Protective Effect of Dinitrosyl Iron Complexes Bound with Hemoglobin on Oxidative Modification by Peroxynitrite.

Dinitrosyl iron complexes (DNICs) are a physiological form of nitric oxide (•NO) in an organism. They are able not only to deposit and transport •NO, but are also to act as antioxidant and antiradical agents. However, the mechanics of hemoglobin-bound DNICs (Hb-DNICs) protecting Hb against peroxynitrite-caused, mediated oxidative modification have not yet been scrutinized. Through EPR spectroscopy we show that Hb-DNICs are destroyed under the peroxynitrite action in a dose-dependent manner. At the same time, DNICs inhibit the oxidation of tryptophan and tyrosine residues and formation of carbonyl derivatives. They also prevent the formation of covalent crosslinks between Hb subunits and degradation of a heme group. These effects can arise from the oxoferryl heme form being reduced, and they can be connected with the ability of DNICs to directly intercept peroxynitrite and free radicals, which emerge due to its homolysis. These data show that DNICs may ensure protection from myocardial ischemia.

Pulmonaria obscura and Pulmonaria officinalis Extracts as Mitigators of Peroxynitrite-Induced Oxidative Stress and Cyclooxygenase-2 Inhibitors-In Vitro and In Silico Studies.

The Pulmonaria species (lungwort) are edible plants and traditional remedies for different disorders of the respiratory system. Our work covers a comparative study on biological actions in human blood plasma and cyclooxygenase-2 (COX-2) -inhibitory properties of plant extracts (i.e., phenolic-rich fractions) originated from aerial parts of P. obscura Dumort. and P. officinalis L. Phytochemical profiling demonstrated the abundance of phenolic acids and their derivatives (over 80% of the isolated fractions). Danshensu conjugates with caffeic acid, i.e., rosmarinic, lithospermic, salvianolic, monardic, shimobashiric and yunnaneic acids were identified as predominant components. The examined extracts (1-100 µg/mL) partly prevented harmful effects of the peroxynitrite-induced oxidative stress in blood plasma (decreased oxidative damage to blood plasma components and improved its non-enzymatic antioxidant capacity). The cellular safety of the extracts was confirmed in experimental models of blood platelets and peripheral blood mononuclear cells. COX-2 inhibitor screening evidently suggested a stronger activity of P. officinalis (IC50 of 13.28 and 7.24 µg/mL, in reaction with synthetic chromogen and physiological substrate (arachidonic acid), respectively). In silico studies on interactions of main components of the Pulmonaria extracts with the COX-2 demonstrated the abilities of ten compounds to bind with the enzyme, including rosmarinic acid, menisdaurin, globoidnan A and salvianolic acid H.

Protective Role of Glutathione against Peroxynitrite-Mediated DNA Damage During Acute Inflammation.

Inflammation is an immune response to protect against various types of infections. When unchecked, acute inflammation can be life-threatening, as seen with the current coronavirus pandemic. Strong oxidants, such as peroxynitrite produced by immune cells, are major mediators of the inflammation-associated pathogenesis. Cellular thiols play important roles in mitigating inflammation-associated macromolecular damage including DNA. Herein, we have demonstrated a role of glutathione (GSH) and other thiols in neutralizing the effect of peroxynitrite-mediated DNA damage through stable GSH-DNA adduct formation. Our observation supports the use of thiol supplements as a potential therapeutic strategy against severe COVID-19 cases and a Phase II (NCT04374461) open-label clinical trial launched in early May 2020 by the Memorial Sloan Kettering Cancer Center.

Pine (Pinus sylvestris L.) bark proanthocyanidins affords prevention of peroxynitrite-induced l-tyrosine nitration, DNA damage and hydroxyl radical formation.

