Burst Of Superoxide Anion Research Articles

Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.

Effects of different LED light wavelengths on the resistance of tomato against Botrytis cinerea and the corresponding physiological mechanisms

New types of light-emitting diode (LED) sources were applied to irradiate Botrytis cinerea mycelium and tomato leaves that were inoculated with B. cinerea to assess the effect of different LED light wavelengths on the infection of tomato with B. cinerea, to determine the optimum light wavelengths to control B. cinerea, and to explore the mechanism of LED influence on the development of gray mold. The results showed that purple light and blue light irradiation significantly inhibited the growth of B. cinerea mycelium, and the inhibition rates were 22.3 and 15.16%, respectively, and purple light exhibited a better inhibitory effect than blue light. The lesion development of B. cinerea on tomato leaves was significantly inhibited upon irradiation with red and purple light with inhibition rates of 32.08 and 36.74%, respectively. Irradiation with red light inhibited the oxidative burst of superoxide anion (O2−) that was caused by infection with B. cinerea, and red light regulated the H2O2 content in the tomato leaf, which increased and rapidly returned to a lower level. In addition, red light irradiation improved the activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in tomato leaves. However, purple light irradiation did not make tomato leaves exhibit this kind of physiological response. Therefore, red light and purple light can suppress gray mold in tomatoes, but the disease suppression mechanisms of these two types of LED light are different. In general, red light suppresses gray mold primarily by regulating the tomato defense mechanism for disease, whereas the suppression of tomato gray mold by purple light can be partially explained by the photo-inhibition of B. cinerea.

Phototherapy-Induced Antitumor Immunity: Long-Term Tumor Suppression EffectsviaPhotoinactivation of Respiratory Chain Oxidase-Triggered Superoxide Anion Burst

Our previous studies have demonstrated that as a mitochondria-targeting cancer phototherapy, high-fluence, low-power laser irradiation (HF-LPLI) results in oxidative damage that induces tumor cell apoptosis. In this study, we focused on the immunological effects of HF-LPLI phototherapy and explored its antitumor immune regulatory mechanism. We found not only that HF-LPLI treatment induced tumor cell apoptosis but also that HF-LPLI-treated apoptotic tumor cells activated macrophages. Due to mitochondrial superoxide anion burst after HF-LPLI treatment, tumor cells displayed a high level of phosphatidylserine oxidation, which mediated the recognition and uptake by macrophages with the subsequent secretion of cytokines and generation of cytotoxic T lymphocytes. In addition, in vivo results showed that HF-LPLI treatment caused leukocyte infiltration into the tumor and efficaciously inhibited tumor growth in an EMT6 tumor model. These phenomena were absent in the respiration-deficient EMT6 tumor model, implying that the HF-LPLI-elicited immunological effects were dependent on the mitochondrial superoxide anion burst. In this study, for the first time, we show that HF-LPLI mediates tumor-killing effects via targeting photoinactivation of respiratory chain oxidase to trigger a superoxide anion burst, leading to a high level of oxidatively modified moieties, which contributes to the phenotypic changes in macrophages and mediates the antitumor immune response. Our results suggest that HF-LPLI may be an effective cancer treatment modality that both eradicates the treated primary tumors and induces an antitumor immune response via photoinactivation of respiratory chain oxidase to trigger superoxide anion burst.

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 p47phox, 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 P47phox 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 p47phox involved in ROS generation.

A Novel Tetrapeptide Derivative Exhibits In Vitro Inhibition of Neutrophil-Derived Reactive Oxygen Species and Lysosomal Enzymes Release

Neutrophil infiltration plays a major role in the pathogenesis of myocardial injury. Oxidative injury is suggested to be a central mechanism of the cellular damage after acute myocardial infarction. This study is pertained to the prognostic role of a tetrapeptide derivative PEP1261 (BOC-Lys(BOC)-Arg-Asp-Ser(tBu)-OtBU), a peptide sequence (39–42) of lactoferrin, studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation, lysosomal enzymes release, and enhanced expression of C proteins. The groundwork experimentation was concerned with the isolation of neutrophils from the normal and acute myocardial infarct rats to find out the efficacy of PEP1261 in the presence of a powerful neutrophil stimulant, phorbol 12-myristate 13 acetate (PMA). Stimulation of neutrophils with PMA resulted in an oxidative burst of superoxide anion and enhanced release of lysosomal enzymes and expression of complement proteins. The present study further demonstrated that the free radicals increase the complement factors in the neutrophils confirming the role of ROS. PEP1261 treatment significantly reduced the levels of superoxide anion and inhibited the release of lysosomal enzymes in the stimulated control and infarct rat neutrophils. This study demonstrated that PEP1261 significantly inhibited the effect on the ROS generation as well as the mRNA synthesis and expression of the complement factors in neutrophils isolated from infarct heart.

