Identification of the structure-dependent toxicity of hepatotoxic pyrrolizidine alkaloids

不饱和吡咯里西啶生物碱（PA）是一类毒性最强的化合物，在多种植物中已发现400多种PA。它们也存在于千里光中。PA对人类和动物有毒。蜂蜜和茶叶中检测到大量PA。食物污染引起的急性PA中毒会对肝脏造成严重损害;长期的亚致死剂量可能会导致累积性损害或癌症。动物实验表明PA是致癌物质。在几种体外和体内试验系统中观察到PA的致突变性。这些化合物的毒性与主要在肝脏中表达的酶的酶促反应有关，并取决于PA的生物利用度。然而，PA产生肝毒性的作用方式尚未确定。基于我们的结果的生物利用度和分子模式的作用，结构不同的PA echimidine，heliotrine，seneciline，和senkirkine，我们假设PA的行为在一个结构依赖性的方式。因此，我们的目的是在该项目中对PA结构和毒性依赖性进行分组。为此，将在具有较大PA组的人肠道屏障的体外模型中进一步研究生物利用度（22）。作为分组的终点，应研究PA的结构依赖性作用模式的ABCB 1依赖性转运/解毒。此外，还应分析PA肝毒性的分子机制。我们已经在我们的转录组学研究中显示了4种结构不同的PA在诱导肝脏基因表达方面的差异。因此，应使用双光子激发显微镜分析小鼠肝毒性诱导的真实的时间体内分析。应在野生型小鼠中使用特异性肝脏标志物研究毒性的结构依赖性诱导，并在Abcb 1敲除小鼠中进一步研究。确定的肝脏毒性作用应与人原代肝细胞的转录组学数据相关。基于这些结果，特定的基因和信号通路将被验证的结构依赖性诱导PA使用定量真实的时间PCR和报告基因assays.This研究的目的是填补空白方面的结构依赖性PA的生物利用度和肝脏的作用模式，形成PA毒性组的基础上，其不同的结构。这些数据将有助于更好地了解急性或慢性PA摄入的毒性后果，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.