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Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling
Published on: March 20, 2018
N-Acetylbenzidine-DNA adduct formation by phorbol 12-myristate-stimulated human polymorphonuclear neutrophils
V M Lakshmi1, F F Hsu, B B Davis
1VA Medical Center, Division of Geriatric Medicine and Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63125-4199, USA.
Chemical Research in Toxicology
|August 24, 2000
Summary
Human white blood cells form N'-(3'-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) adducts through a myeloperoxidase (MPO) dependent pathway. This study elucidates the metabolic mechanisms involved in dGp-ABZ formation in response to benzidine exposure.
Area of Science:
- Biochemistry
- Toxicology
- Cell Biology
Background:
- N'-(3'-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is a significant biomarker in individuals exposed to benzidine.
- Understanding the metabolic pathways of dGp-ABZ formation is crucial for assessing health risks associated with benzidine exposure.
Purpose of the Study:
- To investigate the metabolic pathways responsible for the formation of dGp-ABZ in human peripheral white blood cells.
- To identify the specific enzymes and reactive species involved in this process.
Main Methods:
- Utilized [(3)H]-N-Acetylbenzidine (ABZ) to study its transformation by myeloperoxidase (MPO), hypochlorous acid (HOCl), and human peripheral white blood cells.
- Employed techniques including High-Performance Liquid Chromatography (HPLC), nuclease P(1) hydrolysis, (32)P-postlabeling, and electrospray ionization mass spectrometry (ESI-MS).
- Assessed the effects of inhibitors like taurine, glutathione, ascorbic acid, and DMPO, as well as activators like beta-phorbol 12-myristate 13-acetate (PMA).
Main Results:
- Myeloperoxidase (MPO) and hypochlorous acid (HOCl) were identified as key players in ABZ transformation.
- dGp-ABZ formation was significantly enhanced in the presence of DNA or deoxyguanosine monophosphate (dGp) and stimulated by PMA.
- Neutrophil-derived dGp-ABZ was confirmed to be identical to synthetic standards, indicating a peroxidatic mechanism involving MPO.
Conclusions:
- Human peripheral white blood cells, particularly neutrophils, can form dGp-ABZ via a myeloperoxidase-mediated peroxidatic mechanism.
- The findings provide critical insights into the bioactivation of benzidine and the formation of DNA adducts in exposed individuals.
