Different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 microM or 100 microM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H(2)O(2) generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H(2)O(2) and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca(2+). Instead, 1 mM agmatine generates larger amounts of H(2)O(2) than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca(2+)-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Different behavior of agmatine in liver mitochondria: Inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 μM or 100 μM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H₂O₂ generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H₂O₂ and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca²⁺. Instead, 1 mM agmatine generates larger amounts of H₂O₂ than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca²⁺-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Is alpha-lipoic acid a scavenger of reactive oxygen species in vivo? Evidence for its initiation of stress signaling pathways that promote endogenous antioxidant capacity

The chemical reduction and oxidation (redox) properties of alpha-lipoic acid (LA) suggest that it may have potent antioxidant potential. A significant number of studies now show that LA and its reduced form, dihydrolipoic acid (DHLA), directly scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) species and protect cells against a host of insults where oxidative stress is part of the underlying etiology. However, owing to its limited and transient accumulation in tissues following oral intake, the efficacy of nonprotein-bound LA to function as a physiological antioxidant has been questioned. Herein, we review the evidence that the micronutrient functions of LA may be more as an effector of important cellular stress response pathways that ultimately influence endogenous cellular antioxidant levels and reduce proinflammatory mechanisms. This would promote a sustained improvement in cellular resistance to pathologies where oxidative stress is involved, which would not be forthcoming if LA solely acted as a transient ROS scavenger.     

Superoxide anion: Oncogenic reactive oxygen species?

Recent evidence linking intracellular reactive oxygen species to cell survival and/or proliferation signals has resulted in a paradigm shift from the age-old dogma implicating reactive oxygen species exclusively in cell damage and death. It is now accepted that reactive oxygen species play important roles in normal physiological states and that depending on the species involved the effect could be highly varied. In this regard, the effects of the two major reactive oxygen species, superoxide and hydrogen peroxide have been extensively studied. During normal cell growth a tight balance between the two species is kept under check by the cells' anti-oxidant defense systems. Deficiency or defect in this defense armory is invariably associated with neoplasia, thus rendering the intracellular redox status in a state of imbalance and generating a "pro-oxidant" milieu. A variety of model systems have underscored the relationship between a pro-oxidant state and cancer promotion and progression. In this review, we present evidence to support the hypothesis that the effect of intracellular reactive oxygen species on oncogenesis is dependent on the ratio of intracellular superoxide to hydrogen peroxide in that a predominant increase in superoxide supports cell survival and promotes oncogenesis whereas a tilt in favor of hydrogen peroxide prevents carcinogenesis by facilitating cell death signaling.     

Do reactive oxygen species or does oxygen itself confer obligate anaerobiosis? The case of Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron was examined to determine whether its obligate anaerobiosis is imposed by endogenous reactive oxygen species or by molecular oxygen itself. Previous analyses established that aerated B. thetaiotaomicron loses some enzyme activities due to a high rate of endogenous superoxide formation. However, the present study establishes that another key step in central metabolism is poisoned by molecular oxygen itself. Pyruvate dissimilation was shown to depend upon two enzymes, pyruvate:formate lyase (PFL) and pyruvate:ferredoxin oxidoreductase (PFOR), that lose activity upon aeration. PFL is a glycyl-radical enzyme whose vulnerability to oxygen is already understood. The rate of PFOR damage was unaffected by the level of superoxide or peroxide, showing that molecular oxygen itself is the culprit. The cell cannot repair PFOR, which amplifies the impact of damage. The rates of PFOR and fumarase inactivation are similar, suggesting that superoxide dismutase is calibrated so the oxygen- and superoxide-sensitive enzymes are equally sensitive to aeration. The physiological purpose of PFL and PFOR is to degrade pyruvate without disrupting the redox balance, and they do so using catalytic mechanisms that are intrinsically vulnerable to oxygen. In this way, the anaerobic excellence and oxygen sensitivity of B. thetaiotaomicron are two sides of the same coin.     

