Quantification of the 2-Deoxyribonolactone and Nucleoside 5′-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of γ-Radiation and Fe2+−EDTA,Journal of the American Chemical Society

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Quantification of the 2-Deoxyribonolactone and Nucleoside 5′-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of γ-Radiation and Fe2+−EDTA
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2010-05-05 , DOI: 10.1021/ja910928n
Wan Chan ₁ , Bingzi Chen , Lianrong Wang , Koli Taghizadeh , Michael S Demott , Peter C Dedon

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The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin gamma(1)(I), was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5'-aldehyde per 10(6) nt per microM in accord with its established minor 1'- and major 5'-oxidation chemistry. Calicheamicin unexpectedly caused 1'-oxidation at a low level of 10 2-deoxyribonolactone per 10(6) nt per microM in addition to the expected predominance of 5'-oxidation at 560 nucleoside 5'-aldehyde per 10(6) nt per microM. The two hydroxyl radical-mediated DNA oxidants, gamma-radiation and Fe(2+)-EDTA, produced nucleoside 5'-aldehyde at a frequency of 57 per 10(6) nt per Gy (G-value 74 nmol/J) and 3.5 per 10(6) nt per microM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, gamma-radiation and Fe(2+)-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 10(6) nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 10(6) nt per microM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5'-aldehyde of 9.7 and 73 lesions per 10(6) nt, respectively. Gamma-irradiation of the cells caused increases of 0.045 and 0.22 lesions per 10(6) nt per Gy, respectively, which represents a approximately 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by gamma-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between gamma-radiation and Fe(2+)-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.

中文翻译：

通过同位素稀释气相色谱质谱法定量 DNA 和细胞中 2-脱氧核糖氧化的 2-脱氧核糖内酯和核苷 5'-醛产物：γ-辐射和 Fe2+−EDTA 的差异效应

DNA 中 2-脱氧核糖的氧化已成为 DNA 氧化损伤细胞毒性的关键决定因素，每个碳的氧化都会产生独特的亲电子产物谱。我们开发并验证了一种同位素稀释气相色谱-质谱联用 (GC-MS) 方法，用于严格定量两种主要的 2-脱氧核糖氧化产物：1'-氧化的 2-脱氧核糖内酯脱碱基位点和核苷 5' 5'-氧化化学的-醛。该方法需要消除这些产物分别为 5-亚甲基-2(5H)-呋喃酮 (5MF) 和糠醛，然后用五氟苯肼 (PFPH) 衍生化，添加同位素标记的 PFPH 衍生物作为内标，提取衍生物，并通过 GC-MS 分析进行定量。该方法的精密度和准确性通过含有 2-脱氧核糖内酯和核苷 5'-醛损伤的寡脱氧核苷酸进行验证。此外，在对照研究中利用了烯二炔抗生素、新制癌素和加利车霉素 γ(1)(I) 的明确定义的 2-脱氧核糖氧化化学，新制癌素每 10( 6) nt per microM 根据其已建立的次要 1'-和主要 5'-氧化化学。除了预期的 5'-氧化在 560 核苷 5'-醛每 10(6) nt/microM 的预期优势外，加利车霉素意外地导致 1'-氧化，每 10(6) nt/microM 的低水平为 10 2-脱氧核糖内酯. 两种羟基自由基介导的 DNA 氧化剂，γ-辐射和 Fe(2+)-EDTA，以每 10(6) nt/Gy 57 的频率（G 值 74 nmol/J）和每 10(6) nt/microM 3.5 的频率产生核苷 5'-醛，分别为 40% 和 35%，分别是通过质粒切口测定法测量的总 2-脱氧核糖氧化。然而，γ-辐射和 Fe(2+)-EDTA 产生不同比例的 2-脱氧核糖内酯，分别占总 2-脱氧核糖氧化的 7% 和 24%，频率为每 10(6) nt/Gy 10 个病变 (G -值，13 nmol/J) 和每 10(6) nt/microM 2.4 个病变。TK6 人类淋巴母细胞的研究，其中分析数据针对 DNA 分离过程中持续的损失进行了校正，揭示了 2-脱氧核糖内酯和核苷 5'-醛的背景水平分别为每 10(6) nt 9.7 和 73 个病变。细胞的伽马辐射导致 0.045 和 0 的增加。每 10(6) nt/Gy 分别有 22 处病变，这代表细胞环境的大约 250 倍猝灭效应，类似于以前的研究中观察到的效应。对于纯化的 DNA 和细胞，伽马辐射产生的各种 2-脱氧核糖氧化产物的比例相似。这些结果与溶剂暴露是与 DNA 中 2-脱氧核糖发生羟基自由基反应性的主要决定因素一致，但伽马辐射和 Fe(2+)-EDTA 之间的巨大差异表明，除羟基自由基反应性之外的其他因素控制着 DNA 氧化化学. 对于纯化的 DNA 和细胞，伽马辐射产生的各种 2-脱氧核糖氧化产物的比例相似。这些结果与溶剂暴露是与 DNA 中 2-脱氧核糖发生羟基自由基反应性的主要决定因素一致，但伽马辐射和 Fe(2+)-EDTA 之间的巨大差异表明，除羟基自由基反应性之外的其他因素控制着 DNA 氧化化学. 对于纯化的 DNA 和细胞，伽马辐射产生的各种 2-脱氧核糖氧化产物的比例相似。这些结果与溶剂暴露是与 DNA 中 2-脱氧核糖发生羟基自由基反应性的主要决定因素一致，但伽马辐射和 Fe(2+)-EDTA 之间的巨大差异表明，除羟基自由基反应性之外的其他因素控制着 DNA 氧化化学.

更新日期：2010-05-05

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