Allicin Induces Calcium and Mitochondrial Dysregulation Causing Necrotic Death in Leishmania

Background Allicin has shown antileishmanial activity in vitro and in vivo. However the mechanism of action underlying its antiproliferative effect against Leishmania has been virtually unexplored. In this paper, we present the results obtained in L.infantum and a mechanistic basis is proposed. Methodology/Principal Finding Exposure of the parasites to allicin led to high Ca2+ levels and mitochondrial reactive oxygen species (ROS), collapse of the mitochondrial membrane potential, reduced production of ATP and elevation of cytosolic ROS. The incubation of the promastigotes with SYTOX Green revealed that decrease of ATP was not associated with plasma membrane permeabilization. Annexin V and propidium iodide (PI) staining indicated that allicin did not induce phospholipids exposure on the plasma membrane. Moreover, DNA agarose gel electrophoresis and TUNEL analysis demonstrated that allicin did not provoke DNA fragmentation. Analysis of the cell cycle with PI staining showed that allicin induced cell cycle arrest in the G2/M phase. Conclusions/Significance We conclude that allicin induces dysregulation of calcium homeostasis and oxidative stress, uncontrolled by the antioxidant defense of the cell, which leads to mitochondrial dysfunction and a bioenergetic catastrophe leading to cell necrosis and cell cycle arrest in the premitotic phase.


Introduction
Leishmaniases are vectorial parasitic diseases of mammals, including humans, caused by Leishmania species and present in all inhabited continents. It is estimated that 12 million people are infected, with an annual incidence of 2 million cases, and between ca. 350 million [1] and 3.4 billion people [2] living in areas at risk. It is considered the second most lethal parasitic disease, after malaria, visceralizing species being responsible of 20,000 to 40,000 human deaths per year [3]. In the last years a rise in human prevalence has been found, the disease extending to previously exempt areas. Control of infections relies on chemotherapy but these drugs have several shortcomings including high price, length of treatments and side effects such as toxicity and teratogenicity [4]. Moreover, resistance to the treatment of choice (antimonials) has been reported in endemic areas (e.g. India) [5] and new molecules are needed.
Information in unicellular eukaryotes is scarce although allicin seems to inhibit the expression of silent information regulator 2 (SIR2) gene (ortholog to mammalian SIRT1) [26] thus inhibiting the hyphae formation in the fungus Candida [27]; one of the metabolites of allicin, allyl alcohol, induces oxidative stress in this fungus. Preliminary transmission electron microscopy (TEM) studies of Leishmania promastigotes exposed to allicin showed that the most altered organelle was the mitochondrion [17]. The importance of this organelle in the energetic machinery of eukaryotic cells is critical in Leishmania and other trypanosomatids since they only have a large mitochondrion (ca. 12% of cellular volume) [28] and these organisms exhibit a scarce ability to survive and multiply in anaerobic environments [29].
Results presented indicate that diallyl thiosulfinate induces in Leishmania promastigotes a rapid elevation of cytosolic Ca 2+ levels, high ROS generation, mitochondrial dysfunction with a collapse of the mitochondrial membrane potential (ΔCm). These events lead to a bioenergetic catastrophe with fall of mitochondrial ATP production and cell necrosis with no evidence of apoptotic-like markers.

Parasite culture and maintenance
The canine isolate of L.infantum (MCAN/ES/2001/UCM9) was employed in all the experimental procedures. Promastigotes were routinely cultured in 25 mL culture flasks at 27°C in RPMI 1640 modified medium (Lonza) supplemented with 10% heat-inactivated (30 min at 56°C) fetal bovine serum (TDI Laboratories, Madrid) and 100 U/mL of penicillin plus 100 μg/mL of streptomycin (BioWhittaker). Allicin was obtained as liquid Allisure from Allicin International Ltd. (Rye, East Sussex, UK) at a concentration of 5000 ppm and stored at -80°C until used.

