Use of a plasma focus device to study pulsed x-ray effects on peripheral blood lymphocytes : Analysis of chromosome aberrations

X-ray pulses (full width at half maximum ∼ 90 ns, dose rate ∼ 107 Gy s−1) were used to irradiate the monolayer of peripheral blood mononucleated cells using the PF-2kJ kilojoule plasma focus device. Four different exposure conditions were evaluated using 5, 10, 20, and 40 pulses, with the mean dose measured by TLD-100 being 0.12 ± 0.02 mGy, 0.14 ± 0.03 mGy, 0.22 ± 0.06 mGy, and 0.47 ± 0.09 mGy, respectively. Cytogenetic analysis showed an increase in all types of chromosomal aberrations following exposure to x-ray pulses. The distribution of dicentrics and centric rings was overdispersed after 5, 10, 20, and 40 pulses. Additionally, after 20 and 40 pulses, the presence of tricentric chromosomes is detected. Chromosome aberration frequencies found in this study were always higher than the estimated frequencies of chromosome aberrations using published dose-effect curves for conventional radiation sources. The overdispersion observed, the elevated maximum relative biological effectiveness (RBEM) and the presence of tricentric chromosomes at the relatively low doses of exposure (<0.5 Gy) seem to indicate that low doses of pulsed x-rays of low energy show similar biological effects as those observed for high-LET radiation. X-ray pulses emitted by PF-2kJ were found to be more efficient in inducing chromosome aberrations, even more than α particles

RENEB intercomparisons applying the conventional Dicentric Chromosome Assay (DCA)

Purpose: Two quality controlled inter-laboratory exercises were organized within the EU project ‘Realizing the European Network of Biodosimetry (RENEB)’ to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network. Materials and methods: The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants. Results: The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point. Conclusions: In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners

FANCD2 depletion does not modulate TLS or checkpoint markers after UV irradiation.

<p>A) W.B. showing the extent of PCNA ubiquitination in control and D2 depleted samples after the indicated doses of UV irradiation in U2OS cells. Images belong to lanes within the same gel and correspond to the same exposure. Quantification of Ubi-PCNA levels 6 hours post-UV is shown on the right. B) Percentage of U2OS cells with more than 10 GFP-Pol η foci at the indicated times after UV radiation (5 J/m²). C) W.B. showing phospho-Chk1 (S3545), Chk1, p53 and p21 levels in U2OS transfected with control and FANCD2 siRNAs. Images belong to lanes within the same gel and correspond to the same exposure. Quantifications of p-Chk1, p53 and p21 normalized to KU70 for the 6-hours´ time point are shown on the right. Figure is representative of 3 independent experiments for each panel.</p

FANCD2 facilitates the recruitment of Rad51 to UV-damaged DNA and the activation of sister chromatid exchanges.

<p>A) Schematics and representative image of the recruitment of Rad51 to UV-irradiated sub-nuclear regions (visualized with γH2AX staining). B) Rad51 recruitment to damaged nuclear regions in U2OS cells transfected with control and D2 siRNAs, and C) in PD20 and PD20+D2 samples after UV irradiation (5 J/m²). D) Representative panel and SCE quantification in U2OS cells transfected with control and D2 siRNA (1.5 J/m²). E) 53BP1 and γH2AX focal organization in control and UV-treated cells (5 J/m²) transfected with FANCD2 or control siRNA. F) Focal organization of 53BP1 after UV irradiation (5 J/m²- solid color columns) and MMC treatment (40 ng/ml- striped columns) in U2OS cells transfected with control and D2 siRNA. The percentages of cells with 53BP1 foci in both UV- and MMC-treated samples are expressed as folds compared to untreated cells. Fold increases with respect to controls are shown below in black (UV) and grey (MMC). Significant differences for UV-treatment are shown (for MMC, ***p<0.001 at 24hrs). Figures are representative of 3 independent experiments.</p

FANCD2 is activated but it is not required for cell survival after UV irradiation.

<p>A) Western blot (W.B.) of FANCD2 (D2) in U2OS and PD20 cells expressing D2 (PD20+D2). B) Flow cytometry analysis of U2OS cells transfected with control or D2 siRNA after UV irradiation (5 J/m²) and MMC (40 ng/ml). Samples were collected 72 hours after DNA damage induction C) Clonogenic assay in U2OS cells transfected with control and D2 siRNA and treated with the indicated doses of UV irradiation and MMC. D) Surviva (Cell titer Glo) assay in PD20 and PD20+D2 cells treated with the indicated doses of UV irradiation and MMC. In all cases, the survival rate was calculated with respect to untreated samples within the same curve. For each panel, three independent experiments were analyzed obtaining similar results. For all figures in this manuscript: significance of the differences are: *p<0.1; **p<0.01; ***p<0.001; when the p value is not shown the difference is not statistically significant. Error bars represent SEM (standard error of the mean).</p

Chromosome aberrations caused by UV irradiation of FANCD2-depleted cells are completely reverted by NHEJ inactivation.

<p>A) Pulse field gel electrophoresis showing the levels of DSB formation after 24 hours of UV irradiation in U2OS transfected with the indicated siRNA. Data quantification is shown underneath the PFGE image. B) MN accumulation in binucleated cells; C) gaps and breaks and D) complex chromatidic exchanges. Two independent experiments were analyzed obtaining similar results.</p

FANCD2 prevents gross chromosome rearrangements after UV irradiation.

