Human lymphocyte damage and phosphorylation of H2AX and ATM induced by γ-rays

Objective To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM.Methods Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy.The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively.The micronucleus(MN)was analyzed by CB method to evaluate DNA damage.Results FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses,and the fitting curve was shown as Y=3.96+11.29D-0.45D2.The level of phosphorylated ATM(p-ATM)was not changed significantly by using the same method.Western blot showed that p-ATM protein expression was significandy increased after irradiation compared with sham.irradiated group.The MN assay which represented DNA damage was sensitive to different doses.Conclusions γ-ray irradiation could induce the phosphorylation of H2AX and ATM,which may play an important role in indicating DNA damage.Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage.     

Monte Carlo calculation of the self-absorption of γ-rays in a volume-shaped source connecting a face-type and a well-type NaI-detector

The self-absorption of γ-rays in various sources for photo-peak counting with a 2 × 2 in. face-type and a 3 × 3 in. well-type NaI-detector has been calculated by using the Monte Carlo method.     

Protective effects of Zaoshen capsule on irradiation damage induced by 60Co γ-rays in mice

nificantly (P<0.01). Conclusions Animal experiments demonstrated that Zaoshen capsule had obvious protective effects on hematopoietic and immune systems of irradiated mice.     

Amplification of DNA damage by a γH2AX-targeted radiopharmaceutical

¹¹¹In-DTPA-anti-γH2AX-Tat, which combines an anti-γH2AX antibody with a cell-penetrating peptide, Tat, and the Auger electron-emitting radioisotope, ¹¹¹In, targets the DNA damage signalling protein, γH2AX, and has potential as a probe for imaging DNA damage in vivo. The goal of this study was to investigate whether ¹¹¹In-DTPA-anti-γH2AX-Tat labelled to high specific activity (6 MBq/μg) can amplify treatment-related DNA damage for therapeutic gain.MethodsMDA-MB-468 and MDA-MB-231/H2N (231-H2N) breast cancer cells were incubated with ¹¹¹In-DTPA-anti-γH2AX-Tat (3 MBq, 6 MBq/μg) or a control radioimmunoconjugate, ¹¹¹In-DTPA-mIgG-Tat, and exposed to IR or bleomycin. DNA damage was studied by counting γH2AX foci and by neutral comet assay. Cytotoxicity was evaluated using clonogenic assays. ¹¹¹In-DTPA-anti-γH2AX-Tat was administered intravenously to 231-H2N-xenograft-bearing Balb/c nu/nu mice in tumor growth inhibition studies.ResultsThe number of γH2AX foci was greater after exposure of cells to IR (10 Gy) plus ¹¹¹In-DTPA-anti-γH2AX-Tat compared to IR alone (20.6 ± 2.5 versus 10.4 ± 2.3 foci/cell; P < .001).¹¹¹In-DTPA-anti-γH2AX-Tat resulted in a reduced surviving fraction in cells co-treated with IR (4 Gy) versus IR alone (5.2% ± 0.9% versus 47.8% ± 2.8%; P < .001). Similarly, bleomycin (25–200 μg/mL) plus ¹¹¹In-DTPA-anti-γH2AX-Tat resulted in a lower SF compared to bleomycin alone. The combination of a single exposure to IR (10 Gy) plus ¹¹¹In-DTPA-anti-γH2AX-Tat significantly decreased the growth rate of 231-H2N xenografts in vivo compared to either ¹¹¹In-DTPA-anti-γH2AX-Tat or IR alone (? 0.002 ± 0.004 versus 0.036 ± 0.011 and 0.031 ± 0.014 mm³/day, respectively, P < .001).Conclusion¹¹¹In-DTPA-anti-γH2AX-Tat amplifies anticancer treatment-related DNA damage in vitro and has a potent anti-tumor effect when combined with IR in vivo.     

Elemental analysis using x- and prompt γ-rays induced by 4.7 MeV α particles

A new possibility to use the variable-energy U-120 INPE Cyclotron for elemental analysis of biological and geological samples is presented. Samples were exposed to 4.7 MeV α particles and x- and prompt γ-ray spectra were recorded. The method was tested on hair, spleen, liver, lung, bone and lunar soil samples and was found promising for performing rapid analyses.     

Assessment of DNA damage produced by 125I‐triplex‐forming oligonucleotides in cells

Purpose: Triplex‐forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger‐electron‐emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two ¹²⁵I‐labeled TFOs, one with a single target in the genome and another with multiple targets.

