Destruction of leishmania mexicana amazonensis amastigotes within macrophages in culture by phenazine methosulfate and other electron carriers

Abstract

Exposure of [hamster] macrophages infected with L. mexicana amazonensis to phenazine methosulfate (PMS) resulted in rapid damage and disappearance of the intracellular amastigotes without obvious ill effects to the host cells. The reduction of the percent infection was related to the concentration of PMS and to the duration of the pulse. Most Leishmania disappeared within 2 h of a 2-h pulse with 10 .mu.M of the drug. Pretreatment of the macrophages with PMS followed by removal of the drug before infection did not result in disappearance of the parasites. The pH of the PMS medium markedly influenced the disappearance of Leishmania: maximum effect was obsered at pH 8.0, while the effect was negligible at pH 6.3. The pH effect may be related to pseudobase formation by the PMS cation. Dose-response curves for PMS were similar for resident, elicited or activated macrophages. Observations by time-lapse cinemicrography documented the explosion-like fragmentation of the amastigotes within 1-2 h of exposure of infected macrophages to the drug. Parasite-derived granules and vacuoles scattered within the parasitophorous vacuoles. This early damage to the parasites was confirmed by transmission electron microscopic observations. Infected macrophages incubated with PMS displayed detectable vacuolar fluorescence, indicating that PMS or a metabolite of PMS had access to the vacuoles. A series of other electron carriers, including phenyl methanes, phenazines, oxazines, a xanthene and a naphthoquinone, given continuously for 18 h, also induced the disappearance of the Leishmania. The most potent was crystal violet, active at 70 nM. The presence of apolar substituents enhanced activity; this is probably related to increased permeation of the dyes. PMS, as well as other electron carriers examined, also reduced the growth of Leishmania promastigotes in culture. A direct effect of the drugs on the intracellular amastigotes, involving only a permissive participation of the macrophages, is indicated. The diverse agents probably destroy the amastigotes by redox-cycling generation of active oxygen metabolites at or near the parasites. The effect of the drugs could be mediated by toxic free radical reduction species of drugs or by interference with electron flow or with the intermediary metabolism o Leishmania.