Reaction of peroxynitrite with hyaluronan and related saccharides

The effects of peroxynitrite on hyaluronan has been studied by using an integrated spectroscopical approach, namely electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The reaction has been performed with the polymer, the tetrasaccharide oligomer as well as with the monosaccharides N-acetylglucosamine and glucuronic acid. The outcome of the presence of molecular oxygen and carbon dioxide has been also evaluated. Although ₁H-NMR and ESI-MS experiments did not revealed peroxynitrite-mediated modification of hyaluronan as well as of related saccharides, from spin-trapping EPR experiments it was concluded that peroxynitrite induce the formation of C-centered carbon radicals, most probably by the way of its hydroxyl radical-like reactivity. These EPR data support the oxidative pathway involved in the degradation of hyaluronan, a probable event in the development and progression of rheumatoid arthritis.     

Free-radical degradation of high-molar-mass hyaluronan induced by ascorbate plus cupric ions: evaluation of antioxidative effect of cysteine-derived compounds

Based on our previous findings, the present study has focused on free-radical-mediated degradation of the synovial biopolymer hyaluronan. The degradation was induced in vitro by the Weissberger's system comprising ascorbate plus cupric ions in the presence of oxygen, representing a model of the early phase of acute synovial joint inflammation. The study presents a novel strategy for hyaluronan protection against oxidative degradation with the use of cysteine-derived compounds. In particular, the work objectives were to evaluate potential protective effects of reduced form of L-glutathione, L-cysteine, N-acetyl-L-cysteine, and cysteamine, against free-oxygen-radical-mediated degradation of high-molar-mass hyaluronan in vitro. The hyaluronan degradation was influenced by variable activity of the tested thiol compounds, also in dependence of their concentration applied. It was found that L-glutathione exhibited the most significant protective and chain-breaking antioxidative effect against the hyaluronan degradation. Thiol antioxidative activity, in general, can be influenced by many factors such as various molecule geometry, type of functional groups, radical attack accessibility, redox potential, thiol concentration and pK(a), pH, ionic strength of solution, as well as different ability to interact with transition metals. Antioxidative activity was found to decrease in the following order: L-glutathione, cysteamine, N-acetyl-L-cysteine, and L-cysteine. These findings might be beneficial in future development of potential drugs in the treatment of synovial hyaluronan depletion-derived diseases.     

Degradation of high-molar-mass hyaluronan and characterization of fragments

A sample of high-molar mass hyaluronan was oxidized by seven oxidative systems involving hydrogen peroxide, cupric chloride, ascorbic acid, and sodium hypochlorite in different concentrations and combinations. The process of the oxidative degradation of hyaluronan was monitored by rotational viscometry, while the fragments produced were investigated by size-exclusion chromatography, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and non-isothermal chemiluminometry. The results obtained imply that the degradation of hyaluronan by these oxidative systems, some of which resemble the chemical combinations present in vivo in the inflamed joint, proceeds predominantly via hydroxyl radicals. The hyaluronan fragmentation occurred randomly and produced species with rather narrow and unimodal distribution of molar mass. Oxidative degradation not only reduces the molecular size of hyaluronan but also modifies its component monosaccharides, generating polymer fragments that may have properties substantially different from those of the original macromolecule.     