Measurement of reactive oxygen species (ROS) generation
Intracellular ROS levels were measured using the cell permeable probe H2DCFDA (2',7'dichlorodihydrofluorescein diacetate, Molecular Probes). Experiments were carried out in triplicate following a modified protocol described by Fonseca-Silva et al. [30]. Briefly, 2 x 10 6 /mL mid-log phase promastigotes were incubated at 27°C for 3h in the absence or presence of increasing concentrations of allicin (15-120 μM). Parasites were washed in phosphate saline buffer (PBS) (Lonza), resuspended in 1 mL PBS (2 x 10 6 /mL) and incubated with 20 μM H2DCFDA for 20 min at 37°C, 5% CO 2 . Aliquots of 200 μL/well were transferred to a 96-well solid flat bottomed black microtiter plate (Costar, Corning) and fluorescence intensity was measured in a FLUOstar OPTIMA microplate reader (BMG Labtech) using excitation/ emission wavelength of 500 nm/490 nm. Antimycin A (5 μM) (Sigma) was used as a positive control of ROS generation.

Measurement of free cytosolic calcium
Cytosolic Ca 2+ in promastigotes was monitored using Fluo 4AM dye (Molecular Probes) using a modified protocol previously described [32].

Measurement of cellular ATP levels
Quantification of ATP levels was carried out using the CellTiter-Glo luminescent assay (Promega) [35]. Mid-log phase promastigotes (2 x 10 6 /mL) were incubated at 27°C in RPMI supplemented medium in the presence of 15, 30, 60, 90 and 120 μM allicin for 3 h. Untreated cultures and cultures treated with 20 mM sodium azide (Sigma) were included as controls.
After the drug exposure period, a 30 μL aliquot of the parasite suspension was transferred to 96-well solid white flat bottomed microtiter plates (Costar, Corning) and an equal volume of CellTiter-Glo was added to each well. Plates were incubated in the dark for 10 min at RT and luminescence was measured using a FLUOstar Omega microplate reader (BMG Labtech).
Three independent experiments were carried out in triplicate.

Determination of plasma membrane integrity
Cell membrane permeabilization was determined using the SYTOX Green nucleic acid stain (Molecular Probes) [36] with modifications. Mid-log phase promastigotes were washed twice in HBSS and parasites (2 x 10 6 promastigotes/mL) were incubated (

Determination of trypanothione reductase (TryR) activity in parasite lysates
Assessment of TryR activity was carried out using the colorimetric method described [37]. Leishmania logarithmic promastigotes (2 x 10 6 /mL; 50 mL) were grown in 75 cm 2 culture flasks and exposed to different allicin concentrations (30, 60 and 120 μM) for 3 h at 27°C. Untreated cultures were included as negative controls. The tricyclic neuroleptic drug clomipramine HCl (Sigma) has previously been reported to selectively inhibit TryR [38]. Positive control cultures treated with the TryR inhibitor, clomipramine HCl (Sigma), 10 μM (3h, 27°C) were also included in the experiment. Cells were washed twice in PBS and cultures were concentrated (2 x 10 7 /mL; 1mL/eppendorf). Samples were centrifuged (7826 xg, 5 min, RT) and supernatants were discarded. Pellets were incubated for 15 min at RT with 1 mL of lysis  was prepared at a final concentration of 0.75 nmol/mL. The extraction of thiols was performed in acid media using as extraction solvent 50 μL of 6.3 mM DTPA (0.1% TFA) that was added to microcentrifuge tubes containing the pellets resuspended in PBS. After vortex mixing, Leishmania extracts were immediately frozen in liquid N 2 and thawed three times to fully release cellular content. The supernatant was collected by centrifugation (13,000 rpm, 10 min) and subsequently derivatized. The derivatization procedure followed was based on the method described [39]. Firstly, 10 μL of 20 mM TCEP and 246 μL of 200 mM HEPPS buffer (6.3 mM DTPA, pH 8.2) were mixed to obtain a sulfur reducing solution that was subsequently added to 100 μL aliquots of Leishmania sample extracts or standards; 4 μL of 75 μmol/L DMP was also added as an internal standard. Then, the mixtures were incubated at 45°C in a water bath for 10 min to guarantee the reduced state of thiols before mBBr derivatization. The column was equilibrated with 10% of solvent B for a total of 8 min before next injection. Analyses were performed at a flow rate of 1.0 mL/min and the column temperature was kept at 40°C. The injection volume was 100 μL and all the compounds elute within 30 min. The DAD detector was set at both 290 and 380 nm and the excitation and emission wavelengths of the FLD detector were set at 380 nm and 470 nm, respectively. Quantification was performed using internal calibration and peak area measurements. Multiple analyses were performed using blanks and standards to determine detection limits, response linearity and reproducibility of the protocol.

