Natural Sesquiterpene Lactones Induce Oxidative Stress in Leishmania mexicana

Leishmaniasis is a worldwide parasitic disease, caused by monoflagellate parasites of the genus Leishmania. In the search for more effective agents against these parasites, the identification of molecular targets has been attempted to ensure the efficiency of drugs and to avoid collateral damages on the host's cells. In this work, we have investigated some of the mechanisms of action of a group of natural sesquiterpene lactones that are effective against Leishmania mexicana mexicana promastigotes. We first observed that the antiproliferative effect of mexicanin I (Mxc), dehydroleucodine (DhL), psilostachyin (Psi), and, at lesser extent, psilostachyin C (Psi C) is blocked by 1.5 mM reduced glutathione. The reducing agent was also able to reverse the early effect of the compounds, suggesting that lactones may react with intracellular sulfhydryl groups. Moreover, we have shown that all the sesquiterpene lactones, except Psi C, significantly decreased the endogenous concentration of glutathione within the parasite. Consistent with these findings, the active sesquiterpene lactones increased between 2.7 and 5.4 times the generation of ROS by parasites. These results indicate that the induction of oxidative stress is at least one of the mechanisms of action of DhL, Mxc, and Psi on parasites while Psi C would act by another mechanism.


Introduction
Leishmaniasis is a parasitic disease caused by flagellated parasites of the genus Leishmania and transmitted by phlebotomine sandflies. These parasites exhibit a heteroxenous life cycle, alternating between intracellular amastigotes in the mammalian cells and flagellate promastigotes in the vector.
Leishmaniasis affects about 12 million people worldwide and, according to the World Health Organization (WHO), 2 million of new cases occur annually and 350 million people are considered at risk of contracting leishmaniasis [1]. The clinical forms of the disease depend on the species of Leishmania involved and include local infections of the skin, subcutaneous tissue, and regional lymphatic nodes (cutaneous leishmaniasis); metastatic infections of the oronasal mucosa (mucocutaneous leishmaniasis); and disseminated infection involving visceral organs (visceral leishmaniasis) [2].
Leishmaniasis is distributed worldwide with foci of infection in Central and South America, Southern Europe, North and East Africa, the Middle East and India [3]. In Argentina, this parasitosis affects the northern region of the country with an incidence that has increased over the last two decades [4].
Current drugs used to treat leishmaniasis include pentavalent antimonials, pentamidine, and amphotericin B, which induce serious toxic effects on patients. Parasite resistance to these drugs has also been described. New formulations, such as liposomal amphotericin B and other drugs (miltefosine, paromomycin), have serious drawbacks such as parenteral route of administration, duration of the treatment, teratogenic effects, toxicity, and cost of treatment, which limit their use in endemic areas [5]. Therefore, there is an urgent need for novel candidates to treat this parasitic disease.
One of the most important aspects in antiprotozoal drug discovery is to determine the mechanism of action of the potential candidates and to identify the possible molecular targets upon which these compounds act. Among other mechanisms, it is presumed that sesquiterpene lactones could exert their leishmanicidal activity by the generation of an oxidative environment within the parasite [17,18]. The particular defense mechanism against oxidative stress in trypanosomatids makes parasites susceptible to these kinds of compounds.
In this sense, the aim of the present work was to evaluate the possible effect of four bioactive sesquiterpene lactones: dehydroleucodine (DhL); mexicanin I (Mxc). psilostachyin (Psi), and psilostachyin C (Psi C) on the defense mechanism of Leishmania mexicana mexicana against oxidative stress.

Treatments.
Leishmania mexicana mexicana promastigotes (2 × 10 6 parasites) were incubated with 0.5 g/mL of Mxc, Psi, or Psi C or 2.5 g/mL of DhL, at 25 ∘ C, either in the presence or in the absence of 1.5 mM glutathione (GSH). The concentrations used for each compound were those corresponding to each IC 50 , as previously determined (data not shown). Aliquots of the parasites were collected every 24 h and counted in a Neubauer hemocytometer [16].
In other experiments, parasites were preincubated with the compounds for 30 min and the reducing agent was then added. Alternatively, the lactones were withdrawn after incubation for 1 h and before adding GSH. Controls were carried out in the presence of DMSO (less than 0.05%) which was used to dissolve the compounds.

