3-nitrotyrosine and dihydroethidium

Chronic heart failure is characterized by decreased exercise capacity with early exacerbation of fatigue and dyspnea. Intrinsic skeletal muscle abnormalities can play a role in exercise intolerance. Causal or contributing factors responsible for muscle alterations have not been completely defined. This study evaluated skeletal muscle oxidative stress and NADPH oxidase activity in rats with myocardial infarction (MI) induced heart failure.. Four months after MI, rats were assigned to Sham, MI-C (without treatment), and MI-NAC (treated with N-acetylcysteine) groups. Two months later, echocardiogram showed left ventricular dysfunction in MI-C; NAC attenuated diastolic dysfunction. In soleus muscle, glutathione peroxidase and superoxide dismutase activity was decreased in MI-C and unchanged by NAC. 3-nitrotyrosine was similar in MI-C and Sham, and lower in MI-NAC than MI-C. Total reactive oxygen species (ROS) production was assessed by HPLC analysis of dihydroethidium (DHE) oxidation fluorescent products. The 2-hydroxyethidium (EOH)/DHE ratio did not differ between Sham and MI-C and was higher in MI-NAC. The ethidium/DHE ratio was higher in MI-C than Sham and unchanged by NAC. NADPH oxidase activity was similar in Sham and MI-C and lower in MI-NAC. Gene expression of p47(phox) was lower in MI-C than Sham. NAC decreased NOX4 and p22(phox) expression.. We corroborate the case that oxidative stress is increased in skeletal muscle of heart failure rats and show for the first time that oxidative stress is not related to increased NADPH oxidase activity.

Topics: Acetylcysteine; Animals; Ethidium; Free Radical Scavengers; Glutathione Peroxidase; Heart Failure; Heart Ventricles; Male; Malondialdehyde; Muscle, Skeletal; Myocardial Infarction; NADPH Oxidase 4; NADPH Oxidases; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Superoxide Dismutase; Tyrosine

Secondary degeneration contributes substantially to structural and functional deficits following traumatic injury to the CNS. While it has been proposed that oxidative stress is a feature of secondary degeneration, contributing reactive species and resultant oxidized products have not been clearly identified in vivo. The study is designed to identify contributors to, and consequences of, oxidative stress in a white matter tract vulnerable to secondary degeneration. Partial dorsal transection of the optic nerve (ON) was used to model secondary degeneration in ventral nerve unaffected by the primary injury. Reactive species were assessed using fluorescent labelling and liquid chromatography/tandem mass spectroscopy (LC/MS/MS). Antioxidant enzymes and oxidized products were semi-quantified immunohistochemically. Mitophagy was assessed by electron microscopy. Fluorescent indicators of reactive oxygen and/or nitrogen species increased at 1, 3 and 7days after injury, in ventral ON. LC/MS/MS confirmed increases in reactive species linked to infiltrating microglia/macrophages in dorsal ON. Similarly, immunoreactivity for glutathione peroxidase and haem oxygenase-1 increased in ventral ON at 3 and 7days after injury, respectively. Despite increased antioxidant immunoreactivity, DNA oxidation was evident from 1day, lipid oxidation at 3days, and protein nitration at 7days after injury. Nitrosative and oxidative damage was particularly evident in CC1-positive oligodendrocytes, at times after injury at which structural abnormalities of the Node of Ranvier/paranode complex have been reported. The incidence of mitochondrial autophagic profiles was also significantly increased from 3days. Despite modest increases in antioxidant enzymes, increased reactive species are accompanied by oxidative and nitrosative damage to DNA, lipid and protein, associated with increasing abnormal mitochondria, which together may contribute to the deficits of secondary degeneration.

