Cellular effects of reversed amidines on Trypanosoma cruzi

Abstract

Aromatic diamidines represent a class of DNA minor groove-binding ligands that exhibit high levels of antiparasitic activity. Since the chemotherapy for Chagas' disease is still an unsolved problem and previous reports on diamidines and related analogues show that they have high levels of activity against Trypanosoma cruzi infection both in vitro and in vivo, our present aim was to evaluate the cellular effects in vitro of three reversed amidines (DB889, DB702, and DB786) and one diguanidine (DB711) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas' disease. Our data show that the reversed amidines have higher levels of activity than the diguanidine, with the order of trypanocidal activities being as follows: DB889 > DB702 > DB786 > DB711. Transmission electron microscopy analysis showed that the reversed amidines induced many alterations in the nuclear morphology, swelling of the endoplasmic reticulum and Golgi structures, and consistent damage in the mitochondria and kinetoplasts of the parasites. Interestingly, in trypomastigotes treated with the reversed amidine DB889, multiple axoneme structures (flagellar microtubules) were noted. Flow cytometry analysis confirmed that the treated parasites presented an important loss of the mitochondrial membrane potential, as revealed by a decrease in rhodamine 123 fluorescence. Our results show that the reversed amidines have promising activities against the relevant mammalian forms of T. cruzi and display high trypanocidal effects at very low doses. This is especially the case for DB889, which merits further in vivo evaluation.

FIG. 1.

Structures of the four drugs used in this study.

FIG. 2.

Activities of DB889 (A), DB702 (B), DB786 (C), and DB711 (D) against trypomastigote forms of T. cruzi. The percentage of dead parasites was measured by light microscopy after 2 and 24 h of treatment at 37°C.

FIG. 3.

Transmission electron micrographs (A to H) and flow cytometry analysis (I to M) of trypomastigote forms of T. cruzi treated with the compounds of interest. (A and B) Untreated parasites; (C, D, and H) DB889-treated parasites; (E) DB702-treated parasites; (G and F) DB711-treated parasites. Note the swelling mitochondrion and kinetoplast (C to F and H), the alterations of the nuclear morphology (C, D, and H), and the alterations of the microtubule organization (F). Multiple flagellar structures (C and F) and dilated membrane profiles (G) can be found. (I to M) Histograms of the results of a representative assay displaying the fluorescence intensities of untreated parasites (I) and diamidine-treated parasites after incubation with Rh123: (J) DB889, (K) D702, (L) DB786, and (M) DB711. The high-fluorescence-intensity peaks are marked M1, whereas the low-fluorescence-intensity peaks, which represent decreased mitochondrial membrane potential, are marked M2. k, kinetoplast; m, mitochondrion; n, nucleus; and G, Golgi structure. Bars: 1 μm (A to H) and 0.5 μm (inset, H).

FIG. 4.

Transmission electron micrographs of intracellular amastigotes of T. cruzi. The parasites were untreated (A and B) or were treated with DB889 (C), DB702 (E), and DB786 (G and H). Note the alterations in the nuclear morphology (C), the vacuolization and loss of the cytoplasm components (C, asterisk), disorganization of the kinetoplast (D), the swelling and the presence of low electrondense structures in the mitochondrion (E), the intense vesicular profiles in the flagellar pocket (F and G, arrows), and the disorganization of the subpellicular microtubules (H, arrowhead). n, nucleus; m, mitochondrion; k, kinetoplast; fp, flagellar pocket (fp). Bars, 0.5 μm (A) and 1 μm (B to H).