Methemoglobin oxidation of N-acetylbenzidine to form a sulfinamide.
Aromatic amine sulfinamide adducts of hemoglobin are biomarkers of exposure and evidence for cytochrome P-450 N-hydroxylation. The possible peroxidatic formation of an N-acetylbenzidine (ABZ) sulfinamide adduct by methemoglobin was examined. Following addition of H2O2, 0.06 mM [3H]ABZ was metabolized by methemoglobin. With 0.3 mM glutathione, a new peak was observed, ABZ-SG, representing 17% of the total radioactivity. N'-Hydroxy-N-acetylbenzidine and 4'-nitro-4-acetylaminobiphenyl were not detected. Optimal ABZ-SG formation was observed with 3 uM methemoglobin, 0.1 to 0.3 mM glutathione, and pH 5.5. Higher concentrations of glutathione were inhibitory. Without glutathione, an H2O2-to-ABZ molar ratio of 1:1 resulted in complete metabolism of ABZ. This ratio increased to greater than 2:1 with 0.3 mM glutathione. Nearly complete inhibition of ABZ-SG formation by cyanide (10 mM), ascorbic acid (0.1 mM), 5,5-dimethyl-1-pyrroline N-oxide (50 mM), thiourea (1 mM), and azide (0.3 mM), and the lack of inhibition by mannitol (50 mM) and superoxide dismutase (2 microg) is consistent with a methemoglobin-mediated peroxidatic reaction, which does not involve hydroxyl radical or superoxide. ABZ-SG was identified by electrospray ionization/mass spectrometry as N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide. Conjugate was hydrolyzed by 0.1 N HCl and NaOH, was relatively stable at pH 5.5 and 7.4, and was susceptible to gamma-glutamyltranspeptidase treatment. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The results demonstrate that methemoglobin can catalyze the peroxidatic formation of an ABZ sulfinamide adduct, perhaps by a diimine monocation intermediate.
Veterans Affairs Medical Center, Division of Geriatric Medicine, St. Louis University School of Medicine, St. Louis, Missouri, USA. zensertv@slu.edu
T V
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Zenser
V M
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Lakshmi
F F
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Hsu
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Davis
D001560:Benzidines / Q000302:isolation & purification* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000031:analogs & derivatives / Q000302:isolation & purification* / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008706:Methemoglobin / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction
D001560:Benzidines; C431127:N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide; D016877:Oxidants; D008706:Methemoglobin; D006861:Hydrogen Peroxide; D005978:Glutathione; C034938:N-acetylbenzidine