Generation of reactive oxygen species by the mitochondrial electron transport chain

Generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain (ETC), which is composed of four multiprotein complexes named complex I-IV, is believed to be important in the aging process and in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. Previous studies have identified the ubiquinone of complex III and an unknown component of complex I as the major sites of ROS generation. Here we show that the physiologically relevant ROS generation supported by the complex II substrate succinate occurs at the flavin mononucleotide group (FMN) of complex I through reversed electron transfer, not at the ubiquinone of complex III as commonly believed. Indirect evidence indicates that the unknown ROS-generating site within complex I is also likely to be the FMN group. It is therefore suggested that the major physiologically and pathologically relevant ROS-generating site in mitochondria is limited to the FMN group of complex I. These new insights clarify an elusive target for intervening mitochondrial ROS-related processes or diseases.     

Regulation of acid-base transporters by reactive oxygen species following mitochondrial fragmentation

Mitochondrial morphology is determined by the balance between the opposing processes of fission and fusion, each of which is regulated by a distinct set of proteins. Abnormalities in mitochondrial dynamics have been associated with a variety of diseases, including neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and dominant optic atrophy. Although the genetic determinants of fission and fusion are well recognized, less is known about the mechanism(s) whereby altered morphology contributes to the underlying pathophysiology of these disease states. Previous work from our laboratory identified a role for mitochondrial dynamics in intracellular pH homeostasis in both mammalian cell culture and in the genetic model organism Caenorhabditis elegans. Here we show that the acidification seen in mutant animals that have lost the ability to fuse their mitochondrial inner membrane occurs through a reactive oxygen species (ROS)-dependent mechanism and can be suppressed through the use of pharmacological antioxidants targeted specifically at the mitochondrial matrix. Physiological approaches examining the activity of endogenous mammalian acid-base transport proteins in rat liver Clone 9 cells support the idea that ROS signaling to sodium-proton exchangers contributes to acidification. Because maintaining pH homeostasis is essential for cell function and viability, the results of this work provide new insight into the pathophysiology associated with the loss of inner mitochondrial membrane fusion.     

Regulation of VEGF-induced endothelial cell migration by mitochondrial reactive oxygen species

Endothelial migration is a crucial aspect of a variety of physiologic and pathologic conditions including atherosclerosis and vascular repair. Reactive oxygen species (ROS) function as second messengers during endothelial migration. Multiple intracellular sources of ROS are regulated by cellular context, external stimulus, and the microenvironment. However, the predominant source of ROS during endothelial cell (EC) migration and the mechanisms by which ROS regulate cell migration are incompletely understood. In this study, we tested the hypothesis that mitochondria-derived ROS (mtROS) regulate EC migration. In cultured human umbilical vein endothelial cells, VEGF increased mitochondrial metabolism, promoted mtROS production, and induced cell migration. Either the targeted mitochondrial delivery of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1.     

Functional implications of mitochondrial reactive oxygen species generated by oncogenic viruses

Between 15% and 20% of human cancers are associated with infection by oncogenic viruses. Oncogenic viruses, including HPV, HBV, HCV and HTLV-1, target mitochondria to influence cell proliferation and survival. Oncogenic viral gene products also trigger the production of reactive oxygen species which can elicit oxidative DNA damage and potentiate oncogenic host signaling pathways. Viral oncogenes may also subvert mitochondria quality control mechanisms such as mitophagy and metabolic adaptation pathways to promote virus replication. Here, we will review recent progress on viral regulation of mitophagy and metabolic adaptation and their roles in viral oncogenesis.     

Provinol prevents CsA-induced nephrotoxicity by reducing reactive oxygen species, iNOS, and NF-kB expression.

