Effect of hypoxia on tert-butylhydroperoxide-induced oxidative injury in hepatocytes

Toxicity of t-butylhydroperoxide (t-BuOOH) was studied at different steady state O2 concentrations under conditions at which O2 deficiency alone did not cause cell death. t-BuOOH-induced cell death was more rapid in hypoxic than normoxic cells; the maximal rate of cell death occurred in anoxic cells. t-BuOOH elimination was independent of O2 concentration and was complete within 15 min; t-Butanol was produced at the same rate and was the only product detected by gas chromatography. Measurement of radical production by formation of adducts of the spin-trapping agent N-tert-butylphenylnitrone showed that the amount of radicals trapped was 0.02% of the amount of peroxide added and was the same under anoxic and oxygenated (214 microM O2) conditions. These results show that the O2 dependence of t-BuOOH-induced toxicity is not related to quantitative alterations in its metabolism. Lipid peroxidation was lowest in anoxic cells and increased as the O2 concentration was increased to 1.07 mM O2, showing that enhanced toxicity during hypoxia and anoxia was not due to enhanced lipid peroxidation. In contrast, O2 deficiency impaired the ability of cells to maintain and recovery GSH and NADPH pools after addition of t-BuOOH. GSH was decreased to a greater extent in anoxic cells than in normoxic cells, and the GSH content remained lower in these cells for up to 30 min. This decrease was due both to a decrease in the rate of synthesis and to decreased supply of the NADPH needed for the reduction of GSSG. Taken together, these results show that O2 deficiency has little effect on metabolism of t-BuOOH but impairs the ability of cells to maintain cellular GSH and renders them more susceptible to injury from oxidizing agents. This suggests that oxidative injury under hypoxia or following ischemia may not require a marked stimulation in generation of oxidative species but may occur as a consequence of the impaired ability to tolerate or repair oxidative injury.