Reduced apurinic/apyrimidinic endonuclease 1 activity and increased DNA damage in mitochondria are related to enhanced apoptosis and inflammation in the brain of senescence- accelerated P8 mice (SAMP8).

The senescence- accelerated mouse prone 8 (SAMP8) is a well- characterized animal model of senescence that shows early age- related neurodegeneration with impairment in learning and memory skills when compared with control senescence- resistant mice (SAMR1). In the current study, we investigated whether such impairment could be partly due to changes in mitochondrial DNA (mtDNA) repair capacity and mitochondrial DNA damage in the brain of SAMP8 mice. Besides we studied whether these potential changes were related to modifications in two major processes likely involved in aging and neurodegeneration: apoptosis and inflammation. We observed that the specific activity of one of the main mtDNA repair enzymes, the mitochondrial APE1, showed an age- related reduction in SAMP8 animals, while in SAMR1 mice mitochondrial APE1 increased with age. The reduction in mtAPE1 activity in SAMP8 animals was associated with increased levels of the DNA oxidative damage marker 8oxodG in mtDNA. Our results also indicate that these changes were related to a premature increase in apoptotic events and inflammation in the brain of SAMP8 mice when compared to SAMR1 counterparts. We suggest that the premature neurodegenerative phenotype observed in SAMP8 animals might be due, at least in part, to changes in the processing of mtDNA oxidative damage, which would lead to enhancement of apoptotic and inflammatory processes.

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.

Effects of fasting on oxidative stress in rat liver mitochondria.

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O(2) consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N(epsilon)-malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.

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.

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.

Resveratrol, melatonin, vitamin E, and PBN protect against renal oxidative DNA damage induced by the kidney carcinogen KBrO3.

Free radical scavengers can protect against the genotoxicity induced by chemical carcinogens by decreasing oxidative damage. The protective effect of the antioxidants melatonin, resveratrol, vitamin E, butylated hydroxytoluene and 2-mercaptoethylamine, and the spin-trapping compound alpha-phenyl-N-tert-butyl nitrone (PBN) against oxidative DNA damage was studied in the kidney of rats treated with the kidney-specific carcinogen potassium bromate (KBrO3). KBrO3 was given to rats previously treated with melatonin, resveratrol, PBN, vitamin E, butylated hydroxytoluene, or 2-mercaptoethylamine. Oxidative damage to kidney DNA was estimated 6 hours afterwards by measuring 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG) referred to deoxyguanosine (dG) by means of high performance liquid chromatography with electrochemical-coulometric and ultraviolet detection. Levels of oxo8dG in the renal genomic DNA significantly increased by more than 100% after the KBrO3 treatment. This increase was completely abolished by the treatment with resveratrol and was partially prevented by melatonin, PBN and vitamin E. Resveratrol and PBN also prevented the increase in relative kidney weight induced by KBrO3. These results show that various different antioxidants and a free radical trap, working in either the water-soluble or the lipid-soluble compartments, can prevent the oxidative DNA damage induced in the kidney by the carcinogen KBrO3.

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.

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.

H2O2 production of heart mitochondria and aging rate are slower in canaries and parakeets than in mice: sites of free radical generation and mechanisms involved.

Birds have a maximum longevity (MLSP) much higher than mammals of similar body size in spite of their high metabolic rates. In this study, State 4 and State 3 rates of H2O2 production were lower in canary (MLSP = 24 years) and parakeet (MLSP = 21 years) than in mouse (MLSP = 3.5 years) heart mitochondria. Studies using specific inhibitors of the respiratory chain indicate that free radical generation sites at Complexes I and III are responsible for these differences. Main mechanisms lowering H2O2 production in these birds are a low rate of mitochondrial oxygen consumption in the parakeet and a low mitochondrial free radical leak in the canary. Strong increases in H2O2 production during active respiration (State 3) released by addition of ADP to pyruvate/malate-supplemented mitochondria are avoided in three species because the free radical leak decreases during the transition from State 4 to State 3 respiration. These results, together with those previously obtained in pigeons and in various mammalian species, suggest that the rate of mitochondrial free radical production correlates better with the rate of aging and the MLSP than the metabolic rate. They also suggest that a low rate of mitochondrial H2O2 production is a general characteristic of birds, animals showing very slow aging rates.

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 the degree of fatty acid unsaturation of rat heart mitochondria on their rates of H2O2 production and lipid and protein oxidative damage.

