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Identification of Adducts Formed by Pyridyloxobutylation of Deoxyguanosine and DNA by 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a Chemically Activated Form of Tobacco Specific Carcinogens Mingyao Wang, Guang Cheng, Shana J. Sturla, Yongli Shi, Edward J. McIntee, Peter W. Villalta, Pramod Upadhyaya, and Stephen S. Hecht* University of Minnesota Cancer Center, 420 Delaware Street SE, Mayo Mail Code 806, Minneapolis, Minnesota 55455 Received January 7, 2003

The tobacco specific carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are metabolically activated to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (7), which is known to pyridyloxobutylate DNA. A substantial proportion of the adducts in this DNA releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 11) under various hydrolysis conditions, including neutral thermal hydrolysis. These HPB-releasing DNA adducts have been detected in target tissues of animals treated with NNK and NNN as well as in lung tissue from smokers. Although their presence in pyridyloxobutylated DNA was conclusively demonstrated 15 years ago, their structures have not been previously determined. We investigated this question in the present study by determining the structures of products formed in reactions with dGuo and DNA of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH2OAc, 3), a stable precursor to 7. Reaction mixtures from NNKCH2OAc and dGuo were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LCESI-MS) with selected ion monitoring at m/z 415. A major peak was detected and identified as 7-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (37) by its ESI-MS fragmentation pattern and by neutral thermal hydrolysis, which converted it to 11 and 7-[4-oxo-4-(3-pyridyl)but-1-yl]Gua (26). The latter was identified by comparison to synthetic 26 using LC-ESI-MS with selected ion monitoring at m/z 299, M + 1 of 26. Further evidence was obtained by NaBH4 reduction of 26 to 7-[4-hydroxy-4-(3-pyridyl)but-1-yl]Gua, which was also matched with a standard. Adduct 37 was similarly identified in enzyme hydrolysates of DNA reacted with NNKCH2OAc, accounting for 30-35% of the HPB-releasing adducts in this DNA. Several other adducts resulting from pyridyloxobutylation of the N2- and O6-positions of Gua were also identified as products in the dGuo or DNA reactions by comparison to standards; their concentrations were considerably less than that of 37. These adducts were N2-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (23), N2-[4-oxo-4-(3-pyridyl)but-2-yl]dGuo (25), N2-[2-(3-pyridyl)tetrahydrofuran-2-yl]dGuo (31a) (or its open chain tautomer 31b), and O6-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (10). Adducts 23, 25, and 10 did not release HPB upon neutral thermal hydrolysis. The results of this study provide the first structural identification of an HPB-releasing DNA adduct of the tobacco specific nitrosamines NNK and NNN.

Introduction Cigarette smoking is responsible for 30% of all cancer death and 90% of lung cancer in developed countries (1, 2). It also causes cancers of the oral cavity; naso-, oro-, and hypopharynx; nasal cavity and paranasal sinuses; larynx; esophagus; stomach; pancreas; liver; kidney; ureter; urinary bladder; uterine cervix; and bone marrow (2). Smokeless tobacco products cause oral cavity cancer (3). The tobacco specific nitrosamines NNK1 (Scheme 1) and NNN (Scheme 2) are among the most important carcinogens in tobacco products. Extensive analytical data clearly document the presence of substantial amounts of NNK and NNN not only in cigarette smoke but also * To whom correspondence should be addressed. Tel: 612-624-7604. Fax: 612-626-5135. E-mail: [email protected].

in cured, processed tobacco used to manufacture cigarettes and smokeless tobacco products such as chewing tobacco and snuff (2-6). NNK is likely to play a significant role as a cause of lung cancer in smokers (4, 7). It is a potent systemic pulmonary carcinogen in rodents, inducing tumors of the lung independent of the route of administration (8). Its lung carcinogenicity is particularly 1 Abbreviations: HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; LC-ESIMS, liquid chromatography/electrospray ionization/mass spectrometry; N2-pyridyloxobutyl-dGuo, N2-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo; N2-pyridyloxobut-2-yl-dGuo, N2-[4-oxo-4-(3-pyridyl)but-2-yl]dGuo; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNKCH2OAc, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, N′-nitrosonornicotine; O6pyridyloxobutyl-dGuo, O6-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo; PEITC, 2-phenethyl isothiocyanate; 7-pyridyloxobutyl-Gua, 7-[4-oxo-4-(3-pyridyl)but-1-yl]Gua; 7-pyridyloxobutyl-dGuo, 7-[4-oxo-4-(3-pyridyl)but1-yl]dGuo.

