Interaction of ascaridole, carvacrol, and caryophyllene oxide from essential oil of Chenopodium ambrosioides L. with mitochondria in Leishmania and other eukaryotes

The antileishmanial activity of the essential oil (EO) from Chenopodium ambrosioides L. has been demonstrated in vitro and in animal models, attributed to the major components of the EO. This study focused on the effects of the three major EO compounds carvacrol, caryophyllene oxide (Caryo), and the antileishmanial endoperoxide ascaridole (Asc) on mitochondrial functions in Leishmania tarentolae promastigotes (LtP). EO and Caryo were able to partially inhibit the leishmanial electron transport chain, whereas other components failed to demonstrate a direct immediate effect. Caryo demonstrated inhibition of complex III activity in LtP and in isolated complex III from other species. The formation of superoxide radicals was studied in Leishmania by electron spin resonance spectroscopy in the presence of iron chelators wherein selected compounds failed to trigger a significant immediate additional superoxide production in LtP. However, upon prolonged incubation of Leishmania with Asc and especially in the absence of iron chelators (allowing the activation of Asc), an increased superoxide radical production and significant impairment of mitochondrial coupling in Leishmania was observed. Prolonged incubation with all EO components resulted in thiol depletion. Taken together, the major components of EO mediate their leishmanicidal activity via different mitochondrial targets and time profiles. Further studies are required to elucidate possible synergistic effects of carvacrol and Asc and the influence of minor compounds.

In a series of previous studies, we observed the antileishmanial potential of essential oil (EO) from C. ambrosioides L. in different in vitro and in vivo models (Monzote et al., 2006;Monzote, Garcia, et al., 2014). In parallel, in the chromatogram obtained by gas chromatography/mass spectrometry, we identified three major compounds of the EO (Figure 1), namely, carvacrol (Car) with 62%, ascaridole (Asc) with 22%, and caryophyllene oxide (Caryo) with 5% of total content (Monzote et al., 2006). These three compounds were also identified in EO of Chinese C. ambrosioides L., however, in different percentages (Chu, Hu, & Liu, 2011). In addition to these volatile components, this EO contains also nonvolatile (solvent extractable) pharmacologically active compounds (Shah & Khan, 2017). Car, Caryo, and Asc showed antileishmanial activity, although they were less selective for Leishmania in comparison with mammalian host cells than EO for Leishmania compared with effects on mammalian host cells (Monzote et al., 2006;Monzote, Garcia, et al., 2014). Asc, which is also present in tea tree oil, demonstrated a skin-sensitizing effect in mammals (Chittiboyina, Avonto, & Khan, 2016;Krutz et al., 2015). By the use of iron chelators, it was shown that activation of the endoperoxide Asc in EO by iron is essential for its antiparasitic actions. Nevertheless, differences in the activity profile of Asc and EO have been observed in the system of macrophages/Leishmania.
In addition, it has been demonstrated that one possible toxicological mechanism behind the actions of EO and its major components against mammalian cells is related to mitochondrial dysfunction (Monzote, Stamberg, Staniek, & Gille, 2009). In Leishmania, there have been indications that EO and its major compounds also influence mitochondrial functions (Monzote, Garcia, et al., 2014), but specific mechanisms and targets have not been identified so far.
Herein, we study the effects of EO's main compounds (Asc, Car, and Caryo) on electron transport chain (ETC) complexes I-III in different models (yeast, Leishmania, and mammals) at the molecular level.
Short-term effects as well as long-term effects of Asc have been investigated with the objective to elucidate the role of mitochondrial effects in the EO actions in Leishmania.

