Archives of Medical Research 44 (2013) 415e421

ORIGINAL ARTICLE

Effect of Nitaxozanide and Pyrimethamine on Astrocytes Infected byToxoplasma gondii In Vitro

Ma. de la Luz Galv�an-Ram�ırez,a Judith Marcela Due~nas Jim�enez,a Laura Roc�ıo Rodr�ıguez P�erez,a

Rogelio Troyo-Sanroman,b Mario Ram�ırez-Herrera,a and Trinidad Garc�ıa-Iglesiasc

aLaboratorio de Neurofisiolog�ıa, bDepartamento de Fisiolog�ıa, cLaboratorio de Inmunolog�ıa, Centro Universitario de Ciencias de la Salud,

Universidad de Guadalajara, Jalisco, Mexico

Received for publication January 18, 2013; accepted July 10, 2013 (ARCMED-D-13-00046).

Address reprint re

Researcher Sierra M

Guadalajara, Jalisco,

mlgalvanr@gmail.com

0188-4409/$ - see frohttp://dx.doi.org/10

Background and Aims. T. gondii is a causal agent of encephalitis in immunocompro-mised patients. Pyrimethamine (PYR) has been the treatment of choice for toxoplas-mosis. The aim of this study was to analyze the effect of nitazoxanide andpyrimethamine on astrocytes infected with T. gondii in vitro.

Methods. Rat astrocytes were cultured and infected with T. gondii. The effect of nitazox-anide (10, 20 and 30 mg/mL) and pyrimethamine (7, 10 and 13 mg/mL) on astrocytes in-fected was evaluated at 24 and 48 h post-infection. Tachyzoites and astrocytes weredetected by the immunocytochemical method. T. gondii viability in astrocytes infectedand treated with NTZ and PYR as well as NTZ and PYR citotoxicity on astrocytes in vi-tro were evaluated by the MTT assay.

Results. The number of parasites in astrocytes treated with the drugs was significantlyreduced when compared to control ( p !0.001) at 24 and 48 h. Nitazoxanide produced97% T. gondii death in a concentration of 10 mg/mL in 48 h infected astrocytes. At 48h, the death rate of T. gondii was higher when treated with nitazoxanide than with pyri-methamine. A higher toxicity rate in astrocyte was observed when using pyrimethamineat 40 mg/mL.

Conclusions. Nitazoxanide reduced T. gondii infection more efficiently than pyrimethamineand is not cytotoxic to astrocytes at the administered dose. � 2013 IMSS. Published byElsevier Inc.

Key Words: Effect, Nitazoxanide, Pyrimethamine, Toxoplasma infection, Astrocytes.

Introduction

Toxoplasmosis is a disease caused by an obligate intracel-lular parasite called Toxoplasma gondii discovered byNicolle and Manceaux in 1908. Human infection can occurthrough different mechanisms: oral transmission, bloodtransfusion, via the placenta, organ transplant and by directcontact with the parasites in laboratory accidents and occu-pationally exposed personnel. Toxoplasmosis is a majoropportunistic infection in the central nervous system(CNS) in patients with human immunodeficiency virus

quests to: Ma. de la Luz Galv�an-Ram�ırez, Senior

ojada No. 950 Colonia Independencia C.P. 44340

Mexico; Phone: (þ52) (33) 1058-5313; E-mail:

nt matter. Copyright � 2013 IMSS. Published by Elsevier.1016/j.arcmed.2013.07.002

(HIV) and in other immunosuppressed patients. In this con-dition, the disease manifests as multiple necrotizing lesions,commonly in the deep central nuclei, posterior fossa orlobar regions. Microglial nodule inflammation accompa-nied by extensive necrosis is commonly seen. In humans,proliferating tachyzoites have been detected in glial cellsin patients developing toxoplasmic encephalitis (1). Inaddition, T. gondii bradyzoites were observed in Purkinjecells in the cerebellum of acquired immunodeficiency syn-drome patients (2). Toxoplasma gondii cysts have also beenreported in human astrocytes (3e9). Neurons and astro-cytes in humans can be infected by T. gondii tachyzoites(10). Pyrimethamine (PYR) has been the treatment chosenfor toxoplasmosis since 1952. Toxoplasma and Plasmodiumhave the same susceptibility to PYR (11).

Inc.

416 Galv�an-Ram�ırez et al./ Archives of Medical Research 44 (2013) 415e421

Pyrimethamine is a dihydrofolate dehydrogenase in-hibitor and thus leads to a depletion of dihydro- andtetrahydrofolic acid, followed by an impairment ofnucleotide biosynthesis (12). Considering this actionmechanism, PYR treatment causes severe anemia and,consequently, is always administered simultaneously withfolic acid for preventing this side effect. Furthermore,susceptibility to PYR varies among the different T. gondiistrains (13).

