Abstract

The activity of human liver microsomal cytochrome P450 1A2 (CYP1A2) is readily estimated by following the O-deethylation of [O-ethyl ¹⁴C]phenacetin (PODase). The basis of the assay is the quantitative measurement of [¹⁴C]acetaldehyde, remaining in the supernatant of assay incubates, after extraction of unmetabolized [O-ethyl ¹⁴C]phenacetin with charcoal. In the presence of native human liver microsomes (K_m = 54 ± 27 μM;V_max = 14 ± 2.3 nmol/hr/mg; mean ± SD; N = 3 different livers) and human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP1A2 (K_m = 46 μM;V_max = 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= 0.91; p < 0.001; N = 11) with CYP1A2-selective 7-ethoxyresorufin O-deethylase activity, and was markedly inhibited (≥ 92%) by furafylline (FURA, IC₅₀ = 0.4 μM) and 7,8-benzoflavone (ANF, IC₅₀ = 0.1 μM), 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 (≥ 10·K_i ). 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 ¹⁴C]phenacetin as substrate.