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T Douki

Publications and source records attributed to T Douki.

At least 37 records · Page 2Linked to original sources
PubMed2000

Protection against radiation-induced degradation of DNA bases by polyamines.

Polyamines have been reported to protect DNA against the formation of radiation-induced strand breaks and crosslinks to proteins. The present study was aimed at investigating the protective effect of spermine, spermidine and putrescine against the degradation of DNA bases upon exposure to gamma rays in aerated aqueous solution. The yield of 8-oxo-7,8-dihydroguanine and 5-hydroxycytosine was found to decrease for concentrations of spermine and spermidine greater than 0.1 mM. A protection factor of 10 was observed for a concentration of 1 mM of the latter two polyamines. Putrescine afforded a lower protection. In addition, the formation yield of a series of radiation-induced degradation products of the purine and pyrimidine bases was determined within DNA in the presence or absence of spermine. The protection factor was within the same range for all the lesions measured. The latter observation ruled out the possibility of degradation of DNA by radiation-induced polyamine peroxyl radicals. This was confirmed by studies involving radiolysis of DMSO and decomposition of 2,2'-azobis(2-methyl-propionamidine) as sources of alkylperoxyl radicals. Therefore, it is likely that the polyamine-mediated protection against the radiation-induced degradation of DNA bases is due to the compaction of the DNA structure and the reduction in the accessibility of DNA to .OH rather than by scavenging .OH in the bulk solution or in the vicinity of the DNA.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

2'-Deoxycytidine glycols, a missing link in the free radical-mediated oxidation of DNA.

2'-Deoxycytidine glycols (5,6-dihydroxy-5, 6-dihydro-2'-deoxycytidine) are major products of the hydroxyl radical-induced oxidation of 2'-deoxycytidine resulting from either a Fenton reaction or exposure to ionizing radiation. Because of their instability, however, the glycols have not previously been characterized. Instead, the impetus has been placed on the primary decomposition products of 2'-deoxycytidine glycols, which includes 5-hydroxy-2'-deoxycytidine, 5-hydroxy-2'-deoxyuridine, and 2'-deoxyuridine glycols. Here, we have identified one of the four possible diastereomers of 2'-deoxycytidine glycols by product analyses of decomposition products, (1)H NMR, and mass spectrometry. This glycol was observed to decompose with a half-life of 50 min at 37 degrees C in buffered neutral solutions and preferentially undergo dehydration to 5-hydroxy-2'-deoxycytidine. The rate of decomposition was strongly dependent on pH (2-10) and the concentration of phosphate ion (10-300 mM). Next, we report on the deamination of cytosine glycols to uracil glycols in oxidized DNA using acid hydrolysis and high performance liquid chromatography analysis with electrochemical detection to monitor 5-hydroxycytosine and 5-hydroxyuracil. The results showed that the lifetime of cytosine glycols is greatly enhanced in DNA (34-fold; half-life, 28 h), and that deamination accounts for at least one-third of the total decomposition. The relatively long lifetime of cytosine glycols in DNA suggests that this important class of DNA oxidation products will be significantly involved in repair and mutagenesis processes.

Animals↗
PubMed1999

Hydroxyl radicals and DNA base damage.

Modified purine and pyrimidine bases constitute one of the major classes of hydroxyl-radical-mediated DNA damage together with oligonucleotide strand breaks, DNA-protein cross-links and abasic sites. A comprehensive survey of the main available data on both structural and mechanistic aspects of.OH-induced decomposition pathways of both purine and pyrimidine bases of isolated DNA and model compounds is presented. In this respect, detailed information is provided on both thymine and guanine whereas data are not as complete for adenine and cytosine. The second part of the overview is dedicated to the formation of.OH-induced base lesions within cellular DNA and in vivo situations. Before addressing this major point, the main available methods aimed at singling out.OH-mediated base modifications are critically reviewed. Unfortunately, it is clear that the bulk of the chemical and biochemical assays with the exception of the high performance liquid chromatographic-electrochemical detection (HPLC/ECD) method have suffered from major drawbacks. This explains why there are only a few available accurate data concerning both the qualitative and quantitative aspects of the.OH-induced formation of base damage within cellular DNA. Therefore, major efforts should be devoted to the reassessment of the level of oxidative base damage in cellular DNA using appropriate assays including suitable conditions of DNA extraction.

