Evidence for upregulated repair of oxidatively induced DNA damage in human colorectal cancer

Carcinogenesis may involve overproduction of oxygen-derived species including free radicals, which are capable of damaging DNA and other biomolecules in vivo. Increased DNA damage contributes to genetic instability and promote the development of malignancy. We hypothesized that the repair of oxidatively induced DNA base damage may be modulated in colorectal malignant tumors, resulting in lower levels of DNA base lesions than in surrounding pathologically normal tissues. To test this hypothesis, we investigated oxidatively induced DNA damage in cancerous tissues and their surrounding normal tissues of patients with colorectal cancer. The levels of oxidatively induced DNA lesions such as 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine and (5'S)-8,5'-cyclo-2'-deoxyadenosine were measured by gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution tandem mass spectrometry. We found that the levels of these DNA lesions were significantly lower in cancerous colorectal tissues than those in surrounding non-cancerous tissues. In addition, the level of DNA lesions varied between colon and rectum tissues, being lower in the former than in the latter. The results strongly suggest upregulation of DNA repair in malignant colorectal tumors that may contribute to the resistance to therapeutic agents affecting the disease outcome and patient survival. The type of DNA base lesions identified in this work suggests the upregulation of both base excision and nucleotide excision pathways. Development of DNA repair inhibitors targeting both repair pathways may be considered for selective killing of malignant tumors in colorectal cancer.     

Progress in the analysis of urinary oxidative DNA damage

Oxidative DNA damage has been implicated to be important in the pathogenesis of many diseases, including cancer and heart disease. The assessment of damage in various biological matrices, such as DNA, serum, and urine, is vital to understanding this role and subsequently devising intervention strategies. Despite the numerous techniques to measure oxidative DNA damage products in urine, it remains unclear what these measurements truly represent. Sources of urinary lesions may include the diet, cell death, and, of most interest, DNA repair. Were it possible to exclude the two former contributions, a noninvasive assay for DNA repair would be invaluable in the study of DNA damage and disease. This review highlights that, although progress has been made, significant work remains. Diet, cell death, and repair need continued examination to further elucidate the kinetics of lesion formation and clearance in vivo. Studies from our laboratory and others are making appreciable progress towards the interpretation of urinary lesion measurements along with the development of urinary assays to evaluate DNA repair. Upon establishment of these details, urinary oxidative DNA damage measurements may become more than a reflection of generalized oxidative stress.     

Accumulation of Oxidatively Induced DNA Damage in Human Breast Cancer Cell Lines Following Treatment with Hydrogen Peroxide

Breast cancer is a leading cause of cancer deaths in women. Although the causes of this disease are largely unknown, inefficient repair of oxidatively induced DNA lesions has been thought to play a major role in the transformation of normal breast tissue to malignant breast tissue. Previous studies have revealed higher levels of 8-hydroxyguanine in malignant breast tissue compared to non-malignant breast tissue. Furthermore, some breast cancer cell lines have greatly reduced capacity to repair this lesion suggesting that oxidatively induced DNA lesions may be elevated in breast cancer cells. We used liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of 8-hydroxy-2’-deoxyadenosine, (5’S)-8,5’-cyclo-2’-deoxyadenosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine in MCF-7 and HCC1937 breast cancer cell lines before and after exposure to H2O2 followed by a DNA repair period. We show that H2O2-treated HCC1937 and MCF-7 cell lines accumulate significantly higher levels of these lesions than the untreated cells despite a 1 h repair period. In contrast, the four lesions did not accumulate to any significant level in H2O2-treated non-malignant cell lines, AG11134 and HCC1937BL. Furthermore, MCF-7 and HCC1937 cell lines were deficient in the excision repair of all the four lesions studied. These results suggest that oxidatively induced DNA damage and its repair may be critical in the etiology of breast cancer.     

Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease

Mitochondrial generation of reactive oxygen species (ROS) is increased in mice with fatty livers induced by genetic obesity, chronic consumption of ethanol, or methionine/choline-deficient diets. Both nuclear and mitochondrial (mt) DNA are targets for ROS-induced damage and accumulate hydroxylated bases, such as 8-hydroxy-2'-deoxyguanosine (8-oxoG) and base substitution of adenine with 8-oxoG (A*8-oxoG), that introduce mutations that promote cancer as well as cell death. The mammalian homolog of the bacterial DNA mismatch repair enzyme MutY (MYH) removes A*8-oxoG from nuclear and mtDNA, reduces 8-oxoG accumulation, and restores genomic stability after ROS exposure. Cumulative damage to mtDNA occurs as fatty liver disease progresses. Therefore, differences in hepatic MYH activity may influence the severity of fatty liver disease. To evaluate this hypothesis, we compared mtH2O2 production, MYH expression, oxidative DNA damage, and hepatocyte death in healthy mice and different mouse models of fatty liver disease. The results show that diverse causes of steatohepatitis increase mtROS production, limit repair of mtDNA, and oxidatively damage DNA. However, there are important differences in the DNA repair response to oxidant stress among mouse models of fatty liver disease. Independent of the degree of mtROS generation, models with the least MYH exhibit the greatest accumulation of 8-oxoG and the most hepatocyte death. These findings raise the intriguing possibility that inherited or acquired differences in DNA repair enzyme activity may underlie some of the interindividual differences in fatty liver disease outcomes.     

