Juan Sastre

Mitochondria from females exhibit higher antioxidant gene expression and lower oxidative damage than males

We have investigated the differential mitochondrial oxidative stress between males and females to understand the molecular mechanisms enabling females to live longer than males. Mitochondria are a major source of free radicals in cells. Those from female rats generate half the amount of peroxides than those of males. This does not occur in ovariectomized animals. Estrogen replacement therapy prevents the effect of ovariectomy. Mitochondria from females have higher levels of reduced glutathione than those from males. Those from ovariectomized rats have similar levels to males, and estrogen therapy prevents the fall in glutathione levels that occurs in ovariectomized animals. Oxidative damage to mitochondrial DNA in males is 4-fold higher than that in females. This is due to higher expression and activities of Mn-superoxide dismutase and of glutathione peroxidase in females, which behave as double transgenics overexpressing superoxide dismutase and glutathione peroxidase, conferring protection against free-radical-mediated damage in aging. Moreover, 16S rRNA expression, which decreases significantly with aging, is four times higher in mitochondria from females than in those from males of the same chronological age. The facts reported here provide molecular evidence to explain the different life span in males and females.

Decreasing xanthine oxidase‐mediated oxidative stress prevents useful cellular adaptations to exercise in rats

Reactive oxygen or nitrogen species (RONS) are produced during exercise due, at least in part, to the activation of xanthine oxidase. When exercise is exhaustive they cause tissue damage; however, they may also act as signals inducing specific cellular adaptations to exercise. We have tested this hypothesis by studying the effects of allopurinol‐induced inhibition of RONS production on cell signalling pathways in rats submitted to exhaustive exercise. Exercise caused an activation of mitogen‐activated protein kinases (MAPKs: p38, ERK 1 and ERK 2), which in turn activated nuclear factor κB (NF‐κB) in rat gastrocnemius muscle. This up‐regulated the expression of important enzymes associated with cell defence (superoxide dismutase) and adaptation to exercise (eNOS and iNOS). All these changes were abolished when RONS production was prevented by allopurinol. Thus we report, for the first time, evidence that decreasing RONS formation prevents activation of important signalling pathways, predominantly the MAPK–NF‐κB pathway; consequently the practice of taking antioxidants before exercise may have to be re‐evaluated.

Mitochondrial Oxidative Stress Plays a Key Role in Aging and Apoptosis

Harman first suggested in 1972 that mitochondria might be the biological clock in aging, noting that the rate of oxygen consumption should determine the rate of accumulation of mitochondrial damage produced by free radical reactions. Later in 1980 Miquel and coworkers proposed the mitochondrial theory of cell aging. Mitochondria from postmitotic cells use O2 at a high rate, hence releasing oxygen radicals that exceed the cellular antioxidant defences. The key role of mitochondria in cell aging has been outlined by the degeneration induced in cells microinjected with mitochondria isolated from fibroblasts of old rats, especially by the inverse relationship reported between the rate of mitochondrial production of hydroperoxide and the maximum life span of species. An important change in mitochondrial lipid composition is the age‐related decrease found in cardiolipin content. The concurrent enhancement of lipid peroxidation and oxidative modification of proteins in mitochondria further increases mutations and oxidative damage to mitochondrial DNA (mtDNA) in the aging process. The respiratory enzymes containing the defective mtDNA‐encoded protein subunits may increase the production of reactive oxygen species, which in turn would aggravate the oxidative damage to mitochondria. Moreover, superoxide radicals produced during mitochondrial respiration react with nitric oxide inside mitochondria to yield damaging peroxynitrite. Treatment with certain antioxidants, such as sulphur‐containing antioxidants, vitamins C and E, or the Ginkgo biloba extract EGb 761, protects against the ageassociated oxidative damage to mtDNA and the oxidation of mitochondrial glutathione. Moreover, the EGb 761 extract also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver.

