Oxidatively modified low-density lipoprotein (oxLDL), generated by treatment of LDL with a myeloperoxidase/hydrogen peroxide/nitrite reaction system, elicits oxidative stress in bovine aortic endothelial cells or human umbilical vein endothelial cells, as judged by an increase in 2´,7´ - dichlorofluorescein fluorescence and elevated levels of carbonylated, nitrated, and 2-hydroxynonenal-coupled proteins. These effects were sensitive to apocynin, indicating involvement of NADPH oxidase. Polypeptides of 170 kDa and 140 kDa, carbonylated upon exposure of cells to oxLDL, were identified by immunoprecipitation as EGF receptor and endothelial ·NO synthase, respectively. Immunocytochemical visualization by confocal microscopy revealed highest levels of modified proteins in the perinuclear region, co-localizing with the NADPH oxidase isoform Nox4. Exposure of endothelial cells to oxLDL led to modulation of the expression levels of ·NO synthases; the endothelial isoform (eNOS) was down-regulated via proteasomal degradation, whereas the inducible isoform (iNOS) was up-regulated in an enzymatically active state. eNOS protein was found to be carbonylated as well as nitrated upon exposure of cells to oxLDL. iNOS contributed to the generation of modified proteins as judged by the effects of the selective inhibitor l-NIO. Pretreatment of the cells with (-)-epicatechin, a dietary polyphenol, prevented these hallmarks of oxidative stress elicited by either oxLDL or angiotensin II in an apocynin-like fashion. (-)-Epicatechin and their metabolites inhibited endothelial NADPH oxidase activity in both intact cells and cell homogenates. (-)-Epicatechin is metabolized to methylated derivatives by COMT, which act as apocynin-like inhibitors on Nox. It is proposed that inhibition of endothelial NADPH oxidase is the molecular mechanism underlying the rise in plasma levels of NO and serves as a basis for antiinflammatory actions of this polyphenol on the vascular endothelium.
We have previously shown that hypercholesterolemic LDL receptor knockout (k/o) mice mitochondria possess a low antioxidant capacity due to a large consumption of reducing equivalents from NADPH to sustain high rates of lipogenesis ( Faseb J. 2005;19:278-280). In this work, this k/o mice mitochondrial oxidative stress was further characterized by showing a lower mitochondrial GSH/GSSG ratio and a higher liver content of protein carbonyls as compared to controls. No differences in the activities of the antioxidant enzyme system glutathione reductase/peroxidase were observed. Exogenous catalase prevented the spontaneous oxidation of endogenous NADPH in rotenone poisoned mitochondria isolated from k/o mice indicating that this oxidation is mediated by mitochondrially generated H2O2. The higher rate of ROS production was also prevented by the presence of exogenous isocitrate that maintains NADP fully reduced. The hypothesis that high rates of lipogenesis decreases NADPH/NADP+ ratio due to a decreased content of NADP-linked substrates was supported by two observations: (i) oxygen consumption supported by endogenous NAD(P)-linked substrates was slower in k/o than in control mitochondria, but was similar in the presence of exogenous isocitrate; ii) oral administration of 65 mM sodium citrate/citric acid, during one week, partially restored both the rate of oxygen consumption supported by NAD(P)-linked substrates and the mitochondrial capacity to sustain reduced NADPH. In conclusion, the data demonstrate that the mitochondrial oxidative stress in hypercholesterolemic LDL receptor knockout mice is the result of a low content of mitochondrial NADPH-linked substrates in the intact animal that can be, at least in part, replenished by oral administration of citrate. Supported by FAPESP, CNPq and FAEPEX/UNICAMP.
The objective of this study was to define the role that NADPH oxidase or myeloperoxidase (MPO) plays in host defense in lymphopenic mice. To do this we crossed gp91 phox deficient (gp91 ko) or MPO ko mice with mice deficient in recombinase activating gene-1 (RAG ko) to generate lymphopenic offspring deficient in either NADPH oxidase or MPO. We found that neither gp91 ko, MPO ko mice nor RAG ko mice developed spontaneous infections when raised under specific pathogen free (SPF) conditions and all animals lived normal life spans. In contrast, gp91xRAG double deficient (gp91xRAG DKO) but not MPOxRAG DKO mice developed spontaneous multi-organ bacterial and fungal infections early in life and lived only a few months. Addition of antibiotics to the drinking water attenuated some of the infections and increased modestly the survival of these mice. The enhanced mortality of the gp91xRAG DKO mice was not due to defects in inflammatory cell recruitment or NO synthase (iNOS) activity as the number of elicited PMNs and macrophages as well as PMN and macrophage-derived production of nitric oxide in these mice were similar to wild type, gp91 ko or RAG ko mice. Taken together, our data suggest that that NADPH oxidase but not MPO nor iNOS is required for host defense in lymphopenic mice and that lymphocytes and NADPH oxidase may compensate for each others deficiency in providing resistance to spontaneous infections (Supported by DK64023 and the Yamanouchi USA Foundation).
