ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis

Reactive oxygen species (ROS) have been shown to be toxic but also function as signalling molecules. This biological paradox underlies mechanisms that are important for the integrity and fitness of living organisms and their ageing. The pathways that regulate ROS homeostasis are crucial for mitigating the toxicity of ROS and provide strong evidence about specificity in ROS signalling. By taking advantage of the chemistry of ROS, highly specific mechanisms have evolved that form the basis of oxidant scavenging and ROS signalling systems.     

Mechanisms of oxidant production in esophageal squamous cell and Barrett's cell lines

We hypothesized that differences among individuals in reflux-induced oxidant production by esophageal squamous epithelial cells might contribute to the development of Barrett's esophagus. We studied the effects of acid and bile acids on the production of reactive oxygen species (ROS) in esophageal squamous cell lines derived from gastroesophageal reflux disease patients with (NES-B3T) and without (NES-G2T) Barrett's esophagus and in a Barrett's epithelial cell line (BAR-T). Cells were incubated with an ROS-sensitive probe and exposed to acidic medium, neutral bile acid medium, or acidic bile acid medium. ROS were quantified in the presence and absence of diphenyleneiodonium chloride (DPI, an NADPH oxidase inhibitor), N(G)-monomethyl-l-arginine (l-NMMA, a nitric oxide synthase inhibitor), and rotenone (a mitochondrial electron transport chain inhibitor). Acidic bile acid medium induced ROS production in both squamous cell lines; however, only DPI blocked ROS production by NES-B3T cells, whereas both DPI and l-NMMA blocked ROS production by NES-G2T cells. In BAR-T cells, acidic medium and acidic bile acid medium induced the production of ROS; l-NMMA prevented ROS production after exposure to acidic medium, whereas ROS production induced by acidic bile acid medium was blocked by DPI. These studies demonstrate that there are differences between esophageal squamous cells and Barrett's epithelial cells and between esophageal squamous cells from gastroesophageal reflux disease patients with and without Barrett's esophagus in the mechanisms of oxidant production induced by exposure to acid and bile acids.     

Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide

Free radicals are produced continuously by skeletal muscle fibers. Extracellular release of reactive oxygen species (ROS) and nitric oxide (NO) derivatives has been demonstrated, but little is known about intracellular oxidant regulation. We used a fluorescent oxidant probe, 2',7'-dichlorofluorescin (DCFH), to assess net oxidant activity in passive muscle fiber bundles isolated from mouse diaphragm and studied in vitro. We tested the following three hypotheses. 1) Net oxidant activity is decreased by muscle cooling. 2) CO(2) exposure depresses intracellular oxidant activity. 3) Muscle-derived ROS and NO both contribute to overall oxidant activity. Our results indicate that DCFH oxidation was diminished by cooling muscle fibers from 37 degrees C to 23 degrees C (P < 0.001). The rate of DCFH oxidation correlated positively with CO(2) exposure (0-10%; P < 0.05) and negatively with concurrent changes in pH (7.0-8.5; P < 0.05). Separate exposures to anti-ROS enzymes (superoxide dismutase, 1 kU/ml; catalase, 1 kU/ml), a glutathione peroxidase mimetic (ebselen, 30 microM), NO synthase inhibitors (N(omega)-nitro-l-arginine methyl ester, 1 mM; N(omega)-monomethyl-l-arginine, 1 mM), or an NO scavenger (hemoglobin, 1 microM) each inhibited DCFH oxidation (P < 0.05). Oxidation was increased by hydrogen peroxide, 100 microM, an NO donor (NOC-22, 400 microM), or the substrate for NO synthase (l-arginine, 5 mM). We conclude that net oxidant activity in resting muscle fibers is 1) decreased at subphysiological temperatures, 2) increased by CO(2) exposure, and 3) influenced by muscle-derived ROS and NO derivatives to similar degrees.     

Concentration-dependent dual effects of hydrogen peroxide on insulin signal transduction in H4IIEC hepatocytes

Background

Oxidative stress induced by the accumulation of reactive oxygen species (ROS) has a causal role in the development of insulin resistance, whereas ROS themselves function as intracellular second messengers that promote insulin signal transduction. ROS can act both positively and negatively on insulin signaling, but the molecular mechanisms controlling these dual actions of ROS are not fully understood.

