a Centre for Medical Parasitology, Department of Clinical Microbiology, University Hospital (Rigshospitalet), Copenhagen, Denmark b Department of Molecular Cell Biology, Institute for Medical Microbiology and Immunology, Copenhagen University, Copenhagen, Denmark c Statens Serum Institut, Institute for Medical Microbiology and Immunology, Copenhagen University, Copenhagen, Denmark d Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Copenhagen, Denmark
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Abstract |
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Introduction |
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Our previous studies have shown that licochalcone A, an oxygenated chalcone, has strong antileishmanial activity.6,7 Recently, we reported that 2,4-dimethoxy-4' -butoxychalcone, a novel oxygenated chalcone, exhibited potent activity against human malaria parasite Plasmodium falciparum, in vitro, and rodent parasites Plasmodium berghei and Plasmodium yoelii, in vivo.8 Licochalcone A alters the ultrastructure of the parasite mitochondria and inhibits their function.9 The present study was designed to investigate the antileishmanial activity of a group of new oxygenated chalcones and their mechanism of action. The data indicate that the tested oxygenated chalcones inhibit the in-vitro growth of Leishmania major promastigotes and Leishmania donovani amastigotes, reduce the parasite load in the liver and the spleen of hamsters infected with L. donovani, and alter the ultrastructure and the function of the parasite mitochondria.
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Materials and methods |
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The tested oxygenated chalcones were synthesized in our laboratory and were dissolved in 1% dimethyl sulphoxide (DMSO) in medium 199 to prepare a working solution of 1 g/mL. The structure of the tested oxygenated chalcones is shown in Figure 1.
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Male golden hamsters (Mesocricetus auratus), with a body weight of approximately 50 to 60 g, were used in this study.
Parasite cultures
One strain of L. major (MHOM/IL/67/LRC-L137) and two strains of L. donovani (MHOM/KE/85/NLB 439 and MHOM/SD/00/1SD2D) were used. Promastigotes were cultured in medium 199 containing 10% heat-inactivated fetal calf serum. Incubation and growth of the parasite were carried out at 26°C. Promastigotes were harvested on day four of the culture and used. The axenic amastigotes of MHOM/SD/00/1SD2D were cultured using a modified method originally described by Doyle et al.10 Briefly, after 7 days of culture at 26°C, the promastigotes were centrifuged at 500g for 10 min, supernatant was removed and the pellet was resuspended in Schneider's Drosophila medium with 20% HFCS. The cultures were carried out at 37°C in a 95% air:5% CO2 atmosphere for 7 days.
Effect on promastigotes
The effect of oxygenated chalcones on promastigotes was assessed as described previously. 6 Promastigotes were incubated at 26°C in the presence of different concentrations of oxygenated chalcones, or the medium alone in 96-well flat-bottom microtitre plates (Nunc, Roskilde, Denmark). After 2 h, 1 µCi of 3H-thymidine (Amersham International plc, Amersham, UK) was added to each well. Parasites were harvested 18 h later and 3H-thymidine incorporation was measured. All cultures were performed in triplicate.
Effect on intracellular amastigotes
The study was performed on human peripheral blood monocyte-derived macrophages (MDM) as described previously.6 Briefly, 200 µL of a suspension containing 2x 106 human peripheral blood mononuclear cells were added to each well in 96 well flat-bottom microtitre plates. After 4 h and 3 days of incubation at 37°C in 5% CO2, the old medium was removed and the cells were washed three times and then replaced with fresh medium. After 6 days, 200 µL of 1 x 107/mL stationary phase L. donovani promastigotes were added to each well. After 24 h, the cultures were washed three times and incubated in the medium containing different concentrations of oxygenated chalcones or the medium alone. Three days after infection, macrophages were lysed by 0.01% sodium dodecyl sulphate (SDS) and the cultures were incubated ar 26°C and the amastigotes were left to transform into promastigotes. Parasite growth was determined by adding 1 µCi of 3H-thymidine to each well after 48 h incubation, the parasites were harvested 20 h later, and 3H-thymidine incorporation was measured.