Peroxynitrite is known as a strong deleterious species that may readily trigger several geriatric diseases via injuring cellular constituents. Proanthocyanidins, a biological flavonoids constituent of Pinus sylvestris L. bark, has been attributed a large variety of pharmacological functions to its antioxidant potential. The results revealed that peroxynitrite could cause the generation of hydroxyl radical, the breakage of φX-174 plasmid DNA brand as well as the nitration of L-tyrosine. However, pine (Pinus sylvestris L.) bark proanthocyanidins extracts at low concentration range markedly inhibited the peroxynitrite -induced the formation of open circular DNA form (IC50 = 5.03±0.39 mg/mL). The 3-Nitro-L-tyrosine generated by the reaction of peroxynitrite with L-tyrosine was reduced by PBP (IC50 = 1.01±0.01 mg/mL). Besides, electron spin resonance spectroscopy data indicates that the intensive signal of dimethyl pyridine N-oxide hydroxyl radical adduct from peroxynitrite was reversed by pine bark proanthocyanidins extracts (IC50 =1.02±0.04 mg/mL). Moreover, the obtained data shows that PBP provides more efficient protection against peroxynitrite than that of ascorbic acid. Together, the present study suggests that pine bark proanthocyanidins could exert potent preventive activity against peroxynitrite -elicited cytotoxicity on the biomacromolecules, a study-worthy finding with pharmacological importance.

Peroxynitrite Detoxification by Human Haptoglobin:Hemoglobin Complexes: A Comparative Study.

Haptoglobin (Hp) reacts with dimeric hemoglobin (Hb), shifts the equilibrium in favor of the αß dimer and displays heme-based catalysis. Here, kinetics of peroxynitrite scavenging by ferric human haptoglobin1-1:hemoglobin and haptoglobin2-2:hemoglobin complexes (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively) is reported between pH 6.2 and 8.3 at 20.0 °C. The reactivity of Hp1-1:Hb(III) and Hp2-2:Hb(III) against peroxynitrite is similar to that of tetrameric Hb(III), reflecting the R-like structure of the αß dimers of Hb(III) bound to Hp. To investigate the protective role of Hp1-1:Hb(III) and Hp2-2:Hb(III) against peroxynitrite-mediated nitration, the relative yield of nitro-l-tyrosine formed by the reaction of peroxynitrite with free l-tyrosine was determined. Interestingly, both Hp1-1:Hb(III) and Hp2-2:Hb(III) impair peroxynitrite-mediated nitration of free l-tyrosine. Therefore, Hp:Hb complexes could participate to the detoxification of reactive nitrogen and oxygen species in vivo, contributing to prevent extra-erythrocytic Hb-induced damage during hemolytic crisis.

Cellular mechanisms of peroxynitrite-induced neuronal death.

Peroxynitrite (ONOO-) is a strong biological oxidant formed by the diffusion-limited reaction of nitric oxide (NO-) and superoxide anion (O2-). It has long been theorized that peroxynitrite generation could be the cause in a number of pathological conditions ranging from atherosclerosis to inflammatory, autoimmune, heart and neurodegenerative diseases. Its relatively long biological half-life and high reactivity allows peroxynitrite to oxidize a number of different targets in the cell. In physiologically relevant conditions peroxynitrite can directly react with thiols, or the radical products of peroxynitrite decomposition may indirectly oxidize other cellular components such as lipids, proteins and DNA. Downstream, oxidative modifications caused by peroxynitrite trigger cell death by a variety of mechanisms depending on the concentration of the oxidant. Peroxynitrite stimulates necrosis, apoptosis, autophagy, parthanatos and necroptosis. Here we review the mechanisms activated by peroxynitrite to cause neuronal death.

Neuroprotective Efficacy of Mitochondrial Antioxidant MitoQ in Suppressing Peroxynitrite-Mediated Mitochondrial Dysfunction Inflicted by Lead Toxicity in the Rat Brain.

