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

Flow cytometry analysis of <i>Leishmania amazonensis</i> treated with quercetin.

<p><i>Leishmania amazonensis</i> was cultivated in Schneider's <i>Drosophila</i> medium at 26°C as for 48 h, in the absence or in the presence of 24 µM and 96 µM quercetin. Promastigotes were subsequently labeled with rhodamine 123 (10 µg/ml). Data acquisition and analysis were performed using a FACSCalibur flow cytometer (equipped with the Cell Quest software). A total of 10,000 events were acquired in the regions previously established as those corresponding to each form of <i>L. amazonensis</i>. Positive control was obtained by addition of FCCP (20 µM) for 20 minutes. In the control (absence of quercetin) the same volume of vehicle (DMSO 0.2%) was added to the growth medium.</p

Effect of N-Acetyl-L-cysteine, Reduced Glutathione and Oxidized Glutathione on quercetin-induced cell death (A) and ROS formation (B).

<p><i>L. amazonensis</i> was cultivated in Schneider's <i>Drosophila</i> medium at 26°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014666#s2" target="_blank">Materials and Methods</a> for 48 h in the presence of N-Acetyl-L-cysteine, reduced glutathione and oxidized glutathione in the absence or in the presence of quercetin. N-Acetyl-L-cysteine, reduced glutathione and oxidized glutathione were solubilized in PBS and quercetin was solubilized in DMSO. N-Acetyl-L-cysteine, reduced glutathione and oxidized glutathione were added to the culture to a final concentration of 300 µM, and quercetin was added to the culture to a final concentration of 96 µM. Values shown are the mean±standard error of three different experiments. In the control (absence of quercetin), the same volume of vehicle (DMSO 0.2%) was added to the growth medium. Generation of ROS was measured using fluorescent dye H₂DCFDA as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014666#s2" target="_blank">Materials and Methods</a>. Data are expressed as fold increase in ROS production relative to control. Values shown are the mean±standard error of three different experiments. Positive control for ROS generation was obtained by addition of 20 units/ml glucose oxidase+60 mM glucose for 20 minutes. CTRL - control; NAC - N-Acetyl-L-cysteine; GSH - Reduced Glutathione; GSSG - Oxidized Glutathione, Q−Quercetin and G/GO−Glucose+Glucose oxidase. ** indicates significant difference relative to the control group (<i>p</i><0.01); * indicates significant difference relative to the control group (<i>p</i><0.05).</p

Quercetin induced ROS formation.

<p><i>Leishmania amazonensis</i> was cultivated in Schneider's <i>Drosophila</i> medium at 26°C as for 48 h in the absence or presence of 24 µM or 96 µM quercetin. Generation of ROS was measured using fluorescent dye H₂DCFDA as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014666#s2" target="_blank">Materials and Methods</a>. Data are expressed as fold increase in ROS production relative to control. Values shown are the mean±standard error of three different experiments. Positive control was obtained by addition of 20 units/ml glucose oxidase+60 mM glucose for 20 minutes. * indicates significant difference relative to the control group (<i>p</i><0.05); ** indicates significant difference relative to the control group (<i>p</i><0.01); # indicates significant difference relative to the 24 µM quercetin treated group (<i>p</i><0.05). G/GO−Glucose+Glucose oxidase.</p