Detection and quantification of 1,N6-ethenoadenine in human placental DNA by mass spectrometry
Detection and quantification of 1,N6-ethenoadenine in human placental DNA by mass spectrometry
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DOI：
10.1021/tx990074s
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发表时间：
1999-12-01
影响因子：
4.1
通讯作者：
Chung, FL
中科院分区：
文献类型：
--
作者：
Chen, HJC;Chiang, LC;Chung, FL
已报道环外DNA加合物来源于各种外源性以及内源性来源，如脂质过氧化。其中，1，N-6-乙烯基腺嘌呤（ETADE）先前已检测到未经处理的啮齿类动物和人类的组织DNA的免疫亲和/P-32-后标记方法。本研究报告了通过三种独立测定（即GC/MS、LC/MS和HPLC/荧光）检测和定量相同人胎盘DNA中的内源性epsilon Ade加合物。采用气相色谱/负离子化学电离/质谱（GC/NICI/MS）方法[Chen，H. - J.C.，等人（1998）Chem.Res.Toxicol. 11，1.474]，发现来自商业来源的人胎盘DNA中的Ade水平为每10（6）个Ade碱基2.3个加合物。为了证实这些发现，开发了用于AdodAdo的液相色谱/电喷雾电离串联质谱（LC/ESI/MS/MS）方法。使用该LC/MS测定，在相同的人胎盘DNA中检测到2.5个加合物/10（6）个dAdo核苷的水平的dAdo。在GC/MS和LC/ESI/MS/MS测定中分别加入了稳定同位素的β-Ade和β-dAdo作为内标，因此提供了高特异性、重现性和准确定量。通过HPLC/荧光分析进一步验证了通过质谱法检测到的该人胎盘DNA中相对高水平的β-Ade。采用氯乙醛处理的小牛胸腺DNA通过HPLC/荧光测定法或2，3-环氧-4-羟基壬醛修饰的小牛胸腺DNA通过LC/MS法对GC/MS法进行了验证。在存在抗氧化剂的情况下新鲜分离的人胎盘DNA中的抗氧化剂水平与来自商业来源的DNA中的抗氧化剂水平相似。由于β-Ade是一种潜在的致突变性损伤，采用特异、灵敏的GC/NICI/MS方法分析β-Ade可能为癌症风险评估提供有用的生物标志物。
Exocyclic DNA adducts have been reported to derive from various exogenous as well as endogenous sources, such as lipid peroxidation. Among them, 1,N-6-ethenoadenine (epsilon Ade) has previously been detected in tissue DNA of untreated rodents and humans by an immunoaffinity/P-32-postlabeling method. This study reports detection and quantification of the endogenous epsilon Ade adduct in the same human placental DNA by three independent assays, namely, GC/MS, LC/MS, and HPLC/fluorescence. Using a recently reported gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) method [Chen, H. -J. C., et al. (1998) Chem. Res. Toxicol. 11, 1.474], the level of epsilon Ade in human placental DNA from a commercial source was found to be 2.3 adducts per 10(6) Ade bases. To confirm these findings, a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method was developed for epsilon dAdo. With this LC/MS assay, epsilon dAdo was detected at the level of 2.5 adducts per 10(6) dAdo nucleosides in the same human placental DNA. The stable isotopes of epsilon Ade and epsilon dAdo were added as internal standards in both GC/MS and LC/ESI/MS/MS assays, respectively, and thus provided high specificity, reproducibility, and accurate quantification. The relatively high levels of epsilon Ade in this human placental DNA detected by mass spectrometry were further verified by HPLC/fluorescence analysis. The GC/MS method was validated by the HPLC/fluorescence assay using calf thymus DNA treated with chloroacetaldehyde or by the LC/MS method with 2,3-epoxy-4-hydroxynonanal-modified calf thymus DNA. The epsilon Ade level, in human placental DNA freshly isolated in the presence of an antioxidant was similar to that in DNA from the commercial source. Since epsilon Ade is a potential mutagenic lesion, analysis of epsilon Ade by the specific and sensitive GC/NICI/MS method may provide a useful biomarker in cancer risk assessment.