Abstract
The spiders of the Loxosceles genus (called brown or violin spiders) are of medical relevance in several countries due to the many human envenomation cases reported. The main component of Loxosceles venom is the enzyme sphingomyelinase D (SMase D), which is responsible for the local and systemic effects induced by the whole venom. Here, we investigated the cytotoxic and genotoxic effects caused by Loxosceles laeta venom and SMase D on human keratinocytes to better understand the dermonecrosis development mechanism. Our findings indicate that whole venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage. These effects appear to be dependent on the binding of SMase D to the cell surface, although the complete pathway triggered as a result of the binding still needs to be elucidated. Moreover, after SMase D treatment, we observed the presence of histone γH2AX, suggesting that the cells are undergoing DNA repair. Moreover, when ATR kinase was inhibited, the cell viability of human keratinocytes was decreased. Together, our findings strongly suggest that L. laeta venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage in human keratinocytes. Additionally, the induced DNA damage is repaired through the activation of an apparent ATR-mediated DNA-damage response. This knowledge may contribute to a better understanding of the behaviour of human keratinocytes during cutaneous loxoscelism, a condition that affects thousands of people around the world.
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Acknowledgments
The authors would like to thank the São Paulo Research Foundation (FAPESP)/Center of Toxins, Immune Response and Cell Signaling (CeTICS) and the National Council for Scientific and Technological Development (CNPq) for their support.
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This work was supported by FAPESP/CeTICS under grant 2013/07467-1. MSdS and PHL are fellows from FAPESP (postdoctoral fellowship grants 2014/24170-5 and 2015/17053-5, respectively). DVT and MCE are fellows from CNPq (grants 301358/2017-6 and 306199/2018-1, respectively).
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MSdS, PHL, MCE, and DVT conceived the rationale of the experimental design. MSdS and PHL carried out the experiments. MSdS and PHL performed statistical analyses. MSdS and PHL wrote the manuscript with essential contribution from MCE and DVT. All authors read and approved the final version of the manuscript. DVT and MCE supervised the project.
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da Silva, M.S., Lopes, P.H., Elias, M.C. et al. Cytotoxic and genotoxic effects on human keratinocytes triggered by sphingomyelinase D from Loxosceles venom. Arch Toxicol 94, 3563–3577 (2020). https://doi.org/10.1007/s00204-020-02830-2
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DOI: https://doi.org/10.1007/s00204-020-02830-2