Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.     

Preconditioning with millimolar concentrations of vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide -- H(2)O(2), superoxide anion radical O(2)*- and hydroxyl radical *OH -- the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO* from sodium nitroprusside -- SNP), and peroxynitrite (ONOO(-) from 3-morpholinosydnonimine -- SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 microM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H(2)O(2), O(2)*-, *OH, ONOO(-) and NO*. Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H(2)O(2) and ONOO(-), while NO* additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate - ASC (0.01, 0.1, 1 mM), or N-acetylcysteine - NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.     

抗氧化系统研究新进展

氧化还原系统主要由活性氧、自由基、活性氧生成系统和抗氧化系统组成。大量的研究表明,氧化还原系统在机体多种生物学功能中发挥关键的调节作用。抗氧化系统主要包括酶类抗氧化剂和非酶类抗氧化剂。抗氧化系统一方面可以通过调节活性氧的水平影响各种生物学功能,另一方面各种酶类抗氧化剂和非酶类抗氧化剂本身也可以参与多种生化反应,调节机体功能。近年来的研究表明,机体内除了典型的抗氧化酶,如超氧化物歧化酶和过氧化氢酶等,还存在多种抗氧化新型抗氧化酶,如硫氧还蛋白、谷氧还蛋白和金属基质蛋白酶等。在本文中,我们将回顾近年来的一些文献,综述抗氧化系统的研究新进展,旨在为抗氧化系统的深入研究提供理论基础。     

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.     

Roles of sedentary aging and lifelong physical activity in exchange of glutathione across exercising human skeletal muscle

Reactive oxygen species (ROS) are important signaling molecules with regulatory functions, and in young and adult organisms, the formation of ROS is increased during skeletal muscle contractions. However, ROS can be deleterious to cells when not sufficiently counterbalanced by the antioxidant system. Aging is associated with accumulation of oxidative damage to lipids, DNA, and proteins. Given the pro-oxidant effect of skeletal muscle contractions, this effect of age could be a result of excessive ROS formation. We evaluated the effect of acute exercise on changes in blood redox state across the leg of young (23±1 years) and older (66±2 years) sedentary humans by measuring the whole blood concentration of the reduced (GSH) and oxidized (GSSG) forms of the antioxidant glutathione. To assess the role of physical activity, lifelong physically active older subjects (62±2 years) were included. Exercise increased the venous concentration of GSSG in an intensity-dependent manner in young sedentary subjects, suggesting an exercise-induced increase in ROS formation. In contrast, venous GSSG levels remained unaltered during exercise in the older sedentary and active groups despite a higher skeletal muscle expression of the superoxide-generating enzyme NADPH oxidase. Arterial concentration of GSH and expression of antioxidant enzymes in skeletal muscle of older active subjects were increased. The potential impairment in exercise-induced ROS formation may be an important mechanism underlying skeletal muscle and vascular dysfunction with sedentary aging. Lifelong physical activity upregulates antioxidant systems, which may be one of the mechanisms underlying the lack of exercise-induced increase in GSSG.     

Free radicals and antioxidants in normal physiological functions and human disease

Reactive oxygen species (ROS) and reactive nitrogen species (RNS, e.g. nitric oxide, NO(*)) are well recognised for playing a dual role as both deleterious and beneficial species. ROS and RNS are normally generated by tightly regulated enzymes, such as NO synthase (NOS) and NAD(P)H oxidase isoforms, respectively. Overproduction of ROS (arising either from mitochondrial electron-transport chain or excessive stimulation of NAD(P)H) results in oxidative stress, a deleterious process that can be an important mediator of damage to cell structures, including lipids and membranes, proteins, and DNA. In contrast, beneficial effects of ROS/RNS (e.g. superoxide radical and nitric oxide) occur at low/moderate concentrations and involve physiological roles in cellular responses to noxia, as for example in defence against infectious agents, in the function of a number of cellular signalling pathways, and the induction of a mitogenic response. Ironically, various ROS-mediated actions in fact protect cells against ROS-induced oxidative stress and re-establish or maintain "redox balance" termed also "redox homeostasis". The "two-faced" character of ROS is clearly substantiated. For example, a growing body of evidence shows that ROS within cells act as secondary messengers in intracellular signalling cascades which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. This review will describe the: (i) chemistry and biochemistry of ROS/RNS and sources of free radical generation; (ii) damage to DNA, to proteins, and to lipids by free radicals; (iii) role of antioxidants (e.g. glutathione) in the maintenance of cellular "redox homeostasis"; (iv) overview of ROS-induced signaling pathways; (v) role of ROS in redox regulation of normal physiological functions, as well as (vi) role of ROS in pathophysiological implications of altered redox regulation (human diseases and ageing). Attention is focussed on the ROS/RNS-linked pathogenesis of cancer, cardiovascular disease, atherosclerosis, hypertension, ischemia/reperfusion injury, diabetes mellitus, neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), rheumatoid arthritis, and ageing. Topics of current debate are also reviewed such as the question whether excessive formation of free radicals is a primary cause or a downstream consequence of tissue injury.     

