Clinical aspects of reactive oxygen and nitrogen species

Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolaemia, hypertension, diabetes mellitus and chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species in endothelial and/or smooth muscle cells and the adventitia, and the subsequent decrease in vascular bioavailability of NO. Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include NAD(P)H-oxidase, xanthine oxidase and endothelial nitric oxide synthase in an uncoupled state. Recent studies indicate that endothelial dysfunction of peripheral and coronary resistance and conductance vessels represents a strong and independent risk factor for future cardiovascular events. Ways to reduce endothelial dysfunction include risk-factor modification and treatment with substances that have been shown to reduce oxidative stress and, simultaneously, to stimulate endothelial NO production, such as inhibitors of angiotensin-converting enzyme or the statins. In contrast, in conditions where increased production of reactive oxygen species, such as superoxide, in vascular tissue is established, treatment with NO, e.g. via administration of nitroglycerin, results in a rapid development of endothelial dysfunction, which may worsen the prognosis in patients with established coronary artery disease.     

Roles of reactive oxygen and nitrogen species in pain

Peroxynitrite (PN; ONOO⁻) and its reactive oxygen precursor superoxide (SO; O₂^•−) are critically important in the development of pain of several etiologies including pain associated with chronic use of opiates such as morphine (also known as opiate-induced hyperalgesia and antinociceptive tolerance). This is now an emerging field in which considerable progress has been made in terms of understanding the relative contributions of SO, PN, and nitroxidative stress in pain signaling at the molecular and biochemical levels. Aggressive research in this area is poised to provide the pharmacological basis for development of novel nonnarcotic analgesics that are based upon the unique ability to selectively eliminate SO and/or PN. As we have a better understanding of the roles of SO and PN in pathophysiological settings, targeting PN may be a better therapeutic strategy than targeting SO. This is because, unlike PN, which has no currently known beneficial role, SO may play a significant role in learning and memory [1]. Thus, the best approach may be to spare SO while directly targeting its downstream product, PN. Over the past 15 years, our team has spearheaded research concerning the roles of SO and PN in pain and these results are currently leading to the development of solid therapeutic strategies in this important area.     

Ras regulation by reactive oxygen and nitrogen species

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. We have previously shown that both NO/O(2) (via nitrogen dioxide, (*)NO(2)) and superoxide radical anion (O(2)(*)(-)) promote Ras guanine nucleotide dissociation. We now show that hydrogen peroxide in the presence of transition metals (i.e., H(2)O(2)/transition metals) and peroxynitrite also trigger radical-based Ras guanine nucleotide dissociation. The primary redox-active reaction species derived from H(2)O(2)/transition metals and peroxynitrite is O(2)(*)(-) and (*)NO(2), respectively. A small fraction of hydroxyl radical (OH(*)) is also present in both. We also show that both carbonate radical (CO(3)(*)(-)) and (*)NO(2), derived from the mixture of peroxynitrite and bicarbonate, facilitate Ras guanine nucleotide dissociation. We further demonstrate that NO/O(2) and O(2)(*)(-) promote Ras GDP exchange with GTP in the presence of a radical-quenching agent, ascorbate, or NO, and generation of Ras-GTP promotes high-affinity binding of the Ras-binding domain of Raf-1, a downstream effector of Ras. S-Nitrosylated Ras (Ras-SNO) can be formed when NO serves as a radical-quenching agent, and hydroxyl radical but not (*)NO(2) or O(2)(*)(-) can further react with Ras-SNO to modulate Ras activity in vitro. However, given the lack of redox specificity associated with the high redox potential of OH(*), it is unclear whether this reaction occurs under physiological conditions.     

