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.


Introduction
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 [7][8][9].
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) [12][13][14][15].
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.

Reagents
H 2 DCFDA (29,79-dichlorodihydroflurescein diacetate) was obtained from Invitrogen Molecular Probes (Leiden, The Netherlands). Others reagents were purchased from Merck (São 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.

Parasites
Promastigotes of L. amazonensis (MHOM/BR/LTB0016 strain) were grown at 26uC in Schneider's 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.

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 mM-96 mM) for 24 to 96 at 26uC. The cell density was estimated in a Neubauer chamber and the growth curve was initiated with 1.0610 6 cells/ml. The cell proliferation was verified by the counting of the cell number in a Neubauer chamber. 4.2. JC-1. The cationic JC-1 was used as a probe to determine the mitochondrial membrane potential (DY m ) as described [9]. Promastigotes (1610 6 cells/ml) were cultured for 48 hours in the absence or presence of 24 mM or 96 mM quercetin. Cells were harvested, re-suspended in Hank's Balanced Salt Solution (HBSS) and the cell number was counted in a Neubauer chamber. Promastigotes (2610 6 cells/ml) were incubated with JC-1 (10 mg/ ml) for 10 minutes at 37uC. After washing twice with HBSS, fluorescence was measured spectrofluorometrically at both 530 nm and 590 nm using an excitation wavelength of 480 nm. The ratio of values obtained at 590 nm and 530 nm was plotted as the relative DY m .

Alamar Blue assay
Promastigotes of L. amazonensis (1610 6 cells/ml) were treated for 48 hours with or without different concentration of quercetin (3 mM-96 mM). Cells were harvested, re-suspended in Hank's Balanced Salt Solution (HBSS) and the cell number was counted in a Neubauer chamber. Promastigotes (5610 6 cells/ml) were incubated with Alamar Blue (10% v/v) for 6 hours at 26uC. The absorbance was measured at 570 nm with a spectrophotometer. L. amazonensis cells lysed by addition of 0.1% Triton X-100 were used as positive control.

Measurement of reactive oxygen species (ROS) levels
Intracellular ROS levels were measured in treated and untreated cells. Promastigotes (1610 6 cells/ml) were cultured for 48 hours in the absence or presence of 24 mM or 96 mM quercetin. Promastigotes were then harvested, re-suspended in Phosphate Buffered Saline (PBS) and the cell number was counted in a Neubauer chamber. Promastigotes (2610 6 cells/ml) were incubated with H 2 DCFDA (20 mM) for 20 minutes at 37uC. Fluorescence was measured spectrofluorometrically at 530 nm using an excitation wavelength of 507 nm. For all measurements, basal fluorescence was subtracted. Positive control was obtained by addition of 20 units/ml glucose oxidase+60 mM glucose for 20 minutes.

Statistical analysis
All experiments were performed in triplicate. The data were analyzed statistically using Student's t-test and two-way analysis of variance (ANOVA). In all cases, the level of significance was set at p,0.05 or p,0.01 as indicated. The data are expressed as means 6 standard errors.

