Sulfinamide formation following peroxidatic metabolism of N-acetylbenzidine

Arylamine-hemoglobin conjugates identified as sulfinamides are considered d osimeters for the bioavailability of metabolically formed N-oxidation produ cts. This report considers peroxidation as an alternative pathway for aroma tic amine metabolism and examines horseradish peroxidase metabolism of N-ac etylbenzidine (ABZ) in the presence of glutathione. When 0.06 mM [H-3]ABZ w as incubated with 1 mM glutathione, a decrease in the total extent of metab olism was observed along with detection of a new metabolite (ABZ-SG), repre senting 12% of the total radioactivity. Optimum ABZ-SG formation occurred a t 0.3 mM glutathione with higher concentrations (10 mM) being inhibitory. I n the absence of glutathione, a molar ratio of H2O2 to ABZ of 1:1 resulted in complete metabolism of ABZ. This ratio increased to >2:1 in the presence of 0.3 mM glutathione. N-Oxidation products of ABZ metabolism, such as N'- hydroxy-N-acetylbenzidine, were not detected using a variety of incubation conditions. ABZ-SG was sensitive to gamma-glutamyltranspeptidase, and compl etely hydrolyzed by 0.1 N HCl or 0.1 N NaOH in 10 min at room temperature. ABZ-SG was identified by mass spectrometry and NMR to be N'-(glutathion-S-y l)-N-acetylbenzidine S-oxide. ABZ-SG formation, but not total ABZ metabolis m, was prevented by 0.3 mM NaN3, 50 mM DMPO, 1.0 mM thiourea, and 1.0 mM hi stidine. Cyanide (50 mM) and ascorbic acid (0.1 mM) completely inhibited AB Z metabolism. The lack of effect of 50 mM mannitol and 2 mu g of superoxide dismutase suggests that neither hydroxyl radical nor superoxide is involve d in the reaction. Studies also indicated that molecular oxygen is not a so urce of the sulfinamide oxygen. Formation of an ABZ sulfinamide conjugate w ith hemoglobin was demonstrated. The proposed mechanism for sulfinamide for mation, involving two consecutive one-electron oxidations with subsequent r earrangement to a sulfur-stabilized nitrenium ion, suggests that oxygen may be derived from water. The results demonstrate that while arylamine-hemogl obin conjugates serve as useful biomarkers of exposure, their mechanism of formation may be complex, perhaps involving peroxidation as in the case of N'- (glutathion-S-yl)-N- acetylbenzidine S-oxide.