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 IC(50) 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

The Effect of (-)-Epigallocatechin 3-O- Gallate In Vitro and In Vivo in Leishmania braziliensis: Involvement of Reactive Oxygen Species as a Mechanism of Action

Made available in DSpace on 2015-05-04T16:34:30Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) luiza_gervazonietal_IOC_2014.pdf: 2344832 bytes, checksum: 1ac3495a8c7a441c220acc2131675009 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanossomatídeos. Rio de Janeiro, RJ, Brasil.Background: Leishmaniasis is a parasitic disease associated with extensive mortality and morbidity. The treatment for leishmaniasis is currently based on pentavalent antimonials and amphotericin B; however, these drugs result in numerous adverse side effects. Natural compounds have been used as novel treatments for parasitic diseases. In this paper, we evaluated the effect of (-)-epigallocatechin 3-O-gallate (EGCG) on Leishmania braziliensis in vitro and in vivo and described the mechanism of EGCG action against L. braziliensis promastigotes and intracellular amastigotes. Methodology/Principal Finding: In vitro activity and reactive oxygen species (ROS) measurements were determined during the promastigote and intracellular amastigote life stages. The effect of EGCG on mitochondrial membrane potential (DYm) was assayed using JC-1, and intracellular ATP concentrations were measured using a luciferin-luciferase system. The in vivo experiments were performed in infected BALB/c mice orally treated with EGCG. EGCG reduced promastigote viability and the infection index in a time- and dose-dependent manner, with IC50 values of 278.8 mM and 3.4 mM, respectively, at 72 h and a selectivity index of 149.5. In addition, EGCG induced ROS production in the promastigote and intracellular amastigote, and the effects were reversed by polyethylene glycol (PEG)-catalase. Additionally, EGCG reduced DYm, thereby decreasing intracellular ATP concentrations in promastigotes. Furthermore, EGCG treatment was also effective in vivo, demonstrating oral bioavailability and reduced parasitic loads without altering serological toxicity markers. Conclusions/Significance: In conclusion, our study demonstrates the leishmanicidal effects of EGCG against the two forms of L. braziliensis, the promastigote and amastigote. In addition, EGCG promotes ROS production as a part of its mechanism of action, resulting in decreased DYm and reduced intracellular ATP concentrations. These actions ultimately culminate in parasite death. Furthermore, our data suggest that EGCG is orally effective in the treatment of L. braziliensis-infected BALB/c mice without altering serological toxicity markers

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

Submitted by Sandra Infurna ([email protected]) on 2018-06-26T14:02:36Z No. of bitstreams: 1 jobD_inacio_etal_IOC_2013.pdf: 238824 bytes, checksum: 39f61d8a84e7b02c095b411433569c4a (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-06-26T14:09:27Z (GMT) No. of bitstreams: 1 jobD_inacio_etal_IOC_2013.pdf: 238824 bytes, checksum: 39f61d8a84e7b02c095b411433569c4a (MD5)Made available in DSpace on 2018-06-26T14:09:27Z (GMT). No. of bitstreams: 1 jobD_inacio_etal_IOC_2013.pdf: 238824 bytes, checksum: 39f61d8a84e7b02c095b411433569c4a (MD5) Previous issue date: 2013Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatideos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatideos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatideos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatideos. Rio de Janeiro, RJ. Brasil.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

Effect of Apigenin on Leishmania amazonensis Is Associated with Reactive Oxygen Species Production Followed by Mitochondrial Dysfunction

Submitted by sandra infurna ([email protected]) on 2015-10-20T10:37:42Z No. of bitstreams: 1 fernanda_silva_etal_IOC_2015.pdf: 3831947 bytes, checksum: 66f09a2ff56eb28882271dd922d1a61e (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2015-10-20T10:38:06Z (GMT) No. of bitstreams: 1 fernanda_silva_etal_IOC_2015.pdf: 3831947 bytes, checksum: 66f09a2ff56eb28882271dd922d1a61e (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2015-10-20T10:52:05Z (GMT) No. of bitstreams: 1 fernanda_silva_etal_IOC_2015.pdf: 3831947 bytes, checksum: 66f09a2ff56eb28882271dd922d1a61e (MD5)Made available in DSpace on 2015-10-20T10:53:39Z (GMT). No. of bitstreams: 1 fernanda_silva_etal_IOC_2015.pdf: 3831947 bytes, checksum: 66f09a2ff56eb28882271dd922d1a61e (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Leishmaniasis is an important neglected disease caused by protozoa of the genus Leishmania that affects more than 12 million people worldwide. Leishmaniasis treatment requires the administration of toxic and poorly tolerated drugs, and parasite resistance greatly reduces the efficacy of conventional medications. Apigenin (1), a naturally occurring plant flavone, has a wide range of reported biological effects. In this study, antileishmanial activity of 1 in vitro was investigated, and its mechanism of action against Leishmania amazonensis promastigotes was described. Treatment with 1 for 24 h resulted in concentration-dependent inhibition of cellular proliferation (IC50 = 23.7 μM) and increased reactive oxygen species (ROS) generation. Glutathione and N-acetyl-L-cysteine protected L. amazonensis from the effects of 1 and reduced ROS levels after the treatment. By contrast, oxidized glutathione did not reduce the levels of ROS caused by 1 by not preventing the proliferation inhibition. Apigenin 1 also induced an extensive swelling in parasite mitochondria, leading to an alteration of the mitochondrial membrane potential, rupture of the trans-Golgi network, and cytoplasmic vacuolization. These results demonstrate the leishmanicidal effect of 1 and suggest the involvement of ROS leading to mitochondrial collapse as part of the mechanism of action

