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 N 6-[4-(3-pyridyl)-4-oxo-1-butyl]-2′-deoxyadenosine (N 6-POB-dAdo) and its reduced form N 6-PHB-dAdo formed by NNN 2′-hydroxylation in rat liver and lung DNA. More importantly, we characterized a new dAdo adduct N 6-[4-hydroxy-1-(pyridine-3-yl)­butyl]-2′-deoxyadenosine (N 6-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 N 6-HPB-dAdo and the corresponding isotopically labeled internal standard [pyridine-d 4]N 6-HPB-dAdo were synthesized using a four-step method. Both NMR and high-resolution mass spectrometry data agreed well with the proposed structure of N 6-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 N 6-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 N 6-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.

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

“…5′-AcetoxyNNN was obtained from our accompanying study. 43 Chemical standards of N 6 -Py-THF-dAdo and Py-Py-dN were synthesized using the method described before. 29 Their corresponding isotopically labeled internal standards [ 13 C 10 15 N 5 ]N 6 -Py-THF-dAdo and [pyridine-d 4 ]Py-Py-dN were synthesized as described below, as was Py-Py(OH)-dN.…”

“…33 Purified DNA hydrolysis enzymes and porcine liver esterase were from our accompanying study. 43 All other chemicals and supplies were purchased from Sigma-Aldrich or Fisher Scientific. Milli-Q H 2 O was routinely used unless otherwise mentioned.…”

“…Py-Py(OH)-dN was synthesized as illustrated in Scheme 2, starting with the newly identified dAdo adduct N 6 -HPB-dAdo (20). 43 To a solution of 20 (0.03 mmol, 12 mg) in DMSO (0.5 mL) were added Dess-Martin periodinane (0.033 mmol, 14 mg) and Et 3 N (0.15 mmol, 21 μL). The mixture was stirred at room temperature for 24 h. After reaction, the mixture was subjected directly to reverse phase HPLC purification as described in our accompanying study.…”

“…The mixture was stirred at room temperature for 24 h. After reaction, the mixture was subjected directly to reverse phase HPLC purification as described in our accompanying study. 43 The desired product was collected at a retention time of 27.4 min. However, it was converted to 4 peaks after evaporation of the solvents (MeOH and H 2 O) (Figure S3A).…”

“…DNA samples were prepared essentially as described in our accompanying study. 43 Briefly, when using NaBH 3 CN as the reducing agent, to the isolated DNA (∼50 μg) dissolved in 500 μL of 10 mM sodium succinate buffer containing 5 mM CaCl 2 was added 100 μL 20 mg/mL NaBH 3 CN in the succinate buffer. The mixture was incubated at 37 °C for 1 h. Then the internal standards [pyridine-d 4 ]Py-Py-dN (10 fmol) and [ 15 N 5 ]Py-Py-dI (10 fmol) were added, followed by three purified hydrolytic enzymes: deoxyribonuclease I (0.4 units), phosphodiesterase I (0.1 units), and alkaline phosphatase (0.8 units).…”