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PURPOSE: This article reviews the early history of ionizing radiation-induced sugar damage in DNA in dedication to Prof. Clemens von Sonntag, who recently passed away. It covers the time between 1968 and 1978, during which most of the work on the ionizing radiation-induced damage to polyalcohols, carbohydrates and the 2'-deoxyribose moiety in DNA was performed. Methodologies using gas chromatography-mass spectrometry (GC-MS) were developed to identify and quantify the radiation-induced products that had previously remained elusive. Products were identified by GC-MS either directly or after reduction of samples with NaBH(4) or NaBD(4). Incorporation of deuterium atoms by NaBD(4)-reduction facilitated the identification of aldehyde, keto, carboxyl and deoxy groups in the molecules. Numerous products of a polyalcohol and carbohydrates were identified and quantified. Mechanisms of product formation were proposed. Several products of the 2'-deoxyribose moiety in DNA were identified, indicating that they were released from DNA strand, not bound to it. Alkali labile sites and products still remaining within DNA or bound to DNA as end groups were also elucidated by first reducing irradiated samples with NaBD(4) followed by alkali treatment and GC-MS analysis. CONCLUSION: The knowledge of the products of the 2'-deoxyribose moiety in DNA led to the first mechanistic understanding of various pathways of hydroxyl radical-induced DNA strand breakage. To this date, some of these mechanisms still remain the most-widely studied mechanisms of DNA damage. Prof. von Sonntag's contributions to the understanding of the radiation chemistry of carbohydrates and DNA helped shape this field of science for years to come.

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Dose-response curves were measured for the formation of direct-type DNA products in X-irradiated d(GCACGCGTGC)(2)prepared as dry films and as crystalline powders. Damage to deoxyribose (dRib) was assessed by HPLC measurements of strand break products containing 3' or 5' terminal phosphate and free base release. Base damage was measured using GC/ MS after acid hydrolysis and trimethylsilylation. The yield of trappable radicals was measured at 4 K by EPR of films X-irradiated at 4 K. With exception of those used for EPR, all samples were X-irradiated at room temperature. There was no measurable difference between working under oxygen or under nitrogen. The chemical yields (in units of nmol/J) for trapped radicals, free base release, 8-oxoGua, 8-oxoAde, diHUra and diHThy were G(total)(fr) = 618 +/- 60, G(fbr) = 93 +/- 8, G(8-oxoGua) = 111 +/- 62, G(8-oxoAde) = 4 +/- 3, G(diHUra) = 127 +/- 160, and G(diHThy) = 39 +/- 60, respectively. The yields were determined and the dose-response curves explained by a mechanistic model consisting of three reaction pathways: (1) trappable-radical single-track, (2) trappable-radical multiple-track, and (3) molecular. If the base content is projected from the decamer's GC:AT ratio of 4:1 to a ratio of 1:1, the percentage of the total measured damage (349 nmol/J) would partition as follows: 20 +/- 16% 8-oxoGua, 3 +/- 3% 8-oxoAde, 28 +/- 46% diHThy, 23 +/- 32% diHUra, and 27 +/- 17% dRib damage. With a cautionary note regarding large standard deviations, the projected yield of total damage is higher in CG-rich DNA because C combined with G is more prone to damage than A combined with T, the ratio of base damage to deoxyribose damage is approximately 3:1, the yield of diHUra is comparable to the yield of diHThy, and the yield of 8-oxoAde is not negligible. While the quantity and quality of the data fall short of proving the hypothesized model, the model provides an explanation for the dose-response curves of the more prevalent end products and provides a means of measuring their chemical yields, i.e., their rate of formation at zero dose. Therefore, we believe that this comprehensive analytical approach, combined with the mechanistic model, will prove important in predicting risk due to exposure to low doses and low dose rates of ionizing radiation.

