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Effect of diet on cancer development: is oxidative DNA damage a biomarker?1,2
Free Radical Biology & Medicine, Vol. 32, No. 10, pp. 968 –974, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter
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Serial Review: Oxidative DNA Damage and Repair Guest Editor: Miral Dizdaroglu EFFECT OF DIET ON CANCER DEVELOPMENT: IS OXIDATIVE DNA DAMAGE A BIOMARKER? BARRY HALLIWELL Department of Biochemistry, National University of Singapore, Singapore (Received 11 December 2001; Accepted 21 February 2002)
Abstract—Free radicals and other reactive species are generated in vivo and many of them can cause oxidative damage to DNA. Although there are methodological uncertainties about accurate quantitation of oxidative DNA damage, the levels of such damage that escape immediate repair and persist in DNA appear to be in the range that could contribute significantly to mutation rates in vivo. The observation that diets rich in fruits and vegetables can decrease both oxidative DNA damage and cancer incidence is consistent with this. By contrast, agents increasing oxidative DNA damage usually increase risk of cancer development. Such agents include cigarette smoke, several other carcinogens, and chronic inflammation. Rheumatoid arthritis and diabetes are accompanied by increased oxidative DNA damage but the pattern of increased cancer risk seems unusual. Other uncertainties are the location of oxidative DNA damage within the genome and the variation in rate and level of oxidative damage between different body tissues. In well-nourished human volunteers, fruits and vegetables have been shown to decrease oxidative DNA damage in several studies, but data from short-term human intervention studies suggest that the protective agents are not vitamin C, vitamin E, -carotene, or flavonoids. © 2002 Elsevier Science Inc. Keywords—Oxidative DNA damage, Free radical, 8-Hydroxy-2⬘-deoxyguanosine, Mass spectrometry, Cancer, Diabetes
INTRODUCTION
oxyguanosine, 8OHdG [3], or use mass spectrometry to measure a wide range of DNA damage products [4]? What artifacts can arise in these various methods and how best can they be eliminated? DNA is easily oxidatively damaged during isolation, hydrolysis, and preparation for analysis (reviewed in [5]). At last however, due in part to the efforts of the European Standards Committee on Oxidative DNA Damage, ESCODD [6 –9], different groups in several countries are coming together to review these methodological questions and determine optimal protocols. My own laboratory has contributed to this research [5,10 –12]. However, the purpose of the present paper is not to revisit this issue, but to ask the second fundamental question, is oxidative DNA damage a valid biomarker of the late development of cancer?
It is widely believed that antioxidants help maintain human health by decreasing oxidative damage to key biomolecules. Thus biomarkers (some prefer the term “markers” [1]) of oxidative damage should be useful in establishing which antioxidants are important. Validation of biomarkers requires two different steps [1,2]. One is the analytical validation, including development of procedures, analysis of reference materials, and quality control. The second is the validation of the fact that changes in their level do reflect the later development of disease. These Serial Reviews in Free Radical Biology and Medicine contain many papers discussing what are the best methods to measure oxidative DNA damage. Should we rely on HPLC-based determination of 8-hydroxy-2⬘-de-
WHY SHOULD WE MEASURE OXIDATIVE DNA DAMAGE?
This article is part of a series of reviews on “Oxidative DNA Damage and Repair.” The full list of papers may be found on the homepage of the journal. Address correspondence to: Prof. Barry Halliwell, Department of Biochemistry, National University of Singapore, Singapore 119260, Singapore; Tel: (65) 6874-3247; Fax: (65) 6779-1453; E-Mail: [email protected].
The simple answer is that it is widely believed that oxidative DNA damage over the long human lifespan is a significant contributor to the age-related development 968
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of the major cancers, such as those of the colon, prostate, rectum, and breast [13–16]. If it is, it follows that diets or therapeutic agents that decreased oxidative DNA damage would delay or prevent the onset of cancer. No one to date has proved that oxidative DNA damage is a valid biomarker of subsequent cancer development. To do so would require measuring it in a range of healthy human subjects, subsequently followed over many years to see who develops cancer [17]. Epidemiologists have generally resisted including biomarkers in large human studies, although this view may be beginning to change. We must therefore turn to circumstantial evidence. Oxidative DNA damage is extensive Despite the various methodological problems that have arisen, the data available still collectively suggest that rates and levels of oxidative DNA damage in the human body are biologically significant. The commonest method of assessing “total body” oxidative DNA damage is to quantitate the urinary excretion of 8OHdG [15,18, 19]. Estimates based on 24 h excretion rates suggest an average of at least a few hundred “oxidative hits” per day on the DNA of each of the approximately 5 ⫻ 1013 cells in the human body [15,18 –20]. These values for urinary 8OHdG could be an underestimate of damage, in that they ignore other DNA base damage products that are excreted (see below). They are in any case only an average: some cells may suffer far more damage than others, but few data exist on this point. A second approach to assessing oxidative DNA damage has been to isolate DNA from tissues and measure its content of oxidative damage products. These values are presumably steady-state levels arising from a dynamic equilibrium between rates of oxidative DNA damage and rates of repair of that damage. Unfortunately, here is a major area of disagreement. For example, values for levels of 8-hydroxylated guanine in cellular DNA in some studies are less than 0.1/105 guanines and in others up to 100/105 guanines [3,4,15,21,22]. Again there is a multiplicity of other oxidative DNA base damage products [2] in cellular DNA, some of which may be present in greater amounts than 8OHdG [5]. Unfortunately, measurement techniques for these other base damage products have not yet been subject to the methodological comparisons that are underway [6,7–9] for 8-hydroxyguanine (8OHG) and 8OHdG. For example, there is disagreement over how much 8-hydroxyadenine may be present in DNA [2,23]. There could also be problems with urinary 8OHdG measurements, although they have the advantage that there is no DNA in urine to artifactually oxidize. Methodological problems can occur [23–26], e.g., because urine is a complex fluid containing many compounds that
