Superoxide-generating System Research Articles

Bortezomib is an effective enhancer for chemical probe-dependent superoxide detection.

Various chemical probes for the detection of reactive oxygen species have been developed to examine oxidative stress associated with different pathologies. L-012, a luminol-based chemiluminescent probe, is widely used to detect extracellular superoxide because of its high sensitivity. We herein demonstrated that the co-application of the peptide boronic acid proteasome inhibitor, bortezomib, with L-012 significantly increased its luminescence without affecting the background. More than a 5-fold increase was detected in the total luminescence of L-012 in both NADPH oxidase-expressing cells and the xanthine oxidase-dependent cell-free superoxide generation system, but not in their background. Therefore, bortezomib increased the signal-to-background ratio and improved the detection of low levels of superoxide. The application of MLN2238, another peptide boronic acid proteasome inhibitor, also enhanced the luminescence of L-012. In contrast, carfilzomib, an epoxyketone proteasome inhibitor, did not increase luminescence, suggesting that the effects of bortezomib depend on the chemical structure of the peptide boronic acid, but not on its pharmacological effects. Bortezomib-induced enhancements appeared to be specific to the detection of superoxide because the detection of H2O2 by Amplex Red/HRP was not affected by the application of bortezomib. In the quantitative detection of the superoxide-specific oxidative product 2-hydroxyethidium (2-OH-E+), the application of bortezomib resulted in a 2-fold increase in the level of 2-OH-E+. Therefore, bortezomib sensitizes the detection of superoxide in both cell-based and cell-free systems, highlighting a novel feature of compounds containing the peptide boronic acid as powerful enhancers for the detection of superoxide.

Nitric oxide-dependent biodegradation of graphene oxide reduces inflammation in the gastrointestinal tract.

Understanding the biological fate of graphene-based materials such as graphene oxide (GO) is crucial to assess adverse effects following intentional or inadvertent exposure. Here we provide first evidence of biodegradation of GO in the gastrointestinal tract using zebrafish as a model. Raman mapping was deployed to assess biodegradation. The degradation was blocked upon knockdown of nos2a encoding the inducible nitric oxide synthase (iNOS) or by pharmacological inhibition of NOS using l-NAME, demonstrating that the process was nitric oxide (NO)-dependent. NO-dependent degradation of GO was further confirmed in vitro by combining a superoxide-generating system, xanthine/xanthine oxidase (X/XO), with an NO donor (PAPA NONOate), or by simultaneously producing superoxide and NO by decomposition of SIN-1. Finally, by using the transgenic strain Tg(mpx:eGFP) to visualize the movement of neutrophils, we could show that inhibition of the degradation of GO resulted in increased neutrophil infiltration into the gastrointestinal tract, indicative of inflammation.

The Antioxidant properties of para-Aminobenzoic acid and its sodium salt

The antioxidant properties of para-aminobenzoic acid, a substance from the group of vitamins, and its sodium salt has been found using the reaction of adrenaline autoxidation in an alkaline medium as a superoxide-generating model system. These compounds inhibited the accumulation of adrenochrome, which is a product of adrenaline oxidation, and the formation of superoxide anions, detected by their reaction with nitro blue tetrazolium. Approaches have been developed to produce a true solution of para-aminobenzoic acid and conditions were established to measure the antioxidant activity of para-aminobenzoic acid and its sodium salt. The antioxidant properties of these compounds indicate their possible participation in the redox reactions of the cell and can also be one reason that they are essential.

NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation

ObjectiveCirrhosis downregulates phagocyte oxidant production via their antibacterial superoxide-generating system, NADPH oxidase (NOX2) and increases patients’ susceptibility to infection and mortality rate. To explore novel biochemical parameters that explain susceptibility...

Diverse Functions of Endothelial NO Synthases System: NO and EDH.

