Jayne Moquet

DNA damage foci: Meaning and significance

The discovery of DNA damage response proteins such as γH2AX, ATM, 53BP1, RAD51, and the MRE11/RAD50/NBS1 complex, that accumulate and/or are modified in the vicinity of a chromosomal DNA double‐strand break to form microscopically visible, subnuclear foci, has revolutionized the detection of these lesions and has enabled studies of the cellular machinery that contributes to their repair. Double‐strand breaks are induced directly by a number of physical and chemical agents, including ionizing radiation and radiomimetic drugs, but can also arise as secondary lesions during replication and DNA repair following exposure to a wide range of genotoxins. Here we aim to review the biological meaning and significance of DNA damage foci, looking specifically at a range of different settings in which such markers of DNA damage and repair are being studied and interpreted. Environ. Mol. Mutagen. 56:491–504, 2015.

935 MHz cellular phone radiation. An in vitro study of genotoxicity in human lymphocytes.

Purpose: The possibility of genotoxicity of radiofrequency radiation (RFR) applied alone or in combination with x-rays was investigated in vitro using several assays on human lymphocytes. The chosen specific absorption rate (SAR) values are near the upper limit of actual energy absorption in localized tissue when persons use some cellular telephones. The purpose of the combined exposures was to examine whether RFR might act epigenetically by reducing the fidelity of repair of DNA damage caused by a well-characterized and established mutagen. Methods: Blood specimens from 14 donors were exposed continuously for 24 h to a Global System for Mobile Communications (GSM) basic 935 MHz signal. The signal was applied at two SAR; 1 and 2 W/Kg, alone or combined with a 1-min exposure to 1.0 Gy of 250 kVp x-rays given immediately before or after the RFR. The assays employed were the alkaline comet technique to detect DNA strand breakage, metaphase analyses to detect unstable chromosomal aberrations and sister chromatid exchanges, micronuclei in cytokinesis-blocked binucleate lymphocytes and the nuclear division index to detect alterations in the speed of in vitro cell cycling. Results: By comparison with appropriate sham-exposed and control samples, no effect of RFR alone could be found for any of the assay endpoints. In addition RFR did not modify any measured effects of the x-radiation. Conclusions: This study has used several standard in vitro tests for chromosomal and DNA damage in Go human lymphocytes exposed in vitro to a combination of x-rays and RFR. It has comprehensively examined whether a 24-h continuous exposure to a 935 MHz GSM basic signal delivering SAR of 1 or 2 W/Kg is genotoxic per se or whether, it can influence the genotoxicity of the well-established clastogenic agent; x-radiation. Within the experimental parameters of the study in all instances no effect from the RFR signal was observed.

Comparison of Established and Emerging Biodosimetry Assays

Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3–0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5–4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools.

Intercomparison of translocation and dicentric frequencies between laboratories in a follow-up of the radiological accident in Estonia

Purpose : To perform an interlaboratory comparison of FISH chromosome painting and to study the time-course of translocations and dicentrics in three accident victims exposed to radiation. Also, to use the data in the validation of the FISH technique as a retrospective dosimeter. Materials and methods : Twelve blood samples were collected during 4 years from three subjects exposed to radiation in an accident in Estonia in 1994 involving γ -radiation from a 137Cs source. Two of the subjects were exposed during approximately 7 h, both receiving a protracted dose of about 1 Gy and also localized exposure. The third subject received a protracted whole-body dose of 2.7Gy during 4 weeks as well as a short-term partialbody dose. Preparations from 48-h metaphase cultures were painted by the FISH technique using routine methods and probe cocktails in four laboratories. Samples from each subject were analysed in two different laboratories that used different combinations of whole chromosome probes. The PAINT nomenclature was applied when recording chromosome aberrations. Results : The intercomparison of FISH analysis data showed reasonable similarities between laboratories, the largest discrepancy being 21% in the frequency of two-way translocations in subject 3. Half-time calculations, based on combined data sets from two laboratories, showed that dicentrics decreased rapidly with half-times of approximately 2 years. In all cases, the initial dicentric yields were lower than the initial translocation yields. During the 4-year follow-up, the frequencies of all translocations in cells containing only simple rearrangements fell on average to about 65% of their initial value. Two-way translocations were slightly more persistent than all translocations. The average halftime was about 8 years for two-way translocations and around 6 years for all translocations. Cells containing complex rearrangements were few in number and they disappeared with time. In general, the inclusion of complex cells caused a more rapid fall in aberration yield. Conclusions : In general, the results imply that relatively consistent scoring data were obtained with different chromosome painting protocols. They also support the idea that the reduction of translocations with time is associated with partial-body irradiation.

