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Interest in the role of mitochondria in aging has intensified in recent years. This focus on mitochondria originated in part from the free radical theory of aging, which argues that oxidative damage plays a key role in degenerative senescence. Among the numerous mechanisms known to generate oxidants, leakage of the superoxide anion and hydrogen peroxide from the mitochondrial electron transport chain are of particular interest, due to the correlation between species-specific metabolic rate (“rate of living”) and life span. Phenomenological studies of mitochondrial function long ago noted a decline in mitochondrial function with age, and on-going research continues to add to this body of knowledge. The extranuclear somatic mutation theory of aging proposes that the accumulation of mutations in the mitochondrial genome may be responsible in part for the mitochondrial phenomenology of aging. Recent studies of mitochondrial DNA (mtDNA) deletions have shown that they increase with age in humans and other mammals. Currently, there exist numerous important and fundamental questions surrounding mitochondria and aging. Among these are (1) How important are mitochondrial oxidants in determining overall cellular oxidative stress? (2) What are the mechanisms of mitochondrial oxidant generation? (3) How are lesions and mutations in mtDNA formed? (4) How important are mtDNA lesions and mutations in causing mitochondrial dysfunction? (5) How are mitochondria regulated, and how does this regulation change during aging? (6) What are the dynamics of mitochondrial turnover? (7) What is the relationship between mitochondrial damage and lipofuscinogenesis? (8) What are the relationships among mitochondria, apopotosis, and aging? and (9) How can mitochondrial function (ATP generation and the establishment of a membrane potential) and dysfunction (oxidant generation) be modulated and degenerative senescence thereby treated? If the promulgation of a scientific hypothesis in the popular press is a measure of how well it has captivated the scientific community, then the “mitochondrial free radical theory of aging” has surely been successful. In recent years, on the pages of Scientific American (as well as in scores of daily newspapers and health magazines), mitochondrial oxygen free radicals have been fingered as a major contributor to age-related degeneration. In the scientific literature, the attention being paid to mitochondria has paralleled an overall swell of interest in free radicals themselves, as can be seen in the number of recent review articles devoted to the topic. Also, the burgeoning field of mitochondrial genetics, in particular the elucidation of mitochondrial genetic defects in a number of progressive degenerative diseases, has added to the interest in mitochondria. What is the mitochondrial free radical theory of aging? It entails the following central concepts, which have been put forth in papers from a number of researchers: (1) with increasing age, there is a loss in mitochondrial metabolic and oxidative phosphorylation This work was supported by National Cancer Institute Outstanding Investigator Grant CA39910 and National Institute of Environmental Health Sciences Center Grant ES01896 to B.N. Ames. Tel: 510/642-5163 or -9218; fax: 510/643-7935; e-mail: kbeckman@uclink4.berkeley. edu Tel: 510/642-5165; fax: 510/643-7935; e-mail: bnames@uclink4.berkeley. edu BECKMAN & AMES: MITOCHONDRIAL AGING 119 (OXPHOS) activity, such that cells may become limited by their capacity to generate ATP; (2) concomitantly, there is an increase in mitochondrial free radical generation, due to increased “leakage” of electrons from the electron transport chain, which may damage cellular macromolecules; and (3) the loss of OXPHOS capacity may itself be caused by damage wrought by the oxidants generated by mitochondria themselves. Hypotheses surrounding the involvement of mitochondrial DNA (mtDNA) in aging comprise a corollary to the mitochondrial free radical theory of aging. In short, it has been proposed that mtDNA, which encodes a small number of critical mitochondrial genes and may be exceptionally sensitive to oxidative damage, is a weak link. Damage to mtDNA by mitochondrial oxidants, it has been suggested, may result in faulty mitochondrial proteins, decreased OXPHOS, increased mitochondrial oxidant generation, and increased mtDNA damage. This hypothetical positive feedback loop is generally referred to as the “vicious cycle” (FIG. 1). Evidence that supports the mitochondrial free radical theory, of which there is much, has been discussed in recent research reports and review articles, including a number of articles in this volume. We will not attempt to write another such review here. Rather, we have identified some of the most outstanding unanswered questions. The following is a brief (and by no means complete) list of questions, the resolution of which may push mitochondria farther into the spotlight