Identification of the structure-dependent toxicity of hepatotoxic pyrrolizidine alkaloids

不饱和吡咯里西丁生物碱(PA)是毒性最强的化合物，已在多种植物中发现了400多种PA。在千里光中也发现了它们对人和动物有毒。在蜂蜜和茶叶中检测到大量的苯丙氨酸。由食物污染引起的急性PA中毒会对肝脏造成严重损害；长期亚致死剂量可能会导致累积损害或癌症。动物研究表明，PA具有致癌作用。在几种体外和体内试验系统中观察了PA的致突变性。这些化合物的毒性与主要在肝脏表达的酶的酶反应有关，并取决于PA‘S的生物利用度。然而，PA产生肝毒性的作用模式尚未确定。根据我们对生物利用度的结果以及结构不同的PA儿茶素、向日葵碱、千里光碱和千里光碱的分子作用模式，我们假设PA以结构依赖的方式发挥作用。因此，我们的目标是将依赖于结构和毒性的PA归类到这个项目中。为此，我们将在人体肠道屏障的体外模型中进一步研究PA的生物利用度。作为分组的终点，应研究依赖于ABCB1的转运/解毒作用的依赖于PA的结构模式。此外，还应分析PA肝脏毒性的分子机制。我们已经在转录学研究中表明，4种结构不同的PA在诱导肝脏基因表达方面存在差异。因此，实时分析小鼠肝脏毒性的体内分析应使用双光子激发显微镜进行分析。在野生型小鼠和ABCB1基因敲除小鼠中，应使用特定的肝脏标志物来研究结构依赖性毒性诱导。已确定的对肝脏的毒性作用应与人类原代肝细胞的转录组数据相关联。基于这些结果，具体的基因和信号通路需要分别用实时定量聚合酶链式反应和报告基因检测来验证PA的结构依赖性诱导作用。本研究旨在弥补结构依赖性PA的生物利用度和肝脏作用模式方面的空白，根据PA的不同结构形成PA毒性群。这些数据将有助于更好地了解急性或慢性摄取PA的毒性后果，以加强对人类的保护。

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. PA were detected in substantial amounts into honey and tea. 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 and dependent on PA´s bioavailability. However, the mode of action of PA to generate hepatotoxicity is not yet identified.Based on our results for bioavailability and the molecular mode of action of the structurally different PA echimidine, heliotrine, senecionine, and senkirkine we hypothesize that PA act in a structure-dependent manner. Therefore we aimed to group the PA structure- and toxicity-dependent within this project.For that purpose bioavailability shall be further studied in an in vitro model for the human intestinal barrier with a larger set of PA (22). As endpoint for grouping the ABCB1-dependent transport/detoxification shall be investigated for the structure-dependent mode of action of PA. Additionally, the molecular mechanism of PA hepatic toxicity shall be analyzed. We already showed in our transcriptomic study with the 4 structurally different PA differences in the induction of gene expression in the liver. Therefore, in vivo analysis of the induction of hepatotoxicity in mice in real time shall be analysed using two-photon excitation microscopy. The structure-dependent induction of toxicity shall be investigated using specific liver marker in wildtype mice and in further step in Abcb1 knockout mice. The identified toxic effects on liver shall be correlated with the transcriptomics data from human primary hepatocytes. Based on these results specific genes and signalling pathways shall be verified in regard to a structure-dependent induction by PA using quantitative real time PCR and reporter gene assays, respectively.This study aimed to close gaps in regard to the structure-dependent PA bioavailability and hepatic mode of action to form PA toxicity group on the basis of its different structure. These data shall contribute to a better understanding of the toxic consequences of an acute or chronic PA uptake to enhance the protection of humans.