Abstract
The potential inducing ability of azole antifungal drugs on cytochrome P450 isozymes, in particular the P4504A subfamily, have been investigated in vivo and in vitro. In rat liver, bifonazole (1-(P,α-diphenylbenzyl) imidazole), but not the structurally related clotrimazole, was found to induce the cytochrome P4504A subfamily (similar to clofibrate), based on their relative abilities to induce lauric acid ω-hydroxylase activity, immunochemical reactivity with an antibody raised against P4504A1, and Northern blotting with a cytochrome P4504A1 cDNA probe. Additionally, bifonazole was subsequently shown to be a peroxisome proliferator, inducing peroxisomal palmitoyl-CoA oxidation, trifunctional protein, and associated hepatomegaly. Furthermore, immunoblotting data demonstrated that bifonazole also induces P4501A1 and 2B1/2 proteins, and the P4503A subfamily. By contrast, clotrimazole did not induce the cytochrome P4504A subfamily, but did induce P4502B1/2 proteins and the P4503A subfamily, and to a lesser extent P4501A1. In rat kidney, based on the ability to induce lauric acid w-hydroxylase activity, cytochrome P4504A1 apoprotein, and cytochrome P4504A1 mRNA, bifonazole, but not clotrimazole, was found to be a weak inducer of the cytochrome P4504A subfamily. In addition, bifonazole at the high dose level was able to cause only slight increases in kidney cytochromes P4501A1 and P4502B1/2 proteins, and the P4503A subfamily, as judged by EROD, PROD, and immunoblotting assays. Bifonazole was also able to induce the trifunctional protein (similar to clofibrate), but did not induce peroxisomal palmitoyl-CoA activity. In vitro, using rat hepatocyte primary cultures, clofibrate and all of the other azole antimycotic agents studied (except itraconazole), namely bifonazole, clotrimazole, geniconazole, miconazole, and UK-47265, maintained levels of total cytochrome P450 over the 70 hours culture period as compared to the freshly isolated cells. However, only bifonazole and clofibrate were able to induce lauric acid 11- and 12-hydroxylase activities, and cytochrome P4504A1 apoprotein. The apparent EC50 values for bifonazole and clofibrate for the induction of the 11- and 12-hydroxylation of lauric acid, demonstrated that bifonazole is 160 and 44 times more potent than clofibrate, respectively. In order to explain the inductive effect observed by bifonazole, molecular modelling of the interaction of the test compounds with the putative binding site of the recently isolated receptor, PPAR, were investigated. The results showed a more facile docking of bifonazole and clofibric acid with the PPAR than the other imidazole agents, as reflected in more favourable AG values. Taken collectively, results of the present study further substantiate the close relationship between induction of P4504A1 and peroxisome proliferation, in vivo and in vitro. In addition, the result of molecular modelling studies is not inconsistent with the involvement of the PPAR in P4504A1 induction and peroxisome proliferation.