The effect of aging and increasing ascorbate concentrations on respiratory chain activity in cultured human fibroblasts

The specific activities of Complexes I‐III, II‐III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 µmol 1^?1) were added. Results indicated that ascorbate at 100 µmol 1^?1 was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 µmol 1^?1 however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I‐III and II‐III activities which required 300 µmol 1^?1 ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations. Copyright © 2010 John Wiley & Sons, Ltd.     

Impaired superoxide radical production by bronchoalveolar lavage cells from NO(2)-exposed rats

Production of superoxide radicals is a central property of professional phagocytes used to combat invading microorganisms. Even though the number of macrophages and neutrophils is often increased in the lungs of patients with chronic lung diseases, these patients frequently suffer from bacterially induced exacerbations. To understand the underlying mechanisms, we investigated the production of superoxide radicals by bronchoalveolar lavage (BAL) cells in a rat NO(2) exposure model (10 ppm NO(2) for 1, 3, or 20 days). We showed that cells from NO(2)-exposed animals display a significantly impaired superoxide radical release after zymosan stimulation. The use of specific inhibitors (antimycin or diphenyleneiodonium [DPI]) revealed that the major enzyme systems, NADPH oxidase and complex III of the respiratory chain, are affected. In addition, we investigated gene expression and enzyme activities of antioxidant enzymes. mRNA expression was significantly enhanced for glutathione peroxidase (GPx)-3 and CuZn-superoxide dismutase (SOD) in BAL cells from animals exposed 3 and 20 days, and GPx and SOD enzyme activities were increased in BAL cells from rats exposed 20 days. In conclusion, concomitant occurrence of reduced production and increased scavenging of superoxide radicals resulted in the drastically impaired release of these radicals from BAL cells of NO(2)-exposed rats.     

Role of Oxidative Damage in Friedreich's Ataxia

Plasma malondialdehyde (MDA) levels were raised in Friedreich's ataxia (FRDA) patients. These levels correlated with increasing age and disease duration, suggesting lipid peroxidation increased with disease progression. Using fibroblasts from FRDA patients we observed that GSH levels and aconitase activities were normal, suggesting their antioxidant status was unchanged. When exposed to various agents to increase free radical generation we observed that intracellular superoxide generation induced by paraquat caused enhanced oxidative damage. This correlated with the size of the GAA1 expansion, suggesting decreased frataxin levels may render the cells more vulnerable to mild oxidative stress. More severe oxidative stress induced by hydrogen peroxide caused increased cell death in FRDA fibroblasts but was not significantly different from control cells. We propose that abnormal respiratory chain function and iron accumulation may lead to a progressive increase in oxidative damage, but increased sensitivity to free radicals may not require detectable respiratory chain dysfunction.     

Mitochondrial superoxide production and respiratory activity: Biphasic response to ischemic duration

Long bouts of ischemia are associated with electron transport chain deficits and increases in free radical production. In contrast, little is known regarding the effect of brief ischemia on mitochondrial function and free radical production. This study was undertaken to examine the relationship between the duration of ischemia, effects upon electron transport chain activities, and the mitochondrial production of free radicals. Rat hearts were subjected to increasing ischemic durations, mitochondria were isolated, and superoxide production and electron transport chain activities were measured. Results indicate that even brief ischemic durations induced a significant increase in superoxide production. This rate was maintained with ischemic durations less than 15 min, and then increased further with longer ischemic times. Mechanistically, brief ischemia was accompanied by an increase in NADH oxidase activity, reflected by a specific increase in complex IV activity. In contrast, longer ischemic durations were accompanied by a decrease in NADH oxidase activity, reflected by deficits in complexes I and IV activities.     

Analysis of age-associated changes in mitochondrial free radical generation by rat testis

Throughout spermatogenesis, mitochondria undergo a morphological and functional differentiation. Mitochondria are involved in the production of reactive oxygen species (ROS), considered one of the mediators of ageing. Particularly, lipid peroxidation is regarded as a major phenomenon by which ROS can impair cellular function. In the present study, we examined the production of superoxide anion, superoxide dismutase activity and the effect of Fe²⁺/ascorbate induced-lipid peroxidation on the respiratory chain activities of testis mitochondria throughout the process of spermatogenesis and ageing. Mitochondria from rat testes generated superoxide anion, mainly using NADH as substrate, which increased according to age. The activity of SOD is age-dependent and greatly stimulated during the first wave of spermatogenesis, but decreases in adulthood and old age. TBARS concentration was also markedly increased by ageing. The activity of mitochondrial respiratory chain complexes is differentially affected by oxidative stress induced by iron/ascorbate, succinate-dehydrogenase activity being less vulnerable than that of NADH-dehydrogenase and cytochrome c oxidase. The data suggest that ageing is accompanied by reduced activity of SOD, leading to excessive oxidative stress and enhanced lipid peroxidation that compromises the functionality of the electron transport chain. The data support the concept that mitochondrial function is an important determinant in ageing.     

