Formation of Formaldehyde Adducts in the Reactions of DNA and

Jan 19, 2008 - Guang Cheng, Mingyao Wang, Pramod Upadhyaya, Peter W. Villalta .... Kun Lu , Wenjie Ye , Li Zhou , Leonard B. Collins , Xian Chen , Avr...
0 downloads 0 Views 336KB Size
Download PDF
746

Chem. Res. Toxicol. 2008, 21, 746–751

Formation of Formaldehyde Adducts in the Reactions of DNA and Deoxyribonucleosides with r-Acetates of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N-Nitrosodimethylamine (NDMA) Guang Cheng, Mingyao Wang, Pramod Upadhyaya, Peter W. Villalta, and Stephen S. Hecht* UniVersity of Minnesota Cancer Center, MMC 806, 420 Delaware Street Southeast, Minneapolis, Minnesota 55455 ReceiVed October 24, 2007

The cytochrome P450-mediated R-hydroxylation of the carcinogenic nitrosamines N-nitrosodimethylamine (NDMA, 1), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 6a), and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanol (NNAL, 6b) produces diazonium ions and formaldehyde. The DNA-binding properties of the diazonium ions have been thoroughly characterized, and there is no doubt that they are critical in cancer induction by these nitrosamines. However, the possibility of additional DNA damage via released formaldehyde has not been reported. In this study, we used acetoxymethylmethylnitrosamine (5), 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a), and 4-(acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanol (10b) as stable precursors to the R-hydroxymethylnitrosamines that would be formed in the metabolism of NDMA, NNK, and NNAL. These R-acetates were incubated with calf thymus DNA in the presence of esterase at pH 7.0 and 37 °C. The DNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides, and the hydrolysates were analyzed by liquid chromatographyelectrospray ionization-mass spectrometry-selected ion monitoring for formaldehyde DNA adducts. Convincing evidence for the formation of the formaldehyde adducts N6-hydroxymethyl-dAdo (11), N4hydroxymethyl-dCyd (12), N2-hydroxymethyl-dGuo (13), and the cross-links di-(N6-deoxyadenosyl)methane (14), (N6-deoxyadenosyl-N2-deoxyguanosyl)methane (15), and di-(N2-deoxyguanosyl)methane (16) was obtained in these reactions. These results demonstrate that NDMA, NNK, and NNAL have the potential to be bident carcinogens, damaging DNA through the metabolic formation of both diazonium ions and formaldehyde. Introduction Just over 50 years ago, Magee and Barnes published their classic paper describing the hepatocarcinogenicity in rats of a simple water-soluble compound containing only 11 atomssNnitrosodimethylamine (NDMA, 1, Scheme 1) (1). This remarkable finding spurred research on the carcinogenicity of N-nitrosamines, which established these compounds as arguably the most diverse and potent class of chemical carcinogens. Over 200 N-nitrosamines are carcinogenic in laboratory animals, frequently after administration of very low doses, and at least 30 different species are responsive (2, 3). Many N-nitrosamines are standard model compounds for the induction of tumors at different sites such as the esophagus, lung, pancreas, and bladderswithout the need for embryonic stem cell technology. Two members of this classsthe tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 6a, Scheme 1) and N′-nitrosonornicotine (NNN)sare carcinogenic to humans, according to the International Agency for Research on Cancer (IARC) (4). N-Nitrosamines require metabolic activation to form DNA adducts that are critical for their mutagenic and carcinogenic activity (2). The well-established major pathway is R-hydroxy* To whom correspondence should be addressed. Tel: 612-626-7604. Fax: 612-626-5135. E-mail: [email protected].

