3-nitrotyrosine and Asthma

Since the discovery of nitric oxide (NO), an intracellular signal transmitter, the role of NO has been investigated in various organs. In the respiratory system, NO derived from the constitutive type of NO synthase (cNOS, NOS1, NOS3) induces bronchodilation and pulmonary vasodilatation to maintain homeostasis. In contrast, the roles of excessive NO derived from the inducible type of NOS (iNOS, NOS2) in airway and lung inflammation in inflammatory lung diseases including bronchial asthma and chronic obstructive pulmonary disease (COPD) are controversial. In these inflammatory lung diseases, excessive nitrosative stress has also been observed. In asthma, some reports have shown that nitrosative stress causes airway inflammation, airway hyperresponsiveness, and airway remodeling, which are the features of asthma, whereas others have demonstrated the anti-inflammatory role of NO derived from NOS2. In the case of refractory asthma, more nitrosative stress has been reported to be observed in such airways compared with that in well-controlled asthmatics. In COPD, reactive nitrogen species (RNS), which are NO and NO-related molecules including nitrogen dioxide and peroxynitrite, cause lung inflammation, oxidative stress, activation of matrix metalloproteinase, and inactivation of antiprotease, which are involved in the pathophysiology of the disease. In the present paper, we review the physiological and pathophysiological effects of NO and NO-related molecules in the respiratory system and in inflammatory lung diseases.

Topics: Animals; Asthma; Humans; Inflammation; Lung; Lung Diseases; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; S-Nitrosothiols; Signal Transduction; Tyrosine

Endogenous Nitric Oxide (NO) plays a key role in the physiological regulation of airway functions. In response to various stimuli activated inflammatory cells (e.g., eosinophils and neutrophils) generate oxidants ("oxidative stress") which in conjunction with exaggerated enzymatic release of NO and augmented NO metabolites produce the formation of strong oxidizing reactive nitrogen species, such as peroxynitrite, in various airway diseases including asthma, chronic obstructive pulmonary diseases (COPD), cystic fibrosis and acute respiratory distress syndrome (ARDS). Reactive nitrogen species provoke amplification of inflammatory processes in the airways and lung parenchyma causing DNA damage, inhibition of mitochondrial respiration, protein dysfunction and cell damage ("nitrosative stress"). These effects alter respiratory homeostasis (such as bronchomotor tone and pulmonary surfactant activity) and the long-term persistence of "nitrosative stress" may contribute to the progressive deterioration of pulmonary functions leading to respiratory failure. Recent studies showing that protein nitration can be dynamic and reversible ("denitration mechanisms") open new horizons in the treatment of chronic respiratory diseases affected by the deleterious actions of "nitrosative stress".

Topics: Animals; Asthma; Cystic Fibrosis; Humans; Nitric Oxide; Oxidative Stress; Pulmonary Disease, Chronic Obstructive; Reactive Nitrogen Species; Reactive Oxygen Species; Respiratory System; Tyrosine

Topics: Asthma; Biomarkers; Carbon Monoxide; Chromatography, Gas; Chromatography, High Pressure Liquid; Clinical Enzyme Tests; Enzyme-Linked Immunosorbent Assay; Guanine; Infections; Neoplasms; Nitric Oxide; Pulmonary Disease, Chronic Obstructive; Reference Values; Sepsis; Tyrosine

Nitric oxide (NO), a simple free-radical gas, elicits a diverse range of physiologic and pathophysiologic effects, and plays an important role in pulmonary diseases. Nitrosative stress and nitration of proteins in airway epithelium may be responsible for steroid resistance in asthma and their ineffectiveness in chronic obstructive pulmonary disease (COPD), supporting the potential role of future therapeutic strategies aimed at regulating NO synthesis in asthma and COPD. In this article, we review the potential role of NO modulators (NO synthase inhibitors and NO donors), which, if given on a regular basis, may have clinical benefit in asthma and COPD.

Topics: Asthma; Breath Tests; Gas Chromatography-Mass Spectrometry; Humans; Immunoenzyme Techniques; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Proteomics; Pulmonary Disease, Chronic Obstructive; Reactive Oxygen Species; Tyrosine

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Eosinophils; Humans; Nitrates; Nitric Oxide; Pneumonia; Tyrosine

Allergic asthma is a chronically relapsing inflammatory airway disease with a complex pathophysiology.. This study was undertaken to investigate the potential contribution of NOD2 signaling, proinflammatory cytokines, chitotriosidase (CHIT1) activity, oxidative stress and DNA damage to atopic asthma pathogenesis, as well as to explore their possible role as surrogate noninvasive biomarkers for monitoring asthma severity.. Sixty patients with atopic bronchial asthma who were divided according to asthma severity into 40 mild-moderate, 20 severe atopic asthmatics, in addition to thirty age-matched healthy controls were enrolled in this study. NOD2 expression in PBMCs was assessed by quantitative real-time RT-PCR. DNA damage indices were assessed by alkaline comet assay. Serum IgE, IL-17, IL-8 and 3-Nitrotyrosine levels were estimated by ELISA. Serum CHIT1and GST activities, as well as MDA levels, were measured.. NOD2 mRNA relative expression levels were significantly decreased in atopic asthmatic cases relative to controls with lower values among severe atopic asthmatics. On the other hand, IL-17 and IL-8 serum levels, CHIT1 activity, DNA damage indices and oxidative stress markers were significantly increased in atopic asthmatic cases relative to controls with higher values among severe atopic asthmatics. The change in these parameters correlated significantly with the degree of decline in lung function.. The interplay between NOD2 signaling, proinflammatory cytokines, CHIT1 activity, heightened oxidative stress and DNA damage orchestrates allergic airway inflammation and thus contributing to the pathogenesis of atopic asthma. These parameters qualified for measurement as part of new noninvasive biomarker panels for monitoring asthma severity.