Evidence for attenuated cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine removal in cancer patients

Measurement of the products of oxidatively damaged DNA in urine is a frequently used means by which oxidative stress may be assessed non-invasively. We believe that urinary DNA lesions, in addition to being biomarkers of oxidative stress, can potentially provide more specific information, for example, a reflection of repair activity. We used high-performance liquid chromatography prepurification, with gas chromatography-mass spectrometry (LC-GC-MS) and ELISA to the analysis of a number of oxidative [e.g., 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-guanine, 5-(hydroxymethyl)uracil], non-oxidative (cyclobutane thymine dimers) and oligomeric DNA products in urine. We analysed spot urine samples from 20 healthy subjects, and 20 age- and sex-matched cancer patients. Mononuclear cell DNA 8-oxodG levels were assessed by LC-EC. The data support our proposal that urinary DNA lesion products are predominantly derived from DNA repair. Furthermore, analysis of DNA and urinary 8-oxodG in cancer patients and controls suggested reduced repair activity towards this lesion marker in these patients.     

DNA repair: insights from urinary lesion analysis

Due to various confounding factors, namely dietary contribution and cell death, measurement of urinary 8-oxo-2'-deoxyguanosine (8-oxodG) has long been considered to be no more than a marker of generalised oxidative stress. Indeed, the action of no single enzyme has been reported to excise 8-oxodG from DNA. However, analysis of recent research has suggested that these confounders may be circumvented, which, combined from work from the authors' laboratory, indicates that urinary 8-oxodG has the potential to become a most important marker of oxidative damage to, and repair of, DNA.     

Ischemic preconditioning induces XRCC1, DNA polymerase-beta, and DNA ligase III and correlates with enhanced base excision repair

Neuronal protection induced by ischemic preconditioning has an important role in the reduction of stroke volume and attenuation of neuronal cell death. Ischemic injury is associated with increased oxidative DNA damage, and failure to efficiently repair these oxidatively damaged lesions results in the accumulation of mutations and neuronal cell death. Although the effects of ischemic tolerance can have profound implications, the precise mechanisms mediating this phenomenon remain unclear. The base excision repair (BER) pathway has a major role in the repair of oxidative DNA base damage after ischemic injury. Using a rat model of ischemic preconditioning, we now report that the neuronal protection observed after induction of ischemic tolerance is associated with increased BER. In situ detection of single-strand breaks and apurinic/apyrimidinic sites reduced to baseline levels after reperfusion following ischemic preconditioning. By contrast, no change was seen in the quantity of in situ lesions after reperfusion in non-ischemic preconditioned brain. Induction of the BER proteins XRCC1, DNA polymerase-beta, and DNA ligase III was seen after reperfusion in ischemically conditioned brain. Moreover, an increase in binding between XRCC1 and DNA polymerase-beta was seen under these conditions, as might be expected during formation of functional BER complexes. Using in vitro BER oligonucleotides, we directly demonstrated an increase in total BER capacity of nuclear extracts prepared from ischemic-conditioned brain after reperfusion compared with sham-operated brain. These findings provide direct evidence that increased BER is associated with the neuroprotection induced after ischemic preconditioning, and provides important new mechanistic insight into the important biologic pathways that protect neurons against irreversible ischemic injury.     