Interaction of reactive oxygen and nitrogen species with proteins

Free radicals and reactive oxygen or nitrogen species generated during oxidative stress and as by-products of normal cellular metabolism may damage all types of biological molecules. Proteins are major initial targets in cell. Reactions of a variety of free radicals and reactive oxygen and nitrogen species with proteins can lead to oxidative modifications of proteins such as protein hydroperoxides formation, hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, oxidation of sulfhydryl groups, oxidation of methionine residues, conversion of some amino acid residues into carbonyl groups, cleavage of the polypeptide chain and formation of cross-linking bonds. Such modifications of proteins leading to loss of their function (enzymatic activity), accumulation and inhibition of their degradation have been observed in several human diseases, aging, cell differentiation and apoptosis. Formation of specific protein oxidation products may be used as biomarkers of oxidative stress.     

Effects of reactive oxygen and nitrogen species on cyclooxygenase-1 and -2 activities

The effects of reactive oxygen species (superoxide anion radical--O(2)*-, hydrogen peroxide--H(2)O(2) and hydroxyl radical--*OH; the reaction products of xanthine plus xanthine oxidase system) and reactive nitrogen species [nitric oxide--NO*; from 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene--NOC7 and peroxynitrite--ONOO(-)] on the activities of purified cyclooxygenase (COX)-1 and -2 were studied. Xanthine plus xanthine oxidase suppressed the COX-1 and -2 activities in a xanthine oxidase concentration-dependent fashion. This effect was reversed by addition of catalase to the reactive oxygen species-generating system but not by superoxide dismutase or mannitol, indicating that H(2)O(2) is the responsible metabolite. NOC7 activated the COX-1 activity but inhibited the COX-2 activity at concentrations ranging from 1 to 50 microM. Experiments utilizing a NO* antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide revealed that the observed effects of NOC7 are caused by NO*.ONOO(-), a product of NO* and O(2)*-, both activated and inhibited the COX-1 and -2 activities, depending on ONOO(-) concentration. At a low concentration of ONOO(-) (5 microM) there was enhancement of the COX-1 and -2 activities, but with higher concentrations there was suppression of these two enzyme activities (COX-1, at 200 microM; COX-2, >50 microM). These results suggest that H(2)O(2), NO* and ONOO(-) can have different modulatory effects on the COX-1 and -2 activities.     

Cell signaling by reactive nitrogen and oxygen species in atherosclerosis

The production of reactive oxygen and nitrogen species has been implicated in atherosclerosis principally as means of damaging low-density lipoprotein that in turn initiates the accumulation of cholesterol in macrophages. The diversity of novel oxidative modifications to lipids and proteins recently identified in atherosclerotic lesions has revealed surprising complexity in the mechanisms of oxidative damage and their potential role in atherosclerosis. Oxidative or nitrosative stress does not completely consume intracellular antioxidants leading to cell death as previously thought. Rather, oxidative and nitrosative stress have a more subtle impact on the atherogenic process by modulating intracellular signaling pathways in vascular tissues to affect inflammatory cell adhesion, migration, proliferation, and differentiation. Furthermore, cellular responses can affect the production of nitric oxide, which in turn can strongly influence the nature of oxidative modifications occurring in atherosclerosis. The dynamic interactions between endogenous low concentrations of oxidants or reactive nitrogen species with intracellular signaling pathways may have a general role in processes affecting wound healing to apoptosis, which can provide novel insights into the pathogenesis of atherosclerosis.     

