Spectrofluorometric Analysis of CYP2A6-Catalyzed Coumarin 7-Hydroxylation

Abstract

CYP2A6 has been isolated and purified to apparent homogeneity from human liver (1, 2). The level of CYP2A6 protein expressed in liver is low (∼4% of total hepatic P450 content) (3), although substantial intermdividual variation exists (3–5). This P450 is primarily a hepatic protein, as suggested by its absence from several extrahepatic tissues, including adult human lung, colon, breast, kidney, and placenta microsomes (2). According to experiments with primary cultures of human hepatocytes, CYP2A6 is mducible by phenobarbital, dexamethasone, and rifampm (rifampicin) (6). Experiments with individual cDNA-expressed human P450 forms have indicated that CYP2A6 is a major catalyst of coumarm 7-hydroxylation (7). Among the other recombinant human P450s examined, CYPlAl, lA2, 2C8, 2C9, 2D6, 2E1, 3A3, 3A4, and 3A5 are catalytically inactive with respect to coumarm 7-hydroxylation, whereas CYP2B6 is active at only ∼5% the rate of CYP2A6 (7). Immunoinhibition studies have validated the use of coumarm 7-hydroxylase activity as a diagnostic catalytic marker for human hepatic CYP2A6. Antihuman CYP2A6 IgG inhibits coumarm 7-hydroxylase activity by >90% in human liver microsomes (2), indicating that CYP2A6 is the principal and perhaps the sole catalyst of human liver microsomal coumarin 7-hydroxylase activity. This activity can be inhibited by a variety of compounds, includmg 8-methoxypsoralen (8), tranylcypromme (8), α-naphthoflavone (8–11) and diethyldithiocarbamate (8, 10, 11). This chapter describes a modification (12) of a spectrofluorometric method (13) for the determination of coumarm 7-hydroxylase activity.