lation (adjacent to the N-nitroso group), catalyzed by cytochrome P450 enzymes. Examples are illustrated in Scheme 1 for NDMA (1), NNK (6a), and the NNK metabolite 4-(methylnitrosamino)1-(3-pyridyl)-1-butanol (NNAL, 6b). Intermediates 2 and 7a,b are initially produced, and these spontaneously release reactive diazohydroxides 3 and 8a,b. These diazohydroxides or the corresponding diazonium ions react with DNA, producing adducts such as O6-methyl-dGuo (4) from NDMA, O6-pyridyloxobutyl-dGuo (9a) from NNK, and O6-pyridylhydroxybutyldGuo (9b) from NNAL, along with numerous other adducts. (NNK and NNAL are also metabolically activated by methylene hydroxylation, which is not shown here.) The roles in carcinogenesis of methyl, pyridyloxobutyl-, and pyridylhydroxybutylDNA adducts of NDMA, NNK, NNAL, and other N-nitroso compounds have been extensively studied (2, 5–13). However, there is barely any information in the literature on DNA modification in these reactions by the other product of methyl hydroxylation of N-nitrosomethyl compoundssformaldehyde. Several products have been previously characterized in reactions of formaldehyde with DNA, including hydroxymethyl adducts and cross-links (14–18). Formaldehyde is mutagenic in a variety of different test systems and carcinogenic in laboratory animals (19) and has been described as “carcinogenic to humans” by IARC and “reasonably anticipated to be a human carcinogen” by the U.S. Department of Health and Human

10.1021/tx7003823 CCC: $40.75  2008 American Chemical Society Published on Web 01/19/2008

Formaldehyde-DNA Adducts

Chem. Res. Toxicol., Vol. 21, No. 3, 2008 747

Scheme 1. Generation of Formaldehyde upon Methyl Hydroxylation of NDMA and NNK

Table 1. Formaldehyde Adducts Formed in the Reactions of Formaldehyde or 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a) with Deoxyribonucleosidesa formaldehyde reactant

product

N6-HOCH2-dAdo (11) dAdo-CH2-dAdo (14) dCyd N4-HOCH2-dCyd (12) dGuo N2-HOCH2-dGuo (13) dGuo-CH2-dGuo (16) dAdo + dGuo dAdo-CH2-dGuo (15) dAdo

10a

retention time (min) UV (λmax) [M + H]+ [BH]+ retention time (min) UV (λmax) [M + H]+ [BH]+ 51.3 88.2 19.6 39.8 67.3 69.1

265 272 275 256 257 268

282 515 258 298 547 531

166 399 142 182 431 415

51.2 88.8 21.6 40.4 68.7 NDb

264 271 274 256 257 ND

282 515 258 298 547 ND

166 399 142 182 431 ND

a Formaldehyde or 10a was allowed to react with deoxyribonucleosides as described in the Experimental Procedures, and reaction mixtures were analyzed by HPLC with UV detection and by LC-ESI-SIM. b ND, not done.

Services (19, 20). It is plausible that formaldehyde-DNA adducts could play a role in carcinogenesis by N-nitrosomethyl compounds. Therefore, in this study, we investigated the formation of such adducts in reactions of the R-acetates 5 and 10a,b (Scheme 1) with DNA and deoxyribonucleosides. Compounds 5 and 10a,b are stable precursors to the unstable R-hydroxynitrosamines 2 and 7a,b, formed upon R-hydroxylation of NDMA, NNK, and NNAL.

Experimental Procedures Apparatus and Assay Conditions. LC-ESI-MS analysis of the DNA hydrolysates 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 (Waters Corp., Milford, MA) and equipped with an SPD10 A UV detector (Shimadzu Scientific Instruments, Columbia, MD). UV detection was at 254 nm. A 4.6 mm × 25 cm Supelcosil LC-18-DB column (Supelco, Bellefonte, PA) was eluted isocratically with 5% CH3OH in 40 mM ammonium acetate buffer, pH 6.6, for 10 min and then a gradient from 5 to 35% CH3OH in 60 min at a flow rate of 0.3 mL/min. The ESI source was set as follows: voltage, 2.0 kV; current, 10 µA; and capillary temperature, 250 °C. The same HPLC conditions were used for analysis of reactions of formaldehyde, 5, and 10a with deoxyribonucleosides, except that the flow rate was 0.5 mL/min. UV spectra were obtained with a Waters model 996 photodiode array detector. Chemicals and Enzymes. Acetoxymethylmethylnitrosamine (5) was obtained from the National Cancer Institute Chemical Carcinogen Reference Standards Repository. 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (10b) were synthesized (21). Formaldehyde, 37% by weight in H2O containing 10–15% CH3OH, was obtained from Sigma-Aldrich Chemical Co. (Milwaukee, WI). All other chemicals and enzymes were also purchased from SigmaAldrich. Solvents were acquired from Fisher Scientific (Fairlawn, NJ).

