Poster 129.  Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil.  Abstracts...  J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.488.

 

 

Cytotoxic Action Of Bothrops alternatus Snake Venom In Cultured MDCK Cells

 

Nascimento, J.M.1,2; Collares-Buzato, C.B.3; Hyslop, S.1

 

1 Department of Pharmacology, Faculty of Medical Sciences and Departments of 2 Biochemistry and 3 Histology and Embryology, Institute of Biology, State University of Campinas (UNICAMP), 13083-970, Campinas, SP, Brazil.

 

Bothropssnake venoms are cytotoxic to a variety of cell types in vitro and in vivo. Most of these effects are mediated by enzymes present in the venom, particularly L-amino acid oxidase, metalloproteinases and phospholipase A2. In this work, we investigated the cytotoxicity of Bothrops alternatus venom in cultured Madin-Darby canine kidney (MDCK) cells. Incubation with venom (10 and 100 mg/ml) significantly (p<0.05) decreased the cellular uptake of neutral red dye by 25.5 ± 7% (n=12; mean±SD) and 43.4 ± 12.2% (n=12), respectively, after 1 h, and by 65.3 ± 7.2% (n=12) and 85.3 ± 10% (n=12) after 3 h. The transepithelial electrical resistance (RT) was measured to assess whether the venom altered the epithelial barrier function. Venom (100 mg/ml) decreased the RT across MDCK monolayers from 319.7 ± 52.3 to 52.0 ± 14.5 W/cm2 after 2 h, and after 4 h the RT were zero (n=3 each). 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-to-matrix contact region. These effects were particularly marked at a venom concentration of 100 mg/ml. Staining with the Feulgen reaction revealed a significant (p<0.01) decrease in the percentage of cells undergoing mitosis (decreases of 77.5% and 85.6% with 10 mg/ml and 78.6% and 95.5% with 100 mg of venom/ml after 1 and 3 h, respectively), and an increase in the frequency of altered nuclei. Staining with toluidine blue confirmed these findings. These results indicate that B. alternatus venom is cytostatic and cytotoxic to cultured MDCK cells and could partly explain the nephrotoxicity seen after envenoming by this species.

 

Financial support: FAPESP

 

Correspondence to: jminardi@unicamp.br