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Jean Cadet

Publications and source records attributed to Jean Cadet.

At least 55 records · Page 3Linked to original sources
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↗
PubMed2003

Solution study of the NF-kappaB p50-DNA complex by UV laser protein-DNA cross-linking.

In this study, we describe a new approach for studying protein-DNA interactions in solution. The approach is based on mapping the UV laser-induced protein-DNA cross-links between the amino acids of the protein and the DNA bases that are in direct contact. The approach was applied for studying the solution structure of the human necrosis factor (NF)-kappaB p50 homodimer bound to a 37 base pair DNA. Several points of contact identical to those observed in the NF-kappaB-DNA crystal structure were found between the two biomolecules. Evidence is provided for the occurrence of two new contact points, one for each DNA strand. These new points of contact are located symmetrically a base apart from the extremity of the binding sequence.

Base Sequence↗
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

Excision of 8-methylguanine site-specifically incorporated into oligonucleotide substrates by the AlkA protein of Escherichia coli.

8-Methyl-2'-deoxyguanosine (8-medGuo) has been shown to be a major stable alkylation product of 2'-deoxyguanosine induced by methyl radical attack on DNA. Moreover, by using primer extension assays, the latter DNA modification has recently been reported to be a miscoding lesion by generating G to C and G to T transversions and deletions in vitro. However, no data have been reported up to now, concerning the processing of this C8-alkylated nucleoside by the DNA repair machinery. Therefore, we have investigated the capability of excision of 8-methylguanine (8-meGua) site specifically incorporated into oligonucleotide substrates by several bacterial, yeast and mammalian DNA N-glycosylases. The results show that the 3-methyladenine (3-meAde) DNA glycosylase II (AlkA protein) from Escherichia coli is the only DNA N-glycosylase tested able to remove 8-meGua from double-stranded DNA fragments. Moreover, the activity of AlkA for 8-meGua varied markedly depending on the opposite base in DNA, being the highest with Adenine and Thymine and the lowest with Cytosine and Guanine. The removal of 8-meGua by AlkA protein was compared to that of 7-methylguanine (7-meGua) and hypoxanthine (Hx). The rank of damage as a substrate for AlkA being 7-meGua>8-meGua>Hx. In contrast, the human 3-meAde DNA N-glycosylase (Mpg) is not able to release 8-meGua paired with any of the four DNA bases. We also show that, DNA N-glycosylases involved in the removal of oxidative damage, such as Fpg or Nth proteins from E. coli, Ntg1, Ntg2 or Ogg1 proteins of Saccharomyces cerevisiae, or human Ogg1 do not release 8-meGua placed opposite any of the four DNA bases. Furthermore, HeLa and Chinese hamster ovary (CHO) cell free protein extracts do not show any cleavage activity at 8-meGua paired with adenine or cytosine, which suggests the absence of base excision repair (BER) of this lesion in mammalian cells.

Alkaline Phosphatase↗
PubMed2002

Chemical synthesis and biochemical properties of oligonucleotides that contain the (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-deoxyuridine DNA lesion.

The first chemical synthesis of (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-deoxyuridine [(5'S,5S,6S)-cyclo-5-OH-dHdU], a radiation-induced decomposition product of 2'-deoxycytidine in aerated solution, is reported. Subsequently, 2'-deoxycytidine was incorporated into oligodeoxyribonucleotides with defined sequences by using an optimized system of protection that takes into account the reactivity and stability of the modified building blocks. After deprotection and purification, the chemical composition of the modified DNA fragments was assessed by enzymatic digestions and mass spectrometry measurements. The MS analyses confirmed the presence and integrity of the lesion within the synthesized DNA fragments. In vitro replication and repair studies showed that (5'S,5S,6S)-cyclo-5-OH-dHdU acts as a block for DNA polymerases when inserted into DNA oligomers and is not excised by any of the tested DNA N-glycosylases. Therefore, (5'S,5S,6S)-cyclo-5-OH-dHdU may represent a potential lethal lesion within the cell if it is not removed by the nucleotide excision repair machinery.

Chromatography, High Pressure Liquid↗
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↗
PubMed2002

Cross-linked thymine-purine base tandem lesions: synthesis, characterization, and measurement in gamma-irradiated isolated DNA.

5-(Phenylthiomethyl)-2'-deoxyuridine has been recently shown to be a specific photolabile precursor of 5-(2'-deoxyuridilyl)methyl radical that is involved in the formation of tandem base lesion with vicinal guanine in oxygen-free aqueous solution. The thionucleoside was incorporated by either liquid or solid-phase phosphoramidite synthesis into dinucleoside monophosphates with a 2'-deoxyadenosine residue as the vicinal nucleoside located either at the 3' or 5'-extremity. UV-C irradiation of the modified dinucleoside monophosphate under anaerobic conditions gives rise to cross-linked thymine(CH2-C8)adenine tandem base lesions which were isolated and characterized by (1)H NMR and mass spectrometry analyses. The formation of the latter tandem lesions involved an intramolecular addition of the 5-(2'-deoxyuridilyl)methyl radical to the C8 of the adenine moiety. A sensitive and specific assay aimed at monitoring the formation of the four thymine(CH2-C8)purine adducts, namely d(T Delta G), d(G Delta T), d(T Delta A), d(A Delta T), within DNA, was designed. This was based on a liquid chromatography analysis coupled to tandem mass spectrometry (HPLC-MS/MS) detection of the dinucleoside monophosphates which were quantitatively released from gamma-irradiated DNA and oligodeoxyribonucleotides by enzymatic hydrolysis. The four lesions were detected in both single-stranded oligodeoxyribonucleotide and isolated DNA upon exposure to gamma-radiation in oxygen-free aqueous solution. It was found that the tandem guanine-thymine lesions were produced more efficiently than the adenine-thymine cross-links. Moreover, a significant sequence effect was observed. Thus, the yield of formation of the tandem lesions is higher when the purine base is located at the 5' position of the 5-(2'-deoxyuridilyl)methyl radical.

Animals↗
PubMed2002

Formation of 2'-deoxyuridine hydrates upon exposure of nucleosides to gamma radiation and UVC-irradiation of isolated and cellular DNA.

The two diastereoisomers of 6-hydroxy-5,6-dihydro-2'-deoxyuridine (dUrd hydrates) are produced upon deamination of the related 2'-deoxycytidine derivatives. Liquid chromatography coupled to tandem mass spectrometry was used to quantify dUrd hydrates. Their rate constant of formation within UVC-irradiated solutions of dCyd was first determined. Their formation was also shown to occur within solutions of either dUrd or dCyd exposed to gamma-rays in the absence of oxygen. dUrd hydrates were then quantified within isolated and cellular DNA exposed to UVC light. In both cases, their yield of formation was found to be, at least, 2 orders of magnitude lower than the overall yield of bipyrimidine photoproducts such as cyclobutane dimers and (6-4) photoproducts.

Animals↗
PubMed2002

Radical oxidation of the adenine moiety of nucleoside and DNA: 2-hydroxy-2'-deoxyadenosine is a minor decomposition product.

A method involving high performance liquid chromatography (HPLC) separation associated with tandem mass spectrometry (MS/MS) detection in the multiple reaction monitoring mode was set-up for the measurement of 2-hydroxy-2'-deoxyadenosine (2-OHdAdo). This modified nucleoside, arising from the radical oxidation of 2'deoxyadenosine (dAdo), has been described in the literature as a potential biological marker of the Fenton reaction. Using the specific and sensitive HPLC-MS/MS assay, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 4,6-diamino-5-formamidopyrimidine and 2-hydroxy-2-deoxyadenosine (2-OHdAdo) were measured within 2'-deoxyadenosine and DNA solutions either exposed to gamma-rays or treated under Fenton reaction conditions. It was found that the yield of 2-OHdAdo was low compared to that of 8-oxodAdo under most of the oxidative conditions studied. In particular and in contrast to previous works, the formation of 2-OHdAdo was shown to be a minor process both upon gamma irradiation and under Fenton reaction conditions. However, a significant yield of formation of 2-OHdAdo was observed either upon incubation with high concentrations of Fe2+ ions in the absence of hydrogen peroxide or upon gamma-radiolysis of a nucleoside solution in the presence of the copper/ (o)-phenanthroline complex.

Adenine↗
PubMed2002

Cellular background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up.

The measurement of oxidative damage to cellular DNA is a challenging analytical problem requiring highly sensitive and specific methods. In addition, artefactual DNA oxidation during its extraction and subsequent work-up may give rise to overestimated levels of oxidized DNA bases. In the present study, we have used (18)O-labelled 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) as an internal standard to evaluate the extent of artefactual DNA oxidation during the critical steps preceding the measurement. The labelled oxidized purine nucleoside was specifically generated in cellular DNA using the recently available generator of (18)O-labelled singlet oxygen. Artefactual DNA oxidation that could take place during the work-up increases the level of 8-oxodGuo but not of the (18)O-oxidized nucleoside. Therefore, the ratio between the two compounds, as measured by high performance liquid chromatography coupled to tandem mass spectrometry, allows an unambiguous comparison of different methodologies. The comparison of different DNA extraction protocols led to the conclusion that artefactual DNA oxidation during the extraction step could be minimized if: (i) nuclei are isolated after cell lysis; (ii) desferrioxamine, a transition metal chelator is added to the different extraction buffers; and (iii) sodium iodide (or alternatively guanidine thiocyanate) is used for DNA precipitation. It was also demonstrated that sodium iodide does not decompose the targeted oxidized purine nucleoside. In addition, three different DNA digestion protocols were evaluated and they were found to give rise to similar results. Using the best-studied protocol, the steady-state cellular background level of 8-oxodGuo, in a lymphocyte cell line, was determined to be approximately 0.5 lesions/10(6) DNA nucleosides.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2002

Recent aspects of oxidative DNA damage: guanine lesions, measurement and substrate specificity of DNA repair glycosylases.

