Comparative study of the in vitro cytotoxic activity of two rear-fanged snake venoms against 3T3 fibroblasts

MARIA ELISA PEICHOTO; FLAVIO LUIZ TAVARES; SARAH W. L. JONES; GREGORY DEKREY; STEPHEN P. MACKESSY

Foz do Iguaçu

Congreso; 44º Congresso Brasileiro de Farmacologia e Terapeutica Experimental; 2012

Sociedade Brasileira de Farmacologia e Terapeutica Experimental

Introduction: Relatively few studies have investigated the composition and biological activities of the venoms of rear-fanged snakes. Comparative analyses of these venoms may provide a better understanding of their toxicity and may reveal novel compounds with biomedical applications. In this study, we conducted a comparative analysis of the cytotoxic activity against 3T3 fibroblast cell line of the venom of the South American rear-fanged snakes Philodryas patagoniensis (PpV), and the venom of the North American rear-fanged snake Trimorphodon biscutatus lambda (TblV). Taking into account that one of the most significant differences between both opisthoglyphous "colubrid" snake venoms tested is the presence of phospholipase A2 (PLA2) activity in TblV only (Peichoto et al., Toxicol. Lett. 196S, S347), we also investigated the cytotoxicity induced by trimorphin, a PLA2 from this venom. Methods: Both venoms were dissolved in PBS (pH 7.4) and filtered through a Millipore filter (0.22 µm). Trimorphin from TblV was purified in two chromatographic steps: size exclusion chromatography on a TSKgel G2000 SWXL column (TOSOH Bioscience LLC, Tokyo, Japan) and reversed-phase chromatography on a Protein and Peptide C18 column (VYDAC, Hesperia, CA, USA), using a previously described procedure (Peichoto et al., Toxicon 58, 28, 2011). In order to verify their possible cytotoxic effects, both venoms and the trimorphin fraction were incubated in a culture of 3T3 fibroblasts for the time of 72 hours. Cell proliferation/viability was determined colorimetrically by the MTT (bromuro de 3-(4,5-dimetiltiazol-2-yl)-2,5-difeniltetrazolio) assay, according to the manufacturer's instructions (ATCC, Manassas, VA, USA). Results: Both crude venoms induced cytotoxic activity on 3T3 fibroblasts. PpV induced a dose-dependent response, producing 96.6 ± 5.1% cytotoxicity at a concentration of 12 µg/mL. However, TbLV showed a more complex response, exhibiting an intriguing hormetic effect. Thus, TbLV at a concentration of 24 µg/mL induced cytotoxic activity (96.6 ± 6.3%), but lower concentrations stimulated proliferation of fibroblasts. Trimorphin from TbLV produced an hormetic dose-response effect comparable to the whole venom, inducing a cytotoxic activity of 87.3 ± 9.7% at a concentration of 5 µg/mL. For this reason, this isolated protein may be considered as the main responsible for the hormetic cytotoxicity induced by the whole venom. Discussion: This is the first report showing the in vitro effect of rear-fanged snake venoms on fibroblast proliferation. These findings may help to understand the local tissue damage induced by these venoms, which is characterized by a complex sobreposition of pathophisiologic phenomena, such as hemorrhage, inflammation and repair. Moreover, trimorphin may be considered as a potential tool to study the hormetic effect of cell death and proliferation. Financial Support: A postdoctoral fellowship for MEP by the Fulbright Commission and CONICET is gratefully acknowledged. Additional financial support was provided by CONICET (PIP 114-200801-00088, to MEP) and ANPCyT (PICT-2010-1908, to MEP) from Argentina, and by a Bioscience Discovery Evaluation Grant from the Colorado Office of Economic Development and International Trade (to SPM).