10.1021/tx034003b CCC: $25.00 © 2003 American Chemical Society Published on Web 04/03/2003

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Scheme 1. Overview of the Metabolic Activation of NNK to DNA Adducts

Scheme 2. Overview of the Metabolic Activation of NNN to DNA Adducts

impressive in F344 rats, in which a total dose as low as 6 mg/kg induced lung tumors (9). Even lower doses of NNK caused lung tumors when considered as part of a dose-response trend (9). NNK also induces pancreatic,

liver, and nasal cavity tumors in rats, and when given to rats by oral swabbing together with NNN, the mixture causes tumors of the oral cavity (8). NNN induces esophageal and nasal tumors in rats and is likely to play an important role in tobacco-related esophageal cancer in smokers (8, 10). NNK and NNN are widely acknowledged as causative agents for oral cancer in people who use smokeless tobacco products (8). DNA adduct formation is a key step in the carcinogenic process (11). If the adducts persist, miscoding can occur during DNA replication, leading to permanent mutations and derangement of normal cellular growth control processes. Metabolic activation is a prerequisite for DNA adduct formation by NNK and NNN (8). An overview of NNK metabolism and DNA adduct formation is presented in Scheme 1. Hydroxylation of the carbons R- to the N-nitroso group, catalyzed by cytochromes P450 and other enzymes, is the major pathway of NNK metabolic activation (8). Hydroxylation at the methylene carbon yields intermediate 5, which spontaneously decomposes to keto aldehyde 8 and methanediazohydroxide (9). The latter methylates DNA, probably via a diazonium ion, yielding 7-methylGua (12), O6-methylGua (13), and O4methylThy (14). These adducts have been detected in DNA isolated from tissues of NNK-treated animals (8). Compounds 12 and 13 have also been detected in human lung DNA (12). Compounds 13 and 14 are mutagenic, and there is strong evidence that 13 is important in carcinogenesis by NNK (9, 13, 14). Hydroxylation of NNK at the methyl group produces intermediate 4, which spontaneously loses formaldehyde giving 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (7). This intermediate and related electrophiles generated from it cause pyridyl-

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oxobutylation of DNA, and this persistent modified DNA has been detected in tissues of NNK-treated animals and in humans (8). Analysis of human lung DNA demonstrates that levels of pyridyloxobutylated DNA are higher in lung cancer patients than in controls (15). Pyridyloxobutylated DNA is mutagenic, and a considerable body of data demonstrate that it is critical in carcinogenesis by NNK (8). O6-pyridyloxobutyl-dGuo (10) is one product of this pathway (16). This adduct is highly mutagenic in bacterial and human cells but quantitatively represents a relatively minor component of total DNA pyridyloxobutylation (16, 17). The major adducts in pyridyloxobutylated DNA release HPB (11) upon hydrolysis, but they have not been previously characterized (8). This pathway is discussed further below. NNK is also converted metabolically to NNAL. NNAL is detoxified by glucuronidation but can be further activated by R-hydroxylation yielding methyl and pyridylhydroxybutyl adducts in DNA (18). The metabolic activation of NNN to DNA adducts is summarized in Scheme 2. Hydroxylation at the 2′position gives intermediate 16, which spontaneously ring opens producing 7, the same intermediate formed by methyl hydroxylation of NNK (8). This results in pyridyloxobutylation of DNA by NNN with the formation of HPB-releasing adducts, as observed with NNK. NNN can also be metabolically activated by 5′-hyroxylation yielding intermediates 17 and 18, but adduct formation by this pathway has not been reported. Hydrolysis conditions affect the results obtained upon analysis of pyridyloxobutylated DNA. Mild acid hydrolysis (0.1 N HCl, 80 °C, 0.5 h) of this DNA produces 11 and 22 in a ratio of 8:1 (16). Strong acid hydrolysis (0.8 N HCl, 80 °C, 3 h) converts 22 to 11, and consequently, only HPB is observed when these conditions are used (16, 19). 3-Hydroxy-1-(3-pyridyl)-1-butanone is not detected (19). Neutral thermal hydrolysis (pH 7.0, 100 °C, 1 h) gives mainly 11 and does not release 10, while enzyme hydrolysis produces a variety of products, which usually include HPB (19, 20). These results conclusively demonstrate that there are thermally unstable HPB-releasing adducts present in pyridyloxobutylated DNA, but these adducts, which clearly represent the major adducts in this DNA, have never been characterized (8). In this study, we have investigated the structures of adducts formed by pyridyloxobutylation of dGuo and DNA, focusing on the thermally unstable HPB-releasing adducts. We used NNKCH2OAc (3), a stable precursor to intermediate 4, to generate the pyridyloxobutylating intermediates.

