CONTRIBUTION OF INCREASED GLUTATHIONE CONTENT TO MECHANISMS OF OXIDATIVE STRESS RESISTANCE IN HYDROGEN-PEROXIDE RESISTANT HAMSTER FIBROBLASTS

An H2O2-resistant variant (OC14) of the HA1 Chinese hamster fibroblast cell line, which demonstrates cross resistance to 95% O-2 and a 2-fold increase in total glutathione content, was utilized to investigate mechanisms responsible for cellular resistance to H2O2- and O-2-toxicity. OC14 and HAI cells were pretreated with buthionine sulfoximine (BSO) to deplete total cellular glutathione. Following BSO pretreatment, cells were either placed in 250 mu M BSO to maintain the glutathione depleted condition and challenged with 95% O-2, or challenged with hydrogen peroxide in the absence of BSO. Total glutathione and the activities of CuZn superoxide dismutase, Mn superoxide dismutase, catalase, glutathione peroxidase, and glutathione transferase were evaluated immediately following the BSO pretreatment as well as following 39 to 42 hr of exposure to 250 mu M BSO. BSO treatment did not cause significant decreases in any cellular antioxidant tested, except total glutathione. Glutathione depletion resulted in significant (P < 0.05) sensitization to O-2-toxicity and H2O2-toxicity in both cell lines at every time point tested. However, glutathione depletion did not completely abolish the resistance to either O-2- or H2O2-toxicity demonstrated by OC14 cells, relative to HA1 cells. Also, glutathione depletion did not effect the ability of OC14 cells to metabolize extracellular H2O2. These data indicate that glutathione dependent processes significantly contribute to cellular resistance to acute H2O2- and O-2-toxicity, but are not the only determinants of resistance in cell lines. The contribution of aldehydes formed by lipid peroxidation in mechanisms involved with the sensitization to O-2-toxicity in glutathione depleted cells was tested by measuring the lipid peroxidation byproduct, 4-hydroxy-2-nonenal (4HNE), bound in Schiff-base linkages or in its free form in cell homogenates at 49 hr of 95% O-2-exposure. No significant increase in 4HNE was detected in glutathione depleted cells relative to glutathione competent cells, indicating that glutathione depletion does not sensitize these cells to O-2-toxicity by altering the intracellular accumulation of free or Schiff-base bound 4HNE. (C) 1995 Wiley-hiss, Inc.