Elsevier

Biochemical Pharmacology

Volume 54, Issue 7, 1 October 1997, Pages 761-772
Biochemical Pharmacology

Research paper
Drug metabolism in hepatocyte sandwich cultures of rats and humans

https://doi.org/10.1016/S0006-2952(97)00204-9Get rights and content

Abstract

Adult hepatocytes from rat and man were maintained for 2 weeks between two gel layers in a sandwich configuration to study the influence of this culture technique on the preservation of basal activities of xenobiotic-metabolizing phase I and phase II enzymes. The response of these enzyme activities to an enzyme inducer was investigated using rifampicin (RIF). Basal levels of cytochrome P-450 (CYP) isozymes were characterized by measuring ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and the specific oxidation of testosterone (T). In hepatocytes from untreated rats, CYP isozyme levels, including the major form CYP 2C11, increased during the first 3 days in culture. After this period of recovery, the levels of CYP 2C11, CYP 2A1, and CYP 2B1 decreased, whereas CYP 3A1 increased. In contrast to these dynamic changes, CYP activities such as CYP 1A2 and the major isozyme CYP 3A4 were largely preserved until day 9 in cultures of human hepatocytes. In measuring phase II activities, a distinct increase in glucuronosyltransferase (UDP-GT) activity toward p-nitrophenol (PNP) was found for rat and human hepatocytes over 2 weeks in culture. Sulfotransferase (ST) activity toward PNP showed an initial increase, with a maximum at day 7 and day 9 in culture, respectively, and then decreased until day 14. Glutathione S-transferase (GST) activity decreased constantly during the time of culture. Effects of the enzyme-inducing drug rifampicin on phase I and phase II enzymes were investigated using cultured human hepatocytes. Rifampicin treatment (50 μmol/L) for 7 days resulted in a 3.7-fold induction of CYP 3A4 at day 9 in culture. ECOD activity was increased sixfold and phase II ST activity increased twofold compared to the initial value at day 3. No effect of rifampicin on CYP 3A was found in cultures of rat hepatocytes. These results demonstrate that rat and human hepatocytes preserve the major forms of CYP isozymes and phase II activities and respond to inducing drugs such as rifampicin. The novel hepatocyte sandwich culture is suitable for investigating drug metabolism, drug-drug interactions and enzyme induction.

References (53)

  • MT Donato et al.

    Prolonged expression of biotransformation activities of rat hepatocytes co-cultured with established cell lines

    Toxic In Vitro

    (1990)
  • MT Donato et al.

    Effect of xenobiotics on monooxygenase activities in cultured human hepatocytes

    Biochem Pharmacol

    (1990)
  • EM Suolinna et al.

    Effect of culture age on drug metabolizing enzymes and their induction in primary cultures of rat hepatocytes

    Biochem Pharmacol

    (1986)
  • P Maier et al.

    Effect of periportal- and centrilobular-equivalent oxygen tension on liver specific functions in long-term rat hepatocyte cultures

    Toxic In Vitro

    (1994)
  • DJ Waxman

    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

    (1988)
  • MH Grant et al.

    Human adult hepatocytes in primary mono layer culture: Maintenance of mixed function oxidase and conjugation pathways of drug metabolism

    Biochem Pharmacol

    (1987)
  • M Daujat et al.

    Induction protocols for cytochromes P450IIIA in vivo and in primary cultures of animal and human hepatocytes

    Methods Enzymol

    (1991)
  • CRW Padgham et al.

    The loss of cytochromes P450 (CYPs) in rat liver cell culture is triggered during hepatocyte isolation and again during the first 4 hours of culture

  • JD Schuetz et al.

    Extracellular matrix regulation of multidrug resistance in primary monolayer cultures of adult rat hepatocytes

    Cell Growth Diff

    (1993)
  • A Bader et al.

    Reconstruction of liver tissue in vitro—Geometry of characteristic flat bed-, hollow fiber-, and spouted bed bioreactors with reference to the in vivo liver

    Artificial Organs

    (1995)
  • JC Dunn et al.

    Hepatocyte function and extracellular matrix geometry long-term culture in a sandwich configuration

    FASEB J

    (1989)
  • A Bader et al.

    Use of organotypical cultures of primary hepatocytes to analyze drug biotransformation in man and animals

    Xenobiotica

    (1994)
  • DA Smith

    Species differences in metabolism and pharmaco-kinetics: Are we close to an understanding?

    Drug Metab Rev

    (1991)
  • T Elsdale et al.

    Collagen substrate for studies on cell behaviour

    J Cell Biol

    (1972)
  • MN Berry et al.

    High-yield preparation of isolated rat liver parenchymal cells

    J Cell Biol

    (1969)
  • A Bader et al.

    In vitro imitation of the in vivo three-dimensional microenvironment enables primary hepatocytes to maintain stable metabolic functions

    Cell Transplantat

    (1992)
  • Cited by (0)

    This research was supported in part by the BGVV ZEBET (1328-131) and by the BMBF (BEO 21/11263).

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