Experimental Section Apparatus and Assay Conditions. HPLC was carried out with a Waters Associates (Milford, MA) system equipped with a model 996 photodiode array detector. A 4.6 mm × 25 cm Supelcosil LC-18-DB column (Supelco, Bellefonte, PA) was eluted isocratically with 5% CH3OH in 40 mM ammonium phosphate buffer, pH 6.6, for 10 min and then a gradient from 5 to 35% CH3OH in 60 min. The flow rate was 0.5 mL/min, and detection was by UV (254 nm). LC-ESI-MS was carried out with a Thermo Finnigan LCQ Deca instrument (Themo Finnigan LC/MS Division, San Jose, CA) interfaced with a Waters Alliance 2690 HPLC multisolvent delivery system and equipped with an SPD-10 A UV detector (Shimadzu Scientific Instruments, Columbia, MD). The HPLC column and elution conditions were the same as above, except

Wang et al. that the flow rate was 0.3 mL/min. The ESI source was set as follows: voltage, 2.0 kV; current, 10 µA; capillary temperature, 250 °C. Chemicals and Enzymes. Compounds 3 (21), 10 (16), N2pyridyloxobutyl-dGuo (23) (20), N2-pyridyloxobut-2-yl-dGuo (25) (22), 7-pyridyloxobutyl-Gua (26) (18), and the corresponding pyridylhydroxybutyl adducts O6-pyridylhydroxybutyl-dGuo (27), N2-pyridylhydroxybutyl-dGuo (28), 29, and 7-pyridylhydroxybutyl-Gua (30) (18, 22) were synthesized as described. The synthesis of adduct 32 is described below. O6-[(Trimethylsilyl)ethyl]-2-fluoroinosine (36) was purchased from Chem Genes Corp. (Ashland, MA). Calf thymus DNA, dGuo, and enzymes were obtained from Sigma Chemical Co. (St. Louis, MO). 4-Amino-4-(3-pyridyl)-1-butanol (35). Benzhydrylidene-(3pyridyl)methylamine (33, 1 g, 3.68 mmol) (23) was dissolved in anhydrous THF (50 mL) under N2 and cooled to -78 °C. Lithium diisopropylamide (5.5 mL, 11 mmol) was added dropwise with a syringe over a 10 min period. The reaction mixture turned deep purple as the temperature rose to -40 °C. A solution of 3-bromo-1-propanol (0.47 mL, 5.15 mmol) dissolved in THF (20 mL) was added via syringe over a period of 20 min. The reaction mixture was stirred at -40 °C for 7 h and then allowed to warm to room temperature. The pH of the mixture was adjusted to 6 with 10% HCl, and it was extracted with CHCl3 (4 × 25 mL). The organic solutions were combined, dried (Na2SO4), filtered, and concentrated. The resulting oil 34 was purified by flash column chromatography with elution by ethyl acetate. It was dissolved in ethanol containing hydroxylamine (0.5 M, 5 mL) and allowed to stir at room temperature overnight. The mixture was concentrated under reduced pressure and purified by preparative TLC with elution by CHCl3/MeOH/H2O (5:2:0.25). The lower band (Rf 0.14) was isolated to give 35 (550 mg, 2.3 mmol, 63% yield). 1H NMR (D2O): δ 8.39 (s, 1H, pyr-2H), 8.37 (d, J ) 7.8 Hz, 1H, pyr-6H), 7.80 (d, J ) 7.8 Hz, 1H, pyr-4H), 7.37 (dd, J ) 3.0, 7.8 Hz, 1H, pyr-5H), 4.28 (dd, J ) 6.0, 6.6 Hz, 1H, 4-H), 3.35 (dd, J ) 6.6, 6 Hz, 2H, 1-H), 1.96-1.84 (m, 2H, 3-H), 1.32 (m, 1H, 2-H), 1.17 (m, 1H, 2-H). MS (ESI-MS): m/z 167.1 (M + H). N2-[4-Hydroxy-1-(3-pyridyl)but-1-yl]dGuo (32). Compound 35 was dissolved in a mixture of dimethyl sulfoxide (DMSO; 1 mL) and triethylamine (0.33 mL). Compound 36 (37 mg, 0.1 mmol) was added, and the mixture was stirred at 60 °C for 72 h. The mixture was then cooled and concentrated to dryness under reduced pressure. The residue was purified by preparative TLC with elution by CHCl3/MeOH/H2O (5:2:0.25). The second lowest band (Rf 0.23) was isolated to give 32 (14 mg, 0.034 mmol, 23% yield). 1H NMR (DMSO-d6): δ 10.95 (bs, 1H, dGuo-N1-H), 8.58 (s, 0.5H, pyr-2H), 8.56 (s, 0.5H, pyr-2H), 8.42 (m, 1H, pyr-6H), 7.84 (s, 0.5H, dGuo-C8-H), 7.80 (s, 0.5H, dGuo-C8-H), 7.77 (m, 0.5H, pyr-4H), 7.74 (m, 0.5H, pyr-4H), 7.35 (m, 1H, pyr-5H), 6.11 (dd, J ) 7.2, 5.6 Hz, 0.5H, 1′-H), 6.04 (dd, J ) 7.2, 5.6 Hz, 0.5H, 1′-H), 5.28 (s, 0.5H, 3′-OH), 5.27 (s, 0.5H, 3′-OH), 4.94 (dd, J ) 7.2, 8.0 Hz, 0.5H, 1-H), 4.86 (m, 1.5H, 1-H and 5′-OH), 4.45 (bs, 1H, 1-OH), 4.31 (m, 1H, 3′-H), 3.79 (m, 1H, 4′-H), 3.58 (m, 1H, 5′-Ha), 3.48 (m, 1H, 5′-Hb), 3.37 (m, 2H, 4-H), 2.50 (m, 0.5H, 2′-H), 2.45 (m, 0.5H, 2′-H), 2.12 (m, 0.5H, 2′-H), 2.05 (m, 0.5H, 2′-H), 1.79 (m, 2H, 2-H), 1.48 (m, 1H, 3-H), 1.38 (m, 1H, 3-H). MS (ESI-MS): m/z 417.0 (M + H). Reactions. 1. NNKCH2OAc and dGuo. Compound 3 (66 mg, 0.25 mmol) was allowed to react with dGuo (0.05 mmol) in 5 mL of 0.1 M phosphate buffer, pH 7.0, in the presence of esterase (90 µL, 320 units) at 37 °C for 1 h. The reaction mixture was washed three times with 5 mL of CHCl3, and the aqueous phase was analyzed by HPLC. A portion (200 µL) of the aqueous phase was treated with NaBH4 (2 mg) and analyzed by HPLC. Retention times of adducts were as follows (min): 10, 90.2; 23, 78.0; 25, 79.6, 80.4; 26, 66.9; 27, 87.2; 28, 73.8, 74.2; 29, 68.8, 74.7, 75.4, 78.2; 30, 60.7; 32, 67.8, 69.5. The material eluting from 65 to 80 min in the NaBH4-treated portion was analyzed by LC-ESI-MS for 32. 2. NNKCH2OAc and DNA. Compound 3 (132 mg, 0.5 mmol) was allowed to react with calf thymus DNA (20 mg) in 10 mL



