Ionization generates a large number of secondary low-energy electrons (LEEs) with a most probable energy of approximately 10 eV, which can break DNA bonds by dissociative electron attachment (DEA) and lead to DNA damage. In this study, we investigated radiation damage to dry DNA induced by X rays (1.5 keV) alone on a glass substrate or X rays combined with extra LEEs (average energy of 5.8 eV) emitted from a tantalum (Ta) substrate under an atmosphere of N 2 and standard ambient conditions of temperature and pressure. The targets included calf-thymus DNA and double-stranded synthetic oligonucleotides. We developed analytical methods to measure the release of non-modified DNA bases from DNA and the formation of several base modifications by LC-MS/MS with isotopic dilution for precise quantification. The results show that the yield of non-modified bases as well as base modifications increase by 20–30% when DNA is deposited on a Ta substrate compared to that on a glass substrate. The order of base release (Gua > Ade > Thy ∼ Cyt) agrees well with several theoretical studies indicating that Gua is the most susceptible site toward sugar-phosphate cleavage. The formation of DNA damage by LEEs is explained by DEA leading to the release of non-modified bases involving the initial cleavage of N1-C1′, C3′-O3′ or C5′-O5′ bonds. The yield of base modifications was lower than the release of non-modified bases. The main LEE-induced base modifications include 5,6-dihydrothymine (5,6-dHT), 5,6-dihydrouracil (5-dHU), 5-hydroxymethyluracil (5-HmU) and 5-formyluracil (5-ForU). The formation of base modifications by LEEs can be explained by DEA and cleavage of the C-H bond of the methyl group of Thy (giving 5-HmU and 5-ForU) and by secondary reactions of H atoms and hydride anions that are generated by primary LEE reactions followed by subsequent reaction with Cyt and Thy (giving 5,6-dHU and 5,6-dHT).

A novel technique has been employed to investigate the simultaneous damage to DNA components induced by soft X rays (1.5 keV) and low-energy electrons (0–30 eV) in thin films of thymidine deposited on glass and tantalum substrates and irradiated under atmospheric pressure and temperature. The films were surrounded by either an N 2 or O 2 environment. The formation of four radiation-induced products is reported in this article: base release, 5-hydroxymethyl-2′-deoxyuridine (5-HMdUrd), 5-formyl-2′-deoxyuridine (5-FordUrd) and 5,6-dihydrothymidine (5,6-DHThd). Analysis with LC-MS/MS shows larger damage yields in the samples deposited on tantalum than in those deposited on glass, which is attributed to the interaction of the additional low-energy electrons that are photoemitted from the metal surface. From a comparison of the results obtained from N 2 and O 2 environment, we report a dramatic effect from 6 O 2 : an approximately threefold increase in the yield of products, attributed to the reaction of O 2 with initial carbon-centered thymidine radicals generated in the film during irradiation.

The majority of studies on lethal radiobiological damage have focused on double-strand breaks (DSBs), a type of clustered DNA damage and the evaluation of their toxicity, while other types of clustered DNA damage have received much less attention. The main purpose of this study is to evaluate the contribution of different lesions induced by ionizing radiation to the loss of plasmid DNA functionality. We employed a simple model system comprising E. coli transformed with an irradiated plasmid [pGEM-3Zf (–)] to determine the effect of DSBs and other lesions including base damage and clustered lesions on the functionality (“viability”) of the plasmid. The yields of γ-radiation-induced single-strand breaks (SSBs) and DSBs were measured by gel electrophoresis. We found that the transformation efficiency decreases with radiation dose, but this decrease cannot be explained by the formation of DSBs. For example, at doses of 500 and 700 Gy, the relative transformation efficiency falls from 100% to 53% and 26%, respectively, while only 5.7% and 9.1% of the plasmids contain a DSB. In addition, it is also unlikely that randomly distributed base lesions could explain the loss of functionality of the plasmid, since cells can repair them efficiently. However, clustered lesions other than DSBs, which are difficult to repair and result in the loss of information on both DNA strands, have the potential to induce the loss of plasmid functionality. We therefore measured the yields of γ-radiation-induced base lesions and cluster damage, which are respectively converted into SSBs and DSBs by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). Our data demonstrate that the yield of cluster damage (i.e., lesions that yield DSBs following digestion) is 31 times higher than that of frank DSBs. This finding suggests that frank DSBs make a relatively minor contribution to the loss of DNA functionality induced by ionizing radiation, while other toxic lesions formed at a much higher frequencies than DSBs must be responsible for the loss of plasmid functionality. These lesions may be clustered lesions/locally multiply damaged sites (LMDS), including base damage, SSBs and/or intrastrand and interstrand crosslinks, leading to the loss of vital information in the DNA. Using a mathematical model, we estimate that at least three toxic lesions are required for the inactivation of plasmid functionality, in part because even these complex lesions can be repaired.

