**1. Introduction**

Pyrrolizidine alkaloids comprise a large group of secondary plant compounds occurring ubiquitously in the plant kingdom. They are constitutively produced in about 3% of the world's flowering plants as a protective mechanism against herbivores [1,2]. To date, over 660 structurally different pyrrolizidine alkaloids and their *N*-oxide derivates have been identified, and about half of them are considered to be toxic [3,4]. The PA-associated harmful effects on livestock and humans [5–8] are associated with a double bound in C1,2-position in the necine base. In the following, the abbreviation PA is always used for 1,2-unsaturated PAs. Intoxications in humans by PA-contaminated cereals, herbal teas, and herbal medicines were reported in the United States, India, Tajikistan, Afghanistan, and South Africa [9–14]. PAs can cause severe liver damage characterized by hemorrhagic liver necrosis, ascites, cirrhosis, and the development of the characteristic hepatic sinusoidal obstruction syndrome (HSOS) [6,14,15].

The molecular mechanisms of PA hepatotoxicity are not yet fully elucidated. a wellknown molecular mechanism is the formation of adducts with DNA and proteins by reactive PA metabolites, which are formed by enzymatic conversion in the liver [7]. Further mechanisms at the molecular level, such as interactions of PAs with specific signaling and metabolic pathways, are not completely understood. Recently, a genome-wide expression study in primary human hepatocytes provided evidence for PA-mediated induction of

**Citation:** Waizenegger, J.; Glück, J.; Henricsson, M.; Luckert, C.; Braeuning, A.; Hessel-Pras, S. Pyrrolizidine Alkaloids Disturb Bile Acid Homeostasis in the Human Hepatoma Cell Line HepaRG. *Foods* **2021**, *10*, 161. https://doi.org/ 10.3390/foods10010161

Received: 7 December 2020 Accepted: 11 January 2021 Published: 14 January 2021

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apoptosis and impairment of bile acid homeostasis [16], suggesting a partial involvement of these mechanisms in PA-induced hepatotoxicity. Additionally, increased bile acid levels in blood, as well as disturbance of bile acid secretion, were shown in mice following an acute toxic dose of senecionine [17].

The bile produced in the liver is essential for the absorption and digestion of lipids from the intestinal lumen, as well as for the elimination of xenobiotics, endogenous compounds, and metabolic products (e.g., cholesterol, bilirubin, and hormones) [18,19]. It consists predominantly of water (82%), followed by bile acids, phospholipids, cholesterol, proteins, bilirubin, and electrolytes. Primary bile acids are synthesized from cholesterol in the hepatocytes by various enzymes (including cytochrome P450 monooxygenase 7A1 (CYP7A1), CYP8B1, and CYP27A1) [20,21]. Under physiological conditions, the primary bile acids are conjugated with the amino acids glycine or taurine, followed by secretion by hepatocytes into the bile canaliculi, along with the remaining bile constituents, and storage and concentration in the gallbladder. After release into the duodenum and subsequent absorption and digestion of the food constituents, about 95% of the bile acids are reabsorbed into the liver via blood circulation, thus entering enterohepatic circulation [20,22]. Therefore, hepatic transport proteins are essential for the formation of bile and maintaining bile flow. Sinusoidal (basolateral) transporters are responsible for the uptake of endogenous and exogenous substances into the hepatocytes, and canalicular (apical) transporters mediate the biliary secretion into the bile canaliculi [23,24]. If the secretion of bile acids and consequently the bile flow are impaired, potentially toxic bile components, such as bile acids, bilirubin, and cholesterol, can accumulate, and thus may lead to the damage of hepatic cells [25,26]. In 2013, Vinken et al. proposed an adverse outcome pathway (AOP) for drug-mediated cholestasis [26]. The proposed AOP connects as the primary molecular initiating event the inhibition of the canalicular transporter ATP-binding cassette subfamily B member 11 (ABCB11) with various key events (e.g., bile acid accumulation, regulation of nuclear receptors, induction of inflammation, and oxidative stress) and intermediate steps (e.g., induction of apoptosis/necrosis and gene expression changes) to the adverse outcome, cholestasis.

Based on the proposed AOP for cholestatic liver disease [26] and data suggesting that an impairment of bile acid homeostasis may contribute to the PA-induced hepatotoxicity [16,17], the present study aims to systematically investigate the effect of PA treatment on bile acid homeostasis in the human hepatoma cell line HepaRG. In this regard, possible structure-dependent effects were examined using the four structurally different PAs echimidine, heliotrine, senecionine, and senkirkine (for chemical structures see Figure 1). In addition, two different exposure scenarios were comparatively investigated, namely a single treatment for 24 h and a repeated treatment for 14 days. The human hepatoma cell line HepaRG was used due to its hepatocyte-like morphology, high metabolic activity, and its suitability to investigate bile acid homeostasis [21,27]. Furthermore, HepaRG cells can be cultivated under long-term conditions, enabling a continuous treatment with test substances [28,29].

**Figure 1.** Chemical structure of PAs used in this study. Selected PAs represent the main occurring basic structures of necine base types and structures of esters: heliotrine is a monoester of the heliotridine-type, and senecionine and senkirkine are both cyclic diesters representing retronecineand otonecine-type PAs, respectively. The open-chained diester echimidine belongs to the retronecinetype group.
