Exposing murine macrophages infected th the protozoan parasite Toxoplasma gondii to micromolar concentrations of some cationic electron carriers (dyes), resulted in complete killing of the intracellular parasites at concentrations at which these compounds did not seem toxic for the macrophages. The 50% inhibitory concentrations (with 95% confidence limits) were calculated as 0·26 (0·18-0·37), 1·35 (1-2·25), 0·45 (0·13-1·50), and 1·52 (0·91-2·53) μM for crystal violet, phenazine methosulphate, methylene blue and brilliant cresyl blue, respectively. The effects of these electron carriers did not appear to be the result of an enhancement of the natural antitoxoplasmic activity of the macrophages. None of the tested compounds was active against extracellular Tox. gondii as measured by ability to reinfect murine macrophages; thus, these dyes seem to act primarily on actively metabolizing, intracellular, Tox. gondii. Our data also suggest that the killing effect of the electron carriers was not related to the generation of reactive oxygen intermediates as judged by the inability of scavengers of these intermediates to block the activity against intracellular Tox. gondii. Further studies with related redox compounds would have an interesting chemotherapeutic perspective for treating toxoplasma infection

Chang, Hernan R.

Pechère, Jean-Claude

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Journal of Antimicrobial Chemotherapy (1989) 23, 229-235In-vitro toxoplasmacidal activity of cationic electron carriersHeraan R. Chang and Jean-Clande PecbereDepartment of Microbiology, University of Geneva Medical School,C.M.U., 9 av. de Champel, 1211 Geneva 4, SwitzerlandExposing murine macrophages infected with the protozoan parasite Toxoplasmagondii to micromolar concentrations of some cationic electron carriers (dyes),resulted in complete killing of the intracellular parasites at concentrations at whichthese compounds did not seem toxic for the macrophages. The 50% inhibitoryconcentrations (with 95% confidence limits) were calculated as 0-26 (018-0-37),1-35 (1-2-25), 0-45 (013-1-50), and 1-52 (0-91-2-53) tm for crystal violet, phenazinemethosulphate, methylene blue and brilliant cresyl blue, respectively. The effects ofthese electron carriers did not appear to be the result of an enhancement of thenatural antitoxoplasmic activity of the macrophages. None of the tested compoundswas active against extracellular Tox. gondii as measured by ability to rcinfect murinemacrophages; thus, these dyes seem to act primarily on actively metabolizing,intracellular, Tox. gondii. Our data also suggest that the killing effect of the electroncarriers was not related to the generation of reactive oxygen intermediates as judgedby the inability of scavengers of these intermediates to block the activity againstintracellular Tox. gondii. Further studies with related redox compounds would havean interesting chemotherapeutic perspective for treating toxoplasma infections.IntroductionToxoplasma gondii is a ubiquitous protozoan parasite which can cause disease in man.No satisfactory cidal therapy is available for treating life-threatening toxoplasmainfections which can occur among patients with the Acquired Immune DeficiencySyndrome (AIDS).It seems to be well established that components of the cellular immune response(mononuclear phagocytic system) are the main effectors of defence against this obligateintracellular parasite. Activated macrophages are able to kill Tox. gondii in vitro, andthis killing seems to be associated with the ability of the macrophages to releasereactive oxygen intermediates (ROI) such as superoxide anion, hydrogen peroxide andhydroxyl radical (Nathan et al., 1983; Murray, Spitalny & Nathan, 1985). Studies haveshown that cationic electron carriers, which are thought to undergo redox-cycling andgenerating superoxide anion and hydrogen peroxide (Hassan & Fridovich, 1979), wereable to inhibit intracellular Trypanosoma cruzi and Leishmania spp. (Rabinovitchet al., 1982; Alves & Rabinovitch, 1983; Mauel, Schnyder & Baggjolini, 1984). One ofthese electron carriers, the triarylmethane dye crystal violet (gentian violet), was shownto be active against extracellular Tryp. cruzi in vitro (Nussenzweig et al., 1953). Sincethen, this dye has been used by blood banks in Latin-American endemic countries inan attempt to reduce the transmission of Chagas' disease. The mechanism of action of2290305-7453/89/020229+07 $02.00/0 © 1989 The British Society for Antimicrobial Chemotherapy230 H. R. Chug and J.