Reactions. Formaldehyde and Deoxyribonucleosides. Mixtures of formaldehyde (3.4 mg, 0.1 mmol) and dGuo (5.7 mg, 0.02 mmol), dGuo (5.7 mg) plus dCyd (4.8 mg, 0.02 mmol), dGuo (5.7 mg) plus dAdo (5.3 mg, 0.02 mmol), dAdo (5.3 mg, 0.02 mmol), or dAdo (5.3 mg, 0.02 mmol) plus dCyd (4.8 mg, 0.02 mmol) were allowed to react in 2 mL of phosphate buffer, pH 7.0, at 37 °C for 90 h. The resulting mixtures were washed three times with 2 mL of CHCl3 and analyzed by HPLC-UV or LC-ESI-MS as described for the DNA reactions. Compound 10a and Deoxyribonuceosides. A mixture of 10a (30 mg, 0.1 mmol), hog liver esterase (130 units), and dGuo (5.4 mg, 0.02 mmol) or dAdo (5.0 mg, 0.02 mmol) or dCyd (4.5 mg, 0.02 mmol) was allowed to react in 2 mL of phosphate buffer, pH 7.0, at 37 °C for 1 h and worked up and analyzed as described above. Reaction of 5 with dGuo was similarly performed. Compound 5 and DNA. A mixture of 5 (41 mg, 0.31 mmol) and calf thymus DNA (20 mg) was incubated in 10 mL of pH 7.0 phosphate buffer at 37 °C for 1 h in the presence of hog liver esterase (400 units). The mixture was diluted with 5 mL of H2O and extracted twice with 15 mL of CHCl3/isoamyl alcohol (24:1). The DNA was precipitated by addition of 1 mL of 5 M NaCl and 20 mL of ethanol and then washed four times with 70% aqueous ethanol and ethanol. Compounds 10a and 10b and DNA. A mixture of 10a or 10b (170 mg, 0.64 mmol) and calf thymus DNA (20 mg) was incubated in 4 mL of pH 7.0 phosphate buffer at 37 °C for 1.5 h in the presence of hog liver esterase (500 units). The incubation mixture was extracted, and the DNA was precipitated and washed as above, except that ethyl acetate was also used for extraction. DNA Adduct Analysis. The procedure for enzyme hydrolysis and solid-phase extraction (SPE) was previously described (18). In brief, the modified 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. Hydrolysates were further purified by SPE on C-18 Sep-Pak cartridges (Waters). The cartridge was conditioned with CH3OH, H2O, and Tris-HCl/MgCl2 buffer. The

748

Chem. Res. Toxicol., Vol. 21, No. 3, 2008

Guang et al. Table 2. Formaldehyde-DNA Adducts Formed in the Reaction of Acetoxymethylmethylnitrosamine (5) with DNAa adduct N6-HOCH2-dAdo (11) N4-HOCH2-dCyd (12) N2-HOCH2-dGuo (13) dAdo-CH2-dAdo (14) dAdo-CH2-dGuo (15) dGuo-CH2-dGuo (16)

retention relative time (min) [M + H]+ [BH]+ adduct levelsb 45.2 28.0 40.0 87.1 70.5 59.7

282 258 298 515 531 547

166 142 182 399 415 431

40 2 1 80 2 1

a Compound 5 was allowed to react with DNA as described in the Experimental Procedures, and reaction mixtures were analyzed by LC-ESI-MS-SIM. b Adducts 11-13 were compared to each other, and adducts 14-16 were compared to each other.

Figure 1. Representative chromatograms obtained upon LC-ESI-MSSIM analysis of the reaction of acetoxymethylmethylnitrosamine (5) with dGuo. SIM at (A) m/z 282, [M + H]+ of 7-Me-dGuo and O6Me-dGuo; (B) m/z 298, [M + H]+ of N2-HOCH2-dGuo (13); and (C) m/z 547 [M + H]+ of dGuo-CH2-dGuo (16), was used.

hydrolysate was loaded onto a Sep-Pak and washed with the above buffer and 20, 30, and 100% CH3OH. The three CH3OH-containing fractions were combined and analyzed for DNA adducts of 5; only the 100% CH3OH fraction from the reactions with 10a,b was analyzed. The solvents were removed, and the residue was redissolved in H2O and analyzed by LC-ESI-MS.

Results Previous studies demonstrated that at least six adducts, the hydroxymethyl compounds N6-HOCH2-dAdo (11), N4-HOCH2dCyd (12), N2-HOCH2-dGuo (13) and the cross-links dAdoCH2-dAdo (14), dAdo-CH2-dGuo (15), and dGuo-CH2-dGuo (16), are formed in the reactions of formaldehyde with DNA (14, 15). We prepared these standards by reacting formaldehyde with deoxyribonucleosides. Their UV spectra, summarized in Table 1, were the same as reported in the literature, and their ESI-MS properties were consistent with their structures, showing [M + H]+ ions, which were also the base peaks, and characteristic [(M + H) – dR + H]+ ions, referred to here as [BH]+ ions. Reactions of R-acetate 10a were then carried out with dAdo, dCyd, and dGuo. As shown in Table 1, all of the expected products were formed, as confirmed by comparison of their UV and ESI-MS properties to those of the standards. Reactions of R-acetate 5 with dGuo also produced formaldehyde adducts in addition to the well-established adducts 7-Me-dGuo and O6-Me-dGuo, as shown by LC-ESI-SIM analysis (Figure 1A-C). These results established the principle that formaldehyde adducts could be formed in the reaction of R-acetoxynitrosamines with deoxyribonucleosides. We then investigated reactions with DNA. Table 2 summarizes the results of LC-ESI-MS-SIM analysis of the reaction of R-acetate 5 with DNA. Following enzymatic hydrolysis of the DNA, clear peaks were observed for adducts 11-16 at retention times corresponding to standards, and in each case, the appropriate [M + H]+ and [BH]+ ions were observed. Representative LC-ESI-MS-SIM chromatograms showing the major adductssN6-HOCH2-dAdo and dAdo-CH2-dAdos

formed in this reaction are illustrated in Figure 2A,B. In Figure 2A, peaks were also observed for 7-methyl-dGuo and O6methyl-dGuo, which have the same molecular weight as N6HOCH2-dAdo. We did not attempt to quantify levels of DNA adducts in this study. The relative ratios of the three hydroxymethyl adducts and the three cross-links were estimated based on relative peak areas. As shown in Table 2, the dAdo adducts N6-HOCH2-dAdo and dAdo-CH2-dAdo were the major products observed. In the reactions of the R-acetates 10a,b with DNA, we mainly analyzed the cross-link adducts, since these eluted in the same SPE fraction (100% methanol) as the pyridyloxobutyl- and pyridylhydroxybutyl-DNA adducts formed from these compounds, which were our major focus when these experiments were carried out. As in the case of R-acetate 5, clear LC-ESIMS-SIM evidence was obtained for the presence of adducts. This is illustrated in Figure 3A-C and Figure 4A-C for the reactions of 10a and 10b, respectively, with DNA. Peaks corresponding to N2-HOCH2-dGuo and N6-HOCH2-dAdo were also observed, and the relative ratios of these as well as the cross-link adducts were similar to those shown in Table 2. In some experiments, the reaction mixtures were treated with NaBH4, and in these cases, clear evidence was obtained for the presence of N6-CH3-dAdo, which would be formed by reduction of N6-HOCH2-dAdo (11) (22).

Discussion Our goal in this study was to provide definitive qualitative evidence for the formation of formaldehyde-DNA adducts from R-hydroxymethylnitrosamines, the acknowledged proximate carcinogens of NDMA and related compounds. Using an approach pioneered by Keefer (23), we used the stable R-acetates 5 and 10a,b as precursors to the R-hydroxynitrosamines, which were generated in situ in the presence of DNA. The LC-ESI-MS-SIM data presented here leave no doubt about the identity of the products. For reasons detailed in another paper (22), quantitation of these adducts is not straightforward, and we have restricted our efforts in that respect to in vivo studies. Thus, it is possible that artifact formation could have contributed to the amount of adduct 14 observed here. The results of this study unite two lines of investigation, which originated in the middle of the last century: the metabolism of NDMA and other carcinogenic nitrosamines and the modification of nucleic acids by formaldehyde. Dutton and Heath, in 1956, were the first to demonstrate the metabolism of NDMA to CO2 in rats and mice. They wrote, “These results indicate that dimethylnitrosamine is demethylated, presumably via formaldehyde” (24). This was echoed by Magee and Vandekar in 1958 who observed that “some formaldehyde is produced on incubation of the compound (NDMA) in vitro with

Formaldehyde-DNA Adducts

Chem. Res. Toxicol., Vol. 21, No. 3, 2008 749

Figure 2. Representative chromatograms obtained upon LC-ESI-MS-SIM analysis of an enzymatic hydrolysate of DNA that had been allowed to react with acetoxymethylmethylnitrosamine (5). SIM at (A) m/z 282, [M + H]+ of N6-HOCH2-dAdo (11), and (B) m/z 515, [M + H]+ of dAdo-CH2-dAdo (14), was used.

Figure 3. Representative chromatograms obtained upon LC-ESI-MS-SIM analysis of an enzymatic hydrolysate of DNA that had been allowed to react with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a). SIM at (A) m/z 515, [M + H]+ of dAdo-CH2-dAdo (14); (B) m/z 531, [M + H]+ of dAdo-CH2-dGuo (15); and (C) m/z 547, [M + H]+ of dGuo-CH2-dGuo (16), was used.

rat-liver slices (unpublished results)” (25). Brouwers and Emmelot were apparently the first, in 1960, to actually

demonstrate the production of formaldehyde from NDMA by rat liver microsomes (26). They observed that “NDMA appears

750

Chem. Res. Toxicol., Vol. 21, No. 3, 2008

Guang et al.

Figure 4. Representative chromatograms obtained upon LC-ESI-MS-SIM analysis of an enzymatic hydrolysate of DNA that had been allowed to react with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (10b). SIM at (A) m/z 515, [M + H]+ of dAdo-CH2-dAdo (14); (B) m/z 531, [M + H]+ of dAdo-CH2-dGuo (15); and (C) m/z 547, [M + H]+ of dGuo-CH2-dGuo (16), was used.

to be the first water-soluble compound which is N-demethylated by the microsomal enzyme.” The enzyme-catalyzed formation of formaldehyde from NDMA was confirmed in many studies, and ultimately, measurement of formaldehyde became a standard technique for assessing NDMA metabolism in vitro, and P450 2E1 was identified as a major catalyst (2, 27, 28). The generation of formaldehyde in the metabolism of NNK was likewise observed (5). Reactions of formaldehyde with nucleic acids were also reported in the early 1950s [reviewed by Feldman (29)], but there were no structural identifications of adducts using modern methods until Shapiro and co-workers, in 1980, characterized the cross-linked adducts in RNA and DNA (14). Beland et al. fully characterized hydroxymethyl adducts of

formaldehyde in reactions with deoxyribonucleosides (15). On the basis of these studies, it is clear that R-acetates 5 and 10a,b should release formaldehyde upon hydrolysis and that this formaldehyde should form adducts with DNA, but to our knowledge, this has never been reported previously, the only other example being our recent in vivo study of DNA modification by NDMA and NNK (22). Consistent with the results presented here, we observed the hydroxymethyl adduct 11 and the cross-link adduct 14 in hepatic and pulmonary DNA of rats treated with NDMA and NNK (22). The highest levels were those of adduct 11, found in the hepatic DNA of NDMA- and NNK-treated rats. Lower amounts were detected in the pulmonary DNA of these rats. Levels of cross-

Formaldehyde-DNA Adducts

link adduct 14 were near the detection limit except in the liver of NNK-treated rats. These adducts were also detected, although at lower levels, in DNA of untreated rats, presumably due to endogenous formaldehyde-generating reactions. While the formation of adducts 11 and 14 in rats treated with NDMA and NNK logically follows from the results presented here, it is possible that they may have resulted from indirect mechanisms in which formaldehyde may have been generated as part of the response to the DNA-damaging effects of these carcinogens. A limitation of this study was the relatively high concentrations of R-acetates 5 and 10a,bs31–160 mMsused in the reactions with DNA. Such concentrations of R-hydroxynitrosamines would not likely be formed in vivo from carcinogenic doses of NDMA and NNK. Our goal was only to establish the principle that these adducts could form. Our in vivo data indicate that this principle holds under realistic conditions (18). In summary, the results of this study clearly demonstrate that formaldehyde-DNA adducts can be formed upon R-hydroxylation of the carcinogenic methylnitrosamines NDMA, NNK, and NNAL. These nitrosamines are therefore bident carcinogens, potentially damaging DNA with simultaneous release of two electrophilessa diazonium ion and an aldehydesas discussed by Loeppky (30). These results unify two venerable lines of research to provide a potentially new dimension in nitrosamine carcinogenesis. Acknowledgment. This study was supported by Grant CA81301 from the National Cancer Institute. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the University of Minnesota Cancer Center, supported in part by Grant CA-77598. We thank Bob Carlson for editorial support.

