title: Immunochemical detection of glyoxal DNA damage.
abstract: The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.
authors.affiliation: Division of Chemical Pathology, MRC Centre for Mechanisms of Human Toxicity, University of Leicester, UK. nm14@leicester.ac.uk
authors.forename: N
authors.initials: N
authors.lastname: Mistry
authors.forename: M D
authors.initials: MD
authors.lastname: Evans
authors.forename: H R
authors.initials: HR
authors.lastname: Griffiths
authors.forename: H
authors.initials: H
authors.lastname: Kasai
authors.forename: K E
authors.initials: KE
authors.lastname: Herbert
authors.forename: J
authors.initials: J
authors.lastname: Lunec
mesh_terms: D000818:Animals; D000974:Antibodies, Antinuclear; D001205:Ascorbic Acid / Q000633:toxicity; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000276:immunology; D018736:DNA Adducts / Q000032:analysis* / Q000276:immunology; D004249:DNA Damage; D004797:Enzyme-Linked Immunosorbent Assay; D006037:Glyoxal / Q000031:analogs & derivatives* / Q000032:analysis; D006861:Hydrogen Peroxide / Q000633:toxicity; D007120:Immunochemistry; D066298:In Vitro Techniques; D010084:Oxidation-Reduction; D011817:Rabbits; D017382:Reactive Oxygen Species
chemical_list: D000974:Antibodies, Antinuclear; D018736:DNA Adducts; D017382:Reactive Oxygen Species; D006037:Glyoxal; D004247:DNA; D006861:Hydrogen Peroxide; D001205:Ascorbic Acid