Elizabeth A. Ainsbury

A semi-automated micronucleus-centromere assay to assess low-dose radiation exposure in human lymphocytes

Abstract Purpose: The in vitro micronucleus (MN) assay is a reliable method to assess radiation-induced chromosomal damage in human peripheral blood lymphocytes. It is used to evaluate in vivo radiation over-exposure and to assess in vitro chromosomal radiosensitivity. A limitation of the MN assay is the relatively high and variable spontaneous MN frequency that restricts low-dose estimation to doses of about 0.3 gray (Gy). As radiation-induced MN mainly contain acentric fragments and spontaneous MN originate from lagging chromosomes, both MN types can be distinguished from each other by using fluorescence in situ hybridisation (FISH) with a pan-centromeric probe. The aim of this study was to investigate if the sensitivity, reliability and processing time of the MN assay can be enhanced by combining the automated MN assay with pan-centromere scoring. Materials and methods: Blood samples from 10 healthy donors were irradiated in vitro with low doses of gamma-rays. Dose response curves were determined for fully-automated and semi-automated MN scoring and semi-automated scoring of centromere negative MN (MNCM−). Results: A good correlation was obtained between fully-automated and semi-automated MN scoring (r2 = 0.9973) and between fully automated MN scoring and semi-automated scoring of MNCM− (r2 = 0.998). With the Wilcoxon test, a significant p value was obtained between 0 and 0.2 Gy for the fully-automated MN analysis, between 0 and 0.1 Gy for semi-automated MN analysis and between 0 and 0.05 Gy for semi-automated scoring of MNCM−. Conclusion: The semi-automated micronucleus-centromere assay combines high-speed MN analysis with a more accurate assessment in the low-dose range which makes it of special interest for large-scale radiation applications.

Exposure to low level GSM 935 MHz radiofrequency fields does not induce apoptosis in proliferating or differentiated murine neuroblastoma cells

The aim of this study was to investigate whether radiofrequency (RF) fields characteristic of mobile phones at non-thermal levels can induce apoptosis in murine neuroblastoma (N2a) cells in both proliferating and differentiated states. Cells were exposed continuously for 24 h to one of the three 935-MHz RF signals: global system for mobile communication (GSM) basic, GSM talk and a continuous wave, unmodulated signal; all at a specific energy absorption rate of 2 W kg(-1). The measured increase in temperature of the cells due to the RF fields was around 0.06 degrees C. At a number of time points between 0 and 48 h post-exposure, the cells were assessed for apoptosis under a fluorescence microscope using three independent assays: Annexin V, caspase activation and in situ end-labelling. No statistically significant differences in apoptosis levels were observed between the exposed and sham-exposed cells using the three assays at any time point post-exposure. These data suggest that RF exposures, characteristic of GSM mobile phones, do not significantly affect the apoptosis levels in proliferating and differentiated murine neuroblastoma cell line N2a.

Validation of semi-automatic scoring of dicentric chromosomes after simulation of three different irradiation scenarios.

AbstractLarge scale radiological emergencies require high throughput techniques of biological dosimetry for population triage in order to identify individuals indicated for medical treatment. The dicentric assay is the “gold standard” technique for the performance of biological dosimetry, but it is very time consuming and needs well trained scorers. To increase the throughput of blood samples, semi-automation of dicentric scoring was investigated in the framework of the MULTIBIODOSE EU FP7 project, and dose effect curves were established in six biodosimetry laboratories. To validate these dose effect curves, blood samples from 33 healthy donors (>10 donors/scenario) were irradiated in vitro with 60Co gamma rays simulating three different exposure scenarios: acute whole body, partial body, and protracted exposure, with three different doses for each scenario. All the blood samples were irradiated at Ghent University, Belgium, and then shipped blind coded to the participating laboratories. The blood samples were set up by each lab using their own standard protocols, and metaphase slides were prepared to validate the calibration curves established by semi-automatic dicentric scoring. In order to achieve this, 300 metaphases per sample were captured, and the doses were estimated using the newly formed dose effect curves. After acute uniform exposure, all laboratories were able to distinguish between 0 Gy, 0.5 Gy, 2.0, and 4.0 Gy (p < 0.001), and, in most cases, the dose estimates were within a range of ± 0.5 Gy of the given dose. After protracted exposure, all laboratories were able to distinguish between 1.0 Gy, 2.0 Gy, and 4.0 Gy (p < 0.001), and here also a large number of the dose estimates were within ± 0.5 Gy of the irradiation dose. After simulated partial body exposure, all laboratories were able to distinguish between 2.0 Gy, 4.0 Gy, and 6.0 Gy (p < 0.001). Overdispersion of the dicentric distribution enabled the detection of the partial body samples; however, this result was clearly dose-dependent. For partial body exposures, only a few dose estimates were in the range of ± 0.5 Gy of the given dose, but an improvement could be achieved with higher cell numbers. The new method of semi-automation of the dicentric assay was introduced successfully in a network of six laboratories. It is therefore concluded that this method can be used as a high-throughput screening tool in a large-scale radiation accident.

