Lethal action of the nitrothiazolyl-salicylamide derivative
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ARTICLE IN PRESS
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Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum
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Juliana Tonini Mesquita a , Erika Gracielle Pinto a,b , Noemi Nosomi Taniwaki a , Andres Jimenez Galisteo Jr b , Andre Gustavo Tempone a,∗ a
Department of Parasitology, Instituto Adolfo Lutz, Av. Dr. Arnaldo, 351, 01246-900 São Paulo, SP, Brazil Laboratorio de Protozoologia, Instituto de Medicina Tropical, Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 470, 05403-000 São Paulo, SP, Brazil
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Article history: Received 11 July 2013 Received in revised form 13 September 2013 Accepted 14 September 2013 Available online xxx
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Keywords: Leishmania Nitazoxanide Therapy Oxidative stress Phosphatidylserine Mitochondria
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1. Introduction
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Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileishmanials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileishmanial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZ’s activity against L. infantum was confirmed, producing IC50 values of 42.71 g/mL against promastigotes and 6.78 g/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depolarising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX® Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chromatin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets. © 2013 Published by Elsevier B.V.
Neglected parasitic diseases, such as visceral leishmaniasis (VL), remain an important concern for the public health systems of developing countries because these diseases affect at least 12 million people in 98 countries and territories. VL is associated with poverty, malnutrition and co-infections with HIV and other parasitic diseases (WHO, 2010). The therapeutics for VL are problematic because they exhibit elevated toxicity and require long-term treatment. The increasing parasite resistance to these drugs is also a serious obstacle (Sundar and Chakravarty, 2012). Creating new selective drugs with higher therapeutic indexes and better affordability for developing countries has been a major spotlight for Drug Discovery & Development programmes (WHO, 2010).
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∗ Corresponding author. Tel.: +55 11 3068 2991; fax: +55 11 3068 2890. E-mail address: [email protected] (A.G. Tempone).
Manipulating the cell death pathways in Leishmania (L.) infantum may reveal new treatment opportunities (Andrade-Neto et al., 2011). Therefore, assessing new therapeutic targets creates unique opportunities to discover selective compounds with activity focused against the parasite (Nunes et al., 2013) while avoiding toxicity in mammalian cells. In this context, the redox system is critical for the survival of L. (L.) infantum in macrophages. Drugs that affect metabolism or cause reactive oxygen species (ROS) generation, inhibit the production of ergosterol (Medina et al., 2012) or induce programmed cell death are novel therapeutic advances (Debrabant et al., 2003; Pal and Bandyopadhyay, 2012). Redox imbalance occurs in the parasite when the endogenous antioxidants fail to cope with the excessive ROS, leading to oxidative stress. Therefore, antiparasitic drugs that inhibit vital redox reactions or promote oxidative stress in parasites may be promising candidates (Pal and Bandyopadhyay, 2012). Drug repurposing has been a strategic, efficient and costeffective approach for finding novel and effective antiparasitics for neglected diseases (Ekins et al., 2011). Nitazoxanide (NTZ) is a
0001-706X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.actatropica.2013.09.018
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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synthetic nitrothiazolyl-salicylamide derivative that is approved for treating infectious diarrhoea caused by Cryptosporidium parvum and Giardia lamblia, as well as others parasites such as Giardia intestinalis, Entamoeba histolytica, and Trichomonas vaginalis (Anderson and Curran, 2007). Antileishmanial (Zhang et al., 2010) and antitrypanosomal activities have also been reported for this compound (Navarrete-Vazquez et al., 2011). Using different fluorimetric techniques and transmission electron microscopy, the activity and lethal action of NTZ was evaluated for the first time in L. (L.) infantum to investigate its effects in mitochondria and on the regulation of programmed cell death markers.
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2. Materials and methods
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2.1. Chemicals
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Alamar blue® (resazurin), SYTOX® Green, Mitotracker® Red CM-H2 XROS, the Reactive Oxygen Species H2 DCF-DA and the Annexin-V FITC Apoptosis Kit were purchased from Molecular Probes® (Invitrogen, USA). Dimethyl sulfoxide (DMSO) and methanol (MeOH) were obtained from Merck. M-199 medium, Hank’ Balanced Salt Solution (HBSS), phosphate-buffered saline (PBS), oligomycin, Triton X-100 and nitazoxanide were obtained from Sigma. All other reagents were obtained from Sigma.
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2.2. Parasites and mammalian cells maintenance
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L. (L.) infantum (MHOM/BR/1972/LD) was maintained in Golden hamsters up to approximately 60–70 days post-infection. The promastigotes were maintained in M-199 medium supplemented with 10% calf serum and 0.25% hemin at 24 ◦ C. The amastigotes were obtained from the spleens of previously infected hamsters by differential centrifugation. The macrophages were collected from the peritoneal cavity of BALB/c mice by washing with RPMI-1640 medium supplemented with 10% calf serum and were maintained at 37 ◦ C in a humidified incubator with 5% CO2 . 2.3. Bioassay procedures The golden hamsters were obtained from the animal breeding facility at the Adolfo Lutz Institute, SP, Brazil. The animals were maintained in sterilised cages in a controlled environment, while receiving water and food ad libitum. All of the animal procedures were performed with the approval of the Research Ethics Commission, in agreement with the Guide for the Care and Use of Laboratory Animals from the National Academy of Sciences. The experiments involved the minimum number of animals necessary to produce statistically reproducible results. 2.4. Determination of the 50% inhibitory concentration (IC50 ) against L. (L.) infantum 2.4.1. Promastigotes NTZ was dissolved in DMSO and diluted in M-199 medium in 96-well dark microplates to concentrations up to 100 g/mL. The promastigotes were in the late growth-phase (non-stationary) before they were washed in M-199 medium, counted in a Neubauer hemocytometer, seeded at 1 × 106 /well with a final volume of 200 L and incubated for 48 h at 24 ◦ C. The viability of the promastigotes was detected with rezasurin (0.011% in PBS) (Mikus and Steverding, 2000) followed by a 48 h incubation (20 L/well) under the same conditions. The fluorescence intensity was determined using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader-Molecular Devices® ) with excitation and emission wavelengths of 540 and 595 nm, respectively. The control group consisted of promastigotes incubated with 0.1–0.5% DMSO
(internal control). Miltefosine was used as a standard drug. The cell viability was expressed based on the fluorescence units of the control after normalisation. The data represent the mean of three independent assays (triplicates). 2.4.2. MTT assay The colorimetric diphenyltetrazolium assay (MTT) was performed to assess the viability of the parasites after 24 h. Briefly, MTT (5 mg/mL) was dissolved in PBS, sterilised using 0.22 m membranes, added at 20 L/well and incubated for 4 h at 24 ◦ C. The formazan extraction was performed using 10% SDS for 18 h (80 L/well) at 24 ◦ C, and the optical density (OD) was colorimetrically determined in a plate reader (FilterMax F5 Multi-Mode Microplate Reader) at 550 nm (Corrêa et al., 2011). The data obtained represent the mean of three independent assays. 2.4.3. Intracellular amastigotes Peritoneal macrophages were seeded at 1 × 105 cells/well in 16-wells slide chambers (NUNC® ), infected with the previously isolated (hamster spleen) L. (L.) infantum amastigotes in a 1:10 ratio (macrophage/amastigotes) and treated with NTZ for 120 h at 37 ◦ C in a humidified incubator with 5% CO2 . At the end of the assay, the macrophages were fixed with methanol, stained with Giemsa and observed using light microscopy. The parasite burden was verified using the number of infected macrophages out of 400 cells. The data obtained represent the mean of three independent assays, and each assay was performed in duplicate (Corrêa et al., 2011). 2.4.4. Cytotoxicity against mammalian cell Mouse connective tissue cells (NCTC clone 929) were seeded at 6 × 104 cells/well in 96-well microplates and incubated with test compounds for 24, 48, 72 and 96 h at 37 ◦ C in an incubator with 5% CO2 . Nitazoxanide was dissolved in DMSO and diluted to the highest concentration (200 g/mL); miltefosine and Glucantime® were used as the standards. The viability of the cells was determined using resazurin (Mikus and Steverding, 2000). The data represent the mean of three independent assays (triplicates). The selectivity index was determined by the IC50 against mammalian cells/IC50 against amastigotes (Corrêa et al., 2011). 2.5. Spectrofluorimetric detection of cell membrane permeability Late growth-phase (non-stationary) promastigotes were washed in PBS, seeded at 2 × 106 /well and incubated with 1 M SYTOX® Green for 15 min at 24 ◦ C (Mangoni et al., 2005). NTZ was added at a concentration similar to its IC50 (60 g/mL), and the fluorescence was measured every 20 min (up to 60 min). The maximum permeabilisation was obtained with 0.1% Triton X-100. The fluorescence intensity was determined using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate ReaderMolecular Devices) with excitation and emission wavelengths of 485 and 520 nm, respectively. The following internal controls were used: (i) detection of possible NTZ fluorescence at the appropriate wavelengths, (ii) detection of possible interference by DMSO, (iii) untreated promastigotes and (iv) medium without cells. The samples were examined in triplicate. 2.6. Effect on mitochondrial membrane potential L. (L.) infantum promastigotes (late growth-phase) were washed twice in PBS, seeded at 2 × 106 /well and incubated with NTZ at a concentration close to its IC50 (60 g/mL) for 60 min. MitoTracker® Red CM-H2 XROS (500 nM) was added, and the sample was incubated for 40 min in the dark. The cells were washed twice in HBSS, and the fluorescence was determined using a fluorimetric
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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microplate reader (FilterMax F5 Multi-Mode Microplate ReaderMolecular Devices) with excitation and emission wavelengths of 540 and 595 nm, respectively (Williams et al., 2012). The following internal controls were used: (i) detection of possible NTZ fluorescence at the appropriate wavelengths, (ii) detection of possible interference by DMSO, (iii) untreated promastigotes and (iv) medium without cells. The samples were assessed in triplicate.