Topics: Adult; Asthma; DNA Damage; Female; Gene Expression Regulation, Enzymologic; Hexosaminidases; Humans; Immunoglobulin E; Inflammation; Interleukin-17; Interleukin-8; Leukocytes, Mononuclear; Male; Middle Aged; Nod2 Signaling Adaptor Protein; Oxidation-Reduction; Oxidative Stress; Severity of Illness Index; Tyrosine

Exhaled nitric oxide (FeNO) has been reported to be elevated in the oxidative stress involved in asthmatic patients, and the reaction of nitric oxide (NO) with superoxide anions results in the formation of nitrotyrosine. The purpose of this study was to investigate the effect of inhaled steroid treatment on nitrotyrosine levels collected by exhaled breath condensate (EBC) and on FeNO.. This was a single-blind placebo-controlled study. The lung function, FeNO, and nitrotyrosine levels were evaluated in 10 asthmatic children.. The nitrotyrosine levels were stable during the placebo period (T0 = 1.16 ng/ml versus T1 = 1.05 ng/ml; NS.), whereas they decreased after the treatment with flunisolide (T2 = 1.14 ng/ml versus T3 = 0.88 ng/ml; P < .001). No significant reduction in FeNO levels was observed after placebo treatment (T0 = 38.4 ppb versus T1 = 34.7 ppb, NS.). In contrast, FeNO values decreased significantly being at T3 = 14.9 ppb (T1 versus T3; P = .024).. This study shows that corticosteroid treatment reduces nitrotyrosine levels in EBC of asthmatic subjects.

Topics: Administration, Inhalation; Adolescent; Anti-Asthmatic Agents; Asthma; Breath Tests; Child; Fluocinolone Acetonide; Humans; Lung; Male; Nitric Oxide; Pilot Projects; Respiratory Function Tests; Single-Blind Method; Tyrosine

Toll-like receptors (TLRs) through innate immune system recognize pathogen associated molecular patterns and play an important role in host defense against bacteria, fungi and viruses. TLR-7 is responsible for sensing single stranded nucleic acids of viruses but its activation has been shown to be protective in mouse models of asthma. The NADPH oxidase (NOX) enzymes family mainly produces reactive oxygen species (ROS) in the lung and is involved in regulation of airway inflammation in response to TLRs activation. However, NOX-4 mediated signaling in response to TLR-7 activation in a mouse model of allergic asthma has not been explored previously. Therefore, this study investigated the role TLR-7 activation and downstream oxidant-antioxidant signaling in a murine model of asthma. Mice were sensitized with ovalbumin (OVA) intraperitoneally and treated with TLR-7 agonist, resiquimod (RSQ) intranasally before each OVA challenge from days 14 to 16. Mice were then assessed for airway reactivity, inflammation, and NOX-4 and nuclear factor E2-related factor 2 (Nrf2) related signaling [inducible nitric oxide synthase (iNOS), nitrotyrosine, lipid peroxides and copper/zinc superoxide dismutase (Cu/Zn SOD)]. Treatment with RSQ reduced allergen induced airway reactivity and inflammation. This was paralleled by a decrease in ROS which was due to induction of Nrf2 and Cu/Zn SOD in RSQ treated group. Inhibition of MyD88 reversed RSQ-mediated protective effects on airway reactivity/inflammation due to reduction in Nrf2 signaling. SOD inhibition produced effects similar to MyD88 inhibition. The current study suggests that TLR-7 agonist is beneficial and may be developed into a therapeutic option in allergic asthma.

Topics: Animals; Antioxidants; Asthma; Bronchoalveolar Lavage; Imidazoles; Immunoblotting; Inflammation; Lipid Peroxides; Male; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; NF-E2-Related Factor 2; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Superoxide Dismutase; Toll-Like Receptor 7; Tyrosine

Our objective was to assess the oxidative stress status and analyse the relationship between an oxidant/antioxidant imbalance and the mediator release properties of cord blood basophils from neonates born to mothers with atopic asthma.. Cord blood was collected from the neonates of 16 asthmatic mothers and 18 healthy mothers Basophils were purified and stimulated by Dermatophagoides farinae (Df), hyperosmotic mannitol or peptidoglycan (PGN). Immunoblotting detected nuclear factor κB (NFκB) as a measure of functional receptor response. The linear correlations between IL-4 levels in the supernatants and 3-nitrotyrosine (3-NT), glutathione peroxidase (GSH-Px) in the serum were evaluated.. Compared with the healthy group, the levels of 3-NT in maternal blood and cord blood were significantly higher in allergic asthma group, whereas the GSH-Px activity were lower. After stimulation, cord blood basophils from the neonates born to atopic mothers produced more IL-4 involving NF-κB pathways. There was a significant relationship between the IL-4 levels produced by basophils and 3-NT (or GSH-Px) in cord blood from allergic asthma group.. In asthma, mediator release properties of human basophils induced by environmental allergens and irritants are associated with oxidative stress, which may be one of the pathogenesis of allergic diseases.

Topics: Adult; Asthma; Basophils; Female; Fetal Blood; Glutathione Peroxidase; Humans; Hypersensitivity, Immediate; Infant, Newborn; Interleukin-4; Oxidative Stress; Pregnancy; Pregnancy Complications; Tyrosine

Topics: Adult; Asthma; Chronic Disease; Eosinophils; Female; Humans; Immunohistochemistry; Lung; Macrophages, Alveolar; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type II; Pulmonary Eosinophilia; Tyrosine

Reactive oxygen species produced during allergic inflammation are key players of the pathophysiology of asthma, leading to oxidative tissue injury and inactivation of endogenous manganese superoxide dismutase (MnSOD). On this ground, removal of excess superoxide anion by scavenger molecules would be beneficial and protective. Here we show that a novel manganese(II)-containing polyamine-polycarboxylic compound, termed Mn(II)(Me(2)DO2A), with potent superoxide dismuting properties decreases the respiratory and histopathological lung abnormalities due to allergen inhalation in a model of ovalbumin (OA)-induced allergic asthma-like reaction in sensitized guinea pigs. Severe respiratory dysfunction in response to OA aerosolic challenge arose rapidly in the sensitized animals and was accompanied by bronchoconstriction, alveolar hyperinflation, mast cell activation, increased leukocyte infiltration (evaluated by myeloperoxidase assay), oxidative lung tissue injury (evaluated by the thiobarbituric-acid-reactive substances and nitrotyrosine immunostaining), decay of endogenous MnSOD activity, production of pro-inflammatory prostaglandins, and lung cell apoptosis. Treatment with Mn(II)(Me(2)DO2A) (15mg/kg, given 1h before allergen challenge), but not the inactive congener Zn(II)(Me(2)DO2A) lacking redox-active metal site, significantly attenuated all the above functional, histopathological and biochemical parameters of allergic inflammation and restored the levels of MnSOD activity. In conclusion, our findings support the potential therapeutic use of Mn(II)(Me(2)DO2A) as novel superoxide scavenger drug in asthma and anaphylactic reactions.

