Tumor Necrosis Factor-α Increases the Steady-state Reduction of Cytochrome b of the Mitochondrial Respiratory Chain in Metabolically Inhibited L929 Cells

Abstract

The mechanism of tumor necrosis factor ␣ (TNF␣)-induced cytotoxicity in metabolically inhibited cells is unclear, although some studies have suggested that mitochondrial dysfunction and generation of reactive oxygen species may be involved. Here we studied the effect of TNF␣ on the redox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatment with TNF␣ and cycloheximide (TNF␣/CHX) induced mitochondrial cytochrome c release, increased the steady-state reduction of cytochrome b, and decreased the steady-state reduction of cytochromes cc 1 and aa 3 . TNF␣/CHX treatment also induced lipid peroxidation, intracellular generation of reactive oxygen species, and cell death. Furthermore, as the cells died mitochondrial morphology changed from an orthodox to a hyperdense and condensed and finally to a swollen conformation. Antimycin A, a mitochondrial respiratory chain complex III inhibitor that binds to cytochrome b, blocked the formation of reactive oxygen species, suggesting that the free radicals are generated at the level of cytochrome b. Moreover, antimycin A, when added after 3 h of TNF␣/CHX treatment, arrested the further release of cytochrome c and the cytotoxic response. We propose that the reduced cytochrome b promotes the formation of reactive oxygen species, lipid peroxidation of the cell membrane, and cell death. Tumor necrosis factor ␣ (TNF␣)1 is a cytokine that is cytotoxic against certain tumor cells (1), and this effect is enhanced by cycloheximide (CHX) (2). In the presence of CHX not only is less TNF␣ required, but also cell death occurs in a shorter period of time (3). Although these effects have been described extensively, the molecular mechanisms of action are not well understood (4, 5). The reported ability of antioxidants to protect cells against TNF␣-induced cytotoxicity suggests that mitochondrial dysfunction and generation of reactive oxygen species (ROS) in the mitochondria may play an essential role (6 -11). Oxidative stress can result in severe metabolic dysfunction, including the peroxidation of lipid membranes (12), an increase in cytosolic Ca 2ϩ (13), induction of the mitochondrial permeability transition (14), and DNA damage (reviewed in Refs. 15 and 16).Previous investigations have shown contradictory results regarding whether TNF␣ induces cytotoxicity through necrosis or apoptosis (17). Recent results indicate that TNF␣ induces necrosis in L929 cells, although apoptosis-like features have also been observed (18,19). Regardless of the mode of cell death, however, it has been shown that TNF␣-induced cytotoxicity of L929 cells is mediated by mitochondrial formation of ROS (20,21). In intact mitochondria, three components of the respiratory chain have been found to be involved in the generation of ROS (22, 23); one is located in complex I, and the other two are ubisemiquinone (24) and reduced cytochrome b (25, 26), which are both located in complex III. Furthermore, substantial evidence in other sy...

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