[en] An in vitro experimental model, fetal rat hepatocytes in culture, was metabolically characterized. Several enzymatic activities were expressed in these hepatocytes, namely, testosterone hydroxylations. Hepatocytes cultured up to 3 weeks in the presence of dexamethasone and phenobarbital still expressed some drug-metabolizing enzyme activities (e.g., ECOD). The enzymatic activities were measured both directly on monolayers during culture and on the corresponding harvested and homogenized cells. The results correlate perfectly with each other. The 'on cell' procedure allows us to repeat the assay or to measure several activities on the same cells at different time intervals. The presence of dexamethasone in the culture medium allows the expression and the induction of several cytochrome P450 isoenzymes, namely, those hydroxylating testosterone. This makes the model particularly attractive for induction experiments as well as for metabolic or toxicological studies needing longer treatments.
Thomé, Jean-Pierre ; Université de Liège - ULiège > Département Biologie, Ecologie et Evolution > Ecologie animale et écotoxicologie
Language :
English
Title :
Expression and Induction of Drug-Metabolizing Enzymes in Cultured Fetal Rat Hepatocytes
Publication date :
April 1994
Journal title :
Cell Biology and Toxicology
ISSN :
0742-2091
eISSN :
1573-6822
Publisher :
Kluwer Academic Publishers, Netherlands
Volume :
10
Issue :
2
Pages :
117-25
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
grant of the national impulse program 'Health Hazards' of the Bdgian Ministry of Science and Technology (contract HH46) FRSM - Fonds de la Recherche Scientifique Médicale [BE]
Bollinne A., Kremers P., Kolodjici C., Gielen J. (1987) Long term maintenance of monooxygenase activities in cultured fetal-rat hepatocytes. Cell Differentiation 21:239-46.
Burke M.D., Mayer R.M. (1983) Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450 binding of phenoxazone and a homologous series of its alkyl ethers (alkoxyresorufins). Chem Biol Interact 45:243-58.
Donato M.T., Gómez-Lechón M.J., Castell J.V. (1990) Drug metabolizing enzymes in rat hepatocytes co-cultured with cell lines. In Vitro Cell Dev Biol. 26:1057-62.
Ernster I. (1967) DT diaphorase. Methods Enzymol. 10:309-17.
Greenlee W.F., Poland A. (1978) An improved assay of 7-ethoxycoumarin O-deethylase activity: induction of hepatic enzyme activity in C57B1/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo- p-dioxin. J Pharm Exp Ther. 205:596-605.
Grislain L., Ratanasavanh D., Mocquad M.T. (1988) Primary culture of rat and human hepatocytes as a model system for the evaluation of cytotoxicity and metabolic pathways of the new drug S3341. Liver cells and drugs, A., Guillouzo, Libbey-Inserm, London, Paris; .
Guillouzo A. (1986) Use of isolated and cultured hepatocytes for xenobiotic metabolism and cytotoxicity studies. Isolated and cultured hepatocytes , A., Guillouzo, C., Guguen-Guillouzo, Libbey-Inserm, London, Paris; 313-32.
Iwasaki M., Juvonen R., Lindberg R., Negishi M. (1991) Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450. J Biol Chem. 266:3380-2.
Jaiswal A.K., Haaparanta T., Luc P-V, Schembri J., Adesnik M. (1990) Glucocorticoid regulation of a phenobarbital-inducible cytochrome P-450 gene: The presence of a functional glucocorticoid response element in the 5′-flanking region of the CYP2B2 gene. Nucl Acids Res. 18:4237-42.
Kremers P. (1986) Drug metabolism in cultured fetal hepatocytes. Research in isolated and cultured hepatocytes , A., Guillouzo, C., Guguen-Guillouzo, Libbey-Inserm, London, Paris; 285-312.
Kremers P., Letawe-Goujon F., De Graeve J., Duvivier J., Geilen J. (1983) The expressiqon of different monooxygenases supported by cytochrome P-450 in neonatal rats and in primary fetal hepatocytes culture. Eur J Biochem. 137:603-8.
Kremers P., Negro L., Gielen J. (1990) Rat fetal hepatocytes in culture: a model for metabolic and toxicological studies. Acta Pharm Jugosl. 40:383-93.
Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-5.
Lescoat G., Theze N., Clement B., Guillouzo A., Guguen-Guillouzo C. (1985) Modulation of fetal and neonatal rat hepatocyte functional activity by glucocorticoids in co-culture. Cell Differentiation 16:259-68.
Letawe-Goujon F., Kremers P., Beaune P., Paye M., Gielen J. (1984) Preparation and characterization of monoclonal antibodies against phenobarbital inducible cytochrome P-450. Biochem Biophys Res Commun 119:744-50.
Lowry O.H., Rosenbrough N.J., Farr A.L., Randall R.J. (1962) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-75.
Mathis M., Houser W.H., Bresnick E. (1989) Glucocorticoid regulation of the rat cytochrome P450c (P450IA1) gene: receptor binding with intron. Arch Biochem Biophys 269:93-105.
McMillan J.M., Shaddock J.G., Casciano D.A., Arlotto M.P., Leakey J.E.A. (1991) Differential stability of drug-metabolizing enzyme activities in primary rat hepatocytes, cultured in the absence or presence of dexamethasone. Mutat Res Fundam Mol Mech Mutagen 249:81-92.
Moldeus P. (1987) Comparison of model systems for metabolism. Drug metabolism: from molecules to man , D.J., Benford, J.W., Bridges, G.G., Gibson, Taylor and Francis, London; 309-16.
Paye M., Beaune P., Kremers P. (1984) Quantification of two cytochrome P-450 isoenzymes by an enzyme linked immunoabsorbent assay (ELISA). Biochem Biophys Res Commun 122:137-42.
Schuetz E.G., Li D., Omiecinski C.J. (1988) Regulation of gene-expression in adult-rat hepatocytes cultured on a basement-membrane matrix (review). J Cell Physiol 134:309-23.
Sirica A.E., Pitot H.C. (1980) Drug metabolism and effects of carcinogens in cultured hepatic cells. Pharmacol Rev. 31:205-28.
Towbin H., Staehelin T., Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4.
Van Cantfort J., Goujon F., Gielen J. (1979) Benzopyrene metabolism in rat fetal hepatocytes in culture. Improved methodology and effect of substrate concentration. Chem Biol Interact 28:147-60.
Villa P., Arioli P., Guaitani A. (1991) Mechanism of maintenance of liver-specific functions by DMSO in cultured rat hepatocytes. Exp Cell Res. 194:157-60.
Waxman D.J. (1988) Interactions of hepatic cytochromes P-450 with steroid hormones. Regioselectivity and stereospecificity of steroid metabolism and hormonal regulation of rat P-450 enzyme expression. Biochem Pharmacol. 37:71-84.
Waxman D.J. (1991) P-450 catalyzed steroid hydroxylation: assay and product identification by thin layer chromatography. Methods Enzymol. 206:462-75.
Waxman D.J., Lapenson D.P., Aoyama T., Gelboin H.V., Gonzalez F.J., Korzekwa K. (1991) Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s. Arch Biochem Biophys 290:160-6.
Wortelboer H.M., De Kruif C.A., Van Iersel A.A.J., Falke H.E., Noordhoek J., Blaauboer B. (1990) The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers. Biochem Pharmacol. 40:2525-34.
Wortelboer H.M., De Kruif C.A., Van Iersel A.A.J., Falke H.E., Noordhoek J., Blaauboer B.J. (1991) Comparison of cytochrome P450 isoenzyme profiles in rat liver and hepatocyte cultures. The effects of model inducers on apoproteins and biotransformation activities. Biochem Pharmacol 42:381-90.
Wright M.C., Paine A.J. (1992) Evidence that the loss of rat liver cytochrome P450 in vitro is not solely associated with the use of collagenase, the loss of cell-cell contacts and/or the absence of an extracellular matrix. Biochem Pharmacol 43:237-43.
Yeoh G.C.T., Brighton V.J., Angus D.A., Kraemer M., Vassy J., Chalemeau M.T. (1985) The effect of dexamethasone on albumin production by fetal rat hepatocytes in culture. Eur J Cell Biol 38:157-64.