Mitochondrial ROS metabolism: 10 Years later

The role of mitochondria in oxidative stress is well recognized, but many questions are still to be answered. This article is intended to update our comprehensive review in 2005 by highlighting the progress in understanding of mitochondrial reactive oxygen species (ROS) metabolism over the past 10 years. We review the recently identified or re-appraised sources of ROS generation in mitochondria, such as p66^shc protein, succinate dehydrogenase, and recently discovered properties of the mitochondrial antioxidant system. We also reflect upon some controversies, disputes, and misconceptions that confound the field.     

Reduced ROS-mediated antibiotic resistance and its reverting by glucose in Vibrio alginolyticus

Antibiotic-resistant Vibrio alginolyticus poses a big challenge to human health and food safety. It is urgently needed to understand the mechanisms underlying antibiotic resistance to develop effective approaches for the control. Here we explored the metabolic difference between gentamicin-resistant V. alginolyticus (VA-R_GEN) and gentamicin-sensitive V. alginolyticus (VA-S), and found that the reactive oxygen species (ROS) generation was altered. Compared with VA-S, the ROS content in VA-R_GEN was reduced due to the decreased generation and increased breakdown of ROS. The decreased production of ROS was attributed to the decreased central carbon metabolism, which is associated with the resistance to gentamicin. As such a mechanism, we exogenously administrated VA-R_GEN with the glucose that activated the central carbon metabolism and promoted the generation of ROS, but decreased the breakdown of ROS in VA-R_GEN. The gentamicin-mediated killing was increased with the elevation of the ROS level by a synergistic effect between gentamicin and exogenous glucose. The synergistic effect was inhibited by thiourea, a scavenger of ROS. These results reveal a reduced ROS-mediated antibiotic resistance mechanism and its reversal by exogenous glucose.     

Glutathione and thioredoxin dependent systems in neurodegenerative disease: What can be learned from reverse genetics in mice

Oxidative stress is a major common hallmark of many neurodegenerative disease such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and stroke. Novel concepts in our understanding of oxidative stress indicate that a perturbed redox circuitry could be strongly linked with the onset of such diseases. In this respect, glutathione and thioredoxin dependent antioxidant enzymes play a central role as key regulators due to the fact that a slight dysfunction of any of these enzymes leads to sustained reactive oxygen species (ROS) production. Apart from their classical role as ROS scavengers, some of these enzymes are also able to control post-translational modifications. Therefore, efficient control of ROS production and reversibility of post-translational modifications are critical as improper control of such events may lead to the activation of pathological redox circuits that eventually culminate in neuronal cell death. To dissect the apparently opposing functions of ROS in cell physiology and pathophysiology, a proper working toolkit is mandatory. In vivo modeling is an absolute requirement due to the complexity of redox signaling systems that often contradict data obtained from in vitro approaches. Hence, inducible/conditional knockout mouse models for key redox enzymes are emerging as powerful tools to perturb redox circuitries in a temporal and spatial manner. In this review we address the basics of ROS generation, chemistry and detoxification as well as examples in where applications of mouse models of important enzymes have been successfully applied in the study of neurodegenerative processes. We also highlight the importance of new models to overcome present technical limitations in order to advance in the study of redox processes in the role of neurodegeneration.     

Cytosolic phospholipase A(2), lipoxygenase metabolites, and reactive oxygen species

Reactive oxygen species (ROS) are generated in mammalian cells via both enzymatic and non-enzymatic mechanisms. Although certain ROS production pathways are required for the performance of specific physiological functions, excessive ROS generation is harmful, and has been implicated in the pathogenesis of a number of diseases. Among the ROS-producing enzymes, NADPH oxidase is widely distributed among mammalian cells, and is a crucial source of ROS for physiological and pathological processes. Reactive oxygen species are also generated by arachidonic acid (AA) metabolites, which are released from membrane phospholipids via the activity of cytosolic phospholipase A(2) (cPLA(2)). In this study, we describe recent studies concerning the generation of ROS by AA metabolites. In particular, we have focused on the manner in which AA metabolism via lipoxygenase (LOX) and LOX metabolites contributes to ROS generation. By elucidating the signaling mechanisms that link LOX and LOX metabolites to ROS, we hope to shed light on the variety of physiological and pathological mechanisms associated with LOX metabolism.     