Processing of DNA damage clusters in human cells: current status of knowledge

Eukaryotic cells exposed to DNA damaging agents activate important defensive pathways by inducing multiple proteins involved in DNA repair, cell cycle checkpoint control and potentially apoptosis. After the acceptance of the hypothesis that oxidatively generated clustered DNA lesions (OCDL: closely spaced DNA lesions) can be induced even by low doses of ionizing radiation or even endogenously, and significant advances have been made in the understanding of the biochemistry underlying the repair of closely spaced DNA lesions, many questions still remain unanswered. The major questions that have to be answered in the near future are: 1) how human cells process these types of DNA damage if they repair them at all, 2) under what conditions a double strand break (DSB) may be created during the processing of two closely spaced DNA lesions and 3) what type of repair protein interactions govern the processing of complex DNA damage? The data existing so far on human cells and tissues are very limited and in some cases contradicting. All of them though agree however on the major importance of gaining mechanistic insights on the pathways used by the cell to confront and process complex DNA damage located in a small DNA volume and the need of more in depth analytical studies. We selectively review recently-obtained data on the processing of non-DSB DNA damage clusters in human cells and tissues and discuss the current status of knowledge in the field.     

Monoclonal antibody to single-stranded DNA: a potential tool for DNA repair studies

Growing evidence suggests that DNA repair capacity is an important factor in cancer risk and is therefore essential to assess. Immunochemical assays are amenable to the detection of repair products in complex matrices, such as urine, facilitating noninvasive measurements, although diet and extra-DNA sources of lesion can confound interpretation. The production of single-stranded, lesion-containing DNA oligomers characterises nucleotide excision repair (NER) and hence defines the repair pathway from which a lesion may be derived. Herein we describe the characterisation of a monoclonal antibody which recognises guanine moieties in single-stranded DNA. Application of this antibody in ELISA, demonstrated such oligomers in supernatants from repair-proficient cells post-insult. Testing of urine samples from volunteers demonstrated a relationship between oligomer levels and two urinary DNA damage products, thymine dimers and 8-oxo-2'-deoxyguanosine, supporting our hypothesis that NER gives rise to lesion-containing oligomers which are specific targets for the investigation of DNA repair.     

Bisphenol A Promotes Cell Survival Following Oxidative DNA Damage in Mouse Fibroblasts

Bisphenol A (BPA) is a biologically active industrial chemical used in production of consumer products. BPA has become a target of intense public scrutiny following concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. Recent studies link BPA with the generation of reactive oxygen species, and base excision repair (BER) is responsible for removing oxidatively induced DNA lesions. Yet, the relationship between BPA and BER has yet to be examined. Further, the ubiquitous nature of BPA allows continuous exposure of the human genome concurrent with the normal endogenous and exogenous insults to the genome, and this co-exposure may impact the DNA damage response and repair. To determine the effect of BPA exposure on base excision repair of oxidatively induced DNA damage, cells compromised in double-strand break repair were treated with BPA alone or co-exposed with either potassium bromate (KBrO₃) or laser irradiation as oxidative damaging agents. In experiments with KBrO₃, co-treatment with BPA partially reversed the KBrO₃-induced cytotoxicity observed in these cells, and this was coincident with an increase in guanine base lesions in genomic DNA. The improvement in cell survival and the increase in oxidatively induced DNA base lesions were reminiscent of previous results with alkyl adenine DNA glycosylase-deficient cells, suggesting that BPA may prevent initiation of repair of oxidized base lesions. With laser irradiation-induced DNA damage, treatment with BPA suppressed DNA repair as revealed by several indicators. These results are consistent with the hypothesis that BPA can induce a suppression of oxidized base lesion DNA repair by the base excision repair pathway.     

Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or “short-patch” (SP-BER), and “long-patch” BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase β (Polβ) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP- and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polβ itself. We have developed a new approach for determining the relative contributions of SP- and LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single- and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polβ-deficient cell extracts supplemented with Polλ was ∼80% SP-BER. The results show that recombinant Polλ can contribute to both SP- and LP-BER. However, endogenous Polλ, which is present at a level ˜50% that of Polβ in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.     

DNA Repair: Insights from Urinary Lesion Analysis

Due to various confounding factors, namely dietary contribution and cell death, measurement of urinary 8-oxo-2'-deoxyguanosine (8-oxodG) has long been considered to be no more than a marker of generalised oxidative stress. Indeed, the action of no single enzyme has been reported to excise 8-oxodG from DNA. However, analysis of recent research has suggested that these confounders may be circumvented, which, combined from work from the authors' laboratory, indicates that urinary 8-oxodG has the potential to become a most important marker of oxidative damage to, and repair of, DNA.     

Five repair pathways in one context: chromatin modification during DNA repair.

The eukaryotic cell is faced with more than 10 000 various kinds of DNA lesions per day. Failure to repair such lesions can lead to mutations, genomic instability, or cell death. Therefore, cells have developed 5 major repair pathways in which different kinds of DNA damage can be detected and repaired: homologous recombination, nonhomologous end joining, nucleotide excision repair, base excision repair, and mismatch repair. However, the efficient repair of DNA damage is complicated by the fact that the genomic DNA is packaged through histone and nonhistone proteins into chromatin, a highly condensed structure that hinders DNA accessibility and its subsequent repair. Therefore, the cellular repair machinery has to circumvent this natural barrier to gain access to the damaged site in a timely manner. Repair of DNA lesions in the context of chromatin occurs with the assistance of ATP-dependent chromatin-remodeling enzymes and histone-modifying enzymes, which allow access of the necessary repair factors to the lesion. Here we review recent studies that elucidate the interplay between chromatin modifiers / remodelers and the major DNA repair pathways.     

Influence of the OGG1 Ser326Cys polymorphism on oxidatively damaged DNA and repair activity

Oxidatively damaged DNA base lesions are considered to be mainly repaired by 8-oxoguanine DNA glycosylase (OGG1) mediated pathways. We investigated the effect of the OGG1 Ser326Cys polymorphism on the level and repair of oxidatively damaged DNA in mononuclear blood cells (MNBC) by means of the comet assay. We collected blood samples from 1,019 healthy subjects and genotyped for the OGG1 Ser326Cys polymorphism. We found 49 subjects homozygous for the variant genotype (Cys/Cys) and selected same numbers of age-matched subjects with the heterozygous (Ser/Cys) and homozygous wild-type genotype (Ser/Ser). Carriers of the Cys/Cys genotype had higher levels of formamidopyrimidine DNA glycosylase (FPG) sensitive sites in MNBC (0.31 ± 0.03 lesions/10(6)bp) compared to Ser/Ser (0.19 ± 0.02 lesions/10(6)bp, P<0.01). The level of hOGG1 sensitive sites in MNBC from the Ser326Cys carriers (0.19 ± 0.16 lesions/10(6) bp) was also higher compared to the Ser/Ser genotype (0.11 ± 0.09 lesions/10(6) bp, P<0.05). Still, there was no genotype-related difference in DNA repair incision activity of MNBC extracts on nucleoids with oxidatively damaged DNA induced by Ro19-8022/white light (P=0.20). In addition, there were no differences in the expression of OGG1 (P=0.69), ERCC1 (P=0.62), MUTYH (P=0.85), NEIL1 (P=0.17) or NUDT1 (P=0.48) in whole blood. Our results indicate that the OGG1 Ser326Cys polymorphism has limited influence on the DNA repair incisions by extracts of MNBC, whereas the apparent increased risk of cancer in subjects with the Cys/Cys genotype may be because of higher levels of oxidatively damaged DNA.     

The novel DNA glycosylase,NEIL1, protects mammalian cells from radiation-mediated cell death

DNA damage mediated by reactive oxygen species generates miscoding and blocking lesions that may lead to mutations or cell death. Base excision repair (BER) constitutes a universal mechanism for removing oxidatively damaged bases and restoring the integrity of genomic DNA. In Escherichia coli, the DNA glycosylases Nei, Fpg, and Nth initiate BER of oxidative lesions; OGG1 and NTH1 proteins fulfill a similar function in mammalian cells. Three human genes, designated NEIL1, NEIL2 and NEIL3, encode proteins that contain sequence homologies to Nei and Fpg. We have cloned the corresponding mouse genes and have overexpressed and purified mNeil1, a DNA glycosylase that efficiently removes a wide spectrum of mutagenic and cytotoxic DNA lesions. These lesions include the two cis-thymineglycol(Tg) stereoisomers, guanine- and adenine-derived formamidopyrimidines, and 5,6-dihydrouracil. Two of these lesions, fapyA and 5S,6R thymine glycol, are not excised by mOgg1 or mNth1. We have also used RNA interference technology to establish embryonic stem cell lines deficient in Neil1 protein and showed them to be sensitive to low levels of gamma-irradiation. The results of these studies suggest that Neil1 is an essential component of base excision repair in mammalian cells; its presence may contribute to the redundant repair capacity observed in Ogg1 -/- and Nth1 -/- mice.     

Harmonising measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and urine

Levels of oxidatively damaged cellular DNA and urinary excretion of damaged 2'-deoxyribonuclosides are widely measured in biomonitoring studies examining the role of oxidative stress induced by environmental exposures, lifestyle factors and development of disease. This has promoted efforts to harmonise measurements of oxidised guanine nucleobases by the variety of analytical approaches for DNA and urinary levels of damage, in multi-laboratory trials that are centred in Europe. The large inter-laboratory variation reported of values of oxidatively damaged DNA is reduced by harmonising assay protocols. Recent attention on optimal conditions for the comet assay may lead to better understanding of the most critical steps in procedure, which generate variation in DNA damage levels between laboratories. Measurements of urinary excretion of oxidatively generated 8-oxo-7,8-dihydro-2'-deoxyguanosine also show large differences between different methods, where chromatographic techniques generally show more reliable results than antibody-based methods. In this case, standardising calibrants is aimed at improving within technique agreement.     

Substantial decrease of urinary 8-oxo-7,8-dihydroguanine, a product of the base excision repair pathway, in DNA glycosylase defective mice

Genome integrity is maintained via removal (repair) of DNA lesions and an increased load of such DNA damage has been linked to numerous pathological conditions, including carcinogenesis and ageing. 8-Oxo-7,8-dihydroguanine is one of the most critical lesions of this type. The free 8-oxo-7,8-dihydroguanine produced by the action of a specific DNA glycosylase is a potential source of this compound in urine. To date, there has been no direct, experimental evidence demonstrating that urinary 8-oxo-7,8-dihydroguanine is produced by the base excision repair pathway. For clarification of this issue, we applied a recently developed methodology which involved high performance liquid chromatography pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection to compare the urinary excretion rate of 8-oxo-7,8-dihydroguanine in wild type and OGG1 glycosylase knock out mice. Our study revealed a 26% reduction in urinary level of 8-oxo-7,8-dihydroguanine in OGG1 deficient mice in comparison with the wild type strain. This clearly indicates that the mouse OGG1 glycosylase contributes significantly to the generation of urinary 8-oxo-7,8-dihydroguanine. Therefore, urinary measurements of 8-oxo-7,8-dihydroguanine may be attributed to DNA damage and repair, which in turn suggests that they may be useful in studying associations between DNA repair and disease.     

Repair of exogenous (plasmid) DNA damaged by photoaddition of 8-methoxypsoralen in the yeast Saccharomyces cerevisiae

The contribution of different repair pathways to the repair of 8-methoxypsoralen (8-MOP) plus UVA induced lesions on a centromeric plasmid (YCp50) was investigated in the yeast Saccharomyces cerevisiae using the lithium acetate transformation method. The pathways of excision-resynthesis (RAD1) and recombination (RAD52) were found to be involved in the repair of exogenous as well as of genomic DNA. Mutants in RAD6 and PSO2 genes showed the same transformation efficiency with 8-MOP plus UVA treated plasmid as wild-type cells suggesting that these latter pathways involved in mutagenesis are not operating on plasmid DNA although required for the repair of 8-MOP photoadducts induced in genomic DNA. These results indicate that DNA-repair gene products may be differently involved in the repair of exogenous and endogenous DNA depending on the repair system and the nature of the DNA damage considered.     

Mechanism of metal-mediated DNA damage and apoptosis induced by 6-hydroxydopamine in neuroblastoma SH-SY5Y cells

6-Hydroxydopamine (6-OHDA) is a neurotoxin to produce an animal model of Parkinson's disease. 6-OHDA increased the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), a biomarker of oxidatively damaged DNA, and induced apoptosis in human neuroblastoma SH-SY5Y cells. Iron or copper chelators inhibited 6-OHDA-induced 8-oxodG formation and apoptosis. Thus, iron and copper are involved in the intracellular oxidatively generated damage to DNA, a stimulus for initiating apoptosis. This study examined DNA damage caused by 6-OHDA plus metal ions using (32)P-5'-end-labelled DNA fragments. 6-OHDA increased levels of oxidatively damaged DNA in the presence of Fe(III)EDTA or Cu(II). Cu(II)-mediated DNA damage was stronger than Fe(III)-mediated DNA damage. The spectrophotometric detection of p-quinone and the scopoletin method showed that Cu(II) more effectively accelerated the 6-OHDA auto-oxidation and H(2)O(2) generation than Fe(III)EDTA. This study suggests that copper, as well as iron, may play an important role in 6-OHDA-induced neuronal cell death.     

Measurement of cellular repair activities for oxidative DNA damage

Two related assays capable of determining cell extract repair activities for different oxidative lesions in DNA are described. Both assays measure the incorporation of radiolabeled nucleotides during repair of an oxidatively damaged template in a cell-free system. The assays differ in the type of oxidative damage present in the DNA. In one, singlet oxygen is used to generate predominantly 8-oxo-2'-deoxyguanosine lesions. In the other, hydroxyl radicals are used to generate a broad spectrum of damage including oxidized bases and strand breaks. Assay conditions were adjusted to ensure that radiolabel incorporation was directly proportional to cell extract repair activity. These assays represent sensitive tools for investigating the regulation of repair systems for oxidative DNA damage.