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Objective . To study the effect of dose-rate in the time evolution of chemical yields produced in pure water versus a cellular-like environment for FLASH radiotherapy research. Approach. A version of TOPAS-nBio with Tau-Leaping algorithm was used to simulate the homogenous chemistry stage of water radiolysis using three chemical models: (1) liquid water model that considered scavenging of e aq - , H • by dissolved oxygen; (2) Michaels & Hunt model that considered scavenging of • OH, e aq ‒ , and H • by biomolecules existing in cellular environment; (3) Wardman model that considered model 2) and the non-enzymatic antioxidant glutathione (GSH). H 2 O 2 concentrations at conventional and FLASH dose-rates were compared with published measurements. Model 3) was used to estimate DNA single-strand break (SSB) yields and compared with published data. SSBs were estimated from simulated yields of DNA hydrogen abstraction and attenuation factors to account for the scavenging capacity of the medium. The simulation setup consisted of monoenergetic protons (100 MeV) delivered in pulses at conventional (0.2857Gy s -1 ) and FLASH (500Gy s -1 ) dose rates. Dose varied from 5-20 Gy, and oxygen concentration from 10 µ M-1 mM. Main Results. At the steady state, for model (1), H 2 O 2 concentration differed by 81.5%± 4.0% between FLASH and conventional dose-rates. For models (2) and (3) the differences were within 8.0%± 4.8%, and calculated SSB yields agreed with published data within 3.8%± 1.2%. A maximum oxygen concentration difference of 60% and 50% for models (2) and (3) between conventional and FLASH dose-rates was found between 2 × 10 6 and 9 × 10 13 ps for 20 Gy of absorbed dose. Significance. The findings highlight the importance of developing more advanced cellular models to account for both the chemical and biological factors that comprise the FLASH effect. It was found that differences between pure water and cellular environment models were significant and extrapolating results between the two should be avoided. Observed differences call for further experimental investigation., (© 2025 Institute of Physics and Engineering in Medicine. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

Charged particle radiation can, directly and indirectly, affect cells by breaking DNA strands. This effect includes DNA single-strand breaks (SSB) and DNA double-strand breaks (DSB), which may cause cell death and mitotic failure. Thus, using short-range charged particles such as Auger electrons (AEs) not only leads to the destruction of the target cell but also prevents the nearby healthy cells from exposing to ionizing radiation. In this study, two spherical cells (C and C 2 ) and their cell nucleus, both made of liquid water, were modeled. An atomic DNA model constructed in the Geant4-DNA Monte Carlo (MC) simulation toolkit was placed inside the nucleus of the C and C 2 cells. The number of direct and indirect SSB, DSB, and hybrid DSB (HDSB), caused by some of the most widely-used Auger electron-emitting (AEE) radionuclides, including 99m Tc, 111 In, 123 I, 125 I, and 201 Tl, distributed within different compartments of the C cell, was calculated in the C and C 2 cells, considering the distance between the surface of the two cells ranges from 0 to 5 μm. The present work aimed to investigate the biological effects of AEE radionuclides and their potential for cancer treatment through targeted radiation therapy. The results indicate the impact of 201 Tl > 125 I > 123 I > 111 In > 99m Tc on DNA damage when the target is C (first spherical cell). On the other hand, for C 2 at distances of 0 to 5 μm, the impact of 99m Tc > 123 I > 111 In > 201 Tl > 125 I on DNA damage is observed., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Research involving human and animal rights: This article does not contain any studies with human participants or animals performed by any of the authors., (© 2024. The Author(s).)

The radiosensitization characteristics of gold nanoparticles (GNPs) have been investigated in a single cell irradiated with monoenergetic beams of protons of various energies using TOPAS-nBio, an advanced toolkit of TOPAS. Both direct and indirect effects against single-strand breaks (SSBs) are investigated and their double-strand breaks (DSBs) have been calculated. A single spherical cell interaction with a detailed DNA structure has been modeled and simulated under different conditions such as particle sizes and concentrations of GNPs, their biodistributions and associated proton energies. The physical interaction among protons, suspension water and GNPs has been simulated using a dual physics approach, while the interaction between water radiolysis and OH radicals was considered in the chemical process to save computational time. The present simulations involve irradiating the cell geometry with a dose of 1 Gy. The range of DSBs (Gy-1 Gbp-1) obtained was 2.1 ± 0.09 to 21.74 ± 0.4 for all GNPs of sizes 6-50 nm the proton energies in the range of 5-50 MeV. Regardless of proton energy and GNP size, the calculations showed that the contribution of indirect and hybrid DSBs remains higher in all simulation types than that of direct DSBs. New simulation outcomes of the indirect DSBs illustrate a percentage increase, while we cannot get an increase in the direct and hybrid DSBs in most cases when compared with no GNPs cases. The indirect DSBs provide the highest enhancement factor of 1.89 at 30 nm GNPs in size for 30 MeV protons energy, and the direct and hybrid DSBs indicate a slight increase in enhancement. The work indicates that the use of GNPs increased indirect DNA DSBs, while hybrid DSBs show only a slight increase in enhancement, and no enhancement is shown in direct DNA DSBs. It is significant to consider other mechanisms such as DNA damage repair when investigating DNA damage., (© 2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

Green fluorescent protein (GFP) has opened vast new avenues in studies of live cells and is generally perceived as a benign, nontoxic and harmless fluorescent tag. We demonstrat that excited GFP is capable of inducing substantial DNA damage in cells expressing fusion proteins. In the presence of GFP, even low doses of blue light (12 μJ) induce single strand breaks (SSBs). When the fluorescence of GFP located in the cell nucleus or in the cytoplasm is excited by a much higher dose (17 mJ), DNA double-strand breaks (DSBs) are also induced. Such breaks are induced even when GFP is placed and illuminated in culture medium outside of living cells. We demonstrate that DNA damage is induced by singlet oxygen, which is generated by excited GFP. Although short exposures of live cells to exciting light typically used in fluorescence microscopy induce SSBs but carry little risk of inducing DNA double-strand breaks, larger doses, which may be used in FRAP, FLIM, FCS and super-resolution fluorescence microscopy studies, are capable of inducing not only numerous SSBs but also DSBs., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Exposure to ionizing radiation can induce genetic aberrations via unrepaired DNA strand breaks. To investigate quantitatively the dose-effect relationship at the molecular level, we irradiated dry pBR322 plasmid DNA with 3 MeV protons and assessed fragmentation yields at different radiation doses using long-read sequencing from Oxford Nanopore Technologies. This technology applied to a reference DNA model revealed dose-dependent fragmentation, as evidenced by read length distributions, showing no discernible radiation sensitivity in specific genetic sequences. In addition, we propose a method for directly measuring the single-strand break (SSB) yield. Furthermore, through a comparative study with a collection of previous works on dry DNA irradiation, we show that the irradiation protocol leads to biases in the definition of ionizing sources. We support this scenario by discussing the size distributions of nanopore sequencing reads in the light of Geant4 and Geant4-DNA simulation toolkit predictions. We show that integrating long-read sequencing technologies with advanced Monte Carlo simulations paves a promising path toward advancing our comprehension and prediction of radiation-induced DNA fragmentation., (© 2024. The Author(s).)

