*Article* **Pyrrolizidine Alkaloids Disturb Bile Acid Homeostasis in the Human Hepatoma Cell Line HepaRG**

**Julia Waizenegger 1,2, Josephin Glück 1, Marcus Henricsson 3, Claudia Luckert 1, Albert Braeuning <sup>1</sup> and Stefanie Hessel-Pras 1,\***


**Abstract:** 1,2-unsaturated pyrrolizidine alkaloids (PAs) belong to a group of secondary plant metabolites. Exposure to PA-contaminated feed and food may cause severe hepatotoxicity. a pathway possibly involved in PA toxicity is the disturbance of bile acid homeostasis. Therefore, in this study, the influence of four structurally different PAs on bile acid homeostasis was investigated after single (24 h) and repeated (14 days) exposure using the human hepatoma cell line HepaRG. PAs induce a downregulation of gene expression of various hepatobiliary transporters, enzymes involved in bile acid synthesis, and conjugation, as well as several transcription regulators in HepaRG cells. This repression may lead to a progressive impairment of bile acid homeostasis, having the potential to accumulate toxic bile acids. However, a significant intracellular and extracellular decrease in bile acids was determined, pointing to an overall inhibition of bile acid synthesis and transport. In summary, our data clearly show that PAs structure-dependently impair bile acid homeostasis and secretion by inhibiting the expression of relevant genes involved in bile acid homeostasis. Furthermore, important biliary efflux mechanisms seem to be disturbed due to PA exposure. These mole-cular mechanisms may play an important role in the development of severe liver damage in PA-intoxicated humans.

**Keywords:** pyrrolizidine alkaloids; hepatotoxicity; HepaRG; cholestasis; bile acid
