*2.5. Bile Acid Quantification*

The content of different bile acids was detected via UPLC-MS/MS in the cell culture supernatant and cell lysates. Thus, HepaRG cells were seeded at a density of 0.2 × <sup>10</sup><sup>6</sup> cells per well in 6-well plates. After the normal proliferation and differentiation period of four weeks, the FBS level in the medium was set to 2% for 48 h. DMSO concentration of 1.7% was kept to reach the maximum expression of various CYP enzymes and transporters to ensure a strong metabolic activation of PAs [42]. Before the incubation with PAs, the cells were washed once with PBS (room temperature) to remove the traces of FBS from cell culture medium. The incubation with PAs was conducted with FBS-free differentiation medium to reduce the background signal from bile acids contained in FBS, as described in the study by Sharanek et al. [39]. The volume of incubation medium (differentiation medium containing the respective amount of PAs) was reduced by 50% to 1 mL per well, to increase the concentration of excreted bile acids in the supernatant. Cells were incubated in triplicates to pool the medium and cells of three wells during harvesting, in order to facilitate analytical determination of bile acids present only at low levels. After incubation for 48 h, the medium was collected and stored at −80 ◦C. Cells were washed twice with PBS, trypsinized for 15 min at 37 ◦C and collected (Trypsin-EDTA, Capricorn Scientific, Ebsdorfergrund, Germany). After a second washing step with 1 mL PBS and centrifugation at 250× *g* for 5 min, the supernatant was discarded and the cell pellets were stored at −80 ◦C.

Bile acid analysis was conducted using UPLC coupled to tandem mass spectrometry as described previously [45]. Briefly, bile acids in cell lysates and medium were extracted by adding methanol containing deuterated internal standards. After vortexing and centrifugation, the methanol was evaporated under a stream of nitrogen, the samples were reconstituted in methanol:water [1:1 *v*/*v*] and injected onto the UPLC system (Infinity1290, Agilent Technologies, Palo Alto, CA, USA). Separation was made on a Kinetex C18 column (Phenomenex, Torrance, CA, USA) using H2O and ACN as mobile phases. Detection was made in negative mode using a QTRAP 5500 mass spectrometer (Sciex, Concord, Canada). Three independent experiments were performed.

#### *2.6. Statistical Analysis*

For statistical analysis, SigmaPlot 14.0 software (Systat Software, Erkrath, Germany) was used. Statistically significant differences in a concentration series were calculated using a one-way ANOVA. Following the differences of the treated samples versus the respective solvent control were tested using Dunnett's post hoc analysis. Differences that were statistically significant are indicated by \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

### **3. Results**