The role of mitochondrial oxidative stress in aging

Mitochondria are both a major source of oxidants and a target for their damaging effects, and, therefore, mitochondrial oxidative stress appears to be a cause, rather than a consequence, of cell aging. Oxidative damage in aging is particularly high in specific molecular targets, such as mitochondrial DNA and aconitase, and mitochondrial oxidative stress may drive tissue aging through intrinsic apoptosis. Mitochondrial function and morphology are impaired upon aging, as judged by a decline in membrane potential as well as by an increase in peroxide production and size of the organelles. In view of the age-related decreases in mitochondrial protein synthesis, mitochondrial transcripts, and expression of genes involved in mitochondrial turnover, the rate of this turnover might determine its susceptibility of mitochondria to oxidative damage and mutation, thus controlling the rate of cell aging. In fact, aging is a feature of differentiated somatic cells, especially postmitotic cells such as neurons or muscle cells. The age-associated mitochondrial DNA deletions focally accumulate in brain and skeletal muscle, thus contributing significantly to aging of these postmitotic tissues. Expansion of mitochondrial DNA mutations may occur through mitochondrial complementation. The use of mutants of the mitochondrial electron transport system, as well as knockouts or transgenics of mitochondrial antioxidants or repair enzymes, may provide clear-cut evidence of the precise mitochondrial mechanisms that control the rate of cell aging.

Preterm Resuscitation With Low Oxygen Causes Less Oxidative Stress, Inflammation, and Chronic Lung Disease

OBJECTIVE: The goal was to reduce adverse pulmonary adverse outcomes, oxidative stress, and inflammation in neonates of 24 to 28 weeks of gestation initially resuscitated with fractions of inspired oxygen of 30% or 90%. METHODS: Randomized assignment to receive 30% (N = 37) or 90% (N = 41) oxygen was performed. Targeted oxygen saturation values were 75% at 5 minutes and 85% at 10 minutes. Blood oxidized glutathione (GSSG)/reduced glutathione ratio and urinary o-tyrosine, 8-oxo-dihydroxyguanosine, and isoprostane levels, isofuran elimination, and plasma interleukin 8 and tumor necrosis factor α levels were determined. RESULTS: The low-oxygen group needed fewer days of oxygen supplementation (6 vs 22 days; P < .01) and fewer days of mechanical ventilation (13 vs 27 days; P < .01) and had a lower incidence of bronchopulmonary dysplasia at discharge (15.4% vs 31.7%; P < .05). GSSG/reduced glutathione × 100 ratios at day 1 and 3 were significantly higher in the high-oxygen group (day 1: high-oxygen group: 13.36 ± 5.25; low-oxygen group: 8.46 ± 3.87; P < .01; day 3: high-oxygen group: 8.87 ± 4.40; low-oxygen group: 6.97 ± 3.11; P < .05). Urinary markers of oxidative stress were increased significantly in the high-oxygen group, compared with the low-oxygen group, in the first week after birth. GSSG levels on day 3 and urinary isofuran, o-tyrosine, and 8-hydroxy-2′-deoxyguanosine levels on day 7 were correlated significantly with development of chronic lung disease. CONCLUSIONS: Resuscitation of preterm neonates with 30% oxygen causes less oxidative stress, inflammation, need for oxygen, and risk of bronchopulmonary dysplasia.

Mitochondria, oxidative stress and aging

In the eighties, Miquel and Fleming suggested that mitochondria play a key role in cellular aging. Mitochondria, and specially mitochondrial DNA (mtDNA), are major targets of free radical attack. At present, it is well established that mitochondrial deficits accumulate upon aging due to oxidative damage. Thus, oxidative lesions to mtDNA accumulate with age in human and rodent tissues. Furthermore, levels of oxidative damage to mtDNA are several times higher than those of nuclear DNA. Mitochondrial size increases whereas mitochondrial membrane potential decreases with age in brain and liver. Recently, we have shown that treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and oxidation of mitochondrial glutathione. Moreover, the extract EGb 761 also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver. Thus, mitochondrial aging may be prevented by antioxidants. Furthermore, late onset administration of certain antioxidants is also able to prevent the impairment in physiological performance, particularly motor co-ordination, that occurs upon aging.

