Biomedical subjects

Thierry Douki

Publications and source records attributed to Thierry Douki.

At least 37 records · Page 2Linked to original sources

PubMed2005

Photosensitization of DNA by dipicolinic acid, a major component of spores of Bacillus species.

The DNA in spores of Bacillus species exhibits a relatively novel photochemistry, as 5-thyminyl-5,6-dihydrothymine (spore photoproduct (SP)) is by far the major UV photoproduct whereas cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4PPs) are the major photoproducts in growing cells. Dehydration and more importantly complexation of DNA by alpha/beta-type small, acid-soluble spore proteins (SASP) have been shown to partly explain the photochemistry of spore DNA. The large amount ( approximately 10% of dry weight) of the spore's dipicolinic acid (DPA) also has been shown to play a role in spore DNA photochemistry. In the present work we showed by exposing spores of various strains of B. subtilis to UVC radiation that DPA photosensitizes spore DNA to damage and favors the formation of SP. The same result was obtained in either the presence or absence of the alpha/beta-type SASP that saturate the spore chromosome. Addition of DPA to dry films of isolated DNA or to frozen solutions of thymidine also led to a higher yield of SP and increased ratio of CPDs to 6-4PPs; DPA also significantly increased the yield of CPDs in thymidine exposed to UVC in liquid solution. These observations strongly support a triplet energy transfer between excited DPA and thymine residues. We further conclude that the combined effects of alpha/beta-type SASP and DPA explain the novel photochemistry of DNA in spores of Bacillus species.

Bacillus↗

PubMed2005

Ultraviolet radiation-mediated damage to cellular DNA.

Emphasis is placed in this review article on recent aspects of the photochemistry of cellular DNA in which both the UVB and UVA components of solar radiation are implicated individually or synergistically. Interestingly, further mechanistic insights into the UV-induced formation of DNA photoproducts were gained from the application of new accurate and sensitive chromatographic and enzymic assays aimed at measuring base damage. Thus, each of the twelve possible dimeric photoproducts that are produced at the four main bipyrimidine sites can now be singled out as dinucleoside monophosphates that are enzymatically released from UV-irradiated DNA. This was achieved using a recently developed high-performance liquid chromatography-tandem mass spectrometry assay (HPLC-MS/MS) assay after DNA extraction and appropriate enzymic digestion. Interestingly, a similar photoproduct distribution pattern is observed in both isolated and cellular DNA upon exposure to low doses of either UVC or UVB radiation. This applies more specifically to the DNA of rodent and human cells, the cis-syn cyclobutadithymine being predominant over the two other main photolesions, namely thymine-cytosine pyrimidine (6-4) pyrimidone adduct and the related cyclobutyl dimer. UVA-irradiation was found to generate cyclobutane dimers at TT and to a lower extent at TC sites as a likely result of energy transfer mechanism involving still unknown photoexcited chromophore(s). Oxidative damage to DNA is also induced although less efficiently by UVA-mediated photosensitization processes that mostly involved 1O2 together with a smaller contribution of hydroxyl radical-mediated reactions through initially generated superoxide radicals.

Animals↗

PubMed2005

Enhancement of the in vitro transcription by T7 RNA polymerase of short DNA templates containing oxidative thymine lesions.

5-(Hydroxymethyl)uracil, 5-formyluracil or 5-carboxyuracil can be formed in DNA upon exposure to ionizing radiations. We report the effect of the latter oxidative lesions on the in vitro transcription by T7 RNA polymerase using a single-stranded short model template. Kinetic experiments showed that the transcription yield was increased with the modified template. Due to the unforeseen presence of a hairpin structure within the transcribed RNA, we postulated that we are in the presence of a T7 RNA polymerase pausing signal and that the lesions within this critical DNA structure could interfere with normal regulation of RNA transcription.

Bacteriophage T7↗

PubMed2004

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells.

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2004

MiaB protein is a bifunctional radical-S-adenosylmethionine enzyme involved in thiolation and methylation of tRNA.

