Structure‐dependent relative toxic potencies of selected pyrrolizidine alkaloids
Authors: Gao Lan , Schrenk Dieter 
Published: June 2023

Pyrrolizidine alkaloids are naturally occurring secondary plant metabolites mainly found in plant families of Asteraceae, Boraginaceae, and Fabaceae. Chemically, Pas consist of a pyrrolizidine core bearing hydroxyl groups, the so‐called necine base, and mono‐ or dicarboxylic necine acids bound to the pyrrolizidine core via ester linkages. 1,2‐unsaturated PAs are hepatotoxic, genotoxic, and carcinogenic due to the highly reactive pyrrolic metabolites formed by cytochrome P450 monooxygenases (CYPs) primarily in the liver. The presence of PAs as frequent contaminants in the wide variety of food and feed products has to be considered a relevant safety issue.

Based on the currently available data, the risk assessment of PAs was mainly approached using the two most toxic potent congeners, i.e., lasiocarpine and riddelliine. However, it is well recognized that toxicity is differing significantly between the congeners related to their structural features. The risk of PA‐containing products is indeed overestimated, and a comprehensive risk assessment should take these differences into account.

After analyzing the data of many PAs, Merz and Schrenk derived interim Relative Potency (iREP) factors to present the differences in their toxicity between the sub‐groups of PA congeners concerning their structural features. The use of such iREP factors could probably provide a more scientific basis for PA risk assessment until sufficient experimental analysis of the toxicities of individual congeners is applied. To obtain a better understanding of the relationship between structure and toxicity of PA congeners and provide more evidence for further refinement of relative (toxic) potency factors, data of the in vitro cytotoxicity, genotoxicity, and mutagenicity of diverse individual PA congeners (lasiocarpine, monocrotalineφ, retrorsine, senecionine, seneciphyllineφ, echimidineφ, europineφ, heliotrineφ, indicine, and lycopsamine) has been generated in our project supported by Kooperation Phytopharmaka. Among them, lasiocarpine, retrorsine, senecionine, indicine and lycopsamine have been investigated on my part.

Cytotoxicity was assessed using the Alamar blue assay in primary rat hepatocytes, HepG2 cells, and the HepG2 (CYP3A4) cell line. In HepG2 cells, none of the selected PAs exhibited cytotoxic effects, probably due to the lack of CYPs. In primary rat hepatocytes as well as in HepG2(CYP3A4) cells, a clear structure dependent cytotoxicity could be demonstrated. The role of CYP450 enzymes in metabolic activation was further confirmed using an inhibition assay. A kinetic assay analyzing 7‐benzyloxyresorufin‐O‐ dealkylation (BROD) was used for measuring the activity of CYP450 enzymes. Furthermore, the utilization of a glutathione‐reductase‐DTNB recycling assay indicated that glutathione might not play a critical role in PA‐induced cytotoxicity. A micronucleus test was used for determining the PA‐induced clastogenic genotoxicity. All selected PA congeners exhibited concentration‐dependent toxicity in the HepG2 (CYP3A4) cells. The relative potencies of PA congeners estimated from Alamar blue assay and micronucleus assay are generally consistent with the following ranking: lasiocarpine > senecionine > seneciphylline > retrorsine > heliotrine (?) echimidine > europine = indicine = lycopsamine = monocrotaline. The relative toxic potencies evaluated based on our findings were not completely consistent with the iREP classification previously reported by Merz and Schrenk. Monocrotaline in both assays exhibited considerably lower toxic potency. Echimidine, however, was more toxic than expected. On the other hand, mutagenicity was measured in Ames fluctuation assay with Salmonella typhimurium strains TA98 and TA100. None of the selected PA congeners up to 300 μM showed mutagenic effects despite metabolic activation with S9‐mix.