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Cytoskeletal rearrangement and cell death induced by Bothrops alternatus snake venom in cultured Madin–Darby canine kidney cells

2007; NRC Research Press; Volume: 85; Issue: 5 Linguagem: Inglês

10.1139/o07-067

1208-6002

Juliana Nascimento, Gilberto Carlos Franchi, Alexandre E. Nowill, Carla Beatriz Collares‐Buzato, Stephen Hyslop,

Healthcare and Venom Research

Bothrops snake venoms cause renal damage, with renal failure being the main cause of death in humans bitten by these snakes. In this work, we investigated the cytoskeletal rearrangement and cytotoxicity caused by Bothrops alternatus venom in cultured Madin–Darby canine kidney (MDCK) cells. Incubation with venom (10 and 100 µg/mL) significantly (p <0.05) decreased the cellular uptake of neutral red dye after 1 and 3 h. Venom (100 µg/mL) also markedly decreased the transepithelial electrical resistance (R T ) across MDCK monolayers. Staining with rhodamine-conjugated phalloidin revealed disarray of the cytoskeleton that involved the stress fibers at the basal cell surface and focal adhesion-associated F-actin in the cell–matrix contact region. Feulgen staining showed a significant decrease in the number of cells undergoing mitosis and an increase in the frequency of altered nuclei. Scanning electron microscopy revealed a decrease in the number of microvilli and the presence of cells with a fusiform format. Flow cytometry with annexin V and propidium iodide showed that cell death occurred by necrosis, with little apoptosis, a conclusion supported by the lack of DNA fragmentation characteristic of apoptosis. Pretreating the cells with catalase significantly attenuated the venom-induced loss of viability, indicating a possible involvement of H 2 O 2 in the cellular damage; less protection was observed with superoxide dismutase or N ω -nitro-l-arginine methyl ester. These results indicate that Bothrops alternatus venom is cytotoxic to cultured MDCK cells, possibly via the action of reactive oxygen species. This cytotoxicity could contribute to nephrotoxicity after envenoming by this species.