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Comparison of coumarin-induced toxicity between sandwich-cultured primary rat hepatocytes and rats in vivo: A toxicogenomics approach

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Author: Kienhuis, A.S. · Wortelboer, H.M. · Hoflack, J.C. · Moonen, E.J. · Kleinjans, J.C.S. · Ommen, B. van · Delft, J.H.M. van · Stierum, R.H.
Institution: TNO Kwaliteit van Leven
Source:Drug Metabolism and Disposition, 12, 34, 2083-2090
Identifier: 239699
doi: doi:10.1124/dmd.106.011262
Keywords: Biology · Biomedical Research · beta naphthoflavone · coumarin · cytochrome P450 · dexamethasone · drug metabolite · enzyme inducing agent · phenobarbital · animal cell · animal experiment · animal model · animal tissue · article · controlled study · culture medium · cytotoxicity · drug activation · drug metabolism · enzyme activity · experimental rat · gene expression · histopathology · in vitro study · in vivo study · liver cell · liver toxicity · male · nonhuman · nucleotide sequence · prediction · principal component analysis · priority journal · rat · signal transduction · toxicogenetics · Alanine Transaminase · Animals · Aspartate Aminotransferases · Cell Culture Techniques · Cells, Cultured · Cholesterol · Coumarins · gamma-Glutamyltransferase · Gene Expression Profiling · Hepatocytes · Liver · Male · Oligonucleotide Array Sequence Analysis · Rats · Rats, Wistar · Toxicogenetics


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 hepatocytes were cultured in sandwich configuration in medium containing a mixture of low concentrations of P450 inducers, phenobarbital, dexamethasone, and β-naphthoflavone. The toxicogenomics approach used enabled comparison of similar mechanistic endpoints at the molecular level between in vitro and in vivo conditions, 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 coumarin toward active metabolites, coumarin-induced cytotoxicity, and gene expression modulation were more pronounced in hepatocytes cultured in modified medium compared with hepatocytes cultured in standard medium. In addition, more genes and biological 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, sandwich-cultured hepatocytes maintained in modified medium better represent the situation in vivo compared with hepatocytes cultured in standard medium. Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics.