Vascular superoxide and hydrogen peroxide production and oxidative stress resistance in two closely related rodent species with disparate longevity

Vascular aging is characterized by increased oxidative stress, impaired nitric oxide (NO) bioavailability and enhanced apoptotic cell death. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower production of reactive oxygen species (ROS) and/or superior resistance to oxidative stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), that show a more than twofold difference in maximum lifespan potential (MLSP = 8 and 3.5 years, respectively). We compared interspecies differences in endothelial superoxide (O2-) and hydrogen peroxide (H2O2) production, NAD(P)H oxidase activity, mitochondrial ROS generation, expression of pro- and antioxidant enzymes, NO production, and resistance to oxidative stress-induced apoptosis. In aortas of P. leucopus, NAD(P)H oxidase expression and activity, endothelial and H2O2 production, and ROS generation by mitochondria were less than in mouse vessels. In P. leucopus, there was a more abundant expression of catalase, glutathione peroxidase 1 and hemeoxygenase-1, whereas expression of Cu/Zn-SOD and Mn-SOD was similar in both species. NO production and endothelial nitric oxide synthase expression was greater in P. leucopus. In mouse aortas, treatment with oxidized low-density lipoprotein (oxLDL) elicited substantial oxidative stress, endothelial dysfunction and endothelial apoptosis (assessed by TUNEL assay, DNA fragmentation and caspase 3 activity assays). According to our prediction, vessels of P. leucopus were more resistant to the proapoptotic effects of oxidative stressors (oxLDL and H2O2). Primary fibroblasts from P. leucopus also exhibited less H2O2-induced DNA damage (comet assay) than mouse cells. Thus, increased lifespan potential in P. leucopus is associated with a decreased cellular ROS generation and increased oxidative stress resistance, which accords with the prediction of the oxidative stress hypothesis of aging.     

Effects of aging and hyperoxia on oxidative damage to cytochrome c in the housefly, Musca domestica

Cytochrome c is a component of the mitochondrial electron transport chain, where it transfers electrons from ubiquinol-cytochrome c reductase to cytochrome c oxidase. Autoxidation of some of the components of the electron transport chain is the main source of intracellular O(2)(-*)/H(2)O(2) production in aerobic organisms. Because cytochrome c is located on the outer surface of the inner mitochondrial membrane, it is likely to be constantly exposed to H(2)O(2), secreted by mitochondria into the cytosol. The specific objective of this study was to determine whether cytochrome c in the flight muscle mitochondria of the housefly is oxidatively damaged during aging and/or under severe oxidative stress induced by exposure of flies to 100% oxygen. Results of two independent methods, namely tritiated borohydride labeling for determining carbonylation and mass spectral analysis for the measurement of molecular mass, indicated that neither the carbonyl level nor the molecular mass of cytochrome c was affected by aging or hyperoxia. Thus, either cytochrome c is resistant to oxidative damage in vivo or the oxidized cytochrome c is promptly degraded. These findings also support the concept that protein oxidative damage during aging and under oxidative stress is selective.     

The role of the electron transport SDHC gene on lifespan and cancer

Much attention has been focused on the hypothesis that oxidative damage plays in cellular and organismal aging. It is known that oxygen is initially converted to superoxide anion (O2-), one of reactive oxygen species (ROS), by electron leaked from mainly complex III in the electron transport system present in mitochondria, where it is the major endogenous source of ROS. We have shown that a mutation in a subunit, cytochrome b large subunit (SDHC), of complex II, also results in increasing O2- production and therefore lead to apoptosis and precocious aging in Caenorhabditis elegans. Recently, individuals with an inherited propensity for vascularized head and neck tumors (i.e., paragangliomas) have been demonstrated to contain one of several mutations in complex II. To further explore the role of oxidative stress from mitochondria on apoptosis and cancer, we established a transgenic cell line with a point mutation at the ubiquinone binding region in the SDHC gene. As expected, this mutation increased O2- production from complex II and led to excess apoptosis. Moreover, a significant fraction of the surviving cells from the apoptosis were transformed, as evidenced by increased tumor formation after injection into mice. Oxidative stress results in the damage to the cellular components including mitochondria and, therefore leads to apoptosis. Furthermore, oxidative stress must cause mutations in DNA and leads to cancer. It is suggested that oxidative stress from mitochondria play an important role of both apoptosis, which leads to precocious aging, and cancer.     

Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice

Hypopituitary Ames dwarf mice have low circulating growth hormone (GH)/IGF-I levels, and they have extended longevity and exhibit many symptoms of delayed aging. To elucidate the vascular consequences of Ames dwarfism we compared endothelial O2(-) and H2O2 production, mitochondrial reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and nitric oxide (NO) production in aortas of Ames dwarf and wild-type control mice. In Ames dwarf aortas endothelial O2(-) and H2O2 production and ROS generation by mitochondria were enhanced compared with those in vessels of wild-type mice. In Ames dwarf aortas there was a less abundant expression of Mn-SOD, Cu,Zn-SOD, glutathione peroxidase (GPx)-1, and endothelial nitric oxide synthase (eNOS). NO production and acetylcholine-induced relaxation were also decreased in aortas of Ames dwarf mice. In cultured wild-type mouse aortas and in human coronary arterial endothelial cells treatment with GH and IGF significantly reduced cellular O2(-) and H2O2 production and ROS generation by mitochondria and upregulated expression of Mn-SOD, Cu,Zn-SOD, GPx-1, and eNOS. Thus GH and IGF-I promote antioxidant phenotypic changes in the endothelial cells, whereas Ames dwarfism leads to vascular oxidative stress.     

Prevention of flight activity prolongs the life span of the housefly, Musca domestica, and attenuates the age-associated oxidative damamge to specific mitochondrial proteins

The purpose of this study was to explore the mechanisms by which oxidative stress affects the aging process. The hypothesis that the rate of accumulation of oxidative damage to specific mitochondrial proteins is linked to the life expectancy of animals was tested in the housefly. The rate of oxygen consumption and life expectancy of the flies were experimentally altered by confining the flies in small jars, where they were unable to fly. Prevention of flight activity decreased the rate of oxygen utilization of flies and almost tripled their life span as compared to those permitted to fly. Rate of mitochondrial H(2)O(2) generation at various ages was lower in the low activity flies than in the high activity flies. Oxidative damage to mitochondrial proteins, adenine nucelotide translocase, and aconitase, detected as carbonyl modifications, was attenuated; and the loss in their functional activity occurring with age was retarded in the long-lived low activity flies as compared to the short-lived high activity flies. The two proteins were previously identified to be the only mitochondrial proteins exhibiting age-related increases in carbonylation. Results support the hypothesis that accrual of oxidative damage to specific protein targets and the consequent loss of their function may constitute a mechanism by which oxidative stress controls the aging process.     

Mitochondrial production of pro-oxidants and cellular senescence.

Mitochondria are the major intracellular producers of O2- and H2O2. The level of oxidative stress in cells, as indicated by the in vivo exhalation of alkanes and the concentration of molecular products of oxy-radical reactions, increases during aging in mammals as well as insects. In this paper, we discuss the relationship between mitochondrial generation of O2- and H2O2, and the aging process. The rate of mitochondrial O2- and H2O2 generation increases with age in houseflies and the brain, heart and liver of rat. This rate has been found to correspond to the life expectancy of flies and to the maximum life span potential (MLSP) of six different mammalian species, namely, mouse, rat, guinea pig, rabbit, pig and cow. In contrast, the level of antioxidant defenses provided by activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione concentration neither uniformly declines with age nor corresponds to variations in MLSP of different mammalian species. It is argued that the rate of mitochondrial O2- and H2O2 generation rather than the antioxidant level may act as a longevity determinant.     

Influence of aging and long-term caloric restriction on oxygen radical generation and oxidative DNA damage in rat liver mitochondria

The effect of long-term caloric restriction and aging on the rates of mitochondrial H2O2 production and oxygen consumption as well as on oxidative damage to nuclear (nDNA) and mitochondrial DNA (mtDNA) was studied in rat liver tissue. Long-term caloric restriction significantly decreased H2O2 production of rat liver mitochondria (47% reduction) and significantly reduced oxidative damage to mtDNA (46% reduction) with no changes in nDNA. The decrease in ROS production was located at complex I because it only took place with complex I-linked substrates (pyruvate/malate) but not with complex II-linked substrates (succinate). The mechanism responsible for that decrease in ROS production was not a decrease in mitochondrial oxygen consumption because it did not change after long-term restriction. Instead, the caloric restricted mitochondria released less ROS per unit electron flow, due to a decrease in the reduction degree of the complex I generator. On the other hand, increased ROS production with aging in state 3 was observed in succinate-supplemented mitochondria because old control animals were unable to suppress H2O2 production during the energy transition from state 4 to state 3. The levels of 8-oxodG in mtDNA increased with age in old animals and this increase was abolished by caloric restriction. These results support the idea that caloric restriction reduces the aging rate at least in part by decreasing the rate of mitochondrial ROS production and so, the rate of oxidative attack to biological macromolecules like mtDNA.     

HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise

Exercise causes heat shock (muscle temperatures of up to 45 degrees C, core temperatures of up to 44 degrees C) and oxidative stress (generation of O2- and H2O2), and exercise training promotes mitochondrial biogenesis (2-3-fold increases in muscle mitochondria). The concentrations of at least 15 possible heat shock or oxidative stress proteins (including one with a molecular weight of 70 kDa) were increased, in skeletal muscle, heart, and liver, by exercise. Soleus, plantaris, and extensor digitorum longus (EDL) muscles exhibited differential protein synthetic responses ([3H]leucine incorporation) to heat shock and oxidative stress in vitro but five proteins (particularly a 70 kDa protein and a 106 kDa protein) were common to both stresses. HSP70 mRNA levels were next analyzed by Northern transfer, using a [32P]-labeled HSP70 cDNA probe. HSP70 mRNA levels were increased, in skeletal and cardiac muscle, by exercise and by both heat shock and oxidative stress. Skeletal muscle HSP70 mRNA levels peaked 30-60 min following exercise, and appeared to decline slowly towards control levels by 6 h postexercise. Two distinct HSP70 mRNA species were observed in cardiac muscle; a 2.3 kb mRNA which returned to control levels within 2-3 h postexercise, and a 3.5 kb mRNA species which remained at elevated concentrations for some 6 h postexercise. The induction of HSP70 appears to be a physiological response to the heat shock and oxidative stress of exercise. Exercise hyperthermia may actually cause oxidative stress since we also found that muscle mitochondria undergo progressive uncoupling and increased O2- generation with increasing temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly

The oxidative stress hypothesis of aging predicts that a reduction in the generation of mitochondrial reactive oxygen species (ROS) will decrease oxidative damage and extend life span. Increasing mitochondrial proton leak-dependent state 4 respiration by increasing mitochondrial uncoupling is an intervention postulated to decrease mitochondrial ROS production. When human UCP2 (hUCP2) is targeted to the mitochondria of adult fly neurons, we find an increase in state 4 respiration, a decrease in ROS production, a decrease in oxidative damage, heightened resistance to the free radical generator paraquat, and an extension in life span without compromising fertility or physical activity. Our results demonstrate that neuronal-specific expression of hUCP2 in adult flies decreases cellular oxidative damage and is sufficient to extend life span.     

Age-associated cellular relocation of Sod 1 as a self-defense is a futile mechanism to prevent vascular aging

Vascular aging is characterized by the presence of chronic oxidative stress. Although cytosolic Sod 1 has a key role in the detoxification of superoxide ((*)O(2)(-)), little is known about its importance in vascular aging. We found that inhibition of Sod 1 had no effect on (*)O2- generation. Furthermore, its expression decreased in an age-dependent manner. Interestingly, Sod 1 loses its membrane-association and is also lost from the caveolae with increasing age. Instead, a relocation of Sod 1 to the mitochondria takes place, presumably in an attempt to maintain mitochondrial integrity and to counter-balance age-associated oxidative stress. Unlike Sod 2, which is constitutively expressed in mitochondria to control (*)O2- radical fluxes, Sod 1 is not inactivated by peroxynitrite and is not nitrated as a function of age. These novel insights into oxidative stress-associated vascular aging and the understanding about how redox-systems are regulated in old age may identify new targets to ameliorate aging as the greatest cardiovascular risk factor.     

