Oxidative stress, mitochondrial DNA mutation, and apoptosis in aging

A wide spectrum of alterations in mitochondria and mitochondrial DNA (mtDNA) with aging has been observed in animals and humans. These include (i) decline in mitochondrial respiratory function; (ii) increase in mitochondrial production of reactive oxygen species (ROS) and the extent of oxidative damage to DNA, proteins, and lipids; (iii) accumulation of point mutations and large-scale deletions of mtDNA; and (iv) enhanced apoptosis. Recent studies have provided abundant evidence to substantiate the importance of mitochondrial production of ROS in aging. On the other hand, somatic mtDNA mutations can cause premature aging without increasing ROS production. In this review, we focus on the roles that ROS play in the aging-associated decline of mitochondrial respiratory function, accumulation of mtDNA mutations, apoptosis, and alteration of gene expression profiles. Taking these findings together, we suggest that mitochondrial dysfunction, enhanced oxidative stress, subsequent accumulation of mtDNA mutations, altered expression of a few clusters of genes, and apoptosis are important contributors to human aging.     

The antioxidant Trolox helps recovery from the familial Parkinson's disease-specific mitochondrial deficits caused by PINK1- and DJ-1-deficiency in dopaminergic neuronal cells

The nature of mitochondrial dysfunction in dopaminergic neurons in familial Parkinson's disease (PD) is unknown. We characterized the pathophenotypes of dopaminergic neuronal cells that were deficient in PINK1 or DJ-1, genes with mutations linked to familial PD. Both PINK1- and DJ-1-deficient dopaminergic neurons had the increased production of ROS, severe mitochondrial structural damages and complex I deficits. A striking decrease in complex IV activity was also prominent by the PINK1-deficiency. The complex I deficits were relatively PD-specific and were significantly improved by an antioxidant Trolox. These data suggest that mitochondrial deficits are severe in dopaminergic neurons in familial PD and antioxidant-mediated functional recovery is feasible.     

A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans

The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O^{⋅ –}), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Here we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts as a signal in young mutant animals to trigger changes of gene expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response.     

Mercuric Chloride Induced Ovarian Oxidative Stress by Suppressing Nrf2-Keap1 Signal Pathway and its Downstream Genes in Laying Hens

Over the last decade, there has been an increased concern about the health risks from exposure to arsenic at low doses, because of their neurotoxic effects on the developing brain. The exact mechanism underlying arsenic-induced neurotoxicity during sensitive periods of brain development remains unclear, although enhanced oxidative stresses, leading to mitochondrial dysfunctions might be involved. Here, we highlight the generation of reactive oxygen species (ROS) and oxidative stress which leads to mitochondrial dysfunctions and apoptosis in arsenic-induced developmental neurotoxicity. Here, the administration of sodium arsenite at doses of 2 or 4 mg/kg body weight in female rats from gestational to lactational (GD6-PD21) resulted to increased ROS, led to oxidative stress, and increased the apoptosis in the frontal cortex, hippocampus, and corpus striatum of developing rats on PD22, compared to controls. Enhanced levels of ROS were associated with decreased mitochondrial membrane potential and the activity of mitochondrial complexes, and hampered antioxidant levels. Further, neuronal apoptosis, as measured by changes in the expression of pro-apoptotic (Bax, Caspase-3), anti-apoptotic (Bcl2), and stress marker proteins (p-p38, pJNK) in arsenic-exposed rats, was discussed. The severities of changes were found to more persist in the corpus striatum than in other brain regions of arsenic-exposed rats even after the withdrawal of exposure on PD45 as compared to controls. Therefore, our results indicate that perinatal arsenic exposure leads to abrupt changes in ROS, oxidative stress, and mitochondrial functions and that apoptotic factor in different brain regions of rats might contribute to this arsenic-induced developmental neurotoxicity.     