of molecular gerontology, and will at the very least refine our knowledge of the role of mitochondria in aging. “MITOCHONDRIAL MYSTERIES” How Important Are Mitochondrial Oxidants Relative to Other Endogenous Sources? In support of the mitochondrial free radical theory of aging, it is often written that 1– 2% of electron flow through the mitochondrial electron transport chain (ETC) results in the univalent generation of superoxide (O2 ). Upon what evidence are these statements based? Are mitochondria, rather than other endogenous sources, the cell’s most significant source of oxidants? The initial experiments in this area were straightforward and were reviewed by Chance and co-workers. First, it was shown that isolated mitochondria (from rat and pigeon tissues, generally) generate hydrogen peroxide (H2O2) at a rate equivalent to about 2% of total oxygen consumption. Second, isolated submitochondrial FIGURE 1. The hypothetical “vicious cycle. ” Mutations in mtDNA result in defective mitochondrial proteins, defective electron transport, and ultimately, the generation of mutagenic oxidants. These, in turn, may cause mtDNA damage and mutagenesis, completing the cycle. 120 ANNALS NEW YORK ACADEMY OF SCIENCES particles (from which the O2 -detoxifying enzyme, superoxide dismutase, has been removed) were observed to generate from 1.5 to 2.1 times as much O2 as the isolated organelles generated H2O2, indicating near stoichiometry between the former and the latter. Since two molecules of O2 ultimately dismutate—either spontaneously or with the help of superoxide dismutase—to form H2O2, these experiments resulted in the oftenrepeated mitochondrial “leakage” statistic of 1–2% of electron flow. It is not a criticism of this pioneering work in mitochondrial oxidant generation to stress that one cannot extrapolate from such experiments to the in vivo situation. For one, measurements of mitochondrial H2O2 generation were generally carried out in air-saturated buffer, a condition of higher oxygen tension than exists in vivo, which may overestimate the rate of H2O2 generation (shown to be sensitive to the concentration of oxygen ). Second, mitochondrial H2O2 generation was routinely measured under supraphysiological conditions of such saturating substrates as succinate, a potentially confounding factor recently examined by Hansford, whose experiments suggest that the leakage of O2 from mitochondria may be less dramatic in vivo. When these workers measured H2O2 generation using physiological concentrations of substrates, they measured a rate of generation of H2O2 below 0.1% of oxygen consumption. Therefore, little can be said with certainty about the in vivo generation of oxidants. To what extent does this matter? As Hansford et al. point out, even their lower estimates of mitochondrial H2O2 generation may represent a considerable oxidative stress. Although this is undoubtedly true, until robust and physiologically defensible in vivo estimates have been achieved, perhaps by methods that have yet to be devised, it would be wise to refrain from repeating concrete and potentially erroneous rates of endogenous mitochondrial oxidant generation. As is shown schematically in FIGURE 2, mitochondrial oxidant generation represents only one of numerous proand antioxidant mechanisms whose modulation could accelerate or retard aging, a point that has recently been made by others. Last, despite evidence that the ubisemiquinone site of the electron transport chain can generate O2 , it is not evident that this is the principle site of O2 generation in vivo, and so there exist uncertainties about both the extent and the mechanisms of oxidant generation by mitochondria in vivo. Does Mitochondrial Generation of Oxidants Increase with Age? In addition to these unanswered questions about the relative importance of mitochondrial versus nonmitochondrial oxidants in the overall scheme of oxidative stress, it also remains to be definitively shown that the generation of mitochondrial oxidants increases with age. Although some workers report an age-related increase, 26 others have failed to repeat this work. Although there are numerous studies that show an increase in oxidative stress in older cells, as well as recent studies showing increases in specific oxidative damage to mitochondrial enzymes with age, these phenomena could be the result of any number of physiological changes besides increased mitochondrial oxidant generation (for example, they could easily result from decreased antioxidant defenses). One approach to tackling this question has been to show that the oxidative damage of isolated mitochondria results in increased generation of oxidants, an observation that supports the concept of a positive feedback loop of oxidative damage and oxidant generation in aging. BECKMAN & AMES: MITOCHONDRIAL AGING 121 What Are the Most Significant Targets of Mitochondrial Oxidant Generation? A number of related questions concern the nature of the oxidant species present in mitochondria