:Endothelium-dependent relaxations are predominantly regulated by nitric oxide (NO) in large conduit arteries and by endothelium-dependent hyperpolarization (EDH) in small resistance vessels. Although the nature of EDH factors varies depending on species and vascular beds, we have previously demonstrated that endothelial NO synthases (eNOS)-derived hydrogen peroxide (H2O2) is an EDH factor in animals and humans. This vessel size-dependent contribution of NO and EDH is, at least in part, attributable to the diverse roles of endothelial NOSs system; in large conduit arteries, eNOS mainly serves as a NO-generating system to elicit soluble guanylate cyclase–cyclic guanosine monophosphate-mediated relaxations, whereas in small resistance vessels, it serves as a superoxide-generating system to cause EDH/H2O2-mediated relaxations. Endothelial caveolin-1 may play an important role for the diverse roles of NOSs. Although reactive oxygen species are generally regarded harmful, the physiological roles of H2O2 have attracted much attention as accumulating evidence has shown that endothelium-derived H2O2 contributes to cardiovascular homeostasis. The diverse functions of endothelial NOSs system with NO and EDH/H2O2 could account for a compensatory mechanism in the setting of endothelial dysfunction. In this review, we will briefly summarize the current knowledge on the diverse functions of endothelial NOSs system: NO and EDH/H2O2.

NADPH Oxidase 1, a Novel Molecular Source of ROS in Hippocampal Neuronal Death in Vascular Dementia

Chronic cerebral hypoperfusion (CCH) is a common pathological factor that contributes to neurodegenerative diseases such as vascular dementia (VaD). Although oxidative stress has been strongly implicated in the pathogenesis of VaD, the molecular mechanism underlying the selective vulnerability of hippocampal neurons to oxidative damage remains unknown. We assessed whether the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, a specialized superoxide generation system, plays a role in VaD by permanent ligation of bilateral common carotid arteries in rats. Male Wistar rats (10 weeks of age) were subjected to bilateral occlusion of the common carotid arteries (two-vessel occlusion [2VO]). Nox1 expression gradually increased in hippocampal neurons, starting at 1 week after 2VO and for approximately 15 weeks after 2VO. The levels of superoxide, DNA oxidation, and neuronal death in the CA1 subfield of the hippocampus, as well as consequential cognitive impairment, were increased in 2VO rats. Both inhibition of Nox by apocynin, a putative Nox inhibitor, and adeno-associated virus-mediated Nox1 knockdown significantly reduced 2VO-induced reactive oxygen species generation, oxidative DNA damage, hippocampal neuronal degeneration, and cognitive impairment. We provided evidence that neuronal Nox1 is activated in the hippocampus under CCH, causing oxidative stress and consequential hippocampal neuronal death and cognitive impairment. This evidence implies that Nox1-mediated oxidative stress plays an important role in neuronal cell death and cognitive dysfunction in VaD. Nox1 may serve as a potential therapeutic target for VaD.

Impaired NADPH oxidase activity in peripheral blood lymphocytes of galactosemia patients

Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.

Use of nitro blue tetrazolium in the reaction of adrenaline autooxidation for the determination of superoxide dismutase activity

Addition of nitro blue tetrazolium (NBT) into the reaction of adrenaline autooxidation allows to identify directly superoxide anion formation (02.-) in this superoxide-generating system. The kinetics of formation of adrenochrome and 02.- were compared under the same conditions. Three possible approaches to the use the adrenaline autooxidation reaction for determining the activity of superoxide dismutase (SOD) and revealing the antioxidant properties of various compounds are discussed. Two of these approaches have been described previously: the spectrophotometric method of the registration of adrenochrome, the final product of adrenaline autoxidation, at 347 nm (Sirota, 1999) and the polarographic method, based on determination of oxygen consumption for 02.- formation (Sirota, 2011). Here, a novel approach to this problem is presented; it consists in the spectrophotometric determination of 02.- using NBT. The application of this approach enables one to lower the pH of carbonate buffer from 10.5 to 9.7 and to decrease (4-fold) the amount of added adrenaline, i.e., to generate milder and more effective conditions for revealing and studying the antioxidant properties of examined materials.

Abstract 503: Antioxidant Activity Accounts for the Antiplatelet Effects of the Probucol Derivative Succinobucol