-C. PecMrethese compounds, however, has not been elucidated. Here, the in-vitro activity ofcationic electron carriers against extracellular and intracellular tachyzoites of Tox.gondii was investigated.Materials and methodsAnimalsFemale Swiss-Webster mice (Madorin, FiiUinsdorf, Switzerland), weighing 23 to 25 geach, were used in all experiments.MacrophagesAnimals were killed by CO2 asphyxiation, their resident peritoneal macrophages wereharvested from their peritoneal cavities as described previously (Chang & Pechere,1988), and the concentration of mononuclear cells was adjusted to 2x 106/ml withmedium 199 (M199) supplemented with 10% fetal calf serum (FCS) (pH 7-2). Eachwell of a 96-well tray (Costar, Cambridge, Massachussetts), or each chamber of four-chamber Lab-Tek slides (Lab-Tek Div., Miles Laboratories Inc., Naperville, Illinois),was seeded with 2 x 103 and 2 x 106 cells, respectively. They were incubated for 2-3 hat 37°C in a 5% CO2-95% air humid atmosphere. The nonadherent cells werediscarded by washing twice with prewarmed Hanks' balanced salt solution (HBSS).Electron carriersThe following electron carriers were obtained from Fluka AG, Buchs, Switzerland:brilliant cresyl blue (mol. wt, 385-96), crystal violet (mol. wt, 407-99), methylene blue(mol. wt, 319-86) and phenazine methosulfate (mol. wt, 306-34). They were dissolved inculture medium and sterilized by nitration immediately before use.Scavengers of oxygen intermediatesScavengers of oxygen intermediates (Catterall, Sharma & Remington, 1986) wereobtained from Sigma GmbH, Deisenhofen, West Germany, and used at theconcentrations shown. Superoxide dismutase, 3000 U/mg (2-5 mg/ml) was used forsuperoxide anion; catalase from bovine liver recrystallized twice (2-5 mg/ml) forhydrogen peroxide; and histidinc (10 mM) and diazabicyclooctane (1 ITLM) for singletoxygen radicals. The scavengers of hydroxyl radicals were benzoic acid (10 mM),mannitol (50 mM) and tetramethylurea (25 mM). These scavengers were added to themonolayers immediately after the 1 h challenge with Tox. gondii and 30 min before theaddition of the cationic electron carriers. All these experiments were performed over a24-h period of incubation.Tox. gondii infection and microbicidal assaysCell cultures were challenged for 1 h with 2 x 10s (96-well trays) or 2 x 106 (Lab-Tekslides) RH strain Tox. gondii tachyzoites. After uningested parasites had been removedby washing, standard medium containing reagents was added and the monolayers wereCationk etectroo carriers T. toxoplasma in vitro 231pulsed with 2-5 /iCi of [5-6-3H]uracil (specific activity 49 Ci/mmol; Amersham pic,Buckinghamshire, England). After a 24-h period of incubation the radioactivity wascounted in the acid-precipitable material by filtration procedure (Chang & Pechere,1988). The inhibition of the intracellular Tox. gondii growth rate was thus assessed byusing [3H]uracil, a precursor which is incorporated by Tox. gondii, but not the hostcells (Chang & Pechere, 1988). In addition, the outcome of intracellular infection wasassessed by microscopically counting the number of infected cells and the number ofintracellular parasites per 100 cells in Giemsa-stained preparations. Anantitoxoplasmic effect was indicated by a decrease in the parameters considered at24 h.Effects of electron carriers on extracellular toxoplasmasFresh suspensions of Tox. gondii (3-6 x 106 tachyzoites/ml) were incubated in 5%CO2-95% air at 37°C with the electron carriers for 30min at the desiredconcentrations. The suspensions were centrifuged at 50 g for lOmin, washed withphosphate-buffered saline (pH 7-2), centrifuged again and resuspended in Ml99 with3% FCS. They were