COMPARISON OF METABOLOME ATTENUATION IN MONOLAYER AND THREE DIMENSIONAL HEPATOCYTE CULTURES

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Introduction and aim: Hepatotoxicity is a leading cause of drug withdrawal. Readily available immortalised cell lines do not express the full complement of metabolising enzymes, limiting their use in toxicology. However, spheroid cultures of hepatocytes more closely approximate in vivo architecture and could increase the application of immortalised cells. Here, the aim was to determine the metabolic activity of selected phase I enzymes in HepG2 monolayer and spheroid cultures following induction.

Methods: HepG2 spheroid cultures, using an agarose based 3D Petri Dish (12-series mold, 81 spheroid capacity) were optimised. Major drug metabolising CYP450 isoforms were targeted for induction using a drug cocktail. Cell stocks were passaged in the presence of Phenacetin (CYP1A2, 2.5 uM), Dextromethorphan (CYP2D6, 1.25 uM), Diclofenac (CYP2C9, 5 uM) and Midazolam (CYP3A4, 0.15 uM) for three weeks. Cells were then seeded as monolayers or spheroids in the continuous presence of the drug cocktail for 72h after which Bupropion (CYP2B6, 5 uM) was included in the drug cocktail for an additional 72h. A short term induction control included cell monolayers and spheroids not passaged in the presence of the drug cocktail, exposed only for the final 72h. Parent drug and metabolites were then quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Results: Optimal spheroid size was achieved when seeding 9.6x105 cells/well. Spheroid characterisation demonstrated an increase in protein content with spheroid viability confirmed using flow cytometry and fluorescence imaging. An LC-MS/MS method for the quantification of parent drugs and metabolites was developed and validated on an AB Sciex 4000 QTrap mass spectrometer. Following induction, under the conditions described, neither HepG2 monolayers nor spheroids were able to induce metabolic activity. Parent drugs were present at similar concentrations in monolayer and spheroid samples with no quantifiable increase in drug metabolites.

Conclusion: Exposure to sub-toxic concentrations of known CYP450 enzyme inducers over three weeks was unable to induce HepG2 cells to alter actively metabolising CYP450 enzymes in monolayers or spheroids. This suggests that despite neither culturing as spheroids nor induction with a drug cocktail recover a useful HepG2 cells phenotype and they remain suboptimal for toxicology and metabolic studies.