Douki T, Delatour T, Paganon F, Cadet J
CEA/Département de Recherche Fondamentale sur la Matière Condensée, France.
Chem Res Toxicol. 1996 Oct-Nov;9(7):1145-51. doi: 10.1021/tx960095b.
Oxidized nucleobases represent one of the main classes of damage induced in DNA by ionizing radiation. Emphasis was placed in this work on the measurement of four oxidized pyrimidine bases, including 5-(hydroxymethyl)uracil (5-HMUra), 5-formyluracil (5-ForUra), 5-hydroxycytosine (5-OHCyt), and 5-hydroxyuracil (5-OHUra), in isolated DNA upon exposure to gamma radiation in aerated aqueous solution. For this purpose, both high performance liquid chromatography associated with electrochemical detection (HPLC-EC) and gas chromatography coupled to mass spectrometry (GC-MS) were used. Conditions of hydrolysis of the N-glycosidic bond were carefully checked in order to achieve a quantitative release of the lesions. We showed that 60% formic acid treatment leads to the decomposition of the four lesions studied. On the other hand, hydrolysis based on the use of either 88% formic acid or 70% hydrogen fluoride in pyridine (HF/Pyr) allowed the quantitative release of the modified bases, with the exception of 5-HMUra when the latter reagent was utilized. A dose course study of the radiation-induced formation of 5-HMUra and 5-ForUra in DNA by using the GC-MS assay showed that the latter lesion was produced in a 2.1-fold higher yield than the former one. HF/Pyr and 88% formic acid hydrolysis provided similar results for 5-ForUra, indicating the reliability of both techniques for the measurement of this lesion. For 5-OHUra and 5-OHCyt, the level of modification determined by GC-MS analysis was higher after 88% formic acid treatment than upon HF/Pyr hydrolysis. When DNA was enzymatically digested and analyzed by HPLC-EC for 5-OHdCyd and 5-OHdUrd, the results were very close to those obtained by GC-MS following HF/Pyr treatment. It was concluded that additional amounts of both 5-OHUra and 5-OHCyt are produced during the 88% formic acid treatment from radiation-induced 5,6-saturated pyrimidine precursors. It is likely that cytosine and uracil diols are involved in this reaction. The radiochemical yields of formation (in mumol.J-1) for the products studied are in the following decreasing order: 5-ForUra (0.0083) > 5-OHCyt (0.0046) > 5-HMUra (0.0039) > 5-OHUra (0.0035).
氧化核碱基是电离辐射在DNA中诱导产生的主要损伤类型之一。本研究重点在于测量在充氧水溶液中，经γ辐射处理的分离DNA中的四种氧化嘧啶碱基，包括5 -（羟甲基）尿嘧啶（5 - HMUra）、5 - 甲酰基尿嘧啶（5 - ForUra）、5 - 羟基胞嘧啶（5 - OHCyt）和5 - 羟基尿嘧啶（5 - OHUra）。为此，使用了与电化学检测联用的高效液相色谱法（HPLC - EC）和与质谱联用的气相色谱法（GC - MS）。仔细检查了N - 糖苷键的水解条件，以实现损伤的定量释放。我们发现60%甲酸处理会导致所研究的四种损伤分解。另一方面，基于使用88%甲酸或吡啶中的70%氟化氢（HF/Pyr）进行的水解能够定量释放修饰碱基，但使用后一种试剂时5 - HMUra除外。通过GC - MS分析对DNA中辐射诱导形成的5 - HMUra和5 - ForUra进行剂量过程研究表明，后一种损伤的产生产率比前一种高2.1倍。HF/Pyr和88%甲酸水解对5 - ForUra给出了相似结果，表明这两种技术对于测量这种损伤都是可靠的。对于5 - OHUra和5 - OHCyt，GC - MS分析确定的修饰水平在88%甲酸处理后高于HF/Pyr水解后的水平。当对DNA进行酶切并用HPLC - EC分析5 - OHdCyd和5 - OHdUrd时，结果与HF/Pyr处理后通过GC - MS获得的结果非常接近。得出的结论是，在88%甲酸处理过程中，由辐射诱导的5,6 - 饱和嘧啶前体产生了额外量的5 - OHUra和5 - OHCyt。胞嘧啶和尿嘧啶二醇可能参与了此反应。所研究产物的形成放射化学产率（以μmol·J⁻¹计）按以下降序排列：5 - ForUra（0.0083）> 5 - OHCyt（0.0046）> 5 - HMUra（0.0039）> 5 - OHUra（0.0035）。