Succinobucol is a derivative of probucol with improved partitioning into cells and potent extracellular antioxidant activity. It has antiplatelet effects ex vivo in human blood but it is unclear if this is due to antioxidant activity. We aimed to study the effect of succinobucol on free radical-modulated platelet aggregation and generation of vascular tone. We also studied platelet aggregation ex vivo in rats following acute administration of succinobucol. Rabbit blood was collected and aggregation in whole blood and platelet-rich plasma (PRP) measured using dual-wire impedance. Rabbit iliac arteries were mounted in a small artery wire myograph to measure vessel tone and the effect of free radicals and succinobucol. Rat carotid and jugular vessels were cannulated in anaesthetised animals prior to administration of succinobucol. Blood was withdrawn to measure platelet aggregation 45 minutes following injection of succinobucol. In whole blood and PRP, succinobucol (10-100μM) caused a dose-dependent reduction in collagen-induced platelet aggregation. Aggregation to collagen in PRP was significantly increased by the superoxide-generating system xanthine/xanthine oxidase (X/XO; 29.8% increase, n=4; p&lt;0.05 vs. control) and succinobucol abolished this effect. In denuded rabbit iliac arteries incubated with excess xanthine, addition of XO (3-9mU/mL) induced a dose-dependent relaxation and this was largely abolished by succinobucol at both 10 and 100μM. Injection of 3 doses of succinobucol (50-150mg/kg) in vivo had no effect on mean arterial pressure or heart rate. However, blood removed 45minutes following the last dose of succinobucol showed a significant reduction in aggregation to collagen (reduction of 40.4% compared to control with 5μg/mL collagen; n=5, p&lt;0.05 vs. control). The results of the current study demonstrate that the antiplatelet effects of succinobucol may be attributed to an antioxidant effect in rabbit blood and that succinobucol exerts an antiplatelet effect in vivo for at least 45mins after administration. Furthermore, the antioxidant effects may be able to influence vascular tone at sites of free radical generation in the vascular tree.

NADPH Oxidase 1-Mediated Oxidative Stress Leads to Dopamine Neuron Death in Parkinson's Disease

Oxidative stress has long been considered as a major contributing factor in the pathogenesis of Parkinson's disease. However, molecular sources for reactive oxygen species in Parkinson's disease have not been clearly elucidated. Herein, we sought to investigate whether a superoxide-producing NADPH oxidases (NOXs) are implicated in oxidative stress-mediated dopaminergic neuronal degeneration. Expression of various Nox isoforms and cytoplasmic components were investigated in N27, rat dopaminergic cells. While most of Nox isoforms were constitutively expressed, Nox1 expression was significantly increased after treatment with 6-hydroxydopamine. Rac1, a key regulator in the Nox1 system, was also activated. Striatal injection of 6-hydroxydopamine increased Nox1 expression in dopaminergic neurons in the rat substantia nigra. Interestingly, it was localized into the nucleus, and immunostaining for DNA oxidative stress marker, 8-oxo-dG, was increased. Nox1 expression was also found in the nucleus of dopaminergic neurons in the substantia nigra of Parkinson's disease patients. Adeno-associated virus-mediated Nox1 knockdown or Rac1 inhibition reduced 6-hydroxydopamine-induced oxidative DNA damage and dopaminergic neuronal degeneration significantly. Nox1/Rac1 could serve as a potential therapeutic target for Parkinson's disease. We provide evidence that dopaminergic neurons are equipped with the Nox1/Rac1 superoxide-generating system. Stress-induced Nox1/Rac1 activation causes oxidative DNA damage and neurodegeneration. Reduced dopaminergic neuronal death achieved by targeting Nox1/Rac1, emphasizes the impact of oxidative stress caused by this system on the pathogenesis and therapy in Parkinson's disease.

Role of superoxide and thromboxane receptors in acute angiotensin II-induced vasoconstriction of rabbit vessels

This study explored the hypothesis that a portion of angiotensin II-induced contractions is dependent on superoxide generation and release of a previously unidentified arachidonic acid metabolite that activates vascular smooth muscle thromboxane receptors. Treatment of rabbit aorta or mesentery artery with the thromboxane receptor antagonist SQ29548 (10 μM) reduced angiotensin II-induced contractions (maximal contraction in aorta; control vs. SQ29548: 134 ± 16 vs. 93 ± 10%). A subset of rabbits deficient in vascular thromboxane receptors also displayed decreased contractions to angiotensin II. The superoxide dismutase mimetic Tiron (30 mM) attenuated angiotensin II-induced contractions only in rabbits with functional vascular thromboxane receptors (maximal contraction in aorta; control vs. Tiron: 105 ± 5 vs. 69 ± 11%). Removal of the endothelium or treatment with a nitric oxide synthase inhibitor, nitro-l-arginine (30 μM) did not alter angiotensin II-induced contractions. Tiron and SQ29548 decreased angiotensin II-induced contractions in the denuded aortas by a similar percentage as that observed in intact vessels. The cyclooxygenase inhibitor indomethacin (10 μM) or thromboxane synthase inhibitor dazoxiben (10 μM) had no effect on angiotensin II-induced contractions indicating that the vasoconstrictor was not thromboxane. Angiotensin II increased the formation of a 15-series isoprostane. Isoprostanes are free radical-derived products of arachidonic acid. The unidentified isoprostane increased when vessels were incubated with the superoxide-generating system xanthine/xanthine oxidase. Pretreatment of rabbit aorta with the isoprostane isolated from aortic incubations enhanced angiotensin II-induced contractions. Results suggest the factor activating thromboxane receptors and contributing to angiotensin II vasoconstriction involves the superoxide-mediated generation of a 15-series isoprostane.