Topics: Analysis of Variance; Animals; Chromatography, Liquid; Disease Models, Animal; Ectodysplasins; Ethidium; Female; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Guanine; Microscopy, Electron, Transmission; Mitochondria; Myelin Basic Protein; Nerve Degeneration; Optic Nerve Injuries; Oxidative Stress; Rats; Reactive Oxygen Species; Tandem Mass Spectrometry; Time Factors; Tyrosine

In humans, apolipoprotein E (apoE) is encoded by three major alleles (ε2, ε3, and ε4) and, compared to apoE3, apoE4 increases the risk of developing Alzheimer disease and cognitive impairments following various environmental challenges. Exposure to irradiation, including that of (56)Fe, during space missions poses a significant risk to the central nervous system, and apoE isoform might modulate this risk.. We investigated whether apoE isoform modulates hippocampus-dependent cognitive performance starting 2 weeks after (56)Fe irradiation. Changes in reactive oxygen species (ROS) can affect cognition and are induced by irradiation. Therefore, after cognitive testing, we assessed hippocampal ROS levels in ex vivo brain slices, using the ROS-sensitive fluorescent probe, dihydroethidium (DHE). Brain levels of 3-nitrotyrosine (3-NT), CuZn superoxide dismutase (CuZnSOD), extracellular SOD, and apoE were assessed using Western blotting analysis.. In the water maze, spatial memory retention was impaired by irradiation in apoE2 and apoE4 mice but enhanced by irradiation in apoE3 mice. Irradiation reduced DHE-oxidation levels in the enclosed blade of the dentate gyrus and levels of 3-NT and CuZnSOD in apoE2 but not apoE3 or apoE4 mice. Finally, irradiation increased apoE levels in apoE3 but not apoE2 or apoE4 mice.. The short-term effects of (56)Fe irradiation on hippocampal ROS levels and hippocampus-dependent spatial memory retention are apoE isoform-dependent.

Topics: Animals; Apolipoprotein E2; Apolipoprotein E3; Apolipoprotein E4; Apolipoproteins E; Blotting, Western; Dentate Gyrus; Ethidium; Fluorescent Dyes; Genotype; Hippocampus; Humans; Iron; Male; Maze Learning; Memory Disorders; Mice; Mice, Inbred C57BL; Reactive Oxygen Species; Superoxide Dismutase; Tyrosine

Inflammation and oxidative stress play a crucial role in angiotensin (Ang) II-mediated vascular injury. Angiotensin-converting enzyme 2 (ACE2) has recently been identified as a specific Ang II-degrading enzyme but its role in vascular biology remains elusive. We hypothesized that loss of ACE2 would facilitate Ang II-mediated vascular inflammation and peroxynitrite production. 10-week wildtype (WT, Ace2(+/y)) and ACE2 knockout (ACE2KO, Ace2(-/y)) mice received with mini-osmotic pumps with Ang II (1.5 mg.kg⁻¹.d⁻¹) or saline for 2 weeks. Aortic ACE2 protein was obviously reduced in WT mice in response to Ang II related to increases in profilin-1 protein and plasma levels of Ang II and Ang-(1-7). Loss of ACE2 resulted in greater increases in Ang II-induced mRNA expressions of inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β, and IL-6 without affecting tumor necrosis factor-α in aortas of ACE2KO mice. Furthermore, ACE2 deficiency led to greater increases in Ang II-mediated profilin-1 expression, NADPH oxidase activity, and superoxide and peroxynitrite production in the aortas of ACE2KO mice associated with enhanced phosphorylated levels of Akt, p70S6 kinase, extracellular signal-regulated kinases (ERK1/2) and endothelial nitric oxide synthase (eNOS). Interestingly, daily treatment with AT1 receptor blocker irbesartan (50 mg/kg) significantly prevented Ang II-mediated aortic profilin-1 expression, inflammation, and peroxynitrite production in WT mice with enhanced ACE2 levels and the suppression of the Akt-ERK-eNOS signaling pathways. Our findings reveal that ACE2 deficiency worsens Ang II-mediated aortic inflammation and peroxynitrite production associated with the augmentation of profilin-1 expression and the activation of the Akt-ERK-eNOS signaling, suggesting potential therapeutic approaches by enhancing ACE2 action for patients with vascular diseases.