Reactive oxygen species‐provoked mitochondria‐dependent cell death during ageing of elm (Ulmus pumila L.) seeds

Previous studies have shown that controlled deterioration treatment (CDT) induces programmed cell death in elm (Ulmus pumila L.) seeds, which undergo certain fundamental processes that are comparable to apoptosis in animals. In this study, the essential characteristics of mitochondrial physiology in elm seeds during CDT were identified by cellular ultrastructural analysis, whole‐body optical imaging, Western blotting and semi‐quantitative RT–PCR. The alteration in mitochondrial morphology was an early event during CDT, as indicated by progressive dynamic mitochondrial changes and rupture of the mitochondrial outer membrane; loss of mitochondrial transmembrane potential (Δψ_m) ensued, and mitochondrial ATP levels decreased. The mitochondrial permeability transition pore inhibitor cyclosporine A effectively suppressed these changes during ageing. The in situ localization of production of reactive oxygen species (ROS), and evaluation of the expression of voltage‐dependent anion‐selective channel and cyclophilin D indicated that the levels of mitochondrial permeability transition pore components were positively correlated with ROS production, leading to an imbalance of the cellular redox potential and ultimately to programmed cell death. Pre‐incubation with ascorbic acid slowed loss of mitochondrial Δψ_m, and decreased the effect of CDT on seed viability. However, there were no significant changes in multiple antioxidant elements or chaperones in the mitochondria during early stages of ageing. Our results indicate that CDT induces dynamic changes in mitochondrial physiology via increased ROS production, ultimately resulting in an irreversible loss of seed viability.     

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H₂O₂ in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.     

Parkinson's disease: genetic versus toxin-induced rodent models

Parkinson's disease (PD), a common progressive neurodegenerative disorder, is characterized by degeneration of dopamine neurons in the substantia nigra and neuronal proteinaceous aggregates called Lewy bodies (LBs). The etiology of PD is probably a combination of environmental and genetic factors. Recent progress in molecular genetics has identified several genes causing PD, including alpha-synuclein, leucine-rich repeat kinase 2 (LRRK2), Parkin, DJ-1 and PTEN-induced kinase 1 (PINK1), many of them coding for proteins found in LBs and/or implicated in mitochondrial function. However, the mechanism(s) leading to the development of the disease have not been identified, despite intensive research. Animal models help us to obtain insights into the mechanisms of several symptoms of PD, allowing us to investigate new therapeutic strategies and, in addition, provide an indispensable tool for basic research. As PD does not arise spontaneously in animals, characteristic and specific functional changes have to be induced by administration of toxins or by genetic manipulations. This review will focus on the comparison of three types of rodent animal models used to study different aspects of PD: (a) animal models using neurotoxins; (b) genetically modified mouse models reproducing findings from PD linkage studies or based on ablation of genes necessary for the development and survival of dopamine neurons; and (c) tissue-specific knockouts in mice targeting dopamine neurons. The advantages and disadvantages of these models are discussed.     

Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin

Reactive oxygen species (ROS) are considered a key factor in mitochondrial dysfunction associated with brain aging process. Mitochondrial respiration is an important source of ROS and hence a potential contributor to brain functional changes with aging. In this study, we examined the effect of aging on cytochrome c oxidase activity and other bioenergetic processes such as oxygen consumption, membrane potential and ROS production in rat brain mitochondria. We found a significant age-dependent decline in the cytochrome c oxidase activity which was associated with parallel changes in state 3 respiration, membrane potential and with an increase in H₂O₂ generation. The cytochrome aa₃ content was practically unchanged in mitochondria from young and aged animals. The age-dependent decline of cytochrome c oxidase activity could be restored, in situ, to the level of young animals, by exogenously added cardiolipin. In addition, exposure of brain mitochondria to peroxidized cardiolipin resulted in an inactivation of this enzyme complex. It is suggested that oxidation/depletion of cardiolipin could be responsible, at least in part, for the decline of cytochrome c oxidase and mitochondrial dysfunction in brain aging. Melatonin treatment of old animals largely prevented the age-associated alterations of mitochondrial bioenergetic parameters. These results may prove useful in elucidating the molecular mechanisms underlying mitochondrial dysfunction associated with brain aging process, and may have implications in etiopathology of age-associated neurodegenerative disorders and in the development of potential treatment strategies.     

