Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation

Abstract

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.

NHC-gold compounds mediate immune suppression through induction of AHR-TGFβ1 signalling in vitro and in scurfy mice

“…1B. We used hepatocellular carcinoma cell line HepG2 as a metabolite generator, which expresses the majority of human cytochromes P450 (CYPs) and is widely used in various microfluidic systems 17,18,22,23 . Thus, the compound of interest is metabolized in the chamber 1 (HepG2 cells) and cellular responses to metabolic products can be recorded in the chamber 2.…”

“…1c). We monitored the enzymatic activity of CYP1s in living cells using a commercially available dye Vividye BOMCC as described previously 18 . The result confirmed that resveratrol neutralized MC3-induced enzymatic activity of CYP1s (Supplementary Fig.…”

“…Recently, we established a liver-on-a-chip system consisting of a microfluidic chip equipped with two interconnected chambers to investigate cellular responses to liver-mediated drug metabolites 17,18 . Applying this system, we screened our own small molecules collection including some organometallics 19–21 .…”

Highlight report: Monitoring cytochrome P450 activities in living hepatocytes

“…Recently, Jannick Theobald and Xinlai Cheng from Heidelberg University published a methods' paper how to monitor cytochrome P450 (CYP) activities in living hepatocytes (Theobald et al, 2017[ 15 ]). For this purpose, the authors used substrates that are metabolized by CYP enzymes thereby forming highly fluorescent leaving groups that were quantified by a plate reader.…”

“…For this purpose, the authors used substrates that are metabolized by CYP enzymes thereby forming highly fluorescent leaving groups that were quantified by a plate reader. This technique allows repeated real-time measurements of cultivated hepatocytes over extincted time periods (Theobald et al, 2017[ 15 ]). The monitoring technique was validated by the use of CYP inducers and was applied to characterize differentiating HepaRG cells.…”