Gustavo Augusto Travassos Laranja

Toxicological evaluation by in vitro and in vivo assays of an aqueous extract prepared from Echinodorus macrophyllus leaves

Toxicity of an aqueous extract prepared from Echinodorus macrophyllus dried leaves, a plant used in folk medicine to treat inflammation and kidney malfunctions, was estimated by different bioassays. Mutagenicity of the aqueous extract was evaluated in the Salmonella/microsome assay (TA97a, TA98, TA100 and TA102 strains), with or without metabolic activation. No mutagenic activity (lyophilized extract tested up to 50 mg/plate) could be detected to any of the tester strain. Furthermore, no cytotoxic effect has been observed when a crude extract of E. macrophyllus (up to 7.5 mg/ml) was tested on the exponential growth of hepatoma and normal kidney epithelial cells in culture. Toxicity of E. macrophyllus was also evaluated in male Swiss mice after 6 weeks of continuous ingestion of the aqueous extract in drinking water. Average daily ingested doses were 3, 23 and 297 mg/kg for a lyophilized extract, and 2200 mg/kg for a crude extract, with dose two being equivalent to the daily dose recommended to humans. At the end of the treatment, all animals revealed a deficit in final body weight ranging from 5 to 47%. Biochemical analysis of the plasma revealed some minor alterations indicating subclinical hepatic toxicity. Genotoxic effect on liver, kidney and blood cells has been also evaluated by the comet assay, being negative to liver and blood cells. However, DNA analyses of the kidney cells detected some genotoxic activity for the highest dose tested of E. macrophyllus extract, either lyophilized or crude. On the other hand, exposure dose of 23 mg/kg, equivalent to the daily dose recommended to humans, did not revealed any genotoxic effect and hence this herb seems to be safe to human organism.

Biochemical behaviour of norbixin during in vitro DNA damage induced by reactive oxygen species

Naturally occurring antioxidants such as carotenoids are extensively studied for their potential in reducing the risk for cancer and other chronic diseases. In the present study, the radical-scavenger activity of the food additive norbixin, a water-soluble carotenoid extracted from Bixa orellana seeds and commercialized as annatto, was evaluated under conditions of DNA damage induced by reactive oxygen species, particularly by hydroxyl radicals. The cell-free scavenger activity of norbixin was evaluated using plasmid DNA as target molecule and Sn2+ or Fe2+ as oxidant. The addition of H2O2 enhanced DNA breakage induced by metal ions, particularly Fe2+. Under these conditions, norbixin started to protect plasmid DNA against single- and double-strand breakage at a metal:norbixin ratio of 1:1 (Sn2+) and 1:10 (Fe2+). However, at lower ratios to Sn2+, norbixin enhanced Sn2+-induced DNA breakage (P < 0.05). The ability of norbixin to protect genomic DNA against oxidative damage was assessed in murine fibroblasts submitted to H2O2-induced oxidative stress and the results were evaluated by the comet assay. Under low serum conditions (2 % fetal bovine serum (FBS)), a protective effect of norbixin against H2O2-induced DNA breakage was inversely related to its concentration, a protection ranging from 41 % (10 microm) to 21 % (50 microm). At higher concentrations of norbixin, however, oxidative DNA breakage was still enhanced, even in the presence of a high serum concentration (10 % FBS). Under normal conditions, norbixin per se has no detectable genotoxic or cytotoxic effects on murine fibroblasts. The antimutagenic potential of norbixin against oxidative mutagens was also evaluated by the Salmonella typhimurium assay, with a maximum inhibition of 87 % against the mutagenicity induced by H2O2. Although plasmid DNA and Ames data indicated that norbixin can protect DNA against oxidative damage, it seems to be a risky guardian of genomic DNA as it can also increase the extent of oxidative damage.

Norbixin ingestion did not induce any detectable DNA breakage in liver and kidney but caused a considerable impairment in plasma glucose levels of rats and mice

From the seeds of Bixa orellana are extracted the carotenoids bixin and norbixin that have been widely used for coloring food. In this study, the toxicity of norbixin, purified or not (annatto extract containing 50% norbixin), was investigated in mice and rats after 21 days of ingestion through drinking water. Mice were exposed to doses of 56 and 351 mg/kg (annatto extract) and 0.8, 7.6, 66 and 274 mg/kg (norbixin). Rats were exposed to doses of 0.8, 7.5 and 68 mg/kg (annatto extract) and 0.8, 8.5 and 74 mg/kg (norbixin). In rats, no toxicity was detected by plasma chemistry. In mice, norbixin induced an increase in plasma alanine aminotransferase activity (ALT) while both norbixin and annatto extract induced a decrease in plasma total protein and globulins (P < 0.05). However, no signs of toxicity were detected in liver by histopathological analysis. No enhancement in DNA breakage was detected in liver or kidney from mice treated with annatto pigments, as evaluated by the comet assay. Nevertheless, there was a remarkable effect of norbixin on the glycemia of both rodent species. In rats, norbixin induced hyperglycemia that ranged from 26.9% (8.5 mg/kg norbixin, to 52.6% (74 mg/kg norbixin, P < 0.01) above control levels. In mice, norbixin induced hypoglycemia that ranged from 14.4% (0.8 mg/kg norbixin, P < 0.05) to 21.5% (66 mg/kg norbixin, P < 0.001) below control levels. Rats and mice treated with annatto pigments showed hyperinsulinemia and hypoinsulinemia, respectively indicating that pancreatic beta-cells were functional. More studies should be performed to fully understand of how species-related differences influences the biological fate of norbixin.

Proliferation and differentiation of Trypanosoma cruzi inside its vector have a new trigger: redox status.

Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H2O2 and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. β-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle.

Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production

Abstract Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector‐parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p‐(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria‐targeted antioxidant ((2‐(2,2,6,6 Tetramethylpiperidin‐1‐oxyl‐4‐ylamino)−2‐oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme‐induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously‐indicating the hyperpolarization and increase of potential of the mitochondrial membrane (&Dgr;&PSgr;m). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme‐treated epimastigotes decreased their oxygen consumption, and increased the complex II‐III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme‐induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation. Graphical abstract Figure. No Caption available. HighlightsHeme decreases oxygen consumption inhibiting cytochrome c oxidase activity.Heme increases mitochondrial ROS and mitochondrial membrane potential in T. cruzi epimastigotes.Mitochondrial ROS induced by heme favours epimastigote proliferation.

Trypanosomatid essential metabolic pathway: New approaches about heme fate in Trypanosoma cruzi

Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and depends on hosts for its nutritional needs. Our group has investigated heme (Fe-protoporphyrin IX) internalization and the effects on parasite growth, following the fate of this porphyrin in the parasite. Here, we show that epimastigotes cultivated with heme yielded the compounds α-meso-hydroxyheme, verdoheme and biliverdin (as determined by HPLC), suggesting an active heme degradation pathway in this parasite. Furthermore, through immunoprecipitation and immunoblotting assays of epimastigote extracts, we observed recognition by an antibody against mammalian HO-1. We also detected the localization of the HO-1-like protein in the parasite using immunocytochemistry, with antibody staining primarily in the cytoplasm. Although HO has not been described in the parasites genome, our results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets.