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.

The aim of the present study was to measure the formation of eight base modifications in the DNA of cells exposed to either low-LET ((60)Co gamma rays) or high-LET ((12)C(6+) particles) radiation. For this purpose, a recently optimized HPLC-MS/MS method was used subsequent to DNA extraction and hydrolysis. The background level of the measured modified bases and nucleosides was shown to vary between 0.2 and 2 lesions/10(6) bases. Interestingly, thymidine glycols constitute the main radiation-induced base modifications, with an overall yield of 0.097 and 0.062 lesion/10(6) bases per gray for gamma rays and carbon heavy ions, respectively. Both types of radiations generate four other major degradation products, in the following order of decreasing importance: FapyGua > 5-HmdUrd > 5-FordUrd > 8-oxodGuo. The yields of formation of FapyAde and 8-oxoAde are one order of magnitude lower than those of the related guanine modifications, whereas the radiation-induced generation of 5-OHdUrd was below the limit of detection of the assay. The efficiency for both types of radiation to generate base damage in cellular DNA is low because the highest yield per gray was 0.097 thymine glycols per 10(6) DNA bases. As a striking observation, the yield of formation of the measured DNA lesions was found to be, on average, twofold lower after exposure to high-LET radiation ((12)C(6+)) than after exposure to low-LET gamma radiation. These studies show that the HPLC-MS/MS assay provides an accurate, reliable and sensitive method for measuring cellular DNA base damage.

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.

The hydroxyl radical-mediated formation of two tandem base lesions within DNA including N-(2deoxy-β-D-erythro-pentofuranosyl)formylamine-8-oxo-7,8-dihydro-2′-deoxyguanosine (dβF-8-oxodGuo) and 8-oxodGuo-dβF is reported in this study. A specific enzymatic processing was developed to quantitatively release the tandem lesions as dinucleoside monophosphates from DNA. Then, the resulting hydrolyzed DNA samples were analyzed using liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The simultaneous measurement of the two vicinal lesions was performed in the negative mode using the accurate and sensitive multiple reaction monitoring technique. For this purpose, two characteristic ions arising from the fragmentation of the pseudo-molecular ion [M -H]were monitored. Both dβF-8-oxodGuo and 8-oxodGuo-dβF damage were found to be generated in γ-irradiated DNA as a significant fraction of • OH radical-induced base damage. Interestingly, 8-oxodGuo-dβF was produced in a much higher yield than the reversed sequence lesion. Indirect evidence is provided for the formation of other tandem lesions involving 8-oxodGuo, but that still remain to be fully identified. Insights into the mechanism of formation of the DNA damage were gained from several experiments including DNA photosensitization, γ-irradiation in the presence of iron, and exposure to Fenton reagents. This allowed refinement of the proposed pathways for the formation of dβF-8-oxodGuo and 8-oxodGuo-dβF tandem base damage.

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.

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.

The interactions between uranium and four metalloproteins (Apo-HTf, HSA, MT and Apo-EqSF) were investigated using fluorescence quenching measurements. The combined use of a microplate spectrofluorometer and logarithmic additions of uranium into protein solutions allowed us to define the fluorescence quenching over a wide range of [U]/[Pi] ratios (from 0.05 to 1150) at physiologically relevant conditions of pH. Results showed that fluorescence from the four metalloproteins was quenched by UO(2)(2+). Stoichiometry reactions, fluorescence quenching mechanisms and complexing properties of metalloproteins, i.e. binding constants and binding sites densities, were determined using classic fluorescence quenching methods and curve-fitting software (PROSECE). It was demonstrated that in our test conditions, the metalloprotein complexation by uranium could be simulated by two specific sites (L(1) and L(2)). Results showed that the U(VI)-Apo-HTf complexation constant values (log K(1)=7.7, log K(2)=4.6) were slightly higher than those observed for U(VI)-HSA complex (log K(1)=6.1, log K(2)=4.8), U(VI)-MT complex (log K(1)=6.5, log K(2)=5.6) and U(VI)-Apo-EqsF complex (log K(1)=5.3, log K(2)=3.9). PROSECE fitting studies also showed that the complexing capacities of each protein were different: 550 moles of U(VI) are complexed by Apo-EqSF while only 28, 10 and 5 moles of U(VI) are complexed by Apo-HTf, HSA and MT, respectively.

Data describing the biokinetics of radionuclides after contamination come mainly from experimental acute exposures of laboratory animals and follow-up of incidental exposures of humans. These data were compiled to form reference models that could be used for dose calculation in humans. In case of protracted exposure, the same models are applied, assuming that they are not modified by the duration of exposure. This work aims at testing this hypothesis. It presents new experimental data on retention of uranium after chronic intake, which are compared to values calculated from a biokinetic model that is based on experiments of acute exposure of rats to uranium. Experiments were performed with 56 male Sprague Dawley rats, from which 35 were exposed during their whole adult life to 40 mg L of uranyl nitrate dissolved in mineral water and 21 were kept as controls. Animals were euthanatized at 32, 95, 186, 312, 368, and 570 d after the beginning of contamination. Urine and all tissues were removed, weighted, mineralized, and then analyzed for uranium content by Kinetics Phosphorescence Analysis (KPA) or by ICP-MS. Experimental data showed that uranium accumulated in most organs, following a nonmonotonous pattern. Peaks of activities were observed at 1-3, 10, and 19 mo after the beginning of exposure. Additionally, accumulation was shown to occur in tissues such as teeth and brain that are not usually described as target organs. Comparison with model prediction showed that the accumulation of uranium in target organs after chronic exposure is overestimated by the use of a model designed for acute exposure. These differences indicate that protracted exposure to uranium may induce changes in biokinetic parameters when compared to acute contamination and that calculation of dose resulting from chronic intake of radionuclides may need specific models that are not currently available.