Mechanism of superoxide and hydrogen peroxide generation by human electron-transfer flavoprotein and pathological variants

Reactive oxygen species production by mitochondrial enzymes plays a fundamental role both in cellular signaling and in the progression of dysfunctional states. However, sources of reactive oxygen species and the mechanisms by which enzymes produce these reactive species still remain elusive. We characterized the generation of reactive oxygen species by purified human electron-transfer flavoprotein (ETF), a mitochondrial enzyme that has a central role in the metabolism of lipids, amino acids, and choline. The results showed that ETF produces significant amounts of both superoxide and hydrogen peroxide in the presence of its partner enzyme medium-chain acyl-CoA dehydrogenase (MCAD). ETF-mediated production of reactive oxygen species is partially inhibited at high MCAD/ETF ratios, whereas it is enhanced at high ionic strength. Determination of the reduction potentials of ETF showed that thermodynamic properties of the FAD cofactor are changed upon formation of a complex between ETF and MCAD, supporting the notion that protein:protein interactions modulate the reactivity of the protein with dioxygen. Two pathogenic ETF variants were also studied to determine which factors modulate the reactivity toward molecular oxygen and promote reactive oxygen species production. The results obtained show that destabilized conformations and defective protein:protein interactions increase the ability of ETF to generate reactive oxygen species. A possible role for these processes in mitochondrial dysfunction in metabolic disorders of fatty acid β-oxidation is discussed.     

An apoptotic model for nitrosative stress

Nitric oxide overproduction has been implicated in the pathogenesis of many disorders, including artherosclerosis, neurodegenerative diseases, inflammatory and autoimmune diseases, and cancer. The common view holds that nitric oxide-induced cellular injury is caused by oxidative stress. This theory predicts that interactions between reactive nitrogen species and reactive oxygen species produce powerful oxidants that initiate cell death programs. Cytokine-treated murine macrophages are the prototype of this form of cellular injury. Here we report that generation of reactive nitrogen species upon lipopolysacharide/interferon-gamma stimulation of RAW 264.7 cells is largely divorced from production of reactive oxygen species, and that oxidative stress is not principally responsible for cell death (in this model). Rather, the death program is induced mainly by a nitrosative challenge, characterized by the accrual of nitrosylated proteins without a major alteration in cellular redox state. Moreover, interactions between reactive oxygen and nitrogen species may alter the balance between pathways that yield nitrite and nitrate, without impacting the level of S-nitrosylation or extent of cell death. Our results thus (1) provide new insights into NO-related metabolic pathways, (2) demonstrate that apoptotic injury can be caused by nitrosative mechanisms, and (3) establish a model for nitrosative stress in mammalian cells.     

Mitochondrial damage induced by conditions of oxidative stress

Up to 2% of the oxygen consumed by the mitochondrial respiratory chain undergoes one electron reduction, typically by the semiquinone form of coenzyme Q, to generate the superoxide radical, and subsequently other reactive oxygen species such as hydrogen peroxide and the hydroxyl radical. Under conditions in which mitochondrial generation of reactive oxygen species is increased (such as in the presence of Ca²⁺ ions or when the mitochondrial antioxidant defense mechanisms are compromised), these reactive oxygen species may lead to irreversible damage of mitochondrial DNA, membrane lipids and proteins, resulting in mitochondrial dysfunction and ultimately cell death. The nature of this damage and the cellular conditions in which it occurs are discussed in this review article.     

Invited Commentary:Concepts Related to the Study of Reactive Oxygen and Cardiac Reperfusion Injury

The phenomenon of reperfusion injury remains poorly defined. Questions remain about whether injury occurs in addition to that produced by hypoxia or ischemia. or whether the observed changes simply reflect the unmasking of an underlying injury. Various pathological processes which occur upon the return of oxygen to hypoxic and ischemic heart tissue have been quantitated to assess the extent of reperfusion injury. yet it is not known if they reflect identical or different processes. In addition. the mechanism(s) responsible for these diverse changes may not be the same in the various model systems used to study reperfusion injury. Although reactive oxygen species clearly are formed at reperfusion. conclusive evidence that they are producing injury. particularly during the first seconds. is not available. Several sources of these reactive oxygen species have been proposed but none have been clearly linked with injury in several species or model systems. As research in the field of reperfusion injury continues. it is imperative for scientists to clearly define the system they are using so that studies examining mechanisms of cell lysis at reperfusion are not confused with those assessing the occurrence and mechanisms of damage in addition to that produced by oxygen deprivation.     

