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J L Ravanat

Publications and source records attributed to J L Ravanat.

At least 19 recordsLinked to original sources
PubMed2001

Chlorination of guanosine and other nucleosides by hypochlorous acid and myeloperoxidase of activated human neutrophils. Catalysis by nicotine and trimethylamine.

Activated human neutrophils secrete myeloperoxidase, which generates HOCl from H2O2 and Cl(-). We have found that various (2'-deoxy)nucleosides react with HOCl to form chlorinated (2'-deoxy)nucleosides, including novel 8-chloro(2'-deoxy)guanosine, 5-chloro(2'-deoxy)cytidine, and 8-chloro(2'-deoxy)adenosine formed in yields of 1.6, 1.6, and 0.2%, respectively, when 0.5 mM nucleoside reacted with 0.5 mM HOCl at pH 7.4. The relative chlorination, oxidation, and nitration activities of HOCl, myeloperoxidase, and activated human neutrophils in the presence and absence of nitrite were studied by analyzing 8-chloro-, 8-oxo-7,8-dihydro-, and 8-nitro-guanosine, respectively, using guanosine as a probe. 8-Chloroguanosine was always more easily formed than 8-oxo-7,8-dihydro- or 8-nitro-guanosine. Using electrospray ionization tandem mass spectrometry, we show that several chlorinated nucleosides including 8-chloro(2'-deoxy)guanosine are formed following exposure of isolated DNA or RNA to HOCl. Micromolar concentrations of tertiary amines such as nicotine and trimethylamine dramatically enhanced chlorination of free (2'-deoxy)nucleosides and nucleosides in RNA by HOCl. As the G-463A polymorphism of the MPO gene, which strongly reduces myeloperoxidase mRNA expression, is associated with a reduced risk of lung cancer, chlorination damage of DNA /RNA and nucleosides by myeloperoxidase and its enhancement by nicotine may be important in the pathophysiology of human diseases associated with tobacco habits.

Animals↗
PubMed2001

Singlet oxygen induces oxidation of cellular DNA.

The aim of the present work was to evaluate the potential for (1)O(2) to induce oxidation of cellular DNA. For this purpose cells were incubated in the presence of a water-soluble endoperoxide whose thermal decomposition leads to the formation of singlet oxygen. Thereafter, DNA was extracted and the level of several modified DNA bases was determined by HPLC analysis coupled to a tandem mass spectrometric detection. A significant increase in the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine was observed upon incubation of the cells with the chemical generator of (1)O(2), whereas the level of the other DNA bases measured remained unchanged. To demonstrate that singlet oxygen is directly involved in the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine, the corresponding (18)O-labeled endoperoxide was used. Incubation of the cells with such a generator of (18)O-labeled singlet oxygen results in the formation of (18)O-labeled 8-oxo-7,8-dihydro-2'-deoxyguanosine in the nuclear DNA. This result clearly demonstrates that singlet oxygen, when released within cells, is able to directly oxidize cellular DNA.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

2'-deoxyguanosine oxidation is associated with decrease in the DNA-binding activity of the transcription factor Sp1 in liver and kidney from diabetic and insulin-resistant rats.

Over the years, several lines of evidence have emerged supporting the role of oxidative stress in the development of diabetic complications. This could involve the increase in the production of reactive oxygen species and the decrease in antioxidative defense systems. Modulation of the level of intracellular reactive oxygen species is likely to affect the intracellular redox homeostasis, which is crucial for numerous biological events such as the transcriptional activation of genes. In this work we studied the binding of the redox transcription factors Sp1 and NF-kappaB extracted from kidney and liver of streptozotocin diabetic (STZ) and fructose-fed rats using electrophoretic mobility shift (EMSA) assay. In addition, the level in 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) was assessed within DNA by high performance liquid chromatography with electrochemical detection (HPLC-EC). A decrease in the affinity of Sp1 to DNA was observed in the kidney of STZ rats and fructose-fed rats (15% +/- 8.3 and 54% +/- 6.9, respectively, versus control group set to 100%). This was also found to occur to a lower extent, in the liver. Interestingly, higher levels of 8-oxodGuo, a biomarker of DNA oxidation, were measured in the kidney of diabetic rats. Therefore, the modification in the binding efficiency of Sp1 or NF-kappaB could be related to reactive oxygen species-mediated DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

Direct and indirect effects of UV radiation on DNA and its components.

In this survey, emphasis was placed on the main photoreactions of nucleic acid components, involving both direct and indirect effects. The main UVB- and UVA-induced DNA photoproducts, together with the mechanisms of their formation, are described. Information on the photoproduct distribution within cellular DNA is also provided, taking into account the limitations of the different analytical methods applied to monitor the formation of the DNA damage. Thus, the formation of the main DNA dimeric pyrimidine lesions produced by direct absorption of UVB photons was assessed using a powerful HPLC-tandem mass spectrometry assay. In addition, it was found that UVA photooxidation damage mostly involves the guanine residues of cellular DNA as the result of singlet oxygen generation by still unknown endogenous photosensitizers.