Effect on L. donovani infection in hamsters
The in-vivo activity of 35m4ac and 24m4ac in hamsters infected with L. donovani was examined according to a method described previously.7 On day 0, hamsters received intracardial injections of 1x 109 stationary phase L. donovanipromastigotes in 0.1 mL PBS. One hour after inoculation, five of the hamsters were killed by carbon dioxide asphyxiation. The liver and the spleen from the hamsters were removed and weighed, and impression smears were made. The slides were fixed with absolute methanol, stained with Giemsa stain and examined by light microscopy. Parasite load in the liver and the spleen was determined according the method described by Stauberet al.11 The parasite load in the spleen was also estimated by a 3H-thymidine uptake method as described previously.7 Briefly, the tissues were cut into small pieces and homogenized. The supernatants containing the released parasites were cultured in 15 mL of RPMI 199 containing 10% HFCS in a 25 cm2, 50 mL culture flask (Nunc, Roskilde, Denmark) at 28°C. After 3 days of incubation, 1 mL of the culture supernatant was centrifuged at 1000 r.p.m. for 10 min, washed with medium three times, resuspended in 1 mL of fresh medium and then 200 µL of the culture solution was transferred to round-bottom microtitre plates. The cultures were then pulsed with 1 µCi of 3H-thymidine. After 18 h of incubation the cultures were harvested and 3H-thymidine incorporation was measured. From day one, groups of five hamsters were each injected intraperitoneally with 5 or 20 mg of 35m4ac or 24m4ac per kg body weight in 0.1 mL of PBS od for 6 consecutive days. Five hamsters received the same volume of PBS as negative control and another five hamsters received 400 mg of Pentostam (Sodium Stibogluconate Injection BP, The Wellcome Foundation Ltd., London, UK) per kg body weight as positive control. On day eight, all hamsters were killed by carbon dioxide asphyxiation. Their liver and spleen were removed and weighed, and impression smears were prepared. Parasite load in the liver and the spleen was determined. The parasite load in the spleen was also estimated by 3H-thymidine uptake method.
Ultrastructure studies
Electron microscopic studies were carried out to examine the effect of oxygenated chalcones (35m4ac, 24m4hc, 24m4ac and 34m4ac) on the ultrastructure of the promastigote form of the parasite as described previously.6 Briefly, a suspension of 3 x 106 L. major promastigotes per millilitre was incubated in the presence of either oxygenated chalcones or medium alone for 24 h at 26°C. After incubation, the promastigotes were centrifuged, resuspended in 1 mL of medium, fixed with 3% glutaraldehyde and embedded in Noble agar. After postfixation in OsO4, specimens were stained in 2% uranyl acetate and embedded in Vestopal W. The sections were poststained with magnesium uranyl acetate and lead citrate and were examined in a Philips 201 C electron microscope at 60 KV.
Respiration of the parasite (oxygen consumption and changes of carbon dioxide and pH)
A previously described method was used for this study.9 Briefly, a suspension of 4.95 mL of L. major promastigotes (2 x 10 6/mL) or L. donovani axenic amastigotes (5x 107/mL) were incubated at 26°C in the presence of 50µL of oxygenated chalcones in different concentrations or medium alone in sealed bottles. Oxygen consumption and changes of carbon dioxide and pH were measured at 2, 24, 48 and 72 h after incubation using an acid-base laboratory ABL4 (Radiometer, Copenhagen, Denmark).
Activity of mitochondrial dehydrogenases
The effect of oxygenated chalcones on the activity of parasite mitochondrial dehydrogenases was determined by an MTS method as described previously.12 In this method, the substrate, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulphonyl)-2H-tetrazolium (MTS), is converted into a soluble formazan-like dye-complex by the parasite mitochondrial dehydrogenase. Briefly, 5 x 104 promastigotes were seeded in 96-well flat-bottom microtitre plates and incubated with different concentrations of oxygenated chalcones or medium alone at 26°C. After 24 h, 25 µL MTS/PMS was added to each well and further incubated for 3 h at 37°C, where after the optical densities (ODs at 492 nm) were measured directly using a Titre-Tech 96-well scanner.
