Superoxide, nitric oxide and peroxynitrite production by macrophages under different physiological oxygen tensions.

Macrophages count on two O2-consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O2•-) and nitric oxide (•NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O2•- and •NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO2) normally ranges within 2-14 %, with a typical average pO2 value for most tissues ca. 5 %. Given that O2 is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O2•- and •NO production. Also, O2 is a modulator of the macrophage adaptative response and may influence iNOS expression in a hypoxia inducible factor 1-α (HIF1α-)-dependent manner. However, most of the reported experiments in cellula, analyzing the formation and effects of O2•- and •NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO2; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O2) which are far from the physiological pO2. Here, the role of O2 as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO2 and O2•- and •NO formation rates were measured, obtaining a KM of 26 and 42 µM O2 for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO2, reaching a maximum at ≥ 10 % O2, but even at levels as low as 2 % O2, a substantial formation rate of this oxidant was detected. Indeed, the cytotoxic capacity of immunostimulated macrophages against the intracellular parasite T. cruzi was significant, even at low pO2 values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions.

Nox2-derived superoxide radical is crucial to control acute Trypanosoma cruzi infection.

Trypanosoma cruzi is a flagellated protozoan that undergoes a complex life cycle between hematophagous insects and mammals. In humans, this parasite causes Chagas disease, which in thirty percent of those infected, would result in serious chronic pathologies and even death. Macrophages participate in the first stages of infection, mounting a cytotoxic response which promotes massive oxidative damage to the parasite. On the other hand, T. cruzi is equipped with a robust antioxidant system to repeal the oxidative attack from macrophages. This work was conceived to explicitly assess the role of mammalian cell-derived superoxide radical in a murine model of acute infection by T. cruzi. Macrophages derived from Nox2-deficient (gp91phox-/-) mice produced marginal amounts of superoxide radical and were more susceptible to parasite infection than those derived from wild type (wt) animals. Also, the lack of superoxide radical led to an impairment of parasite differentiation inside gp91phox-/- macrophages. Biochemical or genetic reconstitution of intraphagosomal superoxide radical formation in gp91phox-/- macrophages reverted the lack of control of infection. Along the same line, gp91phox-/- infected mice died shortly after infection. In spite of the higher lethality, parasitemia did not differ between gp91phox-/- and wt animals, recapitulating an observation that has led to conflicting interpretations about the importance of the mammalian oxidative response against T. cruzi. Importantly, gp91phox-/- mice presented higher and disseminated tissue parasitism, as evaluated by both qPCR- and bioimaging-based methodologies. Thus, this work supports that Nox2-derived superoxide radical plays a crucial role to control T. cruzi infection in the early phase of a murine model of Chagas disease.

Arachidonic Acid and Nitroarachidonic: Effects on NADPH Oxidase Activity.

Arachidonic acid (AA) is a polyunsaturated fatty acid that participates in the inflammatory response mainly through bioactive-lipids formation in macrophages and also in the phagocytic NADPH oxidase 2 (NOX2) activation. NOX2 is the enzyme responsible for a huge superoxide formation in macrophages, essential to eliminate pathogens inside the phagosome. The oxidase is an enzymatic complex comprised of a membrane-bound flavocytochrome b 558 (gp91phox/p22phox), three cytosolic subunits (p47phox, p40phox and p67phox) and a Rac-GTPase. The enzyme becomes active when macrophages are exposed to appropriate stimuli that trigger the phosphorylation of cytosolic subunits and its migration to plasmatic membrane to form the active complex. It is proposed that AA stimulates NOX2 activity through AA interaction with different components of the NADPH oxidase complex. In inflammatory conditions, there is an increase in reactive oxygen and nitrogen species that results in the production of nitrated derivatives of AA, such as nitroarachidonic acid (NO2-AA). NO2-AA is capable to inhibit NOX2 activity by interfering with p47phox migration to the membrane without affecting phosphorylation of cytosolic proteins. Also, NO2-AA is capable to interact with protein disulfide isomerase (PDI), which is involved on NOX2 active complex formation. It has been demonstrated that NO2-AA forms a covalent adduct with PDI that could prevent the interaction with NOX2 and it would explain the inhibitory effects of the fatty acid upon NOX2. Together, current data indicate that AA is an important activator of NOX2 formed in the early events of the inflammatory response, leading to a massive production of oxidants that may, in turn, promote NO2-AA formation and shutting down the oxidative burst. Hence, AA and its derivatives could have antagonistic roles on NOX2 activity regulation.

