Formation of spiroiminodihydantoin nucleoside from 8-oxo-7,8-dihydro-2′-deoxyguanosine by nitric oxide under aerobic conditions

When 8-oxo-7,8-dihydro-2′-deoxyguanosine in potassium phosphate buffer of pH 7.4 was bubbled by nitric oxide at room temperature under aerobic conditions, two major products were formed. They were identified as the diastereomers of spiroiminodihydantoin deoxyribonucleoside on the basis of their identical ESI-MS and UV spectra and HPLC retention times with those of the major products in reaction of 8-oxo-7,8-dihydro-2′-deoxyguanosine with hypochlorous acid. A 1000-fold excess of 2′-deoxyguanosine did not inhibit the reaction of 8-oxo-7,8-dihydro-2′-deoxyguanosine with nitric oxide. The results suggest that an 8-oxo-7,8-dihydroguanine moiety formed in DNA may react with nitric oxide in the presence of oxygen molecule generating spiroiminodihydantoin in humans.     

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.     

A New Approach for the Efficient Synthesis of Oligodeoxyribonucleotides Containing the Mutagenic DNA Modification 7,8-Dihydro-8-oxo-2′-deoxyguanosine at Predefined Positions

Abstract

A combination of H-phoshonate and phosphoramidite chemistry has been applied for the automated solid-phase synthesis of oligodeoxyribonucleotides containing 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) residues at predefined positions. The unmodified part of the oligomers has been synthesized by using protected standard phosphoramidites, for the incorporation of 8-oxodG the synthon 2-N-acetyl-5′-0-(4,4′-dimethoxytrityl)-7,8-dihydro-2′-deoxyguanosin-8-one-3′-H-phosphonate, prepared in a five step synthesis via 8-bromo-2′-deoxyguanosine, has been used. This approach combines the advantages of both DNA synthesis strategies in that a high yield of full length oligomers is obtained and unreacted, protected 8-oxodG monomers can be recycled, respectively.     

The effect of the 2-amino group of 7,8-dihydro-8-oxo-2'-deoxyguanosine on translesion synthesis and duplex stability

Replication of DNA containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG) gives rise to G → T transversions. The syn-isomer of the lesion directs misincorporation of 2′-deoxyadenosine (dA) opposite it. We investigated the role of the 2-amino substituent on duplex thermal stability and in replication using 7,8-dihydro-8-oxo-2′-deoxyinosine (OxodI). Oligonucleotides containing OxodI at defined sites were chemically synthesized via solid phase synthesis. Translesion incorporation opposite OxodI was compared with 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG), 2′-deoxyinosine (dI) and 2′-deoxyguanosine (dG) in otherwise identical templates. The Klenow exo⁻ fragment of Escherichia coli DNA polymerase I incorporated 2′-deoxyadenosine (dA) six times more frequently than 2′-deoxycytidine (dC) opposite OxodI. Preferential translesion incorporation of dA was unique to OxodI. UV-melting experiments revealed that DNA containing OxodI opposite dA is more stable than when the modified nucleotide is opposed by dC. These data suggest that while duplex DNA accommodates the 2-amino group in syn-OxodG, this substituent is thermally destabilizing and does not provide a kinetic inducement for replication by Klenow exo⁻.     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine as a biomarker in type 2 diabetes

The increasing prevalence of diabetes together with the associated morbidity and mortality calls for additional preventive and therapeutic strategies. New biomarkers that can be used in therapy control and risk stratification as alternatives to current methods are needed and can facilitate a more individualized and sufficient treatment of diabetes. Evidence derived from both epidemiological and mechanistic studies suggests that oxidative stress has an important role in mediating the pathologies of diabetic complications. A marker of intracellular oxidative stress that potentially could be used as a valuable biomarker in diabetes is the DNA oxidation marker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), which can be assessed noninvasively in the urine, with minimal discomfort for the patient. In this review the analytical validity of 8-oxodG is addressed by highlighting important methodological issues. The available epidemiological evidence regarding urinary 8-oxodG and type 2 diabetes is presented. A possible role for DNA oxidation in cancer development in type 2 diabetes patients is discussed, followed by an evaluation of the potential of urinary 8-oxodG as a clinical biomarker in type 2 diabetes.     