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Bleomycin, a radiomimetic drug currently used in human cancer therapy, is a well known carcinogen. Its toxicity is mostly attributed to its potentiality to induce DNA double strand breaks likely arising from the formation of two vicinal DNA strand breaks, initiated by C4-hydrogen abstraction on the 2-deoxyribose moiety. In this work we demonstrate that such a hydrogen abstraction reaction is able to induce the formation of a clustered DNA lesion, involving a 3' strand break together with a modified sugar residue exhibiting a reactive alpha,beta-unsaturated aldehyde that further reacts with a proximate cytosine base. The lesion thus produced was detected as a mixture of four isomers by HPLC coupled to tandem mass spectrometry subsequent to DNA extraction and enzymatic digestion. The modified nucleosides that constitute new types of cytosine adducts were identified as the likely two pairs of diastereomers of 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-2-hydroxy-3(3-hydroxy-2-oxopropyl)-2,6-dihydroimidazo[1,2-c]-pyrimidin-5(3H)-one as inferred from mass spectrometry and NMR analyses of the chemically synthesized nucleosides. We demonstrate that bleomycin, and to a minor extent ionizing radiation, are able to induce significant amounts of the cytosine damage in cellular DNA. In addition, the repair kinetic of the lesion in a human lymphocyte cell line is rather slow, with a half-life of 10 h. The 2'-deoxycytidine adducts thus characterized that represent the first example of complex DNA lesions isolated and identified in cellular DNA upon one radical hit are likely to play an important role in the toxicity of bleomycin.

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We report that 10-100 eV Ar+ ion irradiation induces severe damage to the biologically relevant sugar molecules D-ribose and 2-deoxy-D-ribose in the condensed phase on a polycrystalline Pt substrate. Ar+ ions with kinetic energies down to 15 eV induce effective decomposition of both sugar molecules, leading to the desorption of abundant cation and anion fragments, including CH3+, C2H3+, C3H3+, H3O+, CHO+, CH3O+, C2H3O+, H-, O-, and OH-, etc. Use of isotopically labelled molecules (5- 13C D-ribose and 1-D D-ribose) reveals the site specificity for some of the fragment origins, and thus the nature of the chemical bond breaking. It is found that all of the chemical bonds in both molecules are vulnerable to ion impact at energies down to 15 eV, particularly both the endo- and exocyclic C-O bonds. In addition to molecular fragmentation, several chemical reactions are also observed. A small amount of O-/O fragments abstract hydrogen to form OH-. It is found that the formation of the H3O+ ion is related to the hydroxyl groups of the sugar molecules, and is associated with additional hydrogen loss from the parent or adjacent molecules via hydrogen abstraction or proton transfer. The formation of several other cation fragments also requires hydrogen abstraction from its parent or an adjacent molecule. These fragmentations and reactions are likely to occur in a real biomedium during ionizing radiation treatment of tumors and thus bear significant radiobiological relevance.

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Emerging evidence points to the importance of deoxyribose oxidation in the toxicity of oxidative DNA damage, including the formation of protein-DNA crosslinks and base adducts. With the goal of understanding the differences in deoxyribose oxidation chemistry known to occur with different oxidants, we have compared the formation of one product of 3'-oxidation of deoxyribose in DNA, 3'-phosphoglycolaldehyde (PGA) residues, in isolated DNA and cells exposed to ionizing radiations. A recently developed gas chromatography/negative chemical ionization mass spectrometry method was used to quantify PGA residues in purified DNA and in human TK6 lymphoblastoid cells exposed to gamma radiation (60Co) and alpha particles (241Am). The level of PGA residues was then correlated with the total quantity of deoxyribose oxidation determined by plasmid topoisomer analysis. Alpha-particle irradiation (0-100 Gy) of purified DNA in 50 mM potassium phosphate (pH 7.4) produced a linear dose response of 0.13 PGA residues per 10(6) nucleotides per gray. When normalized to an estimate of the total number of deoxyribose oxidation events (2.0 per 10(6) nucleotides per gray), PGA formation occurred in 7% (+/-0.5) of deoxyribose oxidation events produced by alpha-particle radiation. In contrast, the efficiency of PGA formation in gamma-irradiated DNA was found to be 1% (+/-0.02), which indicates a shift in the chemistry of deoxyribose oxidation, possibly as a result of the different track structures of the two types of ionizing radiation. Studies with gamma radiation were extended to TK6 cells, in which it was observed that gamma radiation produced a linear dose response of 0.0019 PGA residues per 10(6) nucleotides per gray. This is consistent with an approximately 1000-fold quenching effect in cells, similar to the results of other published studies of oxidative DNA damage in vivo.

