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Electron microscopy has proven to be a reliable and essential tool to determine morphological alterations and target organelles in the investigation of new drugs for Chagas disease. In this review, we focused on evaluating different agents that induce death of Trypanosoma cruzi, i.e. lysophospholipids analogues, naphthoquinones and derivatives, cytoskeletal inhibitors and natural products. Apoptosis-like presents as morphological characteristics DNA fragmentation, membrane blebbing and apoptotic body formation. Autophagy involves autophagosome formation, with the appearance of membranes surrounding organelles and cytosolic structures. Necrosis causes the loss of osmotic balance, an increase of cytoplasmic vacuolization and plasma membrane disruption. Mitochondrion appears as a central checkpoint in both apoptosis and necrosis. Our evidences of ultrastructural changes to T. cruzi treated with the different classes of compounds point to dramatic mitochondrial alterations and similar autophagic phenotypes. Lysophospholipid analogues interfere in the lipid biosynthesis in epimastigotes, altering the amount of both phospholipids and sterols, and consequently the physical properties of the membrane. Naphthoquinone derivatives led to a strong DNA fragmentation in trypomastigotes and to the release of cysteine proteases from reservosomes to cytosol in epimastigotes, starting a proteolytic process which results in parasite death. The susceptibility of reservosomes was also observed in parasites treated with propolis, suggesting impairment of lipid metabolism, compromising membrane fluidity and leading to lysis. The cytoskeletal agents blocked mitosis of epimastigotes, arresting cell cycle and impairing the parasite proliferation. The variety of drug stimuli converge to the same pathway of death suggests an intense cross-talking between the three types of PCD in the protozoa.

Ethanol extract of Bulgarian propolis (Et-Blg) was administered by oral route in doses ranging from 25 to 100mg/kg body weight in experimental Trypanosoma cruzi-infected Swiss mice. Treatment with 50mg Et-Blg/kg body weight/day led to a decrease in parasitemia and showed no hepatic or renal toxic effect. Treatment with Et-Blg led to a decrease in the spleen mass and modulated the initial inflammatory reaction as demonstrated by analysis of the leukocyte profile in peripheral blood, quantification of T cells subsets, and phenotypic markers in the spleen. Preferential expansion of CD8(+) and partial inhibition in the increase of CD4(+)CD69(+) and CD8(+)CD69(+) in CD4(+)CD44(+) and CD8(+)CD44(+) and in the decrease of CD8(+)CD62L in Trypanosoma cruzi-infected mice were also observed. Taken together, our data indicate that treatment of Trypanosoma cruzi-infected mice with Et-Blg interferes with the basic properties of immune cells.

Acetone and ethanol extracts of two Bulgarian propolis samples (Bur and Lov) were investigated by high temperature high resolution gas chromatography coupled to mass spectrometry (HT-HRGC-MS), and their activity against Trypanosoma cruzi was evaluated. The ethanol extracts--Et-Bur and Et-Lov--showed similar composition, with a high content of flavonoids, and strong inhibitory activity against T. cruzi proliferative epimastigotes, which were more susceptible than trypomastigotes. In the presence of blood, the activity of Et-Bur or Et-Lov against trypomastigotes was similar to that of the standard drug, crystal violet. Both extracts also showed similar and significant activity against Staphylococcus aureus and Candida albicans, while being inactive against Escherichia coli. The acetone extract, Ket-Bur, was more active than Et-Bur against both forms of T. cruzi.