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Expression of base excision DNA repair genes as a biomarker of oxidative DNA damage
Cancer Letters 229 (2005) 1–11 www.elsevier.com/locate/canlet
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Expression of base excision DNA repair genes as a biomarker of oxidative DNA damage Christine L. Powell, James A. Swenberg, Ivan Rusyn* Curriculum in Toxicology and Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, NC 27599, USA Received 25 November 2004; accepted 1 December 2004
Abstract Oxidative stress induced DNA damage is considered to be the most common insult affecting the genome. Moreover, it is recognized as a common pathway to mutations and is suggested to play a major role in the development of chronic diseases such as cancer. However, current analytical methods used to detect oxidative DNA damage have been hampered by both technical and biological obstacles. These include spurious oxidation during DNA isolation and processing, and the inherent removal of damaged bases by numerous operating DNA repair systems. The removal of oxidized bases is performed predominantly by the base excision repair (BER) pathway and it has been shown that induction of DNA repair genes occurs in response to oxidative stress. Here, we demonstrate the utility of measuring changes in expression of BER genes as a sensitive in vivo biomarker for oxidative DNA damage. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: DNA damage; Base excision repair (BER); Oxidative stress; Reactive oxygen species (ROS)
1. Introduction Cells living in an oxygen-rich environment are inundated with various endogenous and exogenous sources of reactive oxygen species (ROS). As a consequence of cellular metabolic and biochemical processes such as mitochondrial respiration, boxidation, and cytochrome P450 metabolism, there is a steady production of ROS in the cell [1]. Additionally, inflammation, exposure to ultraviolet radiation, g-irradiation, and the formation of * Corresponding author. Tel./fax: C1 919 843 2596. E-mail address: [email protected] (I. Rusyn).
reactive intermediates from xenobiotic metabolism serve as exogenous mediators of ROS generation [2]. As a result, cells have evolved numerous defense mechanisms to counteract and limit the levels of reactive oxidants and the cellular damage that can ensue [3]. These include enzymatic reduction of ROS by superoxide dismutase, glutathione peroxidase, and catalase, as well as nonenzymatic quenching of ROS by vitamin E, vitamin C, b-carotene, and glutathione [4]. However, oxidative stress can arise when the production of ROS exceeds the cell’s antioxidant capacity, resulting in damage to cellular macromolecules such as DNA, proteins, and lipids [5].
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.12.002
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DNA damage as a result of oxidative stress is considered to be the most common insult affecting the genome [6,7]. DNA is a particularly sensitive cellular target because of the potential to create cumulative mutations that can disrupt cellular homeostasis. Oxidative DNA damage can include chemical and structural modifications to purine and pyrimidine bases and 2 0 -deoxyribose, and the formation of singleand double-strand breaks [8]. Persistent oxidative DNA damage can alter signaling cascades, gene expression, induce or arrest transcription, and cause replication errors and genomic instability. In a given cell, it is estimated that 105 oxidative DNA lesions are formed each day [9]. The exact number of oxidative DNA adducts is unknown but over 100 have been identified thus far; however, whether each of these adducts are produced in measurable amounts in vivo to be biological relevant remains to be determined [10–13]. Oxidative stress-induced mutations are suggested to play a major role in a number of chronic diseases including carcinogenesis, neurodegenerative disorders, and cardiovascular disease [1,8,14]. The development of sensitive analytical methods to detect oxidative DNA damage in vivo at the level of a base or sugar has been hampered by both technical and biological obstacles. Conflicting estimates on the basal levels of a commonly measured marker of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxo-dG), by chromatography were found to be attributed mostly to erroneous experimental oxidation during DNA isolation and processing [15]. Data reported by the European Standards Committee on Oxidative DNA Damage (ESCODD) have shown that it is possible to minimize DNA oxidation during isolation which has brought measured background levels closer in agreement with methods that do not require DNA processing (e.g., Comet assay) [16,17]. Although, the constant removal and repair of oxidized DNA lesions by the numerous operating DNA repair pathways presents another challenge in the utility of these methods to measure DNA adducts [3]. The cell has an elaborate system known as DNA repair to respond to DNA damage that reduces the yield of mutations and chromosomal aberrations [18]. Repair enzymes recognize and remove DNA adducts, correct the DNA sequence, and rejoin strand breaks. The removal and repair of oxidized base lesions is performed predominantly by the base excision repair
(BER) pathway and it has been shown that the induction of DNA repair enzymes occurs in response to oxidative stress [19–21]. Here, we demonstrate that changes in expression of BER genes can be used as a sensitive and artifact-free measure of in vivo oxidative DNA damage.
2. Reactive oxygen species and DNA damage In living cells, ROS are formed continuously as a consequence of both metabolic and biochemical reactions in addition to external factors. These ROS include oxygen radicals such as superoxide (O%K 2 ), hydroxyl (%OH), peroxyl (RO2%), alkoxyl (RO%), and hydroperoxyl (HO2%); and non-radicals that possess strong oxidizing potential or are easily converted to radicals by transition metals that include singlet oxygen (1O2), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), ozone (O3), and peroxynitrite (ONOOK). DNA damage induced by ROS occurs
Scheme 1. Schematic diagram of types of DNA damage that may be induced by reactive oxygen species. Abbreviations: reactive oxygen species, ROS; 4-hydroxy-2-nonenal, 4-HNE; malondialdehyde, MDA; pyrimidopurinone, M1G; etheno-deoxyguanosine, edA; etheno-deoxyadenosine, edA; human 8-oxoguanine DNA glycosylase 1, hOgg1; N-methylpurine DNA glycosylase, MPG; 2,6diamino-4-hydroxy-5-formamidopyrimidine, FapyG; 8-oxo-7,8dihydro-2 0 -guanosine, 8-oxo-G.
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by way of chemical and structural alterations to purine and pyrimidine bases and 2 0 -deoxyribose, and the formation of abasic sites and DNA strand breaks, see Scheme 1 [8]. The interaction of reactive oxidants with DNA can occur in a variety of ways. For instance, some ROS do not interact at all with DNA bases, such as superoxide and hydrogen peroxide [22,23]. Instead, they are believed to elicit their toxicity to DNA by conversion to hydroxyl radicals mediated by transition metal ions (e.g. iron and copper) through Haber-Weiss and Fenton reactions [24,25]. The hydroxyl radical is highly reactive and does not diffuse more than a couple of molecular diameters before reacting with the closest cellular component [25,26]. Therefore, in order to oxidize DNA directly, the hydroxyl radical must be generated immediately adjacent to nucleic acids. An assortment of products could be generated from such reactions since the hydroxyl radical reacts with all bases by either addition or abstraction of hydrogen atoms [22]. The most frequent base lesion produced is by addition of a hydroxyl radical to the C8 position of guanine to produce 8-oxo-dG, a marker commonly measured to assess oxidative stress to DNA. This adduct is a mutagenic lesion that preferentially pairs with adenine rather than cytosine resulting in G:C to T:A transversions following replication [27,28]. Peroxynitrite is a strong DNA oxidizing and nitrating agent that is a product of the coupling reaction of superoxide and nitric oxide. Damage to DNA by peroxynitrite can include strand breaks, base oxidation, deamination of guanine and adenine, and nitration of guanine bases [29–31]. Peroxynitrite has been demonstrated to oxidize purine bases with the formation of oxazolone, 8-oxo-7,8-dihydro-2 0 deoxyadenosine, and 8-oxo-dG [32,33]. Moreover, it has been shown that peroxynitrite is at least a 1000fold more reactive toward 8-oxo-dG than normal 2 0 -deoxyguanosine generating secondary products of 2-deoxy-b-D-erythro-pentofuranosyl derivatives of cyanuric, parabanic, and oxaluric acid [34,35]. Mutations induced by peroxynitrite using pSP189 shuttle vector were predominately G:C to T:A transversions after replication in both bacteria and mammalian cells [36]. Activated macrophages produce both superoxide and nitric oxide; thus, they are a potential source of peroxynitrite. Unlike the
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hydroxyl radical, peroxynitrite has the ability to diffuse across cells providing a potential linkage between chronic inflammation and carcinogenesis. Oxidants can react with the sugar moiety of DNA leading to the formation of apurinic/apyrimidinic (AP) sites and single- and double-strand breaks. The hydroxyl radical can abstract hydrogen atoms from all five carbon atoms of 2 0 -deoxyribose resulting in base loss and/or strand breakage [2]. Oxidative DNA adducts can promote cleavage of the N-glycosidic bond with deoxyribose which can result in the formation of an AP site. Furthermore, deoxyribose oxidation leads to the formation of base propenal and 3-phosphoglyceraldehyde which can react with DNA to form pyrimidopurinone (M1G) and ethenoadducts, respectively [37]. Indirect mutagenicity of DNA can occur by lipid peroxidation, a process involving the oxidation of polyunsaturated fatty acids (PUFAs), the basic component of biological membranes. The process is initiated by abstraction of a hydrogen atom and can be mediated by the hydroxyl, peroxyl, and alkoxyl radicals forming lipid hydroperoxides; however, these species are relatively short-lived [38]. Conversely, in the presence of transition metals the highly biologically reactive carbonyl products, including epoxides and aldehydes [e.g., crotonaldehyde, acrolein, 4-hydroxynonenal (HNE), and malondialdehyde (MDA)], can be produced and then diffuse from site of production and covalently bind to proteins and DNA [39]. These reactive substances damage DNA by forming exocyclic adducts [40,41] which have been shown to have genotoxic and mutagenic effects. For example, HNE can form an etheno-DNA adduct which can promote chromosomal aberrations and sister chromatid exchanges [42,43] whereas, MDA can give rise to M1G that is highly mutagenic resulting in base pair substitutions [42,43].
3. Oxidative DNA damage and cancer Cancer pathogenesis is a multi-step process involving mutations in critical genes required for maintaining cellular homeostasis and the clonal expansion of these mutated cells [44]. The foremost is the ability to induce DNA damage that can lead to mutations if
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replication proceeds without proper repair. Oxidative stress-induced DNA damage can lead to mutations and is suspected to be a major cause of cancer [14,45]. Furthermore, persistent oxidative DNA damage can alter signaling cascades, gene expression, induce or arrest transcription, and increase replication errors and genomic instability, all of which have been described in the progression of cancer development. Chronic inflammation from infection or injury is believed to contribute to about one in four of all cancer cases worldwide [46]. Inflammation activates a variety of immune cells, which induce a number of oxidant-generating enzymes such as NADPH oxidase, inducible nitric oxide synthase, and myeloperoxidase that are capable of producing high concentrations of ROS (e.g., superoxide anion, hydrogen peroxide, nitric oxide, and peroxynitrite). The increased production of ROS can increase the yield of mutations in DNA and also serve as an intracellular signal to promote mitogenesis [47]. A number of viral and microbial diseases such as hepatitis C, Helicobacter pylori, and human papillomavirus are associated with an increased risk of liver, colon, and cervical cancer, respectively [48]. Furthermore, they are all associated with an increase in the DNA adduct, 8-oxo-dG. To reduce the cancer risk, treatment strategies have targeted to alleviate the inflammation (e.g., anti-inflammatory agents such as interferons) or oxidant production (e.g., antioxidants such as ascorbic acid, b-carotene, and a-tocopherol) [49,50]. Chlorinated compounds, metal ions, barbiturates, phorbol esters, aromatic hydrocarbons, and some peroxisome proliferators are chemical carcinogens that have been shown to induce oxidative stress and damage in vitro and in vivo [51]. The mode of action of many chemical carcinogens is by generating ROS through redox cycling via electron transfer groups such as quinones, metal complexes, aromatic nitro compounds, and conjugated imines. Benzene is classified as a human carcinogen and inhalation or dermal occupational exposures have been associated with acute leukemia and lymphoma [52]. While the mechanism of action for benzene is still not clearly understood, the induction of chromosomal aberrations in hematopoietic stem cells is believed to be a critical element. The metabolism of benzene produces a number of phenolic and quinone species that
can undergo a one electron reduction to a very unstable semiquinone that rapidly reduces oxygen to superoxide, which regenerates the quinone and completes one redox cycle. Thus, this continuous cycle leads to an increase in ROS production and potential for oxidative DNA damage. Measurements of the DNA adduct, 8-oxo-dG, have been reported in cell cultures and bone marrow in vivo after treatment with benzene [53]. The presence of multiple pathways of repair for DNA damage demonstrates its important role in maintaining genomic stability. Therefore, it would be expected that reduced enzymatic activity or a defective enzyme in DNA repair would increase the likelihood of mutations and as a result increase risk of disease. Indeed, there are a number of hereditary diseases (e.g., Xeroderma Pigmentosum, Trichothiodystrophy, Cockayne’s Syndrome, and Fanconi’s Anemia), although extremely rare, that are characterized by an increased cancer risk due to deficiencies in nucleotide excision repair (NER). For example, Xeroderma Pigmentosum is a human disease with multiple defects in the NER pathway responsible for the removal of UV radiation-induced DNA damage and thus, individuals with this disease have a 1000-fold increased risk of developing skin cancer compared to the general population. More common are subtle changes in DNA repair phenotype derived from single nucleotide polymorphisms (SNPs) that are increasingly considered as cancer susceptibility genes [54]. An increased risk of esophagus, lung, and prostate cancer has been linked to SNP S326C in the human Ogg1 DNA repair gene, responsible for the removal of oxidized guanines. Another repair gene, XRCC1 with a SNP R194W, has been linked to increased risk of bladder, breast, lung, and stomach cancer.
4. Mechanisms of DNA repair Systems of response to DNA damage that reduce the yield of mutations and chromosomal aberrations in damaged cells are collectively known as DNA repair. Repair enzymes recognize and remove DNA adducts, correct the DNA sequence, and rejoin strand breaks. The cell possesses a number of DNA repair mechanisms to deal with oxidative and alkylated
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Scheme 2. Schematic diagram of base excision repair pathways for removal of oxidized DNA lesions formed as a result of a chemical exposure that causes oxidative stress. The sequence of modifications of DNA is depicted in bold, whereas the molecular events that occur at each step are shown in italics. The gene products that participate at each step of repair are also displayed.