Animals↗
PubMed1999

Modification of DNA bases by photosensitized one-electron oxidation.

PURPOSE: To determine the distribution of base damage within isolated DNA upon oxidation by three type I photosensitizers in aerated aqueous solution. MATERIALS AND METHODS: Aqueous solutions of DNA were exposed to UVA in the presence of riboflavin, benzophenone or menadione. Then, eight modified nucleobases were measured, using HPLC-EC, GC-MS or HPLC with fluorescence detection. RESULTS: The three photosensitizers led to a predominant degradation of guanine bases within DNA. The relative yield of the three main guanine degradation products measured in DNA was similar with the three sensitizers. 8-OxodAdo was also produced in an almost constant yield with respect to its guanine analogue. The yield of oxidized pyrimidines was lower and was found to depend on the photosensitizer used. The results were compared with the yield of photosensitization-induced degradation of the 2'-deoxyribonucleosides. CONCLUSION: The favoured photosensitized degradation of guanine within DNA may be explained by its lower oxidation potential with respect to that of the other bases, together with the occurrence of charge transfer through DNA. The base modification pattern determined in the present work is different from that obtained upon reaction of hydroxyl radicals. Under the latter conditions, pyrimidine oxidation products were generated more efficiently than by photosensitized one-electron oxidation.

Benzophenones↗
PubMed1999

Measurement of DNA base damage in cells exposed to low doses of gamma-radiation: comparison between the HPLC-EC and comet assays.

PURPOSE: Two different methods aimed at measuring DNA damage were compared. Monocytes were exposed to 60Co gamma-rays and the level of DNA damage was determined using either the HPLC-EC or comet assay. MATERIALS AND METHODS: The alkaline comet assay was used in association with the Fpg and Endo III DNA glycosylases to estimate the amount of modified bases together with strand breaks and alkali-labile sites. The HPLC-EC analysis was performed to measure 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) levels in cells. RESULTS: A correlation between these assays allowed the determination of the steady-state level and the yield of Fpg sensitive sites (assimilated to 8-oxodGuo), which were estimated to be 0.18 per 10(6) bp and 0.044 per 10(6) bp per Gy, respectively. Similar levels of Endo III sensitive sites were found. For the strand breaks and alkali-labile sites, the background level was 0.26 per 10(6) bp and the yield 0.123 per 10(6) bp per Gy. DISCUSSION: The modified comet assay appears to be an appropriate tool to estimate DNA base damage in cells exposed to low doses of gamma-radiation.

Chromatography, High Pressure Liquid↗
PubMed1999

Oxidation of guanine in cellular DNA by solar UV radiation: biological role.

The formation of cyclobutane pyrimidine dimers (CPD) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) was investigated in Chinese hamster ovary cells upon exposure to either UVC, UVB, UVA or simulated sunlight (SSL). Two cell lines were used, namely AT3-2 and UVL9, the latter being deficient in nucleotide excision repair and consequently UV sensitive. For all types of radiation, including UVA, CPD were found to be the predominant lesions quantitatively. At the biologically relevant doses used, UVC, UVB and SSL irradiation yielded 8-oxodGuo at a rather low level, whereas UVA radiation produced relatively higher amounts. The formation of CPD was 10(2) and 10(5) more effective upon UVC than UVB and UVA exposure. These yields of formation followed DNA absorption, even in the UVA range. The calculated relative spectral effectiveness in the production of the two lesions showed that efficient induction of 8-oxodGuo upon UVA irradiation was shifted toward longer wavelengths, in comparison with those for CPD formation, in agreement with a photosensitization mechanism. In addition, after exposure to SSL, about 19% and 20% of 8-oxodGuo were produced between 290-320 nm and 320-340 nm, respectively, whereas CPD were essentially (90%) induced in the UVB region. However, the ratio of CPD to 8-oxodGuo greatly differed from one source of light to the other: it was over 100 for UVB but only a few units for UVA source. The extent of 8-oxodGuo and CPD was also compared to the lethality for the different types of radiation. The involvement of 8-oxodGuo in cell killing by solar UV radiation was clearly ruled out. In addition, our previously reported mutation spectra demonstrated that the contribution of 8-oxodGuo in the overall solar UV mutagenic process is very minor.