Urinary 8-oxo-2′-deoxyguanosine — Source,significance and supplements

Oxidative damage to cellular biomolecules, in particular DNA, has been proposed to play an important role in a number of patholgical conditions, including carcinogenesis. A much studied consequence of oxygen-centred radical damage to DNA is 8-oxo-2′-deoxyguanosine (8-oxodG). Using numerous techniques, this lesion has been quantified in various biological matrices, most notably DNA and urine. Until recently, it was understood that urinary 8-oxodG derives solely from DNA repair, although the processes which may yield the modified deoxynucleoside have never been thoroughly discussed. This review suggests that nucleotide excision repair and the action of a specific endonuclease may, in addition to the nucleotide pool, contribute significantly to levels of 8-oxodG in the urine. On this basis, urinary 8-oxodG represents an important biomarker of generalised, cellular oxidative stress. Current data from antioxidant supplementation trials are examined and the potential for such compounds to modulate DNA repair is considered. It is stressed that further work is required to link DNA, serum and urinary levels of 8-oxodG such that the kinetics of formation and clearance may be elucidated, facilitating greater understanding of the role played by oxidative stress in disease.     

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products

Epidemiological studies testing the effect of β-carotene in humans have found a relative risk for lung cancer in smokers supplemented with β-carotene. We investigated the reactions of retinal and β-apo-8′-carotenal, two β-carotene oxidation products, with 2′-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N²-etheno-2′-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with β-carotene or β-carotene oxidation products, significantly increased levels of 1,N²-etheno-2′-deoxyguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of β-carotene oxidation products.     

Lung cancer biomarkers for the assessment of modified risk tobacco products: an oxidative stress perspective

Abstract

Manufacturers have developed prototype cigarettes yielding reduced levels of some tobacco smoke toxicants, when tested using laboratory machine smoking under standardised conditions. For the scientific assessment of modified risk tobacco products, tests that offer objective, reproducible data, which can be obtained in a much shorter time than the requirements of conventional epidemiology are needed. In this review, we consider whether biomarkers of biological effect related to oxidative stress can be used in this role. Based on published data, urinary 8-oxo-7,8-dihydro-2-deoxyguanosine, thymidine glycol, F₂-isoprostanes, serum dehydroascorbic acid to ascorbic acid ratio and carotenoid concentrations show promise, while 4-hydroxynonenal requires further qualification.     

Association between urinary excretion of cortisol and markers of oxidatively damaged DNA and RNA in humans

Chronic psychological stress is associated with accelerated aging, but the underlying biological mechanisms are not known. Prolonged elevations of the stress hormone cortisol is suspected to play a critical role. Through its actions, cortisol may potentially induce oxidatively generated damage to cellular constituents such as DNA and RNA, a phenomenon which has been implicated in aging processes. We investigated the relationship between 24 h excretion of urinary cortisol and markers of oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2′-deoxyguanosine and 8-oxo-7,8-dihydroguanosine, in a sample of 220 elderly men and women (age 65 – 83 years). We found a robust association between the excretion of cortisol and the oxidation markers (R² = 0.15, P<0.001 for both markers). Individuals in the highest quartile of cortisol excretion had a 57% and 61% higher median excretion of the DNA and RNA oxidation marker, respectively, than individuals in the lowest quartile. The finding adds support to the hypothesis that cortisol-induced damage to DNA/RNA is an explanatory factor in the complex relation between stress, aging and disease.     

Inhibition of Rac and Rac-linked functions by 8-oxo-2′-deoxyguanosine in murine macrophages

Rac is a protein involved in the various functions of macrophages (Mφ), including the production of reactive oxygen species (ROS), phagocytosis, chemotaxis and the secretion of cytokines (such as γ-INF). This study tested the effects of nucleosides containing 8-oxoguanine(8-hydroxyguanine) such as 8-oxo-2′-guanosine (8-oxoG) or 8-oxo-2′-deoxyguanosine (8-oxodG), on Rac and the above-listed Rac-associated functions of Mφ using mouse peritoneal Mφ (MpMφ). It is reported that 8-oxodG was able to effectively inhibit Rac and the Rac-associated functions of MpMφ. Compared to 8-oxodG, 8-oxoG showed negligible effects. Furthermore, normal nucleosides such as deoxyguanosine (dG), guanosine (G) and adenosine (A) did not exert any effects. These results suggest that 8-oxodG could be used as a potential tool to modulate the functions of Mφ that are intimately related to various pathological processes.     

Impairment of the Viability of Transformed Chinese Hamster Cells in a Nonsubcultured Culture under the Influence of Exogenous Oxidized Guanoside is Manifested Only in the Stationary Phase of Growth

Despite the fact that oxidation products of nucleotides and nucleosides are markers of oxidative stress, reports of the paradoxical ability of these compounds to protect cells from the harmful effects of reactive oxygen species began to appear more often. Among all nitrogenous bases, guanine is most susceptible to the influence of oxidative stress; therefore, guanosine is oxidized more often than other bases. In the present work, the effect of exogenous 8-oxo-2′-deoxyguanosine on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during cell proliferation slowing down within a single passage and subsequent “aging” in the stationary growth phase) of nonsubcultured transformed Chinese hamster cells was studied. We showed that the nucleoside is rapidly absorbed by the cells from the medium, but it does not affect the growth of the culture, and impairs the viability of the cells in the late stationary growth phase. Thus, no mitogenic or geroprotective effect of 8-oxo-2′-deoxyguanosine was found.     

