Molecular Pharmacology, Vol 19, 140-145, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

The Formation of Arylating and Alkylating Metabolites of Phenacetin in Hamsters and Hamster Liver Microsomes

¹ Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, Department of Medicine and Institute for Lipid Research, Baylor College of Medicine, Houston, Texas 77030, and The Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20205

Phenacetin (4'-ethoxyacetanilide) is metabolized by multiple pathways, some of which can lead to the formation of reactive electrophilic products that may combine with glutathione or tissue macromolecules. Hamster liver microsomes converted ring-labeled [¹⁴C]phenacetin to an arylating metabolite that irreversibly bound to microsomal protein (1.65 ± 0.08 nmoles/mg/10 min). The binding was decreased more than 80% in the presence of 2.5 mM ascorbic acid or 1.0 mM glutathione. Under the same conditions, radiolabel from [ethyl-¹⁴C]phenacetin bound to microsomal protein only to about one-half the extent of the ring label (0.89 ± 0.06 mmol/mg/10 min), although binding was decreased to the same levels as ring-labeled phenacetin by either ascorbate or glutathione. In striking contrast, radiolabel from [acetyl-¹⁴C]phenacetin did not bind to microsomal protein, but rather yielded significant amounts of acetamide and acetic acid. However, both acetyl-and ring-labeled phenacetin formed approximately equivalent amounts of a glutathione conjugate of acetaminophen in the presence of hamster liver microsomes and glutathione, whereas ethyl-labeled phenacetin formed lesser amounts of S-ethyl glutathione. Metabolites found in hamster urine included 3-cysteinylacetaminophen and acetaminophen-3-mercapturate which maintained radiolabel from both ring- and acetyl-labeled phenacetins, and S-ethylcysteine and S-ethyl-N-acetylcysteine which maintained radiolabel from ethyl-labeled phenacetin. Deuterium substitution for hydrogen in the ethyl group of phenacetin showed that no deuterium was lost in the ethyl conjugates derived from phenacetin either in vitro or in vivo. These results show that phenacetin can be metabolized to both reactive arylating and alkylating agents by hamsters and hamster liver microsomes, and indicate that the metabolites involved in sulfur-ether conjugate formation are different from those that bind to tissue macromolecules.