17β-oestradiol up-regulates longevity-related, antioxidant enzyme expression via the ERK1 and ERK2[MAPK]/NFκB cascade

Females live longer than males. Oestrogens protect females against aging by up‐regulating the expression of antioxidant, longevity‐related genes such as glutathione peroxidase (GPx) and Mn‐superoxide dismutase (Mn‐SOD). The mechanism through which oestrogens up‐regulate those enzymes remains unidentified, but may have implications for gender differences in lifespan. We show that physiological concentrations of oestradiol act through oestrogen receptors to reduce peroxide levels in MCF‐7 cells (a mammary gland tumour cell line). Oestradiol increases MAP kinase (MAPK) activation as indicated by ERK1 and ERK2 phosphorylation in MCF‐7 cells, which in turn activates the nuclear factor kappa B (NFκB) signalling pathways as indicated by an increase in the p50 subunit of NFκB in nuclear extracts. Blockade of MAPK and NFκB signalling reduces the antioxidant effect of oestradiol. Finally, we show that activation of MAPK and NFκB by oestrogens drives the expression of the antioxidant enzymes Mn‐SOD and GPx. We conclude that oestradiol sequentially activates MAPK and NFκB following receptor activation to up‐regulate the expression of antioxidant enzymes, providing a cogent explanation for the antioxidant properties of oestrogen and its effects on longevity‐related genes.

A Ginkgo Biloba Extract (EGb 761) Prevents Mitochondrial Aging by Protecting Against Oxidative Stress

The effect of aging on indices of oxidative damage in rat mitochondria and the protective effect of the Ginkgo biloba extract EGb 761 was investigated. Mitochondrial DNA from brain and liver of old rats exhibited oxidative damage that is significantly higher than that from young rats. Mitochondrial glutathione is also more oxidized in old than in young rats. Peroxide formation in mitochondria from old animals was higher than in those from young ones. According to morphological parameters (size and complexity), there are two populations of mitochondria. One is composed of large, highly complex mitochondria, and the other population is smaller and less complex. Brain and liver from old animals had a higher proportion of the large, highly complex mitochondria than seen in organs from young animals. Treatment with the Ginkgo biloba extract EGb 761 partially prevented these morphological changes as well as the indices of oxidative damage observed in brain and liver mitochondria from old animals.

Free Radicals in Exhaustive Physical Exercise: Mechanism of Production, and Protection by Antioxidants

Moderate exercise is a healthy practice. However, exhaustive exercise generates free radicals. This can be evidenced by increases in lipid peroxidation, glutathione oxidation, and oxidative protein damage. It is well known that activity of cytosolic enzymes in blood plasma is increased after exhaustive exercise. This may be taken as a sign of damage to muscle cells. The degree of oxidative stress and of muscle damage does not depend on the absolute intensity of exercise but on the degree of exhaustion of the person who performs exercise. Training partially prevents free radical‐formation in exhaustive exercise. Treatment with antioxidants such as vitamins C or E protects in part against free radical‐mediated damage in exercise. Xanthine oxidase is involved in free‐radical formation in exercise in humans and inhibition of this enzyme with allopurinol decreases oxidative stress and muscle damage associated with exhaustive exercise. Knowledge of the mechanism of free‐radical formation in exercise is important because it will be useful to prevent oxidative stress and damage associated with exhaustive physical activity.

Mechanism of Free Radical Production in Exhaustive Exercise in Humans and Rats; Role of Xanthine Oxidase and Protection by Allopurinol

Exhaustive exercise generates free radicals. However, the source of this oxidative damage remains controversial. The aim of this paper was to study further the mechanism of exercise‐induced production of free radicals. Testing the hypothesis that xanthine oxidase contributes to the production of free radicals during exercise, we found not only that exercise caused an increase in blood xanthine oxidase activity in rats but also that inhibiting xanthine oxidase with allopurinol prevented exercise‐induced oxidation of glutathione in both rats and in humans. Furthermore, inhibiting xanthine oxidase prevented the increases in the plasma activity of cytosolic enzymes (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase) seen after exhaustive exercise. Our results provide evidence that xanthine oxidase is responsible for the free radical production and tissue damage during exhaustive exercise. These findings also suggest that mitochondria play a minor role as a source of free radicals during exhaustive physical exercise.