AJP - Regu Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 287: R1244-R1249, 2004. First published July 22, 2004; doi:10.1152/ajpregu.00226.2004
0363-6119/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/5/R1244    most recent
00226.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (29)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Navarro, A.
Right arrow Articles by Boveris, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Navarro, A.
Right arrow Articles by Boveris, A.

APPETITE, OBESITY, DIGESTION, AND METABOLISM

Rat brain and liver mitochondria develop oxidative stress and lose enzymatic activities on aging

Ana Navarro1 and Alberto Boveris2

1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Cádiz, 11003 Cádiz, Spain; and 2School of Pharmacy and Biochemistry, University of Buenos Aires, C1113AAD Buenos Aires, Argentina

Submitted 5 April 2004 ; accepted in final form 15 July 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The mitochondrial mass of rat brain and liver remained unchanged on aging in young adults, old adults, and senescent animals (28, 60, and 92 wk of age); the values were 15–17 and 29–31 mg protein/g for brain and liver, respectively. The whole aging process was associated with an increased content of the oxidation products, thiobarbituric acid-reactive substances and protein carbonyls, by 61–69% in brain and 36–45% in liver, respectively. The activities of critical enzymes for mitochondrial function, mitochondrial nitric oxide synthase, Mn-superoxide dismutase, complex I, and complex IV, decreased progressively during aging with activity losses of 73, 37, 29, and 28%, respectively, in the brain and 47, 46, 30, and 24% in the liver of senescent rats compared with young adults. Brain mitochondria isolated from aged rats showed increased mitochondrial fragility, as assayed by mitochondrial marker enzyme activities in the postmitochondrial supernatant, and increased volume and water permeability, as assayed by light scattering. Liver mitochondria isolated from young and old rats did not show differences in fragility and water permeability. A subpopulation of brain mitochondria with increased size and fragility was differentiated in aging rats, whereas liver showed a homogeneous mitochondrial population.

mitochondrial nitric oxide synthase; manganese-superoxide dismutase; NADH-cytochrome c reductase; cytochrome oxidase; mitochondrial fragility; oxidative stress

THE MAINTENANCE OR LOSS of mitochondrial function during aging is both an interesting and a controversial issue. On the one hand, mitochondria are brought to attention in aging biology due to 1) the central role of mitochondria in producing chemical energy (ATP) to meet cellular requirements, and 2) the declines of metabolic rate and of physical performance in energy-requiring tasks that are associated with the aging process (29). Mitochondrial function includes the conversion of the chemical redox energy of substrate oxidation: first to a proton electrochemical gradient and then to high-energy phosphate chemical bonds (ATP) in a highly organized and coupled process that requires the physical integrity of the mitochondrial inner membrane. On the other hand, the mitochondrial hypothesis of aging considers mitochondria as the pacemaker of tissue aging due to continuous mitochondrial production of reactive oxygen and nitrogen species (O2–·, H2O2, NO, ONOO–, HO·, ROO·, and 1O2). It is understood that the reactive species, although kept in low steady-state concentrations by antioxidant enzymes, reductants, and quenchers, are able to react and damage biomolecules leading to cumulative oxidative damage (1, 10).

Mitochondria are the main intracellular source of oxidizing free radicals; two free radicals, O2–· and NO, are primarily and continuously produced in these organelles. Other reactive species such as H2O2, ONOO–, HO·, ROO·, and 1O2, derive from the primary production of O2–· and NO. O2–· is generated as a by-product of the respiratory chain electron transfer, mainly by the autoxidation of ubisemiquinone (3, 8). NO is the product of the enzymatic action of mitochondrial NO synthase (mtNOS) (16, 17), a specialized NOS (13) that carries out a classical NOS reaction, requiring NADPH, arginine, O2, and Ca2+/calmodulin for enzyme activity (47).

The free radical chain reactions are started by O2–· and NO and involve a series of reactive oxygen and nitrogen species that are capable of damaging mitochondrial membranes, proteins, and DNA. Mitochondria, the main cellular site of oxygen uptake and oxyradical generation, are also the main target of oxyradical-mediated damage. Cumulative free radical damage contributes to cell and tissue senescence and leads mitochondria to a state of mitochondrial dysfunction with decreased organelle ability to synthesize ATP and to adapt themselves to the destabilizing effects of cellular stress (1, 44). Dysfunctional mitochondria show decreased state 3 oxygen uptake (32) and inner membrane potential (40), and increased O2–· and H2O2 production (45), as well as increased size (40). Dysfunctional mitochondria generate intracellular signals for lysosomal digestion and for apoptosis.