Peroxisomes as a cellular source of reactive nitrogen species signal molecules

Peroxisomes are single membrane-bounded subcellular organelles with an essentially oxidative type of metabolism and are probably the major sites of intracellular H₂O₂ production. These organelles also generate superoxide radicals () and besides catalase they have a complex battery of antioxidative enzymes. In recent years the existence of l-arginine-dependent nitric oxide synthase (NOS) activity and the generation of the reactive nitrogen species (RNS) nitric oxide (NO) have been demonstrated in plant peroxisomes. The inter-cellular and intracellular NO carrier S-nitrosoglutathione (GSNO) can be generated inside peroxisomes and the presence of this RNS has been demonstrated in peroxisomes from several plant species. This review analyzes the available evidence concerning the properties of the NOS activity and the generation of the RNS messengers NO and GSNO in peroxisomes in the context of the cellular function of these organelles as a source of RNS signaling molecules. The important physiological functions displayed by NO and other RNS in intra- and inter-cellular communication in different organisms indicate that more attention should be payed to the RNS signaling function of peroxisomes in human, animal and fungal cells, where it is very likely that similar mechanisms to those found in plant peroxisomes are also operative.     

Unravelling how plants benefit from ROS and NO reactions,while resisting oxidative stress

Background and Aims Reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO), play crucial roles in the signal transduction pathways that regulate plant growth, development and defence responses, providing a nexus of reduction/oxidation (redox) control that impacts on nearly every aspect of plant biology. Here we summarize current knowledge and concepts that lay the foundations of a new vision for ROS/RNS functions – particularly through signalling hubs – for the next decade.Scope Plants have mastered the art of redox control using ROS and RNS as secondary messengers to regulate a diverse range of protein functions through redox-based, post-translational modifications that act as regulators of molecular master-switches. Much current focus concerns the impact of this regulation on local and systemic signalling pathways, as well as understanding how such reactive molecules can be effectively used in the control of plant growth and stress responses.Conclusions The spectre of oxidative stress still overshadows much of our current philosophy and understanding of ROS and RNS functions. While many questions remain to be addressed – for example regarding inter-organellar regulation and communication, the control of hypoxia and how ROS/RNS signalling is used in plant cells, not only to trigger acclimation responses but also to create molecular memories of stress – it is clear that ROS and RNS function as vital signals of living cells.     

Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells

Antioxidants in plant cells mainly include glutathione, ascorbate, tocopherol, proline, betaine and others, which are also information-rich redox buffers and important redox signaling components that interact with cellular compartments. As an unfortunate consequence of aerobic life for higher plants, reactive oxygen species (ROS) are formed by partial reduction of molecular oxygen. The above enzymatic and non-enzymatic antioxidants in higher plant cells can protect their cells from oxidative damage by scavenging ROS. In addition to crucial roles in defense system and as enzyme cofactors, antioxidants influence higher plant growth and development by modifying processes from miotosis and cell elongation to senescence and death. Most importantly, they provide essential information on cellular redox state, and regulate gene expression associated with biotic and abiotic stress responses to optimize defense and survival. An overview of the literature is presented in terms of primary antioxidant free radical scavenging and redox signaling in plant cells. Special attention is given to ROS and ROS-anioxidant interaction as a metabolic interface for different types of signals derived from metabolisms and from the changing environment. This interaction regulates the appropriate induction of acclimation processes or execution of cell death programs, which are the two essential directions for higher plant cells.     

