NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O₂ ^•− source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O₂ ^•− molecule and half a H₂O₂ molecule per NADH molecule, at rates 3 times those observed for XO (29.2 ± 1.6 and 9.38 ± 0.31 min⁻¹, respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 ± 1.36 μM⁻¹ min⁻¹) was found to be higher than that of the XO specificity constant (1.07 ± 0.09 μM⁻¹ min⁻¹). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O₂ ^•− source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O₂ ^•− than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.     

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

Effects of antirheumatic gold compounds on the conversion of xanthine dehydrogenase to oxidase in rabbit liver cytosol in vitro

Effects of auranofin (AUR), aurothioglucose (AuTG) and aurothiomalate (AuTM) on the conversion of xanthine dehydrogenase (XD) to oxidase (XO) in the cytosolic fraction from rabbit liver were examined. AUR had no effect on the conversion of XD to XO at concentrations up to 50 microM, whereas at concentrations ranging from 10 to 25 microM, AuTG and AuTM induced the conversion of XD to XO. The constituents of AuTG and AuTM, aurous ion (Au+), but not mercaptosuccinic acid and 1-thio-beta-D-glucose, converted XD to XO in a similar degree to AuTG and AuTM. This means that Au (I) moiety has an important role in the AuTG- and AuTM-induced conversion of XD to XO. Furthermore, N-acetyl-L-cysteine (NAC) and British anti-Lewisite (BAL) reconverted AuTG and AuTM-induced XO to XD, implying that clinical activity of NAC and BAL against toxic reactions of AuTG and AuTM is partially due to the XO reconversion. These results suggest that AuTG and AuTM have the potential to convert XD to its reactive oxygen species-generating form, XO, and that this effect may be correlated with cytotoxic actions of these drugs.     

Fatty acids as modulators of the cellular production of reactive oxygen species

Long-chain nonesterified ("free") fatty acids (FFA) and some of their derivatives and metabolites can modify intracellular production of reactive oxygen species (ROS), in particular O(2)(-) and H(2)O(2). In mitochondria, FFA exert a dual effect on ROS production. Because of slowing down the rate of electron flow through Complexes I and III of the respiratory chain due to interaction within the complex subunit structure, and between Complexes III and IV due to release of cytochrome c from the inner membrane, FFA increase the rate of ROS generation in the forward mode of electron transport. On the other hand, due to their protonophoric action on the inner mitochondrial membrane ("mild uncoupling effect"), FFA strongly decrease ROS generation in the reverse mode of electron transport. In the plasma membrane of phagocytic neutrophils and a number of other types of cells, polyunsaturated FFA stimulate O(2)(-) generation by NADPH oxidase. These effects of FFA can modulate signaling functions of ROS and be, at least partly, responsible for their proapoptotic effects in several types of cells.     

Peroxisomes and reactive oxygen species,a lasting challenge

Oxidases generating and enzymes scavenging H₂O₂ predestine peroxisomes (PO) to a pivotal organelle in oxygen metabolism. Catalase, the classical marker enzyme of PO, exhibits both catalatic and peroxidatic activity. The latter is responsible for the staining with 3,3′-diamino-benzidine, which greatly facilitated the visualization of the organelle and promoted further studies on PO. d-Amino acid oxidase catalyzes with strict stereospecificity the oxidative deamination of d-amino acids. The oxidase is significantly more active in the kidney than in liver and more in periportal than pericentral rat hepatocytes. Peroxisomes in these tissues differ in their enzyme activity and protein concentration not only in adjacent cells but even within the same one. Moreover, the enzyme appears preferentially concentrated in the central region of the peroxisomal matrix compartment. Urate oxidase, a cuproprotein catalyzing the oxidation of urate to allantoin, is confined to the peroxisomal core, yet is lacking in human PO. Recent experiments revealed that cores in rat hepatocytes appear in close association with the peroxisomal membrane releasing H₂O₂ generated by urate oxidase to the surrounding cytoplasma. Xanthine oxidase is exclusively located to cores, oxidizes xanthine thereby generating H₂O₂ and O₂ ^– radicals. The latter are converted to O₂ and H₂O₂ by CuZn superoxide dismutase, which has been shown recently to be a bona fide peroxisomal protein. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1-4, 2008.     

