Detection and simultaneous quantification of three smoking-related ethylthymidine adducts in human salivary DNA by liquid chromatography tandem mass spectrometry

• First simultaneous analysis of three ethylthymidine adducts in human salivary DNA by mass spectrometry.
• Ethylthymidine adduct levels are higher in smokers than nonsmokers.
• Ethylthymidine adduct levels are associated with cigarette smoking.
• Ethylthymidine adducts are not detectable in nonsmokers.

Smoking cigarette increases levels of certain ethylated DNA adducts in certain tissues and urine. Cigarette smoking is a major risk factor of various cancers and DNA ethylation is involved in smoking-related carcinogenesis. Among the ethylated DNA adducts, O2-ethylthymidine (O2-edT) and the promutagenic O4-ethylthymidine (O4-edT) are poorly repaired and they can accumulate in vivo. Using an accurate, highly sensitive, and quantitative assay based on stable isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry (nanoLC–NSI/MS/MS), O2-edT, N3-edT (N3-ethylthymidine), and O4-edT adducts in human salivary DNA were simultaneous detected and quantified. Saliva is easily accessible and available and it can be a potential target in searching for noninvasive biomarkers. Under the highly selected reaction monitoring (H-SRM) mode, salivary samples from 20 smokers and 13 nonsmokers were analyzed. Starting with 50 μg of DNA isolated from about 3.5 mL of saliva, levels of O2-edT, N3-edT, and O4-edT in 20 smokers’ salivary DNA samples were 5.3 ± 6.2, 4.5 ± 5.7, 4.2 ± 8.0 in 108 normal nucleotides, respectively, while those in 13 nonsmokers were non-detectable. In addition, statistically significant correlations (p < 0.0001) were observed between levels of O2-edT and N3-edT (γ = 0.7388), between levels of O2-edT and O4-edT (γ = 0.8839), and between levels of N3-edT, and O4-edT (γ = 0.7835). To the best of our knowledge, this is the first report of detection and quantification of these three ethylthymidine adducts in human salivary DNA, which might be potential biomarkers for exposure to ethylating agents and possibly for cancer risk assessment.