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1.
Characterization of DNA--protein cross-links induced by oxanine: cellular damage derived from nitric oxide and nitrous acid 总被引:2,自引:0,他引:2
Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion derived from the guanine base with nitric oxide, nitrous acid, or N-nitrosoindoles. It was shown by gel electrophoresis that oxanine mediated the formation of DNA-protein cross-links (DPCs) with DNA-binding proteins and in the cell extract. Although 2'-deoxyoxanosine was shown to react with amines including the N-terminal amino group of glycine, the structures of DNA-protein cross-links induced by oxanine have not been characterized. In this study, we find that the thiol group of the amino acid side chain is reactive toward oxanine, forming a thioester. Two reaction products of oxanine, namely, the thioester and the amide adducts, with the endogenous tripeptide glutathione (GSH) as a model protein were characterized on the basis of their UV, NMR (1H- and 13C-), and mass spectra. Interestingly, the disulfide GSSG also reacts with oxanine, forming the thioester adduct. The thioester and the amide adducts are generated when GSH and GSSG react with oxanine-containing calf thymus DNA, and they might be possible forms of cellular DPCs. Because the repair mechanism of DPCs is not extensively investigated, the characterization of oxanine-derived DPC structures should shed light on their detection in vivo and on their biological consequences. 相似文献
2.
E V Mitsevich Iu A Semin A M Poverenny? Iu G Suchkov 《Biulleten' eksperimental'no? biologii i meditsiny》1979,87(5):466-468
It was shown that aminomethylol compounds formed during reaction of formaldehyde with amino acids and formaldehyde as well exert a pronounced lethal action on E. coli strains with various defects of the DNA repair systems. The correlation between the extent of the DNA depurination caused by in vitro action of diverse aminomethylol derivatives and the inactivation of bacteria by these derivatives is revealed. The data obtained suggest that the inactivating effect of formaldehyde and its aminomethylol derivatives seems likely to be due to the formation of depurinized groups in bacterial DNA rather than to dimerization of purine bases. 相似文献
3.
Mechanistic studies on depurination and apurinic site chain breakage in oligodeoxyribonucleotides. 总被引:2,自引:2,他引:0
Products formed from defined oligodeoxyribonucleotide tetramers (oligonucleotides) by depurination at pH 5.0 and 90 degrees C followed by chain breakage at the resulting apurinic sites (AP sites) were assigned by reversed phase HPLC. Through kinetic analysis, rate constants of depurination and subsequent chain breakage reactions were measured. Depurination of the oligonucleotides with purine bases locating at the terminal positions was several times faster than those with purines at the internal ones. The pKa values for the N7 of the G residues and the activation energies of the depurination were essentially independent of the position of the bases. The frequency factor was found to be responsible for the observed difference of the depurination rates. In contrast, the chain breakage by beta-elimination was several times faster for the AP sites formed at the internal positions than those at the 5'-terminal positions. It is suggested that an electron withdrawing phosphate group attached to the 5'-side of an AP site facilitates the chain cleavage. 相似文献
4.
Purine nucleotides are formed de novo by a widespread biochemical route that may be of monophyletic origin, or are synthesized from preformed purine bases and nucleosides through different salvage pathways. Three monophyletic sets of purine salvage enzymes, each of which catalyzes mechanistically similar reactions, can be identified: (a) adenine-, xanthine-, hypoxanthine- and guanine-phosphoribosyltransferases, which are all homologous among themselves, as well as to nucleoside phosphorylases; (b) adenine deaminase, adenosine deaminase, and adenosine monophophate deaminase; and (c) guanine reductase and inosine monophosphate dehydrogenase. These homologies support the idea that substrate specificity is the outcome of gene duplication, and that the purine nucleotide salvage pathways were assembled by a patchwork process that probably took place before the divergence of the three cell domains (Bacteria, Archaea, and Eucarya). Based on the ability of adenine PRTase to catalyze the condensation of PRPP with 4-aminoimidazole-5-carboxamide (AICA), a simpler scheme of purine nucleotide biosynthesis is presented. This hypothetical route requires the prior evolution of PRPP biosynthesis. Since it has been argued that PRPP, nucleosides, and nucleotides are susceptible to hydrolysis, they are very unlikely prebiotic compounds. If this is the case, it implies that many purine salvage pathways appeared only after the evolution of phosphorylated sugar biosynthetic pathways made ribosides available. 相似文献
5.
