Gustavo Barja

Free radicals and aging

Aging is characterized by decrements in maximum function and accumulation of mitochondrial DNA mutations, which are best observed in organs such as the brain that contain post-mitotic cells. Oxygen radicals are increasingly considered responsible for part of these aging changes. Comparative studies of animals with different aging rates have shown that the rate of mitochondrial oxygen radical generation is directly related to the steady-state level of oxidative damage to mitochondrial DNA and is inversely correlated with maximum longevity in higher vertebrates. The degree of unsaturation of tissue fatty acids also correlates inversely with maximum longevity. These are the two known traits connecting oxidative stress with aging. Furthermore, caloric restriction, which decreases the rate of aging, proportionately decreases mitochondrial oxygen radical generation, especially at complex I. These findings are reviewed, highlighting the results obtained in the brain.

Mitochondrial Oxygen Radical Generation and Leak: Sites of Production in States 4 and 3, Organ Specificity, and Relation to Aging and Longevity

Studies in heart and nonsynaptic brain mitochondria from two mammals and three birds showthat complex I generates oxygen radicals in heart and nonsynaptic brain mitochondria in States4 and 3, whereas complex III does it only in heart mitochondria and only in State 4. Theincrease in oxygen consumption during the State 4 to 3 transition is not accompanied by aproportional increase in oxygen radical generation. This will protect mitochondria and tissuesduring bursts of activity. Comparisons between young and old rodents do not show a consistentpattern of variation in mitochondrial oxygen radical production during aging. However, allthe interspecies comparisons performed to date between different mammals, and betweenmammals and birds, agree that animals with high maximum longevities have low rates ofmitochondrial oxygen radical production, irrespective of the value of their basal specificmetabolic rate. The sites and mechanisms allowing this, the recently described low degree ofmembrane fatty acid unsaturation of longevous animals, and their relation to longevity andaging are discussed.

Caloric restriction decreases mitochondrial free radical generation at complex I and lowers oxidative damage to mitochondrial DNA in the rat heart

The effect of caloric restriction (CR) (40%) on the rates of mitochondrial H2O2 production and oxygen consumption and oxidative damage to nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) was studied for short‐term (6‐wk) and long‐term (1‐year) periods in the heart of young and old rats. Short‐term CR did not change any of the parameters measured. However, long‐term CR significantly decreased the rate of mitochondrial H2O2 generation (by 45%) and significantly lowered oxidative damage to mtDNA (by 30%) without modifying damage to nDNA. The decrease in H2O2 production occurred exclusively at the complex I free radical generator of the respiratory chain. The mechanism allowing that decrease was not a simple decrease in mitochondrial oxygen consumption. Instead, the mitochondria of caloric‐restricted animals released fewer oxygen radicals per unit electron flow in the respiratory chain. This was due to a decrease in the degree of reduction of the complex I generator in caloric‐restricted mitochondria. The results are consistent with the concept that CR decreases the aging rate at least in part by decreasing the rate of mitochondrial oxygen radical generation and then the rate of attack on mtDNA.

The rate of free radical production as a determinant of the rate of aging: evidence from the comparative approach

Abstract The relationship of oxidative stress with maximum life span (MLSP) in different vertebrate species is reviewed. In all animal groups the endogenous levels of enzymatic and non-enzymatic antioxidants in tissues negatively correlate with MLSP and the most longevous animals studied in each group, pigeon or man, show the minimum levels of antioxidants. A possible evolutionary reason for this is that longevous animals produce oxygen radicals at a low rate. This has been analysed at the place where more than 90% of oxygen is consumed in the cell, the mitochondria. All available work agrees that, across species, the longer the life span, the lower the rate of mitochondrial oxygen radical production. This is true even in animal groups that do not conform to the rate of living theory of aging, such as birds. Birds have low rates of mitochondrial oxygen radical production, frequently due to a low free radical leak in their respiratory chain. Possibly the low rate of mitochondrial oxygen radical production of longevous species can decrease oxidative damage at targets important for aging (like mitochondrial DNA) that are situated near the places of free radical generation. A low rate of free radical production can contribute to a low aging rate both in animals that conform to the rate of living (metabolic) theory of aging and in animals with exceptional longevities, like birds and primates. Available research indicates there are at least two main characteristics of longevous species: a high rate of DNA repair together with a low rate of free radical production near DNA. Simultaneous consideration of these two characteristics can explain part of the quantitative differences in longevity between animal species.

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.

Low Mitochondrial Free Radical Production Per Unit O2 Consumption Can Explain the Simultaneous Presence of High Longevity and High Aerobic Metabolic Rate in Birds

Birds are unique since they can combine a high rate of oxygen consumption at rest with a high maximum life span (MLSP). The reasons for this capacity are unknown. A similar situation is present in primates including humans which show MLSPs higher than predicted from their rates of O2 consumption. In this work rates of oxygen radical production and O2 consumption by mitochondria were compared between adult male rats (MLSP = 4 years) and adult pigeons (MLSP = 35 years), animals of similar body size. Both the O2 consumption of the whole animal at rest and the O2 consumption of brain, lung and liver mitochondria were higher in the pigeon than in the rat. Nevertheless, mitochondrial free radical production was 2-4 times lower in pigeon than in rat tissues. This is possible because pigeon mitochondria show a rate of free radical production per unit O2 consumed one order of magnitude lower than rat mitochondria: bird mitochondria show a lower free radical leak at the respiratory chain. This result, described here for the first time, can possibly explain the capacity of birds to simultaneously increase maximum longevity and basal metabolic rate. It also suggests that the main factor relating oxidative stress to aging and longevity is not the rate of oxygen consumption but the rate of oxygen radical production. Previous inconsistencies of the rate of living theory of aging can be explained by a free radical theory of aging which focuses on the rate of oxygen radical production and on local damage to targets relevant for aging situated near the places where free radicals are continuously generated.

