Regulation of cytochrome P-450p by phenobarbital and phenobarbital-like inducers in adult rat hepatocytes in primary monolayer culture and in vivo.
Treatment of rats with phenobarbital increases the hepatic concentration of P-450p, a form of cytochrome P-450 believed to be controlled primarily by a mechanism that stereospecifically recognizes glucocorticoids like dexamethasone and anti-glucocorticoids like pregnenolone-16 alpha-carbonitrile [Schuetz, E.G., & Guzelian, P.S. (1984) J. Biol. Chem. 259, 2007]. To test the possibility that phenobarbital induces P-450p indirectly by increasing the availability of endogenous glucocorticoids in the liver, we added phenobarbital and phenobarbital-like inducers to primary monolayer cultures of adult rat hepatocytes incubated in serum-free medium without glucocorticoids and found stimulated de novo synthesis of P-450p measured as increased incorporation of [3H]leucine into immunoprecipitable P-450p protein. With some of the inducers, notably the organochlorine pesticides chlordane and trans-nonachlor, there was a greater accumulation of P-450p measured on quantitative immunoblots than could be accounted for by the increase in P-450p synthesis. "Pulse-chase" experiments confirmed that these compounds significantly lengthen the half-life of P-450p up to 60 h as compared to the values in control (11 h) or dexamethasone-treated (10 h) cultures. Treatment of rats with chlordane, trans-nonachlor, or other cyclodiene organochlorine pesticides confirmed that these agents increase the concentration of P-450p in liver microsomes analyzed on immunoblots of two-dimensional electrophoretic gels. The time courses of induction in trans-nonachlor-treated rats of P-450p protein and of P-450PB proteins induced by phenobarbital were similar as were the amounts of P-450PB mRNA and P-450p mRNA measured by hybridization to cloned cDNA probes. However, analysis of structure-activity relationships among polychlorinated biphenyls revealed that isomers with two ortho chlorinated positions maximally induced P-450PB whereas isomers with three and four ortho chlorines maximally induced P-450p in rats and in hepatocyte culture, respectively. We conclude that P-450p is induced by the phenobarbital class of inducers through direct contact with the hepatocytes involving decreased degradation of the protein and stimulation of its synthesis in a manner similar but not identical with that of P-450PB.
Schuetz
E G
EG
Wrighton
S A
SA
Safe
S H
SH
Guzelian
P S
PS
D000818:Animals; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology*; D011078:Polychlorinated Biphenyls / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D011078:Polychlorinated Biphenyls; D010634:Phenobarbital