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could co-elute with 8OHdG on HPLC analysis. The amount of 8OHdG in urine seems to be unaffected by diet and 8OHdG is thought not to be metabolized in humans [18,19,27]. Some (possibly all) of the 8OHdG excreted in human urine arises not from DNA, but from oxidation of deoxyguanosine triphosphate (dGTP) or diphosphate (dGDP) in the DNA precursor pool [19,28]. If so, it follows that 8OHdG excretion rates are not a quantitative index of oxidative damage to guanine residues in DNA. The measurement of other products in urine might give additional information. For example, in patients treated with adriamycin, 8OHdG excretion did not change, but there was a significant rise in 5-(hydroxymethyl) uracil content of urine [29]. Several DNA base damage products have been identified in human urine [23,29 –31], although it will be necessary to rule out a confounding effect of diet (absorption and re-excretion of oxidized DNA bases present in foods, e.g., generated by oxidative DNA damage during cooking) before these other products can be used as biomarkers of “whole body” oxidative DNA damage. Several lesions generated by oxidative DNA damage are mutagenic For the sake of argument, let me suggest that the urinary excretion of 8OHdG does represent an approximate biomarker of whole body oxidative DNA damage, and that the sum of all base damage products in DNA is at a steady-state level of at least one modified base per 105 unmolested purines and pyrimidines. These levels of whole body and steady-state oxidative DNA damage are consistent with a mutagenic load theoretically sufficient to promote cancer development. The mutagenicity of 8OHdG in causing GC 3 TA transversions is well established [3]. Several other DNA base oxidation products are mutagenic [32–34], including 2-hydroxyadenine, 5-hydroxycytosine, formyluracil, and 5-hydroxyuracil. In addition, the mutagenicity and other biological consequences of many of the other base oxidation products found in cellular DNA [4] have not been studied in detail and could be significant. It is instructive to make a comparison with levels of DNA adducts of known carcinogens. The concentrations of, for example, benzpyrene-DNA adducts in DNA from malignant tumors taken from smokers have been shown to be 0.65–5.33/106 DNA bases [35,36]. In rat liver, the calculated carcinogen-DNA adduct concentration associated with a 50% incidence of liver cancer ranged from 0.53 to 20.83 adducts/106 nucleotides for a range of carcinogens including aflatoxin and dimethylnitrosamine [37]. In mouse liver the respective figures were in the range of 8.12–55.43 adducts/106 nucleotides for ethylene
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oxide, dimethylnitrosamine, 4 aminobiphenyl, and 2-acetylaminofluorene [37]. If we assume that total DNA base oxidation products are at a level of at least 1 per 105 bases and are on average only one tenth as mutagenic as the above chemical carcinogens, it still seems that the levels that can be measured in cellular DNA represent a threat to the cell, given that the above levels are associated with a 50% cancer incidence. Agents that decrease oxidative DNA damage have an anti-cancer effect Diets rich in fruits and vegetables are associated with decreased incidence of the major human cancers. Multiple mechanisms can account for this, but several studies have shown that administration of fruits and vegetables to healthy human volunteers decreases levels of oxidative DNA damage as assessed using several different methods [38 – 42]. Administration of vitamin E, coenzyme Q10, vitamin C, -carotene or flavonoids could not decrease oxidative DNA damage, at least in the subjects examined, who appeared well nourished, with an intake of vitamins E and C equal to or exceeding current RDA values [5,17,43–51]. Indeed, the lack of effect of -carotene and antioxidant vitamins in decreasing oxidative DNA damage in subjects in advanced countries may help explain why intervention trials with these compounds as anti-cancer agent have given disappointing results [17, 52]. Agents that increase oxidative DNA damage promote cancer development Cigarette smoking is well known to be associated with increased risk of cancer development, and several studies report that it also raises levels of 8OHdG in urine and in cellular DNA [53–55], although conflicting data exist (discussed in [56]). The increased DNA damage may induce repair systems, which could explain the few studies in which rises in steady-state damage levels have not been detected [56]. The rise in 8OHdG in human lung appears to precede cancer development [55]. Many other carcinogens (and possibly all carcinogens) raise levels of oxidative damage prior to cancer development (reviewed in [13,16]). They include not only carcinogens that appear to act primarily by inducing oxidative stress (such as the peroxisome proliferator clofibrate [57,58]) but also more “classical” carcinogens such as dimethylbenzanthracene [59], nickel [60], benzpyrene [61], and asbestos [62]. High-fat diets appear to promote development of certain cancers, e.g., colon and breast, and studies suggest that such diets also increase 8OHdG formation [63– 66]. Chronic inflammation of several tissues, such as liver
and colon, increases cancer risk and also raises levels of oxidative DNA damage [10,67– 69]. In studies of a transgenic mouse model of hepatitis [70], formation of 8OHdG preceded cancer development. In another such model, vitamin E protected against DNA damage and decreased subsequent hepatic tumor formation [71], although in this study oxidative DNA damage was not directly measured. In mice exposed to diesel soot, levels of 80HdG in the lung rose well before tumor development [72]. Knockout mice lacking the MTHI gene encoding 8-oxo-dGTPase showed increased formation of tumors in lung, liver, and stomach at 18 months of life [73]. Increased oxidative DNA damage has also been detected in human benign prostate hyperplasia, although the association of this condition with subsequent cancer development is uncertain [74]. Hence, collectively the evidence, although obviously incomplete, is consistent with the view that increased oxidative DNA damage contributes to subsequent cancer development, whereas plant-rich diets decrease oxidative DNA damage and delay or prevent cancer development. Is this circumstantial evidence strong enough to prove beyond a reasonable doubt that oxidative DNA damage is a biomarker of subsequent cancer development? THE CASE AGAINST
Elevated oxidative DNA damage may not always be associated with increased cancer development There may be cases in which oxidative DNA damage levels are elevated, but cancer development does not ensue. In patients with rheumatoid arthritis (RA), levels of 8OHdG in blood cells [75] are elevated, as are other parameters of oxidative damage [76], yet the data on increased risk of malignancy are conflicting [77]. There appears to be moderately increased risk of hematopoietic, prostate, and lung cancers, but decreased risk for colorectal and gastric cancer [77]. Of course, the frequent use of cyclooxygenase-I and -II inhibitors in the therapy of RA could affect cancer development, given that this enzyme plays a role in tumor growth in some cancer types, such as colon cancer. In patients with type 2 diabetes, elevated levels of 8OHdG [78,79] and other DNA base oxidation products [79] in blood cells, as well as increased urinary excretion of 8OHdG [80], have been reported. Diabetes is associated with increased risk of pancreatic cancer [81], but no clear evidence for a generalized increased cancer risk in diabetics has emerged [81– 84], although such an increased risk cannot be ruled out on the data presently available. It may be that oxidative DNA base damage alone is insufficient to cause cancer development or perhaps damage over only a certain range is effective, excessive