Animals↗
PubMed1999

Purine DNA adducts of 4,5-dioxovaleric acid and 2,4-decadienal.

The present overview describes recent findings on the formation of cyclic adducts of purine DNA bases after reaction with two aldehyde compounds, 4,5-dioxovaleric acid (DOVA) and 2,4-decadlenal (DDE), which are involved in 5-aminolaevulinic acid (ALA) accumulation and lipid peroxidation, respectively. ALA accumulates under pathological conditions and is associated with an increased incidence of liver cancer. The final oxidation product of ALA, DOVA, is an efficient alkylating agent of the guanine moieties in both nucleoside and isolated DNA. Adducts were produced through the formation of a Schiff base involving the N2-amino group of 2'-deoxyguanosine and the ketone function of DOVA, respectively. DDE is an important breakdown product of lipid peroxidation. It is cytotoxic to mammalian cells and is known to be implicated in DNA damage. It can bind to 2'-deoxyadenosine, yielding highly fluorescent products, including 1,N6-etheno-2'-deoxyadenosine and two other, related adducts. The reaction mechanism for the formation of DDE-2'-deoxyadenosine adducts involves epoxidation of DDE and subsequent addition of the resulting reactive intermediates to the N6 amino group of 2'-deoxyadenosine, followed by cyclization at the N1 site. Formation of endogenous DNA adducts may contribute to the genotoxic potential of ALA and DDE.

Aldehydes↗
PubMed1998

Isotope dilution high-performance liquid chromatography-electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine in biological samples.

A sensitive and specific assay aimed at measuring 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) has been developed by associating a reversed-phase liquid chromatographic separation with an electrospray tandem mass spectrometric detection. The HPLC-MS approach in the single ion monitoring (SIM) mode and the HPLC-MS/MS assay in the multiple reaction monitoring (MRM) mode have been compared, using isotopically labeled [M+4] 8-oxodGuo as the internal standard. The limit of detection of 8-oxodGuo was found to be around 5 pmol and 20 fmol for the HPLC-MS and HPLC-MS/MS methods, respectively. The HPLC-MS/MS assay is sensitive enough to allow the determination of the level of 8-oxodGuo in cellular liver DNA and in urine samples.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

Hydroxyl radicals are involved in the oxidation of isolated and cellular DNA bases by 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA) is a heme precursor, pathological accumulation of which is associated with liver cancer. We show that the reactive oxygen species produced upon ALA metal-catalyzed oxidation promote the formation of several radical-induced base degradation products in isolated DNA. The distribution of modified bases is similar to that obtained upon gamma irradiation. This observation strongly suggests the involvement of hydroxyl radicals in the ALA-mediated DNA damage. Increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-hydroxy-2'-deoxycytidine in organ DNA of rats chronically treated with ALA were observed. This is strongly suggestive of the implication of hydroxyl radicals in the ALA-induced degradation of cellular DNA.

Aminolevulinic Acid↗
PubMed1998

DNA alkylation by 4,5-dioxovaleric acid, the final oxidation product of 5-aminolevulinic acid.

The heme precursor 5-aminolevulinic acid (ALA) accumulates under pathological conditions, namely, acute intermittent porphyria (AIP) and tyrosinosis, two diseases that are associated with increased liver cancer incidence. This has been previously linked to an enhanced production of reactive oxygen species generated by a metal-catalyzed ALA oxidation process, which was shown to cause DNA single-strand breaks and guanine oxidation within both isolated and cellular DNA. In the present work, we established that the final oxidation product of ALA, 4,5-dioxovaleric acid (DOVA), is an efficient alkylating agent of the guanine moieties within both nucleoside and isolated DNA. Adducts were produced through the formation of a Schiff base involving the N2-amino group of 2'-deoxyguanosine (dGuo) and the ketone function of DOVA, respectively. The modified dGuo nucleosides were characterized, following reduction into stable secondary amines, by extensive NMR, infrared, and mass spectrometry analyses. A method, based on the use of HPLC with electrochemical detection, was then developed for the sensitive measurement of the DOVA-dGuo adducts. Using this assay, we showed that the guanine moieties of isolated DNA can undergo the same reaction as the free nucleoside. The present data provide additional information on the genotoxic potential of ALA and reinforce the hypothesis that AIP may be involved in the induction of primary liver cell carcinoma.

