Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Oxidative stress and growth-regulating intracellular signaling pathways in cardiac myocytes

The toxic effects of oxidative stress on cells (including cardiac myocytes, the contractile cells of the heart) are well known. However, an increasing body of evidence has suggested that increased production of reactive oxygen species (ROS) promotes cardiac myocyte growth. Thus, ROS may be 'second messenger' molecules in their own right, and growth-promoting neurohumoral agonists might exert their effects by stimulating production of ROS. The authors review the principal growth-promoting intracellular signaling pathways that are activated by ROS in cardiac myocytes, namely the mitogen-activated protein kinase cascades (extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinases, and p38-mitogen-activated protein kinases) and the phosphoinositide 3-kinase/protein kinase B (Akt) pathway. Possible mechanisms are discussed by which these pathways are activated by ROS, including the oxidation of active site cysteinyl residues of protein and lipid phosphatases with their consequent inactivation, the potential involvement of protein kinase C or the apoptosis signal-regulating kinase 1, and the current models for the activation of the guanine nucleotide binding protein Ras.

Baroreflex sensitivity and oxidative stress in the LDL receptor knockout mice

This study alms at observing the effect of low-density lipoprotein (LDL) receptor deficiency in cholesterol blood levels, baroreflex sensitivity (BRS), nitric oxide (NO) bioavailability, and oxidative stress. The lack of LDL receptors in mice significantly increased the cholesterol blood levels (179 +/- 35 vs. 109 +/- 13 mg/dL) in the knockout (KO) mice compared to control. There was no difference in basal mean arterial pressure and heart rate between the groups. However, in KO mice the BRS was significantly attenuated and the antioxidant enzyme activities, measured in erythrocytes and heart, were significantly decreased. On the other hand, the oxidative damage measured by chemiluminescence and carbonyls was increased, while total plasma nitrate levels were lower in KO mice, indicating a decrease in NO availability. In conclusion, these results indicate that the lack of LDL receptor increased cholesterol blood levels, induced oxidative stress and decreased BRS. (C) 2008 Elsevier GmbH. All rights reserved.

Homocysteine Thiolactone Induces Cardiac Dysfunction: Role of Oxidative Stress

This study investigates the cardiac functioning in male Wistar rats after treatments with methionine and homocysteine thiolactone (HcyT). The rats were distributed into 3 groups and treated for 8 weeks. Group I was the control (CO) group, given water, group II was treated with methionine, and group III with HcyT (100 mg/kg). Morphometric and functional cardiac parameters were evaluated by echocardiography. Superoxide dismutase (SOD), catalase, and glutathione S-transferase activities, chemiluminescence, thiobarbituric acid reactive substances, and immunocontent were measured in the myocardium. Hyperhomocysteinemia was observed in rats submitted to the both treatments. The results showed diastolic function was compromised in HcyT group, seen by the increase of E/A (peak velocity of early (E) and late (A) diastolic filling) ratio, decrease in deceleration time of E wave and left ventricular isovolumic relaxation time. Myocardial performance index was increased in HcyT group and was found associated with increased SOD immunocontent. HcyT group demonstrated an increase in SOD, catalase, and glutatione S-transferase activity, and chemiluminescence and thiobarbituric acid reactive substances. Overall, these results indicated that HcyT induces a cardiac dysfunction and could be associated with oxidative stress increase in the myocardium.

Dehydroepiandrosterone protects against oxidative stress-induced endothelial dysfunction in ovariectomized rats

Cardiovascular disease is less frequent in premenopausal women than in age-matched men or postmenopausal women. Moreover, the marked age-related decline in serum dehydroepiandrosterone (DHEA) level has been associated to cardiovascular disease. The aim of this study was to evaluate the effects of DHEA treatment on vascular function in ovariectomized rats. At 8 weeks of age, female Wistar rats were ovariectomized (OVX) or sham (SHAM) operated and 8 weeks after surgery both groups were treated with vehicle or DHEA (10 mg kg-1 week-1) for 3 weeks. Aortic rings were used to evaluate the vasoconstrictor response to phenylephrine (PHE) and the relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP). Tissue reactive oxygen species (ROS) production and SOD, NADPH oxidase and eNOS protein expression were analysed. PHE-induced contraction was increased in aortic rings from OVX compared to SHAM, associated with a reduction in NO bioavailability. Furthermore, the relaxation induced by ACh was reduced in arteries from OVX, while SNP relaxation did not change. The incubation of aortic rings with SOD or apocynin restored the enhanced PHE-contraction and the impaired ACh-relaxation only in OVX. DHEA treatment corrected the increased PHE contraction and the impaired ACh-induced relaxation observed in OVX by an increment in NO bioavailability and decrease in ROS production. Besides, DHEA treatment restores the reduced Cu/Zn-SOD protein expression and eNOS phosphorylation and the increased NADPH oxidase protein expression in the aorta of OVX rats. The present results suggest an important action of DHEA, improving endothelial function in OVX rats by acting as an antioxidant and enhancing the NO bioavailability.