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damage having an anti-cancer effect by promoting apoptosis. Reactive oxygen species could also promote cancer development by accelerating cell proliferation, although higher levels would have the opposite effect, halting proliferation [85]. We may not always be examining DNA from the correct tissue An important factor in explaining the different types of cancer observed in RA, diabetes, and cigarette smokers may be the relative extent of oxidative DNA damage in target tissues. A major problem in studying oxidative DNA damage is the limited availability of human tissues from which to obtain DNA. Most studies are performed on DNA isolated from blood lymphocytes (or sometimes total white cells). Most groups prefer to isolate lymphocytes, since total white cell DNA will include a significant contribution from neutrophils. However, there is no obvious reason why levels of oxidative DNA damage in either cell type should reflect levels in other body tissues (e.g., the pancreas in diabetes). Sperm, buccal cells, placenta, and biopsies of muscle, stomach, skin, colon, and other tissues are other potential sources of DNA, although biopsy samples often yield too little DNA for HPLC- or MS-based methods and we urgently need to develop micro-methods for DNA isolation and analysis. The comet assay may be useful, but has not been validated against rigorous chemical methods. Are we justified in assuming that changes in the amount of oxidative DNA damage in lymphocytes caused, for example, by antioxidant supplementation, are reflected in the tissues in which cancer is most likely to develop later in life (e.g., breast, lung, prostate, rectum, and colon)? More data are needed to answer such questions. Nevertheless, it is encouraging to note that in cigarette smokers and diabetic and RA patients, increased DNA damage can be detected by current methodology in blood cells and urine. Increased 80HdG levels were also observed in leukocytes from subjects with hepatic inflammation caused by hepatitis C virus [86]. Studies on dogs showed that an exercise regime decreased levels of 8OHdG in blood lymphocytes and colon, but not in other tissues [87]. In rats fed high-fat diets, oxidative DNA damage increased in both blood cells and mammary gland [63]. However, studies on rats are consistent with the existence of different steady-state levels of 8OHdG in different tissues [88,89]. Other mutagenic lesions generated by oxidative stress may be important The present article has focused on products of direct oxidative damage to the DNA bases. However,
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the contribution made by the reaction of end-products of lipid peroxidation with the DNA bases to create mutagenic lesions [90 –93] and the mutagenic effects of products generated by attack of reactive species on deoxyribose residues in DNA [94] may be worthy of consideration. Currently available data do not allow us to assess the relative importance of these and other lesions (e.g., DNA-protein cross-links) in promoting cell transformation. Repair rates must be considered Rises in 8OHdG or other DNA base oxidation products are not necessarily due to increased rates of oxidative DNA damage: decreased repair rate is also a possibility. This makes it necessary to be cautious in interpreting urinary excretion rates. For example, an agent that increases 8OHdG excretion might be interpreted as “bad” (seemingly increasing DNA damage) but might in fact be “good” (if it stimulated repair and therefore decreased steady state 8OHdG concentrations in DNA). It has been suggested, for example, that ascorbate may stimulate DNA repair in vivo [95]. The ideal would be to measure both steady state DNA damage and total damage (e.g., by urinary excretion rates). If only one set of measurements can be made, the steady state measurement is arguably preferable, because miscoding induced by oxidized bases is presumably what determines the risk of mutation and in turn the risk of cancer development. The repair process itself is not error free, however, and can introduce mutations, so it could be argued that a greater “throughput” of DNA base oxidation is deleterious even if it does not result in significant rises in the steady state concentrations of DNA base damage products. The genomic location of oxidative DNA damage is unknown The mutagenicity of 8OHdG, and probably that of other lesions, is sequence-specific to some extent, so that the total number of 8OHdG residues is not necessarily proportional to mutation frequency [3,96]. Oxidative damage in “junk DNA” may have no biological consequences, only that fraction of total damage that occurs in genes encoding functional proteins, (such as p53), or in telomeres [97] presumably being important. As yet, we have no clear information on distribution of oxidative DNA damage in the genome, although techniques to investigate this are under development [98]. CONCLUSION
There is no direct and compelling evidence that oxidative DNA damage is a biomarker of subsequent cancer
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development, because studies to address this point have not been done. However, there is considerable circumstantial evidence, but not proof beyond reasonable doubt to support the view that it is a valid biomarker. It follows that agents that decrease the amount of oxidative DNA damage should decrease the risk of subsequent cancer development [17]. We need to study predictive values of oxidative damage levels in healthy human populations. Correlations between intervention-induced changes in levels of oxidative DNA damage and effects of such intervention on subsequent cancer development also need to be studied. It may well be that levels of oxidative DNA damage show only a limited response to diet and are more affected by genetics [99 –101]. Acknowledgement — I am grateful to the Academic Research Fund of The National University of Singapore (grant R-183-000-027-112) for research support.
