Abstract
The major forms of cytochrome P-450 protein induced by phenobarbital (P-450[b]) and 3-methylcholanthrene (P-450[c]) have been isolated, purified to electrophoretic homogeneity and fully characterised with respect to substrate specificity. The purified cytochrome P-450[c] was used as antigen in the preparation of sheep polyclonal antibodies, which were used for the immunological detection, characterisation and quantification of the haemoprotein. The N1-substituted antifungal agents, ketoconazole, miconazole and clotrimazole, have been shown to be potent in vitro inhibitors of both the phenobarbital-induced cytochrome P-450- and the 3-methylcholanthrene-induced cytochrome P-448-dependent rat hepatic mixed-function oxidases. All three drugs were more potent inhibitors of the phenobarbital-induced activities in microsomal systems, as well as in reconstituted systems comprising purified NADPH-cytochrome P-450 reductase and cytochrome P-450[b] or P-450[c]. Ketoconazole was the weakest inhibitor and the least selective for the former haemoprotein. All three antimycotic agents elicited type II difference spectra with microsomes from both phenobarbital- and 3-methylcholanthrene-induced rats, as well as with both purified haemoprotein preparations. Miconazole and clotrimazole, and to a lesser extent ketoconazole, have been shown to decrease the magnitude of the type I spectral perturbation of hexobarbital with phenobarbital-induced microsomes. These observations indicate that, although the primary mechanism of inhibition involves reversible binding to haem, an additional interaction with the apoprotein or substrate (type I) binding site is involved and may contribute to the isoenzyme selectivity displayed by all three compounds. When administered systemically to rats, all three antifungal agents stimulated the hepatic microsomal mixed-function oxidases, particularly those activities associated with the phenobarbital and/or pregnenolone-16a-carbonitrile-induced cytochromes P-450. This was confirmed by Western blotting employing anti-cytochrome P-450[p] (PB[2C]) and anti-cytochrome P-450[b]. Administration of the planar benzimidazole 2-amino-3-methylimidazo-(4,5-f)-quinoline (IQ), a food mutagen and carcinogen, in contrast to the globular antifungal agents, selectively induced the cytochromes P-448, especially the high spin form (cytochrome P-450[d]).