Human pathophysiological concentrations of bile salts induce necrosis in primary human hepatocytes (398.1)

Abstract

Liver injury during cholestasis is thought to develop due to plasma and liver accumulation of toxic bile salts (BS), such as glycochenodeoxycholate (GCDC), which leads to apoptosis. This paradigm relies on the use of cytotoxic concentrations of GCDC to mimic cholestasis in rat hepatocytes or human hepatoma lines. Instead, this study assessed how pathophysiological concentrations of BS measured in cholestatic patients affected primary human hepatocytes (PHH). Individual BS levels were determined by UPLC/MS in 50 patients with extrahepatic cholestasis, with or without concurrent liver injury. Patients with liver injury had increased BS levels in serum, while biliary levels decreased, implicating development of bile infarcts, which were confirmed by histology. Human hepatocytes isolated from resection material, retained functional BS uptake via Na+ ‐dependent and ‐independent transporters, measured by uptake of taurocholate. Exposing PHH to GCDC for 6 hours resulted in little cell death, measured by LDH release, until exposure to concentrations >1mM GCDC. In contrast to data in rat hepatocytes, no dose of GCDC produced an increase in caspase‐3 activity in PHH, and pretreatment with a pan‐caspase inhibitor did not protect. Finally, PHH were treated with complete BS mixtures derived from patient serum or bile. While serum values resulted in no increase in cell death for up to 24 hours after application, bile mixtures caused direct toxicity. These data suggest PHH are more resistant to human BS concentrations than rat hepatocytes, and die by necrosis. Moreover, biliary concentrations of BS are required for acute cell death, implicating biliary infarction as the initiator of injury.