3-nitrotyrosine and ebselen

Oxidative stress is a key driver of vascular dysfunction in diabetes mellitus. Ebselen is a glutathione peroxidase mimetic. A single-site, randomized, double-masked, placebo-controlled, crossover trial was carried out in 26 patients with type 1 or type 2 diabetes to evaluate effects of high-dose ebselen (150 mg po twice daily) administration on oxidative stress and endothelium-dependent vasodilation. Treatment periods were in random order of 4 wk duration, with a 4-wk washout between treatments. Measures of oxidative stress included nitrotyrosine, plasma 8-isoprostanes, and the ratio of reduced to oxidized glutathione. Vascular ultrasound of the brachial artery and plethysmographic measurement of blood flow were used to assess flow-mediated and methacholine-induced endothelium-dependent vasodilation of conduit and resistance vessels, respectively. Ebselen administration did not affect parameters of oxidative stress or conduit artery or forearm arteriolar vascular function compared with placebo treatment. There was no difference in outcome by diabetes type. Ebselen, at the dose and duration evaluated, does not improve the oxidative stress profile, nor does it affect endothelium-dependent vasodilation in patients with diabetes mellitus.

Topics: Adult; Antioxidants; Azoles; Brachial Artery; Case-Control Studies; Cross-Over Studies; Diabetes Mellitus; Dinoprost; Double-Blind Method; Endothelium, Vascular; Female; Forearm; Glutathione; Humans; Isoindoles; Male; Methacholine Chloride; Middle Aged; Organoselenium Compounds; Oxidative Stress; Parasympathomimetics; Plethysmography; Tyrosine; Ultrasonography; Vasodilation

Intracochlear electrical stimulation (ES) generated by cochlear implants (CIs) is used to activate auditory nerves to restore hearing perception in deaf subjects and those with residual hearing who use electroacoustic stimulation (EAS) technology. Approximately 1/3 of EAS recipients experience loss of residual hearing a few months after ES activation, but the underlying mechanism is unknown. Clinical evidence indicates that the loss is related to the previous history of noise-induced hearing loss (NIHL). In this report, we investigated the impact of intracochlear ES on oxidative stress levels and synaptic counts in inner hair cells (IHCs) of the apical, middle and basal regions of guinea pigs with normal hearing (NH) and NIHL. Our results demonstrated that intracochlear ES with an intensity of 6 dB above the thresholds of electrically evoked compound action potentials (ECAPs) could induce the elevation of oxidative stress levels, resulting in a loss of IHC synapses near the electrodes in the basal and middle regions of the NH cochleae. Furthermore, the apical region of cochleae with NIHL were more susceptible to synaptic loss induced by relatively low-intensity ES than that of NH cochleae, resulting from the additional elevation of oxidative stress levels and the reduced antioxidant capability throughout the whole cochlea.

Topics: Action Potentials; Aldehydes; Animals; Antioxidants; Cochlea; Cochlear Implants; Electric Stimulation; Evoked Potentials, Auditory, Brain Stem; Fatty Acids, Unsaturated; Guinea Pigs; Hair Cells, Auditory, Inner; Hearing Loss, Noise-Induced; Hydroxy Acids; Isoindoles; Organoselenium Compounds; Oxidative Stress; Severity of Illness Index; Synapses; Tyrosine