Biological dosimetry assessments of a serious radiation accident in Bulgaria in 2011

In 2011, a serious radiation accident occurred in Stamboliyski, Bulgaria, in an industrial sterilisation facility using very-high-activity (60)Co sources. For the five persons accidentally exposed, biological dosimetry based on dicentric analysis was performed in Sofia and in Paris, where the patients were transferred for treatment. Before completing the chromosomal dose assessment, and for the most exposed person, a preliminary cytogenetic evaluation based on electronically transmitted metaphase images was made. The averaged acute whole-body dose estimates for the five patients ranged from 5.2 to 1.2 Gy, and good agreement was obtained between the two laboratories. The patients were also assessed by their prodromal responses and depressed blood cell counts over the first week. The cytogenetic dose estimates were in good accord with those derived from the blood counts, and both techniques indicated that, for the two most seriously exposed persons both techniques indicated that the initial prodromal reactions had suggested somewhat less severe exposure.

Intestinal tumours induced in ApcMin/+ mice by X-rays and neutrons

Purpose: To compare the development of intestinal adenomas following neutron and X-ray exposure of ApcMin/+ mice (Apc – adenomatous polyposis coli; Min – multiple intestinal neoplasia). Materials and methods: Adult mice were exposed to acute doses of X-rays or fission neutrons. Tumour counting was undertaken 200 days later and samples were taken for Loss of Heterozygosity (LOH) analysis. Results: Tumour numbers (adenomas and microadenomas) increased by 1.4-fold, 1.7-fold, 2.7-fold and 9-fold, after 0.5, 1, 2 and 5 Gy X-rays, respectively, and by 2.4-fold and 5.7-fold, after 0.5 and 1 Gy fission neutrons, respectively. LOH analysis of tumours from neutron-exposed mice showed that 63% had lost Apc and 90% (cf. 53% in controls) had lost D18mit84, a marker for Epb4.1l4a/NBL4 (erythrocyte protein band 4.1-like 4a/novel band 4.1-like 4), known to be involved in the Wnt (wingless-related mouse mammary tumour virus integration site) pathway. Some tumours from neutron-exposed mice appeared to have homozygous loss of some chromosomal markers. Conclusions: X-ray or fission neutron irradiation results in strongly enhanced tumour multiplicities. Comparison of tumour yields indicated a low Relative Biological Effectiveness of around 2–8 for fission neutrons compared with X-rays. LOH in intestinal tumours from neutron-exposed mice appeared to be more complex than previously reported for tumours from X-irradiated mice.

Dose Estimation in Radiation Cytogenetics

Throughout the radiation cytogenetics community, a core group of methods exists to produce an estimate of radiation dose from an observed yield of DNA damage in blood. Mathematical and statistical analysis is extremely important for accurate assessment of data and results, and a number of classical statistical methods are commonly employed. However, the large number of statistical techniques, and the complexity of the methods, can lead to errors in data analysis and misinterpretation of results. Cytogenetics dose estimation software has been developed to simplify mathematical and statistical analysis of cytogenetic data. “Dose Estimate” is a collection of mathematical and statistical methods based on the cytogenetics methods and programs written by Alan Edwards, David Papworth, and others. Details of the biological and mathematical techniques used in the software will be presented, including maximum likelihood estimation of yield curve coefficients for the dicentric or translocation assays. Proposals for ...

Successful Proposals, Fund Raising, and Project Leadership

A brief description of the Fundraising, Grant Writing, and Project Leadership workshop at the Third IUPAP International Conference of Women in Physics is presented. The workshop’s purpose was to assist delegates in developing skills in successful proposal writing, fund raising, and project leadership. Handouts and talks were given with information on funding sources in different regions. Talks on grant writing and leadership by Beverly Hartline and J. Grace Lin led to active participation and brainstorming discussions. Based on the discussions a list of recommendations is outlined in this paper. This information is also available at the IUPAP Working Group of Women in Physics website (http://wgwip.df.uba.ar).