Treatment with PYR and sulfadiazine in newborns in-fected with T. gondii proved to be 67% effective after12 months (14).

Nitazoxanide [2-acetolyloxy-N-(5-nitro-2-thiazolyl)ben-zamide] (NTZ) has been used against helminths, nema-todes, cestodes and trematodes causing infections inhumans. Recent findings employing anaerobic parasiteshave shown that NTZ inhibits pyruvate ferredoxin oxidore-ductase (PFOR), a vital intermediary metabolism enzymein these organisms (15). Thus, NTZ might be the firstexample of an antimicrobial agent targeting the ‘‘activatedcofactor’’ of an enzymatic reaction, rather than its substrateor catalytic sites. This novel mechanism may escapemutation-based drug resistance. It has been reported thatother mechanisms, in addition to the proposed mode of ac-tion involving PFOR, could be responsible for the wideNTZ action spectrum. Modifications in the benzene ringcould also be important in achieving antiparasitic activityagainst Neospora caninum (16).

A great deal of research has been published regardingthe activity of NTZ against intestinal parasites; its overallefficacy against helminth and protozoa ranks from85e97%. NTZ has demonstrated therapeutic efficacyin vitro and in vivo studies as well as in acquired immunedeficiency syndrome (AIDS) patients infected withCryptosporidium parvum (17).

The effects of NTZ and PYR have not been proven inastrocytes infected with T. gondii. This work aims at study-ing the effect of nitazoxanide and pyrimethamine in astro-cytes infected with T. gondii in vitro.

Materials and Methods

Parasites

Six-week-old female Swiss mice weighing 20e25 g wereinjected i.p. with T. gondii (strain RH) 2 � 103 tachyzoitesand sacrificed at 7 days; the infection was maintained innew mice every 3 days and sacrificed for obtaining tachy-zoites in order to infect the astrocytes.

Ethical Aspects

The project was approved by our Institutional ResearchCommittee under Registration No. CI-5408 and by theState of Jalisco Department of Health under Registration

No. 43/UG-JAL/2008. The care and use of experimental an-imals were performed in accordance with the OfficialMexican Norm NOM-062-Z00-1999.

Astrocyte Culture

Eight newborn Wistar rats were sacrificed by decapitation.Brain tissue was obtained by craniotomy and mechanicallydissociated in OPTI-MEM 51985 medium (Invitrogen,Carlsbad, CA) supplemented with a 10% fetal bovineserum (FBS) and a 1% solution of penicillinestreptomycin.Cells were counted by hemocytometer in a 0.3% trypanblue solution for determining the percentage of survival.Cells were plated at 16.7 � 104 on 0.1% poly-L-lysine pre-treated cover slips placed in 24-well plates and maintainedat 37�C for 2 h in a 95% air and 5% CO2 atmosphere. Theculture medium was changed to eliminate unattached neu-rons; glial cells were incubated for 15 additional days inOPTI-MEM medium. Astrocytes were obtained by compe-tition with no proliferating neurons. Cell cultures at 80%confluence were used for the experiments.

Experimental Nitazoxanide and Pyrimethamine Treatmentin Astrocytes In Vitro

On the 15th day of culture, astrocytes (90% confluence)were infected at 24 and 48 h, respectively, with Toxoplasmagondii tachyzoites (103 parasites) and treated with 10, 20and 30 mg/mL NTZ or 7, 10 and 13 mg/mL PYR in a0.1% dimethyl sulfoxide solution. Each dose was evaluatedindependently in six replicates. For immunocytochemicalstudy, infected and uninfected astrocytes were fixed with4% paraformaldehyde for 1 h at room temperature andstored at 4�C in PBS until processed.

Immunocytochemical Method

T. gondii infection in cultured astrocytes was detected bythe immunocytochemical method in consecutive order asfollows. For identifying astrocytes, cells were washedtwice with phosphate buffer solution (0.1 M PBS) for 10min and permeabilized with 0.1% Triton X-100 in PBS(TPBS) for 1 h. Cells were incubated with 3% bovineserum albumin in PBS containing 0.01% sodium azide(blocking solution) for 1 h at 4�C. After washing, the as-trocytes were incubated overnight in rabbit anti-GFAPantibody (1:1000, Dako Corporation, Carpinteria, CA),diluted in a blocking solution and incubated at 4�C. Afterthorough washing with TPBS, the cells were first incu-bated in 10% pre-immune goat serum in a PBS bufferfor 6 h at 4�C and then with Alexa Fluor 594 labeledanti-rabbit IgG (1:2000, Invitrogen, Carlsbad, CA) for 2h at room temperature in darkness.

