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Immuno–spin trapping of DNA radicals

Nature Methods volume 3, pages 123–127 (2006)Cite this article

Abstract

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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Figure 1: Intermediacy of DNA radicals in oxidatively generated damage to DNA by Fenton-type chemistry.
Figure 2: Detection of H2O2-induced, metal-catalyzed calf-thymus DNA nitrone adducts by IST.
Figure 3: Factors modulating Cu(II)-H2O2–induced calf-thymus DNA nitrone adducts and detection of DNA nitrone adducts formed inside functioning cells.

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Acknowledgements

This research was supported by the Intramural Research Program of the National Institutes of Health (NIH) and National Institute of Environmental Health Sciences (NIEHS). We thank M. Mason, A.G. Motten, B. van Houten and S. Wilson for their help in preparing this manuscript, and J.T. Corbett for technical assistance.

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Authors and Affiliations

  1. Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Dr., Research Triangle Park, 27709, North Carolina, USA

    Dario C Ramirez, Sandra E Gomez Mejiba & Ronald P Mason

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Correspondence to Dario C Ramirez.

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The US National Institutes of Health licenses anti-DMPO to three companies, and R.P.M is a recipient of a fraction of the royalties.

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Ramirez, D., Mejiba, S. & Mason, R. Immuno–spin trapping of DNA radicals. Nat Methods 3, 123–127 (2006). https://doi.org/10.1038/nmeth852

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