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1 Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil
2 Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, Brazil
3 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Correspondence
Marcos A. Vannier-Santos
vannier{at}bahia.fiocruz.br
or
marcos.vannier{at}pesquisador.cnpq.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-difluoromethylornithine; FCS, fetal calf serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ODC, ornithine decarboxylase; PA, polyamine; ROS, reactive oxygen species; TBA, thiobarbituric acid; TBARS, TBA-reactive substances |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Clinical manifestations may include single self-healing or disseminated cutaneous lesions, mucocutaneous destruction and visceral commitment, all resulting from Leishmania replication within mononuclear phagocytes (Vannier-Santos et al., 2002). A growing interest by developed countries was prompted by increased frequency of leishmaniasis cases among HIV patients and overseas travellers (Desjeux, 2004).
Antileishmanial chemotherapy is currently limited to a few compounds such as the pentavalent antimonials glucantime (N-methylglucamine antimoniate) and pentostam (sodium stibogluconate), amphotericin B and pentamidine. Pentavalent antimonials have been the first-choice drugs for leishmaniasis treatment since the 1920s. Although they are generally effective against most leishmaniasis cases, severe side effects are often observed. Also, resistance to these compounds is a serious problem in many parts of the world such as India (Ouellette et al., 2004). The high toxicity of the antimonial formulation has been implicated in the death of several patients under treatment (Silva, 2001; Ahasan et al., 1996). Miltefosine was recently added to the therapeutical arsenal and is effective against Indian visceral leishmaniasis (Bhattacharya et al., 2007). Nevertheless miltefosine cure rates in mucosal disease range from only 50 % (Soto et al., 2004) to 70 % (Soto & Toledo, 2007), and 58 % in severe clinical forms. In addition, miltefosine is not as rapid as the antimonials (Soto et al., 2008), not effective in HIV-infected patients (Troya et al., 2008) and permits relapses in diffuse cutaneous forms (Zerpa et al., 2007) as well as displaying limited efficacy against Brazilian cutaneous infection (Tuon et al., 2008). Furthermore, the prolonged treatments and half-life of the compound, especially in an oral therapy with variable adherence, may promote resistance (Dupouy-Camet, 2004; Janvier et al., 2008), and miltefosine-resistant parasites have been readily obtained in vitro (Ouellette et al., 2004). Therefore, new chemotherapeutic agents are required.
The natural polyamines (PAs) putrescine, spermidine and spermine are low-molecular-mass organic cations found in up to millimolar concentrations in eukaryotic and prokaryotic cells and essential for cell growth and differentiation. PA biosynthesis is initiated by the rate-limiting decarboxylation of ornithine to putrescine by ornithine decarboxylase (ODC; EC 4.1.1.17). Successive transfers of aminopropyl groups from decarboxylated S-adenosylmethionine give rise to spermidine and then spermine.
PA analogues have been synthesized and tested for cancer and parasitic infection therapies. Distinct PA synthesis and transport mechanisms have been described in mammalian cells and micro-organisms, providing potential chemotherapeutic targets (Heby et al., 2003; Müller et al., 2001; Bacchi & Yarlett, 2002). Treatment of human Trypanosoma gambiense infection with eflornithine (DL--difluoromethylornithine, DFMO), an irreversible ODC inhibitor, results in successful clearance of both early and late stages of the disease and also lacks severe side effects (Bitonti et al., 1991; McCann et al., 1987). However, the effectiveness of DFMO in Leishmania is controversial. It was reported not to inhibit replication of some species, but it is effective against Leishmania infantum both in vitro and in vivo (Gradoni et al., 1989). The bis(benzyl)polyamine analogues also show significant activity against Leishmania donovani amastigotes in vitro and in vivo (Baumann et al., 1990). We have previously shown that the putrescine analogue 1,4-diamino-2-butanone (DAB) blocks the proliferation of Tritrichononas foetus (Reis et al., 1999) and Trypanosoma cruzi (Menezes et al., 2006). Here we tested the effects of DAB on Leishmania amazonensis proliferation, putrescine uptake and biosynthesis, ultrastructure and macrophage infection. The implications of the findings for the parasite's biology are discussed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Parasites.