Oxidative stress is involved in the development of carotid body (CB) chemosensory potentiation and systemic hypertension induced by chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea. We tested whether peroxynitrite (ONOO(-)), a highly reactive nitrogen species, is involved in the enhanced CB oxygen chemosensitivity and the hypertension during CIH. Accordingly, we studied effects of Ebselen, an ONOO(-) scavenger, on 3-nitrotyrosine immunoreactivity (3-NT-ir) in the CB, the CB chemosensory discharge, and arterial blood pressure (BP) in rats exposed to CIH. Male Sprague-Dawley rats were exposed to CIH (5% O2, 12 times/h, 8 h/day) for 7 days. Ebselen (10 mg/kg/day) was administrated using osmotic minipumps and BP measured with radiotelemetry. Compared to the sham animals, CIH-treated rats showed increased 3-NT-ir within the CB, enhanced CB chemosensory responses to hypoxia, increased BP response to acute hypoxia, and hypertension. Rats treated with Ebselen and exposed to CIH displayed a significant reduction in 3-NT-ir levels (60.8 ± 14.9 versus 22.9 ± 4.2 a.u.), reduced CB chemosensory response to 5% O2 (266.5 ± 13.4 versus 168.6 ± 16.8 Hz), and decreased mean BP (116.9 ± 13.2 versus 82.1 ± 5.1 mmHg). Our results suggest that CIH-induced CB chemosensory potentiation and hypertension are critically dependent on ONOO(-) formation.

Topics: Animals; Azoles; Blood Pressure; Carotid Body; Diastole; Hypertension; Hypoxia; Isoindoles; Male; Neuronal Plasticity; Organoselenium Compounds; Peroxynitrous Acid; Rats, Sprague-Dawley; Systole; Tyrosine

Metabolic syndrome (MetS) is a combination of clinical disorders that together increase the risk for cardiovascular disease and diabetes. SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP.ZF) rats with MetS show impaired nitric oxide-mediated relaxation in coronary and mesenteric arteries, and angiotensin II receptor type 1 blockers protect against dysfunction and oxidative-nitrosative stress independently of metabolic effects. We hypothesize that superoxide contributes to functional deterioration in SHRSP.ZF rats. To test our hypothesis, we studied effects of treatment with tempol, a membrane-permeable radical scavenger, on impaired vasodilation in SHRSP.ZF rats. Tempol did not alter body weight, high blood pressure, or metabolic abnormalities, but prevented impairment of acetylcholine-induced and nitroprusside-induced vasodilation in the coronary and mesenteric arteries. Furthermore, tempol reduced the levels of serum thiobarbituric acid reactive substance (TBARS) and 3-nitrotyrosine content in mesenteric arteries. Systemic administration of tempol elevated the expression of soluble guanylate cyclase (sGC) above basal levels in mesenteric arteries of SHRSP.ZF rats. However, acute treatment with tempol or ebselen, a peroxynitrite scavenger, did not ameliorate impaired relaxation of isolated mesenteric arteries. No nitration of tyrosine residues in sGC was observed; however, sGC mRNA expression levels in the arteries of SHRSP.ZF rats were lower than those in the arteries of Wistar-Kyoto rats. Levels of Thr(496)- and Ser(1177)-phosphorylated endothelial nitric oxide synthase (eNOS) were lower in arteries of SHRSP.ZF rats, and acetylcholine decreased Thr(496)-phosphorylated eNOS levels. These results indicated that prolonged superoxide production, leading to oxidative-nitrosative stress, was associated with impaired vasodilation in SHRSP.ZF rats with MetS. Down-regulated sGC expression may be linked to dysfunction, while reduced NO bioavailability/eNOS activity and modified sGC activity due to superoxide production were excluded as pivotal mechanisms.

Topics: Animals; Azoles; Coronary Vessels; Cyclic N-Oxides; Disease Models, Animal; Down-Regulation; Free Radical Scavengers; Guanylate Cyclase; Heart; Isoindoles; Male; Metabolic Syndrome; Nitric Oxide; Nitric Oxide Synthase Type III; Nitrogen; Organoselenium Compounds; Oxidative Stress; Phosphorylation; Rats; Receptor, Angiotensin, Type 1; RNA, Messenger; Spin Labels; Superoxides; Tyrosine; Vasodilation

