The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an effective lung carcinogen in laboratory animals, and it is believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK leads to the formation of DNA adducts, a critical step in the mechanism of chemical carcinogenesis. In addition to nucleobase DNA adducts, phosphate DNA adducts can also be formed by the NNK-induced pyridyloxobutylation of the oxygen atoms of the internucleotidic phosphodiester linkages. In this study, 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 (CT-DNA). A total of 25 different phosphate adducts were detected in the NNKOAc-treated CT-DNA. An 15N-labelled internal standard was synthesized for one of the most abundant adducts, dCp(4-oxo-4-(3-pyridyl)butyl)dC (CpopC), and the synthesized standard was used to quantify CpopC in the NNKOAc-treated CT-DNA by liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry. The relative levels of other phosphate adducts were determined based on their MS responses compared to CpopC. Formation of phosphate DNA adducts by NNK was further investigated in liver DNA samples from rats (n = 5) treated with NNK (0.1 mmol/kg once daily for 4 days by s.c. injection). Twenty four different phosphate adducts were detected in these samples, with a total adduct level of 20 ± 5.6 pmol/mg DNA. The developed methodology is currently being applied to study the formation of phosphate adducts in rats treated chronically with NNK (5 ppm in drinking water for 10, 30, 50 and 70 weeks). This research provides the first quantitation of phosphate DNA adducts from NNK, and lays the groundwork for establishing a representative array of phosphate adducts as biomarkers of tobacco carcinogen exposure in future mechanistic and in vivo studies.

Analysis of phosphate adducts in hepatic and pulmonary DNA of rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

250th National Meeting (2015)

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