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.

The combined effect of copper and low pH on antioxidant defenses and biochemical parameters in neotropical fish pacu, Piaractus mesopotamicus (Holmberg, 1887)

Copper sulfate is widely used in aquaculture. Exposure to this compound can be harmful to fish, resulting in oxidative metabolism alterations and gill tissue damage. Pacu, Piaractus mesopotamicus, (wt = 43.4 +/- A 3.35 g) were distributed in experimental tanks (n = 10; 180 l) and exposed for 48 h to control (without copper addition), 0.4Cu (0.4 mg l(-1)), 0CupH (without copper addition, pH = 5.0) and 0.4CupH (0.4 mg l(-1), pH = 5.0). In liver and red muscle, the superoxide dismutase (SOD) was responsive to the increases in the aquatic copper. The plasmatic intermediary metabolites and hematological variables in the fish of group 0.4Cu were similar to those of the control group. Conversely, the exposure to 0.4CupH caused an increase in the plasmatic lactate, number of red blood cells (RBC) and hemoglobin (Hb). Plasmatic copper concentration [Cu(p)] increased in group 0.4Cu and 0.4CupH, which is higher in group 0.4CupH, suggests an effect of water pH on the absorbed copper. Exposure to 0.4Cu and 0.4CupH resulted in a reduction in the Na(+)/K(+)-ATPase activity and an increase in metallothionein (MT) in the gills. Exposure to 0CupH caused a decrease in glucose and pyruvate concentrations and an increase in RBC, Hb, and the branchial Na(+)/K(+)-ATPase activity. These responses suggest that the fish triggered mechanisms to revert the blood acidosis, save energy and increase the oxygen uptake. MT was an effective biomarker, responding to copper in different pHs and dissolved oxygen. Combined-factors caused more significant disturbance in the biomarkers than single-factors.

Macrophage suppression following phagocytosis of apoptotic neutrophils is mediated by the S100A9 calcium-binding protein

The clearance of apoptotic cells by phagocytes is a fundamental process during tissue remodeling and resolution of inflammation. In turn, the phagocytosis of apoptotic cells generates signals that suppress pro-inflammatory activation of macrophages. These events occur during the resolution phase of inflammation and therefore the malfunctioning of this process may lead to inflammation-related tissue damage. Here, we demonstrate that the calcium-binding protein S100A9, normally abundant in the cytoplasm of neutrophils and also released by apoptotic neutrophils, is involved in the suppression of macrophages after the uptake of apoptotic neutrophils. Both, spontaneous and induced production of inflammatory species (nitric oxide, hydrogen peroxide and TNF-alpha) as well as the phagocytic activity were inhibited when macrophages were in presence of apoptotic neutrophils, conditioned medium from neutrophil cultures or a peptide corresponding to the C-terminal region of S100A9 protein. On the other hand, macrophages kept in the conditioned medium of neutrophils that was previously depleted of S100A9 were shown to resume the activated status. Finally, we demonstrate that the calcium-binding property of S100A9 might play a role in the suppression process, since the stimulation of intracellular calcium release with ionomycin significantly reversed the effects of the uptake of apoptotic neutrophils in macrophages. In conclusion, we propose that S100A9 is a novel component of the regulatory mechanisms of inflammation, acting side-by-side with other suppressor factors generated upon ingestion of apoptotic cells. (C) 2009 Elsevier GmbH. All rights reserved.

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.

Angiotensin II modulates CD40 expression in vascular smooth muscle cells

The signalling pathway CD40/CD40L (CD40 ligand) plays an important role in atherosclerotic plaque formation and rupture. AngII (angiotensin II), which induces oxidative stress and inflammation, is also implicated in the progression of atherosclerosis. In the present study, we tested the hypothesis that AngII increases CD40/CD40L activity in vascular cells and that ROS (reactive oxygen species) are part of the signalling cascade that controls CD40/CD40L expression. Human CASMCs (coronary artery smooth muscle cells) in culture exposed to IL (interleukin)-1 beta or TNF-alpha (tumour necrosis factor-a) had increased superoxide generation and enhanced CD40 expression, detected by EPR (electron paramagnetic resonance) and immunoblotting respectively. Both phenomena were abolished by previous incubation with membrane-permeant antioxidants or cell transfection with P22(phox) antisense. AngII (50-200 nmol/l) induced an early and sustained increase in CD40 mRNA and protein expression in CASMCs, which was blocked by treatment with antioxidants. Increased CD40 expression led to enhanced activity of the pathway, as AngII-treated cells stimulated with recombinant CD40L released higher amounts of IL-8 and had increased COX-2 (cyclo-oxygenase-2) expression. We conclude that AngII stimulation of vascular cells leads to a ROS-dependent increase in CD40/CD40L signalling pathway activity. This phenomenon may be an important mechanism modulating the arterial injury observed in atherosclerosis-related vasculopathy.