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.

DNA Adducts↗
PubMed2002

Development and application of a novel immunoassay for measuring oxidative DNA damage in the environment.

We developed a facile, cost-effective competitive binding assay for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in DNA, using a polyclonal rabbit antiserum raised against an 8-oxodGuo hapten coupled to bovine serum albumin and radiolabeled synthetic ligand containing multiple 8-oxodGuo residues. This radioimmunoassay (RIA) displays a high affinity for 8-oxodGuo in DNA, with a detection limit of approximately 1 adduct in 10(5) bases of DNA. 8-oxodGuo standards for RIA were quantified by high-performance liquid chromatography and electrochemical detection in DNA diluted in methylene blue and exposed to visible light. As an initial application we quantified 8-oxodGuo in dosimeters deployed at increasing depths in the Southern Ocean during the austral spring of the 1998 field season or at the surface at Palmer Station, Antarctica, throughout the 1999 field season. Cyclobutane pyrimidine dimers (CPD) were quantified using an established RIA. We found that the frequency of both photoproducts decreased with depth. However, CPD induction was attenuated at a faster rate than 8-oxodGuo, correlating with the differential attenuation of solar ultraviolet wavelengths in the water column. CPD induction was closely related with ultraviolet-B radiation (UVB) attenuation, whereas the lower attenuation of 8-oxodGuo suggests that oxidative damage is more closely related to ultraviolet-A radiation (UVA) irradiance. The ratio of 8-oxodGuo: CPD was also found to covary with changes in stratospheric ozone concentrations at Palmer Station. These data demonstrate the usefulness of these assays for environmental photobiology and the potential for their use in studying the relative impacts of UVB versus UVA, including ozone depletion events.

Animals↗
PubMed2002

Is 5-aminolevulinic acid involved in the hepatocellular carcinogenesis of acute intermittent porphyria?

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in acute intermittent porphyria (AIP) due to enzymatic deficiencies in the heme biosynthetic pathway Its accumulation has been associated with several symptoms, such as abdominal pain attacks, neuromuscular weaknesses, neuropsychiatric alterations and increased hepatocellular carcinoma (HCC) incidence. The use of exogenous ALA to elevate porphyrin levels in tumor photodynamic therapy, adds further significance to ALA toxicology. Under ferritin mediated and metal catalyzed oxidation, ALA produces reactive oxygen species that can damage plasmid and isolated DNA in vitro, and increases the steady-state level of 8-oxo-7,8-dihydro-2'-deoxyguanosine in liver, spleen and kidney DNA and 5-hydroxy-2'-deoxycytidine in liver DNA of ALA-treated rats. The in vitro DNA damage could be partially inhibited by SOD, catalase, DTPA, mannitol and melatonin. ALA also promotes the formation of radical-induced base degradation products in isolated DNA. 4,5-Dioxovaleric acid, the final oxidation product of ALA, alkylates guanine moieties within both nucleoside and isolated DNA, producing two diastereoisomeric adducts. Dihydropyrazine derivatives of ALA generated by its dimerization, promote DNA strand-breaks and 8-oxodGuo formation in the presence of Cu2+. Together these results reinforce the hypothesis that the DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1996

Synthesis and Kinetic Study of the Deamination of the Cis Diastereomers of 5,6-Dihydroxy-5,6-dihydro-5-methyl-2'-deoxycytidine.

The main objectives of the present work were the synthesis of the two cis diastereomers of 5,6-dihydroxy-5,6-dihydro-5-methyl-2'-deoxycytidine and the kinetic study of their hydrolytic deamination. The preparation of the two glycols, two main (*)OH-mediated oxidation products of 5-methyl-2'-deoxycytidine, was achieved in two steps. The first one involved the synthesis of the two trans-(5R,6S)- and (5S,6R)-5-bromo-6-hydroxy-5,6-dihydro-5-methyl-2'-deoxycytidine. In a subsequent step, the bromohydrins were specifically converted into the cis-(5S,6S) and (5R,6R) diastereomers of 5,6-dihydroxy-5,6-dihydro-5-methyl-2'-deoxycytidine, respectively, under slightly alkaline conditions. The resulting glycols were purified by reverse phase high performance liquid chromatography and characterized by extensive spectroscopy measurements including (13)C- and (1)H-NMR analyses. Exact mass determination was inferred from high resolution fast atom bombardment mass spectrometry measurements. Circular dichroism spectroscopy confirmed the diastereomeric relationship existing between the pair of glycols. Kinetic study of the deamination of the above glycols was carried out in phosphate buffer solutions (pH 7) at two different temperatures (37 degrees C and 25 degrees C) in order to determine the thermodynamic and kinetic parameters of the reaction.

Journal Article↗
PubMedDate not supplied

Recognition of cyclonucleoside lesions by the Lactococcus lactis FPG protein.

Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases. Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamidopyrimidine-DNA glycosylases (Fpg-Ll) and the cyclonucleosides. This may provide new information on the mechanism involved in the activity of the latter enzyme.

DNA Damage↗