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2.7. Detection of reactive oxygen species (ROS) production
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L. (L.) infantum promastigotes (2 × 106 /well) were washed in HBSS and incubated with NTZ at a concentration close to its IC50 (60 g/mL) for 60 min. H2 DCF-DA (5 M) was added, and the cells were incubated for 15 min. The fluorescence intensity was detected using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader-Molecular Devices) at 485 and 520 nm for excitation and emission, respectively. Oligomycin (20 M) was used at as a positive control. The following internal controls were used: (i) detection of possible fluorescence NTZ at the appropriate wavelengths, (ii) detection of possible interference by DMSO, (iii) untreated promastigotes and (iv) medium without cells. The samples were assessed in triplicate.
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2.8. Flow cytometry analysis of phosphatidyserine (PS) exposure
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The promastigotes (2 × 106 /well) were washed in M-199 medium and incubated with NTZ at a concentration close to its IC50 (60 g/mL) for different periods (12, 16 and 24 h). To assess the PS exposure using FACS, the cells were incubated with FITC-annexin V and propidium iodide according to the manufacturer’ instructions. The cells were evaluated with a BD LSRFortessa flow cytometer (Becton Dickinson® ), and the data were analysed using FlowJo software (Tree Star® , Inc.); at least 10,000 events were acquired per analysis (Farias et al., 2013).
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2.9. Ultrastructural studies
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Transmission electron microscopy images of L. (L.) infantum promastigotes incubated with NTZ were collected after different periods of incubation (1, 3, 6 h). The cells (1 × 107 /well) were incubated with NTZ at 24 ◦ C in 24-well plates before being processed and observed using a JEOL transmission electron microscope (Duarte et al., 1992).
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2.10. Statistical analysis
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The results are represented by the means and standard deviations of replicate samples from at least two independent assays. The IC50 values were calculated using sigmoid dose–response curves in Graph Pad Prism 5.0, and the 95% confidence intervals are included in parentheses. The ANOVA test was used to test for significance (p < 0.05). 3. Results 3.1. NTZ is effective against L. (L.) infantum Promastigotes (Fig. 1) were incubated with nitazoxanide in vitro, and their viability was evaluated using a fluorimetric assay with resazurin. After 48 h, 100% of the parasites were eliminated at the highest concentration (100 g/mL), generating an IC50 value of 42.71 g/mL (95% confidence interval 28.77–63.45 g/mL) (Table 1). Miltefosine was used as the standard drug and generated an IC50 value of 6.69 g/mL (95% confidence interval 6.29–7.11 g/mL). NTZ was incubated with intracellular amastigotes, and after 120 h, no parasites were observed in
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Fig. 1. Evaluation of the mitochondrial membrane potential of Leishmania (L.) infantum incubated with nitazoxanide. MitoTracker Red® was incubated with treated and untreated parasites (control group). The fluorescence was measured using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader) at 540 and 595 nm for excitation and emission, respectively. The chemical structure of nitazoxanide was included (PubMed Compound). *p < 0.05.
the macrophages, revealing an IC50 value of 6.78 g/mL (95% confidence interval 6.51–7.06 g/mL) (Table 1). Miltefosine and Glucantime® were used as the standard drugs and attained IC50 values of 6.87 g/mL (95% confidence interval 4.71–10.01 g/mL) and 22.80 g/mL (95% confidence interval 20.76–25.04 g/mL), respectively (Table 1). 3.2. Mammalian cell cytotoxicity NTZ was incubated for 24, 48, 72 and 96 h with mammalian cells, and the cell viability was assessed using the resazurin assay. Mouse connective tissue cells (NCTC clone 929) were susceptible to NTZ, generating IC50 values in the range of 8–29 g/mL, and selectivity indexes in the range of 1.2–4.3. Miltefosine also affected mammalian cells and had an IC50 value of 49.72 g/mL (95% confidence interval 39.85–63.98 g/mL) (Table 1). 3.3. Permeability of plasma membrane NTZ was incubated with promastigotes, and the plasma membrane permeability was evaluated with the vital dye SYTOX® Green (20 min intervals). NTZ did not interfere with the plasma membrane, even after up to 60 min of incubation; no increase in the fluorescence levels could be detected (data not shown). 3.4. NTZ affects the mitochondria of Leishmania: evaluation of the mitochondrial membrane potential Promastigotes were incubated with NTZ for 60 min, and the mitochondrial membrane potential was monitored using the fluorescent probe Mitotracker Red® . NTZ induced a 100% (p < 0.05) decrease in the fluorescence intensity relative to the untreated promastigotes (Fig. 1). MitoTracker Red® was incubated with untreated parasites as a control, which displayed the parasite’s
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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Table 1 Antileishmanial activity, mammalian cytotoxicity and selectivity index of nitazoxanide. Cell viability was determined using a fluorimetric assay with resazurin. *p < 0.05. Drugs
Nitazoxanide
IC50 (g/mL) 95% C.I. Promastigotes
Amastigotes
Cytotoxicity
S.I.#
42.71* (28.77–63.45)
6.78* (6.51–7.06)
a
4.35 3.08 1.73 1.25 >8.70 7.23
29.51 (22.57–38.58) 20.94 (17.90–24.48) c 11.79 (9.96–13.95) d 8.48 (7.75–9.27) >200 49.72 (39.85–63.98) b
Glucantime® Miltefosine
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nd 6.69 (6.29–7.11)
22.80 (20.76–25.04) 6.87 (4.71–10.01)
IC50 – 50% inhibitory concentration; 95% C.I. – 95% confidence interval; # S.I. – selectivity index; *p < 0.05. a Cytotoxicity performed in 24 h. b Cytotoxicity performed in 48 h. c Cytotoxicity performed in 72 h. d Cytotoxicity performed in 96 h.
typical mitochondrial membrane potential. Fig. 1 also includes NTZ’s chemical structure. 3.5. Reactive oxygen species (ROS) production NTZ was incubated with promastigotes for 60 min, and the ROS production was monitored using the H2 DCF-DA fluorescent probe. NTZ enhanced the production of ROS in L. (L.) infantum 129-fold relative to the untreated cells, displaying levels similar to the positive control, oligomycin (Fig. 2). 3.6. NTZ induces exposure of phosphatidylserine L. (L.) infantum was incubated for 12, 16 and 24 h with NTZ, while the binding of annexin V and propidium iodide was investigated by flow cytometry. After 12 h, 83.9% of the parasites were positive for both annexin V and propidium iodide, 3.97% were positive for only propidium iodide and 0.49% were positive for only annexin V (Fig. 3 D). After 16 h, 93.4% were positive for both annexin V and propidium iodide, 5.9% were positive for only propidium iodide and 0.04% were only positive for only annexin V (Fig. 3E). After 24 h, 97.4% of the parasites were positive for both annexin V and propidium iodide, 2.43% were positive for only propidium iodide and 0.024% were positive for only annexin V (Fig. 3F). Miltefosine was used as a positive control; after 24 h, 99.4% of the parasites were positive for both annexin V and propidium iodide, 0.12% were positive for only propidium iodide and 0.018% were positive for
only annexin V (Fig. 3C). Untreated promastigotes were used as a negative control (24 h), revealing that 6.58% of the parasites were positive for both annexin V and propidium iodide, 2% were positive for only propidium iodide and 1.75% were positive for only annexin V (Fig. 3B). Promastigotes without fluorescent labelling were used as the internal control (Fig. 3A). 3.7. NTZ induces ultrastructural alterations NTZ was incubated with promastigotes for different periods, and the ultrastructural modifications were analysed by transmission electron microscopy. After 1 h of incubation, it was possible to observe a slight mitochondrial swelling relative to the untreated control (Fig. 4B), but the kinetoplast was preserved. Therefore, the plasma membrane of L. (L.) infantum was intact. After 3 h, although no alteration in the parasite’s morphology was observed, an increased number of vacuoles (v) were observed; NTZ also induced the formation of enlarged mitochondria (Fig. 4C). After 6 h of incubation (Fig. 4D), nuclear membrane detachment and chromatin condensation were observed (arrow), along with an increase in plasma membrane blebs (arrow) and a progressive swelling of the mitochondria (m). Untreated parasites are depicted in Fig. 4A, demonstrating preserved structures, such as the nucleus (n), kinetoplast (k) and flagellum (f). 3.8. MTT reduction The mitochondrial oxidative activity of L. (L.) infantum incubated with NTZ (100 g/mL) was evaluated using a tetrazolium assay (MTT). After 24 h of incubation, no MTT reduction was observed in the promastigotes, demonstrating the leishmanicidal activity of the drug (Fig. 5). Miltefosine was used as a standard drug. 4. Discussion
Fig. 2. Reactive oxygen species (ROS) regulation in Leishmania (L.) infantum incubated with nitazoxanide. H2 DCF-DA was incubated with cells, and the fluorescence intensity was detected using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader) at 485 and 520 nm for excitation and emission, respectively. Oligomycin (20 M) was used as a positive control. *p < 0.05.
