A nutritional characteristic of trypanosomatid protozoa is that they need a heme compound as a growth factor. Because of the cytotoxic activity of heme and its structural similarity to cobalamins, we have investigated the in vitro and in vivo effect of vitamin B 12 (or cyanocobalamin) on the different forms of Trypanosoma cruzi. Cyanocobalamin showed a marked antiparasitic activity against epimastigotes (50% inhibitory concentration [IC 50 ], 2.42 M), amastigotes (IC 50 , 10.69 M), and trypomastigotes (IC 50 , 9.46 M). Anti-epimastigote and -trypomastigote values were 1.7 to 4 times lower than those obtained with the reference drug benznidazole (Bnz). We also found that B 12 and hemin do not interact with each other in their modes of action. Our results show that B 12 increases intracellular oxidative activity and stimulates both superoxide dismutase (50%) and ascorbate peroxidase (20%) activities, while the activity of trypanothione reductase was not modified. In addition, we found that the antioxidants dithiothreitol and ascorbic acid increase the susceptibility of the parasite to the cytotoxic action of B 12 . We propose that vitamin B 12 exerts its growth-inhibitory effect through the generation of reactive oxygen species. In an in vivo assay, a significant reduction in the number of circulating parasites was found in T. cruzi-infected mice treated with cyanocobalamin and ascorbic acid. The reduction of parasitemia in benznidazole-treated mice was improved by the addition of these vitamins. According to our results, a combination of B 12 and Bnz should be further investigated due to its potential as a new therapeutic modality for the treatment of Chagas' disease.

A nutritional characteristic of trypanosomatid protozoa is that in vitro they need a haem-compound as a growth factor, which is supplied as haemoglobin, haematin or haemin. Because haemin and related porphyrins are an important source of oxidative stress in biological systems, the effect of haemin on growth, protein content and the antioxidant defence system in Trypanosoma cruzi was evaluated. We have observed that, in epimastigotes grown under different haemin concentrations in the culture medium (0-30 mg/l), 5 mg/l was the haemin concentration yielding optimum growth. Above 15 mg/l there was a clear decrease in growth rate, producing the epimastigote to amastigote transformation. Such morphological change was observed together with a marked injury of the enzymatic machinery of the parasite, leading to diminished protein synthesis as well as lower activity of the antioxidant enzymes (superoxide dismutase, ascorbate peroxidase and trypanothione reductase), reduced total thiol content and a marked increase in the HaemOx-1 activity and expression. The current work demonstrates that there is a correlation between higher haemin concentrations in the culture medium and oxidative damage in the cells. Under these conditions induction of HaemOx-1 would indicate the important role of this enzyme as an antioxidant defence response in Trypanosoma cruzi.

Three thioamide-substituted imidazoquinolinone, which possess a heterocyclic center similar to tryptanthrin and are named C1, C2, and C3, were studied regarding (a) their in vitro anti-Trypanosoma cruzi activity, (b) their cytotoxicity and electrochemical behaviour, and (c) their effect on cell viability, redox state, and mitochondrial function. The assayed compounds showed a significant activity against the proliferative forms, but only C1 showed activity on the trypomastigote form (for C1, IC50  epi = 1.49 μM; IC50  amas = 1.74 μM; and IC50  try = 34.89 μM). The presence of an antioxidant compound such as ascorbic acid or dithiotreitol induced a threefold increase in the antiparasitic activity, whereas glutathione had a dual effect depending on its concentration. Our results indicate that these compounds, which exhibited low toxicity to the host cells, can be reduced inside the parasite by means of the pool of low molecular weight thiols, causing oxidative stress and parasite death by apoptosis. The antiparasitic activity of the compounds studied could be explained by a loss of the capacity of the antioxidant defense system of the parasite to keep its intracellular redox state. C1 could be considered a good candidate for in vivo evaluation.