Methodology/Principal Findings

Here, we directly treated H4IIEC hepatocytes with hydrogen peroxide (H2O2), a representative membrane-permeable oxidant and the most abundant ROS in cells, to identify the key factors determining whether ROS impair or enhance intracellular insulin signaling. Treatment with high concentrations of H2O2 (25–50 µM) for 3 h reduced insulin-stimulated Akt phosphorylation, and increased the phosphorylation of both JNK and its substrate c-Jun. In contrast, lower concentrations of H2O2 (5–10 µM) enhanced insulin-stimulated phosphorylation of Akt. Moreover, lower concentrations suppressed PTP1B activity, suggesting that JNK and phosphatases such as PTP1B may play roles in determining the thresholds for the diametrical effects of H2O2 on cellular insulin signaling. Pretreatment with antioxidant N-acetyl-L-cysteine (10 mM) canceled the signal-promoting action of low H2O2 (5 µM), and it canceled out further impairment of insulin of insulin signaling induced by high H₂O₂ (25 µM).

Conclusions/Significance

Our results demonstrate that depending on its concentration, H2O2 can have the positive or negative effect on insulin signal transduction in H4IIEC hepatocytes, suggesting that the concentration of intracellular ROS may be a major factor in determining whether ROS impair or enhance insulin signaling.     

Free radicals and senescence

There is a significant body of experimental evidence that a rise in intracellular reactive oxygen species (ROS) contributes to senescence. Here we review experiments where entry into senescence has been evaluated in cells whose intracellular ROS levels have been modulated by growth in either high or low ambient oxygen concentrations, or where the cellular antioxidant status has been perturbed. In addition, we discuss the observations that senescence triggered by oncogene expression also appears to be in part mediated by a rise in ROS levels. Finally, we discuss the emerging evidence that in vivo senescence might also be triggered by a rise in cellular oxidant levels. Although these data tend to support a role for ROS in mediating senescence, significant questions remain as to whether ROS act in a random or specific fashion and what precise oxidant species acts as the potential senescence trigger.     

DeltaPsi(m)-Dependent and -independent production of reactive oxygen species by rat brain mitochondria.

Mitochondria are widely believed to be the source of reactive oxygen species (ROS) in a number of neurodegenerative disease states. However, conditions associated with neuronal injury are accompanied by other alterations in mitochondrial physiology, including profound changes in the mitochondrial membrane potential DeltaPsi(m). In this study we have investigated the effects of DeltaPsi(m) on ROS production by rat brain mitochondria using the fluorescent peroxidase substrates scopoletin and Amplex red. The highest rates of mitochondrial ROS generation were observed while mitochondria were respiring on the complex II substrate succinate. Under this condition, the majority of the ROS signal was derived from reverse electron transport to complex I, because it was inhibited by rotenone. This mode of ROS generation is very sensitive to depolarization of DeltaPsi(m), and even the depolarization associated with ATP generation was sufficient to inhibit ROS production. Mitochondria respiring on the complex I substrates, glutamate and malate, produce very little ROS until complex I is inhibited with rotenone, which is also consistent with complex I being the major site of ROS generation. This mode of oxidant production is insensitive to changes in DeltaPsi(m). With both substrates, ubiquinone-derived ROS can be detected, but they represent a more minor component of the overall oxidant signal. These studies demonstrate that rat brain mitochondria can be effective producers of ROS. However, the optimal conditions for ROS generation require either a hyperpolarized membrane potential or a substantial level of complex I inhibition.     

Structural studies of an eukaryotic cambialistic superoxide dismutase purified from the mature seeds of camphor tree

