Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron.
Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 microM Fe(III)-NTA and 100 microM L-ascorbate induced detectable DNA strand breaks after 30 min at 24 degrees C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload.
Toyokuni
S
S
Molecular Biology Branch, Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland 20857.
Sagripanti
J L
JL
D001205:Ascorbic Acid / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects*; D004278:DNA, Superhelical / Q000187:drug effects; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D016166:Free Radical Scavengers; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010957:Plasmids; D013482:Superoxide Dismutase / Q000494:pharmacology
D002614:Chelating Agents; D004278:DNA, Superhelical; D005290:Ferric Compounds; D005296:Ferrous Compounds; D016166:Free Radical Scavengers; D006861:Hydrogen Peroxide; D007501:Iron; D013482:Superoxide Dismutase; D003545:Cysteine; D009571:Nitrilotriacetic Acid; D001205:Ascorbic Acid; C020326:ferric nitrilotriacetate