Covalent binding of the phenacetin metabolite p-nitrosophenetole to protein.

Addition of p-[3H]nitrosophenetole to protein incubations resulted in quantitative binding of radiolabel to protein. Preincubation of protein with N-methylmaleimide to derivatize sulfhydryl groups followed by addition of p-[3H]nitrosophenetole resulted in a 90% decrease in covalent binding. Treatment of the p-[3H]nitrosophenetole protein-bound residue, after extensive solvent washing, with HCl decreased binding by 72% and the corresponding amine, p-phenetidine was detected in the aqueous phase. Inasmuch as incubation of the bound residue with reduced glutathione did not alter protein binding appreciably the involvement of a sulfinanilide S-oxide is suggested. Incubation of p-[3H]phenetidine with microsomal incubation mixtures resulted in NADPH-dependent covalent binding to protein. Treatment of the p-[3H]phenetidine protein-bound residue after extensive solvent washing with HCl decreased binding by 64%. Administration of either p-[3H]phenetidine (500 mg/kg i.p.) or [14C]phenacetin (500 mg/kg i.p.) to mice resulted in covalent binding of radiolabel to protein of liver, lung, kidney, small intestine and blood. Incubation of solvent-washed protein with acid resulted in an approximately 35% decrease in protein binding in lung and blood of both treatment groups.