title: Long-term maintenance of cytochrome P450 activities by rat hepatocyte/3T3 cell co-cultures in heparinized human plasma.
abstract: Little information on the effect of plasma on hepatocyte cytochrome P450 (CYP) activities is currently available. We characterized the effect of plasma on CYPs of hepatocyte-mesenchymal cell co-cultures, which exhibit stable liver specific functions and may be potentially useful for bioartificial liver design. Rat hepatocyte-mouse 3T3-J2 cell co-cultures were maintained for 6 days in medium, and then switched to heparinized human plasma containing 3-methylcholanthrene (3MC; 2 microM), phenobarbital (PB; 1 mM), or no inducer for up to 7 days. CYP activities were measured in situ based on the o-dealkylation of ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD), or benzyloxy- (BROD) resorufin. Plasma alone increased PROD/BROD but not EROD/MROD. The endogenous inducer was in the high molecular weight fraction (>5 kD) of plasma and inhibited by >5 nM okadaic acid and >10 microM dibutyryl cyclic AMP, two inhibitors of PB-inducible CYPs. Furthermore, plasma increased CYP1A1 and CYP2B1/2 mRNA levels. In plasma, 3MC induced EROD/MROD to about 60% of the level induced in culture medium while PB induced PROD/BROD that were three- to 10-fold above levels induced in medium. CYP activities decreased between days 2 and 7 of plasma exposure, but were enhanced by plasma supplementation with amino acids, insulin, glucagon, and hydrocortisone.
authors.affiliation: Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: J
authors.initials: J
authors.lastname: Washizu
authors.forename: F
authors.initials: F
authors.lastname: Berthiaume
authors.forename: Y
authors.initials: Y
authors.lastname: Mokuno
authors.forename: R G
authors.initials: RG
authors.lastname: Tompkins
authors.forename: M
authors.initials: M
authors.lastname: Toner
authors.forename: M L
authors.initials: ML
authors.lastname: Yarmush
mesh_terms: D016475:3T3 Cells; D000818:Animals; D018920:Coculture Techniques / Q000379:methods; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology*; D006493:Heparin; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D008748:Methylcholanthrene; D051379:Mice; D010634:Phenobarbital; D051381:Rats
chemical_list: D003470:Culture Media; D008748:Methylcholanthrene; D006493:Heparin; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital