The superoxide anion donor, potassium superoxide, induces pain and inflammation in mice through production of reactive oxygen species and cyclooxygenase-2

It is currently accepted that superoxide anion (O2•−) is an important mediator in pain and inflammation. The role of superoxide anion in pain and inflammation has been mainly determined indirectly by modulating its production and inactivation. Direct evidence using potassium superoxide (KO2), a superoxide anion donor, demonstrated that it induced thermal hyperalgesia, as assessed by the Hargreaves method. However, it remains to be determined whether KO2 is capable of inducing other inflammatory and nociceptive responses attributed to superoxide anion. Therefore, in the present study, we investigated the nociceptive and inflammatory effects of KO2. The KO2-induced inflammatory responses evaluated in mice were: mechanical hyperalgesia (electronic version of von Frey filaments), thermal hyperalgesia (hot plate), edema (caliper rule), myeloperoxidase activity (colorimetric assay), overt pain-like behaviors (flinches, time spent licking and writhing score), leukocyte recruitment, oxidative stress, and cyclooxygenase-2 mRNA expression (quantitative PCR). Administration of KO2 induced mechanical hyperalgesia, thermal hyperalgesia, paw edema, leukocyte recruitment, the writhing response, paw flinching, and paw licking in a dose-dependent manner. KO2 also induced time-dependent cyclooxygenase-2 mRNA expression in the paw skin. The nociceptive, inflammatory, and oxidative stress components of KO2-induced responses were responsive to morphine (analgesic opioid), quercetin (antioxidant flavonoid), and/or celecoxib (anti-inflammatory cyclooxygenase-2 inhibitor) treatment. In conclusion, the well-established superoxide anion donor KO2 is a valuable tool for studying the mechanisms and pharmacological susceptibilities of superoxide anion-triggered nociceptive and inflammatory responses ranging from mechanical and thermal hyperalgesia to overt pain-like behaviors, edema, and leukocyte recruitment.

Mitochondrial superoxide anion radicals mediate induction of apoptosis in cardiac myoblasts exposed to chronic hypoxia

Both reactive oxygen species (ROS) and ATP depletion may be significant in hypoxia-induced damage and death, either collectively or independently, with high energy requiring, metabolically active cells being the most susceptible to damage. We investigated the kinetics and effects of ROS production in cardiac myoblasts, H9C2 cells, under 2%, 10% and 21% O2 in the presence or absence of apocynin, rotenone and carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. H9C2 cells showed significant loss of viability within 30 min of culture at 2% oxygen which was not due to apoptosis, but was associated with an increase in protein oxidation. However, after 4 h, apoptosis induction was observed at 2% oxygen and also to a lesser extent at 10% oxygen; this was dependent on the levels of mitochondrial superoxide anion radicals determined using dihydroethidine. Hypoxia-induced ROS production and cell death could be rescued by the mitochondrial complex I inhibitor, rotenone, despite further depletion of ATP. In conclusion, a change to superoxide anion radical steady state level was not detectable after 30 min but was evident after 4 h of mild or severe hypoxia. Superoxide anion radicals from the mitochondrion and not ATP depletion is the major cause of apoptotic cell death in cardiac myoblasts under chronic, severe hypoxia.

The bimodal regulation of vascular function by superoxide anion: role of endothelium

Reactive oxygen species (ROS) are implicated in vascular homeostasis. This study investigated whether O2 .

Superoxide anion production by neutrophils is associated with prevalent clinical manifestations in systemic lupus erythematosus

To determine the relation between neutrophil function and the clinical characteristics of systemic lupus erythematosus ( SLE), the superoxide anion (O(2)(-)) production by neutrophils, mediated by Fc gamma R and Fc gamma R/CR cooperation, was studied in 64 SLE patients classified according to their prevalent clinical manifestations. Three clinically distinct patterns were designated: ( 1) manifestations associated with the occurrence of cytotoxic antibodies ( SLE-I group); ( 2) manifestations associated with circulating immune complexes ( IC; SLE-II group), and ( 3) manifestations associated with IC and cytotoxic antibodies ( SLE-III group). O(2)(-) production was evaluated by a lucigenin-dependent chemiluminescent assay in neutrophils stimulated with ICIgG opsonized or not with complement. No difference in O(2)(-) production was observed when neutrophil responses from healthy controls were compared to the unclassified patients. However, when the SLE patient groups were considered, the following differences were observed: ( 1) SLE-I neutrophils showed lower O(2)(-) production mediated by the IgG receptor ( Fc gamma R) with the cooperation of complement receptors ( Fc gamma R/ CR) than observed in the SLE-II, SLE-III, and healthy groups; ( 2) neutrophils from the SLE-II group showed a decreased O(2)(-) production mediated by Fc gamma R/ CR compared to the SLE-III group, ( 3) SLE-III neutrophils produced more O(2)(-) than neutrophils from the SLE-II and control groups, and ( 4) CR showed inefficiency in mediating the O(2)(-) production by neutrophils from the SLE-I group. Comparative experiments on the kinetics of chemiluminescence ( CL; T(max) and CL(max)) disclosed differences only for the SLE- I group. Taken together, these results suggest that differences in oxidative metabolism of neutrophils mediated by Fc gamma R/ CR may reflect an acquired characteristic of disease associated with distinct clinical manifestations.

Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity

As a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain, Neisseria gonorrhoeae requires defence systems to respond to toxic oxygen species such as superoxide. It has been shown that supplementation of media with 100 muM Mn(II) considerably enhanced the resistance of this bacterium to oxidative killing by superoxide. This protection was not associated with the superoxide dismutase enzymes of N. gonorrhoeae. In contrast to previous studies, which suggested that some strains of N. gonorrhoeae might not contain a superoxide dismutase, we identified a sodB gene by genome analysis and confirmed its presence in all strains examined by Southern blotting, but found no evidence for sodA or sodC. A sodB mutant showed very similar susceptibility to superoxide killing to that of wild-type cells, indicating that the Fe-dependent SOD B did not have a major role in resistance to oxidative killing under the conditions tested. The absence of a sodA gene indicated that the Mn-dependent protection against oxidative killing was independent of Mn-dependent SOD A. As a sodB mutant also showed Mn-dependent resistance to oxidative killing, then it is concluded that this resistance is independent of superoxide dismutase enzymes. Resistance to oxidative killing was correlated with accumulation of Mn(II) by the bacterium. We hypothesize that this bacterium uses Mn(II) as a chemical quenching agent in a similar way to the already established process in Lactobacillus plantarum. A search for putative Mn(II) uptake systems identified an ABC cassette-type system (MntABC) with a periplasmic-binding protein (MntC). An mntC mutant was shown to have lowered accumulation of Mn(II) and was also highly susceptible to oxidative killing, even in the presence of added Mn(II). Taken together, these data show that N. gonorrhoeae possesses a Mn(II) uptake system that is critical for resistance to oxidative stress.

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

J Biol Inorg Chem (2004) 9: 839–849 DOI 10.1007/s00775-004-0584-6

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective ATI receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P) H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT(1)-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. (c) 2012 Elsevier Inc. All rights reserved.

Higher macrophage superoxide anion production in coronary artery disease (CAD) patients with Type D personality

BACKGROUND Type D personality (Type D) is an independent psychosocial risk factor for poor cardiac prognosis and increased mortality in patients with cardiovascular disease (CVD), but the involved mechanisms are poorly understood. Macrophages play a pivotal role in atherosclerosis, the process underlying coronary artery disease (CAD). We investigated macrophage superoxide anion production in production in CAD patients with and without Type D. METHODS AND RESULTS We studied 20 male CAD patients with Type D (M:66.7±9.9years) and 20 age-matched male CAD patients without Type D (M:67.7±8.5years). Type D was measured using the DS14 questionnaire with the two subscales 'negative affectivity' and 'social inhibition'. We assessed macrophage superoxide anion production using the WST-1 assay. All analyses were controlled for potential confounders. CAD patients with Type D showed higher superoxide anion production compared to CAD patients without Type D (F(1,38)=15.57, p<0.001). Complementary analyses using the Type D subscales 'negative affectivity' and 'social inhibition', and their interaction as continuous measures, showed that both Type D subscales (negative affectivity: (ß=0.48, p=0.002, R(2)=0.227); social inhibition: (ß=0.46, p=0.003, R(2)=0.208)) and their interaction (ß=0.36, p=0.022, R(2)=0.130) were associated with higher WST-1 reduction scores. Results remained significant when controlling for classical CVD risk factors (i.e. body mass index, mean arterial blood pressure), atherosclerosis severity (i.e. intima media thickness, presence of carotid plaques), and psychological factors (depressive symptom severity, chronic stress). CONCLUSIONS Our results indicate higher macrophage superoxide anion production in CAD patients with Type D compared to those without Type D. This may suggest a mechanism contributing to increased morbidity and mortality in CAD patients with Type D.

Effects of asbestos and silica on superoxide anion production in the guinea pig alveolar macrophage

Asbestos and silica are important industrial hazards. Exposure to these dusts can result in pulmonary fibrosis and, in the case of asbestos, cancer. Although the hazards of asbestos and silica exposure have long been known, the pathogenesis of dust-related disease is not well understood. Both silica and asbestos are thought to alter the function of the alveolar macrophage, but the nature of the biochemical alteration is unknown. Therefore, this study examined the effect of asbestos and silica on the activation pathway of the guinea pig alveolar macrophage. Activation of macrophages by physiological agents results in stimulation of phospholipase C causing phosphatidyl inositol turnover and intracellular calcium mobilization. Phosphatidyl inositol turnover produces diacylglycerol which activates protein kinase C causing superoxide anion production.^ Chrysotile stimulated alveolar macrophages to produce superoxide anion. This stimulation proceeded via phospholipase C, since chrysotile stimulated phosphatidyl inositol turnover and intracellular calcium mobilization. The possible involvement of a coupling protein was evaluated by pretreating cells with pertussis toxin. Pertussis toxin pretreatment partially inhibited chrysotile stimulation, suggesting that chrysotile activates a coupling protein in an non-classical manner. Potential binding sites for chrysotile stimulation were examined using a series of nine lectins. Chrysotile-stimulated superoxide anion production was blocked by pretreatment with lectins which bound to N-acetylglucosamine, but not by lectins which bound to mannose, fucose, or N-acetylgalactosamine. In addition, incubation with the N-acetylglucosamine polymer, chitin, inhibited chrysotile-stimulated superoxide anion production, suggesting that chrysotile stimulated superoxide anion production by binding to N-acetylglucosamine residues.^ On the other hand, silica did not stimulate superoxide anion production. The effect of silica on agonist stimulation of this pathway was examined using two stimulants of superoxide anion production, N-formyl-nle-leu-phe (FNLP, which stimulates through phospholipase C) and phorbol-12,13-dibutyrate (which directly activates protein kinase C). Sublethal doses of silica inhibited FNLP-stimulated superoxide anion production, but did not affect phorbol-12,13-dibutyrate-stimulated superoxide anion production, suggesting that the site of inhibition precedes protein kinase C. This inhibition was not due to cell membrane damage, since cell permeability to calcium-45 and rubidium-86 was not increased. It is concluded that chrysotile binds to N-acetylglucosamine residues on macrophage surface glycoproteins to stimulate the physiological pathway resulting in superoxide anion production. In contrast, silica does not stimulate superoxide anion production, but it did inhibit FNLP-stimulated superoxide anion production. ^

Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production.

Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism by which this protection is mediated is unclear. Since animal studies strongly suggest that production of endothelium-derived relaxing factor is enhanced by estradiol, we have examined the effect of estrogens on nitric oxide (NO) synthase (NOS) activity, protein, and mRNA in cultured bovine aortic endothelial cells. In reporter cells rich in guanylate cyclase, it has been observed that long-term treatment (> or = 24 hr) with ethinylestradiol (EE2) dose-dependently increased guanylate cyclase-activating factor activity in the conditioned medium of endothelial cells. However, conversion of L-[14C]arginine to L-[14C]citrulline by endothelial cell homogenate or quantification of nitrite and nitrate released by intact cells in the conditioned medium did not reveal any change in NOS activity induced by EE2 treatment. Similarly, Western and Northern blot analyses did not reveal any change in the endothelial NOS protein and mRNA content in response to EE2. However, EE2 dose- and time-dependently decreased superoxide anion production in the conditioned medium of endothelial cells with an EC50 value (0.1 nM) close to that which increased guanylate cyclase-activating factor activity (0.5 nM). Both of these effects were completely prevented by the antiestrogens tamoxifen and RU54876. Thus, endothelium exposure to estrogens appears to induce a receptor-mediated antioxidant effect that enhances the biological activity of endothelium-derived NO. These effects could account at least in part for the vascular protective properties of these hormones.

Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures.

N-Methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity may depend, in part, on the generation of nitric oxide (NO.) and superoxide anion (O2.-), which react to form peroxynitrite (OONO-). This form of neurotoxicity is thought to contribute to a final common pathway of injury in a wide variety of acute and chronic neurologic disorders, including focal ischemia, trauma, epilepsy, Huntington disease, Alzheimer disease, amyotrophic lateral scelerosis, AIDS dementia, and other neurodegenerative diseases. Here, we report that exposure of cortical neurons to relatively short durations or low concentrations of NMDA, S-nitrosocysteine, or 3-morpholinosydnonimine, which generate low levels of peroxynitrite, induces a delayed form of neurotoxicity predominated by apoptotic features. Pretreatment with superoxide dismutase and catalase to scavenge O2.- partially prevents the apoptotic process triggered by S-nitrosocysteine or 3-morpholinosydnonimine. In contrast, intense exposure to high concentrations of NMDA or peroxynitrite induces necrotic cell damage characterized by acute swelling and lysis, which cannot be ameliorated by superoxide dismutase and catalase. Thus, depending on the intensity of the initial insult, NMDA or nitric oxide/superoxide can result in either apoptotic or necrotic neuronal cell damage.