S-nitrosothiols transport nitric oxide in vivo, and so-called transnitrosation reactions (i.e. the transfer of the nitroso function from nitrosothiol to thiolate) are believed to be involved in this process. In the present study we examined the N-nitrosotryptophan derivative-dependent nitrosation of thiols, a hitherto ignored possibility for the formation of S-nitrosothiols. The corresponding products were identified by (15)N-NMR spectrometry. The fact that the reaction proceeded under hypoxic conditions as well as in non-aqueous solution strongly indicated the occurrence of a transnitrosation reaction. Interestingly, S-nitrosothiols could only very slowly transnitrosate N-terminal-blocked tryptophan derivatives like melatonin in non-aqueous solution but did not induce such a reaction in water. The indole moiety of the N-nitrosotryptophan derivatives was fully restituted during the reaction with thiols, as demonstrated by both capillary zone electrophoresis and fluorescence spectroscopy. A determination of the Arrhenius parameters demonstrated that the corresponding rate constants were comparable with the ones known for the transfer of the nitroso function from nitrosothiol to thiolate. Thus, N-nitrosotryptophan-dependent nitrosation of thiols may occur in vivo and might offer the possibility of developing a new class of vasodilative drugs. 相似文献
6.
The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [14C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [14C]adenine or [14C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase. 相似文献
7.
Mechanisms for the solvolytic decompositions of nucleoside analogues. X. Acidic hydrolysis of 6-substituted 9-(beta-D-ribofuranosyl)purines. 总被引:2,自引:1,他引:1
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The pH-rate profiles were determined for the acidic hydrolysis of some 6-substituted 9-(beta-D-ribofuranosyl) purines. The product analyses indicated that the reactions generally proceed with formation of purine bases as initial products. However, at low oxonium ion concentrations the hydrolysis of the unsubstituted compound yields 4-amino-5-formamidopyrimidine, instead of purine formed in highly acidic solutions. The rate constants for the spontaneous and oxonium ion catalyzed heterolysis of the protonated substrates were calculated from the acidity constants and the observed rate constants. The dependence of the partial rate constants on the polar nature of the 6-substituents are consistent with rate-limiting formation of free purine bases and glycosyl oxocarbenium ions. No anomerization of the substrates was observed during the course of the hydrolysis. 相似文献
8.
The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [14C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [14C]adenine or [14C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase. 相似文献
9.
Martinez GR Gasparutto D Ravanat JL Cadet J Medeiros MH Di Mascio P 《Free radical biology & medicine》2005,38(11):1491-1500
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. 相似文献
10.
Takashi Utagawa Hirokazu Morisawa Shigeru Yamanaka Akihiro Yamazaki Fumihiro Yoshinaga Yoshio Hirose 《Bioscience, biotechnology, and biochemistry》2013,77(7):2167-2171
A simple method for the synthesis of various purine arabinosides from purine bases and uracil arabinoside by microbial transarabinosylation is described. A wet cell paste of Enterobacter aerogenes AJ 11125 showed a wide substrate specificity range for purine bases. Not only naturally occurring purine bases such as adenine and hypoxanthine but also unnatural bases such as 6-thioguanine and 2-chlorohypoxanthine were catalyzed to give the corresponding purine arabinosides. The enzymatically synthesized purine arabinosides were isolated from the reaction mixtures and identified by physicochemical means. The biological activities of the compounds were investigated and it was found that thioguanine arabinoside and 2-methyladenine arabinoside have potent activity against Hela cells, and their ED50 were 10.5 and 21.5 μg/ml, respectively. 相似文献
11.