Animals↗
PubMed2001

Analysis of fluoroquinolone-mediated photosensitization of 2'-deoxyguanosine, calf thymus and cellular DNA: determination of type-I, type-II and triplet-triplet energy transfer mechanism contribution.

Fluoroquinolone (FQ) antibacterials are known to exhibit photosensitization properties leading to the formation of oxidative damage to DNA. In addition, photoexcited lomefloxacin (Lome) was recently shown to induce the formation of cyclobutane pyrimidine dimers via triplet-triplet energy transfer. The present study is aimed at gaining further insights into the photosensitization mechanisms of several FQ including enoxacin (Enox), Lome, norfloxacin (Norflo) and ofloxacin (Oflo). This was achieved by monitoring the formation of DNA base degradation products upon UVA-mediated photosensitization of 2'-deoxyguanosine, isolated and cellular DNA. Oflo and Norflo act mainly via a Type-II mechanism whereas Lome and, to a lesser extent, Enox behave more like Type-I photosensitizers. However, the extent of oxidative damage was found to be relatively low. In contrast, it was found that cyclobutane thymine dimers represent the major class of damage induced by Enox, Lome and Norflo within isolated and cellular DNA upon UVA irradiation. This striking observation confirms that FQ are able to promote efficient triplet energy transfer to DNA. The levels of photosensitized formation of strand breaks, alkali-labile sites and oxidative damage to cellular DNA, as measured by the comet assay, were confirmed to be rather low. Therefore, we propose that the phototoxic effects of FQ are mostly accounted for energy transfer mechanism rather than by Type-I or -II photosensitization processes.

Animals↗
PubMed2000

Singlet oxygen induces oxidation of cellular DNA.

The aim of the present work was to evaluate the potential for (1)O(2) to induce oxidation of cellular DNA. For this purpose cells were incubated in the presence of a water-soluble endoperoxide whose thermal decomposition leads to the formation of singlet oxygen. Thereafter, DNA was extracted and the level of several modified DNA bases was determined by HPLC analysis coupled to a tandem mass spectrometric detection. A significant increase in the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine was observed upon incubation of the cells with the chemical generator of (1)O(2), whereas the level of the other DNA bases measured remained unchanged. To demonstrate that singlet oxygen is directly involved in the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine, the corresponding (18)O-labeled endoperoxide was used. Incubation of the cells with such a generator of (18)O-labeled singlet oxygen results in the formation of (18)O-labeled 8-oxo-7,8-dihydro-2'-deoxyguanosine in the nuclear DNA. This result clearly demonstrates that singlet oxygen, when released within cells, is able to directly oxidize cellular DNA.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Measurement of the main photooxidation products of 2'-deoxyguanosine using chromatographic methods coupled to mass spectrometry.

Analytical methods were developed for the measurement of the main photooxidation products of 2'-deoxyguanosine (dGuo), arising from either the type I (electron transfer) or the type II (singlet oxygen) photosensitization mechanism. Oxidation of dGuo by a type I mechanism leads to the predominant formation of 2, 2-diamino-5-[2-deoxy-beta-d-erythro-pentofuranosyl)amino]-5(2H)-oxazo lone. On the other hand, the two 4R and 4S diastereomers of 4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine are the main singlet oxygen-mediated dGuo oxidation products. The modified nucleosides were measured by either gas chromatography coupled to mass spectrometry after silylation of the sample or by high-performance liquid chromatography associated to tandem mass spectrometry. In order to improve the accuracy of the assays, isotopically labeled internal standards were synthesized for an isotope dilution mass spectrometry quantitation. The methods were successfully applied to the measurement of methylene blue- and riboflavin-mediated 2'-deoxyguanosine photooxidation reactions. The advantages of the two above-mentioned methods are discussed on the basis of comparative sensitivity and accuracy.

Chromatography, High Pressure Liquid↗
PubMed2000

Oxidative base damage to DNA: specificity of base excision repair enzymes.

