Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells
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GENOTOXICITY AND CARCINOGENICITY
Structure‑dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells Lukas Rutz1 · Lan Gao1 · Jan‑Heiner Küpper2 · Dieter Schrenk1 Received: 12 May 2020 / Accepted: 27 August 2020 © The Author(s) 2020
Abstract 1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 μM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration–response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration–response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs. Keywords Genotoxicity · Liver cells · Micronuclei · Mutagenicity · Pyrrolizidine alkaloids · Relative potencies Abbreviations BMC Benchmark concentration BROD 7-Benzoxyresorufin-O-dealkylase CYP Cytochrome P450 DAPI 4′,6-diamidino-2-phenylindole DMEM Dulbecco’s modified Eagle’s Medium DMSO Dimethyl sulfoxide DSMZ German collection of microorganisms and cell cultures GmbH EC50 Effective concentration 50% Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00204-020-02895-z) contains supplementary material, which is available to authorized users. * Dieter Schrenk [email protected]‑kl.de 1
Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany
Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany
2
EDTA Ethylene diamine tetraacetate EFSA European Food Safety Authority FAU Formazine attenuation unit FCS Fetal calf serum GSH Glutathione γH2AX Gamma-histone H2AX HG High glucose HVOC Hepato-venoocclusive disease HCl
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