Annexin V binding and propidium iodide staining
Changes in the transbilayer arrangement of phospholipids [40] in Leishmania promastigotes was analyzed using the Annexin V-FLUOS Staining kit (Roche). Cells (2 x 10 6 /mL) were incubated at 27°C in the presence of allicin (30-120 μM) for 3, 12, 24 and 48 h. Cells were washed twice in cold PBS and the resulting pellet resuspended in 100 μL of HEPES buffer containing Annexin-V and PI (2 μL of each). Samples were incubated at RT in the dark for 15 min and analyzed by flow cytometry (FACScan, Becton Dickinson) with CellQUEST software. Cultures incubated at 45°C overnight were used as positive death controls.

DNA fragmentation assay by agarose gel electrophoresis
Qualitative analysis of DNA fragmentation was performed by agarose gel electrophoresis of total genomic DNA extracted from untreated and allicin treated promastigotes. Leishmania promastigotes were exposed to allicin (30 μM and 60 μM) for 24, 48 and 72h. After drug exposure cells were washed twice in sterile PBS (1000 xg, 10 min, RT) and the total DNA was extracted from the cell pellet (10 8 promastigotes) using the Apoptotic DNA ladder kit (Roche) following manufacturer's protocol. The DNA was quantified at 260/280 nm using a NanoDrop ND-1000 spectrophotometer. The genomic DNA (5 μg) was run on a 2% agarose gel containing SYBR Safe DNA gel stain for 1 h at 100 V and visualized under UV light using the DNA Molecular Weight marker XIV (Roche).

Statistical analysis
Statistical analysis and graphs were performed with GraphPad Prism 5 software. Statistical significance of differences was determined by one-way and two-way analysis of variance (ANOVA) and Bonferroni post-test. Differences were considered significant at a p value of < 0.05.

Allicin induces cytosolic and mitochondrial ROS production in promastigotes of Leishmania
Allicin triggered the intracellular levels of hydroxyl radicals, hydrogen peroxide and peroxynitrites ( Fig 1A) in the promastigote stage of Leishmania after 3 h exposition. Intracellular ROS generation was concentration dependent and reached over 5-fold production in the presence of 90 μM allicin. Exposition of promastigotes to the estimated EC 50 value (30 μM) elicited a ROS production similar to the value reached by the positive control (5 μM antimycin A). Similarly, allicin effectively induced a strong elevation of superoxide production in mitochondria of treated cells (Fig 1B) as determined by the mitochondrial targeted probe (MitoSox Red). The induction was notable even with moderate allicin concentrations and a 6-fold increase was found with a concentration of 30 μM. It should be indicated that this increase, in the region of the levels reached by the positively treated promastigotes (5 μM antimycin), was maintained with higher allicin concentrations (60-120 μM).
Allicin reduced moderately trypanothione reductase (TryR) activity and increased non-protein thiol levels in Leishmania Trypanothione reductase (TryR) plays a fundamental role in the antioxidant defense of the cells through the reduction of trypanothione, the unique thiol present in Kinetoplastida including Leishmania. Exposure of Leishmania promastigotes to allicin for 3 h provoked a dose-dependent moderate reduction of TryR activity in parasite lysates of treated cells. Residual activity in the presence of the lowest allicin concentration (30 μM) reached a 66.67% of that found in untreated control cultures; 120 μM allicin reduced the TryR activity by 80%, inhibition higher than that induced by the specific inhibitor clomipramine. Remaining TryR present in the leishmanial lysates displayed standard enzymatic kinetics (Fig 2). Non-protein cellular thiols of Leishmania were separated, identified and quantified using HPLC-DAD-FLD. This   (Table 1).

Allicin triggers cytosolic Ca 2+ in Leishmania promastigotes
The cytosolic levels of calcium in L.infantum promastigotes treated with increasing concentrations of allicin (15-120 μM) were determined at different times (0-60 min). For comparative purposes results obtained were normalized using as baseline the fluorescence before adding stimuli. Addition of allicin induced a rapid increase of cytosolic Ca 2+ in a dose-dependent manner for a given post treatment time. Fig 4 shows the results obtained after 20 min. Chelation with 8 mM EGTA inhibited this increase whereas the permeabilization of the cell membrane of

