Induction of DNA strand breaks and DNA-protein cross-links in normal human skin fibroblasts following exposure to 254 nm UV radiation

Levels of DNA strand breaks and DNA-protein cross-links (DPCs) were measured using the alkaline elution assay in normal human skin fibroblasts irradiated with 0-200 J m-2 of 254 nm UV radiation and incubated for 0-24 h. On incubation, the yields of both single-strand breaks (SSBs) and DPCs increased with similar kinetics and remained elevated. In addition, when SSBs were measured under conditions in which DPCs were not eliminated by treatment with proteinase K, a measurable yield of SSBs could not be detected. Hence, the SSBs that form in the UV-irradiated cells following incubation appear to be associated with the DPCs.     

Single,double, and multiple double strand breaks induced in DNA by 3-100 eV electrons

Nonthermal secondary electrons with initial kinetic energies below 100 eV are an abundant transient species created in irradiated cells and thermalize within picoseconds through successive multiple energy loss events. Here we show that below 15 eV such low-energy electrons induce single (SSB) and double (DSB) strand breaks in plasmid DNA exclusively via formation and decay of molecular resonances involving DNA components (base, sugar, hydration water, etc.). Furthermore, the strand break quantum yields (per incident electron) due to resonances occur with intensities similar to those that appear between 25 and 100 eV electron energy, where nonresonant mechanisms related to excitation/ionizations/dissociations are shown to dominate the yields, although with some contribution from multiple scattering electron energy loss events. We also present the first measurements of the electron energy dependence of multiple double strand breaks (MDSB) induced in DNA by electrons with energies below 100 eV. Unlike the SSB and DSB yields, which remain relatively constant above 25 eV, the MDSB yields show a strong monotonic increase above 30 eV, however with intensities at least 1 order of magnitude smaller than the combined SSB and DSB yields. The observation of MDSB above 30 eV is attributed to strand break clusters (nano-tracks) involving multiple successive interactions of one single electron at sites that are distant in primary sequence along the DNA double strand, but are in close contact; such regions exist in supercoiled DNA (as well as cellular DNA) where the double helix crosses itself or is in close proximity to another part of the same DNA molecule.     

Induction of instability in water-in-oil-in-water double emulsions by freeze-thaw cycling

Individual water-in-oil-in-water (W1/O/W2) double-emulsion globules loaded with fluorescently labeled bovine serum albumin (FITC-BSA) were optically monitored within cylindrical capillaries during freeze-thaw cycling. Coalescence of internal aqueous droplets (W1) and external aqueous phase (W2), termed external coalescence, was not observed before or during freezing of the oil phase (O). On the other hand, this instability mechanism was readily promoted during thawing. This realization confirms the previously suggested potential of W1/O/W2 double emulsions to trigger release upon oil thawing and demonstrates that it is a direct result of globule breakage through external coalescence. The presented results also identified a threshold in the relative W1 droplet size above which instability occurred, while smaller droplets remained unperturbed and therefore indicate that optimization of the delivery can be achieved by tuning the size of W1 droplets. In addition, we propose a possible explanation for the occurrence of instability during oil thawing and its dependence on the size of W1 droplets. Because this alternative globule-breakage mechanism simply uses temperature increase (solid-to-liquid-phase transition) as external stimulus, W1/O/W2 double-emulsion delivery systems can be easily tailored by choosing an oil phase with the appropriate phase-transition temperature.     

Enhanced single strand breaks of supercoiled DNA in a matrix of gold nanotubes under X-ray irradiation

This work shows for the first time the potential of cobalt oxide silica (CoO(x)Si) membranes for desalination of brackish (1 wt.% NaCl), seawater (3.5 wt.% NaCl) and brine (7.5-15 wt.% NaCl) concentrations at feed temperatures between 25 and 75 °C. CoO(x)Si xerogels were synthesised via a sol-gel method including TEOS, cobalt nitrate hydrate and peroxide. Initial hydrothermal exposure (<2 days) of xerogels prepared with various pH (3-6) resulted in densification of the xerogel via condensation reactions within the silica matrix, with the xerogel synthesised at pH 5 the most resistant. Subsequent exposure was not found to significantly alter the pore structure of the xerogels, suggesting they were hydrostable and that the pore sizes remained at molecular sieving dimensions. Membranes were then synthesised using identical sol-gel conditions to the xerogel samples and testing showed that elevated feed temperatures resulted in increased water fluxes, whilst increasing the saline feed concentration resulted in decreased water fluxes. The maximum flux observed was 1.8 kg m(-2) h(-1) at 75 °C for a 1 wt.% NaCl feed concentration. The salt rejection was consistently in excess of 99%, independent of either the testing temperature or salt feed concentration.     

