAc and did not significantly increase above 1 mM NNKOAc (Figure 3b). We chose 10 mM NNKOAc exposure for our stable isotope labeling experiments to ensure that the O6-POB-dG adduct yields at 15N-labeled guanines were sufficient for accurate quantitation by HPLC-ESI MS/MS. Further analysis of the concentration-dependent data for 2 mM NDMAOAc and 10 mM NNKOAc treatments (Figure 3) reveals that at these conditions, less than 0.4% of all guanines contained O6-methyl adducts and less than 0.1% of all guanines contained O6-pyridyloxobutyl adducts, indicating “single hit” conditions (less than one adduct per DNA strand). Distribution of N7-Me-G and O6-Me-dG Adducts within a K-ras-Derived DNA Sequence Containing Codon 12. We examined the formation of N7-Me-G and O6-Me-dG at specific guanine nucleobases within a region of the K-ras gene containing codon 12, the major mutational “hotspot” in smoking-induced lung tumors (1924). A series of synthetic 18-oligomers of the sequence 5′-G1G2AG3CTG4G5TG6G7CG8TAG9G10C-3′ (codon 12 ) G4G5T) were prepared, each containing a single [15N3]guanine at positions G3, G4, G5, G6, or G7. The oligonucleotides were annealed to the complementary strands and treated with 2 mM NDMAOAc. The extent of N7Me-G and O6-Me-dG formation at each guanine was determined by stable isotope labeling HPLC-ESI MS as described above (Scheme 2). Because the double-stranded 18-mer 5′-G1G2AG3CTG4G5TG6G7CG8TAG9G10C-3′ contains a total of 13 guanine residues, the theoretical adduct amount produced at each guanine in the absence of any sequence effects is 7.7% (100%/13). However, our results indicate that the formation of N7-Me-G within a K-ras-derived sequence is not uniform (Figure 4). The largest amount of N7-Me-G (15%) originates from G5, closely followed by G7 (13%). Only 7% of N7-Me-G formation takes place at G3, and 9.79.8% of the adducts were formed at G4 and G6 (Figure 4). Statistical analysis indicates that N7-Me-G amounts formed at G5 and G7 are the same (P > 0.2) and are significantly greater than the theoretical value of 7.7% (P < 0.001 and P < 0.0001, respectively). G4 and G6 gave rise to statistically equal levels of N7-Me-G (P ) 1), which were within the theoretical value (P > 0.3 and P > 0.2, respectively). The percentage of N7-Me-G lesions formed at G3 (7%) was also within the theoretical value (P > 0.4) but was significantly less than the adduct yields at G4 and G6 (P < 0.02). In summary, the extent of N7-Me-G formation at all five guanines examined was at or above the theoretical value. The distribution of O6-Me-dG adducts within the same K-ras-derived DNA sequences demonstrated a more

K-ras Gene Sequence Effects on Adduct Formation

Figure 4. Relative formation of N7-Me-G at guanine nucleobases within double-stranded DNA oligonucleotide derived from the K-ras gene: G1G2AG3CTG4G5TG6G7CG8TAG9G10C (codon

12 ) G4G5T). The data were compiled from two separate experiments (N ) 6). The percent reaction at each guanine was calculated from the area ratio of 15N-labeled HPLC-ESI MS peak to the sum of unlabeled and 15Nlabeled HPLC-ESI MS peak areas. The random reaction value was determined from the total number of guanine nucleobases in both DNA strands.

