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 parasite's genome, our results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets.

Melatonin affects conjugation of 4-hydroxynonenal with glutathione in liver of pacu, a hypoxia-tolerant fish.

In cytosol from liver of pacu, Piaractus mesopotamicus, a hypoxia-tolerant fish that dwells in Pantanal, we found an enzyme activity capable of modulating the alkenal 4-hydroxy-2-nonenal (HNE) by conjugating it with glutathione (GST-HNE activity). HNE is a downstream metabolite from the oxidation of polyunsaturated fatty acids by reactive oxygen species arisen from mitochondria of animal cells. HNE production may increase more intensively under oxidative stress. Harmful effects to cell survival may occur when HNE increases over 10(-4) M. Pacus submitted to hypoxia in July (cold season in Pantanal) showed 40% less of this GST-HNE conjugating activity in their liver cytosol. Injecting pacus subjected to hypoxia during the cold season with a summer physiological dose of melatonin caused their liver cytosolic GST-HNE activity to increase up to the levels found in the warm season. From October to March (warm season in Pantanal), pacus are prone to oxidative stress particularly during potamodromous active oxygen-demanding swimming, when they migrate up rivers to spawn. Thus, our findings point out that the higher levels of melatonin in circulation during the summer are important to avoid the increase of 4-HNE inside liver cells of this fish species.

In vitro and in vivo toxicological evaluation of extract and fractions from Baccharis trimera with anti-inflammatory activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Baccharis trimera (Less) DC. (Asteraceae), popularly known in Brazil as "carqueja", have been used in folk medicine to treat gastrointestinal, hepatic and renal diseases, and inflammatory processes as rheumatism. AIM OF THE STUDY: To evaluate the in vitro and in vivo toxicological effects of anti-inflammatory Baccharis trimera aqueous extract and fractions. MATERIALS AND METHODS: Aqueous extract of Baccharis trimera (AEBt) was produced by infusion in boiling water. After lyophylization AEBt was extracted with 80% ethanol, originating the ethanolic supernatant fraction (EFBt) and the aqueous sediment fraction (AFBt). Anti-inflammatory properties of AEBt, EFBt or AFBt (3, 30 or 300 µg/kg b.w.) were evaluated by the carrageenan-induced mouse paw edema using indomethacin (10mg/kg) as positive control. The growth of rat hepatoma cells (HTC) and human embryo kidney epithelial cells (HEK) was determined by protein staining assay. Cytotoxicity was assayed by the tetrazolium salt (MTT) reduction. Cyclosporin was used as reference cytotoxic drug for spleen cells and doxorubicin for HTC and HEK cells. For in vivo toxicological evaluation SW male mice were daily and oral (gavage) treated with extract/fractions at 4.2mg/kg or 42 mg/kg during 15 days. After treatment liver or kidney cells were submitted to comet assay to determine the DNA damage index, and the glutathione S-transferase activity was assayed towards ETHA (class Pi) and CDNB (several classes). Mutagenicity was evaluated by the Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102. RESULTS: The anti-inflammatory effects of EFBt were higher than those of AEBt or AFBt. Mice treatment (3-300 µg/kg) with AFBt reduced the paw edema (3h) at lower levels (29.2-37.3%; P<0.01), than those observed for AEBt (44.7-54.2%; P<0.001), EFBt (49.3-58.2%; P<0.001) or indomethacin (64.6%, P<0.001, 10mg/kg). The growth of kidney cells (HEK) was inhibited by AEBt (IC(50) 182.6 µg/ml), EFBt (IC(50) 78.1 µg/ml) and AFBt (IC(50) 86.2 µg/ml), with lower effects on HTC hepatic cell (IC(50) 308.8 µg/ml, 396.5 µg/ml and 167.9 µg/ml, respectively). As evaluated by MTT test, AFBt exhibited cytotoxicity for HEK cells (IC(50) 372.5 µg/ml), but none for HTC ones; by the way, AFBt stimulated spleen cells (EC(50) 2.2 µg/ml) while cyclosporine, a cytotoxic reference drug inhibited them with IC(50) of 0.42 µg/ml; the IC(50) for doxorubicin for HEK and HTC cells was 0.28 µg/ml and 14.4 µg/ml, respectively, at 96h. No mutagenic potential was observed. Mice treatment with AEBt or AFBt at 42 mg/kg for 15 days altered the kidney relative weight, but not at 4.2mg/kg. Baccharis trimera did not change liver, spleen or popliteal lymph node relative weight. DNA damage index of kidney cells was observed on mice treated with AEBt/AFBt, but not on animals treated with EFBt, while DNA lesions were detected on liver cells only after AFBt treatment. The general activities of hepatic GST and Pi GST were reduced by EFBt and AFBt treatment, respectively. CONCLUSIONS: Baccharis trimera did not show mutagenicity, inhibited the GST activity, a hepatic detoxification enzyme, and induced in vivo (genotoxicity) and in vitro toxicological effects to kidney cells.