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Immuno-Spin Trapping of Protein and DNA Radicals

This article discusses the use of immuno-spin trapping with ESR detection as a specific and universal tool for detecting free radicals in biological systems, with a focus on protein and DNA radicals. The article also explores the development of new technology to study oxidative DNA damage and provides a step-by-step protocol for detecting DNA radicals in various systems.

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Immuno-Spin Trapping of Protein and DNA Radicals

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  1. Ramirez DC, 1 of 21 Immuno-spin Trapping of Protein and DNA Radicals Dario C. Ramirez ramirez1@niehs.nih.gov Free Radical Metabolites Workgroup LPC/NIEHS/NIH/DHHS http://www.niehs.nih.gov

  2. Oxidative Damage to Macromolecules Macromolecule Free radicals Oxidized products Antioxidants Pro-oxidants Repaired Degradated Accumulated Ramirez DC, 2 of 21

  3. hydroxylamine adduct H 5,5-dimethyl-1-pyrroline N-oxide R N reduction (DMPO) radical adduct OH . H R dispropor- + + R H tionation N N . nitrone adduct O O oxidation + H+ R reduction potential –1.92V N oxidation potential +1.63V O Trapping of Radicals with DMPO The specificity of the reaction of nitrone spin traps with free radicals has already made spin trapping with ESR detection the most universal, specific tool for the detection of free radicals in biological systems. Ramirez DC, 3 of 21 Mason, R.P. 2004. Free Radic. Biol. Med. 36: 1214-1223.

  4. Product Secondary antibody conjugated to an enzyme Substrate Anti-DMPO antibody Solid support DMPO ( )-protein/DNA nitrone adducts Heterogeneous Immuno-Spin trapping Assays . Enzyme Linked Immuno-Sorbent Assay (ELISA) . Western blot or Immunoblot/ting . Immuno-Dot/Slot Blot Ramirez DC, 4 of 21

  5. In vitro and in vivo Production and Detection of DNA Nitrone Adducts Ramirez, DC & Mason R.P. Improved Immuno-spin Trapping Analyses of DNA Radicals. Nature Protocols. Coming soon. Fall 2006. This article will bring to Free radical researcher’s bench a step-by-step protocol to detect DNA radicals in tissues, whole cells, organelles, and in chemical systems. Ramirez DC, 5 of 21

  6. Reaction of Hydroxyl Radical with DNA • Hydroxyl radical react with DNA (~109 M-1 s-1) • abstraction • addition • DNA radicals are involved • C- and N-centered radicals in bases (purines and pyrimidines) • C-centered radicals in 2'-deoxyribose • Final consequences • base modifications • abasic sites • strand breaks • tandem-base lesions • DNA-protein cross-links Evans, M.D., Dizdaroglu, M. & Cooke, M. S. 2004. Mutation Research, 567: 1-61. Von Sonntag, C. 1987, The Chemical Basis of Radiation Biology. Tylor & Francis, London, NY. Ramirez DC, 6 of 21

  7. Why Develop New Technology to Study Oxidative DNA Damage? • 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) • . A biomarker of oxidative DNA damage • . Misreading of 8-oxo-dG can lead to G:C T:A transversion • 2. Artifactual 8-oxo-dG formation during • . isolation of DNA • . sample work-up • . analysis • 3. The European Standards Committee on Oxidative DNA Damage (ESCODD) recommended the development of new approaches to detect oxidative DNA damage to bypass the artifactual oxidation of DNA in intact cells. Halliwell, B. & Whiteman, M. 2005. Br. J. Pharmacol. 142: 231-255 Collins, A.R. et al. 2004. Arch. Biochem. Biophys. 423: 57-65 Ramirez DC, 7 of 21

  8. DNA Oxidation and its Investigation DMPO DNA radicals DNA-[8-oxo-dG]• (8-hydroxy-7,8-dihydro-2’- deoxyguanosyl radical) Electron transfer processes DNA radical adducts - e–,- H+ DNA-(sugar)-centered radicals - e–,- H+ DNA nitrone adducts DNA-[8-oxo-dG] (8-oxo-7,8-dihydro-2’- deoxyguanosine) O2 Cleavage of phosphodiester bonds Immuno-spin Trapping Immunochemical and Chromatographic (HPLC-MS/MS, GC/MS, etc.) measurement Electrophoresis Cadet, J. 1999. Hydroxyl radicals and DNA base damage. Mutat. Res. 424: 9-21 Ramirez DC, 8 of 21 Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127

  9. DNA Radicals, 8-oxo-dG, and Site-specific Fragmentation 8-oxo-dG nitrone adducts 4000 * 3000 Agarose gel electrophoresis * Immunoreactivity (RLU) 2000 * * + 500 + 5 + 10 + 50 + 100 + 1,000 * * 1000 DNA + Cu(II) DNA DNA + H2O2 * * * * * [H2O2] (M) 0 0 5 10 50 1 0.5 100 500 1,000 [H2O2] (M) Ramirez DC, 9 of 21 Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127

  10. DMPO Traps DNA Radicals and Prevents 8-oxo-dG Formation and DNA Fragmentation 8-oxo-dG nitrone adducts 3000 Agarose gel electrophoresis 2000 Immunoreactivity (RLU) + 10 + H2O2 + 500 DNA + 100 1000 + Cu(II) [DMPO] (mM) 0 0 100 200 300 400 500 [DMPO] (mM) Ramirez DC, 10 of 21 Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127

