Snake venom L-amino acid oxidases (LAAOs) are flavoproteins, which perform diverse biological activities in the victim such as edema, myotoxicity and cytotoxicity, contributing to the development of clinical symptoms of envenomation. LAAO cytotoxicity has been described, but the temporal cascade of events leading to cell death has not been explored so far. This study evaluates the involvement of LAAO in dermonecrosis in mice and its cytotoxic effects in normal human keratinocytes, the major cell type in the epidermis, a tissue that undergoes extensive necrosis at the snakebite site. Pharmacological inhibition by the antioxidant NAC (N-acetyl cysteine) prevented B. atrox venom-induced necrosis. Consistent with the potential role of oxidative stress in wounding, treatment with purified LAAO decreased keratinocyte viability with an Effective Concentration (EC50) of 5.1 μg/mL. Cytotoxicity caused by LAAO was mediated by H2O2 and treated cells underwent autophagy, followed by apoptosis and necrosis. LAAO induced morphological alterations that precede cell death. Our results show the chronological events leading to cell death and the temporal resolution from autophagy, apoptosis and necrosis as distinct mechanisms triggered by LAAO. Fluorescently-labelled LAAO was efficiently and rapidly internalized by keratinocytes, suggesting that catalysis of intracellular substrates may contribute to LAAO toxicity. A better understanding of LAAO cytotoxicity and its mechanism of action will help to identify potential therapeutic strategies to ameliorate localized snake envenomation symptoms.
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We would like to thank Sileia Gontijo from Fundação Ezequiel Dias for kindly helping with LAAO purification and Stephen Rothery and all staff from Facility for Imaging by Light Microscopy (FILM) from Imperial College London for the assistance in images acquisition and processing. This work was supported by the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES – project “Toxinologia” no.
23038000825/2011-63), by Brazilian National Council for Scientific and Technological Development (CNPq– “Pesquisador Visitante Especial” - PVE no. 71/2013, process: 407266/2013-5) and by funds of the INCTTOX Program of CNPq, the Welcome Trust Pathfinder grant (201054/Z/16/Z) and the Newton Fund/FAPEMIG MRC (number MR/M026310/1).