3.2.2. Enzymes of Cholesterol and Bile Acid Metabolism

While the expression of the genes for the enzymes of HMG-CoA synthesis (HMGCS1/2) was significantly downregulated, no change was detected in the gene expression of the rate-limiting HMGCR. The expression of INSIG1, responsible for the regulation of HMGCR expression via negative feedback was slightly downregulated (maximum reduction to 16% for 21 μM senecionine). BAAT, an enzyme for the amidation of bile acids with taurine and glycine, showed a significant reduced gene expression down to 5.5% of the solvent control. The decrease of the gene expression levels was stronger, the more cytotoxic and higher concentrated the respective PA was. The transcripts of CYP enzymes directly involved in the catabolism of cholesterol or the de novo synthesis of bile acids (CYP7A1, CYP8B1) were strongly downregulated in a structure-dependent manner, while the expression of *CYP27A1* and *CYP39A1*, relevant for the synthesis of secondary bile acids, were not affected at all.

## 3.2.3. Xenobiotic-Metabolizing Enzymes

The expression levels of all investigated genes of xenobiotic-metabolizing enzymes were affected in a structure- and concentration-dependent manner. For *CYP1A1* and *POR*, however, the decrease of mRNA content was rather weak. For these genes we could still detect around 20–30% of the transcript level of the solvent control after the exposure with the strongest regulating PAs. The transcription of the genes encoding the enzymes CYP1A2, CYP2B6, CYP2E1, CYP3A4, SULT2A1 and UGT2B4 was strongly downregulated.

**Figure 3.** *Cont*.

**Figure 3.** Changes in expression of cholestasis-associated genes after PA treatment of HepaRG cells for 24 h. Differentiated HepaRG were treated with PAs in concentrations of 5, 21 and 35 μM. The Ct-values were evaluated according to the 2-ΔΔCt method by normalizing Ct-values of the respective gene to the housekeeping gene β-glucuronidase (GUSB) and by referring to solvent-treated cells (0.35% ACN and 0.5% DMSO). The cutoff for gene expression regulation was set from 67% to 150% of solvent control. Changes in gene expression within this range were considered not to be biologically relevant. The cells of the heat map show the changes in gene expression of the target genes in percent of the solvent control as means of three replicates. Blue color indicates a downregulation and yellow color an upregulation of gene expression. Expression levels below 10% of solvent control are additionally highlighted by white + (+ expression level below 10%; ++ expression level below 5%; +++ expression level below 1%). Statistics: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 (one-way ANOVA followed by Dunnett's post hoc analysis versus the respective solvent control). Mean values, standard deviations and *p*-values are summarized in the supplemental material (Table S3), as well as the sequences of used primers and the full-length names and synonyms of the detected genes (Table S2). Structural characteristics: retronecine (R), heliotrine (H), otonecine (O) or platynecine (P) type; free base (B), monoester (M), open-chained diester (D(o)) or cyclic diester (D(c)).

#### 3.2.4. Transcription Factors

In general, gene expression of transcription factors was less affected by PA treatment in HepaRG cells. For transcription factors typically expressed in the liver (*FXR*, *HNF1A*, *HNF4A*, *LRH-1*, *LXR*, *PPARA*, and *PXR*), a downregulation of expression at the mRNA level down to 14% compared to untreated cells could be detected. No changes were detected for the nuclear receptors *ESR1* and *RXRA*. The gene expression of the constitutive androstane receptor (CAR) was strongly affected by PAs in a structure-dependent manner: a reduction of the CAR transcript down to 0.6% was detected in the treated HepaRG cells for the most cytotoxic PAs senecionine, heliosupine and lasiocarpine.