Cytosolic Fe-superoxide dismutase safeguards Trypanosoma cruzi from macrophage-derived superoxide radical.

Trypanosoma cruzi, the causative agent of Chagas disease (CD), contains exclusively Fe-dependent superoxide dismutases (Fe-SODs). During T. cruzi invasion to macrophages, superoxide radical (O2•-) is produced at the phagosomal compartment toward the internalized parasite via NOX-2 (gp91-phox) activation. In this work, T. cruzi cytosolic Fe-SODB overexpressers (pRIBOTEX-Fe-SODB) exhibited higher resistance to macrophage-dependent killing and enhanced intracellular proliferation compared with wild-type (WT) parasites. The higher infectivity of Fe-SODB overexpressers compared with WT parasites was lost in gp91-phox-/- macrophages, underscoring the role of O2•- in parasite killing. Herein, we studied the entrance of O2•- and its protonated form, perhydroxyl radical [(HO2•); pKa = 4.8], to T. cruzi at the phagosome compartment. At the acidic pH values of the phagosome lumen (pH 5.3 ± 0.1), high steady-state concentrations of O2•- and HO2• were estimated (∼28 and 8 µM, respectively). Phagosomal acidification was crucial for O2•- permeation, because inhibition of the macrophage H+-ATPase proton pump significantly decreased O2•- detection in the internalized parasite. Importantly, O2•- detection, aconitase inactivation, and peroxynitrite generation were lower in Fe-SODB than in WT parasites exposed to external fluxes of O2•- or during macrophage infections. Other mechanisms of O2•- entrance participate at neutral pH values, because the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid decreased O2•- detection. Finally, parasitemia and tissue parasite burden in mice were higher in Fe-SODB-overexpressing parasites, supporting the role of the cytosolic O2•--catabolizing enzyme as a virulence factor for CD.

Fluorescence and chemiluminescence approaches for peroxynitrite detection.

In the last two decades, there has been a significant advance in understanding the biochemistry of peroxynitrite, an endogenously-produced oxidant and nucleophile. Its relevance as a mediator in several pathologic states and the aging process together with its transient character and low steady-state concentration, motivated the development of a variety of techniques for its unambiguous detection and estimation. Among these, fluorescence and chemiluminescence approaches have represented important tools with enhanced sensitivity but usual limited specificity. In this review, we analyze selected examples of molecular probes that permit the detection of peroxynitrite by fluorescence and chemiluminescence, disclosing their mechanism of reaction with either peroxynitrite or peroxynitrite-derived radicals. Indeed, probes have been divided into 1) redox probes that yield products by a free radical mechanism, and 2) electrophilic probes that evolve to products secondary to the nucleophilic attack by peroxynitrite. Overall, boronate-based compounds are emerging as preferred probes for the sensitive and specific detection and quantitation. Moreover, novel strategies involving genetically-modified fluorescent proteins with the incorporation of unnatural amino acids have been recently described as peroxynitrite sensors. This review analyzes the most commonly used fluorescence and chemiluminescence approaches for peroxynitrite detection and provides some guidelines for appropriate experimental design and data interpretation, including how to estimate peroxynitrite formation rates in cells.

Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate.

The specific and sensitive detection of peroxynitrite (ONOO-/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a very simply two-step conversion synthetic route with high purity (>99%) and overall yield (∼42%). Reactivity analysis of Fl-B with relevant biological oxidants including hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×106M-1s-1, a million times faster than the rate constant measured for H2O2 (k=1.7M-1s-1) and 2,700 faster than HOCl (6.2×102M-1s-1) at 37°C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO2, a well-known peroxynitrite reactant. Experimental and simulated kinetic analyses confirm that the main oxidation process of Fl-B takes place with peroxynitrite itself via a direct bimolecular reaction and not with peroxynitrite-derived radicals. Fl-B was successfully applied for the detection of endogenously-generated peroxynitrite by endothelial cells and in macrophage-phagocyted parasites. Moreover, the generated data allowed estimating the actual intracellular flux of peroxynitrite. For instance, ionomycin-stimulated endothelial cells generated peroxynitrite at a rate of ∼ 0.1µMs-1, while immunostimulated macrophages do so in the order of ∼1µMs-1 inside T. cruzi-infected phagosomes. Fl-B revealed not to be toxic in concentrations up to 1mM for 24h. Cellular peroxynitrite detection was achieved by conventional laboratory fluorescence-based methods including flow cytometry and epi-fluorescence microscopy. Fl-B was shown to be more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite.

