Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes

Xenobiotica. 1991 Sep;21(9):1091-106. doi: 10.3109/00498259109039549.


1. The long-term maintenance of metabolism of representative drugs and steroid hormone substrates by cytochromes P-450, and their inducibility, was investigated in primary cultures of adult rat hepatocytes. Collagenase-isolated cells were seeded on collagen-coated tissue culture dishes and cultured in Chee's essential media in the presence or absence of phenobarbital (PB, 0.75 mM, 96 h or continuously) and 3-methylcholanthrene (MC, 5 microM, 48 h) for up to 45 days. 2. Hepatic P-450-dependent metabolism of diazepam to its primary oxidized metabolite was inducible by PB both in vivo (monitored in isolated liver microsomes) and in cultured cells (up to 100% and 400% increases in the formation of temazepam and nordiazepam, respectively, after 25 days in culture). Hepatocyte microsomal androstenedione 16 beta-hydroxylase activity was also induced by PB treatment of the hepatocytes (350-650% increase in 20-day-old cells). 3. Western blot analysis revealed that immunoreactive P-450 form PB-4 (IIB1), which catalysed the N-demethylation of diazepam to yield nordiazepam as well as androstenedione 16 beta-hydroxylation when assayed in a purified enzyme system, was substantially elevated following PB treatment of the cultured cells. Similarly, MC induced 7-ethoxycoumarin O-deethylase activity (up to 2000% increase from 5 to 45 days) as well as immunoreactive P-450c (IA1) in the hepatocyte cultures. 4. These studies demonstrate that cytochrome P-450 activities can be maintained, and also induced, after extended periods of time in hepatocytes cultured using a simple collagen mixture as substrate and a commercially available tissue culture media. This culture system should provide an important tool for further studies of P-450-dependent xenobiotic metabolism in a well-defined, liver-derived cellular system.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 7-Alkoxycoumarin O-Dealkylase / metabolism
  • Animals
  • Blotting, Western
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Coumarins / metabolism
  • Cytochrome P-450 Enzyme System / biosynthesis*
  • Diazepam / metabolism
  • Enzyme Induction / drug effects
  • Liver / drug effects
  • Liver / enzymology*
  • Male
  • Methylcholanthrene / pharmacology
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • Microsomes, Liver / metabolism
  • Mixed Function Oxygenases / biosynthesis*
  • Phenobarbital / pharmacology
  • Rats
  • Rats, Inbred Strains
  • Xenobiotics / pharmacology*


  • Coumarins
  • Xenobiotics
  • 7-ethoxycoumarin
  • Methylcholanthrene
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • 7-Alkoxycoumarin O-Dealkylase
  • Diazepam
  • Phenobarbital