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 (TNFalpha)-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 TNFalpha on the redox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatment with TNFalpha and cycloheximide (TNFalpha/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). TNFalpha/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 TNFalpha/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.
Sánchez-Alcázar
J A
JA
Centro de Investigación and Departamento de Anatomia Patológica, Hospital Universitario "12 de Octubre," Madrid 28041, Spain.
Schneider
E
E
Martínez
M A
MA
Carmona
P
P
Hernández-Muñoz
I
I
Siles
E
E
De La Torre
P
P
Ruiz-Cabello
J
J
García
I
I
Solis-Herruzo
J A
JA
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000968:Antimycin A / Q000494:pharmacology; D003573:Cytochrome b Group / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D003573:Cytochrome b Group; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D000968:Antimycin A