Tumour Necrosis Factor Induced an Early Release of Superoxide and a Late Mitochondrial Membrane Depolarization in L929 Cells

Abstract

Tumour necrosis factor α (TNF) cytotoxicity is mediated, at least in part, by oxidative stress. One of the post-receptor events shortly after the addition of TNF is the generation of the superoxide anion (O₂^–·). In the present study, we attempted to examine the role of O₂^–· in the regulation of mitochondrial membrane potential (ΔΨm) and the release of cytochrome c (cyto c) in L929 cells after stimulation with TNF. Challenge of cells with TNF (50 ng/ml) resulted in an early (30 min after the addition of TNF) increase in the production of O₂^–·. The use of mitochondrial electron transport chain inhibitors such as antimycin A and rotenone could, respectively, potentiate or suppress the TNF-mediated release of O₂^–· and cytotoxicity. TNF also induced a late (>3 h after the addition of TNF) depolarization in the ΔΨm. Reduction in the release of O₂^–· by rotenone (50 µM) or thenoyltrifluoroacetone (250 µM) suppressed both the TNF-mediated ΔΨm depolarization and cyto c release. However, increase in the production of O₂^–· by antimycin A (25 µM) only slightly enhanced the TNF effect in altering the ΔΨm and the release of cyto c. Treating cells with antimycin A alone could not induce a reduction in ΔΨm nor a release of cyto c. Taken together, our results indicate that TNF induced damage in mitochondria in L929 cells. Our data also show that an increase in the production of O₂^–· was important in the TNF cytotoxicity, but was not sufficient to mimic the action of TNF.

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