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Albendazole induces oxidative stress and DNA damage in the parasitic protozoan Giardia duodenalis

The control of Giardia duodenalisinfections is carried out mainly by drugs, among these albendazole (ABZ) is commonly used. Although the cytotoxic effect of ABZ usually involves binding to β-tubulin, it has been suggested that oxidative stress may also play a role in its parasiticidal mechanism. In this work the effect of ABZ in Giardiaclones that are susceptible or resistant to different concentrations (1.35, 8, and 250 μM) of this drug was analyzed. Reactive oxygen species (ROS) were induced by ABZ in susceptible clones and this was associated with a decrease in growth that was alleviated by cysteine supplementation. Remarkably, ABZ-resistant clones exhibited partial cross-resistance to H2O2, whereas a GiardiaH2O2-resistant strain can grow in the presence of ABZ. Lipid oxidation and protein carbonylation in ABZ-treated parasites did not show significant differences as compared to untreated parasites; however, ABZ induced the formation of 8OHdG adducts and DNA degradation, indicating nucleic acid oxidative damage. This was supported by observations of histone H2AX phosphorylation in ABZ-susceptible trophozoites treated with 250 μM ABZ. Flow cytometry analysis showed that ABZ partially arrested cell cycle in drug-susceptible clones at G2/M phase at the expense of cells in G1 phase. Also, ABZ treatment resulted in phosphatidylserine exposure on the parasite surface, an event related to apoptosis. All together these data suggest that ROS induced by ABZ affect Giardiagenetic material through oxidative stress mechanisms and subsequent induction of apoptotic-like events.

Keywords: Giardia duodenalis, albendazole, oxidative stress, DNA damage, apoptosis.

Introduction

Giardia duodenalisis an intestinal parasitic protozoan that causes the infection known as giardiasis which affects about 280 million people around the world; 500,000 new cases are reported each year (Lane and Lloyd, 2002; Plutzer et al., 2010). This parasite is orally transmitted by the ingestion of infective cysts. Once in the host’s stomach excystation occurs and trophozoites emerge. At the duodenum the parasites replicate and colonize this portion of the intestine. Subsequently trophozoites are transported by peristalsis to the jejunum and the ileum where encystation takes place and mature cysts are expelled in the stool (Ankarklev et al., 2010; Watkins and Eckmann,2014). The infection can be asymptomatic or present several clinical manifestations ranging from mild to severe symptoms that include diarrhea, steatorrhea, post-prandialepigastric pain, anorexia, bloating, and flatulence (Rossignol et al., 2012; Nash, 2013). Some infected patients may develop a chronic infection with recurrent diarrhea, steatorrhea, malabsorption, weight loss, and poor growth in children (Plutzer et al., 2010; Watkins and Eckmann, 2014).
The control of this infection is mainly carried out by treatment with chemotherapeutic agents. Among the drugs used are components that belong to 5-nitroimidazoles (e.g., metronidazole) and benzimidazoles (e.g., albendazole) derivatives. Other drugs prescribed against Giardiainclude nitazoxanide, furazolidone, paromomycin, and quinacrine (Tejman-Yarden and Eckmann, 2011; Watkins and Eckmann, 2014). Among these drugs albendazole (ABZ) is given in massive chemotherapy interventions against helminths based on its relative safety, high efficacy, broad spectrum against helminths, and low cost (Rossignol, 2010; Watkins and Eckmann, 2014). Further ABZ has been used against Giardia, particularly when metronidazole refractory cases occur (Lemée et al., 2000; Solaymani-Mohammadi et al., 2010). The side effects of this drug arerare but in some cases anorexia, constipation and neutropenia have been reported (Dayan, 2003). The use of ABZ is contraindicated during pregnancy due to possible teratogenic effects, although such effects have not been entirely confirmed (Gardner and Hill, 2001; Dayan, 2003).
In pharmacokinetic studies it has been determined that after its absorption, ABZ is oxidized to its metabolites, sulphoxide and sulphone, by cytochrome P450 and/or by flavin-dependent oxidases (Dayan, 2003). The production of these metabolites has been reported in Giardiaafter ABZ exposure (Oxberry et al., 2000; Argüello-García et al., 2015). It has also been reported that in helminths and fungi ABZ selectively binds to four β-tubulin sites, preventing its polymerization and affecting microtubule stability which in turn inhibits mobility and transport of molecules within the microorganism (Robinson et al., 2004; Diawara et al., 2013; Watkins and Eckmann, 2014).In helminths, ABZ-resistant parasites harbor hot-spotmutations in β-tubulin encodingdifferent amino acids, particularly at glutamate 198 and phenylalanine 200 (Rossignol,2010; Diawara et al., 2013; Hansen et al., 2013). In Giardia, it has been established that hot-spotamino acid mutations in β-tubulin are absent (Upcroft et al., 1996; Argüello-García et al., 2009) suggesting that the induction of ABZ-resistant phenotypes involves different mechanisms.
Regarding ABZ resistance in Giardia, it has been reported that resistant trophozoites display morphological changes, particularly in the median body, despite the conserved amino acid residues at positions 198 and 200 in β-tubulin (Chavez et al., 1992; Upcroftet al., 1996; Argüello-García et al., 2009). On the other hand, chromosomal rearrangements have been documented in ABZ-treated parasites, although there is no evidence of a gene or group of genes that may be affected during ABZ resistance in this parasite (Upcroft and Upcroft, 2001). Previous studies by our group suggest that diverse metabolic mechanisms may be involved in the ABZ resistance in Giardiathat could include components of antioxidant and energy metabolism as well as cytoskeletal changes in the parasite (Paz-Maldonado et al., 2013).
In this context, recent reports have suggested a direct relationship between the use of ABZ and oxidative stress. In a report in which ABZ was administered to rats in various doses and times, oxidative stress was elicited particularly in hepatocytes (Locatelli et al., 2004). Other studies have also shown the ability of ABZ to induce oxidative stress in sheep liver (Dimitrijević et al., 2012), and ABZ consumption may be correlated with liver damage in humans (Nandi and Sarkar, 2013). In Dicrocoelium dendriticum, a fluke of veterinary and human health importance, an increase in antioxidant enzyme activity after ABZ exposure was identified (Bártíková et al., 2010). However, no reports on oxidative damage due to ABZ in other parasites are available.
To determine in more detail the mode of action of ABZ in Giardia, in this work we have assessed the induction of oxidative stress by ABZ in G. duodenalistrophozoites by monitoring reactive oxygen species (ROS) formation. Results identified ABZ-induced oxidative stress in this protozoan. Oxidative damage to the parasite´s DNA is associated with cell cycle arrest and apoptosis. The consequences of this stress and its possible relationship to ABZ resistance in Giardiaare discussed.

