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.     

A novel protein, Romo1, induces ROS production in the mitochondria

The majority of endogenous reactive oxygen species (ROS) are produced in the mitochondrial respiratory chain. An imbalance in ROS production alters the intracellular redox homeostasis, triggers DNA damage, and contributes to cancer development and progression. This study identified a novel protein, reactive oxygen species modulator 1 (Romo1), which is localized in the mitochondria. Romo1 was found to increase the level of ROS in the cells. Increased Romo1 expression was observed in various cancer cell lines. This suggests that the increased Romo1 expression during cancer progression may cause persistent oxidative stress to tumor cells, which can increase their malignancy.     

Mitochondrial metabolism of reactive oxygen species

For a long time mitochondria have mainly been considered for their role in the aerobic energy production in eukaryotic cells, being the sites of the oxidative phosphorylation, which couples the electron transfer from respiratory substrates to oxygen with the ATP synthesis. Subsequently, it was showed that electron transfer along mitochondrial respiratory chain also leads to the formation of radicals and other reactive oxygen species, commonly indicated as ROS. The finding that such species are able to damage cellular components, suggested mitochondrial involvement in degenerative processes underlying several diseases and aging.More recently, a new role for mitochondria, as a system able to supply protection against cellular oxidative damage, is emerging. Experimental evidence indicates that the systems, evolved to protect mitochondria against endogenously produced ROS, can also scavenge ROS produced by other cellular sources. It is possible that this action, particularly relevant in physio-pathological conditions leading to increased cellular ROS production, is more effective in tissues provided with abundant mitochondrial population. Moreover, the mitochondrial dysfunction, resulting from ROS-induced inactivation of important mitochondrial components, can be attenuated by the cell purification from old ROS-overproducing mitochondria, which are characterized by high susceptibility to oxidative damage. Such an elimination is likely due to two sequential processes, named mitoptosis and mitophagy, which are usually believed to be induced by enhanced mitochondrial ROS generation. However, they could also be elicited by great amounts of ROS produced by other cellular sources and diffusing into mitochondria, leading to the elimination of the old dysfunctional mitochondrial subpopulation.     

Mitochondria,oxidative stress and cell death

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of “free” cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.     

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

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

Role of mitochondrial thiols of different localization in the generation of reactive oxygen species

The role of thiols of the outer and the inner membranes of mitochondria in the regulation of generation of reactive oxygen species (ROS) has been studied. It was found that N-ethylmaleimide (NEM), which penetrates through the mitochondrial membrane and binds thiols to form thioesters, at concentrations from 20 to 250 μM activates the production of superoxide anion and hydrogen peroxide during the oxidation of the substrates of complexes I and II of the respiratory chain. 5′,5′-Dithiobis-(2-nitrobenzoate) (DTNB), which does not penetrate into mitochondria and binds thiols to form disulfides, weakly activates hydrogen peroxide production during the oxidation of NAD-dependent substrates and inhibits the ROS production upon succinate oxidation. DTNB is particularly effective in inhibiting the menadione-induced formation of ROS. The differences in the ROS formation by these reagents are explained by the fact that they influence different thiol-containing proteins and enzymes. As distinct from NEM, which inhibits complex I of the respiratory chain, DTNB has no effect on the respiratory chain of mitochondria but can bind the SH-groups of NADH-quinone oxidoreductase, which is localized in the outer mitochondrial membrane and participates in the redox cycle of menadione. It was also shown that the ability to inhibit the ADP-stimulated respiration, a feature inherent in both reagents, does not significantly contribute to ROS production.     

Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism

The recent knowledge on mitochondria as the substantial source of reactive oxygen species, namely superoxide and hydrogen peroxide efflux from mitochondria, is reviewed, as well as nitric oxide and subsequent peroxynitrite generation in mitochondria and their effects. The reactive oxygen species formation in extramitochondrial locations, in peroxisomes, by cytochrome P450, and NADPH oxidase reaction, is also briefly discussed. Conditions are pointed out under which mitochondria represent the major ROS source for the cell: higher percentage of non-phosphorylating and coupled mitochondria, in vivo oxygen levels leading to increased intensity of the reverse electron transport in the respiratory chain, and nitric oxide effects on the redox state of cytochromes. We formulate hypotheses on the crucial role of ROS generated in mitochondria for the whole cell and organism, in concert with extramitochondrial ROS and antioxidant defense. We hypothesize that a sudden decline of mitochondrial ROS production converts cells or their microenvironment into a “ROS sink” represented by the instantly released excessive capacity of ROS-detoxification mechanisms. A partial but immediate decline of mitochondrial ROS production may be triggered by activation of mitochondrial uncoupling, specifically by activation of recruited or constitutively present uncoupling proteins such as UCP2, which may counterbalance the mild oxidative stress.     

Mechanisms of tumor promotion by reactive oxygen species

This review analyzes the available information concerning mechanisms of non-genotoxic action of reactive oxygen species (ROS) during tumor promotion and pathways of their generation under the influence of chemical compounds. Special attention is given to the ability of ROS to induce pseudohypoxia through inhibition of prolyl oxidase, which is an oxygen sensor in the cell. Functions of HIF-1α as a main contributor to the ROS-induced promotion are analyzed. Data suggest that an unregulated high level of HIF-1α in the cell could induce the development of tumors. Hypothetical possibilities of ROS production under the influence of different environmental pollutants, which are promoters of tumorigenesis, include functioning of cytochrome P450 during oxidation of substrates, functioning of the mitochondrial respiratory chain, and action of peroxisome proliferators.     

Effect of steroid hormones on production of reactive oxygen species in mitochondria

Among the targets of steroid hormones are mitochondria, which as the main source of reactive oxygen species (ROS) in the cell play a central role in the development of various pathologies. We studied the effect of progesterone and its synthetic analogs on mitochondrial ROS production. It was found that progesterone promoted formation of superoxide anion and hydrogen peroxide in mitochondria oxidizing the substrates of complex I of the respiratory chain but did not influence the production of ROS during oxidation of succinate, respiratory chain complex II substrate. Progesterone derivatives—Medroxyprogesterone acetate, Buterol, Acetomepregenol, Megestrol acetate—had different effects on ROS production, depending on their chemical structure. By the stimulation of ROS production in mitochondria (during oxidation of pyruvate + malate), the tested steroids can be arranged in decreasing order as follows: progesterone > Buterol ≈ Acetomepregenol > Medroxyprogesterone acetate = Megestrol acetate. Activation of ROS production by progesterone and by Buterol involves different mechanisms: progesterone acts as an inhibitor of NAD-dependent respiration, while Buterol and Acetomepregenol perhaps form noncovalent complexes by hydrogen bonding of the ester carbonyl at C3 to the SH groups of the respective targets.     

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.     

Mitochondrial role in life and death of the cell

Mitochondria are the major ATP producer of the mammalian cell. Moreover, mitochondria are also the main intracellular source and target of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in human cells. A low level of ROS generated from the respiratory chain was recently proposed to take part in the signaling from mitochondria to the nucleus. Several structural characteristics of mitochondria and the mitochondrial genome enable them to sense and respond to extracellular and intracellular signals or stresses in order to sustain the life of the cell. It has been established that mitochondrial respiratory function declines with age, and that defects in the respiratory chain increase the production of ROS and free radicals in mitochondria. Within a certain concentration range, ROS may induce stress responses of the cell by altering the expression of a number of genes in order to uphold energy metabolism to rescue the cell. However, beyond this threshold, ROS may elicit apoptosis by induction of mitochondrial membrane permeability transition and release of cytochrome c. Intensive research in the past few years has established that mitochondria play a pivotal role in the early phase of apoptosis in mammalian cells. In this article, the role of mitochondria in the determination of life and death of the cell is reviewed on the basis of recent findings gathered from this and other laboratories.     