Given their medical importance, proteases have been studied by diverse approaches and screened for small molecule protease inhibitors. Here, we present a multiplexed microsphere-based protease assay that uses high-throughput flow cytometry to screen for inhibitors of the light chain protease of botulinum neurotoxin type A (BoNTALC). Our assay uses a full-length substrate and several deletion mutants screened in parallel to identify small molecule inhibitors. The use of multiplex flow cytometry has the advantage of using full-length substrates, which contain already identified distal-binding elements for the BoNTALC, and could lead to a new class of BoNTALC inhibitors. In this study, we have screened 880 off patent drugs and bioavailable compounds to identify ebselen as an in vitro inhibitor of BoNTALC. This discovery demonstrates the validity of our microsphere-based approach and illustrates its potential for high-throughput screening for inhibitors of proteases in general.

Topics: Antigens, Bacterial; Azoles; Bacterial Toxins; Botulinum Toxins, Type A; Drug Evaluation, Preclinical; Flow Cytometry; Fluorescence Resonance Energy Transfer; High-Throughput Screening Assays; Isoindoles; Metalloproteases; Microspheres; Organoselenium Compounds; Protease Inhibitors

To investigate the effect of the GPx1-mimetic ebselen on diabetes-associated atherosclerosis and renal injury in a model of increased oxidative stress.. The study was performed using diabetic apolipoprotein E/GPx1 (ApoE(-/-)GPx1(-/-))-double knockout (dKO) mice, a model combining hyperlipidemia and hyperglycemia with increased oxidative stress. Mice were randomized into two groups, one injected with streptozotocin, the other with vehicle, at 8 weeks of age. Groups were further randomized to receive either ebselen or no treatment for 20 weeks.. Ebselen reduced diabetes-associated atherosclerosis in most aortic regions, with the exception of the aortic sinus, and protected dKO mice from renal structural and functional injury. The protective effects of ebselen were associated with a reduction in oxidative stress (hydroperoxides in plasma, 8-isoprostane in urine, nitrotyrosine in the kidney, and 4-hydroxynonenal in the aorta) as well as a reduction in VEGF, CTGF, VCAM-1, MCP-1, and Nox2 after 10 weeks of diabetes in the dKO aorta. Ebselen also significantly reduced the expression of proteins implicated in fibrosis and inflammation in the kidney as well as reducing related key intracellular signaling pathways.. Ebselen has an antiatherosclerotic and renoprotective effect in a model of accelerated diabetic complications in the setting of enhanced oxidative stress. Our data suggest that ebselen effectively repletes the lack of GPx1, and indicate that ebselen may be an effective therapeutic for the treatment of diabetes-related atherosclerosis and nephropathy. Furthermore, this study highlights the feasibility of addressing two diabetic complications with one treatment regimen through the unifying approach of targeted antioxidant therapy.

Topics: Aldehydes; Animals; Antioxidants; Apolipoproteins E; Atherosclerosis; Azoles; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Diabetic Nephropathies; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Isoindoles; Kidney; Male; Mice; Mice, Knockout; Organoselenium Compounds; Reactive Oxygen Species; Tyrosine; Vascular Cell Adhesion Molecule-1

Recently we showed that lack of the antioxidant enzyme glutathione peroxidase-1 (GPx1) accelerates atherosclerosis and upregulates proatherogenic pathways in diabetic apoE/GPx1-deficient double-knockout mice, thereby establishing GPx1 as an important therapeutic target. In vivo studies now investigate ebselen, a seleno-organic GPx1-mimetic, for its potential to reduce diabetes-associated atherosclerosis.. Lesions were significantly increased in diabetic apoE(-/-) aortas (P<0.001) compared with nondiabetic controls after 20 weeks of diabetes. Ebselen-gavage significantly reduced total aortic lesions (P<0.001), with significant regional reductions in the arch (P<0.001), thoracic (P<0.001), and abdominal regions (P<0.05), but not within the aortic sinus of diabetic apoE(-/-) mice. These reductions were accompanied by significantly lower nitrotyrosine and Nox2 levels, reduced proatherogenic cellularity (macrophages and SMCs), and reduced expression of the proatherogenic mediator RAGE. Within the aortic sinus, ebselen reduced nitrotyrosine, Nox2, and VEGF levels but had no effect on RAGE. Studies in HAECs show that ebselen abrogates H(2)O(2)-induced increases in P-IKK, P-JNK, TNF-alpha, and Nox2.. Ebselen reduces atherosclerotic lesions in most regions of diabetic apoE(-/-) aorta, except within the aortic sinus, suggesting its effectiveness as a potential antiatherogenic therapy in diabetic-macrovascular disease. Ebselen may elicit its effect via modulation of transcription factors such as NF-kappaB and AP-1.

