Light-enhanced free radical formation and trypanocidal action of gentian violet (crystal violet).

Transmission of Chagas' disease by transfusion of blood containing Trypanosoma cruzi has often been reported, and gentian violet, a triarylmethane dye, is widely used by blood banks in attempts to eliminate such transmission. In a study of intact trypanosomes, gentian violet was found to undergo a one-electron reduction to produce a carbon-centered free radical as demonstrated by electron spin resonance spectroscopy. Either reduced nicotinamide adenine dinucleotide or the reduced dinucleotide phosphate could serve as a source of reducing equivalents for the production of this free radical by homogenates of Trypanosoma cruzi. The formation of this free radical, and the trypanocidal action of gentian violet, were enhanced by light. The enhanced free radical formation may be the basic cause of the selective toxicity of gentian violet to Trypanosoma cruzi.

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.

Evaluation of the toxicity of 3-allyl-beta-lapachone against Trypanosoma cruzi bloodstream forms.

In vitro incubation of Trypanosoma cruzi (Y strain) with 3-allyl-beta-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 H2O2 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.

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.

Clinical and electrophysiologic characteristics of patients with antidromic circus movement tachycardia in the Wolff-Parkinson-White syndrome.

Antidromic circus movement tachycardia was documented in 36 of 345 consecutive patients with Wolff-Parkinson-White syndrome undergoing detailed electrophysiologic evaluation. Twenty-six patients were men and 10 were women (mean age +/- standard deviation 26 +/- 12 years [range 12 to 45]). Multiple accessory pathways were identified in 12 of these 36 patients (33%). Ten of the patients (67%) with clinically documented antidromic tachycardia had multiple accessory pathways. Dizziness and syncope occurred in 61 and 50% of patients with antidromic circus movement tachycardia. Six patients had clinical documentation of atrial fibrillation, and 4 patients (11%) were resuscitated from ventricular fibrillation. In the 36 patients, 56 distinct antidromic tachycardias were recorded and several different pathways were observed. Orthodromic tachycardia was the most frequently associated arrhythmia (72%). Dual atrioventricular nodal pathways were present in 12 patients (33%); however, atrioventricular nodal tachycardia could be initiated in only 2 of them. Interruption of the accessory pathway was successfully performed in all 20 patients undergoing surgery.

Prevention of transfusion-induced Chagas' disease by amphotericin B.

Amphotericin B, a polyene antibiotic effective against eukaryotic cells, can eliminate the trypomastigote form of Trypanosoma cruzi from blood stored at 4 degrees C. This antitrypanosomal effect can be achieved with a concentration of 3 micrograms/ml within 48 hours. This concentration of amphotericin B does not produce hemolysis over a period of 3 weeks. Amphotericin B methyl ester and nystatin are not effective. Amphotericin B may be considered as a replacement for crystal violet in blood bank blood to prevent transfusion-induced Chagas' disease.

Effect of beta-lapachone on hydrogen peroxide production in Trypanosoma cruzi.

beta-Lapachone, an antimicrobial agent, markedly increase the generation of H2O2 in intact Trypanosoma cruzi epimastigotes (Y strain). Increase in H2O2 was determined by the horseradish peroxidase-H2O2 Compound II formation as well as by a cytochemical technique.

Generation of free radicals from phenazine methosulfate in Trypanosoma cruzi epimastigotes.

A significant growth inhibition of Trypanosoma cruzi epimastigotes by phenazine methosulfate (PMS) was observed in Warren's medium. This toxic activity could be related to the following parameters: a) formation of phenazinium free radical, b) generation of superoxide anion in intact cells incubated with PMS, and c) PMS also increased significantly the rate of O2- generation in epimastigotes mitochondrial and microsomal fractions using NADH as electron donor.

The photodynamic action of rose bengal on Trypanosoma cruzi.

In the presence of light and oxygen, rose bengal causes oxidative damage to Trypanosoma cruzi. The production of lipid hydroperoxides was demonstrated by thin-layer chromatography, and severe ultrastructural alterations compatible with an increased permeability of the cells, which led to gradual osmotic swelling and ultimately to lysis, were observed by electron microscopy. As a result of this treatment, the infectivity of T. cruzi trypomastigotes in mice was abolished. In addition, under anaerobic conditions, rose bengal was found to undergo a one-electron reduction in intact T. cruzi epimastigotes to produce a carbon-centered free radical as demonstrated by electron spin resonance spectroscopy. The formation of this radical was also enhanced by light.

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.

Antibacterial activity of a substance produced by the fungus Pycnoporus sanguineus (Fr.) Murr.

A fraction obtained from the culture fluids of Pycnoporus sanguineus fungus was shown to contain a compound with biological activity against strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and members of the genus Streptococcus. The fraction was clearly more active on Gram-positive cocci than on Gram-negative bacilli.

Prevention of Chagas' disease resulting from blood transfusion by treatment of blood: toxicity and mode of action of gentian violet.

Blood transfusion is the second most important mechanism of transmission of Chagas' disease. Gentian violet, a cationic dye, is currently used in blood banks in endemic areas in attempts to eliminate such transmission. A photodynamic action of gentian violet has been demonstrated in Trypanosoma cruzi. Visible light causes photoreduction of gentian violet to a carbon-centered radical. Under aerobic conditions this free radical autooxidizes generating superoxide anion whose dismutation yields hydrogen peroxide. This photodynamic action of gentian violet is thus probably mediated by the oxygen reduction products. Since irradiation with visible light in the presence of sodium ascorbate reduces the effective dose and time of contact of the dye with T. cruzi-infected blood, a possible application of these findings can be envisaged. In addition to this photodynamic action, an uncoupling effect of gentian violet on mitochondrial oxidative phosphorylation has been described in rat liver and T. cruzi mitochondria. Gentian violet released respiratory control, hindered ATP synthesis, enhanced ATPase activity, released the inhibition of State 3 respiration by oligomycin, and produced swelling of isolated rat liver mitochondria or T. cruzi mitochondria in situ. Taken together, these results indicate that the T. cruzi mitochondrion is the main target of gentian violet toxicity in the dark.

Chemically defined media for growing anaerobic bacteria of the genus Veillonella.

A chemically defined medium for Veillonella parvula and V. alcalescens is described. Some nutritional aspects of the two strains used were examined: the optimum concentration of reducing agents, the requirements for amino acids, diamines, vitamins and other growth factors, and the conditions needed for well balanced nutrition. No specific requirements for single amino acids were observed. A combination of L-cysteine, DL-aspartic acid, L-glutamic acid, L-serine and L-tyrosine, promoted growth. In V. alcalescens, serine could substitute both arginine and tryptophan (or histidine). No growth was obtained with ammonium salts as the sole N source. Decarboxylation of L-ornithine, L-lysine and L-arginine was not demonstrated in the Veillonella parvula strain, which required putrescine or cadaverine for growth. Spermine, spermidine, L-lysine, L-ornithine and L-arginine, could not substitute putrescine in Veillonella parvula. Veillonella alcalescens, which does not require putrescine in the medium, was able to decarboxylate L-ornithine while forming putrescine.

Toxicity of 4-nitroquinoline 1-oxide for Crithidia fasciculata.

Growth inhibition of Crithidia fasciculata by 4-nitroquinoline 1-oxide (NQO) was observed in defined and complex media at 28 C. Aromatic amino acids, cystein, and nicotinic acid, among several other substances, were ineffective in overcoming NQO toxicity. Dicoumarol and bovine albumin reversed NQO inhibition. While bovine albumin probably acted by the extra-cellular binding of NQO, dicoumarol inhibited the activity of DT-diaphorase, which reduces NQO to 4-hydroxyaminonitroquinoline 1-oxide (HAQO). The DT-diaphorase from C. fasciculata had the same characteristics as the enzyme from rat liver. The specific protection by dicoumarol against NQO inhibition suggests that HAQO is the active toxic substance for C. fasciculata.

Generation of superoxide anions and hydrogen peroxide from beta-lapachone in bacteria.

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

Acetate oxidation by bloodstream forms of Trypanosoma cruzi.

Bloodstream forms of Trypanosoma cruzi had a substantial increase in respiration in the presence of acetate. Oxidation of acetate took place via the tricarboxylic acid cycle and involved an antimycin A-sensitive respiratory pathway. Oxygen uptake in the presence of acetate was a sensitive to antimycin A inhibition as was CO2 production. There was a 6--7% residual O2 uptake which was not inhibited by high antimycin concentrations. Human anti-T. cruzi sera had no effect on oxygen uptake.

Ureteral involvement secondary to diverticulitis of the colon.

Ureteral involvement in instances of diverticulitis of the colon is quite unusual. The left ureter is more commonly involved but right sided and bilateral instances have been reported. There are three types of involvement: fistula, stricture and compression, the latter being the most frequent. Roentgenographic characteristics and the effects of various treatment modalities on the affected ureter are presented herein.