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1.
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.  相似文献   

2.
Abstract: Oxidative damage has been implicated in the pathology of Parkinson's disease (PD), e.g., rises in the level of the DNA damage product, 8-hydroxy-2'-deoxyguanosine, have been reported. However, many other products result from oxidative DNA damage, and the pattern of products can be diagnostic of the oxidizing species. Gas chromatography/mass spectrometry was used to examine products of oxidation and deamination of all four DNA bases in control and PD brains. Products were detected in all brain regions examined, both normal and PD. Analysis showed that levels of 8-hydroxyguanine (8-OHG) tended to be elevated and levels of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine) tended to be decreased in PD. The most striking difference was a rise in 8-OHG in PD substantia nigra ( p = 0.0002); rises in other base oxidation/deamination products were not evident, showing that elevation in 8-OHG is unlikely to be due to peroxynitrite (ONOO) or hydroxyl radicals (OH), or to be a prooxidant effect of treatment with l -Dopa. However, some or all of the rise in 8-OHG could be due to a change in 8-OHG/FAPy guanine ratios rather than to an increase in total oxidative guanine damage.  相似文献   

3.
T4 RNA ligase was used to construct a deoxypentanucleotide containing a single 8-hydroxyguanine (7-hydro-8-oxoguanine; G8-OH) residue, which is one of the putatively mutagenic DNA adducts produced by oxidants and ionizing radiation. The pentamer d(GCTAG8-OH)p was prepared by the ligation of a chemically synthesized acceptor molecule, d(GCTA), to an adducted donor, 8-hydroxy-2'-deoxyguanosine 5',3'-bisphosphate. The acceptor was efficiently converted to the reaction product (greater than 95%), and the final product yield was 50%. Following 3'-dephosphorylation, the pentamer was characterized by UV spectroscopy, by high-pressure liquid chromatography, and by gas chromatography-mass spectrometry of the nucleosides released by enzymatic hydrolysis. Both d(GCTAG8-OH) and an unmodified control were 5'-phosphorylated by using [gamma -32P]ATP and incorporated covalently by DNA ligase into a five-base gap at a unique NheI restriction site in the otherwise duplex genome of an M13mp19 derivative. The ligation product contained G8-OH at the 3' residue of an in-frame amber codon (5'-TAG-3') (genome position 6276) of the phage lacZ alpha gene. The adduct was part of a nonsense codon in a unique restriction site in order to facilitate the identification and selection of mutants generated by the replication of the modified genome in Escherichia coli. Both control and adducted pentamers ligated into the genome at 50% of the maximum theoretical efficiency, and nearly all (approximately 90%) of the site-specifically adducted products possessed pentanucleotides that were covalently linked at both 5' and 3' termini. The G8-OH lesion in the NheI site inhibited the cleavage of the site by a 200-fold excess of NheI.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

4.
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   

5.
DNA damage induced by estrogens dispersed in liposomes was investigated. 2-Hydroxyestradiol (2HOE(2)) and 4-hydroxyestradiol (4HOE(2)), but not estrone, estradiol-17beta or estriol, caused strand break of plasmid DNA damage in the presence of ADP-Fe(3+). The catechol structure may be necessary for DNA damage. When DNA was incubated with 2HOE(2) for a long time (24 h), DNA damage was induced even at very low concentrations. Adding hydrogen peroxide markedly enhanced the sensitivity of DNA to the attack by 2HOE(2). Hydroxyl radical (HO.) scavengers strongly inhibited the 2HOE(2)-induced DNA damage, and EDTA partially inhibited DNA damage. However, 2HOE(2) caused 8-hydroxyguanine formation from calf thymus DNA only in the presence of EDTA-Fe(3+), but not ADP-Fe(3+). In addition, deoxyribose, which is a detective molecule of HO(.), was not degraded by 2HOE(2) in the presence of ADP-Fe(3+). Upon adding EDTA 2HOE(2) rapidly degraded deoxyribose. These results suggest that DNA strand break caused by 2HOE(2) in the presence of ADP-Fe(3+) was due to ferryl ion rather than HO(.), whereas 8-hydroxyguanine (8HOG) induced by 2HOE(2) in the presence of EDTA-Fe(3+) was due to HO(.).  相似文献   