Impact of PGC-1α on the topology and rate of superoxide production by the mitochondrial electron transport chain

Reactive oxygen species (ROS) play an important role in normal signaling events and excessive ROS are associated with many pathological conditions. The amount of ROS in cells is dependent on both the production of ROS by the mitochondrial electron transport chain and their removal by ROS-detoxifying enzymes. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial functions and a key regulator of the ROS-detoxifying program. However, the impact of PGC-1α on the topology and rate of superoxide production by the mitochondrial electron transport chain is not known. We report here, using mitochondria from muscle creatine kinase–PGC-1α transgenic mice, that PGC-1α does not affect the topology of ROS production, but increases the capacity of complexes I and III to generate ROS. These changes are associated with increased mitochondrial respiration and content of respiratory chain complexes. When normalizing ROS production to mitochondrial respiration, we find that PGC-1α preserves the percentage of free radical leak by the electron transport chain. Together, these data demonstrate that PGC-1α regulates the intrinsic properties of mitochondria in such a way as to preserve a tight coupling between mitochondrial respiration and ROS production.     

Superoxide anion and hydroxyl radical scavenging activities of vegetable extracts measured using electron spin resonance.

Radical scavenging by reconstituted lyophilized powders of water extracts from 16 common vegetables was measured using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), hydroxyl radicals, (.OH) or superoxide anion radicals (O2.-), as DMPO-OH or DMPO-OOH spin adducts. On a dry weight basis, eggplant, and red, yellow and green bell pepper extracts showed potent superoxide anion radical scavenging activities (SOD-like activities). Ascorbate oxidase- or heat-treatments, decreased SOD-like activities in bell pepper extracts suggesting that ascorbate accounts for much of their free radical scavenging activity. Eggplant epidermis extract exhibited the most potent hydroxyl radical scavenging and SOD-like activities. Eggplant SOD-like activity did not decrease after ascorbate oxidase treatment, but decreased following ultrafiltration demonstrating that SOD-like activity is partially due to high molecular weight substances. Nasunin, an anthocyanin in eggplant epidermis, showed markedly potent superoxide anion radical scavenging activity, while it inhibited hydroxyl radical generation probably by chelating ferrous ion.     

Age-associated decreased activities of mitochondrial electron transport chain complexes in heart and skeletal muscle: role of L-carnitine

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and aging. L-Carnitine (4-N-trimethylammonium-3-hydroxybutric acid) plays an important role in transport of fatty acid from cytoplasm to mitochondria for energy production. Previous studies in our laboratory reported L-carnitine as a free radical scavenger in aged rats. In the present study we focused the effect of L-carnitine on the activities of electron transport chain in young and aged rats. The activities of electron transport chain complexes were found to be significantly decreased in aged rats when compared to young control rats. Supplementation of carnitine to young and aged rats for 14 and 21 days improved the electron transport chain complexes levels in aged rats when compared with young rats in duration dependent manner. No significant changes were observed in young rats. Our result suggested that L-carnitine improved the activities of electron transport chain enzymes there by improving the energy status in aged rats.     

Pathology of urethral fibromuscular system related to parturition-induced stress urinary incontinence and TGF-β1/Smad pathway

The accumulation of octanoic (OA) and decanoic (DA) acids in tissue is the common finding in medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), the most frequent defect of fatty acid oxidation. Affected patients present hypoketotic hypoglycemia, rhabdomyolysis, hepatomegaly, seizures and lethargy, which may progress to coma and death. At present, the pathophysiological mechanisms underlying hepatic and skeletal muscle alterations in affected patients are poorly known. Therefore, in the present work, we investigated the in vitro effects of OA and DA, the accumulating metabolites in MCADD, on various bioenergetics and oxidative stress parameters. It was verified that OA and DA decreased complexes I-III, II-III and IV activities in liver and also inhibit complex IV activity in skeletal muscle. In addition, DA decreased complexes II-III activity in skeletal muscle. We also verified that OA and DA increased TBA-RS levels and carbonyl content in both tissues. Finally, DA, but not OA, significantly decreased GSH levels in rat skeletal muscle. Our present data show that the medium-chain fatty acids that accumulate in MCADD impair electron transfer through respiratory chain and elicit oxidative damage in rat liver and skeletal muscle. It may be therefore presumed that these mechanisms are involved in the pathophysiology of the hepatopathy and rhabdomyolysis presented by MCADD-affected patients.     