Materials and Methods
Trophozoite Cultures, Growth of ABZ-Resistant Clones and Obtention of H2O2-Resistant Trophozoites

iardia duodenalistrophozoites of the WB strain (ATCC#30957) and ABZ-resistant cloneswere maintained in TYI-S-33 medium supplemented with 10% adult bovine serum (HyClone) and antibiotic/antimycotic solution (Thermo, USA) at 37°C (Keister, 1983) in 4.5 mL screw-capped vials. ABZ-resistant trophozoites were selected by continuous subculture under increasing sub-lethal concentrations of ABZ (Sigma cat. A-4673). When parasites were adapted to each increase of drug concentration, cultures were cloned by limiting dilution using the corresponding ABZ concentration (Paz-Maldonado et al., 2013). Trophozoites were sub-cultured twice a week under the continuous presence of drug (for ABZ-resistant clones) and for the ABZ-sensitive clones only in the presence of the vehicle (N, N-dimethylformamide; DMF, Sigma). To obtain the H2O2-resistant parasites (ROX), trophozoites were selected by continuous subculture under increasing sub-lethalconcentrations of H2O2 (Sigma, USA). Vials containing trophozoites were refilled to three-quarter capacity and H2O2-resistant parasites were cultured as described above for ABZ-resistant Giardia. Stock solutions (0.01–25 mM) of ABZ in DMF or DMF alone wereused in all assays. Oxidative stress was induced by exposing the parasites to 100 μM H2O2 and these cultures were used as positive controls (Raj et al., 2013).

