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.     

Formation of the oxidative damage marker 8-hydroxy-2'-deoxyguanosine from the nucleoside 2'-deoxyguanosine: parameter studies and evidence of Fe(II) binding

High-performance liquid chromatography (HPLC) with UV absorption detection was employed to measure the amounts of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) produced from the nucleoside 2'-deoxyguanosine (dG) under varying reaction conditions using iron and H(2)O(2). The results indicate that 8-OH-dG produced from the reaction of iron and H(2)O(2) with dG can undergo reaction with free (i.e., unchelated) Fe(III) and that adding the chelating agent ethylenediaminetetraacetic acid (EDTA) after the reaction prevents this from occurring. It also appears that the free radical species generated by iron-EDTA chelates in pH 7.4 N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (Hepes) buffer is either not formed or unstable in unbuffered aqueous solution. Finally, 8-OH-dG levels are significantly larger when Fe(II) is allowed to bind to the nucleoside dG prior to addition of H(2)O(2). However, production of 8-OH-dG from unbound Fe(II) is also relevant. The results of this work show that differing reaction conditions in vivo, especially at the cellular level, will affect significantly the measured yields of 8-OH-dG. These results also have implications for studies involving DNA and the ability to distinguish between 8-OH-dG produced from free iron and iron bound to both phosphate groups and the DNA base guanine.     

2-Oxoglutarate transport: A protential mechanism for regulating glutamate and tricarboxylic acid cycle intermediates in neurons

2-Oxoglutarate (-ketoglutarate) is transported into synaptosomal and synaptoneurosomal preparations by a Na⁺-dependent, high-affinity process that exhibits complex kinetics, and is differentially modulated by glutamate, glutamine, aspartate, malate, and a soluble, heat-labile substance of high molecular weight present in rat brain extracts. Glutamate and aspartate generally inhibit 2-oxoglutarate uptake, but under certain conditions may increase uptake. Glutamine generally increases 2-oxoglutarate uptake, but under certain conditions may inhibit uptake. One interpretation of our results is that 2-oxoglutarate uptake is mediated primarily by a transporter that exhibits negative cooperativity and possesses three regulatory sites that differentially modulate substrate affinity, V_max, and negative cooperativity. Glutamate, aspartate, malate, and 2-oxoglutarate itself may interact with a site that reduces substrate affinity; whereas glutamine, and possibly glutamate and aspartate, appear to interact with another site that increases V_max. A putative regulatory protein appears to abolish negative cooperativity and increases substrate affinity in the absence of glutamine. Based on the evidence that glutamatergic and GABAergic neurons depend on astrocytes to supply precursors to replenish their neurotransmitter and tricarboxylic acid cycle pools, the uptake of 2-oxoglutarate, presumably into synaptic terminals, may reflect a role for this metabolite in replenishing the transmitter and tricarboxylic acid pools, and a role for the transporter as a site at which these pools are regulated.Abbreviations used AAT aspartate aminotransferase - glu glutamate - gln glutamine - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LDS low-density synaptosomes - OAA oxaloacetate - 2-OG 2-oxoglutarate (-ketoglutarate) - PC pyruvate carboxylase - PDH pyruvate dehydrogenase - TCA tricarboxylic acid Special issue dedicated to Dr. Claude Baxter.     

Formation of a diimino-imidazole nucleoside from 2'-deoxyguanosine by singlet oxygen generated by methylene blue photooxidation

Singlet oxygen ((1)O(2)) is capable of inducing genotoxic, carcinogenic and mutagenic effects. It has previously been reported that the reaction of (1)O(2) with 2'-deoxyguanosine, which is a major target of (1)O(2) among the DNA constituents, leads to formation of various oxidized products including 8-oxo-7,8-dihydro-2'-deoxyguanosine and spiroiminodihydantoin, amino-imidazolone and diamino-oxazolone nucleosides. In addition to these products, we report that a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dD), is formed by reaction of 2'-deoxyguanosine with (1)O(2) generated by irradiation with visible light in the presence of methylene blue under aerobic conditions. Its identification is based on identical chromatographic and spectroscopic data with an authentic compound, which we recently isolated and characterised from the reaction mixture of 2'-deoxyguanosine with reagent HOCl and a myeloperoxidase-H(2)O(2)-Cl(-) system. The yield of dD was increased by D(2)O and decreased by azide. dD was not generated from 8-oxo-7,8-dihydro-2'-deoxyguanosine. These results indicate that dD is generated by (1)O(2) directly from 2'-deoxyguanosine, but not via 8-oxo-7,8-dihydro-2'-deoxyguanosine. dD may play a role in the genotoxicity of singlet oxygen in cells.     