The 60Co gamma ray dose-response for chromosomal aberrations in human lymphocytes analysed by FISH; applicability to biological dosimetry.

PURPOSE To investigate the in vitro dose-response for 60Co irradiated human lymphocytes assayed by FISH, and to consider how this may be applied to retrospective dosimetry. METHOD Blood was irradiated with doses in the range 0.25-4.0 Gy. Cultured lymphocytes were scored for all stable and unstable aberrations involving painted chromosomes 2, 3 and 5 and, in addition, all unstable aberrations in the counterstained chromosomes. A pancentromeric probe was included. RESULTS The relative numbers of painted and full genome dicentrics agreed well with the Lucas hypothesis for calculating genome equivalence. The involvement of each painted chromosome in exchanges agreed with their relative arm lengths. The dose-response relationship fitted well to the linear quadratic model; Y=(0.9 x 10(-2))D+(6.5 x 10(-2))D2 where D is the dose in Gy for the incidence Y, of all one plus two-way translocations in all cells corrected for genome equivalence. Complex rearrangements also became more frequent with increasing dose. A correlation was noted between the distributions of dicentrics and translocations among the cells and this was entirely due to complexes. CONCLUSIONS For retrospective dosimetry it is recommended to use an in vitro dose-response for apparently simple translocations in stable (Cs) cells. To date, acute linear yield coefficients from FISH data carry statistical uncertainties too large for useful application to retrospective dosimetry of persons exposed to chronic or low doses. As an interim measure it is suggested that one may derive a linear term from full genome dicentrics corrected by a factor representing the translocation to dicentric ratio.

Realising the European network of biodosimetry: RENEB—status quo

Creating a sustainable network in biological and retrospective dosimetry that involves a large number of experienced laboratories throughout the European Union (EU) will significantly improve the accident and emergency response capabilities in case of a large-scale radiological emergency. A well-organised cooperative action involving EU laboratories will offer the best chance for fast and trustworthy dose assessments that are urgently needed in an emergency situation. To this end, the EC supports the establishment of a European network in biological dosimetry (RENEB). The RENEB project started in January 2012 involving cooperation of 23 organisations from 16 European countries. The purpose of RENEB is to increase the biodosimetry capacities in case of large-scale radiological emergency scenarios. The progress of the project since its inception is presented, comprising the consolidation process of the network with its operational platform, intercomparison exercises, training activities, proceedings in quality assurance and horizon scanning for new methods and partners. Additionally, the benefit of the network for the radiation research community as a whole is addressed.

A Study to Verify a Reported Excess of Chromosomal Aberrations in Blood Lymphocytes of Namibian Uranium Miners

Abstract Lloyd, D. C., Lucas, J. N., Edwards, A. A., Deng, W., Valente, E., Hone, P. A. and Moquet, J. E. A Study to Verify a Reported Excess of Chromosomal Aberrations in Blood Lymphocytes of Namibian Uranium Miners. Radiat. Res. 155, 807–815 (2001). This report describes a study to verify an earlier report of excess chromosomal damage in the blood lymphocytes of uranium miners. Coded blood samples from 10 miners and 10 controls were analyzed conventionally for unstable aberrations and by FISH for translocations. Conventional analysis, scoring 1000 metaphases per subject, showed no significant difference between miners and controls in the frequencies of chromosome- and chromatid-type aberrations. Investigators at two laboratories undertook FISH analyses, each scoring 4000 metaphases per subject. When the data from each laboratory were examined separately, one found slightly more translocations in the miners while the other found fewer. In neither case was the difference significant at the 95% level of confidence. Combining the data likewise showed no significant excess of damage in the miners. This applied to simple one- and two-way translocations and to cells with complex exchanges. There was no correlation between levels of translocations and total lifetime doses from occupational and/or background irradiation. A borderline significant excess of rogue cells was found in the miners. This may be a chance observation, as these rare, highly abnormal cells are considered to be unrelated to radiation exposure and are probably due to a virus. The overall conclusion is that the frequency of chromosomal damage in the miners did not exceed that in the controls. Therefore, the result of the earlier study was not confirmed.

