INCREASED SARCOLEMMAL PERMEABILITY AS AN EARLY EVENT IN EXPERIMENTAL SEPTIC CARDIOMYOPATHY: A POTENTIAL ROLE FOR OXIDATIVE DAMAGE TO LIPIDS AND PROTEINS

This study describes increased sarcolemmal permeability and myofilamentar damage that occur together with lipid peroxidation and protein nitration in the myocardium in severe sepsis induced by cecal ligation and puncture. Male C57BL/6 mice were submitted to moderate and severe septic injury and sham operation. Using light and laser confocal microscopy, diffuse foci of myocytolysis associated with focal disruption of the actin/myosin contractile apparatus could be seen in hearts with severe septic injury. The myocardial expressions of the sarcomeric proteins myosin and actin were downregulated by both severe and moderate injuries. The detection of albumin staining in the cytoplasm of myocytes to evaluate sarcolemmal permeability provided evidence of severe and mild injury of the plasma membrane in hearts with severe and moderate septic injury, respectively. The administration of a superoxide scavenger caused marked reduction of sarcolemmal permeability, indicating the involvement of free radicals in its genesis. On electron microscopy, these changes were seen to correspond to spread blocks of a few myocytes with fragmentation and dissolution of myofibrils, intracellular edema, and, occasionally, rupture of the sarcolemma. In addition, oxidative damage to lipids, using anti-4-hydroxynonenal, an indicator of oxidative stress and disruption of plasma membrane lipids, and to proteins, using antinitrotyrosine, a stable biomarker of peroxynitrite-mediated protein nitration, was demonstrated. These findings make plausible the hypothesis that increased sarcolemmal permeability might be a primary event in myocardial injury in severe sepsis possibly due to oxidative damage to lipids and proteins that could precede phenotypic changes that characterize a septic cardiomyopathy.

Protective effects of Mangifera indica L extract (Vimang), and its major component mangiferin, on iron-induced oxidative damage to rat serum and liver

In vivo preventive effects of a Mangifera indica L extract (Vimang) or its major component mangiferin on iron overload injury have been studied in rats given respectively, 50, 100, 250 mg kg(-1) body weight of Vimang, or 40 mg kg(-1) body weight of mangiferin, for 7 days prior to, and for 7 days following the administration of toxic amounts of iron-dextran. Both Vimang or mangiferin treatment prevented iron overload in serum as well as liver oxidative stress, decreased serum and liver lipid peroxidation, serum GPx activity, and increased serum and liver GSH, serum SOD and the animals overall antioxidant condition. Serum iron concentration was decreased although at higher doses, Vimang tended to increase it; percent tranferrin saturation, liver weight/body mass ratios, liver iron content was decreased. Treatment increased serum iron-binding capacity and decreased serum levels of aspartate-amine transferase (ASAT) and alanine-amine transferase (ALAT), as well as the number of abnormal Kupffer cells in iron-loaded livers. It is suggested that besides acting as antioxidants, Vimang extract or its mangiferin component decrease liver iron by increasing its excretion. Complementing earlier in vitro results from our group, it appears possible to support the hypothesis that Vimang and mangiferin present therapeutically useful effects in iron overload related diseases. (C) 2007 Elsevier Ltd. All rights reserved.

Low level and sub-chronic exposure to methylmercury induces hypertension in rats: nitric oxide depletion and oxidative damage as possible mechanisms

Increased risk of hypertension after methylmercury (MeHg) exposure has been suggested. However, the underlying mechanisms are not well explored. In this paper, we have analyzed whether sub-chronic exposure to MeHg increases systolic blood pressure even at very low levels. In addition, we analyzed if the methylmercury-induced hypertension is associated with a decreased plasmatic nitric oxide levels and with a dysregulation of the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as the levels of MDA and glutathione. For this study, Wistar rats were treated with methylmercury chloride (100 mu g/kg per day) or vehicle. Total treatment time was 100 days. Malondialdehyde (MDA) and circulating NOx levels and superoxide dismutase (SOD) and catalase (CAT) activities were determined in plasma, whereas glutathione levels were determined in erythrocytes. Our results show that long-term treatment at a low level of MeHg affected systolic blood pressure, increasing and reducing the levels of plasmatic MDA and NOx, respectively. However, the activity of SOD did not decrease in the MeHg exposed group when compared to the control. We found a negative correlation between plasmatic nitrite/nitrate (NOx) levels and systolic blood pressure (r = -0.67; P = 0.001), and a positive correlation between MDA and systolic blood pressure (r = 0.61; P = 0.03), thus suggesting increased inhibition of NO formation with the increase of hypertension. In conclusion, long-term exposure to a low dose of MeHg increases the systolic pressure and is associated, at least in part, with increased production of ROS as judged by increased production of malondialdehyde and depressed NO availability.

Neuronal nicotinic receptors as new targets foramphetamine-induced oxidative damage and neurotoxicity

Amphetamine derivatives such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are drugs widely abused in a recreational context. This has led to concern because of the evidence that they are neurotoxic in animal models and cognitive impairments have been described in heavy abusers. The main targets of these drugs are plasmalemmal and vesicular monoamine transporters, leading to reverse transport and increased monoamine efflux to the synapse. As far as neurotoxicity is concerned, increased reactive oxygen species (ROS) production seems to be one of the main causes. Recent research has demonstrated that blockade of 7 nicotinic acetylcholine receptors (nAChR) inhibits METH- and MDMA-induced ROS production in striatal synaptosomes which is dependent on calcium and on NO-synthase activation. Moreover, 7 nAChR antagonists (methyllycaconitine and memantine) attenuated in vivo the neurotoxicity induced by METH and MDMA, and memantine prevented the cognitive impairment induced by these drugs. Radioligand binding experiments demonstrated that both drugs have affinity to 7 and heteromeric nAChR, with MDMA showing lower Ki values, while fluorescence calcium experiments indicated that MDMA behaves as a partial agonist on 7 and as an antagonist on heteromeric nAChR. Sustained Ca increase led to calpain and caspase-3 activation. In addition, modulatory effects of MDMA on 7 and heteromeric nAChR populations have been found.

Glutathione peroxidase induction protects Saccharomyces cerevisiae sod1deltasod2delta double mutants against oxidative damage

Saccharomyces cerevisiae mutants deficient in superoxide dismutase genes (sod1delta, sod2delta and the double mutant) were subjected to H2O2 stress in the stationary phase. The highest sensitivity was observed in the sod2delta mutant, while the sod1deltasod2delta double mutant was not sensitive. Sod mutants had lower catalase activity (44%) than wild-type cells, independent of H2O2 stress. Untreated cells of sod1deltasod2delta double mutants showed increased glutathione peroxidase activity (126%), while sod1delta had lower activity (77%) than the wild type. Glutathione levels in sod1delta were increased (200-260%) after exposure to various H2O2 concentrations. In addition, the highest malondialdehyde levels could be observed without H2O2 treatment in sod1delta (167%) and sod2delta (225%) mutants. In contrast, the level of malondialdehyde in the sod1deltasod2delta double mutant was indistinguishable from that of the wild type. These results suggest that resistance to H2O2 by sod1deltasod2delta cells depends on the induction of glutathione peroxidase and is independent of catalase, and that glutathione is a primary antioxidant in the defense against H2O2 in stationary phase sod1delta mutants.

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

Hypermethioninemia provokes oxidative damage and histological changes in liver of rats

In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia. (C) 2009 Elsevier Masson SAS. All rights reserved.

5-Lipoxygenase plays a role in the control of parasite burden and contributes to oxidative damage of erythrocytes in murine Chagas` disease

Chagas` disease is accompanied by severe anemia and oxidative stress, which may contribute to mortality. In this study, we investigated the role of 5-lipoxygenase (5-LO) in the control of parasitism and anemia associated with oxidative damage of erythrocytes in experimental Trypanosoma cruzi infection. Wild-type C57BL/6, 129Sv mice treated or not with nordihydroguaiaretic acid (NDGA, 5-LO inhibitor), mice lacking the 5-LO enzyme gene (5-LO(-/-)) and inducible nitric oxide synthase gene (iNOS(-/-)) were infected with the Y strain of T cruzi. impairment of 5-LO resulted in increased numbers of trypomastigote forms in the blood and amastigote forms in the heart of infected mice. We assessed oxidative stress in erythrocytes by measuring oxygen uptake, induction time and chemiluminescence following treatment with tert-butyl hydroperoxide (TBH). Our results show that 5-LO metabolites increased lipid peroxidation levels in erythrocytes during the early phase of murine T cruzi infection. NDGA treatment reduced oxidative damage of erythrocytes in C57BL/6 T cruzi-infected mice but not in C57BL/6 iNOS-/- infected mice, showing that the action of NDGA is dependent on endogenous nitric oxide (NO). In addition, our results show that 5-LO metabolites do not participate directly in the development of anemia in infected mice. We conclude that 5-LO products may not only play a major role in controlling heart tissue parasitism, i.e., host resistance to acute infection with T cruzi in vivo, but in the event of an infection also play an important part in erythrocyte oxidative stress, an NO-dependent effect. (C) 2009 Elsevier B.V. All rights reserved.

Superoxide radical protects liposome-contained cytochrome c against oxidative damage promoted by peroxynitrite and free radicals

The effects of nitrosative species on cyt c structure and peroxidase activity were investigated here in the presence of O(2)(center dot-) and anionic and zwitterionic vesicles. Nitrosative species were generated by 3-morpholinesydnonymine (SIN1) decomposition, using cyt c heme iron and/or molecular oxygen as electron acceptor. Far-and near-UV CD spectra of SIN1-treated cyt c revealed respectively a slight decrease of a-helix content (from 39 to 34%) and changes in the tryptophan structure accompanied by increased fluorescence. The Soret CD spectra displayed a significant decrease of the positive signal at 403 nm. EPR spectra revealed the presence of a low-spin cyt c form (S = 1/2) with g(1) = 2.736, g(2) = 2.465, and g(3) = 2.058 after incubation with SIN1. These data suggest that the concomitant presence of NO(center dot) and O(2)(center dot-) generated from dissolved oxygen, in a system containing cyt c and liposomes, promotes chemical and conformational modi. cations in cyt c, resulting in a hypothetical bis-histidine hexacoordinated heme iron. We also show that, paradoxically, O(2)(center dot-) prevents not only membrane lipoperoxidation by peroxide-derived radicals but also oxidation of cyt c itself due to the ability of O(2)(center dot-) to reduce heme iron. Finally, lipoperoxidation measurements showed that, although it is a more efficient peroxidase, SIN1-treated cyt c is not more effective than native cyt c in promoting damage to anionic liposomes in the presence of tert-ButylOOH, probably due to loss of affinity with negatively charged lipids. (C) 2009 Elsevier Inc. All rights reserved.

Increased SOD1 association with chromatin, DNA damage, p53 activation, and apoptosis in a cellular model of SOD1-linked ALS

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multifactorial and remain unclear. Here we examined DNA damage;p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93) --> Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis. (C) 2010 Elsevier B.V. All rights reserved.

Trypanocidal action of eupomatenoid-5 is related to mitochondrion dysfunction and oxidative damage in Trypanosoma cruzi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Nitric oxide reduces oxidative damage induced by water stress in sunflower plants

Drought is one of the main environmental constraints that can reduce plant yield. Nitric oxide (NO) is a signal molecule involved in plant responses to several environmental stresses. The objective of this study was to investigate the cytoprotective effect of a single foliar application of 0, 1, 10 or 100 µM of the NO donor sodium nitroprusside (SNP) in sunflower plants under water stress. Water stressed plants treated with 1μM SNP showed an increase in the relative water content compared with 0 μM SNP. Drought reduced the shoot dry weight but SNP applications did not result in alleviation of drought effects. Neither drought nor water stress plus SNP applications altered the content of photosynthetic pigments. Stomatal conductance was reduced by drought and this reduction was accompanied by a significant reduction in intercellular CO2 concentration and photosynthesis. Treatment with SNP did not reverse the effect of drought on the gas exchange characteristics. Drought increased the level of malondialdehyde (MDA) and proline and reduced pirogalol peroxidase (PG-POD) activity, but did not affect the activity of superoxide dismutase (SOD). When the water stressed plants were treated with 10 μM SNP, the activity of PG-POD and the content of proline were increased and the level of MDA was decreased. The results show that the adverse effects of water stress on sunflower plants are dependent on the external NO concentration. The action of NO may be explained by its ability to increase the levels of antioxidant compounds and the activity of ROS-scavenging enzymes.