Results and Discussion
Quercetin, an abundant naturally occurring flavonoid, 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 [11,18]. To determine the effect of quercetin on the growth of L. amazonensis, we incubated the parasites with different concentrations of quercetin (3 mM-96 mM) for 24 to 96 hours. Incubation with quercetin inhibited L. amazonensis promastigote growth in a time-and dose-dependent manner (Fig. 1). This inhibitory effect was equal to 66% after 48 hours with 96 mM. The IC 50 for quercetin at 48 hours was 31.4 mM. Total growth arrest was observed after incubating L. amazonensis promastigotes with 96 mM quercetin at 96 hours. This result demonstrates the antileishmanial activity of quercetin against L. amazonensis.
Mitochondria are essential cellular organelles that play a central role in energy metabolism and are critical for the survival of any cell. During oxidative phosphorylation, electrons are moved thorough the mitochondrial respiratory chain, and a proton gradient is established across the inner mitochondrial membrane as the energy source for ATP production. The maintenance of mitochondrial membrane potential (DY m ) is vital for this metabolic process as well as for cell survival [3,19]. Studies have shown that variations in DY m induced by drugs are associated with cell survival in Trypanosoma cruzi [9,16], Leishmania donovani [19] and L. amazonensis [20].
To determine if quercetin induces an alteration in the DY m of L. amazonensis promastigotes, we measured the DY m by using the fluorescent probe rhodamine 123, which accumulates within energized mitochondria, in conjunction with flow cytometry analysis. Figure 2    treatment with a mitochondrial uncoupling agent, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at 20 mM [21,22].
To confirm that quercetin induces DY m collapse, we used JC-1, which is a cationic mitochondrial vital dye. This dye is lipophilic and becomes concentrated in the mitochondria in proportion to the membrane potential such that more dye accumulates in mitochondria with greater DY m . Spectrofluorometric data presented in Fig. 3 showed a marked decrease in relative fluorescence intensity (DY m values) in a dose-dependent manner, indicating depolarization of the membrane potential in cells following treatment with 24 mM or 96 mM of quercetin, with DY m reductions of 42.61% and 77.62%, respectively. Furthermore, cells incubated with 20 mM FCCP had a similar decrease in the relative fluorescence intensity values (79.82% of reduction). Taken together, these data suggest that quercetin inhibit L. amazonensis growth by affecting parasite mitochondrial function.
The reduced fluorescence of rhodamine 123 and JC-1 was not due to measuring membrane potential in the remaining live cells, because the percentage of viable cells was similar in treated and untreated parasites (Fig. 4).
In mammalian cells, increased cellular ROS production has been suggested to be responsible for the depolarization of DY m and subsequent cell death [23,24]. To investigate if the mitochondrial dysfunction observed in L. amazonensis promastigote treated with quercetin is promoted by ROS production, we measured ROS levels using the cell-permeable dye H 2 DCFDA [25][26][27]. The levels of ROS in quercetin-treated cells were 2.4fold and 4.4-fold higher for 24 mM and 96 mM, respectively, compared to the level of ROS in control cells throughout the experiment (Fig. 5). Since glucose oxidase catalyses the oxidation of D-glucose generating H 2 O 2 , it was employed as a positive control. Glucose/glucose oxidase led to an increase in ROS levels compared to control (2.6-folds, compared to the level of ROS in the control).
Reduced glutathione (GSH) is an important molecule for protecting kinetoplastids from ROS or toxic compounds, and intracellular GSH levels can be increased by the antioxidant compound N-Acetyl-L-cysteine (NAC) [28,29]. In L. amazonensis, glutathione is a component of trypanothione, a major antioxidant of this parasite [30]. Thus, we tested if pre-incubation of L. amazonensis promastigotes with GSH or NAC (300 mM) could prevent the inhibitory effect of quercetin and found that these intracellular antioxidants protected L. amazonensis from inhibition by quercetin (Fig. 6A) and reduced the levels of ROS in quercetintreated cells (Fig. 6B). Alternatively, oxidized glutathione (GSSG) did not reduce the levels of ROS and did not protect the growth inhibition promoted by quercetin. Taken together, these results indicate that inhibition of growth and disruption of DY m promoted by quercetin in L. amazonensis is mediated by ROS production which might alter the cellular redox status.
Mitochondria are responsible for respiration and oxidative phosphorylation in eukaryotes, including trypanosomes, and they provide ATP through respiratory-coupled oxidative phosphorylation [31,32]. The mitochondria of Leishmania exhibit a unique structure and are functionally distinct from mammalian mitochondria, making this organelle an exceptional chemotherapeutical target. Here, we demonstrate for the first time that quercetin exerts its antileishmanial effect on L. amazonensis promastigotes by generating ROS and affecting parasite mitochondrial function.
To elucidate the mechanism of cell death induced by quercetin, we chose to measure the mitochondrial membrane potential because previous studies have shown that the single mitochondrion of the kinetoplastid parasite is a good indicator of cellular dysfunction [16,19,33]. L. amazonensis promastigotes were incubated with different concentrations of quercetin for 48 hours, the   shortest time that demonstrated inhibition of cellular growth (66% of inhibition). The concentrations of quercetin used in these experiments were based on the IC 50 for 48 hours (31.4 mM). A decrease in rhodamine 123 fluorescence intensity and the JC-1 fluorescence ratio when L. amazonensis was treated with different concentrations of quercetin suggests increased proton permeability across the inner mitochondrial membrane with increasing quercetin. This could decrease ATP synthesis and result in parasite death. Collapse of DY m was observed in Leishmania spp. [20,[25][26][27] and T. cruzi [16,17,34] after treatment with different drugs.
It has been demonstrated that quercetin inhibited the growth of Leishmania donovani promastigotes, as well as, amastigote in infected macrophages in vitro, induced topoisomerase II mediated linearization of kDNA, causing cell cycle arrest, leading to apoptosis [35]. However, quercetin has been described as a prooxidant, generating ROS which are responsible for cell death in some cancer cells [12,36]. DY m loss can be brought about by ROS added directly in vitro or induced by chemical agents [37,38].
In this work, quercetin increased ROS generation in a dosedependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. Therefore, we suggest that quercetin-induced cell death is attributable to ROS generation could lead to DY m collapse. These results are consistent with findings in L. donovani that the presence of ROS caused mitochondrial depolarization [39].
In conclusion, our study demonstrates, for the first time, ROS production due to quercetin treatment of L. amazonensis and suggests the mechanism of action for quercetin to potently inhibit cell growth in L. amazonensis.