Lead (Pb) is one of the most pollutant metals that accumulate in the brain mitochondria disrupting mitochondrial structure and function. Though oxidative stress mediated by reactive oxygen species remains the most accepted mechanism of Pb neurotoxicity, some reports suggest the involvement of nitric oxide (•NO) and reactive nitrogen species in Pb-induced neurotoxicity. But the impact of Pb neurotoxicity on mitochondrial respiratory enzyme complexes remains unknown with no relevant report highlighting the involvement of peroxynitrite (ONOO-) in it. Herein, we investigated these effects in in vivo rat model by oral application of MitoQ, a known mitochondria-specific antioxidant with ONOO- scavenging activity. Interestingly, MitoQ efficiently alleviated ONOO--mediated mitochondrial complexes II, III and IV inhibition, increased mitochondrial ATP production and restored mitochondrial membrane potential. MitoQ lowered enhanced caspases 3 and 9 activities upon Pb exposure and also suppressed synaptosomal lipid peroxidation and protein oxidation accompanied by diminution of nitrite production and protein-bound 3-nitrotyrosine. To ascertain our in vivo findings on mitochondrial dysfunction, we carried out similar experiments in the presence of different antioxidants and free radical scavengers in the in vitro SHSY5Y cell line model. MitoQ provided better protection compared to mercaptoethylguanidine, N-nitro-L-arginine methyl ester and superoxide dismutase suggesting the predominant involvement of ONOO- compared to •NO and O2•-. However, dimethylsulphoxide and catalase failed to provide protection signifying the noninvolvement of •OH and H2O2 in the process. The better protection provided by MitoQ in SHSY5Y cells can be attributed to the fact that MitoQ targets mitochondria whereas mercaptoethylguanidine, N-nitro-L-arginine methyl ester and superoxide dismutase are known to target mainly cytoplasm and not mitochondria. Taken together the results from the present study clearly brings out the potential of MitoQ against ONOO--induced toxicity upon Pb exposure indicating its therapeutic potential in metal toxicity.

Molecular mechanisms of diabetic retinopathy: potential therapeutic targets.

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults in United States. Research indicates an association between oxidative stress and the development of diabetes complications. However, clinical trials with general antioxidants have failed to prove effective in diabetic patients. Mounting evidence from experimental studies that continue to elucidate the damaging effects of oxidative stress and inflammation in both vascular and neural retina suggest its critical role in the pathogenesis of DR. This review will outline the current management of DR as well as present potential experimental therapeutic interventions, focusing on molecules that link oxidative stress to inflammation to provide potential therapeutic targets for treatment or prevention of DR. Understanding the biochemical changes and the molecular events under diabetic conditions could provide new effective therapeutic tools to combat the disease.

Estradiol attenuates high glucose-induced endothelial nitrotyrosine: role for neuronal nitric oxide synthase.

Hyperglycemia in diabetes causes increased oxidative stress in the vascular endothelium with generation of free radicals such as superoxide. Peroxynitrite, a highly reactive species generated from superoxide and nitric oxide (NO), induces proinflammatory tyrosine nitration of intracellular proteins under such conditions. The female sex hormone estrogen appears to exert protective effects on the nondiabetic endothelium. However, several studies show reduced vascular protection in women with diabetes, suggesting alterations in estrogen signaling under high glucose. In this study, we examined the endothelial effects of estrogen under increasing glucose levels, focusing on nitrotyrosine and peroxynitrite. Human umbilical vein endothelial cells were incubated with normal (5.5 mM) or high (15.5 or 30.5 mM) glucose before addition of estradiol (E2, 1 or 10 nM). Selective NO synthase (NOS) inhibitors were used to determine the role of specific NOS isoforms. Addition of E2 significantly reduced high glucose-induced increase in peroxynitrite and consequently, nitrotyrosine. The superoxide levels were unchanged, suggesting effects on NO generation. Inhibition of neuronal NOS (nNOS) reduced high glucose-induced nitrotyrosine, demonstrating a critical role for this enzyme. E2 increased nNOS activity under normal glucose while decreasing it under high glucose as determined by its phosphorylation status. These data show that nNOS contributes to endothelial peroxynitrite and subsequent nitrotyrosine generation under high glucose, which can be attenuated by E2 through nNOS inhibition. The altered regulation of nNOS by E2 under high glucose is a potential therapeutic target in women with diabetes.

The extract from hop cones (Humulus lupulus) as a modulator of oxidative stress in blood platelets.

