Advertisement

Bioassay of 2′-Deoxyguanosine/8-Hydroxy-2′-Deoxyguanosine by HPLC With Electrochemical/Photodiode Array Detection

  • Kelly S. Williamson
  • Kenneth Hensley
  • Quentin N. Pye
  • Scott Ferrell
  • Robert A. Floyd
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

Living organisms exposed to reactive oxygen species (ROS) on a continual basis will promote oxidative stress, thereby forming mutations in DNA and damage to cells. As an end result, it has been shown that modified forms of damaged DNA can lead to mutagenesis, carcinogenesis, degenerative diseases, cancer, diabetes, and aging (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). With this in mind, the DNA nucleoside 2′-deoxyguanosine (dG) undergoes a hydroxylation reaction at the C-8 position to yield a useful biomarker, 8-hydroxy-2′-deoxyguanosine (8-OH-dG) that has been employed to measure oxidative damage as illustrated in Fig. 1. Other disease/nondisease related events resulting in the formation of this oxidation product, 8-OH-dG, are reported by Möller et al. (5). There have been a wide variety of assays used in the detection of free radical-mediated DNA oxidation products such as: high performance liquid chromatography coupled with electrochemical and ultraviolet detection (HPLC-ECD and HPLC-UV) (2, 3, 4, 5, 6, 7, 8, 9,12,13,16, 17, 18, 19, 20, 21,24), liquid and gas chromatography-mass spectrometry (LC-MS and GC-MS) (4,10,12,22, 23, 24, 25, 26, 27), postlabeling techniques (32P-HPLC [5], 32P-TLC [thin-layer chromatography] [30, 31, 32, 33], or fluorescent probe-HPLC [34]), antibody assays (35, 36, 37), and lastly, HPLC using tandem mass spectrometry (HPLC-MS-MS) (1,23,25,26,28,29). R. A. Floyd, H. Kasai, and others (9,17,18,38,39) were some of the early pioneers for accurately measuring DNA oxidation adducts using HPLC in series with electrochemical, spectrophotometric, or fluoro-metric detection in order to determine the level of oxidative DNA damage in cells.
Fig. 1.

The hydroxylation reaction of dG to yield the 8-OH-dG oxidation product as discussed in the text.