Measurement of Reduced Glutathione.
Endogenous GSH was measured in parasite lysates by using 5,5 -dithiobis-2nitrobenzoic acid (DTNB), according to Beutler et al. [21]. Briefly, parasites (1 × 10 7 cells/mL) were previously incubated with the lactones (10 g/mL) for 3 h at 25 ∘ C then pelleted, lysed with 200 L lysis solution (10% EDTA, 0.5% Triton X-100 in bidistilled water) during 30 min, and centrifuged at 12,000 ×g. Supernatants were mixed with 300 L of solution P (0.2 M HPO 3 , 5 mM EDTA, and 5.1 M NaCl) and centrifuged again at 12,000 ×g. Supernatants were mixed with 800 L of 0.3 M Na 2 HPO 4 , and 200 L of DTNB (in 1% sodium citrate). Absorbances were then measured in a spectrophotometer at 412 nm, and the concentration of GSH was derived from a standard curve.

Statistical Analysis.
Results are presented as mean ± SD. The level of statistical significance was determined by using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test.

Results and Discussion
We had previously reported the antileishmanial activity of Mxc, DhL, Psi, and Psi C (Figure 1 functional group -methylene--lactone present in the sesquiterpene lactones is believed to be responsible for their antiprotozoal activity. However, the presence of other alkylating groups such as , -unsaturated cyclopentenones and other factors, such as lipophilicity, molecular geometry, and chemical environment, may also influence their bioactivity [22]. In this work we have corroborated the antiproliferative effect of the four lactones on L. mexicana mexicana promastigotes and we have demonstrated that this effect was blocked by 1.5 mM GSH ( Figure 2). As these lactones are nonpolar molecules they could easily pass through the parasite's plasmalemma. The blocking effect of GSH might be due to the transformation of the compounds into derivatives unable to traverse the plasmalemma. However, it is more likely that the compounds interfere with the intracellular concentration of GSH, as the antiproliferative effect of lactones can be reversed by GSH when the reducing agent is added 30 min after incubation with the compounds or 1 h after incubation followed by withdrawal of the lactones (Figure 3). In addition, it was observed that DhL, Mxc, and Psi, but not Psi C, reduced the concentration of endogenous GSH (Figure 4). On the other hand, treatment with DhL, Mxc, or Psi, but not Psi C, induced a significant increase of ROS in L. mexicana promastigotes ( Figure 5).
The generation of free radicals in Leishmania by the sesquiterpene lactones would be deleterious for trypanosomatids, as the regulation of oxidative stress is crucial for parasite survival. It is known that sesquiterpene lactones Evidence-Based Complementary and Alternative Medicine 5 react with sulfhydryl groups by the Michael-type addition and therefore could act by inhibiting the activity of enzymes that are vital against oxidative stress (e.g., trypanothione reductase) [17]. This situation could lead to an increase in the level of reactive oxygen species and to parasite damage via the generation of an oxidative burst by a deregulation of the redox balance within the parasite [23]. However, a direct interaction of the compounds with GSH or trypanothione should not be ruled out.
The decrease in the concentration of glutathione within the parasites induced by the sesquiterpene lactones Mxc, DhL, and Psi would lead to an enhancement in the production of reactive oxygen species. These results are in accordance with ROS production and the in vitro leishmanicidal activity, with psilostachyin C being the less active compound against L. mexicana. Given that sesquiterpene lactones can also induce GSH depletion and ROS generation in certain mammalian cells (e.g., tumor cells) [24], these compounds should be improved before use as therapeutic agents against Leishmania.
One vital step in the process of drug development is the identification of the molecular target/s of such drugs. Taking into consideration the data obtained, we can suggest that Mxc, DhL, and Psi were able to affect the defense mechanism against oxidative stress in L. mexicana. This mechanism could be related to inhibition of key enzymes that maintain redox balance in the parasite.
This study must be complemented by further investigations on amastigotes forms of Leishmania and on in vivo models of leishmaniasis.