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PURPOSE: To compare the molecular decomposition of 2-deoxy-D-ribose induced by 0.6 keV electron irradiation or by 0.5keV ultrasoft X-ray irradiation. MATERIALS AND METHODS: A thin film of 2-deoxy-D-ribose was irradiated by two radiation sources: low-energy (approximately 0.6 keV) electrons and ultrasoft X-rays (approximately 0.5 keV). The positive ions that were desorbed from the sample during the irradiation were measured using a quadrupole mass spectrometer. The spectral changes in the X-ray absorption near edge structure (XANES) were also examined after the irradiation. RESULTS AND DISCUSSION: The ions that were desorbed from 2-deoxy-D-ribose due to electron irradiation were mainly H+, CHx+, C2Hx+, CO+, CHxO+, C3Hx+, C2HxO+ and C3sHO+ (x=1, 2, and 3) ions. These ions were the same as those observed in desorption due to ultrasoft X-ray irradiation. The XANES spectral changes induced by electron irradiation showed C-O bond cleavage in the molecule and C=O bond formation in the surface residues. These results show that intensive molecular decomposition of the furanose ring structure was induced by both types of irradiation. It is inferred that these irradiation products are primarily produced by secondary electrons (several tens of eV), which are thought to be generated by both types of irradiation when they are applied to the 2-deoxy-D-ribose sample.

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The physicochemical characteristics of 2-deoxy-D-ribose moieties in DNA strands are important to understand biological radiation stress. So, the X-ray absorption near edge structure (XANES) of 2-deoxy-D-ribose within the energy region around the oxygen K-shell absorption edge was measured. 2-deoxy-D-ribose was exposed to 3 energies of X-rays, i.e., 526.3 eV (below O 1s-->pi*), 537.8 eV (at the absorption peak of O 1s-->sigma*) and 552.6 eV (above O 1s-->sigma*) for given periods. Slight differences in spectral changes were seen in the each irradiation energy, suggesting in fact that the chemical state and following rearranged chemical structure of 2-deoxy-D-ribose may be different between the 3 irradiation energies.

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Oxidative damage as a result of DNA-mediated long-range charge transport occurs readily and at high yield in duplex DNA, and it is of interest whether similar damage can occur in duplex oligonucleotides that include both ribo- and deoxyribonucleotides. Assemblies containing RNA and mixed RNA.DNA strands were constructed containing tethered ethidium as a photooxidant. In photooxidation experiments, long-range oxidative damage to the ribose-containing strand of the oligonucleotide duplexes was examined. Hole injection by photoexcited ethidium followed by radical migration to oxidatively susceptible guanines afforded significant damage on ribose-containing strands at long range ( approximately 35 A). This damage does not differ substantially in yield and location from that found in B-DNA duplexes. No oxidative damage was found upon photooxidation of DNA/RNA duplexes containing tethered metallointercalator, despite the ability of the rhodium complex to promote oxidative damage at a distance in DNA duplexes. This result is attributed to the poor coupling of the rhodium complex into the A-like RNA/DNA duplex. The ability for long-range charge transport to occur in double-stranded nucleic acids of different comformations is considered in light of modeling studies that show interstrand base-base overlap between the opposing, complementary strands that make up RNA/DNA hybrid duplexes. Thus, the possibility of long-range radical migration to effect oxidative damage or signaling may be considered also in the context of transcriptional events.

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Triplet states of deoxyribose are expected to dissociate efficiently into radicals, leading to strand breaks. Such states could be excited by slow secondary electrons (A) or result from ion recombination in spurs containing two or more ion pairs (B). Estimates of the efficiencies of these processes are presented and the mechanisms discussed in the light of recent work with electrons, vacuum ultraviolet (VUV) photons, and X rays. Route B could play a significant role in producing double-strand breaks, while route A may be a better approach to characterizing the process experimentally.

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2'-Deoxy-5-methyleneuridin-5-yl (1) is produced in a variety of DNA damage processes and is believed to result in the formation of lesions that are mutagenic and refractory to enzymatic repair. 2'-Deoxy-5-methyleneuridin-5-yl (1) was independently generated under anaerobic conditions via Norrish Type I photocleavage during Pyrex filtered photolysis of the benzyl ketone 7. The radical (1) exhibits behavior consistent with that of a resonance-stabilized radical. The KIE for hydrogen atom transfer from t-BuSH was found to be 7.3 +/- 1.7. Competition studies between radical recombination and hydrogen atom donors (2,5-dimethyltetrahydrofuran, kTrap = 46.1 +/- 15.4 M(-1) s(-1); propan-2-ol, kTrap = 13.6 +/- 3.5 M(-1) s(-1)) chosen to mimic the carbohydrate components of 2'-deoxyribonucleotides suggest that 2'-deoxy-5-methyleneuridin-5-yl (1) may be able to transfer damage from the nucleobase to the deoxyribose of an adjacent nucleotide in DNA under hypoxic conditions.

