Increase of OH radical yields due to the decomposition of hydrogen peroxide by gold nanoparticles under X-ray irradiation.

We elucidate the decomposition mechanism of hydrogen peroxide, which is formed by water radiolysis, by gold nanoparticles (GNPs) under X-ray irradiation. The variations in yields of hydrogen peroxide generated in the presence of GNPs are evaluated using the Ghormley technique. The increase of yields of OH radicals has been quantified using Ampliflu® Red solutions. Almost all hydrogen peroxide generated by irradiation of <25 Gy is decomposed by GNPs, while the yield of OH radicals increases by 1.6 times. The amount of OH radicals thus obtained is almost equivalent to that of the decomposed hydrogen peroxide. The decomposition of hydrogen peroxide is an essential reaction to produce additional OH radicals efficiently in the vicinity of GNPs.

Revealing the mechanism of damage to the carbonate ester in PADC polymeric nuclear track detector using low-energy electron stimulated desorption.

We investigate the mechanism of damage to the carbonate ester chemical functions in Poly allyl diglycol carbonate (PADC) induced by low-energy electrons (LEEs) of <50 eV, which are major components of the initial secondary products of ionizing radiation. PADC is the world's most widely used polymeric nuclear track detector (PNTD) for swift ion detection. Using diethylene glycol monoethyl ether acetate as a surrogate for PADC, we have measured for irradiation with low-energy electrons (LEEs) of <50 eV, the electron stimulated desorption (ESD) signal of O- from 3-monolayer thick films of DGMEA by time-of-flight mass spectrometry. We find that for electron irradiation at energies >6-9 eV, the instantaneous ESD yield of O- increases with the cumulative number of incident electrons (i.e., fluence), indicating that the additional O- signal derives from an electron-induced DGMEA product. From comparison with ESD measurements from films of acetic acid and acetaldehyde, we identify that the additional desorbed O- signal derives from oxygen atoms originally adjacent to the carbonyl bond in DGMEA. Since LEEs are the predominant secondary particles produced by ionizing radiation, this finding helps to better understand the mechanism of damage to carbonate ester in PADC, which is a key step for latent track formation in PADC.

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.

Methionine oxidation by hydrogen peroxide in peptides and proteins: A theoretical and Raman spectroscopy study.

The oxidation of proteins results in their deterioration via the oxidation of reactive amino acids. Oxidation of the amino acid, methionine plays an important role during biological conditions of oxidative stress, and equally a role in protein stability. In this study the oxidation of the methionine residue using the tripeptide GlyMetGly with respect to hydrogen peroxide has been studied using both Raman spectroscopy and DFT calculations. Spectral modifications following the formation of methionine sulfoxide are shown with the appearance of the SO vibration whilst there is also the modification of the CS vibrations at approximately 700 cm-1. The changes in the intensity of the CS stretching band were used to calculate the kinetic rate constant as 7.9 ±â€¯0.6 × 10-3 dm3 mol-1 s-1. The energy barrier for the reaction. is determined both experimentally and using DFT calculations. The reaction of the dairy protein beta-lactoglobulin with hydrogen peroxide is equally studied using the same technique. The solvent accessible surface area of the methionine residues within the protein were also determined and a comparison of the reaction rate constant and the energy barriers of reaction for the oxidation of the tripeptide and for the protein respectively thus, provides information about the role of the protein environment in the oxidation process.

Single- and Double-Strand Breaks of Dry DNA Exposed to Protons at Bragg-Peak Energies.

Ultrathin layers (<20 nm) of pBR322 plasmid DNA were deposited onto 2.5 µm thick polyester films and exposed to proton Bragg-peak energies (90-3000 keV) at various fluences. A quantitative analysis of radio-induced DNA damage is reported here in terms of single- and double-strand breaks (SSB and DSB, respectively). The corresponding yields as well as G-values and the cross sections exhibit fairly good agreement with the rare available data, stemming from close experimental conditions, namely, based on α particle irradiation. SSB/DSB rates appear to be linear when plotted against linear energy transfer (LET) in the whole energy range studied. All the data present a maximum in the 150-200 keV energy range; as for LET, it peaks at 90 keV. We also show that fragmentation starts to be significant for proton fluences greater than 1 × 1011 cm-2 at the Bragg-peak energies. Finally, we determine the average proton track radial extension, rmax, corresponding to an occupation probability of 100% DSB in the Bragg-peak region. The rmax values determined are in excellent agreement with the radial extensions of proton tracks determined by simulation approaches in water. When plotted as a function of LET, both SSB and DSB cross sections bend back at high LETs.

