Fraunhofer Project Group PZ-Syn, Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany, located at the Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Germany.
Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
Clin Hemorheol Microcirc. 2021;79(1):231-243. doi: 10.3233/CH-219117.
Cell-based in vitro liver models are an important tool in the development and 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 role in catalysing phase-1 biotransformation of pharmaceuticals and xenobiotics. For a comprehensive understanding of the phase-1 biotransformation of drugs, the availability of well-characterized substances for the targeted modulation of in vitro liver models is essential. In 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 without recombinant expression of the most important drug metabolization enzyme CYP3A4.Aim of the study was to identify effective DPI concentrations for CPR/CYP activity modulation and potentially associated dose and time dependent hepatotoxic effects. The cells were treated with DPI doses up to 5,000nM (versus vehicle control) for a maximum of 48 h and subsequently examined for CYP3A4 activity as well as various toxicological relevant parameters such as cell morphology, integrity and viability, intracellular ATP level, and proliferation. Concluding, the experiments revealed a time- and concentration-dependent DPI mediated partial and complete inhibition of CYP3A4 activity in CYP3A4 overexpressing HepG2-cells (HepG2-CYP3A4). Other cell functions, including ATP synthesis and consequently the proliferation were negatively affected in both in vitro cell models. Since neither cell integrity nor cell viability were reduced, the effect of DPI in HepG2 can be assessed as cytostatic rather than cytotoxic.
基于细胞的体外肝模型是药理学和毒理学药物评估中新药物开发和评估的重要工具。肝微粒体酶复合物由细胞色素 P450 氧化还原酶 (CPR) 和细胞色素 P450 单加氧酶 (CYPs) 组成，在催化药物和外源性物质的 I 相生物转化中起决定性作用。为了全面了解药物的 I 相生物转化，需要有经过良好表征的物质来靶向调节体外肝模型。在这项研究中，我们研究了二苯并碘（DPI）抑制 I 相酶活性的能力，以及在有和没有重组表达最重要的药物代谢酶 CYP3A4 的体外 HepG2 细胞模型中的毒理学特征。研究目的是确定有效 DPI 浓度以调节 CPR/CYP 活性，并可能与潜在的剂量和时间相关的肝毒性作用有关。将细胞用 DPI 处理至高达 5000nM（与载体对照相比），最长达 48 小时，随后检查 CYP3A4 活性以及各种与毒性相关的参数，如细胞形态、完整性和活力、细胞内 ATP 水平和增殖。总之，实验结果表明，DPI 介导的 CYP3A4 活性的部分和完全抑制在 CYP3A4 过表达的 HepG2 细胞（HepG2-CYP3A4）中具有时间和浓度依赖性。两种体外细胞模型中的其他细胞功能，包括 ATP 合成和随后的增殖都受到负面影响。由于细胞完整性或细胞活力没有降低，因此可以将 DPI 在 HepG2 中的作用评估为细胞生长抑制而不是细胞毒性。