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Diet and oxidative DNA damage [34] Fujikawa, K.; Kamiya, H.; Kasai, H. The mutations induced by oxidatively damaged nucleotides, 5-formyl-dUTP and 5-hydroxy-dCTP, in Escherichia coli. Nucleic Acids Res. 26:4582– 4587; 1998. [35] Bartsch, H. DNA adducts in human carcinogenesis: etiological relevance and structure-activity relationship. Mutat. Res. 340: 67– 69; 1996. [36] Poirier, M. C.; Weston, A. Human DNA adduct measurements: state of the art. Environ. Health Perspect. 104:883– 893; 1996. [37] Ottender, M.; Lutz, W. K. Correlation of DNA adduct levels with tumor incidence: carcinogenic potency of DNA adducts. Mutat. Res. 424:237–247; 1999. [38] Rehman, A.; Bourne, L. C.; Halliwell, B.; Rice-Evans, C. Tomato consumption modulates oxidative DNA damage in humans. Biochem. Biophys. Res. Commun. 262:828 – 831; 1999. [39] Pool-Zobel, B. L.; Bub, A.; Muller, H.; Wollowski, I.; Rechkemmer, G. Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods. Carcinogenesis 18:1847–1850; 1997. [40] Verhagen, V.; Poulsen, H. E.; Loft, S.; Van Poppel, G.; Willems, M. I.; Van Bladeren, P. J. Reduction of oxidative damage in humans by Brussels sprouts. Carcinogenesis 16:969 –970; 1995. [41] Smith, N. J.; Inserra, P. F.; Watson, R. R.; Wise, J. A.; O’Neill, K. L. Supplementation with fruit and vegetable extracts may decrease DNA damage in the peripheral lymphocytes of an elderly population. Nutr. Res. 1507–1518; 1999. [42] Thompson, H. J.; Heimendinger, J.; Haegele, A.; Sedlacek, S. M.; Gillette, C.; O’Neill, C.; Wolfe, P.; Conry, C. Effect of increased vegetable and fruit consumption on markers of oxidative cellular damage. Carcinogenesis 20:2261–2266; 1999. [43] Von Poppel, G.; Poulsen, H. E.; Loft, S.; Verhagen, V. No influence of beta carotene on oxidative DNA damage in male smokers. J. Natl. Cancer Inst. 37:310 –311; 1995. [44] Beatty, E. R.; England, T. G.; Geissler, C. A.; Aruoma, O. I.; Halliwell, B. Effect of antioxidant vitamin supplementation on markers of DNA damage and plasma antioxidants. Proc. Nutr. Soc. 58:44A; 1999. [45] Prieme, H.; Loft, S.; Nyyssonen, K.; Salonen, J. T.; Poulsen, H. E. No effect of supplementation with vitamin E, ascorbic acid, or coenzyme Q10 on oxidative DNA damage estimated by 8-oxo-7, 8-dihydro-2⬘-deoxyguanosine excretion in smokers. Am. J. Clin. Nutr. 65:503–507; 1997. [46] England, T. G.; Beatty, E.; Rehman, A.; Nourooz-Zadeh, J.; Pereira, P.; O’Reilly, J.; Wiseman, H.; Geissler, C.; Halliwell, B. The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects. Free Radic. Res. 33:115–127; 2000. [47] Podmore, I. D.; Griffiths, H. R.; Herbert, T. E.; Mistry, N.; Mistry, P.; Lunec, J. Vitamin C exhibits prooxidant properties. Nature 392:559; 1998. [48] McCall, M. R.; Frei, B. Can antioxidant vitamins materially reduce oxidative damage in humans? Free Radic. Biol. Med. 26:1034 –1053; 1999. [49] Huang, H. Y.; Helzlsouer, K. J.; Appel, L. J. The effects of vitamin C and vitamin E on oxidative DNA damage: results from a randomised controlled trial. Cancer Epidemiol. Biomarkers Prev. 9:647– 652; 2000. [50] Sarataho, E. P.; Salonen, J. T.; Nyyssonen, K.; Kaikonen, J.; Salonen, R.; Ristonmaa, U.; Diczfalusy, U.; Brigelius-Flohe, R.; Loft, S.; Poulsen, H. E. Long-term effects of vitamin E, vitamin C, and combined supplementation on urinary 7-Hydro-8-oxo-2⬘deoxyguanosine, serum cholesterol oxidation products, and oxidation resistance of lipids in nondepleted men. Arterioscler. Thromb. Vasc. Biol. 20:2087–2093; 2000. [51] Collins, A. R.; Gedik, C. M.; Olmedilla, B.; Southon, S.; Bellizzi, M. Oxidative DNA damage measured in human lymphocytes: large differences between sexes and between countries, and correlations with heart disease mortality rates. FASEB J. 12:1397–1400; 1998. [52] Rowe, P. M. Beta-carotene takes a collective beating. Lancet 347:249; 1996.
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[53] Van Zeeland, A. A.; De Groot, A. J.; Hall, J.; Donato, F. 8-Hydroxydeoxyguanosine in DNA from leukocytes of healthy adults: relationship with cigarette smoking, environmental tobacco smoke, alcohol and coffee consumption. Mutat. Res. 439: 249 –257; 1999. [54] Kiyosawa, H.; Suko, M.; Okudaira, H.; Murata, K.; Miyamoto, T.; Chung, M. H.; Kasai, H.; Nishimura, S. Cigarette smoking induces formation of 8-hydroxydeoxyguanosine, one of the oxidative DNA damages, in human peripheral leukocytes. Free Radic. Res. Commun. 11:23–27; 1990. [55] Asami, S.; Manabe, H.; Miyake, J.; Tsurudome, Y.; Hirano, T.; Yamaguchi, R.; Itoh, H.; Kasai, K. Cigarette smoking induces an increase in oxidative DNA damage, 8-hydroxydeoxyguanosine, in a central site of the human lung. Carcinogenesis 18:1763– 1766; 1997. [56] Nia, A. B.; Van Schooten, F. J.; Schilderman, P. A.; Dekok, T. M.; Haenen, G. R.; Van Herwijnen, M. H.; Van Agen, E.; Pachen, D.; Kleinjans, J. C. A multi-biomarker approach to study the effects of smoking on oxidative DNA damage and repair and antioxidative defense mechanisms. Carcinogenesis 21:395– 401; 2001. [57] Kasai, H.; Okada, Y.; Nishimura, S.; Rao, M. S.; Reddy, J. K. Formation of 8-hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator. Cancer Res. 49:2603–2605; 1989. [58] Qu, B.; Li, Q. T.; Wong, K. P.; Halliwell, B. Mitochondrial damage by the “pro-oxidant” peroxisome proliferator clofibrate. Free Radic. Biol. Med. 27:1095–1102; 1999. [59] Fischer, W. H.; Lutz, W. K. Correlation of individual papilloma latency time with DNA adducts, 8-hydroxy-2⬘-deoxyguanosine, and the rate of DNA synthesis in the epidermis of mice treated with 7,12-dimethylbenz[a]anthracene. Proc. Natl. Acad. Sci. USA 92:5900 –5904; 1995. [60] Kasprzak, K. S.; Diwan, B. A.; Rice, J. M.; Misra, M.; Riggs, C. W.; Olinski, R.; Dizadaroglu, M. Nickel(II)-mediated oxidative DNA base damage in renal and hepatic chromatin of pregnant rats and their fetuses. Possible relevance to carcinogenesis. Chem. Res. Toxicol. 5:809 – 815; 1992. [61] Lee, B. M.; Jang, J. J.; Kim, H. S. Benzo[a]pyrene diol-epoxideI-DNA and oxidative DNA adducts associated with gastric adenocarcinoma. Cancer Lett. 125:61– 68; 1998. [62] Marczynski, B.; Kraus, T.; Rozynek, P.; Raithel, H. J.; Baur, H. Association between 8-hydroxy-2⬘-deoxyguanosine levels in DNA of workers highly exposed to asbestos and their clinical data, occupational and non-occupational confounding factors, and cancer. Mutat. Res. 468:203–212; 2000. [63] Djuric, Z.; Lewis, S. M.; Lu, M. H.; Mayhugh, M.; Tang, N.; Hart, R. W. Effect of varying dietary fat levels on rat growth and oxidative DNA damage. Nutr. Cancer 39 214 –219; 2001. [64] Leighton, F.; Cuevas, A.; Guasch, V.; Perez, D. D.; Strobel, P.; San Martin, A.; Urzua, U.; Diez, M. S.; Foncea, R.; Castillo, O.; Mizon, C.; Espinoza, M. A.; Urquiaga, I.; Rozowski, J.; Maiz, A.; Germain, A. Plasma polyphenols and antioxidants, oxidative DNA damage and endothelial function in a diet and wine intervention study in humans. Drugs Exp. Clin. Res. 25:133–141; 1999. [65] Ichinose, T.; Yajima, Y.; Nagashima, M.; Takenoshita, S.; Nagamachi, Y.; Sagai, M. Lung carcinogenesis and formation of 8hydroxy-deoxyguanosine in mice by diesel exhaust particles. Carcinogenesis 18:185– 192; 1997. [66] Loft, S.; Thorling, E. B.; Poulsen, H. E. High-fat-diet-induced oxidative DNA damage estimated by 8-oxo-7,8-dihydro-2⬘-deoxyguanosine excretion in rats. Free Radic. Res. 29:595– 600; 1998. [67] Ohshima, H.; Bartsch, H. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat. Res. 305:253–264; 1994. [68] Shimoda, R.; Nagashima, M.; Sakamoto, M.; Yamaguchi, N.; Hirohashi, S.; Yokota, J.; Kasai, H. Increased formation of oxidative DNA damage, 8-hydroxydeoxyguanosine, in human livers with chronic hepatitis. Cancer Res. 54:3171–3172; 1994.
