Cytotoxic effect of curcumin on Giardia lamblia trophozoites

Acta Tropica 98 (2006) 152–161

Cytotoxic effect of curcumin on Giardia lamblia trophozoites L. P´erez-Arriaga a , M.L. Mendoza-Maga˜na a,∗ , R. Cort´es-Z´arate b , A. Corona-Rivera c , L. Bobadilla-Morales c , R. Troyo-Sanrom´an a , M.A. Ram´ırez-Herrera a a

c

Departamento de Fisiolog´ıa, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada 950, Guadalajara, Jalisco, CP 44340, M´exico b Departamento de Patolog´ıa, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada 950, Guadalajara, Jalisco, CP 44340, M´exico Instituto de Gen´etica, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada 950, Guadalajara, Jalisco, CP 44340, M´exico Received 2 February 2006; received in revised form 24 March 2006; accepted 30 March 2006 Available online 5 May 2006

Abstract Giardia lamblia is one of the most important worldwide causes of intestinal infections produced by protozoa. Thus, the search for new alternative therapeutic approaches for this parasitic disease is very important. Common drugs used to control and eradicate this infection, frequently exhibit side effects that force patients to abandon treatment. The present work evaluates the anti-protozoan activity of curcumin, the main constituent of turmeric. Axenic G. lamblia (Portland 1 strain) cultures were exposed to different concentrations of curcumin. Its effects were evaluated on parasite growth, adhesion capacity and parasite morphology. We also evaluated the capacity of curcumin to induce an apoptosis-like effect. All curcumin concentrations inhibited trophozoite growth and adhesion in more than 50% in dose and time dependent manner. Morphological changes were described as protrusions formed under the cytoplasmic membrane, deformation due to swelling and cell agglutination. Curcumin induced apoptosis-like nuclear staining in dose and time dependent manner. In conclusion, curcumin exhibited a cytotoxic effect in G. lamblia inhibiting the parasite growth and adherent capacity, induced morphological alterations, provoked apoptosis-like changes. Future in vitro and in vivo experiments are endowed to elucidate the effect of curcumin in an experimental model of G. lamblia infection, analyze the involvement of ion channels in the swelling effect of curcumin during an apparent osmotic deregulation in G. lamblia trophozoites. This will lead to the proposal of the action mechanism of curcumin as well as the description of mechanism involved during the activation process for the apoptotic-like effect. © 2006 Elsevier B.V. All rights reserved. Keywords: Giardia lamblia; Curcumin; Cytotoxicity

1. Introduction

∗ Corresponding author. Tel.: +52 33 3617 7929; fax: +52 33 3617 7929. E-mail address: [email protected] (M.L. Mendoza-Maga˜na).

0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2006.03.005

Human and animal diseases caused by protozoan parasites have great impact on public health (Upcroft and Upcroft, 2001). Giardia lamblia, is one of the most common parasites found in the human intestinal tract which may cause an acute or chronic infection worldwide known as giardiosis. Clinical symptoms include:

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nausea, stomach cramps, diarrhea, vomiting, malabsorption syndrome, steatorrhea, weight loss, headache, low scholar progress, deficiencies in mental and physical development being more intense in childhood (Ridley and Ridley, 1976; Gardner and Hill, 2001; Ortiz et al., 2001). A number of antigiardiosic drugs have been developed to control this infectious disease. Nitroimidazoles, as metronidazole, is known to induce DNA fragmentation and this may affect host cells, thus, its use during pregnancy is not recommended (Oxberry et al., 1994; Campanati and Monteiro-Leal, 2002). Benzimidazoles, as albendazole are drugs that affect parasite cytoskeleton causing parasite detachment from intestinal epithelium (Harder et al., 2001; Samuelson, 1999). Nitrofuran compounds, as furazolidone affect parasite adhesion capacity but not its viability, this drug is approved by FDA (Hoyne et al., 1989). Nitazoxamide is tiazolidic derivative that exhibits similar effects as metronidazole and has been tested for treatment of giardiosis (Ponce-Macotela et al., 2001; Ortiz et al., 2001). All anti-giardiosic drugs above mentioned cause side effects that frequently induce treatment interruption. Curcumin is a yellow–orange dye derived from the rhizome of the plant Curcuma longa. Curcumin (diferuloyl methane, Fig. 1), one of the eleven curcuminoids identified by analytical methods, is the most abundant compound and exerts a variety of well documented effects. It is an anti-tumor agent with antiinflammatory and anti-oxidant properties by inhibition of nuclear factor ␬ B and AP1 transcription which consequently decreases the expression of pro-inflammatory cytokines, inducible nitric oxide synthase, cyclooxygenase and lipoxygenase, which contribute to reduce the inflammation and radical oxidant synthesis (Huang et al., 1997; Chan et al., 1998; Skrzypezak-Jankun et al., 2000; Bhaumik et al., 2000; Lim et al., 2001; Kang et al., 1999; Pan et al., 2000). Curcumin confers anti-oxidant protection against ethanol-induced lipid peroxidation in neuronal cells functioning as a scavenger for oxidant radicals (Rajakrishnan et al., 1999). As anti-cancer, curcumin induces apoptosis in tumoral cell lines, in vivo chemically induced cancer

