Expression of five forms of microsomal cytochrome P-450 in primary cultures of rabbit hepatocytes treated with various classes of inducers.
In order to investigate the expression of five different forms of microsomal cytochrome P-450 including P-450 2 (P450IIBI), 3b (P450IIC3), 3c (P450IIIA4), 4 and 6 (P450IA2 and A1), hepatocytes were isolated from untreated rabbit and maintained in primary monolayer cultures in serum free modified Waymouth medium in the absence and in the presence of various classes of inducers including phenobarbital (PB), rifampicin (RIF), dexamethasone (DEX) and B-naphthoflavone (BNF). In untreated cultures the level of the various forms of P-450, determined by immunoblot with the use of specific antibodies, generally declined with time but at markedly different rates. In cultures treated with the inducers decline of the various forms was either unaffected, reduced, or even reversed, so that 96 hr after plating some of these forms appeared to be induced several-fold with respect to the untreated cultures. The forms 2 and 3c were co-induced by PB, RIF or DEX; as in vivo, BNF induced forms 4 and 6. Induction of forms 2, 3c, 4 and 6 was accompanied by stimulation of related monooxygenase activities, benzphetamine demethylase, progesterone 6B hydroxylase and benzpyrene hydroxylase and ethoxyresorufin deethylase, respectively. In all cases, induction was accompanied by an increased rate of de novo synthesis of the protein, determined by radio-immunoprecipitation assay with the use of specific antibodies on [3H]-Leu labeled cell lysate. Both induction and increased de novo synthesis were time- and inducer concentration-dependent. In cultures treated with RIF or BNF de novo synthesis of P-450 3c or of P-450 4 and 6 was correlated with the level of their specific mRNA quantitated from northern blots probed with either pLM3c-4.1 or pLM6.1, two plasmids containing inserted cDNA coding for P-450 3c or P-450 6, respectively. We conclude from these experiments that rabbit hepatocytes in primary monolayer cultures represent suitable models for studying regulation induction and pharmacological implications of the microsomal cytochromes P-450.
Daujat
M
M
INSERM U 128, Site du CNRS, Montpellier, France.
Pichard
L
L
Dalet
C
C
Larroque
C
C
Bonfils
C
C
Pompon
D
D
Li
D
D
Guzelian
P S
PS
Maurel
P
P
D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010634:Phenobarbital / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012293:Rifampin / Q000494:pharmacology; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D012333:RNA, Messenger; D019324:beta-Naphthoflavone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D012293:Rifampin; D010634:Phenobarbital