Cysteine dietary supplementation reverses the decrease in mitochondrial ROS production at complex I induced by methionine restriction.

It has been described that dietary cysteine reverses many of the beneficial changes induced by methionine restriction in aging rodents. In this investigation male Wistar rats were subjected to diets low in methionine, supplemented with cysteine, or simultaneously low in methionine and supplemented with cysteine. The results obtained in liver showed that cysteine supplementation reverses the decrease in mitochondrial ROS generation induced by methionine restriction at complex I. Methionine restriction also decreased various markers of oxidative and non-oxidative stress on mitochondrial proteins which were not reversed by cysteine. Instead, cysteine supplementation also lowered protein damage in association with decreases in mTOR activation. The results of the present study add the decrease in mitochondrial ROS production to the various beneficial changes induced by methionine restriction that are reversed by cysteine dietary supplementation.

Assessment of performance and egg quality in laying hens of Spanish indigenous breed Black Castellana as compared with a selected white egg-layer strain.

Indigenous animal genetic resources should be preserved because of their well adaptation to the environment, their tolerance to low food availability and their sociocultural importance. The characterization of the quality of the products generated by heritage breeds may bring more arguments to encourage the raising of these animals. This study aimed at evaluating the egg performance and quality of Spanish indigenous Black Castellana (BC) breed as compared with a selected strain (Lohmann LSL-Classic). Four groups of 30 hens were arranged: 1) Lohmann hens fed a control diet; 2) BC hens fed the control diet; 3) Lohmann hens fed a diet including linseed at 70 g/kg (omega-3 diet); 4) BC hens fed the omega-3 diet. Egg production was higher by 12.3% for Lohmann hens but, since BC eggs were heavier by 15.4%, no effect of genetics was found on daily egg mass. Feed intake was higher by 5.0% for BC hens. Nonetheless, no difference was detected for feed conversion ratio. Eggshell was thicker by 6.78% in Lohmann eggs. Haugh units did not differ among freshly laid and stored eggs in Lohmann hens, whereas Haugh units decreased in stored BC eggs (80.5 vs. 76.7 vs. 72.3 at 0, 14, and 30 d of storage). Yolks of BC eggs contained less fat (57.5 vs. 60.8% DM), more protein (32.8 vs. 31.9% DM) and more cholecalciferol (1.25 vs. 1.22 µg/g DM), and showed lower proportion of saturated fatty acids (29.0 vs. 37.0%) and higher proportions of monounsaturated (45.7 vs. 39.6%) and polyunsaturated (25.2 vs. 23.4%) fatty acids. Feeding the omega-3 diet reduced the yolk proportions of saturated (32.5 vs. 33.5%) and monounsaturated (42.0 vs. 43.3%) fatty acids and increased those of polyunsaturated (25.4 vs. 23.2%) and ω-3 (7.05 vs. 2.42%) fatty acids. No effect due to genetics or diet was found on yolk color score or on yolk content in cholesterol, cobalamin, retinol and γ-tocopherol. This study represents the first exhaustive characterization of eggs from Spanish indigenous Black Castellana breed.

Effect of alkaline pretreatment on anaerobic digestion of solid wastes.

The introduction of the anaerobic digestion for the treatment of the organic fraction of municipal solid waste (OFMSW) is currently of special interest. The main difficulty in the treatment of this waste fraction is its biotransformation, due to the complexity of organic material. Therefore, the first step must be its physical, chemical and biological pretreatment for breaking complex molecules into simple monomers, to increase solubilization of organic material and improve the efficiency of the anaerobic treatment in the second step. This paper describes chemical pretreatment based on lime addition (Ca(OH)2), in order to enhance chemical oxygen demand (COD) solubilization, followed by anaerobic digestion of the OFMSW. Laboratory-scale experiments were carried out in completely mixed reactors, 1 L capacity. Optimal conditions for COD solubilization in the first step of pretreatment were 62.0 mEq Ca(OH)2/L for 6.0 h. Under these conditions, 11.5% of the COD was solubilized. The anaerobic digestion efficiency of the OFMSW, with and without pretreatment, was evaluated. The highest methane yield under anaerobic digestion of the pretreated waste was 0.15 m3CH4/kg volatile solids (VS), 172.0% of the control. Under that condition the soluble COD and VS removal were 93.0% and 94.0%, respectively. The results have shown that chemical pretreatment with lime, followed by anaerobic digestion, provides the best results for stabilizing the OFMSW.

Rapamycin reverses age-related increases in mitochondrial ROS production at complex I, oxidative stress, accumulation of mtDNA fragments inside nuclear DNA, and lipofuscin level, and increases autophagy, in the liver of middle-aged mice.

Rapamycin consistently increases longevity in mice although the mechanism of action of this drug is unknown. In the present investigation we studied the effect of rapamycin on mitochondrial oxidative stress at the same dose that is known to increase longevity in mice (14mgofrapamycin/kg of diet). Middle aged mice (16months old) showed significant age-related increases in mitochondrial ROS production at complex I, accumulation of mtDNA fragments inside nuclear DNA, mitochondrial protein lipoxidation, and lipofuscin accumulation compared to young animals (4months old) in the liver. After 7weeks of dietary treatment all those increases were totally or partially (lipofuscin) abolished by rapamycin, middle aged rapamycin-treated animals showing similar levels in those parameters to young animals. The decrease in mitochondrial ROS production was due to qualitative instead of quantitative changes in complex I. The decrease in mitochondrial protein lipoxidation was not due to decreases in the amount of highly oxidizable unsaturated fatty acids. Rapamycin also decreased the amount of RAPTOR (of mTOR complex) and increased the amounts of the PGC1-α and ATG13 proteins. The results are consistent with the possibility that rapamycin increases longevity in mice at least in part by lowering mitochondrial ROS production and increasing autophagy, decreasing the derived final forms of damage accumulated with age which are responsible for increased longevity. The decrease in lipofuscin accumulation induced by rapamycin adds to previous information suggesting that the increase in longevity induced by this drug can be due to a decrease in the rate of aging.

Effect of age on antioxidants and molecular markers of oxidative damage in murine epidermis and dermis.

This is the first study of antioxidants and oxidative-damage-related parameters in epidermis and dermis of the skin as a function of age. The four major antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase), hydrophilic and lipophilic antioxidants, and lipid hydroperoxides were assayed in both epidermis and dermis of young and old hairless mice. Catalase, superoxide dismutase, and glutathione reductase had similar activity levels in young and old animals. Only glutathione peroxidase from epidermis showed an activity decrease due to age. This decrease became apparent when enzyme activity was expressed per mg of total cellular protein. Hydrophilic and lipophilic antioxidants did not change as a function of age, nor did lipid hydroperoxide levels. Both the absolute level of oxidized glutathione and the ratio of oxidized to reduced glutathione were higher in dermis from old mice. These results suggest that skin aging is not accelerated in old age due to a general decrease in the antioxidant capacity of the tissue. The data are compatible, however, with the idea that continuous damage to skin tissue by free radicals occurs throughout an organism's lifetime because scavenging cannot be 100% efficient.

Vitamin E protects guinea pig liver from lipid peroxidation without depressing levels of antioxidants.

Oxidative stress is considered a pathogenic factor in many disorders. The capacity of dietary vitamin E to increase global antioxidant capacity and to decrease lipid peroxidation was studied in the guinea pig, an animal that cannot synthesize ascorbate. Male guinea pigs were subjected for 5 weeks to three diets differing in vitamin E content in the presence of optimum levels of vitamin C: group 15 (15 mg vitamin E/kg diet), group 150 (150 mg/kg), and group 1500 (1500 mg/kg). Hepatic vitamin E increased in the three groups in relation to the level of vitamin E in the diet. The increase in vitamin E between groups 15 and 150 was accompanied by a reduction in sensitivity to enzymatic lipid peroxidation. This did not occur between groups 150 and 1500. The different liver vitamin E concentrations did not affect the antioxidant enzymes superoxide dismutase, catalase, GSH-peroxidase and GSH-reductase, nor the non-enzymatic antioxidants vitamin C, GSH and ascorbate. It is concluded that dietary supplementation with vitamin E, at a level 6 times higher than the minimum daily requirement for guinea pigs, increases protection against hepatic lipid peroxidation without depressing endogenous antioxidant defences. Further increases in vitamin E to megadose levels did not provide additional protection from oxidative stress. The results also suggest that optimum levels of both vitamin C and vitamin E, simultaneously needed for protection against oxidative stress, are much higher than the minimum daily requirements.

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.

Dietary vitamin C decreases endogenous protein oxidative damage, malondialdehyde, and lipid peroxidation and maintains fatty acid unsaturation in the guinea pig liver.

