Comparison of Coumarin-Induced Toxicity between Sandwich-Cultured Primary Rat Hepatocytes and Rats in Vivo: A Toxicogenomics Approach Anne S. Kienhuis, Heleen M. Wortelboer, Jean-Christophe Hoflack, Edwin J. Moonen, Jos C.S. Kleinjans, Ben van Ommen, Joost H. M. van Delft, and Rob H. Stierum Business Unit Biosciences, TNO Quality of Life (Netherlands Organization for Applied Scientific Research), Zeist, The Netherlands (A.S.K., H.M.W., B.v.O., R.H.S.); Department of Health Risk Analysis and Toxicology, University of Maastricht, Maastricht, The Netherlands (A.S.K., E.J.M., J.C.S.K., J.H.M.v.D.); Independent author (J.C.H.) Received June 1, 2006; accepted September 7, 2006 ABSTRACT: Sandwich-cultured primary rat hepatocytes are often used as an in vitro model in toxicology and pharmacology. However, loss of liver-specific functions, in particular, the decline of cytochrome P450 (P450) enzyme activity, limits the value of this model for prediction of in vivo toxicity. In this study, we investigated whether a hepatic in vitro system with improved metabolic competence enhances the predictability for coumarin-induced in vivo toxicity by using a toxicogenomics approach. Therefore, primary rat hepato- cytes were cultured in sandwich configuration in medium contain- ing a mixture of low concentrations of P450 inducers, phenobar- bital, dexamethasone, and -naphthoflavone. The toxicogenomics approach used enabled comparison of similar mechanistic end- points at the molecular level between in vitro and in vivo condi- tions, namely, compound-induced changes in multiple genes and signaling pathways. Toxicant-induced cytotoxic effects and gene expression profiles observed in hepatocytes cultured in modified medium and hepatocytes cultured in standard medium (without inducers) were compared with results from a rat in vivo study. Coumarin was used as a model compound because its toxicity depends on bioactivation by P450 enzymes. Metabolism of cou- marin toward active metabolites, coumarin-induced cytotoxicity, and gene expression modulation were more pronounced in hepa- tocytes cultured in modified medium compared with hepatocytes cultured in standard medium. In addition, more genes and biolog- ical pathways were similarly affected by coumarin in hepatocytes cultured in modified medium and in vivo. In conclusion, these experiments showed that for coumarin-induced toxicity, sand- wich-cultured hepatocytes maintained in modified medium better represent the situation in vivo compared with hepatocytes cultured in standard medium. To assess possible hepatotoxicity, conventional studies rely on the use of animal model systems to examine tissue toxin levels, changes in serum levels of hepatic enzymes, and histopathological changes (Nuwaysir et al., 1999; Waring and Ulrich, 2000). Simple, well established in vitro assays, such as primary hepatocytes, precision-cut liver slices, and hepatic cell lines, are increasingly in demand for identifying potential hepatotoxicity in early stages of investigative toxicology and for decreasing attrition rates of drugs during lead optimization (Dambach et al., 2005). However, extrapolation of in vitro results to the in vivo situation remains a scientific challenge (Guillouzo, 1998). Toxicogenomics, the application of the genomics technologies in toxicology, would be particularly useful in the extrapolation from in vitro experiments to the in vivo situation. Extrapolations can be made at the molecular level, comparing similar mechanistic endpoints, namely compound-induced changes in multiple genes and signaling pathways (Hamadeh et al., 2002; Stierum et al., 2005). Several toxi- cogenomics-based studies have already been performed comparing rat hepatic in vitro models with the situation in vivo (Waring et al., 2001; Boess et al., 2003; Jessen et al., 2003). These studies concluded that, to date, no toxicogenomics-based in vitro system allowed for predic- tion of hepatotoxic responses in vivo. The main limitation of hepatic in vitro assays used in these toxicogenomics-based studies is the loss of liver-specific functions, in particular, cytochrome P450 (P450) monooxygenase activities (Balls et al., 2002; Boess et al., 2003). Extrapolation from these in vitro models to the in vivo situation is Financial support was provided by The Netherlands Organisation for Health Research and Development, program Alternatives to Animal Experiments (3170.0049) and the Dutch Ministry of Economic Affairs. Financial support pro- vided by Servier Nederland B.V. is greatly appreciated. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.011262. ABBREVIATIONS: P450, cytochrome P450; PB, phenobarbital; DEX, dexamethasone; -NF, -naphthoflavone; DMEM, Dulbecco’s modified Eagle’s medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ALAT, alanine aminotransferase; ASAT, aspartate amino- transferase; LDH, lactate dehydrogenase; GGT, -glutamyl transferase; DMSO, dimethyl sulfoxide; o-HPAA, o-hydroxyphenylacetic acid; TIGR, The Institute for Genomic Research; BRB, Biometric Research Branch; PCA, principal component analysis; PC, principal component; CE, coumarin 3,4-epoxide; o-HPA, o-hydroxyphenylacetaldehyde. 0090-9556/06/3412-2083–2090$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 12 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 11262/3153769 DMD 34:2083–2090, 2006 Printed in U.S.A. 2083 at ASPET Journals on May 20, 2016 dmd.aspetjournals.org Downloaded from