Mitochondrial Dysfunction Induced by N-Butyl-1-(4-Dimethylamino)Phenyl-1,2,3,4-Tetrahydro-β-Carboline-3-Carboxamide Is Required for Cell Death of Trypanosoma cruzi

Background Chagas’ disease is caused by the protozoan Trypanosoma cruzi and affects thousands of people worldwide. The available treatments are unsatisfactory, and new drugs must be developed. Our group recently reported the trypanocidal activity of the synthetic compound N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxamide (C4), but the mechanism of action of this compound was unclear. Methodology/Principal Findings We investigated the mechanism of action of C4 against epimastigote and trypomastigote forms of T. cruzi. The results showed alterations in mitochondrial membrane potential, alterations in cell membrane integrity, an increase in the formation of reactive oxygen species, phosphatidylserine exposure, a reduction of cell volume, DNA fragmentation, and the formation of lipid inclusions. Conclusion/Significance These finding suggest that mitochondria are a target of C4, the dysfunction of which can lead to different pathways of cell death.


Introduction
Chagas' disease is a tropical infection caused by Trypanosoma cruzi. Approximately 7-8 million people worldwide are infected by this protozoan, mostly in Latin America. Up to 30% of chronically infected individuals develop cardiac complications [1]. It is found endemically in 21 Latin American countries, and 28 million people are at risk of acquiring this infection around the world [2].
The available treatment for Chagas' disease is based on only two drugs, nifurtimox and benznidazole, which were discovered approximately 40 years ago. Both drugs are only partially effective and have many side effects [3,4]. The search for new drugs must be intensified. Different research groups are investigating the effectiveness of possible trypanocidal agents [5]. Our group demonstrated the in vitro and in vivo effects on T. cruzi of some β-carboline compounds, especially N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxamide (C4) (Fig 1) [6,7]. This compound was effective against the three evolutive forms of T. cruzi. Furthermore, transmission electron microscopy indicated that the mitochondrion is the major organelle affected by this compound in trypanosomatids, such as T. cruzi and Leishmania amazonensis [6,8]. This compound has also been shown to have low toxicity in mammalian cells in vitro and other animal models [6,7].
The present study evaluated biochemical alterations in epimastigote and trypomastigote forms of T. cruzi treated with C4. Flow cytometry, fluorimetry, and fluorescence microscopy were used to investigate cellular and subcellular structures and identify organelles that are affected by C4 treatment. We found that mitochondrial damage may be a possible target for C4 in these parasites, thus providing a better understanding of the mechanism of action of this compound. Based on our results, we suggest that mitochondrial dysfunction induced by C4 can lead to different pathways of cell death in T. cruzi.

Substance preparation
C4 was prepared in DMSO. All of the groups, including the controls were tested at final concentrations of less than 1% DMSO, a concentration that was found not to affect the parasite.

Parasites
The experiments were performed with the Y strain of T. cruzi. Epimastigote forms were grown in Tryptose Liver Infusion (LIT) supplemented with 10% FBS at 28°C for 96 h. Trypomastigote forms were obtained from the supernatant of an infected LLCMK 2 cells monolayer (epithelial cell of monkey kidney; Macaca mulatta) in DMEM supplemented with 2 mM L-glutamine, 10% FBS, 50 units/mL penicillin, and 0.05 mg/mL streptomycin and buffered with sodium bicarbonate in a 5% CO 2 air mixture at 37°C. Sub-confluent cultures of LLCMK 2 cells were infected with 1 × 10 6 trypomastigotes/mL. Extracellular parasites were removed after 24 h. The cells were washed, and these cultures were maintained in DMEM that contained 10% FBS until trypomastigotes emerged from the infected cells.

Mitochondrial membrane potential
Epimastigotes (5 × 10 6 cells/mL treated with 18.0 and 77.0 μM of C4) and trypomastigotes (1 × 10 7 cells/mL treated with 45.0 and 230.0 μM of C4) of T. cruzi were incubated at 28°C and 37°C, respectively, for 3 h. Afterward, the parasites were washed and incubated with 5 μg/mL Rh123 for 15 min to verify mitochondrial membrane potential (ΔCm). CCCP (100.0 μM) was used as a positive control. The data acquisition and analysis were performed using a FACSCalibur flow cytometer (Becton-Dickinson, Rutherford, NJ, USA) equipped with CellQuest software (Joseph Trotter, The Scripps Research Institute, La Jolla, CA, USA). A total of 10,000 events were acquired in the region that was previously established as the one that corresponded to the parasites.
KCl, 120 mM NaCl, 0.7 mM Na 2 HPO 4 , and 1.5 mM NaH 2 PO 4 (pH 7.3). The cells were loaded with 5 μM MitoSOX reagent and incubated for 10 min at room temperature while protected from light. After incubation with MitoSOX reagent, the parasites were washed twice with KH buffer and untreated or treated with 18.0 and 77.0 μM of C4 (for epimastigotes) and 45.0 and 230.0 μM of C4 (for trypomastigotes). Antimycin A (10 μM), which is known to induce superoxide anion (O 2 •− ) production by mitochondria, was used as a positive control. MitoSOX detection was performed using black 96-well plates for 3 h. Fluorescence was measured in a fluorescence microplate reader (Victor X3, PerkinElmer) at an excitation wavelength of 510 nm and emission wavelength of 580 nm [9].