GCN5 Regulates the Superoxide-Generating System in Leukocytes Via Controlling gp91-phox Gene Expression

The superoxide anion (O(2)(-))-generating system is an important mechanism of innate immune response against microbial infection in phagocytes and is involved in signal transduction mediated by various physiological and pathological signals in phagocytes and other cells, including B lymphocytes. The O(2)(-)-generating system is composed of five specific proteins: p22-phox, gp91-phox, p40-phox, p47-phox, p67-phox, and a small G protein, Rac. Little is known regarding epigenetic regulation of the genes constituting the O(2)(-)-generating system. In this study, by analyzing the GCN5 (one of most important histone acetyltransferases)-deficient DT40 cell line, we show that GCN5 deficiency causes loss of the O(2)(-)-generating activity. Interestingly, transcription of the gp91-phox gene was drastically downregulated (to ∼4%) in GCN5-deficient cells. To further study the involvement of GCN5 in transcriptional regulation of gp91-phox, we used in vitro differentiation system of U937 cells. When human monoblastic U937 cells were cultured in the presence of IFN-γ, transcription of gp91-phox was remarkably upregulated, and the cells were differentiated to macrophage-like cells that can produce O(2)(-). Chromatin immunoprecipitation assay using the U937 cells during cultivation with IFN-γ revealed not only that association of GCN5 with the gp91-phox gene promoter was significantly accelerated, but also that GCN5 preferentially elevated acetylation levels of H2BK16 and H3K9 surrounding the promoter. These results suggested that GCN5 regulates the O(2)(-)-generating system in leukocytes via controlling the gp91-phox gene expression as a supervisor. Our findings obtained in this study should be useful in understanding the molecular mechanisms involved in epigenetic regulation of the O(2)(-)-generating system in leukocytes.

Enhanced superoxide and hydrogen peroxide detection in biological assays

Superoxide reductase (SOR) is an enzyme that converts superoxide into hydrogen peroxide at a twofold higher yield than canonical superoxide dismutases (SOD). Superoxide radical detection was investigated using the Amplex red (AR)/peroxidase system to measure the difference in hydrogen peroxide production yield in the presence of SOR or SOD. We found that reduced SOR reacts with the AR oxidation intermediate, a one-electron reduced AR • radical, by reducing this intermediate back to the initial AR leuco compound. Ascorbate also quenched this radical in a concentration-dependent manner and could be used to compete efficiently with SOR; at concentrations of ascorbate higher than 5 µM, SOR no longer interfered with the detection of H 2O 2. By using xanthine/xanthine oxidase as a superoxide-generating system, it was possible to successfully quantify superoxide and hydrogen peroxide in vitro using the AR/peroxidase/SOR system, either by visible absorption or by fluorescence emission, with a considerable low detection limit of 10 nM/min. The use of enzymes with diffusion-limited reactivity toward superoxide substantially increases specificity and detection threshold for superoxide and turns this approach into a powerful system to detect ROS in biological systems.

A case report of chronic granulomatous disease presenting with aspergillus pneumonia in a 2-month old girl

Chronic granulomatous disease (CGD) is an uncommon inherited disorder caused by mutations in any of the genes encoding subunits of the superoxide-generating phagocyte NADPH oxidase system, which is essential for killing catalase producing bacteria and fungi, such as Aspergillus species, Staphylococcus aureus, Serratia marcescens, Nocardia species and Burkholderia cepacia. In case of a history of recurrent or persistent infections, immune deficiency should be investigated. Particularly, in the case of uncommon infections such as aspergillosis in early life, CGD should be considered. We describe here a case of CGD that presented with invasive pulmonary aspergillosis in a 2-month-old girl. We confirmed pulmonary aspergillosis noninvasively through a positive result from the culture of bronchial alveolar lavage fluid, positive serological test for Aspergillus antigen and radiology results. She was successfully treated with Amphotericin B and recombinant IFN-γ initially. Six weeks later after discharge, she was readmitted for pneumonia. Since there were infiltrates on the right lower lung, which were considered as residual lesions, voriconazole therapy was initiated. She showed a favorable response to the treatment and follow-up CT showed regression of the pulmonary infiltrates.