Guinea pigs were fed during 5 weeks with three different levels of vitamin C in the diet: 33 (marginal deficiency), 660, or 13,200 mg of vitamin C per kg of diet. The group fed 660 mg of vitamin C/kg of diet showed strongly reduced levels of protein carbonyls (46% decrease), malondialdehyde (HPLC; 72% decrease), and in vitro production of TBARS (both stimulated with ascorbate-Fe2+ and with NADPH-ADP-Fe2+; 68% and 71% decrease), increased glutathione reductase activity, and increased vitamin C content (48 times higher) in the liver in relation to the group fed 33 mg/kg. The treatment with 660 mg of vitamin C/kg did not decrease any of the antioxidant defenses studied: superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, GSH, vitamin E, or uric acid. Further supplementation with 13,200 mg vitamin C/kg also reduced protein and lipid peroxidation, but decreased hepatic glutathione reductase and uric acid and resulted in a lower body weight of the animals. Both low (33 mg/kg) and very high (13,200 mg/kg) levels of vitamin C decreased body weight, glutathione reductase, and unsaturation of fatty acids in membrane lipids. The results show that a diet supplying an amount of vitamin C 40 times higher than the minimum daily requirement to avoid scurvy increases the global antioxidant capacity and is of protective value against endogenous lipid and protein oxidation in the liver under normal nonstressful conditions.

Increase in heart glutathione redox ratio and total antioxidant capacity and decrease in lipid peroxidation after vitamin E dietary supplementation in guinea pigs.

Dietary treatment with three diets differing in vitamin E, Low E (15 mg of vitamin E/kg diet), Medium E (150 mg/kg), or High E (1,500 mg/kg), resulted in guinea pigs with low (but nondeficient), intermediate, or high heart alpha-tocopherol concentration. Neither the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and reductase, nor the nonenzymatic antioxidants, GSH, ascorbate, and uric acid were homeostatically depressed by increases in heart alpha-tocopherol. Protection from both enzymatic (NADPH dependent) and nonenzymatic (ascorbate-Fe2+) lipid peroxidation was strongly increased by vitamin E supplementation from Low to Medium E whereas no additional gain was obtained from the Medium E to the High E group. The GSH/GSSG and GSH/total glutathione ratios increased as a function of the vitamin E dietary concentration closely resembling the shape of the dependence of heart alpha-tocopherol on dietary vitamin E. The results show the capacity of dietary vitamin E to increase the global antioxidant capacity of the heart and to improve the heart redox status in both the lipid and water-soluble compartments. This capacity occurred at levels six times higher than the minimum daily requirement of vitamin E, even in the presence of optimum dietary vitamin C concentrations and basal unstressed conditions. The need for vitamin E dietary supplementation seems specially important in this tissue due to the low constitutive levels of endogenous enzymatic and nonenzymatic antioxidants present of the mammalian heart in comparison with those of other internal organs.

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.

Thyroid status modulates glycoxidative and lipoxidative modification of tissue proteins.

Steady state protein modification by carbonyl compounds is related to the rate of carbonyl adduct formation and the half-life of the protein. Thyroid hormones are physiologic modulators of both tissue oxidative stress and protein degradation. The levels of the glycation product N(epsilon)-fructoselysine (FL) and those of the oxidation products, N(epsilon)-(carboxymethyl)lysine (CML) and malondialdehyde-lysine (MDA-lys), identified by GC/MS in liver proteins, decreased significantly in hyperthyroid rats, as well as (less acutely) in hypothyroid animals. Immunoblotting of liver proteins for advanced glycation end-products (AGE) is in agreement with the results obtained by GC/MS. Cytosolic proteolytic activity against carboxymethylated foreign proteins measured in vitro was significantly increased in hypo- and hyperthyroidism. Oxidative damage to DNA, estimated as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG), did not show significant differences between groups. The results suggests that the steady state levels of these markers depend on the levels of thyroid hormones, presumably through their combined effects on the rates of protein degradation and oxidative stress, whereas DNA is more protected from oxidative damage.

Aging in brown fat: antioxidant defenses and oxidative stress.

Brown adipose tissue (BAT) responds to physiological stimulation with high rates of mitochondrial O2 consumption, and with high rates of lipid turnover. These are the most susceptible molecules to peroxidation. Thus, it is important to elucidate the changes in antioxidant defenses and lipid peroxidation that occur in this tissue during the lifetime of the individual. It is shown for the first time that during development from young (3 months) to mature adults (9 months) quantitatively important increases of all the antioxidant enzymes (superoxide dismutase, catalase, selenium dependent and independent glutathione peroxidases and glutathione reductase) take place in BAT. This is concordant with the much higher aerobic capacity and sensitivity to in vitro peroxidation of the tissue in mature adults than in the young. During aging (from 9 to 28 months of age), aerobic capacity is clearly reduced. Nevertheless, the sensitivity of BAT to in vitro peroxidation is maintained in old animals and, accordingly, the antioxidant defensive systems do not show important changes either.

