Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response

Abstract

S_rnuary Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-Ul and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines.Keyworid glutathione; radiosensitivity; Tietze assay; high-performance liquid chromatography For at least the past decade the tripeptide sulphydryl compound glutathione (GSH) has been the subject of intensive investigation regarding its role in mediating both radiationand drug-induced responses in living cells. GSH has been proposed to act in a vast number of different cellular processes, amongst them the maintenance of a suitable intracellular reductive environment, protection against harmful xenobiotics, a catalyst of or reactant in several metabolic schemes and an intracellular store of cysteine (Nygaard and Simic, 1983;Mitchell and Russo, 1987). Its potential importance for radiobiology has been recognised ever since the development of the competition model for radiation cell killing, a mathematical consequence of the reaction rates for competing reactions (Alper and Howard-Flanders, 1956). DNA radicals produced by irradiation are considered to be subject to a competition between oxidising (or electronaffinic) agents, leading to damage fixation and ultimate cell death, and reducing species (such as sulphydryl compounds) which, through hydrogen atom donation would facilitate damage repair and so lead to continued cell viability, against a background of the intramolecular decay of DNA radicals to render them non-restorable (Koch, 1983). Since the rate of reaction between oxygen and DNA radicals is far greater than that between GSH and DNA radicals (Bump and Brown, 1990), the competition theory predicts that under normal aerobic conditions the former reaction would dominate, rendering unimportant any changes in GSH content. However GSH may nevertheless play a significant role in the aerobic radiation response, its protective effects mediated through other processes limiting radiation damage, for instance the detoxification of radiation-induced hydroperoxides (Dethmers and Meister, 1981;Biaglow et al., 1984).Cells may be depleted of GSH by treatment with the specific enzyme inhibitor DL-buthionine-S,R-sulphoximine (BSO) which prevents GSH synthesi...

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