Mitochondrial respiratory chain complexes as sources and targets of thiol-based redox-regulation

The respiratory chain of the inner mitochondrial membrane is a unique assembly of protein complexes that transfers the electrons of reducing equivalents extracted from foodstuff to molecular oxygen to generate a proton-motive force as the primary energy source for cellular ATP-synthesis. Recent evidence indicates that redox reactions are also involved in regulating mitochondrial function via redox-modification of specific cysteine-thiol groups in subunits of respiratory chain complexes. Vice versa the generation of reactive oxygen species (ROS) by respiratory chain complexes may have an impact on the mitochondrial redox balance through reversible and irreversible thiol-modification of specific target proteins involved in redox signaling, but also pathophysiological processes. Recent evidence indicates that thiol-based redox regulation of the respiratory chain activity and especially S-nitrosylation of complex I could be a strategy to prevent elevated ROS production, oxidative damage and tissue necrosis during ischemia–reperfusion injury. This review focuses on the thiol-based redox processes involving the respiratory chain as a source as well as a target, including a general overview on mitochondria as highly compartmentalized redox organelles and on methods to investigate the redox state of mitochondrial proteins. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling

Reactive oxygen species (ROS) produced by the mitochondrial respiratory chain can be a redox signal, but whether they affect mitochondrial function is unclear. Here we show that low levels of ROS from the respiratory chain under physiological conditions reversibly modify the thiol redox state of mitochondrial proteins involved in fatty acid and carbohydrate metabolism. As these thiol modifications were specific and occurred without bulk thiol changes, we first had to develop a sensitive technique to identify the small number of proteins modified by endogenous ROS. In this technique, redox difference gel electrophoresis, control, and redox-challenged samples are labeled with different thiol-reactive fluorescent tags and then separated on the same two-dimensional gel, enabling the sensitive detection of thiol redox modifications by changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. Thiol redox modification affected enzyme activity, suggesting that the reversible modification of enzyme activity by ROS from the respiratory chain may be an important and unexplored mode of mitochondrial redox signaling.     

Alternative oxidase present in procyclic Trypanosoma brucei may act to lower the mitochondrial production of superoxide

The mitochondrial electron transfer chain present in the procyclic form of the African trypanosome Trypanosoma brucei contains both cytochrome c oxidase and an alternative oxidase (TAO) as terminal oxidases that reduce oxygen to water. By contrast, the electron transfer chain of the primitive mitochondrion present in the bloodstream form of T. brucei contains only TAO as the terminal oxidase. TAO functions in the bloodstream forms to oxidize the ubiquinol produced by the glycerol-3-phosphate shuttle that results in the oxidation of the reduced nicotinamide adenine dinucleotide phosphate produced by glycolysis. The function, however, of TAO in the procyclic forms is unknown. In this study, we found that inhibition of TAO by the specific inhibitor salicylhydroxamic acid stimulates the formation of reactive oxygen species (ROS) in trypanosome mitochondria, resulting in mitochondrial alteration and increased oxidation of cellular proteins. Moreover, the activity and protein content of TAO in procyclic trypanosomes were increased when cells were incubated in the presence of hydrogen peroxide or antimycin A, the cytochrome bc1 complex inhibitor, which also results in increased ROS production. We suggest that one function of TAO in procyclic cells may be to prevent ROS production by removing excess reducing equivalents and transferring them to oxygen.     

Impact of PGC-1α on the topology and rate of superoxide production by the mitochondrial electron transport chain

Reactive oxygen species (ROS) play an important role in normal signaling events and excessive ROS are associated with many pathological conditions. The amount of ROS in cells is dependent on both the production of ROS by the mitochondrial electron transport chain and their removal by ROS-detoxifying enzymes. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial functions and a key regulator of the ROS-detoxifying program. However, the impact of PGC-1α on the topology and rate of superoxide production by the mitochondrial electron transport chain is not known. We report here, using mitochondria from muscle creatine kinase–PGC-1α transgenic mice, that PGC-1α does not affect the topology of ROS production, but increases the capacity of complexes I and III to generate ROS. These changes are associated with increased mitochondrial respiration and content of respiratory chain complexes. When normalizing ROS production to mitochondrial respiration, we find that PGC-1α preserves the percentage of free radical leak by the electron transport chain. Together, these data demonstrate that PGC-1α regulates the intrinsic properties of mitochondria in such a way as to preserve a tight coupling between mitochondrial respiration and ROS production.     