A formation mechanism for 8-hydroxy-2'-deoxyguanosine mediated by peroxidized 2'-deoxythymidine

The oxidative formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA is closely associated with the induction of degenerative diseases, including cancer. However, the oxidant species participating in the formation of 8-OHdG has yet to be fully clarified. On the basis that peroxyl radicals are a strong candidate for this species, we employed 2,2'-azobis(2-amidinopropane) (AAPH) as a peroxyl radical generator. Exposure of calf thymus DNA to AAPH formed 8-OHdG, but the exposure of 2'-deoxyguanosine (dG) alone did not. From the exposure of various combinations of nucleotides, 8-OHdG was formed only in the presence of dG and thymidine (dT). A mix of dG with an oxidation product of dT, 5-(hydroperoxymethyl)-2'-deoxyuridine, produced 8-OHdG, but the amount formed was small. In contrast, 8-OHdG was produced abundantly by the addition of dG to peroxidized dT with AAPH. Thus, the formation of 8-OHdG was mediated by the peroxidized dT. Instead of artificial AAPH, endogenous peroxyl radicals are known to be lipid peroxides, which are probably the oxidant species for 8-OHdG formation mediated by thymidine in vivo.     

Epigallocatechin gallate markedly enhances formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the reaction of 2'-deoxyguanosine with hypochlorous acid

A tea polyphenol, (-)-epigallocatechin gallate (EGCG), which can scavenge a variety of reactive oxygen species, enhances the yield of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) up to 20-fold in the reaction of 2'-deoxyguanosine with hypochlorous acid (HOCl), compared with the reaction without EGCG. Certain concentrations of EGCG inhibited HOCl-mediated oxidation of 2'-deoxyguanosine to 8-oxo-dG to a limited extent, but efficiently inhibited further oxidation of 8-oxo-dG to spiroiminodihydantoin nucleoside, resulting in the accumulation of 8-oxo-dG in the reaction mixture. Conversely, EGCG inhibited dose-dependently an increase in 8-oxo-dG levels in calf thymus DNA incubated with HOCl. However, addition of HOCl to the DNA preoxidized with an oxidant-generating system (CuCl2, ascorbate, H2O2), led to the extensive loss of 8-oxo-dG due to its further oxidation. EGCG effectively inhibited this HOCl-mediated loss of 8-oxo-dG in the oxidized DNA, resulting in an apparent increase in 8-oxo-dG levels in the oxidized DNA, compared with the levels found without EGCG. The conversion of 8-oxo-dG into other oxidized lesions will inevitably affect recognition by DNA repair enzymes as well as the rates of mutations and DNA synthesis. Thus, our results suggest that as a biomarker of oxidative DNA damage, not only 8-oxo-dG but also the products of its further oxidation should be analyzed.     

Misincorporation of 2'-deoxyoxanosine into DNA: a molecular basis for NO-induced mutagenesis derived from theoretical calculations

A wide range of theoretical methods, including high level ab initio, density functional, self-consistent reaction field, molecular dynamics and thermodynamic integration calculations, have been used to analyze the mutagenic properties of oxanosine. The major tautomeric forms in the gas phase and aqueous solution have been determined. The ability of oxanosine to recognize thymine and cytosine in the gas phase and in the DNA environment has been compared with that of guanine. A physicochemical explanation for the mutagenic properties of oxanosine is suggested.     