Purpose: Interdisciplinary scientific communities have shown large interest to achieve a mechanistic description of radiation-induced biological damage, aiming to predict biological results produced by different radiation quality exposures. Monte Carlo track-structure simulations are suitable and reliable for the study of early DNA damage induction used as input for assessing DNA damage. This study presents the most recent improvements of a Geant4-DNA simulation tool named "dsbandrepair"., Methods: "dsbandrepair" is a Monte Carlo simulation tool based on a previous code (FullSim) that estimates the induction of early DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). It uses DNA geometries generated by the DNAFabric computational tool for simulating the induction of early single-strand breaks (SSBs) and double-strand breaks (DSBs). Moreover, the new tool includes some published radiobiological models for survival fraction and un-rejoined DSB. Its application for a human fibroblast cell and human umbilical vein endothelial cell containing both heterochromatin and euchromatin was conducted. In addition, this new version offers the possibility of using the new IRT-syn method for computing the chemical stage., Results: The direct and indirect strand breaks, SSBs, DSBs, and damage complexity obtained in this work are equivalent to those obtained with the previously published simulation tool when using the same configuration in the physical and chemical stages. Simulation results on survival fraction and un-rejoined DSB are in reasonable agreement with experimental data., Conclusions: "dsbandrepair" is a tool for simulating DNA damage and repair, benchmarked against experimental data. It has been released as an advanced example in Geant4.11.2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Associazione Italiana di Fisica Medica e Sanitaria. Published by Elsevier Ltd. All rights reserved.)

Background . Radiation-induced DNA damages such as Single Strand Break (SSB), Double Strand Break (DSB) and Complex DSB (cDSB) are critical aspects of radiobiology with implications in radiotherapy and radiation protection applications. Materials and Methods . This study presents a thorough investigation into the effects of protons (0.1-100 MeV/u), helium ions (0.13-100 MeV/u) and carbon ions (0.5-480 MeV/u) on DNA of human fibroblast cells using Geant4-DNA track structure code coupled with DBSCAN algorithm and Monte Carlo Damage Simulations (MCDS) code. Geant4-DNA-based simulations consider 1 μ m × 1 μ m × 0.5 μ m water box as the target to calculate energy deposition on event-by-event basis and the three-dimensional coordinates of the interaction location, and then DBSCAN algorithm is used to calculate yields of SSB, DSB and cDSB in human fibroblast cell. The study investigated the influence of Linear Energy Transfer (LET) of protons, helium ions and carbon ions on the yields of DNA damages. Influence of cellular oxygenation on DNA damage patterns is investigated using MCDS code. Results . The study shows that DSB and SSB yields are influenced by the LET of the particles, with distinct trends observed for different particles. The cellular oxygenation is a key factor, with anoxic cells exhibiting reduced SSB and DSB yields, underscoring the intricate relationship between cellular oxygen levels and DNA damage. The study introduced DSB/SSB ratio as an informative metric for evaluating the severity of radiation-induced DNA damage, particularly in higher LET regions. Conclusions . The study highlights the importance of considering particle type, LET, and cellular oxygenation in assessing the biological effects of ionizing radiation., (Creative Commons Attribution license.)

Ultra-high dose rate (UHDR) irradiation has been shown to have a sparing effect on healthy tissue, an effect known as 'FLASH'. This effect has been studied across several radiation modalities, including photons, protons and clinical energy electrons, however, very little data is available for the effect of FLASH with Very High Energy Electrons (VHEE). pBR322 plasmid DNA was used as a biological model to measure DNA damage in response to Very High Energy Electron (VHEE) irradiation at conventional (0.08 Gy/s), intermediate (96 Gy/s) and ultra-high dose rates (UHDR, (2 × 10 9  Gy/s) at the CERN Linear Electron Accelerator (CLEAR) user facility. UHDRs were used to determine if the biological FLASH effect could be measured in the plasmid model, within a hydroxyl scavenging environment. Two different concentrations of the hydroxyl radical scavenger Tris were used in the plasmid environment to alter the proportions of indirect damage, and to replicate a cellular scavenging capacity. Indirect damage refers to the interaction of ionising radiation with molecules and species to generate reactive species which can then attack DNA. UHDR irradiated plasmid was shown to have significantly reduced amounts of damage in comparison to conventionally irradiated, where single strand breaks (SSBs) was used as the biological endpoint. This was the case for both hydroxyl scavenging capacities. A reduced electron energy within the VHEE range was also determined to increase the DNA damage to pBR322 plasmid. Results indicate that the pBR322 plasmid model can be successfully used to explore and test the effect of UHDR regimes on DNA damage. This is the first study to report FLASH sparing with VHEE, with induced damage to pBR322 plasmid DNA as the biological endpoint. UHDR irradiated plasmid had reduced amounts of DNA single-strand breaks (SSBs) in comparison with conventional dose rates. The magnitude of the FLASH sparing was a 27% reduction in SSB frequency in a 10 mM Tris environment and a 16% reduction in a 100 mM Tris environment., (© 2024. The Author(s).)

The antibacterial and cell-proliferative character of atmospheric pressure plasma jets (APPJs) helps in the healing process of chronic wounds. However, control of the plasma-biological target interface remains an open issue. High vacuum ultraviolet/ultraviolet (VUV/UV) radiation and RONS flux from plasma may cause damage of a treated tissue; therefore, controlled interaction is essential. VUV/UV emission from argon APPJs and radiation control with aerosol injection in plasma effluent is the focus of this research. The aerosol effect on radiation is studied by a fluorescent target capable of resolving the plasma oxidation footprint. In addition, DNA damage is evaluated by plasmid DNA radiation assay and cell proliferation assay to assess safety aspects of the plasma jet, the effect of VUV/UV radiation, and its control with aerosol injection. Inevitable emission of VUV/UV radiation from plasmas during treatment is demonstrated in this work. Plasma has no antiproliferative effect on fibroblasts in short treatments ( t < 60 s), while long exposure has a cytotoxic effect, resulting in decreased cell survival. Radiation has no effect on cell survival in the medium due to absorption. However, a strong cytotoxic effect on the attached fibroblasts without the medium is apparent. VUV/UV radiation contributes 70% of the integral plasma effect in induction of single- and double-strand DNA breaks and cytotoxicity of the attached cells without the medium. Survival of the attached cells increases by 10% when aerosol is introduced between plasma and the cells. Injection of aerosol in the plasma effluent can help to control the plasma-cell/tissue interaction. Aerosol droplets in the effluent partially absorb UV emission from the plasma, limiting photon flux in the direction of the biological target. Herein, cold and safe plasma-aerosol treatment and a safe operational mode of treatment are demonstrated in a murine model.