Chem. Res. Toxicol., Vol. 21, No. 3, 2008 751

(11)

(12)

(13)

(14) (15) (16)

(17)

(18)

(19)

(20)

References (1) Magee, P. N., and Barnes, J. M. (1956) The production of malignant primary hepatic tumors in the rat by feeding dimethylnitrosamine. Br. J. Cancer 10, 114–122. (2) Preussmann, R., and Stewart, B. W. (1984) N-Nitroso carcinogens. In Chemical Carcinogens, 2nd ed. (Searle, C. E., Ed.) ACS Monograph 182, Vol. 2, pp 643–828, American Chemical Society, Washington, DC. (3) Hecht, S. S. (2007) N-Nitrosamines. In EnVironmental and Occupational Medicine, 4th ed. (Rom, W. B., and Markowitz, S. B., Eds.) pp 1226–1239, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia. (4) International Agency for Research on Cancer (2008) Smokeless tobacco and tobacco-specific nitrosamines. In IARC Monographs on the EValuation of Carcinogenic Risks to Humans, Vol. 89, in press, IARC, Lyon, France. (5) Hecht, S. S. (1998) Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 11, 559–603. (6) Shuker, D. E., and Bartsch, H. (1994) DNA adducts of nitrosamines. In IARC Sci. Publ. No.125. DNA Adducts: Identification and Biological Significance (Hemminki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Segerback, D., and Bartsch, H., Eds.) pp73–89, IARC, Lyon, France. (7) Kyrtopoulos, S. A., Anderson, L. M., Chhabra, S. K., Souliotis, V. L., Pletsa, V., Valanis, C., and Georgiadis, P. (1997) DNA adducts and the mechanism of carcinogenesis and cytotoxicity of methylating agents of environmental and clinical significance. Cancer Detect. PreV. 21, 391–405. (8) Choi, J. Y., Chowdhury, G., Zang, H., Angel, K. C., Vu, C. C., Peterson, L. A., and Guengerich, F. P. (2006) Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases. J. Biol. Chem. 281, 38244–38256. (9) Lao, Y., Yu, N., Kassie, F., Villalta, P. W., and Hecht, S. S. (2007) Analysis of pyridyloxobutyl DNA adducts in F344 rats chronically treated with (R)- and (S)-N′-nitrosonornicotine. Chem. Res. Toxicol. 20, 246–256. (10) Lao, Y., Yu, N., Kassie, F., Villalta, P. W., and Hecht, S. S. (2007) Formation and accumulation of pyridyloxobutyl DNA adducts in F344 rats chronically treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-

(21)

(22)

(23)

(24) (25) (26)

(27)

(28)

(29) (30)

