Free radical reactions in atherosclerosis; An EPR spectrometry study

The copper catalysed oxidation of homocysteine has been studied by electron paramagnetic resonance (EPR) spectroscopy and spin trapping techniques to determine the nature of free radical species formed under varying experimental conditions. Three radicals; thiyl, alkyl and hydroxyl were detected with hydroxyl being predominant. A reaction mechanism is proposed involving Fenton chemistry. Inclusion of catalase to test for intermediate generation of hydrogen peroxide showed a marked reduction in amount of hydroxyl radical generated. In contrast, the addition of superoxide dismutase showed no significant effect on the level of hydroxyl radical formed. Enhanced radical formation was observed at higher levels of oxygen, an effect which has consequences for differential oxygen levels in arterial and venous systems. Implications are drawn for a higher incidence of atherosclerotic plaque formation in arteries versus veins. © 2006 - IOS Press and the authors. All rights reserved.

Ceramide induces a loss in cytosolic peroxide levels in mononuclear cells

Ceramide (a sphingolipid) and reactive oxygen species are each partly responsible for intracellular signal transduction in response to a variety of agents. It has been reported that ceramide and reactive oxygen species are intimately linked and show reciprocal regulation [Liu, Andreieu-Abadie, Levade, Zhang, Obeid and Hannun (1998) J. Biol. Chem. 273, 11313-11320]. Utilizing synthetic, short-chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide formation or using stimulation of CD95 to induce ceramide formation, we found that the principal redox-altering property of ceramide is to lower the [peroxide]cyt (cytosolic peroxide concentration). Apoptosis of Jurkat T-cells, primary resting and phytohaemagglutinin-activated human peripheral blood T-lymphocytes was preceded by a loss in [peroxide]cyt, as measured by the peroxide-sensitive probe 2′,7′-dichlorofluorescein diacetate (also reflected in a lower rate of superoxide dismutase-inhibitable cytochrome c reduction), and this was not associated with a loss of membrane integrity. Where growth arrest of U937 monocytes was observed without a loss of membrane integrity, the decrease in [peroxide]cyt was of a lower magnitude when compared with that preceding the onset of apoptosis in T-cells. Furthermore, decreasing the cytosolic peroxide level in U937 monocytes before the application of synthetic ceramide by pretreatment with either of the antioxidants N-acetyl cysteine or glutathione conferred apoptosis. However, N-acetyl cysteine or glutathione did not affect the kinetics or magnitude of ceramide-induced apoptosis of Jurkat T-cells. Therefore the primary redox effect of cellular ceramide accumulation is to lower the [peroxide]cyt of both primary and immortalized cells, the magnitude of which dictates the cellular response.

The central role of the neutrophil in ischaemic/reperfusion injury

Inadequate blood flow to an organ, ischaemia, may lead to both local and remote tissue injury characterized by oedema, increased microvascular permeability to protein and degradation of connective tissue components. This damage is probably caused by the accumulation and inappropriate activation of neutrophils which occurs when the tissue is reperfused. To test this hypothesis a number of in vitro models of the sequential stages of ischaemia/reperfusion injury were examined. Methods were initially developed to examine the adhesion of neutrophils to monolayers of a cultured endothelial cell line (ECV304) after periods of hypoxia and reoxygenation. Neutrophil migration in response to factors secreted by the treated endothelial cells was then assessed. The genesis of an inappropriate oxidative burst by the neutrophil upon exposure to endothelial chemoattractants and adhesion molecules was also measured. Finally to appraise how tissue function might be affected by endothelial cell hypoxia the contractility of vascular smooth muscle was examined. Neutrophil adhesion to ECV304 cells, which had been hypoxic for 4 hours and then reoxygenated for 30 minutes, was significantly increased. This response was probably initiated by reactive oxygen species (ROS) generated by the endothelial cells. Blockage of their production by allopurinol reduced the heightened adhesion. Similarly removal of ROS by superoxide dismutase or catalase also attenuated adhesion. ROS generation in turn caused the release of a soluble factor (s) which induced a conformational change on the neutrophil surface allowing it to bind to the intercellular adhesion molecule 1 (ICAM-1) on the endothelial cell. Soluble factor (s) from hypoxia/reoxygenated endothelial cells also had a powerful neutrophil chemoattractant ability. When neutrophils were exposed to both hypoxic/reoxygenated endothelial cells and the soluble factor (s) released by them a large oxidative burst was elicited. This response was greatest immediately after reoxygenation and one hour later was diminishing suggesting at least one of the components involved was labile. Analysis of the supernatant from hypoxic/reoxygenated endothelial cell cultures and studies using inhibitors of secretion suggested platelet activating factor (PAF) may be a major component in this overall sequence of events. Lesser roles for IL-8, TNF and LTB4 were also suggested. The secretory products from hypoxia/reoxygenated endothelial cells also affected smooth muscle contractility having an anti-vasoconstrictor or relaxation property, similar to that exerted by PAF.