DNA strand break dependence on Tris and arginine scavenger concentrations under ultra-soft X-ray irradiation: the contribution of secondary arginine radicals.

In this study, we used a bench-top cold-cathode ultra-soft X-ray (USX) generator to expose aqueous DNA plasmid solutions to low-LET radiation under various scavenging conditions. Single- and double-strand breaks were assessed using classic gel electrophoresis quantification of linear, circular and supercoiled plasmid DNA topologies. With their very low penetration range in water, USX can only interact with matter up to short distances, of the order of 50 µm. We validated a stirring procedure which makes it possible to expose 100 µL of aqueous samples (2 mm thick). The scavenging of OH radicals by Tris buffer was studied at ambient temperature under aerobic conditions and compared to data gathered in the literature. A very good agreement was found with the rare data dealing with DNA plasmid exposed to Al Kα photons at low temperature (T ≤ 277 K), which therefore validated the experimental procedure. The yields for DNA single-strand breaks determined during this study enabled the ratio of indirect to direct effects to be determined at 96.2%, in good agreement with the value of 97.7% stemming from a study based on γ-ray irradiation of frozen solutions of plasmid DNA. Then, arginine was used both to create a "biological-like" chemical environment around the DNA plasmids and as an OH radical scavenger, in vitro. Although arginine has a greater scavenging (protecting) power than Tris, surprisingly, it led to higher rates of strand breakage. Based on the specific binding modes of arginine to DNA, we suggest that the side effects observed are due to the presence of arginine near to, but also inside, the DNA double helix.

Isotopic fractionation of tritium in biological systems.

Isotopic fractionation of tritium is a highly relevant issue in radiation protection and requires certain radioecological considerations. Sound evaluation of this factor is indeed necessary to determine whether environmental compartments are enriched/depleted in tritium or if tritium is, on the contrary, isotopically well-distributed in a given system. The ubiquity of tritium and the standard analytical methods used to assay it may induce biases in both the measurement and the signification that is accorded to the so-called fractionation: based on an exhaustive review of the literature, we show how, sometimes large deviations may appear. It is shown that when comparing the non-exchangeable fraction of organically bound tritium (neOBT) to another fraction of tritium (e.g. tritiated water) the preparation of samples and the measurement of neOBT reported frequently led to underestimation of the ratio of tritium to hydrogen (T/H) in the non-exchangeable compartment by a factor of 5% to 50%. In the present study, corrections are proposed for most of the biological matrices studied so far. Nevertheless, the values of isotopic fractionation reported in the literature remain difficult to compare with each other, especially since the physical quantities and units often vary between authors. Some improvements are proposed to better define what should encompass the concepts of exchangeable and non-exchangeable fractions.

Measurement of tritium in the free water of milk : spotting and quantifying some biases and proposing ways of improvement.

As one of the three natural isotopes of hydrogen, tritium is ubiquitous and may potentially be present in any water or organic molecule that constitutes a biological matrix. Milk is one of the most frequently monitored foodstuffs in the vicinity of chronic release of radionuclides, as it is a very common food product and also because it integrates deposition on large areas of grass or crops at a local scale. Different parameters have been studied to assess their impact on the reliability of tritium measurements in the free water of milk. The volume of the sample, the technique used to extract the water and the level of dehydration modulate the results but in different ways: dispersion of results and under- or over-estimation of the tritium activity. The influence of sample storage and preparation has also been investigated. Methodological improvements of tritium measurements in the free water of milk are proposed. An original fractionation effect during distillation of milk is also described.

Dissociative electron attachment to DNA-diamine thin films: impact of the DNA close environment on the OH- and O- decay channels.