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and in clinical trails (Sharma et al., 2001; Shukla et al., 2003; Lin et al., 2001; Dorai et al., 2001). Curcumin also inhibits TPA-induced protein kinase C activity (Limtrakul et al., 1997). Anti-genotoxic effects have also been documented against mutagenic and carcinogenic compounds (Limtrakul et al., 2001; Huang et al., 1997; Ishizaki et al., 1996). The anti-atherogenic effect of curcumin has been demonstrated in an animal model of experimental atherosclerosis where the most striking fact is the anti-oxidant activity avoiding the oxidation of LDLcholesterol as oxidized LDL-cholestrol is very injurious to blood vessels (Ram´ırez-Tortosa et al., 1999) Curcumin, also posses anti-bacterial (beta-lactamase positive) and anti-fungal activities (Apisariyakul et al., 1995; Wuthi-udomlert et al., 2003). Anti-protozoal activities have been documented in in vitro studies against Leishmania major promastigotes (Rasmusen et al., 2000) and against leishmanial strains and also against amastigote like cells of L. major (Saleheen et al., 2002). In this paper we describe the cytotoxic effect of curcumin against G. lamblia trophozoites, measuring parasite growth, adhesion capacity, viability, morphology and apoptotic-like changes. 2. Materials and methods 2.1. Chemicals Curcumin was purchased from Sigma Chemical Co., St. Louis, MO, purity: 65–70% and is an extract obtained from Curcuma longa. 2.1.1. Cultivation of G. lamblia Portland I strain G. lamblia trophozoites were grown to late logarithmic phase in TYI-S-33 medium supplemented with 0.5% of bovine bile and bovine serum 10% (Diamond et al., 1978). 2.1.2. Growth curve When G. lamblia cultures reached 80% of confluence, trophozoites were detached by chilling on ice cold

Fig. 1. Chemical structure of curcumin (diferuloyl methane).

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water for 30 min. Trophozoites (1.5 × 105 /ml) were seed in plastic flat sided tubes with TYI-S-33 medium and incubated at 37 ◦ C for 170 h. Every 24 h the trophozoites were detached and then counted to obtain the curve of growth for each time. Finally, only three periods of time were considered for experiments (24, 48 and 72 h) of the parasite exposure to curcumin. The following concentrations of curcumin dissolved in ethanol were used: 0.3, 3, 30 and 100 ␮M, these concentrations were determined from previous pilot experiments (unpublished) and also based in concentrations used in in vitro experiments with Leishmania expossed to curcumin (Koide et al., 2002; Saleheen et al., 2002). All assays were performed by triplicate. Every 24 h trophozoites were detached and counted. 2.2. Adhesion capacity This assay was also performed to analyze the adhesion capacity of parasites to the wall of tubes. The same amount of parasites was inoculated in plastic tubes and simultaneously curcumin was added at the same concentrations indicated in growth curve section. The first parasite count was made with detached parasites before chilling, the second parasite count was performed after parasites were chilled and detached. Finally, the results are expressed as the difference between the two measurements. 2.3. Viability curve In this assay 1.5 × 105 /ml parasites were seed in conic plastic tubes and simultaneously exposed to curcumin (0.3, 3, 30 and 100 ␮M) to study the viability of the parasites exposed to curcumin for 24, 48 and 72 h. Afterwards, the parasites were detached and counted. The tubes were centrifuged at 3000 rpm during 10 min, medium was decanted and fresh medium was added. Cultures were further incubated for 48 h and then parasites were detached and counted. 2.4. Morphologic effects of curcumin Culture samples were taken from culture tubes, approximately 15 ␮l were fixed with paraformaldehyde 0.2% fixing solution placed on a slide and mounted with a coverslip. Trophozoites were examined using an inverted microscope Olympus 1 × 50 and the morphological changes were observed and photographic records were stored using the Image-Pro plus program (Media Cybernetics).

