Stefanie Hessel-Pras

1,2-Unsaturated pyrrolizidine alkaloids (PA) induce severe acute and chronic hepatotoxicity. Effects include an impairment of the cell cycle. To elucidate this effect, cell cycle progression was analyzed by flow cytometry, changes in cell and nucleus size, differential gene expression and protein phosphorylation patterns of regulatory key proteins in the Chinese hamster lung fibroblast cell line V79 and/or the human hepatoblastoma cell line HepG2, both overexpressing human CYP3A4 (V79_3A4/HepG2_3A4). Lasiocarpine, a potent PA representative, reduced the cell viability of human CYP3A4-overexpressing cell lines concentration-dependently. Microscopic observation showed a marked increase in cell and nucleus size of V79_3A4 cells after exposure to 10 µM lasiocarpine. In the human CYP3A4-overexpressing cell lines, cells accumulate in G₂/M phase after lasiocarpine treatment. Based on these findings, the gene expression pattern of cell cycle-related genes was investigated in HepG2_3A4 cells showing a decrease of e. g. WEE1, and CHEK1 and an increase of PAK1 and ATM. While results on cell cycle regulation at the level of gene expression are of limited relevance, protein phosphorylation plays an important role. Therefore, we also elucidated the protein phosphorylation status of regulatory key proteins. The results clearly indicate an induction of the DNA damage response and a late G2 arrest. In conclusion, an impairment of the cell cycle was observed. It correlates with the metabolic activation of lasiocarpine and is most likely mediated by adduct formation of the reactive pyrrole esters with DNA, leading to a disruption of cellular homeostasis and genomic instability.

Retrorsine is a hepatotoxic pyrrolizidine alkaloid (PA) found in herbal supplements and medicines, food and livestock feed. Dose-response studies enabling the derivation of a point of departure including a benchmark dose for risk assessment of retrorsine in humans and animals are not available. Addressing this need, a physiologically based toxicokinetic (PBTK) model of retrorsine was developed for mouse and rat. Comprehensive characterization of retrorsine toxicokinetics revealed: both the fraction absorbed from the intestine (78%) and the fraction unbound in plasma (60%) are high, hepatic membrane permeation is dominated by active uptake and not by passive diffusion, liver metabolic clearance is 4-fold higher in rat compared to mouse and renal excretion contributes to 20% of the total clearance. The PBTK model was calibrated with kinetic data from available mouse and rat studies using maximum likelihood estimation. PBTK model evaluation showed convincing goodness-of-fit for hepatic retrorsine and retrorsine-derived DNA adducts. Furthermore, the developed model allowed to translate in vitro liver toxicity data of retrorsine to in vivo dose-response data. Resulting benchmark dose confidence intervals (mg/kg bodyweight) are 24.1–88.5 in mice and 79.9–104 in rats for acute liver toxicity after oral retrorsine intake. As the PBTK model was built to enable extrapolation to different species and other PA congeners, this integrative framework constitutes a flexible tool to address gaps in the risk assessment of PA.

Phytochemicals like pyrrolizidine alkaloids (PAs) can affect the health of humans and animals. PAs can occur for example in tea, honey or herbs. Some PAs are known to be cytotoxic, genotoxic, and carcinogenic. Upon intake of high amounts, hepatotoxic and pneumotoxic effects were observed in humans. This study aims to elucidate different toxicokinetic parameters like the uptake of PAs and their metabolism with in vitro models. We examined the transport rates of differently structured PAs (monoester, open-chained diester, cyclic diester) over a model of the intestinal barrier. After passing the intestinal barrier, PAs reach the liver, where they are metabolized into partially instable electrophilic metabolites interacting with nucleophilic centers. We investigated this process by the usage of human liver, intestinal, and lung microsomal preparations for incubation with different PAs. These results are completed with the detection of apoptosis as indicator for bioactivation of the PAs. Our results show a structure-dependent passage of PAs over the intestinal barrier. PAs are structure-dependently metabolized by liver microsomes and, to a smaller extent, by lung microsomes. The detection of apoptosis of A549 cells treated with lasiocarpine and monocrotaline following bioactivation by human liver or lung microsomes underlines this result. Conclusively, our results help to shape the picture of PA toxicokinetics which could further improve the knowledge of molecular processes leading to observed effects of PAs in vivo.