Determination of Trophozoite Growth

Trophozoite growth was assessed by the fluorescent tracer SYTOX Green according to the manufacturer’s instructions (Invitrogen, USA). Briefly, trophozoites were washed three times in phosphate buffered saline (PBS) then suspended in lysis buffer (6% SDS, 10 mM HEPES) using a Vortex shaker for 10 s. A stock solution of SYTOX Green (5 mM) was added in a 1:5 v/v ratio and incubated for 10 min in the dark. The standard growth curve was obtained using variable numbers of lysed trophozoites and absorbance values of eachsample from non-treated and treated trophozoites were determined in 96-well, black-bottomed microtiter plates using a FACSCalibur reader fitted with 504/525 nm excitation/emission filters (Gerphagnon et al., 2013). Negative control absorbance values were obtained from wells with no cells.
Detection of Reactive Oxigen Species (ROS) in Trophozoites Incubated with ABZ or H2O2

Albendazole-sensitive trophozoites were incubated with ABZ (1.35, 8, and 250 μM), Dimethylformamide (DMF referred as vehicle) or H2O2 (100 μM) as control for oxidative stress, for 16 h at 37°C. ROS formation was assessed by Image-IT LIVE Green Reactive Oxygen Species Detection KitTM according to manufacturer´s instructions (Life Technologies, USA). After incubation, trophozoites were washed in PBS and suspended in 25 μM 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy- H2DCFDA) at 37°C for 30 min. Then Hoechst 33342 was added at a final concentration of 1 μM for 5 min. Cell fluorescence signals were detected at the end of the incubation period in a Beckman FACSCalibur Flow Cytometer or in an optical microscope using the BD FACSComp software.

Determination of Cross-Resistance to ABZ and H2O2 and Protection by Cysteine

The cross resistance between ABZ and H2O2 was evaluated using the ABZ-resistant and H2O2-resistant clones mentioned above. The ABZ-resistant clones (R1.35, R8, R250) were exposed to 0, 25, 50, 75, and 100 μM H2O2 for 24 h at 37°C. Cell number was determined by SYTOX Green. Resistance to ABZ was determined in ROX (H2O2-resistant) parasites which were exposed to 0.05, 0.1, 0.2, 0.4, and 0.8 μM of ABZ for 24 h at 37°C. In control cultures, ABZ or H2O2 were not added. Cell number was also determined using SYTOX Green. For cysteine protection assays, ABZ-sensitive trophozoites were grown in TYI-S-33 medium supplemented with different concentrations of cysteine (0.5, 1, 2, or 4mM). Then, trophozoites were incubated in the presence of 0.2 μM ABZ for 48 h at 37°C. Cell growth was determined by SYTOX Green as indicated above.

Detection of Protein Carbonylation and Lipid Peroxidation
Albendazole-sensitive trophozoites were incubated with DMF, ABZ (1.35, 8, and 250 μM) or H2O2 (100 μM) for 24 h at 37°C. Protein carbonylation was determined using a commercial kit (Protein Carbonyl Assay, Cayman Chemical, USA). Trophozoites were washed with PBS, suspended in lysis solution (50 mM MES, 1 mM EDTA at pH 7.4) and lysed by three cycles of freezing-thawing followed by centrifugation at 10,000 x gfor 10 min.Subsequently the protein was derivatized with dinitrophenylhydrazine (DNPH) for 60 minin the dark, the reaction was stopped with 20% trichloroacetic acid and samples were centrifuged at 10,000 × gfor 10 min. Then the samples were washed three times with ethanol/ethyl acetate solution. Finally, the proteins were suspended in guanidine hydrochloride and the absorbance was determined at 450 nm (Krisko and Radman, 2010). The concentration of protein in the soluble fraction was determined by absorbance at 280 nm.
For lipid peroxidation determination, after incubation with compounds or vehicle a solution of 1-methyl-2-phenylindole in a mixture of acetonitrile/methanol (3:1) was added to trophozoite homogenates. For malondialdehyde (MDA) determination, the reaction was initiated by adding HCl to a 37% v/v final concentration and for the 4-hydroxynonenal (HNE) assay methanesulfonic acid and FeCl3 at 34 μM (final concentration each) were used. The absorbance at 586 nm was measured upon incubation of the reaction mixture at 45°C for 40 min. For each series of assays, the absorbance of a control containing water instead of a sample was always subtracted. For each assay homogenate, a control sample in which the reagent was replaced by acetonitrile/methanol (3:1, v/v) was included. A standard curve of trimethoxypropane was used in all assays (Gérard-Monnier et al., 1998; Orozco-Ibarra et al., 2007).