Are mitochondria a permanent source of reactive oxygen species?

The observation that in isolated mitochondria electrons may leak out of the respiratory chain to form superoxide radicals (O(2)(radical-)) has prompted the assumption that O(2)(radical-) formation is a compulsory by-product of respiration. Since mitochondrial O(2)(radical-) formation under homeostatic conditions could not be demonstrated in situ so far, conclusions drawn from isolated mitochondria must be considered with precaution. The present study reveals a link between electron deviation from the respiratory chain to oxygen and the coupling state in the presence of antimycin A. Another important factor is the analytical system applied for the detection of activated oxygen species. Due to the presence of superoxide dismutase in mitochondria, O(2)(radical-) release cannot be realistically determined in intact mitochondria. We therefore followed the release of the stable dismutation product H(2)O(2) by comparing most frequently used H(2)O(2) detection methods. The possible interaction of the detection systems with the respiratory chain was avoided by a recently developed method, which was compared with conventional methods. Irrespective of the methods applied, the substrates used for respiration and the state of respiration established, intact mitochondria could not be made to release H(2)O(2) from dismutating O(2)(radical-). Although regular mitochondrial respiration is unlikely to supply single electrons for O(2)(radical-) formation our study does not exclude the possibility of the respiratory chain becoming a radical source under certain conditions.     

Phagocyte-derived reactive species: salvation or suicide?

Activated phagocytes produce "reactive oxygen, halogen and nitrogen species" that help to kill some types of microorganism. How these species destroy microorganisms remains, however, an enigma: both direct oxidative damage and indirect damage (whereby reactive species promote the actions of other antibacterial agents) are involved, and no single mechanism is likely to account for the killing of all microorganisms. Phagocyte-derived reactive species are known to injure human tissues and to contribute to inflammation. Recently, however, we have learned that they can also be anti-inflammatory by modulating the immune response. These data have implications for the proposed use of antioxidants to treat inflammation.     

Production of reactive oxygen species and reactive nitrogen species by angiosperm stigmas and pollen: potential signalling crosstalk?

Angiosperm stigmas exhibit high levels of peroxidase activity when receptive to pollen. To explore possible function(s) of this peroxidase activity we investigated amounts of reactive oxygen species (ROS), particularly hydrogen peroxide, in stigmas and pollen. Because nitric oxide (NO) was recently implicated in pollen tube growth, we also investigated amounts of NO in pollen and stigmas. Reactive oxygen species accumulation was assessed with confocal microscopy and light microscopy using ROS probes DCFH2-DA and TMB, respectively. NO was assayed using the NO probe DAF-2DA and confocal microscopy. Stigmas from various different angiosperms were found to accumulate ROS, predominantly H2O2, constitutively. In Senecio squalidus and Arabidopsis thaliana high amounts of ROS/H2O2 were localized to stigmatic papillae. ROS/H2O2 amounts appeared reduced in stigmatic papillae to which pollen grains had adhered. S. squalidus and A. thaliana pollen produced relatively high amounts of NO compared with stigmas; treating stigmas with NO resulted in reduced amounts of stigmatic ROS/H2O2. Constitutive accumulation of ROS/H2O2 appears to be a feature of angiosperm stigmas. This novel finding is discussed in terms of a possible role for stigmatic ROS/H2O2 and pollen-derived NO in pollen-stigma interactions and defence.     

Does underwater rugby stimulate the over-production of reactive oxygen species?

