Effects of carbon-ion radiation on the postnatal development of mice and on the yield of white spots in the mid-ventrum and tail tips

Pregnant female C57BL/10JHir mice were irradiated whole-body at 9 days of gestation with a single acute dose of carbon-ion radiation. The average linear energy transfer (LET) of the carbon ions was 50 keV/microm within a spread-out Bragg peak (SOBP). The effects were studied by scoring changes in the postnatal development of the mice as well as in the pigmentation of the cutaneous coats and tail tips of their offspring 22 days after birth. The percentage of live births was reduced in mice exposed to carbon ions at doses greater than 0.5 Gy. The survival to day 22 was also reduced in mice exposed to carbon ions at doses greater than 0.75 Gy. Moreover, the body weight at day 22 was reduced in mice exposed to carbon ions at doses greater than 0.1 Gy. A comparison of the survival to day 22 after exposure to carbon ions with our previous results for 60Co gamma rays indicated that carbon ions were twice as effective as gamma rays. White spots were found in the mid-ventrum as well as in the tail tips of offspring exposed to carbon ions in utero. The frequency and the size of the white spots in the mid-ventrum and in the tail tips increased as the dose increased. Carbon ions appear to be slightly more effective than the gamma rays used in our previous study. In the ventral white spots, no melanocytes were observed in the epidermis, dermis and hair follicles. These results indicate that prenatal exposure to carbon ions has a greater effect on the postnatal development and survival of mice than does exposure to gamma rays, and that the relative biological effectiveness is greater than that for effects on melanocyte development.     

Mutagenic effects of carbon ions near the range end in plants

To gain insight into the mutagenic effects of accelerated heavy ions in plants, the mutagenic effects of carbon ions near the range end (mean linear energy transfer (LET): 425keV/μm) were compared with the effects of carbon ions penetrating the seeds (mean LET: 113keV/μm). Mutational analysis by plasmid rescue of Escherichia coli rpsL from irradiated Arabidopsis plants showed a 2.7-fold increase in mutant frequency for 113keV/μm carbon ions, whereas no enhancement of mutant frequency was observed for carbon ions near the range end. This suggested that carbon ions near the range end induced mutations that were not recovered by plasmid rescue. An Arabidopsis DNA ligase IV mutant, deficient in non-homologous end-joining repair, showed hyper-sensitivity to both types of carbon-ion irradiation. The difference in radiation sensitivity between the wild type and the repair-deficient mutant was greatly diminished for carbon ions near the range end, suggesting that these ions induce irreparable DNA damage. Mutational analysis of the Arabidopsis GL1 locus showed that while the frequency of generation of glabrous mutant sectors was not different between the two types of carbon-ion irradiation, large deletions (>～30kb) were six times more frequently induced by carbon ions near the range end. When 352keV/μm neon ions were used, these showed a 6.4 times increase in the frequency of induced large deletions compared with the 113keV/μm carbon ions. We suggest that the proportion of large deletions increases with LET in plants, as has been reported for mammalian cells. The nature of mutations induced in plants by carbon ions near the range end is discussed in relation to mutation detection by plasmid rescue and transmissibility to progeny.     

Immobilization of metallothionein to carbon paste electrode surface via anti-MT antibodies and its use for biosensing of silver

In this paper, heavy metal biosensor based on immobilization of metallothionein (MT) to the surface of carbon paste electrode (CPE) via anti-MT-antibodies is reported. First, the evaluation of MT electroactivity was done. The attention was focused on the capturing of MT to the CPE surface. Antibodies incorporated and mixed into carbon paste were stable; even after two weeks the observed changes in signal height were lower than 5%. Further, the interaction of MT with polyclonal chicken antibodies incorporated in carbon paste electrode was determined by square-wave voltammetry. In the voltammogram, two signals--labelled as cys(MT) and W(a)--were observed. The cys(MT) corresponded to -SH moieties of MT and W(a) corresponded to tryptophan residues of chicken antibodies. Time of interaction (300 s) and MT concentration (125 μg/ml) were optimized to suggest a silver(I) ions biosensor. Biosensor (CPE modified with anti-MT antibody) prepared under the optimized conditions was then used for silver(I) ions detection. The detection limit (3 S/N) for silver(I) ions was estimated as 0.5 nM. The proposed biosensor was tested by detection spiking of silver(I) ions in various water samples (from very pure distilled water to rainwater). Recoveries varied from 74 to 104%.     

Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/microm carbon ions, and 200 keV/microm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = approximately 1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G(1) premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.     

Specificity of mutations induced by carbon ions in budding yeast Saccharomyces cerevisiae

To investigate the nature of mutations induced by accelerated ions in eukaryotic cells, the effects of carbon-ion irradiation were compared with those of γ-ray irradiation in the budding yeast Saccharomyces cerevisiae.

The mutational effect and specificity of carbon-ion beams were studied in the URA3 gene of the yeast. Our experiments showed that the carbon ions generated more than 10 times the number of mutations induced by γ-rays, and that the types of base changes induced by carbon ions include transversions (68.7%), transitions (13.7%) and deletions/insertions (17.6%). The transversions were mainly G:C → T:A, and all the transitions were G:C → A:T. In comparison with the surrounding sequence context of mutational base sites, the C residues in the 5′-AC(A/T)-3′ sequence were found to be easily changed. Large deletions and duplications were not observed, whereas ion-induced mutations in Arabidopsis thaliana were mainly short deletions and rearrangements. The remarkable feature of yeast mutations induced by carbon ions was that the mutation sites were localized near the linker regions of nucleosomes, whereas mutations induced by γ-ray irradiation were located uniformly throughout the gene.     

Changes in bone volume after irradiation with carbon ions

The effects of carbon ions on bone volume were studied by histological and morphometrical methods. Anesthetized rats irradiated with three different single doses (15, 22.5, or 30 Gy) of either carbon ions or gamma rays were sacrificed (5 rats/group) at 10 time points together with non-irradiated controls. Carbon ion-induced bone responses were qualitatively and quantitatively different from those induced by gamma irradiation at the same dose level. Irradiation with carbon ions resulted in a dose-dependent increase in bone volume, while gamma irradiation induced loss of bone volume, which was found to be independent of dose in the range studied over the same time course. After irradiation with carbon ions, bones showed thickening of the trabeculae, and the bone marrow became fibrous with fewer vessels. Carbon ion irradiation showed a greater effect on the bone-absorbing capability of osteoclasts than gamma irradiation. These observations suggest that irradiation with carbon ions may produce differential modulation of radiation-induced growth factor expression.     

XPS evidence for the formation of Ni(II) complexes on treated activated carbon

X-ray photoelectron spectroscopy (XPS) was applied to a solid-state investigation on adsorbed systems obtained by contacting different samples of an activated carbon (Chemviron Filtrasorb 400) with solutions containing (a) 1,10-phenanthroline (phen); (b) NiCl₂·6H₂O. (c) Ni²⁺ ions and phen in a 1:3 molar ratio.XPS results on these systems are discussed with particular regard to the chemical state of the ligand (interaction of functional groups with the substrate) and of the metal ions on carbon (assessment of Ni oxidation state; detection of Ni complexes on carbon and information on their stoichiometries and structures). Various effects of successive chemical treatments on the above reported carbon species were also revealed by XPS, such as:(i) the formation of a ‘carbon–ligand’ system (activated carbon with ligand firmly present on it) after acid elution of the activated carbon treated with Ni–phen complexes in solution, and (ii) the adsorption of Ni²⁺ ions on this ‘carbon–ligand’ system, with fomation of Ni–phen complexes on carbon.     

Energetic heavy ions accelerate differentiation in the descendants of irradiated normal human diploid fibroblasts