Reactive oxygen species: influence on cerebral vascular tone.

Reactive oxygen species have multiple effects on vascular cells. Defining the sources and the impact of the various reactive oxygen species within the vessel wall has emerged as a major area of study in vascular biology. This review will focus on recent findings related to effects of reactive oxygen species on cerebral vascular tone. Effects of superoxide radical, hydrogen peroxide, and the reactive nitrogen species peroxynitrite are summarized. Although higher concentrations may be important for cerebral vascular biology in disease, relatively low concentrations of reactive oxygen species may function as signaling molecules involved with normal regulation of cerebral vascular tone. The mechanisms by which reactive oxygen species affect vascular tone may be quite complex, and our understanding of these processes is increasing. Additionally, the role of reactive oxygen species as mediators of endothelium-dependent relaxation is addressed. Finally, the consequences of the molecular interactions of superoxide with nitric oxide and arachidonic acid are discussed.     

Regulation of the primary photosynthesis processes

The mechanisms of primary processes of photosynthesis and macromolecular conformational changes that control the efficiency of primary energy transformation in photosynthesis are discussed. Special attention is focused on the analysis of chlorophyll fluorescence as an integrated parameter indicative of the efficiency and dynamics of primary steps of photosynthesis. Sharp changes in environmental conditions and other unfavorable factors may lead to the distortions of the coupling between consecutive electron transfer steps. As a result, an excess of electrons and/or electronic excitation energy may form at some sites of the electron transport chain. This may lead to the generation of reactive oxygen species responsible for the subsequent oxidative stress. The results of the application of these data in the areas of biotechnology and ecology are demonstrated.     

Invited Commentary:Concepts Related to the Study of Reactive Oxygen and Cardiac Reperfusion Injury

The phenomenon of reperfusion injury remains poorly defined. Questions remain about whether injury occurs in addition to that produced by hypoxia or ischemia. or whether the observed changes simply reflect the unmasking of an underlying injury. Various pathological processes which occur upon the return of oxygen to hypoxic and ischemic heart tissue have been quantitated to assess the extent of reperfusion injury. yet it is not known if they reflect identical or different processes. In addition. the mechanism(s) responsible for these diverse changes may not be the same in the various model systems used to study reperfusion injury. Although reactive oxygen species clearly are formed at reperfusion. conclusive evidence that they are producing injury. particularly during the first seconds. is not available. Several sources of these reactive oxygen species have been proposed but none have been clearly linked with injury in several species or model systems. As research in the field of reperfusion injury continues. it is imperative for scientists to clearly define the system they are using so that studies examining mechanisms of cell lysis at reperfusion are not confused with those assessing the occurrence and mechanisms of damage in addition to that produced by oxygen deprivation.     

Oxidative stress and oxidant signaling in obstructive sleep apnea and associated cardiovascular diseases

Obstructive sleep apnea (OSA) has emerged as a major public health problem and increasing evidence indicates that untreated OSA can lead to the development of various cardiovascular disorders. One important mechanism by which OSA may promote cardiovascular diseases is intermittent hypoxia, in which patients are subjected to repeated episodes of brief oxygen desaturation in the blood, followed by reoxygenation. Such cycles of hypoxia/reoxygenation may result in the generation of reactive oxygen species. Some studies have demonstrated the presence of oxidative stress in OSA patients as well as in animals subjected to intermittent hypoxia. Further, modulations of nitric oxide and biothiol status might also play important roles in the pathogenesis of OSA-associated diseases. Reactive oxygen species and redox events are also involved in the regulation of signal transduction for oxygen-sensing mechanisms. This review summarizes currently available information on the evidence for and against the occurrence of oxidative stress in OSA and the role of reactive oxygen species in cardiovascular changes associated with OSA.     

Advanced glycation endproducts: from precursors to RAGE: round and round we go

The formation of advanced glycation endproducts (AGEs) occurs in diverse settings such as diabetes, aging, renal failure, inflammation and hypoxia. The chief cellular receptor for AGEs, RAGE, transduces the effects of AGEs via signal transduction, at least in part via processes requiring the RAGE cytoplasmic domain binding partner, diaphanous-1 or mDia1. Data suggest that RAGE perpetuates the inflammatory signals initiated by AGEs via multiple mechanisms. AGE–RAGE interaction stimulates generation of reactive oxygen species and inflammation—mechanisms which enhance AGE formation. Further, recent data in type 1 diabetic kidney reveal that deletion of RAGE prevents methylglyoxal accumulation, at least in part via RAGE-dependent regulation of glyoxalase-1, a major enzyme involved in methylglyoxal detoxification. Taken together, these considerations place RAGE in the center of biochemical and molecular stresses that characterize the complications of diabetes and chronic disease. Stopping RAGE-dependent signaling may hold the key to interrupting cycles of cellular perturbation and tissue damage in these disorders.     