6.
One-electron oxidation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) yielded a guanidinohydantoin derivative (dGh) and a spiroiminodihydantoin derivative (dSp), both putatively mutagenic products that may be formed in vivo. The nucleoside dGh was the major product at room temperature, regardless of pH. The results are contrary to previously published model studies using 2',3',5'-triacetoxy-8-oxo-7,8-dihydroguanosine (Luo, W.; Miller, J. G.; Rachlin, E. M.; Burrows, C. J. Org. Lett. 2000, 2, 613; Luo, W.; Miller, J.G.; Rachlin, E.M.; Burrows, C.J. Chem. Res. Toxicol. 2001, 14, 927), who observed a spiroiminodihydantoin derivative as the major product at neutral pH. Clearly, the functional groups attached to the ribose moiety of 8-oxodG influence the oxidation chemistry of the nucleobase derivative. To explore this chemistry in vivo, (14)C-labeled 8-oxodG was synthesized and incubated with growing MCF-7 human breast cancer cells, resulting in the incorporation of the compound into cellular DNA as measured by a novel accelerator mass spectrometry assay.  相似文献   

7.
To investigate the mechanism by which the 8-hydroxyguanine residue in DNA affects the fidelity of DNA replication, the intrinsic properties of this modified base were investigated using an ab initio molecular orbital method. The most stable 8-hydroxyguanine form was revealed to be 6,8-diketo. The addition of an oxygen atom to the 8 position of a guanine base was shown to change the electrostatic potential of the molecule entirely and to give it a negative character. This effect may influence the local structure of 8-hydroxyguanine-containing DNA and the interaction with DNA polymerase, thereby resulting in infidelity of DNA replication.  相似文献   

8.
Consideration of how 15-hydroxyeicosatetraenoic acid (15-HETE) might exert its biological actions led us to investigate the consequences of its incorporation into bovine pulmonary arterial endothelial cell (BPAEC) phospholipids [3H]15(S)-HETE was incorporated mainly (89%) into phosphatidylinositols, predominantly as 1-stearoyl-2-(15-HETE) phosphatidylinositol. By contrast 5(S)- and 12(S)-HETE are incorporated largely into phosphatidylcholine. 15-HETE had a long persistence in the phosphatidylinositols of BPAEC with a half-life of 12 h; its uptake was concentration-dependent, and it accumulated so that 2-(15-HETE) phosphatidylinositol accounted for 10.9% of total phosphatidylinositol after four sequential 1-h incubations of cells with 1 microM 15(S)-HETE. After incubating BPAEC with 15(S)-HETE, stimulation of the cells with bradykinin led to an increase in the levels of 15-HETE. Following addition of bradykinin to cells exposed to [3H]15(S)-HETE, a radiolabeled diacylglycerol was isolated. A mass spectrum of its pentafluorobenzoyl (PFBO) trimethylsilyl (Me3Si) derivative obtained with direct electron capture negative ion chemical ionization mass spectrometry (DNICI/MS) revealed a molecular anion and fragment ions that were identical with those observed with the PFBO/Me3Si derivative of authentic 1-stearoyl-2-(15-HETE) diacylglycerol. There was a lesser quantity of 1-oleoyl-2-(15-HETE) diacylglycerol. An increase in the quantity of 1-stearoyl-2-(15-HETE) diacylglycerol from 6 +/- 1.4 pmol/10(7) cells in the basal state to 12.7 +/- 3.5 after bradykinin was measured by DNICI/MS utilizing a deuterium-labeled analog as an internal standard. Thus, incorporation of 15(S)-HETE into the phosphatidylinositol of these cells led to the release of altered second messengers.  相似文献   

9.
The horseradish-peroxidase(HRP)-catalyzed aerobic oxidation of aldehydes, in particular isobutanal, was used for the oxidative damage of DNA. In isolated calf-thymus DNA, the enzymatic oxidation of isobutanal led to 7,8-dihydro-8-oxoguanine (8-oxoGua) in up to 1.3% yield and appreciable single-strand breaks in supercoiled pBR 322 DNA. For the nucleoside dG, significant amounts of the guanidine-releasing products oxazolone and oxoimidazolidine have been detected, but 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) was not obtained. Only enolizable aldehydes are effective, molecular oxygen is essential, and radical scavengers inhibit efficiently the oxidation. Comparative experiments with 3,3,4,4-tetramethyl-1,2-dioxetane (TMD) revealed that triplet-excited acetone does not play a significant role in this enzymatic DNA oxidation. 2-Hydroperoxy-2-methylpropanal, an intermediate in the HRP-catalyzed aerobic oxidation of isobutanal, does not contribute directly in the observed dG conversion. However, the peroxyl radical derived from the 2-hydroperoxy-2-methylpropanal appears to be active as oxidant because model studies with a structurally related peroxyl radical, produced by HRP-catalyzed one-electron oxidation of 3-hydroperoxy-3-methyl-2-butanone, causes both dG conversion and DNA strand breaks, but to a moderate extent. The active oxidant, as established by control experiments, is the peroxyisobutyric acid, that is efficiently formed through the HRP-catalyzed autoxidation of isobutanal. Still more effective is the acylperoxyl radical, conveniently generated from the peracid by one-electron oxidation by HRP.  相似文献   