Protein Restriction Without Strong Caloric Restriction Decreases Mitochondrial Oxygen Radical Production and Oxidative DNA Damage in Rat Liver

Previous studies have shown that caloric restriction decreases mitochondrial oxygen radical production and oxidative DNA damage in rat organs, which can be linked to the slowing of aging rate induced by this regime. These two characteristics are also typical of long-lived animals. However, it has never been investigated if those decreases are linked to the decrease in the intake of calories themselves or to decreases in specific dietary components. In this study the possible role of the dietary protein was investigated. Using semipurified diets, the ingestion of proteins of Wistar rats was decreased by 40% below that of controls while the other dietary components were ingested at the same level as in animals fed ad libitum. After seven weeks in this regime the liver of the protein restricted animals showed 30–40% decreases in mitochondrial production of reactive oxygen species (ROS) and in oxidative damage to nuclear and mitochondrial DNA. The decreases in ROS generation occurred specifically at complex~I. They also occurred without changes in mitochondrial oxygen consumption. Instead, there was a decrease in the percent free radical leak (the percentage of total electron flow leading to ROS generation in the respiratory chain). These results are strikingly similar to those previously obtained after 40% caloric restriction in the liver of Wistar rats. Thus, the results suggest that part of the decrease in aging rate induced by caloric restriction can be due to the decreased intake of proteins acting through decreases in mitochondrial ROS production and oxidative DNA damage. Interestingly, these tissue oxidative stress-linked parameters can be lowered by restricting only the intake of dietary protein, probably a more feasible option than caloric restriction for adult humans.     

多巴胺能神经元线粒体的氧化损伤与帕金森病

帕金森病(Parkinson’s disease,PD)的一个主要病理特征就是中脑黑质多巴胺能神经元的丧失,目前研究认为该病理变化与多种因素有关,包括蛋白质异常积聚、泛素蛋白酶体系统功能异常、神经炎症、线粒体损伤和氧化应激。在帕金森病人和动物模型中,中脑黑质有着明显的氧化改变。帕金森病的遗传和环境因素均会作用于线粒体,尤其对线粒体呼吸链复合体I有着抑制作用,造成线粒体损伤,产生活性氧(ROS)。活性氧的大量产生造成脂类、蛋白质和DNA的氧化,从而加剧多巴胺能神经元的线粒体和细胞损伤。多巴胺代谢过程中会产生活性氧,该自身代谢特点决定了多巴胺能神经元存在有较高的氧化应激,易受环境因素的影响。因而,线粒体的氧化损伤在帕金森病病理发生中起着重要作用。     

Role of Ceramides and Sphingolipids in Parkinson's Disease

Sphingolipids, including the basic ceramide, are a subset of bioactive lipids that consist of many different species. Sphingolipids are indispensable for proper neuronal function, and an increasing number of studies have emerged on the complexity and importance of these lipids in (almost) all biological processes. These include regulation of mitochondrial function, autophagy, and endosomal trafficking, which are affected in Parkinson’s disease (PD). PD is the second most common neurodegenerative disorder and is characterized by the loss of dopaminergic neurons. Currently, PD cannot be cured due to the lack of knowledge of the exact pathogenesis. Nonetheless, important advances have identified molecular changes in mitochondrial function, autophagy, and endosomal function. Furthermore, recent studies have identified ceramide alterations in patients suffering from PD, and in PD models, suggesting a critical interaction between sphingolipids and related cellular processes in PD. For instance, autosomal recessive forms of PD cause mitochondrial dysfunction, including energy production or mitochondrial clearance, that is directly influenced by manipulating sphingolipids. Additionally, endo-lysosomal recycling is affected by genes that cause autosomal dominant forms of the disease, such as VPS35 and SNCA. Furthermore, endo-lysosomal recycling is crucial for transporting sphingolipids to different cellular compartments where they will execute their functions.This review will discuss mitochondrial dysfunction, defects in autophagy, and abnormal endosomal activity in PD and the role sphingolipids play in these vital molecular processes.     