The plant Humulus lupulus is known as the raw material of the brewing industry. Hop cones, rich in polyphenolic compounds and acyl phloroglucides, are widely used to preserve beer and to give it a characteristic aroma and flavor. Hop cones have long been used for medicinal purposes. In particular, hop preparations were mainly recommended for the treatment of sleeping disorders. The antioxidative action of hop cones, however, is poorly understood. The aim of our present study was to investigate in vitro changes in human blood platelets induced by peroxynitrite (ONOO(-), the compound of particular importance for vascular thrombosis and inflammatory process) in the presence of hop cone extract (Humulus lupulus). The antioxidative action of the extract was also compared with the properties of a well-characterized antioxidative commercial monomeric polyphenol, resveratrol (3,4',5-trihydroxystilbene) in a model system in vitro. Various biomarkers of oxidative/nitrative stress, such as carbonyl groups, 3-nitrotyrosine and thiobarbituric acid reactive substances (TBARS) were estimated. The 3-nitrotyrosine formation and carbonyl group generation was assessed by the use of a competition ELISA test and ELISA test, respectively. Tested plant extract (12.5-50 µg/ml), like resveratrol, significantly inhibited protein carbonylation and nitration in the blood platelets treated with ONOO(-) (0.1 mM). The extract from hop cones, like resveratrol, also caused a distinct reduction of platelet lipid peroxidation induced by ONOO(-). The present results indicate that the hope cone extract has in vitro protective effects against ONOO(-), such as induced oxidative/nitrative damage to the human platelet proteins and lipids. However, in comparative studies the extract was not found to be a more effective antioxidant than the solution of pure resveratrol.

Effects of a potent peroxynitrite decomposition catalyst in murine models of endotoxemia and sepsis.

Excessive free-radical production due to various bacterial components released during bacterial infection has been linked to cell death and tissue injury. Peroxynitrite is a highly reactive oxidant produced by the combination of nitric oxide (NO) and superoxide anion, which has been implicated in cell death and tissue injury in various forms of critical illness. Pharmacological decomposition of peroxynitrite may represent a potential therapeutic approach in diseases associated with the overproduction of NO and superoxide. In the present study, we tested the effect of a potent peroxynitrite decomposition catalyst in murine models of endotoxemia and sepsis. Mice were injected i.p. with LPS 40 mg/kg with or without FP15 [Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin] (0.1, 0.3, 1, 3, or 10 mg/kg per hour). Mice were killed 12 h later, followed by the harvesting of samples from the lung, liver, and gut for malondialdehyde and myeloperoxidase measurements. In other subsets of animals, blood samples were obtained by cardiac puncture at 1.5, 4, and 8 h after LPS administration for cytokine (TNF-α, IL-1ß, and IL-10), nitrite/nitrate, alanine aminotransferase, and blood urea nitrogen measurements. Endotoxemic animals showed an increase in survival from 25% to 80% at the FP15 doses of 0.3 and 1 mg/kg per hour. The same dose of FP15 had no effect on plasma levels of nitrite/nitrate. There was a reduction in liver and lung malondialdehyde in the endotoxemic animals pretreated with FP15, as well as in hepatic myeloperoxidase and biochemical markers of liver and kidney damage (alanine aminotransferase and blood urea nitrogen). In a bacterial model of sepsis induced by cecal ligation and puncture, FP15 treatment (0.3 mg/kg per day) significantly protected against mortality. The current data support the view that peroxynitrite is a critical factor mediating liver, gut, and lung injury in endotoxemia and septic shock: its pharmacological neutralization may be of therapeutic benefit.

[Pathophysiological mechanisms and treatment of septic shock-induced vasoplegia]. / Physiopathologie et traitement de la vasoplégie au cours du sepsis.

The hemodynamics of septic shock is characterized by a primary reduction of vascular tone, which defines vasoplegia. Septic vasoplegia is due to reduced endogenous production of vasopressin, as well as to the overproduction of vasodilating molecules (nitric oxide, prostacyclin, peroxynitrite and kynurenine) and the opening of ATP-sensitive potassium channels. Treatment is supportive and includes primarily alpha-adrenergic catecholamines. Vasopressin may also be useful, although its place is still controversial. Further agents can improve the vascular responsiveness to catecholamines, most notably low doses hydrocortisone, and, to a lesser extent, activated protein C. Further, innovative therapies, based on recent understanding of pathophysiological mechanisms, might become useful agents to treat septic vasoplegia in the future.

Myeloperoxidase interaction with peroxynitrite: chloride deficiency and heme depletion.

Myeloperoxidase (MPO) is a hemoprotein involved in the leukocyte-mediated defense mechanism and uses hydrogen peroxide (H2O2) and chloride (Cl(-)) to produce hypochlorous acid. In human saliva and in hypochloremic alkalosis syndrome occurring in breast-fed infants, the MPO-H2O2 system functions in a lower Cl(-) concentration (10-70 mM) compared to plasma levels (100 mM) as part of the antibacterial defense system. The impact of low Cl(-) concentration and exposure to high peroxynitrite (ONOO(-)) synthesized from cigarette smoke or oxidative stress on MPO function is still unexplored. Rapid mixing of ONOO(-) and MPO caused immediate formation of a transient intermediate MPO Compound II, which then decayed to MPO-Fe(III). Double mixing of MPO with ONOO(-) followed by H2O2 caused immediate formation of Compound II, followed by MPO heme depletion, a process that occurred independent of ONOO(-) concentration. Peroxynitrite/H2O2-mediated MPO heme depletion was confirmed by HPLC analysis, and in-gel heme staining showing 60-70% less heme content compared to the control. A nonreducing denaturing SDS-PAGE showed no fragmentation or degradation of protein. Myeloperoxidase heme loss was completely prevented by preincubation of MPO with saturating amounts of Cl(-). Chloride binding to the active site of MPO constrains ONOO(-) binding by filling the space directly above the heme moiety or by causing a protein conformational change that constricts the distal heme pocket, thus preventing ONOO(-) from binding to MPO heme iron. Peroxynitrite interaction with MPO may serve as a novel mechanism for modulating MPO catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

[Novel antioxidant therapeutic strategies for cardiovascular dysfunction associated with ageing]. / Uj antioxidáns terápiás stratégiák az idoskori cardiovascularis diszfunkció kezelésében.

UNLABELLED: Overproduction of free radicals in ageing tissues induces nitro-oxidative stress, which has been implicated in the functional decline of the cardiovascular system at old age. Toxic oxidants like hydrogen peroxide or peroxynitrite damage proteins and DNA and activate several pathways causing tissue injury, including the poly(ADP-ribose) polymerase (PARP) pathway. AIM: First, we tested whether the inhibition of PARP can improve endothelial dysfunction induced by hydrogen peroxide in an in vitro model of vascular oxidative stress. In turn, our main aim was to investigate the effects of acute PARP inhibition and catalytic peroxynitrite decomposition on ageing-associated cardiac and endothelial dysfunction. METHODS: In vascular reactivity measurements on isolated rat aortic rings, we investigated the endothelium-dependent and -independent vasorelaxation by using acetylcholine and sodium nitroprusside. Endothelial dysfunction was induced by hydrogen peroxide. In the treatment group, rings were preincubated with a PARP-inhibitor. In the in vivo rat model of ageing-associated cardiovascular dysfunction, young and ageing rats were treated with a single dose of PARP-inhibitor, or with the peroxynitrite decomposition catalyst FP15. Using a pressure-conductance catheter, left ventricular pressure-volume analysis was performed to study cardiac function. Endothelium-dependent and -independent relaxation of aortic rings were investigated. Immunohistochemical analysis was performed to study the changes on the cellular and tissue level. RESULTS: In our in vitro model, hydrogen peroxide caused a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings which was significantly improved by PARP-inhibition. Ageing rats showed a marked reduction of cardiac function and loss of endothelium-dependent relaxant responsiveness of aortic rings. Both acute PARP-inhibition and FP15-treatment improved cardiac performance and endothelial function. Immunohistochemistry confirmed enhanced nitro-oxidative stress and PARP-activation in ageing rats, which were reversed in the treatment groups. CONCLUSIONS: Our results demonstrate the importance of endogenous peroxynitrite-overproduction and the activation of the PARP pathway in the age-related functional decline of the cardiovascular system. Catalytic peroxynitrite decomposition and PARP-inhibition may represent novel antioxidant therapeutic utilities to improve ageing-associated cardiovascular dysfunction.