Detection of DNA Fragmentation

Albendazole-sensitive trophozoites were incubated with different concentrations of ABZ (1.35, 8 and 250 μM), DMF or H2O2 (100 μM) for 24 h at 37°C, then washed twice in PBS 1X and incubated overnight at 42°C in a lysis solution (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 150 mM NaCl, 0.4% sodium dodecyl sulfate, and 200 μg/mL proteinase K). RNA wasremoved by incubating samples with 20 mg/mL RNase A at 37°C for 30 min. The lysate was treated with phenol/chloroform (1:1) and nucleic acids were precipitated at -20°C with 0.3 M sodium acetate pH 7 and ethanol. After quantification, the extent of DNA fragmentation was analyzed by electrophoresis on 1% agarose/ethidium bromide gels (Ghosh et al., 2009).

Detection of Oxidative DNA Damage

DNA damage was assessed by immunofluorescence with an anti-8-hydroxydeoxyguanosine (8OHdG) monoclonal antibody (Santa Cruz Technologies, USA). ABZ-sensitive trophozoites treated with DMF, different concentrations of ABZ (1.35, 8, and 250 μM) or H2O2 (100 μM) for 16 h at 37°C were incubated for 1 h at 37°C on poly-L-lysine-coated (2 mg/ml) coverslips, rinsed twice with PBS and fixed with a solution of methanol:acetone (1:1 v/v) at -20°C. Fixed cells were treated with 0.05 N HCl for 5 minon ice, rinsed with PBS and washed with PBS containing 35, 50, and 75% ethanol consecutively for 3 min each time. DNA was denatured in situwith 0.15 N NaOH in 70% ethanol for 4 min. The precipitate was rinsed twice with PBS and incubated with 0.2 μg/ml Hoechst dye for 10 min. Subsequently parasites were washed with PBS containing 75, 50, and 35% ethanol consecutively in the presence of 4% formaldehyde for 2 min each time. The samples were incubated in trypsin solution (49.5 mM Tris base, 1 mM EDTA, 150.7 mM Na2HPO4, 14.9 mM K2HPO4, 0.1% trypsin at pH 7.2) for 10 min at 37°C and washed three times with PBS. Trophozoites were then incubated for 30 min with 1% bovine serum albumin (BSA) to block nonspecific binding and incubated with mouse monoclonal anti- 8-OHdG for 1 h. After a wash with PBS, cells were incubated for 1 h at room temperature with goat anti-mouse IgG coupled to FITC (Santa Cruz Technologies, USA). Samples were analyzed using a Zeiss microscope equipped with epifluorescence illumination as previously described (Suzuki et al., 2006).

Detection of Protein-MDA Adducts and H2AX Phosphorylation by Western Blot

Both protein-MDA adducts and histone H2AX phosphorylation (at ser139) were evaluated by Western blot assays using specific antibodies. In these, ABZ-sensitive trophozoiteswere incubated with DMF, different concentrations of ABZ (1.35, 8, and 250 μM) or H2O2 (100 μM) for 24 h at 37°C, washed with PBS tree times and suspended in lysis buffer. Twenty microgram of protein were analyzed by SDS-PAGE in 12% acrylamide gels for the protein-MDA assay and in 15% acrylamide gels for detection of H2AX phosphorylation. After electrophoresis, gels were transferred to nitrocellulose membranes. Membranes were blocked with PBS containing 0.1% Tween-20 and 1% skim milk for 2 h at 37°C. After washing with Tris-buffered saline (TBS) membranes were incubated with rabbit anti-MDA (Abcam, USA) and rabbit anti-H2AX (Millipore, USA) antibodies for 1 h at room temperature under constant shaking. Membranes were washed and incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG (Thermo, USA). Chemiluminescence detection was performed with the Amersham ECL detection kit according to manufacturer’s instructions (Hofštetrová et al., 2010; Moore et al., 2013).

Identification and Quantification of Apoptotic and Necrotic Cells

The cells undergoing apoptotic or necrotic processes after ABZ- or H2O2 exposure were analyzed by flow cytometry in which fluorescence by annexin V binding (green) and propidium iodide (PI) uptake (red) were quantified. Positioning of quadrants on annexin V/PI dot plots was analyzed according to the following pattern: living cells (annexin V-/PI-), early apoptotic/primary apoptotic cells (annexin V+/PI-), late apoptotic/secondary apoptotic cells (annexin V+/PI+) and necrotic cells (annexin V-/PI+). The assay was carried out using the Annexin V-FITC Apoptosis Detection Kit (BioVision, USA) following the manufacturer´s instructions. Briefly, cells were incubated with DMF, different concentrations of ABZ (1.35, 8, and 250 μM) or H2O2 (100 μM) for 24 h. Then, trophozoites were centrifuged at 440 × gat 4°C and suspended in 500 μl of 1X binding buffer. Cells were then incubated with 5 μl of annexin V–FITC and 5 μl of PI (50 μg/ml) for 5 min in the dark at room temperature. The FITC and PI fluorescence was measured with a FACS Calibur Flow Cytometer equipped with an FL-1 filter (530 nm) and an FL-2 filter (585 nm), respectively, in at least 10,000 events (Ghosh et al., 2009) in each experiment.

