Evaluation of Iranian snake ‘Macrovipera lebetina’ venom cytotoxicity in kidney cell line HEK-293

Background: Envenomation by Macrovipera lebetina (M. lebetina) is characterized by prominent local tissue damage, hemorrhage,abnormalities in the blood coagulation system, necrosis, and edema. However, the main cause of death after a bite by M. lebetina hasbeen attributed to acute renal failure (ARF). It is unclear whether the venom components have a direct or indirect action in causingARF. To investigate this point, we looked at the in vitro effect of M. lebetina crude venom, using cultured human embryonic kidney(HEK-293) mono layers as a model.Methods: The effect of M. lebetina snake venom on HEK-293 growth inhibition was determined by the MTT assay and the neutralred uptake assay. The integrity of the cell membrane through LDH release was measured with the Cytotoxicity Detection Kit.Morphological changes in HEK-293 cells were also evaluated using an inverted microscope.Results: In the MTT assay, crude venom showed a significant cytotoxic effect on HEK-293 cells at 24 hours of exposure and wasconfirmed by the neutral red assay. Also, at 24 hours exposure, crude venom caused a non-significant increase in LDH activity of theculture medium at concentrations above 20 μg/ml. Various morphological abnormalities were observed in cells exposed to the venomand showed loss of their common polygonal shape, appearing as several roughly rounded cells of variable size. The M. lebetina crudevenom induced detachment of cells from the plate.Conclusion: Based on the results obtained in this study, it can be concluded that the Iranian snake M. lebetina venom causes a cytotoxiceffect on kidney tissue not by necrotic mechanism but rather by secondary effects, including hypotension, hemolysis, hemoglobinuria,rhabdomyolysis, myoglobinuria and disseminated intravascular coagulation (DIC), which may lead to ARF.