[The effect of puerarin on prevention of oxidative damage of cultured lens capsule epithelial cells].

OBJECTIVE: To investigate the peroxynitrite damage to the lens epithelial cells (LEC) and the prevention of this damage by puerarin in vitro. METHODS: This paper was experimental study. Rabbit LEC were isolated and cultured and the third or forth passage LEC were used in this experiment The experiment groups included: (1) CONTROL GROUP: Heat-pathogen free saline (NS) 200 microl was added to the medium; (2) ONOO- group: ONOO- 200 microl was added to obtain the terminal concentration at 0. 5 mmol/L; (3) Puerarin group: 5 microg/ml ONOO- and 10 microg/ml puerarin were added simultaneously. Then, the cells were cultured and collected after 6,12 or 24 hours. The nitrotyrosine (NT), a symbol of the ONOO-, was tested with immunofluorescence technique. The expression of NT protein was examined with Western blot method. The cell morphology was observed with light microscope. Cell apoptosis was examined via DNA ladder, flow cytometry and Fas/FasL immunohistochemical staining. These datas were analyzed by one-way-ANOVA and q test. RESULTS: During the 6 to 24 hours of experiment period, green color could be observed in the cell nucleus and cytoplasm of control group. Staining ranged from yellow to brown-yellow, then to brown color were observed in STZ group. Staining ranged from faint green to yellow green or faint green color were observed in puerarin group. Slight expression of nitrotyrosine (NT) could be seen in the control group. A moderate to strong expression of NT was observed at different stages in the STZ group (A = 77.22 +/- 2.44, 145.00 +/- 3.94, 235. 8 +/- 5.97). At 6 hours, a slight expression of NT could be seen in the control group (A = 72.78 +/- 2.64), this increased at 12 hours (A =89. 94 +/- 3.01) and decreased at 24 hours (A = 74. 44 +/- 3.00). With computer photo-analysis, there were significant differences between the control, STZ and puerarin groups at different period during the experiment (q = 78.12, 82.76, 69.98, P <0. 01). In the control group, cell morphology and gene DNA ladder were normal, minor apoptosis could be observed but no expression of Fas/FasL in the membrane and cytoplasm of the cells. Distinctive cell morphology changes and the typical "ladder bands" as well as the expression of Fas/FasL could be observed in STZ group. All of these aspects were comparatively normal in puerarin group. CONCLUSIONS: The LEC apoptosis induced by ONOO- in vitro could be alleviated by puerarin. Fas/FasL cell signal transduction pathway may affect and strengthen the apoptosis process mediated by ONOO-.

An in vitro examination of the antioxidant and anti-inflammatory properties of buckwheat honey.

OBJECTIVE: Hydroxyl radical and hypochlorite anion formed at the wound site from superoxide anion produced by activated polymorphonuclear neutrophils (PMNs) are considered important factors in impaired wound healing. Superoxide anion may also react with nitric oxide produced by macrophages to form peroxynitrite, a third strong oxidant that damages surrounding tissue. In order to select honey for use in wound-healing products, different samples were compared for their capacity to reduce levels of reactive oxygen species (ROS) in vitro. METHOD: Honey samples were tested in assays for inhibition of ROS production by activated human PMNs, antioxidant activity (scavenging of superoxide anion in a cell-free system) and inhibition of human complement (reducing levels of ROS by limiting formation of complement factors that attract and stimulate PMNs). For buckwheat honey (NewYork, US), moisture and free acid content were determined by refractive index measurement and potentiometric titration respectively. Honey constituents other than sugars were investigated by thin layer chromatography, using natural product reagent to detect phenolic compounds. Constituents with antioxidant properties were detected by spraying the chromatogram with DPPH. RESULTS: Although most honey samples were shown to be active, significant differences were observed, with the highly active honey exceeding the activities of samples with minor effects by factors of 4 to 30. Most pronounced activities were found for American buckwheat honey from the state of NewYork. Phenolic constituents of buckwheat honey were shown to have antioxidant activity. CONCLUSION: As buckwheat honey was most effective in reducing ROS levels, it was selected for use in wound-healing products. The major antioxidant properties in buckwheat honey derive from its phenolic constituents, which are present in relatively large amounts. Its phenolic compounds may also exert antibacterial activity, whereas its low pH and high free acid content may assist wound healing.