of 0.1 M phosphate buffer, pH 7.0, in the presence of esterase (110 µL, 400 units) at 37 °C for 1.5 h. The mixture was diluted with 10 mL of H2O and extracted twice with 20 mL of CHCl3/ isoamyl alcohol (24:1) and 20 mL of ethyl acetate. The DNA was precipitated by addition of ethanol and then washed with 70% aqueous ethanol and ethanol sequentially. For enzyme hydrolysis, DNA (2.5-5.0 mg) was dissolved in 1 mL of 10 mM Tris-HCl/5 mM MgCl2 buffer, pH 7.0. The mixture was incubated at 37 °C for 70 min with DNAse I, phosphodiesterase I, and alkaline phosphatase as described (24). For neutral thermal hydrolysis, a portion of the enzyme hydrolysate was heated at 100 °C for 1 h. Acid hydrolysis was carried out with 0.1 N HCl at 80 °C for 1 h. Hydrolysates were further purified by solid phase extraction on C18 Sep-pak cartridges (Waters) as described (25). The products were eluted with CH3OH and analyzed by LC-ESI-MS. Standard curves were constructed with 11 and adduct 26.

Results On the basis of previous work, carbocations 19-21 or closely related electrophiles would likely be involved in DNA alkylation by 3 (21). Gua is generally the most

reactive base in DNA toward electrophiles, and earlier work indicated that pyridyloxobutylation of DNA involved alkylation at Gua (26). Therefore, Gua was selected as the focus of this study. Several standards were available from previous work including 10, first synthesized by Peterson and co-workers (16), 23 (20), 25 (22), and 26 (18). The corresponding pyridylhydroxybutyl adducts 27-30 and the Gua bases 22 and 24 produced by mild acid hydrolysis of 10 and 23 were also available (18). We hypothesized that N2-pyridyltetrahydrofuranyldGuo (31a) would be a major HPB-releasing adduct in pyridyloxobutylated DNA since alkylation of N2 or O6 of dGuo is favored with SN1 type reactants such as 21 (27). On the basis of our work with adducts analogous to 31a but lacking the pyridine ring, we expected that 31a might be unstable at the nucleoside level and would release HPB via the acyclic tautomer, Schiff base 31b (28, 29). However, 31b could be stabilized by treatment of the DNA with NaBH4, which would produce 32 (29). Therefore, we synthesized 32 as shown in Scheme 3. The key intermediate 35 was prepared using methods described by Crooks (23, 30). This was coupled to 36, giving adduct 32 as a mixture of diastereomers. HPLC chromatograms of standard adducts 10, 23, 25, and 26 as well as adducts that would result from NaBH4 treatment (27-30 and 32) are shown in Figure 1. We then proceeded to investigate the reaction of NNKCH2OAc with dGuo, both before and after NaBH4 treatment. A chromatogram obtained upon HPLC analysis of the reaction mixture of NNKCH2OAc and dGuo is illustrated in Figure 2. The complexity of this chromatogram resulted in part from the presence of UV-absorbing sol-

