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Rapid and sensitive detection of hydroxyl radicals formed by activated neutrophils in the presence of chelated iron: Hydroxylation of deoxyguanosine to 8-hydroxydeoxyguanosine

  • Inflammation and Immunomodulation
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Abstract

Hydroxyl radicals (OH) can react with deoxyguanosine (dG) leading to the formation of 8-hydroxydeoxyguanosine (8OHdG). In this study, this has been used to detect the hydroxyl radicals formed when human polymorphonuclear leukocytes (PMNL) are stimulated with phorbol myristate acetate (PMA) in the presence of chelated iron. Reaction mixtures containing PMNL, PMA, dG and Fe-EDTA were incubated at 37°C, and the formation of 8OHdG was analysed with high-performance liquid chromatography and electrochemical detection. 8OHdG formation was detected at PMA concentrations of 2 nM or higher, and half-maximal 8OHdG formation was found at around 6 nM PMA. Stimulation of 500 000 cells with 10 nM PMA for 20 min resulted in a 500 to 1000-fold increase in 8OHdG formation as compared to unstimulated cells. The 8OHdG formation decreased after addition of hydroxyl radical scavengers (sodium benzoate, dimethylsulfoxide, and mannitol) and increased after addition of platelet-activating factor (PAF), an agent known to stimulate the generation of reactive oxygen metabolites in neutrophils. These results demonstrate that hydroxylation of dG to 8OHdG can be used to determine neutrophil-generated hydroxyl radicals in different experimental systems. Since the analysis of 8OHdG is rapid, sensitive and easy, this may have wide applications in inflammation and cancer research.

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References

  1. J. C. Fantone and P. A. Ward,Role of oxygen derived free radicals and metabolites in leucocyte-dependent inflammatory reactions. Am. J. Pathol.107, 397–414 (1982).

    Google Scholar 

  2. G. M. Vercellotti, B. S. van Asbeck and H. S. Jacob,Oxygen radical inducec erythrocyte hemolysis by neutrophils: Critical role of iron and lactoferrin. J. Clin. Invest.76, 956–962 (1985).

    PubMed  Google Scholar 

  3. B. D. Goldstein, G. Wits, M. Amoruso and W. Troll,Protease inhibitors antagonize the action of polymorphonuclear leucocyte oxygen consumption. Biochem. Biophys. Res. Commun.88, 854–860 (1979).

    PubMed  Google Scholar 

  4. L. Fucci, C. N. Oliver, M. J. Coon and E. R. Stadtman,Inactivation of key metabolic enzymes by mixed-function oxidation reactions: Possible implication in protein turnover and ageing. Proc. Natl. Acad. Sci.80, 1521–1525 (1983).

    PubMed  Google Scholar 

  5. K. Kim, S. G. Rhee and E. R. Stadtman,Nonenzymatic cleavage of proteins by reactive oxygen species generated by dithiothreitol and iron. J. Biol. Chem.260, 15394–15397 (1985).

    PubMed  Google Scholar 

  6. M. K. Logani and R. E. Davies,Lipid peroxidation: Biological effects and antioxidants — A review. Lipids15, 485–495 (1979).

    Google Scholar 

  7. K. Frenkel, K. Chrzan, W. Troll, G. W. Teebor and J. J. Steinberg,Radiation-like modification of bases in DNA exposed to tumor promoter-activated polymorphonuclear leucocytes. Cancer Res.46, 5553–5540 (1986).

    PubMed  Google Scholar 

  8. J. H. Jackson, E. Gajewski, I. U. Schraufstatter, P. A. Hyslop, A. F. Fuciarelli, C. G. Cochrane and M. Dizdaroglu,Damage to the bases in DNA induced by stimulated neutrophils. J. Clin. Invest.84, 1644–1649 (1989).

    PubMed  Google Scholar 

  9. R. A. Floyd, J. J. Watson, J. Harris, M. West and P. K. Wong,Formation of 8-hydroxydeoxyguanosine, hydroxyl free radical adduct in DNA in granulocytes exposed to the tumor promoter tetradeconylphorbolacetate. Biochem. Biophys. Res. Commun.137, 841–846 (1986).

