Trans-4-oxo-2-nonenal potently alters mitochondrial function

Alzheimer disease elevates lipid peroxidation in the brain and data indicate that the resulting lipid-aldehydes are pathological effectors of lipid peroxidation. The disposition of 4-substituted nonenals derived from arachidonate (20:4, n-6) and linoleate (18:2, n-6) oxidation is modulated by their protein adduction targets, their metabolism, and the nature of the 4-substitutent. Trans-4-oxo-2-nonenal (4-ONE) has a higher toxicity in some systems than the more commonly studied trans-4-hydroxy-2-nonenal (HNE). In this work, we performed a structure-function analysis of 4-hydroxy/oxoalkenal upon mitochondrial endpoints. We tested the hypotheses that 4-ONE, owing to a highly reactive nature, is more toxic than HNE and that HNE toxicity is enantioselective. We chose to study freshly isolated brain mitochondria because of the role of mitochondrial dysfunction in neurodegenerative disorders. Whereas there was little effect related to HNE chirality, our data indicate that in the mitochondrial environment, the order of toxic potency under most conditions was 4-ONE>HNE. 4-ONE uncoupled mitochondrial respiration at a concentration of 5μM and inhibited aldehyde dehydrogenase 2 (ALDH2) activity with an IC(50) of approximately 0.5μM. The efficacy of altering mitochondrial endpoints was ALDH2 inhibition>respiration=mitochondrial swelling=ALDH5A inhibition>GSH depletion. Thiol-based alkenal scavengers, but not amine-based scavengers, were effective in blocking the effects of 4-ONE upon respiration. Quantum mechanical calculations provided insights into the basis for the elevated reactivity of 4-ONE>HNE. Our data demonstrate that 4-ONE is a potent effector of lipid peroxidation in the mitochondrial environment.     

Acrolein inhibits respiration in isolated brain mitochondria

Lipid peroxidation is elevated in diseased regions of brain in several neurodegenerative diseases. Acrolein (2-propenal) is a major cytotoxic product of lipid peroxidation and its adduction to neuronal proteins has been demonstrated in diseased brain regions from patients with Alzheimer's disease. Mitochondrial abnormalities are implicated in several neurodegenerative disorders, and mitochondria are targets of alkenal adduction in vivo. We examined the effects of acrolein upon multiple endpoints associated with the mitochondrial involvement in neurodegenerative disease. Acrolein inhibited state 3 respiration with an IC(50) of approx. 0.4 micromol/mg protein; however, there was no reduction in activity of complexes I-V. This inhibition was prevented by glutathione and N-acetylcysteine. Acrolein did not alter mitochondrial calcium transporter activity or induce cytochrome c release. These studies indicate that acrolein is a potent inhibitor of brain mitochondrial respiration.     

Neuronal Mitochondrial Toxicity of Malondialdehyde: Inhibitory Effects on Respiratory Function and Enzyme Activities in Rat Brain Mitochondria

Malondialdehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines so as to modify proteins and inactivate enzymes; it can also modify nucleosides so as to cause mutagenicity. Brain mitochondrial dysfunction is a major contributor to aging and neurodegenerative diseases. We hypothesize that MDA accumulated during aging targets mitochondrial enzymes so as to cause further mitochondrial dysfunction and additional contributions to aging and neurodegeneration. Herein, we investigated the neuronal mitochondrial toxic effects of MDA on mitochondrial respiration and activities of enzymes (mitochondrial complexes I–V, α-ketoglutarate dehydrogenase (KGDH) and pyruvate dehydrogenase (PDH)), in isolated rat brain mitochondria. MDA depressed mitochondrial membrane potential, and also showed a dose-dependent inhibition of mitochondrial complex I- and complex II-linked respiration. Complex I and II, and PDH activities were depressed by MDA at ≥0.2 μmol/mg; KGDH and complex V were inhibited by ≥0.4 and ≥1.6 μmol MDA/mg, respectively. However, MDA did not have any toxic effects on complex III and IV activities over the range 0–2 μmol/mg. MDA significantly elevated mitochondrial reactive oxygen species (ROS) and protein carbonyls at 0.2 and 0.002 μmol/mg, respectively. As for the antioxidant defense system, a high dose of MDA slightly decreased mitochondrial GSH and superoxide dismutase. These results demonstrate that MDA causes neuronal mitochondrial dysfunction by directly promoting generation of ROS and modifying mitochondrial proteins. The results suggest that MDA-induced neuronal mitochondrial toxicity may be an important contributing factor to brain aging and neurodegenerative diseases. Special issue article in honor of Dr. Akitane Mori.     

