51.    Cell death in Trypanosoma brucei induced by a thiosemicarbazone involves oxidative stress

Trypanosoma brucei is the causative agent of sleeping sickness in man and nagana in cattle. The lack of a vaccine against these diseases and the notable limitations of drug treatments, mean a significant challenge to the proposed goal of eliminating these neglected diseases as a public health problem. A thiosemicarbazone (TSC1)
emerged as a promising compound since it has been demonstrated that is capable to inhibit T. brucei proliferation and showed no cytotoxic effects on Vero cells. Detection of chemical compounds inducing programmed cell death in parasites and nontoxicity for mammalian cells is of great pharmacological relevance. The present study aims to characterize by flow cytometry, the biochemical and morphological alterations induced by TSC1 in the death of parasite. Mitochondrial membrane potential was measured after incubation of TSC1 and untreated trypanosomes with MitoTracker red CMXRos. The carbonyl cyanide m-chlorophenyl hydrazone was used as a control for a complete depolarization of the inner mitochondrial membrane. In all studies, fluorescence analysis was performed in a FACSCalibur apparatus and a total of 10,000 events were acquired in the region that was previously established as the one that correspond to the parasites. DNA content was measured after permeabilization of trypanosomes and propidium iodide (IP) solution was used for the staining of the nuclei. Necrosis was analysed with IP by the disruption of the plasma membrane. Phosphadidylserine exposure was detected on the outer membrane of parasites using Annexin V. Cell volume was determined using FACS and finally, reactive oxygen species was measured with DCFH-DA. In the various tests carried out, we observed a reduction of mitochondrial membrane potential, an increase in the formation of reactive oxygen species, phosphatidylserine exposure, reduction of cell volume but no significant changes were observed in cell cycle. Altogether, the alterations induced by TSC1 point to a break in the homeostatic balance of oxidative stress that caused the death of parasite through mechanism that likely involve autophagy or apoptosis.