Caracterização de mecanismos citotóxicos induzidos pelo veneno de Bothrops atrox e de sua toxina purificada L-aminoácido oxidase

Abstract

Bothrops spp. are responsible for 90% of snakebites in Brazil. Bothropic venom is a complex mixture of molecules enriched with peptides and proteins. An important component of this venom are L-amino acid oxidases (LAAOs), enzymes that exhibit multiple pharmacological activities such as hemorrhage, edema and cytotoxicity and consequently, contribute to the development of clinical envenomation symptoms. Animal venoms and isolated components characterization contributes for elucidation of the mechanism of action and opens possibilities for treatment improvement. In addition, snake venoms are a rich source of molecules with biotechnological potential which needs to be explored. The aim of this study was to evaluate changes caused by B. atrox venom and LAAO in keratinocytes. LAAO purification was performed through three chromatographic steps: molecular exclusion, ion exchange and heparin affinity. Purified LAAO presented 62.5kDa and was more stable when kept at -80 oC. B. atrox venom and LAAO were able to decrease cell viability in a concentration-dependent manner. Cytotoxic dose was defined as 5.5 g/mL for the venom and 5.1 g/mL for LAAO. LAAO-treated keratinocytes underwent autophagy (after 1.5 hours) followed by apoptosis and necrosis (after 12 and 24 hours). Regarding morphology, B. atrox venom induced cell and colony retraction, cell clusters formation, cell elongation, cadherin and actin disruption. Cells treated with LAAO showed decreased substrate adhesion, abnormal actin extensions and disruption of cadherin and actin bundles. Cytotoxicity and cytoskeleton alterations caused by LAAO were mediated by H2O2, since the addition of catalase decreased the toxic effects observed. It was detected an increase in intracellular ROS after addition of LAAO which corroborates with the possible involvement of H2O2 in these activities. Results obtained in this work contributes to a better understanding of B. atrox venom and LAAO toxicity, helping to elucidate their mechanism of action during envenoming.