Oxidative stress,mitochondrial DNA mutation,and impairment of antioxidant enzymes in aging

Mitochondria do not only produce less ATP, but they also increase the production of reactive oxygen species (ROS) as by-products of aerobic metabolism in the aging tissues of the human and animals. It is now generally accepted that aging-associated respiratory function decline can result in enhanced production of ROS in mitochondria. Moreover, the activities of free radical-scavenging enzymes are altered in the aging process. The concurrent age-related changes of these two systems result in the elevation of oxidative stress in aging tissues. Within a certain concentration range, ROS may induce stress response of the cells by altering expression of respiratory genes to uphold the energy metabolism to rescue the cell. However, beyond the threshold, ROS may cause a wide spectrum of oxidative damage to various cellular components to result in cell death or elicit apoptosis by induction of mitochondrial membrane permeability transition and release of apoptogenic factors such as cytochrome c. Moreover, oxidative damage and large-scale deletion and duplication of mitochondrial DNA (mtDNA) have been found to increase with age in various tissues of the human. Mitochondria act like a biosensor of oxidative stress and they enable cell to undergo changes in aging and age-related diseases. On the other hand, it has recently been demonstrated that impairment in mitochondrial respiration and oxidative phosphorylation elicits an increase in oxidative stress and causes a host of mtDNA rearrangements and deletions. Here, we review work done in the past few years to support our view that oxidative stress and oxidative damage are a result of concurrent accumulation of mtDNA mutations and defective antioxidant enzymes in human aging.     

Proteome analysis of human substantia nigra in Parkinson's disease

Protein expression has been compared in human substantia nigra specimens from Parkinson's disease (PD) patients and from controls, and 44 proteins expressed in this midbrain region were identified by peptide mass fingerprinting. Among them, nine showed changes in their abundance. L and M neurofilament chains are less abundant in PD specimens, whereas peroxiredoxin II, mitochondrial complex III, ATP synthase D chain, complexin I, profilin, L-type calcium channel delta-subunit, and fatty-acid binding protein are significantly more present in PD samples than in controls. Besides the consolidated view of oxidative stress involvement in PD pathogenesis, suggested by overexpression of mitochondrial and reactive oxygen species (ROS)-scavenging proteins, these results indicate a possible potentiation mechanism of afferent signals to substantia nigra following degeneration of dopaminergic neurons.     

Exposure of yeast cells to anoxia induces transient oxidative stress. Implications for the induction of hypoxic genes

The mitochondrial respiratory chain is required for the induction of some yeast hypoxic nuclear genes. Because the respiratory chain produces reactive oxygen species (ROS), which can mediate intracellular signal cascades, we addressed the possibility that ROS are involved in hypoxic gene induction. Recent studies with mammalian cells have produced conflicting results concerning this question. These studies have relied almost exclusively on fluorescent dyes to measure ROS levels. Insofar as ROS are very reactive and inherently unstable, a more reliable method for measuring changes in their intracellular levels is to measure their damage (e.g. the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in DNA, and oxidative protein carbonylation) or to measure the expression of an oxidative stress-induced gene, e.g. SOD1. Here we used these approaches as well as a fluorescent dye, carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA), to determine whether ROS levels change in yeast cells exposed to anoxia. These studies reveal that the level of mitochondrial and cytosolic protein carbonylation, the level of 8-OH-dG in mitochondrial and nuclear DNA, and the expression of SOD1 all increase transiently during a shift to anoxia. These studies also reveal that carboxy-H(2)-DCFDA is an unreliable reporter of ROS levels in yeast cells shifted to anoxia. By using two-dimensional electrophoresis and mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), we have found that specific proteins become carbonylated during a shift to anoxia and that some of these proteins are the same proteins that become carbonylated during peroxidative stress. The mitochondrial respiratory chain is responsible for much of this carbonylation. Together, these findings indicate that yeast cells exposed to anoxia experience transient oxidative stress and raise the possibility that this initiates the induction of hypoxic genes.     

