Potency ranking of pyrrolizidine alkaloids in metabolically competent human liver cancer cells and primary human hepatocytes using a genotoxicity test battery

Abstract

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.

OCT1-dependent uptake of structurally diverse pyrrolizidine alkaloids in human liver cells is crucial for their genotoxic and cytotoxic effects

“…In the present work, we investigated the relevance of OCT1 for the uptake of structurally diverse PAs in metabolically competent human HepG2-CYP3A4 cells, Chinese hamster V79-CYP3A4 cells and PHH. HepG2-CYP3A4 cells were revealed as a very useful model in our previous study, since the genotoxic and cytotoxic potency of structurally diverse PAs was in very good agreement with that obtained in PHH as gold standard (Haas et al 2023 ). Furthermore, HepG2 cells were reported to display OCT1 gene expression, although the expression levels were lower than those in PHH (Herzog et al 2016 ; Rodrigues et al 2009 ).…”

“…This biotransformation step gives rise to dehydro-pyrrolizidine derivatives (dehydro-PAs) and, upon hydrolysis, to (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), which both react with DNA and proteins (Edgar 2014 ; Fu 2017 ). A plethora of studies performed in different liver cell models including human primary hepatocytes demonstrated a structure–toxicity relationship for PAs, supporting the concept of grouping PAs into potency classes (Allemang et al 2018 ; Gao et al 2020 ; Haas et al 2023 ; Hadi et al 2021 ; Lester et al 2019 ; Louisse et al 2019 ; Merz and Schrenk 2016 ; Rutz et al 2020 ).…”

“…Up to date, no study is available that investigated the transport of PAs into PHHs. Based on our obtained results in HepG2-CYP3A4 as well as V79-CYP3A4 cells and our previous study in different human liver cell models (Haas et al 2023 ), we selected lasiocarpine at a concentration of 45 µM for these experiments in PHH. Our results showed that lasiocarpine treatment decreased the viability of PHH to approximately 33% (Fig.…”

“…However, quinidine only partially blocked γH2AX formation triggered by lasiocarpine, whereas it strongly attenuated γH2AX formation caused by heliotrine and riddelliine. This finding might indicate that low intracellular levels of lasiocarpine are sufficient to cause substantial γH2AX formation, bearing in mind that this is an open-chained PA diester with a higher genotoxic potency than the cyclic PA diester riddelliine and the PA monoester heliotrine (Haas et al 2023 ). Furthermore, we analyzed the DNA damage response markers CHK1 and CHK2.…”

“…All cell lines were mycoplasma negative, as demonstrated by routine PCR testing using Venor ® GeM OneStep (Berlin, Germany). Cryopreserved PHH pooled from five Caucasian donors were from Thermo Fisher Scientific (Massachusetts, USA) and were maintained as described recently (Haas et al 2023 ). After attachment of PHH to collagen type I coated plates, the plating medium was replaced with incubation medium containing the test compounds and cells were incubated for 24 h. Collagen type I was obtained from Corning (New York, USA) and prepared as sterile-filtered 50 µg/ mL stock solution in 0.8 M acetic acid for coating.…”