Measurement of 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxo-7,8-dihydroguanine in isolated DNA exposed to gamma radiation in aqueous solution
Measurement of 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxo-7,8-dihydroguanine in isolated DNA exposed to gamma radiation in aqueous solution
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DOI：
10.1093/carcin/18.12.2385
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发表时间：
1997-12-01
期刊：
影响因子：
4.7
通讯作者：
Cadet, J
中科院分区：
文献类型：
--
作者：
Douki, T;Martini, R;Cadet, J
羟基自由基介导的核碱基修饰是电离辐射在 DNA 中诱导的主要有害过程之一，确定分离的 DNA 和细胞 DNA 的潜在机制需要开发用于检测修饰碱基的准确测定方法，电化学检测相关的高效液相色谱 (HPLC-EC) 和气相色谱-质谱联用 (GC-MS) 是用于此目的的两种主要方法。 DNA酶消化后产生8-oxo-7,8-二氢-2'-脱氧鸟苷（8-oxodGuo），为后一步的定量方面提供了证据，此外，设计了GC-MS测定法用于检测2,6-二氨基-4-羟基-5-甲酰胺嘧啶（FapyGua），首次表明广泛使用的DNA热甲酸水解诱导了病变的完全分解，对 4,6-二氨基-5-甲酰胺嘧啶 (FapyAde) 也进行了类似的观察，将一种较温和的 DNA 水解剂吡啶中的 HF 替换为 HCOOH，用于从 DNA 中定量释放 FapyGua，比较了 DNA 水溶液中辐射诱导的 8-oxodGuo 和 FapyGua 的形成，从不同条件下进行的 γ 辐照研究结果推断出机理方面的见解。在气体饱和条件下，清楚地表明两种病变的形成都是由 HO 度介导的。此外，氧气的存在有利于8-oxodGuo的形成，但会损害FapyGua。
Hydroxyl radical-mediated modifications of nucleobases are among the main deleterious processes induced in DNA by ionizing radiation, Determination of the underlying mechanisms in both isolated and cellular DNA requires the development of accurate assays for the detection of modified bases, High-performance liquid chromatography associated with electrochemical detection (HPLC-EC) and gas chromatography coupled to mass spectrometry (GC-MS) are the two main methods used for this purpose, In the present work, HPLC-EC was applied to the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) following enzymatic digestion of DNA, Evidence was provided for the quantitative aspect of the latter step, Moreover, a GC-MS assay was designed for the detection of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), It was first shown that the widely used hot formic acid hydrolysis of DNA induced a complete decomposition of the lesion, A similar observation was also made for 4,6-diamino-5-formamidopyrimidine (FapyAde), A milder DNA hydrolyzing agent, HF in pyridine, was substituted to HCOOH for the quantitative release of FapyGua from DNA, The radiation-induced formation of 8-oxodGuo and FapyGua in aqueous solution of DNA was compared, Insights in the mechanistic aspects were inferred from the results of gamma-irradiation studies carried out under different gas saturating conditions, It was clearly shown that the formation of both lesions is mediated by HO degrees. In addition, the presence of oxygen favors the formation of 8-oxodGuo at the expense of FapyGua.