Estudo da atividade enzimática e dos efeitos do veneno da serpente Bothropoides insularis sobre macrófagos RAW 264.7 in vitro

Abstract

Around the world, there are recorded over 3 million accident involving bites of venomous animals per year, of which 125 to 150 thousand culminate in death. In Brazil, most cases occur with snakes from Bothrops and Bothropoides genus, causing several local and systemic complications, such myotoxicity, disseminated intravascular coagulation, cytotoxicity, acute renal failure and sepsis. Bothropoides insularis is a snake from Queimada Grande Island, whose venom shows pronounced toxicity. However, its effect over cells with defense function remains unclear, as well as how these effects can influence the toxicity observed in vivo. The present study aimed to investigate the cellular changes induced by Bothropoides insularis whole venom (BinsVT) over murine macrophage from RAW 264.7 lineage. In this context, colorimetric tests were performed to determine proteolytic activity and the production of hydrogen peroxide in vitro. The results showed high catalytic activity in both tests, suggesting that the biological effects of this venom may be related to the presence of enzymes such metalloproteinases (svMPs) and L-amino acid oxidases (LAAOs) at concentrations relevant in these experimental conditions. The determination of the cytotoxic potential was conducted by MTT reduction assay, a method of assessing redox metabolism, after 2, 6, 12 and 24 hours of incubation. It was observed cytotoxic effect at high concentrations in a time-dependent way, with cell death more evident at 200 and 100 µg/mL after 12 and 24 hours of treatment. In lower concentrations, there was a gradual increase in cell viability, reaching around 200% of cell viability in groups treated for 24 hours. This result suggests that BinsVT possess proliferative effect over these cells. Then, lactate dehydrogenase (LDH) activity was determined in culture supernatants from experimental groups for investigation of cell lysis induced by BinsVT. It was observed a significant increase in enzymatic activity in all groups, suggesting the coexistence of fractions with cytotoxic and proliferative effect and that concentration and exposure time determine its outcomes. For morphological evaluation of RAW 264.7 cells after exposure to BinsVT, the experiments were performed on the surface of coverslips for staining with May-Grunwald Giemsa method. The experimental groups were analyzed by optical microscopy and the most representative morphological characteristics were photographed. Various morphological changes were observed, such as appearance of cellular debris and bare nuclei, vacuolated cells, reduced cell volume and increased cytoplasmic projections. Finally, the mechanism of cell death induced by BinsVT was assessed by flow cytometry, by staining with propidium iodide (PI) and annexin V-FITC. The analysis revealed the presence of apoptosis and necrosis, and the appearance of doubly labeled cells with Annexin-FITC and PI, indicating the occurrence of late apoptosis. In conclusion, BinsVT has a cytotoxic effect on RAW 264.7 cell, which necrotic and apoptotic mechanisms, besides stimulatory effect on these cells in dose-and time-dependent ways. These effects may be related to the enzymatic activities found in vitro.