Allicin alters the mitochondrial membrane potential of Leishmania
TEM studies showed that allicin induced a range of ultrastructural alterations in Leishmania promastigotes including generalized vacuolization. Most prominent lesions were observed in mitochondrion and kinetoplast with notable deformation, swelling of the organelle and loss of internal integrity (Fig 5). Evaluation of mitochondrial transmembrane potential (ΔCm) of L. infantum promastigotes treated with allicin was carried out using the cationic dye JC-1 and its selective accumulation in mitochondria inversely related to ΔCm. Net negative charge of mitochondrial membrane is a characteristic of healthy cells thus allowing the concentration of the cationic dye. JC-1 aggregates emit red fluorescence at higher potential whereas with membrane potentials below 140 mV remains as a monomer within the cytoplasm emitting green fluorescence. The potential can be altered by some intracellular events (e.g. ROS production) in Leishmania and our results showed an increase in mitochondrial ROS production in the presence of allicin, even at low micromolar concentrations. To asses changes in the ΔCm in the presence of allicin it was considered relevant to determine the ratio between red fluorescence (590 nm or FL-2) and green fluorescence (530 nm or FL-1). Fluorescence intensity as determined by FACS analysis (Fig 6A) showed that the FL-2/FL-1 ratio (red/green fluorescence ratio) was significantly affected, in our experimental conditions, in a dose-related manner since only 15-30 μM allicin induced a 23-30% reduction of the ratio and this value was below 90% with allicin concentrations >90 μM. Furthermore, the depolarization of the mitochondria evidenced by the ΔCm fall was supported by the shift of the Leishmania population towards the right in the FL-1 channel (green fluorescence) as observed by FACS analysis (Fig 6B).  Cellular levels of ATP in Leishmania promastigotes exposed to allicin are reduced without significant cell membrane damage  (Fig 7A). Exposition to allicin ( 90 μM) induced altered permeability of plasma membrane of promastigotes of Leishmania (p<0.001); 120 μM allicin yielded ca. 50% increased permeability from that obtained with the positive control (0.5% Triton X-100) at least as assessed by SYTOX Green internalization (Fig 7B). Interestingly, low concentrations of diallyl thiosulfinate, and in particular in the range of EC 50 values for this parasite stage (ca. 30 μM), did not result in any significant alteration of the cell membrane. These results point towards the diminished intracellular ATP generation observed in treated cells (<60 μM; i.e. drug concentrations that did not induce a notable damage of the plasma membrane) was probably due to allicin.