Recent aspects of oxidative DNA damage: guanine lesions,measurement and substrate specificity of DNA repair glycosylases 总被引:2,自引:0,他引:2
Cadet J Bellon S Berger M Bourdat AG Douki T Duarte V Frelon S Gasparutto D Muller E Ravanat JL Sauvaigo S 《Biological chemistry》2002,383(6):933-943
This review discusses recent aspects of oxidation reactions of DNA and model compounds involving mostly OH radicals, one-electron transfer process and singlet oxygen (1O2). Emphasis is placed on the formation of double DNA lesions involving a purine base on one hand and either a pyrimidine base or a 2-deoxyribose moiety on the other hand. Structural and mechanistic information is also provided on secondary oxidation reactions of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a major DNA marker of oxidative stress. Another major topic which is addressed here deals with recent developments in the measurement of oxidative base damage to cellular DNA. This has been mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents, including UVA and ionizing radiations. In addition, the modified comet assay, which involves the use of bacterial DNA N-glycosylases to reveal two main classes of oxidative base damage, is applicable to isolated cells and is particularly suitable when only small amounts of biological material are available. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision pathways are briefly reviewed. 相似文献
12.
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. 相似文献
13.
Erika Nuka Susumu Tomono Akari Ishisaka Yoji Kato Noriyuki Miyoshi 《Bioscience, biotechnology, and biochemistry》2016,80(10):2007-2013
Lipid peroxidation products react with cellular molecules, such as DNA bases, to form covalent adducts, which are associated with aging and disease processes. Since lipid peroxidation is a complex process and occurs in multiple stages, there might be yet unknown reaction pathways. Here, we analyzed comprehensively 2′-deoxyguanosine (dG) adducts with oxidized arachidonic acid using liquid chromatography–tandem mass spectrometry and found the formation of 7-(2-oxo-hexyl)-etheno-dG as one of the major unidentified adducts. The formation of this adduct was reproduced in the reaction of dG with 2-octenal and predominantly with 4-oxo-2-octenal (OOE). We also found that other 2-alkenals (with five or more carbons) generate corresponding 4-oxo-2-alkenal-type adducts. Importantly, it was found that transition metals enhanced the oxidation of C4-position of 2-octenal, leading to the formation of OOE-dG adduct. These findings demonstrated a new pathway for the formation of 4-oxo-2-alkenals during lipid peroxidation and might provide a mechanism for metal-catalyzed genotoxicity. 相似文献
14.
Highly efficient synthesis of oligodeoxyribonucleotides using alpha-phenyl cinnamoyl group for selective amino protection.
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alpha-phenyl cinnamoyl (alpha-PhCm) group has been found to be highly selective for exocylic amino function of all the three deoxynucleosides viz, 2'-deoxyadenosine, 2'-deoxyguanosine and 2'-deoxycytidine. The stereospecific nature of the group confers stability to the N-protected derivatives of 2'-deoxyadenosine and 2'-deoxyguanosine towards acids thereby minimising depurination. The easy preparation and introduction of the group, stability of the protected monomers, milder conditions for deprotection resulting in negligible side products during synthesis and above all hydrophobicity of the group are the additional advantages. 相似文献
15.
Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.). 总被引:4,自引:0,他引:4
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1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage. 相似文献
16.
The mechanisms of hexavalent chromium(VI) induced DNA damage were unveiled by detecting products of single- and double-stranded DNA in the presence of glutathione. The absence of a detectable hydroxyl radical in the reactions indicates that DNA damage was exclusively by hypervalent chromium species. Polyacrylamide gel electrophoresis (PAGE) experiments with 32-mer single-stranded oligonucleotide and its complementary duplex revealed cleavages largely at purine bases with significant enhancement of such cleavages in the presence of a base. Quantitative estimations of bases released by HPLC before and after enzymatic digestion with exonucleases unequivocally established the excessive release of purine bases. This release was accompanied by the concomitant formation of phosphoglycolate as characterized by liquid chromatography-mass spectrometry (LC-MS). These data connote that the preponderance DNA damage is due to an oxidation specifically at H4' of the ribose moiety leading to the formation of apurinic sites. In addition to the oxidation at H4', DNA oxidation was also initiated through H5' site as evidenced by the identification of furfural. This pathway appears to be non-selective and more abundant for ssDNA as cleavages were observed at both purine and pyrimidine bases. Finally, the detection of guanidinohydantoin as a minor product points the involvement of an oxygen activated hypervalent chromium species, perhaps a peroxo-chromium species. Both major and minor pathways lead to cleavages at purine sites for ds-DNA and are consistent with the observation that DNA cleavage was enhanced in the presence of a base. In contrast, when hydrogen peroxide was added to the reactions, random DNA cleavages were apparent indicating involvement of multiple species including a hydroxyl radical. These data pinpoint mutation mechanisms induced by chromium(VI) in the presence of glutathione due to transversion either by inserting the wrong bases opposite to the apurinic sites during replication or by purine-purine mismatch. 相似文献
17.