Evaluation of genotoxicity and oxidative damage in painters exposed to low levels of toluene

Toluene is an organic solvent used in numerous processes and products, including industrial paints. Toluene neurotoxicity and reproductive toxicity are well recognized: however, its genotoxicity is still under discussion, and toluene is not classified as a carcinogenic solvent. Using the comet assay and the micronucleus test for detection of possible genotoxic effects of toluene, we monitored industrial painters from Rio Grande do Sul, Brazil. The putative involvement of oxidative stress in genetic damage and the influences of age, smoking, alcohol consumption, and exposure time were also assessed. Although all biomarkers of toluene exposure were below the biological exposure limits, painters presented significantly higher DNA damage (comet assay) than the control group; however, in the micronucleus assay, no significant difference was observed. Painters also showed alterations in hepatic enzymes and albumin levels, as well as oxidative damage, suggesting the involvement of oxidative stress. According to multiple linear regression analysis, blood toluene levels may account for the increased DNA damage in painters. In summary, this study showed that low levels of toluene exposure can cause genetic damage, and this is related to oxidative stress, age, and time of exposure. (C) 2012 Elsevier B.V. All rights reserved.

Phytoremediation by poplar: polyphenols polyamines and oxidative damage in the response to heavy metals in in vitro and in hydroponic cultures.

Poplar is considered a good candidate for phytoremediation, but its tolerance to heavy metals has not been fully investigated yet. In the present work, two different culture systems (in vitro and aeroponic/hydroponic) and two different stress tolerant clones of Populus alba (AL22 and Villafranca) were investigated for their total polyphenol and flavonoid content, individual phenolic compounds, polyamine, lipid peroxidation and hydrogen peroxide levels in response to Cu. In AL22 poplar plants cultured in vitro in the presence or absence of 50 μM Cu, total leaves polyphenol and flavonoid content was higher in treated samples than in controls but unaltered in the roots. Equally the same clone, grown under aeroponic conditions and hydroponically treated for 72 h with 100 μM Cu, displayed increased amount of polyphenols and flavonoids in the leaves, in particular chlorogenic acid and quercetin, and no differences in the roots. In exudates from treated roots total polyphenols and flavonoids, in particular catechin and epicatechin, were more abundant than in controls. Polyamine levels show an increase in conjugated putrescine (Put) and spermidine (Spd) was found. In the Villafranca clone, treated with 100 μM Cu for 6, 24 and 72 h, the pattern of polyphenol and flavonoid accumulation was the same as in AL22; in Cu-treated roots these compounds decreased compared with controls while they increased in root exudates. Free polyamine levels rose at 24 and 72 h while only conjugated Put increased at 24 h. Cu-treated Villafranca plants exhibited a higher malondialdehyde production than controls indicative of membrane lipid peroxidation and, therefore, oxidative stress. An in vitro experiment was carried to investigate the antioxidant effect of the polyamine spermidine (Spd). Exogenous Spd, supplied together with 100 μM Cu, reduced the accumulation of polyphenols and flavonoids, MDA and hydrogen peroxide induced by Cu.

Induction of haem oxygenase-1 causes cortical non-haem iron increase in experimental pneumococcal meningitis: evidence that concomitant ferritin up-regulation prevents iron-induced oxidative damage

Desferrioxamine inhibits cortical necrosis in neonatal rats with experimental pneumococcal meningitis, suggesting that iron-induced oxidative damage might be responsible for neuronal damage. We therefore examined the spatial and temporal profile of changes in cortical iron and iron homeostatic proteins during pneumococcal meningitis. Infection was associated with a steady and global increase of non-haem iron in the cortex, particularly in neuronal cell bodies of layer II and V, and in capillary endothelial cells. The non-haem iron increase was associated with induction of haem oxygenase (HO)-1 in neurones, microglia and capillary endothelial cells, whereas HO-2 levels remained unchanged, suggesting that the non-haem iron increase might be the result of HO-1-mediated haem degradation. Indeed, treatment with the haem oxygenase inhibitor tin protoporphyrin (which completely blocked the accumulation of bilirubin detected in HO-1-positive cells) completely prevented the infection-associated non-haem iron increase. The same cells also displayed markedly increased ferritin staining, the increase of which occurred independently of HO activity. At the same time, no increase in DNA/RNA oxidation was observed in infected animals (as assessed by in situ detection of 8-hydroxy[deoxy]guanosine), strongly suggesting that ferritin up-regulation protected the brain from iron-induced oxidative damage. Thus, although pneumococcal meningitis leads to an increase of cortical non-haem iron, protective mechanisms up-regulated in parallel prevent iron-induced oxidative damage. Cortical damage does not appear to be a direct consequence of increased iron, therefore.