Metabolic actions of free radicals: walking the tightrope

Sandeep Raha1, Brian H. Robinson1,2
1Metabolism Research Programme, Hospital for Sick Children, Toronto;
1,2Department of Biochemistry, University of Toronto, Ontario, Canada
Correspondence: Dr Brian H. Robinson, Metabolism Research Programme,
Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
E-mail: bhr@sickkids.on.ca

Introduction
Free radicals and the damage they effect have come to take a central role in a very large number of diseases and biochemical processes. The damaging effects of both oxygen- and nitrogen-derived free radicals with relation to aging and disease propagation have become a very active area of biomedical research [1, 2]. Free radical damage has been implicated in a range of diseases including atherosclerosis, diabetes mellitus, neurodegenerative disorders, hypertension, rheumatoid arthritis [3], and amyotrophic lateral sclerosis [4]. In the majority of these cases, the most destructive species of free radicals are thought to be hydroxyl and peroxynitrite radicals. The former arises as a result of the combination of superoxide and hydrogen peroxide and the latter is formed from the reaction of superoxide with nitric oxide [5]. The primary source of superoxide radical formation is believed to be the mitochondrial electron transport chain. Approximately 1% to 2% of the electrons are lost to oxygen and result in the formation of superoxide [6, 7]. Superoxide formation also causes an accumulation of damage to DNA resulting in a shortening in the telomeres of nuclear DNA [7, 8]. This is hypothesized to be one of the major consequences of cumulative free radical damage during the aging process. This review will focus primarily on the mitochondria as the site of free radical formation, and the organelle where the majority of the “balancing act” associated with the regulation of radicals must occur.
It is estimated that well over 60 million North Americans suffer from cardiovascular diseases. Cardiovascular tissues, because they contain higher numbers of mitochondria and increased levels of respiratory chain components per milligram of mitochondrial protein, are subject to extensive damage resulting from elevated levels of superoxide. Diseases that affect mitochondrial function also impact severely on cardiomyocytes as well as on the surrounding vascular tissues [9, 10]. These include the development of atherosclerosis, hypertension [9], and arterial thrombosis [11]. Under normal conditions the vascular endothelium plays a pivotal role in inhibiting intravascular thrombus formation. Vascular endothelial cells play a crucial role in this pathway by synthesizing various substances such as thrombomodulin, tissue factor-pathway inhibitor, prostacyclin, and tissue plasminogen activator. Disruption of the pathways involved in the release of these substances can affect the antithrombotic properties of the endothelium.
Much of this damage within the cardiovascular system results as a consequence of the activation of a number of apoptotic pathways [12]. Free radicals contribute to the apoptotic process in one of two ways: direct damage of proteins, or the activation of transcription factors culminating in a change in gene expression patterns [13]. Direct damage occurs as a result of oxidative damage to proteins and lipids. Oxidative damage of crucial redox-sensitive proteins can lead to an increase in the production of free radicals and an elevation in the level of damage incurred [14]. Triggering of key transcription factors such as hypoxia-inducible factor (HIF)-1a [15] or nuclear factor kappaB (NFkB) can result in the activation of genes related to the progression of the apoptotic pathway [13]. In addition to these roles, recent observations have demonstrated that free radicals can behave like conventional second messengers that can activate Ca2+ flux from IP3-sensitive and/or ryanodine-sensitive stores directly [16]. This review will focus on the balance between the production of free radicals and the pathways for their removal (Figure 1).

More importantly, the basal level of free radical production that occurs during normal cellular metabolism constitutes an important mechanism of cellular communication (Figure 2). Significant changes beyond these basal levels can result in severe cellular damage leading to cell death. Networks of cellular systems help to maintain this delicate balance. Clearly the cell must “walk a tightrope” between an excess of free radicals, which may cause damage, and a sufficiency of free radicals in order to maintain the ability to evoke gene regulatory function necessary for the induction of protective mechanisms.

Formation of reactive oxygen and nitrogen species
A number of reviews have addressed the contribution of increased oxidative stress towards the progression of cardiovascular disease [17]. In order to better understand this problem, an appreciation of the sources of free radical production within the cardiovascular system is required. Mitochondria are a major source of cellular free radicals and their contribution to oxidative injury in the cardiovascular system becomes important for two reasons. Firstly, cardiomyocytes are enriched in mitochondria because of their metabolic demands. Secondly, ischemic conditions can occur during myocardial infarction. Subsequent reperfusion can result in a dramatic elevation of free radical production [18] and lead to the activation of a host of other regulatory pathways.
Both reactive oxygen and reactive nitrogen species are responsible for damage to cardiovascular tissues. Endothelium-derived relaxing factor, or nitric oxide, is an important signaling molecule produced in a variety of tissues by nitric oxide synthase enzymes of which there are several isoforms [19]. Several groups have reported a mitochondrial form of the enzyme [20, 21].
The superoxide radical has also been shown to be particularly reactive. Aside from mitochondrial sources, a number of other enzyme systems are known to produce reactive oxygen species. These include xanthine oxidase, cytochrome P-450 mono-oxygenases, lipoxygenase, nitric oxide synthase, and reduced nicotinamide adenine dinucleotide phosphate oxidase [9]. Superoxides can react with H2O2 or nitric oxide to form either hydroxyl radicals or peroxynitrite radicals. The hydroxyl radical is extremely reactive, with an estimated lifetime of 10-9 seconds, and is widely known to cause lipid peroxidation and DNA damage [22]. Within the mitochondria, superoxides are thought to originate primarily from nicotinamide adenine dinucleotide-coenzyme Q-oxidoreductase complex (complex I) and ubiquinol-cytochrome c reductase (complex III) [23–25]. It should be borne in mind that there is a basal level of superoxide production, and management of these free radicals dictates the fate of the cells. This constant level of free radical insult is what contributes to the age-dependent damage to cellular elements.

