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Assessing the Metabolic Competence of Sandwich-Cultured Mouse Primary Hepatocytes

Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, The Netherlands (K.M., A.S.K., K.J.J.B., D.G.J.J., J.C.S.K., J.H.M.v.D.); Netherlands Toxicogenomics Center, Maastricht University, Maastricht, The Netherlands (K.M., D.G.J.J., J.C.S.K., J.H.M.v.D.); and Unidad Mixta Hospital La Fe-Advancell, Centro de Investigación Hospital La Fe, Valencia, Spain (A.L.)

Primary human and rat hepatocyte cultures are well established in vitro systems used in toxicological studies. However, whereas transgenic mouse models provide an opportunity for studying mechanisms of toxicity, mouse primary hepatocyte cultures are less well described. The potential usefulness of a mouse hepatocyte-based in vitro model was assessed in this study by investigating time-dependent competence for xenobiotic metabolism and gene expression profiles. Primary mouse hepatocytes, isolated using two-step collagenase perfusion, were cultured in a collagen sandwich configuration. Gene expression profiles and the activities of various cytochrome P450 (P450) enzymes were determined after 0, 42, and 90 h in culture. Principal component analysis of gene expression profiles shows that replicates per time point are similar. Gene expression levels of most phase I biotransformation enzymes decrease to approximately 69 and 57% of the original levels at 42 and 90 h, respectively, whereas enzyme activities for most of the studied P450s decrease to 59 and 34%. The decrease for phase II gene expression is only to 96 and 92% of the original levels at 42 and 90 h, respectively. Pathway analysis reveals initial effects at the level of proteins, external signaling pathways, and energy production. Later effects are observed for transcription, translation, membranes, and cell cycle-related gene sets. These results indicate that the sandwich-cultured primary mouse hepatocyte system is robust and seems to maintain its metabolic competence better than that of the rat hepatocyte system.