Oxidative damage to DNA: formation, measurement and biochemical features

Emphasis is placed in the first part of this survey on mechanistic aspects of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) as the result of exposure to z.rad;OH radical, one-electron oxidants and singlet oxygen (1O(2)) oxidation. It was found that 8-oxoGua, which is generated by either hydration of the guanine radical cation or .OH addition at C8 of the imidazole ring, is a preferential target for further reactions with 1O(2) and one-electron oxidants, including the highly oxidizing oxyl-type guanine radical. Interestingly, tandem base lesions that involve 8-oxoGua and a vicinal formylamine residue were found to be generated within DNA as the result of a single .OH radical hit. The likely mechanism of formation of the latter lesions involves the transient generation of 5-(6)-peroxy-6-(5)-hydroxy-5,6-dihydropyrimidyl radicals that may add to the C8 of a vicinal guanine base before undergoing rearrangement. Another major topic which is addressed deals with recent developments in the measurement of oxidative base damage to cellular DNA. This was mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents including UVA and ionizing radiations. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision and nucleotide excision pathways are briefly reviewed. For this purpose modified oligonucleotides in which cyclopurine, and cyclopyrimidine nucleosides were site-specifically inserted were synthesized.     

Mechanism of oxidative DNA damage repair and relevance to human pathology

Since DNA is prone to oxidative attack cells have evolved multiple protective strategies to prevent the deleterious effects of DNA oxidation. Base excision repair is the major mechanism for repair of DNA base damage by reactive oxygen species but recent evidence indicate that nucleotide excision repair proteins, that are mutated in human syndromes, are involved too. The mechanisms of repair dealing with the direct oxidation of DNA will be reviewed taking as prototype the oxidized base 7,8-dihydro-8-hydroxyguanine. The function of the individual repair components as inferred from model mice indicate that the ablation of two gene functions is mostly required to lead to accumulation of oxidative DNA damage, mutagenesis and cancer development. The recent identification of human diseases associated with mutations in oxidative damage repair show that defects in this pathway may lead to increased cancer but their major causative role seems to be in neurological diseases.     

Free radical-induced damage to DNA: mechanisms and measurement

Free radicals are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells, including DNA. The resulting damage to DNA, which is also called oxidative damage to DNA, is implicated in mutagenesis, carcinogenesis, and aging. Mechanisms of damage involve abstractions and addition reactions by free radicals leading to carbon-centered sugar radicals and OH- or H-adduct radicals of heterocyclic bases. Further reactions of these radicals yield numerous products. Various analytical techniques exist for the measurement of oxidative damage to DNA. Techniques that employ gas chromatography (GC) or liquid chromatography (LC) with mass spectrometry (MS) simultaneously measure numerous products, and provide positive identification and accurate quantification. The measurement of multiple products avoids misleading conclusions that might be drawn from the measurement of a single product, because product levels vary depending on reaction conditions and the redox status of cells. In the past, GC/MS was used for the measurement of modified sugar and bases, and DNA-protein cross-links. Recently, methodologies using LC/tandem MS (LC/MS/MS) and LC/MS techniques were introduced for the measurement of modified nucleosides. Artifacts might occur with the use of any of the measurement techniques. The use of proper experimental conditions might avoid artifactual formation of products in DNA. This article reviews mechanistic aspects of oxidative damage to DNA and recent developments in the measurement of this type of damage using chromatographic and mass spectrometric techniques.     

