Identification of Human Cytochrome P450s Involved in the Formation of All-trans-Retinoic Acid Principal Metabolites

Abstract

Cytochrome P450 (P450)-dependent metabolism of all-trans-retinoic acid (atRA) is important for the expression of its biological activity. Because the human P450s involved in the formation of the principal atRA metabolites have been only partially identified, the purpose of this study was to identify the human P450s involved in atRA metabolism. The use of phenotyped human liver microsomes (n = 16) allowed the identification of the following P450s: 2B6, 2C8, 3A4/5, and 2A6 were involved in the formation of 4-OH-RA and 4-oxo-RA; 2B6, 2C8, and 2A6 correlated with the formation of 18-OH-RA; and 2A6, 2B6, and 3A4/5 activities correlated with 5,6-epoxy-RA formation (30-min incubation, 10 μM atRA, HPLC separation, UV detection 340 nm). The use of 15 cDNA-expressed human P450s from lymphoblast microsomes, showed the formation of 4-OH-RA by CYP3A7 > CYP3A5 > CYP2C18 > CYP2C8 > CYP3A4 > CYP2C9, whereas the 18-OH-RA formation involved CYPs 4A11 > 3A7 > 1A1 > 2C9 > 2C8 > 3A5 > 3A4 >2C18. Kinetic studies identified 3A7 as the most active P450 in the formation of three of the metabolites: for 4-OH-retinoic acid, 3A7 showed aV _max/K _m of 127.7, followed by 3A5 (V _max/K _m = 25.6), 2C8 (V _max/K _m = 24.5), 2C18 (V _max/K _m = 15.8), 3A4 (V _max/K _m = 5.7), 1A1 (V _max/K _m = 5.0), and 4A11 (V _max/K _m = 1.9); for 4-oxo-RA, 3A7 showed aV _max/K _m of 13.4, followed by a 10-fold lower activity for both 2C18 and 4A11 (V _max/K _m = 1.2); and for 18-OH-RA, 3A7 showed aV _max/K _m of 10.5 compared with aV _max/K _m of 2.1 for 4A11 and 2.0 for 2C8. 5,6-Epoxy-RA was only detected at high substrate concentrations in this system (>10 μM), and P450s 2C8, 2C9, and 1A1 were the most active in its formation. The use of embryonic kidney cells (293) stably transfected with human P450 cDNA confirmed the major involvement of P450s 3A7, 1A1, and 2C8 in the oxidation of atRA, and to a lesser extent, 1A2, 2C9, and 3A4. In conclusion, several human P450s involved in atRA metabolism have been identified, the expression of which was shown to direct atRA metabolism toward the formation of specific metabolites. The role of these human P450s in the biological and anticancer effects of atRA remains to be elucidated.