Oxidative stress and inflammatory mediators contribute to endothelial dysfunction in high-fat diet-induced obesity in mice

Objective We investigated the effects of high-fat diet-induced obesity on vascular proinflammatory factors and oxidative stress on endothelium-dependent relaxation of the aorta. Methods Female Swiss mice were submitted to a high-fat diet for 16 weeks. At the end of the experimental period, we evaluated blood pressure, relaxation in response to acetylcholine in aortic rings in the absence and the presence of the superoxide anion scavenger, superoxide dismutase (SOD, 150 U/ml), and the nuclear factor (NF)-kappa B inhibitor, sodium salicylate (5 mmol/l). Aortic protein expression of endothelial nitric oxide synthase, Cu/Zn-SOD, NF-kappa B, I kappa B-alpha, and proinflammatory cytokines were also evaluated. Results Obese mice presented higher systolic and diastolic blood pressure than control mice (P<0.05). The relaxation of aortas to acetylcholine, but not to sodium nitroprusside, was significantly decreased in obese mice and was corrected by both SOD and sodium salicylate (P<0.05). The protein expression of endothelial nitric oxide synthase and Cu/Zn-SOD was significantly decreased in aorta from obese mice (P<0.05). Total p65 NF-kappa B subunit protein expression was not affected by obesity, but the protein expression of NF-kappa B inhibitor I kappa B-alpha was lower in aorta from obese mice (P<0.05). There were no significant differences in the interleukin (IL)-1 beta and IL-6 protein expression between groups. In contrast, the expression of TNF-alpha was significantly increased in aortas from obese mice. Conclusion Our resultssuggest that the reducedantioxidant defense and the local NF-kappa B pathway play an important role in the impairment of endothelium-dependent relaxation in aorta from obese mice. J Hypertens 28: 2111-2119 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Testosterone suppresses oxidative stress in human neutrophils

The in vitro effect of testosterone on human neutrophil function was investigated. Blood neutrophils from healthy male subjects were isolated and treated with 10 nM, 0.1 and 10 mu M testosterone for 24 h. As compared with untreated cells, the testosterone treatment produced a significant decrease of superoxide production as indicated by the measurement of extra- and intracellular superoxide content. An increment in the production of nitric oxide was observed at 0.1 and 10 mu M testosterone concentrations, whereas no effect was found for 10 nM. Intracellular calcium mobilization was significantly increased at 10 nM, whereas it was reduced at 10 mu M testosterone. There was an increase in phagocytic capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with testosterone at 10 mu M. Glutathione reductase activity was increased by testosterone treatment, whereas no effect was observed in other antioxidant enzyme activities. An increase in the content of thiol groups was observed at all testosterone concentrations. Lipid peroxidation in neutrophils evaluated by levels of TBARS was decreased at 10 nM and 0.1 mu M testosterone. These results indicate the antioxidant properties of testosterone in neutrophils as suggested by reduction of superoxide anion production, and lipid peroxidation, and by the increase in nitric oxide production, glutathione reductase activity and the content of thiol groups. Therefore, the plasma levels of testosterone are important regulators of neutrophil function and so of the inflammatory response. Copyright (C) 2010 John Wiley & Sons, Ltd.

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

Peripheral Oxidative Stress Biomarkers in Mild Cognitive Impairment and Alzheimer`s Disease

Oxidative stress has been associated with normal aging and Alzheimer`s disease (AD). However, little is known about oxidative stress in mild cognitive impairment (MCI) patients who present a high risk for developing AD. The aim of this study was to investigate plasma production of the lipid peroxidation marker, malonaldehyde (MDA) and to determine, in erythrocytes, the enzymatic antioxidant activity of catalase, glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) in 33 individuals with MCI, 29 with mild probable AD and 26 healthy aged subjects. GR/GPx activity ratio was calculated to better assess antioxidant defenses. The relationship between oxidative stress and cognitive performance was also evaluated by the Mini Mental State Examination (MMSE). AD patients showed higher MDA levels than both MCI and healthy elderly subjects. MCI subjects also exhibited higher MDA levels compared to controls. Catalase and GPx activity were similar in MCI and healthy individuals but higher in AD. GR activity was lower in MCI and AD patients than in healthy aged subjects. Additionally, GR/GPx ratio was higher in healthy aged subjects, intermediate in MCI and lower in AD patients. No differences in GST activity were detected among the groups. MMSE was negatively associated with MDA levels (r = -0.31, p = 0.028) and positively correlated with GR/GPx ratio in AD patients (r = 0.68, p < 0.001). MDA levels were also negatively correlated to GR/GPx ratio (r = -0.31, p = 0.029) in the AD group. These results suggest that high lipid peroxidation and decreased antioxidant defenses may be present early in cognitive disorders.