Base excision repair (BER) is likely to be the main mechanism involved in the enzymatic restoration of oxidative base lesions within the DNA of both prokaryotic and eukaryotic cells. Emphasis was placed in early studies on the determination of the ability of several bacterial DNA N-glycosylases, including Escherichia coli endonuclease III (endo III) and formamidopyrimidine DNA N-glycosylase (Fpg), to recognize and excise several oxidized pyrimidine and purine bases. More recently, the availability of related DNA repair enzymes from yeast and human has provided new insights into the enzymatic removal of several.OH-mediated modified DNA bases. However, it should be noted that most of the earlier studies have involved globally modified DNA as the substrates. This explains, at least partly, why there is a paucity of accurate kinetic data on the excision rate of most of the modified bases. Interestingly, several oxidized pyrimidine and purine nucleosides have been recently inserted into defined sequence oligonucleotides. The use of the latter substrates, together with overexpressed DNA N-glycosylases, allows detailed studies on the efficiency of the enzymatic release of the modified bases. This was facilitated by the development of accurate chromatographic and mass spectrometric methods aimed at measuring oxidized bases and nucleosides. As one of the main conclusions, it appears that the specificity of both endo III and Fpg proteins is much broader than expected a few years ago.

DNA↗
PubMed2000

High-performance liquid chromatography--tandem mass spectrometry measurement of radiation-induced base damage to isolated and cellular DNA.

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 assessment of radiation-induced degradation products of DNA bases. This sensitive and specific assay is aimed at assessing six oxidized 2'-deoxyribonucleosides and two modified purine bases within both isolated and cellular DNA. For this purpose, stable isotopically labeled internal standards were prepared and used for isotope dilution mass spectrometry measurements. The latter method was validated through a comparison with two other assays, including HPLC associated with electrochemical detection and gas chromatography coupled to mass spectrometry. Using the specific and sensitive HPLC-MS/MS approach, 5,6-dihydroxy-5,6-dihydrothymidine, 5-hydroxy-2'-deoxyuridine, 5-(hydroxymethyl)-2'-deoxyuridine, 5-formyl-2'-deoxyuridine, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 4, 6-diamino-5-formamidopyrimidine, and 2, 6-diamino-4-hydroxy-5-formamidopyrimidine were quantified within both isolated and cellular DNA upon exposure to gamma-radiation.

Cell Line↗
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

Simultaneous determination of five oxidative DNA lesions in human urine.

A method, involving a HPLC prepurification followed by a GC/MS analysis, has been set up for the measurement of nucleic acid oxidation products in human urine. For this purpose, isotopically labeled internal standards have been prepared and used for isotope dilution mass spectrometric detection. Using this approach, four oxidized DNA bases, i.e., 5-hydroxyuracil, 5-(hydroxymethyl)uracil, 8-oxo-7,8-dihydroadenine, and 8-oxo-7,8-dihydroguanine, together with 8-oxo-7,8-dihydro-2'-deoxyguanosine have been simultaneously quantified in human urine samples. The levels of the oxidized nucleic acid constituents, as expressed in picomoles per milliliter, were determined to be, in decreasing order: 8-oxo-7,8-dihydroguanine (583 +/- 376) > 5-(hydroxymethyl)uracil (121 +/- 56) > 5-hydroxyuracil (58 +/- 23) > 8-oxo-7,8-dihydro-2'-deoxyguanosine (30 +/- 15) > 8-oxo-7,8-dihydroadenine (7 +/- 4). Attempts to determine the amount of 5,6-dihydroxy-5,6-dihydrothymine, 5-hydroxycytosine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine using the above HPLC-GC/MS method were unsuccessful.

8-Hydroxy-2'-Deoxyguanosine↗
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

Detection of 8-oxoguanine in cellular DNA using 2,6-diamino-8-oxopurine as an internal standard for high-performance liquid chromatography with electrochemical detection.

The quantitative aspect of the electrochemical detection method to detect 8-oxo-7,8-dihydroguanine (8-oxoGua) has been improved by using an internal standard. In addition, emphasis was placed on the reduction of artifactual oxidation of DNA during isolation and hydrolysis. Nuclear DNA was isolated from rat organs and purified on an anion-exchange column following treatment with proteinase K and RNase. DNA hydrolysis to nucleobases or nucleosides was performed using either formic acid treatment or enzymatic digestion, respectively. The levels of either 8-oxoGua or 8-hydroxy-7,8-dihydro-2'-deoxyguanosine were comparable. For accurate quantification, 2,6-diamino-8-oxopurine [(NH2)2-OH-Pur], added prior to hydrolysis, was used as an internal standard for the high-performance liquid chromatography with electrochemical detection assay. The baseline level of 8-oxoGua in DNA of Sprague-Dawley rats was estimated to be 2 to 5 8-oxoGua residues per 10(6) DNA bases, with slight differences depending on the tissue origin. In agreement with the results of previous observations, the level of the oxidized base in the kidney of animal treated with iron complexed to nitrilotriacetic acid (Fe-NTA) (15 mg/kg) was three- to fourfold higher than that of untreated rats or animals treated with a saline solution, while there was no change in 8-oxoGua levels in the liver and colon of these treated animals.

2-Aminopurine↗