Trypanosomatid Diseases (Molecular Routes to Drug Discovery) || Peroxynitrite as a Cytotoxic Effector Against Trypanosoma Cruzi : Oxidative Killing and Antioxidant Resistance Mechanisms

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<ul><li><p>11Peroxynitrite as a Cytotoxic Effector Against Trypanosoma cruzi :Oxidative Killing and Antioxida nt Resistanc e MechanismsMadia Trujillo, Mara Noel Alvarez, Luca Piacenza, Martn Hugo,Gonzalo Peluffo, and Rafael Radi </p><p>AbstractInside the phagosomes of activated macrophages, Trypanosoma cruzi is exposed toreactive oxygen species formed by NADPH oxidase, namely the superoxide radical(O2</p><p> ) and, through its dismutation, hydrogen peroxide. Moreover, induction ofnitric oxide synthase by pro-inammatory cytokines leads to the production of nitricoxide ( NO). Neither O2</p><p> nor NO are particularly cytotoxic per se, and most oftheir parasiticidal actions rely on their diffusion-controlled reaction to form pero-xynitrite a potent oxidizing and nitrating species with a higher killing capabilitytowards microorganisms. Thus, the antioxidant mechanisms that allow the parasiteto detoxify peroxynitrite and other cytotoxic peroxides constitute an active eld ofinvestigation, since they can provide clues for a rationalized drug design. Moreover,although most research has focused on the formation of reactive species and theirdetoxi cation during acute infections, Chagas is mostly a chronic disease and nodrug is currently available for its treatment at this stage, where the microorganisminfects cardiac muscle and other non-phagocytic cells. The role of peroxynitrite inT. cruzi infectivity and virulence at the chronic stage of the disease remains tobe established.</p><p>Introduction</p><p>Trypanosoma cruzi is an intracellular parasite that causes Chagas disease, alsoknown as American trypanosomiasis, endemic in 21 Latin American countries,where millions of persons are infected and thousands of people die each year of thisillness (http://www.who.int/mediacentre/factsheets/fs340/en/index.html). Macro-phage infection plays a key role during the acute phase of the disease. Inside thephagosomes of activated macrophages, the parasites are exposed to reactive oxygenspecies formed by NADPH oxidase [1,2], namely the superoxide radical (O2</p><p>) and,through its dismutation, hydrogen peroxide (H2O2) [3]. Moreover, induction ofinducible nitric oxide synthase (iNOS) by pro-inammatory cytokines leads to the</p><p> Corresponding Author</p><p>Trypanosomatid Diseases: Molecular Routes to Drug Discovery, First edition. Edited by T. Jger, O. Koch, and L. Floh.# 2013 Wiley-VCH Verlag GmbH &amp; Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH &amp; Co. KGaA.</p><p>j215</p></li><li><p>production of nitric oxide (NO) [46]. Neither O2 nor NO are particularly</p><p>cytotoxic, and most of their parasiticidal actions rely on their fast reaction toform peroxynitrite an oxidizing and nitrating species with a higher capabilityto kill microorganisms [79] (IUPAC recommended names for peroxynitrite anion(ONOO) and its conjugated acid, peroxynitrous acid (ONOOH), are oxoperoxoni-trate (1) and hydrogen oxoperoxonitrate, respectively. The term peroxynitrite isused to refer to the sum of ONOO and ONOOH). Thus, the antioxidant mecha-nisms that allow the parasite to detoxify peroxynitrite and other cytotoxic peroxidesare intensively explored, since they could provide clues for new therapeuticapproaches. Moreover, although most research has focused on the formation ofreactive species and their detoxication during acute infections, Chagas is mostly achronic disease and no drug is currently available for its treatment at this stage,where the microorganism infects cardiac muscle and other non-phagocytic cells[1013]. The roles of reactive oxygen and nitrogen species, in particular peroxyni-trite, in T. cruzi killing and the antioxidant enzymatic mechanisms that allowparasite survival during chronic infections are only starting to be unraveled.</p><p>PeroxynitriteFormationDuringT.cruziMammalianHostCell Interaction</p><p>Once in the mammalian host, metacyclic trypomastigotes infect and replicateintracellularly in different types of cells, macrophages being one of the mostimportant. Phagocytic cells, such as macrophages, have an active role in microbialkilling, linking innate and adaptive immune responses. Several molecules on thehost cell surface have been described to interact with T. cruzi, such as mannose andToll-like receptors (TLRs) [14,15]. Specically, TLR2 and TLR4 have been involvedin the recognition of T. cruzi [16,17], which interact through molecules presentsin the trypomastigote membrane. Parasite recognition leads to an upregulation ofantimicrobial activity, with the production of pro-inammatory cytokines and apositive regulation of macrophage oxidative response [18,19].TLR-mediated phagocytosis is usually accompanied by a respiratory burst,</p><p>accomplished by NOX2, a specialized NADPH oxidase enzymatic complex. NOX2consists of the membrane-bound avocytochrome b gp91phox, which transferselectrons from a cytosolic NADPH to oxygen in the phagocytic vacuole to producelarge quantities of O2</p><p>when activated. Other components of the complex includemembrane-bound p22phox, cytosolic proteins p40phox, p47phox, and p67phox, and thesmall GTPases Rac1 and Rap1; all are translocated to the membrane in atightly regulated way [2022]. Some pathogens, among which are some unicellularparasites, manage to evade NOX2 activation in order to survive in the phagosomeand efciently infect the host. It has been debated whether the invasion processof T. cruzi activates NOX2 [2325]. Results from our group and others haveshown the formation of O2</p><p> during T. cruzi invasion, which occurs on theluminal side of the phagosome and lasts for around 6090min; the magnitude ofthe respiratory burst is comparable with that elicited by other phagocyticstimuli [26,27].