Removal of free radicals
Recent attempts to identify enzymes involved in the regulation of the mitochondrial antioxidant defense system using a proteomics approach resulted in the identification of manganese superoxide dismutase (SOD), peroxiredoxin III, and mitochondrial thioredoxin as proteins regulated by oxidative stress [26]. There exist a number of SOD [27] including copper-zinc SOD, to dissipate superoxides within the cytosol, and manganese SOD that regulate superoxide homeostasis within the mitochondria. It has been demonstrated that removal of manganese SOD (sod2) from rat heart mitochondria results in a significantly greater level of basal superoxide production [20]. The neonatal lethality of the sod2- mouse resulting from the inactivation of iron-sulfur centers within the electron transport chain and citric acid cycle enzymes underscores the importance of manganese SOD [28].
The product of superoxide dismutation is hydrogen peroxide, which can react via the Fenton reaction to form extremely toxic hydroxyl radicals. An extensive network of enzymes exists to facilitate the removal of H2O2. Catalase, thioredoxin reductase, and glutathione peroxidase are primarily responsible for the removal of peroxide. However, there are also specific isoforms of these enzyme systems within the mitochondria for this purpose. Inactivating Gpx1, an isoform expressed in heart and muscle [29], increased mitochondrial H2O2 production and resulted in greater levels of lipid oxidation and decreased mitochondrial energy output.
Thioredoxin peroxidase (peroxiredoxin) also catalyzes the removal of H2O2 generated from cellular metabolism or during the cellular signaling processes. This enzyme relies on thioredoxin as a source for reducing equivalents. There are at least 12 varieties of peroxiredoxin in mammals and these can be subdivided into three distinct subclasses [30]. Peroxiredoxin III is localized to the mitochondria.
The thioredoxin, thioredoxin reductase, and peroxiredoxin system is one that is also involved in maintaining the free radical detoxification in several compartments inside and outside cells. Inhibition of thioredoxin-2, a mitochondrial-specific member of the thioredoxin superfamily, results in a marked increase in intracellular reactive oxygen species [31]. Conversely, overexpression of peroxiredoxin decreases levels of H2O2 which result from tumor necrosis factor-a activation in NIH 3T3 fibroblasts. Moreover, the activation of NFkB by exogenously added H2O2 was attenuated following overexpression of peroxiredoxin II [32].

Free radicals and cellular signaling
There are a number of diseases that are characterized by elevated levels of basal superoxide production. Conventionally, the increased level of free radical production was attributed to cellular dysfunction. However, recent evidence suggests that this shift in the equilibrium level of free radicals may serve to activate other second messenger pathways. The importance of understanding the signaling role of free radicals is underscored in the speculation by Toyokuni et al [33] that the consistently high levels of free radicals produced by cancerous cells have a role in promoting ongoing proliferation. The universality of these signaling molecules is demonstrated in the observation that they are capable of interacting with and regulating the function of membrane receptors, enzymes, or transcription factors.
The reactivity of oxygen and its intermediates towards the activation of a peripheral benzodiazepine receptor (PBR) suggests that it may function as a “superoxide receptor” [34]. These observations were made primarily on the basis of structural homologies between the mitochondrial PBR and the bacterial TspO protein, which is responsible for regulating oxygen sensitivity. It has also been demonstrated that transfection of Jurkat cells with human PBR cDNA exhibited an increased resistance to high levels of H2O2 [35].
Superoxides have also been linked with the activation of manganese SOD via a direct activation of protein kinase C [36]. This was demonstrated by measuring the activity of protein kinase C in the presence of a xanthine/xanthine oxidase-based system to generate superoxides [37]. The activation was postulated to be the result of a free radical-based thiol oxidation and the release of Zn2+ from a cysteine-rich zinc finger domain. This is supported by the observation that the addition of dithioreitol, a reducing agent, prevented the observed increased in protein kinase C activity in the presence of the superoxide-generating system. This highlights recent evidence that the redox regulation of cellular proteins occurs primarily through sulfhydryl (RSH) groups. In most cases these groups are oxidized to form disulfide bonds (RSSR), sulfenic acid (RSOH), sulfinic acid (RSO2H) or sulfonic acid (RSO3H) [38].
Much of the gene transcription that results from exposure of cells to hypoxic conditions, especially in cardiovascular tissues, is likely due to activation by pathways involving free radical-mediated mechanisms. The regulatory effect of both reactive oxygen species and reactive nitrogen species on transcription factors and ultimately on gene expression is an indication as to the importance of their second messenger roles [5]. In general, the effects of gene expression can be divided into a number of different categories including ion transport, apoptosis, transcription, hormone action or neuromodulation. These effects have been well summarized in a number of reviews including that of Allen and Tresini [13]. Reactive oxygen species have been demonstrated to regulate both HIF-1a [39] and NFkB [40]. The interaction of oxygen and/or oxygen intermediates is postulated to affect HIF-1a stability via modulation of the von Hippel-Lindau protein [41]. The regulation of HIF-1a has been shown to be modulated as a function of the changes in reactive oxygen species such as H2O2 [39, 41].
Superoxides may directly regulate a number of very important cardiovascular control points (Figure 2). Protein kinase C activity and the release of Ca2+ from internal stores are important control points for signal transduction for any type of vascular muscle tissue. Superoxides increase both the release of Ca2+ and the activity of protein kinase C. In the case of protein kinase C [37], it was reported that the oxidation of cysteine and the release of Zn2+ was a prerequisite for the activation of the enzyme. In the case of intracellular Ca2+ release [42], the mechanism of action of superoxides was observed to be in a calmodulin-dependent fashion. Other studies have also suggested that reactive nitrogen species, such as nitric oxide, can activate intracellular Ca2+ release by S-nitrosylating a single cysteine residue [43].
The dosage level is the distinguishing factor as whether these radicals are utilized for signaling events or evoke cellular damage.
To date, there are no clear-cut values on the “normal” dosages of free radicals within a cell. Therefore it is difficult to estimate the percentage of the total amount of cellular free radicals required for normal cellular communication. For this reason, constant administration of antioxidant drugs may actually be detrimental to the cell and interfere with its ability to actively recover from situations of oxidative stress.
Clearly, the exact mechanisms for the activation of second messenger pathways by reactive oxygen and nitrogen species will be difficult to elucidate because of the transient nature of these entities. Further detailed research towards the elucidation of the mechanisms of oxygen and nitrogen free radicals in intra and intercellular signaling events will help to more clearly define their role in cellular communication.

