Pyrrolizidine alkaloids (PAs) occur as contaminants in plant-based foods and herbal medicines. Following metabolic activation by cytochrome P450 (CYP) enzymes, PAs induce DNA damage, hepatotoxicity and can cause liver cancer in rodents. There is ample evidence that the chemical structure of PAs determines their toxicity. However, more quantitative genotoxicity data are required, particularly in primary human hepatocytes (PHH). Here, the genotoxicity of eleven structurally different PAs was investigated in human HepG2 liver cells with CYP3A4 overexpression and PHH using an in vitro test battery. Furthermore, the data were subject to benchmark dose (BMD) modeling to derive the genotoxic potency of individual PAs. The cytotoxicity was initially determined in HepG2-CYP3A4 cells, revealing a clear structure–toxicity relationship for the PAs. Importantly, experiments in PHH confirmed the structure-dependent toxicity and cytotoxic potency ranking of the tested PAs. The genotoxicity markers γH2AX and p53 as well as the alkaline Comet assay consistently demonstrated a structure-dependent genotoxicity of PAs in HepG2-CYP3A4 cells, correlating well with their cytotoxic potency. BMD modeling yielded BMD values in the range of 0.1–10 µM for most cyclic and open diesters, followed by the monoesters. While retrorsine showed the highest genotoxic potency, monocrotaline and lycopsamine displayed the lowest genotoxicity. Finally, experiments in PHH corroborated the genotoxic potency ranking, and revealed genotoxic effects even in the absence of detectable cytotoxicity. In conclusion, our findings strongly support the concept of grouping PAs into potency classes and help to pave the way for a broader acceptance of relative potency factors in risk assessment.

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Introduction: Pyrrolizidine alkaloids (PAs) are secondary metabolites that occur as contaminants in various plant-based foods and dietary supplements. Following metabolic activation by CYP450 enzymes, PA cause hepatotoxicity and induce DNA damage that can lead to the development of liver tumours [1]. Several lines of evidence suggest that PAs are not equally toxic and differ in their genotoxic and cytotoxic potential, which is important for their risk assessment in food and phytopharmaceuticals [2,3]. The aim of the study was to detail the relative genotoxic and cytotoxic potential of eleven pyrrolizidine alkaloids in human liver cells depending on their structure and degree of esterification.Methods: CYP3A4-proficient human liver cells were used for metabolic activation of PAs. The cells were incubated with eleven different PAs for 24 h or 72 h. The cytotoxic effects of PAs were determined by the resazurin viability assay and EC50 values were calculated to assess the relative cytotoxicity. The genotoxic potential was investigated using western-blot analysis of the DNA damage markers γH2AX and p53. Data were subject to BMD modelling via PROAST software to derive BMD values for assessing the relative genotoxicity.Results: In general, monoesters such as lycopsamine display the lowest and cyclic diesters including lasiocarpine the strongest cytotoxic effects. The determined EC50 values of the tested PAs are in a broad range from 4 µM up to 500 µM and above. Furthermore, a concentration-dependent formation of γH2AX and p53 was observed. The lowest BMDL values ranging from 0.2 -0.8 µM were obtained for retrorsine, lasiocarpine, seneciphylline and echimidine. Conclusion:The results show a concentration-and structure-dependent toxicity based on the degree of esterification. In addition, p53 was revealed as a similarly sensitive marker of genotoxicity as γH2AX. Our data support the view to classify PAs according to their relative potency, which is relevant for their risk assessment. The data are supplemented by ongoing comet assays.