[en] 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 ·OH radical, one-electron oxidants and singlet oxygen (¹O₂) 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 ¹O₂ 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

[en] Emphasis has been placed in the last decade on the elucidation of the main degradation pathways of isolated DNA mediated by hydroxyl radical (OH) and one-electron oxidation reactions as the result of indirect and direct effects of ionizing radiation respectively. This has led to the isolation and characterization of about 100 oxidized purine and pyrimidine nucleosides if hydroperoxide precursors and diastereomers are included. However, far less information is available on the mechanisms of radiation-induced degradation of bases in cellular DNA mostly due partly to analytical difficulties. It may be reminded that the measurement of oxidized nucleosides and bases in nuclear DNA is still a challenging issue which until recently has been hampered by the use of inappropriate methods such as the GC-MS that have led to overestimated values of the lesions by factors varying between two and three orders of magnitude. At the present, using the accurate and sensitive HPLC/MS/MS assay, 11 single modified nucleosides and bases were found to be generated in cellular DNA upon exposure to gamma rays and heavy ions. This validates several of the OH-mediated oxidation pathways of thymine, guanine and adenine that were previously inferred from model studies. The concomitant decrease in the yields of oxidized bases with the increase in the LET of heavy ions is accounted for by the preponderance of indirect effects in the damaging action of ionizing radiation on DNA. Further evidence for the major role played by .OH was provided by the results of exposure of cells to high intensity 266 nm laser pulses. Under these conditions 8-oxo-7,8-dihydroguanine is mostly produced by biphotonic ionization of DNA nucleobases and subsequent hole migration to guanine bases. It is likely that some of the oxidized bases that have been isolated as single lesions are in fact involved in clustered damage. Interestingly it was recently shown that a single oxidation hit is capable of generating complex lesions in DNA. Thus .OH-mediated abstraction at C4 of the 2-deoxyribose moiety gives rise to DNA strand cleavage together with the formation of a highly reactive aldehyde that undergoes an addition reaction to the amino group of a proximate cytosine, leading to 4 diastereomeric cycloadducts as components of likely interstrand cross-links. It was also shown that the (5'R)- 5',8-cyclo-2'-deoxyadenosine, a tandem lesion, that arises from intramolecular addition of the OH-mediated C5' radical to the C8 position of the adenine moiety is generated in DNA, however, in a low yield upon exposure of cells to gamma radiation. (author)

[en] UVA-mediated oxidative damage to DNA is mostly accounted for by predominant singlet oxygen oxidation of guanine together with a relatively minor implication of OH reactions towards the sugar moieties and the nucleobases. Molecular aspects of radiation-induced damage to cellular DNA are studied

[en] Full text: The measurement of damage induced in cellular DNA by ionizing and solar radiations is of major importance to assess the molecular mode of action and the biological role (mutagenesis, DNA repair) of these genotoxic agents. For this purpose several analytical approaches including immunodetection, post-labeling and chromatographic assays have been designed. However most of them have been shown to suffer from a lack of specificity, sensitivity or quantitative response. It may be noted that the gas-chromatography method in its basal version has been found to lead to overestimated yields of oxidatively generated base lesions by two to three order of magnitude due to the occurrence of artifactual oxidation of the overwhelming purine and pyrimidine bases during the derivatization step of the assay. The advent of HPLC coupled to tandem mass spectrometry operating in the electrospray ionization mode has allowed overcoming most of these drawbacks. Thus, accurate determination of 11 oxidized bases and nucleosides has been achieved in cellular DNA upon exposure to radiation-induced hydroxyl radical and one-electron oxidation agents. This has involved quantitative enzymatic release of lesions from extracted DNA and their accurate detection at the output of the HPLC column using the highly quantitative isotopic dilution technique. Evidence was also provided for the generation of five clustered lesions that all involve a base modification and an altered 2-deoxyribose residue as the result of only one initial radical oxidation hit. These consist of (5'R)-5',8-cyclo-2'-deoxyadenosine and cytosinealdehyde adducts that arise from .OH-mediated hydrogen abstraction at C5 and C4 of the sugar moiety of cellular DNA respectively. The damaging effects of UVA radiation on cellular DNA and human skin were rationalized in terms of predominant ¹O₂-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine. Other relevant types of DNA modifications consist in bipyrimidine photoproducts including cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone lesions and their Dewar valence isomers that are involved in the etiology of skin cancers. HPLC-MS/MS has been also shown to be an appropriate tool to monitor the formation of the above dimeric photoproducts at the four possible bipyrimidine sites in the DNA of isolated cells and from biopsies of human skin explants upon exposure to UVB radiation. The repair kinetic of each photoproduct has been assessed in cells and human skin by HPLC-MS/MS showing differences between the dimeric lesions. (author)

