*Article* **Identification of microRNAs Implicated in Modulating Senecionine-Induced Liver Toxicity in HepaRG Cells**

**Anne-Margarethe Enge, Heike Sprenger , Albert Braeuning and Stefanie Hessel-Pras \***

Department of Food Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany; anne-margarethe.enge@bfr.bund.de (A.-M.E.); heike.sprenger@bfr.bund.de (H.S.); albert.braeuning@bfr.bund.de (A.B.)

**\*** Correspondence: stefanie.hessel-pras@bfr.bund.de; Tel.: +49-30-18412-25203

**Abstract:** 1,2-unsaturated Pyrrolizidine Alkaloids (PAs) are secondary plant metabolites that occur as food contaminants. Upon consumption, they can cause severe liver damage. PAs have been shown to induce apoptosis, to have cytotoxic and genotoxic effects, and to impair bile acid homeostasis in the human hepatoma cell line HepaRG. The major mode of action of PAs is DNA- and protein-adduct formation. Beyond that, nuclear receptor activation has only been observed for one receptor and two PAs, yielding the possibility that other cellular mediators are involved in PA-mediated toxicity. Here, the mode of action of Senecionine (Sc), a prominent and ubiquitous representative of hepatotoxic PAs, was investigated by analyzing 7 hepatic microRNAs (miRNAs) in HepaRG cells. Ultimately, 11 target genes that were predicted with Ingenuity Pathway Analysis software (IPA) were found to be significantly downregulated, while their assigned miRNAs showed significant upregulation of gene expression. According to IPA, these targets are positively correlated with apoptosis and cellular death and are involved in diseases such as hepatocellular carcinoma. Subsequent antagomiRinhibition analysis revealed a significant correlation between PA-induced *miRNA-4434* induction and P21-Activated Kinase-1 (*PAK1*) downregulation. *PAK1* downregulation is usually associated with cell cycle arrest, suggesting a new function of Sc-mediated toxicity in human liver cells.

**Keywords:** cell cycle; IPA; microRNA; *PAK1*; pyrrolizidine alkaloids; senecionine