<p>A) Representative binucleated cell with MN. B) MN accumulation in U2OS cells transfected with control and D2 siRNA after UV irradiation (5 J/m²). C) W.B. showing the levels of Ubi-D2 in PD20, PD20+D2 and GM00637 cells. D) MN accumulation in PD20, PD20+D2 and GM00637 cells after UV irradiation (5 J/m²). E) Gaps + breaks and F) complex chromatidic exchange accumulation in U2OS transfected with control and D2 siRNA after UV irradiation (1.5 J/m²). Three independent experiments were analyzed obtaining similar results.</p

Interlaboratory comparison of dicentric chromosome assay using electronically transmitted images

The bottleneck in data acquisition during biological dosimetry based on a dicentric assay is the need to score dicentrics in a large number of lymphocytes. One way to increase the capacity of a given laboratory is to use the ability of skilled operators from other laboratories. This can be done using image analysis systems and distributing images all around the world. Two exercises were conducted to test the efficiency of such an approach involving 10 laboratories. During the first exercise (E1), the participant laboratories analysed the same images derived from cells exposed to 0.5 and 3 Gy; 100 images were sent to all participants for both doses. Whatever the dose, only about half of the cells were complete with well-spread metaphases suitable for analysis. A coefficient of variation (CV) on the standard deviation of ̃15 % was obtained for both doses. The trueness was better for 3 Gy (0.6 %) than for 0.5 Gy (37.8 %). The number of estimated doses classified as satisfactory according to the z-score was 3 at 0.5 Gy and 8 at 3 Gy for 10 dose estimations. In the second exercise, an emergency situation was tested, each laboratory was required to score a different set of 50 images in 2 d extracted from 500 downloaded images derived from cells exposed to 0.5 Gy. Then the remaining 450 images had to be scored within a week. Using 50 different images, the CVon the estimated doses (79.2 %) was not as good as in E1, probably associated to a lower number of cells analysed (50 vs. 100) or from the fact that laboratories analysed a different set of images. The trueness for the dose was better after scoring 500 cells (22.5 %) than after 50 cells (26.8 %). For the 10 dose estimations, the number of doses classified as satisfactory according to the z-score was 9, for both 50 and 500 cells. Overall, the results obtained support the feasibility of networking using electronically transmitted images. However, before its implementation some issues should be elucidated, such as the number and resolution of the images to be sent, and the harmonisation of the scoring criteria. Additionally, a global website able to be used for the different regional networks, like Share Points, will be desirable to facilitate worldwide communication.Fil: García, O.. Centro de Protección e Higiene de las Radiaciones; CubaFil: Di Giorgio, Marina. Autoridad Regulatoria Nuclear. Gerencia Apoyo Científico Técnico; ArgentinaFil: Vallerga, María Belén. Autoridad Regulatoria Nuclear. Gerencia Apoyo Científico Técnico; ArgentinaFil: Radl, Analía. Autoridad Regulatoria Nuclear. Gerencia Apoyo Científico Técnico; ArgentinaFil: Taja, María Rosa. Autoridad Regulatoria Nuclear. Gerencia Apoyo Científico Técnico; ArgentinaFil: Seoane, Analia Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: de Luca, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Stuck Oliveira, Mónica. Instituto de Radioprotección y Dosimetría; BrasilFil: Valdivia, Patricia. Comision Chilena de Energia Nuclear; ChileFil: Lamadrid, Ana Ilsa. Centro de Proteccion E Higiene de Las Radiaciones; CubaFil: González, Jorge Ernesto. Centro de Proteccion E Higiene de Las Radiaciones; CubaFil: Romero, I.. Centro de Proteccion E Higiene de Las Radiaciones; CubaFil: Mandina, Tania. Centro de Proteccion E Higiene de Las Radiaciones; CubaFil: Pantelias, G.. National Center For Scientific Research ‘demokritos’; GreciaFil: Terzoudi, G.. National Center For Scientific Research ‘demokritos’; GreciaFil: Guerrero Carbajal, Citlalo. Instituto Nacional de Investigaciones Nucleares; MéxicoFil: Arceo Maldonado, Carolina. Instituto Nacional de Investigaciones Nucleares; MéxicoFil: Espinoza, Marco. Instituto Peruano de Energia Nuclear; PerúFil: Oliveros, Nilda. Universidad Nacional Mayor de San Marcos; PerúFil: Martinez Lopez, Wilner. Instituto Investigaciones Biologicas Clemente Estable; UruguayFil: Di Tomasso, Maria Vittoria. Instituto Investigaciones Biologicas Clemente Estable; UruguayFil: Mendez, Leticia Jesica. Instituto Investigaciones Biologicas Clemente Estable; UruguayFil: Puig, Nora Raquel. Universitat Autònoma de Barcelona; EspañaFil: Roy, Laurence. Institut de Radioprotection Et de Surete Nucleaire; FranciaFil: Barquinero, J.F.. Institut de Radioprotection Et de Surete Nucleaire; Franci

RENEB intercomparisons applying the conventional Dicentric Chromosome Assay (DCA)

10.1080/09553002.2016.1233370International Journal of Radiation Biology93120-2