Fernando S. Cruz

Mechanism of nifurtimox toxicity in different forms of Trypanosoma cruzi

Abstract Addition of nifurtimox (a nitrofuran derivative) to NAD(P)H-containing homogenates of Trypanosoma cruzi (epi-, trypo- or amastigote forms) determined the appearance of an e.p.r. spectrum that could be identified as corresponding to the nitroaromatic anion radical. The anion radical signal was observed after an induction period that depended on the oxygen concentration and the pyridine nucleotide in the incubation medium. Incubation of intact T. cruzi forms with nifurtimox also led to the appearance of the anion radical signal. The nitroaromatic anion radical, which is assumed to be the first product of nitroreductase activity, reacted with oxygen under aerobic conditions, as shown by the increased rate of superoxide anion and hydrogen peroxide production after addition of nifurtimox to homogenates of T. cruzi in the presence of NAD(P)H. The nifurtimox-induced peroxide production was higher with T. cruzi amastigotes than with epi- or trypomastigotes.

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.

Trypanosoma cruzi: ultrastructural and metabolic alterations of epimastigotes by β-lapachone.

Abstract β-Lapachone (a 1,2-naphthoquinone derivative) causes ultrastructural and metabolic alterations in Trypanosoma cruzi epimastigotes. In β-lapachone-treated cells 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 are also observed. Concomitant with the ultrastructural alterations was a reduction in the respiratory rate and inhibition of glucose and pyruvate oxidation.

Superoxide anion production and trypanocidal action of naphthoquinones on Trypanosoma cruzi.

1. O2− production is involved in the toxic action of naphthoquinones against Trypanosoma cruzi as is inferred from the correlation between quinone effect upon growth and the rate of O2− release from quinone-treated epimastigotes. 2. Mitochondrial membranes contribute to O2− generation in naphthoquinone-treated T. cruzi epimastigotes as is suggested by the correlation between the release of O2− from intact cells and the formation of O2− from the mitochondrial fraction.

Generation of Superoxide Anions and Hydrogen Peroxide from β-Lapachone in Bacteria

β-Lapachone markedly increased the generation of superoxide anions and hydrogen peroxide by subcellular membranes of Bacillus subtilis and Bacillus stearothermophilus. Peroxide generation by β-lapachone was parallel to the inhibition of growth in both microorganisms.

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.

Photosensitization by the trypanocidal agent crystal violet. Type I versus type II reactions

The photoreduction of crystal violet to a carbon-centered radical was detected directly by electron spin resonance (ESR) spectroscopy under anaerobic conditions. The linewidth (0.9 G) of this radical was less broad than the linewidth (11.0 G) of the free radical obtained in Trypanosoma cruzi incubations. No crystal violet radical could be detected under aerobic conditions. However, crystal violet was found to convert oxygen to superoxide anion and hydrogen peroxide in the presence of light. This superoxide anion and hydrogen peroxide formation was greatly enhanced by reducing agents such as NAD(P)H. In addition, irradiation of crystal violet did not generate detectable amounts of singlet oxygen.

Trypanosoma cruzi: Effect of olivacine on macromolecular synthesis, ultrastructure, and respiration of epimastigotes

Abstract Olivacine (a pyridocarbazole derivative) causes ultrastructural and metabolic alterations in Trypanosoma cruzi epimastigotes. The cytoplasm of cells grown in the presence of olivacine appears vacuolated, and swelling of the mitochondria is observed. Concomitant with the ultrastructural alterations, there was a reduction of the respiratory rate as well as inhibition of pyruvate oxidation. A marked inhibition of protein synthesis and a slower although significant inhibition of DNA, RNA, and lipid synthesis were also observed. The in vivo activity of olivacine does not parallel its in vitro effects suggesting inactivation of the drug by the host.