Chemical and biological consequences of oxidatively damaged guanine in DNA

Abstract

Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after the incubation of 2'-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

Identification of the main oxidation products of 8-methoxy-2'-deoxyguanosine by singlet molecular oxygen

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.     

DNA damage in digestive gland and mantle tissue of the mussel Perna perna

Data concerning the susceptibility of DNA to damage by reactive oxygen and nitrogen species and other endogenous compounds produced by physiological stress in marine organisms is lacking, especially in bivalve mollusks. In this article, we analyzed the background levels of lipid peroxidation (malondialdehyde, MDA), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 1,N2-etheno-2'-deoxyguanosine (1,N2-epsilon dGuo) in digestive gland and mantle tissue of mussels Perna perna collected at a cultivation zone in Florianópolis (Santa Catarina, Brazil). The present data point to the possibility of the use of both 8-oxodGuo and 1,N2-epsilon dGuo as complementary indicators of oxidative stress processes in mussels. A sensitive method coupling high performance liquid chromatography to mass spectrometry was applied for the detection of 1,N2-epsilon dGuo in mussel tissues.     

DNA damage and 2'-deoxyguanosine oxidation induced by S(IV) autoxidation catalyzed by copper(II) tetraglycine complexes: synergistic effect of a second metal ion

S(IV) (SO(2),HSO(3)(-)andSO(3)(2-)) autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0x10(-4)M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0x10(-4)M) in the presence of Ni(II) or Mn(II) (10(-7)-10(-6)M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. SO*(3)(-) and HO* radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide).     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Abstract

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) after the incubation of 2′-deoxyguanosine (dGuo) with ChOOH and Cu²⁺. In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

DNA damage by 5-aminolevulinic and 4,5-dioxovaleric acids in the presence of ferritin

Cellular accumulation of 5-aminolevulinic acid (ALA), the first specific intermediate of heme biosynthesis, is correlated in liver biopsy samples of acute intermittent porphyria affected patients with an increase in the occurrence of hepatic cancers and the formation of ferritin deposits in hepatocytes. 5-Aminolevulinic acid is able to undergo enolization and to be subsequently oxidized in a reaction catalyzed by iron complexes yielding 4,5-dioxovaleric acid (DOVA). The released superoxide radical (O(*-)(2)) is involved in the formation of reactive hydroxyl radical ((*)OH) or related species arising from a Fenton-type reaction mediated by Fe(II) and Cu(I). This leads to DNA oxidation. The metal catalyzed oxidation of ALA may be exalted by the O(*-)(2) and enoyl radical-mediated release of Fe(II) ions from ferritin. We report here the potentiating effect of ferritin on the ALA-mediated cleavage of plasmid DNA and the enhancement of the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo). Plasmid pBR322 was incubated with ALA and varying amounts of purified ferritin. DNA damage was assessed by gel electrophoresis analysis of the open and the linear forms of the plasmid from the native supercoiled structure. Addition of either the DNA compacting polyamine spermidine or the metal chelator ethylenediaminetetraacetic acid (EDTA) inhibited the damage. It was also shown that ALA in the presence of ferritin is able to increase the oxidation of the guanine moiety of monomeric 2'-deoxyguanosine (dGuo) and calf thymus DNA (CTDNA) to form 8-oxodGuo as inferred from high performance liquid chromatography (HPLC) measurements using electrochemical detection. The formation of the adduct dGuo-DOVA was detected in CTDNA upon incubation with ALA and ferritin. In a subsequent investigation, the aldehyde DOVA was also able to induces strand breaks in pBR322 DNA.     