Nature of Inhibition of Mitochondrial Respiratory Complex I by 6-Hydroxydopamine

Abstract: The catecholaminergic neurotoxin 6-hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6-hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6-Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron(III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6-hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6-hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions.     

MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging

In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2(fl/fl) mice). In this study, we used TnIFastCreSod2(fl/fl) mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2(fl/fl) mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2(fl/fl) mice, when compared with control mice. Complex II activity is reduced by 47% in young and by approximately 90% in old TnIFastCreSod2(fl/fl) mice, and was found to be associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2(fl/fl) mice. Complex II-linked mitochondrial Adenosine-Tri-Phosphate (ATP) production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2(fl/fl) mice. Furthermore, in old TnIFastCreSod2(fl/fl) mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains > 2-fold elevated; and oxidative damage (measured as F(2) - isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage, and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2(fl/fl) mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy.     

Endomorphins and morphine limit anoxia-reoxygenation-induced brain mitochondrial dysfunction in the mouse

The protection of brain mitochondria from oxidative stress is an important therapeutic strategy against ischemia-reperfusion injury and neurodegenerative disorders. Isolated brain mitochondria subjected to a 5 min period of anoxia followed by 5 min reoxygenation mirrored the effect of oxidative stress in the brain. The present study attempts to evaluate the protective effects of endomorphin 1 (EM1), endomorphin 2 (EM2), and morphine (Mor) in an in vitro mouse brain mitochondria anoxia-reoxygenation model. Endomorphins (EM1/2) and Mor were added to mitochondria prior to anoxia or reoxygenation. EM1/2 and Mor markedly improved mitochondrial respiratory activity with a decrease in state 4 and increases in state 3, respiratory control ratio (RCR) and the oxidative phosphorylation efficiency (ADP/O ratio), suggesting that they may play a protective role in mitochondria. These drugs inhibited alterations in mitochondrial membrane fluidity, lipoperoxidation, and cardiolipin (CL) release, which indicates protection of the mitochondrial membranes from oxidative damage. The protective effects of these drugs were concentration-dependent. Furthermore, these drugs blocked the enhanced release of cytochrome c (Cyt c), and consequently inhibited the cell apoptosis induced by the release of Cyt c. Our results suggest that EM1/2 and Mor effectively protect brain mitochondria against oxidative stresses induced by in vitro anoxia-reoxygenation and may play an important role in the prevention of deleterious effects during brain ischemia-reperfusion and neurodegenerative diseases.     

Succinobucol versus probucol: Higher efficiency of succinobucol in mitigating 3-NP-induced brain mitochondrial dysfunction and oxidative stress in vitro

This study evaluated and compared the potential protective effects of probucol and succinobucol, two lipid-lowering compounds with anti-inflammatory and antioxidant properties, on oxidative stress and mitochondrial dysfunction induced by 3-nitropropionic acid (3-NP, a succinate dehydrogenase (SDH) inhibitor largely used as model of Huntington's disease) in rat brain mitochondria-enriched synaptosomes. 3-NP caused significant inhibition of mitochondrial complex II activity, induced mitochondrial dysfunction and oxidative stress. Probucol and succinobucol prevented oxidative stress, but only succinobucol was able to prevent the mitochondrial dysfunction induced by 3-NP. Succinobucol, which did not recover complex II inhibition, was able to protect against 3-NP-induced decreased of MTT reduction, indicating that SDH is not the only enzyme responsible for MTT reduction. The present findings suggest that succinobucol might be a novel strategy to slow or halt oxidative events in neurodegenerative conditions.     

