Abstract
The effect of medium formulation, composition of extracellular matrix overlay, and culture dish material on liver microsomal cytochrome P-450 (CYP) 2B induction by phenobarbital (PB) was investigated in primary cultures of rat hepatocytes. When hepatocytes were maintained on Permanox dishes with an overlay of either collagen (type I) or Matrigel, Williams’ E medium was superior to other medium formulations in terms of the magnitude of induction of CYP2B on a per milligram microsomal protein basis. Modified Chee’s medium (MCM) and hepatocyte culture medium were intermediate in their capacity to sustain induction of CYP2B by PB, and Dulbecco’s modified Eagle’s medium was slightly less effective. The overall induction of CYP2B activity by PB was, on average, 50% lower in hepatocytes cultured on polystyrene dishes (LUX). Little or no difference was observed between hepatocytes overlaid with collagen and those overlaid with Matrigel. MCM was superior to Williams’ E medium in terms of the yield of microsomal protein and the ultrastructural features of the hepatocyte monolayers. CYP2B induction by PB was optimal after 3 days of treatment in either medium. CYP1A, CYP3A, and CYP4A activities could be induced in vitro by prototypical inducing agents in hepatocytes cultured on Permanox dishes with MCM and a Matrigel overlay to comparable levels observed in vivo. The results of these studies show that medium formulation and culture vessel material, but not the type of extracellular matrix overlay, have significant effects on the induction of CYP enzymes in cultured rat hepatocytes maintained in a sandwich configuration.
Footnotes
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Send reprint requests to: Dr. Ajay Madan, XenoTech, LLC, 3800 Cambridge, Kansas City, KS 66103.
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↵1 Present address: School of Pharmacy, Division of Drug Delivery and Disposition, CB# 7360, Beard Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
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↵2 Present address: Cerep, Inc., 15318 NE 95th St., Redmond, WA 98052.
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This work was funded in part by National Institutes of Health Grants ES-03765 and GM-37044, Du Pont Pharmaceuticals Company, Pfizer, Inc., and the Procter and Gamble International Program for Animal Alternatives.
- Abbreviations used are::
- CYP
- cytochrome P-450 enzymes
- DMEM
- Dulbecco’s modified Eagle’s medium
- HCM
- hepatocyte culture medium
- ITS+
- insulin, transferrin, selenium, linoleic acid, and BSA supplement
- MCM
- modified Chee’s medium
- PROD
- 7-pentoxyresorufin O-dealkylase
- WEM
- Williams’ E medium
- PB
- phenobarbital
- Received January 22, 1999.
- Accepted April 27, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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