PT  - JOURNAL ARTICLE
AU  - Terry V. Zenser
AU  - Vijaya M. Lakshmi
AU  - Fong Fu Hsu
AU  - Bernard B. Davis
TI  - Methemoglobin Oxidation of <em>N</em>-Acetylbenzidine to Form a Sulfinamide
DP  - 2001 Apr 01
TA  - Drug Metabolism and Disposition
PG  - 401--406
VI  - 29
IP  - 4
4099  - http://dmd.aspetjournals.org/content/29/4/401.short
4100  - http://dmd.aspetjournals.org/content/29/4/401.full
SO  - Drug Metab Dispos2001 Apr 01; 29
AB  - Aromatic amine sulfinamide adducts of hemoglobin are biomarkers of exposure and evidence for cytochrome P-450N-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 μg) 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 asN′-(glutathion-S-yl)-N-acetylbenzidineS-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 γ-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. The American Society for Pharmacology and Experimental Therapeutics