Ionizing radiation-induced genomic instability has been demonstrated in a variety of endpoints such as delayed reproductive death, chromosome instability and mutations, which occurs in the progeny of survivors many generations after the initial insult. Dependence of these effects on the linear energy transfer (LET) of the radiation is incompletely characterized; however, our previous work has shown that delayed reductions in clonogenicity can be most pronounced at LET of 108 keV/microm. To gain insight into potential cellular mechanisms involved in LET-dependent delayed loss of clonogenicity, we investigated morphological changes in colonies arising from normal human diploid fibroblasts exposed to gamma-rays or energetic carbon ions (108 keV/microm). Exposure of confluent cultures to carbon ions was 4-fold more effective at inactivating cellular clonogenic potential and produced more abortive colonies containing reduced number of cells per colony than gamma-rays. Second, colonies were assessed for clonal morphotypic heterogeneity. The yield of differentiated cells was elevated in a dose- and LET-dependent fashion in clonogenic colonies, whereas differentiated cells predominated to a comparable extent irrespective of radiation type or dose in abortive colonies. The incidence of giant or multinucleated cells was also increased but much less frequent than that of differentiated cells. Collectively, our results indicate that carbon ions facilitate differentiation more effectively than gamma-rays as a major response in the progeny of irradiated fibroblasts. Accelerated differentiation may account, at least in part, for dose- and LET-dependent delayed loss of clonogenicity in normal human diploid cells, and could be a defensive mechanism that minimizes further expansion of aberrant cells.     

Chromosome aberrations induced by high-LET carbon ions in radiosensitive and radioresistant tumour cells

Chromosome aberration formation was analysed in two human tumour cell lines displaying different radiosensitivity. Aberrations involving chromosomes 2, 4, and 5 were studied in one radioresistant cell line (WiDr) and in one radiosensitive cell line (MCF-7). Chromosome aberrations were studied by application of single-colour FISH. We studied the effects of monoenergetic 100 MeV/u carbon ions and carbon ions from extended Bragg peak. Chromosome aberrations induced by carbon ions were compared with aberrations induced by standard 200 kV X-rays. In both tumour cell lines, carbon ions induced aberrations more effectively than X-rays. The radioresistance and radiosensitivity of the corresponding cell lines, as observed for X-rays, were also found after carbon ion irradiation. In both cell lines, the typical effects of ion irradiation were an increased proportion of cells containing complex aberrations, and an increased complexity of these complex exchanges. However, comparable effects were induced in MCF-7 cells by a much lower dose than in WiDr cells. Insertions were also induced more efficiently in MCF-7 cells than in WiDr cells.     

NMR spin-lattice relaxation times of intracellular Na-23 on rat livers and related lipid peroxidation following CCl4 intoxication

Liver tissues were isolated from rats acutely intoxicated with carbon tetrachloride, and Na-23 NMR signals were analyzed to investigate the T1 relaxation times of intracellular sodium ions under pathological conditions in presence of the paramagnetic shift reagent (dysprosium tripolyphosphate). We studied the significant increase of T1 found in CCl4 treated rats with respect to controls, which was elsewhere demonstrated as being independent of cell necrosis. Evidence is given that neither fat accumulation nor proliferative processes affect the observed T1 lengthening. When T1 relaxation times were measured in the liver of vitamin E treated rats subsequently intoxicated with carbon tetrachloride, a significative shortening of T1 with respect to CCl4-intoxicated rats was observed. These results were discussed in terms of the antioxidant action exerted by vitamin E, taking into account that peroxidation of microsomal lipids is the key factor in the process of carbon tetrachloride induced liver injury. Furthermore, the observed T1 changes were discussed in terms of the interactions of Na+ with cell membranes and/or the occurrence of viscosity changes.     

Changes in osteoclasts after irradiation with carbon ion particles

We examined the effects of radiation on decreases in osteoclast numbers after regional irradiation of rats with carbon ions and gamma rays. Male Wistar rats were subjected to hind-leg irradiation with carbon ions (290 MeV/u) or gamma rays at doses of 15, 22.5, or 30 Gy. The effects of carbon ions and gamma rays on osteoclasts were studied using histologic and morphometric methods. At doses of 15 Gy and 22.5 Gy, osteoclast numbers increased transiently until day 5 after irradiation and then decreased rapidly in both the carbon ion and gamma ray irradiation groups. The carbon ion group showed reduced osteoclast size compared with the gamma ray group. Carbon ion irradiation had a more marked effect on osteoclast activity, and suppressed maturation to a greater extent than gamma irradiation. These observations suggest that carbon ion irradiation induces differential modulation of osteoclast growth factor expression.     