Induction of strand breaks in DNA films by low energy electrons and soft X-ray under nitrous oxide atmosphere

Five-monolayer (5 ML) plasmid DNA films deposited on glass and tantalum substrates were exposed to Al K_α X-rays of 1.5 keV under gaseous nitrous oxide (N₂O) at atmospheric pressure and temperature. Whereas the damage yields for DNA deposited on glass are due to soft X-rays, those arising from DNA on tantalum are due to both the interaction of low energy photoelectrons from the metal and X-rays. Then, the differences in the yields of damage on glass and tantalum substrates, essentially arises from interaction of essentially low-energy electrons (LEEs) with DNA molecules and the surrounding atmosphere. The G-values (i.e., the number of moles of product per Joule of energy absorbed) for DNA strand breaks induced by LEEs (G_LEE) and the lower limit of G-values for soft X-ray photons (G_XL) were calculated and the results compared to those from previous studies under atmospheric conditions and other ambient gases, such as N₂ and O₂. Under N₂O, the G-values for loss of supercoiled DNA are 103±15 nmol/J for X-rays, and 737±110 nmol/J for LEEs. Compared to corresponding values in an O₂ atmosphere, the effectiveness of X-rays to damage DNA in N₂O is less, but the G value for LEEs in N₂O is more than twice the corresponding value for an oxygenated environment. This result indicates a higher effectiveness for LEEs relative to N₂ and O₂ environments in causing SSB and DSB in an N₂O environment. Thus, the previously observed radiosensitization of cells by N₂O may not be only due to OH radicals but also to the reaction of LEE with N₂O molecules near DNA. The previous experiments with N₂ and O₂ and the present one demonstrate the possibility to investigate damage induced by LEEs to biomolecules under various types of surrounding atmospheres.     

Photoelectron imaging following 2 + 1 multiphoton excitation of HBr

The photodissociation and photoionization dynamics of HBr via low-n Rydberg and ion-pair states was studied by using 2 + 1 REMPI spectroscopy and velocity map imaging of photoelectrons. Two-photon excitation at about 9.4-10 eV was used to prepare rotationally selected excited states. Following absorption of the third photon the unperturbed F (1)Delta(2) and i (3)Delta(2) states ionize directly into the ground vibrational state of the molecular ion according to the Franck-Condon principle and upon preservation of the ion core. In case of the V (1)Sigma(+)(0(+)) ion-pair state and the perturbed E (1)Sigma(+)(0(+)), g (3)Sigma(-)(0(+)), and H (1)Sigma(+)(0(+)) Rydberg states the absorption of the third photon additionally results in a long vibrational progression of HBr(+) in the X (2)Pi state as well as formation of electronically excited atomic photofragments. The vibrational excitation of the molecular ion is explained by autoionization of repulsive superexcited states into the ground state of the molecular ion. In contrast to HCl, the perturbed Rydberg states of HBr show strong participation of the direct ionization process, with ionic core preservation.     

Single and double IR laser multiphoton decomposition of trifluoromethylsilane

Single and double IR laser multiphoton decomposition of trifluoromethylsilane have been studied at 0.1 and 0.3 mbar. The IRMPD products FSiH₃ and C₂H₄ form independently of the wavelength of the laser pulses both in the single and double laser experiments. The decomposition rate is however very sensitive to both the wavenumber of the irradiation and the time delay between the two laser pulses at different wavenumbers.     

Fluorometric analysis of DNA unwinding (FADU) as a method for detecting repair-induced DNA strand breaks in UV-irradiated mammalian cells

Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).     

The effects of secondary structure and O2 on the formation of direct strand breaks upon UV irradiation of 5-bromodeoxyuridine-containing oligonucleotides.

BACKGROUND: 5-Bromodeoxyuridine is a radiosensitizing agent that is currently being evaluated in clinical trials as an adjuvant in the treatment of a variety of cancers. gamma-Radiolysis and UV irradiation of oligonucleotides containing 5-bromodeoxyuridine result in the formation of direct strand breaks at the 5'-adjacent nucleotide by oxidation of the respective deoxyribose. We investigated the effects of DNA secondary structure and O2 on the induction of direct strand breaks in 5-bromodeoxyuridine-containing oligonucleotides. RESULTS: The efficiency of direct strand break formation in duplex DNA is dependent upon O2 and results in fragments containing 3'-phosphate and the labile 3'-ketodeoxyadenosine termini. The ratio of the 3'-termini is also dependent upon O2 and structure. Deuterium product isotope effects and tritium-transfer studies indicate that hydrogen-atom abstraction from the C1'- and C2'-positions occurs in an O2- and structure-dependent manner. CONCLUSIONS: The reaction mechanisms by which DNA containing 5-bromodeoxyuridine is sensitized to damage by UV irradiation are dependent upon whether the substrate is hybridized and upon the presence or absence of O2. Oxygen reduces the efficiency of direct strand break formation in duplex DNA, but does not affect the overall strand damage. It is proposed that the sigma radical abstracts hydrogen atoms from the C1'- and C2'-positions of the 5'-adjacent deoxyribose moiety, whereas the nucleobase peroxyl radical selectively abstracts the C1'-hydrogen atom from this site. This is the second example of DNA damage amplification by a nucleobase peroxyl radical, and might be indicative of a general reaction pattern for this family of reactive intermediates.     