10.
Methylene blue (MB) plus light, in the presence of oxygen, mediates formation of 8-hydroxyguanine in DNA. The yield of 8-hydroxyguanine may be as much as from 2 to 4% of the guanines present. The results presented here show that treatment of supercoiled plasmid DNA with methylene blue plus light causes single-stranded nicks. However, single-stranded nicking occurs approximately 17-fold less frequently than does formation of 8-hydroxyguanine. The nicking rate is reduced in the presence of Mg ion but is not prevented by inhibitors of the iron-catalyzed Fenton reaction or by scavengers of hydroxyl free radicals. Extensive exposure of DNA to light in the presence of MB produces no detectable thiobarbital reactive material thus implicating that single strand nicking does not occur by hydroxyl free radical attack on deoxyribose. Formation of 8-hydroxyguanine is apparently not dependent upon intercalative binding of MB to DNA, since it is formed in polydeoxyguanylic acid.  相似文献   

11.
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.  相似文献   

12.
Methylene blue plus light mediates 8-hydroxyguanine formation in DNA   总被引:14,自引:0,他引:14  
Exposure to methylene blue (MB) plus light mediates formation of large levels of 8-hydroxyguanine in DNA. The amount of 8-hydroxy-2'-deoxyguanosine (8-OHdG) present in DNA increased as the amount of MB concentration increased throughout the 2 to 200 microM range studied and was dependent on light exposure. As the time of light exposure increased so did the 8-OHdG content to levels of about 750 8-OHdG/10(5) deoxyguanosine after 15 min of light exposure when MB was at 20 microM. Even though previous research has demonstrated that hydroxyl free radicals formed from a variety of sources mediate 8-OHdG formation in DNA, inclusion of mannitol, superoxide dismutase, catalase, and desferal in the MB plus light experiments demonstrated that these scavengers of oxygen free radical intermediates or precursors caused either no change or an increase in the 8-OHdG content of DNA exposed to MB plus light. These results appear to rule out the direct role of oxygen free radical intermediates in the primary events involved in the MB plus light mediated formation of 8-OHdG in DNA. Oxygen was essential to cause MB plus light mediated 8-OHdG formation in DNA. It was noted that when the reaction was carried out where the deuterium oxide content had been increased to 100%, the amount of 8-OHdG formed in DNA increased about threefold over that observed when comparable reactions were carried out in pure H2O. Use of the singlet oxygen scavenger 2,5-dimethylfuran has yielded variable results on the MB plus light mediated formation of 8-OHdG in DNA. The data taken collectively clearly indicate that MB plus light mediates 8-OHdG formation in DNA. The D2O data and the requirement for oxygen suggest that singlet oxygen may be an intermediate.  相似文献   

13.
Recent intervention studies revealed that beta-carotene supplement to smokers resulted in a higher incidence of lung cancer. However, the causal mechanisms remain to be clarified. We reported here that vitamin A (retinol) and its derivative (retinal) caused cellular DNA cleavage detected by pulsed field gel electrophoresis. Retinol and retinal significantly induced 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in HL-60 cells but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. Experiments using (32)P-labeled isolated DNA demonstrated that retinol and retinal caused Cu(II)-mediated DNA damage, which was inhibited by catalase. UV-visible spectroscopic and electron spin resonance-trapping studies revealed the generation of superoxide and carbon-centered radicals, respectively. The superoxide generation during autoxidation of retinoids was significantly correlated with the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, although the yield of carbon-centered radicals was not necessarily related to the intensity of DNA damage. These findings suggest that superoxide generated by autoxidation of retinoids was dismutated to H(2)O(2), which was responsible for DNA damage in the presence of endogenous metals. Retinol and retinal have prooxidant abilities, which might lead to carcinogenesis of the supplements of beta-carotene.  相似文献   

