The tobacco-specific
nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) is a potent lung
carcinogen in laboratory animals and is believed to play a key role in the development of
lung cancer in smokers. Metabolic activation of NNK leads to the formation of pyridyloxobutyl
DNA adducts, a critical step in its mechanism of
carcinogenesis. In addition to
DNA nucleobase
adducts, DNA phosphate adducts can be formed by pyridyloxobutylation of the
oxygen atoms of the internucleotidic phosphodiester linkages. We report the use of a liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry technique to characterize 30 novel pyridyloxobutyl
DNA phosphate adducts in
calf thymus DNA (CT-
DNA) treated with
4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (
NNKOAc, 2), a regiochemically activated form of NNK. A (15)N3-labeled internal standard was synthesized for one of the most abundant
phosphate adducts, dCp[4-oxo-4-(3-pyridyl)butyl]dC (CpopC), and this standard was used to quantify CpopC and to estimate the levels of other adducts in the
NNKOAc-treated CT-
DNA. Formation of
DNA phosphate adducts by NNK in vivo was further investigated in rats treated with NNK acutely (0.1 mmol/kg once daily for 4 days by
subcutaneous injection) and chronically (5 ppm in
drinking water for 10, 30, 50, and 70 weeks). This study provides the first comprehensive structural identification and quantitation of a panel of
DNA phosphate adducts of a structurally complex
carcinogen and chemical support for future mechanistic studies of tobacco
carcinogenesis in humans.