title: Variation in apoptosis mechanisms employed by malaria parasites: the roles of inducers, dose dependence and parasite stages.
abstract: BACKGROUND
  Plasmodium berghei ookinetes exhibit an apoptotic phenotype when developing within the mosquito midgut lumen or when cultured in vitro. Markers of apoptosis increase when they are exposed to nitric oxide or reactive oxygen species but high concentrations of hydrogen peroxide cause death without observable signs of apoptosis. Chloroquine and other drugs have been used to induce apoptosis in erythrocytic stages of Plasmodium falciparum and to formulate a putative pathway involving cysteine protease activation and mitochondrial membrane permeabilization; initiated, at least in the case of chloroquine, after its accumulation in the digestive vacuole causes leakage of the vacuole contents. The lack of a digestive vacuole in ookinetes prompted the investigation of the effect of chloroquine and staurosporine on this stage of the life cycle. Finally, the suggestion that apoptosis may have evolved as a strategy employed by ookinetes to increase the fitness of surviving parasites was explored by determining whether increasing the ecological triggers parasite density and nutrient depletion induced apoptosis.
  
  
  METHODS
  Ookinetes were grown in culture then either exposed to hydrogen peroxide, chloroquine or staurosporine, or incubated at different densities and in different media. The proportion of ookinetes displaying positive markers for apoptosis in treated samples was compared with controls and results were analyzed using analysis of variance followed by a Turkey's test, or a Kruskal-Wallis test as appropriate.
  
  
  RESULTS
  Hydrogen peroxide below 50 μM triggered apoptosis but cell membranes were rapidly compromised by higher concentrations, and the mode of death could not be defined. Both chloroquine and staurosporine cause a significant increase in ookinetes with condensed chromatin, caspase-like activity and, in the case of chloroquine, phosphatidylserine translocation and DNA fragmentation (not investigated for staurosporine). However, mitochondrial membrane potential remained intact. No relationship between ookinete density and apoptosis was detected but nutrient depletion significantly increased the proportion of ookinetes with chromatin condensation in four hours.
  
  
  CONCLUSIONS
  It is proposed that both a mitochondrial and an amitochondrial apoptotic pathway may be involved, dependent upon the trigger that induces apoptosis, and that pathways may differ between erythrocytic stages and ookinetes, or between rodent and human malaria parasites.
authors.affiliation: Centre for Applied Entomology and Parasitology, Institute for Science and Technology in Medicine, School of Life Sciences, Keele University, Keele, Staffordshire, ST5 5BG, UK.
authors.forename: Holly
authors.initials: H
authors.lastname: Matthews
authors.forename: Medhat
authors.initials: M
authors.lastname: Ali
authors.forename: Victoria
authors.initials: V
authors.lastname: Carter
authors.forename: Ann
authors.initials: A
authors.lastname: Underhill
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hunt
authors.forename: Hannah
authors.initials: H
authors.lastname: Szor
authors.forename: Hilary
authors.initials: H
authors.lastname: Hurd
mesh_terms: D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002738:Chloroquine / Q000633:toxicity*; D002843:Chromatin / Q000378:metabolism; D053938:DNA Fragmentation; D006861:Hydrogen Peroxide / Q000633:toxicity*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects; D051336:Mitochondrial Membranes / Q000187:drug effects; D010718:Phosphatidylserines / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects*; D019311:Staurosporine / Q000633:toxicity*
chemical_list: D002843:Chromatin; D010718:Phosphatidylserines; D002738:Chloroquine; D006861:Hydrogen Peroxide; D020169:Caspases; D019311:Staurosporine