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Cytochrome P450 activities in pure and co-cultured rat hepatocytes. Effects of model inducers

  • Cellular And Molecular Toxicology
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Summary

The stability and inducibility of several P450 activities (namely, P450 1A1, 2A1, 2B1/2, 2C11, and 3A1) were studied in rat hepatocytes co-cultured with the MS epithelial cell line derived from monkey kidney. The results revealed that these monooxygenase activities were systematically higher in co-cultures than in conventional hepatocyte cultures. Pure cultures showed a rapid loss of monooxygenase activities, which were undetectable after 5 days. In contrast, all isozymes assayed were measurable in co-cultured hepatocytes on Day 7 (about 15 to 40% of the initial activities of Day 0 of culture). The beneficial effects of the co-culture system seemed to be more selective for certain cytochrome P450 isoforms, with P450 1A1 and 3A1 being the best stabilized isozymes after 1 wk. A clear response to inducers was observed in co-cultures, each isozyme showing a different induction pattern. 3-Methylcholanthrene produced a strong increase in P450 1A1 (7-ethoxyresorufin O-deethylase) activity and a low increase in P450 2A1 (testosterone 7α-hydroxylation), whereas no changes were observed in the other activities. Phenobarbital treatment resulted in increases in P450 2B1/2 (7-pentoxyresorufin O-depentylase and 16α- and 16β-hydroxylation of testosterone) activities, while minor effects were observed on P450 3A1 (testosterone 6β-hydroxylation) activity. Dexamethasone markedly increased P450 3A1 (testosterone 6β- and 15β-hydroxylation) activity and, to a lesser extent, P450 2B1/2 (16β-hydroxylation).

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References

  1. Akrawi, M.; Rogiers, V.; Vandenberghe, Y., et al. Maintenance and induction in co-cultured rat hepatocytes of components of the cytochrome P450-mediated mono-oxygenase. Biochem. Pharmacol. 45:1583–1591; 1993.

    Article  PubMed  CAS  Google Scholar 

  2. Begué, J.; Guguen-Guillouzo, C.; Pasdeloup, N., et al. Prolonged maintenance of active cytochrome P-450 in adult rat hepatocyte co-cultured with another liver cell type. Hepatology 4:839–842; 1984.

    Article  PubMed  Google Scholar 

  3. Burke, M. D.; Thompson, S.; Elcombe, C. R., et al. Ethoxy-, pentoxy-, and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem. Pharmacol. 34:3337–3345; 1985.

    Article  PubMed  CAS  Google Scholar 

  4. Castell, J. V.; Gómez-Lechón, M. J. Thein vitro evaluation of the potential risk of hepatotoxicity of drugs. In: Castell, J. V.; Gómez-Lechón, M. J., eds. In vitro alternatives to animal pharmaco-toxicology. Madrid: Farmaindustria; 1992:179–204.

    Google Scholar 

  5. Corlu, A.; Kneip, B.; Lhadi, C., et al. A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes. J. Cell Biol. 115:505–515; 1991.

    Article  PubMed  CAS  Google Scholar 

  6. Donato, M. T.; Gómez-Lechón, M. J.; Castell, J. V. Drug metabolizing enzymes in rat hepatocytes co-cultured with cell lines. In Vitro Cell. Dev. Biol. 26:1057–10623; 1990.

    Article  PubMed  CAS  Google Scholar 

  7. Donato, M. T.; Gómez-Lechón, M. J. Co-cultures of hepatocytes with epithelial-like cell lines: expression of drug-biotransformation activities by hepatocytes. Cell. Bio. Toxicol. 7:1–14; 1991.

    Article  CAS  Google Scholar 

  8. Donato, M. T.; Gómez-Lechón, M. J.; Castell, J. V. A mcroassay for measuring cytochrome P450Ia1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal. Biochem. 213:29–33; 1993.

    Article  PubMed  CAS  Google Scholar 

  9. Donato, M. T.; Gómez-Lechón, M. J.; Castell, J. V. Rat hepatocytes cultured on a monkey kidney cell line: expression of biotransformation and hepatic metabolic activities. Toxicol. In Vitro 5:435–438; 1991.