Alkylation↗
PubMed1998

A novel vicinal lesion obtained from the oxidative photosensitization of TpdG: characterization and mechanistic aspects.

A new type of vicinal base lesion was isolated from the photosensitization of TpdG in aerated aqueous solution. One- and two-dimensional NMR measurements were used together with mass spectrometry to accurately characterize the new adduct. Chemical detection of guanidine provided additional structural information on the base moiety at the 3'-OH terminal end. Altogether the experiments results were indicative of the occurrence of a covalent bonding between the pyrimidine ring on the 5'-OH terminal end and the imidazole ring on the 3'-OH terminal end through a methylene bridge. Photosensitization studies of TpdG, thymidine, and 2'-deoxyguanosine in the presence of either benzophenone, menadione, or riboflavin associated with isotopic labeling experiments using enriched oxygen and water provided relevant information on the mechanism of formation of the adduct. The results of these experiments clearly demonstrated that the initial event leading to the formation of the lesion is the abstraction of a hydrogen atom from the methyl group of the thymine base moiety of TpdG. This is followed by the addition of the methyl-centered radical to the C-4 atom of the guanine ring which gives rise to the vicinal lesion after reaction with molecular oxygen and subsequent rearrangement.

Chromatography, High Pressure Liquid↗
PubMed1998

Formation of 1,N6-etheno-2'-deoxyadenosine adducts by trans,trans-2, 4-Decadienal.

trans,trans-2,4-Decadienal (DDE) is an important breakdown product of lipid peroxidation. This aldehyde is cytotoxic to mammalian cells and is known to be implicated in DNA damage. Therefore, attempts were made in this work to assess the reactivity of DDE with 2'-deoxyadenosine (dAdo). It was shown that DDE is able to bind to 2'-deoxyadenosine, yielding highly fluorescent products. Besides 1, N6-etheno-2'-deoxyadenosine (epsilondAdo), two other related adducts, 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-1,2,3-octanetriol and 1-[3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]-1,2-heptanediol, were isolated by reverse phase high-performance liquid chromatography and characterized on the basis of their UV, fluorescence, nuclear magnetic resonance, and mass spectrometry features. The reaction mechanism for the formation of the DDE-2'-deoxyadenosine adducts involves 2,4-decadienal epoxidation and subsequent addition to the N2 amino group of 2'-deoxyadenosine, followed by cyclization at the N-1 site. Adducts differ by the length of carbon side chain and the number of hydroxyl groups. The present data indicate that DDE can be epoxidized by peroxides, and the resulting products are able to form several adducts with 2'-deoxyadenosine and/or DNA. Endogenous DNA adduct formation can contribute to the already reported high cytotoxicity of DDE to mammalian cells.

Aldehydes↗
PubMed1998

Facts and artifacts in the measurement of oxidative base damage to DNA.

