Immuno-spin trapping of DNA radicals.
The detection of DNA radicals by immuno-spin trapping (IST) is based on the trapping of radicals with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), forming stable nitrone adducts that are then detected using an anti-DMPO serum. DNA radicals are very reactive species, and because they are paramagnetic they have previously been detected only by electron spin resonance (ESR) with or without spin trapping, which is not available in most bioresearch laboratories. IST combines the simplicity, reliability, specificity and sensitivity of spin trapping with heterogeneous immunoassays for the detection of DNA radicals, and complements existing methods for the measurement of oxidatively generated DNA damage. Here we have used IST to demonstrate that DMPO traps Cu(II)-H(2)O(2)-induced DNA radicals in situ and in real time, forming DMPO-DNA nitrone adducts, but preventing both 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) formation and DNA fragmentation. We also applied IST to detect DNA radicals in rat hepatocytes exposed to Cu(II) and H(2)O(2) under nonlethal conditions.
Ramirez
Dario C
DC
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Dr., Research Triangle Park, North Carolina 27709, USA. ramirez1@niehs.nih.gov
Mejiba
Sandra E Gomez
SE
Mason
Ronald P
RP
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002712:Chlorides / Q000737:chemistry; D003300:Copper / Q000737:chemistry / Q000494:pharmacology; D003486:Cyanides / Q000737:chemistry; D003497:Cyclic N-Oxides / Q000737:chemistry / Q000276:immunology; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017666:Deuterium Oxide / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005609:Free Radicals / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D022781:Hepatocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000494:pharmacology; D007118:Immunoassay / Q000379:methods; D009589:Nitrogen Oxides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D019097:Spin Trapping / Q000379:methods*; D013890:Thiourea / Q000737:chemistry; D017967:Zinc Compounds / Q000737:chemistry
D002712:Chlorides; D003486:Cyanides; D003497:Cyclic N-Oxides; D005609:Free Radicals; D009589:Nitrogen Oxides; D017967:Zinc Compounds; C015388:nitrones; D006639:Histidine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D003300:Copper; C016837:zinc chloride; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D006861:Hydrogen Peroxide; D003849:Deoxyguanosine; D005978:Glutathione; D013890:Thiourea; D017666:Deuterium Oxide; D001205:Ascorbic Acid; C029892:cupric chloride