The cells were washed three times in PBS. An unlabeledgoat anti-rabbit antibody (1:100, Sigma Chemical Co.,St. Louis, MO) was added and maintained for 2 h at room

417Nitazoxanide in Astrocytes Infected by T. gondii

temperature in order to block immune rabbit IgG com-plexes that may give a nonspecific reaction. After overnightincubation in rabbit anti-Toxoplasma antibody (1:1500,GenWay Biotech, San Diego, CA), the cells were placedin 10% pre-immune goat serum for 2 h at 4�C. The cellswere additionally incubated with biotin-labeled anti-rabbitIgG (1:1000, Sigma) for 2 h at room temperature. Theywere further incubated with streptavidin-fluorescein conju-gate (1:3000, Sigma) diluted in PBS for 2 h. Finally, digitalimages were obtained and analyzed using a fluorescence in-verted Olympus microscope.

Microscopic Analysis

Astrocytes were evaluated 6-fold for each dose of NTZ andPYR. Twenty microscopic fields were analyzed per group,divided as follows: 1) astrocytes infected with T. gondii ta-chyzoites and treated with NTZ; 2) infected astrocytestreated with PYR; 3) untreated astrocytes infected withT. gondii (control group); and 4) intact astrocytes (non-infected control group). Parasite location was evaluated asintra- or extracellular, and the sum of both provided the to-tal parasite number. The number of infected astrocytes andparasites was analysed on digital images employing an in-verted Olympus IX71 microscope with 100 � magnifica-tion, using the Image Pro-Plus software 6.0 of MediaCybernetics 2.6.

Viability

This test was based on the presence of the enzyme mito-chondrial succinate dehydrogenase in live cells; only viableand early apoptotic cells were capable of reducing the tetra-zolium salt MTT (yellow) resulting in the formation ofwater-insoluble formazan crystals (blue). Dead cells there-fore retained the yellow color of the medium (18).

T. gondii viability in astrocytes infected and treatedwith NTZ and PYR was evaluated by the MTT assay;96-well plates were used to culture astrocytes (5 � 104)following the method previously described. Astrocyteswere infected with Toxoplasma gondii tachyzoites (28,750 parasites/well) and incubated for 24 h at 37�C. Oneassay of cells was treated post-infection for 24 h with10, 20 and 30 mg/mL NTZ and another with 10, 20 and30 mg/mL PYR. Briefly, 50 mL MTT solution (5 mg/mL, Sigma) was added for 2 h and incubated at 37�C.Then, 100 mL 50% dimethyl formamide was added fordissolving the formazan crystals and incubated overnight.T. gondii viability was determined by comparing to un-treated parasites considered 100% viable. Absorbancewas measured at 570 nm in an ELISA plate reader (Bio-lab System). Percent viability was calculated from theoptical density (OD) as follows: ([mean OD infected as-trocytes in well with drug)/mean OD no treated and in-fected astrocytes]) � 100. Results were given as meanviability � SEM.

NTZ and PYR Toxicity on Astrocytes In Vitro

NTZ and PYR toxicity on astrocytes in vitro was evaluatedby the MTT assay. Astrocytes were cultured in 96-wellplates following the previously described method. Astro-cytes not infected with T. gondii were incubated for 24 hat 37�C. One assay of cells was treated with 5, 10, 20,30, 40, 50 and 60 mg/mL NTZ and another with PYR atthe same dose. Formazan crystals were formed by addinga 50-ml MTT (5 mg/mL) solution (Sigma) for 2 h at37�C; 100 ml 50% dimethylformamide was added and incu-bated overnight. Absorbance was measured at 570 nm in anELISA plate reader (Biolab System). NTZ and PYRtoxicity on astrocytes in vitro was determined by compari-son to untreated cells considered 100% viable. Results areshown as mean � SEM.

Statistical Analysis

The analysis of quantitative variables was obtained and ex-pressed as mean � SEM. The control group was considered100% viable. Control was represented by untreated cellsspecifically for the tachyzoite viability study.

The NTZ and PYR toxicity study on astrocytes was per-formed. The experimental groups were compared to thecontrol group. Data were analyzed by ANOVA test and aDunnett’s post-hoc test was performed for multiple compar-isons of the study groups.

