ORIGINAL PAPER

Albendazole and its derivative JVG9 induce encystationon Giardia intestinalis trophozoites

Armando Pérez-Rangel & José Manuel Hernández &

Araceli Castillo-Romero & Lilián Yépez-Mulia & Rafael Castillo &

Francisco Hernández-Luis & Benjamín Nogueda-Torres &

Juan Pedro Luna-Arias & Gerardo Radilla & Gloria León-Avila

Received: 30 October 2012 /Accepted: 13 June 2013 /Published online: 16 July 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract In the present study, we evaluated the effect of analbendazole (ABZ) derivative JVG9 on cultured Giardiaintestinalis. To assess the JVG9 effects, we evaluated thetubulin cytoskeleton by confocal microscopy, and we foundthat the characteristic staining was modified. The scanningelectron microscopy images revealed extremely damagedtrophozoites and cyst-like cells. The confocal images re-vealed that this drug triggered the expression of cyst wallprotein 1 and encystation. We also found that at low doses,AL triggered the encystation process too.

Introduction

Giardia intestinalis is a flagellated parasite that infects thou-sands of people around the world causing an asymptomaticor symptomatic illness known as giardiosis. The parasite hastwo different states, the trophozoite and the cyst. The infec-tion is acquired by ingesting contaminated food or waterwith cysts. Once the cysts reach the stomach, the excystationprocess is triggered. The trophozoites emerge and firmlyattach (through the ventral disk) to the epithelial cells ofthe duodenum and upper jejunum, damaging the microvilli.The disease is characterized by nausea, diarrhea, abdominalpain, flatulence, bloating, weight loss, and vomiting. Inchildren, it provokes iron-deficiency anemia, micronutrientdeficiencies, and growth retardation due to malabsorption(Wolfe 1992; Chavez et al. 1992a). In the small intestine, thetrophozoites replicate and colonize, covering a wide epithe-lial surface. The exposure of the trophozoites to primarybiliary salts as they pass through the jejunum and the reduc-tion of cholesterol in the microenvironment induce theencystation process. During this process, the trophozoiteexpresses specific cyst wall proteins (CWPs), and dramaticmorphological changes due to cytoskeleton rearrangementsoccur (Reiner et al. 1989; Lujan et al. 1997). The cytoskel-eton ofGiardia is constituted by: the ventral disk, the medianbody, four pairs of basal bodies, eight flagella, and the funis(Adam 2001). These structures are mainly formed by micro-tubules (MTs) containing α, β, and γ tubulins. In addition,Giardia has cytoplasmic actin and specific Giardia proteinsnamed giardins (Holberton and Ward 1981; Crossley andHolberton 1983).

Several drugs have been used for giardiosis treatmentsuch as tinidazole, metronidazole, quinacrine, furazolidone,

A. Castillo-Romero :G. Radilla :G. León-Avila (*)Departamento de Zoología, Escuela Nacional de Ciencias Biológicas,Instituto Politécnico Nacional, Carpio y Plan de Ayala,Col. Casco de Santo Tomás, 11340 México D.F., Mexicoe-mail: leonavila60@yahoo.com.mx

A. Pérez-Rangel : J. M. Hernández : J. P. Luna-AriasDepartamento de Biología Celular, Centro de Investigación yEstudios Avanzados del Instituto Politécnico Nacional, Av. IPN2508, Col. San Pedro Zacatenco, 07360 México D.F., Mexico

L. Yépez-MuliaUMAE Hospital de Pediatría, Centro Médico Nacional Siglo XXI(CMN-SXXI), Instituto Mexicano del Seguro Social (IMSS),2° piso, 06720 México D.F., Mexico

R. Castillo : F. Hernández-LuisDepartamento de Farmacia, Facultad de Química, UniversidadNacional Autónoma de México, 04510 México D.F., Mexico

B. Nogueda-TorresDepartamento de Parasitología, Escuela Nacional de CienciasBiológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala,Col. Casco de Santo Tomás, 11340 México D.F., Mexico

Parasitol Res (2013) 112:3251–3257DOI 10.1007/s00436-013-3521-1

nitaxozanide, and albendazole (ABZ) (Upcroft and Upcroft2001; Robertson et al. 2010). The mechanism of action ofABZ is the depolymerization of microtubules as a result of thebinding of the drug to the plus end of the β-tubulin, causingthe “capping effect,” and disassembly of MTs. The ABZ–tubulin interaction is located near the colchicine binding site,a well known microtubule depolymerizing compound(Upcroft et al. 1996b).