The MHOM/Br/75/Josefa strain of Leishmania amazonensis was used throughout this study. It was isolated from a human case of diffuse cutaneous leishmaniasis in Brazil by Dr C. A. Cuba-Cuba (University of Brasília, Brazil) and has been maintained since then, in both axenic culture and hamster (Mesocricetus auratus) footpad inoculation. Promastigotes were maintained at 26 °C in Warren's medium (brain and heart infusion, 20 mg folic acid l–1 and 20 mg haemin l–1) supplemented with 10 % heat-inactivated fetal calf serum (FCS). Amastigotes were obtained from axenic cultures and cultivated as described elsewhere (Borges et al., 1998). These cells were able to infect and induce lesion formation in hamsters.
Macrophages.
Peritoneal macrophages from 6–8-week-old Swiss mice were collected in Hanks' balanced salt solution and plated on glass coverslips in Falcon 24-well tissue culture plates. Cells were allowed to adhere for 60 min at 37 °C in a 5 % CO2 atmosphere, after which the non-adhering cells were removed and fresh medium 199 plus 10 % FCS was added. Cells were cultivated overnight before the assays, as described previously (Borges et al., 1998).
Proliferation assays.
Mid-exponential-phase parasites were harvested and inoculated in fresh culture medium before or after addition of increasing concentrations of the analogue. Aliquots were collected and fixed every 24 h and parasite density was assessed by direct counting in a Neubauer chamber. The mean generation time, g, was determined according to the equation g=(log10 Nt–log10 N0)/log10 2, where t is time and N is the cell number at a defined period of time. The IC50 was determined employing GraphPad Prisma on the 5th day of axenic culture.
Transmission electron microscopy.
Parasites were fixed in 2.5 % glutaraldehyde grade II (Sigma) in 0.1 M sodium cacodylate buffer pH 7.2, post-fixed in 1 % osmium tetroxide and 0.8 % potassium ferricyanide in the same buffer, dehydrated in an acetone series and embedded in Polybed resin (Polysciences). Thin sections were stained with uranyl acetate and lead citrate and observed under a Zeiss 900 transmission electron microscope.
MTT reduction.
Parasites were inoculated (107 cells ml–1) and treated as described under ‘Proliferation assays’ for different periods of time. Treated and untreated cells were washed, reinoculated in fresh culture medium containing 10 % (v/v) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and incubated for an additional 16 h. Cell pellets were solubilized in DMSO, transferred to flat-bottomed 96-well plates and formazan precipitates derived from MTT reduction were determined spectrophotometrically at 540 nm. Wells containing solely MTT and DMSO were employed as controls. MTT reduction by macrophages was performed as above, but in samples with 105 cells incubated for 24 h.
Lipid peroxidation.
Thiobarbituric acid-reactive substances (TBARS) were measured in parasites pre-incubated or not with the analogue for 24 h. After treatment, the cells were washed, resuspended in 200 µl PBS and 200 µl of 1 % TBA in acetic acid was added. Then samples with equal cell numbers were incubated at 95 °C for 3–4 h, cooled, and the absorbance was determined at 532 nm.
Analysis of putrescine content.
Putrescine content was determined as described elsewhere (Seiler & Lamberty, 1973), modified as follows (De Mello et al., 1976). DAB-treated and untreated parasites were washed and incubated with 5 % perchloric acid. Precipitated proteins were removed by centrifugation and 400 µl 112 mM dansyl chloride solution and 20 mg sodium carbonate were added to the supernatants. Samples were incubated overnight at room temperature protected from light and then the reaction was quenched by addition of 100 µl of an aqueous proline solution at 100 mg ml–1. Dansyl derivatives were extracted in benzene and dansylated putrescine was separated by thin-layer chromatography in silica gel using benzene/cyclohexane/methanol (85 : 15 : 2, by vol.) as solvent. A second separation in the same direction was performed using ethyl ether/cyclohexane (90 : 10, v/v). Fluorescent dansylated PAs were identified under UV light. The putrescine spot was removed and extracted in dioxane and its concentration determined spectrofluorimetrically (365 nm excitation and 550 nm emission). This method accurately measured <10 nmol dansylated products.
Ornithine decarboxylase activity.
