Mitochondrial poisons cause depletion of reduced glutathione in isolated hepatocytes

doi:10.1016/0003-9861(92)90498-L

Archives of Biochemistry and Biophysics

Volume 295, Issue 1, 15 May 1992, Pages 132-136

https://doi.org/10.1016/0003-9861(92)90498-L Get rights and content

Abstract

Antimycin A, KCN, and 1-methyl-4-phenylpyridinium ion (MPP+) all produced a marked depletion of cellular GSH levels in freshly isolated hepatocytes. This effect was consistently observed before the onset of cytotoxicity and seemed to be correlated with the loss of cellular ATP induced by these mitochondrial poisons. Concentrations of GSSG remained unchanged both intracellularly and extracellularly, indicating that oxidation was not involved in the events leading to GSH depletion. Approximately 40% of the decrease of intracellular GSH was accounted for by efflux of this tripeptide, assessed by increased formation of cysteinyl-glutathione when hepatocytes were incubated in the presence of 0.2 mm cystine. Therefore, an overall loss of glutathione was observed during incubations with all three inhibitors of mitochondrial function. Addition of 10 mm fructose to the incubation media substantially protected against GSH depletion caused by antimycin A, KCN, and MPP+. These results indicate that energy-dependent mechanisms are involved in the maintenance of intracellular GSH levels, and suggest that GSH depletion may be a general phenomenon associated with impairment of mitochondrial function.

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Decreased glutathione levels associated with increased oxidative stress are a hallmark of numerous neurodegenerative diseases, including Parkinson's disease. GSH is an important molecule that serves as an anti-oxidant and is also a major determinant of cellular redox environment. Previous studies have demonstrated that neurotoxins can cause changes in reduced and oxidized GSH levels; however, information regarding steady state levels remains unexplored. The goal of this study was to characterize changes in cellular GSH levels and its regulatory enzymes in a dopaminergic cell line (N27) following treatment with the Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP⁺). Cellular GSH levels were initially significantly decreased 12 h after treatment, but subsequently recovered to values greater than controls by 24 h. However, oxidized glutathione (GSSG) levels were increased 24 h following treatment, concomitant with a decrease in GSH/GSSG ratio prior to cell death. In accordance with these changes, ROS levels were also increased, confirming the presence of oxidative stress. Decreased enzymatic activities of glutathione reductase and glutamate–cysteine ligase by 20–25% were observed at early time points and partly account for changes in GSH levels after MPP⁺ exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP⁺ treatment was not associated with increased efflux of glutathione to the medium. These data further elucidate the mechanisms underlying GSH depletion in response to the Parkinsonian toxin, MPP⁺.
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^☆: This work was supported by the German Academic Exchange Service (K.M.), the Health Effects Component of the University of California Toxic Substances Program, the California Parkinson's Foundation, NINCDS (R01 NS27365-02), and NIEHS (P30 ES01896).

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Mitochondrial poisons cause depletion of reduced glutathione in isolated hepatocytes☆

Abstract

Biochem. Biophys. Res. Commun

Neurosci. Lett

Neurosci. Lett

J. Biol. Chem

Toxicol. Lett

Life Sci

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Toxicol. Appl. Pharmacol

Anal. Biochem

Biochem. Pharmacol

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Arch. Biochem. Biophys

Biochem. Pharmacol

Arch. Biochem. Biophys