Topics: Allergens; Anaphylaxis; Animals; Anti-Asthmatic Agents; Apoptosis; Asthma; Bronchoconstriction; Caspase 3; Free Radical Scavengers; Guinea Pigs; Male; Mast Cells; Organometallic Compounds; Ovalbumin; Peroxidase; Superoxides; Thiobarbituric Acid Reactive Substances; Tyrosine

The expression of arginase I has been a focus of research into the pathogenesis of experimental asthma, because arginase deprives nitric oxide synthase (NOS) of arginine and therefore participates in the attenuation of bronchodilators such as nitric oxide (NO). The present study used an intranasal mite-induced NC/Nga mouse model of asthma to investigate the contribution of arginase to the asthma pathogenesis, using an arginase inhibitor, N(omega)-hydroxy-nor-l-arginine (nor-NOHA). The treatment with nor-NOHA inhibited the increase in airway hyperresponsiveness (AHR) and the number of eosinophils in bronchoalveolar lavage fluid. NOx levels in the lung were elevated despite suppressed NOS2 mRNA expression. Accompanied by the attenuated activity of arginase, the expression of arginase I at both the mRNA and protein level was downregulated. The levels of mRNA for T helper 2 cytokines such as IL-4, IL-5, and IL-13, and for chemotactants such as eotaxin-1 and eotaxin-2, were reduced. Moreover, the accumulation of inflammatory cells and the ratio of goblet cells in the bronchiole were decreased. The study concluded that the depletion of NO caused by arginase contributes to AHR and inflammation, and direct administration of an arginase inhibitor to the airway may be beneficial and could be of use in treating asthma due to its anti-inflammatory and airway-relaxing effects, although it is not clear whether the anti-inflammatory effect is direct or indirect.

Topics: Allergens; Animals; Antigens, Dermatophagoides; Arginase; Arginine; Asthma; Bronchial Hyperreactivity; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Humans; Isoenzymes; Male; Mice; Nitric Oxide; Nitric Oxide Synthase; Tyrosine

Children with severe allergic asthma have persistent airway inflammation and oxidant stress.. We hypothesized that children with severe allergic asthma would have increased concentrations of the nitric oxide (NO) oxidation products nitrite, nitrate, and nitrotyrosine in the proximal and distal airway epithelial lining fluid (ELF). We further hypothesized that NO oxidation products would be associated with higher exhaled NO values (fraction of exhaled nitric oxide [F(ENO)]), greater allergic sensitization, and lower pulmonary function.. Bronchoalveolar lavage fluid was obtained from 15 children with mild-to-moderate asthma, 30 children with severe allergic asthma, 5 nonasthmatic children, and 20 nonsmoking adults. The bronchoalveolar lavage fluid was divided into proximal and distal portions and nitrite, nitrate, and nitrotyrosine values were quantified.. Children with mild-to-moderate and severe allergic asthma had increased concentrations of nitrite (adult control subjects, 15 +/- 3 micromol/L; pediatric control subjects, 23 +/- 4 micromol/L; subjects with mild-to-moderate asthma, 56 +/- 26 micromol/L; subjects with severe asthma, 74 +/- 18 micromol/L), nitrate (37 +/- 13 vs 145 +/- 38 vs 711 +/- 155 vs 870 +/- 168 micromol/L, respectively) and nitrotyrosine (2 +/- 1 vs 3 +/- 1 vs 9 +/- 3 vs 10 +/- 4 micromol/L, respectively) in the proximal ELF. Similar results were seen in the distal ELF, although the concentrations were significantly lower (P < .05 for each). Although univariate analyses revealed no associations between NO oxidation products and clinical features, multivariate analyses revealed F(ENO) values to be a significant predictor of NO oxidation in asthmatic children.. NO oxidation products are increased in the ELF of asthmatic children. The relationship between F(ENO) values and airway nitrosative stress is complicated and requires further study.

Topics: Adolescent; Adult; Anti-Asthmatic Agents; Asthma; Bronchoalveolar Lavage Fluid; Child; Female; Humans; Male; Multivariate Analysis; Nitrates; Nitric Oxide; Nitrites; Respiratory Mucosa; Spirometry; Tyrosine

To study the possible potential of nitric oxide (NO) in the processes of atopic inflammation, the authors evaluated the intensity of nitrosyling and oxidative stresses in the bronchoalvelar lavage fluid and expired air condensate of patients with bronchial asthma (BA). Chronic inflammation was shown to result in an increase in the processes of nitration and oxidation in worsening BA and to reliably correlate with airway NO-producing functions, by explaining the pathological effects of NO due to the formation of 3-nitrotyrosine and malonic dialdehyde with the existing imbalance in NO metabolism, by intensifying nitrosylating stress. In the authors' opinion, nitrosothiols that are required as a NO donor may be rapidly destroyed or virtually do not form so the peroxynitrite-nitrosothiol ratio may predetermine the final effects of NO.

