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

Almeida-Amaral EE (2011) Reactive Oxygen Species Production and Mitochondrial Dysfunction Contribute to Quercetin Induced Death in Leishmania amazonensis. PLoS ONE 6(2): e14666. doi:10.1371/journal.pone.0014666 Reactive Oxygen Species Production and Mitochondrial Dysfunction Contribute to Quercetin Induced Death in Leishmania amazonensis Fernanda Fonseca-Silva 0 Job D. F. Inacio 0 Marilene M. Canto-Cavalheiro 0 Elmo Eduardo Almeida-Amaral 0 Photini Sinnis, New York University, United States of America 0 Laborato rio de Bioqu mica de Tripanosomatideos, Instituto Oswaldo Cruz (IOC), Fundac a o Oswaldo Cruz (FIOCRUZ) , Rio de Janeiro, Rio de Janeiro , Brazil 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 mM. 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. - Recently, the effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described [1,2]. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Leishmania has a single large mitochondrion which is distributed in branches under the subpelicular microtubes and a specialized region rich in DNA called the kinetoplast [3]. Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide [4]. Treatment of leishmaniasis is based on pentavalent antimonials, drugs developed more than 50 years ago that are toxic and prone to drug resistance [5]. Several drug screens of natural compounds have been successful in discovering novel compounds for treating some parasitic diseases [6]. Extracts obtained from plants, as well as pure compounds including some kinds of flavonoids, have been reported to possess significant antiprotozoan activities [79]. Quercetin is the most common polyphenolic flavonoids present in plants such as onions, ginko biloba and tea and can be absorbed by humans. Quercetin has a wide range of reported biological effects including antioxidant, anti-hypertension, anti-inflammatory, antimicrobial and antiprotozoan activities [10,11], although, the precise molecular mechanism of action of quercetin has not yet been demonstrated. Quercetin can induce the production of superoxide anion, hydrogen peroxide, and other reactive oxygen species (ROS) [1215]. ROS are generated in cells infected by pathogens to combat infection. ROS can also be generated in response to some drugs, and the same principle works for certain antiprotozoan drugs in killing parasites in an infected cell. This property of a drug, to generate ROS to cause the destruction of cellular macromolecular components, is important because this action can be modulated to derive maximal effect. The aim of the present work is to study the antileishmanial activity of quercetin and describe the mechanism of quercetin against promastigotes of Leishmania amazonensis. Our results demonstrate that the effect of the quercetin is associated with ROS production leading to mitochondrial dysfunction, ultimately causing parasite death. Materials and Methods 1. Reagents H2DCFDA (29,79-dichlorodihydroflurescein diacetate) was obtained from Invitrogen Molecular Probes (Leiden, The Netherlands). Others reagents were purchased from Merck (Sao Paulo, Brazil) or Sigma-Aldrich (St Louis, MO). Deionized distilled water was obtained using a MilliQ system of resins (Millipore Corp., Bedford, MA) and was used in the preparation of all solutions. 2. Parasites Promastigotes of L. amazonensis (MHOM/BR/LTB0016 strain) were grown at 26uC in Schneiders Drosophila medium pH 7.2 supplemented with 10% (v/v) heat-inactivated fetal calf serum. The number of parasites was determined by direct counting with a Neubauer chamber. 3. Cell proliferation Promastigotes of L. amazonensis were harvested, washed twice and seed into fresh medium in the absence or in the presence of different concentration of quercetin (3 mM96 mM) for 24 to 96 at 26uC. The cell density was estimated in a Neubauer chamber and the growth curve was initiated with 1.06106 cells/ml. The cell proliferation was verified by the counting of the cell number in a Neubauer chamber. 4. Determination of mitochondrial membrane potential (DYm) 4.1. Flow cytometry studies. Promastigotes of L. amazonensis (16106 cells/ml) were treated for 48 hours with or without 24 mM or 96 mM quercetin and then incubated with 10 mg/ml rhodamine 123 for 20 minutes. Samples were kept on ice until analysis. Data acquisition and analysis were performed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, USA) equipped with the Cell Quest software (Joseph Trotter, Scripps Research Institute, La Jolla, USA). A total of 10,000 events were acquired in the region previously established as corresponding to the parasites. Alterations in the fluorescence for Rh123 were quantified using an index of variation (IV) obtained by the equation (MT2MC)/MC, where MT is the median of fluorescence for treated parasites and MC is the median of control parasites. Negative IV values correspond to depolarization of the mitochondrial membrane [16,17]. 4.2. JC-1. The cationic JC-1 was used as a probe to determine the mitoch (...truncated)