2.5. Detection of apoptosis The G. lamblia trophozoites were observed to determine the presence of apoptosis by a Tunnel method. To observe apoptosis-like changes we used the DNA Fragmentation Detection Kit (Oncogene Research Products). The trophozoites were obtained by centrifugation at 3000 rpm and extended on a poly-l-lysine covered slide. This method allows the recognition of apoptotic nuclei in G. lamblia preparations by Fragment End Labeling (FragEL) of DNA. Terminal deoxynucleotidyl transferase (TdT) binds to exposed 3 -OH ends of DNA fragments generated in response to apoptotic signals and catalyzes the addition of biotin-labeled and unlabeled deoxynucleotides. Biotinylated nucleotides were detected using a streptavidin-horseradish peroxidase (HRP) conjugate. Diaminobenzidine reacts with the labeled DNA fragments generating an insoluble colored substrate allowing the identification of normal and apoptotic cells. The annexin C3Y method allows the detection of phosphatidylserine in apoptotic cells. Phosphatidylserine in non-apoptotic cells cannot bind annexin because it is maintained in the inner sheet of membrane. Annexin is conjugated to C3Y, a fluorescent compound, which fluoresce in orange or reddish color. Viable cells are stained in yellow–green by a fluorescein derivative. Then apoptotic cells exhibit reddish and yellow–green fluorescence and necrotic cells are stained only in red. We used the Annexin C3Y (Sigma Chemicals) following the instructions of the manufacturer. 3. Results 3.1. Effect of curcumin on G. lamblia trophozoite growth After seeding 1.5 × 105 trophozoites/ml, amount determined with a growth curve assay, the maximum amount of parasites was achieved after 72 h of incubation. Thus, the optimal times for analysis are 24, 48 and 72 h after seeding and initial exposure to curcumin. G. lamblia trophozoites growth significantly decreased at all curcumin concentrations and at all times analyzed compared to the vehicle control group. Briefly, 0.3 ␮M of curcumin decreased G. lamblia growth at 24 h 31.7%, at 48 h 52% and at 72 h 52.9%. The concentration of 3 ␮M produced a decrease of 43.3% at 24 h, 83.4% at 48 h and 94.9% at 72 h. A 30 ␮M concentration of curcumin produced a growth inhibition of G. lamblia at 24 h 55%, at 48 h 85.7% and at 72 h 97.4%. Finally, curcumin 100 ␮M produced a growth

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Fig. 2. Effect of curcumin on G. lamblia trophozoites growth in vitro. Trophozoites (1.5 × 105 /ml) were seed in TYI-S-33 medium containing curcumin and incubated at 37 ◦ C for 72 h. Every 24 h the trophozoites were detached and counted in a Neubauer chamber. Each point represents the mean ± S.D. of triplicate determinations.

inhibition at 24 h 57.3%, at 48 h 95.3% and at 72 h 97.5%. These data are graphically depicted in Fig. 2. 3.2. Inhibition of adhesion capacity We found significant increase in adhesion capacity of G. lamblia trophozoites after exposing them to 0.3 ␮M. However, this apparent increase disappeared when parasites were exposed to 30 and 100 ␮M, a remarkable feature in the remaining trophozoites was the lack of mobility and an increase in cell size (not measured). This data are summarized in Table 1. 3.3. Viability This measurement allows to distinguish the effect of curcumin exposition for different times on the capacity of G. lamblia trophozoites to overcome the cytotoxic damage. We found that 30 and 100 ␮M curcumin concentrations completely inhibited trophozoite recovery and multiplication in fresh medium. Meanwhile, when exposing G. lamblia trophozoites to 0.3 ␮M of curcumin in spite the number of parasites decayed they maintained a low capacity to divide. The number of viable trophozoites after exposure to curcumin for 48 h and incubated for further 48 h with fresh medium are shown in Fig. 3.

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Fig. 3. Effect of curcumin on G. lamblia trophozoites viability. Trophozoites (1.5 × 105 /ml) were seed in TYI-S-33 medium containing curcumin and incubated at 37 ◦ C for 48 h, centrifuged and fresh medium was added and then further incubated for 48 h. Every 24 h the trophozoites were detached and counted.

The percentage of viable trophozoites was determined considering as 100% the value obtained from vehicle control group. For the concentration of 0.3 ␮M the percentages were 42.5, 33.9 and 26.7 at 24, 48 and 72 h, respectively. When exposing to 30 ␮M the viability was only 0.5% at 24 h. All other concentrations of curcumin on exposure times gave 0% viability. These data are summarized in Table 2. 3.4. Morphological alterations The morphological alterations induced by exposure to curcumin were strikingly evident since 24 h in all curcumin concentrations. The cell membrane of trophozoites showed irregularities, which varied from mild to huge protrusions; this was noticeable 24 h after exposure. After 48 h the effect was defined as a cellular swelling that brought the trophozoites to adopt a rounded shape, at this point the motility was completely absent. With curcumin 100 ␮M prominent protrusions occurred in the dorsal region near the nucleus forming a prominent vesicle. In contrast, the trophozoites exposed to albendazole underwent morphological alterations resulting in an aberrant morphology. Meanwhile, the trophozoites of the experimental group exposed to ethanol and considered as control did not exhibit morphological alterations (Fig. 4). Table 2 Percent of viable G. lamblia trophozoites after 48 h incubation with curcumin

Table 1 Effect of curcumin on G. lamblia trophozoites adhesion Time (h)

0.3 ␮M

30 ␮M

100 ␮M

Time (h)

0.3 ␮M

30 ␮M

100 ␮M

24 48 72

625.5 112.7 108.4

0.0 6.0 3.0

0.0 0.0 0.0

24 48 72

42.5 33.9 26.7

0.5 0.0 0.0

0.0 0.0 0.0

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Fig. 4. Morphological alterations in G. lamblia trophozoites exposed to different concentrations of curcumin: (A) trophozoites with normal morphology after exposure to ethanol and used as negative control; (B) cells exposed to albendazole as positive control; (C) cells exposed to curcumin 0.3 ␮M; (D) curcumin 30 ␮M; (E) curcumin 100 ␮M. Note the formation of giant vacuoles in (C), (D) and (E) indicated by arrows.

3.5. Apoptosis DNA fragmentation was detected in a dose/response manner, in higher curcumin concentration assays apoptosis-like changes were observed in more then 90% of trophozoite nucleus by the TUNEL assay. In experiments with 3 ␮M, apoptotic changes were detected 6 h after treatment approximately 30% trophozoites were clearly apoptotic, 24 h later about 90% were apoptotic. In some trophozoites were seen as exploded cells, no membrane cases, visibly distinguished and cytoskeleton was observed surrounding the nucleus

(Fig. 5). In control group there was no positively labeled nucleus in G. lamblia trophozoites. Phosphatidylserine was detected as apoptotic signal in G. lamblia trophozoites with Annexin C3Y kit. With this kit, necrotic cells fluoresce in red color, living cells in bright green and apoptotic cells fluoresce in green and red simultaneously. G. lamblia trophozoites exposed to metronidazole fluoresce in red showing cytoplasmic debris dispersed around the cells. Meanwhile, curcumin 0.3 ␮M and 72 h after exposition induced apoptotic-like changes and with curcumin 100 ␮M and 72 h after exposure were clearly necrotic. In both cases cells showed

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Fig. 5. Apoptosis detected by TUNEL method in G. lamblia trophozoites exposed to curcumin: negative control (A); curcumin 0.3 ␮M for 6 h (B); and 24 h (C and D), note in (C) the presence of residual structures (nuclei and cytoskeleton) from a G. lamblia trophozoite that had exploded.

Fig. 6. Detection of phosphatidylserine translocation as indicator of apoptotic event induced by curcumin: (A) G. lamblia trophozoites exposed to ethanol as negative control group, cells are completely viable; (B) cells exposed to metronidazole 10 ␮M; (C) curcumin 0.3 ␮M; (D) curcumin 100 ␮M 72 h of exposure. Note the necrotic event in cells exposed to metronidazole and the apoptotic cells observed when exposed to curcumin in (C) and (D).