Antioxidant enzymes in erythrocytes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and antioxidant sex hormone oestradiol in serum and malondialdehyde (MDA) production as a marker of lipid peroxidation in erythrocytes were investigated in male and female underwater rugby (UWR) players. Results showed that except for GSH-Px activity in female players, all antioxidant enzymes increased significantly (p < 0.05) after an UWR game. It was interesting to note that while the concentration of oestradiol in female players did not change, it increased significantly (p < 0.05) in male players. Lipid peroxidation (LPO) levels in female players as well as oestradiol concentration did not change significantly (p > 0.05) whereas LPO levels in male players increased significantly (p < 0.05) compared to pre-exercise values. In conclusion, the results showed that underwater rugby can stimulate over-production of ROS and antioxidant systems and affect oestradiol levels in male players. Because of increased LPO levels observed in male players, complex antioxidant supplementation including co-factors of antioxidant enzymes such as Cu, Zn, Fe, Se and antioxidant vitamins such as vitamin C and E may be recommended to players before the UWR game.     

Uncoupling proteins: A role in protection against reactive oxygen species—or not?

A physiological function of the original uncoupling protein, UCP1, is well established: UCP1 is the molecular background for nonshivering thermogenesis. The functions of the “novel” UCPs, UCP2 and UCP3, are still not established. Recent discussions imply that all UCPs may play a role in protection against reactive oxygen species (ROS). Here we examine critically the evidence that UCP1, UCP2 and UCP3 are stimulated by ROS (superoxide) or ROS products (4-hydroxy-2-nonenal), and that the UCPs actually diminish oxidative damage. We conclude that, concerning UCP1, it is unlikely that it has such a role; concerning UCP2/UCP3, most evidence for physiologically significant roles in this respect is still circumstantial.     

Post-translational modifications mediated by reactive nitrogen species: Nitrosative stress responses or components of signal transduction pathways?

In animal cells, nitric oxide and NO-derived molecules have been shown to mediate post-translational modifications such as S-nitrosylation and protein tyrosine nitration which are associated with cell signalling and pathological processes, respectively. In plant cells, knowledge of the function of these post-translational modifications under physiological and stress conditions is still very rudimentary. In this addendum, we briefly examine how reactive nitrogen species (RNS) can exert important effects on proteins that could mediate signalling processes in plants.Key words: nitrosative stress, nitric oxide synthase, S-nitrosoglutathione, nitro-tyrosine, post-translational modifications, S-nitrosylation     

Are mitochondrial reactive oxygen species required for autophagy?

Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H₂O₂ was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.     

Are mitochondria a spontaneous and permanent source of reactive oxygen species?

The bioenergetic properties of mitochondria in combination with the high turnover rate of dioxygen qualify these organelles for the formation of reactive oxygen species (ROS). The assumption that mitochondria are the major intracellular source of ROS was essentially based on in vitro experiments with isolated mitochondria. The transfer of these data to the living cell may, however, be incorrect. Artefacts due to the preparation procedure or inadequate detection methods of ROS may lead to false positive results. Inhomogeneous results were found to be due to an interaction of the detection system with components of the respiratory chain which could be avoided by a recently developed non-invasive method. One of the most critical electron transfer steps in the respiratory chain is the electron bifurcation from ubiquinol to the cytochrome bc(1) complex. This electron bifurcation requires the free mobility of the head domain of the Rieske iron-sulfur protein. Inhibition of electron bifurcation by antimycin A causes leakage of single electrons to oxygen which results in the release of ROS. Hindrance of electron bifurcation was also observed following alterations of the physical state of membrane phospholipids in which the cytochrome bc(1) complex is inserted. Irrespective of whether the fluidity of the membrane was elevated or decreased, electron flow rates to the Rieske iron-sulfur protein were drastically reduced. Concomitantly superoxide radicals were released from these mitochondria, strongly suggesting the involvement of the ubiquinol/cytochrome bc(1) redox couple in this process.     

Is mitochondrial generation of reactive oxygen species a trigger for autophagy?