Plant responses to water stress: Role of reactive oxygen species

Terrestrial plants most often encounter drought stress because of erratic rainfall which has become compounded due to present climatic changes.Responses of plants to water stress may be assigned as either injurious change or tolerance index. One of the primary and cardinal changes in response to drought stress is the generation of reactive oxygen species (ROS), which is being considered as the cause of cellular damage. However, recently a signaling role of such ROS in triggering the ROS scavenging system that may confer protection or tolerance against stress is emerging. Such scavenging system consists of antioxidant enzymes like SOD, catalase and peroxidases, and antioxidant compounds like ascorbate, reduced glutathione; a balance between ROS generation and scavenging ultimately determines the oxidative load. As revealed in case of defence against pathogen, signaling via ROS is initiated by NADPH oxidase-catalyzed superoxide generation in the apoplastic space (cell wall) followed by conversion to hydrogen peroxide by the activity of cell wall-localized SOD. Wall peroxidase may also play role in ROS generation for signaling. Hydrogen peroxide may use Ca2+ and MAPK pathway as downstream signaling cascade. Plant hormones associated with stress responses like ABA and ethylene play their role possibly via a cross talk with ROS towards stress tolerance, thus projecting a dual role of ROS under drought stress.     

Ceramide-induced preconditioning involves reactive oxygen species

INTRODUCTION: Ceramide induces programmed cell death and it is thought to contribute to cardiac ischemia/reperfusion (I/R) injury. In contrast, we have demonstrated that administration of low doses of ceramide engenders cardiac preconditioning (PC). Ceramide is known to generate reactive oxygen species (ROS) in cells. Since mechanisms triggering the ceramide-induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective mechanism. METHODS: Using an isolated Langendorff-perfused rat heart model, four groups (n > or = 6 in each group) were considered: Control hearts underwent 30 min index regional ischemia and 120 min of reperfusion. In the ceramide group, hearts were preconditioned with c2-ceramide 1 microM for 7 min followed by 10 min washout prior to the I/R insult. In additional groups, MPG (1 mM), a synthetic antioxidant was given for 15 min alone or bracketing the ceramide perfusion. In each group, infarct size was determined at the end of the reperfusion period and superoxide dismutases (CuZnSOD and MnSOD) and catalase activities were evaluated. RESULTS: Ceramide preconditioning reduced the infarct/area at risk (I/AAR) ratio (8.3 +/- 1.1% for ceramide vs. 36.4 +/- 1.2% for control, p < 0.001). Perfusion with MPG abolished the preconditioning effect of ceramide (I/AAR ratio = 36.7 +/- 4.9%). Ceramide was also associated with a 29% and 38% increase in catalase and CuZnSOD activities, respectively, compared with control group. CONCLUSION: Production of reactive oxygen species following ceramide preconditioning of the ischemic-reperfused heart appears to play a role in the cardioprotective effect of ceramide.     

Revealing acupuncture meridian-like system by reactive oxygen species visualization

To investigate the reactive oxygen species (ROS) distribution in living animal tissues, two ROS indicators, dichlorofluorescin diacetate (DCFH-DA) and MitoSOX™ Red were applied to visualize ROS on the frontal interior abdominal wall of living SD-rats by tail vein injection and local smearing respectively. Revealed was an unexpected ROS distribution pattern. ROS were demonstrated in a few vertical fluorescent lines, which related to neither veins nor nerves but could be almost perfectly superimposable on a standard human acupuncture meridian network. The phenomenon that cells with high ROS content should be aligned in a regular manner is interesting as well as its resemblance to meridian system.     

Crosstalk between calcium and reactive oxygen species signaling in cancer

The interplay between Ca²⁺ and reactive oxygen species (ROS) signaling pathways is well established, with reciprocal regulation occurring at a number of subcellular locations. Many Ca²⁺ channels at the cell surface and intracellular organelles, including the endoplasmic reticulum and mitochondria are regulated by redox modifications. In turn, Ca²⁺ signaling can influence the cellular generation of ROS, from sources such as NADPH oxidases and mitochondria. This relationship has been explored in great depth during the process of apoptosis, where surges of Ca²⁺ and ROS are important mediators of cell death. More recently, coordinated and localized Ca²⁺ and ROS transients appear to play a major role in a vast variety of pro-survival signaling pathways that may be crucial for both physiological and pathophysiological functions. While much work is required to firmly establish this Ca²⁺-ROS relationship in cancer, existing evidence from other disease models suggests this crosstalk is likely of significant importance in tumorigenesis. In this review, we describe the regulation of Ca²⁺ channels and transporters by oxidants and discuss the potential consequences of the ROS-Ca²⁺ interplay in tumor cells.     

