3-ethyladenine and 7-ethylguanine

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.

Topics: Adenine; Adolescent; Adult; Chromatography, High Pressure Liquid; DNA; DNA Adducts; Female; Guanine; Humans; Isotope Labeling; Leukocytes; Male; Reproducibility of Results; Sensitivity and Specificity; Smoking; Spectrometry, Mass, Electrospray Ionization; Statistics, Nonparametric; Tandem Mass Spectrometry

The extreme sensitivity of the developing rat brain to tumor induction by N-ethyl-N-nitrosourea (ENU) has been ascribed to the relatively inefficient repair of the presumed promutagenic lesion O6-ethylguanine from brain DNA. We have compared the brain of the newborn rat with liver, kidney and lung with respect to the repair of other types of DNA lesions that ENU induces, namely single strand breaks and alkali-labile lesions. The induction and repair or loss of these lesions has been analysed by alkaline sucrose gradient sedimentation using both mild as well as strong alkaline hydrolysis conditions. We found that ENU induces few lesions in the DNA of various organs that are detectable as breaks after mild alkaline hydrolysis. 24 h after ENU treatment such lesions are no longer detectable in brain DNA, while they are detectable in the DNA from other organs up to 10 days after ENU treatment. The number of ENU-induced lesions, detectable as breaks after strong alkaline hydrolysis, is far larger. These lesions were persistent in kidney DNA, disappeared slowly from liver DNA (t(1/2) = about 10 days), and more rapidly from brain and lung DNA (t(1/2) = 2-3 days). The latter result seems to be in contradiction with various reports that a large fraction of the ENU-induced alkali-labile lesions are stable in vivo. This difference between our results and those of others might be due to a difference between proliferating and non-proliferating cells. Whether the alkali-labile lesions are removed from brain and lung DNA by a specific repair mechanism or by other causes remains to be investigated.

Topics: Adenine; Alkylating Agents; Animals; Animals, Newborn; Brain; DNA; DNA Damage; DNA Repair; Ethylnitrosourea; Female; Guanine; Hydrogen-Ion Concentration; Kidney; Liver; Lung; Male; Rats; Rats, Sprague-Dawley; Sodium Hydroxide