Time course effects of allicin on Leishmania promastigotes
Previous experiments provided conclusive evidence of the effect of allicin on increased Ca 2+ , ROS generation, both cytosolic and mitochondrial, and a fall of mitochondrial membrane potential and ATP production. These findings suggested that mitochondrion could be the target of diallyl thiosulfinate. However all experiments, with the exception of calcium levels determination, were performed at a fixed incubation time of 3 h. Moreover, mitochondrial alterations were observed after 24 h incubation with allicin concentrations (90 μM) well over the estimated EC 50 value (ca. 30 μM). Therefore a time-course study was carried out to determine the sequence of events and thus the potential primary mechanism of action of allicin (Fig 8).
Results obtained in the time-course study confirmed the previous findings with fixed time experiments (3 h). Allicin elicited a time-and concentration-related alteration of all parameters determined when compared to untreated Leishmania cells. Thus, after 1 h incubation with 30 μM allicin, the estimated EC 50 , cytosolic ROS increased ca. 50% ( Fig 8D) and superoxide levels in mitochondria were elevated over three times the basal values (Fig 8E). In a similar way intracellular calcium levels reached after 1 h > 40% increase over untreated cells (Fig 8A). By its part the fall of ΔCm (Fig 8B) and ATP production (Fig 8C) represented between a 35-40% of the values found in control cells. Alterations were increased with higher allicin concentrations (60 μM). With the exception of ATP production and cytosolic ROS maximum effects were observed after 1 h treatment and remained almost constant for the entire duration of the experiments. Effect of allicin was rapid since treatment of promastigotes with 30 μM and μM 60 allicin induced significant increases (p<0.001) of cytosolic calcium after 15 min and 5 min, respectively. This elevation was accompanied by a clear fall of mitochondrial membrane potential, ATP production and mitochondrial ROS generation with both allicin concentrations tested after 15 min (p< 0.001). By its part cytosolic ROS showed a steady increase along the experiment and their levels were significantly higher from 60 min onwards with 30 μM allicin (p<0.001).
Allicin does not induce externalization of Annexin 5 binding membrane phospholipids in Leishmania, an early marker of apoptosis One of the most frequently used early markers of apoptosis is the externalization of phosphatidylserine (PS) by apoptotic cells. It has been shown that Leishmania promastigotes lack PS Time-course effect of allicin on ROS levels, intracellular Ca 2+ , mitochondrial transmembrane potential and ATP production. Leishmania promastigotes were exposed to allicin (30 μM and 60 μM). Intracellular Ca 2+ levels (A), transmembrane mitochondrial potential (B), ATP production (C), cytosolic ROS (D) and mitochondrial ROS levels (E) were determined at 0, 5, 15, 30, 45, 60, 120 and 180 minutes post-treatment. Methods employed for each determination were those given in Material and Methods. Results are the mean ± S.D. of three determinations. Asterisks represent statistically significant differences with untreated controls (*: p<0.05; ** p<0.001). doi:10.1371/journal.pntd.0004525.g008 although other phospholipid classes are present and could bind Annexin V upon permeabilization [40].  Treatment with allicin of Leishmania promastigotes is not followed by DNA fragmentation No intranucleosomal DNA fragmentation was evident in treated cells with allicin using the dUTP nick-end labelling (TUNEL) method and FACS analysis of cultures of Leishmania promastigotes treated for 48 and 72 h with allicin (30, 60 and 120 μM) (Fig 10A and 10B). There was a shift in the FL1-H axis but this shift was present in all cultures irrespective of the treatment and the concentration of allicin. A small and possibly non-significant apoptotic population was observed after 48 h in the cultures with the highest allicin concentration (>60 μM). In the 72h sample a significant fraction of the population displayed an apparent phenotypic fragmentation pattern. However cellular distribution was similar in both treated and untreated control cultures.
Under our conditions neither allicin (30 and 60 μM) nor miltefosine (40 μM) induced any DNA laddering after incubation for 24, 48 or 72 h. Fig 11 shows a representative analysis after 24 h exposition to allicin. Leishmania DNA from promastigotes treated with both compounds did not show evidence of apoptotic-like laddering.

Allicin induces G 2 /M phase cell cycle arrest of Leishmania promastigotes
To determine the effect of allicin on the progression of the cell cycle of L.infantum, promastigotes were treated with different concentrations of the antileishmanial (30, 60 and 120 μM) at different exposure times (48 and 72 h) and analyzed by flow cytometry using PI staining. Results obtained with early (Annexin V binding), late (DNA fragmentation) and cell cycle analysis did not support an apoptotic-like effect of allicin on Leishmania but rather cell necrosis.