Results

Effect of Nitazoxanide on T. gondii

Twenty-four hour NTZ treatment at 10, 20 and 30 mg/mLdecreased the number of parasites present in astrocytes in28.8, 49.52 and 50.83%, respectively, and when comparedto the control group, a significant difference was observed( p !0.001). When the experimental groups treated at 10,20 and 30 mg/mL NTZ were analysed at 48 h, the percent-age of parasite death was 97.39, 97.14 and 98.4%, respec-tively. A statistically significant difference was observed( p !0.001) when these percentages were compared tothe control group. Furthermore, NTZ did not exert a toxiceffect on astrocytes (Figure 1A).

Effect of Pyrimethamine on T. gondii infection

Twenty-four hour PYR treatment at 7, 10 and 13 mg/mLdecreased the number of parasites present in astrocytesfrom 34.95% to 32.94% and 27.78%, respectively, exhibit-ing a significant difference vs. control ( p !0.001). Whenexperimental groups treated with PYR at 7, 10 and13 mg/mL were analysed after 48 h, the parasite deathpercentage was 63.16%, 79.84% and 80.31%, respectively.Statistically significant differences were observed in com-parison to the control group at 24 and 48 h of treatment( p !0.001) (Figure 1B).

Figure 1. NTZ effect on T. gondii tachyzoites in cultured astrocytes treated with 10, 20 and 30 mg/mL of NTZ, at 24 (Columns 2, 3, 4) and at 48 h (Columns

5, 6, 7). The number of tachyzoites was reduced significantly vs. positive control group ( p !0.001). (A) Treatment with NTZ at 48 h provoked O90%

parasite death. PYR effect on T. gondii tachyzoites in cultured of astrocytes treated with 7, 10 y 13 mg/mL of PYR, at 24 (Columns 2, 3, 4) and 48 h (Columns

5, 6, 7) post-infection. The number of tachyzoites was reduced significantly vs. positive control group ( p !0.001). (B) The PYR treatment during 48 h

provoked |80% parasite death.

418 Galv�an-Ram�ırez et al./ Archives of Medical Research 44 (2013) 415e421

Effect of Drugs on Intra- and Extracellular Parasites

The number of tachyzoites present in astrocyte cultures wasreduced by a 24-h NTZ dose-dependent treatment. Struc-tural damage in infected astrocytes was observed whenthe parasites were treated with 20 mg/mL NTZ (Figure 2).

Figure 2. Effect of NTZ and PYR on T. gondii tachyzoites in cultured astrocytes.

infected and treated with PYR at 7, 10 and 13 mg/mL, respectively, during 24 h. (

control (H) Astrocytes uninfected and treated with PYR (13 mg/mL) and (I) Ast

The high proliferation of tachyzoites was not limited by thisconcentration; however, parasites suffered significant mem-brane fragmentation and T. gondii lyses. At 10 mg/mL, thenumber of intracellular tachyzoites was 6.21, which at 30mg/mL nitazoxanide was reduced to 3.92. At 48 h, nointracellular parasites were observed. The number of

(A) 10 mg/mL, (B) 20 mg/mL, and (C) 30 mg/mL of NTZ. (DeF) Astrocytes

G) Astrocytes infected with tachyzoites of T. gondiii untreated. (H,I) Drugs

rocytes uninfected and treated with NTZ (30 mg /mL).

Figure 3. Average of intra- and extracellular tachyzoites treated with 10, 20 and 30 mg/mL of nitazoxanide at 24 and 48 h. Note that there were not extra-

cellular parasites at 48 h. (A) Intracellular parasites are represented by grey columns and extracellular parasites by black columns. Average of intra- and

extracellular tachyzoites treated with 7, 10 and 13 mg/mL of pyrimethamine at 24 and 48 h. (B) Intracellular parasites are represented by grey columns

and extracellular parasites by black columns.

419Nitazoxanide in Astrocytes Infected by T. gondii

extracellular tachyzoites was reduced by |40% at 24 h;however, observation was limited at 48 h (Figure 3A).The number of intracellular tachyzoites exhibited signifi-cant differences at 7, 10 and 13 mg/mL when treated withpyrimethamine at 24 h ( p !0.01). However, the highestdecrease was observed at 48 h at 10 mg/mL ( p !0.001).A statistically significant difference was found at 48 h inextracellular tachyzoites at different doses (Figure 3B).