Initial yields of DNA double-strand breaks and DNA Fragmentation patterns depend on linear energy transfer in tobacco BY-2 protoplasts irradiated with helium, carbon and neon ions

The ability of ion beams to kill or mutate plant cells is known to depend on the linear energy transfer (LET) of the ions, although the mechanism of damage is poorly understood. In this study, DNA double-strand breaks (DSBs) were quantified by a DNA fragment-size analysis in tobacco protoplasts irradiated with high-LET ions. Tobacco BY-2 protoplasts, as a model of single plant cells, were irradiated with helium, carbon and neon ions having different LETs and with gamma rays. After irradiation, DNA fragments were separated into sizes between 1600 and 6.6 kbp by pulsed-field gel electrophoresis. Information on DNA fragmentation was obtained by staining the gels with SYBR Green I. Initial DSB yields were found to depend on LET, and the highest relative biological effectiveness (about 1.6) was obtained at 124 and 241 keV/microm carbon ions. High-LET carbon and neon ions induced short DNA fragments more efficiently than gamma rays. These results partially explain the large biological effects caused by high-LET ions in plants.     

培养液中钙对碳纳米材料细胞毒性作用机制的同步辐射初步研究

本工作研究了细胞培养液中钙离子和多壁碳纳米管,以及纳米碳黑的相互作用,采用钙荧光标记的显微成像技术和同步辐射X射线荧光技术,研究了HeLa细胞内钙离子浓度的增高及其相关机制。实验结果指出,细胞培养介质中的钙能被碳纳米颗粒(CNPs)大量吸附并载带进入细胞内,因此,CNPs向细胞内输送过量的钙离子可能是造成CNPs细胞毒性的重要原因。本工作为正确检测和评估碳纳米材料细胞毒性提供了新思路。     

LET and ion species dependence for cell killing in normal human skin fibroblasts

We studied the LET and ion species dependence of the RBE for cell killing to clarify the differences in the biological effects caused by the differences in the track structure that result from the different energy depositions for different ions. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon, neon, silicon and iron ions that were generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. Cell killing was measured as reproductive cell death using a colony formation assay. The RBE-LET curves were different for carbon ions and for the other ions. The curve for carbon ions increased steeply up to around 98 keV/microm. The RBE of carbon ions at 98 keV/microm was 4.07. In contrast, the curves for neon, silicon and iron ions had maximum peaks around 180 keV/microm, and the RBEs at the peak position ranged from 3.03 to 3.39. When the RBEs were plotted as a function of Z*2/beta2 (where Z* is the effective charge and beta is the relative velocity of the ion) instead of LET, the discrepancies between the RBE-LET curves for the different ion beams were reduced, but branching of the RBE-Z*2/beta2 curves still remained. When the inactivation cross section was plotted as a function of either LET or Z*2/beta2, it increased with increasing LET. However, the inactivation cross section was always smaller than the geometrical cross section. These results suggest that the differences in the energy deposition track structures of the different ion sources have an effect on cell killing.     

Application of Polypyrrole Multi-Walled Carbon Nanotube Composite Layer for Detection of Mercury,Lead and Iron Ions Using Surface Plasmon Resonance Technique

Polypyrrole multi-walled carbon nanotube composite layers were used to modify the gold layer to measure heavy metal ions using the surface plasmon resonance technique. The new sensor was fabricated to detect trace amounts of mercury (Hg), lead (Pb), and iron (Fe) ions. In the present research, the sensitivity of a polypyrrole multi-walled carbon nanotube composite layer and a polypyrrole layer were compared. The application of polypyrrole multi-walled carbon nanotubes enhanced the sensitivity and accuracy of the sensor for detecting ions in an aqueous solution due to the binding of mercury, lead, and iron ions to the sensing layer. The Hg ion bonded to the sensing layer more strongly than did the Pb and Fe ions. The limitation of the sensor was calculated to be about 0.1 ppm, which produced an angle shift in the region of 0.3° to 0.6°.     

