Heme Impairs Prostaglandin E(2) and TGF-beta Production by Human Mononuclear Cells via Cu/Zn Superoxide Dismutase: Insight into the Pathogenesis of Severe Malaria

In many hemolytic disorders, such as malaria, the release of free heme has been involved in the triggering of oxidative stress and tissue damage. Patients presenting with severe forms of malaria commonly have impaired regulatory responses. Although intriguing, there is scarce data about the involvement of heme on the regulation of immune responses. In this study, we investigated the relation of free heme and the suppression of anti-inflammatory mediators such as PGE(2) and TGF-beta in human vivax malaria. Patients with severe disease presented higher hemolysis and higher plasma concentrations of Cu/Zn superoxide dismutase (SOD-1) and lower concentrations of PGE(2) and TGF-beta than those with mild disease. In addition, there was a positive correlation between SOD-1 concentrations and plasma levels of TNF-alpha. During antimalaria treatment, the concentrations of plasma SOD-1 reduced whereas PGE(2) and TGF-beta increased in the individuals severely ill. Using an in vitro model with human mononuclear cells, we demonstrated that the heme effect on the impairment of the production of PGE(2) and TGF-beta partially involves heme binding to CD14 and depends on the production of SOD-1. Aside from furthering the current knowledge about the pathogenesis of vivax malaria, the present results may represent a general mechanism for hemolytic diseases and could be useful for future studies of therapeutic approaches. The Journal of Immunology, 2010, 185: 1196-1204.

A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity

Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed TrP(32) residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H(2)O and H(2)(18)O, and analyzed by UV-Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp(32) residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp(32), partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo. (C) 2010 Elsevier Inc. All rights reserved.

Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide MOLECULAR INSIGHTS INTO THE GENOME INSTABILITY OF PEROXIREDOXIN-NULL STRAINS

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1 Delta tsa2 Delta) are more resistant to hydrogen peroxide than wildtype (WT) cells and consume it faster under fermentative conditions. Also, tsa1 Delta tsa2 Delta cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1 Delta tsa2 Delta cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu, Zn-superoxide dismutase (Sod1), whose expression and activity increased similar to 5- and 2-fold, respectively, in tsa1 Delta tsa2 Delta compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1 Delta tsa2 Delta cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of (14)C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1 Delta tsa2 Delta cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.

Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheria boydii

The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a saprophytic filamentous fungus recognized as a potent etiologic agent of a wide variety of infections in immunocompromised as well as in immunocompetent patients. Very little is known about the virulence factors expressed by this fungal pathogen. The present review provides an overview of recent discoveries related to the identification and biochemical characterization of potential virulence attributes produced by P. boydii, with special emphasis on surface and released molecules. These structures include polysaccharides (glucans), glycopeptides (peptidorhamnomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases, phosphatases and superoxide dismutase), which have been implicated in some fundamental cellular processes in P. boydii including growth, differentiation and interaction with host molecules. Elucidation of the structure of cell surface components as well as the secreted molecules, especially those that function as virulence determinants, is of great relevance to understand the pathogenic mechanisms of P. boydii.

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.

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.

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.

Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe-SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 angstrom and T. cruzi at 1.9 angstrom resolution, and that from P. falciparum at a higher resolution (2.0 angstrom) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity.

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.

Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)(2)] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38(MAPK)/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N`]copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

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.

Two New Ternary Complexes of Copper(II) with Tetracycline or Doxycycline and 1,10-Phenanthroline and Their Potential as Antitumoral: Cytotoxicity and DNA Cleavage

This paper reports on the synthesis and characterization of two new ternary copper(II) complexes: [Cu(doxy-cycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (1) and [Cu(tetracycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (2). These compounds exhibit a distorted tetragonal geometry around copper, which is coordinated to two bidentate ligands, 1,10-phenanthroline and tetracycline or doxycyline, a water molecule, and a perchlorate ion weakly bonded in the axial positions. In both compounds, copper(II) binds to tetracyclines`. via the oxygen of the hydroxyl group and oxygen of the amide group at ring A and to 1,10-phenanthroline via its two heterocyclic nitrogens. We have evaluated the binding of the new complexes to DNA, their capacity to cleave it, their cytotoxic activity, and uptake in tumoral cells. The complexes bind to DNA preferentially by the major groove, and then cleave its strands by an oxidative mechanism involving the generation of ROS. The cleavage of DNA was inhibited by radical inhibitors and/or trappers such as superoxide dismutase, DMSO, and the copper(I) chelator bathocuproine. The enzyme T4 DNA ligase was not able to relegate the products of DNA cleavage, which indicates that the cleavage does not occur via a hydrolytic mechanism. Both complexes present an expressive plasmid DNA cleavage activity generating single- and double-strand breaks, under mild reaction conditions, and even in the absence of any additional oxidant or reducing agent. In the same experimental conditions, [Cu(phen)(2)](2+) is approximately 100-fold less active than our complexes. These complexes are among the most potent DNA cleavage agents reported so far. Both complexes inhibit the growth of K562 cells With the IC(50) values of 1.93 and 2.59 mu mol L(-1) for compounds I and 2, respectively. The complexes are more active than the free ligands, and their cytotoxic activity correlates with intracellular copper concentration and the number of Cu-DNA adducts formed inside cells.

Fluorimetric study of the pro-oxidant activity of EUK8 in the presence of hydrogen peroxide

The catalase mimetic complex Mn(III)-salen chloride (EUK8) was found to be pro-oxidant under low hydrogen peroxide concentrations. The increase in the fluorescence rate of the probe 1,2,3-dihydrorhodamine (DHR) in solution, as well as the carbonyl content of human serum albumin were found to be maximum at H(2)O(2):EUK8 molar ratios ranging from 0 to 2, supporting previous findings regarding the mechanism of EUK8 catalase activity and the formation of highly oxidative Mn(V)-O(2-) species. This pro-oxidant effect is precluded by the presence of glutathione. Cytotoxicity to HeLa cells, as probed by increased rate of oxidation of intracellular DHR, was not observed. Our findings suggest that the combination of H(2)O(2) and EUK8 at specific molar ratios, in the absence of reductants/antioxidants, induces the oxidation of organic molecules. It is shown that the fluorimetric determination of pro-oxidant activity of metal complexes is more sensitive than the colorimetric quantification of protein carbonyl content. The implications of our findings with respect to the somewhat confusing results arising from in vivo studies of EUK8 and other Mn(III) anti-oxidant metal complexes are discussed.

Effects of Mate Tea (Ilex paraguariensis) Ingestion on mRNA Expression of Antioxidant Enzymes, Lipid Peroxidation, and Total Antioxidant Status in Healthy Young Women

The antioxidant activity of mate tea, the roasted product derived from yerba mate (Ilex paraguarienis), was observed in vitro and in animal models, but studies in humans are lacking. The aim of this study was to investigate the effects of mate tea supplementation on plasma susceptibility to oxidation and on antioxidant enzyme gene expression in healthy nonsmoking women, after acute or prolonged ingestion. We evaluated plasma total antioxidant status (TAS), the kinetics of diene conjugate generation, and thiobarbituric acid reactive substance (TBARS) contents in plasma, as well as mRNA levels of antioxidant gluthatione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). After the supplementation period with mate tea, lipid peroxidation was acutely lowered, an effect that was maintained after prolonged administration. Total antioxidant status and the level of antioxidant enzyme gene expression were also demonstrated after prolonged consumption. These results suggest that regular consumption of mate tea may increase antioxidant defense of the body by multiple mechanisms.