A comparison of arteries and veins in oxidative stress: producers, destroyers, function, and disease

Reactive oxygen species (ROS) are by-products of oxygen metabolism, normally present in low levels inside cells, where they participate in signaling processes. The delicate balance in the continuous cycle of ROS generation and inactivation is maintained by enzymatic and nonenzymatic endogenous systems. Overwhelming production of ROS (by such sources as the mitochondrial electron transport chain, NADPH oxidase, xanthine oxidase, or uncoupled nitric oxide synthase), when inadequately counteracted by destruction through antioxidant systems (such as superoxide dismutase or catalase), leads to a prooxidant state also known as oxidative stress. Increased levels of ROS and markers of oxidative stress have been consistently found in such cardiovascular diseases as atherosclerosis or hypertension, although controversy still exists over the pathophysiological role of oxidative stress in these conditions. ROS can modulate vascular function either by direct oxidative damage or by activating cellular signaling pathways that lead to abnormal contractile, inflammatory, proliferative, or remodeling properties of the blood vessel. Most current research focuses on these processes in arteries, leaving veins, "the other side" of vascular biology, in obscurity. Veins are different structurally and functionally from arteries. Equipped with a smaller smooth muscle layer compared to arteries, but being able to accommodate 70% of the circulating blood volume, veins can modulate cardiovascular homeostasis and contribute significantly to hypertension pathogenesis. Although the reports on the quantitative differences in ROS production in veins compared to arteries had conflicting results, there is a clear qualitative difference in ROS metabolism and utilization between the two vessel types. This review will compare and contrast the current knowledge of ROS metabolism in arteries versus veins in both physiological and pathophysiological conditions. Our understanding of the mechanisms underlying vascular diseases would greatly benefit from a more thorough exploration of the role of veins and venous oxidative stress.     

细菌产生的活性氧及其功能

活性氧(Reactive Oxygen Species,ROS)是需氧生物有氧代谢和专一酶类产生的含氧的、化学活性极强的一类小分子物质。按照其产生机理可分为两大产生途径,其一是呼吸作用中发生的单电子转移产生的ROS,通常认为此途径产生过量的ROS对生物大分子具有极强的氧化损伤,与多种疾病密切相关;其二是由专一酶类产生的少量ROS,一般认为此途径产生的ROS具有杀灭入侵的外来微生物的作用,但近年来大量研究表明,此途径产生的ROS可行使信号分子和基因开关等多种生理功能。同时,生物体自身的抗氧化系统也可直接调控ROS的水平。本文综合分析近年来对细菌中的ROS的研究成果,并对目前存在的问题和未来的发展进行评述。     

Mitochondrial complex III ROS regulate adipocyte differentiation

Adipocyte differentiation is characterized by an increase in mitochondrial metabolism. However, it is not known whether the increase in mitochondrial metabolism is essential for differentiation or a byproduct of the differentiation process. Here, we report that primary human mesenchymal stem cells undergoing differentiation into adipocytes display an early increase in mitochondrial metabolism, biogenesis, and reactive oxygen species (ROS) generation. This early increase in mitochondrial metabolism and ROS generation was dependent on mTORC1 signaling. Mitochondrial-targeted antioxidants inhibited adipocyte differentiation, which was rescued by the addition of exogenous hydrogen peroxide. Genetic manipulation of mitochondrial complex III revealed that ROS generated from this complex is required to initiate adipocyte differentiation. These results indicate that mitochondrial metabolism and ROS generation are not simply a consequence of differentiation but are a causal factor in promoting adipocyte differentiation.     

Non-esterified polyunsaturated fatty acids distinctly modulate the mitochondrial and cellular ROS production in normoxia and hypoxia

