Plasma Damage Control: From Biomolecules to Cells and Skin.

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.

The intrinsic ATPase activity of Mycobacterium tuberculosis UvrC is crucial for its damage-specific DNA incision function.

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.

Radiation Damage in Biomolecules and Cells.

Ionizing radiation is widely used in medicine, both as a diagnostic tool and as a therapeutic agent [...].

Topoisomerase I-driven repair of UV-induced damage in NER-deficient cells.

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.

Bis-tridentate N-Heterocyclic Carbene Ru(II) Complexes are Promising New Agents for Photodynamic Therapy.

Ruthenium(II) complexes developed for photodynamic therapy (PDT) are almost exclusively tris-bidentate systems with C2 or D3 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 (1O2) and other reactive oxygen species. Complexes containing tridentate ligands would be advantageous for biological applications as they are generally achiral (D2d or C2v 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 1O2 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, 1O2 production, and photocytotoxicity. To the best of our knowledge, these complexes are the most potent light-activated bis-tridentate complexes reported.

Strand with mutagenic lesion is preferentially used as a template in the region of a bi-stranded clustered DNA damage site in Escherichia coli.

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.

Computational approach to determine the relative biological effectiveness of fast neutrons using the Geant4-DNA toolkit and a DNA atomic model from the Protein Data Bank.

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 Î³ rays are used as the reference radiation quality. Both strategies succeed in reproducing the behavior of the RBE_{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.

Evaluation of early radiation DNA damage in a fractal cell nucleus model using Geant4-DNA.

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%.

A pilot study of the UVA-photoprotective potential of dehydrosilybin, isosilybin, silychristin, and silydianin on human dermal fibroblasts.

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 contribution of hydrogen peroxide to the radiosensitizing effect of gold nanoparticles.

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 H2O2 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 H2O2 molecules.

Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny.

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.

Protective effects of black tea extract against oxidative DNA damage in human lymphocytes.

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.

Micro-irradiation tools to visualize base excision repair and single-strand break repair.

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.

Breaking DNA strands by extreme-ultraviolet laser pulses in vacuum.

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.

Oxidized extracellular DNA as a stress signal that may modify response to anticancer therapy.

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.

No evidence of persisting unrepaired nuclear DNA single strand breaks 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.

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.

Loss of cellular transformation efficiency induced by DNA irradiation with low-energy (10 eV) electrons.

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.

Impact of α-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel.

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.

Clustered DNA damage on subcellular level: effect of scavengers.

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.

Correlation between energy deposition and molecular damage from Auger electrons: A case study of ultra-low energy (5-18 eV) electron interactions with DNA.

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.