Figure 5. Relative formation of O6-Me-dG at guanine nucleobases within a double-stranded DNA oligonucleotide derived from the K-ras gene: G1G2AG3CTG4G5TG6G7CG8TAG9G10C (codon 12 ) G4G5T). The data were compiled from two separate experiments (N ) 5-6). The percent reaction at each guanine was calculated from the area ratio of 15N-labeled HPLC-ESI MS/ MS peak to the sum of unlabeled and 15N-labeled HPLC-ESI MS/MS peak areas. The random reaction value was determined from the total number of guanine nucleobases in both DNA strands.

pronounced sequence selectivity, with lesion yields differing up to 6-fold within this sequence (Figure 5). As in the case of N7-Me-G adducts, the majority of O6-Me-dG adduct formation occurred at G5 (17.2%) within the frequently mutated K-ras codon 12 (GGT). The reactivity of G5 is significantly greater than the random probability value of 7.7% (P < 0.003). O6-Me-dG levels at G3 (9.9%) and G7 (5.9%) were within the theoretical value (P > 0.1 and P > 0.05, respectively), while the numbers of adducts produced at G4 and G6 (2.8 and 3.7%, respectively) were significantly lower than the random value (P < 0.0001 for each). As was the case with N7-Me-G (Figure 4), O6Me-dG adduct yields from G4 and G6 were statistically the same (P ) 1), consistent with their identical sequence context (TGG). Distribution of O6-POB-dG Adducts within a K-ras-Derived DNA Sequence Containing Codon 12. The distribution of O6-POB-dG adducts within the K-ras-derived sequence (Figure 6) was remarkably similar to that observed for O6-Me-dG (Figure 5). The majority of O6-POB-dG adduct formation (12.4%) occurred at G5 (the second G in codon 12), followed by G3 (8.8%). The reactivity at G5 was significantly greater than the random probability value of 7.7% (P < 0.04), while G3

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Figure 6. Relative formation of O6-POB-dG at guanine nucleobases within double-stranded DNA oligonucleotide derived from K-ras gene sequence: G1G2AG3CTG4G5TG6G7CG8TAG9G10C (codon 12 ) G4G5T). The data were compiled from two separate experiments (N ) 4-5). The percent reaction at each guanine was calculated from the area ratio of 15N-labeled HPLC-ESI MS/ MS peak to the sum of unlabeled and 15N-labeled HPLC-ESI MS/MS peak areas. The random reaction value was determined from the total number of guanine nucleobases in both DNA strands.

adduct yields were within the random value (P > 0.2). Adduct levels produced at G4 (3.3%), G6 (2.8%), and G7 (4.5%) were statistically the same (P ) 1, G4 vs G6 and G4 vs G7; P > 0.3, G6 vs G7) but were significantly lower than the random value (P < 0.02, P < 0.03, and P < 0.02, respectively). Although the percentage of O6-POB-dG adducts originating from G7 is 1.6-fold greater than that at G6, this difference is not statistically significant (P > 0.3). The Kolmogorov-Smirnov test comparing the distributions of O6-Me-dG and O6-POB-dG adducts within the K-ras-derived DNA sequence (Figures 5 and 6, respectively) fails to reject the null hypothesis, indicating that the distribution patterns of the two adducts are not significantly different. These results demonstrate that the relative reactivity of a given guanine within a K-rasderived sequence toward NNK-derived methylating and pyridyloxobutylating agents is the same.

Discussion The initiation of smoking-induced lung cancer is thought to involve the covalent binding of metabolically activated tobacco carcinogens to DNA nucleobases (2). If not repaired before DNA replication, the resulting nucleobase lesions (DNA adducts) can lead to heritable mutations in critical genes. Because tobacco smoke is a complex mixture containing many known carcinogens that are capable of forming a wide variety of DNA adducts (2), it is difficult to link the mutations observed in the lung tumors of smokers to specific types of tobacco carcinogeninduced DNA damage. A thorough examination of the patterns of formation of tobacco carcinogen-DNA adducts within critical genes, e.g., the K-ras protooncogene and the p53 tumor suppressor gene, may provide an insight into the origins of mutational hotspots observed in smoking-induced lung cancer. The present study focused on sequence distribution of DNA lesions produced by DNA reactive metabolites of NNK within a frequently mutated region of K-ras exon 1. Codon 12 of the K-ras protooncogene (GGT) is the major mutational hotspot in smoking-induced lung adenocarcinoma and in NNK-induced mouse lung tumors (19-25). The distribution patterns of DNA adducts induced by simple alkylating agents within K-ras exons 1 and 2 have been previously examined using ligation-

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mediated polymerase chain reaction (LMPCR) in combination with PAGE (29, 30). However, because LMPCRPAGE is limited to nucleobase lesions that can be converted to DNA strand breaks (e.g., N7-Me-G), the distribution patterns of biologically relevant O6-Me-G and O6-pyridyloxobutylguanine adducts could not be examined. Furthermore, the latter approach provides little structural information about the adducts detected. Our laboratory has developed a mass spectrometrybased approach capable of quantifying specific carcinogeninduced DNA lesions at defined sites within a DNA sequence (37). This method has previously been used to determine the distribution patterns of N2-guanine adducts induced by the ultimate carcinogenic species of benzo[a]pyrene, another potent tobacco carcinogen (37). In this approach, stable isotope-labeled [15N3]-guanine nucleobases are incorporated at specific positions within oligodeoxynucleotides representing gene sequences of interest, and the relative adduct yields at different sites are established by HPLC-ESI MS/MS analysis (Scheme 2). The present study has investigated the formation of several NNK-induced DNA lesions, including N7-Me-G, O6-Me-dG, and O6-POB-dG, within a DNA sequence derived from the K-ras protooncogene. All three lesions were overproduced at the second guanine of K-ras codon 12, the major hotspot for both GfA transitions and GfT transversions in smoking-induced pulmonary cancer (Figures 4-6) (20-23). Because previous experiments have shown that O6-POB-dG and O6-Me-dG adducts cause mainly GfA transition mutations (6-9), our data support the role of NNK-induced O6-Me-dG and O6-POBdG adducts in the induction of transition mutations at the second position of K-ras codon 12. Furthermore, abasic sites resulting from the depurination of hydrolytically unstable N7-Me-G may contribute to the GfT transversion mutations observed at the second G of K-ras codon 12 (GGT) (Figure 4) (14, 16, 20-23, 25). In general, sequence preferences for O6-Me-dG formation within the K-ras gene context were GGT (G5) > AGC (G3) > GGC (G7) > TGG (G4, G6) (Figure 5). This order of reactivity is consistent with the previous studies that observed a stimulating effect of 5′-flanking purines on methylation of the target G (26, 27, 31). In contrast, the 5′-flanking pyrimidines generally decrease the O6-MedG yields (26, 27, 31). However, unlike the previous report that observed the same molar yields of O6-Me-dG at the central G within GGT, AGC, and GGC sequence contexts (27), our study demonstrates up to 3-fold differences in reactivity of these sites (e.g., AG3C, GG5T, and GG7C in Figure 5). These discrepancies may result from the differences in DNA sequence selection and/or experimental methodology. Unlike the earlier study that used DNA 24-mers consisting of repeating nucleotides (27), DNA oligomers included in our experiments are derived from the actual K-ras gene sequence. In the latter case, the reactivity of target guanines is potentially affected by more distant neighbors within the sequence. For example, AG3C may be activated by the preceding guanines (GGAG3C, Figure 5). Similar to O6-Me-dG, the highest yields of N7-Me-G lesions are also observed at G5 (GGT), followed by G7 (GGC), then G4 and G6 (TGG), and last, G3 (AGC) (Figure 4). This order of reactivity is consistent with previous studies of DNA alkylation by SN1 type alkylating agents, e.g., NDMAOAc (29), MNU (29, 33), and MNNG (33) that

Ziegel et al.