The catalytic effect of the carcinogen "4-nitroquinoline-N-oxide" on the oxidation of vitamin C.

The carcinogen 4-nitroquinoline-N-oxide was found to mediate the reaction between ascorbate and oxygen. The oxidation of ascorbate was initiated by the production of the nitro radical anion which reacted with oxygen to produce the oxygen superoxide radical anion, peroxide and hydroxyl radical. The production of partially reduced oxygen intermediates resulted in additional reactions with ascorbate. The consumption of oxygen could be either completely blocked by reacting the nitro radical with ferricytochrome c or partially blocked by the combined effects of superoxide dismutase and catalase. The consumption of oxygen could be enhanced by reducing the hydroxyl radicals with dimethylsulfoxide.     

Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin

Mitochondrial DNA (mtDNA) mutations and impaired respiratory function have been demonstrated in various tissues of aged individuals. We hypothesized that age-dependent increase of ROS and free radicals production in mitochondria is associated with the accumulation of large-scale mtDNA deletions. In this study, we first confirmed that the proportion of mtDNA with the 4977 bp deletion in human skin tissues increases with age. We then investigated the 8-hydroxy-2'-deoxyguanosine (8-OH-dG) content in skin tissues and lipid peroxides content of the skin fibroblasts from subjects of different ages. The results showed an age-dependent increase of 8-OH-dG level in the total DNA of skin tissues of the subjects above the age of 60 years. The specific content of malondialdehyde, an end product of lipid peroxidation, was also found to increase with age. On the other hand, we examined the enzyme activities of Cu, Zn-superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPx) in the skin fibroblasts. The activities of Cu,Zn-SOD, catalase and glutathione peroxidase were found to decrease with age. However, the activity of Mn-SOD was increased with age before 60 years but was decreased thereafter. Moreover, the activity ratios of Mn-SOD/catalase and Mn-SOD/GPx exhibited the same pattern of change with age. This indicates that free radical scavenging enzymes can effectively dispose of ROS and free radicals before 60 years of age. However, elevated oxidative stress caused by an imbalance between the production and removal of ROS and free radicals occurred in skin fibroblasts after 60 years of age. Taken together, we suggest that the functional decline of free radical scavenging enzymes and the elevation of oxidative stress may play an important role in eliciting oxidative damage and mutation of mtDNA during the human aging process.     

Intracellular GSH and ascorbate inhibit radical-induced protein chain peroxidation in HL-60 cells

The results of this study suggest that the well-documented loss of GSH and ascorbate in organisms under oxidative stress may be mainly due to their reactions with protein radicals and/or peroxides. Protein hydroperoxides were generated in HL-60 cells exposed to radiation-generated hydroxyl radicals. We found for the first time evidence of chain peroxidation of the proteins in cells, with each hydroxyl radical leading to the formation of about 10 hydroperoxides. Protein peroxidation showed a lag, probably due to the endogenous antioxidant enzymes, with simultaneous loss of the intracellular GSH. Enhancement of the GSH levels by N-acetylcysteine decreased the formation of hydroperoxides, while treatment with l-buthionine sulfoximine had the opposite effect. The effect of variation of GSH levels on the formation of the peroxidized proteins is explained primarily by reduction of the protein hydroperoxides by GSH. Loading of the cells with ascorbate resulted in reduction of the amounts of protein hydroperoxides generated by the radiation, which was proportional to the intracellular ascorbate concentration. In contrast to the GSH, inhibition of protein hydroperoxide formation in the presence of the high (mM) intracellular ascorbate levels achieved was mainly due to the direct scavenging of hydroxyl radicals by the vitamin.     