Inhibition of succinic semialdehyde dehydrogenase activity by alkenal products of lipid peroxidation

Lipid peroxidation causes the generation of the neurotoxic aldehydes acrolein and 4-hydroxy-trans-2-nonenal (HNE). These products are elevated in neurodegenerative diseases and acute CNS trauma. Previous studies demonstrate that mitochondrial class 2 aldehyde dehydrogenase (ALDH2) is susceptible to inactivation by these alkenals. In the liver and brain another mitochondrial aldehyde dehydrogenase, succinic semialdehyde dehydrogenase (SSADH/ALDH5A1), is present. In this study, we tested the hypothesis that aldehyde products of lipid peroxidation inhibit SSADH activity using the endogenous substrate, succinic semialdehyde (SSA, 50 microM). Acrolein potently inhibited SSADH activity (IC(50)=15 microM) in rat brain mitochondrial preparations. This inhibition was of an irreversible and noncompetitive nature. HNE inhibited activity with an IC(50) of 110 microM. Trans-2-hexenal (HEX) and crotonaldehyde (100 microM each) did not inhibit activity. These data suggest that acrolein and HNE disrupt SSA metabolism and may have subsequent effects on CNS neurochemistry.     

Mitochondrial Involvement in Brain Function and Dysfunction: Relevance to Aging,Neurodegenerative Disorders and Longevity

It is becoming increasingly evident that the mitochondrial genome may play a key role in neurodegenerative diseases. Mitochondrial dysfunction is characteristic of several neurodegenerative disorders, and evidence for mitochondria being a site of damage in neurodegenerative disorders is partially based on decreases in respiratory chain complex activities in Parkinson's disease, Alzheimer's disease, and Huntington's disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant balance perturbation, are thought to underlie defects in energy metabolism and induce cellular degeneration. Efficient functioning of maintenance and repair process seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of genes termed vitagenes. A promising approach for the identification of critical gerontogenic processes is represented by the hormesis-like positive effect of stress. In the present review, we discuss the role of energy thresholds in brain mitochondria and their implications in neurodegeneration. We then review the evidence for the role of oxidative stress in modulating the effects of mitochondrial DNA mutations on brain age-related disorders and also discuss new approaches for investigating the mechanisms of lifetime survival and longevity.     

Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria

Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson's disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase-generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson's disease and methamphetamine-induced neurotoxicity.     

Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.     

Oxidation of 4-hydroxy-2-nonenal by succinic semialdehyde dehydrogenase (ALDH5A)

Elevated levels of 4-hydroxy-trans-2-nonenal (HNE) are implicated in the pathogenesis of numerous neurodegenerative disorders. Although well-characterized in the periphery, the mechanisms of detoxification of HNE in the CNS are unclear. HNE is oxidized to a non-toxic metabolite in the rat cerebral cortex by mitochondrial aldehyde dehydrogenases (ALDHs). Two possible ALDH enzymes which might oxidize HNE in CNS mitochondria are ALDH2 and succinic semialdehyde dehydrogenase (SSADH/ALDH5A). It was previously established that hepatic ALDH2 can oxidize HNE. In this work, we tested the hypothesis that SSADH oxidizes HNE. SSADH is critical in the detoxification of the GABA metabolite, succinic semialdehyde (SSA). Recombinant rat SSADH oxidized HNE and other alpha,beta-unsaturated aldehydes. Inhibition and competition studies in rat brain mitochondria showed that SSADH was the predominant oxidizing enzyme for HNE but only contributed a portion of the total oxidizing activity in liver mitochondria. In vivo administration of diethyldithiocarbamate (DEDC) effectively inhibited (86%) ALDH2 activity but not HNE oxidation in liver mitochondria. The data suggest that a relationship between the detoxification of SSA and the neurotoxic aldehyde HNE exists in the CNS. Furthermore, these studies show that multiple hepatic aldehyde dehydrogenases are able to oxidize HNE.     