Parasites were washed and stored at –20 °C in 20 mM Tris/HCl pH 7.1, 50 µM Na4EDTA, 50 µM DTT and 50 µM pyridoxal phosphate until use. Samples were lysed by freeze–thaw cycles and incubated in the presence of L-[1-14C]ornithine hydrochloride (0.12 µCi ml–1; 4.4 kBq ml–1) for 15–90 min at 37 °C. Reaction was terminated by the addition of 10 % trichloroacetic acid and the resulting 14CO2 was adsorbed to 1 M benzethonium hydroxide (Sigma) at 37 °C for 30 min. ODC activity was determined by liquid scintillation. For reversibility assays the parasites were washed to remove the analogue before lysis.
Putrescine uptake.
DAB-treated and untreated promastigotes were collected by centrifugation at 3000 g for 10 min, resuspended in PBS at a concentration of 108 cells ml–1 and incubated with 2.5 mM putrescine, containing [1,4(n)-3H]putrescine dihydrochloride (1 µCi ml–1; 37 kBq ml–1) for 15–60 min at 28 °C. Cells were then extensively washed in ice-cold PBS to remove non-incorporated putrescine, collected in MilliQ water and ruptured by three freeze–thaw cycles. Supernatant samples spotted onto fibreglass filters (Millipore AP25) were air-dried and the radioactivity measured by liquid scintillation. In all experiments the uptake of putrescine by cells was also measured at 0–4 °C. Under this condition the incorporation of the tritiated compound was inhibited by over 85 % compared to the incorporation observed at 28 °C; this level of incorporation was considered as non-specific adsorption of the diamine to the parasite. The net putrescine taken up by the cells was calculated by subtracting the radioactivity observed at 0–4 °C from that at 28 °C.
Infection assays.
DAB-treated and untreated parasites and macrophages were left in contact at 35 °C in a 10 : 1 parasite : macrophage ratio. Macrophage monolayers were incubated with 100 µM DAB prior to or after infection. Coverslips were collected after 1–96 h infection, rinsed in PBS, fixed in Bouin's fixative and stained with Giemsa. The percentage of infected macrophages was determined by counting 1000–2000 cells on each preparation. The association index (AI) was determined by multiplying the percentage of infected cells by the mean number of parasites per cell.
Statistical analysis.
Comparisons between groups were done by the unpaired Student's t-test or one-way analysis of variance (ANOVA) and a posteriori Tukey's tests, by use of Prism 4.0 software (GraphPad). For all tests, differences of P<0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), with an IC50 of approximately 144 µM and up to threefold increase in the generation time. Addition of 10 mM exogenous putrescine or spermidine to the culture medium restored parasite growth at micromolar but not millimolar DAB concentrations, which led to the irreversible loss of viability of most cells (data not shown). Mass effects of excess DAB cannot be ruled out. Growth inhibition induced by DAB was also reversed by analogue removal up to 72 h (data not shown).
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The diminished intracellular putrescine pool could be explained by inhibition of its biosynthesis or transport. Therefore we assayed the parasite ODC activity and [3H]putrescine uptake in the presence or absence of DAB.
Parasite ODC activity
Promastigote ODC activity in homogenates was fully inhibited by 100 µM DAB; the inhibition was reversible, as removing the drug by washing the parasites entirely restored ODC activity (Fig. 2). Nevertheless, promastigotes treated with 100 µM DAB displayed putrescine contents quite similar to untreated cells, possibly due, at least in part, to exogenous putrescine uptake. Therefore we assayed putrescine uptake by parasite cells.
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). However, promastigotes pretreated with 100 µM DAB for 24 h showed 40–50 % enhanced putrescine uptake (Fig. 3A). Similarly, 10 mM DAB blocked putrescine uptake by axenic amastigotes, whereas preincubation of parasites with 100 µM DAB increased putrescine incorporation at least twofold (Fig. 3B). This compensatory effect was blocked by protein synthesis antagonists in both developmental forms (not shown).
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), DAB-treated parasites showed severe damage of the mitochondrion–kinetoplast complex (Fig. 4B–F). The organelle displayed a fenestrate (Fig. 4B) or swollen appearance with a washed-out matrix and disarrangement of the cristae architecture (Fig. 4D, E), culminating in complete mitochondrial destruction. The kinetoplast sometimes showed disorganized k-DNA (Fig. 4C). Myelin-like figures were often observed within cytoplasmic vesicles (not shown) and the flagellar pocket in DAB-treated parasites (Fig. 4F). Vacuoles containing electron-dense material were often observed (not shown).
