ATOVAQUONE, A BROAD-SPECTRUM ANTIPARASITIC DRUG, COLLAPSES MITOCHONDRIAL-MEMBRANE POTENTIAL IN A MALARIAL PARASITE

At present, approaches to studying mitochondrial functions in malarial parasites are quite limited because of the technical difficulties in isolating functional mitochondria in sufficient quantity and purity, W e have developed a flow cytometric assay as an alternate means to stud y mitochondrial functions in intact erythrocytes infected with Plasmod ium yoelii, a rodent malaria parasite, By using a very low concentrati on (2 nM) of a Lipophilic cationic fluorescent probe, 3,3'dihexyloxaca rbocyanine iodide, we were able to measure mitochondrial membrane pote ntial(Delta Psi(m)) in Live intact parasitized erythrocytes through fl ow cytometry, The accumulation of the probe into parasite mitochondria was dependent on the presence of a membrane potential since inclusion of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, dissipat ed the membrane potential and abolished the probe accumulation. We tes ted the effect of standard mitochondrial inhibitors such as myxothiazo le, antimycin, cyanide and rotenone. All of them except rotenone colla psed the Delta Psi(m), and inhibited respiration. The assay was valida ted by comparing the EC(50) of these compounds for inhibiting Delta Ps i(m) and respiration. This assay was used to investigate the effect of various antimalarial drugs such as chloroquine, tetracycline and a br oad spectrum antiparasitic drug atovaquone, We observed that only atov aquone collapsed Delta Psi(m) and inhibited parasite respiration withi n minutes after drug treatment, Furthermore, atovaquone had no effect on mammalian Delta Psi(m). This suggests that atovaquone, shown to inh ibit mitochondrial electron transport, also depolarizes malarial mitoc hondria with consequent cellular damage and death.