EFFECTS OF Crotalus durissus terrificus VENOM ON MITOCHONDRIAL ACTIVITY 

SILVA, N.S.; PRIANTI, A.C.; RIBEIRO, W.; LOPES-MARTINS, R.A.; COGO, J.C.;  PACHECO–SOARES, C.

Serpentário do Centro de Estudos da Natureza (CEN), Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, SP, Brasil.

Snake venoms can affect a variety of intracellular metabolic pathways. In this work, we examined the effects of Crotalus durissus terrificus (South American rattlesnake) venom on the mitochondrial activity of cultured cells. CHO-K1 cells (1x10⁶ cells/ml) were cultured  overnight on sterile coverslipsin 24-well plates (NUNC) containing minimum essential medium supplemented with 5% (v/v) fetal calf serum (FCS), 100 U penicillin/ml and 100 M streptomycin (Gibco) at 37ºC in a humidified 5% CO₂ atmosphere. Venom diluted in PBS was added to the cells (final conc. 5, 10 and 20 mg/ml) which were then incubated as above for up to 60 min. Subsequently, the cells were washed with PBS and then incubated with the fluorescent probe JC-1 (5,5´,6,6´-tetrachloro-1,1´,3,3´,-tetraethyl-benzimidazol-carbocyanine iodide, 0.1 mM) for 10 min. After washing with PBS, the cells were fixed in 4% paraformaldehydein  0.1 M PHEM buffer, pH 6.8, rinsed with PHEM buffer and the coverslips then mounted on slides containing N-propyl-gallate. The slides were analyzed by epi-fluorescence using a LeicaDMLB microscope and photographed with a Leica MPS30 camera. After incubation with 5 mg of venom/ml for 60 min, the cells showed high mitochondrial activity with no change in numbers compared to non-treated cells. At concentrations of 10 and 20 mg/ml, the venom altered mitochondrial activity and caused time-dependent cell death.

Financial support: FVE-UNIVAP, FAPESP.

CORRESPONDENCE TO:

José Carlos Cogo, Rua Pirassununga 141, Apto. 53, São José dos Campos, SP, Brasil, Email: jccogo@univap.br