Abstract
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.
Footnotes
-
Send reprint requests to: Terry V. Zenser, Ph.D., VA Medical Center (GRECC/11G-JB), St. Louis, MO. E-mail:zensertv{at}slu.edu
-
This work was supported by the Department of Veterans Affairs (T.V.Z.) and National Cancer Institute Grant CA72613 (T.V.Z.). Mass spectrometry was performed at the Mass Spectrometry Resource Center, Washington University School of Medicine, through National Institutes of Health Grants RR-00954 and AM-20579.
- Abbreviations used are::
- ABZ
- N-acetylbenzidine
- CAD
- collisionally activated dissociation
- DETAPAC
- diethylenetriaminepentaacetic acid
- DMPO
- 5,5-dimethyl-1-pyrroline N-oxide
- ESI
- electrospray ionization
- MS
- mass spectrometry
- Received April 13, 2000.
- Accepted November 13, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|