DNA lesions, including direct damage reversal (via the enzyme O6-methylguanine-DNA methyltransferase), base and nucleotide excision, and mismatch repair. Mechanisms for repair of strand breaks include non-homologous end-joining and homologous recombination. It is believed that the predominant pathway used for removal of oxidized and many of the alkylated bases is base excision repair (BER), see Scheme 2. The process of BER is initiated by DNA glycosylases [e.g. 8-oxoguanine DNA glycosylase 1 (Ogg1), endonuclease III homolog 1 (NTH1), thymine glycol-DNA-glycosylase (NTH)], which are often promiscuous as far as their substrate specificity is concerned. The glycosylase hydrolyzes the Nglycosylic bond between the oxidized base and sugar moiety thus releasing the free damaged base and giving rise to an AP site. AP endonuclease (APE) acts upon the AP site generating a single strand break by cleaving the phosphodiester backbone 5 0 to the AP site, leaving behind a 3 0 -hydroxyl group and a 5 0 deoxyribose phosphate group (dRP). At this point the
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BER pathway can proceed through two different subpathways: short-patch and long-patch BER. These pathways are differentiated by the enzymes involved and the number of nucleotides removed. Short-patch BER replaces a single nucleotide by polymerase b (Pol b) and the newly synthesized DNA sealed by DNA ligase III/XRCC1 heterodimer [55]. Long-patch BER inserts 2–13 nucleotides by concordant action of Pol b, PCNA, Fen 1, and ligase 1. Even though DNA lesions and misincorporations are dealt with by a complex system of DNA repair enzymes, the process of repair proceeds through several intermediate steps that involve the formation of secondary lesions which are also mutagenic and clastogenic (i.e., abasic sites and single strand breaks) [18]. There are a number of consequences of induction/deficiency in DNA repair that are important for the process of carcinogenesis. Although induction would lead to enhanced repair, it has been suggested that this can be deleterious and promote mutagenesis [56]. If enzymes that act consecutively on different steps of repair are up-regulated unevenly, a state of imbalanced DNA repair might occur and lead to accumulation of both mutagenic and clastogenic lesions [57,58]. It should also be noted that not all polymerases have the same fidelity with some being more prone to introducing incorrect nucleotides [59].
5. Measurements of oxidative DNA damage The revelation of the ability of oxidants to damage DNA and the appreciation of its importance in disease has pressed the need for the development of sensitive analytical methods to detect and quantify levels of DNA adducts. The ability to draw any cause-andeffect conclusions regarding the role of oxidative stress to DNA and a particular chemical agent or disease state depends on knowing the precise endogenous or control levels of adducts that are to be used as biomarkers. Unfortunately, determining the background levels for the most commonly occurring and measured DNA adduct, 8-oxo-dG, has been difficult to ascertain up until recent efforts by ESCODD. Historical reports by enzymatic and chromatographic methods used to assay this particular DNA adduct have basal level estimates spread over three orders of magnitude [60]. Importantly, it was
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Table 1 Methods for measuring oxidative DNA damage Chromatographic (GC-MS/MS, HPLC-ECD, LC-MS/MS) Oxidized purines Oxidized pyrimidines M1G Comet assay Single-strand breaks Oxidized purines Oxidized pyrimidines Slot-blot assays Apurinic/apyrimidinic sites Oxidized purines Oxidized pyrimidines Immunohistochemistry 8-oxo-dG
shown that the grossly inflated values were most likely due to erroneous oxidation during DNA isolation and processing which can be limited by the inclusion of radical scavengers [61]. The introduction of this modification to these standard procedures has brought chromatographic values closer in line with enzymatic methods that do not require DNA isolation (e.g., Comet assay). However, there still remains an approximate seven-fold difference between these two methods in control levels for 8-oxo-dG. It is likely that true endogenous levels for this DNA lesion are approximately 1 per million guanines. Below, we review the most commonly used analytical techniques for measuring oxidative DNA damage in cells and tissues (Table 1). 5.1. Chromatographic methods The most investigated and measured oxidative DNA lesion is 8-oxo-dG. Several chromatographic approaches, including GC-MS, HPLC with electrochemical detection (ECD), and HPLC-MS/MS, have been applied; however, ESCODD has reported discrepancies in the estimates of basal levels of 8-oxo-dG ranging over three orders of magnitude with these techniques [62]. The GC-MS method reports the highest levels of endogenous DNA lesions, which have been attributed to spurious oxidation generated in the source of the instrument. Consequently, this method is no longer recommended in quantifying oxidative DNA damage; however, GC-MS is a valuable tool for identifying DNA adducts.
HPLC-ECD is a highly sensitive method with a limit of detection of approximately 50 fmol or 2 adducts per 106 dG (at least 100 mg of DNA is required for measurements). The 8-oxo-dG is first released from DNA by enzymatic digestion and the DNA hydrolysate is injected into a HPLC for separation and detection by electrochemical detector. Since there are fewer sample manipulation steps compared to GC-MS following DNA digestion, erroneous oxidation is minimized. Technological advances in mass spectrometry in the past decade have led to the emergence of LC-MS/MS as the method of choice for detecting and quantifying DNA adducts. Compared to HPLC-ECD, LC-MS/MS offers several advantages including the ability to provide structural information and the inclusion of internal standards resulting in greater specificity and improved quantitation. Recently, a capillary LCMS/MS method has been developed for the detection and quantification for 8-oxo-dG. This method involves the enzymatic digestion of DNA with 15N5-8-oxo-dG as internal standard and is followed by either isolation of 8-oxo-dG by HPLC (20–50 mg DNA required) or immunoaffinity (2–10 mg DNA required) chromatography. The isolated fraction is injected into the LCMS/MS using electrospray ionization (ESI), and measured by selective reaction monitoring (SRM). Reported endogenous 8-oxo-dG levels in calf thymus DNA, untreated rat liver, and human HeLa cells were consistently between 2 and 3 adducts per 106 dG (Swenberg, unpublished data). The M1G adduct is a secondary product of deoxyribose oxidation or covalent binding of MDA to guanine. This adduct exists in equilibrium between a ring-closed and ring-opened aldehyde form; consequently, this has hindered attempts to measure M1G reliably. This has been recently rectified by stabilizing M1G by reacting it with an aldehyde reactive probe (ARP) to form a ARP-M1G conjugate prior to enzymatic hydrolysis [63]. The conjugate is purified by solid phase extraction (SPE) and detected by LCMS/MS. This method requires about 200 mg of DNA and can readily detect 2 M1G adducts per 108 guanines. 5.2. Comet assay The comet assay, or single cell gel electrophoresis (SCGE), measures DNA strand breaks in whole cells
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without DNA isolation and, when lesion-specific endonucleases are included, allows detection of specific DNA lesions including oxidized and alkylated bases [64,65]. The cells are embedded in a thin layer of agarose, lysed with detergent and high salt. Resultant nucleoids are electrophoresed at high pH, and DNA visualized with DNA binding dye. Breaks in DNA are drawn out of the nucleoid to form a comet tail and the amount of DNA in tail compared to comet head is reflective of the amount of DNA damage. Since this method does not require DNA isolation, it is less prone to spurious oxidation. The addition of lesion-specific enzymes such as formamidopyrimidine glycosylase (FPG) or endonuclease III provides the ability to measure oxidized purines and pyrimidines, respectively. ESCODD has found this method to detect the lowest levels of background DNA damage (0.5 per 106 guanines) [17]; however, this may be due to incomplete enzymatic digestion or clusters of DNA lesions recognized as a single site. Additionally, if this method is to be used quantitatively it requires careful calibration and standardization. 5.3. Slot-blot type assays Slot blot assays for sugar back-bone lesions are based on the ability of an aldehyde reactive probe (ARP) to recognize the open ring structure of 2 0 -deoxyribose formed when a base is lost [66]. Thus, this assay allows the measurement of abasic (AP) sites and with the addition of lesion-specific endonucleases (e.g., FPG or endonuclease III) allows detection of oxidized purines and pyrimidines. The measurement of AP sites is performed on isolated genomic DNA, followed by treatment with ARP, transferred to nitrocellulose membrane, reacted with streptavidin-conjugated horseradish peroxidase, and enzymatic activity measured by chemiluminescence. Quantification is based on an internal standard containing a known number of AP sites. This method has similar drawbacks as the Comet assay in that it can underestimate DNA damage if enzymatic reactions do not go to completion. 5.4. Immunohistochemistry Antibodies specific for stable oxidative DNA adducts can be visualized in individual nuclei of
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cells in paraffin-embedded tissues and a qualitative assessment can be made using imaging software. There are a number of commercially available antibodies to measure oxidative modifications of DNA and proteins, including 8-oxo-dG, HNE, and MDA. Antibodies are limited in specificity if there is cross-reactivity between structurally similar DNA adducts or with other constituents of the cell. Thus, the application of this method must include proper positive and negative controls for accurate interpretation of results.