Protective effect of lycopene on lipid peroxidation and oxidative DNA damage in cell culture

A high incidence of cancer has been correlated with chronic iron overload, and carotenoids are of interest as possible anticarcinogens. We have investigated the effect of lycopene on lipid peroxidation and on the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in CV1-P monkey cells exposed to ferric nitrilotriacetate (Fe-NTA) plus ascorbate. Cells supplemented with lycopene (20 pmol/10(6) cells) showed a reduction of 86% in Fe-NTA/ascorbate-induced lipid peroxidation (TBARS). Levels of 8-oxodGuo rose from 1.59+/-0.09 residues/10(6) dGuo in the control cells to 14.02+/-0.41 residues/10(6) dGuo after incubation with (1:4 mM) Fe-NTA/ascorbate (40 microM). Lycopene supplementation decreased in 77% the 8-oxodGuo levels in Fe-NTA/ascorbate-treated cells. These results indicate that lycopene can protect mammalian cells against membrane and DNA damage and possibly play a protective role against tumor promotion associated with oxidative damage.     

Hydrogen peroxide causes greater oxidation in cellular RNA than in DNA

Human A549 lung epithelial cells were challenged with 18O-labeled hydrogen peroxide ([18O]-H2O2), the total RNA and DNA extracted in parallel, and analyzed for 18O-labeled 8-oxo-7,8-dihydroguanosine ([18O]-8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine ([18O]-8-oxodGuo) respectively, using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-MS/MS). [18O]-H2O2 exposure resulted in dose-response formation of both [18O]-8-oxoGuo and [18O]-8-oxodGuo and 18O-labeling of guanine in RNA was 14-25 times more common than in DNA. Kinetics of formation and subsequent removal of oxidized nucleic acids adducts were also monitored up to 24 h. The A549 showed slow turnover rates of adducts in RNA and DNA giving half-lives of approximately 12.5 h for [18O]-8-oxoGuo in RNA and 20.7 h for [18O]-8-oxodGuo in DNA, respectively.     

Oxidative and alkylating damage in DNA

Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.     

Simultaneous determination of O6-methyl-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, and 1,N6-etheno-2'-deoxyadenosine in DNA using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

O(6)-Methyl-2'-deoxyguanosine (O(6)-mdGuo), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), and 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O(6)-[(2)H(3)]mdGuo was synthesized and used as internal standard. The limits of quantification for O(6)-mdGuo, 8-oxodGuo, and epsilondAdo were 24, 98, and 48 fmol on column, respectively. The method showed linearity in the range 0.24-125 pmol/ml, 0.98-125 pmol/ml, and 0.49-62.5 pmol/ml for the three adducts, respectively. The inter-day precision in the linear concentration range was between 1.7 and 9.3% for O(6)-mdGuo, 10.6 and 28.7% for 8-oxodGuo, and 6.2 and 10.4%, for epsilondAdo. In DNA isolated from liver of untreated 12-week-old female F344 rats, O(6)-mdGuo was above the limit of detection (37 adducts per 10(9) normal nucleosides) but could not be quantified. 8-oxodGuo and epsilondAdo showed background levels of 500 and 130 adducts per 10(9) normal nucleosides, respectively. DNA analyzed 1h after treatment of rats with dimethylnitrosamine by oral gavage of 50 microg/kg b.wt. did not affect the levels of 8-oxodGuo and epsilondAdo but resulted in 200 O(6)-mdGuo adducts per 10(9) normal nucleosides. The method developed will be of use to study the biological significance of exogenous DNA adducts as an increment to background DNA damage and the role of modulating factors, such as DNA repair.     

Novel products generated from 2'-deoxyguanosine by hypochlorous acid or a myeloperoxidase-H2O2-Cl- system: identification of diimino-imidazole and amino-imidazolone nucleosides