</p><p>216j 11 Peroxynitrite as a Cytotoxic Effector Against Trypanosoma cruzi: Oxidative Killing and Antioxidant</p></li><li><p>In the early stage of T. cruzi infection, natural killer and helper T (Th) cells becomeactive when they are presented to peptide antigens exposed on the surface ofantigen-presenting cells. Once activated, they secrete cytokines that regulate or assistin the active immune response. Macrophages exposed to TLR agonists and Th1cytokines (i.e., interferon (IFN)-c, tumor necrosis factor (TNF)-a), namely classi-cally activated macrophages, show alterations in phagocyte physiology with induc-tion of nitric oxide synthase 2 expression, responsible for the prolonged productionof large amounts of NO [4,5] and an enhanced respiratory burst [28,29]. Microbialkilling in activated macrophages relies more heavily on the production of reactiveoxygen and nitrogen intermediates [3035]. Under pro-inammatory conditions,maximal and simultaneous production of O2</p><p> and NOwill occur. NADPH oxidaseassembly in the phagosome membrane results in the formation of O2</p><p> inside thevacuole, whereas NO, a hydrophobic radical, diffuses from the cytosol. Inside thephagosome, the diffusion-controlled reaction between NO and O2</p><p> yields perox-ynitrite, a strong one- and two-electron oxidant, and a precursor of secondaryspecies, which can also lead to one-electron oxidation and nitration reactions, aswe will see below [36].Immunostimulated macrophages are involved in early parasite killing. Many</p><p>years ago, several investigators demonstrated that the ability of T. cruzi to infectand multiply inside mouse macrophages in vitro was inhibited by macrophageactivation [3739]. Thereafter, it was suggested that the ability to produce andrelease H2O2 by activated macrophages was related to an enhanced capacity to killintracellular trypomastigotes [25,40]. Other investigators showed the importanceof NO in infection control [4144]. In addition, in vitro experiments revealed thatbolus peroxynitrite addition is effective in compromising T. cruzi viability [45].Moreover, coculture of the non-infective epimastigote form with resting andactivated macrophages shows that under conditions of macrophage-derivedperoxynitrite formation in the extracellular space, the oxidant was capable ofdiffusing to parasites causing intracellular probe oxidation and strongly inhibitingT. cruzi survival (around 50%) [46].Macrophage infection studies with T. cruzi trypomastigotes were performed to</p><p>evaluate the role of peroxynitrite during parasite internalization. Experiments usingoxidation of 2,7-dichlorodihydrouorescein diacetate (DCFH2) as a probe forperoxynitrite formation indicated that the oxidant was indeed formed by immu-nostimulated macrophages (forming simultaneously O2</p><p> and NO), but not bynaive macrophages (which produce only O2</p><p>) (Figure 11.1a). The amount ofperoxynitrite inside the phagosome was sufcient to damage and kill trypomasti-gotes as evidenced by protein nitroxidative modications detected in internalizedparasites, 60% less amastigote load into macrophages after 24 h, and ultrastructuralalterations in parasites visualized in electron microscopy studies (Figure 11.1b).Notably, overexpression of an enzyme with peroxidase and peroxynitrite reductaseactivity (cytosolic tryparedoxin peroxidase (cTXNPx), see below) signicantly dimin-ished cytotoxicity [27]. The progression of Chagas disease to the chronic phasedepends on parasite persistence in tissues and on the host immune responses[47,48]. Some parasites can evade the initial assault of the immune system and infect</p><p>Peroxynitrite Formation During T. cruziMammalian Host Cell Interaction j217</p></li><li><p>Figure 11.1 Peroxynitrite formation inimmunostimulated-infected macrophages:ultrastructural consequences for T. cruziinside the phagosome. (a) Detection ofperoxynitrite formed during macrophageinfection by T. cruzi trypomastigotes.Unstimulated (T. cruzi) andimmunostimulated (IFN-c/LPS T. cruzi)macrophages were infected withtrypomastigotes (5: 1 parasite/macrophageratio) in the presence of 30 mM DCFH2.The time course of DCFH2 oxidation wasfollowed in a fluorescence plate reader withfilters at lexc 485 nm and lem 520 nm, atpH 7.4 and 37 C. Control (CTL): uninfectedmacrophages. (b) Peroxynitrite exposure of</p><p>phagocytosed T. cruzi results inultrastructural alterations as evidenced bytransmission electron microscopy.Micrographs shown unstimulated (T. cruzi)and activated (T. cruzi IFN-c/LPS) infectedmacrophages at 1 h postinfection;peroxynitrite-producing activatedmacrophages showed marked damage ofT. cruzi organelles and membranedisorganization and disappearance. Arrowsshow normal membrane microtubularstructures in phagocytosed trypomastigotesby resting macrophages and reductions ofmembrane microtubules inside peroxynitrite-producing macrophages. n, T. cruzi nucleus;f, flagellum; r, reservosomes.