Facts and artifacts in the measurement of oxidative base damage to DNA

This short survey is aimed at critically evaluating the main available methods for measuring oxidative base damage within cellular DNA. Emphasis is placed on separative methods which are currently widely applied. These mostly concern high performance liquid chromatography (HPLC) and gas chromatography (GC) associated with sensitive detection techniques such as electrochemistry (EC) and mass spectrometry (MS). In addition, the comparison is extended to 32p-postlabeling methods, immunoassays and measurement of two main classes of oxidative DNA damage within isolated cells. It may be concluded that the HPLC-electrochemical detection (ECD) method, even if restricted to the measurement of only a few electroactive oxidized bases and nucleosides, is the simplest and safest available method at the moment. In contrast, the more versatile GC-MS method, which requires a HPLC pre-purification step in order to prevent artifactual oxidation of overwhelming normal bases to occur during derivatization, is more tedious and its sensitivity may be questionable. Alternative simpler procedures of background prevention for the GC-MS assay, which, however, remain to be validated, include low-temperature for derivatization and addition of antioxidants to the silylating reagents. Interestingly, similar levels of 8-oxo-7,8-dihydroguanine were found in cellular DNA using HPLC-ECD, HPLC-MS/MS and HPLC/32P-postlabeling methods. However, it should be noted that the level of cellular 8-oxodGuo, thus determined, is on average basis 10-fold higher than that was inferred for more indirect measurement involving the use of DNA repair enzymes with methods on isolated cells. Further efforts should be made to resolve this apparent discrepancy. In addition, the question of the biological validation of the non-invasive measurement of oxidized bases and nucleosides in urine is addressed.     

Hydroxyl radicals and DNA base damage

Modified purine and pyrimidine bases constitute one of the major classes of hydroxyl-radical-mediated DNA damage together with oligonucleotide strand breaks, DNA-protein cross-links and abasic sites. A comprehensive survey of the main available data on both structural and mechanistic aspects of.OH-induced decomposition pathways of both purine and pyrimidine bases of isolated DNA and model compounds is presented. In this respect, detailed information is provided on both thymine and guanine whereas data are not as complete for adenine and cytosine. The second part of the overview is dedicated to the formation of.OH-induced base lesions within cellular DNA and in vivo situations. Before addressing this major point, the main available methods aimed at singling out.OH-mediated base modifications are critically reviewed. Unfortunately, it is clear that the bulk of the chemical and biochemical assays with the exception of the high performance liquid chromatographic-electrochemical detection (HPLC/ECD) method have suffered from major drawbacks. This explains why there are only a few available accurate data concerning both the qualitative and quantitative aspects of the.OH-induced formation of base damage within cellular DNA. Therefore, major efforts should be devoted to the reassessment of the level of oxidative base damage in cellular DNA using appropriate assays including suitable conditions of DNA extraction.     

Ultraviolet radiation-mediated damage to cellular DNA

Emphasis is placed in this review article on recent aspects of the photochemistry of cellular DNA in which both the UVB and UVA components of solar radiation are implicated individually or synergistically. Interestingly, further mechanistic insights into the UV-induced formation of DNA photoproducts were gained from the application of new accurate and sensitive chromatographic and enzymic assays aimed at measuring base damage. Thus, each of the twelve possible dimeric photoproducts that are produced at the four main bipyrimidine sites can now be singled out as dinucleoside monophosphates that are enzymatically released from UV-irradiated DNA. This was achieved using a recently developed high-performance liquid chromatography-tandem mass spectrometry assay (HPLC-MS/MS) assay after DNA extraction and appropriate enzymic digestion. Interestingly, a similar photoproduct distribution pattern is observed in both isolated and cellular DNA upon exposure to low doses of either UVC or UVB radiation. This applies more specifically to the DNA of rodent and human cells, the cis-syn cyclobutadithymine being predominant over the two other main photolesions, namely thymine-cytosine pyrimidine (6-4) pyrimidone adduct and the related cyclobutyl dimer. UVA-irradiation was found to generate cyclobutane dimers at TT and to a lower extent at TC sites as a likely result of energy transfer mechanism involving still unknown photoexcited chromophore(s). Oxidative damage to DNA is also induced although less efficiently by UVA-mediated photosensitization processes that mostly involved 1O2 together with a smaller contribution of hydroxyl radical-mediated reactions through initially generated superoxide radicals.     