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The fundamental new universal method of evaluation of the interaction between macroradical and radioprotector (the access window method) is presented here. It's based on the modelling phenomenon of molecule penetration to the active centre of macromolecule structure through the functional groups free "window". The steric modelling of the B-DNA structure fragments allowed to measure the conformation parameters of the intermediate stereocomplex between interacting substancies. Using the thesis about molecule InH mask the interaction process of InH (steric hindered phenol 4-oxy-3,5-di-tret-butyl-alpha-metylbenzylamine, mercaptoethanol, MET, and L-cysteine, CYS) with 2-deoxyribosyl five radicals in DNA was studied. It was determined, that the radioprotection maximum effect on single-strand breakage formation in irradiated cell can reach 2/3 of the total sugar radicals yield and for MET and CYS (with minor mask squares) -87.5, and 91%, accordingly (it agrees with experiment in literature data).

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By steric computer modelling of five types of 2-deoxyribosyl radicals and their molecular products generated in DNA by radiation as a result of R + InH = RH + In reaction the displacement of DNA bases (maximum--for C'1 atoms and minimum for C'3 ones) has been determined. Literature data on the DNA decay in the irradiated cells, and in DNA frozen solutions as well as the data on radiolysis of compounds modelling separate DNA fragments allowed to offer a general scheme of the every 2-deoxyribosyl radical transformation and to calculate a balance between the intermediate and final molecular products (processes) of the 2-deoxyribosyl radiolysis. The DNA conformation change due to the 2-deoxyribosyl stereoisomers formation at C'3 and C'4 atoms is discussed as one of the suggested reasons for genetic radiation mutation.

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The free radicals produced from the irradiation of hydrated DNA with a heavy-ion beam have been investigated by ESR spectroscopy. The dominant free radical species formed after 60 MeV/nucleon (16)O(8+) ion-beam irradiations at low temperatures (77 K) are the same as those previously identified from studies using low-LET radiation, pyrimidine electron-gain radicals and purine electron-loss radicals; however, greater relative amounts of neutral carbon-centered radicals are found with the higher-LET radiation, and a new phosphorus-centered radical is identified. The fraction of neutral carbon radicals is also found to increase along the ion-beam track with the highest amounts found in the Bragg peak. The neutral carbon-centered radicals likely arise in part from the sugar moiety. The G values found for total trapped radicals at 77 K are significantly smaller for the (16)O(8+) ion beam than those found for low-LET radiation. The new phosphorus-centered radical species is identified by its large 31P parallel hyperfine coupling of about 780 G as a phosphoryl radical. This species is produced linearly with dose and is not found in significant amounts in DNA irradiated with low-LET radiation. The phosphoryl radical must be produced by the fragmentation of a P-O bond and suggests the possibility of a direct strand break. The yield of phosphoryl species is small (about 0.1% of all radicals); however, it clearly indicates that new mechanisms of damage which are not significant for low-LET radiation must be considered for high-LET radiation.

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The lethal and mutagenic effects of 3H decay in 2' position of deoxyribose residues in DNA of extracellular lambda phage were studied, [2'-3H]-deoxyadenosine (3H-dA) or [2'-3H]-thymidine (3H-dT) being used as labelled DNA precursors. As estimated by the efficiency of the lethal and mutagenic actions of 3H decay in position 2' was significantly lower than that of the decay in the incorporated 3H-pyrimidines. The genetic effects of 3H decay in 2' position may be attributed to the radiation effect of beta-particles on DNA. In UV-irradiated E. coli cells, with the induced SOS repair, the mutagenic effect of 3H-dA in phage lambda is significantly higher than that of 3H-dT. This is perhaps related to the formation in DNA of AP-sites, resulting from 3H-decay in 2' position, and to the predominant incorporation of adenosine residues opposite to AP-sites during SOS repair.

