Effect of beta-lapachone on superoxide anion and hydrogen peroxide production in Trypanosoma cruzi.
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ABSTRACT:
SUBMITTER: Boveris A
PROVIDER: S-EPMC1186088 | biostudies-other | 1978 Nov
REPOSITORIES: biostudies-other
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Project description:The glutathione peroxidase-glutathione reductase system, an alternative pathway for metabolic utilization of H2O2 [Chance, Sies & Boveris (1979) Physiol. Rev. 59, 527-605], was investigated in Trypanosoma cruzi, an organism lacking catalase and deficient in peroxidase [Boveris & Stoppani (1977) Experientia 33, 1306-1308]. The presence of glutathione (4.9 +/- 0.7 nmol of reduced glutathione/10(8) cells) and NADPH-dependent glutathione reductase (5.3 +/- 0.4 munit/10(8) cells) was demonstrated in the cytosolic fraction of the parasite, but with H2O2 as substrate glutathione peroxidase activity could not be demonstrated in the same extracts. With t-butyl hydroperoxide or cumene hydroperoxide as substrate, a very low NADPH-dependent glutathione peroxidase activity was detected (equivalent to 0.3-0.5 munit of peroxidase/10(8) cells, or about 10% of glutathione reductase activity). Blank reactions of the glutathione peroxidase assay (non-enzymic oxidation of glutathione by hydroperoxides and enzymic oxidation of NADPH) hampered accurate measurement of peroxidase activity. The presence of superoxide dismutase and ascorbate peroxidase activity in, as well as the absence of catalase from, epimastigote extracts was confirmed. Ascorbate peroxidase activity was cyanide-sensitive and heat-labile, but no activity could be demonstrated with diaminobenzidine, pyrogallol or guaiacol as electron donor. The summarized results support the view that T. cruzi epimastigotes lack an adequate enzyme defence against H2O2 and H2O2-related free radicals.
| S-EPMC1161944 | biostudies-other
Project description:Life evolved in an anaerobic world; therefore, fundamental enzymatic mechanisms and biochemical pathways were refined and integrated into metabolism in the absence of any selective pressure to avoid reactivity with oxygen. After photosystem II appeared, environmental oxygen levels rose very slowly. During this time, microorganisms acquired oxygen tolerance by jettisoning enzymes that use glycyl radicals and exposed low-potential iron-sulfur clusters, which can be directly poisoned by oxygen. They also developed mechanisms to defend themselves against superoxide (O(2)()) and hydrogen peroxide, partially reduced oxygen species that are generated as inadvertent by-products of aerobic metabolism. Contemporary organisms have inherited both the vulnerabilities and the defenses of these ancestral microbes. Current research seeks to identify these, and bacteria comprise an exceptionally accessible experimental system that has provided many of the answers. This manuscript reviews recent developments and identifies remaining puzzles.
| S-EPMC3057177 | biostudies-literature
Project description:The photoproduction of superoxide anion radical (O2-•) and hydrogen peroxide (H2O2) in photosystem II (PSII) preparations depending on the damage to the water-oxidizing complex (WOC) was investigated. The light-induced formation of O2-• and H2O2 in the PSII preparations rose with the increased destruction of the WOC. The photoproduction of superoxide both in the PSII preparations holding intact WOC and the samples with damage to the WOC was approximately two times higher than H2O2. The rise of O2-• and H2O2 photoproduction in the PSII preparations in the course of the disassembly of the WOC correlated with the increase in the fraction of the low-potential (LP) Cyt b559. The restoration of electron flow in the Mn-depleted PSII preparations by exogenous electron donors (diphenylcarbazide, Mn2+) suppressed the light-induced formation of O2-• and H2O2. The decrease of O2-• and H2O2 photoproduction upon the restoration of electron transport in the Mn-depleted PSII preparations could be due to the re-conversion of the LP Cyt b559 into higher potential forms. It is supposed that the conversion of the high potential Cyt b559 into its LP form upon damage to the WOC leads to the increase of photoproduction of O2-• and H2O2 in PSII.
