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Purification and Characterization of Cytochrome P-450 Induced by Benz(a)anthracene in Mouse Skin Microsomes

Department of Oral and Maxillofacial Surgery I [T. I., K. T.] and Department of Biochemistry [S. H., M. N., K. O.], Hiroshima University School of Dentistry, Hiroshima 734, Japan

Topical application of benz(a)anthracene to mouse skin elicited a 2-fold increase in cytochrome P-450 content, with accompanying increases in monooxygenase activities such as benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and acetanilide 4-hydroxylation, in the microsomes. A major form of cytochrome P-450 was purified from skin microsomes of mice treated with polycyclic aromatic hydrocarbon. A specific content of 1.95 nmol/mg of protein, which corresponded to 48-fold purification from the microsomes, was observed. The purified protein produced a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a molecular weight of 55,000. Using Western blotting, the band immunochemically cross-reacted with antibody which had been raised against rat liver cytochrome P-450_MC-1. The purified preparation efficiently catalyzed benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation when reconstituted with NADPH-cytochrome P-450 reductase. These activities were inhibited by 7,8-benzoflavone as well as anti-cytochrome P-450_MC-1 antibody, but not by P-450_PS-1 antibody. The results indicate that, in mouse skin microsomes, a cytochrome P-450 induced by benz(a)anthracene is enzymatically and immunochemically similar to rat liver cytochrome P-450_MC-1. It is suggested that this enzyme plays an important role in the activation of carcinogenic polycyclic aromatic hydrocarbons.

¹ To whom requests for reprints should be addressed.

Received 8/ 3/88. Revised 11/11/88. Accepted 11/16/88.