Peroxynitrite is a major trigger of cardiomyocyte apoptosis in vitro and in vivo

Recent evidence indicates that peroxynitrite represents a major cytotoxic effector in heart diseases, but its mechanisms of action are still not known exactly. Notably, the ability of peroxynitrite to trigger cardiomyocyte apoptosis, a crucial mode of cell death in many cardiac conditions, remains poorly defined. We evaluated apoptotic and necrotic cell death in cultured H9C2 cardiomyocytes, following a brief (20 min) exposure to peroxynitrite (50-500 microM). Peroxynitrite-dependent myocardial toxicity was then investigated in a rat model of myocardial ischemia-reperfusion (MIR), where the effects of peroxynitrite were blocked by the superoxide dismutase mimetics and peroxynitrite scavenger Mn(III)-tetrakis(4-benzoic acid) porphyrin (MnTBAP). In vitro, peroxynitrite killed cardiomyocytes mostly through apoptosis (DNA fragmentation, apoptotic nuclear alterations, caspase-3 activation, and PARP cleavage), but not necrosis (propidium iodide staining and LDH release). In vivo, MIR triggered myocardial oxidative stress (malondialdehyde generation), nitrotyrosine formation, neutrophil accumulation, and the cleavage of caspase-3 and PARP, indicating ongoing myocardial apoptosis. MnTBAP suppressed these alterations, allowing a considerable reduction of myocardial injury. Thus, peroxynitrite triggers apoptosis in cardiomyocytes in vitro and in the myocardium in vivo, through a pathway involving caspase-3 activation and the cleavage of PARP. These results provide important novel information on the mechanisms of myocardial toxicity of peroxynitrite.     

Chain scission of hyaluronan by peroxynitrite

The reaction of peroxynitrite with the biopolymer hyaluronan has been studied using stopped-flow techniques combined with detection of molecular weight changes using the combination of gel permeation chromatography and multiangle laser light scattering. From the effect of peroxynitrite on the yield of hyaluronan chain breaks, it was concluded that the chain breaks were caused by hydroxyl radicals which escape a cage containing the *OH NO*(2) radical pair. The yield of free hydroxyl radicals was determined as 5+/-1% (as a proportion of the total peroxynitrite concentration). At high peroxynitrite concentrations, it was observed that the yield of chain breaks leveled out, an effect largely attributable to the scavenging of hydroxyl radicals by nitrite ions present in the peroxynitrite preparation. These experiments also provided some support for a previous proposal that the adduct formed between ONOOH and ONOO(-) might itself produce hydroxyl radicals. The rate of this reaction would have to be of the order of 0.05 s(-1) to produce hydroxyl radical yields that would account quantitatively for chain break yields at high peroxynitrite concentrations. By carrying out experiments at higher hyaluronan concentrations, it was also concluded that an additional yield of chain breaks was produced by the bimolecular reaction of the polymer with ONOOH at a rate constant of about 10 dm(3)mol(-1)s(-1). At 5.3 x 10(-3)mol dm(-3) hyaluronan, this amounted to 3.5% chain breaks (per peroxynitrite concentration). These conclusions support the proposal that the yield of hydroxyl radicals arising from the isomerization of ONOOH to nitrate ions is relatively low.     

Simple primary structure, complex turnover regulation and multiple roles of hyaluronan

Hyaluronan is a major macromolecular polysaccharide component of the extra-cellular matrix that confers structural frameworks for cells. Despite its relatively simple chemical composition, hyaluronan mediates many other important functional aspects including signalling activity during embryonic morphogenesis, cellular regeneration and wound healing. Abnormalities in hyaluronan metabolism have been implicated in many diseases, such as inflammatory disorders, cardiovascular diseases and cancer. To date, it has become increasingly clear that hyaluronan production in vertebrates is tightly regulated by three hyaluronan synthases and that hyaluronan catabolism is regulated by an enzymatic degradation reaction involving several hyaluronidases. Together, these discoveries have provided key insights into the physiological roles of hyaluronan and a deeper understanding of the mechanisms underlying altered hyaluronan turnover in diseases. The central aim of this review article is therefore to highlight the multiple roles of hyaluronan in physiological and pathological states via its complex turnover regulation.     

20S proteasome mediated degradation of DHFR: implications in neurodegenerative disorders

The 20S proteasome is responsible for the degradation of protein substrates implicated in the onset and progression of neurodegenerative disorders, such as alpha-synuclein and tau protein. Here we show that the 20S proteasome isolated from bovine brain directly hydrolyzes, in vitro, the dihydrofolate reductase (DHFR), demonstrated to be involved in the pathogenesis of neurodegenerative diseases. Furthermore, the DHFR susceptibility to proteolysis is enhanced by oxidative conditions induced by peroxynitrite, mimicking the oxidative environment typical of these disorders. The results obtained suggest that the folate metabolism may be impaired by an increased degradation of DHFR, mediated by the 20S proteasome.     

