J. Marill et al., Identification of human cytochrome P450s involved in the formation of all-trans-retinoic acid principal metabolites, MOLEC PHARM, 58(6), 2000, pp. 1341-1348
Cytochrome P450 (P450)-dependent metabolism of all-trans-retinoic acid (atR
A) is important for the expression of its biological activity. Because the
human P450s involved in the formation of the principal atRA metabolites hav
e been only partially identified, the purpose of this study was to identify
the human P450s involved in atRA metabolism. The use of phenotyped human l
iver 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-o
xo-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 muM atRA, HPLC separation, UV detection 340 nm). The use of
15 cDNA-expressed human P450s from lymphoblast microsomes, showed the forma
tion 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>2C
18. Kinetic studies identified 3A7 as the most active P450 in the formation
of three of the metabolites: for 4-OH-retinoic acid, 3A7 showed a V-max/K-
m of 127.7, followed by 3A5 (V-max/ K-m = 25.6), 2C8 (V-max/K-m = 24.5), 2C
18 (V-max/K-m = 15.8), 3A4 (V-max/K-m = 5.7), 1A1 (V-max/K-m = 5.0), and 4A
11 (V-max/K-m = 1.9); for 4-oxo-RA, 3A7 showed a V-max/K-m of 13.4, followe
d by a 10-fold lower activity for both 2C18 and 4A11 (V-max/K-m = 1.2); and
for 18-OH-RA, 3A7 showed a V-max/K-m of 10.5 compared with a V-max/K-m of
2.1 for 4A11 and 2.0 for 2C8. 5,6-Epoxy- RA was only detected at high subst
rate concentrations in this system (>10 muM), and P450s 2C8, 2C9, and 1A1 w
ere the most active in its formation. The use of embryonic kidney cells (29
3) 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 me
tabolism have been identified, the expression of which was shown to direct
atRA metabolism toward the formation of specific metabolites. The role of t
hese human P450s in the biological and anticancer effects of atRA remains t
o be elucidated.