Abstract
Purpose. To examine the potential of cultured human liver slices to predict cytochrome P450 (CYP) inducibility, regarding global and zonal CYP expression, together with drug-induced histologic changes.
Methods. We first assessed whether CYP2B6, 3A4, and 2C9 expression was maintained in cultured liver slices. Cultured hepatocytes were used as the reference culture system. Then we tested the effects of phenobarbital and cyclophosphamide on CYP expression in both models.
Results. Morphologic features are preserved in slices. Basal CYP expression declines with time in culture in both models. Slices display the same region specificity of CYP2B6, 2C9, and 3A4 expression as intact liver. CYP2B6 and 3A4 mRNA, apoprotein, and enzyme-related activities were induced by phenobarbital and cyclophosphamide, whereas CYP2C9 apoprotein was not. Their immunoreactivities were also increased, while their zonal distribution was preserved on slice tissue sections. Microsomal enzyme induction was confirmed by histology.
Conclusions. Cultured human liver slices are an attractive alternative to hepatocyte culture for the prediction of human CYP isoenzyme induction by xenobiotics.
Similar content being viewed by others
REFERENCES
S. Coecke, V. Rogiers, M. Bayliss, J. Castell, J. Doehmer, G. Fabre, J. Fry, A. Kern, and C. Westmoreland. The use of long-term hepatocyte cultures for detecting induction of drug metabolizing enzymes: the current status. ATLA 27:579-638 (1999).
A. J. Gandolfi, J. Wijeweera, and K. Brendel. Use of precision-cut liver slices as an in vitro tool for evaluating liver function. Toxic. Pathol. 24:58-61 (1996).
BG. Lake. C. Charzat, J. M. Tredger, A. B. Renwick, J. A. Beamand, and R. J. Price. Induction of cytochrome P450 isoenzymes in cultured precision-cut rat and human liver slices. Xenobiotica 26:297-306 (1996).
B. G. Lake, S. E. Ball, A. B. Renwick, J. M. Tredger, J. Kao, J. A. Beamand, and R. J. Price. Induction of CYP3A isoforms in precision-cut human liver slices. Xenobiotica 27:1165-1173 (1997).
B. G. Lake, J. M. Tredger, A. B. Renwick, P. T. Barton, and R. J. Price. 3,3′-Diindolylmethane induces CYP1A2 in cultured precision-cut human liver slices. Xenobiotica 28:803-811 (1998).
M. Vandenbranden, S. A. Wrighton, S. Ekins, J. S. Gillespie, S. N. Binkley, B. J. Ring, M. G. Gadberry, D. C. Mullins, S. C. Strom, and C. B. Jensen. Alterations of the catalytic activities of drug-metabolizing enzymes in cultures of human liver slices. Drug Metab. Dispos. 26:1063-1068 (1998).
A. B. Renwick, P. S. Watts, R. J. Edwards, P. T. Barton, I. Guyonnet, R. J. Price, J. M. Tredger, O. Pelkonen, A. R. Boobis, and B. G. Lake. Differential maintenance of cytochrome P450 enzymes in cultured precision-cut human liver slices. Drug Metab. Dispos. 28:1202-1209 (2000).
A. T. Drahushuk, B. P. McGarrigle, K. E. Larsen, J. J. Stegeman, and J. R. Olson. Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture. Carcinogenesis 19:1361-1368 (1998).
R. Glöckner, P. Steinmetzer, C. Drobner, and D. Müller. Use of fresh and cryopreserved human liver slices in toxicology with special reference to in vitro induction of cytochrome P450. Toxicol. In Vitro 13:531-535 (1999).
T. K. H. Chang, L. Yu, P. Maurel, and D. J. Waxman. Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines. Cancer Res. 57:1946-1954 (1997).
L. Gervot, B. Rochat, J.-C. Gautier, F. Bohnenstengel, H. Kroemer, V. de Berardinis, H. Martin, P. Beaune, and I. de Waziers. Human CYP2B6: expression, inducibility and catalytic activities. Pharmacogenetics 9:295-306 (1999).
E. Lecluyse, A. Madan, G. Hamilton, K. Carroll, R. DeHaan, and A. Parkinson. Expression and regulation of cytochrome P450 enzymes in primary cultures of human hepatocytes. J. Biochem. Md. Toxicol. 14:177-188 (2000).
D. Runge, C. Köhler, V. E. Kostribsky, D. Jäger, T. Lehmann, D. M. Runge, U. May, D. B. Stolz, S. C. Strom, W. E. Fleig, and G. K. Michalopoulos. Induction of cytochrome P450 (CYP) 1A1, CYP1A2, and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes. Biochem. Biophys. Res. Commun. 273:333-341 (2000).
