Noninvasive measurement of smoking-associated N³-ethyladenine and N⁷-ethylguanine in human salivary DNA by stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry

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• 作者：Hauh-Jyun C ; y Chen ; Chao-Ray Lin
• 关键词：DNA adduct ; 3-Ethyladenine ; 7-Ethylguanine ; Nanoflow LC&ndash ; MS/MS ; Saliva
• 刊名：Toxicology Letters
• 出版年：10 February, 2014
• 年：2014
• 卷：225
• 期：1
• 页码：27-33
• 全文大小：1146 K

文摘

Evidence showed that ethylating agents are contained in cigarette smoke, which damage DNA producing ethylated DNA adducts, including N³-ethyladenine (3-EtAde) and N⁷-ethylguanine (7-EtGua). These two ethylpurines can be depurinated spontaneously and be repaired by enzymes and they have been detected in human urine. In this study, a highly specific and sensitive assay based on stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was used to measure 3-EtAde and 7-EtGua in human salivary DNA. These ethylpurines were released from DNA by neutral thermal hydrolysis and then enriched by a solid-phase extraction column before nanoLC-NSI/MS/MS analysis. The detection limits (S/N 鈮?#xA0;3) of 3-EtA and 7-EtG were 15 fg (92 amol) and 10 fg (56 amol), respectively, injected on-column. The lower quantification limits of 3-EtAde and 7-EtGua were both 100 fg, i.e. 620 and 560 amol, respectively, corresponding to 9.4 and 8.6 adducts in 10⁹ normal nucleotides, respectively, starting with as little as 20 渭g of DNA isolated from an average of 3 mL of saliva. The mean (卤SD) levels of 3-EtAde in 15 smokers and 15 nonsmokers were 12.6 卤 7.0 and 9.7 卤 5.3 in 10⁸ normal nucleotides, respectively, while those of 7-EtGua were 14.1 卤 8.2 and 3.8 卤 2.8 in 10⁸ normal nucleotides in smokers and nonsmokers, respectively. Levels of 7-EtGua, but not 3-EtAde, were statistically significantly higher in smokers than in nonsmokers (p < 0.0001). Furthermore, salivary 7-EtGua levels are significantly correlated with the number of cigarettes smoked per day as well as with the smoking index. This highly specific and sensitive stable isotope dilution nanoLC-NSI/MS/MS assay might be feasible in measuring 7-EtGua in human salivary DNA as a noninvasive biomarker for DNA damage induced by cigarette smoking.