The Superoxide Dismutase Mimic TEMPOL Protects Cultured Rabbit Lens Epithelial Cells from Hydrogen Peroxide Insult

Abstract

The superoxide dismutase mimic, 4-hydroxy TEMPO (TEMPOL), was used to investigate the mechanism by which H 2O 2 damages cultured rabbit lens epithelial cells and to identify some of the targets of H 2O 2 insult. Most studies aimed at determining the mechanism by which H 2O 2 exerts its cytotoxic effect have used iron chelators to prevent the generation of the damaging hydroxyl radical. Since TEMPOL does not chelate transition metals, we were afforded an additional means of investigating the mechanism by which H 2O 2 exerts its cytotoxicity. Cells at low or high density were cultured in MEM containing 5 mM TEMPOL and exposed to a single sub-lethal dose of 0·05 or 0·5 mM H 2O 2, respectively. Analysis of EPR spectra indicated that TEMPOL was stable in MEM, did not destroy H 2O 2 and penetrated the intracellular fluid. TEMPOL prevented or curtailed the H 2O 2-induced inhibition of cell growth, blebbing of the cell membrane, the decrease in NAD +, the activation of poly ADP-ribose polymerase, an enzyme involved in DNA repair, and limited the induction of single strand breaks in DNA normally brought about by H 2O 2. TEMPOL did not prevent the H 2O 2-induced decrease in reduced glutathione, lactate production, and the activity of glyceraldehyde 3-phosphate dehydrogenase, or the H 2O 2-induced increases in oxidized glutathione and hexose monophosphate shunt activity. Addition of TEMPOL 1-15 min after exposure of cells to H 2O 2 offered partial protection from the inhibition of cell division. TEMPOL at 5 mM did not inhibit cell growth. These results, coupled with our other findings suggest that some of the H 2O 2-induced damage in cultured rabbit LECs is mediated by intracellular redox-active metals involved in the Haber-Weiss cycle. Cellular changes not protected by TEMPOL, including attack of H 2O 2 on the thiol groups of GSH (mediated through glutathione peroxidase) and G3PDH, are likely brought about by H 2O 2 itself and not by reactions of oxygen free-radicals generated from H 2O 2.