**1. Introduction**

Secondary plant compounds have increasingly come into the focus of risk assessment as food contaminants in recent years. One group of these contaminants are the 1,2-unsaturated pyrrolizidine alkaloids (PAs). Humans are exposed to PAs mainly via the consumption of contaminated food. The Federal Institute for Risk Assessment identified in 2013 tea, herbal teas and honey as the main sources for human PA uptake in Western countries [1]. In addition, a consumption of contaminated salad mixes, herbs, flour or cereals can also lead to the uptake of substantial amounts of PA [2–5]. However, there are also some plants used as food that produce PA themselves such as borage. Chen et al. [6] showed that lycopsamine *N*-oxide, lycopsamine and acetyllycopsamine were the main PAs in the sample they studied. Dietary supplements based on plants containing PAs may also contribute to increased exposure to PAs. However, the total intake of PAs through their consumption cannot be estimated yet.

Although the levels of PA in most foodstuffs have been significantly reduced in recent years, exposure via highly contaminated dried herbs or herbal mixtures with PA levels of up to 3000 μg/kg seem to be possible [7].

PAs most commonly enter foods through mechanical harvesting processes, but contamination of honey by pollen from PA-containing plants or the use of PA-containing plants as herbal medicine is also a possible source of exposure [8–10].

**Citation:** Glück, J.; Henricsson, M.; Braeuning, A.; Hessel-Pras, S. The Food Contaminants Pyrrolizidine Alkaloids Disturb Bile Acid Homeostasis Structure-Dependently in the Human Hepatoma Cell Line HepaRG. *Foods* **2021**, *10*, 1114. https://doi.org/10.3390/foods10051114

Academic Editor: Dirk W. Lachenmeier

Received: 22 April 2021 Accepted: 12 May 2021 Published: 18 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

<sup>2</sup> Wallenberg Laboratory, Department of Molecular and Clinical Medicine, Institute of Medicine, University of Gothenburg, 413 45 Gothenburg, Sweden; marcus.henricsson@wlab.gu.se

Since PAs are primarily a random contamination of otherwise PA-free foods, it is not possible to predict which PAs are present in which foods. On the one hand, there are considerable differences in PA content between plant species, and on the other hand, factors such as soil conditions, climate and geographical origin can lead to considerable variations in the composition and quantity of PA within a plant species. Large intra-plant differences between different parts are also possible [11,12]. Therefore, exposure to PAs can only be estimated based on previous studies, but cannot be calculated accurately. Some studies have investigated the PA content in different foods and food supplements, and all the 1,2-unsaturated PAs used in this study were found in foodstuffs. For example, lasiocarpine and senecionine, two of the most toxic PAs examined in our study, were detected in various (herbal) teas and food supplements. Echimidine was also frequently detected [5,13].

After uptake, PAs can cause severe damage to humans and livestock after consumption of contaminated food or feed. Depending on the exposure, acute and chronic liver damage can result, such as liver hardening, ascites and the hepatic sinusoidal obstruction syndrome (HSOS), as well as liver cirrhosis, fibrosis, or liver cancer [14,15]. Due to their widespread distribution in more than 6000 plant species, especially in the *Asteraceae*, *Boraginaceae* and *Fabaceae* families, around the world, PA contamination is not locally limited [3,8,9,16,17].

Currently, more than 660 different PAs and their corresponding N-oxides are known. About half of them are considered to exhibit genotoxic and hepatotoxic properties [18]. Chemically, PAs consist of a necine base, which can be esterified with organic acids at the OH-groups at the C 7 and C 9 positions of the double-ring system. Based on their chemical structure, PAs can be classified into different groups. PAs can be divided according to their corresponding necine base (1-hydroxymethylpyrrolizidine) into platynecine-, heliotridine-, retronecine- and otonecine-type PAs. The different grades of esterification allow a further subdivision into free bases (no esterification), monoesters and open-chained or cyclic diesters (Figure 1) [19].

The PA parent substance and its N-oxide are not very reactive as such, and therefore do not directly induce toxic effects. Due to metabolic activation in the liver, reactive metabolites such as dehydropyrrolizidine alkaloids (DHP) are formed, resulting in DNA and protein adducts [20–22]. For the formation of these metabolites, a double bond at the C 1/C 2 position is necessary. Therefore, the 1,2-saturated platynecine-type PAs are considered to be non-toxic [23,24].

The molecular mode of action of PAs and their metabolites in the liver is not fully understood yet. In previous studies, effects on various intracellular pathways, such as the induction of apoptosis, DNA damage response, and prostanoid synthesis were investigated [25–29]. In a whole-genome microarray analysis by Luckert et al. [30] in primary human hepatocytes, evidence for PA-induced disturbance of bile acid homeostasis was found. In association with PA-induced HSOS, jaundice is often diagnosed. An accumulation of bilirubin is also an indication that the normal pathway of bilirubin degradation and bile flow may be impaired [15,31].

The bile, produced in the liver, is essential for efficient absorption of fats and lipophilic substances in the intestine, as well as for the excretion of metabolites and endogenous substances from the liver. It consists of bile acids, phospholipids, cholesterol, proteins, bilirubin, electrolytes and water. The first and rate-limiting step in the de novo synthesis of bile acids is the 7α-hydroxylation of cholesterol, catalyzed by CYP7A1. The primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) are conjugated very rapidly with the amino acids glycine and taurine after their synthesis. Secondary bile acids are formed by conversion by microorganisms in the intestine and enter the liver after absorption from the intestine [32–36].

A disturbance of bile acid homeostasis can have serious consequences for the liver. Reduced bile flow can cause accumulation of potentially cytotoxic bile acids in the liver, leading to serious cell damage [37–39].

Some recently published studies by Waizenegger et al. [40] and Hessel-Pras et al. [41] described the disturbance of bile homeostasis by selected PAs. Due to the small number of PAs investigated in these studies, predictions on the structure–activity relationship are not applicable. Therefore, in the present study, a set of 22 structurally different PAs was systematically investigated in relation to selected endpoints associated with the disturbance of bile acid homeostasis. The endpoints in the focus of this study include the induction of cytotoxicity, changes in the expression of cholestasis-associated genes and the influence on the levels of intra- and extracellular bile acids in the metabolically competent human hepatoma cell line HepaRG.

**Figure 1.** Overview of different PAs and their structural characteristics sorted by their grade of esterification: (**A**)—free bases; (**B**)—monoesters; (**C**)—open-chained diesters; (**D**)—cyclic diesters. The respective type of necine base is indicated in brackets: H—heliotrine (7S); O—otonecine (7R); P—platynecine (7R); R—retronecine (7R). The only 1,2-saturated PA, platyphylline, is indicated in italics. For cytotoxicity studies, all listed esters and the free bases heliotridine and retronecine were analyzed. The bold and underlined PAs represent the reduced test set for all subsequent experiments.

#### **2. Materials and Methods**