Materials and methods: Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone γ‐H2AX foci formed at the sites of DNA double strand breaks (DSBs).

Results: The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more γ‐H2AX foci per cell than the TFO with a single target.

Conclusion: Since the two methods gave comparable results, measuring the number of γ‐H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.     

Role of DNA methylation in long-term low-dose γ-rays induced adaptive response in human B lymphoblast cells

Abstract

Purpose: With widespread use of ionizing radiation, more attention has been attracted to low-dose radiation (LDR); however, the mechanisms of long-term LDR-induced bio-effects are unclear. Here, we applied human B lymphoblast cell line HMy2.CIR to monitor the effects of long-term LDR and the potential involvement of DNA methylation.

Materials and methods: HMy2.CIR cells were irradiated with 0.032 Gy γ-rays three times per week for 1–4 weeks. Some of these primed cells were further challenged with 2 Gy γ-rays. Cell proliferation, micronuclei formation, gene expression of DNA methyltransferases (DNMT), levels of global genomic DNA methylation and protein expression of methyl CpG binding protein 2 (MeCP2) and heterochromatin protein-1 (HP1) were measured.

Results: Long-term LDR enhanced cell proliferation and clonogenicity and triggered a cellular adaptive response (AR). Furthermore, global genomic DNA methylation was increased in HMy2.CIR cells after long-term LDR, accompanied with an increase of gene expression of DNMT1 and protein expression of MeCP2 and HP1. After treatment with 5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, the long-term LDR-induced global genomic DNA hypermethylation was decreased and the AR was eliminated.

Conclusion: Global genomic DNA hypermethylation accompanied with increases of DNMT1 and MeCP2 expression and heterochromatin formation might be involved in long-term LDR-induced adaptive response.     

Involvement of calcium ion in elevation of mRNA for γ-glutamylcysteine synthetase (γ-GCS) induced by low-dose γ-rays

Purpose : To investigate the mechanism of the intracellular glutathione elevation induced by low-dose gamma-radiation. Materials and methods : RAW 264.7 cells were irradiated with 1-400 cGy γ-rays. Intracellular total glutathione content was determined by DTNB-recycling assay. Expression of mRNA for intracellular glutathione synthesis-related enzymes with or without treatment with various inhibitors of second messengers of gene expression were examined by Northern blot analysis. Results : Expression of mRNA for γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme of the de novo glutathione synthesis pathway, was elevated much more than that of glutathione reductase (GR) mRNA after exposure to 50 cGy γ-rays. The low-dose γ-ray-induced γ-GCS mRNA elevation was abolished by inhibitors of protein kinase C and protein tyrosine kinase, as well as by the calcium ion channel blocker, nifedipine. Calcium-related reagents, such BAPTA/AM and EGTA, chelators of intra- and extracellular Ca 2+ respectively, and a Ca 2+ ionophore (A23187), also strongly blocked the elevation of γ-GCS mRNA expression induced by γ-rays. Conclusions : The increase of intracellular glutathione in RAW 264.7 soon after low-dose gamma-ray exposure mainly occurs through the operation of the de novo pathway, following by the induction of γ-GCS mRNA, for which elevation of intracellular calcium is required.     

DNA radiolysis in DNA–protein complexes: a stochastic simulation of attack by hydroxyl radicals

Purpose: To propose an improved version of RADACK, a stochastic simulation of radiolytic attack on DNA, that takes into account the reactivity of each amino acid of a specifically bound protein with hydroxyl radicals. To apply it to the natural lactose operator–repressor complex taking advantage of recently reported structures. To compare the obtained probabilities of DNA strand break induction with those calculated with the previous versions and with an experimental pattern of strand break probabilities.

Materials and methods: Models of complexes close to the natural ones, derived from crystallography‐ and NMR‐based structures recently available in the PDB databank, were used. The specific chemical reactivity of each amino acid was introduced in the new version of RADACK (the reactivity model). The probabilities of strand break induction by the irradiation of the complex were calculated with this new version as well as with previous ones.

Results: The patterns of probabilities of strand break induction calculated with the improved version of RADACK were partially different from those obtained with previous versions. The patterns obtained for both, using putative models of natural complexes, were consistent with the experimental results, but some discrepancies were suggestive of slight differences between these structures and the real natural system. The crystallographic structure agreed best with the experimental results.

