Hepatocytes are highly specialized polarized liver cells in which most liver-specific functions are performed. Isolation of primary hepatocytes is a set of stressors, among which the enzymatic degradation of intercellular junctions stands out. Due to stress upon isolation, a reversible stress adaptation called pre-apoptotic cell stress response (PACOS) occurs in primary rat hepatocytes and through inactivation of caspase-9 prevents apoptosis triggering through the intrinsic pathway. PACOS is present immediately after isolation and dissipates by day 6 in culture. The aim of our study was to examine/investigate PACOS, its impact on hepatocyte function, hepatocyte response to additional stressors (hypoxia and reoxygenation), and whether precision-cut liver slices can be used as a control to primary hepatocyte cell cultures in the PACOS study. In our work we demonstrated that primary hepatocytes survive in culture and maintain the level of metabolic activity and the ability of urea production. We confirmed the constant occurrence of PACOS in isolated primary hepatocytes. We investigated the induction of apoptosis in primary hepatocytes and demonstrated that it is normally activated through the intrinsic pathway. Precision-cut liver slices can serve as a control to primary hepatocytes with respect to PACOS, as caspase-9 can be efficiently activated in the slices. For the first time, we have shown that the consequences of stress in hepatocyte isolation are two reversible responses to stress: induction of an endogenous antioxidant response and PACOS. Both are triggered by increased ROS, which mediate cell survival and normal function. In addition, we showed that stress-adapted primary hepatocytes are less susceptible to triggering apoptosis after hypoxia/reoxygenation than hepatocytes in which PACOS and antioxidant response are no longer present. Primary hepatocytes are a relevant and, with some limitations, appropriate model to study detoxification, metabolism, and potential therapeutics. Based on the results of our study, we emphasize that stress-adapted hepatocytes respond differently to additional stressors than hepatocytes in vitro and in vivo, in which the stress responses are not induced. Understanding the mechanisms of ROS and stress responses in primary hepatocytes is crucial for planning research, interpreting the results, applying them in vivo and, ultimately, for understanding liver physiology, pathophysiology, and devising interventions for prevention and treatment of liver pathology.
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