Nowadays, ABZ is the most common drug used to treatgiardiosis in México. It has been documented that some drugsare not absolutely effective for giardiosis treatment, and drugresistance is one of the reasons of the non-effectiveness; also,it is difficult to discriminate between “cure followed by rein-fection” and other factors.

Quinacrine has been available since 1930 when it wasintroduced as an antimalarial drug. Giardia lines resistant toquinacrine are maintained at levels that are usually lethalto susceptible strains (5 to 20 mM). Fluorescence studiesdescribed that this drug was incorporated by sensitive cells butwas excluded from resistant trophozoites (Upcroft et al.1996a).

Resistance to metronidazole and other nitroimidazoles hasbeen shown in vitro, and it was suggested to be associatedwith both the negative regulation of pyruvate ferredoxin oxi-doreductase (PFOR) (Smith et al. 1988) and with the decreaseof ferredoxin reduction (Townson et al. 1996).

The non-effectiveness of metronidazole or furazolidonetreatment has been documented in refractory patients. In ad-dition, isolates resistant to these drugs have been obtainedfrom the same patients (Upcroft and Upcroft 2001). On theother hand, Argüello-García et al. (2009) obtained isolates thatwere grown under increasing concentrations of metronidazoleand ABZ. These authors did not find changes in the sequenceof genes coding for β-tubulin and PFOR. However, clonesresistant to metronidazole exhibited increased expressionlevels of pfor mRNA. Furthermore, Jiménez-Cardoso et al.(2009) observed permanent mutations in the β-giardin se-quence in resistant strains, as well as in recovered-sensitiveand resistance-recovered strains to ABZ.

Inadequate use of drugs, incomplete treatment, and theresistant strains justify the search for new chemotherapeuticagents to treat giardiosis. In this work, we analyzed the in vitroeffect of 5-chloro-1H benzimidazole-2-thiol (JVG9), an ABZderivative, on cultured trophozoites. We observed that in

Fig. 1 Effect of JVG9 on the growth of G. intestinalis trophozoites.Trophozoites (5×105/ml) were exposed to different concentrations ofJVG9 (0, 5, 10, 15, and 20 μg/ml) at 24, 48, and 72 h

Fig. 2 Scanning electronmicroscopy images showingmorphological changes causedby JVG9 on G. intestinalistrophozoites. Control: Parasiteswere incubated in TYI-S33medium and DMSO 0.1 % for72 h (a). The cells were cultured inTY-I-S33 medium with 20 μg/mlJVG9 for 72 h (b–d). The arrowsshow the encysting cells

3252 Parasitol Res (2013) 112:3251–3257

addition to causing destruction of trophozoites, JVG9 and alsoABZ induced encystation.

Materials and methods

Culture of Giardia trophozoites and drug treatment

GiardiaWB strain trophozoites were grown axenically in 10-ml culture tubes containing Diamond's TYI-S-33 mediumsupplemented with 10 % bovine serum and 0.5 mg/ml bovinebile, pH 7.1 (Keister 1983) at 37 °C. The effect of the drugswas evaluated on trophozoites (5×105/ml) by adding increas-ing concentrations of JVG9 (0, 5, 10, 15, and 20 μg/ml)dissolved in 0.1 % dimethyl sulfoxide (DMSO). Control cellswere incubated in TYI-S-33 medium with 0.1 % DMSO. Cellproliferation was monitored by counting cell samples fromdifferent periods (24, 48, and 72 h) using a hemocytometer.

Scanning electron microscopy

The trophozoites were incubated for 72 h with JVG9 (20-μg/ml) or ABZ (4 μg/ml), washed twice with PBS, and fixedwith 2.5 % glutaraldehyde in PBS for 1 h. The cells wereattached to cover slips (pre-treated with poly-lysine 1 %) for10 min. Then, the trophozoites were washed with PBS, post-fixed with 1 % osmium tetroxide solution for 1 h, anddehydrated through a graded ethanol series (50–100 %). Sam-ples were dried using the critical point method, covered withgold, and observed by scanning electron microscopy (SEM).

Production of antibodies

Balb/c mice or Wistar rats were injected intraperitoneallywith recombinant CWP1 protein (Castillo-Romero et al.2010) (100 or 200 μg, respectively) at days 0, 15, and 30.The first boost was done with 10:1 protein/TiterMax emul-sion, and the following boosts were done with a 1:1protein/aluminum hydroxide mixture. The sera wereobtained 4 days after the last boost and assayed by ELISAand western blot.

Effect of JVG9 on trophozoites

To evaluate the effect of JVG9 or ABZ on trophozoites, thecells were treated with: TYI-S-33 medium and DMSO (con-trol of growing cells), TYI-S-33 medium and JVG9 (20-μg/ml), TYI-S-33 medium andABZ (4μg/ml), and encystingmedium (control of encysted cells). After 72 h, the cells wereharvested by placing the tubes on ice bath and centrifuged for10 min. The cell pellets were suspended in bidistilled waterand incubated for 4 h. The resistant cysts were suspended inPBS and processed for immunofluorescence.

Immunofluorescence

The trophozoites were harvested, washed twice with sterilePBS, and fixed with 3 % paraformaldehyde in PBS for20 min, then washed twice and suspended in 100 μl ofPBS. A drop of fixed cells was placed on cover slips (pre-treated with poly-L-lysine) and incubated for 30 min to allowcells to attach to the glass surface. The trophozoites then

Fig. 3 JVG9 induces tubulin redistribution in trophozoites. Trophozo-ites incubated in: TYI-S-33 medium and DMSO (control growing cells)(a and b); TYI-S-33 medium and JVG9 (20 μg/ml) (c–f) or encystingmedium without JVG9 (control encysting cells) (g and h). The cellswere harvested and incubated with mouse anti-alpha tubulin, followedby goat anti-mouse-FITC.DIC images correspond to b, d, f and h

Parasitol Res (2013) 112:3251–3257 3253

were permeabilized with 0.5 % Triton X-100 for 15 min andwashed twice with PBS, and nonspecific binding wasblocked with 1 % bovine serum albumin in PBS for 1 h atroom temperature. Next, cells were incubated with rat anti-CWP1 antibody diluted 1:100 or mouse anti-alpha tubulin(Zymed)for 1 h, followed by an incubation for 1 h with goatanti-rat conjugated to fluorescein isothiocyanate (FITC) orTRITC diluted 1:100 in PBS or goat anti-mouse conjugatedto FITC diluted 1:100. The cover slips were washed threetimes in PBS. To stain nuclear material, the cells were incu-bated with TO-PRO-3 staining (Molecular Probes) for 1 minat room temperature. Finally, the cover slips were mountedon glass slides with Vectashield. The images were obtainedusing a Leica confocal microscope then were processed andanalyzed with Leica Lite and Photoshop software.

Results

JVG9 inhibits the growth of Giardia trophozoites

In order to analyze the effect of JVG9 on G. intestinalistrophozoites, the cells were incubated with different

concentrations of the drug. We observed a dose-dependentinhibition of the parasite proliferation (Fig. 1), and suchinhibition was apparent since 24 h of incubation and moreevident at 72 h. The compound at 20 μg/ml inhibited thegrowth of trophozoites (88 %) and has been the most markedinhibitory dose. Meanwhile, the lower concentrations of 5and 10 μg/ml reduced the growth slightly at 24 and 48 h. Onthe other hand, we found that 54 μM was the IC50.

Scanning electron microscopy analysis

To evaluate the effect of JVG9 on the morphology of tro-phozoites, the cells were treated with 20 μg/ml for 72 h(Fig. 2). The SEM images revealed that cells incubated withDMSO presented normal morphology and the ventral disk,flagella, and lateral flange without alterations (Fig. 2a).JVG9 caused several morphological changes; some cellsshowed pores on the dorsal and ventral faces, disk, andmembrane (Fig. 2b); also the dorsal surface exhibited pro-trusions. Various cells were completely misshapen (Fig. 2c).Moreover, during the interaction with JVG, some parasitesapparently were induced to encyst, since we observed round-ed cells like cysts (Fig. 2d).

Fig. 4 JVG9 inducesencystation in G. intestinalis.Trophozoites treated with JVG9(20 μg/ml) were incubated withrat anti-CWP1 followed by goatanti-rat-TRIC (a) and mouseanti-alpha-tubulin followed bygoat anti-mouse-FITC (b); cellsincubated with anti-CWP1followed by goat anti-rat-FITC(d and g). The nuclei werestained with TO PRO (e and h).DIC images correspond to c, f,and i

3254 Parasitol Res (2013) 112:3251–3257

Effect of JVG9 on tubulin distribution

Because cytoskeleton is vital to the preservation of the parasitestructure, we wanted to know if JVG9 as albendazole inter-rupts the microtubule integrity and, as a consequence, if thetypical morphology of the trophozoites might be altered. Incontrol cells, tubulin labeling was on the median body, flagel-la, and ventral disk (Fig. 3a). Cells incubated with JVG9 andstained with anti α-tubulin antibody showed fragmented mi-crotubules and dramatic changes of the label distribution; insome cells, it was observed as dots, in comparison to thecontrol (Fig. 3c). The ventral disk was weakly labeled, andsometimes, the label was not detected. Furthermore, the stain-ing over the ventral disk almost disappeared (Fig. 3c). Somerounded cells like cysts exhibited the tubulins as folded rib-bons (Fig. 3e), as were observed in the control cysts (Fig. 3g).

In order to investigate if the rounded cells observed could becysts, the trophozoites incubated with JVG9 (and after beingsuspended in bidistilled water and incubated for 4 h at 4 ° C)were submitted to double staining using anti-CWP1 antibodyand anti-alpha-tubulin. We observed the CWP1 labeling

at the wall of the rounded cells (the periphery) (Fig. 4a). It waspossible to detect some precysts containing several encystation-specific vesicles stained with the anti-CWP1 (Fig. 4d and g). Inaddition, to know if the cysts became mature, we stained thenuclei with TO-PRO (double-stranded DNA stain) (Chavez-Munguia et al. 2007), and we observed mature cysts with fournuclei and precysts with two nuclei (Fig. 4e and h). Thechanges in the microtubules distribution were observed asdescribed previously (Figs. 3e and 4b).

Albendazole also induced encystation

Since JVG9 is an ABZ derivate compound, we wanted toexplore if ABZ could induce the encystation process as well.Trophozoites were treated with 4.0μg/ml during 72 h (and afterbeing suspended in bidistilled water and incubated for 4 h at 4 °C). Most of the cells were killed, but the SEM analysis of theremaining cells revealed intact cysts (Fig. 5a) and damaged cyst(Fig. 5b). To confirm that those cells were cysts, we performedanti-CWP1 staining, and we corroborate that albendazole alsoinduced encystation as JVG9 did (Fig. 5c and e).

Fig. 5 Albendazole inducesencystation in G. intestinalis.Parasites were incubated withalbendazole 4 μg/ml in TYI-S33medium. SEM images (a and b).Cyst were labeled with anti-CWP1 followed by goat anti-rat-FITC (c and e). DIC imagescorrespond to d and f

Parasitol Res (2013) 112:3251–3257 3255

Discussion

Even though there are various drugs available for giardiosistreatment, not one is 100 % effective. This failure could bedue to cessation of the treatment, inadequate doses, or para-site resistance to the drugs.

Several dramatic morphological changes were describedpreviously in trophozoites treated with ABZ. SEM imagesshowed: deformed trophozoites, perforated membrane, frag-mentation of the ventral disk with dislocation of the microribbons and tubulin, and dispersion of microtubules in thecytoplasm (Cedillo-Rivera et al. 2002; Chavez et al. 1992b).From these observations, our research group designed ABZderivatives in order to find drugs for more effective treatment orsome compounds causing interesting effects on the parasite.

