Oxidation of 2 '-deoxyguanosine by H2O2-ascorbate: evidence against free OH. and thermodynamic support for two-electron reduction of H2O2

Of the four major nucleosides in DNA, 2'-deoxyguanosine (dG) is the most easily oxidized. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by oxidation of free dG, as well as of dG in DNA, by hydrogen peroxide (H2O2) and the reducing agent ascorbate (HAsc(-)) was measured using HPLC with high sensitivity electrochemical detection. High concentrations of the hydroxyl radical (OH.) scavengers PBN, DMPO, pentoxifylline and imidazole had a very limited protective effect on 8-oxodG formation, which provides evidence against free OH. production as the major mechanism. A possible mechanism involves a two-electron reduction of H2O2 with oxidation of HAsc(-) and dG, producing the guanine radical cation (dG(.+;+)) leading to 8-oxodG. This mechanism is, based on changes in Gibbs free energy (DeltaG(0)') at pH 7, much more favourable than OH. production. Catalase from Aspergillus niger and the aminoxyl TEMPO almost completely inhibited 8-oxodG formation, whereas bovine catalase cleaved the free dG molecule. Substantial 8-oxodG formation from free dG occurred even at 0 degreesC.