Induction of cytochrome P-450 by pregnenolone-16 alpha-carbonitrile in primary monolayer cultures of adult rat hepatocytes and in a cell-free translation system.

Abstract

To investigate whether primary cultures of adult rat hepatocytes retain the capacity to respond to inducers of cytochrome P-450 other than polycyclic aromatic compounds, we tested the effects of pregnenolone-16acarbonitrile (PCN) which induces a novel form of the hemoprotein (PCN cytochrome P-450) i n rat liver (Elshourbagy, N. A., and Guzelian, P. S. (1980) J. EioL Chem, 255,1279-1286).Cultured hepatocytes incubated for 72 h in serum-free medium containing IO-' M PCN exhibited increases in the concentration of cytochrome P -450 (1.5-fold) and in the activities of ethylmorphine (4-fold) and aminopyrine (2-fold) N-demethylation as compared to values in incubated control cultures.The cytochrome P-450 induced in culture by PCN matched the PCN cytochrome P-450 purified from PCN-treated rats in the sensitivity of its catalytic activity to formspecific anti-PCN cytochrome P-450 I& in being im-m~oprecipitable with this antibody and in its mobility on polyacrylamide gels (51,000 daltons).Pulse-labeling cells with C3H]leucine and isolation of PCN cytochrome P-450 from solublized cell lysates by immunoprecipitation revealed a striking increase in the relative rate of synthesis of the cytochrome protein 48 (9-fold) and 72 h (30-fold) after PCN was added to the medium of 24-h-old cultures.Moreover, RNA extracted from such PCN-treated cultures and translated in a cell-free system contained at least 6 to 8 times more mRNA activity directing synthesis of immunoprecipitable PCN cytochrome P-450 than did RNA extracted from control cultures.As judged from pulse-chase experiments, PCN cytochrome P-450 protein is degraded rapidly, at a rate 5 times faster than that of total cellular protein.In summary, our results demonstrate unequivoca~y that mature hepatocytes in maintenance culture retain the capacity to carry out inducible, de m u 0 synthesis of a form of cytochrome P-450 found in adult liver.Cytochrome P-450 is a collective term for a group of microsomal hemoproteins, located prominently in the endoplasmic reticulum of the liver, that catalyzes the oxidation of many lipophilic drugs, carcinogens, and environmental chemicals.An important feature of cytochrome P-450 is that it is induc-