The reaction of nitric oxide with 6-hydroxydopamine: implications for Parkinson's disease.

Oxidation of catecholamines is suggested to contribute to oxidative stress in Parkinson's disease. Nitric oxide (*NO) is able to oxidize cyclic compounds like ubiquinol; moreover, recent lines of evidence proposed a direct role of *NO and its by-product peroxynitrite in the pathophysiology of Parkinson's disease. The aim of this study was to analyze the potential reaction between 6-hydroxydopamine, a classic inducer of Parkinson's disease, and *NO. The results showed that *NO reacts with the deprotonated form of 6-hydroxydopamine at pH 7 and 37 degrees C with a second-order rate constant of 1.5 x 10(3) M(-1) x s(-1) as calculated by the rate of *NO decay measured with an amperometric sensor. Accordingly, the rates of formation of 6-hydroxy-dopamine quinone were dependent on *NO concentration. The coincubation of *NO and 6-hydroxydopamine with either bovine serum albumin or alpha-synuclein led to tyrosine nitration of the protein, in a concentration dependent-manner and sensitive to superoxide dismutase. These findings suggest the formation of peroxynitrite during the redox reactions following the interaction of 6-hydroxydopamine with *NO. The implications of this reaction for in vivo models are discussed in terms of the generation of reactive nitrogen and oxygen species within a propagation process that may play a significant role in neurodegenerative diseases.     

Effect of nitrosative stress on Schizosaccharomyces pombe: inactivation of glutathione reductase by peroxynitrite

Oxidative stress has been shown to alter cellular redox status in various cell types. Changes in expressions of several antioxidative and antistress-responsive genes along with activation or inactivation of various proteins were also reported during oxidative insult as well as during nitrosative stress. In the present study, we show the effect of nitrosative stress on cellular redox status of fission yeast Schizosaccharomyces pombe. This is the first report of S-nitrosoglutathione (GSNO) reductase activity in S. pombe and its inactivation by GSNO. We also show the inactivation of glutathione reductase (GR) and glutathione peroxidase in the presence of various reactive nitrogen species in vivo. In addition, we first observe the inactivation of GR by peroxynitrite in vivo using S. pombe cells and also similar observations under in vitro conditions. An immunoreactive band against monoclonal anti-3-nitrotyrosine antibody confirms the modification of GR under in vitro conditions. We also show the effect of nitrosative stress on Deltapap1 cells of S. pombe, which are more sensitive to nitrosative stress, indicating the involvement of Pap1 in the protection against nitrosative stress. Finally, exposure of S. pombe cells to reactive nitrogen species reveals an important role of cellular thiol pool in protection against nitrosative stress.     

Oxidative stress and inhibition of oxidative phosphorylation induced by peroxynitrite and nitrite in rat brain subcellular fractions

Nitrite and nitrate, two endogenous oxides of nitrogen, are toxic in vivo. Furthermore, the reaction of superoxide (produced by all aerobic cells) with nitric oxide (NO) generates peroxynitrite, a potent oxidizing agent, that can cause biological oxidative stress. Using subcellular fractions from rat brain hemispheres we studied oxidative stress induced by these nitrogen compounds with special emphasis on nitrite. The consumption of Vitamin C (ascorbate) and Vitamin E (alpha tocopherol), two of the important nutritional antioxidants, was followed in synaptosomes (nerve-ending particles) and mitochondria along with changes in parameters of mitochondrial oxidative phosphorylation. Nitrite, but not nitrate, oxidized ascorbate without oxidizing alpha tocopherol in both synaptosomes and mitochondria whereas peroxynitrite oxidized both ascorbate and alpha tocopherol. Functionally, both nitrite and peroxynitrite inhibited mitochondrial oxidative phosphorylation. Nitrite was less potent than peroxynitrite when the effects of equal concentrations of the two were compared. However, since nitrite is much more stable than peroxynitrite the impact of nitrite as an oxidant in vivo could be as much or even more significant than peroxynitrite. Nitrate would not have similar action unless it is reduced to nitrite. It is possible that nitrite may impair oxidative phosphorylation through modulating levels of nitric oxide, changing the activity of heme proteins or a mild uncoupling of mitochondria.     