Topics: Administration, Oral; Animals; Antioxidants; Aorta, Abdominal; Aorta, Thoracic; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Azoles; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Endothelial Cells; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Humans; I-kappa B Kinase; Isoindoles; JNK Mitogen-Activated Protein Kinases; Macrophages; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; NADPH Oxidase 2; NADPH Oxidases; Organoselenium Compounds; Phenotype; Phosphorylation; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine; Vascular Endothelial Growth Factor A

Small coronary arteries (SCA) from diabetic rats exhibit enhanced peroxynitrite (ONOO(-)) formation and concurrent impairment of voltage-dependent potassium (K(v)) channel function. However, it is unclear whether ONOO(-) plays a causative role in this impairment. We hypothesized that functional loss of K(v) channels in coronary smooth muscle cells (SMC) in diabetes is due to ONOO(-) with subsequent tyrosine nitration of K(v) channel proteins. Diabetic rats and nondiabetic controls were treated with or without ebselen (Eb) for 4 wk. SCA were prepared for immunohistochemistry (IHC), immunoprecipitation (IP) followed by Western blot (WB), videomicroscopy, and patch-clamp analysis. IHC revealed excess ONOO(-) in SCA from diabetic rats. IP and WB revealed elevated nitration of the K(v)1.2 alpha-subunit and reduced K(v)1.2 protein expression in diabetic rats. Each of these changes was improved in Eb-treated rats. Protein nitration and K(v)1.5 expression were unchanged in SCA from diabetic rats. Forskolin, a direct cAMP activator that induces K(v)1 channel activity, dilated SCA from nondiabetic rats in a correolide (Cor; a selective K(v)1 channel blocker)-sensitive fashion. Cor did not alter the reduced dilation to forskolin in diabetic rats; however, Eb partially restored the Cor-sensitive component of dilation. Basal K(v) current density and response to forskolin were improved in smooth muscle cells from Eb-treated DM rats. We conclude that enhanced nitrosative stress in diabetes mellitus contributes to K(v)1 channel dysfunction in the coronary microcirculation. Eb may be beneficial for the therapeutic treatment of vascular complications in diabetes mellitus.

Topics: Adenylyl Cyclases; Animals; Antioxidants; Azoles; Colforsin; Coronary Vessels; Cyclic AMP; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Enzyme Activators; Isoindoles; Kv1.2 Potassium Channel; Kv1.5 Potassium Channel; Male; Membrane Potentials; Muscle, Smooth, Vascular; Organoselenium Compounds; Oxidative Stress; Peroxynitrous Acid; Potassium Channel Blockers; Protein Subunits; Rats; Rats, Sprague-Dawley; Research Design; Triterpenes; Tyrosine; Vasodilation

