Specific Aim 1. Mechanism of mitochondrially-triggered cell death. We have recently demonstrated that permeabilized mitochondria release apoptotic factors. providing a testable biochemical, molecular and cellular hypothesis for the mitochondrial regulation of cell death. Firstly, we will test the predictions of our model for cell death by measuring the ability of death-inducers to both induce release of apoptotic factors from mitochondria, and the ability of mitochondrial regulators to inhibit such release. Secondly, our hypothesis for the order of events in cell death will be tested with purified microsomes, mitochondria, and nuclei, and combinations thereof. Thirdly in whole cells, we will observe the sequence of pathological events in toxin- induced apoptosis, and measure the time course and efficacy of mitochondrial effectors to apoptosis. Specific Aim 2. Investigation of the cause-and-effect relationships between mitochondrial antioxidant and repair enzymes and age-related mitochondrial DNA mutagenesis. Using the quantitative assay of mutagenesis developed in the first grant period, we will investigate the effect of specific enzymes to protect cells and animals from mtDNA mutagenesis.