We measure the desorption of anions stimulated by the impact of 0-20 eV electrons on highly uniform thin films of plasmid DNA-diaminopropane. The results are accurately correlated with film thickness and composition by AFM and XPS measurements, respectively. Resonant structures in the H(-), O(-), and OH(-) yield functions are attributed to the decay of transient anions into the dissociative electron attachment (DEA) channel. The diamine induces ammonium-phosphate bridges along the DNA backbone, which suppresses the DEA O(-) channel and in counter-part increases considerably the desorption of OH(-). The close environment of the phosphate groups may therefore play an important role in modulating the rate and type of DNA damages induced by low energy electrons.

Absolute cross section for loss of supercoiled topology induced by 10 eV electrons in highly uniform /DNA/1,3-diaminopropane films deposited on highly ordered pyrolitic graphite.

It was recently shown that the affinity of doubly charged, 1-3 diaminopropane (Dap(2+)) for DNA permits the growth on highly ordered pyrolitic graphite (HOPG) substrates, of plasmid DNA films, of known uniform thickness [O. Boulanouar, A. Khatyr, G. Herlem, F. Palmino, L. Sanche, and M. Fromm, J. Phys. Chem. C 115, 21291-21298 (2011)]. Post-irradiation analysis by electrophoresis of such targets confirms that electron impact at 10 eV produces a maximum in the yield of single strand breaks that can be associated with the formation of a DNA(-) transient anion. Using a well-adapted deterministic survival model for the variation of electron damage with fluence and film thickness, we have determined an absolute cross section for strand-break damage by 10 eV electrons and inelastic scattering attenuation length in DNA-Dap complex films.

Nontargeted stressful effects in normal human fibroblast cultures exposed to low fluences of high charge, high energy (HZE) particles: kinetics of biologic responses and significance of secondary radiations.

The induction of nontargeted stressful effects in cell populations exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation. We investigated the up-regulation of stress markers in confluent normal human fibroblast cultures exposed to 1,000 MeV/u iron ions [linear energy transfer (LET) ∼151 keV/µm] or 600 MeV/u silicon ions (LET ∼50 keV/µm) at mean absorbed doses as low as 0.2 cGy, wherein 1-3% of the cells were targeted through the nucleus by a primary particle. Within 24 h postirradiation, significant increases in the levels of phospho-TP53 (serine 15), p21(Waf1) (CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage. than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ∼109 keV/µm) that targets ∼1.6% of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ∼13 keV/µm) by which, on average, ∼13% of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1% of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 µm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles.

Damage induced to DNA by low-energy (0-30 eV) electrons under vacuum and atmospheric conditions.

In this study, we show that it is possible to obtain data on DNA damage induced by low-energy (0-30 eV) electrons under atmospheric conditions. Five monolayer films of plasmid DNA (3197 base pairs) deposited on glass and gold substrates are irradiated with 1.5 keV X-rays in ultrahigh vacuum and under atmospheric conditions. The total damage is analyzed by agarose gel electrophoresis. The damage produced on the glass substrate is attributed to energy absorption from X-rays, whereas that produced on the gold substrate arises from energy absorption from both the X-ray beam and secondary electrons emitted from the gold surface. By analysis of the energy of these secondary electrons, 96% are found to have energies below 30 eV with a distribution peaking at 1.4 eV. The differences in damage yields recorded with the gold and glass substrates is therefore essentially attributed to the interaction of low-energy electrons with DNA under vacuum and hydrated conditions. From these results, the G values for low-energy electrons are determined to be four and six strand breaks per 100 eV, respectively.

Propagation distance of the alpha-particle-induced bystander effect: the role of nuclear traversal and gap junction communication.

When cell populations are exposed to low-dose alpha-particle radiation, a significant fraction of the cells will not be traversed by a radiation track. However, stressful effects occur in both irradiated and bystander cells in the population. Characterizing these effects, and investigating their underlying mechanism(s), is critical to understanding human health risks associated with exposure to alpha particles. To this end, confluent normal human fibroblast cultures were grown on polyethylene terephthalate foil grafted to an ultrathin solid-state nuclear track detector and exposed under non-perturbing conditions to low-fluence alpha particles from a broadbeam irradiator. Irradiated and affected bystander cells were localized with micrometer precision. The stress-responsive protein p21(Waf1) (also known as CDKN1A) was induced in bystander cells within a 100-microm radius from an irradiated cell. The mean propagation distance ranged from 20 to 40 microm around the intranuclear alpha-particle impact point, which corresponds to a set of approximately 30 cells. Nuclear traversal, induced DNA damage, and gap junction communication were critical contributors to propagation of this stressful effect. The strategy described here may be ideal to investigate the size of radiation-affected target and the relative contribution of different cellular organelles to bystander effects induced by energetic particles, which is relevant to radioprotection and cancer radiotherapy.