| EO from C. ambrosioides L. and its main compounds
In this study, we used an aliquot of the original sample (stored at −20°C) for which the type of collection, extraction of EO, and chemical characterization were described previously (Monzote et al., 2006).
Briefly, aerial part of C. ambrosioides L. plant in flowering stage was collected in July, and voucher specimen number (ROIG4639) was assigned at the Experimental Station of Medicinal Plants "Dr Juan Tomás Roig," Cuba. The EO was extracted from fresh material by hydrodistillation in a Clevenger apparatus over 4 hr to yield approximately 1% oil (Monzote et al., 2006). Asc was obtained by chemical synthesis by addition of singlet oxygen to α-terpinene using rose bengal as a photosensitizer (Monzote et al., 2009). Structure and stability of the product was studied by nuclear magnetic resonance (NMR), and FIGURE 1 Chemical structure of main compounds of essential oil from Chenopodium ambrosioides L.: carvacrol (Car, 62%), ascaridole (Asc, 22%), and caryophyllene oxide (Caryo, 5%) according to Monzote et al. (2006) magnetic resonance; Oligo, oligomycin; PBS, phosphate-buffered saline; RCR, respiratory control ratio; ScY, Saccharomyces cerevisiae yeast; ScY-bc 1 , complex bc 1 from submitochondrial particles of Saccharomyces cerevisiae; ScY-SMP, submitochondrial particles from Saccharomyces cerevisiae; SMP, submitochondrial particles; Tris, tris(hydroxymethyl)aminomethane; YEM, yeast extract medium a purity of around 95% was determined. 1 H NMR (400.13 MHz,  Cavalli, Tomi, Bernardini, & Casanova, 2004). The accordance of this NMR data with our previously published data (Monzote et al., 2009) confirms the stability and purity of Asc. Car and Caryo were from Sigma (USA), with a purity of >98% and >95%, respectively.
All products were diluted with DMSO.

| Isolation of mitochondrial fractions from LtP
LtP culture (2,700 ml) was centrifuged at 478 g over 10 min at 4°C (Sorvall RC26 Plus, USA). The supernatant was discarded, and the cell pellet was resuspended in buffer (10 mM Tris-HCl, 0.3 M sucrose, 0.2 mM EDTA, and 0.2% BSA, pH 7.4). Following two repeated washes (478 g, 10 min at 4°C), the resulting cell pellet was incubated in lysis buffer (5 mM Tris-HCl, pH 7.4) for 10 min at 25°C and subsequently homogenized in a Dounce homogenizer. Cell debris was removed by centrifugation (1,005 g, 10 min, 4°C). The supernatant was again centrifuged (13,176 g, 20 min, 4°C) to sediment the mitochondrial fraction (LtP-Mit). The mitochondria were resuspended in 1 ml of buffer (250 mM sucrose, 50 mM KH 2 PO 4 , and 0.2 mM EDTA, pH 7.2) and stored in liquid nitrogen until use.

| Isolation of submitochondrial particles from bovine heart
Bovine heart submitochondrial particles (BH-SMP) were obtained from bovine heart mitochondria by sonication (Nohl & Hegner, 1978) and stored in liquid nitrogen until use.

| Isolation of SMP from yeast
Yeast mitochondria were prepared from Saccharomyces cerevisiae yeast (ScY) strain DBY 747 (Daum, Bohni, & Schatz, 1982). Cells were harvested by centrifugation (1,464 g, 5 min, 20°C), and the pellet was resuspended in buffer I (10 mM Tris and 10 mM DTT, pH 9.4). Following 15 min of incubation at 37°C, cells were centrifuged (1,464 g, 5 min, 20°C) and resuspended in buffer II (1.2 M sorbitol and 20 mM KH 2 PO 4 , pH 7.4). Finally, after a repeated centrifugation (1,464 g, 5 min, 20°C), the weight of the cell pellets was determined. To prepare spheroblasts, pellets were suspended in buffer II complemented with 2 mg zymolyase/g yeast cells. After incubation for 45 min at 28°C, spheroblasts were collected by centrifugation (1,464 g, 5 min, 20°C), resuspended in 30 ml of buffer II, sedimented again (5 min at 1,464 g and 20°C), and homogenized in 30 ml of buffer III (600 mM sorbitol and 20 mM Tris, pH 7.4) using a Wheaton Dounce tissue grinder. Cells and cell debris were removed by two centrifugations (1,464 g, 5 min, 4°C). Mitochondria were finally collected from the supernatant by centrifugation (11,952 g, 10 min, 4°C) and used to prepare ScY submitochondrial particles (ScY-SMP). Mitochondrial pellets were suspended in 5 ml of buffer I (without DTT) and diluted to 25 ml with 10 mM Tris (pH 7.5). The suspension was kept on ice for 20 min followed by a centrifugation (39,500 g, 10 min, 4°C). The pellet was resuspended in 20 ml of sucrose buffer (250 mM sucrose and 10 mM Tris, pH 7.4) and sonicated 18 times for 20 s (Branson sonifier at maximum intensity) with interruptions of 10 s for heat dissipation. Subsequently, the suspension was centrifuged (5,400 g, 10 min, 4°C) to remove mitochondria. ScY-SMP were sedimented from the supernatant by centrifugation (195,000 g, 60 min, 4°C). The obtained ScY-SMP pellet was homogenized in 1.5 ml of buffer (250 mM sucrose, 0.2 mM EDTA, and 50 mM KH 2 PO 4 , pH 7.2) and stored in liquid nitrogen.

