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Apoptosis is now widely recognized as being a distinct process of importance both in normal physiology and pathology. In the current paradigm for apoptotic cell death, the activity of a family of proteases, caspases, related to interleukin-1 -converting enzyme (ICE) orchestrates the multiple downstream events, such as cell shrinkage, membrane blebbing, glutathione (GSH) efflux, and chromatin degradation that constitute apoptosis. Recent studies suggest that mitochondria could be the principle sensor and that the release of mitochondrial factors, such as cytochrome c, is the critical event governing the fate of the cell. One of the most reproducible inducers of apoptosis is mild oxidative stress, although it is unclear how an oxidative stimulus can activate the caspase cascade. Oxidative modification of proteins and lipids has also been observed in cells undergoing apoptosis in response to nonoxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. The caspases themselves are cysteine-dependent enzymes and, as such, appear to be redox sensitive. Indeed, our recent work on hydrogen peroxide– mediated apoptosis suggests that prolonged or excessive oxidative stress can actually prevent caspase activation. A physiological example of this is the NADPH oxidasederived oxidants generated by stimulated neutrophils that prevent caspase activation in these cells. Pursuant to these findings, stimulated neutrophils appear to use a specialized caspase-independent pathway to initiate phosphatidylserine (PS) exposure and subsequent phagocytic clearance. The possible implications of these dual roles for reactive oxygen species in apoptosis, that is, induction and inhibition of caspases, are discussed in the present review. Reactive oxygen species (ROS), also called oxygen free radicals or oxidants, are generated in all aerobic cells. They are produced during normal mitochondrial respiration, they are used by specialized phagocytic cells to destroy invading pathogens, and they are byproducts of the intracellular metabolism of toxic drugs and environmental agents. ROS can react rapidly with a range of biological molecules, making them highly destructive. Fortunately, aerobic cells are endowed with a complex network of antioxidants to scavenge ROS or repair the damage; however, the overwhelming of these defenses, termed oxidative stress, is implicated in numerous disease states. The gradual accumulation of oxidative damage has also been hypothesized to contribute to aging. For example, it was recently reported that in liver cells from old rats the mitochondrial membrane potential declines and mitochondrial heterogeneity and production of oxidants increase. Similar findings have been made in mouse lymphocytes, suggesting that a population of lymphocytes from old mice are more susceptible to activation M. Hampton was supported by the New Zealand Foundation for Research, Science and Technology. Corresponding author: Tel: (46 8) 728 7590; fax: (46 8) 32 90 41; e-mail: sten.orrenius@imm.ki. se Current address: Department of Pathology, Christchurch School of Medicine, P.O. Box 4345, Christchurch, New Zealand. HAMPTON et al.: REDOX REGULATION 329 of the mitochondrial permeability transition, which would be compatible with the suggestion that mitochondrial dysfunction contributes to immunosenescence. Although it is known that acute oxidative stress kills cells, the mode of cell death has been largely ignored. Today, the study of cell death itself is one of the most rapidly growing areas in biomedical research. It is recognized that many compounds, rather than being directly cytotoxic, actually cause sublethal damage that triggers an innate suicide program in the cell. This process, termed apoptosis, involves cell shrinkage, nuclear condensation and fragmentation, and the bundling up of the cellular material into apoptotic bodies. Changes on the outer surface of the plasma membrane, such as the exposure of phosphatidylserine (PS), label these bodies for removal by neighboring cells or phagocytes, without the leakage of cellular material. Apoptosis is essential during development in maintaining tissue homeostasis in the adult organism and also for the removal of damaged, infected, or potentially neoplastic cells. The possible role of increased apoptotic cell death in aging is less well established, although a number of reports suggest that this may well be the case. For example, aging in the circulation is associated with an increased susceptibility to apoptosis in neutrophils, and age-associated immune dysfunction has been suggested to correlate with defects in Tcell apoptosis. Aging has also been reported to be associated with a progressive increase in the proportion of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive hepatocytes, and sensory hair cells in the inner ear exhibit agerelated cell death by apoptosis. Further, myocyte cell death, apoptotic and