Formation of the base modification 8-hydroxyl-2'-deoxyguanosine and DNA fragmentation following seizures induced by systemic kainic acid in the rat

The formation of oxidative DNA damage as a consequence of seizures remains little explored. We therefore investigated the regional and temporal profile of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) formation, a hallmark of oxidative DNA damage and DNA fragmentation in rat brain following seizures induced by systemic kainic acid (KA). Formation of 8-OHdG was determined via HPLC with electrochemical detection, and single- and double-stranded DNA breaks were detected using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated nick end-labeling (TUNEL), respectively. Systemic KA (11 mg/kg) significantly increased levels of 8-OHdG within the thalamus after 2 h, within the amygdala/piriform cortex after 4 h, and within the hippocampus after 8 h. Levels remained elevated up to sevenfold within these areas for 72 h. Smaller increases in 8-OHdG levels were also detected within the parietal cortex and striatum. PANT-positive cells were detected within the thalamus, amygdala/piriform cortex, and hippocampus 24-72 h following KA injection. TUNEL-positive cells appeared within the same brain regions and over a similar time course (24-72 h) but were generally lower in number. The present data suggest oxidative damage to DNA may be an early consequence of epileptic seizures and a possible initiation event in the progression of seizure-induced injury to DNA fragmentation and cell death.     

Reactivity of 2'-deoxyoxanosine, a novel DNA lesion.

2'-Deoxyoxanosine (dOxo) is a novel DNA lesion produced from 2'-deoxyguanosine by the reaction with nitrous acid or nitric oxide. We found that dOxo reacted with glycine under physiological conditions. The product was identified by spectrometric data as an adduct between the six membered ring of dOxo and an amino group of glycine. The adduct was more stable than dOxo under physiological conditions. The incubation of an oligodeoxynucleotide containing dOxo with glycine gave also rise to the adduct. These results suggest that dOxo formed in DNA reacts with amino groups of various compounds around DNA in vivo resulting in the adduct.     

The nitrous acid deamination of glycosides and acetates of 2-amino-2-deoxy-D-glucose

A procedure is described for the nitrous acid deamination of 2-amino-2-deoxy-D-glucose hydrochloride (1), and reduction of the product with buffered borohydride, to afford crystalline 2,5-anhydro-D-mannitol (3) in 71% yield. Similar treatment of the methyl α-pyranoside (4) of 1 gives 59% of crystalline 3, and the same product is obtained in 44% yield from 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-α(or β)-D-glucopyranose hydrochloride (5 or 6) by the deamination-reduction sequence with subsequent deacetylation. These results provide a model, for a nonhydrolytic, depolymerization technique for structural characterization of glycosaminoglycans.     

Formation of adducts in the reaction of glyoxal with 2'-deoxyguanosine and with calf thymus DNA

The reactions of glyoxal with 2′-deoxyguanosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions resulted in the formation of two previously undetected adducts in addition to the known reaction product 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx-dG). The adducts were isolated and purified by reversed-phase liquid chromatography and structurally characterised by UV absorbance, mass spectrometry, ¹H and ¹³C NMR spectroscopy. The hitherto unknown adducts were identified as: 5-carboxymethyl-3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx₂-dG) and N²-(carboxymethyl)-9-(2′-deoxy-β-d-erythro-pentofuranosyl)-purin-6(9H)-one (Gx₁-dG). Both adducts were shown to arise from Gx-dG. Gx-dG and Gx₂-dG were found to be unstable and partly transformed to Gx₁-dG, which is a stable adduct and seems to be the end-product of the glyoxal reaction with 2′-deoxyguanosine. All adducts formed in the reaction of glyoxal with 2′-deoxyguanosine were observed in calf thymus DNA. Also in DNA, Gx₁-dG was the only stable adduct. The transformation of Gx-dG to Gx₁-dG seemed to take place in single-stranded DNA and therefore, Gx₁-dG may be a potentially reliable biomarker for glyoxal exposure and may be involved in the genotoxic properties of the compound.     

