RT Journal Article
SR Electronic
T1 [<em>O</em>-ETHYL <sup>14</sup>C]PHENACETIN<em>O</em>-DEETHYLASE ACTIVITY IN HUMAN LIVER MICROSOMES
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1097
OP 1100
VO 25
IS 9
A1 A. David Rodrigues
A1 Bruce W. Surber
A1 Ye Yao
A1 Shekman L. Wong
A1 Ellen M. Roberts
YR 1997
UL http://dmd.aspetjournals.org/content/25/9/1097.abstract
AB 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 (Km = 54 ± 27 μM;Vmax = 14 ± 2.3 nmol/hr/mg; mean ± SD; N = 3 different livers) and human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP1A2 (Km = 46 μM;Vmax = 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 &lt; 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·Ki ). 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. The American Society for Pharmacology and Experimental Therapeutics