Cyclosporine A (CsA) use is associated with several side effects, the most important of which is nephrotoxicity that includes, as we previously showed, tubular injury and interstitial fibrosis. Recently, many researchers have been interested in minimizing these effects by pharmacological interventions. To do this, we tested whether the administration of a red wine polyphenol, Provinol (PV), prevents the development of CsA-induced nephrotoxicity. Rats were treated for 21 days and divided into four groups: control; group treated with PV (40 mg/kg/day by oral administration in tap water); group treated with CsA (15 mg/kg/day by subcutaneous injection); group treated with CsA plus PV. CsA produced a significant increase of systolic blood pressure; it did not affect urinary output, but caused a significant decrease in creatinine clearance. These side effects were associated with an increase in conjugated dienes, which are lipid peroxidation products, inducible NO-synthase (iNOS), and nuclear factor (NF)-kB, which are involved in antioxidant damage. However, PV prevented these negative effects through a protective mechanism that involved reduction of both oxidative stress and increased iNOS and NF-kB expression induced by CsA. These results provide a pharmacological basis for the beneficial effects of plant-derived polyphenols against CsA-induced renal damage associated with CsA.     

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H₂O₂ release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (α-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1–0.2% of O₂ consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release.     

Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation

Phosphatidic acid (PA) level increases during various stress conditions. However, the physiological roles of this lipid in stress response remain largely unknown. In this study, we report that PA induced leaf cell death and elevated the levels of reactive oxygen species (ROS) in the whole leaf and single cells. To further elucidate the mechanism of PA-induced cell death, we then examined whether Rho-related small G protein (ROP) 2, which enhanced ROS production in an in vitro assay, is involved in PA-induced ROS production and cell death. In response to PA, transgenic leaves of Arabidopsis expressing a constitutively active rop2 mutant exhibited earlier cell death and higher levels of ROS than wild type (WT), whereas those expressing a dominant-negative rop2 mutant exhibited later cell death and lower ROS. However, in the absence of exogenous PA, no spontaneous cell death or elevated ROS was observed in constitutively active rop2 plants, suggesting that the activation of ROP GTPase alone is insufficient to activate the ROP-mediated ROS generation pathway. These results suggest that PA modulates an additional factor required for the active ROP-mediated ROS generation pathway. Therefore, PA may be an important regulator of ROP-regulated ROS generation and the cell death process during various stress and defense responses of plants.     

Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846-39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H(2)O(2) production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H(2)O(2) generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H(2)O(2) production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both DeltaPsi(m) and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.     

Uncoupling proteins and the control of mitochondrial reactive oxygen species production

Reactive oxygen species (ROS), natural by-products of aerobic respiration, are important cell signaling molecules, which left unchecked can severely impair cellular functions and induce cell death. Hence, cells have developed a series of systems to keep ROS in the nontoxic range. Uncoupling proteins (UCPs) 1-3 are mitochondrial anion carrier proteins that are purported to play important roles in minimizing ROS emission from the electron transport chain. The function of UCP1 in this regard is highly contentious. However, UCPs 2 and 3 are generally thought to be activated by ROS or ROS by-products to induce proton leak, thus providing a negative feedback loop for mitochondrial ROS production. In our laboratory, we have not only confirmed that ROS activate UCP2 and UCP3, but also demonstrated that UCP2 and UCP3 are controlled by covalent modification by glutathione. Furthermore, the reversible glutathionylation is required to activate/inhibit UCP2 and UCP3, but not UCP1. Hence, our findings are consistent with the notion that UCPs 2 and 3 are acutely activated by ROS, which then directly modulate the glutathionylation status of the UCP to decrease ROS emission and participate in cell signaling mechanisms.     

MAO-A-induced mitogenic signaling is mediated by reactive oxygen species, MMP-2, and the sphingolipid pathway

