TY - GEN A1 - Rosellini, Matteo A1 - Omer, Ejlal A. A1 - Schulze, Alicia A1 - Ali, Nadeen T. A1 - Boulos, Joelle C. A1 - Marini, Federico A1 - Küpper, Jan-Heiner A1 - Efferth, Thomas T1 - Impact of plastic-related compounds on the gene expression signature of HepG2 cells transfected with CYP3A4 T2 - Archives of Toxicology N2 - The presence of plastic and microplastic within the oceans as well as in marine flora and fauna have caused a multitude of problems that have been the topic of numerous investigations for many years. However, their impact on human health remains largely unknown. Such plastic and microplastic particles have been detected in blood and placenta, underlining their ability to enter the human body. Plastics also contain other compounds, such as plasticizers, antioxidants, or dyes, whose impact on human health is currently being studied. Critical enzymes within the metabolism of endogenous molecules, especially of xenobiotics, are the cytochrome P450 monooxygenases (CYPs). Although their importance in maintaining cellular balance has been confirmed, their interactions with plastics and related products are poorly understood. In this study, the possible relationship between different plastic-related compounds and CYP3A4 as one of the most important CYPs was analyzed using hepatic cells overexpressing this enzyme. Beginning with virtual compound screening and molecular docking of more than 1000 plastic-related compounds, several candidates were identified to interact with CYP3A4. In a second step, RNA-sequencing was used to study in detail the transcriptome-wide gene expression levels affected by the selected compounds. Three candidate molecules ((2,2′-methylenebis(6- tert -butyl-4-methylphenol), 1,1-bis(3,5-di- tert -butyl-2-hydroxyphenyl)ethane, and 2,2′-methylenebis(6-cyclohexyl-4-methylphenol)) had an excellent binding affinity to CYP3A4 in-silico as well as cytotoxic effects and interactions with several metabolic pathways in-vitro. We identified common pathways influenced by all three selected plastic-related compounds. In particular, the suppression of pathways related to mitosis and ‘DNA-templated DNA replication’ which were confirmed by cell cycle analysis and single-cell gel electrophoresis. Furthermore, several mis-regulated metabolic and inflammation-related pathways were identified, suggesting the induction of hepatotoxicity at different levels. These findings imply that these compounds may cause liver problems subsequently affecting the entire organism. KW - Cytotoxicity KW - Ecotoxicity KW - Environmental pollution KW - Hepatotoxicity KW - Marine pollution KW - Microplastic KW - RNA-sequencing KW - Biological Sciences Y1 - 2024 U6 - https://doi.org/10.1007/s00204-023-03648-4 SN - 0340-5761 SN - 1432-0738 VL - 98 IS - 2 SP - 525 EP - 536 PB - Springer Berlin Heidelberg ER - TY - GEN A1 - Schulz, Christian A1 - Stegen, Sarah A1 - Jung, Friedrich A1 - Küpper, Jan-Heiner ED - Lamb, David ED - Syed, Khajamohiddin T1 - Mono-CYP CHO model : a recombinant Chinese hamster ovary cell platform for investigating CYP-specific tamoxifen metabolism T2 - International journal of molecular sciences N2 - The metabolism of drugs and foreign substances in humans typically involves multiple enzymatic steps, particularly in phase-1 biotransformation in the liver, where various cytochrome P450 monooxygenases (CYPs) play crucial roles. This complexity can lead to a wide range of metabolites. Understanding the contributions of individual CYPs and their interactions within these intricate enzyme cascades can be challenging. We recently developed an in vitro biotransformation platform employing various Chinese Hamster Ovarian (CHO) cell clones. These clones express human cytochrome P450 oxidoreductase (CPR), and each is defined by a specific human CYP enzyme expression, thus exhibiting no detectable endogenous CYP enzyme activity (mono-CYP CHO platform). In this study, we investigated whether the mono-CYP CHO platform is a suitable tool for modeling complex drug metabolization reactions in vitro. Tamoxifen (TAM) was selected as a model substance due to its role as a prodrug widely used in breast cancer therapy, where its main active metabolite, endoxifen, arises from a two-step metabolism primarily involving the CYP system. Specifically, the combined activity of CYP3A4 and CYP2D6 is believed to be essential for efficient endoxifen production. However, the physiological metabolization pathway of TAM is more complex and interconnected, and the reasons for TAM’s therapeutic success and variability among patients are not yet fully understood. Analogous to our recently introduced mono-CYP3A4 CHO cells, we generated a CHO cell line expressing human CPR and CYP2D6, including analysis of CYP2D6 expression and specific activity. Comparative studies on the metabolization of TAM were performed with both mono-CYP CHO models individually and in co-culture with intact cells as well as with isolated microsomes. Supernatants were analyzed by HPLC to calculate individual CYP activity for each metabolite. All the picked mono-CYP2D6 clones expressed similar CYP2D6 protein amounts but showed different enzyme activities. Mono-CYP2D6 clone 18 was selected as the most suitable for TAM metabolization based on microsomal activity assays. TAM conversion with mono-CYP2D6 and -3A4 clones, as well as the combination of both, resulted in the formation of the expected main metabolites. Mono-CYP2D6 cells and microsomes produced the highest detected amounts of 4-hydroxytamoxifen and endoxifen, along with N -desmethyltamoxifen and small amounts of N , N -didesmethyltamoxifen. N -desmethyltamoxifen was the only TAM metabolite detected in notable quantities in mono-CYP3A4, while 4-hydroxytamoxifen and endoxifen were present only in trace amounts. In CYP2D6/3A4 co-culture and equal mixtures of both CYP microsomes, all metabolites were detected at concentrations around 50% of those in individual clones, indicating no significant synergistic effects. In conclusion, our mono-CYP CHO model confirmed the essential role of CYP2D6 in synthesizing the active TAM metabolite endoxifen and indicated that CYP2D6 is also involved in producing the by-metabolite N , N -didesmethyltamoxifen. The differences in metabolite spectra between the two mono-CYP models highlight the CYP specificity and sensitivity of our in vitro system. KW - Chinese hamster ovary cells KW - Mono-CYP CHO KW - Liver KW - Phase-1 biotransformation KW - Cytochrome P450 monooxygenase KW - CYP2D6 KW - Tamoxifen KW - Endoxifen KW - DDM-TAM Y1 - 2025 U6 - https://doi.org/10.3390/ijms26093992 SN - 1422-0067 VL - 26 IS - 9 SP - 1 EP - 24 PB - MDPI CY - Basel 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 - TY - GEN A1 - Nerusch, Julia A1 - Schicht, Gerda A1 - Herzog, Natalie A1 - Küpper, Jan-Heiner A1 - Seehofer, Daniel A1 - Damm, Georg T1 - Investigation and distinction of energy metabolism in proliferating hepatocytes and hepatocellular carcinoma cells T2 - Cells N2 - Metabolic rewiring is a hallmark of both hepatic regeneration and malignant transformation, complicating the identification of cancer-specific traits. This study aimed to distinguish the metabolic profiles of proliferating hepatocytes and hepatocellular carcinoma (HCC) cells through integrated analyses of mRNA and protein expression, along with functional characterization. We compared non-malignant Upcyte® hepatocytes (HepaFH3) cultured under proliferative and confluent conditions with primary human hepatocytes, primary human hepatoma cells, and hepatoma cell lines. Proliferating HepaFH3 cells exhibited features of metabolic reprogramming, including elevated glycolysis, increased HIF1A expression, and ketone body accumulation, while maintaining low c-MYC expression and reduced BDH1 levels, distinguishing them from malignant models. In contrast, HCC cells showed upregulation of HK2, c-MYC, and BDH1, reflecting a shift toward aggressive glycolytic and ketolytic metabolism. Functional assays supported the transcript and protein expression data, demonstrating increased glucose uptake, elevated lactate secretion, and reduced glycogen storage in both proliferating and malignant cells. These findings reveal that cancer-like metabolic changes also occur during hepatic regeneration, limiting the diagnostic utility of individual metabolic markers. HepaFH3 cells thus provide a physiologically relevant in vitro model to study regeneration-associated metabolic adaptation and may offer insights that contribute to distinguishing regenerative from malignant processes. Our findings highlight the potential of integrated metabolic profiling in differentiating proliferation from tumorigenesis. KW - Hepatocellular carcinoma KW - HepaFH3 KW - Metabolic reprogramming KW - Glycolysis KW - Ketone body metabolism KW - Liver regeneration KW - Tumor markers KW - HIF1A KW - c-MYC KW - BDH1 Y1 - 2025 U6 - https://doi.org/10.3390/cells14161254 SN - 2073-4409 VL - 14 IS - 16 SP - 1 EP - 26 PB - MDPI CY - Basel ER - 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 - Braune, Steffen A1 - Jung, Conrad G. H. A1 - Küpper, Jan-Heiner A1 - Jung, Friedrich T1 - Arthrospira platensis as protein-rich source for human nutrition T2 - Life N2 - The continuing growth of the world’s population, combined with climate change, poses a growing challenge to ensuring food security in the 21st century. Animal protein, e.g., from beef, is a particularly rich source of protein, but there is by no means enough arable land on earth to transfer the animal meat-rich nutritional style of the early industrialized countries to the global South. A hitherto largely neglected option for the production of proteins is the cultivation of microalgae and cyanobacteria, which already have a long history of use as a human or animal food for their nutritional and environmental merits. In particular, Limnospira platensis (Spirulina and formerly Arthrospira platensis)—a filamentous cyanobacterium—is considered the “food of the future” since it is a viable source of vegan protein. In this manuscript, we review the scientific literature as well as national and intergovernmental agency statements regarding the quality and quantity of AP-based proteins. The content of AP protein is d KW - Arthrospira platensis KW - Limnospira platensis KW - Protein quality KW - Protein quantity Y1 - 2025 U6 - https://doi.org/10.3390/life15121789 SN - 2075-1729 VL - 15 IS - 12 SP - 1 EP - 15 PB - MDPI CY - Basel ER -