Isolation and Characterization of a Novel Product, 2‘-Deoxyoxanosine, from 2‘-Deoxyguanosine, Oligodeoxynucleotide, and Calf Thymus DNA Treated by Nitrous Acid and Nitric Oxide

Suzuki, Toshinori; Yamaoka, Ryohei; Nishi, Masatoshi; Ide, Hiroshi; Makino, Keisuke

doi:10.1021/ja952550g

Cited by 108 publications

(120 citation statements)

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“…Preparation of dOTP and Oxa-containing DNA-Oxanine is a newly identified deamination product of guanine (11) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies suggested that xanthine in DNA is prone to spontaneous depurination because of the instability of the CЈ-N glycosidic bond (1,7); however, a more recent study indicates that Xan is a stable lesion in DNA under physiological conditions (8,9). Treatment of deoxyguanosine or DNA by nitrous acid, nitric oxide, or 1-nitrosoindole-3-acetonitrile also yields an intracyclic guanine deamination product, oxanine (Oxa), in which the N 1 -nitrogen is substituted by an oxygen atom (10,11). A chemical pathway leading to the formation of xanthine and oxanine from guanine by nitrous acid treatment has been proposed based on the isolation of a diazoate intermediate (12).…”

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confidence: 99%

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Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

Hitchcock

Dong

Connor

et al. 2004

Journal of Biological Chemistry

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Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N 1 -nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase ␤, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency ϳ80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxacontaining DNA as well. Indeed, AAG can also remove 1,N 6 -ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.

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“…Preparation of dOTP and Oxa-containing DNA-Oxanine is a newly identified deamination product of guanine (11) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

Hitchcock

Dong

Connor

et al. 2004

Journal of Biological Chemistry

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“…Quantification of the standard is performed by UV spectroscopy using an extinction coefficient of 10,300 M -1 cm -1 at 260 nm. 42,43 dX and dO standards are synthesized by a modification of the method of Suzuki et al 45 Briefly, incubate 10 mM 2′-deoxyguanosine or [ 15 N 5 ]-2′-deoxyguanosine with 100 mM NaNO 2 in 0.3 M sodium acetate buffer (pH 3.7) at 37 °C for 6 h. dX and dO are purified by HPLC using a LUNA C18 reversed-phase column (250 × 3 mm, 5 μm particle size, 100 Å pore size; Phenomenex, Torrance, CA) with elution performed at a flow rate of 0.4 ml/min with 1% acetonitrile in 50 mM ammonium acetate (pH 7.4) for the first 5 min, followed by a linear gradient of 1-25% acetonitrile for 5 min; holding at 25% for 10 min; then a reversal of the gradient to 1% for 5 min; and finally eluting at 1% acetonitrile over the last 5 min. Fractions containing the products are dried under vacuum and redissolved in water followed by desalting on a second HPLC system consisting of a Haisil HL C18 reversed phase column (250 × 4.6 mm, 5 μm particle size, 100 Å pore size; Higgins Analytic Inc, Mountain View, CA) eluted with 5% acetonitrile in water at a flow rate of 0.4 ml/min, with elution confirmed using commercially available standards.…”

Section: Synthesis Of Internal Standardsmentioning

confidence: 99%

Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry

et al. 2008

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The analysis of damage products as biomarkers of inflammation has been hampered by a poor understanding of the chemical biology of inflammation, the lack of sensitive analytical methods, and a focus on single chemicals as surrogates for inflammation. To overcome these problems, we developed a general and sensitive liquid chromatographic tandem mass spectrometry (LC/MS-MS) method to quantify, in a single DNA sample, the nucleoside forms of seven DNA lesions reflecting the range of chemistries associated with inflammation: 2′-deoxyuridine, 2′-deoxyxanthosine, and 2′-deoxyinosine from nitrosative deamination; 8-oxo-2′-deoxyguanosine from oxidation; and 1,N 2 -etheno-2′-deoxyguanosine, 1,N 6 -etheno-2′-deoxyadenosine, and 3,N 4 -etheno-2′-deoxycytidine arising from reaction of DNA with lipid peroxidation products. Using DNA purified from cells or tissues under conditions that minimize artifacts, individual nucleosides are purified by HPLC and quantified by isotope-dilution, electrospray ionization LC/MS-MS. The method can be applied to other DNA damage products and requires 4-6 days to complete depending upon the number of samples. Search terms