Hypochlorous acid (HOCl), generated by myeloperoxidase from H2O2 and Cl-, plays an important role in host defense and inflammatory tissue injury. We report here the identification of products generated from 2'-deoxyguanosine (dGuo) with HOCl. When 1 mM dGuo and 1 mM HOCl were reacted at pH 7.4 and 37 degrees C for 15 min and the reaction was terminated with N-acetylcysteine (N-AcCys), two products were generated in addition to 8-chloro-2'-deoxyguanosine (8-Cl-dGuo). One was identified as an amino-imidazolone nucleoside (dIz), a previously reported product of dGuo with other oxidation systems. The other was identified as a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dDiz) by spectrometric measurements. The yields were 1.4% dDiz, 0.6% dIz and 2.4% 8-Cl-dGuo, with 61.5% unreacted dGuo. Precursors of dDiz and dIz containing a chlorine atom were found in the reaction solution in the absence of termination by N-AcCys. dDiz, dIz and 8-Cl-dGuo were also formed from the reaction of dGuo with myeloperoxidase in the presence of H2O2 and Cl- under mildly acidic conditions. These results imply that dDiz and dIz are generated from dGuo via chlorination by electrophilic attack of HOCl and subsequent dechlorination by N-AcCys. These products may play a role in cytotoxic and/or genotoxic effects of HOCl.     

Measurement of the main photooxidation products of 2'-deoxyguanosine using chromatographic methods coupled to mass spectrometry

Analytical methods were developed for the measurement of the main photooxidation products of 2'-deoxyguanosine (dGuo), arising from either the type I (electron transfer) or the type II (singlet oxygen) photosensitization mechanism. Oxidation of dGuo by a type I mechanism leads to the predominant formation of 2, 2-diamino-5-[2-deoxy-beta-d-erythro-pentofuranosyl)amino]-5(2H)-oxazo lone. On the other hand, the two 4R and 4S diastereomers of 4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine are the main singlet oxygen-mediated dGuo oxidation products. The modified nucleosides were measured by either gas chromatography coupled to mass spectrometry after silylation of the sample or by high-performance liquid chromatography associated to tandem mass spectrometry. In order to improve the accuracy of the assays, isotopically labeled internal standards were synthesized for an isotope dilution mass spectrometry quantitation. The methods were successfully applied to the measurement of methylene blue- and riboflavin-mediated 2'-deoxyguanosine photooxidation reactions. The advantages of the two above-mentioned methods are discussed on the basis of comparative sensitivity and accuracy.     

Oxidised guanidinohydantoin (Ghox) and spiroiminodihydantoin (Sp) are major products of iron- and copper-mediated 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine oxidation

8-Oxo-7,8-dihydroguanine (8-oxoGua), an important biomarker of DNA damage in oxidatively generated stress, is highly reactive towards further oxidation. Much work has been carried out to investigate the oxidation products of 8-oxoGua by one-electron oxidants, singlet oxygen, and peroxynitrite. This report details for the first time, the iron- and copper-mediated Fenton oxidation of 8-oxoGua and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). Oxidised guanidinohydantoin (Gh(ox)) was detected as the major product of oxidation of 8-oxoGua with iron or copper and hydrogen peroxide, both at pH 7 and pH 11. Oxaluric acid was identified as a final product of 8-oxoGua oxidation. 8-oxodGuo was subjected to oxidation under the same conditions as 8-oxoGua. However, dGh(ox) was not generated. Instead, spiroiminodihydantoin (Sp) was detected as the major product for both iron and copper mediated oxidation at pH 7. It was proposed that the oxidation of 8-oxoGua was initiated by its one-electron oxidation by the metal species, which leads to the reactive intermediate 8-oxoGua (+), which readily undergoes further oxidation. The product of 8-oxoGua and 8-oxodGuo oxidation was determined by the 2'-deoxyribose moiety of the 8-oxodGuo, not whether copper or iron was the metal involved in the oxidation.     

8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in human urine do not depend on diet

In the present study, we used the method involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in human urine. The mean levels of 8-oxoGua and 8-oxodGuo in the urine samples of the subjects on unrestricted diet were respectively 1.87 nmol/kg 24 h (±0.90) and 0.83 nmol/kg 24h (±0.49), and in the case of the groups studied, they did not depend on the applied diet. The sum of the amounts of both compounds in urine can give information about the formation rate of 8-oxoGua in cellular DNA. It is also likely that the levels of modified nucleo-base/side in urine sample are reflective of the involvement of different repair pathways responsible for the removal of 8-oxodGuo from DNA, namely base excision repair (BER) and nucleotide excision repair (NER).     