Viral mutation accelerated by nitric oxide production during infection in vivo.

Nitric oxide (NO), superoxide (O(2)(-)), and their reaction product peroxynitrite (ONOO(-)) are generated in excess during a host's response against viral infection, and contribute to viral pathogenesis by promoting oxidative stress and tissue injury. Here we demonstrate that NO and peroxynitrite greatly accelerates the mutation of Sendai virus (SeV), a nonsegmented negative-strand RNA virus, by using green fluorescent protein (GFP) inserted into and expressed by a recombinant SeV (GFP-SeV) as an indicator for mutation. GFP-SeV mutation frequencies were much higher in the wild-type mice than in those lacking inducible NO synthase, suggesting that mutation of the virus in vivo is NO dependent. High levels of NO and NO-mediated oxidative stress were induced by GFP-SeV infection in the lung of the wild-type mice, but not in the iNOS-deficient mice, as evidenced by electron spin resonance spectroscopy and immunohistochemical analysis for nitrotyrosine formation as well as histopathological examination. Furthermore, peroxynitrite, an NO-derived reactive nitrogen intermediate, enhanced viral mutation in vitro. These results indicate that the oxidative stress induced by NO produced during the natural course of viral infection increases mutation, expands the quasispecies spectrum, and facilitates evolution of RNA viruses.     

Free‐Radical Degradation of High‐Molar‐Mass Hyaluronan Induced by Ascorbate plus Cupric Ions: Evaluation of Antioxidative Effect of Cysteine‐Derived Compounds

Based on our previous findings, the present study has focused on free‐radical‐mediated degradation of the synovial biopolymer hyaluronan. The degradation was induced in vitro by the Weissberger's system comprising ascorbate plus cupric ions in the presence of oxygen, representing a model of the early phase of acute synovial joint inflammation. The study presents a novel strategy for hyaluronan protection against oxidative degradation with the use of cysteine‐derived compounds. In particular, the work objectives were to evaluate potential protective effects of reduced form of L ‐glutathione, L ‐cysteine, N‐acetyl‐L ‐cysteine, and cysteamine, against free‐oxygen‐radical‐mediated degradation of high‐molar‐mass hyaluronan in vitro. The hyaluronan degradation was influenced by variable activity of the tested thiol compounds, also in dependence of their concentration applied. It was found that L ‐glutathione exhibited the most significant protective and chain‐breaking antioxidative effect against the hyaluronan degradation. Thiol antioxidative activity, in general, can be influenced by many factors such as various molecule geometry, type of functional groups, radical attack accessibility, redox potential, thiol concentration and pK_a, pH, ionic strength of solution, as well as different ability to interact with transition metals. Antioxidative activity was found to decrease in the following order: L ‐glutathione, cysteamine, N‐acetyl‐L ‐cysteine, and L ‐cysteine. These findings might be beneficial in future development of potential drugs in the treatment of synovial hyaluronan depletion‐derived diseases.     

