In vitroInhibition byN-Acetylcysteine of Oxidative DNA Modifications Detected by32P Postlabeling

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Abstract
Reactive oxygen species are involved in the pathogen-esis 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 chromatcgraphic 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 PI (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 (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 electrophore-sis, 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.