![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Discipline of Nutrition, The University of Auckland, Auckland, New Zealand
* To whom correspondence should be addressed. E-mail: ferguson{at}auckland.ac.nz.
A range of cellular processes, external factors, and/or disease states can lead to the formation of reactive oxygen species (ROS)4 and/or reactive nitrogen species (RNS), and these themselves have ongoing (and usually detrimental) effects in humans (Fig. 1). For example, chronic inflammatory processes produce an excess of ROS as well as DNA-reactive aldehydes including trans-4-hydroxy-2-nonenal and malondialdehyde from lipid peroxidation (LPO) (1). There is evidence that individuals with chronic inflammatory conditions, including chronic pancreatitis, ulcerative colitis, and Crohn's disease, show elevated levels of oxidative damage including modified DNA bases in various organs, as compared with the normal population (2). They are also at increased risk of cancer. Therefore, dietary modifications that reduce the steady-state levels of these modified bases may be likely to protect against cancer.
|
|
In considering the ability of a given individual or population group to cope with oxidative stress, one obvious method is to measure levels of free radical scavengers in the plasma. Examples that have been commonly studied thus include all the plant-related chemicals identified above, at the indicated levels to be expected in plasma: ascorbic acid, 20–80 µM; vitamin E, 50 µM; carotenoids, 0.1–0.4 µM; plant polyphenols, 0.01–0.1 µM; and isothiocyanates, 0.1–1 µM. It is also possible to measure the ability of plasma to scavenge free radicals using, for example, Trolox equivalent antioxidant activity (TEAC) or oxygen radical absorbance activity (ORAC) assays (8). However, this gives only a part of the picture.
A considerable number of biomarker studies have assayed excretory products in the urine. For example, etheno-DNA adducts can be measured using an immuno-enriched HPLC fluorescence method with a sensitivity of 
5 fmol/ml (9). Similarly, 7-hydroxy-8-oxo-2'-deoxyguanosine (8-OxodG) (or the corresponding base 8-oxoGua) is a commonly formed modified DNA base product that can be detected in urine after oxidative stress, has been shown to be mutagenic in vivo, and can be measured with high sensitivity (10). Thus, measures of urinary levels of this modified base have been supported as a biomarker of the total extent of oxidative damage to DNA, and a reduction in urinary excretion rates of this molecule interpreted as revealing dietary protection (10). Such methods take advantage of the fact that there is cellular turnover of such molecules. However, reduced excretion of these markers could result either from reduced formation of oxidized DNA bases (likely to be beneficial) or from reduced repair of such damage (likely to be detrimental).
Although such urinary assays have the advantage of being noninvasive, more direct measures of DNA damage in other tissue types may be more informative. A significant number of human monitoring studies have estimated 8-oxodG in cells, especially white blood cells including leukocytes, lymphocytes, or mononuclear blood cells (WBC), using electron capture detection with chromatographic techniques (HPLC-EC), gas chromatography with mass spectrometry (GC-MS), antibody-based immunoassays, or enzymic detection by bacterial glycosylase and endonuclease enzymes (11). However, validation studies to compare results from different laboratories have revealed large variations in the level of 8-oxodG detected by these different assays, and the European Standardization Committee on Oxidative DNA Damage recommended that published studies containing data on 8-oxodG and 8-oxoGua in WBC be reassessed (11).
The single cell gel electrophoresis (or COMET) assay provides a measure of either single- or double-strand DNA breaks (or other damage) at the level of the single cell. Although peripheral blood lymphocytes may be easily accessed, buccal cells and/or cells harvested from urine or feces may be more informative and are increasingly being applied in biomarker studies (12). Oxidatively altered bases can be detected using an enzyme-modified version that includes a DNA digestion step using either DNA glycosylase or endonuclease enzymes (13,14). Oxidized purines, including 8-oxodG, can be detected after incubation with formamidopyrimidine DNA glycosylase (FPG), whereas oxidized pyrimidines are revealed by endonuclease III (ENDOIII). A further variant of the technique exposes WBC ex vivo to DNA-breaking agents such as hydrogen peroxide (H2O2) or bleomycin, as a measure of the way in which a dietary regimen has enhanced the innate ability of the cells to repair DNA damage (15). We ourselves have applied such methods in estimating the ability of selenium supplementation to reduce susceptibility to cancer in the New Zealand population (7).
