3-nitrotyrosine and dihydrorhodamine-123

N-acetylcysteine (NAC) is known as an antioxidant and used for mucus viscosity reduction. However, this drug prevents or induces cell death depending on the cell type. The response of steroidogenic luteal cells to NAC is unknown. Our data shows that NAC can behave as an antioxidant or prooxidant in dependency on the concentration and mitochondrial energization. NAC elevated the flowcytometric-measured portion of hypodiploid (dying) cells. This rise was completely abolished by aurintricarboxylic acid, an inhibitor of topoisomerase II. NAC increased the secretion of nitric oxide and cellular nitrotyrosine. An image analysis indicated that cells pretreated with NAC and loaded with DHR showed a fluorescent structure probably elicited by the oxidative product of DHR, rhodamine 123 that sequesters mitochondrially. Pretreating luteal cells with NAC or adding NAC directly to mitochondrial fractions followed by assessing the mitochondrial transmembrane potential difference (Deltapsi) by the JC-1 technique demonstrated a marked decrease in Deltapsi. A protonophore restored Deltapsi and rotenone (an inhibitor of respiratory chain complex I) inhibited mitochondrial recovering. Thus, in steroidogenic luteal cells from healthy mature corpus luteum, NAC impairs cellular survival by interfering with mitochondrial metabolism. The protonophore-induced recovering of NAC-provoked decrease in Deltapsi indicates that an ATP synthase-favored route of H(+) re-entry to the matrix is essentially switched off by NAC while other respiratory chain complexes remain intact. These data may be important for therapeutic timing of treatments with NAC. (c) 2010 International Society for Advancement of Cytometry.

Topics: Acetylcysteine; Animals; Aurintricarboxylic Acid; Biomarkers; Cattle; Cell Nucleus; Cell Survival; Female; Flow Cytometry; Fluorescence; Intracellular Space; Lipid Peroxides; Luteal Cells; Membrane Potential, Mitochondrial; Mitochondria; Molecular Imaging; Oxidation-Reduction; Progesterone; Reactive Oxygen Species; Rhodamines; Signal Transduction; Steroids; Time Factors; Tyrosine

The ability of the sulfur-containing compounds glutathione (GSH), glutathione disulphide (GSSG), S-methylglutathione (GSMe), lipoic acid (LA), and dihydrolipoic acid (DHLA) to protect against hypochlorous acid (HOCl)-mediated damage and peroxynitrite (ONOOH)-induced damage has been compared. Protective activity was assessed in competition assays by monitoring several detectors, i.e. dihydrorhodamine-123 (DHR-123) oxidation, alpha(1)-antiproteinase (alpha(1)-AP) inactivation, and glutathione S-transferase P1-1 (GST-P1-1) inactivation. In addition, nitration of tyrosine was measured to assess protection of the sulfur-containing compounds against ONOOH. For protection against HOCl, the efficacy of the antioxidant was controlled by the ratio of the reaction rates of the antioxidant and the detector molecule with the oxidant. The rank order of the activity of the antioxidants (GSH > DHLA approximately LA approximately GSMe > GSSG) appeared to be independent of the detector used. However, the rank order of the antioxidants against ONOOH-induced damage is strongly dependent on the detector. LA was 40 times less active than GSH in the inhibition of ONOOH-induced DHR-123 oxidation, whereas LA was 20 times more active than GSH in preventing the inhibition of GST-P1-1 by ONOOH. This points to different molecular mechanisms of ONOOH damage to DHR-123 compared with ONOOH damage to GST-P1-1. LA is a poor antioxidant in protecting against the form of ONOOH damage involved in DHR-123 oxidation. In the form of ONOOH toxicity involved in GST-P1-1 inhibition, LA is the most potent sulfur-containing antioxidant in our series. It is proposed that an intermediate product in which both sulfur atoms of LA have reacted is involved in the reaction of ONOOH with LA. The high potency of LA to protect GST-P1-1 against ONOOH might be of therapeutic interest.

Topics: Antioxidants; Dose-Response Relationship, Drug; Glutathione; Glutathione S-Transferase pi; Glutathione Transferase; Hypochlorous Acid; Inhibitory Concentration 50; Isoenzymes; Models, Biological; Models, Chemical; Nitric Oxide; Oxygen; Peroxynitrous Acid; Potassium; Protein Binding; Rhodamines; Sulfur; Thioctic Acid; Tyrosine