| Determination of protein and cell concentrations
The protein concentration of mitochondrial preparations was determined by the Biuret method (Lowry, Rosebrough, Farr, & Randall, 1951). The number of LtP was determined by optical density at 600 nm (HITACHI U-1100 Spectrophotometer, Japan). The cell broth was diluted 1:10 with culture medium and measured against a blank of culture medium. The cell number was calculated using the formula C Cell (10 6 /ml) = OD 600nm * 0.969 * 124 (Fritsche, Sitz, Weiland, Breitling, & Pohl, 2007). Two replicates of each culture were performed.
2.7 | Activity of EO compounds on ETC complexes in LtP-Mit and BH-SMP 2.7.1 | Influence on NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II) activities

| Influence on ubiquinol:cytochrome c oxidoreductase activity
To measure the ubiquinol:cyt c 3+ oxidoreductase (complex III) activity, the reduction of 100 μM cyt c 3+ at 550 nm using 540 nm as reference was monitored in the presence of the artificial substrate decylubiquinol (dUQH 2 , 75 μM), which was prepared from dUQ by reduction (Müllebner et al., 2010). The dUQH 2 :cyt c 3+ oxidoreductase activities of 40 μg protein/ml of LtP-Mit or 3.2 μg protein/ml of BH-SMP were measured in 1 ml of buffer containing 250 mM sucrose, 50 mM KH 2 PO 4 , 0.2 mM EDTA (pH 7.2), 2 mM KCN, and 4 mM NaN 3 . Respective EO compounds were added 50 s after starting the time scan, and the reaction was started after 100 s with dUQH 2 and was monitored for additional 150 s. The activity of noninhibited dUQH 2 :cyt c 3+ oxidoreductase activity was measured in the presence of the vehicle for the respective inhibitor (DMSO).
All inhibitor concentrations were tested in triplicate. The reduction rates for cyt c 3+ were calculated from the time trace of the absorption difference at 550 − 540 nm (ε 550-540 nm = 19 mmol −1 L cm −1 ). Reduction rates in the presence of DMSO (maximum amount that was introduced by test compound stocks) were set to 100%, and the remaining activities in the presence of EO compounds were expressed in %. Three replicates were measured for each concentration.

| Influence of EO compounds on isolated bc 1 complex
To determine the influence of EO compounds on purified bc 1 complex, the dUQH 2 :cyt c 3+ oxidoreductase activities were measured as described. In this case, concentrations of 21.3 μg/ml of ScY-bc 1 or 1.6 μg/ml of BH-bc 1 were used.    The OD 600 nm was determined for cell count, and the measured fluorescence intensity was normalized to the cell number. Three replicate measurements were performed for each condition.

| Statistical analysis
The median of inhibitory concentration (IC 50 ) value was determined from nonlinear concentration-response curves using Origin ® Program Version 6.1 and expressed as the mean ± standard deviation. Statistically significant differences of p < .05 were identified using Student's t test.

| Antileishmanial activity of EO components
Viability assays for LtP resulted in IC 50 values for Asc of 24.5 ± 3.0 μM, Car of 11.6 ± 3.4 μM, and Caryo of 36.0 ± 17.6 μM (n = 3). For EO, an IC 50 value of 19.1 ± 6.1 μg/ml was determined. This indicates that all major EO components possibly contribute to the antileishmanial action of EO.

| Inhibition of mitochondrial complexes
In general, no strong inhibition was observed for complexes I and II (Table 1). However, complex III inhibition of LtP-Mit by Caryo FIGURE 2 Influence of major components of essential oil from Chenopodium ambrosioides L. on the cellular oxygen consumption of Leishmania tarentolae promastigotes (LtP). Oxygen consumption of LtP (72-100 × 10 6 cells/ml) was assessed by a Clark-type electrode in airsaturated medium containing 14.6 mM glucose. Increasing concentrations of compounds were added subsequently using DMSO as vehicle. At 1% DMSO (highest final concentration), O 2 consumption of LtP was inhibited by 1.74 ± 9.46%. Data are means ± standard deviation of four independent experiments. Asc = Ascaridole; Car = carvacrol; Caryo = caryophyllene oxide confirmed its interference at this site. In contrast, for BH-SMP, the inhibitory effect of Caryo was weaker. Asc and Car showed no strong inhibition in the studied concentration ranges suggesting that they have no specific targets in the ETC of Leishmania and mammals (Table 1).
In a next step, we extended our experiments to isolated ScY-bc 1 and BH-bc 1 (

| Influence on cellular superoxide radical production
In a next experiment, we studied superoxide radical formation in LtP by an ESR method using cyclic hydroxyl amine (CMH), which is converted upon one-electron transfer reaction with superoxide radicals to a stable nitroxyl radical (CM • , Figure 3a). As can be seen (Figure 3b), LtP significantly triggered CMH oxidation in comparison with buffer. Also, the positive control with AA, a well-known trigger of mitochondrial superoxide formation, showed an increase of CM • formation in LtP. Asc, Car, and Caryo only slightly increased CM • formation. At first view, it appears puzzling that Asc, which is known to trigger formation of carbon-centered radicals (Geroldinger et al., 2017), does not trigger superoxide formation in this assay.

| Activation of Asc and cellular superoxide radical production
The dose-dependent increase of CMH oxidation triggered by Asc becomes increasingly effective upon prolonged incubation and in the absence of iron chelators (Figure 4) suggesting that superoxide radical formation by Asc does not occur immediately and is enhanced by the availability of iron allowing for Asc activation.

| Influence on mitochondrial coupling
Although Asc is not a direct inhibitor of ETC in LtP (Figure 2), a decrease in the membrane potential of Leishmania amazonensis promastigotes (LaP), as assessed by JC-1, was observed after 72 hr of incubation (Monzote, Garcia, et al., 2014), and Asc was shown to produce radicals in LtP (Geroldinger et al., 2017). Accordingly, the long-term effects of Asc on mitochondrial coupling in LtP were examined by measuring the respiration of LtP with a Clark-type electrode.
Upon addition of Oligo (inhibitor of ATP synthase), oxygen consumption stimulated by ATP production was blocked ( Figure 5), and inclusion of the uncoupler CCCP yielded the maximally uncoupled respiration. The quotient of both rates, the RCR, is an indication of the ability of LtP to respond to increased ATP demands due to stress conditions. Therefore, a high RCR is indicative of a healthy cell, and a decreased RCR reflects an impaired stress response.
In a first experimental series, we tested the immediate uncoupling effect of EO major components in 96-well OxoPlates. Neither Car nor Asc (Figure 6a