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[69] Tardieu, D.; Jaeg, J. P.; Deloly, A.; Corpet, D. E.; Cadet, J.; Petit, C. R. The COX-2 inhibitor nimesulide suppresses superoxide and 8-hydroxy-deoxyguanosine formation, and stimulates apoptosis in mucosa during early colonic inflammation in rats. Carcinogenesis 21:973–976; 2000. [70] Hagen, T. M.; Huang, S.; Curnutte, J.; Fowler, P.; Martinez, V.; Wehr, C. M.; Ames, B. N.; Chisari, F. V. Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 91:12808 –12812; 1994. [71] Factor, V. M.; Laskowska, D.; Jensen, M. R.; Woitach, J. T.; Popescu, N. C.; Thorgeirsson, S. S. Vitamin E reduces chromosomal damage and inhibits hepatic tumour formation in a transgenic mouse model Proc. Natl. Acad. Sci. USA 97:2196 –2201; 2000. [72] Iwai, K.; Adachi, S.; Takahashi, M.; Moller, L.; Udagawa, T.; Mizuno, S.; Sugawa, I. Early oxidative DNA damages and late development of lung cancer in diesel exhaust-exposed rats. Environ. Res. 84:255–264; 2000. [73] Tsuzuki, T.; Egashira, A.; Igarashi, H.; Iwakuma, T.; Nakatsuru, Y.; Taminaga, Y.; Kawate, H.; Nakao, K.; Nakamvia, H.; Ide, F.; Kura, S.; Nakabeppu, Y.; Katsuki, M.; Ishikawa, T.; Sekiguchi, M. Spontaneous tumorigenesis in mice defective in the in MTH1 gene encoding 8-oxo-dGTPase. Proc. Natl. Acad. Sci. USA 98:11456 –11461; 2001. [74] Olinski, R.; Zastawny, T. H.; Foksinski, M.; Barecki, A.; Dizdaroglu, M. DNA base modifications and antioxidant enzyme activities in human benign prostatic hyperplasia. Free Radic. Biol. Med. 18:807– 813; 1995. [75] Bashir, S.; Harris, G.; Denman, M. A.; Blake, D. R.; Winyard, P. G. Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases. Ann. Rheum. Dis. 52:659 – 666; 1993. [76] Halliwell, B. Oxygen radicals, nitric oxide and human inflammatory joint disease. Ann. Rheum. Dis. 54:505–510; 1995. [77] Thomas, E.; Brewster, D. H.; Black, R. J.; Macfarlane, G. J. Risk of malignancy among patients with rheumatic conditions. Int. J. Cancer 88:497–502; 2000. [78] Dandona, P.; Thusu, K.; Cook, S.; Snyder, B.; Makowski, J.; Armstrong, D. Oxidative damage to DNA in diabetes mellitus. Lancet 347:444 – 445; 1996. [79] Rehman, A.; Nourooz-Zadeh, J.; Moller, W.; Nicotera, T.; Tritschler, H.; Pereira, P.; Halliwell, B. Increased oxidative damage to all DNA bases in patients with type II diabetes mellitus. FEBS Lett. 448:120 –122; 1999. [80] Leinonen, J.; Lehtimaki, T.; Toyokuni, S.; Okada, K.; Tanaka, T.; Hia, H.; Ochi, H.; Laippala, P.; Rantalaiho, V.; Wirta, O.; Pasternack, A.; Alho, H. New biomarker evidence of oxidative DNA damage in patients with non-insulin-dependent diabetes mellitus. FEBS Lett. 417:150 –152; 1997. [81] De Meo, M. T. Pancreatic cancer and sugar diabetes. Nutr. Rev. 59:112–115; 2001. [82] Shoff, S. M.; Newcomb, P. A. Diabetes, body size, and risk of endometrial cancer. Am. J. Epidemiol. 148:234 –240; 1998. [83] Will, J. C.; Galuska, D. A.; Vinicor, F.; Calle, E. E. Colorectal cancer: another complication of diabetes mellitus? Am. J. Epidemiol. 147:816 – 825; 1998. [84] Mori, M.; Nishida, T.; Sugiyama, T.; Komai, K.; Yakushiji, M.; Fukuda, K.; Tanaka, T.; Yokoyama, M.; Sugimori, H. Anthropometric and other risk factors for ovarian cancer in a casecontrol study. Jpn. J. Cancer Res. 89:246 –253; 1998.
