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Analysis of Ethylated Thymidine Adducts in Human Leukocyte DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography–Nanospray Ionization Tandem Mass Spectrometry
- Chen, Hauh-Jyun Candy, Wang, Yi-Ching, Lin, Wen-Peng
- Analytical chemistry 2012 v.84 no.5 pp. 2521-2527
- DNA adducts, biomarkers, detection limit, humans, ionization, isotope dilution technique, leukocytes, monitoring, neoplasms, nucleotides, risk assessment, risk factors, smoking (habit), stable isotopes, tandem mass spectrometry, thymidine, tissues, urine
- Studies showed that levels of ethylated DNA adducts in certain tissues and urine are higher in smokers than in nonsmokers. Because cigarette smoking is a major risk factor of various cancers, DNA ethylation might play an important role in cigarette smoke-induced cancer formation. Among the ethylated DNA adducts, O²-ethylthymidine (O²-edT) and O⁴-ethylthymidine (O⁴-edT) are poorly repaired and are accumulated in the body. In addition, O⁴-edT possesses promutagenic properties. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of O²-edT, N³-edT (N³-ethylthymidine), and O⁴-edT adducts by isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry (nanoLC–NSI/MS/MS). Under the highly selected reaction monitoring (H-SRM) mode, the detection limit of O²-edT, N³-edT, and O⁴-edT injected on-column was 5.0, 10, and 10 fg, respectively. The quantification limit for the entire assay was 50, 100, and 100 fg of O²-edT, N³-edT, and O⁴-edT, respectively, corresponding to 1.1, 2.3, and 2.3 adducts in 10⁹ normal nucleotides, respectively, starting with 50 μg of DNA (from 1.5–2.0 mL of blood). Levels of O²-edT, N³-edT, and O⁴-edT in 20 smokers’ leukocyte DNA were 44.8 ± 52.0, 41.1 ± 43.8, 48.3 ± 53.9 in 10⁸ normal nucleotides, while those in 20 nonsmokers were 0.19 ± 0.87, 4.1 ± 13.3, and 1.0 ± 2.9, respectively. Levels of O²-edT, N³-edT, and O⁴-edT in human leukocyte DNA are all significantly higher in smokers than in nonsmokers, with pvalues of 0.0004, 0.0009, and 0.0004, respectively. Furthermore, levels of O²-edT show a statistically significant association (γ = 0.4789, p = 0.0327) with the smoking index in smokers. In the 40 leukocyte DNA samples, the extremely significant statistical correlations (p < 0.0001) are observed between levels of O²-edT and O⁴-edT (γ = 0.9896), between levels of O²-edT and N³-edT (γ = 0.9840), and between levels of N³-edT and O⁴-edT (γ = 0.9901). To our knowledge, this is the first mass spectrometry-based assay for ethylated thymidine adducts. Using this assay, the three ethylated thymidine adducts were detected and quantified for the first time. Therefore, this highly sensitive, specific, and accurate assay should be clinically feasible for simultaneous quantification of the three ethylated thymidine adducts as potential biomarkers for exposure to ethylating agents and for cancer risk assessment.