3-nitrotyrosine and Hyperoxia

Hyperoxia (ventilation with FIO2 = 1.0) has vasoconstrictor properties, in particular in the coronary vascular bed, and, hence, may promote cardiac dysfunction. However, we previously showed that hyperoxia attenuated myocardial injury during resuscitation from hemorrhage in swine with coronary artery disease. Therefore, we tested the hypothesis whether hyperoxia would also mitigate myocardial injury and improve heart function in the absence of chronic cardiovascular comorbidity.After 3 h of hemorrhage (removal of 30% of the calculated blood volume and subsequent titration of mean arterial pressure to 40 mm Hg) 19 anesthetized, mechanically ventilated, and instrumented pigs received FIO2 = 0.3(control) or hyperoxia(FIO2 = 1.0) during the first 24 h. Before, at the end of and every 12 h after shock, hemodynamics, blood gases, metabolism, cytokines, and cardiac function (pulmonary artery thermodilution, left ventricular pressure-conductance catheterization) were recorded. At 48 h, cardiac tissue was harvested for western blotting, immunohistochemistry, and mitochondrial respiration.Except for higher left ventricular end-diastolic pressures at 24 h (hyperoxia 21 (17;24), control 17 (15;18) mm Hg; P = 0.046), hyperoxia affected neither left ventricular function cardiac injury (max. Troponin I at 12 h: hyperoxia:9 (6;23), control:17 (11;24) ng mL; P = 0.395), nor plasma cytokines (except for interleukin-1β: hyperoxia 10 (10;10) and 10 (10;10)/control 14 (10;22), 12 (10;15) pg mL, P = 0.023 and 0.021 at 12 and 24 h, respectively), oxidation and nitrosative stress, and mitochondrial respiration. However, hyperoxia decreased cardiac tissue three-nitrotyrosine formation (P < 0.001) and inducible nitric oxide synthase expression (P = 0.016). Ultimately, survival did not differ significantly either.In conclusion, in contrast to our previous study in swine with coronary artery disease, hyperoxia did not beneficially affect cardiac function or tissue injury in healthy swine, but was devoid of deleterious side effects.

Topics: Animals; Gene Expression Regulation, Enzymologic; Hyperoxia; Myocardium; Nitric Oxide Synthase Type II; Resuscitation; Shock, Hemorrhagic; Swine; Tyrosine; Ventricular Function, Left

We previously demonstrated beneficial effects of 22 h of hyperoxia following near-lethal porcine hemorrhagic shock, whereas therapeutic hypothermia was detrimental. Therefore, we investigated whether shorter exposure to hyperoxia (12 h) would still improve organ function, and whether 12 h of hypothermia with subsequent rewarming could avoid deleterious effects after less severe hemorrhagic shock.Twenty-seven anesthetized and surgically instrumented pigs underwent 3 h of hemorrhagic shock by removal of 30% of the blood volume and titration of the mean arterial blood pressure (MAP) to 40 mm Hg. Post-shock, pigs were randomly assigned to control, hyperoxia (FIO2 100% for 12 h) or hypothermia group (34°C core temperature for 12 h with subsequent rewarming). Before, at the end of shock, after 12 and 23 h of resuscitation, data sets comprising hemodynamics, blood gases, and parameters of inflammation and organ function were acquired. Postmortem, kidney samples were collected for immunohistochemistry and western blotting.Hyperoxia exerted neither beneficial nor detrimental effects. In contrast, mortality in the hypothermia group was significantly higher compared with controls (67% vs. 11%). Hypothermia impaired circulation (MAP 64 (57;89) mm Hg vs. 104 (98; 114) mm Hg) resulting in metabolic acidosis (lactate 11.0 (6.6;13.6) mmol L vs. 1.0 (0.8;1.5) mmol L) and reduced creatinine clearance (26 (9;61) mL min vs. 77 (52;80) mL min) compared to the control group after 12 h of resuscitation. Impaired kidney function coincided with increased renal 3-nitrotyrosine formation and extravascular albumin accumulation.In conclusion, hyperoxia proved to be safe during resuscitation from hemorrhagic shock. The lacking organ-protective effects of hyperoxia compared to resuscitation from near-lethal hemorrhage suggest a dependence of the effectiveness of hyperoxia from shock severity. In line with our previous report, therapeutic hypothermia (and rewarming) was confirmed to be detrimental most likely due to vascular barrier dysfunction.

Topics: Animals; Blood Gas Analysis; Hemodynamics; Hyperoxia; Hypothermia, Induced; Nitric Oxide Synthase Type II; Oxidative Stress; Shock, Hemorrhagic; Swine; Tyrosine

Preterm infants often require supplemental oxygen due to lung immaturity, but hyperoxia can contribute to an increased risk of respiratory illness later in life. Our aim was to compare the effects of mild and moderate levels of neonatal hyperoxia on markers of pulmonary oxidative stress and inflammation and on lung architecture; both immediate and persistent effects were assessed. Neonatal mice (C57BL6/J) were raised in either room air (21% O2), mild (40% O2), or moderate (65% O2) hyperoxia from birth until postnatal day 7 (P7d). The mice were killed at either P7d (immediate effects) or lived in air until adulthood (P56d, persistent effects). We enumerated macrophages in lung tissue at P7d and immune cells in bronchoalveolar lavage fluid (BALF) at P56d. At P7d and P56d, we assessed pulmonary oxidative stress [heme oxygenase-1 (HO-1) and nitrotyrosine staining] and lung architecture. The data were interrogated for sex differences. At P7d, HO-1 gene expression was greater in the 65% O2 group than in the 21% O2 group. At P56d, the area of nitrotyrosine staining and number of immune cells were greater in the 40% O2 and 65% O2 groups relative to the 21% O2 group. Exposure to 65% O2, but not 40% O2, led to larger alveoli and lower tissue fraction in the short term and to persistently fewer bronchiolar-alveolar attachments. Exposure to 40% O2 or 65% O2 causes persistent increases in pulmonary oxidative stress and immune cells, suggesting chronic inflammation within the adult lung. Unlike 65% O2, 40% O2 does not affect lung architecture.

