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Clinical Hemorheology and Microcirculation 79 (2021) 23143 DOI 10.3233/CH-219117 IOS PressInhibition of phase-1 biotransformation and cytostatic effects of diphenyleneiodonium on hepatoblastoma cell line HepG2 as well as a CYP3A4-overexpressing HepG2 cell cloneChristian Schulza , Friedrich Jungb and Jan-Heiner Kpperb, uFraunhofer Project Group PZ-Syn, Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany, positioned at the Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Germany b Institute of Biotechnology, Brandenburg University of Technologies Cottbus-Senftenberg, Senftenberg, GermanyaAbstract. Cell-based in vitro liver models are an essential tool within the improvement and Orthopoxvirus Gene ID evaluation of new drugs in pharmacological and toxicological drug assessment. Hepatic microsomal enzyme complexes, consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs), play a decisive function in catalysing phase-1 biotransformation of pharmaceuticals and xenobiotics. For any complete understanding from the phase-1 biotransformation of drugs, the availability of well-characterized substances for the targeted modulation of in vitro liver models is essential. Within this study, we investigated diphenyleneiodonium (DPI) for its ability to inhibit phase-1 enzyme activity and further its toxicological profile in an in vitro HepG2 cell model with and with no recombinant expression on the most significant drug metabolization enzyme CYP3A4. Aim on the study was to recognize efficient DPI concentrations for CPR/CYP activity modulation and potentially associated dose and time dependent hepatotoxic effects. The cells have been treated with DPI doses up to five,000 nM (versus automobile manage) to get a maximum of 48 h and subsequently examined for CYP3A4 activity at the same time as various toxicological relevant parameters including cell morphology, integrity and viability, intracellular ATP level, and proliferation. Concluding, the experiments revealed a time- and concentration-dependent DPI mediated partial and full inhibition of CYP3A4 activity in CYP3A4 overexpressing HepG2-cells (HepG2-CYP3A4). Other cell functions, such as ATP synthesis and consequently the ADC Linker Chemical site proliferation were negatively impacted in both in vitro cell models. Since neither cell integrity nor cell viability had been reduced, the impact of DPI in HepG2 could be assessed as cytostatic as opposed to cytotoxic. Keyword phrases: Phase-1, biotransformation, CYP, cytochrome P450 monooxygenase, CYP3A4, diphenyleneiodonium, DPI, HepG2, HepG2-CYP3A4, hepatocytes, NADPH-cytochrome P450 oxidoreductase, POR, CPR1. Introduction In humans, the liver may be the major organ for the metabolization and elimination of pharmaceuticals and xenobiotics on account of the higher expression of phase-1 and -2 enzymes in hepatocytes [1]. Because of this, hepatocytes are the topic of intensive research efforts, and in vitro systems based on these cells areCorresponding author: Jan-Heiner Kpper, Institute of Biotechnology, Brandenburg.