necrotic in nature, constitutes an important determinant of the aging process. However, in contrast to these reports, a recent study of apoptosis in mice following gamma irradiation has found a striking age-dependent decrease in radiation-induced apoptosis in splenic lymphocytes but not in colorectal epithelial cells, and Zhou et al. showed a decrease in Fasmediated apoptosis in thymocytes and peripheral T cells of aged mice. Thus, the overall relationship between apoptosis and aging is still unclear. Apoptosis can be triggered by numerous factors, including receptor-mediated signals, removal of growth factors, and cell damage. Each of these stimuli has its own specific pathway that leads to activation of the apoptotic process; however, all appear to converge at a highly conserved sequence of events. The caspases are a central component of this apoptotic program. This family of cysteine-dependent enzymes cleaves its target substrates on the carboxyl side of aspartate residues. Caspases exist in the cytoplasm as inactive zymogens (procaspases). Conversion to the active form involves zymogen proteolysis and assembly and joining of two identical heterodimers. Caspases cleave a defined number of target proteins, and these changes lead to the characteristic features of apoptosis and organized disassembly of the cell. Caspase activity has been detected at an early stage in numerous models of apoptosis, and specific peptide inhibitors of the caspases can block almost all of the subsequent apoptotic changes. The ability of caspases to cleave and activate other cytoplasmic procaspases sets off a destructive cascade of events. Although this proteolytic cascade is likely to be common among different models of apoptosis, the sequence of events leading to activation of the first caspases will vary depending on the apoptotic stimulus. The best characterized systems are the cytotoxic T-lymphocyte component granzyme B, which can directly cleave specific procaspases, and the Fas (CD95/APO-1) surface receptor, which sequesters and activates procaspases via interactions between intracellular “death domains. ” 330 ANNALS NEW YORK ACADEMY OF SCIENCES The majority of other models seem more complex. The challenge of contemporary apoptosis research will be to elucidate the sequence of events leading to caspase activation in these systems. ACTIVATION OF APOPTOSIS BY ROS Incubating cells with exogenous oxidants, or adding redox-active compounds, can trigger apoptosis. How then does a broad-spectrum biological oxidant initiate apoptosis? One possibility is that extensive oxidation of cellular components could trigger general protection systems, and these sensors detect the damage and activate the apoptotic machinery. A classic example of this is p53 detection of DNA damage. An alternate mechanism is that oxidants could activate specific signaling pathways that lead to apoptosis. Low-level oxidative stress is already known to activate different protein kinase pathways and transcription factors, and the ability of nitric oxide to rapidly react with the heme group of guanylate cyclase has been used as a message in several cellular pathways. We have recently focused on the ability of hydrogen peroxide to trigger apoptosis in Jurkat T lymphocytes. Hydrogen peroxide is a relatively ineffective oxidant, and we suspected that selective pathways were involved in hydrogen peroxide–mediated apoptosis. Initial experiments showed that 50 μM hydrogen peroxide (10cells/mL) optimally triggered caspase activation and subsequent apoptosis. This occurred several hours after the initial bolus of hydrogen peroxide had been consumed by the cells, and addition of hydrogen peroxide to cytoplasmic extracts does not activate the caspases directly (Hampton, unpublished data), indicating that other factors were involved in the pathway to activation. Mitochondria are receiving considerable attention in apoptosis research for their ability to release proapoptotic factors. In particular, the export of cytochrome c to the cytoplasm has been detected in various systems, and a groundbreaking study showed that cytochrome c, in combination with other cytosolic factors, is capable of triggering the caspase cascade. Kluck et al. reported cytochrome c release from peroxide-treated T lymphoblastoid CEM cells, and in our Jurkat cell model cytochrome c was detected in the cytoplasm within one to two hours after treatment with hydrogen peroxide, which was at least one hour before significant caspase activation could be detected. Specific inhibition of the as yet unknown export mechanism will be important to determine the necessity of cytochrome c release for hydrogen peroxide–mediated caspase activation. Another important issue is to determine the initial target of the hydrogen peroxide and how this initiates cytochrome c export. Mitochondria are well known targets of oxidative stress, with alterations in cellular calcium metabolism as a prominent result of o