Roberto Docampo

Presence of a Plant-like Proton-pumping Pyrophosphatase in Acidocalcisomes of Trypanosoma cruzi

The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper demonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyrophosphate promoted organellar acidification in permeabilized amastigotes, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ions and pyrophosphate analogs, as is the plant activity. Separation of epimastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity measured both by proton uptake and phosphate release but lacked markers for mitochondria, lysosomes, glycosomes, cytosol, and plasma membrane. Antiserum raised against specific sequences of the plant V-H+-PPase cross-reacted with a T. cruziprotein, which was also detectable in the dense Percoll fraction. The organelles in this fraction appeared by electron microscopy to consist mainly of acidocalcisomes (acidic calcium storage organelles). This identification was confirmed by x-ray microanalysis. Immunofluorescence and immunoelectron microscopy indicated that the V-H+-PPase was located in the plasma membrane and acidocalcisomes of the three different forms of the parasite. Pyrophosphate was able to drive calcium uptake in permeabilized T. cruzi. This uptake depended upon a proton gradient and was reversed by a specific V-H+-PPase inhibitor. Our results imply that the phylogenetic distribution of V-H+-PPases is much wider than previously perceived but that the enzyme has a unique subcellular location in trypanosomes.

Different behaviors of benznidazole as free radical generator with mammalian and Trypanosoma cruzi microsomal preparations

Abstract Benznidazole (a nitroimidazole derivative used for the treatment of Chagas disease) is reduced by rat liver microsomes to the nitro anion radical, as indicated by ESR spectroscopy. Addition of benznidazole to rat liver microsomes produced an increase of electron flow from NADPH to molecular oxygen, and generation of both superoxide anion and hydrogen peroxide. The benznidazole-stimulated O2 consumption and O2⨪ formation was greatly inhibited by NADP+ and p-chloromercuribenzoate but not by SKF-525-A and metyrapone. The former inhibitions indicated the involvement of NADPH-cytochrome P-450 (c) reductase, while the lack of inhibition by SKF-525-A and metyrapone ruled out any major role for cytochrome P-450 in benznidazole reduction. In contrast to nifurtimox, a nitrofuran derivative ( R. Docampo and A. O. M. Stoppani, 1979 , Arch. Biochem. Biophys.197, 317–321), benznidazole was not reduced to the nitro anion radical, nor did it stimulate oxygen consumption, O2⨪ production, and H2O2 generation by Trypanosoma cruzi cells or microsomal fractions. A different mechanism of benznidazole toxicity in T. cruzi and the mammalian host is postulated.

Lipid peroxidation and the generation of free radicals, superoxide anion, and hydrogen peroxide in β-lapachone-treated Trypanosoma cruzi epimastigotes

Abstract β-Lapachone, an antimicrobial agent, was reduced by Trypanosoma cruzi epimastigotes to a semiquinone radical. It markedly increased the generation of superoxide anion and hydrogen peroxide in intact cells. Using NADH as electron donor, β-lapachone also increased significantly the rate of H2O2 generation in epimastigote homogenates. Incubation of epimastigotes with β-lapachone stimulated lipid peroxidation.

β-lapachone enhancement of lipid peroxidation and superoxide anion and hydrogen peroxide formation by Sarcoma 180 ascites tumor cells

Abstract Addition of β-lapachone, an o -naphthoquinone endowed with antitumor properties for Sarcoma 180 cells, induced the formation of a semiquinone radical. β-Lapachone was able to stimulate Superoxide anion and hydrogen peroxide production by the mitochondrial fraction supplemented with NADH. β-Lapachone also increased O − 2 and H 2 O 2 production by the microsomal fraction with NADPH as reductant. Cyanide-insensitive NADH and NADPH oxidations by the mitochondrial and microsomal fractions (quinone reductase activity) were stimulated to about the same extent by β-lapachone. Incubation of sarcoma cells with β-lapachone stimulated lipid peroxidation and resulted in a decrease in the viability of the cells. The toxicity of β-lapachone to tumor cells was reduced by incubation of the cells with the free radical scavenger, α-tocopherol. The basic mechanism of the biological action of β-lapachone in sarcoma cells seems to be: (a) reduction at the mitochondrial and microsomal membranes with generation of the semiquinone form, (b) autoxidation of the semiquinone free radical with primary production of O − 2 , (c) production of H 2 O 2 via Superoxide dismutase reaction and generation of HO· from the reaction of O − 2 and H 2 O 2 with subsequent stimulation of lipid peroxidation and decreased viability of the cells.

Evaluation of the toxicity of 3-allyl-β-lapachone against Trypanosma cruzi bloodstream forms

Abstract In vitro incubation of Trypanosoma cruzi (Y strain) with 3-allyl-β-lapachone was followed by: (1) growth inhibition of epimastigotes, (2) damage to cellular membranes, especially of the mitochondria, alterations in the chromatin structure and swelling of mitochondria, (3) increase in the respiratory rate, (4) increase in the rate of H 2 O 2 generation by the epimastigotes, (5) increase of the rate of lipid peroxidation as detected by malonyldialdehyde formation, (6) decrease or total disappearance of trypomastigotes from mouse-infected blood. This drug might therefore be useful in preventing transmission of Chagas disease during blood transfusion. It is not, however, active against infections in mice.