The daily rhythms of mitochondrial gene expression and oxidative stress regulation are altered by aging in the mouse liver

The circadian clock regulates many cellular processes, notably including the cell cycle, metabolism and aging. Mitochondria play essential roles in metabolism and are the major sites of reactive oxygen species (ROS) production in the cell. The clock regulates mitochondrial functions by driving daily changes in NAD⁺ levels and Sirt3 activity. In addition to this central route, in the present study, we find that the expression of some mitochondrial genes is also rhythmic in the liver, and that there rhythms are disrupted by the Clock^Δ19 mutation in young mice, suggesting that they are regulated by the core circadian oscillator. Related to this observation, we also find that the regulation of oxidative stress is rhythmic in the liver. Since mitochondria and ROS play important roles in aging, and mitochondrial functions are also disturbed by aging, these related observations prompt the compelling hypothesis that circadian oscillators influence aging by regulating ROS in mitochondria. During aging, the expression rhythms of some mitochondrial genes were altered in the liver and the temporal regulation over the dynamics of mitochondrial oxidative stress was disrupted. However, the expression of clock genes was not affected. Our results suggested that mitochondrial functions are combinatorially regulated by the clock and other age-dependent mechanism(s), and that aging disrupts mitochondrial rhythms through mechanisms downstream of the clock.     

Impaired hepatic function and central dopaminergic denervation in a rodent model of Parkinson's disease: A self-perpetuating crosstalk?

In Parkinson's disease (PD), aside from the central lesion, involvement of visceral organs has been proposed as part of the complex clinical picture of the disease. The issue is still poorly understood and relatively unexplored. In this study we used a classic rodent model of nigrostriatal degeneration, induced by the intrastriatal injection of 6-hydroxydopamine (6-OHDA), to investigate whether and how a PD-like central dopaminergic denervation may influence hepatic functions. Rats received an intrastriatal injection of 6-OHDA or saline (sham), and blood, cerebrospinal fluid, liver and brain samples were obtained for up to 8 weeks after surgery. Specimens were analyzed for changes in cytokine and thyroid hormone levels, as well as liver mitochondrial alterations. Hepatic mitochondria isolated from animals bearing extended nigrostriatal lesion displayed increased ROS production, while membrane potential (ΔΨ) and ATP production were significantly decreased. Reduced ATP production correlated with nigral neuronal loss. Thyroid hormone levels were significantly increased in serum of PD rats compared to sham animals while steady expression of selected cytokines was detected in all groups. Hepatic enzyme functions were comparable in all animals. Our study indicates for the first time that in a rodent model of PD, hepatic mitochondria dysfunctions arise as a consequence of nigrostriatal degeneration, and that thyroid hormone represents a key interface in this CNS-liver interaction. Liver plays a fundamental detoxifying function and a better understanding of PD-related hepatic mitochondrial alterations, which might further promote neurodegeneration, may represent an important step for the development of novel therapeutic strategies.     

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H 2O 2 in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.     

PLA(2) dependence of diaphragm mitochondrial formation of reactive oxygen species.