Acknowledgments
We wish to thank the Heart and Stroke Association of Canada for its financial support.

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Department of Medicine, Division of Cardiology, Emory University, 1639 Pierce Drive, WMB 319, Atlanta, GA 30322, USA. tfukai@emory.edu

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Lankenau Medical Research Center, Thomas Jefferson University, Wynnewood, PA 19106, USA.

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Division of Neurogerontology, Andrus Gerontology Center and Programs in Neurobiology and Molecular Biology, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA.

Oxidative stress appears to play an important role in degeneration of dopaminergic neurons of the substantia nigra (SN) associated with Parkinson's disease (PD). The SN of early PD patients have dramatically decreased levels of the thiol tripeptide glutathione (GSH). GSH plays multiple roles in the nervous system both as an antioxidant and a redox modulator. We have generated dopaminergic PC12 cell lines in which levels of GSH can be inducibly down-regulated via doxycycline induction of antisense messages against both the heavy and light subunits of gamma-glutamyl-cysteine synthetase, the rate-limiting enzyme in glutathione synthesis. Down-regulation of glutamyl-cysteine synthetase results in reduction in mitochondrial GSH levels, increased oxidative stress, and decreased mitochondrial function. Interestingly, decreases in mitochondrial activities in GSH-depleted PC12 cells appears to be because of a selective inhibition of complex I activity as a result of thiol oxidation. These results suggest that the early observed GSH losses in the SN may be directly responsible for the noted decreases in complex I activity and the subsequent mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with PD.

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Oxygen Signaling and Lung Membrane Transport Groups, Center for Research into Human Development, Tayside Institute of Child Health, Faculty of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, Scotland.

The O(2) and redox-sensitive transcription factors hypoxia inducible factor-1alpha (HIF-1alpha) and nuclear factor-kappaB (NF-kappaB) are differentially regulated in the alveolar epithelium over fetal to neonatal oxygen tensions. We have used fetal alveolar type II epithelial cells to monitor their regulation in association with redox responsiveness to antioxidant pretreatment in vitro. N-Acetyl-l-cysteine, a glutathione (GSH) precursor and a potent scavenger of reactive oxygen species, induced HIF-1alpha and ameliorated NF-kappaB nuclear abundance and DNA binding activity, respectively, in a dose-dependent manner. Analysis of variations in glutathione homeostasis at ascending DeltapO(2) regimen with N-acetyl-(L)-cysteine reveals increased GSH at the expense of the oxidized form of glutathione (GSSG), thereby shifting GSH/GSSG into reduction equilibrium. Pyrrolidine dithiocarbamate (PDTC), which exerts both antioxidant and pro-oxidant effects, provoked a substantial increase in HIF-1alpha nuclear abundance, with no apparent effect on its activation. PDTC reduced NF-kappaB nuclear abundance and its inhibitory effects on binding activity are dose-dependent. Assessment of glutathione homeostasis with PDTC shows increasing levels of GSSG at the expense of GSH, lowering GSH/GSSG in favor of an oxidative equilibrium. Our results indicate the hypoxic activation of HIF-1alpha and the hyperoxic induction of NF-kappaB in the fetal epithelium is redox-sensitive and, thus, tightly regulated by the GSH/GSSG equilibrium. This highlights glutathione as a key regulatory component for determining genetic responsiveness to oxidant/antioxidant imbalance in normal lung development and pathophysiological conditions.

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Waypa GB, Schumacker PT.

Department of Medicine MC6026, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA.

The identity of the O(2) sensor underlying the hypoxic pulmonary vasoconstriction (HPV) response has been sought for more than 50 years. Recently, the mitochondria have again come into sharp focus as the cellular organelle responsible for triggering the events that culminate in pulmonary artery constriction. Studies from different laboratories propose two disparate models to explain how mitochondria react to a decrease in P(O(2)). One model proposes that hypoxia slows or inhibits mitochondrial electron transport resulting in the accumulation of reducing equivalents and a decrease in the generation of reactive oxygen species (ROS). This is proposed to activate a redox-sensitive pathway leading to pulmonary vasoconstriction. A second and opposing model suggests that hypoxia triggers a paradoxical increase in mitochondrial ROS generation. This increase would then lead to the activation of an oxidant-sensitive signaling transduction pathway leading to HPV. This article summarizes the potential involvement of mitochondria in these two very different models. Copyright 2002 Elsevier Science B.V.