Formation of 8-nitroguanine from 2'-deoxyguanosine by NO/O2 system.

We investigated the reaction of 2'-deoxyguanosine (dGuo) with NO/O2 gas mixture under physiological condition and detected 8-nitroguanine, which is known as a novel DNA lesion caused by peroxynitrite (ONOO-). The yield increased with increase in the ratio of O2 and pH. The reaction mechanism is discussed.     

Repair and replication of oxidized DNA bases using modified oligodeoxyribonucleotides

Modified oligodeoxyribonucleotides (ODNs) are powerful tools to assess the biological significance of oxidized lesions to DNA. For this purpose, we developed original synthetical pathways for the site-specific insertion of several oxidized bases into DNA fragments. Thus, the chemical solid-phase synthesis of ODNs using original strategies of protection and mild conditions of deprotection, as well as a specific post-oxidation approach of an unique nucleoside residue within the sequence have been applied. These two approaches of preparation allowed us to have access to a set of modified ODNs that contain a single modified nucleoside, i.e., N-(2-deoxy-beta-D-erythro-pentofuranosyl)formylamine (dF), 5-hydroxy-2'-deoxycytidine (5-OHdCyd), thymidine glycol (dTg), 5,6-dihydrothymidine (DHdThd), 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)-amino]-5(2H)- oxazolone (dZ), N-(2-deoxy-beta-D-erythro-pentofuranosyl)cyanuric acid (dY), 5',8-cyclo-2'-deoxyguanosine (cyclodGuo) and 5',8-cyclo-2'-deoxyadenosine (cyclodAdo). The substrates were used to investigate recognition and removal of the lesions by bacterial DNA N-glycosylases, including endonuclease III (endo III) and Fapy glycosylase (Fpg). In addition, the DNA polymerase-mediated nucleotide incorporation opposite the damage was determined using modified ODNs as templates.     

The level of 8-oxo-7,8-dihydro-2′-deoxyguanosine is positively correlated with the size of the labile iron pool in human lymphocytes

It appears that the labile iron pool (LIP, low molecular weight iron) presence in cells can result in the production of reactive oxygen species (ROS). ROS may be responsible for the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in cellular DNA. In the present study we report on the relationship between LIP and the endogenous level of 8-oxodGuo in human lymphocytes. Good correlation has been determined between LIP and the oxidatively modified nucleoside. This in turn points out the possibility that under physiological condition there is the availability of LIP for catalyzing Fenton-type reactions in close proximity to cellular DNA. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0335-x.     

Antioxidant ebselen reduces oxidative damage in focal cerebral ischemia

The antioxidant and neuroprotective potential of the glutathione peroxidase mimic ebselen has been investigated in experimental stroke. Intravenous ebselen (1 mg/kg/h) or vehicle infusion was started 45 min before permanent middle cerebral artery occlusion in the rat, and continued until the end of the experiment. The topography and extent of oxidative damage to the brain was assessed immunohistochemically using an antibody for DNA damage that identified hydroxylated products of 2'-deoxyguanosine (8-OHdG/8-oxodGuo) and an antibody for lipid peroxidation that identified the 4-hydroxynonenal histidine adduct (4-HNE). Ischemic damage was mapped and evaluated with standard histopathology. In the vehicle-treated rats immunopositive staining for both 8-oxodGuo and 4-HNE extended beyond the boundary of ischemic damage. In ebselen-treated rats, the extent of tissue immunopositive for 8-oxodGuo, and 4-HNE was less than that demonstrating ischemic damage confirming the antioxidant mechanism of action in vivo. In addition, ebselen treatment induced a 28% reduction in cortical ischemic damage (p <.02).