In this study we characterized the dysfunctional state of brain and liver mitochondria associated with normal aging by assaying oxidative stress indicators, mitochondrial enzyme activities that behaved as aging indicators, and mitochondrial size, fragility, and passive water permeability.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Wistar rats in the stages of young adults, aged adults and senescent, 28, 60, and 92 wk of age, were used (n = 20 in each group). The animals were siblings grown and aged at the Department of Experimental Animals of the University of Cádiz, housed in groups of five animals and kept at 24 ± 1°C with 12:12-h light-dark cycles, with full access to water and food. Animal experiments were carried out in accordance with the 86/609/CEE European Community regulations and the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.

Isolation of mitochondria. Brain and liver mitochondria were isolated from the whole organs homogenized in 0.23 M mannitol, 0.07 M sucrose, 15 mM MOPS-KOH (pH 7.2) at a ratio of 9 ml of homogenization medium/1 g of tissue in a Potter homogenizer with a Teflon pestle. The homogenate was centrifuged at 700 g for 10 min and the supernatant at 8,000 g for 10 min to precipitate mitochondria that were washed in the same conditions (34, 35). Mitochondrial suspensions, containing ~20 mg protein/ml, were used immediately after isolation for swelling studies or frozen in liquid N2 and kept at –80°C. Mitochondrial samples, twice thawed and frozen, were homogenized by passage through 15/10 tuberculin needles; the resulting submitochondrial membranes were used for the determination of enzyme activities and oxidative stress markers. Protein content of samples was determined using the Folin reagent and BSA as standard.

Mitochondrial electron transfer activities. The electron transfer activities of complexes I-III, II-III, and IV were determined spectrophotometrically at 30°C in 100 mM phosphate buffer (pH 7.4) (34). For NADH-cytochrome c reductase (complexes I-III) and succinate-cytochrome c reductase (complexes II-III) activities, samples were added with 0.2 mM NADH or 20 mM succinate as substrates, 0.1 mM cytochrome c3+, and 1 mM KCN, and the enzymatic activity was determined at 550 nm ({epsilon} = 19 mM–1·cm–1) and expressed as nanomoles cytochrome c reduced per milligram protein. Cytochrome oxidase (complex IV) activity was determined in the same phosphate buffer added with 0.1 mM cytochrome c2+, which was prepared by reduction with NaBH4 and HCl. The rate of cytochrome c oxidation was calculated as the first-order reaction constant per milligram protein and expressed as nanomoles cytochrome c oxidized at 20 µM cytochrome c and at 1 mg protein/ml, which gives rates of the order of physiological activities (34, 35).

Mitochondrial superoxide dismutase activity (Mn-superoxide dismutase). This enzymatic activity was determined in tissue homogenates and isolated mitochondria by the adrenochrome assay in a reaction medium containing 1 mM epinephrine, 1 mM KCN, and 50 mM glycine/KOH (pH 10.0). One Misra-Fridovich unit of enzyme activity, 50% inhibition of the rate of adrenochrome formation, is equivalent to 11 pmol of superoxide dismutase (SOD) active center (18, 30).

mtNOS activity. Mitochondrial NO production was determined by the oxyhemoglobin (HbO2) oxidation assay as described elsewhere (2). The reaction medium consisted of 0.1 mM NADPH, 0.2 mM arginine, 1 mM CaCl2, 4 µM Cu,Zn-SOD, 0.1 µM catalase, and 25 µM HbO2 heme, in 50 mM phosphate buffer at pH 7.2 (liver) and at pH 5.8 (brain). A diode array sensitive spectrophotometer (Agilent, model 8453) was used to follow the absorbance change at 577 nm with a reference wavelength at the isosbestic point of 591 nm ({epsilon}577–591 = 11.2 mM–1·cm–1). NO production was calculated from the absorbance change that was inhibited by 2 mM NG-methyl-L-arginine, usually 86–94%, and expressed in nanomoles NO per minutes per milligram protein.

Biochemical markers of oxidative stress. The mitochondrial content of the oxidation products, thiobarbituric acid-reactive substances (TBARS) and protein carbonyls, was determined as previously described (15, 34, 38).