volysis products of NNKCH2OAc. Nevertheless, we were able to isolate peaks corresponding in retention time to 23, 25, and 10. Adduct 25 was previously identified as a product of the reaction of a pyridyloxobutylating agent with dGuo (22). Consistent with those results, its UV and MS were the same as those of standard 25. The UV spectra and MS of the peaks marked 23 and 10 were also essentially the same as those of standard adducts 23 and 10 (Figures 3A-D and 4A-D). Treatment of 23 and 10 with NaBH4 shifted their retention times to those of 28 and 27, respectively. The UV spectra and MS of these products also matched those of the standards (data not shown). These results demonstrate that 10, 23, and 25 are products of the reaction of NNKCH2OAc with dGuo.

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Scheme 3. Synthesis of 32

Figure 2. Chromatogram obtained upon HPLC analysis of the reaction mixture of NNKCH2OAc and dGuo. The peaks marked 23, 25, and 10 are adducts 23, 25, and 10, respectively.

selected ion monitoring at m/z 417, which is M + H of 32. Two peaks corresponding in retention time to diastereomers of 32 (confirmed by coinjection) were observed as illustrated in Figure 6. The MS of these peaks was weak, although ions at m/z 417 and m/z 301, the major ions in the spectra of diastereomers of 32, were detected. LC-ESI-MS also confirmed the presence of adducts 28 and 29 in this fraction (Figure 6). These results indicate that a relatively small amount of 31a or b is produced in the reaction of NNKCH2OAc and dGuo. These results did not support our hypothesis that adduct 31a was a major HPB-releasing adduct. We then used LC-ESI-MS with selected ion monitoring at m/z 415, the molecular ion of 7-pyridyloxobutyl-dGuo (37), to further investigate this reaction. Adduct 37 would be another likely candidate as a major HPB-releasing adduct. Selected ion monitoring at m/z 415 would also detect M + H of 10, 23, and 25. The results of this analysis are illustrated in Figure 7. The peaks marked 10, 23, and 25 corresponded to adducts 10, 23, and 25, respectively, which have already been identified by the data described above. The peak marked 37 was identified as adduct 37 by the following data. Its ESI-MS showed

Figure 1. Chromatograms obtained upon HPLC analysis of (A) standard adducts 10, 23, 25, and 26 and (B) adducts 27-30 and 32, which would be formed after NaBH4 reduction.

We then investigated the presence of 31a. The reaction mixture was treated with NaBH4, concentrated, and analyzed by HPLC with UV detection. This produced the chromatogram illustrated in Figure 5. The peaks marked 32, 28, 29, and 27 correspond in retention time to standard adducts 32, 28, 29, and 27, respectively. Adducts 32 and 28 appear as pairs of diastereomers while four diastereomers of adduct 29 are evident. Fractions 1-3 were collected and analyzed by LC-ESI-MS, with

a pseudo molecular ion at m/z 415, a base peak at m/z 299 (M - deoxyribose + H)+, and a peak at m/z 148 corresponding to [3-Pyr(CO)(CH2)3]+. MS/MS of m/z 415 produced a daughter ion at m/z 299 and MS/MS of m/z 299 produced daughter ions at m/z 152 (Gua + H)+ and m/z 148. Therefore, peak 37 clearly was a pyridyloxobutyl-dGuo. Neutral thermal hydrolysis of the NNKCH2OAc-dGuo reaction mixture caused the disappearance of peak 37, with an increase in the amount of HPB, as monitored by LC-ESI-MS with selected ion monitoring. The peaks corresponding to 10, 23, and 25 were unaf-



Figure 3. UV spectra of (A) reference 23, (B) 10, and (C and D) products isolated from the reaction of NNKCH2OAc with dGuo.