</p><p>218j 11 Peroxynitrite as a Cytotoxic Effector Against Trypanosoma cruzi: Oxidative Killing and Antioxidant</p></li><li><p>non-phagocytic cells in organs such as the heart and the digestive tract. Indeed,chagasic cardiomyopathy is characterized by the presence of amastigote nests in thecardiac ber [49]. T. cruzi invasion to cardiomyocytes triggers the production ofinammatory mediators (TNF-a, interleukin (IL)-1b) and the induction of iNOSwithin the cells [50,51]. They can also be exposed to NO diffusing from inamma-tory cells. Experiments using knockout mice for IFN-c, IL-12, and NOS isoformsclearly demonstrated a key role of the IL-12/IFN-c/iNOS axis in the control ofT. cruzi infection and for the outcome in the disease [52,53]. Moreover, cardiomyo-cytes and other non-phagocytic cells possess sources of O2</p><p> such as the mitochon-drial electron transport chain or uncoupled nitric oxide synthases [54,55]. Thesuperoxide radical could also be formed inside the parasite from its own electrontransport chain or other metabolic processes as well as by redox cycling of anti-chagasic drugs [5658]. It is therefore tempting to speculate that peroxynitrite couldbe formed also in infected non-phagocytic cells or even inside the parasite and that itcould be implicated in the control of the infection. Host-nitrated proteins have beenidentied in heart lesions in a mouse model of Chagas disease [59,60]. However,formation of peroxynitrite within or reaching the parasite from external sources andits possible role in controlling proliferation during the chronic stage of the disease isstill to be demonstrated.</p><p>Peroxynitrite Diffusion and Reactivity with T. cruzi Targets</p><p>Peroxynitrous acid has a pKa of 6.56.8 [6163] and therefore the anionic form ismore abundant than its conjugated acid at physiological pH. Homolysis of theperoxo bond (k 0.9 s1, pH 7.4, 37 C) makes peroxynitrous acid decay, giving riseto hydroxyl (OH) and nitrogen dioxide (NO2) radicals at approximately 30% yields[64,65]. Both radicals may participate in secondary, indirect reactions, resulting inthe oxidation, hydroxylation, or nitration of different targets. In biological systems,however, most peroxynitrite will react directly with different biomolecules beforedecomposing. Low-molecular-weight and protein thiols, metal centers, and carbondioxide (CO2) constitute themain targets for peroxynitrite in vivo (for a recent review,see [66]). The direct reaction with thiols involves a two-electron oxidation of thedeprotonated form of the thiol by peroxynitrous acid, yielding nitrite and a sulfenicacid intermediate that, in the presence of another accessible thiol group, results indisulde formation [67,68]. In the case of metal centers, such as those in hemeproteins or other metal-containing proteins, mechanisms of reaction with perox-ynitrite include one- and two-electron oxidations as well as metal-catalyzed isomeri-zation to nitrate [6973]. Metal oxidation may result in the formation of strongoxidative species such as compound 1 or 2 of heme peroxidases or other oxo-metalcomplexes, which may participate in nitro-oxidative target modications [74].Moreover, the peroxynitrite anion reacts with CO2 (k 4.6 104M1 s1, pH 7.4,37 C) forming a transient intermediate that homolyzes to NO2 and carbonateradical (CO3</p><p>) in around 35% yields [7577]; radicals that in turn can participate inindirect oxidation and/or nitration reactions [74].</p><p>Peroxynitrite Diffusion and Reactivity with T. cruzi Targets j219</p></li><li><p>The main low-molecular-weight thiol in trypanosomatids, trypanothione (theterm trypanothione (T(SH)2) is used for bis(glutathionyl) spermidine and itsdisulde is dened as trypanothione disulde (TS2) [78]) is oxidized by peroxyni-trite with a rate constant of 7200M1 s1, pH 7.4, 37 C [79,80], as expected for alow-molecular-weight dithiol with a pKa of 7.4 [81,82]. It also reacts with differentthiol-containing proteins of the parasite, including the fast-reacting peroxidaticthiols in peroxiredoxins (Prxs) [83] (see below), and with metal-containing proteinssuch as ascorbate peroxidase and iron-dependent superoxide dismutases (SODs) Aand B (Hugo et al., unpublished andMartinez et al., unpublished results). Expositionto peroxynitrite also leads to the nitration of parasite proteins, which is higher whenintracellular thiol content decrease [84]. It must be taken into account that, at leastduring acute infections, most peroxynitrite is formed outside the parasite, inside thephagosome of activated macrophages, and that it must diffuse to and through th...</p></li></ul>

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