Formation of protein S-nitrosylation by reactive oxygen species

Abstract

In the present study, the formation of whole cellular S-nitrosylated proteins (protein-SNOs) by the reactive oxygen species (ROS), hydrogen peroxide (H₂O₂), and superoxide (O₂^??) is demonstrated. A spectrum of protein cysteine oxidative modifications was detected upon incubation of serum-starved mouse embryonic fibroblasts with increasing concentrations of exogenous H₂O₂, ranging from exclusive protein-SNOs at low concentrations to a mixture of protein-SNOs and other protein oxidation at higher concentrations to exclusively non-SNO protein oxidation at the highest concentrations of the oxidant used. Furthermore, formation of protein-SNOs was also detected upon inhibition of the antioxidant protein Cu/Zn superoxide dismutase that results in an increase in intracellular concentration of O₂^??. These results were further validated using the phosphatase and tensin homologue, PTEN, as a model of a protein sensitive to oxidative modifications. The formation of protein-SNOs by H₂O₂ and O₂^?? was prevented by the NO scavenger, c-PTIO, as well as the peroxinitrite decomposition catalyst, FETPPS, and correlated with the production or the consumption of nitric oxide (NO), respectively. These data suggest that the formation of protein-SNOs by H₂O₂ or O₂^?? requires the presence or the production of NO and involves the formation of the nitrosylating intermediate, peroxinitrite.     

Biochemical reactivity of melatonin with reactive oxygen and nitrogen species

Melatonin (N-acetyl-5-methoxytryptamine), an endogenously produced indole found throughout the animal kingdom, was recently reported, using a variety of techniques, to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. Initially, melatonin was discovered to directly scavenge the high toxic hydroxyl radical (*OH). The methods used to prove the interaction of melatonin with the *OH included the generation of the radical using Fenton reagents or the ultraviolet photolysis of hydrogen peroxide (H202) with the use of spin-trapping agents, followed by electron spin resonance (ESR) spectroscopy, pulse radiolysis followed by ESR, and several spectrofluorometric and chemical (salicylate trapping in vivo) methodologies. One product of the reaction of melatonin with the *OH was identified as cyclic 3-hydroxymelatonin (3-OHM) using high-performance liquid chromatography with electrochemical (HPLC-EC) detection, electron ionization mass spectrometry (EIMS), proton nuclear magnetic resonance (1H NMR) and COSY 1H NMR. Cyclic 3-OHM appears in the urine of humans and other mammals and in rat urine its concentration increases when melatonin is given exogenously or after an imposed oxidative stress (exposure to ionizing radiation). Urinary cyclic 3-OHM levels are believed to be a biomarker (footprint molecule) of in vivo *OH production and its scavenging by melatonin. Although the data are less complete, besides the *OH, melatonin in cell-free systems has been shown to directly scavenge H2O2, singlet oxygen (1O2) and nitric oxide (NO*), with little or no ability to scavenge the superoxide anion radical (O2*-) In vitro, melatonin also directly detoxifies the peroxynitrite anion (ONOO-) and/or peroxynitrous acid (ONOOH), or the activated form of this molecule, ONOOH*; the product of the latter interaction is proposed to be 6-OHM. How these in vitro findings relate to the in vivo antioxidant actions of melatonin remains to be established. The ability of melatonin to scavenge the lipid peroxyl radical (LOO*) is debated. The weight of the evidence is that melatonin is probably not a classic chain-breaking antioxidant, since its ability to scavenge the LOO* seems weak. Its ability to reduce lipid peroxidation may stem from its function as a preventive antioxidant (scavenging initiating radicals), or yet unidentified actions. In sum, in vitro melatonin acts as a direct free radical scavenger with the ability to detoxify both reactive oxygen and reactive nitrogen species; in vivo, it is an effective pharmacological agent in reducing oxidative damage under conditions in which excessive free radical generation is believed to be involved.     

Antimicrobial reactive oxygen and nitrogen species: concepts and controversies

Phagocyte-derived reactive oxygen and nitrogen species are of crucial importance for host resistance to microbial pathogens. Decades of research have provided a detailed understanding of the regulation, generation and actions of these molecular mediators, as well as their roles in resisting infection. However, differences of opinion remain with regard to their host specificity, cell biology, sources and interactions with one another or with myeloperoxidase and granule proteases. More than a century after Metchnikoff first described phagocytosis, and more than four decades after the discovery of the burst of oxygen consumption that is associated with microbial killing, the seemingly elementary question of how phagocytes inhibit, kill and degrade microorganisms remains controversial. This review updates the reader on these concepts and the topical questions in the field.     