During the constant search for novel targets in L. (L.) infantum, the pursuit of drugs that affect mitochondrial metabolism might be an interesting strategy to find new therapeutic compounds that are active against protozoans. The survival of L. (L.) infantum depends on the proper behaviour of its mitochondria; in particular, this organism contains only a single mitochondrion, and therefore, drugs affecting mitochondria could explore the low compensation capacity of the injured organelle. The synthetic nitrothiazolylsalicylamide derivative, nitazoxanide (NTZ), is an orally delivered drug that is used for treating infectious diarrhoea caused by C. parvum and G. lamblia (Anderson and Curran, 2007); NTZ was studied against the etiologic agent of American Visceral Leishmaniasis, L. (L.) infantum. The promastigotes of L. (L.) infantum were susceptible to NTZ at levels to the standard drug miltefosine. Despite its toxicity towards mammalian cells, NTZ eliminated the intracellular
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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Fig. 3. Evaluation of the phosphatydilserine exposure using flow cytometry data for Leishmania (L.) infantum incubated with nitazoxanide. The cells were labelled with FITCannexin V and propidium iodide and analysed in a BD LSRFortessa flow cytometer (Becton Dickinson® ). The data were analysed using the FlowJo software (Tree Star® , Inc.) and at least 10,000 events were acquired per analysis. (A) untreated and unlabelled parasites (control 24 h); (B) negative control (untreated 24 h); (C) miltefosine (positive control 24 h); (D) nitazoxanide (12 h); (E) nitazoxanide (16 h); (F) nitazoxanide (24 h).
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amastigotes with effectiveness similar to the standard drug miltefosine; it was also 3.3-fold more effective than the standard drug Glucantime® . NTZ has also demonstrated activity against Leishmania donovani promastigotes (Zhang et al., 2010) with a similar IC50 value, but this is the first description of its activity against the clinically relevant stage of the parasite (the intracellular amastigotes). NTZ is therefore a promising antileishmanial drug candidate. Zhang et al. (2010) also demonstrated that oral NZT (200 mg/kg) reduced approximately 85% of the parasite load of L. donovani in a mouse model. New synthetic derivatives have also been explored, demonstrating that NTZ might be a promising scaffold for drug design (Zhang et al., 2010).
To investigate the permeability of NTZ-treated L. (L.) infantum’s plasma membranes, a spectrofluorimetric assay using SYTOX® Green was performed. SYTOX® Green is a green-fluorescent nuclear and chromosome counter stain that is impermeant towards live cells and exhibits >500-fold fluorescence enhancement after binding nucleic acids (Mangoni et al., 2005). Our data clearly demonstrated that NTZ did not alter the permeability of L. (L.) infantum’s plasma membrane after 60 min of incubation. Viability tests have been used to confirm the potential of drug candidates. The reduction of the yellow mitochondrial dehydrogenase substrate, methyl-tetrazolium (MTT), to form the purple water-insoluble formazan was used as a cell death marker (Tada
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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Fig. 4. Transmission electron microscopy images of Leishmania (L.) infantum promastigotes treated with nitazoxanide. (A) Control (untreated parasites); (B) nitazoxanide 1 h; (C) nitazoxanide 3 h; (D) nitazoxanide 6 h; k – kinetoplast, n – nucleus, f – flagellum, v – vacuoles, L – lipids.
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et al., 1986; Tempone et al., 2011). Leishmania-treated parasites exhibited no MTT oxidation after 24 h of treatment with NTZ, suggesting the drug had a leishmanicidal effect. Studying the lethal action of drugs in parasites could provide important information about novel drug targets. Because NTZ had antileishmanial potential, its lethal action was investigated in L. (L.) infantum promastigotes. The mitochondrial membrane potential is crucial for ATP generation in the respiratory chain (Joshi and Bakowska, 2011). In the absence of properly functioning mitochondria, the cells stop synthesising ATP from their mitochondrial
Fig. 5. Evaluation of the mitochondrial oxidative activity of Leishmania (L.) infantum promastigotes incubated for 24 h with NTZ. The colorimetric assay of MTT was read at 550 nm. Miltefosine was used as a standard drug; *p < 0.05.
source, leading to cell death (Joshi and Bakowska, 2011). MitoTracker Red® CM-H2 XROS is a reduced, nonfluorescent probe that fluoresces after its oxidation in the mitochondria of live cells; its accumulation depends on the membrane potential (Williams et al., 2012). In our assays, NTZ significantly (p < 0.05) depolarised the mitochondrial membrane potential of L. (L.) infantum because the fluorescence became undetectable after 60 min of incubation. Hoffman et al. (2007) demonstrated that NTZ intercepts pyruvate oxidation by inhibiting pyruvate: ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reaction in T. vaginalis, E. histolytica, G. intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni. Oxidising pyruvate during glycolysis is essential for generating acetyl-CoA and consequently ATP. Considering that a significant loss of mitochondrial membrane potential depletes cells of energy (Joshi and Bakowska, 2011), our data suggest that the depolarised mitochondrial membrane potential induced by NTZ might have contributed to L. (L.) infantum death. Furthermore, mitochondria are the main target of injury after stresses leading to programmed cell death and necrosis. Moreover, this event verifies that depolarising the mitochondrial membrane potential is a pre-apoptotic event (Joshi and Bakowska, 2011). Mitochondrial membrane potential reflects the pumping of hydrogen ions across the inner membrane during electron transport and oxidative phosphorylation. Under physiological conditions, oxidative phosphorylation releases reactive oxygen species (ROS) equivalent to 3 to 5% of total oxygen consumed (Boonstra and Post, 2004). To evaluate the ROS regulation in L. (L.) infantum treated with NTZ, the cell-permeant H2 DCF-DA was used as a probe. H2 DCF-DA is a chemically reduced form of fluorescein that is used
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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as an ROS indicator in cells; after its acetate groups are cleaved by intracellular esterases and oxidation, it is converted to the highly fluorescent 2 ,7 -dichlorofluorescein (DCF) (Williams et al., 2012). Our data revealed that NTZ induced a rapid up-regulation of ROS in L. (L.) infantum, resulting in a fluorescence intensity that was approximately 129-fold higher than that of the negative control (untreated cells). Oligomycin has been widely used to up-regulate ROS in L. (L.) infantum parasites (Carvalho et al., 2011). Drugs that inhibit oxidative phosphorylation rapidly upregulate ROS production (Mattiazzi et al., 2004). Reactive oxygen species are important signalling molecules, and their accumulation under pathological conditions leads to oxidative stress. Furthermore, one of the causes of mitochondrial ROS generation is dysfunction in the mitochondrial respiratory chain (Mehta and Shaha, 2004). Due to the intense mitochondrial swelling in L. (L.) infantum induced by NTZ (ultrastructural studies), the depolarisation of the mitochondrial membrane potential and the increased ROS generation, we suggest that the lethal action of NTZ in L. (L.) infantum involves oxidative stress and contributes to cell death. Apoptosis-like programmed cell death (PCD) has been described in unicellular protists, including Plasmodium, Trypanosoma and Leishmania. Furthermore, in protists, this process shares some morphological features with apoptosis in multicellular organisms, including chromosomal condensation, nuclear DNA fragmentation, cell shrinkage, loss of mitochondrial membrane potential, the formation of apoptotic bodies, and the externalisation of phosphatidylserine (Kaczanowski et al., 2011; Reece et al., 2011). During the search for apoptotic markers, flow cytometry analyses were performed using annexin V and propidium iodide. Annexin V has been used to detect the externalisation of phosphatidylserine in apoptotic cells as a recombinant protein conjugated to a greenfluorescent probe and propidium iodide (PI) to stain necrotic cells with red fluorescence (dos Santos et al., 2013). In our assays, NTZ induced a time-dependent externalisation of PS in L. (L.) infantum promastigotes that could be detected within 6 h of treatment and resulted in more than 97% of the cells being labelled with annexin V after 24 h. Moreover, L. (L.) infantum parasites were simultaneously labelled with annexin V and PI; these doubly labelled cells (annexin V and PI) might indicate late stage apoptosis (Kulkarni et al., 2009). These data also suggest that the plasma membrane permeability was altered in L. (L.) infantum within 6 h of incubation with NTZ, permitting the internalisation and binding of PI to stain nucleic acids. Miltefosine was used as a positive control for PSexternalisation in L. (L.) infantum; in these studies, this compound generated 99.4% of parasites positive for both annexin V and propidium iodide, corroborating data published elsewhere (Paris et al., 2004; Marinho et al., 2011). Untreated parasites were also labelled for both annexin V and propidium iodide (6.58% of parasites), confirming that the parasites underwent an apoptosis-like process in the stationary phase culture of promastigotes (Lee et al., 2002). Finally, our transmission electron microscopy studies of NTZ-treated parasites clearly revealed ultrastructural alterations compatible with programmed cell death (Jiménez-Ruiz et al., 2010): rounding-up of the cell, pyknosis, chromatin condensation, plasma membrane blebbing and mitochondrial swelling. Considering our additional data revealing the late stage alteration of plasma membrane (PI labelling), PS-externalisation (annexin V labelling), depolarisation of mitochondrial membrane potential (MitoTracker labelling) and ROS up-regulation, we suggest that aside from oxidative stress, NTZ’s lethal action against L. (L.) infantum might involve programmed cell death; however, further assays must be performed to confirm an apoptosis-like death process. Manipulating cell death pathways in L. (L.) infantum may offer new opportunities for disease control. Considering the lethal effects of NTZ in L. (L.) infantum, further drug design studies using NTZ as scaffold might assist the selection of novel and effective drugs.