[85] Burdon, R. H. Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic. Biol. Med. 18:775– 794; 1995. [86] Cardin, R.; Saccoccio, G.; Masulti, F.; Bellentani, S.; Farinati, F.; Tiribelli, C. DNA oxidative damage in leukocytes correlates with the severity of HCV-related liver disease: validation in an open population study. J. Hepatol. 34:587–592; 2001. [87] Okamura, K.; Doi, T.; Sakurai, M.; Hamada, K.; Yoshioka, Y.; Sumida, S.; Sugawa-Ketoyama, Y. Effect of endurance exercise on the tissue 8-hydroxy-deoxyguanosine content in dogs. Free Radic. Res. 26:523–528; 1997. [88] Nakae, D.; Akai, H.; Kishida, H.; Kusuoka, O.; Tsutsumi, M.; Konishi, Y. Age and organ dependent spontaneous generation of nuclear 8-hydroxydeoxyguanosine in male Fischer 344 rats. Lab. Invest. 80:249 –261; 2000. [89] Devanaboyina, U.; Gupta, R. C. Sensitive detection of 8-hydroxy-2⬘-deoxyguanosine in DNA by 32P-postlabeling assay and the basal levels in rat tissues. Carcinogenesis 17:917–924; 1996. [90] Chaudhary, A. K.; Nokubo, M.; Reddy, G. R.; Yeola, S. N.; Morrow, J. D.; Blair, I. A.; Marnett, L. J. Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science 265:1580 –1582; 1994. [91] Barbin, A. Etheno-adduct-forming chemicals: from mutagenicity testing to tumor mutation spectra. Mutat. Res. 462:55– 69; 2000. [92] Nair, J.; Carmichael, P. L.; Fernando, R. C.; Phillips, D. H.; Strain, A. J.; Bartsch, H. Lipid peroxidation-induced ethenoDNA adducts in the liver of patients with the genetic metal storage disorders Wilson’s disease and primary hemochromatosis. Cancer Epidemiol. Biomarkers Prev. 7:435– 440; 1998. [93] Burcham, P. C. Genotoxic lipid peroxidation products: their DNA damaging properties and role in formation of endogenous DNA adducts. Mutagenesis 13:287–305; 1998. [94] Von Sonntag, C. The chemical basis of radiation biology. London: Taylor and Francis; 1987. [95] Cooke, M. S.; Evans, M. D.; Podmore, I. D.; Herbert, K. E.; Mistry, N.; Mistry, P.; Hickenbotham, P. T.; Hussieni, A.; Griffiths, H. R.; Lunec, J. Novel repair action of vitamin C upon in vivo oxidative DNA damage. FEBS Lett. 439:363–367; 1998. [96] Grollman, A. P.; Moriya, M. Mutagenesis by 8-oxoguanine: an enemy within. Trends Genet. 9:246 –249; 1993. [97] Oikawa, S.; Kawanishi, S. Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening. FEBS Lett. 453:365–368; 1999. [98] Rodriguez, H.; Akman, S. A.; Holmquist, G. P.; Wilson, G. L.; Driggers, W. J.; Le Doux, S. P. Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology. Methods 22:148 –156; 2000. [99] Halliwell, B. Antioxidant defence mechanisms; from the beginning to the end (of the beginning). Free Radic. Res. 31:261–272; 1999. [100] Forsberg, L.; de Faire, U.; Morgenstern, R. Oxidative stress, human genetic variation and disease. Arch. Biochem. Biophys. 389:84 –93; 2001. [101] Mitrunen, K.; Sillanpaa, P.; Kataja, V.; Eskelinen, M.; Kosma, V. M.; Benhamou, S.; Uusitupa, M.; Hirvonen, A. Association between manganese superoxide dismutase (MnSOD) gene polymorphism and breast cancer risk. Carcinogenesis 22:827– 829; 2001.
PII S0891-5849(02)00808-0
Serial Review: Oxidative DNA Damage and Repair Guest Editor: Miral Dizdaroglu EFFECT OF DIET ON CANCER DEVELOPMENT: IS OXIDATIVE DNA DAMAGE A BIOMARKER? BARRY HALLIWELL Department of Biochemistry, National University of Singapore, Singapore (Received 11 December 2001; Accepted 21 February 2002)
Abstract—Free radicals and other reactive species are generated in vivo and many of them can cause oxidative damage to DNA. Although there are methodological uncertainties about accurate quantitation of oxidative DNA damage, the levels of such damage that escape immediate repair and persist in DNA appear to be in the range that could contribute significantly to mutation rates in vivo. The observation that diets rich in fruits and vegetables can decrease both oxidative DNA damage and cancer incidence is consistent with this. By contrast, agents increasing oxidative DNA damage usually increase risk of cancer development. Such agents include cigarette smoke, several other carcinogens, and chronic inflammation. Rheumatoid arthritis and diabetes are accompanied by increased oxidative DNA damage but the pattern of increased cancer risk seems unusual. Other uncertainties are the location of oxidative DNA damage within the genome and the variation in rate and level of oxidative damage between different body tissues. In well-nourished human volunteers, fruits and vegetables have been shown to decrease oxidative DNA damage in several studies, but data from short-term human intervention studies suggest that the protective agents are not vitamin C, vitamin E, -carotene, or flavonoids. © 2002 Elsevier Science Inc. Keywords—Oxidative DNA damage, Free radical, 8-Hydroxy-2⬘-deoxyguanosine, Mass spectrometry, Cancer, Diabetes
INTRODUCTION
oxyguanosine, 8OHdG [3], or use mass spectrometry to measure a wide range of DNA damage products [4]? What artifacts can arise in these various methods and how best can they be eliminated? DNA is easily oxidatively damaged during isolation, hydrolysis, and preparation for analysis (reviewed in [5]). At last however, due in part to the efforts of the European Standards Committee on Oxidative DNA Damage, ESCODD [6 –9], different groups in several countries are coming together to review these methodological questions and determine optimal protocols. My own laboratory has contributed to this research [5,10 –12]. However, the purpose of the present paper is not to revisit this issue, but to ask the second fundamental question, is oxidative DNA damage a valid biomarker of the late development of cancer?
It is widely believed that antioxidants help maintain human health by decreasing oxidative damage to key biomolecules. Thus biomarkers (some prefer the term “markers” [1]) of oxidative damage should be useful in establishing which antioxidants are important. Validation of biomarkers requires two different steps [1,2]. One is the analytical validation, including development of procedures, analysis of reference materials, and quality control. The second is the validation of the fact that changes in their level do reflect the later development of disease. These Serial Reviews in Free Radical Biology and Medicine contain many papers discussing what are the best methods to measure oxidative DNA damage. Should we rely on HPLC-based determination of 8-hydroxy-2⬘-de-
WHY SHOULD WE MEASURE OXIDATIVE DNA DAMAGE?
This article is part of a series of reviews on “Oxidative DNA Damage and Repair.” The full list of papers may be found on the homepage of the journal. Address correspondence to: Prof. Barry Halliwell, Department of Biochemistry, National University of Singapore, Singapore 119260, Singapore; Tel: (65) 6874-3247; Fax: (65) 6779-1453; E-Mail: [email protected].