The last biosynthetic step for 2-methylthio-N(6)-isopentenyl-adenosine (ms(2)i(6)A), present at position 37 in some tRNAs, consists of the methylthiolation of the isopentenyl-adenosine (i(6)A) precursor. In this work we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. We show that a synthetic i(6)A-containing RNA corresponding to the anticodon stem loop of tRNA(Phe) is also a substrate. This study demonstrates that MiaB protein is a bifunctional system, involved in both thiolation and methylation of i(6)A. In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation ofa5'-deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. The origin of the sulfur atom is discussed.

Adenosine↗

PubMed2004

Predominance of the 1,N2-propano 2'-deoxyguanosine adduct among 4-hydroxy-2-nonenal-induced DNA lesions.

4-Hydroxy-2-nonenal (HNE), one of the main aldehydic compounds released during lipid peroxidation, has been proposed to react with DNA bases in cells. Several classes of DNA lesions involving addition of either HNE or its 2,3-epoxide (epox-HNE) have been identified. In the present work, HPLC associated with tandem mass spectrometry was used to determine the pattern of HNE-induced DNA lesions. First, adducts were quantified within isolated DNA treated with HNE under peroxidizing conditions. The 1,N2-propano-2'-deoxyguanosine adduct of HNE (HNE-dGuo) was found to be the major lesion under all conditions studied. 1,N6-Ethenoadenine and 1,N2-ethenoguanine together with their (1,2-dihydroxyheptyl)-substituted derivatives, which all arise from the reaction of epox-HNE with DNA, were produced in significantly lower yields, even in the presence of 20 mM H2O2. The pyrimidopurinone malondialdehyde-2'-deoxyguanosine adduct was also found to be produced, although in very low yield. Similar results were obtained in cultured human monocytes incubated with HNE, because the HNE-dGuo adduct represented more than 95% of the overall adducts to DNA. In addition, the former lesion was poorly repaired, in contrast to 1,N2-ethenoguanine and, to a lesser extent, 1,N6-ethenoadenine. Altogether, these results suggest than HNE-dGuo may represent the best biomarker of the genotoxic effects of HNE.

Animals↗

PubMed2004

The upstream oxylipin profile of Arabidopsis thaliana: a tool to scan for oxidative stresses.

Various physiological imbalances lead to reactive oxygen species (ROS) overproduction and/or increases in lipoxygenase (LOX) activities, both events ending in lipid peroxidation of polyunsaturated fatty acids (PUFAs). Besides the quantification of such a process, the development of tools is necessary in order to allow the identification of the primary cause of its development and localization. A biochemical method assessing 9 LOX, 13 LOX and ROS-mediated peroxidation of membrane-bound and free PUFAs has been improved. The assay is based on the analysis of hydroxy fatty acids derived from PUFA hydroperoxides by both the straight and chiral phase high-performance liquid chromatography. Besides the upstream products of peroxidation of the 18:2 and 18:3 PUFAs, products coming from the 16:3 were characterized and their steady-state level quantified. Moreover, the observation that the relative amounts of the ROS-mediated peroxidation isomers of 18:3 were constant in leaves allowed us to circumvent the chiral analyses for the discrimination and quantification of 9 LOX, 13 LOX and ROS-mediated processes in routine experiments. The methodology has been successfully applied to decipher lipid peroxidation in Arabidopsis leaves submitted to biotic and abiotic stresses. We provide evidence of the relative timing of enzymatic and non-enzymatic lipid peroxidation processes. The 13 LOX pathway is activated early whatever the nature of the stress, leading to the peroxidation of chloroplast lipids. Under cadmium stress, the 9 LOX pathway added to the 13 LOX one. ROS-mediated peroxidation was mainly driven by light and always appeared as a late process.

Arabidopsis↗

PubMed2004

Unrepaired cyclobutane pyrimidine dimers do not prevent proliferation of UV-B-irradiated cultured human fibroblasts.