The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM–3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with 60 Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 m M tris in an N 2 atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N 2 O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 m M tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing radiation.

Low-energy electrons (LEE) induce single- and double-strand breaks in DNA. To investigate the mechanism of LEE-induced DNA damage, nucleotides and short oligonucleotide were irradiated with monoenergetic electrons in the solid state and the modifications were observed by chemical analyses. With 10 eV electrons and TpTpT as the target, approximately one-third of the total damage of TpTpT involves cleavage of the phosphodiester-sugar bond (C-O) and the N-glycosidic bond (C-N). Here we focus on the remaining two-thirds of the damage. The major products were observed to elute between TpT and TpTpT on the HPLC chromatogram. Of these products, three modifications were identified as XpTpT, TpXpT and TpTpX, where X = 5,6-dihydrothymine, on the basis of comparison with standard compounds using HPLC and mass spectrometry. These results suggest that 5,6-dihydrothymine is a major product of the reaction of LEE with DNA.

Zheng, Y. and Sanche, L. Gold Nanoparticles Enhance DNA Damage Induced by Anti-cancer Drugs and Radiation. Radiat. Res. 172, 114-119 (2009). The chemotherapeutic agent cisplatin was chemically linked to pGEM-3Zf(-) plasmid DNA to produce a cisplatin-DNA complex, Gold nanoparticles, which bind electrostatically to pure DNA, could also be added to this complex. Dry films of pure plasmid DNA and DNA-cisplatin, DNA-gold nanoparticles and DNA-cisplatin-gold nanoparticles complexes were bombarded by 60 keV electrons. The yields of single- and double-strand breaks were measured as a function of exposure by electrophoresis. From a comparison of such yields from the different type of films, we found that the binding of only one gold nanoparticle to a plasmid-cisplatin complex containing 3197 base pairs increases by a factor of 3 the efficiency of the chemotherapeutic agent cisplatin to produce double-strand breaks in irradiated DNA. Furthermore, adding two cisplatin molecules and one gold nanoparticle to DNA enhances radiation-induced DSBs by a factor of 7.5. A number of phenomena could contribute to this huge enhancement, including the higher density of low-energy electrons and reactive species around the gold nanoparticles and the weakening of bonds adjacent to cisplatin in the DNA backbone. The addition of gold nanoparticles to cisplatin and other platinum agents may therefore provide interesting avenues of research to improve the treatment of cancer by concomitant chemoradiation.

Zheng, Y., Hunting, D. J., Ayotte, P. and Sanche, L. Radiosensitization of DNA by Gold Nanoparticles Irradiated with High-Energy Electrons. Radiat. Res. 168, 19–27 (2008). Thin films of pGEM-3Zf(−) plasmid DNA were bombarded by 60 keV electrons with and without gold nanoparticles. DNA single- and double-strand breaks (SSBs and DSBs) were measured by agarose gel electrophoresis. From transmission electron micrographs, the gold nanoparticles were found to be closely linked to DNA scaffolds, probably as a result of electrostatic binding. The probabilities for formation of SSBs and DSBs from exposure of 1:1 and 2:1 gold nanoparticle:plasmid mixtures to fast electrons increase by a factor of about 2.5 compared to neat DNA samples. For monolayer DNA adsorbed on a thick gold substrate, the damage increases by an order of magnitude. The results suggest that the enhancement of radiosensitivity is due to the production of additional low-energy secondary electrons caused by the increased absorption of ionizing radiation energy by the metal, in the form of gold nanoparticles or of a thick gold substrate. Since short-range low-energy secondary electrons are produced in large amounts by any type of ionizing radiation, and since on average only one gold nanoparticle per DNA molecule is needed to increase damage considerably, targeting the DNA of cancer cells with gold nanoparticles may offer a novel approach that is generally applicable to radiotherapy treatments.