Influences of Organic Solvents on CYPMPO-Electron Spin Resonance Spectra in In Vitro Radical Generating Systems

Estimation of radical scavenging capacity of lipophilic antioxidants by electron spin resonance (ESR) in vitro is a challenging issue due to their poor solubility in aqueous radical generating and measuring systems. Water-miscible organic solvents are used for this purpose. A novel radical trapping agent, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), that has practical advantages over well-known trapping agents was synthesized. However, no available data for the influence of solvents in an ESR system that uses CYPMPO has been presented. The influences of six water-miscible organic solvents, acetonitrile (AcN), acetone, dimethyl sulfoxide (DMSO), ethanol, polyethylene glycol (PEG), and dimethoxyethane (DME), on ESR responses to Fenton Fe(2+)/H (2)O(2 )OH· and hypoxanthine/xanthine oxidase superoxide generation systems in vitro were studied. Reduction of the ESR signal to CYPMPO-OH· adducts by 55.86 ± 5.95 and 83.17 ± 2.50% compared with the control was observed in the presence of AcN and acetone, respectively, at a final concentration of 5% (v/v). AcN of less than 1% had minimal effects. DMSO, ethanol, PEG and DME at 5% (v/v) strongly inhibited the ESR signals and/or caused derangement in the signal patterns. The six water-miscible solvents at 5% (v/v) had no influence on the ESR spectra of CYPMPO-superoxide adducts. From these results, AcN, at less than 1% (v/v), is a useful water-miscible organic solvent for assessing radical scavenging capacities of lipophilic compounds in the CYPMPO-Fenton Fe(2+)/H(2)O(2) OH· reaction system in an ESR assay. Any of the solvents used in the present study can be used in a hypoxanthine/xanthine oxidase superoxide generation system.

Hypoxia-generated superoxide induces the development of the adhesion phenotype

Adhesion fibroblasts exhibit higher TGF-β1 and type I collagen expression as compared to normal peritoneal fibroblasts. Furthermore, exposure of normal peritoneal fibroblasts to hypoxia results in an irreversible increase in TGF-β1 and type I collagen. We postulated that the mechanism by which hypoxia induced the adhesion phenotype is through the production of superoxide either directly or through the formation of peroxynitrite. To test this hypothesis, normal peritoneal and adhesion fibroblasts were treated with superoxide dismutase (SOD), a superoxide scavenger, and xanthine/xanthine oxidase, a superoxide-generating system, under normoxic and hypoxic conditions. Also, cells were treated with peroxynitrite. TGF-β1 and type I collagen expression was determined before and after all treatments using real-time RT/PCR. Hypoxia treatment resulted in a time-dependent increase in TGF-β1 and type I collagen mRNA levels in both normal peritoneal and adhesion fibroblasts. Similarly, treatment with xanthine oxidase, to endogenously generate superoxide, resulted in higher mRNA levels of TGF-β1 and type I collagen in both normal peritoneal and adhesion fibroblasts. In contrast, treatment with SOD, to scavenge endogenous superoxide, resulted in a decrease in TGF-β1 and type I collagen expression in adhesion fibroblasts to levels seen in normal peritoneal fibroblasts; no effect on the expression of these molecules was seen in normal peritoneal fibroblasts. Exposure to hypoxia in the presence of SOD had no effect on mRNA levels of TGF-β1 and type I collagen in either normal peritoneal or adhesion fibroblasts. Peroxynitrite treatment alone significantly induced both adhesion phenotype markers. In conclusion, hypoxia, through the production of superoxide, causes normal peritoneal fibroblasts to acquire the adhesion phenotype. Scavenging superoxide, even in the presence of hypoxia, prevented the development of the adhesion phenotype. These findings further support the central role of free radicals in the development of adhesions.