Energy deposition mechanisms and biochemical aspects of DNA strand breaks by ionizing radiation

A theoretical model based on physical, chemical, and biochemical mechanisms has been presented to evaluate the yields of DNA strand breaks (single and double) as a function of linear energy transfer (LET ) or ?dE/dx. Energetic heavy charged particles are considered explicitly to provide a general theory for low- as well as for high-LET radiation. There are three main features of the calculation: (a) track structure considerations for the energy deposition pattern, (b) three-dimensional structure of DNA molecules to provide information on the exact location of damage, and (c) a Monte-Carlo scheme to simulate the diffusion processes of water radicals. To avoid the complexities of a cellular medium, an aqueous solution of DNA is considered in the calculation. When the results of the calculations are compared with experimental measurements of the yields of strand breaks in mammalian DNA (exposed in a cellular complex), reasonable agreement is obtained. However, only those experimental data have been compared where there were no enzyme repair processes. The method of calculation has also been extended to study breaks in higher-order structures of DNA molecules such as chromatin. Specific limitations of the present model have been pointed out for making further improvements.     

Induction of strand breaks by low-energy electrons (8-68 eV) in a self-assembled monolayer of oligonucleotides: effective cross sections and attenuation lengths

Self-assembled monolayers of 5'-32P-labeled 3'-thiolated oligonucleotides chemisorbed on gold were bombarded by low-energy electrons (LEE) of 8-68 eV. Shorter 5'-32P-oligonucleotides produced by LEE-induced strand breaks were separated with denaturing polyacrylamide gel electrophoresis and quantified by phosphor imaging. The yields of short oligonucleotides (y) decrease exponentially with their length (n), following the equation y=ae-bn, where a and b are constants, which are related to the average effective cross section per nucleotide for DNA strand break (sigmaeff) and the attenuation length (AL=1b) of LEE, respectively. The AL decreases with LEE energies from 2.5+/-0.6 nm at 8 eV to 0.8+/-0.1 nm at 68 eV, whereas sigmaeff increases from (3+/-1)x10(-18) to (5.1+/-1.6)x10(-17) cm2 within the same energy range. The energy dependence of sigmaeff shows a resonance peak of (2.8+/-0.9)x10(-17) cm2 at 18 eV superimposed on a monotonically rising curve. Transient electron attachment to a sigma* anion state of the deoxyribose group, followed by dipolar dissociation into H- and the corresponding positive-ion radical, leading to C-O bond cleavage, is proposed to account for this maximum.     

Detection of telomere damage as a result of strand breaks in telomeric and subtelomeric DNA

Telomeres are the tandem repetitive DNA sequences at both ends of a chromosome with a repeating unit of TTAGGG. The integrity of a telomere is crucial to chromosomal stability and cellular viability. Damages to telomere DNA disrupt telomere integrity and accelerate telomere shortening. We describe a method for the assessment of strand breaks in the telomere/subtelomere region in cultured cells. Cells were embedded in agarose plugs and subjected to lysis and alkaline treatment to relax the DNA double helix. The telomere fragments as the result of strand breaks in the telomere/subtelomere region were then separated from the genomic DNA by electrophoresis, blotted onto membranes, and detected by a probe specific to the telomere sequence. Because of the large content of the telomere in human cells and the fact that telomere DNA is much more prone to damage than the bulk genomic DNA, the analysis may serve as a good indication of general DNA damage as well.     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

Only complete rejoining of DNA strand breaks after UVC allows K562 cell proliferation and DMSO induction of erythropoiesis