Plant responses to water stress: Role of reactive oxygen species

Terrestrial plants most often encounter drought stress because of erratic rainfall which has become compounded due to present climatic changes.Responses of plants to water stress may be assigned as either injurious change or tolerance index. One of the primary and cardinal changes in response to drought stress is the generation of reactive oxygen species (ROS), which is being considered as the cause of cellular damage. However, recently a signaling role of such ROS in triggering the ROS scavenging system that may confer protection or tolerance against stress is emerging. Such scavenging system consists of antioxidant enzymes like SOD, catalase and peroxidases, and antioxidant compounds like ascorbate, reduced glutathione; a balance between ROS generation and scavenging ultimately determines the oxidative load. As revealed in case of defence against pathogen, signaling via ROS is initiated by NADPH oxidase-catalyzed superoxide generation in the apoplastic space (cell wall) followed by conversion to hydrogen peroxide by the activity of cell wall-localized SOD. Wall peroxidase may also play role in ROS generation for signaling. Hydrogen peroxide may use Ca2+ and MAPK pathway as downstream signaling cascade. Plant hormones associated with stress responses like ABA and ethylene play their role possibly via a cross talk with ROS towards stress tolerance, thus projecting a dual role of ROS under drought stress.     

Dynamic determination of Ox-LDL-induced oxidative/nitrosative stress in single macrophage by using fluorescent probes

Increased oxidative/nitrosative stress, resulting from generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears to play an important role in the inflammatory responses to atherosclerosis. By using MitoTracker Orange CM-H(2)TMRos, CM-H(2)DCFDA (DCF-DA), Dihydrorhodamine 123 (DHR123), DAF-FM, Dihydroethidium (DHE) and JC-1 alone or in all combinations of red and green probes, the present study was designed to monitor the ROS and RNS generation in acute exposure of single monocyte U937-derived macrophage to oxidized low density lipoprotein (Ox-LDL). Acute Ox-LDL (100 microg/ml) treatment increased time-dependently production of intracellular nitric oxide (NO), superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)), and decreased mitochondrial membrane potential (Deltapsi) in single cell. Pretreatment of aminoguanidine (an inhibitor of inducible nitric oxide synthase (iNOS), 10 microM) and vitamin C (an antioxidant agent, 100 microM) for 2h, reduced significantly the Ox-LDL-induced increase of NO and O2*-, and vitamin C completely inhibited increase of intracellular NO and O2*-. In contrast to aminoguanidine, Vitamin C pretreatment significantly prevented Ox-LDL-induced overproduction of NO and O2*- (P<0.01), indicating that antioxidant may be more effective in therapeutic application than iNOS inhibitor in dysfunction of ROS/RNS. By demonstrating a complex imbalance of ROS/RNS via fluorescent probes in acute exposure of single cell to Ox-LDL, oxidative/nitrosative stress might be more detected in the early atherosclerotic lesions.     

Combination of Ascorbate and α-Tocopherol as a Preventive Therapy against Structural and Functional Defects of Erythrocytes in Visceral Leishmaniasis

The redox unbalance in erythrocytes has been found to contribute significantly in the development of anemia in visceral leishmaniasis (VL). The present study revealed enhanced production of reactive oxygen species (ROS) and gradual depletion of α-tocopherol and ascorbate in the erythrocytes of infected animals. The response of erythrocytes to chronic treatment with antioxidants was studied in hamsters during leishmanial infection. Treatment with a combination of α-tocopherol and ascorbate proved to be the most effective preventive for the proteolytic degradation of erythrocyte membrane. Erythrocytes from infected animals were thermally more sensitive compared to the control ones. Combination of both antioxidants was most successful in resisting heat induced structural defects in the cells. Cross-linking of membrane proteins subsequent to oxidative damage in the red cells was accompanied by the formation of high molecular weight protein band at the top of the resolving gel in the presence of the cross-linking agent dimethyladepimidate (DMA). Marked inhibition of cross-linking was observed with combination of both antioxidants. Treatment with α-tocopherol and ascorbate together could withstand osmotic lysis of erythrocytes in the infected animals very efficiently. Decreased hemoglobin (Hb) level was successfully replenished and was coupled with significant increase in the life span of red cells after treating the animals with both antioxidants. Results indicate better efficacy of the combination therapy with α-tocopherol and ascorbate in protecting the erythrocytes from structural and functional damages during leishmanial infection.     