In the present study, our experiments were carried out toevaluate the effects of the derivative JVG9 on G. intestinalis.The SEM images of JVG9-treated cells also showed damagedparasites (Fig. 2b and c) as reported previously, but sometrophozoites became spherical, suggesting cyst appearance orinduction of encystation (Fig. 2d). By confocal immunofluo-rescence, the cells labeled with anti-tubulin showed damaged orrearrangements of microtubules in trophozoites and sphericalforms (Fig. 3), but the anti-CWP1 allowed us to confirm thelabel at the cyst wall (Fig. 4). The corresponding confocalimages suggested that the encystation-specific vesicles werepresent (Fig.4d). In addition, we found cysts in the cells treatedwith albendazole (Fig. 5). All the above findings indicated thatJVG9 and ABZ triggered the encystation process. However,Hausen et al. 2009 reported that ABZ inhibited the in vitroencystation process, because he used encysting medium con-taining albendazole; therefore, our result cannot be comparedbecause we did not use the same conditions; we used only TYI-S33 medium with albendazole. The explanation to our resultmight be that the JVG9 exposition kills trophozoites or inducesstress conditions driving cytoskeleton modifications; thesechanges may trigger a signal transduction for encystation, butif these serial processes are not so fast, the cysts will bedamaged. This possibility also explains the results obtainedby Corrêa and Benchimol (2006) who observed that the treat-ment of trophozoites with cytochalasin B or D (which promotesdepolymerization of actin) induced encystation. Another possi-ble explanation is based on the finding that ABZ arrests thehepatocellular carcinoma cells in G2 phase (Pourgholami et al.2001; Patel et al. 2011), and encystation starts in this phase(Bernander et al. 2001).

The efficacy of ABZ is 35–96 % (Robertson et al. 2010);the lower percentage (35 %) can be explained if we assumethat a small number of trophozoites can encyst, and if some ofthose cysts are mature, the patient could suffer a self-infectionand apparently the ABZ treatment could be ineffectual. Ge-netic variability has been reported among the Giardia iso-lations. This variability can influence the epidemiology,

virulence, and drug susceptibility, and then the findings re-ported in this paper also contribute to explain the drug effi-ciency. On the other hand, this drug could help understand themolecular mechanism of the encystation.

We think that it is vital to investigate the genes expressedin Giardia during the incubation with JVG9 and ABZ at lowdoses as well as determine the proteins that can be phosphor-ylated during this process.

Acknowledgments The authors thank the Electron Microscopy Unitof CINVESTAV for all the facilities. This investigation was supportedby grants SIP2010726, SIP20110886, SIP20121394, SIP20130645,CONACyT 167376, and ICyTDF scholarships granted to Pérez-Rangeland Castillo-Romero.

References

Adam RD (2001) Biology of Giardia lamblia. Clin Microbiol Rev14:447–475

Argüello-García R, Cruz-Soto M, Romero-Montoya L, Ortega-PierresG (2009) In vitro resistance to 5 nitroimidazoles and benzimid-azoles in Giardia duodenalis: variability and variation in geneexpression. Infect Genet Evol 96:1057–1064

Bernander R, Palm JE, Svärd SG (2001) Genome ploidy in differentstages of the Giardia lamblia life cycle. Cell Microbiol 3:55–62

Castillo-Romero A, Leon-Avila G, Wang CC, Perez Rangel A,Camacho Nuez M, Garcia Tovar C, Ayala-Sumuano JT, Luna-Arias JP, Hernandez JM (2010) Rab11 and actin cytoskeletonparticipate in Giardia lamblia encystation, guiding the specificvesicles to the cyst wall. PLoS Negl Trop Dis 4(6):e697

Cedillo-Rivera R, Chavez B, Gonzalez-Robles A, Tapia A, Yepez-Mulia L (2002) In vitro effect of nitazoxanide againts Entamoebahistolytica, Giardia intestinalis and Trichomonas vaginalis tro-phozoites. J Eukariotic Microbiol 49:2001–2008

