N-acetylbenzidine-DNA adduct formation by phorbol 12-myristate-stimulated human polymorphonuclear neutrophils

N'-(3'-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is the ma jor adduct in exfoliated urothelial cells and in peripheral white blood cel ls of workers exposed to benzidine. This study was designed to assess the m etabolic pathways leading to dGp-ABZ formation in human peripheral white bl ood cells. [H-3]-N-Acetylbenzidine (ABZ) transformation was assessed using myeloperoxidase (MPO), hypochlorous acid (HOCl), and human peripheral white blood cells in the absence and presence of DNA or dGp. MPO metabolism requ ired H2O2, but not NaCl. While transformation by HOCl was completely inhibi ted by 10 mM taurine, the level of metabolism of ABZ by MPO was only reduce d 56%. Transformation by either MPO or HOCl was inhibited by 100 mM DMPO, 1 mM glutathione, and 1 mM ascorbic acid. Glutathione formed a new product w ith MPO, but not with HOCl. Previously identified oxidation products of ABZ , N'-hydroxy-N-acetylbenzidine or 4'-nitro-4-acetylaminobiphenyl, were not detected. with DNR, or dcp present, a new product was observed that corresp onded to synthetic dGp-ABZ in its HPLC elution profile, in nuclease P-1 hyd rolysis to dG-ABZ, and in P-32-postlabeling analysis. The HOCl-derived addu ct was identified by electrospray ionization mass spectrometry, with collis ion-activated dissociation, as dGp-ABZ. Metabolism of [H-3]ABZ by periphera l blood cells was stimulated about 3-fold with 30 ng/mL beta-phorbol 12-myr istate 13-acetate (PMA). Using P-32-postlabeling, dGp-ABZ was detected only in the presence of PMA and its level was increased more than 300-fold if e ither 0.7 mg/mL DNA or dGp was present. Indomethacin (0.1 mM) did not alter adduct: formation. With dGp, dGp-ABZ formation could be detected with as l ittle as 0.12 x 10(6) neutrophils. Using specific chromatographic and enzym atic techniques, neutrophil-derived dGp-ABZ was identical to the synthetic standard. Thus, these results are consistent with human polymorphonuclear n eutrophils forming dGp-ABZ by a peroxidatic mechanism involving MPO.