14.
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.  相似文献   

15.
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   

16.
Mutations in breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 predispose women to a high risk of these cancers. Here, we show that lymphoblasts of women with BRCA1 mutations who had been diagnosed with breast cancer are deficient in the repair of some products of oxidative DNA damage, namely, 8-hydroxy-2'-deoxyguanosine and 8,5'-cyclopurine-2'-deoxynucleosides. Cultured lymphoblasts from 10 individuals with BRCA1 mutations and those from 5 control individuals were exposed to 5 Gy of ionizing radiation to induce oxidative DNA damage and then allowed to repair this damage. DNA samples isolated from these cells were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure 8-hydroxy-2'-deoxyguanosine, (5'-S)-8,5'-cyclo-2'-deoxyadenosine, (5'-R)-8,5'-cyclo-2'-deoxyguanosine, and (5'-S)-8,5'-cyclo-2'-deoxyguanosine. After irradiation and a subsequent period of repair, no significant accumulation of these lesions was observed in the DNA from control cells. In contrast, cells with BRCA1 mutations accumulated statistically significant levels of these lesions in their DNA, providing evidence of a deficiency in DNA repair. In addition, a commonly used breast tumor cell line exhibited the same effect when compared to a relevant control cell line. The data suggest that BRCA1 plays a role in cellular repair of oxidatively induced DNA lesions. The failure of cells with BRCA1 mutations to repair 8,5'-cyclopurine-2'-deoxynucleosides indicates the involvement of BRCA1 in nucleotide-excision repair of oxidative DNA damage. This work suggest that accumulation of these lesions may lead to a high rate of mutations and to deleterious changes in gene expression, increasing breast cancer risk and contributing to breast carcinogenesis.  相似文献   

17.
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.  相似文献   

18.
Potosensitized formation of 8-hydroxyguanine in DNA by riboflavin was observed. A reaction mechanism involving guanine radical cation and hydration reaction was proposed. This hypothesis was confirmed by the incorporation of [18O]-atom within guanine moiety in isotopic experiments using [18O]-H2O. Photosensitized formation of oh8Gua by riboflavin was also observed in cellular DNA.  相似文献   

19.
DNA damage induced by oxygen radicals, e.g., hydroxyl radicals generated in living cells either by cellular metabolism or external agents such as ionizing radiations, appears to play an important role in mutagenesis, carcinogenesis, and aging. Elucidation of the chemical nature of such DNA lesions at biologically significant quantities is required for the assessment of their biological consequences and repair. For this purpose, a sensitive method using gas chromatography-mass spectrometry with the selected-ion-monitoring technique (GC-MS/SIM) was developed in the present work. DNA was exposed to hydroxyl radicals and hydrogen atoms produced by ionizing radiation in N2O-saturated aqueous solution. DNA samples were subsequently hydrolyzed with formic acid, trimethylsilylated, and analyzed by GC-MS/SIM. Characteristic ions from previously known mass spectra of DNA base products as their trimethylsilyl derivatives were recorded and the area counts of each ion were integrated. From these acquired data, a partial mass spectrum of each product was generated and then compared with those of authentic materials. This technique permitted the detection and characterization of a large number of free radical-induced based products of DNA, i.e., 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-hydroxycytosine, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine, simultaneously in a single sample after radiation doses from 0.1 to 10 Gy. Detectable amounts of the base products were found to be as low as approximately 10 fmol per injection.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

20.
Aberrant oxidation is a property of many tumor cells. Oxidation of DNA precursors, i.e., deoxynucleotide triphosphates (dNTPs), as well as DNA is a major cause of genome instability. Here, we report that human DNA polymerase eta (h Poleta) incorporates oxidized dNTPs, i.e., 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), into DNA in an erroneous and efficient manner, thereby inducing various types of mutations during in vitro gap-filling DNA synthesis. When 2-OH-dATP was present at a concentration equal to those of the four normal dNTPs in the reaction mixture, DNA synthesis by h Poleta enhanced the frequency of G-to-T transversions eight-fold higher than that of the transversions in control where only the normal dNTPs were present. When 8-OH-dGTP was present at an equimolar concentration to the normal dNTPs, it enhanced the frequency of A-to-C transversions 17-fold higher than the control. It also increased the frequency of C-to-A transversions about two-fold. These results suggest that h Poleta incorporates 2-OH-dATP opposite template G and incorporates 8-OH-dGTP opposite template A and slightly opposite template C during DNA synthesis. Besides base substitutions, h Poleta enhanced the frequency of single-base frameshifts and deletions with the size of more than 100 base pairs when 8-OH-dGTP was present in the reaction mixture. Since h Poleta is present in replication foci even without exogenous DNA damage, we suggest that h Poleta may be involved in induction of various types of mutations through the erroneous and efficient incorporation of oxidized dNTPs into DNA in human cells.  相似文献   

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