Mónica López-Torres

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.

Effect of Short-Term Caloric Restriction on H2O2 Production and Oxidative DNA Damage in Rat Liver Mitochondria and Location of the Free Radical Source

Oxygen free radicals (ROS) of mitochondrial origin seem to be involved in aging. Whereas in other tissues complexes I or III of the respiratory chain contain the ROS generators, in this study we find that rat liver mitochondria generate oxygen radicals at complexes I, II, and III. Short-term (6 weeks) caloric restriction significantly decreased H2O2 production in rat liver mitochondria. This decrease in ROS production was located at complex I because it occurred with complex I-linked substrates (pyruvate/malate), but did not reach statistical significance with the complex II-linked substrate succinate. The mechanism responsible for the lowered ROS production was not a decrease in oxygen consumption. Instead, the mitochondria of caloric-restricted animals released less ROS per unit electron flow. This was due to a decrease in the degree of reduction of the complex I generator. Furthermore, oxidative damage to mitochondrial and nuclear DNA was also decreased in the liver by short-term caloric restriction. The results agree with the idea that caloric restriction delays aging, at least in part, by decreasing the rate of mitochondrial ROS generation and thus the rate of attack to molecules, like DNA, highly relevant for the accumulation of age-dependent changes.

Maximum life span in vertebrates: relationship with liver antioxidant enzymes, glutathione system, ascorbate, urate, sensitivity to peroxidation, true malondialdehyde, in vivo H2O2, and basal and maximum aerobic capacity.

In order to help clarify whether free radicals are implicated or not in the evolution of maximum life span (MLSP) of animals, a comprehensive study was performed in the liver of various vertebrate species. Strongly significant negative correlations against MLSP were found for hepatic catalase, Se-dependent and -independent glutathione peroxidases, and GSH, whereas superoxide dismutase, glutathione reductase, ascorbate, uric acid, GSSG/GSH, in vitro peroxidation (TBA-RS), and in vivo steady-state H2O2 concentration in the liver did not correlate with MLSP. Superoxide dismutase, catalase, glutathione peroxidase, and GSH results were in agreement with those independently reported by other authors, whereas the rest of our data are reported for the first time. Potential limitations arising from the use of animals of different vertebrate Classes were counterbalanced by the possibility to study animals with very different MLSPs and life energy potentials. Furthermore, the results agreed with previous data obtained using only mammals. Since liver GSSG/GSH, peroxidation, and specially H2O2 concentration were similar in species with widely different MLSPs, it is suggested that the decrease in enzymatic H2O2 detoxifying capacity of longevous species represents an evolutionary co-adaptation with a smaller in vivo rate of free radical generation. We propose the possibility that maximum longevity was increased during vertebrate evolution by lowering the rate of free radical recycling in the tissues.

EFFECT OF THYROID STATUS ON LIPID COMPOSITION AND PEROXIDATION IN THE MOUSE LIVER

In order to analyze the possible relationship between metabolic rate and oxidative stress, OF1 female mice were rendered hyper- or hypothyroid for 4-5 weeks by administration of 0.0012% L-thyroxine (T4) or 0.05% 6-n-propyl-2-thiouracil (PTU), respectively, in their drinking water. Treatment with T4 resulted in increased basal metabolic rate measured by oxygen consumption and liver cytochrome oxidase activity without altering the glutathione redox system. Endogenous lipid peroxidation, sensitivity to lipid peroxidation and fatty acid unsaturation were decreased in the hyperthyroid group. Hypothyroidism also decreased phosphatidylcholine and cardiolipin fatty acid unsaturation but not in total lipids, and thus lipid peroxidation was not altered. Cardiolipin, a mainly mitochondrial lipid, was the most profoundly altered fraction by both hyper- and hypothyroidism. It is suggested that the lipid changes observed in hyperthyroid animals can protect them against an increased oxidative attack to tissue lipids due to their increased mitochondrial activities.

A comparative study of free radicals in vertebrates--II. Non-enzymatic antioxidants and oxidative stress.

1. The three main non-enzymatic endogenous soluble antioxidants and three estimators of oxidative stress were measured in the liver, lung and brain of seven animal species of different vertebrate classes. 2. The more concentrated antioxidant was GSH, followed by ascorbate and finally by uric acid. Liver showed higher levels of GSH and uric acid than the other two organs in the majority of the species. 3. GSSG/GSH ratio was highest in lung, probably due to the high pO2 prevalent in the tissue. Nevertheless, this did not result in higher tissue peroxidation, suggesting that the lung antioxidants are capable of coping with a high tissue pO2. 4. Tissue peroxidation was maximal in the brain when assayed by the TBA test, but this was not confirmed by HPLC of malondialdehyde (MDA). HPLC resulted in much lower MDA values than TBA.

Simultaneous induction of sod, glutathione reductase, GSH, and ascorbate in liver and kidney correlates with survival during aging

Catalase was continuously inhibited with aminotriazole in the liver and kidney during 33 months in large populations of old and young frogs in order to study the effects of the modification of the tissue antioxidant/prooxidant balance on the life span of a vertebrate species showing an oxygen consumption rate similar to that of humans. Free-radical-related parameters were measured during three consecutive years at 2.5, 14.5, and 26.5 months of experimentation. Aging per se did not decrease antioxidant enzymes and did not increase peroxidation (thiobarbituric acid positive substances, or high-pressure liquid chromatography [HPLC]-malondialdehyde), either cross sectionally or longitudinally. Long-term catalase inhibition leads to time-dependent increases (100-900%) of endogenous superoxide dismutase, GSH, ascorbate, and especially glutathione reductase at 2.5 and 14.5 months of experimentation. This was positively correlated with a higher survival of treated animals (91% in treated versus 46% in controls at 14.5 months of experimentation). The loss of those inductions after 26.5 months leads to a sharp increase in mortality rate. The results show for the first time that simultaneous induction of various tissue antioxidant enzymes and nonenzymatic antioxidants can increase the mean life span of a vertebrate animal. It is concluded that the tissue antioxidant/prooxidant balance is a strong determinant of mean life span.

A comparative study of free radicals in vertebrates—I. Antioxidant enzymes

1. Five antioxidant enzymes and cytochrome oxidase were measured in three vital organs of seven animal species of different vertebrate classes. 2. Minimal superoxide dismutase activities were found in the brain of homeotherms and in the lung of amphibia. Catalase (CAT) was maximal in liver and minimal in brain. 3. Possession of both Se dependent and independent glutathione peroxidase (GPx) is widespread in vertebrate organs. Similarities in tissue distribution were found among enzymes which use hydroperoxides (Se and non-Se GPx and CAT) or glutathione (both GPx and glutathione reductase) as substrates. 4. The results also suggest that the high aerobic capacity of the liver strongly influences the activities of the antioxidant enzymes in this tissue across vertebrate species, whereas other factors such as tissue pO2 can be more important in the lung.