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Acknowledgements The authors thank the M.Sc. Nahiara Esteves Zorgi for assistance with flow cytometry and Ms. Matilia Nascimento and Vicente Duarte for laboratory assistance. This work was supported by a grant (2012/18756-1) from the São Paulo Research Foundation (FAPESP). The authors are grateful to the CNPq scientific award given to A.G.T and the scholarships from FAPESP and CNPq awarded to E.G.P and J.T.M, respectively.
References Anderson, V.R., Curran, M.P., 2007. Nitazoxanide: a review of its use in the treatment of gastrointestinal infections. Drugs 67, 1947–1967. Andrade-Neto, V.V., Cicco, N.N., Cunha-Junior, E.F., Canto-Cavalheiro, M.M., Atella, G.C., Torres-Santos, E.C., 2011. The pharmacological inhibition of sterol biosynthesis in Leishmania is counteracted by enhancement of LDL endocytosis. Acta Trop. 119, 194–198. Boonstra, J., Post, J.A., 2004. Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 4, 1–13. Carvalho, L., Luque-Ortega, J.R., López-Martín, C., Castanys, S., Rivas, L., Gamarro, F., 2011. The 8-aminoquinoline analogue sitamaquine causes oxidative stress in Leishmania donovani promastigotes by targeting succinate dehydrogenase. Antimicrob. Agents Chemother. 55, 4204–4210. Corrêa, D.S., Tempone, A.G., Reimão, J.Q., Taniwaki, N.N., Romoff, P., Fávero, O.A., Sartorelli, P., Mecchi, M.C., Lago, J.H., 2011. Anti-leishmanial and anti-trypanosomal potential of polygodial isolated from stem barks of Drimys brasiliensis Miers (Winteraceae). Parasitol. Res. 109, 231–236. Debrabant, A., Lee, N., Bertholet, S., Duncan, R., Nakhasi, H.L., 2003. Programmed cell death in trypanosomatids and other unicellular organisms. Int. J. Parasitol. 33, 257–267. dos Santos, M.G., Muxel, S.M., Zampieri, R.A., Pomorski, T.G., Floeter-Winter, L.M., 2013. Transbilayer dynamics of phospholipids in the plasma membrane of the Leishmania genus. PLoS ONE 8, e55604. Duarte, M.I., Mariano, O.N., Takakura, C.F., Everson, D., Corbett, C.E., 1992. A fast method for processing biologic material for electron microscopic diagnosis in infectious disease. Ultrastruct. Pathol. 16, 475–482. Ekins, S., Williams, A.J., Krasowski, M.D., Freundlich, J.S., 2011. In silico repositioning of approved drugs for rare and neglected diseases. Drug Discov. Today 16, 298–310. Farias, L.H., Rodrigues, A.P., Silveira, F.T., Seabra, S.H., DaMatta, R.A., Saraiva, E.M., Silva, E.O., 2013. Phosphatidylserine exposure and surface sugars in two Leishmania (Viannia) braziliensis strains involved in cutaneous and mucocutaneous leishmaniasis. J. Infect. Dis. 207, 537–543. Hoffman, P.S., Sisson, G., Croxen, M.A., Welch, K., Harman, W.D., Cremades, N., Morash, M.G., 2007. Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of Helicobacter pylori, selected anaerobic bacteria and parasites, and Campylobacter jejuni. Antimicrob. Agents Chemother. 51, 868–876. Jiménez-Ruiz, A., Alzate, J.F., Macleod, E.T., Lüder, C.G., Fasel, N., Hurd, H., 2010. Apoptotic markers in protozoan parasites. Parasit. Vectors 9, 3–104. Joshi, D.C., Bakowska, J.C., 2011. Determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons. J. Vis. Exp. 23, 51. Kaczanowski, S., Sajid, M., Reece, S.E., 2011. Evolution of apoptosis-like programmed cell death in unicellular protozoan parasites. Parasit. Vectors 25, 4–44. Kulkarni, M.M., McMaster, W.R., Kamysz, W., McGwire, B.S., 2009. Antimicrobial peptide-induced apoptotic death of Leishmania results from calcium-dependent, caspase-independent mitochondrial toxicity. J. Biol. Chem. 284, 15496–15504. Lee, N., Bertholet, S., Debrabant, A., Muller, J., Duncan, R., Nakhasi, H.L., 2002. Programmed cell death in the unicellular protozoan parasite Leishmania. Cell Death Differ. 9, 53–64. Mangoni, M.L., Saugar, J.M., Dellisanti, M., Barra, D., Simmaco, M., Rivas, L., 2005. Temporins, small antimicrobial peptides with leishmanicidal activity. J. Biol. Chem. 280, 984–990. Marinho, F.deA., Gonc¸alves, K.C., Oliveira, S.S., Oliveira, A.C., Bellio, M., d’Avila-Levy, C.M., Santos, A.L., Branquinha, M.H., 2011. Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes. Mem. Inst. Oswaldo Cruz 106, 507–509. Mattiazzi, M., Vijayvergiya, C., Gajewski, C.D., DeVivo, D.C., Lenaz, G., Wiedmann, M., Manfredi, G., 2004. The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants. Hum. Mol. Genet. 13, 869–879. Medina, J.M., Rodrigues, J.C., De Souza, W., Atella, G.C., Barrabin, H., 2012. Tomatidine promotes the inhibition of 24-alkylated sterol biosynthesis and mitochondrial dysfunction in Leishmania amazonensis promastigotes. Parasitology 139, 1253–1265. Mehta, A., Shaha, C., 2004. Apoptotic death in Leishmania donovani promastigotes in response to respiratory chain inhibition: complex II inhibition results in increased pentamidine cytotoxicity. J. Biol. Chem. 279, 11798–11813. Mikus, J., Steverding, D., 2000. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. Parasitol. Int. 48, 265–269. Navarrete-Vazquez, G., Chávez-Silva, F., Argotte-Ramos, R., Rodríguez-Gutiérrez ˜ Mdel, C., Chan-Bacab, M.J., Cedillo-Rivera, R., Moo-Puc, R., Hernández-Nunez,
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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E., 2011. Synthesis of benzologues of Nitazoxanide and Tizoxanide: a comparative study of their in vitro broad-spectrum antiprotozoal activity. Bioorg. Med. Chem. Lett. 21, 3168–3171. Nunes, D.C., Figueira, M.M., Lopes, D.S., De Souza, D.L., Izidoro, L.F., Ferro, E.A., Souza, M.A., Rodrigues, R.S., Rodrigues, V.M., Yoneyama, K.A., 2013. BnSP-7 toxin, a basic phospholipase A2 from Bothrops pauloensis snake venom, interferes with proliferation, ultrastructure and infectivity of Leishmania (Leishmania) amazonensis. Parasitology 140, 844–854. Pal, C., Bandyopadhyay, U., 2012. Redox-active antiparasitic drugs. Antioxid. Redox Signal. 17, 555–582. Paris, C., Loiseau, P.M., Bories, C., Bréard, J., 2004. Miltefosine induces apoptosis-like death in Leishmania donovani promastigotes. Antimicrob. Agents Chemother. 48, 852–859. Reece, S.E., Pollitt, L.C., Colegrave, N., Gardner, A., 2011. The meaning of death: evolution and ecology of apoptosis in protozoan parasites. PLoS Pathog. 7, e1002320.
Sundar, S., Chakravarty, J., 2012. Recent advances in the diagnosis and treatment of kala-azar. Natl. Med. J. India 25, 85–89. Tada, H., Shiho, O., Kuroshima, K., 1986. An improved colorimetric assay for interleukin 2. J. Immunol. Methods 93, 157–165. Tempone, A.G., Martins de Oliveira, C., Berlinck, R.G., 2011. Current approaches to discover marine antileishmanial natural products. Planta Med. 77, 572–585. Williams, R.A., Smith, T.K., Cull, B., Mottram, J.C., Coombs, G.H., 2012. ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major. PLoS Pathog. 8, e1002695. WHO, 2010. Control of the leishmaniasis. Technical Report Series 949. World Health Organization. Zhang, R., Shang, L., Jin, H., Ma, C., Wu, Y., Liu, Q., Xia, Z., Wei, F., Zhu, X.Q., Gao, H., 2010. In vitro and in vivo antileishmanial efficacy of nitazoxanide against Leishmania donovani. Parasitol. Res. 107, 75–79.
