Influence of extracellular matrix overlay and medium formulation on the induction of cytochrome P-450 2B enzymes in primary cultures of rat hepatocytes.
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.
Department of Pharmacology, Toxicology and Therapeutics, Center for Environmental and Occupational Health, University of Kansas Medical Center, Kansas City, Kansas, USA.
E
E
LeCluyse
P
P
Bullock
A
A
Madan
K
K
Carroll
A
A
Parkinson
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D002478:Cells, Cultured; D003470:Culture Media; D019362:Cytochrome P-450 CYP2B1 / Q000096:biosynthesis* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D005109:Extracellular Matrix / Q000201:enzymology* / Q000648:ultrastructure; D018757:GABA Modulators / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D010634:Phenobarbital / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D051381:Rats; D013250:Steroid Hydroxylases / Q000096:biosynthesis*
D003470:Culture Media; D018757:GABA Modulators; D011506:Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; C025619:steroid 16-beta-hydroxylase; D010634:Phenobarbital