Mitochondria in exercise-induced oxidative stress

In recent years it has been suggested that reactive oxygen species (ROS) are involved in the damage to muscle and other tissues induced by acute exercise. Despite the small availability of direct evidence for ROS production during exercise, there is an abundance of literature providing indirect support that oxidative stress occurs during exercise. The electron transport associated with the mitochondrial respiratory chain is considered the major process leading to ROS production at rest and during exercise. It is widely assumed that during exercise the increased electron flow through the mitochondrial electron transport chain leads to an increased rate of ROS production. On the other hand, results obtained by in vitro experiments indicate that mitochondrial ROS production is lower in state 3 (ADP-stimulated) than in state 4 (basal) respiration. It is possible, however, that factors, such as temperature, that are modified in vivo during intense physical activity induce changes (uncoupling associated with loss of cytochrome oxidase activity) leading to increased ROS production. The mitochondrial respiratory chain could also be a potential source of ROS in tissues, such as liver, kidney and nonworking muscles, that during exercise undergo partial ischemia because of reduced blood supply. Sufficient oxygen is available to interact with the increasingly reduced respiratory chain and enhance the ROS generation. At the cessation of exercise, blood flow to hypoxic tissues resumes leading to their reoxygenation. This mimics the ischemia-reperfusion phenomenon, which is known to cause excessive production of free radicals. Apart from a theoretical rise in ROS, there is little evidence that exercise-induced oxidative stress is due to its increased mitochondrial generation. On the other hand, if mitochondrial production of ROS supplies a remarkable contribution to exercise-induced oxidative stress, mitochondria should be a primary target of oxidative damage. Unfortunately, there are controversial reports concerning the exercise effects on structural and functional characteristics of mitochondria. However, the isolation of mitochondrial fractions by differential centrifugation has shown that the amount of damaged mitochondria, recovered in the lightest fraction, is remarkably increased by long-lasting exercise.     

Stress,Aging, and Brain Oxidative Damage

Stress may contribute to aging acceleration and age-related degenerative diseases. Stress and adaptation to stress require numerous homeostatic adjustments including hormones, neurotransmitters, oxidants, and other mediators. The stress-induced hormones, neurotransmitters, and oxidants all have beneficial, but also harmful effects if out of balance. Therefore, the homeostasis of stress and adaptation should be governed by the hormone balance, neurotransmitter balance, and oxidant balance, as well as the interactions among these substances. The imbalance and the over-interaction of these balances may ultimately cause increased oxidant generation and oxidative damage to biomolecules. This increased oxidative damage may add to the oxidant burden associated with normal aerobic metabolism, which in itself, generates oxidants, causes accumulation of oxidative damage in mitochondria, and contributes to normal aging. Therefore, the stress-associated increase of oxidative damage may, in part, contribute to stress-associated aging acceleration and age-related neurodegenerative diseases.     

Oxidative stress in human aging and mitochondrial disease-consequences of defective mitochondrial respiration and impaired antioxidant enzyme system

Respiratory function of mitochondria is compromised in aging human tissues and severely impaired in the patients with mitochondrial disease. A wide spectrum of mitochondrial DNA (mtDNA) mutations has been established to associate with mitochondrial diseases. Some of these mtDNA mutations also occur in various human tissues in an age-dependent manner. These mtDNA mutations cause defects in the respiratory chain due to impairment of the gene expression and structure of respiratory chain polypeptides that are encoded by the mitochondrial genome. Since defective mitochondria generate more reactive oxygen species (ROS) such as O2- and H2O2 via electron leak, we hypothesized that oxidative stress is a contributory factor for aging and mitochondrial disease. This hypothesis has been supported by the findings that oxidative stress and oxidative damage in tissues and culture cells are increased in elderly subjects and patients with mitochondrial diseases. Another line of supporting evidence is our recent finding that the enzyme activities of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) decrease with age in skin fibroblasts. By contrast, Mn-SOD activity increases up to 65 years of age and then slightly declines thereafter. On the other hand, we observed that the RNA, protein and activity levels of Mn-SOD are increased two- to three-fold in skin fibroblasts of the patients with CPEO syndrome but are dramatically decreased in patients with MELAS or MERRF syndrome. However, the other antioxidant enzymes did not change in the same manner. The imbalance in the expression of these antioxidant enzymes indicates that the production of ROS is in excess of their removal, which in turn may elicit an elevation of oxidative stress in the fibroblasts. Indeed, it was found that intracellular levels of H2O2 and oxidative damage to DNA and lipids in skin fibroblasts from elderly subjects or patients with mitochondrial diseases are significantly increased as compared to those of age-matched controls. Furthermore, Mn-SOD or GPx-1 gene knockout mice were found to display neurological disorders and enhanced oxidative damage similar to those observed in the patients with mitochondrial disease. These observations are reviewed in this article to support that oxidative stress elicited by defective respiratory function and impaired antioxidant enzyme system plays a key role in the pathophysiology of mitochondrial disease and human aging.     