Antioxidant and antiaggregatory effects of an extract from Conyza canadensis on blood platelets in vitro.

The antioxidative activity of the polysaccharide extract from Conyza canadensis in blood platelets treated with peroxynitrite (ONOO-) was studied. Peroxynitrite as a strong biological oxidant has toxic effects on blood platelets and induces the oxidation of thiols, carbonylation and nitration of platelet proteins and lipid peroxidation. Therefore, the aim of our study was to assess if the natural extract from herbal plant, Conyza Canadensis, may protect platelet proteins against nitrative and oxidative damage induced by ONOO-. In our study we measured oxidative damage of platelet proteins induced by peroxynitrite and protectory effects of this extract by estimation of the level of carbonyl groups and nitrotyrosine (a marker of platelet protein nitration). We also used cytochrome c reduction method to test the ability of this extract to change O2-* generation in platelets. Moreover, we determined the effects of the extract on blood platelet aggregation induced by ADP. We observed that the extract from Conyza canadensis distinctly reduced oxidation and nitration of proteins in blood platelets treated with ONOO-(0.1mM) and O2-* production in these cells. The extract from Conyza canadensis also inhibited platelet aggregation. The ability of the extract to decrease O2-* generation in blood platelets supports the importance of free radicals in platelet functions, including aggregation process. The present study suggests that the natural polysaccharide extract from Conyza canadensis has antiaggregatory and antioxidative activities, and therefore may be beneficial in the prevention of peroxynitrite-related diseases, such as cardiovascular or inflammatory diseases.

Role of peroxynitrite in the pathogenesis of cardiovascular complications of diabetes.

Hyperglycemic episodes, which complicate even well-controlled cases of diabetes, lead to increased polyol pathway flux, activation of protein kinase C and accelerated non-enzymatic formation of advanced glycation end products. Many of these pathways become activated in response to the production of superoxide anion. Superoxide can interact with nitric oxide, forming the potent cytotoxin peroxynitrite. Peroxynitrite attacks various biomolecules in the vascular endothelium, vascular smooth muscle and myocardium, eventually leading to cardiovascular dysfunction via multiple mechanisms. This review focuses on emerging evidence suggesting that peroxynitrite plays a key role in the pathogenesis of the cardiovascular complications of diabetes, which underlie the development and progression of diabetic retinopathy, neuropathy and nephropathy.

Tobacco-related-compound-induced nitrosative stress injury in the hamster cheek pouch.

The nitric oxide radical (*NO) released from tobacco-related compounds induces DNA damage, protein modifications, and cellular toxicity through the formation of peroxynitrite (ONOO-), the reaction product of *NO and the oxygen radical, superoxide. We hypothesize that tobacco-related compounds are cytotoxic and induce quantifiable DNA single-strand breaks in immortalized hamster cheek pouch (POII) cells, and that an amino acid marker of ONOO- injury, namely, 3-nitrotyrosine (3-NT), is detectable in hamster cheek pouch tissues chronically exposed to these compounds. We observed a dose-dependent decrease in POII cell viability with increasing tobacco-related compound concentrations, as well as a dose-dependent increase in DNA strand breaks. Semi-quantitative immunohistochemistry showed intense 3-NT immunoreactivity in hamster tissues treated with tobacco-related compounds compared with controls (p < 0.005). Our results suggest that tobacco-related compounds, including nicotine, are genotoxic, and that 3-NT is a quantifiable marker of ONOO- damage in intact hamster cheek pouch tissues.

Nitrosative stress and potassium channel-mediated neuronal apoptosis: is zinc the link?

Nitrosative stress has been implicated in a large number of neurological disorders. The molecular mechanisms underlying the neuronal injury associated with this stimulus, however, are not clearly understood. Emerging evidence suggests that the liberation of intracellular zinc as well as over-activation of potassium channels may be two important components of nitrosative stress-induced neuronal death.