To ensure genome stability, bacteria have evolved a network of DNA repair mechanisms; among them, the UvrABC-dependent nucleotide excision repair (NER) pathway is essential for the incision of a variety of bulky adducts generated by exogenous chemicals, UV radiation and by-products of cellular metabolism. However, very little is known about the enzymatic properties of Mycobacterium tuberculosis UvrABC excinuclease complex. Furthermore, the biochemical properties of Escherichia coli UvrC (EcUvrC) are not well understood (compared to UvrA and UvrB), perhaps due to its limited availability and/or activity instability in vitro. In addition, homology modelling of M. tuberculosis UvrC (MtUvrC) revealed the presence of a putative ATP-binding pocket, although its function remains unknown. To elucidate the biochemical properties of UvrC, we constructed and purified wild-type MtUvrC and its eight variants harbouring mutations within the ATP-binding pocket. The data from DNA-binding studies suggest that MtUvrC exhibits high-affinity for duplex DNA containing a bubble or fluorescein-dT moiety, over fluorescein-adducted single-stranded DNA. Most notably, MtUvrC has an intrinsic UvrB-independent ATPase activity, which drives dual incision of the damaged DNA strand. In contrast, EcUvrC is devoid of ATPase activity; however, it retains the ability to bind ATP at levels comparable to that of MtUvrC. The ATPase-deficient variants map to residues lining the MtUvrC ATP-binding pocket. Further analysis of these variants revealed separation of function between ATPase and DNA-binding activities in MtUvrC. Altogether, these findings reveal functional diversity of the bacterial NER machinery and a paradigm for the evolution of a catalytic scaffold in UvrC., (© 2020 Federation of European Biochemical Societies.)

Ruthenium(II) complexes developed for photodynamic therapy (PDT) are almost exclusively tris-bidentate systems with C 2 or D 3 symmetry. This is due to the fact that this structural framework commonly produces long-lived excited states, which, in turn, allow for the generation of large amounts of singlet oxygen ( 1 O 2 ) and other reactive oxygen species. Complexes containing tridentate ligands would be advantageous for biological applications as they are generally achiral (D 2d or C 2v symmetry), which eliminates the possibility of multiple isomers which could exhibit potentially different interactions with chiral biological entities. However, Ru(II) complexes containing tridentate ligands are rarely studied as candidates for photobiological applications, such as PDT, since they almost exclusively exhibit low quantum yields and very short excited-state lifetimes and, thus, are not capable of generating sufficient 1 O 2 or engaging in electron transfer reactions. Here, we report a proof-of-concept approach to make bis-tridentate Ru(II) complexes useful for PDT applications by altering their photophysical properties through the inclusion of N-heterocyclic carbene (NHC) ligands. Three NHC and two terpyridine ligands were studied to evaluate the effects of structural and photophysical modulations of bis-substituted Ru(II) complexes. The NHC complexes were found to have superior excited-state lifetimes, 1 O 2 production, and photocytotoxicity. To the best of our knowledge, these complexes are the most potent light-activated bis-tridentate complexes reported.

Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1., Competing Interests: The authors declare no competing interest.

The damaging potential of ionizing radiation arises largely from the generation of clustered DNA damage sites within cells. Previous studies using synthetic DNA lesions have demonstrated that models of clustered DNA damage exhibit enhanced mutagenic potential of the comprising lesions. However, little is known regarding the processes that lead to mutations in these sites, apart from the fact that base excision repair of lesions within the cluster is compromised. Unique features of the mutation frequencies within bi-stranded clusters have led researchers to speculate that the strand containing the mutagenic lesion is preferentially used as the template for DNA synthesis. To gain further insights into the processing of clustered DNA damage sites, we used a plasmid-based assay in E. coli cells. Our findings revealed that the strand containing a mutagenic lesion within a bi-stranded clustered DNA damage site is frequently used as the template. This suggests the presence of an, as yet unknown, strand synthesis process that is unrelated to base excision repair, and that this process plays an important role in mutagenesis. The length of the region of strand preference was found to be determined by DNA polymerase I.

The exposure of naked unprotected skin to solar radiation may result in numerous acute and chronic undesirable effects. Evidence suggests that silymarin, a standardized extract from Silybum marianum (L.) Gaertn. seeds, and its major component silybin suppress UVB-induced skin damage. Here, we aimed to investigate the UVA-protective effects of silymarin's less abundant flavonolignans, specifically isosilybin (ISB), silychristin (SC), silydianin (SD), and 2,3-dehydrosilybin (DHSB). Normal human dermal fibroblasts (NHDF) pre-treated for 1 h with flavonolignans were then exposed to UVA light using a solar simulator. Their effects on reactive oxygen species (ROS), carbonylated proteins and glutathione (GSH) level, caspase-3 activity, single-strand breaks' (SSBs) formation and protein level of matrix metalloproteinase-1 (MMP-1), heme oxygenase-1 (HO-1), and heat shock protein (HSP70) were evaluated. The most pronounced preventative potential was found for DHSB, a minor component of silymarin, and SC, the second most abundant flavonolignan in silymarin. They had significant effects on most of the studied parameters. Meanwhile, a photoprotective effect of SC was mostly found at double the concentration of DHSB. ISB and SD protected against GSH depletion, the generation of ROS, carbonylated proteins and SSBs, and caspase-3 activation, but had no significant effect on MMP-1, HO-1, or HSP70. In summary, DHSB and to a lesser extent other silymarin flavonolignans are potent UVA-protective compounds. However, due to the in vitro phototoxic potential of DHSB published elsewhere, further studies are needed to exclude phototoxicity for humans as well as to confirm our results on human skin ex vivo and in vivo.

The advancement of multidisciplinary research fields dealing with ionising radiation induced biological damage - radiobiology, radiation physics, radiation protection and, in particular, medical physics - requires a clear mechanistic understanding of how cellular damage is induced by ionising radiation. Monte Carlo (MC) simulations provide a promising approach for the mechanistic simulation of radiation transport and radiation chemistry, towards the in silico simulation of early biological damage. We have recently developed a fully integrated MC simulation that calculates early single strand breaks (SSBs) and double strand breaks (DSBs) in a fractal chromatin based human cell nucleus model. The results of this simulation are almost equivalent to past MC simulations when considering direct/indirect strand break fraction, DSB yields and fragment distribution. The simulation results agree with experimental data on DSB yields within 13.6% on average and fragment distributions agree within an average of 34.8%., (Copyright © 2019 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.)

This study proposes an innovative approach to estimate relative biological effectiveness (RBE) of fast neutrons using the Geant4 toolkit. The Geant4-DNA version cannot track heavy ions below 0.5 MeV/nucleon. In order to explore the impact of this issue, secondary particles are simulated instead of the primary low-energy neutrons. The Evaluated Nuclear Data File library is used to determine the cross sections for the elastic and inelastic interactions of neutrons with water and to find the contribution of each secondary particle spectrum. Two strategies are investigated in order to find the best possible approach and results. The first one takes into account only light particles, protons produced from elastic scattering, and α particles from inelastic scattering. Geantino particles are shot instead of heavy ions; hence all heavy ions are considered in the simulations, though their physical effects on DNA not. The second strategy takes into account all the heavy and light ions, although heavy ions cannot be tracked down to very low energies (E<0.5 MeV/nucleon). Our model is based on the combination of an atomic resolution DNA geometrical model and a Monte Carlo simulation toolkit for tracking particles. The atomic coordinates of the DNA double helix are extracted from the Protein Data Bank. Since secondary particle spectra are used instead of simulating the interaction of neutrons explicitly, this method reduces the computation times dramatically. Double-strand break induction is used as the end point for the estimation of the RBE of fast neutrons. ^{60}Co γ rays are used as the reference radiation quality. Both strategies succeed in reproducing the behavior of the RBE&#95;{max} as a function of the incident neutron energy ranging from 0.1 to 14 MeV, including the position of its peak. A comparison of the behavior of the two strategies shows that for neutrons with energies less than 0.7 MeV, the effect of heavy ions would not be very significant, but above 0.7 MeV, heavy ions have an important role in neutron RBE.