usually target the central guanine in a run of three or more Gs and at the 3′-guanine in a GG sequence (29, 33). In contrast, SN2 type alkylating agents, e.g., DMS, produce more N7-Me-G at the 5′- guanine in a run of several Gs and show little sequence selectivity in GG dinucleotides (29, 33). Several explanations have been proposed for the increased reactivity of SN1 type agents toward the 3′-guanine within GG runs, including electronic and steric effects, the nature of the alkylating agent, and a regioselective alkylation mechanism that involves the formation of a tetrahedral intermediate by the attack of imidourea at the 5′ guanine (29, 33, 44-46). Electronic factors alone cannot explain the reactivity order of different guanines within the K-ras-derived sequence toward NNK metabolites (Figures 4-6). Computational studies indicate that over 70% of the highest occupied molecular orbital is localized on the 5′-guanine within 5′-GG-3′ dinucleotides (47, 48), increasing its nucleophilicity as compared with the 3′-guanine. Our observation of marked similarities between the adduction patterns observed with relatively compact NDMAOAc (Figure 5) and the bulky NNKOAc (Figure 6) rules out steric effects as the major factor in defining the relative reactivities of the neighboring guanines toward activated NNK species. NDMAOAc in the presence of esterase is unlikely to form a tetrahedral intermediate analogous to that proposed for MNU (46). Further studies are warranted to elucidate the mechanisms of regioselectivity of NNK metabolites. Importantly, the distribution patterns of N7 and O6-Me-G within K-ras gene-derived sequence (Figures 4 and 5) are not the same, indicating that N7-Me-G cannot be used to predict the distribution of biologically relevant O6-Me-dG lesions. To our knowledge, this study is the first paper on how DNA sequence context influences the formation of O6POB-dG lesions. The distribution patterns of O6-Me-dG and O6-POB-dG within a K-ras-derived gene sequence (Figures 5 and 6) are statistically the same, consistent with the similar nature of the two alkylating species of NNK (Scheme 1). Cloutier and associates have previously examined the distribution of NNKOAc-induced DNA damage along the p53, H-ras, K-ras, and N-ras genes (30). These authors (30) observed little difference between the reactivity of analogous K-ras guanines toward NNKOAc (30). However, it should be noted that the earlier study (30) was limited to DNA strand breaks and did not detect biologically relevant O6-alkylguanine lesions. Considering the remarkable similarities between the distribution of the O6-alkylguanine lesions induced by methylating and pyridyloxobutylating agents (Figures 5 and 6), it is worth mentioning their notable dissimilarity with the patterns of DNA adduct formation by BPDE within the same K-ras gene-derived DNA sequence (37). Unlike model NNK diazohydroxides, BPDE targeted the first, rather than second, guanine of codon 12 (GGT) (37). These results indicate that sequence specificity for DNA alkylation by metabolically activated tobacco carcinogens is not an intrinsic property of the DNA duplex itself but rather is strongly dependent on the identity of the reactive species. The characteristic damage patterns generated by the active metabolites of tobacco carcinogens are potentially useful in linking the mutations observed in lung cancer to specific types of DNA adducts. For example, the highest yields of O6-Me-dG and O6-POB-dG were ob-

K-ras Gene Sequence Effects on Adduct Formation

served at the second guanine of K-ras codon 12, a known hotspot for GfA transition mutations (19-24), suggesting that these genetic changes may originate from polymerase bypass of NNK-induced nucleobase lesions. It should be noted, however, that in addition to the initial adduct formation step, other factors could also influence the mutational spectra, including the relative repair rates, mispairing efficiency, and mutation selection. Studies are in progress at our laboratory to evaluate the role of DNA repair in shaping the mutational spectra of NNK and other tobacco carcinogens.

Acknowledgment. We thank Prof. Lisa Peterson at the University of Minnesota for O6-pyridyloxobutylguanine standard; Prof. Stephen Hecht for helpful discussions and comments on the paper; Dr. Pramod Upadhyaya at the University of Minnesota for providing NNKOAc; Dr. Peter Villalta for assistance with the TSQ mass spectrometer; Susan Schulte and Shelby Li at the Biostatistics Core of the University of Minnesota Cancer Center for the statistical analysis of our data; and Agilent Technologies for providing the 1100 Capillary HPLC/ion trap system. Funding for this research was from the University of Minnesota Cancer Center, the University of Minnesota Transdisciplinary Tobacco Research Center, Minnesota Medical Foundation, University of Minnesota Graduate School, the Ziagen drug development grant from the University of Minnesota Department of Medicinal Chemistry, and the National Cancer Institute (CA095039).

