Comparison of cytochrome P450 isoenzyme profiles in rat liver and hepatocyte cultures. The effects of model inducers on apoproteins and biotransformation activities.
The metabolic profile of seven subfamilies of cytochrome P450 (P450IA, IIA, IIB, IIC, IIE, IIIA, IVA) was studied in rat liver (in vivo) and in primary hepatocyte cultures (in vitro) after treatment with various inducers. The dealkylation of 7-ethoxyresorufin (EROD) and 7-pentoxyresorufin (PROD), aniline 4-hydroxylation and the regio- and stereoselective hydroxylation of testosterone were measured to characterize the isoenzyme pattern in intact hepatocytes and in liver microsomes. Occurrence of isoenzyme apoproteins was determined using Western blotting. Primary cultures of rat hepatocytes retain the capacity to respond to inducers of isoenzymes belonging to six different subfamilies (P450IA, IIA, IIB, IIC, IIIA and IVA). Treatment of cells with beta-naphthoflavone revealed a P450-activity profile similar to in vivo, namely a highly induced EROD (P450IA1), a small enhancement of testosterone 7 alpha-hydroxylation (P450IIA) and a marked reduction in 2 alpha- and 16 alpha-hydroxylation (P450IIC11). Exposure of cultured cells to phenobarbital resulted in a higher testosterone 16 beta-hydroxylation (reflecting P450IIB), though to a lesser extent than in vivo. The induction of P450IIIA due to both phenobarbital and dexamethasone, as mirrored by 6 beta- and 15 beta-hydroxylation of testosterone, was the same in cultured hepatocytes and in vivo. Treatment of cells with clofibric acid resulted in an induction profile similar to the one observed in liver microsomes from clofibrate-treated rats: the apoprotein P450IVA as well as the apoprotein P450IIB1/2 and its associated activities (PROD and testosterone 16 beta-hydroxylation) were induced. Isoniazid, a known in vivo inducer of P450IIE1 and aniline 4-hydroxylation, did not change any of the determined P450-dependent activities in vitro.
Wortelboer
H M
HM
UTOX, Research Institute of Toxicology (RITOX), University of Utrecht, The Netherlands.
de Kruif
C A
CA
van Iersel
A A
AA
Falke
H E
HE
Noordhoek
J
J
Blaauboer
B J
BJ
D000818:Animals; D001059:Apoproteins / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases; D001571:Benzoflavones / Q000494:pharmacology; D001711:Biotransformation; D015153:Blotting, Western; D002478:Cells, Cultured; D002994:Clofibrate / Q000494:pharmacology; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction; D007527:Isoenzymes / Q000096:biosynthesis*; D007538:Isoniazid / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D010088:Oxidoreductases / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism; D019324:beta-Naphthoflavone
D001059:Apoproteins; D001571:Benzoflavones; D007527:Isoenzymes; D019324:beta-Naphthoflavone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D019363:Cytochrome P-450 CYP1A1; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; C022724:testosterone 7-alpha-hydroxylase, hamster; D002994:Clofibrate; D007538:Isoniazid; D010634:Phenobarbital