Plasmid DNA in aerated aqueous solution is used as a probe to determine whose of the reactive oxygen species (ROS) generated after absorption of ultra-soft X-rays (USX) take part in biomolecule damage in the presence and in absence of Gold Nano-Particles (GNP) and specific scavengers. Citrate-coated GNPs with core sizes of 6, 10 and 25 nm are synthetized and characterized, especially in terms of plasmon band shift, ζ-potential and hydrodynamic radii (respectively 9, 21 and 30 nm). We confirm the radiosensitizing effect of GNP and show that the SSB number per plasmid increases when, for a same mass of gold element, the core size of the gold nanoparticles decreases. Hydroxyl radicals (OH) are scavenged using the positively-charged 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) and the neutral dimethyl sulfoxide (DMSO) molecules. Due to both negatively-charged environments of DNA and GNP, at identical scavenging capacity, TRIS is more effective at quenching OH than DMSO. The strong radiosensitizing effect of hydroxyl radicals is confirmed. Methanoate anions are then used to transform OH into hydrogen peroxide; the latter being known to be non-aggressive regarding DNA in the absence of easily oxidable metallic ions (Fenton reactions). Surprisingly, in the presence of GNP, high DNA damage yields are observed even though hydrogen peroxide might not be hold as responsible. Conversely, the radiosensitizing effect of GNP is not observed anymore when H 2 O 2 is scavenged using pyruvate ions. We demonstrate that hydrogen peroxide constitutes quite unexpectedly a hidden stock of OH which are activated at the surface of the GNP by decomposition of H 2 O 2 molecules., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The epidermis undergoes constant renewal during its lifetime. This is possible due to a special population of keratinocyte stem cells (KSCs) located at the basal layer. These cells are surrounded by their direct progeny, keratinocyte progenitors or transient amplifying cells (TAs), which arise from cell division. Skin is exposed every day to sun radiation; in particular, UVA radiation penetrates through the epidermis and induces damage to KSCs and TAs. Although keratinocytes in the basal layer are the most likely skin carcinomas and/or photoaging cells of origin, surprisingly few studies have addressed the specific responses of these cells to UV radiation. In this study, we showed for the first time that keratinocyte stem cells were more resistant to UVA irradiation than their direct progeny, transient amplifying cells. Using both the MTT assay and clonogenic assay, we found that KSCs were more photo-resistant compared to TAs after exposure to different doses of UVA (from 0 to 50 J/cm2). Moreover, KSCs had a greater ability to reconstruct human epidermis (RHE) after UVA exposure compared with TAs. Finally, investigations of DNA repair using the comet assay showed that DNA single-strand breaks and thymine dimers were repaired quicker and more efficiently in KSCs compared with TAs. In a previous work, we showed that the same stem cell population was more resistant to ionizing radiation, another carcinogenic agent. Collectively, our results combined with other observations demonstrate that keratinocyte stem cells, which are responsible for epidermal renewal throughout life, are equipped with an efficient arsenal against several genotoxic agents. Our future work will try to identify the factors or signaling pathways that are responsible for this differential photo-sensitivity and DNA repair capacity between KSCs and TAs., Competing Interests: E.M, N.B and F.D. are employees of Gattefosse. O.D. is hospital practitioner at Civil Hospitals of Lyon. W.R. is Prof. at University Grenoble Alpes. The financial support from Gattefosse does not alter the authors’ adherence to all PLOS ONE policies on sharing data and materials and there are no restrictions on sharing of data and/or materials.

The aim of the present study was to examine the genoprotective and radioprotective effects of black tea extract (BTE) against the induction of single strand DNA breaks in human lymphocytes subjected to hydrogen peroxide (H2O2) or gamma-rays (2 Gy dose). Lymphocytes were incubated with or without different concentrations of BTE (0.005-500 µg/ml) for 30 min, followed by treatment with or without H2O2 (0.088 µmol/l) for 5 min. To examine the radioprotective effect of BTE, the lymphocytes were incubated with or without BTE for 30 and 60 min prior to and following in vitro irradiation. Oxidative damage to DNA was monitored using a comet assay. BTE at lower concentrations prevented H2O2-induced DNA damage. An increase in BTE concentrations resulted in increased formation of single strand DNA breaks. BTE also exerted significant protective effects against gamma radiation-induced total DNA damage in healthy lymphocytes during their 30 or 60 min incubation with BTE prior to or following irradiation. Therefore, the protective effect of BTE against irradiation was time-dependent. The results contribute to the research on potential beneficial effects of natural compounds, such as BTE, in cancer and its protective effects of normal tissue during radiation therapy.

Microscopy and micro-irradiation imaging techniques have significantly advanced our knowledge of DNA damage tolerance and the assembly of DNA repair proteins at the sites of damage. While these tools have been extensively applied to the study of nucleotide excision repair and double-strand break repair, their application to the repair of oxidatively-induced base lesions and single-strand breaks is just beginning to yield new insights. This review will focus on examining micro-irradiation techniques reported to create base lesions and single-strand breaks; these lesions are considered to be primarily addressed by proteins involved in the base excision repair (BER) pathway. By examining conditions for generating these DNA lesions and reviewing information on the assembly and dissociation of repair complexes at the induced lesion sites, we hope to promote further investigations into BER and to stimulate further development and enhancement of these techniques for the study of BER., (Published by Elsevier B.V.)

Ionizing radiation induces a variety of DNA damages including single-strand breaks (SSBs), double-strand breaks (DSBs), abasic sites, modified sugars, and bases. Most theoretical and experimental studies have been focused on DNA strand scissions, in particular production of DNA double-strand breaks. DSBs have been proven to be a key damage at a molecular level responsible for the formation of chromosomal aberrations, leading often to cell death. We have studied the nature of DNA damage induced directly by the pulsed 46.9-nm (26.5 eV) radiation provided by an extreme ultraviolet (XUV) capillary-discharge Ne-like Ar laser (CDL). Doses up to 45 kGy were delivered with a repetition rate of 3 Hz. We studied the dependence of the yield of SSBs and DSBs of a simple model of DNA molecule (pBR322) on the CDL pulse fluence. Agarose gel electrophoresis method was used for determination of both SSB and DSB yields. The action cross sections of the single- and double-strand breaks of pBR322 plasmid DNA in solid state were determined. We observed an increase in the efficiency of strand-break induction in the supercoiled DNA as a function of laser pulse fluence. Results are compared to those acquired at synchrotron radiation facilities and other sources of extreme-ultraviolet and soft x-ray radiation.