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a clear loss of normal morphology and cell swelling (Fig. 6). 4. Discussion Turmeric is a spice traditionally used in Asian gastronomy. Curcumin is the most abundant curcuminoid extracted from this spice. This compound has been exhaustively studied since the early 1980’s. The most striking biological effects of curcumin include antioxidant, anti-inflammatory, anti-genotoxic, anti-tumoral and anti-bacterial effects (Ara´ujo and Leon, 2001). Recently, an anti-parasitic in vitro activity against Leishmania has been described (Saleheen et al., 2002; Koide et al., 2002). Its lack of utility in in vivo infection models has been reported for leishmanial infection, as curcumin is able to exacerbate the infection (Chan et al., 2005). The host defense mechanisms to control protozoan infections vary depending whether the parasite is an obligate intracellular pathogen or not. In the case of intracellular parasites one of the most important defense mechanisms is the generation of reactive oxygen species and inflammation. Thus, if curcumin is thought to be used to eliminate the previous kind of parasites, curcumin’s anti-oxidant and anti-inflammatory activities could be detrimental for disease outcome. The pharmacodynamical distribution of curcumin, when administrated by oral route, is quite advantageous, since 35% of the ingested curcumin remains in the intestinal tract and is eliminated through feces (Ram´ırezTortosa et al., 1999). Thus, its utility to kill extracellular intestinal protozoans could be emphasized. In the case of G. lamblia trophozoites cultured in vitro, we found that curcumin exerts profound effects in parasite growth, viability, adhesion capacity and morphology. It also induced apoptosis, which is not a common feature studied in parasites. In the assays performed to analyze Giardia growth in presence of curcumin, all concentrations induced trophozoite growth decline, but 0.3 ␮M, in which, at 48 h post-exposure growth seemed to enter to a stationary phase, this could be due to the limited damage induced by curcumin and posterior adaptation to survive in such condition. However, when analyzing the viability curve, the exposure to curcumin 0.3 ␮M for 48 h and subsequent incubation with fresh medium seemed to induce a more severe effect which could be interpreted as an incapability of treated trophozoites to reestablish the microaerophilic conditions in fresh medium, consequently provoking marked cell death. Nevertheless, other possible explanations could be plausible for this effect.

Comparing curcumin’s effects with other antigiardiosic drug effects we found similarities as well as differences, for example: metronidazole induces parasite growth decrease in similar drug concentrations as curcumin does. Curcumin also induces viability decrease similarly as metronidazole (Oxberry et al., 1994; Campanati and Monteiro-Leal, 2002). G. lamblia adhesion capacity is markedly increased when the parasite is exposed to our lowest curcumin concentration (0.3 ␮M) 24 h post-exposure, however, after 48 h and 72 h post-exposure its adhesion capacity decreases to almost a normal percentage remaining similar to control group. A possible explanation for this kind of response could be that G. lamblia trophozoites sense an aggressive change in culture conditions and they respond immobilizing their cell bodies attached to the available surface, it also could be an early and incomplete stimulus for encystation. In contrast with higher curcumin concentrations, adhesion capacity was significantly decreased at all post-exposure times analyzed compared against vehicle control group. In the case of metronidazole effect, the adhesion capacity of G. lamblia is affected with an IC50 of 17.22 ␮g/ml (Meloni et al., 1990). The most important similarity was found in morphological alterations because both compounds induced the formation of swelled vesicles (Campanati and Monteiro-Leal, 2002), but curcumin’s effect was more intense in the concentrations of 30 and 100 ␮M than the effect reported for metronidazole and nitazoxanide (Campanati and Monteiro-Leal, 2002; Adagu et al., 2002; M¨uller et al., 2006) this may be due to the sensitivity of the targeted molecules for both drugs in the membrane of the G. lamblia trophozoite. When G. lamblia trophozoites or other microaerophilic microorganisms are exposed to metronidazole their sensitivity depends mainly on the expression of pyruvate ferredoxin oxireductase or other ferredoxins acting as electron donors to form the reduced metronidazole derivate which in turn can induce DNA fragmentation (Samuelson, 1999). As the DNA fragmentation effect has been documented in rodent models of carcinogenicity, it constitutes the basis for contraindication against its use during pregnancy. This seems to be the most important mechanism of action, however, other early changes may occur before DNA fragmentation, we assume this is because there could be other explanations for the swelling effect elsewhere reported (Campanati and Monteiro-Leal, 2002). To explain curcumin’s swelling effect, we suspect that a drastic osmotic disturbance occurs in G. lamblia membrane and could be one of the first events directly related