The simple answer is that it is widely believed that oxidative DNA damage over the long human lifespan is a significant contributor to the age-related development 968
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of the major cancers, such as those of the colon, prostate, rectum, and breast [13–16]. If it is, it follows that diets or therapeutic agents that decreased oxidative DNA damage would delay or prevent the onset of cancer. No one to date has proved that oxidative DNA damage is a valid biomarker of subsequent cancer development. To do so would require measuring it in a range of healthy human subjects, subsequently followed over many years to see who develops cancer [17]. Epidemiologists have generally resisted including biomarkers in large human studies, although this view may be beginning to change. We must therefore turn to circumstantial evidence. Oxidative DNA damage is extensive Despite the various methodological problems that have arisen, the data available still collectively suggest that rates and levels of oxidative DNA damage in the human body are biologically significant. The commonest method of assessing “total body” oxidative DNA damage is to quantitate the urinary excretion of 8OHdG [15,18, 19]. Estimates based on 24 h excretion rates suggest an average of at least a few hundred “oxidative hits” per day on the DNA of each of the approximately 5 ⫻ 1013 cells in the human body [15,18 –20]. These values for urinary 8OHdG could be an underestimate of damage, in that they ignore other DNA base damage products that are excreted (see below). They are in any case only an average: some cells may suffer far more damage than others, but few data exist on this point. A second approach to assessing oxidative DNA damage has been to isolate DNA from tissues and measure its content of oxidative damage products. These values are presumably steady-state levels arising from a dynamic equilibrium between rates of oxidative DNA damage and rates of repair of that damage. Unfortunately, here is a major area of disagreement. For example, values for levels of 8-hydroxylated guanine in cellular DNA in some studies are less than 0.1/105 guanines and in others up to 100/105 guanines [3,4,15,21,22]. Again there is a multiplicity of other oxidative DNA base damage products [2] in cellular DNA, some of which may be present in greater amounts than 8OHdG [5]. Unfortunately, measurement techniques for these other base damage products have not yet been subject to the methodological comparisons that are underway [6,7–9] for 8-hydroxyguanine (8OHG) and 8OHdG. For example, there is disagreement over how much 8-hydroxyadenine may be present in DNA [2,23]. There could also be problems with urinary 8OHdG measurements, although they have the advantage that there is no DNA in urine to artifactually oxidize. Methodological problems can occur [23–26], e.g., because urine is a complex fluid containing many compounds that
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could co-elute with 8OHdG on HPLC analysis. The amount of 8OHdG in urine seems to be unaffected by diet and 8OHdG is thought not to be metabolized in humans [18,19,27]. Some (possibly all) of the 8OHdG excreted in human urine arises not from DNA, but from oxidation of deoxyguanosine triphosphate (dGTP) or diphosphate (dGDP) in the DNA precursor pool [19,28]. If so, it follows that 8OHdG excretion rates are not a quantitative index of oxidative damage to guanine residues in DNA. The measurement of other products in urine might give additional information. For example, in patients treated with adriamycin, 8OHdG excretion did not change, but there was a significant rise in 5-(hydroxymethyl) uracil content of urine [29]. Several DNA base damage products have been identified in human urine [23,29 –31], although it will be necessary to rule out a confounding effect of diet (absorption and re-excretion of oxidized DNA bases present in foods, e.g., generated by oxidative DNA damage during cooking) before these other products can be used as biomarkers of “whole body” oxidative DNA damage. Several lesions generated by oxidative DNA damage are mutagenic For the sake of argument, let me suggest that the urinary excretion of 8OHdG does represent an approximate biomarker of whole body oxidative DNA damage, and that the sum of all base damage products in DNA is at a steady-state level of at least one modified base per 105 unmolested purines and pyrimidines. These levels of whole body and steady-state oxidative DNA damage are consistent with a mutagenic load theoretically sufficient to promote cancer development. The mutagenicity of 8OHdG in causing GC 3 TA transversions is well established [3]. Several other DNA base oxidation products are mutagenic [32–34], including 2-hydroxyadenine, 5-hydroxycytosine, formyluracil, and 5-hydroxyuracil. In addition, the mutagenicity and other biological consequences of many of the other base oxidation products found in cellular DNA [4] have not been studied in detail and could be significant. It is instructive to make a comparison with levels of DNA adducts of known carcinogens. The concentrations of, for example, benzpyrene-DNA adducts in DNA from malignant tumors taken from smokers have been shown to be 0.65–5.33/106 DNA bases [35,36]. In rat liver, the calculated carcinogen-DNA adduct concentration associated with a 50% incidence of liver cancer ranged from 0.53 to 20.83 adducts/106 nucleotides for a range of carcinogens including aflatoxin and dimethylnitrosamine [37]. In mouse liver the respective figures were in the range of 8.12–55.43 adducts/106 nucleotides for ethylene
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oxide, dimethylnitrosamine, 4 aminobiphenyl, and 2-acetylaminofluorene [37]. If we assume that total DNA base oxidation products are at a level of at least 1 per 105 bases and are on average only one tenth as mutagenic as the above chemical carcinogens, it still seems that the levels that can be measured in cellular DNA represent a threat to the cell, given that the above levels are associated with a 50% cancer incidence. Agents that decrease oxidative DNA damage have an anti-cancer effect Diets rich in fruits and vegetables are associated with decreased incidence of the major human cancers. Multiple mechanisms can account for this, but several studies have shown that administration of fruits and vegetables to healthy human volunteers decreases levels of oxidative DNA damage as assessed using several different methods [38 – 42]. Administration of vitamin E, coenzyme Q10, vitamin C, -carotene or flavonoids could not decrease oxidative DNA damage, at least in the subjects examined, who appeared well nourished, with an intake of vitamins E and C equal to or exceeding current RDA values [5,17,43–51]. Indeed, the lack of effect of -carotene and antioxidant vitamins in decreasing oxidative DNA damage in subjects in advanced countries may help explain why intervention trials with these compounds as anti-cancer agent have given disappointing results [17, 52]. Agents that increase oxidative DNA damage promote cancer development Cigarette smoking is well known to be associated with increased risk of cancer development, and several studies report that it also raises levels of 8OHdG in urine and in cellular DNA [53–55], although conflicting data exist (discussed in [56]). The increased DNA damage may induce repair systems, which could explain the few studies in which rises in steady-state damage levels have not been detected [56]. The rise in 8OHdG in human lung appears to precede cancer development [55]. Many other carcinogens (and possibly all carcinogens) raise levels of oxidative damage prior to cancer development (reviewed in [13,16]). They include not only carcinogens that appear to act primarily by inducing oxidative stress (such as the peroxisome proliferator clofibrate [57,58]) but also more “classical” carcinogens such as dimethylbenzanthracene [59], nickel [60], benzpyrene [61], and asbestos [62]. High-fat diets appear to promote development of certain cancers, e.g., colon and breast, and studies suggest that such diets also increase 8OHdG formation [63– 66]. Chronic inflammation of several tissues, such as liver
