Marilene M. Canto-Cavalheiro

Reactive Oxygen Species Production and Mitochondrial Dysfunction Contribute to Quercetin Induced Death in Leishmania amazonensis

Background Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Quercetin has generated considerable interest as a pharmaceutical compound with a wide range of therapeutic activities. One such activity is exhibited against the bloodstream parasite Trypanosoma brucei and amastigotes of Leishmania donovani. However, the mechanism of protozoan action of quercetin has not been studied. Methodology/Principal Findings In the present study, we report here the mechanism for the antileishmanial activity of quercetin against Leishmania amazonensis promastigotes. Quercetin inhibited L. amazonensis promastigote growth in a dose- and time- dependent manner beginning at 48 hours of treatment and with maximum growth inhibition observed at 96 hours. The IC50 for quercetin at 48 hours was 31.4 µM. Quercetin increased ROS generation in a dose-dependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. In addition, quercetin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential. Conclusions/Significance The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Quercetin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. Taken together, our results demonstrate that quercetin eventually exerts its antileishmanial effect on L. amazonensis promastigotes due to the generation of ROS and disrupted parasite mitochondrial function.

Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis

The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) and serum samples of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients positive and tested 98.4% of the serum samples of the VL suspects positive. In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania chagasi and found 85.3% of the serum samples of the suspected patients positive. Although the rK39 dipstick is more rapid and user friendlier than the DAT, the latter has a superior sensitivity and specificity. Furthermore, the reagents used for DAT do not require cold storage, whereas the buffer of the rK39 must be stored at 4oC. Therefore, the DAT is the most suitable test for the sero-diagnosis of VL under field conditions.

A review of antimalarial plants used in traditional medicine in communities in Portuguese-Speaking countries: Brazil, Mozambique, Cape Verde, Guinea-Bissau, São Tomé and Príncipe and Angola

The isolation of bioactive compounds from medicinal plants, based on traditional use or ethnomedical data, is a highly promising potential approach for identifying new and effective antimalarial drug candidates. The purpose of this review was to create a compilation of the phytochemical studies on medicinal plants used to treat malaria in traditional medicine from the Community of Portuguese-Speaking Countries (CPSC): Angola, Brazil, Cape Verde, Guinea-Bissau, Mozambique and São Tomé and Príncipe. In addition, this review aimed to show that there are several medicinal plants popularly used in these countries for which few scientific studies are available. The primary approach compared the antimalarial activity of native species used in each country with its extracts, fractions and isolated substances. In this context, data shown here could be a tool to help researchers from these regions establish a scientific and technical network on the subject for the CPSC where malaria is a public health problem.

Reactive oxygen species production by quercetin causes the death of Leishmania amazonensis intracellular amastigotes.

The present study reports the mechanism of the antileishmanial activity of quercetin against the intracellular amastigote form of Leishmania amazonensis. Treatment with 1 reduced the infection index in L. amazonensis-infected macrophages in a dose-dependent manner, with an IC₅₀ value of 3.4 μM and a selectivity index of 16.8, and additionally increased ROS generation also in a dose-dependent manner. Quercetin has been described as a pro-oxidant that induces the production of reactive oxygen species, which can cause cell death. Taken together, these results suggest that ROS production plays a role in the mechanism of action of 1 in the control of intracellular amastigotes of L. amazonensis.

Mitochondrial damage contribute to epigallocatechin-3-gallate induced death in Leishmania amazonensis.

Epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, has been reported to have antiproliferative effects on Trypanosoma cruzi however, the mechanism of protozoan action of EGCG has not been studied. In the present study, we demonstrate the mechanism for the antileishmanial activity of EGCG against Leishmania amazonensis promastigotes. Incubation with EGCG significantly inhibited L. amazonensis promastigote proliferation in a time- and dose-dependent manner. The IC(50) for EGCG at 120 h was 0.063 mM. Ultrastructural alterations of the mitochondria were observed in promastigote treated with EGCG, being the organelle injury reinforced by the decrease in rhodamine 123 fluorescence. The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. These data suggest mitochondrial collapse as a part of the EGCG mechanism of action and demonstrate the leishmanicidal effect of EGCG.

Effectiveness of the local or oral delivery of the novel naphthopterocarpanquinone LQB-118 against cutaneous leishmaniasis

OBJECTIVES This paper describes the antileishmanial properties of LQB-118, a new compound designed by molecular hybridization, orally active in Leishmania amazonensis-infected BALB/c mice. METHODS In vitro antileishmanial activity was determined in L. amazonensis-infected macrophages. For in vivo studies, LQB-118 was administered intralesionally (15 μg/kg/day, five times a week), intraperitoneally (4.5 mg/kg/day, five times a week) or orally (4.5 mg/kg/day, five times a week) to L. amazonensis-infected BALB/c mice throughout experiments lasting 85 or 105 days. At the end of the experiments, serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine were measured as toxicological parameters. RESULTS LQB-118 was active against intracellular amastigotes of L. amazonensis [50% inhibitory concentration (IC(50)) 1.4 μM] and significantly less so against macrophages (IC(50) 18.5 μM). LQB-118 administered intralesionally, intraperitoneally or orally was found to control both lesion and parasite growth in L. amazonensis-infected BALB/c mice, without altering serological markers of toxicity. CONCLUSIONS These results demonstrate that the molecular hybridization of a naphthoquinone core to pterocarpan yielded a novel antileishmanial compound that was locally and orally active in an experimental cutaneous leishmaniasis model.

