The temporal relationship between mitochondrial membrane permeabilization and reactive oxygen species production during apoptosis remains unknown. We analyzed the rate of superoxide production of human breast carcinoma cells expressing a cytochrome c-green fluorescent protein (cyt-c-GFP) fusion protein at the single-cell level during apoptosis. In cells treated with the proapoptotic agents staurosporine (3 M) or tumor necrosis factor-␣ (100 ng/ml), the release of cyt-c-GFP was individually set

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... or each cell, and the majority of the fusion protein was released in less than 10 min. Prior to the release of the fusion protein, cells demonstrated a constant rate of superoxide production determined with the probe hydroethidine. After the release was completed, the superoxide concentration increased rapidly to a level more than 3-fold above baseline. Treatment with the broad spectrum caspase inhibitor z-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk; 200 M) did not alter the kinetics of the cyt-c-GFP release but significantly reduced superoxide concentration after the release of cyt-c-GFP. Interestingly, treatment with z-VAD-fmk also reduced the increase in superoxide concentration in response to menadione in the absence of mitochondrial cyt-c-GFP release. Mitochondrial depolarization with the protonophore carbonyl cyanide ptrifluoromethoxy-phenylhydrazone per se did not trigger cyt-c-GFP release or an increase in superoxide production. Our data suggest that mitochondria increase their superoxide production during apoptosis directly after the quantitative release of soluble intermembrane proteins and demonstrate novel antioxidative effects of the commonly used caspase inhibitor z-VAD-fmk.