Topics: Animals; Animals, Newborn; Bronchoalveolar Lavage Fluid; Female; Heme Oxygenase-1; Hyperoxia; Inflammation; Macrophages, Alveolar; Membrane Proteins; Mice; Mice, Inbred C57BL; Oxidative Stress; Oxygen; Pulmonary Alveoli; Tyrosine

Central nervous system oxygen toxicity (CNS-OT) can occur in humans at pressures above 2atmospheres absolute (ATA), and above 4.5ATA in the rat. Pulmonary oxygen toxicity appears at pressures above 0.5ATA. We hypothesized that exposure to mild HBO following extreme exposure might provide protection against CNS, but not pulmonary oxygen toxicity. We measured the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and nitrotyrosine and nNOS levels in the brain and lung in the following groups: (1) Sham rats, no pressure exposure (SHAM); (2) Exposure to 6ATA oxygen for 60% of latency to CNS-OT (60%LT); (3) Exposure to 6ATA for 60% of latency to CNS-OT, followed by 20min at 2.5ATA for recovery (REC); (4) Exposure to 6ATA for 60% of latency to CNS-OT, followed by 20min at 2.5ATA oxygen and a subsequent increase in pressure to 6ATA until the appearance of convulsions (CONV); (5) Control rats exposed to 6ATA until the appearance of convulsions (C). SOD and CAT activity were reduced in both brain and lung in the REC group. GPX activity was reduced in the hippocampus in the REC group, but not in the cortex or the lung. nNOS levels were reduced in the hippocampus in the REC group. Contrary to our hypothesis, no difference was observed between the brain and the lung for the factors investigated. We suggest that at 2.5ATA and above, CNS and pulmonary oxygen toxicity may share similar mechanisms.

Topics: Animals; Catalase; Cerebral Cortex; Glutathione Peroxidase; Hippocampus; Hyperoxia; Lung; Male; Nitric Oxide Synthase Type I; Pressure; Rats, Sprague-Dawley; Seizures; Superoxide Dismutase; Tyrosine

Hyperoxia causes oxidative stress. Breath-hold diving is associated with transient hyperoxia followed by hypoxia and a build-up of carbon dioxide (CO₂), chest-wall compression and significant haemodynamic changes. This study analyses variations in plasma oxidative stress markers after a series of repetitive breath-hold dives.. Thirteen breath-hold divers were asked to perform repetitive breath-hold dives to 20 metres' depth to a cumulative breath-hold time of approximately 20 minutes over an hour in the open sea. Plasma nitric oxide (NO), peroxinitrites (ONOO⁻) and thiols (R-SH) were measured before and after the dive sequence.. Circulating NO significantly increased after successive breath-hold dives (169.1 ± 58.26% of pre-dive values; P = 0.0002). Peroxinitrites doubled after the dives (207.2 ± 78.31% of pre-dive values; P = 0.0012). Thiols were significantly reduced (69.88 ± 19.23% of pre-dive values; P = 0.0002).. NO may be produced by physical effort during breath-hold diving. Physical exercise, the transient hyperoxia followed by hypoxia and CO₂ accumulation would all contribute to the increased levels of superoxide anions (O₂²⁻). Since interaction of O₂²⁻ with NO forms ONOO⁻, this reaction is favoured and the production of thiol groups is reduced. Oxidative stress is, thus, present in breath-hold diving.

Topics: Adult; Biomarkers; Breath Holding; Diving; Humans; Hyperoxia; Hypoxia; Male; Nitric Oxide; Nitrites; Oxidative Stress; Sulfhydryl Compounds; Time Factors; Tyrosine

The effect of hyperoxygenation at reperfusion, particularly in the setting of cardiac arrest, remains unclear. This issue was studied in a prolonged cardiac arrest model consisting of 25 min cardiac arrest in a rat resuscitated with cardiopulmonary bypass (CPB). The objective of this study was to determine the effect of hyperoxygenation following prolonged cardiac arrest resuscitation on mitochondrial and cardiac function.. Male Sprague-Dawley rats (400-450 g) were anesthetized with ketamine and xylazine and instrumented for closed chest cardiopulmonary bypass (CPB). Following a 25-min KCl-induced cardiac arrest, the animals were resuscitated by CPB with 100% oxygen. Three minutes after successful return of spontaneous circulation (ROSC), the animals received either normoxemic reperfusion (CPB with 40-50% oxygen) or hyperoxemic reperfusion (CPB with 100% oxygen) for 1 h. Post-resuscitation hemodynamics, cardiac function, mitochondrial function and immunostaining of 3-nitrotyrosine were compared between the two different treatment groups.. At 1 h after ROSC, the hyperoxemic reperfusion group had a significant higher mean arterial pressure, less metabolic acidosis and better diastolic function than the normoxemic reperfusion group. Cardiac mitochondria from the hyperoxemic reperfusion group had a higher respiratory control ratio (RCR) and cardiac tissue showed less nitroxidative stress compared to the normoxemic reperfusion group.. One hour of hyperoxemic reperfusion after 25 min of cardiac arrest in an in vivo CPB model resulted in significant short-term improvement in myocardial and mitochondrial function compared with 1h of normoxemic reperfusion. This myocardial response may differ from previously reported post-arrest hyperoxia mediated effects following shorter arrest times.

Topics: Analysis of Variance; Animals; Blood Gas Analysis; Cardiopulmonary Bypass; Heart Arrest; Hemodynamics; Hyperoxia; Immunohistochemistry; Male; Mitochondria, Heart; Oxygen Inhalation Therapy; Rats; Rats, Sprague-Dawley; Time Factors; Tyrosine

Prematurely born infants are often treated with supraphysiologic amounts of oxygen, which is associated with lung injury and the development of diseases such as bronchopulmonary dysplasia. Complimentary responses between the lung and liver during the course of hyperoxic lung injury have been studied in adult animals, but little is known about this relationship in neonates. These studies tested the hypothesis that oxidant stress occurs in the livers of newborn mice in response to continuous hyperoxia exposure. Greater levels of glutathione disulfide and nitrotyrosine were detected in lung tissues but not liver tissues from newborn mice exposed to hyperoxia than in room air-exposed controls. However, early increases in 5-lipoxygenase and cyclooxygenases-2 protein levels and increases in total hydroxyeicosatetraenoic acid and prostaglandin levels were observed in the liver tissues of hyperoxia-exposed pups. These studies indicate that free radical oxidation occurs in the lungs of newborn pups exposed to hyperoxia, and alterations in lipid metabolism could be a primary response in the liver tissues. The findings of this study identify possible new mechanisms associated with hyperoxic lung injury in a newborn model of bronchopulmonary dysplasia and thus open opportunities for research.