Sensitivity of antioxidant-deficient yeast to hypochlorite and chlorite

Sodium hypochlorite and sodium chlorite are commonly used as disinfectants, and understanding the mechanisms of microbial resistance to these compounds is of considerable importance. In this study, the role of oxidative stress and antioxidant enzymes in the sensitivity of the yeast Saccharomyces cerevisiae to hypochlorite and chlorite was studied. Yeast mutants lacking Cu-Zn superoxide dismutase, but not mutants deficient in cytoplasmic and peroxisomal catalase, were hypersensitive to the action of both hypochlorite and chlorite. Both compounds depleted cellular glutathione, induced the production of reactive oxygen species and decreased the viability of the cells. The toxicity of hypochlorite and chlorite was abolished by hypoxic and anoxic conditions and ameliorated by thiol antioxidants and ascorbate. The results demonstrated that the action of hypochlorite and chlorite involves the formation of superoxide and peroxide and that SOD1 is protective, probably by limiting the formation of hydroxyl radicals and damage to proteins.

Palmitate promotes monocyte atherogenicity via de novo ceramide synthesis

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. This study investigates the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Incubation of human U937 and THP-1 monocytes with palmitate for 24h increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300µM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300µM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis.

A preliminary investigation into the impact of a pesticide combination on human neuronal and glial cell lines in vitro

Many pesticides are used increasingly in combinations during crop protection and their stability ensures the presence of such combinations in foodstuffs. The effects of three fungicides, pyrimethanil, cyprodinil and fludioxonil, were investigated together and separately on U251 and SH-SY5Y cells, which can be representative of human CNS glial and neuronal cells respectively. Over 48h, all three agents showed significant reductions in cellular ATP, at concentrations that were more than tenfold lower than those which significantly impaired cellular viability. The effects on energy metabolism were reflected in their marked toxic effects on mitochondrial membrane potential. In addition, evidence of oxidative stress was seen in terms of a fall in cellular thiols coupled with increases in the expression of enzymes associated with reactive species formation, such as GSH peroxidase and superoxide dismutase. The glial cell line showed significant responsiveness to the toxin challenge in terms of changes in antioxidant gene expression, although the neuronal SH-SY5Y line exhibited greater vulnerability to toxicity, which was reflected in significant increases in caspase-3 expression, which is indicative of the initiation of apoptosis. Cyprodinil was the most toxic agent individually, although oxidative stress-related enzyme gene expression increases appeared to demonstrate some degree of synergy in the presence of the combination of agents. This report suggests that the impact of some pesticides, both individually and in combinations, merits further study in terms of their impact on human cellular health.

Decreased NADPH oxidase expression and antioxidant activity in cachectic skeletal muscle

Background - Cancer cachexia is the progressive loss of skeletal muscle protein that contributes significantly to cancer morbidity and mortality. Evidence of antioxidant attenuation and the presence of oxidised proteins in patients with cancer cachexia indicate a role for oxidative stress. The level of oxidative stress in tissues is determined by an imbalance between reactive oxygen species production and antioxidant activity. This study aimed to investigate the superoxide generating NADPH oxidase (NOX) enzyme and antioxidant enzyme systems in murine adenocarcinoma tumour-bearing cachectic mice. Methods - Superoxide levels, mRNA levels of NOX enzyme subunits and the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidise (GPx) and catalase was measured in the skeletal muscle of mice with cancer and cancer cachexia. Protein expression levels of NOX enzyme subunits and antioxidant enzyme activity was also measured in the same muscle samples. Results - Superoxide levels increased 1.4-fold in the muscle of mice with cancer cachexia, and this was associated with a decrease in mRNA of NOX enzyme subunits, NOX2, p40phox and p67phox along with the antioxidant enzymes SOD1, SOD2 and GPx. Cancer cachexia was also associated with a 1.3-fold decrease in SOD1 and 2.0-fold decrease in GPx enzyme activity. Conclusion - Despite increased superoxide levels in cachectic skeletal muscle, NOX enzyme subunits, NOX2, p40phox and p67phox, were downregulated along with the expression and activity of the antioxidant enzymes. Therefore, the increased superoxide levels in cachectic skeletal muscle may be attributed to the reduction in the activity of endogenous antioxidant enzymes.

Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts; the roles of reactive oxygen and nitrogen species

Adaptive mechanisms involving upregulation of cytoprotective genes under the control of transcription factors such as Nrf2 exist to protect cells from permanent damage and dysfunction under stress conditions. Here we explore of the hypothesis that Nrf2 activation by reactive oxygen and nitrogen species modulates cytotoxicity during hypoxia (H) with and without reoxygenation (H/R) in H9C2 cardiomyoblasts. Using MnTBap as a cell permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger and L-NAME as an inhibitor of nitric oxide synthase (NOS), we have shown that MnTBap inhibited the cytotoxic effects of hypoxic stress with and without reoxygenation. However, L-NAME only afforded protection during H. Under reoxygenation, conditions, cytotoxicity was increased by the presence of L-NAME. Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R. The increased cytotoxicity and inhibition of Nrf2 activation by the presence of L-NAME during reoxygenation suggests that NOS activity plays an important role in cell survival at least in part via Nrf2-independent pathways. In contrast, O2 -• scavenging by MnTBap prevented both toxicity and Nrf2 activation during H and H/R implying that toxicity is largely dependent on O2 -.To confirm the importance of Nrf2 for myoblast metabolism, Nrf2 knockdown with siRNA reduced cell survival by 50% during 4h hypoxia with and without 2h of reoxygenation and although cellular glutathione (GSH) was depleted during H and H/R, GSH loss was not exacerbated by Nrf2 knockdown. These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage. © 2013 The Authors.

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on express ion/activity of the main DDS phase-II- metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxiclation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.

Clinical oxidative stress during leprosy multidrug therapy:impact of dapsone oxidation

This study aims to assess the oxidative stress in leprosy patients under multidrug therapy (MDT; dapsone, clofazimine and rifampicin), evaluating the nitric oxide (NO) concentration, catalase (CAT) and superoxide dismutase (SOD) activities, glutathione (GSH) levels, total antioxidant capacity, lipid peroxidation, and methemoglobin formation. For this, we analyzed 23 leprosy patients and 20 healthy individuals from the Amazon region, Brazil, aged between 20 and 45 years. Blood sampling enabled the evaluation of leprosy patients prior to starting multidrug therapy (called MDT 0) and until the third month of multidrug therapy (MDT 3). With regard to dapsone (DDS) plasma levels, we showed that there was no statistical difference in drug plasma levels between multibacillary (0.518±0.029 μg/mL) and paucibacillary (0.662±0.123 μg/mL) patients. The methemoglobin levels and numbers of Heinz bodies were significantly enhanced after the third MDTsupervised dose, but this treatment did not significantly change the lipid peroxidation and NO levels in these leprosy patients. In addition, CAT activity was significantly reduced in MDT-treated leprosy patients, while GSH content was increased in these patients. However, SOD and Trolox equivalent antioxidant capacity levels were similar in patients with and without treatment. These data suggest that MDT can reduce the activity of some antioxidant enzyme and influence ROS accumulation, which may induce hematological changes, such as methemoglobinemia in patients with leprosy. We also explored some redox mechanisms associated with DDS and its main oxidative metabolite DDS-NHOH and we explored the possible binding of DDS to the active site of CYP2C19 with the aid of molecular modeling software. © 2014 Schalcher et al.

In vitro protective effect and antioxidant mechanism of Resveratrol induced by Dapsone Hydroxylamine in human cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDSNHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10-1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.

Macrophage polarisation by fatty acids is PPARgamma-dependent

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. We investigated the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Palmitate increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300µM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300µM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis. We have also explored whether specific dietary fatty acids drive monocyte to macrophage polarisation via metabolic pathways. Here we show that monocytes pre-incubated with the saturated fatty acid palmitate increase production of inflammatory cytokines such as TNFa and IL-6 in response to a phorbol myristate differentiation trigger. This increases mitochondrial superoxide production, reduces dependency on oxidative phosphorylation through ceramide-dependent inhibition of PPARgamma activity and increases TNFa production, again via a mechanism that requires ceramide production.

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.

Dehdyroepiandrosterone sulfate directly activates protein kinase C-β to increase human neutrophil superoxide generation

Dehydroepiandrosterone sulfate (DHEAS) is the most abundant steroid in the human circulation and is secreted by the adrenals in an age-dependent fashion, with maximum levels during the third decade and very low levels in old age. DHEAS is considered an inactive metabolite, whereas cleavage of the sulfate group generates dehydroepiandrosterone (DHEA), a crucial sex steroid precursor. However, here we show that DHEAS, but not DHEA, increases superoxide generation in primed human neutrophils in a dose-dependent fashion, thereby impacting on a key bactericidal mechanism. This effect was not prevented by coincubation with androgen and estrogen receptor antagonists but was reversed by the protein kinase C inhibitor Bisindolylmaleimide 1. Moreover, we found that neutrophils are unique among leukocytes in expressing an organic anion-transporting polypeptide D, able to mediate active DHEAS influx transport whereas they did not express steroid sulfatase that activates DHEAS to DHEA. A specific receptor for DHEAS has not yet been identified, but we show that DHEAS directly activated recombinant protein kinase C-ß (PKC-ß) in a cell-free assay. Enhanced PKC-ß activation by DHEAS resulted in increased phosphorylation of p47phox, a crucial component of the active reduced nicotinamide adenine dinucleotide phosphate complex responsible for neutrophil superoxide generation. Our results demonstrate that PKC-ß acts as an intracellular receptor for DHEAS in human neutrophils, a signaling mechanism entirely distinct from the role of DHEA as sex steroid precursor and with important implications for immunesenescence, which includes reduced neutrophil superoxide generation in response to pathogens.