The effects of catalase gene overexpression on life span and resistance to oxidative stress in transgenic Drosophila melanogaster.

Oxygen free radicals and hydroperoxides have been postulated to play a causal role in the aging process, implying that antioxidant enzymes may act as longevity determinants. Catalase (H2O2:H2O2 oxidoreductase; EC1.11.1.6) is the sole enzyme involved in the elimination of H2O2 in Drosophila melanogaster; glutathione peroxidase being absent. A genomic fragment containing the Drosophila catalase gene was used to construct transgenic Drosophila lines by means of P element-mediated transformation. Enhanced levels of catalase (up to 80%) did not prolong the life span of flies, nor did they provide improved protection against oxidative stress induced by hyperoxia or paraquat treatment. However, enhanced resistance to hydrogen peroxide was observed in the overexpressors.     

Overexpression of Mn-containing superoxide dismutase in transgenic Drosophila melanogaster.

The general objective of this study was to examine the role of mitochondria in the aging process. Two alternative hypotheses were tested: (i) that overexpression of Mn superoxide dismutase (Mn SOD) in the mitochondria of Drosophila melanogaster would slow the accrual of oxidative damage and prolong survival or (ii) that there is an evolved optimum level of superoxide anion radical, such that overexpression of Mn SOD would have deleterious or neutral effects. Microinjection and mobilization of a transgene, which contained a 9-kb genomic sequence encoding Mn SOD, produced 15 experimental lines overexpressing Mn SOD by 5-116% relative to the parental y w strain. Comparisons between these lines and control lines containing inserted vector sequences alone indicated that the mean longevity of the experimental lines was decreased by 4-5% relative to controls. There were no compensatory changes in the metabolic rate, level of physical activity, or the levels of other antioxidants, namely Cu-Zn SOD, catalase, and glutathione. There were no differences between groups in rates of mitochondrial hydrogen peroxide release, protein oxidative damage, or resistance to 100% oxygen or starvation conditions. The experimental lines had a marginally increased resistance to moderate heat stress. These results are consistent with the existence of an optimum level of Mn SOD activity which minimizes oxidative stress. The naturally evolved level of Mn SOD activity in Drosophila appears to be near the optimum required under normal conditions, although the optimum may be shifted to a higher level under more stressful conditions.     

Catalase, a target of glycation damage in rat liver mitochondria with aging

Aging is characterized by progressive decline of major cell functions, associated with accumulation of altered macromolecules, particularly proteins. This deterioration parallels age-related dysfunction of mitochondria, thought to be a major determinant of this decline in cell function, since these organelles are both the main sources of reactive oxygen species and targets for their damaging effects. To investigate the link between glycation damages that accumulate with aging and the status of mitochondrial antioxidant enzymes, we identified, by mass spectrometry after two dimensional-gel electrophoresis and western blotting, advanced glycation end product-modified matrix proteins in rat liver mitochondria. Catalase appeared to be the only antioxidant enzyme markedly glycated in old rats. Immunogold labeling performed on isolated mitochondria confirmed the mitochondrial matrix location of this enzyme. The content of catalase protein in mitochondrial extract increased with aging whereas the catalase activity was not significantly modified, in spite of a significant increase rate of glycation. Treatment of catalase with the glycating agent fructose led to significant time-dependent inactivation of the enzyme, while methylglyoxal had no noticeable effect. Catalase was co-identified with unglycated glutathione peroxidase-1 in the mitochondrial extracts. Taken together, these results indicate that both anti-oxidant enzymes catalase and glutathione peroxidase-1 housed in liver mitochondria, exhibited a differential sensitivity to glycation; moreover, they lend support to the hypothesis that glycation damages targeting catalase with aging may severely affect its activity, suggesting a link between glycation stress and the age-related decline in antioxidant defense in the mitochondria.     

Age-associated changes in oxidative damage and the activity of antioxidant enzymes in rats with inherited overgeneration of free radicals

Reactive oxygen species have been hypothesized to play an important role in the process of aging. To investigate the correlation between oxidative stress and accumulation of protein and DNA damage, we have compared the age-dependent levels of protein carbonyl groups and the activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase in cytosol and mitochondrial extracts from liver cells of Wistar and OXYS rats. The latter strain is characterized by increased sensitivity to free radicals. Faster age-dependent increase in the level of protein carbonyl groups was found in OXYS as compared with Wistar rats. A complicated enzyme-specific pattern of age-dependent changes in the activities of antioxidant enzymes was observed. Long-term uptake of dietary supplements Mirtilene forte (extract from the fruits of Vaccinium myrtillus L.) or Adrusen zinco (vitamin E complex with zinc, copper, selenium and omega-3 polyunsaturated fatty acids) sharply decreased the level of protein oxidation in cytosol and mitochondrial extracts of hepatocytes of Wistar and of OXYS rats. Both dietary supplements increased the activity of catalase in the liver mitochondria of OXYS rats. Our results are in agreement with the shorter life-span of OXYS and with the mitochondrial theory of aging, which postulates that accumulation of DNA and protein lesions leads to mitochondrial dysfunction and accelerates the process of aging.     

Transcriptional regulation of the Drosophila catalase gene by the DRE/DREF system

Reactive oxygen species (ROS) cause oxidative stress and aging. The catalase gene is a key component of the cellular antioxidant defense network. However, the molecular mechanisms that regulate catalase gene expression are poorly understood. In this study, we have identified a DNA replication-related element (DRE; 5'-TATCGATA) in the 5'-flanking region of the Drosophila catalase gene. Gel mobility shift assays revealed that a previously identified factor called DREF (DRE- binding factor) binds to the DRE sequence in the Drosophila catalase gene. We used site-directed mutagenesis and in vitro transient transfection assays to establish that expression of the catalase gene is regulated by DREF through the DRE site. To explore the role of DRE/DREF in vivo, we established transgenic flies carrying a catalase-lacZ fusion gene with or without mutation in the DRE. The beta-galactosidase expression patterns of these reporter transgenic lines demonstrated that the catalase gene is upregulated by DREF through the DRE sequence. In addition, we observed suppression of the ectopic DREF-induced rough eye phenotype by a catalase amorphic Cat(n1) allele, indicating that DREF activity is modulated by the intracellular redox state. These results indicate that the DRE/DREF system is a key regulator of catalase gene expression and provide evidence of cross-talk between the DRE/DREF system and the antioxidant defense system.     

Sex differences in survival and mitochondrial bioenergetics during aging in Drosophila

The goal of this study is to test the role of mitochondria and of mitochondrial metabolism in determining the processes that influence aging of female and male Drosophila. We observe that Drosophila simulans females tended to have shorter lifespan, higher levels of hydrogen peroxide production and significantly lower levels of catalase but not superoxide dismutase compared to males. In contrast, mammalian females tend to be longer lived, have lower rates of reactive oxygen species production and higher antioxidant activity. In both Drosophila and mammals, mitochondria extracted from females consume a higher quantity of oxygen when provided with adenosine diphosphate and have a greater mtDNA copy number than males. Combined, these data illustrate important similarities between the parameters that influence aging and mitochondrial metabolism in Drosophila and in mammals but also show surprising differences.     

Attenuation of Rotenone-Induced Mitochondrial Oxidative Damage and Neurotoxicty in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Supplemented with Creatine

Creatine (Cr), an ergogenic nutritional supplement is demonstrated to possess bioenergetic, antiexcitotoxic and antioxidant properties. This study investigated the neuroprotective effects of Cr against rotenone induced oxidative stress, mortality and neurotoxicty in Drosophila melanogaster. We found significant diminution in the endogenous levels of oxidative markers in whole body homogenates of flies exposed to Cr (2–10 mM). Cr supplementation resulted in reduced mortality in flies exposed to rotenone (500 μM) and better performance in a negative geotaxis assay. Further Cr (10 mM) markedly offset rotenone induced mitochondrial oxidative stress, completely restored the GSH levels, nitric oxide levels, activity of Mn-SOD and dopamine depletion. In an oxidative stress bioassay, flies given Cr prophylaxis exhibited marked resistance to paraquat exposure. These data allow us to hypothesize that the neuroprotective action of Cr in Drosophila may be related to its direct antioxidant activity and ability to abrogate rotenone induced mitochondrial oxidative stress.     

