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Vol. 27, Issue 8, 860-865, August 1999

Involvement of CYP2E1 as A Low-Affinity Enzyme in Phenacetin O-Deethylation in Human Liver Microsomes

Kaoru Kobayashi, Miki Nakajima, Kanako Oshima, Noriaki Shimada, Tsuyoshi Yokoi, and Kan Chiba

Laboratory of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University, Chiba (K.K., K.O., K.C.); Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa (M.N., T.Y.); and Materials Technology Research Laboratories, Daiichi Pure Chemicals Co. Ltd., Ibaraki (N.S.), Japan

Phenacetin O-deethylation (POD) exhibits biphasic kinetics in human liver microsomes. Although cytochrome P-450 (CYP) 1A2 is responsible for the high-affinity component of POD, the enzyme(s) that catalyzes the low-affinity reaction is still unknown. We examined the roles of human CYPs in POD by using human liver microsomes and recombinant CYPs from baculovirus-infected insect cells. Of the recombinant CYPs studied, CYP1A2 showed the highest POD activity. CYP1A1, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 also showed POD activity at 500 µM phenacetin. KM values of recombinant CYP1A2 and CYP2E1 (28 ± 2 µM and 785 ± 125 µM, respectively) were similar to those of the high- and low-affinity components of POD in pooled human liver microsomes (15 ± 5 and 894 ± 189 µM, respectively). Fluvoxamine (10 µM) and anti-CYP1A2 antibodies potently inhibited POD activity at 500 µM phenacetin in pooled human liver microsomes to 22.8 and 34.2% of controls, respectively. CYP2E1 inhibitors diethyldithiocarbamate and aniline also reduced POD activity. The combination of fluvoxamine (10 µM) and aniline (1 mM) further inhibited the residual POD activity not inhibited by fluvoxamine alone. Microsomal POD activity in 12 human livers in the absence of fluvoxamine was correlated with immunoquantified CYP1A2 levels (r = 0.961, p < .001) and, in the presence of 10 µM fluvoxamine, was correlated with immunoquantified CYP2E1 levels (r = 0.589, p < .01) or chlorzoxazone 6-hydroxylase activity (r = 0.823, p < .001). These results suggest that CYP2E1 is responsible for the low-affinity component of POD in human liver microsomes.

Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics


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Copyright © 1999 by the American Society for Pharmacology and Experimental Therapeutics.