Light scattering and passive mitochondrial swelling. Light scattering and hypotonically induced mitochondrial swelling were determined in 90 mM KCl, 20 mM MOPS-KOH (pH 7.2) at 1 mg protein/ml by the absorbance changes that were followed spectrophotometrically at 540 nm. The absorption change ({Delta}A) after the addition of mitochondria to the suspension medium and the initial rate (1 min) of mitochondrial swelling ({Delta}A/min) were recorded.

Mitochondrial mass in the tissue. The content of mitochondria (mitochondrial mass) of the whole organs was calculated from the ratios of cytochrome oxidase and Mn-SOD activities in organ homogenates and in isolated mitochondria (41).

Fraction of marker mitochondrial activities isolated in the mitochondrial fraction. The mitochondrial electron transfer activities, NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome oxidase, were determined, as described before, in the whole organ homogenate, in the isolated mitochondrial fraction, and in the postmitochondrial supernatant in each individual animal (n = 8). The percentage of organ mitochondria isolated by the utilized isolation procedure was calculated as the fraction of homogenate activity centrifuged down in the isolated mitochondrial fraction (see list in Table 5). The sum of the marker enzyme activities in isolated mitochondria and in the postmitochondrial supernatant accounted for the following percentages of homogenate enzyme activities: NADH-cytochrome c reductase, 96–114%; succinate-cytochrome c reductase, 90–95%; and cytochrome oxidase, 94–98%. The fraction of mitochondrial activity in the postmitochondrial supernatant is a direct indication of mitochondrial fragility and rupture during the homogenization procedure.


View this table:
[in this window]
[in a new window]
  		Table 5. Fraction of organ mitochondrial electron transfer activities isolated in the mitochondrial fraction (inverse relationship with mitochondrial fragility)

 
Statistics. The numbers in tables and figures indicate mean values ± SE. Differences between groups were analyzed by Dunnett's bilateral post hoc test after significant one-way ANOVA. A P value of <0.05 was considered to be biologically significant. Statistical analyses were carried out using a statistical package (SPSS 11.5 for Windows).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Aging and mitochondrial mass in brain and liver. The content of mitochondria in brain and liver, calculated from the activities of the mitochondrial marker enzymes cytochrome oxidase and Mn-SOD, was 15.2–17.0 and 29.2–31.0 mg mitochondrial protein/g of organ for brain and liver and did not show differences in the aging animals (Table 1). The mitochondrial mass was markedly lower (~52%) in the brain compared with the liver.


View this table:
[in this window]
[in a new window]
  		Table 1. Mitochondrial content in the brain and liver of aging rats

 
Aging and oxidative stress markers. Protein carbonyls and TBARS increased significantly in brain and liver mitochondria on rat aging from 28 to 92 wk of age as an indication of progressive mitochondrial oxidative stress (Table 2). In brain mitochondria, protein carbonyls increased by 42 and 69% at 60 and 92 wk of age and in liver mitochondria by 26 and 36% at the same ages. TBARS were a similarly sensitive indicator of mitochondrial oxidative stress: in brain, they augmented by 47 and 61% at 60 and 92 wk of age, and in liver by 24 and 45% at the same ages.


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of aging on oxidative stress markers of rat brain and liver mitochondria

 
Aging, mtNOS, and Mn-SOD activities. The activities of these two physiologically important mitochondrial enzymes markedly and progressively decreased on aging (Table 3). In brain, mtNOS activity decreased to 27% and Mn-SOD decreased to 62% at 92 wk of age. In liver, the same activities were similarly affected by aging; mtNOS and Mn-SOD activities decreased to 53% and to 50% at 92 wk of age. A significant positive correlation was found between the activities of these two enzymes in brain (r2 = 0.99, P < 0.05) and liver (r2 = 0.99, P < 0.05).


View this table:
[in this window]
[in a new window]
  		Table 3. NOS and Mn-SOD activities in the brain and liver mitochondria of aging rats

 
Aging and the mitochondrial electron transfer activities. Aging decreased NADH-cytochrome c reductase activities (complexes I-III) in brain and liver. The age-dependent loss in activity was 29–30% for brain and liver at 92 wk of age (Table 4). Cytochrome oxidase (complex IV) activity was also progressively lost on aging; activities diminished by 28% in brain and by 43% in liver at 92 wk of rat age (Table 4). At variance, aging did not modify succinate-cytochrome c reductase activities (complexes II-III) in both organs (Table 4), indicating a selective effect of aging on mitochondrial electron transfer activities.