Inorganic Arsenic Activates Reduced NADPH Oxidase in Human Primary Macrophages through a Rho Kinase/p38 Kinase Pathway

Inorganic arsenic is an immunotoxic environmental contaminant to which millions of humans are chronically exposed. We recently demonstrated that human primary macrophages constituted a critical target for arsenic trioxide (As(2)O(3)), an inorganic trivalent form. To specify the effects of arsenic on macrophage phenotype, we investigated in the present study whether As(2)O(3) could regulate the activity of NADPH oxidase, a major superoxide-generating enzymatic system in human phagocytes. Our results show that superoxide levels were significantly increased in a time-dependent manner in blood monocyte-derived macrophages treated with 1 muM As(2)O(3) for 72 h. Concomitantly, As(2)O(3) induced phosphorylation and membrane translocation of the NADPH oxidase subunit p47(phox) and it also increased translocation of Rac1 and p67(phox). Apocynin, a selective inhibitor of NADPH oxidases, prevented both p47(phox) translocation and superoxide production. NADPH oxidase activation was preceded by phosphorylation of p38-kinase in As(2)O(3)-treated macrophages. The p38-kinase inhibitor SB-203580 prevented phosphorylation and translocation of p47(phox) and subsequent superoxide production. Pretreatment of macrophages with the Rho-kinase inhibitor Y-27632 was found to mimic inhibitory effects of SB-203580 and to prevent As(2)O(3)-induced phosphorylation of p38 kinase. Treatment with As(2)O(3) also resulted in an increased secretion of the proinflammatory chemokine CCL18 that was fully inhibited by both apocynin and SB-203580. Taken together, our results demonstrate that As(2)O(3) induced a marked activation of NADPH oxidase in human macrophages, likely through stimulation of a Rho-kinase/p38-kinase pathway, and which may contribute to some of the deleterious effects of inorganic arsenic on macrophage phenotype.

Evaluation of preservation solutions by ESR-spectroscopy: Superior effects of University of Wisconsin over Histidine–Tryptophan–Ketoglutarate in reducing renal reactive oxygen species

Despite the causative role of oxidative stress in renal ischemia-reperfusion (I-R) injury effects of preservation solutions on reactive oxygen species (ROS) release have not been sufficiently evaluated. We compared the effects of most common solutions in kidney transplantation, University of Wisconsin (UW) and Histidine-Tryptophan-Ketoglutarate (HTK). ROS formation in isolated perfused rat kidney was detected by electron spin resonance spectroscopy using spin label 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Donor kidneys from Lewis rats were pretreated with saline (controls), in therapeutic groups, kidneys underwent 18 h of cold storage (CS) preserved by HTK or UW solution. Experimental protocol included a stabilization period followed by additional I-R. Kidneys preserved by HTK produced highest ROS values in the control period after CS, whereas levels in UW and control group did not vary significantly. A peak release induced by additional I-R was also significantly highest in HTK kidneys, and UW did not differ from controls. During reperfusion, levels in HTK exceeded control and UW values. Renal vascular resistance, caspase-3-activity, and tissue hydration were enhanced in HTK compared with UW group, whereas ATP concentration was less reduced in UW-preserved tissue. These data show the greater antioxidative potential of UW solution, which also attenuated organ impairment after CS in the early reperfusion period.

Synthetic Peroxisome Proliferator-Activated Receptor-γ Agonists Restore Impaired Vasorelaxation via ATP-Sensitive K+ Channels by High Glucose

The present study was designed to examine whether in the human artery, synthetic peroxisome proliferator-activated receptor (PPAR)-gamma agonists restore vasorelaxation as well as hyperpolarization via ATP-sensitive K+ channels impaired by the high concentration of D-glucose and whether the restoration may be mediated by the antioxidant capacity of these agents. The isometric force and membrane potential of human omental arteries without endothelium were recorded. The production rate of superoxide was evaluated using a superoxide-generating system with xanthine-xanthine oxidase in the absence of smooth muscle cells. Glibenclamide abolished vasorelaxation and hyperpolarization in response to levcromakalim. Addition of D-glucose (20 mM) but not L-glucose (20 mM) reduced this vasorelaxation and hyperpolarization. Synthetic PPAR-gamma agonists (troglitazone and rosiglitazone) and/or an inhibitor of superoxide generation (4,5-dihydroxy-1,3-benzene-disulfonic acid, Tiron), but not a PPAR-alpha agonist (fenofibrate), restored vasorelaxation and hyperpolarization in response to levcromakalim in arteries treated with D-glucose. Troglitazone and rosiglitazone, but not fenofibrate, decreased the production rate of superoxide without affecting uric acid generation. These findings suggest that synthetic PPAR-gamma agonists recover the function of ATP-sensitive K+ channels reduced by the high concentration of glucose in human vascular smooth muscle cells and that the effect of these agonists may be mediated in part by their antioxidant capacity.

Superoxide-dependent Oxidation of Melatonin by Myeloperoxidase

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.