[en] The Non-ionizing radiation section of the French Society of Radiation Protection (SFRP) organized a technical meeting on the current knowledge of UV mutagenicity mechanisms, on professional exposures and on the risks linked with artificial tanning and their prevention. This document brings together the 3 available presentations (slides) of the talks given at the meeting: 1 - UV induction of DNA photoproducts: recent data (Thierry DOUKI, CEA Grenoble); 2 - Professional exposure to UV radiations (Mathieu BONIOL, IPRI); 3 - Artificial tanning: a major but avoidable public health problem (Jean-Francois DORE, Centre de Recherche en Cancerologie)

[en] The main oxidative decomposition pathways of purine and pyrimidine nucleobases associated with the direct (·OH radical) and indirect effects (one-electron oxidation) of ionizing radiation have been elucidated in isolated DNA and model compounds. Guanine that exhibits the lowest ionization potential among nucleic acid components has recently received a great deal of attention. Thus, the main one-electron oxidation and ·OH-mediated decomposition products of guanine that include 2,2,4-triamino-5-(2H)-oxazolone, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxo-7,8-dihydroguanine (8-oxoGua) have been identified. The complexity of the radical oxidation reactions induced in DNA by ·OH is illustrated by the characterization of 8-oxoGua as part of tandem base modifications in the biopolymer. Thus, formylamine (FO), that is a radical oxidation product of both cytosine and thymine is generated adjacent either 3'- or 5'- to 8-oxoGua . A likely mechanism for the formation of the FO-8-oxoGua and 8-oxoGua-FO tandem base damage involves the transient generation of 5(6)-hydroperoxyl-6-(5)-hydroxy-5,6-dihydrothymyl radicals which are able to add to the vicinal guanine moiety at C8. The two tandem base lesions were inserted into defined sequence DNA fragments using an optimized version of the solid phase phosphoramidate method for biochemical type of experiments. It was found that the 8-oxoGua and FO lesions are still preferential substrates for the Fpg and endo III DNA base excision repair proteins respectively. Work is in progress to assess whether the latter vicinal base modifications could be excised from the DNA chain by enzymes involved in the nucleotide excision repair pathway as it was recently shown to be the case for the (5'R) and (5'S) diastereomers of 5',8-cyclo-2'-deoxyadenosine. Emphasis was recently placed on the development of sensitive chemical and biochemical assays aimed at monitoring the formation of oxidized purine and pyrimidine bases within cellular DNA. For more than a decade, the level of oxidized bases and particularly of 8-oxoGua, a non specific but predominant DNA oxidation decomposition product, has been overestimated. The origins of the drawbacks are now identified. In fact artifactual oxidation of nucleobases may occur during several steps including the silylation reaction prior to gas chromatography mass spectrometry analysis, the DNA extraction and the subsequent work-up prior to chromatographic analysis. The use of chelating agents and NaI in the so-called chaotropic DNA extraction method was found to significantly prevent the formation of the background level of oxidized bases in control cellular samples. HPLC separations coupled either to electrochemical detection or to the more versatile tandem-mass spectrometry electrospray ionization technique appear to be appropriate tools when the levels of induced damage is close to 1 lesion in 106 normal bases. Interestingly, up to 9 oxidized nucleosides including 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyadenosine, the four cis and trans diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine, 5-formyl-2'-deoxyuridine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-hydroxy-2'-deoxycytidine were measured in the DNA of neoplastic human monocytes exposed to ionizing radiation. The association of base excision DNA repair enzymes including bacterial Fpg and endo III with the comet assay represents a better alternative for assessing levels of base damage within the range of 1 to 5 lesions per 107 bases. The basal level of Fpg- and endo III-sensitive sites was found to be similar, close to 2.1 per 107 bases, within the DNA of human monocytes. The latter frequency has to be compared with that of strand breaks and alkali-labile sites (2.9 per 107 bases). Interestingly, the measurement of oxidative base damage to cellular DNA was performed for doses of ionizing radiation as low as 2 Gy. The yields of the different classes of damage per Gy and 107 bases are the following: 0.48 Fpg-sensitive sites, 0.53 endo III-sensitive sites and 1.30 strand breaks (direct nicks and alkali-labile sites). (authors)

No abstract available

[en] This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and ³²P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.

[en] Emphasis is placed in this short review on recent developments concerning several aspects of the chemical and biochemical effects of ionizing radiation on both isolated and cellular DNA. This includes the mechanism of formation of single and tandem DNA lesions upon one-electron oxidation and one hydroxyl radical hit only. Information is also provided on the specificity of DNA repair enzymes and the measurement of radiation-induced damage in cellular DNA

[en] 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