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to the mechanism of action, thus, this effect deserves further study. Apoptosis induced by anti-parasitic drugs has been barely studied in protozoan parasites. One of the reports in literature refer this metronidazole effect in Blastocystis hominis as inducing apoptotic-like changes describing and identifying cell shrinkage, darkened cell bodies and condensed cytoplasm (Nasirudeen et al., 2004), however, no images are shown of parasite nucleus displaying DNA fragmentation. Additionally, when phosphatidylserine was detected in outer leaf sheet of the parasite membrane the results are shown in graphics obtained from flow cytometry assays. In our results we show photographic evidences of the apoptotic nucleus of G. lamblia detected by the TUNEL method as well as the externalization of phosphatidylserine in parasite’s membrane identified by the Annexin CY3 method, both effects are attributable to curcumin since vehicle exposed trophozoites are negative for these apoptosis markers. These effects might be consequences of the activation of apoptotic mechanisms that may be exclusive for microorganisms lacking mitochondria as else were reported (Chose et al., 2003). Recently, a membrane-active synthetic ether-lipid analogue called miltefosine was originally developed for the treatment of cutaneous metastasis of mammary carcinomas, was successfully tested for treatment of visceral leishmaniasis and this drug has been proven as an inductor of apoptotic-like effect in promastigote and amastigote forms of Leishmania donovani. The phenomena was detected by nuclear DNA condensation, DNA fragmentation and in situ labeling of DNA fragments (Verma and Dey, 2004). Albendazole induces morphological changes producing aberrant morphology in G. lamblia trophozoites, such morphological changes are explained because albendazole damages trophozoites cytoskeleton and pleomorphic figures are induced (Ch´avez et al., 1992). Furazolidone does not affect G. lamblia morphology, instead, it induces trophozoite detachment from intestinal epithelium and trophozoites are flushed away by peristaltic movements so they do not have time to initiate an encystation process and trophozoites do not survive outside the host. This effect is provoked by interruption of enzymatic machinery that produces energy for the trophozoite and the ventral disc does not work without energy (Gardner and Hill, 2001). Exposing G. lamblia trophozoites to curcumin, suggest the possibility of direct cytotoxic damage affecting parasite growth, adherence, viability and morphology which are evident in our results. Another important approach is the induction of a process leading to DNA

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fragmentation in a so-called apoptotic event, which in turn may be a consequence rather than a central effect. The swelling process is, to us, the most important feature that curcumin has on G. lamblia, because volume regulation results impaired in the exposed trophozoites, thus, membrane function should be involved. We are performing new experiments to elucidate early changes induced by curcumin in G. lamblia membrane affecting volume regulation and in comparative studies we will include classical anti-giardiosic drug effects. Up to date, no direct estimations have been made with respect to electrophysiological properties of G. lamblia membrane. In an indirect estimation using the fluorescent compound bis-oxonol (bis-1,3-dibutylbarbituric acid trimethine oxonol, DiBAC4 ), a K+ current has been reported to participate in G. lamblia membrane function and an ion channel should be involved in volume regulation (Biagini et al., 2000). Also oxidative and nitrosative stress depolarizes the plasma membrane inducing an influx of DiBAC4 and may work as an early indicator of membrane perturbation caused by cytotoxic agents which may lead to cell death by necrotic or apoptotic mechanisms (Lloyd et al., 2004). Thus, our experiments to study the electrophysiology of this parasite by patch clamp are underway to confirm the nature of ion currents involved in early damage of G. lamblia trophozoites by curcumin or other natural or synthetic antiprotozoal drugs. Acknowledgements This research work was supported by Universidad de Guadalajara, PROPESTI/PITI/65 2004, and represents the Doctoral Thesis of P´erez-Arriaga L. Ram´ırez-Herrera M.A., P´erez-Arriaga L. and Cort´es Z´arate R. contributed equally for this research work. References Adagu, I.S., Nolder, D., Warhurst, D.C., Rossignol, J.-F., 2002. In vitro activity of nitazoxanide and related compounds against isolates of Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. J. Antimicrob. Chemother. 49, 103–111. Apisariyakul, A., Vanittanakom, N., Buddhasukh, D., 1995. Antifungal activity of turmeric oil extracted from Curcuma longa (Zingiberaceae). J. Ethnopharmacol. 49, 163–169. Ara´ujo, C.A.C., Leon, L.L., 2001. Biological activities of Curcuma longa L. Mem. Inst. Oswaldo Cruz Rio de Janeiro 96, 723–728. Bhaumik, S., Jyothi, M.D., Khar, A., 2000. Differential modulation of nitric oxide production by curcumin in host macrophages and NK cells. FEBS Lett. 483, 78–82. Biagini, G.A., Lloyd, D., Kirk, K., Edwards, M.R., 2000. The membrane potential of Giardia intestinalis. FEMS Microbiol. Lett. 192, 153–157.