fected by neutral thermal hydrolysis. The same results were obtained when peak 37 was collected and subjected to neutral thermal hydrolysis: it disappeared with the formation of 11, as determined by matching the retention time and MS of the product to that of standard 11. This reaction was also analyzed by LC-ESI-MS with selected ion monitoring at m/z 299, which is M + H of 26. The results demonstrated the appearance of a peak with identical retention time (by coinjection) as 26. The MS and MS/MS of this peak were also the same as those of 26. Moreover, treatment with NaBH4 converted this material to 30, established by matching HPLC retention times and MS. These results conclusively demonstrate that the peak marked 37 in Figure 7 is 37 and that upon neutral thermal hydrolysis this adduct is converted to 26 and 11 (Scheme 4). The ratio of 26 to 11 formed upon neutral thermal hydrolysis of isolated 37 was approximately 1:1. The half-life of 37 at 37 °C was 3 h. Chromatograms obtained upon LC-ESI-MS analysis of an enzymatic hydrolysate of DNA that had been allowed to react with NNKCH2OAc are illustrated in Figure 8AF. The left panels A, C, and E show the results of selected ion monitoring of m/z 415, 166, and 299, respectively, before neutral thermal hydrolysis, while the right panels B, D, and F show the results after neutral thermal hydrolysis. In Figure 8A, the peaks marked 37, 23, and 10 correspond in retention time to adducts 37, 23, and 10, respectively. The MS of peak 37 matched that of adduct 37 identified in the NNKCH2OAc-dGuo reaction. The structures of adducts 23 and 10 were confirmed by comparison of their MS to those of standards, by NaBH4 reduction of the hydrolysate and comparison to adducts 28 and 27, respectively, and by mild acid hydrolysis and comparison to 24 and 22, respectively. In all cases,

Figure 4. ESI-MS of (A) reference 23, (B) 10, and (C and D) products isolated from the reaction of NNKCH2OAc with dGuo.

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Figure 7. LC-ESI-MS analysis with selected ion monitoring at m/z 415 of the reaction mixture of NNKCH2OAc and dGuo. The peaks marked 37, 23, 25, and 10 were identified as 37, 23, 25, and 10, respectively, as described in the text.

Figure 5. Chromatogram obtained upon HPLC analysis of the reaction mixture of NNKCH2OAc and dGuo, which was then treated with NaBH4. The peaks marked 27, 28, 29, and 32 correspond to adducts 27, 28, 29, and 32, respectively. Fractions F-1-F-3 were collected as indicated for LC-ESI-MS analysis.

Figure 6. LC-ESI-MS analysis with selected ion monitoring at m/z 417 of HPLC fraction 1 of Figure 5 from the reaction mixture of NNKCH2OAc and dGuo that was subsequently treated with NaBH4. The indicated peaks had retention times the same as those of standard adducts 28, 29, and 32. One isomer of adduct 29 coeluted with the first diastereomer of adduct 32 (not shown by arrow).

retention times and MS data matched. Minor amounts of adduct 25 were also detected in this DNA by LC-ESIMS with selected ion monitoring (data not shown). Figure 8C shows a peak corresponding to 11, which has M + H of m/z 166. The retention time of this peak was slightly longer than that of peak 37 in Figure 8A. HPB likely was formed by partial decomposition of unstable adducts during the enzymatic hydrolysis, as observed previously. Figure 8E shows a peak corresponding in retention time to adduct 37. This is due to the m/z 299 fragment in the MS of 37. Similarly, the peaks marked 23 and 10 are due to the m/z 299 fragments in the MS of adducts 23 and 10, respectively. The retention time of the peak marked 26 is the same as that of adduct 26, while the peak marked × is an unknown. MS data for peak × indicates that it is a pyridyloxobutyl-Gua derivative. In Figure 8B, it is evident that neutral thermal hydrolysis caused complete disappearance of 37, while having no effect on 23 and 10. In tandem, the area under the 11 peak increased 10-fold (Figure 8D). The peak in Figure 8E corresponding to 37 also disappeared upon

neutral thermal hydrolysis (see Figure 8F), while the area under the peak corresponding to 26 increased 4-fold. The retention time and MS of peak 26 were identical to those of standard 26. NaBH4 reduction of each of the compounds represented by peaks 26 and × produced new peaks with retention times different from one another. The product of reduction of 26 matched the retention time of 30. Similar results were obtained when enzyme hydrolysates of DNA that had been allowed to react with NNKCH2OAc were subjected to acid hydrolysis conditions. Peak 37 disappeared with increases in HPB and in the peak corresponding to 26. Under these conditions, adducts 10 and 23 were converted to the corresponding bases 22 and 24. These results are summarized in Scheme 4. Hydrolysis of 37 occurs both by attack of H2O on carbon 1 of the pyridyloxobutyl group and on the 1′-carbon of the deoxyribose ring. The former mode of hydrolysis produces 11 and dGuo (detected by LC-ESI-MS) while the latter gives adduct 26 and deoxyribose or its decomposition products. Treatment of the DNA with NaBH4, followed by enzyme hydrolysis and analysis by LC-ESI-MS, provided no evidence for the presence of adduct 32. Relative amounts of the various products in the DNA reactions were compared based on areas under the peaks in the LC-ESI-MS chromatograms. In enzyme hydrolysates of DNA, the ratio of 37 to 10 was 2.8:1 and the ratio of 37 to 23 was 14:1. Adduct 37 comprised 30-35% of the thermally unstable HPB-releasing adducts in pyridyloxobutylated DNA.