Nitric oxide diffusion to red blood cells limits extracellular, but not intraphagosomal, peroxynitrite formation by macrophages.

Macrophage-derived nitric oxide ((•)NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by (•)NO itself or (•)NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O(2)(•-)). In vivo, the facile extracellular diffusion of (•)NO as well as different competing consumption routes limit its bioavailability for the reaction with O(2)(•-) and, hence, peroxynitrite formation. In this work, we evaluated the extent by which (•)NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O(2)(•-) and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of (•)NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of (•)NO and O(2)(•-)-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of (•)NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O(2)(•-) reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of (•)NO and O(2)(•-)-forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, (•)NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.

Peroxynitrite, a potent macrophage-derived oxidizing cytotoxin to combat invading pathogens.

Macrophages are among the first cellular actors facing the invasion of microorganisms. These cells are able to internalize pathogens and destroy them by means of toxic mediators, many of which are produced enzymatically and have strong oxidizing capacity. Indeed, macrophages count on the NADPH oxidase complex activity, which is triggered during pathogen invasion and leads to the production of superoxide radical inside the phagosome. At the same time, the induction of nitric oxide synthase results in the production of nitric oxide in the cytosol which is able to readily diffuse to the phagocytic vacuole. Superoxide radical and nitric oxide react at diffusion controlled rates with each other inside the phagosome to yield peroxynitrite, a powerful oxidant capable to kill micro-organisms. Peroxynitrite toxicity resides on oxidations and nitrations of biomolecules in the target cell. The central role of peroxynitrite as a key effector molecule in the control of infections has been proven in a wide number of models. However, some microorganisms and virulent strains adapt to survive inside the potentially hostile oxidizing microenvironment of the phagosome by either impeding peroxynitrite formation or rapidly detoxifying it once formed. In this context, the outcome of the infection process is a result of the interplay between the macrophage-derived oxidizing cytotoxins such as peroxynitrite and the antioxidant defense machinery of the invading pathogens.

Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages.

Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2(•-)) due to the NADPH-dependent univalent reduction of oxygen to O2(•-) by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O2(•-) in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO2 can modulate macrophage activation by inhibiting O2(•-) formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO2 decreased O2(•-) production in a dose- (IC50=4.1±1.8 µM AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40(phox) and p47(phox)), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47(phox) was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.

Trypanosoma cruzi antioxidant enzymes as virulence factors in Chagas disease.

SIGNIFICANCE: Chagas disease (CD) affects several million people in Latin America and is spreading beyond its classical boundaries due to the migration of infected host and insect vectors, HIV co-infection, and blood transfusion. The current therapy is not adequate for treatment of the chronic phase of CD, and new drugs are warranted. RECENT ADVANCES: Trypanosoma cruzi is equipped with a specialized and complex network of antioxidant enzymes that are located at different subcellular compartments which defend the parasite against host oxidative assaults. Recently, strong evidence has emerged which indicates that enzyme components of the T. cruzi antioxidant network (cytosolic and mitochondrial peroxiredoxins and trypanothione synthetase) in naturally occurring strains act as a virulence factor for CD. This precept is recapitulated with the observed increased resistance of T. cruzi peroxirredoxins overexpressers to in vivo or in vitro nitroxidative stress conditions. In addition, the modulation of mitochondrial superoxide radical levels by iron superoxide dismutase (FeSODA) influences parasite programmed cell death, underscoring the role of this enzyme in parasite survival. CRITICAL ISSUES: The unraveling of the biological significance of FeSODs in T. cruzi programmed cell death in the context of chronic infection in CD is still under examination. FUTURE DIRECTIONS: The role of the antioxidant enzymes in the pathogenesis of CD, including parasite virulence and persistence, and their feasibility as pharmacological targets justifies further investigation.

Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized Trypanosoma cruzi: consequences for oxidative killing and role of microbial peroxiredoxins in infectivity.

Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide ((•)NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO(-) + ONOOH) formation, a strong oxidant arising from the reaction of (•)NO with superoxide radical (O(2)(-)). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O(2)(-) during a 60-90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained (•)NO production (∼24 h). The major mechanism for infection control via reactive species formation occurred when (•)NO and O(2)() were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite.

Fighting the oxidative assault: the Trypanosoma cruzi journey to infection.

Activation of professional phagocytes with the concomitant generation of oxidant species is a medullar innate immune process for the control of acute Trypanosoma cruzi infection. Recent data reinforce the hypothesis that parasites more prepared to deal with the host-oxidative assault are more efficient for the establishment of Chagas disease. For instance, parasites overexpressing peroxiredoxins are more resistant to macrophage-derived peroxynitrite, a key cytotoxic oxidant produced in the phagosome towards the internalized parasite. Differentiation to the infective metacyclic trypomastigote is accompanied by an increased expression of antioxidant enzymes. Moreover, augmented antioxidant enzyme expression and activities correlate with higher parasite virulence in experimental infections. The potency of the parasite antioxidant armamentarium influences the final fate of the Trypanosoma cruzi journey to macrophage invasion at the onset of infection.

Peroxiredoxins play a major role in protecting Trypanosoma cruzi against macrophage- and endogenously-derived peroxynitrite.

There is increasing evidence that Trypanosoma cruzi antioxidant enzymes play a key immune evasion role by protecting the parasite against macrophage-derived reactive oxygen and nitrogen species. Using T. cruzi transformed to overexpress the peroxiredoxins TcCPX (T. cruzi cytosolic tryparedoxin peroxidase) and TcMPX (T. cruzi mitochondrial tryparedoxin peroxidase), we found that both cell lines readily detoxify cytotoxic and diffusible reactive oxygen and nitrogen species generated in vitro or released by activated macrophages. Parasites transformed to overexpress TcAPX (T. cruzi ascorbate-dependent haemoperoxidase) were also more resistant to H2O2 challenge, but unlike TcMPX and TcCPX overexpressing lines, the TcAPX overexpressing parasites were not resistant to peroxynitrite. Whereas isolated tryparedoxin peroxidases react rapidly (k=7.2 x 10(5) M(-1) x s(-1)) and reduce peroxynitrite to nitrite, our results demonstrate that both TcMPX and TcCPX peroxiredoxins also efficiently decompose exogenous- and endogenously-generated peroxynitrite in intact cells. The degree of protection provided by TcCPX against peroxynitrite challenge results in higher parasite proliferation rates, and is demonstrated by inhibition of intracellular redox-sensitive fluorescence probe oxidation, protein 3-nitrotyrosine and protein-DMPO (5,5-dimethylpyrroline-N-oxide) adduct formation. Additionally, peroxynitrite-mediated over-oxidation of the peroxidatic cysteine residue of peroxiredoxins was greatly decreased in TcCPX overexpressing cells. The protective effects generated by TcCPX and TcMPX after oxidant challenge were lost by mutation of the peroxidatic cysteine residue in both enzymes. We also observed that there is less peroxynitrite-dependent 3-nitrotyrosine formation in infective metacyclic trypomastigotes than in non-infective epimastigotes. Together with recent reports of up-regulation of antioxidant enzymes during metacyclogenesis, our results identify components of the antioxidant enzyme network of T. cruzi as virulence factors of emerging importance.

Reaction of the carbonate radical with the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide in chemical and cellular systems: pulse radiolysis, electron paramagnetic resonance, and kinetic-competition studies.

