以穩定同位素稀釋奈升流速液相層析奈電噴灑游離串聯式質譜法在人類唾液DNA和尿液中定量乙基嘌呤加成產物

Quantitative Assay for Ethylpurine Adducts in Human Salivary DNA and in Urine by Stable Isotope Dilution nanoLC-Nanospray Ionization Tandem Mass Spectrometry

林朝瑞(Lin, Chao Ruei)

指導教授 ： 陳皓君

國立中正大學/理學院/化學暨生物化學系/碩士(2013年)

摘要

香煙中含有許多烷基化試劑，研究顯示抽菸會導致DNA受到乙基化試劑的傷害而產生DNA乙基化加成產物，如3-乙基腺嘌呤和7-乙基鳥糞嘌。在文獻中只有研究是利用人類唾液DNA去分析吸菸者與非吸菸者間，DNA遭受tobacco carcinogen phenylimidazo[4,5-b]pyridine (PhIP)修飾的程度，結果顯示在吸菸者可量測到的加成產物dG-C8-PhIP平均高於非吸菸者。在本研究中，我們使用高特異性與靈敏度的奈升流速液相層析奈電噴灑游離串聯式質譜法搭配穩定同位素稀釋法來量測人類唾液DNA與尿液中的乙基化加成產物。在人類唾液DNA部分，我們利用中性熱水解方法使3-乙基腺嘌呤和7-乙基鳥糞嘌呤從唾液DNA上被釋出。尿液部分則是經由高速離心後收集包含這些乙基化加成產物的上清液。之後再透過固相萃取管柱的樣品前處理步驟來純化這些乙基化加成產物。利用本分析方法所得之3-乙基腺嘌呤及7-乙基鳥糞嘌呤的偵測極限分別為92和56 amol。而3-乙基腺嘌呤及7-乙基鳥糞嘌呤的定量極限分別為310和280 amol。在14位吸菸者的唾液DNA含量為，108個正常核苷酸中有12.5 ± 7.2個3-乙基腺嘌呤與14.0 ± 8.5個7-乙基鳥糞嘌呤；在15位非吸菸者的含量為，108個正常核苷酸中有9.7 ± 5.3個3-乙基腺嘌呤與3.8 ± 2.8個7-乙基鳥糞嘌呤。比較此二加成產物在吸菸者與非吸菸者唾液DNA之含量，其7-乙基鳥糞嘌呤之p值小於0.05。在尿液樣品部分，3-乙基腺嘌呤及7-乙基鳥糞嘌呤的定量極限分別為620和560 amol。在20位吸菸者的人類尿液樣品中，3-乙基腺嘌呤與7-乙基鳥糞嘌呤含量分別為66.5 ± 28.6 pg/mL與18.5 ± 14.2 pg/mL；在20位非吸菸者中，3-乙基腺嘌呤與7-乙基鳥糞嘌呤含量則有3.5 ± 3.8 pg/mL與2.4 ± 3.0 pg/mL。比較此二加成產物在吸菸者與非吸菸者尿液樣品之含量，其3-乙基腺嘌呤與7-乙基鳥糞嘌呤之p值皆小於0.05。藉由此分析方法來評估DNA受到乙基化損害的程度，可比較出3-乙基腺嘌呤和7-乙基鳥糞嘌呤與吸菸相關疾病的相關性，做為癌症風險評估的生化指標。

關鍵字

7-乙基鳥糞嘌呤 ； 3-乙基腺嘌呤 ； 人類唾液DNA

並列摘要

Cigarette smoke contains many alkylating agents which damage DNA producing DNA adducts, such as N3-ethyladenine (3-EtA) and N7-ethylguanine (7-EtG). Cells of the oral cavity have been employed to measure DNA adducts that modified by tobacco carcinogen phenylimidazo[4,5-b]pyridine (PhIP) following exposure to tobacco smoke and levels of dG-C8-PhIP in smokers were higher than in nonsmoker. In this study, a highly specific and sensitive assay based on stable isotope dilution nanoLC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was used to measure 3-EtA and 7-EtG in human salivary DNA and urine. Neutral thermal hydrolysis was used to release 3-EtA and 7-EtG from DNA, while the supernatant of urine containing adducts was obtained after centrifugation. These adducts from the supernatant of DNA hydrolysate and urine were enriched by a reversed-phase solid-phase extraction column before nanoLC-NSI/MS/MS analysis. The on-column detection limits (S/N ≥ 3) of 3-EtA and 7-EtG were 92 and 56 amol, respectively. The quantification limits of 3-EtA and 7-EtG were 310 and 280 amol, respectively. In human salivary DNA, levels of 3-EtA and 7-EtG in 14 smokers were 12.5 ± 7.2 and 14.0 ± 8.5 in 108 normal nucleotides, respectively. In 15 nonsmokers, levels of 3-EtA and 7-EtG were 9.7 ± 5.3 and 3.8 ± 2.8 in 108 normal nucleotides, respectively. The level of 7-EtG was statistically significantly higher in smokers than in nonsmokers (p < 0.05). In human urinary samples, adduct concentrations of 3-EtA and 7-EtG in 20 smokers were 66.5 ± 28.6 and 18.5 ± 14.2 pg/mL urine, respectively. In 20 nonsmokers, adduct concentrations of 3-EtA and 7-EtG were 3.5 ± 3.8 and 2.4 ± 3.0 pg/mL urine, respectively. The concentrations of 3-EtA and 7-EtG were statistically significantly higher in smokers than in nonsmokers (p < 0.05). This highly specific and sensitive assay based on stable isotope dilution nanoLC-NSI/MS/MS assay should be clinically useful in assessing the possibility of measuring ethylpurines in human salivary DNA and in urine as risk biomarkers for smoking-related cancers.

並列關鍵字

Human Salivary DNA ； 7-ethylguanine ； 3-ethyladenine

參考文獻

2. Hemminki, K., Nucleic acid adducts of chemical carcinogens and mutagens. Arch. Toxicol. 1983, 52, 249-285.

3. Dipple, A., DNA adducts of chemical carcinogens. Carcinogenesis 1995, 16, 437-441.

4. Singh, R.; Farmer, P. B., Liquid chromatography-electrospray ionization-mass spectrometry: the future of DNA adduct detection. Carcinogenesis 2006, 27, 178-196.

9. Stephany, R. W.; Freudenthal, J.; Egmond, E.; Gramberg, L. G.; Schuller, P. L., Mass spectrometric quantification of traces of volatile N-nitrosamines in meat products. J. Agric. Food Chem. 1976, 24, 536-539.

10. Hecht, S. S., DNA adduct formation from tobacco-specific N-nitrosamines. Mutat. Res. 1999, 424, 127-142.