The effect of EGCG on <i>L. braziliensis</i> promastigotes.

<p><i>L. braziliensis</i> was cultivated in Schneider's <i>Drosophila</i> medium at 26°C for 72 h in the absence or presence of EGCG (62.5–500 µM). The number of parasites was determined by direct counting using a Neubauer chamber. In the control (absence of EGCG), the same volume of PBS (solvent of EGCG) was added to the growth medium. The values are presented as the mean ± standard error of three different experiments.</p

EGCG induces H₂O₂ formation.

<p><i>Leishmania braziliensis</i> was cultivated in Schneider's <i>Drosophila</i> medium at 26°C as for 72 h in the absence or presence of EGCG (62.5–500 µM). H₂O₂ was measured using Amplex red as described in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003093#s2" target="_blank">Materials and Methods</a> (Panel A). The data are expressed as the fold increase in H₂O₂ production relative to the control. The values presented are the mean ± standard error of three different experiments. * indicates a significant difference relative to the control group (<i>p</i><0.05); ** indicates a significant difference relative to the control group (<i>p</i><0.01). Panel B: Correlation between the H₂O₂ production and inhibition of <i>L. braziliensis</i> viability by EGCG (R² = 0.975).</p

<i>In vivo</i> leishmanicidal effect of EGCG in <i>L. braziliensis</i>-infected BALB/c mice.

<p>The right ears of the mice were infected intradermally with 2×10⁶<i>L. braziliensis</i> promastigotes. Panel A: Lesion development in the animals administered oral EGCG (100 mg/kg/day; closed square) or the control group orally administered sterile PBS (vehicle of EGCG; closed circle) once a day seven times a week. Arrow represents the initiation of treatment. Inset: Lesion development in animals that were administered oral EGCG (100 mg/kg/day; closed square) and the control groups, which were orally administered sterile PBS (vehicle; closed circle) or treated with intraperitoneal injections of meglumine antimoniate (30 mg/kg/day; open triangle) once a day seven times a week. The arrow represents the initiation of treatment. Panel B: Macroscopic evaluation of lesions (arrowhead) in untreated mice (left column), EGCG-treated mice (medium column), and meglumine antimoniate-treated mice (right column) at the end of the experiment (day 32). The arrowhead represents the lesion. Panel C: Parasite burden of <i>L. braziliensis</i>-infected BALB/c mice untreated or treated with EGCG (100 mg/kg/day) or meglumine antimoniate (30 mg/kg/day). Ear parasite loads were determined via a limiting dilution assay. Panels D–F: Toxicity parameters for the kidneys and liver. At the end of experiment, the mice were euthanized, and serum samples were collected for colorimetric determination of aspartate aminotransferase (AST) (panel D), alanine aminotransferase (ALT) (panel E), and creatinine (panel F) concentrations as parameters of liver and kidney toxicity. Data are expressed as the mean ± standard error, <i>n</i> = 5 ears. [*** indicates a significant differences relative to the control group (<i>p</i><0.001)]. (CTRL, control; antimonial, meglumine antimoniate).</p

The effect of EGCG on mitochondrial membrane potential in <i>Leishmania braziliensis</i>.

<p><i>Leishmania braziliensis</i> was cultivated in Schneider's <i>Drosophila</i> medium at 26°C for 72 h in the absence or presence of 62.5–500 µM EGCG. Promastigotes were labeled with the potentiometric probe JC-1 (10 µg/ml). The positive control was treated with FCCP (20 µM) for 20 minutes. In the control (absence of EGCG), the same volume of vehicle (PBS) was added to the growth medium. Dose-dependent alterations in relative ΔΨ_m values are expressed as the ratio of the fluorescence measurements at 590 nm (for J-aggregate) versus 530 nm (for J-monomer). The data are expressed as the means ± standard errors of three different experiments. * indicates a significant difference relative to the control group (<i>p</i><0.05); ** indicates a significant difference relative to the control group (<i>p</i><0.01).</p