Evaluation of Nile red accumulation
Epimastigotes (1 × 10 6 cells/mL treated with 18.0 and 77.0 μM of C4) and trypomastigotes (1 × 10 7 cells/mL treated with 45.0 and 230.0 μM of C4) of T. cruzi were incubated at 28°C and 37°C, respectively, for 24 h. After treatment, the parasites were washed twice in PBS, pH 7.4, and incubated with 10 μg/mL of Nile red in the dark for 30 min. Fluorescence was measured in a fluorescence microplate reader (Victor X3, PerkinElmer) and analyzed using an Olympus BX51 fluorescence microscope at an excitation wavelength of 485 nm and emission wavelength of 535 nm. The images were captured using an Olympus UC30 camera.

Exposure of phosphatidylserine
Phosphatidylserine exposure was detected using annexin-V FITC, a calcium-dependent phospholipid binding protein. Epimastigotes (5 × 10 6 cells/mL treated with 18.0 and 77.0 μM of C4) and trypomastigotes (1 × 10 7 cells/mL treated with 45.0 and 230.0 μM of C4) of T. cruzi were incubated at 28°C and 37°C, respectively, for 3 h. Afterward, the cells were washed and resuspended in 100 μL of binding buffer (140 mM NaCl, 5 mM CaCl 2 , and 10 mM HEPES-Na, pH 7.4), followed by the addition of 5 μL annexin-V FITC for 15 min at room temperature. Binding buffer (400 μL) and 0.2 μg/mL PI were then added. Data acquisition and analysis were performed using a FACSCalibur flow cytometer equipped with CellQuest software. A total of 10,000 events were acquired in the region that was previously established as the one that corresponded to the parasites. The following analyzes were performed: cells apoptotic (annexin Vpositive-FL1, but PI-negative-FL2), late apoptotic cells (annexin V-positive-FL1, but PIpositive-FL2) and cells in necrosis (annexin V-negative-FL1, but PI-positive-FL2) [10].
37°C, respectively, for 3 h. Afterward, the protozoa were collected by centrifugation, washed twice in PBS, and resuspended in PBS. Data acquisition and analysis were performed using a FACSCalibur flow cytometer equipped with CellQuest software. A total of 10,000 events were acquired in the region that was previously established as the one that corresponded to the parasites. Histograms and analyses were performed using CellQuest software. Forward light scatter (FSC-H) was considered to represent cell volume.

Evaluation of DNA fragmentation
DNA double-strand ruptures were analyzed in situ using a TUNEL kit. Epimastigotes (1 × 10 6 cells/mL) were treated with 18.0 and 77.0 μM of C4 for 24 h at 28°C, after the cells were subjected to the TUNEL assay according to the manufacturer's instructions. Actinomycin D (10.0 μg/mL) was used as a positive control. Fluorescence was observed in an Olympus BX51 fluorescence microscope, and pictures were captured with an Olympus UC30 camera.

Statistical analysis
The data that are shown in the graphs are expressed as mean ± standard error (SE) of at least three independent experiments. The data were analyzed using two-way and one-way analysis of variance (ANOVA), with significant differences among means identified using the Bonferroni and Tukey post hoc tests. Values of p 0.05 were considered statistically significant. The statistical analysis was performed using GraphPad software.

C4 induces mitochondrial depolarization
Based on previous studies that reported the effect of C4 on T. cruzi mitochondria [6], we evaluated ΔCm in C4-treated cells by flow cytometry. The histograms showed a noticeably pronounced loss of ΔCm in both the epimastigote and trypomastigote forms of T. cruzi at the highest concentrations assayed after 3 h of treatment, with > 60.0% reductions of ΔCm compared with the control group (Fig 2A). The positive control CCCP decreased ΔCm by 50.7% and 76.3% in epimastigotes and trypomastigotes, respectively (data not shown). C4, this increase was 84.0% and 230.0%, respectively, with 3 h of incubation. The positive control (AA) also increased mitochondrial O 2 •− production (data not shown).

C4 increases total reactive oxygen species
In addition to mitochondrial O 2 •− production, we evaluated the production of reactive oxygen species (ROS) in C4-treated parasites. Fig 2C shows that C4 significantly increased total ROS production at both forms of T. cruzi after 24 h of treatment compared with the control group.
In epimastigotes that were treated with 18.0 and 77.0 μM of C4, the increase in total ROS was 60.0% and 68.0%, respectively. In trypomastigotes that were treated with 45.0 and 230.0 μM of C4, the increase was 32.0% and 92.0%, respectively. The positive control (H 2 O 2 ) also increased total ROS production (data not shown).

C4 induces lipid body formation
Epimastigotes and trypomastigotes of T. cruzi that were treated for 24 h with C4 exhibited the presence of many lipid bodies marked with Nile red. Two assays showed this alteration: (i) fluorescence microscopy revealed the presence of lipid bodies, and (ii) the fluorimetric assay quantified this accumulation. These assays showed a concentration-dependent increase in the number of lipid bodies (Fig 2D), with an increase > 50% for epimastigotes and trypomastigotes at both concentrations tested.