View this table:
[in this window]
[in a new window]
  		Table 4. Mitochondrial electron transfer activities in the brain and liver mitochondria of aging rats

 
Aging and mitochondrial fragility. In brain, the percentage of organ mitochondrial marker enzyme activities (NADH -> cytochrome c3+; succinate -> cytochrome c3+; and cytochrome c2+ -> O2) isolated in the mitochondrial fraction was significantly lower in senescent rats (18–19%) compared with young adults (28–29%) and with old adults (21–23%) (Table 5). The whole age-dependent loss in marker activities in isolated brain mitochondria was ~35%. In liver, at variance, the percentages of the same marker activities isolated in the mitochondrial fraction were similar, in the 35–38% range, for young and old adult animals and for senescent rats (Table 5).

A direct estimation of mitochondrial size by light scattering indicated an increased size (less light scattering and absorption) in brain mitochondria isolated from senescent rats ({Delta}A = 0.751) compared with the ones isolated from young adults ({Delta}A = 0.948), with intermediate sizes in old adults ({Delta}A = 0.864) (Fig. 1). At variance, there were no significant changes in light scattering associated with aging in liver mitochondria ({Delta}A = 0.810–0.848).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 1. Mitochondrial light scattering and hypotonic swelling. Mitochondria were suspended in 90 mM KCl at 1 mg protein/ml, and light scattering was followed spectrophotometrically at 540 nm. Left: the determination procedure; n = 8 animals in each group. A, absorption; {Delta}A, absorption change. *P < 0.05.

 
In agreement with the above-referenced results, the passive rate of mitochondrial swelling in a hypotonic medium was significantly faster in brain mitochondria isolated from 92-wk-old rats ({Delta}A/min = 0.096) than in the organelles isolated from 28- and 60-wk-old rats ({Delta}A/min = 0.063 and 0.080) (Fig. 1). Again at variance, in the liver, the rate of passive mitochondrial swelling was similar for mitochondria isolated from senescent animals and from young adult rats ({Delta}A/min = 0.051–0.055) (Fig. 1).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Reduction of the mitochondrial capacity to produce ATP and oxidative stress in the organs and tissues of old mammals are the two underlying concepts of the mitochondrial hypothesis of aging (1, 43, 49). The mentioned reduction in ATP production is equally possible through a reduction of the mitochondrial mass in the cells or through a decrease in the specific rate of mitochondrial ATP synthesis, referred to a unit of mitochondrial mass (for instance, mg of mitochondrial protein). The data reported here indicate that the first possibility is not supported experimentally, the mitochondrial contents of brain and liver were found not affected by aging, and then the latter possibility seems the right one.

Mitochondria isolated from both organs of aged animals showed an increased content of oxidation products, decreased electron transfer activities, and increased mitochondrial size. Those features, added to decreased rates of O2 uptake and ATP synthesis and to decreased membrane potential, describe the condition of dysfunctional mitochondria.

The observed increase in oxidation products in rat brain and liver mitochondria isolated from aging animals, which describes an oxidative stress condition, agrees with previous reports of increased protein carbonyls (14, 34, 46), TBARS (34), ROOH, and 8-hydroxy-deoxy-guanosine (40, 42). Interestingly, cellular free radicals and oxidants produced at faster rates in the oxidative stress situation exhibit selectivity in damaging mitochondrial macromolecules and impairing membrane functions (43). A series of mitochondrial enzymes, membrane bound or matrix contained, have been consistently reported with lower activities in aging. However, it is not clear if the loss of enzyme activity is due to enzyme inactivation by free radicals and oxidation products or to a decreased expression.

The observed decreased enzyme activities are mtNOS, Mn-SOD, NADH-cytochrome c reductase, and cytochrome oxidase. The decrease in brain mtNOS activity associated with aging was the most marked of the observed decreases of mitochondrial enzyme activities, pointing to mtNOS as a marker of tissue aging (33) and suggesting a loss in the regulatory roles of mitochondrial NO as a critical mechanism in aging (5, 7).

