TY - GEN A1 - Carlsson, Max J. A1 - Herzog, Natalie A1 - Felske, Christina A1 - Ackermann, Gabriel A1 - Regier, Alexander A1 - Wittmann, Simon A1 - Fernández Cereijo, Raúl A1 - Sturla, Shana J. A1 - Küpper, Jan-Heiner A1 - Fahrer, Jörg T1 - The DNA repair protein MGMT protects against the genotoxicity of N-nitrosodimethylamine, but Not N-nitrosodiethanolamine and N-nitrosomethylaniline, in human HepG2 liver cells with CYP2E1 expression T2 - Chemical research in toxicology N2 - N-nitrosamines are genotoxic contaminants that occur in the diet, consumer products, and the environment. More recently, N-nitrosamines were also detected as drug impurities. After uptake, N-nitrosamines undergo metabolic activation by cytochrome P450 monooxygenases (CYPs), resulting in DNA damage and tumor formation. In this study, the genotoxicity and cytotoxicity of three N-nitrosamines with structurally distinct substituents, N-nitrosodimethylamine (NDMA), N-nitrosodiethanolamine (NDELA) and N-nitrosomethylaniline (NMA), were analyzed in human HepG2 liver cell models proficient or deficient in CYP2E1 biotransformation. Furthermore, the impact of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) was investigated. The novel genetically engineered HepG2-CYP2E1 cell line strongly expressed CYP2E1, which was not detectable in wildtype (WT) HepG2 cells. We then confirmed that the CYP2E1 substrate NDMA caused O6-methyldesoxyguanosine adducts and DNA strand breaks in a CYP2E1-dependent manner, leading to cytotoxicity. By the same approach, we demonstrated that NDELA induced DNA strand breaks in HepG2-CYP2E1 cells, whereas no effect was observed for NMA. However, NMA was revealed to cause DNA cross-links. Furthermore, both NDELA and NMA were cytotoxic in HepG2-CYP2E1 cells, but not in WT cells. Subsequently, the pharmacological MGMT inhibitor O6-benzylguanine was used to deplete MGMT in both HepG2 cell models. MGMT inhibition clearly increased DNA strand break levels due to NDMA exposure, whereas DNA strand break formation by NDELA and NMA were not affected by inhibiting MGMT. In line with these findings, the clastogenic effects of NDMA were potentiated in the absence of MGMT. In contrast to that, NDELA- and NMA-induced clastogenicity was not influenced by MGMT inhibition. Taken together, our study revealed that all three structurally diverse N-nitrosamines are cytotoxic and clastogenic in a CYP2E1-dependent manner, while only NDMA and NDELA caused DNA strand breaks. Furthermore, we demonstrated for the first time that DNA repair by MGMT does not confer protection against NDELA and NMA-triggered DNA strand break induction and clastogenicity. KW - Adducts KW - Assays KW - Genetics KW - Toxicity KW - Transition metals Y1 - 2025 U6 - https://doi.org/10.1021/acs.chemrestox.5c00133 SN - 0893-228X VL - 38 IS - 6 SP - 1134 EP - 1146 PB - American Chemical Society (ACS) CY - New York, NY ER - TY - GEN A1 - Haas, M. A1 - Wirachowski, K. A1 - Küpper, Jan-Heiner A1 - Schrenk, Dieter A1 - Fahrer, J. T1 - Structure‐dependent genotoxicity and cytotoxicity of eleven pyrrolizidine alkaloids in human liver cells with CYP3A4 overexpression T2 - Lebensmittelchemie : Volume 75: Vorträge und Poster des 49. Deutschen Lebensmittelchemikertags Y1 - 2021 U6 - https://doi.org/10.1002/lemi.202158135 SN - 0937-1478 SN - 1521-3811 SP - S134 ER - TY - GEN A1 - Ackermann, G. A1 - Peil, M. A1 - Quarz, C. A1 - Schmidt, A. A1 - Halaczkiewicz, M. A1 - Thomas, A. D. A1 - Stegmüller, S. A1 - Richling, E. A1 - Manolikakes, G. A1 - Christmann, M. A1 - Küpper, Jan-Heiner A1 - Schrenk, D. A1 - Fahrer, J. T1 - Molecular dosimetry of estragole and 1′-hydroxyestragole-induced DNA adduct formation, clastogenicity and cytotoxicity in human liver cell models T2 - Archives of toxicology N2 - The phenylpropene estragole (ES) is found in essential oils of herbs and spices, such as bitter fennel and basil. Humans are exposed to ES through the diet and phytomedicines. After its absorption, ES undergoes metabolic activation by CYP1A2 and SULT1A1 in the liver, which can give rise to DNA adducts and hepatocarcinogenesis. Until now, quantitative genotoxicity data for ES in human liver cells are scarce, correlating DNA adduct levels with critical effects such as clastogenicity. Here, we used human HepG2 and HepG2-CYP1A2 cells as well as primary human hepatocytes (PHH) to study the genotoxic, clastogenic and cytotoxic potential of ES and its crucial metabolite 1′-hydroxyestragole (1′OH-ES). In addition, primary rat hepatocytes (PRH) were used for selected endpoints. Treatment of HepG2-CYP1A2 cells with ES (0–2 mM) led to the concentration-dependent formation of E3′- N 2 -dG adducts. Apart from a moderate γH2AX induction, neither p53 accumulation nor cytotoxicity was observed. However, clastogenicity was demonstrated at ES concentrations ≥ 1 mM. Incubation of HepG2 cells with 1′OH-ES (0—35 µM) led to 10–50-fold higher E3′- N 2 -dG adduct levels compared to equimolar ES concentrations. Furthermore, 1′OH-ES caused γH2AX formation, p53 accumulation and cytotoxicity, which was confirmed in PHH. In agreement, 1′OH-ES induced clastogenicity at concentrations ≥ 25 µM. Molecular dosimetry revealed that a certain E3′- N 2 -dG adduct level is required to trigger clastogenicity and cytotoxicity. This was confirmed by Benchmark Concentration (BMC) modelling, showing that the BMC for clastogenicity is 12–17-fold higher than the respective BMC for DNA adduct formation. Our data indicate that a threshold level of DNA adducts is required, both in rat and human liver cells, to trigger markers of clastogenicity. These levels are unlikely to be reached in humans following chronic ES exposure through phytomedicines or the diet. KW - Plant toxin KW - Phenylpropanoids KW - Estragole KW - Human hepatocytes KW - Liver damage KW - Cytotoxicity KW - Genotoxicity KW - Clastogenicity KW - Concentration–response modelling Y1 - 2025 U6 - https://doi.org/10.1007/s00204-025-04084-2 SN - 0340-5761 SN - 1432-0738 VL - 99 IS - 9 SP - 3769 EP - 3785 PB - Springer CY - Berlin ; Heidelberg ER -