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Biomedical subjects

J Cadet

Publications and source records attributed to J Cadet.

At least 19 recordsLinked to original sources
PubMed1999

Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: kinetic and mechanistic aspects.

Oligonucleotides that contain a single modified pyrimidine, i.e., thymine glycol (Tg), 5,6-dihydrothymine (DHT), and 5-hydroxycytosine (5-OHC) were synthesized in order to investigate the substrate specificity and the excision mechanism of two Escherichia coli repair enzymes: endonuclease III and formamidopyrimidine DNA glycosylase (Fpg). Three techniques of analysis were employed. A gas chromatography-mass spectrometry (GC-MS) assay with HPLC prepurification was used to quantify the release of the modified bases, while polyacrylamide gel electrophoresis and matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) provided insights into the mechanism of oligonucleotide cleavage. Values of Vm/Km constants lead to the conclusion that the substrates are processed by endonuclease III with the following preference: Tg >> 5-OHC > DHT. This confirms that Tg is an excellent substrate for endonuclease III. Fpg-mediated cleavage of the 5-OHC-containing oligonucleotide is processed at the same rate than endonuclease III. Furthermore, Fpg was found to have a little but relevant activity on DHT-containing oligonucleotide, thus broadening the substrate specificity of this enzyme to a new modified pyrimidine. While 5-OHC-containing oligonucleotides are cleaved by the two enzymes, no or a small amount of the modified base was found to be released, as determined by GC-MS. From these data it may be suggested that 5-OHC could be modified during its enzymatic excision. Finally, MALDI-MS analyses shed new light on the mechanism of action of endonuclease III: the molecular masses of the repaired fragments of 5-OHC- and DHT-containing oligonucleotides showed that endonuclease III cleaves the DNA backbone mainly through a hydrolytic process and that no beta-elimination product was detected.

Cytosine↗
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

Synthesis and enzymatic processing of oligodeoxynucleotides containing tandem base damage.

Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage. One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method. For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed. The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry. The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA. In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates. The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported.

Alkaline Phosphatase↗
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↗
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

Urinary excretion of 5-(hydroxymethyl) uracil in healthy volunteers: effect of active and passive tobacco smoke.

The urinary excretion of 5-(hydroxymethyl)uracil (5-HMUra), one of the major oxidative modifications of thymine, was investigated in 134 healthy volunteers living in North Italy. Overnight urine was collected, and a questionnaire was completed on smoking habits and exposure to environmental tobacco smoke (ETS). 5-HMUra was analyzed by GC/MS, following urine purification by HPLC. 5-HMUra excretion showed an approximately normal distribution, ranging from 0.08 to 0.84 (mean 0.44) nmoles/kg/8 hr and from 3.2 to 18.7 (mean 8.5) nmoles/mmoles creatinine. 5-HMUra excretion was significantly higher in women than in men and in smokers than in non-smokers when results were expressed as the ratio to creatinine. Slightly higher levels of 5-HMUra excretion, expressed as nmoles/mmoles creatinine, were also found in subjects highly exposed to ETS, monitored either as the number of hours of exposure or as the number of smokers in the workplace and at home. Our results show that the urinary excretion of 5-HMUra is higher than that of other oxidized nucleobases, including 8-oxo-7,8-dihydroguanine, and can be slightly modified by environmental factors such as tobacco smoke. These findings suggest that measurement of urinary excretion of 5-HMUra could be useful as a biomarker of oxidative DNA damage and repair, though further research is needed to support these data.

Adult↗
PubMed1998

Gas chromatography-mass spectrometry with high-performance liquid chromatography prepurification for monitoring the endonuclease III-mediated excision of 5-hydroxy-5,6-dihydrothymine and 5,6-dihydrothymine from gamma-irradiated DNA.

The endonuclease III from Escherichia coli is a repair enzyme which exhibits both a glycosylase and an endonuclease function. The activity of the enzyme can be assayed by measuring the released targeted bases in solution from a sample of modified DNA. In the present study, gas chromatography-mass spectrometry was used together with an HPLC prepurification step in order to single out the released bases. The prepurification was found to enhance the specificity and the sensitivity of the assay. Thus, the overall method allowed us to analyze separately 5-hydroxy-5,6-dihydrothymine from the cis and trans isomers of 6-hydroxy-5,6-dihydrothymine. Examples of application of the assay are provided with the measurement of the E. coli endonuclease III-mediated excision of 5-hydroxy-5,6-dihydrothymine and 5,6-dihydrothymine from samples of gamma-irradiated DNA in the presence of cysteine.