6. Expression of base excision DNA repair genes as a biomarker for oxidative DNA damage While many analytical techniques that assess oxidative DNA damage at the level of base or sugar are vulnerable to technical difficulties, the expression of BER genes, a biological response to DNA damage, holds promise as a sensitive in vivo biomarker. Moreover, because this pathway encompasses broad specificity and multiple routes of repair, it allows greater sensitivity in the ability to detect oxidative DNA damage compared to previously mentioned analytical techniques. The tireless effort of DNA repair to remove both endogenous and exogenous DNA lesions provides a potential biomarker of DNA damage that only recently has begun to be recognized. A number of non-genotoxic carcinogens are known to induce the production of free radicals and it is proposed that oxidative stress to DNA is one of the modes of action of such chemicals [67,68]; however, evidence for persistent oxidative DNA damage after treatment with such chemicals has remained elusive. For example, peroxisome proliferators, a class of nongenotoxic rodent carcinogens [69], have been the center of controversy for decades over the role of oxidative stress in the mechanism of liver carcinogenesis. These chemicals have been shown to produce ROS using electron spin resonance spectroscopy [47]; however, results on direct adduct measurements have been interpreted with caution due to large discrepancies in reports from different laboratories. Furthermore, it has been shown that the commonly measured end points for oxidative DNA damage (e.g., 8-oxodG, strand breaks, and AP sites) in liver tissue are not sensitive endpoints in this model [70]. One potential
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conclusion that can be drawn from the confusing literature on this subject is that there is no consistent evidence for oxidative DNA damage after treatment with peroxisome proliferators. However, it could also mean that the analytical approaches used were not sensitive enough to detect subtle increases in damage, or the lesions that were considered were not formed in vivo. Conversely, a concordant induction of genes specific to long-patch BER DNA repair (e.g., glycosylases, AP endonuclease, PCNA, Pol b, FEN1, and Lig1) was detected in livers of peroxisome proliferator-treated mice and it was shown that this endpoint can be successfully used in further mechanistic studies of the sources for DNA-damaging oxidants. Chronic inflammation is a risk factor for a number of cancers. It has been suggested that the low steady state level of ROS production by oxidant producing enzymes of phagocytic cells over time results in increased mutagenesis. For instance, individuals infected with either hepatitis B or C virus are at higher risk of developing hepatocellular carcinoma (HCC). Jungst et al., hypothesized that the surrounding tissue of liver carcinomas would be characterized by increased oxidative DNA damage associated with inflammatory infiltration [71]. A significant increase in 8-oxo-dG DNA adducts for non-tumor tissue with moderate inflammation compared to the adjacent carcinoma was reported. Alongside the presence of oxidative DNA damage was a significant increase in the expression of the DNA repair enzyme hMYH, responsible for the removal of mis-incorporated adenines from 8-oxo-dG base pairs. Thus, the increased expression of hMYH appears to corroborate the rise in 8-oxo-dG adducts in non-tumor tissue and links chronic inflammation with oxidative stressinduced DNA damage for liver cancer. Ulcerative colitis (UC) was also associated with oxidative stress and colon cancer. It has been recently reported that the activity of two base excision-repair enzymes, AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease, was increased in colon epithelium undergoing elevated inflammation in patients with UC [72]. Furthermore, such increases in DNA repair genes were mechanistically linked to microsatellite instability, thus providing a convincing link between
oxidant-induced upregulation of DNA repair and accumulation of mutations in vivo. Diesel exhaust particles (DEP) in ambient air are suspected to contribute to lung cancer and cardiopulmonary diseases mediated through the production of ROS and establishment of oxidative stress [73,74]. Diesel particles consist of a variety of mutagenic and carcinogenic chemicals, including polyaromatic hydrocarbons, aldehydes, quinones, and metals capable of generating ROS through Fenton reaction and redox cycling [75,76]. Additionally, DEP promotes an inflammatory response in the lung causing the release of ROS from macrophages [77]. The mechanism for DEP-induced lung carcinogenesis in rodents is unknown at this time but it is suspected that ROS are involved. Tsurudome et al., investigated the time dependent changes in the levels of 8-oxo-dG, its repair, and induction of Ogg1 expression in rats exposed to a single intra-tracheal administration of DEP [78]. Five days after the initial exposure, there was an induction of Ogg1 mRNA levels but no change was observed for 8-oxo-dG adducts or in Ogg1 repair activity. Thus, the expression of the DNA repair gene, Ogg1, provides a marker of cellular oxidative stress that was unobtainable by other methods for this model.
7. Conclusions In summary, the ability to associate oxidative stress to DNA with chemical or biological exposures that generate chronic low-level increases in ROS can be difficult to discern with numerous operating DNA repair systems. Furthermore, spurious oxidation that arises during DNA isolation and processing increases this difficulty when trying to observe minute, but important, changes in DNA adducts compared to controls. Chronic low-level exposures and the resultant chronic low-level increase in ROS could have significant long-term effects, but it has been difficult to elucidate the linkage among exposure, effect, and disease. BER gene expression provides several advantages over current methods, including technical ease, accessibility, sensitivity, and foremost an ex vivo artifactfree marker of oxidative DNA damage.
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Acknowledgements We thank Mr. Yo-Chan Jeong for providing Scheme 1. Supported, in part, by grants from NIH ES07126, ES11391, ES11660 and ES07126.