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“…Of particular interest is the observation of Suzuki et al that deamination of guanine by nitrous acid in vitro partitions to form both xanthine and oxanine (2′-deoxyoxanosine; dO; ref. 6), which complicates the decades-old assumption of a simple guaninediazonium ion intermediate common to xanthine and dG-dG cross-links. Suzuki et al originally described the formation of dO in reactions of nucleosides and DNA with nitrite under acidic (pH < 4) conditions (6,7) and subsequently described the physicochemical and biological properties of dO (e.g., refs.…”

Section: Introductionmentioning

confidence: 99%

“…6), which complicates the decades-old assumption of a simple guaninediazonium ion intermediate common to xanthine and dG-dG cross-links. Suzuki et al originally described the formation of dO in reactions of nucleosides and DNA with nitrite under acidic (pH < 4) conditions (6,7) and subsequently described the physicochemical and biological properties of dO (e.g., refs. [8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Relatively Small Increases in the Steady-State Levels of Nucleobase Deamination Products in DNA from Human TK6 Cells Exposed to Toxic Levels of Nitric Oxide

Dong

Dedon

2005

Chem. Res. Toxicol.

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Nitric oxide (NO) is a physiologically important molecule that has been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While the complicated chemistry of NO-mediated genotoxicity has been extensively study in vitro, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem by exposing human TK6 lymphoblastoid cells to controlled steady-state concentrations of 1.75 or 0.65 μM NO along with 186 μM O 2 in a recently developed reactor that avoids the anomalous gas-phase chemistry of NO and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nucleobase deamination products was defined using a recently developed liquid chromatography/mass spectrometry (LC/MS) method and the results were correlated with cytotoxicity and apoptosis. A series of control experiments revealed the necessity of using dC and dA deaminase inhibitors to avoid adventitious formation of 2′-deoxyuridine (dU) and 2′-deoxyinosine (dI), respectively, during DNA isolation and processing. Exposure of TK6 cells to 1.75 μM NO and 186 μM O 2 for 12 hr (1260 μM•min dose) resulted in 32% loss of cell viability measured immediately after exposure and 87% cytotoxicity after a 24 hr recovery period. The same exposure resulted in 3.5-, 3.8-, and 4.1-fold increases in dX, dI and dU, respectively, to reach the following levels: dX, 7 (±1) per 10 6 nt; dI, 25 (±2.1) per 10 6 nt; and dU, 40 (±3.8) per 10 6 per nt. dO was not detected above the limit of detection of 6 lesions per 10 7 nt in 50 μg of DNA. A 12 hr exposure to 0.65 μM NO and 190 μM O 2 (468 μM•min dose) caused 1.7-, 1.8-, and 2.0-fold increases in dX, dI, and dU, respectively, accompanied by a ∼15 (±3.6) % reduction in cell viability immediately after exposure. Again, dO was not detected. These results reveal modest increases in the steady-state levels of DNA deamination products in cells exposed to relatively cytotoxic levels of NO. This could result from limited nitrosative chemistry in nuclear DNA in cells exposed to NO or high levels of formation balanced by rapid repair of nucleobase deamination lesions in DNA.

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Isolation and Characterization of a Novel Product, 2‘-Deoxyoxanosine, from 2‘-Deoxyguanosine, Oligodeoxynucleotide, and Calf Thymus DNA Treated by Nitrous Acid and Nitric Oxide

Cited by 108 publications

References 17 publications

Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry

Relatively Small Increases in the Steady-State Levels of Nucleobase Deamination Products in DNA from Human TK6 Cells Exposed to Toxic Levels of Nitric Oxide

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