The cytokinesis-blocked micronucleus assay provides a measure of chromosome breakage and/or aneuploidy (16). This method is increasingly being used as an endpoint in human biomonitoring studies and may be applied to a range of tissues. A series of validation studies have compared the reproducibility of the method in various laboratories and are currently validating its usefulness in prediction of cancer risk.
In summary, biomarker approaches appear useful in assessing antioxidant activity of dietary components and may provide information of relevance to cancer protection. There is a need for agreement in study design, including timing of interventions, washout periods, etc. New technologies are also applying genomic, transcriptomic, and proteomic approaches to the various available tissues. It is important to recognize, however, that even some of the original exponents of the significance of oxidative damage in humans are now questioning whether it is the antioxidant properties or other effects of the relevant molecules that may be leading to cancer protection (17). New study approaches may be necessary in the near future.
 |
FOOTNOTES |
|---|
2 This work was supported by grants from the Auckland Cancer Society. The contents are solely the responsibility of the authors.
3 Author disclosure: no relationships to disclose.
4 Abbreviations used: FPG, formamidopyrimidine DNA glycosylase; LPO, lipoid peroxidation; ORAC, oxygen radical absorbance activity; RNS, reactive nitrogen species; ROS, reactive oxygen species; TEAC, Trolox equivalent antioxidant activity.
|
LITERATURE CITED |
|---|
 TOP LITERATURE CITED |
|---|
1. Halliwell B. Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans. Free Radic Res. 1996;25:57–74.[Medline]
2. D'Odorico A, Bortolan S, Cardin R, D'Inca R, Martines D, Ferronato A, Sturniolo GC. Reduced plasma antioxidant concentrations and increased oxidative DNA damage in inflammatory bowel disease. Scand J Gastroenterol. 2001;36:1289–94.[Medline]
3. Ferguson LR. Role of plant polyphenols in genomic stability. Mutat Res. 2001;475:89–111.[Medline]
4. Zhang Y. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. Mutat Res. 2004;555:173–90.[Medline]
5. Philpott M, Gould KS, Markham KR, Lewthwaite SL, Ferguson LR. Enhanced coloration reveals high antioxidant potential in new sweetpotato cultivars. J Sci Food Agric. 2003;83:1076–82.
6. The Alpha-Tocopherol Beta-Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med. 1994;330:1029–35.
7. Karunasinghe N, Ryan J, Tuckey J, Masters J, Jamieson M, Clarke LC, Marshall JR, Ferguson LR. DNA stability and serum selenium levels in a high risk group for prostate cancer. Cancer Epidemiol Biomarkers Prev. 2004;13:391–7.
8. Prior RL, Wu X, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem. 2005;53:4290–302.[Medline]
9. Bartsch H, Nair J. Oxidative stress and lipid peroxidation-derived DNA-lesions in inflammation driven carcinogenesis. Cancer Detect Prev. 2004;28:385–91.[Medline]
10. Loft S, Vistisen K, Ewertz M, Tjonneland A, Overvad K, Poulsen HE. Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index. Carcinogenesis. 1992;13:2241–7.
11. ESCODD (European Standards Committee on Oxidative DNA Damage). Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories: an approach to consensus. Carcinogenesis. 2002;23:2129–33.
12. Szeto YT, Benzie IFF, Collins AR, Choi SW, Cheng CY, Yow CMN, Tse MMY. A buccal cell model comet assay: Development and evaluation for human biomonitoring and nutritional studies. Mutat Res. 2005;578:371–81.[Medline]
13. Collins AR, Duthie SJ, Dobson VL. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis. 1993;14:1733–5.
14. Collins AR. Assays for oxidative stress and antioxidant status: applications to research into the biological effectiveness of polyphenols. Am J Clin Nutr. 2005;81:261S–7S.
15. Panayiotidis M, Collins AR. Ex vivo assessment of lymphocyte antioxidant status using the comet assay. Free Radic Res. 1997;27:533–7.[Medline]
16. Bonassi S, Neri M, Lando C, Ceppi M, Lin YP, Chang WSP, Holland N, Kirsch-Volders M, Zeiger E, et al. Effect of smoking habit on the frequency of micronuclei in human lymphocytes: results from the Human MicroNucleus project. Mutat Res. 2003;543:155–66.[Medline]
17. Halliwell B, Rafter J, Jenner A. Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not? Am J Clin Nutr. 2005;81:268S–76S.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