Note Added after ASAP Posting This article was released ASAP on 03/22/2003. Subsequently, one correction was made in the Introduction, a hyphen was removed from a compound in the Discussion, and a grant number was added to the Acknowledgment. The article was reposted to the Web on 03/28/2003.

References (1) Hecht, S. S., Chen, C. B., Ohmori, T., and Hoffmann, D. (1980) Comparative carcinogenicity in F344 rats of the tobacco-specific nitrosamines, N′-nitrosonornicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res. 40, 298-302. (2) Hecht, S. S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl. Cancer Inst. 91, 1194-1210. (3) Hecht, S. S. (1998) Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 11, 559-603. (4) Hecht, S. S. (1999) DNA adduct formation from tobacco-specific N-nitrosamines. Mutat. Res. 424, 127-142. (5) Thun, M. J., Lally, C. A., Flannery, J. T., Calle, E. E., Flanders, W. D., and Heath, C. W., Jr. (1997) Cigarette smoking and changes in the histopathology of lung cancer. J. Natl. Cancer Inst. 89, 1580-1586. (6) Loechler, E. L., Green, C. L., and Essigmann, J. M. (1984) In vivo mutagenesis by O6-methylguanine built into a unique site in a viral genome. Proc. Natl. Acad. Sci. U.S.A. 81, 6271-6275. (7) Eadie, J. S., Conrad, M., Toorchen, D., and Topal, M. D. (1984) Mechanism of mutagenesis by O6-methylguanine. Nature 308, 201-203. (8) Singer, B., and Essigmann, J. M. (1991) Site-specific mutagenesis: retrospective and prospective. Carcinogenesis 12, 949-955. (9) Pauly, G. T., Peterson, L. A., and Moschel, R. C. (2002) Mutagenesis by O6-[4-oxo-4-(3-pyridyl)butyl]guanine in Esherichia coli and human cells. Chem. Res. Toxicol. 15, 165-169. (10) Liu, X. K., Spratt, T. E., Murphy, S. E., and Peterson, L. A. (1996) Pyridyloxobutylation of guanine residues by 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone generates substrates of O6alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 9, 949953. (11) Peterson, L. A., Liu, X. K., and Hecht, S. S. (1993) Pyridyloxobutyl DNA adducts inhibit the repair of O6-methylguanine. Cancer Res. 53, 2780-2785.

