@misc{HaasAckermannKuepperetal., author = {Haas, Manuel and Ackermann, Gabriel and K{\"u}pper, Jan-Heiner and Glatt, Hansruedi and Schrenk, Dieter and Fahrer, J{\"o}rg}, title = {OCT1-dependent uptake of structurally diverse pyrrolizidine alkaloids in human liver cells is crucial for their genotoxic and cytotoxic effects}, series = {Archives of Toxicology}, volume = {97}, journal = {Archives of Toxicology}, number = {12}, issn = {1432-0738}, doi = {10.1007/s00204-023-03591-4}, pages = {3259 -- 3271}, abstract = {Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors D-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.}, language = {en} } @misc{SchulzHerzogKubicketal., author = {Schulz, Christian and Herzog, Natalie and Kubick, Stefan and Jung, Friedrich and K{\"u}pper, Jan-Heiner}, title = {Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies}, series = {Cells}, volume = {12}, journal = {Cells}, number = {17}, issn = {2073-4409}, doi = {10.3390/cells12172140}, abstract = {In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising "humanised" in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine.}, language = {en} } @misc{KruegerGengeKoehlerLaubeetal., author = {Kr{\"u}ger-Genge, Anne and K{\"o}hler, Susanne and Laube, Markus and Haileka, Vanessa and Lemm, Sandy and Majchrzak, Karolina and Kammerer, Sarah and Schulz, Christian and Storsberg, Joachim and Pietzsch, Jens and K{\"u}pper, Jan-Heiner and Jung, Friedrich}, title = {Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation—Relevance to Thrombosis}, series = {Cells}, volume = {12}, journal = {Cells}, number = {15}, issn = {2073-4409}, doi = {10.3390/cells12151965}, abstract = {Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.}, language = {en} } @misc{HaasWirachowskiThiboletal., author = {Haas, Manuel and Wirachowski, Karina and Thibol, Lea and K{\"u}pper, Jan-Heiner and Schrenk, Dieter and Fahrer, J{\"o}rg}, title = {Potency ranking of pyrrolizidine alkaloids in metabolically competent human liver cancer cells and primary human hepatocytes using a genotoxicity test battery}, series = {Archives of Toxicology}, volume = {97}, journal = {Archives of Toxicology}, number = {5}, issn = {0340-5761}, doi = {10.1007/s00204-023-03482-8}, pages = {1413 -- 1428}, 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.}, language = {en} } @misc{JungWaldeckPetricketal., author = {Jung, Conrad H. G. and Waldeck, Peter and Petrick, Ingolf and Akinwunmi, Mosunmol and Braune, Steffen and Jung, Friedrich and K{\"u}pper, Jan-Heiner}, title = {Light-induced changes in the morphology and fluorescence of Arthrospira platensis}, series = {Clinical Hemorheology and Microcirculation}, volume = {9}, journal = {Clinical Hemorheology and Microcirculation}, number = {1}, issn = {1386-0291}, doi = {10.3233/JCB-239001}, pages = {71 -- 77}, abstract = {The cyanobacterium Arthrospira platensis is a well-known source of bioactive substances. Growth and the generation of bioactive ingredients of Arthrospira platensis depend mainly on the quantity of light in a controlled environment. Photoinhibition is a time, nutrient, and light intensity-dependent decrease in photosynthetic efficiency. However, too strong illumination can induce two harmful effects: (1) photoinhibition as a reduction in photosynthetic rate and (2) photooxidation which can have lethal effects on the cells, and which can lead to total loss of the culture. The results of this microscopical study demonstrate, that under the procedures described and very high photon flux den-sities, not only a decrease in the photosynthetic efficiency but beyond, also destruction of Arthrospira platensis can occur.}, language = {en} } @misc{RoselliniSchulzeOmeretal., author = {Rosellini, Matteo and Schulze, Alicia and Omer, Ejlal A. and Ali, Nadeen T. and Marini, Federico and K{\"u}pper, Jan-Heiner and Efferth, Thomas}, title = {The effect of plastic-related compounds on transcriptome-wide gene expression on CYP2C19-overexpressing HepG2 cells}, series = {Molecules}, volume = {28}, journal = {Molecules}, number = {16}, issn = {1420-3049}, doi = {10.3390/molecules28165952}, abstract = {In recent years, plastic and especially microplastic in the oceans have caused huge problems to marine flora and fauna. Recently, such particles have also been detected in blood, breast milk, and placenta, underlining their ability to enter the human