Chromatography, High Pressure Liquid↗
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

Use of the single-cell gel electrophoresis assay for the immunofluorescent detection of specific DNA damage.

The single-cell gel electrophoresis assay or comet assay is now a widely used method to assess the level of DNA damage in irradiated or chemically modified cells. We propose an adaptation of the currently applied protocol, aimed at singling out a defined modified base, using an immunodetection approach. After the electrophoresis step, the DNA tail moment was measured using ethidium bromide. Simultaneously, cyclobutane pyrimidine dimers (CPDs), the targeted lesions, were revealed by an indirect immunofluorescence detection using a specific monoclonal antibody. The assay was validated on human fibroblasts exposed to UVB light. The dose-response curves were established, showing a linear increase of the antibody response with the dose between 1000 and 10,000 J/m2. The detection limit of the method was 500 J/m2. Digestion of the CPDs, induced at 3000 J/m2, with T4 endonuclease V led to a marked decrease of the antibody response, confirming the specificity of the assay. A preliminary repair experiment is reported in which the tail moment of the comets together with the antibody response are measured, showing the disappearance of 80% of the antibody fixation sites within 48 h.

Antibodies, Monoclonal↗
PubMed1998

Repair and mutagenic potency of 8-oxoG:A and 8-oxoG:C base pairs in mammalian cells.

Replication of the oxidative lesion 8-oxo-7,8-dihydroguanine (GO) leads to the formation of both 8-oxo-7,8-dihydroguanine:adenine (GO:A) and 8-oxo-7,8-di-hydroguanine:cytosine (GO:C) pairs. The repair and mutagenic potency of these two kinds of base pairs were studied in simian COS7 and human MRC5V1 cells using the shuttle vector technology. Shuttle vectors carrying a unique GO residue opposite either a C or an A were constructed, then transfected into recipient mammalian cells. DNA repair resulting in G:C pairs and mutation frequency, were determined using resistance to digestion by the Ngo MI restriction enzyme for screening and DNA sequencing of suspect mutants. Results showed that the GO:C mismatch was well repaired since almost no mutations were detected in the plasmid progeny obtained 72 h after cell transfection. The GO:A pair was poorly repaired since only 32-34% of the plasmid progeny contained G:C whereas two thirds contained A:T at the original site. Repair kinetics measured with a non-replicating vector deleted by 13 bp at the SV40 replication origin, showed that GO:A was slowly repaired. Only 30% of the mispairs were corrected in 12 h. During this time 100% of the plasmids containing GO:A pairs were replicated as seen by the replication kinetics in a vector with an intact SV40 replication origin. These results show that, under our experimental conditions, replication is occurring before completion of DNA repair which explains the high mutagenic potency of the GO:A mispair.

Animals↗
PubMed1998

Solution structure of N-(2-deoxy-D-erythro-pentofuranosyl)urea frameshifts, one intrahelical and the other extrahelical, by nuclear magnetic resonance and molecular dynamics.

The presence of a N-(2-deoxy-D-erythro pentofuranosyl)urea (henceforth referred to as deoxyribosylurea) residue, ring fragmentation product of a thymine, in a frameshift situation in the sequence 5'd(AGGACCACG).d(CGTGGurTCCT) has been studied by 1H and 31P nuclear magnetic resonance and molecular dynamics. At equilibrium, two species are found in slow exchange. We observe that the deoxyribosylurea residue can be either intra- or extrahelical within structures which otherwise do not deviate strongly from that of a B-DNA as observed by NMR. Our study suggests that this is determined by the nature and number of hydrogen bonds which this residue can form as a function of two possible isomers. There are two possible structures for the urea side chain, either cis or trans for the urido bond which significantly changes the hydrogen bonding geometry of the residue. In the intrahelical species, the cis isomer can form two good hydrogen bonds with the bases on the opposite strand in the intrahelical species, A4 and C5, which is not the case for the trans isomer. This results in a kink in the helical axis. For the major extrahelical species, the situation is reversed. The trans isomer is able to form two good hydrogen bonds, with G13 on the same strand and A7 on the opposite strand. For the extrahelical species, the cis isomer can form only one hydrogen bond. In this major structure the NMR data show that the bases which are on either side of the deoxyribosylurea residue in the sequence, G14 and T16, are stacked over each other in a way similar to a normal B-DNA structure. This requires the formation of a loop for the backbone between these two residues. This loop can belong to one of two families, right- or left-handed. In a previous study of an abasic frameshift [Cuniasse et al. (1989) Biochemistry 28, 2018-2026], a left-handed loop was observed, whereas in this study a right-handed loop is found for the first time in solution. The deoxyribosylurea residue lies in the minor groove and can form both an intra- and an interstrand hydrogen bond.

Computer Simulation↗
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

Polyclonal antibodies to adenine N1-oxide: characterization and use for the measurement of DNA damage.

Adenine N1-oxide is a DNA lesion whose formation involves the specific oxidation of the adenine base by hydrogen peroxide under nonradical conditions. The damage may be measured using a HPLC/32P-postlabeling method, which however cannot be used for routine analysis. We propose herein as an alternative an immunological assay which allows a rapid evaluation of the level of adenine N1-oxide in DNA exposed to oxidative stress. Two polyclonal antibodies were raised using two different strategies for the coupling of the hapten to the protein. The first approach is based on the universal method of Erlanger and Beiser, whereas the preparation of the second antigen involves the conjugation of a morpholino derivative of adenosine N1-oxide to the carrier protein. The affinity and the specificity of those antibodies were determined by competitive enzyme-linked immunosorbent assay. The antibody obtained by the traditional method shows some cross-reactivity with normal nucleotides, whereas for the other antiserum, the selectivity was found to be higher. Therefore, this polyclonal antibody was used to quantify the level of adenine N1-oxide in calf thymus DNA oxidized either by m-chloroperbenzoic acid or by hydrogen peroxide. The detection limit of the assay is four residues of adenine N1-oxide per 10(6) normal bases. The level of adenine N1-oxide in nonmodified DNA was lower than the detection limit of the assay, whereas in mCPB- and H2O2-modified DNA, it could be up to 14 and 0.7 adenine N1-oxide residues per 10(4) normal bases, respectively.

Adenine↗
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

The direct effect of heavy ions and electrons on thymidine in the solid state.

PURPOSE: To study the direct effect of heavy ions and electrons on thymidine. MATERIALS AND METHODS: The thymidine samples in the solid state were exposed to a beam of O7+ heavy ions with an energy of 10.6 MeV/u (LET approximately 500keV/microm) and to electrons of 2MeV (LET approximately 0.18keV/microm). The major decomposition products of thymidine were purified by high performance liquid chromatography (HPLC) and identified by extensive spectrometric measurements (UV, mass spectroscopy, 1H and 13C NMR). RESULTS: The main degradation products of thymidine were isolated and characterized. Reaction mechanisms, involving transient radical species, are proposed to explain the heavy ion-mediated formation of the modified products. Furthermore, a semi-quantitative comparison of the modifications induced within thymidine by the two types of radiations was performed. CONCLUSION: Several new radiation-induced thymidine decomposition products have been isolated and characterized. The comparison of the effects induced by heavy ions and electrons on thymidine in the solid state clearly indicates several significant differences in the mechanisms of action. A relative increase in the extent of the modifications of the sugar moiety with respect to those of the base is observed with the heavy ions by comparison with electrons.

Chromatography, High Pressure Liquid↗
PubMed1998

Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture.

Ultraviolet A radiation (UVA, 320-400 nm) is mutagenic and induces genomic damage to skin cells. N-acetyl-cysteine (NAC), selenium and zinc have been shown to have antioxidant properties and to exhibit protective effects against UVA cytotoxicity. The present work attempts to delineate the effect of these compounds on genomic integrity of human skin fibroblasts exposed to UVA radiation using the single cell gel electrophoresis (SCGE) or Comet assay. The cells were incubated with NAC (5 mM), sodium selenite (0.6 microM) or zinc chloride (100 microM). Then cells were embedded in low melting point agarose, and immediately submitted to UVA fluences ranging from 1 to 6J/cm2. In the Comet assay, the tail moment increased by 45% (1 J/cm2) to 89% (6J/cm2) in non-supplemented cells (p)<0.01). DNA damage was significantly prevented by NAC, Se and Zn, with a similar efficiency from 1 to 4J/cm2 (p < 0.05). For the highest UVA dose (6J/cm2), Se and Zn were more effective than NAC (p < 0.01).

Acetylcysteine↗