Chem. Res. Toxicol., Vol. 16, No. 4, 2003 549 (12) Peterson, L. A., Thomson, N. M., Crankshaw, D. L., Donaldson, E. E., and Kenney, P. J. (2001) Interactions between methylating and pyridyloxobutylating agents in A/J mouse lungs: implications for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis. Cancer Res. 61, 5757-5763. (13) Singer, B., and Grunberger, D. (1983) Molecular Biology of Mutagens and Carcinogens, Plenum Press, New York and London. (14) Gentil, A., Cabral-Neto, J. B., Mariage-Samson, R., Margot, A., Imbach, J. L., Rayner, B., and Sarasin, A. (1992) Mutagenicity of a unique apurinic/apyrimidinic site in mammalian cells. J. Mol. Biol. 227, 981-984. (15) Takeshita, M., and Eisenberg, W. (1994) Mechanism of mutation on DNA templates containing synthetic abasic sites: study with a double strand vector. Nucleic Acids Res. 22, 1897-1902. (16) Avkin, S., Adar, S., Blander, G., and Livneh, Z. (2002) Quantitative measurement of translesion replication in human cells: evidence for bypass of abasic sites by a replicative DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 99, 3764-3769. (17) Belinsky, S. A., Foley, J. F., White, C. M., Anderson, M. W., and Maronpot, R. R. (1990) Dose-response relationship between O6methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone. Cancer Res. 50, 3772-3780. (18) Peterson, L. A., and Hecht, S. S. (1991) O6-methylguanine is a critical determinant of 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone tumorigenesis in A/J mouse lung. Cancer Res. 51, 55575564. (19) Rodenhuis, S., Slebos, R. J., Boot, A. J., Evers, S. G., Mooi, W. J., Wagenaar, S. S., van Bodegom, P. C., and Bos, J. L. (1988) Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung. Cancer Res. 48, 5738-5741. (20) Rodenhuis, S., and Slebos, R. J. (1992) Clinical significance of ras oncogene activation in human lung cancer. Cancer Res. 52, 2665s-2669s. (21) Westra, W. H., Baas, I. O., Hruban, R. H., Askin, F. B., Wilson, K., Offerhaus, G. J., and Slebos, R. J. (1996) K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res. 56, 2224-2228. (22) Westra, W. H., Slebos, R. J., Offerhaus, G. J., Goodman, S. N., Evers, S. G., Kensler, T. W., Askin, F. B., Rodenhuis, S., and Hruban, R. H. (1993) K-ras oncogene activation in lung adenocarcinomas from former smokers. Evidence that K-ras mutations are an early and irreversible event in the development of adenocarcinoma of the lung. Cancer 72, 432-438. (23) Mills, N. E., Fishman, C. L., Rom, W. N., Dubin, N., and Jacobson, D. R. (1995) Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma. Cancer Res. 55, 1444-1447. (24) Slebos, R. J., Hruban, R. H., Dalesio, O., Mooi, W. J., Offerhaus, G. J., and Rodenhuis, S. (1991) Relationship between K-ras oncogene activation and smoking in adenocarcinoma of the human lung. J. Natl. Cancer Inst. 83, 1024-1027. (25) Ronai, Z. A., Gradia, S., Peterson, L. A., and Hecht, S. S. (1993) G to A transitions and G to T transversions in codon 12 of the Ki-ras oncogene isolated from mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and related DNA methylating and pyridyloxobutylating agents. Carcinogenesis 14, 2419-2422. (26) Dolan, M. E., Oplinger, M., and Pegg, A. E. (1988) Sequence specificity of guanine alkylation and repair. Carcinogenesis 9, 2139-2143. (27) Sendowski, K., and Rajewsky, M. F. (1991) DNA sequence dependence of guanine-O6 alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea. Mutat. Res. 250, 153160. (28) Pegg, A. E., Oplinger, M., and Dolan, M. E. (1988) Investigation of sequence specificity in formation of O6-methylguanine and its repair using oligodeoxynucleotide substrates. J. Cell. Biochem. Suppl. 12A, 274. (29) Cloutier, J. F., Drouin, R., and Castonguay, A. (1999) Treatment of human cells with N-Nitroso(acetoxymethyl)methylamine: distribution patterns of piperidine-sensitive DNA damage at the nucleotide level of resolution are related to the sequence context. Chem. Res. Toxicol. 12, 840-849. (30) Cloutier, J. F., Drouin, R., Weinfeld, M., O’Connor, T. R., and Castonguay, A. (2001) Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA. J. Mol. Biol. 313, 539-557. (31) Richardson, F. C., Boucheron, J. A., Skopek, T. R., and Swenberg, J. A. (1989) Formation of O6-methyldeoxyguanosine at specific

550

(32)

(33)

(34) (35)

(36)

(37)

(38) (39)

