PENTOSTAM INDUCES RESISTANCE TO ANTIMONY AND THE PRESERVATIVE CHLOROCRESOL IN LEISHMANIA-DONOVANI PROMASTIGOTES AND AXENICALLY GROWN AMASTIGOTES

An axenic amastigote culture system was utilized to directly assess th e stage-specific antileishmanial effects of antimony on amastigotes of Leishmania donovani devoid of the macrophage host cell, Pentostam, wh ich contains antimony in the form of sodium stibogluconate and the pre servative chlorocresol, was used, Cell density was quantified by measu ring the activity of the stable enzyme ornithine decarboxylase. Dose-r esponse curve analyses show that Leishmania promastigotes are suscepti ble to Pentostam, with the 50% inhibitory concentration (IC50) being 1 04 mu g/ml, while amastigotes are more susceptible, with the IC50 bein g 24 mu g/ml. Promastigotes and amastigotes are also susceptible to ch lorocresol, with IC(50)s being 1.27 and 1.82 mu g/ml, respectively. Gi ven that promastigotes are insensitive to antimony, these results sugg est that the increased susceptibility of amastigotes to Pentostam is d ue to the stage-specific activity of sodium stibogluconate. To further study this phenomenon, spontaneous resistance to Pentostam was induce d in L. donovani promastigotes by increasing the concentration of Pent ostam in the growth medium in a stepwise fashion, Two mutants, Ld1S.04 and Ld1S.20, grew at 0.4 and 2.0 mg of Pentostam per mi, respectively , Promastigotes of these mutants were 11 and 21 times, respectively, m ore resistant to Pentostam than the wild type. Amastigotes were 40 and 148 times, respectively, more resistant than the wild type, The mutan ts were also chlorocresol resistant; promastigotes, were 6 and 9 times , respectively, more resistant than the wild type, and amastigotes wer e 14 and 35 times, respectively, more resistant than the wild type, Th ese data show that resistance to Pentostam induced in antimony-insensi tive promastigotes is manifested in amastigotes as resistance both to pentavalent antimony and to chlorocresol, The axenic amastigote system is a unique tool which enables direct evaluation of the activity of a ntileishmanial compounds on the amastigote devoid of its host cell.