Protection from oxidative insult in glutathione depleted lens epithelial cells

It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine , compounds that limit the availability of Fe+2 and prevent the generation of hydroxyl radicals, protect cultured rabbit lens epithelial cells from H2 O2-induced damage. In view of the importance of glutathione as an antioxida nt and the decrease in GSH that is known to accompany most forms of catarac t, we investigated whether these compounds protected cultured lens epitheli al cells from H2O2 when 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 sulfoxi mine (BSO), an inhibitor of glutathione biosynthesis, reduced the glutathio ne content to an average of 15-20 % of the control values without a concomi tant increase in oxidized glutathione. Morphological changes were assessed by phase contrast and electron microscopy. In order to assess growth, cells in 5 mi serum-free MEM were exposed to an initial concentration of 0.05 mM H2O2 (for 50,000 cells) or 2 doses of 0.5 mM H2O2 (for 800,000 cells). Aft er exposure to H2O2, medium was replaced with MEM plus 8% rabbit serum; cel ls 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 0.0 5 or 0.5 mM H2O2 the H2O2 was cytotoxic, whereas cells treated with H2O2 al one remained viable but showed inhibited proliferation. An unexpected findi ng was that cells continued to remove H2O2 from the medium at normal rates even when the GSH level was reduced. Cells treated with CDNB or BSO alone e xhibited morphological and growth properties comparable to untreated cells. Cells treated with CDNB or BSO and then with H2O2 exhibited decreased cell -to-cell contact, nuclear shrinkage, and arborization when viewed with phas e-contrast microscopy and showed extensive nuclear and cytoplasmic degenera tion at the EM level. Cell death was determined by dye exclusion and confir med by video microscopy. When cells were treated with CDNB or BSO and subse quently treated with TEMPOL, n-propyl gallate or deferoxamine and then chal lenged with H2O2 cytotoxicity was prevented and the cells were capable of g rowth. The data show that H2O2 was not lethal to glutathione-depleted lens epithelial cells when they were treated with compounds that prevented the g eneration of reactive oxygen species. In addition, the results indicate tha t GSH has an important protective role independent of its ability to decomp ose H2O2 via glutathione peroxidase, (C) 1999 Academic Press.