Nature Methods 3, 123 - 127 (2006)
Published online: 23 January 2006; | doi:10.1038/nmeth852
Immuno–spin trapping of DNA radicalsDario C Ramirez, Sandra E Gomez Mejiba
& Ronald P Mason
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.
Correspondence should be addressed to Dario C Ramirez ramirez1@niehs.nih.gov 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)-H2O2–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 H2O2 under nonlethal conditions.
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