Autophagy is a conserved lysosomal degradation pathway that has been extensively studied in recent years. However, the mechanism of autophagy induction is still not clear. Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial mediated apoptosis is the production of reactive oxygen species (ROS). Recently, several studies have indicated that ROS may be also involved in induction of autophagy. ROS are molecules or ions that are formed by the incomplete one-electron reduction of oxygen, including superoxide (O2 (*-)), hydrogen peroxide (H2O2), hydroxyl radical ((*)OH), nitric oxide (NO), and peroxynitrite (ONOO-). Our recent studies provide strong evidences for the involvement of mitochondrially-generated ROS production in the induction of autophagy as determined by the formation of autophagosomes and autolysosomes. This was accomplished through treatment with mitochondrial toxins that inhibit the electron transport chain in transformed and cancer cells. In addition, we have determined that H2O2 and 2-methoxyestradiol (inhibitor of superoxide dismutases and electron transport chain) induce autophagy leading to cell death. In contrast, normal astrocytes fail to induce autophagy following treatment with mitochondrial toxins. Herein, we discuss several important points of our studies and provide a model for mitochondrially-induced autophagic cell death mediated by ROS.     

Controversy of free radical hypothesis: reactive oxygen species--cause or consequence of tissue injury?

For a decade or two, the hypothesis of causality of various disorders by reactive oxygen species (ROS), due to their potentially harmful effect towards cellular constituents, is one of the most frequently cited in biomedical sciences. In fact, the ROS-mediated alterations of biomacromolecules are considered to be essential events in the etiopathogenesis of those diseases where involvement of ROS has been indicated. ROS easily react in vitro with most biological molecules, causing their degradation and destruction. This may implicitly suggest that, when excessively produced in vivo, ROS are deleterious to integral components of the cell and cause their dysfunctions. Some experimental data indicate that ROS-mediated lipid peroxidation, protein oxidation and oxidative alterations to nucleic acids are crucial events of unfavorable actions of ROS. Yet the most convincing evidence, i.e. unambiguous inhibition of tissue injury by pretreatment with antioxidants, has not been provided. On the contrary, there are quite a few papers reporting failure in applying antioxidants to heal those pathologies where the causal role of ROS was supposed. Other papers reported serious complications arising from antioxidant therapy, which is quite in contradiction to its expected effect. On the other hand, an increasing number of recent findings have provided evidence of a key role of ROS in both intracellular signaling and intercellular communication, processes involved in maintaining homeostasis. Hence, some investigators consider excessive production of ROS to be rather a "smoke after the fire" than "a deleterious fire" itself, suggesting the occurrence of overproduced ROS as being the consequence of some primary damage. The present paper aims at summarizing some pros and cons of various opinions with an attempt to help better understand the involvement of ROS in tissue injury.     

The antioxidant systems vis-à-vis reactive oxygen species during plant–pathogen interaction

Plant resistance to pathogens requires the activation of complex metabolic pathways in the infected cells, aimed at recognizing pathogen presence and hindering its propagation within plant tissues. In spite of this both compatible and incompatible responses induce alterations in plant metabolism, only in the latter the plant is able to efficiently block pathogen penetration without suffering excessive damage. One of the most studied incompatible responses is based on the hypersensitive response (HR), in which cells surrounding the site of pathogen penetration switch on genes encoding for phytoalexin synthesis and other pathogenesis related proteins before activating programmed cell death (PCD). The production of reactive oxygen species (ROS) is a key event in HR. Several enzymatic systems have been proposed to be responsible for the oxidative burst characterizing HR. In this review, the involvement of antioxidant redox systems, in particular those related to ascorbate (ASC) and glutathione (GSH), in activating both compatible and incompatible plant responses is analysed. Increasing lines of evidence indicate that alterations in the levels and/or redox state of ASC and/or GSH, as well as in the activity of their redox enzymes, occur during the HR programme. These alterations do not seem to be a mere consequence of the oxidative stress induced by the massive ROS production, but they are induced as part of the transduction pathways triggering defence responses and PCD. The possibility that ASC and GSH systems are links in a redox signalling chain activating defence strategies is also discussed.     

Mitochondria as source of reactive oxygen species under oxidative stress. Study with novel mitochondria-targeted antioxidants — the “Skulachev-ion” derivatives

Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1–2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of “bad” mitochondria in cell physiology is discussed.