Topics: Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; Aorta; Blotting, Western; Chemokine CCL2; Ethidium; Inflammation; Interleukin-1beta; Interleukin-6; Mice; Mice, Knockout; NADPH Oxidases; Peptidyl-Dipeptidase A; Peroxynitrous Acid; Profilins; Real-Time Polymerase Chain Reaction; Tyrosine

Superoxide has been shown to play a major role in ventricular remodeling and arrhythmias after myocardial infarction. However, the source of increased myocardial superoxide production and the role of superoxide in sympathetic innervation remain to be further characterized. Male Wistar rats, after coronary artery ligation, were randomized to vehicle, allopurinol, or apocynin for 4weeks. To determine the role of peroxynitrite in sympathetic reinnervation, we also used 3-morpholinosydnonimine (a peroxynitrite generator). The postinfarction period was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine, xanthine oxidase activity, NADPH oxidase activity, and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Sympathetic hyperinnervation was blunted after administration of allopurinol. Arrhythmic scores in the allopurinol-treated infarcted rats were significantly lower than those in vehicle. For similar levels of ventricular remodeling, apocynin had no beneficial effects on oxidative stress, sympathetic hyperinnervation, or arrhythmia vulnerability. Allopurinol-treated hearts had significantly decreased nerve growth factor expression, which was substantially increased after coadministration of 3-morpholinosydnonimine. These results indicate that xanthine oxidase but not NADPH oxidase largely mediates superoxide production after myocardial infarction. Xanthine oxidase inhibition ameliorates sympathetic innervation and arrhythmias possibly via inhibition of the peroxynitrite-mediated nerve growth factor pathway.

Topics: Acetophenones; Allopurinol; Animals; Arrhythmias, Cardiac; Coronary Vessels; Ethidium; Male; Molsidomine; Myocardial Infarction; Myocardium; NADPH Oxidases; Nerve Growth Factor; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Wistar; Superoxides; Sympathetic Nervous System; Tyrosine; Xanthine Oxidase

Mice lacking Cu,Zn superoxide dismutase (SOD1) show accelerated, age-related loss of muscle mass. Lack of SOD1 may lead to increased superoxide, reduced nitric oxide (NO), and increased peroxynitrite, each of which could initiate muscle fiber loss. Single muscle fibers from flexor digitorum brevis of wild-type (WT) and Sod1(-/-) mice were loaded with NO-sensitive (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, DAF-FM) and superoxide-sensitive (dihydroethidium, DHE) probes. Gastrocnemius muscles were analyzed for SOD enzymes, nitric oxide synthases (NOS), and 3-nitrotyrosine (3-NT) content. A lack of SOD1 did not increase superoxide availability at rest because no increase in ethidium or 2-hydroxyethidium (2-HE) formation from DHE was seen in fibers from Sod1(-/-) mice compared with those from WT mice. Fibers from Sod1(-/-) mice had decreased NO availability (decreased DAF-FM fluorescence), increased 3-NT in muscle proteins indicating increased peroxynitrite formation and increased content of peroxiredoxin V (a peroxynitrite reductase), compared with WT mice. Muscle fibers from Sod1(-/-) mice showed substantially reduced generation of superoxide in response to contractions compared with fibers from WT mice. Inhibition of NOS did not affect DHE oxidation in fibers from WT or Sod1(-/-) mice at rest or during contractions, but transgenic mice overexpressing nNOS showed increased DAF-FM fluorescence and reduced DHE oxidation in resting muscle fibers. It is concluded that formation of peroxynitrite in muscle fibers is a major effect of lack of SOD1 in Sod1(-/-) mice and may contribute to fiber loss in this model, and that NO regulates superoxide availability and peroxynitrite formation in muscle.

Topics: Aging; Animals; Blotting, Western; Carbonic Anhydrase III; Electric Stimulation; Ethidium; Fluoresceins; Fluorescence; Isometric Contraction; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Muscle, Skeletal; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Peroxiredoxins; Peroxynitrous Acid; Reactive Oxygen Species; Superoxide Dismutase; Superoxide Dismutase-1; Superoxides; Tyrosine