body, presumably via the food chain and other yet-unknown mechanisms. In addition, plastic contains plasticizers, antioxidants, or lubricants, whose impact on human health is also under investigation. At the cellular level, the most important enzymes involved in the metabolism of xenobiotic compounds are the cytochrome P450 monooxygenases (CYPs). Despite their extensive characterization in the maintenance of cellular balance, their interactions with plastic and related products are unexplored. In this study, the possible interactions between several plastic-related compounds and one of the most important cytochromes, CYP2C19, were analyzed. By applying virtual compound screening and molecular docking to more than 1000 commercially available plastic-related compounds, we identified candidates that are likely to interact with this protein. A growth inhibition assay confirmed their cytotoxic activity on a CYP2C19-transfected hepatic cell line. Subsequently, we studied the effect of the selected compounds on the transcriptome-wide gene expression level by conducting RNA sequencing. Three candidate molecules were identified, i.e., 2,2′-methylene bis(6-tert-butyl-4-methylphenol), 1,1-bis(3,5-di-tert-butyl-2-hydroxyphenyl) ethane, and 2,2′-methylene bis(6-cyclohexyl-4-methylphenol)), which bound with a high affinity to CYP2C19 in silico. They exerted a profound cytotoxicity in vitro and interacted with several metabolic pathways, of which the 'cholesterol biosynthesis process' was the most affected. In addition, other affected pathways involved mitosis, DNA replication, and inflammation, suggesting an increase in hepatotoxicity. These results indicate that plastic-related compounds could damage the liver by affecting several molecular pathways.}, language = {en} } @misc{KammererNowakMantkeetal., author = {Kammerer, Sarah and Nowak, Elisabeth and Mantke, Ren{\´e} and Jung, Friedrich and K{\"u}pper, Jan-Heiner}, title = {In vitro simulation of the liver first-pass effect with biotransformation-competent HepG2 cells to study effects of MG-132 on liver and cancer cells}, series = {Clinical Hemorheology and Microcirculation}, volume = {86}, journal = {Clinical Hemorheology and Microcirculation}, number = {1-2}, issn = {1386-0291}, doi = {10.3233/CH-238108}, pages = {159 -- 168}, abstract = {BACKGROUND: Liver biotransformation is the major route for drug metabolism in humans, often catalysed by cytochrome P450 (CYP) enzymes. This first-pass effect can lead to hepatotoxicity and influences the bioavailability of drugs. OBJECTIVE: We aimed to establish in vitro culture systems simulating the liver first-pass to study effects of the proteasome inhibitor MG-132 simultaneously on hepatocytes and cancer cells. METHODS: The first-pass effect was simulated by conditioned medium transfer (CMT) from pre-treated HepG2 CYP3A4-overexpressing cells to either pancreatic cancer cell line PANC-1 or primary colon cancer cells, and by indirect co-culture (CC) of liver and cancer cells in a shared medium compartment. Experimental proteasome inhibitor MG-132 was used as test substance as it is detoxified by CYP3A4. RESULTS: Cancer cells showed higher viabilities in the first-pass simulation by CMT and CC formats when compared to monocultures indicating effective detoxification of MG-132 by HepG2 CYP3A4-overexpressing cells. HepG2-CYP3A4 cells showed reduced viabilites after treatment with MG-132. CONCLUSIONS: We successfully established two different culture systems to simulate the liver first-pass effect in vitro. Such systems easily allow to study drug effects simultaneously on liver and on target cancer cells. They are of great value in pre-clinical cancer research, pharmaceutical research and drug development.}, language = {en} } @misc{NghinaunyeWaldeckJungetal., author = {Nghinaunye, Theopolina and Waldeck, Peter and Jung, Conrad H. G. and K{\"u}pper, Jan-Heiner and Jung, Friedrich and Braune, Steffen}, title = {Response of Arthrospira platensis to different temperatures regarding growth and biochemical composition}, series = {Clinical Hemorheology and Microcirculation}, journal = {Clinical Hemorheology and Microcirculation}, issn = {1386-0291}, doi = {10.3233/CH-238104}, pages = {1 -- 7}, abstract = {The growth of cyanobacteria can vary considerably depending on the ambient temperature. Since the optimal growth temperature for Arthrospira platensis (strain SAG21.99) is not yet known, this was investigated in the present study. The study revealed that a process temperature of 30°C seems to be optimal for the Arthrospira strain SAG21.99 cultivation in terms of a maximum biomass productivity. This was also true for the phycocyanin content which was at 30°C significantly higher than at 20 or 40°C.