Previous comparative studies have shown that long-lived animals have lower fatty acid double bond content in their mitochondrial membranes than short-lived ones. In order to ascertain whether this trait protects mitochondria by decreasing lipid and protein oxidation and oxygen radical generation, the double bond content of rat heart mitochondrial membranes was manipulated by chronic feeding with semi-purified AIN-93G diets rich in highly unsaturated (UNSAT) or saturated (SAT) oils. UNSAT rat heart mitochondria had significantly higher double bond content and lipid peroxidation than SAT mitochondria. They also showed increased levels of the markers of protein oxidative damage malondialdehyde-lysine, protein carbonyls, and N(e)-(carboxymethyl)lysine adducts. Basal rates of mitochondrial oxygen radical generation were not modified by the degree of fatty acid unsaturation, but the rates of H2O2 generation stimulated by antimycin A were higher in UNSAT than in SAT mitochondria. These results demonstrate that increasing the degree of fatty acid unsaturation of heart mitochondria increases oxidative damage to their lipids and proteins, and can also increase their rates of mitochondrial oxygen radical generation in situations in which the degree of reduction of Complex III is higher than normal. These observations strengthen the notion that the relatively low double bond content of the membranes of long-lived animals could have evolved to protect them from oxidative damage.

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.

Double bond content of phospholipids and lipid peroxidation negatively correlate with maximum longevity in the heart of mammals.

Free radical damage is currently considered a main determinant of the rate of aging. Unsaturated fatty acids are the tissue macromolecules most sensitive to oxidative damage. Therefore, the presence of relatively low degrees of fatty acid unsaturation is expected in the tissues of longevous animals. In agreement with this prediction, fatty acid analyses of heart phospholipids in eight mammals ranging in maximum life span (MLSP) from 3.5 to 46 years showed that their total number of double bonds is negatively correlated with MLSP (r = -0.78, P < 0.02). The low double content of longevous mammals was not due to a low polyunsaturated fatty acid content. Instead, it was mainly due to a redistribution between types of polyunsaturated fatty acids from the highly unsaturated docosahexaenoic acid (22:6n-3) to the less unsaturated linoleic acid (18:2n-6) in longevous animals (r = -0.89, P < 0.003 for 22:6n-3 and r = 0.91, P < 0.002 for 18:2n-6 versus MLSP), where n = number of different animals in each species. This redistribution suggests that one of the mechanisms responsible for the low number of fatty acid double bonds is the presence of low desaturase activities in longevous animals, although other causing factors must be involved. In agreement with the low degree of fatty acid unsaturation of longevous mammals, the sensitivity to lipid peroxidation (r = -0.87; P < 0.005) and the in vivo lipid peroxidation (r = -0.86, P < 0.005) in the heart were also negatively correlated with MLSP across species. These results, together with previous ones obtained in rodents, birds, and humans, suggest that the low degree of tissue fatty acid unsaturation of longevous homeothermic animals could have been selected during evolution to protect the tissues against oxidative damage.

A low degree of fatty acid unsaturation leads to lower lipid peroxidation and lipoxidation-derived protein modification in heart mitochondria of the longevous pigeon than in the short-lived rat.

Birds have a maximum longevity (MLSP) much greater than mammals of similar metabolic rate and body size. Thus, they are ideal models to identify longevity characteristics not linked to low metabolic rates. In this investigation, we show that the fatty acid double bond content of total lipids and phosphatidylcholine, phosphatidylethanolamine and cardiolipin fractions of heart mitochondria is intrinsically lower in pigeons (MLSP = 35 years) than in rats (MLSP = 4 years). This is mainly due to a lower content of the most highly unsaturated docosahexaenoic acid (22:6n-3) and in some fractions arachidonic acid (20:4n-6). The lower double bond content leads to a lower sensitivity to in vitro lipid peroxidation, and is associated with a lower concentration of lipid peroxidation products in vivo, and a lower level of malondialdehyde-lysine protein adducts in heart mitochondria of pigeons than rats. These results, together with those previously obtained in other species or tissues, suggest that a low degree of fatty acid unsaturation is a general characteristic of longevous homeothermic vertebrate animals both when they have low metabolic rates (mammals of large body size) or high metabolic rates (small sized birds). This constitutive trait helps to protect their tissues and mitochondria against lipid peroxidation and oxidative protein modification and can be a factor contributing to their slow rate of aging. The results also show, for the first time in a physiological model, that lipid peroxidizability is related to lipoxidative protein damage.

Localization of the site of oxygen radical generation inside the complex I of heart and nonsynaptic brain mammalian mitochondria.

Mitochondrial production of oxygen radicals seems to be involved in many diseases and aging. Recent studies clearly showed that a substantial part of the free radical generation of rodent mitochondria comes from complex I. It is thus important to further localize the free radical generator site within this respiratory complex. In this study, superoxide production by heart and nonsynaptic brain submitochondrial particles from up to seven mammalian species, showing different longevities, were studied under different conditions. The results, taking together, show that rotenone stimulates NADH-supported superoxide generation, confirming that complex I is a source of oxygen radicals in mammals, in general. The rotenone-stimulated NADH-supported superoxide production of the heart and nonsynaptic brain mammalian submitochondrial particles was inhibited both by p-chloromercuribenzoate and by ethoxyformic anhydride. These results localize the complex I oxygen radical generator between the ferricyanide and the ubiquinone reduction site, making iron-sulfur centers possible candidates, although unstable semiquinones can not be discarded. The results also indicate that the previously described inverse correlation between rates of mitochondrial oxygen radical generation and mammalian longevity operates through mechanisms dependent on the presence of intact functional mitochondria.

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.