The Food Contaminants Pyrrolizidine Alkaloids Disturb Bile Acid Homeostasis Structure-Dependently in the Human Hepatoma Cell Line HepaRG.

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.

Pyrrolizidine Alkaloids Disturb Bile Acid Homeostasis in the Human Hepatoma Cell Line HepaRG.

1,2-unsaturated pyrrolizidine alkaloids (PAs) belong to a group of secondary plant metabolites. Exposure to PA-contaminated feed and food may cause severe hepatotoxicity. A pathway possibly involved in PA toxicity is the disturbance of bile acid homeostasis. Therefore, in this study, the influence of four structurally different PAs on bile acid homeostasis was investigated after single (24 h) and repeated (14 days) exposure using the human hepatoma cell line HepaRG. PAs induce a downregulation of gene expression of various hepatobiliary transporters, enzymes involved in bile acid synthesis, and conjugation, as well as several transcription regulators in HepaRG cells. This repression may lead to a progressive impairment of bile acid homeostasis, having the potential to accumulate toxic bile acids. However, a significant intracellular and extracellular decrease in bile acids was determined, pointing to an overall inhibition of bile acid synthesis and transport. In summary, our data clearly show that PAs structure-dependently impair bile acid homeostasis and secretion by inhibiting the expression of relevant genes involved in bile acid homeostasis. Furthermore, important biliary efflux mechanisms seem to be disturbed due to PA exposure. These mole-cular mechanisms may play an important role in the development of severe liver damage in PA-intoxicated humans.

Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner.

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure-activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

In silico genotoxicity and carcinogenicity prediction for food-relevant secondary plant metabolites.

Humans are exposed to thousands of different secondary plant metabolites which may have beneficial health effects, but numerous compounds may also have toxic potential. In the present study we have examined the genotoxic and carcinogenic potential of 609 food-relevant phytochemicals by using computer models for toxicity prediction. We developed a scoring method and combined the results of different models to increase the predictive power. A combination of the VEGA models SARpy, KNN, ISS, and CAESAR, and of the LAZAR model "Salmonella typhimurium" for genotoxicity prediction performed better than the single models regarding specificity and accuracy. Statistical evaluation of the combined model for carcinogenicity prediction was not possible due to the low number of substances suitable for model validation. The in silico results of the present exercise will be useful for priority setting purposes regarding future risk assessment of secondary plant metabolites. Based on our analysis, (-)-asimilobine, aloin, annoretine, chrysothrone, coptisine, elymoclavine, and thalicminine were predicted to be genotoxic with high probability and may therefore be selected for subsequent experimental genotoxicity testing. Moreover, the class of pyrrolizidine alkaloids is suggested to be a high priority subject for further studies as these substances have been predicted to be carcinogenic with high probability.