}, language = {en} } @misc{JungNghinaunyeWaldecketal., author = {Jung, Conrad H. G. and Nghinaunye, Theopolina and Waldeck, Peter and Braune, Steffen and Petrick, Ingolf and K{\"u}pper, Jan-Heiner and Jung, Friedrich}, title = {Decarbonization of Arthrospira platensis production by using atmospheric CO2 as an exclusive carbon source: proof of principle}, series = {International Journal of Environmental Science and Technology}, journal = {International Journal of Environmental Science and Technology}, issn = {1735-2630}, doi = {10.1007/s13762-023-05215-x}, abstract = {There is an urgent need to develop technologies for removing CO2 from the atmosphere to combat climate change. Microalgae and cyanobacteria, such as Arthrospira platensis (AP), have shown promise due to their high photoautotrophic biomass production. Conventional AP culture media are supplemented with high concentrations of NaHCO3 since AP utilizes as a carbon source. These culture conditions result in significant amounts of CO2 escaping into the atmosphere, instead of being sequestered during cultivation. Here, we investigated whether ambient air (0.042\% CO2) can be used for growing AP in a culture medium lacking a fossil-based carbon source. AP was cultured in 2 L glass bioreactors containing: (1) Zarrouk medium with 16.8 g/L NaHCO3 and aeration with 0.236 vvm air with 2\% CO2 ("NaHCO3/CO2-based") to compensate carbon loss due to CO2 outgassing, and (2) Zarrouk medium without NaHCO3 and a gas flow with ambient air (0.926 vvm) as the only carbon source ("air-based"). The air-based production resulted in the biofixation of 3.78 gCO2/L during the linear growth phase. With NaHCO3/CO2-based production, a comparable amount of 3.42 gCO2/L was obtained while 659.12 g of CO2 was released into the atmosphere. Total protein, phycocyanin, chlorophyll-a, and carotenoids were present in similar or increased amounts in AP produced by the air-based method. We concluded that cultivation of AP with Zarrouk medium lacking NaHCO3 but using ambient air with atmospheric CO2 as the only carbon source is possible without reducing productivity. These results improve our understanding of how atmospheric CO2 can be reduced by culturing AP.}, language = {en} } @misc{KruegerGengeHarbBrauneetal., author = {Kr{\"u}ger-Genge, Anne and Harb, Kudor and Braune, Steffen and Jung, Conrad H. G. and Westphal, Sophia and B{\"a}r, Stefanie and Mauger, Olivia and K{\"u}pper, Jan-Heiner and Jung, Friedrich}, title = {Effects of arthrospira platensis on human Uumbilical vein endothelial cells}, series = {Life}, volume = {14}, journal = {Life}, number = {10}, editor = {Georgianos, Panagiotis}, publisher = {MDPI}, issn = {2075-1729}, doi = {10.3390/life14101253}, pages = {19}, abstract = {Atherosclerosis is initiated by injury or damage to the vascular endothelial cell monolayer. Therefore, the early repair of the damaged vascular endothelium by a proliferation of neighbouring endothelial cells is important to prevent atherosclerosis and thrombotic events. Arthrospira platensis (AP) has been used as a dietary supplement, mainly due to its high content of vitamins, minerals, amino acids, and pigments such as chlorophylls, carotenoids, and phycocyanin, ingredients with antioxidant, anti-inflammatory, and anti-thrombotic properties. Therefore, in this prospective, placebo-controlled, data-driven, sample-size-estimated in vitro study, we tested whether an aqueous extract of AP at different concentrations (50, 100, and 200 µg/mL) had an effect on the different cellular parameters of human umbilical vein endothelial cells. Therefore, cell impedance measurement and cell proliferation were measured to investigate the monolayer formation. In addition, cell viability, integrity, and metabolism were analysed to evaluate singular cellular functions, especially the antithrombotic state. Furthermore, cell-cell and cell-substrate interactions were observed. The highest proliferation was achieved after the addition of 100 µg/mL. This was consistently confirmed by two independent optical experiments in cell cultures 48 h and 85 h after seeding and additionally by an indirect test. At this concentration, the activation or dysfunction of HUVECs was completely prevented, as confirmed by prostacyclin and interleukin-6 levels. In conclusion, in this study, AP induced a significant increase in HUVEC proliferation without inducing an inflammatory response but altered the hemostasiological balance in favour of prostacyclin over thromboxane, thereby creating an antithrombotic state. Thus, APE could be applied in the future as an accelerator of endothelial cell proliferation after, e.g., stent placement or atherosclerosis.