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Mini-review
Expression of base excision DNA repair genes as a biomarker of oxidative DNA damage Christine L. Powell, James A. Swenberg, Ivan Rusyn* Curriculum in Toxicology and Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, NC 27599, USA Received 25 November 2004; accepted 1 December 2004
Abstract Oxidative stress induced DNA damage is considered to be the most common insult affecting the genome. Moreover, it is recognized as a common pathway to mutations and is suggested to play a major role in the development of chronic diseases such as cancer. However, current analytical methods used to detect oxidative DNA damage have been hampered by both technical and biological obstacles. These include spurious oxidation during DNA isolation and processing, and the inherent removal of damaged bases by numerous operating DNA repair systems. The removal of oxidized bases is performed predominantly by the base excision repair (BER) pathway and it has been shown that induction of DNA repair genes occurs in response to oxidative stress. Here, we demonstrate the utility of measuring changes in expression of BER genes as a sensitive in vivo biomarker for oxidative DNA damage. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: DNA damage; Base excision repair (BER); Oxidative stress; Reactive oxygen species (ROS)
1. Introduction Cells living in an oxygen-rich environment are inundated with various endogenous and exogenous sources of reactive oxygen species (ROS). As a consequence of cellular metabolic and biochemical processes such as mitochondrial respiration, boxidation, and cytochrome P450 metabolism, there is a steady production of ROS in the cell [1]. Additionally, inflammation, exposure to ultraviolet radiation, g-irradiation, and the formation of * Corresponding author. Tel./fax: C1 919 843 2596. E-mail address: [email protected] (I. Rusyn).
reactive intermediates from xenobiotic metabolism serve as exogenous mediators of ROS generation [2]. As a result, cells have evolved numerous defense mechanisms to counteract and limit the levels of reactive oxidants and the cellular damage that can ensue [3]. These include enzymatic reduction of ROS by superoxide dismutase, glutathione peroxidase, and catalase, as well as nonenzymatic quenching of ROS by vitamin E, vitamin C, b-carotene, and glutathione [4]. However, oxidative stress can arise when the production of ROS exceeds the cell’s antioxidant capacity, resulting in damage to cellular macromolecules such as DNA, proteins, and lipids [5].
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.12.002
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DNA damage as a result of oxidative stress is considered to be the most common insult affecting the genome [6,7]. DNA is a particularly sensitive cellular target because of the potential to create cumulative mutations that can disrupt cellular homeostasis. Oxidative DNA damage can include chemical and structural modifications to purine and pyrimidine bases and 2 0 -deoxyribose, and the formation of singleand double-strand breaks [8]. Persistent oxidative DNA damage can alter signaling cascades, gene expression, induce or arrest transcription, and cause replication errors and genomic instability. In a given cell, it is estimated that 105 oxidative DNA lesions are formed each day [9]. The exact number of oxidative DNA adducts is unknown but over 100 have been identified thus far; however, whether each of these adducts are produced in measurable amounts in vivo to be biological relevant remains to be determined [10–13]. Oxidative stress-induced mutations are suggested to play a major role in a number of chronic diseases including carcinogenesis, neurodegenerative disorders, and cardiovascular disease [1,8,14]. The development of sensitive analytical methods to detect oxidative DNA damage in vivo at the level of a base or sugar has been hampered by both technical and biological obstacles. Conflicting estimates on the basal levels of a commonly measured marker of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxo-dG), by chromatography were found to be attributed mostly to erroneous experimental oxidation during DNA isolation and processing [15]. Data reported by the European Standards Committee on Oxidative DNA Damage (ESCODD) have shown that it is possible to minimize DNA oxidation during isolation which has brought measured background levels closer in agreement with methods that do not require DNA processing (e.g., Comet assay) [16,17]. Although, the constant removal and repair of oxidized DNA lesions by the numerous operating DNA repair pathways presents another challenge in the utility of these methods to measure DNA adducts [3]. The cell has an elaborate system known as DNA repair to respond to DNA damage that reduces the yield of mutations and chromosomal aberrations [18]. Repair enzymes recognize and remove DNA adducts, correct the DNA sequence, and rejoin strand breaks. The removal and repair of oxidized base lesions is performed predominantly by the base excision repair
(BER) pathway and it has been shown that the induction of DNA repair enzymes occurs in response to oxidative stress [19–21]. Here, we demonstrate that changes in expression of BER genes can be used as a sensitive and artifact-free measure of in vivo oxidative DNA damage.
2. Reactive oxygen species and DNA damage In living cells, ROS are formed continuously as a consequence of both metabolic and biochemical reactions in addition to external factors. These ROS include oxygen radicals such as superoxide (O%K 2 ), hydroxyl (%OH), peroxyl (RO2%), alkoxyl (RO%), and hydroperoxyl (HO2%); and non-radicals that possess strong oxidizing potential or are easily converted to radicals by transition metals that include singlet oxygen (1O2), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), ozone (O3), and peroxynitrite (ONOOK). DNA damage induced by ROS occurs
Scheme 1. Schematic diagram of types of DNA damage that may be induced by reactive oxygen species. Abbreviations: reactive oxygen species, ROS; 4-hydroxy-2-nonenal, 4-HNE; malondialdehyde, MDA; pyrimidopurinone, M1G; etheno-deoxyguanosine, edA; etheno-deoxyadenosine, edA; human 8-oxoguanine DNA glycosylase 1, hOgg1; N-methylpurine DNA glycosylase, MPG; 2,6diamino-4-hydroxy-5-formamidopyrimidine, FapyG; 8-oxo-7,8dihydro-2 0 -guanosine, 8-oxo-G.