| S-EPMC6784185 | biostudies-literature
Project description:The production of H(2)O(2) by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H(2)O(2). However, matrix enzymes such as the glutathione peroxidases can consume H(2)O(2) and compete with efflux of H(2)O(2), causing an underestimation of superoxide production. To assess this underestimate, we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90% as a consequence of pretreatment with 1-chloro-2,4-dintrobenzene (CDNB). The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H(2)O(2), consistent with decreased peroxidase capacity, but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H(2)O(2) formation from matrix-directed superoxide by up to two-fold from several sites of production, as defined by substrates and electron transport inhibitors, over a wide range of control rates, from 0.2-2.5 nmol H(2)O(2) min(-1) mg protein(-1). Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submitochondrial particles. The data indicate that the increased H(2)O(2) efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic correction curve was constructed, making H(2)O(2) efflux a more quantitative measure of matrix superoxide production. For rat muscle mitochondria, the correction equation was: CDNB-pretreated rate = control rate + [1.43 x (control rate)]/(0.55 + control rate). These results have significant ramifications for the rates and topology of superoxide production by isolated mitochondria.
| S-EPMC2891561 | biostudies-literature
Project description:The goal of this study was to identify whether heat-shock protein 90 (Hsp90) regulates the production of superoxide and other reactive oxygen species from the NADPH oxidases (Nox). We found that pharmacological and genetic inhibition of Hsp90 directly reduced Nox5-derived superoxide without secondarily modifying signaling events. Coimmunoprecipitation and bioluminescence resonance energy transfer studies suggest that the C-terminus of Nox5 binds to Hsp90. Long-term Hsp90 inhibition reduced Nox5 expression and provides further evidence that Nox5 is an Hsp90 client protein. Inhibitors of Hsp90 also reduced superoxide from Nox1, Nox2 (neutrophils), and Nox3. However, Nox4, which emits only hydrogen peroxide, was unaffected by Hsp90 inhibitors. Hydrogen peroxide production from the other Nox enzymes was not affected by short-term inhibition of Hsp90, but long-term inhibition reduced production of all reactive oxygen species coincident with loss of enzyme expression. Expression of chimeric Nox enzymes consisting of N-terminal Nox1 or Nox3 and C-terminal Nox4 resulted in only hydrogen peroxide formation that was insensitive to Hsp90 inhibitors. We conclude that Hsp90 binds to the C-terminus of Noxes1-3 and 5 and is necessary for enzyme stability and superoxide production. Hsp90 does not bind to the C-terminus of Nox4 and is not required for hydrogen peroxide formation.
| S-EPMC3085945 | biostudies-literature
Project description:Herein, we investigated the effects of new cofactors and inhibitors on an oxidative cleavage of DNA catalysis, known as a pistol-like DNAzyme (PLDz), to discuss its catalytic mechanism. PLDz performed its catalytic activity in the presence of ascorbic acid (AA), in which Cu2+ promoted, whereas Fe2+ significantly inhibited the catalytic function. Since Fe2+/AA-generated hydroxyl radicals are efficient on DNA damage, implying that oxidative cleavage of PLDz had no relation with hydroxyl radical. Subsequently, we used Fe2+/H2O2 and Cu2+/H2O2 to identify the role of hydroxyl radicals in PLDz catalysis. Data showed that PLDz lost its activity with Fe2+/H2O2, but exhibited significant cleavage with Cu2+/H2O2. Because Fe2+/H2O2 and Cu2+/H2O2 are popular reagents to generate hydroxyl radicals and the latter also produces superoxide anions, we excluded the possibility that hydroxyl radical participated in oxidative cleavage and confirmed that superoxide anion was involved in PLDz catalysis. Moreover, pyrogallol, riboflavin and hypoxanthine/xanthine oxidase with superoxide anion and hydrogen peroxide generation also induced self-cleavage of PLDz, where catalase inhibited but superoxide dismutase promoted the catalysis, suggesting that hydrogen peroxide played an essential role in PLDz catalysis. Therefore, we proposed a catalytic mechanism of PLDz in which superoxide anion and hydrogen peroxide mediated an oxidative cleavage process.