Topics: Acute Lung Injury; Animals; Animals, Newborn; Arachidonate 5-Lipoxygenase; Coenzyme A; Cyclooxygenase 2; Disease Models, Animal; Glutathione Disulfide; Hydroxyeicosatetraenoic Acids; Hyperoxia; Liver; Lung; Mice; Mice, Inbred C3H; Oxidative Stress; Prostaglandins; Time Factors; Tyrosine

Diabetic retinopathy and retinopathy of prematurity are blinding disorders that follow a pathological pattern of ischemic retinopathy and affect premature infants and working-age adults. Yet, the treatment options are limited to laser photocoagulation. The goal of this study is to elucidate the molecular mechanism and examine the therapeutic effects of inhibiting tyrosine nitration on protecting early retinal vascular cell death and late neovascularization in the ischemic retinopathy model. Ischemic retinopathy was developed by exposing neonatal mice to 75% oxygen [postnatal day (p) 7-p12] followed by normoxia (21% oxygen) (p12-p17). Peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTPPS) (1 mg/kg), the nitration inhibitor epicatechin (10 mg/kg) or the thiol donor N-acetylcysteine (NAC, 150 mg/kg) were administered (p7-p12) or (p7-p17). Vascular endothelial cells were incubated at hyperoxia (40% oxygen) or normoxia (21% oxygen) for 48 h. Vascular density was determined in retinal flat mounts labeled with isolectin B4. Expression of vascular endothelial growth factor, caspase-3, and poly(ADP ribose) polymerase (PARP), activation of Akt and p38 mitogen-activated protein kinase (MAPK), and tyrosine nitration of the phosphatidylinositol (PI) 3-kinase p85 subunit were analyzed by Western blot. Hyperoxia-induced peroxynitrite caused endothelial cell apoptosis as indicated by expression of cleaved caspase-3 and PARP leading to vaso-obliteration. These effects were associated with significant tyrosine nitration of the p85 subunit of PI 3-kinase, decreased Akt activation, and enhanced p38 MAPK activation. Blocking tyrosine nitration of PI 3-kinase with epicatechin or NAC restored Akt phosphorylation, and inhibited vaso-obliteration at p12 and neovascularization at p17 comparable with FeTPPS. Early inhibition of tyrosine nitration with use of epicatechin or NAC can represent safe and effective vascular-protective agents in ischemic retinopathy.

Topics: Acetylcysteine; Animals; Animals, Newborn; Apoptosis; Blotting, Western; Catechin; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Glutathione; Hyperoxia; Hypoxia; Ischemia; Lipid Peroxidation; Metalloporphyrins; Mice; Mice, Inbred C57BL; Peroxynitrous Acid; Protective Agents; Retinal Neovascularization; Retinal Vessels; Tyrosine

Inhaled nitric oxide is being evaluated as a preventative therapy for patients at risk for bronchopulmonary dysplasia (BPD). Nitric oxide (NO), in the presence of superoxide, forms peroxynitrite, which reacts with tyrosine residues on proteins to form 3-nitrotyrosine (3-NT). However, NO can also act as an antioxidant and was recently found to improve the oxidative balance in preterm infants. Thus, we tested the hypothesis that the addition of a therapeutically relevant concentration (10 ppm) of NO to a hyperoxic exposure would lead to decreased 3-NT formation in the lung. FVB mouse pups were exposed to either room air (21% O(2)) or >95% O(2) with or without 10 ppm NO within 24 h of birth. In the first set of studies, body weights and survival were monitored for 7 days, and exposure to >95% O(2) resulted in impaired weight gain and near 100% mortality by 7 days. However, the mortality occurred earlier in pups exposed to >95% O(2) + NO than in pups exposed to >95% O(2) alone. In a second set of studies, lungs were harvested at 72 h. Immunohistochemistry of the lungs at 72 h revealed that the addition of NO decreased alveolar, bronchial, and vascular 3-NT staining in pups exposed to both room air and hyperoxia. The lung nitrite levels were higher in animals exposed to >95% oxygen + NO than in animals exposed to >95% oxygen alone. The protein levels of myeloperoxidase, monocyte chemotactic protein-1, and intracellular adhesion molecule-1 were assessed after 72 h of exposure and found to be greatest in the lungs of pups exposed to >95% O(2). This hyperoxia-induced protein expression was significantly attenuated by the addition of 10 ppm NO. We propose that in the presence of >95% O(2), peroxynitrite formation results in protein nitration; however, adding excess NO to the >95% O(2) exposure prevents 3-NT formation by NO reacting with peroxynitrite to produce nitrite and NO(2). We speculate that the decreased protein nitration observed with the addition of NO may be a potential mechanism limiting hyperoxic lung injury.

Topics: Animals; Chemokine CCL2; Hyperoxia; Intercellular Adhesion Molecule-1; Lung; Lung Injury; Mice; Nitric Oxide; Oxygen; Peroxidase; Tyrosine

Overproduction of neuronal nitric oxide synthase (nNOS)-derived NO is detrimental during cerebral ischemia. Normobaric hyperoxia (NBO) has been shown to be neuroprotective, extending the therapeutic time window for ischemic stroke, but the mechanism is not fully understood. In the present study, using a rat model of ischemic stroke, we investigated the effect of early NBO treatment on neuronal NO production. Male Sprague-Dawley rats were given normoxia (30% O(2)) or NBO (95% O(2)) during 10, 30, 60 or 90min filament occlusion of the middle cerebral artery. NO(x)(-) (nitrite plus nitrate) and 3-nitrotyrosine were measured in the ischemic cortex. Ischemia caused a rapid increase in the production of NO(x)(-), with a peak at 10min after ischemia onset, then gradually declining to the baseline level at 60min. NBO treatment delayed the NO(x)(-) production peak to 30min and attenuated the total amount of NO(x)(-). Ischemia also increased 3-nitrotyrosine formation, which was significantly reduced by NBO treatment. Inhibition of nNOS by pre-treatment with 7-nitroindazole had similar effect as NBO treatment on NO(x)(-) and 3-nitrotyrosine production, and when combined with NBO, no further reduction in NO production was observed. Furthermore, NBO treatment significantly decreased brain infarct volume. Taken together, our findings demonstrate that delaying and attenuating the early NO release from nNOS may be an important mechanism accounting for NBO's neuroprotection.

