title: Simultaneous quantitative analysis of N3-ethyladenine and N7-ethylguanine in human leukocyte deoxyribonucleic acid by stable isotope dilution capillary liquid chromatography-nanospray ionization tandem mass spectrometry.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan. chehjc@ccu.edu.tw
authors.forename: Hauh-Jyun Candy
authors.initials: HJ
authors.lastname: Chen
authors.forename: Yen-Fu
authors.initials: YF
authors.lastname: Liu
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000097:blood; D000293:Adolescent; D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000097:blood; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives* / Q000097:blood; D006801:Humans; D007553:Isotope Labeling; D007962:Leukocytes / Q000737:chemistry*; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012907:Smoking / Q000097:blood; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D018709:Statistics, Nonparametric; D053719:Tandem Mass Spectrometry
chemical_list: D018736:DNA Adducts; C022007:7-ethylguanine; C022595:3-ethyladenine; D006147:Guanine; D004247:DNA; D000225:Adenine