The degradation of biogenic amines by monoamine oxidase A (MAO-A) generates reactive oxygen species (ROS) which participate in serotonin and tyramine signaling. This study aimed to investigate the role of ROS in the mitogenic signaling activated during tyramine and serotonin oxidation by MAO-A in smooth muscle cells (SMC). Incubation of SMC with serotonin or tyramine induced intracellular ROS generation, and a signaling cascade involving metalloproteases and the neutral sphingomyelinase-2 (nSMase2, the initial step of the sphingolipid pathway), ERK1/2 phosphorylation, and DNA synthesis. Silencing MAO-A by siRNA, pharmacological MAO-A inhibitors (pargyline and Ro41-1049), and the antioxidant/ROS scavenger butylated hydroxytoluene (BHT) inhibited the signaling cascade, suggesting that ROS generated during tyramine oxidation by MAO-A are required. The MMP inhibitor Batimastat, MMP2-specific siRNA, and MMP2 deletion (MMP2(-/-) fibroblasts) blocked nSMase activation and SMC proliferation, suggesting a role for MMP2 in this signaling pathway. Silencing nSMase2 by siRNA did not inhibit ROS generation and MMP2 activation, but blocked SMC proliferation induced by tyramine, suggesting that nSMase2 is downstream MMP2. These findings demonstrate that H(2)O(2)-generated during tyramine oxidation by MAO-A triggers a stress-induced mitogenic signaling via the MMP2/sphingolipid pathway, which could participate in excessive remodeling and alteration of the vascular wall.     

Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway

In previous research, a thiamine-auxotrophic yeast for alpha-ketoglutaric acid (KGA) overproduction was screened in our laboratory and named Yarrowia lipolytica WSH-Z06 (CCTCC no. M207143). However, the high concentration of by-products (mainly pyruvate) limited its application on an industrial scale. To enhance KGA production and reduce pyruvate (PA) accumulation, the pyruvate carboxylation pathway was regulated. By overexpressing the pyruvate carboxylase genes ScPYC1 from Saccharomyces cerevisiae and RoPYC2 from Rhizopus oryzae in Y. lipolytica WSH-Z06, the yields of KGA in Y. lipolytica-ScPYC1 and Y. lipolytica-RoPYC2 increased by 24.5 and 35.3?%, and the yields of PA decreased by 51.9 and 69.8?% in shake flasks, respectively. These changes in the expression levels and activities of key intracellular enzymes showed that enhancing the pyruvate carboxylation pathway had successfully redistributed the carbon flux from PA to KGA. Finally, by controlling the pH in a 3-L fermenter, the maximum concentration of KGA in Y. lipolytica-RoPYC2 reached 62.5?g?L^?1 with an evident decrease in PA yield from 35.2 to 13.5?g?L^?1.     

Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies.

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen (1O2), as shown by the inhibitory effect of sodium azide, a specific 1O2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein-stimulated photoprocess. In addition to 1O2, hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers. The calcein-photosensitized alterations of mitochondria as a potential source of artifacts in confocal microscopy studies of cells were considered. Irradiation of isolated mitochondria with visible light (500-600 nm) in the presence of calcein induced opening of the permeability transition (PT) pore. The extent of the mitochondrial membrane photodamage, however, was modulated by the nature of the calcein environment. Thus, pore opening was triggered at short irradiation times and low dye concentrations when calcein was dissolved in the bulk medium. On the contrary, calcein concentrated in the matrix space was rather inefficient as photosensitizer even at concentrations 10 times higher than those present in the external medium.     

Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

Background  

Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells.     

Mitochondria: omega-3 in the route of mitochondrial reactive oxygen species

Mitochondria are the main organelles that produce reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage to macromolecules, including lipids, and can damage cellular membrane structure and functions. Mitochondria, the main target of ROS-induced damage, are equipped with a network of antioxidants that control ROS production. Dietary intake of omega-3 polyunsaturated fatty acids (ω3PUFAs) and consequently the increase in ω3PUFA content of membrane lipids may be disadvantageous to the health because ROS-induced oxidative peroxidation of ω3PUFAs within membrane phospholipids can lead to the formation of toxic products. Mitochondrial control of lipid peroxidation is one of the mechanisms that protect cell against oxidative damage. This review discusses the role of mitochondria in ROS generation and the mechanisms by which it regulates ROS production. The susceptibility to peroxidation of PUFAs by ROS raises the question of the adverse effects of ω3PUFA dietary supplementation on embryonic development and prenatal developmental outcomes.     

