Mass Spectrometric Analysis of Tobacco-Specific Nitrosamine-DNA Adducts in Smokers and Nonsmokers

A gas chromatography, negative ion chemical ionization mass spectrometry (GC-NICI-MS) based assay for tobacco-specific nitrosamine adducts of DNA is described. The assay is based on the observation that acid hydrolysis of DNA from animals treated with tobacco-specific nitrosamines releases 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB). HPB and the internal standard [4,4-D₂]HPB are derivatized with pentafluorobenzoyl chloride and the resulting HPB-penta-fluorobenzoate is purified by high-performance liquid chromatography prior to GC-NICI-MS analysis. DNA from human peripheral lung and tracheobronchial tissue, collected at autopsy, was analyzed for acid-released HPB. The mean HPB level (fmol/mg of DNA) for peripheral lung DNA was 11 ± 16 (SD, n = 9) for smokers and 0.9 ± 2.3 (n = 8) for nonsmokers. Mean adduct levels in tracheobronchus were 16 ± 18 (n = 4) for smokers and 0.9 ± 1.7 (n = 4) for nonsmokers. These are the first measurements of tobacco-specific nitrosamine-DNA adducts in humans. Further studies comparing the levels of DNA and globin adducts will provide a better understanding of the metabolic activation of tobacco-specific nitrosamines in humans and may provide a more accurate indication of an individual’s risk of developing tobacco-related cancer.