Li, X., Sanche, L. and Sevilla, M.D Base Release in Nucleosides Induced by Low-Energy Electrons: A DFT Study. Radiat. Res. 165, 721–729 (2006). Low-energy electrons are known to induce strand breaks and base damage in DNA and RNA through fragmentation of molecular bonding. Recently the glycosidic bond cleavage of nucleosides by low-energy electrons has been reported. These experimental results call for a theoretical investigation of the strength of the C 1 ′–N link in nucleosides (dA, dC and dT) between the base and deoxyribose before and after electron attachment. Through density functional theory (DFT) calculations, we compare the C 1 ′–N bond strength, i.e., the bond dissociation energy of the neutral and its anionic radical, and find that an excess electron effectively weakens the C 1 ′– N bond strength in nucleosides by 61–75 kcal/mol in the gas phase and 76–83 kcal/mol in the solvated environment. As a result, electron-induced fragmentation of the C 1 ′–N bond in the gas phase is exergonic for dA (Δ G = −14 kcal/mol) and for dT (Δ G = −6 kcal/mol) and is endergonic (Δ G = +1 kcal/ mol) only for dC. In the gas phase all the anionic nucleosides are found to be in valence states. Solvation is found to increase the exergonic nature by an additional 20 kcal, making the fragmentation both exothermic and exergonic for all nucleoside anion radicals. Thus C 1 ′–N bond breaking in nucleoside anion radicals is found to be thermodynamically favorable both in the gas phase and under solvation. The activation barrier for the C 1 ′–N bond breaking process was found to be about 20 kcal/mol in every case examined, suggesting that a 1 eV electron would induce spontaneous cleavage of the bond and that stabilized anion radicals on the DNA strand would undergo base release at only a modest rate at room temperature. These results suggest that base release from nucleosides and DNA is an expected consequence of low-energy electron-induced damage but that the high barrier would inhibit this process in the stable anion radicals.

Panajotovic, R., Martin, F., Cloutier, P., Hunting, D. and Sanche, L. Effective Cross Sections for Production of Single-Strand Breaks in Plasmid DNA by 0.1 to 4.7 eV Electrons. Radiat. Res. 165, 452–459 (2006). We determined effective cross sections for production of single-strand breaks (SSBs) in plasmid DNA [pGEM 3Zf(-)] by electrons of 10 eV and energies between 0.1 and 4.7 eV. After purification and lyophilization on a chemically clean tantalum foil, dry plasmid DNA samples were transferred into a high-vacuum chamber and bombarded by a monoenergetic electron beam. The amount of the circular relaxed DNA in the samples was separated from undamaged molecules and quantified using agarose gel electrophoresis. The effective cross sections were derived from the slope of the yield as a function of exposure and had values in the range of 10 −15 – 10 −14 cm 2 , giving an effective cross section of the order of 10 −18 cm 2 per nucleotide. Their strong variation with incident electron energy and the resonant enhancement at 1 eV suggest that considerable damage is inflicted by very low-energy electrons to DNA, and it indicates the important role of π* shape resonances in the bond-breaking process. Furthermore, the fact that the energy threshold for SSB production is practically zero implies that the sensitivity of DNA to electron impact is universal and is not limited to any particular energy range.

Cai, Z., Cloutier, P., Hunting, D. and Sanche, L. Enhanced DNA Damage Induced by Secondary Electron Emission from a Tantalum Surface Exposed to Soft X Rays. Radiat. Res. 165, 365–371 (2006). Both thick and thin films of pGEM®-3Zf(-) plasmid DNA deposited on a tantalum foil were exposed to soft X rays (effective energy of 14.8 keV) for various times in air under a relative humidity of 45% (Γ ≈ 6, where Γ is the number of water molecules per nucleotide) and 84% (Γ ≈ 21), respectively. For a thick film, the DNA damage was induced chiefly by X-ray photons. For a thin film of DNA, X-ray-induced secondary electrons emitted from the tantalum result in a substantial increase in DNA damages. Different forms of plasmid DNA were separated and quantified by agarose gel electrophoresis and laser scanning. The exposure curves for the formation of nicked circular (single-strand break, SSB), linear (double-strand break, DSB), and interduplex crosslink forms 1 and 2 were obtained for both thick and thin films of DNA. The secondary electron enhancement factor for SSBs, DSBs and crosslinks of the thin film of DNA were derived to be 3.8 ± 0.5, 2.9 ± 0.7 and 7 ± 3 at Γ ≈ 6 and 6.0 ± 0.8, 7 ± 1 and 3.9 ± 0.9 at Γ ≈ 21, respectively. This study provides a molecular basis for understanding the enhanced biological effects at interfaces during diagnostic X-ray examination and radiotherapy.