Topics: Asthma; Biomarkers; Bronchoalveolar Lavage Fluid; Catalase; Glutathione; Humans; Malondialdehyde; Nitric Oxide; Nitrites; Respiratory System; Superoxide Dismutase; Tyrosine

Most asthma is mild and moderate and can be well controlled by low-dose inhaled steroid with or without bronchodilators. However, 5% to 10% of patients with asthma have more troublesome disease despite using such medication. Recent reports showed that nitrative stress induced tissue remodeling in vitro, which is associated with a component of refractoriness in asthma. However, there is no report that nitrative stress is involved in refractory asthma.. The aim of this study is to evaluate whether patients with refractory asthma have more nitrative stress.. Ten healthy subjects, 10 patients with well-controlled asthma, and 8 patients with refractory asthma took part in the current study. Exhaled nitric oxide, xanthine oxidase activity in the supernatant of the sputum, immunostaining for the inducible type of nitric oxide synthase, and 3-nitrotyrosine in induced sputum from the subjects were assessed.. All nitrative markers including exhaled nitric oxide (P < .01), immunopositivities for inducible nitric oxide synthase (P < .01), xanthine oxidase activities (P < .01), and 3-nitrotyrosine (P < .01) in sputum from the refractory asthma group were enhanced compared with the well-controlled group. All these nitrative markers in the sputum had a significant negative correlation with the %FEV(1) values (P < .01).. These results suggested that patients with refractory asthma have more nitrative stress in their airways compared with patients with well-controlled asthma.

Topics: Adult; Aged; Asthma; Biomarkers; Bronchodilator Agents; Drug Resistance; Exhalation; Female; Forced Expiratory Volume; Humans; Immunohistochemistry; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type II; Sputum; Staining and Labeling; Steroids; Tyrosine; Vital Capacity; Xanthine Oxidase

Topics: Acute Disease; Animals; Asthma; beta-N-Acetylhexosaminidases; Biomarkers; Bronchoalveolar Lavage Fluid; Calgranulin B; Electrophoresis, Gel, Two-Dimensional; Humans; Hypersensitivity, Immediate; Lectins; Lung; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Mice; Mice, Knockout; Protein Processing, Post-Translational; Proteomics; Subtraction Technique; Th2 Cells; Tyrosine

A(2A) adenosine receptor (A(2A)AR) has potent anti-inflammatory properties, which may be important in the regulation of airway reactivity and inflammation. Inflammatory cells that possess A(2A)AR also produce nitrosative stress, which is associated with pathophysiology of asthma, so we hypothesized that A(2A)AR deficiency may lead to increased airway reactivity and inflammation through nitrosative stress. Thus the present study was carried out to investigate the role of A(2A)AR on airway reactivity, inflammation, NF-kappaB signaling, and nitrosative stress in A(2A)AR knockout (KO) and wild-type (WT) mice using our murine model of asthma. Animals were sensitized intraperitoneally on days 1 and 6 with 200 microg of ragweed, followed by aerosolized challenges with 0.5% ragweed on days 11, 12, and 13, twice a day. On day 14, airway reactivity to methacholine was assessed as enhanced pause (Penh) using whole body plethysmography followed by bronchoalveolar lavage (BAL) and lung collection for various analyses. Allergen challenge caused a significant decrease in expression of A(2A)AR in A(2A) WT sensitized mice, with A(2A)AR expression being undetected in A(2A) KO sensitized group leading to decreased lung cAMP levels in both groups. A(2A)AR deletion/downregulation led to an increase in Penh to methacholine and influx of total cells, eosinophils, lymphocytes, and neutrophils in BAL with highest values in A(2A) KO sensitized group. A(2A) KO sensitized group further had increased NF-kappaB expression and nitrosative stress compared with WT sensitized group. These data suggest that A(2A)AR deficiency leads to airway inflammation and airway hyperresponsiveness, possibly via involvement of nitrosative stress in this model of asthma.

Topics: Ambrosia; Animals; Asthma; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Cyclic AMP; Disease Models, Animal; I-kappa B Proteins; Leukocytes; Lipid Peroxidation; Male; Methacholine Chloride; Mice; Mice, Inbred Strains; Mice, Knockout; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase Type II; Phosphorylation; Plethysmography, Whole Body; Pneumonia; Reactive Nitrogen Species; Receptor, Adenosine A2A; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor RelA; Tyrosine

Changes in the expression of arginase and their association with nitrosative stress were investigated using an asthmatic model previously established in NC/Nga mice with mite extract. Mite crude extract (100 microg/day) from Dermatophagoides farinae was administered intranasally for 5 consecutive days (day 0-4), and a single challenge was performed on day 11. On day 12, upregulation of the mRNA expression of inducible types of nitric oxide synthase (iNOS) and increases in immunohistochemical staining for iNOS and nitrotyrosine were observed. However, the level of nitrite + nitrate was unchanged. An increase in enzymatic activity, upregulation of mRNA expression, and immunostaining for arginase I was detected in the lung tissue and serum. Moreover, increases in both arginase I and II were revealed by immunoblotting. Goblet cell hyperplasia in bronchial epithelial cells and increasing collagen synthesis around the bronchus were also observed. These results suggested that an increase in arginase may lead to decreased availability of arginine for nitric oxide synthase and may contribute to the remodeling of the lung.

Topics: Animals; Arginase; Asthma; Collagen; Cytokines; Gene Expression Regulation, Enzymologic; Goblet Cells; Immunohistochemistry; Isoenzymes; Male; Mice; Nitrates; Nitric Oxide Synthase Type II; Nitrites; RNA, Messenger; Tyrosine; Up-Regulation

Asthmatic patients have high exhaled nitric oxide (NO) levels. NO-mediated inflammatory actions are mainly due to NO conversion into reactive nitrogen species, which can lead to nitrotyrosine formation. The aim of this study was to assess 3-nitrotyrosine (3-NT) levels in exhaled breath condensate (EBC) of asthmatic and healthy children and to investigate whether there is any relationship with exhaled NO (FE(NO)) and lung function.. The study included 20 asthmatic children (10 steroid-naive with intermittent asthma, 10 steroid-treated with unstable persistent asthma) and 18 healthy controls. They underwent FE(NO) measurement, EBC collection and spirometry. 3-NT was measured by a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in isotopic dilution.. The median EBC concentration of 3-NT (expressed as nitrotyrosine/tyrosine ratio x 100) in asthmatic children was fivefold higher than in healthy subjects [0.23% (0.12-0.32) vs 0.04% (0.02-0.06), P < 0.001] with no difference between steroid-naive and unstable steroid-treated asthmatic patients. FE(NO) levels were higher in asthmatic [44.6 ppb (36.0-66.0)] than in healthy children [7.5 ppb (6.0-8.8), P < 0.001]. No correlation was found among 3-NT, FE(NO) and lung function parameters.. Nitrotyrosine is high in EBC of asthmatic children and could be considered as a noninvasive marker of nitrosative events in the airways.

