Detoxification of H 2O 2 by cultured rabbit lens epithelial cells: Participation of the glutathione redox cycle

Abstract

Although it has been shown that cultured rabbit lenses can adequately defend against the 0·03–0·05 m m level of H 2O 2 normally found in aqueous humor, the contribution of the epithelium in this process has not been well defined. In the present study, the peroxide-detoxifying ability of the epithelium is evaluated in cultured rabbit lens cells established from 4–6-day-old rabbits and compared to that of skin fibroblasts from rabbits of the same age. When cells were cultured in medium containing H 2O 2, the concentration of peroxide rapidly decreased; however, various concentrations could be maintained for 3-hr periods by using glucose oxidase to enzymically generate H 2O 2. At an extracellular level of 0·03 m m H 2O 2, the rate of detoxification of peroxide by epithelial cells was 2 μmol H 2O 2 (8 × 10 5 cells) −1 3 hr −1, twice as fast as that for fibroblasts. Epithelial cells contained a high level of reduced glutathione (GSH) equal to 36 nmol (8 × 10 5 cells) −1, twice that present in the fibroblasts. The concentration of GSH in 8 × 10 5 epithelial cells, a number of cells normally present in one intact rabbit lens epithelium, remained constant during 3 hr of exposure to H 2O 2 levels as high as 0·03 m m, even though the amount of H 2O 2 taken up under these conditions was sufficient to oxidize completely the cellular GSH every 2 min. In contrast, the GSH content of fibroblasts declined at levels of peroxide above 0·01 m m. Participation of the glutathione redox cycle in the H 2O 2-detoxification process was demonstrated from studies of hexose monophosphate shunt (HMPS) activity as measured by oxidation of [1- 14C]-labeled glucose. The oxidation of [1- 14C]-glucose in epithelial cells was stimulated 13 times that of controls during exposure to 0·04–0·05 m m H 2O 2, while the corresponding increase in oxidation of [6- 14C]-labeled glucose was only 1·6 times. In contrast, maximum shunt activity in fibroblasts occurred at 0·03–0·04 m m H 2O 2 and was six times the control value. The growth potential of the cells following a 3-hr exposure to H 2O 2 was also used as a measure of oxidant toxicity in both cell types. Concentrations of H 2O 2 up to 0·03 m m had no effect on the growth of 8 × 10 5 epithelial cells but did diminish the growth of the same number of fibroblasts. Cell density was found to be an important parameter in the ability of the cells to tolerate H 2O 2, and it was noted that isolated cells were more susceptible to H 2O 2 insult than contiguous cells. The overall results of the investigation indicate that the lens epithelium and the glutathione redox cycle have important roles in protecting the lens against H 2O 2-induced damage.