Synthesis and antileishmanial evaluation of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazole derivatives

A series of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (4a-g) and 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (5a-g) were synthesized and evaluated in vitro against three Leishmania species: L. amazonensis, L. braziliensis and L. infantum (L. chagasi syn.). The cytotoxicity was assessed. Among the derivatives examined, six compounds emerged as the most active on promastigotes forms of L. amazonensis with IC(50) values ranging from 15 to 60 μM. The reference drug pentamidine presented IC(50)=10 μM. However, these new compounds were less cytotoxic than pentamidine. Based on these results, the more promising derivative 5d was tested further in vivo. This compound showed inhibition of the progression of cutaneous lesions in CBA mice infected with L. amazonensis relative to an untreated control.

In vitro and in vivo effects of (-)-epigallocatechin 3-O-gallate on Leishmania amazonensis.

(-)-Epigallocatechin 3-O-gallate (1), the most abundant flavanol in green tea, has been reported to have antiproliferative effects on Trypanosoma cruzi. The present study reports the effects in vitro and in vivo of 1 on Leishmania amazonensis. L. amazonensis-infected macrophages treated with 1 exhibited a significant reduction of the infection index in a dose-dependent manner, with an IC50 value of 1.6 μM. Oral administration of 1 on L. amazonensis-infected BALB/c mice (30 mg/kg/day) resulted in a decrease in the lesion size and parasite burden, without altering serological markers of toxicity. These data demonstrate the in vitro and in vivo leishmanicidal effects of compound 1.

The pharmacological inhibition of sterol biosynthesis in Leishmania is counteracted by enhancement of LDL endocytosis

Leishmania parasites, despite being able to synthesize their own sterols, acquire and accumulate significant amounts of cholesterol through low density lipoprotein (LDL) particle endocytosis. The role of this system in Leishmania amazonensis promastigotes under pharmacological pressure by sterol biosynthesis inhibitors (SBIs) was investigated. First, thin layer chromatography demonstrated that L. amazonensis promastigotes, in response to ergosterol biosynthesis inhibition by treatment with 4.0 and 6.0 μM ketoconazole or miconazole, accumulate up to two times more cholesterol than controls. The treatment of promastigotes with ketoconazole and simvastatin, two SBIs with non-related mechanisms of action, showed that both drugs induce increases in (125)I-LDL endocytosis in a dose-dependent manner, indicating that the accumulation of exogenous cholesterol is due to the enhancement of LDL uptake. Finally, it was demonstrated that L. amazonensis promastigotes were rendered more susceptible to treatment with SBIs (ketoconazole, miconazole, simvastatin and terbinafine) in the absence of exogenous cholesterol sources, with a reduction of the IC50s of about 50% in three of the four tested drugs. These results show that the exogenous cholesterol uptake system in L. amazonensis plays a role as a compensatory mechanism in response to the presence of SBIs, suggesting that it may be a potential pharmacological target.

In vitro sodium nitroprusside‐mediated toxicity towards Leishmania amazonensis promastigotes and axenic amastigotes

Leishmania parasites survive despite exposure to the toxic nitrosative oxidants during phagocytosis by the host cell. In this work, the authors investigated comparatively the resistance of Leishmania amazonensis promastigotes and axenic amastigotes to a relatively strong nitrosating agent that acts as a nitric oxide (NO) donor, sodium nitroprusside (SNP). Results demonstrate that SNP is able to decrease, in vitro, the number of L. amazonensis promastigotes and axenic amastigotes in a dose‐dependent maner. Promastigotes, cultured in the presence of 0.25, 0.5, and 1 mmol L−1 SNP for 24 h showed about 75% growth inhibition, and 97–100% when the cultures were treated with >2 mmol L−1 SNP. In contrast, when axenic amastigotes were growing in the presence of 0.25–8 mM SNP added to the culture medium, 50% was the maximum of growth inhibition observed. Treated promastigotes presented reduced motility and became round in shape further confirming the leishmanicidal activity of SNP. On the other hand, axenic amastigotes, besides being much more resistant to SNP‐mediated cytotoxicity, did not show marked morphological alteration when incubated for 24 h, until 8 mM concentrations of this nitrosating agent were used. The cytotoxicity toward L. amazonensis was attenuated by reduced glutathione (GSH), supporting the view that SNP‐mediated toxicity triggered multiple oxidative mechanisms, including oxidation of thiols groups and metal‐independent oxidation of biomolecules to free radical intermediates. Copyright