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The effects of metyrapone and nicotinamide, two substituted pyridines, were studied in cultured chick embryo hepatocytes, a system characterized by preserved inducibility of cytochrome P-450 hemoproteins. Both metyrapone and nicotinamide caused a dose-dependent increase in cytochrome P-450 concentration. Their inducing potencies differed by one to two orders of magnitude and correlated with the known difference in the binding affinity of these two pyridines to cytochrome P-450. The increase of cytochrome P-450 concentration after metyrapone and nicotinamide was additive to the induction of cytochrome P-450 by phenobarbital and β-naphthoflavone and was abolished by cycloheximide. Treatment of hepatocyte cultures with metyrapone resulted in an increase in a microsomal protein with an apparent mol. wt of 52,000. In addition, induction of cytochrome P-450 by the substituted pyridines was associated with enhanced 5-aminolevulinate synthase, the rate-limiting enzyme of heme biosynthesis. These data suggest that in cultured chick embryo hepatocytes the substituted pyridines metyrapone and nicotinamide induce cytochrome P-450.

Twelve homologous and regioisomeric pyridylalkanamides were examined spectrally for their binding affinity to cytochrome P-450 in phenobarbital- and 3-methylcholanthrene-induced rat liver microsomes. The pKs values were calcuated by the Lineweaver-Burk method andby non-linear analysis using both a one ligand-one acceptor and a one ligand-two acceptor model. The latter model best fits most of the data, conforming that two pKs values exist for most derivatives in the 3-pyridyl and 4-pyridyl series. Structure-binding relationships are discussed. The two binding constants are hypothesized to arise from a dual mode of binding to the ferric ion. At low ligand concentrations, binding to hexacoordinated cytochrome P-450 occurs and involves displacement of an endogenous 6th ligand; at higher concentrations, the ligands bind to the pentacoordinated P-450, resulting in a high-to-low spin shift.