Mechanisms of free radical-induced damage to DNA

Abstract

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5′-cyclopurine-2′-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.     

Identification and quantification of (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines in human urine as putative biomarkers of oxidatively induced damage to DNA

Biomarkers of oxidatively induced DNA damage are of great interest and can potentially be used for the early detection of disease, monitoring the progression of disease and determining the efficacy of therapy. The present work deals with the measurement in human urine of (5′R)-8,5′-cyclo-2′-deoxyadenosine (R-cdA) and (5′S)-8,5′-cyclo-2′-deoxyadenosine (S-cdA). These modified nucleosides had hitherto not been considered or investigated to be present in urine as possible biomarkers of oxidatively induced DNA damage. Urine samples were collected from volunteers, purified and analyzed by LC-MS/MS with isotope-dilution. R-cdA and S-cdA were detected in urine and quantified. Creatinine levels were also measured. In addition, we measured 8-hydroxy-2′-deoxyguanosine that is commonly used as a biomarker. This study shows, for the first time, that R-cdA and S-cdA exist in human urine and can be identified and quantified by LC-MS/MS. We propose that R-cdA and S-cdA may be well-suited biomarkers for disease processes such as carcinogenesis.     

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Commentary the Measurement of Oxidative Damage to DNA by HPLC and GC/MS Techniques

Oxidative damage to DNA has been measured by quantitating 8-hydroxy-2′-deoxyguanosine (8-OHdGuo) after enzymic digestion of DNA, followed by HPLC separation and electrochemical detection. Alternatively, 8-hydroxyguanine (and a wide range of other base-derived products of free radical attack) may be measured after acidic hydrolysis of DNA or chromatin, followed by derivatization and gas-chromatography/mass spectrometry. Both techniques have comparable sensitivity, but GC/MS enables determination of a wide variety of chemical changes to all four DNA bases and it can be applied to DNA-protein complexes. However, the two techniques do not always give similar results. Potential reasons for this are discussed. Greater attention to methodological questions is required before using measurement of 8-OHdGuo as a “routine” marker of oxidative DNA damage in vivo.     

Hydroxyl Free Radical Adduct of Deoxyguanosine: Sensitive Detection and Mechanisms of Formation

DNA or 2-deoxyguanosine reacts with hydroxyl free radical to form 8-hydroxy-deoxyguanosine (8-OH-dG). We found that 8-OH-dG can be effectively separated from deoxyguanosine by high pressure liquid chromatography and very sensitively detected using electrochemical detection. The sensitivity by electrochemical detection is about one-thousand fold enhanced over optical detection. Utilizing deoxyguanosine in bicarbonate buffer it was found that ferrous ion, but not ferric ion, was effective in forming 8-OH-dG. The hydroxyl free radical scavenging agents, thiourea and ethanol, were very effective in quenching Fe(11) mediated 8-OH-dG formation, but superoxide dismutase had very little effect.     

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis

Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences.     

Identification of DNA repair and damage induced proteins from Neurospora crassa

Summary The response of Neurospora crassa to DNA damage induced by UV irradiation has been studied using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Whole cell extracts of irradiated and untreated cultures were compared. Five polypeptides that show changes in response to DNA damage have been identified.Several mutagen sensitive strains of Neurospora were also tested for polypeptide changes on 2-D PAGE. Profiles of whole cell extracts of these mutant strains were compared to wild type. Two changes were observed in the meiotic mutant, mei-3 and one change was detected in the excision repair mutant, upr-1. Two changes were also detected in the allelic mutants, uvs-3 and nuh-4. Profiles of uvs-3 and nuh-4 revealed one polypeptide that was missing and another polypeptide which appeared to shift to a more basis position. This same shift was detected in wild type after induction by UV irradiation or heat shock.     