Laboratory Intercomparison of the Dicentric Chromosome Analysis Assay

The study design and obtained results represent an intercomparison of various laboratories performing dose assessment using the dicentric chromosome analysis (DCA) as a diagnostic triage tool for individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25–5 Gy) as well as blind samples (0.1–6.4 Gy) were sent to the participants. DCA was performed according to established protocols. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) was calculated and radiation doses were merged into four triage categories reflecting clinical aspects to calculate accuracy, sensitivity and specificity. The earliest report time was 2.4 days after sample arrival. DCA dose estimates were reported with high and comparable accuracy, with MAD values ranging between 0.16–0.5 Gy for both manual and automated scoring. No significant differences were found for dose estimates based either on 20, 30, 40 or 50 cells, suggesting that the scored number of cells can be reduced from 50 to 20 without loss of precision of triage dose estimates, at least for homogenous exposure scenarios. Triage categories of clinical significance could be discriminated efficiently using both scoring procedures.

Inter- and intra-laboratory comparison of a multibiodosimetric approach to triage in a simulated, large scale radiation emergency

Abstract Purpose: The European Unions Seventh Framework Programme-funded project ‘Multi-disciplinary biodosimetric tools to manage high scale radiological casualties’ (MULTIBIODOSE) has developed a multiparametric approach to radiation biodosimetry, with a particular emphasis on triage of large numbers of potentially exposed individuals following accidental exposures. In November 2012, an emergency exercise took place which tested the capabilities of the MULTIBIODOSE project partners. The exercise described here had a dual purpose: Intercomparison of (i) three biodosimetric assays, and (ii) the capabilities of the seven laboratories, with regards to provision of triage status for suspected radiation exposed individuals. Materials and methods: Three biological dosimetry tools – the dicentric, micronucleus and gamma-H2AX (the phosphorylated form of member X of histone H2A, in response to DNA double-strand breaks) foci assays – were tested, in addition to provision of the triage status results (low exposure: < 1 Gy; medium exposure: 1–2 Gy; high exposure: > 2 Gy) by the MULTIBIODOSE software. The exercise was run in two modes: An initial triage categorisation of samples (based on the first dose estimates for each assay received from each laboratory) followed by collation of the full set of estimated doses (all the results from all modes of each assay carried out by the participating laboratories) calculated using as many modes of operation as possible of the different assays developed during the project. Simulated acute whole body and partial body exposures were included. Results: The results of the initial triage categorisation and the full comparison of assays and methods within and between laboratories are presented here. Conclusions: The data demonstrate that the MULTIBIODOSE approach of applying multiparametric tools to radiation emergencies is valid and effective.

Interlaboratory Variation in Scoring Dicentric Chromosomes in a Case of Partial-Body X-Ray Exposure: Implications for Biodosimetry Networking and Cytogenetic “Triage Mode” Scoring

Abstract Ainsbury, E. A., Livingston, G. K., Abbott, M. G., Moquet, J. E., Hone, P. A., Jenkins, M. S., Christensen, D. M., Lloyd, D. C. and Rothkamm, K. Interlaboratory Variation in Scoring Dicentric Chromosomes in a Case of Partial-Body X-Ray Exposure: Implications for Biodosimetry Networking and Cytogenetic “Triage Mode” Scoring. The international radiation biodosimetry community has recently been engaged in activities focused on establishing cooperative networks for biodosimetric triage for radiation emergency scenarios involving mass casualties. To this end, there have been several recent publications in the literature regarding the potential for shared scoring in such an accident or incident. We present details from a medical irradiation case where two independently validated laboratories found very different yields of dicentric chromosome aberrations. The potential reasons for this disparity are discussed, and the actual reason is identified as being the partial-body nature of the radiation exposure combined with differing criteria for metaphase selection. In the context of the recent networking activity, this report is intended to highlight the fact that shared scoring may produce inconsistencies and that further validation of the scoring protocols and experimental techniques may be required before the networks are prepared to deal satisfactorily with a radiological or nuclear emergency. Also, the findings presented here clearly demonstrate the limitations of the dicentric assay for estimating radiation doses after partial-body exposures and bring into question the usefulness of rapid “triage mode” scoring in such exposure scenarios.