Short Communication [O-ETHYL C]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]phenacetin (PODase). The basis of the assay is the quantitative measurement of [C]acetaldehyde, remaining in the supernatant of assay incubates, after extraction of unmetabolized [O-ethyl C]phenacetin with charcoal. In the presence of native human liver microsomes (Km 5 54 6 27 mM; Vmax 5 14 6 2.3 nmol/hr/mg; mean 6 SD; N 5 3 different livers) and human Blymphoblastoid cell microsomes containing cDNA-expressed CYP1A2 (Km 5 46 mM; Vmax 5 55 nmol/hr/nmol CYP), PODase activity conformed to monophasic Michaelis-Menten kinetics. Furthermore, PODase activity in a panel of microsomes prepared from a series of human livers was significantly correlated (r 5 0.91; p < 0.001; N 5 11) with CYP1A2-selective 7-ethoxyresorufin O-deethylase activity, and was markedly inhibited (> 92%) by furafylline (FURA, IC50 5 0.4 mM) and 7,8-benzoflavone (ANF, IC50 5 0.1 mM), two well known CYP1A2 inhibitors. Inhibitors selective for other forms of CYP (e.g. CYP3A, CYP2C, CYP2D6, CYP2E1) elicited a marginal effect (< 17% inhibition) at relatively high concentrations (> 10zKi). It is concluded that the inhibition of human liver microsomal CYP1A2 activity can be readily determined by using a charcoal-based radiometric method employing [O-ethyl C]phenac-