Viability of T. gondii treated with Nitazoxanide andPyrimethamine

The survival of T. gondii tachyzoites was reduced in a similarway by the effect of nitazoxanide and pyrimethamine at 24and 48 h, using 10, 20 and 30 mg/mL. The maximum deathof T. gondiiwas obtained at 24 h with nitazoxanide at dose of30 mg/mL and for pyrimethamine at 10 mg/mL. A significantdifference was found ( p !0.001) when doses werecompared vs. control (Figure 4A). The maximum death oftachyzoites was obtained at 48 h post-treatment with a dose

Figure 4. Therapeutic effect of nitazoxanide and pyrimethamine against T. gondii

for 24 h (A) and 48 h (B). Efficacy was determined by MTT test. Control indicat

had higher efficacy than pyrimethanmine at all concentrations used. Results show

cells (black columns).

of 20 mg/mL for nitazoxanide and 30 mg/mL for pyrimeth-amine. No statistically significant differences between doseswere observed for both drugs; however, when compared tocontrol a significant difference was found ( p !0.001)(Figure 4B).

Nitazoxanide and Pyrimethamine Toxicity in Astrocytes

Nitazoxanide and pyrimethamine toxicity was determinedby MTT test in astrocytes in vitro. The highest nitazoxanidetoxicity was observed at 50 mg/mL and for pyrimethamineat 40 mg/mL in astrocytes (Figure 5).

Discussion

NTZ is a wide-spectrum drug that has been used for treatingintestinal parasite infections with excellent results (19). Inour results, NTZ decreased Toxoplasma gondii viabilitymore efficiently in a dose- and time-dependent manner thanPYR. In previous studies conducted in our laboratory, we

in astrocytes infected. Cells were treated with 10, 20 and 30 mg/mL of drug

es percentage viability (100%) of T. gondii without treatment; nitazoxanide

mean � SEM and significant differences. p !0.001 compared to control

Figure 5. Percentage viability of astrocytes (MTT test) after 24 h of incu-

bation with nitazoxanide or pyrimethamine in a range of concentration of

5e60 m/mL. Control indicates percentage viability of untreated cells

(100%). Results are showed as mean � SEM. Exclusively at 50 mg/mL

of PYR was observed a significant difference of p !0.05 compared with

the untreated control cells.

420 Galv�an-Ram�ırez et al./ Archives of Medical Research 44 (2013) 415e421

observed that NTZ at 10 mg/kg of weight was more effi-cient than PYR in controlling T. gondii infectionin vivo (19). NTZ showed excellent efficacy for regulatingC. parvum infection in vitro. T. gondii belongs to the Api-complexa family as well as C. parvum (20). Moreover, thisis the first time that NTZ has been evaluated as a treatmentfor T. gondii in astrocytes in vitro.

Toxoplasma gondii causes encephalitis in congenitaltoxoplasmosis and immunocompromised patients. In thisstudy we evaluated the effect of NTZ on astrocytesin vitro infected with Toxoplasma gondii due to the impor-tant role that these cells have on brain toxoplasmosis.Moreover, T. gondii causes severe damage in CNS with apronounced effect on glial cells such as microglia and astro-cytes (4e7).

In this study, NTZ at doses employed in uninfected cellsdid not alter astrocyte morphology. We observed that at 60mg/mL of NTZ, the survival of astrocytes was near 90%,suggesting that NTZ is not toxic to astrocytes in vitro.Regarding PYR cytotoxicity, we observed that a 40 mg/mL dose exerted the highest damage to astrocytes; there-fore, PYR is more toxic than NTZ.

The action mechanisms of NTZ on astrocytes infectedwith T. gondii are unknown. In cells infected with T. gondiitachyzoites, the parasites prevent apoptosis by caspase inac-tivation and NF-kB activation and thus also avoid humanquinone oxidoreductase (NQO1) expression (21).

It has recently been shown that NTZ inhibits NQO1enzymatic activity, probably acting upstream in the rapamy-cin complex 1 (mTORC1) mammalian target for stimulatingautophagy (22). On the other hand, new evidences revealthat T. gondii is degraded directly in astrocyte cytoplasm.Autophagosomes have been observed aggregating aroundthe disrupted vacuoles, suggesting that autophagosomesmay be stimulated by the disrupting vacuoles and involvedin eliminating the parasite in the cytoplasm (23).

The role of astrocytes in the defense against Toxoplasmais not clear. The most prominent effector mechanismsagainst Toxoplasma are activating nitric oxide synthase(iNOS) and inducing indoleamine 2,3-dioxygenase (IDO)(24). Although these mechanisms are insufficient for inhib-iting the growth of parasite, other mechanisms can be play acritical role in death and clearance of T. gondii associatedwith nitazoxanide in the astrocyte.

In conclusion, our results suggest that NTZ is more effi-cient than PYR for inhibiting T. gondii growth in astrocytesin vitro. Nevertheless, further studies in vivo are requiredfor examining the effect of NTZ in T. gondii infection. Ifthese study results are successful, clinical assays of thisdrug could subsequently be designed.

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