Discussion The results of this study clearly demonstrate that 37 is a thermally unstable HPB-releasing adduct in DNA reacted with the pyridyloxobutylating agent NNKCH2OAc. These results represent the first characterization of a thermally unstable HPB-releasing adduct, the presence of which has been inferred from indirect evidence for 15 years. These findings represent a significant advance in our understanding of DNA pyridyloxobutylation by metabolically activated forms of the important carcinogenic tobacco specific nitrosamines NNK and NNN. The presence of HPB-releasing DNA adducts was first reported in 1988 (19). DNA isolated from the livers of rats treated with a single dose of NNK or NNN released HPB upon strong acid or neutral thermal hydrolysis. We



Figure 8. Chromatograms obtained upon LC-ESI-MS analysis of enzymatic hydrolysates of DNA that had been allowed to react with NNKCH2OAc. Selected ion monitoring was carried out at m/z 415 (A, B), m/z 166 (C, D), and m/z 299 (E, F) before (A, C, E) or after (B, D, F) neutral thermal hydrolysis. The numbered peaks were identified as 37, 23, 10, 26, and 11, respectively, by the data described in the text. The peak marked × has not been identified.

Scheme 4. Hydrolysis of Adduct 37 to 11 and 26

speculated that an HPB-releasing adduct might be a 7-pyridyloxobutylated dGuo, but no conclusive evidence could be obtained until the present study. HPB-releasing DNA adducts were also detected upon strong acid hydrolysis of liver DNA from rats treated with NNAL (31), presumably through reconversion of NNAL to NNK followed by R-hydroxylation, and in enzymatic hydrolysates of DNA isolated from livers of NNK-treated rats or from rat nasal mucosa cultured with NNK or NNN (20). These data demonstrated the presence of substantial amounts of thermally unstable HPB-releasing adducts in pyridyloxobutylated DNA in vivo. In subsequent studies discussed here, strong acid hydrolysis was used. Therefore, the HPB-releasing adducts include both quantitatively major thermally unstable adducts and 10. Treatment of mice with NNK, NNN, or NNKCH2OAc produced HPB-releasing adducts in pulmonary and/or hepatic DNA (13, 32). These adducts were not observed in mice treated with HPB, nor did HPB itself bind to DNA (19, 32). These results conclusively demonstrated that the HPB-releasing adducts resulted from pyridyloxobutylation of DNA, not Schiff base forma-

tion from HPB. A dose-response study in rats demonstrated that HPB-releasing adducts were higher in lung than in liver DNA at low doses, but the reverse was observed at higher doses (33). Higher levels of HPBreleasing adducts in pulmonary rather than in hepatic DNA were also observed in a recent study in which NNK was administered to rats in the drinking water under conditions the same as those used for lung tumor induction (34). These results are consistent with the lung tumorigenicity of low doses of NNK in the rat. The in vivo stability of HPB-releasing adducts was examined in rats treated with NNK (35). In lung and liver, HPBreleasing adducts disappeared in a multiphasic manner with initial half-lives of 38 and 50 h, respectively. They were still detectable 4 weeks after a single NNK treatment. HPB-releasing adducts were detected in the nasal mucosa of rats treated with NNK or NNN (36). Comparisons of adduct levels among deuterated NNK derivatives strongly support an important role for DNA pyridyloxobutylation in rat nasal carcinogenesis by NNK and NNN. HPB-releasing adducts were also found in rat esophageal DNA cultured with NNN but not with NNK,