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.

Lung antioxidant enzymes, peroxidation, glutathione system and oxygen consumption in catalase inactivated young and old Rana perezi frogs.

In the lung of Rana perezi no differences as a function of age have been found for any of the five major antioxidant enzymes, reduced (GSH), oxidized (GSSG) or glutathione ratio (GSSG/GSH), oxygen consumption (VO2) and for in vivo or in vitro stimulated tissue peroxidation. This frog shows a moderate rate of oxygen consumption and a life span substantially longer than that of rats and mice. Chronic (2.5 months) catalase depletion in the lung did not affect survival or any additional antioxidant enzyme, GSH, GSSG or in vivo and in vitro lung peroxidation in any age group. Only the GSSG/GSH ratio and the VO2 were elevated in catalase depleted old but not young frogs. After comparison of these results with those obtained in other animal species by other authors we suggest the possibility that decreases in antioxidant capacity in old age be restricted to species with high basal metabolic rates. Nevertheless, scavenging of oxygen radicals can not be 100% effective in any species. Thus, aging can still be due to the continuous presence of small concentrations of O2 radicals in the tissues throughout the life span in animals with either high or low metabolic rates.

Lung glutathione reductase induction in aging catalase-depleted frogs correlates with early survival throughout the life span.

A comprehensive experimental study on free radical-related parameters was performed in the lung throughout the life span of 220 initially young or old frogs. No age related differences were found transversely or longitudinally for lung superoxide dismutase, catalase, Se-dependent and -independent glutathione peroxidases, glutathione reductase, GSH, GSSG, or GSSG/GSH ratio. Continuous catalase depletion with aminotriazole led to glutathione reductase induction in the lung after 14.5 months of experimentation. This was accompanied by a great increase in survival rate of treated animals in relation to controls (especially in the old group). After 26.5 months of experimentation, glutathione reductase induction was lost and GSSG/GSH values tended to increase. This was followed by a 3-month long period of acute decrease in survival rate of treated animals. It is suggested that a high antioxidant/prooxidant balance is of protective value against causes of early death and can possibly be used in the future (when appropriately controlled) to increase the number of healthy years of the normal life span.

Low fatty acid unsaturation protects against lipid peroxidation in liver mitochondria from long-lived species: the pigeon and human case.

Birds have a much higher maximum longevity (MLSP) than mammals of similar metabolic rate. Recent data showed that pigeon mitochondria produce oxygen radicals at a rate much slower than rat mitochondria, in spite of showing similar levels of oxygen consumption (Free Rad. Res., 21 (1994) 317-328). Since oxidative damage from and to mitochondria seems important in relation to aging and longevity, and mitochondrial membranes are situated at the place where oxygen radicals are generated, we studied protein and lipid peroxidation and fatty acid composition of the three main membrane phospholipids of liver mitochondria from rats (MLSP = 4 years) and pigeons (MLSP = 35 years). It was found that pigeon mitochondria show lower levels of fatty acid unsaturation than rat mitochondria in the three lipid fractions, mainly due to a substitution of highly unsaturated fatty acids (20:4 and 22:6) by linoleic acid (18:2), and that these mitochondria are more resistant to lipid peroxidation. Previous research has also obtained exactly the same major difference in fatty acid composition in human mitochondria when compared to those of rat. Thus, present information suggests that the liver mitochondrial membranes of especially long-lived species show both a low level of free radical production and a low degree of fatty acid unsaturation as important constitutive protective traits to slow down aging.

Caloric and carbohydrate restriction in the kidney: effects on free radical metabolism.

Carbohydrate restriction and caloric restriction (60% restriction of calories in relation to controls in both cases) were imposed on OF1 mice during 8 weeks in their growing phase. The three groups of animals ingested the same amount of vitamins and minerals. Kidney ascorbate strongly decreased in both restriction groups. Nevertheless, global caloric restriction significantly increased kidney antioxidant glutathione (GSH)/oxidized glutathione (GSSG) ratio, a sign of a reduced kidney oxidative stress. Increased glutathione peroxidase and cytochrome oxidase activities and decreased in vivo peroxidation were found in the kidney when the restriction was performed by substituting carbohydrates by nonnutritive bulk. No significant changes were observed for superoxide dismutase, catalase, glutathione reductase, glutathione, uric acid, malondialdehyde (HPLC), or in vitro sensitivity to peroxidation in the kidney. The results, reported for the first time in this tissue, show that short-term caloric restriction can increase the capacity for enzymatic decomposition of hydroperoxides and can decrease oxidative stress in the kidney, thus suggesting a role for free radical metabolism in the caloric restriction phenomenon.