Mitochondrial Free Radical Production and Aging in Mammals and Birdsa

ABSTRACT: The mitochondrial rate of oxygen radical (ROS) production is negatively correlated with maximum life span potential (MLSP) in mammals following the rate of living theory. In order to know if this relationship is more than circumstantial, homeothermic vertebrates with MLSP different from that predicted by the body size and metabolic rate of the majority of mammals (like birds and primates) must be studied. Birds are unique because they combine a high rate of basal oxygen consumption with a high MLSP. Heart, brain, and lung mitochondrial ROS production and free radical leak (percent of total electron flow directed to ROS production) are lower in three species of birds of different orders than in mammals of similar body size and metabolic rate. This suggests that the capacity to show a low rate of ROS production is a general characteristic of birds. Using substrates and inhibitors specific for different segments of the respiratory chain, the main ROS generator site (responsible for those bird‐mammalian differences) in state 4 has been localized at complexes I and III in heart mitochondria and only at complex I in nonsynaptic brain mitochondria. In state 3, complex I is the only generator in both tissues. The results also suggest that the iron‐sulphur centers are the ROS generators of complex I. A general mechanism that allows pigeon mitochondria to show a low rate of ROS production can be the capacity to maintain a low degree of reduction of the ROS generator site. In heart mitochondria, this is supplemented with a low rate of oxygen consumption physiologically compensated with a comparatively higher heart size. A low rate of free radical production near DNA, together with a high rate of DNA repair, can be responsible for the slow rate of accumulation of DNA damage and thus the slow aging rate of longevous animals.

Rate of generation of oxidative stress-related damage and animal longevity.

Comparative studies about the relationship between endogenous antioxidant and pro-oxidant factors and maximum longevity of different animal species are reviewed. The majority of studies on antioxidant supplementation indicate that it can increase mean survival without changing maximum longevity. On the other hand, endogenous antioxidants are negatively correlated with maximum longevity. The same is true for the rates of mitochondrial oxygen radical generation, oxidative damage to mitochondrial DNA, and the degree of fatty acid unsaturation of cellular membranes in postmitotic tissues. The lower rate of mitochondrial oxygen radical generation of long-lived animals in relation to that of short-lived ones can be a primary cause of their slow aging rate. This is secondarily complemented in long-lived animals with low rates of lipid peroxidation due to their low degrees of fatty acid unsaturation. These two traits suggest that the rate of generation of endogenous oxidative damage determines, at least in part, the rate of aging in animals.

Sites and mechanisms responsible for the low rate of free radical production of heart mitochondria in the long-lived pigeon

Basal (substrate alone) and maximum rates of H2O2 production, oxygen consumption and free radical leak in the respiratory chain were higher in heart mitochondria of the short-lived rat (4 years) than in the long-lived pigeon (35 years). This suggests that the low free radical production of pigeon heart mitochondria is due in part to both a low electron flow and a low percent leak of electrons out of sequence in the respiratory chain. Thenoyltrifluoroacetone did not increase H2O2 production with succinate either in rats or pigeons. Mitochondrial H2O2 production was higher with pyruvate/malate than with succinate in both animal species. Rotenone and antimycin A increased H2O2 production with pyruvate/malate to the maximum levels observed in each species. Addition of myxothiazol to antimycin A-treated mitochondria supplemented with pyruvate/malate decreased H2O2 production in both species. All the combinations of inhibitors added with pyruvate/malate resulted in higher rates of H2O2 production in rats than in pigeons. When succinate instead of pyruvate/malate was used as substrate, rotenone and thenoyltrifluoroacetone decreased mitochondrial H2O2 production in the rat and did not change it in the pigeon. The results indicate that Complexes I and III are the main H2O2 generators of heart mitochondria in rats and pigeons and that both Complexes are responsible for the low H2O2 production of the bird. p-Chloromercuribenzoate and ethoxyformic anhydride strongly inhibited the H2O2 production induced by rotenone with pyruvate/malate in both species. This suggests that the free radical generator of Complex I is located after the ferricyanide reduction site, between the ethoxyformic and the rotenone-sensitive sites.

Endogenous oxidative stress: relationship to aging, longevity and caloric restriction.

Available studies are consistent with the possibility that oxygen radicals endogenously produced by mitochondria are causally involved in the determination of the rate of aging in homeothermic vertebrates. Oxidative damage to tissue macromolecules seems to increase during aging. The rate of mitochondrial oxygen radical generation of post-mitotic tissues is negatively correlated with animal longevity. In agreement with this, long-lived animals show lower levels of oxidative damage in their mitochondrial DNA (mtDNA) than short-lived ones, whereas this does not occur in nuclear DNA (nDNA). Caloric restriction, which decreases the rate of aging, also decreases mitochondrial oxygen radical generation and oxidative damage to mitochondrial DNA. This decrease in free radical generation occurs in complex I and is due to a decrease in the degree of electronic reduction of the complex I free radical generator, similarly to what has been described in various cases in long-lived animals. These results suggest that similar mechanisms have been used to extend longevity through decreases in oxidative stress in caloric restriction and during the evolution of species with different longevities.