    Article  CAS  PubMed  Google Scholar 

  10. Forster, U.; Luippold, G.; Schwartz, L. R. Induction of monooxygenase and UDP-glucuronyltransferase activities in primary cultures of rat hepatocytes. Drug. Metab. Dispos. 4:353–360; 1986.

    Google Scholar 

  11. Grant, M. H.; Melvin, M. A. L.; Shaw, P., et al. Studies on the maintenance of cytochromes P450 and b5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes. FEBS Lett. 190:99–103; 1985.

    Article  PubMed  CAS  Google Scholar 

  12. Gómez-Lechón, M. J.; López, P.; Castell, J. V. Biochemical functionality and recovery of hepatocytes after deep-freezing storage. In Vitro 20:826–832; 1984.

    Article  PubMed  Google Scholar 

  13. Holme, J. A.; Hepatocytes. Xenobiotic metabolism and toxicity in primary monolayer cultures of hepatocytes. NIPH Ann. 8:49–63; 1985.

    PubMed  CAS  Google Scholar 

  14. Holme, J. A.; Soderlund, E.; Dybing, E. Drug metabolism activities of isolated rat hepatocytes in monolayer culture. Acta Pharmacol. Toxicol. 52:348–356; 1983.

    Article  CAS  Google Scholar 

  15. Iwasaki, K.; Lum, P. Y.; Ioannides, C., et al. Induction of cytochrome P-448 activity as exemplified by the O-deethylation of ethoxyresorufin. Effects of dose, sex, tissue and animal species. Biochem. Pharmacol. 35:3879–3884; 1986.

    Article  PubMed  CAS  Google Scholar 

  16. Kuri-Harcuch, W.; Mendoza-Figueroa, T. Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions. Differentiation 41:148–157; 1989.

    Article  PubMed  CAS  Google Scholar 

  17. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L., et al. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.

    PubMed  CAS  Google Scholar 

  18. Michalopoulos, G.; Rusell, F.; Biles, C. Primary cultures of hepatocytes on human fibroblasts. In Vitro 15:796–806; 1979.

    Article  PubMed  CAS  Google Scholar 

  19. Morin, O.; Normand, C. Long-term maintenance of hepatocyte functional activity in co-culture: requirements for sinusoidal endothelial cells and dexamethasone. J. Cell Physiol. 129:103–110; 1986.

    Article  PubMed  CAS  Google Scholar 

  20. Neimann, C.; Gauthier, J. C.; Richert, L., et al. Rat adult hepatocytes in primary pure and mixed monolayer culture. Comparison of the maintenance of mixed function oxidase and conjugation pathways of drug metabolism. Biochem. Pharmacol. 42:373–379; 1991.

    Article  Google Scholar 

  21. Rogiers, V.; Vandenberghe, Y.; Callaerts, A., et al. Phase I and phase II xenobiotic biotransformation in cultures and co-cultures of adult rat hepatocytes. Biochem. Pharmacol. 40:1701–1706; 1990.

    Article  PubMed  CAS  Google Scholar 

  22. Ryan, D. E.; Levin, W. Purification and characterization of hepatic microsomal cytochrome P-450. Pharmac. Ther. 45:153–239; 1990.

    Article  CAS  Google Scholar 

  23. Saad, B.; Scholl, A.; Thomas, H., et al. Crude liver membrane as substrate preserve liver-specific functions in long-term, serum-free rat hepatocyte cultures. In Vitro Cell. Dev. Biol. 29A:32–40; 1993.

    Article  PubMed  CAS  Google Scholar 

  24. Schuetz, E. G.; Li, D.; Omiecinski, C. J., et al. Regulation of gene expression in adult rat hepatocytes cultured on basement membrane matrix. J. Cell Physiol. 134:309–323; 1988.

    Article  PubMed  CAS  Google Scholar 

  25. Sidhu, J. S.; Farin, F. M.; Omiecinski, C. J. Influence of extracellular matrix overlay on phenobarbital-mediated induction of CYP2B1, 2B2, and 3A1 genes in primary adult rat hepatocyte culture. Arch. Biochim. Biophys. 301:103–113; 1993.

