Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action - PubMed (original) (raw)

Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action

D Sereno et al. Antimicrob Agents Chemother. 1998 Dec.

Free PMC article

Abstract

The mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly understood. In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three Leishmania species, supporting the idea of an in vivo metabolic conversion of Sb(V) into Sb(III). We report that amastigotes of Leishmania infantum cultured under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concentrations [IC50s], about 1.8 mg/ml and 22 microg/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we first induced in vitro Sb(III) resistance by direct drug pressure on axenically grown amastigotes of L. infantum. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodium stibogluconate plus m-chlorocresol (Pentostam). The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 were 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT). In accordance with the hypothesis described above, we found that intracellular chemoresistant amastigotes were resistant to meglumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resistance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate. This lack of cross-resistance is probably due to the presence in this reagent of m-chlorocresol, which we found to be more toxic than Sb(III) to L. infantum amastigotes (IC50s, of 0.54 and 1.32 microg/ml, respectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compounds, which in turn became readily toxic to the Leishmania amastigote stage.

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Figures

FIG. 1

Susceptibilities of THP-1 cells to drugs. The in vitro susceptibilities of THP-1 cells to sodium stibogluconate (Pentostam) (○), _m_-chlorocresol (□), and potassium antimonyl tartrate (◊) were ascertained by the MTT-based in vitro micromethod. Results are expressed as means ± standard deviations of triplicate experiments.

FIG. 2

Toxicity of meglumine to _L. infantum_wild-type and chemoresistant amastigotes in THP-1 cells. Host cells were infected with chemoresistant (LdiR2 [◊], LdiR10 [○], and LdiR20 [▵]) or wild-type (LdiWT [□]) amastigotes at a cell-parasite ratio of 1:5 in medium supplemented with 10% FCS after differentiation with PMA. Infected cells were exposed to drugs for 5 days. The percentage of growth (A) and the percentage of infected macrophages (B) were evaluated. The results are the means of duplicate experiments.

FIG. 3

Toxicity of _m_-chlorocresol to L. infantum amastigotes in THP-1 cells. Human cell lines were infected with wild-type (LdiWT) or chemoresistant (LdiR20) amastigotes at a cell-parasite ratio of 1:5 in medium supplemented with 10% FCS after differentiation with PMA. Infected cells were exposed to the drugs for 5 days. The percentage of growth inhibition of wild-type (┌) and the LdiR20 chemoresistant variant (□) (A) and the percentage of macrophages infected by the wild-type ( formula image ) and the chemoresistant LdiR20 parasites () (B) were evaluated. The results are the means of duplicate experiments.

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Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action - PubMed (original) (raw)