Review ArticleDietary antioxidants and beneficial effect on oxidatively damaged DNA
Introduction
The cells of the human body are continuously attacked by reactive oxygen species generated as natural by-products of the normal cellular energy production, from daily activities such as exhaustive exercise, or from metabolism of xenobiotics. Although not directly substantiated in cohort studies, it is speculated that oxidative stress causes or aggravates many diseases such as cancer, diabetes, and coronary heart disease [1]. Humans have a rather complex network of defense barriers that protect the cellular biomolecules against oxidation by eliminating reactive oxygen species. The dietary intake of antioxidants is thought to play a major role in this network. Antioxidant is a widely used term that is difficult to define clearly in biological systems; we use the term antioxidant to broadly denote any substance that prevents oxidation of biomolecules either directly by scavenging reactive oxygen species or indirectly by upregulating the antioxidant defense or DNA repair systems. The indirect antioxidant effect may be evoked by xenobiotics or components in vegetables that are not scavengers or even considered harmful; e.g., isothiocyanates are oxidants that stimulate cellular antioxidant proteins and detoxification enzymes [2]. Antioxidants such as vitamin C, vitamin E, carotenoids, and flavonoids have been identified in many natural food products [3]. Natural products also contain mixtures of other antioxidants and bioactive substances with unknown antioxidant properties. The antioxidant activity can be tested in vitro or in animal experimental models, but this is associated with a number of uncertainties when extrapolating to humans. The most relevant way to explore antioxidant effects in humans is supplementation trials although this often is restricted to the use of surrogate tissues such as white blood cells (WBC) and urine.
Descriptive epidemiological studies indicate low risk of epithelial cancers, particularly in the airways and upper gastrointestinal tracts of people with high dietary intake of vegetables and fruits. However, double-blind epidemiological intervention studies are not feasible for antioxidant-rich foods and a series of intervention studies with antioxidant supplements in tablets, such as β-carotene (β-CT) and vitamin E, have been negative with respect to prevention of cancer [4], [5], [6], [7]. An alternative approach to investigating antioxidant effects has been the use of biomarkers of oxidatively damaged DNA. This is based on the mechanistic rationale that dietary antioxidants inhibit the oxidation of DNA. With the possibility of detecting 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), the first of many antioxidant supplementation studies with focus on this lesion in WBC appeared in the beginning of the 1990s [8]. At the same time, reliable detection of urinary 8-oxodG excretion was achieved [9], and this was soon followed by antioxidant supplementation studies using urinary 8-oxodG excretion as key biomarker. However, by far the most popular method in antioxidant intervention trials has been the comet assay that detects DNA strand breaks (SB). An enzyme-modified version of the comet assay has been developed to detect oxidatively altered nucleobases by including a DNA digestion step with DNA glycosylase or endonuclease enzymes [10]. Oxidized purines, including 8-oxodG, can be detected by formamidopyrimidine DNA glycosylase (FPG) and oxidized pyrimidines by endonuclease III (ENDOIII). Ex vivo exposure of cells to DNA breaking agents such as H2O2 or ionizing radiation has been used as a semiquantitative measurement of the donor's antioxidative status. This modification of the comet assay is based on the notion that the intracellular content of antioxidants will inhibit DNA breakage.
8-OxodG (or 8-oxo-7,8-dihydroguanine (8-oxoGua) which is the corresponding base) is one of the most easily formed oxidatively damaged DNA lesions and can be detected in both urine and tissue after oxidative stress [11]. In biomonitoring studies the preferred assays for detection of oxidatively damaged DNA have been chromatographic techniques (HPLC-EC), gas chromatography with mass spectrometry (GC-MS), antibody-based immunoassays, and enzymic detection by bacterial glycosylase and endonuclease enzymes [12]. There has been large variation in the levels of 8-oxodG detected by these assays that to some extent can be explained by spurious generation of 8-oxodG in the chromatographic methods [13]. In particular, the silylation reaction required for the GC-MS analysis is associated with severe spurious oxidation, which increases the level of oxidized bases compared to the HPLC measurements. Recently, the addition of metal chelators and antioxidants and the use of sodium iodide precipitation have reduced the spurious oxidation of DNA during HPLC analysis [14], [15]. The use of isotopically labeled internal standards allows sensitive and accurate detection of several oxidized nucleosides and bases by mass spectrometry [15], [16]. It has been suggested that the background level of 8-oxodG in normal human cells is 0.3–4.2 lesions per 106 dG, where the lower value represents enzymic detection and the higher value represents HPLC measurements [17]. In the light of these results the European Standards Committee on Oxidative DNA Damage recommended that published studies containing data on 8-oxodG and 8-oxoGua in WBC should be carefully reassessed [18]. For other types of oxidatively damaged DNA, standardization of laboratory procedures and agreement on basal levels are also warranted, although similar levels of SB,ENDOIII, and FPG sites have been obtained in WBC of humans [19].