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PMID: 12126697 [PubMed - indexed for MEDLINE]

 
The oxidative stress hypothesis of congestive heart failure: radical thoughts.

Mak S, Newton GE.

Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada.

There is extensive experimental evidence from in vitro and animal experiments that congestive heart failure (CHF) is a state of oxidative stress. Moreover, in animal models, the development of CHF is accompanied by changes in the antioxidant defense mechanisms of the myocardium as well as evidence of oxidative myocardial injury. This has led to the hypothesis that oxidative stress may be a mechanism of disease progression in CHF. Indeed, many patients consume antioxidant supplements making the assumption that no harm will result and, possibly, that this therapy will yield some clinical benefits. The focus of this review is to examine the oxidative stress hypothesis of CHF as it pertains to humans. To date, human studies that have sought evidence for a role of oxidative stress in patients with CHF have fallen short of providing strong support for this hypothesis. Studies that have demonstrated an association between oxidant stress and CHF are small and are hindered by methodologic limitations that diminish the impact of their conclusions. Randomized trials of antioxidant supplementation for CHF are scarce, and to our knowledge no study yet convincingly demonstrates any benefit from consuming antioxidant supplements. Therefore, the available evidence is insufficient to support or negate the oxidative stress hypothesis of CHF and the use of antioxidants cannot be recommended as a specific therapy for this condition.

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Reactive species mechanisms of cellular hypoxia-reoxygenation injury.

Li C, Jackson RM.

Department of Veterans Affairs Medical Center, Birmingham 35233, USA.

Exacerbation of hypoxic injury after restoration of oxygenation (reoxygenation) is an important mechanism of cellular injury in transplantation and in myocardial, hepatic, intestinal, cerebral, renal, and other ischemic syndromes. Cellular hypoxia and reoxygenation are two essential elements of ischemia-reperfusion injury. Activated neutrophils contribute to vascular reperfusion injury, yet posthypoxic cellular injury occurs in the absence of inflammatory cells through mechanisms involving reactive oxygen (ROS) or nitrogen species (RNS). Xanthine oxidase (XO) produces ROS in some reoxygenated cells, but other intracellular sources of ROS are abundant, and XO is not required for reoxygenation injury. Hypoxic or reoxygenated mitochondria may produce excess superoxide (O) and release H(2)O(2), a diffusible long-lived oxidant that can activate signaling pathways or react vicinally with proteins and lipid membranes. This review focuses on the specific roles of ROS and RNS in the cellular response to hypoxia and subsequent cytolytic injury during reoxygenation.

Publication Types:

• Review
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PMID: 11788333 [PubMed - indexed for MEDLINE]

 
Mitochondria: regulators of signal transduction by reactive oxygen and nitrogen species.

Brookes PS, Levonen AL, Shiva S, Sarti P, Darley-Usmar VM.

Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

The functional role of mitochondria in cell physiology has previously centered around metabolism, with oxidative phosphorylation playing a pivotal role. Recently, however, this perspective has changed significantly with the realization that mitochondria are active participants in signal transduction pathways, not simply the passive recipients of injunctions from the rest of the cell. In this review the emerging role of the mitochondrion in cell signaling is discussed in the context of cytochrome c release, hydrogen peroxide formation from the respiratory chain, and the nitric oxide-cytochrome c oxidase signaling pathway. Copyright 2002 Elsevier Science Inc.

Publication Types:

• Review
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PMID: 12208364 [PubMed - indexed for MEDLINE]

 
Production of nitric oxide by mitochondria.

Giulivi C, Poderoso JJ, Boveris A.

Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033, USA. cgiulivi@hsc.usc.edu

The production of NO. by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Percoll-purified rat liver mitochondria exhibited a negligible contamination with other subcellular fractions (1-4%) and high degree of functionality (respiratory control ratio = 5-6). Toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of nitric oxide synthase (NOS) incubated with the spin trap N-methyl-D-glucamine-dithiocarbamate-FeII produced a signal ascribed to the NO. spin adduct (g = 2.04; aN = 12.5 G). The intensity of the signal increased with time, protein concentration, and L-Arg, and decreased with the addition of the NOS inhibitor NG-monomethyl-L-arginine. Intact mitochondria, mitochondrial homogenates, and submitochondrial particles produced NO. (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO. x (min.mg protein)-1, respectively, with a Km for L-Arg of 5-7 microM. Comparison of the rates of NO. production obtained with homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. This study demonstrates that mitochondria are a source of NO., the production of which may effect energy metabolism, O2 consumption, and O2 free radical formation.

PMID: 9556586 [PubMed - indexed for MEDLINE]

 
Nitric oxide synthase activity in mitochondria.

Ghafourifar P, Richter C.

Laboratory of Biochemistry I, Swiss Federal Institute of Tehchnology, (ETH), Zurich, Switzerland.