Fe(II) phthalocyanine catalyzed oxidation of dGMP by molecular oxygen

We show that iron(II)-phthalocyanines are able to catalyze guanosine oxidation by molecular oxygen in the presence of reducing agents such as ascorbic acid and 2-mercaptoethanol. The products of 5′-monophosphate-2′-deoxyguanosine (dGMP) oxidation were directly analyzed using the HPLC-ESI/MS method. The main oxidation products were 5′-phospho-2′-deoxy-8-oxo-7,8-dihydroguanine and the 1,N2-glyoxal adduct of the 5′-monophosphate-2′-deoxyguanosine.     

Synthesis and Characterization of Oligodeoxyribonucleotides Containing Tandem Base Damage

Abstract

Both N(2-deoxy-β-D-erythro-pentofuranosyl)-formylamine (dβF) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) were introduced in synthetic oligonucleotides at a vicinal position via the solid phase phosphoramidite method in order to investigate the biological and structural significance of such a tandem lesion. Further experiments aimed at determining the enzymatic repair by both E. coli endonuclease III (Endo III) and Fapy-glycosylase (Fpg) were carried out with these synthetic substrates.     

Analysis of urinary 8-oxo-7,8-dihydro-purine-2’-deoxyribonucleosides by LC-MS/MS and improved ELISA

Non-invasive monitoring of oxidative stress is highly desirable. Urinary 7,8-8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) is a biologically relevant and convenient analytical target. However, immunoassays can over-estimate levels of urinary 8-oxodG. Measurement of more than one DNA oxidation product in urine would be advantageous in terms of mechanistic information. Urines samples were analysed for 8-oxodG by solid-phase extraction/LC-MS/MS and ELISA. The solid-phase extraction/LC-MS/MS assay was also applied to the analysis of urinary 7,8-dihydro-8-oxo-2’-deoxyadenosine (8-oxodA). Concurring with previous reports, urinary 8-oxodG measured by ELISA was significantly higher than levels measured by LC-MS/MS. However, apparent improvement in the specificity of the commercially available Japanese Institute for the Control of Ageing (JaICA) ELISA brought mean LC-MS/MS and ELISA measurements of urinary 8-oxodG into agreement. Urinary 8-oxodA was undetectable in all urines, despite efficient recovery by solid phase extraction. Exploitation of the advantages of ELISA may be enhanced by a simple modification to the assay procedure, although chromatographic techniques still remain the ‘gold standard’ techniques for analysis of urinary 8-oxodG. Urinary 8-oxodA is either not present or below the limit of detection of the instrumentation.     

Complement-dependent NADPH oxidase enzyme activation in renal ischemia/reperfusion injury

NADPH oxidase plays a central role in mediating oxidative stress during heart, liver, and lung ischemia/reperfusion injury, but limited information is available about NADPH oxidase in renal ischemia/reperfusion injury. Our aim was to investigate the activation of NADPH oxidase in a swine model of renal ischemia/reperfusion damage. We induced renal ischemia/reperfusion in 10 pigs, treating 5 of them with human recombinant C1 inhibitor, and we collected kidney biopsies before ischemia and 15, 30, and 60 min after reperfusion. Ischemia/reperfusion induced a significant increase in NADPH oxidase 4 (NOX-4) expression at the tubular level, an upregulation of NOX-2 expression in infiltrating monocytes and myeloid dendritic cells, and 8-oxo-7,8-dihydro-2′-deoxyguanosine synthesis along with a marked upregulation of NADPH-dependent superoxide generation. This burden of oxidative stress was associated with an increase in tubular and interstitial expression of the myofibroblast marker α-smooth muscle actin (α-SMA). Interestingly, NOX-4 and NOX-2 expression and the overall NADPH oxidase activity as well as α-SMA expression and 8-oxo-7,8-dihydro-2′-deoxyguanosine synthesis were strongly reduced in C1-inhibitor-treated animals. In vitro, when we incubated tubular cells with the anaphylotoxin C3a, we observed an enhanced NADPH oxidase activity and α-SMA protein expression, which were both abolished by NOX-4 silencing. In conclusion, our findings suggest that NADPH oxidase is activated during ischemia/reperfusion in a complement-dependent manner and may play a potential role in the pathogenesis of progressive renal damage in this setting.     