Oxidants may play a role in hypoxia-induced respiratory muscle dysfunction. In the present study we hypothesized that hypoxia-induced impairment in diaphragm contractility is associated with elevated peroxynitrite generation. In addition, we hypothesized that strenuous contractility of the diaphragm increases peroxynitrite formation. In vitro force-frequency relationship, isotonic fatigability, and nitrotyrosine levels were assessed under hypoxic (Po(2) approximately 6.5 kPa) and hyperoxic (Po(2) approximately 88.2 kPa) control conditions and also in the presence of authentic peroxynitrite (60 min), ebselen (60 min), and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine acetate (L-NMMA) (90 min). A hypoxia-induced downward shift of the force-frequency relationship was associated with elevated nitrotyrosine level in the diaphragm. During hypoxia, both ebselen and L-NMMA decreased nitrotyrosine levels but did not affect force generation. Strenuous contractions impaired force generation but did not affect nitrotyrosine levels in the diaphragm during hypoxia. But under hyperoxic conditions, fatiguing contractions were associated with elevated diaphragm nitrotyrosine levels. Under hyperoxic conditions exogenous peroxynitrite impaired force generation and increased nitrotyrosine level. These studies show that hypoxia-induced impairment in diaphragm contractility is associated with increased diaphragm protein nitration, but no causal relationship was found between diaphragm nitrotyrosine formation and in vitro force generation.

Topics: Animals; Azoles; Diaphragm; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Isoindoles; Lipid Peroxidation; Male; Muscle Contraction; Muscle Fatigue; omega-N-Methylarginine; Organoselenium Compounds; Peroxynitrous Acid; Rats; Rats, Wistar; Tyrosine

Although the accelerated atherosclerosis and premature aging of the cardiovascular system in patients with metabolic syndrome have been appreciated, the mechanisms of their development and potential therapeutic interventions remain unresolved. Our previous studies implicated advanced glycosylation end products in development of premature senescence preventable with a peroxynitrite scavenger, ebselen. Therefore, the effect of ebselen on endothelial senescence and vasculopathy in a model of metabolic syndrome--Zucker diabetic rats (ZDF)--was investigated. Ebselen decreased the abundance of 3-nitrotyrosine-modified proteins in ZDF rats. A 6-fold increase in the number of senescent endothelial cells in 22-week-old ZDF was prevented by ebselen. Development of vasculopathy, as collectively judged by the acetylcholine-induced vasorelaxation, NO production, angiogenic competence, and number of circulating microparticles, was almost completely prevented when ebselen was administered from 8 to 22 weeks and partially reversed when the treatment interval was 13 to 22 weeks. In conclusion, premature senescence of endothelial cells is progressively rampant in ZDF rats and is associated with the signs of severe vasculopathy. In addition, prevention of premature senescence of vascular endothelium through controlled decrease in nitrotyrosine formation was chronologically associated with the amelioration of vasculopathy, lending support to the idea of the pathogenetic role of premature senescence of endothelial cells in diabetic macrovasculopathy.

Topics: Acetylcholine; Animals; Antioxidants; Aorta, Thoracic; Azoles; Cell Cycle Proteins; Cellular Senescence; Diabetes Mellitus; Endothelial Cells; Endothelium, Vascular; In Vitro Techniques; Isoindoles; Microcirculation; Muscle, Skeletal; Neovascularization, Physiologic; Nitric Oxide; Obesity; Organoselenium Compounds; Rats; Rats, Zucker; Tyrosine; Vasodilation; Vasodilator Agents