In the present study we show the existence of a functional nitric oxide synthase (NOS) in rat liver mitochondria. The enzyme uses L-arginine (L-arg) to produce nitric oxide (NO) and L-citrulline, and is Ca2+-dependent. L-Arg analogues, N(omega)monomethyl-L-arg and N(omega)-nitro-L-arg, inhibit the enzyme, and D-arginine is not a substrate for it. We found mitochondrial NOS (mtNOS) activity associated with the inner mitochondrial membrane but not with the matrix fraction. In intact, succinate-energized mitochondria, the enzyme is constitutively active and exerts substantial control over mitochondrial respiration and membrane potential. The activity is further stimulated when Ca2+ is taken up by mitochondria. We suggest that the existence of mtNOS and its Ca2+ dependence are highly relevant for mitochondrial functioning.

PMID: 9428730 [PubMed - indexed for MEDLINE]

 
Control of oxygen free radical formation from mitochondrial complex I: roles for protein kinase A and pyruvate dehydrogenase kinase.

Raha S, Myint AT, Johnstone L, Robinson BH.

The Hospital for Sick Children, Metabolism Research Programme, Toronto, ON, Canada.

Human NADH CoQ oxidoreductase is composed of a total of 43 subunits and has been demonstrated to be a major site for the production of superoxide by mitochondria. Incubation of rat heart mitochondria with ATP resulted in the phosphorylation of two mitochondrial membrane proteins, one with a M(r) of 6 kDa consistent with the NDUFA1 (MWFE), and one at 18kDa consistent with either NDUFS4 (AQDQ) or NDUFB7 (B18). Phosphorylation of both subunits was enhanced by cAMP derivatives and protein kinase A (PKA) and was inhibited by PKA inhibitors (PKAi). When mitochondrial membranes were incubated with pyruvate dehydrogenase kinase, phosphorylation of an 18kDa protein but not a 6kDa protein was observed. NADH cytochrome c reductase activity was decreased and superoxide production rates with NADH as substrate were increased. On the other hand, with protein kinase A-driven phosphorylation, NADH cytochrome c reductase was increased and superoxide production decreased. Overall there was a 4-fold variation in electron transport rates observable at the extremes of these phosphorylation events. This suggests that electron flow through complex I and the production of oxygen free radicals can be regulated by phosphorylation events. In light of these observations we discuss a potential model for the dual regulation of complex I and the production of oxygen free radicals by both PKA and PDH kinase.

PMID: 11864782 [PubMed - indexed for MEDLINE]

 
Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase.

Raha S, McEachern GE, Myint AT, Robinson BH.

Hospital for Sick Children, Metabolism Research Programme, Toronto, ON, Canada.

In this report we show that ubiquinone cytochrome c reductase (complex III) from isolated rat heart mitochondria when inhibited with antimycin A, produces a large amount of superoxide as measured by the chemiluminescent probe coelenterazine. When mitochondria are inhibited with myxothiazol or stigmatellin, there is no detectable formation of superoxide. The antimycin A-sensitive free radical production can be dramatically reduced using either myxothiazol or stigmatellin. This suggests that the antimycin A-sensitive generation of superoxides originates primarily from the Q(o) semiubiquinone. When manganese superoxide dismutase depleted submitochondrial particles (SMP) were inhibited with myxothiazol or stigmatellin, a large superoxide signal was observed. These two inhibitors likely increase the concentration of the Q(i) semiquinone at the N center. The antimycin A-sensitive signal can, in the case of both the mitochondria and the SMP, be dissipated by the addition of copper zinc superoxide dismutase, suggesting that the measured coelenterazine signal was a result of superoxide production. Taken together, this data suggests that free radicals generated from the Q(i) species are more effectively eliminated by MnSOD in intact mitochondria.

PMID: 10980405 [PubMed - indexed for MEDLINE]

 
Mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase.

Pitkanen S, Robinson BH.

Department of Pediatrics, University of Toronto, Ontario, Canada.

Mitochondria were isolated from skin fibroblast cultures derived from healthy individuals (controls) and from a group patients with complex I (NADH-CoQ reductase) deficiency of the mitochondrial respiratory chain. The complex I deficient patients included those with fatal infantile lactic acidosis (FILA), cardiomyopathy with cataracts (CC), hepatopathy with tubulopathy (HT), Leigh's disease (LD), cataracts and developmental delay (CD), and lactic acidemia in the neonatal period followed by mild symptoms (MS). Production of superoxide radicals, on addition of NADH, were measured using the luminometric probe lucigenin with isolated fibroblast mitochondrial membranes. Superoxide production rates were highest with CD and decreased in the order CD >> MS > LD > control > HT > FILA = CC. The quantity of Mn-superoxide dismutase (MnSOD), as measured by ELISA techniques, however, was highest in CC and FILA and lowest in CD. Plots of MnSOD quantity versus superoxide production showed an inverse relationship for most conditions with complex I deficiency. We hypothesize that oxygen radical production is increased when complex I activity is compromised. However, the observed superoxide production rates are modulated by the variant induction of MnSOD which decreases the rates, sometimes below those seen in control fibroblast mitochondria. In turn, we show that the variant induction of MnSOD is most likely a function of the change in the redox state of the cell experienced rather than a result of the complex I defect per se.

PMID: 8755643 [PubMed - indexed for MEDLINE]

 
Mitochondrial disease in superoxide dismutase 2 mutant mice.

Melov S, Coskun P, Patel M, Tuinstra R, Cottrell B, Jun AS, Zastawny TH, Dizdaroglu M, Goodman SI, Huang TT, Miziorko H, Epstein CJ, Wallace DC.

Center for Molecular Medicine, Emory University, Atlanta, GA 30322, USA.

