Ultrastructural alterations induced by nifurtimox and another nitro derivative on epimastigotes of Trypanosoma cruzi.

We report the ultrastructural alterations induced on epimastigotes by nifurtimox and 5-nitro-2-thienyl-malononitrile (5NO2TM), a novel compound with anti-Trypanosoma cruzi activity. Parasites treated with concentrations of nifurtimox lower than usually employed for this kind of study showed vacuolisation, alterations of the mitochondria, the nucleus and the ribosomes. 5NO2TM caused the same kind of damage, but to a greater degree. This result correlates with the fact that cultures treated with this compound experienced a greater loss of viability.

In vitro effects of anti-Trypanosoma cruzi compounds on HSP60 levels.

The effect of nifurtimox and 5-nitro-2-thienylmalononitrile (5NO2TM), a novel compound with anti-Trypanosoma cruzi activity, upon vitality and HSP60 immunoreactivity of epimastigotes, has been determined. Both products showed no activity against epimastigotes at 0.1 microg/ml, while at 0.5 and 1 microg/ml, after 24 h of incubation, densities of these groups were significantly reduced, when compared to controls. An enhancement of HSP60 immunoreactivity was observed after 24 h in groups treated with 0.5 and 1 microg/ml nifurtimox. On the other hand, 5NO2TM had no effect.

In vitro screening of american plant extracts on Trypanosoma cruzi and trichomonas vaginalis.

From the beginning of this decade and with the revival of the phytotherapy, biological research about immunomodulatory, anti-inflammatory and antiprotozoal effects of Central and South American plants have been in progress. Our objective was to determine the antiprotozoal activity of 79 extracts from different plant families, including Asteraceae, Araceae, Moraceae, Solanaceae, Rhamnaceae, Zingiberaceae, Leguminosae and Sapotaceae. Once matching with herbarium specimens authenticated the plants, selected parts were separated, dried carefully and reduced to powder. Most of the screened extracts were aqueous. Two protozoa with different metabolic pathways, Trypanosoma cruzi and Trichomonas vaginalis were used as experimental models. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose (LIT). Anti-Trichomonas activity was determined over cultures of the parasite in Diamond medium. In both cases, microscopic counting of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the cytocidal and cytostatic activities respect to control cultures. Of the nine extracts that showed antiprotozoal activity, those from Mikania cordifolia and Philodendron bipinnatifidum were then fractionated, and again, were assayed the organic and aqueous phases obtained.

Setting of a colorimetric method to determine the viability of Trypanosoma cruzi epimastigotes.

The method most commonly used in screening of drugs for the treatment of Chagas' disease, microscopic counting of viable trypanosomes, is time-consuming, labor-intensive, and dependent on the observer. Although the tetrazolium dye [MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is comparatively quick and accurate, it requires careful attention in design as well as in interpretation of the results. Therefore, we examined under various conditions the sensitivity and specificity of the MTT assay versus microscopic counting for determination of the viability of Trypanosoma cruzi for drug-screening purposes. We tested different concentrations of MTT in phenazine methosulfate (PMS) against T. cruzi epimastigotes of the Y strain in different stages of logarithmic growth. In our model, in tests of benznidazole and nifurtimox the optimal concentration of MTT was 2.5 mg/ml of PMS and the optimal incubation period was 75 min. This method detected parasite concentrations of approx. 500,000 epimastigotes/ml (P<0.01), and the linear correlation between absorbance values and numbers of epimastigotes per milliliter was very strong (approx. R = 0.99). The present MTT assay results in faster determination of the activity of compounds, is more objective, and enables testing of several drugs simultaneously.