Hydroxylation of deoxyguanosine in DNA by copper and thiols.

DNA was incubated with glutathione (GSH) and copper and then assayed for 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 production; however, higher ratios did not generate 8-OHdG. A combination of GSH and Cu(I) (10:1) was used to determine if DNA oxidation occurred upon the addition of H2O2. No increase in 8-OHdG was noted until the concentration of H2O2 was almost half that of GSH, and then a substantial increase of 8-OHdG was detected. The stoichiometry of thiol oxidation 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 cysteine and Cu(I) was used instead of GSH and Cu(I), there was considerable hydroxylation of deoxyguanosine. The glycyl carboxyl, the gamma-glutamate carboxyl, and the amine of GSH 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 indicated that the thiol ligand of GSH was essential for inhibition of Cu-dependent damage, while the carboxyl groups and the amine were not essential ligands. In conclusion, GSH can catalyze the in vitro hydroxylation of deoxyguanosine when the ratio of GSH to Cu is low, however, when the ratio is high GSH is an effective antioxidant.