  11. CO2 Enhances DNA Radicals, but Prevents 8-oxo-dG and DNA Fragmentation by Copper-catalyzed Oxidations anti-8-oxo-dG anti-DMPO 4500 * * 3600 * * 2700 Immunoreactivity (RLU) 1800 † † 900 † 0 PB + 6.25 + 12.5 + 25.0 + 50.0 [BB] (mM) BB: (bi)carbonate Ramirez DC, 11 of 21 Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127

  12. CO2 + HO2— DNA + Cu(II) DTPA or BCDS H2O2 HOOCO2— DNA-[Cu(I)/(II)] (DNA-bound copper) Catalase or KCN DNA-[Cu(I)] / HOOCO2— H2O2 DNA-[Cu(II)] + H2O CO3• – DNA-[Cu(I)O, Cu(II)/•OH or Cu(III)] (Hydroxyl radical-like species) Oxidation, but not fragmentation Oxidation and fragmentation DNA radicals Role of CO2 in Copper-catalyzed Damage to DNA Ramirez DC, 12 of 21 Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127

  13. Some Applications Detection of DNA Radicals in Functioning Cells by Immuno-spin Trapping Ramirez DC, 13 of 21 Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127

  14. Why Has Spin Trapping in vivo Been so Challenging? DMPO (232 mg/100g b.wt.) or over 20 mM average concentration was not lethal. Both gross pathology and histopathological examination of the major organs were essentially negative with no obvious evidence of cellular damage being observed. Schaefer C.F., Janzen, E.G., West, M.S., Poyer, J.L., Kosanke, S.D.: Free Radical Bio. Med. 21: 427-436, 1996 Ramirez DC, 14 of 21

  15. DNA Nitrone Adducts are Stable to DNA Extraction Procedure Known calf-thymus DNA nitrone adducts are addedto tissue homogenates (4mg proteins /ml) Added Recovered 1500 1000 • DNA is extracted • Phenol • Chlorophorm/Phenol/isoamyl alcohol • Chloroform/isoamilic • Ethanol precipitation DNA nitrone adducts (RLU) 500 0 Lung Liver Intestine Calf-thymus DNA nitrone adductsare recovered Ramirez DC, 15 of 21

  16. Copper-catalyzed DNA Nitrone Adducts in Rat Hepatocytes Hepatocyte nuclei Whole hepatocytes PB 25 mM BB/PB 3000 1000 anti-DMPO anti-8-oxo-dG 2000 Immunoreactivity 500 Anti-DMPO immunoreactivity (RLU) 1000 0 0 0 1 5 10 25 50 0 0.1 0.5 1 m [Cu(II)] ( M) [H O ] (mM) 2 2 Immuno-slot blot PB 25 mM BB/PB 0 0.1 0.5 1 H2O2 (mM) Ramirez DC, 16 of 21 Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127

  17. DNA Nitrone Adducts Induced by Copper-mediated Paraquat (PQ) Toxicity in Rats DMPO (1 g/Kg) intraperitoneal 1500 Cu(II) and/or PQ given by intragastric intubation Intestine Liver 1000 DMPO (1 g/Kg) intraperitoneal Lung DNA nitrone adducts (RLU) 500 Sacrifice and tissues are removed 0 Saline Cu(II) PQ PQ/Cu(II) DNA extraction and ELISA analysis Based on: Kadiiska, M.B. & Mason, R.P. 2002. Spectrochemica Acta Part A, 58: 1227-1239 Ramirez DC, 17 of 21

  18. Production and Detection of Protein Nitrone Adducts Ramirez DC, 18 of 21

  19. Suggested References on Immuno-spin Trapping of Protein Radicals • Mason R. P. 2004. Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with Immuno-spin trapping. Free Radic. Biol. Med. 36: 1214-1223. • Ramirez, D.C., Gomez Mejiba, S.E. & Mason, R.P. 2005. Mechanism of Hydrogen Peroxide-induced Cu,Zn-superoxide dismutase-centered radical formation as explored by immuno-spin trapping: the role of copper- and carbonate radical anion-mediated oxidations. Free Radic. Biol. Med. 38: 201-214. • Ramirez, D.C. & Mason, R.P. 2005. Immuno-spin trapping: Detection of protein-centered radicals. In: Current Protocols in Toxicology, Suppl. 24, 17.7.1-17.7.18, John Wiley & Sons, Inc. • Ramirez, D.C, Gomez Mejiba, S.E. & Mason, R.P. 2005. Copper-catalyzed protein oxidation and its modulation by carbon dioxide. J. Biol. Chem. 280: 27402-27411. • Deterding, L.J., Ramirez, D.C., Dubin, J.R., Mason, R.P. & Tomer, K.B. 2004. Identification of free radicals on hemoglobin from self-peroxidation using mass spectrometry and immuno-spin trapping. J. Biol. Chem. 279: 11600-11607. Ramirez DC, 19 of 21

  20. Conclusions • ESR spin-trapping applications in biological research has been limited • . expensive instrumentation • . specialized technicians • 2. Immuno-spin trapping assays are heterogeneous • 3. DMPO traps intermediate species • 4. Oxygen is an spin trap and competes with DMPO • DMPO prevents final oxidation consequences • Nitrone adducts stability • 7. Immuno-spin trapping is a simple, reliable, economic, and specific method to detect oxidatively generated radicals within isolated and cellular proteins and DNA. Ramirez DC, 20 of 21

  21. Acknowledgments Dr. Ronald P. Mason M.S. Sandra E. Gomez Mejiba M.S. Jean Corbett Dr. Leesa Deterding Thanks Free Radical Metabolites Workgroup for building day-after-day such an excellent environment to work doing science !!! Ramirez DC, 21 of 21

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