Interactions between oligoamines and superoxide anion

1- Oligoamines and EDTA inhibited the reduction of cytochrome-C and nitrobule tetrazolium (NBT) induced by the hypoxanthine/xanthine oxidase superoxide anion generating system in the following order of effectiveness: putrescine > diaminopropane > spermidine > EDTA > spermine > cadaverine. 2- Oligoamines and EDTA did not affect the rate of urate formation from the hypoxanthine/xanthine oxidase system. 3- Oligoamines and EDTA inhibited the reduction of cytochrome-C induced by stimulated PMNL's in the same order of effectiveness as mentioned before. 4- Oligoamines and EDTA inhibited luminol dependent stimulated PMNL's chemiluminescence. 5- Oligoamines and EDTA inhibited the aerobic photoreduction of NBT. 6- Oligoamines-copper sulphate complexes inhibited the reduction of cytochrome-C induced by the hypoxanthine/xanthine oxidase system more effectively than oligoamines or copper sulphate individually. 7- Superoxide anion, hydrogen peroxide and hydroxyl radical induced breakdown of isolated intact guinea pig liver lysosomes. 8- Oligoamines and EDTA protected isolated intact guinea pig liver lysosomes from the lytic effect of superoxide anion generated either by the hypoxanthine/xanthine oxidase system or by stimulated PMNL's. 9- Oligoamines and EDTA have no stabilizing effect on isolated intact guinea pig liver lysosomes. 10- The uptake of oligoamines by lysosomes was in the following order: putrescine > spermidine > spermine. 11- Oligoamines were metabolised into aldehyde compounds either by the hypoxanthine/xanthine oxidase system or stimulated PMNL's. 12- Oligoamines and EDTA have no effect on the activities of free lysosomal enzymes (acid phosphatase and -glucosaminidase). 13- Oligoamines and EDTA inhibited lipid peroxidation in guinea pig liver lysosomes induced either by the hypoxanthine/xanthine oxidase or ascorbic acid-ferrous sulphate. 14- Oligoamines and EDTA have no effect on the release of PGE_2 from stimulated peritoneal guinea pig PMNL's. 15- Oligoamines increased the uptake of (^3H)thymidine and (^3H)leucine by stimulated peritoneal guinea pig macrophages in the following order of effectiveness: spermine > spermidine > putrescine > cadaverine. 16- PGE_2, dibutyryl Cyclic AMP, and theophylline inhibited luminol dependent stimulated peritoneal guinea pig PMNL's chemiluminescence.

Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 μmol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 .−) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative ρ0 strain, lacking mitochondrial DNA, was used. The ρ0 strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 .− precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.

Role of oxidative stress in plasticity of Dendritic cells

Dendritic cells (DCs) are antigen presenting cells with an unique ability to induce primary immune responses. Different DCs subsets with an intrinsic capacity to polarise Tcells have been described: myeloid (Th1) and lymphoid (Th2). Plasticity is defined as DCs capacity to polarise T cells independent of the DCs origin. We investigated the potential role played by oxidants such as superoxide anion (·O2-), in the plasticity of DCs, measured by the induction of a specific DCs subset, cytokine release and antigen presentation. Furthermore, we are interested in the amplification of immune response analysed by the exosomes production after oxidative stress and LPS stimulation. Recently, we have demonstrated that exposure of cells to superoxide anions resulted in the activation of DC2 profile. To analyse the role of oxidative stress in DCs subsets, we used BDCA-1 and BDCA-2 antibodies, which identify myeloid and plasmacytoid DCs respectively. Freshly isolated monocytes have shown to be BDCA-1-, but BDCA-2+ populations. During 6 days culture up-regulation of BDCA-1, but a down-regulation of BDCA-2 were observed, giving a clear myeloid population. When DC were stimulated with superoxide anions or LPS, we have observed that both down regulate the expression of BDCA-1 when compared to immature DC. Antigen presentation was markedly altered according to the periodicity used, and antigens and oxidants exposures. Using DCs trapped in collagen "matrix" after LPS activation we were able to quantify DCs-exosomes (small membrane vesicles ~50-100 nm in diameter) by reconstruction pictures in three dimensions. Using double vital staining we have found that exosomes from activated DCs can fuse with the membrane of resting DCs. Understanding the capacity of DCs to integrate external signals we will be able to unravel and control Tcells-polarisation triggering a specific immune response or tolerance. We will be able also to understand the amplification role of DCs-exosomes in remote not yet activated DCs.