This study was carried out to elucidate whether the protective activity of (-)-epicatechin 3-O-gallate (ECg) against excessive peroxynitrite (ONOO(-)) production, is distinct from the activity of several well-known free radical inhibitors, the ONOO(-) inhibitors ebselen and uric acid, the superoxide anion (O(2)(-)) scavenger copper zinc superoxide dismutase (CuZnSOD) and the selective inducible nitric oxide synthase inhibitor L-N(6)-(1-iminoethyl)lysine hydrochloride (L-NIL). To generate ONOO(-), male Wistar rats (n = 6/group) were subjected to ischaemia-reperfusion process together with lipopolysaccharide (LPS) injection. Although ECg did not scavenge the ONOO(-) precursors nitric oxide (NO) and O(2)(-), it reduced the 3-nitrotyrosine level, a property similar to that of uric acid, but distinct from L-NIL. In addition, the elevation in myeloperoxidase activity was reversed by the administration of ECg, uric acid and SOD, but not by that of L-NIL. Furthermore, ECg was the more potent scavenger of the ONOO(-) decomposition product, the hydroxyl radical (*OH), than any other free radical inhibitor tested. The LPS plus ischaemia-reperfusion process resulted in renal dysfunction, estimated by measuring the parameters of renal function--serum urea nitrogen and creatinine levels. However, administration of ECg ameliorated renal dysfunction more than that of the other free radical inhibitors. Moreover, ECg reduced the excessive uric acid level, while the others did not, suggesting a property of ECg distinct from the others. Furthermore, proteinuria, which was demonstrated by the low- and high-molecular weight (LMW and HMW) protein bands of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern, caused by LPS plus ischaemia-reperfusion, was attenuated by administration of ECg and L-NIL, after which the HMW band intensities decreased and LMW protein bands were absent. This study indicates that, in an in-vivo model of ONOO(-) generation, ECg, L-NIL and uric acid exert stronger protective activity against ONOO(-)-induced oxidative damage than SOD and ebselen, and that the mechanism whereby ECg protects against ONOO(-) is distinct from that of L-NIL or uric acid.

Topics: Animals; Azoles; Blood Urea Nitrogen; Catechin; Copper; Creatinine; Disease Models, Animal; Dose-Response Relationship, Drug; Free Radical Scavengers; Isoindoles; Japan; Kidney; Lipopolysaccharides; Lysine; Male; Nitric Oxide; Organoselenium Compounds; Peroxidase; Phytotherapy; Plant Extracts; Plant Roots; Proteinuria; Rats; Rats, Wistar; Reperfusion Injury; Rheum; Superoxide Dismutase; Tyrosine; Uric Acid; Zinc

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

Nitric oxide (NO) mediates apoptosis induction in fibroblasts with constitutive src or induced ras oncogene expression, whereas nontransformed parental cells and revertants are not affected. This direct link between the transformed phenotype and sensitivity to NO-mediated apoptosis induction seems to be based on the recently described extracellular superoxide anion generation by transformed cells, as NO-mediated apoptosis induction in transformed cells is inhibited by extracellular superoxide dismutase (SOD), by SOD mimetics and by apocynin, an inhibitor of NADPH oxidase. Furthermore, nonresponsive nontransformed cells can be rendered sensitive for NO-mediated apoptosis induction when they are supplemented with xanthine oxidase/xanthine as an extracellular source for superoxide anions. As superoxide anions and NO readily interact in a diffusion-controlled reaction to generate peroxynitrite, peroxynitrite seems to be the responsible apoptosis inducer in NO-mediated apoptosis induction. In line with this conclusion, NO-mediated apoptosis induction in superoxide anion-generating transformed cells is inhibited by the peroxynitrite scavengers ebselen and FeTPPS. Moreover, direct application of peroxynitrite induces apoptosis both in transformed and nontransformed cells, indicating that peroxynitrite is no selective apoptosis inducer per se, but that selective apoptosis induction in transformed cells by NO is achieved through selective peroxynitrite generation. The interaction of NO with target cell derived superoxide anions represents a novel concept for selective apoptosis induction in transformed cells. This mechanism may be the basis for selective apoptosis induction by natural antitumor systems (like macrophages, natural killer cells, granulocytes) that utilize NO for antitumor action. Apoptosis induction mediated by NO involves mitochondrial depolarization and is blocked by Bcl-2 overexpression.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Azoles; Biomarkers; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; Fibroblasts; Genes, src; Glutathione; Isoindoles; Kinetics; Mitochondria; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroprusside; Organoselenium Compounds; Rats; S-Nitrosoglutathione; Spermine; Tyrosine