Cai, Z., Cloutier, P., Sanche, L. and Hunting, D. DNA Interduplex Crosslinks Induced by Al Kα X Rays under Vacuum. Radiat. Res. 164, 173–179 (2005). Dry pGEM®-3Zf(-) plasmid DNA was exposed to Al kα X rays (1.5 keV) for various times in an ultra-high vacuum chamber with mean absorbed dose rates ranging from 1.8 to 41.7 Gy s −1 . The different forms of plasmid DNA were separated by neutral agarose gel electrophoresis and quantified by staining and laser scanning. In addition to the bands for supercoiled, nicked circular, linear and concatameric forms of plasmid DNA, two additional bands were observed in X-irradiated samples; these migrated at rates similar to those for 8-kb and >10-kb linear double-stranded DNA. Digestion of irradiated DNA with the restriction enzymes Eco R1 and Pvu I suggested that the two slowly migrating bands were interduplex crosslinked DNA. Alkaline agarose gel electrophoresis of irradiated DNA digested with Eco R1 confirmed that the interduplex crosslink was covalent. Exposure–response curves were determined for the formation of nicked circular, linear and interduplex crosslinked DNA as well as for the loss of supercoiled and concatameric DNA. Formation and loss of these species were independent of absorbed dose rate over a 20-fold range. The G values for DNA single-strand breaks, double-strand breaks and crosslinks were determined to be 62 ± 6, 5.6 ± 0.6 and 16 ± 4 nmol J −1 , respectively. The formation of DNA interduplex crosslinks appears to be due to single event. The mechanism responsible for the formation of DNA interduplex crosslinks is discussed with emphasis on its implications in vivo.

Abdoul-Carime, H., Dugal, P-C. and Sanche, L. Damage Induced by 1–30 eV Electrons on Thymine- and Bromouracil-Substituted Oligonucleotides. The impact of low-energy (1–30 eV) electrons on self-assembled monolayers of heterogeneous oligonucleotides chemisorbed on a gold surface has been investigated by mass spectrometry of desorbed neutral species in an attempt to understand the consequences of secondary electron damage in a short sequence of a DNA single strand. We demonstrate that the most intense observable neutral species (CN, OCN and/or H 2 NCN) desorbed from Cy 6 -Th 3 and Cy 6 -(BrdU) 3 oligos are related to primary fragmentation of the bases induced by electron impact. The dependence of the neutral species desorption on electron energy shows typical signatures of dissociative electron attachment initiated by the formation of shape- and core-excited resonances (i.e. single-electron and two-electron–one-hole transitory anions, respectively). Substitution of dTh by BrdU increases the production of neutral fragments by as much as a factor of about 3 for the entire electron energy range. When the distribution of secondary electrons along radiation tracks in H 2 O is taken into account, we show that the probability for electron damage to heterogeneous oligonucleotides is enhanced by a factor of 2.5–3 for electron energies below 20 eV for both sensitized and unsensitized strands.

Radiation-induced damage to homo-oligonucleotides is investigated by electron-stimulated desorption of neutral fragments from chemisorbed organic films. Six and 12 mers of cytidine phosphate (poly dCs) and thymidine phosphate (poly dTs) are chemisorbed from various solutions onto a crystalline gold substrate by a thiol modification at the 3′ end and are irradiated under ultra-high vacuum conditions with 5-25 eV electrons. The mass selected neutral desorption yields consist mainly of fragments of the DNA bases, i.e. CN and OCN (and/or <tex-math>${\rm H}_{2}{\rm HCN}$</tex-math> for poly dCs) from both poly dCs and poly dTs, indicating that the electrons interact specifically via fragmentation of the aromatic ring of either of the bases. Other heavier fragments are also detected such as <tex-math>${\rm H}_{3}{\rm CC}-{\rm CO}$</tex-math> from poly dTs. The yields generally process a threshold near 5 eV and a broad maximum around 12-13 eV incident electron energy. Dissociative electron attachments as well as electronically excited neutral or cation states are believed to be responsible for the various desorption yields. The latter yields are consistently larger for oligos chemisorbed from water and acetone solutions, compared to methanol solution. The invariance of the fragment yield intensities with oligo length suggests that the molecules are likely to adsorb almost parallel to the surface.

The radiosensitization properties of 5-halouracils (5-FU, 5-BrU and 5-IU), i.e. the enhanced sensitivity of biological media containing these compounds to ionizing radiation, have been studied using surface science methods. We show that soft X rays and near 0 eV electrons both induce dissociation of 5-halouracils into a halogen anion and a uracilyl radical. The yield of anions from 5-FU is much smaller than that from the bromo- and iodo-analogs. We explain the high anion yields in 5-BrU and 5-IU with dissociative electron attachment (DEA) of near 0 eV electrons. The thermodynamic threshold for DEA to 5-FU is near 2 eV and therefore prohibits dissociation by near 0 eV electrons.