Antimonial-Mediated DNA Fragmentation in <em>Leishmania infantum</em> Amastigotes

D Sereno

P Holzmuller

I Mangot

Gérard Cuny

A Ouaissi

J Lemesre

Laboratoire de Biologie Parasitaire,^{CJF-INSERM N°96/04,}^{and Laboratoire de Parasitologie et Entomologie Moléculaire,}^{Centre IRD (Institut de Recherche pour le Développement), 34032 Montpellier Cedex 1, France}

^{Corresponding author. Mailing address: Laboratoire de Biologie Parasitaire, IRD, 911 Av. Agropolis, BP 5045, 34032 Montpellier Cedex 1, France. Phone: (33) 04 67 41 62 20. Fax: (33) 04 67 54 78 00. E-mail:}rf.dri.lpm@ersemeL.

Received 2000 Nov 30; Revisions requested 2001 Jan 25; Accepted 2001 Apr 2.

Abstract

The basic treatment of leishmaniasis consists in the administration of pentavalent antimonials. The mechanisms that contribute to pentavalent antimonial toxicity against the intracellular stage of the parasite (i.e., amastigote) are still unknown. In this study, the combined use of several techniques including DNA fragmentation assay and in situ and cytofluorometry terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling methods and YOPRO-1 staining allowed us to demonstrate that potassium antimonyl tartrate, an Sb(III)-containing drug, was able to induce cell death associated with DNA fragmentation in axenic amastigotes of Leishmania infantum at low concentrations (10 μg/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 μg/ml). Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase-1, caspase-3, calpain, cysteine protease, or proteasome activation. Altogether, our results demonstrate that the antileishmanial toxicity of Sb(III) antimonials is associated with parasite oligonucleosomal DNA fragmentation, indicative of the occurrence of late events in the overall process of apoptosis. The elucidation of the biochemical pathways leading to cell death could allow the isolation of new therapeutic targets.

Abstract

Leishmaniasis is a significant cause of morbidity and mortality in several countries. A vertebrate host is infected with flagellated extracellular promastigote forms via the bite of a sand fly. Promastigotes are rapidly transformed into nonflagellated amastigotes dividing actively within the mononuclear phagocytes of the vertebrate host. The basic treatment consists in the administration of sodium stibogluconate (Pentostam), meglumine (Glucantime), pentamidine, or amphotericin B. Treatment failure, especially for kala-azar, mucosal leishmaniasis, and diffuse cutaneous leishmaniasis is becoming a common problem in many areas where leishmaniasis is endemic. Immunological, physiological, or pharmacological deficiencies in the host are possible explanations for variations in clinical response (29). But there is evidence that inherent lack of susceptibility and (or) the development of resistance can also contribute to parasite unresponsiveness to drugs (13, 18, 23, 28, 39, 40). The mode of action of pentavalent antimonials remains poorly understood (3, 4, 5). An in vivo metabolic conversion of pentavalent antimonial [Sb(V)] into trivalent ones [Sb(III)] was suggested more than 50 years ago by Goodwin and Page (15, 16). This hypothesis was supported by the high toxicity of trivalent antimony against both parasite stages of different Leishmania species (10, 14, 26, 31, 34). Recently, we and other investigators have shown that axenically grown amastigotes of Leishmania represent a powerful model to investigate drug activity on the active and dividing population of the mammalian parasite stage (7, 34). We have shown that potassium antimonyl tartrate [containing Sb(III)] was generally more toxic than pentavalent antimony [Sb(V)] for both parasite stages of different Leishmania species and demonstrated that the extracellular amastigotes of Leishmania infantum were the Leishmania species most susceptible to Sb(III) (35). Moreover, in vitro-selected Sb(III)-resistant axenic amastigotes expressed a strong cross-resistance to meglumine when growing in THP-1 cells (37). A stage-specific susceptibility of amastigotes towards antimonials has also been proposed. This hypothesis is based on the assumption that amastigotes of Leishmania donovani are able to reduce pentavalent antimonial into a trivalent one (11, 12).

There are now increasing numbers of reports of single-celled organisms that kill themselves by a mechanism whose activation is not obligatory but can be used in threatening situations (i.e., apoptosis) (2). Drugs, toxins, and physical injuries could also provoke apoptosis in mammalian cells (1, 9, 41). Interestingly, arsenite-mediated apoptosis has been characterized and extensively studied in mammalian cells (8, 20, 24, 43, 44). As antimonials share several chemical properties with arsenicals, trivalent antimonial-mediated apoptosis has been studied and reported in NB4 and NB4R4 cells (27). In order to more precisely clarify the mode of action of antimonials against the amastigote forms of L. infantum, we have investigated the type of cell death induced by antimonials.

In this study, we demonstrate that the cell death mediated by antimonials presents some features previously shown to be induced by heat shock in the promastigote forms of Leishmania amazonensis (25), by antibiotic G418 in the epimastigote forms of Trypanosoma cruzi (1), and by reactive oxygen species in Trypanosoma brucei (30, 45). Trivalent antimonials (tartar emetic) species were able to kill amastigotes with a cell death phenotype presenting some homologies with the programmed cell death observed in metazoans (i.e., DNA fragmentation). The term apoptosis, which was originally defined purely on morphological grounds, has been recently redefined as “caspase-mediated cell death with associated apoptotic morphology” (32, 42). Our study suggests that nuclease activation does not depend on caspase-1, caspase-3, calpain, cystein protease, or proteasome activation. These results suggest that the cell death pathway involved in antimonial toxicity should be different from those involved in metazoan apoptosis. The implication of these observations on the antimonial mode of action is discussed.

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