Toxicology in Vitro 21 (2007) 892–901 www.elsevier.com/locate/toxinvit 0887-2333/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.01.010 A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression proWling A.S. Kienhuis a,b , H.M. Wortelboer a , W.J. Maas a , M. van Herwijnen b , J.C.S. Kleinjans b , J.H.M. van Delft b , R.H. Stierum a,¤ a Business Unit Biosciences, TNO Quality of Life, P.O. Box 360, 3700 AJ, Zeist, The Netherlands b Department of Health Risk Analysis and Toxicology, University of Maastricht, Maastricht, The Netherlands Received 24 August 2006; accepted 13 January 2007 Available online 20 January 2007 Abstract A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Conse- quently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte-based in vitro system was developed with special focus on metabolic competence. Therefore, a mixture of CYP450 inducers, phenobarbital, dexamethasone and -naphthoXavone, was added to culture medium of sandwich-cultured rat hepatocytes. The resulting modiWed model was evaluated by comparing its genome-wide expression proWles with liver and a standard model without the inducer mixture. Metabolic capacity for CYP450 enzymes showed that the modiWed model resembled more closely the in vivo situation. Gene expression results revealed large diVerences between in vivo and both in vitro models. The slight diVerences between the two sandwich models were predominantly represented by gene expression changes in CYP450s. Importantly, in the modiWed model, expression ratios of the phase I and the majority of phase II genes more closely resembled liver in vivo. The CYP450 enzyme activities cor- responded with gene expression data. In conclusion, for toxicological applications using sandwich-cultured hepatocytes, the modiWed model may be preferred.  2007 Elsevier Ltd. All rights reserved. Keywords: Primary rat hepatocytes; In vitro models; Sandwich-culture; Metabolic competence; Gene expression proWling 1. Introduction Toxicological research extensively relies on data obtained from in vivo studies. As in vivo biological complex- ity cannot be eVectively modelled in vitro yet, replacement of these in vivo studies is likely to be a long-term prospect (Snodin, 2002). Nevertheless, in vitro assays represent a valuable tool for investigating mechanisms of toxicology (Davila et al., 1998). Traditionally, as the liver is the main organ for metabolism of many compounds, hepatocyte sys- tems are frequently used for in vitro investigations (Blaaub- oer et al., 1994; Davila et al., 1998). Evident shortcomings of rat liver in vitro systems are represented by the rapid decline of liver speciWc functions, in particular of cytochrome P450 (CYP450) enzyme activi- ties (Balls et al., 2002; Boess et al., 2003). Therefore, the in vitro models have limited relevance for the in vivo situa- tion with respect to chemicals for which metabolism depends on the CYP450 enzyme system. Many options to maintain liver speciWc functions in hepatocyte-based in vitro systems have been investigated. Three interrelated elements of hepatic in vitro systems, namely, extracellular Abbreviations: CYP450, cytochrome P450; PB, phenobarbital; DEX, dexamethasone; -NF, -naphthoXavone; DMEM, Dulbecco’s modiWed Eagle’s medium; FCS, fetal calf serum; PBS, phosphate buVered saline; KHB, Krebs Henseleit buVer; CTP, cytosine triphosphate; DMSO, dimethylsulfoxide; MMLV-RT, molony murine leukemia virus-reverse transcriptase; PCA, principal component analysis; BRB, Biometric Re- search Branch; GO, gene ontology; GenMAPP, Gene Map Annotator and Pathway ProWler; PC, principal component. * Corresponding author. Tel.: +31 30 694 4545; fax: +31 30 696 0264. E-mail address: rob.stierum@tno.nl (R.H. Stierum).