Contraction-induced respiratory muscle fatigue and sepsis-related reductions in respiratory muscle force-generating capacity are mediated, at least in part, by reactive oxygen species (ROS). The subcellular sources and mechanisms of generation of ROS in these conditions are incompletely understood. We postulated that the physiological changes associated with muscle contraction (i.e., increases in calcium and ADP concentration) stimulate mitochondrial generation of ROS by a phospholipase A(2) (PLA(2))-modulated process and that sepsis enhances muscle generation of ROS by upregulating PLA(2) activity. To test these hypotheses, we examined H(2)O(2) generation by diaphragm mitochondria isolated from saline-treated control and endotoxin-treated septic animals in the presence and absence of calcium and ADP; we also assessed the effect of PLA(2) inhibitors on H(2)O(2) formation. We found that 1) calcium and ADP stimulated H(2)O(2) formation by diaphragm mitochondria from both control and septic animals; 2) mitochondria from septic animals demonstrated substantially higher H(2)O(2) formation than mitochondria from control animals under basal, calcium-stimulated, and ADP-stimulated conditions; and 3) inhibitors of 14-kDa PLA(2) blocked the enhanced H(2)O(2) generation in all conditions. We also found that administration of arachidonic acid (the principal metabolic product of PLA(2) activation) increased mitochondrial H(2)O(2) formation by interacting with complex I of the electron transport chain. These data suggest that diaphragm mitochondrial ROS formation during contraction and sepsis may be critically dependent on PLA(2) activation.     

Aging in vertebrates,and the effect of caloric restriction: a mitochondrial free radical production–DNA damage mechanism?

Oxygen is toxic to aerobic animals because it is univalently reduced inside cells to oxygen free radicals. Studies dealing with the relationship between oxidative stress and aging in different vertebrate species and in caloric-restricted rodents are discussed in this review. Healthy tissues mainly produce reactive oxygen species (ROS) at mitochondria. These ROS can damage cellular lipids, proteins and, most importantly, DNA. Although antioxidants help to control this oxidative stress in cells in general, they do not decrease the rate of aging, because their concentrations are lower in long- than in short-lived animals and because increasing antioxidant levels does not increase vertebrate maximum longevity. However, long-lived homeothermic vertebrates consistently have lower rates of mitochondrial ROS production and lower levels of steady-state oxidative damage in their mitochondrial DNA than short-lived ones. Caloric-restricted rodents also show lower levels of these two key parameters than controls fed ad libitum. The decrease in mitochondrial ROS generation of the restricted animals has been recently localized at complex I and the mechanism involved is related to the degree of electronic reduction of the complex I ROS generator. Strikingly, the same site and mechanism have been found when comparing a long- with a short-lived animal species. It is suggested that a low rate of mitochondrial ROS generation extends lifespan both in long-lived and in caloric-restricted animals by determining the rate of oxidative attack and accumulation of somatic mutations in mitochondrial DNA.     

Bardoxolone-methyl inhibits migration and metabolism in MCF7 cells

Bardoxolone-methyl (BAR) is reported to have anti-inflammatory, anti-proliferative and anti-fibrotic effects. BAR activates Nrf2 and may ameliorate oxidative stress through induction of antioxidant genes. However, off-target effects, probably concentration and NFkB-dependent, have limited the clinical use of BAR. Nrf2 regulates expression of antioxidant and mitochondrial genes and has been proposed as a target for both obesity and breast cancer. Therefore, we explored whether BAR can alter migration and proliferation in the MCF7 cell line and whether metabolic function is affected by BAR. Incubation with BAR caused a time-dependent migratory inhibition and an associated decrease in mitochondrial respiration. Both migratory and mitochondrial inhibition by BAR were further enhanced in the presence of fatty acids. In addition to the activation of Nrf2, BAR altered the expression of target mRNA GCLC and UCP1. After 24?h, BAR inhibited both glycolytic capacity, reserve (p?p?N-acetyl cysteine. The fatty acid, palmitate, increased mitochondrial ROS, impaired migration and oxidative phosphorylation but palmitate toxicity towards MCF7 could not be inhibited by N-acetyl cysteine suggesting that they exert effects through different pathways. BAR-activated AKT, induced DNA damage and inhibited cell proliferation. When the proteasome was inhibited, there was loss of BAR-mediated changes in p65 phosphorylation and SOD2 expression suggesting non-canonical NFkB signaling effects. These data suggest that BAR-induced ROS are important in inhibiting MCF7 migration and metabolism by negatively affecting glycolytic capacity and mitochondrial function.