Comparison of metabalome attenuation in monolayer and three dimensional hepatocyte cultures

Abstract

Introduction: Drugs have been removed from the market for years due to toxicity screening. The highest attrition from the market can be seen by drugs that cause hepatotoxicity. Most drugs are metabolised by phase I cytochrome enzymes with five cytochromes metabolising approximately 95% of drugs. One of the most used screening platforms is the immortalised hepatocyte cancer cell line: HepG2. As the common model displays almost no phase I metabolomic competency, HepG2 cells are under investigation for prediction of hepatotoxicity via phase I enzymes using both an affordable and reliable methodology. Multicellular spheroid cell models have been shown to be closer to physiological conditions than monolayer cell cultures. Thus, the aim of the study was to determine the degree to which long term exposure to a tailored cytochrome inducing drug cocktail could enhance cytochrome P450 activity, in both traditional monolayer and spheroid cell cultures, at the level of the metabolome Methods and materials A suspension spheroid model was used with an agarose mould which, post-optimization, formed 81 spheroids per well. Protein quantification, live-dead microscopy and cell cycle analysis was used to determine the cell viability and culture time. The sulfurhodamine B assay was used to determine the IC50 of each drug individually as well as the combined IC50 of the cocktail for induction. Cells were cultured in presence of an induction cocktail, containing five prototypical cytochrome inducers, continuously for three weeks in monolayers. Cells were then cultured in monolayer and spheroid formats for a further six days. The liquid chromatography mass spectrometry method was optimised by first optimising the chromatography then the mass spectrometer parameters. A single method was developed for liquid chromatography tandem mass spectrometry analysis of 10 analytes in one consecutive run. Cell culture supernatant was collected, and the developed method used to determine the change in parent drug versus metabolites. Results and discussion During the optimisation of the spheroids, cells were shown to be compromised by day seven. Therefore, the cells were only cultured for a total time of six days within the spheroid format, to ensure viability. Only two of the drugs appeared to be metabolised. Dextromethorphan was metabolised more in the monolayer format and midazolam was metabolised more in the spheroid format. The overall phase I metabolic capacity of the HepG2 cells were not increased by long-term culture in the presence of a drug cocktail when cultured in either the monolayer or the three-dimensional spheroid format. Conclusion Exposure to the drug cocktail containing phenacetin, dextromethorphan, diclofenac, midazolam and bupropion was assessed using the validated method established during this study. Metabolism was not consistent for the enzyme-combination, as implemented in this study, as only certain drugs were metabolised sufficiently to quantitate the metabolites. It remains unclear whether or not the measured metabolism is because of actual enzyme induction, from inherent metabolism potential or from further problems regarding abnormalities of other inherent metabolism (such as possible phase III transporter inactivity) of the cells.a