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: Modulation by culture conditions

Summary Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells were prelabeled with⁵¹Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus seemed more sensitive to this oxidant stress. The effect of cell culture age, as indicated by population doubling level (PDL), was examined. Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response of PDL 15 cells. Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived free radicals than did higher PDL cells. Specific activities of the antioxidant enzymes catalase, managanese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low and high PDL fibroblasts and endothelial cells. Antioxidant enzyme specific activities could only partially explain the differences in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells. Cell culture medium composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially reduced species of oxygen, i.e. superoxide, hydrogen peroxide, and hydroxyl radical. Serum content of medium was important in modulating free radical generation; superoxide production rates decreased 32%, H₂O₂ became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum. The medium protein and iron content also modulated free radical generation. The data suggest that cell culture media constituents, cell type, and cell culture age greatly affect in vitro response of cells subjected to oxidant stress. Research supported by American Lung Association Fellowship Training Grant and Research Training Grant, the R. J. Reynolds Corporation, and National Institutes of Health Grants HL29784 and 1 HL 23805.     

A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders.

The beta-oxidation of stearic acid and of alpha- and gamma-methyl isoprenoid-derived fatty acids (pristanic and tetramethylheptadecanoic acids, respectively) was investigated in normal skin fibroblasts and in fibroblasts from patients with inherited defects in peroxisomal biogenesis. Stearic acid beta-oxidation by normal fibroblast homogenates was several-fold greater compared to the oxidation of the two branched chain fatty acids. The effect of phosphatidylcholine, alpha-cyclodextrin, and bovine serum albumin on the three activities suggests that different enzymes are involved in the beta-oxidation of straight chain and branched chain fatty acids. Homogenates of fibroblasts from patients with a deficiency in peroxisomes (Zellweger syndrome and infantile Refsum's disease) showed a normal ability to beta-oxidize stearic acid, but the oxidation of pristanic and tetramethylheptadecanoic acid was decreased. Concomitantly, 14CO2 production from the branched chain fatty acids by Zellweger fibroblasts in culture (but not from stearic acid) was greatly diminished. The Zellweger fibroblasts also showed a marked reduction in the amount of water-soluble metabolites from the radiolabeled branched chain fatty acids that are released into the culture medium. The data presented indicate that the oxidation of alpha- and gamma-methyl isoprenoid-derived fatty acids takes place largely in peroxisomes in human skin fibroblasts.     

Generation of radical anions of nitrofurantoin, misonidazole, and metronidazole by ascorbate

Nitrofurantoin, misonidazole, and metronidazole were reduced to their corresponding nitro anion radicals by ascorbate in anaerobic solutions at high pH. The nitrofurantoin anion radical could be detected at neutral pH. In neutral solutions, the nitro anion radicals of misonidazole and metronidazole were too unstable to be observed by electron spin resonance spectroscopy. At neutral pH, solutions containing ascorbate, nitrofurantoin, or misonidazole consumed oxygen. The addition of superoxide dismutase, catalase, or both superoxide dismutase and catalase decreased the rate of oxygen consumption. These results show that nitro anion radicals are formed by reduction with ascorbate, and superoxide anion radical and hydrogen peroxide are produced by reactions of these radicals with oxygen.     

A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals

The metal-mediated site-specific mechanism for free radical-induced biological damage is reviewed. According to this mechanism, cooper- or iron-binding sites on macromolecules serve as centers for repeated production of hydroxyl radicals that are generated via the Fenton reaction. The aberrations induced by superoxide, ascorbate, isouramil, and paraquat are summarized. An illustrative example is the enhancement of double-strand breaks by ascorbate/copper. Prevention of the site-specific free radical damage can be accomplished by using selective chelators for iron and copper, by displacing these redox-active metals with other redox-inactive metals such as zinc, by introducing high concentrations of hydroxyl radicals scavengers and spin trapping agents, and by applying protective enzymes that remove superoxide or hydrogen peroxide. Histidine is a special agent that can intervene in free radical reactions in variety of modes. In biological systems, there are traces of copper and iron that are at high enough levels to catalyze free-radical reactions, and account for such deleterious processes. In the human body Fe/Cu = 80/1 (w/w). Nevertheless, both (free) copper and iron are soluble enough, and the rate constants of their reduced forms with hydrogen peroxide are sufficiently high to suggest that they might be important mediators of free radical toxicity.     

