Background:
Pyrrolizidine alkaloids (PAs) are secondary metabolites occurring in a wide range of plant species. Some 1,2-unsaturated PAs exert toxic effects through metabolic activation which form the corresponding dehydropyrrolizidine derivatives, primarily in the liver, catalyzed by cytochrome P450 monooxygenases. Due to their hepatotoxicity, genotoxicity and carcinogenicity, the accidental presence of PAs in food, feed and herbal medicinal products can be, depending on the dose, a cause for safety concerns.
Objectives:
In order to assess potential risks and confirm the connection between structure and in vitro toxicity [1], we generate data firstly concerning cytotoxicity of some food-relevant PAs. In addition, we also wanted to test in vitro the role CYP3A plays in metabolic activation of PA-induced cytotoxicity.
Methods:
After 24 h and 48 h exposure, cytotoxicity of the selected PAs was determined at concentrations ranging from 1 to 300 µM by the Alamar blue assay in primary rat hepatocytes and in the human HepG2 cell line. A kinetic assay analyzing 7-benzyloxyresorufin-O-dealkylation (BROD) was used for measuring the activity of CYP3A enzymes.
Results:
A structure dependent cytotoxicity was demonstrated with rat hepatocytes in primary culture. Lasiocarpine, an open-chained di-ester with 7S-structure, proved to be the most cytotoxic followed by the other di-esters echimidine, retrorsine, seneciphylline and senecionine. The mono-esters heliotrine, indicine, europine and lycopsamine were much less cytotoxic. On the contrary, failure to detect cytotoxicity in HepG2 cells is possibly due to the lack of CYP3As. In primary rat hepatocytes, the CYP3A activity decreased rapidly during the culture, therefore, the time to incubation significantly affects the cytotoxicity. These data confirm that CYP3A plays a critical role in PA-induced toxicity.
Reference:
[1] Merz, KH and Schrenk D. Toxicology letters 2016, 263: 44-57.
Acknowledgement:
The study is supported by Kooperation Phytopharmaka and by DFG, Bonn, Germany