Phenotypical and functional characterization of a HepG2 cell clone stably overexpressing cytochrome P450 (CYP) 2C9

We aimed to generate a HepG2 cell clone stably overexpressing cytochrome P450 (CYP) 2C9 together with an empty vector (EV) control cell clone for pharmacological and toxicological studies of substances with CYP2C9-mediated biotransformation.

A new HepG2 cell clone was generated by lentiviral transduction to functionally overexpress human CYP2C9. We found high CYP2C9 transcript and protein levels based on qRT-PCR, Western blot and immunofluorescence in the cell clone. Most importantly, specific enzyme activities of 62.9 ± 2.6 pmol 4-hydroxydiclofenac/min/10⁶ cells as analyzed by diclofenac conversion via liquid chromatography-mass spectrometry were found in HepG2-CYP2C9 cells. CYP2C9 enzyme activity could successfully be inhibited by the application of the pan-CYP inhibitor 1-aminobenzotriazole or the CYP2C9-specific inhibitor sulfaphenazole. No changes in morphology or population doubling times were detected when comparing the clone to parental HepG2 cells. The corresponding EV control cell line was detected negative for CYP2C9 expression in all experimental setups and did not show any difference to the parental HepG2 cells. Thus, the newly established HepG2-CYP2C9 cell line is an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP2C9.