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Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum
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Juliana Tonini Mesquita a , Erika Gracielle Pinto a,b , Noemi Nosomi Taniwaki a , Andres Jimenez Galisteo Jr b , Andre Gustavo Tempone a,∗ a
Department of Parasitology, Instituto Adolfo Lutz, Av. Dr. Arnaldo, 351, 01246-900 São Paulo, SP, Brazil Laboratorio de Protozoologia, Instituto de Medicina Tropical, Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 470, 05403-000 São Paulo, SP, Brazil
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Article history: Received 11 July 2013 Received in revised form 13 September 2013 Accepted 14 September 2013 Available online xxx
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Keywords: Leishmania Nitazoxanide Therapy Oxidative stress Phosphatidylserine Mitochondria
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1. Introduction
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Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileishmanials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileishmanial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZ’s activity against L. infantum was confirmed, producing IC50 values of 42.71 g/mL against promastigotes and 6.78 g/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depolarising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX® Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chromatin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets. © 2013 Published by Elsevier B.V.
Neglected parasitic diseases, such as visceral leishmaniasis (VL), remain an important concern for the public health systems of developing countries because these diseases affect at least 12 million people in 98 countries and territories. VL is associated with poverty, malnutrition and co-infections with HIV and other parasitic diseases (WHO, 2010). The therapeutics for VL are problematic because they exhibit elevated toxicity and require long-term treatment. The increasing parasite resistance to these drugs is also a serious obstacle (Sundar and Chakravarty, 2012). Creating new selective drugs with higher therapeutic indexes and better affordability for developing countries has been a major spotlight for Drug Discovery & Development programmes (WHO, 2010).
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∗ Corresponding author. Tel.: +55 11 3068 2991; fax: +55 11 3068 2890. E-mail address: [email protected] (A.G. Tempone).
Manipulating the cell death pathways in Leishmania (L.) infantum may reveal new treatment opportunities (Andrade-Neto et al., 2011). Therefore, assessing new therapeutic targets creates unique opportunities to discover selective compounds with activity focused against the parasite (Nunes et al., 2013) while avoiding toxicity in mammalian cells. In this context, the redox system is critical for the survival of L. (L.) infantum in macrophages. Drugs that affect metabolism or cause reactive oxygen species (ROS) generation, inhibit the production of ergosterol (Medina et al., 2012) or induce programmed cell death are novel therapeutic advances (Debrabant et al., 2003; Pal and Bandyopadhyay, 2012). Redox imbalance occurs in the parasite when the endogenous antioxidants fail to cope with the excessive ROS, leading to oxidative stress. Therefore, antiparasitic drugs that inhibit vital redox reactions or promote oxidative stress in parasites may be promising candidates (Pal and Bandyopadhyay, 2012). Drug repurposing has been a strategic, efficient and costeffective approach for finding novel and effective antiparasitics for neglected diseases (Ekins et al., 2011). Nitazoxanide (NTZ) is a
0001-706X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.actatropica.2013.09.018
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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synthetic nitrothiazolyl-salicylamide derivative that is approved for treating infectious diarrhoea caused by Cryptosporidium parvum and Giardia lamblia, as well as others parasites such as Giardia intestinalis, Entamoeba histolytica, and Trichomonas vaginalis (Anderson and Curran, 2007). Antileishmanial (Zhang et al., 2010) and antitrypanosomal activities have also been reported for this compound (Navarrete-Vazquez et al., 2011). Using different fluorimetric techniques and transmission electron microscopy, the activity and lethal action of NTZ was evaluated for the first time in L. (L.) infantum to investigate its effects in mitochondria and on the regulation of programmed cell death markers.
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2. Materials and methods
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Alamar blue® (resazurin), SYTOX® Green, Mitotracker® Red CM-H2 XROS, the Reactive Oxygen Species H2 DCF-DA and the Annexin-V FITC Apoptosis Kit were purchased from Molecular Probes® (Invitrogen, USA). Dimethyl sulfoxide (DMSO) and methanol (MeOH) were obtained from Merck. M-199 medium, Hank’ Balanced Salt Solution (HBSS), phosphate-buffered saline (PBS), oligomycin, Triton X-100 and nitazoxanide were obtained from Sigma. All other reagents were obtained from Sigma.
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2.2. Parasites and mammalian cells maintenance
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L. (L.) infantum (MHOM/BR/1972/LD) was maintained in Golden hamsters up to approximately 60–70 days post-infection. The promastigotes were maintained in M-199 medium supplemented with 10% calf serum and 0.25% hemin at 24 ◦ C. The amastigotes were obtained from the spleens of previously infected hamsters by differential centrifugation. The macrophages were collected from the peritoneal cavity of BALB/c mice by washing with RPMI-1640 medium supplemented with 10% calf serum and were maintained at 37 ◦ C in a humidified incubator with 5% CO2 . 2.3. Bioassay procedures The golden hamsters were obtained from the animal breeding facility at the Adolfo Lutz Institute, SP, Brazil. The animals were maintained in sterilised cages in a controlled environment, while receiving water and food ad libitum. All of the animal procedures were performed with the approval of the Research Ethics Commission, in agreement with the Guide for the Care and Use of Laboratory Animals from the National Academy of Sciences. The experiments involved the minimum number of animals necessary to produce statistically reproducible results. 2.4. Determination of the 50% inhibitory concentration (IC50 ) against L. (L.) infantum 2.4.1. Promastigotes NTZ was dissolved in DMSO and diluted in M-199 medium in 96-well dark microplates to concentrations up to 100 g/mL. The promastigotes were in the late growth-phase (non-stationary) before they were washed in M-199 medium, counted in a Neubauer hemocytometer, seeded at 1 × 106 /well with a final volume of 200 L and incubated for 48 h at 24 ◦ C. The viability of the promastigotes was detected with rezasurin (0.011% in PBS) (Mikus and Steverding, 2000) followed by a 48 h incubation (20 L/well) under the same conditions. The fluorescence intensity was determined using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader-Molecular Devices® ) with excitation and emission wavelengths of 540 and 595 nm, respectively. The control group consisted of promastigotes incubated with 0.1–0.5% DMSO
(internal control). Miltefosine was used as a standard drug. The cell viability was expressed based on the fluorescence units of the control after normalisation. The data represent the mean of three independent assays (triplicates). 2.4.2. MTT assay The colorimetric diphenyltetrazolium assay (MTT) was performed to assess the viability of the parasites after 24 h. Briefly, MTT (5 mg/mL) was dissolved in PBS, sterilised using 0.22 m membranes, added at 20 L/well and incubated for 4 h at 24 ◦ C. The formazan extraction was performed using 10% SDS for 18 h (80 L/well) at 24 ◦ C, and the optical density (OD) was colorimetrically determined in a plate reader (FilterMax F5 Multi-Mode Microplate Reader) at 550 nm (Corrêa et al., 2011). The data obtained represent the mean of three independent assays. 2.4.3. Intracellular amastigotes Peritoneal macrophages were seeded at 1 × 105 cells/well in 16-wells slide chambers (NUNC® ), infected with the previously isolated (hamster spleen) L. (L.) infantum amastigotes in a 1:10 ratio (macrophage/amastigotes) and treated with NTZ for 120 h at 37 ◦ C in a humidified incubator with 5% CO2 . At the end of the assay, the macrophages were fixed with methanol, stained with Giemsa and observed using light microscopy. The parasite burden was verified using the number of infected macrophages out of 400 cells. The data obtained represent the mean of three independent assays, and each assay was performed in duplicate (Corrêa et al., 2011). 2.4.4. Cytotoxicity against mammalian cell Mouse connective tissue cells (NCTC clone 929) were seeded at 6 × 104 cells/well in 96-well microplates and incubated with test compounds for 24, 48, 72 and 96 h at 37 ◦ C in an incubator with 5% CO2 . Nitazoxanide was dissolved in DMSO and diluted to the highest concentration (200 g/mL); miltefosine and Glucantime® were used as the standards. The viability of the cells was determined using resazurin (Mikus and Steverding, 2000). The data represent the mean of three independent assays (triplicates). The selectivity index was determined by the IC50 against mammalian cells/IC50 against amastigotes (Corrêa et al., 2011). 2.5. Spectrofluorimetric detection of cell membrane permeability Late growth-phase (non-stationary) promastigotes were washed in PBS, seeded at 2 × 106 /well and incubated with 1 M SYTOX® Green for 15 min at 24 ◦ C (Mangoni et al., 2005). NTZ was added at a concentration similar to its IC50 (60 g/mL), and the fluorescence was measured every 20 min (up to 60 min). The maximum permeabilisation was obtained with 0.1% Triton X-100. The fluorescence intensity was determined using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate ReaderMolecular Devices) with excitation and emission wavelengths of 485 and 520 nm, respectively. The following internal controls were used: (i) detection of possible NTZ fluorescence at the appropriate wavelengths, (ii) detection of possible interference by DMSO, (iii) untreated promastigotes and (iv) medium without cells. The samples were examined in triplicate. 2.6. Effect on mitochondrial membrane potential L. (L.) infantum promastigotes (late growth-phase) were washed twice in PBS, seeded at 2 × 106 /well and incubated with NTZ at a concentration close to its IC50 (60 g/mL) for 60 min. MitoTracker® Red CM-H2 XROS (500 nM) was added, and the sample was incubated for 40 min in the dark. The cells were washed twice in HBSS, and the fluorescence was determined using a fluorimetric
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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microplate reader (FilterMax F5 Multi-Mode Microplate ReaderMolecular Devices) with excitation and emission wavelengths of 540 and 595 nm, respectively (Williams et al., 2012). The following internal controls were used: (i) detection of possible NTZ fluorescence at the appropriate wavelengths, (ii) detection of possible interference by DMSO, (iii) untreated promastigotes and (iv) medium without cells. The samples were assessed in triplicate.