used to challenge macrophage monolayers for [3H]uracilexperiments and for Giemsa-stained preparations.Effect of electron carriers on antitoxoplasmic activity of resident macrophagesMouse macrophage monolayers were exposed for 24 h to the electron carriers at thedesired concentrations. At the end of the incubation period, the monolayers werewashed three times with prewarmed HBSS and challenged with freshly harvested Tox.gondii for 1 h. Assessment of Tox. gondii inhibition was made after a 24-h incubationperiod in medium without the electron carriers.Assessment of toxicity on macrophagesAdherent macrophage monolayer cells were incubated for 24 h with differentconcentrations of the electron carriers and their viability was assessed as describedpreviously (Chang & Pechere, 1988), by the trypan blue dye exclusion test. In addition,macrophage monolayers were examined in Giemsa-stained preparations, after 24 hincubation, for the number of cells per high power field and assessed for theirmorphological characteristics-.StatisticsData are reported as the mean ± standard error of the mean (SEM). The 50%inhibitory concentrations (IC30), the 95% confidence limits, and the IC90 w e r ecalculated by probit analysis following the method of Litchfield & Wilcoxon (1949).differences between values were analyzed by the Student's r-test. A P value <0-05 wasconsidered significant.ResultsActivity of electron carriers on intracellular toxoplasmasInfected murine peritoneal macrophages were exposed to increasing amounts of theelectron carriers in concentrations from 0001 to 35/ZM, and the growth rate of232 H. R. Chang and J.-C. PecbereTable I. Activity of cationic electron carriers against intracellular Tox.gondii as assessed by [3H]uracil assayTreatment of infectedmacrophagesCrystal violetPhenazinemethosulphateMethylene blueBrilliant cresyl blue50% Inhibitoryconcentration (IC50)(jot) (95% fiducialrange)0-26 (018-0-37)1-35 (1-00-2-25)0-45 (0 13-1-50)1-52 (0-91-2-53)90% Inhibitoryconcentration (IC,0)0-432-382-383-24intracellular Tox. gondii was assessed by measuring the uptake of [3H]uracil over aperiod of 24 h. Micromolar concentrations of all electron carriers tested were able torestrict the intracellular multiplication of the parasite. This allowed calculation of theirICS0 and IC90 as shown in Table I. Table n summarizes the results of experimentsperformed with microscopically assessed preparations. All electron carriers at theirrespective IC90, according to the [3H]uracil incorporation studies, significantly reducedthe number of infected cells and the number of toxoplasmas per 100 cells, and athigher concentrations allowed complete eradication of the parasites from themacrophages. These effects were seen at concentrations which were not toxic for themacrophages according to the trypan blue dye exclusion test and morphologicalcriteria. By raising the concentrations, toxic effects on the cells however, appeared withincreased concentrations of crystal violet, phenazine methosulphate and methylene blue(Table II).Effect of electron carriers on extracellular toxoplasmasThe electron carriers did not alter the viability of extracellular Tox. gondii, assessed bytheir ability to reinfect macrophage cultures (Table III).Table II. Activity of cationic electron carriers against intracellular Tox. gondii as assessed bymicroscopyTreatment of infectedmacrophages withelectron carriers (jot)Medium only (control)Crystal violetPhenazinemethosulphateMethylene blueBrilliant cresyl blue(0-43)(2-38)(5)(2-38)(15)(3.24)(31)%ofinfectedcells56±40*8±1»0i7±1»0*5±1»0*Number oftoxoplasmasper 100 cells717±80*16±T0*11 ± 1 '0*11±1'0*Minimal concentrationfor toxicity to macrophagemonolayers (/u*)"0-55730No toxicity up to 35Results are the mean ±SEM of three experiment!.'Assessed by trypan blue dye exclusion and morphological criteria.