This short survey is aimed at critically evaluating the main available methods for measuring oxidative base damage within cellular DNA. Emphasis is placed on separative methods which are currently widely applied. These mostly concern high performance liquid chromatography (HPLC) and gas chromatography (GC) associated with sensitive detection techniques such as electrochemistry (EC) and mass spectrometry (MS). In addition, the comparison is extended to 32p-postlabeling methods, immunoassays and measurement of two main classes of oxidative DNA damage within isolated cells. It may be concluded that the HPLC-electrochemical detection (ECD) method, even if restricted to the measurement of only a few electroactive oxidized bases and nucleosides, is the simplest and safest available method at the moment. In contrast, the more versatile GC-MS method, which requires a HPLC pre-purification step in order to prevent artifactual oxidation of overwhelming normal bases to occur during derivatization, is more tedious and its sensitivity may be questionable. Alternative simpler procedures of background prevention for the GC-MS assay, which, however, remain to be validated, include low-temperature for derivatization and addition of antioxidants to the silylating reagents. Interestingly, similar levels of 8-oxo-7,8-dihydroguanine were found in cellular DNA using HPLC-ECD, HPLC-MS/MS and HPLC/32P-postlabeling methods. However, it should be noted that the level of cellular 8-oxodGuo, thus determined, is on average basis 10-fold higher than that was inferred for more indirect measurement involving the use of DNA repair enzymes with methods on isolated cells. Further efforts should be made to resolve this apparent discrepancy. In addition, the question of the biological validation of the non-invasive measurement of oxidized bases and nucleosides in urine is addressed.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Measurement of 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxo-7,8-dihydroguanine in isolated DNA exposed to gamma radiation in aqueous solution.

Hydroxyl radical-mediated modifications of nucleobases are among the main deleterious processes induced in DNA by ionizing radiation. Determination of the underlying mechanisms in both isolated and cellular DNA requires the development of accurate assays for the detection of modified bases. High-performance liquid chromatography associated with electrochemical detection (HPLC-EC) and gas chromatography coupled to mass spectrometry (GC-MS) are the two main methods used for this purpose. In the present work, HPLC-EC was applied to the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) following enzymatic digestion of DNA. Evidence was provided for the quantitative aspect of the latter step. Moreover, a GC-MS assay was designed for the detection of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). It was first shown that the widely used hot formic acid hydrolysis of DNA induced a complete decomposition of the lesion. A similar observation was also made for 4,6-diamino-5-formamidopyrimidine (FapyAde). A milder DNA hydrolyzing agent, HF in pyridine, was substituted to HCOOH for the quantitative release of FapyGua from DNA. The radiation-induced formation of 8-oxodGuo and FapyGua in aqueous solution of DNA was compared. Insights in the mechanistic aspects were inferred from the results of gamma-irradiation studies carried out under different gas saturating conditions. It was clearly shown that the formation of both lesions is mediated by HO.. In addition, the presence of oxygen favors the formation of 8-oxodGuo at the expense of FapyGua.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Far-UV-induced dimeric photoproducts in short oligonucleotides: sequence effects.

Cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone adducts represent the two major classes of far-UV-induced DNA photoproducts. Because of the lack of appropriate detection methods for each individual photoproduct, little is known about the effect of the sequence on their formation. In the present work, the photoproduct distribution obtained upon exposure of a series of dinucleoside monophosphates to 254 nm light was determined. In the latter model compounds, the presence of a cytosine, located at either the 5'- or the 3'-side of a thymine moiety, led to the preferential formation of (6-4) adducts, whereas the cis-syn cyclobutane dimer was the main thymine-thymine photoproduct. In contrast, the yield of dimeric photoproducts, and particularly of (6-4) photoadducts, was very low upon irradiation of the cytosine-cytosine dinucleoside monophosphate. However, substitution of cytosine by uracil led to an increase in the yield of (6-4) photoproduct. It was also shown that the presence of a phosphate group at the 5'- end of a thymine-thymine dinucleoside monophosphate does not modify the photoproduct distribution. As an extension of the studies on dinucleoside monophosphates, the trinucleotide TpdCpT was used as a more relevant DNA model. The yields of formation of the thymine-cytosine and cytosine-thymine (6-4) photoproducts were in a 5:1 ratio, very close to the value obtained upon photolysis of the related dinucleoside monophosphates. The characterization of the two TpdCpT (6-4) adducts was based on 1H NMR, UV and mass spectroscopy analyses. Additional evidence for the structures was inferred from the analysis of the enzymatic digestion products of the (6-4) adducts of TpdCpT with phosphodiesterases. The latter enzymes were shown to induce the quantitative release of the photoproduct as a modified dinucleoside monophosphate in a highly sequence-specific manner.

Dimerization↗
PubMed1997

Artifacts associated with the measurement of oxidized DNA bases.

In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay.

Artifacts↗