Mitochondrial nitric oxide in the signaling of cell integrated responses

Mitochondria are the specialized organelles for energy metabolism, but, as a typical example of system biology, they also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or oppositely promote cell arrest and programmed cell death by a limited number of oxidative or nitrosative reactions. These reactions are influenced by matrix nitric oxide (NO) steady-state concentration, either from local production or by gas diffusion to mitochondria from the canonical sources. Likewise, in a range of approximately 30-200 nM, NO turns mitochondrial O(2) utilization down by binding to cytochrome oxidase and elicits a burst of superoxide anion and hydrogen peroxide that diffuses outside mitochondria. Depending on NO levels and antioxidant defenses, more or less H(2)O(2) accumulates in cytosol and nucleus, and the resulting redox grading contributes to dual activation of proliferating and proapoptotic cascades, like ERK1/2 or p38 MAPK. Moreover, these sequential activating pathways participate in rat liver and brain development and in thyroid modulation of mitochondrial metabolism and contribute to hypothyroid phenotype through complex I nitration. On the contrary, lack of NO disrupts pathways like S-nitrosylation or H(2)O(2) production and likewise is a gateway to disease in amyotrophic lateral sclerosis with superoxide dismutase 1 mutations or to cancer proliferation.

Synthesis and characterization of a novel class of reducing agents that are highly neuroprotective for retinal ganglion cells

Retinal ganglion cells (RGCs) undergo apoptosis after axonal injury, in part regulated by an intracellular superoxide anion burst, for which the target(s) are unknown. Shifting the RGC redox state towards reduction and preventing sulfhydryl oxidation is neuroprotective in vitro and in vivo, implying that one or more sulfhydryls on one or more critical proteins may be involved. We synthesized novel borane-protected analogues of the reductant tris(2-carboxyethyl)phosphine (TCEP) with the intent of increasing cell permeability and improving chemical stability, and tested their ability to increase RGC survival in vitro. Retinal ganglion cells of postnatal day 2–4 Long–Evans rats were retrogradely labeled with 4′,6-diamidino-2-phenylindole (DAPI). At postnatal days 11–13 the animals were sacrificed, the retinas enzymatically dissociated and plated on poly- l-lysine-coated 96-well flat-bottomed tissue culture plates for 72 h in Neurobasal-A, B27 supplement lacking antioxidants, and TCEP, bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1), (3-propionic acid methyl ester)diphenylphosphine borane complex (PB2), or three commercially available phosphines. Viable DAPI-positive RGCs were identified by calcein-AM staining. At 72 h, PB1 was effective at rescuing acutely axotomized RGCs at concentrations from 1 nM to 100 μM. RGC survival with 1 nM PB1 was 174 ± 12% of control ( p = 0.002). Another compound, PB2, rescued RGCs at 10 pM (177 ± 24%; p = 0.006) and 10 nM (251 ± 34%; p = 0.004) at 72 h. A PAMPA assay demonstrated that PB1 and PB2 were substantially more permeable than TCEP. These data demonstrate that modified reductants are effective RGC neuroprotectants at picomolar-nanomolar concentrations. We propose that these novel molecules may act by inhibiting the sulfhydryl oxidation effect of an intracellular superoxide burst.

Critical Role of Mitochondrial Glutathione in the Survival of Hepatocytes during Hypoxia

Hypoxia is known to stimulate reactive oxygen species (ROS) generation. Because reduced glutathione (GSH) is compartmentalized in cytosol and mitochondria, we examined the specific role of mitochondrial GSH (mGSH) in the survival of hepatocytes during hypoxia (5% O2). 5% O2 stimulated ROS in HepG2 cells and cultured rat hepatocytes. Mitochondrial complex I and II inhibitors prevented this effect, whereas inhibition of nitric oxide synthesis with Nomega-nitro-L-arginine methyl ester hydrochloride or the peroxynitrite scavenger uric acid did not. Depletion of GSH stores in both cytosol and mitochondria enhanced the susceptibility of HepG2 cells or primary rat hepatocytes to 5% O2 exposure. However, this sensitization was abrogated by preventing mitochondrial ROS generation by complex I and II inhibition. Moreover, selective mGSH depletion by (R,S)-3-hydroxy-4-pentenoate that spared cytosol GSH levels sensitized rat hepatocytes to hypoxia because of enhanced ROS generation. GSH restoration by GSH ethyl ester or by blocking mitochondrial electron flow at complex I and II rescued (R,S)-3-hydroxy-4-pentenoate-treated hepatocytes to hypoxia-induced cell death. Thus, mGSH controls the survival of hepatocytes during hypoxia through the regulation of mitochondrial generation of oxidative stress.

