Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes.
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.
Jauregui
H O
HO
Department of Pathology and Oncology, Rhode Island Hospital, Providence.
Ng
S F
SF
Gann
K L
KL
Waxman
D J
DJ
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D003975:Diazepam / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000096:biosynthesis*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D015262:Xenobiotics / Q000494:pharmacology*
D003374:Coumarins; D015262:Xenobiotics; C017299:7-ethoxycoumarin; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase; D003975:Diazepam; D010634:Phenobarbital