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), whereas 24 h incubations led to significantly diminished peroxidation (Fig. 5B). Furthermore, putrescine addition was able to prevent short-term 1 mM DAB effects, but not 10 mM or 24 h-induced inhibition.
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). MTT reduction by macrophages was not affected by DAB treatment (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and particularly antileishmanial activities (Baumann et al., 1991; Balaña-Fouce et al., 1991; Mukhopadhyay & Madhubala, 1995). The putrescine analogue DAB has been tested in different micro-organisms (Calvo-Méndez et al., 1993; Arteaga-Nieto et al., 1996; Reis et al., 1999; Garcia et al., 2005; Menezes et al., 2006) and was effective against DFMO-resistant parasites (Calvo-Méndez et al., 1993; Reis et al., 1999). DAB has also been shown to be a powerful ODC inhibitor in a number of organisms (Calvo-Méndez et al., 1993; Arteaga-Nieto et al., 1996). In our study, DAB displayed a dose-dependent reversible antiproliferative activity in vitro that was competitively reversed by the addition of exogenous putrescine or spermidine. Moreover removal of the analogue readily restored ODC activity. Similarly PAs can modulate murine ODC activity post-transcriptionally (van Daalen Wetters et al., 1989). Although 100 µM DAB completely blocked ODC activity in L. amazonensis it had no effect on cellular putrescine contents. Moreover, 10 mM DAB reduced the diamine concentration in parasite cells by only 46 %. The different sensitivities of ODC and whole cells may be due, at least in part, to the presence of PAs in the culture medium and/or inefficient DAB uptake.
The antiproliferative effect of DAB may be due, at least in part, to destruction of mitochondria as reported for spermine analogues (Stevens et al., 1977), consistent with the observed reduction in O2 consumption in DFMO-treated Trypanosoma brucei (McCann et al., 1981) and with gross mitochondrial ultrastructural alterations in DAB-treated T. cruzi (Menezes et al., 2006). We have previously shown that DAB leads to the destruction of the trichomonad redox organelle (Reis et al., 1999), suggesting a conserved mode of action of DAB in unrelated parasitic protozoa.
Antimicrobial compounds targeting mitochondria are valuable targets for chemotherapy (Kita et al., 2003) and may be particularly relevant against trypanosomatid parasites, in which the glycolytic pathway depends on a cyanide-insensitive glycerol-3-phosphate oxidase system in the inner mitochondrial membrane (Clarkson et al., 1989). In addition, pentamidine resistance in Leishmania involves drug extrusion from the mitochondria (Basselin et al., 2002). In this regard, the PA transporter LmPOTI of Leishmania major is inhibited by pentamidine (Hasne & Ullman, 2005). In addition putrescine may stabilize T. cruzi mitochondrial membranes (Giffin et al., 1986).
PA analogues do inhibit leishmanial proliferation and cause mitochondrial potential changes and modifications compatible with apoptosis (Tavares et al., 2005). Several lines of evidence indicate that oxidative stress is associated with mitochondrial disruption in several species. Interestingly the bis-naphthalimidopropyl putrescine analogue was the most active PA derivative against Leishmania, particularly in the amastigote form. Mitochondrial enlargement and disruption of internal membrane and cristae were also reported in DFMO-treated Trypanosoma rhodesiense (De Gee et al., 1984). Therefore, ROS generation in the mitochondrion might explain the kDNA disorganization observed here, since these organic cations protect the DNA from oxidative species (Khan et al., 1992). Moreover, as PAs downregulate lipid peroxidation induced by pro-oxidant compounds (Tadolini, 1988; Bellé et al., 2004) the enhanced peroxidation described above, assessed by TBARS production, probably results from increased ROS in the mitochondrion. The diminished lipid peroxidation observed after treatment with 10 mM DAB might be due to mitoptosis, or ‘programmed mitochondrial destruction’ a process in which the ROS-producing mitochondria are selected for destruction as an antioxidant defence (Skulachev, 1998). Interestingly, this DAB concentration did not significantly affect macrophage viability (not shown). The MTT reduction by mitochondria was markedly inhibited by DAB, corroborating the respiratory activity loss and the mitochondrial swelling observed here, which may lead to disruption of the organelle. After destruction, the mitochondria may be degraded by autophagy (reviewed by Skulachev, 1998) and therefore lipid peroxidation would not be detectable. Spermine and spermidine, but not putrescine, can reverse lipid peroxidation in T. cruzi (Hernandez et al., 2006). Therefore the effects of DAB may be due, at least in part, to inhibition of the conversion of putrescine to spermidine, which is required for the synthesis of the major antioxidant molecule of trypanosomatid parasites, trypanothione [N1,N8-bis(glutathionyl)spermidine].