Mutagenic and carcinogenic UV-B radiation is known to damage DNA mostly through the formation of bipyrimidine photoproducts, including cyclobutane dimers (CPD) and (6-4) photoproducts ((6-4) PP). Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of thymine-thymine (TT) and thymine-cytosine (TC) CPD and (6-4) PP in the DNA of cultured human dermal fibroblasts. A major observation was that the rate of repair of the photoproducts did not depend on the identity of the modified pyrimidines. In addition, removal of CPD was found to significantly decrease with increasing applied UV-B dose, whereas (6-4) PP were efficiently repaired within less than 24 h, irrespective of the dose. As a result, a relatively large amount of CPD remained in the genome 48 h after the irradiation. Because the overall applied doses (<500 J m(-2)) were chosen to induce moderate cytotoxicity, fibroblasts could recover their proliferation capacities after transitory cell cycle arrest, as shown by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and flow cytometry analysis. It could thus be concluded that UV-B-irradiated cultured primary human fibroblasts normally proliferate 48 h after irradiation despite the presence of high levels of CPD in their genome. These observations emphasize the role of CPD in the mutagenic effects of UV-B.

Bromodeoxyuridine↗

PubMed2004

Interexperimental and interindividual variations of DNA repair capacities after UV-B and UV-C irradiations of human keratinocytes and fibroblasts.

DNA repair plays a central role in the cellular response to UV. In this work we have studied the response of skin cells (i.e. fibroblasts and keratinocytes) from the same or from different individuals after both ultraviolet-B (UV-B) and ultraviolet-C (UV-C) irradiations using the comet assay to characterize the specific cellular response to UV-induced DNA damage. Cells were irradiated with increasing doses of UV-B or UV-C. To study the UV dose dependency of initial steps of DNA repair, namely recognition and incision at DNA damage level, the comet assay was performed, under alkaline conditions, 60 min after UV irradiation to allow detection of DNA strand breaks. Comparative analysis of tail moment values after UV exposure of cells from the same or from different individuals showed interexperimental and interindividual variations, implying that repeated assays are necessary to characterize the individual DNA repair capacity. With increasing doses of UV in keratinocytes, a plateau was rapidly reached after irradiation, whereas in fibroblasts a linear dose-effect relationship was observed. These interindividual variations associated with cellular specificity in DNA response may be of significance in skin cell and individual susceptibility toward UV-induced carcinogenesis.

Cells, Cultured↗

PubMed2004

Radiation-induced DNA damage: formation, measurement, and biochemical features.

Most of the reactions induced by *OH radicals (indirect effects) and by one-electron oxidation (direct effects) as the result of exposure to ionizing radiation may be described for the four main DNA nucleobases. Relevant information is now available on the formation of single and tandem base lesions implicating guanine as the most susceptible DNA component to the deleterious effects of ionizing radiation. In contrast, there is still a paucity of information on the radiation-induced formation of base damage within cellular DNA. This is mostly a result of difficulties associated with the measurement of oxidized purine and pyrimidine bases that appear to be generated in very low yields. This is illustrated by the measurement of low amounts of E. coli formamidopyrimidine glycosylase- and endonuclease-III-sensitive sites in the DNA of neoplastic monocytes upon exposure to gamma rays (48 and 53 per 10(9) bases and per Gy, respectively) using a modified comet assay (the overall number of strand breaks and alkali-labile sites was estimated to be 130 per 10(9) bases and per Gy). More specifically, the level of several radiation-induced modified bases, including thymine glycols, 5-formyluracil, 5-(hydroxymethyl)uracil, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroadenine, together with related formamidopyrimidine derivatives was assessed using the suitable HPLC-MS/MS method. Information is also provided on the substrate specificity of DNA repair enzymes and the mutagenic potential of base lesions using site-specific modified oligonucleotides as the probes.

Animals↗

PubMed2003

Oxidative damage to DNA: formation, measurement and biochemical features.