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by way of chemical and structural alterations to purine and pyrimidine bases and 2 0 -deoxyribose, and the formation of abasic sites and DNA strand breaks, see Scheme 1 [8]. The interaction of reactive oxidants with DNA can occur in a variety of ways. For instance, some ROS do not interact at all with DNA bases, such as superoxide and hydrogen peroxide [22,23]. Instead, they are believed to elicit their toxicity to DNA by conversion to hydroxyl radicals mediated by transition metal ions (e.g. iron and copper) through Haber-Weiss and Fenton reactions [24,25]. The hydroxyl radical is highly reactive and does not diffuse more than a couple of molecular diameters before reacting with the closest cellular component [25,26]. Therefore, in order to oxidize DNA directly, the hydroxyl radical must be generated immediately adjacent to nucleic acids. An assortment of products could be generated from such reactions since the hydroxyl radical reacts with all bases by either addition or abstraction of hydrogen atoms [22]. The most frequent base lesion produced is by addition of a hydroxyl radical to the C8 position of guanine to produce 8-oxo-dG, a marker commonly measured to assess oxidative stress to DNA. This adduct is a mutagenic lesion that preferentially pairs with adenine rather than cytosine resulting in G:C to T:A transversions following replication [27,28]. Peroxynitrite is a strong DNA oxidizing and nitrating agent that is a product of the coupling reaction of superoxide and nitric oxide. Damage to DNA by peroxynitrite can include strand breaks, base oxidation, deamination of guanine and adenine, and nitration of guanine bases [29–31]. Peroxynitrite has been demonstrated to oxidize purine bases with the formation of oxazolone, 8-oxo-7,8-dihydro-2 0 deoxyadenosine, and 8-oxo-dG [32,33]. Moreover, it has been shown that peroxynitrite is at least a 1000fold more reactive toward 8-oxo-dG than normal 2 0 -deoxyguanosine generating secondary products of 2-deoxy-b-D-erythro-pentofuranosyl derivatives of cyanuric, parabanic, and oxaluric acid [34,35]. Mutations induced by peroxynitrite using pSP189 shuttle vector were predominately G:C to T:A transversions after replication in both bacteria and mammalian cells [36]. Activated macrophages produce both superoxide and nitric oxide; thus, they are a potential source of peroxynitrite. Unlike the
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hydroxyl radical, peroxynitrite has the ability to diffuse across cells providing a potential linkage between chronic inflammation and carcinogenesis. Oxidants can react with the sugar moiety of DNA leading to the formation of apurinic/apyrimidinic (AP) sites and single- and double-strand breaks. The hydroxyl radical can abstract hydrogen atoms from all five carbon atoms of 2 0 -deoxyribose resulting in base loss and/or strand breakage [2]. Oxidative DNA adducts can promote cleavage of the N-glycosidic bond with deoxyribose which can result in the formation of an AP site. Furthermore, deoxyribose oxidation leads to the formation of base propenal and 3-phosphoglyceraldehyde which can react with DNA to form pyrimidopurinone (M1G) and ethenoadducts, respectively [37]. Indirect mutagenicity of DNA can occur by lipid peroxidation, a process involving the oxidation of polyunsaturated fatty acids (PUFAs), the basic component of biological membranes. The process is initiated by abstraction of a hydrogen atom and can be mediated by the hydroxyl, peroxyl, and alkoxyl radicals forming lipid hydroperoxides; however, these species are relatively short-lived [38]. Conversely, in the presence of transition metals the highly biologically reactive carbonyl products, including epoxides and aldehydes [e.g., crotonaldehyde, acrolein, 4-hydroxynonenal (HNE), and malondialdehyde (MDA)], can be produced and then diffuse from site of production and covalently bind to proteins and DNA [39]. These reactive substances damage DNA by forming exocyclic adducts [40,41] which have been shown to have genotoxic and mutagenic effects. For example, HNE can form an etheno-DNA adduct which can promote chromosomal aberrations and sister chromatid exchanges [42,43] whereas, MDA can give rise to M1G that is highly mutagenic resulting in base pair substitutions [42,43].
3. Oxidative DNA damage and cancer Cancer pathogenesis is a multi-step process involving mutations in critical genes required for maintaining cellular homeostasis and the clonal expansion of these mutated cells [44]. The foremost is the ability to induce DNA damage that can lead to mutations if
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replication proceeds without proper repair. Oxidative stress-induced DNA damage can lead to mutations and is suspected to be a major cause of cancer [14,45]. Furthermore, persistent oxidative DNA damage can alter signaling cascades, gene expression, induce or arrest transcription, and increase replication errors and genomic instability, all of which have been described in the progression of cancer development. Chronic inflammation from infection or injury is believed to contribute to about one in four of all cancer cases worldwide [46]. Inflammation activates a variety of immune cells, which induce a number of oxidant-generating enzymes such as NADPH oxidase, inducible nitric oxide synthase, and myeloperoxidase that are capable of producing high concentrations of ROS (e.g., superoxide anion, hydrogen peroxide, nitric oxide, and peroxynitrite). The increased production of ROS can increase the yield of mutations in DNA and also serve as an intracellular signal to promote mitogenesis [47]. A number of viral and microbial diseases such as hepatitis C, Helicobacter pylori, and human papillomavirus are associated with an increased risk of liver, colon, and cervical cancer, respectively [48]. Furthermore, they are all associated with an increase in the DNA adduct, 8-oxo-dG. To reduce the cancer risk, treatment strategies have targeted to alleviate the inflammation (e.g., anti-inflammatory agents such as interferons) or oxidant production (e.g., antioxidants such as ascorbic acid, b-carotene, and a-tocopherol) [49,50]. Chlorinated compounds, metal ions, barbiturates, phorbol esters, aromatic hydrocarbons, and some peroxisome proliferators are chemical carcinogens that have been shown to induce oxidative stress and damage in vitro and in vivo [51]. The mode of action of many chemical carcinogens is by generating ROS through redox cycling via electron transfer groups such as quinones, metal complexes, aromatic nitro compounds, and conjugated imines. Benzene is classified as a human carcinogen and inhalation or dermal occupational exposures have been associated with acute leukemia and lymphoma [52]. While the mechanism of action for benzene is still not clearly understood, the induction of chromosomal aberrations in hematopoietic stem cells is believed to be a critical element. The metabolism of benzene produces a number of phenolic and quinone species that
can undergo a one electron reduction to a very unstable semiquinone that rapidly reduces oxygen to superoxide, which regenerates the quinone and completes one redox cycle. Thus, this continuous cycle leads to an increase in ROS production and potential for oxidative DNA damage. Measurements of the DNA adduct, 8-oxo-dG, have been reported in cell cultures and bone marrow in vivo after treatment with benzene [53]. The presence of multiple pathways of repair for DNA damage demonstrates its important role in maintaining genomic stability. Therefore, it would be expected that reduced enzymatic activity or a defective enzyme in DNA repair would increase the likelihood of mutations and as a result increase risk of disease. Indeed, there are a number of hereditary diseases (e.g., Xeroderma Pigmentosum, Trichothiodystrophy, Cockayne’s Syndrome, and Fanconi’s Anemia), although extremely rare, that are characterized by an increased cancer risk due to deficiencies in nucleotide excision repair (NER). For example, Xeroderma Pigmentosum is a human disease with multiple defects in the NER pathway responsible for the removal of UV radiation-induced DNA damage and thus, individuals with this disease have a 1000-fold increased risk of developing skin cancer compared to the general population. More common are subtle changes in DNA repair phenotype derived from single nucleotide polymorphisms (SNPs) that are increasingly considered as cancer susceptibility genes [54]. An increased risk of esophagus, lung, and prostate cancer has been linked to SNP S326C in the human Ogg1 DNA repair gene, responsible for the removal of oxidized guanines. Another repair gene, XRCC1 with a SNP R194W, has been linked to increased risk of bladder, breast, lung, and stomach cancer.
4. Mechanisms of DNA repair Systems of response to DNA damage that reduce the yield of mutations and chromosomal aberrations in damaged cells are collectively known as DNA repair. Repair enzymes recognize and remove DNA adducts, correct the DNA sequence, and rejoin strand breaks. The cell possesses a number of DNA repair mechanisms to deal with oxidative and alkylated
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Scheme 2. Schematic diagram of base excision repair pathways for removal of oxidized DNA lesions formed as a result of a chemical exposure that causes oxidative stress. The sequence of modifications of DNA is depicted in bold, whereas the molecular events that occur at each step are shown in italics. The gene products that participate at each step of repair are also displayed.
