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

Christian Schulz; Natalie Herzog; Stefan Kubick; Friedrich Jung; Jan-Heiner Küpper

doi:10.3390/cells12172140

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

Cells. 2023 Aug 24;12(17):2140. doi: 10.3390/cells12172140.

Authors

Christian Schulz^{1

2

3}, Natalie Herzog², Stefan Kubick^{3

4

5}, Friedrich Jung², Jan-Heiner Küpper^{1

2}

Affiliations

¹ Fraunhofer Project Group PZ-Syn, Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB) Located at the Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
² Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
³ Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany.
⁴ Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
⁵ Faculty of Health Sciences, Joint Faculty of the Brandenburg University of Technology Cottbus-Senftenberg, The Brandenburg Medical School Theodor Fontane and the University of Potsdam, Potsdam, Germany.

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.

Keywords: CHO-K1; CPR; CYP450; Chinese hamster ovary cells; NADPH P450 oxidoreductase; cytochrome P450 monooxygenase; liver; phase-1 biotransformation; serum-free; suspension cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Biotransformation
CHO Cells
Cricetinae
Cricetulus
Cytochrome P-450 CYP3A* / genetics
Cytochrome P-450 Enzyme System*
Humans

Substances

Cytochrome P-450 CYP3A
Cytochrome P-450 Enzyme System

Grants and funding

This research was funded by grants of the Ministerium für Wirtschaft, Forschung und Kultur (MWFK, state of Brandenburg, Germany) for the Fraunhofer Project Group “Pilzbasierte zellfreie Synthese-Plattformen–PZ-Syn” (project number 22-F241-03-FhG/005/001).