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Structure-activity relationship of hepatotoxic pyrrolizidine alkaloids
Project
Project code: BfR-LMS-08-1322-591
Contract period: 01.03.2014
- 31.12.2016
Purpose of research: Applied research
Unsaturated pyrrolizidine alkaloids (PA) belong to the most toxic compounds; more than 400 PA have been described in a wide range of plants. They are also found in Senecio vulgaris L. PA are toxic to humans and animals. Acute PA poisoning via food contamination causes severe damage to the liver; long-term, sub-lethal doses may cause cumulative damage or cancer. Animal studies revealed that PA are carcinogenic. Mutagenicity of PA was observed in several in vitro and in vivo test systems. The toxicity of these compounds is associated with enzymatic reactions by enzymes dominantly expressed in the liver. However, the mode of action of PA to generate hepatotoxicity is not yet identified. Research results concerning the molecular mode of action of PA that were gained in the BfR research project “development of a hepatic in vitro test system for the sensitive detection of pyrrolizidine alkaloids” (1322-454) clearly show a structure-activity relationship of the studied PA heliotrine, echimidine, senecionine and senkirkine. These four PA represent all possible structural types of PA. They cover the three existing necic acid structure types (monoester, diester, cyclic diester) as well as the most frequent necic bases (retronecine, heliotridine, otonecine type). Thus, these PA are suitable for the investigation of structure-activity relationships. In 2014 structure-activity relationships of PA shall be investigated on three levels. First, the structure dependent transport of PA over the gastrointestinal barrier shall be analysed by using the Caco-2 cell model. In contrast to cyclic PA the non-cyclic PA heliotrine and echimidine show a MDR1-dependent transport. This structure-activity relationship shall be validated with other members of both structure classes. In the second part, structure-activity relationships shall be investigated on gene expression level based on the whole genome µ-array data that were generated in the BfR research project 1322-454. The third part deals with analyses on protein level. Here, interactions of PA with different receptors shall be analysed by employing reporter gene assays. Furthermore, the development of a suitable in vitro hepatic cell model that is needed for the analysis of hepatocellular mode of actions of PA is planned. These studies should help to elucidate the mode of action of the strong hepatotoxicity in humans and gain knowledge about the oral bioavailability of PA.
Results: To analyze the structure-activity relationship of the molecular mode of action of hepatotoxic pyrrolizidine alkaloids (PA) the four different PA heliotrine, echimidine, senecionine and senkirkine were chosen as model PA. They represent all three occurring necic acid structures (monoester, diester, cyclic diester) as well as the most common necine bases (retronecine, heliotridine, otonecine). In 2014 the structure dependent transport of PA over the gastrointestinal barrier was analyzed using the Caco-2 cell modell. The proclaimed hypothesis that the transport of PA over the epithelial layer is structure-dependent and that monoesters as well as diesters are transported back into the intestinal lumen via an active efflux could only partly be confirmed. For the monoesters intermedine and lycopsamine and the diester echimidine an active ABCB1 mediated efflux could be confirmed, however for the diester lasiocarpine and the cyclic diesters no efflux after co-exposure could be observed. Indeed, the transport seems to be structure dependent, although the structure-transport relationship remains to be clarified for open-chained diester. Furthermore, microarray gene expression data from a former BfR research project (1322-454) could be verified in HepaRG cells. Thus, these cells pose a suitable model for future analyses concerning the structure dependent gene regulation by PA in human hepatocytes. In contrast, the 3D cultivation of HepG2 cells proved to be an unsuitable in vitro hepatocyte model. First analyses in different 3D systems (collagen sandwich, Matrigel, Alvetex plates) raise the question of the usefulness of the employed 3D cultivation systems as the observed increases in gene expression of liver specific marker proteins, xenobiotic metabolizing enzymes and xenobiotic metabolism associated nuclear receptors and transporters could also be detected over time in the 2D control monolayer. The structure dependent interaction of the four PA with different nuclear receptors was only analyzed for VDR using a VDR mediated CYP3A4 reporter gene assay. No effects could be observed. Furthermore, numerous additional reporter gene assays were acquired, developed and already partly established and optimized for future analyses. These reporter gene assays shall help to elucidate the structure dependent interaction of PA with nuclear receptors and cellular signaling pathways.
Section overview
Subjects
- Toxicology
