Estudo citoquímico de enzimas mitocondriais de Trypanosoma cruzi em culturas de tecido

Abstract

In order to locate the activity of rnitochondrial enzymes in Trypanosoma cruzi in tissue cultures a cytochemical study at light and electron rnicroscopic levels was performed using chick embryo heart rnuscle cultures infected by the Y strain of the parasite. Tetrazolium salts were used as hidrogen aceptors of the reaction to locace Succinate Dehydrogenase (E.C. 1.3.99.1), Isocitrate Dehydrogenase (E.C. 1.1.42), a Glycerophosphate Dehydrogenase (E.C. 1.1.2.1), b Hydroxybutyrate Dehydrogenase (E.C. 1.1.1.30) and NADPH Tetrazolium Redutase. Light microscopy showed an intense reaction. Blue granules (NBT salt) were seen within the cytoplasm of the cells in the region of the nucleus. In parasitized cells they were seen throughout the cytoplasm not occupied by parasites. Isocitrate Dehydrogenase, Succinate Dehydrogenase and NADPH Tetrazoliurn Redutase activity was revealed as 5 to 7 granules in the spherornastigotes (amastigote) and epimastigotes and as 8 to 15 granules in the trypomastigotes. At the ultrastructural level the electrondense product (DS NBT salt) was concentrated in the host cell rnitochondria at the region of the cristae; and inner membrane. In the intracellular forms of the parasite the reaction also occurs at the cristae and inner rnembrane in all regions of the mitochondria. For the lipid pathway enzymes a Glycerophosphate Dehydrogenase and B Hydroxybutyrate Dehydrogenase the reaction was mainly located at the inner membrane of the mitochondria. Few cristae were seen with the reaction. Cytochrome Oxidase (E.C. 1.9.31.1) activity was best revealed with the use of diaminobenzidine (DAB) as hydrogen donor and sodium cacodylate (0.1M, pH 7.2) as buffer. At the host cells and intracellular parasites the activity was observed in the form of brown granules in the cytoplasm and as an electrondense product at the cristae and inner membrane of the mitochondria.