Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitroimidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H2O2 as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H2O2 as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H2O2 as substrate.