Hydrogen peroxide-mediated toxicity for Leishmania donovani chagasi promastigotes: Role of hydroxyl radical and protection by heat shock

Leishmania must survive despite exposure to the toxic oxidant hydrogen peroxide (H₂O₂) during phagocytosis by macrophages. We investigated the mechanism of H₂O₂ toxicity for L, donovani chagasi promastigotes, and factors responsible for their relative H₂O₂ resistance. There was a dose-dependent toxic effect of H₂O₂ for promastigotes isolated during logarithmic phase of growth. In contrast, stationary phase promastigotes were less susceptible to H₂O₂ toxicity, and more infectious for BALB/c mice. By spin trapping we found that hydroxyl radical (·OH) was generated after exposure of promastigotes to H₂O₂, and the amount of ·OH was greater with log-phase than with stationary-phase promastigotes. ·OH was generated after the addition of H₂O₂ to the cytosol but not the membranes of fractionated promastigotes, and the magnitude of ·OH was greater in log than in stationary promastigote ćytosol. Deferoxamine inhibition suggested that intracellular promastigote iron catalyzes ·OH formation via the Fenton reaction. Furthermore, exposure of log-phase promastigotes to heat shock induced a relative H₂O₂-resistant state, which was not associated with a decrease in · OH formation but which required ongoing transcription. Thus, growth to stationary phase and heat shock both induce a state of relative H₂O₂ resistance, but these are probably due to different resistance mechanisms.