Peroxynitrite (ONOO-), formed from the reaction of superoxide (.O2-) and nitric oxide (NO), is a potent oxidant that contributes to the oxidation of various cellular constituents, including lipids, amino acids, sulfhydryls and nucleotides. It can cause cellular injury, such as DNA fragmentation and apoptotic cell death. ONOO- toxicity is also reported to be involved in inflammatory and neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and atherosclerosis. Moreover, the necessity for a strong ONOO- scavenger is important because of the lack of endogenous enzymes that protect against the damage caused by ONOO-. The aim of this study was to evaluate the ability of natural products to scavenge ONOO-. We tested various plant extracts for their ONOO- scavenging activity. Among them, extract from Cassia tora, which is well known as an oriental herb in traditional medicine, showed potent ONOO- scavenging activity. Further analysis identified the phenolic active components, alaternin and nor-rubrofusarin glucose, as potent ONOO- scavengers. Spectrophotometric analysis demonstrated that alaternin and nor-rubrofusarin glucose led to a decrease in the ONOO- -mediated nitration of tyrosine through electron donation. In bovine serum albumin, alaternin, but not nor-rubrofusarin glucose, showed significant inhibition of ONOO- -mediated nitration in a dose-dependent manner. We believe alaternin can be developed as an effective ONOO- scavenger for the prevention of ONOO- -associated diseases.

Topics: Cassia; Emodin; Free Radical Scavengers; Glucosides; Nitric Oxide; Oxidation-Reduction; Peroxynitrous Acid; Plants, Medicinal; Pyrones; Rhodamines; Serum Albumin, Bovine; Spectrophotometry; Superoxides; Tyrosine

The protective effect of (-)-epicatechin 3-O-galate (ECg) against peroxynitrite (ONOO-)-mediated damage was examined using an animal model and a cell culture system. In rats subjected to lipopolysaccharide (LPS) administration plus ischemia-reperfusion, the plasma 3-nitrotyrosine level an indicator of ONOO- production in vivo, was elevated, whereas it declined significantly and dose-dependently after the oral administration of ECg at doses of 10 and 20 micromoles/kg body weight/day for 20 days prior to the process. Moreover, oral administration of ECg significantly enhanced the activities of the antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, and the antioxidant glutathione, showing enhancement of the biological defense system against the damage induced by ONOO-. In addition, the significant increase in the renal mitochondrial thiobarbituric acid-reactive substance level of LPS and ischemic-reperfused control rats was attenuated in rats given ECg. Furthermore, the elevations in the plasma urea nitrogen and creatinine (Cr) levels and the urinary methylguanidine/Cr ratio induced by the procedure were attenuated markedly after oral administration of ECg, implying amelioration of renal impairment. The addition of ECg (25 or 125 microM) prior to 3-morpholinosydnonimine (SIN-1, 800 microM) exposure reduced ONOO- formation and increased the viability of cultured renal epithelial (LLC-PK1) cells in a dose-dependent manner. In particular, ECg inhibited ONOO(-)-mediated apoptotic cell death, which was confirmed by decreases in the DNA fragmentation rate and the presence of apoptotic morphological changes, i.e. small nuclei and nuclear fragmentation. Furthermore, adding ECg before SIN-1 treatment regulated the cell cycle by enhancing G2/M phase arrest. This study provides evidence that ECg has protective activity against the renal damage induced by excessive ONOO- in cellular and in vivo systems.

Topics: Animals; Antioxidants; Caspases; Catechin; Cell Cycle; Cell Line; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Glutathione; Glutathione Peroxidase; Kidney; Lipid Peroxides; Lipopolysaccharides; LLC-PK1 Cells; Male; Molsidomine; Peroxynitrous Acid; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Rhodamines; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Time Factors; Tyrosine

Serum levels of uric acid (UA), an inhibitor of peroxynitrite- (ONOO-) related chemical reactions, became elevated approximately 30 million years ago in hominid evolution. During a similar time frame, higher mammals lost the ability to synthesize another important radical scavenger, ascorbic acid (AA), leading to the suggestion that UA may have replaced AA as an antioxidant. However, in vivo treatment with AA does not protect against the development of experimental allergic encephalomyelitis (EAE), a disease that has been associated with the activity of ONOO- and is inhibited by UA. When compared in vitro, UA and AA were found to have similar capacities to inhibit the nitrating properties of ONOO-. However UA and AA had different capacities to prevent ONOO- -mediated oxidation, especially in the presence of iron ion (Fe3+). While UA at physiological concentrations effectively blocked dihydrorhodamine-123 oxidation in the presence of Fe3+, AA did not, regardless of whether the source of ONOO- was synthetic ONOO-, SIN-1, or RAW 264.7 cells. AA also potentiated lipid peroxidation in vivo and in vitro. In conclusion, the superior protective properties of UA in EAE may be related to its ability to neutralize the oxidative properties of ONOO- in the presence of free iron ions.