Chem. Res. Toxicol., Vol. 16, No. 4, 2003 sites in a synthetic oligonucleotide designed to resemble a known mutagenic hotspot. J. Biol. Chem. 264, 838-841. Wurdeman, R. L., and Gold, B. (1988) The effect of DNA sequence, ionic strength, and cationic DNA affinity binders on the methylation of DNA by N-methyl-N-nitrosourea. Chem. Res. Toxicol. 1, 146-147. Shoukry, S., Anderson, M. W., and Glickman, B. W. (1993) Use of fluorescently tagged DNA and an automated DNA sequencer for the comparison of the sequence selectivity of SN1 and SN2 alkylating agents. Carcinogenesis 14, 155-157. Delaney, J. C., and Essigmann, J. M. (2001) Effect of sequence context on O(6)-methylguanine repair and replication in vivo. Biochemistry 40, 14968-14975. Topal, M. D., Eadie, J. S., and Conrad, M. (1986) O6-methylguanine mutation and repair is nonuniform. Selection for DNA most interactive with O6-methylguanine. J. Biol. Chem. 261, 98799885. Georgiadis, P., Smith, C. A., and Swann, P. F. (1991) Nitrosamine-induced cancer: selective repair and conformational differences between O6-methylguanine residues in different positions in and around codon 12 of rat H-ras. Cancer Res. 51, 58435850. Tretyakova, N., Matter, B., Jones, R., and Shallop, A. (2002) Formation of Benzo[a]pyrene Diol Epoxide-DNA Adducts at Specific Guanines within K-ras and p53 Gene Sequences: Stable Isotope-Labeling Mass Spectrometry Approach. Biochemistry 41, 9535-9544. Sansone, E. B., Tewary, Y. B. (1978) The Permiability of Laboratory Gloves to Selected Nitrosamines, IARC Scientific Publishers. Zhao, H., Pagano, A. R., Wang, W., Shallop, A., Gaffney, B., and Jones, R. A. (1997) Use of a 13C Atom to Differentiate Two 15Nlabeled Nucleosides. Syntheses of [15NH2]-Adenosine, [1,NH215N2]-, and [2-13C-1,NH2-15N2]Guanosine, and [1,7,NH2-15N3]and [2-13C-1,7,NH2-15N3]-2′-Deoxyguanosine. J. Org. Chem. 62, 7832-7835.

Ziegel et al. (40) Pfeifer, G. P. (2000) p53 mutational spectra and the role of methylated CpG sequences. Mutat. Res 450, 155-166. (41) Denissenko, M. F., Chen, J. X., Tang, M. S., and Pfeifer, G. P. (1997) Cytosine methylation determines hot spots of DNA damage in the human P53 gene. Proc. Natl. Acad. Sci U.S.A. 94, 38933898. (42) Pfeifer, G. P., Drouin, R., and Holmquist, G. P. (1993) Detection of DNA adducts at the DNA sequence level by ligation-mediated PCR. Mutat. Res. 288, 39-46. (43) Tretyakova, N., Matter, B., Ogdie, A., Wishnok, J. S., and Tannenbaum, S. R. (2001) Locating Nucleobase Lesions within DNA Sequences by MALDI-TOF Mass Spectral Analysis of Exonuclease Ladders. Chem. Res. Toxicol. 14, 1058-1070. (44) Liang, G., Encell, L., Nelson, M. G., Switzer, C., Shuker, D. E. G., and Gold, B. (1995) Role of Electrostatics in the SequencSelective Reaction of Charged Alkylating Agents with DNA. J Am. Chem Soc. 117, 10135-10136. (45) Dande, P., Liang, G., Chen, F. X., Roberts, C., Nelson, M. G., Hashimoto, H., Switzer, C., and Gold, B. (1997) Regioselective effect of zwitterionic DNA substitutions on DNA alkylation: evidence for a strong side chain orientational preference. Biochemistry 36, 6024-6032. (46) Buckley, N. (1987) A regioselective mechanism for mutagenesis and oncogenesis caused by alkylnitrosourea sequence-specific DNA alkylation. J. Am. Chem. Soc. 109, 7918-7920. (47) Saito, I., Takayama, M., Nakamura, T., Sugiyama, H., Komeda, Y., and Iwasaki, M. (1995) The most electron-donating sites in duplex DNA: guanine-guanine stacking rule. Nucleic Acids Symp. Ser., 191-192. (48) Nakatani, K., Dohno, C., and Saito, I. (1999) Chemistry of sequence-dependent remote guanine oxidation: photoreaction of duplex DNA containing cyanobenzophenone-substituted uridine. J. Am. Chem. Soc. 121, 10854-10855.

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