Quantitation by Liquid Chromatography-Nanoelectrospray Ionization-High Resolution Tandem Mass Spectrometry of Methyl and Ethyl DNA Adducts in Oral Cells from Cigarette Smokers and Nonsmokers of the Shanghai Cohort Study.

We used liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) to quantify DNA adducts released from human oral cell DNA upon neutral thermal hydrolysis followed by acid hydrolysis. The assay was applied to 80 buccal cell samples selected from those collected in the Shanghai Cohort Study, a prospective epidemiology study of 18,244 Chinese men 45-64 years old who resided in Shanghai, China when the samples were collected in 2001-2003. The DNA adducts quantified were 3-methyladenine (3-Me-Ade), 3-ethyladenine (3-Et-Ade), and 7-ethylguanine (7-Et-Gua). The method used hydrolysis of DNA samples containing the stable isotope labeled internal standards, solid phase extraction for adduct enrichment, and analysis by LC-NSI-HRMS/MS. Accuracy and precision of the analytical method were established with detection limits of 10-20 amol on column. Median levels of 3-Me-Ade -187 adducts/10⁹ nucleotides in smokers and 129 adducts/10⁹ nucleotides in nonsmokers; and 7-Et-Gua -49 adducts/10⁹ nucleotides in smokers and 21 adducts/10⁹ nucleotides in nonsmokers─were significantly higher in smokers than in nonsmokers (both P values <0.01). Levels of 3-Et-Ade -50 adducts/10⁹ nucleotides in smokers and 43 adducts/10⁹ nucleotides in nonsmokers - were not significantly different. These results demonstrate the applicability of a highly sensitive LC-NSI-HRMS/MS method for the analysis of human oral cell DNA for adducts released by neutral thermal and acid hydrolysis and show the significant effects of cigarette smoking on levels of 3-Me-Ade and 7-Et-Gua in this DNA. This is apparently the first study to characterize 3-Me-Ade in intact DNA isolated from any human tissue.