*3.1. PA-Induced Cytotoxic Effects in HepaRG Cells*

To elucidate the structure-dependent effects of PAs on HepaRG cells and to establish a suitable concentration range for further investigations, cell viability studies were performed. Metabolically competent HepaRG cells were treated with the 22 PAs for 24 h at six different concentrations ranging from 0.1 to 250 μM. The upper concentration limit resulted from the low PA solubility and the resulting high concentration of solvent in the incubation medium (max. 2.5% ACN). The high concentration of 1.7% DMSO in the medium was chosen to obtain the highest possible activity of xenobiotic-metabolizing CYP enzymes for effective bioactivation of PAs [42].

The results of the MTT assay are summarized in the heat map in Figure 2. For better overview, the PAs were sorted in descending order according to cell viability detected at the highest concentration. Clear differences in cell viability became obvious at a concentration of 250 μM, varying between 100 and 41%. Upon closer examination, it can be seen that

among the less cytotoxic PAs (cell viability > 80% at 250 μM), the free bases heliotridine and retronecine, as well as monoesters, such as lycopsamine, indicine, intermdine, rinderine, echinatine and europine, were mainly represented. Moderate (cell viability between 80 and 60%) to strong toxicity (cell viability < 60%) was more likely to be induced by openchained and cyclic diesters. Platyphylline, which is actually considered to be non-toxic, nevertheless led to a statistically significant reduced cell viability of 88% and 81% at the highest concentrations of 100 μM and 250 μM, respectively. Corresponding cytotoxicity data for HepaRG cells with a reduced DMSO content of only 0.5% have been published previously [25]. The levels of the effects here turned out to be somewhat smaller, but the order of PAs by strength of induced cytotoxicity is comparable.

**Figure 2.** Decreasing viability of HepaRG cells 24 h after PA exposure. Cell viability was measured by the MTT assay. The mean values out of three biological replicates with three technical replicates each were normalized to the solvent control (SC, 2.5% ACN, 1.7% DMSO). Triton X-100 (0.05%) was used as positive control (PC). The heat map shows the cell viabilities in percent of the solvent control. Blue color indicates a decrease in cell viability and yellow indicates an increase. Statistics: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 (one-way ANOVA followed by Dunnett's post hoc test versus the solvent control). Mean values, standard deviations and *p*-values can be found in the supplemental material. Abbreviations for structural characteristics of the PAs: 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. PAs Affect Structure-Dependently Expression of Genes Involved in Bile Acid Homeostasis*

Effects of PAs on the regulation of cholestasis-associated gene expression were elucidated with a test set of 14 selected PAs in which representatives of all structural groups were present. qPCR was used to investigate the expression of 45 genes associated with cholesterol and bile acid metabolism. HepaRG cells were treated with PAs at concentrations of 5, 21, and 35 μM for 24 h. These concentrations were chosen to induce no or only weak cytotoxic effects. For the analysis of gene expression, the DMSO concentration was lowered from 1.7% to 0.5% to ensure inducibility of gene expression.

In Figure 3 the induction of gene expression in percent of the solvent control is represented as the heat map. Further details such as means and standard deviations are summarized in the supplemental material (Table S3). For better comparability of the results for the different endpoints tested, the PAs were always sorted according to their cytotoxicityinducing potential, as shown in Figure 2. The analyzed genes were classified related to the function of their corresponding proteins into the groups transport proteins, xenobioticmetabolizing enzymes, transcription factors, and enzymes of cholesterol metabolism.

In the heat map, it can be clearly seen that the expression of all investigated genes was downregulated or unchanged without an exception. A significant upregulation of expression was not detected for any of the examined genes. In addition, it is noticeable that the effects, particularly in the case of the xenobiotic-metabolizing enzymes, were sometimes extremely strong with a reduction of the expression down to around 0.02% (*CYP7A1*, senecionine, 35 μM). Summarizing the gene expression data, it can additionally be concluded that PAs showing the strongest effects on cell viability also have more pronounced effects on the regulation of gene expression.
