Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan.
Anal Chem. 2012 Mar 6;84(5):2521-7. doi: 10.1021/ac203405y. Epub 2012 Feb 13.
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, O(2)-ethylthymidine (O(2)-edT) and O(4)-ethylthymidine (O(4)-edT) are poorly repaired and are accumulated in the body. In addition, O(4)-edT possesses promutagenic properties. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of O(2)-edT, N(3)-edT (N(3)-ethylthymidine), and O(4)-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 O(2)-edT, N(3)-edT, and O(4)-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 O(2)-edT, N(3)-edT, and O(4)-edT, respectively, corresponding to 1.1, 2.3, and 2.3 adducts in 10(9) normal nucleotides, respectively, starting with 50 μg of DNA (from 1.5-2.0 mL of blood). Levels of O(2)-edT, N(3)-edT, and O(4)-edT in 20 smokers' leukocyte DNA were 44.8 ± 52.0, 41.1 ± 43.8, 48.3 ± 53.9 in 10(8) normal nucleotides, while those in 20 nonsmokers were 0.19 ± 0.87, 4.1 ± 13.3, and 1.0 ± 2.9, respectively. Levels of O(2)-edT, N(3)-edT, and O(4)-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 O(2)-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 O(2)-edT and O(4)-edT (γ = 0.9896), between levels of O(2)-edT and N(3)-edT (γ = 0.9840), and between levels of N(3)-edT and O(4)-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.
研究表明，某些组织和尿液中的乙基化 DNA 加合物水平在吸烟者中高于非吸烟者。由于吸烟是各种癌症的主要危险因素，因此 DNA 乙基化可能在香烟烟雾诱导的癌症形成中发挥重要作用。在乙基化 DNA 加合物中，O(2)-乙基胸腺嘧啶（O(2)-edT）和 O(4)-乙基胸腺嘧啶（O(4)-edT）的修复能力较差，并且在体内积累。此外，O(4)-edT 具有致突变特性。在这项研究中，我们开发了一种高度敏感、准确和定量的分析方法，通过同位素稀释纳米流液相色谱-纳喷雾电离串联质谱法（nanoLC-NSI/MS/MS）同时检测和定量 O(2)-edT、N(3)-edT（N(3)-乙基胸腺嘧啶）和 O(4)-edT 加合物。在高选择反应监测（H-SRM）模式下，O(2)-edT、N(3)-edT 和 O(4)-edT 注入柱上的检测限分别为 5.0、10 和 10 fg。整个分析的定量限分别为 50、100 和 100 fg 的 O(2)-edT、N(3)-edT 和 O(4)-edT，分别对应于 10(9)个正常核苷酸中的 1.1、2.3 和 2.3 个加合物，起始量为 50 μg DNA（来自 1.5-2.0 mL 血液）。20 名吸烟者白细胞 DNA 中的 O(2)-edT、N(3)-edT 和 O(4)-edT 水平分别为 44.8 ± 52.0、41.1 ± 43.8 和 48.3 ± 53.9 个/10(8)个正常核苷酸，而 20 名非吸烟者的 O(2)-edT、N(3)-edT 和 O(4)-edT 水平分别为 0.19 ± 0.87、4.1 ± 13.3 和 1.0 ± 2.9。吸烟者白细胞 DNA 中的 O(2)-edT、N(3)-edT 和 O(4)-edT 水平均明显高于非吸烟者，p 值分别为 0.0004、0.0009 和 0.0004。此外，O(2)-edT 水平与吸烟者的吸烟指数呈显著统计学关联（γ=0.4789，p=0.0327）。在 40 个白细胞 DNA 样本中，O(2)-edT 和 O(4)-edT（γ=0.9896）、O(2)-edT 和 N(3)-edT（γ=0.9840）以及 N(3)-edT 和 O(4)-edT（γ=0.9901）之间观察到极显著的统计学相关性（p<0.0001）。据我们所知，这是第一个基于质谱的乙基化胸腺嘧啶加合物分析方法。使用该分析方法，首次检测和定量了三种乙基化胸腺嘧啶加合物。因此，这种高度敏感、特异性和准确的分析方法在临床上应该可行，可用于同时定量三种乙基化胸腺嘧啶加合物，作为接触乙基化剂的潜在生物标志物和癌症风险评估。