An increase in the levels of oxidation is a universal feature of genomic DNA of irradiated or aged or even malignant cells. In case of apoptotic death of stressed cells, oxidized DNA can be released in circulation (cfDNA). According to the results of the studies performed in vitro by our group and other researchers, the oxidized cfDNA serves as a biomarker for a stress and a stress signal that is transmitted from the "stressed" area i.e. irradiated cells or cells with deficient anti-oxidant defenses to distant (bystander) cells. In recipient cells, oxidized DNA stimulates biosynthesis of ROS that is followed up by an increase in the number of single strand and double strand breaks (SSBs and DSBs), and activation of DNA Damage Response (DDR) pathway. Effects of oxidized DNA are considered similar to that of irradiation. It seems that downstream effects of irradiation, in part, depend on the release of oxidized DNA fragments that mediate the effects in distant cells. The responses of normal and tumor cell to oxidized DNA may differ. It seems that tumor cells are more sensitive to oxidized DNA-dependent DNA damage, while developing pronounced adaptive response. This may suggest that in chemotherapy or irradiation-treated human body, the release of oxidized DNA from dying cancer cells may give a boost to remaining malignant cells by augmenting their survival and stress resistance. Further studies of the effects of oxidized DNA in both in vitro and in vivo systems are warranted., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in biological matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (-)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap(2+)). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to <10% using previous methods, which is sufficient for the subsequent determination of their functionality. Upon LEE irradiation, the fraction of functional plasmids decreased exponentially with increasing electron fluence, while LEE-induced isolated base damage, frank DSB, and non DSB-cluster damage increased linearly with fluence. While DSBs can be toxic, their levels were too low to explain the loss of plasmid functionality observed upon LEE irradiation. Similarly, non-DSB cluster damage, revealed by transforming cluster damage into DSBs by digestion with repair enzymes, also occurred relatively infrequently. The exact nature of the lethal damage remains unknown, but it is probably a form of compact cluster damage in which the lesions are too close to be revealed by purified repair enzymes. In addition, this damage is either not repaired or is misrepaired by E. coli, since it results in plasmid inactivation, when they contain an average of three lesions. Comparison with previous results from a similar experiment performed with γ-irradiated plasmids indicates that the type of clustered DNA lesions, created directly on cellular DNA by LEEs, may be more difficult to repair than those produced by other species from radiolysis.

Clustered DNA damages are induced by ionizing radiation, particularly of high linear energy transfer (LET). Compared to isolated DNA damage sites, their biological effects can be more severe. We investigated a clustered DNA damage induced by high LET radiation (C 290 MeV u(-1) and Fe 500 MeV u(-1)) in pBR322 plasmid DNA. The plasmid is dissolved in pure water or in aqueous solution of one of the three scavengers (coumarin-3-carboxylic acid, dimethylsulfoxide, and glycylglycine). The yield of double strand breaks (DSB) induced in the DNA plasmid-scavenger system by heavy ion radiation was found to decrease with increasing scavenging capacity due to reaction with hydroxyl radical, linearly with high correlation coefficients. The yield of non-DSB clusters was found to occur twice as much as the DSB. Their decrease with increasing scavenging capacity had lower linear correlation coefficients. This indicates that the yield of non-DSB clusters depends on more factors, which are likely connected to the chemical properties of individual scavengers.

Background: It has been hypothesized in the literature that exposure to extremely low frequency electromagnetic fields (50 or 60 Hz) may lead to human health effects such as childhood leukemia or brain tumors. In a previous study investigating multiple types of cells from brain and kidney of the mouse (Acta Neuropathologica 2004; 107: 257-264), we found increased unrepaired nuclear DNA single strand breaks (nDNA SSB) only in epithelial cells of the choroid plexus in the brain using autoradiographic methods after a continuous eight-week 50 Hz magnetic field (MF) exposure of adult mice with flux density of 1.5 mT., Methods: In the present study we tested the hypothesis that MF exposure with lower flux densities (0.1 mT, i.e., the actual exposure limit for the population in most European countries, and 1.0 mT) shows similar results to those in the previous study. Experiments and data analysis were carried out in a similar way as in our previous study., Results: Continuous eight-week 50 Hz MF exposure with 0.1 mT or 1.0 mT did not result in increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice. MF exposure with 1.0 mT led to reduced unscheduled DNA synthesis (UDS) in epithelial cells in the choroid plexus of the fourth ventricle in the brain (EC-CP) and epithelial cells of the cortical collecting duct in the kidney, as well as to reduced mtDNA synthesis in neurons of the caudate nucleus in the brain and in EC-CP., Conclusion: No evidence was found for increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice after continuous eight-week 50 Hz magnetic field exposure with flux density of 0.1 mT or 1.0 mT.

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/²¹²Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/²¹²Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that ²¹²Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.

The potency of UVA radiation, representing 90% of solar UV light reaching the earth's surface, to induce human skin cancer is the subject of continuing controversy. This study was undertaken to investigate the role of reactive oxygen species in DNA damage produced by the exposure of human cells to UVA radiation. This knowledge is important for better understanding of UV-induced carcinogenesis. We measured DNA single-strand breaks and alkali-labile sites in human lymphocytes exposed ex vivo to various doses of 365-nm UV photons compared to X-rays and hydrogen peroxide using the comet assay. We demonstrated that the UVA-induced DNA damage increased in a linear dose-dependent manner. The rate of DNA single-strand breaks and alkali-labile sites after exposure to 1J/cm(2) was similar to the rate induced by exposure to 1 Gy of X-rays or 25 μM hydrogen peroxide. The presence of either the hydroxyl radical scavenger dimethyl sulfoxide or the singlet oxygen quencher sodium azide resulted in a significant reduction in the UVA-induced DNA damage, suggesting a role for these reactive oxygen species in mediating UVA-induced DNA single-strand breaks and alkali-labile sites. We also showed that chromatin relaxation due to hypertonic conditions resulted in increased damage in both untreated and UVA-treated cells. The effect was the most significant in the presence of 0.5M Na(+), implying a role for histone H1. Our data suggest that the majority of DNA single-strand breaks and alkali-labile sites after exposure of human lymphocytes to UVA are produced by reactive oxygen species (the hydroxyl radical and singlet oxygen) and that the state of chromatin may substantially contribute to the outcome of such exposures., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H2AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H2O2 and converted H2O2-induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation.

Purpose: The present study introduces a new method to establish a direct correlation between biologically related physical parameters (i.e., stopping and damaging cross sections, respectively) for an Auger-electron emitting radionuclide decaying within a target molecule (e.g., DNA), so as to evaluate the efficacy of the radionuclide at the molecular level. These parameters can be applied to the dosimetry of Auger electrons and the quantification of their biological effects, which are the main criteria to assess the therapeutic efficacy of Auger-electron emitting radionuclides., Methods: Absorbed dose and stopping cross section for the Auger electrons of 5-18 eV emitted by(125)I within DNA were determined by developing a nanodosimetric model. The molecular damages induced by these Auger electrons were investigated by measuring damaging cross section, including that for the formation of DNA single- and double-strand breaks. Nanoscale films of pure plasmid DNA were prepared via the freeze-drying technique and subsequently irradiated with low-energy electrons at various fluences. The damaging cross sections were determined by employing a molecular survival model to the measured exposure-response curves for induction of DNA strand breaks., Results: For a single decay of(125)I within DNA, the Auger electrons of 5-18 eV deposit the energies of 12.1 and 9.1 eV within a 4.2-nm(3) volume of a hydrated or dry DNA, which results in the absorbed doses of 270 and 210 kGy, respectively. DNA bases have a major contribution to the deposited energies. Ten-electronvolt and high linear energy transfer 100-eV electrons have a similar cross section for the formation of DNA double-strand break, while 100-eV electrons are twice as efficient as 10 eV in the induction of single-strand break., Conclusions: Ultra-low-energy electrons (<18 eV) substantially contribute to the absorbed dose and to the molecular damage from Auger-electron emitting radionuclides; hence, they should be considered in the dosimetry calculation of such radionuclides. Moreover, absorbed dose is not an appropriate physical parameter for nanodosimetry. Instead, stopping cross section, which describes the probability of energy deposition in a target molecule can be an appropriate nanodosimetric parameter. The stopping cross section is correlated with a damaging cross section (e.g., cross section for the double-strand break formation) to quantify the number of each specific lesion in a target molecule for each nuclear decay of a single Auger-electron emitting radionuclide.

