Detoxification of H2O2 by cultured rabbit lens epithelial cells: participation of the glutathione redox cycle.

Although it has been shown that cultured rabbit lenses can adequately defend against the 0.03-0.05 mM level of H2O2 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 H2O2, the concentration of peroxide rapidly decreased; however, various concentrations could be maintained for 3-hr periods by using glucose oxidase to enzymically generate H2O2. At an extracellular level of 0.03 mM H2O2, the rate of detoxification of peroxide by epithelial cells was 2 mumol H2O2 (8 x 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 x 10(5) cells)-1, twice that present in the fibroblasts. The concentration of GSH in 8 x 10(5) epithelial cells, a number of cells normally present in one intact rabbit lens epithelium, remained constant during 3 hr of exposure to H2O2 levels as high as 0.03 mM, even though the amount of H2O2 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 mM. Participation of the glutathione redox cycle in the H2O2-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 mM H2O2, 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 mM H2O2 and was six times the control value. The growth potential of the cells following a 3-hr exposure to H2O2 was also used as a measure of oxidant toxicity in both cell types. Concentrations of H2O2 up to 0.03 mM had no effect on the growth of 8 x 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 H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)