Occupational airborne contamination in South Brazil: 2. Oxidative stress detected in the blood of workers of incineration of hospital residues

One of the most useful methods for elimination of solid residues of health services (SRHS) is incineration. However, it also provokes the emission of several hazardous air pollutants such as heavy metals, furans and dioxins, which produce reactive oxygen species and oxidative stress. The present study, which is parallel to an accompanied paper (Avila Jr. et al., this issue), investigated several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of vitamin E, lipoperoxidation = TBARS, reduced glutathione = GSH, oxidized glutathione = GSSG, and activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in three different groups (n = 20 each) exposed to airborne contamination associated with incineration of SRHS: workers directly (ca. 100 m from the incinerator) and indirectly exposed (residents living ca. 5 km the incineration site), and controls (non-exposed subjects). TBARS and GSSG levels were increased whilst GSH, TG and alpha-tocopherol contents were decreased in workers and residents compared to controls. Increased GST and CAT activities and decreased GPx activities were detected in exposed subjects compared to controls, while GR did not show any difference among the groups. In conclusion, subjects directly or indirectly exposed to SRHS are facing an oxidative insult and health risk regarding fly ashes contamination from SRHS incineration.

Total phenolic content and free radical scavenging activities of methanolic extract powders of tropical fruit residues

Methanolic extract powders of acerola, passion fruit and pineapple industrial residues, including pulp, seeds and peel, altogether (except for acerola) devoid of seeds, were screened for antioxidant capacity. The total phenolic contents (TPCs) of the extract powders were compared with their radical-scavenging activities (RSA) against both DPPH(center dot) and superoxide anion (O(2)(center dot-)) radicals, and their protective effect against liposome peroxidation, triggered by peroxyl radical. Lipid peroxidation was followed by the fluorescence decay of the probe, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C(11)-BODIPY(581/591)). The TPCs of acerola, passion fruit and pineapple extract powders were (94.6 +/- 7.4); (41.2 +/- 4.2) and (9.1 +/- 1.3) mg of gallic acid equivalents g(-1) of dry extract, respectively. Acerola showed the best RSA-DPPH(center dot) scores, whereas passion fruit was more protective on the RSA-O(2)(center dot-) system. Together with the protective effects against lipid peroxidation (rate of BODIPY decay) which, were similar for acerola and passion fruit extracts, these data suggest that the methanolic extracts of acerola and passion fruit residues may be useful as antioxidant supplements, particularly the acerola extract, due to its high phenolic content. (C) 2008 Elsevier Ltd. All rights reserved

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the Uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release. (C) 2009 Elsevier Inc. All rights reserved.

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier. (C) 2009 Elsevier Inc. All rights reserved.

Peroxymonocarbonate and Carbonate Radical Displace the Hydroxyl-like Oxidant in the Sod1 Peroxidase Activity under Physiological Conditions