There is an intense discussion about the subcellular origin of the generation of reactive oxygen species (ROS) under hypoxia. Since this fundamental question can be addressed only in a cellular system, the O(2) -sensing rat pheochromocytoma (PC12) cells were used. Severe hypoxia is known to elevate non-esterified fatty acids. Therefore, the site(s) of ROS generation were studied in cells which we simultaneously exposed to hypoxia (1% oxygen) and free fatty acids (FFA). We obtained the following results: (i) at hypoxia, ROS generation increases in PC12 cells but not in mitochondria isolated therefrom. (ii) Non-esterified polyunsaturated fatty acids (PUFA) enhance the ROS release from PC12 cells as well as from mitochondria, both in normoxia and in hypoxia. (iii) PUFA-induced ROS generation by PC12 cells is not decreased either by inhibitors of the cell membrane NAD(P)H oxidase or inhibitors impairing the PUFA metabolism. (iv) PUFA-induced ROS generation of mitochondria is paralleled by a decline of the NADH-cytochrome c reductase activity (reflecting combined enzymatic activity of complex I plus III). (v) Mitochondrial superoxide indicator (MitoSOXred)-loaded cells exposed to PUFA exhibit increased fluorescence indicating mitochondrial ROS generation. In conclusion, elevated PUFA levels enhance cellular ROS level in hypoxia, most likely by impairing the electron flux within the respiratory chain. Thus, we propose that PUFAs are likely to act as important extrinsic factor to enhance the mitochondria-associated intracellular ROS signaling in hypoxia.     

Role of mitochondria in reactive oxygen species generation and removal; relevance to signaling and programmed cell death

Reactive oxygen species (ROS) are universal products of aerobic metabolism, which can be also produced in stress conditions. In eukaryotic cells, mitochondria are the main source of ROS. The main mitochondrial sites of ROS formation are electron carriers of respiratory chain. However, there are also other enzymatic sites capable of ROS generation in different mitochondrial compartments. Reactive oxygen species can cause serious damage to many biological macromolecules, such as proteins, lipids and nucleic acids, which oxidation leads to a lost of their biological properties and eventually to a cell death. Mitochondria, which are also exposed to harmful ROS action, have a defense system that decreases ROS production (first line of defense) or removes generated ROS (second line of defense). Mitochondrial antioxidant system involves proteins that decrease ROS formation, enzymes that directly react with ROS, and non-enzymatic antioxidants that also remove ROS and other oxygen derivatives. Mitochondrial ROS can also act as signal messengers and modify operation of many routes in different cell compartments. Mitochondrial ROS are also important in execution of programmed cell death.     

Mitochondrial reactive oxygen species-mediated signaling in endothelial cells

Once thought of as toxic by-products of cellular metabolism, reactive oxygen species (ROS) have been implicated in a large variety of cell-signaling processes. Several enzymatic systems contribute to ROS production in vascular endothelial cells, including NA(D)PH oxidase, xanthine oxidase, uncoupled endothelial nitric oxide synthase, and the mitochondrial electron transport chain. The respiratory chain is the major source of ROS in most mammalian cells, but the role of mitochondria-derived ROS in vascular cell signaling has received little attention. A new paradigm has evolved in recent years postulating that, in addition to producing ATP, mitochondria also play a key role in cell signaling and regulate a variety of cellular functions. This review focuses on the emerging role of mitochondrial ROS as signaling molecules in vascular endothelial cells. Specifically, we discuss some recent findings that indicate that mitochondrial ROS regulate vascular endothelial function, focusing on major sites of ROS production in endothelial mitochondria, factors modulating mitochondrial ROS production, the physiological and clinical implications of endothelial mitochondrial ROS, and methodological considerations in the study of mitochondrial contribution to vascular ROS generation.     

Reactive oxygen species in cell signaling

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of "oxidative stress" is thought to contribute to the pathogenesis of a number of human diseases including those of the lung. Recent studies have also implicated ROS that are generated by specialized plasma membrane oxidases in normal physiological signaling by growth factors and cytokines. In this review, we examine the evidence for ligand-induced generation of ROS, its cellular sources, and the signaling pathways that are activated. Emerging concepts on the mechanisms of signal transduction by ROS that involve alterations in cellular redox state and oxidative modifications of proteins are also discussed.     

Endoplasmic reticulum stress and Oxidative stress in the pathogenesis of Non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. The underlying causes of the disease progression in NAFLD are unclear. Recent evidences suggest endoplasmic reticulum stress in the development of lipid droplets (steatosis) and subsequent generation of reactive oxygen species (ROS) in the progression to non-alcoholic steatohepatitis (NASH). The signalling pathway activated by disruption of endoplasmic reticulum (ER) homoeostasis, called as unfolded protein response, is linked with membrane biosynthesis, insulin action, inflammation and apoptosis. ROS are important mediators of inflammation. Protein folding in ER is linked to ROS. Therefore understanding the basic mechanisms that lead to ER stress and ROS in NAFLD have become the topics of immense interest. The present review focuses on the role of ER stress and ROS in the pathogenesis of NAFLD. We also highlight the cross talk between ER stress and oxidative stress which suggest and encourage the development of therapeutics for NAFLD. Further we have reviewed various strategies used for the management of NAFLD/NASH and limitations of such strategies. Our review therefore highlights the need for newer strategies with regards to ER stress and oxidative stress.     

Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha, although the events through which TNF-alpha induces ROS generation are not yet well characterized. We therefore investigated selected candidates likely to mediate TNF-alpha-induced ROS generation. Consistent with the role of Rac in that process, stable expression of Rac(Asn-17), a dominant negative Rac1 mutant, completely blocked TNF-alpha-induced ROS generation. To understand better the mediators downstream of Rac, we investigated the involvement of cytosolic phospholipase A(2) (cPLA(2)) activation and metabolism of the resultant arachidonic acid (AA) by 5-lipoxygenase (5-LO). TNF-alpha-induced ROS generation was blocked by inhibition of cPLA(2) or 5-LO, but not cyclooxygenase, suggesting that TNF-alpha-induced ROS generation is dependent on synthesis of AA and its subsequent metabolism to leukotrienes. Consistent with that hypothesis, TNF-alpha Rac-dependently stimulated endogenous production of leukotriene B(4) (LTB(4)), while exogenous application of LTB(4) increased levels of ROS. In contrast, application of leukotrienes C(4), D(4), and E(4) or prostaglandin E(2) had little effect. Our findings suggest that LTB(4) production by 5-LO is situated downstream of the Rac-cPLA(2) cascade, and we conclude that Rac, cPLA(2), and LTB(4) play pivotal roles in the ROS-generating cascade triggered by TNF-alpha.     

Metal contaminants promote degradation of lipid/DNA complexes during lyophilization

Oxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe(2+)) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage.     

A Novel Endogenous Indole Protects Rodent Mitochondria and Extends Rotifer Lifespan

Aging is a multi-factorial process, however, it is generally accepted that reactive oxygen species (ROS) are significant contributors. Mitochondria are important players in the aging process because they produce most of the cellular ROS. Despite the strength of the free-radical hypothesis, the use of free radical scavengers to delay aging has generated mixed results in vertebrate models, and clinical evidence of efficacy is lacking. This is in part due to the production of pro-oxidant metabolites by many antioxidants while scavenging ROS, which counteract their potentially beneficial effects. As such, a more effective approach is to enhance mitochondrial metabolism by reducing electron leakage with attendant reduction of ROS generation. Here, we report on the actions of a novel endogenous indole derivative, indolepropionamide (IPAM), which is similar in structure to melatonin. Our results suggest that IPAM binds to the rate-limiting component of oxidative phosphorylation in complex I of the respiratory chain and acts as a stabilizer of energy metabolism, thereby reducing ROS production. IPAM reversed the age-dependent decline of mitochondrial energetic capacity and increased rotifer lifespan, and it may, in fact, constitute a novel endogenous anti-aging substance of physiological importance.     

Suofu Qin's work on studies of cell survival signaling in cancer and epithelial cells

Reactive oxygen species (ROS) encompass a variety of diverse chemical species including superoxide anions, hydrogen peroxide, hydroxyl radicals and peroxynitrite, which are mainly produced via mitochondrial oxidative metabolism, enzymatic reactions, and light-initiated lipid peroxidation. Over-production of ROS and/or decrease in the antioxidant capacity cause cells to undergo oxidative stress that damages cellular macromolecules such as proteins, lipids, and DNA. Oxidative stress is associated with ageing and the development of age-related diseases such as cancer and age-related macular degeneration. ROS activate signaling pathways that promote cell survival or lead to cell death, depending on the source and site of ROS production, the specific ROS generated, the concentration and kinetics of ROS generation, and the cell types being challenged. However, how the nature and compartmentalization of ROS contribute to the pathogenesis of individual diseases is poorly understood. Consequently, it is crucial to gain a comprehensive understanding of the molecular bases of cell oxidative stress signaling, which will then provide novel therapeutic opportunities to interfere with disease progression via targeting specific signaling pathways. Currently, Dr. Qin's work is focused on inflammatory and oxidative stress responses using the retinal pigment epithelial (RPE) cells as a model. The study of RPE cell inflammatory and oxidative stress responses has successfully led to a better understanding of RPE cell biology and identification of potential therapeutic targets.