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Background: The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in people who use tobacco products. NNK undergoes cytochrome P450s-catalyzed metabolic activation – an important step in its mechanism of carcinogenesis – to electrophilic intermediates which react with DNA nucleobases and form pyridyloxobutyl and methyl DNA base adducts. Another important metabolic pathway of NNK is its conversion to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is similarly metabolized to reactive intermediates, which react with DNA and form pyridylhydroxybutyl and methyl DNA base adducts that have been characterized previously. We have also demonstrated the formation of pyridyloxobutyl DNA phosphate adducts due to the reaction of NNK-derived intermediates with the oxygen of the internucleotide phosphodiester linkages in the DNA. The goal of this study was to investigate the potential formation of pyridylhydroxybutyl and methyl DNA phosphate adducts in rats upon treatment with NNK. Methods: Male F-344 rats were treated chronically with 5 ppm of NNK in their drinking water for 10, 30, 50 and 70 weeks. DNA was isolated from the lung tissues, enzymatically digested, purified and analyzed by using a novel liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Results: In the rat lung, we characterized and quantified 107 structurally unique pyridylhydroxybutyl DNA phosphate adducts. We also detected and quantified 21 methyl DNA phosphate adducts in the same tissue. Together with 30 pyridyloxobutyl phosphate adducts characterized previously, a total of 158 DNA phosphate adducts were formed due to metabolic activation of NNK. The levels of pyridyloxobutyl, pyridylhydroxybutyl and methyl phosphate adducts were 218–475, 3880–7900 and 2290–4510 fmol/mg DNA over the course of the study, which accounted for 2-3%, 30-47% and 14-29% of all the measured DNA phosphate and base adducts, respectively (Fig. 1). Some of the pyridylhydroxybutyl phosphate adducts persisted in the rat lung for over 70 weeks, suggesting that they could be potential biomarkers of chronic exposure to NNK and NNAL. Conclusion: This study provides a comprehensive structural identification and quantitation of a panel of DNA phosphate adducts formed upon metabolic activation of a structurally complex carcinogen NNK. These newly characterized adducts could be used in future studies of tobacco carcinogenesis.
Figure 1. Relative levels of DNA phosphate and base adducts in lung DNA of rats treated with 5 ppm of NNK in drinking water for 10, 30, 50 or 70 weeks.