Luminal A type breast cancer was suitable for Photodynamic therapy (PDT) as its strong adhesion ability, low malignancy and easily being exposed to laser. To examine the novel photosensitizer agent 5-5-(4-N, N-diacetoxylphenyl-10, 15, 20-tetraphenylporphyrin)(DTPP) mediate PDT in breast cancer cell, Luminal A type breast cancer MCF-7 cells were used in this study, various concentrations of DTPP (0, 2, 4, 6, 8, 10, 12, 15, 20, 25, 30 μg/mL) and different time intervals (0, 0.5, 1, 2, 4, 6, 8 min) of laser exposure at 650 nm wavelength (power of 20 mW) were tested in PDT. The survival rates of MCF-7 cells were measured using a sensitive cell proliferation assay (MTT) to establish optimal semilethal dose and optimal time exposure, a further study of effects on cytoskeleton and apoptosis were also performed. Cell cycle and apoptosis variation were assayed by flow cytometry. Microtubule, microfilament, and nuclei were observed using laser scanning confocal microscopy. Oncoproteins Bcl-2, beta-tubulin, and beta-catenin were detected by means of electrophoresis. The novel DTPP showed an efficient growth inhibition of MCF-7 during PDT, effective combinations in MCF-7 cells were shown to be 4 μg mL(-1) PS irradiated for 8 min at least or 15 μg mL(-1) irradiated for 2 min at least. Microtubule, microfilament, and nucleus staining demonstrated that cytoskeletal collapse occurs at 0.5 h after PDT. Bcl-2 and skeleton adhesion proteins beta-catenin were reduced in the level of expression; whereas, skeleton proteins beta-tubulin and actin maintained similar levels of expression 12 h after PDT. These results provided a better understanding of DTPP-PDT in MCF-7 cells.

Astrocytes, the most common cell type in the brain, play a principal role in the repair of damaged brain tissues during external radiotherapy of brain tumours. As a downstream gene of p53, the effects of Krüppel-like factor 4 (KLF4) in response to X-ray-induced DNA damage in astrocytes are unclear. In the present study, KLF4 expression was upregulated after the exposure of astrocytes isolated from the murine brain. Inhibition of KLF4 expression using lentiviral transduction produced less double-strand DNA breaks (DSB) determined by a neutral comet assay and flow cytometric analysis of phosphorylated histone family 2A variant and more single-strand DNA breaks (SSB) determined by a basic comet assay when the astrocytes were exposed to 4 Gy of X-ray radiation. These data suggest that radiation exposure of the tissues around brain tumour during radiation therapy causes KLF4 overexpression in astrocytes, which induces more DSB and reduces SSB. This causes the adverse effects of radiation therapy in the treatment of brain tumours., (Copyright © 2013 John Wiley & Sons, Ltd.)

It is shown that exposure of 365 nm UV radiation at doses of 10, 20 and 50 kJ/m2 induces a dose-dependent increase in DNA single-strand breaks and alkali-labile sites (SSB and ALS) detected by comet and halo assays in human blood lymphocyte nucleoids. Adding 10% dimethyl sulfoxide (DMSO) reduces the SSB and ALS yields--in 3 times. A strong drop in the output of UV-A-induced SSB and ALS in lymphocyte nucleoids in the presence of DMSO shows the leading role of *OH radicals in this DNA damage formation under exposure to 365-nm UV-radiation.

The mechanism responsible for the remarkable photomutagenicity of fluoroquinolone (FQ) antibiotics remains unknown. For this reason, it was considered worthwhile to study in detail the interactions between DNA and a dihalogenated FQ such as lomefloxacin (LFX; one of the most photomutagenic FQs) and its N-acetyl derivative ALFX. Studies of photosensitized DNA damage by (A)LFX, such as formation of DNA single-strand breaks (SSBs), together with pulse radiolysis, laser flash photolysis, and absorption and fluorescence measurements, have shown the important effects of the cationic character of the piperazinyl ring on the affinity of this type of drug for DNA. Hence, the formation of SSBs was detected for LFX, whereas ALFX and ciprofloxacin (a monofluorated FQ) needed a considerably larger dose of light to produce some damage. In this context, it was determined that the association constant (Ka) for the binding of LFX to DNA is ca. 2×10(3)M(-1), whereas in the case of ALFX it is only ca. 0.5×10(3)M(-1). This important difference is attributed to an association between the cationic peripheral ring of LFX and the phosphate moieties of DNA and justifies the DNA SSB results. The analysis of the transient species detected and the photomixtures has allowed us to establish the intermolecular processes involved in the photolysis of FQ in the presence of DNA and 2'-deoxyguanosine (dGuo). Interestingly, although a covalent binding of the dihalogenated FQ to dGuo occurs, the photodegradation of FQ…DNA complexes did not reveal any significant covalent attachment. Another remarkable outcome of this study was that (A)LFX radical anions, intermediates required for the onset of DNA damage, were detected by pulse radiolysis but not by laser flash photolysis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ultraviolet (UV)-induced DNA damage are removed by nucleotide excision repair (NER) or can be tolerated by specialized translesion synthesis (TLS) polymerases, such as Polη. TLS may act at stalled replication forks or through an S-phase independent gap-filling mechanism. After UVC irradiation, Polη-deficient (XP-V) human cells were arrested in early S-phase and exhibited both single-strand DNA (ssDNA) and prolonged replication fork stalling, as detected by DNA fiber assay. In contrast, NER deficiency in XP-C cells caused no apparent defect in S-phase progression despite the accumulation of ssDNA and a G2-phase arrest. These data indicate that while Polη is essential for DNA synthesis at ongoing damaged replication forks, NER deficiency might unmask the involvement of tolerance pathway through a gap-filling mechanism. ATR knock down by siRNA or caffeine addition provoked increased cell death in both XP-V and XP-C cells exposed to low-dose of UVC, underscoring the involvement of ATR/Chk1 pathway in both DNA damage tolerance mechanisms. We generated a unique human cell line deficient in XPC and Polη proteins, which exhibited both S- and G2-phase arrest after UVC irradiation, consistent with both single deficiencies. In these XP-C/Polη(KD) cells, UVC-induced replicative intermediates may collapse into double-strand breaks, leading to cell death. In conclusion, both TLS at stalled replication forks and gap-filling are active mechanisms for the tolerance of UVC-induced DNA damage in human cells and the preference for one or another pathway depends on the cellular genotype., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Purpose: This study focuses on the effects of high-field (3T) magnetic resonance imaging (MRI) scans on the DNA integrity of human leukocytes in vitro in order to validate the study where genotoxic effects were obtained and published by Lee et al., Materials and Methods: The scanning protocol and exposure situation were the same as those used under routine clinical brain MRI scan. Peripheral blood samples from healthy non-smoking male donors were exposed to electromagnetic fields (EMF) produced by 3T magnetic resonance imaging equipment for 0, 22, 45, 67, and 89 min during the scanning procedure. Samples of positive control were exposed to ionizing radiation (4 Gy of (60)Co-γ). Single breaks of DNA in leukocytes were detected by single-cell gel electrophoresis (Comet assay). Chromosome breakage, chromosome loss and micronuclei formations were detected by a micronucleus test (MN). Three independent experiments were performed., Results: The data of comet tail DNA%, olive tail moment and micronucleus frequency showed no DNA damages due to MRI exposure., Conclusions: The results of the Comet assay and the micronucleus test indicate that the applied exposure of MRI does not appear to produce breaks in the DNA and has no significant effect on DNA integrity.

