Citotoxicity of baltergin on endothelial cells

VAN DE VELDE, ANDREA C.; GAY CLAUDIA C.; LEIVA, LAURA C.A.; BUSTILLO SOLEDAD

Chascomús

Congreso; XVII Jornadas de la Sociedad Argentina de Biología; 2014

Sociedad Argentina de Biología

The most important events associated to the development of local tissue damage in viperid snakebites depend on a limited number of components. Metalloproteinases type P-III are multiple domain enzymes whose principal toxic effects are due to disruption of the hemostatic system. Hemorrhage, a relevant local manifestation in envenomation by Bothrops genus, may result from a direct action of these metalloproteinases upon extracellular matrix components. In this work, the mechanism was investigated on a target cell line, thus cytotoxic activity on endothelial cells induced by baltergin, a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom, was evaluated. Briefly, cells (tEnd cell line) were exposed to different concentrations of baltergin (50-200 g/ml) for 3 h at 37ºC-5% CO2, toxicity was quantitatively assayed by crystal violet method. The percentage of cell detachment was registered and its DC50 was calculated. Cytolisis was determined by release of the cytosolic enzyme lactate dehydrogenase (LDH). Acridine orange?ethidium bromide double staining was performed to evaluate morphological alterations. Results indicate that the metalloproteinase induces a dose-dependent detachment of cells, DC50= 173.17μg/mL, with no cell lysis (LDH not detected). Fluorescence analysis showed typical features of apoptosis, chromatin condensation and nuclear fragmentation, cell shrinkage and membrane blebbing. Therefore, baltergin trigger cell detaching from the surrounding extracellular matrix and possibly induce a particular type of apoptosis denominated "anoikis".