Involvement of the superoxide anion in sulphoxidation

Sulphoxidation of compounds capable of undergoing biological sulphoxidation has been demonstrated in a model system (NADH–phenazine methosulphate–O2), known to generate superoxide anions (O2-). Addition of superoxide dismutase to this system results in complete inhibition, suggesting the involvement of O2- in sulphoxidation.

Mechanism of hydroxylation of aromatic compounds: Evidence for the involvement of superoxide anion in a model system

Hydroxylation of aromatic compounds was observed in NADH-phenazine methosulfate-O2 model system known to generate superoxide anions (Image ). Addition of superoxide dismutase prepared from ovine erythrocytes to this hydroxylating system resulted in complete inhibition, suggesting an involvement of Image in aromatic hydroxylations.

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.

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.

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.

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 .

Functional effect of the C242T polymorphism in the NAD(P)H oxidase p22phox gene on vascular superoxide production in atherosclerosis.

BACKGROUND:

Increased superoxide anion production increases oxidative stress and reduces nitric oxide bioactivity in vascular disease states. NAD(P)H oxidase is an important source of superoxide in human blood vessels, and some studies suggest a possible association between polymorphisms in the NAD(P)H oxidase CYBA gene and atherosclerosis; however, no functional data address this hypothesis. We examined the relationships between the CYBA C242T polymorphism and direct measurements of superoxide production in human blood vessels.

METHODS AND RESULTS:

Vascular NAD(P)H oxidase activity was determined in human saphenous veins obtained from 110 patients with coronary artery disease and identified risk factors. Immunoblotting, reverse-transcription polymerase chain reaction, and DNA sequencing showed that p22phox protein, mRNA, and 242C/T allelic variants are expressed in human blood vessels. Vascular superoxide production, both basal and NADH-stimulated, was highly variable between patients, but the presence of the CYBA 242T allele was associated with significantly reduced vascular NAD(P)H oxidase activity, independent of other clinical risk factors for atherosclerosis.

CONCLUSIONS:

Association of the CYBA 242T allele with reduced NAD(P)H oxidase activity in human blood vessels suggests that genetic variation in NAD(P)H oxidase components may play a significant role in modulating superoxide production in human atherosclerosis.