Pathophysiological implications of mitochondrial oxidative stress mediated by mitochondriotropic agents and polyamines: the role of tyrosine phosphorylation

Mitochondria, once merely considered as the “powerhouse” of cells, as they generate more than 90 % of cellular ATP, are now known to play a central role in many metabolic processes, including oxidative stress and apoptosis. More than 40 known human diseases are the result of excessive production of reactive oxygen species (ROS), bioenergetic collapse and dysregulated apoptosis. Mitochondria are the main source of ROS in cells, due to the activity of the respiratory chain. In normal physiological conditions, ROS generation is limited by the anti-oxidant enzymatic systems in mitochondria. However, disregulation of the activity of these enzymes or interaction of respiratory complexes with mitochondriotropic agents may lead to a rise in ROS concentrations, resulting in oxidative stress, mitochondrial permeability transition (MPT) induction and triggering of the apoptotic pathway. ROS concentration is also increased by the activity of amine oxidases located inside and outside mitochondria, with oxidation of biogenic amines and polyamines. However, it should also be recalled that, depending on its concentration, the polyamine spermine can also protect against stress caused by ROS scavenging. In higher organisms, cell signaling pathways are the main regulators in energy production, since they act at the level of mitochondrial oxidative phosphorylation and participate in the induction of the MPT. Thus, respiratory complexes, ATP synthase and transition pore components are the targets of tyrosine kinases and phosphatases. Increased ROS may also regulate the tyrosine phosphorylation of target proteins by activating Src kinases or phosphatases, preventing or inducing a number of pathological states.     

The role of mitochondria in protection of the heart by preconditioning

A prolonged period of ischaemia followed by reperfusion irreversibly damages the heart. Such reperfusion injury (RI) involves opening of the mitochondrial permeability transition pore (MPTP) under the conditions of calcium overload and oxidative stress that accompany reperfusion. Protection from MPTP opening and hence RI can be mediated by ischaemic preconditioning (IP) where the prolonged ischaemic period is preceded by one or more brief (2-5 min) cycles of ischaemia and reperfusion. Following a brief overview of the molecular characterisation and regulation of the MPTP, the proposed mechanisms by which IP reduces pore opening are reviewed including the potential roles for reactive oxygen species (ROS), protein kinase cascades, and mitochondrial potassium channels. It is proposed that IP-mediated inhibition of MPTP opening at reperfusion does not involve direct phosphorylation of mitochondrial proteins, but rather reflects diminished oxidative stress during prolonged ischaemia and reperfusion. This causes less oxidation of critical thiol groups on the MPTP that are known to sensitise pore opening to calcium. The mechanisms by which ROS levels are decreased in the IP hearts during prolonged ischaemia and reperfusion are not known, but appear to require activation of protein kinase Cepsilon, either by receptor-mediated events or through transient increases in ROS during the IP protocol. Other signalling pathways may show cross-talk with this primary mechanism, but we suggest that a role for mitochondrial potassium channels is unlikely. The evidence for their activity in isolated mitochondria and cardiac myocytes is reviewed and the lack of specificity of the pharmacological agents used to implicate them in IP is noted. Some K(+) channel openers uncouple mitochondria and others inhibit respiratory chain complexes, and their ability to produce ROS and precondition hearts is mimicked by bona fide uncouplers and respiratory chain inhibitors. IP may also provide continuing protection during reperfusion by preventing a cascade of MPTP-induced ROS production followed by further MPTP opening. This phase of protection may involve survival kinase pathways such as Akt and glycogen synthase kinase 3 (GSK3) either increasing ROS removal or reducing mitochondrial ROS production.