Oxidative stress has been implicated in many diseases. The chief source of reactive oxygen species within the cell is the mitochondrion. We have characterized a variety of the biochemical and metabolic effects of inactivation of the mouse gene for the mitochondrial superoxide dismutase (CD1-Sod2(tm1Cje)). The Sod2 mutant mice exhibit a tissue-specific inhibition of the respiratory chain enzymes NADH-dehydrogenase (complex I) and succinate dehydrogenase (complex II), inactivation of the tricarboxylic acid cycle enzyme aconitase, development of a urine organic aciduria in conjunction with a partial defect in 3-hydroxy-3-methylglutaryl-CoA lyase, and accumulation of oxidative DNA damage. These results indicate that the increase in mitochondrial reactive oxygen species can result in biochemical aberrations with features reminiscent of mitochondrial myopathy, Friedreich ataxia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

PMID: 9927656 [PubMed - indexed for MEDLINE]

 
Mitochondrial oxidative stress in mice lacking the glutathione peroxidase-1 gene.

Esposito LA, Kokoszka JE, Waymire KG, Cottrell B, MacGregor GR, Wallace DC.

Center for Molecular Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.

Oxidative stress resulting from mitochondrially derived reactive oxygen species (ROS) has been hypothesized to damage mitochondrial oxidative phosphorylation (OXPHOS) and to be a factor in aging and degenerative disease. If this hypothesis is correct, then genetically inactivating potential mitochondrial antioxidant enzymes such as glutathione peroxidase-1 (Gpx1; EC 1.11.1.9) should increase mitochondrial ROS production and decrease OXPHOS function. To determine the expression pattern of Gpx1, isoform-specific antibodies were generated and mutant mice were prepared in which the Gpx1 protein was substituted for by beta-galactosidase, driven by the Gpx1 promoter. These experiments revealed that Gpx1 is highly expressed in both the mitochondria and the cytosol of the liver and kidney, but poorly expressed in heart and muscle. To determine the physiological importance of Gpx1, mice lacking Gpx1 were generated by targeted mutagenesis in mouse ES cells. Homozygous mutant Gpx1(tm1Mgr) mice have 20% less body weight than normal animals and increased levels of lipid peroxides in the liver. Moreover, the liver mitochondria were found to release markedly increased hydrogen peroxide, a Gpx1 substrate, and have decreased mitochondrial respiratory control ratio and power output index. Hence, genetic inactivation of Gpx1 resulted in growth retardation, presumably due in part to reduced mitochondrial energy production as a product of increased oxidative stress.

PMID: 10754271 [PubMed - indexed for MEDLINE]

 
Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis.

Tanaka T, Hosoi F, Yamaguchi-Iwai Y, Nakamura H, Masutani H, Ueda S, Nishiyama A, Takeda S, Wada H, Spyrou G, Yodoi J.

Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.

Thioredoxin-2 (Trx-2) is a mitochondria-specific member of the thioredoxin superfamily. Mitochondria have a crucial role in the signal transduction for apoptosis. To investigate the biological significance of Trx-2, we cloned chicken TRX-2 cDNA and generated clones of the conditional Trx-2-deficient cells using chicken B-cell line, DT40. Here we show that TRX-2 is an essential gene and that Trx-2-deficient cells undergo apoptosis upon repression of the TRX-2 transgene, showing an accumulation of intracellular reactive oxygen species (ROS). Cytochrome c is released from mitochondria, while caspase-9 and caspase-3, but not caspase-8, are activated upon inhibition of the TRX-2 transgene. In addition, Trx-2 and cytochrome c are co-immunoprecipitated in an in vitro assay. These results suggest that mitochondrial Trx-2 is essential for cell viability, playing a crucial role in the scavenging ROS in mitochondria and regulating the mitochondrial apoptosis signaling pathway.

PMID: 11927553 [PubMed - indexed for MEDLINE]

 
Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-alpha.

Kang SW, Chae HZ, Seo MS, Kim K, Baines IC, Rhee SG.

Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.

Mammalian tissues express three immunologically distinct peroxiredoxin (Prx) proteins (Prx I, II, and III), which are the products of distinct genes. With the use of recombinant proteins Prx I, II, and III, all have now been shown to possess peroxidase activity and to rely on Trx as a source of reducing equivalents for the reduction of H2O2. Prx I and II are cytosolic proteins, whereas Prx III is localized in mitochondria. Transient overexpression of Prx I or II in cultured cells showed that they were able to eliminate the intracellular H2O2 generated in response to growth factors. Moreover, the activation of nuclear factor kappaB (NFkappaB) induced by extracellularly added H2O2 or tumor necrosis factor-alpha was blocked by overproduction of Prx II. These results suggest that, together with glutathione peroxidase and catalase, Prx enzymes likely play an important role in eliminating peroxides generated during metabolism. In addition, Prx I and II might participate in the signaling cascades of growth factors and tumor necrosis factor-alpha by regulating the intracellular concentration of H2O2.

PMID: 9497357 [PubMed - indexed for MEDLINE]

 
Persistent oxidative stress in cancer.

Toyokuni S, Okamoto K, Yodoi J, Hiai H.

Department of Pathology, Faculty of Medicine, Kyoto University, Japan.

DNA of cancers such as renal cell carcinoma and mammary invasive ductal carcinoma, is persistently exposed to more oxidative stress than that of adjacent normal tissue. We suggest that the concept of 'persistent oxidative stress in cancer' may open up a new research area, explaining part of the characteristic tumor biology of cancer such as activated transcription factors and proto-oncogenes, genomic instability, chemotherapy-resistance, invasion and metastasis.

