MECHANISM OF 3-(GLUTATHIONE-S-YL)-BENZIDINE FORMATION

The formation of thioether conjugates is an important mechanism for in activation of carcinogens. 3-(Glutathion-S-yl)-benzidine (BZ-SG) forma tion prevents benzidinediimine and peroxidase-mediated benzidine bindi ng to DNA. Benzidinediimine is the two-electron oxidized product of be nzidine thought to be the reactive intermediate involved in peroxidase -mediated binding of benzidine to DNA. Diimine interacts with benzidin e to form a dimeric complex known as the charge-transfer complex. The latter is in equilibrium with the cation radical. This study evaluated the mechanism by which BZ-SG forms. Benzidinediimine was synthesized and used to study the formation of BZ-SG. With 0.05 mM benzidinediimin e, BZ-SG formation was optimum at pH 4.5 and with glutathione at 0.05 to 0.1 mM. By monitoring specific absorption spectra, the reduction of benzidinediimine at pH 4.5 was evaluated. The t(1/2) for diimine deca y (425 nm) and maximum absorbance of the charge-transfer complex (600 nm) were each at approximately 5 min. Within 10 min, the maximum amoun t of benzidine had formed from diimine. BZ-SG formation followed the d ecay of diimine. The relationship between benzidinediimine and benzidi ne, with respect to BZ-SG formation, was assessed at a fixed concentra tion of glutathione (0.05 mM) and a fixed total concentration of amine and diimine (0.05 mM). In three separate experiments, each of these t hree components was radiolabeled independent of the other two componen ts. Experiments with [H-3]glutathione indicated that conjugate formati on was dependent upon diimine, and not benzidine. With [H-3]benzidined iimine or [H-3]benzidine, two different calculations were necessary to assess conjugate formation. For [H-3]benzidinediimine, the calculatio n considered that only the radiolabeled diimine formed conjugate, whil e with [3H]benzidine, a specific activity calculation was necessary to demonstrate that conjugate formation was dependent upon diimine. With 0.05 mM [H-3]benzidine, horseradish peroxidase-catalyzed formation of BZ-SG was optimum between 0.05 and 0.0625 mM H2O2. The latter is cons istent with conversion of benzidine to diimine before formation of BZ- SG. Specific inhibitors and the absence of oxygen uptake indicated the lack of involvement of cation, thiyl, and carbon-centered radicals. T he results are consistent with the existence of the charge-transfer co mplex and with benzidinediimine reacting with glutathione to form BZ-S G. (C) 1994 Academic Press, Inc.