Discussion
Our results showed that allicin, at sublethal concentrations (ca. EC 50 ), induced an elevation of intracellular Ca 2+ levels. In most eukaryotic cells a major signaling function of this cation in the cytosolic compartment is played when its levels are elevated. In Leishmania Ca 2+ is maintained at very low levels [41] and the fine tuning of its intracellular levels is critical for cell homeostasis [42]. The high levels of intracellular Ca 2+ observed in Leishmania exposed to allicin probably came from intracellular calcium stores and particularly mitochondrion, since no variations in the plasma membrane permeability were found at least as assessed by SYTOX Green internalization. There is a tight connection between oxidative stress and intracellular Ca 2+ in all organisms including Leishmania [43][44][45]. Although ROS are present in normal cells playing a significant role as signaling messengers [24] the overproduction is linked to oxidative stress, mitochondrial dysfunction and cell death [46,47]. Oxidative stress can disrupt the intracellular calcium translocation [48] among calcium stores (e.g. mitochondrion, acidocalcisomes, endoplasmic reticulum). Our results showed that 30 μM allicin induced an increase of mitochondrial and cytosolic ROS levels. To cope with the damaging oxidative stress Leishmania relies mainly on trypanothione (= bis glutathionyl spermidine) [49] since these Kinetoplastida are devoid of catalase and classical selenium containing GSH peroxidase and no glutathione reductase is present [50]. However, trypanothione reductase (TryR) activity was only moderately inhibited in the presence of allicin and, expectedly, thiols (cysteine, glutathione and trypanothione) levels increased. These findings, besides confirming the interaction of allicin with thiols [19], suggested that the molecule did not provoke a collapse of the trypanothione reducing system and therefore cell death of allicin-treated Leishmania was not primarily related to this mechanism of action.
ROS generation and increase of cytosolic Ca 2+ were accompanied by the fall of mitochondrial membrane potential (ΔCm) and reduced ATP production in the fixed time (3 h) experiments carried out. The high levels of superoxide anion (O 2 -) inside the mitochondrion and their apparent saturation (> 30 μM allicin) besides the extensive morphological alterations observed in TEM suggested that this organelle could be the primary target of allicin. However, TEM results were obtained with promastigotes exposed to high allicin concentrations for 24 h. Allicin can easily cross cell membranes [19] and surely these events should take place very rapidly after exposition to the molecule. Time course studies carried out did show a rapid and significant elevation of cytosolic Ca 2+ after 5 min incubation with 60 μM allicin and a 20% increase with 30 μM after 15 min. This elevation was followed by a parallel fall of ATP production, mitochondrial membrane depolarization, and superoxide levels whereas a delayed and non saturated cytosolic ROS production was found up to 3 h. Calcium is a key regulator of mitochondrial function and acts within the organelle to stimulate ATP synthesis [51]. This is critical since an estimated 70% of all energy requirements in Leishmania are fulfilled by oxidative phosphorylation in mitochondria [35] and they have a single mitochondrion and poor ability to multiply in anaerobic environments [28,29]. In contrast to other trypanosomatids TCA cycle in Leishmania promastigotes has major anabolic function by anaplerosis and glycosomal and mitochondrial metabolism is tightly coupled [52]. No determination of the glutamate /glutamine ratio in allicin-treated cells has been carried out by us and the involvement of glycosomes in the mechanism of action could also be considered. However, mitochondrial oxidative ATP generation, but not the glycolytic ATP generation, is inhibited by sodium azide [35] and in our time course experiments 30 μM allicin reduced ATP production by 30% and 60% after 15 min and 1 hour incubation, respectively. Thus our results suggest that allicin induced an increase in cytosolic Ca 2+ levels this leading to high superoxide levels, ΔCm depolarization with dysfunction of oxidative phosphorylation and subsequent reduction of ATP mitochondrial synthesis. These interconnected events induce a mitochondrial collapse with swelling and loss of organelle integrity and, finally, a cellular energetic catastrophe. Nonetheless, there are open questions and further investigation to validate the proposed mechanism of action is needed. Increase in cytosolic Ca 2+ due to the influx from the extracellular milieu cannot be excluded and experimentation using calcium chelating agents and antioxidants prior to or in conjunction with the allicin treatment is needed. Mitochondrial membrane collapse (MMC), and consequently the lack of functionality of mitochondria, is an irreversible cellular dysfunction leading, depending on the relative weight of the bioenergetic catastrophe/protease and endonuclease activation, to cell necrosis or apoptosis respectively [53]. Apparently allicin is able to induce apoptosis in some cancer cell lines (L-929, SGC-7901) [23,24]. ATP availability, after mitochondrial membrane depolarization, determines the switch from apoptosis (no fall of ATP) to cell necrosis (low ATP) [54][55][56]. In our case there was a net fall of ATP production. Some apoptotic-like deaths have been described in Leishmania [44,57] although cell deaths mechanisms in parasitic protozoa, in particular apoptosis-like, are a controversial issue [58]. Despite the variations found in programmed cell death our results did not show any conclusive evidence of apoptotic-like death since DNA fragmentation was not observed (DNA laddering, TUNEL assay), no phospholipids (Annexin V/PI staining) were exposed and there was a cell cycle arrest at G 2 /M phase (PI staining). On these grounds, the death type found in Leishmania treated with allicin is compatible with cell necrosis.
Additional experiments would precisely determine the relative role of the events and the participation of other organelles but available data point towards the rapid induction of high Ca 2+ levels and mitochondrial ROS in Leishmania by exposure to allicin. The impairment of the redox balance induced the mitochondrial membrane depolarization (ΔCm) with dysfunction of TCA and reduction of ATP production. These events, possibly through a feed-back process, lead to the collapse of mitochondrion with loss of integrity and finally a cellular energetic catastrophe leading to the necrotic death of Leishmania promastigotes with cell arrest at the premitotic phase (G 2 /M). Present results were obtained with promastigotes and further research should confirm that these mechanisms are also involved in the antileishmanial activity of allicin on the actual stage causing leishmaniasis, amastigotes.