Topics: Animals; Brain Ischemia; Cerebral Infarction; Hyperoxia; Ischemic Attack, Transient; Male; Middle Cerebral Artery; Nitrates; Nitric Acid; Nitrites; Rats; Rats, Sprague-Dawley; Tyrosine

Increased oxidative stress is associated with perinatal asphyxia and respiratory distress in the newborn period. Induction of nuclear factor erythroid 2 p45-related factor (Nrf2) has been shown to decrease oxidative stress through the regulation of specific gene pathways. We hypothesized that Nrf2 attenuates mortality and alveolar growth inhibition in newborn mice exposed to hyperoxia. Nrf2(+/+) and Nrf2(-/-) newborn mice were exposed to hyperoxia at 24 h. Survival was significantly less in Nrf2(-/-) mice exposed to 72 h of hyperoxia and returned to room air (P < 0.0001) and in Nrf2(-/-) mice exposed to hyperoxia for 8 continuous days (P < 0.005). To determine the response of Nrf2 target genes to hyperoxia, glutathione peroxidase 2 (Gpx2) and NAD(P)H:quinone oxidoreductase (NQO1) expression was measured from lung of newborn mice using real-time PCR. In the Nrf2(+/+) mice, significant induction of lung Gpx2 and NQO1 above room air controls was found with hyperoxia. In contrast, Nrf2(-/-) mice had minimal induction of lung Gpx2 and NQO1 with hyperoxia. Expression of p21 and IL-6, genes not regulated by Nrf2, were also measured. IL-6 expression in Nrf2(-/-) lung was markedly induced by 72 h of hyperoxia in contrast to the Nrf2(+/+) mice. p21 was induced in both Nrf2(+/+) and Nrf2(-/-) lung by hyperoxia. Mean linear intercept (MLI) and mean chord length (MCL) were significantly increased in 14-day-old Nrf2(-/-) mice previously exposed to hyperoxia compared with Nrf2(+/+) mice. The percentage of surfactant protein C (Sp-c(+)) type 2 alveolar cells in 14-day-old Nrf2(-/-) mice exposed to neonatal hyperoxia was also significantly less than Nrf2(+/+) mice (P < 0.02). In summary, these findings indicate that Nrf2 increases survival in newborn mice exposed to hyperoxia and that Nrf2 may help attenuate alveolar growth inhibition caused by hyperoxia exposure.

Topics: Animals; Animals, Newborn; Cell Death; Cell Proliferation; Glutathione Peroxidase; Hyperoxia; In Situ Nick-End Labeling; Inhalation Exposure; Mice; NAD(P)H Dehydrogenase (Quinone); NADPH Dehydrogenase; NF-E2-Related Factor 2; Oxidative Stress; Pulmonary Alveoli; Pulmonary Surfactant-Associated Protein C; Survival Analysis; Tyrosine

Hyperoxia disrupts postnatal lung development in part through inducing inflammation. To determine the contribution of leukocyte-derived reactive oxygen species, we exposed newborn wild-type and NADPH oxidase p47(phox) subunit null (p47(phox-/-)) mice to air or acute hyperoxia (95% O(2)) for up to 11 days. Hyperoxia-induced pulmonary neutrophil influx was similar in wild-type and p47(-/-) mice at postnatal days (P) 7 and 11. Macrophages were decreased in wild-type hyperoxia-exposed mice compared with p47(phox-/-) mice at P11. Hyperoxia impaired type II alveolar epithelial cell and bronchiolar epithelial cell proliferation, but depression of type II cell proliferation was significantly less in p47(-/-) mice at P3 and P7, when inflammation was minimal. We found reciprocal results for the expression of the cell cycle inhibitor p21(cip/waf) in type II cells, which was induced in 95% O(2)-exposed wild-type mice, but significantly less in p47(phox-/-) littermates at P7. Despite partial preservation of type II cell proliferation, deletion of p47(phox) did not prevent the major adverse effects of hyperoxia on alveolar development estimated by morphometry at P11, but hyperoxia impairment of elastin deposition at alveolar septal crests was significantly worse in wild-type vs. p47(phox-/-) mice at P11. Since we found that p47(phox) is expressed in a subset of alveolar epithelial cells, its deletion may protect postnatal type II alveolar epithelial proliferation from hyperoxia through effects on epithelial as well as phagocyte-generated superoxide.

Topics: Aging; Air; Animals; Animals, Newborn; Body Weight; Bronchoalveolar Lavage Fluid; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Epithelial Cells; Gene Deletion; Hyperoxia; Mice; NADPH Oxidases; Oxygen; Pneumonia; Protein Transport; Pulmonary Alveoli; Pulmonary Surfactant-Associated Protein C; Survival Analysis; Tyrosine

