The Effect on the K m for Radiosensitization at 0 degrees C of Thiol Depletion by Diethylmaleate Pretreatment: Quantitative Differences Found Using the Radiation Sensitizing Agent Misonidazole or Oxygen

Abstract

Pretreatment of V79-WNRE cells [Chinese hamster lung] with 150 .mu.M diethylmaleate for 1 h at 37.degree. C caused a decrease in intracellular glutathione levels to .apprx. 10-15% of control levels (0.5 vs. 5.0 nmol/106 cells). The cells could be washed free of diethylmaleate and held at 0.degree. C for several hours without toxicity and with no increase in glutathione concentration, although the glutathione concentration rapidly increased to normal levels at higher temperatures. Survival curves were determined as a function of O2 or misonidazole concentration (the latter in the absence of O2). A new thin-film technique was used to avoid changes in O2 concentration because of radiochemical or cellular O2 consumption. Glutathione depletion itself caused a small but consistent radiosensitization of hypoxic cells (dose enhancement ratio of 1.2). However, glutathione depletion caused a profound change in the radiosensitizing efficiency of misonidazole, with a decrease in Km of about 2-fold from 0.6 to 0.09 mM. In contrast, only a 2.5-fold decrease was found in the Km for radiosensitization by O2 with diethylmaleate pretreatment. These results suggest a fundamental problem with the conventional theory of radiosensitivity whereby a first-order competition for reaction with target radicals between radical-fixing vs. radical-repairing species must be considered. It also suggests difficulties in the interpretation of glutathione as the only endogenous protective species.