Inflammation is one of the leading causes of the many pathological states associated with oxidative stress. A crucial role in the development of inflammation-induced oxidative stress is played by reactive oxidant species (ROS), which are very difficult to detect in vivo. One of the most sensitive and definitive methods in the detection of ROS is electron spin resonance, especially as used in conjunction with spin trapping. Unfortunately, the commonly used nitrone spin traps have a very low efficacy for trapping superoxide radicals, and their radical adducts are not stable. To address this deficiency, we have developed negatively charged cyclic hydroxylamines such as 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) for the detection of reactive oxidant species as a diagnostic tool for extracellular inflammation-induced oxidative stress. We used inflammation induced by a bacterial endotoxin lipopolysaccharide (LPS) as a model. ROS formation was tested in cultured macrophages, in blood and in vivo. PP-H reacts with reactive oxidant species generating the stable nitroxide radical 4-phosphonooxy-TEMPO. It was shown that a 5-h treatment of macrophages with LPS (1 microg/ml) leads to a threefold increase in superoxide formation as demonstrated using superoxide dismutase. Formation of reactive oxidant species 5 h after LPS (1 mg/kg) treatment of Fischer rats was analyzed in arterial blood; formation of reactive oxidant species in LPS-treated animals increased by a factor of 2.2 and was dependent upon the LPS dose. Diphenyleneiodonium (0.1 mM) inhibited formation of LPS-stimulated reactive oxidant species by 80%. We suggest that this test could be used as a noninvasive diagnostic tool for inflammation-induced oxidative stress.     

Antimicrobial peptides increase tolerance to oxidant stress in Drosophila melanogaster

It is well appreciated that reactive oxygen species (ROS) are deleterious to mammals, including humans, especially when generated in abnormally large quantities from cellular metabolism. Whereas the mechanisms leading to the production of ROS are rather well delineated, the mechanisms underlying tissue susceptibility or tolerance to oxidant stress remain elusive. Through an experimental selection over many generations, we have previously generated Drosophila melanogaster flies that tolerate tremendous oxidant stress and have shown that the family of antimicrobial peptides (AMPs) is over-represented in these tolerant flies. Furthermore, we have also demonstrated that overexpression of even one AMP at a time (e.g. Diptericin) allows wild-type flies to survive much better in hyperoxia. In this study, we used a number of experimental approaches to investigate the potential mechanisms underlying hyperoxia tolerance in flies with AMP overexpression. We demonstrate that flies with Diptericin overexpression resist oxidative stress by increasing antioxidant enzyme activities and preventing an increase in ROS levels after hyperoxia. Depleting the GSH pool using buthionine sulfoximine limits fly survival, thus confirming that enhanced survival observed in these flies is related to improved redox homeostasis. We conclude that 1) AMPs play an important role in tolerance to oxidant stress, 2) overexpression of Diptericin changes the cellular redox balance between oxidant and antioxidant, and 3) this change in redox balance plays an important role in survival in hyperoxia.     

Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis.

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant pro-apoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 - but not cathepsins B or D - causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.     

Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100-1000 μM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50-100 μM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.     

Activity of grape extracts from Greek varieties of Vitis vinifera against mutagenicity induced by bleomycin and hydrogen peroxide in Salmonella typhimurium strain TA102

Several in vivo and in vitro studies have shown that grape extracts could prevent certain steps in carcinogenesis and a few mechanisms have been proposed for this activity. In this study, the potential antimutagenic activity of methanolic and aqueous extracts from two Greek grape varieties of Vitis vinifera against DNA damage induced by reactive oxygen species (ROS) was assessed as a potential novel chemopreventive mechanism, using Salmonella typhimurium strain TA102. The two grape varieties were Assyrtiko (white grapes) and Mandilaria (red grapes), while the oxidant mutagens used were bleomycin (BLM) and hydrogen peroxide (H(2)O(2)). Since it has been considered that polyphenols present in grapes are their most potent biologically active compounds, we also tested the effects of polyphenol-rich fractions as well as some of the more common grape polyphenols on the activity of the two test mutagens. These polyphenols were quercetin, (+)-catechin, (-)-epicatechin, trans-resveratrol, gallic acid and protocatechuic acid. Almost all extracts showed inhibitory activity against both mutagens. On the other hand, polyphenol-rich fractions as well as individual polyphenols at concentrations found in the extracts either did not diminish or did enhance the activity of the mutagens. These results suggest that the protection of DNA from mutations induced by ROS may be one of the mechanisms accounting for the chemopreventive activity of grape extracts. However, it seems that this protective activity may not be attributed to polyphenols but rather to a synergism of many compounds in the grapes.     

