Selectivity screening of cytotoxicity evoked by viper venoms and their toxins after nanofractionation

Cytotoxicity is a major pathological effect that can occur during snakebite envenoming. To better understand the underlying biochemical and molecular mechanisms behind snake venom-induced cytotoxicity, it is essential to use appropriate in vitro tools for bioassaying cytotoxicity evoked by snake venoms. Identifying the toxins causing cytotoxicity is also important in this regard, particularly in the context of developing more effective snakebite treatments. Cytotoxicity induced by venom toxins can result in local pathologies in snakebite victims, which can result in long-term morbidity, and is frequently observed after bites by medically important vipers. In the present study, we optimized and applied an analytical cytotoxicity profiling platform for in vitro cytotoxicity assessment of viper venoms. Using four cell lines (RPTEC/TERT1, HepaRG, iPSC-EC, HaCat), we applied an imaging analysis assay together with resazurin reduction to identify the mechanisms of cytotoxicity at the level of cell necrosis, extracellular matrix (ECM) degradation and/or cell apoptosis. Strong cytotoxic peaks are consistent with ECM-associated cytotoxic effects, as reflected by pronounced reductions in cell area and monolayer integrity. These cytotoxicity bioassays were integrated into nanofractionation analytics and high throughput venomics, which allowed for the identification of viper venom cytotoxins at the biological and chemical levels. Venom profiling showed ECM degradation as the main cytotoxic mechanism, except for Daboia russelii, which induced necrosis and apoptosis in three cell lines. Cytotoxicity largely disappeared after reversed-phase separation, prompting use of non-denaturing SEC in nanofractionation analytics, which revealed strong cytotoxic peaks for Bothrops jararaca and Calloselasma rhodostoma in RPTEC/TERT1 cells. The methodology presented here combined analytical and biochemical tools allowing rapid cytotoxicity profiling of viper venom toxins in parallel with toxin identification.