Emphasis is placed in the first part of this survey on mechanistic aspects of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) as the result of exposure to z.rad;OH radical, one-electron oxidants and singlet oxygen (1O(2)) oxidation. It was found that 8-oxoGua, which is generated by either hydration of the guanine radical cation or .OH addition at C8 of the imidazole ring, is a preferential target for further reactions with 1O(2) and one-electron oxidants, including the highly oxidizing oxyl-type guanine radical. Interestingly, tandem base lesions that involve 8-oxoGua and a vicinal formylamine residue were found to be generated within DNA as the result of a single .OH radical hit. The likely mechanism of formation of the latter lesions involves the transient generation of 5-(6)-peroxy-6-(5)-hydroxy-5,6-dihydropyrimidyl radicals that may add to the C8 of a vicinal guanine base before undergoing rearrangement. Another major topic which is addressed deals with recent developments in the measurement of oxidative base damage to cellular DNA. This was 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. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision and nucleotide excision pathways are briefly reviewed. For this purpose modified oligonucleotides in which cyclopurine, and cyclopyrimidine nucleosides were site-specifically inserted were synthesized.

Animals↗

PubMed2003

Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation.

Exposure to solar UV radiation gives rise to mutations that may lead to skin cancer. UVA (320-340 nm) constitutes the large majority of solar UV radiation but is less effective than UVB (290-320 nm) at damaging DNA. Although UVA has been implicated in photocarcinogenesis, its contribution to sunlight mutagenesis has not been elucidated, and DNA damage produced by UVA remains poorly characterized. We employed HPLC-MS/MS and alkaline agarose gel electrophoresis in conjunction with the use of specific DNA repair proteins to determine the distribution of the various classes and types of DNA lesions, including bipyrimidine photoproducts, in Chinese hamster ovary cells exposed to pure UVA radiation, as well as UVB and simulated sunlight (lambda > 295 nm) for comparison. At UVA doses compatible with human exposure, oxidative DNA lesions are not the major type of damage induced by UVA. Indeed, single-strand breaks, oxidized pyrimidines, oxidized purines (essentially 8-oxo-7,8-dihydroguanine), and cyclobutane pyrimidine dimers (CPDs) are formed in a 1:1:3:10 ratio. In addition, we demonstrate that, in contrast to UVB and sunlight, UVA generates CPDs with a large predominance of TT CPDs, which strongly suggests that they are formed via a photosensitized triplet energy transfer. Moreover, UVA induces neither (6-4) photoproducts nor their Dewar isomers via direct absorption. We also show that UVA photons contained in sunlight, rather than UVB, are implicated in the photoisomerization of (6-4) photoproducts, a quickly repaired damage, into poorly repaired and highly mutagenic Dewar photoproducts. Altogether, our data shed new light on the deleterious effect of UVA.

Animals↗

PubMed2003

Inter-strand photoproducts are produced in high yield within A-DNA exposed to UVC radiation.

Far-UV irradiation of DNA leads to the dimerization of pyrimidine bases, resulting in the formation of cyclobutane type dimers and (6-4) photoproducts. In the dry state, an additional thymine dimeric photolesion, the spore photoproduct, is also generated. While most photoproducts are expected to be produced between adjacent pyrimidines, little attention has been paid to lesions involving bases located on different DNA strands. Using HPLC- mass spectrometry analysis of enzymatically digested DNA, we observed that, in the dry state, inter-strand dimeric photoproducts represented 30% of the total yield of dimeric thymine lesions. The major inter-strand damage was found to be the spore photoproduct. Formation of inter-strand lesions in significant yield could be obtained in solution upon modification of the DNA conformation as the result of the addition of large amounts of ethanol. In both cases, DNA is in the A-form, which is characterized by a high compaction, likely to favor inter-strand photoreactions. Since the latter DNA conformation is also predominant in bacterial spores, the formation and repair of dimeric photoproducts involving thymine bases located on different DNA strands may thus be relevant in terms of deleterious effects of UV radiation to the latter microorganisms.

Chromatography, High Pressure Liquid↗

PubMed2003

Formation of a methide derivative upon photolysis of thymidine bromohydrins.

