Detection and imaging of the free radical DNA in cells—Site-specific radical formation induced by Fenton chemistry and its repair in cellular DNA as seen by electron spin resonance, immuno-spin trapping and confocal microscopy

摘要

Oxidative stress-related damage to the DNA macromolecule produces lesions that are implicated in various diseases. To understand damage to DNA, it is important to study the free radical reactions causing the damage. Measurement of DNA damage has been a matter of debate as most of the available methods measure the end product of a sequence of events and provide limited information on the initial free radical formation. We report a measurement of free radical damage in DNA induced by a Cu(II)-H₂O₂ oxidizing system using immuno-spin trapping supplemented with electron paramagnetic resonance. In this investigation, the short-lived radical generated is trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) immediately upon formation. The DMPO adduct formed is initially electron paramagnetic resonance active, but is subsequently oxidized to the stable nitrone adduct, which can be detected and visualized by immuno-spin trapping and has the potential to be further characterized by other analytical techniques. The radical was found to be located on the 2′-deoxyadenosine (dAdo) moiety of DNA. The nitrone adduct was repaired on a time scale consistent with DNA repair. In vivo experiments for the purpose of detecting DMPO–DNA nitrone adducts should be conducted over a range of time in order to avoid missing adducts due to the repair processes.