Immunochemical detection of glyoxal DNA damage.
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.
Mistry
N
N
Division of Chemical Pathology, MRC Centre for Mechanisms of Human Toxicity, University of Leicester, UK. nm14@leicester.ac.uk
Evans
M D
MD
Griffiths
H R
HR
Kasai
H
H
Herbert
K E
KE
Lunec
J
J
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
D000974:Antibodies, Antinuclear; D018736:DNA Adducts; D017382:Reactive Oxygen Species; D006037:Glyoxal; D004247:DNA; D006861:Hydrogen Peroxide; D001205:Ascorbic Acid