Statistical analysis
A paired two-tailed t-test was used for analysis of the data. Values of P < 0.05 were considered significant.
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Results |
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Figure 2 shows the effect of the oxygenated chalcones on the in-vitro growth of L. major promastigotes. All 10 oxygenated chalcones exhibited a clear concentration-dependent inhibitory effect. In comparison with the control, the oxygenated chalcones showed a significant inhibitory effect on the in-vitro growth of promastigotes at concentrations of 1.5 µM and above (P <0.05).
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In the hamster model, the total number of L. donovani parasites (as determined by the method of Stauber et al.11) in the liver were reduced by 97% and 84% when animals received intraperitoneal administration of 20 and 5 mg of 35m4ac per kg of body weight for 6 days (P <0.05; Figure 4a). The parasite load in the liver was also reduced by 88% and 70% when animals received intraperitoneal administration of 20 and 5 mg of 24m4ac per kg of body weight. 35m4ac and 24m4ac also significantly reduced the parasite load in the spleen as determined by both Stauber' s method and 3H-thymidine uptake method (P < 0.05; Figure 4b and c). When the pentavalent antimony, Pentostam was administered intraperitoneally at a dosage of 400 mg per kg of body weight for 6 days, the parasite load in the liver was reduced by 94%.
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Electron microscopic studies were carried out to determine the possible ultrastructural changes of the parasite mitochondria after incubation with the oxygenated chalcones. Figure 5 shows representative samples of the effect of 35m4ac, 24m4hc and 24m4ac on the ultrastructure of the mitochondria of L. major promastigotes. The unaffected control promastigotes contained slender and dense mitochondria (Figure 5a). After adding 30 µM of different oxygenated chalcones, the mitochondria showed different degrees of enlargement, and a disarray, with irregularity and a loss of the cristae (Figure 5b, c and d). These changes did not differ from the effect of 34m4ac (data not shown).
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Figure 6 shows that 24m4ac, 24m4hc, 35m4ac and 34m4ac inhibited the respiration of L. major promastigotes in a concentration-dependent manner, as revealed by inhibition of the oxygen consumption of the parasites, the accumulation of CO2 and the decline of pH in the parasite culture. Among the four oxygenated chalcones, 34m4ac exhibited the strongest inhibitory effect on the respiration of the promastigotes, and the other three oxygenated chalcones inhibited the respiration of the parasite to almost the same degree. Licochalcone A also exhibited a strong inhibitory effect on the oxygen consumption, the accumulation of CO2, and the decline of pH in L. donovani axenic amasitigotes (data not shown).
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Figure 7 shows a concentration-dependent inhibitory effect of some oxygenated chalcones on the activity of the mitochondria dehydrogenase in L. major promastigotes. The P values for the effect of most oxygenated chalcones (except 24mc, 25m4ac and 26m4ac) on the activity of the mitochondria dehydrogenase at concentrations of 1.5 µM and greater in comparison with the effect of the control were <0.05. The P values for the effect of 24mc and 26m4ac on the enzyme activity at concentrations of 3 µM and greater in comparison with the effect of the control were <0.05. At concentrations of 15 and 30 µM, the P values for the effect of 25m4ac in comparison with the effect of the control were <0.05.
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Figure 8 shows the comparison of the effect of six oxygenated chalcones on the in-vitro growth ofL. major promastigotes measured by 3H-thymidine incorporation and on the activity of the mitochondrial dehydrogenase of the parasite. Licochalcone A, 24mc and 34m4ac inhibited the 3H-thymidine incorporation and the activity of the mitochondrial dehydrogenase to the same degree. The inhibitory effects of 24m4ac and 35m4ac on the 3H-thymidine incorporation were slightly stronger than that on the activity of the mitochondrial dehydrogenase. At concentrations of 15 and 30 µM, 24m4hc exhibited a significantly stronger inhibitory effect on 3H-thymidine incorporation than on the mitochondrial dehydrogenase.
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Discussion |
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Similar to licochalcone A, all the tested oxygenated chalcones showed inhibition of the in-vitro growth of L. major promastigotes measured by 3H-thymidine incorporation in a concentration-dependent manner. The six oxygenated chalcones also exhibited a clear concentration-dependent inhibitory effect on the in-vitro growth of L. donovaniamastigotes in human MDM. These data imply that the DNA synthesis in the parasites was inhibited. At low concentrations, the inhibitoryeffect of the oxygenated chalcones on amastigotes was stronger than that on promastigotes. This effect could be a consequence of a direct action of chalcones on amastigotes, or interference with defence mechanism of amastigotes in the hostile environment of the macrophage. The third possibility could be that chalcones activate macrophages to kill amastigotes. Nevertheless, the important aspect of this finding is that the intracellular amastigote form of the parasite which is present in the in-vivo environment is more susceptible to these compounds. Treatment of hamsters infected with L. donovani with intraperitoneal administration of 35m4ac and 24m4ac at dosages of 5 and 20 mg/kg of body weight per day for 6 consecutive days resulted in a significant reduction of parasite load in the liver and the spleen of treated animals.
The studies on the ultrastructure of the L. major promastigotes showed that four oxygenated chalcones, 35m4ac, 24m4hc, 24m4ac and 34m4ac, destroyed the mitochondria while the other organelles of the parasite appeared normal. In a concentration-dependent manner, oxygenated chalcones inhibited the respiration of L. major promastigotes and L. donovani axenic amasitigotes, as shown by inhibition of O2 consumption and CO 2 production by the parasites. Oxygenated chalcones also inhibited the activity of the mitochondrial dehydrogenase of L. major promastigotes in a concentration-dependent manner. The mitochondrial dehydrogenase activity measured in this assay includes the activity of many dehydrogenases in mitochondrion, such as malate dehydrogenase and succinate dehydrogenase, and has been shown to be mitochondria specific.12 These findings indicate that the enzymes of the parasite respiratory chain might be the target for oxygenated chalcones.
The understanding of the mechanism of action will help the development of oxygenated chalcones into new antileishmanial drugs. In order to elucidate the mechanism of action of the oxygenated chalcones further, we have compared the effect of six oxygenated chalcones on the in-vitro growth of L. major promastigotes measured by 3H-thymidine incorporation into DNA and on the activity of the mitochondrial dehydrogenase of the parasite. Three of the oxygenated chalcones inhibited 3H-thymidine incorporation and the mitochondrial dehydrogenase to the same degree, indicating that the effect on DNA replication could be secondary to the effect on the mitochondrial dehydrogenase. In the other three oxygenated chalcones there was a disassociation between the effect on 3H-thymidine incorporation and on mitochondrial dehydrogenase. These findings indicate that the inhibition of the mitochondrial dehydrogenase of the parasite was probably not the only mechanism responsible for the effect of these chalcones on DNA replication. It has been reported that the trypanocidal activity of quinones is probably caused by their effect on specific enzymes, involved in DNA synthesis, or cell membrane. 13,14 Quinones bind easily to proteinic blood components, with interaction of the quinone moiety with basic free NH 2 residues, leading to the fixation of quinone to the protein. Oxygenated chalcones also easily bind to protein, and they might bind to specific enzymes, influencing function of mitochondria or DNA synthesis and resulting in the death of parasite. Li et al. have shown that some chalcones inhibit cysteine proteinases in malaria parasites P. falciparum.15 Our preliminary results indicate that the oxygenated chalcones described in this communication do not act through the cysteine proteinases of Leishmania spp. parasites (data not shown ).
Studies on the energy metabolism pathway in the mitochondria and other biochemical aspects of the parasite are warranted to clarify the exact mechanism of action of the oxygenated chalcones. Further elucidation of the mechanism of action of the oxygenated chalcones is important for the development of these compounds into a new class of antiparasitic drugs and could also help to design new antiparasitic drugs which act on some specific targets only present in the parasite.
In conclusion, the oxygenated chalcones exhibit a strong antileishmanial activity both in vitro and in vivo. This activity might be the result of interference with the function of the parasite mitochondria.
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Acknowledgments |
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Notes |
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References |
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Received 9 July 1998; returned 20 November 1998; revised 14 December 1998; accepted 14 February 1999