The purpose of the present research is to examine whether human hair root cells can be used for dose assessment after in vitro exposure to ionizing radiation. Hair root samples plucked from random head regions were collected from 5 healthy human subjects. Some of these hair samples were used as control and some were irradiated with 0.5-5Gy of gamma ray using a Cs-137 gamma irradiator at a dose rate of 0.14Gy/s. DNA damage (single-strand breaks) was determined in hair root cells of these samples using the comet assay technique. The comet assay parameters, tail length (TL) and tail moment (TM), showed a significant increase (p<.05) in single-strand DNA breaks in hair roots cells of the exposed samples compared to control. A linear dose-effect relationship was observed when tail moment or tail length was plotted against the log of the radiation dose. This research suggests a possible use of human hair root cell DNA damage as a biomarker especially for low dose radiation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The DNA double strand break repair in plants is preferentially by non homologous end joining (NHEJ) pathway. A key protein of NHEJ pathway is Ku70. We have identified Ku70 homolog (VvKu70) from grapevine genome database. In this report we characterize a Ku70 homologue from Vitis vinifera cv. Mango. The VvKu70 expression was found to increase strongly in response to gamma radiation. The transcript level of VvKu70 was found to increase up to 36 h in gamma irradiated shoots of grapevine. The expression of VvKu70 was found in many organs like stem, leaves and roots. A GFP fused VvKu70 protein was found to be nuclear localized which indicates that the VvKu70 is a nuclear localized protein. The VvKu70 identified by in silico approaches is present as a single copy number in V. vinifera cv. Mango genome. The VvKu70-GFP fused protein possesses ATPase activity and fails to bind dsDNA but binds ssDNA.

Repair of DNA bulky lesions often involves multiple repair pathways such as nucleotide-excision repair, translesion DNA synthesis (TLS), and homologous recombination (HR). Although there is considerable information about individual pathways, little is known about the complex interactions or extent to which damage in single strands, such as the damage generated by UV, can result in double-strand breaks (DSBs) and/or generate HR. We investigated the consequences of UV-induced lesions in nonreplicating G2 cells of budding yeast. In contrast to WT cells, there was a dramatic increase in ssDNA gaps for cells deficient in the TLS polymerases η (Rad30) and ζ (Rev3). Surprisingly, repair in TLS-deficient G2 cells required HR repair genes RAD51 and RAD52, directly revealing a redundancy of TLS and HR functions in repair of ssDNAs. Using a physical assay that detects recombination between circular sister chromatids within a few hours after UV, we show an approximate three-fold increase in recombinants in the TLS mutants over that in WT cells. The recombination, which required RAD51 and RAD52, does not appear to be caused by DSBs, because a dose of ionizing radiation producing 20 times more DSBs was much less efficient than UV in producing recombinants. Thus, in addition to revealing TLS and HR functional redundancy, we establish that UV-induced recombination in TLS mutants is not attributable to DSBs. These findings suggest that ssDNA that might originate during the repair of closely opposed lesions or of ssDNA-containing lesions or from uncoupled replication may drive recombination directly in various species, including humans.

Effect of quantum mechanical tunneling on single strand breaks induced by low energy electron (LEE) has been investigated in a modeled gas phase system, 2'-deoxycytidine-3'-monophosphate (3'-dCMPH). The potential energy curves for the sugar-phosphate C-O (3' C-O) bond cleavage have been generated using second order Møller-Plesset perturbation theory at the 6-31+G(d) accuracy level. Results from the electronic structure theory calculations in conjunction with our time dependent calculations for the 3' C-O bond rupture in 3'-dCMPH using local complex potential based time dependent wave packet approach show significant quantum tunneling of the 3' C-O bond from the bound vibrational states above 1 eV of the anionic potential energy curve. A comparison of the fragmentation profile with that of our earlier gas phase investigations based on Hartree-Fock and density functional theory--Becke, 3-parameter, Lee-Yang-Parr methods with 6-31+G(d) basis set is also provided. Further, inspection of the singly occupied molecular orbitals generated at different 3' C-O bond lengths clearly indicates the electron transfer from the low lying base-π(∗) shape resonance state to the phosphate P = O π(∗) orbital of the DNA backbone during the strand breaks. The decisive step during LEE induced strand breaks follows via "charge induced dissociation" (CID) for the metastable anion formed below 1 eV, whereas quantum mechanical tunnel-ing is out-weighted the CID mechanism for the LEE above 1 eV.

Using a modified DNA-halo method single-strand breaks and DNA alkaline-labile site induction were stud- ied in human peripheral blood lymphocytes after a short-term (up to 10 min) exposure in vitro to X-rays, hy- drogen peroxide and long-wave ultraviolet light (365 ± 10 nm). It was shown that the dose-effect dependence in thee X-ray dose range of 0.3-2 Gy approximates by a linear function of y = 0.25 + 0.42x (R2 = 0.98), where y is a DNA-halo index in standardized units, x--a radiation dose in Gy. The effect of "saturation" was ob- served in the range of 2-5 Gy. Under exposure to hydrogen peroxide up to a concentration of 25 μmol/L, the dose-effect is described by a linear function y = 0.23 + 0.033x (R2 = 0.96), where y is the DNA-halo index in standardized units, x--hydrogen peroxide concentration in μmol/L. UV exposure induced a linear in- crease of the DNA-halo index in the dose range of 2-10 kJ/m2 (y = 0.26 + 0.032x (R2 = 0.99), where y is theDNA-halo index in standardized units, x--a radiation dose in kJ/m2). In summary, the described modi- fication of the DNA-halo method provides a simple, sensitive, well reproducible and rapid assay for the anal- ysis of DNA single-strand breaks and alkaline-labile sites in living cells.

Radiation-induced DNA damage initiates a series of overlapping responses that include DNA damage recognition and repair, induction of cell cycle checkpoints, senescence and/or apoptosis. This study assessed the DNA damage response and whole genome expression profile in two mammalian cell lines (HEK and U87) in response to (5-{4-methylpiperazin-1-yl}-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl] benzimidazole) DMA and ionizing radiation. DMA has been shown to act as a potent radiation protector, yielding significant levels of protection, i.e., 20.9% in HEK cells and 21.2% in U87 cells. Our findings revealed treatment with DMA significantly reduced γ-H2AX, 53BP1 and Rad51 foci formation after irradiation. MAP kinase, WNT signaling and p53 pathways were found to be activated in DMA-treated cells. In addition, the DNA damage response genes, HSP70, HSPD1, PRDX1, PRX, CALR, NPM, UBC, and SET showed differential regulation in DMA, DMA + radiation and radiation-treated cells. The data suggest that DMA-influenced repertoire of repair proteins, which are an indispensable part of the cell, interplay with each other to reduce DNA damage and maintain the genomic integrity of the cell.