Formation of reactive oxygen species following bioactivation of gentamicin

The present study investigated the ability of gentamicin to catalyze free radical reactions and probed the underlying mechanisms by hydroethidine imaging, oxygen consumption, and reduction of cytochrome c. In Epstein-Barr virus-transformed lymphoblastoid cells, a respiratory burst was induced by phorbol ester and detected by hydroethidine, a fluorescent indicator of superoxide radical. The addition of gentamicin increased the fluorescence two-fold while gentamicin did not produce fluorescence in the absence of phorbol ester. In membrane preparations, gentamicin did not enhance NADPH consumption ruling out a direct activation of NADPH oxidase. The formation of reactive oxygen species by gentamicin was additionally supported by experiments that showed gentamicin increased oxygen consumption two-fold in intact cells and a cell-free system. In addition, generation of superoxide was indicated by the gentamicin-stimulated reduction of cytochrome c. The stimulation by gentamicin depended upon the presence of iron (Fe^II/Fe^III) and of arachidonic acid as an electron donor. These results support the hypothesis that an iron-gentamicin complex can increase reactive oxygen species in nonenzymatic and in biological systems. The requirement for a reductive activation in intact cells (e.g., by a respiratory burst) is interpreted as the conversion of an inactive Fe^III-gentamicin to a redox-active Fe^II-gentamicin complex.     

Metal chelators react also with reactive oxygen and nitrogen species

Popular chelators (desferrioxamine, SIH, EDTA, EGTA, DTPA, and NTA) were demonstrated to have antioxidant properties, being able to reduce ABTS radical cation and react with peroxyl radicals, peroxynitrite, and hypochlorite. Desferrioxamine and SIH were most potent antioxidants in all cases. These results point to the necessity of a careful interpretation of experiments in which the inhibition of free radical reactions by antioxidants is used as a proof of involvement of metal ions in a reaction.     

Role of reactive oxygen and nitrogen species in the vascular responses to inflammation

Inflammation is a complex and potentially life-threatening condition that involves the participation of a variety of chemical mediators, signaling pathways, and cell types. The microcirculation, which is critical for the initiation and perpetuation of an inflammatory response, exhibits several characteristic functional and structural changes in response to inflammation. These include vasomotor dysfunction (impaired vessel dilation and constriction), the adhesion and transendothelial migration of leukocytes, endothelial barrier dysfunction (increased vascular permeability), blood vessel proliferation (angiogenesis), and enhanced thrombus formation. These diverse responses of the microvasculature largely reflect the endothelial cell dysfunction that accompanies inflammation and the central role of these cells in modulating processes as varied as blood flow regulation, angiogenesis, and thrombogenesis. The importance of endothelial cells in inflammation-induced vascular dysfunction is also predicated on the ability of these cells to produce and respond to reactive oxygen and nitrogen species. Inflammation seems to upset the balance between nitric oxide and superoxide within (and surrounding) endothelial cells, which is necessary for normal vessel function. This review is focused on defining the molecular targets in the vessel wall that interact with reactive oxygen species and nitric oxide to produce the characteristic functional and structural changes that occur in response to inflammation. This analysis of the literature is consistent with the view that reactive oxygen and nitrogen species contribute significantly to the diverse vascular responses in inflammation and supports efforts that are directed at targeting these highly reactive species to maintain normal vascular health in pathological conditions that are associated with acute or chronic inflammation.     

The role of reactive oxygen and nitrogen species in cellular iron metabolism

The catalytic role of iron in the Haber-Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.     