and colon, increases cancer risk and also raises levels of oxidative DNA damage [10,67– 69]. In studies of a transgenic mouse model of hepatitis [70], formation of 8OHdG preceded cancer development. In another such model, vitamin E protected against DNA damage and decreased subsequent hepatic tumor formation [71], although in this study oxidative DNA damage was not directly measured. In mice exposed to diesel soot, levels of 80HdG in the lung rose well before tumor development [72]. Knockout mice lacking the MTHI gene encoding 8-oxo-dGTPase showed increased formation of tumors in lung, liver, and stomach at 18 months of life [73]. Increased oxidative DNA damage has also been detected in human benign prostate hyperplasia, although the association of this condition with subsequent cancer development is uncertain [74]. Hence, collectively the evidence, although obviously incomplete, is consistent with the view that increased oxidative DNA damage contributes to subsequent cancer development, whereas plant-rich diets decrease oxidative DNA damage and delay or prevent cancer development. Is this circumstantial evidence strong enough to prove beyond a reasonable doubt that oxidative DNA damage is a biomarker of subsequent cancer development? THE CASE AGAINST
Elevated oxidative DNA damage may not always be associated with increased cancer development There may be cases in which oxidative DNA damage levels are elevated, but cancer development does not ensue. In patients with rheumatoid arthritis (RA), levels of 8OHdG in blood cells [75] are elevated, as are other parameters of oxidative damage [76], yet the data on increased risk of malignancy are conflicting [77]. There appears to be moderately increased risk of hematopoietic, prostate, and lung cancers, but decreased risk for colorectal and gastric cancer [77]. Of course, the frequent use of cyclooxygenase-I and -II inhibitors in the therapy of RA could affect cancer development, given that this enzyme plays a role in tumor growth in some cancer types, such as colon cancer. In patients with type 2 diabetes, elevated levels of 8OHdG [78,79] and other DNA base oxidation products [79] in blood cells, as well as increased urinary excretion of 8OHdG [80], have been reported. Diabetes is associated with increased risk of pancreatic cancer [81], but no clear evidence for a generalized increased cancer risk in diabetics has emerged [81– 84], although such an increased risk cannot be ruled out on the data presently available. It may be that oxidative DNA base damage alone is insufficient to cause cancer development or perhaps damage over only a certain range is effective, excessive
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damage having an anti-cancer effect by promoting apoptosis. Reactive oxygen species could also promote cancer development by accelerating cell proliferation, although higher levels would have the opposite effect, halting proliferation [85]. We may not always be examining DNA from the correct tissue An important factor in explaining the different types of cancer observed in RA, diabetes, and cigarette smokers may be the relative extent of oxidative DNA damage in target tissues. A major problem in studying oxidative DNA damage is the limited availability of human tissues from which to obtain DNA. Most studies are performed on DNA isolated from blood lymphocytes (or sometimes total white cells). Most groups prefer to isolate lymphocytes, since total white cell DNA will include a significant contribution from neutrophils. However, there is no obvious reason why levels of oxidative DNA damage in either cell type should reflect levels in other body tissues (e.g., the pancreas in diabetes). Sperm, buccal cells, placenta, and biopsies of muscle, stomach, skin, colon, and other tissues are other potential sources of DNA, although biopsy samples often yield too little DNA for HPLC- or MS-based methods and we urgently need to develop micro-methods for DNA isolation and analysis. The comet assay may be useful, but has not been validated against rigorous chemical methods. Are we justified in assuming that changes in the amount of oxidative DNA damage in lymphocytes caused, for example, by antioxidant supplementation, are reflected in the tissues in which cancer is most likely to develop later in life (e.g., breast, lung, prostate, rectum, and colon)? More data are needed to answer such questions. Nevertheless, it is encouraging to note that in cigarette smokers and diabetic and RA patients, increased DNA damage can be detected by current methodology in blood cells and urine. Increased 80HdG levels were also observed in leukocytes from subjects with hepatic inflammation caused by hepatitis C virus [86]. Studies on dogs showed that an exercise regime decreased levels of 8OHdG in blood lymphocytes and colon, but not in other tissues [87]. In rats fed high-fat diets, oxidative DNA damage increased in both blood cells and mammary gland [63]. However, studies on rats are consistent with the existence of different steady-state levels of 8OHdG in different tissues [88,89]. Other mutagenic lesions generated by oxidative stress may be important The present article has focused on products of direct oxidative damage to the DNA bases. However,
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the contribution made by the reaction of end-products of lipid peroxidation with the DNA bases to create mutagenic lesions [90 –93] and the mutagenic effects of products generated by attack of reactive species on deoxyribose residues in DNA [94] may be worthy of consideration. Currently available data do not allow us to assess the relative importance of these and other lesions (e.g., DNA-protein cross-links) in promoting cell transformation. Repair rates must be considered Rises in 8OHdG or other DNA base oxidation products are not necessarily due to increased rates of oxidative DNA damage: decreased repair rate is also a possibility. This makes it necessary to be cautious in interpreting urinary excretion rates. For example, an agent that increases 8OHdG excretion might be interpreted as “bad” (seemingly increasing DNA damage) but might in fact be “good” (if it stimulated repair and therefore decreased steady state 8OHdG concentrations in DNA). It has been suggested, for example, that ascorbate may stimulate DNA repair in vivo [95]. The ideal would be to measure both steady state DNA damage and total damage (e.g., by urinary excretion rates). If only one set of measurements can be made, the steady state measurement is arguably preferable, because miscoding induced by oxidized bases is presumably what determines the risk of mutation and in turn the risk of cancer development. The repair process itself is not error free, however, and can introduce mutations, so it could be argued that a greater “throughput” of DNA base oxidation is deleterious even if it does not result in significant rises in the steady state concentrations of DNA base damage products. The genomic location of oxidative DNA damage is unknown The mutagenicity of 8OHdG, and probably that of other lesions, is sequence-specific to some extent, so that the total number of 8OHdG residues is not necessarily proportional to mutation frequency [3,96]. Oxidative damage in “junk DNA” may have no biological consequences, only that fraction of total damage that occurs in genes encoding functional proteins, (such as p53), or in telomeres [97] presumably being important. As yet, we have no clear information on distribution of oxidative DNA damage in the genome, although techniques to investigate this are under development [98]. CONCLUSION
There is no direct and compelling evidence that oxidative DNA damage is a biomarker of subsequent cancer
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development, because studies to address this point have not been done. However, there is considerable circumstantial evidence, but not proof beyond reasonable doubt to support the view that it is a valid biomarker. It follows that agents that decrease the amount of oxidative DNA damage should decrease the risk of subsequent cancer development [17]. We need to study predictive values of oxidative damage levels in healthy human populations. Correlations between intervention-induced changes in levels of oxidative DNA damage and effects of such intervention on subsequent cancer development also need to be studied. It may well be that levels of oxidative DNA damage show only a limited response to diet and are more affected by genetics [99 –101]. Acknowledgement — I am grateful to the Academic Research Fund of The National University of Singapore (grant R-183-000-027-112) for research support.
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