Relationship between antioxidants, lipid peroxidation and aging.

Experiments performed on species as different as flies, rats and frogs are not conclusive about the possibility that antioxidant defenses decrease in old animals. Even when these decreases are found, their physiological meaning is far from clear. Furthermore, a constancy of antioxidant capacity in old age is consistent with the fact that aging is a progressive phenomenon which occurs at a rather constant rate from the mature young to the very old animal, without showing a great acceleration rate in the aged. Nevertheless, experimental results strongly suggest that the maintenance of an appropriate antioxidant/prooxidant balance does have an important role in maintaining health in the aging animal. It is possible that the continuous presence of small amounts of free radicals in the adult tissues of both mature adults and old animals is an important factor in aging (a progressive phenomenon) and susceptibility to disease. Since, similarly to what occurs in procariota, the whole antioxidant system seems to be under homeostatic control in vertebrates, it is imperative to perform comprehensive and detailed studies on the effects of carefully controlled doses of antioxidants on biomarkers of health as well as on the different endogenous cellular antioxidant and prooxidant systems. These studies should have as a final goal the knowledge of which doses of antioxidants are high enough to increase antioxidant protection but low enough to avoid feedback depression of other endogenous antioxidants; this could further improve the health state of humans situated in the middle and last phases of their life span.

Effect of thyroid hormones on mitochondrial oxygen free radical production and DNA oxidative damage in the rat heart.

Mitochondria seem to be involved in oxygen radical damage and aging. However, the possible relationships between oxygen consumption and oxygen radical production by functional mitochondria, and oxidative DNA damage, have not been studied previously. In order to analyze these relationships, male Wistar rats of 12 weeks of age were rendered hyper- and hypothyroid by chronic T(3) and 6-n-propyl-2-thiouracil treatments, respectively. Hypothyroidism decreased heart mitochondrial H(2)O(2) production in States 4 (to 51% of controls; P<0.05) and 3 (to 21% of controls; P<0.05). In agreement with this, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) decreased in the heart genomic DNA of hypothyroid animals to 40% of controls (P<0.001). Studies with respiratory inhibitors showed that the decrease in oxygen radical generation observed in hypothyroidism occurred at Complex III (mainly) and at Complex I; that decrease was due to the presence of a lower free radical leak in the respiratory chain (P<0.05). Hyperthyroidism did not significantly change heart mitochondrial H(2)O(2) production since the increase in State 4 oxygen consumption in comparison with control and hypothyroid animals (P<0.05) was compensated by a decrease in the free radical leak in relation to control animals (P<0.05). In agreement with this, heart 8-oxodG was not changed in hyperthyroid animals. The lack of increase in H(2)O(2) production per unit of mitochondrial protein will protect mitochondria themselves against self-inflicted damage during hyperthyroidism.

Effect of time of restriction on the decrease in mitochondrial H2O2 production and oxidative DNA damage in the heart of food-restricted rats.

In the present study, the question if medium-term (4 months) caloric restriction (40%) decreases mitochondrial H2O2 production and oxidative DNA damage was investigated. Caloric restriction (CR) is the only experimental manipulation that increases maximum life span. Previous long-term CR studies have showed that CR decreases the mitochondrial rate of free radical production in diverse tissues and species. Those studies agree with the idea that the superior longevity of the restricted animals can be partly due to their lower mitochondrial rate of free radical generation. However, caloric restriction effects strongly depend on implementation time. Previous studies have shown that the decrease induced by CR on oxygen radical generation and oxidative damage to mitochondrial DNA occurs after 1 year but not after 6 weeks of restriction. In the present investigation, mitochondrial H2O2 production did not change in medium-term (4 months) caloric restricted animals, and, in agreement with that, no differences were found in either mitochondrial or nuclear oxidative DNA damage between restricted and ad libitum-fed animals. These results confirm the importance of the time of CR implementation, and show that time longer than 4 months is needed to decrease the mitochondrial rate of free radical generation and the oxidative damage to mtDNA in the rat heart.