Mitochondrial dynamics in human NADH:ubiquinone oxidoreductase deficiency

Mitochondrial NADH:ubiquinone oxidoreductase or complex I (CI) is a frequently affected enzyme in cases of mitochondrial disorders. However, the cytopathological mechanism of the associated pediatric syndromes is poorly understood. Evidence in the literature suggests a connection between mitochondrial metabolism and morphology. Previous quantitative analysis of mitochondrial structure in cultured fibroblasts of 14 patients revealed that mitochondria were fragmented and/or less branched in patients with severe CI deficiency. These patient cells also displayed greatly increased levels of reactive oxygen species (ROS) and marked aberrations in mitochondrial and cellular Ca²⁺/ATP handling upon hormone stimulation. Here, we discuss the interrelationship between these parameters and demonstrate that the hormone-induced increase in mitochondrial Ca²⁺ and ATP concentration, as well as the rate of cytosolic Ca²⁺ removal, are not related to mitochondrial length and/or degree of branching, but decrease as a function of the number of mitochondria per cell. This suggests that the amount of mitochondria, and not their shape, is important for Ca²⁺-induced stimulation of mitochondrial ATP generation to feed cytosolic ATP-demanding processes.     

Inflammasome Activation by Altered Proteostasis

The association between altered proteostasis and inflammatory disorders has been increasingly recognized, but the underlying mechanisms are not well understood. In this study, we show that deficiency of either autophagy or sequestosome 1 (p62 or SQSTM) led to inflammasome hyperactivation in response to LPS and ATP in primary macrophages and in mice in vivo. Importantly, induction of protein misfolding by puromycin, thapsigargin, or geldanamycin resulted in inflammasome activation that was more pronounced in autophagy- or p62-deficient macrophages. Accumulation of misfolded proteins caused inflammasome activation by inducing generation of nonmitochondrial reactive oxygen species and lysosomal damage, leading to release of cathepsin B. Our results suggest that altered proteostasis results in inflammasome activation and thus provide mechanisms for the association of altered proteostasis with inflammatory disorders.     

The role of free radicals in asbestos-induced diseases.

Asbestos exposure causes pulmonary fibrosis and malignant neoplasms by mechanisms that remain uncertain. In this review, we explore the evidence supporting the hypothesis that free radicals and other reactive oxygen species (ROS) are an important mechanism by which asbestos mediates tissue damage. There appears to be at least two principal mechanisms by which asbestos can induce ROS production; one operates in cell-free systems and the other involves mediation by phagocytic cells. Asbestos and other synthetic mineral fibers can generate free radicals in cell-free systems containing atmospheric oxygen. In particular, the hydroxyl radical often appears to be involved, and the iron content of the fibers has an important role in the generation of this reactive radical. However, asbestos also appears to catalyze electron transfer reactions that do not require iron. Iron chelators either inhibit or augment asbestos-catalyzed generation of the hydroxyl radical and/or pathological changes, depending on the chelator and the nature of the asbestos sample used. The second principal mechanism for asbestos-induced ROS generation involves the activation of phagocytic cells. A variety of mineral fibers have been shown to augment the release of reactive oxygen intermediates from phagocytic cells such as neutrophils and alveolar macrophages. The molecular mechanisms involved are unclear but may involve incomplete phagocytosis with subsequent oxidant release, stimulation of the phospholipase C pathway, and/or IgG-fragment receptor activation. Reactive oxygen species are important mediators of asbestos-induced toxicity to a number of pulmonary cells including alveolar macrophages, epithelial cells, mesothelial cells, and endothelial cells. Reactive oxygen species may contribute to the well-known synergistic effects of asbestos and cigarette smoke on the lung, and the reasons for this synergy are discussed. We conclude that there is strong evidence supporting the premise that reactive oxygen species and/or free radicals contribute to asbestos-induced and cigarette smoke/asbestos-induced lung injury and that strategies aimed at reducing the oxidant stress on pulmonary cells may attenuate the deleterious effects of asbestos.     

Role of reactive oxygen species in intestinal diseases.

