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 N 6-[4-hydroxy-1-(pyridine-3-yl)­butyl]-2′-deoxyadenosine (N 6-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, N 6-[5-(3-pyridyl)­tetrahydrofuran-2-yl]-2′-deoxyadenosine (N 6-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 N 6-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-d 4]­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.

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

“…Further study suggested that N 6 -PHB-dAdo was formed by the reduction of N 6 -[5-(3-pyridyl)­tetrahydrofuran-2-yl]-2′-deoxyadenosine ( N 6 -Py-THF-dAdo) (Scheme S3), which had been previously observed only at the nucleoside level, for example, in the reaction of 5′-acetoxyNNN with dAdo . However, our present results described in the accompanying manuscript suggested that it is also present in calf thymus DNA incubated with 5′-acetoxyNNN but not in liver and lung DNA of rats treated with NNN …”

“…The precursor compound of N 6 -HPB-dAdo is considered to be N 6 -[4-oxo-1-(pyridine-3-yl)­butyl]-2′-deoxyadenosine ( N 6 -OPB-dAdo), in equilibration with 6-[2-(3-pyridyl)- N -pyrrolidinyl-5-hydroxy]-2′-deoxynebularine (Py-Py­(OH)-dN), which has been observed in vitro and in vivo (Scheme S3). No such peak was observed in the untreated (Figure A) or NNKOAc-treated calf thymus DNA (Figure G). This strongly indicates that N 6 -HPB-dAdo is specifically formed by NNN metabolism.…”

Mass Spectrometric Quantitation of N′-Nitrosonornicotine-1N-oxide in the Urine of Cigarette Smokers and Smokeless Tobacco Users

“…On the basis of our previous LC-NSI-HRMS/MS methods for DNA adduct quantitation, , an optimized method was developed for the urinary NNN- N -oxide assay. It is common to use MS 2 transitions for quantitation with the Orbitrap detector since high specificity can be readily achieved using a tight mass tolerance (5 ppm) filter.…”

“…Using the current protocol, the assay nominal recovery (recovery plus ion suppression) was 29 ± 8% (n = 4) by spiking [pyridine- MS Method Development. On the basis of our previous LC-NSI-HRMS/MS methods for DNA adduct quantitation, 30,31 an optimized method was developed for the urinary NNN-N-oxide assay. It is common to use MS 2 transitions for quantitation with the Orbitrap detector since high specificity can be readily achieved using a tight mass tolerance (5 ppm) filter.…”

Multiscale computational investigations of the translesion synthesis bypass of tobacco-derived DNA adducts: critical insights that complement experimental biochemical studies

“…In fact, computational work has already played a vital role in explaining the mutational signatures associated with tobacco exposure. Other NNK-derived lesions that should be investigated in the future include the larger POB/PHB adducts at C 127,155,156 or A, [157][158][159] as well as phosphate [160][161][162][163] lesions. Other adducts, such as those formed from combustion products (1,3butadiene (EB-GII)) 164,165 and acrolein (g-OH-Acr-dG), 166,167 found in tobacco smoke would also be of interest.…”