*3.5. Effects of PAs on Bile Acid Content*

Amounts of primary and conjugated bile acids were measured in HepaRG cells and supernatants after treatment for 48 h with 5, 21, or 35 μM of PA. The primary bile acids synthesized in the liver are cholic and chenodeoxycholic acid, which are further conjugated with glycine or taurine. Within the UPLC/MS method used, the following bile acids were measured: primary bile acids, such as cholic acid and chenodeoxycholic acid, as well as conjugated bile acids, such as taurohyocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycohyocholic acid, glycocholic acid, glycochenodeoxycholic acid, and glycodeoxycholic acid.

For a better comparison of the intracellular and extracellular effects, total bile acid amounts were referred to the solvent control (0.35% ACN), which was set as 100%. The sum of the determined bile acids in each treatment group is depicted in Figure 6. an overall concentration-dependent decrease of bile acids was observed intra- and extracellularly after PA incubation. However, the strongest decrease occurred for the retronecine-type PAs senecionine and echimidine. The treatment of HepaRG cells with the known cholestasis inducer cyclosporine a also resulted in a decrease to 50% and 20% of the extra- and intracellular bile acid content, respectively.

**Figure 6.** Sum of intra- and extracellular bile acid content after PA treatment. Differentiated HepaRG cells were treated under serum-free conditions for 48 h with PAs, as indicated in the figure. Bile acids were quantified as described in the material and methods section. The sum of bile acids was calculated and normalized to solvent control (1.7% DMSO, 0.35% ACN) set as 100%. As the control, cells were treated with 20 μM of known cholestasis inducer cyclosporine A, resulting in a decrease of bile acid amounts to 50% ± 15 in the medium and 20% ± 12 in the cells. Statistical differences were evaluated using one-way ANOVA followed by Dunnett's test: \*\*\* *p* < 0.001.