The reported effects of aging on Mn-SOD activity have not being consistent. For simplicity we will restrict the consideration to brain, cerebrospinal fluid, and liver. Aging was reported as associated or causing an increased SOD activity (6, 11, 26, 35, 37, 49) and decreased SOD (Table 3 and Refs. 9, 21, 28, 31, 34, 39, 48). Moreover, SOD activity was not affected by aging (23, 36) or was modified in different ways in different tissues (12, 22, 24, 27). Differences may be due in part to the method used for enzyme determination and to interferences. We observed increased Mn-SOD and Cu,Zn-SOD activities in mouse brain and liver in aging (33, 35) using the classical xanthine oxidase/cytochrome c assay (43) and decreased Mn-SOD and Cu,Zn-SOD in mouse and rat brain and liver in aging by using the adrenochrome assay (Table 3; Ref. 34).

Concerning the electron transfer activities of the mitochondrial respiratory chain, complexes I and IV (NADH-ubiquinone reductase and cytochrome oxidase) were found with reduced activity (Table 4) in aged animals, in agreement with previous observations (25, 34, 35). The decreased activity of NADH-dehydrogenase in brain and liver of old rats, by ~30%, is close to the limit of a tolerable functional damage in terms of minimal energy production. In the liver of young rats and under physiological conditions, ATP is provided by the respiration of ~36% of the mitochondrial mass in metabolic state 3 (4). Under conditions of increased ATP demand, liver mitochondria will be able to increase ATP synthesis by ~2.8 times by switching more mitochondria from the resting state 4 to the active state 3. Aged hepatocytes, with a 30% reduction in NADH-dehydrogenase activity, will be able to increase ATP production only by 1.9 times by switching mitochondria from state 4 to state 3, due to the decreased NADH-dehydrogenase activity. The situation is clearly worst in the brain due to the lower mitochondrial mass; aged neurons may be close to being unable to respond to any increased ATP demands.

Light scattering studies indicated that brain mitochondria isolated from aged animals had a larger volume, in agreement with the report by Sastre et al. (40), and an increased water permeability when challenged by an hypotonic medium. Instead, liver mitochondria did not show such difference between young and old animals. A partial loss of the selective permeability of the inner membrane of brain mitochondria in old animals, as indicated by the increase in water permeability, is consistent with an increased H+ permeability, in turn associated with uncoupling of oxidative phosphorylation, increase in state 4 respiration, and decreases in respiratory control and membrane potential, as reported in old animals (20, 32, 40).

The relative fragility of tissue mitochondria was estimated by comparing the fraction of mitochondrial marker enzymes recovered in the mitochondrial fraction after similarly processing (equal tissue disruption procedure and equal shearing forces in the homogenization procedure) tissues from young adults, old adults, and senescent animals. The greater the percentage of marker enzyme activity recovered in isolated mitochondria, the less the mitochondrial fragility or rupture during homogenization. The observed age-dependent decrease in marker activities in isolated brain mitochondria (35%) can be understood as a subpopulation of one-third of brain mitochondrial mass (mg mitochondrial protein/g of brain tissue) with increased fragility, a result in quantitative agreement with the data of Sastre et al. (40) that reported an increased mitochondrial size with age. Most brain cells age at the same time as the animal ages, and the persistent neurogenesis is spatially restricted and quantitatively negligible (19). Thus most neurons are long-living cells and exhibit a slow turnover time for their mitochondria and mitochondrial components, two factors that contribute to the cellular accumulation of dysfunctional mitochondria.

Liver mitochondria isolated from young and old animals did not exhibit a significant difference in fragility. In hepatocytes, mitochondria are continuously subjected to protein and phospholipid turnover and to elimination of the dysfunctional organelles. The cellular population of damaged mitochondria is likely to become determinant in the signaling for apoptosis as hepatocytes reach their lifespan limit.

An estimation of half-life (t1/2) of several mitochondrial proteins or mitochondrial fractions gives an estimation of 7–10 days for liver mitochondria but of 4–6 wk for brain mitochondria. This slower turnover time of brain mitochondria makes possible the detection of a fraction of dysfunctional mitochondria with increased fragility, which may correspond to subpopulations of enlarged mitochondria in morphological or flow cytometry studies.