| Influence on low molecular thiols
As an indicator of oxidative stress, the status of low molecular thiols was assessed by the CMFDA method (Sarkar et al., 2009) wherein CMFDA is intracellularly deacetylated to CMF, which is then converted (by glutathione S-transferase activity) to a low molecular Results were expressed as median inhibitory concentration (IC 50 ) ± SD of five independent experiments. Asc = ascaridole; BH-bc 1 = cytochrome bc 1 complex purified from Bos taurus; Car = carvacrol; Caryo = caryophyllene oxide; EO = essential oil; ScY-bc 1 = cytochrome bc 1 complex purified from Saccharomyces cerevisiae; SD = standard deviation. Note. IC 50 > value: In these cases, the IC 50 was not determined because only an inhibition <50% was observed at highest concentration. In parentheses, % of inhibition at highest concentration tested. Results were expressed as mean ± SD or as percentage of three independent experiments. Asc = ascaridole; BH-SMP = submitochondrial particles from bovine heart; Car = carvacrol; Caryo = caryophyllene oxide; EO = essential oil; LtP-Mit = mitochondrial crude fraction of Leishmania tarentolae; SD = standard deviation. fluorescent thiol-MF adduct. The rate of this adduct formation is expected to be proportional to the intracellular low molecular thiol level. Control experiments without LtP yielded no significant rates, whereas untreated control LtP showed strong fluorescence evolution.
Incubation with EO components showed a decrease for all products, especially for Caryo, which might be a link to its mitochondrial effects ( Figure 8).

| DISCUSSION
EO prepared from Cuban C. ambrosioides L. plants was analyzed by gas chromatography/mass spectrometry showing that Asc, Car, and Caryo are the main components of the EO (Monzote et al., 2006). Independent studies on EO from Chinese C. ambrosioides L. also listed Asc, Car, and Caryo as detected components, though in different amounts (Chu et al., 2011). It was shown that the EO composition from C. ambrosioides L. can vary widely (Jesus et al., 2017;Soares et al., 2017 (Monzote, Garcia, et al., 2014). In mammalian mitochondria, except for Caryo, no direct effect of EO on mitochondrial ETC with respect to oxygen consumption was observed (Monzote et al., 2009).
In this study, the influence of EO main compounds on complexes of the mitochondrial ETC as possible targets for antileishmanial drugs was explored. Biological model systems have greatly facilitated the understanding of drug actions. In our study, besides whole LtP cells,

FIGURE 4
Formation of superoxide radicals in Leishmania tarentolae promastigotes (LtP) triggered by ascaridole (Asc) increased with Asc concentration, incubation time, and iron availability. Superoxide radical formation was assessed by reaction of 400 μM CMH in suspensions containing 5 × 10 8 LtP/ml in phosphate-buffered saline with 15 mM glucose and 100 μM DFO and 25 μM DTPA (white bars). Immediate effects (0-hr incubation) of Asc in LtP in the presence of chelators (DFO/DTPA). After 1-hr incubation (light grey) and after 1-hr incubation in the absence of iron chelators (dark grey). Data represent mean ± standard deviation of quadruplicate experiments. * Significant differences versus LtP on the level p < .05. Bu = buffer controls without LtP   Lainson & Shaw, 1987), and is not pathogenic for humans (Raymond et al., 2012). L. tarentolae has been widely used in pharmacological studies for (a) the screening of natural and synthetic products (Taylor et al., 2010), (b) the purification and characterization of proteins that are used for the screening of drugs with potential antileishmanial activity (Fritsche et al., 2007;Yakovich, Ragone, Alfonzo, Sackett, & Werbovetz, 2006), and (c) the amplification of genes involved in the resistance to certain antileishmanial drugs such as amphotericin B (Singh, Papadopoulou, & Ouellette, 2001) and sodium stibogluconate (Haimeur & Ouellette, 1998 (Monzote, Garcia, et al., 2014) and LtP (Geroldinger et al., 2017), the quantitative outcome of viability assays may strongly depend on the cell number to drug ratio, detection method and even on assay medium and premature activation of Asc in media. Our current studies explore this systematically. From the listed IC 50 values for LtP in this work, it can be concluded that it is at least likely that all three major components (and also possibly nonstudied trace compounds) are involved in the antileishmanial action of EO.

