MYELOPEROXIDASE ACTIVATION OF N ACETYLBENZIDINE

Washington University, Saint Louis MO

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Abstract

In workers exposed to benzidine, N'-(3'-monophospho-deoxyguanosin-8 -yl)-N-acetylbenzidine (1A) was detected in exfoliated bladder cells and peripheral white blood cells. Because both cell types exhibit substantial peroxidatic activity, this was considered a likely mechanism for activation of N-acetylbenzidine (ABZ). For this reason, myeloperoxidase (MPO) metabolism of 3H-ABZ (0.06 mM) was assessed with analysis by HPLC. Substantial metabolism of ABZ by MPO was observed with a 2-fold increase elicited by inclusion of 100 mM NaCl in the reaction mixture. Taurine (10 mM) reduced metabolism in the presence, but not the absence of NaCl. Azide (1 mM), cyanide (10 mM), and ascorbic acid (1 mM) dramatically inhibited metabolism. With glutathione (0.1 mM), a new metabolite was observed which was sensitive to (-glutamyl transpeptidase and hydrolyzed to ABZ with mild acid or base treatment (0.1 N HCl or 0.1 N NaOH for 10 min). In the presence of dGp, MPO or 0.3 mM NaOC l produced an adduct whose formation was prevented by the presence of either glutathione or vitamin C. ESI/MS/MS analysis demonstrated this adduct to be 1A. Thus, ABZ is a reducing co-substrate for MPO and a reductant for OCl-. MPO may be responsible for the activation of ABZ by peripheral white blood cells to form 1A.