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)-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.