Fat intake reverses the beneficial effects of low caloric intake on skeletal muscle mitochondrial H(2)O(2) production

Food restriction is the most effective modulator of oxidative stress and it is believed that a reduction in caloric intake per se is responsible for the reduced generation of reactive oxygen species (ROS) by mitochondria. Hydrogen peroxide (H(2)O(2)) generation and oxygen consumption (O(2)) by skeletal muscle mitochondria were determined in a peculiar strain of rats (Lou/C) characterized by a self-low-caloric intake and a dietary preference for fat. These rats were fed either with a standard high-carbohydrate (HC) or a high-fat (HF) diet and the results were compared to those measured in Wistar rats fed a HC diet. H(2)O(2) production was significantly reduced in Lou/C rats fed a HC diet; this effect was not due to a lower O(2) consumption but rather to a decrease in rotenone-sensitive NADH-ubiquinone oxidoreductase activity and increased expression of uncoupling proteins 2 and 3. The reduced H(2)O(2) generation displayed by Lou/C rats was accompanied by a significant inhibition of permeability transition pore (PTP) opening. H(2)O(2) production was restored and PTP inhibition was relieved when Lou/C rats were allowed to eat a HF diet, suggesting that the reduced oxidative stress provided by low caloric intake is lost when fat proportion in the diet is increased.     

Age-specific decrease in aerobic efficiency associated with increase in oxygen free radical production in Drosophila melanogaster

This study was designed to test the free radical theory of aging by using Drosophila melanogaster as a model system. Oxygen free radicals are generated by mitochondria during the process of normal oxidative metabolism. Age-specific measurements of oxygen consumption, heat production and anti-oxidant enzyme activity were obtained from two inbred lines of male flies, one selected for longevity and one normal-lived. The findings of this study demonstrate that although oxygen consumption remains relatively constant over the majority of the life span of each line of flies, aerobic efficiency declines with advancing age. This loss of aerobic efficiency manifests itself as a decline in total body metabolism as measured by heat production, and appears to be associated with an age-specific increase in damage inflicted upon mitochondria by oxygen free radicals.     

Translocation of deltaPKC to mitochondria during cardiac reperfusion enhances superoxide anion production and induces loss in mitochondrial function

Activation of the delta-isoform of protein kinase C (deltaPKC) by certain conditions of oxidative stress results in translocation of the kinase to the mitochondria leading to release of cytochrome c and the induction of apoptosis. In the current study, the effects of myocardial reperfusion-induced deltaPKC translocation on mitochondrial function were assessed. Mitochondria isolated from hearts that had undergone ischemia (30 min) followed by reperfusion (15 min) exhibited a significant increase in the rate of superoxide anion (O(2)(-)) generation. This was associated with the translocation of deltaPKC to the mitochondria within the first 5 min of reperfusion. deltaPKC translocation occurred exclusively during reperfusion and could be mimicked by infusion of intact hearts with H(2)O(2) suggesting redox-dependent activation during reperfusion. Infusion of a peptide inhibitor (deltaV(1-1)) specific to the delta-isoform of PKC significantly reduced reperfusion-induced increases in mitochondrial O(2)(-) generation. Finally, the decline in mitochondrial respiratory activity evident upon prolonged reperfusion (120min) was completely prevented by inhibition of deltaPKC translocation. Thus, deltaPKC represents a cytosolic redox-sensitive molecule that plays an important role in amplification of O(2)(-) production and subsequent declines in mitochondrial function during reperfusion.     

Oxidative stress increased respiration and generation of reactive oxygen species, resulting in ATP depletion, opening of mitochondrial permeability transition, and programmed cell death

Mitochondria constitute a major source of reactive oxygen species and have been proposed to integrate the cellular responses to stress. In animals, it was shown that mitochondria can trigger apoptosis from diverse stimuli through the opening of MTP, which allows the release of the apoptosis-inducing factor and translocation of cytochrome c into the cytosol. Here, we analyzed the role of the mitochondria in the generation of oxidative burst and induction of programmed cell death in response to brief or continuous oxidative stress in Arabidopsis cells. Oxidative stress increased mitochondrial electron transport, resulting in amplification of H(2)O(2) production, depletion of ATP, and cell death. The increased generation of H(2)O(2) also caused the opening of the MTP and the release of cytochrome c from mitochondria. The release of cytochrome c and cell death were prevented by a serine/cysteine protease inhibitor, Pefablock. However, addition of inhibitor only partially inhibited the H(2)O(2) amplification and the MTP opening, suggesting that protease activation is a necessary step in the cell death pathway after mitochondrial damage.     

