title: Hypochlorous acid-mediated activation of N-acetylbenzidine to form N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine.
abstract: Hypochlorous acid (HOCl), a chemically reactive oxidant, is an important component of the inflammatory response and may contribute to carcinogenesis. This study assessed the possible activation of N-acetylbenzidine (ABZ) by HOCI to form a specific DNA adduct, N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. HOCl was incubated with 0.06 mM 3H-ABZ, and transformation assessed by HPLC. Similar results were observed at pH 5.5 or 7.4. A linear increase in transformation was observed from 0.025 to 0.1 mM HOCl with up to 80% of ABZ changed. Approximately, 2 nmoles of HOCI oxidized 1 nmole of ABZ. N-oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected. Oxidation of ABZ was prevented by taurine, DMPO, glutathione, and ascorbic acid, whereas mannitol was without effect. Results are consistent with a radical mechanism. In the presence of 2'-deoxyguanosine 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 (>80% remaining) at 70 degrees C in pH 5.5 (60 min) and 7.4 (240 min). Electrospray mass spectrometry indicated that dGp-ABZ was N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine, and this was confirmed by NMR. 32P-postlabeling in combination with TLC and HPLC determined that the adduct made by either HOCl or prostaglandin H synthase oxidation of ABZ in the presence of dGp or DNA was dGp-ABZ. Thus, HOCI activates ABZ to form dGp-ABZ and may be responsible for the presence of this adduct in peripheral white blood cells from workers exposed to benzidine. Reaction of ABZ with HOCl provides an easy, convenient method for preparing dGp-ABZ.
authors.affiliation: VA Medical Center, Department of Biochemistry, St. Louis University School of Medicine, Missouri, USA.
authors.forename: V M
authors.initials: VM
authors.lastname: Lakshmi
authors.forename: F F
authors.initials: FF
authors.lastname: Hsu
authors.forename: A E
authors.initials: AE
authors.lastname: McGarry
authors.forename: B B
authors.initials: BB
authors.lastname: Davis
authors.forename: T V
authors.initials: TV
authors.lastname: Zenser
mesh_terms: D000818:Animals; D001560:Benzidines / Q000378:metabolism*; D002273:Carcinogens / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000187:drug effects*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006997:Hypochlorous Acid / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D015394:Molecular Structure
chemical_list: D001560:Benzidines; D002273:Carcinogens; D018736:DNA Adducts; C112791:N'-(3'-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine; D006997:Hypochlorous Acid; D004247:DNA; D003849:Deoxyguanosine; C034938:N-acetylbenzidine