Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells

The mechanism of tumor necrosis factor alpha (TNF alpha)-induced cytotoxici ty 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 alpha on the red ox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatmen t with TNF alpha and cycloheximide (TNF alpha/CHX) induced mitochondrial cy tochrome c release, increased the steady-state reduction of cytochrome b, a nd decreased the steady-state reduction of cytochromes cc(1) and aa(3). TNF alpha/CHX treatment also induced lipid peroxidation, intracellular generat ion of reactive oxygen species, and cell death. Furthermore, as the cells d ied mitochondrial morphology changed from an orthodox to a hyperdense and c ondensed and finally to a swollen conformation. Antimycin A, a mitochondria l respiratory chain complex III inhibitor that binds to cytochrome b, block ed the formation of reactive oxygen species, suggesting that the free radic als are generated at the level of cytochrome b. Moreover, antimycin A, when added after 3 h of TNF alpha/CHX treatment, arrested the further release o f cytochrome c and the cytotoxic response. We propose that the reduced cyto chrome b promotes the formation of reactive oxygen species, lipid peroxidat ion of the cell membrane, and cell death.