Bcl-2 family proteins regulate mitochondrial reactive oxygen production and protect against oxidative stress

Bcl-2 family proteins protect against a variety of forms of cell death, including acute oxidative stress. Previous studies have shown that overexpression of the antiapoptotic protein Bcl-2 increases cellular redox capacity. Here we report that cell lines transfected with Bcl-2 paradoxically exhibit increased rates of mitochondrial H(2)O(2) generation. Using isolated mitochondria, we determined that increased H(2)O(2) release results from the oxidation of reduced nicotinamide adenine dinucleotide-linked substrates. Antiapoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 also increase mitochondrial H(2)O(2) release when overexpressed. Chronic exposure of cells to low levels of the mitochondrial uncoupler carbonyl cyanide 4-(triflouromethoxy)phenylhydrazone reduced the rate of H(2)O(2) production by Bcl-xL overexpressing cells, resulting in a decreased ability to remove exogenous H(2)O(2) and enhanced cell death under conditions of acute oxidative stress. Our results indicate that chronic and mild elevations in H(2)O(2) release from Bcl-2, Bcl-xL, and Mcl-1 overexpressing mitochondria lead to enhanced cellular antioxidant defense and protection against death caused by acute oxidative stress.     

Changes in antioxidant status of myocardium during oxidative stress under the influence of coenzyme Q<Subscript>10</Subscript>

Changes in myocardium were studied during oxidative stress induced by infusion of hydrogen peroxide in the coronary vessels of isolated rat heart. Moderate concentrations of H2O2 increased the heart rate but decreased the contractile force, whereas higher concentrations of H2O2 decreased both parameters and increased the end diastolic pressure. The effect of H2O2 was stable, cumulative, and was associated with disturbance in respiration of mitochondria, increased production of ROS in them, and decrease in activities of antioxidant enzymes in the myocardium. Changes in the antioxidant status of the myocardium induced by long-term addition of coenzyme Q(10) into food was accompanied by decrease in the negative inotropic effect of H2O2, whereas the levels of superoxide dismutase and glutathione peroxidase after oxidative stress were virtually unchanged. The activities of these enzymes displayed a high positive correlation with the cardiac function. The findings suggest that coenzyme Q(10) should increase resistance of the myocardium to oxidative stress not only by a direct antioxidant mechanism but also indirectly, due to increased protection of antioxidant enzymes.     

Ectopic expression of catalase in Drosophila mitochondria increases stress resistance but not longevity

The goal of this study was to test the hypothesis that the rate of mitochondrial oxidant production governs the aging process of the fruit fly, Drosophila melanogaster. Catalase, an antioxidative enzyme expressed in the cytosol and peroxisomes of Drosophila, was targetted ectopically to the mitochondrial matrix by fusion of a leader peptide derived from ornithine aminotransferase with its N-terminus. The presence of the transgene encoding this fusion protein was associated with moderate (35 +/- 13%) increases in total catalase activity in most lines, and measurable levels of catalase activity in the mitochondria (30-140 U/mg protein). There was no impact on the life span of the flies at 25 degrees C, even in an exceptional line with a 149% increase in total catalase activity, and there was a small decrease in longevity at 29 degrees C. There were no compensatory changes in the rate of metabolism or physical activity, or in the levels of other major antioxidants, suggesting that the aging process was largely unaffected. Resistance to exogenous hydrogen peroxide, paraquat, and cold stress was enhanced, but there was no appreciable effect on resistance to hyperoxia. The results demonstrate the importance of mitochondrial antioxidant levels in the resistance to oxidative stress at the organismal level, and illustrate that different effects on aging and stress resistance may ensue from a single treatment. The main inferences drawn are that: (i) levels of stress resistance may neither be a cause nor a reliable indicator of the rate of aging, and (ii) bolstering antioxidant levels in Drosophila may not delay or slow down the aging process.