The data reported here show that mitochondria isolated from the organs of aged animals are also aged in terms of cytosolic and mitochondrial oxidative stress and losses of enzymatic activities. The observation recalls the challenging question of why the differentiated cells of systemic organs are subjected to aging whereas the cells of the germ line and the continuously proliferative stem cells, which sustain a population turnover in most tissues (except brain), are free from the aging process.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Grants FIS 02–1354 from Ministerio de Sanidad y Consumo de España and by Plan Andaluz de Investigación 2000–03 (CTS-194).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Navarro, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Plaza Fragela 9, 11003 Cádiz, Spain (E-mail: ana.navarro{at}uca.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Beckman KB and Ames BN. The free radical theory of aging matures. Physiol Rev 78: 547–581, 1998.[Abstract/Free Full Text]
  2. Boveris A, Arnaiz SL, Bustamante J, Alvarez S, Valdez L, Boveris AD, and Navarro A. Pharmacological regulation of mitochondrial nitric oxide synthase. Methods Enzymol 359: 328–339, 2002.[Web of Science][Medline]
  3. Boveris A and Cadenas E. Mitochondrial production of superoxide anions and its relationship to the antimycin insensitive respiration. FEBS Lett 54: 311–314, 1975.[CrossRef][Web of Science][Medline]
  4. Boveris A, Costa LE, and Cadenas E. The roles of mitochondria, free radicals, and antioxidants. In: Understanding the Process of Aging, edited by Cadenas E and Packer L. New York: Dekker, 1999, p. 1–16.
  5. Boveris A, Valdez LB, Alvarez S, Zaobornyj T, Boveris AD, and Navarro A. Kidney mitochondrial nitric oxide synthase. Antioxid Redox Signal 5: 265–271, 2003.[CrossRef][Web of Science][Medline]
  6. Bracco F, Scarpa M, Rigo A, and Battistin L. Determination of superoxide dismutase activity by the polarographic method of catalytic currents in the cerebrospinal fluid of aging brain and neurologic degenerative diseases. Proc Soc Exp Biol Med 196: 36–41, 1991.[CrossRef][Medline]
  7. Brown GC. Cell biology. NO says yes to mitochondria. Science 299: 838–839, 2003.[Abstract/Free Full Text]
  8. Cadenas E and Boveris A. Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria. Biochem J 188: 31–37, 1980.[Web of Science][Medline]
  9. Cand F and Verdetti J. Superoxide dismutase, glutathione peroxidase, catalase, and lipid peroxidation in the major organs of the aging rats. Free Radic Biol Med 7: 59–63, 1989.[CrossRef][Web of Science][Medline]
  10. Chance B, Sies H, and Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 59: 527–605, 1979.[Free Full Text]
  11. Cristiano F, de Haan JB, Iannello RC, and Kola I. Changes in the levels of enzymes which modulate the antioxidant balance occur during aging and correlate with cellular damage. Mech Ageing Dev 80: 93–105, 1995.[CrossRef][Web of Science][Medline]
  12. De Haan JB, Cristiano F, Iannello RC, and Kola I. Cu/Zn-superoxide dismutase and glutathione peroxidase during aging. Biochem Mol Biol Int 35: 1281–1297, 1995.[Web of Science][Medline]
  13. Elfering SL, Sarkela TM, and Giulivi C. Biochemistry of mitochondrial nitric-oxide synthase. J Biol Chem 277: 38079–38086, 2002.[Abstract/Free Full Text]
  14. Forster MJ, Dubey A, Dawson KM, Stutts WA, Lal H, and Sohal RS. Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain. Proc Natl Acad Sci USA 93: 4765–4769, 1996.[Abstract/Free Full Text]
  15. Fraga CG, Leibovitz BE, and Tappel AL. Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes. Free Radic Biol Med 4: 155–161, 1988.[CrossRef][Web of Science][Medline]
  16. Ghafourifar P and Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett 418: 291–296, 1997.[CrossRef][Web of Science][Medline]
  17. Giulivi C, Poderoso JJ, and Boveris A. Production of nitric oxide by mitochondria. J Biol Chem 273: 11038–11043, 1998.[Abstract/Free Full Text]
  18. Gonzalez-Flecha B, Cutrin JC, and Boveris A. Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion. J Clin Invest 91: 456–464, 1993.[Web of Science][Medline]
  19. Gould E and McEwen BS. Neuronal birth and death. Curr Opin Neurobiol 3: 676–682, 1993.[CrossRef][Medline]