Metabolic effects of ganglioside GM1 on PC12 cells in oxidative stress depend on modulation of activity of tyrosine kinase Trk of receptors

There have been obtained evidences that not only GM1, but also other main brain gangliosides (GD1a, GD1b, and GT1b) increase viability of cells of the neuronal line PC12 under action of H₂O₂. By the example of GM1 and GD1a, gangliosides have been shown to produce a protective effect on PC12 cells under conditions of oxidative stress both at micro- and nanomolar concentrations that are physiological concentrations of gangliosides in cerebrospinal fluid. For the first time, GM1 at nanomolar concentrations was shown to decrease the H₂O₂-induced formation of reactive oxygen species (ROS). It was found that in the presence of inhibitor of tyrosine kinase Trk of receptors K-252a, GM1 at concentrations of 10 μM and 10 nM lost its ability to produce such metabolic effects as a decrease of ROS accumulation and of the degree of oxidative inactivation of Na⁺,K⁺-ATPase in PC12 cells, as well as ceased to increase viability of these cells under conditions of oxidative stress. The dependence of protective and metabolic effects of gangliosides GM1 in PC12 cells treated with H₂O₂ on modulation of activity of activity of tyrosine kinase Trk receptors (i.e., from the same signal system) agrees with concept about the essential role of oxidant effect of GM1 in its increase of cell viability.     

The role of free radicals in asbestos-induced diseases.

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

Curcumin induces concentration-dependent alterations in mitochondrial function through ROS in C2C12 mouse myoblasts

Curcumin exhibits antioxidant properties in normal cells where the uptake is low, unlike in tumor cells where uptake is high and curcumin increases reactive oxygen species (ROS) production and cell death. Mitochondria are the main source and primary target of cellular ROS. We hypothesized that curcumin would regulate cellular redox status and mitochondrial function, depending on cell sensitivity and/or curcumin concentration in normal cells. We examined the differences between low and high concentrations of curcumin, with specific attention focused on ROS levels, mitochondrial function, and cell viability in mouse C2C12 myoblast under normal and simulated conditions of diabetes. Cells incubated with high concentrations of curcumin (10–50 μM) resulted in decreased cell viability and sustained robust increases in ROS levels. Mechanistic studies showed that increased ROS levels in cells incubated with 20 μM curcumin induced opening of mitochondrial permeability transition pores and subsequent release of cytochrome c, activation of caspases 9 and 3/7, and apoptotic cell death. Low concentrations of curcumin (1–5 μM) did not affect cell viability, but induced a mild increase in ROS levels, which peaked at 2 hr after the treatment. Incubation with 5 μM curcumin also induced ROS-dependent increases in mitochondrial mass and membrane potential. Finally, pretreatment with 5 μM curcumin prevented high glucose-induced oxidative cell injury. Our study suggests that mitochondria respond differentially depending on curcumin concentration-dependent induction of ROS. The end result is either cell protection or death. Curcumin may be an effective therapeutic target for diabetes and other mitochondrial diseases when used in low concentrations.     

Loss of tafazzin in yeast leads to increased oxidative stress during respiratory growth

The tafazzin (TAZ) gene is highly conserved from yeast to humans, and the yeast taz1 null mutant shows alterations in cardiolipin (CL) metabolism, mitochondrial dysfunction and stabilization of supercomplexes similar to those found in Barth syndrome, a human disorder resulting from loss of tafazzin. We have previously shown that the yeast tafazzin mutant taz1Delta, which cannot remodel CL, is ethanol-sensitive at elevated temperature. In the current report, we show that in response to ethanol, CL mutants taz1Delta as well as crd1Delta, which cannot synthesize CL, exhibited increased protein carbonylation, an indicator of reactive oxygen species (ROS). The increase in ROS is most likely not due to defective oxidant defence systems, as the CL mutants do not display sensitivity to paraquat, menadione or hydrogen peroxide (H2O2). Ethanol sensitivity and increased protein carbonylation in the taz1Delta mutant but not in crd1Delta can be rescued by supplementation with oleic acid, suggesting that oleoyl-CL and/or oleoyl-monolyso-CL enables growth of taz1Delta in ethanol by decreasing oxidative stress. Our findings of increased oxidative stress in the taz1Delta mutant during respiratory growth may have important implications for understanding the pathogenesis of Barth syndrome.     

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.