Publication Types:

• Review
• Review, Tutorial

PMID: 7821417 [PubMed - indexed for MEDLINE]

 
Induction of MnSOD gene by arachidonic acid is mediated by reactive oxygen species and p38 MAPK signaling pathway in human HepG2 hepatoma cells.

Bianchi A, Becuwe P, Franck P, Dauca M.

Laboratoire de Biologie Cellulaire du Developpement, Universite Henri Poincare-Nancy I, Faculte des Sciences, Vandoeuvre-les-Nancy, France.

Metabolism of arachidonic acid (AA) is known to induce in different cell types an oxidative stress via the production of reactive oxygen species. As these latter may be scavenged by antioxidant enzymes as manganese and copper/zinc-dependent superoxide dismutase (MnSOD and Cu/ZnSOD, respectively), we investigated the effects of AA on their expression in human HepG2 hepatoma cells. RT-PCR and Western blot data revealed that AA induced an increase in the MnSOD, but not Cu/ZnSOD, expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in MnSOD activity. The AA-induced MnSOD expression required de novo transcription as demonstrated by cotreatment of HepG2 cells with AA and actinomycin D. The fact that MnSOD expression was not induced when HepG2 cells were cultured with 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolizable analog of AA, or with different inhibitors of the AA metabolism pathways suggested that the metabolism of AA was required. Further investigations into the mechanisms by which AA induced MnSOD expression showed that superoxide anions released from AA metabolism act as second messengers via a signal-controlling pathway involving protein kinase C and p38 mitogen activated protein kinase (MAPK). These results define a novel role of p38 MAPK dependent-pathway in the regulation of MnSOD gene.

PMID: 12031898 [PubMed - indexed for MEDLINE]

 
Superoxide-induced stimulation of protein kinase C via thiol modification and modulation of zinc content.

Knapp LT, Klann E.

Department of Neuroscience and the Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

We investigated the effects of mild oxidation on protein kinase C (PKC) using the xanthine/xanthine oxidase system of generating superoxide. Exposure of various PKC preparations to superoxide stimulated the autonomous activity of PKC. Similarly, thiol oxidation increased autonomous PKC activity, consistent with the notion that superoxide stimulates PKC via thiol oxidation. The superoxide-induced stimulation of PKC activity was partially reversed by reducing agents, suggesting that disulfide bond formation contributed to the oxidative stimulation of PKC. In addition, superoxide increased the autonomous activity of the alpha, beta(II), epsilon, and zeta PKC isoforms, all of which contain at least one cysteine-rich region. Taken together, our observations suggested that superoxide interacts with PKC at the cysteine-rich region, zinc finger motif of the enzyme. Therefore, we examined the effects of superoxide on this region by testing the hypothesis that superoxide stimulates PKC by promoting the release of zinc from PKC. We found that a zinc chelator stimulated the autonomous activity of PKC and that superoxide induced zinc release from an PKC-enriched enzyme preparation. In addition, oxidized PKC contained significantly less zinc than reduced PKC. Finally, we have isolated a persistent, autonomously active PKC by DEAE-cellulose column chromatography from hippocampal slices incubated with superoxide. Taken together, these data suggest that superoxide stimulates autonomous PKC activity via thiol oxidation and release of zinc from cysteine-rich region of PKC.

PMID: 10823825 [PubMed - indexed for MEDLINE]

 
Redox regulation of cellular signalling.

Kamata H, Hirata H.

Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, Japan. h_kamata@sci.himeji-tech.ac.jp

Extracellular stimuli elicit a variety of responses, such as cell proliferation and differentiation, through the cellular signalling system. Binding of growth factors to the respective receptor leads to the activation of receptor tyrosine kinases, which in turn stimulate downstream signalling systems such as mitogen-activated protein (MAP) kinases, phospholipase Cgamma (PLCgamma) and phosphatidylinositol 3-kinase. These biochemical reactions finally reach the nucleus, resulting in gene expression mediated by the activation of several transcription factors. Recent studies have revealed that cellular signalling pathways are regulated by the intracellular redox state. Generation of reactive oxygen species (ROS), such as H2O2, leads to the activation of protein tyrosine kinases followed by the stimulation of downstream signalling systems including MAP kinase and PLCgamma. The activation of PLCgamma by oxidative radical stress elevates the cellular Ca2+ levels by flux from the intracellular Ca2+ pool and from the extracellular space. Such reactions in the upstream signalling cascade, in concert, result in the activation of several transcription factors. On the other hand, reductants generally suppress the upstream signalling cascade resulting in the suppression of transcription factors. However, it is well known that cysteine residues in a reduced state are essential for the activity of many transcription factors. In fact, in vitro, oxidation of NFkappaB results in its activation, whereas reductants promote its activity. Thus, cellular signalling pathways are generally subjected to dual redox regulation in which redox has opposite effects on upstream signalling systems and downstream transcription factors. Not only are the cellular signalling pathways subjected to redox regulation, but also the signalling systems regulate the cellular redox state. When cells are activated by extracellular stimuli, the cells produce ROS, which in turn stimulate other cellular signalling pathways, indicating that ROS act as second messengers. It is thus evident that there is cross talk between the cellular signalling system and the cellular redox state. Cell death and life also are subjected to such dual redox regulation and cross talk. Death signals induce apoptosis through the activation of caspases in the cells. Oxidative radical stress induces the activation of caspases, whereas the oxidation of caspases results in their inactivation. Furthermore, some cell-death signals induce the production of ROS in the cells, and the ROS produced in turn stimulate the cell-death machinery. All this evidence shows that the cell's fate is determined by cross talk between the cellular signalling pathways and the cellular redox state through a complicated regulation mechanism.

