Povey A C, Wilson V L, Weston A, Doan V T, Wood M L, Essigmann J M, Shields P G
Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
IARC Sci Publ. 1993(124):105-14.
Human exposure to reactive oxygen species is unavoidable and has been implicated in the etiology of a number of human diseases. This exposure results in the formation of various modified DNA bases: the promutagenic lesion 8-hydroxydeoxyguanosine (8OHdG), in particular, is a major product. We have developed an assay using ion-pair HPLC and 32P-postlabelling to quantify 8OHdG in human DNA with high specificity and sensitivity. An internal standard is used to account for variations in labelling efficiency. Chemically synthesized 8OHdG 3'-monophosphate and 5'-monophosphate standards were used to optimize the HPLC-32P-postlabelling and TLC separative steps, respectively. The assay was validated using known ratios of 8OHdG to normal nucleotides. The limit of detection is in the range of one 8OHdG residue per 10(6)-10(7) dG residues. Using this procedure, 8OHdG levels of 16-35 8OHdG adducts per 10(5) dG residues have been found in leukocytes isolated from patients who received 180-200 cGy of ionizing radiation. These levels were 2-4-fold greater than those found in an unexposed individual. Since 8OHdG may be formed during DNA extraction and digestion, current procedures for measuring background levels are discussed.
人类不可避免地会接触到活性氧物种，这与许多人类疾病的病因有关。这种接触会导致各种修饰的DNA碱基形成：特别是前诱变损伤8-羟基脱氧鸟苷（8OHdG）是主要产物。我们开发了一种使用离子对高效液相色谱法和32P后标记法的检测方法，以高特异性和灵敏度定量人DNA中的8OHdG。使用内标来解释标记效率的变化。化学合成的8OHdG 3'-单磷酸酯和5'-单磷酸酯标准品分别用于优化HPLC-3×2P后标记法和薄层色谱分离步骤。该检测方法使用已知的8OHdG与正常核苷酸的比例进行了验证。检测限为每10(6)-10(7)个dG残基中有一个8OHdG残基。使用该方法，在接受180-200 cGy电离辐射的患者分离的白细胞中，发现每10(5)个dG残基的8OHdG水平为16-35个8OHdG加合物。这些水平比未暴露个体中的水平高2-4倍。由于8OHdG可能在DNA提取和消化过程中形成，因此讨论了测量背景水平的现行方法。
