2-Acetamido-p-benzoquinone: a reactive arylating metabolite of 3'-hydroxyacetanilide

The covalent binding to protein of 3'-hydroxyacetanilide (3HAA), its primary metabolite 2',5'-dihydroxyacetanilide (2,5DHAA), and a putative secondary metabolite thereof, 2-acetamido-p-benzoquinone (APBQ), was studied in hepatic microsomal preparations from phenobarbital-pretreated mice. All compounds were found to bind irreversibly to microsomal protein, APBQ being by far the most effective member of the group. In the case of 3HAA, binding was dependent upon the presence in incubation media of the co-factor NADPH, indicating that metabolism of 3HAA was necessary for the generation of a reactive intermediate. In contrast, NADPH decreased by more than 2-fold the binding of both 2,5DHAA and APBQ. The free radical spin-trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) did not reduce the binding of 3HAA to protein. These results support the contention that metabolic activation of 3HAA is a two-step process which involves initial aromatic hydroxylation to give the substituted hydroquinone, 2,5DHAA, followed by a second oxidation reaction (which may not be enzyme-mediated) to produce the benzoquinone derivative, APBQ. This quinone is a reactive, electrophilic intermediate which may either undergo reduction back to 2,5DHAA or bind covalently to cellular macromolecules.