HYDROXYLATION OF DEOXYGUANOSINE IN DNA BY COPPER AND THIOLS

DNA was incubated with glutathione (GSH) and copper and then assayed f or 8-hydroxydeoxyguanosine (8-OHdG) in order to better understand the antioxidant and prooxidant characteristics of GSH in copper-dependent DNA damage. Ratios of GSH to Cu(II) less than 3 resulted in 8-OHdG pro duction; however, higher ratios did not generate 8-OHdG. A combination of GSH and Cu(I) (10:1) was used to determine if DNA oxidation occurr ed upon the addition of H2O2. No increase in 8-OHdG was noted until th e concentration of H2O2 was almost half that of GSH, and then a substa ntial increase of 8-OHdG was detected. The stoichiometry of thiol oxid ation by H2O2 was 2 mol GSH oxidized per 1 mol H2O2. Oxidation of Cu(I ) was not detected until most of the thiol had been oxidized. When cys teine and Cu(I) was used instead of GSH and Cu(I), there was considera ble hydroxylation of deoxyguanosine. The glycyl carboxyl, the gamma-gl utamate carboxyl, and the amine of G8H were altered to determine their role in the peptide's ability to inhibit Cu-dependent damage. In the presence of Cu(I), H2O2, and DNA, these GSH analogs behaved similarly to GSH. However, when S-methylglutathione was used in this system, it was very effective at promoting oxidative damage to DNA. This indicate d that the thiol ligand of GSH was essential for inhibition of Cu-depe ndent damage, while the carboxyl groups and the amine were not essenti al ligands. In conclusion, GSH can catalyze the in vitro hydroxylation of deoxyguanosine when the ratio of GSH to Cu is low, however, when t he ratio is high GSH is an effective antioxidant. (C) 1995 Academic Pr ess, Inc.