It is well known that reactive oxygen metabolites are generated during several pathologies, and that they are able to disturb many cellular processes and eventually lead to cellular injury. After intestinal ischemia, reactive oxygen species are produced when the ischemic tissue is reperfused. The enzyme xanthine oxidase is thought to play a key role in this process. As a result of this oxygen radical production, the permeability of the endothelium and the mucosa increases, allowing infiltration of inflammatory leukocytes into the ischemic area. Moreover, reactive oxygen species are also indirectly involved in leukocyte activation. In turn, these inflammatory cells respond with the production of oxygen radicals, which play an important role in the development of tissue injury. Thus, intestinal ischemia and reperfusion evokes an inflammatory response. Also during chronic intestinal inflammatory diseases, reactive oxygen metabolites are proposed to play an important role in the pathology. Scavenging of reactive oxygen species will thus be beneficial in these disorders.     

Reactive oxygen and ischemia/reperfusion injury of the liver

Pharmacological experiments suggested that reactive oxygen species contribute to ischemia-reperfusion injury of the liver. Since there is no evidence that quantitatively sufficient amounts of reactive oxygen are generated intracellularly to overwhelm the strong antioxidant defense mechanisms in the liver and cause parenchymal cell injury, the role of reactive oxygen in the pathogenesis remains controversial. This paper reviews the data and conclusions obtained with pharmacological intervention studies in vivo, the sources of reactive oxygen in the liver as well as the growing evidence for the importance of liver macrophages (Kupffer cells) and infiltrating neutrophils in the pathogenesis. A comprehensive hypothesis is presented that focuses on the extracellular generation of reactive oxygen in the hepatic sinusoids, where Kupffer cell-derived reactive oxygen species seem to be involved in the initial vascular and parenchymal cell injury and indirectly also in the recruitment of neutrophils into the liver. Reactive oxygen species may also contribute to the subsequent neutrophil-dependent injury phase as one of the toxic mediators released by these inflammatory cells.     

The cold but not hard fats in ectotherms: consequences of lipid restructuring on susceptibility of biological membranes to peroxidation,a review

The production of reactive oxygen species is a regular feature of life in the presence of oxygen. Some reactive oxygen species possess sufficient energy to initiate lipid peroxidation in biological membranes, self-propagating reactions with the potential to damage membranes by altering their physical properties and ultimately their function. Two of the most prominent patterns of lipid restructuring in membranes of ectotherms involve contents of polyunsaturated fatty acids and ratios of the abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine. Since polyunsaturated fatty acids and phosphatidylethanolamine are particularly vulnerable to oxidation, it is likely that higher contents of these lipids at low body temperature elevate the inherent susceptibility of membranes to lipid peroxidation. Although membranes from animals living at low body temperatures may be more prone to oxidation, the generation of reactive oxygen species and lipid peroxidation are sensitive to temperature. These scenarios raise the possibility that membrane susceptibility to lipid peroxidation is conserved at physiological temperatures. Reduced levels of polyunsaturated fatty acids and phosphatidylethanolamine may protect membranes at warm temperatures from deleterious oxidations when rates of reactive oxygen species production and lipid peroxidation are relatively high. At low temperatures, enhanced susceptibility may ensure sufficient lipid peroxidation for cellular processes that require lipid oxidation products.     

Protection Against Ischemic Brain Injury by Inhibition of Mitochondrial Oxidative Stress

Mitochondria are both targets and sources of oxidative stress. This dual relationship is particularly evident in experimental paradigms modeling ischemic brain injury. One mitochondrial metabolic enzyme that is particularly sensitive to oxidative inactivation is pyruvate dehydrogenase. This reaction is extremely important in the adult CNS that relies very heavily on carbohydrate metabolism, as it represents the sole bridge between anaerobic and aerobic metabolism. Oxidative injury to this enzyme and to other metabolic enzymes proximal to the electron transport chain may be responsible for the oxidized shift in cellular redox state that is observed during approximately the first hour of cerebral reperfusion. In addition to impairing cerebral energy metabolism, oxidative stress is a potent activator of apoptosis. The mechanisms responsible for this activation are poorly understood but likely involve the expression of p53 and possibly direct effects of reactive oxygen species on mitochondrial membrane proteins and lipids. Mitochondria also normally generate reactive oxygen species and contribute significantly to the elevated net production of these destructive agents during reperfusion. Approaches to inhibiting pathologic mitochondrial generation of reactive oxygen species include mild uncoupling, pharmacologic inhibition of the membrane permeability transition, and simply lowering the concentration of inspired oxygen. Antideath mitochondrial proteins of the Bcl-2 family also confer cellular resistance to oxidative stress, paradoxically through stimulation of mitochondrial free radical generation and secondary upregulation of antioxidant gene expression.     