Impact of bulk and surface properties of some biocompatible hydrophobic polymers on the stability of methylene chloride-in-water mini-emulsions used to prepare nanoparticles by emulsification-solvent evaporation.

The emulsifying and stabilizing ability of several hydrophobic (insoluble in water and soluble in volatile organic solvents) polymers, such as Eudragit RL, Eudragit RS, PLGA, PCL, and their mixtures, with regard to the methylene chloride (MC)-in-water mini-emulsions, has been compared to the viscosity of MC solutions and to the properties of adsorption and spread monolayers of these polymers. Eudragits RS and RL contain approximately 2.5 and approximately 5 mol% of pendent cationic trimethylammonium (TMA) groups per approximately 164 g/mol segments, whereas PLGA and PCL contain 1 and 2 polar carbonyl groups per 130 and 114 g/mol, respectively. The electrostatic attraction between the dipoles, formed by TMA groups and the condensed counter ions in the MC solutions, leads to the contraction of macromolecular coils of Eudragits, whereas the PLGA and PCL macromolecules, interacting by low polar carbonyl groups (with dipole moment mu = 2.7 D) retain more extended conformation in MC. This explains why the characteristic viscosities [eta] of MC solutions are much lower for the former polymers ( approximately 0.1 dL/g) with regard to PLGA and PCL solutions whose [eta] is equal to 0.3 and 0.6 dL/g, respectively. The ionization of TMA groups in contact with the water phase leads to the irreversible adsorption of Eudragits at the MC/water interface and to high decrease of the interfacial tension gamma (down to 4 mN/m for the 5% MC solutions). Whereas PLGA and PCL possessing low polar carbonyl groups adsorb poorly at the MC/water interface exhibiting gamma congruent with 28 mN/m. Higher stability of spread monolayers of Eudragits (pi* approximately 40 mN/m) with regard to PLGA and PCL (pi* < 20 mN/m) correlates well with higher interfacial activity of the former with regard to the later. The higher surface potential DeltaV of Eudragits (0.9 V) with regard to PLGA (0.3 V) and PCL (0.4V) is explained by the formation of electric double layer (DL) by the former, whereas the later contribute to the DeltaV only by cumulative dipole moments of carbonyl groups. The experimental values of surface potentials correlate well with the Gouy-Chapman model of the DL and the Helmholtz model of the monolayer. The ensemble of experimental results leads to the conclusion that higher emulsifying and stabilizing ability of Eudragits with regard to PLGA and PCL is due to higher adsorption activity of the former which form the corona of polymeric chains with ionized TMA groups around the droplets. It can be postulated that Eudragit polymers have good surface active properties which may allow manufacturing of biocompatible nanoparticles by emulsification-solvent evaporation method without surfactants.

Production and validation of CR-39-based dishes for alpha-particle radiobiological experiments.

The study of radiobiological effects induced in vitro by low fluences of alpha particles would be significantly enhanced if the precise localization of each particle track in the cell monolayer was known. From this perspective, we developed a new method based on tailor-made UV-radiation-cured CR-39, the production of which is described. Its validation both as a petri dish and as solid-state nuclear track detectors is demonstrated. With respect to the demands on solid-state nuclear track detectors in such experiments, these biologically compatible detectors have a controlled micrometric thickness that allows them to be crossed by the alpha particles. In this study, we present a method for obtaining 10-mum-thick CR-39, its chemical characterization, and its properties as a solid-state nuclear track detector under the environmental conditions of radiobiological experiments. The experimental studies performed with 3.5 MeV alpha particles show that their transmitted energy is sufficient enough to cross the entire cellular volume. Under optimal conditions, etched tracks are clearly defined 2 h after etching. Moreover, the UV-radiation-cured CR-39 represents an essentially zero background that is due to the short time between the production and use of the polymer. Under a confocal microscope, this thin solid-state nuclear track detector allows the precise localization of the impact parameter at the subcellular level.