Topics: Albumins; Animals; Antioxidants; Ascorbic Acid; Blood-Brain Barrier; Cell Line; Encephalomyelitis, Autoimmune, Experimental; Free Radicals; Immunohistochemistry; Iron; Lipid Peroxidation; Mice; Molsidomine; Myelin Sheath; Nitric Oxide; Oxygen; Peroxynitrous Acid; Rhodamines; Time Factors; Tyrosine; Uric Acid

The aim of the present study was to investigate the protective effect of the peroxynitrite decomposition catalyst 5,10,15, 20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrinato iron (III) (FeTMPS) in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malonaldehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin localized mainly in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats and stained with antibody to ICAM-1 showed a diffuse staining. Administration of FeTMPS significantly reduced ischemia/reperfusion injury in the bowel, and reduced lipid and the production of peroxynitrite during reperfusion. Treatment with PN catalyst also markedly reduced the intensity and degree of P-selectin and ICAM-1 staining in tissue sections from SAO-shocked rats and improved survival. Our results clearly demonstrate that peroxynitrite decomposition catalysts exert a protective effect in SAO and that this effect may be due to inhibition of the expression of adhesion molecules and the tissue damage associated with peroxynitrite-related pathways.

Topics: Animals; Blood Pressure; Catalysis; Celiac Artery; Disease Models, Animal; Endothelium, Vascular; Ferric Compounds; Ileum; Intercellular Adhesion Molecule-1; Leukocyte Count; Lipid Peroxidation; Male; Malondialdehyde; Metalloporphyrins; Nitrates; Nitric Oxide; Nitrites; Oxidative Stress; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Rhodamines; Splanchnic Circulation; Tyrosine

Peroxynitrite is a mediator molecule in inflammation, and its biological properties are being studied extensively. Flavonoids, which are natural plant constituents, protect against peroxynitrite and thereby could play an anti-inflammatory role. Procyanidin oligomers of different sizes (monomer through nonamer), isolated from the seeds of Theobroma cacao, were recently examined for their ability to protect against peroxynitrite-dependent oxidation of dihydrorhodamine 123 and nitration of tyrosine and were found to be effective in attenuating these reactions. The tetramer was particularly efficient at protecting against oxidation and nitration reactions. Epicatechin oligomers found in cocoa powder and chocolate may be a potent dietary source for defense against peroxynitrite.

Topics: Biflavonoids; Cacao; Catechin; Chromatography, High Pressure Liquid; Drug Interactions; Nitrates; Oxidants; Oxidation-Reduction; Proanthocyanidins; Rhodamines; Structure-Activity Relationship; Tyrosine

In the present study, we evaluated the impact of the lack of the gene for inducible nitric oxide synthase (iNOS) on oxidation, tyrosine nitration and cytotoxicity reactions triggered by immunostimulation. In mice injected with E. coli endotoxin (bacterial lipopolysaccharide, LPS, 50 mg/kg i.p.), there was a significant increase in the degree of oxidation of dihydrorhodamine 123 to rhodamine 123. This response was attenuated by inhibition of NO biosynthesis with NG-methyl-L-arginine (L-NMA, 30 mg/kg i.p.). In mice lacking functional iNOS gene (iNOS knock-out mice), the degree of the LPS-induced, L-NMA inhibitable increase in dihydrorhodamine oxidation was decreased, but not completely abolished. LPS stimulation induced a marked increase in the immunoreactivity for nitrotyrosine (an indicator of peroxynitrite formation), as measured in the aorta and lung. An L-NMA inhibitable increase in nitrotyrosine staining induced by LPS was also observed in the tissues of the iNOS knockout animals. LPS treatment induced the appearance of DNA single strand breakage and a suppression of mitochondrial respiration in peritoneal macrophages ex vivo. A significant degree of LPS-induced DNA single strand breakage and suppression of mitochondrial respiration was still observed in the peritoneal macrophages obtained from the iNOS knockout animals. Macrophages from wild-type mice stimulated with LPS and interferon-gamma suppressed the proliferation of various target cells (P815 mastocytoma, L929 fibrosarcoma and embryonic lung fibroblast cell line): this effect was abolished by in vitro treatment with L-NMA (1 mM). Macrophages from the iNOS knockout animals exhibited a reduced degree of target cell cytostatic activity. The remainder of the cytostasis in iNOS knockout macrophages was abolished by preventing cell contact and neutralizing tumor necrosis factor á. The present results demonstrate that the lack of iNOS gene does not fully abolish oxidation, tyrosine nitration and cytostatic activity in response to immunostimulation. The current findings may have implications for the development of NO-based approaches for the experimental therapy of inflammation.

Topics: Animals; Cell Division; Cells, Cultured; DNA Damage; Enzyme Inhibitors; Interferon-gamma; Lipopolysaccharides; Macrophage Activation; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Nitroarginine; Oxidation-Reduction; Rhodamine 123; Rhodamines; Tyrosine