Hypochlorous acid-mediated activation of N-acetylbenzidine to form N '-(3 '-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine

Hypochlorous acid (HOCl), a chemically reactive oxidant, is an important co mponent of the inflammatory response and may contribute to carcinogenesis. This study assessed the possible activation of N-acetylbenzidine (ABZ) by H OCl to form a specific DNA adduct, N'-(3' -monophospho-deoxyguanosin-8-yl)- N-acetylbenzidine. HOCl was incubated with 0.06 mM H-3-ABZ, and transformat ion assessed by HPLC. Similar results were observed at pH 5.5 or 7.4. A Lin ear increase in transformation was observed from 0.025 to 0.1 mM HOCl with up to 80% of ABZ changed. Approximately, 2 nmoles of HOCl oxidized 1 nmole of ABZ. N-oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetyl benzidine, were not detected. Oxidation of ABZ was prevented by taurine, DM PO, glutathione, and ascorbic acid, whereas mannitol was without effect. Re sults are consistent with a radical mechanism. In the presence of 2'-deoxyg uanosine 3'-monophosphate (dGp), a new product (dGp-ABZ) was observed. The same adduct was observed with DNA. dGp-ABZ was found to be quite stable (>8 0% remaining) at 70 degrees C in pH 5.5 (60 min) and 7.4 (240 min). Electro spray mass spectrometry indicated that dGp-ABZ was N'-(3' -monophospho-deox yguanosin-8-yl)-N-acetylbenzidine, and this was confirmed by NMR. P-32-post labeling in combination with TLC and HPLC determined that the adduct made b y either HOCl or prostaglandin H synthase oxidation of ABZ in the presence of dGp or DNA was dGp-ABZ. Thus, HOCl activates ABZ to form dGp-ABZ and may be responsible for the presence of this adduct in peripheral white blood c ells from workers exposed to benzidine. Reaction of ABZ with HOCl provides an easy, convenient method for preparing dGp-ABZ.