A study of the sensitivity ofLeishmania donovanipromastigotes and amastigotes to hydrogen peroxide. I. Differences in sensitivity correlate with parasite-mediated removal of hydrogen peroxide

Abstract

The sensitivities of promastigotes and amastigotes ofLeishmania donovanito reagent or glucose oxidase-generated hydrogen peroxide (H2O2) were examined in a phagocyte-free system and compared with direct measurements of loss of H2O2due to reaction with the parasite. Using a combined fluorescence dye uptake/dye exclusion viability assay in conjunction with motility and transformation data it was shown that log-phase promastigotes harvested from recently transformed cultures were intermediate in their H2O2sensitivity between amastigotes and log-phase promastigotes harvested from long-term subcultures. It was also observed that, while promastigotes are equally sensitive to either form of H2O2stress, amastigotes are more resistant to single larger amounts of reagent H2O2than to equivalent amounts of H2O2generated over a 1 h period. In each case the respective LD50values obtained for each form of the parasite under each type of H2O2stress correlated with saturation of their ability to remove H2O2from the phagocyte-free system. For both promastigotes and amastigotes there was always a time delay after removal of either form of H2O2stress before H2O2-mediated damage to membranes became apparent. The results suggest that the differential responses of promastigotes and amastigotes to different forms of H2O2stress may depend upon different H2O2scavenging mechanisms examined in more detail in the accompanying paper.