| 11:45 - 12:10 | Oral Presentation | 06.Inflammation, Oxidants and Myeloperoxidase in Disease
Oxidant Production by Myeloperoxidase
Kettle, AJ 1(*); Chapman, AL 1; Senthilmohan, R 1; Harwood, DT 1; Peskin, A 1
Myeloperoxidase and eosinophil peroxidase use hydrogen peroxide to oxidize halides and thiocyanate to their respective hypohalous acids. Myeloperoxidase produces mainly hypochlorous acid and hypothiocyanite. Hypobromous acid and hypothiocyanite are the major products of eosinophil peroxidase. We have investigated the ability of myeloperoxidase to produce hypobromous acid in the presence of physiological concentrations of chloride and bromide. In accord with previous studies, between pH 5 and 7, myeloperoxidase converted about 90% of available hydrogen peroxide to hypochlorous acid and the remainder to hypobromous acid. Above pH 7, there was an abrupt rise in the yield of hypobromous acid. At pH 7.8 it accounted for 40% of the hydrogen peroxide. Bromide, at physiological concentrations, promoted a dramatic increase in bromination of human serum albumin catalyzed by myeloperoxidase. The level of 3-bromotyrosine increased to 16-fold greater than that for 3-chlorotyrosine. In either reaction system, reagent hypochlorous acid did not oxidize bromide to hypobromous acid. Therefore, transhalogenation was not a substantive route to either taurine bromamine or 3-bromotyrosine. We are currently investigating the mechanism by which myeloperoxidase oxidizes bromide in the presence of a vast excess of chloride. It is likely that myeloperoxidase forms a chlorinating intermediate that is responsible for the formation of hypobromous acid. The existence of this intermediate will have important ramifications for oxidant production within neutrophil phagosomes where bacteria are killed. It will also influence oxidative reactions during inflammation. We conclude that hypobromous acid can be a major oxidant produced by myeloperoxidase and that 3-bromotyrosine cannot be used as a specific biomarker for eosinophil peroxidase.
As intracellular parasite M. tuberculosis depends on an efficient antioxidant system to survive in the oxidant environment of phagocytes. Apart from a heme-type catalase/peroxidase it contains several peroxiredoxin-type peroxidases. Two of the latter, AhpC and TPx, are reduced by thioredoxins. AhpC and more efficiently TPx reduce H2O2, organic hydroperoxides and peroxynitrite. Mechanistically, AhpC is a typical 2-Cys-peroxiredoxin, while TPx acts as a 1-Cys-peroxiredoxin. The system for detoxification of organic hydroperoxides and peroxynitrite in Mycobacteria is thus dominated by thioredoxin, whereas in the mammalian host it is achieved by the glutathione pathway. Furthermore, thioredoxin reductase of M.tuberculosis belongs to a subfamily that substantially differs from the homologous mammalian thioredoxin reductases which, in contrast to the bacterial ones, work with selenium catalysis. This fundamental difference in the defence systems opens up the therapeutic perspective to selectively inhibit the mycobacterial system to strengthen the innate immune response without affecting the antioxidant defence of the host. On this basis, strategies to develop novel tuberculostatics will be discussed.
Superoxide dismutases (SODs) are a ubiquitous group of metalloenzymes that play a central role in antioxidant defence. They function by removing superoxide anions from the cellular environment via a dismutation reaction. To date, four distinct SODs have been characterised in Trypanosoma brucei. Each isoform uses iron as a co-factor, in contrast to human SODs that rely on manganese or copper/zinc for activity. Two of the T. brucei SODs are predominantly glycosomal (SODB1 and SODB2), whereas the other two are targeted to the mitochondrion (SODA and SODC). RNA interference experiments demonstrated that the glycosomal isoforms are essential to bloodstream form parasites. However, because of the high level of sequence identity (92%) between the SODB1 and B2 genes, it is not possible to determine if one or both isoforms are functionally required. To dissect this further, we generated null mutants of both SODB1 and SODB2. Results demonstrate that bloodstream form T. brucei remain viable following deletion of both copies of SODB1, provided that expression of SODB2 is not disrupted, and following deletion of both copies of SODB2, provided that expression of SODB1 is not perturbed. Intriguingly, SODB1 null mutant cells were hypersensitive to the trypanocidal agents nifurtimox and benznidazole thus providing direct evidence of an association between drug-induced superoxide formation and nitroheterocycle activity.