  20. Hagen TM, Wehr CM, and Ames BN. Mitochondrial decay in aging. Reversal through supplementation of acetyl-L-carnitine and N-tert-butyl-{alpha}-phenyl-nitrone. Ann NY Acad Sci 854: 214–223, 1998.[CrossRef][Web of Science][Medline]
  21. Inal ME, Kanbak G, and Sunal E. Antioxidant enzyme activities and malondialdehyde levels related to aging. Clin Chim Acta 305: 75–80, 2001.[CrossRef][Web of Science][Medline]
  22. Ji LL, Dillon D, and Wu E. Alteration of antioxidant enzymes with aging in rat skeletal muscle and liver. Am J Physiol Regul Integr Comp Physiol 258: R918–R923, 1990.[Abstract/Free Full Text]
  23. Kasapoglu M and Ozben T. Alterations of antioxidant enzymes and oxidative stress markers in aging. Exp Gerontol 36: 209–220, 2001.[CrossRef][Web of Science][Medline]
  24. Lammi-Keefe CJ, Swan PB, and Hegarty PV. Copper-zinc and manganese superoxide dismutase activities in cardiac and skeletal muscles during aging in male rats. Gerontology 30: 153–158, 1984.[Web of Science][Medline]
  25. Lenaz G, D'Aurelio M, Merlo PM, Genova ML, Ventura B, Bovina C, Formiggini G, and Parenti CG. Mitochondrial bioenergetics in aging. Biochim Biophys Acta 1459: 397–404, 2000.[Medline]
  26. Leutner S, Eckert A, and Muller WE. ROS generation, lipid peroxidation and antioxidant enzyme activities in the aging brain. J Neural Transm 108: 955–967, 2001.[CrossRef][Web of Science][Medline]
  27. Lopez-Torres M, Perez-Campo R, Rojas C, Cadenas S, and Barja G. Simultaneous induction of sod, glutathione reductase, GSH, and ascorbate in liver and kidney correlates with survival during aging. Free Radic Biol Med 15: 133–142, 1993.[CrossRef][Web of Science][Medline]
  28. Maria CS, Revilla E, Ayala A, de la Cruz CP, and Machado A. Changes in the histidine residues of Cu/Zn superoxide dismutase during aging. FEBS Lett 374: 85–88, 1995.[CrossRef][Web of Science][Medline]
  29. McCarter RJM. Energy utilization. Handbook of Physiology. Aging. Bethesda, MD: Am. Physiol. Soc., 1995, sect. 11, chapt. 6, p. 95–118.
  30. Misra HP and Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247: 3170–3175, 1972.[Abstract/Free Full Text]
  31. Mo JQ, Hom DG, and Andersen JK. Decreases in protective enzymes correlates with increased oxidative damage in the aging mouse brain. Mech Ageing Dev 81: 73–82, 1995.[CrossRef][Web of Science][Medline]
  32. Nakahara H, Kanno T, Inai Y, Utsumi K, Hiramatsu M, Mori A, and Packer L. Mitochondrial dysfunction in the senescence accelerated mouse (SAM). Free Radic Biol Med 24: 85–92, 1998.[CrossRef][Web of Science][Medline]
  33. Navarro A. Mitochondrial enzyme activities as biochemical markers of aging. Mol Aspects Med 25: 37–48, 2004.[CrossRef][Medline]
  34. Navarro A, Gomez C, Lopez-Cepero JM, and Boveris A. Beneficial effects of moderate exercise on mice aging: survival, behavior, oxidative stress, and mitochondrial electron transfer. Am J Physiol Regul Integr Comp Physiol 286: R505–R511, 2004.[Abstract/Free Full Text]
  35. Navarro A, Sanchez Del Pino MJ, Gomez C, Peralta JL, and Boveris A. Behavioral dysfunction, brain oxidative stress, and impaired mitochondrial electron transfer in aging mice. Am J Physiol Regul Integr Comp Physiol 282: R985–R992, 2002.[Abstract/Free Full Text]
  36. Nohl H, Hegner D, and Summer KH. Responses of mitochondrial superoxide dismutase, catalase and glutathione peroxidase activities to aging. Mech Ageing Dev 11: 145–151, 1979.[CrossRef][Web of Science][Medline]
  37. Okabe T, Hamaguchi K, Inafuku T, and Hara M. Aging and superoxide dismutase activity in cerebrospinal fluid. J Neurol Sci 141: 100–104, 1996.[CrossRef][Web of Science][Medline]
  38. Oliver CN, Ahn BW, Moerman EJ, Goldstein S, and Stadtman ER. Age-related changes in oxidized proteins. J Biol Chem 262: 5488–5491, 1987.[Abstract/Free Full Text]
  39. Sandhu SK and Kaur G. Alterations in oxidative stress scavenger system in aging rat brain and lymphocytes. Biogerontology 3: 161–173, 2002.[CrossRef][Web of Science][Medline]
  40. Sastre J, Millan A, Garcia dlA, Pla R, Juan G, Pallardo O'Connor E, Martin JA, Droy-Lefaix MT, and Vina J. A Ginkgo biloba extract (EGb 761) prevents mitochondrial aging by protecting against oxidative stress. Free Radic Biol Med 24: 298–304, 1998.[CrossRef][Web of Science][Medline]