Extracellular superoxide concentration increases following cerebral hypoxia but does not affect cerebral blood flow

Abnormalities of cerebral blood flow during and following hypoxia and ischemia contribute to the progression of tissue injury. Oxidative stress during and following hypoxia is known to markedly increase superoxide anion concentration. There is conflicting evidence that the concentration of superoxide anion regulates cerebral blood flow through its effect on vascular tone, although difficulties in measurement of superoxide anion complicate these studies. In order to test the hypothesis that changes in cerebral blood flow during and following hypoxia are due to changes in extracellular superoxide anion levels, we examined tissue oxygen levels by fiberoptic oximetry and superoxide anion levels using a previously validated cytochrome c coated electrode on the cortical surface and correlated these measurements to cerebral blood flow measured by laser Doppler in rats subjected to 20min of hypoxia followed by hyperoxic reoxygenation recovery. The results showed a burst of superoxide anion with the onset of reoxygenation that temporally correlated with a transient peak in tissue oxygen tension lasting 10min. and was eliminated by pretreatment with Cu–Zn superoxide dismutase conjugated to polyethylene glycol. Cerebral blood flow did not differ during hypoxia or recovery in the polyethylene glycol conjugated superoxide dismutase and control treatment groups. This study demonstrated no effect of increased superoxide anion concentration on cerebral blood flow during hyperoxic recovery following hypoxia.

Regulation of superoxide anion production by NADPH oxidase in monocytes/macrophages: contributions to atherosclerosis.

Monocyte extravasation into the vessel wall has been shown to be a critical step in the development of atherosclerosis. Upon activation, monocytes produce a burst of superoxide anion due to activation of the NADPH oxidase enzyme complex. Monocyte-derived superoxide anion contributes to oxidant stress in inflammatory sites, is required for monocyte-mediated LDL oxidation, and alters basic cell functions such as adhesion and proliferation. We hypothesize that monocyte-derived superoxide anion production contributes to atherosclerotic lesion formation. In this brief review, we summarize our current understanding of the signal transduction pathways regulating NADPH oxidase activation and related superoxide anion production in activated human monocytes. Novel pathways are identified that may serve as future targets for therapeutic intervention in this pathogenic process. The contributions of superoxide anion and NADPH oxidase to atherogenesis are discussed. Future experiments are needed to clarify the exact role of NADPH oxidase-derived superoxide anion in atherogenesis, particularly that derived from monocytes.

Superoxide anion burst and taxol production induced by Ce 4+ in suspension cultures of Taxus cuspidata

Generation of reactive oxygen species (ROS) induced by Ce 4+ in suspension cultures of Taxus cuspidata was investigated. The burst of superoxide anions (O 2 −) occurred rapidly after the addition of Ce 4+ and reached maximum at 4.3 h, while the total level of the cellular reactive oxygen species maintained unchanged. The intracellular superoxide dismutase (SOD) and catalase (CAT) were activated while the intra/extracellular peroxidases (PODs) were inhibited accompanying the O 2 − burst. The pretreatment of the suspension cultures with diphenylene iodonium (DPI), a suicide inhibitor of the NADPH oxidase, blocked the O 2 − burst, inhibiting the cell apoptosis and taxol production induced by Ce 4+. These results show that NADPH oxidase played a key role in O 2 − burst and O 2 − served as a mediator of Ce 4+ for cell apoptosis and taxol production. The pretreatments of the suspension cultures with anthracene-9-carboxylate, an ion-channel blocker, nifedipine, a Ca 2+-channel blocker, neomycin, a phospholipase C (PLC) inhibitor, or suramin, a G-protein inhibitor, decreased O 2 − burst induced by Ce 4+. It is thus inferred that Ce 4+-induced O 2 − burst, which mediated cell apoptosis and taxol production by activating the ion-channels, PLC, G-proteins and NADPH oxidase.

Parathyroid hormone induces superoxide anion burst in the osteoclast: evidence for the direct instantaneous activation of the osteoclast by the hormone.