Comparison of Results from Different Laboratories in Measuring 8-oxo-2′-deoxyguanosine in Synthetic Oligonucleotides

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up in 1997 to resolve methodological problems and to reach agreement on the basal level of 8-oxo-2'-deoxyguanosine (8-oxodG) in biological samples. In the present ESCODD trial 6 samples of 8-oxodG-containing oligonucleotides with different ratios of 8-oxodG/2'-deoxyadenosine (dAdo) were sent to 25 laboratories throughout Europe. The methods used were HPLC with electrochemical detection (amperometric or coulometric), GC-MS or LC-MS-MS. The LC-MS-MS and the coulometric HPLC analyses gave 8-oxodG concentrations within 53 and 73% of expected values, respectively, whereas the amperometric HPLC and GC-MS consistently overestimated the 8-oxodG concentration by several fold. As the oligonucleotides contained no 2'-deoxyguanosine (dGuo), this was not due to artificial oxidation. On the contrary, in most cases the concentrations of dAdo and thymidine (dThd), used as estimates for non-oxidised DNA bases were underestimated, though a few laboratories overestimated the lowest concentration samples containing 8 and 20 &#119 M, respectively. In one-third of the reported results, the ratio of 8-oxodG/10 5 dAdo was within 25% of the calculated value in the oligonucleotide samples and in half of the results the coefficient of variation in duplicate samples was less than 10%. The coefficients of variation were higher for the dAdo concentrations than for 8-oxodG. Our findings strongly indicate that careful quality control must be applied to the analytical procedures for 8-oxodG and very importantly also to the procedures for non-modified 2'-deoxyribonucleosides. We recommend the use of synthetic oligonucleotides for this purpose.     

Effects of 8-halo-7-deaza-2′-deoxyguanosine triphosphate on DNA synthesis by DNA polymerases and cell proliferation

8-OxodG (8-oxo-2′-deoxyguanosine) is representative of nucleoside damage and shows a genotoxicity. To significantly reveal the contributions of 7-NH and C8-oxygen to the mutagenic effect of 8-oxodG by DNA polymerases, we evaluated the effects of the 8-halo-7-deaza-dG (8-halogenated 7-deaza-2′-deoxyguanosine) derivatives by DNA polymerases. 8-Halo-7-deaza-dGTPs were poorly incorporated by both KF(exo⁻) and human DNA polymerase β opposite dC or dA into the template DNA. Furthermore, it was found that KF(exo⁻) was very sensitive to the introduction of the C8-halogen, while polymerase β can accommodate the C8-halogen resulting in an efficient dCTP insertion opposite the 8-halo-7-deaza-dG in the template DNA. These results indicate that strong hydrogen bonding between 7-NH in the 8-oxo-G nucleobase and 1-N in the adenine at the active site of the DNA polymerase is required for the mutagenic effects. Whereas, I-deaza-dGTP shows an antiproliferative effect for the HeLa cells, suggesting that it could become a candidate as a new antitumor agent.     

Time And Concentration Dependence Of Fenton-Induced Oxidation Of dG

The influence of incubation time and Fenton reagent concentrations was investigated on the oxidation of 2′-deoxyguanosine. The compounds identified and quantified, through use of an LC-MS/MS system, were 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8,5′-cyclo-2′-deoxyguanosine (8,5′cyclodG) and the secondary oxidation products guanidinohydantoin and dehydro-guanidinohydantoin. 8-oxodG and 8,5′cyclodG formed very quickly, reaching a maximum rapidly, but with 8-oxodG a rapid decline occurred thereafter due to its further oxidation into the secondary products, which formed more slowly. Due to the better stability, 8,5′cyclodG correlated better with the general level of oxidation than 8-oxodG. The results emphasize the advantages of measuring other oxidation adducts than 8-oxodG alone.     

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.     

Exogenous 8-oxo-7,8-dihydro-2′-deoxyguanosine: Biomedical properties,mechanisms of action,and therapeutic potential

Abstract—8-Oxo-7,8-dihydroguanine (8-oxo-G) is a key biomarker of oxidative damage to DNA in cells, and its genotox- icity is well-studied. In recent years, it has been confirmed experimentally that free 8-oxo-G and molecules containing it are not merely inert products of DNA repair or degradation, but they are actively involved in intracellular signaling. In this review, data are systematized indicating that free 8-oxo-G and oxidized (containing 8-oxo-G) extracellular DNA function in the body as mediators of stress signaling and initiate inflammatory and immune responses to maintain homeostasis under the action of external pathogens, whereas exogenous 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) exhibits pro- nounced antiinflammatory and antioxidant properties. This review describes known action mechanisms of oxidized guanine and 8-oxo-G-containing molecules. Prospects for their use as a therapeutic target are considered, as well as a pharmaceu- tical agent for treatment of a wide range of diseases whose pathogenesis is significantly contributed to by inflammation and oxidative stress.