Effect of a ß-lapachone-derived naphthoimidazole on Trypanosoma cruzi: identification of target organelles

Rubem F. S. Menna-Barreto1, Andrea Henriques-Pons1, Antônio V. Pinto2, José A. Morgado-Diaz3, Maurilio J. Soares1 and Solange L. De Castro1,*

1 Dept. de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil; 2 Núcleo de Pesquisas em Produtos Naturais, Centro de Ciências da Saúde, UFRJ, Rio de Janeiro, RJ, Brazil; 3 Divisão de Biologia Celular, Instituto Nacional do Câncer, Rio de Janeiro, RJ, Brazil


* Corresponding author. Tel: +55-21-25984330; Fax: +55-21-2604434; Email: solange{at}ioc.fiocruz.br

Received 14 January 2005; returned 15 May 2005; revised 24 May 2005; accepted 7 October 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Investigation of the mode of action of the naphthoimidazole N1, obtained from the reaction of ß-lapachone with benzaldehyde, which among 45 semi-synthetic derivatives of naphthoquinones isolated from Tabebuia sp. was one of the most active compounds against Trypanosoma cruzi trypomastigotes.

Methods: Quantification of the effect of N1 against the proliferative forms of T. cruzi, and investigation of potential targets in the parasite using electron microscopy and flow cytometry techniques.

Results: N1 presented the following order of activity: amastigotes > trypomastigotes > epimastigotes. The effect on intracellular forms was ~25 times higher than on macrophages and heart muscle cells. N1-treated parasites presented an abnormal chromatin condensation and mitochondrial damage. In epimastigotes, alterations of reservosomes were observed, and in trypomastigotes, a decrease in the electron density of acidocalcisomes was observed. In epimastigotes, the naphthoimidazole inhibited the activity of succinate cytochrome c reductase. Labelling with rhodamine 123 or Acridine Orange was decreased in both forms treated with N1.

Conclusions: The results suggest that epimastigotes, reservosomes, mitochondrion, and nucleus contain N1 targets. In trypomastigotes, in which reservosomes are absent, the organelles affected by the compound were also the mitochondrion and nucleus, as well as acidocalcisomes, in which the decrease in electron density could be due to the use of polyphosphate as an alternative energy supply.

Keywords: T. cruzi , chemotherapy , naphthoquinones , parasites


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Naphthoquinones are widely distributed in the plant kingdom. Their molecular structures endow them with redox properties, which could interfere in biological oxidative processes. Plants containing naphthoquinones have been employed, mainly among native Amerindian populations, for the treatment of a number of diseases.1,2

Lapachol and ß-lapachone are bioactive naphthoquinones isolated from trees of the Bignoniaceae and Verbanaceae families, which are abundant in tropical rain forests. In Brazil there are >46 types of such woods, popularly known as ‘ipês’ (Tabebuia sp.). Biological activity of these quinones has been reported against viruses and pathogenic micro-organisms, as well as anti-inflammatory and cytotoxic properties.3 The anti-tumour activity of ß-lapachone has been a subject of intense research, this compound being considered by several researchers as a candidate for assays in cancer chemotherapy.4 The cytotoxicity of this naphthoquinone is related to inhibition of topoisomerases and induction of apoptosis.57

Chagas' disease, caused by Trypanosoma cruzi and endemic in Latin America, affects ~15 million people.8 Efforts have been addressed to replace the only available drug for treatment of this disease, benznidazole, which presents severe side effects and variable efficacy.9 It has been shown that ß-lapachone is active against T. cruzi and its mode of action was associated with generation of free radicals and to inhibition of macromolecule biosynthesis.1012 Our group developed alternative synthetic routes based on the reactivity of quinoidal carbonyl towards nucleophilic agents.1317 From the reaction of Tabebuia sp. naphthoquinones with 30 aromatic and 15 aliphatic aldehydes in the presence of ammonium acetate, we synthesized 45 derivatives comprising naphthoimidazoles and naphthoxazoles and assayed against T. cruzi trypomastigotes.3,1518 One of the most active compounds against the parasite was a ß-lapachone-derived naphthoimidazole with a phenyl moiety linked to the imidazole ring, named as N1. In the present work, we have investigated the effect of N1 against all three evolutive forms of T. cruzi, possible targets in the parasite and the drug toxicity to mammalian cells by light, fluorescence and electron microscopy, together with flow cytometry and biochemical assays.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Parasites and cell cultures

All experiments were performed with the Y strain of T. cruzi. Epimastigote forms were maintained axenically at 28°C with weekly transfers in LIT medium, and harvested during the exponential phase of growth (5-day-old culture forms). Bloodstream trypomastigotes were obtained from infected albino Swiss mice at the peak of parasitaemia. Amastigotes were collected from the supernatant of trypomastigote-infected J774G-8 macrophage cultures.

Mouse peritoneal macrophages were obtained from Swiss mice. Primary cultures of mouse embryo heart muscle cells (HMCs) were prepared as previously described.19 Briefly, the hearts of 18-day-old mouse embryos were fragmented and dissociated with trypsin and collagenase in phosphate buffered saline (PBS), pH 7.2. Thereafter, the cells were resuspended in Dulbecco's modified Eagle medium (Sigma–Aldrich, St Louis, USA), supplemented with horse and fetal calf sera chicken embryo extract, CaCl2 and L-glutamine, plated onto gelatin-coated glass coverslips in 24-well plates (Nunc Inc., IL, USA) (105 cells/well) and maintained at 37°C in 5% CO2 atmosphere.

Synthesis of the naphthoimidazole

The compound was obtained from the reaction of ß-lapachone with benzaldehyde in the presence of ammonium acetate. The reaction mixture was refluxed for 2 h and after precipitation in water, it was purified by column chromatography and recrystallization in hexane/ethyl acetate (9 : 1, v/v).15 Physical and spectral analysis characterized the compound N1 as 4,5-dihydro-6,6-dimethyl-6H-2-(phenyl)-pyran [b-4,3]naphth[1,2-d]imidazole) (Figure 1). A stock solution of N1 was prepared in dimethylsulphoxide (Merck, Darmstadt, Germany) at a concentration of 100 mM.



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Figure 1. Chemical structures: (a) ß-lapachone; (b) N1.

 
Direct effect on T. cruzi

Bloodstream trypomastigotes were resuspended to a concentration of 10 x 106 cells/mL in Dulbecco's modified Eagle medium plus 10% fetal calf serum (DMES). This suspension (100 µL) was added to the same volume of N1, previously prepared at twice the desired final concentrations. Alternatively, experiments were performed with trypomastigotes resuspended in mouse blood (5 x 106 cells/mL), where 196 µL was incubated with 4 µL of N1 (50x the final concentration). For tissue culture-derived amastigotes, the protocol was similar to that described for trypomastigotes in DMES. For both forms, incubation was performed in 96-well microplates (Nunc Inc., IL, USA) at 37°C for 1 day. Effect of N1 on epimastigote proliferation was monitored for up to 4 days in 24-well plates at 28°C in LIT medium. The naphthoimidazole was assayed in the range of 1–200 µM and the final concentration of DMSO did not exceed 0.2%, which caused no damage to the parasites. Cell counts were performed in a Neubauer chamber and the activity of N1 was expressed as IC50, corresponding to the concentration that leads to lysis of 50% of the parasites.

Effect on mammalian cells infected by T. cruzi

Peritoneal macrophages in DMES were added to 24-well plates (106 cells/well) and maintained at 37°C. After 1 day, the cultures were washed and infected with bloodstream trypomastigotes (ratio 10 : 1 parasite/host cell). After 1 h of interaction, the non-internalized parasites were removed by washing with PBS and fresh DMES—with or without N1—was added to the cultures and changed every 2 days. The experiments with HMC were performed in similar conditions, but the interaction time was 24 h. At specific times the infection levels were quantified using a Zeiss Axioplan microscope (Oberkochen, Germany).

Effect at the ultrastructural level

Epimastigotes and trypomastigotes were treated with N1 for 1 day and the corresponding controls were processed for ultrastructural analysis. For scanning electron microscopy (SEM), the parasites were adhered to poly-L-lysine-coated coverslips, fixed with 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.2) at room temperature for 40 min and post-fixed with a solution of 1% OsO4, 0.8% potassium ferricyanide and 2.5 mM CaCl2 in the same buffer for 20 min. The cells were dehydrated in an ascending acetone series and dried by the critical point method with CO2. The samples were mounted on aluminum stubs, coated with a 20 nm thick gold layer and examined in a Zeiss DSM940 scanning electron microscope. For transmission electron microscopy (TEM), after washing in PBS the parasites were fixed, post-fixed and dehydrated as described above, and embedded in PolyBed 812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Zeiss EM10C transmission electron microscope.

Effect on mitochondrion and acidic compartments of T. cruzi

Epimastigotes (maintained in LIT medium at 28°C) and trypomastigotes (maintained in DMES medium at 37°C) were treated (at 5 x 106 cells/mL) with 2.5–35 µM N1 for 1 day. The parasites were then incubated for 15 min with 30 µg/mL propidium iodide (PI) plus 10 µg/mL rhodamine 123 (Rh123), or else with 10 µg/mL Acridine Orange (AO). The material was kept on ice until analysis. Data acquisition and analysis were performed using a FACSCalibur flow cytometer (Becton-Dickinson, CA, USA) equipped with the Cell Quest software (Joseph Trotter, Scripps Research Institute, CA, USA). A total of 10 000 events were acquired in the region previously established as that corresponding to the parasites. Alterations in the fluorescence for Rh123 or AO were quantified using an index of variation (IV) obtained by the equation (MT – MC)/MC, where MT is the median of fluorescence for treated parasites and MC that of control parasites. Negative IV values correspond to depolarization of the mitochondrial membrane (Rh123) or increase in pH of acidic compartments (AO).

Epimastigotes treated with N1 and labelled with Rh123 + PI or AO, as described above, were washed in PBS and added to 0.01% gelatin-covered glass coverslips for 15 min at 28°C. Image acquisition was performed with a Zeiss ZVS47 video camera and processed using the software Adobe Premiere 6.0 (Adobe Systems Inc., IL, USA).

Effect on succinate cytochrome c reductase activity of T. cruzi

Untreated and treated epimastigotes (50 µM N1) were maintained at 28°C for 1 day, and harvested by centrifugation at 3000 g for 10 min in a refrigerated centrifuge (Sorvall, model super 21, NC, USA). Thereafter, the parasites were washed in PBS (pH 7.2), resuspended in 0.1 M phosphate buffer (pH 7.4) and disrupted on ice by sonication with 25 cycles of 1 s with intervals of 1 s using an Ultrasonic Processor (Sonics Inc, model CV33, CT, USA). Parasite disruption was monitored by light microscopy. Protein concentration was determined by a commercial protein assay kit (Bio-Rad, CA, USA), using BSA as a standard and following the manufacturer's instructions.

Succinate cytochrome c reductase activity was measured as previously described.20 The reaction mixture contained 0.2 M phosphate buffer (pH 7.4), 0.003 M EDTA (pH 7.4), 0.6 M succinic acid adjusted to pH 7.4 with NaOH, and 0.001 M cytochrome c. The specific activity was measured by the reduction of cytochrome c at 30°C using a 550 nm filter.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect against T. cruzi and infected host cells

We determined the activity of N1 against trypomastigotes (in the absence and presence of blood), epimastigotes, tissue culture-derived and intracellular amastigotes (Figure 2). The corresponding IC50 values are displayed in Table 1. For analysis of the effect on intracellular forms, we used peritoneal macrophages and HMC as host cells. Treatment of both infected primary cultures with the naphthoimidazole caused an inhibitory effect on the infection (Table 2). This effect was observed at 1–4 days of treatment with 5–10 µM N1. As judged by light microscopy, a 4 day treatment with N1 led to damage to HMC at concentrations >100 µM and to macrophages >200 µM.



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Figure 2. Effect of N1 on T. cruzi: (a) trypomastigotes in DMES; (b) trypomastigotes in blood; (c) proliferation of epimastigotes; (d) tissue culture-derived amastigotes; (e) amastigotes internalized in macrophages; (f) amastigotes internalized in HMC.

 

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Table 1. IC50 values expressed in µM for the effect of N1 against different forms of T. cruzi

 

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Table 2. Effect of N1 on the percentage infection of mammalian cells by T. cruzi

 
Ultrastructural analysis

The parasites were treated with 20–40 µM N1 for 1 day and analysed by TEM and SEM. In epimastigotes, the naphthoimidazole caused swelling of the mitochondrion, disorganization of the reservosome morphology and abnormal chromatin condensation (Figure 3c–e). It also caused alterations in the parasite morphology, including body retraction and flagellar detachment (Figure 3f). In trypomastigotes, damage to the mitochondrion, kDNA and chromatin was induced by N1, as well as loss of acidocalcisome electron density (Figure 4c). Morphological alterations in trypomastigotes were similar to those observed in epimastigotes (Figure 4d), besides contortion of the parasite body (Figure 4e).



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Figure 3. Ultrastructural alterations in T. cruzi epimastigotes treated with N1 for 1 day: (a and b) control parasites showing the typical elongated body, with terminal flagellum and normal morphology of reservosomes (R), glycosomes (g), mitochondrion (M), nucleus (N) and kinetoplast (k); (c) 20 µM caused swelling of the mitochondrion (star), disorganization of reservosomes (asterisk) and abnormal chromatin condensation (arrow); (d–f) 40 µM led to disorganization of reservosomes (asterisks) and abnormal chromatin distribution (arrow). Alterations of the parasite morphology with body retraction (white star) and flagellar detachment (white arrowhead) were also observed. Bars: 0.5 µm.

 


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Figure 4. Ultrastructural alterations in T. cruzi trypomastigotes treated with N1 for 1 day: (a and b) control parasites with typical morphology, showing normal kinetoplast (k) and acidocalcisomes (Ac), inset = detail of kinetoplast (k) and mitochondrion (M); (c) 40 µM led to kDNA disorganization (star), swelling of the mitochondrion (asterisk), decrease in the electron density of acidocalcisomes (arrowhead) and abnormal chromatin condensation (arrow), while the nucleolus presents its normal aspect (n), inset = detail of abnormal kinetoplast; (d and e) 25 µM induced retraction (arrow) and contortion (arrowhead) of the parasite body, as well as flagellar detachment (large arrow). E, erythrocyte. Bars, 2 µm.

 
Flow cytometry, fluorescence microscopy and biochemical analysis

Labelling of N1-treated epimastigotes and trypomastigotes with PI showed no significant permeabilization of the plasma membrane in concentrations of the naphthoimidazole from 1 to 40 µM, with a percentage of PI-positive cells of ~5%, similar to that of untreated parasites. When Rh123 or AO was employed, a decrease in fluorescence was observed in epimastigotes. The IV values fluctuated up to –0.68 for Rh123 and to –0.40 for AO (Table 3). This variation was also observed in trypomastigotes, where the IV values (Table 4) oscillated up to –0.59 (Rh123) or –0.55 (AO). In accordance with the flow cytometry data, fluorescence microscopy showed a lower fluorescence level in treated parasites in both forms (data not shown).


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Table 3. Flow cytometry analysis of T. cruzi epimastigotesa treated with N1 and labelled with Rh123 or AO

 

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Table 4. Flow cytometry analysis of T. cruzi trypomastigotesa treated with N1 and labelled with Rh123 or AO

 
Treatment of epimastigotes with 50 µM N1 for 1 day led to a reduction of 17.2 ± 4.1% in the activity of the respiratory chain enzyme succinate cytochrome c reductase, as compared with the activity of control parasites.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although quinoidal substances are important sources of heterocycles, there are only a few reports about the reactivity of 1,2-quinoidal carbonyls towards nucleophilic reagents. Through the reaction of ß-lapachone with 30 aromatic aldehydes, a series of 30 pyran[b-4,3]naphtho[1,2-d]imidazoles with substituted aromatic rings at the 2-position was synthesized.3,15,16,18 Among these naphthoimidazoles, 19 derivatives (63.3%) were more active than the standard trypanocidal compound Crystal Violet. Previous assays performed in the presence of 5% blood at 4°C reported that N1 was 10.6 times more active than the original quinone, ß-lapachone.15

N1 was active against bloodstream trypomastigotes, and inhibited the proliferation of epimastigotes, extra- and intracellular amastigotes of T. cruzi. The effect on both amastigote forms occurred at similar concentrations, and the parasite was much more susceptible to the naphthoimidazole than mammalian cells. After 4 days of treatment, damage to macrophages and HMC was observed only above 100 µM, but only 4 µM N1 (a 25-fold lower dose) was needed to reduce the host cell infection by 50%.

The presence of blood led to a 1.7-fold decrease in the activity of N1 against trypomastigotes when compared with experiments performed in culture medium alone, both at 37°C. Inactivation by blood has been already reported for ß-lapachone and other naphthoquinones.11 In experiments performed at 4°C, addition of 5% blood led to a four times reduction in the trypanocidal effect of ß-lapachone.15 It was suggested that interaction of naphthoquinones with serum proteins reduces the amount of free compound.11 In accordance with this suggestion, the formation has been demonstrated of a covalent bond between the quinoidal carbonyl and free NH2 from primary amines;13,14 a reaction that could also occur with amine groups from proteins. Another possibility for such inactivation is the reduction of 1,2-naphthoquinones by blood DT-diaphorase, leading to hydroquinones, which could be converted to non-toxic conjugates.21 Deactivation of the activity of quinones and naphthoimidazoles by blood requires more investigation, as possibly different mechanisms are operating, depending on the structure of each compound.

Comparison of the activity of N1 against the three forms of the parasite showed the following order of activity: amastigotes > trypomastigotes > epimastigotes, indicating that the vertebrate forms of T. cruzi are more prone to its action than epimastigotes.

Treatment of epimastigotes and trypomastigotes with N1 induced an abnormal chromatin condensation and mitochondrial damage, with preservation of the plasma membrane. By SEM progressive alterations in parasite morphology were observed. In epimastigotes the compound led to morphological alterations in reservosomes, whereas in trypomastigotes a decrease in the electron density of acidocalcisomes was observed.

Swelling of the mitochondrion prompted us to treat both forms of T. cruzi with N1 for 1 day, to label them with Rh123 and PI and to analyse by flow cytometry and fluorescence microscopy. At concentrations up to 35 µM N1 led to a gradual decrease in Rh123 fluorescence, suggesting interference with the proton electrochemical potential gradient of the mitochondrial membrane. The reduced retention of Rh123 was not due to plasma membrane permeabilization, since in our experimental conditions the percent of PI-positive cells was similar for untreated and treated parasites. Reduction of succinate cytochrome c reductase activity in epimastigotes suggests that N1 interferes with the energy production and reinforces the hypothesis that the mitochondrion is one of the initial targets of this naphthoimidazole.

AO, a well-known probe for acidic compartments, is used to label reservosomes22,23 and acidocalcisomes.24 Treatment of epimastigotes and trypomastigotes with N1 led to a decrease in AO fluorescence. This result together with the reservosome disorganization observed by TEM in epimastigotes suggest that this organelle is a target for this naphthoimidazole, which possibly interferes with the endocytic pathway of this parasite, since one of the major functions of reservosomes is the accumulation of endocytosed macromolecules. Reservosomes are absent in trypomastigotes,25 and thus the decrease in AO fluorescence possibly indicates alterations in acidocalcisomes. These organelles are rich in polyphosphates, and this form of phosphorous represents an alternative energy source24 besides the mitochondrion. Thus, it could be suggested that mitochondrial damage induced by N1 leads to polyphosphate hydrolysis as an alternative energy supply, resulting in a decrease in acidocalcisome electron density.

The high activity of naphthoimidazoles, especially N1, against T. cruzi when compared with other classes of compounds synthesized from ß-lapachone may be related to the basic imidazole moiety also present in several substances with trypanocidal activity,2628 as is also the case of benznidazole.

The structure of N1 does not endow this naphthoimidazole the capacity of free radical generation as observed with naphthoquinones.10,29 Although the present results suggest that mitochondrion, acidocalcisomes and reservosomes are targets of N1, the alterations in chromatin organization together with the polycyclic planar topology of this naphthoimidazole encourage us to further investigate its effect at the level of DNA, the mechanism of parasite death and to perform in vivo experiments.


    Acknowledgements
 
We thank Mr Marcos Meuser for his excellent technical work. This work was supported by grants from CNPq, FIOCRUZ and INCA.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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