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Putative defense mechanisms of Trypanosoma cruzi against heme oxidative stress
Nogueira, N.P.A.*, Laranja, G. A.T. ¶, Eitler, R. B. ¶, Monteiro-Leal, L. H. §, Daulmau, S. R. ¶, Paes, M. C. ¶
* Instituto de Bioquímica Médica – UFRJ, ¶ Departamento de Bioquímica – UERJ, § Departamento de Histologia e Embriologia - UERJ
Trypanosoma cruzi epimastigotes propagates in the triatomine insect crop. These hematophagous insects usually ingest in a single meal about 10mM heme bound to hemoglobin. Heme is a powerful generator of reactive oxygen species (ROS), which can damage a variety of biomolecules. Our group has demonstrated that the addition of heme in the medium (0 to 1mM) increased the parasite proliferation in a dose-dependent manner. In this work we investigated the contribution of the antioxidant enzyme superoxide dismutase (SOD) and the low molecular mass antioxidant glutathione (GSH) to the parasite survival in the presence of different concentrations of heme. GSH is an important precursor of trypanothione. In order to access this hypothesis we added to the medium diethyldithiocarbamate (DETC), a superoxide dismutase inhibitor, or buthionine sulfoximine (BSO), the inhibitor of gamma-glutamylcysteine synthase (gamma-ECS). T. cruzi epimastigotes Dm28c strain, at the exponentional phase, were maintained in BHI supplemented with 10% FCS at 28ºC without addition of heme for 14 days. Afterwards cells were incubated in the absence or in the presence of different concentrations of heme, 5µM of DETC or 2mM of BSO, as well as 5mM GSH. Growth of epimastigotes for 5 days was evaluated in the presence of these drugs. A drastic decrease in parasites growth was observed when treated with 2mM of BSO and heme with the addition of 5mM GSH being able to reverse this effect. The treatment with DETC also greatly decreased epimastigotes proliferation caused by heme addition. ROS formation was accessed by microscopy and flow cytometry analysis of CM-H2DCFDA fluorescence. We observed a slight increase of basal ROS formation in the presence of 30µM of heme, and a drastic increase of ROS with the addition of 100µM of heme. Pre incubation with 5mM DETC showed an increment in ROS formation. The incubation of parasites with BSO also increased ROS formation, which was reversed in the presence of GSH. Our data suggest that, T. cruzi likely developed effective mechanisms to efficienly cope with high concentrations of heme found in its enviroment and these defenses are involved with GSH and SOD activity. Supported by FAPERJ, CNPQ, CAPES
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