Analysis of modified bases in DNA by stable isotope dilution gas chromatography-mass spectrometry: 5-Methylcytosine

A sensitive assay for 5-methylcytosine in DNA has been developed based on stable isotope dilution gas chromatography-mass spectrometry with selected ion monitoring. 5-([²H₃]-Methyl)cytosine and [methyl-²H₃]thymine have been synthesized as internal standards for analysis of DNA following acid digestion, conversion of pyrimidines to volatile t-butyldimethylsilyl derivatives, and separation in 3 min by gas chromatography. Submicrogram amounts of DNA have been analyzed for 5-methylcytosine content in the range 0.02–1.5 mol%. The estimated limit of quantitative measurement is 0.3 pmol of methylated base in a DNA hydrolysate. The method is compared with other techniques for quantitative measurement of methylated bases in DNA, and 5-methylcytosine levels and precision of analysis for calf thymus, pBR322, and ΦX-174 DNAs are reported and compared with literature values. The method can readily be adapted to the accurate high-sensitivity analysis of other methylated bases in DNA.     

Facts about the artifacts in the measurement of oxidative DNA base damage by gas chromatography-mass spectrometry

Recently, several papers reported an artifactual formation of a number of modified bases from intact DNA bases during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC/MS). These reports dealt with 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra) and 5-formyluracil that represent only a small percentage of the 20 or so modified DNA bases that can be analyzed by GC/MS. Removal of intact DNA bases by prepurification of calf thymus DNA hydrolysates using HPLC was shown to prevent artifactual formation of these modified bases during derivatization. It needs to be emphasized that the procedures for hydrolysis of DNA and derivatization of DNA hydrolysates used in these papers substantially differed from the established procedures previously described. Furthermore, a large number of relevant papers reporting the levels of these modified bases in DNA of various sources have been ignored. Interestingly, the levels of modified bases reported in the literature were not as high as those reported prior to prepurification. Most values for the level of 5-OH-Cyt were even lower than the level measured after prepurification. Levels of 8-OH-Ade were quite close to, or even the same as, or smaller than the level reported after prepurification. The same holds true for 5-OHMeUra and 8-OH-Gua. All these facts raise the question of the validity of the claims about the measurement of these modified DNA bases by GC/MS.

A recent paper reported a complete destruction of 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) by formic acid under the conditions of DNA hydrolysis prior to GC/MS. The complete destruction of FapyGua and FapyAde by formic acid is in disagreement with the data on these compounds in the literature. These two compounds were measured by GC/MS following formic acid hydrolysis for many years in our laboratory and by other researchers with no difficulties. These facts clearly raise the question of the validity of the claims made about the previous measurements of these compounds by GC/MS.     

DNA damage repair is unaffected by mimicked heterozygous levels of BRCA2 in HT-29 cells

Functional loss of both alleles of the breast cancer susceptibility gene, BRCA2, facilitates tumorigenesis. However, the direct effects of BRCA2 heterozygosity remain unclear. Here, BRCA2 heterozygosity was mimicked in HT-29 colon cells by reducing levels of BRCA2 through stable RNA interference. No difference in RAD51 subcellular localization and focus formation was observed between control and mimicked heterozygous cell lines. DNA repair ability, as measured by colony survival following mitomycin C treatment and ultraviolet radiation exposure, was also unaffected by reduced levels of BRCA2. Interestingly, the growth rate of the mimicked BRCA2 heterozygous cell line was significantly lower than that of control cells. Increased expression of p53 in the mimicked heterozygous cells was observed, perhaps in response to BRCA2 deficiency. Levels of p27 were also found to be slightly increased in cells with reduced BRCA2, perhaps contributing to the slower growth rate. Overall, these results suggest that tumors are unlikely to arise directly from BRCA2 heterozygous cells without other genetic events such as loss of the wild-type BRCA2 allele and/or loss of p53 function or other cell cycle inhibitors.     

Cell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1

Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving gamma-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation.     

BRCA1和BRCA2与DNA损伤修复及转录调控

乳腺癌和卵巢癌敏感基因BRCA1和BRCA2与同源重组,DNA损伤修复,胚胎生长,转录调控及遍在蛋白化有关,其中,BRCA1和BRCA2在DNA损伤修复和转录调控中功能的确定,将有助于探讨和阐明两者的肿瘤抑制功能及其机理,作者将综述近年来有关BRCA1和BRCA2在DNA损伤修复和转录调控中功能研究的最新进展。     

Oxidative DNA damage induced by nitrotyrosine, a biomarker of inflammation

Inflammation has been postulated as a risk factor for several cancers. 3-Nitrotyrosine is a biochemical marker for inflammation. We investigated the ability of nitrotyrosine and nitrotyrosine-containing peptides (nitroY-peptide) to induce DNA damage by the experiments using 32P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and an HPLC-electrochemical detector. Nitrotyrosine and nitroY-peptide caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction. Catalase inhibited DNA damage, suggesting the involvement of H2O2. Nitrotyrosine and nitroY-peptide increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, an indicator of oxidative DNA damage. Nitrotyrosine-containing peptides of histone induced 8-oxodG formation more efficiently than free nitrotyrosine. We propose the possibility that nitrotyrosine-induced H2O2 formation and DNA damage contribute to inflammation-associated carcinogenesis.     

DNA binding and oxidative DNA damage induced by a quercetin copper(II) complex: potential mechanism of its antitumor properties

The interaction of a quercetin copper(II) complex with DNA was investigated using UV–vis spectra, fluorescence measurement, viscosity measurement, agarose gel electrophoresis, and thiobarbituric acid reactive substances assay. The results indicate that the quercetin copper(II) complex can promote the cleavage of plasmid DNA, producing single and double DNA strand breaks, and intercalate into the stacked base pairs of DNA. Moreover, the complex can induce oxidative DNA damage involving generation of reactive oxygen species such as H₂O₂ and Cu(I)OOH. In addition, the cytotoxicity experiments carried out with A549 cells confirmed its apoptosis-inducing activity. And we also demonstrate that the levels of survivin protein expression in A549 cells decreased, and that relative activity of caspase-3 increased significantly after treatment with the complex. So our results suggest that the antitumor mechanism of the quercetin copper(II) complex involves not only its oxidative DNA damage with generation of reactive oxygen species but also its specific interaction with DNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Suppression of UVC-induced cell damage and enhancement of DNA repair by the fermented milk, Kefir

An aqueous extract of Kefir, fermented milk originally produced in the Caucasus mountains, suppressed morphological changes of human melanoma HMV-1 and SK-MEL cells and human normal fibroblastTIG-1 cells caused by UVC-irradiation, suggesting that UV damage can be suppressed by the Kefir extract. The addition of the Kefir extract after UVC-irradiation of HVM-1 cells resulted in a remarkable decrease in intracellular reactive oxygen species (ROS) which had been increased by UVC irradiation. The Kefir extract also stimulated unscheduled DNA synthesis and suppressed UVC-induced apoptosis of HMV-1 cells. A colony formation assay revealed that the Kefir extract rescued HMV-1 cells from cell death caused by UVC irradiation. The Kefir extract, as well as methyl methanethiosulfonate which is known to enhance the nucleotide excision repair (NER) activity, exhibited strong thymine dimer repair-enhancing activity. Epigalocatechin exhibited a weak NER activity but vitamins A, C, and E and catechin showed no NER activity. The thymine dimer repair-enhancing factors in the Kefir extract were heat-stable and assumed to be molecules with a molecular weight of less than 5000. The treatment of HMV-1 cells with the Kefir extract during or before UVC- irradiation also prevented the generation of ROS and thymine dimmer, and suppressed the apoptosis of HMV-1 cells, suggesting that application of Kefir can prevent UV damage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genotoxicity of streptozotocin in normal and cancer cells and its modulation by free radical scavengers

Streptozotocin (STZ) is an antibiotic which can be used to induce diabetes in experimental animals in order to have an insight into pathogenesis of this disease. To use STZ as a diabetogenic substance, its molecular mode of action should be elucidated. Using the alkaline comet assay, we showed that STZ at concentrations in the range 0.01-100 micromol/L induced DNA damage in normal human lymphocytes and HeLa cancer cells in a dose-dependent manner. Lymphocytes were able to remove damage to their DNA within a 30-min repair incubation, whereas HeLa cells completed the repair in 60 min. Vitamins C and E at 10 and 50 micromol/L diminished the extent of DNA damage induced by 50 micromol/L STZ. Pretreatment of the lymphocytes with the nitrone spin trap, alpha-(4-pyridil-1-oxide)-N-tert-butylnitrone (POBN) or ebselen, which mimics glutathione peroxidase, or pyrrolidine dithiocarbamate (PDTC) reduced the extent of DNA damage evoked by STZ. The cells exposed to STZ and treated with endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. These results suggest that free radicals may be involved in the formation of DNA lesions induced by streptozotocin. The drug can also alkylate DNA bases. This broad range of DNA damage induced by STZ indicates that the drug may seriously affect genomic stability in normal and pathological cells.     

Distinct mechanisms of DNA damage in apoptosis induced by quercetin and luteolin

Quercetin has been reported to have carcinogenic effects. However, both quercetin and luteolin have anti-cancer activity. To clarify the mechanism underlying the carcinogenic effects of quercetin, we compared DNA damage occurring during apoptosis induced by quercetin with that occuring during apoptosis induced by luteolin. Both quercetin and luteolin similarly induced DNA cleavage with subsequent DNA ladder formation, characteristics of apoptosis, in HL-60 cells. In HP 100 cells, an H₂O₂-resistant clone of HL-60 cells, the extent of DNA cleavage and DNA ladder formation induced by quercetin was less than that in HL-60 cells, whereas differences between the two cell types were minimal after treatment with luteolin. In addition, quercetin increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in HL-60 cells but not in HP 100 cells. Luteolin did not increase 8-oxodG formation, but inhibited topoisomerase II (topo II) activity of nuclear extract more strongly than quercetin and cleaved DNA by forming a luteolin-topo II-DNA ternary complex. These results suggest that quercetin induces H₂O₂-mediated DNA damage, resulting in apoptosis or mutations, whereas luteolin induces apoptosis via topo II-mediated DNA cleavage. The H₂O₂-mediated DNA damage may be related to the carcinogenic effects of quercetin.     

Protective effect of quercetin on hyperglycemia,oxidative stress and DNA damage in alloxan induced type 2 diabetic mice

Aims

Quercetin is a natural polyphenolic flavonoid and acts as a quencher for reactive oxygen species generated by any physical or chemical action. In type 2 diabetes mellitus (T2DM) the basic characteristic feature is hyperglycemia which leads to complications involving oxidative stress. In view of this, the present study was conducted to examine the effect of quercetin in T2DM.

Main methods

A total of 18 mice were divided into three groups, vis control, diabetic and diabetic treated with quercetin. Fasting blood glucose (FBG) levels and anti-oxidant enzyme activity were assayed. Creatinine, urea, lipid peroxidation, GLUT4 expression and DNA damage were also measured.

Key findings

A significant decrease in FBG level and liver and kidney marker enzymes was observed in the quercetin treated group as compared to the diabetic one. Glutathione, SOD, catalase, and glutathione-S-transferase levels were also found to be increased on quercetin supplementation. Thiobarbituric acid-reactive substance level was decreased while GLUT4 expression levels were increased in the treated group. DNA damage was also affected positively by quercetin when subjected with single cell alkaline gel electrophoresis. Thus, we may suggest an anti-oxidant potential and protective effect of quercetin in T2DM mice.

Significance

From this study, we conclude that quercetin ameliorates hyperglycemia and oxidative stress, by blunting free radical induced toxicity in T2DM.     

HBXIP,a binding protein of HBx,regulates maintenance of the G2/M phase checkpoint induced by DNA damage and enhances sensitivity to doxorubicin-induced cytotoxicity

To maintain the integrity of the genome, cells need to detect and repair DNA damage before they complete cell division. Hepatitis B x-interacting protein (HBXIP), a binding protein of HBx (Hepatitis B virus × protein), is aberrantly overexpressed in human cancer cells and show to promote cell proliferation and inhibit apoptosis. The present study is designed to investigate the role of HBXIP on the DNA damage response. Our results show that HBXIP acts as an important regulator of G2/M checkpoint in response to DNA damage. HBXIP knockdown increases phospho-histone H2AX expression and foci formation after treatment with ionizing radiation (IR). HBXIP regulates the ATM-Chk2 pathway following DNA damage. Depletion of HBXIP abrogates IR-induced G2/M cell cycle checkpoints, accompanying decrease the expression of phospho-Cdc25C, phospho-Cdc2 (Tyr15) and p27. We also show that downregulation of HBXIP expression sensitizes cancer cells to chemotherapy, as evidenced by an increase in apoptosis and cleavage of caspase-3 and caspase-9. Our data suggest that HBXIP can function as a mediator protein for DNA damage response signals to activate the G2/M checkpoint to maintain genome integrity and prevent cell death.