FIGURE 6
Mitochondrial uncoupling in Leishmania tarentolae promastigotes (LtP) in the presence of major components of essential oil from Chenopodium ambrosioides L. Uncoupling was assessed by stimulation of oxygen consumption in the presence of the ATP synthase inhibitor oligomycin (Oligo). LtP cells (1 × 10 8 /ml) in brain heart infusion medium were supplemented with Oligo (5 μM) and decreasing concentrations of ascaridole (Asc), carvacrol (Car), caryophyllene oxide (Caryo), and the uncoupler CCCP. Oxygen consumption was assessed in 96-well OxoPlates for 40 min at 26°C. Oxygen consumption rates were normalized to the respiration of non-inhibited Leishmania (LtP = 100%). Data represent mean ± standard deviation of 3-4 experiments. * Significant differences versus LtP + Oligo on the level p < .05 Indirect effects, which do not occur immediately after drug exposure but after several hours, may be caused by drug metabolites (rather rare), the intrinsic pathway of apoptosis triggered by nonmitochondrial targets, lipid peroxidation of mitochondrial membranes, and other processes.
A hallmark of drug actions on mitochondria is inhibition of the mitochondrial ETC (Chan, Truong, Shangari, & O'Brien, 2005). Therefore, oxygen uptake by LtP and its inhibition by EO major compounds were studied (Figure 2 Due to the inherent relationship between generation of reactive oxygen species and respiratory chain inhibition, complex III was described as the main source of superoxide radicals in both mammals and Leishmania species (Carvalho et al., 2010;Dawson, Gores, Nieminen, Herman, & Lemasters, 1993;Garcia-Ruiz, Colell, Morales, Kaplowitz, & Fernandez-Checa, 1995). Mitochondrial inhibition is sometimes (depending on the site of inhibition) accompanied by increased mitochondrial superoxide production. In addition, impairment of the mitochondrial ETC by lipid peroxidation and protein oxidation may trigger mitochondria to produce more superoxide radicals. Also NADPH oxidases, P450 oxidases, or xanthine oxidases as well as low molecular weight iron and ascorbate (which are both present in Leishmania) are known nonmitochondrial superoxide radical sources. We studied superoxide production in LtP by the CMH/ ESR method, which is highly specific for superoxide except the interference with Fe 3+ (Dikalov, Skatchkov, & Bassenge, 1997). Therefore, these assays are usually performed in the presence of iron chelators to prevent this side reaction. This, however, has the limitation that under these assay conditions, we can only assess the effects of nonactivated Asc because Asc needs iron for its pharmacological action.
In these experiments (Figure 3), both negative buffer control (lacking LtP) and positive control (in the presence of the complex III inhibitor AA) showed that the detection system is working. All three EO compounds slightly increased the superoxide radical formation in LtP in the assays time frame of about 15 min.

