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.

Free radicals as potential antitumour agents

The aim of this work was to use extremely low concentrations of free radical generating compounds as a 'catalyst' to trigger endogenous free radical chain reactions in the host and to selectively eliminate neoplastic cells in the host. To test the hypothesis, a number of free radical generating compounds were screened on several malignant cell lines in vitro to select model compounds that were used against tumour models in vivo. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and its derivatives were selected at the model compounds for in vivo experiments in view of their high cytotoxic potency against several malignant cell lines in vitro. The water soluble derivative, 2,2-diphenyl-1-(2', 4'-dinitro-6'-sulphophenyl) hydrazyl (DDSH) given by subcutaneous injections demonstrated significant antitumour activities against the MAC 16 murine colon adenocarcinoma implanted subcutaneously in male NMRI mice at nanomolar concentration range. 40-60% of long term survival of over 60 days was achieved (compared with control survival of 20 days) with total tumour elimination. This compound was also active against both P388 leukaemia in male BDF1 mice and TLX5 lymphoid tumour in male CBA/CA mice at a similar concentration range. However, some of these animals died suddenly after treatment with no evidence of disease present at post mortem. The cause of death was unknown but thought to be related to the treatment. There was significant increase in serum level of malondialdehyde (MDA) following treatment, but did not correlate to the antitumour activities of these compounds. Induction of supcroxide dismutase (SOD), and glutathione peroxidase (GPx) occurred around day 8 after the administration of DDSH. Histological sections of MAC16 tumours showed areas of extensive massive haemorrhagic necrosis and vascular collapse associated with perivascular cell death following the administration of nanomolar concentration of DDSH which was probably compatible with the effects of free radicals. It was concluded that the antitumour activities of these compounds may be related to free radical and cytokine production.

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.

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.

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.

Redox Regulation of Ras Proteins in Dictyostelium discoideum

Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide. The main objective of this project was to determine the mechanism of superoxide mediated Ras regulation in vivo. To accomplish the main objective, we cloned, and in some cases, mutated different Ras proteins and later determined their activity in wild type and sodC- cells. RasC and RasD showed normal activation in sodC- cells, however RasG and RasS displayed high Ras activity. These last two Ras proteins contain the NKC118D motif inside the nucleotide binding region. A mutation of cysteine118 to alanine in RasG rendered the protein less active in sodC- than the wild type RasG protein and a mutation alanine118 to cysteine in RasD conferred redox sensitivity to this small GTPase. Additionally, the propensity of RasG to be targeted by superoxide was evident when the environment of wild type cells was manipulated to induce the internal generation of superoxide through changes in the extracellular ion levels mainly magnesium. Lack of magnesium ions increased the intracellular level of superoxide and severely hampered directional cell migration. Chemotaxis of cells expressing RasG was negatively impacted by the absence of magnesium ions; however rasG- cells did not seem to be affected in their ability to perform chemotaxis. The last experiment implies that RasG is an important mediator of cell signaling during oxidative stress, responsible for preventing cells from continuing their developmental program. Our study suggests that the cysteine residue in the NKCD motif is essential for mediating the redox sensitivity of Ras proteins in Dictyostelium and that RasG is an essential mediator of the response to oxidative stress in this organism.

Effects of ocean acidification on the swimming ability, development and biochemical responses of sand smelt larvae

Ocean acidification, recognized as a major threat to marine ecosystems, has developed into one of the fastest growing fields of research in marine sciences. Several studies on fish larval stages point to abnormal behaviours, malformations and increased mortality rates as a result of exposure to increased levels of CO2. However, other studies fail to recognize any consequence, suggesting species-specific sensitivity to increased levels of CO2, highlighting the need of further research. In this study we investigated the effects of exposure to elevated pCO2 on behaviour, development, oxidative stress and energy metabolism of sand smelt larvae, Atherina presbyter. Larvae were caught at Arrábida Marine Park (Portugal) and exposed to different pCO2 levels (control: 600 µatm, pH = 8.03; medium: 1000 µatm, pH = 7.85; high: 1800 µatm, pH = 7.64) up to 15 days, after which critical swimming speed (Ucrit), morphometric traits and biochemical biomarkers were determined. Measured biomarkers were related with: 1) oxidative stress-superoxide dismutase and catalase enzyme activities, levels of lipid peroxidation and DNA damage, and levels of superoxide anion production; 2) energy metabolism - total carbohydrate levels, electron transport system activity, lactate dehydrogenase and isocitrate dehydrogenase enzyme activities. Swimming speed was not affected by treatment, but exposure to increasing levels of pCO2 leads to higher energetic costs and morphometric changes, with larger larvae in high pCO2 treatment and smaller larvae in medium pCO2 treatment. The efficient antioxidant response capacity and increase in energetic metabolism only registered at the medium pCO2 treatment may indicate that at higher pCO2 levels the capacity of larvae to restore their internal balance can be impaired. Our findings illustrate the need of using multiple approaches to explore the consequences of future pCO2 levels on organisms.