Nitric oxide and cell signaling; modulation of redox tone and protein modification

Summary. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have an impact on many cellular processes, often serving as signal transducers in both physiological and pathological situations. These small molecules can act as ligands for receptors as is the case for nitric oxide and guanylate cyclase. However, they can also modify proteins, changing their function and establishing a baseline for other signals in a process that we have termed redox tone. In this review, we discuss the different mechanisms of redox cell signaling, and give specific examples of RNS participation in cell signaling via classical and redox tone pathways.     

Selenium vitaminology: The connection between selenium,vitamin C,vitamin E,and ergothioneine

Selenium is connected to three small molecule antioxidant compounds, ascorbate, α-tocopherol, and ergothioneine. Ascorbate and α-tocopherol are true vitamins, while ergothioneine is a “vitamin-like” compound. Here we review how selenium is connected to all three. Selenium and vitamin E work together as a team to prevent lipid peroxidation. Vitamin E quenches lipid hydroperoxyl radicals and the resulting lipid hydroperoxide is then converted to the lipid alcohol by selenocysteine-containing glutathione peroxidase. Ascorbate reduces the resulting α-tocopheroxyl radical in this reaction back to α-tocopherol with concomitant production of the ascorbyl radical. The ascorbyl radical can be reduced back to ascorbate by selenocysteine-containing thioredoxin reductase. Ergothioneine and ascorbate are both water soluble, small molecule reductants that can reduce free radicals and redox-active metals. Thioredoxin reductase can reduce oxidized forms of ergothioneine. While the biological significance of this is not yet realized, this discovery underscores the centrality of selenium to all three antioxidants.     

Bordetella bronchiseptica responses to physiological reactive nitrogen and oxygen stresses

Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-microM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens.     

Stabilization of superoxide dismutase by acetyl-l-carnitine in human brain endothelium during alcohol exposure: novel protective approach

Oxidative damage of the endothelium disrupts the integrity of the blood-brain barrier (BBB). We have shown before that alcohol exposure increases the levels of reactive oxygen species (ROS; superoxide and hydroxyl radical) and nitric oxide (NO) in brain endothelial cells by activating NADPH oxidase and inducible nitric oxide synthase. We hypothesize that impairment of antioxidant systems, such as a reduction in catalase and superoxide dismutase (SOD) activity, by ethanol exposure may elevate the levels of ROS/NO in endothelium, resulting in BBB damage. This study examines whether stabilization of antioxidant enzyme activity results in suppression of ROS levels by anti-inflammatory agents. To address this idea, we determined the effects of ethanol on the kinetic profile of SOD and catalase activity and ROS/NO generation in primary human brain endothelial cells (hBECs). We observed an enhanced production of ROS and NO levels due to the metabolism of ethanol in hBECs. Similar increases were found after exposure of hBECs to acetaldehyde, the major metabolite of ethanol. Ethanol simultaneously augmented ROS generation and the activity of antioxidative enzymes. SOD activity was increased for a much longer period of time than catalase activity. A decline in SOD activity and protein levels preceded elevation of oxidant levels. SOD stabilization by the antioxidant and mitochondria-protecting agent acetyl-L-carnitine (ALC) and the anti-inflammatory agent rosiglitazone suppressed ROS levels, with a marginal increase in NO levels. Mitochondrial membrane protein damage and decreased membrane potential after ethanol exposure indicated mitochondrial injury. These changes were prevented by ALC. Our findings suggest the counteracting mechanisms of oxidants and antioxidants during alcohol-induced oxidative stress at the BBB. The presence of enzymatic stabilizers favors the ROS-neutralizing antioxidant redox of the BBB, suggesting an underlying protective mechanism of NO for brain vascular tone and vasodilation.