In response to UV light, single-stranded DNA intermediates coated with replication protein A (RPA) are generated, which trigger the ATR-Chk1 checkpoint pathway. Recruitment and/or activation of several checkpoint proteins at the damaged sites is important for the subsequent cell cycle arrest. Surprisingly, upon UV irradiation, Rad9 and RPA only minimally accumulate at DNA lesions in G2 phase, suggesting that only a few single-stranded DNA intermediates are generated. Also, little phosphorylated Chk1 is observed in G2 phase after UV-irradiation, and UV light fails to elicit efficient accumulation of typical DNA damage response proteins at sites of damage in this phase. By contrast, p38 MAPK is phosphorylated in G2 phase cells after UV damage. Interestingly, despite the lack of an obvious activation of the ATR-Chk1 pathway, only the combined inhibition of the ATR- and p38-dependent pathways results in a complete abrogation of the UV-induced G2/M arrest. This suggests that UV light induces less hazardous lesions in G2 phase or that lesions created in this phase are less efficiently processed, resulting in a low activation of the ATR-Chk1 pathway. UV-induced G2 checkpoint activation in this situation therefore relies on signalling via the p38 MAPK and ATR-Chk1 signalling cascades.

This study aimed to analyze the phototoxic mechanism and photostability of quinine in human skin cell line A375 under ambient intensities of UVA (320-400 nm). Photosensitized quinine produced a photoproduct 6-methoxy-quinoline-4-ylmethyl-oxonium identified through LC-MS/MS. Generation of (1)O2, O2(•-), and (•)OH was measured and further substantiated through their respective quenchers. Photosensitized Quinine (Q) caused degradation of 2-deoxyguanosine, the most sensitive nucleotide to UV radiation. The intracellular ROS was increased in a concentration-dependent manner. Significant reduction in metabolic status measured in terms of cell viability (54%) at 25 μg mL(-1) was observed through MTT assay. Results of MTT assay accord NRU assay. Single strand DNA breaks and apoptosis were increased significantly (P < 0.01) as observed through comet assay and EB/AO double staining. Photosensitized quinine caused cells to arrest in G2 phase of cell cycle and induced apoptosis (5.08%) as revealed through FACS. Real-Time PCR showed upregulation of p21 (4.56 folds) and p53 (2.811 folds) genes expression. Thus, our study suggests that generation of reactive oxygen species by quinine under ambient intensity of UVA may result into deleterious phototoxic effects among human population., (© 2013 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2013 The American Society of Photobiology.)

We developed a colorimetric method to specifically detect single-strand DNA breaks using gold nanoparticles. In our assay, broken DNA cannot stabilize gold nanoparticles to prevent salt-induced aggregation as good as intact DNA can, and this effect can be easily observed with the naked eye as a red-to-purple color change.

Purpose: In this work the number of DNA single-strand breaks (SSB) and double-strand breaks (DSB) due to direct and indirect effects of Auger electrons from incorporated (123)I and (125)I have been calculated by using the Geant4-DNA toolkit. We have performed and compared the calculations for several cases: (125)I versus (123)I, source positions and direct versus indirect breaks to study the capability of the Geant4-DNA in calculations of DNA damage yields., Materials and Methods: Two different simple geometries of a 41 base pair of B-DNA have been simulated. The location of (123)I has been considered to be in (123)IdUrd and three different locations for (125)I., Results: The results showed that the simpler geometry is sufficient for direct break calculations while indirect damage yield is more sensitive to the helical shape of DNA. For (123)I Auger electrons, the average number of DSB due to the direct hits is almost twice the DSB due to the indirect hits. Furthermore, a comparison between the average number of SSB or DSB caused by Auger electrons of (125)I and (123)I in (125)IdUrd and (123)IdUrd shows that (125)I is 1.5 times more effective than (123)I per decay., Conclusions: The results are in reasonable agreement with previous experimental and theoretical results which shows the applicability of the Geant-DNA toolkit in nanodosimetry calculations which benefits from the open-source accessibility with the advantage that the DNA models used in this work enable us to save the computational time. Also, the results showed that the simpler geometry is suitable for direct break calculations, while for the indirect damage yield, the more precise model is preferred.

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ~170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20-30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair.

In cells exposed to ionizing radiation, double-strand breaks (DSBs) form within clustered damage sites from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that DSBs form promptly and are immediately detected and processed by the cellular DNA damage response apparatus. However, DSBs also form by delayed chemical conversion of thermally-labile sugar lesions (TLSL) to breaks. We recently reported that conversion of thermally-labile sugar lesions to breaks occurs in cells maintained at physiological temperatures. Here, we investigate the influence of radiation quality on the formation of thermally-labile sugar lesions dependent DSBs. We show that, although the yields of total DSBs are very similar after exposure to neutrons and X rays, the yields of thermally-labile sugar lesions dependent DSBs from neutrons are decreased in comparison to that from X rays. Thus, the yields of prompt DSBs for neutrons are greater than for X rays. Notably, after neutron irradiation the decreased yield of thermally-labile sugar lesion dependent DSBs is strongly cell line dependent, likely reflecting subtle differences in DNA organization. We propose that the higher ionization density of neutrons generates with higher probability prompt DSBs within ionization clusters and renders the ensuing chemical evolution of thermally-labile sugar lesions inconsequential to DNA integrity. Modification of thermally-labile sugar lesion evolution may define novel radiation protection strategies aiming at decreasing DSB formation by chemically preserving thermally-labile sugar lesions until other DSB contributing lesions within the clustered damage site are removed by non-DSB repair pathways.

Purpose: We studied the response of human embryonic stem cells (hESC) to the β-emitter (131)I, which affects the entire cell and to the Auger electron emitter (125)I-deoxyuridine ((125)I-dU), primarily affecting the deoxyribonuleic acid (DNA). The effects were also studied in keratinocytes as a prototype for somatic cells., Methods: HESC (H1) and human keratinocytes (HaCaT, human) were exposed to (125)I-dU (5 × 10(-5) - 5 MBq/ml) and (131)I-iodide (5 × 10(-5) - 12.5 MBq/ml) and apoptosis was measured by DNA-fragmentation. Cell morphology was studied by light microscopy and electron microscopy. Transcriptional profiling was done on the Agilent oligonucleotide microarray platform., Results: Auger-process induced no apoptosis but a strong transcriptional response in hESC. In contrast, HaCaT cells showed a pronounced induction of apoptosis but only a moderate transcriptional response. Transcriptional response of hESC was similar after (125)I-dU and (131)I treatments, whereas HaCaT cells expressed a much more pronounced response to (125)I-dU than to (131)I. A striking radiation-induced down-regulation of pluripotency genes was observed in hESC whereas in keratinocytes the enriched gene annotations were related primarily to apoptosis, cell division and proliferation., Conclusions: Human embryonic stem cells respond to ionizing radiation by (125)I-dU and (131)I in a different way compared to keratinocytes. Transcriptional response and gene expression appear to facilitate an escape from programmed cell death by striking a new path which probably leads to cell differentiation.