Keywords

Methylene Blue Continual Basis Early Pioneer Picomole Level Respective Retention Time 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Podmore, I. D., Cooper, D., Evans, M. D., Wood, M., and Lunec, J. (2000) Simultaneous measurement of 8-oxo-2′-deoxyguanosine and 8-oxo-2′-deoxyadenosine by HPLC-MS/MS. Biochem. Biophys. Res. Commun. 277, 764–770.CrossRefGoogle Scholar
  2. 2.
    Halliwell, B. and Gutteridge, J. M. C. (1999) Free Radicals in Biology and Medicine, 3rd ed., Oxford University Press, Oxford, UK, pp. 388–393.Google Scholar
  3. 3.
    Beckman, K. B., Saljoughi, S., Mashiyama, S. T., and Ames, B. N. (2000) A simpler, more robust method for the analysis of 8-oxoguanine in DNA. Free Radic. Biol. Med. 29, 357–367.PubMedCrossRefGoogle Scholar
  4. 4.
    Douki, T. and Cadet, J. (1999) Modification of DNA bases by photosensitized one-electron oxidation. Int. J. Radiat. Biol. 75, 571–581.PubMedCrossRefGoogle Scholar
  5. 5.
    Möller, L., Hofer, T., and Zeisig, M. (1998) Methodological considerations and factors affecting 8-hydroxy-2′-deoxyguanosine analysis. Free Radic. Res. 29, 511–524.PubMedCrossRefGoogle Scholar
  6. 6.
    Loft, S., Deng, X. S., Tuo, J., Wellejus, A., Sorensen, M., and Poulsen, H. E. (1998) Experimental study of oxidative DNA damage. Free Radic. Res. 29, 525–539.PubMedCrossRefGoogle Scholar
  7. 7.
    Hofer, T. and Möller, L. (1998) Reduction of oxidation during the preparation of DNA and analysis of 8-hydroxy-2′-deoxyguanosine. Chem. Res. Toxicol. 11, 882–887.PubMedCrossRefGoogle Scholar
  8. 8.
    Möller, L. and Hofer, T. (1997) [32P]ATP mediates formation of 8-hydroxy-2′-deoxyguanosine from 2′-deoxyguanosine, a possible problem in the 32P-postlabeling assay. Carcinogenesis 18, 2415–2419.PubMedCrossRefGoogle Scholar
  9. 9.
    Maidt, M. L. and Floyd, R. A. (1996) in Free Radicals: A Practical Approach. (Punchard, N. A. and Kelly, F. J., eds.), Oxford University Press, London, 14, 201–209.Google Scholar
  10. 10.
    Douki, T., Delatour, T., Bianchini, F., and Cadet, J. (1996) Observation and prevention of an artefactual formation of oxidized DNA bases and nucleosides in the GC-EIMS method. Carcinogenesis 17, 347–353.PubMedCrossRefGoogle Scholar
  11. 11.
    Ames, B. N., Shigenaga, M. K., and Hagen, T. M. (1993) Oxidants, antioxidants, and degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90, 7915–7922.PubMedCrossRefGoogle Scholar
  12. 12.
    Ravanat, J.-L., Turesky, R. J., Gremaud, E., Trudel, L. J., and Stadler, R. H. (1995) Determination of 8-oxoguanine in DNA by gas chromatography-mass spectrometry and HPLC-electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography-mass spectrometry assay. Chem. Res. Toxicol. 8, 1039–1045.PubMedCrossRefGoogle Scholar
  13. 13.
    Haegele, A. D., Briggs, S. P., and Thompson, H. J. (1994) Antioxidant status and dietary lipid unsaturation modulate oxidative DNA damage. Free Radic. Biol. Med. 16, 111–115.PubMedCrossRefGoogle Scholar
  14. 14.
    Tesfamariam, B. (1994) Free radicals in diabetic endothelial cell dysfunction. Free Radic. Biol. Med. 16, 383–391.PubMedCrossRefGoogle Scholar
  15. 15.
    Hayakawa, M. and Kuzuya, F. (1990) Free radicals and diabetes mellitus. Nippon Ronen Igakkai Zasshi. 27, 149–154.PubMedGoogle Scholar
  16. 16.
    Ramon, O., Wong, H.-K., Joyeux, M., Riondel, J., Halimi, S., Ravanat, J.-L., et al. (2001) 2′-deoxyguanosine oxidation is associated with the decrease in the DNA-binding activity of the transcription factor SP1 in liver and kidney from diabetic and insulin-resistant rats. Free Radic. Biol. Med. 30, 107–118.PubMedCrossRefGoogle Scholar
  17. 17.
    Floyd, R. A., Watson, J. J., Wong, P. K., Altmiller, D. H., and Rickard, R. C. (1986) Hydroxyl free radical adduct of deoxyguanosine: sensitive detection and mechanisms of formation. Free Radic. Res. Commun. 1, 163–172.PubMedCrossRefGoogle Scholar
  18. 18.
    Kasai, H., Tanooka, H., and Nishimura, S. (1984) Formation of 8-hydroxyguanine residues in DNA by x-irradiation. Gann 75, 1037–1039.PubMedGoogle Scholar
  19. 19.
    Evans, M. D. (2000) Measurement of 8-oxo-2′-deoxyguanosine in cellular DNA by high performance liquid chromatography-electrochemical detection, in Measuring In Vivo Oxidative Damage: A Practical Approach. (Lunec, J. and Griffiths, H. R., eds.), Wiley, NY, pp. 53–61.Google Scholar
  20. 20.
    Helbock, H. J., Beckman, K. B., Shigenaga, M. K., Walter, P. B., Woodall, A. A., et al. (1998) DNA oxidation matters: The HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc. Natl. Acad. Sci. USA 95, 288–293.PubMedCrossRefGoogle Scholar
  21. 21.
    Shigenaga, M. K., Aboujaoude, E. N., Chen, Q., and Ames, B. N. (1994) Assays of oxidative DNA damage biomarkers of 8-oxo-2′-deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by high-performance liquid chromatography with electrochemical detection. Methods Enzymol. 234, 16–33.PubMedCrossRefGoogle Scholar