In vitro effects of inorganic lead on isolated rat brain mitochondrial respiration

The effects of lead acetate on respiration in cerebral and cerebellar mitochondria from immature and adult rats were studied polarographically. With all substrates low lead concentrations produced an increase in respiration. Higher concentrations produced an inhibition of both this lead-induced respiration and ADP-dependent (State 3) respiration. Lead-induced respiration required inorganic phosphate and was inhibited by oligomycin, suggesting a coupling to oxidative phosphorylation. Inhibition of respiration was produced by much lower lead concentrations with NAD-linked citric acid cycle substrates than with succinate or -glycerophosphate. In partially disrupted mitochondria, NAD-linked substrate oxidation was inhibited at lead concentrations which did not affect NADH oxidation. Thus, in brain mitochondria the NAD-linked dehydrogenases, located in the matrix space, were more sensitive to inhibition by lead than were inner membrane enzymes. All in vitro lead effects on mitochondrial respiration were comparable in cerebral and cerebellar mitochondria isolated from both immature and adult rats.     

Acute and severe hypobaric hypoxia increases oxidative stress and impairs mitochondrial function in mouse skeletal muscle.

Severe high-altitude hypoxia exposure is considered a triggering stimulus for redox disturbances at distinct levels of cellular organization. The effect of an in vivo acute and severe hypobaric hypoxic insult (48 h at a pressure equivalent to 8,500 m) on oxidative damage and respiratory function was analyzed in skeletal muscle mitochondria isolated from vitamin E-supplemented (60 mg/kg ip, 3 times/wk for 3 wk) and nonsupplemented mice. Forty male mice were randomly divided into four groups: control + placebo, hypoxia + placebo (H + P), control + vitamin E, and hypoxia + vitamin E. Significant increases in mitochondrial heat shock protein 60 expression and protein carbonyls group levels and decreases in aconitase activity and sulfhydryl group content were found in the H + P group when compared with the control + placebo group. Mitochondrial respiration was significantly impaired in animals from the H + P group, as demonstrated by decreased state 3 respiratory control ratio and ADP-to-oxygen ratio and by increased state 4 with both complex I- and II-linked substrates. Using malate + pyruvate as substrates, hypoxia decreased the respiratory rate in the presence of carbonyl cyanide m-chlorophenylhydrazone and also stimulated oligomycin-inhibited respiration. However, vitamin E treatment attenuated the effect of hypoxia on the mitochondrial levels of heat shock protein 60 and markers of oxidative stress. Vitamin E was also able to prevent most mitochondrial alterations induced by hypobaric hypoxia. In conclusion, hypobaric hypoxia increases mitochondrial oxidative stress while decreasing mitochondrial capacity for oxidative phosphorylation. Vitamin E was an effective preventive agent, which further supports the oxidative character of mitochondrial dysfunction induced by hypoxia.     

Differential Toxicity of 6-Hydroxydopamine in SH-SY5Y Human Neuroblastoma Cells and Rat Brain Mitochondria: Protective Role of Catalase and Superoxide Dismutase

Oxidative stress and mitochondrial dysfunction are two pathophysiological factors often associated with the neurodegenerative process involved in Parkinson's disease (PD). Although, 6-hydroxydopamine (6-OHDA) is able to cause dopaminergic neurodegeneration in experimental models of PD by an oxidative stress-mediated process, the underlying molecular mechanism remains unclear. It has been established that some antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) are often altered in PD, which suggests a potential role of these enzymes in the onset and/or development of this multifactorial syndrome. In this study we have used high-resolution respirometry to evaluate the effect of 6-OHDA on mitochondrial respiration of isolated rat brain mitochondria and the lactate dehydrogenase cytotoxicity assay to assess the percentage of cell death induced by 6-OHDA in human neuroblastoma cell line SH-SY5Y. Our results show that 6-OHDA affects mitochondrial respiration by causing a reduction in both respiratory control ratio (IC(50)?=?200?±?15?nM) and state 3 respiration (IC(50)?=?192?±?17?nM), with no significant effects on state 4(o). An inhibition in the activity of both complex I and V was also observed. 6-OHDA also caused cellular death in human neuroblastoma SH-SY5Y cells (IC(50)?=?100?±?9?μM). Both SOD and CAT have been shown to protect against the toxic effects caused by 6-OHDA on mitochondrial respiration. However, whereas SOD protects against 6-OHDA-induced cellular death, CAT enhances its cytotoxicity. The here reported data suggest that both superoxide anion and hydroperoxyl radical could account for 6-OHDA toxicity. Furthermore, factors reducing the rate of 6-OHDA autoxidation to its p-quinone appear to enhance its cytotoxicity.     