Efeito anti-inflamatório e antioxidante do extrato hidroalcóolico de Turnera subulata na colite ulcerativa induzida por ácido acético em ratos

Inflammatory bowel diseases is composed by a set of chronic and inflammatory disorders, among them is ulcerative colitis (UC). UC treatment is based on anti-inflammatory administration; however, this group of drugs clearly leads to development of undesirable side effects, what stimulate the search for new therapies alternatives. The aim of this study was to evaluate the effect of hydroalcholic Turnera subulata extract on acetic acid-induced acute UC in rats. UC was induced by 1 mL injection of 4% acetic acid via rectal in Wistar mouse. 42 animals were distributed among 6 experimental groups: Control, UC, Sulfasalazine 500 mg/Kg/day (SSZ), T. subulata 50mg/Kg/day (TS 50), T. subulata 100mg/Kg/day (TS 100), T. subulata 200mg/Kg/day (TS 200). Throughout the experiment, body weight, food and water ingestion was daily evaluated. At the end of the experiment, the animals were euthanized and a colon fragment was observed by macroscopic analysis. Colon fragments were also collected for microscopic analysis and oxidative stress evaluation. The means from each group was compared by ANOVA test with a significance level of 5% (p<0.05) using GraphPad Prism Software. As results, we can clearly observe that SSZ group had the greater body weight decrease among the groups throughout the experiments, 14.78%, as well as, the lowest food intake, 6.23 g of food/day. The animals treated with T. subulata extracts showed no important body weight loss when compared to control. UC group showed the highest tissue damage macroscope score, 6.5, while TS 50 showed the lowest tissue damage score: 1. Microscope evaluation showed the presence of edema, haemorraghia and ulceration in all group of animals, except for Control. Nevertheless, TS 50 showed the lowest inflammatory damage among all groups. Oxidative stress analysis revealed that T. subulata treatment modulate catalase and superoxide dismutase activity, we also observed a decrease in protein and lipid peroxidation in response to extract administration. Taken together, these results shows that T. subulata extract exerts anti-inflammatory and anti-oxidant effects on experimental UC.

Efeito anti-inflamatório e antioxidante do extrato hidroalcóolico de Turnera subulata na colite ulcerativa induzida por ácido acético em ratos

Inflammatory bowel diseases is composed by a set of chronic and inflammatory disorders, among them is ulcerative colitis (UC). UC treatment is based on anti-inflammatory administration; however, this group of drugs clearly leads to development of undesirable side effects, what stimulate the search for new therapies alternatives. The aim of this study was to evaluate the effect of hydroalcholic Turnera subulata extract on acetic acid-induced acute UC in rats. UC was induced by 1 mL injection of 4% acetic acid via rectal in Wistar mouse. 42 animals were distributed among 6 experimental groups: Control, UC, Sulfasalazine 500 mg/Kg/day (SSZ), T. subulata 50mg/Kg/day (TS 50), T. subulata 100mg/Kg/day (TS 100), T. subulata 200mg/Kg/day (TS 200). Throughout the experiment, body weight, food and water ingestion was daily evaluated. At the end of the experiment, the animals were euthanized and a colon fragment was observed by macroscopic analysis. Colon fragments were also collected for microscopic analysis and oxidative stress evaluation. The means from each group was compared by ANOVA test with a significance level of 5% (p<0.05) using GraphPad Prism Software. As results, we can clearly observe that SSZ group had the greater body weight decrease among the groups throughout the experiments, 14.78%, as well as, the lowest food intake, 6.23 g of food/day. The animals treated with T. subulata extracts showed no important body weight loss when compared to control. UC group showed the highest tissue damage macroscope score, 6.5, while TS 50 showed the lowest tissue damage score: 1. Microscope evaluation showed the presence of edema, haemorraghia and ulceration in all group of animals, except for Control. Nevertheless, TS 50 showed the lowest inflammatory damage among all groups. Oxidative stress analysis revealed that T. subulata treatment modulate catalase and superoxide dismutase activity, we also observed a decrease in protein and lipid peroxidation in response to extract administration. Taken together, these results shows that T. subulata extract exerts anti-inflammatory and anti-oxidant effects on experimental UC.

L'effet pathologique du monoxyde d'azote est diminué dans les myocytes cardiaques hypertrophiés

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

L'effet pathologique du monoxyde d'azote est diminué dans les myocytes cardiaques hypertrophiés

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.