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 antagomiR-inhibition 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.

Scope: 1,2‐unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites that are found in many plant species throughout the world. They are of concern for risk assessment as consumption of contaminated foodstuff can cause severe liver damage. Of late, transporter‐mediated uptake and transport has advanced as a vital determinant of PA toxicity. In this study, the authors investigate a transporter‐mediated uptake of PAs and its implications in PA toxicity.Methods and Results: We show that transporter expression levels are significantly affected by treatment with the PAs senecionine (Sc) and retrorsine (Re) in the human hepatoma cell line HepaRG. Furthermore, the specific contribution to PA uptake of the two transporters Na⁺/taurocholate co‐transporting polypeptide (SLC10A1) and organic cation transporter I (SLC22A1), both belonging to the heterogeneous solute carrier super family, is investigated by means of a siRNA‐mediated knockdown approach. Knockdown of both uptake transporters result in reduced uptake of Re and Sc in a time‐dependent manner and attenuated PA‐mediated cytotoxic effects in HepaRG cells.Conclusion: Our results confirm previous findings of active transport mechanisms of PAs into hepatocytes and highlight the importance of toxicokinetic studies for the risk assessment of PAs.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites synthesized by a wide range of plants as protection against herbivores. These toxins are found worldwide and pose a threat to human health. PAs induce acute effects like hepatic sinusoidal obstruction syndrome and pulmonary arterial hypertension. Moreover, chronic exposure to low doses can induce cancer and liver cirrhosis in laboratory animals. The mechanisms causing hepatotoxicity have been investigated previously. However, toxic effects in the lung are less well understood, and especially data on the correlation effects with individual chemical structures of different PAs are lacking. The present study focuses on the identification of gene expression changes in vivo in rat lungs after exposure to six structurally different PAs (echimidine, heliotrine, lasiocarpine, senecionine, senkirkine, and platyphylline). Rats were treated by gavage with daily doses of 3.3 mg PA/kg bodyweight for 28 days and transcriptional changes in the lung and kidney were investigated by whole-genome microarray analysis. The results were compared with recently published data on gene regulation in the liver. Using bioinformatics data mining, we identified inflammatory responses as a predominant feature in rat lungs. By comparison, in liver, early molecular consequences to PAs were characterized by alterations in cell-cycle regulation and DNA damage response. Our results provide, for the first time, information about early molecular effects in lung tissue after subacute exposure to PAs, and demonstrates tissue-specificity of PA-induced molecular effects.

Pyrrolizidine alkaloids (PAs) are a group of secondary plant metabolites being contained in various plant species. The consumption of contaminated food can lead to acute intoxications in humans and exert severe hepatotoxicity. The development of jaundice and elevated bile acid concentrations in blood have been reported in acute human PA intoxication, indicating a connection between PA exposure and the induction of cholestasis. Additionally, it is considered that differences in toxicity of individual PAs is based on their individual chemical structures. Therefore, we aimed to elucidate the structure-dependent disturbance of bile acid homeostasis by PAs in the human hepatoma cell line HepaRG. A set of 14 different PAs, including representatives of all major structural characteristics, namely, the four different necine bases retronecine, heliotridine, otonecine and platynecine and different grades of esterification, was analyzed in regard to the expression of genes involved in bile acid synthesis, metabolism and transport. Additionally, intra- and extracellular bile acid levels were analyzed after PA treatment. In summary, our data show significant structure-dependent effects of PAs on bile acid homeostasis. Especially PAs of diester type caused the strongest dysregulation of expression of genes associated with cholestasis and led to a strong decrease of intra- and extracellular bile acid concentrations.