| S-EPMC5428237 | biostudies-literature
Project description: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.
| S-EPMC6500117 | biostudies-literature
Project description: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.
| S-EPMC8397891 | biostudies-literature
Project description:BackgroundComponents of the antioxidant defense system in Trypanosoma cruzi are potential targets for new drug development. Superoxide dismutases (SODs) constitute key components of antioxidant defense systems, removing excess superoxide anions by converting them into oxygen and hydrogen peroxide. The main goal of the present study was to investigate the genes coding for iron superoxide dismutase (FeSOD) in T. cruzi strains from an evolutionary perspective.MethodsIn this study, molecular biology methods and phylogenetic studies were combined with drug assays. The FeSOD-A and FeSOD-B genes of 35 T. cruzi strains, belonging to six discrete typing units (Tcl-TcVI), from different hosts and geographical regions were amplified by PCR and sequenced using the Sanger method. Evolutionary trees were reconstructed based on Bayesian inference and maximum likelihood methods. Drugs that potentially interacted with T. cruzi FeSODs were identified and tested against the parasites.ResultsOur results suggest that T. cruzi FeSOD types are members of distinct families. Gene copies of FeSOD-A (n = 2), FeSOD-B (n = 4) and FeSOD-C (n = 4) were identified in the genome of the T. cruzi reference clone CL Brener. Phylogenetic inference supported the presence of two functional variants of each FeSOD type across the T. cruzi strains. Phylogenetic trees revealed a monophyletic group of FeSOD genes of T. cruzi TcIV strains in both distinct genes. Altogether, our results support the hypothesis that gene duplication followed by divergence shaped the evolution of T. cruzi FeSODs. Two drugs, mangafodipir and polaprezinc, that potentially interact with T. cruzi FeSODs were identified and tested in vitro against amastigotes and trypomastigotes: mangafodipir had a low trypanocidal effect and polaprezinc was inactive.ConclusionsOur study contributes to a better understanding of the molecular biodiversity of T. cruzi FeSODs. Herein we provide a successful approach to the study of gene/protein families as potential drug targets.
| S-EPMC9169349 | biostudies-literature
Project description:Mitochondrial reactive oxygen species (ROS) play important roles in cellular signaling; however, certain pathological conditions such as ischemia/reperfusion (I/R) injury disrupt ROS homeostasis and contribute to cell death. A major impediment to developing therapeutic measures against oxidative stress-induced cellular damage is the lack of a quantitative framework to identify the specific sources and regulatory mechanisms of mitochondrial ROS production. We developed a thermodynamically consistent, mass-and-charge balanced, kinetic model of mitochondrial ROS homeostasis focused on redox sites of electron transport chain complexes I, II, and III. The model was calibrated and corroborated using comprehensive data sets relevant to ROS homeostasis. The model predicts that complex I ROS production dominates other sources under conditions favoring a high membrane potential with elevated nicotinamide adenine dinucleotide (NADH) and ubiquinol (QH2) levels. In general, complex I contributes to significant levels of ROS production under pathological conditions, while complexes II and III are responsible for basal levels of ROS production, especially when QH2 levels are elevated. The model also reveals that hydrogen peroxide production by complex I underlies the non-linear relationship between ROS emission and O2 at low O2 concentrations. Lastly, the model highlights the need to quantify scavenging system activity under different conditions to establish a complete picture of mitochondrial ROS homeostasis. In summary, we describe the individual contributions of the electron transport system complex redox sites to total ROS emission in mitochondria respiring under various combinations of NADH- and Q-linked respiratory fuels under varying workloads.
| S-EPMC8788716 | biostudies-literature