DNA lesions, including direct damage reversal (via the enzyme O6-methylguanine-DNA methyltransferase), base and nucleotide excision, and mismatch repair. Mechanisms for repair of strand breaks include non-homologous end-joining and homologous recombination. It is believed that the predominant pathway used for removal of oxidized and many of the alkylated bases is base excision repair (BER), see Scheme 2. The process of BER is initiated by DNA glycosylases [e.g. 8-oxoguanine DNA glycosylase 1 (Ogg1), endonuclease III homolog 1 (NTH1), thymine glycol-DNA-glycosylase (NTH)], which are often promiscuous as far as their substrate specificity is concerned. The glycosylase hydrolyzes the Nglycosylic bond between the oxidized base and sugar moiety thus releasing the free damaged base and giving rise to an AP site. AP endonuclease (APE) acts upon the AP site generating a single strand break by cleaving the phosphodiester backbone 5 0 to the AP site, leaving behind a 3 0 -hydroxyl group and a 5 0 deoxyribose phosphate group (dRP). At this point the
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BER pathway can proceed through two different subpathways: short-patch and long-patch BER. These pathways are differentiated by the enzymes involved and the number of nucleotides removed. Short-patch BER replaces a single nucleotide by polymerase b (Pol b) and the newly synthesized DNA sealed by DNA ligase III/XRCC1 heterodimer [55]. Long-patch BER inserts 2–13 nucleotides by concordant action of Pol b, PCNA, Fen 1, and ligase 1. Even though DNA lesions and misincorporations are dealt with by a complex system of DNA repair enzymes, the process of repair proceeds through several intermediate steps that involve the formation of secondary lesions which are also mutagenic and clastogenic (i.e., abasic sites and single strand breaks) [18]. There are a number of consequences of induction/deficiency in DNA repair that are important for the process of carcinogenesis. Although induction would lead to enhanced repair, it has been suggested that this can be deleterious and promote mutagenesis [56]. If enzymes that act consecutively on different steps of repair are up-regulated unevenly, a state of imbalanced DNA repair might occur and lead to accumulation of both mutagenic and clastogenic lesions [57,58]. It should also be noted that not all polymerases have the same fidelity with some being more prone to introducing incorrect nucleotides [59].
5. Measurements of oxidative DNA damage The revelation of the ability of oxidants to damage DNA and the appreciation of its importance in disease has pressed the need for the development of sensitive analytical methods to detect and quantify levels of DNA adducts. The ability to draw any cause-andeffect conclusions regarding the role of oxidative stress to DNA and a particular chemical agent or disease state depends on knowing the precise endogenous or control levels of adducts that are to be used as biomarkers. Unfortunately, determining the background levels for the most commonly occurring and measured DNA adduct, 8-oxo-dG, has been difficult to ascertain up until recent efforts by ESCODD. Historical reports by enzymatic and chromatographic methods used to assay this particular DNA adduct have basal level estimates spread over three orders of magnitude [60]. Importantly, it was
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Table 1 Methods for measuring oxidative DNA damage Chromatographic (GC-MS/MS, HPLC-ECD, LC-MS/MS) Oxidized purines Oxidized pyrimidines M1G Comet assay Single-strand breaks Oxidized purines Oxidized pyrimidines Slot-blot assays Apurinic/apyrimidinic sites Oxidized purines Oxidized pyrimidines Immunohistochemistry 8-oxo-dG
shown that the grossly inflated values were most likely due to erroneous oxidation during DNA isolation and processing which can be limited by the inclusion of radical scavengers [61]. The introduction of this modification to these standard procedures has brought chromatographic values closer in line with enzymatic methods that do not require DNA isolation (e.g., Comet assay). However, there still remains an approximate seven-fold difference between these two methods in control levels for 8-oxo-dG. It is likely that true endogenous levels for this DNA lesion are approximately 1 per million guanines. Below, we review the most commonly used analytical techniques for measuring oxidative DNA damage in cells and tissues (Table 1). 5.1. Chromatographic methods The most investigated and measured oxidative DNA lesion is 8-oxo-dG. Several chromatographic approaches, including GC-MS, HPLC with electrochemical detection (ECD), and HPLC-MS/MS, have been applied; however, ESCODD has reported discrepancies in the estimates of basal levels of 8-oxo-dG ranging over three orders of magnitude with these techniques [62]. The GC-MS method reports the highest levels of endogenous DNA lesions, which have been attributed to spurious oxidation generated in the source of the instrument. Consequently, this method is no longer recommended in quantifying oxidative DNA damage; however, GC-MS is a valuable tool for identifying DNA adducts.
HPLC-ECD is a highly sensitive method with a limit of detection of approximately 50 fmol or 2 adducts per 106 dG (at least 100 mg of DNA is required for measurements). The 8-oxo-dG is first released from DNA by enzymatic digestion and the DNA hydrolysate is injected into a HPLC for separation and detection by electrochemical detector. Since there are fewer sample manipulation steps compared to GC-MS following DNA digestion, erroneous oxidation is minimized. Technological advances in mass spectrometry in the past decade have led to the emergence of LC-MS/MS as the method of choice for detecting and quantifying DNA adducts. Compared to HPLC-ECD, LC-MS/MS offers several advantages including the ability to provide structural information and the inclusion of internal standards resulting in greater specificity and improved quantitation. Recently, a capillary LCMS/MS method has been developed for the detection and quantification for 8-oxo-dG. This method involves the enzymatic digestion of DNA with 15N5-8-oxo-dG as internal standard and is followed by either isolation of 8-oxo-dG by HPLC (20–50 mg DNA required) or immunoaffinity (2–10 mg DNA required) chromatography. The isolated fraction is injected into the LCMS/MS using electrospray ionization (ESI), and measured by selective reaction monitoring (SRM). Reported endogenous 8-oxo-dG levels in calf thymus DNA, untreated rat liver, and human HeLa cells were consistently between 2 and 3 adducts per 106 dG (Swenberg, unpublished data). The M1G adduct is a secondary product of deoxyribose oxidation or covalent binding of MDA to guanine. This adduct exists in equilibrium between a ring-closed and ring-opened aldehyde form; consequently, this has hindered attempts to measure M1G reliably. This has been recently rectified by stabilizing M1G by reacting it with an aldehyde reactive probe (ARP) to form a ARP-M1G conjugate prior to enzymatic hydrolysis [63]. The conjugate is purified by solid phase extraction (SPE) and detected by LCMS/MS. This method requires about 200 mg of DNA and can readily detect 2 M1G adducts per 108 guanines. 5.2. Comet assay The comet assay, or single cell gel electrophoresis (SCGE), measures DNA strand breaks in whole cells
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without DNA isolation and, when lesion-specific endonucleases are included, allows detection of specific DNA lesions including oxidized and alkylated bases [64,65]. The cells are embedded in a thin layer of agarose, lysed with detergent and high salt. Resultant nucleoids are electrophoresed at high pH, and DNA visualized with DNA binding dye. Breaks in DNA are drawn out of the nucleoid to form a comet tail and the amount of DNA in tail compared to comet head is reflective of the amount of DNA damage. Since this method does not require DNA isolation, it is less prone to spurious oxidation. The addition of lesion-specific enzymes such as formamidopyrimidine glycosylase (FPG) or endonuclease III provides the ability to measure oxidized purines and pyrimidines, respectively. ESCODD has found this method to detect the lowest levels of background DNA damage (0.5 per 106 guanines) [17]; however, this may be due to incomplete enzymatic digestion or clusters of DNA lesions recognized as a single site. Additionally, if this method is to be used quantitatively it requires careful calibration and standardization. 5.3. Slot-blot type assays Slot blot assays for sugar back-bone lesions are based on the ability of an aldehyde reactive probe (ARP) to recognize the open ring structure of 2 0 -deoxyribose formed when a base is lost [66]. Thus, this assay allows the measurement of abasic (AP) sites and with the addition of lesion-specific endonucleases (e.g., FPG or endonuclease III) allows detection of oxidized purines and pyrimidines. The measurement of AP sites is performed on isolated genomic DNA, followed by treatment with ARP, transferred to nitrocellulose membrane, reacted with streptavidin-conjugated horseradish peroxidase, and enzymatic activity measured by chemiluminescence. Quantification is based on an internal standard containing a known number of AP sites. This method has similar drawbacks as the Comet assay in that it can underestimate DNA damage if enzymatic reactions do not go to completion. 5.4. Immunohistochemistry Antibodies specific for stable oxidative DNA adducts can be visualized in individual nuclei of
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cells in paraffin-embedded tissues and a qualitative assessment can be made using imaging software. There are a number of commercially available antibodies to measure oxidative modifications of DNA and proteins, including 8-oxo-dG, HNE, and MDA. Antibodies are limited in specificity if there is cross-reactivity between structurally similar DNA adducts or with other constituents of the cell. Thus, the application of this method must include proper positive and negative controls for accurate interpretation of results.
