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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-450mediated drug oxidation is metabolized primarily by the glutathione peroxidase system. [ABSTRACT FROM AUTHOR]

Turnover of cellular glulathione in isolated rat kidney cells was studied using cystine or methionine as sulfur donor. In the absence of any sulfur donor a continuous decrease of intracellular reduced glutathione (GSH) during incubation of the cells was observed. This decrease was abolished in the presence of cystine and, as indicated by incorporation of 35S, there was also a rapid synthesis of GSH. In the presence of a γ-glutamyltransferase inhibitor, the synthesis of intracellular GSH was accompanied by an accumulation of extracellular cysteine-glutathione mixed disulfide whereas only minor amounts of GSH and glutathione disulfide could be detected. The intracellular levels of both the cysteine-glutathione and glutathione disulfides were at all time points very low. Even though the uptake of cysteine was rapid and not rate-limiting for GSH synthesis, almost no cystine could be detected intracellularly. An increasing intracellular cysteine concentration was however observed, indicating a paid reduction of cysteine. In contrast of cysteine, methionine did not protect from the loss of intracellular GSH and only a low rate of incorporation of 35S into GSH was observed. Methionine was rapidly taken up into the cells but was apparently converted to cysteine only a very limited extent. This is most likely due to a low activity of the enzyme cystathionase since neither homocystine nor cystathionine was very effective in supporting GSH synthesis. [ABSTRACT FROM AUTHOR]

Lipid peroxidation was initiated by the addition of either ADP- complexed Fe3+ or cumene hydroperoxide to isolated rat hepatocytes and the resultant biochemical and morphological alterations investigated. As previously observed with microsomes, malonaldehyde formation was associated with the inactivation of glucose-6-phosphatase. Inhibition of microsomal oxidative drug metabolism was correlated with the release and subsequent inactivation of NADPH-cytochrome c reductase, whereas cytochrome P-450 destruction occurred only in the presence of high concentrations of the organic hydroperoxide which were associated with extensive malonaldehyde formation. Under these conditions there were also marked ultrastructural alterations in the hepatocytes which were not apparent after incubation in the presence of iron (≤187 μM Fe3+). The latter treatment was, however, associated with moderate biochemical effects such as glucose-6-phosphatase inactivation and increased membrane permeability. The cellular defence system against lipid peroxidation is discussed and it is concluded that the isolated liver cell system provides a valuable tool for the study of lipid peroxidation and its pathological implications. [ABSTRACT FROM AUTHOR]

Isolated rat liver cells were found to catalyze the cytochrome P-450 dependent, oxidative metabolism of alprenolol [1-(2-allylphenoxy)-3-isopropyiaminopropanol] at a rate similar to that obtained with the isolated microsomal fraction in the presence of a NADPH-generating system. Alprenolol was rapidly token up into the liver cells and the apparent Michaelis constant was similar to that obtained with liver microsomes. Further, inhibitors of drug metabolism such as SKF 525-A and metyrapone showed similar inhibitory patterns in the cellular and microsomal systems. With liver cells isolated from control rats, NADPH generation from endogenous substrates was sufficient to support optimal alprenolol metabolism, whereas the addition of glucose or lactate stimulated this rate in liver cells isolated from starved, phenobarbital-treated rats. Inhibitors of mitochondrial respiration such as rotenone, antimycin A and KCN and the uncoupler carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone inhibited cellular alprenolol metabolism, presumably by lowering the ATP/ADP ratio thereby interfering with NADPH generation via the malic enzyme system; this effect was more pronounced in liver cells from starved rats. Furthermore, in analogy with previous findings, the rate of gluconeogenesis from lactate was inhibited during alprenolol metabolism; this effect in turn was most pronounced in liver cells from phenobarbital-treated rats. The findings indicate that under certain conditions, the level of cyroplasmic NADPH may become rate-limiting for drug metabolism linked to cytochrome P-450 and support the previous assumption that in fed rats, generation of cytoplasmic NADPH is mainly via the pentose phosphate shunt whereas the malic enzyme system becomes more important for this purpose during starvation. [ABSTRACT FROM AUTHOR]

Cells isolated from rat lung by protease digestion were found to catalyze the reduced glutathione (GSH) conjugation of 1-chloro-2,4-dinitrobenzene. The rate of conjugation was stimulated severalfold in the presence of GSH, indicating uptake and utilization of extracellular GSH by the lung cells. The stimulation was dependent on the GSH concentration and not due to a spontaneous nonenzymatic reaction or to extracellular GSH-transferase activity. Conjugation of 1-chloro-2,4-dinitrobenzene was also measured using isolated perfused rat lung. The conjugation, which was linear for a longer time than with the isolated cells, was also stimulated in the presence of GSH in the perfusion medium. The results indicate the ability of rat lung to utilize extracellular GSH. [ABSTRACT FROM AUTHOR]