Topics: Adolescent; Anti-Asthmatic Agents; Asthma; Biomarkers; Breath Tests; Case-Control Studies; Child; Female; Humans; Male; Nitric Oxide; Probability; Reference Values; Respiratory Function Tests; Sensitivity and Specificity; Severity of Illness Index; Spirometry; Tyrosine

Reactive oxygen species and reactive nitrogen species produced by epithelial and inflammatory cells are key mediators of the chronic airway inflammation of asthma. Detection of 3-nitrotyrosine in the asthmatic lung confirms the presence of increased reactive oxygen and nitrogen species, but the lack of identification of modified proteins has hindered an understanding of the potential mechanistic contributions of nitration/oxidation to airway inflammation. In this study, we applied a proteomic approach, using nitrotyrosine as a marker, to evaluate the oxidation of proteins in the allergen-induced murine model of asthma. Over 30 different proteins were targets of nitration following allergen challenge, including the antioxidant enzyme catalase. Oxidative modification and loss of catalase enzyme function were seen in this model. Subsequent investigation of human bronchoalveolar lavage fluid revealed that catalase activity was reduced in asthma by up to 50% relative to healthy controls. Analysis of catalase isolated from asthmatic airway epithelial cells revealed increased amounts of several protein oxidation markers, including chloro- and nitrotyrosine, linking oxidative modification to the reduced activity in vivo. Parallel in vitro studies using reactive chlorinating species revealed that catalase inactivation is accompanied by the oxidation of a specific cysteine (Cys(377)). Taken together, these studies provide evidence of multiple ongoing and profound oxidative reactions in asthmatic airways, with one early downstream consequence being catalase inactivation. Loss of catalase activity likely amplifies oxidative stress, contributing to the chronic inflammatory state of the asthmatic airway.

Topics: Adult; Animals; Asthma; Catalase; Cell Line; Electrophoresis, Gel, Two-Dimensional; Enzyme Activation; Epithelial Cells; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Ovalbumin; Oxidation-Reduction; Proteome; Proteomics; Reactive Nitrogen Species; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tyrosine

3-Nitrotyrosine (3-NT) is considered as a marker of oxidative stress, which occurs during inflammation. Since 3-NT levels in exhaled breath condensate (EBC) are very low, we applied a specific and sensitive gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) method and high performance liquid chromatography (HPLC) with electrochemical detection for the analysis of free 3-NT in EBC. A total of 42 children (aged 5-17 years) were enrolled in this study, including children with asthma (n=12), cystic fibrosis (n=12), and healthy controls (n=18). Additionally, 14 healthy non-smoking adults (aged 18-59 years) were included. An EcoScreen system was used for the collection of EBC samples. Free 3-NT levels in EBC ranged from 0.54-6.8 nM. Median (interquartile range) concentrations (nM) were similar in all groups: 1.46 (0.97-2.49) in healthy adults, 2.51 (1.22-3.51) in healthy children, 1.46 (0.88-2.02) in children with asthma, and 1.97 (1.37-2.35) in CF children, respectively (p=0.24, Kruskall-Walis test). No difference was found between the children with airway disease and age-matched healthy controls. In healthy subjects, there was no effect of age on 3-NT concentrations. HPLC analyses provided similar concentration ranges for EBC 3-NT when compared with GC-NICI-MS. Our study has clearly demonstrated that free 3-NT in EBC fails as a marker for oxidative stress in children with stable CF and asthma.

Topics: Adolescent; Adult; Asthma; Biomarkers; Breath Tests; Child; Child, Preschool; Cystic Fibrosis; Exhalation; Female; Humans; Male; Middle Aged; Oxidative Stress; Tyrosine

Reactive nitrogen species can cause oxidative modifications of certain amino acid residues in proteins, notably the modification of tyrosine to 3-nitrotyrosine (3-NT), which is a potentially useful marker of oxidative stress. Since lung diseases are associated with airway inflammation and oxidative stress, quantification of 3-NT in exhaled breath condensate (EBC) may provide a non-invasive means for monitoring ongoing inflammatory processes. 3-NT-like immunoreactivity has previously been detected in EBC, but no definitive evidence for the presence of 3-NT in EBC is available. Here, a method based on gas chromatography/negative ion chemical ionization/tandem mass spectrometry was established for the quantification of free 3-NT in EBC. The detection limit was 0.56 pM (corresponding to 3.0 amol microl(-1) sample injected) and the method was found to give linear results (r2 > 0.999) in the concentration range of 0-5.0 nM. The coefficient of variation (CV) for within-day and between-day precision were 11 and 12%, respectively. No artifactual nitration was observed during sample processing. The method was applied to study subjects with asthma (n = 8), and healthy subjects (n = 10), but only a slight non-significant increase in 3-NT levels was found in the former group (median [interquartile ranges]; 99 [50-547] amol s(-1) vs. 75 [35-147] amol s(-1)). No correlation with exhaled nitric oxide (NO), pulmonary function or EBC levels of total protein was observed. The 3-NT levels were much lower compared to previously reported levels, based on immunochemical measurements. The method does not allow the simultaneous quantification of tyrosine in samples.