6. Expression of base excision DNA repair genes as a biomarker for oxidative DNA damage While many analytical techniques that assess oxidative DNA damage at the level of base or sugar are vulnerable to technical difficulties, the expression of BER genes, a biological response to DNA damage, holds promise as a sensitive in vivo biomarker. Moreover, because this pathway encompasses broad specificity and multiple routes of repair, it allows greater sensitivity in the ability to detect oxidative DNA damage compared to previously mentioned analytical techniques. The tireless effort of DNA repair to remove both endogenous and exogenous DNA lesions provides a potential biomarker of DNA damage that only recently has begun to be recognized. A number of non-genotoxic carcinogens are known to induce the production of free radicals and it is proposed that oxidative stress to DNA is one of the modes of action of such chemicals [67,68]; however, evidence for persistent oxidative DNA damage after treatment with such chemicals has remained elusive. For example, peroxisome proliferators, a class of nongenotoxic rodent carcinogens [69], have been the center of controversy for decades over the role of oxidative stress in the mechanism of liver carcinogenesis. These chemicals have been shown to produce ROS using electron spin resonance spectroscopy [47]; however, results on direct adduct measurements have been interpreted with caution due to large discrepancies in reports from different laboratories. Furthermore, it has been shown that the commonly measured end points for oxidative DNA damage (e.g., 8-oxodG, strand breaks, and AP sites) in liver tissue are not sensitive endpoints in this model [70]. One potential
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conclusion that can be drawn from the confusing literature on this subject is that there is no consistent evidence for oxidative DNA damage after treatment with peroxisome proliferators. However, it could also mean that the analytical approaches used were not sensitive enough to detect subtle increases in damage, or the lesions that were considered were not formed in vivo. Conversely, a concordant induction of genes specific to long-patch BER DNA repair (e.g., glycosylases, AP endonuclease, PCNA, Pol b, FEN1, and Lig1) was detected in livers of peroxisome proliferator-treated mice and it was shown that this endpoint can be successfully used in further mechanistic studies of the sources for DNA-damaging oxidants. Chronic inflammation is a risk factor for a number of cancers. It has been suggested that the low steady state level of ROS production by oxidant producing enzymes of phagocytic cells over time results in increased mutagenesis. For instance, individuals infected with either hepatitis B or C virus are at higher risk of developing hepatocellular carcinoma (HCC). Jungst et al., hypothesized that the surrounding tissue of liver carcinomas would be characterized by increased oxidative DNA damage associated with inflammatory infiltration [71]. A significant increase in 8-oxo-dG DNA adducts for non-tumor tissue with moderate inflammation compared to the adjacent carcinoma was reported. Alongside the presence of oxidative DNA damage was a significant increase in the expression of the DNA repair enzyme hMYH, responsible for the removal of mis-incorporated adenines from 8-oxo-dG base pairs. Thus, the increased expression of hMYH appears to corroborate the rise in 8-oxo-dG adducts in non-tumor tissue and links chronic inflammation with oxidative stressinduced DNA damage for liver cancer. Ulcerative colitis (UC) was also associated with oxidative stress and colon cancer. It has been recently reported that the activity of two base excision-repair enzymes, AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease, was increased in colon epithelium undergoing elevated inflammation in patients with UC [72]. Furthermore, such increases in DNA repair genes were mechanistically linked to microsatellite instability, thus providing a convincing link between
oxidant-induced upregulation of DNA repair and accumulation of mutations in vivo. Diesel exhaust particles (DEP) in ambient air are suspected to contribute to lung cancer and cardiopulmonary diseases mediated through the production of ROS and establishment of oxidative stress [73,74]. Diesel particles consist of a variety of mutagenic and carcinogenic chemicals, including polyaromatic hydrocarbons, aldehydes, quinones, and metals capable of generating ROS through Fenton reaction and redox cycling [75,76]. Additionally, DEP promotes an inflammatory response in the lung causing the release of ROS from macrophages [77]. The mechanism for DEP-induced lung carcinogenesis in rodents is unknown at this time but it is suspected that ROS are involved. Tsurudome et al., investigated the time dependent changes in the levels of 8-oxo-dG, its repair, and induction of Ogg1 expression in rats exposed to a single intra-tracheal administration of DEP [78]. Five days after the initial exposure, there was an induction of Ogg1 mRNA levels but no change was observed for 8-oxo-dG adducts or in Ogg1 repair activity. Thus, the expression of the DNA repair gene, Ogg1, provides a marker of cellular oxidative stress that was unobtainable by other methods for this model.
7. Conclusions In summary, the ability to associate oxidative stress to DNA with chemical or biological exposures that generate chronic low-level increases in ROS can be difficult to discern with numerous operating DNA repair systems. Furthermore, spurious oxidation that arises during DNA isolation and processing increases this difficulty when trying to observe minute, but important, changes in DNA adducts compared to controls. Chronic low-level exposures and the resultant chronic low-level increase in ROS could have significant long-term effects, but it has been difficult to elucidate the linkage among exposure, effect, and disease. BER gene expression provides several advantages over current methods, including technical ease, accessibility, sensitivity, and foremost an ex vivo artifactfree marker of oxidative DNA damage.
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Acknowledgements We thank Mr. Yo-Chan Jeong for providing Scheme 1. Supported, in part, by grants from NIH ES07126, ES11391, ES11660 and ES07126.
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