**1. Introduction**

Pyrrolizidine alkaloids are the most common natural toxins with over 660 different chemical structures. They are present in more than 6000 plant species worldwide and can occur as contaminants in food and feed, posing a possible health risk for humans and livestock [1]. For example, they can be found in products such as tea, honey, culinary herbs, dietary supplements or herbal preparations such as traditional medicine [2–5]. It must be stressed that the exposure to pyrrolizidine alkaloids can occur via a wide variety of foodstuff, which increases the likelihood of ingestion. Because 1,2-unsaturated Pyrrolizidine Alkaloids (PAs) have genotoxic properties and are considered to be carcinogenic, no safe threshold value such as the TDI (Tolerable Daily Intake) can be derived. Therefore, it is recommended to apply the ALARA principle (As Low As Reasonably Achievable) to prevent unnecessary exposure [6].

PAs are known to require metabolic activation in the liver, usually provided by members of the cytochrome P450 superfamily [7]. The hepatotoxic metabolites may cause acute toxic effects such as Hepatic Sinusoidal Obstruction Syndrome (HSOS), resulting in ascites, hepatomegaly, and ultimately death. Chronic intoxication may lead to megalocytosis, liver cirrhosis and cancer [7–16]. To a lesser extent, PAs are also pneumotoxic, most likely due to systemic distribution of reactive metabolites to the lung [17].

Pyrrolizidine alkaloids consist of one so-called necine base, which is esterified with one or two aliphatic mono- or dicarboxylic acids (necic acids). Accordingly, they are classified

**Citation:** Enge, A.-M.; Sprenger, H.; Braeuning, A.; Hessel-Pras, S. Identification of microRNAs Implicated in Modulating Senecionine-Induced Liver Toxicity in HepaRG Cells. *Foods* **2022**, *11*, 532. https://doi.org/10.3390/ foods11040532

Academic Editor: Junsoo Lee

Received: 18 January 2022 Accepted: 8 February 2022 Published: 12 February 2022

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into different structure types, depending on the structure of their necine base (retronecine-, heliotridine-, otonecine-, and platynecine-type) and further into monoesters, open-chained diesters and cyclic diesters [18].

Until today, many in vitro studies have been conducted showing that PAs cause apoptosis [19,20], cytotoxicity [21], and genotoxicity [22] in a structure-dependent manner. Moreover, they were shown to disturb bile acid homeostasis in the human hepatoma cell line HepaRG [23,24]. Upon PA treatment, it is consistently observed that the expression pattern of genes relevant for apoptosis, bile acid homeostasis, and metabolism is highly deregulated in liver cells in vivo and in vitro [21,23,25]. Nonetheless, some ambiguities remain regarding the underlying molecular mechanisms of PA-mediated hepatotoxicity. The major molecular mode of action is the binding of PA metabolites to DNA and proteins to form adducts via alkylation [7]. Nevertheless, interactions between PAs and specific regulatory elements may pose additional molecular mechanisms leading to deregulation of cellular signaling and disturbance of metabolic pathways. Therefore, PA-mediated nuclear receptor activation was investigated, mainly because nuclear receptors are typical targets in xenobiotic-induced toxicity. However, no PA-induced activation could be observed, with the exception of Pregnane X Receptor (PXR), which was exclusively activated by the PAs echimidine and Lasiocarpine (Lc), resulting in the activation of its target CYtochrome P450 monooxygenase (CYP) 3A4 [26]. Therefore, it appears likely that cellular signaling pathways and mediators other than nuclear receptor interference are involved in mediating the effects of PAs. miRNAs are small, non-coding, 21- to 25-nucleotide-long RNA molecules that regulate a wide variety of physiological processes such as cell growth, development, apoptosis, differentiation, and carcinogenesis at the post-transcriptional level [27,28]. In animals, miRNAs bind their target mRNAs in the 3- -UnTranslated Region (UTR). Depending on the complementarity of base pairing between the miRNA and the mRNA target, the binding of the miRNA either leads to mRNA cleavage or translational repression. It is postulated that translational repression is the predominant mechanism by which metazoan miRNAs negatively regulate their targets [29]. Many miRNAs are crucial regulators of bile acid homeostasis, lipid and glucose metabolism, inflammation, apoptosis, and proliferation [30–32], for example, and are deregulated in many liver diseases [32,33]. Notably, CYP7A1, the rate-limiting enzyme in bile acid synthesis, is regulated by two miRNAs, with one of them being the most abundant miRNA in the liver [34]. Interestingly, a correlation between chronic exposure to the PA riddelliine and an altered miRNA expression pattern in the liver has been observed in a 12-week feeding study with rats [35]. Moreover, an integrative analysis studying the miRNA-mRNA interaction after acute incubation with the PA Monocrotaline (Mc) in high doses found the phagosome signaling pathway to be a relevant molecular mechanism of PA-induced HSOS in mice [36]. Some miRNAs are solely expressed in a tissue-specific manner; others circulate in body fluids such as blood or urine and have been proposed as non-invasive biomarkers for disease prediction and progression. For example, circulating miRNAs in blood samples from patients suffering from PA-induced HSOS were positively correlated with the severity of liver injury and progression of HSOS [37]. Additionally, patients suffering from intrahepatic cholestasis showed elevated miRNA levels in blood and liver tissue [33]. Therefore, miRNA signatures are currently discussed as early-stage biomarkers for HSOS and other liver-specific injuries [28,38].

The present study aimed to examine the role of hepatic miRNAs in the regulation of PA-induced hepatotoxicity in HepaRG cells. For this purpose, relevant hepatic miRNAs were discovered that are sensible to PA treatment. With quantitative analysis and Ingenuity Pathway Analysis (IPA), an evidence-based tool for target and pathway prediction, a selection of target genes was further subjected to analysis to identify the potential engaged molecular functions and biological pathways involved in PA-mediated toxicity in human liver cells.