Despite being one of the most important antioxidant defenses, Cu,Zn-superoxide dismutase (Sod1) has been frequently associated with harmful effects, including neurotoxicity. This toxicity has been attributed to immature forms of Sod1 and extraneous catalytic activities. Among these, the ability of Sod1 to function as a peroxidase may be particularly relevant because it is increased in bicarbonate buffer and produces the reactive carbonate radical. Despite many studies, how this radical forms remains unknown. To address this question, we systematically studied hSod1 peroxidase activity in the presence of nitrite, formate, and bicarbonate-carbon dioxide. Kinetic analyses of hydrogen peroxide consumption and of nitrite, formate, and bicarbonate-carbon dioxide oxidation showed that the Sod1-bound hydroxyl-like oxidant functions in the presence of nitrite and formate. In the presence of bicarbonate-carbon dioxide, this oxidant is replaced by peroxymonocarbonate, which is then reduced to the carbonate radical. Peroxymonocarbonate intermediacy was evidenced by (13)C NMR experiments showing line broadening of its peak in the presence of Zn,ZnSod1. In agreement, peroxymonocarbonate was docked into the hSod1 active site, where it interacted with the conserved Arg(143). Also, a reaction between peroxymonocarbonate and Cu(I)Sod1 was demonstrated by stopped-flow experiments. Kinetic simulations indicated that peroxymonocarbonate is produced during Sod1 turnover and not in bulk solution. In the presence of bicarbonate-carbon dioxide, sustained hSod1-mediated oxidations occurred with low steady-state concentrations of hydrogen peroxide (4-10 mu M). Thus, carbonate radical formation through peroxymonocarbonate may be a key event in Sod1-induced toxicity.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB`s cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.

Aminoacetone, a putative endogenous source of methylglyoxal, causes oxidative stress and death to insulin-producing RINm5f cells

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4+ ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O-2(center dot-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 mu M) and Fe3+ ion (100 mu M) addition to the cell cultures had no effect, whereas Cu2+ (5.0-100 mu M) significantly increased cell death. Supplementation of the AA- and Cu2+-containing culture medium with antioxidants, such as catalase (5.0 mu M), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu2+ ions (10-50 mu M) relative to control cultures. This may imply higher activity of antioxidant enzymes C, in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) Plus Cu2+ (100 mu M) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

Inhibition of myeloperoxidase-mediated protein nitration by tempol: Kinetics, mechanism, and implications

Despite the therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetra-methyl-1-piperidinyloxy) and related nitroxides as antioxidants, their effects on peroxidase-mediated protein tyrosine nitration remain unexplored. This posttranslational protein modification is a biomarker of nitric oxide-derived oxidants, and, relevantly, it parallels tissue injury in animal models of inflammation and is attenuated by tempol treatment. Here, we examine tempol effects on ribonuclease (RNase) nitration mediated by myeloperoxidase (MPO), a mammalian enzyme that plays a central role in various inflammatory processes.. Some experiments were also performed with horseradish peroxidase (HRP). We show that tempol efficiently inhibits peroxidase-mediated RNase nitration. For instance, 10 mu M tempol was able to inhibit by 90% the yield of 290 mu M 3-nitrotyrosine produced from 370 mu M RNase. The effect of tempol was not completely catalytic because part of it was consumed by recombination with RNase-tyrosyl radicals. The second-order rate constant of the reaction of tempol with MPO compound I and 11 were determined by stopped-flow kinetics as 3.3 x 10(6) and 2.6 x 10(4) M-1 s(-1), respectively (pH 7.4, 25 degrees C); the corresponding HRP constants were orders of magnitude smaller. Time-dependent hydrogen peroxide and nitrite consumption and oxygen production in the incubations were quantified experimentally and modeled by kinetic simulations. The results indicate that tempol inhibits peroxidase-mediated RNase nitration mainly because of its reaction with nitrogen dioxide to produce the oxammonium cation, which, in turn, recycles back to tempol by reacting with hydrogen peroxide and superoxide radical to produce oxygen and regenerate nitrite. The implications for nitroxide antioxidant mechanisms are discussed.

Tryptophan oxidation by singlet molecular oxygen [O-2 ((1)Delta(g))]: Mechanistic studies using O-18-labeled hydroperoxides, mass spectrometry, and light emission measurements

Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O-2 ((1)Delta(g))]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using O-18-labeled hydroperoxides ((WOOH)-O-18-O-18), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from (WOOH)-O-18-O-18 decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species.

Radical acetylation of 2`-deoxyguanosine and L-histidine coupled to the reaction of diacetyl with peroxynitrite in aerated medium

Diacetyl, like other alpha-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant (k(2) = 1.0 X 10(4) M-1 s(-1)) when compared with monocarbonyl substrates (k(2) < 10(3) M-1 s(-1)). Phosphate ions (100-500 MM) do not affect the rate of spontaneous peroxynitrite decay, but the H2PO4- anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum (a(N) = a(H) = 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2-nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either L-histidine or 2`-deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS"". These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.