624


consistent with their carcinogenic activities toward this tissue (37). A correlation was observed between levels of HPB-releasing adducts in type II cells of lung and lung tumor incidence in rats treated with various doses of NNK (38). Furthermore, inhibition of HPB-releasing adduct formation by PEITC in type II cells of rat lung correlated with inhibition of NNK induced pulmonary tumorigenicity by PEITC (38). Collectively, these results provide convincing evidence that HPB-releasing DNA adducts are crucial in carcinogenesis by NNK and NNN. Pyridyloxobutylated DNA has been quantified in human lung by strong acid hydrolysis followed by extraction and GC-MS analysis (39). Using this methodology, we showed that levels of HPB-releasing DNA adducts in human lung were higher in smokers than in nonsmokers (39). HPB-releasing DNA adducts were not detected in a subsequent study using smaller DNA samples (40). Recently, however, a relatively large study clearly confirmed the presence of HPB-releasing DNA adducts in human lung and found significantly higher levels in lung cancer patients than in controls (15). In these studies as well as in the animal studies described above, adduct 37 would likely comprise a substantial portion of the HPBreleasing DNA adducts formed in vivo. 4-(Carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone and NNKCH2OAc are mutagenic in bacterial and mammalian cells (41-43). Lung tumors in A/J mice treated with NNKCH2OAc contained equal proportions of G f A and G f T mutations in codon 12 (44). These results demonstrate the mutagenicity of pyridyloxobutylated DNA. Site specific mutagenesis studies showed that 10 is mutagenic in both bacterial and mammalian cells, producing mainly G f A mutations (17). The G f T mutations observed in mice treated with NNKCH2OAc could be caused by adduct 37. Depurination of 37 with formation of 26 would lead to an abasic site, and such sites are known to cause G f T mutations (45). Analysis of NNKCH2OAc solvolysis products indicated that there was considerable involvement of electrophiles related to carbocations 19-21 (21). Therefore, adducts resulting from reactions with these electrophiles should be formed, consistent with our results. We expected to find greater amounts of adduct 31a than observed based on the generalizations of Moschel and co-workers, who proposed that soft electrophiles, which react by an SN1 mechanism, preferentially alkylate N2- of dGuo (27). We cannot exclude the possibility that there are adducts resulting from reaction of 21 at other positions of dGuo or with other DNA bases. However, it does not appear that the unknown adduct × (Figure 8E) is derived from a cyclic dGuo adduct because it readily undergoes NaBH4 reduction to a pyridylhydroxybutyl-Gua. LC-ESI-MS was critical for the identification of 37. HPLC with UV detection does not distinguish between products of solvolysis of NNKCH2OAc, which are UVabsorbing due to the pyridine ring, and adducts formed by reaction of NNKCH2OAc with DNA bases. As shown in Figure 2, complex chromatograms were obtained because even minor solvolysis products are likely formed in higher yields than most adducts. The selectivity of LCESI-MS with selected ion monitoring produced clean chromatograms as shown in Figures 6-8. This allowed us to distinguish adduct 37 from HPB (Figure 7A,C), despite their very similar retention times in our HPLC system. The similar HPLC elution properties of 37 and

Wang et al. Scheme 5. Proposed Mechanism of Formation of 11 upon Hydrolysis of Adduct 37

HPB might explain some of the difficulties in observing 37 in previous studies, even when radiolabeled NNK was used. As shown in Scheme 4, neutral thermal hydrolysis of adduct 37 produced both 11 and depurinated adduct 26. The formation of HPB in this reaction is different from results reported for simple 7-alkyl-dGuo derivatives, which give mainly the corresponding depurinated adduct (e.g., 12 from 7-methyl-dGuo) upon neutral thermal hydrolysis (46, 47). Our results can be readily explained as shown in Scheme 5. Anchimeric assistance by the carbonyl oxygen of adduct 37 results in cleavage of the pyridyloxobutyl-dGuo bond with formation of intermediate 21. Reaction of 21 with H2O produces the cyclic tautomer 38, which is unstable at pH 7.0 with respect to its open chain form, HPB (21). Similar results have been observed in studies of the major DNA adduct of aflatoxin B1 epoxide (48). Hydrolysis of this 7-substituted dGuo adduct gives both the corresponding dihydrodiol of aflatoxin B1 and the aflatoxin B1-Gua adduct. The latter is excreted in urine and has been used as a biomarker of aflatoxin B1 uptake and metabolic activation (49). A similar strategy can be envisioned for adduct 26. In summary, our results demonstrate that pyridyloxobutylated DNA contains 37, which comprises 30-35% of the previously unidentified thermally unstable HPBreleasing adducts known to be present in this DNA. At least one other thermally unstable HPB-releasing adduct, the precursor to peak ×, is also present in this DNA. On the basis of MS data, this material is a pyridyloxobutyldGuo. We have also observed pyridyloxobutylation of other DNA bases in this study, and their identity is a subject of ongoing investigations in our laboratory. Our data confirm Peterson’s observation that adduct 10 is present in pyridyloxobutylated DNA (16) and demonstrate the presence of N2-pyridyloxobutyl adducts as well. A recent paper also provides evidence for pyridyloxobutyl phosphate adducts in hepatic DNA isolated from NNKtreated mice (50). Collectively, the results of these ongoing studies will be critical in further assessing the mutagenic and carcinogenic consequences of DNA pyridyloxobutylation by NNK and NNN in laboratory animals and humans. We are currently analyzing DNA from NNK-treated rats for adduct 37.


Acknowledgment. This study was supported by Grant CA-81301 from the National Cancer Institute. S.S.H. is an American Cancer Society Research Professor, supported by ACS Grant RP-00-138.


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TX034003B

Identification of Adducts Formed by Pyridyloxobutylation of

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