The tobacco-specific N-nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are potent carcinogens. Both require metabolic activation to exert their carcinogenic effects, and DNA adduct formation is central to their mechanisms of carcinogenesis. DNA adducts formed by the NNK- and NNN-induced pyridyloxobutylation of DNA nucleobases have been well characterized.  In addition, pyridyloxobutylation of DNA may also occur at the oxygen atoms of the internucleotidic phosphodiester linkages. Such adducts were previously detected in NNK-treated mouse liver by employing transalkylation approach; however, phosphate adducts have never been directly identified and characterized. In the current study, identification of possible phosphate DNA adducts formed by tobacco-specific nitrosamines was performed. We used 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), which is a regiochemically activated form of NNK, as a model compound and reacted it with calf thymus DNA in the presence of esterase at 37�C for 16 h. The treated DNA was isolated and enzymatically hydrolyzed at 37�C for 48 h followed by centrifugal filtration (30K) and further purification by Strata-X solid-phase extraction. Analysis was performed using liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high resolution tandem mass spectrometry (HRMS/MS) under full scan and product ion scan modes. Based on the accurate mass measurement of precursor and fragment ions at a mass tolerance of 5 ppm, 12 phosphate adducts were detected and characterized for the first time, including three major ones, dCp(4-oxo-4-(3-pyridyl)butyl)dC, dAp(4-oxo-4-(3-pyridyl)butyl)dC and dTp(4-oxo-4-(3-pyridyl)butyl)dC. We further synthesized dCp(4-oxo-4-(3-pyridyl)butyl)dC by reacting NNKOAc with dCpdC dinucleotide, and the structure of this adduct was characterized. This study provides the first structural identification of phosphate DNA adducts of the tobacco-specific nitrosamines and chemical support for the future mechanistic studies of tobacco carcinogenesis in laboratory animals and humans. Studies of phosphate DNA adduct formation in vivo by tobacco nitrosamines are in progress in our laboratory.

Identification of phosphate adducts formed by pyridyloxobutylation of DNA by 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a chemically activated form of tobacco specific carcinogens

248th National Meeting (2014)

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