DNA strand breaks are early intermediates of the repair of UVC-induced DNA damage, however, since they severely impair cellular activities, their presence should be limited in time. In this study, the effects of incomplete repair of UVC-induced DNA strand breaks are investigated on K562 cell growth and the induction of erythroid differentiation by addition of DMSO to the cell culture medium. The kinetics were followed after UV irradiation by single cell gel electrophoresis, and in total cell population by alkaline or neutral agarose gel electrophoresis. Shortly after exposure, an extensive fragmentation occurred in DNA; DNA double strand breaks were negatively correlated with recovery time for DNA integrity. DNA damage induced by UVC 9J/m2 rapidly triggered necrosis in a large fraction of irradiated K562 cells, and only 40% of treated cells resumed growth at a very low rate within 24h of culture. The addition of DMSO to the culture medium of cells 15min after UVC, when DNA strand break repair was not yet complete, produced apoptosis in >70% of surviving cells, as determined by TUNEL assay. Conversely, if DMSO was added when the resealing of DNA strand breaks was complete, surviving K562 cells retained full growth capacity, and their progeny underwent erythroid differentiation with normal levels of erythroid proteins, delta-aminolevulinic acid dehydrase and hemoglobin. This study shows that the extent of DNA strand break repair influences cell proliferation and the DMSO induced erythroid program, and the same UVC dose can have opposite effects depending on cellular status.     

The effect of photofrin on DNA strand breaks and base oxidation in HaCaT keratinocytes: a comet assay study

Photodynamic therapy (PDT) kills cells via the production of singlet oxygen and other reactive oxygen species. PDT causes chromosomal damage and mutation to cultured cells. However, DNA damage does not contribute to the phototoxic effect. To study the effect of Photofrin-PDT-induced DNA damage, we used the comet assay in combination with endonuclease III and formamidopyrimidine DNA glycosylase and a human keratinocyte cell line to investigate photogenotoxicity and its prevention by tocopherol (TOC). This study shows that PDT induced DNA damage in HaCaT cells at doses allowing cells to survive 7 days after irradiation. alpha-TOC did not prevent the acute cell lysis caused by Photofrin-PDT but did prevent Photofrin-PDT-induced DNA damage. However, the concentration of TOC that conferred protection (100 microM) was higher than is detected in human serum. Base oxidation was also measured using the comet assay. Although TOC could prevent frank DNA strand breaks caused by PDT, it was unable to decrease the level of base oxidation as revealed by enzyme-sensitive sites. It is suggested that the potential genotoxic risk from laser-PDT could be low, and that topical micro-TOC at a high concentration may be useful in preventing some types of DNA damage without preventing acute photolysis after Photofrin-PDT.     

Pentaerythritol tetrakis(4-iodo-2,3,5,6-tetrafluorophenyl) ether: a tecton for the self-assembly of double strand 1D infinite chains

The p-iodotetrafluorophenyl motif has been appended to the four alcoholic groups of pentaerythritol to give the corresponding tetraether 1, which works as an effective tecton in halogen bonding based crystal engineering. In fact, in the solution and the solid phases, the halogen bonding drives the self-assembly of this ether with primary, secondary, and tertiary amines as well as with pyridine derivatives. Co-crystals are isolated where 1 invariably works as a tetradentate halogen bonding donor. Double strand, 1D, infinite chains are formed where the nitrogen substituted motifs are pinned in positions that fulfil Schmidt's requirements for solid phase photocycloaddition reactions. Quantitative yields and complete stereoselectivity have been obtained in the cycloaddition reaction.     

Solid-state self-assembly of polymeric double helicates leading to linear arrays of silver(I) ions and reversible strand/double helix interconversion in solution

Reaction of a bent py-hyz-pym-hyz-pym 1 and of a linear py-hyz-py-hyz-pym 3 (py=pyridine; pym=pyrimidine; hyz=hydrazone) ligand strands with silver(I) tetrafluoroborate in CH(3)NO(2) generates double-helical dinuclear 2 and trinuclear 4 complexes. These complexes form polymeric, highly ordered solid-state structures, with wirelike, linear continuous or discontinuous polycationic Ag(n) (+) arrays with Ag--Ag distances of 2.78 to 4.42 A. Ligand 5, an isomer of 1, is found to yield a [2x2] grid-type complex 6. Titration experiments reveal the formation of linear rack-type dinuclear species from 1 and 5. Acid-base modulated, reversible interconversion between strand 1 and double helicate 2 may be achieved by using tren as a competing complexing agent (tren=N(CH(2)CH(2)NH(2))(3)). Progressive addition of silver(I) ions to a 1:1 mixture of 1 and 5 leads to the preferential formation of the double helicate 2 over the grid complex 6, illustrating a process of self-organisation with selection of the correct ligand.     

Photofragmentation study of hexamethyldisiloxane following core ionization and direct double ionization

X-ray photoelectron spectroscopy and Auger spectroscopy studies of gas-phase hexamethyldisiloxane (HMDSO) are presented. The photodissociation of this molecule is studied using various experimental coincidence techniques. We compare the fragmentation pathways observed after core ionization followed by Auger decay and after valence double photoionization of the molecule. A strongly selective production of the doubly charged tetramethyldisiloxane ion is observed in the low binding-energy regions. Theoretical calculations are carried out to tentatively explain the stability of the produced dication.