A brief survey of databases such as PubMed reveals a multitude of papers that describe the effect of antioxidants in humans. Here we have reviewed the effects of antioxidant supplements and antioxidant-rich foods on oxidatively damaged DNA in WBC and urine. We use the term WBC although this may refer to leukocytes, lymphocytes, or mononuclear blood cells. We have included intervention trials and cross-sectional studies (correlation analysis or comparisons of different populations). We have limited the survey to encompass studies that included data measured either by the comet assay (or related assays) or by 8-oxodG/8-oxoGua. Data from assays that require growth of cells, e.g., micronuclei and chromosome aberrations are not included in the survey. Other types of oxidatively generated DNA lesions have been investigated in very few studies and potential problems related to methodologically generated spurious oxidation limits the interpretation of results for some lesions, e.g., oxidatively generated adenine lesions. It is now possible to reliably measure 8-oxodG and 4,6-diamino-5-formamidopyrimidine in cellular DNA by HPLC coupled to tandem mass spectrometry [20]. Application of such recent methodological achievements in future antioxidant intervention trials provides the benefit of reliable measurement of oxidatively generated adenine lesions and other oxidized nucleobases.
Section snippets
Identification of publications
The publications were identified by searches in PubMed/Medline, EMBASE, and Web of Science databases. We searched for papers that contained data from antioxidant intervention trials with endpoints for the comet or related assays and for oxidized guanines (8-oxoGua or 8-oxodG) measured in WBC or urine. In addition, we searched for cross-sectional studies containing correlations between oxidatively damaged DNA and antioxidant concentrations or antioxidant intake. The search produced 139 potential
Interpretation
A brief survey of databases such as PubMed indicates that the literature is loaded with antioxidant intervention studies that have investigated the effects of oxidatively damaged DNA in tissues, WBC, or urine of humans. The purpose of this report was to bring attention to the fact that the studies differ in quality and that some of the reports should be interpreted with caution. To the best of our knowledge, we have cited all the antioxidant intervention studies related to oxidatively damaged
Comments
Fruits and vegetables are widely acknowledged to be healthy due to a number of mechanisms, including protection against reactive oxygen species. There are numerous experimental studies published in support for this hypothesis each year. Many of these studies are in vitro experiments and animal experimental models that are difficult to extrapolate to humans. This survey shows that ingestion of antioxidants is associated with lower levels of oxidatively damaged DNA and ex vivo sensitivity in WBC
Acknowledgment
The authors were supported by grants from the Danish Medical Research Council.
Peter Møller became Master of Science in 1995. In 2000 and 2005 he received a PhD degree and a doctorate in medical sciences, respectively. He was employed at the Danish National Institute of Occupational Health in 1995–1999. Since 1999 he has been employed at the University of Copenhagen, currently as associate professor. Major research interests include the relationship between environmental exposures and oxidative stress with special attention to the relationship between DNA damage and DNA
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Peter Møller became Master of Science in 1995. In 2000 and 2005 he received a PhD degree and a doctorate in medical sciences, respectively. He was employed at the Danish National Institute of Occupational Health in 1995–1999. Since 1999 he has been employed at the University of Copenhagen, currently as associate professor. Major research interests include the relationship between environmental exposures and oxidative stress with special attention to the relationship between DNA damage and DNA repair.
Steffen Loft received an MD in 1980 and worked clinically for 6 years. He received a doctorate in medical sciences in 1990. Since 1998 he has been full professor of environmental health and department chairman at the University of Copenhagen. His research interests focus on oxidatively damaged DNA in relation to risk of cancer, diet, personal exposure and health effects of air pollution and endocrine disruptors by means of biomarkers and animal models.