160

L. P´erez-Arriaga et al. / Acta Tropica 98 (2006) 152–161

Campanati, L., Monteiro-Leal, L.H., 2002. The effects of the antiprotozoal drugs metronidazole and furazolidone on trophozoites of Giardia lamblia (P1 strain). Parasitol. Rev. 88, 80–85. Chan, M.M.-Y., Huang, H.-I., Fenton, M.R., Fong, D., 1998. In vivo inhibition of nitric oxide synthase gene expression by curcumin; a cancer preventive natural product with anti-inflamatory properties. Biochem. Pharmacol. 55, 1955–1962. Chan, M.M-Y., Adapala, N.S., Fong, D., 2005. Curcumin overcomes the inhibitory effect of nitric oxide on Leishmania. Parasitol. Res. 96, 49–56. Ch´avez, B., Espinosa-Cantellano, M., Cedillo-Rivera, R., Ram´ırez, A., Mart´ınez-Palomo, A., 1992. Effects of albendazole on Entamoeba histolytica and Giardia lamblia trophozoites. Arch. Med. Res. 23, 63–67. Chose, O., Sarde, C., No¨el, C., Gerbod, D., Jim´enez, J., Brenner, C., Capron, M., Viscogliosi, E., Roseto, A., 2003. Cell death in protist without mitochondria. Ann. N. Y. Acad. Sci. 1010, 121–125. Diamond, L.S., Harlow, D., Cunnick, C.C., 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72, 431–432. Dorai, T., Cao, Y.C., Dorai, B., Buttyan, R., Katz, A.E., 2001. Therapeutic potential of curcumin in human prostate cancer. III. Curcumin inhibits proliferation, induces apoptosis, and inhibits angiogenesis of LNCaP prostate cancer cells in vivo. Prostate 47, 293–303. Gardner, T.B., Hill, D.R., 2001. Treatment of giardiasis. Clin. Microbiol. Rev. 14, 114–128. Harder, A., Greif, G., Haberkorn, A., 2001. Chemotherapeutic approaches to protozoa: Giardia, Trichomonas and Entamoeba—current level of knowledge and outlook. Parasitol. Res. 87, 785–786. Hoyne, G.F., Boreham, P.F.L., Parsons, P.G., Nard, C., Biggs, B., 1989. The effects of drugs on the cell cycle of Giardia intestinalis. Parasitology 99, 333–339. Huang, M.T., Ma, W., Yen, P., Xie, J.G., Han, J., Frenkel, K., Grunberg, D., Conney, A.H., 1997. Inhibitory effects of topical application of low doses of curcumin on 12-O-tetradecanoyl-phorbol-13-acetateinduced tumor promotion and oxidized DNA bases in mouse epidermis. Carcinogenesis 18, 83–88. Ishizaki, C., Oguro, T., Yoshida, T., Wen, C.O., Sueki, H., Iijima, M., 1996. Enhancing effect of ultraviolet A on ornithine decarboxylase induction and dermatitis evoked by 12-O-tetradecanoylphorbol13-acetate and its inhibition by curcumin in mouse skin. Dermatology 193, 311–317. Kang, B.Y., Chung, S.W., Chung, W.J., Im, S.Y., Hwuang, S.Y., Kim, T.S., 1999. Inhibition of interleukin-12 production in lipopolysaccharide-activated macrophages by curcumin. Eur. J. Pharmacol. 384, 191–195. Koide, T., Nose, M., Ogihara, Y., Yabu, Y., Ohta, N., 2002. Leishmanicidal effect of curcumin in vitro. Biol. Pharm. Bull. 25, 131–133. Lim, G.P., Chu, T., Yang, F., Beech, W., Frautschy, S.A., Cole, G.M., 2001. The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse. J. Neurosci. 21, 8370–8377. Limtrakul, P., Lipigorngoson, S., Namwong, O., Apisariyakul, A., Dunn, F.W., 1997. Inhibitory effect of dietary curcumin on skin carcinogenesis in mice. Cancer Lett. 116, 197–203. Limtrakul, P., Anuchapreeda, S., Lipogorngoson, S., Dunn, F.W., 2001. Inhibition of carcinogenic induced c-Ha-ras and c-fos protooncogenes expresion by dietary curcumin. BMC Cancer 1, 1. Lin, C.C., Ho, C.T., Huang, M.T., 2001. Mechanistic studies on the inhibitory action of dietary dibenzoylmethane, a beta-diketone ana-

