The role of metal ion binding in the antioxidant mechanisms of reduced and oxidized glutathione in metal-mediated oxidative DNA damage

Abstract

The antioxidant activity of glutathione in its reduced (GSH) and oxidized (GSSG) forms against metal-mediated oxidative DNA damage was studied by monitoring production of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) from calf-thymus DNA. GSH and GSSG were combined with Fe(II) and Cu(II) before and after addition of DNA to investigate the role of metal coordination in the antioxidant mechanism. The antioxidant behavior of GSH and GSSG was also compared to the known radical scavenger DMSO. GSH and GSSG lower oxidative DNA damage for Fe(II) and Cu(II) reactions. GSH only exhibited appreciable antioxidant behavior when combined with Fe(II) prior to adding DNA, and GSH and GSSG were slightly more effective against Cu(II)-mediated damage when combined with Cu(II) prior to adding DNA. Raman spectra of GSH in the presence of Cu(II) indicate that Cu(II) oxidizes GSH and raises the possibility that the antioxidant activity of GSH against Cu(II) reactions may be attributed to its ability to form GSSG. No evidence of GSH oxidation in the presence of Fe(II) was observed. The fluorescent probe dichlorofluorescein diacetate (DCF-DA) shows that the presence of GSH (for Cu(II) reactions) and GSSG (for Fe(II) and Cu(II) reactions) lowers levels of reactive oxygen species (ROS) in bulk solution. Overall, the results suggest that the mechanism of antioxidant activity for GSH and GSSG against Fe(II) and Cu(II)-mediated oxidative damage involves metal coordination, and isothermal titration calorimetry (ITC) studies of the Cu(II)–GSSG system show an enthalpically favored complexation reaction with an apparent 1 : 1 stoichiometry.