  22. 22.
    Halliwell, B. and Dizdaroglu, M. (1992) The measurement of oxidative damage to DNA by HPLC and GC/MS techniques. Free Radic. Res Commun. 16, 75–87.PubMedCrossRefGoogle Scholar
  23. 23.
    Serrano, J., Palmeria, C. M., Wallace, K. B., and Kuehl, D. W. (1996) Determination of 8-hydroxydeoxyguanosine in biological tissue by liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry. Rapid Commun. Mass Spectrom. 10, 1789–1791.PubMedCrossRefGoogle Scholar
  24. 24.
    Douki, T., Delatour, T., Paganon, F., and Cadet, J. (1996) Measurement of oxidative damage at pyrimidine bases in γ-irradiated DNA. Chem. Res. Toxicol. 9, 1145–1151.PubMedCrossRefGoogle Scholar
  25. 25.
    Ravanat, J.-L., Duretz, B., Guiller, A., Douki, T., and Cadet, J. (1998) Isotopic dilution high-performance liquid chromatography-electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in biological samples. J. Chromatogr. B. 715, 349–356.CrossRefGoogle Scholar
  26. 26.
    Renner, T., Fechner, T., and Scherer, G. (2000) Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2′-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadruple mass detection. J. Chromatogr. B. 738, 311–317.CrossRefGoogle Scholar
  27. 27.
    Dizdaroglu, M. (1994) Chemical determination of oxidative DNA damage by gas chromatography-mass spectrometry. Methods Enzymol. 234, 3–16.PubMedCrossRefGoogle Scholar
  28. 28.
    Poulsen, H. E., Weimann, A., and Loft, S., (1999) Methods to detect DNA damage by free radicals: Relation to exercise. Proc. Nutr. Soc. 58, 1007–1014.PubMedCrossRefGoogle Scholar
  29. 29.
    Kasai, H. (1997) Analysis of a form of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat. Res. 387, 147–163.PubMedCrossRefGoogle Scholar
  30. 30.
    Randerath, K., Reddy, M. V., and Gupta, R. C. (1981) 32P-Labelling test for DNA damage. Proc. Natl. Acad. Sci. USA 78, 6126–6129.PubMedCrossRefGoogle Scholar
  31. 31.
    Gupta, R. C., Reddy, M. V., and Randerath, K. (1982) 32P-Postlabelling analysis of nonradioactive aromatic carcinogen-DNA adducts. Carcinogenesis 3, 1081–1092.PubMedCrossRefGoogle Scholar
  32. 32.
    Beach, A. C. and Gupta, R. C. (1992) Human biomonitoring and the 32P-postlabelling assay. Carcinogenesis 13, 1053–1074.PubMedCrossRefGoogle Scholar
  33. 33.
    Devanaboyina, U. and Gupta, R. C. (1996) Sensitive detection of 8-hydroxy-2′-deoxyguanosine in DNA by 32P-postlabeling assay and the basal levels in rat tissue. Carcinogenesis 17, 917–924.PubMedCrossRefGoogle Scholar
  34. 34.
    Sharma, M., Box, H. C., and Paul, C. R. (1990) Detection and quantitation of 8-hydroxydeoxyguanosine 5′-monophosphate in X-irradiated calf thymus by fluorescent postlabeling. Biochem. Biophys. Res. Commun. 167, 419–424.PubMedCrossRefGoogle Scholar
  35. 35.
    Degan, P., Shigenaga, M. K., Park, E.-M., Alperin, P. E., and Ames, B. N. (1991) Immunoaffinity isolation of urinary 8-hydroxy-2′-deoxyguanosine and 8-hydroxy-guanine and quantitation of 8-hydroxy-2′-deoxyguanosine in DNA by polyclonal antibodies. Carcinogenesis 12, 865–871.PubMedCrossRefGoogle Scholar
  36. 36.
    Musaratt, J. and Wani, A. A. (1994) Quantitative immunoanalysis of promutagenic 8-hydroxy-2′-deoxyguanosine in oxidized DNA. Carcinogenesis 15, 2037–2043.CrossRefGoogle Scholar
  37. 37.
    Yarborough, A., Zhang, Y. J., Hus, T. M., and Santella, R. M. (1996) Immunoperoxidase detection of 8-hydroxydeoxyguanosine in aflatoxin B1 treated rat liver and human mucosal cells. Cancer Res. 56, 683–688.PubMedGoogle Scholar
  38. 38.
    Kasai, H. and Nishimura, S. (1986) Hydroxylation of guanine in nucleosides and DNA at the C-8 position by heated glucose and oxygen radical-forming agents. Environ. Health Perspect. 67, 111–116.PubMedCrossRefGoogle Scholar
  39. 39.
    Kasai, H., Hayami, H., Yamaizumi, Z., Saito, H., and Nishimura, S. (1984) Detection and identification of mutagens and carcinogens as their adducts with guanosine derivatives. Nucleic Acids Res. 12, 2127–2136.PubMedCrossRefGoogle Scholar
  40. 40.
    Schneider, J. E., Jr., Pye, Q. N., and Floyd, R. A. (1999) Qβ bacteriophage photoinactivated by methylene blue plus light involves inactivation of its genomic RNA. Photochem. Photobiol. 70, 902–909.PubMedCrossRefGoogle Scholar
  41. 41.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar

Copyright information

© Humana Press Inc.,Totowa, NJ 2003

Authors and Affiliations

  • Kelly S. Williamson
    • 1
  • Kenneth Hensley
    • 1
  • Quentin N. Pye
    • 1
  • Scott Ferrell
    • 1
  • Robert A. Floyd
    • 1
  1. 1.Free Radical Biology and Aging ProgramOklahoma Medical Research FoundationOklahoma City

Personalised recommendations