Characteristics of Ca2+ transport by Trypanosoma cruzi mitochondria in situ☆

The use of digitonin to permeabilize Trypanosoma cruzi plasma membrane has allowed the study of Ca2+ transport and oxidative phosphorylation in mitochondria in situ (R. Docampo and A. E. Vercesi (1989) J. Biol. Chem. 264, 108-111). The present results show that these mitochondria are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Addition of Ca2+ or ethylene glycol bis (beta-aminoethyl ether) N-N-tetraacetic acid to these preparations under steady-state conditions was followed by Ca2+ uptake or release, respectively, tending to restore the original Ca2+ set point at about 0.9 microM. In addition, large amounts of Ca2+ were retained by T. cruzi mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butyl hydroperoxide, diamide, and the 1,2-naphthoquinone beta-lapachone. However, when ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine in the presence of antimycin A was used as subtrate, beta-lapachone caused pyridine nucleotide oxidation, and Ca2+ accumulation by these mitochondria was considerably lower than in control preparations, this effect being dose-dependent.

Enhancement of the cytotoxicity of crystal violet against Trypanosoma cruzi in the blood by ascorbate

Blood transfusion is the second most important mechanism of transmission of Chagas disease, and crystal violet is currently used in blood banks in endemic areas in attempts to eliminate such transmission. A photodynamic action of crystal violet against Trypanosoma cruzi trypomastigotes in blood has been detected. This action was enhanced by addition of sodium ascorbate. Photoirradiation of whole blood containing crystal violet increased the concentration of ascorbyl radical and the generation of superoxide anion. Similar results were observed in incubations containing ascorbate and crystal violet in the absence of blood. Hydrogen peroxide generation was also detected in these incubations, thus confirming redox cycling of crystal violet under aerobic conditions. Since photoirradiation and addition of sodium ascorbate reduces significantly the effective dose and time of contact of crystal violet with T. cruzi-infected blood, a possible practical application of these findings is envisaged.

Ultrastructural alterations and peroxide formation induced by naphthoquinones in different stages ofTrypanosoma cruzi

SummaryAddition of β-lapachone, ano-naphthoquinone with bactericidal, cytotoxic, and trypanocidal activities, toTrypanosoma cruzi epimastigote and amastigote stages induced the release of O2− and H2O2 from the whole cells into the suspending medium. In the presence of reduced nicotinamide adenine dinucleotide as reductant β-lapachone was also able to stimulate O2− and H2O2 production by homogenates of these stages. Electron micrographs showed that in β-lapachone-treated amastigotes and trypomastigotes, the chromatin is arranged in patches, clearly differing from the normal pattern of chromatin distribution.Alterations of the nuclear, mitochondrial, and cytoplasmic membranes, as well as swelling of the mitochondrion were also observed.

The mitochondrion of Trypanosoma cruzi is a target of crystal violet toxicity

The first morphological alteration observed in Trypanosoma cruzi different stages upon incubation with crystal violet was mitochondrial swelling. The use of digitonin to solubilize T. cruzi plasma membrane allowed the demonstration of an uncoupling action of crystal violet on epimastigote mitochondria in situ. Low concentrations of crystal violet (20-50 microM) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 0.5 microM) uncoupled the respiratory control mechanism. The inhibition of State 3 respiration by oligomycin was released by crystal violet or FFCCP. Crystal violet released respiratory control, and enhanced ATPase activity of digitonin-permeabilized epimastigotes. Higher concentrations of crystal violet inhibited mitochondrial respiration. The uncoupled effect of crystal violet was stimulated by inorganic phosphate. In addition, crystal violet inhibited endongenous and glucose-stimulated respiration of the intact epimastigotes, and inhibited the Mg2+-ATPase in the epimastigote mitochondrial fractions. The inhibition of this Mg2+-ATPase increased up to pH 9.0 and decreased with increasing protein concentration. These data indicate that the T. cruzi mitochondrion is apparently the main target of crystal violet toxicity.

Reduction of 5-Nitroimidazoles, Nitrofurazone, and 2,4-Dinitrophenol to their Free Radical Metabolites by Tritrichomonas Foetus Hydrogenosomes

Oxidative decarboxylation of pyruvate is catalyzed in trichomonads by pyruvate:ferredoxin oxidoreductase (PFO). This enzyme is localized in characteristic membrane-bound organelles that are termed hydrogenosomes on the basis of their biochemical characteristics. The main metabolic function of hydrogenosomes is the conversion of pyruvate to acetate via acetyl-CoA, accompanied by substrate level phosphorylation coupled to production of molecular hydrogen (Figure 1).