DNA N-glycosylase deficient mice: a tale of redundancy

The generation of mouse models of base excision repair deficiency has resulted in a re-examination of the cellular defence mechanisms that exist to counteract oxidative base damage. Contrary to exhibiting various detrimental effects of the gene disruption, the different strains of DNA-N-glycosylase deficient mice have proved to be remarkably resilient to the loss of the major activities that catalyse the removal of oxidised bases from DNA. Indeed, with a few exceptions, there is little evidence for the accumulation of oxidised bases in tissues and organs of the glycosylase knockout mice, even in older animals. This is highly suggestive of hitherto unknown backup mechanisms for dealing with the removal of oxidative base damage from genomic DNA. Results from both a genomics-based approach and biochemical analyses of cell free extracts from DNA glycosylase knockout mice have indicated that this is so and there is increasing evidence of several novel DNA glycosylase/AP lyases in mammalian cells that are capable of acting on oxidised bases in vitro. This, in parallel with other repair mechanisms involving mismatch repair, the Cockayne syndrome B protein and the efficient and accurate bypass of replication blocking lesions by a battery of translesion DNA polymerases, may explain the lack of severe phenotype observed for the DNA glycosylase deficient mice discussed in this article.     

Chemical and biochemical postlabeling methods for singling out specific oxidative DNA lesions.

A survey of the main available chemical and biochemical postlabeling assays for measuring oxidative DNA damage is reported. Two main approaches, radio and fluorescent postlabeling, have been used in order to reach a high level of sensitivity of detection. This is required for the measurement of DNA damage within cells and tissues upon exposure to agents of oxidative stress. Most of the methods are based on liquid chromatographic separation of defined DNA modifications following either acidic hydrolysis or enzymic digestion of DNA. In a subsequent step, the isolated base or sugar damages are either radiolabeled or made fluorescent by chemical or enzymatic reactions. Emphasis is placed on the recently developed high performance liquid chromatographic 32P-postlabeling assay, which allows the specific and sensitive measurement of various base damages including adenine N-1 oxide and 5-hydroxymethyluracil at the level of one modification per 10(7) normal bases in a sample size of 1 microgram of DNA. Examples of application of radioactive postlabeling to the measurement of DNA base damage following exposure of human cells to oxidizing agents including hydrogen peroxide and UVA radiation are provided.     

Oxidation by DNA charge transport damages conserved sequence block II, a regulatory element in mitochondrial DNA

Sites of oxidative damage in mitochondrial DNA have been identified on the basis of DNA-mediated charge transport. Our goal is to understand which sites in mitochondrial DNA are prone to oxidation at long range and whether such oxidative damage correlates with cancerous transformation. Here we show that a primer extension reaction can be used to monitor directly oxidative damage to authentic mitochondrial DNA through photoreactions with a rhodium intercalator. The complex [Rh(phi)2bpy]Cl3 (phi = 9,10-phenanthrenequinone diimine) binds to DNA without sequence specificity and, upon photoactivation, either promotes strand breaks directly at the binding site or promotes one-electron oxidative damage; comparing the sites of base oxidation to direct strand breaks reveals the oxidative damage that arises from a distance through DNA-mediated charge transport. Significantly, base oxidation by charge transport overlaps with known mutational hot spots associated with cancers at nucleotides surrounding positions 263 and 303; the latter is known as conserved sequence block II and is vital to DNA replication. Since DNA base oxidation at conserved sequence block II should weaken the ability of damaged mitochondrial genomes to be replicated, DNA-mediated charge transport may provide a protection mechanism for excluding damaged DNA.     

Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement

Oxidation reactions that involve several oxygen and nitrogen reactive species together with nucleobase radical cations give rise among various classes of lesions to modified bases. About 70 of oxidized nucleosides that include diastereomeric forms have been characterized in mechanistic studies involving isolated DNA and related model compounds. However, only eight modified bases have been accurately measured within cellular DNA upon exposure to either gamma or UVA radiations. Emphasis is placed in this survey on recent developments of HPLC associated with tandem mass spectrometry (MS/MS) operating in the mild electrospray ionization mode. Interestingly, the HPLC-MS/MS assay in the multiple reaction monitoring mode appears to be the more sensitive and accurate method currently available for singling out several oxidized nucleosides including 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 5-formyl-2'-deoxyuridine, 5-(hydroxymethyl-2'-deoxyuridine, 5-hydroxy-2'-deoxyuridine, and the four diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine within isolated and cellular DNA. However, one limitation of the assay that also applied to all chromatographic methods is the slight side-oxidation of normal bases during DNA extraction and subsequent work-up. This explains why the combined use of DNA repair glycosylases with either the comet assay or the alkaline elution technique is a better alternative to monitor the formation of low levels of oxidized bases within cellular DNA.     

8-Oxoguanine induces intramolecular DNA damage but free 8-oxoguanine protects intermolecular DNA from oxidative stress

7,8-Dihydro-8-oxoguanine (8-oxoguanine; 8-oxo-G), one of the major oxidative DNA adducts, is highly susceptible to further oxidation by radicals. We confirmed the higher reactivity of 8-oxo-G toward reactive oxygen (singlet oxygen and hydroxyl radical) or nitrogen (peroxynitrite) species as compared to unmodified base. In this study, we raised the question about the effect of this high reactivity toward radicals on intramolecular and intermolecular DNA damage. We found that the amount of intact nucleoside in oligodeoxynucleotide containing 8-oxo-G decreased more by various radicals at higher levels of 8-oxo-G incorporation, and that the oligodeoxynucleotide damage and plasmid cleavage by hydroxyl radical were inhibited in the presence of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG). We conclude that 8-oxo-G within DNA induces intramolecular DNA base damage, but that free 8-oxo-G protects intermolecular DNA from oxidative stress. These results suggest that 8-oxo-G within DNA must be rapidly released to protect DNA from overall oxidative damage.     

Peroxyl radical mediated oxidative DNA base damage: implications for lipid peroxidation induced mutagenesis

Endogenous DNA damage induced by lipid peroxidation is believed to play a critical role in carcinogenesis. Lipid peroxidation generates free radical intermediates (primarily peroxyl radicals, ROO(*)) and electrophilic aldehydes as the principal genotoxicants. Although detailed information is available on the role of aldehyde base adducts in mutagenesis and carcinogenesis, the contribution of peroxyl radical mediated DNA base damage is less well understood. In the present study we have mapped oxidative base damage induced by peroxyl radicals in the supF tRNA gene and correlated this information with peroxidation-induced mutations in several human fibroblast cell lines. Nearly identical patterns of oxidative base damage were obtained from reaction of DNA with either peroxidizing arachidonic acid (20:4omega6) or peroxyl radicals generated by thermolysis of ABIP in the presence of oxygen. Oxidative base damage primarily occurred at G and C. Transversions at GC base pairs in the supF gene were the major base substitution detected in all cell lines. Peroxyl radical induced tandem mutations were also observed. Many mutation hot spots coincided with sites of mapped oxidative lesions, although in some cases hot spots occurred adjacent to the damaged base. Evidence is presented for the involvement of 8-oxodG in the oxidation of DNA by ROO(*). These results are used to interpret some key features of previously published mutation spectra induced by lipid peroxidation in human cells.     

Further studies of KMnO4 oxidation of synthetic DNAs containing oxidatively damaged bases.

Recently we found that KMnO4 oxidation of DNA oligomers containing a 7,8-dihydro-8-oxoguanine (8-oxo-G) residue induces damage to the neighboring base residues; other modified bases, 7,8-dihydro-8-oxoadenine (8-oxo-A) and 5-hydroxyuracil (5-oh-U), show similar behavior in DNA. The present study indicated that the ability to induce damage, which could also occur by the oxidation of a 5-oh-C residue, was low as in the case of 5-oh-U. On the other hand, in order to examine the pathways and the intermediates for the oxidative degradation of 8-oxo-A, we have carried out the KMnO4 oxidation using an 8-oxo-2'-deoxyadenosine derivative as a model and have determined the structures of the three major products.     