Exercise by lifelong voluntary wheel running reduces subsarcolemmal and interfibrillar mitochondrial hydrogen peroxide production in the heart

Evidence suggests that mitochondrial dysfunction and oxidant production, in association with an accumulation of oxidative damage, contribute to the aging process. Regular physical activity can delay the onset of morbidity, increase mean lifespan, and reduce the risk of developing several pathological states. No studies have examined age-related changes in oxidant production and oxidative stress in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria in combination with lifelong exercise. Therefore, we investigated whether long-term voluntary wheel running in Fischer 344 rats altered hydrogen peroxide (H2O2) production, antioxidant defenses, and oxidative damage in cardiac SSM and IFM. At 10-11 wk of age, rats were randomly assigned to one of two groups: sedentary and 8% food restriction (sedentary; n = 20) or wheel running and 8% food restriction (runners; n = 20); rats were killed at 24 mo of age. After the age of 6 mo, running activity was maintained at an average of 1,145 +/- 248 m/day. Daily energy expenditure determined by doubly labeled water technique showed that runners expended on average approximately 70% more energy per day than the sedentary rats. Long-term voluntary wheel running significantly reduced H2O2 production from both SSM (-10.0%) and IFM (-9.6%) and increased daily energy expenditure (kJ/day) significantly in runners compared with sedentary controls. Additionally, MnSOD activity was significantly lowered in SSM and IFM from wheel runners, which may reflect a reduction in mitochondrial superoxide production. Activities of the other major antioxidant enzymes (glutathione peroxidase and catalase) and glutathione levels were not altered by wheel running. Despite the reduction in mitochondrial oxidant production, no significant differences in oxidative stress levels (4-hydroxy-2-nonenal-modified proteins, protein carbonyls, and malondialdehyde) were detected between the two groups. The health benefits of chronic exercise may be, at least partially, due to a reduction in mitochondrial oxidant production; however, we could not detect a significant reduction in several selected parameters of oxidative stress.     

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.     

Alterations of Antioxidant Enzymes and Oxidative Damage to Macromolecules in Different Organs of Rats During Aging

Oxygen free radicals have been hypothesized to play an important role in the aging process. To investigate the correlation between the oxidative stress and aging, we have determined the levels of oxidative protein damage and lipid peroxidation in the brain and liver, and activities of antioxidant enzymes in the brain, liver, heart, kidney, and serum from the Fisher 344 rats at ages of 1, 6, 12, 18, and 24 months. The results showed that the level of oxidative protein damage (measured as carbonyl content) in the brain and liver was significantly higher in older animals than in young animals. No statistical difference was observed in the lipid peroxidation of the liver and brain between young and old animals. The activities of antioxidant enzymes in most tissues displayed an age-dependent decline. Superoxide dismutases in the heart, kidney, and serum, glutathione peroxidase activities in the serum and kidney, and catalase activities in the brain, liver, and kidney, significantly decreased during aging. Cytochrome c oxidase, an enzyme involved in electron transport in mitochondria, initially increased, but subsequently decreased in the aged brain, whereas no significant alteration was observed in the liver mitochondrial antioxidant enzymes. The present studies suggest that the accumulation of oxidized proteins during aging is most likely to be linked with an age-related decline of antioxidant enzyme activities, whereas lipid peroxidation is less sensitive to predict the aging process.     

The effects of reactive oxygen species on amphibian aging

To clarify the role of reactive oxygen species (ROS) in the aging process of amphibians, antioxidant enzyme activity and indexes of ROS damage were investigated biochemically using the livers of 3- and 10-year-old Rana nigromaculata frog males and females. Findings revealed no significant difference in survival rate between males and females. Antioxidant enzyme activity displayed an age-related decline. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activity in 10-year-old liver decreased 40-80% from 3-year-old liver levels. In contrast, urate oxidase activity in the 10-year-old liver increased more than 200% from 3-year-old liver levels. At the same time levels of ROS damage, including the concentration of inorganic peroxide and thiobarbituric acid reactive substances (TBARS), greatly increased with age. Liver catalase from 10-year-old frogs proved to be more susceptible to aminotriazole and urea, losing approximately 80% of its original activity after 30 min of treatment. It seems likely that liver catalase in older frogs has diverged from liver catalase in younger frogs through oxidative modification. These findings suggest that a decrease in the activity of antioxidant enzymes over time results in increased levels of ROS damage in the livers of older frogs.     