Formation of phospholipid hydroperoxides and its inhibition by alpha-tocopherol in rat brain synaptosomes induced by peroxynitrite

Peroxynitrite resulted from the reaction of nitric oxide and superoxide anion has been implicated in the genesis of neurotoxicity. In this study, the oxidation of phospholipids in rat brain synaptosomes induced by peroxynitrite generated from 3-morpholinosydnonimine (SIN-1) was studied in vitro. The formation and accumulation of phospholipid hydroperoxides, including phosphatidylcholine hydroperoxide (PCOOH) and phosphatidyl-ethanolamine hydroperoxide (PEOOH) in rat brain synaptosomes induced by peroxynitrite, were observed. PEOOH and PCOOH were formed rapidly and SIN-1 concentration-dependently. The hydroperoxides formed in synaptosomes were unstable and it was suggested that phospholipase A2 played a role in degradation of the hydroperoxides. The endogenous alpha-tocopherol acted as a potent antioxidant. It was oxidized very rapidly and concentration-dependently by SIN-1 to alpha-tocopheryl quinone. Furthermore, uric acid was found to be an effective antioxidant in inhibiting oxidative damage to synaptosomal lipids induced by SIN-1. The results provide direct evidence to show that peroxynitrite can not only deplete alpha-tocopherol, but also cause production of phospholipid hydroperoxides resulting in disrupted brain tissue.     

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/l peroxynitrite additionally increased S-nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (-)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/l)-treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.     

The role of carbon dioxide in free radical reactions of the organism

Carbon dioxide interacts both with reactive nitrogen species and reactive oxygen species. In the presence of superoxide, NO reacts to form peroxynitrite that reacts with CO2 to give nitrosoperoxycarbonate. This compound rearranges to nitrocarbonate which is prone to further reactions. In an aqueous environment, the most probable reaction is hydrolysis producing carbonate and nitrate. Thus the net effect of CO2 is scavenging of peroxynitrite and prevention of nitration and oxidative damage. However, in a nonpolar environment of membranes, nitrocarbonate undergoes other reactions leading to nitration of proteins and oxidative damage. When NO reacts with oxygen in the absence of superoxide, a nitrating species N2O3 is formed. CO2 interacts with N2O3 to produce a nitrosyl compound that, under physiological pH, is hydrolyzed to nitrous and carbonic acid. In this way, CO2 also prevents nitration reactions. CO2 protects superoxide dismutase against oxidative damage induced by hydrogen peroxide. However, in this reaction carbonate radicals are formed which can propagate the oxidative damage. It was found that hypercapnia in vivo protects against the damaging effects of ischemia or hypoxia. Several mechanisms have been suggested to explain the protective role of CO2 in vivo. The most significant appears to be stabilization of the iron-transferrin complex which prevents the involvement of iron ions in the initiation of free radical reactions.     

Age-associated changes in erythrocyte glutathione peroxidase activity: correlation with total antioxidant potential

Oxidative stress is believed to play a central role in aging and age-associated diseases. It leads to oxidative changes in human red blood cells (RBCs) in vivo and in vitro. In this study, we evaluated the oxidative damage to the erythrocytes during aging in the humans using RBC as a model, by measuring the cytosolic antioxidant enzyme glutathione peroxidase (GPx) activity. GPx activity was found to be significantly decreased as a function of human age and positively correlated with total antioxidant capacity, while negatively correlated with SOD activity. Thus, results of the present study showed involvement of oxidative stress as one of the risk factors, which can initiate and/or promote human aging.     

Aurothiomalate as Preventive and Chain‐Breaking Antioxidant in Radical Degradation of High‐Molar‐Mass Hyaluronan

The potential anti‐ or pro‐oxidative effects of a disease‐modifying antirheumatic drug, aurothiomalate, to protect high‐molar‐mass hyaluronan against radical degradation were investigated along with L ‐glutathione – tested in similar functions. Hyaluronan degradation was induced by the oxidative system Cu^II plus ascorbate known as the Weissberger's oxidative system. The time‐ and dose‐dependent changes of the dynamic viscosity of the hyaluronan solutions were studied by the method of rotational viscometry. Additionally, the antioxidative activity of aurothiomalate expressed as a radical‐scavenging capacity based on a decolorization 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) assay was inspected. At the higher concentrations tested, L ‐glutathione showed excellent scavenging of ^.OH and peroxyl‐type radicals, however, at the lowest concentration applied, its pro‐oxidative effect was revealed. The effects of aurothiomalate on hyaluronan degradation were similar to that of L ‐glutathione, however, at the lowest concentration tested, no significant pro‐oxidant effect was observed.     