Publication Types:

• Review
• Review, Academic

PMID: 10206339 [PubMed - indexed for MEDLINE]

 
Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing.

Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, Schumacker PT.

Department of Medicine, The University of Chicago, IL 60637, USA.

During hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1alpha protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (rho(0) cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H(2)O(2) stabilizes HIF-1alpha protein during normoxia and activates luciferase expression in wild-type and rho(0) cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1alpha stabilization during hypoxia.

PMID: 10833514 [PubMed - indexed for MEDLINE]

 
A NF-kappaB p65 subunit is indispensable for activating manganese superoxide: dismutase gene transcription mediated by tumor necrosis factor-alpha.

Maehara K, Hasegawa T, Isobe KI.

Department of Basic Gerontology, National Institute for Longevity Sciences, Obu, Aichi, 474-8522 Japan.

Expression of the manganese superoxide dismutase (Mn-SOD) is induced by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and lipopolysaccharide (LPS). Recently, a TNF-responsive element (TNFRE) was identified within the second intron of the murine Mn-SOD gene. The 5' CCAAT/enhancer binding protein (C/EBP)-related region within the TNFRE was responsive to TNF, whereas the 3' NF-kappaB-related region alone was not. This report describes the minimal promoter region of the Mn-SOD gene and investigates the cis-acting elements and trans-acting factors responsible for TNF-alpha-induced Mn-SOD gene expression. Reporter plasmid transfection studies demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn-SOD gene through the intronic enhancer region. Electrophoretic mobility shift assays demonstrated that after TNF-alpha stimulation, p50 and p65 NF-kappaB subunits bound specifically to the newly identified NF-kappaB transcription factor-binding site, distinct from the previously described NF-kappaB site, within the intronic enhancer region. In addition, site-directed mutagenesis and cotransfection studies demonstrated that the NF-kappaB p65 subunit enhanced the transcriptional activity of the Mn-SOD gene through the newly identified NF-kappaB site. These results show that a NF-kappaB p65 subunit is mainly involved in the molecular mechanisms controlling TNF-alpha-mediated Mn-SOD gene transcription. Copyright 2000 Wiley-Liss, Inc.

PMID: 10760955 [PubMed - indexed for MEDLINE]

 
Superoxide anion radical-triggered Ca2+ release from cardiac sarcoplasmic reticulum through ryanodine receptor Ca2+ channel.

Kawakami M, Okabe E.

Department of Pharmacology and ESR Laboratory, Kanagawa Dental College, Yokosuka, Kanagawa 238, Japan.

The ryanodine receptor Ca2+ channel (RyRC) constitutes the Ca2+-release pathway in sarcoplasmic reticulum (SR) of cardiac muscle. A direct mechanical and a Ca2+-triggered mechanism (Ca2+-induced Ca2+ release) have been proposed to explain the in situ activation of Ca2+ release in cardiac muscle. A variety of chemical oxidants have been shown to activate RyRC; however, the role of modification induced by oxygen-derived free radicals in pathological states of the muscle remains to be elucidated. It has been hypothesized that oxygen-derived free radicals initiate Ca2+-mediated functional changes in or damage to cardiac muscle by acting on the SR and promoting an increase in Ca2+ release. We confirmed that superoxide anion radical (O2-) generated from hypoxanthine-xanthine oxidase reaction decreases calmodulin content and increases 45Ca2+ efflux from the heavy fraction of canine cardiac SR vesicles; hypoxanthine-xanthine oxidase also decreases Ca2+ free within the intravesicular space of the SR with no effect on Ca2+-ATPase activity. Current fluctuations through single Ca2+-release channels have been monitored after incorporation into planar phospholipid bilayers. We demonstrate that activation of the channel by O2- is dependent of the presence of calmodulin and identified calmodulin as a functional mediator of O2--triggered Ca2+ release through the RyRC. For the first time, we show that O2- stimulates Ca2+ release from heavy SR vesicles and suggest the importance of accessory proteins such as calmodulin in modulating the effect of O2-. The decreased calmodulin content induced by oxygen-derived free radicals, especially O2-, is a likely mechanism of accumulation of cytosolic Ca2+ (due to increased Ca2+ release from SR) after reperfusion of the ischemic heart.

PMID: 9495817 [PubMed - indexed for MEDLINE]

 
The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions.

Eu JP, Sun J, Xu L, Stamler JS, Meissner G.

Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.

Ion channels have been studied extensively in ambient O2 tension (pO2), whereas tissue PO2 is much lower. The skeletal muscle calcium release channel/ryanodine receptor (RyR1) is one prominent example. Here we report that PO2 dynamically controls the redox state of 6-8 out of 50 thiols in each RyR1 subunit and thereby tunes the response to NO. At physiological pO2, nanomolar NO activates the channel by S-nitrosylating a single cysteine residue. Among sarcoplasmic reticulum proteins, S-nitrosylation is specific to RyR1 and its effect on the channel is calmodulin dependent. Neither activation nor S-nitrosylation of the channel occurs at ambient PO2. The demonstration that channel cysteine residues subserve coupled O2 sensor and NO regulatory functions and that these operate through the prototypic allosteric effector calmodulin may have general implications for the regulation of redox-related systems.

PMID: 10966111 [PubMed - indexed for MEDLINE]