Reaction of bromine with thymidine in aqueous solution produces, in high yield, the corresponding 5-bromo-6-hydroxy-5,6-dihydroderivative (thymidine bromohydrins). UVC photolysis of thymidine bromohydrins gives rise to a reactive intermediate that is converted into 5-(hydroxymethyl)-2'-deoxyuridine upon incubation in water. When the former compound is left in methanol, ethanol, or propanol, the corresponding 5-alkoxymethyl derivatives are produced. The proposed structure for the primary photolysis product of thymidine bromohydrins is a methide derivative of the thymine ring. This compound could be an interesting intermediate in the synthesis of methyl-substituted thymidine.

Alcohols↗

PubMed2003

Hydroxyl radical is not the main reactive species involved in the degradation of DNA bases by copper in the presence of hydrogen peroxide.

Copper is an important biological metal that tightly binds to DNA. Its reaction with endogenously generated hydrogen peroxide may thus lead to the formation of DNA damage. To gain insights into the underlying mechanisms, a comparative study of the damage produced within isolated DNA upon exposure to gamma-radiation in aqueous solution, a source of hydroxyl radicals, and incubation with Cu(I) or Cu(II) complexes in the presence of hydrogen peroxide was carried out. Several relevant base modifications were quantified by HPLC-tandem mass spectrometry. It was first shown that addition of copper ions only slightly modified the profile of radiation-induced lesions within DNA. However, the distribution of base modifications was drastically different upon incubation of DNA with Cu(I) or Cu(II) complexes in the presence of H(2)O(2). Indeed, guanine degradation products were produced in much higher yield than lesions of the other bases. These observations are rationalized in terms of the occurrence of one electron oxidation with Cu(I) complexes, as confirmed by the study of the degradation of free thymidine. In contrast, the formation of the sole 8-oxo-7,8-dihydroguanine upon incubation of DNA with Cu(II) ions and H(2)O(2) strongly suggests the production of singlet oxygen as the predominant reactive oxygen species.

Animals↗

PubMed2003

Formation of the spore photoproduct and other dimeric lesions between adjacent pyrimidines in UVC-irradiated dry DNA.

Far-UV irradiation of DNA leads to the formation of several types of dimeric lesions between adjacent pyrimidine bases including cyclobutane dimers, (6-4) photoproducts and Dewar valence isomers In the dry state, an additional specific thymine lesion, the spore photoproduct, is produced. We designed an HPLC-tandem mass spectrometry assay for the detection of the latter lesion. This technique that does not require radio-labelling of DNA allowed the simultaneous quantification of the spore photoproduct and other pyrimidine dimeric photoproducts. Using this approach, the complete distribution of bipyrimidine lesions within UVC-irradiated dry DNA was determined.

Animals↗

PubMed2002

Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement.

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.

Artifacts↗

PubMed2002

DNA tandem lesions containing 8-oxo-7,8-dihydroguanine and formamido residues arise from intramolecular addition of thymine peroxyl radical to guanine.

Exposure of aerated aqueous solutions of dinucleoside monophosphates bearing both a pyrimidine base and a guanine residue to ionizing radiation leads to the formation of 8-oxo-7,8-dihydroguanine/formamido tandem lesions (8-oxodG/dF). Recent evidence for the formation of the latter damage within isolated DNA emphasized the possible biological relevance of this class of lesions. Therefore, an extensive mechanistic study of the formation of 8-oxodG/dF was carried out with thymine and guanine containing dinucleoside monophosphates (dGpdT and dTpdG). First, the peroxyl radical-induced degradation of guanine within dGpdT and dTpdG was studied in order to assess the possibility of intramolecular electron transfer between guanine and thymine peroxyl radicals. Then, the formation of a series of tandem lesions involving a formamido residue, thymine glycols, 8-oxodG, and oxazolone was monitored within aerated aqueous solutions of dTpdG and dGpdT exposed to gamma-radiation. The absence of formation of tandem lesions other than 8-oxodG/dF in significant yield led to us propose a new mechanism involving addition of the thymine peroxyl radical to the guanine moiety. This received support from (18)O labeling experiments.

8-Hydroxy-2'-Deoxyguanosine↗