The effect of reactive oxygen species on cardiomyocyte differentiation of pluripotent stem cells

The coordination of metabolic shift with genetic circuits is critical to cell specification, but the metabolic mechanisms that drive cardiac development are largely unknown. Reactive oxygen species (ROS) are not only the by-product of mitochondrial metabolism, but play a critical role in signalling cascade of cardiac development as a second messenger. Various levels of ROS appear differential and even oppose effect on selfrenewal and cardiac differentiation of pluripotent stem cells (PSCs) at each stage of differentiation. The intracellular ROS and redox balance are meticulous regulated by several systems of ROS generation and scavenging, among which mitochondria and the NADPH oxidase (NOX) are major sources of intracellular ROS involved in cardiomyocyte differentiation. Some critical signalling modulators are activated or inactivated by oxidation, suggesting ROS can be involved in regulation of cell fate through these downstream targets. In this review, the literatures about major sources of ROS, the effect of ROS level on cardiac differentiation of PSCs, as well as the underlying mechanism of ROS in the control of cardiac fate of PSC are summarised and discussed.     

Novel Nox inhibitor of oxLDL-induced reactive oxygen species formation in human endothelial cells

In this study, we investigated effects of a novel NAD(P)H oxidase (Nox)-inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870) on oxidized low-density lipoprotein (oxLDL)-mediated reactive oxygen species (ROS) formation in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were cultured to confluence and ROS formation was induced with 50microg/ml oxLDL for 2h. ROS formation was detected by chemiluminescence (CL) using the Diogenes reagent. OxLDL induced ROS formation in human endothelial cells (171+/-12%; n=10, P<0.05 vs. control). This augmented ROS formation in response to oxLDL was completely inhibited by the Nox inhibitor VAS2870 (101+/-9%; n=7, P<0.05 vs. oxLDL). Similar results were obtained with superoxide dismutase (91+/-7%; n=7, P<0.05 vs. oxLDL). However, the Nox4 mRNA expression level was neither changed by oxLDL nor VAS2870. We conclude that VAS2870 could provide a novel strategy to inhibit the augmented endothelial superoxide anion formation in response to cardiovascular risk factors.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis

Observations of apoptosis in virtual anaerobiosis have raised doubts on the significance of reactive oxygen species in the cascade of events of programmed cell death. This work presents evidence that cells and mitochondrial preparations produce similar levels of hydrogen peroxide under either aerobic or virtually anaerobic conditions. These levels are relevant to the increased production of radicals induced by a ceramide analog that promotes apoptosis. This ceramide acts at center o of mitochondrial complex III.     

Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production

Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Arjunolic acid ameliorates reactive oxygen species via inhibition of p47phox-serine phosphorylation and mitochondrial dysfunction

Impaired cardiovascular function during acute myocardial infarction (MI) is partly associated with recruitment of activated polymorphonuclear neutrophils. The protective role of arjunolic acid (AA; 2,3,23-trihydroxy olean-12-en-28-oic acid) is studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation. Neutrophils were isolated from normal and acute MI mice to find out the efficacy of AA in reducing oxidative stress. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) resulted in an oxidative burst of superoxide anion (O₂⁻) and enhanced release of lysosomal enzymes. The treatment of neutrophils with PMA induced phosphorylation of Ser345 on p47^phox, a cytosolic component of NADPH oxidase. Furthermore, we observed activated ERK induced phosphorylation of Ser345 in MI neutrophils. Treatment with AA significantly inhibited the phosphorylation of P47^phox and ERK in the stimulated controls and MI neutrophils. Oxidative phosphorylation activities in MI cells were lower than in control, while the glycolysis rates were elevated in MI cells compared to the control. In addition, we observed AA decreased intracellular oxidative stress and reduced the levels of O₂⁻ in neutrophils. This study therefore identifies targets for AA in activated neutrophils mediated by the MAPK pathway on p47^phox involved in ROS generation.     

线粒体电子传递链电子漏的化学发光测定

本实验用差速离心法分离正常大鼠肝脏和心肌线粒体 ,以lucigenin (探测超氧阴离子 )与luminol (探测过氧化氢 )为探剂 ,用化学发光法测定METC电子漏的生成。在反应体系中加入外源底物 ,其发光强度明显高于空白对照 (体系中无线粒体 )。在肝线粒体体系中 ,无论是lucigenin还是luminol诱发的发光 ,琥珀酸底物引起的发光强均要高于丙酮酸 /苹果酸引起的发光强度。在心肌线粒体 luminol体系中也有与肝线粒体相似的结果 ,在心肌线粒体 lucigenin体系中 ,加入外源底物丙酮酸 /苹果酸诱发的发光强度高于琥珀酸诱发的发光强度     

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.