DNA oxidation by charge transport in mitochondria

Sites of oxidative DNA damage in functioning mitochondria have been identified using a rhodium photooxidant as a probe. Here we show that a primer extension reaction can be used to monitor oxidative DNA damage directly in functioning mitochondria after photoreaction with a rhodium intercalator that penetrates the intact mitochondrial membrane. The complex [Rh(phi)2bpy]Cl3 (phi = 9,10-phenanthrenequinonediimine) binds to DNA within the mitochondria and, upon irradiation, initiates DNA oxidation reactions. Significantly, piperidine treatment of the mitochondria leads to protein-dependent primer extension stops spaced every approximately 20 base pairs. Hence, within the mitochondria, the DNA is well covered and packaged by proteins. Photolysis of the mitochondria containing [Rh(phi)2bpy]3+ leads to oxidative DNA damage at positions 260 and 298; both are mutational hot spots associated with cancers. The latter position is the 5'-nucleotide of conserved sequence block II and is critical to replication of the mitochondrial DNA. The oxidative damage is found to be DNA-mediated, utilizing a charge transport mechanism, as the Rh binding sites are spatially separated from the oxidation-prone regions. This long-range DNA-mediated oxidation occurs despite protein association. Indeed, the oxidation of the mitochondrial DNA leads not only to specific oxidative lesions, but also to a corresponding change in the protein-induced stops in the primer extension. Mitochondrial DNA damage promotes specific changes in protein-DNA contacts and is thus sensed by the mitochondrial protein machinery.     

Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells

Exposure to exogenous and endogenous sources cause oxidative damage to cellular macromolecules, including DNA. This results in gradual accumulation of oxidative DNA base lesions, and in order to maintain genomic stability we must have effective systems to repair this kind of damage. The accumulation of lesions is most dramatic in the mitochondrial DNA, and this may cause dysfunction and loss of cellular energy production. Base excision DNA repair (BER) is the major pathway that removes oxidative DNA base lesions, and while we know much about its mechanism in the nuclear DNA, little is yet known about this pathway in mitochondria. While nuclear BER decreases with age, the mitochondrial DNA repair may increase with age. This increase is not enough to prevent the gradual accumulation of lesions in the mitochondrial DNA with age. Accumulation of DNA lesions with age may be the underlying cause for age-associated diseases including cancer.     

Depleted uranium-catalyzed oxidative DNA damage: absence of significant alpha particle decay

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. DU possesses both a radiological (alpha particle) and a chemical (metal) component. Since DU has a low-specific activity in comparison to natural uranium, it is not considered to be a significant radiological hazard. In the current study we demonstrate that DU can generate oxidative DNA damage and can also catalyze reactions that induce hydroxyl radicals in the absence of significant alpha particle decay. Experiments were conducted under conditions in which chemical generation of hydroxyl radicals was calculated to exceed the radiolytic generation by one million-fold. The data showed that markers of oxidative DNA base damage, thymine glycol and 8-deoxyguanosine could be induced from DU-catalyzed reactions of hydrogen peroxide and ascorbate similarly to those occurring in the presence of iron catalysts. DU was 6-fold more efficient than iron at catalyzing the oxidation of ascorbate at pH 7. These data not only demonstrate that DU at pH 7 can induced oxidative DNA damage in the absence of significant alpha particle decay, but also suggest that DU can induce carcinogenic lesions, e.g. oxidative DNA lesions, through interaction with a cellular oxygen species.     

Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerability of polymorphonuclear leukocytes

Since oxidative stress is thought to play an important role in the pathogenesis and complications of diabetes, we used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as FPG (formamidopyrimidine DNA glycosylase)-sensitive sites, in peripheral blood cells (PBC) from type 2 diabetes patients and healthy controls. Oxidative DNA damage in leukocytes was increased in diabetic compared to normal subjects. However, no differences in the levels of DNA damage in isolated lymphocytes were found between the two groups. These data indicate a higher vulnerability to oxidative damage of polymorphonuclear as compared to mononuclear leukocytes in type 2 diabetes. Thus, the measurement of oxidative DNA damage in leukocytes by means of the comet assay is a suitable marker for the evaluation of systemic oxidative stress in diabetic patients.