}, language = {en} } @misc{RoselliniOmerSchulzeetal., author = {Rosellini, Matteo and Omer, Ejlal A. and Schulze, Alicia and Ali, Nadeen T. and Boulos, Joelle C. and Marini, Federico and K{\"u}pper, Jan-Heiner and Efferth, Thomas}, title = {Impact of plastic-related compounds on the gene expression signature of HepG2 cells transfected with CYP3A4}, series = {Archives of Toxicology}, volume = {98}, journal = {Archives of Toxicology}, number = {2}, publisher = {Springer Berlin Heidelberg}, issn = {0340-5761}, doi = {10.1007/s00204-023-03648-4}, pages = {525 -- 536}, abstract = {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.}, language = {en} } @misc{SchulzStegenJungetal., author = {Schulz, Christian and Stegen, Sarah and Jung, Friedrich and K{\"u}pper, Jan-Heiner}, title = {Mono-CYP CHO model : a recombinant Chinese hamster ovary cell platform for investigating CYP-specific tamoxifen metabolism}, series = {International journal of molecular sciences}, volume = {26}, journal = {International journal of molecular sciences}, number = {9}, editor = {Lamb, David and Syed, Khajamohiddin}, publisher = {MDPI}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms26093992}, pages = {1 -- 24}, abstract = {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.}, language = {en} } @misc{AckermannPeilQuarzetal., author = {Ackermann, G. and Peil, M. and Quarz, C. and Schmidt, A. and Halaczkiewicz, M. and Thomas, A. D. and Stegm{\"u}ller, S. and Richling, E. and Manolikakes, G. and Christmann, M. and K{\"u}pper, Jan-Heiner and Schrenk, D. and Fahrer, J.}, title = {Molecular dosimetry of estragole and 1′-hydroxyestragole-induced DNA adduct formation, clastogenicity and cytotoxicity in human liver cell models}, series = {Archives of toxicology}, volume = {99}, journal = {Archives of toxicology}, number = {9}, publisher = {Springer}, address = {Berlin ; Heidelberg}, issn = {0340-5761}, doi = {10.1007/s00204-025-04084-2}, pages = {3769 -- 3785}, abstract = {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.}, language = {en} } @misc{NeruschSchichtHerzogetal., author = {Nerusch, Julia and Schicht, Gerda and Herzog, Natalie and K{\"u}pper, Jan-Heiner and Seehofer, Daniel and Damm, Georg}, title = {Investigation and distinction of energy metabolism in proliferating hepatocytes and hepatocellular carcinoma cells}, series = {Cells}, volume = {14}, journal = {Cells}, number = {16}, publisher = {MDPI}, address = {Basel}, issn = {2073-4409}, doi = {10.3390/cells14161254}, pages = {1 -- 26}, abstract = {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.}, language = {en} } @misc{CarlssonHerzogFelskeetal., author = {Carlsson, Max J. and Herzog, Natalie and Felske, Christina and Ackermann, Gabriel and Regier, Alexander and Wittmann, Simon and Fern{\´a}ndez Cereijo, Ra{\´u}l and Sturla, Shana J. and K{\"u}pper, Jan-Heiner and Fahrer, J{\"o}rg}, title = {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}, series = {Chemical research in toxicology}, volume = {38}, journal = {Chemical research in toxicology}, number = {6}, publisher = {American Chemical Society (ACS)}, address = {New York, NY}, issn = {0893-228X}, doi = {10.1021/acs.chemrestox.5c00133}, pages = {1134 -- 1146}, abstract = {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.}, language = {en} } @misc{BrauneJungKuepperetal., author = {Braune, Steffen and Jung, Conrad G. H. and K{\"u}pper, Jan-Heiner and Jung, Friedrich}, title = {Arthrospira platensis as protein-rich source for human nutrition}, series = {Life}, volume = {15}, journal = {Life}, number = {12}, publisher = {MDPI}, address = {Basel}, issn = {2075-1729}, doi = {10.3390/life15121789}, pages = {1 -- 15}, abstract = {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}, language = {en} }