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2.7. Detection of reactive oxygen species (ROS) production
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L. (L.) infantum promastigotes (2 × 106 /well) were washed in HBSS and incubated with NTZ at a concentration close to its IC50 (60 g/mL) for 60 min. H2 DCF-DA (5 M) was added, and the cells were incubated for 15 min. The fluorescence intensity was detected using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader-Molecular Devices) at 485 and 520 nm for excitation and emission, respectively. Oligomycin (20 M) was used at as a positive control. The following internal controls were used: (i) detection of possible fluorescence NTZ at the appropriate wavelengths, (ii) detection of possible interference by DMSO, (iii) untreated promastigotes and (iv) medium without cells. The samples were assessed in triplicate.
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2.8. Flow cytometry analysis of phosphatidyserine (PS) exposure
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The promastigotes (2 × 106 /well) were washed in M-199 medium and incubated with NTZ at a concentration close to its IC50 (60 g/mL) for different periods (12, 16 and 24 h). To assess the PS exposure using FACS, the cells were incubated with FITC-annexin V and propidium iodide according to the manufacturer’ instructions. The cells were evaluated with a BD LSRFortessa flow cytometer (Becton Dickinson® ), and the data were analysed using FlowJo software (Tree Star® , Inc.); at least 10,000 events were acquired per analysis (Farias et al., 2013).
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Transmission electron microscopy images of L. (L.) infantum promastigotes incubated with NTZ were collected after different periods of incubation (1, 3, 6 h). The cells (1 × 107 /well) were incubated with NTZ at 24 ◦ C in 24-well plates before being processed and observed using a JEOL transmission electron microscope (Duarte et al., 1992).
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The results are represented by the means and standard deviations of replicate samples from at least two independent assays. The IC50 values were calculated using sigmoid dose–response curves in Graph Pad Prism 5.0, and the 95% confidence intervals are included in parentheses. The ANOVA test was used to test for significance (p < 0.05). 3. Results 3.1. NTZ is effective against L. (L.) infantum Promastigotes (Fig. 1) were incubated with nitazoxanide in vitro, and their viability was evaluated using a fluorimetric assay with resazurin. After 48 h, 100% of the parasites were eliminated at the highest concentration (100 g/mL), generating an IC50 value of 42.71 g/mL (95% confidence interval 28.77–63.45 g/mL) (Table 1). Miltefosine was used as the standard drug and generated an IC50 value of 6.69 g/mL (95% confidence interval 6.29–7.11 g/mL). NTZ was incubated with intracellular amastigotes, and after 120 h, no parasites were observed in
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Fig. 1. Evaluation of the mitochondrial membrane potential of Leishmania (L.) infantum incubated with nitazoxanide. MitoTracker Red® was incubated with treated and untreated parasites (control group). The fluorescence was measured using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader) at 540 and 595 nm for excitation and emission, respectively. The chemical structure of nitazoxanide was included (PubMed Compound). *p < 0.05.
the macrophages, revealing an IC50 value of 6.78 g/mL (95% confidence interval 6.51–7.06 g/mL) (Table 1). Miltefosine and Glucantime® were used as the standard drugs and attained IC50 values of 6.87 g/mL (95% confidence interval 4.71–10.01 g/mL) and 22.80 g/mL (95% confidence interval 20.76–25.04 g/mL), respectively (Table 1). 3.2. Mammalian cell cytotoxicity NTZ was incubated for 24, 48, 72 and 96 h with mammalian cells, and the cell viability was assessed using the resazurin assay. Mouse connective tissue cells (NCTC clone 929) were susceptible to NTZ, generating IC50 values in the range of 8–29 g/mL, and selectivity indexes in the range of 1.2–4.3. Miltefosine also affected mammalian cells and had an IC50 value of 49.72 g/mL (95% confidence interval 39.85–63.98 g/mL) (Table 1). 3.3. Permeability of plasma membrane NTZ was incubated with promastigotes, and the plasma membrane permeability was evaluated with the vital dye SYTOX® Green (20 min intervals). NTZ did not interfere with the plasma membrane, even after up to 60 min of incubation; no increase in the fluorescence levels could be detected (data not shown). 3.4. NTZ affects the mitochondria of Leishmania: evaluation of the mitochondrial membrane potential Promastigotes were incubated with NTZ for 60 min, and the mitochondrial membrane potential was monitored using the fluorescent probe Mitotracker Red® . NTZ induced a 100% (p < 0.05) decrease in the fluorescence intensity relative to the untreated promastigotes (Fig. 1). MitoTracker Red® was incubated with untreated parasites as a control, which displayed the parasite’s
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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Table 1 Antileishmanial activity, mammalian cytotoxicity and selectivity index of nitazoxanide. Cell viability was determined using a fluorimetric assay with resazurin. *p < 0.05. Drugs
Nitazoxanide
IC50 (g/mL) 95% C.I. Promastigotes
Amastigotes
Cytotoxicity
S.I.#
42.71* (28.77–63.45)
6.78* (6.51–7.06)
a
4.35 3.08 1.73 1.25 >8.70 7.23
29.51 (22.57–38.58) 20.94 (17.90–24.48) c 11.79 (9.96–13.95) d 8.48 (7.75–9.27) >200 49.72 (39.85–63.98) b
Glucantime® Miltefosine
Q3
256 257
258
259 260 261 262 263
264
265 266 267 268 269 270 271 272 273 274 275 276 277 278
nd 6.69 (6.29–7.11)
22.80 (20.76–25.04) 6.87 (4.71–10.01)
IC50 – 50% inhibitory concentration; 95% C.I. – 95% confidence interval; # S.I. – selectivity index; *p < 0.05. a Cytotoxicity performed in 24 h. b Cytotoxicity performed in 48 h. c Cytotoxicity performed in 72 h. d Cytotoxicity performed in 96 h.
typical mitochondrial membrane potential. Fig. 1 also includes NTZ’s chemical structure. 3.5. Reactive oxygen species (ROS) production NTZ was incubated with promastigotes for 60 min, and the ROS production was monitored using the H2 DCF-DA fluorescent probe. NTZ enhanced the production of ROS in L. (L.) infantum 129-fold relative to the untreated cells, displaying levels similar to the positive control, oligomycin (Fig. 2). 3.6. NTZ induces exposure of phosphatidylserine L. (L.) infantum was incubated for 12, 16 and 24 h with NTZ, while the binding of annexin V and propidium iodide was investigated by flow cytometry. After 12 h, 83.9% of the parasites were positive for both annexin V and propidium iodide, 3.97% were positive for only propidium iodide and 0.49% were positive for only annexin V (Fig. 3 D). After 16 h, 93.4% were positive for both annexin V and propidium iodide, 5.9% were positive for only propidium iodide and 0.04% were only positive for only annexin V (Fig. 3E). After 24 h, 97.4% of the parasites were positive for both annexin V and propidium iodide, 2.43% were positive for only propidium iodide and 0.024% were positive for only annexin V (Fig. 3F). Miltefosine was used as a positive control; after 24 h, 99.4% of the parasites were positive for both annexin V and propidium iodide, 0.12% were positive for only propidium iodide and 0.018% were positive for
only annexin V (Fig. 3C). Untreated promastigotes were used as a negative control (24 h), revealing that 6.58% of the parasites were positive for both annexin V and propidium iodide, 2% were positive for only propidium iodide and 1.75% were positive for only annexin V (Fig. 3B). Promastigotes without fluorescent labelling were used as the internal control (Fig. 3A). 3.7. NTZ induces ultrastructural alterations NTZ was incubated with promastigotes for different periods, and the ultrastructural modifications were analysed by transmission electron microscopy. After 1 h of incubation, it was possible to observe a slight mitochondrial swelling relative to the untreated control (Fig. 4B), but the kinetoplast was preserved. Therefore, the plasma membrane of L. (L.) infantum was intact. After 3 h, although no alteration in the parasite’s morphology was observed, an increased number of vacuoles (v) were observed; NTZ also induced the formation of enlarged mitochondria (Fig. 4C). After 6 h of incubation (Fig. 4D), nuclear membrane detachment and chromatin condensation were observed (arrow), along with an increase in plasma membrane blebs (arrow) and a progressive swelling of the mitochondria (m). Untreated parasites are depicted in Fig. 4A, demonstrating preserved structures, such as the nucleus (n), kinetoplast (k) and flagellum (f). 3.8. MTT reduction The mitochondrial oxidative activity of L. (L.) infantum incubated with NTZ (100 g/mL) was evaluated using a tetrazolium assay (MTT). After 24 h of incubation, no MTT reduction was observed in the promastigotes, demonstrating the leishmanicidal activity of the drug (Fig. 5). Miltefosine was used as a standard drug. 4. Discussion
Fig. 2. Reactive oxygen species (ROS) regulation in Leishmania (L.) infantum incubated with nitazoxanide. H2 DCF-DA was incubated with cells, and the fluorescence intensity was detected using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader) at 485 and 520 nm for excitation and emission, respectively. Oligomycin (20 M) was used as a positive control. *p < 0.05.
