Identification of an N'-Nitrosonornicotine-Specific Deoxyadenosine Adduct in Rat Liver and Lung DNA

Abstract

The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are considered to be two of the most important carcinogens in unburned tobacco and its smoke. They readily cause tumors in laboratory animals and are classified as "carcinogenic to humans" by the International Agency for Research on Cancer. DNA adduct formation by these two carcinogens is believed to play a critical role in tobacco carcinogenesis. Among all the DNA adducts formed by NNN and NNK, 2'-deoxyadenosine (dAdo)-derived adducts have not been fully characterized. In the study reported here, we characterized the formation of N⁶-[4-(3-pyridyl)-4-oxo-1-butyl]-2'-deoxyadenosine (N⁶-POB-dAdo) and its reduced form N⁶-PHB-dAdo formed by NNN 2'-hydroxylation in rat liver and lung DNA. More importantly, we characterized a new dAdo adduct N⁶-[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (N⁶-HPB-dAdo) formed after NaBH₃CN or NaBH₄ reduction both in vitro in calf thymus DNA reacted with 5'-acetoxy-N'-nitrosonornicotine and in vivo in rat liver and lung upon treatment with NNN. This adduct was specifically formed by NNN 5'-hydroxylation. Chemical standards of N⁶-HPB-dAdo and the corresponding isotopically labeled internal standard [pyridine-d₄]N⁶-HPB-dAdo were synthesized using a four-step method. Both NMR and high-resolution mass spectrometry data agreed well with the proposed structure of N⁶-HPB-dAdo. The new adduct coeluted with the synthesized internal standard under various LC conditions. Its product ion patterns of MS² and MS³ transitions were also consistent with the proposed fragmentation patterns. Chromatographic resolution of the two diastereomers of N⁶-HPB-dAdo was successfully achieved. Quantitation suggested a dose-dependent response of the levels of this new adduct in the liver and lung of rats treated with NNN. However, its level was lower than that of 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxyinosine, a previously reported dGuo adduct that is also formed from NNN 5'-hydroxylation. The identification of N⁶-HPB-dAdo in this study leads to new insights pertinent to the mechanism of carcinogenesis by NNN and to the development of biomarkers of NNN metabolic activation.

Figure 1.

Summary of previously investigated pyridyl DNA base adducts formed by NNN and NNK metabolism in vitro and in vivo. dR = 2′-deoxyribose.

Figure 2.

Representative chromatograms and MS² product ion spectra of N⁶-HPB-dAdo and [pyridine-D₄]N⁶-HPB-dAdo versus N⁶-PHB-dAdo and [¹⁵N₅]N⁶-PHB-dAdo. All the results agreed well with the proposed fragmentation patterns of the four chemical standards. The ion m/z 173.53 was a co-eluting interfering ion.

Figure 3.

Representative extracted product ion chromatograms of MS² transitions for the analysis of N⁶-HPB-dAdo and N⁶-PHB-dAdo formation in DNA samples upon NaBH₄ reduction. (A) Blank calf thymus DNA, (B) calf thymus DNA incubated with 5′-acetoxyNNN, (C) liver DNA of rat from the control group, (D) liver DNA of rats treated with 500 ppm racemic NNN for 3 weeks, (E) lung DNA of rat from the control group, (F) lung DNA of rats treated with 500 ppm racemic NNN for 3 weeks, (G) calf thymus DNA incubated with NNKOAc, and (H) lung DNA of rats treated with 5 ppm NNK for 70 weeks.

Scheme 1.

Mechanisms of NNN and NNK metabolic activation leading to dAdo-derived adducts in DNA.

Scheme 2.

Synthetic routes for N⁶-HPB-dAdo and [pyridine-D₄]N⁶-HPB-dAdo.