4-hydroxynonenal and neurodegenerative diseases

The development of oxidative stress, in which production of highly reactive oxygen species (ROS) overwhelms antioxidant defenses, is a feature of many neurological diseases: ischemic, inflammatory, metabolic and degenerative. Oxidative stress is increasingly implicated in a number of neurodegenerative disorders characterized by abnormal filament accumulation or deposition of abnormal forms of specific proteins in affected neurons, like Alzheimer's disease (AD), Pick's disease, Lewy bodies related diseases, amyotrophic lateral sclerosis (ALS), and Huntington disease. Causes of neuronal death in neurodegenerative diseases are multifactorial. In some familiar cases of ALS mutation in the gene for Cu/Zn superoxide dismutase (SOD1) can be identified. In other neurodegenerative diseases ROS have some, usually not clear, role in early pathogenesis or implications on neuronal death in advanced stages of illness. The effects of oxidative stress on "post-mitotic cells", such as neurons may be cumulative, hence, it is often unclear whether oxidative damage is a cause or consequence of neurodegeneration. Peroxidation of cellular membrane lipids, or circulating lipoprotein molecules generates highly reactive aldehydes among which one of most important is 4-hydroxynonenal (HNE). The presence of HNE is increased in brain tissue and cerebrospinal fluid of AD patients, and in spinal cord of ALS patients. Immunohistochemical studies show presence of HNE in neurofibrilary tangles and in senile plaques in AD, in the cytoplasm of the residual motor neurons in sporadic ALS, in Lewy bodies in neocortical and brain stem neurons in Parkinson's disease (PD) and in diffuse Lewy bodies disease (DLBD). Thus, increased levels of HNE in neurodegenerative disorders and immunohistochemical distribution of HNE in brain tissue indicate pathophysiological role of oxidative stress in these diseases, and especially HNE in formation of abnormal filament deposites.     

Complex I and cytochrome c are molecular targets of flavonoids that inhibit hydrogen peroxide production by mitochondria

Flavonoids can protect cells from different insults that lead to mitochondria-mediated cell death, and epidemiological data show that some of these compounds attenuate the progression of diseases associated with oxidative stress and mitochondrial dysfunction. In this work, a screening of 5 flavonoids representing major subclasses showed that they display different effects on H?O? production by mitochondria isolated from rat brain and heart. Quercetin, kaempferol and epicatechin are potent inhibitors of H?O? production by mitochondria from both tissues (IC?? approximately 1-2 μM), even when H?O? production rate was stimulated by the mitochondrial inhibitors rotenone and antimycin A. Although the rate of oxygen consumption was unaffected by concentrations up to 10 μM of these flavonoids, quercetin, kaempferol and apigenin inhibited complex I activity, while up to 100 μM epicatechin produced less than 20% inhibition. The extent of this inhibition was found to be dependent on the concentration of coenzyme Q in the medium, suggesting competition between the flavonoids and ubiquinone for close binding sites in the complex. In contrast, these flavonoids did not significantly inhibit the activity of complexes II and III, and did not affect the redox state of complex IV. However, we have found that epicatechin, quercetin and kaempferol are able to stoichiometrically reduce purified cytochrome c. Our results reveal that mitochondria are a plausible main target of flavonoids mediating, at least in part, their reported preventive actions against oxidative stress and mitochondrial dysfunction-associated pathologies.     