Leukotriene B(4) inhibits neutrophil apoptosis via NADPH oxidase activity: redox control of NF-κB pathway and mitochondrial stability

Leukotriene B(4), an arachidonic acid-derived lipid mediator, is a known proinflammatory agent that has a direct effect upon neutrophil physiology, inducing reactive oxygen species generation by the NADPH oxidase complex and impairing neutrophil spontaneous apoptosis, which in turn may corroborate to the onset of chronic inflammation. Despite those facts, a direct link between inhibition of neutrophil spontaneous apoptosis and NADPH oxidase activation by leukotriene B(4) has not been addressed so far. In this study, we aim to elucidate the putative role of NADPH oxidase-derived reactive oxygen species in leukotriene B(4)-induced anti-apoptotic effect. Our results indicate that NADPH oxidase-derived reactive oxygen species are critical to leukotriene B(4) pro-survival effect on neutrophils. This effect also relies on redox modulation of nuclear factor kappaB signaling pathway. We have also observed that LTB(4)-induced Bad degradation and mitochondrial stability require NADPH oxidase activity. All together, our results strongly suggest that LTB(4)-induced anti-apoptotic effect in neutrophils occurs in a reactive oxygen species-dependent manner. We do believe that a better knowledge of the molecular mechanisms underlying neutrophil spontaneous apoptosis may contribute to the development of more successful strategies to control chronic inflammatory conditions such as rheumatoid arthritis.     

The H-ATP synthase: A gate to ROS-mediated cell death or cell survival

Cellular oxidative stress results from the increased generation of reactive oxygen species and/or the dysfunction of the antioxidant systems. Most intracellular reactive oxygen species derive from superoxide radical although the majority of the biological effects of reactive oxygen species are mediated by hydrogen peroxide. In this contribution we overview the major cellular sites of reactive oxygen species production, with special emphasis in the mitochondrial pathways. Reactive oxygen species regulate signaling pathways involved in promoting survival and cell death, proliferation, metabolic regulation, the activation of the antioxidant response, the control of iron metabolism and Ca^2 + signaling. The reversible oxidation of cysteines in transducers of reactive oxygen species is the primary mechanism of regulation of the activity of these proteins. Next, we present the mitochondrial H⁺-ATP synthase as a core hub in energy and cell death regulation, defining both the rate of energy metabolism and the reactive oxygen species-mediated cell death in response to chemotherapy. Two main mechanisms that affect the expression and activity of the H⁺-ATP synthase down-regulate oxidative phosphorylation in prevalent human carcinomas. In this context, we emphasize the prominent role played by the ATPase Inhibitory Factor 1 in human carcinogenesis as an inhibitor of the H⁺-ATP synthase activity and a mediator of cell survival. The ATPase Inhibitory Factor 1 promotes metabolic rewiring to an enhanced aerobic glycolysis and the subsequent production of mitochondrial reactive oxygen species. The generated reactive oxygen species are able to reprogram the nucleus to support tumor development by arresting cell death. Overall, we discuss the cross-talk between reactive oxygen species signaling and mitochondrial function that is crucial in determining the cellular fate. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease

The inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) are a collection of chronic idiopathic inflammatory disorders of the intestine and/or colon. Although the pathophysiology of IBD is not known with certainty, a growing body of experimental and clinical data suggests that chronic gut inflammation may result from a dysregulated immune response to normal bacterial antigens. This uncontrolled immune system activation results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. It is thought that some of the intestinal and/or colonic injury and dysfunction observed in IBD is due to elaboration of these reactive species. This review summarizes the current state-of-knowledge of the role of reactive oxygen species and nitric oxide in the pathophysiology of IBD.     

Reactive oxygen species generation and signaling in plants

The introduction of molecular oxygen into the atmosphere was accompanied by the generation of reactive oxygen species (ROS) as side products of many biochemical reactions. ROS are permanently generated in plastids, peroxisomes, mitochiondria, the cytosol and the apoplast. Imbalance between ROS generation and safe detoxification generates oxidative stress and the accumulating ROS are harmful for the plants. On the other hand, specific ROS function as signaling molecules and activate signal transduction processes in response to various stresses. Here, we summarize the generation of ROS in the different cellular compartments and the signaling processes which are induced by ROS.Keyword: reactive oxygen species, signal transduction, plastids