Abstract
Species differences in mitochondrial apoptosis control can bias cytotoxicity endpoints and hazard-relevant mechanistic inference in preclinical test systems that rely on intrinsic-apoptosis readouts. We benchmarked steady-state localization and outer mitochondrial membrane (OMM) assemblies of the BCL-2 effectors BAX and BAK across mammals and combined this with functional apoptosis assays in bovine hepatocytes. Across species, bovine cells most closely recapitulated the human baseline state, whereas murine systems displayed distinct BAX/BAK partitioning and reduced capture of mitochondrial BAX in high-molecular-weight OMM assemblies. Functionally, bovine BFH12 cells showed minimal apoptotic response to the BH3 mimetic ABT-737 as a single agent, while responding robustly to doxorubicin and actinomycin D, as assessed by cytochrome c release, caspase processing and activity, and Annexin V/PI cytometry. These findings identify species-dependent regulation of BAX/BAK retrotranslocation and OMM assembly states as a baseline variable that shapes intrinsic-apoptosis pharmacodynamics. Incorporating bovine systems as a complementary non-rodent in vitro platform may improve model selection and pharmacodynamic readouts for compounds expected to engage mitochondrial apoptosis, thereby reducing avoidable translational misclassification in apoptosis-focused pipelines.