Aging sensitizes toward ROS formation and lipid peroxidation in PS1M146L transgenic mice

Mutations in the presenilins (PS) account for the majority of familial Alzheimer disease (FAD) cases. To test the hypothesis that oxidative stress can underlie the deleterious effects of presenilin mutations, we analyzed lipid peroxidation products (4-hydroxynonenal (HNE) and malondialdehyde) and antioxidant defenses in brain tissue and levels of reactive oxygen species (ROS) in splenic lymphocytes from transgenic mice bearing human PS1 with the M146L mutation (PS1M146L) compared to those from mice transgenic for wild-type human PS1 (PS1wt) and nontransgenic littermate control mice. In brain tissue, HNE levels were increased only in aged (19-22 months) PS1M146L transgenic animals compared to PS1wt mice and not in young (3-4 months) or middle-aged mice (13-15 months). Similarly, in splenic lymphocytes expressing the transgenic PS1 proteins, mitochondrial and cytosolic ROS levels were elevated to 142.1 and 120.5% relative to controls only in cells from aged PS1M146L animals. Additionally, brain tissue HNE levels were positively correlated with mitochondrial ROS levels in splenic lymphocytes, indicating that oxidative stress can be detected in different tissues of PS1 transgenic mice. Antioxidant defenses (activities of antioxidant enzymes Cu/Zn-SOD, GPx, or GR) or susceptibility to in vitro oxidative stimulation was unaltered. In summary, these results demonstrate that the PS1M146L mutation increases mitochondrial ROS formation and oxidative damage in aged mice. Hence, oxidative stress caused by the combined effects of aging and PS1 mutations may be causative for triggering neurodegenerative events in FAD patients.     

Inhibition of brain mitochondrial respiration by dopamine: involvement of H(2)O(2) and hydroxyl radicals but not glutathione-protein-mixed disulfides

Examination of the downstream mediators responsible for inhibition of mitochondrial respiration by dopamine (DA) was investigated. Consistent with findings reported by others, exposure of rat brain mitochondria to 0.5 mm DA for 15 min at 30 degrees C inhibited pyruvate/glutamate/malate-supported state-3 respiration by 20%. Inhibition was prevented in the presence of pargyline and clorgyline demonstrating that mitochondrial inhibition arose from products formed following MAO metabolism and could include hydrogen peroxide (H(2) O(2) ), hydroxyl radical, oxidized glutathione (GSSG) or glutathione-protein mixed disulfides (PrSSG). As with DA, direct incubation of intact mitochondria with H(2) O(2) (100 microm) significantly inhibited state-3 respiration. In contrast, incubation with GSSG (1 mm) had no effect on O(2) consumption. Exposure of mitochondria to 1 mm GSSG resulted in a 3.3-fold increase in PrSSG formation compared with 1.4- and 1.5-fold increases in the presence of 100 microm H(2) O(2) or 0.5 mm DA, respectively, suggesting a dissociation between PrSSG formation and effects on respiration. The lack of inhibition of respiration by GSSG could not be accounted for by inadequate delivery of GSSG into mitochondria as increases in PrSSG levels in both membrane-bound (2-fold) and intramatrix (3.5-fold) protein compartments were observed. Furthermore, GSSG was without effect on electron transport chain activities in freeze-thawed brain mitochondria or in pig heart electron transport particles (ETP). In contrast, H(2) O(2) showed differential effects on inhibition of respiration supported by different substrates with a sensitivity of succinate > pyruvate/malate > glutamate/malate. NADH oxidase and succinate oxidase activities in freeze-thawed mitochondria were inhibited with IC(50) approximately 2-3-fold higher than in intact mitochondria. ETPs, however, were relatively insensitive to H(2) O(2). Co-administration of desferrioxamine with H(2) O(2) had no effect on complex I-associated inhibition in intact mitochondria, but attenuated inhibition of rotenone-sensitive NADH oxidase activity by 70% in freeze-thawed mitochondria. The results show that DA-associated inhibition of respiration is dependent on MAO and that H(2) O(2) and its downstream hydroxyl radical rather than increased GSSG and subsequent PrSSG formation mediate the effects.     

Stanozolol treatment decreases the mitochondrial ROS generation and oxidative stress induced by acute exercise in rat skeletal muscle

Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.