Role of Exogenous Adenosine Triphosphate in Catabolic and Synthetic Activities of Chlamydia psittaci 总被引:13,自引:6,他引:7
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The synthetic activities of isolated cells of the meningopneumonitis strain (MN) of Chlamydia psittaci were investigated and further observations were made on their catabolic reactions. These observations included the demonstration of CO(2) production from aspartate in the presence of pyruvate and the formation of pyruvate from glucose-6-phosphate. Both reactions were enhanced by added adenosine triphosphate (ATP). Of a large number of compounds tested, only glucose-6-phosphate, pyruvate, aspartate, and isoleucine were shown to furnish carbons that were incorporated into molecules precipitated by trichloroacetic acid. The reactions with pyruvate, aspartate, and isoleucine were dependent entirely, or almost entirely, on added ATP, and the reaction with glucose-6-phosphate was enhanced by ATP. Except for CO(2), which greatly stimulated the reactions, the addition of a number of other compounds or a combination of compounds, such as cofactors, amino acids, and purine and pyrimidine bases, did not greatly affect incorporation. About 95% of the activity of the trichloroacetic acid precipitates was recovered in the chloroform-methanol soluble fraction. 相似文献
18.
Hitchcock TM Dong L Connor EE Meira LB Samson LD Wyatt MD Cao W 《The Journal of biological chemistry》2004,279(37):38177-38183
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 beta, 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 approximately 80% 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 Oxa-containing 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. 相似文献
19.
In this study, we apply a hybrid DFT functional, MPW1LYP, to make a comparison between MeSNO and MeSeNO. Due to the mesomeric
effect and negative hyperconjugation, Se-nitrososelenols seem to be more unstable than S-nitrosothiols regarding unimolecular
decomposition. Interestingly, however, the barrier of the transnitrosation reaction of MeSeNO is larger than that of MeSNO,
disregarding nucleophiles in the gas phase. Using the polarizable continuum model to consider the water solvent effect, the
transnitrosation reactions of MeXNO and YMe− (X = S, Se; Y = S, Se) are found to undergo concerted reactions, in sharp contrast to the two-step reaction pathways concluded
in the gas phase. Moreover, the barriers of the transnitrosation reactions of MeSNO for nucleophiles SMe− and SeMe− from the gas phase to the aqueous solution are found to be decreased, while the transnitrosation reactions of MeSeNO are
essentially barrierless in aqueous solution. 相似文献
20.
Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement 总被引:4,自引:0,他引:4
Cadet J Douki T Frelon S Sauvaigo S Pouget JP Ravanat JL 《Free radical biology & medicine》2002,33(4):441-449
Oxidation reactions that involve several oxygen and nitrogen reactive species together with nucleobase radical cations give rise among various classes of lesions to modified bases. About 70 of oxidized nucleosides that include diastereomeric forms have been characterized in mechanistic studies involving isolated DNA and related model compounds. However, only eight modified bases have been accurately measured within cellular DNA upon exposure to either gamma or UVA radiations. Emphasis is placed in this survey on recent developments of HPLC associated with tandem mass spectrometry (MS/MS) operating in the mild electrospray ionization mode. Interestingly, the HPLC-MS/MS assay in the multiple reaction monitoring mode appears to be the more sensitive and accurate method currently available for singling out several oxidized nucleosides including 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 5-formyl-2'-deoxyuridine, 5-(hydroxymethyl-2'-deoxyuridine, 5-hydroxy-2'-deoxyuridine, and the four diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine within isolated and cellular DNA. However, one limitation of the assay that also applied to all chromatographic methods is the slight side-oxidation of normal bases during DNA extraction and subsequent work-up. This explains why the combined use of DNA repair glycosylases with either the comet assay or the alkaline elution technique is a better alternative to monitor the formation of low levels of oxidized bases within cellular DNA. 相似文献