Carbonate radicals (CO3-) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite-carbon dioxide adduct (ONOOCO2-), and enzymatic activities, i.e., peroxidase activity of CuZnSOD and xanthine oxidase turnover in the presence of bicarbonate. It has been reported that the spin-trap DMPO reacts with CO3(-) to yield transient species to yield finally the DMPO-OH spin adduct. In this study, the kinetics of reaction of CO3(-) with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 x 10(6) M(-1) s(-1). A Fenton system, composed of Fe(II)-DTPA plus H2O2, generated OH that was trapped by DMPO; the presence of 50-500 mM bicarbonate, expected to convert OH to CO3(-), markedly inhibited DMPO-OH formation. This was demonstrated to be due mainly to a fast reaction of CO3(-) with FeII-DTPA (k=6.1 x 10(8) M(-1) s(-1)), supported by kinetic analysis. Generation of CO3(-) by the Fenton system was further proved by analysis of tyrosine oxidation products: the presence of bicarbonate caused a dose-dependent inhibition of 3,4-dihydroxiphenylalanine with a concomitant increase of 3,3'-dityrosine yields, and the presence of DMPO inhibited tyrosine oxidation, in agreement with the rate constants with OH or CO3(-). Similarly, the formation of CO3(-) by CuZnSOD/H(2)O(2)/bicarbonate and peroxynitrite-carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO3(-) with DMPO to yield DMPO-OH was shown in peroxynitrite-forming macrophages. In conclusion, CO3(-) reacts quite rapidly with DMPO and may contribute to DMPO-OH yields in chemical and cellular systems; in turn, the extent of oxidation of other target molecules (such as tyrosine) by CO3(-) will be sensitive to the presence of DMPO.

Mitochondrial superoxide radicals mediate programmed cell death in Trypanosoma cruzi: cytoprotective action of mitochondrial iron superoxide dismutase overexpression.

Trypanosoma cruzi undergo PCD (programmed cell death) under appropriate stimuli, the mechanisms of which remain to be established. In the present study, we show that stimulation of PCD in T. cruzi epimastigotes by FHS (fresh human serum) results in rapid (<1 h) externalization of phosphatidylserine and depletion of the low molecular mass thiols dihydrotrypanothione and glutathione. Concomitantly, enhanced generation of oxidants was established by EPR and immuno-spin trapping of radicals using DMPO (5,5-dimethylpyrroline-N-oxide) and augmentation of the glucose flux through the pentose phosphate pathway. In the early period (<20 min), changes in mitochondrial membrane potential and inhibition of respiration, probably due to the impairment of ADP/ATP exchange with the cytosol, were observed, conditions that favour the generation of O2*-. Accelerated rates of mitochondrial O2*- production were detected by the inactivation of the redox-sensitive mitochondrial aconitase and by oxidation of a mitochondrial-targeted probe (MitoSOX). Importantly, parasites overexpressing mitochondrial FeSOD (iron superoxide dismutase) were more resistant to the PCD stimulus, unambiguously indicating the participation of mitochondrial O2*- in the signalling process. In summary, FHS-induced PCD in T. cruzi involves mitochondrial dysfunction that causes enhanced O(2)(*-) formation, which leads to cellular oxidative stress conditions that trigger the initiation of PCD cascades; moreover, overexpression of mitochondrial FeSOD, which is also observed during metacyclogenesis, resulted in cytoprotective effects.

Macrophage-derived peroxynitrite diffusion and toxicity to Trypanosoma cruzi.

We studied the capacity of macrophage-derived peroxynitrite to diffuse into and exert cytotoxicity against Trypanosoma cruzi, the causative agent of Chagas' disease. In two types of macrophage-T. cruzi co-cultures, one with a fixed separation distance between source and target cells, and another involving cell-to-cell interactions, peroxynitrite resulted in significant oxidation of intracellular dihydrorhodamine and inhibition of [(3)H]thymidine incorporation in T. cruzi, which were not observed by superoxide or nitric oxide alone. The effects were attenuated in the presence of bicarbonate, in agreement with the extracellular consumption of peroxynitrite by its fast reaction with CO(2). However, studies using different T. cruzi densities, which allow to modify average diffusion distances of exogenously added peroxynitrite to target cells, indicate that at distances <5 microm, the diffusion process outcompetes the reaction with CO(2) and that the levels of peroxynitrite formed by macrophages would be sufficient to cause toxicity to T. cruzi during cell-to-cell contact and/or inside the phagosome.

L-arginine metabolism during interaction of Trypanosoma cruzi with host cells.

Trypanosoma cruzi invades a diversity of nucleated cells in the mammalian host. Macrophages are among the first cells to be parasitized and, after activation by inflammatory stimuli, they participate in the control of infection. However, some parasites manage to evade the immune response and establish a chronic infection in differentiated cells. L-arginine is located at the crossroads of divergent routes that produce metabolites, including nitric oxide and polyamines, which influence the outcome (i.e. resolution or progression) of infection. This article discusses the fate and actions of L-arginine-derived biomolecules formed both in the host and in the parasite during T. cruzi-host-cell interactions.