Investigation of 2'-Deoxyadenosine-Derived Adducts Specifically Formed in Rat Liver and Lung DNA by N'-Nitrosonornicotine Metabolism

Abstract

The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as "carcinogenic to humans" (Group 1). To exert its carcinogenicity, NNN requires metabolic activation to form reactive intermediates which alkylate DNA. Previous studies have identified cytochrome P450-catalyzed 2'-hydroxylation and 5'-hydroxylation of NNN as major metabolic pathways, with preferential activation through the 5'-hydroxylation pathway in some cultured human tissues and patas monkeys. So far, the only DNA adducts identified from NNN 5'-hydroxylation in rat tissues are 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxynebularine (Py-Py-dN), and N⁶-[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (N⁶-HPB-dAdo) after reduction. To expand the DNA adduct panel formed by NNN 5'-hydroxylation and identify possible activation biomarkers of NNN metabolism, we investigated the formation of dAdo-derived adducts using a new highly sensitive and specific liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Two types of NNN-specific dAdo-derived adducts, N⁶-[5-(3-pyridyl)tetrahydrofuran-2-yl]-2'-deoxyadenosine (N⁶-Py-THF-dAdo) and 6-[2-(3-pyridyl)-N-pyrrolidinyl-5-hydroxy]-2'-deoxynebularine (Py-Py(OH)-dN), were observed for the first time in calf thymus DNA incubated with 5'-acetoxyNNN. More importantly, Py-Py(OH)-dN was also observed in relatively high abundance in the liver and lung DNA of rats treated with racemic NNN in the drinking water for 3 weeks. These new adducts were characterized using authentic synthesized standards. Both NMR and MS data agreed well with the proposed structures of N⁶-Py-THF-dAdo and Py-Py(OH)-dN. Reduction of Py-Py(OH)-dN by NaBH₃CN led to the formation of Py-Py-dN both in vitro and in vivo, which was confirmed by its isotopically labeled internal standard [pyridine-d₄]Py-Py-dN. The NNN-specific dAdo adducts Py-THF-dAdo and Py-Py(OH)-dN formed by NNN 5'-hydroxylation provide a more comprehensive understanding of the mechanism of DNA adduct formation by NNN.

Figure 1.

Summary of DNA adducts formed by NNN 5′-hydroxylation in vitro and in vivo. dR = 2′-deoxyribose. Structures of oxonium ion 8 and diazonium ion 9 are shown in Scheme 1.

Figure 2.

Representative chromatograms and MS² and MS³ product ion spectra of Py-Py(OH)-dN. The fragmentation patterns of MS² and MS³ transitions of Py-Py(OH)-dN agreed well with proposed patterns.

Figure 3.

Representative extracted product ion chromatograms of MS² transitions for the analysis of N⁶-Py-THF-dAdo and Py-Py(OH)-dN formation in calf thymus DNA incubated with 5′-acetoxyNNN. Four diastereomer peaks of N⁶-Py-THF-dAdo were observed from the MS² transition of 399.2 → 283.1302, co-eluting with the synthesized internal standard [¹³C₁₀¹⁵N₅]N⁶-Py-THF-dAdo. However, a broad peak nearly co-eluting with N⁶-Py-THF-dAdo was observed. It can form a unique MS² fragment ion 265.1195, which suggests its structure to be Py-Py(OH)-dN (see Figure 2 for the structure and fragmentation pattern of Py-Py(OH)-dN).

Figure 4.

Py-Py(OH)-dN was formed in calf thymus DNA incubated with 5′-acetoxyNNN and in the liver and lung DNA of rats treated with 500 ppm racemic NNN in the drinking water for 3 weeks. Synthesized Py-Py(OH)-dN spiked into the DNA samples co-eluted with the observed peak. No such Py-Py(OH)-dN peak was observed in an untreated control calf thymus DNA sample.

Figure 5.

Py-Py(OH)-dN (in red) but not N⁶-Py-THF-dAdo (in blue) was observed in the lung DNA of rats treated with 500 ppm racemic NNN for 3 weeks. LC conditions are critical for resolving N⁶-Py-THF-dAdo peaks from the massive peak of Py-Py(OH)-dN. (A) LC conditions depicted in Table S1 can separate the 4 diastereomers of N⁶-Py-THF-dAdo and partially resolve it from Py-Py(OH)-dN, however causing a significantly broad peak of Py-Py(OH)-dN. (B) LC conditions depicted in Table S2 afford a sharper peak of Py-Py(OH)-dN but fail to resolve N⁶-Py-THF-dAdo.

Figure 6.

Py-Py-dN was clearly observed in the lung DNA of rats reduced by NaBH₃CN but not NaBH₄. (A) Untreated calf thymus DNA reduced with 2 mg NaBH₃CN; (B) lung DNA of rats treated with 500 ppm NNN reduced with 2 mg NaBH₃CN; (C) lung DNA of rats treated with 500 ppm NNN reduced with 2 mg NaBH₄.

Scheme 1.

Mechanisms of dAdo-derived adduct formation from NNN and NNK metabolic activation catalyzed by cytochrome P450 enzymes.

Scheme 2.

Synthetic route for Py-Py(OH)-dN (14).