The microsomal metabolism and site of covalent binding to protein of 3'-hydroxyacetanilide, a nonhepatotoxic positional isomer of acetaminophen.

Incubations of 3'-hydroxyacetanilide (3HAA) with hepatic microsomal preparations from phenobarbital-pretreated mice led to the formation of three products of aromatic hydroxylation, viz. 2',5'-, 3',4'-, and 2',3'-dihydroxyacetanilide, which were identified by GC/MS techniques and quantified by GLC analysis. NADPH-dependent covalent binding of radioactivity from [14C]3HAA to microsomal protein took place at almost four times the rate at which [14C]acetaminophen became irreversibly bound to protein under the same experimental conditions. This binding was inhibited by the addition to incubation media of ascorbate, glutathione, and the soluble proteins bovine serum albumin and bovine alpha s1-casein, but not by superoxide dismutase. Radioactivity from [14C]3HAA also became covalently bound to the added soluble proteins, the extent of which was greatest when the proteins contained a high content of free -SH groups. From an analysis of the effect of ascorbate and glutathione on both the covalent binding of 14C to protein and the production of the noncovalently bound products of 3HAA metabolism, it is concluded that reactive intermediates most likely derive from further oxidation of the primary 3HAA metabolites to electrophilic semiquinone and/or quinone species. Sulfhydryl groups appear to be the principal sites on protein at which covalent binding of these reactive metabolites of 3HAA takes place, a feature shared by the reactive species generated during acetaminophen metabolism.