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Supplement: Free Radicals: The Pros and Cons of Antioxidants

DNA Oxidation Products, Antioxidant Status, and Cancer Prevention¹

Colorado State University, Fort Collins, CO 80523

²To whom correspondence should be addressed. E-mail: henry.thompson{at}colostate.edu.

KEY WORDS: • antioxidant • biomarkers • cancer • DNA oxidation • 8-hydroxy-2-deoxyguanosine

	EXPANDED ABSTRACT

TOP EXPANDED ABSTRACT LITERATURE CITED

 
Oxygen is essential for life but can also harm cells. This is because active forms of oxygen such as hydroxyl (OH|$$^·) and superoxide (O₂|$$^·) radicals, hydrogen peroxide (H₂O₂), and singlet oxygen (¹O₂) arise as by-products and intermediates of aerobic metabolism and during oxidative stress (1). Directly or indirectly, these chemical species of oxygen, as well as reactive species of nitrogen, can transiently or permanently damage nucleic acids, lipids, and proteins. Oxidative damage to these cellular macromolecules is implicated in the genesis of several diseases, including cancer (2). Consequently, the measurement of antioxidant status (i.e., the balance between prooxidants and antioxidants) via the measurement of levels of oxidatively damaged molecules, antioxidant chemicals, and/or the capacity to inhibit oxidative damage (antioxidant capacity assays), is increasingly being considered for use as a surrogate biomarker for cancer risk (3). This presentation focuses on the potential for measures of DNA oxidation to serve as antioxidant-status biomarkers that reflect cancer risk and, by implication, cancer prevention.

DNA oxidation and cancer

Reactive species of oxygen or nitrogen induce all forms of DNA damage, including base modifications, base-free (apurinic and apyrimidinic) sites, strand breakage, and DNA-protein crosslinks, but the specific spectrum of products depends on the reactive species involved (4). It appears that the most prevalent product of DNA oxidation that is detected in genomic DNA in mammalian cells is 8-hydroxy-2-deoxyguanosine (8-OHdG) (4). 8-OHdG is a promutagenic base modification that is rapidly removed from DNA by base excision repair mechanisms, although some evidence of removal by transcription-coupled and replication-associated repair also is reported (5). The half-life of 8-OHdG in mammalian DNA is estimated to be 11 min (6). If DNA is replicated prior to repair of this base modification, 8-OHdG can cause G to T transversion mutations (7). This type of mutation is reported in genes whose dysfunction is involved in the genesis of cancer. An example of the linkage between G to T transversion mutations and cancer is their occurrence in codons of the p53 gene, diminishing its tumor-suppressing activity (8). Mutations in the p53 gene occur in >50% of human cancers. Although direct evidence of a cause-and-effect relation between DNA oxidation and carcinogenesis has not yet been reported, the indirect evidence referenced here provides a strong rationale for the evaluation of 8-OHdG as a candidate biomarker for assessing antioxidant status and cancer risk.

Selection of biomarkers for DNA oxidation

There are substantial questions regarding which tissues and/or body fluids should be selected for assessment of 8-OHdG and which analytical method should be used to measure levels of this modified nucleoside. These issues are reviewed by Halliwell (9). At this time, the best case can be made for measuring steady-state levels of 8-OHdG in lymphocytes isolated from peripheral blood. In order to avoid the artifactual formation of 8-OHdG by direct chemical analysis, DNA should be purified from isolated nuclei using sodium iodide to precipitate the DNA (10). Following enzymatic digestion of the purified DNA to nucleosides, HPLC separation using either electrochemical detection (HPLC-ECD) or detection via tandem mass spectrometry (HPLC-MS/MS) is recommended. When these procedures are used, the cellular background levels of 8-OHdG are observed to be 0.5 residues per million bases (2 8-OHdG per million dG) (4,10).

As an alterative to the HPLC-ECD or HPLC-MS/MS method of analysis, reliable estimates of oxidized purines or pyridimines can be obtained using a single cell gel electrophoresis assay in the presence or absence of a glycosylase (formamidopyrimidine glycosylase) or an endonuclease (endonuclease III), respectively (11). This method is referred to as the Comet assay. This indirect approach to assessing DNA oxidation has several advantages, including the requirement for a small amount of sample, a reduction in the time required for analysis, and the decreased potential for introduction of artifactual oxidation of DNA during sample preparation and analysis. However, drawbacks of the Comet assay include its lack of specificity, the difficulty in calibrating the assay relative to chemical equivalents of oxidative damage that are measured, and the considerable variability that can exist in assay results among laboratories. Moreover, levels of DNA oxidation are estimated to be lower in mammalian DNA when determined by the Comet assay versus HPLC-ECD, raising the question of whether the Comet assay underestimates levels of DNA oxidation due to incomplete digestion of DNA by the glycosylase or endonuclease. Nonetheless, the results of the Comet assay and HPLC-ECD are reported to be correlated (4).

Exceptional caution

It is important to note that exceptional caution must be exercised in interpreting reports in the literature regarding DNA oxidation, because cellular background levels of DNA oxidation products determined using a variety of methods vary by 3 orders of magnitude, primarily due to the artifactual formation of DNA oxidation products during analysis. Such differences clearly obscure the ability to detect physiologically and/or pathophysiologically relevant levels of 8-OHdG and their modulation by antioxidants.

Gaps in knowledge

Currently, there are many unanswered questions in this field of investigation. Several gaps in knowledge appear critical to address as work progresses on the antioxidant–cancer prevention hypothesis. They include defining the interrelations among markers of DNA oxidation measured in an individual’s urine, peripheral lymphocytes, and the target organ of interest; identification of the interrelations among markers of DNA, lipid, and protein oxidation measured in a particular tissue or body fluid and of DNA oxidation with measurements of antioxidant concentration and/or antioxidant capacity; elucidation of the effect of functional polymorphisms of genes involved in oxidant formation, antioxidant protection, and in the repair of DNA oxidation on markers of antioxidant status; determination of whether antioxidant treatment needs to be tailored to the genotype of the individual; and proof in principal that DNA oxidation causes specific mutations (gene and site specific), and that these mutations contribute to the occurrence of cancer.

	FOOTNOTES

 
¹ Presented as part of the conference "Free Radicals: The Pros and Cons of Antioxidants," held June 26–27 in Bethesda, MD. This conference was sponsored by the Division of Cancer Prevention (DCP) and the Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Department of Health and Human Services (DHHS); the National Center for Complementary and Alternative Medicine (NCCAM), NIH, DHHS; the Office of Dietary Supplements (ODS), NIH, DHHS; the American Society for Nutritional Science; and the American Institute for Cancer Research and supported by the DCP, NCCAM, and ODS. Guest editors for the supplement publication were Harold E. Seifried, National Cancer Institute, NIH; Barbara Sorkin, NCCAM, NIH; and Rebecca Costello, ODS, NIH.

	LITERATURE CITED

TOP EXPANDED ABSTRACT LITERATURE CITED

 

1. Halliwell, B. & Gutteridge, J.M.C. (1999) Free Radical in Biology and Medicine 1999 Oxford Science Publications New York, NY.

2. Institute, of, Medicine, National, Academy, of, Sciences, Food, Nutrition, Board, Panel, on, Dietary & Antioxidants, and, Related Compounds (2000) Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids 2000 National Academies Press Washington, DC.

3. Mayne, S. T. (2003) Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research. J. Nutr. 133:933S-940S.[Abstract/Free Full Text]

4. Cadet, J., Douki, T., Frelon, S., Sauvaigo, S., Pouget, J. P. & Ravanat, J. L. (2002) Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement. Free Radic. Biol. Med. 33:441-449.[Medline]

5. Hazra, T. K., Izumi, T., Kow, Y. W. & Mitra, S. (2003) The discovery of a new family of mammalian enzymes for repair of oxidatively damaged DNA, and its physiological implications. Carcinogenesis 24:155-157.[Abstract/Free Full Text]

6. Hamilton, M. L., Guo, Z., Fuller, C. D., Van Remmen, H., Ward, W. F., Austad, S. N., Troyer, D. A., Thompson, I. & Richardson, A. (2001) A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA. Nucl. Acids Res. 29:2117-2126.[Abstract/Free Full Text]

7. Henderson, P. T., Delaney, J. C., Gu, F., Tannenbaum, S. R. & Essigmann, J. M. (2002) Oxidation of 7,8-dihydro-8-oxoguanine affords lesions that are potent sources of replication errors in vivo. Biochemistry 41:914-921.[Medline]

8. Hussain, S. P., Raja, K., Amstad, P. A., Sawyer, M., Trudel, L. J., Wogan, G. N., Hofseth, L. J., Shields, P. G. & Billiar, T. R., et al (2000) Increased p53 mutation load in nontumorous human liver of Wilson disease and hemochromatosis: oxyradical overload diseases. Proc. Natl. Acad. Sci. U.S.A. 97:12770-12775.[Abstract/Free Full Text]

9. Halliwell, B. (2000) Why and how should we measure oxidative DNA damage in nutritional studies?. How far have we come? Am. J. Clin. Nutr. 72:1082-1087.

10. Ravanat, J. L., Douki, T., Duez, P., Gremaud, E., Herbert, K., Hofer, T., Lasserre, L., Saint-Pierre, C., Favier, A. & Cadet, J. (2002) Cellular background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up. Carcinogenesis 23:1911-1918.[Abstract/Free Full Text]

11. Sauvaigo, S., Petec-Calin, C., Caillat, S., Odin, F. & Cadet, J. (2002) Comet assay coupled to repair enzymes for the detection of oxidative damage to DNA induced by low doses of gamma-radiation: use of YOYO-1, low-background slides, and optimized electrophoresis conditions. Anal. Biochem. 303:107-109.[Medline]

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