Topics: Adult; Aged; Asthma; Biomarkers; Case-Control Studies; Exhalation; Female; Gas Chromatography-Mass Spectrometry; Humans; Inflammation; Male; Middle Aged; Oxidative Stress; Reproducibility of Results; Sensitivity and Specificity; Tyrosine

Oxidative damage by reactive nitrogen species is linked to the pathogenesis of numerous inflammatory disorders, including atherosclerosis. 3-Nitrotyrosine (NO2Tyr), a posttranslational modification of proteins generated by reactive nitrogen species, serves as a "molecular fingerprint" for protein modification by nitric oxide (NO)-derived oxidants. Studies demonstrate that systemic levels of protein-bound NO2Tyr serve as an independent predictor of cardiovascular risks and are modulated by statin therapy. Measurement of NO2Tyr in biological matrices may thus serve both as a quantitative index of nitrative stress in vivo and an important new prognostic marker of clinical relevance. Analytical methods for the accurate detection and quantification of trace levels of NO2Tyr in biological tissues and fluids are, thus, of considerable interest. Here, we describe a rapid, sensitive, and specific method for the quantification of NO2Tyr in biological matrices using readily available benchtop ion-trap mass spectrometry instrumentation (e.g., LCQDeca) combined with high-performance liquid chromatography (HPLC) interface. Through judicious use of stable isotopically labeled precursors as synthetic internal standards, the tandem mass spectrometric method described simultaneously adjusts for potential intrapreparative sample losses and monitors potential artifactual generation of NO2Tyr during processing. The described method permits rapid and reproducible quantification of NO2Tyr in biological and clinical specimens at the 100 fmol on column detection limit and should prove useful for studies defining the impact of reactive nitrogen species in cardiovascular disease and other inflammatory disorders.

Topics: Animals; Asthma; Chromatography, High Pressure Liquid; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidation-Reduction; Proteins; Spectrometry, Mass, Electrospray Ionization; Tyrosine

Because reactive nitrogen species (RNS) have potent inflammatory activity, they may be involved in the inflammatory process in pulmonary diseases. We recently reported increased numbers of 3-nitrotyrosine immunopositive cells, which are evidences of RNS production, in the sputum of patients with chronic obstructive pulmonary disease (COPD) and patients with asthma compared with healthy subjects. In the present study, we attempted to quantify this protein nitration in the airways by means of high-performance liquid chromatography (HPLC) used together with an electrochemical detection system that we developed. Sputum samples were obtained from 15 stable COPD patients, 9 asthmatic patients and 7 healthy subjects by using hypertonic saline inhalation. The values for the molar ratio of protein-bound 3-nitrotyrosine/tyrosine in patients with asthma (4.31 +/- 1.13 x 10(-6), p < 0.05) and patients with COPD (3.04 +/- 0.36 x 10(-6), p < 0.01) were significantly higher than those in healthy subjects (1.37 +/- 0.19 x 10(-6)). The levels of protein-bound 3-nitrotyrosine in the airways were not significantly different in asthmatic patients and COPD patients. A significant negative correlation was found between values for protein-bound 3-nitrotyrosine/tyrosine and % FEV1 values in patients with COPD (r = -0.53, p < 0.05) but not in patients with asthma. These results suggest that our HPLC-electrochemical method is useful for quantifying RNS production in human airways. More importantly, they show that increased RNS production in the airways seems to contribute in a critical way to the pathogenesis of COPD, and that the effects of RNS in airways may differ in asthma and COPD.

Topics: Aged; Asthma; Case-Control Studies; Chromatography, High Pressure Liquid; Electrochemistry; Female; Forced Expiratory Volume; Humans; Male; Middle Aged; Nitric Oxide; Proteins; Pulmonary Disease, Chronic Obstructive; Reference Values; Sensitivity and Specificity; Sputum; Tyrosine

Poly(ADP-ribose) polymerase (PARP) plays an important role in tissue injury in conditions associated with oxidative stress and inflammation. Because asthma is a chronic inflammatory disorder of the airways, we designed the present experimental study to evaluate the effects of PARP inhibition on allergen-induced asthma-like reaction in ovalbumin-sensitized guinea pigs. Cough and dyspnea in response to ovalbumin aerosol were absent in naive guinea pigs, whereas they became severe in the sensitized animals. In the latter ones, ovalbumin aerosol also induced a rapid increase in PARP activity, bronchiolar constriction, pulmonary air space inflation, mast cell degranulation, poly(ADP-ribose) and nitrotyrosine immunostaining, myeloperoxidase activity, and malondialdehyde in lung tissue, as well as a rise in the amounts of nitrites and tumor necrosis factor-alpha in bronchoalveolar lavage fluid. Pretreatment with the PARP inhibitors 3-aminobenzamide (10 mg/kg b.wt.) or 5-aminoisoquinolinone (0.5 mg/kg b.wt.) given i.p. 3 h before ovalbumin challenge significantly reduced the severity of cough and the occurrence of dyspnea and delayed the onset of respiratory abnormalities. Both PARP inhibitors were also able to prevent the above morphological and biochemical changes of lung tissue or bronchoalveolar lavage fluid induced by ovalbumin challenge. Conversely, p-aminobenzoic acid, the inactive analog of 3-aminobenzamide, had no effects.