butanone and enantiomers of its metabolite, 4-(methylnitrosamino)1-(3-pyridyl)-1-butanol. Chem. Res. Toxicol. 20, 235–245. Upadhyaya, P., Sturla, S., Tretyakova, N., Ziegel, R., Villalta, P. W., Wang, M., and Hecht, S. S. (2003) Identification of adducts produced by the reaction of 4-(acetoxymethynitrosamino)-1-(3-pyridyl)-1-butanol with deoxyguanosine and DNA. Chem. Res. Toxicol. 16, 180–190. Wang, M., Cheng, G., Sturla, S. J., Shi, Y., McIntee, E. J., Villalta, P. W., Upadhyaya, P., and Hecht, S. S. (2003) 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. Chem. Res. Toxicol. 16, 616–626. Hecht, S. S., Villalta, P. W., Sturla, S. J., Cheng, G., Yu, N., Upadhyaya, P., and Wang, M. (2004) Identification of O2-substituted pyrimidine adducts formed in reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1–butanone and 4-(acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanol with DNA. Chem. Res. Toxicol. 17, 588–597. Chaw, Y. F., Crane, L. E., Lange, P., and Shapiro, R. (1980) Isolation and identification of cross-links from formaldehyde-treated nucleic acids. Biochemistry 19, 5525–5531. Beland, F. A., Fullerton, N. F., and Heflich, R. H. (1984) Rapid isolation, hydrolysis and chromatography of formaldehyde-modified DNA. J. Chromatogr. 308, 121–131. Huang, H., and Hopkins, P. B. (1993) DNA interstrand cross-linking by formaldehyde: nucleotide sequence preference and covalent structure of the predominant cross-link formed in synthetic oligonucleotides. J. Am. Chem. Soc. 115, 9402–9408. Huang, H., Solomon, M. S., and Hopkins, P. B. (1992) Formaldehyde preferentially interstrand cross-links duplex DNA through deoxyguanosine residues at the sequence 5′-d(AT). J. Am. Chem. Soc. 114, 9240–9241. Cheng, G., Shi, Y., Sturla, S., Jalas, J., McIntee, E. J., Villalta, P. W., Wang, M., and Hecht, S. S. (2003) Reactions of formaldehyde plus acetaldehyde with deoxyguanosine and DNA: Formation of cyclic deoxyguanosine adducts and formaldehyde cross-links. Chem. Res. Toxicol. 16, 145–152. International Agency for Research on Cancer (2006) Formaldehyde, 2-butoxyethanol and 1-tert-butoxypropan-2-ol. In IARC Monographs on the EValuation of Carcinogenic Risks to Humans, Vol. 88, pp 39– 325, IARC, Lyon, France. U.S. Department of Health and Human Services (2004) 11th Report on Carcinogens, U.S. Government Printing Office, Washington, DC. Spratt, T. E., Peterson, L. A., Confer, W. L., and Hecht, S. S. (1990) Solvolysis of model compounds for R-hydroxylation of N′-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: Evidence for a cyclic oxonium ion intermediate in the alkylation of nucleophiles. Chem. Res. Toxicol. 3, 350–356. Wang, M., Cheng, G., Villalta, P. W., and Hecht, S. S. (2007) Development of liquid chromatography electrospray ionization tandem mass spectrometry methods for analysis of DNA adducts of formaldehyde and their application to rats treated with N-nitrosodimethylamine or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Chem. Res. Toxicol. 20, 1141–1148. Roller, P. P., Shimp, D. R., and Keefer, L. K. (1975) Synthesis and solvolysis of methyl(acetoxymethyl)nitrosamine. Solution chemistry of the presumed carcinogenic metabolite of dimethylnitrosamine. Tetrahedron Lett. 25, 2065–2068. Dutton, A. H., and Heath, D. F. (1956) Demethylation of dimethylnitrosamine in rats and mice. Nature 178, 644. Magee, P. N., and Vandekar, M. (1958) Toxic liver injury; the metabolism of dimethylnitrosamine in Vitro. Biochem. J. 70, 600– 605. Brouwers, J. A., and Emmelot, P. (1960) Microsomal N-demethylation and the effect of the hepatic carcinogen dimethylnitrosamine on amino acid incorporation into the proteins of rat livers and hepatomas. Exp. Cell Res. 19, 467–474. Keefer, L. K., Kroeger-Koepke, M. B., Ishizaki, H., Michejda, C. J., Saavedra, J. E., Hrabie, J. A., Yang, C. S., and Roller, P. P. (1990) Stereoselectivity in the microsomal conversion of N-nitrosodimethylamine to formaldehyde. Chem. Res. Toxicol. 3, 540–544. Yoo, J. S., Ishizaki, H., and Yang, C. S. (1991) Enzyme kinetics of N-nitrosodimethylamine demethylase in rodents and humans. In ReleVance to Human Cancer of N-Nitroso Compounds, Tobacco Smoke and Mycotoxins (O’Neill, I. K., Chen, J. C., and Bartsch, H., Eds.) pp 366–369, IARC Publications, Lyon, France. Feldman, M. Y. (1973) Reactions of nucleic acids and nucleoproteins with formaldehyde. Prog. Nucleic Acid Res. Mol. Biol. 13, 1–49. Loeppky, R. N., and Goelzer, P. (2002) Microsome-mediated oxidation of N-nitrosodiethanolamine (NDELA), a bident carcinogen. Chem. Res. Toxicol. 15, 457–469.

TX7003823