The genome of the pathogen Trypanosoma brucei encodes three putative monothiol glutaredoxins (proteins that possess a single cysteine residue at their putative active sites; 1-C-Grxs). 1-C-Grx-1 and 2 are single domain monothiol glutaredoxins and harbour putative mitochondrial targeting sequences, while 1-C-Grx3 contains an additional thioredoxin-like domain and has a predicted cytosolic location. The recombinant tag-free forms of the three proteins presented different oligomeric conformations under native conditions. In vitro and in vivo analysis of the redox state of 1-C-Grx1 suggested that the protein undergoes conformational changes upon oxidation and exists in an equilibrium between reduced an oxidized (intramolecular disulfide) species, respectively. 1-C-Grxs are differentialy expressed in both life stages of T. brucei and immunofluorescence confirmed the mitochondrial compartmentalization of 1-C-Grx1. Several attempts to down regulate 1-C-Grx1 expression by dsRNAi or to delete the gene in bloodstream and procyclic parasites failed. In conditional null mutants of procyclic cells, depletion of 1-C-Grx1 to 30% of the level found in wildtype cells was not associated to growth impairment and neither triggers the up-regulation of the other 1-C-Grxs. Overexpression of 1-C-Grx1 in both parasite forms did not yield any phenotype under optimal growth conditions. However, bloodstream parasites overexpressing 1-C-Grx1 displayed a reduced growth rate when cultivated in the presence of the iron chelator deferoxamine, and an enhanced sensitivity against H2O2 but, strikingly, not towards menadione (a generator of O2-). Oxidative stress up-regulated the expression of 1-C-Grx1 while iron-depletion shouted-off. Recombinant 1-C-Grx1 and 2 form mixed disulfides with tryparedoxin, indicating that this parasite specific oxidoreductase is the physiological reductant of the proteins. Taking together, these data strongly indicate: i) the lack of functional redundancy among trypanosomatids 1-C-Grxs, ii) the involvement of 1-C-Grx1 in iron- and redox-metabolism and iii) an essential, although yet elusive, role of 1-C-Grx1 in the parasite´ mitochondrion.
Palabras clave: mitochondria, oxidative stress, iron sulfur cluster
Leishmania infantum causes visceral leishmaniasis, and is present in the Mediterranean areas of Europe. This protozoan parasite has two different life forms and both are likely to have to withstand stress induced by endogenously produced reactive oxygen and nitrogen species. However, to give rise to a productive infection, they might also have to deal with ROS/RNS produced by their mammalian hosts. Promastigote phagocytosis has been referred to signal for the assemblage of the NADPH oxidase on the macrophage membrane and, consequently, to induce O2•- production. On the other hand, macrophage activation by Th1 cytokines such as IFN-ã, is linked to induction of iNOS and, therefore, associated with •NO production. A concomitant production of O2•- and •NO leads to the formation of peroxynitrite a strong oxidant and a cytotoxic molecule. In vitro, all these molecules are lethal to parasites so, to be able to thrive, they must detoxify them. We are interested in investigating how the parasite performs this. As other trypanosomatids, Leishmania possess ROS/RNS detoxification cascades dependent on trypanothione, the trypanosomatid’s specific thiol. The last components of these cascades are peroxiredoxins of the typical two-cysteine subtype, designated as tryparedoxin peroxidases. We have verified that Leishmania recombinant TXNPxs are able to reduce both H2O2 and peroxynitrite. Based on those observations, we have generated L.infantum parasites overexpressing the three different TXNPx encoded in the genome, two cytosolic and one mithocondrial, with the intent to test if they would also detoxify peroxynitrite in the context of the parasite. Here, we show that this is indeed the case, mainly in what refers the cells overexpressing the cytosolic enzymes. In order to see whether these tryparedoxin peroxidases would play a role in parasite resistance to macrophage produced ROS/RNS, experiments are underway comparing the capacity of the different parasite lines to infect macrophages.
The microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during infection with Leishmania or Trypanosoma cruzi. However, we have found that mice deficient in nitric oxide synthase 2 (NOS2-/-) are less susceptible to infection with L. major than mice deficient in IFN-g (GKO), suggesting that there is an IFN-g dependent but NOS2 independent mechanism of resistance to this parasite. In order to investigate this issue, we infected NADPH phagocyte oxidase deficient mice (gp91phox -/-) with L. major or T. cruzi and followed the course of infection. We found that the course of infection in gp91phox-/- mice did not differ significantly from the course of infection in wild-type mice. In contrast, when infected gp91phox-/- mice with T. cruzi all mice succumbed to infection between day 15 and 21, while wild-type mice did not. Interestingly, gp91phox-/- mice have similar or reduced parasitemia and similar levels of IFN-g and TNF in serum and spleen cell culture supernatant when compared to wild-type controls. Further investigation demonstrated increased serum levels of NOx at day 15 of infection. The association between this free RNS and mortality is under investigation, but we speculate that the high levels of NO in sera of p91phox-/- mice induce tissue damage and shock. This issue is currently under investigation. Supported by CAPES, CNPq and FAPEMIG.
Palabras clave: Trypanosoma cruzi , NADPH oxidase, NO