Electrochemical investigation of metal ion interactions of a ferrocene deoxyuridine conjugate

The metal ion interactions with ferrocene-modified deoxyuridine (FcdU) have been studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) at a glassy carbon electrode in aqueous medium. Binding constants (K_n+) were determined from voltammetric data, i.e. shifts in potential and changes in limiting current with FcdU and after the addition of different metal ions. Electrospray ionization (ESI) mass spectra and matrix-assisted laser desorption/ionization-time flight mass spectrometry (MALDI-TOF MS) of FcdU recorded in presence of metal ions suggest that FcdU forms stable complexes with Cd²⁺.     

Adsorption of heavy metal ions from aqueous solutions by activated carbon prepared from apricot stone

Apricot stones were carbonised and activated after treatment with sulphuric acid (1:1) at 200 degrees C for 24 h. The ability of the activated carbon to remove Ni(II), Co(II), Cd(II), Cu(II), Pb(II), Cr(III) and Cr(VI) ions from aqueous solutions by adsorption was investigated. Batch adsorption experiments were conducted to observe the effect of pH (1-6) on the activated carbon. The adsorptions of these metals were found to be dependent on solution pH. Highest adsorption occurred at 1-2 for Cr(VI) and 3-6 for the rest of the metal ions, respectively. Adsorption capacities for the metal ions were obtained in the descending order of Cr(VI) > Cd(II) > Co(II) > Cr(III) > Ni(II) > Cu(II) > Pb(II) for the activated carbon prepared from apricot stone (ASAC).     

黄土塬区苹果园土壤有机碳分布特征

以黄土塬区塬面和梁坡梯田5、10、15a和20a苹果园为对象,在行间距果树1.0、1.5m和2.0m处用土钻法分层采集0-100cm土样,LiquiTOCⅡ测定样品土壤有机碳(Soil Organic Carbon,SOC)含量,分析两种地形条件下各龄果园SOC的分布特征。结果表明:塬面5、10、15a和20a果园SOC分别为6.39、6.46、6.66g/kg和6.47g/kg,梁坡分别为5.83、6.05、6.54g/kg和6.09g/kg,两种地形条件下同龄果园SOC差异显著(P0.05),但龄间SOC消长趋势相似,均有15a的20a的10a的5a的;水平方向上,5a果园SOC沿树干向外增大,10a果园减小,15a和20a果园变化较小,塬面和梁坡同龄果园间SOC水平分布格局较一致;垂直方向上,同梁坡相比,塬面果园4个层次(0-10cm、10-20cm、20-50cm和50-100cm)的平均SOC较高;梁坡果园50-100cm土层的SOC龄间差异较大,20-50cm土层龄间差异较小,塬面果园50-100cm土层的SOC龄间变化较小;在"纯果园"利用阶段,果园利用方式并未引起SOC下降,深层SOC有明显的积累效应。     

Assessing the influence of historic net and gross land changes on the carbon fluxes of Europe

Legacy effects of land cover/use on carbon fluxes require considering both present and past land cover/use change dynamics. To assess past land use dynamics, model‐based reconstructions of historic land cover/use are needed. Most historic reconstructions consider only the net area difference between two time steps (net changes) instead of accounting for all area gains and losses (gross changes). Studies about the impact of gross and net land change accounting methods on the carbon balance are still lacking. In this study, we assessed historic changes in carbon in soils for five land cover/use types and of carbon in above‐ground biomass of forests. The assessment focused on Europe for the period 1950 to 2010 with decadal time steps at 1‐km spatial resolution using a bookkeeping approach. To assess the implications of gross land change data, we also used net land changes for comparison. Main contributors to carbon sequestration between 1950 and 2010 were afforestation and cropland abandonment leading to 14.6 PgC sequestered carbon (of which 7.6 PgC was in forest biomass). Sequestration was highest for old‐growth forest areas. A sequestration dip was reached during the 1970s due to changes in forest management practices. Main contributors to carbon emissions were deforestation (1.7 PgC) and stable cropland areas on peaty soils (0.8 PgC). In total, net fluxes summed up to 203 TgC yr^?1 (98 TgC yr^?1 in forest biomass and 105 TgC yr^?1 in soils). For areas that were subject to land changes in both reconstructions (35% of total area), the differences in carbon fluxes were about 68%. Overall for Europe the difference between accounting for either gross or net land changes led to 7% difference (up to 11% per decade) in carbon fluxes with systematically higher fluxes for gross land change data.