The formation of myelin-like figures from endoplasmic reticulum cisternae may be indicative of autophagy. It is reasonable to suppose that parasites may undergo necrotic death before apoptosis, since no ultrastructural evidence of apoptosis was observed here. Another possibility is that accumulation of DAB by the parasite may lead to the production of H2O2 as a result of oxidation; nevertheless it is not known whether DAB can be a substrate for amine oxidases (Calcabrini et al., 2002).
The appearance of vesicles with an electron-dense core, presumably acidocalcisomes, may result from microbicidal drug-induced autophagy (Vannier-Santos et al., 1995, 1999) and it was reported that this organelle may have a lysosome-related origin in Leishmania amastigotes (Zhang et al., 2005).
Putrescine is by far the most common PA/diamine in Leishmania, while trace amounts of spermine have been detected (Balaña-Fouce et al., 1991). Its uptake is stage- and cell cycle-regulated (Gonzalez et al., 1992), suggesting its importance in cell growth and differentiation. Interestingly, although not essential, addition of putrescine to ODC-deficient L. donovani mutants restores parasite growth more rapidly and to a greater extent than PAs such as spermidine (Jiang et al., 1999). Furthermore the addition of this diamine leads to higher concentrations of trypanothione.
DAB significantly inhibited both ODC activity and putrescine uptake. This analogue acts as an ODC inhibitor in other parasitic protozoa (Calvo-Méndez et al., 1993; Arteaga-Nieto et al., 1996) but the mechanism has not been elucidated. It is known that, unlike mammalian cells, PAs only slightly downregulate Leishmania ODC activity (Mukhopadhyay & Madhubala, 1995) and the enzyme was unaffected by up to 1 mM putrescine (Hanson et al., 1992). Leishmania parasites not only synthesize putrescine and spermidine, but also accumulate extracellular PAs via specific transport systems. Energy-dependent putrescine and spermidine transporters have been described in L. donovani, L. mexicana (Basselin et al., 2000) and L. major (Hasne & Ullman, 2005). PA transport in protozoa may furnish potential targets for parasitic disease chemotherapy (Reguera et al., 2005). A PA permease was identified and characterized in L. major (Hasne & Ullman, 2005). These authors showed that the protein, termed LmPOT1, transports putrescine and spermidine, and transfected in T. brucei can stimulate putrescine uptake. Conceivably, DAB enters the cell via a putrescine transporter; this would explain inhibition of putrescine uptake by competition with an excess of the analogue. Nevertheless, long-term pre-treatment with DAB (24 h) increased the capacity of putrescine uptake twofold when cells were incubated with the amine after DAB removal, in a protein synthesis-dependent manner. Similarly, upregulation of PA transport was observed after DFMO treatment in L. donovani (Kandpal & Tekwani, 1997) and human cells (Redgate et al., 2001) and may provide an alternative PA source in deprived cells. Interestingly DFMO induced a tenfold increase in putrescine transport in L. infantum (Balaña-Fouce et al., 1991). Such compensatory homeostatic mechanisms must be considered in the search for PA analogues for antileishmanial chemotherapy.
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ACKNOWLEDGEMENTS |
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Edited by: J. Tachezy
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Received 16 October 2007;
revised 3 June 2008;
accepted 9 June 2008.
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). Parasites pretreated with 100 µM DAB for 60 min and washed to remove the analogue resulted in enzymic activity (
) similar to control samples (
), indicating that the inhibition is readily reversible. Data are presented as means±SD of at least three independent experiments.