FIGURE 8
Influence of major components of essential oil from Chenopodium ambrosioides L. on the low molecular thiol status of Leishmania tarentolae promastigotes (LtP) after 5 hr incubation at 26°C in phosphate-buffered saline/glucose medium. Low molecular thiol status was assessed by measuring the rate of fluorescence evolution over 1 hr from the conjugation of CMF (arising from 5 μM CMFDA) to low molecular thiols in LtP (1 × 10 7 cells/ml) in phosphatebuffered saline/glucose. Results represent mean ± standard deviation of three experiments. * Significant differences versus LtP on the level p < .05. Asc = Ascaridole; Bu = buffer; Car = carvacrol; Caryo = caryophyllene oxide FIGURE 7 Effects of ascaridole (Asc) on mitochondrial coupling in Leishmania tarentolae promastigotes (LtP) upon prolonged incubation. Different cell batches with 1 × 10 8 LtP/ml in Schneider's medium plus 6 μM hemin were incubated in culture tubes either with DMSO (vehicle for Asc; LtP) or with 200 μM Asc (LtP + Asc). From these culture stocks, aliquots were taken for O 2 consumption measurements at 0, 6, and 24 hr. Mean cell counts during measurements were adjusted with medium to approximately 1-2 × 10 8 LtP/ml. Respiratory control ratios were calculated as the ratios of O 2 consumption rates in the presence of 5 μM oligomycin plus 0.5 μM CCCP to oligomycininhibited O 2 consumption rates as shown in Figure 5. Data represent mean ± standard deviation of four independent experiments. * Significant differences versus LtP on the level p < .05 In the genus Leishmania, different low molecular weight thiol antioxidants are present: glutathione, trypanothione, cysteine, and ovothiol (Romao et al., 2006). In addition, Leishmania can synthesize ascorbate as a powerful antioxidant (Manhas, Anand, Tripathi, & Madhubala, 2014). Therefore, we assessed the redox state of thiols in the presence of Asc, Car, and Caryo by a CMFDA assay (Sarkar et al., 2009)  In our study, Car shows neither mitochondrial inhibition nor mitochondrial uncoupling. Car is a phenol like the well-known uncoupler 2,4-dinitrophenol. However, the pKa value of 2,4-dinitrophenol is around 4, whereas for normal (non-nitro-substituted) phenols like Car, the pKa value is around 10 (Rappoport & Frankel, 1967).
This makes an action of Car as protonophore not very likely under physiological conditions. Others have shown that Car, upon prolonged incubation with superoxide radicals, forms rather stable ESR signals, which cannot be assigned to simple phenoxyl radicals (Deighton, Glidewell, Deans, & Goodman, 1993). The complex ESR signals suggest the presence of large conjugated systems, which could arise from oligomerized Car oxidation products. These trace products could have potential redox-cycling properties causing additional harm to Leishmania. In our experiments, we used nonoxidized Car and therefore did not study these effects.
To address the situation including Asc activation, we performed experiments for Asc with prolonged incubation times and in the presence and the absence of iron chelators (Figure 4). The results clearly show that activation of Asc takes time and is strongly enhanced in the absence of iron chelators. In addition, it was demonstrated that Asc has no immediate direct effect on mitochondria but increases superoxide radical formation after activation.
This prompted us to study Asc effects on mitochondrial coupling ( Figures 5-7). A major mitochondrial function in LtP is the generation of ATP, which can be impaired by inhibition of the ETC or by uncoupling of ETC from ATP synthase function. The latter is often triggered by breaking down the proton gradient across the inner mitochondrial membrane (driving ATP synthesis) by proton-shuttling drugs or increased proton permeability of the inner mitochondrial membrane. Increased proton permeability can be mediated by radical-triggered membrane lipid peroxidation. Coupling reflects the ability of mitochondria to adapt ATP production to ATP demands.
Stress conditions such as treatment with antileishmanial drugs may directly or indirectly increase the ATP demand, usually triggering mitochondria to produce more ATP. Conversely, a decreased mitochondrial coupling impairs this stress response mechanism.
As shown, Asc has no immediate direct uncoupling effect, but after prolonged incubation (probably via Asc activation), mitochondrial coupling is impaired in LtP.
EO from C. ambrosioides L. is a complex mixture with a variety of possible pharmacological mechanisms. In addition, there are numerous possibilities for pharmacological interactions as demonstrated for Asc and Car in a previous publication . The three main compounds are responsible for some but certainly not for all effects (Figure 9). Among these compounds, only Caryo has direct inhibitory effects on complex III in Leishmania and other eukaryotic cells. Car has no inhibiting effects on ETC but similarly impairs LtP viability. Asc has the most complex mechanism. It has no direct inhibiting effect on mitochondrial ETC and no immediate uncoupling effect in LtP. However, upon activation by iron, Asc impairs mitochondrial coupling and triggers superoxide radical formation in LtP. This suggests that impairment of mitochondrial coupling in Leishmania by prolonged incubation with Asc ( Figure 7) is not the primary mode of action but a downstream event of rather selective activation of Asc in Leishmania (Geroldinger et al., 2017). Further studies are required to elucidate possible synergistic effects of Car and Asc.
These findings suggest that Asc, Car, and Caryo mediate their leishmanicidal activity via different targets in mitochondria and in other parts of the cell and that different mitochondrial effects are seen after different times of exposure.