    Article  CAS  Google Scholar 

  26. Simmons, D. L.; Mcquiddy, P.; Kasper, C. B. Induction of the hepatic mixed-function oxidase system by synthetic glucocorticoids: transcriptional and post-transcriptional regulation. J. Biol. Chem. 262:326–332; 1987.

    PubMed  CAS  Google Scholar 

  27. Sinclair, P. R.; Schuetz, E. G.; Bement, W. J., et al. Role of heme in phenobarbital induction of cytochromes P450 and 5-aminolevulinate synthase in cultured rat hepatocytes maintained on an extracellular matrix. Arch. Biochim. Biophys. 282:386–392; 1990.

    Article  CAS  Google Scholar 

  28. Sonderfan, A. J.; Arlotto, M. P.; Dutton, D. R., et al. Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P450. Arch. Biochem. Biophys. 255:27–41; 1987.

    Article  PubMed  CAS  Google Scholar 

  29. Suolinna, E. M. Isolation and culture of liver cells and their use in the biochemical research of xenobiotics. Med. Biol. 60:237–254; 1982.

    PubMed  CAS  Google Scholar 

  30. Turner, N. A.; Wilson, N. M.; Jefcoate, C. R., et al. The expression and metabolic activity of cytochrome P450 isozymes in control and phenobarbital-induced primary cultures of rat hepatocytes. Arch. Biochem. Biophys. 263:204–215; 1988.

    Article  PubMed  CAS  Google Scholar 

  31. Utesch, D.; Molitor, E.; Platt, K. L., et al. Differential stabilization of cytochrome P450 isoenzymes in primary cultures of adult rat liver parenchymal cells. In Vitro Cell. Dev. Biol. 27A:858–863; 1991.

    Article  PubMed  CAS  Google Scholar 

  32. Utesch, D.; Oesch, F. Dependency of the in vitro stabilization of differentiated functions in liver parenchymal cells on the type of cell line used for co-culture. In Vitro Cell. Dev. Biol. 28A:193–198; 1992.

    Article  PubMed  CAS  Google Scholar 

  33. Waxman, D. J.; Ko, A.; Walsh, C. Regioselecty and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver. J. Biol. Chem. 258:11937–11947; 1983.

    PubMed  CAS  Google Scholar 

  34. Waxman, D. J. Interactions of hepatic cytochromes P-450 with steroid hormones: regioselectivity and steroselectivity of steroid metabolism and hormonal regulation of rat P-450 enzymes expression. Biochem. Pharmacol. 37:71–84; 1988.

    Article  PubMed  CAS  Google Scholar 

  35. Wood, A. W.; Ryan, D. E.; Thomas, P. E., et al. Regio-and stereoselectivity metabolism of two C19 steroids by five highly purified and reconstituted rat hepatic cytochrome P450 isozymes. J. Biol. Chem. 258:8839–8847; 1983.

    PubMed  CAS  Google Scholar 

  36. Wortelboer, H. M.; de Kruif, C. A.; van Iersel, A. A. J., et al. Comparison of cytochrome P450 isozyme profiles in rat liver and hepatocyte cultures. The effects of model inducers on apoproteins and biotransformation activities. Biochem. Pharmacol. 42:381–390; 1991.

    Article  PubMed  CAS  Google Scholar 

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  1. Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, Avda. Campanar 21, E-46009, Valencia, Spain

    M. Teresa Donato, José V. Castell & M. José Gómez-Lechón

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  1. M. Teresa Donato
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  2. José V. Castell
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  3. M. José Gómez-Lechón
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Donato, M.T., Castell, J.V. & Gómez-Lechón, M.J. Cytochrome P450 activities in pure and co-cultured rat hepatocytes. Effects of model inducers. In Vitro Cell Dev Biol - Animal 30, 825–832 (1994). https://doi.org/10.1007/BF02639392

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  • DOI: https://doi.org/10.1007/BF02639392

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