Effects of cyclooxygenase inhibitors on parasite burden, anemia and oxidative stress in murine Trypanosoma cruzi infection

Prostaglandins are known to be produced by macrophages when challenged with Trypanosoma cruzi, the etiological agent of Chagas` disease. It is not known whether these lipid mediators play a role in oxidative stress in host defenses against this important protozoan parasite. In this study, we demonstrated that inducible cyclooxygenase-mediated prostaglandin production is a key chemical mediator in the control of parasite burden and erythrocyte oxidative stress during T. cruzi infection in C57BL/6 and BALB/c mice, prototype hosts for the study of resistance and susceptibility in murine Chagas` disease. The results suggested the existence of at least two mechanisms of oxidative stress, dependent or independent with regard to the nitric oxide and cyclooxygenase pathway, where one or the other is more evident depending on the mouse strain.

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

Leishmania amazonensis META2 protein confers protection against heat shock and oxidative stress

The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock. (C) 2010 Elsevier Inc. All rights reserved.

Effects of aerobic exercise on the blood pressure, oxidative stress and eNOS gene polymorphism in pre-hypertensive older people

The polymorphisms of endothelial nitric oxide synthase (eNOS) are associated with reduced eNOS activity. Aerobic exercise training (AEX) may influence resting nitric oxide (NO) production, oxidative stress and blood pressure. The purpose of this study was to investigate the effect of AEX on the relationship among blood pressure, eNOS gene polymorphism and oxidative stress in pre-hypertensive older people. 118 pre-hypertensive subjects (59 +/- A 6 years) had blood samples collected after a 12 h overnight fast for assessing plasma NO metabolites (NOx) assays, thiobarbituric acid reactive substances (T-BARS) and superoxide dismutase activity (ecSOD). eNOS polymorphism (T-786C and G-894T) was done by standard PCR methods. All people were divided according to the genotype results (G1: TT/GG, G2: TT/GT + TT, G3: TC + CC/GG, G4: TC + CC/GT + TT). All parameters were measured before and after 6 months of AEX (70% of VO(2 max)). At baseline, no difference was found in systolic and diastolic blood pressure, ecSOD and T-BARS activity. Plasma NOx levels were significantly different between G1 (19 +/- A 1 mu M) and G4 (14.2 +/- A 0.6 mu M) and between G2 (20.1 +/- A 1.7 mu M) and G4 (14.2 +/- A 0.6 mu M). Therefore, reduced NOx concentration in G4 group occurred only when the polymorphisms were associated, suggesting that these results are more related to genetic factors than NO-scavenging effect. After AEX, the G4 increased NOx values (17.2 +/- A 1.2 mu M) and decreased blood pressure. G1, G3 and G4 decreased T-BARS levels. These results suggest the AEX can modulate the NOx concentration, eNOS activity and the relationship among eNOS gene polymorphism, oxidative stress and blood pressure especially in C (T-786C) and T (G-894T) allele carriers.

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H(2)O(2) and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

Oxidative stress protection of Trypanosomes requires selenophosphate synthase

Selenoproteins are characterized by the incorporation of at least one amino acid selenocysteine (Sec-U) encoded by in-frame UGA stop codons. These proteins, as well as the components of the Sec synthesis pathway, are present in members of the bacteria, archaea and eukaryote domains. Although not a ubiquitous pathway in all organisms, it was also identified in several protozoa, including the Kinetoplastida. Genetic evidence has indicated that the pathway is non-essential to the survival of Trypanosoma growing in non-stressed conditions. By analyzing the effects of RNA interference of the Trypanosoma brucei selenophosphate synthetase SPS2, we found a requirement under sub-optimal growth conditions. The present work shows that SPS2 is involved in oxidative stress protection of the parasite and its absence severely hampers the parasite survival in the presence of an oxidizing environment that results in an apoptotic-like phenotype and cell death. (C) 2011 Elsevier B.V. All rights reserved.