Institute of Pharmacy and Molecular Biotechnology, Pharmaceutical Biology, Heidelberg University, Im Neuenheimer Feld 364, 69120, Heidelberg, Germany.
Department of Medicine II, Section Molecular Hepatology, Alcohol Associated Diseases, Medical Faculty Mannheim at Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167, Mannheim, Germany.
Arch Toxicol. 2018 Mar;92(3):1133-1149. doi: 10.1007/s00204-017-2128-1. Epub 2017 Dec 5.
The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug-drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems.
肝细胞的代谢活性是药物活性的核心前提，也是药物相互作用的关键因素。这种代谢中的核心作用在很大程度上取决于细胞色素 P450（CYP450）酶家族的活性，该活性不仅依赖于肝实质细胞的成熟，而且还受到药物和化学物质暴露的调控。在这里，我们报告了使用 VividDye 荧光 CYP450 底物来直接测量和连续监测活肝细胞中的代谢活性。我们观察到对通过芳香烃受体和药物组合起作用的既定和假定的 CYP450 诱导剂的反应具有时间和剂量依赖性。通过每天重复添加 VividDye 荧光底物，我们展示了 VividDye 用于监测肝实质细胞成熟和去分化的新应用。尽管缺乏对个别 CYP450 同工酶的高特异性，但我们的方法能够在不破坏培养的情况下连续监测活细胞中的代谢活性。我们的测定可以整合到体外肝模拟模型中进行在线监测，从而提高这些组织模型系统的可靠性。