Bradykinin enhances reactive oxygen species generation, mitochondrial injury, and cell death induced by ATP depletion--a role of the phospholipase C-Ca(2+) pathway

This study aimed to study the effect of bradykinin on reactive oxygen species (ROS) generation, mitochondrial injury, and cell death induced by ATP depletion in cell culture. Renal tubular cells were subjected to ATP depletion. Cell death was evaluated with LDH release, sub-G0/G1 fraction, Hoechst staining, and annexin V binding assay. ROS generation, mitochondrial membrane potential (DeltaPsi(m)), and intramitochondrial calcium were evaluated with flow cytometry. Translocation of cytochrome c and activation of apoptotic protein were analyzed with cell fractionating and Western blotting. Intracellular calcium was measured with a spectrofluorometer. Bradykinin enhanced cellular LDH release, apoptosis, generation of superoxide, and hydrogen peroxide induced by ATP depletion. Bradykinin also enhanced the loss of DeltaPsi(m), translocation of cytochrome c into cytosol, and activation of apoptotic protein. The intracellular/mitochondrial calcium was higher in bradykinin-treated cells. All these effects were reversed by coadministration with bradykinin B2 receptor (B2R) antagonist. Besides, blocking the phospholipase C (PLC) could reverse the synergistic effect of bradykinin with ATP depletion on ROS generation, mitochondrial damage, accumulation of intracellular/mitochondrial calcium, and apoptosis. Activation of B2R aggravates ROS generation, mitochondrial damage, and cell death induced by ATP depletion. These effects may act through the PLC-Ca(2+) signaling pathway.     

Reactive oxygen species and the mitochondrial signaling pathway of cell death

Reactive oxygen species (ROS) are produced as a by-product of cellular metabolic pathways and function as a critical second messenger in a variety of intracellular signaling pathways. Thus, a defect or deficiency in the anti-oxidant defense system on the one hand and/or the excessive intracellular generation of ROS on the other renders a cell oxidatively stressed. As a consequence, direct or indirect involvement of ROS in numerous diseases has been documented. In most of these cases, the deleterious effect of ROS is a function of activation of intracellular cell-death circuitry. To that end, involvement of ROS at different phases of the apoptotic pathway, such as induction of mitochondrial permeability transition and release of mitochondrial death amplification factors, activation of intracellular caspases and DNA damage, has been clearly established. For instance, the ROS-induced alteration of constitutive mitochondrial proteins, such as the voltage-dependent anion channel (VDAC) and/or the adenine nucleotide translocase (ANT) can induce the pro-apoptotic mitochondrial membrane permabilization. Not only do these observations provide insight into the intricate mechanisms underlying a variety of disease states, but they also present novel opportunities for the design and development of more effective therapeutic strategies.     

Modulation of mitochondrial membrane potential and reactive oxygen species production by copper in astrocytes

In monolayers of cultured rat astrocytes a number of agents that induce oxidative stress act synergistically with exposure to copper leading to rapid depolarization of the mitochondrial membrane potential (Psi m) and increased reactive oxygen species (ROS) production. Copper sensitized astrocytes to the action of menadione, an intracellular generator of superoxide anion radical, exogenous hydrogen peroxide (H2O2) and rotenone, an inhibitor of mitochondrial electron transport chain complex I. However, significant differences were observed in the ability to modulate the copper-enhanced oxidative stress depending on which stressor was used. The inhibitor of mitochondrial permeability transition cyclosporin A attenuated the effect of copper and rotenone, but had no protective action in the case of H2O2/copper and menadione/copper combinations. The H2O2 scavenger pyruvate was effective at protecting mitochondria against damage associated with the combined exposure to H2O2/copper and menadione/copper but not to the rotenone/copper combination. The antioxidant Trolox was ineffective at protecting against any of these actions and indeed had a damaging effect when combined with copper. The membrane-permeable copper chelator neocuproine combined with sensitizing concentrations of menadione caused a decrease in Psi m, mimicking the action of copper. Penicillamine, a membrane-impermeable copper chelator, was effective at reducing copper sensitization. Endogenous copper, mobilized during periods of oxidative stress, may play a role in the pathophysiology of brain injury. Our results suggest that this might be particularly dangerous in dysfunctional conditions in which the mitochondrial electron transport chain is compromised.