Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes.

The effects of menadione (2-methyl-1,4-naphthoquinone) metabolism on intracellular soluble and protein-bound thiols were investigated in freshly isolated rat hepatocytes. Menadione was found to cause a dose-dependent decrease in intracellular glutathione (GSH) level by three different mechanisms: (a) Oxidation of GSH to glutathione disulfide (GSSG) accounted for 75% of the total GSH loss; (b) About 15% of the cellular GSH reacted directly with menadione to produce a GSH-menadione conjugate which, once formed, was excreted by the cells into the medium; (c) A small amount of GSH (approximately 10%) was recovered by reductive treatment of cell protein with NaBH4, indicating that GSH-protein mixed disulfides were also formed as a result of menadione metabolism. Incubation of hepatocytes with high concentrations of menadione (greater than 200 microM) also induced a marked decrease in protein sulfhydryl groups; this was due to arylation as well as oxidation. Binding of menadione represented, however, a relatively small fraction of the total loss of cellular sulfhydryl groups, since it was possible to recover about 80% of the protein thiols by reductive treatments which did not affect protein binding. This suggests that the loss of protein sulfhydryl groups, like that of GSH, was mainly a result of oxidative processes occurring within the cell during the metabolism of menadione.

Oxidation of glutathione during hydroperoxide metabolism. A study using isolated hepatocytes and the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea.

In the present study freshly isolated rat hepatocytes treated with the glutathione reductase inhibitor BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) were used to investigate the metabolism of tert-butyl hydroperoxide and of hydrogen peroxide formed in different intracellular compartments. Glycolate, benzylamine and hexobarbital were used to stimulate H2O2 production in the peroxisomal, mitochondrial and endoplasmic reticular/cytosolic compartments, respectively. Our results support previous findings that catabolism of H2O2 formed in the mitochondrial and cytosolic compartments occurs predominantly by the glutathione peroxidase system, whereas H2O2 generated within the peroxisomes is metabolized by catalase. They further reveal that the capacity of uninhibited glutathione reductase to reduce glutathione disulfide, formed during hydroperoxide metabolism by glutathione peroxidase, is high and that a decreased NADPH/NADP+ redox level, rather than insufficient reductase activity, is responsible for the accumulation and subsequent excretion of cellular glutathione disulfide observed during hydroperoxide metabolism. Finally, our results demonstrate that H2O2 generated during cytochrome P-450-mediated drug oxidation is metabolized primarily by the glutathione peroxidase system.