logue of curcumin, on 7m12-dimethylbenz(a)antracene-induced mammary tumorogenesis. Proc. Natl. Sci. Counc. Repub. Chin. B 25, 158–165. Lloyd, D., Harris, J.C., Biagini, G.A., Hughes, M.R., Maroulis, S., Bernard, C., Wadley, R.B., Edwards, M.R., 2004. The plasma membrane of microaerophilic protist: oxidative and nitrosative stress. Microbiology 150, 1183–1190. Meloni, B.P., Thompson, R.C.A., Reynoldson, J.A., Seville, P., 1990. Albendazole: a more effective antigiardial agent in vitro than metronidazole or tinidazole. Trans. R. Soc. Trop. Med. Hyg. 84, 375–379. M¨uller, J., R¨uhle, G., M¨uller, N., Rossignol, J.-F., Hemphill, A., 2006. In vitro effects of Thiazolides on Giardia lamblia WB Clone C6 cultured axenically and in coculture with Caco2 Cells. Antimicrob. Agents Chemother. 50, 162–170. Nasirudeen, A.M.A., Yap, E.H., Mulkit, S., Kevin, S.W.T., 2004. Metronidazole induces programmed cell death in the protozoan parasite Blastocystis hominis. Microbiology 150, 33–43. Ortiz, J.J., Ayoub, A., Gargala, G., Chegne, N.L., Favennec, L., 2001. Randomized clinical study of nitazoxanide compared to metronidazole in the treatment of symtomatic gisrdiasis in children from northern Peru. Aliment. Pharmacol. Ther. 15, 1409– 1415. Oxberry, M.E., Thompson, R.C.A., Reynolds, J.A., 1994. Evaluation of the effect of albendazole and metronidazole on the Ultra structure of Giardia duodenalis, Trichomonas vaginalis and Spironucleous muris using transmission electron microscopy. Int. J. Parasitol. 24, 695–703. Pan, M.H., Lin-Shiau, S.Y., Lin, J.K., 2000. Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IkappaB kinase and NFkappaB activation in macrophages. Biochem. Pharmacol. 60, 1665–1676. Ponce-Macotela, M., G´omez-Gardu˜no, J., Gonz´alez-Maciel, A., Reynoso-Robles, R., Anislado-Tolentino, V., Martinez–Gordillo, M.N., 2001. Determinaci´on in vitro de la susceptibilidad a la nitazoxanida de cuatro aislados de Giardia duodenalis obtenidos de diferentes hu´espedes. Rev. Invest. Clin. 53, 41–45. Rajakrishnan, V., Viswanathan, K.N., Menon, V.P., 1999. Neuroprotective role of curcumin form Curcuma Longa on ethanol-induced brain damage. Phytother. Res. 13, 571–574. Ram´ırez-Tortosa, M.C., Mesa, M.D., Aguilera, M.C., Quiles, J.L., Baro, L., Ram´ırez-Tortosa, C.L., Mart´ınez-Victoria, E., Gil, A., 1999. Oral administration of a turmeric extract inhibits LDL oxidation and has hypocholesterolemic effects in rabbits with experimental atherosclerosis. Atherosclerosis 147, 371–378. Rasmusen, H.B., Christensen, S.B., Kuist, L.P., Karazami, A., 2000. A simple and efficient separation of the curcumins. The antiprotozoal constituents of Curcuma longa. Planta Med. 66, 396– 398. Ridley, M.J., Ridley, D.S., 1976. Serum antibodies and jejunal histology in giardiosis associated with malabsortion. J. Clin. Pathol. 29, 30–34. Saleheen, D., Syed, A.A., Khalid, A., Anwar, A.S., Ajmal, A., Muhammad, M.Y., 2002. Latent activity of curcumin against leismaniasis in vitro. Biol. Pharm. Bull. 25, 386–389. Samuelson, J., 1999. Minireview: why metronidazole is active against both bacteria and parasites. Antimicrob. Agents Chemother. 43, 1533–1541. Sharma, R.A., McLelland, H.R., Hill, K.A., Ireson, C.R., Euden, S.A., Manson, M.M., Pirmo, H.M., Marnett, L.J., Gescher, A.J., Steward, W.P., 2001. Pharmacodinamic and pharmacokinetic study of oral

L. P´erez-Arriaga et al. / Acta Tropica 98 (2006) 152–161 curcuma extract in patient with colorectal cancer. Clin. Cancer Res. 7, 1894–1900. Shukla, Y., Arora, A., Taneja, P., 2003. Antigenotoxic potential of certain dietary constituents. Teratog. Carcinog. Mutagen. 23, 323–335. Skrzypezak-Jankun, E., McCabe, N.P., Selman, S.H., Jankun, J., 2000. Curcumin inhibits lipoxygenase by binding to its central cavity: theoretical and X-ray evidence. Int. J. Mol. Med. 6, 521–526.

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Upcroft, P., Upcroft, J.A., 2001. Drug target and mechanisms of resistance in the aerobic protozoa. Clin. Microbiol. Rev., 150–164. Verma, N.K., Dey, C.S., 2004. Possible mechanism of miltefosinemediated death of Leishmania donovani. Antimicrob. Agents Chemother. 48, 3010–3015. Wuthi-udomlert, M., Grisanapan, W., Lauanratana, O., Caichompoo, W., 2003. Antifungal activity of Curcuma longa grown in Thailand. Southeast Asian J. Trop. Med. Public Health 31, 178–182.