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

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

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

EGCG-induced ROS formation in <i>Leishmania</i>-infected macrophages.

<p>Non-<i>Leishmania</i>-infected macrophages and <i>Leishmania</i>-infected macrophages were incubated in the absence or presence of EGCG (12 µM) for 24 h. ROS were measured using the fluorescent dye H₂DCFDA 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>. The data are expressed as fluorescence intensity units (FIU). The values presented are the mean ± standard error of three different experiments. The positive control was treated with 20 units/ml glucose oxidase and 60 mM glucose for 30 minutes. * indicates a significant difference relative to <i>Leishmania</i>-infected macrophages (<i>p</i><0.05). MØ, non-<i>Leishmania</i>-infected macrophages; MØ+EGCG, non-<i>Leishmania</i>-infected macrophages treated with EGCG 12 µM; MØ inf, <i>Leishmania</i>-infected macrophages; MØ inf+EGCG, <i>Leishmania</i>-infected macrophages treated with EGCG 12 µM; G/GO, glucose+glucose oxidase.</p

Reduced intracellular ATP concentrations in EGCG-treated <i>L. braziliensis</i> promastigotes.

<p>Promastigotes were incubated with EGCG for 72 h. Intracellular ATP concentrations were measured using a bioluminescence assay 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>. The results are expressed as a percentage of the control. The intracellular ATP concentration of the control (184.5 nmol×10⁻⁷ cells) was set as 100%. The values presented are the mean ± standard error of three different experiments.</p

The effect of PEG-catalase on EGCG-induced cell death (A) and H₂O₂ formation (B).

<p><i>L. braziliensis</i> was cultivated in Schneider's <i>Drosophila</i> medium at 26°C for 72 h with PEG-catalase in the absence or presence of EGCG 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>. Final concentrations of 500 U/ml PEG-catalase and 500 µM EGCG were added to the culture. The values presented are the mean ± standard error of three different experiments. In the control (absence of EGCG), the same volume of vehicle (PBS) was added to the growth medium. H₂O₂ was measured with 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>. The data are expressed as the fold increase in H₂O₂ production relative to the control. The values are presented as the mean ± standard error of three different experiments. CTRL, control; PEGCAT, 500 U/ml Peg-catalase. * indicates a significant difference relative to the control group (<i>p</i><0.05); # indicates a significant difference relative to the EGCG-treated group (<i>p</i><0.05).</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

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