Mechanism for the changes in levels of glutathione upon exposure of cultured mammalian cells to tertiary-butylhydroperoxide and diamide

Abstract

Qualitative and quantitative changes associated with cellular glutatione (GSH) in response to oxidants were investigated in cultured Chinese hamster V79 cells. Incubation of cells with benzoylperoxide (BZP), tert-butylhydroperoxide (t-BuOOH), hydrogen peroxide or diamide for 1 h reduced the level of total GSH (GSH + GSSG). Among the oxidants, t-BuOOH and diamide caused an increase in levels of glutathione disulfide (GSSG) and a resultant increase in the ratio of the level of GSSG to the level of total GSH, suggestive of the induction within the cells of a pro-oxidant state by the oxidants. o-Phenanthroline, a chelator of divalent ion, almost completely suppressed the decrease in levels of total GSH caused by t-BuOOH while it did not suppressed either increases in levels of GSSG or increases in the ratio of the levels of GSSG to that of total GSH caused by the hydroperoxide. These results suggest that reactive oxygen radicals are involved in the decrease in levels of GSH by treatment with t-BuOOH but not in the increase in the level of GSSG. After treatment with either t-BuOOH or diamide for 1 h, the level of GSH rapidly increased to more than twice the control level during 15–45 min of post-treatment incubation. o-Phenanthroline almost completely suppressed the increase in levels of GSH caused by t-BuOOH, while it did not affect the changes caused by diamide, suggesting a difference between the mechanisms by which t-BuOOH and diamide cause increases in levels of GSH. It seems likely that reactive oxygen radicals participate not only in the decrease in levels of GSH caused by t-BuOOH but also in the rapid increase that occurs after such treatment. Hence, the first decrease in levels of GSH by the hydroperoxide may be causally related to the latter increase. The amount of [³⁵S]-cysteine taken up by cells after treatment with t-BuOOH was about one half of that taken up by control cells. By contrast, the rate of incorporation of radioactive cysteine into acid-soluble material increased to more than twice that of the controls after treatment with t-BuOOH. The increase in the rate of incorporation of [³⁵S]cysteine into acid-soluble material caused by t-BuOOH was not a consequence of inhibition by the hydroperoxide of utilization of cysteine for protein synthesis. Inhibition of protein synthesis by cycloheximide caused neither an increase in the incorporation of cysteine into acid-soluble material nor an increase in rate of biosynthesis of GSH. Incorporation of radioactive cysteine into the cysteine moiety of GSH and the disappearance of the radioactivity from the cysteine fraction were enhanced after treatment with t-BuOOH. These data indicate that biosynthesis of GSH de novo is enhanced by t-BuOOH.