  41. Scarpa A and Graziotti P. Mechanisms for intracellular calcium regulation in heart. I. Stopped-flow measurements of Ca2+ uptake by cardiac mitochondria. J Gen Physiol 62: 756–772, 1973.[Abstract/Free Full Text]
  42. Shigenaga MK, Hagen TM, and Ames BN. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci USA 91: 10771–10778, 1994.[Abstract/Free Full Text]
  43. Sohal RS. Role of oxidative stress and protein oxidation in the aging process. Free Radic Biol Med 33: 37–44, 2002.[CrossRef][Web of Science][Medline]
  44. Sohal RS, Ku HH, Agarwal S, Forster MJ, and Lal H. Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Ageing Dev 74: 121–133, 1994.[CrossRef][Web of Science][Medline]
  45. Sohal RS, Svensson I, and Brunk UT. Hydrogen peroxide production by liver mitochondria in different species. Mech Ageing Dev 53: 209–215, 1990.[CrossRef][Web of Science][Medline]
  46. Stadtman ER. Importance of individuality in oxidative stress and aging. Free Radic Biol Med 33: 597–604, 2002.[CrossRef][Web of Science][Medline]
  47. Tatoyan A and Giulivi C. Purification and characterization of a nitric-oxide synthase from rat liver mitochondria. J Biol Chem 273: 11044–11048, 1998.[Abstract/Free Full Text]
  48. Tiana L, Caib Q, and Wei H. Alterations of antioxidant enzymes and oxidative damage to macromolecules in different organs of rats during aging. Free Radic Biol Med 24: 1477–1484, 1998.[CrossRef][Web of Science][Medline]
  49. Vina J, Sastre J, Pallardo F, and Borras C. Mitochondrial theory of aging: importance to explain why females live longer than males. Antioxid Redox Signal 5: 549–556, 2003.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. L. Haak, G. R. Buettner, D. R. Spitz, and K. C. Kregel
Aging augments mitochondrial susceptibility to heat stress
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2009; 296(3): R812 - R820.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. Navarro, J. M. Lopez-Cepero, M. J. Bandez, M.-J. Sanchez-Pino, C. Gomez, E. Cadenas, and A. Boveris
Hippocampal mitochondrial dysfunction in rat aging
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2008; 294(2): R501 - R509.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Navarro and A. Boveris
The mitochondrial energy transduction system and the aging process
Am J Physiol Cell Physiol, February 1, 2007; 292(2): C670 - C686.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
K. C. Kregel and H. J. Zhang
An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R18 - R36.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
A. Cahill, S. Hershman, A. Davies, and P. Sykora
Ethanol feeding enhances age-related deterioration of the rat hepatic mitochondrion
Am J Physiol Gastrointest Liver Physiol, December 1, 2005; 289(6): G1115 - G1123.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. Navarro, R. Torrejon, M. J. Bandez, J. M. Lopez-Cepero, and A. Boveris
Mitochondrial function and mitochondria-induced apoptosis in an overstimulated rat ovarian cycle
Am J Physiol Endocrinol Metab, December 1, 2005; 289(6): E1101 - E1109.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. Navarro, C. Gomez, M.-J. Sanchez-Pino, H. Gonzalez, M. J. Bandez, A. D. Boveris, and A. Boveris
Vitamin E at high doses improves survival, neurological performance, and brain mitochondrial function in aging male mice
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1392 - R1399.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
T. Zaobornyj, L. B. Valdez, P. La Padula, L. E. Costa, and A. Boveris
Effect of sustained hypobaric hypoxia during maturation and aging on rat myocardium. II. mtNOS activity
J Appl Physiol, June 1, 2005; 98(6): 2370 - 2375.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. Navarro and A. Boveris
Hypoxia exacerbates macrophage mitochondrial damage in endotoxic shock
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2005; 288(2): R354 - R355.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/5/R1244    most recent
00226.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (29)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Navarro, A.
Right arrow Articles by Boveris, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Navarro, A.
Right arrow Articles by Boveris, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.