Topics: Allergens; Animals; Anti-Asthmatic Agents; Asthma; Benzamides; Bronchoalveolar Lavage Fluid; Enzyme Inhibitors; Guinea Pigs; Immunohistochemistry; Isoquinolines; Lung; Male; Malondialdehyde; Mast Cells; Nitric Oxide; Ovalbumin; Peroxidase; Poly(ADP-ribose) Polymerase Inhibitors; Respiratory Mechanics; Tumor Necrosis Factor-alpha; Tyrosine

Although ebselen, a seleno-organic compound, inhibits inflammation in various animal models, its efficacy as an anti-asthma drug remains to be clarified. In this study, we investigated the inhibitory effect of ebselen on a guinea pig asthma model. Ebselen was orally administered at dosages of 1-20 mg/kg 2 h before an ovalbumin (OA) challenge, and then airway responses, airway inflammation, the generation of superoxide, H(2)O(2), and nitrotyrosine, and the induction of inducible nitric oxide synthase (iNOS) were evaluated. Sensitized animals challenged with OA aerosol showed dual airflow limitations, i.e., immediate and late airway responses (IAR and LAR). Ebselen significantly inhibited LAR at dosages greater than 10 mg/kg, but did not inhibit IAR at any dosage. Bronchoalveolar lavage (BAL) examination showed that airway inflammation was significantly suppressed by ebselen at 10 mg/kg. The generation of superoxide and H(2)O(2) occurred on endothelial cells of LAR bronchi, and was inhibited by 10 mg/kg of ebselen. Superoxide generation was inhibited by diphenyleneiodonium chloride (DPI), a NAD(P)H oxidase inhibitor, but not by allopurinol, a xanthine oxidase inhibitor. Immunoreactivities for iNOS and nitrotyrosine were also observed on endothelial cells of LAR bronchi and were abolished in ebselen-treated animals. The present findings suggest that ebselen can be applied as a new therapeutic agent for asthma. The possible mechanisms by which ebselen inhibits LAR likely involve suppression of oxidant formation and iNOS induction in endothelial cells.

Topics: Airway Resistance; Animals; Antineoplastic Agents, Alkylating; Antioxidants; Area Under Curve; Asthma; Azoles; Bronchitis; Bronchoalveolar Lavage Fluid; Cyclophosphamide; Disease Models, Animal; Endothelium, Vascular; Enzyme Activation; Female; Guinea Pigs; Hydrogen Peroxide; Immunoenzyme Techniques; Isoindoles; Lung; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organoselenium Compounds; Ovalbumin; Peptide Fragments; Rabbits; Reactive Nitrogen Species; Reactive Oxygen Species; Respiratory Function Tests; Respiratory Hypersensitivity; Superoxides; Tyrosine

A wealth of evidence supports increased NO (NO.) in asthma, but its roles are unknown. To investigate how NO participates in inflammatory airway events in asthma, we measured NO. and NO. chemical reaction products [nitrite, nitrate, S-nitrosothiols (SNO), and nitrotyrosine] before, immediately and 48 h after bronchoscopic antigen (Ag) challenge of the peripheral airways in atopic asthmatic individuals and nonatopic healthy controls. Strikingly, NO(3)(-) was the only NO. derivative to increase during the immediate Ag-induced asthmatic response and continued to increase over 2-fold at 48 h after Ag challenge in contrast to controls [P < 0.05]. NO(2)(-) was not affected by Ag challenge at 10 min or 48 h after Ag challenge. Although SNO was not detectable in asthmatic airways at baseline or immediately after Ag, SNO increased during the late response to levels found in healthy controls. A model of NO. dynamics derived from the current findings predicts that NO. may have harmful effects through formation of peroxynitrite, but also subserves an antioxidant role by consuming reactive oxygen species during the immediate asthmatic response, whereas nitrosylation during the late asthmatic response generates SNO, safe reservoirs for removal of toxic NO. derivatives.

Topics: Adult; Antigens; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Case-Control Studies; Female; Humans; Immunohistochemistry; Male; Middle Aged; Nitric Oxide; Tyrosine

To explore the possible role of eosinophils in NO-mediated tissue injury, we studied a murine model of allergic asthma. Male A/J mice were sensitized and challenged intranasally with ovalbumin (OVA). Following challenge, the number of eosinophils in bronchoalveolar lavage fluid (BALF) increased from 0.4% of total cells at baseline (0.02 x 10(4) cells/ml) to 60.2% at 48 h after the challenge (9.34 x 10(4) cells/ml). The rise in eosinophil count was accompanied by a 40.3% increase in total NO(2-) plus NO(3-) (NO(x)) in BALF. This in turn was accompanied by expression of inducible NO synthase (NOS II) in airway epithelial and inflammatory cells, as well as by evidence of staining for 3-nitrotyrosine (3NT) in peribronchial inflammatory cells and at the epithelial surface. Both NO(x) production and 3NT were significantly reduced by pretreatment of the challenged mice with the highly specific NOS II inhibitor N-3-aminomethyl-benzyl-acetamidine-dihydrochloride (1400W), as well as by the nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). L-NAME and 1400W also reduced the number of BALF eosinophils (37.2% and 61.5%, respectively, as compared with the control value), suggesting that NO production by NOS II contributes to eosinophil recruitment. To further examine the role of eosinophils, we pretreated additional mice with an anti-interleukin (IL)-5 antibody, which reduced BALF eosinophilia following OVA challenge by 90.1%. In concert with the decrease in eosinophils, the anti-IL-5 antibody reduced NO(x) in BALF almost to the baseline value, and decreased the number of 3NT-positive cells in the peribronchial region by 74.4%. Western blot analysis of protein extracted from whole lung confirmed the reduction in tyrosine nitration by anti-IL-5 antibody. These findings indicate that NO and eosinophilic inflammation are closely coupled, and suggest that eosinophils are an important source of tyrosine nitration.

Topics: Analysis of Variance; Animals; Antibodies; Asthma; Blotting, Western; Bronchoalveolar Lavage Fluid; Eosinophils; Interleukin-5; Lung; Male; Mice; Mice, Inbred Strains; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrosation; Ovalbumin; Time Factors; Tyrosine

Evidence supporting increased nitric oxide (NO) in asthma is substantial, although the cellular and molecular mechanisms leading to increased NO are not known. Here, we provide a clear picture of the events regulating NO synthesis in the human asthmatic airway in vivo. We show that human airway epithelium has abundant expression of NO synthase II (NOSII) due to continuous transcriptional activation of the gene in vivo. Individuals with asthma have higher than normal NO concentrations and increased NOSII mRNA and protein due to transcriptional regulation through activation of Stat1. NOSII mRNA expression decreases in asthmatics receiving inhaled corticosteroid, treatment effective in reducing inflammation in asthmatic airways. In addition to transcriptional mechanisms, post-translational events contribute to increased NO synthesis. Specifically, high output production of NO is fueled by a previously unsuspected increase in the NOS substrate, l -arginine, in airway epithelial cells of asthmatic individuals. Finally, nitration of proteins in airway epithelium provide evidence of functional consequences of increased NO. In conclusion, these studies define multiple mechanisms that function coordinately to support high level NO synthesis in the asthmatic airway. These findings represent a crucial cornerstone for future therapeutic strategies aimed at regulating NO synthesis in asthma.

Topics: Adult; Alternative Splicing; Arginine; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; DNA-Binding Proteins; Epithelial Cells; Female; Fluocinolone Acetonide; Humans; Interferon-gamma; Janus Kinase 1; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Reactive Oxygen Species; RNA, Messenger; STAT1 Transcription Factor; Trans-Activators; Transcription, Genetic; Tyrosine

Peroxynitrite, nitrogen dioxide, and other reactive nitrogen species (RNS) that are formed in the reaction of nitric oxide (NO) with superoxide anion, and in peroxidase-dependent mechanisms, have a potent inflammatory action. These molecules may therefore increase in number and have a role in inflammatory airway diseases. In the present study, we quantified RNS using immunostaining of nitrotyrosine and inducible NO synthase (iNOS) in airway inflammatory cells obtained by the induced sputum technique, and also quantified the exhaled NO concentration in subjects with chronic obstructive pulmonary disease (COPD), subjects with asthma, and healthy subjects (HS). Immunoreactivity for iNOS observed in the airway inflammatory cells was significantly and similarly higher in subjects with COPD and asthma than in HS, although exhaled NO levels were increased only in subjects with asthma. Inflammatory cells showed obvious nitrotyrosine immunoreactivity in subjects with COPD and to a lesser extent in those with asthma, but not in HS. There was a significant negative correlation between the percent predicted values of FEV(1) and the amount of nitrotyrosine formation in subjects with COPD, but not in those with asthma and HS. These results suggest that: (1) RNS may be involved in the pathobiology of the airway inflammatory and obstructive process in COPD; and (2) NO produced in the airways, presumably via iNOS, is consumed by its reaction with superoxide anion and/or peroxidase-dependent mechanisms.

Topics: Adult; Asthma; Cell Count; Female; Forced Expiratory Volume; Humans; Immunohistochemistry; Lung; Lung Diseases, Obstructive; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Sputum; Tyrosine

The reaction of nitric oxide (NO) and superoxide anions (O(2)(-)) in the airway results in the formation of peroxynitrite, a highly reactive oxidant species. Peroxynitrite reacts with tyrosine residues in proteins to form the stable product nitrotyrosine. We investigated whether nitrotyrosine in exhaled breath condensates may be increased in patients with asthma. Four groups of nonsmoking subjects were studied. We measured exhaled NO, nitrotyrosine, and leukotrienes concentrations in breath condensate in healthy nonatopic subjects (n = 15) and in patients with mild asthma (steroid naive, n = 15), moderate asthma (inhaled steroid treatment, n = 12), and severe asthma (oral steroid treatment, n = 12). Exhaled NO was increased significantly in patients with mild (19.2 +/- 2.7 ppb, p < 0.01) and moderate asthma (14.0 +/- 1.53 ppb, p < 0.05), as compared with normal control (6.58 +/- 0.61 ppb). The levels of LTC(4)/D(4)/E(4) and LTB(4) were increased significantly in patients with moderate and severe asthma treated with steroids. Nitrotyrosine concentrations were detectable (6.3 +/- 0.8 ng/ml) in breath condensate of normal subjects, and were increased significantly in patients with mild asthma (15.3 +/- 2.0 ng/ml, p < 0.01). However, the levels of nitrotyrosine in exhaled condensate were lower in patients with moderate (5.0 +/- 0.6 ng/ml) and severe asthma (3.3 +/- 0.6 ng/ml, p < 0.05). There was a significant correlation between nitrotyrosine in breath condensate and exhaled NO in patients with mild asthma (r = 0.65, p < 0.05). We conclude that nitrotyrosine formation in exhaled breath condensates may be a marker of oxidative stress in airways of asthma.

Topics: Adult; Asthma; Breath Tests; Female; Humans; Leukotrienes; Lung; Male; Middle Aged; Nitric Oxide; Oxidative Stress; Tyrosine

Recent evidence has shown that nitric oxide (NO) levels are increased in asthmatic airways. Although the role of NO in asthma is unknown, reactive metabolites of NO may lead to nitrotyrosine formation and promote airway dysfunction.. The aim of this study was to determine whether nitrotyrosine, as a marker of nitrating species, could be found in the airways and lung parenchyma of subjects with asthma who died of status asthmaticus or other nonrespiratory causes.. Lung tissue specimens were obtained from 5 patients who died of status asthmaticus, 2 asthmatic patients who died of nonrespiratory causes, and 6 nonasthmatic control subjects who died of nonrespiratory causes. Lung sections were stained for immunofluorescence with use of an antinitrotyrosine antibody, followed by a indiocarbocyanine (Cy5, Jackson Immunochemicals, Westgrove, Pa)-conjugated secondary antibody.. Nonasthmatic lungs showed little or no nitrotyrosine staining, whereas asthmatic lungs demonstrated significantly more staining of nitrotyrosine residues distributed in both the airways and lung parenchyma.. This study demonstrates the presence of nitrotyrosine, and hence evidence of formation of nitrating species, in the airways and lung parenchyma of patients with asthma who died of status asthmaticus or other nonrespiratory causes. This finding supports the concept that widespread airway and parenchymal inflammation occurs in asthma, and, more specifically, that NO and its reactive metabolites may play a pathophysiologic role in asthma.

Topics: Adolescent; Adult; Asthma; Bronchi; Female; Fluorescent Antibody Technique; Free Radicals; Humans; Lung; Male; Middle Aged; Models, Immunological; Nitric Oxide; Nitrogen Compounds; Status Asthmaticus; Tyrosine