Conclusions: A new version of RADACK was validated that took into account the reactivity of atoms in both DNA and protein. The putative modelled structures of a natural lactose operator–repressor complex were discussed.     

Calculation of electron dose to target cells in a complex environment by Monte Carlo code “CELLDOSE”

Background  We used the Monte Carlo code “CELLDOSE” to assess the dose received by specific target cells from electron emissions in a complex environment. ¹³¹I in a simulated thyroid was used as a model. Methods  Thyroid follicles were represented by 170 μm diameter spherical units made of a lumen of 150 μm diameter containing colloidal matter and a peripheral layer of 10 μm thick thyroid cells. Neighbouring follicles are 4 μm apart. ¹³¹I was assumed to be homogeneously distributed in the lumen and absent in cells. We firstly assessed electron dose distribution in a single follicle. Then, we expanded the simulation by progressively adding neighbouring layers of follicles, so to reassess the electron dose to this single follicle implemented with the contribution of the added layers. Results  Electron dose gradient around a point source showed that the ¹³¹I electron dose is close to zero after 2,100 μm. Therefore, we studied all contributions to the central follicle deriving from follicles within 12 orders of neighbourhood (15,624 follicles surrounding the central follicle). The dose to colloid of the single follicle was twice as high as the dose to thyroid cells. Even when all neighbours were taken into account, the dose in the central follicle remained heterogeneous. For a 1-Gy average dose to tissue, the dose to colloidal matter was 1.168 Gy, the dose to thyroid cells was 0.982 Gy, and the dose to the inter-follicular tissue was 0.895 Gy. Analysis of the different contributions to thyroid cell dose showed that 17.3% of the dose derived from the colloidal matter of their own follicle, while the remaining 82.7% was delivered by the surrounding follicles. On the basis of these data, it is shown that when different follicles contain different concentrations of ¹³¹I, the impact in terms of cell dose heterogeneity can be important. Conclusion  By means of ¹³¹I in the thyroid as a theoretical model, we showed how a Monte Carlo code can be used to map electron dose deposit and build up the dose to target cells in a complex multi-source environment. This approach can be of considerable interest for comparing different radiopharmaceuticals as therapy agents in oncology.     

Computer evaluation of direct and indirect damage induced by free and DNA‐bound Iodine‐125 in the chromatin fibre

Purpose: When Iodine‐125 decays within chromatin, several in vivo experiments have shown that the radiobiological effects are caused mainly by indirect mechanisms and that more than one DNA double strand break (DSB) is produced per decay. We present calculations to evaluate the contribution of direct and indirect effects of radiation tracks to produce DNA damage induced by bound and free I‐125 in a model of chromatin DNA.

Materials and methods: A solenoid model of chromatin with 18 nucleosomal elements placed in bulk water (more than 600,000 atoms) is used where the initial I‐125 decay takes place. All physical and chemical events initiated by Auger and X‐rays were taken into account. The yields of single strand breaks (SSB) and DSB were derived using direct effects on DNA and indirect reactions of all radical species generated in the radiolysis of the bulk water.

Results: The distribution of damage complexity for free and DNA–bound I‐125 is presented. We obtained more than 1.3 DSB per decay, with nearly equal contributions from direct and indirect effects. However, for the most complex type of damage, located at the decay site, the direct effect is about 70% of the total number. To show the protective effect of histones, simulations were carried out with and without the presence of histones.     

Dose per unit cumulated activity (S-values) for e− and beta emitting radionuclides in cancer cell models calculated by Monte Carlo simulation

Cell dosimetry is relevant regarding new efforts in specific molecular radiotherapy using Auger, CE and beta emitters. Absorbed dose in cells can be obtained by means of the dose per unit cumulated activity (S-values), together with the activity distribution. In this work, Monte Carlo simulation codes PENELOPE and MCNPX were used to obtain cellular S-values for point and extended sources of electrons and beta emitting radionuclides in the nucleus of breast (MDA-MB231, MCF7) and prostate (PC3) cancer cell models.     

