Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan.
J Chromatogr A. 2013 Jan 4;1271(1):86-94. doi: 10.1016/j.chroma.2012.11.033. Epub 2012 Nov 22.
Cigarette smoke contains ethylating agents which damage DNA producing ethylated DNA adducts, such as N(3)-ethyladenine (3-EtAde), N(7)-ethylguanine (7-EtGua), and regioisomers of ethylthymine. Among them, 3-EtAde and 7-EtGua are present in human urine and their levels are higher in smokers than in nonsmokers. The amount of ethylated DNA adducts in tissue DNA represents the steady-state levels of DNA adducts resulting from the ethylating agent after repair in vivo. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of 3-EtAde and 7-EtGua by stable isotope dilution capillary liquid chromatography-nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS). Under the highly selective reaction monitoring (H-SRM) mode, the detection limit of 3-EtAde and 7-EtGua injected on-column was 5.0 fg (31 amol) and 10 fg (56 amol), respectively. The quantification limit for the entire assay was 50 and 100 fg of 3-EtAde and 7-EtGua, corresponding to 4.7 and 8.6 adducts in 10(9) normal nucleotides, respectively, starting with 20 μg of DNA isolated from <1 mL of blood and injecting an equivalent of 4 μg of DNA on-column. The mean (±SD) levels of 3-EtAde and 7-EtGua in leukocyte DNA from 20 smokers were 16.0±7.8 and 9.7±8.3 in 10(8) normal nucleotides, respectively, which were statistically significantly higher than those of 5.4±2.6 3-EtAde and 0.3±0.8 7-EtGua in 10(8) normal nucleotides from 20 nonsmokers (p<0.0001). The levels of 3-EtAde and 7-EtGua in these 40 leukocyte DNA samples are positively correlated (γ=0.6970, p<0.0001). Furthermore, there are statistically significant associations between the number of cigarettes smoked per day, as well as the smoking index, and the levels of 3-EtAde and 7-EtGua. Levels of 3-EtAde and 7-EtGua are compared to those of ethylthymidine adducts. To our knowledge, this is the first assay for simultaneous quantification of 3-EtAde and 7-EtGua in the same DNA sample and is the first report of 3-EtAde in human DNA. This highly sensitive and specific stable isotope dilution capLC-NSI/MS/MS assay should be useful in measuring 3-EtAde and 7-EtGua in human leukocyte DNA as potential biomarkers for smoking-related cancers.
香烟烟雾中含有烷基化剂，会破坏 DNA 并产生烷基化 DNA 加合物，如 N(3)-乙基腺嘌呤（3-EtAde）、N(7)-乙基鸟嘌呤（7-EtGua）和乙基胸腺嘧啶的位置异构体。其中，3-EtAde 和 7-EtGua 存在于人尿液中，其在吸烟者中的水平高于非吸烟者。组织 DNA 中烷基化 DNA 加合物的量代表体内修复后烷基化剂产生的 DNA 加合物的稳态水平。在这项研究中，我们开发了一种高度敏感、准确和定量的方法，通过稳定同位素稀释毛细管液相色谱-纳喷雾电离串联质谱（capLC-NSI/MS/MS）同时检测和定量 3-EtAde 和 7-EtGua。在高选择性反应监测（H-SRM）模式下，3-EtAde 和 7-EtGua 的柱上进样检测限分别为 5.0 fg（31 amol）和 10 fg（56 amol）。整个测定的定量限为 3-EtAde 和 7-EtGua 的 50 和 100 fg，分别对应于从 <1 mL 血液中分离的 20 μg DNA 起始时 10(9)个正常核苷酸中的 4.7 和 8.6 个加合物，以及柱上进样等效的 4 μg DNA。20 名吸烟者白细胞 DNA 中 3-EtAde 和 7-EtGua 的平均（±SD）水平分别为 10(8)个正常核苷酸中的 16.0±7.8 和 9.7±8.3，统计学上明显高于 20 名非吸烟者白细胞 DNA 中的 5.4±2.6 3-EtAde 和 0.3±0.8 7-EtGua（p<0.0001）。这 40 个白细胞 DNA 样本中 3-EtAde 和 7-EtGua 的水平呈正相关（γ=0.6970，p<0.0001）。此外，每天吸烟的支数以及吸烟指数与 3-EtAde 和 7-EtGua 的水平呈统计学显著关联。3-EtAde 和 7-EtGua 的水平与乙基胸腺嘧啶加合物的水平进行了比较。据我们所知，这是首次在同一 DNA 样本中同时定量检测 3-EtAde 和 7-EtGua，也是首次在人 DNA 中检测到 3-EtAde。这种高度敏感和特异的稳定同位素稀释 capLC-NSI/MS/MS 测定法应可用于测量人白细胞 DNA 中的 3-EtAde 和 7-EtGua，作为与吸烟相关癌症的潜在生物标志物。