    PubMed  Google Scholar 

  10. E. Shacter, E. J. Beecham, J. M. Covey, K. W. Kohn and M. Potter,Activated neutrophils induce prolonged DNA damage in neighboring cells. Carcinogenesis9, 2297–2304 (1988).

    PubMed  Google Scholar 

  11. A. B. Weitberg,Effects of combination of antioxidants on phagocyte-induced sister-chromatide exchanges. Mutat. Res.224, 1–4 (1989).

    PubMed  Google Scholar 

  12. M. Barak, S. Ulitzur and D. Merzbach,Phagocytosis-induced mutagenesis in bacteria. Mutat. Res.121, 7–16 (1983).

    PubMed  Google Scholar 

  13. S. A. Weitzman, A. B. Weitberg, M. Destrempes, S. A. Latt and T. P. Stossel,Stimulated human phagocytes produce cytogenetic changes in cultured mammalian cells. New Engl. J. Med.308, 26–30 (1983).

    PubMed  Google Scholar 

  14. S. A. Weitzman, A. B. Weitberg, E. P. Clark and T. P. Stossel,Phagocytes as carcinogens: Malignant transformation produced by human neutrophils. Science227, 1231–1233 (1985).

    PubMed  Google Scholar 

  15. S. J. Weiss, P. K. Rustagi and A. F. LoBuglio,Human granulocyte generation of hydroxyl radical. J. Exp. Med.147, 316–322 (1978).

    PubMed  Google Scholar 

  16. A. I. Tauber and B. M. Babior,Evidence for hydroxyl radical production by human neutrophils. J. Clin. Invest.60, 374–379 (1977).

    PubMed  Google Scholar 

  17. Y. Miyachi, A. Yoshioka, S. Imamura and Y. Niwa,Decreased hydroxyl radical generation from polymorphonuclear leucocytes in the presence of d-penicillamine and thiopronine. J. Clin. Lab. Immunol.22, 81–84 (1987).

    PubMed  Google Scholar 

  18. M. R. Green, H. A. O. Hill, J. Okolow-Zubkowska and A. V. Segal,The production of hydroxyl and superoxide radicals by stimulated human neutrophils — Measurements by EPR spectrometry. FEBS Lett.100, 23–26 (1979).

    PubMed  Google Scholar 

  19. B. E. Britigan, G. M. Rosen, Y. Chai and M. S. Cohen,Do human neutrophils make hydroxyl radicals?. J. Biol. Chem.261, 4426–4431 (1986).

    PubMed  Google Scholar 

  20. T. R. Green, J. H. Fellman and A. L. Eicher,Myeloperoxidase oxidation of sulfur-centered and benzoic acid hydroxyl radical scavengers. FEBS Lett.192, 33–36 (1985).

    PubMed  Google Scholar 

  21. R. A. Greenwald, S. W. Rush, S. A. Moak and Z. Weitz,Conversion of superoxide generated by polymorphonuclear leucocytes to hydroxyl radical: A direct spectrophotometric detection system based on degradation of deoxyribose. Free Rad. Biol. Med.6, 385–392 (1989).

    PubMed  Google Scholar 

  22. R. A. Floyd, R. Henderson, J. J. Watson and P. K. WongUse of salicylate with high pressure liquid chromatography and electrochemical detection (LCED) as a sensitive measure of hydroxyl radicals in adriamycine-treated rats. Free Rad. Biol. Med.2, 13–18 (1986).

    Google Scholar 

  23. S. Fujimoto, S. Ishimitsu, H. Kanazawa, T. Mizutani, A. Ohara and T. Hayakawa,Hydroxylation of phenylalanine by stimulated polymorphonuclear leucocytes — Attempts to detect hydroxyl radicals. Agric. Biol. Chem.51, 2851–2853 (1987).

    Google Scholar 

  24. H. Kaur, I. Fagerheim, M. Grootveld, A. Puppo and B. Halliwell,Aromatic hydroxylation of phenylalanine as an assay for hydroxyl radicals: Application to activated human neutrophils and to heme protein leghemoglobin. Anal. Biochem.172, 360–367 (1988).

    PubMed  Google Scholar 

  25. G. A. Higgs, J. A. Salmon, B. Henderson and J. R. Vane,Pharmacokinetics of aspirin and salicylate in relation to inhibition of arachidonate cyclooxygenase and antiinflammatory activity. Proc. Natl. Acad. Sci. USA84, 1417–1420 (1987).

