Quantitative Analysis of Multiple Exocyclic DNA Adducts in Human Salivary DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography-Nanospray Ionization Tandem Mass Spectrometry

Exocyclic DNA adducts, including 1,N-2-propano-2'-deoxypanosine derived from acrolein (AdG) and crotonaldehyde (CdG) and the three lipid peroxidation-related etheno adducts 1,N-6-etheno-2'-deoxyadenosine (epsilon dAdo), 3, N-4-etheno-2'-deoxycytidine (epsilon dCyt), and 1,N-2-etheno-2'-deoxyguanosine (1,N-2-epsilon dGuo), play an important role in cancer formation and they are associated with oxidative-stress-induced DNA damage. Saliva is an easily accessible and available biological fluid and a potential target of noninvasive biomarkers. In this study, a highly sensitive and specific assay based on isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) is developed for simultaneous detection and quantification of these five adducts in human salivary DNA. The levels of AdG, CdG, epsilon dAdo, epsilon dCyd, and 1,N-2-epsilon dGuo, measured in 27 human salivary DNA samples from healthy volunteers, were determined as 104 +/- 50, 7.6 +/- 12, 99 +/- 50, 72 +/- 49, 391 +/- 198 (mean +/- SD) in 10(8) normal nucleotides, respectively, starting with 25 mu g of DNA isolated from an average of 3 mL of saliva. Statistically significant correlations were found between levels of epsilon dAdo and epsilon dCyd (gamma = 0.8007, p < 0.0001), between levels of epsilon dAdo and 1,N-2-epsilon dGuo (gamma = 0.6778, p = 0.0001), between levels of epsilon dCyd and 1,N-2-epsilon dGuo (gamma = 0.5643, p = 0.0022), between levels of AdG and 1,N-2-epsilon dGuo (gamma = 0.5756, p = 0.0017), and between levels of AdG and epsilon dAdo (gamma = 0.3969, p = 0.0404). Only 5 mu g of DNA sample was analyzed for simultaneous quantification of these adducts. The easy accessibility and availability of saliva and the requirement for the small amount of DNA samples make this nanoLC-NSI/MS/MS assay clinically feasible in assessing the possibility of measuring 1,N-2-propano-2'-deoxyguanosine and etheno adducts levels in human salivary DNA as noninvasive biomarkers for DNA damage resulting from oxidative stress and for evaluating their roles in cancer formation and prevention.