The role of metal ion binding in generating 8-hydroxy-2'-deoxyguanosine from the nucleoside 2'-deoxyguanosine and the nucleotide 2'-deoxyguanosine-5'-monophosphate

The metal ions Cu(II), Fe(II), and Cr(III) were allowed to react with H(2)O(2) in the presence of either the mononucleoside 2'-deoxyguanosine (dG) or the mononucleotide 2'-deoxyguanosine-5'-monophosphate (dGMP). The percentage of reacted dG or dGMP that formed the oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was monitored. Oxidative damage from reactions involving Cu(II) appear dependent on an interaction between copper and N7 on the guanine base. Any interactions involving the phosphate group have little additional effect on overall oxidative damage or 8-OH-dG production. Reactions involving Fe(II) seem very dependent on an interaction that may involve both N7 on the guanine base and the phosphate group. This interaction may slow oxidation of Fe(II) to Fe(III) in solution, keeping iron in a readily available form to undergo the Fenton reaction. Chromium(III) appears to interact with the phosphate group of dGMP, resulting in significant overall oxidative damage. However, production of 8-OH-dG appears to be very dependent on the ability of Cr(III) to interact with N7 on the guanine base, an interaction that seems to be weak for both the mononucleoside and mononucleotide.     

Free-radical-induced formation of an 8,5'-cyclo-2'-deoxyguanosine moiety in deoxyribonucleic acid.

Isolation and identification of a novel .OH-induced product, namely an 8,5'-cyclo-2'-deoxyguanosine moiety, in DNA and 2'-deoxyguanosine are described. .OH radicals were generated in dilute aqueous solutions by gamma-irradiation. Analyses of 2'-deoxyguanosine and enzymic hydrolysates of DNA by gas chromatography-mass spectrometry (g.c.-m.s.) after trimethylsilylation showed the presence of 8,5-cyclo-2'-deoxyguanosine on the basis of its fragment ions. This product was isolated by h.p.l.c. Its u.v. and n.m.r. spectra taken were in agreement with the structure suggested by its mass spectrum. Exact masses of the typical ions from the mass spectrum of the trimethylsilyl derivative of this product were measured by high-resolution m.s. The values found were in excellent agreement with the theoretical mass derived from the suggested fragmentation patterns. Both (5'R)- and (5'S)-epimers of 8,5'-cyclo-2'-deoxyguanosine were observed. These two diastereomers were separated from each other by g.c. as well as by h.p.l.c. The assignment of the epimers was accomplished on the basis of the n.m.r. data. The formation of 8,5'-cyclo-2'-deoxyguanosine was suppressed by the presence of O2 in the solutions. The use of g.c.-m.s. with the selected-ion monitoring technique facilitated the detection of 8,5'-cyclo-2'-deoxyguanosine in DNA at radiation doses as low as 1 Gy. Its mechanism of formation probably involves hydrogen atom abstraction by .OH radicals from the C-5' of the 2'-deoxyguanosine moiety followed by intramolecular cyclization with the formation of a covalent bond between the C-5' and C-8 and subsequent oxidation of the resulting N-7-centred radical.     

Spin trapping of nitrogen dioxide and of radicals generated from nitrous acid

Spin trapping of nitrogen dioxide radical by several nitrones has been studied. The reaction results in the formation of persistent acyl nitroxides, after the oxidation of the intermediate spin adducts having an -ONO group on C-2 atom. The intermediate is effectively detected when DEPMPO is used as the spin trap. The reaction between PBN or 5,7-di-tert-butyl-3,3-dimethyl indoline N-oxide with nitrous acid gives the corresponding acyl nitroxide only when oxygen is present in the reaction milieu.

On the other hand, nitroso spin traps do not trap ^⋅NO₂ confirming that the unpaired electron of nitrogen dioxide is localized on the oxygen atom.     

Regioselective 1-alkylation of 2'-deoxyguanosine

A method has been found for the regioselective alkylation of the nitrogen at the 1-position of 2'-deoxyguanosine. This consists in the reaction, in tetrahydrofuran solution, of a fully protected form of dG, namely the 3'5'-O-bis(tert-butyldimethylsilyl)-N2-dimethylaminomethylene derivative, with an alkyl halide in the presence of cesium carbonate. The yields of these previously unavailable derivatives of 2'-deoxyguanosine range from good to excellent. Confirmation of the structure of these substances comes from a comparison of their spectroscopic properties with those of the known 1-methyl homologue. In particular, the UV spectra of these new derivatives and the known 1-methyl homologue are essentially identical.