S. Gerbal-Chaloin, J.-M. Pascussi, L. Pichard-Garcia, M. Daujat, F. Waechter, J.-M. Fabre, N. Carrère, and P. Maurel. Induction of CYP2C genes in human hepatocytes in primary culture. Drug Metab. Dispos. 29:242-251 (2001).
F. Morel, P. H. Beaune, D. Ratanasavanh, J.-P. Flinois, C. S. Yang, F. P. Guengerich, and A. Guillouzo. Expression of cytochrome P-450 enzymes in cultured human hepatocytes. Eur. J. Biochem. 191:437-444 (1990).
C. Belloc, S. Baird, J. Cosme, S. Lecoeur, J.-C. Gautier, D. Challine, I. de Waziers, J. P. Flinois, and P. H. Beaune. Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies. Toxicology 106:207-219 (1996).
D. M. Stresser and D. Kupfer. Monospecific antipeptide antibody to cytochrome P-450 2B6. Drug Metab. Dispos. 27:517-525 (1999).
F. Ballet, M.-E. Bouma, S.-R. Wang, N. Amit, J. Marais, and R. Infante. Isolation, culture and charaterization of adult human hepatocytes from surgical liver biopsies. Hepatology 4:849-854 (1984).
J. George, B. Goodwin, C. Liddle, M. Tapner, and G. C. Farrell. Time-dependent expression of cytochrome P450 genes in primary cultures of well-differentiated human hepatocytes. J. Lab. Clin. Med. 129:638-648 (1997).
Z. Abdel-Razzak, P. Loyer, A. Fautrel, J.-C. Gautier, L. Corcos, B. Turlin, P. Beaune, and A. Guillouzo. Cytokines down-regulates expression of major enzymes in adult human hepatocytes in primary culture. Mol. Pharmacol. 44:707-715 (1993).
F. Lakehal, D. Wendum, V. Barbu, L. Becquemont, R. Poupon, P. Balladur, L. Hannoun, F. Ballet, P. H. Beaune, and C. Housset. Phase I and phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium. Hepatology 30:1498-1506 (1999).
I. H. Hanna, J. R. Reed, F. P. Guengerich, and P. F. Hollenberg. Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver. Arch. Biochem. Biophys. 376:206-216 (2000).
H. Yamazaki and T. Shimada. Progesterone and testosterone hydroxylation by cytochromes P450 2C19, 2C9, and 3A4 in human liver microsomes. Arch. Biochem. Biophys. 346:161-169 (1997).
C. Gopinath, D. E. Prentice, and D. J. Lewis. Atlas of experimental toxicological pathology. In: G. A. Gresham, ed. Current Histopathology, Vol. 13. MTP Press, Norwell, Massachusetts 1987, pp. 53-54.
E. G. Schuetz, J. D. Schuetz, S. C. Strom, M. T. Thompson, R. A. Fisher, D. T. Molowa, D. Li, and P. S. Guzelian. Regulation of human liver cytochrome P-450 in family 3A in primary and continuous culture of human hepatocytes. Hepatology 18:1254-1262 (1993).
D. Ratanasavanh, P. Beaune, F. Morel, J.-P. Flinois, F. P. Guengerich, and A. Guillouzo. Intralobular distribution and quantitation of cytochrome P-450 enzymes in human liver as a function of age. Hepatology 13:1142-1151 (1991).
G. I. Murray and M. D. Burke. Immunohistochemistry of drug-metabolizing enzymes. Biochem. Pharmacol. 50:895-903 (1995).
J. B. Houston and D. J. Carlile. Prediction of hepatic clearance from microsomes, hepatocytes, and liver slices. Drug Metab. Rev. 29:891-922 (1997).
N. Milosevic, H. Schawalder, and P. Maier. Kupffer cell-mediated differential down regulation of cytochrome P450 metabolism in rat hepatocytes. Eur. J. Pharmacol. 368:75-87 (1999).
M. Parola, G. Robino, R. Bordone, G. Leonarduzzi, A. Casini, M. Pinzani, E. Neve, G. Bellomo, M. U. Dianzani, M. Ingelman-Sundberg, and E. Albano. Detection of cytochrome P4503A (CYP3A) in human hepatic stellate cells. Biochem. Biophys. Res. Commun. 238:420-424 (1997).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Martin, H., Sarsat, JP., de Waziers, I. et al. Induction of Cytochrome P450 2B6 and 3A4 Expression by Phenobarbital and Cyclophosphamide in Cultured Human Liver Slices. Pharm Res 20, 557–568 (2003). https://doi.org/10.1023/A:1023234429596
Issue Date:
DOI: https://doi.org/10.1023/A:1023234429596