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 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 end-points 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.
Footnotes
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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 provided by Servier Nederland B.V. is greatly appreciated.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.011262.
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ABBREVIATIONS: P450, cytochrome P450; PB, phenobarbital; DEX, dexamethasone; β-NF, β-naphthoflavone; DMEM, Dulbecco's modified Eagle's medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; LDH, lactate dehydrogenase; GGT, γ-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.
- Received June 1, 2006.
- Accepted September 7, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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