Generation of reactive metabolites of N-hydroxy-phenacetin by glucuronidation and sulfation

Abstract

Several xenobiotics exert their toxic effects in mammals through the formation of reactive metabolites that combine with cellular macromolecules. Thus, N-hydroxy-2-acetylaminofluorene becomes covalently bound to various cellular macromolecules after sulfation of the N-hydroxy group. A method is presented for the indirect measurement of the rate of formation of the N, O-sulfate conjugate of this compound, which is too unstable to be measured directly. In this assay 3′, 5′-adenosine diphosphate and p-nitrophenylsulfate were used as a 3′-phosphoadenosine 5′-phosphosulfate-generating system (PAPS-GS); the release of p-nitrophenol (which was measured spectrophotometrically) was used to estimate the sulfation rate of N-hydroxy-2-acetylaminofluorene. The PAPS-GS was also used in studying the role of N- O-sulfate conjugates as intermediates in the formation of reactive metabolites that become covalently bound. Using this method we found that N-hydroxy-phenacetin became rapidly covalently bound after sulfation of the N-hydroxy group. N-hydroxy-2-acetylaminonaphthalene also became covalently bound after sulfation, but the N- O-sulfate derivatives of N-hydroxy- p-chloroacetanilide and N-hydroxy-acetanilide did not bind covalently, although their rates of sulfation were similar to that of N-hydroxy-phenacetin. Glucuronidation of the N-hydroxy group of N-hydroxy-phenacetin resulted in a glucuronide conjugate that was bound covalently to protein at pH 7.4, but at a slower rate than the N- O-sulfate conjugate. The N- O-glucuronides of the other N-hydroxy- N-arylacetamides investigated did not become covalently bound to protein at pH 7.4. Characteristics of the conjugation of N-hydroxy-phenacetin, and of the covalent binding of its conjugates, were determined.