Hypoxia resultant from haemorrhagic shock is the primary cause of kidney damage. Application of normobaric hyperoxia therapy (NHT) is an acceptable treatment for acute haemorrhagic shock. We investigated the effect of NHT on amelioration of haemorrhagic shock-induced rat renal failure.. Twenty-four Sprague-Dawley rats were subjected to gradual blood withdrawal/reperfusion, followed by 12-h, 24-h or 48-h NHT. Verification/monitoring of intrarenal hypoxia was performed using Hypoxyprobe-TM-1. Subsequently, cystatin C, urea and creatinine were assessed in serum by a Hitachi autoanalyser, and NO, 3-nitro-tyrosine, STAT-8-isoprostane and NF-kB in renal medullae and cortices by specific ELISAs.. In rats subjected to haemorrhagic shock, 12- to 48-h NHT significantly reduced intrarenal Hypoxyprobe-TM-1 stained areas and attenuated augmentation of urea, creatinine and cystatin C. Haemorrhagic shock resulted in a 10-fold drop of intrarenal NO availability. 12-h and 24-h, but not 48-h, NHT significantly increased cortical/medullar NO synthesis, the latter, however, not approaching the pre-shock values. Significant shock-induced accumulation of STAT-8-isoprostane and 3-nitro-tyrosine was further exacerbated by NHT. Haemorrhagic shock activated NF-kB in ischaemic tissues, which was not attenuated by NHT.. (1) 12- to 48-h NHT decreased intrarenal hypoxia signs and ameliorated deterioration of renal functions in a rat model of haemorrhagic shock-induced renal failure. (2) 12- to 24 h NHT improved bioavailability of NO in cortices/medullae of kidneys recuperating from haemorrhagic shock. (3) If any anti-inflammatory activities were stimulated by NHT, they would not be mediated via the NF-kB pathway. (4) Despite NHT-associated elevation of reactive oxygen species (ROS), early oxygen supply proved mandatory for effective recuperation of ischaemic kidney from detrimental consequences of haemorrhagic shock.

Topics: Acute Kidney Injury; Animals; Blood Pressure; Creatinine; Cystatin C; Cystatins; Dinoprostone; Disease Models, Animal; Hyperoxia; Kidney; Male; Nitric Oxide; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Shock, Hemorrhagic; Transcription Factor RelA; Tyrosine; Urea

High-concentration oxygen therapy is used to treat tissue hypoxia, but hyperoxia causes lung injury. Overproduction of nitric oxide by nitric oxide synthase (NOS) is thought to promote hyperoxic lung injury. The present study was conducted to examine the role of inducible nitric oxide synthase (iNOS) in hyperoxic lung injury in mice.. Mice were exposed to >98% oxygen for 72 h, and ONO-1714 (0.05 mg/kg) (ONO) was subcutaneously administered to block iNOS. Hyperoxia significantly increased total cell count, protein concentration, and nitrites/nitrates in the bronchoalveolar lavage fluid and proinflammatory cytokines in the lung tissue. ONO significantly prevented the increases in all of these variables. ONO suppressed histologic evidence of lung injury. ONO markedly inhibited iNOS protein expression and nitrotyrosine production in lung homogenates. After exposure to hyperoxia, alveolar epithelial cells stained positively for 8-hydroxy-2'-deoxyguanosine, a proper marker of oxidative DNA damage by reactive oxygen species. ONO attenuated this finding.. NOS play important roles in the pathogenesis of hyperoxic lung injury. Selective iNOS inhibitors may be useful for the treatment of hyperoxic lung injury.

Topics: Amidines; Animals; Blotting, Western; Bronchoalveolar Lavage Fluid; Cell Differentiation; Cytokines; Drug Administration Schedule; Enzyme Inhibitors; Heterocyclic Compounds, 2-Ring; Hyperoxia; Immunohistochemistry; Injections, Subcutaneous; Lung; Lung Diseases; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Nitrogen Oxides; Tyrosine

To clarify the role of macrophage class A scavenger receptors (SR-A, CD204) in oxidative lung injury, we examined lung tissue of SR-A deficient (SR-A(-/-)) and wild-type (SR-A(+/+)) mice in response to hyperoxic treatment. Protein levels of bronchoalveolar lavage fluid (BALF) and pulmonary oedema (wet : dry weight ratios) were higher in SR-A(-/-) mice than those in SR-A(+/+) mice. Cumulative survival was significantly decreased in SR-A(-/-) mice. However, there were no differences in BALF macrophage and neutrophil count between the two groups. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that messenger RNA (mRNA) levels of the inducible nitric oxide synthase (iNOS) were increased during hyperoxic injury, and this increase was more prominent in SR-A(-/-) mice. Expression levels of iNOS in alveolar macrophages after hyperoxia in vivo and in vitro were higher in SR-A(-/-) macrophages compared with SR-A(+/+) macrophages. Immunohistochemistry using anti-nitrotyrosine antibodies revealed distinctive oxidative stress in the injured lung in both groups, but it was more remarkable in the SR-A(-/-) mice. After hyperoxic treatment, pulmonary mRNA levels of tumour necrosis factor-alpha(TNF-alpha) were elevated more rapidly in SR-A(-/-) mice than in SR-A(+/+) mice. Together these results suggest that SR-A expression attenuates hyperoxia-induced lung injury by reducing macrophage activation.

Topics: Animals; Blotting, Western; Bronchoalveolar Lavage Fluid; Hyperoxia; Immunohistochemistry; Macrophage Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase Type II; Oxidative Stress; Respiratory Distress Syndrome; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scavenger Receptors, Class A; Tumor Necrosis Factor-alpha; Tyrosine

Pulmonary manifestations of oxygen toxicity were studied and quantified in rats breathing >98% O(2) at 1, 1.5, 2, 2.5, and 3 ATA to test our hypothesis that different patterns of pulmonary injury would emerge, reflecting a role for central nervous system (CNS) excitation by hyperbaric oxygen. At 1.5 atmosphere absolute (ATA) and below, the well-recognized pattern of diffuse pulmonary damage developed slowly with an extensive inflammatory response and destruction of the alveolar-capillary barrier leading to edema, impaired gas exchange, respiratory failure, and death; the severity of these effects increased with time over the 56-h period of observation. At higher inspired O(2) pressures, 2-3 ATA, pulmonary injury was greatly accelerated but less inflammatory in character, and events in the brain were a prelude to a distinct lung pathology. The CNS-mediated component of this lung injury could be attenuated by selective inhibition of neuronal nitric oxide synthase (nNOS) or by unilateral transection of the vagus nerve. We propose that extrapulmonary, neurogenic events predominate in the pathogenesis of acute pulmonary oxygen toxicity in hyperbaric oxygenation, as nNOS activity drives lung injury by modulating the output of central autonomic pathways.

