Mechanisms of direct radiation damage in DNA, based on a study of the yields of base damage, deoxyribose damage, and trapped radicals in d(GCACGCGTGC)(2).

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.

Differential oxidation of deoxyribose in DNA by gamma and alpha-particle radiation.

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.

Radiation damage in DNA: possible role of higher triplet states.

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.

Intramolecular H atom abstraction from the sugar moiety by thymine radicals in oligo- and polydeoxynucleotides.

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.

Electron spin resonance of DNA irradiated with a heavy-ion beam ([16]O[8+]): evidence for damage to the deoxyribose phosphate backbone.

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.

Decomposition of 2-deoxy-D-ribose by irradiation with 0.6 keV electrons and by 0.5 keV ultrasoft X-rays.

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.

Induction of sister-chromatid exchanges and chromosome aberrations by gamma-irradiated nucleic acid constituents in CHO cells.

Chinese hamster ovary (CHO) cells were exposed to different concentrations of hydrogen peroxide and to gamma-irradiated, oxygenated solutions of thymine, thymidine and 2-deoxy-D-ribose. By using a modified BrdUrd-labelling method sister-chromatid exchanges (SCEs) and chromosomal aberrations were scored in the same cell populations, one cycle after treatment. Irradiated, oxygenated solutions of 2-deoxy-D-ribose clearly induced SCEs and chromosomal aberrations. In comparison, hydrogen peroxide and irradiated solutions of thymine and thymidine were less effective in CHO cells.

Mutagenicity of gamma-irradiated oxygenated and deoxygenated solutions of 2-deoxy-D-ribose and D-ribose in Salmonella typhimurium.

Solutions of 2-deoxy-D-ribose and D-ribose were gamma-irradiated under different experimental conditions and tested for mutagenicity, with and without preincubation, in Salmonella typhimurium. The irradiated sugar solutions were mutagenic in the tester strains TA100 and TA98. Except for malonaldehyde (MDA), which is not mutagenic in the concentrations produced radiolytically, the relative mutagenicities of the individual radiolytic products are unknown. With irradiated solutions of 2-deoxy-D-ribose, a relationship was found between the level of non-MDA aldehydes and the mutagenicity in TA100. Heating the irradiated solutions of 2-deoxy-D-ribose resulted in a temperature-dependent reduction of the mutagenicity. Autoclaved, non-irradiated solutions of 2-deoxy-D-ribose were not mutagenic in the Salmonella test.

[Efficacy of radioprotectors and gene mutations following 2-deoxyribosyl damage. Computer modeling]. / Effektivnost' radioprotektorov i gennye mutatsii pri povrezhdenii 2-dezoksiribozila. Komp'iuternoe modelirovanie.

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).

[Interactions of inhibitor of free radical reactions and 2-deoxyribosyl macroradicals]. / O vzaimodeistvii veshchestva-ingibitora svobodnoradikal'nykh reaktsii s makroradikalami 2-dezoksiribozila.

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.

[Abiogenic nucleoside synthesis in the presence of phosphorus salts]. / Abiogennyi sintez nukleozidov v prisutstvii fosfornykh solei.

By means of UV-spectroscopy, gel-filtration, ion-exchange and thin layer chromatography it has been shown that the action of ionizing radiation on the mixture of dry preparations of adenine and deoxyribose in the presence of the K, Na and Ca phosphates results in the formation of nucleoside-like substances. The phosphates catalyze or inhibit the nucleoside synthesis but they are not phosphorylating agents. The data obtained indicate the reality of abiogenic synthesis of nucleoside-like substances from the mixture of dry preparations of adenine and deoxyribose in the lithosphere during chemical evolution.

Clemens von Sonntag and the early history of radiation-induced sugar damage in DNA.

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.

Oxidation of the sugar moiety of DNA by ionizing radiation or bleomycin could induce the formation of a cluster DNA lesion.

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.

10-100 eV Ar+ ion induced damage to D-ribose and 2-deoxy-D-ribose molecules in condensed phase.

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.