Chavez B, Espinoza-Cantellano M, Cedillo-Rivera R, Ramirez A,Martinez-Palomo A (1992a) Effects of albendazole on Entamoebahistolytica andGiardia lamblia trophozoites. ArchMedRes 23:63–67

Chavez B, Cedillo-Rivera R, Martinez-Palomo A (1992b) Giardialamblia: ultrastuctural study of the in vitro effect of benzimid-azoles. J Protozool 39:510–515

Chavez-Munguia B, Omana-Molina M, Gonzalez-Lazaro M, Gonzalez-Robles A, Cedillo-Rivera R, Bonilla P, Martinez-Palomo A (2007)Ultrastructure of cyst differentiation in parasitic protozoa. ParasitolRes 100:1169–1175

Corrêa G, Benchimol M (2006) Giardia lamblia behavior under cyto-chalasins treatment. Parasitol Res 98:250–256

Crossley R, Holberton DV (1983) Characterization of proteins from thecytoskeleton of Giardia lamblia. J Cell Sci 59:81–103

Hausen MA, de Oliveira PR, Campanati L, de Carvalho JJ, de CarvalhoL, Barbosa HS (2009) Giardia lamblia: a report of drug effectsunder cell differentiation. Parasitol Res 105:789–796

Holberton D, Ward AP (1981) Isolation of the cytoskeleton fromGiardia tubulin and a low-molecular-weight protein associatedwith microribbon structures. J Cell Sci 47:139–166

Jiménez-Cardoso E, Eligio-García L, Cortés-Campos A, Flores-LunaA, Valencia-Mayoral P, Lozada-Chávez I (2009) Changes in beta-giardin sequence of Giardia intestinalis sensitive and resistant toalbendazole strains. Parasitol Res 105:25–33

Keister DB (1983) Axenic culture of Giardia lamblia in TYI-S-33 medi-um supplementedwith bile. Trans R Soc TropMedHyg 77:487–488

Lujan HD,Mowatt MR, Nash TE (1997)Mechanisms ofGiardia lambliadifferentiation into cysts. Microbiol Mol Biol Rev 61:294–304

3256 Parasitol Res (2013) 112:3251–3257

Patel K, Doudican N, Schiff PB, Orlow S (2011) Albendazole sensitizescancer cells to ionizing radiation. Radiat Oncol 6:160

Pourgholami MH, Woon L, Almajd R, Akther J, Bowery P, Morris DL(2001) In vitro and in vivo suppression of growth of hepatocellularcarcinoma cells by albendazole. Cancer Lett 165:43–49

Reiner DS, Douglas H, Gillin FD (1989) Identification and localizationof cyst-specific antigens of Giardia lamblia. Infect Immun57:963–968

Robertson LJ, Hanevik K, Escobedo AA, Morch K, Langeland N(2010) Giardiasis—why do the symptoms sometimes never stop.Trends Parasitol 26:75–82

Smith NC, Bryant C, Boreham PF (1988) Possible roles for pyruvate:ferredoxin oxidoreductase and thiol-dependent peroxidase and

reductase activities in resistance to nitroheterocyclic drugs in Giar-dia intestinalis. Int J Parasitol 18:991–997

Townson SM, Upcroft JA, Upcroft P (1996) Characterisation andpurification of pyruvate:ferredoxin oxidoreductase from Giardiaduodenalis. Mol Biochem Parasitol 79:183–193

Upcroft JA, Campbell RW, Upcroft (1996a) Quinacrine-resistant Giar-dia duodenalis. Parasitology 112:309–313

Upcroft JA, Chen N, Upcroft P (1996b)Mapping variations chromosomehomologues of different Giardia strains. Mol Biochem Parasitol76:135–143

Upcroft P, Upcroft JA (2001) Drug targets and mechanism of resistancein the anaerobic protozoa. Clin Microbiol Rev 14:150–164

Wolfe MS (1992) Giardiasis. Clin Microbiol Rev 5:93–100

Parasitol Res (2013) 112:3251–3257 3257