The question of whether depletion of glutathione (GSH) could affect the synthesis of stress proteins was investigated in Hep G2 cells. Cells were exposed to BSO/DEM at 37°C to deplete glutathione. When 95% of the glutathione was depleted cells were washed, and BSO was added to cells previously exposed to BSO/DEM; then the cells were incubated at 37, 38.5, or 39°C for 4 h. Two-dimensional PAGE analysis of GSH-depleted cells incubated at 37°C indicated increased synthesis of heme oxygenase and a polypeptide tentatively identified as hsp-70B′. Depletion of GSH did not affect the cellular concentration of hsp-70 as assessed by Western immunoblotting, yet Northern blot analysis indicated that hsp-70 mRNA was increased in GSH-depleted cells. Incubation of GSH-replete cells at 38.5°C did not appear to enhance the amount of hsp-70 mRNA or the relative rate of hsp-70 synthesis. In contrast, incubation of GSH-depleted cells at 38.5°C elevated steady-state hsp-70 mRNA levels and the rate of hsp-70 synthesis relative to total protein synthesis. Depletion of GSH also increased the relative rate of hsp-70 synthesis at 39°C. These results suggest that the synthesis of stress proteins can be affected by glutathione concentrations.

The relationship between the intracellular glutathione (GSH) concentration and the aerobic radiation response was studied in Chinese hamster ovary cells. Various degrees of GSH depletion were produced by exposure to buthionine sulfoximine (BSO) and/or diethyl maleate (DEM). Diethyl maleate did not act as a classical radiosensitizer under the experimental conditions employed, nor did exposure to DEM/BSO nonspecifically affect protein thiols as measured by thiol blotting. Dose-response curves were obtained using cells irradiated in the absence or presence of DEM/BSO, which decreased GSH levels by 90-95%. Exposure to DEM/BSO did not affect the formation of DNA single-strand breaks or DNA-protein crosslinks measured immediately after irradiation performed at ice temperatures. Analysis of survival curves indicated that the $D_{{\rm q}}$ was decreased by 18% when GSH depletion occurred prior to, during, and after irradiation. The DEM/BSO exposure did not affect D 0 . To study postirradiation conditions, cells were exposed to 10 μM DEM prior to and during irradiation, which was performed at ice temperatures. Levels of GSH were depleted by 75% by this protocol. Immediately after irradiation, the cells were rapidly warmed by the addition of 37°C growth medium containing either 10 or 90 μM DEM. Addition of 10 μM DEM after irradiation did not affect the degree of depletion, which remained constant at 75%. In contrast, GSH depletion was increased to 90% 10 min after addition of the 90 μM DEM. Addition of 90 μM DEM after irradiation produced a statistically significant difference in survival compared to addition of 10 μM DEM. In a second depletion protocol, cells were exposed to 100 μM DEM at room temperature for 5 min, irradiated, incubated at 37°C for 1 h, washed, and then incubated in 50 μM BSO for 24 h. This depletion protocol reduced survival by a factor of 2.6 compared to cells not exposed to the combination of DEM/BSO. Survival was not affected if the cells were exposed to the DEM or BSO alone. This was interpreted to indicate that survival was not affected by GSH depletion occurring after irradiation unless depletion was rapid and sustained. The rate of repair of sublethal and potentially lethal damage was measured and found to be independent of the DEM/BSO exposure. These experimental results in addition to previous ones (Freeman and Meredith, Int. J. Radiat. Oncol. Biol. Phys. 13, 1371-1375, 1987) were interpreted to indicate that under aerobic conditions GSH depletion may alter the expression of radiation damage by affecting metabolic fixation.

Chinese hamster ovary cells were exposed to FeSO 4 or FeCl 3 during a 43°C heat shock. Concentrations of iron, which were not toxic when cells were incubated at 37°C, became toxic in a dose-dependent fashion during hyperthermia treatment. The iron chelator EDTA, which supports oxidation/reduction reactions, promoted hyperthermia-induced iron cytotoxicity while the iron chelator desferrioxamine, which has been shown to inhibit iron redox cycling, inhibited cytotoxicity. The presence of exogenous superoxide dismutase, catalase, or mannitol during hyperthermia treatment did not inhibit iron toxicity. Depletion of intracellular glutathione by diethylmaleate increased hyperthermia-induced iron toxicity by 76%. These data are interpreted to mean that heat shock promotes intracellular oxidative damage and intracellular glutathione is necessary for protection.

We tested the hypothesis that depletion of intracellular glutathione (GSH) during heat shock results in protein thiol oxidation, thereby increasing thermal sensitivity. Depletion of GSH was accomplished using a combination of diethylmaleate and buthionine sulfoximine and protein sulfhydryls were measured using two independent methods. Chinese hamster ovary (CHO) cells were solubilized in polyacrylamide gel electrophoresis (PAGE) sample buffer containing 3-(N-maleimido-propionyl) biocytin, separated by sodium dodecyl sulfate (SDS)-PAGE, electroluted onto nitrocellulose, and visualized via avidin-alkaline phosphatase staining. A second method utilized 5,5′-dithiobis(2-nitrobenzoic acid) to measure protein solubilized in SDS. The results indicate that when CHO cells are heated at 43°C GSH depletion can increase thermal sensitivity but does not cause nonspecific protein thiol oxidation at this temperature or at 37°C.

Chinese hamster ovary (CHO) cells were exposed to various concentrations of diethylmaleate (DEM) during a 42°C incubation to determine if glutathione (GSH) compartmentalization was a factor in modification of thermal sensitivity. Cytoplasmic and mitochondrial GSH were isolated from CHO cells immediately after a hyperthermic treatment consisting of 2 h at 42°C. Under these experimental conditions differential GSH depletion between the cytosol and mitochondrial compartments were observed. For example, 12 μM DEM was needed to deplete cytoplasmic GSH by 50% compared to 24 μM DEM needed to deplete mitochondrial GSH to the same level. Further, an In-In plot of the relative cytosolic GSH concentration vs the DEM concentration indicated a linear relationship (slope = -1.0). In contrast, the mitochondrial GSH plot exhibited a shoulder followed by a linear removal (slope = -0.90). Essentially the two linear curves were parallel. Analysis of thermal dose-response curves for cells exposed to between 10 and 100 μM DEM indicated that cell survival was unaffected by the addition of DEM until a critical concentration was surpassed. This threshold response was interpreted to mean that mitochondrial GSH depletion was the limiting factor.

Chinese hamster ovary (CHO) cells were exposed to a 43°C, 15-min heat shock to study the relationship between protein synthesis and the development of thermotolerance. The 43°C heat shock triggered the synthesis of three protein families having molecular weights of 110,000, 90,000, and 65,000 (HSP). These proteins were synthesized at 37 and 46°C. This heat shock also induced the development of thermotolerance, which was measured by incubating the cells at 46°C 4 h after the 43°C heat treatment. CHO cells were also exposed to 20 μg/ml of cycloheximide for 30 min at 37°C, 15 min at 43°C, and 4 h at 37°C. This treatment inhibited the enhanced synthesis of the M r 110,000, 90,000, and 65,000 proteins. The cycloheximide was then washed out and the cells were incubated at 46°C. HSP synthesis did not recover during the 46°C incubation. This cycloheximide treatment also partially inhibited the development of thermotolerance. These results suggest that for CHO cells to express thermotolerance when exposed to the supralethal temperature of 46°C protein synthesis is necessary.