Induction of Chromosome Aberrations and Cell Killing in Syrian Hamster Fibroblasts by γ-rays,X-rays and Fast Neutrons

Summary

Chromosome aberrations were scored in BHK21 C13 Syrian hamster fibroblasts, exposed to ⁶⁰Co γ-rays, 250 kV X-rays, 15 MeV neutrons or neutrons of mean energy 2·1 MeV produced from the ⁹Be(d, n)¹⁰B reaction. The cells were irradiated in stationary phase, where they are concentrated in the G1 phase of the cell cycle. Within experimental uncertainty there was no detectable difference between the responses to ⁶⁰Co γ-rays and to 250 kV X-rays. The r.b.e. for the production of dicentrics, based on the ‘one-hit’ component of response, was (5 ± 2) for the 15 MeV neutrons and (12 ± 5) for the 2·1 MeV neutrons. For each radiation, a graph of the proportion of cells without a dicentric, centric ring or acentric fragment corresponded closely to the survival curve for stationary-phase cells obtained in the same experiment.     

Fast and accurate approach to γ-spectrum modelling: A validation study with a shielded/unshielded voluminous uranium sample

γ-Spectrometry is a basic and widely applied method in nuclear security and nuclear safeguards areas. For addressing the growing needs in education and training, in research and development as well as in practical γ-spectrometry in these areas, Monte Carlo based web-accessible γ-spectrum modelling tools have been developed for the European Commission’s Nucleonica portal at www.nucleonica.net. A recent validation study has demonstrated a good performance of the implemented simulation approach for modelling realistic γ-spectra from shielded and unshielded point-like sources. In the present work this approach has been further tested with the use of a 0.2 kg voluminous 4.46 wt% enriched uranium reference material and a portable 10% HPGe detector. The simulations for shielded and unshielded measurement conditions revealed a good agreement with the experimentally acquired γ-spectra, both for the detection efficiency values and for the overall spectrum shape and intensity in the energy range up to 2.6 MeV.     

DNA damage in cultured skin microvascular endothelial cells exposed to gamma rays and treated by the combination pentoxifylline and α-tocopherol

Purpose: This in vitro study aims at evaluating the effect of the combination of pentoxifylline (PTX) and trolox (Tx), the water-soluble analogue of α-tocopherol, on the oxidative state and DNA damage in dermal microvascular endothelial cells exposed to doses up to 10 Gy of ionizing radiation.

Materials and methods: Confluent primary cultures of dermal endothelial cells were gamma irradiated at 3 and 10 Gy, and 0.5 mM of both drugs, PTX and Tx, was added either before (15 min) or after (30 min or 24 h) irradiation. Reactive oxygen species (ROS), measured by the dichlorodihydrofluorescein diacetate assay, and DNA damage, assessed by the comet and micronucleus assays, were measured at different times after exposure (0 – 21 days).

Results: The PTX/Tx treatment decreased the early and delayed peak of ROS production by a factor of 2.8 in 10 Gy-irradiated cells immediately after irradiation and the basal level by a factor of 2 in non-irradiated control cells. Moreover, the level of DNA strand breaks, as measured by the comet assay, was shown to be reduced by half immediately after irradiation when the PTX/Tx treatment was added 15 min before irradiation. However, unexpectedly, it was decreased to a similar extent when the drugs were added 30 min after radiation exposure. This reduction was accompanied by a 2.2- and 3.6-fold higher yield in the micronuclei (MN) frequency observed on days 10 and 14 post-irradiation, respectively.

Conclusion: These results suggest that oxidative stress and DNA damage induced in dermal microvascular endothelial cells by radiation can be modulated by early PTX/Tx treatment. These drugs acted not only as radical scavengers, but they were also responsible for the increased MN frequency in 10 Gy-irradiated cells. Thus, these drugs may cause a possible interference with DNA repair processes.     

Beta–gamma coincidence counting efficiency and energy resolution optimization by Geant4 Monte Carlo simulations for a phoswich well detector

A single-channel phoswich well detector has been assessed and analysed in order to improve beta–gamma coincidence measurement sensitivity of ^131mXe and ^133mXe. This newly designed phoswich well detector consists of a plastic cell (BC-404) embedded in a CsI(Tl) crystal coupled to a photomultiplier tube (PMT). It can be used to distinguish 30.0-keV X-ray signals of ^131mXe and ^133mXe using their unique coincidence signatures between the conversion electrons (CEs) and the 30.0-keV X-rays. The optimum coincidence efficiency signal depends on the energy resolutions of the two CE peaks, which could be affected by relative positions of the plastic cell to the CsI(Tl) because the embedded plastic cell would interrupt scintillation light path from the CsI(Tl) crystal to the PMT. In this study, several relative positions between the embedded plastic cell and the CsI(Tl) crystal have been evaluated using Monte Carlo modeling for its effects on coincidence detection efficiency and X-ray and CE energy resolutions. The results indicate that the energy resolution and beta–gamma coincidence counting efficiency of X-ray and CE depend significantly on the plastic cell locations inside the CsI(Tl). The degraded X-ray and CE peak energy resolutions due to light collection efficiency deterioration by the embedded cell can be minimised. The optimum of CE and X-ray energy resolution, beta–gamma coincidence efficiency as well as the ease of manufacturing could be achieved by varying the embedded plastic cell positions inside the CsI(Tl) and consequently setting the most efficient geometry.     