During the constant search for novel targets in L. (L.) infantum, the pursuit of drugs that affect mitochondrial metabolism might be an interesting strategy to find new therapeutic compounds that are active against protozoans. The survival of L. (L.) infantum depends on the proper behaviour of its mitochondria; in particular, this organism contains only a single mitochondrion, and therefore, drugs affecting mitochondria could explore the low compensation capacity of the injured organelle. The synthetic nitrothiazolylsalicylamide derivative, nitazoxanide (NTZ), is an orally delivered drug that is used for treating infectious diarrhoea caused by C. parvum and G. lamblia (Anderson and Curran, 2007); NTZ was studied against the etiologic agent of American Visceral Leishmaniasis, L. (L.) infantum. The promastigotes of L. (L.) infantum were susceptible to NTZ at levels to the standard drug miltefosine. Despite its toxicity towards mammalian cells, NTZ eliminated the intracellular
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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Fig. 3. Evaluation of the phosphatydilserine exposure using flow cytometry data for Leishmania (L.) infantum incubated with nitazoxanide. The cells were labelled with FITCannexin V and propidium iodide and analysed in a BD LSRFortessa flow cytometer (Becton Dickinson® ). The data were analysed using the FlowJo software (Tree Star® , Inc.) and at least 10,000 events were acquired per analysis. (A) untreated and unlabelled parasites (control 24 h); (B) negative control (untreated 24 h); (C) miltefosine (positive control 24 h); (D) nitazoxanide (12 h); (E) nitazoxanide (16 h); (F) nitazoxanide (24 h).
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amastigotes with effectiveness similar to the standard drug miltefosine; it was also 3.3-fold more effective than the standard drug Glucantime® . NTZ has also demonstrated activity against Leishmania donovani promastigotes (Zhang et al., 2010) with a similar IC50 value, but this is the first description of its activity against the clinically relevant stage of the parasite (the intracellular amastigotes). NTZ is therefore a promising antileishmanial drug candidate. Zhang et al. (2010) also demonstrated that oral NZT (200 mg/kg) reduced approximately 85% of the parasite load of L. donovani in a mouse model. New synthetic derivatives have also been explored, demonstrating that NTZ might be a promising scaffold for drug design (Zhang et al., 2010).
To investigate the permeability of NTZ-treated L. (L.) infantum’s plasma membranes, a spectrofluorimetric assay using SYTOX® Green was performed. SYTOX® Green is a green-fluorescent nuclear and chromosome counter stain that is impermeant towards live cells and exhibits >500-fold fluorescence enhancement after binding nucleic acids (Mangoni et al., 2005). Our data clearly demonstrated that NTZ did not alter the permeability of L. (L.) infantum’s plasma membrane after 60 min of incubation. Viability tests have been used to confirm the potential of drug candidates. The reduction of the yellow mitochondrial dehydrogenase substrate, methyl-tetrazolium (MTT), to form the purple water-insoluble formazan was used as a cell death marker (Tada
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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Fig. 4. Transmission electron microscopy images of Leishmania (L.) infantum promastigotes treated with nitazoxanide. (A) Control (untreated parasites); (B) nitazoxanide 1 h; (C) nitazoxanide 3 h; (D) nitazoxanide 6 h; k – kinetoplast, n – nucleus, f – flagellum, v – vacuoles, L – lipids.
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et al., 1986; Tempone et al., 2011). Leishmania-treated parasites exhibited no MTT oxidation after 24 h of treatment with NTZ, suggesting the drug had a leishmanicidal effect. Studying the lethal action of drugs in parasites could provide important information about novel drug targets. Because NTZ had antileishmanial potential, its lethal action was investigated in L. (L.) infantum promastigotes. The mitochondrial membrane potential is crucial for ATP generation in the respiratory chain (Joshi and Bakowska, 2011). In the absence of properly functioning mitochondria, the cells stop synthesising ATP from their mitochondrial
Fig. 5. Evaluation of the mitochondrial oxidative activity of Leishmania (L.) infantum promastigotes incubated for 24 h with NTZ. The colorimetric assay of MTT was read at 550 nm. Miltefosine was used as a standard drug; *p < 0.05.
source, leading to cell death (Joshi and Bakowska, 2011). MitoTracker Red® CM-H2 XROS is a reduced, nonfluorescent probe that fluoresces after its oxidation in the mitochondria of live cells; its accumulation depends on the membrane potential (Williams et al., 2012). In our assays, NTZ significantly (p < 0.05) depolarised the mitochondrial membrane potential of L. (L.) infantum because the fluorescence became undetectable after 60 min of incubation. Hoffman et al. (2007) demonstrated that NTZ intercepts pyruvate oxidation by inhibiting pyruvate: ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reaction in T. vaginalis, E. histolytica, G. intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni. Oxidising pyruvate during glycolysis is essential for generating acetyl-CoA and consequently ATP. Considering that a significant loss of mitochondrial membrane potential depletes cells of energy (Joshi and Bakowska, 2011), our data suggest that the depolarised mitochondrial membrane potential induced by NTZ might have contributed to L. (L.) infantum death. Furthermore, mitochondria are the main target of injury after stresses leading to programmed cell death and necrosis. Moreover, this event verifies that depolarising the mitochondrial membrane potential is a pre-apoptotic event (Joshi and Bakowska, 2011). Mitochondrial membrane potential reflects the pumping of hydrogen ions across the inner membrane during electron transport and oxidative phosphorylation. Under physiological conditions, oxidative phosphorylation releases reactive oxygen species (ROS) equivalent to 3 to 5% of total oxygen consumed (Boonstra and Post, 2004). To evaluate the ROS regulation in L. (L.) infantum treated with NTZ, the cell-permeant H2 DCF-DA was used as a probe. H2 DCF-DA is a chemically reduced form of fluorescein that is used
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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as an ROS indicator in cells; after its acetate groups are cleaved by intracellular esterases and oxidation, it is converted to the highly fluorescent 2 ,7 -dichlorofluorescein (DCF) (Williams et al., 2012). Our data revealed that NTZ induced a rapid up-regulation of ROS in L. (L.) infantum, resulting in a fluorescence intensity that was approximately 129-fold higher than that of the negative control (untreated cells). Oligomycin has been widely used to up-regulate ROS in L. (L.) infantum parasites (Carvalho et al., 2011). Drugs that inhibit oxidative phosphorylation rapidly upregulate ROS production (Mattiazzi et al., 2004). Reactive oxygen species are important signalling molecules, and their accumulation under pathological conditions leads to oxidative stress. Furthermore, one of the causes of mitochondrial ROS generation is dysfunction in the mitochondrial respiratory chain (Mehta and Shaha, 2004). Due to the intense mitochondrial swelling in L. (L.) infantum induced by NTZ (ultrastructural studies), the depolarisation of the mitochondrial membrane potential and the increased ROS generation, we suggest that the lethal action of NTZ in L. (L.) infantum involves oxidative stress and contributes to cell death. Apoptosis-like programmed cell death (PCD) has been described in unicellular protists, including Plasmodium, Trypanosoma and Leishmania. Furthermore, in protists, this process shares some morphological features with apoptosis in multicellular organisms, including chromosomal condensation, nuclear DNA fragmentation, cell shrinkage, loss of mitochondrial membrane potential, the formation of apoptotic bodies, and the externalisation of phosphatidylserine (Kaczanowski et al., 2011; Reece et al., 2011). During the search for apoptotic markers, flow cytometry analyses were performed using annexin V and propidium iodide. Annexin V has been used to detect the externalisation of phosphatidylserine in apoptotic cells as a recombinant protein conjugated to a greenfluorescent probe and propidium iodide (PI) to stain necrotic cells with red fluorescence (dos Santos et al., 2013). In our assays, NTZ induced a time-dependent externalisation of PS in L. (L.) infantum promastigotes that could be detected within 6 h of treatment and resulted in more than 97% of the cells being labelled with annexin V after 24 h. Moreover, L. (L.) infantum parasites were simultaneously labelled with annexin V and PI; these doubly labelled cells (annexin V and PI) might indicate late stage apoptosis (Kulkarni et al., 2009). These data also suggest that the plasma membrane permeability was altered in L. (L.) infantum within 6 h of incubation with NTZ, permitting the internalisation and binding of PI to stain nucleic acids. Miltefosine was used as a positive control for PSexternalisation in L. (L.) infantum; in these studies, this compound generated 99.4% of parasites positive for both annexin V and propidium iodide, corroborating data published elsewhere (Paris et al., 2004; Marinho et al., 2011). Untreated parasites were also labelled for both annexin V and propidium iodide (6.58% of parasites), confirming that the parasites underwent an apoptosis-like process in the stationary phase culture of promastigotes (Lee et al., 2002). Finally, our transmission electron microscopy studies of NTZ-treated parasites clearly revealed ultrastructural alterations compatible with programmed cell death (Jiménez-Ruiz et al., 2010): rounding-up of the cell, pyknosis, chromatin condensation, plasma membrane blebbing and mitochondrial swelling. Considering our additional data revealing the late stage alteration of plasma membrane (PI labelling), PS-externalisation (annexin V labelling), depolarisation of mitochondrial membrane potential (MitoTracker labelling) and ROS up-regulation, we suggest that aside from oxidative stress, NTZ’s lethal action against L. (L.) infantum might involve programmed cell death; however, further assays must be performed to confirm an apoptosis-like death process. Manipulating cell death pathways in L. (L.) infantum may offer new opportunities for disease control. Considering the lethal effects of NTZ in L. (L.) infantum, further drug design studies using NTZ as scaffold might assist the selection of novel and effective drugs.
