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Cancer Prevention Laboratory, Colorado State University, Fort Collins, CO 80523
* To whom correspondence should be addressed. E-mail: henry.thompson{at}colostate.edu.
Causality
Considerable indirect evidence exists indicating that oxidation of nucleic acids can play a causal role in the carcinogenic process (1). Because of its propensity for attack by reactive oxygen species, products of guanine oxidation have been the most extensively investigated, and the mutagenic potential of guanine oxidation products has been well characterized (2). Of particular interest is evidence that 8-hydroxy-2-deoxyguanosine (8-oxo-dG), the most prevalent promutagenic oxidation product of guanine, can give rise to G-to-T transversion mutations in key genes known to be involved in the development of cancer (3,4). Evidence has recently been published documenting the following: 1) the accumulation of 8-oxo-dG in some tissues of knockout mice lacking 2 of the DNA glycosylases that excise this adduct from genomic DNA; 2) a multifold increase in cancer rates in some of the tissues in which 8-oxo-dG accumulates; and 3) a high frequency of occurrence, within those cancers, of the predicted G-to-T transversion mutations that activate an oncogene (K-ras) associated with cancer development in those tissues (5,6). Collectively, these observations provide a strong basis for the hypothesis that the concentration of 8-oxo-dG in genomic DNA is a biomarker for cancer risk. The rationale underlying this hypothesis is that higher concentrations of this promutagenic lesion in cellular DNA favor higher rates of mutation and that higher rates of mutation over time increase the risk for cancer. Although the evidence is mixed, numerous reports indicate that lifestyle can modulate steady-state levels of 8-oxo-dG as well as other DNA oxidation products (7,8).
Measurement
There are important theoretical and practical issues to consider in the design of preclinical and clinical studies in which 8-oxo-dG is the endpoint of interest. Excellent reviews of these issues have recently been published (9,10). Of the many technical difficulties to be resolved, those related to the method of sample acquisition have received minimal attention but could influence results (11), and those dealing with analyte analysis continue to be the most difficult to resolve. The goals of analysis validation are summarized as follows: 1) to process tissue samples to obtain a snapshot in time of equilibrium concentrations of 8-oxo-dG; 2) to reproducibly prevent the adventitious oxidation of guanine; and 3) to establish methods for assay validation and calibration that can be uniformly adapted by laboratories assessing DNA oxidation products.
Assessment
Because of the measurement difficulties referenced above, only limited attention has been directed to biologically important questions related to cancer risk assessment. One such question relates to the identification, in an apparently healthy population, of the steady-state concentration of 8-oxo-dG that should be considered to reflect increased risk for cancer and/or other chronic diseases; that is, when should we be concerned about a healthy person's oxidative stress status (7)? A related question that has not yet been given much attention but that could substantively affect the approach to DNA oxidation-related risk assessment is whether chronic levels of oxidation or the response to an acute bout of oxidation, an event that may be as common as an exercise session at a health club, is more important relative to cancer risk? If the response to acute bouts of oxidative stress is determined to be important, some of the same methods used to measure DNA oxidation could be adapted to risk assessment, but they would be applied to determine DNA damage sensitivity and DNA damage repair capacity.
Moving forward
Although the appeal of measuring concentrations of 8-oxo-dG is strong, unanswered issues related to sample processing, lingering problems with adventitious oxidation of guanine, and the amount of sample required for analysis indicate that there is need for additional methodological work before widespread use of this analyte as a cancer risk biomarker can be considered. On the other hand, single-cell gel electrophoresis analyses of DNA damage that are generally referred to as the comet assay, although lacking in specificity, do offer the advantage of reduced levels of adventitious oxidation of DNA, the requirement for small amounts of sample, rapid sample processing, and the ability to measure both steady-state levels of DNA oxidation as well as DNA damage sensitivity and DNA damage repair capacity (12,13). Despite existing questions about comet analysis methodology and assay calibration and validation, the application of this approach for cancer risk assessment merits serious consideration.
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FOOTNOTES |
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2 Author disclosure: no relationships to disclose.
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LITERATURE CITED |
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 TOP LITERATURE CITED |
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1. Poulsen HE. Oxidative DNA modifications. Exp Toxicol Pathol. 2005;57: Suppl 1:161–9.[Medline]
2. Cadet J, Douki T, Frelon S, Sauvaigo S, Pouget JP, Ravanat JL. Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement. Free Radic Biol Med. 2002;33:441–9.[Medline]
3. Henderson PT, Delaney JC, Gu F, Tannenbaum SR, Essigmann JM. Oxidation of 7,8-dihydro-8-oxoguanine affords lesions that are potent sources of replication errors in vivo. Biochemistry. 2002;41:914–21.[Medline]
4. Hsu GW, Ober M, Carell T, Beese LS. Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. Nature. 2004;431:217–21.[Medline]
5. Xie Y, Yang H, Cunanan C, Okamoto K, Shibata D, Pan J, Barnes DE, Lindahl T, McIlhatton M, et al. Deficiencies in mouse Myh and Ogg1 result in tumor predisposition and G to T mutations in codon 12 of the K-ras oncogene in lung tumors. Cancer Res. 2004;64:3096–102.
6. Russo MT, De LG, Degan P, Parlanti E, Dogliotti E, Barnes DE, Lindahl T, Yang H, Miller JH, Bignami M. Accumulation of the oxidative base lesion 8-hydroxyguanine in DNA of tumor-prone mice defective in both the Myh and Ogg1 DNA glycosylases. Cancer Res. 2004;64:4411–4.
7. Thompson HJ, Heimendinger J, Gillette C, Sedlacek SM, Haegele A, O'Neill C, Wolfe P. In vivo investigation of changes in biomarkers of oxidative stress induced by plant food rich diets. J Agric Food Chem. 2005;53:6126–32.[Medline]
8. Halliwell B. Why and how should we measure oxidative DNA damage in nutritional studies? How far have we come? Am J Clin Nutr. 2000;72:1082–7.
9. Collins AR, Cadet J, Moller L, Poulsen HE, Vina J. Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells? Arch Biochem Biophys. 2004;423:57–65.[Medline]
10. Gedik CM, Collins A. Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study. FASEB J. 2005;19:82–4.
11. Hamilton ML, Guo Z, Fuller CD, Van Remmen H, Ward WF, Austad SN, Troyer DA, Thompson I, Richardson A. A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA. Nucleic Acids Res. 2001;29:2117–26.
12. Hininger I, Chollat-Namy A, Sauvaigo S, Osman M, Faure H, Cadet J, Favier A, Roussel AM. Assessment of DNA damage by comet assay on frozen total blood: method and evaluation in smokers and non-smokers. Mutat Res. 2004;558:75–80.[Medline]
13. Sauvaigo S, Petec-Calin C, Caillat S, Odin F, Cadet J. 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. 2002;303:107–9.[Medline]
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