In vitro Inhibition by N-Acetylcysteine of Oxidative DNA Modifications Detected by ³²P Postlabeling

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 P_I (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 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.

Key Words:

• Reactive oxygen species
• hydrogen peroxide
• hydroxyl radical
• N-acetylcysteine
• DNA damage
• ³²P post-labeling