Different responses of tobacco antioxidant enzymes to light and chilling stress

The effect of elevated light treatment (25 degrees C, PPFD 360 mumol m-2 sec-1) or chilling temperatures combined with elevated light (5 degrees C, PPFD 360 mumol m-2 sec-1) on the activity of six antioxidant enzymes, guaiacol peroxidases, and glutathione peroxidase (GPx, EC 1.11.1.9) protein accumulation were studied in tobacco Nicotiana tabacum cv. Petit Havana SR1. Both treatments caused no photooxidative damage, but chilling caused a transient wilting. The light treatment increased the activities of ascorbate peroxidase (APx, EC 1.11.1.11) and guaiacol peroxidases while catalase (EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were unchanged. In contrast, chilling treatment did not increase any of the antioxidant enzyme activities, but decreased catalase and to a lesser extent DHAR activities. Glutathione peroxidase protein levels increased sporadically under light treatment and constantly under chilling. Both chilling and light stress caused induction of glutathione synthesis and accumulation of oxidised glutathione, although the predominant part of the glutathione pool remained in the reduced form. Antioxidant enzymes from the chilling treated plants were measured at both 25 degrees C and 5 degrees C. Measurements at 5 degrees C revealed a 3-fold reduction in catalase activity, compared with that measured at 25 degrees C, indicating that the overall reduction in catalase after four days of chilling was approximately 10-fold. The overall reduction in activity for the other antioxidant enzymes after four days of chilling was 2-fold for GR and APx, 1.5-fold for MDHAR, 3.5-fold for DHAR. The activity of SOD was the same at 25 and 5 degrees C. These results indicate that catalase and DHAR are most strongly affected by the chilling treatment and may be the rate-limiting factor of the antioxidant system at low temperatures.     

Two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress

Cardiac mitochondria are composed of two distinct subpopulations: one beneath the sarcolemma (subsarcolemmal mitochondria: SSM), and another along the myofilaments (interfibrillary mitochondria: IFM). Previous studies suggest a preferential loss of IFM function with age; however, the age-related changes in oxidative stress in these mitochondrial subpopulations have not been examined. To this end, the changes in mitochondrial antioxidant capacity, oxidant output, and oxidative damage to Complex IV in IFM and SSM from young and old rats were studied. Results show no apparent differences in any parameters examined between IFM and SSM from young rats. However, relative to young, only IFM from old rats had a significantly higher rate of oxidant production and a decline in mitochondrial ascorbate levels and GSH redox status. The age-related decline in mitochondrial antioxidant capacity in IFM was accompanied by a marked loss in glutaredoxin and GSSG reductase activities, suggesting a diminished reductive capacity in IFM with age. Moreover, the loss in Complex IV activity was limited to the IFM of old rats, which was accompanied by a 4-fold increase in 4-hydroxynonenal-modified Complex IV. Thus, mitochondrial decay is not uniform and further indicates that myofibrils may be uniquely under oxidative stress in the aging heart.     

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.     

Altered levels of primary antioxidant enzymes in progeria skin fibroblasts

Free radicals are involved in the aging process. In this study, the profile of primary antioxidant enzymes that scavenge reactive oxygen species (ROS) was examined for the first time in human skin fibroblasts from progeria, a premature aging disease. Altered levels of antioxidant enzymes were found in progeria cells. Basal levels of MnSOD were decreased in progeria cells as well as a blunted induction in response to chronic stress. This change may contribute to the accelerated aging process in progeria cells. In contrast, the levels of CuZnSOD showed no progeria-related change. Two H2O2 removing enzymes demonstrated a significant reduction in progeria cells: only 50% of normal CAT activity and 30% of normal GPX activity can be detected in progeria cells. This diminished H2O2 removing capacity in progeria cells may lead to an imbalance of intracellular ROS and therefore may play an important role in the development of progeria.