Izzotti A, Orlando M, Gasparini L, Scatolini L, Cartiglia C, Tulimiero L, De Flora S
Institute of Hygiene and Preventive Medicine, University of Genoa, Italy.
Free Radic Res. 1998 Feb;28(2):165-78. doi: 10.3109/10715769809065802.
Reactive oxygen species are involved in the pathogenesis of cancer and other chronic degenerative diseases through a variety of mechanisms, including DNA damage. We investigated by 32p and 33P postlabeling analyses the nucleotidic modifications induced in vitro by treating calf thymus DNA with H2O2 and CuSO4, interacting in a Fenton type reaction. Six different enrichment procedures and three chromatographic systems were comparatively assayed. The chromatographic system using phosphate/urea, which is more suitable for detecting bulky DNA adducts, was rather insensitive. In contrast, the system using acetic acid/ammonium formate revealed high levels of mononucleotidic modifications. In terms of ratio of adduct levels in treated and untreated DNA, the enrichment procedures ranked as follows: nuclease P1 (19.6), no enrichment (18.3), digestion to trinucleotides (17.6), digestion to monophosphate mononucleotides (8.4), digestion to dinucleotides (3.4), and extraction with butanol (<1.0). The system using formic acid/ammonium formate was quite efficient in detecting 8-hydroxy-2'-deoxyguanosine. Labeling with 33p further enhanced the sensitivity of the method. The oxidative damage was so intense to produce a strong DNA fragmentation detectable by agarose gel electrophoresis, and nucleotidic modifications were more intense when DNA fragmentation was greater. The DNA alterations produced by H2O2 alone were significantly lower than those produced following reaction of H2O2 with CuSO4. The thiol N-acetylcysteine (NAC) was quite efficient in inhibiting both nucleotidic modifications and DNA fragmentation produced in vitro by either H2O2 or the .OH generating system. These results support at a molecular level the findings of previous studies showing the ability of NAC to inhibit the genotoxicity of peroxides and of reactive oxygen species generated by electron transfer reactions.
活性氧通过多种机制参与癌症和其他慢性退行性疾病的发病过程，包括DNA损伤。我们通过32P和33P后标记分析，研究了在Fenton型反应中，用H2O2和CuSO4处理小牛胸腺DNA体外诱导的核苷酸修饰。比较分析了六种不同的富集程序和三种色谱系统。使用磷酸盐/尿素的色谱系统更适合检测大分子DNA加合物，但灵敏度较低。相比之下，使用乙酸/甲酸铵的系统显示出高水平的单核苷酸修饰。就处理后和未处理DNA的加合物水平之比而言，富集程序的排名如下：核酸酶P1（19.6）、无富集（18.3）、消化为三核苷酸（17.6）、消化为单磷酸单核苷酸（8.4）、消化为二核苷酸（3.4）和用丁醇萃取（<1.0）。使用甲酸/甲酸铵的系统在检测8-羟基-2'-脱氧鸟苷方面相当有效。用33P标记进一步提高了该方法的灵敏度。氧化损伤非常严重，可通过琼脂糖凝胶电泳检测到强烈的DNA片段化，并且当DNA片段化程度更大时，核苷酸修饰更强烈。单独由H2O2产生的DNA改变明显低于H2O2与CuSO4反应后产生的改变。硫醇N-乙酰半胱氨酸（NAC）在抑制由H2O2或·OH生成系统体外产生的核苷酸修饰和DNA片段化方面相当有效。这些结果在分子水平上支持了先前研究的发现，即NAC能够抑制过氧化物和电子转移反应产生的活性氧的遗传毒性。