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In the living cell, ionizing radiation can cause DNA damage by the direct effect (ionization of DNA) and the indirect effect (reaction of radicals formed in the neighborhood of DNA with DNA, e.g., OH, eaq-, H, protein- and glutathione-derived radicals). Properties of the base radical cations have been studied in model systems using SO4- radical to oxidize the nucleobases in aqueous solution. The pKa values of some nucleobase radical cations are reported, so are the ensuing reactions of the thymidine radical cation with water. The products of reactions are compared with those formed by OH radical attack. The reaction of eaq- with the nucleobases yields radical anions. Protonation at heteroatom sites and at carbon are discussed, and some recent results regarding the electron transfer to adjacent nucleobases as well as to 5-bromouracil are reported. A brief account is given on the reaction of carbon-centered radicals with the nucleobases. These reactions may mimic the reactions of protein-derived radicals with DNA. Glutathione is present in cells at rather high concentrations and is expected to act as an H- or electron-donor in repairing radiation-induced DNA damage (chemical repair). As thiyl radicals are known to also undergo the reverse reaction, i.e., H-abstraction from suitable solutes, some experiments are reported which probe this type of reaction with dilute DNA solutions. In some polynucleotides radical transfer from the base radical to the sugar moiety occurs with the consequence of strand breakage and base release. Some currently held mechanistic concepts are discussed. Attention is drawn to some important open questions which should be addressed in the near future.

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The radiation damage to Deoxyribose was studied with a view to identify the damaging species. Our results indicate that H, eaq-, CO2- do not cause any appreciable damage in the absence of metal compounds and .OH is the sole damaging entity. Iron compounds sensitize very little O2- damage and CO2- damage could not be sensitized. In N2-saturated solutions metal compounds increase the damage by converting eaq- into deleterious .OH.

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Hydroxyl radical addition to uracil (U) has been suggested to lead to strand breaks in polyuridylic acid, an occurrence attributed in part to H atom abstraction by .U-OH radicals from the ribose moiety [D.G.E. Lemaire et al., Int. J. Radiat. Biol. 45, 351-358 (1984)]. We have investigated this particular reaction by means of the hydroxyl radical-induced products of thymine (T), pT, TpT, TpTpT, polythymidylic acid (poly-T), (T + dR) poly-dA.poly-T, and a mixture of T and 2-deoxyribose (dR). The major monomeric product of .T-OH in TpT, TpTpT, poly-T, and poly-dA.poly-T was found to be 5-hydroxy-6-hydrothymine (H-T-OH), while that in T, pT, and T plus dR was thymine glycol (HO-T-OH). These results indicated that the intramolecular H atom abstraction from a nearby sugar (in this case, deoxyribose) moiety by base radicals, i.e., .T-OH, occurs in oligo- and polydeoxynucleotides of T. In poly-T, the yield of H-T-OH is not much greater than in TpT or TpTpT, indicating that the abstraction of an H atom from the sugar moiety of a nucleotide subunit further than two nucleotides along the chain may not be significant. Additionally, a corresponding decrease in the yield of HO-T-OH with an increase in the yield of H-T-OH suggests that the formations of these two types of thymine products are competitive.

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The degradation of 2-deoxyribose to thiobarbituric acid-reactive material was investigated with two hydroxyl-radical-generating systems: (i) a defined gamma-radiolysis method and (ii) incubation with FeSO4 in phosphate buffer. In each case the thiobarbituric acid-reactive material can be accounted for by malondialdehyde, as measured by an h.p.l.c. method for free malondialdehyde. In the radiolysis system there is a large post-irradiation increase in free malondialdehyde if iron ions are added to the samples. It is proposed that this is due to iron ions catalysing the formation of hydroxyl radicals from radiolytically generated H2O2 as well as stimulating the breakdown of an intermediate deoxyribose degradation product. A mechanism for the formation of malondialdehyde during deoxyribose degradation is proposed.

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Hydroxy radicals induce cross-linking between phenylalanine (Phe) and 2-deoxyribose (dR) via formation of corresponding free radical intermediates. The cross-linked products were separated and identified by capillary gas chromatography-mass spectrometry. When phenylalanine and 2-deoxyribose radicals were generated in a 1:1 ratio, the predominant interaction was between Phe and dR radicals while the Phe-Phe and dR-dR cross-links were less abundant. The newly discovered cross-link between 2-deoxyribose and phenylalanine may serve as a model for radiation or free radical induced cross-linking between DNA and proteins and in general between sugar moieties and amino acids.

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