We have shown that superoxide anion (O2-) production by the osteoclast can be used as an index of the osteoclast activity since the agents that inhibit and stimulate the osteoclast also diminish and stimulate O2- production respectively. Therefore, we have investigated the mechanism of parathyroid hormone (PTH)-mediated stimulation of osteoclast function in terms of its effect on O2- generation. The determination of O2- generation was carried out by employing cytochrome c immobilised on a surface-modified gold electrode. The basal level of free radical production by the osteoblast-like cells (ROS 17/2.8) was 10(4)-fold lower than by osteoclasts cultured on bone. PTH had no acute effect on free radical production by the osteoblasts. The exposure of the osteoclasts cultured on bone to PTH led to a dramatic and immediate stimulation of O2- generation which was unaffected by the presence of ROS 17/2.8 cells. The osteoclasts co-cultured with ROS 17/2.8 cells and exposed to PTH for 3 h were also found to produce greater stimulation of O2- than the osteoclasts exposed to PTH alone. A competitive leukotriene D4 antagonist REV 5901, which also inhibits 5-lipoxygenase, did not block O2- generation by osteoclasts cultured alone or in the presence of osteoblasts. Therefore, we conclude that PTH directly stimulates osteoclasts to produce O2-; this may be the main mode of activation of the osteoclasts, although an osteoblast-mediated effect of the hormone cannot be ruled out.

The role of prostaglandin H synthase-mediated metabolism in the induction of oxidative DNA damage by BHA metabolites.

The carcinogenicity of the phenolic food antioxidant butylated hydroxyanisole may be related to its oxidative biotransformation in vivo. In order to determine the ability of BHA, 2-tert-butyl(1,4)hydroquinone (TBHQ) and 2-tert-butyl(1,4)paraquinone (TBQ) to induce oxidative DNA damage, biological inactivation of single-stranded bacteriophage phi X-174 DNA, as well as induction of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in dG by these compounds was studied in vitro, in the presence and absence of peroxidases. Both test systems showed that BHA and TBQ (probably due to lack of reductase activity in vitro) were not capable of inducting oxidative DNA damage. TBHQ, however, appeared to be a strong inactivator of phage DNA as well as a potent inducer of 8-oxodG formation. Addition of radical scavengers showed that this damage was due to formation of superoxide anion, hydrogen peroxide and hydroxyl radicals. Addition of iron chelators and metal ions showed that the one-electron oxidations of TBHQ via the semiquinone radical into TBQ are toxic via the formation of oxygen radicals and are not directly due to the hydroquinone itself or the formation of semiquinone radicals. Although peroxidation of TBHQ by prostaglandin H synthase (PHS) is indicated to result in a superoxide anion burst, this is not accompanied by an increase in oxidative DNA damage in vitro. This might be due to the use of hydrogen peroxide as a substrate by PHS itself, consequently resulting in less formation of hydroxyl radicals. Oxidation of TBHQ by lipoxygenases showed that no semiquinone radicals or oxygen radicals were formed, probably due to a two-electron oxidation of TBHQ directly into TBQ. The present results indicate that metabolic activation of BHA yielding reactive oxygen species may induce a carcinogenic potential, since the BHA metabolite TBHQ, appeared to be a strong inducer of oxidative DNA damage.

Enhanced Generation of O<sub>2</sub><sup>–</sup> by Human Neutrophils via a Complement iC3b/Mac-1 Interaction

There is evidence for a tumor necrosis factor alpha (TNF alpha)-initiated and CD11b/CD18-dependent burst of superoxide anion (O2-) and hydrogen peroxide production by human polymorphonuclear leukocytes which are adherent to surfaces bearing a variety of proteins. In the current studies neutrophils were stimulated with opsonized (by fresh human serum) zymosan particles in the presence of cytochalasin B, to prevent internalization of particles and to simulate the interaction of neutrophils with protein-bearing surfaces. Under these conditions, the cells demonstrated 2.9-fold greater production of O2- when compared to nonopsonized zymosan particles. Heat inactivation or cobra venom factor treatment of human serum prior to opsonization resulted in 98% and 66% reductions, respectively, in O2- responses. C3 and factor B were required for this response, since sera deficient in either component caused 56 and 68% reductions, respectively, in O2- production. Sera deficient in Clq, C2, C4, C5, C6, C7 or C9 showed no defect in their ability to enhance O2- responses to zymosan particles. Monoclonal antibody to iC3b, but not monoclonal antibodies to C3c or C3d, caused a 29% reduction (p < 0.01) in O2- generation. Antibodies to CD18 (R15.7) or CD11b (CL44 and 60.1) reduced the incremental production of O2- by 76, 71 and 77%, respectively. Two antibodies directed against CD11a as well as the isotype-matched control (MOPC 21) were without effects. These data suggest that, in this model of neutrophil activation, the pathway for O2- generation is a Mac-1 (but not LFA-1)-dependent pathway and also requires iC3b. These findings may be relevant to complement-mediated, neutrophil-dependent vascular injury in vivo.

Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury

In this lecture, evidence is presented to support the following hypothesis regarding the roles of xanthine oxidase-derived oxidants and granulocytes in ischemia-reperfusion-induced microvascular injury. During the ischemic period, ATP is catabolized to yield hypoxanthine. The hypoxic stress also triggers the conversion of NAD-reducing xanthine dehydrogenase to the oxygen radical-producing xanthine oxidase. During reperfusion, molecular oxygen is reintroduced into the tissue where it reacts with hypoxanthine and xanthine oxidase to produce a burst of superoxide anion and hydrogen peroxide. In the presence of iron, superoxide anion and hydrogen peroxide react via the Haber-Weiss reaction to form hydroxyl radicals. This highly reactive and cytotoxic radical then initiates lipid peroxidation of cell membrane components and the subsequent release of substances that attract, activate, and promote the adherence of granulocytes to microvascular endothelium. The adherent granulocytes then cause further endothelial cell injury via the release of superoxide and various proteases.

Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury.

In this lecture, evidence is presented to support the following hypothesis regarding the roles of xanthine oxidase-derived oxidants and granulocytes in ischemia-reperfusion-induced microvascular injury. During the ischemic period, ATP is catabolized to yield hypoxanthine. The hypoxic stress also triggers the conversion of NAD-reducing xanthine dehydrogenase to the oxygen radical-producing xanthine oxidase. During reperfusion, molecular oxygen is reintroduced into the tissue where it reacts with hypoxanthine and xanthine oxidase to produce a burst of superoxide anion and hydrogen peroxide. In the presence of iron, superoxide anion and hydrogen peroxide react via the Haber-Weiss reaction to form hydroxyl radicals. This highly reactive and cytotoxic radical then initiates lipid peroxidation of cell membrane components and the subsequent release of substances that attract, activate, and promote the adherence of granulocytes to microvascular endothelium. The adherent granulocytes then cause further endothelial cell injury via the release of superoxide and various proteases.

Mechanisms of oxidant-mediated microvascular injury following reperfusion of the ischemic intestine.

Based on work from our laboratory and studies by others, we propose the following hypothesis to explain the interaction among xanthine oxidase, PMNs, and tissue injury in the postischemic small intestine (Figure 2). During the ischemic period, ATP is catabolized to yield hypoxanthine. The hypoxic stress also triggers the conversion of NAD-reducing xanthine dehydrogenase to the oxygen radical-producing xanthine oxidase via a protease. When the intestine is reperfused, molecular oxygen is reintroduced into the tissue where it reacts with hypoxanthine and xanthine oxidase to produce a burst of superoxide anion and hydrogen peroxide. In the presence of ferric iron, superoxide anion and hydrogen peroxide react via the Haber-Weiss reaction to form hydroxyl radicals. This highly reactive and cytoxic free radical then initiates lipid peroxidation of cell membrane components and the subsequent release of substances that activate, attract, and promote the adherence of PMN to microvascular endothelium. The adherent PMN then causes further endothelial cell injury via the release of superoxide and various proteases.

Polymorphonuclear Leukocyte-Mediated, Antibody-Dependent, Cellular Cytotoxicity against Tumor Cells: Dependence on Oxygen and the Respiratory Burst

Experiments were done to determine 1) whether the respiratory burst of superoxide anion (O2-) production in polymorphonuclear leukocytes (PMN) is triggered during antibody-dependent killing of tumor cells and 2) whether O2- production is essential for cytotoxicity. Three parameters of the respiratory burst (1-14C-glucose oxidation, oxygen consumption, and O2- release) were increased 2.5- to 7.3-fold during killing of antibody-primed tumor cells by human PMN. Added catalase and superoxide dismutase did not inhibit lysis, possibly because these enzymes were unable to diffuse into the inter-plasma-membrane space between killer and target cells. Evidence for an O2- requirement for cytotoxicity was the fact that concentrations of amobarbital or phenylbutazone sufficient to inhibit the cyanide-insensitive respiration of PMN also inhibited cytotoxicity. Also, hypoxic conditions inhibited cytotoxicity from 29 to 73%. The requirement for oxygen was most likely related to O2- generation and not mitochondrial respiration since cyanide and azide, which inhibit mitochondrial respiration, increased cytotoxicity.