Mass spectrometry of protein modifications by reactive oxygen and nitrogen species

The modification of proteins by reactive oxygen and nitrogen species plays an important role in various biologic processes involving protein activation and inactivation, protein translocation and turnover during signal transduction, stress response, proliferation, and apoptosis. Recent advances in protein and peptide separation and mass spectrometry provide increasingly sophisticated tools for the quantitative analysis of such protein modifications, which are absolutely necessary for their correlation with biologic phenomena. The present review focuses specifically on the qualitative and quantitative mass spectrometric analysis of the most common protein modifications caused by reactive oxygen and nitrogen species in vivo and in vitro and details a case study on a membrane protein the sarco/endoplasmic reticulum Ca-ATPase (SERCA).     

Oxidative depolymerization of polysaccharides by reactive oxygen/nitrogen species

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are constantly produced and are tightly regulated to maintain a redox balance (or homeostasis) together with antioxidants (e.g. superoxide dismutase and glutathione) under normal physiological circumstances. These ROS/RNS have been shown to be critical for various biological events including signal transduction, aging, apoptosis, and development. Despite the known beneficial effects, an overproduction of ROS/RNS in the cases of receptor-mediated stimulation and disease-induced oxidative stress can inflict severe tissue damage. In particular, these ROS/RNS are capable of degrading macromolecules including proteins, lipids and nucleic acids as well as polysaccharides, and presumably lead to their dysfunction. The purpose of this review is to highlight (1) chemical mechanisms related to cell-free and cell-based depolymerization of polysaccharides initiated by individual oxidative species; (2) the effect of ROS/RNS-mediated depolymerization on the successive cleavage of the glycosidic linkage of polysaccharides by glycoside hydrolases; and (3) the potential biological outcome of ROS/RNS-mediated depolymerization of polysaccharides.     

The interdependence of the reactive species of oxygen, nitrogen, and carbon

This mini-review tries to summarize the main interdependences between the free radicals of oxygen, nitrogen, and carbon. Also, the main metabolic pathways for these radical species are described, as well as how these affect their interaction and functional implications. Emphasis is made on the metabolic disturbances induced by stressing aggressions that produce radical species. In this way, cellular oxidative imbalances created by the superiority of reactive oxygen species over the antioxidant systems produce both activation of nitroxide synthases and the oxidation of terminal nitrogen from l-arginine, as well as the metabolization of heme until carbon monoxide by nitric oxide-activated hemoxygenase. Also, multiple cellular protein and nucleoprotein alterations determined by these three kinds of radical species are completed by the involvement of hydrogen sulfide, which results from the degradation of l-cysteine by cistationine-γ-lyase. In this way, sufficient experimental data tend to demonstrate the involvement of hydrogen sulfide and other thiol derivatives in the interrelations between oxygen, nitrogen, and carbon, which results in a true radical cascade. Thus, oxidative stress, together with nitrosative and carbonilic stress, may constitute a central point where other factors of vulnerability meet, and their interactions could have an important impact in many modern diseases. Considering that the actions of reactive species can be most of the time corrected, future studies need to establish the therapeutical importance of various agents which modulate oxidative, nitrosative, or carbonilic stress.     

Determination of reactive oxygen and nitrogen species in rat aorta using the dichlorofluorescein assay

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH₂) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH₂-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.     

Formation of reactive oxygen species in monocytes at adhesion to glass

Processes of oxygen activation in monocytes stimulated with adhesion to glass were studied by methods of luminol-dependent and lucigen-independent chemiluminescence. It was shown that monocyte chemiluminescence was caused by cell adhesion to glass surface. Generation of reactive oxygen species at monocyte adhesion to glass was dependent on calcium ion concentration in the medium. The increase in the level of cytosolic calcium, as the extracellular calcium concentration elevated, was accompanied by the activation of phospholipase A2, 5-lypoxygenase and cycloxygenases. Magnesium ions exerted no influence on oxygen activation by cells. Incubation of cells in glucose-free medium, or the addition of glycolysis blocker (2-deoxy-D-glucose) to cell suspension led to a decrease in chemiluminescence intensity. By means of inhibitory analysis, it has been established that processes of oxygen activation are related to arachidonic acid metabolism, and depend on the activity of phospholipase A2.