The Role of Reactive Oxygen Species in Crotalus atrox Venom-induced Cell Death
Venomous snake bites impact humans all around the world. Anti-venom treatments mitigate systemic effects such as vascular hemorrhage, platelet aggregation inhibition, and the activation of inflammatory mediators. However, hemorrhagic snake venom also causes a loss of cellular adhesion to extracellular matrix components resulting in massive local tissue damage. To better understand the mechanism in which venom induces local tissue damage, human embryonic kidney cells (HEKS) were grown on PEI then stimulated with 500μg/ml Crotalus atrox (CA) venom for 4 and 10 hours. Alamar Blue assays were used to measure cell viability and results suggest a 15±8.6% (p<0.05) and 59±10.7% (p<0.05) reduction in cell viability at 4 and 10 hours, respectively. Cells stimulated with 500μg/ml venom for 10 hours stained 98±2.2% (p<0.05) positive for Trypan blue, suggesting the venom reduces membrane integrity. Identical treatment in the presence of 200 units PEG-catalase (PC) increased viability by 37±5.7% (p>0.001) compared to cells stimulated with venom alone. 2’,7’-Dichlorofluorescin-diacetate (DCF-DA) was used to quantify reactive oxygen species during venom stimulation. HEK cells stimulated with 50μg/ml Crotalus atrox venom resulted in a 336-fold increase in ROS-induced fluorescence between 1 and 2 hours (p<0.001). Pre-treating the cells with 200 Units peg-catalase for 2 hours before venom stimulation resulted in 425-fold decrease in ROS-induced fluorescence that persisted over the 4 hour stimulation period (p<0.0001). Peg-catalase resulted in a greater decrease in fluorescence over time than other treatments including N-acetyl cysteine (NAC), SOD1 inhibitor LCS-1, and NOX inhibitor VAS2870. This suggests hydrogen peroxide is 3 produced during venom induced injury and plays a critical role in venom mediated cell death.
reactive oxygen species, oxidative stress, anoikis, snake venom, Biology, Integrative Biology
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