C4 induces phosphatidylserine exposure
Increases in ROS can lead to apoptosis-like cell death. Apoptosis is characterized by biochemical alterations, including phosphatidylserine exposure [11,12]. We evaluated whether C4 induces phosphatidylserine exposure. As shown in Fig 3A, epimastigote and trypomastigote forms that were treated with C4 exhibited an increase in annexin-V fluorescence intensity after 3 h of treatment compared with the untreated parasites, indicating phosphatidylserine exposure. The histograms showed a > 30.0% increase in the intensity of annexin-V fluorescence at both concentrations tested for trypomastigote forms (Fig 3: e and f). For epimastigote forms, at the higher concentration, annexin-V fluorescence was observed in approximately 40.0% of the parasites (Fig 3: b and c).

C4 decreases cell volume
The present results indicate that C4 induced phosphatidylserine exposure, and we explored the action of this compound on the apoptosis cell death pathway. We performed additional experiments to evaluate cell shrinkage, a hallmark of apoptotic death [12,13]. As shown in Fig 3B, a decrease in cell volume was observed in trypomastigotes at both concentrations of C4 tested after 3 h, with reductions of approximately 90.0% (Fig 3B: b). For epimastigotes, at the higher concentration, we observed a decrease in cell volume in approximately 20.0% of the parasites (Fig 3B: a).

C4 induces DNA fragmentation
Continuing the same line of reasoning, we then evaluated possible cell death by apoptosis, reflected by DNA fragmentation, using the TUNEL assay. Fig 3C illustrates

C4 induces alterations in cell membrane integrity
Previous work also demonstrated the effect of C4 on the cell membrane [6]. We further evaluated the effect of C4 on membrane integrity in epimastigote and trypomastigote forms of T. cruzi. C4 affected membrane integrity in both forms of T. cruzi after 3 h of treatment compared with untreated parasites. The histograms showed an increase in the intensity of PI fluorescence at both concentrations tested (49.34% and 73.64% PI-positive parasites in Fig 3D: e and f), mainly for trypomastigotes, indicating alterations in cell membrane integrity. In epimastigotes at the higher C4 concentrations, approximately 27% of the parasites were PI-positive (Fig 3D:  c). The positive control (digitonin) increased fluorescence by 41.02% and 93.82% in epimastigotes and trypomastigotes, respectively (data not shown).
Discussion β-carbolines have presented numerous biological properties, such as antimicrobial [14], antitumoral [15], antiviral [16] and antiparasitic [6][7][8] effects. In our recent studies, we demonstrated the in vitro and in vivo activity of C4 against T. cruzi [6,7]. Additionally, C4 induced low cytotoxicity, with a selective index higher to the parasites than for mammalian cells [6]. In the present study, we focused on elucidating the mechanism of action of C4 in the cell death of epimastigotes and trypomastigotes of T. cruzi. Our previous study reported ultrastructural alterations, especially in the mitochondria, in parasites that were treated with C4 [6]. The present results confirmed that mitochondria are a target of C4, reflected by the depolarization of ΔCm and increase in the production of mitochondrial ROS and formation of lipid droplets in parasites treated with C4. Changes in ΔCm are associated with opening of the permeability transition pore (PTP) in the mitochondrial membrane [17,18]. Thus, C4 might induce the opening of PTP in the mitochondrial membrane, leading to activation of the apoptotic pathway [19]. This programmed cell death is commonly characterized by different morphological characteristics, such as exposure of phosphatidylserine residues on the external leaflet of the cell membrane, decrease of cell volume and DNA fragmentation [20]. In addition, analysis of red Nile showed an increase of lipid bodies in the cytoplasm, which may indicate that the C4 changes the content of phospholipids and sterols of T. cruzi, and is strongly related to mitochondrial dysfunction [21]. Previous studies with an alkyl phosphocholine-dinitroaniline hybrid molecule [22] and antifungal azoles [23] showed similar results.
Besides acting in the mitochondrial membrane, the C4 also acts on the plasmatic membrane of the parasites. This can be seen in the results obtained starting the labeling of parasites with PI, and also by increase in population of PI-positive parasites/annexin-V negative in the upper left quadrant in relation to the control. Morphological alterations in the plasma membrane are features of cell death by necrosis [20]. However, the increase of cellular ROS production might induce different mechanisms of cell death including both apoptosis and necrosis [24] which can occur in the same population of cells. Similar results have been described for other compounds (e.g., eupomatenoid-5 in T. cruzi parasites [25] and 4-nitrobenzaldehyde thiosemicarbazone, derived from S-limonene in L. amazonensis [26].
Our results demonstrated that C4 can induce several changes in the parasites that lead to cell death either by apoptosis or necrosis [12,[27][28][29]. Our results suggest that mitochondria are one of the target organelles that may be involved in the increase in ROS production through the electron transport chain, which affects cellular structures and induces parasite death. Altogether, the present results suggest that new chemotherapeutic agents can be developed for the treatment of Chagas' disease.