L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes

Fernanda Costal-Oliveira,Dan E Vivas-Ruiz,Stephanie Stransky,Armando Yarlequé,Dayane L Naves De Souza,Carlos Chávez-Olórtegui,Eladio Flores Sanchez,Clara Guerra-Duarte,Vania M M Braga

doi:10.1038/s41598-018-37435-4

Abstract

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.

Highlights

Snakebites constitute a public health problem worldwide and are considered a priority neglected tropical disease by the World Health Organization[1]
Mice injected with B. atrox venom presented dermonecrosis, in addition to hemorrhage (Supplementary Fig. S1) whilst animals that received B. atrox venom + N-acetyl cysteine (NAC) did not present any of these symptoms
These data indicate that blocking oxidative stress strongly prevented the wounding caused at the venom injected site, suggesting that L-amino acid oxidases (LAAOs)’s contribution to local oxidative events may underlie tissue injury caused by B. atrox venom

Summary

Introduction

Snakebites constitute a public health problem worldwide and are considered a priority neglected tropical disease by the World Health Organization[1]. Cell death mechanisms (i.e. autophagy, apoptosis or necrosis) are classified according to morphological structure, enzymological criteria, functional aspects or immunological characteristics. Interplay between these distinct pathways is often observed[32]. Autophagy has been previously reported as a cellular response against snake toxins and, in some cases, to contribute to apoptotic cell death[34,35]. Cell death by apoptosis or necrosis has been reported in cells treated with specific snake toxins such as lectin, LAAO and metalloproteinases[17,36,37,38]. Organelle membrane damage releases proteolytic enzymes from lysosomes and lead to cell destruction[39]

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Results

Conclusion