Topics: Animals; Behavior, Animal; Blood Gas Analysis; Body Fluids; Bronchoalveolar Lavage Fluid; Hyperoxia; L-Lactate Dehydrogenase; Lung; Lung Diseases; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Oxygen; Pneumonia; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Survival Analysis; Tyrosine; Vagotomy

Hypoxia impairs skeletal muscle function, but the precise mechanisms are incompletely understood. In hypoxic rat diaphragm muscle, generation of peroxynitrite is elevated. Peroxynitrite and other reactive nitrogen species have been shown to impair contractility of skinned muscle fibers, reflecting contractile protein dysfunction. We hypothesized that hypoxia induces contractile protein dysfunction and that reactive nitrogen species are involved. In addition, we hypothesized that muscle reoxygenation reverses contractile protein dysfunction. In vitro contractility of rat soleus muscle bundles was studied after 30 min of hyperoxia (Po2 approximately 90 kPa), hypoxia (Po2 approximately 5 kPa), hypoxia + 30 microM N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), hyperoxia + 30 microM L-NMMA, and hypoxia (30 min) + reoxygenation (15 min). One part of the muscle bundle was used for single fiber contractile measurements and the other part for nitrotyrosine detection. In skinned single fibers, maximal Ca2+-activated specific force (Fmax), fraction of strongly attached cross bridges (alphafs), rate constant of force redevelopment (ktr), and myofibrillar Ca2+ sensitivity were determined. Thirty minutes of hypoxia reduced muscle bundle contractility. In the hypoxic group, single fiber Fmax, alphafs, and ktr were significantly reduced compared with hyperoxic, L-NMMA, and reoxygenation groups. Myofibrillar Ca2+ sensitivity was not different between groups. Nitrotyrosine levels were increased in hypoxia compared with all other groups. We concluded that acute hypoxia induces dysfunction of skinned muscle fibers, reflecting contractile protein dysfunction. In addition, our data indicate that reactive nitrogen species play a role in hypoxia-induced contractile protein dysfunction. Reoxygenation of the muscle bundle partially restores bundle contractility but completely reverses contractile protein dysfunction.

Topics: Animals; Enzyme Inhibitors; Hyperoxia; Hypoxia; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle Fibers, Skeletal; Muscle, Skeletal; omega-N-Methylarginine; Oxygen; Partial Pressure; Rats; Rats, Wistar; Reactive Oxygen Species; Tyrosine

Inflammation contributes greatly to the pathogenesis of bronchopulmonary dysplasia. In previous studies, we showed that blocking neutrophil influx by treatment with SB265610, a selective CXCR2 antagonist, could partly reduce superoxide accumulation and preserve alveolar development in 60% O(2)-exposed newborn rats. The purpose of this study was to further investigate the role of neutrophils in the formation of reactive oxygen and nitrogen species mediating hyperoxia-impaired lung development. We found that hydroxyl radical formation and lipid peroxidation in rat lungs were significantly increased during 60% O(2) exposure. These increases were attenuated by the administration of SB265610. In addition, SB265610 largely inhibited protein nitration induced by hyperoxia. SB265610 partly prevented the hyperoxia-enhanced bronchoalveolar lavage (BAL) protein content in 60% O(2)-exposed animals. Our results demonstrate that neutrophils have a pivotal role in hydroxyl radical formation, lipid peroxidation and protein nitration. Taken together with our previous studies, the present findings show that blocking neutrophil influx protects alveolar development and improves lung function in part by preventing reactive oxygen/nitrogen species accumulation.

Topics: Animals; Animals, Newborn; Dinoprost; Hydroxyl Radical; Hyperoxia; Lung; Neutrophils; Oxygen; Rats; Reactive Nitrogen Species; Reactive Oxygen Species; Receptors, Interleukin-8B; Tyrosine

Although nitrated proteins have been repeatedly used as markers of lung injury, little is known about their formation and metabolism under hyperoxia. We therefore measured 3-nitrotyrosine (3NTYR) concentrations in lung tissue and serum of rats with carrageenan-induced pneumonia exposed to hyperoxia. Twenty-nine Wistar male rats were assigned to one of 4 groups. Two experimental groups were treated by intratracheal application of carrageenan (0.5 ml of 0.7 % solution) and then one was exposed to hyperoxia for 7 days (FIO2 0.8), the other to air. Rats of two control groups breathed either hyperoxic gas mixture or air for 7 days. At the end of exposure the ventilation was determined in anesthetized, intubated animals in which 3NTYR concentrations were measured in the lung tissue and nitrites and nitrates (NOx) were estimated in the serum. Carrageenan instillation increased 3NTYR concentrations in lung tissue (carrageenan-normoxic group 147+/-7 pmol/g protein, control 90+/-10 pmol/g protein) and NOx concentration in the serum (carrageenan-normoxic group 126+/-13 ppb, control 78+/-9 ppb). Hyperoxia had no effect on lung tissue 3NTYR concentration in controls (control-hyperoxic 100+/-14 pmol/g protein) but blocked the increase of lung tissue 3NTYR in carrageenan-treated rats (carrageenan-hyperoxic 82+/-13 pmol/g protein), increased NOx in serum (control-hyperoxic 127+/-19 ppb) and decreased serum concentration of 3NTYR in both hyperoxic groups (carrageenan-hyperoxic 51+/-5 pmol/g protein, control-hyperoxic 67+/-7 pmol/g protein, carrageenan-normoxic 82+/-9 pmol/g protein, control 91+/-7 pmol/g protein). The results suggest that hyperoxia affects nitration of tyrosine residues, probably by increasing 3NTYR degradation.

Topics: Animals; Carrageenan; Hyperoxia; Lung; Male; Pneumonia; Rats; Rats, Wistar; Tyrosine

Relative hyperoxia is a condition frequently encountered in premature infants, either spontaneously or during treatment in the Neonatal Intensive Care Unit. The effects of high inspiratory oxygen concentrations on immature brain cells and their signaling cascades are largely unknown. The aim of the study was to investigate the effect of hyperoxia on the amount and topographic distribution of iNOS-expression (inducible nitric oxide synthase) in the immature rat brain, and to localize hyperoxia-induced formation of peroxynitrite as a potential marker of cellular damage to immature cerebral structures. Seven-day-old Wistar rat pups were exposed to >80% oxygen for 24 h and were then transcardially perfused. Following paraformaldehyde fixation, brains were paraffin-embedded and immunohistochemically stained for iNOS and nitrotyrosine. iNOS protein was quantified by Western blot; iNOS mRNA expression was studied by RT-PCR. Total brain iNOS mRNA was up-regulated, demonstrating a peak at 6 h following the onset of hyperoxia. Immunohistochemical staining was predominantly observed in microglial cells of hippocampus and frontal cortex with some iNOS reactivity in endothelial and perivascular cells. Nitrotyrosine staining was positive in apical dendrites of neurons in the frontal cortex. There was no positive staining for iNOS or nitrotyrosine in control animals. Hyperoxia causes iNOS mRNA and protein up-regulation in microglial cells of the immature rat brain. Positive neuronal nitrotyrosine staining indicates formation of peroxynitrite with potential deleterious effects for immature cellular structures in the neonatal brain.

Topics: Animals; Blotting, Western; Brain; Brain Diseases; Hyperoxia; Immunohistochemistry; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Tyrosine

Several studies have suggested that exposure to hyperoxia causes lung injury through increased generation of reactive oxygen and nitrogen species. The present study was aimed to investigate the effects of hyperoxia exposure on protein nitration in lungs. Rats were exposed to hyperoxia (>95%) for 48, 60, and 72 h. Histopathological analysis showed a dramatic change in the severity of lung injury in terms of edema and hemorrhage between 48- and 60-h exposure times. Western blot for nitrotyrosine showed that several proteins with molecular masses of 29-66 kDa were nitrated in hyperoxic lung tissues. Immunohistochemical analyses indicate nitrotyrosine staining of alveolar epithelial and interstitial regions. Furthermore, immunoprecipitation followed by Western blot revealed the nitration of surfactant protein A and t1alpha, proteins specific for alveolar epithelial type II and type I cells, respectively. The increased myeloperoxidase (MPO) activity and total nitrite levels in bronchoalveolar lavage and lung tissue homogenates were observed in hyperoxic lungs. Neutrophils and macrophages isolated from the hyperoxia-exposed rats, when cocultured with a rat lung epithelial L2 cell line, caused a significant protein nitration in L2 cells. Inclusion of nitrite further increased the protein nitration. These studies suggest that protein nitration during hyperoxia may be mediated in part by MPO generated from activated phagocytic cells, and such protein modifications may contribute to hyperoxia-mediated lung injury.

Topics: Amino Acid Sequence; Animals; Bronchoalveolar Lavage Fluid; Cell Line; Edema; Hemorrhage; Hyperoxia; Lung; Macrophages, Alveolar; Neutrophils; Nitrates; Nitrites; Peptide Fragments; Peroxidase; Proteins; Pulmonary Surfactant-Associated Protein A; Rats; Respiratory Mucosa; Time Factors; Tyrosine

Hyperoxia causes a transient decrease in CBF, followed by a later rise. The mediators of these effects are not known. We used mice lacking endothelial or neuronal nitric oxide synthase (NOS) isoforms (eNOS-/- and nNOS-/- mice) to study the roles of the NOS isoforms in mediating changes in cerebral vascular tone in response to hyperoxia. Resting regional cerebral blood flow (rCBF) did not differ between wild type (WT), eNOS-/- mice, and nNOS-/- mice. eNOS-/- mice showed decreased cerebrovascular reactivities to NG-nitro-L-arginine methyl ester (L-NAME), PAPA NONOate, acetylcholine (Ach), and SOD1. In response to hyperbaric oxygen (HBO2) at 5 ATA, WT and nNOS-/- mice showed decreases in rCBF over 30 minutes, but eNOS-/- mice did not. After 60 minutes HBO2, rCBF increased more in WT mice than in eNOS-/- or nNOS-/- mice. Brain NO-metabolites (NOx) decreased in WT and eNOS-/- mice within 30 minutes of HBO2, but after 45 minutes, NOx rose above control levels, whereas they did not change in nNOS-/- mice. Brain 3NT increased during HBO2 in WT and eNOS-/- but did not change in nNOS-/- mice. These results suggest that modulation of eNOS-derived NO by HBO2 is responsible for the early vasoconstriction responses, whereas late HBO2-induced vasodilation depends upon both eNOS and nNOS.

Topics: Animals; Cerebrovascular Circulation; Hyperbaric Oxygenation; Hyperoxia; Mice; Mice, Inbred C57BL; Mice, Knockout; Microdialysis; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Tyrosine; Vasoconstriction

gamma-Glutamyl transferase (GGT) is critical to glutathione homeostasis by providing substrates for glutathione synthesis. We hypothesized that loss of GGT would cause oxidant stress in the lung. We compared the lungs of GGT(enu1) mice, a genetic model of GGT deficiency, with normal mice in normoxia to study this hypothesis. We found GGT promoter 3 (P3) alone expressed in normal lung but GGT P3 plus P1, an oxidant-inducible GGT promoter, in GGT(enu1) lung. Glutathione content was barely decreased in GGT(enu1) lung homogenate and elevated nearly twofold in epithelial lining fluid, but the fraction of oxidized glutathione was increased three- and fourfold, respectively. Glutathione content in GGT(enu1) alveolar macrophages was decreased nearly sixfold, and the oxidized glutathione fraction was increased sevenfold. Immunohistochemical studies showed glutathione deficiency together with an intense signal for 3-nitrotyrosine in nonciliated bronchiolar epithelial (Clara) cells and expression of heme oxygenase-1 in the vasculature only in GGT(enu1) lung. When GGT(enu1) mice were exposed to hyperoxia, survival was decreased by 25% from control because of accelerated formation of vascular pulmonary edema, widespread oxidant stress in the epithelium, diffuse depletion of glutathione, and severe bronchiolar cellular injury. These data indicate a critical role for GGT in lung glutathione homeostasis and antioxidant defense in normoxia and hyperoxia.

Topics: Animals; Antibody Specificity; Female; gamma-Glutamyltransferase; Glutathione; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hyperoxia; Lung; Lung Diseases; Male; Membrane Proteins; Mice; Mice, Mutant Strains; Oxidative Stress; Oxygen; RNA, Messenger; Survival Rate; Tyrosine

Exposure of premature human infants to hyperoxia results in the obliteration of developing retina capillaries, leading to a vision-threatening retinopathy termed retinopathy of prematurity (ROP). The authors hypothesized that this process may be mediated in part by endothelial nitric oxide (NO)-derived oxidants such as peroxynitrite and tested this hypothesis in a mouse model of ROP.. Normal mice, mice treated with the nitric oxide synthase (NOS) inhibitor N:(G)-nitro-L-arginine (L-NNA), and knockout mice carrying a homozygous targeted disruption of the gene for endothelial NOS (eNOS) were studied in an experimental model of ROP. Retinas were compared for extent of capillary obliteration in hyperoxia, vascular endothelial growth factor (VEGF) expression, nitrotyrosine formation, and vitreous neovascularization.. Oxygen-induced retinal vaso-obliteration was significantly reduced by L-NNA treatment (43% decrease from controls). The eNOS-deficient mice showed a similar reduction in vaso-obliteration (46% decrease from controls), and vitreous neovascularization was also substantially reduced (threefold decrease). Retinal nitrotyrosine formation, a measure of in situ peroxynitrite modification of proteins, was significantly elevated in normal mice during hyperoxia, in a spatial and temporal pattern consistent with a role in oxygen-induced vaso-obliteration. This was not seen in eNOS-deficient mice. VEGF expression was similar in both groups of mice, although suppression in hyperoxia was slightly blunted in eNOS-deficient mice.. These data suggest a role for NO and peroxynitrite in the pathogenesis of ROP. Therapies aimed at modulation of eNOS activity may have therapeutic potential for preventing ROP.

Topics: Animals; Endothelial Growth Factors; Enzyme Inhibitors; Humans; Hyperoxia; Infant, Newborn; Lymphokines; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Tyrosine; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Effective lung repair after acute injury requires elimination of proliferating mesenchymal and inflammatory cells without inducing an acute inflammatory response or disturbing concomitant repair of lung microvasculature. Previous studies have shown that endogenous NO regulates programmed cell death in fibroblasts and can modulate wound fibroblast synthetic function. We hypothesized that exposure of human lung fibroblasts to NO gas would decrease viability and induce apoptotic cell death. Primary cultures of normal human lung fibroblasts were exposed for 4 h to room air (RA), 80% oxygen, NO (at either 20 or 50 ppm) blended with RA, or NO blended with 80% O(2), then incubated for 24 to 72 h. Cell viability was determined by fluorescence viability/cytotoxicity assay and DNA fragmentation by TUNEL assay. Peroxynitrite formation was assessed using immunoblotting for S-nitrosotyrosine. NO plus O(2) induced significant cell death at 20 and 50 ppm NO when compared to either RA or O(2) alone at both 24 and 72 h (p < 0.05). Incubation with superoxide dismutase (SOD), catalase (CAT) or SOD + CAT significantly decreased cell death in fibroblasts treated with NO(20)/O(2) and NO(50)/O(2) compared with controls (p < 0.05). NO(20)/O(2) and NO(50)/O(2) exposure significantly increased TUNEL mean fluorescence intensity (MFI), consistent with increased DNA fragmentation, compared to RA at 24 and 72 h (p < 0.05). Antioxidants decreased MFI in cells exposed to NO(20)/O(2) (CAT and SOD + CAT) compared to controls at 24 h (p < 0.05). Western blot analysis for S-nitrosotyrosine showed increased signal intensity in fibroblasts exposed to NO at 20 and 50 ppm plus O(2) compared to RA or O(2) alone. Incubation with SOD + CAT reduced signal intensity for peroxynitrite in cells exposed to NO(20)/O(2). We conclude that NO in hyperoxic conditions induces fibroblast cell death and DNA fragmentation, which could be partially mediated by peroxynitrite synthesis.

Topics: Antioxidants; Catalase; Cell Survival; Cells, Cultured; DNA Fragmentation; Fibroblasts; Humans; Hyperoxia; In Situ Nick-End Labeling; Lung; Nitrates; Nitric Oxide; Oxygen; Superoxides; Tyrosine

The effect of hyperoxia on nitric oxide (NO) production in intact animals is unknown. We described the effects of hyperoxia on inducible nitric oxide synthase (iNOS) expression and NO production in the lungs of rats exposed to high concentrations of oxygen. Animals were placed in sealed Plexiglas chambers and were exposed to either 85% oxygen (hyperoxic group) or 21% oxygen (negative control group). Animals were anesthetized after 24 and 72 h of exposure and were ventilated via a tracheotomy. We measured NO production in exhaled air (E(NO)) by chemiluminescence. The lungs were then harvested and processed for detection of iNOS by immunohistochemistry and Western blotting analysis. The same experiments were repeated in animals exposed to hyperoxia for 72 h after they were infused with L-arginine. We used rats that were injected intraperitoneally with Escherichia coli lipopolysaccharide to induce septic shock as a positive control group. Hyperoxia and septic shock induced expression of iNOS in the lung. However, E(NO) was elevated only in septic shock rats but was normal in the hyperoxic group. Exogenous infusion of L-arginine after hyperoxia did not increase E(NO). To exclude the possibility that in the hyperoxic group NO was scavenged by oxygen radicals to form peroxynitrite, lungs were studied by immunohistochemistry for the detection of nitrotyrosine. Nitrotyrosine was found in septic shock animals but not in the hyperoxic group, further suggesting that NO is not synthesized in rats exposed to hyperoxia. We conclude that hyperoxia induces iNOS expression in the lung without an increase in NO concentration in the exhaled air.

Topics: Animals; Blotting, Western; Hyperoxia; Immunohistochemistry; Lipopolysaccharides; Luminescent Measurements; Lung; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Respiration; Shock, Septic; Time Factors; Tyrosine