Abstract

Cigarette smoke contains ethylating agents which damage DNA producing ethylated DNA adducts, such as N3-ethyladenine (3-EtAde), N7-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 5.0fg (31amol) and 10fg (56amol), respectively. The quantification limit for the entire assay was 50 and 100fg of 3-EtAde and 7-EtGua, corresponding to 4.7 and 8.6 adducts in 109 normal nucleotides, respectively, starting with 20μg of DNA isolated from <1mL of blood and injecting an equivalent of 4μg 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 108 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 108 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.

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