*P<0-01, compared to control; 'P< 0-001, compared to control; 'P < 0-0001, compared to control;'P < 0-00001, compared to control.Cationk electron carrier* T. toxopUsma in ritro 233Tabte m . Effect of cationic electron carriers on extracellular Tox. gondii as assessed bysubculturePretreatment withelectron carriers (JM)Extracellular Tox. gondiiNoneCrystal violetPhenazine methosulpbateMethylene blueBrilliant cresyl blue(0-43)(2-38)(2-38)(3.24)% of infectedcells49±148±253±251±148±1Number of toxoplasmasper 100 macrophages629±6611±7665±9640±5614±8Incorporation ofpHJuradl*10094±8-398±9199 ±6-896 ±7-5Results are the mean ±SEM of three experiments."Expressed as percentage of intracellular inhibition; (100-cpm in test/cpm in control) x 100.Effect on anti-toxoplasma activity of resident macrophagesIn order to test whether or not these compounds produced some degree of macrophageactivation, we incubated resident macrophage monolayers with the different electroncarriers for 24 h, and challenged them with Tox. gondii. None of the tested electroncarriers was able to enhance the antitoxoplasmic activity of the resident mousemacrophages (Table IV).Effect of scavengers of oxygen intermediatesWe further investigated the effect of scavengers of oxygen intermediates on the activityof the cationic electron carriers at their corresponding IC90. The scavengers of ROIdid not block the ability of electron carriers to kill intracellular Tox. gondii at theconcentrations tested (Table V).DiscussionThese studies showed that electron carriers were toxoplasmacidal in vitro atmicromolar concentrations; the parasites were completely eradicated from themacrophages at some of the concentrations tested. The combination of pyrimethamineTable IV. Effect of cationic electron carriers on antitoxoplasmic activity of macrophagesPretrcatment withelectron carriers (jot)Resident mouse macrophagesNoneCrystal violetPhenazine methosulphateMethylene blueBrilliant cresyl blue(0-43)• (2-38)(238)(3-24)% of infectedcells47±145±251±247±350±2Number of toxoplasmasper 100 macrophages597 ±8591 ±7628±9594±6583 ±12Incorporation of['HJuracU*10095 ±3-893 ±7-694±7-897 ±5-3Results are the mean ±SEM of three experiments.•Expressed as percentage of intracellular inhibition: (100-cpm in test/cpm in control) x 100.234 H. R. Chang and J.-C PtcMrcTable V. Effects of scavengers of oxygen intermediates on the activity of cationic electroncarriers against intracellular Tox. goruUfTreatment of infectedmacrophages*Crystal Phenazine Methylene BrilliantNone violet methosulphate blue cresyl blueMedium only 100Catalase (2-5 mg/ml) 102±5-7Superoxide dismutase (2-5 mg/ml) 96±6-9Histidine (10 HIM) 97 ±7-9Diazabicyclooctane (mM) 94 ±9-7Mannitol (50 mu) 99 ±5-2Benzoic acid (25 mM) 97 ±6-8Tetramethylurea (25 mM) 98 ± 5-38-5 ±1-34-9 ±0-74-9 ±0-8100±l-36-8 ±1-910-7±2-l5-7 ±0-95-2+1-216-8±2-321-3±2-724-8 ± 3 021-9±2-418-1 ±2-613-0±3-5ll-2±l-6130±l-215-5±3-823-5±l-O17-3±3-216-8 ±2-419-7 ±3-519-7±3-618-3±2-l19-4 ±1-816-9±3-519 4 ± 1 025-5±31140 ±2-213-7±0-517-5±0-817-8±l-013-9 + 0-3"Measured by [3H]uracil assay, and expressed as percentage of intracelhilar inhibition as stated inTable III and IV.*The electron carriers were added at their respective 90% inhibitory concentrations as shown in Table I.and sulphadiazine, investigated in a similar system, was also active against Tox. goiuiiiat the micromolar level, but exerted less killing effect (Chang & Pechere, 1988).The electron carriers could have stimulated the macrophages to kill the parasites orcould have acted directly on the intracellular parasites. Regarding a macrophage-mediated mechanism, some studies have suggested that the intracellular killing ofleishmania by electron carriers, excepting crystal violet, correlated with the stimulationof the hexose monophosphate shunt, and with the generation of ROI (Maud et al.,1984). Here, stimulation of the release of ROI was unlikely since the antitoxoplasmaactivity of the electron carriers was not altered by the scavengers of ROI. Pretreatmentof macrophages with electron carriers did not enhance the antitoxoplasmic activity ofthese cells, an observation which also tips the balance in favour of a direct antiparasiticeffect. Since the electron carriers were not active against extracellular Tox. gondii, butonly against actively growing intracellular parasites, an interference with the parasites'metabolism by