Manganese and iron porphyrins catalyze peroxynitrite decomposition and simultaneously increase nitration and oxidant yield: implications for their use as peroxynitrite scavengers in vivo.

Twelve substituted metalloporphyrins (MPs), some of which have been previously characterized with respect to superoxide dismutase and peroxynitrite decomposing activities, were evaluated for their ability to scavenge peroxynitrite in vitro at 37 degrees C. Because the overall effectiveness of MPs as catalytic peroxynitrite scavengers is a function of (1) how fast they react with peroxynitrite, (2) how fast they cycle back to the starting compound, and (3) how well they contain or quench the reactive intermediates generated, all of these properties were evaluated and compared directly under the same conditions. Of the various MPs tested, only the iron and manganese porphyrins showed significant reactivity with peroxynitrite. The Mn(IV) intermediates resulting from oxidation by peroxynitrite were relatively stable and rereduction to the Mn(III) forms was rate-limiting to catalytic decomposition of peroxynitrite. However, in the presence of oxidizeable substrates like phenolics, rereduction of Mn(IV) forms occurred very rapidly and both the Mn- and Fe-porphyrins catalyzed nitration and oxidation by peroxynitrite. Mn- and Fe-porphyrins enhanced the yield of nitrated phenolics by peroxynitrite as much as 5-fold at pH 7.4 and up to 12-fold at pH 9. 1, while total oxidative yield was more than doubled. Nitration enhancement by MPs was effectively inhibited by ascorbate, glutathione, or serum, although much higher concentrations of ascorbate were required to inhibit nitration catalyzed by either Mn or Fe tetramethylpyridyl porphyrin. Catalysis of peroxynitrite nitration by MPs appears to proceed via a radical-mediated reaction mechanism whereby the phenolic substrate rapidly reduces Mn(IV) = O or Fe[IV] = O to the +3 state to yield phenoxyl radical which then combines with the other primary product, nitrogen dioxide. Based on the rate constants and the proposed reaction mechanism, it is reasonable to suggest that Mn and Fe porphyrins could detoxify peroxynitrite in vivo by efficiently trapping the relatively unreactive peroxynitrite anion and, in effect, channeling it into a single reaction pathway which could then be more effectively scavenged by cellular reductants like ascorbate.     

A multipumping flow system for in vitro screening of peroxynitrite scavengers

Peroxynitrite anion is a reactive nitrogen species formed in vivo by the rapid, controlled diffusion reaction between nitric oxide and superoxide radicals. By reacting with several biological molecules, peroxynitrite may cause important cellular and tissue deleterious effects, which have been associated with many diseases. In this work, an automated flow-based procedure for the in vitro generation of peroxynitrite and subsequent screening of the scavenging activity of selected compounds is developed. This procedure involves a multipumping flow system (MPFS) and exploits the ability of compounds such as lipoic acid, dihydrolipoic acid, cysteine, reduced glutathione, oxidized glutathione, sulindac, and sulindac sulfone to inhibit the chemiluminescent reaction of luminol with peroxynitrite under physiological simulated conditions. Peroxynitrite was generated in the MPFS by the online reaction of acidified hydrogen peroxide with nitrite, followed by a subsequent stabilization by merging with a sodium hydroxide solution to rapidly quench the developing reaction. The pulsed flow and the timed synchronized insertion of sample and reagent solutions provided by the MPFS ensure the establishment of the reaction zone only inside the flow cell, thus allowing maximum chemiluminescence emission detection. The results obtained for the assayed compounds show that, with the exception of oxidized glutathione, all are highly potent scavengers of peroxynitrite at the studied concentrations.