Differential roles of cytochromes P450 2D1, 2C11, and 1A1/2 in the hydroxylation of bufuralol by rat liver microsomes

Abstract

Bufuralol hydroxylation activities of liver microsomal cytochrome P450 (P450) enzymes were studied in the rat; the reaction has been used widely in determining levels of liver microsomal P450 2D6, which shows debrisoquine-type genetic polymorphism in humans. Liver microsomes catalyzed the conversion of bufuralol to 1'-hydroxybufuralol and a structurally unidentified metabolite (termed here as M-1) in the presence of an NADPH-generating system and molecular oxygen. Bufuralol 1'-hydroxylation activities catalyzed by the liver microsomes were not increased in rats treated with several P450 inducers, whereas beta-naphthoflavone treatment (and to a lesser extent that of isosafrole) caused a significant induction of M-1 formation. The major role of P450 1A1/2 in M-1 formation was confirmed by catalytic inhibition with anti-P450 antibodies and alpha-naphthoflavone in liver microsomes of beta-naphthoflavone-treated rats, and by reconstitution experiments containing P450 1A1 and 1A2. Among nine forms of purified rat P450 enzymes studied in the reconstituted system, P450 2C11 displayed the highest activities for bufuralol 1'-hydroxylation, followed by P450 1A1 and P450 2D1. A female-specific form of P450 2C12 did not catalyze bufuralol 1'-hydroxylation. In liver microsomes of male rats, however, P450 2D1 was the dominant enzyme because only anti-P450 2D1 antibodies, and not anti-P450 2C11 and anti-P450 1A1, inhibited the bufuralol hydroxylation activities, and a specific P450 2D1 inhibitor, quinine, caused a dramatic decrease in the hydroxylation activities. The major contribution of P450 2D1 in the bufuralol 1'-hydroxylation activities was also supported by a kinetic analysis of the reconstituted system; P450 2D1 enzyme had a very low Km value (8.4 microM) as compared with those of P450 2C11 (Km = 83 microM) and P450 1A1 (Km = 230 microM). Thus, the present results suggested that different P450 enzymes are involved in the hydroxylation of bufuralol in rat liver microsomes, and the kinetic analysis, as well as immunoinhibition and chemical inhibition experiments, may be of great importance for determining the major roles of P450 enzymes in drug hydroxylation reactions.