Peroxynitrite (ONOO(-)), which is generated from nitric oxide (NO) and superoxide anion (O(2)(.-)) under pathological conditions, plays an important role in pathophysiological processes. Activation of matrix metalloproteinases (MMPs) contributes to tumor angiogenesis and metastasis. NO mediates the enhanced vascular permeability and retention (EPR) effect in solid tumors, and ONOO(-)activates proMMP to MMP in vitro. In this study, we examined the role of ONOO(-)in the EPR effect in solid tumors and normal tissues as related to MMP activation. Authentic ONOO(-), at 50 nmol or higher concentrations, induced the enhanced vascular permeability in normal dorsal skin of mice. ONOO(-)scavengers ebselen and uric acid significantly suppressed the EPR effect in mouse sarcoma 180 (S-180) tumors. Indirect evidence for formation of ONOO(-)in S-180 and mouse colon adenocarcinoma (C-38) tumors included strong immunostaining for nitrotyrosine in the tumor tissue, predominantly surrounding the tumor vessels. MMP inhibitor BE16627B (66.6 mg / kg i.v., given 2 times) or SI-27 (10 mg / kg i.p., given 2 times) significantly suppressed the ONOO(-)-induced EPR effect in S-180 tumors and in normal skin. Soybean trypsin inhibitor (Kunitz type), broad-spectrum proteinase inhibitor ovomacroglobulin, and bradykinin receptor antagonist HOE 140 also significantly suppressed the ONOO(-)-induced EPR effect in normal skin tissues. These data suggest that ONOO(-)may be involved in and promote the EPR effect in tumors, which could be mediated partly through activation of MMPs and a subsequent proteinase cascade to generate potent vasoactive mediators such as bradykinin.

Topics: Adenocarcinoma; Animals; Azoles; Bradykinin; Capillary Permeability; Collagenases; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Precursors; Free Radical Scavengers; Gelatinases; Guinea Pigs; Isoindoles; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Nitrates; Oligopeptides; Organoselenium Compounds; Protease Inhibitors; Sarcoma 180; Skin; Tyrosine; Uric Acid

Generation of reactive oxygen species and nitric oxide in hypoxia-reperfusion injury may form a cytotoxic metabolite, peroxynitrite, which is capable of causing lipid peroxidation and DNA damage. This study was designed to examine the contribution of oxidative and nitrosative stress to the renal damage in ischemic acute renal failure (iARF). iARF was initiated in rats by 45-min renal artery clamping. This resulted in lipid peroxidation, DNA damage, and nitrotyrosine modification confirmed both by Western and immunohistochemical analyses. Three groups of animals were randomly treated with an inhibitor of inducible nitric oxide synthase (NOS), L-N(6)-(1-iminoethyl)lysine (L-Nil), cell-permeable lecithinized superoxide dismutase (SOD), or both. Each treatment resulted in amelioration of renal dysfunction, as well as reduced nitrotyrosine formation, lipid peroxidation, and DNA damage, thus suggesting that peroxynitrite rather than superoxide anion is responsible for lipid peroxidation and DNA damage. Therefore, in a separate series of experiments, a scavenger of peroxynitrite, ebselen, was administered before the reperfusion period. This treatment resulted in a comparable degree of amelioration of iARF. In conclusion, the present study provides the first attempt to elucidate the role of peroxynitrite in initiation of the cascade of lipid peroxidation and DNA damage to ischemic kidneys. The results demonstrate that L-Nil, lecithinized SOD, and ebselen treatments improve renal function due to their suppression of peroxynitrite production or its scavenging, consequently preventing lipid peroxidation and oxidative DNA damage.

Topics: Animals; Azoles; Blotting, Western; Cell Line; Cyclic N-Oxides; DNA Damage; Enzyme Inhibitors; Free Radical Scavengers; Immunohistochemistry; Ischemia; Isoindoles; Kidney; Lipid Peroxidation; Lysine; Macrophages; Male; Mice; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Organoselenium Compounds; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spin Labels; Superoxide Dismutase; Tyrosine