Protection from Oxidative Insult in Glutathione Depleted Lens Epithelial Cells

Abstract

It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine, compounds that limit the availability of Fe+2and prevent the generation of hydroxyl radicals, protect cultured rabbit lens epithelial cells from H2O2-induced damage. In view of the importance of glutathione as an antioxidant and the decrease in GSH that is known to accompany most forms of cataract, we investigated whether these compounds protected cultured lens epithelial cells from H2O2when the cells were artificially depleted of glutathione. Treatment of lens epithelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound that irreversibly binds to glutathione, or buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis, reduced the glutathione content to an average of 15–20% of the control values without a concomitant increase in oxidized glutathione. Morphological changes were assessed by phase contrast and electron microscopy. In order to assess growth, cells in 5 ml serum-free MEM were exposed to an initial concentration of 0.05 mmH2O2(for 50,000 cells) or 2 doses of 0.5 mmH2O2(for 800,000 cells). After exposure to H2O2, medium was replaced with MEM plus 8% rabbit serum; cells were fed on days 3 and 6 and counted on day 7.When 50,000 or 800,000 cells with decreased glutathione were exposed to 0.05 or 0.5 mmH2O2the H2O2was cytotoxic, whereas cells treated with H2O2alone remained viable but showed inhibited proliferation. An unexpected finding was that cells continued to remove H2O2from the medium at normal rates even when the GSH level was reduced. Cells treated with CDNB or BSO alone exhibited morphological and growth properties comparable to untreated cells. Cells treated with CDNB or BSO and then with H2O2exhibited decreased cell-to-cell contact, nuclear shrinkage, and arborization when viewed with phase-contrast microscopy and showed extensive nuclear and cytoplasmic degeneration at the EM level. Cell death was determined by dye exclusion and confirmed by video microscopy. When cells were treated with CDNB or BSO and subsequently treated with TEMPOL, n-propyl gallate or deferoxamine and then challenged with H2O2cytotoxicity was prevented and the cells were capable of growth. The data show that H2O2was not lethal to glutathione-depleted lens epithelial cells when they were treated with compounds that prevented the generation of reactive oxygen species. In addition, the results indicate that GSH has an important protective role independent of its ability to decompose H2O2via glutathione peroxidase.