Determination of Cell Cycle Stages in G. duodenalisTrophozoites Exposed to ABZ

To determine the proportions of trophozoites at the different cycle stages, nuclear staining with PI was coupled to flow cytometry. In brief, ABZ-sensitive trophozoites were exposed to different concentrations of ABZ (1.35, 8, and 250 μM) for 4 h at 37°C, washed with PBS and fixed 30 min with 70% ethanol in PBS. Then cells were washed again and incubated in PBS containing 0.1 mg/mL RNAase overnight at 4°C. Finally cell pellets were washed, stained with PI (1 μM in PBS), washed and resuspended in small volume (200–300 μL) for analysis in a FACS Calibur Flow Cytometer in at least 10,000 events per sample. The histogram areas were identified as reported by Reaume et al. (2013).

Statistical Analyses

All the data were obtained from at least three experiments and where indicated the results are expressed as mean ± SD. Inter-group variation was assessed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Statistical significance was determined if p≤ 0.05.

Results
Intracellular ROS Formation

In some reports using animal models ABZ was shown to produce oxidative damage (Locatelli et al., 2004; Bártíková et al., 2010). In G. duodenalisoxidative stress damage has been induced using pro-oxidant compounds such as H2O2 (Ghosh et al., 2009; Raj et al., 2013) in which intracellular ROS formation hallmarks this phenomenon. ABZ-exposed parasites showed greater ROS signals than parasites not exposed to the drug(Figure 1, top panels). The effect was mainly detected at the highest ABZ concentration tested (250 μM), however, ROS formation could be determined by flow cytometry at lower concentrations (Figure 1, bottom panels).

FIGURE 1.Reactive oxygen species (ROS) are produced in Giardia duodenalistrophozoites exposed to Albendazole (ABZ).

ABZ-sensitive Giardiastrain (WB) was exposed to vehicle (DMF, A) and to 1.35 μM (B), 8 μM (C), or 250 μM (D)of ABZ for 8 h at 37°C, then DCFDH was added to monitor ROS production. In these experiments a controlof intracellular ROS production was included. In this WB trophozoites were incubated with 100 μM H2O2 (E). Trophozoites micrographs are as follows: top panels bright field(BF), middle panel trophozoites’ nuclei stained with Hoechst and lower panel trophozoites stained with DCFDH. The cells showed increased ROS production by ABZ treatment in comparison to control cells with no drug. Bottom panels are ROS production monitored by flow cytometry, the shift of fluorescence in Xaxis indicates ROS production by live trophozoites (determined by Trypan blue exclusion) at the highest ABZ concentration. The table shows population percent in M1 (negative to ROS) and M2 (positive to ROS) according to flow cytometry data. Micrographs are from representative results of at least three experiments performed with independent batch cultures.
To determine the localization of intracellular ROS formation within the trophozoites confocal microscopy was used. In these experiments the typical altered morphology caused by benzimidazoles (Paz-Maldonado et al., 2013) was observed in ABZ-treated trophozoites (Figure 2Atop panel c). In these cells ROS formation was also evident in most cells as determined by fluorescent staining (Figure 2Atop panel d). When individual cells were observed the trophozoites´nuclei were determined as the primary site of ROS formation (Figure 2B) as judged by fluorescent staining at low ABZ concentrations used (1.35 μM and 8 μM). At the highest drug concentration used (250 μM) there was a widespread distribution of ROS throughout the trophozoite cytoplasm (Figure 2Bmiddle panel).

FIGURE 2.Intracellular localization of ROS production in G. dudodenalistrophozoites exposed to ABZ.