    PubMed  Google Scholar 

  26. J. S. Bomalski, F. F. Hirata and M. A. Clark,Aspirin inhibits phospholipase C. Biochem. Biophys. Res. Commun.139, 115–121 (1986).

    PubMed  Google Scholar 

  27. B. J. deVries, W. B. van den Berg and L. B. A. de Putte,Salicylate-induced depletion of endogeneous inorganic sulfate; potential role in the suppression of sulfated glycosaminoglycan synthesis in murine articular cartilage. Arth Rheum.28, 922–929 (1985).

    Google Scholar 

  28. J. A. Rosier and C. H. Van Peteghem,Detection of trace amounts of 8-hydroxy-2′-deoxyguanosine in commercial 2′-deoxyguanosine by means of HPLC analysis and electrochemical analysis. J. Liquid Chrom.11, 1293–1298 (1988).

    Google Scholar 

  29. H. Kasai and S. Nishimura,Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucleic Acid Res.12, 2137–2145 (1984).

    PubMed  Google Scholar 

  30. A. I. Tauber, D. B. Brettler, E. A. Kennington and P. M. Blumberg,Reaction of neutrophil phorbol ester receptor occupancy and NADPH-oxidase activity. Blood60, 333–339 (1982).

    PubMed  Google Scholar 

  31. R. A. Floyd, J. J. Watson, P. K. Wong, D. H. Altmiller and R. C. Rickard,Hydroxyl radical free adduct of deoxyguanosine: Sensitive detection and mechanism of formation. Free Rad. Res. Comms.1, 163–172 (1986).

    Google Scholar 

  32. C. C. Winterbourn,Myeloperoxidase as an effective inhibitor of hydroxyl radical production: Implications for the oxidative reactions in neutrophils. J. Clin. Invest.78, 545–550 (1986).

    PubMed  Google Scholar 

  33. B. E. Britigan, D. J. Hassett, G. M. Rosen, D. R. Hamill and M. S. Cohen,Neutrophil degranulation inhibits potential hydroxyl-radical formation. Biochem. J.264, 447–455 (1989).

    PubMed  Google Scholar 

  34. S. Pou, M. S. Cohen, B. E. Britigan and G. M. Rosen,Spin trapping and human neutrophils: Limits for detection of hydroxyl radical. J. Biol. Chem.264, 12299–12302 (1989).

    PubMed  Google Scholar 

  35. B. Halliwell,Superoxide dependent formation of hydroxyl radicals in the presence of iron salts is a feasible source of hydroxyl radicals in vivo. FEBS Lett.205, 461–462 (1982).

    Google Scholar 

  36. J. O. Shaw, R. N. Pinckard, K. S. Ferrigini, L. M. McManus and D. J. Hanahan,Activation of human neutrophils with 1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet-activating factor). J. Immunol.127, 1250–1255 (1981).

    PubMed  Google Scholar 

  37. E. Jouvin-Marche, B. Poitevin and J. Benveniste,Platelet-activating factor (PAF-acether), an activator of neutrophil functions, Agents and Actions12, 716–720 (1982).

    PubMed  Google Scholar 

  38. J. C. Gay,Priming of neutrophil oxidative responses by platelet-activating factor. J. Lipid Mediat.2, S161-S175 (1990).

    PubMed  Google Scholar 

  39. G. F. Weber,The measurement of oxygen-derived free radicals and related substances in medicine. J. Clin. Chem. Biochem.28, 569–603 (1990).

    Google Scholar 

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  1. Departments of Occupational Medicine and Clinical Chemistry, Faculty of Health Sciences, S-581 85, Linköping, Sweden

    P. Leanderson & C. Tagesson

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  1. P. Leanderson
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  2. C. Tagesson
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Leanderson, P., Tagesson, C. Rapid and sensitive detection of hydroxyl radicals formed by activated neutrophils in the presence of chelated iron: Hydroxylation of deoxyguanosine to 8-hydroxydeoxyguanosine. Agents and Actions 36, 50–57 (1992). https://doi.org/10.1007/BF01991228

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  • DOI: https://doi.org/10.1007/BF01991228

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