Low-dose hypersensitivity of V79 cells under exposure to γ-rays and 4 He ions of different energies: survival and chromosome aberrations

Purpose : To examine the low-dose sensitivity of V79 cells under exposure to γ-rays and 4 He ions of different energies. Materials and methods : Cell survival and cytogenetic analysis using the Giemsa technique were studied following irradiation to doses of 0-3 Gy at the INFN-LNL facilities. Results : Low-dose hyper-radiosensitivity (HRS) of V79 was demonstrated after irradiation with γ-rays and α -particles of various linear energy transfers (LET) (58.9, 79.3 and 101.7 keV μ m -1) . Cytogenetic analysis showed an LET dependence of aberrations at a dose of 1Gy; the frequency of chromatid fragments appeared to vary with the number of α -particles traversing the cell nucleus. The results of both studies fit together to give a better understanding of so-called 'induced radioresistance' phenomenon. Conclusions : The mechanism of induced cellular radioresistance appears to be initiated after a certain amount of energy is deposited in the cell nucleus. This amount depends on both radiation quality and the number of particles traversing the cell.     

Radiation Quality of Tritium β-rays: Microdosimetric Distributions for Nanometer-size Targets in a Medium Containing Tritiated Water

Summary

To elucidate the characteristics of the action of tritium β-rays, the following parameters are derived: (a) electron slowing down spectra of primary electrons (β-rays) and δ-rays in a medium containing tritiated water; and (b) frequency distributions for the microdosimetric quantity j (number of effective primary events per track per target), f_j, for nanometer-size targets exposed to tritiated water. Features of the radiation quality of tritium β-rays are discussed by comparing the present results with those for ⁶⁰Co γ-rays and 7 MeV electrons. It is concluded that, although tritium β-rays, ⁶⁰Co γ-rays, and 7 MeV electrons are classified as the same low l.e.t. radiation, the radiation quality of tritium β-rays is considerably different from those of ⁶⁰Co γ-rays and 7 MeV electrons, and has specific features such as a high average l.e.t., a small total electron fluence per unit absorbed dose, and a different microdosimetric distribution, f_j, for nanometer-size targets.     

Reduced chromosome aberration complexity in normal human bronchial epithelial cells exposed to low-LET γ-rays and high-LET α-particles

Abstract

Purpose: Cells of the lung are at risk from exposure to low and moderate doses of ionizing radiation from a range of environmental and medical sources. To help assess human health risks from such exposures, a better understanding of the frequency and types of chromosome aberration initially-induced in human lung cell types is required to link initial DNA damage and rearrangements with transmission potential and, to assess how this varies with radiation quality.

Materials and methods: We exposed normal human bronchial lung epithelial (NHBE) cells in vitro to 0.5 and 1 Gy low-linear energy transfer (LET) γ-rays and a low fluence of high-LET α-particles and assayed for chromosome aberrations in premature chromosome condensation (PCC) spreads by 24-color multiplex-fluorescence in situ hybridization (M-FISH).

Results: Both simple and complex aberrations were induced in a LET and dose-dependent manner; however, the frequency and complexity observed were reduced in comparison to that previously reported in spherical cell types after exposure to comparable doses or fluence of radiation. Approximately 1–2% of all exposed cells were categorized as being capable of transmitting radiation-induced chromosomal damage to future NHBE cell generations, irrespective of dose.

Conclusion: One possible mechanistic explanation for this reduced complexity is the differing geometric organization of chromosome territories within ellipsoid nuclei compared to spherical nuclei. This study highlights the need to better understand the role of nuclear organization in the formation of exchange aberrations and, the influence three-dimensional (3D) tissue architecture may have on this in vivo.