WB Giardiatrophozoites were exposed to DMF or to the indicated ABZ concentrations (from left to right: 1.35, 8, and 250 μM) for 8 h at 37°C. Cells were then incubated with DCFDH. Trophozoite micrographs are as follows: (A)top panel: representative images of trophozoites exposed to DMF (a,b) or to 250 μM ABZ (c,d) and then incubated with DCFDH. Morphological changes in trophozoites (c BF) and ROS localization (d epifluorescence illumination) are evident in ABZ treated cells. (B)Images of representative individual cells. Top panel trophozoites´ nuclei stained with Hoechst, middle panel trophozoites incubated with DCFDH (epifluorescence illumination) and lower panel merged cell images. At the lowest concentrations, ROS production is restricted to nuclei, whereas at the highest ABZ concentration, this is detected all over the cytoplasm. The micrographs are representative of at least three independent experiments.

Cross Resistance to ABZ and H2O2 in GiardiaTrophozites

The ABZ-resistant clones, namely R1.35, R.8, and R.250 (Argüello-García et al., 2009; Paz-Maldonado et al., 2013) were used to determine whether cross-resistance to classical oxidative stressor (H2O2) and ABZ was induced in the resistant trophozoites. For this purpose the ABZ-resistant clones were incubated under increasing concentrations of H2O2, and cell growth was determined. In general the resistant clonesR1.35 and R.250 showed a tendency to increased resistance to H2O2-induced death in comparison to the ABZ-susceptible WB strain (Figure 3A). A special case is the R8 resistant strain which frequently behave, in this and other studies, as a “transition state” between low and high ABZ resistance depending on the parameter that is evaluated (see also Figure 6A; Argüello-García et al., 2009; Paz-Maldonado et al., 2013)

FIGURE 3.Cross-resistance to ABZ and H2O2 and cysteine (Cys) protection in trophozoites exposed to ABZ.

(A)Cross-resistance to H2O2 was evaluated in the ABZ-resistant clones R1.35 (black bars), R.8 (horizontal lined bars), and R.250 (vertical lined bars) previously obtained in our group (Paz-Maldonado et al., 2013). Parasites were exposed to the indicated H2O2 concentrations for 24 h and the cell numberwas determined by SYTOX Green. As control, wild type WB strain (white bars) was incubated with the indicated H2O2 concentration. (B)Cross-resistance to ABZ in a H2O2-resistant G. dudodenalis.A H2O2-resistant Giardia strain (ROX) was obtained by sub-culturing the WB strain in increasing H2O2 concentration for 6 months. Control WB (white bars) and ROX (gray bars) trophozoites were exposed to the indicated increasing ABZ concentrations for 24 h and the cell number was determined using the fluorescent tracer SYTOX Green. The ROX strain showed cross-resistance to ABZ. (C)Cysteine protection of Giardiatrophozoites exposed to ABZ. WB trophozoites were incubated in growth medium with 0.5 mM (white bar), 1 mM (lined up to the right bar), 2 mM (lined up tothe left bar), or 4 mM (boxed bar) of cysteine for 24 h and then trophozoites were further incubated with 0.2 μM ABZ for 48 h. In all graphs the results are the mean ± SD of at least three independent experiments. In graphs (B,C)∗ indicates p≤ 0.05 by ANOVA and Tukey´s analysis in which values obtained with WB trophozoites were compared with values obtained in ROX trophozoites exposed to the different ABZ concentrations (B). (C)Values obtained in WB trophozoites treated with 0.5 mM cysteine were compared to values obtained in WB trophozoites treated with different cysteine concentrations and then exposed to ABZ.
Resistance to H2O2 was achieved by using continuous trophozoite subcultures under increasing concentrations of H2O2. Following this procedure for approximately 6 months, a strain displaying resistance to 75 μM H2O2 was obtained (ROX). This strain was incubated in the presence of ABZ showing a significantly higher growth rate as compared with the H2O2-susceptible WB strain (Figure 3B).

Cysteine Increases Tolerance to ABZ in the Drug Treated Trophozoites
Giardia duodenalispossesses a bacterial-like antioxidant metabolism in which cysteineis the major antioxidant thiol instead of glutathione. To test if increased levels of extracellular cysteine may confer increased tolerance to the drug in ABZ-susceptible trophozoites, these clones were incubated in culture media containing different cysteine concentrations and subsequently ABZ at 0.2 μM was added and after 48 h-incubation trophozoite growth was measured. As a result, parasites incubated at cysteine concentrations higher than 1 mM showed higher growth after ABZ exposure than those incubated at lower concentrations of this amino acid (Figure 3C). These results suggest that cysteine may help to contend the ROS damage upon ABZ exposure.

Damage to Biomolecules after ABZ Exposure in G.