Measuring the quantity and activity of mitochondrial electron transport chain complexes in tissues of central nervous system using blue native polyacrylamide gel electrophoresis

Mitochondrial dysfunction and degeneration are associated with many neurodegenerative disorders. A dysfunctional mitochondrial electron transport chain (ETC) impairs ATP production and accelerates the generation of free radicals. To evaluate mitochondrial function, reliable methods are needed. Conventional spectrophotometric assays may not eliminate interference from nonspecific enzyme activities and do not measure quantities of specific ETC complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to resolve mitochondrial ETC complexes. Combined with histochemical staining, it has also been applied to measure ETC enzyme activities in muscles. The current study is to determine (1) whether BN-PAGE can be used to detect ETC complexes from different regions of the central nervous system (CNS) and (2) the quantitative range of BN-PAGE in measuring the amounts and activities of different ETC complexes. By systematically varying the protein amount and the time of histochemical reactions, we have found linear ranges comparable to spectrophotometric assays for measuring enzyme activities of several ETC complexes. In addition, we found linear ranges for measuring protein quantities in several ETC complexes. These results demonstrate that BN-PAGE can be used to measure the amount and activity of the ETC enzymes from the nerve tissues and, thus, can be applied to evaluate the functional changes of mitochondria in neurodegenerative disorders.     

Antioxidants and Manganese Deficiency in Needles of Norway Spruce (Picea abies L.) Trees

Chlorotic and green needles from Norway spruce (Picea abies L.) trees were sampled in the Calcareous Bavarian Alps in winter. The needles were used for analysis of the mineral and pigment contents, the levels of antioxidants (ascorbate, glutathione), and the activities of protective enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate radical reductase, dehydroascorbate reductase, glutathione reductase). In addition, the activities of two respiratory enzymes (glucose-6-phosphate dehydrogenase, NAD-malate dehydrogenase), which might provide the NADPH necessary for functioning of the antioxidative system, were determined. We found that chlorotic needles were severely manganese deficient (3 to 6 micrograms Mn per gram dry weight as compared with up to 190 micrograms Mn per gram dry weight in green needles) but had a similar dry weight to fresh weight ratio, had a similar protein content, and showed no evidence for enhanced lipid peroxidation as compared with green needles. In chlorotic needles, the level of total ascorbate and the activities of superoxide dismutase, monodehydroascorbate radical reductase, NAD-malate dehydrogenase, and glucose-6-phosphate dehydrogenase were significantly increased, whereas the levels of ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, and glutathione were not affected. The ratio of ascorbate to dehydroascorbate was similar in both green and chlorotic needles. These results suggest that in spruce needles monodehydroascorbate radical reductase is the key enzyme involved in maintaining ascorbate in its reduced state. The reductant necessary for this process may have been supplied at the expense of photosynthate.     

Evidence that the inhibitory effects of guanidinoacetate on the activities of the respiratory chain, Na+,K+-ATPase and creatine kinase can be differentially prevented by taurine and vitamins E and C administration in rat striatum in vivo

Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of guanidinoacetate (GAA) and depletion of creatine. Affected patients present epilepsy and mental retardation whose etiopathogeny is unclear. In a previous study we showed that instrastriatal administration of GAA caused a reduction of Na(+),K(+)-ATPase and creatine kinase (CK) activities, as well as an increase in TBARS (an index of lipid peroxidation). In the present study we investigated the in vitro and in vivo effects of GAA on glucose uptake from [U-(14)C] acetate (citric acid cycle activity) and on the activities of complexes II, II-III, III and IV of the respiratory chain in striatum of rats. Results showed that 50 and 100 microM GAA (in vitro studies) and GAA administration (in vivo studies) significantly inhibited complexes II and II-III, respectively, but did not alter complexes III and IV, as well as CO(2) production. We also studied the influence of taurine or vitamins E and C on the inhibitory effects caused by intrastriatal administration of GAA on complexes II and II-III, Na(+),K(+)-ATPase and CK activities, and on TBARS in rat striatum. Pre-treatment with taurine and vitamins E and C revealed that taurine prevents the effects of intrastriatal administration of GAA on the inhibition of complex II, complex II-III, and Na(+),K(+)-ATPase activities. Vitamins E and C prevent the effects of intrastriatal administration of GAA on the inhibition of CK and Na(+),K(+)-ATPase activities, and on the increase of TBARS. The data suggest that GAA in vivo and in vitro treatment disturbs important parameters of striatum energy metabolism and that oxidative damage may be mediating these effects. It is presumed that defects in striatum bioenergetics might be involved in the pathophysiology of striatum damage characteristic of patients with GAMT-deficiency.