[O-ETHYL C-14]PHENACETIN O-DEETHYLASE ACTIVITY IN HUMAN LIVER-MICROSOMES

The activity of human liver microsomal cytochrome P450 1A2 (CYP1A2) is readily estimated by following the O-deethylation of [O-ethyl C-14]ph enacetin (PODase). The basis of the assay is the quantitative measurem ent of [C-14]acetaldehyde, remaining in the supernatant of assay incub ates, after extraction of unmetabolized [O-ethyl C-14]phenacetin with charcoal. In the presence of native human liver microsomes (K-m = 54 /- 27 mu M; V-max = 14 +/- 2.3 nmol/hr/mg; mean +/- SD; N = 3 differen t livers) and human B-lymphoblastoid cell microsomes containing cDNA-e xpressed CYP1A2 (K-m = 46 mu M; V-max = 55 nmol/hr/nmol CYP), PODase a ctivity conformed to monophasic Michaelis-Menten kinetics. Furthermore , PODase activity in a panel of microsomes prepared from a series of h uman livers was significantly correlated (r = 0.91; p < 0.001; N = 11) with CYP1A2-selective 7-ethoxyresorufin O-deethylase activity, and wa s markedly inhibited (greater than or equal to 92%) by furafylline (FU RA, IC50 = 0.4 mu M) and 7,8-benzoflavone (ANF(1) IC50 = 0.1 mu M), tw o well known CYP1P2 inhibitors. Inhibitors selective for other forms o f CYP (e.g. CYP3A, CYP2C, CYP2D6, CYP2E1) elicited a marginal effect ( less than or equal to 17% inhibition) at relatively high concentration s (greater than or equal to 10 K-i). It is concluded that the inhibiti on of human liver microsomal CYP1A2 activity can be readily determined by using a charcoal-based radiometric method employing [O-ethyl C-14] phenacetin as substrate.