the electron carriers is suggested. Some arguments for direct damage tothe intracellular parasites can be found elsewhere. Phenazine methosulphate has beenfound to accumulate in the parasitophorous vacuoles of macrophages infected withleishmania (Rabinovitch et al., 1982). A mechanism for a direct anti-Tryp. cruzi effecthas been proposed for crystal violet, consisting in a photodynamic action apparentlymediated by the formation of carbon-centred free radicals in the parasites themselves(Docampo et al., 1983). An alternative mechanism could be direct oxidative damage tothe parasites. In this view, a correlation of between toxoplasmacidal action and theredox potential of the electron carriers would have been expected. This was not thecase, here, nor with other intracellular parasites also susceptible to the electron carriers(Rabinovitch et al., 1982; Alves & Rabinovitch, 1983; Mauel et al., 1984). However,such correlation is likely to be obscured by other structural requirements for drugpermeation (Rabinovitch et al., 1983; Mauel et al., 1984).In conclusion, we have shown that these electron carriers are toxoplasmacidal, butthe mechanisms of their impressive activity remain unclear. Further investigation iswarranted with these and related compounds because of their interestingchemotherapeutic perspective.Catioolc electron carriers T. toxoplasma in vitro 235AcknowledgementsThis work was supported in part by grant 3.221-085 from the Fonds National Suissede la Recherche Scientifique and in part by grant G4/88 from the Finanz-Pool 3RFoundation.ReferencesAJvcs, M. J. M. & Rabinovitch, M. (1983). Destruction of intracellular Trypanosoma cruzi aftertreatment of infected macrophages with cationk electron carriers. Infection and Immunity39, 435-8.Catterall, J. R., Sharma, S. D. & Remington, J. S. (1986). Oxygen-independent killing byalveolar macrophages. Journal of Experimental Medicine 163, 1113-31.Chang, H. R. & Pechere, J.-C. F. (1988). In vitro effects of four macrolides (roxithromycin,spiramycin, azithromycin [CP-62,993], and A-56268) on Toxoplasma gondii. AntimicrobialAgents and Chemotherapy 32, 524-9.Docampo, R., Moreno, S. N. J., Muniz, R. P. A., Cruz, F. S. & Mason, R. P. (1983). Light-enhanced free radical formation and trypanocidal action of gentian violet (crystal violet).Science 220, 1292-5.Hassan, H. M. & Fridovich, I. (1979). Intracellular production of superoxide radical and ofhydrogen peroxide by redox active compounds. Archives of Biochemistry and Biophysics196, 385-95.litchfield, J. T. & Wikoxon, F. (1949). Simplified method of evaluating dose-effect experiments.Journal of Pharmacology and Experimental Therapeutics 96, 99-113.Mauel, J., Schnyder, J. & Baggiolini, M. (1984). Intracellular parasite killing induced by electroncarriers. II. Correlation between parasite killing and the induction of oxidative events inmacrophages. Molecular and Biochemical Parasitology 13, 97-110.Murray, H. W., Spitalny, G. L. & Nathan, C. F. (1985). Activation of mouse peritonealmacrophages in vitro and in vivo by interferon-y. Journal of Immunology 134, 1619-22.Nathan, C. F., Murray, H. W., Wiebe, M. E. & Rubin, B. Y. (1983). Identification ofinterferon-y as the lymphokine which activates human macrophage oxidative metabolismand antimicrobial activity. Journal of Experimental Medicine 158, 670-89.Nussenzweig, V., Sonntag, R., Biancalana, A., Pedreira de Freitas, J. L., Amado Neto, V. &Kloetzel, J. (1953) Acaode corantes trifenil-metanicos sobre o Trypanosoma cruzi 'in vitro'.Emprego de violeta de genciana na profilaxia da transmissao da molestia de Chagas portransfusSo de sangue. Hospital (Rio de Janeiro) 44, 731-44.Rabinovitch, M., Dedet, J.-P., Ryter, A., Robincaux, R., Topper, G. & Brunet, E. (1982).Destruction of Leishmania mexicana amazonensis amastigotes within macrophages inculture by phenazine methosulfate and other electron carriers. Journal of ExperimentalMedicine 155,415-31.(Received 14 September 1988; accepted 12 October 1988)

In-vitro toxoplasmacidal activity of cationic electron carriers

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