7
Acknowledgements The authors thank the M.Sc. Nahiara Esteves Zorgi for assistance with flow cytometry and Ms. Matilia Nascimento and Vicente Duarte for laboratory assistance. This work was supported by a grant (2012/18756-1) from the São Paulo Research Foundation (FAPESP). The authors are grateful to the CNPq scientific award given to A.G.T and the scholarships from FAPESP and CNPq awarded to E.G.P and J.T.M, respectively.
References Anderson, V.R., Curran, M.P., 2007. Nitazoxanide: a review of its use in the treatment of gastrointestinal infections. Drugs 67, 1947–1967. Andrade-Neto, V.V., Cicco, N.N., Cunha-Junior, E.F., Canto-Cavalheiro, M.M., Atella, G.C., Torres-Santos, E.C., 2011. The pharmacological inhibition of sterol biosynthesis in Leishmania is counteracted by enhancement of LDL endocytosis. Acta Trop. 119, 194–198. Boonstra, J., Post, J.A., 2004. Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 4, 1–13. Carvalho, L., Luque-Ortega, J.R., López-Martín, C., Castanys, S., Rivas, L., Gamarro, F., 2011. The 8-aminoquinoline analogue sitamaquine causes oxidative stress in Leishmania donovani promastigotes by targeting succinate dehydrogenase. Antimicrob. Agents Chemother. 55, 4204–4210. Corrêa, D.S., Tempone, A.G., Reimão, J.Q., Taniwaki, N.N., Romoff, P., Fávero, O.A., Sartorelli, P., Mecchi, M.C., Lago, J.H., 2011. Anti-leishmanial and anti-trypanosomal potential of polygodial isolated from stem barks of Drimys brasiliensis Miers (Winteraceae). Parasitol. Res. 109, 231–236. Debrabant, A., Lee, N., Bertholet, S., Duncan, R., Nakhasi, H.L., 2003. Programmed cell death in trypanosomatids and other unicellular organisms. Int. J. Parasitol. 33, 257–267. dos Santos, M.G., Muxel, S.M., Zampieri, R.A., Pomorski, T.G., Floeter-Winter, L.M., 2013. Transbilayer dynamics of phospholipids in the plasma membrane of the Leishmania genus. PLoS ONE 8, e55604. Duarte, M.I., Mariano, O.N., Takakura, C.F., Everson, D., Corbett, C.E., 1992. A fast method for processing biologic material for electron microscopic diagnosis in infectious disease. Ultrastruct. Pathol. 16, 475–482. Ekins, S., Williams, A.J., Krasowski, M.D., Freundlich, J.S., 2011. In silico repositioning of approved drugs for rare and neglected diseases. Drug Discov. Today 16, 298–310. Farias, L.H., Rodrigues, A.P., Silveira, F.T., Seabra, S.H., DaMatta, R.A., Saraiva, E.M., Silva, E.O., 2013. Phosphatidylserine exposure and surface sugars in two Leishmania (Viannia) braziliensis strains involved in cutaneous and mucocutaneous leishmaniasis. J. Infect. Dis. 207, 537–543. Hoffman, P.S., Sisson, G., Croxen, M.A., Welch, K., Harman, W.D., Cremades, N., Morash, M.G., 2007. Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of Helicobacter pylori, selected anaerobic bacteria and parasites, and Campylobacter jejuni. Antimicrob. Agents Chemother. 51, 868–876. Jiménez-Ruiz, A., Alzate, J.F., Macleod, E.T., Lüder, C.G., Fasel, N., Hurd, H., 2010. Apoptotic markers in protozoan parasites. Parasit. Vectors 9, 3–104. Joshi, D.C., Bakowska, J.C., 2011. Determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons. J. Vis. Exp. 23, 51. Kaczanowski, S., Sajid, M., Reece, S.E., 2011. Evolution of apoptosis-like programmed cell death in unicellular protozoan parasites. Parasit. Vectors 25, 4–44. Kulkarni, M.M., McMaster, W.R., Kamysz, W., McGwire, B.S., 2009. Antimicrobial peptide-induced apoptotic death of Leishmania results from calcium-dependent, caspase-independent mitochondrial toxicity. J. Biol. Chem. 284, 15496–15504. Lee, N., Bertholet, S., Debrabant, A., Muller, J., Duncan, R., Nakhasi, H.L., 2002. Programmed cell death in the unicellular protozoan parasite Leishmania. Cell Death Differ. 9, 53–64. Mangoni, M.L., Saugar, J.M., Dellisanti, M., Barra, D., Simmaco, M., Rivas, L., 2005. Temporins, small antimicrobial peptides with leishmanicidal activity. J. Biol. Chem. 280, 984–990. Marinho, F.deA., Gonc¸alves, K.C., Oliveira, S.S., Oliveira, A.C., Bellio, M., d’Avila-Levy, C.M., Santos, A.L., Branquinha, M.H., 2011. Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes. Mem. Inst. Oswaldo Cruz 106, 507–509. Mattiazzi, M., Vijayvergiya, C., Gajewski, C.D., DeVivo, D.C., Lenaz, G., Wiedmann, M., Manfredi, G., 2004. The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants. Hum. Mol. Genet. 13, 869–879. Medina, J.M., Rodrigues, J.C., De Souza, W., Atella, G.C., Barrabin, H., 2012. Tomatidine promotes the inhibition of 24-alkylated sterol biosynthesis and mitochondrial dysfunction in Leishmania amazonensis promastigotes. Parasitology 139, 1253–1265. Mehta, A., Shaha, C., 2004. Apoptotic death in Leishmania donovani promastigotes in response to respiratory chain inhibition: complex II inhibition results in increased pentamidine cytotoxicity. J. Biol. Chem. 279, 11798–11813. Mikus, J., Steverding, D., 2000. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. Parasitol. Int. 48, 265–269. Navarrete-Vazquez, G., Chávez-Silva, F., Argotte-Ramos, R., Rodríguez-Gutiérrez ˜ Mdel, C., Chan-Bacab, M.J., Cedillo-Rivera, R., Moo-Puc, R., Hernández-Nunez,
Please cite this article in press as: Mesquita, J.T., et al., Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.09.018
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531 532 533 534 535 536 537 538 539 540 541 542 543 544
ARTICLE IN PRESS J.T. Mesquita et al. / Acta Tropica xxx (2013) xxx–xxx
E., 2011. Synthesis of benzologues of Nitazoxanide and Tizoxanide: a comparative study of their in vitro broad-spectrum antiprotozoal activity. Bioorg. Med. Chem. Lett. 21, 3168–3171. Nunes, D.C., Figueira, M.M., Lopes, D.S., De Souza, D.L., Izidoro, L.F., Ferro, E.A., Souza, M.A., Rodrigues, R.S., Rodrigues, V.M., Yoneyama, K.A., 2013. BnSP-7 toxin, a basic phospholipase A2 from Bothrops pauloensis snake venom, interferes with proliferation, ultrastructure and infectivity of Leishmania (Leishmania) amazonensis. Parasitology 140, 844–854. Pal, C., Bandyopadhyay, U., 2012. Redox-active antiparasitic drugs. Antioxid. Redox Signal. 17, 555–582. Paris, C., Loiseau, P.M., Bories, C., Bréard, J., 2004. Miltefosine induces apoptosis-like death in Leishmania donovani promastigotes. Antimicrob. Agents Chemother. 48, 852–859. Reece, S.E., Pollitt, L.C., Colegrave, N., Gardner, A., 2011. The meaning of death: evolution and ecology of apoptosis in protozoan parasites. PLoS Pathog. 7, e1002320.
Sundar, S., Chakravarty, J., 2012. Recent advances in the diagnosis and treatment of kala-azar. Natl. Med. J. India 25, 85–89. Tada, H., Shiho, O., Kuroshima, K., 1986. An improved colorimetric assay for interleukin 2. J. Immunol. Methods 93, 157–165. Tempone, A.G., Martins de Oliveira, C., Berlinck, R.G., 2011. Current approaches to discover marine antileishmanial natural products. Planta Med. 77, 572–585. Williams, R.A., Smith, T.K., Cull, B., Mottram, J.C., Coombs, G.H., 2012. ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major. PLoS Pathog. 8, e1002695. WHO, 2010. Control of the leishmaniasis. Technical Report Series 949. World Health Organization. Zhang, R., Shang, L., Jin, H., Ma, C., Wu, Y., Liu, Q., Xia, Z., Wei, F., Zhu, X.Q., Gao, H., 2010. In vitro and in vivo antileishmanial efficacy of nitazoxanide against Leishmania donovani. Parasitol. Res. 107, 75–79.
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