Tempol and deferoxamine protect cultured rabbit lens epithelial cells from H2O2 insult: insight into the mechanism of H2O2-induced injury.

Abstract

In order to investigate the mechanism by which H2O2 damages the epithelium, 8 x 10(5) rabbit lens epithelial cells were treated with TEMPOL or deferoxamine and exposed to a single sublethal dose of 0.5 mM H2O2. TEMPOL is a SOD mimic, has a characteristic EPR spectrum and is metal independent. EPR spectra indicated that TEMPOL was not destroyed by H2O2, catalyzed the destruction of the superoxide anion, and penetrated the cells. Cells treated with H2O2 showed membrane blebbing, growth inhibition, an increase in GSSG, a dose-dependent decrease in GSH, ATP, NAD+, and in the activity of G3PDH, and in lactate production. H2O2 stimulated the hexose mono-phosphate shunt and induced single strand breaks in DNA. Treatment with TEMPOL or deferoxamine prevented or curtailed H2O2-induced inhibition of growth, the decrease in NAD+, the induction of single strand breaks in DNA, and membrane blebbing, but not the other biochemical parameters investigated. Both TEMPOL and deferoxamine prevent Fe+2-mediated generation of the damaging hydroxyl radical. TEMPOL reacts with superoxide and thus prevents it from recycling Fe+3 to Fe+2. It also oxidizes DNA-Fe+2 to DNA-Fe+3. Deferoxamine chelates intracellular Fe+3 and prevents its reduction to Fe+2. These compounds which limit the availability of Fe+2 by different means indicate that transition metals (including those bound to DNA) mediate certain of the damaging effects of H2O2.