title: Noninvasive measurement of smoking-associated N(3)-ethyladenine and N(7)-ethylguanine in human salivary DNA by stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry.
abstract: Evidence showed that ethylating agents are contained in cigarette smoke, which damage DNA producing ethylated DNA adducts, including N(3)-ethyladenine (3-EtAde) and N(7)-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≥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 10(9) normal nucleotides, respectively, starting with as little as 20 μg of DNA isolated from an average of 3 mL of saliva. The mean (±SD) levels of 3-EtAde in 15 smokers and 15 nonsmokers were 12.6±7.0 and 9.7±5.3 in 10(8) normal nucleotides, respectively, while those of 7-EtGua were 14.1±8.2 and 3.8±2.8 in 10(8) 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.
authors.affiliation: Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan. Electronic address: chehjc@ccu.edu.tw.
authors.forename: Hauh-Jyun Candy
authors.initials: HJ
authors.lastname: Chen
authors.affiliation: Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan.
authors.forename: Chao-Ray
authors.initials: CR
authors.lastname: Lin
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism*; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007201:Indicator Dilution Techniques; D057230:Limit of Detection; D008297:Male; D036103:Nanotechnology; D015203:Reproducibility of Results; D012463:Saliva / Q000378:metabolism*; D012907:Smoking / Q000009:adverse effects / Q000235:genetics / Q000378:metabolism*; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry; D055815:Young Adult
chemical_list: D015415:Biomarkers; D018736:DNA Adducts; C580785:N7-ethylguanine; C022595:3-ethyladenine; D006147:Guanine; D000225:Adenine
keywords: 3-Ethyladenine; 7-Ethylguanine; DNA adduct; Nanoflow LC–MS/MS; Saliva